Blood Culture

To ensure the blood specimen is not contaminated with normal skin flora.

Defat the skin with 70% alcohol wipe, apply iodine tincture or chlorhexidine and allow to dry

Blood volume; higher blood volume increases probability that lab will recover the organism, making total volume more important than number of specimens.

Adults: optimally 2 sets of cultures using 10-20 mL per culture; infants and children: 1-5 mL per culture (volume drawn can be dependent on patient's weight).

Collect blood before antibiotics are given to patient is most optimal; 2-3 culture sets (1 set = 1 aerobic & 1 anaerobic bottle) are sufficient if volume is adequate and may vary depending on cases (i.e. if blood is collected pre or post antibiotic).

• Generally 2 or 3 sets of blood cultures are collected an hour apart to account for intermittent sepsis;
• transport blood cultures at room temperature;
• incubate bottles at 35° to 37°C.

Organisms are difficult to recover from clotted specimens, so blood must be treated with an anticoagulant.

Sodium polyanethol sulfonate (SPS) is an anticoagulant that is anti-phagocyte and anti-complement, inhibits some antibiotics, and inhibits all Peptostreptococcus anaerobius and some strains of Neisseria species, Gardnerella vaginalis, and Streptococcus milliformis.

• Citrate (blue top),
• heparin (green top),
• oxalate (gray top),
• EDTA (ethylenediaminetetraacetic acid) (lavender top).

Dilution of blood to medium is a factor; traditionally a 1:10 ratio of blood to medium is used, but enhanced recovery occurs with a 1:5 ratio in conventional blood cultures. (commercial/automated systems specify their own appropriate dilutions)

Blood culture media typically contain nutrient broth and anticoagulant; varying broths include trypticase soy broth, brain-heart infusion, peptone-supplemented broth, thioglycollate broth, and Columbia or Brucella broth.

Conventional blood cultures are incubated in a stationary incubator and bottles are visually examined for signs of growth such as hemolysis, gas, clumping, and turbidity.

Bottles are periodically blind subcultured to CHOC at 24 and 48 hours, cultures are signed out at 7 days, and specific rule-outs such as Brucella are held for 21 days.

It's a biphasic (both liquid and solid media), self-contained subculture system with a conventional blood bottle attached to a paddle (chamber with slide coated with agar).

The agar paddle contains CHOC, MAC, and malt agar; it is periodically bathed in the blood-broth mixture, examined for growth daily, subcultured at 24 and 48 hours, and a larger volume of broth is subcultured.

Isolator/Isostat is a lysis centrifugation system that is good for intracellular, fastidious, or slow growing organisms.

Instead of regular blood culture media, blood is collected in media containing saponin which lyses RBC & WBC, and SPS & EDTA are used as anticoagulants.

The tube is centrifuged, the supernatant is discarded, and the sediment is vortexed and placed on appropriate solid agar.

Instrumentation allows for continuous blood monitoring, is more hands-off, increases sensitivity, and reduces the need to blind subculture.

Each instrument system uses different methodologies and algorithms to flag for positive blood culture growth.

It uses colorimetric detection of CO₂ by detecting an increase in CO₂ via generation of free hydrogen ions, which cause a color change in the sensor.

It uses TSB (tryptic soy broth) and SPS (sodium polyanethol sulfonate).

Uses fluorescent light detection of CO₂; increased CO₂ generates free hydrogen ions causing a decrease in pH, which in turn increases fluorescent output of the sensor, utilizing an LED sensor read by a photodetector.

It uses a soybean casein digest broth and is the instrument used in the UCLA.

Detects pressure changes; decreased pressure indicates O₂ consumption, while increased pressure indicates production of CO₂, H₂, and N₂.

The aerobic bottle uses soycasein-peptone broth, and the anaerobic bottle uses protease-peptone broth.

Positive blood cultures are high priority and should be processed as quickly as possible, with identification and antimicrobial susceptibility tests performed.

A Gram stain smear is the first step; it determines the morphotype of the organism and guides setup of appropriate media/plates for the morphotype seen.

GPC in clusters → BAP; GPC in pairs/chains → BAP/CHOC; GNR → BAP/MAC; PLGPR/GPR → BAP/CHOC; small GNR/GNCB → BAP/CHOC/MAC.

Repeat Gram stain one more time and confirm with Acridine Orange (fluorescent stain) to visualize small or faintly staining organisms.

Perform blind subculture on BAP/CHOC/THIO and hold for 72 hours; if anaerobic bottle, also subculture to Brucella Blood Agar Plate and incubate anaerobically; return blood culture bottle to instrument within 3 hours.

No; not all bottles flagged positive by the instrument are true positive.

An increase in metabolically active cells that produce CO₂, such as leukocytosis or leukemia, which typically produce linear or flat graphs rather than the logarithmic graph seen with true bacterial growth.

Overfilling blood culture bottles or obstruction of the instrument's ability to read the bottle, including labels obscuring the detector or a bottle defect.

BCID systems provide rapid molecular blood culture diagnostics by testing the direct blood specimen from the bottle, including Luminex Verigene, BioFire FilmArray, and Accelerate Pheno System (can perform sensi on GNR).

BCID provides rapid turnaround time for identification (about 2 hours), allowing more targeted antibiotic therapy or rule-out of potential contaminants such as CoNS or SA.

BCID systems can be costly, may detect non-viable nucleic acid (FDA class 2 recall due to blood culture media contaminated with DNA on non-viable organism), and are limited by the target organism available on the panel.

The cobas® eplex system developed by GenMark.

One of three panels is selected based on the Gram stain result: BCID-GP (gram positive panel), BCID-GN (gram negative panel), or BCID-FP (fungal panel).

Can also detect resistance markers such as mecA, vanA, and CTX-M.

Early identification of the organism is of utmost importance; prior to BCID, conventional biochemicals were set up directly from blood cultures to shorten time to identification.

• GPCCL: perform tube coagulase by inoculating blood directly into rabbit plasma tube; if SA, perform PBP2a for MRSA.
• GPCPr/Ch: use vancomycin 30 mcg and P disk, 6.5% NaCl, and bile-esculin slant by placing Va and P disks on BAP already plated with blood and inoculating blood directly onto 6.5% NaCl tube and bile-esculin slant.

• Small GPR: inoculate blood directly onto TSI slant and stab the butt.
• GNR: perform TSI, API20E, and preliminary Kirby-Bauer disk diffusion using the same TSI procedure, inoculating a few drops of blood into 5 mL saline for API20E, and inoculating MH with 4 drops of blood, streaking and applying antibiotics per usual MH protocol.

• Gram-negative rods
• Enterococcus species
• Staphylococcus aureus
• Streptococcus pneumoniae
• Haemophilus influenzae
• Streptococcus pyogenes
• Streptococcus agalactiae
• Large colonies beta-hemolytic Streptococci
• Listeria monocytogenes

Perform full species-level identification and antimicrobial susceptibility testing.

• Viridans group Streptococcus
• Coagulase negative Staphylococcus, not lugdunensis
• Corynebacterium species, not jeikeium
• Bacillus species, not anthracis
• Propionibacterium (Cutibacterium) acnes

Perform full identification and susceptibility testing if recovered from multiple blood culture sets; exception: perform full ID and susceptibility on all isolates from patients ≤ 3 months old regardless of number of positive sets.

It is a facultative intracellular pathogen (lives in phagocytes) and its colony morphology and biochemical reactions can be confused with group B streptococci.

Transmission occurs via contaminated food, especially unpasteurized milk and dairy, with outbreaks linked to soft cheese, ice cream, and milk.

High-risk patients include
• Pregnant women and fetuses - Nonspecific, mild to moderate symptoms but may lead to sepsis and meningitis
• Neonates - Listeria can cross placenta, acquire infection "in utero" resulting in abortion, stillbirth, premature birth, septicemia, or meningitis
• Elderly and immunocompromised (advanced malignancy, renal transplant, diabetes) - mainly causes meningitis and septicemia

Carrier rate is 1-5%.

• Gram stain: Small "ditpheroid-like", coccobacillary, non-sporeforming GPR. (Pairs may be mistaken for Streptococci)

On BAP: small translucent gray colonies with a narrow zone of beta-hemolysis; on McBride agar: colonies appear blue-green.

Biochemicals:
• Catalase (+)
• Motile: Optimal at 25°C (Tumbling motility on wet mount from 2-6 hour broth culture at RT and Umbrella motility on semisolid agar at RT)
• CAMP (+)
• Hippurate (+)
• Esculin (+)

The Anton (ocular) test assesses pathogenicity/virulence; inoculation of the organism into a rabbit's eye causes purulent conjunctivitis.

Cold enrichment enhances recovery of Listeria and Yersinia and is used for epidemiological studies of contaminated specimens.

Although optimum growth is 30-37 °C, Listeria can grow at 4 °C; inoculated UVM-Listeria Selective Enrichment Broth is refrigerated at 4 °C for 3 months, subcultured weekly for 4 weeks, then monthly for the next 2 months.

Sodium hippurate hydrolysis produces glycine, which is detected by ninhydrin; in the deaminated glycine-ninhydrin reaction, ammonia reacts with residual and reduced ninhydrin to form a blue-purple condensation complex.

Procedure:
1. Add 0.1mL sterile water to plastic test tube (glass test tubes may result in false negative)
2. Emulsify 1-3 colonies (more colonies are needed for smaller organisms such as Gardnerella vaginalis)
3. Drop hippurate disk in mixture
4. Incubate for 2 hours at 35-37C
5. Add 2 drops of ninhydrin
6. Mix and reincubate for 30 minutes
7. Interpretation
Positive: Blue-purple color
Negative: No change or light gray
***(Run positive and negative QC to correlate color change (GBS + and GAS -)

Campylobacter jejuni, Group B Streptococcus, Listeria monocytogenes, and Gardnerella vaginalis.

CAMP (Christie, Atkins, and Munch-Peterson) test detects production of a diffusible extracellular hemolytic protein (CAMP factor) by GBS and Listeria monocytogenes that acts synergistically with beta-lysin of Staphylococcus aureus to enhance lysis of RBCs.

Streak the organism to be tested perpendicular to a streak of Staphylococcus aureus ATCC 25923 on BAP within 2 mm and incubate overnight at 35-37 °C.

• Positive: enhanced zone of hemolysis adjacent to where the two streak lines meet (arrowhead may appear but not always);
• negative: no enhancement;
***Some strains of GAS may have a positive CAMP reaction, test should be limited to colonies characteristic of GBS (small zone of hemolysis)

Erysipelothrix rhusiopathiae is primarily an animal pathogen that infects a wide variety of animals, especially swine.

• In swine it causes erysipelas, acquired by ingestion of contaminated food and water with multiplication in tonsils and gut leading to bloodstream spread;
• In humans it causes erysipeloid, a localized cutaneous infection with red skin lesions that may disseminate and cause bacteremia and endocarditis.

Humans become infected through contact with animals or animal products; infection is occupational, commonly affecting butchers, fishermen, and veterinarians.

For skin lesions, collect skin biopsies; for sepsis, collect blood cultures.

• Gram Stain: short slender GPR , may be slightly pleomorphic,
• easily decolorizes
• GS performed from solid media may appear filamentous

• Alpha or non-hemolytic on BAP
• Smooth colonies - small convex circular
• Rough colonies - larger, matte with fimbriated edge

Biochemicals:
• Catalase (-)
• Produce H2S in TSI
• Non-motile

Corynebacterium jeikeium causes nosocomial infections in immunocompromised patients, including bacteremia and bacterial endocarditis, and is associated with indwelling devices.

• It can cause community and nosocomially acquired bacteremia, sepsis, valve endocarditis, meningitis, osteomyelitis, and tissue infections;
• It is highly resistant to most gram-positive antibiotics but is sensitive to vancomycin.

Small coccobacillary pleomorphic gram-positive rods (PLGPR).

• Small pinpoint gray-to-white, non-hemolytic colonies;
• Lipophilic and will not grow on CHOC.

Biochemicals:
•Catalase (+)
•Motility (-)
•Urea (-)
•Nitrate (-)
•Glucose (+)

Normal respiratory and genitourinary tract flora.

It is a common cause of subacute endocarditis, such as damaged valves after dental surgery involving infected gums, reproductive or urinary (genitourinary tract) surgery or operations on the gastrointestinal tract (e.g. GI endoscopy, sigmoidoscopy or in those with artificial heart valves.

Causes approximately 30-40% of endocarditis cases and may also cause dental cavities, wound infections, and brain abscesses.

• Streptococcus mitis/oralis group
• Streptococcus sanguinis group
• Streptococcus salivarius group
• Streptococcus mutans group

Streptococcus anginosus (milleri) group, which is associated with abscesses and other pyogenic infections, and Streptococcus gallolyticus (formerly Streptococcus bovis), where Streptococcus gallolyticus subsp. gallolyticus bacteremia is linked to the presence of colonic cancer.

Perform full identification using API Strep on critical/sterile sites or when isolated from multiple blood culture bottles

When not from critical sites or multiple blood cultures, biochemical identification as viridans group Streptococcus versus Streptococcus anginosus group is sufficient.

It is a major cause of neonatal and perinatal bacteremia and meningitis, Neonates acquire it during passage through birth canal (may acquire it "in-utero" before delivery)

It is part of the normal vaginal flora and rectum and is usually asymptomatic.

May also cause variety of infections in adults, including UTI, skin and soft tissue infections, postpartum fever, and sepsis. Usually in patients with risk factors such as catheters, diabetes, or advanced age.

Enterococcus species mostly cause UTIs.

Enterococcus enter blood stream from UTI, abdominal or pelvic abscess, wounds, and IV devices; patients are usually elderly, immunocompromised, or have underlying diseases.

Speciation is more important in blood cultures because different Enterococcus species can have different antimicrobial susceptibility patterns.

Fully ID with API Strep

• Enterococcus faecalis - Gamma hemolytic (usually)
- Ampicillin S, Synercid R
• Enterococcus faecium - Alpha hemolytic
- Ampicillin R, Synercid S

Enterococcus gallinarum and Enterococcus casseliflavus/flavescens have intrinsically elevated vancomycin results.

It is a major cause of community-acquired pneumonia and a leading cause of bacteremia and sepsis in adults and children.

• In children, it commonly causes Otitis media(middle ear infection) + pneumonia leading to bacteremia;
• In adults, pneumonia may progress to bacteremia.

It is a common cause of meningitis in adults, infants, and toddlers (Meningitis as a result of bacteremia), with most serious infections occurring in infants <3 years old and adults >65 years old.

Virulence is attributable to polysaccharide capsule (resists intracellular killing)

It is the most common agent of prosthetic valve endocarditis and the most common agent of bacteremia in immunocompromised patients.

It reaches the blood via vascular access devices such as catheters and is the most common isolate of IV catheters causing Catheter-Related Bloodstream Infection (CRBI).

IV catheters are the source of bacteremia; catheter tips can be submitted for culture by rolling the catheter tip on a blood agar plate and reporting identification with colony count.

It is also the most common cause of infection of CSF shunts and CSF catheters.

It causes endocarditis, bacteremia, and abscesses.

Beta-hemolytic with white colonies (Must be differentiated from other coagulase negative Staphylococcus)

0.5 mL Moeller decarboxylase broth base and 0.5 mL Moeller decarboxylase broth with ornithine.

• Inoculate both broths with colonies from an overnight culture to create a heavy suspension,
• overlay suspensions with mineral oil to prevent surface oxidation leading to false positives,
• incubate for 5 hours in a 35 °C heat block.

Interpretation:
• POS: broth with ornithine = purple, plain broth = yellow
• NEG: both remain yellow
• INVALID: base broth is purple

HACEK organisms are fastidious gram-negative coccobacilli associated with infective endocarditis.

• Haemophilus species
• Aggregatibacter species
• Cardiobacterium hominis
• Eikenella corrodens
• Kingella spp.

They require media supplemented with hemin (X factor) and/or NAD (V factor) and enriched incubation with 5-10% CO₂.

They do not grow on traditional enteric media such as MAC.

Up to 14 days with conventional blood culture methods and about 5 days in continuous monitoring systems.

Full identification is performed using the RAPID NH panel.

Hib is the most pathogenic type of H. influenzae, is one of six polysaccharide capsule types, and has an available vaccine.

It is part of the normal oral flora and can cause epiglottitis (resulting in obstruction), otitis media, sinusitis, bronchitis, and pneumonia.

• In the pre-vaccine era, H. influenzae was the leading cause of bacteremia and meningitis in children ages 1 month-2yrs;
• nasopharyngeal colonization and otitis media can lead to meningitis; adult meningitis is uncommon;
• meningitis is frequent and presents early in children.

• Increased risk of invasive disease if not vaccinated
• Nasopharyngeal colonization / respiratory infection/otitis media -> enters blood -> meninges
• Can be found in elderly with underlying diseases (head trauma or neurosurgery)

Small pleomorphic gram-negative coccobacilli.

On CHOC: smooth, round, tan colonies (some strains mucoid); will not grow on BAP or MAC but will grow on horse or rabbit BAP.

Biochemical:
• Oxidase and catalase variable
• Require both X and V factor
• Growth only around XV disk (not X, V)
• Growth on Rabbit BAP, no hemolysis
• Satellite Test (+) on BAP
• Satellite Test (-) on Nutrient Agar
• Beta-lactamase positive (30-40%)

Full identification can be performed using the RAPID NH panel.

Enterobacterales and non-fermenting gram-negative rods.

They can be present in any type of infectious disease, and Salmonella typhi causes typhoid fever.

Pseudomonas aeruginosa is the most commonly isolated non-fermenter and infections occur primarily in immunocompromised patients.

It can spread from the GI tract or respiratory tract to the blood and infect heart valves and prosthetic heart valves, especially in IV drug users who inject drugs contaminated with PSA from the environment.

They are found on human mucous membranes and in the environment; infections are rare but usually occur in immunocompromised patients.

They can cause endocarditis, bacteremia, osteomyelitis, and wound infections.

Gram Stain:
• Pediococcus = GPCCl
• Leuconostoc = GPCPrCh

They are alpha-hemolytic and resemble viridans group streptococci and enterococci.

Biochemical:
• Catalase (-)
• Vancomycin resistant - helps differentiate from most Str. viridans and Enterococci

They are nutritionally variant streptococci (NVS) and require pyridoxal (vitamin B6) for growth.

Upper respiratory tract, urogenital tract, and gastrointestinal tract.

Endocarditis, postpartum sepsis, and many other systemic infections.

Gram-positive cocci in pairs and chains (GPCPrCh).

Colonial morphology:
• BAP: No Growth
• CHOC: Viridans Strep-like (small colonies with greening zone)

Biochemical:
• Catalase (-)
• Satellite Test (+) (on BAP)
• Full ID with API Strep, if needed. Otherwise sufficient to ID as NVS

Haemophilus species and nutritionally variant streptococci (NVS).

Hemin; heat stable; supplied by blood agar.

V factor: NAD, heat-labile (supplied by yeast, potato extract and certain bacteria (e.g. Staphyloccci) - Bacteria that needs V factor will form colonies near SA

X factor present in blood agar, strains that need both X and V factors will statellite SA

• X factor not present (no blood)
• Organisms that satellite around Staphylococcus only requires V factor

Growth of colonies adjacent to Staphylococcus aureus streak.

Procedure:
• Streak Haemophilus or suspected NVS across the surface of an agar plate (e.g., BAP).
• Streak Staphylococcus aureus ATCC 25923 perpendicular to the initial streak

Incubate overnight in CO2
• Positive: faint growth or tiny colonies seen near the SA streak
• Negative: no growth around SA streak

Brucellosis (undulant fever); infectious pathogen that requires the use of biological safety level 3 precautions for all manipulations of specimens and cultures

A zoonotic disease where domestic animals serve as reservoirs and an important veterinary pathogen.

By ingestion of unpasteurized contaminated milk or cheese, iInhalation (aerosolization-primary mechanism of transmission in laboratories), or direct contact via skin abrasions and mucous membranes (including the conjunctiva)

Handling infected animals (dairy farmers, slaughterhouse workers, meat inspectors, veterinarians), ingestion of contaminated animal products (especially imported unpasteurized goat cheese in the U.S.), and handling Brucella cultures (laboratorians).

Aggressive vaccination in cattle =↓ risk of infection, ↓ #s in US after 1975

Ranges from asymptomatic infection to serious debilitating disease, and may present as systemic or localized infection.

Intermittent fevers, chills, weakness malaise, body aches, sweating, headache, and weight loss (Lymphadenopathy and splenomegaly are common)

Arthritis, spondylitis, and endocarditis.

Death is uncommon and is most often associated with endocarditis.

Blood and bone marrow aspirates.

BacT/Alert, BACTEC, and ESP (Biphasic Castenada bottles-ok)

Incubation: growing-incubate at least 21 days (New automated Blood culture systems: 10 days)

Gram Stain: Small gram negative coccobacilli or short rods arranged singly or in pairs and short chains. "Fine sand" appearance (Usually poorly stained (resists counterstain)-stain with safranin for ~3 minutes)

• After 48 hr of incubation colonies appear small, convex, smooth, translucent, nonhemolytic
• After 4-5 days colonies are 2-7mm, spheroidal, moist, slightly opalescent, slightly yellow or bluish-white; may become brownish with age

• Facultative, intracellular organism
• Aerobic to capnophilic, no growth under strict anaerobic conditions
• Oxidase (+), urea +, non-motile

Category B Select Agent of Bioterrorism

Gram Stain: Small GNCB
• Usually positive in blood bottles at/after 3 days of incubation
• UCLA protocol: tape all cultures positive for GNCB on 3rd day

Colony: Poorly growing at 24 hours on BAP CHOC, NG on MAC (May require CO2 for initial growth)

Send to public health, if unable to rule-out:
Grows on BAP without need to satellite
• Oxidase +
• Catalase +
• Urea +

• Coxiella burnettii
• Chlamydophila species
• Rickettsia species
• Tropheryma whipplei (Whipple's disease)