Clinical Bacteriology - 09 Anaerobic Bacteriology

CLINICAL BACTERIOLOGY ANAEROBIC BACTERIOLOGY OUTLINE • • • Introduction The Anaerobes o The Overview and General Considerations o Anaerobic Organisms o Tests for Identification Anaerobes o General Characteristics o Anaerobic Media o Gram Stain o Gram Positive Bacilli o Gram Positive Cocci o Gram Negative Cocci o Gram Negative Bacilli o Fluorescence under Long-Wave UV light o Bilophilia wadsworthia INTRODUCTION • The following genera : Actinomyces , Bifidobacterium and Clostridium can grow in the presence of either reduced air or atmospheric oxygen but grow best under anaerobic conditions. Most anaerobic bacteria that cause infections in humans are also part of our normal flora. The ecology of these organisms is such that various species and genera exhibit preferences for the body sites that they inhabit. Other pathogenic anaerobes like Clostridium botulinum and Clostridium tetani are soil and environmental inhabitants and are not considered part of the normal flora. The majority of anaerobic infections occurs when a patient's normal flora gain access to a sterile site as a result o disruption of some anatomic barrier. The types of infections and diseases in humans that are caused by anaerobic bacteria span a wide spectrum. Certain species, such as C. botulinum and C. tetani produce some of the most potent toxins known. In contrast, specific virulence factors for the organism most commonly encountered infections i.e. B. fragilis group, C. difficile are not well understood. THE ANAEROBES THE OVERVIEW AND GENERAL CONSIDERATIONS Mode of Acquisition (e.g., malignancy, bite wound and other soft tissue infections, diabetes, burns, immunosuppressive therapy, aspiration, etc.) Examples and gynecologic infections, and gangrene (i.e., clostridial myonecrosis). Organisms most commonly encountered in these infections include Bacteroides fragilis group, Prevotella spp., Porphyromonas spp., Fusobacterium nucleatum, Peptostreptococcus, and Clostridium perfringens Contamination of existing wound Tetanus (Clostridium tetani), gas or puncture by objects gangrene (Clostridium contaminated with toxigenic perfringens, and, less Clostridium spp commonly, C. septicum, C. novyi, and others) Ingestion of preformed toxins in Botulism (Clostridium vegetable or meat-based foods botulinum), and other clostridial food poisonings (C. perfringens) Colonization of gastrointestinal Infant botulism (C. botulinum) tract with potent toxin-producing organism Person-to-person spread Nosocomial spread of Clostridium difficile–induced diarrhea and pseudomembranous colitis; bitewound infections caused by variety of anaerobic species CLINICAL SPECIMENS SUITABLE FOR ANAEROBIC CULTURE • • • • • • • • • GENERAL CHARACTERISTICS • • • Some aerotolerant organisms (5% O2), such as Actinomyces spp., Bifidobacterium spp., and Clostridium spp., Lack superoxide dismutase and catalase Most of the anaerobic bacteria that cause infections in humans are also part of our normal flora ACQUISITION OF ANAEROBIC INFECTIONS AND DISEASES Mode of Acquisition Endogenous strains of normal flora gain access to normally sterile sites, usually as result of one or more predisposing factors that compromise normal anatomic barriers (e.g., surgery or accidental trauma) or alter other host defense mechanisms Examples Wide variety of infections involving several anatomic locations, including bacteremia, head and neck infections, dental and orofacial infections, pneumonia and other infections of the thoracic cavity, intraabdominal and obstetric • Bile Biopsy of endometrial tissue obtained with an endometrial suction curette (Pipelle; Unimar, Wilton, Connecticut) Blood Bone marrow Bronchial washings obtained with a doublelumen plugged catheter Cerebrospinal fluid Culdocentesis aspirate 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 (the best specimens are from loculated or walled-off lesions) Percutaneous (direct) 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 Transtracheal aspirate Uterine contents (if collected using a protected swab) CLINICAL SPECIMENS UNSUITABLE FOR ANAEROBIC CULTURE • • • • • • • Bronchial washing or brush (unless collected with a doublelumen 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 Medium Peptone-yeast extract glucose broth (PYG) Thioglycollate broth SPECIMEN TRANSPORT AND PROCESSING • • • • Stools should be placed in an anaerobic transport container if culture is to be performed. Specimens for toxin assay may be collected in leak-proof containers and may be stored for up to 3 days at 4° C or frozen at –70° C. Specimens for anaerobic culture may be processed on the open bench-top with incubation in anaerobic jars or pouches or in an anaerobic chamber Anaerobic jars, chambers, and holding jars • with foul odor With sulfur granules (Actinomyces spp., Propionibacterium spp., or Eubacterium nodatum); brick red fluorescence under long wavelength ultraviolet (UV) light (Prevotella or Porphyromonas spp). • • Medium Anaerobic blood agar Bacteroides bile esculin agar (BBE) Laked kanamycinvancomycin blood agar (LKV) Components/Comments May be prepared with Columbia, Schaedler, CDC, Brucella or brain-heart infusion base supplemented with 5% sheep blood, 0.5% yeast extract, hemin, Lcystine, and vitamin K1 Trypticase soy agar base with ferric ammonium citrate and hemin; bile salts and gentamicin act as inhibitors • • COMMON ANAEROBIC MEDIA Primary Purpose Nonselective medium for isolation of anaerobes and facultative anaerobes Selective and differential for Bacteroides fragilis group; good for presumptive identification Selective for isolation of Prevotella and Bacteroides spp Brucella agar base with kanamycin (75 μg/ mL), vancomycin (7.5 μg/mL), vitamin K1, (10 μg/mL), and 5% laked blood Anaerobic Nutrient agar base, 5% blood, Selective for phenylethyl phenylethyl alcohol inhibition of enteric alcohol agar gram-negative rods (PEA) and swarming by some clostridia Egg-yolk agar Egg yolk base Nonselective for (EYA) determination of lecithinase and lipase production by clostridia and fusobacteria Cycloserine Egg yolk base with fructose, Selective for cefoxitin fructose cycloserine (500 mg/L), and Clostridium difficile agar (CCFA) cefoxitin (16 mg/L); neutral red indicator Cooked meat Solid meat particles initiate Nonselective for (also called growth of bacteria; reducing cultivation of chopped meat) substances lower oxidationanaerobic broth reduction potential (Eh) organisms; w/ addition of glucose, can be used for gasliquid chromatography Primary Purpose Nonselective for cultivation of anaerobic bacteria for gas-liquid chromatography Pancreatic digest of casein, Nonselective for soy broth, and glucose enrich cultivation of growth of most bacteria. anaerobes, as well Thioglycollate and agar as facultative reduce Eh. May be anaerobes and supplemented with hemin and aerobes vitamin K1 ANAEROBIC ORGANISMS MACROSCOPIC CHARACTERISTICS • • Components/Comments Peptone base, yeast extract, glucose, cysteine (reducing agent), resazurin (oxygen tension indicator), salts • • • • • Clostridium spp. o Discussed in the previous modules. o Gram positive anaerobic organism Actinomyces spp. o A. israelii, A. meyeri, A. naeslundii, A. odontolyticus o No well-characterrized virulence factors o A.viscosus and A. naeslundii- involved in periodontal disease and dental caries. Propionibacterium spp. o No definitive virulence factors known o Assoc. with inflammatory process in acne but only rarely Bifidobacterium spp. o Glistening colonies o Forked end in microscope o No definitive virulence factors known o Not commonly found in clinical specimens. Usually encountered in mixed infections with pelvis or abdomen Eubacterium spp. o No definitive virulence factors known o Usually associated with mixed infections of abdomen, pelvis, or genitourinary tract Mobiluncus spp. o Organisms are found in the vagina and have been associated with bacterial vaginosis Bacteroides fragilis o Sensitive to bile o Resistant to most antibiotics o Common anaerobe in GIT Veilonella spp. o G(-) cocci o Red fluorescence in UV light Fusobacterium spp. o G(-) Bacilli with bizarre formations are seen o Can cause Vincent disease or Acute necrotizing ulcerative gingivitis ( May cause by other anaerobic bacteria ( Treponema) – also known as Trench mouth o F. necrophorum – Lemierre disease – acute jugular vein septic thrombophlebitis associated with infectious mononucleosis o F. nucleatum – thin rod with tapered ends ( needle shape morphology) TESTS FOR IDENTIFICATION • Centers for Disease Control and Prevention (CDC) Anaerobic Blood Agar o For general growth of all anaerobes. • Bacteroides Bile Esculin (BBE) Agar o Selective and differential o Medium used to culture and presumptively identify Bacteroides fragilis • Kanamycin-Vancomycin Laked Sheep Blood (KVLB) Agar o Enriched selective medium for isolation of slowly growing anaerobes such a Prevotella and Bacteroides o Laked blood enhances pigment formation • Phenylethyl alcohol (PEA) Agar o Enriched and selective medium used to grow most anaerobes, including Clostridium and Bacteroides o Inhibits the growth of facultative, anaerobic, gramnegative bacilli (ex: Enterobacteriaceae) • Columbia-Colistin-Nalidixic Agar with 5% Sheep Blood o Inhibits gram negative organisms and is used to grow most gram-positive anaerobes and facultative anaerobes ANAEROBES GENERAL CHARACTERISTICS • • • • • Anaerobic bacteria comprise most normal flora of the mucous membranes Suspect anaerobic bacteria in the following situations: Foul odor (from gas production) and necrotic tissue Anaerobic body sites, abscesses, and wounds Surgical specimens ANAEROBIC MEDIA • • • Media contain supplements that enhance anaerobic growth. Vitamin K is added to enhance the growth of Prevotella and Porphyromonas Hemin is an added enrichment for Bacteroides and Prevotella • Egg yolk Agar o Used to detect proteolytic enzymes (lipase and lecithinase) produced by Clostridium o Lecithinase activity poduces an opaque zone from the cleavage of lecithin releasing insoluble fats (diglyceride) o Lipase cleaves lipids, releasing glycerol, which floats to the top of the medium producing a blue-green sheen (mother-of-pearl) on the agar surface • • • • Aerotolerance testing: Before attempting to identify a possible anaerobic bacterium, it first must be demonstrated to be an obligate anaerobe. A colony is inoculated to an anaerobic blood agar plate, which is incubated anaerobically, and to a chocolate agar plate incubated under conditions of increased CO2 Isolated growing only on the plate incubated anaerobically are obligate anaerobes. Broth with reducing agents o Thioglycolate and Cooked (or Chopped) Meat ▪ Can be used to grow anaerobic bacteria GRAM STAIN o • • • Resazurin ▪ an oxidation-reduction indicator, may be added. Indicator is pink in the presence of oxygen and colorless when reduced. Solid media must be placed in anaerobic conditions in order for obligate anaerobes to grow Commonly used systems include anaerobic GasPak jars and bags and anaerobic hoods. In the presence of palladium, a catalyst, the following reaction occurs: 2H2O + O2 -> 2H2O2 An oxidation-reduction indicator (EH) must be used to determine if anaerobic conditions have been met. Methylene blue is the most commonly used oxidationreduction indicator. When anaerobic conditions are achieved, the methylene blue indicator will turn from blue (oxidized) to white, indicating reduction. GRAM POSITIVE BACILLI ANAEROBIC Actinomyces • • • • • Normal flora of animal and human mucous membranes A. Israelii causes abdomen and chest infections and pelvic actinomycosis in women with intrauterine devices, is the most common pathogen; causative agent of LUMPY JAW Exudate contains sulfur granules dense clumps of bacteria Gram positive bacilli with a beaded appearance, often filamentous Colony morphology: Smooth to molar toothlike morphology Propionibacterium • • • • • Species include P.acnes and P.propionicus Often called anaerobic diphtheroids Propionibacterium acnes – most often found as skin contaminant in blood cultures, anaerobic normal skin flora Normal flora of the skin, mouth and GI tract Rarely pathogenic; Catalase and Indole positive GRAM POSITIVE COCCI Peptococcus • • • The only species is P.Niger Catalase positive Produces olive-green colonies that become black Peptostreptococcus • • P.anaerobius, inhibited by sodium polyanethol sulfonate (SPS) P.magnus was renamed Finegoldia magna and P.asaccharolyticus was renamed Peptoniphilus asaccharolytica Mobiluncus • • • • Associated with bacterial vaginosis, pelvic inflammatory disease and abdominal infections Curved bacilli Motile, catalase and indole negative Inhibited by vancomycin GRAM NEGATIVE COCCI Veilonella • • • • Small, gram negative cocci Reduces nitrates to nitrite, does not ferment any carbohydrates Inhibited by kanamycin and colistin but resistant to vancomycin Red fluorescence under UV light GRAM NEGATIVE BACILLI Bifidobacterium Bacteroides and Prevotella • • Mostly nonpathogenic normal oral and intestinal flora Eggerthella / Eubacterium • Mostly nonpathogenic normal oral and intestinal flora GS morphology: Pale, pleomorphic gram-negative coccobacilli with bipolar staining Bacteroides fragilis • • • • • • • Nonpigmented bacillus responsible for MOST Anaerobic infections Virulence Factor: polysaccharide capsule Major normal flora of the colon; Causes INTRAABDOMINAL ABSCESS (extremely virulent) Causes infections by gaining entry into normally sterile body sites, especially after surgery, trauma, or disease NONHEMOLYTIC on anaerobic blood agar Produces brown to black colonies on BBE Agar Growth in 20% bile, catalase positive, Lipase negative, Bile-esculin positive, Lecithinase negative, Gelatinase negative FLUORESCENCE UNDER LONG-WAVE ULTRAVIOLET LIGHT Prevotella melaninogenica • • • • Pigmented saccharolytic gram negative bacilli Normal floral of the oropharynx, nose, GI and urogenital tracts Causes head, neck and lower respiratory tract infections Young colonies appear tan and exhibit brick-red fluorescence under UV light. Older colonies are brown to black Porphyromonas • • • • • Asaccharolytic or weak fermenters, pigmented colonies, gram negative bacilli Normal floral of the oropharynx, nose, GI and urogenital tracts Causes infections of the head, neck, oral cavity and urogenital tract Brick-red fluorescence under UV light Will NOT grow on KVLB agar and are inhibited by bile, vancomycin, penicillin and rifamin. However, they are resistant to kanamycin • • • • • • • • Bilophilia wadsworthia • • • • • • • Asaccharolytic or weak fermenters, nonpigmented colonies, gram negative bacilli. Normal floral of the upper respiratory and GI tracts Causes pulmonary, blood, sinus and dental infections in addition to brain abscesses. Many infections are associated with metastatic conditions. Identifying characteristics: Colony morphology - Opalescent with speckles, Indole and lipase negative, Relatively Biochemically Inactive, Inhibited by kanamycin and colistin, Resistant to vancomycin Two important spp: F.nucleatum (causes serious pulmonary infections; agent of ASPIRATION PNEUMONIA) and F.necrophorum (lung and liver abscesses and arthritis). F.nucleatum is the MORE COMMON isolate but F.necrophorum causes MORE SERIOUS infections. Bile resistant anaerobe that will grow on BBE Agar With a characteristic “fish-eye appearance” May also grow on KVLB Agar Clostridium • • • • • • • Fusobacterium • Prevotella (pigmented) - Brick red P.bivia, P.disiens - Light orange to pink (coral) Porphyromonas - Brick red (some no fluorescence) Fusobacterium - Chartreuse Veilonella - Red but fades rapidly Clostridium ramosum - Red C.innocum, C.difficile - Chartreuse Eggerthella lenta - Red or no fluorescence The species of this genus are obligate anaerobes, catalase-negative, Gram-positive, spore-forming bacilli Frequently encountered in exogenous anaerobic infections or intoxications The toxins produced by the species are acquired through ingestion or open wounds that have been contaminated with soil Virulence contributors: collagenase, hyaluronidase, lecithinase (cell destruction), and phospholipase Species: C. perfringens, C. novyi, C. septicum, C. bistolyticum, C. bifermenrans, C. sordellii, C. innocuum, C. botulinum, and. C. tetani Histotoxic Clostridium specius that cause myonecrosis: C. perfringens, C. septicum, C. bistolyticum, C. biferementans, C. novyi Distinguishing characteristics of the Clostridium species: o They form endospores anaerobically o They are motile with peritrichous flagella except for C. perfringens, C. ramosum, c. innocuum o They have swollen sporangia except for C. perfringens, C. bifermentans o They are non-encapsulated expect for C. perfringens o They have a single hemolytic reaction except for C. perfringens o They have carbohydrate fermenters except for C. tetani, C. histolyticum Clostridium perfringens • • • • • Gas gangrene bacillus Most commonly isolated member of Clostridium in blood cultures Virulence factor: α-toxin and enterotoxin Microscopy: “Boxcar-shaped” bacilli with oral, subterminal spores Culture: o BAP - colonies are dome-shaped and grayish-white with double zones of hemolysis (alpha and beta zones) o • • Litmus milk - colonies exhibit a stormy fermentation of milk Biochemical test: very fermentative o (+) Lecithinase - detected using egg yolk agar (EYA) o (+) Nagler test - lecithovitellin reaction on EYA o (+) Reverse CAMP test - formation of an “arrowheadshaped” zone of hemolysis towards the test organism Related Infection and Disease o Gas gangrene (Myonecrosis) ▪ A life-threatening destruction of muscle and other tissues ▪ Accompanied by bullae (fluid-filled blisters), pain, swelling, serous discharge, discoloration, and tissue necroses o Clostridial necrotizing enteritis or enteritis necroticans ▪ Caused by the ingestion of β-enterotoxin in contaminated food ▪ Improperly stored food allows the germination of the spores and growth of vegetative bacteria ▪ Symptoms: blood diarrhea and abdominal pain • • • • • Clostridium tetani • • • • • • • • Tack head bacillus A soil and environmental inhabitant The endospores are found in the soil, dust, and feces or many farm animals Virulence factor: Tetanospasmin (neurotoxin) Microscopy: cells are with terminal spores and swollen sporangia that have a “drumstick” or “tennis-racket” appearance Culture: BAP - colonies exhibit a slow, anaerobic, heavy, smooth, and swarming growth and have a matte surface with a narrow zone of β-hemolysis Tetanospasmin o An endopeptidase that selectively cleaves the synaptic vesicle membrane protein, synaptobrevin o Causes tension or cramping and twisting in skeletal muscles that surround the wound, and tightness od the jaw muscles Tetanus o Characterized by trismus or lockjaw and risus sardonicus or distorted grin o Occurs when the organism (spore) enters an open wound and spreads a potent toxin that mediates generalized muscle spasms o Symptoms: muscular rigidity (jaws, neck, lumbar region), difficulty in swallowing, rigidity of the abdomen, chest, back, and limbs o Incubation period: 3-21 days; the long incubation period is related to the distance from the injury to the central nervous system (CNS) o Tetanus neonatorum is contracted through the use of contaminated instruments on newborns Clostridium botulinum • • • • “Canned food” bacillus Found in soil and aquatic sediments A potential bioterrorism agent Characterized by the presence of subterminal spores Virulence factor: Botulism toxin - a neurotoxin that is considered as one of the most potent natural toxins known to man Culture: BAP - β-hemolytic colonies Related infection: Botulism Botulism toxin o Selectively cleaves the synaptic cesicle membrane protein, synaptobrevin, thus preventing exocytosis and the release of the neurotransmitter, acetylcholine o Requires a small amount to produce paralysis and death o Botulism antigens: A to G (7 antigenic types) o The antigens that cause human diseases are A, B, E Botulism o Characterized by double or blurred vision, impaired speech, difficulty in swallowing, weakness, and paralysis o There are 2 types of botulism ▪ Foodborne botulism ▪ Infant botulism o Foodborne botulism ▪ Results from the ingestion of preformed toxin in preserved of meat-based food ▪ Commonly caused by botulism toxin A o Infant botulism ▪ An actual infection caused by ingesting the organism from honey or through breastfeeding for infants Clostridium difficile • • • • • • • The most common cause of antibiotic-associated diarrhea and pseudomembranous colitis (bloody diarrhea with necrosis of colonic mucosa) Acquired in the hospitals by individuals who are receiving antibiotics An “infection control dilemma” among hospitalized patients Ferments fructose-producing formic acid that changes the color of medium from pink to yellow Virulence factor: Toxin A (enterotoxin) and toxin B (cytotoxin) Microscopy: chains up to 6 cells that are aligned from end to end with oval, subterminal endospores Culture o Cycloserine-cefoxitin-fructose agar (CCFA) colonies exhibit a yellow color and a “ground-glass” appearance o BAP - colonies exhibit a “horse stable” odor; are nonhemolytic; and produce a fluorescent chartreuse under a long UV light wave