SECTION 1 High Yield Microbiology 1 Bacteriology MORGAN A. PENCE Definitions Obligate/strict anaerobe: an organism that grows only in the absence of oxygen (e.g., Bacteroides fragilis). Spirochete Aerobe: an organism that lives and grows in the presence : spiral-shaped bacterium; neither gram-positive of oxygen. nor gram-negative. Aerotolerant anaerobe: an organism that shows signifi- cantly better growth in the absence of oxygen but may Gram Stain show limited growth in the presence of oxygen (e.g., • Principal stain used in bacteriology. Clostridium tertium, many Actinomyces spp.). • Distinguishes gram-positive bacteria from gram-negative Anaerobe : an organism that can live in the absence of oxy- bacteria. gen. Bacillus/bacilli: rod-shaped bacteria (e.g., gram-negative Method bacilli); not to be confused with the genus Bacillus. • A portion of a specimen or bacterial growth is applied to Coccus/cocci: spherical/round bacteria. a slide and dried. Coryneform: “club-shaped” or resembling Chinese letters; • Specimen is fixed to slide by methanol (preferred) or heat description of a Gram stain morphology consistent with (can distort morphology). Corynebacterium and related genera. • Crystal violet is added to the slide. Diphtheroid: clinical microbiology-speak for coryneform • Iodine is added and forms a complex with crystal violet gram-positive rods (Corynebacterium and related genera). that binds to the thick peptidoglycan layer of gram-posi- Gram-negative: bacteria that do not retain the purple color tive cell walls. of the crystal violet in the Gram stain due to the presence • Acetone-alcohol solution is added, which washes away of a thin peptidoglycan cell wall; gram-negative bacteria the crystal violet–iodine complexes in gram-negative appear pink due to the safranin counter stain. cells walls due to thin layer of peptidoglycan. Gram-positive: bacteria that retain the purple color of the • Safranin counter-stain is added to stain gram-negative crystal violet in the Gram stain due to the presence of a bacteria. thick peptidoglycan cell wall. • Slide is viewed on low power to quantitate polymorpho- Gram-variable: bacteria that partially retain the purple nuclear cells (PMNs) and epithelial cells and on high color of the crystal violet in the Gram stain; most com- power to quantitate bacteria. monly seen with Bacillus spp., Clostridium spp., Acineto- bacter spp., Streptococcus pneumoniae. Classification of Bacteria Microaerophile: an organism that requires a low level of oxygen for growth, increased oxygen may inhibit growth Classification is based on growth pattern (aerobic vs. anaer- (e.g., Campylobacter spp.). obic), Gram stain reaction, and morphology (Figs. 1.1–1.3, Nonfermenters: gram-negative rods that do not utilize Tables 1.1 and 1.2). glucose for growth (e.g., Pseudomonas, Achromobacter, Acinetobacter, etc.). Note: Nonfermenters are not the Blood Cultures same as non-lactose-fermenting gram-negative rods (re- Collection ferring to the lack of reaction/lactose utilization on Mac- Conkey or other lactose-containing agar). The two terms • Proper skin preparation/disinfection is essential to pre- are sometimes used interchangeably, which is incorrect. vent contaminated blood cultures. Obligate aerobe: an organism that grows only in the pres- • One blood culture set is composed of one aerobic bottle ence of oxygen (e.g., Pseudomonas aeruginosa). and one anaerobic bottle. 1 2 SECTION 1 High Yield Microbiology Aerobes Gram Gram positive negative Cocci Rods/Bacilli Cocci Rods/Bacilli Enterobacteriaceae Staphylococcus Bacillus Neisseria (E. coli, etc.) Nonfermenters Moraxella Streptococcus Corynebacterium (Pseudomonas, catarrhalis Acinetobacter, etc.) Enterococcus Listeria Pasteurella Granulicatella/ Erysipelothrix HACEK organisms Abiotrophia • Fig. 1.1 Classification of aerobic bacteria. • Even if anaerobes are low on differential, an anaerobic • Bacillus. bottle should still be collected. → Most bacteria grow • Coagulase-negative staphylococci (not Staphylococcus faster in the anaerobic bottle. lugdunensis). • Collect ≥2 sets from separate sites before administration • Coryneform gram-positive rods. of antibiotics. • Nonpathogenic Neisseria. • Collect up to three blood cultures per day if intermittent • Viridans group streptococci (more likely to be true bacteremia is suspected (due to undrained abscesses, etc.). pathogens in hematology/oncology patients). • Organisms associated with gastrointestinal neoplasia Volume • Streptococcus gallolyticus, Streptococcus infantarius, Streptococcus alactolyticus, Streptococcus lutetiensis, Volume is the most important factor for successful blood Streptococcus equinus (formerly Streptococcus bovis group). cultures. • Clostridium septicum • Most septic patients have 1–5 CFU/mL in their blood. • Also associated with hematologic malignancy. • Bacteremia may be missed by drawing too little blood. • Swarming morphology on agar. • Adults: 20 mL should be collected per set (10 mL per • Causes of false-negative blood cultures bottle). • Too little volume collected. • Pediatrics: Weight-based and age-based guidelines exist. • Administration of antimicrobials prior to collection. • Weight-based guidelines recommend collecting • Organism that does not grow in standard blood cul- 1%–4% of the patient’s total blood volume. ture bottles (Bartonella, etc.). • If pediatric bottles are used, a maximum of 5 mL can be added to each bottle. Specimens for Bacteriology Culture Interpretation • The success of a culture depends on the quality of the • Common contaminants specimen submitted! • Anaerobic gram-positive cocci. • Garbage in → garbage out. CHAPTER 1 Bacteriology 3 Anaerobes Gram Gram positive negative Cocci Rods/Bacilli Cocci Rods/Bacilli Peptostreptococcus Clostridium Veillonella Bacteroides anaerobius Parvimonas Actinomyces Fusobacterium micra Finegoldia Cutibacterium Prevotella magna (Propionibacterium) Staphylococcus Eggerthella/ Porphyromonas saccharolyticus Eubacterium • Fig. 1.2 Classification of anaerobic bacteria. Routine Cultures Miscellaneous bacteria • The more specimen, the better! • There is a common misconception that the microbiology lab only needs a small amount of specimen. Obligate intracellular Spirochetes • Tissues and aspirates/fluids are preferred over swabs. bacteria • If a swab is the only option, a flocked swab, such as the ESwab, is preferred. • Traditional rayon swabs only release 3 of every 100 Chlamydia/ bacteria onto a plate. Borrelia Chlamydophila • For wound specimens, attention to skin decontamina- tion is critical. • Specimens should be taken from the advancing mar- gin of the lesion. Coxiella • Do not send superficial swabs of decubitus ulcers. Leptospira Anaerobic Cultures Rickettsiales • Sites with normal anaerobic flora are not appropriate for (including anaerobic culture. Examples include, but are not limited Treponema Rickettsia, Orientia, to: Ehrlichia, and Anaplasma) • Mouth. • Throat and nasopharyngeal swabs. • Fig. 1.3 Classification of miscellaneous bacteria. • Stool. 4 SECTION 1 High Yield Microbiology TABLE 1.1 Gram Stain Interpretation Gram Stain Result Possible Organisms Figures Gram-positive cocci 1. Staphylococcus (including in clusters Staphylococcus aureus and coagulase-negative staphylococci) 2. Micrococcus (often found in tetrads) 3. Aerococcus 4. Rothia (formerly Stomatococcus) Gram-positive cocci 1. Streptococcus pneumoniae (may in pairs (lancet- stain gram-variable or gram- shaped) negative) Gram-positive cocci 1. Viridans group streptococci in pairs and (including some isolates of S. chains pneumoniae) 2. Beta-hemolytic streptococci (e.g., Streptococcus pyogenes) 3. Enterococcus 4. Abiotrophia and Granulicatella (formerly known as nutritionally variant streptococci) Continued CHAPTER 1 Bacteriology 5 TABLE 1.1 Gram Stain Interpretation—cont’d Gram Stain Result Possible Organisms Figures Gram-positive rods 1. Bacillus (may be big and boxy, – regular may have spores) 2. Clostridium (may be big and boxy, may have spores) 3. Listeria (may appear coccobacillary) 4. Lactobacillus 5. Eggerthella Gram-positive rods 1. Corynebacterium – coryneform 2. Cutibacterium (formerly Propionibacterium) 3. Actinomyces (sulfur granules may be seen on histopathology) 4. Erysipelothrix Beaded gram- 1. Mycobacterium positive rods 2. Nocardia 3. Actinomyces (beaded morphology is more commonly seen on histopathology) Continued 6 SECTION 1 High Yield Microbiology TABLE 1.1 Gram Stain Interpretation—cont’d Gram Stain Result Possible Organisms Figures Branching, 1. Nocardia filamentous gram- 2. Streptomyces positive rods 3. Gordonia 4. Tsukamurella Gram-variable rods 1. Bacillus (and related genera: Paenibacillus, Lysinibacillus) 2. Clostridium 3. Gardnerella vaginalis 4. Leptotrichia Gram-negative cocci 1. Veillonella 2. Acidaminococcus 3. Megasphaera CHAPTER 1 Bacteriology 7 TABLE 1.1 Gram Stain Interpretation—cont’d Gram Stain Result Possible Organisms Figures Gram-negative 1. Neisseria (including Neisseria diplococci meningitidis and Neisseria gonorrhoeae) 2. Moraxella catarrhalis Gram-negative 1. Haemophilus coccobacilli 2. Acinetobacter (may stain gram- variable) 3. Aggregatibacter 4. Moraxella 5. Pasteurella multocida 6. Bacteroides 7. Francisella tularensis (tularemia) 8. Brucella Tiny gram-negative 1. Francisella tularensis (tularemia) rods/“junky” 2. Brucella Gram stain Continued 8 SECTION 1 High Yield Microbiology TABLE 1.1 Gram Stain Interpretation—cont’d Gram Stain Result Possible Organisms Figures Gram-negative rods 1. Enterobacteriaceae 2. Nonfermenters