MLS Review - Bacteriology - Medically-Important Bacteria

MEDICAL LABORATORY SCIENCE REVIEW • CLINICAL BACTERIOLOGY: MEDICALLY-IMPORTANT BACTERIA OUTLINE • • • • • • • • Family Mycoplasmataceae Rickettsia Species Chlamydia / Chlamydophila Spirochetes Anaerobes Bordetella Brucella Jellison Type A • • • • • • • • Legionella HACEK Non-fermenters Enterobacteriaceae Salmonella & Shigella Vibrios Plesiomonas & Plantoae Campylobacter & Helicobacter • • BACTERIOLOGY FAMILY MYCOPLASMATACEAE • • • • • • • • • • • • Generally referred to as Mycoplasma or Mollicutes Genera: Mycoplasma & Ureaplasma o Species: M. hominis, M. pneumoniae, U. urealyticum Characteristics o Lack cell wall ▪ Gram staining is not performed o Susceptible to antibiotics classified as cell wall inhibitors (beta lactams like penicillin & cephalosporins) o Mycoplasma are small living organisms measuring 0.2-0.3 micra o Slow growers except for M. hominis (exceptionally rapid growing) o Facultative anaerobes expect M. pneumoniae (aerobic) o Highly fastidious and require complex media for growth (with cholesterol and fatty acids) Classified under class Mollicutes/Family Mycoplasmataceae o Requires complex media with cholesterol & fatty acids o Shows susceptibility to beta-lactams (penicillin & cephalosporins) M. pneumoniae - Formerly referred to as Pleuropneumoniae, like organism / Eaton agent o First mycoplasma to be isolated o Initially it was called as PPLO/Eaton agent o Cause of Primary atypical pneumonia / walking pneumonia / community-acquired atypical pneumonia M. hominis & U. urealyticum - Causes NGU / nongonococcal urethritis o An agent of PID M. hominis - Mycoplasma species (+) on SBA and Chocolate agar Acridine orange - applicable fluorescent stain for Mollicutes o Non-specific for mollicutes SPF Broth - Ideal culture media for M. pneumoniae and M. hominis A8 agar - Solid media to recover M. hominis & Ureaplasma species U. urealyticum - Previously called T-strain Mycoplasma o T - means tiny o May develops small colonies that are difficult to see Genus Ehrlichia - Member of Anaplasmataceae, that infects WBC’s o May form Mulberry like bodies within phagosomes o Anaplasmataceae - initially Rickettsia o Ehrlichia multiply in phagosomes of host WBC o Rickettsia multiply in cytoplasm of endothelial cells Anaplasma phagocytophilium, Ehrlichia phagocytophilium - cause human granulocytic anaplasmosis o Arthropod-transmitted by ticks (Genus Ixodes) Ehrlichia chafiesnsis - cause human monocytic ehrlichiosis o Arthropod-transmitted ▪ Primary vector: lone star ticks Coxiella burnstii - Generally, not transmitted by arthropods o Initially under Family Rickettsiaseae ▪ It is not arthropod-borne o Potential bioterrorism agent o Modes of transmission ▪ Inhalation transmission of dried birthing fluids of several animals ▪ Ingestions of unpasteurized milk o Causes ▪ Q fever (Query) RICKETTSIA SPECIES • • • • • • • Genus Rickettsia & Orientia Known to cause Rickettsial infections o May develop fever, rashes, headaches o Detect through serological tests (Weil-Felix Test) ▪ Uses antigens obtained from genus Proteus ▪ Weil-Felix Test - non-specific for Rickettsia since it does not use specific Rickettsial antigens R. akari - known to cause Rickettsial pox Orienta tsutsugamushi – cause scrub typhus R. prowazekii o Epidemic typhus- louse borne ▪ Requires vector ▪ Relapse will no longer require vector o Brill Zinsser — no vector ▪ Repeat of epidemic typhus R. typhi - Murine typhus R. rickettsii - cause rocky mountain spotted fever CHLAMYDIA / CHLAMYDOPHILA • • Significant species o C. trachomatis - still Chlamydia o 2 species became Chlamydophila ▪ C. pneumoniae ▪ C. psittacii Characteristics o Obligate intracellular organisms o Can be cultivate but require cell/tissue culture o Sensitive to Sulfa drugs o Cycle involves 2 distinct forms ▪ Elementary Body - infectious form • Round or pear-shaped o Pear - C. pneumoniae o Round - C. trachomatis, C. psittacii • Have spore-like features • Resistant to environmental physical stress • Outer membrane - similar to gram-negative organisms Contributory to its resistance o Major Outer Membrane Protein (MOMP) - most prominent component ▪ Reticulated body - non-infectious form/reproductive form 1 • • • • • • C. trachomatis - Trachoma (TRIC) o TRIC - trachoma inclusion conjunctivitis ▪ Chronic eye infection considered as number 1 cause of preventable blindess o LGV - lymphogranuloma venereum ▪ Sexually transmitted / venereal acquired o Considered as cause of Reiter syndrome ▪ Aka reactive arthritis Requires media for viral culture (tissue/cell culture) Cell Culture for Chlamydophila - McCoy cells, HeLa, Buffalo green monkey Nucleic Acid Amplification Test (NAAT) - Currently the Gold standard for C. trachomatis detection o Before NAAT, cell culture was the gold standard Chlamydophila pneumoniae - respiratory pathogen o Previously known as TWAR Strain (Taiwan Acute Respiratory Strain) o Agent of pneumonia o Pear shaped EB Chlamydophila psittacii - respiratory pathogen o Cause Parrot fever, ornithosis, Psittacosis ▪ Acquired through contact with infected birds and their droppings SPIROCHETES • • • • • Leptospira • • • • • • • • • • • Treponema Spiral organisms Gram-negative organisms Motile due to their axial filaments or periplasmic flagella presenting corkscrew motility Generally cannot be visualized using brightfield microscope, darkfield microscope is required o Silver stains may also be used Genus: Borrelia, Treponema, Leptospira • Borrelia • • • • • • • • • • • Multiply by binary fission Tightly coiled with 1 or both ends bent to form a hook Use of Fletcher's media Requires Darkfield, Phase contrast microscopes / Immunofluorescent microscopy Aseptic meningitis - Hallmark of immune phase of Leptospirosis Ways to detect leptospirosis: Culture, sero test, microscopy Fletcher’s media - for genus Leptospira Microscopic agglutination test - Serologic gold standard for Leptospirosis detection Darkfield microscope - used to detect tightly-coiled appearance of Leptospira L. interrogans - causative agent of human and animal leptospirosis o Anicteric Leptospirosis phase - septicemia, high fever, headache o Icteric Leptospirosis phase - involvement of liver and kidney, ▪ Blood & CSF - required for 1st week ▪ Urine - required for 2nd week Weil’s Disease - severe form of leptospirosis Multiply by binary fission Can be visualized using brightfield/light microscope Arthropod borne o Requires vector for transmission Blood spirochetes Loosely coiled Can be cultivated Use of Kelly medium for culture Sero test & Examination of Giemsa stained smears Borrelia burgdorferi o Vector: Ticks (lxodes spp), Deer ticks, Black legged ticks o Lyme Disease ▪ Tick-borne relapsing fever ▪ Has 3 stages ▪ Stage 1 - Bull’s eye rash at the bite site (ECM Erythema chronicum migrans) ▪ Stage 2 - dissemination of ECM to other parts of the body ▪ Stage 3 - chronic arthritis, development of neurologic symptoms Borrelia recurrentis o Vector: Pediculus spp.: P. humanus capitis (head louse), P. humanis corporis (body louse) o Louse-borne relapsing fever Borrelia duttoni, Borrelia parker, Borrelia hermsii o Ornithodoros Ticks (Soft ticks) o Cause Tick-borne relapsing fever • • Treponema pallidum o Multiply by transverse fission o Consisting of 4-14 regularly spaced coils/Organism o Non-culturable in vitro, viability can be maintained alive using testicular chancre of rabbits o Agent of syphilis/ great pox/ French disease o Acquired through sexual contact o Other MOT: blood transfusion, vertical transmission (mother to fetus) o Syphilis ▪ Primary stage - patient develops lesions in genitals (hard chancre/hunterian chancre) • Hard chancre/hunterian chancre - primary syphilitic lesions) ▪ Secondary stage - presence of extragenital lesions outside the genital (Condyloma latum) • Condyloma latum - extragenital lesions during secondary stage of syphilis ▪ Latent stage - absence of symptoms/lesions positive for blood tests ▪ Tertiary stage - lesions develop in deep organs of the body (Gumma’s) • Gumma’s - tertiary lesions o Non-venereal Treponema ▪ T. pallidum sp. pertenue - Yaws (Frambesia) ▪ T. pallidum sp. carateum - Pinta ▪ T. pallidum sp. endemicium - cause Bejel/ Endemicum syphilis ▪ Characterized by presence of lesions ▪ MOT: direct contact with infected lesions T. cuniculi - Venereal spirochetosis (STD) T. vincentii - Trench mouth / Vincent’s, stomatitis 2 o ANAEROBES • • • • • • • • In the lab, anaerobic culture is not usually performed It is challenging to culture anaerobes o During incubation, ensure that organisms will not be exposed to air GASPAK Jar - equipment used to provide an anerobic environment o Palladium catalyst - component of Gaspak Jar that ensures oxygen free environment o Methylene Blue or Resazurin - Indicators in Gaspak ▪ In presence of oxygen • Methylene blue appear blue • Resazurin appear pink ▪ Colorless - Color in the absence of air Macroscopic exam o One way to identify anaerobes in the lab o Gross examination of the specimen o Examine specimen for foul odor, black color, fluorescence, sulfur granules o C. dificile; Fusobacterium, Porphyromonas anaerobes that emit foul odor o Porphyromonas & Prevotella - produce brick-red fluorescence under UV ▪ Gram-negative anaerobic bacilli ▪ Produce black pigment o Veilonella - produce red fluorescence under UV that rapidly fades o Fusobacterium nucleatum - produce chartreuse fluorescence (yellow fluorescence) o Actinomyces, Propionibacterium, Cutibacterium acnes, Eubacterium nodatum - anaerobic organisms with sulfur granules ▪ Some Propionibacterium have become Cutibacterium • Only P. acnes became C. acnes Microscopic exam o Performed for anaerobes with unique morphology that can be diagnostic of particular organism o Fusobacterium nucleatum - microscopically with tapered ends appearing like toothpick o Bifidobacterium - with “bifid” ends/appearing like bone-shaped dog biscuit o Bacteroides fragilis - anaerobic bacilli that assume safety pin appearance and may show resistance to Kanamycin, Vancomycin, & Colisitin in Potency disk test ▪ Bile-tolerant ▪ Cause of aspiration pneumonia o Propionibacterium spp. - anaerobic diphtheroid Culture o Examine colony morphology Spot indole test - rapid test to detect C. acnes o Reagent: p-dimethylaminocinnamaldehyde ▪ Same reagent used in PYR test (for S. pyogenes) o (+) blue/green = Cutibacterium acnes o (-) pink/orange = Propionibacterium spp. Potency disk test o Purpose: To verify/check gram staining result of anaerobic bacteria ▪ Not necessarily is susceptible/resistant to certain antibiotics used o Similar with antibiotic susceptibility test ▪ Uses several antibiotics • • • • • • • Antibiotics in Potency Disk Test: ▪ Kanamycin (1000 ug) ▪ Vancomycin (5 ug) ▪ Colistin (10 ug) o Results in Potency Disk Test: (Kana-Van-Col) ▪ RRR - B. fragilis ▪ SRS - Fusobacterium, B. ureolyticus., Veilonella ▪ SSR - Clostridium ▪ RSR - Prophyromonas, P. anaerobius ▪ RRS - Prevotella SPS-sensitive - P. anaerobius o 0.025% SPS - anticoagulant used in bacterial cultures ▪ Sodium Polyanethol Sulfonate ▪ Disadvantage: sensitive/inhibitory to P. anaerobius ▪ Add 1% gelatin as remedy to ciounteract inhibitory effect of SPS Spore-forming anaerobes - C. tetani and C. septicum o Show swarming on BAP Actinomyces israelii - can develop colonies appearing like molar tooth o Can cause actinomycosis of jaw (Lumpy Jaw) o Actinomycosis - Affects soft tissues Peptococcus niger - anaerobic staphylococcus Peptostreptococcus - anaerobic streptococcus Prevotella - important component of dental biofilm Thioglycolate - versatile media o Allows growth of a number of organism including anaerobes, facultative anaerobes, aerobes o Sign of growth: turbidity o Anaerobes - growth at the bottom o Facultative anaerobes - diffuse growth all throughout the media o Aerobes - growth towards surface BORDETELLA • • • • • Gram-negative strict aerobes Significant species: B. pertussis, B. bronchispetica, B. parapertussis o Respiratory pathogens Media for Bordetella o Potato blood glycerol agar - traditional o Regan Lowe - recommended Motile Bordetella - B. bronchiseptica Non-motile Bordetella - B. pertussis, B. parapertussis JELLISON TYPE A • F. tularensis o Potential agent of bioterrorism o Unable to grow in ordinary media o Requires special media for growth ▪ With Special requirement for cystine, cysteine, thiosulfate o Uses BCGA (Blood cysteine glucose agar) o Cause tularemia, market men, rabbit fever, deerfly fever, Lemming disease, water rat trapper disease BRUCELLA • • • • Brucella - Genus considered as animal flora, usually found in placental tissues Can cause abortion in animals Strict aerobes Agents of bioterrorism 3 • • • • • • • • • Animal hosts of Brucella o B. abortus (Bang’s bacillus) - cattles o B. melitensis - goat o B. suis - swine/pigs o B. canis - dogs B. canis - brucella species not classified as bioterrorism agent and not detected thru SAT (Serum agglutination test) SAT - Rapid rest for Brucellosis detection o Significant titer = 1:160 Brucellosis o Aka Mediterranean fever/Malta fever/Undulant fever, Gibraltar fever o Febrile disease Blood - Optimal specimen for Brucellosis detection Routine blood culture is 7 days o For detection of Brucellosis, incubation period is 3-4 weeks o SAT is perform for rapid detection Brucella is inhibited by dyes B. abortus - inhibited by Thionine B. suis and B. canis - inhibited by Basic Fuchsin • • • • LEGIONELLA • • Legionnaire’s disease and Pontiac fever - caused by L. pneumophila o Legionnaire’s disease - a form of pneumonia o Pontiac fever - febrile disease, non-pneumonic Buffered Charcoal yeast extract (BCYE) - media for L. pneumophila • • HACEK • • • • • • • • Causative agents of SBE (subacute bacterial endocarditis) o Usual cause are Virdians strep (gram pos cocci) o Gram negative causative agents: HACEK H. parainfluenzae - Normal flora of the oral cavity Aggregatibacter aphropilus - previously under haemophilus Aggregatibacter actinomycetemcomitans o Star-shaped colonies o Grows together with Actinomyces israelii Cardiobacterium hominis - rosette arrangement on gram-stained smears Eikenella corrodens o Normal flora o Human bite wound/clenched fist wound o In culture ▪ Pits and Corrodes agar ▪ Colonies with bleach like odor o Assacharolytic (non-saccharolytic) Kingella kingae - twitching motility o Can infect bones and heart Pasteruella multocida o Animal bite wound o Colonies with musty odor NON-FERMENTERS • • • • • • Significant genus of Non-fermenters o Genus Pseudomonas o Genus Stenotrophomonas o Genus Burkholderia o Genus Acinetobacter P. aeruginosa o #1 ICU isolate ▪ Common in hospital environment o Agent of pneumonia in those with cystic fibrosis o Cause jacuzzi/hot tub syndrome ▪ Can survive in wet environments o Causes ecthyma gangrenosum o Strict aerobe, oxidase (+), beta-hemolytic o Produces colonies with and odor similar to an overripe grapes, corn taco/corn tortilla like odor o Cetrimide agar - Media for initial isolation of P. aeruginosa ▪ Cetrimide is toxic to many bacteria but not to P. aeruginosa Acinetobacter o Non-motile and oxidase (-) Burkholderia o Cause respiratory diseases o B. mallei - Glander’s bacillus ▪ Cause Glander’s disease o B. pseudomallei - aka Whitmore’s bacillus ▪ Cause Mellioidosis, Vietnam time bomb ▪ Colonies with earthy odor on ashdown media Wrinkled-colonies - P. stutzeri and B. pseudomallei Transfusion associated septicemia - P. fluorescence and P. putida o Can only develop pyoverdine o P. aeruginosa - can develop pyocyanin and pyoverdine Gelatin Hydrolysis Test - differential test for P. fluorescens (+) and P. putida (-) OF Media o OF - oxidative fermentative o Detects fermentation & acid production o OF Media of Hugh and Leifson ▪ High carbohydrate but low peptone content o Glucose - fermentable carbohydrate o Two tubes are used ▪ Open/ aerobic tube ▪ Closed/ anaerobic tube o Results ▪ Fermenter - (+) in both open & closed tubes • Produce acid with or without air ▪ Oxidizer/non-fermenter - (+) in open & (-) in closed • Produce acid only in presence of air ▪ Non-oxidizer/non-saccharolytic - (-) in both open & closed tubes o Bromthymol blue - indicator in OF media of Hugh and Leifson ▪ (+) yellow ▪ (-) green/blue green o Phenol Red - indicator in OF media - CDC Method ▪ (+) yellow ▪ (-) red AKA oxidizers Capable of producing acid only under aerobic conditions Not normal flora Cause nosocomial infections (common in hospitals) 4 o ENTEROBACTERIACEAE • • • • • • • • • Facultative anaerobes, fermenters Triple Sugar Iron (TSI) - Usual media to detect acid production of Enterobacteriaceae o Contains three fermentable CHO ▪ 1 part glucose ▪ 10 parts lactose ▪ 10 parts sucrose o Contains pH indicator (phenol red) o Detects production of H2S ▪ Indicator: ferrous sulfate and sodium thiosulfate o Tube media dispensed as slant and butt ▪ Note change in color in slant and butt o Acid/A (+) - yellow o Alkaline/K (-) - red o Red slant and butt = K/K = non-fermenter o Red slant & yellow butt = K/A = non-lactose fermenter ▪ Eg. Shigella (exc. S. sonnei), Proteus, Providencia, Morganella o Yellow slant & butt = A/A = lactose fermenter ▪ Eg. E. coli, Enterobacter, Klebsiella ▪ Late lactose fermenters: Citrobacter, Yersinia enterocolitica, Shigella sonnei OF Media - Usual media to detect acid production of nonfermenters o Increased CHO and low peptone content o Usual is OF Media of Hugh and Leifson K/K - TSI result of non-fermenters o Pseudomonas aeruginosa - Strict aerobe, only produce acid in air A/A or K/A - TSI results of Enterobacteriaceae o Enterobacteriaceae - glucose (+) (+) in both open & closed tubes - OF media result of Enterobacteriaceae (+) in open tube & (-) in closed tube - OF media result of non-fermenters (P. aeruginosa) K/A - TSI Result of non-lactose fermenters A/A - TSI result of rapid lactose fermenters • • • • • • • • PLESIOMONAS & PLANTOEA • • • SALMONELLA & SHIGELLA • • • Not normal flora Both are intestinal pathogens, agents of diarrheal diseases Tests that will differentiate Salmonella-Shigella o Motility test, H2S production, Malonate and Decarboxylase tests Salmonella Shigella Motility Motile Nonmotile H2S Production (+) (-) Malonate and Decarboxylase test (+) (-) VIBRIOS • • • • • Generally halophilic except. V. cholerae & V. mimicus o Requires increased salt concentration String test (+) o Reagent: sodium desoxycolate Sensitive Vibriostatic Agent (0/129 agent) Aeromonas - string test neg, resistant to vibriostatic agents Thiosulfate Citrate Bile Salt (TCBS) - plated media selective differential media for Vibrios o Contains sucrose (CHO), thymol blue & bromthymol blue (indictor) Sucrose fermenters - yellow colonies ▪ V. alginolyticus, V. cholerae, V. fluvialis, V. furnissi, V. metschnikovii o Non-sucrose fermenters - green colonies ▪ V. mimicus, V. parahaemolyticus, V. vulnificus o Selective - Only Vibrios grow in TCBS o Differential - differentiate sucrose and non-sucrose fermenters Alkaline peptone water - enrichment media for Vibrios V. cholerae, V. mimicus - non-halophilic Vibrio V. cholerae o Most significant Vibrio o Causes cholera ▪ Diarrheal disease caharacterized by production of rice watery stool V. parahaemolyticus o 2nd most common Vibrio o Causes Summer Diarrhea in Japan o (+) Kanagawa phenomenon ▪ Beta-hemolysis due to TDH ▪ TDH - Thermostable direct hemolysin ▪ Appears in Wagatsuma agar o Wagatsuma Agar - high salt mannitol ▪ media to demonstrate Kanagawa phenomenon V. alginolyticus - least virulent o Occupational hazard in continuous contact with sea water (fishermen, sailor) (+) oxidase - differentiate Vibrio from Enterobacteriaceae (-) inositol fermentation - differentiate Vibrio from Plesiomonas CHO Fermentation mechanism - Differentiate Vibrio from Pseudomonas o Vibrios are glucose positive, lactose negative (exc. V. vulnificus) Plantoea agglomerans - triple decarboxylase (-) Plesiomonas spp. - triple decarboxylase (+) Decarboxylase test o Purpose: detect ability of organism to produce enzyme decarboxylase which remove carboxyl group from amino acids ▪ Removes cadaverine from lysine, detect ornithine from putrescine from ornithine, and remove citrulline from arginines o Uses Moeller’s broth or LIA o Moeller’s broth - base media ▪ Contains glucose (CHO), bromcresol purple (pH indicator) ▪ (+) purple ▪ (-) yellow o Amino acids: Lysine (cadaverine), Ornithine (putrescine), and Arginine (citrulline) CAMPYLOBACTER & HELICOBACTER • • Campylobacter species: C. jejuni, C. coli o Appear like wings of seagulls ▪ S-shaped organisms o Show darting motility o Capnophilic (↑ CO2) & microaerophilic Helicobacter pylori - Virulence factor = urease production 5