J. Med. Microbiol. - Vol. 46 (1997), 979-986 0 1997 The Pathological Society of Great Britain and Ireland

REVIEW ARTICLE

Resistance to p-lactam in Bacteroides SPP.

R. EDWARDS

Division of Microbiology and Infectious Diseases and PHLS Laboratory, University Hospital, Queen's Medical Centre, Nottingham NG7 2UH

Bacteroides spp., particularly B. fragilis, are well-recognised bacterial pathogens. Production of the typical P-lactamases of Bacteroides restricts the therapeutic use of P-lactam agents mainly to the P-lactamase inhibitor combinations and . These compounds have the advantage of broad-spectrum activity and the ability to combat polymicrobial infections. Resistance of Bacteroides spp. to P-lactam antibiotics appears to be increasing, largely because of an overall increase in P-lactamase activity. There has been a rise in the prevalence of isolates showing high-level production of typical Bacteroides P-lactamases and an increase in reports other potent p-lactamase types. In the case of B. fragilis, metallo-enzymes are a particular threat to current therapeutic practice, as they are not inhibited by common P-lactamase inhibitors and are able to hydrolyse carbapenems. The presence of permeability barriers may confer low-level p-lactam resistance and supplement the effect of P-lactamase activity. There are also sporadic reports of loss of /?-lactam activity because of reduced affinity of the -binding proteins.

Introduction The role of Bacteroides spp. in infection

The pathogenic potential of gram-negative, non-spor- Anaerobic bacteria involved in infections are com- ulating, anaerobic bacilli has been recognised since the monly derived from complex normal flora and, there- late 19th century [l]. Infections caused by these fore, are found usually in association with other organisms are now known to be common and can bacterial types. The major reservoir of bacteroides in sometimes be life-threatening. Bacteroides spp. (mem- the human body is the colon. The numerically bers of the former B. fragilis group) are the anaerobes dominant Bacteroides spp. in the normal colonic flora encountered most fi-equently in clinical specimens. are B. distasonis, B. vulgatus and B. thetaiotaomicron, They are recognised as being among the most resistant with only 5% of the cultivable colonic bacteria of the anaerobes to antimicrobial agents, including p- comprising B. fragilis [4], although this figure has lactam antibiotics, and it is of concern that the number been challenged as being an under-estimate [5]. B. of isolates that display resistance is increasing [2, 31. fragilis, because of its unique virulence properties, is associated more commonly with infections than are the This review focuses on the importance of Bacteroides other Bacteroides spp. It is the most important spp. in human disease and the role of p-lactam pathogen among anaerobic bacteria, accounting for c. compounds in treating these infections. The degree of 25% of all anaerobic bacteria isolated from clinical resistance of clinical Bacteroides isolates to p-lactam specimens [31. antibiotics is discussed and the most effective p- lactam agents are high-lighted. Mechanisms of resis- The most common infections with Bacteroides spp. in tance to these compounds, including degra- general, and B. fragilis in particular, are intra-abdom- dation, reduced permeability, and lack of inal abscesses, peritonitis and wound infections antibiotic binding to lethal target sites, are examined. associated with the large intestine [6]. Bacteroides spp. are also principal pathogens of the female genital tract and pelvic abscesses [7, 81, and are implicated in anaerobic pulmonary infections [3, 91. B. fragilis is Received 17 March 1997; accepted 28 April 1997. implicated frequently in brain abscesses secondary to Corresponding author: Dr R. Edwards. otitis media [lo], and is a major pathogen in cases of 980 R. EDWARDS

diabetic foot ulcer [3, 111. Bacteraemia is commonly commonly used in combination with an anti-aerobe associated with B. fragilis infections, but endocarditis antibiotic, normally a or an aminoglyco- caused by Bacteroides spp. is rare [ 121. side. Another possible option for prophylaxis and the treatment of bacteroides infections is the use of single broad-spectrum compounds. These would be simpler to Susceptibility of Bacteroides spp. to p-lactam administer and may be preferable financially. Candi- antibiotics dates for such antibiotics include the carbapenems and , although the latter, as mentioned pre- Resistance of a wide range of bacterial pathogens to viously, are intrinsically less active. The combinations antimicrobial agents is a growing problem, and includes of with P-lactamase inhibitors are also the reduced effectiveness of p-lactam compounds against useful therapeutic preparations to combat these infec- Bacteroides spp. [ 131. Bacteroides spp. are, almost tions. invariably, relatively insusceptible to ; this is in contrast to most other clinically important anaerobic bacteria, including Prevotella spp. and Mechanisms of resistance to p-lactam Porphyromonas spp. [14]. The other penicillins and the antibiotics (excluding the related cephamycins and oxa-) generally also show low activity towards To inhibit gram-negative bacteria, p-lactam antibiotics

Bacteroides spp. [ 15- 171. However, bacteroides are must be able to penetrate the outer membrane, pass more susceptible to some of the acylureidopenicillins, through the periplasmic space, and arrive with undimin- such as [ 181. Full susceptibility to benzyl- ished potency at the penicillin-binding proteins (PBPs) in penicillin and other p-lactam agents can normally be the cytoplasmic membrane. Co-valent binding with the restored by addition of the P-lactamase inhibitors PBPs has then to occur for the lethal affect to take place. , and [ 191. Mechanisms of resistance that interfere with this chain of events are: blockage of transport of the agent into the The cephamycins are more active than conventional cell; inactivation of the antibiotic, usually within the cephalosporins against Bacteroides spp. [ 181. Low- periplasmic space; and alteration of target sites. level resistance to (MIC of 32 mg/L) was recognised in bacteroides immediately after the intro- Reduced antibiotic penetration duction of this antibiotic in the late 1970s [20]; high- level resistance (MIC of 3 64 mg/L) was first The ability of an antibiotic to reach its site of action is documented in 1983 [21]. However, resistance is a prerequisite for drug action. In gram-negative uncommon; in a European study published in 1992, bacteria, the outer membrane presents a physical 3% of Bacteroides isolates were resistant to cefoxitin barrier to the penetration of antibiotics into the peri- at a break point of 32 mg/L [22]. Bacteroides spp. are plasmic space. The outer-membrane protein (OMP) also susceptible to the oxa- , which composition of Bacteroides spp. is complex [31, 321 exhibits greater in-vitro activity than cefoxitin [ 181. In and, until recently, no specific porin molecules had general, B. fragiZis is more susceptible to these been identified in bacteroides. However, since 1992, antibiotics than other Bacteroides spp. [23, 241. studies with liposome assays have revealed pore- forming ability of fractions isolated from the outer Potent activity against Bacteroides spp. is displayed by membranes of strains of B. distasonis and B. fragilis the carbapenems, with MIC5Os of and [33 -351. of 0.06-0.12 mg/L [25, 261; comparable activity has been shown with the recently developed The ability of p-lactam compounds to permeate the compound [27]. resistance is low, outer membrane of gram-negative bacteria is depen- with < 2% of Bacteroides isolates reported as resistant dent on the physicochemical properties of the [22, 28, 291, while < 10% of isolates show reduced antibiotic [36]. With B. fragilis, the rank order of susceptibility (imipenem MIC of 1-4 mg/L) [30]. The permeative ability of selected p-lactam antibiotics was carbapenems, together with the clavulanate-potentiated reported by Cuchural et al. [37], with penicillins, are the most active p-lactam compounds the most rapid, followed by imipenem, , against Bacteroides spp. [ 18, 19, 22, 28, 291. cefoxitin, cephalothin and latamoxef. Investigation of factors influencing permeability yielded results similar to those described for Escherichia coli, in which P-Lactam antibiotics in the treatment of increases in negative charge and molecular mass were Bacteroides infections associated with decreased antibiotic uptake [36]. However, unlike the situation in E. coli, increased As most infections involving Bacteroides spp. occur in drug hydrophobicity was associated with increased combination with aerobic pathogens, antibiotic therapy uptake by B. fragilis. It has been proposed that the must be directed at both the anaerobic and aerobic different effects of hydrophobicity on permeability are components. In these situations, metronidazole is caused by dissimilarities in the lipopolysaccharide /?-LACTAM RESISTANCE IN BACTEROIDES SPP. 98 1

(LPS) composition. The contribution of LPS to the mediated hydrolysis of the p-lactam ring, which barrier effect has been demonstrated with E. coli [38], renders the antibiotic inactive. Production of elevated but not with Bacteroides spp. [39]. amounts of p-lactamase by Bacteroides strains is associated with high-level resistance to p-lactam anti- The role of permeability barriers in p-lactam resis- biotics [47]. tance in Bacteroides spp. has been observed indirectly. Dornbusch et al. [40] examined strains of B. fragilis The first report of the penicillin-destroying activity of with decreased susceptibility to cefoxitin and found no B. fragilis was by Garrod in 1955 [48], who showed correlation between P-lactamase production and resis- that two of 31 strains examined were able to destroy tance to cephamycins. It was postulated that changes penicillin in solution. The production of P-lactamases in the cell wall, causing decreased penetration of the by 14 penicillin-resistant strains in a collection of 29 antibiotic, could be a resistance factor. isolates of B. fragilis was documented in 1968 [49]. It is now recognised that most Bacteroides strains (c. Results from crypticity measurements (the ratio of P- 900/) produce small amounts of a constitutive P- lactamase activity of broken cells to the activity of lactamase [19, 501. B. distasonis isolates are asso- intact cells), with cephaloridine as substrate, indicate ciated with the least amount of P-lactamase produc- that limited outer-membrane permeability to p-lactam tion, with 58-80% of strains displaying p-lactamase antibiotics contributes to resistance in certain strains activity [14, 511. of B. fragilis [4 11. Also, P-lactamase-mediated imipe- nem resistance in B. fragilis has been shown to be The proportion of isolates of Bacteroides spp. that are associated with a barrier to drug permeation, as capable of high-level P-lactamase production appears determined by crypticity measurements [42]. In to be increasing. In 1977, Olsson et al. [50] reported contrast, a recent study showed no correlation between that 6% of B. fragilis isolates produced elevated the levels of imipenem resistance of B. fragilis amounts of P-lactamase. Ten years later, 25% of B. isolates and crypticity or other permeability measure- fragilis group isolates from the USA produced high ments that take into account kinetic factors involved levels of P-lactamase [52]. In Nottingham, the per- in drug permeation (personal unpublished results). centage of clinical Bacteroides isolates displaying raised p-lactamase production increased from 17% to EDTA has been used to increase the permeability of 25% between 1986 and 1995 [30]. In those cases in the cell wall in an attempt to assess the importance of which elevated levels of typical P-lactamases are impermeability of this wall in terms of antibiotic found, insertion elements may be responsible for resistance [43]. The addition of EDTA enhanced the increased expression of the cepA gene that controls activity of significantly against p-lacta- production of these enzymes [53]. mase-negative, as opposed to P-lactamase-positive, bacteroides, indicating that cell impermeability was a The first detailed characterisation of a P-lactamase major mechanism of resistance to cefoperazone in 0- produced by a strain of B. fragilis exhibiting high- lactamase-negative isolates. Also, an increase in the level resistance was provided by Anderson susceptibility of bacteroides to cephalosporins in the and Sykes [54]. The enzyme was shown to be a presence of EDTA has been observed, suggesting a cephalosporinase rather than a penicillinase, it was not role for a permeability barrier in addition to /3- inducible, and its production correlated with high-level lactamase, with the exception of B. distasonis, which resistance to p-lactam antibiotics. Further characterisa- showed only a permeability barrier [44]. tion of the common P-lactamases from B. fragilis has shown them to have a molecular mass of 30-40 kDa, Evidence of an association between cefoxitin resis- and to be inhibited by , pCMB and tance and altered porin proteins in Bacteroides spp. clavulanic acid [55-571. Cefoxitin, latamoxef and was put forward by Piddock and Wise [45]. They imipenem are typically resistant to hydrolysis by these described two cefoxitin-resistant strains, one of which enzymes [58]. P-Lactamases from B. fragilis, B. was B. fragilis and the other B. thetaiotaomicron, in thetaiotaomicron, B. vulgatus and B. uniformis show which resistance was not P-lactamase mediated, but species-specific differences in terms of substrate related to alterations in OMP profiles together with profile and PI values that range between 4.9 for B. PBP changes. An OMP, possibly a porin protein, of c. fragilis and 4.25 for B. thetaiotaomicron [59]. It is 50 kDa was absent from both strains. Other studies now recognised that most Bacteroides isolates produce have shown conflicting evidence of the association at least one P-lactamase with activity that is species- between reduced susceptibility to carbapenems and specific, although they share the general characteristics reduced expression or loss of OMPs [39, 461. described previously [47, 601.

These properties differ from those of P-lactamases P-Lactamases from aerobic bacteria. However, Bacteroides p-lacta- The most important mechanism of resistance to P- mases do bear some resemblance to class I enzymes lactam antibiotics in Bacteroides spp. is P-lactamase- of the Richmond and Sykes scheme, except in terms 982 R. EDWARDS of inhibition by pCMB and clavulanic acid [59, 611; such as Stenotrophomonas maltophilia and Aeromonas an additional class (VI) of this scheme was put hydrophila [ 751. Other clinical isolates of B. fragilis forward by Neu [62] to accommodate p-lactamases have been described that produce EDTA-sensitive, from Bacteroides spp. In the most recent differential clavulanic acid-resistant carbapenemases, although scheme developed by Bush, Jacoby and Medeiros, these showed lower levels of resistance to imipenem, typical P-lactamases produced by B. fragilis have been ranging from reduced susceptibility to intermediate placed in group 2e [63]. Rogers et al. [64] resistance (MICs of 0.25-32 mg/L) [76, 771. characterised the cephalosporinase gene cepA from B. fragilis that exhibits high specific activity and Gene sequencing and DNA probes have confirmed the showed that these typical P-lactamases form a distinct presence of highly homologous ccrA or c$A genes in subgroup of Ambler molecular class A [65]. Similarly, clinical isolates of B. fragilis from the USA, UK and a clavulanate-sensitive cephalosporinase produced by France that produce metallo-P-lactamase [78-8 11. B. vulgatus, which was capable of slow degradation of DNA sequence analysis has shown that metallo$- cefoxitin, appears to belong to a new class-A lactamases produced by three B. jragilis strains, for homology group [66]. Analysis of P-lactamase from which the MICs of imipenem varied over a 16-fold B. uniformis revealed a species-specific Bush group 2e range, differed at five amino-acid residues [82]. These enzyme that belongs to Ambler class A [67]. However, enzymes were over-expressed, purified and their an unusual P-lactamase from B. unformis strain B kinetic values for a variety of p-lactam antibiotics 371, which appears to belong to group 2e, has been determined. By these means, it was found that the five reported. This P-lactamase showed a substrate profile amino-acid substitutions affected the hydrolysing that resembles enzymes of Ambler class C, except for activity of these P-lactamases only modestly and that its inhibition by clavulanic acid [68]; class C enzymes the differences in susceptibilities were a reflection of have not been encountered previously in Bacteroides the level of gene expression. High-level resistance SPP. among strains harbouring the c$A gene is, therefore, a consequence of enhanced metallo-p-lactamase produc- Other types of p-lactamase, apart from the common tion rather than enzyme differences or permeability group 2e enzymes, have been reported in bacteroides. factors [821 (personal unpublished results). Sat0 et al. [69] described a B. fragilis p-lactamase that was a potent penicillinase, had weak cephalospor- Podglajen et ul. [83] showed with a DNA probe that inase activity, was inhibited by pCMB and had an 2.2% of the B. fragilis clinical isolates studied carried isoelectric point of 6.9. This enzyme has been the c$A gene. In two-thirds of these, the cJiA genes designated a member of Bush group 2d [63]. Also, were ‘silent’ and were associated with strains of atypical p-lactamases from strains of Bacteroides spp., reduced susceptibility (imipenem MICs of d 2 mg/L). such as B. fragilis strain TAL4170 and B. uniformis Selection of imipenem resistance by single step strain 2986, have been reported that inactivate mutation, associated with zinc P-lactamases, has been cefoxitin, show diverse PI values and exhibit various detected in B. fragilis isolates that appeared initially to levels of susceptibility to clavulanic acid [20, 211. be moderately susceptible to imipenem [8 11. Expres- sion and resistance rely on the presence of an A distinct class of B. fragilis p-lactamase that insertion sequence upstream of the c$A gene, and inactivated a wide range of p-lactam substrates usually this arrangement can occur spontaneously at a considered stable to hydrolysis, including cephamycins frequency of c. lop7 [81, 841. The implications of and carbapenems, was reported in 1986 by Cuchural these observations are disturbing. It would seem that et al. [42]. These enzymes were inhibited by the ion B. fragilis strains that possess the ‘silent’ c$A gene, chelator EDTA, and zinc ions completely reversed this and appear susceptible to carbapenems, have the inhibition. Clavulanic acid and sulbactam did not potential to convert to high-level p-lactam resistance, inhibit activity of these P-lactamases [70]. Metallo-p- including resistance to carbapenems. Indeed, this lactamases from other B. fragilis strains with similar situation has been observed recently in vivo, with a properties have been described subsequently [ 7 1, 72). B. fragilis strain developing resistance during treat- Hedberg et al. [73] also characterised an imipenem- ment with imipenem of a patient in Nottingham [85]. hydrolysing B. fragilis metallo-P-lactamase, albeit with Paradoxically, we have been unable to generate a substrate profile that differed from those described resistant mutants in vitro from this or other strains previously [72] while other characteristics such as that appeared likely candidates for such conversion. inhibition profiles and physical properties were similar. All these enzymes caused substantial resistance to The occurrence of metallo-P-lactamases in isolates of imipenem (MICs of 3 100 mg/L), had PI values in B. fragilis pre-dates the widespread use of carbape- the range 4.5-5.2 and a molecular mass of 25-33 kDa nems [86]. These broad-spectum enzymes may have when determined by SDS-PAGE [74]. These metallo- provided protection from common p- lactam antibiotics P-lactamases belong to Ambler’s molecular class B used before 1987, although they hjrdrolyse cefoxitin and the Bush hnctional group 3 [63, 701, and are less efficiently than carbapenems. Interestingly, B. similar to those produced by other bacterial species fragilis is the only Bacteroides spp. in which metallo- P-LACTAM RESISTANCE IN BACTEROIDES SPP. 983

p-lactamases have so far been reported. Several of B. fragilis that contained a metallo-p-lactamase attempts in Nottingham have failed to detect these gene on a small plasmid transferable by conjugation. enzymes in other gram-negative anaerobes. Strains This situation increases greatly the potential for spread carrying the cJiA gene have been separated by of the enzyme. molecular typing into a distinct genotype of B. fragilis with a particular OMP profile [39, 871. Altered PBPs Other types of imipenemases produced by Bacteroides In many species of aerobic gram-negative bacteria, spp. have been described. In 1983, Yotsuji et al. [88] modified PBP affinity has been shown to result in reported a potent P-lactamase produced by a B. fragilis resistance to p-lactam antibiotics [93]. However, there strain with a similar substrate profile to that described is less evidence of changes in particular PBPs as a by Cuchural et al. [42], i.e., capable of hydrolysing resistance mechanism in Bacteroides spp. Bacteroides derivatives and imipenem, and not suscep- are naturally resistant to some p-lactam antibiotics, tible to clavulanic acid. However, EDTA inhibition was including and , because of the not mentioned in this report and this enzyme has been poor affinity of these compounds for the PBPs; assigned to group 4 of the Bush classification scheme binds poorly or undetectably to all PBPs [63]. Also, imipenem resistance (MIC of 16 mg/L) in of B. fragilis [94]. a B. distasonis isolate has been attributed to the combination of production of an unusual imipenem- Accounts of the number and molecular mass of PBPs inactivating serine-b-lactarnase and impermeability of Bacteroides spp. are conflicting, although three [89]. This enzyme, with a molecular mass of high molecular mass PBPs are found consistently and 160 kDa, was inhibited by clavulanic acid and others of lower molecular mass are seen sporadically sulbactam, but not by EDTA. [95-991. Analysis of PBPs from fully sensitive B. fragilis strains in our laboratory yielded results Further attempts have been made to classify the wide broadly similar to those of Wexler and Halebian variety of p-lactamases encountered in bacteroides [96] and Yotsuji et al. [99]; three PBPs of 91, 80 and [76, 771. P-Lactamases produced in raised amounts by 69 kDa were universal, while two others of 63 and clinical Bacteroides isolates have been characterised 47 kDa were detected occasionally [ 1001. The PBPs of according to their antibiotic degradation, inhibitor Bacteroides spp. differ from those of E. coli in terms profiles and specific activity. Two types equated with of their affinity for p-lactam antibiotics and in the the typical p-lactamases of Bush group 2e and the morphological consequences of inhibition of these metallo-P-lactamases of group 3. A third type was proteins. The primary target in bacteroides for most p- produced by strains that exhibited resistance to lactam antibiotics is PBP 2, which is involved in benzylpenicillin and cefoxitin, and reduced suscept- septation and corresponds to PBP 3 of E. coli. The ibility to imipenem; they also displayed intermediate PBP 1 complex is usually the secondary target site or high specific activity. These enzymes showed and is associated with cell elongation, corresponding similar inhibition profiles and hydrolysed p-lacta- to PBP 1 of E. coli. Compounds such as imipenem mase-stable compounds other than imipenem. Others and meropenem bind initially to PBP 3, causing round showed reduced susceptibility to cefoxitin and imipe- cells, and then to PBP 2. PBP 3 in bacteroides is, nem, but these enzymes were unable to hydrolyse therefore, equivalent to PBP 2 in E. coli and is these antibiotics. involved in cell shape. Imipenem also binds to PBP 1 at concentrations correlating with the MIC [95, 981. p-Lactamase production in bacteroides has occasion- ally been found to be transferable. Most genes for p- Several workers have reported an association between lactamase production in bacteroides are located on the reduced affinity of p-lactam compounds for the PBPs chromosome, but the penicillinase belonging to group of Bacteroides spp. and resistance. Georgopapadakou 2d has been transferred from a B. fragilis strain to et al. [98] observed reduced affinity of piperacillin, susceptible strains of B. fragilis and B. vulgatus by in- cefoperazone, cefotaxime, and imipenem vitro filter mating, and this transfer was considered to for PBP 2 in a resistant strain of B. fragilis. Changes be plasmid-mediated [69]. Transmission of resistance in the affinity of PBP 1 or PBP 2 in laboratory- to cefoxitin has also been reported. B. fragilis strain derived mutants have also been correlated with a TAL4 170 was shown to transfer P-lactamase-mediated decrease in susceptibility to cefoxitin [45]. Decreased cefoxitin resistance to a susceptible B. fragilis affinity for PBP 3, and not p-lactamase hydrolysis or recipient by conjugation [90]. Also, cefoxitin resis- membrane permeation, has been implicated as the tance transfer has been demonstrated in a strain of B. important factor in the resistance to ceftezole, thetaiotaomicron, although the precise resistance and cephalothin of a B. fragilis strain [99]. mechanism for this isolate was not determined [91]. Also, the affinity of piperacillin for PBP 1 was Although genes coding for metallo-P-lactamases of B. reduced in a resistant strain of B. uniformis, as was fragilis have been shown to be present on the the binding of cephalothin and cephaloridine to PBP 4 chromosome [79], Bandoh et al. [92] reported a strain [loll. 984 R. EDWARDS

Wexler and Halebian [96] reported changes in both 10. Ingham HR, Selkon JB, Roxby CM. Bacteriological study of the PBP 1 complex and the affinity of one of the PBP otogenic cerebral abscesses: chemotherapeutic role of metro- nidazole. BMJ 1977; 2: 991-993. 1 proteins for cefoxitin between cefoxitin-sensitive and 11. Louie TJ, Bartlett JG, Tally FP, Gorbach SL. Aerobic and cefoxitin-resistant strains of B. thetaiotaomicron. anaerobic bacteria in diabetic foot ulcers. Ann Intern Med Resistant stains of B. uniformis also showed changes 1976; 85: 461-463. 12. Spencer RC. Anaerobic bacteraemia. In: Duerden BI, Draser in the PBP 1 complex in comparison with sensitive BS (eds) Anaerobes in human disease. London, Edward strains. Furthermore, a laboratory-derived cefoxitin- Arnold. 1991: 324-342. resistant mutant of B. distasonis displayed reduced 13. Rasmussen BA, Bush K, Tally FP. Antibiotic resistance in Bacteroides. Clin Infect Dis 1993; 16 Suppl 4: S390-S400. binding to the PBP 1 complex compared with its wild- 14. Wexler HM, Finegold SM. Antimicrobial resistance in type parent and cefoxitin-sensitive revertant. No Bacteroides. J Antimicrob Chemother 1987; 19: 143- 146. obvious changes in OMP profiles were detected that 15. Sutter VL, Finegold SM. Susceptibility of anaerobic bacteria to 23 antimicrobial agents. Antimicrob Agents Chemother might indicate changes in permeability. In a recent 1976; 10: 736-752. study of B. fragilis strains that do not produce 16. Rolfe RD, Finegold SM. Comparative in vitro activity of new carbapenemase, resistance to imipenem was not beta-lactam antibiotics against anaerobic bacteria. Antimicrob Agents Chemother 1981; 20: 600-609. associated with loss of high molecular mass PBPs, 17. Clarke AM, Zemcov SJ. Comparative in-vitro activity of but with reduced binding of imipenem to these PBPs, temocillin (BRL 17421), a new penicillin. J Antimicrob together with the appearance of a new low molecular Chemother 1983; 11: 319-324. 18. Cuchural GJ, Tally FP, Jacobus NV et al. Comparative mass PBP [loo]. activities of newer p-lactam agents against members of the Bacteroides fragilis group. Antimicrob Agents Chemother 1990; 34: 479-480. Concluding remarks 19. Jacobs MR, Spangler SK, Appelbaum PC. Beta-lactamase production and susceptibility of US and European anaerobic gram-negative bacilli to beta-lactams and other agents. Eur J In common with other bacterial genera, resistance Clin Microbiol Infect Dis 1992; 11; 1081-1093. among the bacteroides will continue to increase with 20. Olsson-Liljequist B, Dornbusch K, Nord CE. Characterization antibiotic use. The activity of p-lactam compounds is of three different /3-lactamases from the Bacteroides fragilis group. Antimicrob Agents Chemother 1980; 18: 220-225. particularly under threat, as most strains possess p- 21. Cuchural GJ, Tally FP, Jacobus W, Marsh PK, Mayhew Jw. lactamases and increased production of these enzymes Cefoxitin inactivation by Bacteroides fragilis. Antimicrob enhances resistance to some commonly used agents. Agents Chemother 1983; 24: 936-940. 22. Phillips I, King A, Nord CE, Hoffstedt B. Antibiotic The potential for wider distribution of other potent p- sensitivity of the Bacteroides fragilis group in Europe. lactamases, including the metallo-/3-lactamases, is also European Study Group. Eur J Clin Microbiol Infect Dis worrying. The possibility of conversion of the metallo- 1992; 11: 292-304. 23. Jacobus NV, Curchural GJ, Tally FP. In-vitro susceptibility of P-lactamase gene from ‘silent’ to full expression, with the Bacteroides fragilis group and the inoculum effect of corresponding resistance to carbapenems and common the newer P-lactam antibiotics on this group of organisms. /3-lactamase inhibitor combinations, is of particular J Antimicrob Chemother 1989; 24: 675-682. 24. Betriu C, Campos E, Cabronero C, Rodriguez-Avial C, Picazo concern. There is a need to monitor the degree of JJ. Susceptibilities of species of the Bacteroides fragilis group resistance among clinical isolates of Bacteroides spp., to 10 antimicrobial agents. Antimicrob Agents Chemother together with P-lactamase types and levels of produc- 1990; 34: 671-673. 25. Martin DA, Saunders CV, Marier RL. N-formimidoyl tion. Also, prudent use of highly active antibiotics is (MK 0787): in vitro activity against anaerobic necessary to extend their usehl life. bacteria. Antimicrob Agents Chemother 1982; 21: 168- 169. 26. Edwards JR, Turner PJ, Wannop C, Withell EW, Grindey AJ, Nairn K. In vitro antibacterial activity of SM-7338, a carbapenem antibiotic with stability to dehydropeptidase I. References Antimicrob Agents Chemother 1989; 33: 215-222. 27. Aldridge ICE, Morice N, Schiro DD. In vitro activity of 1. Veillon A, Zuber A. Recherches sur quelques microbes Biopenem (L-627), a new carbapenern, against anaerobes. strictement anaerobies et leur r6le en pathologie. Arch Mkd Antimicrob Agents Chemother 1994; 38: 889-893. Exper Anat Path 1898; 10: 517-545. 28. Betriu C, Cabronero C, Gomez M, Picazo JJ. Changes in the 2. Finegold SM. Pathogenic anaerobes. Arch Intern Med 1982; susceptibility of Bacteroides fragilis group organisms to 142: 1988-1992. various antimicrobial agents 1979- 1989. Eur J Clin Micro- 3. Hedberg M, Nord CE. Antimicrobial-resistant anaerobic biol Infect Dis 1992; 11: 352-356. bacteria in human infections. Med Microbiol Lett 1996; 5: 29. Goldstein EJC, Citron DM, Cherubin CE, Hillier SL. 295-304. Comparative susceptibility of the Bacteroides fragilis group 4. McGowan K, Gorbach SL. Anaerobes in mixed infections. species and other anaerobic bacteria to meropenem, imipe- J Infect Dis 1981; 144: 181-186. nem, piperacillin, cefoxitin, ampicillidsulbactam, clindamycin 5. Namavar F, Theunissen EBM, Verweij-Van Vught AMJJ et al. and metronidazole. J Antimicrob Chemother 1993; 31: Epidemiology of the Bacteroides fragilis group in the colonic 363-372. flora in 10 patients with colonic cancer. J Med Microbiol 30. Edwards R, Thirlwell D, Greenwood D. Changes in P-lactam 1989; 29: 171-176. antibiotic susceptibility and P-lactamase production of clinical 6. Finegold SM. Anaerobic infections in humans: an overview. isolates of Bacteroides and Prevotella species over a 9 year Anaerobe 1995; 1: 3-9. period. J Antimicrob Chemother 1996; 37: 636-638. 7. Gorbach SL, Bartlett JG. Medical progress: anaerobic 31. Diedrich DL, Martin AE. Outer membrane proteins of infections. 1. N Engl J Med 1974; 290: 1177-1 184. Bacteroides fragilis group. Curr Microbiol 1981; 6: 85-88. 8. Thadepalli H, Gorbach SL, Keith L. Anaerobic infections of 32. Kotarski SF, Salyers AA. Isolation and characterization of the female genital tract - bacteriologic and therapeutic outer membranes of Bacteroides thetaiotaomicron grown on aspects. Am J Obstet Gynecol 1973; 117: 1034-1040. different carbohydrates. J Bacteriol 1984; 158: 102- 109. 9. Bartlett JG, Finegold SM. Anaerobic infections of the lung 33. Wexler HM, Getty C, Fisher G. The isolation and and pleural space. Am Rev Respir Dis 1974; 110: 56-77. characterisation of a major outer-membrane protein from P-LACTAM RESISTANCE IN BACTEROIDES SPP. 985

Bacteroides distasonis. J Med Microbiol 1992; 37: 165-175. 58. Brown JE, Del Bene VE, Collins CD. In vitro activity of N- 34. Wexler HM, Getty C. The isolation and characterisation of a formimidoyl thienamycin, moxalactam and other new beta- porin protein from Bacteroides fragilis. Anaerobe 1996; 2: lactam agents against Bacteroides fragilis: contribution of 305-312. beta-lactamase to resistance. Antimicrob Agents Chemother 35. Kanazawa K, Kobayshi Y, Nakano M, Sakurai M, Gotoh N, 1981; 19: 248-252. Nishino T. Identification of three porins in the outer 59. Timewell R, Taylor E, Phillips I. The P-lactamases of membrane of Bacteroides fragilis. FEMS Micmbiol Lett Bacteroides species. J Antimicrob Chemother 198I ; 7: 1995; 127: 181-186. 137-146. 36. Yoshimura F, Nikaido H. Diffusion of p-lactam antibiotics 60. Tajima M, Sawa K, Watanabe K, Ueno K. The P-lactamases through the porin channels of Escherichia coli K-12. of genus Bacteroides. J Antibiot 1983; 36: 423-428. Antimicrob Agents Chemother 1985; 27: 84-92. 61. Richmond MH, Sykes RB. The p-lactamases of gram- 37. Curchural GJ, Hurlbut S, Malamy MH, Tally FP. Permeability negative bacteria and their possible physiological role. Adv to P-lactams in Bacteroides fragilis. J Antimicrob Chemother Microb Physiol 1973; 9: 31-88. 1988; 22: 785-790. 62. Neu HC. Antibiotic inactivating enzymes and bacterial 38. Hiruma R, Yamaguchi A, Sawai T. The effect of lipopoly- resistance. In: Lorian V (ed) Antibiotics in laboratory saccharide on lipid bilayer permeability of 8-lactam anti- medicine. Baltimore, Williams and Wilkins. 1986: 757-789. biotics. FEBS Lett 1984; 170: 268-272. 63. Bush K, Jacoby GA, Medeiros AA. A functional classification 39. Edwards R, Greenwood D. Outer membrane analysis of B. scheme for P-lactamases and its correlation with molecular ,fragilis strains with reduced susceptibility to imipenem. In: structure. Antimicrob Agents Chemother 1995; 39: 1211- Duerden BI, Wade WG, Brazier JS, Wren WG, Hudson MJ 1233. (eds) Medical and dental aspects of anaerobes. Northwood 64. Rogers MB, Parker AC, Smith CJ. Cloning and characteriza- Middlesex, Science Reviews. 1993: 307-309. tion of the endogenous cephalosporinase gene, cepA, from 40. Dornbusch K, Olsson-Liljequist B, Nord CE. Antibacterial Bacteroides fragilis reveals a new subgroup of Ambler Class activity of new P-lactam antibiotics on cefoxitin-resistant A P-lactamases. Antimicrob Agents Chemother 1993; 37: strains of Bacteroides fragilis. J Antimicrob Chemother 1980; 2391 -2400. 6: 207-216. 65. Ambler RP. The structure of p-lactamases. Philos Trans R 41. Olsson B, Dornbusch K, Nord CE. Factors contributing to Soc London B. Biol Sci 1980; 289: 321-331. resistance to beta-lactam antibiotics in Bacteroides fragilis. 66. Parker AC, Smith CJ. Genetic and biochemical analysis of a Antimicrob Agents Chemother 1979; 15: 263-268. novel ambler class A P-lactamase responsible for cefoxitin 42. Cuchural GJ, Malamy MH, Tally FP. P-Lactamase-mediated resistance in Bacteroides species. Antimicrob Agents Che- imipenem resistance in Bacteroides fragilis. Antimicrob mother 1993; 37: 1028-1036. Agents Chemother 1986; 30: 645-648. 67. Smith CJ, Bennett TK, Parker AC. Molecular and genetic 43. Crosby MA, Gump DW. Activity of cefoperazone and two P- analysis of the Bacteroides uniformis cephalosporinase gene, lactamase inhibitors, sulbactam and clavulanic acid, against cblA, encoding the species-specific P-lactamases. Antimicrob Bacteroides spp. correlated with P-lactamase production. Agents Chemother 1994; 38: 17 1I - 1715. Antimicrob Agents Chemother 1982; 22: 398-405. 68. Hedberg M, Lindqvist L, Bergman T, Nord CE. Purification 44. Malouin F, Lamothe F. The role of 8-lactamases and the and characterization of a new P-lactamase from Bacteroides permeability barrier on the activity of cephalosporins against uniformis. Antimicrob Agents Chemother 1995; 39: 1458- members of the Bacteroides fragilis group. Can J Microbiol 1461. 1987; 33: 262-266. 69. Sat0 K, Matsuura Y, Inoue M, Mitsuhashi S. Properties of a 45. Piddock LJ, Wise R. Cefoxitin resistance in Bacteroides new pencillinase type produced by Bacteroides fragilis. species: evidence indicating two mechanisms causing decreased Antimicrob Agents Chemother 1982; 22: 579-584. susceptibility. J Antimicrob Chemother 1987; 19: 161-170. 70. Yang Y, Rasmussen BA, Bush K. Biochemical characteriza- 46. Iaconis JP, Nadler HL, Sheikh W. Potential factors influencing tion of the metallo-P-lactamase CcrA from Bacteroides carbapenem activity against Bacteroides fragilis group fragilis TAL3636. Antimicrob Agents Chemother 1992; 36: isolates. Clin Infect Dis 1995; 20 Suppl 2: S361-S363. 1155-1 157. 47. Nord CE, Hedberg M. Resistance to P-lactam antibiotics in 71. Ajiki Y, Koga T, Ohya S et al. P-Lactamase produced by a anaerobic bacteria. Rev Infect Dis 1990; 12 Suppl 2: S231- highly P-lactam-resistant strain of Bacteroides fragilis: an S234. obstacle to the chemotherapy of experimental mixed infec- 48. Garrod LP. Sensitivity of four species of Bacteroides to tions. J Antimicrob Chemother 1991; 28: 537-546. antibiotics. BMJ 1955; ii: 1529- 153 1. 72. Bandoh K, Muto Y, Watanabe K, Katoh N, Ueno K. 49. Pinkus G, Veto G, Braude AI. Bacteroides penicillinase. Biochemical properties and purification of metallo-p-lacta- J Bacteriol 1968; 96: 1437-1438. mase from Bactemides fragilis. Antimicrob Agents Chemother 50. Olsson B, Dornbusch K, Nord CE. Susceptibility to beta- 1991; 35: 371-372. lactam antibiotics and production of beta-lactamases in 73. Hedberg M, Edlund C, Lindqvist L, Rylander M, Nord CE. Bacteroides fragilis. Med Microbiol Immunol 1977; 163: Purification and characterization of an imipenem hydrolysing 1 83- 194. metallo-P-lactamase from Bacteroides fragilis. J Antimicrob 51. Dias MBS, Jacobus NV, Tally FP. In-vitro activity of Chemother 1992; 29: 105-113. cefoperazone-sulbactam against Bacteroides species. J Anti- 74. Livermore DM. Carbapenemases: the next generation of P- microb Chemother 1986; 18: 467-471. lactamases? ASM News 1993; 59: 129-134. 52. Cornick NA, Cuchural GJ, Snydman DR et al. The 75. Livermore DM. Carbapenemases. J Antimicrob Chemother antimicrobial susceptibility patterns of the Bacteroides fragilis 1992; 29: 609-612. group in the United States, 1987. J Antimicrob Chemother 76. Eley A, Greenwood D. Characterization of 8-lactamases in 1990; 25: 101 1-1019. clinical isolates of Bacteroides. J Antimicrob Chemother 53. Rogers MB, Bennett TK, Payne CM, Smith CJ. Insertional 1986; 18: 325-333. activation of cepA leads to high-level beta-lactamase expres- 77. Edwards R, Greenwood D. An investigation of P-lactamases sion in Bacteroides fragilis clinical isolates. J Bacteriol 1994; from clinical isolates of Bacteroides species. J Med Microbiol 176: 4376-4384. 1992; 36: 89-95. 54. Anderson JD, Sykes RB. Characterisation of a p-lactamase 78. Rasmussen BA, Gluzman Y, Tally FP. Cloning and sequen- obtained from a strain of Bacteroides fragilis resistant to P- cing of the class B P-lactamase gene (ccrA) from Bacteroides lactam antibiotics. J Med Microbiol 1973; 6: 201-206. fragilis TAL3636. Antimicrob Agents Chemother 1990; 34: 55. Olsson B, Nord C-E, Wadstrom T. Formation of P-lactamase 1590-1592. in Bacteroides fragilis: cell-bound and extracellular activity. 79. Thompson JS, Malamy NH. Sequencing the gene for an Antimicrob Agents Chemother 1976; 9: 727-735. imipenem-cefoxitin-hydrolyzingenzyme (CfiA) from Bacter- 56. Wise R. Clavulanic acid and susceptibility of Bacteroides oides fragilis TAL2480 reveals strong similarity between CfiA fragilis to penicillin. Lancet 1977; 2: 145. and Bacillus cereus P-lactamase 11. J Bacteriol 1990; 172: 57. Britz ML, Wilkinson RG. Purification and properties of a 2584-2593. beta-lactamase from Bacteroides fragilis. Antimicrob Agents 80. Rasmussen BA, Gluzman Y, Tally FP. Escherichia coli Chemother 1978; 13: 373-382. chromosomal mutations that permit direct cloning of the 986 R. EDWARDS

Bacteroides fyagilis metallo-a-lactamase gene, ccrA. Mol 90. Cuchural GJ, Tally FP, Storey JR, Malamy MH. Transfer Microbiol 1991; 5: 1211-1219. of p-lactamase-associated cefoxitin resistance in Bacter- 81. Podglajen I, Breuil J, Bordon F, Gutmann L, Collatz E. A oides fragilis. Antimicrob Agents ('hemother 1986; 29: silent carbapenemase gene in strains of Bacteroides fragilis 918-920. can be expressed after a one-step mutation. FEMS Microbiol 9 1. Rashtchian A, Dubes GR, Booth SJ. Transferable resistance to Lett 1992; 91: 21-30. cefoxitin in Bacteroides thetaiotaomicron. Antimicrob Agents 82. Rasmussen BA, Yang Y, Jacobus N, Bush K. Contribution of Chemother 1982; 22: 701-703. enzymatic properties, cell permeability, and enzyme expres- 92. Bandoh K, Watanabe K, Muto Y, Tanaka Y, Kato N, Ueno K. sion to microbiological activities of p-lactams in three Conjugal transfer of imipenem resistance in Bacteroides Bacteroides fragilis isolates that harbor a metallo-P-lactamase fragilis. J Antibiot 1992; 45: 542-547 gene. Antimicrob Agents Chemother 1994; 38: 21 16-2120. 93. Malouin F, Bryan LE. Modification of penicillin-binding 83. Podglajen I, Breuil J, Coutrot A, Gutmann L, Collatz E,. proteins as mechanisms of /3-lactam resistance. Antimicrob Incidence of the carbapenem (Cpm) resistance gene cfiA and Agents Chemother 1986; 30: 1-5. variability of its genomic environment in Cpm-resistant and 94. Georgopapadakou NH, Smith SA, Sykes RB. Mode of action -susceptible clinical isolates of Bacteroides fragilis. In: of aztreonam. Antimicrob Agents Chemother 1982; 21 : Abstracts of the 32nd Interscience Conference on Antimicro- 950-956. bial Agents and Chemotherapy, 1992. Abstract 583: 208. 95. Piddock LJ, Wise R. Properties of pencillin-binding proteins 84. Podglejen I, Bred J, Collatz E. Insertion of a novel DNA of four species of the genus Bacteroides. Antimicrob Agents sequence, IS 1 186, upstream of the silent carbapenemase gene Chemother 1986; 29: 825-832. cJiA, promotes expression of carbapenem resistance in clinical 96. Wexler HM, Halebian S. Alterations to the penicillin-binding isolates of Bacteroides fragilis. Mol Microbiol 1994; 12: proteins in the Bacteroides fragilis group: a mechanism for 105-1 14. non-p-lactamase mediated cefoxitin resistance. J Antimicrob 85. Turner P, Edwards R, Weston V, Gazis A, Ispahani r), Chemother 1990; 26: 7-20. Greenwood D. Simultaneous resistance to metronidazole, co- 97. Botta GA, Privitera G, Menozzi MG. Penicillin-binding amoxiclav, and imipenem in a clinical isolate of Bacteroides proteins in Bacteroides fragilis and their affinities for several frugilis. Lancet 1995; 345: 1275-1277. new cephalosporins. J Antimicrob Chemother 1983; 11: 86. Khushi T, Payne DJ, Fosberry A, Reading C. Production of 325-33 1. metal dependent P-lactamases by clinical strains of Bacter- 98. Georgopapadakou NH, Smith SA, Sykes RB. Penicillin- oides fragilis isolated before 1987. J Antimicrob Chemother binding proteins in Bacteroides fragilis. J Antibiot 1983; 1996; 37: 345-350. 36: 907-910. 87. Podglajen I, Bred J, Casin I, Collatz E. Genotypic 99. Yotsuji A, Mitsuyama J, Hori R et al. Mechanism of action of identification of two groups within the species Bucteroides cephalosporins and resistance caused by decreased affinity for Jkagilis by ribotyping and by analysis of PCR-generated penicillin-binding proteins in Bacteroides fragilis. Antimicrob fragment patterns and insertion sequence content. J Bacteriol Agents Chemother 1988; 32: 1848-1853. 1995; 177: 5270-5275. 100. Edwards R, Greenwood D. Mechanisms responsible for 88. Yotsuji A, Minami S, Inoue M, Mitsuhashi S. Properties of reduced susceptibility to imipenem in Bacteroides fragilis. novel P-lactamase produced by Bacteroides fragilis. Antz- J Antimicrob Chemother 1996; 38: 94 1-951. microb Agents Chemother 1983; 24: 925-929. 101. Hedberg M, Bush K, Bradford PA et al. The role of 89. Hurlbut S, Cuchural GJ, Tally FP. Imipenem resistance in penicillin-binding-proteins for 8-lactam resistance in a P- Bacteroides distasonis mediated by a novel P-lactamase. lactamase producing Bacteroides unij2wmis strain. Anaerobe Antimicrob Agents Chemother 1990; 34: 117-120. 1996; 2: 111-115.