Virulence of Anaerobic Bacteria and the Role of Capsule

PATHOGENICITY OF ANAEROBIC BACTERIA

Most anaerobic infections are pyogenic and arise from the normal flora of the skin, oropharynx, the large intestine, or the female genital tract. Such infections typically involve multiple species of bacteria, some strict anaerobes, some strict aerobes and others that are facultative anaerobes (i.e., able to grow aerobically or anaerobically). The polymicrobial nature of infections involving anaerobic bacteria is apparent in infections of the respiratory tract, abdomen, pelvis, and soft tissue, where the number of isolates in an infectious site varies between two and five (1-3). The contributing role of anaerobes in these infections has been often questioned (4).

In the past, it was thought that treating the aerobic component of the infectious flora to cure the infection was sufficient (4). This simplistic attitude was based on the assumption that anaerobes are dependent on the aerobic and facultative components of the infection to lower the Po2 of their environment (5) and to provide them with essential metabolic by-products (6). Therefore, elimination of the aerobic and facultative flora would deprive the anaerobes of that support, and hence, they would be eliminated by the host defenses. However, substantial clinical and laboratory data exist that disproves this hypothesis and demonstrates the importance of anaerobes as pathogens in single or polymicrobial infections.

Some of the uncertainty regarding the role of anaerobes was clarified following several important observations: anaerobes often may be present in infection in pure culture as the only isolate or as part of a polymicrobial infection involving only anaerobic bacteria. They have also been recovered as the sole isolate in bacteremias (7).

The factors that determine the outcome of an anaerobic infection are the balance between the bacterial and host factors. The bacterial factors include the inoculum size, the virulence, and synergistic potential of the infecting organisms, while the opposing host factors include the host defense, breaks in the anatomic barriers, and reduction in the oxidation-reduction potential.

The major virulence factors of anaerobes are: their ability to adhere and invade epithelial surfaces; the production of toxins, enzymes, or other pathogenic factors; the production of superoxide dismutase and catalase, immunoglobulin proteases; and coagulation promoting and spreading factors (such as hyaluronidase, collagenase, and fibrinolysin), and with the presence of surface constituents such as capsular polysaccharide or lipopolysaccharide. Adherence of bacterial to epithelial cells is the first essential step of colonization or infection. Bacteroides fragilis adherence is mitigated through a pili-like structure, their capsule, and lectin-like adhesions. Prevotella melaninogenica attaches to certain gram-positive organisms, with cervicular epithelium. Fusobacterium nucleatum also attach to that epithelium. Porphyromonas gingivalis possesses fimbria that assists bacterial attachment.

The immune system is active in protection against anaerobic infection. Anaerobes activate complement directly, thus attracting polymorphonuclear leukocytes. Anaerobes are susceptible to killing by macrophages and are killed by oxidative and monoxidative mechanisms intracellularly. Both humoral and cell-mediated immune mechanisms actively protect the host from anaerobes. These include circulating antibodies and complement that have been shown to protect from experimental bacteremia, and T-lymphocytes that resist abscess formation (8).

Anaerobes can adversely affect the cellular and humoral immunity. Some can deplete or bind opsonins that bind to aerobes, thus preventing their oposonization (9); they can suppress the activity of polymorphonuclear leukocytes, macrophages, and lymphocytes (8); and neutrophils killing ability can be inhibited by short chain fatty acids produced by B. fragilis and other anaerobic gram-negative bacilli (AGNB) (10). B. fragilis can also interact with peritoneal macrophages inducing procoagulant activity and fibrin deposition that impairs clearance of the infecting organisms (11).

The ability of several anaerobes to possess a capsule was found to be an important virulence factor.

Factors that enhance the virulence of anaerobes include mucosal damage, oxidation-reduction potential drop, and the presence of hemoglobin or blood in an infected site. However, this chapter will be devoted only to the role of capsule as a virulence factor..

Clinical and animal studies showed bacterial synergy between anaerobic and aerobic or other anaerobic bacteria (12,13). Data derived from therapy of mixed infection also provided support for the importance of anaerobic bacteria. Polymicrobial infection involving aerobic and anaerobic bacteria responded to therapy directed at the eradication of only the anaerobic component of the infection with either metronidazole or clindamycin (14). However, for complete eradication of the infection, animal and patient studies have demonstrated that unless therapy is directed against both aerobic and anaerobic bacteria, the untreated organisms will survive (15-18). Bartlett et al. (15) demonstrated in an intra-abdominal abscess model in rats that combined therapy of clindamycin and gentamicin was needed to prevent mortality caused by Escherichia coli sepsis and abscesses caused by B. fragilis. Thadepalli et al. (16) showed that in patients with intra-abdominal trauma, clindamycin and kanamycin were superior to cephalothin and kanamycin in preventing septic complications. This principle of double coverage against aerobes and anaerobes has since then been proven to be the golden standard of therapy in numerous studies (17,18) using combination therapy (clindamycin, metronida-zole, or cefoxitin plus an aminoglycoside) and single agent therapy with agents effective against both aerobes and anaerobes such as cefoxitin (19) or imipenem (20). A similar approach was found essential in the management of pelvic inflammatory disease in adults (21), and chronic otitis media (22) and chronic sinusitis (23) in children, where mixed aerobic-anaerobic flora were recovered from the majority of cases.

SYNERGY BETWEEN ANAEROBIC AND AEROBIC OR FACULTATIVE BACTERIA

Polymicrobial infections are known to be more pathogenic for experimental animals than those involving single organisms (5).

Several studies documented the synergistic effect of mixtures of aerobic and anaerobic bacteria in experimental infection. Altemeier (13) demonstrated the pathogenicity of bacterial isolates recovered from peritoneal cultures after appendiceal rupture. Pure cultures of individual isolates were relatively innocuous when implanted subcutaneously in animals, but combinations of facultative and anaerobic strains manifested increased virulence. Similar observations were reported by Meleney et al. (24) and Hite et al. (25).

Brook et al. (26) evaluated the synergistic potentials between aerobic and anaerobic bacteria commonly recovered in clinical infections. Each bacterium was inoculated subcutaneously alone or mixed with another organism into mice, and synergistic effects were determined by observing abscess formation and animal mortality. The tested bacteria included encapsulated Bacteroides spp., Prevotella, Fusobacterium spp., Clostridium spp., and anaerobic cocci. Facultative and anaerobic bacteria included Staphylococcus aureus, Pseudomonas aeruginosa, E. coli, Klebsiella pneumoniae, and Proteus mirabilis. In many combinations, the anaerobes significantly enhanced the virulence of each of the five aerobes. The most virulent combinations were between P. aeruginosa or S. aureus and anaerobic cocci or AGNB.

Enhancement of growth of aerobic and facultative bacteria was also apparent when they were co-inoculated into mice and a subcutaneous abscess was formed. Streptococcus pyogenes, E. coli, S. aureus, K. pneumoniae, and P. aeruginosa were enhanced by B. fragilis, P. melaninogenica (27,28) Peptostreptococcus spp. (29,30), Fusobacterium spp. (31,32), and Clostridium spp. (33), except Clostridium difficile. Although mutual enhancement of growth of both aerobic and anaerobic bacteria was noticed, the number of aerobic and facultative bacteria was increased many folds more than their anaerobic counterparts. Exceptions to the mutual enhancement were noticed in combinations between organisms that are generally not recovered together in mixed infections, such as Enterococcus faecalis and P. melaninogenica (28). The above observations suggest that the aerobic and facultative bacterial benefit even more than do the anaerobes from their symbiosis.

The demonstration of the synergistic potentials of anaerobic bacteria commonly recovered in polymicrobial infections provide further support for their pathogenic role in these infections. Several hypotheses have been proposed to explain microbial synergy in mixed infections (30). When this phenomenon occurs in mixtures of aerobic and anaerobic flora, it may be due to protection from phagocytosis and intracellular killing (11,30), production of essential growth factors (6), and lowering of oxidation-reduction potentials in host tissues (5). Obligate anaerobes can interfere with the phagocytosis and killing of aerobic bacteria (35). The ability of human polymorphonuclear leukocytes to phagocytose and kill P. mirabilis was impaired in vitro when the human serum used to opsonize the target bacterium was pretreated with live or dead organisms of various AGNB (34). Porphyromonas gingivalis cells or supernatant culture fluid was shown to possess the greatest inhibitory effect among the AGNB (35). Supernatants of cultures of B. fragilis group, pigmented Prevotella and Porphyromonas, and P. gingivalis were capable of inhibiting the chemotaxis of leukocytes to the chemotactic factors of P. mirabilis (36).

Bacteria may also provide nutrients for each other. Klebsiella spp. produces succinate, which supports P. assacharolytica (37), and oral diphtheroids produced vitamin K which is a growth factor for P. melaninogenica (38).

Another possible mechanism that explains the synergistic effect of aerobic-anaerobic combinations is the lowering of local oxygen concentrations and the oxidation-reduction potential by the aerobic bacteria. The resultant physical conditions are appropriate for replication and invasion by the anaerobic component of the infection. Such environmental factors are known to be critical for anaerobic growth in vitro and may apply with equal relevance to in vivo experimental animal studies. Mergenhagen et al. noted that the infecting dose of anaerobic cocci was significantly lowered when the inoculum was supplemented with chemical reducing agents (5). A similar effect may be produced by facultative bacteria, which may provide the proper conditions for establishing an anaerobic infection at a previously well-oxygenated site.

CAPSULE FORMATION IN EXPERIMENTAL MIXED INFECTIONS

An important virulence factor of Bacteroides spp. is the possession of a capsule. Several studies demonstrated the pathogenicity of encapsulated anaerobes and their ability to induce abscesses when injected alone in animals. Onderdonk et al. (39) correlated the virulence of B. fragilis strains with the presence of capsule, and Simon et al. (40) described decreased phagocytosis of the encapsulated B. fragilis. Capsular material from P. melaninogenica also inhibits phagocytosis and phagocytic killing of other microorganisms in an in vitro system (41). Tofte et al. (42), Jones and Gemmel (34), and Ingham et al. (32) have shown that both phagocytic uptake and killing of facultative species were impaired by encapsulated Bacteroides spp.

The presence of capsule in B. fragilis was shown to provide the organism with growth advantage in vivo over unencapsulated isolates (43). Furthermore, encapsulated strains survived better in vitro than unencapsulated variants when they were grown in an aerobic environment. Thus, the presence of a capsule apparently enables a strain of Bacteroides to resist exposure to oxygen as well as host defenses. Another mechanism of protection is the inhibition of polymorphonuclear migration caused by the production of succinic acid by Bacteroides spp. (11).

The ability of the aerobic component in mixed infections to enhance the appearance of encapsulated anaerobic bacteria in these infections was studied in an abscess model in mice. The anaerobic bacteria with which they were inoculated were those commonly recovered in mixed infections.

Pigmented Prevotella and Porphyromonas spp. (44), Prevotella bivia (45), B. fragilis group (46), and anaerobic and facultative gram-positive cocci (AFGPC) (47) did not induce abscess when isolates that contained only a small number of encapsulated organisms (< 1%) were inoculated. However, when these relatively nonencapsulated isolates were inoculated, mixed with abscess-forming viable or nonviable bacteria ("helpers"), the Bacteroides, Prevotella, Porphyromonas, and AFGPC survived in the abscess and became heavily encapsulated (> 50% of organisms had a capsule). Thereafter, these heavily encapsulated anaerobic isolates were able to induce abscesses when injected alone (Fig. 1). Of interest is the observed appearance of pili along with encapsulation in the B. fragilis group after co-inoculation with K. pneumoniae (46).

Most of the "helper" strains were encapsulated; although several of the strains were not encapsulated, and they were able to induce abscesses when inoculated alone. The "helper" organisms used in conjunction with pigmented Prevotella and Porphyromonas, and AFGPC were S. aureus, S. pyogenes, Haemophilus influenzae, P. aeruginosa, E. coli, K. pneumoniae, and AGNB (44,47). For the B. fragilis group, these organisms were E. coli, K. pneumoniae, S. aureus, S. pyogenes, and Enterococcus spp. (46). Neisseria gonorrhoeae was chosen as a helper for B. fragilis, and Prevotella and Porphyromonas spp. (45). Of interest is the observed inability of N. gonorrhoeae strains to survive in intra-abdominal abscesses and also their disappearance from abscesses within five days of inoculation with AGNB and P. bivia (45).

The virulence of Fusobacterium spp. was also associated with the presence of a capsule. Only encapsulated strains of F. nucleatum, Fusobacterium necrophorum, and Fusobacterium varium were able to induce abscesses when inoculated alone (31). However, following passage in animals of nonencapsulated strains, none of these organisms acquired a capsule.

The presence of a thick granular cell wall (300-360 A) before animal passage was associated with virulence of Clostridium spp. (33). Such structure was observed before inoculation into animals, only in Clostridium perfringens, and Clostridium butyricum, the only organisms capable of inducing an abscess when inoculated alone. This structure was observed in other Clostridium species only after their co-inoculation with encapsulated AGNB or K. pneumoniae.

However, other undetermined factors may also contribute to the induction of an abscess, since most isolates of C. difficile were not able to produce an abscess even though they possessed a thick wall.

The selection of encapsulated AGNB and AFGPC with the assistance of other encapsulated or nonencapsulated but abscess-forming aerobic or anaerobic organisms may explain the conversion into pathogens of non-pathogenic organisms that are part of the normal

Bacteroides fragilis group

Bacteroides fragilis group

Bacterial Encapsulation
FIGURE 1 Encapsulation cycle of B. fragilis group after passage in mice. "Helper" is viable bacteria or formalized bacteria or capsular material.

host flora or are concomitant pathogens. Although such a phenomenon was not observed in Fusobacterium spp., the presence of a capsule in these organisms was a prerequisite for induction of abscesses. Some Clostridium spp. also manifested cell wall changes after animal passage that could be associated with increased virulence. Although the exact nature and chemical composition of the capsule or external cell wall may be different in each of the anaerobic species studied, the changes that were observed tended to follow similar patterns.

The mechanism that is responsible for the observed phenomenon is yet unknown, and may be due to either genetic transformation or a process of selection.

ROLE OF A CAPSULE OF BACTEROIDES SPP. AND ANAEROBIC COCCI IN BACTEREMIA

Anaerobic bacteremia account for 5% to 15% of cases of bacteremia (1,4), and are especially prevalent in polymicrobial bacteremia, associated with abscesses (7).

The role of possession of capsular material in the systemic spread of AGNB and AFGPC was investigated in mice following subcutaneous inoculation of encapsulated strains alone or in combination with aerobic or anaerobic facultative bacteria (48). Encapsulated anaerobes were isolated more frequently from infected animal blood, spleen, liver, and kidney than were nonencapsulated organisms.

After inoculation with a single encapsulated anaerobic strain, encapsulated organisms were recovered in 163 of 420 (39%) animals, whereas nonencapsulated anaerobes were recovered in only 14 of 420 (3%) animals. Following inoculation of B. fragilis mixed with aerobic or facultative flora, encapsulated B. fragilis was isolated more often and for longer periods of time than was the nonencapsulated strain. Furthermore, encapsulated B. fragilis was recovered more often after inoculation with other flora than it was when inoculated alone.

Therefore, encapsulated strains were found to be more virulent than their nonencapsulated strains. These data highlight the importance of encapsulated AGNB and AFGPC in increasing the mortality associated with bacteremia and the spread to different organs. A similar pathogenic quality was observed in other bacterial species, such as Streptococcus pneumoniae (49) and H. influenzae (50), where the encapsulated strains showed greater ability for systemic spread.

SIGNIFICANCE OF ANAEROBIC BACTERIA IN MIXED INFECTION WITH OTHER FLORA

Although anaerobic bacteria often are recovered mixed with other aerobic and facultative flora, their exact role in these infections and their relative contribution to the pathogenic process are unknown. The relative importance of the organisms present in the abscess caused by two bacteria (an aerobe and an anaerobe) and the effect of encapsulation on the relationship were determined by comparing the abscess sizes in (i) mice treated with antibiotics directed against one or both organisms and (ii) nontreated animals (27,31,33,34,47).

As judged by selective antimicrobial therapy, the possession of a capsule in most mixed infections involving AGNB generally made these organisms more important than their aerobic counterparts. In almost all instances, the aerobic counterparts in the infection were more important than nonencapsulated AGNB (27). Encapsulated members of the pigmented Prevotella and Porphyromonas were almost always more important in mixed infections than their aerobic counterparts (S. pyogenes, S. pneumoniae, K. pneumoniae, H. influenzae, and S. aureus). Encapsulated B. fragilis group organisms were found to be more important than or as important as E. coli and enterococci and less important than S. aureus, S. pyogenes, and K. pneumoniae.

In contrast to AGNB, encapsulated AFGPC were found more often to be less important than their aerobic counterparts (47). Clostridium spp. and Fusobacterium spp. were found to be less or equally important to enteric gram-negative rods (31-33). Although Fusobacterium spp., AFGPC, and Clostridium spp. were generally equal to or less important than their aerobic counterpart, variations in the relationship existed. However, as determined by the abscess size, most of the anaerobic organisms enhanced mixed infection.

ENCAPSULATED ANAEROBIC BACTERIA IN CLINICAL INFECTIONS

In an attempt to define the important pathogens among the isolates recovered from clinical specimens, Brook et al. studied the virulence and importance of encapsulated bacterial isolates recovered from 13 clinical abscesses (51). This was done by injecting each of the 35 isolates (30 anaerobes and 5 aerobes) subcutaneously into mice alone or in all possible combinations with the other isolates recovered from the same abscess. The ability of each isolate to induce and/or survive in a subcutaneous abscess was determined. Sixteen of the isolates were encapsulated; 15 of them were able to cause abscesses by themselves and were recovered from the abscesses even when inoculated alone. The other organisms, which were not encapsulated, were not able to induce abscesses when inoculated alone. However, some were able to survive when injected with encapsulated strains. Therefore, the possession of a capsule by an organism was associated with increased virulence, compared with the same organism's nonencapsulated counterparts, and might have allowed some of the other accompanying organisms to survive. We found this phenomenon to occur in AGNB, Prevotella spp. anaerobic gram-positive cocci, Clostridium spp., and E. coli. Detection of a capsule in a clinical isolate may therefore suggest a pathogenic role of the organism in the infection.

Three studies support the importance of encapsulated anaerobic organisms in respiratory infections (52-54). The presence of encapsulated and abscess-forming organisms that belong to the pigmented Prevotella and Porphyromonas spp. (previously called B. melaninogenicus group) was investigated in 25 children with acute tonsillitis and in 23 children without tonsillar inflammation (control) (52). Encapsulated pigmented Prevotella and Porphyromonas were found in 23 of 25 children with acute tonsillitis, compared with 5 of 23 controls (p < 0.001). Subcutaneous inoculation into mice of the Prevotella and Porphyromonas strains that had been isolated from patients with tonsillitis produced abscesses in 17 of 25 instances, compared with 9 of 23 controls (p < 0.05). These findings suggest a possible pathogenic role for pigmented Prevotella and Porphyromonas spp. in acute tonsillar infection, and also suggest the importance of encapsulation in the pathogenesis of the infection.

In another study (53), the presence of encapsulated AGNB (Prevotella and Porphyromonas spp., and fragilis group) and anaerobic gram-positive cocci was investigated in 182 patients with chronic orofacial infections and in the pharynx of 26 individuals without inflammation (Table 1). Forty-nine of the patients had chronic otitis media, 45 had cervical

TABLE 1 Encapsulated Anaerobic Bacteria in Children with Abscesses and Chronic Inflammation Compared with Controls (Number of Strains Isolated)

Clinical diagnosis (number of samples)

Pigmented Prevotella and Porphyromonas

Prevotella oralis

Bacteroides fragilis group

Peptostrepto-coccus spp.

Total

Chronic otitis media (n=

15/19

4/6

7/10

19/25

45/60 (75%)a

48)

Chronic mastoiditis

9/11

2/2

3/3

11/15

25/31 (81%)a

(n=24)

Chronic sinusitis

10/14

3/5

16/20

29/39 (74%)a

("=37)

Peritonsillar abscess (n=

21/23

3/5

16/22

40/50 (80%)a

16)

Periapical abscess (n=

8/9

3/3

10/12

21/24 (87%)a

12)

10/12 (83%)b

Cervical lymphadenitis

4/4

6/8

(n=45)

Total number in all

67/80 (84%)a

15/21(71%)°

10/13 (77%)

78/102 (76%)c

170/216 (79%)a

infected sitesd

(n =182)

Pharyngeal culture (n=

8/35 (25%)

4/13 (31%)

22/48 (46%)

34/96 (35%)

26) (control)

°p<0.05, respectively, when compared to control. dEncapsulated/total (%). Source: From Ref. 53.

lymphadenitis, 37 had chronic sinusitis, 24 had chronic mastoiditis, 10 had peritonsillar abscesses, and 12 had periodontal abscesses. One hundred seventy of the 216 (79%) isolates of Prevotella and Porphyromonas, B. fragilis group, and anaerobic cocci were found to be encapsulated in patients with chronic infections, compared to only 34 of 96 (35%) controls

The presence of encapsulated and piliated AGNB (mostly B. fragilis group and pigmented Prevotella and Porphyromonas) was investigated in isolates from blood, abscesses and normal flora (54). Of the strains of AGNB isolated, 45 of 54 (83%) recovered from blood and 31 of 40 (78%) found in abscesses were encapsulated. In contrast, only 7 of 71 (10%) similar strains isolated from the faeces or pharynx of healthy persons were encapsulated {p <0.001). Pili were observed in 3 of 54 (6%) of strains isolated from blood, 30 of 40 (75%) of those recovered from abscesses (p <0.001), and 49 of 71 (69%) of those found in normal flora (p <0.001) (Fig. 2 shows only B fragilis group). The predominance of encapsulated forms in all strains of AGNB in blood as well as in abscesses suggests an increased virulence of these compared with nonencapsulated isolates. In contrast, the presence of pili in AGNB recovered mostly from abscesses and normal flora suggests that this structure may play a role in the ability of these organisms to adhere to mucous membranes and may interfere with their ability to spread systematically. These findings illustrate the morphologic differences that may be observed in AGNB from various anatomic sites.

The predominance of encapsulated Bacteroides, Prevotella, and Porphyromonas spp. recovered from blood and abscesses compared with their rate of encapsulation in the normal flora of the pharynx and faeces suggests an increased virulence of these strains as compared to nonencapsulated strains. In contrast to the emergence of encapsulated AGNB in blood and abscesses, the presence of pili was less frequent in such strains recovered from blood. The rate of piliated strains was high among those recovered from abscesses.

Since most B. fragilis, and Prevotella and Porphyromonas spp. recovered from infected sites probably originate from the predominantly nonencapsulated endogenous flora of mucous membranes, they may express their capsules only during the inflammatory process. The frequent recovery of encapsulated AGNB in such conditions illustrates their increased virulence as compared to their nonencapsulated counterparts.

Complete eradication of experimental AGNB infection by means of metronidazole was not achieved when these organisms were encapsulated (10). Once the organisms become encapsulated, eradication of AGNB infection becomes difficult. Therapy of infections involving nonencapsulated AGNB, however, was more efficacious. Early treatment of anaerobic infections may therefore prevent the emergence of encapsulated AGNB, and subsequent bacteraemia.

The recovery of a greater number of encapsulated anaerobic organisms in patients with orofacial infections, abscesses, and blood provides support for the potential pathogenic role of encapsulated organisms. Early and vigorous antimicrobial therapy, directed at both aerobic and anaerobic bacteria present in these mixed infections, may abort the infection before the emergence of encapsulated strains that contribute to the chronicity of the infection.

Gastrointestinal lumen

Intraabdominal abscesses

Blood

Blood

FIGURE 2 Dynamics of pili and capsule of B. fragilis group. Source: From Ref. 54.

CONCLUSION

The recovery of a greater number of encapsulated anaerobic organisms in patients with acute and chronic infections provides further support for the potential pathogenic role of these organisms. Detection of the presence of a capsule in a clinical isolate may add importance to the organisms' possible role as a pathogen in the infection. The demonstration of the importance of encapsulated organisms in mixed infection may justify directing therapy in such infections against these potential pathogens. Early and vigorous antimicrobial therapy, directed at both aerobic and anaerobic bacteria present in these mixed infections, may abort the infection before the emergence of encapsulated strains that contribute to the chronicity of the infection.

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  • tekle filmon
    How reduced environment promote virulence of anaerobes?
    10 months ago

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