Anaerobic Bacterial Infections and Their Lab Diagnosis

Anaerobic bacterial infections and their lab diagnosis What Are Anaerobic Microorganisms • Anaerobic microorganisms are widespread and very important. • They do not have cytochrome system for oxygen metabolism and hence are unable to neutralize toxic oxygen metabolites (Superoxide anion, hydrogen peroxide,Hydroxyl radical). • In addition, oxygen has a high affinity for organic compounds containing nitrogen, hydrogen, carbon, and sulfur, which interfere with normal biologic activity. 2 Characteristics of anaerobes Obligate anaerobic bacteria Aerotolerant anaerobes Microaerophilic bacteria Grow in the absence of free oxygen, fails to multiply in the presence of oxygen. Do not utilize oxygen for Requires lower level of oxygen growth, show limited or that present in atmosphere to scanty growth in presence of survive (<21% O₂; typically 2oxygen. 10% O₂) They shows good growth under anaerobic conditions. Completely lack superoxide dismutase and catalase enzymes. They posses small amount of superoxide dismutase and peroxidase(but lack catalase). Organism require O₂ as a terminal electron acceptor. Bacteroides fragilis, Clostridium perfringens, C.novyi, Porphyromonas, Fusobacterium, Prevotella. C. carnis, C. histolyticum, C. tertium, Actinomyces spp., Bifidobacterium Campylobacter jejuni (grow in 5% O₂) Classification of anaerobes Gram positive Spore-Forming Bacilli Non-Spore-Forming Bacilli Gram negative Clostridium species (botulinum, difficile, perfringens, septicum, sordellii, tetani etc.) Actinomyces, Arcanobacterium, Bifidobacterium, Eubacterium, Lactobacillus, Methanobacterium, Mobiluncus, Propionibacterium etc. Cocci Peptostreptococcus, Micromonas, Peptococcus etc. Bacilli Bacteroides, Borrelia, Butyrivibrio, Capnocytophaga, Campylobacter, Fusobacterium, Leptotrichia, Porphyromonas, Prevotella, Treponema etc. Cocci Acidaminococcus, Megasphaera, Based on fermentation of carbohydrates SACCHAROLYTIC ASACCHAROLYTIC • PREVOTELLA P.intermedia P. nigrescens P. melaninogenica • BACTEROIDS • PORPHYROMONAS P. asaccharolytica FACTORS RESPONSIBLE FOR THEIR VIRULENCE 1. Lipopolysaccharide - promotes abscess formation, enhanced coagulation 2. Polysaccharide capsule - correlated with abscess production 3. Enzymes a. Collagenase b. Heparinase - develop thrombophlebitis & septic emboli 4. Short chained fatty acids a. Butyrate- seen in dental plaque b. Succinic acid – reduces phagocytic killing Anaerobes as a part of normal flora Anatomical site Bacteria/g or ml Anaerobic/aerobic ratio Common anaerobic normal flora Mouth Saliva 10 ⁸ - 10⁹ 1:1 Tooth surface 10¹⁰ - 10¹¹ 1:1 Gingiva 10¹¹- 10¹² 10³:1 Anaerobic cocci Actinomyces Fusobacterium Bifidobacterium Prevotella Spirochetes Gastointestinal tract Stomach 0 - 10⁵ 1:1 Lactobacillus Jejunum/ileum 10⁴ - 10⁷ 1:1 Terminal ileum and colon 10¹¹ -10¹² 10³:1 Anaerobic cocci Bacteroides fragilis Fusobacterium 7 Prevotalla Famale genital tract Vagina 10⁷ - 10⁹ 10³:1 Anaerobic cocci Lactobacillus Prevotella Bifidobacterium 3:2 Propionibacterium Skin Skin 8 Common Human Anaerobic Infections Dr.T.V.Rao MD 9 Sites and Infection produced by Anaerobes 10 CLOSTRIDIA • Gram positive bacilli, endspore-forming, obligate anaerobic (or aerotolerant), catalase-negative • Present as normal flora in the intestine of humans and animals, female genital tract and oral mucosa. • Also found in soil, fresh water, marine water, decaying vegetation, animal matter and sewage; thus play a major role in recycling of organic matter. • Several species associated with human disease. 11 Clostridium perfringens  It is capsulated, non-motile, gram positive bacillus (boxcar-shaped).  Spores seldom seen in vivo or in vitro  Produces several toxins • Four major toxins- alpha (lecithinase), beta, epsilon and iota. • Enterotoxin  Causes a spectrum of human diseases • Bacteraemia • Gas gangrene (Myonecrosis) • Food poisoning • Enteritis necroticans (necrotizing enteritis) - gas gangrene of the bowel/ Pigbel/ Darmbrand Clostridium tetani • Small motile spore forming gram positive bacillus with round terminal spores (drum stick appearance). • Causes tetanus. • It produces two exotoxins Tetanolysin: it is heat labile and oxygen labile hemolysin. It plays no role in the pathogenesis of tetanus. Tetanospasmin: • It is a neurotoxin responsible for disease manifestations. • It is oxygen stable but heat labile. • It gets toxoid spontaneously or by formaldehyde. • Acts by blocking release of inhibitory neurotransmitters (eg. GABA and glycine) 14 Clostridium difficile • Gram positive, spore-forming bacillus • Associated with prolonged antimicrobial use (eg. cephalosporins, clindamycin, ampicilline and fluoroquinolones ) • Produces 2 toxins Toxin A -enterotoxin & Toxin B –cytotoxin • Causes diarrhea and pseudomembranous colitis. 16 Clostridium botulinum  Anaerobic gram positive bacillus with subterminal spore.  Produces powerful neurotoxin ‘botulinum toxin’, which typed in to 8 serotypes - A, B, C1, C2, D, E, F & G.  Human disease described with types A, B & E.  After entry of botulinum toxin, it is transported via blood to peripheral cholinergic nerve terminals.  Toxin internalized and prevents release of acetyl choline from vesicles, leading to flaccid paralysis.  Clinical • Food borne botulism (weakness, dizziness, ocular palsy and progressive flaccid paralysis) • Infant botulism (floppy baby) • Wound botulism Gram positive non-sporing anaerobic bacilli Actinomyces  Strict anaerobic Gram positive, non-motile, non sporing bacilli typically arranged in chains or branching filaments.  Normal flora of upper respiratory tract, GI tract and female genital tract.  Produce disease when mucosal barrier is breached (eg: following dental extraction or surgery)  Clinical Manifestations• Cervicofacial actinomycosis • Abdominal form – due to spillage of flora secondary to bowel surgery • Pelvic form – following IUCDs insertion • Brain abscess and soft tissue infection • Dental caries 20 Bifidobacterium species • Gram positive, non-motile, pleomorphic bacilli that frequently exhibit branching. • Normal flora of mouth and gut and are mostly non pathogenic. • Usually encountered in mixed infections of the pelvis or abdomen. Lactobacillus species • They are part of normal flora of mouth, gut and vagina. • Associated with advanced dental carries and also has been identified in endocarditis and bacteremia. ANAEROBIC GRAM NEGATIVE BACILLI • Bacteroides, Prevotolla, Porphyromonas and Fusobacterium. • Present in GI tract -form large component of normal flora. Bacteroides fragalis – -most commonly encountered in anaerobic infection. -virulence factors - capsule, LPS, endotoxin and enzymes • Clinical - Endogenous infections • Peritonitis following bowel injury and PID • Lung abscess and brain abscess • Pelvic and genitourinary infections • Skin and soft tissue infection • Diarrhea Prevotella  It has many species which can be grouped into-pigmented (P. melaninogenica) & nonpigmented (P.denticola and P. buccalis).  Pigmented species produces hemin derived black or brown colored colonies which appear red fluorescence when exposed to UV light.  Causes – Gingivitis and periodontitis Sinusitis Brain and lung abscess Bacterial vaginosis Porphyromonas  They are asaccharolytic and pigmented.  Responsible for periodontal disease and dental root canal infections. 24 Fusobacterium species • Long, thin spindle shaped bacilli with pointed ends. • Normal inhabitant of mouth. • Causes oral infection and pleuropulmonary sepsis. • F. nucleatum is agent of Lemierre’s syndrome (thrombophlebitis of internal jugular vein) 25 Anaerobic gram positive cocci Peptostreptococcus • Gram positive cocci in pairs or chains. • They are normal flora of skin, mouth, intestine and vagina. • They can cause various clinical infection such as puerperal sepsis, skin and soft tissue infections and brain abscess. Anaerobic gram negative cocci Veillonella species  Small gram negative cocci occuring in pairs or short chain.  Normal flora of skin, mouth and vagina.  May be involved in mixed infection.  Organism have been isolated increasingly infections, including meningitis, osteomyelitis, endocarditis, bacteremia, and prosthetic infection. Lab Diagnosis of Anaerobes Clinical Specimens Suitable for Anaerobic Culture • Bile • Blood • Bone marrow • Bronchial washings obtained with a double-lumen plugged catheter • Cerebrospinal fluid • Decubitus ulcer (if obtained from base of the lesion after thorough debridement of the ulcer’s surface) • Fluid from normally sterile site (e.g. joint) • Material aspirated from abscesses • Transtracheal aspirate • Percutaneous lung aspirate or biopsy • Peritoneal (ascitic) fluid • Sulfur granules from a draining fistula • Suprapubic bladder aspirate • Thoracentesis (pleural) fluid • Tissue obtained at biopsy or autopsy 28 Clinical Specimens Unsuitable for Anaerobic Culture • Bronchial washing or brush (unless collected with a double-lumen plugged catheter) • Coughed (expectorated) sputum • Feces (except for Clostridium difficile) • Gastric or small-bowel contents (except in blind loop syndrome) • Ileostomy or colostomy drainage • Nasopharyngeal swab • Rectal swab • Secretions obtained by nasotracheal or orotracheal suction • Swab of superficial (open) skin lesion • Throat swab • Urethral swab • Vaginal or cervical swab • Voided or catheterized urine 29 Specimen Collection and Transport • Proper collection and transport of specimens is required as atmospheric oxygen can cause lethal effect. • Anaerobes are often present in large numbers as normal flora on mucosal surfaces, even minimal contamination of a specimen can produce misleading results. Three kinds of anaerobic transport systems are available A. Anaerobic transport system for liquid specimens – It’s a rubber-stoppered collection vial containing an agar indicator system. The vial is gassed out with oxygenfree CO2 or nitrogen. The specimen (pus, body fluid, or other liquid material) is injected through the rubber stopper after all air has been expelled from the syringe or needle. B. anaerobic transport system for swab specimen A sterile pack contains a sterile swab and an oxygen free inner tube. After the specimen has been collected, the swab is inserted back into the inner tube. Agar on the bottom of the outer tube contains an oxygen tension indicator. C. Anaerobic transport system for tissue specimen Tissue is placed in small amount of saline to keep it moist. Then inserted into a self-contained, atmosphere-generating anaerobic bag for transportation. This system is called GasPack pouch. Put samples in room temperature while waiting for delivery to the laboratory. Some anaerobes are killed by refrigeration. Macroscopic Examination of Specimens The specimens should be inspected for characteristics that strongly indicate the presence of anaerobes: (1) Foul odour (2) Sulfur granules (associated with Actinomyces spp., Propionibacterium spp., or Eubacterium nodatum) (3) Brick red fluorescence under long wavelength ultraviolet (UV) light (associated with pigmented Prevotella or Porphyromonas spp). Some clinical hints also should be considered  Location (infection in proximity to a mucosal surface)  Gas in tissues  Negative aerobic cultures of specimens whose gram stains show organisms and pus cells.  Endocarditis with (-) blood culture  Infection associated with malignancy  Black discoloration  Blood containing exudates  Bacteremic feature with jaundice  Human bites DIRECT DETECTION METHODS GRAM STAINING • The Gram stain is an important rapid tool for anaerobic bacteriology. • Standard Gram stain procedures and reagents are used, except that the safranin counterstain is left on for 3 to 5 minutes. • Stain poorly with safranin, As an alternative, 0.5% aqueous basic fuchsin can be used as the counterstain. ANTIGEN DETECTION • The cytotoxin (toxin B) of Clostridium difficile can be detected by using a tissue culture assay. • Latex particle agglutination tests or enzyme-linked immunosorbent assays (ELISA) to detect toxin A and toxin B. • Enzyme immunoassay for glutamate dehydrogenase antigen found in toxigenic strain of C. difficile. Testing for anaerobes in Routine Practice • Thiogycolate broth tubes can be used to test whether an unknown organism is aerobic/ anaerobic (obligate/facultative) Specimen Processing Specimens for anaerobic culture may be processed in the biologic safety cabinet, after which they are incubated in • McIntose and filde’s anaerobic jar • GasPack system • Anoxomat system • Anaerobic glove box • Pre-reduced anaerobically sterilized (PRAS) media McIntosh and Filde’s anaerobic jar • Most effective and popular method. • Consists of a metal or glass jar with a metal lid with a screw (to close airtight), pressure gauge and two openings (inlet and outlet). • Removal of residual oxygen is done by using palladium catalyst, which is suspended from the inner side of the lid. • It helps in removing residual oxygen by combining with hydrogen to form water. [O₂ + H₂ catalyst H₂O] Anoxomat • Automated equipment which evacuates the air from jar and replaces by hydrogen gas from a cylinder. • Same catalyst is used here to remove the traces of oxygen. • Easier to operate than McIntosh jar method and claims to be highly effective for creating anaerobiosis. GasPak System • Most commonly used method for anaerobiosis, simple to perform and is perfect for a laboratory having less sample load. • Oxygen is removed by chemical reactions in contrast to evacuation and replacement technique used in McIntosh Filde’s jar. • Sachet containing sodium bicarbonate and sodium borohydride which react chemically in presence of water, to produce hydrogen and CO2 gas. • Traces of oxygen is removed by using same catalyst (aluminium pellets coated with palladium) placed below the jar. Indicator of anaerobiosis – • Chemical indicator- Reduced methylene blue remains colourless in anaerobic conditions, but turns blue on exposure to oxygen. • Biological indicator-Plate inoculated with Pseudomonas is incubated along with other inoculated plates for anaerobic culture. Absence of growth of Pseudomonas (which is an obligate aerobe) indicates that perfect anaerobiosis has been achieved. Gas-pak anaerobic system Anaerobic glove box • Also called anaerobic chamber. • Self-contained anaerobic system that allows microbiologists to process the specimen and perform most bacteriological techniques for isolation and identification of anaerobic bacteria without exposure to oxygen. PRAS (Pre-Reduced, Anaerobically Sterilized) PRAS media are prepared entirely under oxygen-free conditions from initial sterilization to packaging in sealed foil packages. By reducing agents • Oxygen in culture media can be reduced by various reducing agents such as glucose, thioglycollate, cooked meat pieces, cysteine and ascorbic acid. • Robertson cooked meat broth is the most widely used anaerobic culture medium which uses chopped meat particles (beef heart) as reducing agent. 45 Common Anaerobic Media Medium Components/Comments Primary Purpose Anaerobic blood agar (Ana BA) Prepared with columbia base or BHI base supplemented with 5% sheep blood, 0.5% yeast extract, hemin, L-cystine, and vitamin K1. Non selective medium for isolation of anaerobes and facultative anaerobes Bacteroides bile esculin agar (BBE) Trypticase soy agar base with ferric ammonium citrate and hemin; bile salts and gentamicin act as inhibitors Selective and differential for Bacteroides fragilis group; good for presumptive identification Laked kanamycin- vancomycin (LKV) Trypticase soy agar base with ferric ammonium citrate and hemin; bile salts and gentamicin act as inhibitors Selective and differential for Bacteroides fragilis group; good for presumptive identification Anaerobic phenylethyl alcohol agar (PEA) Nutrient agar base, 5% blood, phenylethyl alcohol Selective for inhibition of enteric gram-negative rods and swarming by some clostridia Egg yolk agar (EYA) Egg yolk base Nonselective for determination of lecithinase and lipase production by clostridia and fusobacteria Cycloserine cefoxitin fructose agar (CCFA) Egg yolk base with fructose, cycloserine (500 mg/L), and cefoxitin (16 mg/L); neutral red indicator Selective for Clostridium difficile Cooked meat (also called chopped meat) broth Solid meat particles initiate growth of bacteria; reducing substances lower oxidationreduction potential. Nonselective for cultivation of anaerobic organisms; with addition of glucose, can be used for gas-liquid chromatography Peptone–yeast extract– glucose broth (PYG) Peptone base, yeast extract, glucose, cysteine (reducing agent), resazurin (oxygen tension indicator), salts Nonselective for cultivation of anaerobic bacteria for gas-liquid chromatography Thioglycollate broth Pancreatic digest of casein, soy broth, and glucose to enrich growth of most bacteria. Nonselective for cultivation of anaerobes, facultative anaerobes, and aerobes Colonial Appearance of common anaerobic bacteria on anaerobic blood agar plate Clostridium botulinum Gray-white; circular to irregular; usually beta-hemolytic. Clostridium tetani Gray; matte surface, irregular to rhizoid margin, translucent, flat; narrow zone of beta-hemolysis; may swarm over agar surface. Clostridium perfringens Gray to grayish yellow; circular, glossy, dome shaped, entire, translucent; double zone of betahemolysis. Clostridium difficile Large, white, circular, matte to glossy, convex, opaque; nonhemolytic; fluoresces yellow-green. CCFA (cycloserine cefoxitin fructose agar)Selective media Yellow, ground-glass colony Actinomyces spp. Colonies of Actinomyces israelii white, opaque, and may resemble a “molar tooth”; Actinomyces odontolyticus turns red after several days. Bifidobacterium spp. Small, white, convex, shiny, with irregular edge. Lactobacillus spp. Resemble Lactobacillus spp. colonies on aerobic blood or chocolate agar, except colonies are usually larger when incubated anaerobically. Bacteroides fragilis White to gray, circular, entire, convex, translucent to semiopaque; nonhemolytic BBE (At 48 hr.) colonies are .1 mm, circular, entire, raised, and either (1) low convex, dark gray, friable, and surrounded by a dark gray zone (esculin hydrolysis) and sometimes a precipitate (bile) or (2) Glistening, convex, light to dark gray, and surrounded by a gray zone. Prevotella spp. • Ana BA - Dark center with gray to light brown edges; circular, entire, convex, smooth, shiny; nonhemolytic; fluoresces brick red. • LKV - Black pigment. Porphyromonas spp. • Ana BA - Dark brown to black; more mucoid than Prevotella spp.; except for Porphyromonas gingivalis, fluoresces brick red. Fusobacterium spp. Ana BA - Circular; entire or irregular edge, convex or slightly umbonate, smooth, translucent; nonhemolytic. BBE - >1 mm in diameter, flat and irregular. Veillonella spp. Ana BA - Small, almost transparent; grayish white; smooth, entire, opaque, butyrous; may show red fluorescence under UV light (360 nm). IDENTIFICATION OF ANAEROBES • Determination of genus level is done by gram staning, cellular morphology and gas chromatogyaphy. • For anaerobic gram-negative rode tolerance of bile salt and gentian violet, antibiotic resistance and fermentation of glucose are also done. • Species determination is based on fermentation of sugars & other biochemical characteristics. Identification of anaerobic gram-negative bacilli Test Bacteroids Prevotella Porphyromonas Fusobacterium Antibiotic resistance Neomycine Kanamycine Penicilline R R R S R S/R S/R R S S S S Tolerance of Bile salt Gentian violet + - - -(+) -(+) V + + + -(+) V Fermentation of glucose Algorithm for isolation and identification of anaerobic bacteria Direct Gram stain of clinical specimen Streak for isolation Anaerobic (BBE, LKV, PEA, BA, CHOC, THIO, EYA) Examine individual colonies for distinctive morphologies Gram stain and subculture to BA, CHOC 1. Gram-negative add kanamycin, vancomycin, or colistin disk in first quadrant, nitrate disk in second quadrant 2. Gram-positive cocci add nitrate and SPS 3. Gram-positive rod add nitrate • Incubate anaerobically 37°C for 24 to 48 Hours • Aerotolerance Test: Subculture organisms to a CHOC plate, incubate 37°C for 24 to 48 hours in 5% CO2 to detect slow growing aerobes such as Capnocytophaga, Eikenella, and Actinobacillus spp. Rapid Identification Methods • Nucleic acid–based testing - include polymerase chain reaction (PCR), multiplex PCR, and oligonucleotide microarrays. • More recently, matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDI-TOF MS) has been used to identify anaerobes from clinical specimens . Antibiotic Sensitivity Testing • The antibiotic susceptibility profile is determined by the micro tube broth dilution method. • Many species of anaerobes are resistant to penicillin, and some are resistant to clindamycin and other commonly used antibiotics. 58 Treatment Organism Group Therapeutic Options Bacteroides fragilis group, other Bacteroides spp., Porphyromonas spp., Prevotella spp., and Fusobacterium spp. Beta-lactam/beta-lactamase– inhibitor combinations, imipenem, metronidazole, and chloramphenicol, Cefoxitin (cephalosporin) Moxifloxacin (fluoroquinolone) Clostridium spp. Penicillins, with or without beta- lactamase– inhibitor combinations and imipenem; metronidazole or vancomycin for Clostridium difficile–induced gastrointestinal disease; antimicrobial therapy is not indicated for botulism and Clostridium perfringens food poisoning Actinomyces spp., Propionibacterium spp., Bifidobacterium spp., Eubacterium spp. Penicillins, with or without beta- lactamase– inhibitor combinations, imipenem, cefotaxime, and ceftizoxime Peptostreptococcus spp., and Peptococcus niger Penicillins, cephalosporins, imipenem, vancomycin, clindamycin, and chloramphenicol