Gram-negative anaerobic pathogens

Gram-Negative Anaerobic Organisms constitute an important group of pathogens that predominate in many infectious processes. These organisms generally cause disease subsequent to the breakdown of mucosal barriers and the leakage of indigenous flora into normally sterile sites of the body. The predominance of anaerobes in numerous clinical syndromes can be attributed to the elaboration of a variety of virulence factors, the ability to resist oxygenated microenvironments, synergy with other bacteria, and resistance to certain antibiotics. 

I. NORMAL FLORA AND EPIDEMIOLOGY 

Hundreds of species of anaerobic organisms make up the human microflora. Mucosal surfaces such as the oral cavity, gastrointestinal tract, and female genital tract are the major reservoirs for this group of organisms. Infections involving anaerobes are polymicrobial and usually result from the disruption of mucosal surfaces and the subsequent infiltration of resident flora. However, gram-negative anaerobic bacilli are the most commonly isolated anaerobes from clinical infections. The clinically important gram-negative anaerobic bacilli belong to the genera Bacteroides, Fusobacterium, Porphyromonas, Prevotella, and Bilophila (Table 47.1). Anaerobes normally reside in abundance as part of the oral flora, with concentrations ranging from 109/ml in saliva to 1012/ml in gingival scrapings. The indigenous oral anaerobic flora primarily comprise Prevotella and Porphyromonas species, with Fusobacterium and Bacteroides (non-Bacteroides fragilis group) present in fewer numbers. Aerobic or microaerophilic and anaerobic bacteria reside in approximately equal numbers in the oral cavity. Anaerobic bacteria are present only in low numbers under the normally acidic conditions in the stomach and upper intestine. In people with decreased gastric acidity, the microflora of the stomach resemble that of the oral cavity. The upper intestine contains relatively few organisms until the distal ileum, where the flora begins to resemble that of the colon. In the colon, there are up to 1012 organisms/stool, with anaerobes outnumbering aerobes by approximately 1000 : 1. Bacteroides and Fusobacterium are the predominant gram-negative species in the colonic flora. The Bacteroides species present in the colon are often earlier classification as subspecies of B. fragilis. This group encompasses several species, including B. fragilis, B. thetaiotaomicron, B. ovatus, B. vulgatus, B. uniformis, and B. distasonis (although the last organism may be reclassified as a Porphyromonas species). 

These Bacteroides species are all considered to be penicillin resistant, whereas the Fusobacterium species remain penicillin sensitive. Prevotella, Bacteroides, and Fusobacterium are part of normal vaginal flora (106 organisms/g secretions). The most common isolates from clinical specimens are P. bivia and P. disiens, although B. fragilis is also frequently isolated from this site. Bacteroides species are found in approximately 50% of women, whereas B. fragilis is found in _15% of this population. Bilophila species are found in normal stool specimens and are occasionally part of the normal oral or vaginal flora. B. wadsworthia exhibits a fairly broad resistance to _-lactam antibiotics and is of interest because of its reported occurrence in human infections. Anaerobic gram-negative bacilli reside on mucosal surfaces and cause infection following the contamination of normally sterile sites. For example, intraabdominal infection develops after fecal spillage into the peritoneum as a result of trauma or necrosis of the intact bowel wall, and severe infections of the head and neck may arise from an abscessed tooth. It is remarkable that, despite the identification of scores of anaerobic species in normal flora, relatively few species seem to play a role in infection. After the contamination of normally sterile sites by mucosal microflora, the relatively few anaerobic bacteria that survive are those few that have resisted changes in oxidation reduction potential and host defense mechanisms. The hallmark of infection due to these gram-negative anaerobic bacteria is abscess formation, although some sepsis syndromes have been described. Typically, abscesses form at sites of direct bacterial contamination, although distant abscesses resulting from hematogenous spread are not uncommon with the more virulent anaerobes. 

II. CLINICAL SYNDROMES CAUSED BY ANAEROBES 

A. Anaerobic infections of the mouth, head, and neck Anaerobes contribute to infection associated with periodontal disease and to disseminated infection arising from the oral cavity. Locally, infection of the periodontal area may extend into the mandible, causing osteomyelitis of the maxillary sinuses or infection of the submental or submandibular spaces. Other infections associated with oral anaerobes include gingivitis, acute necrotizing ulcerative mucositis, acute necrotizing infections of the pharynx, Ludwig’s angina, fascial infections of the head and neck, sinusitis, and otitis. B. Pleuropulmonary infections Anaerobes are associated with aspiration pneumonia, lung abscesses, and empyema. In these cases, the organisms isolated generally reflect the oral flora, and more than one bacterial species is routinely involved. For example, in anaerobic lung abscesses, it is not uncommon to find up to 10 species of organisms. C. Intra-abdominal infections Infections originating from colonic sites, such as intraabdominal abscesses, most frequently involve Bacteroides species, among which B. fragilis is the most common isolate. B. fragilis has also been associated with watery diarrhea in a few studies. Enterotoxinproducing strains are more prevalent in patients with diarrhea than in control groups. Secondary bacterial peritonitis arises when organisms from the intestine contaminate the peritoneum. 

The terminal ileum and colon are the most common sites of origin because of the high numbers of organisms they contain. Patients typically develop acute peritonitis following contamination, and intraperitoneal abscesses may result.   D. Pelvic infections Anaerobes are encountered in pelvic abscess, septic abortion, endometritis, tubovarian abscess, pelvic inflammatory disease, and postoperative infections. In addition to the major anaerobic gram-negative isolates already mentioned, P. melaninogenica, clostridia, and peptostreptococci are commonly found in infected pelvic sites. These organisms are most often isolated when infection is not due to sexually transmitted agents. E. Central nervous system infections Either a single species or a mixture of anaerobic or aerobic bacteria may be found in brain abscesses; prominent among the anaerobes are Fusobacterium, Bacteroides, and anaerobic gram-positive cocci. Anaerobic brain abscesses may arise by hematogenous dissemination from an infected distant site or by direct extension from otitis, sinusitis, or tooth infection. F. Skin and soft-tissue infections Bacteroides species are found in necrotizing fasciitis, usually as part of a mixed anaerobic–aerobic infection. Approximately five species are typically isolated, with a 3 : 2 ratio of anaerobes to aerobes. These infections usually occur at sites that can be contaminated from oral secretions or feces; they may spread rapidly and be very destructive. Gas may be found in the infected tissues. G. Bone and joint infections These infections typically arise from infected adjacent soft-tissue sites. Both osteomyelitis of bone and septic arthritis are seen. Fusobacterium species are the most common gram-negative anaerobes isolated from infected joints, whereas infected bone may yield a wider variety of isolates. H. Bacteremia Anaerobic organisms, particularly B. fragilis, have been detected in up to 5% of blood cultures. When B. fragilis is isolated, patients are frequently ill with rigors and fever. 

III. PATHOGENESIS 

Infections involving gram-negative anaerobes are generally due to the breakdown of a mucosal barrier and the subsequent leakage of indigenous flora into closed spaces or tissue. The introduction of bacteria into otherwise sterile sites leads to a polymicrobial infection in which certain organisms predominate. These include B. fragilis, Prevotella species, Fusobacterium species, and Porphyromonas species. Although some of these organisms are numerically dominant in the normal flora, others (such as B. fragilis) make up a much smaller proportion. The greater ability of these organisms to cause disease more often than numerically dominant anaerobes usually indicates the possession of one or more virulence factors. These factors include the ability to evade host defenses, adhere to cell surfaces, produce toxins or enzymes, or display surface structures that contribute to pathogenic potential. A. Synergy The ability of different anaerobic bacteria to act synergistically during polymicrobial infection has been described but remains poorly characterized. It has been postulated that facultative organisms function in part to lower the oxidation–reduction potential in the microenvironment and that this change allows the propagation of obligate anaerobes. Conversely, studies have shown that anaerobes, including B. fragilis, can produce compounds such as succinic acid and shortchain fatty acids that inhibit the ability of phagocytes to clear facultative organisms. Further, it is clear that facultative and obligate anaerobes synergistically potentiate abscess formation in experimental models. B. Role of B. fragilis capsular polysaccharide in abscess induction The anaerobe most commonly isolated from clinical infections is B. fragilis. The most frequent sites of isolation of B. fragilis are the bloodstream and abscesses associated with intraabdominal sepsis. The characteristic host response to B. fragilis infections is the development of intraabdominal abscesses. Although the development of an abscess limits the initial spread of the organism, the host usually cannot resolve the abscess, therefore requiring surgical drainage. The high frequency of abscess formation associated with B. fragilis led to studies investigating this organism’s pathogenic potential in relevant animal models of disease. This work identified the capsular polysaccharide as the major virulence factor of B. fragilis and defined the capsule’s role in the induction of abscesses in an animal model of intraabdominal sepsis. 

Further studies have delineated the structural attributes of the B. fragilis capsular polysaccharide that promotes abscess formation in animals. The capsule of strain NCTC 9343 comprises two distinct ionically linked polymers, termed PS A and PS B. Each of these purified polymers induces abscess formation when implanted with sterilized cecal contents (SCC) and barium sulfate as an adjuvant into the peritonea of rats. Historically, this adjuvant is included to simulate the spillage of colonic contents that occurs in intraabdominal sepsis. The implantation of PS Awithout SCC does not induce abscess formation in animals. PS A is the more potent of the two polysaccharides; less than 1_g is required for abscess induction in 50% of challenged animals. The structures of both saccharides have been elucidated. Each polymer consists of repeating units whose possession of both positively and negatively charged groups is rare among bacterial polysaccharides. The ability of PS A and PS B to induce abscesses in animals depends on the presence of these charged groups. Numerous fecal and clinical isolates of B. fragilis have been examined, and this dual polysaccharide motif has been found in every strain. The capsule of B. fragilis probably acts to regulate the host response within the peritoneal cavity to initiate the steps leading to abscess formation. Studies in mice have shown that the capsule mediates bacterial adherence to primary mesothelial cell cultures in vitro. In addition, the capsule promotes the release of the proinflammatory cytokines TNF-_ and IL-1_, as well as the chemokine IL-8, from macrophages and neutrophils. 

The release of TNF-_ from peritoneal macrophages potentiates the increase of cell-adhesion molecules such as ICAM-1 on mesothelial cell surfaces, and this potentiated response in turn leads to an increase in the binding of neutrophils to these cells. These events are the first steps leading to the accumulation of neutrophils at inflamed sites within the peritoneal cavity and are likely to lead to the formation of abscesses at these sites. C. Abscess formation and T cells T cells are critical in the development of intraabdominal abscesses, but little is known about the mechanisms of cell-mediated immunity underlying this host response. Attempts to define the immunologic events leading to abscess formation have been made in athymic or T cell-depleted animals. The results from these studies show that T cells are required for the formation of abscesses following bacterial challenge of animals. Studies by Sawyer et al. (1995) have documented a role for CD4_ T cells in the regulation of abscess formation. B. fragilis produces a host of virulence factors that allow this organism to predominate in disease. Although the lipopolysaccharide (LPS) of B. fragilis possesses little biologic activity, this organism synthesizes pili, fimbriae, and hemagglutinins that aid in attachment to host-cell surfaces. In addition, Bacteroides species produce many enzymes and toxins that contribute to pathogenicity. Enzymes such as neuraminidase, protease, glycoside hydrolases, and superoxide dismutases are all produced by B. fragilis. Recent work has shown that this organism produces an enterotoxin with specific effects on host cells in vitro. This toxin, termed BFT, is a metalloprotease that is cytopathic to intestinal epithelial cells and induces fluid secretion and tissue damage in ligated intestinal loops of experimental animals. 

Strains of B. fragilis associated with diarrhea in children (termed enterotoxigenic B. fragilis, or ETBF) produce a heatlabile 20-kDa protein toxin. BFT specifically cleaves the extracellular domain of E-cadherin, a glycoprotein found on the surface of eukaryotic cells. The pathogenesis of P. gingivalis relies on a broad range of virulence factors. This organism is a prominent etiologic agent in adult periodontitis. The progression of this disease is hypothesized to be related to the production of a variety of enzymes (particularly proteolytic enzymes), fimbriae, capsular polysaccharide, LPS, hemagglutinin, and hemolytic activity. P. gingivalis has been shown to invade and replicate host cells, a mechanism that may facilitate its spread. A class of trypsin-like cysteine proteases, termed gingipains, have recently been implicated as a major virulence factor contributing to the tissue destruction that is the hallmark of periodontal disease. The capsular polysaccharide of P. gingivalis acts as a potent virulence factor facilitating a spreading infection in mice greater than that seen with unencapsulated strains. The LPS of P. gingivalis has been implicated in the initiation and development of periodontal disease. It has been shown that the LPS activates human gingival fibroblasts to release IL-6 via CD14 receptors on host cells. In addition, neutrophils stimulated with P. gingivalis LPS release IL-8. F. necrophorum causes numerous necrotic conditions (necrobacillosis) and human oral infections. 

Several toxins, such as leukotoxin, endotoxin, and hemolysin, have been implicated as virulence factors. Among these, leukotoxin and endotoxin are believed to be the most important. F. nucleatum is a major contributor to gingival inflammation and is isolated from sites of periodontitis. This organism can co-aggregate with other oral bacteria to promote attachment to plaque; in addition, it produces several adhesins that facilitate attachment. Both F. nucleatum and F. necrophorum produce a potent LPS that is responsible for the release of numerous proinflammatory cytokines and other inflammatory mediators.   Virulence factors associated with Prevotella species are poorly defined. The organism’s ability to interact with other anaerobes has been reported. Among their prominent virulence traits is the production of proteases and metabolic products, such as volatile fatty acids and amines. This group of organisms is particularly noted for secretion of IgA proteases. The degradation of IgA produced by mucosal surfaces allows Prevotella to evade this first line of host defense. A study has demonstrated that P. intermedia can invade oral epithelial cells and that antibody specific for fimbriae from this organism inhibits invasion. 

IV. IMMUNITY 

Although relatively little is known about immunity to anaerobic organisms in general, the immune response to B. fragilis has been studied in detail. Prior treatment of animals with PS A from this organism prevents the formation of intraabdominal abscesses after challenge with B. fragilis or other abscess-inducing bacteria. This protection depends on the presence of positively charged amino and negatively charged carboxyl groups associated with the saccharide’s repeating unit structure. Attempts to define the immunologic events regulating abscess formation have suggested an important role for cell-mediated immunity. Studies on rats have shown that administration of PS A shortly before or even after bacterial challenge protects against abscess formation induced by a heterologous array of organisms. This protective activity is dependent on T cells. In other words, these studies suggested that PS A elicits a rapid, broadly protective immunomodulatory response that is dependent on cell-mediated immunity. The capsular polysaccharide of B. fragilis has also been shown to inhibit opsonophagocytosis, and an antibody specific for its capsule activates both the classical and alternative pathways of the complement system. Significant increases in antibody titers in patients with B. fragilis bacteremia have been reported, but hyperimmune globulin generated in animals specific for B. fragilis does not protect against abscesses. Studies have shown that specific capsular antibody does reduce the incidence of bacteremia in experimentally infected animals. Immunity to P. gingivalis infections has been described and can be generated to various degrees in animals models by the capsular polysaccharide, LPS, hemagglutinin, or gingipains. 

The involvement of T cells in regulating the immune response to this organism has also been demonstrated. Immune T cells derived from mucosal and systemic tissues of rats given live P. gingivalis yielded an increase in serum and salivary responses compared to control animals. These results indicate a role for serum IgG and salivary IgA in protection against periodontal disease in which a balance between Th1 and Th2-like cells occurs in humoral immune responses to P. gingivalis. In a recent clinical study, patients with periodontal disease develop a significant antibody response to P. gingivalis, but this response does not eliminate infection. Relatively little is known about the host immune response to Fusobacterium species and Prevotella species. F. nucleatum produces a protein that inhibits T cell activation in vitro by arresting cells in the mid-G1 phase of the cell. It is hypothesized that the suppressive effects of this protein enhance the virulence of F. nucleatum. In addition, F. nucleatum or its purified outer membrane can induce a potent humoral response in mice. Several investigators have attempted to investigate mechanisms of protective immunity against F. necrophorum, but potential immunogens isolated from the organism have not afforded satisfactory protection. In studies of the host response to P. intermedia, a polysaccharide surface component exerted a strong mitogenic effect on splenocytes and a cytokineinducing effect on peritoneal macrophages from both C3H/HeJ and C3H/HeN mice; this polysaccharide also stimulated human gingival fibroblasts to produce cytokines. The immunization of nonhuman primates with P. intermedia resulted in the production of significantly elevated levels of specific IgG. The level of serum IgA antibody also increased. Finally, coculture of P. intermedia with T cells significantly upregulated the expression of specific T-cell receptor-variable regions, a result suggesting that this organism has significant impact on T cells. 

V. GENETICS 

Knowledge of the genetic makeup of the gramnegative anaerobic pathogens lags far behind their aerobic counterparts. Sequence for only a few hundred nonredundant genes have been deposited in the databases for all these genera combined. An accurate understanding of the genetic makeup of these organisms awaits complete genome sequencing. The G_C content of these bacteria shows some dissimilarity among genera, with variation for Bacteroides species reported at 41–46%, Porphyromonas species 41–45%, Prevotella species 39–51%, and Fusobacterium species 26–34%. Little genetic information is available for Bilophila. The only reported sequence for this genus is that of the 16S rDNA, which demonstrates the relatedness of this organism to other sulfur-reducing organisms such as the Desulfovibrio species. Similarly, few genes aside from rDNA have been sequenced from Fusobacterium and few factors implicated in virulence of the human Prevotella species have been characterized genetically. A large proportion of the genes sequenced from Porphyromonas encode the various and diverse types of proteases or hemagglutinins produced by these organisms that are involved in virulence. In addition, mutational analysis of fimA demonstrated that fimbriae are essential for the interaction of the organism with human gingival tissue cells. The most extensive area of genetic research in Bacteroides is the study of antibiotic resistance and the elements involved in the transfer of resistance genes. Aside from this area of research, the genetic analysis of other virulence factors are being performed. The gene encoding the metalloprotease toxin (bft) of B. fragilis is contained on a pathogenicity island that is present only in enterotoxigenic strains. Other products involved in aerotolerance of B. fragilis, such as catalase and superoxide dismutase, have also been studied at the molecular level. 

VI. GENETICS OF ANTIBIOTIC RESISTANCE BY BACTEROIDES 

Antibiotic resistance is an increasing problem in the treatment of Bacteroides infections: The bacteria continue to acquire genes that make them resistant to multiple antibiotics. A drastic increase in resistance to antibiotics such as tetracycline, cephalosporins, and clindamycin over the last 2 decades has necessitated the use of carbapenems, metronidazole, and _-lactamase inhibitors. Resistance to these latter agents, however, is also increasing. The genetic elements responsible for the evolution of antibiotic resistance in Bacteroides are the topic of this section. A. _-Lactamases The majority of Bacteroides species display some level of resistance to _-lactam antibiotics. The genes encoding the enzymes responsible for resistance vary among the Bacteroides species. The best studied are the _-lactamases of B. fragilis. Two distinct classes of _-lactamases have been described in B. fragilis, the active-site serine enzymes encoded by cepA and the metallo-_-lactamases encoded by cfiA. Any given B. fragilis strain contains only one of these _-lactamaseencoding genes, and taxonomic investigations have revealed that cfiA_strains and cepA_strains form two genotypically distinct groups. Although these groups cannot be differentiated phenotypically, cfiA_ strains exhibit a distinctive and homogeneous ribotype, can be distinguished from cepA_strains by arbitrarily primed PCR, and preferentially contain most of the insertion-sequence (IS) elements described for B. fragilis (i.e., IS 4351, IS 942, and IS 1186). 1. cepA The most prevalent _-lactamase of B. fragilis is the active-site serine enzyme encoded by the chromosomal cepA. This cephalosporinase does not confer protection against the carbapenems (such as imipenem) and is sensitive to the action of _-lactamase inhibitors. Not all cepA_strains produce a high level of the cephalosporinase. The analysis of the sequence of cepA from seven B. fragilis strains that produce high or low levels of cephalosporinase demonstrated that the cepA coding regions for all strains were identical. Therefore, structural differences in the enzyme do not account for the differing levels of resistance conferred by cepA_strains. Rather, the production of the enzyme is regulated at the transcriptional level by sequences upstream of cepA. 2. cfiA Only approximately 3% of B. fragilis strains contain cfiA, which encodes a metallo-_-lactamase that varies in properties, substrate specificity, and activity from the cepA gene product. The emergence of cfiA has been closely monitored, as the enzyme it encodes is not affected by _-lactamase inhibitors and is active against a variety of _-lactams, including carbapenems. Only one-third of cfiA_strains have been reported to produce the enzyme; the inability of other cfiA_strains to do so is due to the lack of a promoter driving the transcription of cfiA. These silent cfiA genes become active when an IS element (IS 1168 or IS 1186) is inserted into the chromosome just upstream of cfiA. 

These IS elements contain outward-oriented promoters that drive the transcription of cfiA, leading to a 100-fold increase in the amount of _-lactamase. Given the small fraction of B. fragilis strains that produce the metallo-_-lactamase, carbapenems are still effective for the treatment of Bacteroides infections. B. Conjugative elements involved in the transfer of antibiotic resistance Unlike the _-lactamase genes of B. fragilis, the resistance of Bacteroides to clindamycin, tetracycline, and 5-nitroimidazole is conferred by genes carried on elements that are self-transmissible. Both conjugative transposons and conjugative plasmids are involved in the transfer of these antibiotic-resistance genes in Bacteroides. 1. Conjugative transposons The conjugative transposons of Bacteroides are 70–80-kb elements that are normally integrated into the chromosome or on a plasmid. In addition to carrying genes for resistance to tetracycline and clindamycin, conjugative transposons contain all the necessary genes for their excision and transfer. Once the transposon has been excised from the chromosome, the element forms a covalently closed circle. Transfer of a single strand of the transposon then begins at oriT through a mating pore to the recipient cell. The single strand is replicated in the recipient cell and integrated into the chromosome in an orientation- and site-specific manner. Several factors account for the ability of the conjugative transposons to propagate antibiotic-resistance genes so successfully. Their broad host range allows for their transfer between species that are only distantly related. 

The conjugative transposons can also mediate the mobilization of coresident plasmids and the excision and mobilization of unlinked chromosomal segments of 10–12 kb, termed nonreplicating Bacteroides units (NBUs). These elements contain an origin of transfer that permits their transfer by the mating pore of the conjugative transposon. Therefore, conjugative transposons allow for the spread of antibiotic- resistance genes contained on unlinked elements. Lastly, the transfer capabilities of the conjugative transposons are inducible by subinhibitory concentrations of tetracycline as described later. 2. Conjugative plasmids Of the several conjugative plasmids described in Bacteroides, some contain genes conferring resistance to clindamycin or the 5-nitroimidazoles. The regions involved in transfer have been studied for many Bacteroides conjugative plasmids and usually involve the products of one to three mob genes. As are the conjugative transposons, the conjugative plasmids have been transferred across genera; however, their range is probably more restricted than that of the conjugative transposons, as recognition of the origin of replication is necessary for maintenance. Because conjugative plasmids can be mobilized by coresident conjugative transposons, the spread of 5-nitroimidazole resistance conferred by genes on conjugative plasmids can be induced by tetracycline pretreatment of cells containing conjugative transposons. Therefore, the use of tetracycline may lead to the spread of resistance to various antibiotics. C. Antibiotic resistance genes contained on conjugative elements 1. Tetracycline resistance Tetracycline, once effective against Bacteroides infections, now encounters resistance in the majority of clinical isolates. Most tetracyline-resistant Bacteroides contain the gene tetQ, which is believed to confer widespread resistance among Bacteroides strains. 

Although the tetQ product is most similar to the TetM and TetO classes of resistance mediated by ribosomal protection, the degree of similarity is low enough (40%) to merit a separate class of ribosomal resistance genes. The gene tetQ is carried by the conjugative transposons of Bacteroides. What is unique about these conjugative transposons is that their transfer is increased by 100- to 1000-fold by pretreatment of the donor with subinhibitory concentrations of tetracycline. This finding led to the discovery of three regulatory genes downstream of tetQ on the conjugative transposon. The corresponding gene products are probably involved in the tetracycline-dependent transcriptional activation of genes involved in transfer. Two other tetracycline-resistance determinants have been identified in Bacteroides. tetX, first cloned in 1991, encodes a product that inactivates tetracycline. An additional tetracycline-resistance determinant was found on a Bacteroides transposon that leads to tetracycline efflux. Neither of these products actually confers resistance to tetracycline in Bacteroides, and both are probably remnants of DNA transfer from other organisms. 2. Clindamycin resistance Two reports have shown the frequency of clindamycin resistance among Bacteroides species at various institutions to be as high as 21.7% and 42.7%. The first clindamycin-resistance gene from Bacteroides (ermF) was sequenced in 1986. Since then, two additional genes have been sequenced and found to encode products that are 98% identical to the ermF product. These genes are contained on transposons, conjugative transposons, and conjugative plasmids, which accounts for their widespread distribution among Bacteroides strains. The mechanism of resistance conferred by the ermF product involves neither inactivation nor efflux of the drug. Instead, resistance to clindamycin occurs at the product with erm genes from gram-positive bacteria suggests that the resistance is mediated by methylation of 23S rRNA, which prevents binding of the antibiotic. The clindamycin resistance gene ermG, which was recently sequenced from a Bacteroides conjugative transposon, encodes a product that is only 46% similar to the ermF product. The ermG product is extremely similar to the erm products of gram-positive organisms, whose functions as 23S rRNA methylases have been established. 

Therefore, Bacteroides species contain two distinct genes, probably of different origins, that both confer resistance to clindamycin by the same mechanism. 3. 5-Nitroimidazole resistance The vast majority of Bacteroides strains are sensitive to the 5-nitroimidazole antibiotics, and metronidazole remains the drug of choice for the treatment of Bacteroides infections. However, genes conferring resistance to 5-nitroimidazoles have been described in various Bacteroides species. Four distinct resistance genes have been sequenced (nimA–nimD), the products of which exhibit 67–91% similarity. nimA, nimC, and nimD are present on conjugative plasmids, and nimB is present in the chromosome of a B. fragilis clinical isolate. As are the other three nim genes, nimB is transferable by conjugation to other B. fragilis strains. IS elements are present just upstream of each of the nim genes. Some of these IS elements are highly homologous to IS 1168 and IS 1186, which control the transcription of cfiA of B. fragilis. It is likely that transcription of the nim genes, as with cfiA, is controlled by outward-oriented promoters the IS elements. The mechanism of the resistance conferred by the nimA product has been studied. The gene probably encodes a 5-nitroimidazole reductase that prevents the formation of the toxic form of the drug.