REVIEW OF CLINICAL MICROBIOLOGY PART II MJ Weintraub, DVM

Cell wall/envelope structure GN, GP, AF & wall-less Genome Morphology, arrangements, capsule/slime layer, flagella, fimbriae, pilus Horizontal gene transfer mechanisms 70s ribosome 1 Circular, double stranded DNA chromosome/genome Plasmids: extrachromosomal, circular, dsDNA, self-replicating Prokaryotes Bacteria

Eukaryotes Fungi, Protozoa & Helminths Organelles 80s ribosome Genome in nucleus Protozoa flagella, cilia, pseudopods or non-motile Some cysts, trophozoites Fungi Dimorphism Chitin in cell wall Ergosterols plasma membranes Some capsules Helminths Developmental stages if any

Viruses & Prions Viruses Intracellular obligate parasites Acellular agent RNA or DNA genomes Capsid &/or envelope Bacteriophages Prions Infectious pieces of protein

Common Stains - Bacteria Gram Understand technical errors GN & GP Endospores clear within cell Acid Fast AF red & non-AF blue Ziehl Neelsen (heat) Kinyoun (cold-detergent) Acridine Orange - fluochrome Bacteria difficult id in a specimen blood

COCCI Single or pairs (diplococci) Chains (streptococci) Clusters (staphylococci) tetrads BACILLI 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

Common Stains - Bacteria Methylene Blue Metachromatic granules Corynebacterium diphtheriae AF counterstain Endospore Red/pink endospore green-living cell

Common Stains - Fungi Lactophenol Cotton Blue (LCB) Medically important fungi Calcofluor White fluorochrome Fungi bright apple-green or blue-white India ink Cryptococcus neoformans capsule

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

Types of Media Selective media Contains additives that inhibit growth of some bacteria but supports others to grow MacConkey, CNA, Mannitol Salt Differential 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

Media CategoryUsed For Sheep Blood Agar Enriched Good for fastidious organisms, most organisms will grow, interpret hemolysis ChocolateVery EnrichedVery 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 fermentation Eosin-Methylene Blue Agar (EMB) Selective & DifferentialSelective for GNRs, differential for lactose & sucrose fermentation (Enterobacteriaceae & oth GNRs) Hektoen Enteric Agar (HE) Highly Selective & Differential Selective for enteric pathogens, differential for sucrose & lactose fermentation H 2 S Enhances growth of Salmonella & Shigella Xylose-Lysine Desoxycholate Agar (XLD) Highly Selective & Differential Selective & GNR & enhances growth of Shigella differential for sucrose, lactose & xylose fermentation H 2 S & lysine (decarboxylation) Used to ID Salmonella & Shigella

Media CategoryUsed For Colistin & Nalidixic Acid Blood Agar (Columbia CNA) SelectiveSelective for GPC, read hemolysis Phenylethyl Alcohol agarSelectiveSelective for GPC Modified 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 hominis Campylobacter Blood Agar (CAMPY) Selective & EnrichedCampylobacter spp. Cefsulodin-Irgasan-Novobiocin (CIN) SelectiveYersinia enterocolitica Thiosulfate Citrate Bile Sucrose Salts (TCBS) Selective & Differential Vibrio spp. Differentiates Vibrio spp. based on sucrose fermentation

MediaCategory Used For Buffered Charcoal-Yeast Extract Agar (BCYE) EnrichedLegionella spp. Fletcher Semisolid MediumEnrichedLeptospira spp. Loeffler Coagulated Serum Slant EnrichedClostridium diphtheriae Tellurite Blood Agar with or/without Cystine Selective, differential & enriched Corynebacterium diphtheriae Tinsdale Agar Selective and differentialClostridium diphtheriae Bordet-Gengou Blood AgarSelective and enrichedBordetella pertussis and B. parapertussis Reagan-Lowe MediumSelectiveBordetella pertussis Mannitol Salt AgarSelective and differential Selective for staphylococci and differential for mannitol fermentation Bile Esculin Agar Selective and differential Selective for Group D Strep and Entercoccus spp.

Media CategoryUsed For Blood Agar AnaerobeEnrichedFastidious anaerobic bacteria Kanamycin & Vancomycin Laked Blood Agar (KVLB) Selective & Enriched Bacteroides spp. & Prevotella spp. Bacteroides Bile Esculin agar (BBE) Selective & DifferentialBacteroides fragilis group Phenylethyl Alcohol agar Anaerobic Selective & EnrichedAnaerobes Egg Yolk agar (EYA)DifferentialClostridium spp. Lecithinase, lipase & protease activity Cylcoserine Cefoxitin Fructose agar (CCFA) Selective & DifferentialClostridium difficile Thioglycolate Broth (THIO)All purpose mediaBack-up broth Todd Hewitt Broth & LIM brothSelectiveGroup B streptococci Chopped Meat BrothCultivation of anaerobes Selenite BrothEnrichmentSalmonella spp. & Shigella spp.

JEMBEC transport system Neisseria gonorrhoeae Sodium bicarbonate & sodium citrate Inhibition of contaminating bacteria and fungi

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

Atmospheric requirements Obligate aerobe require oxygen for growth Aerotolerant anaerobe can survive in presence of O 2 but do not use O 2 in metabolism Obligate anaerobe cannot grow in presence of oxygen Facultative anaerobe grow either with or without oxygen Most opportunistic pathogens Capnophilic Best at increased CO 2 Capnophilic bacteria need 5% -10% CO 2 Microaerophilic reduced level of oxygen to grow Ex. Campylobacter spp. requires 5% to 6% O2

Fermentation & Respiration Bacteria use biochemical pathways to catabolize CH 2 O & produce energy Respiration (Oxidation) efficient energy-generating process Obligate aerobes & facultative anaerobes Fermentation Anaerobic process Obligate & facultative anaerobes Less efficient in energy generation

Fermentation vs. Oxidation Used in biochemical testing based on: presence mixture of end products accumulates in medium Changes in pH Change in pH KIA, TSI, CTA CH 2 O, HE Products of process: Methyl Red mixed acids & VP - acetoin NOTE: if evaluating a CH 2 O & not glucose, no glucose should be in the media Bacteria preferentially ferment glucose 1 st

Lactose Fermentation LF, NLF & sLF Key in ID of Enterobacteriaceae 1 st Step -galactoside permease transports lactose across cell wall 2 nd Step -galactosidase breaks galactoside bond releases glucose all organisms that ferment lactose ferment glucose sLF fermenters lack or have a weak galactoside permease ONPG test to id sLF as does not require galactoside permease

Staphylococcus spp. (+) Catalase Non-motile Non-spore-forming Most are facultative anaerobes except S. saccharolyticus S. aureus S. epidermidis S. saprophyticus S. lugdunensis More virulent, may have mecA gene

Staphylococcus spp. Isolation SBA Thioglycollate broth Selective 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 hemolytic

COAGULASE TESTS SLIDE TEST Cell-bound coagulase (Clumping Factor) enzyme that clots human, rabbit, or pig plasma If (+) reported as Staphylococcus aureus If (-) the tube test must be done TUBE 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

Coagulase (+) Staphylococcus aureus (-) other staphylococci (CoNS) S. lugdunensis (+) Cellbound (CF) coagulase & (-) free (staphylocoagulase) Novobiocin on CoNS urine isolates (R) -Staphylococcus saprophyticus (S) other CoNS Hemolysis : Staphylococcus aureus No hemolysis: S. epidermidis & S. saprophyticus

Micrococcus spp. GPC, tetrads, irregular clusters (+) catalase Micrococcus spp.Staphylococcus spp. Oxidase(+)(-) BacitracinSR LysostaphinRS Coagulase(-)varies

Staphylococcus aureus must perform antimicrobial susceptibility on all isolates Most penicillin(R) beta lactamases MRSA, HA-MRSA, CA-MRSA Vancomycin VISA & VRSA MRSA KEY TEST Oxacillin susceptibility but now Cefoxtin (better inducer of mecA-mediated resistance) is preferred GOLD STANDARD Detection mecA gene same day tests: Nucleic Acid probes or PCR D TEST Macrolide resistance Inducible clindamycin resistance presence of erythromycin

Streptococcus spp. GPC, chains, pairs (-) catalase Most facultative anaerobes Streptococcus spp. Enterococci spp. LANCEFIELD CLASSIFICATION C carbohydrate on cell wall A, B, C, D, F, G & N most common

LancefieldHemolysisTerm S. pyogenes A, wide zoneGroup A strep S. agalactiae B narrow zoneGroup B strep S.dysgalactiae, S. equiCGroup C strep S. bovis groupD or nonenonenterococcus Enterococcus faecalis & E. faecium D, or noneenterococcus S. pneumoniaeNo grouppneumococcus Anginosus group, mutans group, mitis group, salivarius group A, C, F, G, N, or --, or noneviridans strep

Susceptibility Tests Bacitracin identify 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 strep Groups A & B are resistant to SXT Optochin susceptibility used on hemolytic strep ethylhydrocupreine hydrochloride Streptococcus pneumoniae is sensitive to optochin

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 hemolytic strep (+) Streptococcus agalactiae (GBS) PYR hydrolysis test Substrate is L-pyrrolidonyl--naphthylamide & L-pyroglutamic acid- naphthylamide (+) Streptococcus pyogenes & Enterococcus spp. (-) other streptococci more specific for Group A strep than bacitracin (+) Aerococcus spp. & Gemella spp

CAMP test CAMP factor PRES ID Group B strep Enhanced hemolysis -hemolytic Staphylococcus aureus & CAMP factor producing - hemolytic or some nonhemolytic Group B strep (+) arrow-shaped hemolysis pattern

Bile Esculin Grow in presence of 40% bile Hydrolyze esculin black complex (+) Group D streptococcus & Enterococcus spp. (-) viridans Strep 6.5% sodium chloride broth (+) Enterococcus spp. (-) Group D Leucine AminoPeptidase test (LAP) (+) Streptococcus & Enterococcus spp. & Pediococcus spp. (-) Aerococcus spp. & Leuconostoc spp. Enterococcus faecalis

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

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

Streptococcus agalactiae invasive 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 gestation Laboratory Grayish white colonies, small zone of hemolysis (-) catalase (+) Hippurate hydrolysis (+) CAMP test

PRES ID of hemolytic streptococci -hemolytic colonies Bacitracin Susceptible Resistant CAMP Group B Streptococcus agalactiae Possibly group D or hemolytic; not group A, B Group A Streptococcus pyogenes +-

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 - or no hemolysis (+) Bile Esculin (-) 6.5% NaCl (-) PYR

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 or or no hemolysis May have pseudocatalase reaction (+) Bile Esculin (+) 6.5% NaCl (+) PYR

PRES ID of Group D strep & Enterococcus , , or no hemolysis in SBA Bile Esculin 6.5% NaCl Non-hemolytic streptococci Enterococcus spp. PYR (+) Group D streptococci PYR (-) + - +-

Streptococcus pneumoniae pneumococcus Normal RT flora Pneumonia, sinusitis, otitis media, bacteremia, meningitis & 30% acute septicemia Quellung reaction capsule swelling allows ID of Streptococcus pneumoniae & specific serotypes treat with penicillin but some strains show resistance & are treated with erythromycin or chloramphenicol Vaccine childhood & > 50 yrs.

Streptococcus pneumoniae Laboratory diagnosis GPC - lancet shaped & usually have capsule SBA or Choc Some require increased CO 2 Large zone of hemolysis round, glistening, wet, mucoid, dome shaped & with time collapse of colony's center Optochin (S) S. pneumoniae (R) other hemolytic streptococci Bile solubility (+) S. pneumoniae (-) other hemolytic strep

viridans streptococci flora URT, female genital tract & gi tract S. anginosus group, S.mutans group, S. mitis group, S. salivarius group & S. bovis group Low 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 hemolysis (-) Bile solubility (R) optochin

Abiotrophia spp. & Granulicatella spp. Nutritionally Variant Streptococci (NVS) Satellite colonies @ other bacteria Bacteremia, endocarditis, otitis media Should be suspected in blood cultures Laboratory GPC resembling strep Do not grow on subculture Subculture Staphyloccous aureus streak Satellitism

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

Neisseria gonorrhoeae urogenital tract infections & conjunctiva Disseminated infections joints, meningitis Specimen: malesurethra; femalesendocervix & rectum, pharynx & joint fluid Best chance for recovery is direct plating of specimen to gonococcal- selective media incubate at 35 O C in 3-5% CO 2 PCR assay N. gonorrhoeae vs. Chlamydia spp.

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

Neisseria gonorrhoeae Requires chocolate agar (+) selective agents Thayer-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

Neisseria gonorrhoeae PRES ID 1.Small, gray/tan, transluscent raised colonies 24-48 hrs. 2.Gram stain to verify GNDC kidney bean-shaped GNRs 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 3.(+) Oxidase Must be done on all isolates Antimicrobial resistance All strains must be tested for -lactamase production Must be performed on primary culture plate as plasmid can be lost on subculture

Definitive Identification Other oxidase (+) GNDC, can be recovered from gonococcal selective media - Neisseria cinerea, N. meningitidis, Moraxella catarrhalis CH 2 O Utilization Cystine Trypticase Agar (CTA) (+) Glucose fermentation & (-) Maltose, sucrose & lactose fermentation Chromogenic enzymes Multitest Matrix assisted Laser Desorption/Ionization Time of Flight (MALDI Tof) Non-culture Nucleic Acid Amplification Test (NAATs) Neisseria gonorrhoeae

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

Moraxella catarrhalis GNDC 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 CH 2 O degradation tests (+) DNase (+) Butyrate esterase

Fastidious GNR HACEK Haemophilus spp. Aggregatibacter spp. (formerly Actinobacillus) Cardiobacterium hominis Eikenella corrodens Kingella spp. Capnocytophaga spp. Pasteurella spp. Brucella spp. Francisella spp. Legionella spp. Bordetella spp.

HACEK Group Characteristics GNR/CB Capnophilic Important cause of endocarditis flora of oral cavity allows for introduction into blood stream Haemophilus spp. GNCB, pleomorphic Most nonpathogenic or opportunistic pathogens X 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

Haemophilus spp. Hemolysis on 5% horses or rabbits blood to differentiate SBA only X factor available V factor dependent do not grow on SBA Chocolate agar Lysed 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

Haemophilus influenzae Meningitis decrease frequency due to vaccine (Hib) Epiglottitis Haemophilus aegyptius - acute contagious conjunctivitis pinkeye H. influenzae biogroup aegyptius -conjunctivitis H. ducreyi - Chancroid painful, soft highly 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

Haemophilus spp. Culture Does not grow on SBA, grows on Chocolate H. ducreyi Special media, hold for at least 7 days at 33 O 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 smear GNCB to long filaments H. ducreyi school of fish formation H. influenzae intra & extracellular

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

Aggregatibacter aphrophilus X-dependent & X-independent strains (-) catalase granular, yellow with center opaque zone colonies on Choc Aggregatibacter actinomycetemcomitans o (+) catalase o BA star 4-6 points in center 100x Cardiobacterium hominis GNR or GV forms rosettes Slow grow SBA & CHOC may see pitting

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

o K ingella spp. o Often follow viral infection or poor oral care or oral surgery o GN CB/short rods with squared ends, in pairs or short chains, tend resists decolorization o Nutritionally fastidious o twitching motility (no flagella) o Capnocytophaga Spp. Resemble HACEK Normal flora mouth & oral cavities GNR - Thin & fusiform Fastidious, 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 o

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

Brucella spp. Zoonosis Brucellosis, Undulant fever, Mediterranean, Crimean & Malta Fevers aerosol, 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 Agent Easily disseminated & moderate morbidity but low mortality BSL 3 lab lab exposure results in 30%-100% acquiring infection Blood & bone marrow cultures performed with acute & convalescent sera for serologic testing

Brucella spp. Laboratory Tiny, GNR/CB facultative intracellular pathogen in phagocytes Grow aerobically on Choc & BA in 18 hrs - hold 4 days (also TM or Martin Lewis) Smooth, raised & translucent colonies 50% grow on MAC (-) motility (+)oxidase (+) catalase (+) Nitrate reduction (+) urease

Francisella spp. Zoonosis tularemia or rabbit fever ingestion, inhalation, arthropod bite or contact infected tissues Category A agent - easily disseminated (ID 50 = 50), high mortality rates BSL 3 Laboratory diagnosis tiny, poor staining GNCB NG 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

Legionella pneumonophila Weakly staining tiny GNR, pleomorphic Intracellular (phagocytes) & extracellular Legionnaires disease & Pontiac fever Laboratory Suspect if NG on SBA Acid wash sputum & plate on selective media for best isolation Subculture on BYCE & BYCE with L-cysteine 3-5 days ground-glass center of gray & granular with pink/light blue periphery of colony As age - gray/white or blue/green, convex, glistening colonies Urine for antigen detection Detects L. pneumophilia serogroup 1 soluble antigen

Bordetella pertussis Whooping cough One most highly communicable diseases of childhood DPT vaccine Laboratory Tiny GNR or GNCB Slow growing 3-5 days Regan-Lowe Charcoal media Young colonies - mercury droplets, as age turn whitish gray Bordet-Gengou Potato Infusion agar Small, white, domed pearls B. pertussis & B. parapertussis hemolytic Nucleic acid detection by PCR Primary rapid diagnostic tool Nasopharyngeal swabs

ENTEROBACTERIACEAE

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

STOOL CULTURES Enhance Salmonella spp. & Shigella spp. growth HE Agar lactose & sucrose in media LF &/or SFyellow colonies NLF & NSF green colonies H2S black centers XLD Xylose-Lysine Deoxycholate (lactose, sucrose & xylose) LF &/or SF &/or XFyellow colonies NLF & NSF & NXF - red colonies H2S black centers Salmonella spp. (+) lysine decarboxylase

Enterobacteriaceae Identification (-) oxidase except Plesiomonas shigelloides (+) glucose fermentation (+) NO3 reduction (+) motile except Shigella spp., Klebsiella spp. & *Yersinia spp. *Yersinia spp. (+) motile at 25 0 C

KIA or TSI PRES ID of Enterics KIA = lactose in slant & glucose & H2S in butt & +/- gas TSIA= lactose & sucrose in slant; glucose & H2S in butt & +/- gas Phenol red indicator alkaline pH= red & acid pH=yellow Slant=aerobic & butt=anaerobic NOTE: Enterobacteriaceae if A/A or K/A Not an Enterobacteriaceae if K/K or A/K

TSIA & KIA Interpretation

ONPG o-Nitrophenyl-p-D-galactopyranoside Determines if slow LF CH 2 O 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. group DecarboxylaseTests Decarboxylase remove carboxyl group of an amino acid Lysine, ornithine & arginine

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

Indole Kovacs or Ehrlich Degradation products of amino acid tryptophan Add paradimethylaminobenzaldehyde -red color SIM H2S production Indole production Motility Malonate use sodium malonate as a sole carbon source Motility

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

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

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 antigens Slide Test 1 drop antisera onto slide & add saline emulsion containing isolate. If (-) may be due to capsule & requires emulsion heated to 100 0 C to remove capsule & then re-test Enteric Pathogens

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

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

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

KLEBSIELLA TRIBE LF Klebsiella spp., Enterobacter spp., Serratia spp., Pantoea spp., Cronobacter spp. & Hafnia spp. (-) H 2 S (+) urease few (-) MR (+) VP (+) Citrate most (-) indole most KES most IMViC - - + + except K. oxytoca +-++

Klebsiella spp. LF K. pneumoniae Nosocomial Frequent cause of LRT infections wound 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

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

Serratia spp. SLF Serratia marcescens S. liquefaciens S. rubidaea S. marcescens Opportunistic pathogen nosocomial outbreaks UT ; RT, bacteremia-nurseries, cardiac surgery & burn units Resistant to a wide range of antimicrobials Susceptibility test MUST be performed on every isolate some pink pigment at 30 0 C & look like a LF MAC at 18 hr even though SLF

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

Proteus spp., Morganella spp. & Providencia spp. NLF Normal intestinal flora - Opportunistic pathogens Differentiate from other Enterobacteriaceae (+) PAD (+) Lysine deaminase Proteus spp. Proteus mirabilis Proteus vulgaris urine, wounds, ear s & bacteremia swarming colonies on non-selective media -SBA burned chocolate odor (+) H2S production

PPM Group Morganella morganii May UTI & diarrhea Providencia spp. P. rettgeri UTIs & caused occasional nosocomial outbreaks P. stuartii nosocomial outbreaks burn units & been isolated from urine cannot differentiate between Morganella & Providencia spp. with standard tests

P. mirabilisP. vulgarisMorganella morganii Providenica rettgeri Indole-+++ Methyl Red ++++ VP+ (50%) --- Citrate+ 65%) --+ Motility++++ Urea++++ TSI Alk/AcidAcid/Acid Ferment sucrose Alk/Acid KIA Alk/Acid H2SH2S++-- PAD++++

Edwardsiella tarda NLF Bacteremia & wound infections (-) Urea (+) Lysine decarboxylase (+) H 2 S (+) Indole (-) Citrate Citrobacter spp. LF (+) urea slowly (+) citrate (+) methyl red C. freundii UTI, pneumonias, intra-abdominal abscesses (+) H 2 S & some do not ferment lactose & can resemble Salmonella when isolated from stools

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 & XLD IMViC - + - + TSI = K/A, no gas, (+) H 2 S (-) PAD (-) Urease (+) lysine decarboxylase

Shigella spp. NLF True Pathogen that causes bacillary dysentery S. dysenteriae (group A) S. flexneri (group B) S. boydii (group C) S. sonnei (group D) most common isolate US S. dysenteriae blood, mucous & pus in stool Media MAC, HE & XLD SS agar - inhibits GP, most coliforms & swarming Proteus spp. LF (pink) from NLF & H 2 S production

Shigella spp. NLF (-) motile (+) glucose fermentation without gas except some S. flexneri (+) gas (-) Urea (-) H 2 S (-) lysine decarboxylase S. sonnei (+) ornithine decarboxylase SLF after 48 hrs & see pink colonies on Mac (+) ONPG

Yersinia spp. NLF Y. pestis plague GNR short, plump Wayson Stain - bipolar staining safety-pin Grows at 37 O C but likes 25-30 O C best Class A Bioterrorism Agent (-) urease (-) motility at 25 0 C & 35 0 C Y. pseudotuberculosis - Similar to plague Pseudotubercles (caseous swellings) (+) motility at 25 O C & (-) at 35 O C (+) urease

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

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REVIEW OF CLINICAL MICROBIOLOGY PART II MJ Weintraub, DVM