Aerobic Gram Positive Bacilli PDF

Aerobic Gram Positive Bacilli RAQUEL M. FERNANDEZ, RMT, MSPH Aerobic Gram positive Bacilli Spore former: Bacillus Non-spore former: Corynebacterium, Arcanobacterium, Rhodococcus, Listeria, Erysipelothrix, Gardnerella, Rothia, Branching non-spore former: Actinomycetes, Nocardia Some...

Aerobic Gram Positive Bacilli RAQUEL M. FERNANDEZ, RMT, MSPH Aerobic Gram positive Bacilli Spore former: Bacillus Non-spore former: Corynebacterium, Arcanobacterium, Rhodococcus, Listeria, Erysipelothrix, Gardnerella, Rothia, Branching non-spore former: Actinomycetes, Nocardia Some are rarely encountered but cause significant disease Most are considered contaminants or commensals Non-Spore Forming, Non-Branching Catalase Positive Bacilli Corynebacterium More than 60 species , 40 of which are clinically significant Most are normal microbiota of skin and mucous membranes Cell walls contain m-DAP All are cat+ and non-motile Divided into lipophilic and non-lipophilic Lipophilic are fastidious and grow slowly (at least 48h) on standard media On Grams stain are slightly curved, club-shaped bacilli Corynebacterium Coryneform-like isolates are unable to be identified to the species level without 16s rRNA sequencing Species include: C. bovis, C. ulcerans, C. xerosis, C. jeikeium, C. pseudodiphtheriticum, C. pseudotuberculosis Corynebacterium diphtheriae VIRULENCE FACTORS. Diphtheria toxin. Produced by strains infected with lysogenic β-phage, which carries the tox gene Non-toxigenic strains can be converted to tox+ by infection with appropriate β-phage Only toxin producing C. diphtheria causes diphtheria Composed of 2 fragments A and B linked together by a disulfide bridge Fragment A is responsible for cytotoxicity and Fragment B binds to receptors on the eukaryotic cells Potent and lethal in humans in 130ng/kg of body weight Acts by blocking protein synthesis Excreted by the bacterial cell and is non-toxic until exposed to trypsin Clinical Significance: Respiratory diphtheria Humans are the only natural host for this organism The bacteria is carried on the URT and spreads by droplet or hand to mouth contact Incubation 2-5 days Illness begins gradually and is characterized by low-grade fever, malaise and mild sore throat The most common site of infection is the tonsils or pharynx Organisms rapidly multiply on epithelial cells Clinical Significance: Respiratory diphtheria Necrotic cell and exudate forms pseudomembrane Toxin causes demyelinating peripheral neuritis which may result in paralysis following the acute illness Clinical Significance: Cutaneous diphtheria Prevalent in the tropics Toxin is also absorbed systematically Consists of non-healing ulcers with a dirty gray membrane Treatment is by administration of anti-toxin produced in horses Drug of choice is P or E Lab DX MICROSCOPY CULTURE TOXIGENECITY TESTS Microscopy Highly pleomorphic gram positive bacillus that appears in palisades or as individual cells lying at sharp angles to another in V and L forms Club-shaped swellings and beaded forms are common Organism often stain irregularly when stained with methylene blue The metachromatic areas of the cell stain more intensely than other parts called Babes-Ernst granules Babes-Ernst granules are nutrient reserves Culture Characteristics Grow best at aerobic conditions at 37°C Multiplication can occur within the range of 15-40°C Require 8 essential amino acids Better growth is usually obtained on a medium containing blood or serum May have very small zone of β-hemolysis CTBA, Loeffler’s serum or Pai agars can be used Cat+ and non-motile C. diphtheria is urease negative, ferments glu and maltose with acid but no gas, reduces nitrate CTBA A modification of Tinsdale medium Contains sheep RBC, bovine serum, cysteine and potassium tellurite Both selective and differential Form black or brownish colonies from the reduction of tellurite A brown halo surrounding the colony due to cystinase activity are produce by C. diphtheria, C. ulcerans and C. pseudotuberculosis Toxigenecity test Addition of iron to iron-starved culture inhibits toxin production quickly In vitro: ELEK TEST Other methods include ELISA, immunochromatographic strip assays, PCR Listeria monocytogenes General characteristics Comprises 6 species, only L. monocytogenes and L. ivanovii are pathogenic Found in the environment from soil, water, vegetation and animal products such as raw milk, cheese, poultry and processed meats Has been isolated from crustaceans, flies and ticks Causes serious infection primarily of neonates, pregnant women, the elderly and immunocompromised host Virulence factors Listeriolysin O (hemolysin) Catalase superoxide dismutase Phospholipase C Surface protein p60 Clinical infections Listeriosis in pregnant women Most commonly in the 3rd trimester responsible for spontaneous abortion and stillbirth Flu-like illness with fever, headache and myalgia Often infection is self-limited because infection is eliminated when birth occurs Disease in the newborn Serious disease, fatality up to 50% 2 Forms: early onset and late onset Early Onset: intra-uterine infection or infection shortly after birth. Sepsis is often the result. Late Onset: several days to weeks after birth. Meningitis is often the manifestation. Disease in Immunocompromised Most commonly CNS infection and endocarditis Infection is acquired by eating food contaminated with L. monocytogenes Amp is the drug of choice Lab DX MICROSCOPY. Appears as gram positive coccobacillus found singly, in short chains and in palisades CULTURE. Grows well on SBA and chocolate agar, nutrient agar, BHIB and Thio broth. Colonies are small, round, smooth and translucent Surrounded by a narrow zone of β-hemolysis Growth occurs over a wide range (0.5-45°C); Optimum: 30-35°C Cold enrichment may help IDENTIFICATION. Catalase+, tumbling motility in wet smear, umbrella pattern in motility medium at room temp. but not at 35°C Lab DX IDENTIFICATION. Hippurate hydrolysis +, BE +, CAMP + Block type of hemolysis on CAMP Produces acid from glucose VP and MR + Non-Spore Forming, Non-Branching Catalase Negative Bacilli General Characteristics 3 species: rhusiopathiae, tonsillarum and inopinata E. rhusiopathiae is the only pathogenic Gram positive, catalase negative, non-spore-forming, pleomorphic rod with a tendency to form long filaments Found worldwide in animals swine, birds and fish Usual route is through the cuts and scratches on the skin Organism is resistant to salting, pickling and smoking Erysipelothrix rhusiopathiae: General characteristics Gram positive, non–spore-forming, pleomorphic rods (can produce long filaments) Distributed in nature Can cause disease in animals (swine, turkey, sheep); swine is the main reservoir Humans acquire the infection through occupational exposure, such as cuts & scratches (fish handlers, animal products) Erysipelothrix rhusiopathiae: Clinical Infections Erysipeloid Self-limiting localized infection at the site of inoculation Produces painful swelling, usually on the hands or fingers Heals within 3 to 4 weeks Treat with penicillin, cephalosporin, erythromycin Endocarditis May occur in those who have had valve replacements Disseminated infections may occur, but rarely due to exacerbation of erysipeloid lesions Laboratory Diagnosis: Erysipelothrix rhusiopathiae Colony Morphology CO2 is required Grows on blood or chocolate agar Colonies may appear gray or translucent, pinpoint Alpha hemolysis or nonhemolytic Laboratory Diagnosis: Erysipelothrix rhusiopathiae Microscopic Morphology Pleomorphic, Gram-positive thin rods that may form long filaments or short rods Arranged singly, in short chains, or in a V shape Decolorizes easily so it may appear gram variable Laboratory Diagnosis: Erysipelothrix rhusiopathiae Identification Catalase, nitrate, urease negative Non-motile Production of H2S on TSI VP negative, does not HOH Esculin Test tube brush growth in semisolid motility media 2 colony types: Pin point, non- hemolytic, glistening colonies or the larger, rough colonies with matte, curled and irregular edges Characteristics of Corynebacterium, Listeria, and Erysipelothrix Gardnerella vaginalis Member of the normal flora of the female genital tract Associated with bacterial vaginosis Foul odor Vaginal pH > 4.5 Laboratory Diagnosis: Gardnerella vaginalis Wet Prep Look for clue cells Large epithelials with various bacterial types on edges Gram stain Small, thin Gram variable rods Laboratory Diagnosis: Gardnerella vaginalis Cultural Characteristics Growth on BAP, CA No growth on MAC Human blood bilayer tween “V” agar Beta-hemolytic Requires a CO2 environment Catalase negative  Gardnerella Vaginitis Gram-variable-staining rod, facultative anaerobic bacteria (actually has a Gram-positive cell wall, but because the cell wall is so thin it can appear either Gram-positive or Gram- negative under the microscope). Small (1-1.5 µm diameter) non-spore forming, non-motile coccobacilli. Previously classified as Haemophilus vaginalis and afterwards as Corynebacterium vaginalis.  Gardnerella Vaginitis Growth: grows as small, circular, convex, gray colonies on chocolate agar; it also grows on HBT agar. Can cause bacterial vaginosis in some women as a result of a disruption in the normal vaginal microbiota  Can be isolated from other Areas Typically isolated in genital cultures. May also be detected in other samples from blood, urine, and pharynx  Morphology Small, Gram negative, on motile Pleomorphic rod which shows metachromatic granules Presence of Clue cells  Gram Stain still the simple in diagnosis of Bacterial Vaginosis The Centers for Disease Control and Prevention (2010) also recommend the gram stain as the gold standard for diagnosis of bacterial vaginosis  Culturing Grows on Blood and Chocolate Agar Hemolytic colonies on Human and Rabbit blood agar, Catalase – Oxidase -  Symptoms Up to 50% of women diagnosed with bacterial vaginosis do not have symptoms. In others, it causes an unpleasant "fishy" vaginal odor and a yellow or white vaginal discharge. For some women, these symptoms are especially bothersome during or after intercourse.  Observation of Vaginal Discharge The discharge seen in bacterial vaginosis tends to be thinner than the "cheesy," thick discharge seen in vaginal yeast (Candida) infections. Bacterial vaginosis usually does not cause significant irritation of the vulva or pain during intercourse. If you have these symptoms, your doctor will check for other possible causes.  No perfect test There is no perfect test, but if you have three of the following four criteria, it is highly likely that you have bacterial vaginosis: 1 White, thin, coating on your vaginal walls during the pelvic exam 44  Diagnosis 2 pH test of vaginal discharge that shows low acidity (pH greater than 4.5) 3 Fishy odor when a sample of vaginal discharge is combined with a drop of potassium hydroxide on a glass slide (the "whiff test")  Clue cells 4 Clue cells (vaginal skin cells that are coated with bacteria) visible on microscopic exam of vaginal fluid  Newer methods in diagnosis of Genital Infections DNA probes have been developed to directly detect the presence of Candida, Trichomonas and Gardnerella, thus providing a more objective diagnosis. Since Gardnerella is a normal part of the vaginal flora, the DNA probe test is designed to be relatively insensitive, detecting only pathogenic levels of Gardnerella. The Affirm VP III Microbial Identification System (Becton Dickinson) is a commercially available DNA probe office-based test kit that simultaneously detects the presence of Gardnerella, Trichomonas and Candida. 47  Treatment Commonly treat bacterial vaginosis with metronidazole (Flagyl or MetroGel-Vaginal) or clindamycin (Cleocin). Either can be taken by mouth or applied as a vaginal cream or gel. However, the U.S. Centers for Disease Control and Prevention (CDC) recommends that all pregnant women with symptoms be treated with oral medications because the medications are safe and work better than vaginal creams or gels.  Treatment Studies show that a seven-day treatment with oral metronidazole or a five-day treatment with metronidazole vaginal gel is equally effective in non-pregnant women. Clindamycin vaginal cream is slightly less effective than either type of metronidazole.  Complications The bacterial vaginosis has been associated with the development of pelvic inflammatory disease and other infections after endometrial biopsy, surgical abortion, hysterectomy, intrauterine device placement, Caesarean section and uterine curettage. Arcanobacterium The genus contains 6 species of which 3 are medically important: A. haemolyticum A. pyogenes A. bernardiae All are catalase negative, pleiomorphic rods that can branch Frequently, a black opaque dot is observed on the agar when scraped Pits agar Arcanobacterium haemolyticum Diseases associated include: Pharyngitis Desquamation of skin of the hands and feet Soft tissue infections, sepsis, endocarditis Produces small colonies with narrow zone of β-hemolysis after 24-48 hours of incubation Lipase+, Lecithinase+ Pen Resistant, Erythromycin is the drug of choice  Clinically important Gram positive bacilli Spore forming Acid-fast bacilli 1. Bacillus (O2) 1.Mycobacterium 2. Clostridium (O2) 2.Nocardia Non spore forming 1.Listeria Non-acid-fast branching 2. Erysipelothrix filamenous bacilli 3.Corynebacterium 1.Actinomyces 4. Propionibacterium Spore Forming, Non-Branching Catalase Positive Bacilli  Gram-Positive Spore-Forming Bacilli Gram positive, motile, rod shaped -Bacillus  GENUS BACILLUS Aerobic Catalase positive Not fastidious  1.BACILLUS Bacillus anthracis – Human pathogen – Isolation also considered to be clinically significant – Zoonosis Bacillus cereus – Environmental organism – Contaminates food – Common cause of food poisoning Bacillus (Geobacillus) stearothermophilus – Tolerates very high temperatures – Used for quality control of autoclaves  a.Bacillus anthracis Large bacilli of 3-5 m Single or paired in clinical isolates Polypeptide capsule and exotoxins Highly resistant central spores Anthrax toxin Consists of 3 proteins: PA, LF, and EF PA serves as the binding molecule for EF and LF PA + EF = Edema PA + LF = Death  Anthrax Spore of B.anthracis Duration of life (> 60 years) soil Medusa head colonies of Bacillus anthracis  Anthrax Zoonotic disease of herbivorous livestock Anthrax - Epidemiology  Anthrax Pathogenesis and clinical presentations Cutaneous Cutaneous anthrax anthrax About About20% 20%mortality mortality Virulence factors Capsule (antiphagocytic) Toxin (oedema & death) Inhalation anthrax Inhalation anthrax Gastrointestinal Gastrointestinal anthrax anthrax Highmortality High mortality High Highmortality mortality  Cutaneous anthrax « malignant pustule » Incubation 2-3 days erythematous papule Increasingly necrotic  later ruptures to form a painless black eschar  Gastro-intestinal anthrax Contaminated meat Asia, Africa Cardiovascular collapse within few hours and death  Pulmonary anthrax Toxemia, capillary thrombosis, cardiovascular shock  Injectional anthrax Injectional anthrax is characterized by soft tissue infection associated with “skin popping” or other forms of injection drug use Results from the direct injection of the spores into tissue Can be associated with necrotizing fasciitis, organ failure, shock, coma, and meningitis, Has a much higher rate of mortality than cutaneous Have not been associated with black eschar formation  Bioterrorism and Anthrax bacillus  Anthrax - Diagnosis Specimen – Aspirate or swab from cutaneous lesion – Blood culture – Sputum Laboratory investigation – Gram stain – Culture – Identification of isolate Anthrax – treatment and prevention Penicillin (Tetracycline /chloramphenicol) Erythromycin,Clindamycin Prevention – Vaccination of animal herds – Proper disposal of carcasses (burning or decontamination before burial) Active immunization with live attenuated bacilli  b. Bacillus cereus Large, motile, saprophytic bacillus Heat resistant spores Airborne and dust-borne contaminants Pre formed heat and acid stable toxin (Emetic syndrome) Heat labile enterotoxin (Diarrhoeal disease) Multiply readily in cooked foods (Rice, potato and meat) Lab diagnosis – Demonstration of large number of bacilli in food Bacillus cereus clinical presentation Gastroenteritis Gastroenteritis EMETIC FORM DIARRHOEAL FORM Incubation period > 6 hours Incubation period < 6 hours Diarrhoea Severe vomiting Lasts 20-36 hours Lasts 8-10 hours Non-spore-forming, Branching Aerobic Gram Positive Bacilli: Aerobic Actinomycetes and Nocardia Nocardia Aerobic, branched, beaded gram positive bacilli Beads are not usually spaced at consistent intervals Finely beaded, branching rods, weakly acid-fast Grow on standard, non-selective media after a week or more Commonly found in soil Infections resemble fungi and usually occur in immunocompromised patients Species include: N. asteroides, N. brasiliensis, N. farcinica and N. nova Virulence factors No virulence factors have been identified Correlated with alterations in the cell wall components Produces SOD and catalase Produces iron-chelating compound called nocobactin Clinical infections 2 routes: pulmonary and cutaneous Pulmonary by inhalation of the organism present in dust or soil usually in immunocompromised persons Mortality rate is high and those who survive often suffer significant damage Pulmonary infections Commonly caused by N. farcinica and N. ciriacigeorgica Common manifestation is confluent bronchopneumonia Disease progresses more rapidly than TB and is measured in months rather than years In acute form, time course is a matter of weeks Initial lesion starts with a focus of pneumonitis that advances to necrosis There is little inflammatory response or scarring, no encapsulation of the abscess and no granuloma formation, no sulfur granules or sinus tract formation Cutaneous infections Occurs following inoculation in the skin and SQ tissues, usually skin and feet N. brasiliensis is the most frequent cause Lesions are termed actinomycotic mycetomas Pus may be pigmented and contain sulfur granules (appearing yellow or orange hence the term) Lab DX MICROSCOPY. Beaded branching filaments often seen in sputum and exudates or aspirates from skin and abscesses Weakly acid fast (0.5-1% sulfuric acid) Granules (when separated from pus) can be seen in wet mounts from cutaneous infection CULTURE Oxidative type metabolism Do not require specific growth factors, grow well on common media Growth occurs after 3-6 days TM may be used Lab DX CULTURE Colonies are chalky, matte, velvety appearance and might be pigmented Can have dry, crumbly appearance Colonies may reveal presence of aerial hyphae IDENTIFICATION. Substrate hydrolysis (casein, tyrosine, xanthine) CHO utilization Antimicrobial susceptibility profile Fatty acid analysis by HPLC 16s rRNA gene sequencing Treatment Drainage and surgery along with antimicrobials R to Pen but susceptible to sulfonamides Lab results are ABSOLUTELY essential for proper antimicrobial treatment Other Genera of the Actinomycetes Actinomadura Streptomyces Gordonia Tsukamurella Rhodococcus Trophyrema whipplei MYCOBACTERIA MYCOBACTERIUM Aerobic bacilli –non spore forming non motile Cell wall –rich in lipids Acid-fast bacilli Very slow growing  MYCOBACTERIA ASSOCIATED WITH HUMAN DISEASE Mycobacterium Environmental contaminant Reservoir M tuberculosis No Human M bovis No Human, cattle M leprae No Humn M kansasii Rarely Water, cattle M marinum Rarely Fish, water M scrofulaceum Possibly Soil, water M avium Possibly Soil, water, birds intracellulare M ulcerans No Unknown M fortuitum Yes Soil, water, animals M chelonae Yes Soil, water, animals  CLASSIFICATION OF MYCOBACTERIA ASSOCIATED WITH HUMAN DISEASE Mycobacterium Clinical significance Pigmentation Growth Unclassified M tuberculosis , M bovis Strict pathogens No No M ulcerans M leprae Strict pathogen - - Runyon Group 1 M marinum , kansasii Usually pathogenic Photochromogens slow Runyon Group 2 M scrofulaceum Rarely pathogenic Scotochromogens slow Runyon Group 3 M avium intracellulare Pathogenic in No slow immunocompromised Runyon Group 4 M fortuitum, M chelonae Rarely pathogenic No ‘rapid’ Mycobacterium tuberculosis Causes tuberculosis Classic human disease Pathogenesis Transmission Clinical presentations Diagnosis Treatment Prevention  Pathogenesis Inhaled aerosols Engulfed by alveolar macrophages Bacilli replicate Macrophages die Infected macrophages migrate local lymph nodes Develop Ghon’s focus Primary complex Cell mediated immune response stops cycle of destruction and spread Viable but non replicating bacilli present in macrophages EVIDENCE OF INFECTION WITH M TUBERCULOSIS Chest x-ray / positive skin test  CLINICAL PRESENTATION Pulmonary tuberculosis Primary complex Asymptomatic HEALS Acute pulmonary disease REACTIVATION Systemic spread Post-primary tuberculosis Aymptomatic /symptomatic LATER DISEASE MILIARY TUBERCULOSIS Renal / CNS etc Pulmonary meningitis  DIAGNOSIS Pulmonary tuberculosis 1 1 Primary complex Asymptomatic HEALS 2 3 Acute pulmonary disease REACTIVATION Systemic spread Post-primary tuberculosis Aymptomatic /symptomatic LATER DISEASE MILIARY TUBERCULOSIS 3 Renal / CNS etc Pulmonary 3 meningitis  DIAGNOSIS 1. Evidence of infection a. Chest x-ray - hilar lymphadenopathy calcification of primary focus/LN b. Delayed hypersensitivity response to purified protein derivative (PPD) MANTOUX /HEAF TEST 2. Evidence of active disease a. Sputum for AFB positive 3. Evidence of active disease a. Indirect evidence of infection (Mantoux) b. Direct evidence of infection PCR / culture c. Histo-pathological evidence TREATMENT Anti-tuberculous drugs INAH Rifampicin Ethambutol Pyrazinamide DOT Multi-drug resistant tuberculosis PREVENTION Incidence declined before availability of anti-tuberculous drugs Improved social conditions - housing /nutrition Case detection & treatment Contact tracing Evidence of infection / disease Treatment of infected / diseased contacts ROLE OF IMMUNIZATION BCG (bacillus Calmette Guerin) Mycobacterium leprae Acid fast bacilli Strict human pathogens Cannot be cultivated in-vitro Armadillo’s used for obtaining M leprae Transmission - ? Air borne Low infectivity - prolonged contact required Spectrum of clinical presentations dependent on host –parasite interactions Borderline Borderline Tuberculoid Lepromatous Tuberculoid lepromatous Non tuberculous mycobacteria M KANSASII  Infection of respiratory compromised hosts Present like pulmonary tuberculosis Treatment – resistant to anti TB drugs M SCROFULACEUM  Infection of cervical lymph nodes Presents as cervical lymphadenopathy Treatment – surgery Non tuberculous mycobacteria M avium intracellulare Immuno competent host Infection of cervical lymph nodes Presents as cervical lymphadenopathy Treatment – surgery M avium intracellulare Immuno deficient host  severe RTI AIDS severe GI infection septicaemia Non tuberculous mycobacteria M ulcerans  ‘Buruli’ ulcer  Prolonged incubation required for growth M fortuitum / M chelonei  Injection related abscesses Associated with sternal wound infections following cardo-thoracic surgery