Demetrio L. Valle Jr., MD, MSc., FPSP, FASCP, IFCAP

Anatomic and Clinical Pathologist

OUTLINEDIAGNOSTIC BACTERIOLOGY

Blood Cerebrospinal FluidUrineStoolUpper respiratory tractLower respiratory tractPus and exudatesUpdates in Multidrug Resistance Bacteria

BLOOD Expected Organisms

Bacteroides fragilis Brucella Burkholderia pseudomallei Candida albicans and Cryptococcus neoformans Haemophilus influenzae Neisseria meningitidis Non-fermenters other than Pseudomonas aeruginosa Other Enterobacteriaceae Pseudomonas aeruginosa Salmonella typhi and non-typhi Staphylococcus aureus Streptococci (S. pyogenes, S. pneumoniae, viridans

streptococci)

Blood - Media and diagnostic reagentsCOLLECTION MEDIAAEROBIC - Tryptic soy broth (TSB) can be

replaced by any rich broth, e.g. Brain–heart infusion broth

ANAEROBIC- thioglycollate broth or Schaedler broth or Wilkins–Chalgren anaerobe broth

ISOLATION MEDIABlood agar, chocolate agar and MacConkey

agar

Tryptic soy broth (TSB) Thioglycollate broth

Blood - Media and diagnostic reagentsDIAGNOSTIC REAGENTS

Bacitracin disc (Group A β- Streptococci – S. pyogenes –susceptible)

Coagulase plasma (S. aureus) β-Lactamase test reagent (detection of Beta-lactamase enzyme)** Optochin disc (differentiate S. pneumoniae from other α hemolytic

strep) Oxidase reagent Salmonella agglutinating antisera V and XV factors (Identification of Hemophilus influenzae) Haemophilus influenzae type b antiserum Neisseria meningitidis agglutinating serum (polyvalent andspecific groups A, B, C)** penicillin, cephalosporins, cephamycins and carbapenems

Oxidase Test (N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride) used to detect the presence of oxidase enzymes

produced by a variety of bacteria. oxidase test can be used to differentiate between

genera :Moraxella (+) and Neisseria (+) from Acinetobacter

(-) Aeromonas (+), Plesiomonas shigelloides (+), and

Vibrio (V+) from other Enterobacteriaceae (-) Burkholderia gladioli (-) and B. mallei (V) from B.

cepacia (+) and B. pseudomallei (+) Pseudomonas aeruginosa , Neisseria gonorrhoeae

and Campylobacter jejuni are oxidase-positive pathogens

Oxidase Test V (Heme) & X (NAD) factor test

BACTEREMIAPresence of bacteria in the blood streamCaused by:

Post-operative complicationsIntravascular cathetersLocalized infection

In-patient mortality – 20%Shock and organ failure – 90%

BACTEREMIAEarly detection 77%Differentiation between Gram positive or

Gram negative is one of the most important factors.

WHOLE BLOOD CULTURE

Most sensitive and the gold standard.

It requires incubation, subculturing, biochemical tests.

3-7 days TAT

http://www.flickr.com/photos/bjorn_banan/161825278/

METHODS TO IMPROVE BLOOD CULTURE YIELDSKIN PREPARATIONSKIN PREPARATION

Strict aseptic technique (Chandrasekar & Brown, 1994).

Povidone iodine versus 2% tincture of iodine (Little et al., 1999)Tincture of iodine -> significant reduction in

skin flora contamination due to the faster onset of action

0.5% Alcoholic chlorhexidine (Mimoz et al., 1999)Reduced the incidence of blood culture

contamination15 to 30 seconds

METHODS TO IMPROVE BLOOD CULTURE YIELD

TIMING OF BLOOD EXTRACTIONTIMING OF BLOOD EXTRACTIONContinuous Bacteremia Intermittent Bacteremia

Chandrasekar & Brown, 1994 drawing multiple blood culture sets in 24 hour

period have been shown to detect intermittent bacteremia

Li et al., 1994 Similar yields if samples were collected within

2 hours or within 24 hoursMylotte & Tayana, 2000

Patients with antibiotics, samples drawn close to the time antibiotic conc. have reached low levels.

METHODS TO IMPROVE BLOOD CULTURE YIELD

VOLUME OF BLOODVOLUME OF BLOODIs the most important factor ( Shafazand &

Weinacker, 2002)At least 10 mL, provide the highest yield

and lowest number of false-negative blood culture results (Mermel and Maki, 1993).

Extracting more than 30 mL of blood does not improve the sensitivity of blood and contributes to iatrogenic causes of anemia (Weinstein et al., 1983)

METHODS TO IMPROVE BLOOD CULTURE YIELD

ANTIBIOTIC TREATMENTANTIBIOTIC TREATMENTSignificant decrease the yield of blood

cultures (Chandrasekar & Brown, 1994; Leibovici, 1991)

10 mL per 100 mL of culture broth dilutes the concentrations of antibiotics and neutralizing serum bactericidal activity in the culture (Washington & Ilstrup, 1996).

Antibiotic-absorbent resins (BacT/Alert, Biomerioux, France).

Antimicrobial removal device (BACTEC,Beckton Dickinson, MD).

Common causes of bacteremia

Processing of blood culturesIncubation SubculturesFinal processing Antimicrobial susceptibility testing (AST)Detection 0f contaminants

Incubation timeManual

35-37⁰CInspected twice a day for at least 3 days for signs of microbial

growthSedimented red bloodSigns of microbial growth

a floccular deposit on top of the blood layer uniform or subsurface turbidity hemolysis coagulation of the broth a surface pellicle production of gas white grains on the surface or deep in the blood layer.

Perform gram stain

Subculturesfor Gram-negative rods: MacConkey agar,

Kligler iron agar, motilityindole–urease (MIU) medium, Simmons citrate agar;

for small Gram-negative rods: blood agar;for staphylococci: blood agar, mannitol salt

agar;for streptococci: blood agar with optochin,

bacitracin, and tellurite discs, sheep blood agar for the CAMP test, and bile–esculin agar.

SubcultureMicroorganisms may grow without producing

turbidity or visible alteration of the broth. e.g. S. Pneumoniae (autolysis and die rapidly)Subculture in chocolate agar after 18-24 hours

incubation. After 7 days of incubation without growth –

inoculate in thioglycollate broth (incubate for another 3 days)

Antimicrobial susceptibility testingTime is gold in BLOOD CULTURE. Gram stain result – gram positive cocci

(Staph) or gram negative rodsSwab dipped into the turbid broth swab

inoculate in Mueller-Hinton medium (95% of cases correlate with the standardized test)

ContaminantsAseptic skin preparationAseptic procedures during inoculation and

subculturesUsual contaminants

S. epidermidis, P. acnes, diphtheroids, Acinetobacter spp. and Bacillus spp.

True bacteremia if the same organism grows in two bottles of

the same blood specimen;if the same organism grows in cultures from

more than one specimen; if growth is rapid (within 48 hours);if different isolates of one species show the

same biotypes and antimicrobial-susceptibility profiles.