Anaerobic Bacteria

DEPT. OF MICROBIOLOGY MEDICAL FACULTY, PADJADJARAN UNIVERSITY

Anaerobic bacteria ?
Before 1978 : A group of bacteria which cannot grow in the presence of oxygen Why ? Some anaerobic bacteria can grow on mucosal surfaces Anaerobic bacteria can caused brain infection or caused bacteremia After 1978 : Bacteria which can grow in a negative / low oxidation-reduction (redox) potential (Eh)

Aerobic Bacteria
A. Obligate Aerobic Bacteria

Require molecular oxygen as a terminal electron

acceptor and cannot grow without it. They generate energy oxidatively and are unable to generate sufficient energy for growth by fermentation reactions. Examples include Micrococcus lutea and Nocardia asteroides.

B. Microaerophilic Bacteria

Require oxygen as a terminal electron acceptor for growth but cannot grow on the surface of solid media in an aerobic incubator. They grow minimally, if
at all, under anaerobic conditions.

Anaerobic Bacteria
A. Facultative - can grow either:

Oxidatively - using oxygen as terminal electron

acceptor

Anaerobically - using fermentation

Ex. - Streptococci, Enterobacteriaceae-E. coli

B. Strict (Obligate) Anaerobes Cannot grow in the presence of oxygen

Carry out fermentation where the final hydrogen acceptor is an organic molecule

Some anaerobes produce Superoxide Dismutase

Levels correlate with O2 Sensitivity

CLOSTRIDIAL GROUP

..... 1978 ........NON CLOSTRIDIAL GROUP

caused Tetanus caused Botulism caused Gas - gangrene

Bacteroides spp. Fusobacterium Peptostreptococcus

Normal flora in

Mouth cavity Respiratory tract Intestinal tract Urogenital tract Skin

ANAEROBIC BACTERIA
will grow in a negative / low redox potential (Eh) most of the anaerobic bacteria that cause infections in human are also part of normal flora other pathogenic anaerobes are soil and environmental inhabitans are not considered part of human flora e.g Cl. tetani and Cl. botulinum the majority of anaerobic infections occur when a normal flora of the patient gains access to a sterile site as a result of disruption of some anatomic barrier

Clues to Anaerobic Infection

1. Infections in continuity to mucosal surfaces 2. Infections with tissue necrosis and abscess formation 3. Putrid odor 4. Gas in tissues 5. Polymicrobial flora 6. Failure to grow in the laboratory

Anaerobes Commonly Encountered in Infection

Hundreds of anaerobes are part of normal human flora Only few cause significant infections Account for 2/3 of clinically significant anaerobic infections:

Bacteroides fragilis group Prevotella and Porphyromonas Fusobacterium nucleatum

Peptostreptococcus

Clostridium perfringens

Pathogenic species have virulence factors that allow survival outside their niche and to cause disease

PATHOGENESIS
The type of infections and diseases are caused by anaerobic bacteria span a wide spectrum : Produce some of the most potent toxins e.g Cl. tetani and Cl. botulinum Specific virulence factors e.g B. fragilis group, Cl. difficile Most anaerobic infections involved a mixture of anaerobic and facultatively anaerobic organisms

Virulence Factors
1. Anti-phagocytic capsule

Also promote abscess formation

2. Tissue destructive enzymes

B. fragilis produces variety of enzymes (lipases,
proteases, collagenases) that destroy tissue Abscess Formation

3. Beta-lactamase production

B. fragilis protect themselves and other species in

mixed infections

4. Superoxide dismutase production

Protects bacteria from toxic O2 radicals as they move out of usual niche

Characteristics of Anaerobic Infections

1. Most pathogenic anaerobes usually commensals
1. Originate from our own flora

are

2. Predisposing Conditions
1. Breeches in the mucocutaneous barrier displace normal flora 2. Compromised vascular supply Decrease 3. Trauma with tissue destruction Redox 4. Antecedent infection Potential

Characteristics of Anaerobic Infections

3. Complex Flora Multiple species
Abdominal

Infection Average of 5 species 3 anaerobic 2 aerobic Less complex then nl flora Fecal flora 400 different species Those predominant in stool are not infecting species Veillonella, Bifidobacterium rarely pathogenic Species uniquely suited to cause infection predominate

Synergistic Mixture of Aerobes & Anaerobes

E. coli Consume O2
Allow

growth of anaerobes Anaerobes promote growth of other bacteria by being antiphagocytic and producing B-lactamases

SPECIMENS COLLECTION AND TRANSPORT

The proper collection and transport of specimens for anaerobic culture cannot be overemphasized Material for anaerobic culture is best obtained by tissue biopsy or by aspiration using a needle and syringe Use of swabs is a poor alternative If a swab must be used, it should be from an oxygen free transport system

SPECIAL COLLECTION INSTRUCTIONS FOR SOME CLOSTRIDIAL ILLNESSES

Foodborne :

C. perfringens and C. botulinum, C. difficile, and C. septicum

Food and fecal specimens : C. perfringens food poisoning

Serum, feces, vomitus, or gastric contents : Botulism

Serum and stool : Infant botulism

Serum, stool and tissue biopsy : Wound botulism Feces : for C. difficile culture Liquid or unformed solid stool : for toxin assay Formed stool or rectal swabs : to detect carriers Stool should be placed in anaerobic transport container

- A crucial factor in the final success of anaerobic cultures is the transport of the specimen, the lethal effect of atmospheric oxygen must be prevented until the specimen can be processed in the laboratory
- Recapping a syringe and transporting the needle and syringe to the laboratory is no longer acceptable because of safety concerns involving needle stick injuries

Therefore, even aspirates must be injected into some of type of oxygen free transport tube or vial.

THREE DIFFERENT KINDS OF ANAEROBIC TRANSPORT SYSTEM

rubber stoppered collection vial an oxygen free atmosphere an anaerobic pouch (Gas-Pak Pouch)

SPECIMENS PROCESSING
may be processed on the open bench-top with incubation in anaerobic jars or pouches or in an anaerobic chamber

Anaerobic conditions can be setup by two different methods : The easiest method uses a commercially available hydrogen or CO2 generator envelope or the evacuation replacement method can be used

PATHOGENESIS AND SPECTRUM OF DISEASES

Organisms
C. perfringens

Virulence factor
several exotoxin, alfa toxin is important (11 other toxins) enterotoxin tetanospasmin, neurotoxic (disrupts nerve impulses to muscles neurotoxins Toxin A (enterotoxin) Toxin B (cytotoxin)

Spectrum of disease and infections

Gas-gangrene, destruction of muscle and other tissues Food poisoning, abdominal cramps, diarrhea, vomiting Tetanus (lockjaw), muscle spasms, respiratory failure Botulism, paralysis of respiratory and other essential muscle groups Wound botulism Antibiotic Associated Diarrhea (AAD), diarrhea Pseudomembranous Colitis (PMC) Pseudomembran Associated with inflammatory process in acne Most commonly encountered in anaerobic infections, often mixed with other anaerobic or facultatively anaerobic organisms Most often found mixed with other anaerobic or facultatively anaerobic organisms Mixed infection

C. tetani C. botulinum C. difficile

Propionibacterium spp. Bacteroides fragilis

groups

No definitive virulence factors known capsules, endotoxin, succinic acid, various enzymes No definitive virulence factors known No definitive virulence factors known

Fusobacterium spp. Peptostreptococcus Fusobacterium spp. Veillonella spp.

CLOSTRIDIUM TETANI
)

General characteristics : is a Gram positive, spore forming anaerobe possesses a terminal spore, resulting in a characteristic tennis racquet or drum stick morphologic appearance spores are ubiquitous in soil

Pathogenicity Toxigenicity is mediated by a large plasmid The organism secretes an exotoxin tetanospasmin

Clinical disease :
- Infection follows minor trauma (laceration or puncture) or occurs as umbilical cord stump infection in a neonate (Tetanus neonatorum) - Manifestations include muscle stiffness, tetanospasms of lockjaw and back arching, and short, frequent spasms of voluntary muscles - Death occurs after several weeks from exhaustion and respiratory failure - Intrauterine infection : Septic abortion, Post abortal tetanus, Tetanus post partum

Treatment : o The patient should be hospitalized and treatment begun without waiting for definitive diagnosis o Antitoxins are effective only if toxins have not yet bound to tissues o Antitoxin and penicillin should be given, tissue should be debrided, a quiet and dark o environment should be provided to minimize external stimuli that can induce spasms

Prevention :
- DTP vaccine A booster should be given every 10 years, for major trauma a booster should be given if the patient has not had one within the last 5 years - Booster should be given to pregnant women to stimulate maternal antibodies That will protect the newborn

CLOSTRIDIUM BOTULINUM
General characteristics :

is a Gram positive, spore forming anaerobic rod requires a low redox potential within tissues spores are ubiquitous in soil and are highly resistant to environmental conditions

Pathogenicity :

produces a potent exotoxin that act at the myoneural junction to produce flaccid muscle paralysis due to suppression of acethylcholine release from the axon terminals of peripheral nerves

Clinical manifestations :
1. Food poisoning

Clinical findings include : nausea, vomiting, dizziness, cranial palsy, double vision, swallowing and speech problems, muscle weakness, respiratory paralysis, and death (in 20 % of cases)

2. Intestinal (infant) botulism - The exotoxin disseminates, causing constipation, generalized weakness, and loss of head and limb control (resulting in a floppy appearance) - This type of infection rarely is fatal 3. Wound infection, manifestations similar to those of soft tissue wound

Laboratory Diagnosis : The presence of the toxin in food, stool, blood and vomitus Treatment : - for food poisoning : give antitoxin and supportive measures for respiratory control, stomach lavage and enemas Antibiotics should not be given and the caregiver should act rapidly - for intestinal botulism in infant only supportive care is needed

Prevention :
give antitoxin to all person who are contaminated food, even if symptoms have not developed heat food to 80o C - 100o C for 10 minutes to inactivate the toxin (but not the spores) use proper sterilization techniques for home canning

CLOSTRIDIUM PERFRINGENS (Clostridium welchii )

General characteristics :
- is an anaerobic, spore forming Gram positive rod - spores can be central or subterminal and are relatively heat resistant - produces 12 exotoxins causing food poisoning - has soil as a natural habitat - contamination can occur in home canned goods, smoked fish, and honey

Pathogenicity - produces alpha toxin - produces 11 other toxins or enzymes that damage eukaryotic cells - Produces an enterotoxin associated with food poisoning

Clinical manifestation :
C. perfringens causes two types of infection :
1. Soft tissue (muscle) wound infection, with two consequences : a. Anaerobic cellulitis, causing destruction of traumatized tissue only b. Myonecrosis (gas gangrene) Other clostridia may be associated with gas gangrene are : C. septicum, C. novyi, C. histolyticum, C. sordellii 2. Food poisoning contaminated food following ingestion of

Treatment
- For anaerobic cellulitis

Penicillin and additional antibiotics are given to prevent secondary bacterial infections, Necrotic tissue should be debrided - For myonecrosis Penicillin and antitoxin are given Necrotic tissue is debrided. Surgery is likely - For food poisoning Treatment usually is not necessary because the infection is self limiting

CLOSTRIDIUM DIFFICILE
General characteristics : - is a Gram positive, spore forming, anaerobic rod - the normal intestinal flora of infants and some adults

Pathogenicity : - has many strains that are resistant to antibiotics - antibiotic treatment kills organisms normally restricting growth of C. difficile resulting in overgrowth of the latter produce two toxins : an enterotoxin and a cytotoxin

Clinical manifestations :
Severe gastroenteritis Pseudomembranous colitis Antibiotic assciated colitis

Treatment :
is with vancomycin or metronidazole Fluid and electrolyte status should be monitored

NON CLOSTRIDIAL GROUP - Endogenous - Chronic - Secondary infection - Non severe toxemia

- Normal flora
- Mixed infection

BACTEROIDES GROUP
B. fragilis s.s B. fragilis s.s B. thetaiotaiomicron s.s B. ovatus s.s B. vulgatus

Prevotella (Bacteroides) melaninogenicus s.s melaninogenicus intermedius sacharolyticus B. corrodens B. oralis B. capillosum

BACTEROIDES FRAGILIS
General characteristics :
- is a Gram negative, anaerobic rod, usually pleomorphic - grows rapidly under anaerobic conditions and is stimulated by bile - contains level of superoxide dismutase & catalase - is found in the female genital tract but rarely in the oral cavity - usually causes polymicrobic infections - possess a beta-lactamase that destroys penicillins and cephalosporins

Pathogenicity :
possesses a capsules that inhibits phagocytosis - has a weak endotoxin and no exotoxin - possesses a collagenase and hyaluronidase, which aids its spread

Clinical manifestation
- Gastrointestinal abscesses after damage to mucosal barriers - Pelvic inflammatory disease - Brain abscesses and cellulitis - are not communicable or transmissible

Treatment : Tetracyclines is generally ineffective Metronidazole, clindamycin, kanamycin and chloramphenicol are also suggested as therapeutic agents

PREVOTELLA MELANINOGENICUS
(formerly Bacteroides melaninogenicus)

General characteristics
- a small Gram negative coccobacillus, occasional long forms - has a distinctive black colonial appearance on agar - the normal flora of the mouth - low numbers in the gastrointestinal and genitoyrinary tracts

Pathogenicity
possesses collagenase a potent endotoxin and a

Clinical manifestation
- causes lung abscesses - causes infections of the female genital tract Treatment

- Metronidazole and Clindamycin - Carbapenems are the most potent betalactam

FUSOBACTERIUM SPP.
General characteristics : - Gram negative, anaerobic, non sporeforming, non motile, long filamentous rod - produce copious amounts of butytic acid and lower levels of acetic and propionic acids - as commensals of the upper respiratory and gastrointestinal tracts

Manifestation of infections

Infections of the oral cavity (F. nucleatum, F. periodonticum, F. naviforme, F. simiae) Mixed infection of the head and neck region (F. nucleatum), including : dental abscesses infection of the central nervous system Intra abdominal infections, perirectal abscesses, osteomyelitis, decubituts (F. mortiferum, F. necrogene, F. varium, together with F. nucleatum)

Treatment

susceptible to metronidazole and Chloramphenicol

Anaerobic Gram Negative Cocci

Veillonella

V. parvula V. atypica V. dispar

V. parvula

normal flora:

Acidaminococcu s fermenatans Megasphaera alspenii Pathogenic

role-??

Mouth Upper airways GI tract Vagina

Part of mixed anaerobic infections

GRAM COCCI

NEGATIVE

ANAEROBIC

General characteristics :

- as part of the normal flora of the gastrointestinal tract - mixed anaerobic infections
Species :

Veillonella alcalescens purvilla Aidominococcus spp.

GRAM POSITIVE ANAEROB COCCI

General characteristics : the normal microbial flora of the mouth, gastrointestinal tract, genitourinary tract and skin - Anaerobic cocci are isolated from infections in various parts of the body, particularly from abscesses (e.g : Dental abcesses, Periodontal diseases, Abdominal infection, Genitourinary tract infections, Decubitus ulcer, Osteomielytis) Species :

Peptococcus spp., Peptostreptococcus spp.

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