Microbio Lec 8 - Bacteroides Actinomyces Propionibacterium

MICROBIOLOGY LECTURE 8 – Anaerobic Bacteria Notes from Lecture USTMED ’07 Sec C - AsM •

Introduction to Anaerobes Generalities • gram (+) or gram (-) • spore forming or non-spore forming • cocci, bacilli, comma-shaped, spiral

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intolerance to O2, grow best at low or negative Eh absence of: cytochrome system, superoxide dismutase, catalase normal flora: skin, mucosa, mouth, GIT

Virulence Factors • lipopolysaccharide capsule

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Bacteroides

enzymes: collagenase, heparinase, lecithinase metabolic end products: fatty acids in some strains: endotoxin infection results: when they contaminate sterile sites

Clinical Infections • usually insidious & may become chronic • frequently produces putrid odor in infected material • gas may be present in tissue or in loculations • most infections are due to moderately obligate anaerobes • Eg. Peritonitis, pneumonitis, UTI, URTI, soft tissue infections (cellulites, necrotizing fascitis), septicemia

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An anaerobic atmosphere can also be created in an anaerobic pouch that hold only two plates or in an anaerobic jar for three or more plates.

Epidemiology  Major component of human microbial flora  B. fragilis: most commonly associated with pleuro-pulmonary, intraabdominal & genital infections  Prominent role in human disease: attributed to enhanced virulence

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Clinical Syndromes  Cardinal features of Bacteroides infections • Endogenic • Polymicrobic mixture • Abscess formation

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Laboratory Diagnosis  Microscopy: faintly staining, pleomorphic, gram (-) bacilli

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Treatment • surgical debridement & resection of necrotic tissue • antibiotics: metronidazole, imipenem, clindamycin, cefoxitin, piperacillin

Bacteroides Prevotella Porphyromonas Fusobacteria

Spot Indole disk test : a plain filter paper is placed on an area of growth on a medium containing tryptophan. After 5 min. a drop of paradimethylaminocinnamaldehyde is placed on the disk. A greenish color indicates the presence of indole(Fusobacterium,Propionibacterium, Porphyromonas, Prevotella, and Peptostreptococcus spp.

Culture: collection & transport= O2-free system Biochemical ID: activity of enzymes, metabolic by-products Others: resistance to: kana, vanco, colistin Growth in 20% bile

Tx, Prevention, Control  Main approach: antibiotics with surgical intervention  Antibiotics: • B-lactam group: cefoxitin, imipenem; • beta lactamase inhibitors • metronidazole  Prophylaxis: diagnostic & surgical procedures  Endogenous spread: virtually impossible to control

Classification 1. Gram negative bacilli • • • •

O2 tolerance: capable of surviving prolonged exposure to O2

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Gas pack anaerobic jar system contains hydrogen and CO2 generator envelope, a disposable methylene blue indicator and a catalyst basket in the lid

Gas pack anaerobic pouch: bag is oxygen impermeable and contains its own gasgenerating kit and cold catalyst

Glycolipid: has little or no endotoxin activity – low or loss of pyrogenic activity Other species: B. ovatus, B. vulgatus

Virulence Factors  Capsule: antiphagocytic & promotes abscess formation  Lipopolysaccharide: can stimulate leukocyte migration & chemotaxis  Agglutinins: role not known  Enzymes: hyaluronidase, collagenase, neuraminidase, etc

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Laboratory Diagnosis • specimen collection: aspirated or tissue specimen preferred than swab • sensitivity testing: should be done in life threatening infections Prereduced anaerobic plates can be held in an anaerobic holding jar for a short time before and after inoculation

physiology & structure  B. fragilis: most important member  Pleomorphic in size and shape  Most members: grow rapidly in culture

Bacteroides fragilis on Brucella blood agar: non-selective medium supplemented by Vit. K1 and hemin

Bacteroides fragilis on Bacteroides bile esculin agar(BBE): Bacteroides fragilis group is resistant to bile salts; gray to black colonies due to hydrolysis of esculin in the medium Growth of Bacteroides fragilis on Bacteroides bile esculin agar

Bacteroides fragilis in an abscess

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History of an invasive dental procedure or oral trauma

Thoracic • Generally have a history of aspiration with establishment of disease in the lungs and then spread to adjoining tissue  Abdominal • Most commonly preceded by surgery or trauma to the bowel  Pelvic • Can be a secondary manifestation of abdominal actinomycosis or • Could be a primary infection in women with IUD  CNS • Usually represent secondary spread form another focus Clinical syndromes  Majority: cervicofacial  Maybe acute pyogenic or slowly evolving painless process  Cervicofacial: swelling with fibrosis & scarring and open draining sinus tracts along the angle of jaw and neck  Thoracic: non-specific  CNS: most common manifestation: solitary abscess Laboratory diagnosis  Microscopy: sulphur-granules (thin), gram (-) bacilli along the periphery of the granules  Culture: anaerobic conditions  Colonies: white with domed surface  Biochemical media Tx, Prevention, Control  Surgical debridement  Antibiotics: penicillin (DOC), tetra, erythro, clinda  Good oral hygiene  Prophylactic AB: GI surgery 

Nitrate disk test: nitrate infiltrated filter paper is placed onto the inoculum and after 48 hrs incubation, drops of nitrate reagents, sulfanilic acid and alpha-napthylamine are added to the disk. A red color indicates the presence of nitrite

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Prevotella o Nonspore forming o May appear as slender rods or coccobacilli o Eg. P. melanogenica, P.bivia, P. disiens

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Porphyromonas o Non-spore forming o Normal flora of mouth

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Fusobacteria o Appear as long, thin filaments with pointed ends o Colonies are hemolytic on blood agar

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Gram positive bacilli • • • • • • • •

Actinomyces Lactobacillus Propionibacterium Eubacterium Bifidobacterium Rothia Mobiluncus (CLOSTRIDIUM)

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Clostridia on Eggyolk agar: lecithinase-opaque white precipitate extending from the colony into the medium(A); Lipase-iridescent sheen on the surface of colony(B) Clostridium perfringens on Brucella blood agar: double zone beta hemolysis; large colonies with peaked centers and irregular edges after 48 hrs incubation

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Sulfur granule collected from sinus tract in patient with actinomycosis

Gram stain of Clostridium spp.: gram variable, long, thin, parallel sided, some with swollen ends indicative of spore formation

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Actinomyces o Physiology & Structure  Pleomorphic  Facultative or strict anaerobe  Grows slowly in culture  Typically form delicate filamentous hyphae  Lack mitochondria & nuclear membrane  Inhibited by penicillin o Pathogenesis  Definition: characterized by multiple abscesses connected by sinus tracts o Epidemiology  No evidence of person to person spread  No disease originating form external source  All age group affected  No seasonal nor occupational predilection  A. israelii – most commonly encountered o Disease classification  Cervicofacial • Seen in patients with poor oral hygiene

Pelvic actinomycosis

Molar tooth appearance of Actinomyces israelii after incubation for 1 week

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Propionibacterium: Generalities o Small, gm (-) bacilli, frequently arranged in short chain or clumps o Commonly found: skin, conjuctiva ext. ear, oropharynx, female genital tract o Physiology: anaerobic or aerotolerant, nonmotile, catalase (+) ferment carbohydrates o 2 commonly isolated species  P. acnes Disease caused by P. acnes o Acne: teenagers & young adults o Opportunistic infections: patients with prosthetic devices & IV line Pathophysiology

Stimulate an inflammatory response o Production of low MW peptides: attract WBC to sebaceous follicles Treatment o Antibiotic: topical & oral o

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P. propionicus Pathophysiology o Causes abscesses, lacrimal canaliculitis, actinomycosis

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Mobiluncus o Gram variable or gram (-) curved, nonsporing bacilli o But classified as gram (+): • Have gram (+) cell wall • Lack endotoxin • Susceptible to: vanco, clinda, erythro, ampi o Physiology  Obligate anaerobes  Fastidious, slow growing even on enriched media  2 species of medical importance: • M. curtsii • M. mulieris  Abundant in women with bacterial vaginosis

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Other gram (+), anaerobic bacilli o Bifidobacterium & Eubacterium  Commonly found in the large intestine o Lactobacillus  Normal flora of urethra & female genital tract, maintains acidic pH, rarely cause disease o Rothia: oropharynx

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Gram positive cocci • Peptostreptococcus spp. • Peptococcus spp. • Etc

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Gram negative cocci • Veillonella, Acidaminacoccus, Megashaera • Isolated from human infections • Isolates from oropharynx & colon: low degree of virulence & represent fewer than 1% of all anaerobic isolates • Isolates: generally present in mixtures & clinical signigicance difficult to assess • Specific treatment: often not necessary

Diseases associated with Peptostreptococcus, Actinomyces, Propionibacterium, Mobiluncus

Sodium polyanethol sulfonate(SPS) disk test: Peptococcus anaerobius is the only gram positive coccus that is inhibited by SPS

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more than 25% of all anaerobic isolates in clinical specimens:

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Peptostreptococcus o Gram (+) cocci o Normal flora: oral cavity, GIT, GUT skin o Infections: pleuropulmonary infections, sinusitis, brain abscesses, intraabdominal sepsis, pelvic infections (endometritis, pelvic abscess, salphingitis), soft tissue infections, endocarditis, osteomyelitis o Most infections are polymicrobial mixtures of anaerobic & aerobic bacteria o Only about 1% of all anaerobic bacteremias are due to gram (+) cocci, with majority caused by Peptostreptococci from genital tract of women o Bone and joint infections are usually associated with surgical procedure o Laboratory Diagnosis  Complicated by 3 factors • Contaminants • Transport of media should be O2-free • Specimen should be cultured on enriched media for 5-7 days o Treatment  Usually susceptible to • Penicillin, • cephalosporins, • imipenem, • chloramphenicol

Intermediate susceptibility to: • Clinda • Erythro • Tetra • Metro Specific therapy: indicated for monomicrobic infections

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