JAC Advance Access first published online on January 8, 2008
This version published online on January 22, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkm508
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Linkage of acquired quinolone resistance (qnrS1) and metallo-β-lactamase (blaVIM-1) genes in multiple species of Enterobacteriaceae from Bolzano, Italy
1 Laboratorio Interaziendale di Microbiologia e Virologia, Via Amba Alagi 5, I-39100 Bolzano, Italy 2 Antibiotic Resistance Monitoring and Reference Laboratory, Health Protection Agency, Centre for Infections, 61 Colindale Avenue, London NW9 5EQ, UK
* Corresponding author. Tel: +44-20-8327-7255; Fax: +44-20-8327-6264; E-mail: neil.woodford{at}hpa.org.uk
Received 12 October 2007; returned 24 November 2007; revised 30 November 2007; accepted 2 December 2007
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Abstract |
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Objectives: Twenty-four of 209 oxyimino-cephalosporin- and/or aztreonam-resistant Enterobacteriaceae collected around Bolzano had reduced susceptibility or resistance to carbapenems and gave positive metallo-β-lactamase (MBL) tests. Their resistance mechanisms were investigated.
Methods: Resistances were identified by Vitek 2 and MIC tests and isolates were genotyped by PFGE. Resistance genes were identified by PCR and sequencing, and plasmids were transferred by conjugation and/or transformation. Plasmid-borne genes were identified by Southern blotting, and their genetic surroundings were investigated by PCR mapping.
Results: The 24 isolates with positive EDTA/imipenem synergy tests had blaVIM-1 carried on 40–150 kb plasmids. Imipenem MICs ranged from 2 to >32 mg/L, while those of meropenem and ertapenem were lower. The isolates included a clonal cluster of 10 Klebsiella pneumoniae, two other K. pneumoniae isolates, and diverse isolates of Escherichia coli (seven), Klebsiella oxytoca (three) and Citrobacter freundii (two). Six MBL producers were aztreonam-susceptible; the 18 aztreonam-resistant isolates had co-resident extended-spectrum β-lactamases. blaVIM-1 occurred as the first cassette in class 1 integrons, with aacA4 as the second cassette. Quinolone resistance gene qnrS1 was detected in 21 of 24 (87.5%) blaVIM-1-positive isolates versus 14 of 185 (7.6%) blaVIM-negative isolates (P < 0.0001), with 13 of the latter belonging to a clonal cluster of E. coli. qnrS1 was located on the same plasmids as blaVIM-1 and aacA4, but was not closely linked, as judged by PCR mapping.
Conclusions: blaVIM-1 has become disseminated among enterobacteria in a small Italian town. The frequent association of genes conferring carbapenem, aminoglycoside and quinolone resistance on single plasmids will facilitate co-selection.
Key Words: carbapenemases , plasmids , DNA gyrase , fluoroquinolones
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Introduction |
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Clinical Enterobacteriaceae show increasing rates of resistance to cephalosporins, fluoroquinolones and aminoglycosides. Resistance to oxyimino-cephalosporins often reflects production of extended-spectrum β-lactamases (ESBLs), particularly CTX-M types.1 These trends mean that carbapenems are fast becoming the preferred agents for treatment of serious infections caused by Enterobacteriaceae. Disturbingly, however, resistance to carbapenems can arise too, via: (i) combinations of an ESBL and impermeability,2 (ii) production of non-metallo carbapenemases, such as the KPC types3 and (iii) production of metallo-β-lactamases (MBLs) of the VIM and IMP families.3,4 The MBLs, which are inhibited by EDTA, hydrolyse cephalosporins and penicillins in addition to carbapenems but, unlike KPC types, are not active versus aztreonam. The genes for VIM and IMP MBLs generally are located within class 1 integrons, which can be embedded in transposons which, in turn, can be carried on plasmids, allowing great mobility.3 The first VIM metallo-β-lactamase determinant was reported in 1997 in a Pseudomonas aeruginosa clinical isolate from Verona, Italy.5 Subsequently blaVIM has been found repeatedly in Italy and elsewhere, principally in P. aeruginosa, but occasionally in Enterobacteriaceae.
In the present study, we investigated resistance mechanisms in 209 cephalosporin-resistant isolates of Enterobacteriaceae from patients in Bolzano, a town in Northern Italy with a population of 100 000. About 10% of these isolates showed some reduction in carbapenem susceptibility, and most were also resistant to quinolones and aminoglycosides, and emphasis was placed on these isolates. We therefore sought genes encoding carbapenemases, ESBLs and also genes responsible for reduced fluoroquinolone susceptibility, including the qnr alleles6–8 and aac(6')-Ib-cr.9
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Bacterial identification and susceptibility testing
We examined 209 non-duplicate enterobacterial isolates, collected during 2005–07, that were resistant to one or more of cefotaxime, ceftazidime, cefepime and aztreonam, as determined by the Vitek 2 system with GN09 or GN13 cards (bioMérieux, Marcy l’Étoile, France). These isolates were from in- and outpatients of the Bolzano Regional Hospital, apart from single isolates collected at other healthcare facilities in the Province of Bolzano. The collection comprised 107 Escherichia coli, 37 Klebsiella oxytoca, 26 Klebsiella pneumoniae, 23 Proteus mirabilis, 1 Proteus vulgaris, 4 Citrobacter freundii, 1 Citrobacter koseri, 4 Enterobacter cloacae, 4 Serratia marcescens and 2 Morganella morganii, all as identified by the Vitek 2 system. One hundred and twenty-two isolates were from urine samples, 25 from blood and 14 from the lower respiratory tract.
Double disc synergy tests (DDS) for ESBLs were carried out according to CLSI criteria,10 with an amoxicillin/clavulanic acid (20 µg/10 µg) disc 30 mm apart (centre to centre) from ceftazidime (30 µg), cefotaxime (30 µg), cefpodoxime (10 µg), cefepime (30 µg) and aztreonam (30 µg) discs. For MBL-positive isolates, the synergy test for amoxicillin/clavulanic acid and aztreonam was repeated with the discs 20 mm apart. Cefotaxime–cefotaxime/clavulanic acid, ceftazidime–ceftazidime/clavulanic acid and cefepime–cefepime/clavulanic acid ESBL Etests and imipenem–imipenem/EDTA Etests for MBL detection were used, following methods in the manufacturer’s manual (AB Biodisk, Solna, Sweden). MICs for MBL producers were confirmed by agar dilution, according to BSAC guidelines.11
Multiplex PCR for blaCTX-M genes was performed with published primers.12 Detection of blaVIM-1/2-type MBL genes was performed by using consensus primers,13 and positive results were confirmed with blaVIM-1-type-specific primers.14 Published primers were used to amplify the class 1 integrase gene,15 the sulI gene,16 and 5' and 3' conserved integron regions,17 the qnrA,6 qnrB7 and qnrS8 alleles, and the aminoglycoside resistance genes aadA118 and aacA419 [the latter is synonymous with aac(6')-Ib], with the allelic variant aac(6')-Ib-cr detected by PCR-RFLP (where RLFP stands for restriction fragment length polymorphism).9,19 Long-range PCR (Expand Long Template PCR System, Roche, Basel, Switzerland) was used to examine any linkage between blaVIM-1 and qnrS1.
Sequencing of PCR products was performed with the primers used for amplification. In all cases, products were first purified with the Geneclean® Turbo for PCR Kit (Q-BIOgene, Cambridge, UK) and sequencing was performed with the GenomeLabTM Dye Terminator Cycle Sequencing using Quick Start Kit with a Beckman Coulter CEQ 8000 Genetic Analysis System (Beckman Coulter, High Wycombe, UK).
Isolates were compared by PFGE of XbaI-digested genomic DNA,20 with banding patterns analysed using BioNumerics software (BioNumerics, Sint-Martens-Latem, Belgium). Isolates were considered to be clonally related if there was 
85% similarity.21
Construction of transformants and transconjugants
MegaX E. coli DH10B T1 electrocompetent cells (Invitrogen, Paisley, UK) were transformed by electroporation with a Bio-Rad GenePulser II electroporator at 2.0 kV, 200 
and 25 µF using plasmids extracted22 and precipitated twice with ethanol. Transformants were selected on LB agar containing 2 mg/L cefotaxime.
Conjugation was performed using both transformants and rifampicin-susceptible clinical isolates as donors and the rifampicin-resistant E. coli J53-2 strain as a recipient. Transconjugants were selected on LB agar with 4 mg/L cefotaxime and 60 mg/L rifampicin. The transfer frequency was calculated as the number of transconjugants per input donor cell.
Plasmid DNA was extracted,22,23 separated on agarose gels and then transferred to nylon membranes (Hybond N, Amersham Biosciences, Buckinghamshire, UK). Probes were prepared by incorporating digoxigenin-11-dUTP into PCR products of blaVIM-1 (primers VIM-1a/VIM-1b)14 and qnrS1 (primers qnrSF/qnrSR)8 according to the manufacturer's directions (Roche Diagnostics, Mannheim, Germany). The probes were hybridized with the nylon membrane overnight at 70ºC and hybrids were detected with anti-digoxigenin-alkaline phosphatase and the reagent NBT/BCIP (Roche).
Resistance plasmids were extracted from transformants and digested with EcoRI, BamHI and SacI (Roche). The resulting fragments were purified with the GENECLEAN®SPIN Kit (Q-BIOgene, Cambridge, UK), digested further with EcoRV and HindIII (Roche) and separated by electrophoresis on 1% agarose gels. Southern blotting and hybridization of the RFLP fragments with blaVIM- and qnrS-specific probes were performed as described for whole plasmids.
Multiple sequences were aligned with Clustal W (http://www.ebi.ac.uk/clustalw/index.html) and homology searches were carried out using BLAST (http://www.ncbi.nlm.nih.gov/).
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Antibiotic susceptibilities and detection of blaCTX-M and blaVIM
Of 209 oxyimino-cephalosporin-resistant enterobacterial isolates collected in Bolzano, 198 (95%), including 106/107 E. coli tested, were positive in one or more phenotypic ESBL test. Ninety-four of these isolates tested positive for blaCTX-M (Table 1); these included 88 E. coli. Ninety-two isolates had blaCTX-M group 1 genes, while single E. coli and K. pneumoniae isolates had blaCTX-M group 9 genes.
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More surprisingly, 24 isolates appeared phenotypically to be MBL producers based on synergy between imipenem and EDTA (Table 1). These comprised 12 K. pneumoniae, 7 E. coli, 3 K. oxytoca and 2 C. freundii. All were resistant to oxyimino-cephalosporins, piperacillin/tazobactam and cefoxitin and showed weak synergy, at most, with cephalosporin/clavulanate combinations (Table 2). MICs of imipenem ranged from 2 to >32 mg/L, but were reduced to
1 mg/L in the presence of 320 mg/L EDTA. MICs of meropenem and ertapenem generally were lower than those of imipenem, but were above those for typical MBL-negative isolates. Aztreonam MICs were bimodally distributed, clustering either 0.12–0.5 or 32 mg/L. Organisms in the latter group showed aztreonam/clavulanate synergy in double disc tests, implying concurrent production of ESBLs.
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These 24 phenotypically MBL-positive isolates were variously from outpatients (three E. coli), a care-of-the-elderly home (one E. coli) and wards of the Bolzano Regional Hospital (General Medicine Unit, Paediatrics, Nephrology, Geriatrics, Gynaecology, Haematology, Vascular Surgery, Intensive Care Unit, Emergency Unit) over a 20 month period. Eleven were from urine samples (six E. coli, two K. pneumoniae, two K. oxytoca and one C. freundii), four were from blood (three K. pneumoniae and one K. oxytoca), three were from respiratory specimens (two K. pneumoniae and one C. freundii) and the remaining six were variously from peritoneal fluid, a central venous catheter, vaginal secretions, an ulcer, a wound and the oral mucosa.
blaVIM-type genes were detected by PCR in all 24 phenotypically MBL-positive isolates, but in none of the 185 isolates that were phenotypically negative for MBLs (Table 1). Sequencing of the amplicons from nine representative isolates confirmed classical blaVIM-1 in all cases.
PCR mapping was performed for all 24 blaVIM-positive isolates and, in all cases, blaVIM-1 was found to be the first gene within a class 1 integron, followed by aacA4 (Figure 1). Sequencing of the aacA4 genes, encoding AAC(6')-Ib, from four representative isolates revealed a Leu119Ser substitution compared with GenBank sequence AAA26550 [GenBank] , with isolate E. coli 108 also having a Gln118Leu substitution. The regions downstream of blaVIM and aacA4 were not further investigated, but the 3'-CS regions and sulI genes were missing. The only exception was K. oxytoca strain 37, which had a second integron with aacA4 as the first gene and aadA1 as the last gene within the variable region amplified; the intervening approx. 1.5 kb region was not investigated; PCR indicated that sulI was present.
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Plasmidic quinolone resistance genes
Twenty-one of 24 blaVIM-1 positive isolates were positive by PCR for a qnrS allele (Table 1), whereas all were negative for qnrA and qnrB. Sequencing of eight qnrS alleles consistently identified qnrS1. Two of the seven blaVIM-1-positive E. coli strains also had both the classical aac(6')-Ib allele and the aac(6')-Ib-cr variant, as indicated by PCR-RFLP (Table 1). The high prevalence of qnrS1 prompted testing of the 185 blaVIM-negative isolates. This screening led to detection of qnrS alleles in 13 of 100 blaVIM-negative, ESBL-positive E. coli isolates, and in one of two M. morganii isolates (Table 1).
The relatedness of the 24 blaVIM-1-positive isolates was assessed by PFGE, as was that of the 13 blaVIM-negative, qnrS-positive E. coli isolates, and (as a control group) 11 blaVIM- and qnrS-negative E. coli isolates. Ten of the 12 blaVIM-1-positive K. pneumoniae isolates, all also positive for qnrS1, belonged to a single cluster, whereas the seven blaVIM-1-positive E. coli, three K. oxytoca and two C. freundii isolates had little obvious relatedness (data not shown). The 13 qnrS-positive, blaVIM-negative E. coli clustered together, with 87% similarity; four qnrS- and blaVIM-negative isolates also formed part of this cluster.
Southern blotting of plasmid preparations from the seven blaVIM-1-positive E. coli isolates located blaVIM-1 on large plasmids of 60–150 kb (Figure 2). Among the Klebsiella and C. freundii isolates, the probe consistently hybridized with 
40–60 kb plasmids. However, variation of plasmid size was noted even within isolates belonging to the K. pneumoniae cluster (data not shown). In 17 of 21 isolates, including in the 10 clonal K. pneumoniae, hybridization confirmed that blaVIM-1 and qnrS1 were carried by the same plasmids; for the four remaining isolates, the qnrS1 signal was very weak, precluding confident assessment. Long extension time PCR was used in attempts to amplify the regions spanning from blaVIM-1 to qnrS1, but no products were obtained, suggesting that these genes were well separated.
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Transfer of blaVIM-1 and qnrS1 plasmids
To further investigate linkage of blaVIM-1 and qnrS1 genes, plasmids from five blaVIM-1/qnrS1-positive Enterobacteriaceae that lacked an ESBL (i.e. which were aztreonam-susceptible) were transformed into E. coli strain DH10B. Around 10 000 transformant colonies were obtained in each experiment and one clone from each experiment was analysed further. In parallel studies, transconjugants were obtained in broth matings using the clinical isolates E. coli 108, K. oxytoca 35 and C. freundii 24, or their E. coli DH10B transformants, as the donor strains. Transfer frequencies ranged from 5 x 10–5 to 5 x 10–4. Transconjugants were not obtained using the isolates K. pneumoniae 25 and C. freundii 25, or their transformants, as donors.
The MICs for a representative transconjugant are shown in Table 3. Plasmid-encoded gene products caused 256-fold increases in the MICs of third-generation cephalosporins (also fourth-generation cephalosporins; data not shown), and these values were not reduced by clavulanate. Rises in carbapenem MICs were 8- to 66-fold, to 0.25–8 mg/L, with values lowest for ertapenem and highest for imipenem. By contrast, plasmid acquisition had little effect on aztreonam MICs, which remained 0.25 mg/L. Among quinolones, nalidixic acid showed the least pronounced increases in MIC (4–8-fold), followed by ofloxacin (8–16-fold), ciprofloxacin, levofloxacin and moxifloxacin (32–125-fold) (Table 3; data not shown). The transformants and transconjugants also showed 16–32-fold MIC increases for tobramycin and kanamycin, 4–8-fold for netilmicin and gentamicin and, at most, a 2-fold increase for amikacin.
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RFLP-typing revealed very similar digestion patterns for the plasmids transformed from three of the five isolates, with 10 of 14 bands identical (Figure 3). Hybridization showed that the blaVIM-1 and qnrS1 genes were located on different fragments, but the same hybridization patterns were seen in all three isolates. Two of these, E. coli 108 and K. oxytoca 35, were from urines of a single paediatric patient and the third, C. freundii 24, was from a bronchial aspirate from a patient in a different hospital ward.
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Between January 2005 and April 2007, 24 enterobacterial isolates with VIM-1 MBL were isolated in the Bolzano Regional Hospital, Italy. They comprised 7 E. coli, 12 K. pneumoniae, 3 K. oxytoca and 2 C. freundii. These isolates, like other MBL-producing Enterobacteriaceae,24 varied in their level of carbapenem resistance. Eighteen also had ESBLs and, unlike those without ESBLs, were also resistant to aztreonam; 10 of the 12 K. pneumoniae isolates were clonally related, whereas no close relatedness could be found for the other isolates. The 10 clonal K. pneumoniae isolates were collected from 9 wards over 16 months. These data follow a few previous reports of MBLs in Enterobacteriaceae in Italy.25–27 There are also reports of P. aeruginosa with VIM and IMP enzymes in Italy.28,29 The blaVIM-1 genes in the Bolzano isolates were localized on plasmids, which varied in size between 40 and 150 kb, and with variation seen even among the clonal K. pneumoniae isolates. Conjugative transfer of these plasmids was demonstrated from strains of three different species; the plasmids had very similar RFLP patterns and shared restriction fragments that carried qnrS1 and blaVIM-1 genes.
These results indicate that blaVIM-1 is disseminating horizontally among different strains and species within the Bolzano Regional Hospital, with evidence also of strain spread. We have provided evidence of plasmid transfer but, allowing the variation in plasmid size, more complex rearrangements must also be occurring. As is typical, blaVIM-1 was carried within integrons and the integron structure in the different isolates was similar or identical. Thus, all of the blaVIM-1-harbouring integrons were of class 1 and had blaVIM-1 and aacA4 in the first and second positions, respectively. This arrangement corresponds to that within the blaVIM-1-positive P. aeruginosa type strain isolated in 1997 in Verona,5 and in other Italian blaVIM-1-harbouring integrons.30–32 Nevertheless, the integron structure in the present isolates was unusual, with respect to the absence of sulI, which normally is present in blaVIM integrons from Italian isolates.
Production of VIM-1 enzyme was associated with significant decreases in susceptibility to different β-lactam antibiotics, including carbapenems, though, as many others have found, resistance was marginal at current breakpoints.26 Aztreonam retained activity. The MICs of imipenem were much more affected by the VIM-1 metallo-β-lactamase than those of meropenem and, especially, ertapenem. Similar relative effects have been reported for VIM-1 enzyme by others24 and contrast with the behaviour of KPC enzymes33 and combinations of ESBL plus impermeability,2 which have their greatest effect on ertapenem.
A further striking feature of this study is the distribution of qnrS1, and its linkage on VIM-1-encoding plasmids. Enterobacterial isolates, primarily E. cloacae, with qnr genes have been reported worldwide and probably are grossly underdetected among quinolone-resistant bacteria. In Europe, qnrS has been reported mainly in Salmonella isolates in France,34 Spain,35 Denmark,36 the UK37 and Turkey,38 but not previously in Italy. Here, qnrS1 was found in 21/24 VIM-positive isolates and unsequenced qnrS alleles also in 13/100 ESBL-positive clonal E. coli isolates. Authors in Taiwan have previously found a high prevalence of qnr (78.6%) among producers of IMP-8 metallo-β-lactamase, and 39.6% among isolates that only had ESBLs.39
Among (fluoro)quinolones, nalidixic acid showed the least pronounced MIC increases following acquisition of plasmids encoding QnrS1, followed by ofloxacin, ciprofloxacin, levofloxacin and moxifloxacin. Nevertheless, qnrS1-harbouring plasmids led to intermediately resistant phenotypes for nalidixic acid, but MICs of ciprofloxacin and levofloxacin remained within the susceptible range, as reported by others.6 The donor bacteria exhibited greater resistance to (fluoro)quinolones than E. coli transformants and transconjugants, suggesting the presence of additional resistance mechanisms, probably chromosomal mutations; the acetyltransferase gene, aac(6')-Ib-cr,9 was present in two VIM-1-producing E. coli isolates.
The transformants and transconjugants showed 16–32-fold MIC increases for kanamycin and tobramycin, 4–8-fold for netilmicin and gentamicin and 2-fold at most for amikacin. AAC(6')-Ib, encoded by aacA4, generally confers resistance to amikacin, but not to gentamicin. However, the Leu119Ser substitution that was detected in four sequenced isolates has been associated with the loss of amikacin resistance and increased gentamicin MICs.40,41
To conclude, related plasmids encoding linked resistance to carbapenems (blaVIM-1), aminoglycosides [aacA4, encoding AAC(6')-Ib] and quinolones (qnrS1) were found in clinical isolates belonging to four enterobacterial species in a small Italian town. Any of the drugs might therefore co-select resistance to all, favouring further dissemination. Since many of these bacteria were resistant to all good antibiotics the extent of this threat cannot be overestimated.
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This work was undertaken by R. A. in partial fulfilllment of the requirements for an MSc degree course. No external funding was provided for its completion.
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D. M. L. and N. W. have each received research grants and accepted speaking engagements/conference invitations from various companies. D. M. L. holds shares in various pharmaceutical companies within a diversified portfolio. Neither D. M. L. nor N. W. is aware of any conflicts of interest with the content of the current paper. Other authors have no conflicts of interest to declare.
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Acknowledgements |
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We would like to express our gratitude to Katie Hopkins and Michael Hornsey for providing primers and to Matthew Ellington, Edi Karisik, Marina Warner and Rachel Pike for valuable technical advice and assistance. We express our thanks to Gianna De Fina, Dietmar Alber, Beatrice Stefani and Stefania Fracasso for the help in the collection of strains and antimicrobial susceptibility testing.
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References |
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