Bacteriology Flashcards
Sites that should not be cultured for anaerobes
stool
skin
vagina
urethra
oropharynx
Characteristics of blood culture
Each bottle has 10 ml blood (adults)
2 bottle set (1 aerobic and 1 anaerobic) collected from one site
multiple sets obtained from multiple sites: need 3 sets
Cell wall in gram + and - organisms
- Gram positive: thick peptidoglycan cell wall without outer membrane; retains purple crystal violet/iodine stain during decolorization
- Gram negative: thin peptidoglycan cell wall with outer membrane; lose crystal violet/iodine during decolorization and are visualized with the red safranin stain
Appearance of the following organisms on gram stain
- Strep pyogenes
- Nonpyogenic strep
- Strep pneumoniae
- Enterococcus species
- Neisseria
- vibrio
- campylobacter
- yersinia
- legionella
Gram positive cocci:
- pairs and short chains
- long chains
- lancet shaped diplococci
- diplococci
Gram negative cocci
Neisseria - coffee bean shaped diplococci
Gram negative rods
- curved, comma shaped, short
- curved, thin (seagulls or corkscrew)
- closed safety pin
- invisible in primary smear
Pleomorphic gram positive rods
- Aerobic
- Aerotolerant
- Anaerobic, nonspore forming
- Aerobic
- Corynebacterium
- Other coryneforms (e.g., Rothia, arthrobacter)
- Non-nocardia actinomycetes
- Aerotolerant
- Actinomyces
- Bifidobacterium
- Anaerobic, nonspore forming
- Propionibacterium
Small to medium gram positive rods
- Aerobic
- Aerotolerant
- Anaerobic, nonspore forming
- Anaerobic, spore forming
- Aerobic
- Listeria
- Erysipelothrix
- Aerotolerant
- Lactobacillus strains
- Anaerobic, non spore forming
- Eubacterium
- Lactobacillus
- Anaerobic, spore forming
- Clostridium ramosum
Large gram positive rods
- Aerobic
- Anaerobic, nonspore forming
- Anaerobic, spore forming
- Aerobic
- Bacillus
- Anaerobic, nonspore forming
- Clostridium perfringens
- Anaerobic, spore forming
- Other clostridia
Branching, filamentous aerobic and anaerobic nonspore forming gram positive rods
Aerobic:
Nocardia
Anaerobic, nonspore forming:
Actinomyces israelii
Acid Fast Stains
Mycobateria:
- cell wall structure of gram positive bacteria but contain mycolic acids, which repel crystal violet/iodine; thus are usually gram invisible
- dye penetrates hydrophobic barrier to stain mycolic acids in cell wall
- Ziehl-Neelsen: red carbol fuschin dye containing phenol is heated so it can penetrate cell wall; strong acid (3% HCl) decolorizes
- Kinyoun (cold technique): higher concentration of phenol and no heat; otherwise same as ZN
-
Fite (modified) acid-fast: weaker decolorizing reagent; good for:
- nocardia
- cryptosporidium
- isospora
- cyclospora
- sarcocystis
- legionella
Alternative stain for acid fast bacilli
Fluorescent auramine rhodamine stain
Buffered charcoal yeast agar
Legionella grows because media has cysteine and iron
Thioglycolate broth
Cultivation of bacteria, including microaerophilic and obligately anaerobic bacteria; oxygen tension decreases at bottom of tube, allowing for growth of anaerobes
MAC agar
- bile salts and crystal violet inhibit growth of Gram+ bacteria and allow for growth of Gram-
- Lactose fermentation results in pink color
Eosin methylene blue agar
- aniline dyes inhibit growth of gram+ bacteria and allow for growth of gram negative rods
- lactose fermentation results in purple-black colonies with a green metalic sheen
- differentiates lactose fermenting and non-fermenting enterics
campylobacter blood agar
contains cephalothin, vanc, trimethoprim, amphotericin B, polymyxin B
allows for recovery of campylobacter
Hektoen enteric agar
- Bile salts and the dyes bromthymol blue and acid fuchsin inhibit Gram+ and some Gram- strains
- recover salmonella and shigella better than MAC
- lactose and/or sucrose fermentation results in yellow or orange color, otherwise translucent
- H2S production results in black coloration
Selenite broth
Sodium selenite inhibits growth of gram+ bacteria and many enterics other than salmonella
- allows for enrichment of salmonella
Thiosulfate citrate bile salts sucrose agar (TCBS)
- Bile salts inhibit gram+ bacteria
- Alkaline pH inhibits most enterics and enhances growth of vibrio species
- sucrose fermentation results in yellow colonies
- differentiated between sucrose fermenting vibrio (e.g., V cholerae) and nonfermenters (other vibrio)
Cefsulodin-Irgasan-novobiocin (CIN) agar
- antimicrobial and crystal violet inhibit most gram negative bacteria other than yersinia
- recover yersinia
- mannitol fermentation results in bull’s eye colonies (colorless with red center), otherwise translucent
- differentiates between manitol fermenters (yersinia enterocolitica) and nonfermenters (other yersinia)
Anaerobic colistin nalidixic acid (CNA) agar
- antimicrobials inhibit gram negative bacteria
- recover anaerobic streptococci; blood in agar allows differentiation based on hemolytic reactions
Lim broth
antimicrobials inhibit gram negative bacteria
- recovers Group B streptococci (agalactiae)
Regan-Low medium
contains antimicrobials that allow for selective growth of Bordetella pertussis and bordetella parapertussis
Thayer-martin medium
Allows for growth of Neisseria
Vanc, colistin, nystatin, and SXT block growth of orgs
Salmonella-shigella agar
- Lactose fermentation results in pink or red colonies
- H2S production makes black color
- good for enterics
Motility test
Semisolid
Motile organisms show growth spreading away from the line of inoculation, clouding the agar
Nonmotile organisms remain in the stabbed inoculation site, so the rest of the agar remains clear
Bacteria that grow optimally at the following temperatures:
- 25-40 degrees
- 42 degrees
- 37 degrees
- 4 degrees
- 25-40
- Yersinia enterocolitica
- pseudomonas fluorescens and putida
- 42
- campylobacter jejuni and coli
- other pseudomonas species
- 37
- Listeria monocytogenes grows optimally at 37 degrees, but is motile at 25 degrees
- 4:
- Listeria monocytogenes can multiple at 4 degrees
Catalase test
- purpose
- how
- avoid?
- catalase positive organisms?
- categorizes gram positive cocci: differentiating staphylococci (catalase positive) from streptococci and enterococci (catalase negative)
- 3% H2O2
- avoid contaminating with blood agar: red blood cells make catalase
- Other catalase positive orgs:
- Campylobacter
- Listeria
- Bacillus
Coagulase test
- types
- purpose
- Distinguish coag positive staph (Staph aureus) from other staph
- Types
-
Tube coagulase test
- detects free (secreted) coagulase and is the definitive test
- isolate + plasma in a tube; free coagulase complexes with coagulase reacting factor (CRF) in plasma to convert fibrinogen to fibrin, forming a clot
- examine at 4 and 24 hours (some S aureus make fibrinolysin that dissolves clot giving false negative after 24 hours and others produced delayed reaction that give false negative if only examined after 4 hours)
-
Slide coagulase test: detects bound coagulase (i.e., clumping factor); reaction is immediate
- false negative results when there is a free, not bound, coagulase
- Staph lugdunensis and S schleiferi produce bound, not free coagulase, so are positive with slide test and negative with tube test
- Strep intermedius is coag +
-
Tube coagulase test
Novobiocin susceptibilty test
S saprophyticus is novobiocin resistant; S epidermidis and other coagulase negative staph are susceptible
Optochin test (P disk)
S pneumoniae is susceptible to optochin; viridans strep are resistant
CAMP test
- identifies streptococci agalactiae and listeria monocytogenes
- CAMP factor is produced by both of the above
- CAMP factor increases staph beta hemolysin
- isolate sreaked at perpendicular angle to a S aureus streak; rectangular enhancement of beta hemolysis at intersection is seen with listeria and arrowhead shaped increase in hemolysis is seen with GBS
- Turicella otitidis is also positive, but is non-hemolytic unlike listeria which is beta hemolytic
PYR test
- identifies enterococci and group A strep
- enzymatically hydrolyze PYR, yielding beta naphthylamide with a red color change
Bile esculin test
- identifies enterococci (also tolerant to 6.5% NaCl) and group D streptococci (S bovis)
- both able to hydrolyze esculin in presence of bile
- bile esculin agar slant inoculated and incubated for 24 hours; diffuse blackening of the medium indicates esculin hydrolysis
- rapid spot test versions of this assay available
Oxidase test
- Classifies gram negatives
- positve:
- Neisseria
- Moraxella
- Pseudomonas
- Legionella
- Brucella
- Aeromonas
- Pasteurella
- curved gram negative rods (campylobacter, vibrio, and helicobacter)
- [one can hallucinate on PCP a VAN drive by an OX named “Bruce”
- negative
- enterobacteriaceae
- the reagent (P phenylenediamine) is oxidized by cytochrome oxidase, forming iodophenol blue
Indole test
Identifies E coli (positive)
- Determines organism’s ability to produce indole via deamination of tryptophan, using Kovac reagent, with which indole combines to produce a pink color
- H influenzae and Pasteurella are also positive, but they are lactose non-fermenters
Rapid urease test
- identifies proteus and H pylori
- splitting (hydrolysis) of urea alkalinizes the medium, leading to color change (red or pink)
- many bacteria have urease activity, but few can degrade it within 4 hours and are designated “rapid urease positive” organisms
Beta glucuronidase enzyme activity
- presumptive ID of E coli members of the anginosus group strep
- fluorometric substrate (4-methylumbelliferyl-beta-D-glucuronide) is used, which makes a fluorescent compound and can be added to MacConkey
- E coli 0157:H7 does not have this activity
Hippurate hydrolysis test
- presumptive ID of
- campylobacter jejuni
- listeria monocytogenes
- Streptococcus agalactiae
- gardenerella vaginalis
- legionella pneumophila
- All of the above are hippurate hydrolysis positive
- Uses ninhydrin reagent; blue = positive
Lysozyme test
- ID nocardia
- most bacteria are inhibited by lysozyme
- growth in lysozyme containing broth medium indicative of nocardia
Anaerobic gram+ cocci
- organisms
- colonizes what sites?
- diseases
- Peptostreptococcus
- Finegoldia
- Peptococcus
- Parvimonas
- Anaerococcus
Commensal flora of the oropharynx, GI, vagina, skin
- endogenous polymicrobial infections (e.g., abscess of the skin, soft tissues, lung, or brain)
- aspiration pneumonia
- necrotizing pneumonia
- empyema
- diabetic foot infections
- crepitant cellulitis
- synergistic gangrene or necrotizing fasciitis
- septic abortion
- intraabdominal infections
Staphylococcus
Colony morphology and gram stain
Creamy colonies
Grapelike clusters and tetrads
S aureus
- biochemicals
- resistance mechanisms
- susceptible to what abx?
- ID of MRSA
- coagulase positive
- most express a beta lactamase, conferring resistance to penicillin, ampicillin, and early generation cephalosporins
- Susceptible to nafcillin, dicloxacillin, or oxacillin
- MRSA - carries mecA gene, which expresses PBP2A, a penicillin target with little affinity for the drug
- MRSA ID
- disk diffusion (cefoxitin)
- DNA hybridization probes to detect mecA
- PBP2A latex agglutination
Coagulase negative staphylococci
S epidermidis
- skin commensal
- bloodstream infection related to IV catheter, prosthetic vascular grafts, or prosthetic cardiac valves
- prosthetic joint infections
- CNS infection related to ventricular shunts
S saprophyticus
- uropathogen
- novobiocin resistant
S lugdunensis
- skin commensal
- infections similar in severity to S aureus
- positive by slide coagulase test, but negative by tube coagulase test
- PYR+
- ornithine decarboxylase+
Strep pyogenes
- colony morphology
- gram stain
- biochemicals
- colonizes
- infections
- treatment
- beta hemolytic (wide)
- catalase negative
- gram+ cocci single, in pairs and short chains
- susceptible to bacitracin (A disk)
- PYR+
- Lancefield group A antigens
- colonizes oropharynx and skin
- Infections
- pharyngitis
- retropharyngeal abscess
- bacteremia
-
skin and soft tissue infections
- impetigo
- erysipelas
- necrotizing fasciitis
- toxin mediated illnesses (toxic shock syndrome and scarlet fever)
- immune mediated disease (acute rheumatic fever, poststreptococcal glomerulonephritis)
- pediatric autoimmune neuropsychiatric disorders
- Likelihood of ARF decreases with treatment, but poststrep GN does not improve
Streptococcus dysgalactiae
- PYR-
- bacitracin resistant
- Lancefield group C or G
Jones criteria
Major
- carditis
- polyarthritis
- chorea
- erythema marginatum
- subcutaneous nodules
- gram negative cocci
Minor
- arthralgia
- fever
- increased ESR or CRP
- prolonged PR interval
Streptococcus agalactiae
- gram stain
- colony morphology
- biochemicals
- gram+ cocci in pairs and short chains
- gray white colonies
- Beta hemolytic with narrow zone of hemolysis
- CAMP test positive
- Hippurate hydrolysis positive
- Pregnant women screened at 35-37 weeks (culture or molecular methods following enrichment broth step)
Beta hemolytic strep
GAS (wide zone of hemolysis) and GBS (narrow zone of hemolysis)
alpha hemolytic streptococci
Streptococcus pneumoniae
- Lancet shaped, gram+ diplococci
- susceptible to optochin (P disk)
- infectious strains have polysaccharide capsule that results in smooth colonies
- Diseases
- community acquired pneumonia
- bacteremia
- meningitis
- otitis media
- sinusitis
- spontaneous bacterial peritonitis
Viridans streptococci
- S mitis group: native valve endocarditis
- S mutans group: dental caries can cause endocarditis
- S salivarius: bacteremia and endocarditis
- S anginosus: abscesses (particularly S intermedius)
Nonhemolytic, catalase negative gram+ cocci
Enterococcus
Streptococcus bovis
Enterococcus
- gram stain
- colony morphology
- biochemicals
- Lancefield group
- colonize what site?
- diseases
- Resistance patterns and mechanisms
- Diplococci in short chains
- small, gray, nonhemolytic colonies
- hydrolyze bile esculin (esculin gets hydrolyzed in presence of bile)
- Lancefield Group D
- normal inhabitants of lower GI
- Diseases:
- UTI (often nosocomial and related to catheters)
- bacteremia
- endocarditis
- intraabdominal and pelvic infections
- E faecalis: susceptible to vancomycin
-
E faecium resistant to:
- vancomycin (due to acquisition of vanA, vanB, or vanD gene clusters)
- penicillin, ampicilin, and carbapenem due to altered PBP
- E gallinarum and E casseliflavus have intrinsic, low level vanc resistance due to chromosomal vanC gene
Streptococcus bovis group
- gram stain
- colony morphology
- biochemicals
- diseases
- colonizes
- subgroup associated with malignancy
- gram+ cocci in pairs or short chains
- gray white, nonhemolytic colonies
- Lancefield group D
- hydrolyze bile esculin
- PYR negative
- cannot grow in 6.5% NaCl
- Colonizes the GI tract
- Diseases: bacteremia and endocarditis
- S bovis gallolyticus subgroup is associated with colonic malignancy
Anaerobic gram negative cocci
Veillonella and other gram negative cocci are normal inhabitants of oral cavity, GI, GU, respiratory tracts
(sometimes found in polymicrobial anaerobic infections)
Neisseria species
- gram stain
- culture agar
- other biochemicals
- fastidious, requiring incubation in CO2 at 35 degrees
- intracellular coffee bean shaped gram negative diplococci
- cultured on chocolate agar with antibiotics to inhibit indigenous flora (Thayer-Martin, Martin-Lewis, or New York City agar)
- cotton swabs inhibit growth
- oxidase positive
Neisseria gonorrheae
- how to identify
- rate of asymptomatic men and women
- infections
- identified by gram stain and colony morphology + biochemical reactions or hybridization probes
- 10% of affected males and 50% of affected females asymptomatic
- Infections:
- epididymitis
- PID
- urethritis in women
- disseminated infection manifests as Fitz Hugh Curtis syndrome or purulent arthritis
Neisseria meningitides
- how to identify
- serogroups defined by ___
- diseases caused by what serogroups
- vaccine includes which serogroups
- colonization
- infections
- identified by culture
- capsular polysaccharide antigens define serogroups
- serogroups B, C, and Y cause majority of disease in the US
- serogroup B is not in the tetravalent vaccines
- nasopharyngeal carriage is common
- invasive infection is most often meningitis
Moraxella catarrhalis
- gram stain
- culture morphology
- colonizes
- biochemical
- diseases
- gram stain similar to neisseria; can be coccobacillary
- oxidase positive
- DNAse positive (unlike neisseria)
- grows well on blood agar and does “hockey puck”
- colonizer of upper respiratory tract
- diseases:
- otitis media
- COPD exacerbation
Kingella kingae
- gram negative coccobacillus
- pairs and short chains
- HACEK organism
- Diseases
- endocarditis in adults
- septic arthritis in kids
- osteomyelitis in kids
- occult bacteremia (in kids?)
Anaerobic spore forming gram positive rods
Clostridium species
Nonspore forming anaerobic gram positive rods
Actinomyces israelii
Propionibacterium
Aerobic gram+ rods spore formers
Bacillus species
Aerobic gram+ rods non-spore formers
Listeria monocytogenes
Erysipelothrix rhusiopathiae
Nocardia
Rhodococcus equi
Corynebacterium diphtheriae
Tropheryma whipplei
C perfringens
- morphology
- ID
- Large boxcar shape gram+ rod, anaerobic
- short chains
- double zone of beta hemolysis
- lecithinase activity
Clostridium septicum
- large, gram+ rod in chains
- swarm rather than form discrete colonies
- bacteremia related to colonic carcinoma or other GI pathology
Botulism
- source
- diagnosis
- Botulism dx relies on detection of toxin in serum, stool, vomitus, or food
- raw honey = source of infant botulism
Clostridium tetani - tetanus diagnosis
Clinical diagnosis; no useful tests for toxin available
Clostridium difficile
- culture medium
- colony morphology and other characteristics
- gram stain
- disease and diagnosis
- culture in cycloserine cefoxitin egg yolk fructose agar (CCFA) under anaerobic conditions
- CCFA colonies are yellow with ground glass appearance, and fluoresce under UV light
- “horse manure” odor
- Gram stain shows thin, regularly shaped gram+ or gram variable rods sometimes with subterminal and free spores
- nontoxigenic strains of C difficile are common in the bowel, so must isolate the toxin to show disease:
- cytotoxic culture is gold standard, but outdated
- molecular tests for toxin A and toxin B genes (toxin B much more likely to cause disease)
- ELISA for stool toxin relatively insensitive; ELISA for stool glutamate dehydrogenase (GDH) more sensitive
- Severe C difficile colitis associated with BI/NAP1/027 strain; can detect toxin genes and mutated form of tcdC gene that typifies the 027 strain
Actinomyces
- gram stain
- culture conditions
- diseases
- Anaerobic gram+ rod, non-spore former
- branching, filamentous
- A israelii forms white cerebriform (molar tooth) colonies
- same gram stain (almost) as nocardia
- unlike nocardia, actinomyces are anaerobes and not acid-fast
- disease
- cervicofacial actinomycosis as a result of trauma
- thoracic actinomycosis 2/2 aspiration
- IUDs
- intraabdominal actinomycosis related to appendicitis, diverticulitis, or surgery
Proprionibacterium
- anaerobic gram+ rod, non-spore forming
- diphtheroid gram stain morphology
- prionic acid production
- disease
- skin commensal causing prosthetic joint infection
- infection of other foreign bodies
Bacillius anthracis
- gram stain
- colony morphology
- biochemical
- motility
- diseases
- forms chains and subterminal spores
- nonhemolytic colonies with irregular borders (Medusa heads) that stand up when teased with a loop
- catalase+
- nonmotile
- Edema factor, protective factor, lethal factor
- Disease
- cutaneous anthrax (inoculation of spores into skin wound while handling animals or hides)
- GI anthrax causes ulcers
- inhalation anthrax (woolsorter disease): widened mediastinum on AP film caused by hemorrhagic mediastinitus
Bacillus cereus
- chains and demonstrates subterminal spores
- large, beta hemolytic colonies
- nontenacious colonies (do not stand up when teased)
- motile
- foodborne illness: Asian rice (lack of refrigeration)
Listereria monocytogenes
- gram stain
- biochemicals
- temperature
- motility
- diseases
- Aerobic gram+ rod, non-spore former
- small colonies in primary culture after 1-2 days
- beta hemolytic with narrow zone of hemolysis like GBS
- gram stain shows short, nonbranching gram+ rods, single or in short chains
- catalase+
- CAMP+: rectangular zone of enhanced hemolysis at Staph intersection with the listeria
- temperature dependent tumbling motility in wet mounts (best at room temperature)
- semisolid agar motility: umbrella shaped motility
- can grow at 4 degrees (can contaminate refrigerated foods
- Disease
- chorioamnionitis
- neonate may have granulomatosis infantiseptica - multiple abscesses
- elderly/IC: bacteremia, meningitis, encephalitis