Things to be covered• Introduction• Types of anaerobes• Anaerobic organism & their classification• Human infections by anaerobes• Methods of diagnosis• Anaerobic Culture techniques• Pitfalls in anaerobic bacteriology• Summary• References
Introduction• Anaerobic bacteriology has always been a time consuming & expensive process. Because culture & identification of anaerobes is typically slow.• Major problems is that most infections involving anaerobes are mixed.• Aside from time factor, how much data are useful to the clinician? Is the clinician interested in accurate speciation or will general identification, with susceptibility data.• Rapid diagnostic procedures may be employed for presumptive or definitive identification.
Types of anaerobes• Obligate anaerobic bacteria- Are those bacteria that can grow in the absence of free oxygen, but fails to multiply in the presence of oxygen on the surface of nutritionally adequate solid media incubated in room air or in a CO2 incubator (containing 5-10% CO2). eg.- Bacteroides fragilis, Clostridium perfringens, C. novyi, Porphyromonas, Fusobacterium.• Aerotolerant anaerobes- Anaerobic bacteria that will show limited or scanty growth on agar in room air or in a 5-10% CO2 incubator, but show good growth under anaerobic conditions. eg.- C. carnis, C. histolyticum, C. tertium etc.• Microaerophilic bacteria- Organism require O2 as a terminal electron acceptor, yet these do not grow on the surface of solid media in an aerobic incubator (21% O2) & grow minimally if at all under anaerobic condition eg.- Campylobacter. jejuni (grow in 5% O2).
it is essential to isolate and identify anaerobic bacteria because1) These are associated with high morbidity & mortality.2) Treatment varies with bacterial species involved.• Currently >3/4th of anaerobes isolated from different clinical specimens are Bacteroides fragilis group, Prevotella, Porphyromonas, Fusobacterium, anaerobic cocci, and the anaerobic gram-positive, non-spore forming rods.• Most of them are resistant to penicillin and its analogues; they are resistant to many cephalosporins including third gen., tetracyclines, aminoglycosides also emergence of resistance to newer quinolones, and clindamycins.
• Anaerobes causes infections involving virtually every organ & anatomic region of the body.• Most deep seated abscesses and necrotizing lesions are polymicrobial, and may include obligate aerobes, facultative anaerobes, or microaerophiles• Within past few decades, however, endogenous anaerobic infections have become far more common. As; Laboratory recovery of anaerobes is improved Compromised host immune response due to immunosuppressive drugs.
Anaerobic infection• Abscess of any organ• Actinomycosis• Aspiration pneumonia• Complication of appendicitis or cholecystitis• Dental & periodontal infection• Endocarditis• Meningitis, usually following brain abscess• Otitis media, sinusitis• Necrotizing pneumonia• Osteomyelitis,• Peritonitis,
Table. shows incidence of anaerobes in various infectionsS. N. Type of infection Incidence (%)1. Lung abscess, necrotizing pneumonia 62-932. Bacteremia 6-103. Brain abscess 60-894. Chronic sinusitis 525. Thoracic empyema 766. Intra abdominal/pelvic abscess 60-1007. Perirectal abscess 758. Gas gangrene 85-959. Post appendectomy 40 Finegold SM et al. 2000
CLINICAL ISOLATION PROFILE• Gram negative bacilli followed by gram positive cocci .
ANAEROBIC GRAM NEGATIVE BACILLI• Bacteroides ( most common )• Prevotella• Porphyromonas• Fusobacterium• BilophilaAll non motile
1. Bacteroides group• B. fragilis (most common)• B.thetaiotaomicron
•Gram negative bacilli with rounded ends•1.5-9 µm vs 0.5-0.8µm•Nonmotile•Broth culture : pleomorphic with vacuoles•Capsules
Colony on CDC anaerobic blood agar• Non hemolytic• Semi opaque• Grey colony with concentric whorls inside
Key features…of this species• Growth enhanced by bile• Disc technique: (R) : Penicillin Kanamycin, Colistin Vancomycin (S): Rifampin
Regarding biochemicals… • All are sachharolytic • Fermentation patterns along with indole help to differentiate among species (para dimethyl aminocinamaldehyde INDOLE RHAMNOSE FERMENTATIONB.fragilis - -B.thetaiotaomicron + +
SOME DIFFERENTIAL CHARACTERISTICS OF SUB-SPECIES OF B. FRAGILISSub-species Rhamnose Trehalose Mannitol IndoleOvatus + + + +Thetaiotamicron + +/ - - +Distasonis +/ - + - -Vulgatus + - - -Fragilis - - - -
Pigmented anaerobic bacilli are no longer classified in the genus Bacteroides Prevotella-porphyromonas group 2nd most common group Next to bacteroides
Based on fermentation of carbohydratesSACCHAROLYTIC ASACCHAROLYTIC• PREVOTELLA • PORPHYROMONAS• P.intermedia • P. asaccharolytica• P. nigrescens• P. melaninognica
Porphyromonas• Tan to buff colonies : brown-black pigment• Brick-red fluorescence (UV)• Inhibited by bile• Disc technique : (R) : Kanamycin (S): Penicillin Rifampin• Failure to grow in Kanamycin-Vancomycin BA as Vancomycin inhibits growth.
Definitive identification ofPorphyromonas to species level is difficult. Rapid ID 32 A system or Rapid ANA II determines enzyme activities Within 4 hrs
Prevotella• Brown to black colonies on BA• Brick –red fluoescence (Long wave UV)• Produce indole• Ferments glucose etc.
P. intermedia resembles P. nigerscens which can be sort out by using egg-yolk agar Lipase produced by P. intermedia
Prevotella- porphyromonas group• 2nd most common group( anaerobic bacteria )• Normal microflora of oropharynx,GIT,GU syst.• Lesions like oro-facial origin & anaerobic pluropulmonary infections they out number the Bacteroides group .• Like B . fragilis group they produce β- lactamases .
Contd..• Gram stain: short coccobacilli 0.6-1µm × 0.3-0.4 µm• Brick-red fluorescence (UV)• Brown-black pigment• Inhibited by bile• Disc test: (S) Penicillin , Rifampin . (R) Kanamycin
Major problem faced with these pigmented groups• Fastidious• Slow growing (2 days - 3 weeks )• Some times may even fail to produce pigment
Clinical syndromes…• Like Prevotella-Porphyromonas group a/w anaerobic pleuropulmonary infections i.e. aspiration pneumonia, lung abscess necrotizing pneumonia, thoracic empyema.• Brain abscess, chronic sinusitis, metastatic osteomyelitis, septic arthritis, liver abscess, intra abdominal infections.
Species..• F. nucleatum (most common)• F. necrophorum• F. mortiferum• F. varium
Fusobacterium nucleatum….• Patients with neutropenia & mucositis following chemotherapy at high risk.• Direct M/S: characteristic spindle shaped cell i.e. long(5-10µm) filamentous tapered ends.Whereas most other species donot have fusiform shape ; rather rounded ends.
Contd..• Anaerobic BA : 1-2 mm in diameter with characteristic internal flecking referred as: crystalline internal structures (CIS) speckled opalescence
• Biochemically : inert• Electrophoresis (DNA) : 3 sub species but clinical significance not known.
Lemiere’s syndrome ( necrobacillosis)• Life threatening• Should be suspected in young patients with septic thrombophlebitis of internal jugular veins following URTI.• 12-25 yr healthy people• Oropharyngeal infections (tosillitis,peritonsilar abscess, pharyngeal abscess) followed by anaerobic septicemia & subsequent metastatic complications (lung , joints)
Contd..• Direct M/S : curved forms & spherical areas with in cells.• On LD egg yolk agar: iridescent sheen(lipase).• Three biovars i.e. A, B, C . clinical significance not known
DIFFERENTIAL CHARACTERISTICS OF SOME FUSOBACTERIUM SPECIESSpecies Aesculin Malt Lact Suc Growth indol hydrolysis in bile Other Resistant to RifampicinF. morteferum + + + - + - +F. varium - - - - + + +F. nucleatum - - - - - + -F. - - - - - + - Lipolytinecrophorum
Antibiotic susceptibility….• Resistant to erythromycin , tetracyclin , aztreonam , co-trimoxazole & aminoglycosides.• However sensitive to : metronidazole , clindamycin chloramphenicol nearly all β- lactam agents
F. mortiferum & F. varium• May produce β- lactamases• Coccoid to filamentous with spherical swelling near centre or one end . (2-10µm ×0.5-2µm)• BA : 1-2 mm diameter fried - egg appearance• Resistant to rifampin separates it from not only other fusobacterium species but also from Bacteroides,Prevotella- Porphyromonas group
They can be differrentiated by …. Esculin Lactose hydrolysis fermentationF. mortiferum + vF. varium - -
Lab. Diagnosis contd.. Microscopy• Sulphur granules are stained with Gram and ZN staining using 1% sulphuric acid for decolourization• gram positive hyphal elements with branching, surrounded by a peripheral zone of swollen radiating clubs• sun ray appearance• Sulphur granules and mycelia in tissue sections can also be identified by direct fluorescence microscopy
Culture• The sulphur granules inoculated on BHIA, BA and thioglycollate broth• Incubation is both aerobic and anaerobic with 5-10% CO2 at 35-37°C for 14 days• Colonies of A. israelii are 0.5-2mm in diameter, white or grey white smooth, entire or lobulated resembling molar tooth
Actinomycosis : Lab. Diagnosis Identification• Microscopy• Direct fluorescent antibody tests• Gel immunodiffusion Biopsy : H & E staining Molecular tests : DNA probes and PCR
SOME DIFFERENTIAL CHARACTERISTICS OF ACTINOMYCES AND ARACHNIAOrganism Growth in Spider Red Starch Aesculin Propionic Air Air ANO2+ macrocolony hydrolysis hydrolysis acid (wide +CO2 CO2 microcolony on BA zone) producedA. israelli - Slight + + - - + -A. + + + - + - +/- -odontolyticusA. eriksonii - - + - - - - -A. bovis Slight + + - - + + -A. naeslundii + + + - - - +/ - -Arachnia Slight + - - - +propionica Slight +
Treatment• Surgical excision with Penicillin for 1 year.
Propionibacterium acnes• Contamination of blood cultures• Endocarditis,CNS shunt infection• Diptheroid appeaance• Anaerobic• Produce indole, catalase & propionic acid
Lactobacillus species• Vagina• Endocarditis,peritonitis• Many can grow aerobically• Growth in rogosa’s selective tomato agar juice• Gm + ve uniform bacilli in chains• Catalase –ve• (R) : Vancomycin
Anaerobic cocci 2nd most common groupencountered next to anaerobic GNR.
Anaerobic gram positive cocci• FAMILY: Peptococcaceae• GENUS: Peptococcus Peptosreptococcus (most common) Ruminococcus SarcinaExcept for peptococcus niger all former species of genus peptococcus were transferred to genus peptostreptococcus
Clinically significant species• Peptostreptococcus anaerobius (Ѳ by SPS) pueperal sepsis,wound infection,abscess… aerotolerant grow well in 10 % CO2. KANAMYCINPeptostreptococcus RanaerobiusPeptostreptococcus Sasaccharolyticus
Gas- Liquid Chromatography• Use of gas-liquid chromatography (GLC) to detect anaerobes in exudates & body fluids has been developed.• A major amount of butyric acid in a specimen that contains only thin, pointed, gram-negative rods would suggest Fusobacterium spp.• A major peak of succinate & the presence of only gram-negative rods would suggest Bacteroides spp., Prevotella spp.• A major propionate peak in a positive blood culture containing pleomorphic, non spore forming gram-positive rods would be most consistent with Propionibacterium spp.• However, direct GLC provides only presumptive clues, & should be interpreted cautiously in polymicrobial infections.
PCR• PCR amplification procedure appear promising, but are not well commercialized.• Anaerobes identified by colony PCR and sequencing of the 16S rRNA gene using universal primers (LiPuma et al. 1999).
Rapid methods for diagnosis of anaerobes• Two rapid systems are available for quick diagnosis of anaerobes.1) RapID ANA by Innovative diagnostic systems2) AnIDENT by Analytal Products, Inc.• These both systems rely on preformed enzymes and only four hours of aerobic incubation is required.• Disadvantage is costly, and variable response.
Antibiotic susceptibility testing• AST is not required in every anaerobic isolates but done in1. Organism of known variability in susceptibility pattern, eg- B. fragilis2. Organism isolated in pure culture.3. Organism from seriously ill pt.4. Organism from pt. undergoing long-term antibiotic therapy.5. Organism from pt. failing to respond to empirical therapy.6. For epidemiological purposes.
Pitfalls in anaerobic bacteriology• Failure to bypass normal flora in collecting specimens.• Failure to setup anaerobic culture promptly from specimens.• Gram stain not prepared directly from clinical specimens• Use of inadequate commercial media.• Failure to use supplement in media eg.- Vitamin K1 for B. fragilis.• Failure to use selective media.• Failure to use a good anaerobic jar.• Failure to monitor catalyst.• Exposure of atmospheric gases during processing.• Inaccurate identification & speciation.• Failure to determine whether organism is a true anaerobes or not etc.
Summary• Many anaerobes grow more slowly than facultative or aerobic bacteria & since clinical specimens yielding anaerobic bacteria commonly contain several organisms.• Limited knowledge of infections caused by anaerobes or colonization of anaerobes.• Limited labs. doing culture & identification.• Culture is time consuming in most of the cases.• Automated systems is costly for anaerobiosis.• Except for few anaerobes, no rapid detection methods/systems is available.• No well formulated, universally accepted lab. protocol are available except Wadsworth Anaerobic Bacteriology Manual (fourth ed.) 1986.• This field of bacteriology should need more exploration.
Gaspak• Method of choice for preparing anaerobic jars. It is available as disposable envelope, containing chemicals which generate H 2 & CO2 on addition of water.• After the inoculated plates are kept in the jar, Gaspak envelope, with water added, is placed inside & the lid screwed tight.• Presence of a cold catalyst in the envelope permits combination of H2 & O2 to produce an anaerobic environment.• Gaspak is simple, effective, & eliminates the need for drawing a vacuum & adding H2.• Indicator should be used for verification of anaerobic environment.• Reduced Methylene blue is used as indicator.
Anaerobic Jar Techniques- Jars areused primarily with primary plated media orsubculture plates.Oxoid jar has a metal lid, valves & apressure gauge.It can be used either as an evacuation-replacement jar or, it can be used with adisposable gas generator (Gaspak).
Contd…• Introduction of gas mixture containing H2 into a jar is followed by catalytic conversion of the O2 in the jar with H2 to water, thus establishing anaerobiosis.• Catalyst composed of palladium-coated aluminum pellets. These can be inactivated by excess moisture & H2S produced by anaerobic bacteria.• So, they should be reactivated after each use by heating the basket or sachet of pellets to 160°C in a drying oven for 1.5 to 2 hrs.
Newer anaerobic systems• Recently, anaerobic gas-generating systems have been introduced that don’t require either catalyst or the addition of water to activate these systems.• AnaeroPack, absorbs O2 and generates CO2, but doesn’t generate H2.• It appear to be an excellent alternative to the GasPak and other established anaerobic incubation systems.• Another type of commercially available catalyst free-system ie. Anaerocult (Merck, Germany), makes use of iron filings in a sachet to which water is added, producing an O2 free, CO2-rich atmosphere.
Anaerobic Glove Box System• Self contained system that allows to process specimens & perform test for isolation & identification without exposure to O2.• Glove boxes suitable for cultivation, can be constructed from various materials, including steel, acrylic plastic, vinyl plastic or fiberglass.• Economical to operate b/c it permits the use of conventional plating media & cost of gases for operation of the system is minimal.• Once setup, the major expense is for the 85% N 2, 10%H2, 5%CO2 gas mixture used to replace the air in the entry lock when materials are passed into the glove box chamber.
Roll Streak System• It uses PRAS media prepared in tubes with rubber stoppers.• Tubes of agar media are cooled in a rolling machine after autoclaving, which results in a thin coating of the inner surfaces of the tubes with solidified medium.• Both PRAS liquid media & roll streak tubes requires addition of a reducing agent, such as L-cystine-hydrochloride, which is added just before autoclaving to maintain a low oxidation-reduction potential.• All inoculating & subculturing of the PRAS solid & liquid media are performed under a stream of O2-free CO2, which minimizes exposure to air & help to maintain a reduced oxidation-reduction potential in the media before & after growth.
Anaerobic Disposable Plastic Bags• Anaerobic bag system (BD Microbiology), and AnaeroPouch (Mitsubishi), Anaerogen (Oxoid), & Anaerocult P (Merck) etc.• Anaerobic Bag (BD) consists of a clear-plastic bag, an H 2-CO2 gas generator that generates an atmosphere when water is added to it, cold palladium catalyst pellets, & a resazurin indicator.• Bag is heat sealed following activation of the generator to permit maintenance of anaerobic conditions.• But in AnaeroPouch & Anaerocult achieve anaerobiosis, without catalyst, to remove O2 from the atmosphere & generate CO2.• O2 removed by combining with iron powder to form iron oxides.• These are used as alternative to anaerobic jar or glove box system.
Anaerobic Holding Jar• Convenient adjunct to the jar & glove box systems that allows primary plating, inspection of cultures, & subcultures of colonies at the bench with only minimal exposure to atmosphere.• Inexpensive, commercial-grade N2 can be used in the holding jar system.• Open the small needle valve & set to gas tank regulator at prescribed pressure.• Alternatively, CO2 passed through a tube of heated copper catalyst (Sargent furnace) can be used in the holding jars instead of N 2.
Use of syringe methods for anaerobiosis• Tubes are almost same as used in roll streak method.• Tubes containing prereduced medium are prepared by exclusion of oxygen with the desired gas, and a standard quantity, 4.5 ml, of reduced, anaerobic agar medium is dispensed into each tube.• This permits a 10x dilution by the addition of 0.5 ml of inoculum.• Relatively simple laboratory set-up is required.• Tubes of melted agar used for culturing are held in a 46° C water bath.• Cylinder that delivers the desired gas, passed through a reduced hot copper column (to remove oxygen).
Syringe methods for anaerobiosis FIG. A) Removal of dead air space from syringe and needle. B) Removal of clinical specimen in holding medium.
DIFFERENTIAL CHARACTERISTICS OF SOME BACTEROIDES SPECIES Species Aesculin Glu Malt Lact Suc Growt hydrolysi h in Resistant to Other s bile Penic- Kana- Rifam- illin mycin picin B. fragilis + + + + + + -/+ + + + B. -/ + +/ - +/ - +/- +/- - +/- - + - Prote melaninog olytic, enicus black fluore scent colon y B. oralis + + + + + - - - + - B. + + - - - + - + + - capillosus B. - - - - - + - + praeacutu s B. - - - - - + - - - - Oxid corrodens ase +ve; pittin g of agar
Contd…• Spectrum of infection from deep seated abscesses (Abdominal, pelvic, brain, thoracic etc.), soft tissue infection (Bite wound, diabetic ulcer, cutaneous abscess, decubitus ulcer, gas gangrene, breast abscess, perirectal abscess), dental abscess, periodontal abscess, aspiration pneumonia, bronchiectasis, vulvovaginal abscess, septic abortion, bacteremia, otitis media, neck space infection, and to ocular infection etc.
Collection & transport of specimens• Decontaminate skin & mucus membrane properly before sample collection from these sites.• Surgical soap scrub should be used, followed by application of 70% ethyl or isopropyl alcohol, then iodine for 1 min.• A needle & syringe should be used whenever possible for collecting specimens for anaerobic culture.• Once collected, precaution should be taken to protect them from oxygen exposure & deliver them promptly to lab.
Isolation of Anaerobic bacteria• Proper selection, collection, and transport : most important stepSelection of specimens for culture- With few exceptions, all materials collected from sites not harboring an indegenous flora, such as body fluids other than urine, exudates from deep abscesses, FNAs, and tissue biopsies, should be cultured for anaerobic bacteria.• However, since anaerobes normally inhabit the skin and mucous membranes as a part of the normal indigenous flora, following samples should not be accepted for anaerobic culture.
Specimens that should not be cultured for anaerobic bacteria• Gingival swabs• Throat or nasopharyngeal swabs• Sputum or bronchoscopic specimens• Gastric contents, small bowel contents, feces, rectal swabs etc.• Surfaces of decubitus ulcers, swab samples of encrusted wall of abscesses, mucosal lining etc.• Material adjacent to skin or mucus membranes other than the above• Voided urine• Vaginal or cervical swabs.• Wound or lesions that will respond to I & D.
A) Direct examination of clinical materials• A foul odor, purulent appearance of fluid specimens, & the presence of necrotic tissue & gas or sulfur granules are valuable for suspicion of anaerobes.• Background & cellular characteristics of smear, Gram reaction; size, shape, number, arrangement of bacteria, presence of spores, & their position, filaments with spherical bodies, pointed ends, and granular forms recorded.• Acridine orange stains are useful for detecting bacteria in blood cultures, CSF, pleural fluid, joint fluid, and exudates.
Methods for diagnosis of anaerobic infectionsA) Direct examination of specimens and stainingB) CultureC) Metabolic product detection by gas-liquid chromatographyD) Molecular methods like PCR etc.E) Rapid systems.
B) Anaerobic culture techniques• Anaerobic bacteria differ in their requirements of & sensitivity to oxygen.Robertson’s cooked meat medium- Probably the most widely used fluid medium for anaerobes.• It contains fat free-minced cooked bullock meat in broth (either peptone infusion/thioglycollate broth). It indicates saccharolytic (eg. Clostridium perfringens.) or proteolytic (C. histolyticum, C. tetani) activities, by the meat turned red or black, respectively.• Other Culture media. Schaedler Broth (Oxoid), Brain-Heart Infusion Broth (Oxoid), Wilkins-Chalgren Anaerobe Broth/agar (Oxoid) and Thioglycollate Broth.
Contd…Blood culture techniques- The mortality associated with an anaerobic bloodstream infection is high.Liquid media- Some commercially available media contain Sodium polyanethol sulfonate (SPS), which has been reported to enhance the recovery of anaerobes, but may be inhibitory to Peptosreptococcus anaerobius. This effect can be overcome by addition of 1.2% gelatin.• eg- Tryptic soy broth, thiol broth, Columbia broth, trypticase soy broth, and thioglycollate medium, PRAS with brain-heart infusion yeast extract broth, supplemented peptone broth, radiometric method.
Selection & use of media• Media used for recovering anaerobes from specimens should include nonselective, selective, and enrichment types.1) CDC Anaerobe blood agar- Trypticase soy agar, 5% sheep blood; yeast extract, hemin, vitamin K1, L-cystine for anaerobes requiring additional growth factors. Acts as nonselective BA plating media for primary isolation of all anaerobes found in clinical specimens.2) Phenylethyl alcohol blood agar- Above contents + 2.5 gm/L of Phenylethyl alcohol (for inhibition of swarming of Proteus spp.). Used for selective isolation of anaerobes from infected material containing mixture of bacteria.3) Kanamycin-vancomycin blood agar- Above + 100 mg/L of kanamycin & 7.5 mg/L of vancomycin. Used for selective isolation of most Bacteroides spp., Prevotella spp., Fusobacterium spp., and Veillonella spp. from specimens containing mixed aerobes & anaerobes.
Contd…4) Paromomycin-vancomycin blood agar- Above (3) + 100mg/L of paromomycin substituted for kanamycin. Used for selective isolation of B. fragilis group (pig. & nonpigmented ie. Prevotella spp.), Fusobacterium spp., Veillonella spp.5) Cycloserine-cefoxitin fructose agar- Trypticase soy base, fructose, neutral red as indicator, cycloserine (500mg/L), and cefoxitin (16mg/L). Used for selective isolation of C. difficile from stool specimen.6) Enriched thioglycollate medium- BBL-0135C formula thioglycollate medium (without indicator) with hemin and vitamin K 1. Used for primary isolation of Actinomycetes.
Anaerobic systems for cultivation• Anaerobic jars with disposable gas generators• Evacuation replacement jars• Anaerobic gloves box techniques,• Roll tube & roll streak tube with prereduced anaerobically sterilized (PRAS) media etc.• Anoxamat
Peptostreptococcus• P.anaerobius : (R) to peniciillin-G
Peptococcus + Coccal Singly, pairs, short chains and clumpsPeptostrept + Coccal Singly, pairsococcus and in chainsVeillonella _ Coccal Pairs, short chains and irregular clumps
Methods used for AST1. Broth dilution methods- a) Macrodilution method b) Microdilution method2. Broth disk method3. Agar dilution method a) Wadsworth method b) Approved reference method4. beta-lactamase testing- Required in Bacteroides fragilis Pigmented Bacteroides F. nucleatum
Gas- Liquid ChromatographyGas-liquid chromatograms of bile acids in the nonamidate and glycine-conjugatefractions after a piperidinohydroxypropyl dextran gel (PHP GEL) column.