A recent fixation of cfiA genes in a monophyletic cluster of Bacteroides fragilis is correlated with the presence of multiple insertion elements

Abstract

Small-subunit ribosomal DNA sequences of 16 strains of Bacteroides fragilis were determined and compared with previously published sequences. Three phylogenetic methods (the neighbor-joining, maximum-likelihood, and maximum-parsimony methods) as well as a bootstrap analysis were used to assess the robustness of each topology. All phylogenetic analyses demonstrated that the B. fragilis strains were clearly divided into two robust monophyletic units which corresponded to the cfiA-negative and cfiA-positive groups. Strains of two previously identified DNA homology groups separated similarly into these two monophyletic units. According to the intensity of the hybridization signal with a cfiA probe, the cfiA-positive cluster could be further divided into two groups. This difference might reflect the existence of two, probably closely related cfiA-type genes. In the strongly hybridizing cfiA-positive strains, the gene is capable of conferring high-level resistance to the carbapenems and to most ␤-lactamase inhibitors as well, while in the weakly hybridizing cfiA-positive strains, only the latter type of resistance is known to occur. The presence of the cfiA-type genes within a monophyletic cluster of B. fragilis that apparently represents only a minority of the species B. fragilis is suggestive of a recent acquisition. The fact that this cluster is also the predominant pool of all known B. fragilis insertion elements, which have been found to play an important role in the expression of carbapenem resistance, raises the possibility that both genetic determinants, i.e., the resistance gene(s) and insertion elements, may have coevolved.Bacteroides fragilis, the anaerobic bacterial species most frequently isolated from human infections (3, 4), shows little phenotypic variability but can nevertheless be assigned to different genotypic groups (11,22). DNA homology studies led to the distinction of two homology groups, called I and II (11). DNA relatedness between B. fragilis ATCC 25285 T , representative of homology group I, and strains of homology group II ranged from 64 to 72%, while intragroup relatedness ranged from 80 to 90% (11). About 80% of B. fragilis strains isolated in clinical studies can be assigned to homology group I (11). Although strains from homology groups I and II are phenotypically indistinguishable (12), strains of homology group II lack susceptibility to the synergistic effect of such ␤-lactamase inhibitors as clavulanic acid, sulbactam, and tazobactam (2, 40), which are potent inhibitors of the vast majority of the ␤-lactamases produced by clinical isolates of B. fragilis (3,4,10,20,40). These are active-site serine enzymes, loosely related to those of Ambler's class A (1, 17, 28). Probing with cfiA gene sequences led to the distinction within B. fragilis of the cfiAnegative and cfiA-positive groups, representing ca. 3 and 97%, respectively, of the clinical B. fragilis isolates (22, 23). The cfiA gene, which has been searched for only in B. fragilis, encodes a metallo-␤-lactamase (5, 21, 25, 37), i.e...

Identification of two genetic groups in Bacteroides fragilis by multilocus enzyme electrophoresis: distribution of antibiotic resistance (cfiA, cepA) and enterotoxin (bft) encoding genes The GenBank accession numbers for the sequences determined in this work are AF197508–AF197534.

“…The second group was characterized by the absence of the cfiA gene and of the associated insertion sequences, the frequent presence of the cepA gene, and a higher genetic heterogeneity (Podglajen et al, 1995 ;Ruimy et al, 1996). By including strains obtained from Johnson (1978) in their 16S rRNA sequence comparison, Ruimy et al (1996) showed that the two groups described above could be related to the DNA homology groups II and I, respectively. Despite these findings, there is as yet no information dealing with the genetic relationship between invasive and noninvasive strains, human and animal strains and strains of different geographical origin.…”

“…Three specific insertion sequence elements, IS1186, IS942 and IS4351, providing the promoter region for the cfiA gene, were shown to be confined to this group (Podglajen et al, 1995). The second group was characterized by the absence of the cfiA gene and of the associated insertion sequences, the frequent presence of the cepA gene, and a higher genetic heterogeneity (Podglajen et al, 1995 ;Ruimy et al, 1996). By including strains obtained from Johnson (1978) in their 16S rRNA sequence comparison, Ruimy et al (1996) showed that the two groups described above could be related to the DNA homology groups II and I, respectively.…”

“…Similarly, two genotypically distinct B. fragilis groups have been identified on the basis of ribotyping, restriction fragment length polymorphism (RFLP), PCR-generated fingerprinting, insertion sequence (IS) content and 16S rRNA sequence alignments (Moraes et al, 1999 ;Kleivdal & Hofstad, 1995 ;Podglajen et al, 1995 ;Ruimy et al, 1996 ;Smith & Callihan, 1992). One group was characterized by the presence of the cfiA gene (encoding a metallo-β-lactamase of Ambler's class B) and the absence of the cepA gene (encoding a β-lactamase of Ambler's class A).…”

recA and glnA sequences separate the Bacteroides fragilis population into two genetic divisions associated with the antibiotic resistance genotypes cepA and cfiA

“…1 and 2). Ruimy et al [4], using a 729-bp internal fragment of the c®A sequence as a probe, demonstrated two types of c®A genes. One type was characterised by a strong hybridisation signal and the other reacted only weakly; this hybridisation difference might have been due to the existence of the two alleles found in the present study.…”

“…Occasionally, some strains produce an enterotoxin [1,2]. Studies with multilocus enzyme electrophoresis (MLEE) [3] and other molecular techniques ± particularly 16S rRNA sequencing [4] and DNA±DNA hybridisation [5] ± have shown the separation of the population into two groups presenting an extensive genetic distance one from the other (divisions I and II). Division I was characterised by the frequent presence of the cepA gene (encoding a serine-â-lactamase of Ambler's class A) and the absence of the c®A gene (encoding a metallo-â-lactamase of Ambler's class B).…”