JAC Advance Access originally published online on February 21, 2008
Journal of Antimicrobial Chemotherapy 2008 61(5):999-1002; doi:10.1093/jac/dkn068
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Original research |
Surveillance for plasmid-mediated quinolone resistance determinants in Enterobacteriaceae within the Calgary Health Region, Canada: the emergence of aac(6')-Ib-cr
1 Division of Microbiology, Calgary Laboratory Services, University of Calgary, #9, 3535 Research Road NW, Calgary, Alberta, Canada T2L 2K8 2 Department of Pathology and Laboratory Medicine, University of Calgary, #9, 3535 Research Road NW, Calgary, Alberta, Canada T2L 2K8 3 Department of Microbiology and Infectious Diseases, University of Calgary, #9, 3535 Research Road NW, Calgary, Alberta, Canada T2L 2K8 4 Department of Medicine, University of Calgary, #9, 3535 Research Road NW, Calgary, Alberta, Canada T2L 2K8
* Corresponding author. Division of Microbiology, Calgary Laboratory Services, #9, 3535 Research Road NW, Calgary, Alberta, Canada T2L 2K8. Tel: +1-403-770-3309; Fax: +1-403-770-3347; E-mail: johann.pitout{at}cls.ab.ca
Received 19 December 2007; returned 22 January 2008; revised 25 January 2008; accepted 28 January 2008
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Abstract |
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Objectives: A study was designed to determine the prevalence of plasmid-mediated quinolone resistance (PMQR) determinants among clinical isolates of Enterobacteriaceae from the Calgary Health Region (CHR).
Methods: During January and February 2004 and January and February 2007, 564 non-repeat isolates of Escherichia coli, Klebsiella spp., Proteus mirabilis and Morganella morganii resistant to ciprofloxacin and/or tobramycin were screened for PMQR determinants using a multiplex PCR for qnrA, qnrS and qnrB and aac(6')-Ib genes; aac(6')-Ib-cr was further identified by digestion with BstF5I.
Results: In 2004, 6/139 (4%) of the resistant E. coli were positive for aac(6')-Ib-cr. In 2007, 53/398 (13%) were positive for aac(6')-Ib-cr, 3/398 (0.8%) were positive for qnrS, and one isolate was positive for both. All the isolates were present in urines and the majority [40/63 (63%)] were submitted from community collection sites; 8 (13%) isolates co-produced AmpC β-lactamases and 34 (54%) co-produced CTX-M-15. aac(6')-Ib-cr was present in one Klebsiella pneumoniae and one P. mirabilis, whereas one isolate of K. pneumoniae was positive for both aac(6')-Ib-cr and qnrB.
Conclusions: Our results showed that isolates with aac(6')-Ib-cr, often associated with CTX-M-15, are emerging among fluoroquinolone-resistant E. coli in the CHR. Our study suggests that surveillance for PMQR determinants should be undertaken on a regular basis.
Keywords: PMQR , CTX-M-15 , Escherichia coli
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Introduction |
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Recently, plasmid-mediated quinolone resistance (PMQR) due to the production of pentapeptide repeat proteins has been described in clinical isolates of Enterobacteriaceae from different parts of the world.1 These proteins, named QnrA, QnrS and QnrB, block the action of fluoroquinolones on bacterial topoisomerases, and the genes responsible for producing these enzymes are associated with class 1 integrons.2
A different mechanism of transferable quinolone resistance that involved the enzymatic inactivation of certain quinolones was first reported in 2006.2 This is due to a variant of an aminoglycoside-modifying enzyme aac(6')-Ib, named aac(6')-Ib-cr. The parent enzyme and its variant provide resistance to the following aminoglycosides: kanamycin, tobramycin and amikacin. The variant enzyme aac(6')-Ib-cr has the additional ability to inactivate fluoroquinolones with an unprotected amino nitrogen on the piperazine ring and includes norfloxacin and ciprofloxacin.
Several recent surveillance studies from different parts of the world have shown that resistance to the fluoroquinolones among Enterobacteriaceae (especially Escherichia coli) has increased dramatically worldwide, especially during the past 5 years. It is not known whether PMQR is responsible for the rapid increase in fluoroquinolone resistance as studies describing the prevalence of PMQR determinants have mostly included selected bacterial populations such as nosocomial, extended-spectrum β-lactamase (ESBL)-3 or AmpC β-lactamase-producing isolates.4 There are no data available regarding the prevalence of PMQR determinants among bacteria in large healthcare regions, serving a population with a significant proportion of community isolates.
The Calgary Health Region (CHR) is such a region that provides all publicly funded healthcare services to the 1.2 million people residing in the cities of Calgary and Airdrie and numerous adjacent communities covering an area of 37 000 km2. Acute care is provided principally through one paediatric and three large adult hospitals. A centralized laboratory (Calgary Laboratory Services) performs routine clinical microbiology services for both the community clinics and hospitals within the CHR. We investigated the prevalence of PMQR determinants in Enterobacteriaceae isolated in the CHR during January and February 2004 and again during January and February 2007.
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Materials and methods |
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Bacterial isolates
Consecutive non-duplicate isolates of ciprofloxacin- and/or tobramycin-resistant Enterobacteriaceae collected at Calgary Laboratory Services during January and February 2004 and January and February 2007 were included in this study. Isolates were identified to the species level with Vitek 2 (Vitek AMS; bioMérieux Vitek Systems Inc., Hazelwood, MO, USA).
Antimicrobial susceptibility testing
MICs of the following drugs were determined by Vitek 2 (Vitek AMS; bioMérieux Vitek Systems Inc.): cefalotin, amoxicillin/clavulanate, imipenem, gentamicin, tobramycin, amikacin, co-trimoxazole, nitrofurantoin and ciprofloxacin. Throughout the study, results were interpreted using the CLSI criteria for broth dilution.5 The quality control strains used for this part of the study were E. coli ATCC 25922 and E. coli ATCC 35218.
The amplification of the qnrA, qnrS and qnrB genes was undertaken with multiplex PCR, as described previously.6 aac(6')-Ib was amplified in a separate PCR reaction using primers and conditions previously described.7 The variant aac(6')-Ib-cr was further identified by digestion with BstF5I (New England Biolabs, Ipswich, MA, USA). The quality control strains used for this part of the study were 16-00 with qnrA, Kp15 with qnrB, S2 with qnrS and 78-01 with aac(6')-Ib-cr.
Screening for and confirmation of ESBLs and AmpC β-lactamases
The presence of ESBLs was evaluated in clinical isolates of E. coli, Klebsiella spp. and Proteus mirabilis by using the CLSI criteria for ESBL screening and disc confirmation tests.5 The presence of AmpC β-lactamases was evaluated in clinical isolates of E. coli, Klebsiella spp. and P. mirabilis by using cefoxitin resistance (on Vitek 2) as a screen, followed by a confirmation with cefotetan discs with and without boronic acid.8
β-Lactamase gene identification
The ESBL-positive isolates investigated for the presence of CTX-M β-lactamases and isolates that tested positive for blaCTX-Ms underwent additional amplification using group-specific primers for CTX-M groups 1, 2, 8, 14 and 25, respectively.9 The blaCTX-M was identified by automated sequencing using primers and conditions, as described previously.9
Pulsed-field gel electrophoresis
Typing was performed with pulsed-field gel electrophoresis (PFGE) following the extraction of genomic DNA and digestion with XbaI using the standardized E. coli (O157:H7) protocol established by the Centres for Disease and Prevention, Atlanta, GA, USA.10 The subsequent PFGE analyses were performed on a CHEF-MAPPER apparatus (Bio-Rad Laboratories, Hercules, CA, USA). DNA relatedness was calculated on the basis of the Dice coefficient, and isolates were considered to be genetically related if the Dice coefficient correlation was 80% or greater, which corresponds to the ‘possibly related (four to six bands difference)’ criteria of Tenover et al.11
Plasmid fingerprints were performed with HpaI using protocols and conditions previously described12 and classified according to their incompatibility group by a PCR-based replicon-typing scheme.13
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Results |
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Bacterial isolates
During 2004, 2895 E. coli were isolated, of which 121 (4%) were ciprofloxacin-resistant and 84 (3%) were tobramycin-resistant [139 (5%) isolates were resistant to both]. In addition, 5 of 392 (1%) Klebsiella spp. were ciprofloxacin-resistant, 1 of 392 (0.3%) Klebsiella spp. were tobramycin-resistant, 3 of 139 (2%) P. mirabilis were ciprofloxacin-resistant and 1 of 14 (7%) Morganella morganii were ciprofloxacin-resistant (Table 1). During 2007, 4352 E. coli were isolated, of which 346 (8%) were ciprofloxacin-resistant and 223 (5%) were tobramycin-resistant [398 (9%) isolates were resistant to both]. In addition, 7 of 389 (2%) Klebsiella spp. were ciprofloxacin-resistant, 2 of 389 (0.5%) Klebsiella spp. were tobramycin-resistant, 10 of 233 (4%) P. mirabilis were ciprofloxacin-resistant and 1 of 25 (4%) M. morganii were ciprofloxacin-resistant (Table 1).
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PMQR determinants
In 2004, 6/139 (4%) of the resistant E. coli produced aac(6')-Ib-cr; whereas 53/398 (13%) were positive in 2007, 3/398 (0.8%) were positive for qnrS, and 1 isolate was positive for both aac(6')-Ib-cr and qnrS (Table 1). The qnr determinants were not present in 2004. All the E. coli isolates (2004 and 2007) were present in urine and the majority [40/63 (63%)] were submitted from community collection sites. aac(6')-Ib-cr was present in one Klebsiella pneumoniae and one P. mirabilis, whereas one isolate of K. pneumoniae was positive for both aac(6')-Ib-cr and qnrB (Table 1). PMQR determinants were not present among the Klebsiella spp. or P. mirabilis collection from 2004 or from M. morganii. The isolates with PMQR determinates that were resistant to ciprofloxacin (33/66) had a ciprofloxacin MIC of >4 mg/L.
Antimicrobial susceptibility testing
Of the 63 E. coli isolates with PMQR determinants, 50 (79%) were resistant to cefalotin, 52 (82%) to amoxicillin/clavulanate, 22 (35%) to gentamicin, 56 (89%) to tobramycin, 2 (3%) to amikacin, 34 (54%) to co-trimoxazole, 19 (30%) to nitrofurantoin and 57 (90%) to ciprofloxacin; none of the isolates was resistant to imipenem. The Klebsiella spp. and P. mirabilis with PMQR determinants were resistant to cefalotin, amoxicillin/clavulanate, tobramycin, co-trimoxazole, ciprofloxacin and nitrofurantoin.
β-Lactamase gene identification
Of the 63 E. coli isolates with PMQR determinants, 8 (13%) isolates co-produced AmpC β-lactamases and 34 (54%) co-produced ESBLs (Table 1). All the isolates producing ESBLs or AmpC β-lactamases were positive for aac(6')-Ib-cr. The ESBL-producing E. coli (2 from 2004 and 32 from 2007) produced CTX-M-15. ESBLs or AmpC β-lactamases were not present among the Klebsiella spp. or P. mirabilis with PMQR determinants (Table 1).
Pulsed-field gel electrophoresis
PFGE identified two closely related groups of E. coli producing CTX-M-15 [designated 15A (n = 22) and 15AR (n = 5) (i.e. related to A)]. The 15A and 15AR isolates formed separate clusters with >80% similar PFGE profiles. The 15AR isolates exhibited >60% similarity of profiles to 15A, suggesting that 15AR is related to 15A. The remaining seven CTX-M-15-producing and five aac(6')-Ib-cr-positive E. coli (i.e. ESBL-negative) were not related to clusters 15A, 15AR or to each other and were designated 15NR. These clusters were previously reported in a molecular epidemiology study.9
Five strains of CTX-M-15 belonging to 15A (n = 2), 15AR (n = 2) and 15NR (n = 1) were selected for the plasmid analysis. The analysed plasmids belonged to the narrow host range incompatibility group IncFII and showed the replicon FII alone (15NR) or in association with FIA (15A and 15AR). The sizes ranged from 85 to 120 kb.
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Discussion |
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The widespread infiltration of qnr determinants and aac(6')-Ib-cr could potentially fuel the rapid development of fluoroquinolone resistance. This was recently documented in a clinical setting when a fluoroquinolone-susceptible E. coli with qnrA developed chromosomal mutations in the topoisomerase genes with subsequent high-level resistance after 5 days of norfloxacin therapy.14
Our study compared the prevalence of PMQR determinants in Enterobacteriaceae over two separate periods, 3 years apart. We chose to screen all ciprofloxacin- and/or tobramycin-resistant isolates; however, not all isolates with qnr determinants are necessarily resistant to these two agents and it is possible that some isolates with qnr determinants were missed. Our results showed a substantial increase in ciprofloxacin resistance in E. coli isolated in the CHR during the two different periods: 121/2895 (4%) isolates from 2004 when compared with 346/4352 (8%) isolates during the same months in 2007 (Table 1). There was an absence of qnr determinants in 2004 and the presence of these determinants remained low in 2007 (Table 1). Our study showed that E. coli with aac(6')-Ib-cr are emerging among fluoroquinolone-resistant isolates; 52/346 (15%) from 2007 were positive for aac(6')-Ib-cr when compared with 5/121 (4%) from 2004. Two isolates from 2007 and one from 2004 tested susceptible to ciprofloxacin. Nearly 70% of E. coli with aac(6')-Ib-cr were isolated from urines submitted to community patient care centres.
Of particular interest was that the majority of E. coli positive for aac(6')-Ib-cr [34/60 (57%)] co-produced CTX-M-15. E. coli producing CTX-M-15 associated with aac(6')-Ib-cr is causing a pandemic and, since 2005, it has become the most common type of ESBL isolated in the CHR.9 It seems that the influx of these clonally related ESBL producers is partly responsible for the increase in aac(6')-Ib-cr.
The prevalence of ciprofloxacin resistance among other members of the Enterobacteriaceae isolated in the CHR remains relatively low. This was also reflected in the low prevalence of PMQR determinants among these species (Table 1).
Robicsek et al.2 in a recent review suggested that ciprofloxacin will emerge as a suboptimal ‘workhorse’ fluoroquinolone where the prevalence of aac(6')-Ib-cr is high. Our results show that E. coli with aac(6')-Ib-cr is emerging as an important cause of fluoroquinolone resistance in the CHR and this is partly due to the influx of clonally related CTX-M-15-producing isolates co-producing aac(6')-Ib-cr. Our study suggests that surveillance for PMQR determinants should be undertaken on a regular basis to monitor the increase in the bacteria harbouring them. This has clinical implications such as the possibility of selection of high-level fluoroquinolone resistance and evaluating the use of ciprofloxacin within the CHR. Future surveillance studies for PMQR determinants should also include ciprofloxacin-intermediate and -susceptible bacteria as these isolates might be inappropriately treated with the quinolones.
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This work was supported by a grant from the University of Calgary Dean's Starter Grant (#75-4777) and Y. W. was employed through the Biomedical Technology summer student programme funded by Calgary Laboratory Services and University of Calgary.
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J. D. D. P., D. L. C. and D. B. G. have previously received research grants from Merck, AstraZeneca and Wyeth.
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Acknowledgements |
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We thank Terry Ross, Calgary Laboratory Services, Calgary, Alberta, for her technical support of this study and Laurent Poirel for providing the control strains Kp15 and S2.
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References |
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1 Nordmann P, Poirel L. Emergence of plasmid-mediated resistance to quinolones in Enterobacteriaceae. J Antimicrob Chemother (2005) 56:463–9.
2 Robicsek A, Jacoby GA, Hooper DC. The worldwide emergence of plasmid-mediated quinolone resistance. Lancet Infect Dis (2006) 6:629–40.[CrossRef][Web of Science][Medline]
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Rodriguez-Martinez JM, Pascual A, Garcia I, et al. Detection of the plasmid-mediated quinolone resistance determinant qnr among clinical isolates of Klebsiella pneumoniae producing AmpC-type β-lactamase. J Antimicrob Chemother (2003) 52:703–6.
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Boyd DA, Tyler S, Christianson S, et al. Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum β-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob Agents Chemother (2004) 48:3758–64.
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Poirel L, Pitout JD, Calvo L, et al. In vivo selection of fluoroquinolone-resistant Escherichia coli isolates expressing plasmid-mediated quinolone resistance and expanded-spectrum β-lactamase. Antimicrob Agents Chemother (2006) 50:1525–7.
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