JAC Advance Access originally published online on February 20, 2008
Journal of Antimicrobial Chemotherapy 2008 61(5):1029-1032; doi:10.1093/jac/dkn056
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Original research |
Increase in β-lactam-resistant Proteus mirabilis strains due to CTX-M- and CMY-type as well as new VEB- and inhibitor-resistant TEM-type β-lactamases
1 Servei de Microbiologia, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain 2 Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Barcelona, Spain
* Correspondence address. Servei de Microbiologia, Hospital de la Santa Creu i Sant Pau, Av. Sant Antoni Ma Claret, 167, 08025 Barcelona, Spain. Tel: +34-93-2919069; Fax: +34-93-2919070; E-mail: fnavarror{at}santpau.es
Received 26 September 2007; returned 22 October 2007; revised 22 January 2008; accepted 23 January 2008
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Abstract |
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Objectives: The aim of this study was to characterize the different inhibitor-resistant TEM β-lactamases, extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases implicated in β-lactam resistance in Proteus mirabilis, which has increased over recent years.
Methods: From February 2000 to December 2005, 1423 clinical isolates of P. mirabilis were collected. The AmpC phenotype was checked by means of a double-disc synergy test using cloxacillin as an inhibitor of AmpC enzymes. The production of ESBL was assessed by the double-disc synergy method and by Etest ESBL. Analytical isoelectric focusing, determination of kinetic constants, conjugation, PCR and a sequencing strategy were used to characterize the enzymes. The possible relationships between isolates were analysed by PFGE.
Results and conclusions: Twenty-five of 1423 isolates were found to display intermediate or full resistance to co-amoxiclav, cefotaxime or ceftazidime. Seventeen isolates had reduced susceptibility to co-amoxiclav; of these, seven produced TEM-110, eight produced the new TEM-159, one the new TEM-160 and one TEM-1. Five isolates producing TEM-110, TEM-159 or TEM-160 enzymes shared the same PFGE profile. Three isolates produced an ESBL, CTX-M-1, CTX-M-32 and the new variant, VEB-4. Finally, five isolates with an AmpC phenotype produced CMY-2, two with the same PFGE profile. Our data emphasize the diversity of β-lactamases found in this species.
Keywords: antimicrobial resistance surveillance , Enterobacteriaceae , mechanisms of resistance , ESBLs , antimicrobial resistance mechanisms , extended-spectrum β-lactamases , resistance epidemiology
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Introduction |
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Proteus mirabilis is the second most common cause of urinary tract infections and is also an important cause of nosocomial infections. Wild-type strains of this species are usually susceptible to β-lactams because they do not express a chromosomally encoded AmpC cephalosporinase.1 However, a broad repertoire of acquired β-lactamases has been reported in this species, including broad- and extended-spectrum β-lactamases, and AmpC enzymes.2 Amoxicillin resistance in P. mirabilis is mainly due to the penicillinases TEM-1 and TEM-2, with the latter being more frequent in this species.3 The inhibitor-resistant phenotype is due to the production of inhibitor-resistant TEM (IRT) β-lactamases (TEM-44, TEM-65, TEM-73 and TEM-74).3 Extended-spectrum β-lactamase (ESBL)-producing P. mirabilis has increased in several geographical locations; the most predominant are TEM-type enzymes1,3 and CTX-M enzymes1–5 with some VEB- and PER-types less frequent.1,3,4,6 Finally, several authors also describe the acquisition of plasmid-mediated AmpC β-lactamases, mainly derived from the CMY/LAT group.1–4,6,7
In this paper, we report an increase in β-lactam resistance in P. mirabilis isolates obtained in a survey between 2000 and 2005 in a tertiary care hospital. In addition, we have identified novel IRT β-lactamases and VEB-4, a novel VEB-type ESBL.
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Materials and methods |
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Bacterial isolates and susceptibility test
A total of 1423 clinically relevant P. mirabilis isolates were obtained from routine cultures at the Hospital de la Santa Creu i Sant Pau from February 2000 to December 2005. Only one isolate per patient was considered. The susceptibility pattern to β-lactams was determined by the disc diffusion method according to the CLSI guidelines.8 The isolates selected for this study were those showing intermediate or full resistance to co-amoxiclav, cefotaxime or ceftazidime according to the CLSI.8 All selected isolates were also tested by a double-disc synergy test based on the utilization of cloxacillin as inhibitor of AmpC enzymes and of the presence of scattered colonies in the halo.9 Briefly, a disc of cloxacillin (500 µg, Neo-Sensitabs, Rosco Diagnostica S/A, Taastrup, Denmark) was placed 2.5 cm from ceftazidime and cefotaxime discs. An enhanced zone of inhibition between any cephalosporin disc and cloxacillin disc was interpreted as AmpC production. The production of ESBL was assessed by the double-disc synergy method and by Etest ESBL strips (AB Biodisk, Solna, Sweden). The MIC of co-amoxiclav was determined by Etest (AB Biodisk).
β-Lactamases were characterized by analytical isoelectric focusing and determination of kinetic constants as described previously.6,10 PCR and sequencing were used to characterize β-lactamase genes with primers described previously for blaTEM, blaSHV, blaCTX-M, blaPER, blaCMY, blaDHA, blaACC, blaMIR and blaFOX.6,9,10 The primers VEB-F 5'-GTT AGC GGT AAT TTA ACC AGA TAG-3' (position 3517–3540, accession no. AF205943 [GenBank] ) and VEB-B 5'-CGG TTT GGG CTA TGG GCA G-3' (position 4587–4569, accession no. AF205943 [GenBank] ) were used to amplify a 1070 bp fragment containing the entire blaVEB gene. DNA sequencing of PCR products was performed by Macrogen (Macrogen Inc., Seoul, Korea). Nucleotide and amino acid sequences were analysed via the Internet using the BLAST program (www.ncbi.nlm.nih.gov).
Filter mating experiments were performed with all isolates carrying TEM-110, TEM-159, TEM-160, ESBL and CMY-2 as donors and Escherichia coli Hb101 (kanamycin-azide resistant, lactos-negative and plasmid-free) as the recipient. Donor and recipient strains at logarithmic phase were grown in Trypticase soy broth and were mixed and incubated on solid medium at 37°C for 20 h. Transconjugants were selected on Mueller–Hinton medium supplemented with ampicillin (100 mg/L) or cefoxitin (10 mg/L) or ceftazidime (10 mg/L) and sodium azide (13 mM).
The clonal relationships between P. mirabilis clinical isolates were analysed by comparing PFGE profiles of genomic DNA digested with NotI (GE Healthcare Bioscience). Electrophoresis was performed with a CHEF DRIII System (Bio-Rad, Richmond, CA, USA) with the following electrophoretic conditions: linear pulse time ramp from 1 to 30 s, run time 8 h, angle 120° and gradient 6 V/cm, and linear pulse time ramp from 30 to 70 s, run time 16 h, angle 120° and gradient 6 V/cm.
Nucleotide sequence accession number
The following β-lactamase genes were submitted to the GenBank nucleotide sequence database under accession nos EF136376 [GenBank] (blaTEM-159), EF136377 [GenBank] (blaTEM-160) and EF136375 [GenBank] (blaVEB-4).
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Results and discussion |
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P. mirabilis, which lacks intrinsic chromosomal β-lactamase genes, is entirely dependent upon acquisition of different β-lactamase genes to express a β-lactamase-mediated resistance phenotype.1–7 During the present survey (2000–2005), 1423 P. mirabilis isolates were studied to determine the mechanisms implicated in co-amoxiclav and third-generation cephalosporin resistance. Among these isolates, 640 (45%) were resistant to ampicillin, 17 (1.2%) showed intermediate or full resistance to co-amoxiclav, 3 (0.2%) to third-generation cephalosporins (all Etest ESBL were positive) and 5 (0.4%) to both co-amoxiclav and third-generation cephalosporins (all double-disc synergy tests with cloxacillin were positive). Although these percentages were low compared with data from other studies,1–5 they imply a notable increase in our hospital. From 2000 to 2005, resistance to co-amoxiclav and third-generation cephalosporins increased from 0.7% to 3.2% and from 0.3% to 1.6%, respectively.
Seven of 17 isolates with intermediate or full resistance to co-amoxiclav produced TEM-110 enzyme (GenBank accession no. AY072920
[GenBank]
), 8 produced TEM-159 (EF136376
[GenBank]
), 1 TEM-160 (EF136377
[GenBank]
) and 1 TEM-1 (Table 1). The TEM-110 enzyme had a pI of 5.4, and the concentration of clavulanic acid giving 50% inhibition (IC50) ranged between 0.42 and 1.7 µM, with 0.03 µM being the IC50 for the TEM-1 control strain. The MICs of co-amoxiclav for these isolates ranged between 16 and 64 mg/L. As all isolates were cefalotin-resistant, reduced susceptibility to the co-amoxiclav combination was probably due to hyperproduction of this enzyme, although mechanisms such as altered permeability could play a role. Only two strains, AY072920
[GenBank]
and AY130283
[GenBank]
, with TEM-110 have been described, both in Klebsiella sp. No transconjugants were obtained in transfer experiments. The novel TEM-159 β-lactamase had a pI of 5.4 and an IC50 range between 2.5 to 17.1 µM, similar to those concentrations found in E. coli for IRT enzymes.10 The MICs of co-amoxiclav for these isolates were 
128 mg/L, showing a high degree of resistance. Transconjugants were obtained from N1285 and N1438 strains with a conjugation frequency of 2.6 x 10–3 and 1.8 x 10–4 transconjugants per recipient, respectively. Aminoglycoside resistance co-transferred was different in each case. In the case of isolate N1285, resistance to streptomycin, gentamicin and tobramycin was co-transferred, whereas in the case of isolate N1438, resistance to streptomycin, kanamycin, gentamicin and tobramycin was co-transferred. Resistance to tetracycline and chloramphenicol (in one case) was also co-transferred. The novel TEM-160 enzyme presented a pI of 5.6 (as TEM-2) and the IC50 was 0.85 µM; the MIC of co-amoxiclav for the producer isolate was 32 mg/L. No transconjugants were obtained in transfer experiments. Finally, as the remaining isolate produced TEM-1 enzyme, possible hyperproduction associated with other mechanisms could be the cause of its resistance to co-amoxiclav. In the literature, amoxicillin resistance in P. mirabilis is due to the plasmid-mediated penicillinases, mainly TEM-2 or its derivatives.3 Nucleotide sequence analysis revealed that blaTEM-110, blaTEM-159 and blaTEM-160 genes showed 99.4%, 99.3% and 99.7% identity with blaTEM-2, respectively, and all three presented 99.2% identity with blaTEM-1.
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Two ESBL-producing P. mirabilis isolates had a CTX-M enzyme, one CTX-M-1 and one CTX-M-32. The CTX-M-1 enzyme has been described in P. mirabilis in one isolate from Greece,5 but we believe that this is the first time that CTX-M-32 ESBL has been found in this species. Transconjugants were obtained from the CTX-M-32-producing strain N2072, with a conjugation frequency of 6.7 x 10–4. Resistance to aminoglycosides (kanamycin and streptomycin) was co-transferred, whereas tetracycline and co-trimoxazole resistance was not. No transconjugants were obtained from the CTX-M-1-producing donor strain. A third ESBL-producing isolate produced the new variant VEB-4 (EF136375 [GenBank] ), which differs from VEB-1 by two amino acid substitutions, T25A and T104M. Again, no transconjugants were obtained in transfer experiments with this strain. VEB ESBLs are not closely related to any of the established families of β-lactamases. A nosocomial outbreak of P. mirabilis with a VEB-1 ESBL was recently described in a Korean hospital.4
All five P. mirabilis isolates with an AmpC phenotype produced CMY-2 enzyme. The presence of scattered colonies located near the edge of the inhibition halo of cefoxitin, cefotaxime, ceftazidime and aztreonam, and synergy using a double-disc test with cloxacillin was observed in all these isolates. Transconjugants were obtained from isolates 88-Hb, N1353 and N1471, with conjugation frequencies of 1.8 x 10–4, 5.5 x 10–6 and 2.7 x 10–5, respectively. Resistance to aminoglycosides (gentamicin, tobramycin, kanamycin and streptomycin), tetracycline and chloramphenicol (except isolate N1353) were co-transferred in all cases. Transferable class C β-lactamases have been identified throughout the world, and although the prevalence of the different types of enzymes may vary according to geographical region, CMY-2 occurs most frequently in our locale.7
The 25 P. mirabilis isolates studied in detail were from urine (17 isolates), wound (4 isolates), blood (3 isolates) and vaginal (1 isolate) specimens. Of these, 71.4% corresponded to outpatient samples. PFGE was used to rule out relatedness between the 25 strains. Five isolates with different TEM-type enzymes showed the same PFGE profile (Table 1): two of them produced TEM-110, two TEM-159 and TEM-160; these were isolated in 2002–2005 and had different associated resistances. Although all were fluoroquinolone-, co-trimoxazole- and tetracycline-resistant, susceptibility to chloramphenicol and aminoglycosides (gentamicin, tobramycin, kanamycin, amikacin, streptomycin and netilmicin) was variable. Two of the CMY-2-producing isolates shared the same PFGE pattern (data not shown); both were isolated within 3 months, but had different associated resistance patterns.
In summary, β-lactam resistance in P. mirabilis has increased in our setting in recent years. Our data emphasize the diversity of the β-lactamases found in this species. Three newly acquired β-lactamases, TEM-159, TEM-160 and VEB-4, were identified, and the CTX-M-32 ESBL was found for the first time in P. mirabilis. Moreover, this is the first time that a P. mirabilis CTX-M-1-producing strain has been isolated in Spain.
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Funding |
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This work was partially supported by Red Española de Investigación en Patología Infecciosa (REIPI C03/14) and Red Española de Investigación en Patología Infecciosa (REIPI RD06/0008), from the Ministry of Health and Consumption of Spain.
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Transparency declarations |
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None to declare.
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Acknowledgements |
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We thank Carolyn Newey for English assistance.
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References |
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2
D'Andrea MM, Nucleo E, Luzzaro F, et al. CMY-16, a novel acquired AmpC-type β-lactamase of the CMY/LAT lineage in multifocal monophyletic isolates of Proteus mirabilis from Northern Italy. Antimicrob Agents Chemother (2006) 50:618–24.
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Navarro F, Pérez-Trallero E, Marimón JM, et al. CMY-2-producing Salmonella enterica, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis and Escherichia coli strains isolated in Spain (October 1999–December 2000). J Antimicrob Chemother (2001) 48:383–9.
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