J Antimicrob Chemother 2014; 69: 1492 – 1496 doi:10.1093/jac/dku011 Advance Access publication 4 February 2014
High diversity of plasmids harbouring blaCMY-2 among clinical Escherichia coli isolates from humans and companion animals in the upper Midwestern USA Valeria Bortolaia1*, Katrine H. Hansen1, Christine A. Nielsen1, Thomas R. Fritsche2,3 and Luca Guardabassi1 1
Department of Veterinary Disease Biology, University of Copenhagen, Frederiksberg C, Denmark; 2Division of Laboratory Medicine, Marshfield Clinic, Marshfield, WI, USA; 3Department of Microbiology, University of Wisconsin, La Crosse, WI, USA *Corresponding author. Tel: +45-35332770; Fax: +45-35332755; E-mail: [email protected]
Received 9 October 2013; returned 14 November 2013; revised 3 January 2014; accepted 7 January 2014 Objectives: To determine the population structure and genetic relatedness of plasmids encoding CMY-2 b-lactamase in clinical Escherichia coli from humans and companion animals within a defined geographical area. Methods: In total, 42 human and 73 companion animal isolates displaying an AmpC phenotype were isolated at a regional diagnostic reference laboratory in the upper Midwestern USA during 2009–11. Following PCR screening for transferable AmpC genes and plasmid transformation, blaCMY-2-positive plasmids were characterized by S1 nuclease PFGE, PCR-based replicon typing, antimicrobial susceptibility testing of transformants, conjugation experiments, plasmid multilocus sequence typing and restriction fragment length polymorphism. Results: blaCMY-2 occurred in 6 (14%), 56 (86%) and 6 (75%) isolates from humans, dogs and cats, respectively. Usually plasmids carrying blaCMY-2 were conjugative (78%) and did not contain additional resistance genes (82%). The replicon types were IncI1 (52%), IncA/C (13%), IncFII (10%), IncI2 (5%), IncL/M (3%), IncB/O (2%) or nontypeable (15%). Related IncI1/ST12 plasmids were detected in one human and five canine isolates, while the remaining plasmids did not show similarity across host species. A novel epidemiological linkage of blaCMY-2 with IncL/M plasmids and a new CMY gene variant (blaCMY-108) were found in human isolates. Conclusions: This study is one of the first One Health attempts to compare plasmids encoding CMY-2 b-lactamase among clinical isolates from humans and companion animals in the same region. The results indicate an unforeseen heterogeneity of plasmid backgrounds and suggest limited exchange between the two populations, in which blaCMY-2 occurred at very different frequencies and was harboured by distinct plasmid types. Keywords: AmpC, antimicrobial resistance, dogs, cats
Introduction CMY-2 is the most commonly reported plasmid-encoded AmpC b-lactamase in Escherichia coli and other Enterobacteriaceae from different host species worldwide.1 Although less frequently reported compared with extended-spectrum b-lactamases (ESBLs), CMY-2 is clinically relevant since, like ESBLs, it inactivates third-generation cephalosporins and may mediate resistance to carbapenems in combination with loss of outer membrane porins.2 A recent study emphasized the possible risk of transmission of blaCMY-2 from poultry to humans.3 Current knowledge about possible exchange of plasmids harbouring blaCMY-2 between humans and animals is limited to studies examining bacterial isolates from humans, food-producing animals and food.4 – 6 Despite the widespread occurrence of blaCMY-2 in E. coli isolates from dogs and cats
worldwide,7 – 9 zoonotic spread of this b-lactam resistance determinant from companion animals has received scarce attention to date. To shed light on this potential zoonotic risk, population structure and genetic relatedness of plasmids carrying blaCMY-2 were investigated in clinical E. coli isolated from humans, dogs and cats within a defined geographical area.
Methods E. coli displaying an AmpC phenotype (i.e. resistance to cefoxitin and thirdgeneration cephalosporins and susceptibility to cefepime) were isolated from community-dwelling human patients (n ¼ 42), dogs (n ¼ 65) and cats (n¼8) at the clinical laboratories of the Marshfield Clinic, Wisconsin, USA, between 2009 and 2011. The clinical laboratories serve hospitals, clinics and veterinary clinics throughout Wisconsin and the upper Midwest. Transferable AmpC genes were identified by multiplex PCR10
# The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
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and confirmed by singleplex PCR11 and sequencing (Macrogen Inc.). The sequence of a novel blaCMY variant (GenBank accession number KF564 648) was determined using forward primers ISEcp1/F and ecpF212 and reverse primer blaCMY/R (5′ -ATACCCGGAATAGCCTGCTC-3′ ), and forward primer blaCMY/F (5′ -GGGTGCATAAAACGGGATCC-3′ ) and reverse primer blc/R (5′ -GACAACCAGGAATGCAGC-3′ ). Plasmid DNA was isolated from blaCMY-2-positive isolates by alkaline lysis and transformed into electrocompetent Genehog E. coli (Invitrogen). Transformants were selected on BHI agar (Merck) supplemented with cefotaxime (2 mg/L), tested by colony PCR to confirm the presence of blaCMY-2 and by S1 nuclease PFGE to determine plasmid content and size.13 Transformants positive for blaCMY-2 and carrying a single plasmid were further characterized by PCR-based replicon typing (PBRT; Diatheva) and plasmid multilocus sequence typing (pMLST) (http://pubmlst.org/ plasmid/). Plasmid types shared by different host species were further analysed by restriction fragment length polymorphism (pRFLP) using FastDigestw EcoRV and FastDigestw PstI enzymes (Thermo Scientific). Susceptibility to ciprofloxacin, gentamicin, imipenem, trimethoprim/sulfamethoxazole and tetracycline was determined for all transformants by disc diffusion according to CLSI standards.14,15 Transferability of blaCMY-2-positive plasmids was tested by filter-mating experiments using rifampicin-resistant E. coli K12 J62-2 as recipient. Transconjugants were selected on BHI agar containing cefotaxime (2 mg/L) and rifampicin (50 mg/L), and tested by PCR to confirm the presence of blaCMY-2.
Results Six (14%), 56 (86%) and 6 (75%) isolates from humans, dogs and cats, respectively, were positive for blaCMY-2. Furthermore, two (5%) human isolates harboured a novel CMY gene variant, which was assigned blaCMY-108 (http://www.lahey.org/studies). No acquired AmpC genes were detected in the remaining 45 isolates. A plasmid origin was demonstrated for 93% of the blaCMY-2 -positive isolates and 53 (78%) of the plasmids were transferable by conjugation. In contrast, blaCMY-108 was most likely located on the chromosome since repeated transformation and conjugation attempts were unsuccessful. Three transformants harboured multiple plasmids and were not further analysed. The remaining 60 plasmids ranged in size from 45 to 104 kb and harboured IncI1 (n ¼ 31; 52%), IncA/C (n ¼ 8; 13%), IncFII (n ¼ 6; 10%), IncI2 (n ¼ 3; 5%), IncL/M (n ¼ 2; 3%) and IncB/O (n ¼ 1; 2%) replicons. Nine (15%) plasmids were non-typeable by PBRT. IncI1 was detected in all three host species, IncFII in both humans and dogs, IncL/M in humans only, and IncA/C, IncI2, IncB/O and non-typeable plasmids in dogs only (Table 1). Subtyping of IncI1 plasmids showed six known sequence types (ST2, ST12, ST18, ST20, ST23 and ST26) and six new sequence types (ST120, ST121, ST122, ST123, ST124 and ST125) resulting both from novel combinations of known alleles and detection of new alleles. Four (13%) IncI1 plasmids were not typeable as the sogS allele was not detected. Analysis of combinations of the remaining alleles showed that: (i) two such plasmids were singlelocus variants (SLVs) of ST12, having the same allele variants; (ii) one plasmid was an SLV of ST2; and (iii) one plasmid had nearest matches in the pMLST database in only three loci (Table 1). IncI1/ ST12 was identified in nine isolates from all three host species. One human and four canine plasmids yielded indistinguishable RFLP profiles using PstI but the human plasmid differed by three bands using EcoRV (Figure S1, available as Supplementary data at JAC Online). RFLP subtyping of IncFII plasmids revealed the presence of indistinguishable plasmids in two canine isolates, while
the remaining four plasmids yielded unique restriction profiles (Figure S2, available as Supplementary data at JAC Online). Co-transfer of resistance to non-b-lactam antimicrobials was observed for 18% of the plasmids. All IncA/C plasmids co-transferred resistance to tetracyclines, and three IncI1 plasmids co-transferred resistance to trimethoprim/sulfamethoxazole (Table 1). Plasmids belonging to other incompatibility groups and non-typeable plasmids did not transfer resistance to any of the antimicrobials tested.
Discussion To the best of our knowledge, this study is the first One Health attempt to compare plasmids harbouring blaCMY-2 in E. coli isolated from human and companion animal infections within a defined geographical area. Notably, blaCMY-2 occurred at significantly higher frequency in companion animal isolates (85%) than in human isolates (14%), confirming that this resistance determinant is common in canine and feline E. coli infections in the USA.8 The higher prevalence of blaCMY-2 in companion animals compared with humans could reflect the patterns of b-lactam consumption in small animal veterinary practice, where use of first-generation cephalosporins and amoxicillin/clavulanic acid is widespread. It has been previously hypothesized that the intensive use of cefalexin may have favoured dissemination of blaCMY-2 in the dog population.7 The plasmid population was heterogeneous, with IncI1 plasmids being predominant in nearly half of the isolates tested. Most incompatibility groups detected in this study, especially IncA/C and IncFII, have been previously associated with CMY-2 in different host species worldwide.2,16 However, we also detected a novel association with the IncL/M plasmid backbone in two human isolates and rare associations with IncI2 and IncB/O plasmid backbones in canine isolates. The authors are aware of single reports of blaCMY-2 on an IncI2 plasmid in a porcine Salmonella Heidelberg strain from the USA17 and on IncB/O plasmids in poultry isolates from Japan.18 The high diversity of plasmid types carrying blaCMY-2 emphasizes the mobility of this b-lactamaseencoding gene, which has previously been associated with ISEcp1-mediated transposition.19 Genetic relatedness across host species was limited to IncI1/ ST12 plasmids of 90 kb, which have been reported in E. coli and Salmonella isolates from humans and poultry in diverse countries.4,5 Nevertheless, the only IncI1/ST12 plasmid originating from a human patient could be distinguished from related plasmids of canine origin by EcoRV RFLP. An SLV of ST12 was found in another human patient and in a dog. These plasmids may have originated from a common ancestor, although they were clearly distinguishable by size. A limitation of the study is that isolates producing both AmpC b-lactamase and ESBL might be excluded on the basis of the criteria used for strain selection. However, our focus on AmpC b-lactamase enabled analysis of a relatively large number of CMY-encoding plasmids (n¼ 68) compared with previous studies on plasmids mediating cephalosporin resistance in E. coli of animal origin.6 This approach, focused on AmpC b-lactamase, resulted in a variety of original findings. A novel CMY b-lactamaseencoding gene designated as blaCMY-108 was detected in two human isolates. Based on amino acid identity, this novel
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Table 1. Characteristics of blaCMY-2-harbouring plasmids in clinical E. coli isolates from humans, dogs and cats
Isolate ID 2011/34/44 2011/37/26 2011/37/22 2011/37/05 2011/37/36 2011/37/41 2011/36/81 2011/34/35 2011/33/01 2011/37/01 2011/37/33 2011/37/35 2011/37/08 2011/34/56 2011/37/40 2011/34/53 2011/37/27 2011/25/62 2011/34/19 2011/34/41 2011/34/48 2011/36/75 2011/37/14 2011/37/29 2011/37/25 2011/34/52 2011/37/18 2011/34/51 2011/37/24 2011/37/30 2011/34/15 2011/34/16 2011/34/31 2011/37/07 2011/37/06 2011/37/02 2011/34/33 2011/37/31 2011/34/54 2011/37/11 2011/37/12 2011/33/36 2011/34/20 2011/34/21 2010/21/71 2011/37/09 2011/37/15 2011/34/18 2011/34/57 2011/37/34 2011/33/46 2011/34/37
Host species
Isolation sample
PBRT
canine canine canine canine canine canine canine canine human human canine canine canine canine canine feline canine human canine canine canine canine canine canine canine canine canine canine canine feline canine canine canine canine canine canine canine feline canine feline canine human canine canine canine canine canine canine canine human human canine
urine abscess perianal transtracheal urine urine urine urine urine urine skin urine urine urine urine urine urine urine abdominal fluid urine urine urine urine urine endotracheal ear ear urine urine urine ear urine urine urine ear urine urine urine ear urine drainage urine urine urine urine ear urine urine urine urine urine brain
A/C A/C A/C A/C A/C A/C A/C A/C FII FII FII FII FII FII I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I2 I2 I2 B/O L/M L/M NT
Size (kb) 80 90 90 90 90 90 104 104 54 54 54 54 65 65 80 80 80 90 90 90 90 90 90 90 80 104 104 104 104 80 90 104 80 80 104 80 104 80 80 90 90 54 75 80 80 65 65 65 80 54 54 45
Co-transfer of resistance TET TET TET TET TET TET TET TET none none none none none none none none SXT none none none none none none SXT none none none none none none none none none none none SXT none none none none none none none none none none none none none none none none
pMLSTa
2 12 12 12 12 12 12 12 12 12 18 18 18 18 18 20 20 20 23 23 26 120 121 122 123 124 125 NTc NTd NTc NTe
pRFLP (PstI)b
pRFLP (EcoRV)b
A B C C D E
1 2 3 3 4 5
I H F F1 F F F F G
10 8 7 6 6 6 6 6a 9
Conjugation positive negative negative negative positive positive positive positive positive positive positive positive positive positive positive negative negative positive positive positive positive positive positive positive negative negative positive positive positive positive positive positive positive positive negative positive positive positive positive positive positive negative negative negative positive positive positive positive positive positive positive positive Continued
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Table 1. Continued
Isolate ID 2011/34/38 2011/34/50 2011/34/34 2011/34/40 2011/34/43 2011/37/43 2011/37/16 2011/34/29 2011/37/21 2011/34/13 2011/34/32 2011/34/55 2011/36/74 2011/37/23 2011/34/17 2011/37/28
Host species canine canine canine canine canine canine canine canine feline canine feline canine canine canine canine canine
Isolation sample
PBRT
Size (kb)
Co-transfer of resistance
brain urine urine urine urine ear urine urine urine urine urine ear skin urine urine urine
NT NT NT NT NT NT NT NT ND1 ND1 ND1 ND2 ND2 ND2 ND2 ND2
45 45 45 45 45 80 90 90 104, 20 54, 28, 33 45, 65 ND2 ND2 ND2 ND2 ND2
none none none none none none none none ND1 ND1 ND1 ND2 ND2 ND2 ND2 ND2
pMLSTa
pRFLP (PstI)b
pRFLP (EcoRV)b
Conjugation positive positive positive positive positive positive positive positive positive negative positive positive positive negative negative negative
NT, non-typeable; ND1, not determined as more than one plasmid was present in the transformant; ND2, not determined as the plasmid could not be transformed; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline. a pMLST was performed on IncI1 plasmids. b pRFLP was performed on plasmid types shared by different host species. c SLV of ST12. d Novel combination of the remaining allele variants repI1, 1; ardA, 3; trbA, 13; pilL, 3. e SLV of ST2.
b-lactamase was more closely related to CMY-39 and CMY-80 (98.7%) than to CMY-2 (97.6%). The findings of plasmid types that had not (IncL/M) or had only rarely (IncI2 and IncB/O) been associated with CMY-2 previously and of several new IncI1 pMLST types and non-typeable plasmids indicate that the diversity of plasmid backgrounds was only partly explored prior to this study. The high level of genetic diversity observed in plasmids encoding CMY-2 represents an evolutionary advantage that could facilitate further spread of this b-lactam resistance determinant in the years to come as a consequence of the widespread use of b-lactams in both human and veterinary medicine. Co-selection by antimicrobials other than b-lactams seems to play a minor role since only a minority of plasmids harbouring blaCMY-2 carried additional resistance genes. In conclusion, this study shows that plasmids carrying blaCMY-2 in clinical E. coli isolates from humans, dogs and cats in the Midwestern USA are highly heterogeneous, suggesting limited exchange between companion animals and humans. However, the design of this study was not particularly suited to the detection of zoonotic transmission of blaCMY-2 due to the low proportion of human CMY-2-producing isolates, the long period and the wide geographical area covered by the study. We did not examine the genetic background of CMY-2-producers because blaCMY-2 is usually not linked to specific E. coli lineages20 and primarily spreads by horizontal transfer of plasmids. Moreover, strain typing would have not added information on zoonotic transmission since animal and human isolates were epidemiologically unrelated on the basis of their plasmid content.
Acknowledgements We thank Ljudmila Troianova for dedicated technical assistance.
Funding This work was supported by grant HEALTH-F3-2011-282004 (EvoTAR) from the European Union. Surveillance collection support was made possible by the Steve J. Miller Endowment in Rural and Agricultural Health and Safety from the National Farm Medicine Center of the Marshfield Clinic Research Foundation.
Transparency declarations None to declare.
Supplementary data Figures S1 and S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
References 1 Jacoby GA. AmpC b-lactamases. Clin Microbiol Rev 2009; 22: 161– 82. 2 Naseer U, Haldorsen B, Simonsen GS et al. Sporadic occurrence of CMY-2-producing multidrug-resistant Escherichia coli of ST-complexes 38 and 448, and ST131 in Norway. Clin Microbiol Infect 2010; 16: 171– 8.
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3 Bo¨rjesson S, Jernberg C, Brolund A et al. Characterization of plasmidmediated AmpC-producing E. coli from Swedish broilers and association with human clinical isolates. Clin Microbiol Infect 2013; 19: E309–11. 4 Mataseje LF, Baudry PJ, Zhanel GG et al. Comparison of CMY-2 plasmids isolated from human, animal and environmental Escherichia coli and Salmonella spp. from Canada. Diagn Microbiol Infect Dis 2010; 67: 387–91. 5 Accogli M, Fortini D, Giufre` M et al. IncI1 plasmids associated with the spread of CMY-2, CTX-M-1 and SHV-12 in Escherichia coli of animal and human origin. Clin Microbiol Infect 2013; 19: E238– 40. 6 Dierikx C, van der Goot J, Fabri T et al. Extended-spectrum b-lactamaseand AmpC b-lactamase-producing Escherichia coli in Dutch broilers and broiler farmers. J Antimicrob Chemother 2013; 68: 60–7. 7 Damborg P, Gaustad IB, Olsen JE et al. Selection of CMY-2 producing Escherichia coli in the faecal flora of dogs treated with cephalexin. Vet Microbiol 2011; 151: 404–8. 8 Shaheen BW, Nayak R, Foley AL et al. Molecular characterization of resistance to extended-spectrum cephalosporins in clinical Escherichia coli isolates from companion animals in the United States. Antimicrob Agents Chemother 2011; 55: 5666 –75. 9 Tamang MD, Nam HM, Jang GC et al. Molecular characterization of extended-spectrum b-lactamase-producing and plasmid-mediated AmpC b-lactamase-producing Escherichia coli isolated from stray dogs in South Korea. Antimicrob Agents Chemother 2012; 56: 2705 –12. 10 Pe´rez-Pe´rez FJ, Hanson ND. Detection of plasmid-mediated AmpC b-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 2002; 40: 2153– 62. 11 Dierikx C, van Essen-Zandbergen A, Veldman K et al. Increased detection of extended-spectrum b-lactamase-producing Salmonella enterica and Escherichia coli isolates from poultry. Vet Microbiol 2010; 145: 273– 8.
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12 D’Andrea MM, Literacka E, Zioga A et al. Evolution and spread of multidrug-resistant Proteus mirabilis clone with chromosomal AmpC-type cephalosporinase in Europe. Antimicrob Agents Chemother 2011; 55: 2735– 42. 13 Barton BM, Harding GP, Zuccarelli AJ. A general method for detecting and sizing large plasmids. Anal Biochem 1995; 226: 235–40. 14 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated From Animals—Third Edition: Approved Standard M31-A3. CLSI, Wayne, PA, USA, 2008. 15 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-third Informational Supplement M100-S23. CLSI, Wayne, PA, USA, 2013. 16 Carattoli A. Resistance plasmid families in Enterobacteriaceae. Antimicrob Agents Chemother 2009; 53: 2227– 38. 17 Han J, Lynne AM, David DE et al. DNA sequence analysis of plasmids from multidrug resistant Salmonella enterica serotype Heidelberg isolates. PLoS One 2012; 7: e51160. 18 Hiki M, Usui M, Kojima A et al. Diversity of plasmid replicons encoding the blaCMY-2 gene in broad-spectrum cephalosporin-resistant Escherichia coli from livestock animals in Japan. Foodborne Pathog Dis 2013; 10: 243– 9. 19 Kang MS, Besser TE, Call DR. Variability in the region downstream of the blaCMY-2 b-lactamase gene in Escherichia coli and Salmonella enterica plasmids. Antimicrob Agents Chemother 2006; 50: 1590– 3. 20 Ewers C, Bethe A, Semmler T et al. Extended-spectrum b-lactamaseproducing and AmpC-producing Escherichia coli from livestock and companion animals, and their putative impact on public health: a global perspective. Clin Microbiol Infect 2012; 18: 646–55.
High diversity of plasmids harbouring blaCMY-2 among clinical Escherichia coli isolates from humans and companion animals in the upper Midwestern USA Valeria Bortolaia1*, Katrine H. Hansen1, Christine A. Nielsen1, Thomas R. Fritsche2,3 and Luca Guardabassi1 1
Department of Veterinary Disease Biology, University of Copenhagen, Frederiksberg C, Denmark; 2Division of Laboratory Medicine, Marshfield Clinic, Marshfield, WI, USA; 3Department of Microbiology, University of Wisconsin, La Crosse, WI, USA *Corresponding author. Tel: +45-35332770; Fax: +45-35332755; E-mail: [email protected]
Received 9 October 2013; returned 14 November 2013; revised 3 January 2014; accepted 7 January 2014 Objectives: To determine the population structure and genetic relatedness of plasmids encoding CMY-2 b-lactamase in clinical Escherichia coli from humans and companion animals within a defined geographical area. Methods: In total, 42 human and 73 companion animal isolates displaying an AmpC phenotype were isolated at a regional diagnostic reference laboratory in the upper Midwestern USA during 2009–11. Following PCR screening for transferable AmpC genes and plasmid transformation, blaCMY-2-positive plasmids were characterized by S1 nuclease PFGE, PCR-based replicon typing, antimicrobial susceptibility testing of transformants, conjugation experiments, plasmid multilocus sequence typing and restriction fragment length polymorphism. Results: blaCMY-2 occurred in 6 (14%), 56 (86%) and 6 (75%) isolates from humans, dogs and cats, respectively. Usually plasmids carrying blaCMY-2 were conjugative (78%) and did not contain additional resistance genes (82%). The replicon types were IncI1 (52%), IncA/C (13%), IncFII (10%), IncI2 (5%), IncL/M (3%), IncB/O (2%) or nontypeable (15%). Related IncI1/ST12 plasmids were detected in one human and five canine isolates, while the remaining plasmids did not show similarity across host species. A novel epidemiological linkage of blaCMY-2 with IncL/M plasmids and a new CMY gene variant (blaCMY-108) were found in human isolates. Conclusions: This study is one of the first One Health attempts to compare plasmids encoding CMY-2 b-lactamase among clinical isolates from humans and companion animals in the same region. The results indicate an unforeseen heterogeneity of plasmid backgrounds and suggest limited exchange between the two populations, in which blaCMY-2 occurred at very different frequencies and was harboured by distinct plasmid types. Keywords: AmpC, antimicrobial resistance, dogs, cats
Introduction CMY-2 is the most commonly reported plasmid-encoded AmpC b-lactamase in Escherichia coli and other Enterobacteriaceae from different host species worldwide.1 Although less frequently reported compared with extended-spectrum b-lactamases (ESBLs), CMY-2 is clinically relevant since, like ESBLs, it inactivates third-generation cephalosporins and may mediate resistance to carbapenems in combination with loss of outer membrane porins.2 A recent study emphasized the possible risk of transmission of blaCMY-2 from poultry to humans.3 Current knowledge about possible exchange of plasmids harbouring blaCMY-2 between humans and animals is limited to studies examining bacterial isolates from humans, food-producing animals and food.4 – 6 Despite the widespread occurrence of blaCMY-2 in E. coli isolates from dogs and cats
worldwide,7 – 9 zoonotic spread of this b-lactam resistance determinant from companion animals has received scarce attention to date. To shed light on this potential zoonotic risk, population structure and genetic relatedness of plasmids carrying blaCMY-2 were investigated in clinical E. coli isolated from humans, dogs and cats within a defined geographical area.
Methods E. coli displaying an AmpC phenotype (i.e. resistance to cefoxitin and thirdgeneration cephalosporins and susceptibility to cefepime) were isolated from community-dwelling human patients (n ¼ 42), dogs (n ¼ 65) and cats (n¼8) at the clinical laboratories of the Marshfield Clinic, Wisconsin, USA, between 2009 and 2011. The clinical laboratories serve hospitals, clinics and veterinary clinics throughout Wisconsin and the upper Midwest. Transferable AmpC genes were identified by multiplex PCR10
# The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected]
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and confirmed by singleplex PCR11 and sequencing (Macrogen Inc.). The sequence of a novel blaCMY variant (GenBank accession number KF564 648) was determined using forward primers ISEcp1/F and ecpF212 and reverse primer blaCMY/R (5′ -ATACCCGGAATAGCCTGCTC-3′ ), and forward primer blaCMY/F (5′ -GGGTGCATAAAACGGGATCC-3′ ) and reverse primer blc/R (5′ -GACAACCAGGAATGCAGC-3′ ). Plasmid DNA was isolated from blaCMY-2-positive isolates by alkaline lysis and transformed into electrocompetent Genehog E. coli (Invitrogen). Transformants were selected on BHI agar (Merck) supplemented with cefotaxime (2 mg/L), tested by colony PCR to confirm the presence of blaCMY-2 and by S1 nuclease PFGE to determine plasmid content and size.13 Transformants positive for blaCMY-2 and carrying a single plasmid were further characterized by PCR-based replicon typing (PBRT; Diatheva) and plasmid multilocus sequence typing (pMLST) (http://pubmlst.org/ plasmid/). Plasmid types shared by different host species were further analysed by restriction fragment length polymorphism (pRFLP) using FastDigestw EcoRV and FastDigestw PstI enzymes (Thermo Scientific). Susceptibility to ciprofloxacin, gentamicin, imipenem, trimethoprim/sulfamethoxazole and tetracycline was determined for all transformants by disc diffusion according to CLSI standards.14,15 Transferability of blaCMY-2-positive plasmids was tested by filter-mating experiments using rifampicin-resistant E. coli K12 J62-2 as recipient. Transconjugants were selected on BHI agar containing cefotaxime (2 mg/L) and rifampicin (50 mg/L), and tested by PCR to confirm the presence of blaCMY-2.
Results Six (14%), 56 (86%) and 6 (75%) isolates from humans, dogs and cats, respectively, were positive for blaCMY-2. Furthermore, two (5%) human isolates harboured a novel CMY gene variant, which was assigned blaCMY-108 (http://www.lahey.org/studies). No acquired AmpC genes were detected in the remaining 45 isolates. A plasmid origin was demonstrated for 93% of the blaCMY-2 -positive isolates and 53 (78%) of the plasmids were transferable by conjugation. In contrast, blaCMY-108 was most likely located on the chromosome since repeated transformation and conjugation attempts were unsuccessful. Three transformants harboured multiple plasmids and were not further analysed. The remaining 60 plasmids ranged in size from 45 to 104 kb and harboured IncI1 (n ¼ 31; 52%), IncA/C (n ¼ 8; 13%), IncFII (n ¼ 6; 10%), IncI2 (n ¼ 3; 5%), IncL/M (n ¼ 2; 3%) and IncB/O (n ¼ 1; 2%) replicons. Nine (15%) plasmids were non-typeable by PBRT. IncI1 was detected in all three host species, IncFII in both humans and dogs, IncL/M in humans only, and IncA/C, IncI2, IncB/O and non-typeable plasmids in dogs only (Table 1). Subtyping of IncI1 plasmids showed six known sequence types (ST2, ST12, ST18, ST20, ST23 and ST26) and six new sequence types (ST120, ST121, ST122, ST123, ST124 and ST125) resulting both from novel combinations of known alleles and detection of new alleles. Four (13%) IncI1 plasmids were not typeable as the sogS allele was not detected. Analysis of combinations of the remaining alleles showed that: (i) two such plasmids were singlelocus variants (SLVs) of ST12, having the same allele variants; (ii) one plasmid was an SLV of ST2; and (iii) one plasmid had nearest matches in the pMLST database in only three loci (Table 1). IncI1/ ST12 was identified in nine isolates from all three host species. One human and four canine plasmids yielded indistinguishable RFLP profiles using PstI but the human plasmid differed by three bands using EcoRV (Figure S1, available as Supplementary data at JAC Online). RFLP subtyping of IncFII plasmids revealed the presence of indistinguishable plasmids in two canine isolates, while
the remaining four plasmids yielded unique restriction profiles (Figure S2, available as Supplementary data at JAC Online). Co-transfer of resistance to non-b-lactam antimicrobials was observed for 18% of the plasmids. All IncA/C plasmids co-transferred resistance to tetracyclines, and three IncI1 plasmids co-transferred resistance to trimethoprim/sulfamethoxazole (Table 1). Plasmids belonging to other incompatibility groups and non-typeable plasmids did not transfer resistance to any of the antimicrobials tested.
Discussion To the best of our knowledge, this study is the first One Health attempt to compare plasmids harbouring blaCMY-2 in E. coli isolated from human and companion animal infections within a defined geographical area. Notably, blaCMY-2 occurred at significantly higher frequency in companion animal isolates (85%) than in human isolates (14%), confirming that this resistance determinant is common in canine and feline E. coli infections in the USA.8 The higher prevalence of blaCMY-2 in companion animals compared with humans could reflect the patterns of b-lactam consumption in small animal veterinary practice, where use of first-generation cephalosporins and amoxicillin/clavulanic acid is widespread. It has been previously hypothesized that the intensive use of cefalexin may have favoured dissemination of blaCMY-2 in the dog population.7 The plasmid population was heterogeneous, with IncI1 plasmids being predominant in nearly half of the isolates tested. Most incompatibility groups detected in this study, especially IncA/C and IncFII, have been previously associated with CMY-2 in different host species worldwide.2,16 However, we also detected a novel association with the IncL/M plasmid backbone in two human isolates and rare associations with IncI2 and IncB/O plasmid backbones in canine isolates. The authors are aware of single reports of blaCMY-2 on an IncI2 plasmid in a porcine Salmonella Heidelberg strain from the USA17 and on IncB/O plasmids in poultry isolates from Japan.18 The high diversity of plasmid types carrying blaCMY-2 emphasizes the mobility of this b-lactamaseencoding gene, which has previously been associated with ISEcp1-mediated transposition.19 Genetic relatedness across host species was limited to IncI1/ ST12 plasmids of 90 kb, which have been reported in E. coli and Salmonella isolates from humans and poultry in diverse countries.4,5 Nevertheless, the only IncI1/ST12 plasmid originating from a human patient could be distinguished from related plasmids of canine origin by EcoRV RFLP. An SLV of ST12 was found in another human patient and in a dog. These plasmids may have originated from a common ancestor, although they were clearly distinguishable by size. A limitation of the study is that isolates producing both AmpC b-lactamase and ESBL might be excluded on the basis of the criteria used for strain selection. However, our focus on AmpC b-lactamase enabled analysis of a relatively large number of CMY-encoding plasmids (n¼ 68) compared with previous studies on plasmids mediating cephalosporin resistance in E. coli of animal origin.6 This approach, focused on AmpC b-lactamase, resulted in a variety of original findings. A novel CMY b-lactamaseencoding gene designated as blaCMY-108 was detected in two human isolates. Based on amino acid identity, this novel
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Table 1. Characteristics of blaCMY-2-harbouring plasmids in clinical E. coli isolates from humans, dogs and cats
Isolate ID 2011/34/44 2011/37/26 2011/37/22 2011/37/05 2011/37/36 2011/37/41 2011/36/81 2011/34/35 2011/33/01 2011/37/01 2011/37/33 2011/37/35 2011/37/08 2011/34/56 2011/37/40 2011/34/53 2011/37/27 2011/25/62 2011/34/19 2011/34/41 2011/34/48 2011/36/75 2011/37/14 2011/37/29 2011/37/25 2011/34/52 2011/37/18 2011/34/51 2011/37/24 2011/37/30 2011/34/15 2011/34/16 2011/34/31 2011/37/07 2011/37/06 2011/37/02 2011/34/33 2011/37/31 2011/34/54 2011/37/11 2011/37/12 2011/33/36 2011/34/20 2011/34/21 2010/21/71 2011/37/09 2011/37/15 2011/34/18 2011/34/57 2011/37/34 2011/33/46 2011/34/37
Host species
Isolation sample
PBRT
canine canine canine canine canine canine canine canine human human canine canine canine canine canine feline canine human canine canine canine canine canine canine canine canine canine canine canine feline canine canine canine canine canine canine canine feline canine feline canine human canine canine canine canine canine canine canine human human canine
urine abscess perianal transtracheal urine urine urine urine urine urine skin urine urine urine urine urine urine urine abdominal fluid urine urine urine urine urine endotracheal ear ear urine urine urine ear urine urine urine ear urine urine urine ear urine drainage urine urine urine urine ear urine urine urine urine urine brain
A/C A/C A/C A/C A/C A/C A/C A/C FII FII FII FII FII FII I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I1 I2 I2 I2 B/O L/M L/M NT
Size (kb) 80 90 90 90 90 90 104 104 54 54 54 54 65 65 80 80 80 90 90 90 90 90 90 90 80 104 104 104 104 80 90 104 80 80 104 80 104 80 80 90 90 54 75 80 80 65 65 65 80 54 54 45
Co-transfer of resistance TET TET TET TET TET TET TET TET none none none none none none none none SXT none none none none none none SXT none none none none none none none none none none none SXT none none none none none none none none none none none none none none none none
pMLSTa
2 12 12 12 12 12 12 12 12 12 18 18 18 18 18 20 20 20 23 23 26 120 121 122 123 124 125 NTc NTd NTc NTe
pRFLP (PstI)b
pRFLP (EcoRV)b
A B C C D E
1 2 3 3 4 5
I H F F1 F F F F G
10 8 7 6 6 6 6 6a 9
Conjugation positive negative negative negative positive positive positive positive positive positive positive positive positive positive positive negative negative positive positive positive positive positive positive positive negative negative positive positive positive positive positive positive positive positive negative positive positive positive positive positive positive negative negative negative positive positive positive positive positive positive positive positive Continued
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blaCMY-2-harbouring plasmids in clinical E. coli, USA
Table 1. Continued
Isolate ID 2011/34/38 2011/34/50 2011/34/34 2011/34/40 2011/34/43 2011/37/43 2011/37/16 2011/34/29 2011/37/21 2011/34/13 2011/34/32 2011/34/55 2011/36/74 2011/37/23 2011/34/17 2011/37/28
Host species canine canine canine canine canine canine canine canine feline canine feline canine canine canine canine canine
Isolation sample
PBRT
Size (kb)
Co-transfer of resistance
brain urine urine urine urine ear urine urine urine urine urine ear skin urine urine urine
NT NT NT NT NT NT NT NT ND1 ND1 ND1 ND2 ND2 ND2 ND2 ND2
45 45 45 45 45 80 90 90 104, 20 54, 28, 33 45, 65 ND2 ND2 ND2 ND2 ND2
none none none none none none none none ND1 ND1 ND1 ND2 ND2 ND2 ND2 ND2
pMLSTa
pRFLP (PstI)b
pRFLP (EcoRV)b
Conjugation positive positive positive positive positive positive positive positive positive negative positive positive positive negative negative negative
NT, non-typeable; ND1, not determined as more than one plasmid was present in the transformant; ND2, not determined as the plasmid could not be transformed; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline. a pMLST was performed on IncI1 plasmids. b pRFLP was performed on plasmid types shared by different host species. c SLV of ST12. d Novel combination of the remaining allele variants repI1, 1; ardA, 3; trbA, 13; pilL, 3. e SLV of ST2.
b-lactamase was more closely related to CMY-39 and CMY-80 (98.7%) than to CMY-2 (97.6%). The findings of plasmid types that had not (IncL/M) or had only rarely (IncI2 and IncB/O) been associated with CMY-2 previously and of several new IncI1 pMLST types and non-typeable plasmids indicate that the diversity of plasmid backgrounds was only partly explored prior to this study. The high level of genetic diversity observed in plasmids encoding CMY-2 represents an evolutionary advantage that could facilitate further spread of this b-lactam resistance determinant in the years to come as a consequence of the widespread use of b-lactams in both human and veterinary medicine. Co-selection by antimicrobials other than b-lactams seems to play a minor role since only a minority of plasmids harbouring blaCMY-2 carried additional resistance genes. In conclusion, this study shows that plasmids carrying blaCMY-2 in clinical E. coli isolates from humans, dogs and cats in the Midwestern USA are highly heterogeneous, suggesting limited exchange between companion animals and humans. However, the design of this study was not particularly suited to the detection of zoonotic transmission of blaCMY-2 due to the low proportion of human CMY-2-producing isolates, the long period and the wide geographical area covered by the study. We did not examine the genetic background of CMY-2-producers because blaCMY-2 is usually not linked to specific E. coli lineages20 and primarily spreads by horizontal transfer of plasmids. Moreover, strain typing would have not added information on zoonotic transmission since animal and human isolates were epidemiologically unrelated on the basis of their plasmid content.
Acknowledgements We thank Ljudmila Troianova for dedicated technical assistance.
Funding This work was supported by grant HEALTH-F3-2011-282004 (EvoTAR) from the European Union. Surveillance collection support was made possible by the Steve J. Miller Endowment in Rural and Agricultural Health and Safety from the National Farm Medicine Center of the Marshfield Clinic Research Foundation.
Transparency declarations None to declare.
Supplementary data Figures S1 and S2 are available as Supplementary data at JAC Online (http://jac.oxfordjournals.org/).
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