Textbook of Microbiology and Immunology, 2e by Parija chap-26


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Textbook of Microbiology and Immunology, 2e by Parija chap-26

  1. 1. 26 43 Neisseria Mycobacterium Leprae many as hundred cocci. Smears from the pus sample show Introduction the intracellular kidney-shaped cocci, typically arranged in pairs with concave sides facing each other. The genus Neisseria consists of Gram-negative, aerobic, ■ Freshly isolated bacteria may be capsulated. They do not nonsporing, nonmotile cocci, typically arranged in pairs form endospores. (diplococci) with adjacent sides flattened together. The bacteria ■ They are nonmotile. belonging to this genus are oxidase positive and mostly catalase positive. They ferment sugars with production of acid but no gas. ◗ Culture The genus Neisseria consists of 10 species. Neisseria gonorrhoeae and Neisseria meningitidis are the two important species that cause N. gonorrhoeae is a fastidious coccus. It requires complex media human infections. These two species are strictly pathogens for for growth. The cocci grow on enriched media, such as blood or humans, whereas the other Neisseria species are commensals of chocolate agar. These cannot grow on ordinary media, such as the mouth and upper respiratory tract, and hence cause oppor- nutrient agar or Mueller–Hinton agar. They are aerobes but can tunistic infections. Human infections caused by Neisseria are also grow anaerobically. They grow optimally at a temperature listed in Table 26-1. range of 35–36°C. They fail to grow at temperature less than 25°C or greater than 37°C. The growth of bacteria is enhanced by incubation in humid atmosphere supplemented with Neisseria gonorrhoeae 5–10% CO2. 1. Blood agar: On blood agar at 24 hours, N. gonorrhoeae N. gonorrhoeae is a strict human pathogen. It is the causative produces convex small colonies measuring 0.6 ϫ 1.4 ␮m agent of gonorrhea, one of the most common sexually trans- in diameter. These colonies are translucent with entire mitted disease worldwide. Gonococci when transmitted non- edges and finely granular surface. They are soft and eas- sexually from the mother’s genital tract to the newborn during ily emulsifiable. Gonococci are inhibited by fatty acids birth cause ophthalmia neonatorum. and trace metals present in the digested products of pep- tone found in the blood agar. Addition of soluble starch Properties of the Bacteria to the media neutralizes the toxic effects of the fatty acids. ◗ Morphology 2. Selective media: Thayer Martin medium (chocolate N. gonorrhoeae shows following features: agar medium containing antibiotics, such as colistin, nystatin, and vancomycin) and modified New York City ■ N. gonorrhoeae are Gram-negative and aerobic diplococci. medium (a translucent medium containing vancomycin, They are mostly intracellular—found within the polymor- colistin, trimethoprim, and either nystatin or amphoteri- phonuclear (PMN) leukocytes—and some cells contain as cin B) are selective media used for isolation of gonococci from the clinical specimens containing mixed micro- Human infections caused by Neisseria bial flora. In these media, the growth of contaminat- TABLE 26-1 species ing bacteria is suppressed including that of commensal Bacteria Diseases Neisseria. On these media, N. gonorrhoeae produces small, Neisseria gonorrhoeae Gonorrhea, disseminated gonococcal translucent, and convex colonies, which are soft and eas- infections, ophthalmia neonatorum; ily friable. Four types of colonies of gonococci have been and other gonococcal diseases: anorectal recognized. gonorrhea, gonococcal pharyngitis, ■ These are T1, T2, T3, and T4. and acute perihepatitis ■ Types 1 and 2 are small and are brown pigmented colo- Neisseria meningitidis Meningitis and meningococcemia; other nies. The strains producing these colonies possess pili, meningococcal diseases: meningococcal pneumonia, septic arthritis, purulent are virulent and cause acute cases of gonorrhea. ■ Types 3 and 4 are large and are nonpigmented colonies. pericarditis, and endophthalmitis The cocci producing these colonies do not possess pili Other Neisseria species Opportunistic infections and are avirulent.Chap-26.indd 201 5/14/2012 1:30:29 PM
  2. 2. 202 BACTERIOLOGY 3. Transport medium: Stuart’s transport medium is used growth and metabolism of the cocci. They promote intake of for the collection and transport of clinical specimens iron by binding hemoglobin, transferrin, and lactoferrin. These to the laboratory for isolation and demonstration of proteins are of three types: N. gonorrhoeae. ■ The Por proteins ■ The Opa proteins ◗ Biochemical reactions ■ The Rmp proteins N. gonorrhoeae shows following features: The Por proteins: The Por proteins, earlier known as protein ■ Gonococci ferment glucose with the production of acid but I, are porin proteins that form pores or channels in the outer no gas. membranes. Por proteins are of two types: Por-A and Por-B, ■ They do not ferment maltose, lactose, sucrose, or fructose. each with a variety of antigenic variations. Strains producing This is an important feature to differentiate N. gonorrhoeae Por-A proteins are commonly associated with disseminated dis- from N. meningitidis. N. gonorrhoeae utilizes glucose only, ease because these proteins prevent killing of gonococci in the whereas N. meningitidis utilizes both glucose and maltose. serum by the serum complement components. The antigenic ■ They do not reduce nitrates, and they do not produce variations observed in Por proteins form the basis for the sero- hydrogen sulfide. type classification of N. gonorrhoeae. ■ They are oxidase and catalase positive. The Opa proteins: These proteins, also known as opacity protein, were formerly known as protein II. These proteins are ◗ Other properties found in the membrane and mediate adherence of the bacteria Susceptibility to physical and chemical agents: The to each other, and also to the eukaryotic cells. Strains produc-Section III gonococci are highly delicate bacteria. They die rapidly on ing Opa proteins produce opaque colonies in culture. drying. They are also killed by soap, and many other disinfec- The Rmp proteins: These proteins, also known as reduction tants, such as phenol, chlorhexidine, and hexachlorophene and modifiable proteins, were formerly known as protein III. These antiseptics. They are killed at a temperature as low as 25°C. are proteins found in the outer membrane of gonococci and Freeze drying or storing in liquid nitrogen are the most effec- lead to the production of antibodies that block serum bacteri- tive methods for storage of gonococci for a longer period. cidal activity against gonococci.Chapter 26 Cell Wall Components and Antigenic ◗ Other important gonococcal proteins Lipo-oligosaccharide (LOS) is another major antigen present Properties in the cell wall of the bacteria. This antigen consists of lipid The cell wall of N. gonorrhoeae like any other Gram-negative A and oligosaccharide similar to that of lipopolysaccharide bacteria consists of three layers: outer cell surface, middle pep- (LPS) of Gram-negative bacteria. However, LOS does not show tidoglycan layer, and inner cytoplasmic membrane. These con- antigenic variation as found in LPS. LOS possesses endotoxic tain following proteins (Fig. 26-1). activity. IgA1 protease and beta-lactamase are the other impor- tant proteins. IgA1 protease degrades secretory IgA1, whereas ◗ Outer membrane proteins beta-lactamase degrades beta-lactam rings in the penicillin. The outer membrane proteins (OMP) are present in the outer membrane. They mediate the uptake of iron essential for Pathogenesis and Immunity N. gonorrhoeae causes disease both by multiplying in tissues and Lipopolysaccharide by causing inflammation. The bacteria do not produce any (Endotoxin) toxins. Peptidoglycan Protein I ◗ Virulence factors Pilus N. gonorrhoeae produces several virulence factors as mentioned below (Table 26-2): Capsule: N. gonorrhoeae does not form a true carbohydrate IgA protease capsule unlike N. meningitidis. Instead, it forms a polyphos- phate capsule, which is loosely associated with its cell surface. Cytoplasmic membrane Capsule is most evident in freshly isolated gonococci and is antiphagocytic. It prevents phagocytosis of the gonococci. Cytoplasm Pili: Pili are hair-like structures that extend from the cyto- plasmic membrane through the outer membrane. The pili are FIG. 26-1. Schematic diagram of Neisseria gonorrhoeae. composed of the proteins known as pilins, which are repeating Chap-26.indd 202 5/14/2012 1:30:30 PM
  3. 3. NEISSERIA 203 adherence to phagocytes. The Opa proteins also facilitate sub- TABLE 26-2 Virulence factors of Neisseria gonorrhoeae sequent migration of gonococci into the epithelial cells. The Virulence factors Biological functions Por proteins inhibit phagolysosome fusion in the phagocytes, thereby protecting the phagocytosed bacteria from intracellu- Capsule Prevents phagocytosis lar killing. Production of beta-lactamase (penicillinase) by the Pili Mediate attachment of gonococci to bacteria also contributes to the invasion. nonciliated epithelial cell; prevent ingestion The host response is characterized by infiltration with leu- and killing of gonococci by neutrophils kocytes, followed by epithelial sloughing, formation of micro- Por proteins Confer resistance to serum killing of abscesses in the submucosa, and production of purulent pus. gonococci by preventing fusion of The LOS of gonococcal cell wall stimulates the production phagolysome in neutrophils of tumor necrosis factor alpha (TNF-␣) and other inflamma- Opa proteins Mediate bacterial adherence to each other, tory responses which contribute to most of the symptoms asso- and to the eukaryotic cells ciated with gonococcal infection. Rmp proteins Produce antibodies that block serum bactericidal activity against gonococci ◗ Host immunity Lipo-oligosaccharide Possesses endotoxic activity of the bacteria (LOS) The main host defense mechanisms against gonococci are anti- bodies (IgA and IgG), complement, and neutrophils. Antibody IgA protease Destroys IgA immunoglobulin response to gonococci is characterized by the production of Beta-lactamase Degrades beta-lactam rings in the penicillin serum IgG antibodies. IgG3 is the predominant immunoglobu- Plasmids Plasmid-borne virulence determinants are lin. Antibody response is strong against Opa proteins and LOS, associated with antimicrobial resistance whereas it is minimal against Por proteins. Antibodies to LOS Section III cause activation of complement, thus producing a chemotac- tic effect on neutrophils. Gonococcal infection does not confer protein subunits. The expression of protein pilin is controlled protection against reinfection. Repeated gonococcal infections by P gene complex. The pilins of all the strains of gonococci are occur due to the antigenic changes of the pili and outer mem- antigenically different. There is a marked antigenic variation brane proteins. Persons with a deficiency of the late-acting in gonococcal pili as a result of chromosomal rearrangement. complement components (C6–C9) are at a risk of disseminated More than 100 serotypes are known. The pili are important infections. Chapter 26 virulence factors: ■ They play an important role in the virulence of the bacteria. Clinical Syndromes They mediate attachment of gonococci to nonciliated epi- N. gonorrhoeae cause following clinical syndromes (Fig. 26-2): thelial cells. (a) gonorrhea, (b) disseminated gonococcal infections (DGI), ■ They also contribute to virulence by preventing ingestion (c) ophthalmia neonatorum, and (d) other gonococcal diseases. and killing of gonococci by neutrophils. Other virulence factors: These include: ◗ Gonorrhea Gonorrhea is a sexually transmitted disease. It is primarily a ■ Por protein of outer membrane protein (OMP) confers resis- genital infection restricted to the urethra in men and cervix in tance to serum killing of gonococci by preventing fusion of women. The incubation period varies from 2 to 8 days. phagolysosome in neutrophils. ■ Opa proteins mediate bacterial adherence of bacteria to each Gonorrhea in men: A symptomatic acute infection is seen in other and to the eukaryotic cells. approximately 95% of all infected men. Urethritis is the major ■ Rmp proteins produce antibodies that block serum bacteri- clinical manifestation, with burning micturition and serous cidal activity against gonococci. urethral discharge as the initial manifestation. Subsequently, the discharge becomes more profuse, purulent, and even blood- Lipo-oligosaccharide of the bacteria possesses endotoxic tinged. Acute epididymitis, prostatitis, and periurethral abscess activity. are rare, but are noted gonococcal complications in men. Gonorrhea in women: In women, endocervix is the primary ◗ Pathogenesis of gonorrhea site (80–90%) of infection because gonococci invade only the N. gonorrhoeae causes disease first by attaching themselves to endocervical columnar epithelial cells. The bacteria cannot mucosal cells. Subsequently, they enter the cells and multiply infect the squamous epithelial cells in the vagina of postpu- inside the cells and pass through the cells into the subepithe- bescent women. Urethra (80%), rectum (40%), and pharynx lial space, thereby establishing the infection. Pili help in attach- (10–20%) are the other sites of infection in women. The infec- ment of gonococci to mucosal surfaces and also contribute tion is mostly asymptomatic in women. The presence of vagi- to the resistance by preventing ingestion and killing by PMN nal discharge, dysuria, dyspareunia, and mild lower abdominal leukocytes. The outer membrane proteins, such as Opa proteins, pain are the common symptoms in symptomatic women. In facilitate adherence between gonococci and also increase 10–20% of infected women, the primary infection may spreadChap-26.indd 203 5/14/2012 1:30:30 PM
  4. 4. 204 BACTERIOLOGY Key Points The strains that cause DGI are characterized by their: Pharyngitis ■ Resistance to bactericidal action of serum, ■ Marked sensitivity to penicillin, and Disseminated infection ■ Auxotropism for arginine, hypoxanthine, and uracil (for growth they require these substances in the medium). ◗ Ophthalmia neonatorum Skin Ophthalmia neonatorum is a nonsexually transmitted infec- tion caused by N. gonorrhoeae. This is a condition of bilateral conjunctivitis of a neonate born by vaginal delivery to an infected mother. However, transmission to the newborn can also occur in utero or in the postpartum period. Pain in the eyes, Anorectal infection redness, and purulent discharge are the common symptoms. Blindness is an important complication of this condition. Genital infection Gonococci can cause permanent injury to the eye in a very short time; hence prompt recognition and treatment of the condi- Arthritis tion are very essential to avoid blindness.Section III ◗ Other gonococcal infections These include the following: ■ Anorectal gonorrhea and gonococcal pharyngitis occur in FIG. 26-2. Sites of infection caused by Neisseria gonorrhoeae. homosexual men following rectal intercourse or by oro- genital contact, respectively. Pharyngitis is most commonly acquired during orogenital contact. Pharyngitis often isChapter 26 from urethra and cervix to cause ascending genital infections asymptomatic, however, it may present as exudative pharyn- including salpingitis, tubo-ovarian abscess, and pelvic inflam- gitis with cervical lymphadenopathy. matory disease (PID). ■ Purulent gonococcal conjunctivitis occurs in adults follow- ■ Pelvic inflammatory disease (PID) is the most important ing autoinoculation of gonococci into the conjunctival sac complication in females following gonococcal infection. from a primary site of infection, such as the genitals. The Increased vaginal discharge or purulent urethral discharge, conjunctivitis may rapidly progress to panophthalmitis and dysuria, lower abdominal pain, and intermenstrual bleeding loss of the eye unless promptly treated. are the common symptoms of the PID. Tubal scarring, ecto- ■ Acute perihepatitis (Fitz–Hugh and Curtis syndrome) occurs pic pregnancy, and infertility are the major complications in due to the direct extension of N. gonorrhoeae or Chlamydia women following PID. trachomatis from the fallopian tube to the liver capsule and ■ Gonococcal vulvovaginitis occurs in prepubertal girls through overlying peritoneum. sexual contact. Epidemiology ◗ Disseminated gonococcal infections Disseminated gonococcal infection (DGI) occurs because of ◗ Geographical distribution hematogenous dissemination of gonococci from the primary site of infection. The symptoms vary greatly from patient to Gonococcal infection is reported throughout the world. patient. Arthritis-dermatitis syndrome is the classic presenta- However, the incidence is much lower in the European coun- tion of DGI. Joint or tendon pain is most common in the early tries, and this condition has virtually been eliminated in stage of infection. Migratory polyarthralgia, especially of the Sweden. The highest incidence of gonorrhea and its complica- knees, elbows, and more distal joints, and also tenosynovitis are tions occurs in developing countries. The median prevalence of the common symptoms. The skin lesions include maculopapu- gonorrhea in pregnant women has been estimated to be 4% in lar to pustular lesions often with a hemorrhagic component. Asia, 5% in Latin America, and 10% in Africa. Septic arthritis, especially of the knee, is the next stage of DGI. During this stage, skin lesions usually disappear and blood cul- ◗ Habitat tures for gonococci are always negative. The DGI is mostly seen N. gonorrhoeae is exclusively a human pathogen. The gonococci in untreated asymptomatic women and in persons with comple- are only found in infected conditions. In infected women, the ment deficiency. gonococci are most commonly found in the endocervix, and in Chap-26.indd 204 5/14/2012 1:30:30 PM
  5. 5. NEISSERIA 205 infected men found in the urethra. In both men and women present at the meatus, urethral specimens are collected by gonococci can also be found in the pharynx, rectum, and eyes. inserting and rotating a small swab 2–3 cm into the ure- The gonococci are not found as normal human flora in the thra. A calcium alginate or Rayon swab on a metal shaft mucosa of the urethra, cervix, or vagina. is usually used for this purpose. ■ In chronic infection, since urethral discharge is less, the ◗ Reservoir, source, and transmission of infection exudate after prostatic massage or morning drop of secretion Only humans, especially asymptomatic infected men and and urine are also examined for the cocci. Rectal specimens women, are reservoirs of infections. Asymptomatic carriage is are frequently useful for demonstration of gonococci in more common in women than in men. Purulent urethra or cer- asymptomatic women and in homosexual and bisexual men. vical discharge is the most common source of infection. The ■ Samples are collected from all possible mucosal sites, such infection is transmitted: as pharynx, urethra, cervix, and rectum, and from blood and synovial fluid in patients with possible DGI. ■ Primarily by sexual contact. N. gonorrhoeae infection occurs following mucosal inoculation during vaginal, anal, or oral After collection, the specimens are transported and pro- sexual contact. Increased sexual contact with infected part- cessed immediately in the laboratory. If delay is unavoidable, ners increases the risk of acquiring the infection. specimens are collected and transported to the laboratory in a ■ Less frequently, by nonsexual contact. Ophthalmia neona- transport medium, such as Stuart’s transport medium. torum is acquired nonsexually. This infection occurs follow- ing a conjunctival inoculation during vaginal delivery. Less ◗ Microscopy frequently, the disease is transmitted through rectum, oro- Gram stain of urethral exudates: The presence of four or pharynx, or through the birth canal. more polymorphonuclear (PMN) leukocytes per oil-immersion Section III Fomites do not play any role in transmission of the disease, field in Gram-stained urethral exudate smear is diagnostic of because gonococci die rapidly outside the human body. urethritis: Strain typing: Strains of N. gonorrhoeae can be typed by ■ Demonstration of typical Gram-negative intracellular (a) auxotyping and (b) serotyping. diplococci is characteristic of N. gonorrhoeae (Fig. 26-3, Color ■ Auxotyping is based on addition of specific nutrients and Photo 22). cofactors in the medium for the growth of gonococci. There ■ Gram stain helps in the presumptive diagnosis of the gonococcal infection. Chapter 26 are over 30 auxotypes. The most common auxotypes are prototrophic or wild type (Proto), praline-requiring type ■ It is more than 90% sensitive and 98% specific for the diagno- (Pro), and the strains requiring arginine, hypoxanthine, and sis of gonococcal infection in symptomatic males. However, uracil (AHU). in asymptomatic males, the sensitivity of the Gram stain is ■ Serotyping is based on the OMP “Porin”, which is further only 60% or less. divided into serovars (e.g., 1A-4, 1B-12) based on agglutina- In women, presence of more than 10 PMN per high-power tion with a panel of monoclonal antibodies. field on an endocervical smear is suggestive of cervicitis. Gram stain of endocervical smears is less sensitive (50–60%) Laboratory Diagnosis and 82–90% specific in both symptomatic and asymptomatic women. Laboratory diagnosis of gonococcal infection depends on dem- Gram stain is not a sensitive method for detection of gono- onstration of N. gonorrhoeae at the site of infection. cocci in patients with anorectal gonorrhea, pharyngitis, and ◗ Specimens The genital (urethral discharge, cervical discharge, etc.), rectal, and pharyngeal specimens are collected for the isolation and identification of gonococci. Intracellular gonococci ■ In acute gonococcal infection, urethral discharge in males and cervical discharge in females are the specimens of choice. High vaginal swab in females is not satisfactory. ■ When collecting specimens, such as endocervical discharge in women, the cervix is first cleaned of the exudate; a swab is then placed into the external os and rotated for several seconds. ■ In males, discharge present at the meatus is collected for examination. The meatus is first cleaned with gauze soaked in saline. The urethral discharge is then col- FIG. 26-3. Gram-negative intracellular Neisseria gonorrhoeae in lected with the help of a platinum loop. If no discharge is Gram-stained smear of pus exudate (ϫ1000).Chap-26.indd 205 5/14/2012 1:30:30 PM
  6. 6. 206 BACTERIOLOGY skin lesions. Specificity is also less because commensal Neisseria ◗ Detection of gonococcal antigen species in the oropharynx and gastrointestinal tract can be con- The gonococcal antigens can be detected by both direct fluo- fused with those of N. gonorrhoeae. rescent antibody (DFA) test and direct enzyme-immunoassays Wet mount examination of centrifuged deposit of urine (EIA) in urethral discharge and endocervical discharge as well sample: In men, the urine sample, preferably 10–15 mL of as in other clinical specimens. early morning (the first) voided urine, is collected and centri- The DFA using fluorescein-conjugated monoclonal fuged and examined under high power. The demonstration of antibodies is a rapid and useful method for demonstration 10 or more PMN in the centrifuged urine under high power is of gonococcal antigens in clinical specimens. The EIA using suggestive of urethritis. polyclonal antigonococcal antibodies are also used for the detection of gonococcal antigens in clinical specimens. ◗ Culture Isolation of N. gonorrhoeae from clinical specimens by culture ◗ Serodiagnosis confirms the diagnosis of gonorrhea. Genital, rectal, and pha- The serological tests are done to detect gonococcal antigens or ryngeal specimens are inoculated on a nonselective medium specific anti-gonococcal antibodies in the serum for diagno- (e.g., blood agar or chocolate agar) and on a selective medium sis of gonorrhea. ELISA and RIA (radioimmunoassays) using (e.g., Modified Thayer Martin medium). whole cell lysates, pilus proteins, and LPS antigens of the gono- The colonies of gonococci on chocolate agar after 48 hours of cocci demonstrate antibodies in the serum. incubation at 35–36°C in the presence of 5–10% CO2 are small, These serological tests are not recommended for routine round, translucent, and convex with finely granular surface. use. These are used only in specific situations, such as chronic On Thayer Martin medium, the colonies show similar mor-Section III gonorrhea, gonococcal arthritis, etc. phology as that on chocolate agar. The mixed microbial flora present in the clinical specimens is suppressed by the selective media. However, the vancomycin present in the selective media inhibits some strains of gonococci. Molecular Diagnosis DNA probes (Gen probe) are commercially available for the direct detection of bacteria in the genital and other clinical ◗ Identification of bacteria specimens.Chapter 26 N. gonorrhoeae are identified by the characteristics listed in Box ■ These probes are specific for nucleic acid of N. gonorrhoeae 26-1. They are differentiated from N. meningitidis and other and are sensitive and rapid. Neisseria species by a variety of tests (Table 26-3). ■ The results become available within 2–4 hours, but these tests are highly expensive. Identifying features of Box 26-1 Neisseria gonorrhoeae Treatment 1. Gram-negative diplococci. 2. On blood agar, produces translucent colonies with entire edge Sulfonamides were used as early as in 1935 for treatment and granular surface. of gonorrhea. In the beginning, all the strains of gono- 3. Oxidase test positive. cocci were sensitive to sulfonamides but subsequently, 4. Catalase test positive. they developed resistance to these antibiotics. Penicillin 5. Ferments glucose with production of acid. 6. Does not ferment maltose or sucrose. is the drug of choice for penicillin-sensitive strains of N. gonorrhoeae. TABLE 26-3 Differential characteristics of Neisseria species Neisseria species Growth on Production of acid from BA at 22°C CHA NA at 35°C Thayer Martin Glucose Maltose Sucrose Lactose medium Neisseria gonorrhoeae Ϫ Ϫ Ϫ ϩ ϩ Ϫ Ϫ Ϫ Neisseria meningitidis Ϫ Ϫ V ϩ ϩ ϩ Ϫ Ϫ Neisseria lactamica V V ϩ ϩ ϩ ϩ Ϫ ϩ Neisseria sicca ϩ ϩ ϩ Ϫ ϩ ϩ ϩ Ϫ V ϭ variable. Chap-26.indd 206 5/14/2012 1:30:31 PM
  7. 7. NEISSERIA 207 Penicillin-resistant strains of N. gonorrhoeae: Initially, gono- cocci were highly sensitive to penicillin (minimal inhibitory con- Neisseria meningitidis centration, or MIC, 0.005 U/mL). However, since 1957, strains of gonococci with decreased sensitivity (MIC Ͼ0.1 U/mL) N. meningitidis causes a spectrum of diseases ranging from to penicillin have been documented. The concentration of peni- meningococcemia (which is rapidly fatal) to a transient bac- cillin required to inhibit the growth of gonococci has increased teremia (which is relatively benign). It is also the second most by many folds and is now considerably higher (2.4–4.8 MU). common cause of community-acquired meningitis in adults. ■ Most of them are beta-lactamase (penicillinase) producing by the virtue of plasmid transmission. These strains show Properties of the Bacteria high level of resistance to penicillin. ◗ Morphology ■ Some strains of N. gonorrhoeae not producing beta-lactamase but yet showing resistance to penicillin have also been reported. N. meningitidis shows following features: This resistance is mediated chromosomally and is of low level. ■ N. meningitidis are Gram-negative, spherical, or oval cocci arranged in pairs with the adjacent sides flattened. The cocci Resistance to other antibiotics: Chromosomal-mediated are generally intracellular in PMN in smears from pus cells resistance to other antibiotics, such as tetracycline, erythromy- and other specimens. cin, and aminoglycosides, has also been reported. ■ They measure 0.6–0.8 ␮m in diameter. ■ Tetracyclines are no longer given for gonococcal infection ■ Freshly isolated bacteria are usually capsulated. because of the prevalence of tetracycline resistance. ■ They are nonmotile and nonsporing. ■ Resistance to ciprofloxacin has also been increasingly docu- Section III mented in Southeast Asia, Africa, and Australia. ◗ Culture Meningococci are strict aerobes. They grow optimally at a tem- perature between 36°C and 39°C and optimum pH of 7.4–7.6. Key Points Their growth is enhanced by incubation in a moist atmosphere Alternative drugs in cases of penicillin resistance or in in the presence of 5% CO2. penicillin-allergic individuals: Ceftriaxone, cefixime, cip- Meningococci are fastidious bacteria with complex nutri- rofloxacin, or ofloxacin are the alternative drugs in cases of tional requirements. They do not grow on ordinary media, but penicillin resistance or in penicillin-allergic individuals. A sin- Chapter 26 gle-dose regimen of any of these antibiotics is given as an initial grow well on the medium enriched with blood or serum, such therapy in uncomplicated urethritis, cervicitis, or rectal or pha- as blood agar, chocolate agar, and Mueller–Hinton agar. The ryngeal infections in adults. A single dose of ceftriaxone 125 mg blood or serum promotes growth of bacteria by neutralizing intramuscularly or cefixime (400 mg), ciprofloxacin (500 mg), inhibitory substances found in the media rather than by pro- or ofloxacin (400 mg) as a single dose orally is also effective. viding additional requirements. 1. Blood agar: On blood agar, N. meningitidis produces small, Immediate saline irrigation and intravenous ceftriaxone round (1–2 mm in diameter), convex, gray, and translucent are effective for treatment of gonococcal conjunctivitis. Local nonpigmented colonies with entire edges after 24 hours of application of 0.5% of erythromycin ophthalmic ointment or incubation. At 48 hours, the colonies become larger with 1% tetracycline or 1% silver nitrate ointment is effective for an opaque raised center and crenated with transparent treatment of gonococcal ophthalmia neonatorum. margin. It does not produce any hemolysis on blood agar. PID as such is a mixed infection of gonococci, Chlamydia, Strains of meningococci with large polysaccharide capsule and other facultative anaerobic pathogens. The treatment, appear as mucoid colonies. Meningococci produce large therefore, is by broad-spectrum antibiotics to cover all infect- colonies on chocolate agar. ing organisms. 2. Selective media: Thayer Martin medium with antibiot- ics (vancomycin, colistin, nystatin, and trimethoprim) and New York City medium are the selective media commonly Prevention and Control used for the isolation of the bacteria from clinical speci- Currently, there is no effective vaccine available against mens containing mixed bacterial flora. N. gonorrhoeae. Chemoprophylaxis by the prophylactic use of penicillin is also ineffective and may promote the development ◗ Biochemical reactions of resistant strains. Therefore, (a) health education, (b) early N. meningitidis shows following reactions: detection of cases, (c) tracing of contacts, and (d) follow-up of screening of sexual contacts is important in the prevention of ■ N. meningitidis is oxidase and catalase positive. These two gonorrheal epidemics. Furthermore, the prevention of gonor- tests are important biochemical markers for preliminary rhea involves the promotion of safe sex and individual counsel- identification of this organism. Alcaligenes spp., Aeromonas ing. Gonococcal conjunctivitis in the newborns is prevented by spp., Vibrio spp., Campylobacter spp., and Pseudomonas spp. using erythromycin ointment. are the other bacteria that are oxidase positive.Chap-26.indd 207 5/14/2012 1:30:31 PM
  8. 8. 208 BACTERIOLOGY Virulence factors of Neisseria Identifying features of TABLE 26-4 meningitidis Box 26-2 Neisseria meningitidis Virulence factors Biological functions 1. Gram-negative diplococci arranged in pairs. Capsule Prevents phagocytosis 2. On blood agar, produces convex, gray, and translucent colonies. LOS endotoxin Causes damage of the blood vessels 3. Oxidase test positive. associated with meningococcal infections 4. Catalase test positive. 5. Ferments glucose and maltose with production of acid. IgA protease Destroys IgA immunoglobulin, thereby helps 6. Does not ferment sucrose or lactose. gonococci to attach to the epithelial cells of the upper respiratory tract Lipooligosaccharides Stimulates release of TNF-␣, which results in ■ Oxidase test: The test can be performed in two ways: In the host cell damage first method, 1% solution of oxidase reagent (tetramethyl paraphenylene-diamine-dihydrocholoride) is poured on the culture media; the Neisseria colonies turn deep purple. Pathogenesis and Immunity In the second method, a few colonies of Neisseria are rubbed with a glass rod on a strip of filter paper moistened with N. meningitidis colonizes the human nasopharynx, and under oxidase reagent. A deep purple color develops immediately. specific conditions, invades the blood stream and then reaches ■ N. meningitidis ferments glucose and maltose with acid but the brain, causing meningitis. no gas. It does not ferment sucrose or lactose. Fermentation tests are required for final identification of Neisseria species ◗ Virulence factors (Box 26-2). It does not produce hydrogen sulphide and doesSection III N. meningitidis has three important virulence factors, which are not reduce nitrates. responsible for causing disease. These are (a) capsular polysac- charide, (b) LOS endotoxin, and (c) IgA protease (Table 26-4). ◗ Other properties Capsular polysaccharide: N. meningitidis is surrounded by Susceptibility to physical and chemical agents: N. menin- a prominent polysaccharide capsule that is antiphagocytic. gitidis are highly delicate organisms. They are highly sensitive to The capsule is an important virulence factor, which contrib- heat, desiccation, and disinfectants. utes to the virulence by inhibiting phagocytosis. The capsule protects meningococci from destruction by the leukocytes.Chapter 26 Inside the phagocytic vesicle of the leukocyte, they survive Cell Wall Components and Antigenic intracellular death, multiply, and then migrate to subepithe- Structure lial spaces. The cell wall of pathogenic meningococci contains a toxic LPS LOS endotoxin: LOS endotoxin is present in the outer or endotoxin. The meningococcal endotoxin is chemically membrane of N. meningitidis. It is responsible for damage of identical to the endotoxin of enteric bacilli. the blood vessels associated with meningococcal infections. The endotoxin comprises two antigenic determinant compo- nents: (a) a protein component and (b) a carbohydrate compo- ◗ Antigenic structure nent. The continuous production and release of endotoxin by Depending on group-specific capsular polysaccharide anti- N. meningitidis cause severe endotoxin reaction, seen in patients gens, meningococci are subdivided into 13 serogroups (A, B, C, with meningococcal disease. D, X, Y, Z, W135, 29E, H, I, K, and L). IgA protease: IgA protease is the other important virulence ■ Meningococci belonging to group A, B, and C are factor. The enzyme acts by clearing the secretory IgA, thus help- responsible for most of the epidemics and outbreaks of ing the bacteria to attach to the epithelial cells of the upper meningitis. respiratory tract. ■ Group Y and group W135 meningococci cause disease more commonly than groups X and Z. ■ Meningococci that lack group-specific antigens are consid- ◗ Pathogenesis of meningitis ered nonpathogenic. Initially, N. meningitidis causes a localized infection by coloniz- ing the nasopharynx. From this site, the meningococci invade Each serogroup includes many serotypes. The classification of the submucosa by circumventing the host defense mecha- the isolates of meningococcal serogroups into their serotypes is nisms and gain access to the central nervous system (CNS). based on the differences in the proteins in the outer membrane Meningococci reach CNS by the following ways: and in the oligosaccharide part of LOS. For example, Group A meningococci has a single serotype, whereas group B and C 1. Invasion of blood stream: This is the most common meningococci consist of many serotypes. Serotyping of strains mode of spread of meningococci. Once inside the blood is useful for the identification of virulent strains for epidemio- stream, the meningococci escape the immune surveillance logical studies. (e.g., antibodies, complement-mediated bacterial killing, Chap-26.indd 208 5/14/2012 1:30:31 PM
  9. 9. NEISSERIA 209 neutrophil phagocytosis) of the host and subsequently ◗ Meningococcemia reach distant sites including the CNS. The specific mecha- Meningococcemia with or without meningitis is a life-threatening nism by which the meningococci reach the subarachnoid condition. The condition presents as an acute fever with pete- space still remains to be clearly understood. chial rash. Small petechial rashes are continuously found on 2. Direct contiguous spread: Meningococci can also the trunk and lower extremities; subsequently the rashes may reach the CNS by direct contiguous spread from naso- coalesce to form large hemorrhagic lesions. pharynx. Inside CNS, the bacteria multiply and survive Waterhouse–Friderichsen syndrome is an overwhelming sys- because host defense mechanisms (such as immuno- temic infection caused by N. meningitidis. This condition is globulins, neutrophils, and complement) appear to have characterized by severe disseminated intravascular coagula- limited role in controlling multiplication of the bacte- tion, shock, and multisystem failure including destruction ria. Uncontrolled multiplication of bacteria continues in of adrenal glands. The condition, associated with circulatory the CSF, which subsequently causes a cascade of men- collapse with intravascular coagulation, is invariably fatal. It is ingeal inflammation. Meningococcal infection of the most commonly seen in persons suffering from deficiency of nasopharynx is usually subclinical. Asymptomatic naso- C5–C9 components of the complement. The vascular damage pharyngeal carriage of meningococci is of short dura- seen in this condition is caused primarily by the action of LOS tion and resolves within several weeks. In a few persons, meningococci invade the circulation and cause clinical endotoxin present in the meningococci. disease. ◗ Other syndromes ◗ Host immunity Nonsuppurative arthritis, usually of the knee joint, is seen in Section III The presence of meningococci in the nasopharynx induces approximately 10% of the patients with meningococcal dis- a humoral antibody response, and most people acquire ease. This condition is observed within the first 48 hours of immunity to meningococcal disease by age of 20 years. treatment and is believed to be immunologically mediated. Maternal antibodies provide protection to infants for the first Recurrent meningococcal meningitis is another condi- 3–6 months of life. Later, colonization with nonpathogenic tion which is associated with hereditary deficiency of various meningococci appears to produce cross-reacting, protective components of complement system. antibodies. Other conditions include meningococcal pneumonia (which probably results from the aspiration of the organisms), septic Chapter 26 Specific IgG antibodies are produced against meningococcal polysaccharides in combination with the complement medi- arthritis, purulent pericarditis, and endophthalmitis. ate bactericidal activity against the meningococci. Individuals lacking the bactericidal antibodies and those suffering from Epidemiology complement deficiencies—such as C5, C6, C7, or C8 compo- nents of the complement—show increased susceptibility to ◗ Geographical distribution meningococcal disease. An episode of meningitis confers group-specific immunity, Meningococcal disease occurs worldwide. N. meningitidis sero- but a second episode may be caused by another meningococcal group A usually causes epidemics, serogroup B causes both serogroup. epidemics and outbreaks, while serogroup C mostly causes localized outbreaks. Endemic meningitis is more common in children below the age of 5 years and in elderly people. Large Clinical Syndromes outbreaks of meningococcal disease have occurred in central African countries with attack rate as high as 400–500 cases per N. meningitidis causes the following conditions: (a) meningitis, 100,000 population. Epidemics of meningococcal disease have (b) meningococcemia, and (c) other syndromes. occurred in many parts of the world. Meningococci of group A are associated with diseases in ◗ Meningitis underdeveloped countries; meningococci of group B, C, or Y Meningococcal meningitis caused by N. meningitidis is most are responsible for most (90%) of the cases of meningococcal common in children and young adults. It is a febrile illness diseases in the developed countries (Table 26-5). of short duration characterized by headache and stiff neck. Lethargy or drowsiness is frequent. Confusion, agitated delir- ium, and stupor are rarer. Mental obtundation, stupor, and ◗ Habitat coma due to increased intracranial pressure are some of the N. meningitidis is primarily a pathogen of humans. Meningococci noted complications at the end stage of the disease. are found in the nasopharynx and oral cavity. Asymptomatic Prognosis of meningitis is good, and the patients recover carriage of N. meningitidis varies from as low as 1% to as high as completely on immediate treatment with appropriate antimi- 40% in the population. The carriage rates are highest in school- crobial therapy. However, prognosis is bad in comatose patients going children, in young adults, and in the population with low and in patients with local neurological findings. economic status.Chap-26.indd 209 5/14/2012 1:30:31 PM
  10. 10. 210 BACTERIOLOGY Epidemiology of Neisseria meningitidis 2. The second part of CSF is used for direct culture. TABLE 26-5 3. The third part is incubated overnight with an equal vol- serogroups Serogroups Disease ume of glucose broth and then subcultured onto the blood agar and chocolate agar. A Meningococcal disease in underdeveloped countries B Meningitis and meningococcemia; most (Ͼ90%) cases of meningitis in developed countries ◗ Microscopy C Meningitis and meningococcemia; most (Ͼ90%) cases Gram staining of the CSF is a very useful method for detec- of meningitis in developed countries tion of meningococci. Meningococci are seen as Gram-negative Y Meningococcal pneumonia diplococci present mainly inside the leukocytes and some may W135 Meningococcal pneumonia even be present extracellularly. These cocci can be demon- strated in the CSF in approximately 50% of the patients with meningococcal meningitis. In fulminant meningococcemia, ◗ Reservoir, source, and transmission of infection Gram staining of the peripheral blood buffy coat may reveal Human is the only reservoir of meningococcal infection. Gram-negative diplococci. Nasopharyngeal secretion is the most common source of infec- tion. Meningococci are transmitted by airborne droplets of ◗ Culture infected nasopharyngeal secretions (the most common source of Isolation of N. meningitidis from the CSF, blood, and other infection). Family members living in crowded conditions or the clinical specimens by culture confirms the diagnosis of menin- people who live in close populations (such as military barracks gococcal infection. The CSF is inoculated immediately on a and prisons) and older people are more susceptible to infection.Section III nonselective medium, such as blood agar or chocolate agar, and incubated at 35–36°C under 5% CO2 for 18–24 hours. The colo- Laboratory Diagnosis nies of meningococci are small, round, translucent, and convex Laboratory diagnosis depends on demonstration of meningo- with a smooth glistening surface. cocci in clinical specimens by microscopy and culture. Blood is inoculated immediately into blood culture bottles containing either glucose broth or sodium taurocholate broth and incubated at 35–36°C. Subcultures are made on blood ◗ Specimens agar or chocolate agar from these broths and are reincubatedChapter 26 Cerebrospinal fluid and blood are the specimens of choice overnight at 35–36°C in the presence of 5% CO2. The cultures for demonstration of meningococci in the early stage of men- should be incubated for 4–7 days with daily subculture. Blood ingitis. Nasopharyngeal swabs are useful to detect carriers. culture is often positive during early stage of meningitis and in The CSF is collected by lumbar puncture and blood by veni- meningococcemia. puncture in strict aseptic conditions. CSF is never refrigerated as Haemophilus influenzae, another agent of meningitis, may die at the cold temperature. CSF specimens are transported ◗ Other specimens immediately to laboratory for processing. Similarly, blood Other specimens, such as nasopharyngeal swabs and petechial is collected in blood culture media containing either glucose exudates are processed in a similar way as described earlier broth or sodium taurocholate broth. Nasopharyngeal speci- for CSF. mens are collected using sterile swabs and are transported in Stuart’s transport medium to the laboratory. ◗ Identification of bacteria CSF N. meningitidis are identified by the characteristics listed in Box Meningococcal meningitis produces various inflammatory 26-2. Serogrouping of the bacterial isolates grown on culture is car- changes in the CSF: ried out by slide agglutination with specific hyperimmune serum. ■ The CSF in bacterial meningitis is more turbid. ■ It contains more than 1000 WBC/␮L, and the cells are pre- ◗ Antigen detection dominantly PMN cells. Detection of soluble polysaccharide antigen in the CSF is a ■ The total protein content is increased. The total glucose useful method for diagnosis of meningococcal meningitis. level, which is normally 60% of simultaneous blood glucose Counter-current immunoelectrophoresis, latex agglutination level, is lowered (hypoglycorrhachia). test, and bacterial coagglutination test using specific antibod- ■ The intracranial pressure may be elevated. ies are the rapid tests frequently used to detect the soluble antigen in the CSF. Antigen detection is useful when bacteria CSF received in the laboratory is processed in three parts: are scanty in the CSF. However, antigen detection is not use- 1. First part is centrifuged and smear is prepared from the ful in the meningitis caused by Group B meningococci because deposit for Gram staining. The supernatant is tested for N. meningitidis serogroup B is relatively nonimmunogenic and meningococcal antigens. does not react with specific antibodies. Chap-26.indd 210 5/14/2012 1:30:31 PM
  11. 11. NEISSERIA 211 ◗ Serodiagnosis Vaccines Indirect hemagglutination test and ELISA are useful for the Quadrivalent meningococcal polysaccharide vaccine demonstration of antibodies against specific polysaccharide (MPSV4): It has been shown to be highly effective in prevent- antigen in the serum. Serodiagnosis is useful in the cases of ing disease caused by A, C, Y, and W135 serogroups of meningo- chronic meningococcal infection where cultures have proved cocci. The vaccine is given intramuscularly. Use of this vaccine negative for meningococci. is indicated for population at risk during outbreak of infection caused by one of these serogroups of meningococci. These vac- cines developed against group A, C, Y, and W135 are poorly Molecular Diagnosis immunogenic under 2 years of age. These vaccines, however, pro- PCR has been used for detection of N. meningitidis DNA in duce good antibody response in children above 2 years of age. clinical specimens. The test is useful to detect small amounts Tetravalent meningococcal polysaccharide-protein conju- of meningococcal DNA in CSF. It is a more sensitive test for gate vaccine (MCV4): Recently, in 2005, MCV4 is being used diagnosis of meningococcal meningitis than the culture. The for the persons aged 11–55 years for vaccination against menin- high cost of the test and the expertise necessary to operate a gococci in the United States. This is recommended for groups PCR assay are the disadvantages of the test. The test, there- of population at risk, which include (a) military recruits, (b) fore, is only used in a large-scale outbreak when a number travelers to areas hyperendemic or epidemic for meningococ- of specimens are to be analyzed, and in a tertiary healthcare cal disease, (c) patients with anatomic or functional asplenia, center. (d) patients with terminal complement deficiency, and (e) microbiologists who are routinely exposed to meningococci. No vaccine against group B meningococci is available because group B meningococcal capsular antigen is not immunogenic. Treatment However, of late some progress has been made in preparation of vaccine for group B meningococci. The vaccine, which is at the Prompt and specific antimicrobial therapy of meningococce- Section III experimental stage, consists of outer membrane proteins that mia or meningococcal meningitis is most crucial. Intravenous are capable of inducing group-specific bactericidal antibodies. penicillin G is the recommended drug for the treatment of meningococcal disease. The MIC of penicillin usually ranges from 0.01 to 0.05 ␮g/mL against meningococcal isolates. Other Neisseria Species Chloramphenicol, rifampicin, erythromycin, tetracycline, and cephalosporins (ceftriaxone, cefotaxime, and cefuroxime) Other species of the genus Neisseria rarely cause human disease. are useful in treatment of bacterial meningitis. Ceftriaxone has They are found as part of normal bacterial flora mostly of the Chapter 26 an additional advantage of eradicating the nasopharyngeal car- respiratory tract. These commensal Neisseria are Neisseria flave- riage of meningococci. Chloramphenicol is useful for patients scens, Neisseria sicca, Neisseria lactamica, and Neisseria subflava. The who are allergic to penicillin. commensal Neisseria differ from pathogenic Neisseria species by Meningococci are not susceptible to vancomycin and poly- following properties: myxin. Meningococci resistant to sulfadiazine (MIC Ն0.128 ␮g/mL) have been documented recently. ■ They can grow on ordinary agar not enriched with blood and serum and they can also grow at 22°C. Prevention and Control ■ They do not require high percentage of CO2 for their growth. ■ They produce greenish yellow or yellow colonies on the media. This includes chemoprophylaxis and vaccines. N. flavescens and N. sicca have been associated with isolated ◗ Chemoprophylaxis cases of meningitis, osteomyelitis, acute otitis media, and acute sinusitis. But true incidence of respiratory tract infection Antimicrobial chemoprophylaxis of close contacts is the key caused by these Neisseria species is not known. Most of these factor for preventing secondary cases of sporadic meningo- strains are susceptible to penicillins. coccal disease. Person-to-person transmission can be inter- N. lactamica is frequently isolated from the nasopharynx and rupted by administration of antibiotics, which eradicate the is a nonvirulent Neisseria; however, it is closely related to patho- asymptomatic nasopharyngeal carrier state. Sulfonamides, genic Neisseria. rifampin, minocycline, ciprofloxacin, and ceftriaxone are the Neisseria catarrhalis—which was later designated as Branhamella drugs frequently used to eradicate meningococci from the catarrhalis and is now renamed as Moraxella catarrhalis—is a com- nasopharynx. However, ciprofloxacin is not recommended for mensal of the upper respiratory tract and, occasionally, is found children, because it has been found to cause cartilage damage in female genital tract. It is a recognized respiratory opportu- in immature experimental animals. nistic pathogen in immunocompromised host and hospital- ized people. M. catarrhalis is multidrug resistant and grows on ◗ Immunoprophylaxis ordinary media, such as nutrient agar and MacConkey agar. It Immunoprophylaxis by vaccination with group-specific menin- causes infections (e.g., otitis media, maxillary sinusitis, menin- gococcal capsular polysaccharides of groups A, C, Y, and W135 gitis, septic arthritis, endocarditis, sepsis, etc.) in immunocom- meningococci is very much useful for prevention of meningo- promised patients and in children. Some strains are susceptible coccal disease. to cephalosporins, chloramphenicol, and tetracycline.Chap-26.indd 211 5/14/2012 1:30:31 PM
  12. 12. 212 BACTERIOLOGY CASE STUDY A 22-year-old female complained of lower abdominal pain on and off for the last 3 months. She complained of a feeling of heaviness in the pelvis and pain during sexual intercourse. On examination, a tender mass was found to the right side during examination. Gram staining of cervical swab showed plenty of pus cells and a few Gram-negative cocci. She gave a history of allergy to penicillins. ■ Which is the most likely genital infection the patient is suffering from? ■ Which is the most likely bacterium to cause this genital condition? ■ What other diseases are caused by this bacterium? ■ How you will confirm diagnosis of this condition in the laboratory? ■ What antibiotics you can use in this patient for treatment of the condition?Section IIIChapter 26 Chap-26.indd 212 5/14/2012 1:30:31 PM