Microbio Final Review

Obligate pathogen

* Always causes disease
* T. pallidum, B. anthracis, etc

Opportunistic pathogen

* Normal flora causing disease due to immune status or getting somewhere it isn’t supposed to be
* NO Normal flora in sterile sites
* Normal flora in GI, GU, Skin, Oral cavity and Respiratory tract

How can we tell if an organism is a contaminant vs true infection?

* Colony count (Few vs Many) and occurrence in repeat culture

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Specimen - swabs

* Transport media to prevent drying
* Cotton can be toxic

Specimen - urine

* Sterile cup or gray top tube(boric acid)
* Clean catch midstream; first morning
* Catheter line NOT bag
* Suprapubic

Specimen - syringe

NO NEEDLE

Specimen

Sterile cups

Specimen - stool

Non-sterile container OK

Specimen - blood culture bottles

Set = aerobic & anaerobic

Blood cultures

* 2+ sets can help us determine if infection vs contamination
* Disinfecting site is important!
* Common contaminants are P. acnes, S. epidermidis, Micrococcus species and Corynebacterium species

Blood cultures - bottle

* ==1:10 ratio of blood to culture media==
* Anticoagulant (SPS, charcoal)
* Antibiotic removal device
* 10% sucrose :osmotic stabilizer

Blood cultures - automated systems

* Detect CO2
* BACTEC FX
* BacT/ALERT
* VersaTREK

Blood cultures - BACTEC FX

Fluorescence

Blood cultures - BacT/ALERT

Colormetric (pH)

Blood cultures - VersaTREK

Gas in headspace of bottle

Plating specimen

* Streak for isolation
* 4 quadrant streak allows for quantitation of organism growth
* How do we do CFU counts?
* Urine cultures BAP and MAC struck for isolation
* 0.001 calibrated loop, multiply colony by 1000
* Quantitate colony from BAP ONLY

Microaerophilic

Requires very little free oxygen

Capnophilic

Requires increased CO2

Anaerobic

Lives in the absence of free oxygen

Strict (obligate) anaerobe

Will die in the presence of oxygen

Facultative anaerobe

Aerobe that can grow without oxygen (anaerobic condition)

Aerotolerant

Anaerobe unaffected by oxygen

BSL 1

Agents not known to cause disease in healthy individuals

BSL 2

Associated with human disease

BSL 3

Negative pressure; agents with potential aerosol transmission

BSL 4

Dangerous agents; full body suit

Biosafety cabinet I

Specimen contamination possible, unfiltered air into cabinet

Biosafety cabinet II

Laminar flow: air is recirculated

Biosafety cabinet III

2 HEPA filters, gas tight

Chemical disinfection

Alcohol, aldehydes, halogens, chlorine, quaternary ammonium compounds, metals

Physical disinfection

Boil, Pasteurization, Inspissation

Chemical sterilization

Gas vapor sterilants (Ethylene oxide), Liquid gas sterilization

Physical sterilization

Steam – Autoclave, Dry Heat – Oven, Ionizing and nonionizing radiation

What is the most likely identification of a mucoid, lactose-fermenting colony isolated on MacConkey agar that is indole negative, citrate positive and nonmotile?

Klebsiella pneumoniae

A gram negative rod was isolated from a stool culture. It produced colorless colonies on EMB and red colonies on XLD. In TSI, it produced a yellow butt and a pink slant with no gas or H2S. It was nonmotile, negative for indole, VP, citrate, urea and phenylalanine deaminase. It was positive for methyl red. What is the presumptive identification?

Shigella

O/F Media - What is in the tube?

* Glucose
* Peptones – smaller concentration to help determine oxidizer since they produce weak acids; prevents the use of peptones to prevent seeing pH change
* pH indicator

TSI - what sugars?

* Lactose, glucose and sucrose; lactose and sucrose 10:1 to glucose

TSI - what can we detect?

H2S, Gas and Sugar fermentation

TSI

Aerobic slant & anaerobic butt

TSI - K/A

Glucose fermenter only

TSI - A/A

Glucose AND lactose or sucrose

TSI - hydrogen sulfide

ONLY seen if the butt of the tube is acidic (==YELLOW==)

TSI - E. coli

A/A with gas

TSI - Shigella

K/A

TSI - Salmonella

K/A with H2S

TSI - P. aeruginosa

K/K

LIA slant

* Determines lysine decarboxylation; glucose fermentation at the butt of the tube and if organism decarboxylates lysine the butt turns ==purple== (alkaline pH)
* Remember the enzyme needs an anaerobic AND acidic environment to decarboxylate
* Lysine deamination results in a ==wine slant==
* For H2S to be seen, the butt of the tube has to be ALKALINE (==purple==)

LIA - Proteus

W/Y

LIA - Salmonella

K/K with H2S

GPC

S. aureus

* Coagulase positive
* Staph latex positive 
* Protein A  and clumping factor
* MSA agar: yellow
* Test for VISA/VRSA
* Vancomycin Screen agar: Confirm with broth microdilution


* Test for MRSA
* Oxacillin screening agar
* Cefoxitin
* mecA: PBP2a



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CNS

* Coagulase negative
* Staph latex negative
* MSA: Red
* S. epidermidis
* Contaminant
* Catheter/indwelling device infections - Biofilm
* Nosocomial UTI
* Source: Urine
* Novobiocin for S. saprophyticus

S. aureus

Skin and wound infections

S. aureus - Exfoliative toxin

Staphylococcal scalded skin syndrome

S. aureus - TSST-1

* Toxic shock syndrome
* Tampons

S. aureus - enterotoxin

* Food poisoning
* Occurs 2-8 hours after consumption

S. aureus - cytolytic toxins

* Alpha, beta, gamma, and delta
* Alpha and beta lyse RBC’s
* Gamma  associated with Panton-Valentine leucocidin (PVL) : prevents phagocytosis of PMNs

S. aureus - Protein A

Binds Fc region of IgG, neutralizing antibody

S. aureus - Enzymes

facilitate spread

GPC - Rothia species

* Sticky!
* Seen in respiratory cultures
* Opportunistic pathogen

GPC - Micrococcus species

* YELLOW colony
* Microdase test (modified oxidase test)
* Gram Stain: tetrads
* Normal flora, contaminant

Streptococcus

* Treat with Penicllin
* Erythromycin if allergic
* Increased CO2 enhances growth
* Hemolysis?
* Alpha, beta, gamma hemolysis
* Gamma
* PYR(quicker!), 6.5% Nacl and BEA determine if Viridans streptococci or Enterococcus

Enterococcus

* Nosocomial infections: UTI, bactermia
* PYR, BEA, 6.5% NaCl (+)
* VRE
* Vancomycin screen agar
* CHROMagar: PINK (E. faecium) or BLUE (E. faecalis if catalase negative)
* Serious infections treated with an aminoglycoside and penicillin - synergistic

Streptococcus - Alpha hemolysis

* P disc or Bile solubility
* S. pneumoniae vs Viridans streptococcus
* “S” and soluble vs “R” not soluble
* Could it be Enterococcus?
* PYR, 6.5% NaCl, Bile Esculin
* Weird ones:
* Aerococcus: GS=more tetrads than chains but looks alpha
* Leuconostoc and Pediococcus: Intrinsically resistant to Vancomycin

S. pneumoniae

* Lancet-shaped
* Mucoid or dimpled (autolysis)
* Capsule most important virulence factor
* Pneumonia - #1 cause; otitis media; meningitis; bacteremia
* Bile soluble (+) and Optochin (S)

Viridans streptococci

* #1 cause of bacterial endocarditis
* Normal flora
* Can cause oral infections
* Hemolysis: alpha or gamma
* PYR (=) and P disk (R)

Streptococcus - Beta hemolysis

* PYR (quicker!) and/or Bacitracin (A disk) help us determine GAS or not
* GDS associated with colon cancer
* PYR and 6.5% NaCl (=)

S. pyogenes

* Toxins and infections
* Bacterial pharyngitis and tonsillitis (strep throat)
* Skin infections: Impetigo, Erysipelas & Cellulitis
* Scarlet fever – Spe’s
* Necrotizing fasciitis “flesh-eating”
* Streptococcal toxic shock syndrome
* Sequelae
* Rheumatic fever: damage to heart valves
* Acute glomerulonephritis

S. pyogenes - M protein

most important virulence factor

S. pyogenes - Streptolysin O

* oxygen labile
* anaerobic
* highly immunogenic (ASO test)

S. pyogenes - Streptolysis S

oxygen stable – see hemolysis of RBCs in agar

S. agalactiae - Capsule

most important virulence factor

S. agalactiae

* Newborn infections: pneumonia, sepsis, meningitis
* Lim broth
* Carrot broth

S. pyogenes

* Large hemolysis and small colony
* A disk “S”
* PYR (+)
* CAMP(=)
* BEA (=)
* Latex A (+)

S. agalactiae

* Small zone of hemolysis compared to colony size
* A disk “R”
* PYR (=)
* CAMP (+)
* Hippurate hydrolysis (+)
* Latex B (+)

Nutritionally deficient strep

* Abiotrophia and Granulicatella
* Gram Stain: GPC in chains in a blood culture but does not grow on BAP
* Grows on CHOC
* Satellite test

B. cereus

* Infections
* food poisoning (8-16 hours) diarrheal enterotoxin OR emetic enterotoxin (Fried rice)
* Ocular infections
* Ground glass, beta hemolysis, motile

B. anthracis

* Infections – how do spores enter?
* Cutaneous anthrax – (eschar)
* Inhalation anthrax – (woolsorter’s disease)
* Gastrointestinal anthrax
* Toxins
* PA + EF = Edema toxin
* PA + LF = Lethal toxin
* Egg-white/medusa head, non-motile, gamma hemolysis

GPR - Corynebacterium

* C. diphtheriae - Diphtheria toxin
* Gray pseudomembrane
* Elek tests for toxin
* C. urealyticum – urea positive; found in urine
* Pallisading, Chinese letters, Catalase (+)

GPR - Listeria

* Listeriosis – contaminated food products
* Pregnant women, newborns, immunocompromised
* Beta hemolysis, Tumbling/Umbrella motility, BEA (+), CAMP (+), Hippurate hydrolysis (+), Catalase (+)

GPR - Erysipelothrix rhusiopathiae

* Infection by a cut
* Fish handlers disease - Erysipeloid
* H2S+, Catalase =, can look alpha, ppt colony

GPR - G. vaginalis

* Bacterial vaginosis – clue cells
* PPT colony, Catalase =

GPR - Branching

* Nocardia – Partial PAF, chalky colony
* Streptomyces – PAF =, waxy colony

GPR - Rhodococcus

Pink mucoid colonies

Cardinal Rules for Enterobacteriaceae

* Gram negative rods
* All ferment glucose
* All reduce nitrate to nitrites
* All are oxidase negative
* Except Plesiomonas
* All motile at body temperature except
* Shigella
* Klebsiella
* Yersinia

Enterobacteriaceae - H antigen

* Flagellar antigen
* Responsible for motility; heat stable

Enterobacteriaceae - O antigen

* Somatic antigen
* Heat stable antigen located in the cell wall (LPS)

Enterobacteriaceae - K antigen

* Capsular antigen
* Heat labile: Add heat if isolate didn’t type and try again
* K1 antigen of E. coli, Vi antigen of Salmonella typhi

E. coli

* IMVC: ++- -
* Motile

E. coli - UTI, GI - Enterotoxigenic

* travelers diarrhea

E. coli - UTI, GI - Enterohemorrhagic

* EHEC 0157:H7 associated with Hemolytic Uremic Syndrome (HUS) and Shiga toxigenic E. coli (STEC)

Klebsiella, Enterobacter, Serratia

* IMVC: - - + +

Klebsiella

* Not motile
* Mucoid
* ==K. oxytoca: indole positive==
* ODC =

Enterobacter

* Motile
* Can also be mucoid
* ODC +

Serratia

DNAse +

Enterobacteriaceae - Morganella morganii

* UTI
* PAD +

Enterobacteriaceae - Providencia

PAD +