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SECTION 1 High Yield 1 Bacteriology

MORGAN A. PENCE

Definitions Obligate/strict anaerobe: an organism that grows only in the absence of (e.g., fragilis).

Spirochete Aerobe: an organism that lives and grows in the presence : spiral-shaped bacterium; neither gram-positive of oxygen. nor gram-negative. : an organism that shows signifi- cantly better growth in the absence of oxygen but may show limited growth in the presence of oxygen (e.g., • Principal stain used in bacteriology. tertium, many spp.). • Distinguishes gram-positive from gram-negative Anaerobe : an organism that can live in the absence of oxy- bacteria. gen. /: rod-shaped bacteria (e.g., gram-negative Method bacilli); not to be confused with the Bacillus. • A portion of a specimen or bacterial growth is applied to /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). 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 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 ; 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., pneumoniae. Classification of Bacteria : 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., 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., , 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. : an organism that grows only in the pres- • One set is composed of one aerobic bottle ence of oxygen (e.g., ). and one anaerobic bottle.

1 2 SECTION 1 High Yield Microbiology

Aerobes

Gram Gram positive negative

Cocci Rods/Bacilli Cocci Rods/Bacilli

Enterobacteriaceae Bacillus Neisseria (E. coli, etc.)

Nonfermenters Moraxella Streptococcus Corynebacterium (Pseudomonas, catarrhalis Acinetobacter, etc.)

Enterococcus Pasteurella

Granulicatella/ Erysipelothrix HACEK organisms

• 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 • -negative staphylococci (not Staphylococcus faster in the anaerobic bottle. lugdunensis). • Collect ≥2 sets from separate sites before administration • Coryneform gram-positive rods. of . • 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 , etc.). 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 (formerly group). cultures. •  • 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 (, 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 / 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 / bacteria onto a plate. • 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. Anaerobic Cultures • Sites with normal anaerobic flora are not appropriate for (including anaerobic culture. Examples include, but are not limited , 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 and coagulase-negative staphylococci) 2. Micrococcus (often found in tetrads) 3. 4. Rothia (formerly Stomatococcus)

Gram-positive cocci 1. (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., ) 3. 4. Abiotrophia and (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 ) 2. Clostridium (may be big and boxy, may have spores) 3. Listeria (may appear coccobacillary) 4. 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. 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: , Lysinibacillus) 2. Clostridium 3. 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 ) 2.

Gram-negative 1. coccobacilli 2. Acinetobacter (may stain gram- variable) 3. 4. Moraxella 5. 6. Bacteroides 7. tularensis () 8. Brucella

Tiny gram-negative 1. (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. 2. Nonfermenters (i.e., Pseudomonas, Achromobacter, Acinetobacter, etc.) 3. HACEK organisms 4. Pasteurella 5. Anaerobic gram-negative rods 6. Bacillus (although gram-positive, it can stain gram-variable or gram- negative) 7. Clostridium (although gram- positive, it can stain gram-variable or gram-negative)

Gram-negative rods 1. – fusiform 2. Leptotrichia 3. Capnocytophaga

Gram-negative rods 1. Campylobacter – curved 2. 3. Arcobacter 4. Anaerobiospirillum 5. (comma-shaped)

(From Inokuchi R, Ishida T, Maeda J, Nakajima S, Yahagi N, Matsumoto A. Am J Emerg Med. 2014;32(7):812e.1.)

HACEK, Haemophilus, Aggregatibacter, , , . CHAPTER 1 Bacteriology 9

TABLE TABLE Notable Nomenclature Changes/ Gram Stain Buzzwords 1.2 1.3 Reclassifications When You Hear This Think This Former Name Current Name Gram-negative diplococci Aggregatibacter – CSF actinomycetemcomitans actinomycetemcomitans Gram-negative diplococci Neisseria gonorrhoeae Actinobaculum schaalii Actinotignum schaalii – genital source, urine, joint fluid Bacteroides distasonis Parabacteroides distasonis Gram-negative diplococci Moraxella catarrhalis Clostridium difficile difficile – respiratory source aerogenes aerogenes Big and boxy Bacillus (Be careful: intrinsic Clostridium resistance is still the same as Enterobacter spp.) Gram-variable Acinetobacter Bacillus Enterobacter amnigenus Lelliottia amnigena Clostridium Enterobacter gergoviae Leptotrichia Streptococcus pneumoniae Aggregatibacter aphrophilus Lancet-shaped S. pneumoniae Aggregatibacter segnis Seagull-shaped/gull wings Campylobacter spp. Anaerococcus (only Peptostreptococcus Atopobium Spores Bacillus and related genera remaining is P. magna (Paenibacillus, Lysinibacillus) anaerobius) Parvimonas micra Clostridium Tiny gram-negative rods Brucella Slackia Francisella Propionibacterium acnes Streptococcus “milleri” group group (never a confirmed (composed of S. anginosus, taxonomic name) Streptococcus • Superficial wound specimens, including superficial intermedius, decubitus ulcer specimens. Streptococcus • Urine (unless collected by suprapubic aspirate). constellatus) • Vaginal and cervical swabs. • The following specimens are acceptable for anaerobic culture: • Blood. • Deep wounds/abscesses collection by aspirate or • Stool pathogens begin to die within minutes of surgically. passage. • Deep sinus specimens. • If raw stool is submitted, sensitivity is decreased, espe- • Sterile body fluids. cially for and Campylobacter. • Tissues. Anaerobic Transport Associations • To ensure recovery of anaerobes, specimens submitted Arthropod-Borne Pathogens (Table 1.7) for anaerobic culture must be transported to the lab in anaerobic transport: • Clinically important arthropod vectors include , lice, • Anaerobic transport vials. mites, and ticks. • Capped syringe with air removed. • Arthropods can transmit a variety of , • Tissue specimens that are 1 cm3 or larger can be sub- including bacteria, , and parasites. mitted in a sterile cup. • Swabs are not preferred, but if necessary, an anaerobic Biothreat (BT) Agents (Table 1.8) swab must be used. • Due to the highly infectious nature of these organisms, Stool Cultures the clinical microbiology laboratory should be notified when a biothreat agent is suspected. The lab can then • Stool must be placed into Cary–Blair or modified Cary– take the necessary precautions to prevent exposure of Blair transport media immediately upon passage. laboratory personnel to the infectious agent. 10 SECTION 1 High Yield Microbiology

TABLE 1.4 Routine Bacteriologic Media – Aerobic Cultures Media/Primary Use What Grows? What Doesn’t Grow Blood agar/demonstration of Majority of pathogens – gram-positive and • Haemophilus gram-negative • Some Aggregatibacter spp. • Some Neisseria gonorrhoeae isolates • Francisella • Granulicatella/Abiotrophia • Organisms that do not grow in routine bacterial cultures (Table 1.14) Almost all organisms, including fastidious • Some Corynebacterium do not grow organisms as well on chocolate agar as on blood Organisms that grow only on chocolate agar agar: • Organisms that do not grow in routine • Haemophilus bacterial cultures (Table 1.14) • Some Aggregatibacter spp. • Some N. gonorrhoeae isolates • Francisella • Granulicatella/Abiotrophia MacConkey agar/Lactose- Nonfastidious gram-negative rods • Gram-positive organisms (some • Enterobacteriaceae Bacillus may have breakthrough • Most nonfermenters growth) • Fastidious gram-negative organisms, including, but not limited to: • Brucella • HACEK organisms • Francisella • Moraxella • Neisseria • Pasteurella

• Most clinical labs are “sentinel labs,” and it is their job to • Eikenella corrodens. “recognize, rule out, and refer” any suspected BT agent. • Kingella. If a BT agent cannot be ruled out, the isolate should be • Bartonella. sent to the local lab. • Brucella. • Sentinel labs should not attempt to definitively identify • Chlamydia/Chlamydophila. suspected BT agents. • . • Suspected BT agents should not be subjected to • Legionella. MALDI-TOF or tested on automated identification • . platforms (see Identification section below). • Tropheryma whipplei.

Important Pathogens in Bite Wounds (Human Important Pathogens in Cystic Fibrosis or Animal) Patients • Anaerobic oral flora Prevotella,( etc.). • Staphylococcus aureus. • Eikenella. • Achromobacter spp. • Pasteurella. • cepacia. • Staphylococcus intermedius/pseudintermedius. • Inquilinus limosus. • Streptococcus anginosus group. • Pandoraea spp. • P. aeruginosa. Important Bacterial Pathogens in Culture- • Stenotrophomonas maltophilia. Negative • Other nonfermenters • HACEK organisms (less likely to be culture-negative due to good recovery in current blood culture systems) Important Pathogens in • Haemophilus. • Aggregatibacter. • Anaerobes. • Cardiobacterium. • Enterobacteriaceae (neonates). CHAPTER 1 Bacteriology 11

TABLE 1.5 Biochemical Characteristics Biochemical Positive Negative Alpha-hemolysis (greening of the agar) • Viridans group streptococci • Staphylococcus (including Streptococcus pneumoniae) • Some enterococci Beta-hemolysis • Staphylococcus aureus • Most coagulase-negative • Beta-hemolytic streptococci staphylococci • (double zone of • Viridans group streptococci (with the hemolysis) exception of S. anginosus group, • Aeromonas which can be beta-hemolytic) • coli • Pseudomonas aeruginosa (used primarily for gram- • Staphylococcus • Enterococcus positives) • Bacillus • Streptococcus • Corynebacterium • Aerococcus • Actinomyces • Clostridium Coagulase • S. aureus • All other Staphylococcus spp. • Staphylococcus intermedius • Staphylococcus pseudintermedius • Staphylococcus schleiferi Oxidase (gram-negatives) • Aeromonas • All Enterobacteriaceae except P. • Moraxella shigelloides • Neisseria • Acinetobacter • Pasteurella • Stenotrophomonas maltophilia • • Pseudomonas (except P. luteola and P. oryzihabitans) Indole (gram-negatives) • Aeromonas • Acinetobacter • E. coli • Pseudomonas • Morganella morganii • Plesiomonas shigelloides • • Providencia • Pasteurella

• . • In addition to the bacterial zoonotic pathogens in Table • Moraxella catarrhalis. 1.10, many viruses, fungi, and parasites also cause zoonoses. • P. aeruginosa. • S. aureus. Gastrointestinal • S. pneumoniae. • S. pyogenes. : Bacterial Associations (Table 1.11) Emerging Pathogens (Table 1.9) • Many bacteria and bacterial toxins are associated with contaminated or undercooked foods. • Several “emerging” pathogens have been reported in • The CDC estimates that there are 48 million cases of recent years. foodborne illness each year, resulting in 128,000 hospi- • While some of the organisms may truly represent emerg- talizations and 3,000 deaths. ing pathogens, it is likely that many of the organisms were previously misidentified due to the limitations of Stool Pathogens biochemical identification methods. • Stool culture is the gold standard but is being replaced Zoonotic Pathogens (Table 1.10) by culture-independent tests such as antigen testing and molecular testing. • Zoonoses are infections that are naturally transmitted • If using culture, check with performing lab to see which between animals and humans. pathogens are being screened. 12 SECTION 1 High Yield Microbiology aeruginosa Think This: Pseudomonas Picture This: Picture Biochemical Buzzwords Green pigment on Green MacConkey agar

• Beta-hemolytic • Metallic • sheen • Oxidase positive • Beta-hemolytic • Oxidase • negative Indole • positive Lactose fermenting Gram-negative rod – Gram-negative rod – Gram-negative rod When You Hear This: When You 1.6 TABLE TABLE CHAPTER 1 Bacteriology 13 aureus Think This: Staphylococcus Picture This: Picture Biochemical Buzzwords—cont’d

clusters – • Beta-hemolytic • Yellow pigment • Catalase positive When You Hear This: When You Gram-positive cocci in 1.6 TABLE TABLE 14 SECTION 1 High Yield Microbiology

TABLE • STEC that produce 2 or Shiga toxins 1 1.7 Associations with Arthropod-Borne Bacteria and 2 are more associated with severe disease than STEC that produce only Shiga toxin 1. Associated Associated Arthropod Pathogens Diseases Fleas Murine Identification Methods Several organisms have recently undergone significant name Lice changes (Table 1.3). Biochemical-Based Methods (Tables 1.4, 1.5, 1.6) Mites • Biochemical methods consist of simple spot tests, such Ticks Anaplasma as catalase, oxidase, coagulase, as well as more complex phagocytophilum biochemical testing. Relapsing fever • Spot tests allow presumptive identification or identifica- Borrelia parkeri Relapsing fever tion of certain organisms, such as S. aureus and S. pyo- Borrelia turicatae Relapsing fever genes, in minutes. Ehrlichia spp. Francisella tularensis Tularemia • For most bacteria, biochemical methods require hours, African tick-bite fever occasionally days. • Biochemically-inert organisms (intrinsically or those found in , etc.) are difficult to identify by -bio Rocky Mountain chemical methods. spotted fever • Commercial instruments/databases are not up to date with the most recent classification changes or the newest microbes. •  and Shigella performed by all labs. Matrix-Assisted Laser Desorption/Ionization • Some labs screen for Aeromonas and Plesiomonas. Time-of-Flight Mass Spectrometry (MALDI- • Some labs add a special agar for Campylobacter. • Campylobacter culture only detects Campylobacter TOF MS) jejuni and . • Identification based on protein content→ primarily ribo- • Other are inhibited. somal proteins due to their relative abundance in the • Shiga toxin-producing E. coli (STEC) bacterial cell. • Some labs use only MacConkey with sorbitol for • Only a single colony is required for identification. detection of E. coli O157. • Method: • Some labs use an immunoassay to detect all • Colony is added to a target plate and covered with serotypes of STEC. matrix. • Some labs do both (recommended by CDC). • Colony/matrix is shot with a laser, which ionizes the • Stool must be processed within 30 minutes or placed in proteins. transport buffer. • Ionized proteins travel through a vacuum and hit a • Shigella and Campylobacter begin dying upon passage. detector at a rate proportional to their mass/charge • Antigen testing ratio. • Campylobacter antigen: detects C. jejuni, C. coli. • This creates a spectrum or fingerprint, which is com- • O157 antigen: poor positive predictive value in low pared to a database to determine an identification. incidence settings. • Fast, accurate identification of aerobes and anaerobes • PCR/molecular within minutes. • Most common are multiplex gastrointestinal (GI) panels. • Second most accurate identification method after sequencing. • GI panel targets vary by manufacturer. • Much more accurate than biochemical methods for • Increased sensitivity compared to culture. certain bacterial groups. • Also detect dead bacteria; results may remain positive 1–4 weeks after . 16S rDNA Sequencing of Bacterial Isolates • Shiga-toxin testing (EIA or PCR) • Detects Shiga toxin 1 and Shiga toxin 2 associated • Partial (more common) or complete sequencing of the with STEC. 16S rDNA gene. • Detection does NOT imply Shigella infection. • 16S gene alone is not sufficient for all bacterial genera. • Does not provide information about serotype → how- • Some genera require additional sequencing targets for ever, serotype is only needed for public health reasons complete identification. → not clinically useful since any serotype can cause • Usually limited to reference labs or large academic labs. hemolytic uremic syndrome (HUS). • Turnaround time is usually slow.