Infections caused by anaerobic bacteria are common, and may be serious and life-threatening. Anaerobes predominant in the bacterial flora of normal human skin and mucous membranes, and are a common cause of bacterial infections of endogenous origin. Infections due to anaerobes can evolve all body systems and sites. The predominate ones include: abdominal, pelvic, respiratory, and skin and soft tissues infections. Because of their fastidious nature, they are difficult to isolate and are often overlooked. Failure to direct therapy against these organisms often leads to clinical failures. Their isolation requires appropriate methods of collection, transportation and cultivation of specimens. Treatment of anaerobic bacterial infection is complicated by the slow growth of these organisms, which makes diagnosis in the laboratory only possible after several days, by their often polymicrobial nature and by the growing resistance of anaerobic bacteria to antimicrobial agents.

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Microbiology and Virulence




MICROBIOLOGY AND VIRULENCE



Anaerobic bacteria do not grow on solid media in the presence of room air (10% carbon dioxide and 18% oxygen), whereas facultative anaerobic bacteria grow both in the presence and in the absence of air. Microaerophilic bacteria grow poorly or not at all aerobically, but grow better under 10% carbon dioxide or anaerobically. Anaerobes can be divided into strict anaerobes that are unable to grow in the presence of more than 0.5% oxygen and moderate anaerobes that are capable of growing at between 2 and 8% oxygen.1 Anaerobes generally do not possess catalase, but some produce superoxide dismutase that can protect them from oxygen.

The clinically important anaerobic bacteria are six genera of Gram-negative rods (Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Bilophila and Sutterella), Gram-positive cocci (primarily Peptostreptococcus), Gram-positive spore-forming (Clostridium) and nonspore-forming bacilli (Actinomyces, Propionibacterium, Eubacterium, Lactobacillus and Bifidobacterium), and Gram-negative cocci (mainly Veillonella) (Table. 1).1-3 The frequency of recovery of anaerobic bacterial strains differs in various infectious sites ( Table. 2). Mixed infections caused by numerous aerobic and anaerobic organisms are observed commonly in clinical situations.2

The taxonomy of anaerobic bacteria has changed in recent years because of their improved characterization using genetic studies.1,3 The ability to differentiate between similar strains enables better characterization of type of infection and predicted antimicrobial susceptibility. The species of anaerobes most frequently isolated from clinical infections are Bacteroides, anaerobic Gram-positive cocci, Clostridium, Fusobacterium, Gram-positive bacilli (Eubacterium, Lactobacillus, Propionibacterium, Actinomyces and Bifidobacterium) and Gram-negative cocci (Veillonella and Acidaminococcus).1 Approximately 95% of the anaerobes isolated from clinical infections are members of these genera. The remaining isolates belong to species not yet described, that usually can be assigned to the appropriate genus on the basis of morphologic characteristics and fermentation products. The frequency of recovery of anaerobic strains differs in various infectious sites.

Gram-positive spore-forming bacilli
Anaerobic spore-forming bacilli belong to the genus Clostridium. Morphologically, the clostridia are highly pleomorphic, ranging from short, thick bacilli to long filamentous forms, and are either ramrod straight or slightly curved. The clostridia found most frequently in clinical infections are Clostridium perfringens , Clostridium septicum, Clostridium ramosum, Clostridium novyi, Clostridium sordellii, Clostridium histolyticum, Clostridium fallax, Clostridium bifermentans and Clostridium innocuum.


Clostridium perfringens:


Clostridium perfringens, the most commonly recovered Clostridia, is an inhabitant of soil and of intestinal contents of humans and animals. It elaborates a number of necrotizing extracellular toxins,4 and is easily isolated and identified. It can be characterized in direct smears of a purulent exudate by the presence of stout Gram-variable rods of varying length, frequently surrounded by a capsule. It can cause a devastating illness with high mortality. Bacteremia is associated with extensive tissue necrosis, hemolytic anemia and renal failure.
Clostridium septicum isolation is associated with malignancy. The intestinal tract is thought to be the source of the organism, and most of the isolates are recovered from blood and subcutaneous tissue.

Clostridium botulinum is associated with food poisoning, and rarely with wound infections. Proteolytic strains of types A and B have been reported from food poisoning and wound infections. Infant botulism occurs with types A, B and F.4 Disease caused by C. botulinum is usually an intoxication produced by ingestion of contaminated food (uncooked meat, poorly processed fish, improperly canned vegetables) containing a highly potent neurotoxin.4 Infection of a wound with C. botulinum occurs rarely and can produce botulism.

Clostridium difficile is the causative agent of antibiotic-associated and spontaneous diarrhea and colitis.3 Clostridium tetani is found in soil and is rarely isolated from human feces. Infections are a result of contamination of wounds with soil containing spores.4 The spores will germinate in devitalized tissue and produce the neurotoxin that is responsible for the clinical findings.


Clostridium difficile


Gram-positive, nonspore-forming bacilli
Anaerobic, Gram-positive, nonspore-forming rods comprise part of the gingival crevices microflora, gastrointestinal tract, vagina and skin. Several distinct genera are recognized: Actinomyces, Arcanobacterium, Atopobium, Bifidobacterium, Eubacterium, Lactobacillus, Mobiluncus, Propionibacterium, and Pseudoramibacter. The most frequanly recovered species are Propionibacterium, Eubacterium, Bifidobacterium, and Lactobacillus.
The Actinomyces, Arcanobacterium, and Bifidobacterium are Gram-positive, pleomorphic, anaerobic to microaerophilic bacilli.


Eubacterium nodatum


Actinomyces israelii, Actinomyces naeslundii and Propionibacterium propionicum are normal inhabitants of the human mouth and throat and are the most frequent cause of actinomycosis. The organisms have been recovered from intracranial abscesses, chronic mastoiditis, aspiration pneumonia and peritonitis.2 The lesions of actinomycosis occur most commonly in the tissues of the face, neck, lungs, pleura and ileocecal regions. Bone, pericardial, anorectal lesions and bacteremia are less common.




Actinomyces israelii






Most Eubacterium and anaerobic lactobacilli are non-pathogenic and are isolated in the oral, vaginal and gastrointestinal flora. They organisms are usually recovered in infections associated with predisposing or underlying conditions that include malignancy, previous surgery, immunodeficiency, diabetes mellitus, the presence of foreign body, dental extraction and broad spectrum antibiotic therapy.5

Propionibacterium spp. ordinarily are not pathogens, but can be associated with implanted cardiac prostheses or central nervous system shunts or as a cause of endocarditis on previously damaged valves. They are recovered from parotid and dental infections, brain abscess, post intracranial surgical, conjunctivitis associated with contact lens, peritonitis, and pulmonary infections. The most common species, Propionibacterium acnes, is isolated from blood cultures but is associated only rarely with bacteremia or endocarditis. Because these organisms are part of the normal skin flora, they are common laboratory contaminants or may grow in blood cultures from skin contamination. P. acnes can cause shunt infections,6 and play a role in the pathogenesis of acne vulgaris.


Propionibacterium acnes 


Gram-negative bacilli
Bacteroides fragilis group are the most frequently recovered species of Bacteroidaceae. B. fragilis group are resistant to penicillins, mostly through the production of beta-lactamase. They include 10 members the most commonly isolated are B. fragilis, Bacteroides thetaiotaomicron, Bacteroides distasonis, Bacteroides ovatus and Bacteroides vulgatus. They are part of the normal gastrointestinal flora2,3 and predominate in intra-abdominal and other infections that originate from the gut flora (i.e. perirectal abscesses, decubitus ulcers).2




Bacteroides fragilis

  


Pigmented Prevotella (Prevotella melaninogenica and Prevotella intermedia) and Porphyromonas spp. (Porphyromonas asaccharolytica), and nonpigmented Prevotella spp. (Prevotella oralis, Prevotella oris) are part of the oral and vaginal flora and predominant in respiratory infections and their complications. These include aspiration pneumonia, lung abscess, chronic otitis media, chronic sinusitis, abscesses around the oral cavity, human bites, paronychia, brain abscesses and osteomyelitis.2 Prevotella bivia and Prevotella disiens are important isolates from obstetric and gynecologic infections.

Fusobacterium species
Fusobacterium spp. are long and thin organisms with tapered ends, and have typical fusiform morphology. The predominate species in infections are Fusobacterium nucleatum, Fusobacterium necrophorum, Fusobacterium mortiferum and Fusobacterium varium. F. nucleatum is associated with oral, pulmonary and intracranial infections.1,2,7 Fusobacterium spp. are frequently isolated from abscesses, obstetric and gynecologic infections, blood and wounds.



 Fusobacterium nucleatum

,



A growing resistance of Gram-negative anaerobic bacilli (GNAB) to penicillins occured in the past decade.8 These include Prevotella, Porphyromonas, and Fusobacterium spp. The main mechanism of resistance is through the production of the enzyme beta-lactamase. Testing for antimicrobial susceptibility and production of beta-lactamase of all Gram-negative anaerobic bacilli can assist in the selection of proper antimicrobials.

The recovery rates of the different AGNB in infected sites are similar to their distributions in the normal flora.2 B. fragilis group were more often isolates in sites proximal to the gastrointestinal tract (abdomen, bile), pigmented Prevotella spp. are prevalent in infections proximal to the oral cavity (bones, sinuses, chest), and P. bivia and P. disiens are often isolates in obstetric and gynecologic infections. This mode of distribution allows for empiric choice of antimicrobials for the therapy of infections in these sites.

Gram-positive cocci
The species most commonly isolated are Peptostreptococcus magnus, Peptostreptococcus asaccharolyticus, Peptostreptococcus anaerobius, Peptostreptococcus prevotii and Peptostreptococcus micros. Additional anaerobic cocci include Coprococcus, Peptococcus, Ruminococcus, sarcina, and Staphylococcus saccharolyticus. They are part of the oral, upper respiratory tract, intestinal, vaginal and skin flora.




Peptostreptococcus spp. 




Cocci are isolated in all types of infections but predominant in respiratory infections (including chronic sinusitis, mastoiditis, acute and chronic otitis media, aspiration pneumonia and lung abscess), and necrotizing, subcutaneous and soft-tissue infections.7 They are generally recovered mixed with other aerobic or anaerobic organisms, but in many cases including  bacteremia, they are the only pathogens recovered.

Microaerophilic gram-positive cocci include Streptococcus anginosus, Streptococcus constellatus, Streptococcus intermedius, and Gemella morbillorum. They are important in chronic sinusitis, brain abscess, and obstetric and gynecologic infections.9

Gram-negative cocci
There are three genera of anaerobic Gram-negative cocci: Veillonella, Acidaminococcus and Megasphaera. There are two species of Veillonella and one each of the other genera. Veillonella is the most frequently isolated genera and is part of the oral, vaginal and small intestine flora.10




Veillonella spp.

 


Virulence factors

Anaerobic infections are often polymicrobial and evolve both aerobic and anaerobic bacteria. Anaerobes contribute to the severity of infection through their synergy with their aerobic counterparts and with each other.11 Generally most infections start with an aerobic bacteria and with time as the tissue conditions become anaerobic anaerobes become predominate. Anaerobes generally take longer than aerobes to become virulent, because some of their major virulence factors (i.e. the production of a capsule) are expressed only in chronic infection.12




The infective process 



Anaerobes possess several virulence factors that assist them to adhere and invade. These include surface structures (such as capsule polysaccharide or lipopolysaccharide), production of superoxide dismutase, catalase, immunoglobulin proteases, coagulation promoters, spreading factors (such as hyaluronidase, collagenase and fibrinolysin), adherence factors , and the production of  toxins.13 Other factors include mucosal damage, oxidation-reduction potential drop and the presence of hemoglobin or blood in an infected site.






Virulence factors of anaerobic bacteria










Electron microscope picture of encapsulated Bacteroides fragilis 


An indirect pathogenic role of AGNB their production of the enzyme beta-lactamase. (Fig. below). Beta-lactamase-producing bacteria (BLPB) can protect not only themselves but also other organisms from the activity of penicillins. This can occur when the enzyme beta-lactamase is secreted into the infected tissue or abscess fluid in sufficient quantities to degrade penicillin.14







Protection of penicillin susceptible bacteria from penicillin by beta-lactamse producing bacteria


In-vitro and in-vivo studies have demonstrated protection of penicillin-susceptible bacteria from penicillin by aerobic and anaerobic BLPB (i.e. protection of group A streptococci by Staphylococcus aureus or Bacteroides spp.).14 The predominant anaerobic BLPB are pigmented Prevotella and Porphyromonas, Bacteroides and Fusobacterium spp.

REFERENCES

1.   Summanen P, Baron EJ, Ciron DM, et al. Wadsworth anaerobic bacteriology manual. 6th ed. Belmont, CA: Star Publishing; 2002.
2.   Finegold SM. Anaerobic bacteria in human disease. Orlando, FL: Academic Press Inc; 1977.
3.  Jousimies H, Summanen P.Recent taxonomic changes and terminology update of clinically significant anaerobic gram-negative bacteria (excluding spirochetes). Clin Infect Dis. 1;35(Suppl 1):S17-21, 2002.     
4.   Hatheway CL. Toxogenic clostridia. Clin Microbiol Rev 1990;3:66–98.
5.  Brook I, Frazier EH : Significant recovery of nonsporulating anaerobic rods from clinical specimens. Clin Infect Dis. 1993 ;16:476-80.
6.   Brook I, Frazier EH. Infections caused by Propionibacterium species. Rev Infect Dis 1991;13:819–22.
7. Brook. Recovery of anaerobic bacteria from clinical specimens in 12 years at two military hospitals.
J Clin Microbiol. 1988 ;26:1181-8.
8.   Rosenblatt JE, Brook I. Clinical relevance of susceptibility testing of anaerobic bacteria. Clin Infect Dis 1993;16(suppl 4):S446–8.
9.   Brook I, Frazier EH. Microaerophilic streptococci as a significant pathogen: a twelve-year review. J Med 1994;25:129–44.
10. Brook I, Frazier EH. Infections caused by Veillonella species. Infect Dis Clin Pract 1992;1:377–381.
11. Brook I. Enhancement of growth of aerobic and facultative bacteria in mixed infections with Bacteroides sp. Infect Immun 1985;50:929–31.
12. Brook I, Myhal LA, Dorsey HC. Encapsulation and pilus formation of Bacteroides sp. J Infect 1991;25:251–7.
13. Hofstad T. Virulence determinants in non-spore-forming anaerobic bacteria. Scand J Infect Dis 1989;(suppl62):15–24.
14. Brook I. The role of beta-lactamase-producing bacteria in the persistence of streptococcal tonsillar infection. Rev Infect Dis 1984;6:601–7.








Table 1:  Predominant recovered anaerobic bacteria



GRAM-POSITIVE COCCI
Peptostreptococcus spp., P. magnus, P. asaccharolyticus, P. prevotii, P. intermedius, P. anaerobius, P. micros
Microaerophilic streptococci (not true anaerobes)

GRAM-POSITIVE NON-SPORE-FORMING RODS
Propionibacterium acnes
Propionibacterium propionicum
Eubacterium lentum
Bifidobacterium eriksonii
Bifidobacterium dentium
Actinomyces species:  A. isrealii, A. naeslundii, A. viscosus, A. odontolyticus, A. meyerii
Arachnia propionica

GRAM-POSITIVE RODS
Clostridium species:  C. perfringens, C. ramosum, C. septicum, C. novyi, C. histolytica, C. sporogenes, C. difficile, C. bifermentans, C. butyricum, C. innocuum, C. sordellii, C. botulinum, C. tetani

GRAM-NEGATIVE RODS
Bacteroides fragilis group:  B. fragilis, B. thetaiotaomicron, B. distasonis, B. vulgatus, B. ovatus, B. uniformis
Other BacteroidesB. gracilis, B. ureolyticus
Pigmented Prevotella spp.:  P. melaninogenica, P. intermedia, P. denticola, P. loescheii, P. corporis, P. nigrescens
Other Prevotella spp.:  P. oris, P. buccae, P. oralis group, (P. oralis, P. buccalis, P. veroralis), P. bivia, P. disiens
Porphyromonas spp:  P. asaccharolytica, P. gingivalis, P. endodontalis
Fusobacterium species:  F. nucleatum, F. necrophorum, F. gonidiaformans, F. naviforme, F. mortiferum, F. varium





Table 2:  Anaerobic bacteria most frequently encountered in clinical specimens





Organism
Infectious site


GRAM-POSITIVE COCCI

Peptostreptococcus sp.
Respiratory tract, intraabdominal and soft tissue infections
Microaerophilic streptococci*
Sinusitis, brain abscesses


GRAM-POSITIVE RODS

Non-spore-forming

Actinomyces sp.
Intracranial abscesses, chronic mastoiditis, aspiration pneumonia, head and neck infections
Propionibacterium acnes
Shunt infections (cardiac, intracranial), infections associated with foreign body
Bifidobacterium sp.
Chronic otitis media, cervical lymphadenitis, abdominal infections


SPORE-FORMING

Clostridium sp.

            C. perfringens
Soft tissue infection, sepsis, food poisoning
            C. septicum
Sepsis, neutropenic enterocolitis
            C. difficile
Colitis, antibiotic-associated diarrheal disease
            C. botulinum
botulism
            C. tetani
Tetanus
            C. ramosum
Soft tissue infections


GRAM-NEGATIVE RODS

B. fragilis group
            (B. fragilis, B. thetaiotamicron)
Intraabdominal and female genital tract infections, sepsis, neonatal infections
Pigmented Prevotella and Porphyromonas
Orofacial infections, aspiration pneumonia, periodontitis
P. oralis
Orofacial infections
P. oris-buccae
Orofacial infections, intraabdominal infections
B. bivius, B. disiens
Female genital tract infections
Fusobacterium sp.

            F. nucleatum
Orofacial and respiratory tract infections, brain abscesses, bacteremia
            F. necrophorum
Aspiration pneumonia, bacteremia
*Not obligate anaerobes.