Lec 11 Anaerobic Bacteria.pdf

MODULE 11. ANAEROBIC BACTERIA SPORE-FORMERS 1. Clostridium perfringens...

MODULE 11. ANAEROBIC BACTERIA SPORE-FORMERS 1. Clostridium perfringens 2. Clostridium botulinum 3. Clostridium tetani 4. Clostridium difficile 5. Clostridium septicum 6. Clostridium sordellii 7. Other Clostridium spp These are organisms that require a reduced oxygen tension for growth and fail to grow on solid media in the presence of oxygen. ❑ Polymicrobic ❑ Involving more than one bacterial species ❑ 2 Types of Anaerobic Infections: a. Exogenous- are soil and environmental inhabitants (pathogenic anaerobes/not microbiota) Food-borne botulism, tetanus, infant botulism, etc. b. Endogenous -exhibit preferences for the body sites they inhabit (microbiota) Arise within the mouth, intestinal tract or female genital tract ANAEROBIC GRAM-POSITIVE SPORE-FORMING BACILLI CLOSTRIDIA Gram-Positive Bacilli Endospore-Forming, Obligately Anaerobic (Or Aerotolerant), Catalase-negative, spores are not present on Gram stain, the ethanol shock spore or heat shock spore test can separate this group from the non– spore-forming anaerobic bacilli Motile and have peritrichous flagella Except C. perfringens, C. ramosum and C. innocuum Non-encapsulated Except C. perfringens Box-car appearance Have a single hemolytic reaction Except C. perfringens Have swollen sporangia Except for C. perfringens and C. bifermentans Carbohydrate fermenters Except C. tetani and C. histolyticum Clostridium Groups Group I - Gas Gangrene Group Group II -Clostridium tetani Group III - Clostridium botulinum Group IV - Clostridium difficile Group V - Miscellaneous clostridium Clostridium perfringens Formerly known as C. welchii Also known as bacillus aerogenes capsulatus / gas gangrene Sporulation (bacterial endospore bacillus / Frankel’s bacillus formation) Most commonly isolated member of Clostridium in blood cultures o Non-motile o Obligatory anaerobe o Only encapsulated clostridia o Spores are absent in solid media & thio o Endospore: subterminal (spores, structures that are derived from the vegetative cell under conditions of nutrient deprivation and that allows survival under harsh environmental conditions) Causes: o Myonecrosis (gas gangrene) - gas in tissues in gangrene o Pork poisoning (pig bell) o Enteritis Necroticans ▪ Type C food poisoning that causes necrosis of the jejenum Virulence Factors: 1. Alpha toxin (Lecithinase, phospholipase C) – Most important toxin – A hemolysin, anti-WBC and platelet 2. Beta toxin (Dambrand pigbel) – Produced by type C (key factor in necrotizing colitis) 3. Lambda toxin – A weak proteolytic toxin 4. Theta toxin – Responsible for beta hemolysis on anaerobic BAP 5. Kappa toxin – A collagenase (involve in the breakdown of gelatin) 6. Mu toxin – Hyaluronidase 7. Nu toxin – An RNAse 8. Epsilon and iota toxin – Leads to increased capillary permeability 9. Enterotoxin Similar to Vibrio cholerae enterotoxin 10. Neuramidase CULTURE Double zone of ß hemolysis on BAP Inner: Complete hemolysis (theta toxin) Outer: Complete hemolysis (α toxin/ lecithinase) Milk media: stormy fermentation Gas production Stormy Fermentation of Litmus Milk. The tube on the left shows fermentation; the Clostridium perfringens on anaerobic tube on the right is negative blood agar. for stormy fermentation. Used Note double zone of beta-hemolysis for the identification of Clostridium species. Biochemical Characteristics 1. Does not ferment xylose 2. (+) Lecithinase test Opaque halo around colonies on egg yolk agar 3. (+) Nagler reaction Ppt’n around colonies on the side without antitoxin & no ppt’n on side with antitoxin Lecithinase degrades the lecithin present in the egg yolks and releases insoluble diglycerides, resulting in 4. (+) Reverse CAMP the formation of a white opaque zone of precipitation Arrowhead zone of hemolysis with S. (zone of opalescence ) in the medium surrounding agalactiae (?) growth extending away from the bacterial growth, Clostridium perfringens, is a "bow tie" or reversed arrow zone of enhanced hemolysis at the junction of the two cultures A, Reverse CAMPpositive Clostridium perfringens and (B) reverse CAMPnegative Clostridium septicum streaked at right angles to (C) assessing bacteria’s ability to produce a Streptococcus agalactiae toxic, phospholipase enzyme called (group B) ‘lecithinase’. Clostridium tetani aka Tack Head bacillus – Spores: terminal & swollen – drumstick/lollipop/tennis racket appearance – Swarming colonies on BAP Exhibits Wasserman-Takaki phenomenon Does not ferment any carbohydrates (non-fermenter) Tetanus Intoxication caused by C. tetani Tetanus typically occurs in nonimmunized persons within the first 2 weeks following a traumatically acquired puncture, laceration, or abrasion Clinical manifestations: a. Trismus (lock jaw) - sustained tetanic spasms of the muscles of mastication. b. Risus sardonicus / Sardonic smile- abnormal, sustained spasm of the facial muscles that appears to produce grinning c. Opisthotonus – abnormal posture due to spastic contraction of the extensor muscles of the neck, trunk, and lower extremities Tetanospasmin (TeNT)- A neurotoxin causing spasmodic contraction (spastic paralysis) - transported to gangliosides in the CNS via the lymphatics and bloodstream, and by migration through the perineural spaces of peripheral nerves. Clostridium botulinum aka Canned good bacillus / Von Ermengen’s Bacillus / Bacillus botulinus Heat resistant spores that are oval and subterminal β hemolytic Potential bioterrorism agent! Causes: Wound botulism (least common) Food botulism (preformed toxin) Infant botulism / Floppy baby syndrome Ingestion of spores & produce toxin in GIT In babies fed with honey Botulinum / Botulism toxin (Botox) One of the most potent exotoxins o A neurotoxin causing flaccid paralysis - prevents the release of the neurotransmitter acetylcholine from axon endings at the neuromuscular junction, thus causing flaccid paralysis o Uses: Botox injections work by weakening or paralyzing certain muscles or by blocking certain nerves. 1. Temporary smoothing of facial wrinkles and improving your appearance 2. Severe underarm sweating  3. Cervical dystonia - a neurological disorder that causes severe neck and shoulder muscle contractions 4. Blepharospasm - uncontrollable blinking 5. Strabismus - misaligned eyes 6. Overactive bladder Clostridium difficile Characteristics: 1. Spores: oval & subterminal 2. On BAP: fluoresce yellow green colonies with horse stable odor 3. On CCFA (Cycloserine-cefoxitin egg yolk fructose agar) – yellow ground glass colonies Major cause of nosocomial diarrhea Toxins: Toxin A and Toxin B Most common cause of antibiotic-associated diarrhea and Pseudomembranous colitis Overgrowth of C. difficile in GIT as a side effect of antibiotics (Clindamycin, Ampicillin and Cephalosporin) Clostridium difficile should be considered a cause of diarrhea in patients on antibiotic therapy or hospitalized for more than 3 days. Specimen: Stool for the detection of toxin and history of antibiotic medication Miscellaneous Clostridia 1. Clostridium ramosum – The normal flora of the large bowel – Important agent in intra- abdominal infections following trauma 2. Clostridium septicum – Cause of bacteremia associated with malignancies Colon and breast cancer Leukemia – Produce oval and subterminal spores  Anaerobic Gram-negative Non-spore-forming Bacilli Non-SPORE-FORMERS Actinomyces israelii Bifidobacterium spp. Lactobacillus spp. Mobiluncus spp. Propionibacterium spp. Actinomyces naeslundii Actinomyces odontolyticus Other Actinomyces spp. Atopobium minutum Atopobium parvulum Collinsella aerofaciens Eggerthella lenta Eubacterium spp. Actinomyces General characteristics Branching & beaded Has sulfur granules Facultative anaerobe Actinomyces colonies form branched Found as normal flora on the mucosal surfaces of the human networks of hyphae, so that their digestive tract and urogenital tract and on the skin macro-morphology shows similarity to fungi, but they are bacteria. Individual similar appearance with Nocardia, but Nocardia are acid fast cells are rod-shaped. Actinomyces israeli Most common cause of Actinomycosis (Lumpy jaws) Part of the indigenous microbiota of the oral cavity Culture: o Brain Heart Infusion Agar ▪ Molar tooth colonies ▪ Bread-crumb like, raspberry or smooth colonies o Thioglycollate: ▪ Fluff ball colonies Young cultures: “Spider-like” and “wooly” colonies Old cultures: “Raspberry” or “Molar tooth” colonies Actinomyces spp. 1. Actinomyces naeslundii Fluff balls with sulfur granules 2. Actinomyces viscosus Pink flocculent colonies in Thioglycollate 3. Actinomyces odontilyticus Red colored colonies on BAP Bifidobacterium Diphtheroidal and long branching rods Bifurcated or forked ends o Resemble the shape of a “dog bone” Normal flora of GI and GU tract Distinguishing characteristics o Small, white, shiny and convex colonies Bifidobacterium dentium o Formerly B. eriksonii Acidophilic Probiotics: Food and Agriculture Organization of the United Nations (FAO) defines probiotics as "live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host." Used in the food industry to relieve and treat many intestinal disorders. Some supplements contains probiotic bifidus BL which supports healthy immune and digestive systems. Two principal kinds of probiotic bacteria are members of the genera Lactobacillus and Bifidobacterium Eubacterium Normal flora of GIT and mouth Pleomorphic rods to coccobacilli forms Distinguishing characteristics o Fluorescent chartreuse color o Produce ↑ butyric acid Species o E. alactolyticum sea-gull-wing shape forms Now Pseudoramibacter alactolyticus o E. nodatum filamantous & branching o E. lentum The good side! most common Eubacteria 1. Probiotics help treat now Eggerthella lenta diarrhea in children 2. Prevent vaginal and urinary tract infections LACTOBACILLUS 3. Treat symptoms of Lactobacillus acidophilus Irritable Bowel syndrome (IBS) aka Doderlain bacilli 4. Prevent colds and flu Most abundant normal flora of vagina in children o Non-pathogenic: has little clinical significance 5. Reduce occurrence Produces lactic acid of bladder cancer Grows on tomato juice agar Lactobacillus gasseri appears to be the main species of lactobacilli that inhabits the human gastrointestinal tract. Lowers pH, suppresses overgrowth of mobiluncus, prevotella and G. vaginalis Lactobacillus casei strain Shirota Yakult contains Mobiluncus spp. Strictly anaerobic Causative agent of bacterial vaginosis Gram staining of vaginal secretions: o Gram-variable, curved rods with tapered ends Rarely isolated in the clinical laboratory because vaginal secretions are not acceptable specimens for anaerobic culture. Propionibacterium “spidery” Produce large amounts of propionic acid (major) & acetic acid Normal flora of skin, GIT, URT (nares) Frequent contaminants of blood & other body fluids culture Microscopy: o Diptheroid-like o Palisades, picket-fence, Chinese letters arrangement Propionibacterium acnes o Infections related to heart valves & prosthetic devices o Plays a role in acne (acne vulgaris) Short rods; may appear “spidery” ANAEROBIC GRAM-NEGATIVE BACILLI GRAM-NEGATIVE BACILLI Bacteroides fragilis group Fusobacterium spp. Porphyromonas spp. Prevotella spp. Other Bacteroides spp. Bacteroides ureolyticus Leptotrichia spp. Bilophila wadsworthia Sutterella wadsworthensis Bacteroides Obligate anaerobes Non-spore-forming o Rods, helical, straight or curved Either motile or non-motile Metabolize carbohydrates, peptones or metabolic intermediates o Produce organic acids Indigenous flora of mucosal surfaces Bacteroides fragilis Most common bacteroides isolate that causes infection A normal fecal flora o Most abundant organism in the intestinal tract Bacteroides can be found as normal flora throughout much of the gastrointestinal and genitourinary tracts. Infections caused by the complex include abdominal abscess, pelvic abscess, bacteremia, and brain abscess. Characteristics o Growth stimulated by 20% bile o Pleomorphic o Encapsulated o Non-motile Bacteroides ureolyticus A saccharolytic, Reduces nitrate Requires formate and fumarate for growth in broth culture. Disk pattern o Same with Fusobacteria but different colony morphology o small, translucent to transparent colonies Fusobacteriaceae The Fusobacteriaceae family includes the genera:  o Fusobacterium o Leptotrichia o Sneathia Typically isolated from the oral cavity as integral components of dental biofilms Fusobacterium nucleatum Most common Fusobacterium isolate that causes infection plays a role in the pathogenesis of periodontal disease o Fusobacterium necrophorum – 2nd most common isolate Normal flora of the mouth Characteristics: o Scattered wheat straw on GS o Colonies described as “bread crumb-like” o Alpha-hemolytic o Susceptible with kanamycin Porphyromonas and Prevotella Pigment producing gram negative bacilli Indigenous in the gingival crevice o most can cause dental infection & can occur in infections involving the head, neck & URT o Some normally found in the lower genital tract Characteristics: o Colonies on BA are β-hemolytic and pigmented tan to black o Vitamin K and hemin are required for growth o All are sensitive to bile Porphyromonas gingivalis – Anaerobic pathogenic oral bacterium – Major etiological agent of severe forms of periodontal disease. Prevotella melanogenica – Formerly Bacteroides melaninogenicus – fluoresces brick red under UV light @ 366nm Periodontal Disease The word "periodontal" literally means "around the tooth“ aka “Gum disease“ or “Pyorrhea" o An ongoing bacterial infection in the gums and bone around your teeth o Inflammation can destroy the bone around your teeth. This results in tooth loss ▪ 75% of all adult tooth loss is due to periodontal infection. Leptotrichia spp. Very large, fusiform rods with one pointed end and one blunt end Colony Morphology: o large, gray, convoluted o “Raspberry-like” colonies They are most often isolated from the oral cavity or urogenital tract Protobacteria Bilophila wadsworthia o an anaerobic, o asaccharolytic o bile-resistant o gram-negative rod.  o Strongly Catalase -positive Sutterella wadsworthensis o asaccharolytic, o bile-resistant, o Short o gram-negative rod. GRAM-NEGATIVE COCCI Anaerobic Gram Positive Cocci Peptococcus Peptostreptococcus Veillonella spp. GRAM-POSITIVE COCCI Acidaminococcus Megasphaera Veillonella spp. Peptococcus Commonly known as “Anaerobic Staphylococcus” Part of the flora of the mouth, upper respiratory tract, and large intestine Causes soft tissue infection and bacteremias P. magnus is the species most often recovered from clinical specimens Peptococcus niger Gram positive cocci Paired singly, pairs or in tetrads Colony morphology o Small black and shiny colonies Peptostreptococcus Commonly referred to as “Anaerobic Streptococci” Commensal organisms in humans, living predominantly in the mouth, skin, gastrointestinal, vagina and urinary tracts Under immunosuppressed conditions these organisms can become pathogenic/septicemic Can cause brain, liver, breast, and lung abscesses Peptostreptococcus anaerobius Appears as large coccobacilli in chains Unique in its resistance to Sodium polyanethol sulfonate (SPS) Resistant to kanamycin and colistin Anaerobic Gram Negative Cocci Acidaminococcus & Megasphaera Large cocci in groups or in packets Stains gram-positive but has gram-negative cell wall (?) Similar to Veillonella but is nitrate (-) Veilonella Gram-negative cocci arranged in pairs, short chains or irregular clumps Rare cause of human infection. Highly sensitive to oxygen – Glove box is used Veillonella parvula – Anaerobic gram-negative diplococci in masses – Produce red fluorescence under UV light – Does not require blood for growth Note: BA, Sheep blood agar; BBE, Bacteroides bile esculin agar; CHOC, chocolate agar; EYA, egg yolk agar (for suspected Clostridium spp.); LKV, laked kanamycin-vancomycin agar; PEA, phenylethyl agar; SPS, sodium polyanethol sulfonate; THIO, thioglycollate enrichment broth (should be examined daily and incubated for up to 7 days if no growth is identified on primary media; subculture to anaerobic media if growth is detected in broth culture).