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Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422
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Sample Chapter Microbiology for Nurses 3e by Sood To Order Call Sms at 91-8527622422

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  • 1. C H A P T E R24The CocciL E A R N I N G O B J E C T I V E SAfter reading this chapter, the student should be able to:• Describe the morphology, culture, biochemical reactions, diseases, and methods of treatmentand prevention of the following cocci bacteria: staphylococcus, streptococcus, enterococcus,pneumococcus, meningococcus and gonococcus.C H A P T E R O U T L I N EIntroduction 148Staphylococcus aureus 149Streptococcus 153Streptococcus pyogenes 154Enterococcus 157Pneumococcus 159Meningococcus 161Gonococcus 163Things to Remember 166Revision Questions 166INTRODUCTIONStaphylococci are the commonest cause of localized suppurative lesions in man. There are manyspecies of medical importance in genus Staphylococcus. Of these, Staphylococcus aureus isdescribed hereunder.Chapter-24.indd 148Chapter-24.indd 148 5/28/2013 11:00:49 AM5/28/2013 11:00:49 AM
  • 2. THE COCCI 149Staphylococcus aureusMorphologyThe genus name has originated from the Greek word, Staphyle meaning ‘a bunch of grapes’.Staphylococci are spherical Gram-positive cocci arranged in grapelike clusters (Fig. 24.1a and b).They are nonmotile, aerobic or facultatively anaerobic. During binary fission, the staphylococcidivide along both longitudinal and horizontal planes, with daughter cells tending to remain in closeproximity leading to the formation of characteristic clusters.(a) (b)FIGURE 24.1 (a) Staphylococci (in clusters). (b) Staphylococci (Gram-positive cocci in clusters).CultureThey grow on most laboratory media, yielding circular, smooth and opaque colonies within atemperature range of 10–42ºC, the optimal temperature and pH being 37ºC and 7.4–7.6,respectively.Solid MediaNutrient agar After 12–24 hour incubation, the colo-nies are 2–4 mm in diameter (pinhead), circular,smooth, convex and easily emulsifiable. Most of thestrains produce golden yellow pigment. The pigment isnot diffusible into the medium.Blood agar Same as in nutrient agar along with thecharacteristic beta-haemolysis seen around the growth(Fig. 24.2).MacConkey’s agar Colonies are small and pink dueto lactose fermentation.Mannitol salt agar This is both a selective and anindicator medium for staphylococcus. Yellow-colouredcolonies are seen on this medium due to fermentationof mannitol by most strains of Staphylococcus aureus.FIGURE 24.2 Growth of S. aureus onblood agar.Chapter-24.indd 149Chapter-24.indd 149 5/28/2013 11:00:49 AM5/28/2013 11:00:49 AM
  • 3. 150 MICROBIOLOGY FOR NURSESLiquid MediaUniform turbidity is produced in peptone water and nutrient broth.Biochemical ReactionsStaphylococcus aureus is catalase positive and oxidase negative. It ferments a number of sugarsproducing acid without gas.The following characteristics help to distinguish a pathogenic strain of staphylococcus fromother nonpathogenic strains:1. Beta-type haemolysis on blood agar2. Production of a golden yellow pigment3. Coagulase production4. Mannitol fermentation5. Gelatin liquefaction6. Phosphatase production7. Production of deoxyribonuclease8. Tellurite reductionResistanceStaphylococci are more resistant among the nonsporing bacteria. They survive in dried pus for 2–3months. Most staphylococci are killed at 62ºC for 30 minutes but some may require 80ºC for 1hour.Antigenic StructureCapsuleSome strains of Staphylococcus aureus possess capsule and inhibit phagocytosis. The capsule iscomposed of polysaccharide. Capsulated strains tend to be more virulent.PeptidoglycanPeptidoglycan is a polysaccharide polymer that provides rigidity to the cell wall.Teichoic AcidIt is a major antigenic determinant of all strains of Staphylococcus aureus.Protein AIt is a cell wall component of most strains of Staphylococcus aureus, especially Cowan I strains(also used in coagglutination tests).Chapter-24.indd 150Chapter-24.indd 150 5/28/2013 11:00:49 AM5/28/2013 11:00:49 AM
  • 4. THE COCCI 151Toxins and EnzymesStaphylococcus aureus forms a number of toxins and enzymes. They are important virulence factorsof the organism for producing a disease in the host.ToxinsToxins include haemolysins, enterotoxins (the toxins responsible for staphylococcal food poisoning),leucocidin, toxic shock syndrome toxin (TSST) and exfoliative (epidermolytic) toxin.EnzymesIt produces a number of enzymes that are related to the virulence: coagulase, phosphatase anddeoxyribonuclease.Coagulase It has a property to clot human or rabbit plasma. Coagulase is secreted free into theculture medium or bound to the cell wall.Staphylococci are divided (based on the production of enzyme coagulase) into Staphylococcusaureus (coagulase producing) and the coagulase-negative staphylococci (coagulase nonproduc-ers). Detection of coagulase can be done by slide or tube method.Slide coagulation test This test detects the bound coagulase. The growth from agar plate isemulsified in two drops of normal saline on a slide. After ruling out spontaneous agglutination, adrop of undiluted human or rabbit plasma is added to one of the suspension and mixed gently.Prompt clumping of the suspension occurs with coagulase-positive strains.Tube coagulase test: This test detects the free coagulase. 0.1 mL of overnight broth culture oran agar culture suspension of the organism is added to 0.5 mL of diluted (1:5) rabbit or humanplasma in a test tube. Diluted plasma alone, in another test tube, acts as the control. The tubes areincubated at 37ºC for 3–6 hours. In a positive test, the plasma clots and does not flow when the tubeis inverted.DiseasesStaphylococcus aureus is recovered from a variety of infections, e.g. cutaneous skin lesions likefuruncles and carbuncles, breast abscesses, wound infections, pneumonia and acute osteomyelitis.It is the most common cause of acute pyogenic infections in man. Invasion of the blood streamfrom these sites can lead to meningitis, endocarditis and renal abscesses. Staphylococcus aureusis also implicated in food poisoning and antibiotic-induced pseudomembranous colitis. In case offood poisoning, enterotoxins are elaborated by the organisms growing in food. This food, wheningested by the patient, causes symptoms of poisoning. At least six different enterotoxins havebeen identified and the types of food usually responsible are milk and milk products.Another toxin-mediated disease produced by S. aureus is the staphylococcal scalded skin syn-drome (SSSS). This clinical condition is seen primarily in children less than 4 years of age and iscaused by an exfoliative toxin called exfoliatin.Yet another toxin-mediated staphylococcal disease is the toxic shock syndrome (TSS). It is anacute febrile exanthematous illness involving multiple systems with the potential of developinghypotension, shock and profound cardiovascular collapse but negative blood cultures. Although theChapter-24.indd 151Chapter-24.indd 151 5/28/2013 11:00:49 AM5/28/2013 11:00:49 AM
  • 5. 152 MICROBIOLOGY FOR NURSESdisease was first recognized among children with focal staphylococcal infection, the group at thehighest risk is the young women using tampons during menstrual periods.However, this organism can also be recovered from the anterior nares, perineum, umbilicalstump and other skin sites from as many as 10–30 per cent of healthy people and a significantlygreater percentage of people in the hospital setting. This is called the carrier state and can serve asa reservoir of infection.The coagulase-negative staphylococcus is implicated as a cause of infection in immunocompro-mised host. It is a common cause of bacteraemia in neutropenic patients and of infections related toindwelling catheters, shunts and prosthetic devices and, less commonly, a cause of urinary tractinfection in young, sexually active women.Bacteriophage TypingStrains of Staphylococcus aureus may be distinguished by their susceptibility to differentbacteriophages. An internationally accepted set of 23 phages is employed. The strains to be typedare grown in nutrient agar to produce a lawn culture. After drying, the phages of basic set areapplied in a fixed dose (routine test dose – RTD). After overnight incubation, the culture will beobserved for lyses by the phages. The phage type is designated according to the phage capable oflysing the bacterial strain. Thus a strain of phage type 54/80/82 is the one that is lysed only byphages 54, 80 and 82.Phage typing is important in epidemiological studies of staphylococcal infections.Antibiotic SensitivityMost strains of Staphylococcus aureus were originally sensitive to penicillin. Soon after penicillincame to be used clinically, resistant strains began to emerge. Penicillin resistance in staphylococcimay be mediated by plasmid or chromosome.1. Production of beta-lactamase (penicillinase), which is plasmid encoded and transmitted bytransduction or conjugation. Beta-lactamase inactivates penicillin by splitting the beta-lactamring.2. Reduction in affinity of penicillin-binding protein (PBP) of the staphylococcal cell wall forbeta-lactam antibiotics. This change is normally chromosomal in nature. This type of resistancealso occurs in beta-lactamase-resistant penicillin such as methicillin, nafcillin and oxacillin.They may cause outbreaks of hospital infection. These strains are called methicillin-resistantStaphylococcus aureus (MRSA). Vancomycin or teicoplanin is used for the treatment ofinfections with MRSA.Laboratory DiagnosisIt can be pus from a suppurative lesion, blood from a case of septicemia, sputum in case ofpneumonia, etc. The sample received for diagnosis depends on the nature of lesion. Directmicroscopic examination of the smear after Gram staining followed by culture on suitable mediasuch as blood agar is essential. However, for the isolation of staphylococcus from materials suchas faeces, food and dust, a primary enrichment in salt-containing media such as cooked meat brothwith 10 per cent sodium chloride, followed by plating on selective media such as mannitol saltagar is helpful.Chapter-24.indd 152Chapter-24.indd 152 5/28/2013 11:00:50 AM5/28/2013 11:00:50 AM
  • 6. THE COCCI 153Diagnosis of the carriers can be made by using enrichment cultures in broth such as cooked meatbroth with 10 per cent sodium chloride because the plate cultures alone may fail to detect as manyas 60 per cent of infected or colonized individuals. Alternatively, one can use mannitol salt agarinstead of blood agar and incubate the plates for 7 days to detect the organism.Once staphylococci are isolated, coagulase test and antibiotic susceptibility testing can be done.TreatmentBenzyl penicillin is a very effective antimicrobial, if the strain is sensitive to it. Methicillin andcloxacillin are effective against penicillinase-producing strains. Vancomycin is used in the treatmentof infections with MRSA strains. Many are also sensitive to netilmicin.Catheter-related sepsis can be treated successfully after the removal of the implicated catheterfollowed by antibiotic therapy for at least 10 days.Preventive MeasuresIn order to prevent transmission of nosocomial infection, it is important to treat all the carrier siteswith local application of mupirocin ointment (2%) and cohorting of caregivers and patients.STREPTOCOCCUSMorphologyStreptococci are Gram-positive cocci that tend to grow in chains in liquid media (Fig. 24.3a andb). They are derived from the word streptos meaning ‘twisted’ or ‘coiled’. While streptococci formpart of the normal human flora of humans and animals, they are also associated with severaldevastating diseases.(a) (b)FIGURE 24.3 (a) Streptococci (in chains). (b) Streptococci (Gram-positive cocci in chains).Their classification is based on morphology and haemolysis produced in 5 per cent sheep bloodagar.Chapter-24.indd 153Chapter-24.indd 153 5/28/2013 11:00:50 AM5/28/2013 11:00:50 AM
  • 7. 154 MICROBIOLOGY FOR NURSESThree types of haemolysis are produced:1. Alpha haemolysis: They produce greenish discolouration around the colonies, which is due topartial haemolysis. The zone of lysis is small (1–2 mm wide) with presence of unlysed erythro-cytes. Alpha haemolysis is seen with viridians group of streptococci and pneumococci.2. Beta haemolysis: These streptococci produce a clear, colourless zone of complete haemolysis(2–4 mm wide) around the colonies, within which erythrocytes are completely lysed. The lysisof erythrocytes is due to the production of two types of streptolysin by the organism: streptolysinO and streptolysin S. Most streptococci fall into this group.3. Gamma haemolysis (nonhaemolysis): Streptococcus faecalis (now called Enterococcusfaecalis) is a typical example.Beta-haemolytic streptococci have been grouped based on the carbohydrate C antigen in the cellwall into 20 Lancefield groups (from A to V, except I and J). Group A streptococcus (GAS) isknown as Streptococcus pyogenes. GAS is most important and is responsible for many importanthuman infections. These may be further subdivided into types based on the protein (M, T and R)antigens present on the cell surface. The M protein is the most important of these. Over 80 M typeshave been described.Streptococcus pyogenesCultureThey are aerobes and facultative anaerobes, growing bestat a temperature of 37°C (range 22–42°C). These are mostexacting in nutritive requirements, growth occurring onlyin media containing blood or serum. On the blood agar,after overnight incubation, the colonies are small (0.5–1.0mm, pinpoint), circular, semitransparent, low convex witha wide zone of beta-haemolysis around them (Fig. 24.4).Growth and haemolysis are promoted by the presence of10 per cent CO2 in the environment. Selective mediacontaining 1:500,000 crystal violet (crystal violet bloodagar) permit growth of streptococci but inhibit otherbacteria, especially staphylococci.In liquid media, such as glucose broth, growth occurs asa granular turbidity with a powdery deposit. Bacterialchains being heavier settle down as deposit.Biochemical ReactionsStreptococci are catalase negative.FIGURE 24.4 Growth of S. pyogeneson blood agar.Chapter-24.indd 154Chapter-24.indd 154 5/28/2013 11:00:50 AM5/28/2013 11:00:50 AM
  • 8. THE COCCI 155Antigenic Structure (Fig. 24.5)Capsular hyaluronic acid• : Capsule may be present on groups A and C streptococci. The capsulewhen present inhibits phagocytosis.Cell wall• : The cell wall is composed of an outer layer of protein (fimbria containing protein) andlipoteichoic acid, a middle layer of group-specific C carbohydrate and inner layer of peptidogly-can (mucoprotein). The peptidoglycan is responsible for cell wall rigidity. Serological groupingof streptococci depends on the C carbohydrate.Type-specific antigen• : The outer part of the cell wall contains protein antigens. Streptococcuspyogenes is further subdivided on the basis of their surface proteins M, T and R.Toxins and EnzymesStreptococcus pyogenes produces several exotoxins and enzymes, which contributes to its virulence.ToxinsHaemolysins Streptococci produce two types of haemolysins: streptolysin O and streptolysin S.Streptolysin O is so named because it is oxygen labile. It is inactivated in the presence of oxygen.Streptolysin S is an oxygen-stable haemolysin and is responsible for the haemolysis seen aroundthe colonies of streptococci on the surface of blood agar plates.Pyrogenic Exotoxins (erythrogenic toxins) This toxin is responsible for the rash of the scarletfever.FIGURE 24.5 Antigenic structure of Streptococcus pyogenes.CytoplasmCytoplasmic membranePeptidoglycanGroup specific carbohydrateProtein or lipoteichoic acidCapsuleCell wallPilusChapter-24.indd 155Chapter-24.indd 155 5/28/2013 11:00:50 AM5/28/2013 11:00:50 AM
  • 9. 156 MICROBIOLOGY FOR NURSESEnzymesStreptokinase (fibrinolysin) Streptokinase facilitates the spread of infection by breaking downthe fibrin barrier around the lesion.Deoxyribonucleases (streptodornase) Group A streptococci elaborate four antigenically distinctdeoxyribonucleases (DNase): A, B, C and D, of which type B is the most antigenic in man.Nicotinamide adenine dinucleotidase (NADase) NADase acts on the coenzyme NAD and liber-ates nicotinamide from the molecule. It is believed to be leucotoxic.Hyaluronidase It breaks down hyaluronic acid of the tissues and favours spread of streptococcallesion along intercellular spaces.DiseasesStreptococcus pyogenes or GAS produces pyogenic infections in man, with a tendency to spreadlocally in contrast to the localized nature of staphylococcal lesions. The most common suppurativestreptococcal infection is the sore throat. This is primarily caused by GAS, although groups C andG may also be responsible for it. There is also a special variety of streptococcal sore throat that isaccompanied by erythematous rash called scarlet fever. Scarlet fever is not seen in India.Streptococcus is also an important cause of puerperal sepsis and pyoderma. It may also lead toabscesses in internal organs and meningitis.Nonsuppurative sequelae of S. pyogenes or GAS include acute rheumatic fever (ARF) and acuteglomerulonephritis (AGN). Rheumatic fever may occur in up to 3 per cent of individuals duringepidemic pharyngitis. (Nephritis is caused by only a few ‘nephritogenic’ M types, i.e. 4, 12, 49, 52and 55. No definite ‘rheumatogenic’ types are known but certain types, i.e. 5, 18 and 24, have beenisolated from outbreaks of rheumatic fever.)In the 1980s, there have been numerous reports of invasive infection caused by GAS. GAS wasrecovered from skin and wound cultures taken from virtually all patients, and the majority ofpatients had positive blood cultures. The case definition of streptococcal TSS includes signs andsymptoms that are due to multiorgan failure and are similar to those of staphylococcal TSS. Insome patients, necrotizing fasciitis is a prominent feature.Group B streptococci (Streptococcus agalactiae) are the single most common aetiological agentof neonatal sepsis and meningitis. They can also cause infections in adults leading to puerperalsepsis, urinary tract infection, osteomyelitis and wound infection.Group D streptococci (enterococci) are discussed under a separate heading.Laboratory DiagnosisDiagnosis of acute infections is always based on culture. The sample received for diagnosis dependson the nature of the lesion. The smear is examined for Gram-positive cocci in chains. It is importantto remember that smears are of no value in infection of throat and genitalia, where streptococciform a part of normal flora. The samples are plated on blood agar and incubated at 37ºC under5–10 per cent carbon dioxide. In case of delay, the swabs can be transported on filter paper stripswhere the bacteria can survive for 10–15 days. After isolation on blood agar, streptococci can begrouped.Chapter-24.indd 156Chapter-24.indd 156 5/28/2013 11:00:50 AM5/28/2013 11:00:50 AM
  • 10. THE COCCI 157The diagnosis of nonsuppurative sequelae is based on the demonstration of antibodies (serol-ogy). The most commonly used test is the antistreptolysin O (ASO) titration. ASO titres more than200 IU are indicative of prior streptococcal infection. High levels are usually found in ARF.Antideoxyribonuclease B (ADNase B) estimation is also done, especially to establish the retrospec-tive diagnosis of streptococcal pyoderma.TreatmentPenicillin is still the drug of choice for most streptococcal infections. In case of patientshypersensitive to penicillin, erythromycin may be used.Preventive MeasuresAntibiotic prophylaxis is indicated only in the prevention of rheumatic fever. Long-termadministration of penicillin is recommended in children who have developed early signs ofrheumatic fever. Streptococcal vaccines are under development.ENTEROCOCCUSEnterococci constitute the normal faecal flora of all warm-blooded animals, including humans.E. faecalis and E. faecium are the most common human isolates among the enterococci. Theenterococci are uniquely antibiotic resistant: they are resistant to penicillin, aminoglycosides,cephalosporins, clindamycin and, most disturbingly, vancomycin (i.e. VRE). One of the majorconcerns today is the emergence of pan-resistant E. faecium.Once considered harmless commensals of the intestinal tract, they now rank among the leadingcauses of problematic and often costly nosocomial infections.MorphologyEnterococci are Gram-positive cocci, oval in shape, either arranged in pairs at an angle, or in shortchains.CultureIt has ability to grow under a wide range of conditions. They typically grow in 6.5 per cent NaCl,at temperatures from 10ºC to 45ºC and in the presence of 40 per cent bile (pH 9.6). On MacConkey’smedium they produce tiny, deep pink colonies. They are relatively heat resistant, surviving 60ºCfor 30 minutes. They may or may not be haemolytic on blood agar.Biochemical ReactionsThey can be identified by its ability to ferment mannitol, sucrose, sorbitol and aesculin and to growon tellurite blood agar producing black colonies.Chapter-24.indd 157Chapter-24.indd 157 5/28/2013 11:00:50 AM5/28/2013 11:00:50 AM
  • 11. 158 MICROBIOLOGY FOR NURSESToxins and EnzymesTraits that enhance the virulence include cytolysin, aggregation substance, adhesions, extracellularsuperoxide (ESO), extracellular surface protein (ESP), haemolysin and gelatinase.DiseasesE. faecalis and E. faecium cause bacteraemia, surgical wound infections, urinary tract infectionsand endocarditis in hospitalized hosts. While E. faecalis remains the predominant species in theclinical infections, E. faecium isolates are increasing in proportion. This trend is particularly truefor blood isolates.Recently identified risk factors for acquisition of VRE include prolonged hospitalization, priorantibiotic use and serious underlying illness.Laboratory DiagnosisIt is important to promptly and accurately detect enterococci to prevent its spread. (The mainreservoir of VRE is the hospitalized patients with gastrointestinal carriage.) It is important toperform surveillance cultures in high-risk areas. Rectal swabs or spontaneously passed stoolsamples are appropriate for culture. In cases with established infection, appropriate sample maybe collected. Recognition of enterococci is based on morphology and its ability to grow under awide range of conditions. They typically grow in 6.5 per cent NaCl, at temperatures from 10 to45°C and in the presence of 40 per cent bile. Identification of enterococci to species level is usefulfor epidemiological investigations and for predicting resistance, because E. faecium is 10 timesmore likely to be vancomycin-resistant than E. faecalis.TreatmentThe treatment of enterococcal infections is typically tough, and sometimes, troublesome. In general,therapy for severe enterococcal infections includes an agent that acts on cell wall in combinationwith an aminoglycoside. The optimal therapy for patients with VRE remains unknown.Preventive MeasuresThe prevention and control of VRE within health care institutions is complex and requires amultidisciplinary approach involving cooperation of all persons involved in direct patient care.VRE are capable of prolonged survival on hands and environmental surfaces; therefore all patientsinfected or colonized with this bacterium should be placed in isolation. Health care providers forthese patients should strictly follow hand washing and barrier precautions. During an outbreak, itmay be appropriate to cohort staff and patients so that personnel do not contaminate noncolonizedpatients.In addition prudent use of antimicrobial agents is mandatory. Vaccines for enterococcal infec-tions are under development.Chapter-24.indd 158Chapter-24.indd 158 5/28/2013 11:00:50 AM5/28/2013 11:00:50 AM
  • 12. THE COCCI 159PNEUMOCOCCUSPneumococcus or Streptococcus pneumoniae are the bile-sensitive streptococci. They form thenormal and nasopharyngeal flora in 10–15 per cent of healthy adults and up to 60 per cent ofhealthy children.MorphologyThey are small, Gram-positive and slightly elongated cocci, arranged in pairs (diplococci), withone end broad or rounded in apposition and other pointed (flame shaped), typically lanceolate inappearance (Fig. 24.6a). They are surrounded by a polysaccharide capsule. The capsule can bedemonstrated as a clear halo in India ink preparation (Figs. 24.6b).(a) (b)FIGURE 24.6 (a) Pneumococci (Gram-positive diplococci). (b) Pneumococci in India ink preparation.CulturePneumococci are aerobes and facultative anaerobes,and their culture requires complex media such asblood or chocolate agar. They produce smooth ormucoid colonies and alpha-haemolysis(Fig. 24.7). Growth is improved by the presence of5–10 per cent CO2. After overnight incubation, thecharacteristic alpha haemolytic colonies can be seen.On further incubation, the colonies become flat, withraised edges and central umbonation (draughtsmanappearance).Biochemical ReactionsPneumococci ferment several sugars with productionof acid only. Fermentation is tested in Hiss’s serumwater. Fermentation of inulin by pneumococci is ofgreat value to differentiate them from streptococci.FIGURE 24.7 Growth of S. pneumoniaeon blood agar.Chapter-24.indd 159Chapter-24.indd 159 5/28/2013 11:00:50 AM5/28/2013 11:00:50 AM
  • 13. 160 MICROBIOLOGY FOR NURSESPneumococci are soluble in bile. When 2 per cent sodium deoxycholate solution is added to abroth culture, the culture clears due to the lysis of the cocci. Alternatively, if a loopful of 10 per centsodium deoxycholate solution is placed on pneumococcus colony, lysis of colony occurs within afew minutes (bile solubility test). Pneumococci are catalase and oxidase negative.ResistancePneumococci are delicate organisms and are destroyed at 52°C for 15 minutes. They are destroyedby the routine antiseptics.Pneumococci are sensitive to optochin (ethylhydrocupreine hydrochloride) in a concentration of1/500,000. When an optochin disc (5 μg) is applied on blood agar plate incubated with pneumo-cocci, a wide zone (14 mm or more) of inhibition occurs on incubation (optochin-sensitivity test).Antigenic StructureThe most important antigen of pneumococcus is capsular polysaccharide. Other antigens includesomatic M protein and a group-specific cell wall carbohydrate.Capsular PolysaccharideAll pathogenic (smooth) strains form rather large capsules, this being their major virulence factor.Rough strains lack a capsule and are nonvirulent. Capsules help the streptococci escape phagocytosis,which is the major host defence in pyogenic infections. They contain a polysaccharide antigencalled the specific soluble substance (SSS) that varies chemically among the pneumococcal types.So far 90 different capsular types (specified by numerals 1, 2, 3, …) have been identified.Antibodies to capsular polysaccharide antigens provide serotype-specific immunity.DiseasesPneumonia caused by S. pneumoniae is the most common type of community-acquired pneumonia(CAP). It is the second most common cause of acute bacterial meningitis and can also causebacteremia. In addition, it is the aetiological agent of otitis media, conjunctivitis, sinusitis, empyemaand peritonitis. The risk of disease is highest among young children, older adults (elderly), smokersand patients with predisposing conditions such as asplenia, chronic medical conditions orimmunosuppressive illness.Laboratory DiagnosisThe clinical sample (sputum, cerebrospinal fluid, etc.) can be Gram stained and examined for thepresence of Gram-positive diplococci. The sample is then inoculated on the blood agar plate andincubated at 37°C under 5–10 per cent carbon dioxide.After overnight incubation, the characteristicalpha-haemolytic colonies can be seen. On further incubation, draughtsman appearance may beseen. These colonies when stained by Gram’s method show typical morphology, and are bilesoluble, optochin sensitive, catalase negative and oxidase negative. (The bile solubility test is basedon the presence of enzymes, which when activated by surface-active agents such as sodiumdeoxycholate, lead to the lysis of organisms.) Although the identification of S. pneumoniae can bemade rapidly with the Quellung test, this test is no longer done. (Quellung test: When the specificChapter-24.indd 160Chapter-24.indd 160 5/28/2013 11:00:50 AM5/28/2013 11:00:50 AM
  • 14. THE COCCI 161capsular antibody is added either to the clinical sample containing the organism or the cultureisolate, the capsules become swollen and refractile because of antibody attachment.)TreatmentPenicillin is the drug of choice, but approximately 5 per cent of all S. pneumoniae strains are nowbeing reported to be relatively resistant to penicillin. Erythromycin can be used in penicillin allergicpatients. Pneumococcal resistance to quinolones is relatively low.Preventive MeasuresA vaccine containing a cocktail of 23 different serotypes (Pneumovax – PPV 23) is recommendedfor patients with chronic respiratory disease, those without spleen, elderly patients andimmunosuppressed patients. Recently, a conjugate pneumococcal vaccine (made by attaching cell-surface polysaccharides from seven strains to a genetically modified nontoxic form of diphtheriatoxin protein) has been introduced for use in children (Prevnar – PCV7). More recently the13-valent pneumococcal protein conjugate vaccine (PCV13) has been approved for use in childrenand adults.MENINGOCOCCUSMeningococcus or Neisseria meningitidis is normally carried in the throat of approximately 40 percent individuals. It may also be seen in the genital tract where its presence is of no pathologicalsignificance.MorphologyThey are Gram-negative, spherical or oval cocci, arranged in pairs with adjacent sides flattened.They are nonmotile and nonsporing. They are intracellular when isolated from lesion.CultureThey do not grow on ordinary media but have exacting growth requirements. Growth occurs onmedia enriched with blood or serum. Blood agar, chocolate agar and Muller–Hinton are commonlyused media.The optimum temperature and pH for growth are 35–36°C and 7.4–7.6, respectively. A moistenvironment with 5–10 per cent CO2 is must for the growth to occur.On solid media the colonies are small (1 mm in diameter), round, convex, grey, translucent andwith entire edges. In liquid media, it produces granular turbidity.Biochemical ReactionsN. meningitides is catalase and oxidase positive. The prompt oxidase reaction helps to identifyneisseria (both meningococci and goncocci) in mixed cultures. Glucose and maltose are fermentedwith acid production but no gas (gonococci ferment glucose but not maltose). They do not fermentlactose or sucrose. Indole and H2S are not produced and nitrates are not reduced.Chapter-24.indd 161Chapter-24.indd 161 5/28/2013 11:00:51 AM5/28/2013 11:00:51 AM
  • 15. 162 MICROBIOLOGY FOR NURSESResistanceThey are very delicate organisms, being highly susceptible to heat, desiccation and disinfectants.They are susceptible to penicillin, ampicillin, chloramphenicol, macrolides and ciprofloxacin.Antigenic StructureBased on the capsular polysaccharide, 13 serogroups of N. meningitidis have been identified: A,B, C, D, X, Y, Z, W135, 29E, H, I, K and L. Meningococci of groups A and C are more frequentlyassociated with epidemics of meningitis although group B is also now implicated.Group B polysaccharide is a very poor immunogen for humans.DiseasesN. meningitidis causes pyogenic meningitis with or without septicaemia after an incubation periodof about 4 days. Haemorrhagic manifestations may be seen in the form of petechial rash involvingthe skin and the mucosa. Rarely it has been associated with septic arthritis and carditis.Laboratory DiagnosisDiagnosis of acute bacterial meningitis can be made by the examination of cerebrospinal fluid aswell as blood during the early stages of illness. Gram staining of the centrifuged deposit of CSFdemonstrates intracellular as well as extracellular Gram-negative diplococci (Fig. 24.8).The supernatant can be used for the demonstration of meningococcal antigen. For confirmation,the sample is plated onto blood agar and heated blood agar incubated at 37°C in 5–10 per centcarbon dioxide. After overnight incubation, demonstration of oxidase-positive colonies showingcharacteristic morphology and biochemicals (i.e. utilization of both, glucose and maltose) help inidentification. Serogrouping is performed by slide agglutination test. Meningococci can also besometimes demonstrated in the petechial lesions.Demonstration of N. meningitidis in the nasopharynx helps in the detection of carriers.TreatmentThey are sensitive to penicillin, chloramphenicol, rifampicin and cephalosporins.FIGURE 24.8 Meningococci (intracellular and extracellular Gram-negative diplococci).Chapter-24.indd 162Chapter-24.indd 162 5/28/2013 11:00:51 AM5/28/2013 11:00:51 AM
  • 16. THE COCCI 163Preventive MeasuresChemoprophylaxis:A4-dayregimenofciprofloxacin/ofloxacin/cefixime/sulphadiazine/rifampicinhas been recommended because of the similar duration of the average incubation period of thedisease.Immunoprophylaxis: Polysaccharide vaccine against serogroups A, C, Y and W135 of N.meningitidis is available in monovalent, bivalent and tetravalent forms. The biggest limitation ofthis vaccine is unsatisfactory immune response below the age of 2 years, which is the highestsusceptible age group. The vaccine also plays no role in established nasopharyngeal carriers. Thereis no vaccine against serogroup B currently.GONOCOCCUSGonococcus or Neisseria gonorrhoeae is the aetiological agent of gonorrhoea, which is asexually transmitted infection (STI). Man is the only natural host for Neisseria gonorrhoeae.Gonorrhoea has been identified as a co-factor in HIV transmission. In the presence of gonorrhoea,HIV transmission increases by a factor of 3–5. The new association provides an important reasonfor proper and timely treatment of gonorrhoea. Further, untreated gonococcal infection can causepelvic inflammatory disease (PID), which may lead to chronic pelvic pain, ectopic pregnancyand infertility.MorphologyThey are Gram-negative oval cocci arranged in pairs (diplococci) with adjacent sides concave(kidney shaped). In smear from purulent material, they are intracellular within polymorphs, as wellas extracellular (Fig. 24.9).FIGURE 24.9 Gonococci (intracellular and extracellular Gram-negative diplococci).Chapter-24.indd 163Chapter-24.indd 163 5/28/2013 11:00:51 AM5/28/2013 11:00:51 AM
  • 17. 164 MICROBIOLOGY FOR NURSESCultureThis organism is more difficult to grow than N.meningitidis. They are aerobic, but many growanaerobically as well. They grow best at 35–36°Cin presence of 5–10 per cent CO2 and at pH7.2–7.6. They require an enriched medium likechocolate agar for their growth. A popularselective medium is Thayer–Martin medium withantibiotics (VCNT – vancomycin, colistin, nysta-tin and trimethoprim).After 24 hours of incubation, colonies aresmall, round, grey, translucent and convex withfinely granular surface. They are easily emulsifi-able (Fig. 24.10).Biochemical ReactionsN. gonorrhoeae is oxidase positive. They fermentglucose (with acid production only) and not maltose.Antigenic StructureThe antigenic structure is complex. The surface structure of N. gonorrhoeae includes thefollowing:Capsule:• It is polyphosphate not polysaccharide.Pili:• Pili enhance attachment of the organism to host cell and act as virulence factor.Lipopolysaccharide (LPS):• Toxicity in gonorrhoea is largely due to the endotoxic effect of thiscomponent.Proteins:• Outer-membrane proteins are the porins.ResistanceGonococci are very delicate organisms. They are strict parasites and die rapidly outside the humanhost.Gonococci are readily killed by heat, drying and antiseptics. Earlier they were highly susceptibleto sulphonamides and penicillin but have steadily developed resistance to many antibiotics. PPNG(penicillinase-producing Neisseria gonorrhoeae) strains are resistant to penicillin due to produc-tion of beta-lactamase (penicillinase) enzyme by these strains.FIGURE 24.10 Growth of N. gonorrhoea onchocolate agar.Chapter-24.indd 164Chapter-24.indd 164 5/28/2013 11:00:51 AM5/28/2013 11:00:51 AM
  • 18. THE COCCI 165DiseasesGonorrhoea presents as lower genital tract infection leading to urethritis, cervicitis, extending tosalpingitis, endometritis and related sequelae in women; urethritis and epididymitis in men; andproctitis, pharyngitis, conjunctivitis and disseminated gonococcal infection (DGI) in both the sexes.However, majority of women together with a proportion of men infected with N. gonorrhoeae areasymptomatic. N. gonorrhoeae can also lead to gonococcal ophthalmia, a nonvenereal infection innewborns, which results from direct infection during passage through the birth canal.Laboratory DiagnosisDiagnosis depends on the combination of clinical symptoms and laboratory findings. The presenceof mucopurulent endocervical or urethral exudate on physical examination is suggestive ofgonococcal infection. Further, demonstration of typical Gram-negative diplococci (Fig. 24.9) onGram stain of urethral discharge (in men) and endocervical secretions (in women) along withgrowth on selective media, e.g. modified Thayer–Martin provides presumptive diagnosis.Confirmation of the identity of the isolates is done by biochemical testing (gonococci ferment onlyglucose).Polymerase chain reaction (PCR) is being used for diagnosis of gonorrhoea in most developedcountries. However, culture is essential for surveillance of antimicrobial susceptibility.TreatmentN. gonorrhoeae has been reported to develop resistance to multiple classes of antimicrobials,including penicillin, tetracycline and quinolones. Reliance is placed on extended spectrumcephalosporins (ESCs) such as ceftriaxone (injectable) and cefixime (oral) for first-line treatmentof gonorrhoea. As per the latest guidelines uncomplicated gonococcal infections of the cervix,urethra and rectum should be treated with a combination of ESCs plus azithromycin/deoxycycline.DGI is also treated with ceftriaxone.Preventive MeasuresThe basis of primary prevention is health education/promotion. Condom promotion has had a verysignificant impact. Secondary prevention is concerned with the promotion of health care seekingbehaviour and case management, which basically includes detection of cases, contact tracing andprompt treatment.Chapter-24.indd 165Chapter-24.indd 165 5/28/2013 11:00:51 AM5/28/2013 11:00:51 AM
  • 19. 166 MICROBIOLOGY FOR NURSESThings to Remember1. Staphylococci are the commonest cause of suppurative lesions in man.2. Staphylococci are divided based on the production of enzyme coagulase into coagulase producers(S. aureus) and nonproducers (coaulase-negative staphylococci).3. Management of methicillin-resistant S. aureus (MRSA) infection is of global concern.4. The most common suppurative streptococcal infection in man is sore throat.5. Nonsupperative sequelae of S. pyogenes or GAS includes acute rheumatic fever (ARF) and acuteglomerulonephritis (AGN).6. Serological grouping of streptococci depends on C carbodydrate in cell wall.7. Enterococci are a leading cause of nosocomial infections.8. The precautions and control of vancomycin-resistant enterococci (VRE) in hospitals is important.9. S. pneumoniae causes serious infections such as pneumonia, bacteremia and meningitis throughoutthe world.10. Recently, the conjugate pneumococcal vaccine (PCV13) has been introduced for use in childrenand adults.11. N. meningitidis causes pyogenic meningitis in all ages, but is most common in children and youngadults.12. N. gonorrheae is the aetiological agent of gonorrhoea, the second most common bacterial STI inthe world.13. Gonorrhoea presents as lower genital tract infection although asymptomatic infections arecommon.14. N. gonorrheae has developed resistance to multiple classes of antimicrobials.REVISION QUESTIONS1. Write short notes on the following:(a) Coagulase test for staphylococci(b) MRSA(c) Classification of staphylococci(d) Antigenic structure of Staphylococcus pyogenes(e) VRE(f) Pneumococcal vaccines(g) Laboratory diagnosis of meningococcal meningitis(h) Laboratory diagnosis of gonorrhoeaChapter-24.indd 166Chapter-24.indd 166 5/28/2013 11:00:51 AM5/28/2013 11:00:51 AM
  • 20. C H A P T E R25The Bacilli and the VibriosL E A R N I N G O B J E C T I V E SAfter reading this chapter, the reader should be able to:• Discuss the morphology, culture characteristics, biochemical reactions, pathogenicity,prevention and treatment of the bacilli and the vibrios.C H A P T E R O U T L I N EBacilli Associated with the Respiratory Tract 167Bacilli Associated with Intestinal Canal 176Acid-Fast Bacilli 186Spore-Bearing Bacilli 190Bacilli Derived from Infected Animals 198The Vibrios 201Bacteroides 205Things to Remember 205Revision Questions 206BACILLI ASSOCIATED WITH THE RESPIRATORY TRACTDiphtheria Bacillus (Corynebacterium diphtheriae)Corynebacterium diphtheriae causes diphtheria in humans. Diphtheria is an infection of the upperrespiratory tract that is characterized by necrosis and sloughing of epithelial mucosa, producing adiphtheritic (pseudo) membrane, which may extend from the anterior nasal mucosa to the bronchi,but is most often limited to the tonsillar and peritonsillar areas. The name is derived from the tough,leathery pseudomembrane formed in the disease (diphtheros, meaning ‘leather’). C. diphtheriaeChapter-25.indd 167Chapter-25.indd 167 5/30/2013 2:27:26 PM5/30/2013 2:27:26 PM
  • 21. 168 MICROBIOLOGY FOR NURSESproduces a very potent exotoxin (a heat-labile protein), which is active against a variety of tissuesincluding the myocardium and the nerve endings.MorphologyC. diphtheriae are Gram-positive, irregularly stainedbacilli pleomorphic in nature. They are nonsporing,noncapsulated and nonmotile. Granules composed ofpolymetaphosphate are seen in the cells. When stainedwith Loeffler’s methylene blue, the granules take up abluish purple colour and hence they are calledmetachromatic granules. They are also called volutin orBabes Ernst granules. Special stains, such as Albert’sstain, are used to demonstrate granules in the cells,which are seen as bluish black against a green cytoplasm.The bacilli are arranged at various angles to each other,in the smear resembling the letters V or L. This has beentermed as the Chinese letter (Fig. 25.1) or cuneiformarrangement.Culture CharacteristicsGrowth is scanty (poor or not grown) on ordinary media. Enrichment of the media with bloodserum or egg is essential for good growth.The media employed for cultivation of the diphtheria bacilli are Loeffler’s serum slope andtellurite blood agar, although it grows on ordinary blood agar too.Loeffler’s serum slope Diphtheria bacilli grow rapidly in 6–8 hours. Colonies are small, circular,white and opaque.Tellurite blood agar This is a selective mediumfor the growth of diphtheria bacilli. The organ-isms grow slowly and colonies may take 2 daysto appear. They reduce tellurite to metallic tellu-rium, which is incorporated in the colonies, pro-viding them a black or grey colour (Fig. 25.2).Based on colonial morphology on the telluritemedium and other properties, three types of diph-theria bacilli have been identified: gravis, inter-medius and mitis.Biochemical ReactionsDiphtheria bacilli ferment glucose, galactose,maltose and dextrin with the production of acidbut no gas. It is necessary to employ Hiss’s serumwater for testing sugar fermentation. Unlikeintermedius and mitis, the gravis type fermentsstarch and glycogen.FIGURE 25.1 Corynebacteriumdiphtheriae (Chinese letter arrangement).FIGURE 25.2 Growth of C. diphtheriae onpotassium telluric blood agar.Chapter-25.indd 168Chapter-25.indd 168 5/30/2013 2:27:27 PM5/30/2013 2:27:27 PM
  • 22. THE BACILLI AND THE VIBRIOS 169ToxinsVirulent strains of diphtheria bacilli produce very powerful exotoxins. The pathogenic effects ofthe bacillus are due to the toxins.The diphtheria toxin is a protein with molecular weight of about 62,000. It consists of two frag-ments A and B of molecular weight 24,000 and 38,000, respectively. Both fragments are necessaryfor the toxicity.All the enzymatic activity of the toxin is present in fragment A. Fragment B is responsible forbinding the toxin to the cells. The diphtheria toxin acts by inhibiting protein synthesis. Diphtheriatoxin can be toxoided.PathogenicityC. diphtheriae causes diphtheria, which is a toxaemia having an incubation period of 3–4 days.Although the bacteria remain confined to the site of entry (which is most commonly the faucialarea), the systemic effects are due to the production of the toxin. The disease may present astoxaemia with marked cervical adenitis (bull neck) or with haemorrhagic manifestations. Theremay be complications in the form of asphyxia or postdiphtheritic paralysis.Laboratory DiagnosisLaboratory confirmation of diphtheria is necessary for the initiation of control measures and forepidemiological purposes but not for treatment of individual cases. Treatment starts on the basesof clinical diagnosis alone.Two swabs from the lesion are collected: one for microscopic examination(following Gram and Albert’s stain) and the other for culture. For culture, the swabs are incubatedon Loeffler’s serum slope, tellurite blood agar and ordinary blood agar. The blood agar plate isused for differentiating streptococcal and staphylococcal pharyngitis, which may simulatediphtheria.Suspected growth on different media is confirmed by microscopy and appropriate biochemicaltests. In addition, the growth is tested for the production of toxin described below.Virulence test In order to complete the bacteriological diagnosis of diphtheria bacillus, testing forvirulence or toxigenicity should be performed. Virulence testing can be performed by in vivo or invitro methods.In vivo methodSubcutaneous test The growth from an overnight culture on Loeffler’s slope is emulsified in 2–5mL broth and 0.8 mL of the emulsion injected subcutaneously into two guinea pigs, one of whichhas been protected with 500 units of diphtheria antitoxin 18–24 hours previously. If the strain isvirulent, i.e. toxin producing, the unprotected animal will die within 4 days, showing pleural andperitoneal exudates and enlarged haemorrhagic adrenals, which is the pathogenic feature.Intracutaneous test 0.1 mL of broth emulsion is inoculated intracutaneously into two guinea pigs(or rabbits). One animal acts as the control and is protected by 500 units of diphtheria antitoxin theprevious day. The other animal is given 50 units of antitoxin intraperitoneally 4 hours after the skintest, in order to prevent death. If the strain is toxigenic, inflammatory reaction at the site of injectionis seen progressing to necrosis in 48–72 hours in the test animal while the control animal shows nochange. An advantage in the intracutaneous test is that multiple strains can be tested at a time on apair of animals and the animals do not die.Chapter-25.indd 169Chapter-25.indd 169 5/30/2013 2:27:27 PM5/30/2013 2:27:27 PM
  • 23. 170 MICROBIOLOGY FOR NURSESIn vitro methodElek’s gel precipitation test This is an immunodiffusion test. A rectangular strip of filter paperimpregnated with diphtheria antitoxin (1000 unit/mL) is placed on the surface of a 20 per cent nor-mal horse serum agar plate while the medium is still fluid. When the agar solidifies, the test strainis streaked at right angle to the filter paper strip. The positive and negative controls are also put up.The plate is incubated at 37ºC for 24–48 hours. The toxin produced by the bacterial growth diffusesin the agar and produces a line of precipitation where it meets the antitoxin at the optimum concen-tration (positive). Nontoxigenic strains will not produce any precipitation line (negative).Tissue culture test The toxigenicity of diphtheria bacilli can be demonstrated by incorporating thetest strain into the agar overlay of cell culture monolayer. If the strain is toxigenic, the toxin willdiffuse into the cells below and kill them.ProphylaxisActive immunization Active immunization starts at 6 weeks of age by toxoid in combination withtetanus toxoid and pertussis vaccine (DPT, triple vaccine). Three doses are given by intramuscularroute at an interval of 4–6 weeks. Booster doses are given at 18 months and at 5 years. Diphtheriatoxoid combined with tetanus toxoid (DT) is used for booster dose at 5 years.Passive immunization 500–1000 units of antitoxin (antidiphtheric serum – ADS) is administeredsubcutaneously as an emergency measure when a suspected case of diphtheria presents in paediat-ric department.Schick test Susceptibility of a person to diphtheria can be detected by using this test. This test isemployed in older children and adults and is an example of neutralization test (toxin–antitoxin).This test is negative in an immune individual, which means that he does not require vaccination.Method A test dose of 0.2 mL diphtheria toxin is injected intradermally on one forearm and asimilar amount of inactivated toxin is injected on the other forearm. Results are read after 1, 4 and7 days and interpreted as follows:1. Positive reaction: An area of swelling and erythema appears at the site of injection of toxinafter 24–48 hours. The control forearm injected with inactivated toxin will show no reaction. Apositive test signifies that the person is susceptible to diphtheria. It implies lack of circulatingantitoxin.2. Negative reaction: There is no reaction in any forearm (control and test). This indicates thatthe toxin has been neutralized by the antitoxin present in the circulation and the person isimmune to diphtheria.3. Pseudoreaction: In this, erythema occurs in both forearms within 6–24 hours and disappearscompletely within 4 days. This implies that the individual is immune to diphtheria but at thesame time hypersensitive to its components.4. Combined reaction: In this, the initial reaction is similar to that of pseudoreaction. Buterythema disappears in the control forearm within 4 days while it progresses in the test forearmto a typical positive reaction. This indicates that the individual is susceptible to diphtheria andat the same time hypersensitive to its components.Chapter-25.indd 170Chapter-25.indd 170 5/30/2013 2:27:27 PM5/30/2013 2:27:27 PM
  • 24. THE BACILLI AND THE VIBRIOS 171TreatmentTreatment of diphtheria is to be initiated as quickly as possible following clinical suspicion.Penicillin is the drug of choice, although it has to be supplemented with antitoxin therapy.Diphtheroids Commensal corynebacteria are normally present in the skin, throat, conjunctiva andother areas of the body. These may sometimes be mistaken for C. diphtheriae and are called diph-theroids. The common example of diphtheroids is C. xerosis (found in the conjunctival sac).Features that distinguish C. diphtheriae from diphtheroids are outlined in Table 25.1.TABLE 25.1 Distinguishing Features of C. diphtheriae and DiphtheroidsS. no. Feature C. diphtheriae Diphtheroids1. Morphology Metachromatic granules presentChinese letter patternPleomorphism seenFew or absentPalisade arrangementPleomorphism absent2. Cultural characteristics Grow on enriched media Can grow on ordinary media3. Biochemical reactions Ferments glucose only and not sucrose Ferments both glucose and sucrose4. Toxin production Positive NegativeHaemophilus influenzaeHaemophilus means blood loving, reflecting its requirement for substances that are supplied bythe addition of blood to the nutrient agar medium for its growth. It was originally namedHaemophilusinfluenzae because it was thought to cause influenza, which is now known to be a viral infection.(It was later discovered that H. influenzae acts as a secondary invader following the infection ofthe respiratory tract by a virus.) It is present as a commensal in the throat and nasopharynx of60–80 per cent of healthy individuals. The majority of bacilli are noncapsulate and only 3–4 percent of individuals harbour capsulated strains.MorphologyIt is a Gram-negative, nonmotile organism that tends to be pleomorphic. It is nonsporing andnon–acid fast.Culture CharacteristicsH. influenzae has fastidious growth requirements. It requires X and V factors: The X factor is aniron-containing porphyrin, which is required for the synthesis of enzymes involved in respirationand V factor is NAD (nicotinamide adenine dinucleotide) or NAD phosphate, which acts as ahydrogen acceptor in the metabolism of the cell. The V factor is present in red blood cells and isalso synthesized by staphylococci.Although it grows on blood agar, the growth is scanty, as the V factor is not freely available. Vfactor is released from erythrocytes on heating. Therefore, chocolate agar or heated blood agar is amedium of choice as it contains accessory growth factors like X and V. The optimum temperaturefor growth is 35–37ºC. Some strains require 5–10 per cent CO2.Chapter-25.indd 171Chapter-25.indd 171 5/30/2013 2:27:27 PM5/30/2013 2:27:27 PM
  • 25. 172 MICROBIOLOGY FOR NURSESSatellitism When S. aureus is streaked across a plate ofblood agar on which a specimen containing H. influenzaehas been inoculated, after overnight incubation, the colo-nies of H. influenzae will be large and well developedalongside the streak of staphylococcus, and smaller fartheraway. This phenomenon is known as satellitism and dem-onstrates the dependence of H. influenzae on the V factor(Fig. 25.3).Biochemical ReactionsBiochemical reactions are not helpful in its identification.It is catalase and oxidase positive.Antigenic StructureThe capsulated strains have been classified into six types, designated types a to f, based on capsularantigens that are polysaccharide in nature. Type b strains account for most invasive infections.Type b antigens can be detected in body fluids including the urine of infected patient.Noncapsulated strains cannot be typed and are called nontypable strains.PathogenicityThe capsulated strains of H. influenzae act as primary pathogens leading to meningitis in childrenbetween 2 months and 3 years of age, laryngoepiglottitis in children older than 2 years otitis media,pneumonia, endocarditis and arthritis. The noncapsulated strains are usually associated withsecondary infections of respiratory tract in the adults.Laboratory DiagnosisAs H. influenzae is very sensitive to low temperature, therefore, clinical specimens suspected tobe containing it should never be refrigerated. The specimen collected for Gram stain and culturedepends on the site of infection. The presence of pleomorphic Gram-negative bacteria in CSF thatdo not stain well should suggest (to the microbiologist) the possibility of haemophilus meningitis.A Quellung test using the type b antiserum, as already described for S. pneumoniae, can be usedfor rapid identification of H. influenzae. The sample should be plated onto heated blood agar orblood agar streaked with S. aureus. The plates are incubated at 37ºC in an environment containing5–10 per cent carbon dioxide. The colonies of H. influenzae are larger, closer to the streak andbecome smaller as they go away from it—a phenomenon called satellitism, as already described(Fig. 25.3).Capsular polysaccharide antigen may be present in the urine or CSF and can be demonstratedwith specific antiserum.The isolation of H. influenzae from sputum samples should be interpreted with caution as it isliable to be contaminated with throat secretions.FIGURE 25.3 Haemophilusinfluenzae demonstrating satellitism.Chapter-25.indd 172Chapter-25.indd 172 5/30/2013 2:27:27 PM5/30/2013 2:27:27 PM
  • 26. THE BACILLI AND THE VIBRIOS 173TreatmentChloramphenicol is the drug of choice for haemophilus meningitis. In case of chloramphenicolresistance, a cephalosporin is used. Ampicillin/amoxicillin is used for severe respiratory tractinfections. Alternatively, amoxicillin/clavulanic acid, 2nd- or 3rd-generation oral cephalosporin,ciprofloxacin or azithromycin may be used.Preventive MeasuresImmunity in H. influenzae is type specific. A conjugate vaccine against H. influenzae type b hasbeen very successful in reducing the incidence of invasive disease. It is widely used.Whooping Cough Bacillus (Bordetella pertussis)Whooping cough is an acute infectious disease caused by Bordetella pertussis, Bordetellabronchiseptica and in a small percentage of cases, by B. parapertussis. It usually affects youngchildren.MorphologyB. pertussis is small, ovoid, Gram-negative coccobacillus. It is nonmotile, nonsporing andcapsulated. However, it tends to lose the capsule on repeated cultivation.Culture CharacteristicsB. pertussis is strictly aerobic and requires complex media for its growth. Bordet–Gengou (BG)medium/charcoal blood agar are used for its growth. B. pertussis is slow growing and may takeup to 5 days to grow on artificial medium. Colonies on BG medium are small, opaque, greyishwhite and refractile, resembling bisected pearls or mercury drops.Biochemical ReactionB. pertussis is biochemically inert. It is oxidase positive and usually produces catalase.PathogenicityWhooping cough is a disease that is clinically characterized by an insidious onset with mild feverand irritating cough, gradually becoming paroxysmal with a characteristic whoop (violent bouts ofcough followed by inrush of air into almost empty lungs). The incubation period of the disease is1–2 weeks. The disease is characterized by three stages: catarrhal, paroxysmal and convalescent.The first stage is the stage of maximum infectivity and the disease lasts for about 8 weeks.The major complications of the disease are bronchopneumonia and lung collapse. The pressureeffects during violent coughing can lead to subconjunctival haemorrhage and cerebral haemorrhageleading to convulsions and coma.Laboratory DiagnosisThe ideal specimen is the nasopharyngeal aspirate/swab.Chapter-25.indd 173Chapter-25.indd 173 5/30/2013 2:27:27 PM5/30/2013 2:27:27 PM
  • 27. 174 MICROBIOLOGY FOR NURSESMicroscopy Fluorescent antibody technique is useful for the rapid diagnosis of pertussis in respi-ratory secretion.Culture Laboratory diagnosis can be established by examination of samples from the upper respi-ratory tract during the early stage of the disease. Samples are plated onto BG medium/charcoalblood agar.Identification can be confirmed by the slide agglutination method.TreatmentErythromycin is the drug of choice but is useful only if initiated within first 10 days of the disease.Alternatively, tetracycline or cotrimoxazole may be used.Preventive MeasuresIsolation of infected patient is important in reducing the secondary attack rate. Primary preventioncan be achieved by the immunization of the infant with DPT vaccine (three doses) followed by abooster at 18–24 months. An acellular vaccine is also available.Legionnaire’s Disease Bacillus (Legionella pneumophila)Legionella is the causative agent of Legionnaire’s disease. It lives in the aquatic habitats and canbe isolated from water in airconditioning cooling tower and airconditioning condensate. It wasdiscovered in 1976 by CDC scientists who were investigating an epidemic of pneumonia amongPennsylvania State American Legion members attending a convention in Philadelphia.MorphologyIt is a Gram-negative bacterium with a tendency to form filaments.Culture CharacteristicsIt has fastidious growth requirement. It can be grown in the Freeley–Gorman (FG) medium.Colonies are tiny and appear in 4–10 days. Alternatively, buffered charcoal yeast extract (BCYE)agar may be used.Biochemical ReactionsIt is catalase positive and weakly oxidase positive. Other tests such as sugar fermentation arenegative.PathogenicityLegionella pneumophila gives rise to two distinct clinical patterns. One is the Legionnaire’s disease,which manifests primarily as a severe consolidated pneumonia. It may either be epidemic orsporadic. The second, milder form of illness is the Pontiac fever, which is characterized by fever,Chapter-25.indd 174Chapter-25.indd 174 5/30/2013 2:27:27 PM5/30/2013 2:27:27 PM
  • 28. THE BACILLI AND THE VIBRIOS 175myalgia and headache but no pneumonia. Immunocompromised patients and smokers are at anincreased risk of acquiring Legionnaire’s disease.Laboratory DiagnosisRapid diagnosis of Legionnaire’s disease can be done on respiratory tract secretions using the directfluorescent antibody (DFA) test. The sample can be cultured onto FG medium or BCYE agar. Theorganism can also be isolated from environmental samples. Serology plays an important part indiagnosis because of difficulties in culturing. Antigen detection in urine sample is a commonlyused method to establish diagnosis.TreatmentErythromycin has been found to be useful in the treatment of this infection.PreventionChlorination and heating of water, and cleaning can help control the multiplication of Legionellain water and airconditioning systems.Klebsiella sp. (Bacillus mucosus capsulatus)They are members of the family Enterobacteriaceae. It is the second most populous member ofthe aerobic bacterial flora (after Escherichia coli) of human intestine. The different species areKlebsiella pneumoniae, Klebsiella ozaenae and Klebsiella rhinoscleromatis (based on biochemicalreactions), and there are over 80 serotypes (based on capsular antigens).MorphologyKlebsiella sp. are Gram negative, nonmotile bacilli with a well-defined polysaccharide capsule.Culture CharacteristicsKlebsiella grows well on ordinary media at optimumtemperature of 37ºC in 18–24 hours. On MacConkey’sagar, the colonies appear large, mucoid and pink dueto lactose fermentation. They are recognized on culturemedia due to formation of characteristic mucoidcolonies, which is due to the secretion of loose solubleslime by bacteria (Fig. 25.4).Biochemical ReactionsThey ferment sugars (glucose, lactose, sucrose,mannitol) with production of acid and gas. They areurease positive, indole negative, methyl red (MR)negative, Voges–Proskauer (VP) positive and citratepositive (IMViC– –++). They are urease producers(positive).FIGURE 25.4 Klebsiella sp. onMacConkey’s agar.Chapter-25.indd 175Chapter-25.indd 175 5/30/2013 2:27:27 PM5/30/2013 2:27:27 PM
  • 29. 176 MICROBIOLOGY FOR NURSESAntigenic StructureOver 80 serotypes have been recognized based on their capsular (K) antigens.PathogenicityKlebsiella pneumoniae is one of the most common causes of pneumonia in hospitalized patientsand causes many other nosocomial infections like urinary tract infection (UTI), septicaemia andrarely diarrhoea.Klebsiella ozaenae is associated with a disease characterized by foul-smelling nasal discharge.Klebsiella rhinoscleromatis causes rhinoscleroma, a chronic granulomatous hypertrophy of thenose.Laboratory DiagnosisSputum is the most commonly collected sample for the diagnosis of pneumonia. The patient shouldbe instructed to provide a deep coughed specimen (a good quality specimen will yield less than10 squamous epithelial cells per low power field). The samples are plated on blood agar andMacConkey’s medium and colonies obtained after overnight incubation are confirmed bybiochemical reactions. In suspected cases of UTI, urine sample is collected and processed.TreatmentEnvironmental strains of Klebsiella sp. are susceptible to a wide range of antibiotics but multipleresistances are common among the hospital strains.PreventionNosocomial transmission of infection can be controlled by following a strict infection controlprotocol, including sterilization of equipment and disinfection.The multiplicity of serotypes hinders the vaccine development.BACILLI ASSOCIATED WITH INTESTINAL CANALEscherichia coliE. coli belongs to the bacterial family Enterobacteriaceae, which by definition includes Gram-negative bacilli (Fig. 25.5), which are motile by peritrichous flagella or nonmotile, reduce nitratesto nitrites, are oxidase negative and, with a few exceptions, catalase positive. They ferment glucosewith the production of acid or acid and gas, grow luxuriantly on ordinary laboratory media andare facultatively anaerobic bacilli. E. coli is a parasite living only in the human or animal intestine.Once it is voided in the faeces, it remains viable in the environment only for a few days. Therefore,the detection of E. coli in water is taken as an evidence of recent pollution with human or animalfaeces.MorphologyE. coli is a Gram-negative straight rod and is usually motile. It is a non-spore-forming andnoncapsulated bacterium.Chapter-25.indd 176Chapter-25.indd 176 5/30/2013 2:27:27 PM5/30/2013 2:27:27 PM
  • 30. THE BACILLI AND THE VIBRIOS 177FIGURE 25.5 Escherichia coli (Gram-negative bacilli).CultureIt is an aerobe and facultative anaerobe, and grows onordinary culture medium at the optimum temperature of37ºC in 18–24 hours. On MacConkey’s medium, coloniesare pink due to lactose fermentation (Fig. 25.6). In liquidmedium, growth occurs as uniform turbidity.Biochemical ReactionsThey ferment glucose, lactose, mannitol and maltosewith production of acid and gas. They do not fermentsucrose. Indole and MR reaction are positive but VP andcitrate utilization tests are negative (IMViC++– –). Ureais not hydrolysed.Antigenic StructureSerotyping of E. coli is based on three antigens: thesomatic antigen O, the capsular antigen K and theflagellar antigen H. Several different serotypes of E. coli are found in the normal intestine. Thenormal colon strains belong to the early O group (1, 2, 3, etc.), while the enteropathogenic strainsbelong to the later O group (55, 111, etc.).ToxinsAs in the case of all other Gram-negative bacteria, E. coli also possesses endotoxic activityassociated with the O antigen. Beside this, it also produces the following:Enterotoxins Enterotoxigenic strains of E. coli (ETEC) produce one or both of the two enterotox-ins, a heat-labile toxin (LT) and a heat-stable toxin (ST).Haemolysin Some strains of E. coli produce a haemolysin, which can lyse erythrocyte of somespecies.FIGURE 25.6 Lactose fermentingcolonies of E. coli.Chapter-25.indd 177Chapter-25.indd 177 5/30/2013 2:27:27 PM5/30/2013 2:27:27 PM
  • 31. 178 MICROBIOLOGY FOR NURSESShiga-like toxin (SLT) Its properties are similar to shiga toxin produced by Shigella dysenteriaetype 1. It is also known as verocytotoxin (VT).PathogenicityE. coli is the most common cause of UTIs (cystitis, pyelitis and pyelonephritis). It is frequentlyassociated with pyogenic infections such as meningitis in neonates, wound infections, cholecystitis,peritonitis and septicaemia. It also causes gastroenteritis although the commensal strains do notact as primary pathogens in the intestine. Certain serotypes of E. coli that cause gastroenteritishave been identified. They can be divided into five distinct groups: enteropathogenic E. coli,(EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enterohaemorrhagicE. coli (EHEC) and enteroadherent E. coli (EAEC).Laboratory DiagnosisDiagnosis of diarrhoea caused by E. coli is based on plating the stool samples on MacConkey’smedium. This is followed by confirmation by biochemical and serological parameters. A modifiedMacConkey’s medium in which lactose is replaced by sorbitol has been employed for the detectionof EHEC as these do not ferment sorbitol, unlike most other E. coli strains. The diagnosis of UTIis made by collecting the clean-catch midstream urine (MSU) followed by its culture bysemiquantitative technique.Technique This technique employs the use of a standard loop that transfers a fixed volume ofurine to the culture medium. The number of colonies growing after overnight incubation are thensuitably interpreted.Interpretation Kass (1956) gave a criterion for active bacterial infection of urinary tract, and isreferred to as Kass’s concept of significant bacteriuria. According to this:Significant bacteriuria: When bacterial count is higher than 105per mL of a single species.Insignificant growth: Counts less than 103bacteria per mL; these are regarded as contaminants.Isolation of E. coli is followed by antibiotic susceptibility determination.TreatmentUTI is commonly managed with ciprofloxacin, nitrofurantoin or aminoglycosides. However,E. coli tends to develop resistance to multiple antibiotics; therefore antibiotic susceptibility testingis necessary.Preventive MeasuresUTI caused by E. coli may be precipitated by urinary obstruction due to prostatic enlargement,pregnancy or calculi, which need to be addressed, if possible.Typhoid and Paratyphoid Bacilli (Salmonella Typhi, Salmonella Paratyphi A, B, C)The typhoid and paratyphoid bacilli constitute the enteric fever group of Salmonella. The genushas more than 2200 serotypes. The serotypes that cause enteric fever in man include SalmonellaTyphi, Salmonella Paratyphi A, Salmonella Paratyphi B and less commonly, Salmonella ParatyphiC and Salmonella sendai. Enteric fever is endemic in all parts of India and on average more thanChapter-25.indd 178Chapter-25.indd 178 5/30/2013 2:27:27 PM5/30/2013 2:27:27 PM
  • 32. THE BACILLI AND THE VIBRIOS 1793,00,000 cases of enteric fever are reported each year (S. Typhi is the most common aetiologicalagent, but fever due to S. Paratyphi A is on a rise in India). These are exclusively or primarilyhuman parasites. In fact, man acts as the reservoir of infection through the cases and carriers.(Patients who continue to shed typhoid bacilli in faeces for 3 weeks to 3 months after clinical cureare called convalescent carriers; those who shed bacilli for more than 3 months but less than 1 yearare called temporary carriers and those who shed bacilli for more than 1 year are called chroniccarriers.) The main sources of infection are faeces and urine of cases or carriers, althoughcontaminated food and water act as secondary sources.The genus Salmonella belongs to the family Enterobacteriaceae.MorphologySalmonellae are Gram-negative bacilli. They are motile, nonsporing and noncapsulated.CultureThey are aerobic and facultative anaerobic, and growonordinaryculturemediaattheoptimumtemperatureof 37ºC. On MacConkey’s agar and deoxycholatecitrate agar (DCA), colonies are colourless due tononlactose fermentation (Fig. 25.7). The colonies are2–3 mm in diameter, circular, translucent, low convexand smooth. On xylose lysine deoxycholate (XLD)salmonellae produce red colonies with black centresdue to production of H2S.Selenite F broth and tetrathionate broth (TTB) arecommonly used as enrichment media for the isola-tion of the organism from faeces.Biochemical ReactionsThey ferment glucose, mannitol and maltose, formingacid and gas except S. Typhi, which produce onlyacid. They do not ferment lactose or sucrose. Indoleis not produced. Most salmonellae produce H2S in triple sugar iron (TSI) agar. They utilize citrate(except S. Typhi and S. Paratyphi A). Urea is not split (they are urease negative).Antigenic StructureSalmonellae possess the following antigens based on which they are classified and identified:(1) flagellar antigen H, (2) somatic antigen O and (3) a surface antigen Vi, found in somespecies.The H antigen is strongly immunogenic while O antigen is less immunogenic.The O and H antigens form the basis of serotyping of salmonellae (Kauffmann–White scheme).On the other hand, the Vi antigen is poorly immunogenic and only low titres of antibody are pro-duced following infection, and the persistence of these antibodies indicates the development of acarrier state.FIGURE 25.7 Non-lactose-fermentingcolonies of S. Typhi on MacConkey’s agar.Chapter-25.indd 179Chapter-25.indd 179 5/30/2013 2:27:27 PM5/30/2013 2:27:27 PM
  • 33. 180 MICROBIOLOGY FOR NURSESPathogenicityThe term enteric fever includes both typhoid fever caused by S. Typhi and paratyphoid fever causedby S. Paratyphi types A, B and C. It is a systemic infection characterized by continuous fever,relative bradycardia, constitutional symptoms and toxaemia. The disease is transmitted via thefaecal–oral route or urine–oral route. The incubation period of the disease is about 2 weeks. Theminimum infective dose is 102–105bacilli.The ulceration of bowel during the course of infection may lead to intestinal perforation andhaemorrhage. A chronic carrier state may develop in 2–5 per cent of cases. In most chronic carriers,the organisms persist in the gallbladder and biliary tract.Laboratory DiagnosisDiagnosis of enteric fever consists of (1) isolation of bacilli, (2) demonstration of antibody and(3) demonstration of antigen in urine.Isolation of bacilli This can be done by culture of specimens like blood, bone marrow aspirates,faeces, urine, duodenal fluid, etc. Choice of specimen depends on the duration of illness. Bloodculture forms the mainstay of diagnosis.Blood culture The culture of blood during the early part of illness, preferably within the first2 weeks, is useful. 5 mL of blood is collected and inoculated into 50 mL of bile broth/brain–heartinfusion broth. After overnight incubation, it is subcultured on to MacConkey’s medium. Theappearance of pale, non-lactose-fermenting colonies is suggestive and can be confirmed by bio-chemical and serological parameters.Clot culture It is an alternative to blood culture. 5 mL of blood is withdrawn aseptically into asterile container and allowed to clot. The serum isseparated and used for the Widal test, while the clotis aseptically transferred to the culture bottle.Faeces culture Faeces cultures are generally posi-tive after the 2nd week of illness (Fig. 25.8). Faecalsamples are inoculated into enrichment media suchas Selenite F broth, followed by subculture onselective media.Urine culture Urine culture is less useful than theother samples and repeated sampling may berequired to yield results.Demonstration of antibodyWidal test This is an agglutination test for detec-tion of agglutinins (H and O) in patients with entericfever. The antibodies appear in the serum at the endof first week and rise sharply during the 3rd weekof illness (Fig. 25.8). An antibody titre of 1:100 or100Positivity(%)806040201 2 3 4 5Week of infectionFIGURE 25.8 Laboratory diagnosis ofenteric fever. Per cent positivity during differ-ent stages of illness (from 1st to 5th week). ••,blood culture; oo, Widal test; and ++, faecesculture.Chapter-25.indd 180Chapter-25.indd 180 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 34. THE BACILLI AND THE VIBRIOS 181more for O agglutinins and 1:200 or more for H agglutinins is regarded as significant. If a sampleis collected during early illness then a repeat sample 7–10 days later is collected to demonstrate arise in antibody titre. It is necessary to obtain information on the distribution of agglutinin levels in‘normal sera’ in different areas in order to determine the cut-off titres of the test in a particular geo-graphic region.Demonstration of antigen in urine Vi antigen of S. Typhi is consistently present in the blood inthe early phase of the disease, and also in the urine of patients. This antigen can be detected usingcounterimmunoelectrophoresis (CIE) or ELISA system.TreatmentChloramphenicol was initially the drug of choice for the treatment of enteric fever. Thereafter,amoxicillin/ampicillin and cotrimoxazole were used effectively till late 1980s. Ciprofloxacin,which was used during the 1990s, has now limited utility.The first-line treatment today comprises the 3rd-generation cephalosporins.Preventive MeasuresIn order to control enteric fever, it is important to control the reservoir of infection, which impliesthe early detection and treatment of cases and carriers. Carriers must not be allowed to work asfood handlers. In addition, proper disposal of wastes, health education, and quality control of watersupply and vaccines play an important role.The vaccines available for the prevention of typhoid fever include TAB vaccine, live oral Ty21avaccine and Vi polysaccharide vaccine. There is no vaccine for the prevention of paratyphoid fever.The use of TAB vaccine is no longer recommended by the WHO.Food Poisoning BacilliFood poisoning is an acute gastroenteritis caused by the ingestion of food or drink contaminatedwith either living organisms or their toxin. It has three main characteristics:1. There is usually a history of ingestion of common food.2. Involvement of many persons at a time.3. There is a similarity in signs and symptoms in most of the cases.The types of food poisoning include the following:1. Salmonella food poisoning caused by S. typhimurium/S. choleraesuis/S. enteritidis and otherSalmonella sp. through contaminated meat, eggs, milk and milk products.2. Staphylococcal food poisoning caused by the release of enterotoxin by S. aureus throughcontaminated custard, milk and other milk products.3. Botulism caused by Clostridium botulinum through canned foods.4. Food poisoning caused by Clostridium perfringens through the ingestion of poultry, meat andmeat products.5. Bacillus cereus food poisoning caused by ingestion of meat and vegetables (diarrhoeal form)or fried rice (emetic form).Chapter-25.indd 181Chapter-25.indd 181 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 35. 182 MICROBIOLOGY FOR NURSES6. Food poisoning caused by Vibrio parahaemolyticus through the ingestion of marine life.(Less common causes include ETEC, EIEC, EHEC and Campylobacter jejuni.)In India, the most common form of food poisoning is caused by Salmonella sp.DiseasesThe predominant feature of all forms of food poisoning is the presence of gastrointestinal symptomsexcept botulism that is characterized by diplopia, blurring of vision, dysphagia and muscle weakness.The incubation period ranges from 1 to 24 hours.DiagnosisDiagnosis is usually clinical. For laboratory confirmation, stool samples, vomitus and remnants offood are usually collected and inoculated into suitable media and incubated aerobically andanaerobically. (Quantitative assessment of bacteria is important in some situations.)TreatmentTreatment is usually symptomatic, although antitoxin is of considerable value in prophylaxis ofbotulism.Preventive MeasuresIt is important to lay emphasis on food sanitation and health education. In addition to this, acontinuous surveillance programme involving the laboratory analysis of food samples from eatingjoints will avoid outbreaks.Dysentery Bacilli (Shigella sp.)Dysentery is a clinical condition characterized by the passage of loose stools with blood and mucus.The bacilli causing dysentery belong to the genus Shigella. This genus belongs to the familyEnterobacteriaceae.They are divided into four serogroups (Shigella dysenteriae, Shigella flexneri, Shigella boydiiand Shigella sonnei), which are further divided into serotypes. Man is the only natural host forShigella and the disease is transmitted by the faecal–oral route.Shigellosis is of major concern in developing countries of the world because of poor sanitationand low hygiene standards. In India, S. flexneri has always been predominant species followed byS. dysenteriae, S. sonnei and S. boydii.MorphologyShigellae are Gram-negative, nonmotile, noncapsulated and nonsporing short bacilli.Culture CharacteristicsThey are aerobic and facultative anaerobes and can grow on ordinary media and the optimumtemperature for growth is 37ºC. Colonies on MacConkey’s agar and DCA are colourless (NLF)except in case of Shigella sonnei, which form pink colonies due to late lactose fermentation. XLDis the most frequently used selective media for the isolation of shigellae.Chapter-25.indd 182Chapter-25.indd 182 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 36. THE BACILLI AND THE VIBRIOS 183Biochemical ReactionsShigella dysenteriae (subgroup A) is the only subgroup that is mannitol nonfermenting. It is alsothe only member of the family that does not form catalase. Shigella sonnie ferments lactose andsucrose late. It is indole negative and decarboxylates ornithine. Broadly, Shigella flexineri andShigella boydii ferment mannitol with acid production, and are lactose and sucrose nonfermenting.Production of indole is variable.PathogenicityDysentery is characteristic of shigellosis but diarrhoea is often common and precedes it. Theincubation period of the disease on an average is 2 days. The minimum infective dose is low, i.e.10–100 bacilli. The severity of disease may vary from acute fulminating dysentery to mild diarrhoea.It may be associated with gripping pain and tenesmus. Fever and vomiting may or may not bepresent. The bacilli infect the epithelial cells of the villi in the large intestine leaving behindtransverse superficial ulcers. Complications are most often seen with S. dysenteriae type 1 in theform of arthritis, toxic neuritis and haemolytic uraemic syndrome (HUS).Laboratory DiagnosisAn ideal specimen is a direct swab of an ulcer taken by sigmoidoscopic examination. Alternatively,stool samples may be collected and directly plated onto selective media like DCA and XLD agar,immediately. In case of delay, the sample can be transported in a suitable medium such as bufferedglycerol saline. Confirmation can be done by biochemical and serological characterization usingpolyvalent and monovalent antisera.TreatmentTreatment tends to be symptomatic. Antibiotics tend to prolong the excretion of shigellae and,therefore, should be avoided except in serious illness. Nalidixic acid and ciprofloxacin has shownto be life saving in such situations. Shigella dysenteriae type 1 has been shown to be associatedwith multiple-drug resistance.Preventive MeasuresProper disposal of wastes, quality control of water supply, health education and control of flypopulation play an important role. Vaccines for Shigella, however, are still in the experimentalstage of development.Proteus sp.Proteus sp. are free-living saprophytes found in the soil, water and vegetation, and are also seenas commensals in the intestine of man. They belong to the family Enterobacteriaceae but differfrom the other members in being urease positive and phenylalanine deaminase positive.MorphologyThey are Gram-negative rods but pleomorphism is common with coccid forms and even longfilaments. They are actively motile.Chapter-25.indd 183Chapter-25.indd 183 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 37. 184 MICROBIOLOGY FOR NURSESCulture CharacteristicsThey are aerobic and facultatively anaerobic, and growwell on ordinary media. Most strains produce swarmingon blood agar media (Fig. 25.9). Swarming does not occuron MacConkey’s medium, on which non-lactose-ferment-ing colonies are seen. Growth on culture media emits acharacteristic fishy or seminal odour.Biochemical ReactionsThe distinctive character of this genus is the ability ofdeamination of phenylalanine to phenylpyruvic acid (PPA).They are urease positive, MR positive and VP negative.All ferment glucose producing acid and gas (with fewexceptions). Lactose is not fermented. They are speciatedbased on indole, H2S production, citrate utilization andornithine.Antigenic StructureThe bacilli possess somatic (O) antigen and flagellar (H) antigens. They form the basis forserotyping.PathogenicityIt is most often associated with the urinary tract infection (UTI). The UTI caused by Proteus sp.is particularly serious because of calculus formation occurring as a result of precipitation ofmagnesium phosphate due to the alkalinity caused as a result of ammonia released by it. It is alsoassociated with pyogenic infections, especially that of burns. It can cause infantile diarrhoea. Italso plays an important role in nosocomial infections.Laboratory DiagnosisDiagnosis can be established by isolating Proteus sp. from urine and other relevant samples. Theswarming colonies on blood agar (Fig. 25.9) can be confirmed by biochemical reactions. Growthof Proteus sp. emits a characteristic fishy or seminal odour.TreatmentMost strains are sensitive to aminoglycosides such as gentamicin, amikacin and also to cepha-losporins. Nitrofurantoin is useful in the treatment of UTI.PreventionIt is important to follow aseptic techniques during instrumentation of the urinary tract to preventinfection.FIGURE 25.9 Blood agar withswarming growth of Proteus sp.Chapter-25.indd 184Chapter-25.indd 184 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 38. THE BACILLI AND THE VIBRIOS 185Pseudomonas sp.Pseudomonas sp. may occur as free-living saprophytes, commensals or pathogens. There are morethan 200 species of Pseudomonas, but Pseudomonas aeruginosa is of particular interest not onlybecause of its resistance to multiple antibiotics but also because of its prevalence in the hospitalenvironment and its ability to cause severe, sometimes untreatable, hospital infections. Two otherspecies of Pseudomonas, i.e. P. mallei and P. pseudomallei, can cause severe infections in man,though infrequently. P. pseudomallei is now called Burkholderia pseudomallei.P. aeruginosa can survive and multiply in the moist areas of the hospital such as sinks, respira-tors and humidifiers. Another interesting feature is its ability to resist chemical disinfectants. Infact, cetrimide and Dettol have been incorporated in the media for the selective isolation of thisorganism. All these factors help the organism to establish itself in the hospital environment.MorphologyThey are slender Gram-negative bacilli that are actively motile by a polar flagellum. They arenonsporing and noncapsulated.Culture CharacteristicsThey are strict aerobes and grow well on ordinary media like nutrient broth and nutrient agar. (Theorganism is unique as it has the ability to grow even in distilled water.) The optimum temperaturefor growth is 37ºC. Colonies on nutrient agar are smooth, large, translucent, low convex, 2–4 mmin diameter. P. aeruginosa most frequently produce greenish blue pigment (Fig. 25.10). In liquidmedia (peptone water) it forms a turbidity with a surface pellicle.Biochemical ReactionsAll strains of P. aeruginosa are oxidase positive and utilize only glucose oxidatively with acidproduction.PathogenicityP. aeruginosa causes infections mainly in thehospitalized patients. It also causes infections in thecommunity, like acute otitis media. In hospitals, it isfrequently associated with UTI and infections of thewounds, burns and eye. It can also cause septicaemiain immunocompromised patients. It also causesnotorious respiratory infections in cases with cysticfibrosis and is a common cause of ventilator-associatedpneumonia (VAP).Laboratory DiagnosisDiagnosis is established by the isolation of the organismfrom clinical specimens. P. aeruginosa grows well onordinary laboratory media but pigment production isbest appreciated on nutrient agar (Fig. 25.10). It isrecognized on solid media by its typical colonialFIGURE 25.10 Pigment-producing strainof P. aeruginosa on nutrient agar plate.Chapter-25.indd 185Chapter-25.indd 185 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 39. 186 MICROBIOLOGY FOR NURSESappearance, a characteristic sweet, musty odour, pigment (when present) and a positive oxidasereaction. In broth, it forms a dense turbidity with a surface pellicle. It is actively motile and growswell at 42ºC.TreatmentP. aeruginosa is famous for its resistance to commonly used antibiotics. The antibiotics likely tobe effective include aminoglycosides such as amikacin, tobramycin; newer ureidopenicillins suchas piperacillin, quinolones; and third-generation cephalosporins. The beta-lactam/beta-lactamaseinhibitor combination is useful for the treatment of hospital-associated infections.PreventionThe best way of minimizing hospital infections is to eliminate the source of infection and tointerrupt the means of spread of this organism in the hospital environment through the use ofstandard precautions.Correction of underlying disorders is helpful. Pseudomonas vaccines are under development.ACID-FAST BACILLITubercle Bacillus (Mycobacterium tuberculosis)Mycobacterium means fungus-like bacterium. It was named so because of its tendency to sometimesform branching filaments. It was identified by Robert Koch in 1882. Infection with M. tuberculosisis a public health problem of tremendous magnitude world wide. It contributes to 3 million deathseach year, and perhaps one-third of the world’s population is infected with M. tuberculosis. Expertsclaim that M. tuberculosis claims more human lives than any other infectious microorganism.Estimated TB incidence in some parts of southeast Asia (WHO, 2011) ranges from 187 to 192cases per 100,000 population. What has made things worse is the lethal combination of HIV andTB. The people infected with HIV have a higher risk of developing clinical forms of TB, eitherfrom activation of dormant endogenous foci or from exogenous infection. Further, the HIV-infectedTB patients provide an ideal condition for mutation of strain to drug-resistant forms. This addsanother dimension to the problem, i.e. the emergence of multidrug-resistant TB (MDR-TB).MorphologyIt is Gram-positive, though it does not stain readily by Gram stain. It is also called acid-fast bacillus(AFB) because once stained, it resists decolourization by dilute mineral acids. These bacilli arenonsporing, noncapsulated and nonmotile. Ziehl–Neelsen staining is useful to study the morphologyof these organisms. With this stain, tubercle bacilli are seen bright red (acid fast), while the tissuecells and other organisms are stained blue (Fig. 3.6). In M. tuberculosis beaded or barred formsare frequently seen. They may also be stained using fluorescent dyes (e.g. auramine O).Culture CharacteristicsLowenstein–Jensen (LJ) medium is the conventional medium and used commonly for isolation ofthis bacterium. This medium consists of beaten eggs, asparagine, mineral salts, malachite greenChapter-25.indd 186Chapter-25.indd 186 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 40. THE BACILLI AND THE VIBRIOS 187and glycerol or sodium pyruvate. The tubercle bacilliusually grow in 2–8 weeks with an average of 6–8 weeks.The inoculated media may be held for up to 12 weeksbefore being discarded as negative.The colonies are rough, tough and buff coloured on LJmedium and are described as eugonic because of their lux-uriant character (Fig. 25.11). Smear is prepared from anisolated colony and stained with Ziehl–Neelsen technique.Confirmation is done by biochemical reactions. Niacin testand nitrate reduction test are positive in M. tuberculosis.Thereafter, drug susceptibility test on drug-incorporatedmedia may be performed. The whole procedure takes about3 months for the result to be available.Recently, new rapid culture and drug susceptibility tests,namely, Bactec-460 (a radiometric method) and mycobac-terial growth indicator tube (MGIT) have been developed.PathogenicityM. tuberculosis causes TB, a chronic infectious disease that primarily affects the lungs (pulmonaryTB). It may also affect the meninges, bones, joints, skin and intestine (extrapulmonary TB). Rarely,the primary infection may lead to haematogenous spread and the development of miliary TB.The most common source of infection is the human case whose sputum is positive for M. tuber-culosis and who has never been treated or was partially treated. Once the bacilli are shed from suchan open case of TB, they remain viable for weeks in dust. Infection of another host depends onseveral factors such as the age and resistance of the host, virulence, dose and portal of entry of thebacillus. Therefore, the incubation period may be weeks, months or years.Laboratory DiagnosisSputum is the specimen most often collected and processed, but other specimens such as inducedsputum, fluids collected by gastric or bronchial lavage, urine, tissue from any organ, spinal fluid,blood and aspirates from lesions may be collected. All specimens from nonsterile sites are similarin that they must be decontaminated before culture.TABLE 25.2 Grading of AFB SmearsObservation Result GradeMore than 10 AFB per OIF in at least 20 fields Positive 3+1–10 AFB per OIF Positive 2+10–99 AFB per 100 OIF Positive 1+1–9 AFB per 100 OIF Scanty Record exactnumber seenNo AFB per 100 OIF Negative –Abbreviations: AFB, acid-fast bacillus; OIF, oil immersion field.FIGURE 25.11 LJ medium – withoutand with growth.Chapter-25.indd 187Chapter-25.indd 187 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 41. 188 MICROBIOLOGY FOR NURSESDirect microscopy The smears are stained by Ziehl–Neelsen method (see Fig. 3.6) or fluoro-chrome stain and examined under the microscope. Smears may be made directly from untreatedspecimen or from concentrated specimen following liquefaction, decontamination and centrifuga-tion. Smears of concentrates are prepared most often when the specimen is to be cultured too. If itis not to be cultured, add an equal volume of a 5 per cent hypochlorite solution such as commonhousehold bleach for a few minutes to kill bacilli before making the smear. The smears must beexamined carefully and results graded as per revised national TB control programme (RNTCP)guidelines (Table 25.2).Sputum smear microscopy is the mainstay of diagnosis of TB. All patients with a cough of 3weeks or more should undergo three sputum diagnostic examinations for AFB.Culture Culture is a very sensitive method for detection of tubercle bacilli. The conventional cul-ture involves the inoculation of specimen (concentrated) on two bottles of solid LJ medium forprimary growth of M. tuberculosis, followed by identification of M. tuberculosis species. The labo-ratory investigations for drug susceptibility tests are not freely available.New rapid culture and drug susceptibility tests methods can be used (as described above) wherefacilities for same are available.Serology is of no value in the diagnosis of TB. Tuberculin test (Mantoux) has been invalidatedas a diagnostic tool because of its limitations.Molecular MethodsThe advanced molecular test, such as polymerase chain reaction (PCR), is being used. The test ishighly sensitive, specific and rapid. However, it is expensive and at present not freely available forroutine use.TreatmentThe classical long-course conventional treatment regime lasts for as long as 18 months and includesstreptomycin, isoniazid and thiacetazone. Directly observed treatment – short course (DOTS),which is now followed, is based on administration of isoniazid, rifampicin, pyrazinamide andstreptomycin/ethambutol for 6–7 months.DOTS Plus treatment of MDR-TB consists of addition of two or more drugs to the existing drugregimen. These drugs include kanamycin, ethionamide, quinolones, clofazimine and para-amin-osalicylic acid (PAS). However, treatment of MDR-TB is difficult, very expensive and toxic.Preventive MeasuresEarly detection of cases and sources of infection, and their prompt treatment are essential.Immunization with BCG, the live attenuated vaccine, has been found to be very useful in pre-venting severe forms of childhood TB, i.e. tuberculous meningitis and miliary TB. In addition,there are about 100 TB vaccines in experimental stages of development.Leprosy Bacillus (Mycobacterium leprae)M. leprae causes Hansen’s disease or leprosy, which continues to be a major public health problemin many parts of the world. In ancient times, it was known as kushtha roga, and was attributed tocurse from Gods.Chapter-25.indd 188Chapter-25.indd 188 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 42. THE BACILLI AND THE VIBRIOS 189MorphologyM. leprae is a slender, slightly curved or straight bacillus. It is an AFB like the tubercle bacillus,but it is less acid fast (5% sulphuric acid is used instead of 20%). They are seen in human host,both intracellularly and extracellularly and also form the characteristic clumps or globi. It ispossible to distinguish between live and dead bacilli in smears because the former are uniformlystained and the latter, fragmented. The percentage of solid staining bacilli is referred to asmorphological index (MI).Culture CharacteristicsIt has not so far been possible to cultivate lepra bacilli either in artificial media or in tissue culture.The animal model susceptible to infection is the nine-banded armadillo (Dasypus novemcinctus).PathogenicityLeprosy is a chronic granulomatus infection caused by M. leprae, which primarily involves theperipheral nerves, skin and nasal mucosa. It is exclusively a human disease with a long incubationperiod ranging from 2 to 5 years. According to the internationally accepted classification of leprosyby Ridley and Jopling, it has been divided into five groups: tuberculoid (TT), borderline tuberculoid(BT), borderline (BB), borderline lepromatous (BL) and lepromatous (LL). The outcome ofinfection depends on the cell-mediated immunity (CMI) of the individual. In lepromatous leprosy,there is complete breakdown of CMI, and lepromin test is negative. It is usually the BL and LLcases that act as a source of infection in the community.The disease is transmitted by contact, which may be direct (e.g. skin to skin) or indirect (e.g. byfomites). This disease does not kill, but causes severe, progressive and permanent physical disabil-ity in the form of nasal depression, loss of fingers or toes, and damage to internal organs.Laboratory DiagnosisDiagnosis of leprosy is clinical. AFB can be demonstrated in patients of lepromatous leprosy butnot in tuberculoid form. Smears are made from various sites such as nasal mucosa, ear lobules andskin. They are stained by ZN method using 5 per cent sulphuric acid. The specimen containingleprosy bacilli may be injected into the foot pad of mice to reproduce the characteristic lesions.The nine-banded armadillo is susceptible to experimental infection. Newer methods like macrophageculture have also been developed.The skin test, i.e. the lepromin test, is useful for classification of leprosy and to assess theresponse to treatment. Several other serological tests like ELISA for the detection of antibodies tophenolic glycolipid (PGL), an antigen unique to M. leprae, have been developed.ImmunityInfection with lepra bacilli induces both humoral and cellular immune responses. Humoral antibodiesare not able to destroy the lepra bacilli whereas cellular immunity is capable of destroying them.The type of leprosy in an individual, i.e. TT, BT, BB, BL, LL, is determined by the status of cell-mediated immunity in him. When it is adequate, the lesions are of the tuberculoid type. When CMIis deficient, the lepromatous type of disease develops.Chapter-25.indd 189Chapter-25.indd 189 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 43. 190 MICROBIOLOGY FOR NURSESLepromin Test The lepromin test is carried out by the intradermal injection of 0.1 mL of lepromin.The response to lepromin is typically biphasic consisting of an early and a late reaction.1. Early reaction of Fernandez consists of erythema and induration developing in 24–48 hoursand usually remains for 3–5 days.2. Late reaction of Mitsuda appears 1–2 weeks after the injection, reaching a peak in 4 weeks.The reaction appears in the form of a nodule that may ulcerate.The lepromin test is employed for classifying the lesion of leprosy. The test is positive in tuber-culoid, while it is negative in lepromatous leprosy. Further, it helps to assess prognosis and responseto treatment. A positive test implies good prognosis and response to drugs being administered.TreatmentFor the purpose of treatment, leprosy cases have been divided into paucibacillary (PB) group(includes only the smear-negative BB, BT and TT patients) and multibacillary (MB) group (includesany smear-positive patient).For PB group, dapsone (daily) and rifampicin (once a month) are recommended for at least 6months. For MB group, dapsone (daily), rifampicin (once a month) and clofazimine (daily) arerecommended for at least 2 years. The PB and MB patients are to be followed up for 2 years and 5years, respectively, after the completion of treatment.PreventionEarly diagnosis and treatment of cases is important. Immunoprophylaxis in the form of BCGvaccine has been shown to provide some protection against clinical leprosy. A leprosy vaccine tobe used as an adjunct to standard multidrug therapy has been approved for use in India. It isprepared from a killed nonpathogenic strain of Mycobacterium.SPORE-BEARING BACILLITetanus Bacillus (Clostridium tetani)C. tetani is found abundantly in soil, dust and the intestine of man and animals. The spores of C.tetani are able to survive in soil for several years. C. tetani causes tetanus, a serious illness.MorphologyClostridium tetani is a Gram-positive, spore-bearing bacillus. The spores of C. tetani are terminalin location, thereby imparting the organism the characteristic drum-stick appearance (Fig. 25.12aand b).Chapter-25.indd 190Chapter-25.indd 190 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 44. THE BACILLI AND THE VIBRIOS 191Culture CharacteristicsIt is an obligate anaerobe that grows on ordinary media. The optimum temperature for growth is37ºC. It grows well in Robertson cooked meat (RCM) broth, thioglycollate broth and blood agar.In RCM broth, growth occurs as turbidity along with little gas formation. On blood agar, it exhibitsa swarming growth.Biochemical ReactionsC. tetani has slightly proteolytic, but demonstrates no saccharolytic property. It does not fermentany sugars.ToxinsC. tetani produces at least two distinct toxins: a haemolysin (tetanolysin) and a powerful neurotoxin(tetanospasmin). Tetanolysin may act as a leucotoxin, although its exact pathogenic role is notknown. Tetanospasmin is the toxin responsible for the clinical manifestation of the disease.PathogenicityC. tetani is the causative agent of tetanus. The incubation period of the disease ranges from 6 to12 days. It is a disease characterized by muscular rigidity as well as painful paroxysmal spasmsof the voluntary muscles. The disease typically involves the facial muscles (risus sardonicus), themuscles of the back (opisthotonus) and the masseters (trismus).It is associated with high mortality, especially in cases of tetanus neonatorum. Tetanus neonato-rum is a result of unhealthy social practices like cutting of the umbilical cord with unsterile instru-ments or the application of cow dung on the umbilical stump. In adults, the disease usually resultsfrom the contamination of the wound with tetanus bacilli and it is the deep puncture wounds thatare more susceptible.The disease may sometimes be localized to the ear in the form of otitis media (otogenictetanus).(a) (b)FIGURE 25.12 Clostridium tetani (drum-stick) appearance.Chapter-25.indd 191Chapter-25.indd 191 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 45. 192 MICROBIOLOGY FOR NURSESLaboratory DiagnosisDiagnosis of tetanus is basically clinical, although one can demonstrate C. tetani by microscopy,culture or animal inoculation. It grows well in RCM broth and isolation is more likely if excisedbits of tissue are taken from the necrotic depths of the wound. Once isolated, the toxin can bedemonstrated by inoculating culture into the root of the tail of a mouse.TreatmentIt is important to control spasms and maintain airway and nutrition. ATS (antitetanus serum) maybe administered to neutralize the toxin. Patients who recover from the disease should be immunizedwith a full course of tetanus toxoid.PreventionIt is a preventable disease. Neonatal tetanus can be prevented by immunizing the pregnant motherwith two doses of tetanus toxoid and by following clean delivery practices.In case of wounds, proper cleaning is essential. Further course of action depends on the type ofwound and the immune status of the individual. In a nonimmune individual, antibiotics like penicil-lin are combined with ATS and the tetanus toxoid (three doses). In an immune individual, one doseof tetanus toxoid and antibiotics suffice. In a partially immune patient, one dose of tetanus toxoid,ATS and antibiotics are required.Gas Gangrene Bacilli [C. welchii (perfringens), C. septicum and C. oedematiens (novyi)]These clostridia are widely distributed in the environment: in soil, water and dust. They form apart of normal flora of the intestine of man and animals, where they are found in large numbers.C. perfringens is the most important of the clostridia causing gas gangrene. This may be seen asthe sole aetiological agent, but more frequently it is seen in association with other clostridial speciesand even nonclostridial anaerobes and facultative aerobes.MorphologyC. perfringens are Gram-positive, stout, pleomorphic bacilli having central or subterminal spores.The spores of C. perfringens are rarely seen in culture or material from the lesions, and theirabsence is one of the characteristic morphological features of C. perfringens (Fig. 25.13).FIGURE 25.13 Clostridium perfringens (stout Gram-positive bacilli without spores).Chapter-25.indd 192Chapter-25.indd 192 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 46. THE BACILLI AND THE VIBRIOS 193Culture CharacteristicsIt is an anaerobe that grows in RCM broth, thioglycollate broth and blood agar. The optimumtemperature for growth is 37ºC. On blood agar, colonies show a target haemolysis, resulting froma narrow zone of complete haemolysis caused by theta toxin and a much wider zone of incompletehaemolysis due to alpha toxin.In RCM broth, the meat peaces turn pink but are not digested.Biochemical ReactionsGlucose, maltose, lactose and sucrose are fermented with the production of acid and gas. In litmusmilk, lactose fermentation leads to formation of acid, which changes the colour of litmus from blueto red. The acid coagulates the casein (acid clot) and the clotted milk is disrupted due to vigorousgas production; this is known as stormy fermentation.ClassificationC. perfringens is classified into five types, i.e. A–E, based on the toxins produced. Type A of C.perfringens is most frequently encountered in cases of gas gangrene.ToxinC. perfringens produces at least 12 distinct toxins, of which the four major toxins are alpha, beta,epsilon and iota toxins.Alpha (α) ToxinThis is the most important toxin and is responsible for the profound toxaemia of gas gangrene.Chemically, it is a phospholipidase (lecithinase C), which splits the lecithin in the medium tophosphorylcholine and diglyceride, which is responsible for the opacity seen around the coloniesbut not in the half containing antitoxin (Nagler reaction; Fig. 25.14) due to specific neutralizationof the alpha toxin.PathogenicityThese bacilli cause gas gangrene. The incubation period of the disease varies from a few hours to6 days. Gas gangrene is a clinical condition characterized by rapidly spreading myonecrosis thatusually follows severe trauma. The infection can be of either endogenous or exogenous inorigin.The C. perfringens multiply at the site and elaborate various toxins and other extracellular prod-ucts (collagenases and proteases) that damage the tissue further.Clinically, the disease is characterized by pain, tenderness and oedema of the affected part, whichmay become crepitus due to accumulation of gas. This is accompanied by profound features oftoxaemia.Laboratory DiagnosisAlthough the diagnosis is clinical, laboratory confirmation is based on microscopy, culture,demonstration of toxigenic characters and, less often, by animal pathogenicity testing.Chapter-25.indd 193Chapter-25.indd 193 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 47. 194 MICROBIOLOGY FOR NURSESMicroscopy Gram stain of the smear from the affected site typically shows the absence or distor-tion of white cells and other cells and the presence of large, stout, fat, Gram-positive rods withblunt ends and without spores. More often, bacteriology appears polymicrobial showing Gram-positive cocci and Gram-negative bacilli in addition to the structures mentioned above.Culture Most strains produce target haemolysis on blood agar, exhibit stormy fermentation and apositive Nagler’s test.Nagler’s test is performed on serum or egg yolk agar with C. perfringens antitoxin spread on onehalf of the plate (Fig. 25.14). This is a rapid method for demonstrating C. perfringens in clinicalspecimens.AntitoxinpresentNoantitoxinFIGURE 25.14 Nagler reaction.Treatment and PreventionPenicillin G is the drug of choice, although metronidazole can also be used effectively. Antibioticsare accompanied by aggressive surgical debridement and drainage along with the administrationof hyperbaric oxygen. Passive immunization with anti–gas gangrene serum may be used in caseswith extensively soiled wounds.Botulism Bacillus (Clostridium botulinum)C. botulinum, like other Clostridium sp., is found extensively distributed in nature. It is strictlyanaerobic and produces a very powerful neurotoxin, which is probably the most toxic proteinknown.MorphologyIt is a Gram-positive bacillus producing subterminal, oval, bulging spores giving it a tennis racketappearance.Culture CharacteristicsC. botulinum is a strict anaerobe and can grow on ordinary media. Optimum temperature for growthis 35oC. Commonly used media include blood agar and RCM broth.ToxinC. botulinum produced a powerful exotoxin (neurotoxin), which is responsible for its pathogenicity.The toxin attaches to the individual motor nerve terminals, preventing the release of acetylcholineChapter-25.indd 194Chapter-25.indd 194 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 48. THE BACILLI AND THE VIBRIOS 195and causing passive paralysis. This leads to diplopia, dysphagia, respiratory paralysis and othermotor paralyses. Death occurs due to respiratory failure. Food suspected to be contaminated withbotulinum toxin can be rendered completely safe by pressure cooking or boiling for 20 minutes.Eight serologically distinct types have been recognized (types A to H).PathogenicityC. botulinum is the primary agent of botulism. The three manifestations of the disease are foodbotulism, wound botulism and infant botulism. Food botulism occurs with the ingestion of preformedtoxin in the contaminated preserved food such as fish and meat. The incubation period of the diseaseis usually 12–36 hours. It is usually associated with canned food that exhibits signs of spoilage. Itmanifests in the form of thirst, vomiting, constipation, difficulty in breathing and swallowing. Thereis no diarrhoea. Wound botulism is a rare condition. Infant botulism occurs with C. botulinumcolonization of the infant’s gastrointestinal tract due to ingestion of food (e.g. honey) contaminatedwith spores.Laboratory DiagnosisIt may be possible to demonstrate Gram-positive, sporing bacilli in faeces, food, vomitus and othersamples. The most conclusive proof is the detection and specific neutralization of toxin in patient’sserum by toxin/antitoxin tests in mice.Treatment and PreventionSupportive therapy is important. Proper preservation and canning of food play a very significantrole in preventing botulism.During an outbreak, a prophylactic dose of antitoxin should be administered to all those whohave consumed the suspected food article.Bacillus of Pseudomembranous Colitis (Clostridium difficile)Clostridium difficile was so named because of the unusual difficulty in isolating it.MorphologyIt is a typical Gram-positive rod with subterminal spores.Culture CharacteristicsIt grows on selective medium such as cycloserine–cefoxitin fructose agar (CCFA), producingyellow ground-glass colonies.ToxinMost strains of C. difficile produce at least two toxins: cytotoxin, which is cytopathic for mosttissue culture cell lines and an enterotoxin, which promotes fluid secretion and destroys intestinallining cells. The colitis is due to the active multiplication of C. difficile and the production of theenterotoxin.Chapter-25.indd 195Chapter-25.indd 195 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 49. 196 MICROBIOLOGY FOR NURSESPathogenicityC. difficile causes colitis, with or without membrane formation, which is a complication of antibiotictherapy (antibiotic- associated colitis). Antimicrobial agents implicated include beta-lactams,lincomycin and clindamycin.Laboratory DiagnosisIsolation of bacilli Clostridium difficile can be isolated from the stool on CCFA medium.C. difficile does not produce spores on CCFA.Demonstration of toxin Toxin can be demonstrated in the faeces by its characteristic effect oncertain cell lines. ELISA can also be used for the demonstration of toxin.Treatment and PreventionRestricting the use of clindamycin and lincomycin may help. Oral vancomycin and metronidazoleare used to treat C. difficile colitis.Anthrax Bacillus (Bacillus anthracis)Bacillus anthracis is a microorganism of considerable historical importance. Robert Koch cultivatedit in pure culture in vitro, inoculated it into the animals and induced anthrax and, thus, establishedthe Koch’s postulates. It was also grown by Louis Pasteur and was made the first effective bacterialvaccine.Animal anthrax is recognized in nearly all parts of India. The spores of B. anthracis are shed byanimals dying or dead from anthrax, into the environment. The bacilli do not sporulate in vivo, butdo so when discharged from the dying animal. The dormant spores may then survive in the environ-ment for long periods. Animals, such as sheep and cattle ingest spores while grazing, or inhalethem. As animals develop anthrax, the cycle repeats itself. Man is accidentally infected either byinhalation or by ingestion of spores.Human anthrax has been reported from limited geographic locations in India. Since the 1990s,there have been reports of increasing incidence of human anthrax. Therefore, it is now recognizedas one of the emerging bacterial diseases in India.MorphologyIt is a sporogenous, large, nonmotile, Gram-positive, rod-shaped bacterium surrounded by acapsule. It is non–acid fast. The capsular material can be demonstrated by polychrome methyleneblue (M’Fadyean’s reaction).Culture CharacteristicsB. anthracis is an aerobe and facultative anaerobe, with optimum temperature for growth being35–37oC. It grows well on ordinary media.Nutrient agar Colonies are round, 2–3 mm in diameter, raised, opaque and greyish white. Underlow power of the microscope, the edge of colony is found to be composed of long, interlacingchains of bacilli, resembling locks of matted hair, referred to as the medusa-head appearance.Chapter-25.indd 196Chapter-25.indd 196 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 50. THE BACILLI AND THE VIBRIOS 197Blood agar The colonies are usually nonhaemolytic.PLET medium This is a selective medium comprising of heart infusion agar with polymyxin,lysozyme, ethylene diamine tetraacetic acid (EDTA) and thallous acetate.ToxinThe disease manifestations are due to a three component protein exotoxin consisting of oedemafactor, lethal factor and protective antigen.PathogenicityB. anthracis is the causative agent of anthrax.Anthrax is a zoonotic illness. It is transmitted to humansby contact with infected animals or contaminated animal products, e.g. wool, bones, hides, etc. Inhumans, the disease may manifest as cutaneous malignant pustule (hide porter’s disease), pulmonary(wool sorter’s disease), gastrointestinal infections or as meningitis. All cases may be associated withfatal septicaemia. Clinical manifestations include haemorrhagic pneumonia, haemorrhagic meningi-tis, bloody diarrhoea and septicaemia. All are associated with a high mortality rate.Based on what is known about the disease, combined with some reasonable assumptions, B.anthracis has been put in the category of top biological weapons.Laboratory DiagnosisDiagnosis of anthrax is established by microscopic examination (of the material from a malignantpustule, sometimes in sputum from pulmonary anthrax, and also in the blood in the septicaemicstage of all forms of the infection), culture, animal inoculation and detection of antigen in theinfected tissue.Microscopy Gram-positive bacilli with positive M’Fadyean’s reaction allow a presumptive diag-nosis to be made. A fluorescent antibody technique has also been used to demonstrate B. anthracisin smears.Culture It grows well on ordinary media such as nutrient agar giving a medusa-head appearanceunder the low-power microscope.Animal inoculation B. anthracis can also be isolated from the infected tissue by rubbing it onshaven skin of a guinea pig.B. anthracis should be handled in a category 3 biosafety cabinet.TreatmentPenicillin is the drug of choice. In case of penicillin allergy, erythromycin, chloramphenicol anddoxycycline may be used. Antibiotics have no effect on the toxin.PreventionHuman anthrax can be prevented by proper sterilization of animal products like wool and skin. Inaddition, the carcasses of animals suspected to have died of anthrax are buried at least 9–12 feetbelow the ground in quicklime.An anthrax vaccine has been designed to protect agricultural workers against subcutaneousinfection.Animal anthrax can be prevented by active immunization with a spore vaccine (Sterne vaccine).Chapter-25.indd 197Chapter-25.indd 197 5/30/2013 2:27:28 PM5/30/2013 2:27:28 PM
  • 51. 198 MICROBIOLOGY FOR NURSESBACILLI DERIVED FROM INFECTED ANIMALSPlague Bacillus (Yersinia pestis)Plague is caused by Yersinia pestis. In India, large plague outbreaks occurred during the first halfof the 20th century. In addition, in August 1994, outbreaks of bubonic and pneumonic plague havebeen reported in India.Yersinia pestis is the cause of wild, sylvatic plague in many species of wild rodents in variousparts of the world. Rodents are in fact the primary hosts of Y. pestis and infection is usually trans-mitted by fleas. Some rodents are relatively resistant to disease; they act as permanent reservoirhosts maintaining the sylvatic or wild rodent plague activity.When commensal rodents (those predominantly dependent on humans for food) get infected,outbreaks (epizootics) of murine plague occur. Sick and dying rats may fall off roof (rat fall) andunusually large numbers of rat carcasses may be found in human habitats. As the body temperatureof the carcasses fall, fleas escape and find other warm-blooded hosts such as dogs and humans.Thus rural or urban plague activity depends upon the densities of wild and commensal rodents aswell as the fleas on them plus the presence of sylvatic plague focus in the region (reservoir).Human plague arises either as a zoonosis through the transfer of infection from rats to manthrough the bites of infected rat fleas (Xenopsylla cheopis) resulting in bubonic plague, or throughdroplet infection that gives rise to the highly contagious pneumonic plague that spreads from per-son to person independently of rats and fleas.Septicaemic plague may also occur either primarily or as a complication of the other two forms.MorphologyIt is a short, thick coccobacillus with rounded ends which onstaining give a characteristic bipolar (safety pin) appearance(Fig. 25.15).It is Gram-negative, although Gram-staining does not yieldsatisfactory results. It forms capsules in living tissues and lessreadily in cultures.Culture CharacteristicsThe organism is aerobic and facultatively anaerobic. Theoptimum temperature for growth is 27oC. It grows on ordinarymedia, and ghee broth is used to demonstrate some growthcharacteristics.Nutrient agar Colonies are small, delicate and transparentafter 24–48 hours of incubation.Blood agar Colonies are small, delicate, transparent, nonhaemolytic and dark brown.Ghee broth If grown in a flask of broth with oil or ghee floated on the top (ghee broth), a charac-teristic growth occurs, which hangs down into the surface, resembling stalactites (stalactitegrowth).FIGURE 25.15 Yersinia pestis(safety pin appearance).Chapter-25.indd 198Chapter-25.indd 198 5/30/2013 2:27:29 PM5/30/2013 2:27:29 PM
  • 52. THE BACILLI AND THE VIBRIOS 199Biochemical ReactionsY. pestis ferments glucose, mannitol and maltose with the production of acid but no gas. Lactoseand sucrose are not fermented. It is catalase positive, indole negative, urease negative and citratenegative.PathogenicityPlague is a severe and highly fatal disease. This infection is carried by rodents and is transmittedby fleas.Bubonic plague Most human plague is the bubonic form, which results from the bites of theinfected fleas. The incubation period of plague ranges from 1 to 7 days and is characterized byrapid onset of fever, chills, headache, malaise, myalgias and prostration. In particular, bubonicplague is characterized by painful swelling of lymph nodes (buboes) in the inguinal, axillary orcervical regions.Pneumonic plague It is characterized by cough and dyspnoea. This form is highly contagiousresulting in explosive outbreaks.Septicaemic plague Septicaemic plague may result in fulminant Gram-negative shock withoutlocalized signs of infection. Multiple clinical presentations can occur in one patient.Another form of disease is pestis minor, a mild disease characterized by fever and buboes.Laboratory DiagnosisFor all suspected cases, appropriate diagnostic specimens include blood for culture and serumantibodies for suspected pneumonic cases, sputum samples; and for suspected bubonic cases,aspirates from affected lymph nodes. Diagnosis is established by microscopy, culture and animalinoculation. Smears are made and stained by Wayson’s stain to demonstrate the characteristicbipolar staining. It grows well on ordinary laboratory media although special media like ghee brothmay be used. It is pathogenic to common laboratory animals like rats, mice and guinea pigs.TreatmentStreptomycin is the preferred drug for the treatment of plague, but gentamicin, tetracyclines andchloramphenicol are also effective.PreventionPreventive measures include: controlling rodents by using suitable rodenticides (warfarin, zincphosphide, arsenic trioxide, cyanogens, etc.), trapping and killing; applying insect repellents;avoiding the handling of dead or sick animals; administering prophylactic antibiotics.Brucellosis Bacilli (Brucella abortus, Brucella melitensis and Brucella suis)Brucella was so named in the honour of Bruce, who grew it from the spleen and blood of apatient.Chapter-25.indd 199Chapter-25.indd 199 5/30/2013 2:27:29 PM5/30/2013 2:27:29 PM
  • 53. 200 MICROBIOLOGY FOR NURSESBrucellosis is one of the serious diseases affecting the livestock of our country. It is a zoonosis,primarily affecting goats, sheep, cattle, buffaloes, pigs and other animals, and transmitted to manthrough contact with infected animals or their products. Man acquires Brucella infection by inges-tion, contact, inhalation and accidental inoculation. In animals, it primarily affects the reproductivetract leading to abortion, stillbirths, infertility, etc. In man, it may take the form of a prolonged,often vague, sometimes febrile illness.MorphologyThe genus Brucella consists of Gram-negative, coccobacilli or short rods arranged singly or rarelyin short chains.Culture CharacteristicsThey are aerobic and grow poorly on ordinary media. The media employed for culture are liverinfusion broth and tryptose broth. In liquid media, it shows uniform growth. The addition ofbacitracin, cycloheximide and polymyxin makes these media selective. On solid media, coloniesare small, moist and translucent.Biochemical ReactionThey are catalase and oxidase positive. No sugars are ordinarily fermented. It rapidly hydrolysesurea (urease positive).PathogenicityBrucellosis has different clinical manifestations: asymptomatic, acute, chronic, relapsing andlocalized. Asymptomatic brucellosis is usually occupational. The acute illness presents as feverwith malaise, chills, nocturnal sweats and weakness. Some patients develop a nonarticular arthralgiaaffecting the knee, ankle, shoulder or elbow. Infection with B. abortus is usually milder than thatwith B. suis or B. melitensis.Chronic brucellosis is difficult to diagnose. There maybe loss of body weight, enlargement of lymph nodes,splenomegaly and hepatomegaly.Laboratory DiagnosisDiagnosis of human brucellosis consists of (1) isolation ofbacilli (2) demonstration of antibody.Blood culture This is the most definitive method for thediagnosis of brucellosis. Blood is inoculated into a bottleof trypticase–soy broth or liver infusion broth and incu-bated at 37oC under 5–10 per cent CO2. Subcultures aremade on solid media every 3–5 days, and should not bedeclared negative in less than 4–8 weeks. The CastanedaLiquidmediaSolidmediaFIGURE 25.16 Castaneda’s medium.Chapter-25.indd 200Chapter-25.indd 200 5/30/2013 2:27:29 PM5/30/2013 2:27:29 PM
  • 54. THE BACILLI AND THE VIBRIOS 201method of blood culture is recommended. In this, both the solid and liquid media are available inthe same bottle. For subculture, the bottle is simply tilted so that the broth flows on the surface ofagar. It is then again incubated in the upright position, and colonies appear on slant (Fig. 25.16).This method minimizes the chances of contamination and risk of infection to laboratory workers.Demonstration of antibody This is a tube agglutination test (also called standard tube agglutina-tion test) in which equal volumes of serial dilutions of patient’s serum and the standardized antigen(a killed Brucella suspension) are mixed and incubated at 37ºC for 24 hours. The agglutination titreis expressed in International Units. A titre of more than 1:160 is indicative of infection.TreatmentTetracycline is used either alone or in combination with streptomycin.PreventionStrict hygienic and sanitary measures such as proper disposal of afterbirth and excreta of animalscan reduce the incidence of brucellosis. In addition, adequate heat treatment of all products ofanimal origin and use of protective clothing is helpful.A vaccine is also available.THE VIBRIOSCholera Vibrio (Vibrio cholerae)Vibrio cholerae causes cholera.It was John Snow in 1855 who epidemiologically proved that spoiled water from the broad streetpump in London spread the disease. But it was in 1884 that Robert Koch isolated the poison postmortem from the intestinal contents of cholera victims and called it comma bacillus.Cholera has been endemic in the Ganges and the Brahmaputra deltas in Bengal from very earlytimes. Before 1817, cholera was almost exclusively confined to the state of Bengal. But between1817 and 1923, there were six pandemics caused by the strains of classical biotype, and each fol-lowed a marked exacerbation of the cholera situation in Bengal. The seventh pandemic that startedin 1961 is the first to be caused by vibrios of El Tor biotype and is still continuing.MorphologyV. cholerae is a Gram-negative, curved or comma-shaped, nonsporing and noncapsulated rod. Theyare actively motile; the motility is termed as darting type.Culture CharacteristicsV. cholerae is strongly aerobic. They grow well in alkaline medium, and growth is inhibited belowa pH of 6.8. They also grow on ordinary media.Ordinary MediaNutrient agar After overnight growth, colonies are moist, translucent, round discs about 1–2 mmin diameter, with a bluish tinge in transmitted light. The growth has a distinctive odour.Chapter-25.indd 201Chapter-25.indd 201 5/30/2013 2:27:29 PM5/30/2013 2:27:29 PM
  • 55. 202 MICROBIOLOGY FOR NURSESMacConkey’s agar The colonies are colourless at first, but become reddish on prolonged incuba-tion because of late lactose fermentation.Blood agar The colonies may show clearing due to haemodigestion.Peptone water Growth occurs in about 6 hours with a fine surface pellicle.Special Media These include the following:Transport or holding media, e.g. Venkatraman–Ramakrishnan (VR) medium and Cary–Blairmedium.Enrichment media, e.g. alkaline peptone water (APW) and Monsur’s taurocholate tellurite peptonewater.Selective media, e.g. alkaline bile salt agar (BSA). Monsur’s gelatin taurocholate trypticase telluriteagar (GTTA) medium, and thiosulphate citrate bile sucrose (TCBS) agar – all have a pH(8.2–8.6).Biochemical ReactionsIt is catalase and oxidase positive. Carbohydrate breakdown is fermentative, producing acid, butno gas. It ferments glucose, mannitol, sucrose, maltose and mannose, but not lactose, though lactosemay be split very slowly (late lactose fermenting). It is indole positive and reduces nitrates tonitrites. It is MR and urease negative. Gelatin is liquefied. It decarboxylates lysine and ornithine.VP reaction and haemolysis of sheep erythrocytes are positive in El Tor biotype, but negative inclassical biotype.Antigenic StructureV. cholerae contain somatic ‘O’and flagellar ‘H’antigens. The H antigen is shared by all the strains.They are serologically classified on the basis of their polysaccharide O antigens (~150 are described)and the O group I (V. cholerae O1) can be further categorized into two biotypes: Classical and ElTor. On the basis of minor O antigen (A, B, C), V. cholerae O1 are subdivided into three serotypes:Ogawa (AB), Inaba (AC) and Hikojima (ABC).ToxinV. cholerae produces exotoxin (enterotoxin). The enterotoxin is also called cholera toxin (CT). Itis composed of two fractions: A (active) and B (binding). It acts by the cyclic adenosine 3′5′monophosphate (cAMP) pathway. This leads to increased levels of cAMP in the gut epithelial cells(enterocytes), resulting in the active secretion of large volumes of fluid into the intestinal lumen.Besides this, two other toxins are produced: zonula occludens toxin (zot) and accessory choleraenterotoxin (ace).PathogenicityCholera is a diarrhoeal disease that affects only man. The incubation period varies from less than24 hours to about 5 days. The minimum infective dose is 108–1010bacilli. The illness is characterizedby profuse watery diarrhoea and vomiting that may lead to hypovolaemic shock and death in lessthan 24 hours.Chapter-25.indd 202Chapter-25.indd 202 5/30/2013 2:27:29 PM5/30/2013 2:27:29 PM
  • 56. THE BACILLI AND THE VIBRIOS 203The diarrhoeal stool has a typical rice water appearance, with flecks of mucus in a colourlessfluid. The disease manifestation is due to the production of toxins.Laboratory DiagnosisMicroscopy Stool samples should reach the laboratory immediately so that a hanging-drop prepa-ration can be made to demonstrate darting motility. In case of delay, the sample may be transportedin transport/enrichment media such as APW.Culture The enrichment media should be incubated for 6–8 hours including the transit time andthen plated onto selective media such as TCBS agar and nonselective media. Vibrio cholera formsyellow on TCBS due to sucrose fermentation (see Fig. 5.4). The direct plating of stool sample canalso be done. After overnight incubation, the colonies suggestive of vibrio are subjected to oxidasetest, biochemical reactions and agglutination with cholera O subgroup 1 serum.TreatmentPrompt and adequate replacement of fluids and electrolytes is essential. Oral tetracycline therapyis useful in severe cases.PreventionIt is important to remember that cholera cannot be completely eradicated because there exists anenvironmental reservoir of V. cholerae, but it can be controlled through improved sanitary measuresand supply of safe drinking water.Today the emphasis has shifted towards the development of oral vaccines. Two promisingvaccines include killed whole-cell/B subunit combination vaccine, and a live attenuated vaccine.V. cholerae O139 syn. Bengal As mentioned earlier, there have been seven pandemics due toV. cholerae serogroup O1, the first six with the classical biotype and the seventh with El Tor. V.cholerae non-O1 serogroups were not known to cause the epidemic of diarrhoea. They wereknown to cause sporadic cases and small outbreaks of diarrhoea and extraintestinal infec-tions.However, in 1992, an epidemic of cholera-like disease due to a V. cholerae non-O1 serogroupbroke out in Chennai. In the next few months, it spread to other south Indian cities and reachedKolkata and, subsequently, Bangladesh. The disease affected thousands of individuals, mainlyadults, and caused many deaths indicating that the population was not immune to the organism.This was identified as V. cholerae O139.Halophilic VibriosVibrios that have a high requirement of sodium chloride are called halophilic vibrios. Their naturalhabitat is sea water and marine life. Some halophilic vibrios that cause diseases in man areV. parahaemolyticus, V. alginolyticus and V. vulnificus.Chapter-25.indd 203Chapter-25.indd 203 5/30/2013 2:27:29 PM5/30/2013 2:27:29 PM
  • 57. 204 MICROBIOLOGY FOR NURSESCampylobacters (Campylobacter jejuni, Campylobacter coli)Campylobacters are small curved Gram-negative rods, (comma-shaped, S-shaped or gull-wingsforms) with polar flagella. They resemble vibrios in their morphology, polar flagellation andoxidase positive nature, but differ in being microaerophilic, not fermenting sugars and having adifferent genetic make-up.PathogenicityCampylobacter jejuni and Campylobacter coli cause diarrhoea in cattle and pigs, respectively, butalso often cause diarrhoea, sometimes with septicaemia, in man.Outbreaks of infection have been attributed to the consumption of milk, water and meat.Laboratory DiagnosisCampylobacters can be isolated from the faeces of the patient using selective media such asSkirrows. Their exceptionally small size enables them to pass through filters fine enough to retainmost other kinds of bacteria. This characteristic was exploited earlier for their isolation fromcontaminated materials. The plates are incubated for 48 hours at 42–43ºC in a microaerophilicatmosphere. The droplet-like colonies of campylobacters can be confirmed by Gram stain, positiveoxidase test and demonstration of darting motility on a hanging-drop preparation. Speciation canbe done based on catalase production, sensitivity to nalidixic acid and hippurate hydrolysis.TreatmentIt is largely supportive. However, a few cases, such as those with long persistence of symptoms,frequent bloody diarrhoea or high fever, may require antimicrobials. In such cases, erythromycinis the drug of choice.PreventionEmphasis on food sanitation and safe drinking water supplies is important.Helicobacter pyloriThis curved Gram-negative bacillus was previously called Campylobacter pylori. The generalcharacters are similar to the campylobacters but differ from them in having not one, but four tofive polar flagella. It causes gastritis, duodenitis and peptic ulceration. The mode of transmissionof this organism is unknown. Diagnosis can be established by taking endoscopic biopsy of thegastric mucosa from the antrum. The specimen is Gram stained and cultured on campylobacter-selective medium in microaerophilic conditions at 37ºC. H. pylori is slow growing and is identifiedpresumptively by typical morphology and positive results for oxidase, catalase and rapid ureasetests.Chapter-25.indd 204Chapter-25.indd 204 5/30/2013 2:27:29 PM5/30/2013 2:27:29 PM
  • 58. THE BACILLI AND THE VIBRIOS 205TreatmentCombination therapy is used.BACTEROIDESBacteroides sp. are the most important group of anaerobes that cause human infection. They consistof two well-defined groups: Bacteroides fragilis group and Bacteroides melaninogenicus/oralisgroup. These are nonsporing, anaerobic, Gram-negative bacilli that may appear as slender rods orcoccobacilli. They are a part of the normal flora of the intestine, the female genital tract and upperrespiratory tract.Bacteroides sp. are often associated with abdominal, brain and lung abscesses, pelvic inflamma-tory disease and postoperative infection. It is important to remember that anaerobic infections ofthe lungs, brain and abdomen are mostly polymicrobial, i.e. the Bacteroides sp. is often associatedwith other bacteria. Classification of Bacteroides sp. is based on colonial (B. melaninogenicus hasblack/brown pigmented colonies) and biochemical features and on characteristic short-chain fattyacid patterns in gas chromatography.The Bacteroides sp. responds well to metronidazole, chloramphenicol and tetracycline.Things to Remember1. The bacilli commonly associated with the respiratory tract included C. diphtheriae, H. influenzaeand L. pneumophila.2. The bacilli commonly associated with the intestinal canal include E. coli, typhoid and paratyphoidbacilli, the different food poisoning bacilli, dysentery bacilli and Proteus sp.3. P. aeruginosa is notorious not only because of its resistance to multiple antibiotics but also becauseof its prevalence in the hospital environment and its ability to cause severe, sometimes untreatable,hospital infections.4. The two acid-fast bacilli of human importance include the M. tuberculosis and M. leprae.5. The different Clostridium species, i.e. C. tetani, C. welchii, C. septicum, and C. novyi are widelydistributed in the environment.6. Animal anthrax is recognized in nearly all parts of India while human anthrax has been reportedfrom limited geographical locations.7. B. anthracis has been put in the category of top biological weapons.8. Some of the important zoonotic diseases include anthrax, plague and brucellosis.9. Six pandemics in the past have been caused by the classical biotype of V. cholerae 01 while theseventh by the El Tor biotype, which is still continuing.Chapter-25.indd 205Chapter-25.indd 205 5/30/2013 2:27:29 PM5/30/2013 2:27:29 PM
  • 59. 206 MICROBIOLOGY FOR NURSESREVISION QUESTIONS1. Write short notes on the following:(a) Schick test(b) Distinguishing features of C. diphtheriae and diphtheroids(c) Satellitism in H. influenzae(d) Laboratory diagnosis of typhoid fever(e) Laboratory diagnosis of bacillary dysentery(f) Laboratory diagnosis of pulmonary tuberculosis(g) Lepromin test(h) Laboratory diagnosis of gas gangrene(i) Nagler’s reaction(j) Prevention of tetanus(k) El Tor vibrios(l) Cholera toxin(m) Vibrio cholerae O139Chapter-25.indd 206Chapter-25.indd 206 5/30/2013 2:27:29 PM5/30/2013 2:27:29 PM

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