Gram Positive Rods Corynebacterium Bacillus Listeria Erysipelothrix Corynebacterium • Over 20 species, most are saprophytic • Human normal flora, especially skin and nares • Most grow well on 5% Sheep’s blood agar • Gamma hemolytic gray colonies • Catalase enzyme positive • “Diphtheroid” morphology, Gram positive rods in Chinese letter forms • No spores produced Corynebacterium diphtheriae • Agent of Diphtheria • Forms tough oropharyngeal membrane (Diphtheroid is Greek for leather), bleeds when attempt to remove Elek plate • Phage mediated exotoxin is distributed from the membrane causing respiratory paralysis, heart, nervous system, and kidney damage • Non-toxin producing strains cutaneous ulcers and lesions • Diagnosis: culture, toxin detection (Elek plate), serology, and PCR to study toxin genes • Grows well on 5% Sheep’s blood agar, gray gamma hemolytic colonies, catalase enzyme positive • Selective medium Cysteine Tellurite agar / black colonies • Metachromatic storage granules when grown on Loeffler’s media and stained with methylene blue Metachromatic granules • Treatment: Anti-toxin and Erythromycin or Penicillin Other Corynebacterium spp. • Corynebacterium jeikeium – • Normal human skin flora / thrives on lipid • Infects patients with indwelling plastic catheters and devices and can lead to bacteremia • Biofilms are formed on the plastic, protecting the organisms from antibiotic treatment • Susceptible to vancomycin and tetracycline • Corynebacterium urealyticum – • Urease positive • Cause of urinary tract infection in post renal transplants + • Resistant to many antibiotics but vancomycin susceptible Red is (+) for Urease reaction Bacillus species • Large Gram positive rods with square ends • Can be easily over-decolorized and appear reddish on Gram stain • Spores produced which cause clearing in bacillus on Gram stain • Catalase enzyme positive Variable staining / not clearly Spores/ clear areas in Gram positive bacillus Bacillus anthracis • Agent of anthrax in herbivores • Category A agent – priority pathogen, potential agent of bioterrorism • Virulence factors: anthrax toxin and capsular polypeptide • Infections: • Wool sorter’s disease – acquired from handling contaminated hides, produces a unique black eschar skin lesion • Systemic infections: Pneumonia, sepsis, and meningitis • Irregular shape to colony border on 5% Sheep’s blood agar (BAP) Medusa head colonies Gamma (no) hemolysis on BAP Non-motile Susceptible to penicillin Bacillus cereus • Most common species causing infection • Saprophytic environmental organism • Nosocomial and opportunistic infections, particularly in immunocompromised patients, and patients with indwelling or implanted devices • Can be a contaminate in blood cultures • Skin infections in Intravenous drug users • Associated with food poisoning due to emetic toxin production and diarrhea from diarrheal toxin • Gram positive rod with spores • Dull gray colony producing beta hemolysis on BAP • Catalase positive • Motile • Resistant to penicillin Listeria monocytogenes • Small Gram positive rod / no spores produced • Catalase enzyme positive • Grows well on BAP producing subtle beta hemolysis • Motility provides identifying information • More motile at 25˚C than 35˚C • Tumbling motility on wet mount examination • Umbrella motility when inoculated into tubed media • Bacteremia and meningitis in immune suppressed, pregnancy, neonates, and elderly • Culture or molecular amplification methods for diagnosis • Grows well at 4˚C / acquire infection from refrigerated foods • Non pasteurized cheese and milk products, Deli case foods • Ampicillin drug of choice • Intrinsic resistance to Cephalosporin antibiotics Erysipelothrix rhusiopathiae H2S production detected on Triple sugar iron agar (TSI) • Small pleomorphic Gram positive rod • No spores produced • Catalase enzyme negative • Alpha hemolytic colony on 5% sheep’s blood agar plate • Only Gram positive rod that produces hydrogen sulfide (H2S) MALDI-TOF is necessary for this difficult to identify bacteria – Infections from nature and animals, mostly swine, so is an occupational disease of farmers and butchers – Virulence factor: capsule – Soft tissue infection and bacteremia +/- endocarditis – Intrinsic resistance to Vancomycin Erysipelas – lacy skin lesions of swine Gram negative bacilli • Escherichia coli • Normal flora in human intestine • #1 cause of UTI [@80% of cases] Lactose Green sheen on • Bacteremia, neonatal meningitis, EMB agar and abdominal infections fermentor Indole positive • MacConkey agar / Lactose fermenter • Spot indole reaction = positive / turns robin’s egg blue • Detects breakdown of tryptophan from growth on BAP • Eosin methylene blue agar (EMB) green sheen produced • Pathogen of diarrhea: • Enterotoxigenic (ETEC) E. coli cause of traveler’s diarrhea • Enterohemorrhagic E. coli (EHEC) (such as 0157:H7) • Bloody diarrhea acquired from eating undercooked beef • Pathogenicity from Shiga toxin production • Hemolytic uremic syndrome (HUS) can result with hemolytic anemia, thrombocytopenia, and renal failure] particularly in young children • Culture on MacConkey agar with sorbitol (not lactose) / does NOT ferment sorbitol/ most all E. coli except EHEC ferment sorbitol Enteric GNRs that ferment Lactose Enterobacter species • Enterobacter cloacae complex, most common species • Environmental GNRs with low pathogenicity • Usually infects a compromised host • Enterobacter (Cronobacter) sakazakii associated with neonatal meningitis Klebsiella species • K. pneumoniae most common species • Mucoid colony due to capsule production • Currant jelly sputum produced in alcoholics due to blood mixed with Klebsiella capsular polysaccharide in sputum Enteric GNRs that do NOT Ferment Lactose! • Proteus species Colonies swarm in layers on agar surface • Proteus vulgaris – spot indole positive • Proteus mirabilis – spot indole negative • Normal flora in intestine - Common cause of UTI and abdominal infections • Serratia marcescens • Produces red pigmentation which can intensify at room temp • Environmental contaminate • Causes infection most usually in • Immune suppressed • Ventilator associated pneumonia • Bacteremia Triple Sugar Iron Agar (TSI)– Used to detect fermentation of glucose, lactose and/or sucrose and production of hydrogen sulfide [H2S] in GNRs CHO Fermentation= yellow medium Gas production= Disruption of the agar No CHO fermentation = Red medium 1 2 3 4 H2S 1. Glu/lac/suc 2. Glucose 3. Glucose 4. No CHO fermented fermented fermented fermentation with gas with H2S Non fermenter • Salmonella species • Diarrhea with +/- fever and PMNs in the stool • Infection from eating contaminated food (raw eggs, poultry, ground beef or dairy) or direct contact with a sick animal – Must ingest large #’s of organisms to make you ill (1,000,000 bacteria), normal levels of stomach acid is protective • MacConkey agar - does not ferment lactose • Produces hydrogen sulfide on selective agars • Motile • Identification based on biochemical reactions and serologic typing • Kaufman White serologic typing for speciation of Salmonella • O Somatic (cell wall) antigen – Salmonella group “B” • H flagellar antigens – 2 phases [h1 & h2] • Vi capsular antigen – Salmonella typhi only Salmonella typhi • Typhoid fever – high fever and sepsis, no diarrhea • Human pathogen - most cases in US (75%) from international travel • Post typhoid fever there can be carriage in gallbladder with passage in feces. Can transmit by bad hygiene and food preparation • Ingested organisms enter the bowel, move into the blood stream and eventually the bone marrow • Diagnosis: Blood cultures and in late stages (>1 month) bone marrow culture • Vi capsular antigen unique to S. typhi • Moustache of Hydrogen sulfide (H2S) produced in TSI medium slant Shigella • Diarrhea, +/-vomiting, fluid loss, PMNs and blood in stool • Infection: Human to human transmission /control with good hand hygiene • Ingestion of low #’s of organisms make you ill [10 – 100 bacteria] • No lactose fermentation on agar • Non motile • No Hydrogen sulfide (H2S) produced • 4 species based on somatic (cell wall) antigen • S. boydii Group C • S. dysenteriae Group A • S. flexneri Group B • S. sonnei Group D Salmonella Shigella Agar (SS agar) Hektoen agar – Shigella are colorless due to lactose Salmonella produces H2S [Hydrogen sulfide] not being fermented. Salmonella does producing black colonies not ferment lactose, but H2S produced Shigella – green colonies by Salmonella turning the colony black Normal flora – orange colored due to fermentation of lactose (E. coli) Normal Flora Lactose fermented Salmonella H2S Shigella Salmonella Shigella Salmonella Non-Lactose fermenter Yersinia enterocolitica • Major reservoir – swine • Humans infected by eating raw or undercooked pork • Infections: • Diarrhea • Septicemia in patients with iron overload syndromes • Mesenteric adenitis – Infection symptomatic for right lower abdominal pain which mimics appendicitis • Infected blood products from transfusion have been reported • Oxidase = neg, Indole = neg, Urease = pos, Grows well at 4 °C • CIN agar (Cefsulodin-irgasan-novobiocin) selective agar for Y. enterocolitica Yersinia pestis • Plague, Category A agent – call public health! • Obligate flea/ rodent/ flea cycle in nature • Human infection usually from a rat flea bite - leads to Bubonic plague that is infection of the lymphatic system, forms painful buboes (lymph node swelling) at site of the bite • Hemorrhagic lymph nodes spread Y. pestis into blood stream • Pneumonia develops from blood