Bacteriology - Aerobic Gram-Positive Bacilli PDF

3rd Year 1st Semester MT6320 MT6320: BACTERIOLOGY LECTURE UNIT #: AEROBIC GRAM-POSITIVE BACILLI...

3rd Year 1st Semester MT6320 MT6320: BACTERIOLOGY LECTURE UNIT #: AEROBIC GRAM-POSITIVE BACILLI AEROBIC GRAM-POSITIVE BACILLI Aerobic G(+) bacilli are categorized either: Spore forming o Bacillus – Catalase (+) Non-spore forming o Branching ▪ Nocardia – Catalase (+), Partially Acid- fast o Non-branching ▪ Catalase (+) Corynebacterium (Non-motile) Listeria ▪ Catalase (-) Erysipelothrix (Non-motile) Gardnerella SPORE-FORMING NON-BRANCHING CATALASE POSITIVE GRAM-POSITIVE BACILLI FAMILY BACILLACEAE BACILLUS SPP. LABORATORY DIAGNOSIS Generalities Microscopy FOR B. ANTHRACIS o G(+) spore forming bacilli o Bamboo fishing poles appearance w/endospores, Specimen: blood, lung tissue, CSF o Aerobic or facultative anaerobes square ended o Sputum is not recommended for pulmonary anthrax, blood o Catalase (+) o GS: G(+) square ended bacilli surrounded by a clear is better (not pneumonia) ▪ Spore-forming Catalase (-) is Clostridium halo (capsulated) or G(V):violet rods o Processing: BSL II (since they are anaerobic) o Fulton Schaeffer: red-veg cells; green-spore o Culture: BSL III o All are motile, and most are beta-hemolytic except B. ▪ Spores appear colorless Y-hemolytic, w/ beaten egg-white or tenacious consistencies for B. anthracis Biochemical/Other Tests anthracis on BAP o Most are saprophytes, laboratory contaminants or o Gelatin liquefaction Non-motile on SIM normal flora of GIT of animals o Catalase (+) CNA is not recommended as it inhibits anthrax ▪ Found almost everywhere (also as normal o For anthrax Growth in 7% NaCl flora) Wet mount preparation/hanging drop method SIM Capsule Staining - M Fadeyan stain (w/ Methylene blue), India ink o Common laboratory contaminant Direct Fluorescent Antibody Assay Penicillin susceptibility Other Bacillus species Nucleic Acid Amplification Test Ascoli precipitation test o B. thurigiensis – insect pathogen Rapid tests o (+) White ring – Anthrax antigens in animal tissue ▪ Production of parasporal crystals AST: Penicillin; Gentamycin, Erythromycin, chloramphenicol o Thermoprecipitation test using boiled tissue ▪ In GMO-plants String of Pearl Tests – Use of MHA w/ 0.05 – 0.5 units Penicillin ▪ Vector-control (a larvicide) o For B. anthracis (susceptible to penicillin) o B. subtillis, B. pumilis, B. sphaercius o Loss of cell wall leads to this shape ROLD AND MATHYEW | 3EMT  3rd Year 1st Semester MT6320 ▪ B. subtilis – "Hay bacillus" Growth on Polymyxin Lysozyme EDTA Thallous Acetate (PLET) o Usually contaminants Medium o Especially for highly-contaminated samples w/ B. anthracis Catalase (+) SIM Clear structures are spores Culture o SBAP, PEA o Colonies: gray, raised w/irregular curled margin & whirling projections “medusa head” Beaten egg-white colonies India ink stain Ascoli precipitation test o Prior to inoculation, specimens that may contain spore- forming bacilli may be subjected to heat-shock or alcohol-shock treatment ▪ To remove the non-spore forming bacteria present o Growth in Egg Yolk Agar ▪ Detection of lecithinase production String of Pearls rxn PLET Medium ▪ (+) Opaque zone surrounding the colonies FOR B. CEREUS Specimen: implicated food, stool o Stool can be used provided B. cereus is significantly high B-hemolytic on BAP Motile on SIM For B. cereus; if >105 cells/gram are present and no other pathogen; infection is confirmed Mannitol Egg Yolk and Polymyxin B Agar (MAYBA) – B. cereus Medusa Head (EYA) White opacity = Lecithinase (+) o PEMBA – B. cereus agar o B. cereus: implicated food, stool ▪ Stool can be used provided B. cereus is significantly high BACILLUS ANTHRACIS BACILLUS CEREUS Anthrax bacillus ANTHRAX “fried rice poisoning” or Chinese food poisoning Y-hemolytic, Non-Motile Associated w/ cattle, hides, goat hairs B-hemolytic, Motile Most pathogenic species of bacillus Occupational hazard (woolsorter’s disease and ragpicker’s Normal flora, small amt. found in stool Largest pathogenic bacilli disease) Common cause of food poisoning and opportunistic infection Reservoir: soil (not a normal flora) Humans: get infected by direct contact w/infected animals: fibers, o 2 forms: Diarrheal and Emetic (vomiting) Produces spore in the presence of oxygen wool, hair Virulence Factors: Emetic (cereulide) & Diarrheal enterotoxin, Bioterror agent handled/cultured in BSL-3, but can be processed Treatment: hemolysins in BSL-2 (in BSC) o Vancomycin, Clindamycin o Emetic – heat-stable Virulence Factors ANTHRAX FORMS o Diarrheal – heat-labile o Capsule: poly gamma D-glutamic acid Cutaneous form MOT: wounds are contaminated w/ Spores: resist pasteurization ▪ Enhanced in the presence of Carbon Dioxide spores (from infected animals) SBAP – aerobic o Anthrax toxin: S/S: lesion forms on the site of Found in the environment: soil, water ▪ Protein/Protective antigen (PA) – for binding inoculation that develops into black Opportunistic infections: eye infections, may also be associated w/ ▪ edema factor (EF) – swelling (PA + EF) eschar endocarditis, meningitis, septicemia ▪ lethal factor (LF) – apoptosis (PA + LF) o Black eschar - Necrotic lesion or Malignant pustule ROLD AND MATHYEW | 3EMT  3rd Year 1st Semester MT6320 ▪ for the anthrax toxin to be activated, a specific May disseminate into lymphatic DIARRHEAL FORM EMETIC FORM factor must be synergistic with PA system (lymphadenopathy) Implicated Food Ingestion of meat Ingestion of fried or o Plasmids Treatable products, poultry, boiled rice most common but least severe form vegetables, sauces, Inhalation/ Woolsorter’s/Ragpickers disease pastas pulmonary form MOT: Inhalation of spores (from Incubation Period Pasta, longer (8-16 hours) Shorter (1-5 hours) sheep's wool) Diarrhea More common Less common S/S: initial: mild fever, malaise, fatigue Vomiting Less common More common (URTI-like), for 2-5 days Response when Diarrhea Vomiting Sudden severe phase: respiratory fed to rhesus distress (dyspnea, cyanosis) monkey o Not pneumonia Spread post-911 More severe can lead to death Gastrointestinal MOT: ingestion of spores >> spores form inoculated on intestinal lesions S/S: abdominal pain, nausea, anorexia Higher fatality compared to cutaneous Oropharyngeal Injectional MOT: direct injection of spores to Anthrax tissue Newest form Associated w/ drug use (heroin)/ “skin popping” Can lead to necrotizing fasciitis, organ failure, shock, coma, and meningitis ROLD AND MATHYEW | 3EMT  3rd Year 1st Semester MT6320 NONSPORE-FORMING, NON-BRANCHING, CATALASE POSITIVE BACILLI FAMILY CORYNEBACTERIACIAE CORYNEBACTERIUM LABORATORY DIAGNOSIS Generalities: Specimen: nasopharyngeal and throat swabs, skin/wound TESTS FOR TOXIGENECITY Pleomorphic (many shapes) G(+) bacilli lesions Definitive method for diagnosis (not culture or microscopy) since o May appear club shaped, Chinese character, picket fence, X or Microscopy: it is toxin producing Y, snapping arrangement o G(+) Highly pleomorphic bacilli In-vivo: Guinea pig inoculation o Diphtheroid appearance: used to describe non-C. diphtheriae o Metachromatic granules/Babes-Ernst In-vitro: (Modified) Elek test species Granules/Volutin o Immunodiffusion test ▪ club shaped, coryneform ▪ Loeffler’s alkaline methylene blue, Albert’s o pH = 7.8 – 8, 10mm between samples Aerobic/Facultative anaerobe, catalase (+) stain, Neisser’s Stain o Do on culture medium w/ serum, low amt. of Iron (for Nonmotile, nonencapsulated, nonsporeforming o Presence of nutrient reserves toxin demonstration) and Tellurite Cell wall contains meso-DAP Culture o Inoculate perpendicularly o Similar to mycolic acid o BAP, CAP, Cysteine Tellurite Blood Agar (CTBA), o (R) Line of identity – same toxins in #1 and #2 o Based on 16s rRNA, it is related to Mycobacteria Tinsdale medium, Loeffler's Serum Agar/Pai's Slant o (+) result: fine precipitin lines at 45°angle Widely distributed in the environment: soil, water o CTBA = preferred medium Lipophilic (Fastidious) members are clinically significant – 2 days to grow o Loeffler's Serum Slant = Best inoculation for o C. diphtheriae & C. jeukeium morphology, and granule production of bacteria o Non-lipophilic members are also present ▪ Pai's Agar is also good for this Most are fermenters of glucose except C. jeukeium, C. o Culture usually has tellurite to inhibit other pseudodiphteriticum, C. urealyticium bacteria PCR Most are commensal species of the mucus membranes except C. C. diphtheriae: requires amino acids for growth (cysteine) Schick's Test – skin test for diphtheria susceptibility diphtheriae o like dick's test for scarlet fever Some are Urease (+) except C. diphtheriae Vero cell cytotoxicity assay – gold standard for diphtheroid toxin CORYNEBACTERIUM DIPHTHERIAE COLONY TYPES OF CORYNEBACTERIUM DIPTHERIAE Klebs Loeffler’s Bacillus Most virulent sp (toxin producing) DIPTHERIA FORMS Virulence Factors Respiratory MOT: Droplets, close contact o Diphtheria toxin (tox gene) – powerful exotoxin form More common in children ▪ Exotoxin produced by strains with lysogenic Beta Acute, contagious phage that carries the tox gene Site of infection: tonsils, pharynx Gained through transduction with Production of diptherial toxin leading to corynephage pseudomembrane of mucus membrane ▪ Iron is necessary for toxin production o Leading to respiratory ▪ Fragment A – cytotoxic obstruction ▪ Fragment B – mediates entry o Toxin can damage heart kidneys On CTBA: Black/Gray/Gun Metal Colonies after 2 days Infects humans - Diphtheria peripheral nerves o Mercury drop – Bordetella pertussis Clinical infections Death due to cardiac failure BAP (Left): Narrow zone of B-hemolysis o Mild sore throat, Low-grade fever, body malaise Bull’s neck appearance ROLD AND MATHYEW | 3EMT  3rd Year 1st Semester MT6320 Isolated from human nasopharynx; not a normal flora Necrosis Tinsdale Medium (Right): Black colonies with brown halo (due to Possess metachromatic granules/Babes-Ernst Granules/Volutin Cutaneous MOT: Direct contact tellurite redn.) o Not exclusive to C. diphtheriae form Common in the tropics Colony types of C. diphtheriae o Loeffler’s alkaline methylene blue (LAMB) Systemic complications are less common o Mitis – fried egg appearance on BAP; bleach like odor o Albert’s stain Ecthyma diptheriticum - Nonhealing on CTBA o Neisser’s Stain ulcers with dirty gray membrane o Intermedius – small colonies on BAP; black colonies w/ Treatment: Penicillin, Erythromycin gray borders on CTBA o Gravis – largest colony type o Belfanti Albert’s stain (L), Loeffler’s (R) OTHER SPECIES OF CORYNEBACTERIUM C. amycolatum C. xerosis C. diptheriae C.ulcerans C.pseudotuberculosis Catalase All + Most frequently isolated species from humans Opportunistic infection Motility Nonmotile Normal flora skin C. urealyticum CRBA Medium & Black colonies w/ Black colonies w/ Brown Halo Modified Tinsdale Brown Halo Associated w/ prosthetic joint infection & endocarditis Most commonly associated with UTIs (on M. Tinsdale: C. jeikeium Rapid Urease (+) Brown-black w/ gray- brown halo) Group JK (Johnson and Kay) C. pseudotuberculosis Nitrate Redn. + - +/V Multi-drug resistant species Rare cause of lymphadenitis in humans following contact CAMP - Reverse Reverse Urease - + + Associated w/ nosocomial infection w/infected livestock Gelatinase - + - Associated w/ endocarditis, pneumonia, peritonitis Rare cause of lymphadenitis in humans Glucose All + Fermentation Lipophilic Reverse CAMP (+) Maltose Fermentation + + + C. ulcerans C. psudodiphtheriticum Pyrazinamidase (-) + + Esculin Hydrolysis (-) (-) (-) Produces diphtheria-like toxin Normal flora skin, mucus membranes Lipophilic +/- (-) (-) Disease: Cause RTI, endocarditis, UTIs, wound infections o humans: diphtheria-like COVID associated pneumonia infection o cattle: mastitis (mammary gland) Reverse CAMP (+) – Arrow head w/o hemolysis (bowtie formation) FAMILY LISTERIACIAE LISTERIA MONOCYTOGENES LABORATORY DIAGNOSIS ROLD AND MATHYEW | 3EMT  3rd Year 1st Semester MT6320 Generalities: LISTERIOSIS Specimen: blood, CSF, swab of lesions o G(+) coccobacillus in singles, chains, palisade MOT: ingestion of contaminated meat, poultry, coleslaw; Microscopy ▪ May appear cocci in chain vertical transmission o GS: may be mistaken for group B-Streptococcus, o Aerobic/Facultative anaerobe Infection of neonates, pregnant, immunocompromised Corynebacterium o Grows best at 25-35C, but can grow at 4C Pregnant; s/s: flu-like illness leading to spontaneous abortion o Motile: “Tumbling Motility” o Fermenter (glucose, trehalose, salicin) and stillborn Culture: BAP, CAP, nutrient agar, and broth BHI, thioglycolate o Recovered from environment: Fetus: granulomatosis infantiseptica o 30– 35C, 4C (cold enrichment) ▪ soil, water, sewage, fecal matter, animal products like Newborn: meningitis, sepsis (very serious, high fatality rate) o Colonies: small, round, smooth, raw milk Immunocompromised: CNS infection, endocarditis o translucent Beta hemolytic o Isolated from crustaceans, flies, ticks, domestic animals LABORATORY RESULTS o SIM: inverted Christmas tree or umbrella pattern at o Human and Animal Pathogen room temperature o Facultative Intracellular Pathogen o CAMP: "block hemolysis" Virulence factor Biochemical tests: o Listeriolysin O – hemolysin-like o Catalase (+) Oxidase (-) o Act A o VP & Bile Esculin Hydrolysis (+) o Siderophores – related to iron chelating o CAMP (+) o Internalins – survival of bacteria inside cell o Growth in 6.5% NaCl o Catalase, Superoxide, dismutase, Phospholipase C, Protein p60 Pathogenicity test: Ocular test of Anton o Rabbit/guinea pig – installation of culture on conjunctival sac o (+) result: keratoconjunctivitis w/in 24hrs Gram Stain (Upper-L), SIM (Upper-R) w/ Tumbling Motility, CAMP Test /w (Bottom) NON-SPOREFORMING, NON-BRANCHING, CATALASE NEGATIVE BACILLI FAMILY ERYSIPELOTRICHIDAE ERYSIPELOTHRIX RHUSIOPATHIAE LABORATORY DIAGNOSIS Generalities: ERYSIPELOID Specimen: tissue biopsy, aspirates from skin lesions, blood o Pleomorphic G(+) bacilli that forms long filaments Localized skin infection sample/culture (due to sepsis) o Aerobic/facultative anaerobe Painful lesions seen on hands or Fingers Microscopy: thin, G(+), V-form o Veterinary infection and occupational hazard (meat, poultry, Resembles streptococcal erysipelas Culture: BAP, (CAP), nutrient broth, Columbia w/ CNA fish handling) o BAP: small, non-hemolytic o Cuts and skin scratches Causes septicemia, endocarditis o Non-motile o Non-motile and non-hemolytic Biochemical Tests: Virulence Factors: capsule, neuraminidase and hyaluronidase o Catalase (-), nitrate (-), VP (-), urease (-) o Oxidase and H2S (+) Test tube brush growth/pipe cleaner on gelatin at 22C FAMILY BIFIDOBACTERIACIAE GARDNERELLA VAGINALIS LABORATORY DIAGNOSIS ROLD AND MATHYEW | 3EMT  3rd Year 1st Semester MT6320 Generalities BACTERIAL VAGINOSIS Specimen: Vaginal Secretion o Gram Variable (+/-) coccobacilli Malodorous discharge and increase Microscopy: G(+), sometimes gram variable/G(V) coccobacilli, ▪ Thin peptidoglycan in vaginal pH presence of clue cells o Nonmotile, Catalase and Oxidase (-) o Becomes more basic w/ o Clue cells = squamous cells covered w/ cocconacilli o Normal flora of the urogenital tract more bacteria Culture: Human Blood Bilayer Tween Agar, CAP, CNA, V Agar Causes UTIs o Beta hemolytic colonies on human and rabbit BAP STDs o Gamma hemolytic colonies on SBAP Mobiluncus spp. is also associated o Non-motile with this disease (not only Gardenerella spp.) Biochemical Test o Catalase (-) o Whiff test or 10% KOH Test = (+) Fishy Amine Odor o Nugent Score - Assessing presence of the following during infection: ▪ Gardnerella – increased ▪ Mobiluncus – increased ▪ Lactobacillus acidophilus – decreased NON-SPOREFORMING, BRANCHING, AEROBIC ACTINOMYCETES FAMILY NOCARDIACEAE NOCARDIA SPP. LABORATORY DIAGNOSIS Generalities: Pulmonary MOT: Inhalation/Airborne Specimen: biopsy, drainage material, BAL, lung tissue, CSF, blood o G(+) bacilli, thin branching filaments(beads) Infection Chronic, confluent Bronchopneumonia o Aerobic Sputum: thick and purulent o Weakly/Partially acid fast No sulfur granules present o (some) 48-72 hours growth -weeks Caused by Nocardia asteroides complex (N. o Found in soil, water farcinia and N. cyriacigeorgica) o Mostly affects immunocompromised o Associated with AIDS patients ▪ Cause brain abscess, cutaneous skin lesions, Cutaneous MOT: Inoculation of the skin or subcutaneous Microscopy pulmonary infections and actinomycotic Infection tissue o G(+), branching bacilli mycetomas (granulomatous lesions) ▪ Actinomycotic Mycetomas May be mistaken for Gram (+) cocci; Beads so Possible Virulence Factors: Toxins, Extracellular proteins, o May involve the bone not touch each other nocobactin o Filamentous branching isolate Found by the leg, jaw, feet areas Species of Nocardia: o Weakly/Partially acid fast Chronic granulomatous of the skin, o N.asteroides ▪ Demonstrated by Modified Kinyoun Method (1% subcutaneous tissue due to outdoor activities o N.brasiliensi H2SO4) Caused by N. braziliensis o N.caviae ▪ Standard AFS is too strong for this Actinomycotic mycetomas may also be caused by actinomycetes o Silver staining can be done to demonstrate Nocadia Treatment spp o Wet mount: presence of granules on cutaneous tissue o Resistant to Penicillin o Susceptible to Sulfonamide ROLD AND MATHYEW | 3EMT  3rd Year 1st Semester MT6320 Culture: Grows on common nonselective medium (longer incubation @ 3-6 days) o Grow at 22C – 37C o SDA w/ 10% CO2 = Bumpy, velvety, yellow-orange colonies o Can also grow on Lowenstein-Jensen Medium Biochemical Tests o Catalase (+) OTHER ACTINOMYCETES RHODOCCUS EQUI GORDONIA STREPTOMYCES G(+) branching coccobacilli/diphtheroid G(+) bacilli G(+) branching filamentous bacilli Partially acid fast Nocardiofrom – grow mycelial forms that fragment to rod or Non-acid fast RTI among animals cocci Saprophytes Salmon pink pigment on colonies o Mycelial forms – Hyphal-like Causes actinomycotic mycetoma Beta hemolytic, Catalase (+) Non-motile Source of streptomycin Partially acid fast Can grow in water agar TROPHERYMA WHIPPLEI LABORATORY DIAGNOSIS G(+) aerobic bacilli; actinomycete WHIPPLE’S DISEASE Specimen: endoscopic specimens Facultative intracellular pathogen Affect middle aged people Microscopy: Rod-shaped bacteria o In monocytes or macrophages Affect monocytes and macrophages of intestinal tract inside macrophages Common among middle aged men Diarrhea, weight loss, malabsorption, abdominal pain o Periodic Acid-Shiff Fatal when untreated o Histology is done in lieu of bacterial culture Unculturable Detected also by Molecular techniques (16S rRNA) or cell culture ROLD AND MATHYEW | 3EMT