ANAEROBIC BACTERIOLOGY

Dr.T.V.Rao MD

Dr.T.V.Rao MD

What Are Anaerobic Microorganisms

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Anaerobic microorganisms are widespread and very important Do not require oxygen for growth - often extremely toxic
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The Requirements for Growth: Related to Oxygen

Oxygen (O2)

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Anaerobes differ from Aerobic Bacteria

Anaerobes generate energy by fermentation Lack the capacity to utilize O2 as a terminal hydrogen acceptor Some are sensitive to O2 concentration as low as 0.5% O2 Most can survive in 3%-5% O2 A few can grow poorly in the presence of air aero tolerant anaerobes Many are members of the normal flora created by presence of facultative anaerobes
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DEFINITIONS
OBLIGAETE ANAEROBE
Lack superoxide dismutase and/or catalase toxic radicals formed by oxidative enzymes kill organisms

AERO-TOLERANT ANAEROBES
survive in presence of oxygen Do not use oxygen for energy requirements

FACULTATIVE ANAEROBES
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Anaerobes and Oxygen

Anaerobes generate energy by fermentation Lack the capacity to utilize O2 as a terminal hydrogen acceptor Some are sensitive to O2 concentration as low as 0.5% O2 Most can survive in 3%-5% O2 A few can grow poorly in the presence of air aero tolerant anaerobes Many are members of the normal flora created by presence of facultative anaerobes
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Anaerobic and Aerobic Respiration

Reaction name
Aerobic Respiration

Reduct. Oxid. Reaction Stoichiometry CHO O2 C6H12O6 + 6O2 ==> 6CO2 + 6H2O
NO3-

kcal/ mol 686

Nitrate Reduction CHO

CHO + NO3- + H+ ==> 649 CO2+ N2+ H2O

Sulfate Reduction CHO

Methanogenesis CHO or H2

SO42- 2CHO + SO42-+2H+ => 2CO2+ H+ 2H2O

CO2

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4H2 + CO2 ==> CH4 + 8.3 2H2O Dr.T.V.Rao MD 7

FACTORS THAT INHIBIT THE GROWTH OF ANAEROBES BY OXYGEN

1.

Toxic compounds are produced e.g. H2O2 , Superoxide's 2. Absence of catalase & Superoxide dismutase 3. Oxidation of essential sulfhydryl groups in enzymes without sufficient reducing power to regenerate them
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Strict Anaerobic Bacteria

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Obligate (strict) anaerobes - oxygen is

toxic to these organisms, do not use oxygen as terminal electron acceptor. Archaea such as methanogens and Bacteria, e.g Clostridia, Bacteriodes etc. etc.
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ROS production during respiration

O2 + e- => O2 O2- + e- + 2H+ => H2O2 H2O2 + e- + H+ => H2O + OH. OH. + e- + H+ => H2O superoxide

anion
hydrogen peroxide Hydroxyl radical water
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Oxygen Toxicity
Oxygen is used by aerobic and facultatively anaerobic organisms as its strong oxidising ability makes it an excellent electron acceptor

During the stepwise reduction of oxygen, which takes place in respiration toxic and highly reactive intermediates are produced reactive oxygen species (ROS).
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FACTORS THAT INHIBIT THE GROWTH OF ANAEROBES BY OXYGEN

1.

Toxic compounds are produced e.g. H2O2 , Superoxide's 2. Absence of catalase & Superoxide dismutase 3. Oxidation of essential sulfhydryl groups in enzymes without sufficient reducing power to regenerate them
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Chemical Dynamics in Anaerobic Bacteria

Organisms that use O2 have developed defence mechanisms to protect themselves from these toxic forms of oxygen enzymes Catalase: H2O2 + H2O2 => 2H2O + O2 Peroxidase: H2O2 + NADH + H+ => 2H2O + NAD+ Superoxide dismutase: O2- + O2- + 2H+ => H2O2 + O2

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Anaerobic environments exist in Nature too

Anaerobic environments (low reduction potential) include: Sediments of lakes, rivers and oceans; bogs, marshes, flooded soils, intestinal tract of animals; oral cavity of animals, deep underground areas, e.g. oil packets and some aquifers Anaerobes also important in some infections, e.g. C. tetanii and C. perfringens important in deep puncture wound infections
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ANAEROBES OF CLINICAL IMPORTANCE

CLOSTRIDIA
C tetani; C perfringens; C difficile; C botulinum

BACTEROIDES
B fragilis; Prevotella Porphyromonas

ACTINOMYCES FUSOBACTERIUM ANAEROBIC STREPTOCOCCI

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FACTORS RESPONSIBLE FOR THEIR VIRULENCE

1. Lipopolysaccharide

- promotes abscess formation, enhanced coagulation 2. Polysaccharide capsule - correlated with abscess production 3. Enzymes a. Collagenase b. Heparinize * develop thrombophlebitis & septic emboli 4. Short chained fatty acids a. Butyrate- seen in dental plaque b. succinic acid reduces phagocytic killing
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Multiplication of the opportunistic pathogens is facilitated by: 1. Inhibition of phagocytosis & intracellular killing by PMN in the presence of Bacteroides by: a. competition of opsonins b. inhibition by capsular materials 2. Protection of antibiotic susceptibility strains in mixtures thru destruction by the lactamases 3. Utilization of O2 by facultative species that aids in producing a suitable environment for growth of anaerobe
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CLINICAL MANIFESTATION
Clinical finding suggestive of Anaerobic infection
1. odor 2. tissue 3. location 4. necrotic tissue 5. endocarditis with (-) blood culture 6. infection associated with malignancy 7. black discoloration 8. blood containing exudates 9. associated with sulfur granules 10. Bacteremic feature with jaundice 11. human bites
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Dr.T.V.Rao MD

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Anaerobic Bacteria of Medical Interest

MORPHOLOGY Spore forming Non-spore forming bacilli (+) GRAM STAIN (+) GENUS Clostridium Actinomycetes, Bifidobacterium,Eubacterium,Propionibacerium, Mobilncus,Lactobacillus

(-)

Bacteroides,Fusobacterium Prevotella,Porphyromonas

Non-sporefoming cocci

(+)

Peptococcus, Pepto-streptococcus Streptococcus

(-)

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Veilonella

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Pathogenesis of anaerobic infections

Contamination of site with spores Factors which promote anaerobiasis
crush injuries with interruption of blood supply, contamination with foreign bodies (dirt), tissue damage

Germination of spores Toxin release Binding of toxin to receptor Resulting effect produces symptom(s) of disease
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Gram-positive anaerobes
Actinomyces (head, neck, pelvic infections; aspiration pneumonia) Bifid bacterium (ear infections, abdominal infections) Clostridium (gas, gangrene, food poisoning, tetanus, pseudomembranous colitis) Peptostreptococcus (oral, respiratory, and intra-abdominal infections) Propionibacterium (shunt infections)
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Gram-negative anaerobes
Bactericides (the most commonly found anaerobes in cultures; intra-abdominal infections, rectal abscesses, soft tissue infections, liver infection) Fusobacterium (abscesses, wound infections, pulmonary and intracranial infections) Porphyromonas (aspiration pneumonia, periodontitis) Prevotella (intra-abdominal infections, soft tissue infections)
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FACTORS RESPONSIBLE FOR THEIR VIRULENCE

1. Lipopolysaccharide

- promotes abscess formation, enhanced coagulation 2. Polysaccharide capsule - correlated with abscess production 3. Enzymes a. Collagenase b. Heparinase * develop thrombophlebitis & septic emboli 4. Short chained fatty acids a. Butyrate- seen in dental plaque b. succinic acid reduces phagocytic killing
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Common Human Anaerobic Infections

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CLOSTRIDIA
Gram positive spore forming bacilli ubiquitous
intestines of man and animals animal and human faeces contaminated soil and water

Several species associated with human disease

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Clostridium perfringens
Large rectangular Gram positive bacillus Spores seldom seen in vivo or in vitro non motile Produces several toxins
alpha (lecithinase), beta, epsilon ...... enterotoxin

Causes a spectrum of human diseases

Bacteraemia Myonecrosis food poisoning enteritis necrotica (pig bel)
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Clostridium tetani
Small motile spore forming gram positive bacillus with round terminal spores Causes tetanus Pathogenesis:
produces tetanospasmin during stationary phase which is released when cell lysis occurs heavy chain binds to ganglioside on neuronal membranes toxin internalized and moves from peripheral to central nervous system by retrograde axonal transport crosses synapse and localized within vesicles acts by blocking release of inhibitory neurotransmittors (eg GABA)
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Clostridium tetani
Small motile spore forming gram positive bacillus with round terminal spores Causes tetanus Pathogenesis:
produces tetanospasmin during stationary phase which is released when cell lysis occurs heavy chain binds to ganglioside on neuronal membranes toxin internalized and moves from peripheral to central nervous system by retrograde axonal transport crosses synapse and localized within vesicles acts by blocking release of inhibitory neurotransmitters (eg GABA) Dr.T.V.Rao MD 30

TETANUS
Clinical syndromes due to unregulated excitatory synaptic activity resulting in spastic paralysis

Generalised tetanus Neonatal tetanus localized tetanus

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Prevention and treatment

Active immunization with tetanus toxoid Wound toilet and active/passive immunization of risk injuries
management of wound tetanus toxoid Anti-tetanus serum (ARS -horse serum) or Human Tetanus ImmunoGlobulin (HTIG) Penicillin or Metronidazole

Management of patient with tetanus

reduce stimuli respiratory and CVS support
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Clostridium difficile
Associated with human disease in mid-1970s Found in human GIT in small numbers With antibiotic use, increase in number in GIT
Clindamycin, ampicillin, cephalosporins .......

Produces 2 entero toxins

Toxin A -enterotoxin & Toxin B -cytotoxin

Diagnosis
Detection of toxins in stools, culture of organism

Clinical - AAC Treatment

Pseudomembranous colitis

omit antibiotic if possible oral vancomycin (125mg qds or metronidazole

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 Associated with human disease in mid-1970s Found in human GIT in small numbers With antibiotic use, increase in number in GIT
Clindamycin, ampicillin, cephalosporins .......

Clostridium difficle

Produces 2 entero toxins

Toxin A -enterotoxin & Toxin B -cytotoxin

Diagnosis
Detection of toxins in stools, culture of organism

Clinical - AAC Treatment

Pseudomembranous colitis

omit antibiotic if possible oral vancomycin (125mg qds or metronidazole Dr.T.V.Rao MD

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Clostridium botulinum
Fastidious spore forming anaerobic gram positive bacillus Produces 8 antigenically distinct toxins Human disease described with types A, B & E Heavy chain binds to ganglioside receptor Toxin internalized and prevents release of acetyl choline from vesicles Clinical
Food borne botulism (weakness, dizziness, ocular palsy and progressive flaccid paralysis) infant botulism (floppy baby) wound botulism
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ANAEROBIC GRAM NEGATIVE BACILLI

Bacteroides, Prevotolla, Porphyromonas and Fusobacterium Present in GI tract -form large component of normal flora >80% of human infections associated with B fragilis
virulence factors - capsule, LPS, agglutinins and enzymes

Clinical - Endogenous infections

Intra-abdominal pyogenic infections pleuro-pulmonary infctions genital infection
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ACTINOMYCES
Strict anaerobic Gram positive bacilli typically arranged in hyphae which fragment into short bacilli Normal flora of upper respiratory tract, GI tract and female genital tract. Low virulence produce disease when mucosal barrier is breached (eg: following dental trauma or surgery) ENDOGENOUS Establishes chronic infection that spreads through normal anatomical barriers Clinical -cervicofacial, abdominal and thoracic Diagnosis:
Gram stain of sulpher granules culture

Treatment - surgery and long term penicillin

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LABORATORY DIAGNOSIS A. COLLECTION

Anaerobes are endogenous in nature I. Appropriate specimens for anaerobic culture : 1. pus 2. pleural fluid 3. urine 4. pulmonary secretions 5. uterine secretions or sinus tract material
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Aspiration is ideal Avoid Swabs

II. Collection by needle

aspiration is preferable than swab culture because of a. better survival of pathogen b. greater quantity of specimen c. less contamination with extraneous organism are often achieved
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HANDLING
If a swab must be used, a 2 tube system must be used
1st tube contains swab in O2 free CO2
2nd tube contains PRAS (pre-reduced anaerobically sterilized culture media)

Specimen should be placed in anaerobic transport device with gas mixture

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Isolation
Gram stain should be done in the laboratory :
a. choice of appropriate media & methods for culture b. quality control for the types of bacteria that laboratory culture reveal
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A solid or liquid medium maybe used & must provide an anaerobic environment Anaerobic Culture

System A. ANAEROBIC JAR

1. Candle Jar

- reduces O2 environment - only CO2 tension 2. Gas Pak Jar a. Palladium aluminum coated pellets - catalyst - chemically reduces O2 - reacts with residual O2 in the presence of H2 to form H2O
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b. Gas Pak envelope - generates CO2 & H2 gases c. Methylene blue strip - indicator blue (+) O2 white (-) O2

II. Anaerobic Glove Chamber

- close system - used for premature babies - e.g. incubator

III. Roll Tube

- has a pedal gas ( CO2 & H2 ) would come out - place test tube directly to the outlet
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IDENTIFICATION
Plates are checked at > 18-24 hours for faster growing species like Cl. Perfringens & B.fragilis & daily thereafter up to

> 5-7 days for slowly growing species like

Actinomyces, Eubacterium & propionibacterium Genus is determined by
- gram stain, cellular morphology, Gas-liquid chromotography Species determination is based on fermentation of sugars & other biochemical determination
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ANAEROBIC GRAM NEGATIVE BACILLI

Bactericides, Prevotolla, Porphyromonas and Fusobacterium Present in GI tract -form large component of normal flora >80% of human infections associated with B fragilis
virulence factors - capsule, LPS, agglutinins and enzymes

Clinical - Endogenous infections

Intra-abdominal pyogenic infections pleuro-pulmonary infections genital infection
Dr.T.V.Rao MD 45

ACTINOMYCES
Strict anaerobic Gram positive bacilli typically arranged in hyphae which fragment into short bacilli Normal flora of upper respiratory tract, GI tract and female genital tract. Low virulence produce disease when mucosal barrier is breached (eg: following dental trauma or surgery) ENDOGENOUS Establishes chronic infection that spreads through normal anatomical barriers Clinical -cervicofacial, abdominal and thoracic Diagnosis:
Gram stain of sulpher granules culture
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Culturing of anaerobes need special skills

Culture of anaerobes is extremely difficult due to the need to exclude oxygen, slow growth and complex growth requirements Molecular methods based on DNA analysis and direct microscopy have shown that we are largely ignorant of the microbial world and previously unknown
diversity has been discovered
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Culture methods
Anaerobes differ in their sensitivity to oxygen and the culture methods employed reflect this some are simple and suitable for less sensitive organisms, others more complex but necessary for fastidious anaerobes Vessels filled to the top with culture medium can be used for organisms not too sensitive
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Appropriate Specimens for Anaerobic Cultures

The Microbiologists understanding of basic anaerobic bacteriology is critical in the interpretation of an anaerobic culture result for the diagnosis and treatment of anaerobic infection. Since anaerobes from part of the normal bacterial flora of the skin and mucous membrane, proper selection and collection of clinical specimens for the laboratory diagnosis of an anaerobic infection critical factors that will determine the clinical significance of the culture results
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Acceptable Specimens
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Specimens for anaerobic cultures are ideally biopsy samples of needle aspirates.

Anaerobic swabs are discouraged but sometimes cannot be avoided. Swabs are the

least desirable because of the small amount of the specimen and effect of drying. There is a greater chance of contamination with normal micro flora
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The accepted specimens for anaerobic processing are as follows:

Sites CNS Dental/ENT

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Acceptable specimen

CSF, abscess, tissue Abscess, aspirates, tissues

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The accepted specimens for anaerobic processing are as follows:

Local abscess
Pulmonary

Needle aspirates Trans tracheal aspirates, lung aspirates, pleural fluid, tissue, Protected bronchial washing

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The accepted specimens for anaerobic processing are as follows

Abdominal Urinary tract Genital tract Ulcers/wounds
Abdominal Abscess aspirate, fluid and tissues Suprapubic bladder aspirate Culdocentesis specimen, endometrial swabs

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Others

Aspirate/swab pus from deep pockets or from under skin flaps that have been decontaminated Deep tissue or bone lesions, blood, bone marrow, synovial fluid, tissues

Handling other Critical Specimens

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Specimens that are normally sterile, such as blood, CSF and synovial fluid, should be collected aseptically to prevent contamination by skin flora. In general, the best materials for anaerobic cultures are obtained by needle aspiration and able tissue biopsy.
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Unacceptable Specimens
Exudates, swabs from burns, wounds and skin abscesses are generally unacceptable for anaerobic cultures. Cysts and abscess are contaminated with normal anaerobic flora. Gastric contents, small bowel contents, feces, colo-cutaneous fistula and colostomy contents should not be cultured for anaerobic bacteria. Voided and catheterized urine are contaminated with distal urethral anaerobes and are therefore unacceptable for anaerobic cultures.

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Interpretation by Physicians and Microbiologists

The physician who collected the specimen can best evaluate the anaerobic culture result. Interpretation of the result should be correlated with the clinical findings and how the specimen was collected. Clinical signs suggesting possible infection with anaerobes include the following: 1. Foul smelling discharge 2. Infection in proximity to a mucosal surface 3. Gas in tissues 4. Negative aerobic cultures of specimens whose gram stains show organisms and pus cells.
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Limitation with Culturing the Specimens

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Respiratory specimens that are generally rejected for anaerobic cultures include nasal and throat swabs, sputum and suction specimens; e.g. nasotracheal, tracheal and endotracheal aspirates collected by suction and unprotected bronchial washing. These specimens are contaminated with oral flora anaerobes.
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Diagnosis
Myonecrosis
clinical Gram stain of exudate - typical organisms no pus cells Culture -growth of C perfringens (and/or other clostridia associated with this clinical condition)

Food poisoning
abdominal pain, diarrhea and vomiting 8-18 hours after a suspect meal. Self limiting

Enteritis necroticans
severe abdominal pain, bloody diarrhoea , shock and peritonitis (C perfringens type C)

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Basic needs in Anaerobic Medium

Most common adaptation of media is the addition of a reducing agent, e.g. Thiglyclolate, cysteine Acts to reduce the oxygen to water, brings down the redox potential -300mV or less. Can add a redox indicator such as rezazurin, pink in the presence of oxygen - colourless in its absence
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Testing for anaerobes in Routine Practice

Deep culture tubes can be used to test whether an unknown organism is anaerobic/facultative or aerobic Thiglyclolate added to culture medium, oxygen only found near top where it can diffuse from air -pattern of colony formation characteristic of organisms
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Why Needle Aspiration Preferred for Anaerobic Bacteria

II. Collection by needle

aspiration is preferable than swab culture because of a. better survival of pathogen b. greater quantity of specimen c. less contamination with extraneous organism are often achieved
Dr.T.V.Rao MD

HANDLING
If a swab must be used, a 2 tube system must be used 1st tube contains swab in O2 free CO2 2nd tube contains PRAS (pre-reduced anaerobically sterilized culture media
Specimen should be placed in anaerobic transport device with gas mixture

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HANDING AND TRANSPORT OF CLINICAL SPECIMENS

The basic principles to remember are proper collection of specimens to avoid contamination with the normal microbial flora and prompt transport to the laboratory where immediate processing is done. Interpreting anaerobic culture result should be easy if proper collection and transport of the specimens have been assured
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Transporting
Anaerobic transport tubes and/or devices should always be available at the OR and ER. Specimens should be placed in leak-proof container with tight fitting caps. Of course, proper label for identification with date and time of collection should accompany all specimens submitted for culture. Put samples in room temperature while waiting for delivery to the laboratory. Some anaerobes are killed by refrigeration.
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Basic Information with Gram Staining

Gram stain should be done in the laboratory :

a choice of appropriate media & methods for culture b. quality control for the types of bacteria that laboratory culture reveal
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Gram stain can be Guiding factor Interpret with caution and Expertise
The gram stain result is helpful because bacteria present in the smear should be present in the culture. Specimens from intraabdominal and genital infections usually yield polymicrobial cultures of aerobes and anaerobes. Some aspirates/abscesses may contain more than one anaerobe. These should all be corrected with the gram stain result.
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Interpretation of Gram Staining

Gram staining is performed on the specimen at the time of culture. While infections can be caused by aerobic or anaerobic bacteria or a mixture of both, some infections have a high probability of being caused by anaerobic bacteria. These infections include brain abscesses, lung abscesses, aspiration pneumonia, and dental infections. Anaerobic organisms can often be suspected because many anaerobes have characteristic microscopic morphology (appearance)
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Anaerobic culturing Needs Define Chemicals and Environment

Pyrogallic acid-sodium hydroxide method can be used, again relies on a chemical reaction to generate an anaerobic environment, but a catalyst rather than a reducing agent Anaerobic jars (GasPak System) are sued to incubate plates in an anaerobic atmosphere, useful if brief exposure to oxygen is not lethal
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Anaerobic Culture Methods

Production of a vacuum Displacement of Oxygen with other gases Absorption of Oxygen by chemical or biological methods By using reducing agents
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McIntosh & Fildes Jar

Hydrogen gas is passed in Catalyst helps to combine Hydrogen & O2 Reduced Methylene blue remains colorless if anaerobiosis is achieved
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McIntosh & Fildes anaerobic Jar

Stout glass or metal jar with a lid Lid has an inlet for gas,outlet&2 terminals Alumina pellets coated with palladium (catalyst) - under the lid Inoculated plates kept inside the jar Lid is clamped tight Air is evacuated
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P. aeruginosa

Strict aerobe

Enterococcus Facultative Grows aerobic or anaerobic.

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Bacteriodes fragilis

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Obligate Anaerobes needs Optimal Methods

Obligate anaerobes can be culture in special reducing media such as sodium Thiglyclolate or in anaerobe chambers and handled in anaerobe hoods.
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Displacement of Oxygen
By inert gases like Hydrogen, Nitrogen, Carbon dioxide or Helium Use of lighted candle Use up Oxygen, but some Oxygen is left behind Vacuum decicator Unsatisfactory
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Absorption of O2 by Chemical method

Pyrogallol Chromium and sulphuric acid Gas-pak -available commercially

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By reducing agents
Thiglyclolate broth Robertsons Cooked Meat (RCM) broth contains nutrient broth with pieces of fat-free minced cooked meat of ox heart.
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McIntosh & Fildes anaerobic Jar

Stout glass or metal jar with a lid Lid has an inlet for gas,outlet&2 terminals Alumina pellets coated with palladium (catalyst) - under the lid Inoculated plates kept inside the jar Lid is clamped tight Air is evacuated
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A solid or liquid medium maybe used & must provide an anaerobic environment Anaerobic Culture System

A.

ANAEROBIC JAR
1. Candle Jar
- reduces O2 environment - only CO2 tension

2. Gas Pak Jar a. Palladium aluminum coated pellets - catalyst - chemically reduces O2 - reacts with residual O2 in the presence of H2 to form H2O
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Culture of strict anaerobes

For culture of strict anaerobes all traces of oxygen must be removed from medium and for many organisms sample must be kept entirely anaerobic during manipulations
Methanogenic archaea from rumen and sewage treatment plants killed by even a brief exposure to O2 Medium usually boiled during preparation and reducing agent added, stored under O2-free atmosphere
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Choosing the Optimal Media

Broth and solid media should both be inoculated. The culture media should include anaerobic blood agar plates enriched with substances such as brain-heart infusion, yeast extract, amino acids, and vitamin K; a selective medium such as kanamycinvancomycin (KV) blood agar or laked blood agar; and a broth such as brain heart infusion broth with Thiglyclolate or other reducing agent.

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Media chosen according to our needs

The choice of media depends upon the type of specimen. Some commonly used media include prereduced peptone-yeast extract-glucose broth which is suitable for analysis of volatile products by gas chromatography; egg yolk agar for detection of lecithinase activity of Clostridium spp.; cycloserine-cefoxitin-fructose agar (CCFA) for isolation of Clostridium difficile from stool; and Bacteroides bile esculin agar for isolation of the Bacteroides fragilis group.
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Antibiotic Sensitivity Testing

.The antibiotic susceptibility profile is determined by the micro tube broth dilution method. Many species of anaerobes are resistant to penicillin, and some are resistant to clindamycin and other commonly used antibiotics
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TREATMENT
- Susceptibility testing should be done

- surgical drainage & resection of necrotic tissue - most are resistant to aminoglycosides - for Bacteroides group, if resistant to Penicillin & Cephalosporin, they may use: a. Clindamycin b. Metronidazole c. Chloramphenicol
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