REVIEW of Clinical Microbiology - PowerPoint Presentation

1. REVIEW of Clinical MicrobiologyPart IIMJ Weintraub, DVM

2. Cell wall/envelope structure GN, GP, AF & wall-lessGenome Morphology, arrangements, capsule/slime layer, flagella, fimbriae, pilusHorizontal gene transfer mechanisms70s ribosome1 Circular, double stranded DNA chromosome/genomePlasmids: extrachromosomal, circular, dsDNA, self-replicating Prokaryotes Bacteria

3. Eukaryotes Fungi, Protozoa & Helminths Organelles80s ribosomeGenome in nucleusProtozoaflagella, cilia, pseudopods or non-motileSome cysts, trophozoitesFungiDimorphismChitin in cell wall Ergosterols plasma membranesSome capsulesHelminthsDevelopmental stages if any

4. Viruses & PrionsVirusesIntracellular obligate parasitesAcellular agentRNA or DNA genomesCapsid &/or envelopeBacteriophages PrionsInfectious pieces of protein

5. Common Stains - BacteriaGram Understand technical errorsGN & GPEndospores – clear within cellAcid FastAF – red & non-AF – blueZiehl Neelsen (heat)Kinyoun (cold-detergent)Acridine Orange - fluochromeBacteria difficult id in a specimen – blood

6. COCCISingle or pairs (diplococci) Chains (streptococci) Clusters (staphylococci) tetradsBACILLI vary size & length Ends may be square or rounded Long & filamentous Coccobacilli -very short Fusiform -tapered or pointed ends Curved Pleomorphic -species varies shape within pure culture Single or chains or palisades SPIROCHETES -helical turns Vary in length & number

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8. Common Stains - BacteriaMethylene BlueMetachromatic granules Corynebacterium diphtheriaeAF counterstainEndospore Red/pink endospore green-living cell

9. Common Stains - FungiLactophenol Cotton Blue (LCB)Medically important fungiCalcofluor White – fluorochromeFungi – bright apple-green or blue-whiteIndia inkCryptococcus neoformans capsule

10. Types of Growth Media Minimal media Contents are simple Not usually used in clinical microbiology lab Nutrient media Complex and made of extracts of meat or soy beans Ex. Nutrient broth, Trypticase Soy Broth, TSA agar Enriched media Contains added growth factors, such as blood, vitamins, and yeast extract Ex. Blood agar & Chocolate agar

11. Types of MediaSelective media Contains additives that inhibit growth of some bacteria but supports others to grow MacConkey, CNA, Mannitol SaltDifferential media See metabolic differences between groups or species of bacteria MacConkey, Mannitol Salt Transport medium Holding medium Preserves viability of microorganisms in specimen but does not allow multiplication Ex. Stuart broth, Cary-Blair

12. MediaCategoryUsed ForSheep Blood AgarEnriched Good for fastidious organisms, most organisms will grow, interpret hemolysisChocolate Very Enriched Very Enriched Good for highly fastidious organisms, Phenylethyl Alcohol Agar Selective Selective for GPC Haemophilus Maconkey AgarSelective & DifferentialSelective for GN, differential LF and NLF (Enterobacteriaceae & oth GNRs)Maconkey-Sorbitol Agar (SMAC)Selective & DifferentialE. coli O157:H7, differential for sorbitol fermentationEosin-Methylene Blue Agar(EMB)Selective & DifferentialSelective for GNRs, differential for lactose & sucrose fermentation(Enterobacteriaceae & oth GNRs)Hektoen Enteric Agar (HE)Highly Selective & DifferentialSelective for enteric pathogens, differential for sucrose & lactose fermentationH2S Enhances growth of Salmonella & ShigellaXylose-Lysine Desoxycholate Agar(XLD)Highly Selective & DifferentialSelective & GNR & enhances growth of Shigelladifferential for sucrose, lactose & xylose fermentationH2S & lysine (decarboxylation)Used to ID Salmonella & Shigella

13. MediaCategoryUsed ForColistin & Nalidixic Acid Blood Agar (Columbia CNA)SelectiveSelective for GPC, read hemolysisPhenylethyl Alcohol agarSelective Selective for GPCModified Thayer MartinSelective & EnrichedNeisseria gonorrhoeae (vancomycin, colistin, nystatin, thrimethoprim)Martin LewisSelective & EnrichedNeisseria gonorrhoeae (vancomycin, colistin, nystatin, thrimethoprim)New York City agar (NYC)Selective & EnrichedNeisseria gonorrhoeae, Ureaplasma urealyticum & Mycoplasma hominisCampylobacter Blood Agar (CAMPY)Selective & EnrichedCampylobacter spp.Cefsulodin-Irgasan-Novobiocin (CIN)SelectiveYersinia enterocoliticaThiosulfate Citrate Bile Sucrose Salts (TCBS)Selective & DifferentialVibrio spp. Differentiates Vibrio spp. based on sucrose fermentation

14. MediaCategoryUsed ForBuffered Charcoal-Yeast Extract Agar (BCYE)Enriched Legionella spp. Fletcher Semisolid Medium Enriched Leptospira spp. Loeffler Coagulated Serum SlantEnriched Clostridium diphtheriae Tellurite Blood Agar with or/without Cystine Selective, differential & enrichedCorynebacterium diphtheriae Tinsdale AgarSelective and differentialClostridium diphtheriae Bordet-Gengou Blood Agar Selective and enriched Bordetella pertussis and B. parapertussis Reagan-Lowe Medium Selective Bordetella pertussis Mannitol Salt Agar Selective and differentialSelective for staphylococci and differential for mannitol fermentationBile Esculin AgarSelective and differentialSelective for Group D Strep and Entercoccus spp.

15. MediaCategoryUsed ForBlood Agar – AnaerobeEnrichedFastidious anaerobic bacteriaKanamycin & Vancomycin Laked Blood Agar (KVLB)Selective & EnrichedBacteroides spp. & Prevotella spp.Bacteroides Bile Esculin agar (BBE)Selective & DifferentialBacteroides fragilis groupPhenylethyl Alcohol agarAnaerobic Selective & EnrichedAnaerobes Egg Yolk agar (EYA)Differential Clostridium spp. Lecithinase, lipase & protease activityCylcoserine Cefoxitin Fructose agar (CCFA)Selective & DifferentialClostridium difficileThioglycolate Broth (THIO)All purpose mediaBack-up brothTodd Hewitt Broth & LIM brothSelectiveGroup B streptococciChopped Meat BrothCultivation of anaerobesSelenite BrothEnrichmentSalmonella spp. & Shigella spp.

16. JEMBEC transport systemNeisseria gonorrhoeaeSodium bicarbonate & sodium citrateInhibition of contaminating bacteria and fungi

17. Environmental FactorspH Most pathogenic bacteria grow best at a neutral pH media 7.0 – 7.5 Temperature Categories Psychrophiles – optimal growth 10 to 20O C Mesophiles – optimal growth 20 to 40O C Thermophiles – optimal growth 50 to 60O C

18. Atmospheric requirements Obligate aerobe require oxygen for growth Aerotolerant anaerobe can survive in presence of O2 but do not use O2 in metabolism Obligate anaerobe cannot grow in presence of oxygen Facultative anaerobe grow either with or without oxygen Most opportunistic pathogensCapnophilic Best at increased CO2 Capnophilic bacteria need 5% -10% CO2 Microaerophilic reduced level of oxygen to grow Ex. Campylobacter spp. – requires 5% to 6% O2

19. Fermentation & Respiration Bacteria use biochemical pathways to catabolize CH2O & produce energy Respiration (Oxidation) efficient energy-generating process Obligate aerobes & facultative anaerobes Fermentation Anaerobic process Obligate & facultative anaerobes Less efficient in energy generation

20. Fermentation vs. OxidationUsed in biochemical testing based on:presence mixture of end products accumulates in medium Changes in pHChange in pH – KIA, TSI, CTA CH2O, HEProducts of process: Methyl Red – mixed acids & VP - acetoinNOTE: if evaluating a CH2O & not glucose, no glucose should be in the mediaBacteria preferentially ferment glucose 1st

21. Lactose FermentationLF, NLF & sLFKey in ID of Enterobacteriaceae 1st Step ß-galactoside permease transports lactose across cell wall2nd Step ß-galactosidase breaks galactoside bond releases glucose all organisms that ferment lactose ferment glucose sLF fermenters lack or have a weak β galactoside permeaseONPG test to id sLF as does not require β galactoside permease

22. GPC

23. Staphylococcus spp.(+) Catalase Non-motile Non-spore-forming Most are facultative anaerobes • except S. saccharolyticus S. aureusS. epidermidisS. saprophyticusS. lugdunensisMore virulent, may have mecA gene

24. Staphylococcus spp.IsolationSBAThioglycollate brothSelective media □ Mannitol Salt agar □ Columbia Colistin Nalidixic Acid agar (CNA) □ Phenylethyl alcohol agar (PEA) General characteristics GPC, in clusters Colonies - creamy, white, golden & may be “buttery-looking” Some β hemolytic

25. COAGULASE TESTSSLIDE TESTCell-bound coagulase (Clumping Factor)enzyme that clots human, rabbit, or pig plasma If (+) reported as Staphylococcus aureusIf (-) the tube test must be doneTUBE TEST Free coagulase (staphylocoagulase) extracellular enzyme that causes clot to form when bacterial are incubated in plasma If (+) reported as Staphylococcus aureus 5% of S. aureus do not produce cell-bound coagulase (CF)If (-) reported as CoNS

26. Coagulase(+) Staphylococcus aureus(-) other staphylococci (CoNS)S. lugdunensis(+) Cellbound (CF) coagulase & (-) free (staphylocoagulase)Novobiocin on CoNS urine isolates(R) -Staphylococcus saprophyticus (S) other CoNSHemolysisβ: Staphylococcus aureusNo hemolysis: S. epidermidis & S. saprophyticus

27. Micrococcus spp.GPC, tetrads, irregular clusters(+) catalaseMicrococcus spp.Staphylococcus spp. Oxidase(+)(-)BacitracinSRLysostaphinRSCoagulase(-)varies

28. Staphylococcus aureus must perform antimicrobial susceptibility on all isolatesMost penicillin(R) – beta lactamases MRSA, HA-MRSA, CA-MRSAVancomycin VISA & VRSAMRSA KEY TESTOxacillin susceptibility but now Cefoxtin (better inducer of mecA-mediated resistance) is preferredGOLD STANDARDDetection mecA gene – same day tests: Nucleic Acid probes or PCRD TESTMacrolide resistanceInducible clindamycin resistance presence of erythromycin

29. Streptococcus spp.GPC, chains, pairs(-) catalaseMost facultative anaerobesStreptococcus spp.Enterococci spp.LANCEFIELD CLASSIFICATIONC carbohydrate on cell wallA, B, C, D, F, G & N – most common

30. LancefieldHemolysisTermS. pyogenes A ß, wide zone Group A strep S. agalactiae B ß – narrow zoneGroup B strep S.dysgalactiae, S. equi C ß Group C strep S. bovis group D α or none nonenterococcus Enterococcus faecalis & E. faecium D α, ß or none enterococcus S. pneumoniae No groupα pneumococcus Anginosus group, mutans group, mitis group, salivarius group A, C, F, G, N, or --­α, ß or none viridans strep

31. Susceptibility TestsBacitracinidentify Group A strep Low concentration 0.04 U selectively inhibits growth of Group A strep Sulfamethoxazole & Trimethoprim (SXT) used with bacitracin to improve accuracy Group A strepGroups A & B are resistant to SXT Optochin susceptibility used on α hemolytic strep ethylhydrocupreine hydrochloride Streptococcus pneumoniae is sensitive to optochin

32. Bile Solubility (sodium deoxycholate & sodium taurocholate) Correlates well with optochin susceptibility (+) Streptococcus pneumoniae in solution lyses & solution clears (-) viridans streptococci do not lyse Hippurate hydrolysis Differentiate Group B strep from other ß hemolytic strep (+) Streptococcus agalactiae (GBS) PYR hydrolysis test Substrate is L-pyrrolidonyl-ß-naphthylamide & L­-pyroglutamic acid-ß naphthylamide (+) Streptococcus pyogenes & Enterococcus spp.(-) other streptococcimore specific for Group A strep than bacitracin (+) Aerococcus spp. & Gemella spp

33. CAMP test CAMP factorPRES ID Group B strep Enhanced hemolysis ß-hemolytic Staphylococcus aureus & CAMP factor producing β- hemolytic or some non-hemolytic Group B strep(+) arrow-shaped hemolysis pattern

34. Bile Esculin Grow in presence of 40% bile Hydrolyze esculin – black complex (+) Group D streptococcus & Enterococcus spp. (-) viridans Strep6.5% sodium chloride broth (+) Enterococcus spp.(-) Group DLeucine AminoPeptidase test (LAP) (+) Streptococcus & Enterococcus spp. & Pediococcus spp. (-) Aerococcus spp. & Leuconostoc spp.Enterococcus faecalis

35. Susceptibility Testing Penicillin drug of choice for most streptococcal infections Penicillin resistant organisms  Some Streptococcus pneumoniae & viridans streptococci  Vancomycin Effective against GPC organismsVancomycin resistance now being encountered Enterococci & some streptococci are showing resistance

36. Streptococcus pyogenes (Group A) Pharyngitis & tonsillitis Scarlet fever, skin infections, and other septic infections Rheumatic fever, acute glomerulonephritis Laboratory Pinpoint colonies, ß hemolysis (+) PYR (S) Bacitracin (-) Hippurate hydrolysis(-) CAMP(-) Bile esculin (-) 6.5% NaCl

37. Streptococcus agalactiaeinvasive disease in newborns Elderly, immunodeficient & women who become ill after childbirth or abortion All pregnant women should be screened for GBS (group B Stept) at 35-37 weeks gestationLaboratoryGrayish white colonies, small zone of ß hemolysis (-) catalase (+) Hippurate hydrolysis (+) CAMP test

38. PRES ID of ß hemolytic streptococciß-hemolytic coloniesBacitracinSusceptibleResistantCAMPGroup BStreptococcus agalactiaePossibly group D or ß hemolytic; not group A, BGroup AStreptococcus pyogenes+-

39. Group D streptococci Streptococcus bovis group group D antigen endocarditis, UTI, abscesses, wound infections & associated bacteremia resulting from presence of gi tumors Penicillin(S) Group D (R) Enterococcus spp., usually Laboratory small, translucent colonies - α or no hemolysis (+) Bile Esculin (-) 6.5% NaCl (-) PYR

40. Enterococcus spp.Normal flora in intestinal tract Enterococcus faecalis – most common isolate Enterococcus faecium Group D antigen UTIs followed by bacteremia & endocarditis Show resistance to many antibiotics VRE – Vancomycin Resistant Enterococcus Laboratory Β or α or no hemolysisMay have pseudocatalase reaction (+) Bile Esculin (+) 6.5% NaCl (+) PYR

41. PRES ID of Group D strep & Enterococcusß, α, or no hemolysis in SBABile Esculin6.5% NaClNon-hemolytic streptococciEnterococcus spp.PYR (+)Group D streptococciPYR (-)+-+-

42. Streptococcus pneumoniae pneumococcusNormal RT floraPneumonia, sinusitis, otitis media, bacteremia, meningitis & 30% acute septicemiaQuellung reaction – capsule swelling allows ID of Streptococcus pneumoniae & specific serotypestreat with penicillin but some strains show resistance & are treated with erythromycin or chloramphenicol Vaccine – childhood & > 50 yrs.

43. Streptococcus pneumoniae Laboratory diagnosis GPC - lancet shaped & usually have capsuleSBA or Choc Some require increased CO2 Large zone of α hemolysis round, glistening, wet, mucoid, dome shaped & with time collapse of colony's center Optochin (S) S. pneumoniae(R) other α hemolytic streptococciBile solubility (+) S. pneumoniae(-) other α hemolytic strep

44.

45. viridans streptococci flora URT, female genital tract & gi tractS. anginosus group, S.mutans group, S. mitis group, S. salivarius group & S. bovis groupLow Virulence, opportunist Most common cause of subacute bacterial endocarditis gingivitis, dental caries, meningitis, abscesses, osteomyelitis & empyema  Penicillin but some resistant strains reported Laboratory SBA may show zone of α hemolysis (-) Bile solubility(R) optochin

46. Abiotrophia spp. & Granulicatella spp.Nutritionally Variant Streptococci (NVS)Satellite colonies @ other bacteriaBacteremia, endocarditis, otitis mediaShould be suspected in blood cultures  LaboratoryGPC resembling strep Do not grow on subculture SubcultureStaphyloccous aureus streak Satellitism

47. GNC

48. Neisseria spp.Aerobic, non-motile, non-spore-forming GNDC(+) Oxidase (+) CatalaseExcept for: N. elongata GNR (-) catalase N. weaveri GNR (+)catalase Capnophilic fastidious organisms, need enriched media & moisture for optimal recoveryNeisseria gonorrhoeae (true pathogen); N. meningitidis (commensal URT of carriers) & other Neisseria spp. (opportunistic pathogens)

49. Neisseria gonorrhoeae urogenital tract infections & conjunctivaDisseminated infections – joints, meningitisSpecimen: males–urethra; females–endocervix & rectum, pharynx & joint fluid Best chance for recovery is direct plating of specimen to gonococcal-selective media incubate at 35O C in 3-5% CO2 PCR assay N. gonorrhoeae vs. Chlamydia spp.

50. Neisseria gonorrhoeae Direct Microscopic exam urogenital specimens Not recommended for pharyngeal specimens Evidence of gonococcal infection GNDC intracellular from symptomatic male with discharge correlates at a rate of 95% with culture Females have vaginal commensal flora that resemble gonococci – culture is necessary Gram stain >5 PMNs/field but no bacteria may suggest Non-Gonococcal Urethritis (NGU)• NGU rule out: Chlamydia trachomatis & Ureaplasma urealyticum

51. Neisseria gonorrhoeae Requires chocolate agar (+) selective agentsThayer-Martin (TM) Vancomycin (GP) , Colistin (GN), Nystatin(fungi)• Modified Thayer-Martin (MTM) Vancomycin, Colistin, Nystatin, Trimethoprim (Proteus spp.)Martin Lewis (ML) Vancomycin,Colistin, Anisomycin (fungi), Trimethoprim New York City (NYC) Vancomycin, Colistin, Amphotericin B (fungi), Trimethoprim

52. Neisseria gonorrhoeae PRES IDSmall, gray/tan, transluscent raised colonies 24-48 hrs.Gram stain to verify GNDC kidney bean-shapedGNRs Kingella spp. & Acinetobacter spp. may grow on gonococcal media ,may look similar, to differentiate, inoculated onto plate with 10 unit penicillin disc, growth on edge of zone of incubation to evaluate morphology(+) Oxidase Must be done on all isolates Antimicrobial resistance All strains must be tested for ß-lactamase production Must be performed on primary culture plate as plasmid can be lost on subculture

53. Definitive Identification Other oxidase (+) GNDC, can be recovered from gonococcal selective media - Neisseria cinerea, N. meningitidis, Moraxella catarrhalis CH2O UtilizationCystine Trypticase Agar (CTA) (+) Glucose fermentation & (-) Maltose, sucrose & lactose fermentationChromogenic enzymesMultitestMatrix assisted Laser Desorption/Ionization Time of Flight (MALDI Tof)Non-cultureNucleic Acid Amplification Test (NAATs)Neisseria gonorrhoeae

54. Neisseria meningitidismeningococcusmeningitis, meningococcemia, rare pneumonia, purulent arthritis & endophthalmitis nasopharynx & oropharynx of 3-30% of asymptomatic individualsLaboratory CSF, blood, nasopharyngeal swabs & aspirates, sputum & urogenital sites Direct exam intracellular & extracellular GNDC & may have capsuleHelps to concentrate to specimen grows on gonococcal-selective agars, SBA & Chocsmall, gray, sometimes mucoid, convex colonies (+) Oxidase (+) Catalase CTA sugar:s (+) glucose & maltose fermentation & (-) sucrose & lactose

55.  Moraxella catarrhalisGNDC Normal commensal RT, opportunistic pathogen Pneumonia, sinusitis, otitis media & systemic disease Laboratory Grows on SBA & Chocolate smooth, opaque, gray to white colonies - “hockey puck” Usually inhibited by colistin in gonococcal selective media (+) Oxidase (+) Catalase Asaccharolytic in CH2O degradation tests •  (+) DNase (+) Butyrate esterase

56. Fastidious GNRHACEKHaemophilus spp. Aggregatibacter spp. (formerly Actinobacillus)Cardiobacterium hominisEikenella corrodensKingella spp.Capnocytophaga spp.Pasteurella spp.Brucella spp.Francisella spp.Legionella spp.Bordetella spp.

57. HACEK Group Characteristics GNR/CBCapnophilic Important cause of endocarditis flora of oral cavity – allows for introduction into blood streamHaemophilus spp.GNCB, pleomorphicMost nonpathogenic or opportunistic pathogensX factor-hemin & V factor (NAD) “para” only V factor H. influenzae H. parainfluenzae H. haemolyticus H. parahaemolyticus H. aphrophilus H. paraphrophilus H. aegyptius H. ducreyi

58. Haemophilus spp. Hemolysis on 5% horse’s or rabbit’s blood to differentiateSBA only X factor available V factor dependent do not grow on SBA Chocolate agarLysed rbcs by heat results in containing both X & V factors Satellitism Staphylococcus aureus, Streptococcus pneumoniae, Neisseria spp. produce V factor as a byproduct of metabolism

59. Haemophilus influenzaeMeningitis – decrease frequency due to vaccine (Hib)Epiglottitis • Haemophilus aegyptius - acute contagious conjunctivitis “pinkeye” • H. influenzae biogroup aegyptius -conjunctivitis • H. ducreyi - Chancroid – painful, softhighly communicable STI Genital Ulcer Disease (GUD) Laboratory Almost any specimen but commonly blood, CSF, middle-ear exudates, joint fluids, URT & LRT specimens, swabs conjunctival swabs, vaginal swabs, abscess drainage Prompt transportation & processing is critical

60. Haemophilus spp. Culture Does not grow on SBA, grows on Chocolate H. ducreyi Special media, hold for at least 7 days at 33O C H. aegyptius Special media, hold for at least 4 days Colony morphology On chocolate translucent, tannish, moist, smooth, convex colonies with “mousy” or “bleach-like” odor Microscopic morphology on direct smearGNCB to long filaments H. ducreyi “school of fish” formation H. influenzae – intra & extracellular

61. Haemophilus spp. 1st clue to a Haemophilus spp. No Growth (NG) on SBA, MAC or other enteric agars GNCB, pleomorphic on Chocolate agar X & V factor testPorphyrin test (+) Porphyrin heme producing will fluoresce(-) Porphyrin non-heme producing will not fluoresceMany strains produce ß-lactamase – rapid testsChromogenic cephalosporin test Acidometric test If (+) strain is resistant to ampicillin & amoxicillin

62. Aggregatibacter aphrophilusX-dependent & X-independent strains(-) catalasegranular, yellow with center opaque zone colonies on ChocAggregatibacter actinomycetemcomitans(+) catalaseBA “star” 4-6 points in center 100xCardiobacterium hominisGNR or GV – forms rosettesSlow grow SBA & CHOC – may see pitting

63. Eikenella corrodens flora mouth & giUsually polymicrobial infection, often trauma - human fights or bites GNCB(-) motility (+) oxidase (-) Catalaseasaccharolytic often produce yellow pigmented colonies@45% pit the agar (if see 1st think Eikenella spp.)Bleach-like odor NG on Mac or EMB

64. Kingella spp.Often follow viral infection or poor oral care or oral surgeryGN CB/short rods with squared ends, in pairs or short chains, tend resists decolorizationNutritionally fastidious“twitching” motility (no flagella)Capnocytophaga Spp.Resemble HACEK Normal flora mouth & oral cavities GNR - Thin & fusiformFastidious, pitting but fermentative May see gliding motility on agar surfaces Often yellow-orange pigment Isolated from blood from granulocytopenic patients who have oral ulcers, juvenile periodontal disease, and endocarditis

65. Pasteurella multocida Most common isolated Pasteurella spp.Zoonosis GNCB, pleomorphic - Bipolar staining Laboratory SBA nonhemolytic (+) oxidase(+) catalaseweak TSI (glucose fermentation weak acid production)(+) ODC(+) Indole(+) UreaseDoes not grow on Mac

66. Brucella spp. Zoonosis Brucellosis, Undulant fever, Mediterranean, Crimean & Malta Feversaerosol, percutaneous & oral routes of exposure remittent fevers, fluctuate regularly at consistent intervals & persist for days, months, or years B. melitensis most common cause of brucellosis Category B Select Biological AgentEasily disseminated & moderate morbidity but low mortalityBSL 3 lab – lab exposure results in 30%-100% acquiring infectionBlood & bone marrow cultures performed with acute & convalescent sera for serologic testing

67. Brucella spp. LaboratoryTiny, GNR/CB facultative intracellular pathogen in phagocytesGrow aerobically on Choc & BA in 18 hrs - hold 4 days (also TM or Martin Lewis)Smooth, raised & translucent colonies50% grow on MAC(-) motility(+)oxidase(+) catalase (+) Nitrate reduction(+) urease

68. Francisella spp. Zoonosis –tularemia or “rabbit fever” ingestion, inhalation, arthropod bite or contact infected tissues Category A agent - easily disseminated (ID50 = 50), high mortality rates BSL 3Laboratory diagnosis tiny, poor staining GNCBNG on Mac & may initially grow on SBA CHOC, MTM, BCYE with cysteine for 36-48 hours Smooth, gray-white, raised colonies (+) catalase - weak(-) Oxidase(-) urease (+) beta-lactamase test(-) X & V factors

69. Legionella pneumonophila Weakly staining tiny GNR, pleomorphicIntracellular (phagocytes) & extracellularLegionnaires’ disease & Pontiac fever LaboratorySuspect if NG on SBA Acid wash sputum & plate on selective media for best isolationSubculture on BYCE & BYCE with L-cysteine3-5 days “ground-glass” center of gray & granular with pink/light blue periphery of colonyAs age - gray/white or blue/green, convex, glistening coloniesUrine for antigen detection Detects L. pneumophilia serogroup 1 soluble antigen

70. Bordetella pertussis Whooping coughOne most highly communicable diseases of childhood DPT vaccineLaboratoryTiny GNR or GNCBSlow growing 3-5 daysRegan-Lowe Charcoal media Young colonies - mercury droplets, as age turn whitish gray Bordet-Gengou Potato Infusion agarSmall, white, domed “pearls”B. pertussis & B. parapertussis – hemolytic Nucleic acid detection by PCR Primary rapid diagnostic tool Nasopharyngeal swabs

71. enterobacteriaceae

72. ENTEROBACTERIACEAE cultureSBA or Choc - Large, grayish, smooth colonies, some hemolytic MacConkey (MAC) - lactose in mediaLF -pink colonies & NLF –tan/colorless coloniesEosin-Methylene Blue (EMB) lactose & sucrose in media LF- Pink colonies E. coli blue-black/green sheen coloniesNLF & NSF -translucent colonies

73. STOOL CULTURESEnhance Salmonella spp. & Shigella spp. growthHE Agar lactose & sucrose in mediaLF &/or SF–yellow coloniesNLF & NSF –green colonies H2S – black centers XLD Xylose-Lysine Deoxycholate (lactose, sucrose & xylose)LF &/or SF &/or XF–yellow coloniesNLF & NSF & NXF - red colonies H2S – black centers Salmonella spp. (+) lysine decarboxylase

74. Enterobacteriaceae Identification (-) oxidase except Plesiomonas shigelloides(+) glucose fermentation(+) NO3 reduction(+) motile except Shigella spp. , Klebsiella spp. & *Yersinia spp.*Yersinia spp. (+) motile at 250 C

75. KIA or TSI PRES ID of EntericsKIA = lactose in slant & glucose & H2S in butt & +/- gasTSIA= lactose & sucrose in slant; glucose & H2S in butt & +/- gasPhenol red indicator alkaline pH= red & acid pH=yellowSlant=aerobic & butt=anaerobicNOTE:Enterobacteriaceae if A/A or K/ANot an Enterobacteriaceae if K/K or A/K

76. TSIA & KIA Interpretation

77. ONPG o-Nitrophenyl-p-D-galactopyranoside Determines if slow LFCH2O fermentation ability to ferment a specific 1% concentration of carbohydrate MRVP Methyl red (MR) Mixed acid fermentation pathway (strong acids)Vogues-Proskauer (VP) Butylene glycol pathway byproduct acetoin (acetylmethylcarbinol)(+) Klebsiella spp., Enterobacter spp. Serratia spp. & Hafnia sp. groupDecarboxylaseTests Decarboxylase remove carboxyl group of an amino acidLysine, ornithine & arginine

78. PAD Phenylalanine Deaminase deaminates phenylalanine to phenylpyruvic acid (+) PPM group (Proteus, Providencia & Morganella)Citrate Utilization Test Utilize sodium citrate as a sole carbon source DNase Extracellular DNase produced by some bacteria (+) most Serratia spp.Gelatin Liquefaction Proteolytic enzymes break down gelatin into amino acids

79. IndoleKovacs or Ehrlich Degradation products of amino acid tryptophan Add paradimethylaminobenzaldehyde -red color SIMH2S productionIndole productionMotilityMalonate  use sodium malonate as a sole carbon source Motility

80. Nitrate reduction - NO3 to NO2 or to N (gas) 1st Step - add sulfanilic acid & N,N-dimethy-α-naphthylamine(+) Red If no color change go to 2nd Step - add zinc (-) Red and (+) No color change •Cytochrome oxidase Oxidizes reduced cytochrome with molecular oxygen

81. Lysine Iron agar (Slant/Butt)Purple/purple = (+) Lysine decarboxylasePurple/yellow butt = (-) Lysine decarboxylase Cherry red/ yellow butt = (+) Lysine deaminase H2S production masks (+) lysine decarboxylase reactionUreaseBreaks down urea to NH3 which decreases pH

82. Salmonella, Shigella, Aeromonas, Campylobacter, Yersinia, Vibrio and E. coli 0157:H7, Plesiomonas shigelloides • All fecal specimens should be routinely screened for Salmonella, Shigella, and Campylobacter Many labs routinely screen for E. coli 0157:H7 Serologic groupings for Salmonella spp. & Shigella spp.Based on O antigensSlide Test1 drop antisera onto slide & add saline emulsion containing isolate. If (-) may be due to capsule & requires emulsion heated to 1000 C to remove capsule & then re-testEnteric Pathogens

83. Escherichia coli LF MAC – LF, bile precipitationSBA – may appear ß-hemolytic EMB – green metallic sheen IMViC + + - -TSI= A/A (+) gas(-) for DNAse; urease & PAD

84. Escherichia coli LF Most common cause UTIs Meningeal (especially newborn) Gastrointestinal infections Wound Bacteremia Five major categories of diarrheogenic E. coli Enteropathogenic (EPEC) Enterotoxigenic (ETEC) Enteroinvasive (EIEC) Enteroadherent Enterohemorrhagic (EHEC) serotype 0157:H7 produces Verotoxins I and II

85. Enterohemorrhagic E. coli (EHEC) 0157:H7 strain SMAC  MAC with sorbitol instead of lactose (SMAC) E. coli 0157:H7 does not ferment sorbitol MUG Screen for E. coli 0157:H7 Rarely produces Beta -glucuronidse 92% of other strains do produce it SMAC (-) &/or (-) MUG Serotype to confirm E. coli 0157:H7 with anti-serum

86. KLEBSIELLA TRIBE LF Klebsiella spp., Enterobacter spp., Serratia spp., Pantoea spp., Cronobacter spp. & Hafnia spp.(-) H2S (+) urease few (-) MR (+) VP(+) Citrate most (-) indole mostKES – most IMViC - - + + except K. oxytoca +-++

87. Klebsiella spp. LF K. pneumoniae NosocomialFrequent cause of LRT infectionswound infections, UTI, bacteremia K. oxytoca infections similar to K. pneumoniae IMViC - - + + except K. oxytoca indole (+)TSIA = A/A, no H2S, no gas (-) motility (+) urea(-) ornithine decarboxylase

88. Enterobacter spp. LF Enterobacter aerogenes E. cloacae Isolated from wounds, urine, blood & CSF Mucoid colonies (+) motilityIMViC - - + +TSI A/A, no H2S, no gasE. aerogenes = (-) urease & (-) arginine decarboxylaseE. cloacae = (+) urease (65%) & (+) arginine decarboxylase

89. Serratia spp. SLFSerratia marcescens S. liquefaciens S. rubidaea S. marcescens Opportunistic pathogennosocomial outbreaks – UT ; RT, bacteremia-nurseries, cardiac surgery & burn unitsResistant to a wide range of antimicrobialsSusceptibility test MUST be performed on every isolatesome pink pigment at 300 C & look like a LF MAC at 18 hr even though SLF

90. Serratia spp. sLFSerratia marcescens TSI = A/A (Ferments sucrose) KIA – K/A in the 1st 18-24 hrs & after that A/A(+) ONPG to id SLFIMViC - - + +(+) DNase (+) Motility

91. Proteus spp., Morganella spp. & Providencia spp. NLFNormal intestinal flora - Opportunistic pathogens Differentiate from other Enterobacteriaceae (+) PAD(+) Lysine deaminaseProteus spp.Proteus mirabilis Proteus vulgarisurine, wounds, ear s & bacteremia swarming colonies on non-selective media -SBA “burned chocolate” odor (+) H2S production

92. PPM GroupMorganella morganii May UTI & diarrhea Providencia spp. P. rettgeri UTIs & caused occasional nosocomial outbreaks P. stuartii nosocomial outbreaks burn units & been isolated from urinecannot differentiate between Morganella & Providencia spp. with standard tests

93. P. mirabilisP. vulgarisMorganella morganiiProvidenica rettgeriIndole-+++Methyl Red++++VP+ (50%)---Citrate+ 65%)--+Motility++++Urea ++++TSIAlk/AcidAcid/AcidFerment sucroseAlk/AcidAlk/AcidKIAAlk/AcidAlk/AcidAlk/AcidAlk/AcidH2S++--PAD++++

94. Edwardsiella tarda NLF Bacteremia & wound infections(-) Urea (+) Lysine decarboxylase(+) H2S (+) Indole (-) Citrate Citrobacter spp. LF (+) urea slowly (+) citrate (+) methyl red C. freundii UTI, pneumonias, intra-abdominal abscesses (+) H2S & some do not ferment lactose & can resemble Salmonella when isolated from stools

95. Salmonella spp.Typhoid fever Most severe form of enteric fever is caused by S. serotype Typhi Other enteric fevers - S. serotype Paratyphi, S. serotype Choleraesuis Non-typhoidal bacteremia Carrier state MAC, HE & XLDIMViC - + - +TSI = K/A, no gas, (+) H2S(-) PAD(-) Urease (+) lysine decarboxylase

96. Shigella spp. NLFTrue Pathogen that causes bacillary dysentery S. dysenteriae (group A) S. flexneri (group B) S. boydii (group C) S. sonnei (group D) – most common isolate USS. dysenteriae blood, mucous & pus in stool MediaMAC, HE & XLDSS agar - inhibits GP, most coliforms & swarming Proteus spp.LF (pink) from NLF & H2S production

97. Shigella spp. NLF(-) motile (+) glucose fermentation without gas except some S. flexneri (+) gas(-) Urea (-) H2S(-) lysine decarboxylase • S. sonnei (+) ornithine decarboxylaseSLF after 48 hrs & see pink colonies on Mac(+) ONPG

98. Yersinia spp. NLFY. pestis – plague GNR short, plumpWayson Stain - bipolar staining “safety-pin”Grows at 37OC but likes 25-30O C best Class A Bioterrorism Agent (-) urease(-) motility at 250 C & 350 CY. pseudotuberculosis - Similar to plaguePseudotubercles (caseous swellings)(+) motility at 25OC & (-) at 35O C (+) urease

99. Yersinia enterocolitica NLFacute enteritis; appendicitis-like syndrome, arthritis & erythema nodosum contaminated meat, esp. pork (chitterlings), vacuum-packed deli meet,Optimal growth 25 – 30O C (+) motility at 25OC & (-) at 35O C TSI A/A (-) H2S ferments sucrose• CIN Selective Mediacefsulodin, irgasan, novobiocin, bile salts & CV inhibitoryInhibits normal colon organisms better than Mac Yersinia-selective agar (YSA) base- add mannitol to make differential(+) Mannitol fermentation colony has red center