1 UPRES n°EA2392, Faculté de Médecine, UFR Saint-Antoine, Université Paris 6; 2 Laboratoire de bactériologie, Hôpital Saint Antoine, 27 rue Chaligny, 75012 Paris Cedex 12; 3 Laboratoire de bactériologie Hôpital Tenon, Assistance Publique-Hôpitaux de Paris, France
Received 1 July 2004; returned 20 July 2004; revised 24 August 2004; accepted 27 August 2004
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Abstract |
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Methods: The 57 isolates studied were recovered from clinical specimens (n=23) or from rectal swabs (n=34) during a 26-month period. Antibiotic susceptibility patterns were determined using standard agar diffusion and dilution methods including the synergy test between extended-spectrum cephalosporins and clavulanic acid. ERIC-2 PCR and pulsed-field gel electrophoresis (PFGE) methods were used to study the clonal relatedness of the strains. Plasmid-mediated and chromosomal ß-lactamases were characterized by mating and specific bla gene amplification and sequencing.
Results: Four different antibiotic resistance patterns were identified whereas ERIC-2 PCR and PFGE revealed six main profiles. Extended-spectrum ß-lactamases (ESBLs) were found in 32 strains: TEM-7 (n=26), TEM-129 (n=1), TEM-3 (n=4), SHV-2 (n=1). The new TEM-type ß-lactamase, TEM-129, differed from TEM-7 by one mutation (Glu-104Lys). All TEM-7 or TEM-129 producers were genetically related. Twenty-five other strains with identical ERIC-2 PCR and PFGE profiles harboured a blaOXY-2 gene different from the reference gene: 24 strains displayed one substitution (Ala-237
Ser) in the KTG motif and one strain, highly resistant to ceftazidime, showed an additional substitution (Pro-167
Ser).
Conclusions: The study demonstrated that the majority of strains (n=52) harbouring the OXY-2-type ß-lactamase corresponded to two clones. The first clone (n=27) corresponded to ESBL-producing strains. The second clone (n=25) displayed extended-spectrum activity of the chromosomal ß-lactamase.
Keywords: OXY-2 ß-lactamase , TEM-7 , TEM-129
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Introduction |
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The ß-lactamases of K. oxytoca have been divided into two main groups: blaOXY-1 and blaOXY-2.6,7 These two ß-lactamases have been placed in functional group 2be in Bush's scheme and in class A of Ambler's classification.8,9 These two genes share 87% nucleotide sequence identity. Each ß-lactamase group is represented by at least four different forms according to their pI values from 7.1 to 8.8 and 5.2 to 6.8 for OXY-1 and OXY-2, respectively.6 Two other groups of K. oxytoca genes have recently been reported and named blaOXY-3 and blaOXY-4.10 The nucleotide sequence of the blaOXY-4 gene is 95% identical to that of the blaOXY-1 gene. The bla genes display the STFK and KTG sequences typically found in ß-lactamases possessing a serine active site.9
Clinical isolates of Klebsiella spp. including K. oxytoca, resistant to broad-spectrum cephalosporins and aztreonam, have been increasingly reported and are due to the acquisition of plasmids encoding extended-spectrum ß-lactamases (ESBLs).11,12 In addition, K. oxytoca isolates that overproduce the chromosomally-encoded ß-lactamase have been found to be resistant to broad-spectrum cephalosporins (e.g. cefotaxime and ceftriaxone) and monobactams.3,1315 Although ß-lactamase production is not regulated, some mutations in the promoter region cause its overproduction. Various mutations have been reported in the 35 and 10 promoter regions.1517 Strains that overproduce ß-lactamase are resistant to cefuroxime, ceftriaxone and aztreonam. In contrast, these strains are not resistant to ceftazidime, distinguishing ß-lactamase overproducers from strains of K. oxytoca with plasmid-borne ESBLs.5 A strain of K. oxytoca that produces a chromosomally-encoded ß-lactamase conferring resistance to ceftazidime was recently reported.18
We report an outbreak involving strains of K. oxytoca with a typical ESBL and strains with an unusual resistance pattern that is consistent with extended-spectrum activity of the chromosomal ß-lactamase.
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Materials and methods |
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The 57 clinical isolates of K. oxytoca that were studied displayed a reduced susceptibility or a resistance to ceftazidime by the disc diffusion method (diameters 20 mm). They were isolated in the different units of the surgery ward at the Saint Antoine Hospital between April 2001 and June 2003. These strains were identified using the API 20E system (bioMérieux, Marcy l'Étoile, France). Twenty-three strains were isolated from clinical specimens and 34 from rectal swabs that are carried out in some units of our hospital to detect patients colonized with plasmid-borne ESBL-producing strains of Enterobacteriaceae. Rectal swabs are inoculated on Drigalski agar (Bio-Rad, Marnes la Coquette, France) containing 2 mg/L of ceftazidime.
Two strains of K. oxytoca producing the blaOXY-2 gene from our laboratory collection were used as reference strains in the susceptibility testing: strain HER is a wild-type strain and strain WEI is a strain overproducing the blaOXY-2 gene with a stronger promoter (G to A transition in the fifth base of the 10 consensus sequence).17
Escherichia coli K12 J53-2 (pro met RifR) and E. coli DH10B (Invitrogen SARL, Cergy Pontoise, France) were, respectively, used for conjugation assays and cloning experiments.
Antibiotic susceptibility and synergy testing
Minimum inhibitory concentrations (MICs) of several ß-lactams were determined by the standard agar dilution method with MuellerHinton agar (Bio-Rad). Inocula (104105 cfu per spot) were delivered with a multipoint inoculator. Resistance to other antibiotics, such as aminoglycosides and quinolones, was determined by the disc diffusion method on MuellerHinton agar according to the recommendations of the Comité de l'Antibiogramme de la Société Française de Microbiologie (http://www.sfm.asso.fr). Antibiotic discs for agar tests were obtained from Bio-Rad. Class A ESBLs were detected by the synergy test between ceftazidime or cefotaxime and co-amoxiclav as recommended by Livermore5 and also between aztreonam or cefepime and co-amoxiclav according to the French recommendations (http://www.sfm.asso.fr).
Enterobacterial repetitive intergenic consensus (ERIC) PCR
DNA was extracted using the Qiagen Mini Kit (Qiagen, Coutaboeuf, France). ERIC-PCR was carried out with the primer ERIC-2 as described previously.19 The primer sequence was 5'-AAGTAAGTGACTGGGGTGACGC-3'. The reaction was carried out in a GeneAmp PCR system 9700 (PE Applied Biosystems, Foster City, CA, USA). The PCR conditions were as follows: 3 min at 95°C, 40 cycles of 30 s at 92°C, 1 min at 40°C, and 8 min at 72°C, and final extension of 16 min at 72°C. The resulting products were run in a 1.5% agarose gel.
Pulsed-field gel electrophoresis (PFGE) analysis
Genomic DNA, prepared as described previously and digested with Xbal (Ozyme, New England Biolabs Inc., Saint Quentin en Yvelines, France), was subjected to PFGE with the CHEF DRIII device (Bio-Rad).20 DNA fragments were separated in a 1% (w/v) agarose gel in 0.5x Tris/borate/EDTA buffer at 200 V for 20 h, with pulse times ranging from 5 to 30 s. The resulting restriction patterns were interpreted as described by Tenover et al.21
Conjugation
Mating experiments were carried out by mixing equal volumes (1 mL) of exponentially growing cultures of each test strain and E. coli K12 J53-2 (met pro RifR) in brain heart infusion (BHI) broth (Difco, Detroit, MI, USA) and incubating the mixture for 3 h at 37°C. ß-Lactam-resistant E. coli transconjugants were selected on Drigalski agar (Bio-Rad) supplemented with rifampicin (256 mg/L) and ceftazidime (1, 4 or 16 mg/L) or rifampicin (256 mg/L) and ticarcillin (16 mg/L).
PCR amplification and molecular characterization of transferable ß-lactamases: TEM-type, SHV-type and CTX-M-type
The genes coding for the following transferable ß-lactamases (TEM-, SHV-, CTX-M-type enzymes) were detected by PCR amplification from genomic DNA. The oligonucleotide PCR primers specific for the ß-lactamase genes are listed in Table 1. The resulting PCR products were sequenced by the Sanger method using an ABI 373A DNA sequencer (PE Applied Biosystems).22
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PCR amplification and sequencing of the chromosomal OXY ß-lactamases
The putative class A ß-lactamase gene from the 57 strains of K. oxytoca was amplified using degenerate oligonucleotide primers based on the consensus sequences of the known blaOXY-1 (accession no. Z30177) and blaOXY-2 (accession no. Z49084) genes. The primers used, KO1 and KO2 (Table 1), are, respectively, located at positions 179197 and 11661184 on the blaOXY-2 gene. PCR conditions were as follows: 5 min at 94°C, 35 cycles of 1 min at 95°C, 1 min at 56°C, and 1 min at 72°C, and final extension of 7 min at 74°C. The resulting PCR products were sequenced as described above.
The blaOXY gene sequences of our isolates were compared with those of reference strains SL781 (blaOXY-1 no. Z30177) and SL911 (blaOXY-2 no. Z49084).
Plasmid DNA analysis
Plasmid DNA was extracted by the alkaline lysis method.26 Plasmid DNA was detected by electrophoresis in a 0.8% agarose gel. The molecular sizes of plasmid DNAs were estimated by comparison with the following plasmids of known sizes from the Institute Pasteur Collection: RP4 (54 kb), pCFF04 (85 kb) and pIP173 (125.8 kb).
Plasmid DNA was purified from transconjugant cells with the Qiagen Plasmid Midi Kit (Qiagen), according to the manufacturer's recommendations. For fingerprinting analysis, plasmid DNA was digested with EcoRI or BamHI (Ozyme) and subjected to electrophoresis in a 1% agarose gel at 80 V for 3 h.
Cloning of blaOXY genes
The ß-lactamase genes from the two K. oxytoca strains isolated from the same patient [one of which showed antibiotic pattern 3 (1547) and the other that showed pattern 4 (16944)] and a wild-type strain of K. oxytoca (HER) were amplified by PCR using primers CL1 and CL2. Amplification products were cloned using the TOPO XL kit (Invitrogen) according to the manufacturer's instructions.
To obtain stable clones, inserts were then subcloned using pACYC 184 and E. coli DH10B (Invitrogen SARL).27 PCR fragments obtained using the primers 21M13 and 21M13 reverse were cut with HindIII and XbaI and ligated in the EcoRI and XbaI sites of pACYC 184. E. coli DH10B was transformed by electroporation. The transformants harbouring the recombinant OXY-2-encoding plasmids were selected on MuellerHinton agar supplemented with amoxicillin (50 mg/L) and chloramphenicol (25 mg/L).
Nucleotide accession numbers
The nucleotide sequences described in this paper have been submitted to EMBL-GenBank under accession no. AJ746225 for TEM-129, no. AJ746226 for K. oxytoca 1547 and no. AJ746227 for K. oxytoca 16944.
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Results and discussion |
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The characteristics of the 57 strains of K. oxytoca isolated from 56 patients are listed in Table 2. These strains could be divided into four main ß-lactam resistance patterns. Thirty-one strains (pattern 1) showed a typical positive synergy test between cefotaxime, ceftazidime, aztreonam or cefepime and clavulanic acid, consistent with an ESBL. All these strains were resistant to gentamicin, tobramycin, netilmicin and sulfamethoxazole. All but one (strain 3701) were highly resistant to fluoroquinolones.
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Compared with the strains HER and WEI used as reference strains, 24 strains displayed decreased susceptibility to ceftazidime by the disc diffusion method (inhibition zone 20 mm) with negative synergy test (pattern 3). The MICs of ceftazidime ranged from 2 to 8 mg/L (MIC50 8 mg/L). These strains were resistant to kanamycin and showed reduced susceptibility to tobramycinconsistent with the presence of APH3'and were resistant to sulfamethoxazole and fluoroquinolones.
Finally, one strain (16944, pattern 4) was highly resistant to ceftazidime with a negative synergy test. This strain presented the same resistance pattern to other antibiotics as pattern 3.
Strains 1547 (antibiotic resistance pattern 3) and 16944 (antibiotic resistance pattern 4) were consecutively isolated from the same patient. These strains differed in terms of susceptibility to ceftazidime: MICs were 2 and >32 mg/L for strains 1547 and 16944, respectively. The strain that was highly resistant to ceftazidime (16944) appeared more susceptible to piperacillin plus tazobactam, cefotaxime and cefepime than strain 1547.
ERIC-2 PCR which is a rapid typing method divided the 57 strains into six different profiles (A, B, C, D, E and F) (Table 2).19,28 The 24 strains with resistance pattern 3 and the one pattern 4 strain displayed profile A. The pattern 1 strains were divided up as follows: profiles B (26 strains), C (two strains), D (one strain), E (one strain) and F (one strain). Finally, the pattern 2 strain also displayed the ERIC-PCR profile B.
We used the PFGE method to confirm the ERIC-PCR results. Indeed, this method has been used for epidemiological studies and is one of the most discriminative genotyping methods for typing various bacterial genera including Klebsiella spp.4,20,29,30 Genomic DNA from 14 strains, representative of each ERIC-2 PCR profile, was subjected to PFGE analysis. Six major profiles (I, II, III, IV, V, VI) and subtypes differing by less than three bands were identified (Figure 1).21 The PFGE results correlated well with those obtained using the ERIC-2 PCR typing method (Table 2).
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Mating was carried out for 15 strains selected on the basis of their susceptibility patterns and their ERIC-2 PCR profile: five profile A strains, five profile B strains, the two profile C strains and one strain with each of the other profiles (D, E and F).
Resistance to broad-spectrum ß-lactams was transferred to E. coli J53 from all the antibiotic pattern 1 strains (ERIC-2 profiles B, C, D, E and F).
In contrast, for the five profile A strains tested, resistance to ceftazidime was not transferred while transconjugants with a typical penicillinase phenotype were obtained on agar containing ticarcillin. These results indicate that the bla gene responsible for the reduced susceptibility (pattern 3) or the resistance (pattern 4) to ceftazidime of the profile A strains was located on the chromosome.
PCR amplification and sequence of the TEM-type and SHV-type ß-lactamases
A PCR product was obtained using primers OT3 and OT4, designed to amplify a TEM-type ß-lactamase, for 56 of the 57 strains. Twenty-five strains were found to carry the TEM-1 ß-lactamase, which is the ß-lactamase most commonly found in Enterobacteriaceae. All except one (1459) of the profile A strains were shown to carry the TEM-1 ß-lactamase. The only strain of profile F (3701) harboured the TEM-1 ß-lactamase. The TEM-3 and TEM-7 ß-lactamases were found in four and 26 strains, respectively. A new TEM-type ß-lactamase, named TEM-129 was identified (GenBank accession no. AJ746225). This new TEM-type ß-lactamase was characterized by one mutation (Glu-104Lys) compared with the TEM-7 ß-lactamase and conferred a high level of resistance to ceftazidime. The fact that all the strains producing TEM-7 and TEM-129 had identical ERIC2-PCR and PFGE profiles strongly suggests that TEM-129 is derived from TEM-7 ß-lactamase. The typical ESBLs TEM-3 and TEM-7 were identified in strains that were positive for the typical synergy test with the agar diffusion method and that could be transferred to E. coli J53-2 by conjugation. The only strain (3701) that was susceptible to ciprofloxacin was shown to carry two types of ß-lactamase: a TEM-1 ß-lactamase and a SHV-2 ß-lactamase, both of which were transferred by conjugation. These results are not surprising as ESBLs first emerged in France in 1984 and a large number of TEM-1, TEM-2 and SHV derived enzymes have since been reported.12
Finally, the CTX-M-type ß-lactamases, which are an emerging group of enzymes with a typical ESBL-resistance phenotype but that are neither TEM nor SHV derivatives, were not found in our collection of K. oxytoca.31,32
Plasmid DNA analysis
Analysis of E. coli transconjugants expressing the TEM-3 ß-lactamase revealed the presence of one plasmid with an estimated molecular size of 85 kb, and digestion with EcoRI or BamHI generated identical restriction patterns (data not shown). These results are consistent with plasmid dissemination in different strains of K. oxytoca.
In addition, the plasmids, with an estimated molecular size >100 kb, isolated from E. coli transconjugants harbouring TEM-7 or TEM-129, yielded indistinguishable restriction patterns (Figure 2). This result is consistent with the hypothesis that TEM-129 originated from TEM-7 ß-lactamase.
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To identify the resistance mechanism in strains with no transferable resistance to extended-spectrum cephalosporins, we amplified the coding and promoter regions with the primers KO1 and KO2. All the strains gave PCR products of the expected size.
Sequence analysis indicated that all the 35 boxes in the promoters of the 57 strains of K. oxytoca were 100% identical to those from wild-type strains of K. oxytoca.15,17 In contrast, with the exception of the five strains with ERIC-2 PCR profiles C (n=2), D (n=1), E (n=1) and F (n=1), two different mutations were observed in the 10 promoter region of all strains (Table 2). Compared with the wild-type promoter sequence, the 10 hexamer sequence exhibited a G-to-T substitution at the first position for the 25 profile A strains and a G-to-A substitution in the fifth position for the 27 profile B strains. These mutations have been previously reported to create stronger promoters.17
Analysis of the sequences of blaOXY genes indicated that five strains (ERIC-2 PCR profiles C, D, E, F) harboured the typical OXY-1-type ß-lactamase whereas the 27 profile B strains harboured the typical OXY-2-type ß-lactamase.6
Finally, the sequences of the 25 profile A strains, including the 24 strains with reduced susceptibility to ceftazidime (resistance pattern 3) and strain 16944 (resistance pattern 4), were very similar to the blaOXY-2 group. However, 24 of these strains differed from the reference blaOXY-2 gene (GenBank accession no. Z49084) by only one mutation whereas strain 16944 differed by two mutations. The first mutation results in a Ala-237Ser substitution, which affects the KTG motif (GenBank accession no. AJ46226) and the second one is responsible for the substitution Pro-167
Ser in the omega loop (GenBank accession no. AJ46227).
Only two OXY-2 variants have been reported in K. oxytoca. The first conferred resistance to inhibitors and was due to a serine to glycine substitution at Ambler position 130.33 More recently, Mammeri et al.18 reported the in vivo selection of a K. oxytoca strain resistant to ceftazidime and described a single nucleotide substitution at position 167.
The substitution located at position 237 could be responsible for the reduced susceptibility to ceftazidime. It has been hypothesized that the extended-spectrum hydrolytic properties towards cefuroxime and extended cephalosporins are in part due to a serine residue located at position 237. This is the case for some TEM-type and SHV-type ESBLs as well as for CTX-M variants and chromosomally-encoded ß-lactamases from Kluyvera ascorbata (KLUA-1), Kluyvera cryocrescens (KLUC-1), Kluyvera georgiana (KLUG-1), Proteus vulgaris (RO104), Serratia fonticola (CUV), Citrobacter sedlakii (Sed-1), Citrobacter diversus (CdiA), Rahnella aquatilis (RHAN-1) and Erwinia persinica (ERP-1), which all possess a serine residue at position 237 whereas an alanine residue is found at position 237 in enzymes like TEM-1, TEM-2, LEN-1, OXY-1, OXY-2 and HER-1.12,15,32,3442
The second substitution in the omega loop is identical to that reported by Mammeri et al.18 In our study, the highly ceftazidime-resistant strain (16944) was also selected in vivo. The Pro-167Ser substitution has also been found at the same Ambler position among CTX-M ESBLs (CTX-M-18 and CTX-M-19) and conferred resistance to ceftazidime.43
However, comparison of the sequences of the two strains described by Mammeri et al. (GenBank accession no. AY303806.1 and AY303807.1) shows that the corresponding strains also possess the Ala-237Ser substitution, like our strains.18
Cloning experiments yielded recombinant strains of E. coli DH10B (pACYC 184) from the three strains tested. Sequences of the PCR products obtained from E. coli DH10B transformants were identical to those of the parental strains of K. oxytoca. However, transformants from strains 1547 and 16944 appeared to be more susceptible than parental strains (Table 3).
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Acknowledgements |
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Footnotes |
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References |
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