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Molecular characterization of imipenem-resistant, cfiA-positive Bacteroides fragilis isolates from the USA, Hungary and Kuwait

¹Institute of Clinical Microbiology, University of Szeged, Szeged, Hungary ²Department of Medicine, Microbiology and Immunology, Loyola University Chicago, Maywood, and Department of Medicine, VA Hospital, Hines, USA ³Division of Microbiology, University Hospital, Queen's Medical Centre, Nottingham, UK ⁴Department of Microbiology, Faculty of Medicine, University of Kuwait, Kuwait ⁵`Vitality’ Laboratory, Biological Research Center of Hungarian Academy of Sciences, Szeged, Hungary

Correspondence József Sóki soki{at}mlab.szote.u-szeged.hu

Received September 4, 2003
Accepted January 23, 2004

Fifteen Bacteroides fragilis isolates from the USA, Hungary and Kuwait were examined for carbapenem resistance, for carbapenemase activity and, with the use of various PCR-based methods and nucleotide sequencing, for cfiA genes and activating insertion sequence (IS) elements. All the B. fragilis isolates were cfiA-positive, 10 of the cfiA genes being upregulated by IS elements that are already known. Of these 10, one was of a novel type (designated IS943) and two further ones (IS614B and IS614C) were suspected hybrids of IS612, IS614 and IS942. There were five cfiA-positive imipenem-resistant B. fragilis isolates with elevated imipenem MICs (minimal inhibitory concentration) that harboured no IS insertion upstream of the cfiA gene, but produced carbapenemase; these isolates might possess a novel activation mechanism. On the basis of the available phenotypic and genotypic evidence, the present data suggest that there are at least two cfiA activation mechanisms among B. fragilis isolates.

The GenBank accession numbers for the sequences of conserved regions upstream of cfiA in B. fragilis 21216, 388/1, 388/2, 19841 and 16997 are AY373492, AY373493, AY373494, AY373495 and AY374121, respectively. The GenBank accession number for the nucleotide sequence of insertion sequence element IS943 is AF519175.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Species of the genus Bacteroides sensu stricto (or the Bacteroides fragilis group), the most significant anaerobic pathogens and important intestinal commensalists, possess the most numerous resistance mechanisms and highest resistance percentages among all human pathogenic anaerobes (Rasmussen et al., 1993). The antibiotic therapy used to combat Bacteroides includes nitroimidazoles, carbapenems and the fourth-generation fluoroquinolones, alone or in combination with anti-aerobic drugs if needed. However, strains resistant to these antibiotics have been isolated in several countries (Rasmussen et al., 1993). Carbapenem-resistant Bacteroides isolates were first reported in Japan and subsequently in the USA (Yotsuji et al., 1983; Cuchural et al., 1986). This resistance was shown to be due to an EDTA-sensitive, clavulanic acid-insensitive, Zn²⁺-requiring metallo-ß-lactamase, together with the presence of a permeability barrier (Cuchural et al., 1986; Thompson & Malamy, 1990). Cloning of the genes (cfiA or ccrA) revealed that they were similar to the metallo-ß-lactamase type II Blm protein of Bacillus cereus (Rasmussen et al., 1990; Thompson & Malamy, 1990) and belong in Ambler Class B and functional group 3a (Bush et al., 1995). Further characterization of cfiA-positive B. fragilis strains demonstrated that the cfiA gene is not expressed highly in most isolates in the Bacteroides system, and imipenem-susceptible strains may possess the cfiA gene. However, strains with these ‘silent’ cfiA genes can convert to resistant ones via a one-step mutation into the upstream region of cfiA genes by insertion sequence (IS) elements (Podglajen et al., 1992). The roles of such IS elements (e.g. IS614, IS1186, IS942 and IS1169) have been well documented (Podglajen et al., 1994; Rasmussen & Kovacs, 1991; Edwards & Read, 2000; Kato et al., 2003) and it has been proven that they harbour multiple outward-oriented Bacteroides and Escherichia coli promoters capable of driving the expression of cfiA (Bayley et al., 2000; Podglajen et al., 2001).

The cfiA-positive B. fragilis isolates are not only intriguing from the aspect of the special expression mechanism; typing methods have furnished evidence that they also comprise a distinct subgroup of the species, which seems to be identical to the chromosomal DNA homology group II of B. fragilis established by Johnson (Johnson, 1978; Podglajen et al., 1995; Ruimy et al., 1996; Gutacker et al., 2000).

Despite the fact that the roles of the silent cfiA gene and this grouping are not well known, imipenem-resistant B. fragilis strains have been isolated in several countries and the prevalences of the cfiA-positive strains have also been estimated (see Podglajen et al., 1995; Edwards et al., 1999; Yamazoe et al., 1999; Sóki et al., 2000 and das Gracas Silva E Souza et al., 2000 for France, UK, Japan, Hungary and Brazil, respectively). These findings have revealed the very low rate of expression (1 %) and the low prevalence of the overall cfiA positivity (2–7 %). To date, only phenotypic tests have been used to measure the frequency of imipenem-resistant B. fragilis strains (0.2 %) in the USA (Snydman et al., 1999).

The aim of the present study was to examine the resistance mechanisms of novel imipenem-resistant Bacteroides isolates from the USA, Hungary and Kuwait by molecular methods.

	METHODS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Bacterial strains, growth conditions and measurement of antibiotic susceptibility and specific carbapenemase activity.

Fifteen selected B. fragilis strains were examined that were positive for the cfiA gene and had at least elevated imipenem MICs (>1 µg ml^–1). Ten of these isolates were from the USA (from various hospitals in Chicago), three were newly isolated from Hungary (hospitals in Székesfehérvár, Gyula and Budapest) and two were from Kuwait. Their sources and the important antibiotic susceptibilities are listed in Table 1. The original identifications by conventional biochemical methods were confirmed by means of the ATB System (bioMérieux). The test isolates were stored and cultivated as previously described (Sóki et al., 2000); for liquid cultures, brain heart infusion broth supplemented with 0.5 % (w/v) yeast extract, 5 mg haemin l^–1 and 1 mg vitamin K₁ l^–1 was used. Antibiotic susceptibilities were determined by means of Etests, under the conditions recommended by the supplier (AB Biodisc) and the NCCLS (2001), with B. fragilis ATCC 25285^T (also known as NCTC 9343^T) or Bacteroides thetaiotaomicron ATCC 29741^T as the control organism. For PCR detection of the cfiA genes, B. fragilis TAL3636 (a metallo-ß-lactamase producer) and B. fragilis ATCC 23745^T (NCTC 9344^T, cfiA-negative) were used as controls.

Specific carbapenemase activities were determined as previously described (Edwards et al., 1999). Carbapenemase was inhibited by preincubating cell extracts with 25 mM EDTA for 1 h at 4 °C before measurements.

PCR experiments.

PCR templates were prepared by boiling 0.5 McFarland turbidity cell suspensions in water, or by the method of Wilson (1994). The concentrations of the chromosomal DNA samples in this latter method were determined by UV spectrophotometry at 260 and 280 nm and, for PCR amplification, they were diluted to 5 ng µl^–1.

Regular amplification mixtures (50 µl) for PCR contained 1xReadyMix solution [10 mM Tris/HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl₂, 0.001 % gelatin, 0.2 mM dNTP mix, stabilizers and 1.25 U Taq polymerase (Sigma)], 0.7 µM of each primer and 5 µl (approx. 25 ng) template DNA. Primer sequences are presented in Table 2.

The PCR strategy included (i) PCR to detect the cfiA gene (primers cfiA1 and cfiA2), (ii) PCR to detect known IS elements (primers starting with IS and the appropriate numbering), (iii) ‘PCR-mapping’ –a PCR step to determine whether the IS element was upstream of the cfiA; this included a forward primer specific to an insertion sequence and a reverse primer at either the 5' (primer Up2) or 3' (primer cfiA2) end of cfiA and (iv) upstream PCR to sequence the region upstream of cfiA; this included a forward primer common to the upstream regions of cfiA genes (primer G) and a reverse primer from the 5' region of cfiA (primer Up2), as previously described (Podglajen et al., 1994, 1995; Edwards & Read, 2000). The PCR strategy and the positions of primers are depicted in Fig. 1.

View larger version (6K): [in this window] [in a new window]   Fig. 1. Schematic diagram of the PCR strategy. cfiA and the IS element are indicated by bars, and the 5' and 3' flanking sequences of cfiA by lines. The possible IS insertion is depicted by lines pointing to this region within the 5' upstream region. Positions of primers are represented by arrows and the primer designation is given, with the exception of IS primers, where general designations are given. cfiA1 and cfiA2 primers served for amplification of the resistance gene, different IS forward and IS reverse primers (Table 2) for amplification of different IS elements, IS forward and Up2 or cfiA2 for ‘PCR-mapping’ of the IS elements and G and Up2 to amplify the 5' region of cfiA to determine whether IS elements are present or not.

The amplification protocol included 35 cycles of 30 s at 94 °C, 1 min at 50 °C and 1 min at 72 °C (steps for cfiA amplification), 35 cycles of 30 s at 94 °C, 1 min at 48 °C and 1 min at 72 °C (steps for IS element detection), 35 cycles of 30 s at 94 °C, 1 min at 50 °C and 3 min at 72 °C (steps for PCR-mapping of IS elements) and 35 cycles of 30 s at 94 °C, 1 min at 52 °C and 3 min at 72 °C (steps for amplification of the upstream regions of the cfiA genes), with 5 min at 94 °C as starting denaturation and 10 min at 72 °C as the final extension step for all amplification cycles.

PCR for IS943 sequencing and agarose gel electrophoresis.

For amplification of the IS943 element upstream of a cfiA gene in the B. fragilis 6729 isolate, a thermostable DNA polymerase mixture (KlenTaq LA; Sigma) was used in 100 µl total volume, using 20 ng chromosomal DNA, with the following cycling parameters: 1 min starting denaturation at 95 °C, 35 cycles of 30 s at 94 °C, 15 s at 55 °C, 4 min at 68 °C and a 10 min final elongation step at 68 °C, under the conditions recommended by the supplier.

PCR products were analysed on 0.8–1.5 % agarose gels containing 0.5 µg ethidium bromide ml^–1 in TBE buffer (45 mM Tris/borate, 1 mM EDTA, pH 8.26), using a 4 V cm^–1 voltage gradient, together with molecular mass markers from Sigma and Fermentas, visualized with UV light and recorded with a Kodak EDAS290 camera and the Kodak 1D 3.5 image analysis system (Eastman Kodak).

Sequencing of PCR fragments of IS elements and nucleotide and amino acid sequence analysis.

PCR fragments were purified by extraction from agarose gels (QIAquick gel extraction kit; Qiagen) or precipitated with 0.5 vol. ammonium acetate (7.5 M) and 2.5 vols absolute ethanol. DNA sequencing was performed with an automated sequencer (ABI PRISM model 373; Applied Biosystems) with the AmpliTaq FS DyeDeoxy terminator cycle sequencing kit (Applied Biosystems). The different sequence reads from the primer walking of IS943 were assembled and analysed by the Lasergene suite (DNAStar). The DNA sequences obtained were compared with nucleotide sequences in GenBank at the Internet site of the National Library of Biotechnical Information (http://www.ncbi.nlm.nih.gov/) by means of the BLAST client program (Altschul et al., 1997).

The IS943 nucleotide sequence was deposited in GenBank under the accession number AF519175; the accession numbers for the sequences upstream of the cfiA genes in B. fragilis strains 388/1, 388/2, 16997, 19841 and 21216 were, respectively, AY373493, AY373494, AY374121, AY373495 and AY373492.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Phenotypic characterization of the B. fragilis isolates

From among 30 Bacteroides isolates in our collection that exhibited at least elevated imipenem MICs (1 µg ml^–1), the antibiotic-resistance mechanisms of the 15 cfiA-positive B. fragilis isolates were examined and are reported here. Antibiotic susceptibility data on these isolates from the USA, Hungary and Kuwait were determined for cefoxitin, amoxycillin/clavulanic acid, clindamycin, metronidazole and imipenem by the Etest method (Table 1). All but four isolates were resistant to imipenem (MICs 16 µg ml^–1), with the remaining four having elevated imipenem MICs (1–8 µg ml^–1). All 15 isolates produced detectable levels of carbapenemase activity (Table 1), with 100 % inhibition by EDTA. The enzymatic activities correlated with imipenem resistance with the exceptions of B. fragilis 19841 and 21216.

Molecular examination of the test isolates

Examples of PCR experiments are indicated in Fig. 2. Ten of the cfiA-positive isolates harboured IS elements. Of these, two (B. fragilis 1672 and 5652) harboured IS elements for which we had primers, upstream of their cfiA genes as revealed by IS element PCR and ‘PCR-mapping’ (Fig. 2b, lanes 1–4, Table 1). In B. fragilis 4729, the simple PCR-detected IS1169 was not observed at < 280 bp upstream of the cfiA gene (Fig. 2b, lanes 5 and 6), while, in B. fragilis 1672, IS4351 was additionally detected in the genome, but not upstream of cfiA.

View larger version (34K): [in this window] [in a new window]   Fig. 2. Illustration of the results of PCR experiments. (a) cfiA PCR with cfiA1 and cfiA2 primers. Lanes 1–9: B. fragilis 1776, 2523, 6729, 4729, 1672, 388/1, 19997, TAL3636 and NCTC 9344, respectively. Lane M, 100 bp ladder (Sigma). (b) ‘PCR-mapping'. Lanes: 1 and 2, B. fragilis 1672; 3 and 4, B. fragilis 5652; 5 and 6, B. fragilis 4729; with primer IS1186A (forward) in lanes 1 and 2, primer IS1169/1 (forward) in lanes 3–6, primer Up2 (reverse) in lanes 1, 3 and 5, and primer cfiA2 (reverse) in lanes 2, 4 and 6. Lane M, 1 kb ladder (Sigma). (c) Upstream region amplification with G and Up2 primers. Lanes 1–9: B. fragilis 6729, 2944, 6721, 2523, 1776, 5396, 2680, 388/1 and 388/2. Lane M, 100 bp ladder (Fermentas). The agarose concentration for all three gels was 0.8 %.

For eight cfiA-positive isolates, including B. fragilis 4729, PCR amplification of the upstream regions of the cfiA genes using primers G and Up2 gave 1.3–1.8 kb fragments (representative bands in Fig. 2c, lanes 1–7), and subsequent sequencing of the approx. 500 bp ends of the PCR fragments was applied to identify IS elements via the following criteria: similarity to IS element sequences deposited in GenBank and characteristic inverted repeats and target site duplications (Table 1). Thus, insertions of IS614-like elements could be demonstrated in four cases and those of IS1187, an IS1187 isoform, IS1169, an IS613 isoform and a novel molecule, IS943, in one case each (Table 1). In five strains, no IS element was found upstream of the cfiA genes (see below).

The inter-relationships between three of the four IS614-like elements in four isolates were also identified along the terminal approx. 500 bp; the DNA sequences were identical along the known part of the IS elements, and thus this sequence was termed IS614B, while the remaining one displayed a different similarity profile, and was therefore named IS614C. In addition, in these latter IS element types the base mismatches relative to IS614 and IS612 were distributed unevenly and, accordingly, mosaic or hybrid structures were suspected of attaining the best similarities to IS612, IS614 and also IS942. To investigate this idea, the full nucleotide sequences of these IS element types are currently being determined in our laboratories. These hybrid/mosaic IS forms may be similar in occurrence; this has been referred to in reports concerning the mosaicity of IS elements: ISS1SC, a streptococcal (Streptococcus thermophilus CNRZ368) IS element, displayed shared regions with two IS elements from other lactic acid bacteria (Bourgoin et al., 1996) and IS3 was also mosaic in different enterobacterial strains (Lawrence et al., 1992).

The IS elements found upstream of cfiA genes in our test isolates, which were mainly from the USA, were similar or identical to those found in France (IS1186, IS1187, IS1188 and IS942) (Podglajen et al., 1994, 1995, 2001), Japan (IS612, IS613, IS614, IS615 and IS616) (Kato et al., 2003) or Hungary (IS942) (Sóki et al., 2001), confirming their worldwide distribution. To date, the only known IS element upstream of cfiA of two B. fragilis isolates from the USA was IS942 (Thompson & Malamy, 1990; Rasmussen & Kovacs, 1991). In our collection, IS942 was absent, but IS614B displayed the highest abundance, which may be a result of the highest frequency of this element in cfiA-positive B. fragilis strains, at least in the USA.

Imipenem-resistant B. fragilis isolates without activating IS elements

In five isolates, B. fragilis 388/1, 388/2, 16997, 19841 and 21216, the sizes of the upstream fragments obtained by primers G and Up2 were 280 bp (Table 1 and representative results in Fig. 2c, lanes 8 and 9), as in silent strains, despite the fact that they had elevated imipenem MICs or were imipenem-resistant and produced detectable levels of imipenemase activity (Table 1).

The 280 bp upstream regions of cfiA were sequenced on both strands of the five isolates to reveal differences that might explain the resistant phenotype. All five upstream regions displayed differences from that of B. fragilis BFr81, a susceptible prototype strain (Podglajen et al., 1994), and the regions of B. fragilis 388/1 and 388/2 showed an identical sequence, while the regions in B. fragilis 16997 and 21216 differed from the previous two, but also exhibited an identical sequence.

For the latter two strains, B. fragilis 16997 and 21216, a particular CT transition could make a hypothetical promoter more active (Fig. 3a), since these sequences were most similar to the Bacteroides promoter consensus (Bayley et al., 2000) (Fig. 3a), and this might explain the higher imipenem MICs of these two strains in this group. However, other mechanisms should be considered to explain the modest MIC increase and carbapenemase activities of the three other strains, and the function of the mutant promoter in B. fragilis 16997 and 21216 should be confirmed experimentally in the future.

View larger version (13K): [in this window] [in a new window]   Fig. 3. Comparison of deduced nucleotide and amino acid sequences with the consensus sequence. (a) Hypothetical promoter regions of IS-less, cfiA-positive B. fragilis strains 16997 and 21216. Matching bases are indicated by colons and mismatches by upper-case letters; dashes represent no nucleotide at that position. Ribosome-binding site (RBS) and the starting methionine of cfiA are in bold and underlined. The nucleotide sequence of the susceptible B. fragilis BFr81 as a negative control is shown above. (b) similarity of the Tn943 DDE motif to the IS4 family consensus (Mahillon & Chandler, 1998). The Bacteroides consensus promoter (Bayley et al., 2000) is shown and matching bases are indicated in bold and underlined. Important amino acids are capitalized, while identical amino acids are denoted by bold underlined letters.

Such carbapenem-resistant B. fragilis strains, or strains with elevated cabapenem MICs having IS-less cfiA genes, were reported earlier (Edwards & Read, 2000) and a faecal meropenem-resistant strain, but with an IS-less cfiA gene, has also been found (Edwards & Yazdani, 2001). For such strains, the outer-membrane protein profile was also examined (Edwards & Greenwood, 1997) and a difference between cfiA-positive and cfiA-negative strains was detected, with no difference between cfiA-positive strains that had elevated imipenem MICs or were fully resistant; this situation with the different outer-membrane protein profile could be similar and might help to explain the lack of promoter mutation in three of our strains with the lowest impenem MICs. These latter data, together with the present findings, draw attention to the possible emergence of these B. fragilis strains with a novel type of cfiA activation.

Identification and sequencing of a novel IS element in a faecal B. fragilis isolate

For B. fragilis 6729, the terminal nucleotide sequences from the upstream region of cfiA, obtained by primers G and Up2, indicated a novel molecule, and the full sequence was determined by primer walking on both strands of a PCR fragment amplified by a polymerase mixture (KlenTaq LA; Sigma) having proofreading activity. The full DNA sequence confirmed that it is a novel IS element; the criteria for identification were similar to those applied in a previous section. It coded for a single, 479 aa protein, an IS type integrase, Tp943, as the BLAST search also indicated. This newly identified IS element was denoted IS943; its length is 1557 bp and it has a 46 % G+C content. Its nucleotide sequence did not show higher (>70 %) similarity to any records in the GenBank nucleotide sequence database. However, the BLAST program detected limited similarity to the following transposases in the SWISSPROT protein sequence database: the orf-9 product of Tn4555 in Bacteroides vulgatus CLA341 (E = 1e–59, 33/52) (Tribble et al., 1999), a transposase of Lactobacillus delbruckii subsp. bulgaricus (E = 5e–13, 28/45) (Lapierre et al., 2002), an undetermined protein of Desulfitobacterium hafniense (E = 2e–4, 25/48) (GenBank AAL87775.1), InsA of Streptomyces netropsis (E = 0.01, 22/38) (GenBank AAD45539.1) and a transposase of IS231E of Bacillus thuringiensis subsp. finitimus (E = 0.18, 27/42) (Rezsohazy et al., 1992), where E is the expect value, i.e. the probability of the chance occurrence of the found matches, and the percentage identity/similarity in the most similar segments indicated by the program are also shown in parentheses (Altschul et al., 1997).

Comparison of the Tp943 sequence with the consensus of the conserved amino acids in the active centre(s), also known as the DDE motif, of each IS group (Mahillon & Chandler, 1998) placed it in the IS4 family (Fig. 3b).

The highest similarity to orf-9, a coding region with an unidentified function in B. vulgatus Tn4555 (Tribble et al., 1999), however, prompted us to suspect not only amino acid and functional, but also structural similarity. A 17 nt long inverted repeat set was found in the Tn4555 nucleotide sequence at positions 9259–9275 and 10907–10923 bordering orf-9. In our opinion, this structure (the inverted repeats and orf-9, encoding a protein similar to IS transposases) could be another IS element hitchhiking on a mobilizable transposon (e.g. Tn4555); thus, on the basis of the similarity of their transposases, IS943 and this latter may represent a subtype of IS4 family elements.

The present data confirm the role of cfiA activation of B. fragilis strains by a number of diverse IS element types that were already known, or possibly hybrids of known types, or entirely new (IS943), in addition to reporting members of an IS-less activation mechanism in B. fragilis strains. The exact mechanism of the latter remains to be examined in more detail.

	ACKNOWLEDGEMENTS

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This work was supported by the Hungarian National Research Foundation (OTKA) through grants T032385 to E. N. and T037475 to J. S. We thank Zoltán Lajos, Erika Molnár and Lenke Szikra for supplying the B. fragilis isolates 16997, 21216 and 19841, respectively.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES