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Bacteriology 2017 Update

This document provides definitions and information about various types of bacteria. It begins with definitions of obligate aerobes, anaerobes, and facultative anaerobes. It then discusses specimen collection and transport media as well as agar media and identification methods like Gram stain, MALDI-TOF, and biochemical tests. Key gram-positive cocci like Staphylococcus, Streptococcus, and Enterococcus are summarized along with their identification and pathogenic characteristics.

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Bacteriology Review Update 2017
Beginning definitions • Obligate Aerobe – require high level of oxygen (20%) to grow • Obligate Anaerobe –>30 min of oxygen exposure can be deadly • Facultative anaerobes – grow in aerobic and anaerobic conditions, most so-named “aerobic” bacteria are actually facultative • Microaerophilic – Grow better with reduced oxygen and elevated carbon dioxide % • Aerotolerant anaerobes– anaerobe is not killed by prolonged exposure to oxygen – example: Clostridium tertium • Lag Phase - >24 hrs old, growth is slowing, not appropriate for biochemical or susceptibility testing • Log Phase – Exponential growth – appropriate for all testing • Stationary phase – appropriate for transporting specimens
Specimen collection for Aerobic Bacteriology Throat / Wound collection using swab 1. Use swabs made of polyester or sponge like material, do not use a cotton swab, traps bacteria /potentially toxic 2. After specimen collection, swab placed in Stuart’s or Amies transport media (buffer solution with peptones) 3. These transport media preserve bacteria viability but do not promote growth of bacteria Urine collection – two methods suggested 1. Boric acid to place bacteria in stationary phase 2. Refrigerate within one hour Purpose to maintain original colony count and maintain viability of organisms Tissues/Sterile body fluids – Sterile containers
Gram stain Procedure 1 minute Rinse Primary stain Mordant 1 minute Rinse 5-10 seconds Rinse Decolorization Counter stain 1 minute Rinse Peptidoglycan in positive cell wall traps the Crystal violet, hence blue color Prepare thin film of specimen on glass slide Heat or methanol
Gram positive cocci in pairs and chains Streptococcus Gram Positive cocci in clusters Staphylococcus Gram positive rod most like a Bacillus species Gram negative bacilli, enteric Gram negative rod – Fusiform Shaped – Fusobacterium species Visit the Gram stain tutorial for more Gram stains
Assess quality of Sputum for Bacterial culture with Gram stain • Sputum specimen is rejected for bacterial culture, if sputum judged to be “spit” not actually a deep cough specimen • Perform Gram stain of (?) sputum specimen • If <=25 epithelial cells – acceptable (right pix) • If >= 25 epithelial cells /field (left pix) • Sputum is judged to be spit / not a sputum specimen • Bacterial culture is not performed / it is rejected • Request made for a new “deep cough” specimen Bad Sputum Good Sputum 10X objective 10X objective
Most used Agar Media - BacteriologyBlood agar- 5% sheep’s blood agar • Gauge hemolytic reaction of bacteria (alpha, beta, gamma) • Culture a variety of Gram positive, Gram negative bacteria and Yeast Chocolate agar • “Carmelized” blood agar – more enriched than blood agar medium. • Will support the growth of the same organisms as 5% Sheep’s blood agar Plus more fastidious bacteria - like Haemophilus influenzae and Neisseria gonorrhoeae
Most used agar media - Bacteriology • MacConkey agar – Supports growth of Gram negative rods (GNR), and determines ability to ferment lactose, described as Selective and Differential media • Selective - crystal violet inhibits Gram positive organisms • Differential agar - Lactose fermentation can be determined • Lactose fermentation = pink (neutral red indicator) • Non-lactose fermentation = no color • After media is plated, incubate in aerobic incubator at 35˚ C for 24 – 72 hrs
MALDI-TOF Mass Spectrometry – newest wide spread identification method –Matrix-assisted laser desorption/ionization – time of flight –Identification by analyzing proteins in bacteria –Replaced many/most biochemical tests for bacterial ID
MALDI-TOF • Laser is fired at the matrix/sample • Laser energy is absorbed by the matrix and converted to heat energy and ionizes the sample. • Positive ions (proteins)are accelerated through a vacuum by an applied electrical field. • The time taken for the proteins to reach the detector depends on their mass/charge ratio (m/z) and create spectrograph. • Each bacterial species has a different protein composition, thus giving rise to a specific mass spectrograph. • The mass spectrograph produced by a sample is then compared with many thousands stored in a spectrograph database to see which one it most closely matches. Thus an identification is achieved.
Gram Positive Cocci Staphylococcus Streptococcus Enterococcus
Staphylococcus Gram positive cocci in clusters due to bound coagulase or “clumping factor” All Staphylococcus are Positive for Catalase Enzyme Staphylococcus aureus Coagulase Negative Staph Coagulase Enzyme Positive Coagulase Enzyme Negative +/- Yellow colony Beta hemolysis White colony Most not hemolytic
Catalase Enzyme Reaction Negative Positive Bacteria added to Hydrogen Peroxide Blood has innate catalase activity and will give false positive catalase reaction
Rabbit plasma inoculated with organism Incubate at 35˚C Read at 4 hours and if negative read again at 24 hours Negative tube coagulase =no clot Coagulase negative Staph Positive tube coagulase [clot at either 4 or 24 hrs] Staph aureus Tube Coagulase Reaction
Staphylococcus aureus • Primary virulence factor: • Protein A – surface protein, ability to bind immunoglobulin and combat the immune response • Diseases and associated toxin: • Toxic shock syndrome (TSST-1 toxin) • Scalded skin syndrome (Exfoliatin (SSS) toxin) • Soft tissue infection (Panton valentine leucocidin toxin – PVL) • Food poisoning / Enterotoxins – stable to heating at 100*C for 30 minutes • Bacteremia and endocarditis • Septic arthritis – Primary cause • Toxins act as super antigens, recruit host defense cells that liberate cytokines with systemic effects
Unique Susceptibility Issues MRSA =Methicillin resistant Staphylococcus aureus –Altered Penicillin binding protein (PBP2) produced by the mecA gene codes for resistance to oxacillin/methicillin /nafcillin resistance –Cefoxitin susceptibility testing is considered a more sensitive indicator of resistance to oxacillin and provides better detection of MRSA –All MRSA are considered (reported) as resistant to semisythetic penicillins and cephalosporins –Vancomycin becomes a drug of choice KB disk resistant
The “D” Test • Is Staph aureus really susceptible to ClindamycinIs Staph aureus really susceptible to Clindamycin • Why?? During therapy, S aureus isolates resistant toWhy?? During therapy, S aureus isolates resistant to Erythromycin possess enzymes capable of inducingErythromycin possess enzymes capable of inducing Clindamycin resistanceClindamycin resistance • Kirby Bauer zone around Clindamycin will be blunted to form aKirby Bauer zone around Clindamycin will be blunted to form a “D” if Clindamycin can be induced by Erythromycin to be“D” if Clindamycin can be induced by Erythromycin to be resistant – so calledresistant – so called INDUCIBLE RESISTANCEINDUCIBLE RESISTANCE.. • Clindamycin should be reported as resistant by clindamycinClindamycin should be reported as resistant by clindamycin induction and not used for therapy.induction and not used for therapy. D test positive Inducible resistance D test negative Clindamycin can be used for therapy
Nares is primary colonization site for MRSA and the most common surveillance site Culture methods for surveillance: ChromAgar is commonly used - It is a selective and differential media containing chromogenic substrates that turn a specific color to ID Staph aureus. Cefoxitin is added to the medium to select for only MRSA. Molecular assays (MA) can also be used to screen nares for MRSA. MAs increase the sensitivity of detection (@5-10%) but increases laboratory costs. Mupiricin can be used to nares eradicate carriage Staphylococcus aureus (MRSA) Hospital Epidemiology Issue: Nares surveillance for MRSA
Coagulase negative Staph (CNS) @ 15 species infect humans • Staph epidermidis – most common –Major aerobic component of normal skin flora –Common cause of subacute bacterial endocarditis –Pathogenicity from cell adhesion factors forming biofilm on biologics and plastics like heart valves, Catheters and replacement joints making treatment difficult • Staph saprophyticus – –Urinary tract infection in the child bearing age female, –This CNS adheres in greater #’s to epithelial cells –Novobiocin resistant by KB disk test White non-hemolytic colony resistant
CNS & Related Cocci • Staphylococcus hemolyticus – • Can cause line related sepsis • Hemolytic on blood agar • Do not confuse with Staph aureus • Staphylococcus lugdunensis • ? greater pathogenicity than other CNS species • Abscesses and bacteremia • PYR test positive = pink • Micrococcus species • Mustard yellow colony – environmental contaminate • Catalase positive / Gram positive cocci in tetrads • Tube coagulase negative • Does not ferment glucose • Staphylococcus ferment glucose Po s NegPYR TEST
Streptococcus Gram positive cocci in chains and pairs Catalase enzyme negative Three groups based on hemolytic pattern on sheep’s blood agar • Alpha – greening of agar, partial hemolysis of RBCs • Viridans Streptococcus, Streptococcus pneumoniae, Granulicatella/Abiotrophia • Beta – clearing of agar, complete hemolysis of RBCs • Beta hemolytic Streptococcus groups A - G • Gamma – no clearing of agar, intact RBCs • Streptococcus bovis (Strep gallolyticus)
Beta Hemolytic Streptococc us • Beta Streptococcus are grouped using the “C” carbohydrate (CHO) in the bacteria cell wall • Identifies Beta Strep groups – A, B, C, F, and G that are the ones most commonly seen in human infections • Lancefield grouping system uses the “C” CHO in the cell wall of Streptococcus as the antigen in a slide agglutination test with monoclonal antibody (A<B<C<D<F<G) attached to latex beads See below slide for a positive and negative latex reaction. • Immunologic typing is more accurate than biochemical testing to group Beta hemolytic Streptococcus + -
Streptococcus pyogenes • Genus/species name for Group A beta Streptococcus [GAS] Biochemical identification: • Bacitracin KB sensitivity test – inhibited, no growth @ disk • This test is not specific for Group A , it x-reacts with group C • “A” disk is the Bacitracin disk – stands for group “A” Strep • PYR (pyrrolidonyl arylmidase) reaction • Organism spotted on moist disk • 2 min – RT incubation • Add Cinnamaldehyde reagent • Look for color reaction • Pink = positive = Strep pyogenes • This test is not exclusive for Strep pyogenes – Enterococcus and Staph lugdunensis are PYR positive • Therapy : Penicillin or Cephalosporin antibiotics No resistance reported to these agents PYR
• Streptolysin O and Streptolysin S toxins • Comprise the ASO titer determination • Cell toxins lead to evasion from the immune system • O toxin is oxygen labile S toxin is oxygen stable • When both are active - most hemolysis is apparent on 5% Sheep’s blood agar Primary virulence factors: M Protein – prevents phagocytosis Capsule – hyaluronic capsule protects from phagocytosis
Streptococcus pyogenes Infections •Diseases – • Pharyngitis • Impetigo (1) • Erysipelas (2) • Cellulitis (3) • Necrotizing fascitis (4) • Puerperal sepsis • Toxic Shock • Scarlet fever (5) 1 2 3 4 5
Sequelae of Strep pyogenes Infections Rheumatic fever • Cause: inadequately treated Strep throat • Rise in the 1990s due to EIA tests being used as the only diagnostic test for pharyngitis – EIA tests are only 60-70% sensitive – and led to children not receiving antimicrobial therapy. Negative EIA specimens must be confirmed by culture • Similarity between the proteins of Strep A and muscle tissue cause immune system confusion which leads to immune system to attack heart, joint, and bones • Anti-streptolysin O serum test measures the amount of antibodies produced against Streptococcus group A Glomerulonephritis • 10-14 days following skin infection or pharyngitis • Renal disease with inflammation of the glomeruli • ASO titers will be positive • Usually resolves with therapy
Streptococcus agalactiae (Group B) • Biochemical tests for identification: –Camp test – performed using a Staph aureus strain with Camp factor streaked perpendicular to group B Strep, incubate 24 hr. / intensifies toxin production and produces arrow shaped hemolysis –Rapid hippurate hydrolysis - 4 hr identification of GBS – Hippurate disk Saline inoculated with Group B Strep Ninhydrin reagent added for color formation • Positive = Purple pos Arrow Staph aureus Strep group B
Strep agalactiae [GBS] • Pathogen of the elderly – Bacteremia and urinary tract infection • Pathogen of neonate – in utero or perinatal organism acquisition during birthing process • Early onset – within 7 days • Late 7 – 28 days from birth process. • Treatment: Penicillin or Cephalosporin (3rd generation) • Pregnant women carry organism in the cervix and/or rectal area. • All pregnant must be tested at 35 – 37 weeks of pregnancy. • Enrichment methods for GBS are standard of practice and must be used • Swab placed into LIM broth – incubate for 18 hours at 35 ˚C then subculture onto Blood agar. This broth can also be used as an enrichment method to enrich for molecular testing. • Carrot enrichment broth turns orange with growth of group B Strep Carrot Broth
Streptococcus bovis (gallolyticus) Streptococcus gallolyticus ssp. gallolyticus formerly S. bovis biotype 1 associated with endocarditis and colon cancer (73%) when isolated from blood cultures Strep gallolyticus ssp. pasteurianus formerly S. bovis biotype II- associated with neonatal meningitis Biochemical reactions: Both species have similar biochemical reactions 6.5% NaCL
• Two most common species • E. faecium • E. faecalis • No well defined virulence factors • Possesses the Group “D” CHO in the cell wall • Biochemical tests: • Bile esculin positive – growth and black precipitin • Growth in 6.5% salt • PYR positive (Group A beta Strep and Staph lugdunensis also positive)* • E. faecium = arabinose fermentation positive • E. faecalis = arabinose fermentation negative Enterococcus Bile esculin PYR + - Neg Pos PYR
Enterococcus • Variety of infections / Pathogen of opportunity • Intestinal normal flora • Cause UTI, bacteremia, and abdominal infections • Antimicrobial therapy: • Natural resistance to cephalosporin antibiotics • Ampicillin plus Aminoglycoside can be synergistic for therapy in cases of endocarditis • Unique susceptibility issues • Acquired resistance to vancomycin known as vancomycin resistant enterococcus or VRE • Van A (E. faecium) resistance genes • Van B (E. faecalis) resistance genes
Alpha hemolytic Streptococcus Streptococcus pneumoniae Gram stain = Gram positive bullet (lancet) shaped cocci in pairs Polysaccharide capsule = virulence factor, antiphagocytic Identification: • Bile soluble – colonies dissolve in sodium deoxycholate (bile) • Optochin sensitive – 14mm or greater zone of inhibition around the 6mm optochin disk – “P” = pneumococcus Inhibition >=14mm NO Inhibition Colonies dissolved
Streptococcus pneumoniae • Upper and Lower respiratory tract infection, sepsis and meningitis • Multi-serotype vaccine aids preventing invasive infections • Susceptibility issues: • Acquired Resistance to Penicillin due to Penicillin binding proteins (PBP) • Minimum inhibitory concentration (MIC) testing necessary to detect resistance to penicillin • Testing of CSF isolate a result of • Penicillin = >2 mcg/ml is resistant • Therapy – • If susceptible 1st line therapy is either Penicillin or 3rd generation CephalosporinMIC MIC
Viridans Streptococcus Several species of alpha hemolytic Streptococcus are NF in mouth and upper respiratory tract S. mutans S. salivarius S. sanguis S. mitis • Bile esculin slant = negative • Not bile soluble • Optochin resistant with zone size <=13 mm • Ethylhydrocupreine hydrochloride • Cause 30 – 40% cases of sub acute endocarditis/native valve • Can cause abscess and various infections throughout the body in the immune suppressed host • Variable susceptibility patterns, some isolates with elevated MICs to Penicillin Viridans Streptococcus
Viridans Streptococcus unique species • Streptococcus anginosis group: includes • S. anginosus S. constellatus S. intermedius • Normal flora in human mouth • More virulent than “normal” viridans Streptococcus, perhaps due to capsule • Cause deep tissue abscess, bacteremia, endocarditis, intra abdominal infections… • Variable susceptibilities – so best to do susceptibility testing
Nutritionally Variant Streptococcus • Vitamin B6 (pyridoxal) deficient – • will not grow on media without B6 supplementation • Will grow in a patient blood culture bottle due to vitamin B6 in patient’s blood • Will not subculture onto 5% Sheep’s blood agar plate (no vit B6) • Will grow with Staph aureus streak (supplies vitamin B6) • Nutritionally variant Strep will satellite @ S. aureus streak • Two species: • Abiotrophia spp • Granulicatella spp • Endocarditis – • More destructive to valve than than “regular” viridans Strep • Higher MIC’s to Penicillin Satellite streptococcus Next to S. aureus streak
Rare Opportunistic Gram pos cocci• Aerococcus ureae – Gram positive cocci in pairs and clusters • Alpha hemolysis , difficult to identify • Urinary tract pathogen • Rothia mucilaginosa – Gram positive cocco-baccilli • Neutropenia and gut problems predispose to infection • Normal flora in the oral cavity and upper respiratory tract • Pathogen in dental caries and periodontal disease • Bacteremia with endocarditis • Gemella morbillorum– • Easily over decolorized gram positive in pairs – requires Co2 to grow • Normal flora in oral cavity • Bacteremia with endocarditis • Leuconostoc – Gram positive cocci in chains • Intrinsic resistance to vancomycin – bacteremia in immune suppressed • Watch out! Do not confuse with VRE
Gram Negative Cocci Neisseria Moraxella
Gram Negative Cocci • Neisseria species and Moraxella catarrhalis • Small kidney bean shaped cocci in pairs • Oxidase enzyme positive • CTA (Cysteine Trypticase Agar) carbohydrate fermentations for identification – glucose, maltose, lactose, sucrose • N. gonorrhoeae Gluc + Mal - Lac - Suc - • N. meningitidis Gluc + Mal + Lac - Suc - • N. lactamica Gluc + Mal + Lac+ Suc- • M. catarrhalis All negative Dna’ase + • N. gonorrhoae will NOT grow on 5% Sheep’s blood agar • N. meningitids will grow on 5% Sheep’s blood agar
Miniature CHO fermentation reactions for Neisseria and Moraxella catarrhalis Compare (+) yellow reactions to negative (red) control well + + + Oxidase enzyme spot test: Detects presence of enzyme cytochrome oxidase Add reagent N,N dimethyl-p-phylenediamine oxalate to filter paper with organism positive = purple/ blue control Full size tube CHO fermentation test
Neisseria meningitidis • Meningitis in children and young adults • Hallmark is petichiae (organisms crowd into capillaries) tissue necrosis and DIC (endotoxin) /infection can be rapidly fatal • Carriage in Nasopharynx / Susceptible to Penicillin • Capsular polysaccharide is the primary virulence factor • Complement deficiencies in 7,8,and 9 can predispose to chronic type disease • Adrenal necrosis is referred to Waterhouse Friderichsen syndrome • Type C is the current endemic strain in the USA – 1*problem in college Freshmen living in dorm Type B is the classic epidemic strain • Immunization available for all serotypes
Neisseria gonorrhoeae • Acute urethritis, endocervix, ocular, rectal, oropharynx, septic arthritis • 10 – 20 % female ascend to PID but only 0.5% disseminate • Gram stain of urethral discharge useful for male diagnosis, however, cervix Gram stain not specific for females • Transport – charcoal swabs, Eswab, No refrigeration • Media: Selective Thayer Martin or Martin Lewis agar • Amplification methods [PCR] increase sensitivity of detection and superior to culture – standard of practice • Produces a beta lactamase enzyme and also Chromosomal resistance : Therapy: Ceftriaxone + Azithromycin or Doxycycline to prevent resistance
Neisseria gonorrhoeae gram negative diplococcus – intra and extra cellular Acinetobacter species – Cocci are larger and more round than Neisseria, not usually in pairs – normal flora in female genital tract Primary reason why female Gram stains Cannot be trusted.
Molecular Testing for NeisseriaMolecular Testing for Neisseria gonorrhea and Chlamydiagonorrhea and Chlamydia trachomatistrachomatis • Amplification of DNA by Polymerase Chain ReactionAmplification of DNA by Polymerase Chain Reaction (PCR) is the Standard of practice(PCR) is the Standard of practice • Urine, Cervix, and Urethral most often testedUrine, Cervix, and Urethral most often tested • More sensitive than culture based systemsMore sensitive than culture based systems • Sensitivity/Specificity @ 96%/99%Sensitivity/Specificity @ 96%/99% • Female: most sensitive specimen is cervixFemale: most sensitive specimen is cervix • Female urine @ 10 – 15% less sensitiveFemale urine @ 10 – 15% less sensitive • Males virtual equal sensitivity with urine and urethralMales virtual equal sensitivity with urine and urethral C. trachomatis culture - Iodine staining of inclusions in McCoy cell culture – previous method of choice Elementary bodies in fluorescent antibody stain of C. trachomatis infected cell
Moraxella catarrhalis • Pneumonia, ocular, sinusitis, otitis media • Gram stain of sputum can be helpful in diagnosis of Moraxella pneumonia • Polys with gram negative diplo-cocci • Hockey puck colony – able to push around on agar surface • Oxidase enzyme positive • DNA’ase enzyme positive • Resistant to ampicillin by beta lactamase enzyme production – Cefinase test can be used • Therapy: Augmentin or 2nd or 3rd generation Cephalosporin
Gram Positive Rods Corynebacteria Bacillus Listeria Erysipelothrix
Corynebacterium •Over 20 species, most not pathogenic •Gamma hemolytic gray colonies •Catalase + •Diphtheroid morphology – Gram positive Chinese letter forms •No spores produced
Corynebacterium diphtheriae• Agent of Diphtheria • Diphtheritic adherent pseudo membrane produced in throat • Phage mediated exotoxin is distributed from the membrane causing respiratory paralysis (virulence factor) • Toxin detected by Elek immuno-diffusion test • Grows well on 5% Sheep’s BAP • Selective medium Cysteine Tellurite agar • Produces black colonies with brown halos • Grow on egg containing Loeffler medium then stain with methylene blue to observe metachromatic granules - colorful storage granules characteristic of C. diphtheria Elek plate Tellurite agar Metachromatic granules
Other Corynebacterium • Corynebacterium jeikeium – • normal skin flora bacteria / thrives on lipid • Infects patients with plastic catheters and indwelling devices by tunneling into the device from the skin • Biofilms are formed on the plastic, protecting the organism from antibiotic therapy • Very resistant to most antibiotics • Susceptible to vancomycin and tetracycline only • Corynebacterium urealyticum – • Rapidly urease positive diphtheroid • Urinary tract infection in post renal transplants • Resistant to many antibiotics – vancomycin susceptible Red is (+) for Urease reaction
Bacillus species •Large gram positive rods with square ends - boxcar •Can over-decolorize easily and appear reddish •Spores produced – causes clearing in rod •Catalase enzyme positive •Most are motile spores
Bacillus anthracis • Anthrax – Category A agent – highest threat to public health and bioterrorism • Natural infection of herbivores – infected animals can have fatal infection and contaminate the environment for years • Virulence factors: anthrax toxin and capsular polypeptide • Skin is the most common presentation -Woolsorter’s disease – acquired from handling imported hides • Unique black eschar skin lesions • Pneumonia, sepsis, and meningitis uncommon
Bacillus • Bacillus anthracis culture: • Medusa head colonies on BAP No hemolysis on 5% Sheep’s blood agar Non-motile Penicillin susceptible With these reactions – Alert public health department for assistance • Bacillus cereus – • Food poisoning with rapid onset (1-6 hr) vomiting • Preformed emetic toxin in food • Fried rice is one of the most common food sources • Traumatic wound infection from contaminated soil • Beta hemolytic colony on Sheep’s blood agar • Motile
Listeria monocytogenes • Small gram positive rod/ no spores • Catalase positive* • Subtle beta hemolysis on Blood agar • Tumbling motility on wet mount • Umbrella motility in tube media • More motile at 25˚C than 35˚C • Cold loving – Grows well at 4˚C and reason why it is abundant in refrigerated foods like dairy and deli case meats • Infections: Bacteremia pregnant & still births • Infections: Bacteremia and CNS in immune suppressed • Ampicillin / intrinsic resistance to Cephalosporins
Erysipelothrix rhusiopathiae • Small gram positive rod • Catalase enzyme negative • Alpha hemolysis on blood agar • Only G+R that produces hydrogen sulfide (H2S) in a TSI agar slant –Human infections acquired from swine with erysipelas or poultry Infections most common in butchers –Bacteremia uncommon • most common in drug addict • High % endocarditis /extensive damage –Intrinsic resistance to Vancomycin H2S production on Triple sugar iron agar (TSI) Erysipelas – lacy skin lesions of swine
Gram negative bacilli Enterics – ferment Glucose Non fermenters – do not ferment Glucose Fastidious
Gram negative bacillus MacConkey agar Lactose fermenter Lactose non fermenter Gram stain enteric bacilli – plump No lactose fermentation Lactose fermentation
Enteric GNRs / ferment lactose • Escherichia coli • Major aerobic NF in intestine • #1 cause of UTI [@80%] • Bacteremia, neonatal meningitis • Abdominal infections Biochemicals: • Spot indole reaction = positive • Green sheen produced on EMB agar (Eosin methylene blue) • Pathogen of diarrhea • Enterotoxigenic E. coli – Traveler’s diarrhea • Enterohemorrhagic E. coli [0157:H7] • Bloody diarrhea usually acquired from eating undercooked cow meat from an infected cow • HUS – hemolytic uremic syndrome [hemolytic anemia, thrombocytopenia, and renal failure] particularly in children • Does not ferment sorbitol – most other E. coli types ferment Sorbitol Green sheen on EMB agar Mucoid colony Indole positive Robin’s egg blue
Enteric GNRs that ferment Lactose•Enterobacter species • Enterobacter cloacae and E. aerogenes most common • Environmental organisms with low pathogenicity • Enterobacter (Cronobacter) sakazakii associated with neonatal meningitis •Klebsiella species – • K. pneumoniae most common • Mucoid colony • Currant jelly sputum in alcoholics due to blood mixed with capsular polysaccharide in sputum
Enterics that do NOT ferment lactose! •Proteus species Colonies swarm in layers on agar surface • Proteus vulgaris – indole positive • Proteus mirabilis – indole negative - Common in UTI, abdominal infections •Serratia marcescens • Produces red pigmentation - intensifies at room temp • Causes infection in • Immune suppressed • Ventilator associated pneumonia • Bacteremia
Glu/lac/suc fermented with gas Glucose fermented only Glucose fermented with H2S No CHO fermentation Non fermenter Triple Sugar Iron Agar – Media for GNR to detect fermentation of glucose, lactose and/or Sucrose and production of hydrogen sulfide [H2S] Fermentation= yellow Gas = Disruption of the agar H2S No fermentation = red
Salmonella species • Salmonella species • Diarrhea with +/- fever – polys in the stool • Infection from food –Eggs, meats and contaminated uncooked vegetables, –must ingest large #’s of organisms to make you ill (1,00,000 bacteria), stomach acid is protective • Does not ferment lactose/ produces Hydrogen sulfide • Selective agars for growth: SS and Hektoen • Identification based on biochemical reactions and serologic typing • Kaufman White serologic typing for speciation of Salmonella • O Somatic (cell wall) antigen – Salmonella group “B” • H flagellar antigens – 2 phases [h1 & h2] • Vi capsular antigen – S. typhi only
Salmonella Shigella Agar (SS agar) Salmonella and Shigella are colorless due to lactose not being fermented – H2S produced by Salmonella Hektoen agar – Salmonella produces H2S [Hydrogen sulfide] producing black colonies Shigella – green colonies Normal flora – orange colored due to fermentation of lactose (E. coli)
Salmonella typhi • Typhoid fever – fever, sepsis, high fatality rate • Vi capsular antigen - surrounds the D cell wall antigen • Boil solution of S. typhi for 15 minutes to destroy the Vi capsular antigen and expose the cell wall D antigen • Serotyping can then be performed for the D antigen • Moustache of H2S produced in TSI tube • Carrier state post typhus in gallbladder • Ingested organism enters the bowel then in @ 1 week moves on to the Blood stream & Bone Marrow –
Shigella • Diarrhea, +/-vomiting, fluid loss, polys and blood in stool , tenesmus, usually treated with Quinolones • Infection : Human to human transmission /control with good hygiene • Low #’s of organisms to make you ill [10 – 100 bacteria] • Non motile and No H2S produced ( – Green colonies on Hektoen agar • Does not ferment lactose • 4 species based on somatic antigen • S. boydii Group C • S. dysenteriae Group A • S. flexneri Group B • S. sonnei Group D
Yersinia enterocolitica •Major reservoir – swine •Humans infected by drinking non pasteurized milk •Infections: • Diarrhea • Septicemia in iron overload syndromes • Mesenteric adenitis – RLQ pain which mimics appendicitis • Infected blood products from transfusion •Grows well at 4 °C (like Listeria) •CIN agar (Cefsulodin-irgasan-novobiocin) is selective agar to grow Y. enterocolitica
Yersinia pestis - Plague • Category A agent • Obligate flea/ rodent/ flea cycle in nature • In human, a rat flea bite - leads to Bubonic plague, infection of the lymphatic system, painful buboes (lymph node swelling) at site of the bite • Hemorrhagic lymph nodes spread to blood • Pneumonic form from blood stream infection • Fatality >=50% • Endemic in Southwestern USA • Grows on blood agar • catalase +, oxidase - • Bipolar staining “safety pin”
Glucose Fermenters Oxidase Positive •Vibrio cholera • Natural environment is saltwater • Halophilic (salt loving) – enhances growth • Rice water diarrheal stool from mucus flecks • Virulence due to enterotoxin production – –Receptor on epithelial cell in small bowel – –activates adenyl cyclase – –increases cAMP with hyper secretion of NaCl and H20 – death from dehydration and metabolic acidosis • Curved “C” shape Gram negative rod • Selective media – thio citrate bile sucrose agar, yellow color due to sucrose fermentation TCBS Agar
Other Vibrio species •Vibrio parahaemolyticus • Diarrhea from ingestion of raw /oysters • Usually self limited, worse in immune suppressed • TCBS medium = green = sucrose fermentation negative •Vibrio vulnificus • Ingestion of shellfish raised in coastal waters • Infections: • Diarrhea • Skin infection from injury exposed to coastal waters • Bacteremia - leads to formation of painful skin lesions on lower extremities with muscle necrosis / This form in patients with pre liver disease – increased serum iron - 50% fatality rate
Classic Gram Negative Rod Gram Stains Campylobacte r Vibrio species Enteric gram negative rod Sea gull wings C shaped Plumb and rectangular
GNRs - Do not ferment Glucose Oxidase negative • Acinetobacter species – isolated from hospital environment and human skin, large gram negative cocco-bacilli • Ac. baumannii a major nosocomial pathogen • Glucose oxidizer • Acquires antibiotic resistance with pressure • Ac. lwoffi – • glucose non oxidizer • Stenotrophomonas maltophilia • Rapid maltose oxidizer • Long Gram negative bacillus • Naturally resistant to many antibiotics • Gun metal gray pigment • Nosocomial pathogen – super-colonizer when on long term Imipenem therapy due to natural resistance to Imipenem
Glucose non-fermenter Oxidase Positive Pseudomonas aeruginosa • Fluorescent & blue-green pigment (pyocyanin) • Grape-like odor • Growth at 42˚C • Ps fluorescens/putida group– no growth at 42°C • Major pathogen of cystic fibrosis • Mucoid strains [polysaccharide capsule] • In combination with Burkholderia cepacia can cause major lung damage • Nosocomial pathogen – associated with water & moisture • Intrinsically resistant to many antibiotics
Glucose Non Fermenters/oxidase + • Burkholderia cepacia • Low virulence – found in the environment • Problem organism in cystic fibrosis: infection causes extensive lung damage • Dry , yellow colony • Oxidase positive • Esculin positive • Chryseobacterium (Elizabethkingia) meningosepticum • Infections: • Newborns: fatal meningitis and septicemia in the newborn • Elderly/immune suppressed: bacteremia • Low virulence • Environmental source – water • Yellow colony, oxidase and indole positive
Haemophilus influenzae • Variety of infections • Transmission – close contact/secretions • Virulence factor – capsular polysaccharide • Small pleomorphic Gram negative rod • Requires 2 nutritional factors for growth: • X = hemin • V= NAD (nicotinamide adenine dinucleotide) • Grows on chocolate agar (has X and V factor) • Will not grow on 5% sheep’s blood agar • Requires high level C0₂ [5 – 8%] for growth • Effective vaccine targets the H. influenza type B (HIB) – effectively eliminated most childhood invasive infections • Resistance to Ampicillin due to beta lactamase enzyme [20 %], 3rd gen Cephalosporin becomes the antibiotic of choice
Test for Beta lactamaseTest for Beta lactamase DetectionDetection • Add bacteria to filter paper impregnated with Nitrocefin orAdd bacteria to filter paper impregnated with Nitrocefin or Cefinase (yellow colored/chromogenic cephalosporin)Cefinase (yellow colored/chromogenic cephalosporin) • Incubate at room temp for @ 1 minuteIncubate at room temp for @ 1 minute • Positive result is a color change fromPositive result is a color change from yellow to redyellow to red –– • Beta lactamase enzyme breaks down beta lactam ring ofBeta lactamase enzyme breaks down beta lactam ring of Nitrocefin to a hydrolyzed red end productNitrocefin to a hydrolyzed red end product • Detects resistance to Amp/Pen/1Detects resistance to Amp/Pen/1StSt gen Cephalosporin ingen Cephalosporin in Haemophilus species, N. gonorrhea , Moraxella catarrhalis,Haemophilus species, N. gonorrhea , Moraxella catarrhalis, and anaerobic Gram negative rodsand anaerobic Gram negative rods • Does NOT detect the ESBL enzyme of enteric GNRsDoes NOT detect the ESBL enzyme of enteric GNRs
Satellite phenomena Small pleomorphic GNR On Gram stain Growth on Chocolate agar Growth between X and V strips Staph aureus supplies the X and V factors required
More Haemophilus species • H. parainfluenza – • requires V (NAD) factor only • Usually normal flora in the upper respiratory tract • However, one of the HACEK organisms of endocarditis • H. (Aggregatibacter) aphrophilus – (new taxonomy) • No X and V factor requirements for growth • Infections: abscesses (liver, lung, brain) & endocarditis • H. ducreyi – • Requires X factor • Cause of Chancroid - venereal disease • “school of fish” appearance on stains
HACEK group • Oral flora organism that due to poor detention or dental procedures become agents of endocarditis • Fastidious Gram negative coccobacilli • 5 -10% of community acquired native valve endocarditis unrelated to IV drug use • Require 2-4 days to grow in patient blood cultures • Haemophilus species = oxidase (-) catalase (-) • Aggregatibacter (Actinobacillus) oxidase (-) catalase (+) • Cardiobacterium hominis = oxidase (+) • Eikinella corrodens = oxidase (+), colony pits BAP • Kingella kingii = oxidase (+), hemolytic on BAP, also cause of septic joint infection in small children
Bordetella pertussis •Whooping cough –three stages of disease – (1) Prodromal – flu like disease – most contagious stage – (2) Catarrhal - cough - with classic whoop in small children – Pertussis Toxin adheres to bronchial epithelial cells and cough continues until toxin wears off – can be months – (3) Paroxysmal - recovery phase • Tiny Gram negative coccobacillus • Human pathogen - Inhabits Nasopharynx • Peripheral blood - Lymphocytosis with atypical large, irregular and deeply basophilic lymphocytes • Charcoal media for transport and culture – Regan Lowe Charcoal agar / growth in 3-5days • PCR more sensitive than culture for diagnosis • Reservoir for infection – young adults due to waning immunity. Reason behind initiative for booster shots in young adults
Pasteurella multocida/canis • Bite wound infections from cats and dogs • Human pneumonia from close cat and dog contact • Infections can disseminate to blood stream • Normal flora in many animals (zoonotic) • Small Gram negative coccobacilli • Growth on 5% Sheep’s blood agar • Non hemolytic grey colony • Will not grow on MacConkey agar (big clue) • Oxidase Positive • One of very few GNR genera that is sensitive to penicillin
Capnocytophaga species • Fusiform shaped Gram negative rods – very pleomorphic • Fingerlike projections from colonies “Gliding” • Dependence on CO2 incubation for growth • Oxidase negative, Catalase negative • Normal mouth flora in humans and animals • C. canimorsus – Associated with dog bites – high % of these bite infections lead to bacteremia and endocarditis • Capnocytophaga species (many) in human • Infect mouth ulcers induced by chemotherapy • Bacteremia
Brucella species • Disease – Brucellosis, fever of unknown origin, significant joint pain, intracellular pathogen of the RES system • Specimens: Blood and bone marrow (granuloma formation) • Serology • Small Gram negative coccobacilli – difficult/slow to grow in culture • Old: Castaneda biphasic blood culture held for 21 days • Current: Automated Blood culture systems with growth @ 5 -6 days • Laboratory acquired Brucellosis is common • Zoonosis – ingestion of raw milk, animal exposure, inhalation • Brucella species dependent upon animal species the patient was exposed: • B. abortus – raw cow milk • B. melitensis – raw goat milk, feta cheese • B. suis – pigs • B. canis - dogs Granuloma in bone marrow
Campylobacter spp. • “Curved” Gram negative rod • C. jejuni – Diarrhea, one of most common causes in US, bacteremia in HIV and immune suppressed • Ingestion of undercooked poultry / juice contaminating raw food • Culture requires selective blood agar with antibiotics / Campy-BAP, Skirrows BAP • Incubate at 42˚C in microaerophilic atmosphere (high CO₂, low O₂) • Sequelae - Significant % Guillain-Barre syndrome • C fetus – Bacteremia in the immune suppressed host. Source cattle and sheep. • Temperature tolerance aids in identification C. jejuni – grows at 37˚C and 42˚, hippurate hydrolysis positive C. fetus - grows at 37˚C and 25˚C hippurate hydrolysis negative
• Reservoir – rabbits, rodents, ticks and flies. Humans infected by insect bites or directly from exposure to animal blood • Associated with skinning rabbits with bare hands • Bacteria can penetrate small breaks in skin: • cause painful skin lesions – • enlarged lymph nodes – • leading to bacteremia (ulceroglandular tularemia) • Pneumonia • Great hazard to lab workers by aerolization • Fastidious small gram negative cocco-bacillus • Culture media requires cysteine* Francisella tularensis
Helicobacter pylori • Small curved Gram negative bacilli • Acute gastritis – can progress to gastric adenocarcinoma • Human to human transmission/ fecal - oral route • Rapid and strong urease enzyme – used for detection directly from gastric antrum biopsy tissue • Difficult to grow in culture • Stool antigen/diagnosis and test of cure • Serum antibody for past exposure • Organism stained by silver stains in GI • Treatment – Antibiotics and acid suppression Histologic exam of gastric biopsy Hematoxylin-eosin stain or Warthin-Starry Silver stain
Legionella • Legionella pneumophila [6 serotypes] • Pulmonary disease – strong association with water • Requires cysteine in culture medium for growth • Buffered Charcoal Yeast Extract agar/ 3-5 days • Will not show on Gram stain of specimens • Use silver impregnation stains in tissue • Urinary antigen test detects L. pneumophila type I infection – the most common cause of infection • Treatment: Erythromycin (macrolide) BCY E
Bacteria without cell walls • Mycoplasma spp and Ureaplasma spp – cell membranes only! Media contain sterols to protect the membrane • Lack of peptidoglycan cell wall – means unable to Gram stain, no colonies produced on agar, cannot be treated by antibiotics that act by inhibiting cell wall formation • M. pneumoniae – • Community acquired pneumonia • PCR for diagnosis • Presence of cold agglutinins • Genital mycoplasmas • M. hominis – fried egg on agar surface, vaginitis, cervicitis, postpartum sepsis, neonatal infections , pre rupture of membranes • Ureaplasma urealyticum – Dark metal-type appearance, rapid urea hydrolysis in broth, NGU & upper genital tract infection, spontaneous abortion, neonatal infections • PCR current method for diagnosis UreaplasmaMycoplasma hominis
Unusual and difficult to grow • Bartonella henselae • Cat scratch disease – exposure to cat and cat excrement • Bacillary angiomatosis – vascular skin lesion +/- invasion / HIV • Bartonella quintana – cause of trench fever/ vector is the body louse • Diagnose Bartonella by Serology/PCR • Chlamydia trachomatis Serovars L1,L2,& L3 Lymphogranuloma venereum lymphatics and lymph nodes involved • Chlamydia pneumoniae (TWAR agent)- Pneumonia • Chlamydia psittaci- psittacosis, pneumonia, exotic parrot exposure • Diagnosis: Serology and PCR assays
Unusual and difficult to grow •Ehrlichiosis – Rickettsia bacteria • Zoonotic intracellular pathogen • Vector – Ixodes tick *(hard tick) • 2 Genera cause Ehrlichiosis • Anaplasma spp, inclusion in the PMN • Ehrlichia spp inclusion in the Monocytes • Fever, leukopenia, thrombocytopenia, • Elevated serum aminotransferases, • No rash (differs Rocky Mountain Spotted Fever) •Found in south central, southeast , midwest US •PCR, serology, and exam of blood smear for diagnosis
Spirochetes • Borrelia burgdorferi - Lyme’s disease • Primarily found in NE part of US • Vector = Ixodes tick • Acute disease: Fatigue, headache, fever, and rash • Can have progression to chronic disease • Diagnosis: Serology and PCR • Borrelia recurrentis - Relapsing fever • High fever (relapsing) with thrombocytopenia • Vector: body louse • Diagnosis: Blood smear observe spirochete
Spirochetes • Treponema pallidum • Syphilis • RPR and VDRL for antibody detection • Molecular methods • Brachyspira – • intestinal spirochete found on the brush border of the intestine, • ?? Role in disease • Leptospira interrogans – Leptospirosis • Fever with rash and renal involvement • Urine from rats and other animals contaminate water supplies Darkfield from chancre lesion (1) Leptospirosis – Shepherd’s crook (2) Presence of spirochete in a renal tubule (1) (2)
Unusual Bacterial diseases •Granuloma inguinale • Klebsiella (Calymmatobacterium) granulomatis • Rare STD – causes ulcerative genital lesions •Streptobacillus moniliformis • Rat bite fever or Haverhill fever • Infection from rat bite • L form - cell wall deficient bacteria • Inhibited by SPS in blood culture media • Needs serum supplementation to grow, will not grow on routine bacteriology media • Detailed history aids diagnosis
Bacterial vaginosis •A mixed anaerobic/aerobic bacterial infection • The normal balance of vaginal flora is disrupted • Rather benign infection except in pregnancy • Discharge: Fishy like odor of discharge, alkaline pH >=4.5 • Usual NF organism of Lactobacillus are overgrown by: • Gardnerella vaginalis (aerobic Gram variable rod) • Mobiluncus (anaerobic curved Gram negative rod) • Increased #s Gardnerella vaginalis is the bacterial marker for infection – Human blood agar media of choice, Beta hemolytic, Gram variable staining • Clue Cells are diagnostic and more specific than growth in culture • Molecular probe assays and amplification assays available as part of “women’s health” screening panels Clue Cell
• Anaerobic infections can occur in virtually every any organ or region of the body • Polymicrobial –with both aerobic and anaerobic species • Endogenous organisms / usually normal flora • Increase in numbers and cause infection due to trauma, vascular or tissue necrosis cutting off the oxygen supply to the involved tissue • Treatment: Usually surgery to restore oxygen and remove necrotic tissue plus antibiotics • Anaerobic culture collection • Gel containing swabs • ESwab • Evacuated vials (port o cult)/ oxygen free • Do not refrigerate specimens – absorb oxygen in cold temperatures and can more rapidly kill anaerobes Anaerobic Bacteria
Anaerobic culturing • PRAS media – pre reduced anaerobically sterile • Media packaged in oxygen free environment • Most common media used • CDC anaerobic enriched blood agar • Kanamycin-vancomycin blood agar • Bile esculin agar • Thioglycollate broth • Chopped meat glucose broth • Anaerobic chambers – perform all culture work in an oxygen free environment • Anaerobic gas pack jars – for anaerobic incubation of agar plates • Wet pack – add 10 ml water to hydrogen and CO2 generating envelope/ requires palladium coated catalysts – generates heat • Dry pack – (Anaeropack) Absorbs O2 and generates CO2 PRAS
Anaerobic Gram Negative Rods Unusual shapes and sometimes poorly staining by Gram
Bacteroides fragilis group Pleomorphic irregular staining Gram negative rod •Grows in the presence of bile •Growth on Bile esculin media is black •Resistant to Penicillin and Kanamycin •Normal flora in GI tract •Infections: Related to the bowel/ GI abscess • foul smelling/gas produced •B. fragilis group • B. fragilis – most common species • B. ovatus • B. thetaiotamicron ( indole reaction positive) • B. uniformis • B. vulgatus • Resistant to Penicillin by beta lactamase enzyme • Metronidazole is antibiotic of choice Growth on bile/esculin media Black pigment from Esculin production
Prevotella and Prophyromonas –Pleomorphic Gram negative rod –Normal flora in the upper respiratory tract –Infections: respiratory tract abscesses –Will not grow in the presence of bile –Will not turn black on esculin media –Unique** Brick red fluorescence and black pigment formed on Blood agar after one week of incubation
Fusobacterium spp. • F. nucleatum Long thin gram negative bacilli – spindle shaped, pointed ends • Normal flora upper respiratory tract • Infections: mouth and respiratory tract abscess formation and liver abscess • Vincent’s angina – necrotizing oral infection caused by Fusobacterium species and spirochetes • F. necrophorum – pleomorphic gram negative bacilli that form filaments or chains of rods • Lemierre’s syndrome - oropharyngeal infection, leads to thrombosis in jugular vein, septicemia, with high fatality rate
Anaerobic Gram Positive Rods
Clostridium species • Gram positive bacilli (boxcar shaped ) form spores • Bacilli may over-decolorize and appear red • Clostridium perfringens • Infections: Food poisoining, necrotic tissue abscesses (Clostridial myonecrosis), bacteremia, cholecystitis • Most common anaerobic G+R in intestine • Double zone of beta hemolysis on BAP • Lecithinase produced on egg yolk agar • Reverse camp test positive Lecithinase Reverse Camp Test
• Clostridium botulinum – Botulism –Adult disease – Preformed heat labile neurotoxin ingested is mass produced or in home-canned foods –Infant disease - spore ingested from nature or product produced in nature such as honey or household dust, neurotoxin produced in gut • Begins with constipation and difficult sucking bottle –Both forms are ife threatening neuroparalytic disease • Clostridium tetani - Tetanus • Gram stained cells appear like Tennis racket • Tetanospasmin toxin enters by penetrating skin injury • Spastic contractions of voluntary muscles, hyper-reflexia, lock jaw (trismus) • Immunization to prevent
•C. septicum – • Bacteremia or Gas Gangrene in patient with underlying malignancy • Hematogenous spread from GI tract – no trauma necessary •C. difficile – • Disease: antibiotic associated colitis, pseudomembranous colitis from toxin production • Toxin A – enterotoxin causing fluid accumulation • Toxin B – potent cell cytotoxin – primary virulence • Binary toxin – so called Nap1 strain is produces larger amount of toxin • Diagnosis of infection: • EIA methods [toxin A/B] are insensitive but detect active toxin • PCR methods [toxin A/B] are more sensitive, detect toxin genes • Culture – Cycloserine, Cefoxitin, Fructose Agar [CCFA] • Infants have @ 70% colonization rate – do not test
Actinomyces •Branching gram positive bacilli - do not form spores • Aerotolerant, but grow best anaerobic •Normal flora oral, GI, vagina, skin •Infections: usu. oral/facial (lumpy jaw) •Form sulfur granules in tissue • Can be found on normal tonsil •Actinomyces israelii – associated with oral, thoracic, and abdominal infections, IUD infections Bread crumb colonies in broth Molar tooth colony on agar plates Penicillin susceptible.
Branching Gram positive rods of Actinomyces – antler like Molar tooth colony Sulfur granule Clumps of Actionomyces
Propionibacterium acnes • Pleomorphic Gram positive rod • Catalase positive, Indole positive • Normal flora - skin, oral, GU and GI • Contaminate in blood cultures from skin NF • Pathogen of acne vulgaris • Opportunistic pathogen: cerebral shunt infections and other endovascular and neurosurgical infections • Firmly established as significant cause of prosthetic joint infection – particularly shoulder joints • Cultures should be held up 7- 14 days • Therapy - Ampicillin

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Bacteriology 2017 Update

  • 1.
  • 2.
    Beginning definitions • ObligateAerobe – require high level of oxygen (20%) to grow • Obligate Anaerobe –>30 min of oxygen exposure can be deadly • Facultative anaerobes – grow in aerobic and anaerobic conditions, most so-named “aerobic” bacteria are actually facultative • Microaerophilic – Grow better with reduced oxygen and elevated carbon dioxide % • Aerotolerant anaerobes– anaerobe is not killed by prolonged exposure to oxygen – example: Clostridium tertium • Lag Phase - >24 hrs old, growth is slowing, not appropriate for biochemical or susceptibility testing • Log Phase – Exponential growth – appropriate for all testing • Stationary phase – appropriate for transporting specimens
  • 3.
    Specimen collection for AerobicBacteriology Throat / Wound collection using swab 1. Use swabs made of polyester or sponge like material, do not use a cotton swab, traps bacteria /potentially toxic 2. After specimen collection, swab placed in Stuart’s or Amies transport media (buffer solution with peptones) 3. These transport media preserve bacteria viability but do not promote growth of bacteria Urine collection – two methods suggested 1. Boric acid to place bacteria in stationary phase 2. Refrigerate within one hour Purpose to maintain original colony count and maintain viability of organisms Tissues/Sterile body fluids – Sterile containers
  • 4.
    Gram stain Procedure 1minute Rinse Primary stain Mordant 1 minute Rinse 5-10 seconds Rinse Decolorization Counter stain 1 minute Rinse Peptidoglycan in positive cell wall traps the Crystal violet, hence blue color Prepare thin film of specimen on glass slide Heat or methanol
  • 5.
    Gram positive cocci inpairs and chains Streptococcus Gram Positive cocci in clusters Staphylococcus Gram positive rod most like a Bacillus species Gram negative bacilli, enteric Gram negative rod – Fusiform Shaped – Fusobacterium species Visit the Gram stain tutorial for more Gram stains
  • 6.
    Assess quality ofSputum for Bacterial culture with Gram stain • Sputum specimen is rejected for bacterial culture, if sputum judged to be “spit” not actually a deep cough specimen • Perform Gram stain of (?) sputum specimen • If <=25 epithelial cells – acceptable (right pix) • If >= 25 epithelial cells /field (left pix) • Sputum is judged to be spit / not a sputum specimen • Bacterial culture is not performed / it is rejected • Request made for a new “deep cough” specimen Bad Sputum Good Sputum 10X objective 10X objective
  • 7.
    Most used AgarMedia - BacteriologyBlood agar- 5% sheep’s blood agar • Gauge hemolytic reaction of bacteria (alpha, beta, gamma) • Culture a variety of Gram positive, Gram negative bacteria and Yeast Chocolate agar • “Carmelized” blood agar – more enriched than blood agar medium. • Will support the growth of the same organisms as 5% Sheep’s blood agar Plus more fastidious bacteria - like Haemophilus influenzae and Neisseria gonorrhoeae
  • 8.
    Most used agarmedia - Bacteriology • MacConkey agar – Supports growth of Gram negative rods (GNR), and determines ability to ferment lactose, described as Selective and Differential media • Selective - crystal violet inhibits Gram positive organisms • Differential agar - Lactose fermentation can be determined • Lactose fermentation = pink (neutral red indicator) • Non-lactose fermentation = no color • After media is plated, incubate in aerobic incubator at 35˚ C for 24 – 72 hrs
  • 9.
    MALDI-TOF Mass Spectrometry– newest wide spread identification method –Matrix-assisted laser desorption/ionization – time of flight –Identification by analyzing proteins in bacteria –Replaced many/most biochemical tests for bacterial ID
  • 10.
    MALDI-TOF • Laser isfired at the matrix/sample • Laser energy is absorbed by the matrix and converted to heat energy and ionizes the sample. • Positive ions (proteins)are accelerated through a vacuum by an applied electrical field. • The time taken for the proteins to reach the detector depends on their mass/charge ratio (m/z) and create spectrograph. • Each bacterial species has a different protein composition, thus giving rise to a specific mass spectrograph. • The mass spectrograph produced by a sample is then compared with many thousands stored in a spectrograph database to see which one it most closely matches. Thus an identification is achieved.
  • 11.
  • 12.
    Staphylococcus Gram positive cocciin clusters due to bound coagulase or “clumping factor” All Staphylococcus are Positive for Catalase Enzyme Staphylococcus aureus Coagulase Negative Staph Coagulase Enzyme Positive Coagulase Enzyme Negative +/- Yellow colony Beta hemolysis White colony Most not hemolytic
  • 13.
    Catalase Enzyme Reaction NegativePositive Bacteria added to Hydrogen Peroxide Blood has innate catalase activity and will give false positive catalase reaction
  • 14.
    Rabbit plasma inoculatedwith organism Incubate at 35˚C Read at 4 hours and if negative read again at 24 hours Negative tube coagulase =no clot Coagulase negative Staph Positive tube coagulase [clot at either 4 or 24 hrs] Staph aureus Tube Coagulase Reaction
  • 15.
    Staphylococcus aureus • Primaryvirulence factor: • Protein A – surface protein, ability to bind immunoglobulin and combat the immune response • Diseases and associated toxin: • Toxic shock syndrome (TSST-1 toxin) • Scalded skin syndrome (Exfoliatin (SSS) toxin) • Soft tissue infection (Panton valentine leucocidin toxin – PVL) • Food poisoning / Enterotoxins – stable to heating at 100*C for 30 minutes • Bacteremia and endocarditis • Septic arthritis – Primary cause • Toxins act as super antigens, recruit host defense cells that liberate cytokines with systemic effects
  • 16.
    Unique Susceptibility Issues MRSA=Methicillin resistant Staphylococcus aureus –Altered Penicillin binding protein (PBP2) produced by the mecA gene codes for resistance to oxacillin/methicillin /nafcillin resistance –Cefoxitin susceptibility testing is considered a more sensitive indicator of resistance to oxacillin and provides better detection of MRSA –All MRSA are considered (reported) as resistant to semisythetic penicillins and cephalosporins –Vancomycin becomes a drug of choice KB disk resistant
  • 17.
    The “D” Test •Is Staph aureus really susceptible to ClindamycinIs Staph aureus really susceptible to Clindamycin • Why?? During therapy, S aureus isolates resistant toWhy?? During therapy, S aureus isolates resistant to Erythromycin possess enzymes capable of inducingErythromycin possess enzymes capable of inducing Clindamycin resistanceClindamycin resistance • Kirby Bauer zone around Clindamycin will be blunted to form aKirby Bauer zone around Clindamycin will be blunted to form a “D” if Clindamycin can be induced by Erythromycin to be“D” if Clindamycin can be induced by Erythromycin to be resistant – so calledresistant – so called INDUCIBLE RESISTANCEINDUCIBLE RESISTANCE.. • Clindamycin should be reported as resistant by clindamycinClindamycin should be reported as resistant by clindamycin induction and not used for therapy.induction and not used for therapy. D test positive Inducible resistance D test negative Clindamycin can be used for therapy
  • 18.
    Nares is primarycolonization site for MRSA and the most common surveillance site Culture methods for surveillance: ChromAgar is commonly used - It is a selective and differential media containing chromogenic substrates that turn a specific color to ID Staph aureus. Cefoxitin is added to the medium to select for only MRSA. Molecular assays (MA) can also be used to screen nares for MRSA. MAs increase the sensitivity of detection (@5-10%) but increases laboratory costs. Mupiricin can be used to nares eradicate carriage Staphylococcus aureus (MRSA) Hospital Epidemiology Issue: Nares surveillance for MRSA
  • 19.
    Coagulase negative Staph(CNS) @ 15 species infect humans • Staph epidermidis – most common –Major aerobic component of normal skin flora –Common cause of subacute bacterial endocarditis –Pathogenicity from cell adhesion factors forming biofilm on biologics and plastics like heart valves, Catheters and replacement joints making treatment difficult • Staph saprophyticus – –Urinary tract infection in the child bearing age female, –This CNS adheres in greater #’s to epithelial cells –Novobiocin resistant by KB disk test White non-hemolytic colony resistant
  • 20.
    CNS & RelatedCocci • Staphylococcus hemolyticus – • Can cause line related sepsis • Hemolytic on blood agar • Do not confuse with Staph aureus • Staphylococcus lugdunensis • ? greater pathogenicity than other CNS species • Abscesses and bacteremia • PYR test positive = pink • Micrococcus species • Mustard yellow colony – environmental contaminate • Catalase positive / Gram positive cocci in tetrads • Tube coagulase negative • Does not ferment glucose • Staphylococcus ferment glucose Po s NegPYR TEST
  • 21.
    Streptococcus Gram positive cocciin chains and pairs Catalase enzyme negative Three groups based on hemolytic pattern on sheep’s blood agar • Alpha – greening of agar, partial hemolysis of RBCs • Viridans Streptococcus, Streptococcus pneumoniae, Granulicatella/Abiotrophia • Beta – clearing of agar, complete hemolysis of RBCs • Beta hemolytic Streptococcus groups A - G • Gamma – no clearing of agar, intact RBCs • Streptococcus bovis (Strep gallolyticus)
  • 22.
    Beta Hemolytic Streptococc us • Beta Streptococcusare grouped using the “C” carbohydrate (CHO) in the bacteria cell wall • Identifies Beta Strep groups – A, B, C, F, and G that are the ones most commonly seen in human infections • Lancefield grouping system uses the “C” CHO in the cell wall of Streptococcus as the antigen in a slide agglutination test with monoclonal antibody (A<B<C<D<F<G) attached to latex beads See below slide for a positive and negative latex reaction. • Immunologic typing is more accurate than biochemical testing to group Beta hemolytic Streptococcus + -
  • 23.
    Streptococcus pyogenes • Genus/speciesname for Group A beta Streptococcus [GAS] Biochemical identification: • Bacitracin KB sensitivity test – inhibited, no growth @ disk • This test is not specific for Group A , it x-reacts with group C • “A” disk is the Bacitracin disk – stands for group “A” Strep • PYR (pyrrolidonyl arylmidase) reaction • Organism spotted on moist disk • 2 min – RT incubation • Add Cinnamaldehyde reagent • Look for color reaction • Pink = positive = Strep pyogenes • This test is not exclusive for Strep pyogenes – Enterococcus and Staph lugdunensis are PYR positive • Therapy : Penicillin or Cephalosporin antibiotics No resistance reported to these agents PYR
  • 24.
    • Streptolysin Oand Streptolysin S toxins • Comprise the ASO titer determination • Cell toxins lead to evasion from the immune system • O toxin is oxygen labile S toxin is oxygen stable • When both are active - most hemolysis is apparent on 5% Sheep’s blood agar Primary virulence factors: M Protein – prevents phagocytosis Capsule – hyaluronic capsule protects from phagocytosis
  • 25.
    Streptococcus pyogenes Infections •Diseases – •Pharyngitis • Impetigo (1) • Erysipelas (2) • Cellulitis (3) • Necrotizing fascitis (4) • Puerperal sepsis • Toxic Shock • Scarlet fever (5) 1 2 3 4 5
  • 26.
    Sequelae of Streppyogenes Infections Rheumatic fever • Cause: inadequately treated Strep throat • Rise in the 1990s due to EIA tests being used as the only diagnostic test for pharyngitis – EIA tests are only 60-70% sensitive – and led to children not receiving antimicrobial therapy. Negative EIA specimens must be confirmed by culture • Similarity between the proteins of Strep A and muscle tissue cause immune system confusion which leads to immune system to attack heart, joint, and bones • Anti-streptolysin O serum test measures the amount of antibodies produced against Streptococcus group A Glomerulonephritis • 10-14 days following skin infection or pharyngitis • Renal disease with inflammation of the glomeruli • ASO titers will be positive • Usually resolves with therapy
  • 27.
    Streptococcus agalactiae (Group B) •Biochemical tests for identification: –Camp test – performed using a Staph aureus strain with Camp factor streaked perpendicular to group B Strep, incubate 24 hr. / intensifies toxin production and produces arrow shaped hemolysis –Rapid hippurate hydrolysis - 4 hr identification of GBS – Hippurate disk Saline inoculated with Group B Strep Ninhydrin reagent added for color formation • Positive = Purple pos Arrow Staph aureus Strep group B
  • 28.
    Strep agalactiae [GBS] •Pathogen of the elderly – Bacteremia and urinary tract infection • Pathogen of neonate – in utero or perinatal organism acquisition during birthing process • Early onset – within 7 days • Late 7 – 28 days from birth process. • Treatment: Penicillin or Cephalosporin (3rd generation) • Pregnant women carry organism in the cervix and/or rectal area. • All pregnant must be tested at 35 – 37 weeks of pregnancy. • Enrichment methods for GBS are standard of practice and must be used • Swab placed into LIM broth – incubate for 18 hours at 35 ˚C then subculture onto Blood agar. This broth can also be used as an enrichment method to enrich for molecular testing. • Carrot enrichment broth turns orange with growth of group B Strep Carrot Broth
  • 29.
    Streptococcus bovis (gallolyticus) Streptococcus gallolyticusssp. gallolyticus formerly S. bovis biotype 1 associated with endocarditis and colon cancer (73%) when isolated from blood cultures Strep gallolyticus ssp. pasteurianus formerly S. bovis biotype II- associated with neonatal meningitis Biochemical reactions: Both species have similar biochemical reactions 6.5% NaCL
  • 30.
    • Two mostcommon species • E. faecium • E. faecalis • No well defined virulence factors • Possesses the Group “D” CHO in the cell wall • Biochemical tests: • Bile esculin positive – growth and black precipitin • Growth in 6.5% salt • PYR positive (Group A beta Strep and Staph lugdunensis also positive)* • E. faecium = arabinose fermentation positive • E. faecalis = arabinose fermentation negative Enterococcus Bile esculin PYR + - Neg Pos PYR
  • 31.
    Enterococcus • Variety ofinfections / Pathogen of opportunity • Intestinal normal flora • Cause UTI, bacteremia, and abdominal infections • Antimicrobial therapy: • Natural resistance to cephalosporin antibiotics • Ampicillin plus Aminoglycoside can be synergistic for therapy in cases of endocarditis • Unique susceptibility issues • Acquired resistance to vancomycin known as vancomycin resistant enterococcus or VRE • Van A (E. faecium) resistance genes • Van B (E. faecalis) resistance genes
  • 32.
    Alpha hemolytic Streptococcus Streptococcus pneumoniae Gramstain = Gram positive bullet (lancet) shaped cocci in pairs Polysaccharide capsule = virulence factor, antiphagocytic Identification: • Bile soluble – colonies dissolve in sodium deoxycholate (bile) • Optochin sensitive – 14mm or greater zone of inhibition around the 6mm optochin disk – “P” = pneumococcus Inhibition >=14mm NO Inhibition Colonies dissolved
  • 33.
    Streptococcus pneumoniae • Upperand Lower respiratory tract infection, sepsis and meningitis • Multi-serotype vaccine aids preventing invasive infections • Susceptibility issues: • Acquired Resistance to Penicillin due to Penicillin binding proteins (PBP) • Minimum inhibitory concentration (MIC) testing necessary to detect resistance to penicillin • Testing of CSF isolate a result of • Penicillin = >2 mcg/ml is resistant • Therapy – • If susceptible 1st line therapy is either Penicillin or 3rd generation CephalosporinMIC MIC
  • 34.
    Viridans Streptococcus Several speciesof alpha hemolytic Streptococcus are NF in mouth and upper respiratory tract S. mutans S. salivarius S. sanguis S. mitis • Bile esculin slant = negative • Not bile soluble • Optochin resistant with zone size <=13 mm • Ethylhydrocupreine hydrochloride • Cause 30 – 40% cases of sub acute endocarditis/native valve • Can cause abscess and various infections throughout the body in the immune suppressed host • Variable susceptibility patterns, some isolates with elevated MICs to Penicillin Viridans Streptococcus
  • 35.
    Viridans Streptococcus unique species •Streptococcus anginosis group: includes • S. anginosus S. constellatus S. intermedius • Normal flora in human mouth • More virulent than “normal” viridans Streptococcus, perhaps due to capsule • Cause deep tissue abscess, bacteremia, endocarditis, intra abdominal infections… • Variable susceptibilities – so best to do susceptibility testing
  • 36.
    Nutritionally Variant Streptococcus • VitaminB6 (pyridoxal) deficient – • will not grow on media without B6 supplementation • Will grow in a patient blood culture bottle due to vitamin B6 in patient’s blood • Will not subculture onto 5% Sheep’s blood agar plate (no vit B6) • Will grow with Staph aureus streak (supplies vitamin B6) • Nutritionally variant Strep will satellite @ S. aureus streak • Two species: • Abiotrophia spp • Granulicatella spp • Endocarditis – • More destructive to valve than than “regular” viridans Strep • Higher MIC’s to Penicillin Satellite streptococcus Next to S. aureus streak
  • 37.
    Rare Opportunistic Grampos cocci• Aerococcus ureae – Gram positive cocci in pairs and clusters • Alpha hemolysis , difficult to identify • Urinary tract pathogen • Rothia mucilaginosa – Gram positive cocco-baccilli • Neutropenia and gut problems predispose to infection • Normal flora in the oral cavity and upper respiratory tract • Pathogen in dental caries and periodontal disease • Bacteremia with endocarditis • Gemella morbillorum– • Easily over decolorized gram positive in pairs – requires Co2 to grow • Normal flora in oral cavity • Bacteremia with endocarditis • Leuconostoc – Gram positive cocci in chains • Intrinsic resistance to vancomycin – bacteremia in immune suppressed • Watch out! Do not confuse with VRE
  • 38.
  • 39.
    Gram Negative Cocci •Neisseria species and Moraxella catarrhalis • Small kidney bean shaped cocci in pairs • Oxidase enzyme positive • CTA (Cysteine Trypticase Agar) carbohydrate fermentations for identification – glucose, maltose, lactose, sucrose • N. gonorrhoeae Gluc + Mal - Lac - Suc - • N. meningitidis Gluc + Mal + Lac - Suc - • N. lactamica Gluc + Mal + Lac+ Suc- • M. catarrhalis All negative Dna’ase + • N. gonorrhoae will NOT grow on 5% Sheep’s blood agar • N. meningitids will grow on 5% Sheep’s blood agar
  • 40.
    Miniature CHO fermentationreactions for Neisseria and Moraxella catarrhalis Compare (+) yellow reactions to negative (red) control well + + + Oxidase enzyme spot test: Detects presence of enzyme cytochrome oxidase Add reagent N,N dimethyl-p-phylenediamine oxalate to filter paper with organism positive = purple/ blue control Full size tube CHO fermentation test
  • 41.
    Neisseria meningitidis • Meningitisin children and young adults • Hallmark is petichiae (organisms crowd into capillaries) tissue necrosis and DIC (endotoxin) /infection can be rapidly fatal • Carriage in Nasopharynx / Susceptible to Penicillin • Capsular polysaccharide is the primary virulence factor • Complement deficiencies in 7,8,and 9 can predispose to chronic type disease • Adrenal necrosis is referred to Waterhouse Friderichsen syndrome • Type C is the current endemic strain in the USA – 1*problem in college Freshmen living in dorm Type B is the classic epidemic strain • Immunization available for all serotypes
  • 42.
    Neisseria gonorrhoeae • Acuteurethritis, endocervix, ocular, rectal, oropharynx, septic arthritis • 10 – 20 % female ascend to PID but only 0.5% disseminate • Gram stain of urethral discharge useful for male diagnosis, however, cervix Gram stain not specific for females • Transport – charcoal swabs, Eswab, No refrigeration • Media: Selective Thayer Martin or Martin Lewis agar • Amplification methods [PCR] increase sensitivity of detection and superior to culture – standard of practice • Produces a beta lactamase enzyme and also Chromosomal resistance : Therapy: Ceftriaxone + Azithromycin or Doxycycline to prevent resistance
  • 43.
    Neisseria gonorrhoeae gram negative diplococcus– intra and extra cellular Acinetobacter species – Cocci are larger and more round than Neisseria, not usually in pairs – normal flora in female genital tract Primary reason why female Gram stains Cannot be trusted.
  • 44.
    Molecular Testing forNeisseriaMolecular Testing for Neisseria gonorrhea and Chlamydiagonorrhea and Chlamydia trachomatistrachomatis • Amplification of DNA by Polymerase Chain ReactionAmplification of DNA by Polymerase Chain Reaction (PCR) is the Standard of practice(PCR) is the Standard of practice • Urine, Cervix, and Urethral most often testedUrine, Cervix, and Urethral most often tested • More sensitive than culture based systemsMore sensitive than culture based systems • Sensitivity/Specificity @ 96%/99%Sensitivity/Specificity @ 96%/99% • Female: most sensitive specimen is cervixFemale: most sensitive specimen is cervix • Female urine @ 10 – 15% less sensitiveFemale urine @ 10 – 15% less sensitive • Males virtual equal sensitivity with urine and urethralMales virtual equal sensitivity with urine and urethral C. trachomatis culture - Iodine staining of inclusions in McCoy cell culture – previous method of choice Elementary bodies in fluorescent antibody stain of C. trachomatis infected cell
  • 45.
    Moraxella catarrhalis • Pneumonia,ocular, sinusitis, otitis media • Gram stain of sputum can be helpful in diagnosis of Moraxella pneumonia • Polys with gram negative diplo-cocci • Hockey puck colony – able to push around on agar surface • Oxidase enzyme positive • DNA’ase enzyme positive • Resistant to ampicillin by beta lactamase enzyme production – Cefinase test can be used • Therapy: Augmentin or 2nd or 3rd generation Cephalosporin
  • 46.
  • 47.
    Corynebacterium •Over 20 species,most not pathogenic •Gamma hemolytic gray colonies •Catalase + •Diphtheroid morphology – Gram positive Chinese letter forms •No spores produced
  • 48.
    Corynebacterium diphtheriae• Agent ofDiphtheria • Diphtheritic adherent pseudo membrane produced in throat • Phage mediated exotoxin is distributed from the membrane causing respiratory paralysis (virulence factor) • Toxin detected by Elek immuno-diffusion test • Grows well on 5% Sheep’s BAP • Selective medium Cysteine Tellurite agar • Produces black colonies with brown halos • Grow on egg containing Loeffler medium then stain with methylene blue to observe metachromatic granules - colorful storage granules characteristic of C. diphtheria Elek plate Tellurite agar Metachromatic granules
  • 49.
    Other Corynebacterium • Corynebacteriumjeikeium – • normal skin flora bacteria / thrives on lipid • Infects patients with plastic catheters and indwelling devices by tunneling into the device from the skin • Biofilms are formed on the plastic, protecting the organism from antibiotic therapy • Very resistant to most antibiotics • Susceptible to vancomycin and tetracycline only • Corynebacterium urealyticum – • Rapidly urease positive diphtheroid • Urinary tract infection in post renal transplants • Resistant to many antibiotics – vancomycin susceptible Red is (+) for Urease reaction
  • 50.
    Bacillus species •Large grampositive rods with square ends - boxcar •Can over-decolorize easily and appear reddish •Spores produced – causes clearing in rod •Catalase enzyme positive •Most are motile spores
  • 51.
    Bacillus anthracis • Anthrax– Category A agent – highest threat to public health and bioterrorism • Natural infection of herbivores – infected animals can have fatal infection and contaminate the environment for years • Virulence factors: anthrax toxin and capsular polypeptide • Skin is the most common presentation -Woolsorter’s disease – acquired from handling imported hides • Unique black eschar skin lesions • Pneumonia, sepsis, and meningitis uncommon
  • 52.
    Bacillus • Bacillus anthracisculture: • Medusa head colonies on BAP No hemolysis on 5% Sheep’s blood agar Non-motile Penicillin susceptible With these reactions – Alert public health department for assistance • Bacillus cereus – • Food poisoning with rapid onset (1-6 hr) vomiting • Preformed emetic toxin in food • Fried rice is one of the most common food sources • Traumatic wound infection from contaminated soil • Beta hemolytic colony on Sheep’s blood agar • Motile
  • 53.
    Listeria monocytogenes • Smallgram positive rod/ no spores • Catalase positive* • Subtle beta hemolysis on Blood agar • Tumbling motility on wet mount • Umbrella motility in tube media • More motile at 25˚C than 35˚C • Cold loving – Grows well at 4˚C and reason why it is abundant in refrigerated foods like dairy and deli case meats • Infections: Bacteremia pregnant & still births • Infections: Bacteremia and CNS in immune suppressed • Ampicillin / intrinsic resistance to Cephalosporins
  • 54.
    Erysipelothrix rhusiopathiae • Smallgram positive rod • Catalase enzyme negative • Alpha hemolysis on blood agar • Only G+R that produces hydrogen sulfide (H2S) in a TSI agar slant –Human infections acquired from swine with erysipelas or poultry Infections most common in butchers –Bacteremia uncommon • most common in drug addict • High % endocarditis /extensive damage –Intrinsic resistance to Vancomycin H2S production on Triple sugar iron agar (TSI) Erysipelas – lacy skin lesions of swine
  • 55.
    Gram negative bacilli Enterics– ferment Glucose Non fermenters – do not ferment Glucose Fastidious
  • 56.
    Gram negative bacillus MacConkeyagar Lactose fermenter Lactose non fermenter Gram stain enteric bacilli – plump No lactose fermentation Lactose fermentation
  • 57.
    Enteric GNRs /ferment lactose • Escherichia coli • Major aerobic NF in intestine • #1 cause of UTI [@80%] • Bacteremia, neonatal meningitis • Abdominal infections Biochemicals: • Spot indole reaction = positive • Green sheen produced on EMB agar (Eosin methylene blue) • Pathogen of diarrhea • Enterotoxigenic E. coli – Traveler’s diarrhea • Enterohemorrhagic E. coli [0157:H7] • Bloody diarrhea usually acquired from eating undercooked cow meat from an infected cow • HUS – hemolytic uremic syndrome [hemolytic anemia, thrombocytopenia, and renal failure] particularly in children • Does not ferment sorbitol – most other E. coli types ferment Sorbitol Green sheen on EMB agar Mucoid colony Indole positive Robin’s egg blue
  • 58.
    Enteric GNRs thatferment Lactose•Enterobacter species • Enterobacter cloacae and E. aerogenes most common • Environmental organisms with low pathogenicity • Enterobacter (Cronobacter) sakazakii associated with neonatal meningitis •Klebsiella species – • K. pneumoniae most common • Mucoid colony • Currant jelly sputum in alcoholics due to blood mixed with capsular polysaccharide in sputum
  • 59.
    Enterics that doNOT ferment lactose! •Proteus species Colonies swarm in layers on agar surface • Proteus vulgaris – indole positive • Proteus mirabilis – indole negative - Common in UTI, abdominal infections •Serratia marcescens • Produces red pigmentation - intensifies at room temp • Causes infection in • Immune suppressed • Ventilator associated pneumonia • Bacteremia
  • 60.
    Glu/lac/suc fermented with gas Glucose fermented only Glucose fermented with H2S NoCHO fermentation Non fermenter Triple Sugar Iron Agar – Media for GNR to detect fermentation of glucose, lactose and/or Sucrose and production of hydrogen sulfide [H2S] Fermentation= yellow Gas = Disruption of the agar H2S No fermentation = red
  • 61.
    Salmonella species • Salmonellaspecies • Diarrhea with +/- fever – polys in the stool • Infection from food –Eggs, meats and contaminated uncooked vegetables, –must ingest large #’s of organisms to make you ill (1,00,000 bacteria), stomach acid is protective • Does not ferment lactose/ produces Hydrogen sulfide • Selective agars for growth: SS and Hektoen • Identification based on biochemical reactions and serologic typing • Kaufman White serologic typing for speciation of Salmonella • O Somatic (cell wall) antigen – Salmonella group “B” • H flagellar antigens – 2 phases [h1 & h2] • Vi capsular antigen – S. typhi only
  • 62.
    Salmonella Shigella Agar (SSagar) Salmonella and Shigella are colorless due to lactose not being fermented – H2S produced by Salmonella Hektoen agar – Salmonella produces H2S [Hydrogen sulfide] producing black colonies Shigella – green colonies Normal flora – orange colored due to fermentation of lactose (E. coli)
  • 63.
    Salmonella typhi • Typhoidfever – fever, sepsis, high fatality rate • Vi capsular antigen - surrounds the D cell wall antigen • Boil solution of S. typhi for 15 minutes to destroy the Vi capsular antigen and expose the cell wall D antigen • Serotyping can then be performed for the D antigen • Moustache of H2S produced in TSI tube • Carrier state post typhus in gallbladder • Ingested organism enters the bowel then in @ 1 week moves on to the Blood stream & Bone Marrow –
  • 64.
    Shigella • Diarrhea, +/-vomiting,fluid loss, polys and blood in stool , tenesmus, usually treated with Quinolones • Infection : Human to human transmission /control with good hygiene • Low #’s of organisms to make you ill [10 – 100 bacteria] • Non motile and No H2S produced ( – Green colonies on Hektoen agar • Does not ferment lactose • 4 species based on somatic antigen • S. boydii Group C • S. dysenteriae Group A • S. flexneri Group B • S. sonnei Group D
  • 65.
    Yersinia enterocolitica •Major reservoir– swine •Humans infected by drinking non pasteurized milk •Infections: • Diarrhea • Septicemia in iron overload syndromes • Mesenteric adenitis – RLQ pain which mimics appendicitis • Infected blood products from transfusion •Grows well at 4 °C (like Listeria) •CIN agar (Cefsulodin-irgasan-novobiocin) is selective agar to grow Y. enterocolitica
  • 66.
    Yersinia pestis -Plague • Category A agent • Obligate flea/ rodent/ flea cycle in nature • In human, a rat flea bite - leads to Bubonic plague, infection of the lymphatic system, painful buboes (lymph node swelling) at site of the bite • Hemorrhagic lymph nodes spread to blood • Pneumonic form from blood stream infection • Fatality >=50% • Endemic in Southwestern USA • Grows on blood agar • catalase +, oxidase - • Bipolar staining “safety pin”
  • 67.
    Glucose Fermenters Oxidase Positive •Vibriocholera • Natural environment is saltwater • Halophilic (salt loving) – enhances growth • Rice water diarrheal stool from mucus flecks • Virulence due to enterotoxin production – –Receptor on epithelial cell in small bowel – –activates adenyl cyclase – –increases cAMP with hyper secretion of NaCl and H20 – death from dehydration and metabolic acidosis • Curved “C” shape Gram negative rod • Selective media – thio citrate bile sucrose agar, yellow color due to sucrose fermentation TCBS Agar
  • 68.
    Other Vibrio species •Vibrioparahaemolyticus • Diarrhea from ingestion of raw /oysters • Usually self limited, worse in immune suppressed • TCBS medium = green = sucrose fermentation negative •Vibrio vulnificus • Ingestion of shellfish raised in coastal waters • Infections: • Diarrhea • Skin infection from injury exposed to coastal waters • Bacteremia - leads to formation of painful skin lesions on lower extremities with muscle necrosis / This form in patients with pre liver disease – increased serum iron - 50% fatality rate
  • 69.
    Classic Gram NegativeRod Gram Stains Campylobacte r Vibrio species Enteric gram negative rod Sea gull wings C shaped Plumb and rectangular
  • 70.
    GNRs - Donot ferment Glucose Oxidase negative • Acinetobacter species – isolated from hospital environment and human skin, large gram negative cocco-bacilli • Ac. baumannii a major nosocomial pathogen • Glucose oxidizer • Acquires antibiotic resistance with pressure • Ac. lwoffi – • glucose non oxidizer • Stenotrophomonas maltophilia • Rapid maltose oxidizer • Long Gram negative bacillus • Naturally resistant to many antibiotics • Gun metal gray pigment • Nosocomial pathogen – super-colonizer when on long term Imipenem therapy due to natural resistance to Imipenem
  • 71.
    Glucose non-fermenter Oxidase Positive Pseudomonasaeruginosa • Fluorescent & blue-green pigment (pyocyanin) • Grape-like odor • Growth at 42˚C • Ps fluorescens/putida group– no growth at 42°C • Major pathogen of cystic fibrosis • Mucoid strains [polysaccharide capsule] • In combination with Burkholderia cepacia can cause major lung damage • Nosocomial pathogen – associated with water & moisture • Intrinsically resistant to many antibiotics
  • 72.
    Glucose Non Fermenters/oxidase + •Burkholderia cepacia • Low virulence – found in the environment • Problem organism in cystic fibrosis: infection causes extensive lung damage • Dry , yellow colony • Oxidase positive • Esculin positive • Chryseobacterium (Elizabethkingia) meningosepticum • Infections: • Newborns: fatal meningitis and septicemia in the newborn • Elderly/immune suppressed: bacteremia • Low virulence • Environmental source – water • Yellow colony, oxidase and indole positive
  • 73.
    Haemophilus influenzae • Varietyof infections • Transmission – close contact/secretions • Virulence factor – capsular polysaccharide • Small pleomorphic Gram negative rod • Requires 2 nutritional factors for growth: • X = hemin • V= NAD (nicotinamide adenine dinucleotide) • Grows on chocolate agar (has X and V factor) • Will not grow on 5% sheep’s blood agar • Requires high level C0₂ [5 – 8%] for growth • Effective vaccine targets the H. influenza type B (HIB) – effectively eliminated most childhood invasive infections • Resistance to Ampicillin due to beta lactamase enzyme [20 %], 3rd gen Cephalosporin becomes the antibiotic of choice
  • 74.
    Test for BetalactamaseTest for Beta lactamase DetectionDetection • Add bacteria to filter paper impregnated with Nitrocefin orAdd bacteria to filter paper impregnated with Nitrocefin or Cefinase (yellow colored/chromogenic cephalosporin)Cefinase (yellow colored/chromogenic cephalosporin) • Incubate at room temp for @ 1 minuteIncubate at room temp for @ 1 minute • Positive result is a color change fromPositive result is a color change from yellow to redyellow to red –– • Beta lactamase enzyme breaks down beta lactam ring ofBeta lactamase enzyme breaks down beta lactam ring of Nitrocefin to a hydrolyzed red end productNitrocefin to a hydrolyzed red end product • Detects resistance to Amp/Pen/1Detects resistance to Amp/Pen/1StSt gen Cephalosporin ingen Cephalosporin in Haemophilus species, N. gonorrhea , Moraxella catarrhalis,Haemophilus species, N. gonorrhea , Moraxella catarrhalis, and anaerobic Gram negative rodsand anaerobic Gram negative rods • Does NOT detect the ESBL enzyme of enteric GNRsDoes NOT detect the ESBL enzyme of enteric GNRs
  • 75.
    Satellite phenomena Small pleomorphicGNR On Gram stain Growth on Chocolate agar Growth between X and V strips Staph aureus supplies the X and V factors required
  • 76.
    More Haemophilus species •H. parainfluenza – • requires V (NAD) factor only • Usually normal flora in the upper respiratory tract • However, one of the HACEK organisms of endocarditis • H. (Aggregatibacter) aphrophilus – (new taxonomy) • No X and V factor requirements for growth • Infections: abscesses (liver, lung, brain) & endocarditis • H. ducreyi – • Requires X factor • Cause of Chancroid - venereal disease • “school of fish” appearance on stains
  • 77.
    HACEK group • Oralflora organism that due to poor detention or dental procedures become agents of endocarditis • Fastidious Gram negative coccobacilli • 5 -10% of community acquired native valve endocarditis unrelated to IV drug use • Require 2-4 days to grow in patient blood cultures • Haemophilus species = oxidase (-) catalase (-) • Aggregatibacter (Actinobacillus) oxidase (-) catalase (+) • Cardiobacterium hominis = oxidase (+) • Eikinella corrodens = oxidase (+), colony pits BAP • Kingella kingii = oxidase (+), hemolytic on BAP, also cause of septic joint infection in small children
  • 78.
    Bordetella pertussis •Whooping cough–three stages of disease – (1) Prodromal – flu like disease – most contagious stage – (2) Catarrhal - cough - with classic whoop in small children – Pertussis Toxin adheres to bronchial epithelial cells and cough continues until toxin wears off – can be months – (3) Paroxysmal - recovery phase • Tiny Gram negative coccobacillus • Human pathogen - Inhabits Nasopharynx • Peripheral blood - Lymphocytosis with atypical large, irregular and deeply basophilic lymphocytes • Charcoal media for transport and culture – Regan Lowe Charcoal agar / growth in 3-5days • PCR more sensitive than culture for diagnosis • Reservoir for infection – young adults due to waning immunity. Reason behind initiative for booster shots in young adults
  • 79.
    Pasteurella multocida/canis • Bitewound infections from cats and dogs • Human pneumonia from close cat and dog contact • Infections can disseminate to blood stream • Normal flora in many animals (zoonotic) • Small Gram negative coccobacilli • Growth on 5% Sheep’s blood agar • Non hemolytic grey colony • Will not grow on MacConkey agar (big clue) • Oxidase Positive • One of very few GNR genera that is sensitive to penicillin
  • 80.
    Capnocytophaga species • Fusiformshaped Gram negative rods – very pleomorphic • Fingerlike projections from colonies “Gliding” • Dependence on CO2 incubation for growth • Oxidase negative, Catalase negative • Normal mouth flora in humans and animals • C. canimorsus – Associated with dog bites – high % of these bite infections lead to bacteremia and endocarditis • Capnocytophaga species (many) in human • Infect mouth ulcers induced by chemotherapy • Bacteremia
  • 81.
    Brucella species • Disease– Brucellosis, fever of unknown origin, significant joint pain, intracellular pathogen of the RES system • Specimens: Blood and bone marrow (granuloma formation) • Serology • Small Gram negative coccobacilli – difficult/slow to grow in culture • Old: Castaneda biphasic blood culture held for 21 days • Current: Automated Blood culture systems with growth @ 5 -6 days • Laboratory acquired Brucellosis is common • Zoonosis – ingestion of raw milk, animal exposure, inhalation • Brucella species dependent upon animal species the patient was exposed: • B. abortus – raw cow milk • B. melitensis – raw goat milk, feta cheese • B. suis – pigs • B. canis - dogs Granuloma in bone marrow
  • 82.
    Campylobacter spp. • “Curved”Gram negative rod • C. jejuni – Diarrhea, one of most common causes in US, bacteremia in HIV and immune suppressed • Ingestion of undercooked poultry / juice contaminating raw food • Culture requires selective blood agar with antibiotics / Campy-BAP, Skirrows BAP • Incubate at 42˚C in microaerophilic atmosphere (high CO₂, low O₂) • Sequelae - Significant % Guillain-Barre syndrome • C fetus – Bacteremia in the immune suppressed host. Source cattle and sheep. • Temperature tolerance aids in identification C. jejuni – grows at 37˚C and 42˚, hippurate hydrolysis positive C. fetus - grows at 37˚C and 25˚C hippurate hydrolysis negative
  • 83.
    • Reservoir –rabbits, rodents, ticks and flies. Humans infected by insect bites or directly from exposure to animal blood • Associated with skinning rabbits with bare hands • Bacteria can penetrate small breaks in skin: • cause painful skin lesions – • enlarged lymph nodes – • leading to bacteremia (ulceroglandular tularemia) • Pneumonia • Great hazard to lab workers by aerolization • Fastidious small gram negative cocco-bacillus • Culture media requires cysteine* Francisella tularensis
  • 84.
    Helicobacter pylori • Smallcurved Gram negative bacilli • Acute gastritis – can progress to gastric adenocarcinoma • Human to human transmission/ fecal - oral route • Rapid and strong urease enzyme – used for detection directly from gastric antrum biopsy tissue • Difficult to grow in culture • Stool antigen/diagnosis and test of cure • Serum antibody for past exposure • Organism stained by silver stains in GI • Treatment – Antibiotics and acid suppression Histologic exam of gastric biopsy Hematoxylin-eosin stain or Warthin-Starry Silver stain
  • 85.
    Legionella • Legionella pneumophila[6 serotypes] • Pulmonary disease – strong association with water • Requires cysteine in culture medium for growth • Buffered Charcoal Yeast Extract agar/ 3-5 days • Will not show on Gram stain of specimens • Use silver impregnation stains in tissue • Urinary antigen test detects L. pneumophila type I infection – the most common cause of infection • Treatment: Erythromycin (macrolide) BCY E
  • 86.
    Bacteria without cellwalls • Mycoplasma spp and Ureaplasma spp – cell membranes only! Media contain sterols to protect the membrane • Lack of peptidoglycan cell wall – means unable to Gram stain, no colonies produced on agar, cannot be treated by antibiotics that act by inhibiting cell wall formation • M. pneumoniae – • Community acquired pneumonia • PCR for diagnosis • Presence of cold agglutinins • Genital mycoplasmas • M. hominis – fried egg on agar surface, vaginitis, cervicitis, postpartum sepsis, neonatal infections , pre rupture of membranes • Ureaplasma urealyticum – Dark metal-type appearance, rapid urea hydrolysis in broth, NGU & upper genital tract infection, spontaneous abortion, neonatal infections • PCR current method for diagnosis UreaplasmaMycoplasma hominis
  • 87.
    Unusual and difficultto grow • Bartonella henselae • Cat scratch disease – exposure to cat and cat excrement • Bacillary angiomatosis – vascular skin lesion +/- invasion / HIV • Bartonella quintana – cause of trench fever/ vector is the body louse • Diagnose Bartonella by Serology/PCR • Chlamydia trachomatis Serovars L1,L2,& L3 Lymphogranuloma venereum lymphatics and lymph nodes involved • Chlamydia pneumoniae (TWAR agent)- Pneumonia • Chlamydia psittaci- psittacosis, pneumonia, exotic parrot exposure • Diagnosis: Serology and PCR assays
  • 88.
    Unusual and difficultto grow •Ehrlichiosis – Rickettsia bacteria • Zoonotic intracellular pathogen • Vector – Ixodes tick *(hard tick) • 2 Genera cause Ehrlichiosis • Anaplasma spp, inclusion in the PMN • Ehrlichia spp inclusion in the Monocytes • Fever, leukopenia, thrombocytopenia, • Elevated serum aminotransferases, • No rash (differs Rocky Mountain Spotted Fever) •Found in south central, southeast , midwest US •PCR, serology, and exam of blood smear for diagnosis
  • 89.
    Spirochetes • Borrelia burgdorferi- Lyme’s disease • Primarily found in NE part of US • Vector = Ixodes tick • Acute disease: Fatigue, headache, fever, and rash • Can have progression to chronic disease • Diagnosis: Serology and PCR • Borrelia recurrentis - Relapsing fever • High fever (relapsing) with thrombocytopenia • Vector: body louse • Diagnosis: Blood smear observe spirochete
  • 90.
    Spirochetes • Treponema pallidum •Syphilis • RPR and VDRL for antibody detection • Molecular methods • Brachyspira – • intestinal spirochete found on the brush border of the intestine, • ?? Role in disease • Leptospira interrogans – Leptospirosis • Fever with rash and renal involvement • Urine from rats and other animals contaminate water supplies Darkfield from chancre lesion (1) Leptospirosis – Shepherd’s crook (2) Presence of spirochete in a renal tubule (1) (2)
  • 91.
    Unusual Bacterial diseases •Granulomainguinale • Klebsiella (Calymmatobacterium) granulomatis • Rare STD – causes ulcerative genital lesions •Streptobacillus moniliformis • Rat bite fever or Haverhill fever • Infection from rat bite • L form - cell wall deficient bacteria • Inhibited by SPS in blood culture media • Needs serum supplementation to grow, will not grow on routine bacteriology media • Detailed history aids diagnosis
  • 92.
    Bacterial vaginosis •A mixedanaerobic/aerobic bacterial infection • The normal balance of vaginal flora is disrupted • Rather benign infection except in pregnancy • Discharge: Fishy like odor of discharge, alkaline pH >=4.5 • Usual NF organism of Lactobacillus are overgrown by: • Gardnerella vaginalis (aerobic Gram variable rod) • Mobiluncus (anaerobic curved Gram negative rod) • Increased #s Gardnerella vaginalis is the bacterial marker for infection – Human blood agar media of choice, Beta hemolytic, Gram variable staining • Clue Cells are diagnostic and more specific than growth in culture • Molecular probe assays and amplification assays available as part of “women’s health” screening panels Clue Cell
  • 93.
    • Anaerobic infectionscan occur in virtually every any organ or region of the body • Polymicrobial –with both aerobic and anaerobic species • Endogenous organisms / usually normal flora • Increase in numbers and cause infection due to trauma, vascular or tissue necrosis cutting off the oxygen supply to the involved tissue • Treatment: Usually surgery to restore oxygen and remove necrotic tissue plus antibiotics • Anaerobic culture collection • Gel containing swabs • ESwab • Evacuated vials (port o cult)/ oxygen free • Do not refrigerate specimens – absorb oxygen in cold temperatures and can more rapidly kill anaerobes Anaerobic Bacteria
  • 94.
    Anaerobic culturing • PRASmedia – pre reduced anaerobically sterile • Media packaged in oxygen free environment • Most common media used • CDC anaerobic enriched blood agar • Kanamycin-vancomycin blood agar • Bile esculin agar • Thioglycollate broth • Chopped meat glucose broth • Anaerobic chambers – perform all culture work in an oxygen free environment • Anaerobic gas pack jars – for anaerobic incubation of agar plates • Wet pack – add 10 ml water to hydrogen and CO2 generating envelope/ requires palladium coated catalysts – generates heat • Dry pack – (Anaeropack) Absorbs O2 and generates CO2 PRAS
  • 95.
    Anaerobic Gram Negative Rods Unusualshapes and sometimes poorly staining by Gram
  • 96.
    Bacteroides fragilis group Pleomorphicirregular staining Gram negative rod •Grows in the presence of bile •Growth on Bile esculin media is black •Resistant to Penicillin and Kanamycin •Normal flora in GI tract •Infections: Related to the bowel/ GI abscess • foul smelling/gas produced •B. fragilis group • B. fragilis – most common species • B. ovatus • B. thetaiotamicron ( indole reaction positive) • B. uniformis • B. vulgatus • Resistant to Penicillin by beta lactamase enzyme • Metronidazole is antibiotic of choice Growth on bile/esculin media Black pigment from Esculin production
  • 97.
    Prevotella and Prophyromonas –PleomorphicGram negative rod –Normal flora in the upper respiratory tract –Infections: respiratory tract abscesses –Will not grow in the presence of bile –Will not turn black on esculin media –Unique** Brick red fluorescence and black pigment formed on Blood agar after one week of incubation
  • 98.
    Fusobacterium spp. • F.nucleatum Long thin gram negative bacilli – spindle shaped, pointed ends • Normal flora upper respiratory tract • Infections: mouth and respiratory tract abscess formation and liver abscess • Vincent’s angina – necrotizing oral infection caused by Fusobacterium species and spirochetes • F. necrophorum – pleomorphic gram negative bacilli that form filaments or chains of rods • Lemierre’s syndrome - oropharyngeal infection, leads to thrombosis in jugular vein, septicemia, with high fatality rate
  • 99.
  • 100.
    Clostridium species • Grampositive bacilli (boxcar shaped ) form spores • Bacilli may over-decolorize and appear red • Clostridium perfringens • Infections: Food poisoining, necrotic tissue abscesses (Clostridial myonecrosis), bacteremia, cholecystitis • Most common anaerobic G+R in intestine • Double zone of beta hemolysis on BAP • Lecithinase produced on egg yolk agar • Reverse camp test positive Lecithinase Reverse Camp Test
  • 101.
    • Clostridium botulinum– Botulism –Adult disease – Preformed heat labile neurotoxin ingested is mass produced or in home-canned foods –Infant disease - spore ingested from nature or product produced in nature such as honey or household dust, neurotoxin produced in gut • Begins with constipation and difficult sucking bottle –Both forms are ife threatening neuroparalytic disease • Clostridium tetani - Tetanus • Gram stained cells appear like Tennis racket • Tetanospasmin toxin enters by penetrating skin injury • Spastic contractions of voluntary muscles, hyper-reflexia, lock jaw (trismus) • Immunization to prevent
  • 102.
    •C. septicum – •Bacteremia or Gas Gangrene in patient with underlying malignancy • Hematogenous spread from GI tract – no trauma necessary •C. difficile – • Disease: antibiotic associated colitis, pseudomembranous colitis from toxin production • Toxin A – enterotoxin causing fluid accumulation • Toxin B – potent cell cytotoxin – primary virulence • Binary toxin – so called Nap1 strain is produces larger amount of toxin • Diagnosis of infection: • EIA methods [toxin A/B] are insensitive but detect active toxin • PCR methods [toxin A/B] are more sensitive, detect toxin genes • Culture – Cycloserine, Cefoxitin, Fructose Agar [CCFA] • Infants have @ 70% colonization rate – do not test
  • 103.
    Actinomyces •Branching gram positivebacilli - do not form spores • Aerotolerant, but grow best anaerobic •Normal flora oral, GI, vagina, skin •Infections: usu. oral/facial (lumpy jaw) •Form sulfur granules in tissue • Can be found on normal tonsil •Actinomyces israelii – associated with oral, thoracic, and abdominal infections, IUD infections Bread crumb colonies in broth Molar tooth colony on agar plates Penicillin susceptible.
  • 104.
    Branching Gram positive rodsof Actinomyces – antler like Molar tooth colony Sulfur granule Clumps of Actionomyces
  • 105.
    Propionibacterium acnes • PleomorphicGram positive rod • Catalase positive, Indole positive • Normal flora - skin, oral, GU and GI • Contaminate in blood cultures from skin NF • Pathogen of acne vulgaris • Opportunistic pathogen: cerebral shunt infections and other endovascular and neurosurgical infections • Firmly established as significant cause of prosthetic joint infection – particularly shoulder joints • Cultures should be held up 7- 14 days • Therapy - Ampicillin