Anaerobes and Anaerobic Bacteriology PDF

Anaerobes and Anaerobic Bacteriology RAQUEL M. FERNANDEZ, RMT, MSPH What are anaerobes  Are bacteria able to replicate in the absence of oxygen  Oxygen are often extremely toxic for them Types of anaerobes  Obligate anaerobes (occasionally called aerophobes) do not...

Anaerobes and Anaerobic Bacteriology RAQUEL M. FERNANDEZ, RMT, MSPH What are anaerobes  Are bacteria able to replicate in the absence of oxygen  Oxygen are often extremely toxic for them Types of anaerobes  Obligate anaerobes (occasionally called aerophobes) do not have SOD and catalase  Aerotolerant anaerobes  Facultative anaerobes (or facultative aerobes) Strict anaerobes  Obligate (strict) anaerobes - oxygen is toxic to these organisms, do not use oxygen as terminal electron acceptor.  Archaea such as methanogens and Bacteria, e.g Clostridia, Bacteriodes etc. etc. Aerotolerant anaerobes  Lactobacillus  Propionibacterium Exogenous vs Endogenous Anaerobic infection Exogenous Endogenous  Caused by anaerobes that exist  Caused by anaerobes that exist outside the bodies of animals inside the bodies of animals  Usually caused by gram positive  More commonly encountered in spore-forming bacilli human infections Requirements for Growth Detoxification of Oxygen radicals Oxygen toxicity  O2 + e- => O2- superoxide anion  O2- + e- + 2H+ => H2O2 hydrogen peroxide  H2O2 + e- + H+ => H2O + OH. Hydroxyl radical  OH. + e- + H+ => H2O water Where are anaerobes found? Anaerobic environments (low reduction potential) include:  Sediments of lakes, rivers and oceans; bogs, marshes, flooded soils,, deep underground areas, e.g. oil packets and some aquifers  Components of microbiota of humans and other animals  ENDOGENOUS (intestinal tract of animals; oral cavity of animals)  EXOGENOUS (exist outside the bodies of animals) General characteristics  Anaerobes generate energy by fermentation  Lack the capacity to utilize O2 as a terminal hydrogen acceptor  Some are sensitive to O2 concentration as low as 0.5% O2  Most can survive in 3%-5% O2  A few can grow poorly in the presence of air  aerotolerant anaerobes  Many are members of the normal flora created by presence of facultative anaerobes Anaerobes of Clinical Importance  CLOSTRIDIA  C tetani; C perfringens; C difficile; C botulinum  BACTEROIDES  B fragilis;  Prevotella  Porphyromonas  ACTINOMYCES  FUSOBACTERIUM  ANAEROBIC STREPTOCOCCI Anaerobes Morphology Anaerobes Morphology Virulence Factors 1. Lipopolysaccharide - promotes abscess formation, enhanced coagulation 2. Polysaccharide capsule - correlated with abscess production 3. Enzymes a. Collagenase b. Heparinize * develop thrombophlebitis & septic emboli 4. Short chained fatty acids a. Butyrate- seen in dental plaque b. succinic acid – reduces phagocytic killing Factors that Predispose Patients to Anaerobic Infections  Trauma to mucous membranes or skin  Vascular stasis  Decreased oxygenation of tissue leading to tissue necrosis and decrease of redox potential of tissue Clinical Manifestation Suggestive of Anaerobic Infection  Odor  black discoloration  tissue  blood containing exudates  location  associated with sulfur  necrotic tissue granules  endocarditis with (-) blood culture  Bacteremic feature with  infection associated with jaundice malignancy  human bites Important Clinical Infections with Gram Positive, Spore-Forming Anaerobic Bacilli  CLOSTRIDIUM Anaerobic nonmotile Sporing Catalase negative Gram positive Diameter of the spore is larger than the cell resemble a spindle Clostridium is derived from Kloster meaning spindle  Spores  Saprophytes - Most Some are opportunists - tetanus/gas gangrene/food poisoning Cl. perfringens - commensal of the intestine Cl. sporogenes - – Can invade the intestine after the death  CLASSIFICATION BASED ON THE TYPE OF DISEASE PRODUCED A. Tetanus Cl. tetani - Present in soil B. Gas gangrene – Established Cl. perfringens ‘gut’ organism Cl. septicum Cl. novyi - Less pathogenic Cl. histolyticum Cl. fallax - Doubtful Cl. bifermentans Cl. sporogenes  C. Food poisoning 1. Gastroenterritis - Cl perfringens Type A 2. Botulism - Cl. botulinum/ Soil 3. Pig-bel Cl. perfringens type C D. Acute colitis - Cl. difficile / gut’ organism (pseudomembranous colitis) – Commonest cause of ‘nosocomial’ diarrhoea CLASSIFICATION BASED ON THE TYPE OF DISEASE PRODUCED  E. Bacteremia. Commonly caused by C. perfringens. C. septicum, when present is a marker of GI malignancy  Role of clostridia in infection and disease Wound and tissue infections *Myonecrosis *Antibiotic-associated colitis *Tetanus Food intoxication of perfringens and botulism varieties.  Gas gangrene Anaerobic cellulitis or myonecrosis Predisposing factors: surgical incisions Contaminated by compound fractures Spores from the Body or diabetic ulcers environment septic abortions puncture and gunshot wounds crushing injuries  Lecithinase C (alpha toxin) Dead tissue, blood clots, foreign matter aerobic organisms In an injury DEVELOP ANAEROBIC CONDITION (Exogenous infection) Germination of spores Gas gangrene Rupture of RBC, edema, necrosis, gas production, toxemia, myositis Crepitus  C Perfringens C histolyticum C septicum C novyii C Perfringens Alpha toxin (lecithinase) Collagenase Hyaluronidase DNAse  Clinical signs Pain Edema Bloody exudate in the lesion Fever Tachycardia Blackened necrotic tissue filled with bubbles of gas Antibiotic-Associated Colitis Clostridium difficile C. difficile PSEUDOMEMBRANOUS COLITIS Virulence factors Enterotoxin (Toxin A) Cytotoxin (Toxin B) Management Discontinue antibiotics Diagnosis Ampi/Tetra/Clinda Oral Clinical suspicion metronidazole Culture of faeces Oral vancomycin Detection of toxin  TETANUS (Lock-jaw) Cause tetanus in both man and animals disease which effect the nervous system of the host. - Agricultural workers and gardeners and are more prone because the spores are present in the soil & intestine of animals. - At birth under unhygienic conditions baby’s can get – tetanus neonatorum.  Clostridium tetani Neuromuscular disease Entrance of spores through accidental puncture wounds – burns – Umbilical stumps – Frostbite – Crushed body parts  Soil/Intestine/Vagina Drum stick appearance Motile with peritrichous flagella Obligatory anaerobes Grow on Robertson’s cooked medium Clostridium tetani  Toxins Tetanolysin - heat and oxygen labile/lyse RBC/ Tetanospasmin - heat and oxygen stable/highly lethal (for mice 0.0000001 mg) dies within 1 - 2 days get easily neutralize with antitoxin  – What happens Toxin acts at the synaptic junction – prevent the synthesis of acetylcholine. Thus, prevents synaptic transmission.  GABA GLYCINE  Spores germinate -------toxin-----motor nerve endings--------along the motor neurones of the peripheral nerve to the anterior horn cells- -----local tetanus (in the proximity of the wound). Ascending tetanus – when toxins spreads upwards along the spinal cord towards C.N.S. Gives generalized spasms. Descending tetanus – when toxin is given IV , spasms will appear in the muscles of the head, neck and spreads downwards.  Clinical symptoms Incubation period: 4-10 days Early symptom is trismus (lock jaw) – spasms of the masseter muscle - difficulty in opening of the mouth and masticating - rigidity spreads to muscles of the face, neck and truck - risus sardonicus –The semblance of a grin caused by facial spasm especially in tetanus at the angle of the mouth - back is usually slightly curved (Opisthonotus ?) - In severe cases violent spasms will last for few seconds to 3-4 mins. - If convulsions appear soon after the initial symptoms, it is very serious. - The spasms gradually intensify and patient may die of –.Paralysis of respiratory muscles and respiratory collapse - fatality rate is 10-70% Trismus Opisthotonos  Clostridium tetani Clinical diagnosis Treatment: – Symptomatic treatment cleansing and removing the afflicted tissue, Penicillin or tetracycline Muscle relaxants assitance of respiration( sometimes tracheostomy) - 10, 000 units of human tetanus immunoglobulin (HTIG) Prevention and control a. Immunization - HTIG 250 - 500 units (to immune patients only) b. To non-immune toxoid followed by HITG The recommended vaccination series for 1- to – 3-month- old babies: three injections given 2 months apart, Booster doses about 1 and 4 years later. Protection against neonatal tetanus: Vaccination of pregnant women  Clostridial food poisoning C. perfringens, type A Carriers for food poisoning strains Survival of heat resistant spores in meals (meat, fish, beans etc.) Sporulation in gut - Short IP and watery diarrhoea, acute abdominal pain and vomiting for 24-48 hours Beta toxin production in C. prerfringens type C – Necrotizing enteritis(Pig bell)  BOTULISM Sausage 8 toxins (A-G) Food borne botulism (IP 1-2 days) Infant botulism Wound botulism (IP > 4 days) Diagnosis Isolation of organism in food/faeces Detection of toxin in faeces / serum C. botulinum Produces Botulism World wide distribution Found in soil and occasionally in animal feces Sporese are highly heat resistant ,withstand 100C for 3-5 hrs.(120C for 5-10min) Heat resistance is reduced by acid pH or high salt concentrations Toxin Released during growth and autolysis of bacteria. It is found in 8 antigenic varieties.A-G The principle cause for human disease A,B,E/F A,B - Variety of foods E - Fish products C - Limberneck in birds D - botulism in mammals Toxin is neurotoxic protein Destroyed by heating at 100C for 20 mins. Action :Block release of Acetylecholine at synapses and NMJ causing flaccid paralysis. Pathogenecity Illness is not an infection. Botulism is an intoxication resulting from the ingestion of food in which C.botulinum has produced toxin.  Botulism Incubation period 12-36 hours Diplopia Dysphagia and dysphonia Paralysis of muscles and respiratory system Mortality rate is 65-70% Botox  Infant botulism Spores germinate in the body and produce infection Raw honey has been implicated in some cases. Spores are common in dust and soil Spores germinate in the intestine and give off neurotoxin  Infant botulism Flaccid paralysis Weak sucking response Generalized loss of tone Weakness and cachexia Respiratory complications  Wound botulism The spores enter a wound or puncture much as in tetanus The symptoms are similar to those of food born botulism More common in drug abusers  Treatment and prevention of botulism -The CDC provides a source of type A, B, and E trivalent antitoxins -Respiratory and cardiac support -Penicillin - Attention to home-preserved foods - Addition of preservatives (sodium nitrate, salt and vinegar) - Toxin in sensitive to 100C for few minutes Important Clinical Infections with Gram Positive Non-spore-forming Anaerobic Bacilli Actinomycosis  Caused by Actinomyces israelii, Propionibacterium and Bifidobacterium  Chronic granulomatous infectious disease  Characterized by the development of sinus tracts and fistulae which erupt to the surface and drain pus that may contain “sulfur granules”  Most common site is the maxillary region and the female genital tract Bacterial Vaginosis  Result of shift in vaginal biota resulting in the overgrowth of other endogenous anaerobes such as Mobiluncus spp., Bacteroides spp., Prevotella spp., anaerobic gram positive cocci, Gardnerella vaginalis.  Gray-white, homogenous, malodorous vaginal discharge with little or no discomfort and no inflammation  Diagnosed based on clinical appearance and a Grams stain of the vaginal secretions Lactobacillus  Gram positive highly  Help protect the health of female genital tract by pleiomorphic bacilli producing lactic acid from glycogen  Appear as coccoid or  Lactobacilli lowers vaginal spiral-shaped organisms pH which suppresses  Aerotolerant anaerobes overgrowth of Mobiluncus, Prevotella and Gardnerella  Over 100 species of which  Associated with L. acidophilus endocarditis, the most predominates common clinical disease it can cause  Widely distributed in nature and foods  Normal biota in the mouth, GIT, and female genital tract Lactobacillus acidophilus  Colonies vary greatly with species  Pin-point alpha hemolytic colonies on SBA ; medium size, gray color and rough on other media  catalase –  Frequently resistant to cephalosphorins  Treatment is usually P with an aminoglycoside Important Clinical Infections with Anaerobic Gram Negative Bacilli  Peritoneal infections  Septicemia  Abscesses such as diabetic foot ulcers, decubitus pressure sores Important Clinical Infections with Anaerobic cocci  Veilonella and Finegoldia magna are pathogens  Finegoldia magna is the most pathogenic anaerobic cocci and most often isolated in pure culture  Inhabit the oral cavity  Seen in brain abscesses, lung abscess, gingivitis, periodontal diseases  Often associated with polymicrobic infections  Ocassionaly recovered from blood cultures or following orthopedic surgery Specimen Selection, Collection, Transport and Processing for Anaerobic Cultures Appropriate Specimens  Pus  Pleural fluid  SPA urine  Pulmonary secretions  Uterine secretions  Sinus tract material Collection If a swab must be used, a 2 tube system must be used  1st tube contains swab in O2 free CO2  2nd tube contains PRAS (pre-reduced anaerobically sterilized culture media) Specimen should be placed in anaerobic transport device with gas mixture Transport  Must be transported and processed as rapidly as possible with minimum exposure to oxygen  Specimens should remain at room temperature, not refrigerated Processing for Culture  Once specimen arrives in the lab. it should be placed immediately in the anaerobic chamber  Macroscopic examination of the specimens  Preparation of Gram’s stained smears  Inoculation of appropriate pre- reduced plates and tubed media  Anaerobic incubation of inoculated media Lab clues to Diagnosis of Anaerobic infections See you at the Lab for Culture and Identification Methods for Anaerobes!