Demetrio L. Valle Jr., MD, MSc.

, FPSP, FASCP, IFCAP Anatomic and Clinical Pathologist

OUTLINE
y DIAGNOSTIC BACTERIOLOGY y Blood y Cerebrospinal Fluid y Urine y Stool y Upper respiratory tract y Lower respiratory tract y Pus and exudates y Updates in Multidrug Resistance Bacteria

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

Blood - Media and diagnostic reagents
COLLECTION MEDIA y AEROBIC - Tryptic soy broth (TSB) can be replaced by any rich broth, e.g. Brain heart infusion broth y ANAEROBIC- thioglycollate broth or Schaedler broth or Wilkins Chalgren anaerobe broth ISOLATION MEDIA y Blood agar, chocolate agar and MacConkey agar

Tryptic soy broth (TSB)

Thioglycollate broth

Blood - Media and diagnostic reagents
y DIAGNOSTIC REAGENTS
y Bacitracin disc (Group A - Streptococci y Coagulase plasma (S. aureus) y

S. pyogenes susceptible)

-Lactamase test reagent (detection of Beta-lactamase enzyme)** y Optochin disc (differentiate S. pneumoniae from other hemolytic strep) y Oxidase reagent y Salmonella agglutinating antisera y V and XV factors (Identification of Hemophilus influenzae) y Haemophilus influenzae type b antiserum y Neisseria meningitidis agglutinating serum (polyvalent and specific groups A, B, C) ** penicillin, cephalosporins, cephamycins and carbapenems

Oxidase Test (N,N,N',N'-tetramethyl-pphenylenediamine dihydrochloride)
y used to detect the presence of oxidase enzymes produced by a variety of bacteria. y oxidase test can be used to differentiate between genera : y Moraxella (+) and Neisseria (+) from Acinetobacter (-) y Aeromonas (+), Plesiomonas shigelloides (+), and Vibrio (V+) from other Enterobacteriaceae (-) y Burkholderia gladioli (-) and B. mallei (V) from B. cepacia (+) and B. pseudomallei (+) y Pseudomonas aeruginosa , Neisseria gonorrhoeae and Campylobacter jejuni are oxidase-positive pathogens

Oxidase Test

V (Heme) & X (NAD) factor test

BACTEREMIA
y Presence of bacteria in the blood stream y Caused by: y Post-operative complications y Intravascular catheters y Localized infection y In-patient mortality 20% y Shock and organ failure

90%

BACTEREMIA
y Early detection

77% y Differentiation between Gram positive or Gram negative is one of the most important factors.

WHOLE BLOOD CULTURE
y Most sensitive and the gold standard. y It requires incubation, subculturing, biochemical tests. y 3-7 days TAT

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METHODS TO IMPROVE BLOOD CULTURE YIELD
SKIN PREPARATION
y Strict aseptic technique (Chandrasekar & Brown, 1994). y Povidone iodine versus 2% tincture of iodine (Little et

al., 1999) y Tincture of iodine -> significant reduction in skin flora contamination due to the faster onset of action y 0.5% Alcoholic chlorhexidine (Mimoz et al., 1999) y Reduced the incidence of blood culture contamination y 15 to 30 seconds

METHODS TO IMPROVE BLOOD CULTURE YIELD
TIMING OF BLOOD EXTRACTION y Continuous Bacteremia y Intermittent Bacteremia y Chandrasekar & Brown, 1994
y

drawing multiple blood culture sets in 24 hour period have been shown to detect intermittent bacteremia Similar yields if samples were collected within 2 hours or within 24 hours Patients with antibiotics, samples drawn close to the time antibiotic conc. have reached low levels.

y

Li et al., 1994
y

y

Mylotte & Tayana, 2000
y

METHODS TO IMPROVE BLOOD CULTURE YIELD
VOLUME OF BLOOD y Is the most important factor ( Shafazand & Weinacker, 2002) y At least 10 mL, provide the highest yield and lowest number of false-negative blood culture results (Mermel and Maki, 1993). y 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 TREATMENT
y

y

y

y

Significant 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 cultures
y Incubation y Subcultures y Final processing y Antimicrobial susceptibility testing (AST) y Detection 0f contaminants

Incubation time
y Manual y 35-37 C y Inspected twice a day for at least 3 days for signs of microbial growth y Sedimented red blood y Signs of microbial growth
y y y y y y y

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.

y Perform gram stain

Subcultures
y for Gram-negative rods: MacConkey agar, Kligler iron agar, motilityindole urease (MIU) medium, Simmons citrate agar; y for small Gram-negative rods: blood agar; y for staphylococci: blood agar, mannitol salt agar; y for streptococci: blood agar with optochin, bacitracin, and tellurite discs, sheep blood agar for the CAMP test, and bile esculin agar.

Subculture
y Microorganisms may grow without producing turbidity or visible alteration of the broth. y e.g. S. Pneumoniae (autolysis and die rapidly) y Subculture in chocolate agar after 18-24 hours incubation. y After 7 days of incubation without growth inoculate in thioglycollate broth (incubate for another 3 days)

Antimicrobial susceptibility testing
y Time is gold in BLOOD CULTURE. y Gram stain result negative rods

gram positive cocci (Staph) or gram

y Swab dipped into the turbid broth swab inoculate in Mueller-Hinton medium (95% of cases correlate with the standardized test)

Contaminants
y Aseptic skin preparation y Aseptic procedures during inoculation and subcultures y Usual contaminants y S. epidermidis, P. acnes, diphtheroids, Acinetobacter spp. and Bacillus spp.

True bacteremia
y if the same organism grows in two bottles of the same blood specimen; y if the same organism grows in cultures from more than one specimen; y if growth is rapid (within 48 hours); y if different isolates of one species show the same biotypes and antimicrobial-susceptibility profiles.

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