anaerobic bacteria

Non sporing anaerobes

Dr Dipankar Pattnaik

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).

Anaerobic bacteria- Classification

A) Gram-negative bacilli (curved, spirals & spirochete forms)- Bacteroides, Borrelia, Butyrivibrio, Capnocytophaga, Campylobacter, Fusobacterium, Leptotrichia, Porphyromonas, Prevotella, Treponema etc.

B) Gram-positive cocci- Anaerococcus, Coprococcus, Micromonas, Peptococcus, Peptostreptococcus, Streptococcus, Gemella etc.

C) Nonsporing Gram-positive bacilli- Actinomyces, Arcanobacterium, Bifidobacterium, Eubacterium, Lactobacillus, Methanobacterium, Mobiluncus, Propionibacterium etc.

D) Gram-negative cocci- Acidaminococcus, Anaeroglobus, Veillonella etc.

it is essential to isolate and identify anaerobic bacteria because

1) 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 infections

S. N. Type of infection Incidence (%)

1. Lung abscess, necrotizing pneumonia 62-93

2. Bacteremia 6-10

3. Brain abscess 60-89

4. Chronic sinusitis 52

5. Thoracic empyema 76

6. Intra abdominal/pelvic abscess 60-100

7. Perirectal abscess 75

8. Gas gangrene 85-95

9. 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 0.5-0.8 µm

•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

Presumpto Quadrant Plates 1,2,3

Quadrant plate & Anaerobic plate

Bacteroides fragilis

Regarding biochemicals…

• All are sachharolytic • Fermentation patterns along with indole

help to differentiate among species (para dimethyl aminocinamaldehyde

INDOLE RHAMNOSE FERMENTATION

B.fragilis - -B.thetaiotaomicron + +

B .thetaiotamicron

B. ureolyticus

• DISC TECHNIQUE• Sensitive : Penicillin Rifanpin Kanamycin • UREASE

Sub-species Rhamnose Trehalose Mannitol Indole

Ovatus + + + +

Thetaiotamicron + +/ - - +

Distasonis +/ - + - -

Vulgatus + - - -

Fragilis - - - -

SOME DIFFERENTIAL CHARACTERISTICS OF SUB-SPECIES OF B. FRAGILIS

Pigmented anaerobic bacilli are no longer classified in the genus

Bacteroides

Prevotella-porphyromonas group2nd most common group

Next to bacteroides

Based on fermentation of carbohydrates

SACCHAROLYTIC• PREVOTELLA• P.intermedia• P. nigrescens• P. melaninognica

ASACCHAROLYTIC• PORPHYROMONAS• P. asaccharolytica

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 of Porphyromonas 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.

COCCO BACILLI

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

Fusobacterium

• Normal habitat : upper respiratory tract gastrointestinal tract genitourinary tract

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.

Fusobacterium necrophorum….

• Lemiere’s syndrome ( necrobacillosis)• Postanginal septicemia• Liver abscess

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

Species Aesculin

hydrolysis

Malt Lact Suc Growth

in bile

indol

Resistant to

Other

Rifampicin

F. morteferum + + + - + - +

F. varium - - - - + + +

F. nucleatum - - - - - + -

F.

necrophorum

- - - - - + - Lipolyti

DIFFERENTIAL CHARACTERISTICS OF SOME FUSOBACTERIUM SPECIES

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 hydrolysis

Lactose fermentation

F. mortiferum + v

F. varium - -

F .murtiferum

ACTINOMYCES

Morphology•Gram positive bacilli (0.5-1m)

•Nonmotile

•Nonsporing

•Non-acidfast

•Filamentous (break up into coccoid and bacillary forms)

• Exhibit true branching

Actinomycosis

• Caused by A. israelii• Chronic suppurative and granulomatous disease • Three types : Cervicofacial (mc) Thoracic Abdominal

Characterised by :

• formation of sinus tracts from suppurative lesions• presence of pus • sulphur granules• colonies

Lab. Diagnosis

Samples :• Pus Bronchial secretions Sputum etc

• Sulphur granules

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

Organism Growth in Spider

microcolony

Red

macrocolony

on BA

Starch

hydrolysis(wide zone)

Aesculin

hydrolysis

Propionic

acid

produced

Air Air

+CO2

ANO2+

CO2

A. israelli - Slight + + - - + -

A.

odontolyticus

+ + + - + - + / - -

A. eriksonii - - + - - - - -

A. bovis Slight + + - - + + -

A. naeslundii + + + - - - +/ - -

Arachnia

propionica

Slight

Slight

+

+ - - - +

SOME DIFFERENTIAL CHARACTERISTICS OF ACTINOMYCES AND ARACHNIA

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 group encountered 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.

KANAMYCIN

Peptostreptococcus anaerobius

R

Peptostreptococcus asaccharolyticus

S

• Peptococcus niger (black pigment)• Gemella morbillorum

Anaerobic gram negative cocci

• FAMILY: Veillonellaceae• GENUS: Veillonella (most common) Acidaminococcus Megaspaera

Veillonella

• V. parvula (mc species) life threatening Endocarditis

To be remembered regarding Antibiotic susceptibility of anaerobic

cocci• P. anaerobius (R) to Penicillin-G.• Microaerophillic streptococci Streptococcus intermedius (R) Metronidazole.• Veillonella : (R) to Vancomycin• Drugs C/I: Aminoglycosides, Aztreonam Co-trimoxazole Fluoroquinolones

Contd.. Drugs active against • Metronidazole (a/e Strep.intermedius)• Clindamycin• β-lactam drugs including Penicillin (except Cefperazone,Cefotaxime,Cefotetan)• Chloramphenicol • Imipenem• Piperacillin – Tazobactam• Newer Fluroquinolones (moxi/gati)• Linezolid

Double zone β- hemolysis

Catalase

C. perfringes + -

Propionibacterium - +

GRAM POSITIVE RODS

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 systems

2) 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 in

1. Organism of known variability in susceptibility pattern, eg- B. fragilis

2. 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.

Thank you

Gaspak• Method of choice for preparing anaerobic jars. It is available as

disposable envelope, containing chemicals which generate H2 &

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 are

used primarily with primary plated media or

subculture plates.

Oxoid jar has a metal lid, valves & a

pressure gauge.

It can be used either as an evacuation-

replacement jar or, it can be used with a

disposable 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% N2, 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 H2-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 N2.

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.

Species Aesculin hydrolysis

Glu Malt Lact Suc Growth in bile

Resistant to Other

Penic-illin

Kana-mycin

Rifam-picin

B. fragilis + + + + + + -/+ + + +B. melaninogenicus

-/ + +/ - +/ - +/- +/- - +/- - + - Proteolytic,black fluorescent colony

B. oralis + + + + + - - - + -B. capillosus

+ + - - - + - + + -

B. praeacutus

- - - - - + - +

B. corrodens

- - - - - + - - - - Oxidase +ve; pitting of agar

DIFFERENTIAL CHARACTERISTICS OF SOME BACTEROIDES SPECIES

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

step

Selection 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 infections

A) Direct examination of specimens and staining

B) Culture

C) Metabolic product detection by gas-liquid chromatography

D) 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.

Contd…• Various automated anaerobic blood culture bottles ; Bactec Plus

Anaerobic/F, Bactec Lytic/10 Anaerobic F, Bactec Anaerobic F,

BacT/Alert, BacT/Alert FN, ESP 80N Anaerobic Broth, ESP 40N

Anaerobic Broth.

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 K1.

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

Anoxomat

Anaerobic Culture Methods• Anaerobic

jar• Chemical

reactions remove oxygen

McIntosh & Fildes Jar GasPak Jar System

Anaerobic Glove Box System

Roll Streak System

FIG. A) Side view of roll tube, butyl rubber closure and screw-on cap. B) Top view of closed roll tube.

Anaerobic Disposable Plastic Bags

presumptive tests

Peptostreptococcus

• P.anaerobius : (R) to peniciillin-G

Peptococcus + Coccal Singly, pairs, short chains and clumps

Peptostreptococcus

+ Coccal Singly, pairs and in chains

Veillonella _ Coccal Pairs, short chains and irregular clumps

Methods used for AST1. Broth dilution methods-

a) Macrodilution method

b) Microdilution method

2. Broth disk method

3. Agar dilution method

a) Wadsworth method

b) Approved reference method

4. beta-lactamase testing- Required in Bacteroides fragilis

Pigmented Bacteroides

F. nucleatum

Gas- Liquid Chromatography

Gas-liquid chromatograms of bile acids in the nonamidate and glycine-conjugate fractions after a piperidinohydroxypropyl dextran gel (PHP GEL) column.