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Anaerobic Bacteriology

Anaerobic Bacteriology

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Anaerobic Bacteriology
Anaerobic Bacteriology

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ANAEROBIC BACTERIOLOGY

Dr.T.V.Rao MD

Dr.T.V.Rao MD

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What Are Anaerobic Microorganisms
2

• Anaerobic microorganisms are widespread and very important • Do not require oxygen for growth - often extremely toxic
Dr.T.V.Rao MD

The Requirements for Growth: Related to Oxygen
• Oxygen (O2)

Dr.T.V.Rao MD

3 Table 6.1

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
Dr.T.V.Rao MD 4

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
Dr.T.V.Rao MD 5

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
Dr.T.V.Rao MD 6

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

•190

•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
Dr.T.V.Rao MD 8

Strict Anaerobic Bacteria
9

• 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.
Dr.T.V.Rao MD

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
Dr.T.V.Rao MD 10

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).
Dr.T.V.Rao MD 11

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
Dr.T.V.Rao MD 12

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

Dr.T.V.Rao MD

13

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
Dr.T.V.Rao MD 14

ANAEROBES OF CLINICAL IMPORTANCE
• CLOSTRIDIA
– C tetani; C perfringens; C difficile; C botulinum

• BACTEROIDES
– B fragilis; – Prevotella – Porphyromonas

• ACTINOMYCES • FUSOBACTERIUM • ANAEROBIC STREPTOCOCCI
Dr.T.V.Rao MD 15

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
Dr.T.V.Rao MD 16

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 ßlactamases 3. Utilization of O2 by facultative species that aids in producing a suitable environment for growth of anaerobe
Dr.T.V.Rao MD 17

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
Dr.T.V.Rao MD 18

Dr.T.V.Rao MD

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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

(-)

Dr.T.V.Rao MD

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
Dr.T.V.Rao MD

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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)
Dr.T.V.Rao MD 23

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)
Dr.T.V.Rao MD 24

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
Dr.T.V.Rao MD 25

Common Human Anaerobic Infections

Dr.T.V.Rao MD

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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

Dr.T.V.Rao MD

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)
Dr.T.V.Rao MD 28

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)
Dr.T.V.Rao MD 29

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
Dr.T.V.Rao MD 31

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
Dr.T.V.Rao MD 32

Clostridium difficile
• Associated with human disease in mid-1970’s • 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
Dr.T.V.Rao MD 33

• Associated with human disease in mid-1970’s • 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

34

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
Dr.T.V.Rao MD 35

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
Dr.T.V.Rao MD 36

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
Dr.T.V.Rao MD 37

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
Dr.T.V.Rao MD 38

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
Dr.T.V.Rao MD 39

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
Dr.T.V.Rao MD 40

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
Dr.T.V.Rao MD 41

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
Dr.T.V.Rao MD 42

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
Dr.T.V.Rao MD 43

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
Dr.T.V.Rao MD 44

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
Dr.T.V.Rao MD 46

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
Dr.T.V.Rao MD

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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
Dr.T.V.Rao MD

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
Dr.T.V.Rao MD 49

Acceptable Specimens
50

• 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
Dr.T.V.Rao MD

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The accepted specimens for anaerobic processing are as follows:

• Sites • CNS • Dental/ENT
Dr.T.V.Rao MD

• Acceptable specimen

• CSF, abscess, tissue Abscess, aspirates, tissues

52

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

Dr.T.V.Rao MD

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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
• • • •
Dr.T.V.Rao MD

• 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
54

• 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.
Dr.T.V.Rao MD

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.

Dr.T.V.Rao MD

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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.
Dr.T.V.Rao MD 56

Limitation with Culturing the Specimens
57

• 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.
Dr.T.V.Rao MD

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)

Dr.T.V.Rao MD

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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
Dr.T.V.Rao MD 59

60

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
Dr.T.V.Rao MD

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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

Dr.T.V.Rao MD

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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
Dr.T.V.Rao MD 63

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.
Dr.T.V.Rao MD 64

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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
Dr.T.V.Rao MD

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)
Dr.T.V.Rao MD 67

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
Dr.T.V.Rao MD 68

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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
Dr.T.V.Rao MD

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McIntosh & Filde’s Jar
• Hydrogen gas is passed in • •Catalyst helps to combine Hydrogen & O2 • •Reduced Methylene blue remains colorless if anaerobiosis is achieved
Dr.T.V.Rao MD

McIntosh & Filde’s 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
Dr.T.V.Rao MD 71

P. aeruginosa

Strict aerobe

Enterococcus Facultative Grows aerobic or anaerobic.

Dr.T.V.Rao MD

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.
Dr.T.V.Rao MD

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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
Dr.T.V.Rao MD

Absorption of O2 by Chemical method

• Pyrogallol • •Chromium and sulphuric acid • •Gas-pak • -available commercially
Dr.T.V.Rao MD 75

By reducing agents
• Thiglyclolate broth • •Robertson’s Cooked Meat (RCM) broth • contains nutrient broth with pieces of fat-free minced cooked meat of ox heart.
Dr.T.V.Rao MD 76

McIntosh & Filde’s 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
Dr.T.V.Rao MD 77

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
Dr.T.V.Rao MD 78

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
Dr.T.V.Rao MD 79

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.
Dr.T.V.Rao MD 81

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
Dr.T.V.Rao MD 82

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
Dr.T.V.Rao MD 83

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• The programme is Created by Dr.T.V.Rao MD for ‘ e ‘ learning resources to Microbiologists
• Email • doctortvrao@gmail.com

Dr.T.V.Rao MD

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