Molecular Characterization of Resistance to Extended-Spectrum ...

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2011, p. 5666–5675 0066-4804/11/$12.00 doi:10.1128/AAC.00656-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Vol. 55, No. 12

Molecular Characterization of Resistance to Extended-Spectrum Cephalosporins in Clinical Escherichia coli Isolates from Companion Animals in the United States䌤† Bashar W. Shaheen,1* Rajesh Nayak,1 Steven L. Foley,1 Ohgew Kweon,1 Joanna Deck,1 Miseon Park,1 Fatemeh Rafii,1 and Dawn M. Boothe2 Division of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas,1 and Department of Anatomy, Physiology and Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, Alabama2 Received 12 May 2011/Returned for modification 16 August 2011/Accepted 6 September 2011

Resistance to extended-spectrum cephalosporins (ESC) among members of the family Enterobacteriaceae occurs worldwide; however, little is known about ESC resistance in Escherichia coli strains from companion animals. Clinical isolates of E. coli were collected from veterinary diagnostic laboratories throughout the United States from 2008 to 2009. E. coli isolates (n ⴝ 54) with reduced susceptibility to ceftazidime or cefotaxime (MIC > 16 ␮g/ml) and extended-spectrum-␤-lactamase (ESBL) phenotypes were analyzed. PCR and sequencing were used to detect mutations in ESBL-encoding genes and the regulatory region of the chromosomal gene ampC. Conjugation experiments and plasmid identification were conducted to examine the transferability of resistance to ESCs. All isolates carried the blaCTX-M-1-group ␤-lactamase genes in addition to one or more of the following ␤-lactamase genes: blaTEM, blaSHV-3, blaCMY-2, blaCTX-M-14-like, and blaOXA-1. Different blaTEM sequence variants were detected in some isolates (n ⴝ 40). Three isolates harbored a blaTEM-181 gene with a novel mutation resulting in an Ala184Val substitution. Approximately 78% of the isolates had mutations in promoter/attenuator regions of the chromosomal gene ampC, one of which was a novel insertion of adenine between bases ⴚ28 and ⴚ29. Plasmids ranging in size from 11 to 233 kbp were detected in the isolates, with a common plasmid size of 93 kbp identified in 60% of isolates. Plasmid-mediated transfer of ␤-lactamase genes increased the MICs (>16-fold) of ESCs for transconjugants. Replicon typing among isolates revealed the predominance of IncI and IncFIA plasmids, followed by IncFIB plasmids. This study shows the emergence of conjugative plasmid-borne ESBLs among E. coli strains from companion animals in the United States, which may compromise the effective therapeutic use of ESCs in veterinary medicine. ␤-lactamase inhibitors (e.g., clavulanic acid) (28). Most ESBLs and ␤-lactamase inhibitor-resistant ␤-lactamases are derived from the classical TEM-1, TEM-2, and SHV-1 enzymes as a result of amino acid substitutions in their sequences (2). The plasmid-encoded CTX-M family members, which confer high levels of resistance to ESCs, have been reported to be found worldwide in E. coli strains isolated from human and other animal sources (1, 3, 4, 9, 26). CTX-M ␤-lactamases exhibit greater activity against cefotaxime than ceftazidime (1). Class C ␤-lactamase and plasmid-encoded AmpC-like CMY ␤-lactamases are also widely distributed in ESC-resistant E. coli strains of animal origin (27, 48, 50). Unlike CTX-M enzymes, CMY enzymes exhibit activity against cephamycins, such as cefoxitin, in addition to oxyimino-cephalosporins and cannot be inactivated by clavulanate (2, 26). Resistance to ESCs also is associated with hyperproduction of chromosomal AmpC ␤-lactamases by either gene amplification or mutations in the promoter or the attenuator region of the chromosomal ampC gene (3, 4, 35). Other possible mechanisms are the class D OXA ESBLs (2, 28) and changes in membrane permeability (31). ESBLs in clinical strains of E. coli isolated from humans and food-producing animals have been characterized in various studies (3, 4, 20, 30, 33, 35, 47), but few studies have been performed on E. coli isolates from dogs or cats (26). E. coli strains from companion animals producing CTX-M-1, CTXM-15, CMY-2, and SHV-12 have been found in Europe (7,

Escherichia coli is one of the primary causes of urinary tract infections and pyometra, particularly in dogs (11, 23). Drugresistant E. coli strains have been associated with urinary tract and other nosocomial infections in veterinary hospitals (14). ␤-Lactams are widely used in veterinary medicine to treat infections caused by E. coli in dogs and cats (26). This widespread use may have contributed to the substantial increase in the emergence of E. coli resistance to ␤-lactams in companion animals over the last several decades (26, 42, 43). The emergence of ␤-lactam-resistant bacteria in companion animals and the transfer of resistant isolates to humans poses a potential serious risk to public health (22). Of particular concern are bacteria from these animals with resistance to extended-spectrum cephalosporins (ESCs), such as ceftiofur and ceftriaxone, which are critical in veterinary and human medicine for treatment of bacterial infections (2). Resistance to ESCs in E. coli is mediated primarily by production of class A extended-spectrum ␤-lactamases (ESBLs), which hydrolyze oxyimino-cephalosporins but are not active against cephamycins and carbapenems and can be inhibited by * Corresponding author. Mailing address: Division of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079. Phone: (870) 543-7599. Fax: (870) 543-7307. E-mail: [email protected]. † Supplemental material for this article may be found at http://aac .asm.org/. 䌤 Published ahead of print on 26 September 2011. 5666

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15–17), whereas strains with CTX-M-14 were reported in Chile (34). Recently, CTX-M-14, CTX-M-15, and SHV-12 ESBLs have been identified in canine and feline E. coli isolates associated with urinary tract infections (UTIs) in the United States (37). Knowledge about the diversity and prevalence of resistance to broad-spectrum ␤-lactams among Enterobacteriaceae from companion animals in the United States remains limited. In this study, we evaluated the prevalence of resistance to these drugs among canine and feline isolates of E. coli from veterinary clinics in the United States and characterized the mechanisms of resistance to ESCs by class A and C ␤-lactamases. We have also determined the resistance phenotypes, the genetic relationships among isolates, and the role of plasmids in resistance development and horizontal gene transfer. MATERIALS AND METHODS Bacterial isolates and antimicrobial susceptibility testing. Canine and feline clinical isolates of E. coli were acquired from clinical veterinary microbiology laboratories in California, Illinois, Massachusetts, North Carolina, and Ohio (IDEXX Reference Laboratories, Inc., Westbrook, ME) between May 2008 and May 2009 (n ⫽ 944). Isolates were initially cultured from samples received from veterinary practitioners after collection from dogs or cats with presumed infections. These isolates were isolated from urine, wound, ear, genital tract, anal sac, nasal structure, and soft tissue samples. Bacterial isolates were shipped overnight to the Clinical Pharmacology Laboratory at Auburn University as pure cultures on Trypticase soy agar slants. Upon arrival of the isolates, their identity was confirmed by plating them on MacConkey and CHROMagar agar plates and by indole testing using Kova´cs reagent (Remel/Thermo Fisher Scientific, Lenexa, KS). The isolates were grown on Trypticase soy agar (TSA; Difco, Detroit, MI) and stored in Trypticase soy broth (Difco) containing 25% glycerol at ⫺80°C. Isolates were classified into four geographical regions based on origin: South (North Carolina), West (California), Midwest (Ohio and Illinois), and Northeast (Massachusetts). Antimicrobial susceptibility testing was performed for all isolates (n ⫽ 944) using custom microdilution susceptibility plates according to the manufacturer’s protocol (Trek Diagnostic Systems, Inc., Cleveland, OH). A panel of 15 antimicrobial agents was tested (see Table S1 in the supplemental material). The MICs were recorded using the Sensititre Vizion system (Trek Diagnostic Systems). E. coli ATCC 25922 was used for quality control. The results were interpreted by CLSI guidelines (13). Multidrug resistance (MDR) was defined as resistance to two or more drug classes (the class of ␤-lactams includes three subclasses: penicillins, cephalosporins [i.e., narrow, expanded, and broad spectrum], and carbapenems). Isolates that exhibited at least an 8-fold increase in their MICs (per CLSI guidelines) of either ceftazidime or cefotaxime alone versus the MIC of the same drug tested in combination with clavulanic acid were identified as potential ESBL producers (13). The susceptibilities of the isolates to ceftazidime or cefotaxime were determined, and a total of 54 isolates, which expressed the ESBL phenotype (n ⫽ 29) per CLSI guidelines or for which the MIC of ceftazidime or cefotaxime was ⱖ16 ␮g/ml (n ⫽ 25), were selected as presumptive ESBL producer strains for this study. PFGE. All 54 isolates were analyzed for genetic relatedness by pulsed-field gel electrophoresis (PFGE) using the XbaI restriction enzyme according to the CDC PulseNet protocol (http://www.cdc.gov/pulsenet/protocols.htm). The DNA fingerprints were analyzed with BioNumerics software (version 6.0; Applied Maths, Austin, TX). Matching and dendrogram analysis of the PFGE patterns were conducted using unweighted pair group method with arithmetic averages (UPGMA) analysis and the Dice correlation coefficient. The Dice band-based similarity coefficient, with a band position tolerance of 1% and an optimization of 1.5%, was used for clustering. PCR analysis of genes conferring resistance to ESCs. The ␤-lactamase genes blaTEM, blaCMY, blaSHV, blaOXA, and blaCTX-M were detected by PCR, using specific primers and conditions previously described (see Table S2 in the supplemental material). Bacterial DNA was extracted by the boiling method (44) and used as the template for PCR. Appropriate positive (E. coli CVM 15103 [blaTEM], CVM 1290 [blaCMY], CVM 15104 [blaSHV], CVM 40118 [blaOXA], and CVM 35778 [blaCTX-M]) and negative controls were included in each PCR. Amplicons from CTX-M genes (group 1 and group 14) were sequenced using the

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appropriate primers (see Table S2) and dideoxy terminators in an automated DNA sequencer (ABI 310; Applied Biosystems, Foster City, CA). The sequences were analyzed using a BLAST search (http://www.ncbi.nlm.nih.gov/guide/dna -rna/) and compared with previously reported sequences for ␤-lactamases (http: //www.lahey.org/Studies). Sequence alignments were performed using Vector NTI Advance 11 software (Invitrogen, Carlsbad, CA). In silico structural analysis. The crystal structures of wild-type TEM-1 (PDB ID, 1ERO) and mutant (A184V; PDB ID, 1JTD) were used for computational analysis in this study. The databases RCSB PDB (http://www.rcsb.org/pdb/home /home.do) and PDBsum (http://www.ebi.ac.uk/pdbsum/) provided the experimentally determined PDB structure files and their at-a-glance structural overview. The three-dimensional homology model for wild-type protein (Ala-184) was generated using SWISS-MODEL (http://swissmodel.expasy.org/workspace /index.php) with the PDB 1JTD (A184V) as the template. SuperPose (version 1.0) was used for structural superposition and RMSD (root mean square deviation) calculation of the wild-type and mutant proteins. To predict changes in protein stability upon the point mutation, CUPSAT (http://cupsat.tu-bs.de/) was adopted. PyMOL (0.99RC6) (http://www.pymol.org) was used for visualization of structure figures. Sequencing of the regulatory region of the chromosomal ampC gene. PCR amplification and sequencing of a 191-bp fragment of the promoter-and-attenuator region of the chromosomal ampC gene were performed using the primers ampC-F (5⬘-AATGGGTTTTCTACGGTCTG-3⬘) and ampC-R (5⬘-GGGCAG CAAATGTGGAGCAA-3⬘) (5). Multisequence alignment was performed to compare the sequences and detect mutations in the promoters and attenuator regions with sequences of the same region in the E. coli K-12 ampC gene, using Vector NTI Advance 11 software (Invitrogen). Plasmid characterization: S1 nuclease digestion and plasmid Inc/Rep typing. PFGE with S1 nuclease digestion of whole genomic DNA (Promega, Madison, WI) was performed for all 54 isolates (46) to detect and estimate the size of large plasmids. The XbaI-digested DNA from a strain of Salmonella serotype Braenderup H9812 was used as a molecular weight standard. A dendrogram of the plasmid profiles was generated by using BioNumerics software and Dice coefficients for band matching with a 2% position tolerance and an optimization of 1% used for generating a dendrogram. Plasmid Inc/Rep typing, including FIA, FIB, FIC, HI1, HI2, I1-I␥, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FIIA, was performed as described previously (6). Transfer of resistance genes by conjugation. Conjugation experiments were performed by plate mating to determine if plasmid coding for CTX-M, TEM, and CMY-2 enzymes was transferable from ESBL-resistant phenotypes. Nine donors, representative of different ␤-lactamase enzymes, were used for mating with a ␤-lactam-susceptible recipient strain of E. coli, J53 AZr, which is resistant to sodium azide. Cells from late exponential phase of the donors and recipient were overcrossed on blood agar plate and incubated at 37°C for 18 h. Transconjugants were selected on TSA medium supplemented with 300 ␮g/ml sodium azide and 2 ␮g/ml cefotaxime or 32 ␮g/ml ampicillin. Microdilution susceptibility testing was performed to determine the MICs for the transconjugants. PCR amplifications and plasmid replicon typing were performed on the DNA extracted from the transconjugants to determine the presence of ESBL genes and incompatibility groups, respectively. Hybridization using Southern blotting. The plasmids from donors and transconjugants were separated by PFGE with S1 nuclease digestion (S1-PFGE) (46). The resolved plasmids were transferred to nylon membranes (GE Healthcare, Little Chalfont, England) by capillary transfer and hybridized to specific probes generated from the purified PCR products, using the oligonucleotide primers shown in Table S2 in the supplemental material. A BrightStar psoralenbiotin nonisotopic labeling kit (Ambion, Inc., Austin, TX) was used to label the PCR products, which were then used to detect the genes for CTX-M-15, CMY-2, or TEM in the plasmid DNA of the bacterial isolates. The hybridization steps were performed according to the manufacturer’s protocol (Ambion, Inc.).

RESULTS Susceptibility of ESBL-producing E. coli strains. Of the 944 E. coli strains isolated from companion animals with UTIs and other infections, 54 (⬃6%) exhibited reduced susceptibility to ceftazidime or cefotaxime (MIC ⱖ 16 ␮g/ml). Of these, 29 (⬃54%) were confirmed as ESBL-producing E. coli strains according to CLSI guidelines (13). Thus, the prevalence of ESBL-producing E. coli causing clinical infections in companion animals was 3%. The majority of ESBL-producing E. coli

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isolates were recovered from urine specimens (35%, 19/54) (Fig. 1). The MIC50s and MIC90s (␮g/ml) of ␤-lactams for ESBL-producing E. coli strains were as follows: ampicillin, ⬎256 and ⬎256; amoxicillin-clavulanic acid, 64 and 64; cephalothin, ⬎1,024 and ⬎1,024; cefoxitin, 128 and 256; cefpodoxime, 256 and ⬎256; ceftazidime, 32 and 256; cefotaxime, 32 and ⬎1,024; and meropenem, 0.5 and 4. All isolates had high MIC90s toward all ␤-lactams, including narrow-, expanded-, and broad-spectrum cephalosporins, but only four isolates (NCTR-929, -926, -938, and -941) exhibited resistance to carbapenems (i.e., meropenem) (Fig. 1). Among isolates producing ESBL, the frequencies of resistance to other drugs were as follows: enrofloxacin, 92%; gentamicin, 44%; chloramphenicol, 44%; doxycycline, 82%; and trimethoprim-sulfamethoxazole, 44% (Fig. 1). All but one strain, NCTR-936, of ESBLproducing E. coli exhibited the MDR phenotype (Fig. 1). Molecular characterization of ESBL genes. The ESC genes blaTEM, blaSHV, blaCTX-M-1 group, blaCMY, and blaOXA-10 were detected in 74%, 17%, 100%, 89%, and 30% of ESBL-producing E. coli, respectively. The blaOXA-2 group was not detected in any isolates. Two variants of blaCTX-M genes were identified in 63% of the isolates. Sequencing of blaCTX-M subgroups revealed blaCTX-M encoding CTX-M-15 as being most prevalent (78%), whereas blaCTX-M encoding CTX-M-14 and CTXM-24 was found in 20% (n ⫽ 11) and 42% (n ⫽ 23) of the isolates, respectively (Fig. 1). Sequencing of the blaTEM gene revealed blaTEM-1 (n ⫽ 33) and two inhibitor-resistant TEM (IRT) ␤-lactamase-encoding genes, blaTEM-33 (IRT-5) (n ⫽ 3) and blaTEM-30 (IRT-2) (n ⫽ 1) (Fig. 1). Additionally, a novel blaTEM gene, named blaTEM-181, resulting from a Ala184Val mutation, was detected in three clinical isolates, NCTR-920, -926, and -927. All ESBL-producing E. coli isolates in which blaSHV, blaOXA-10, and blaCMY were amplified by PCR produced SHV-3, OXA-1, and CMY-2 variants, respectively (Fig. 1). Mutations in the ampC promoter-attenuator region in the ESBL-producing E. coli isolates. Mutations in the promoter and attenuator regions of the chromosomal ampC gene were found in ⬃78% (42/54) of isolates (Fig. 1). These mutations were detected in association with ESBL genes, either alone at positions ⫹58 and ⫺28 or in the following combinations: ⫺18, ⫺1, ⫹20, and ⫹58; ⫺18, ⫺1, and ⫹58; ⫺28 and ⫹58; ⫺28, ⫹34 and ⫹58; ⫺32 and ⫹58; ⫺28, ⫹22, ⫹26, ⫹27, and ⫹32; and ⫹22, ⫹26, ⫹27, and ⫹32. The distance between the ⫺35 and ⫺10 boxes of the ampC gene, which also plays an important role in strengthening the ampC promoter (8), was 16 bp in all isolates. However, one novel insertion of adenine between bases ⫺28 and ⫺29, which increased the distance between the conserved regions to 17 bp, was detected in the ampC promoter region of isolate NCTR-929. PFGE typing. PFGE subtyping of all 54 ESBL-producing E. coli isolates generated 51 XbaI-PFGE patterns (Fig. 1). No

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FIG. 2. Tertiary structure of the class A TEM-1 (1JTD; Val-184). The locations and side chains of the mutation are highlighted. The diagram was drawn with PyMOL (0.99RC6).

genetic relatedness was found among the isolates carrying different ESBLs by PFGE at the cutoff value of ⱖ90% (Fig. 1). However, three distinct PFGE clusters, each containing two closely related isolates (ⱖ93%), were identified (Fig. 1). Isolates in two clusters carried the same resistance determinants. In one cluster, isolates NCTR-920 and -922 displayed the same ESBL resistance genes and profiles, with the exception of fosfomycin resistance in NCTR-922. Isolates NCTR-926 and -927, in the second cluster, also exhibited the same ESBL resistance genes and phenotypes, with the exception of meropenem resistance in NCTR-926. However, the third cluster, comprised of NCTR-911 and -935, had different antimicrobial susceptibility profiles and resistance gene determinants (Fig. 1). In silico structural analysis of the wild-type (TEM-1 [A184]) and mutant (TEM-181 [A184V]) enzymes. As shown in Fig. 2, amino acid residue 184 is located in the ␣-helix H10 (position 182 to 196 in 1JTD), surrounded by the ␣-helices H8 (144 to 154) and H2 (71 to 86) and the ␤-sheet of five antiparallel strands. In the mutant strains NCTR-920, -926, and -927, the side chain of Val-184 of the helix is totally exposed on the enzyme surface, with a solvent accessibility of 24.5%. On the other hand, in the corresponding helix of the enzyme of the wild type, the Ala-184 shows 20.5% solvent accessibility. The distance between the residue Val-184 and the reactive Ser-70 of the catalytic site at the N terminus of the ␣-helix H2 is ⬃17 Å, measured from C␣ to C␣. To verify the protein stability upon the point mutation in both the wild type (Ala184) and the mutant (Val-184), ⌬⌬G values were analyzed. Interestingly, the overall stability change, calculated using the atom and torsion angle potentials together, was increased as a

FIG. 1. Dendrographic analysis of XbaI PFGE data for canine and feline clinical isolates of Escherichia coli isolates from the United States. Black, gray, and white squares represent resistant, intermediate, and susceptible isolates, respectively. Abbreviations: CA, California; NC, North Carolina; IL, Illinois; MA, Massachusetts; OH, Ohio; ND, not determined. The boxes identify three distinct PFGE clusters, each with two closely related (ⱖ93%) isolates that display either the same (NCTR-920 and -922; NCTR-926 and -927) or different (NCTR-911 and -935) ESBL-resistant genes and different susceptibility profiles.

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FIG. 3. Dendrographic analysis of plasmid replicons from clinical E. coli isolates originating from companion animals and subjected to PFGE with S1 nuclease digestion. Plasmid sizes are in kbp. Abbreviations; A, ampicillin; X, amoxicillin-clavulanic acid; C, cephalothin; F, fosfomycin; O, cefoxitin; P, cefpodoxime; T, cefotaxime; Z, ceftazidime; M, meropenem; E, enrofloxacin; D, doxycycline; H, chloramphenicol; G, gentamicin; S, trimethoprim-sulfamethoxazole.

result of the conversion of Ala to Val, with a predicted ⌬⌬G of 0.6 kcal/mol. On the other hand, a Val-to-Ala conversion decreased stability by ⫺2.7 kcal/mol. The RMSD (␣ carbons) of the wild-type and mutant proteins was 0.06 Å, indicating no significant structural change. Plasmid characterization of the ESBL-producing isolates. S1-PFGE analysis revealed plasmids ranging from 11 kbp to 233 kbp (Fig. 3). Common plasmids with approximate sizes of

93 and 55 kbp were identified in 60% (n ⫽ 32) and 43% (n ⫽ 23) of isolates, respectively. The S1-PFGE dendrogram separated the plasmids into 32 distinct clusters, based on a ⱖ90% cutoff (Fig. 3). There were 12 distinct S1-PFGE clusters with ⱖ2 indistinguishable isolates. Of these, one large cluster contained six indistinguishable isolates from different animals with two large plasmids, 93 kbp and 55 kbp. The plasmid replicons IncI1 and IncFIA were most prev-


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alent (96%, 52/54), followed by IncFIB (87%) among E. coli isolates (Fig. 3). Replicon typing identified IncB/O (n ⫽ 2), FIC (n ⫽ 10), N (n ⫽ 1), Y (n ⫽ 10), K/B (n ⫽ 1), and HI1 (n ⫽ 1) replicons. Conjugation experiments. A subset of 27 randomly selected donor E. coli strains were conjugated with a recipient strain, E. coli J53 AZr. After mating, a total of nine (33%) of transconjugants were selected for further analysis based on resistance to ampicillin (32 ␮g/ml) or cefotaxime (2 ␮g/ml). The MICs for different ␤-lactams and ESBL genes detected by PCR in the donors and transconjugants are shown in Table 1. All blaCMY and blaCTX-M-15 and two blaTEM genes were transferred and detected by PCR in the transconjugants. In addition to ampicillin or cefotaxime, all donors and their transconjugants were resistant to amoxicillin-clavulanic acid, cephalothin, cefazolin, cefoxitin, cefpodoxime, and ceftiofur. Resistance to ceftazidime (MIC ⱖ 32 ␮g/ml) was observed in six isolates and their respective transconjugants (Table 1). All transconjugants exhibited an increase of at least 16-fold in MICs of cefotaxime and ceftazidime compared to the recipient, E. coli J53 AZr, while clavulanic acid did not alter sensitivity of the transconjugants to these drugs. Resistance to cefepime was not transferred to the transconjugant (Trans918) (Table 1). All transconjugants remained susceptible to enrofloxacin, gentamicin, chloramphenicol, doxycycline and trimethoprim-sulfamethoxazole (data not shown). Hybridization experiments. Following electrophoresis, plasmids detected by PFGE with S1 nuclease digestion were used for Southern blotting. Probes were used to localize the blaCMY-2 and blaCTX-M-15 genes in a subset of donors (NCTR890, -916, -917, -918, -920, and -927) and their transconjugants (Fig. 4). Plasmid DNA from these isolates yielded strong hybridization signals with the blaCMY-2 gene probe (Fig. 4). Each of these six donors had a unique plasmid profile and harbored two or three large plasmids (size range, 50 to 150 kbp), whereas only one large plasmid (size range, 50 to 93 kbp) was detected in transconjugants (Fig. 4B). The blaCMY-2 probe hybridized with plasmids of the same size in donors and transconjugants. Similarly, the blaCTX-M-15 probe hybridized with the same-size blaCMY-2-positive plasmids, suggesting a possible cointegration event with the blaCMY-2 gene into the same large plasmid. The blaTEM probe from five of the six donors (NCTR-890, -916, -920, -926, and -927) hybridized to plasmids of 150, 93, 55, 55, and 55 kbp, respectively (Fig. 5). No hybridization signal was detected in the transconjugants (Fig. 5B). Plasmids that hybridized to the blaTEM probe were different from those that hybridized to blaCMY-2 and blaCTX-M-15 probes (Fig. 4B and 5B). DISCUSSION Our study shows that ESBL-producing E. coli strains can be isolated from companion animals in the United States. Most ESBL-producing E. coli strains exhibited the MDR phenotype. Diverse ␤-lactamase families associated with high-spectrum activity against ␤-lactams were found in isolates and CTX-M, mainly blaCTX-M-1-group, was endemic in the United States. In some isolates we also found a novel blaTEM gene, blaTEM-181, with a mutation resulting in the conversion of Ala184 to Val. We also detected novel mutations in the promoter-attenuator

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region of ampC. Resistance genes encoding ␤-lactamases were located on transferable-conjugative plasmids, with different incompatibility groups conferring high MICs to ESCs. Data on the prevalence and characterization of ESBL-producing bacteria causing infections in companion animals in the United States are limited (26). Our results showed that the incidence of resistance to ESCs in 944 clinical isolates was similar to that reported from two studies in Europe (17). In our study, nearly 6% of isolates exhibited reduced susceptibility to ESCs. Of these, 3% (29 of 944) of the isolates produced ESBLs. The incidence of ESBL-producing strains reported in China and in one study from the United States were 68% (69 of 101) and 40% (60 of 150), respectively (29, 37). The differences may reflect the greater number of isolates screened for ESBLs in our study (29, 37). The PFGE results revealed a high degree of variation in antimicrobial resistance patterns among the 54 isolates. This result demonstrates the huge genetic heterogeneity among E. coli isolates containing transferable ESC-resistance genes, suggesting that the dissemination of ESBL genes is not due to the presence of a single clone (1, 3). Among the 29 ESBL-producing isolates, most harbored several ESBL genes known to increase the spectrum of activity of ESBL enzymes and confer high-level resistance to ␤-lactams (Fig. 1; Table 1). Interestingly, 78% of isolates selected for this study carried blaCTX-M (i.e., blaCTX-M-15). blaCTX-M-15 was first reported in 6% of E. coli isolates collected from canines and felines with UTIs in the United States (37). The CTX-M-1 group has been documented in companion animals worldwide (7, 15–17, 34). Of particular public health concern is the emergence of the CTX-M-15positive B2-O25:H4-ST131 clone in E. coli strains from companion animals (17, 36, 41). The other enzyme conferring ␤-lactam resistance in 74% of our isolates was a TEM-type ␤-lactamase (74%) (Fig. 1); the blaTEM-1 gene was the most prominent (82.5%). The blaTEM-1 variant was previously identified in clinical E. coli isolates from companion animals in Europe but not in the United States (16, 18, 40). Although the TEM-1 variant is not considered an ESBL, its clinical importance relies on its potential to undergo mutations to increase its activity against other ␤-lactams (10, 25). We have identified two IRT ␤-lactamase-encoding genes, blaTEM-33 (IRT-5) and blaTEM-30 (IRT-2), from four E. coli isolates (NCTR-890, -914, -917, and -918). The fact that blaTEM gene variants are more commonly detected in human E. coli isolates may reflect the fact that more studies have been performed with E. coli strains from humans than from companion animals (5, 12, 33). The new blaTEM-181 gene found in this study carried one mutation, which led to the Ala184Val substitution. Isolates NCTR-920, -926, and -927, harboring blaTEM-181, not only were resistant to aminopenicillins but also exhibited high-level (MIC, 64 to 128 ␮g/ml) resistance to ceftazidime, a broad-spectrum cephalosporin (Fig. 1). The effect of the amino acid substitution in this new variant is unclear in terms of the enzyme function. Ala and Val are nonpolar amino acids with alkyl side chains. Hence, the switch of Ala to Val at position 184 does not significantly change the polarity of ␣-helix H10. Ala is one of the best helix-forming residues in combination with lysine, glutamine, and methionine, whose side chains have straight-chain aliphatic regions, whereas the ␤-branched Val is invariably a poor helix former (38). How-

2 2 2

1

1 ⱕ0.5 2 1 2 2 ⱕ0.5 ⱕ0.5 1

NCTR-926 NCTR-920 NCTR-916

Recipient J53Az

Transconjugants Trans-927 Trans-929 Trans-917 Trans-918 Trans-890 Trans-900 Trans-926 Trans-920 Trans-916

>32 >32 >32 32 >32 32 >32 >32 >32

8

>32 >32 >32

>32 >32 >32 32 >32 >32

>32 >32 >32 >32 >32 >32 >32 >32 >32

ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1

ⱕ1

ⱕ8

>16 >16 >16 >16 >16 >16 >16 >16 >16

ⱕ1 ⱕ1 ⱕ1

4

FOT

8 8 16 4 16 4 8 8 16

ⱕ0.25

8 16 32

>16 ⱕ1 8 >16 ⱕ1 >64 >16 ⱕ1 16 >16 >16 >64 >16 4 32 >16 ⱕ1 8

FEP

>32 >16 >32 >16 >32 >16

>32 >32 >32 >32 >32 >32

AUG AMP FAZ

>32 >32 >32 >32 >32 >32

64 64 >64 64 64 32 64 64 64

ⱕ4 >32 >32 >32 >32 >32 >32 >32 >32 >32

1

64 >32 >64 >32 >64 >32

64 >64 >64 >64 >64 >64

FOX POD

32 32 32 8 32 8 32 16 64

ⱕ0.25

32 32 64

32 128 32 32 128 32

TAZ

8 >8 >8 8 >8 >8 >8 >8 >8

0.5

>8 >8 >8

8 >8 >8 >8 >8 >8

TIO

MIC (␮g/ml) of antimicrobial agent

16 16 16 8 32 8 16 16 16

ⱕ0.25

16 32 32

16 >64 16 >64 64 16

AXO

16 8 16

8 64 8 16 16 16

F/C

32 16 16

16 64 16 16 32 32

T/C

>16 >16 >16 >16 >16 >16 >16 >16 >16

4 8 8 2 8 4 16 4 16

8 16 16 8 16 4 16 8 8

ⱕ8 ⱕ0.12 ⱕ0.12

>16 >16 >16

>16 >16 >16 >16 >16 >16

CEP

ⴙ ⫺ ⫺ ⫺ ⫺ ⫺ ⴙ ⫺ ⫺

⫺

ⴙ ⴙ ⴙ

ⴙ ⴙ ⴙ ⴙ ⴙ ⴙ

ⴙ ⴙ ⴙ ⴙ ⴙ ⴙ ⴙ ⴙ ⴙ

⫺

ⴙ ⴙ ⴙ


ⴙ ⴙ ⴙ ⴙ ⴙ ⴙ ⴙ ⴙ ⴙ

⫺

ⴙ ⴙ ⴙ


⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫺

ⴙ ⴙ ⴙ


⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫺

⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⴙ ⫺ ⫺

CTX-M-I CTX-M-I4 TEM CMY like OXA like group group

Presence or absence of:

93 55 55 93 55 100 93 93 64

ND

55, 93 55, 93 37, 64, 93

55, 93 120, 55 55, 93 40, 55, 93 50, 140, 150 100

Plasmid size(s) (kbp)

I1 I1 I1 I1 I1 I1 I1 I1 I1

ND

I1, FIC, FIA I1, FIC, FIA I1, FIA, FIB I1, FIA I1, FIA, FIB I1, FIC, FIA, FIB I1, FIA, FIB I1 I1, FIA

Replicon type(s)

Plasmids

SHAHEEN ET AL.

a AUG, amoxicillin-clavulanic acid; AMP, ampicillin; TIO, ceftiofur; AXO, ceftriaxone; CEP, cephalothin; FOX, cefoxitin; TAZ, ceftazidime; FAZ, cefazolin; FEP, cefepime; T/C, ceftazidime/clavulanic acid; F/C, cefotaxime/clavulanic acid; FOX, cefoxitin; FOT, cefotaxime; POD, cefpodoxime. ND, not determined. Numbers in bold are resistance breakpoints or MICs which increased at least 16-fold relative to those for the recipient (E. coli J53 AZr).

4 >64 4 8 2 2

AMI

Donors NCTR-927 NCTR-929 NCTR-917 NCTR-918 NCTR-890 NCTR-900

Group and isolate

TABLE 1. Antimicrobial susceptibility profiles of E. coli isolates used in conjugation experimentsa

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5673

FIG. 4. PFGE of plasmid DNA of donors and transconjugants (A) and Southern hybridization of plasmid DNA from donors and transconjugants with a blaCMY-2 probe (B). Lanes A to F, donors NCTR-890, -916, -917, -918, -920, and -927, respectively; lanes 1 to 6, transconjugants Trans-890, -916, -917, -918, -920, and -927, respectively.

FIG. 5. PFGE of plasmid DNA of donors and transconjugants (A) and Southern hybridization of plasmid DNA from donors and transconjugants with a blaTEM probe (B). Lanes A to F, donors NCTR890, -916, -918, -920, -926, and -927, respectively; lanes 1 to 6, transconjugants Trans-890, -916, -918, -920, -926, and -927, respectively.

ever, an Ala-to-Val mutation on ␣-helix H10 increased rather than decreased the overall stability of the mutant TEM-181. The stability effect caused by a difference in intrinsic secondary structure propensity is probably superseded by other energetic effects related to tertiary contacts. Moreover, Val184 is located 17.44 Å from reactive Ser-70 of the catalytic site (Fig. 2), and no significant structural changes were caused by the substitution, suggesting that substitution of this residue (Val-184) may not contribute directly to the substrate profile and catalytic parameters of the ␤-lactamase. Therefore, as suggested in the case of the Met182Thr mutation (45), the Ala184Val substitution might function as a suppressor of folding of TEM or stability defects resulting from mutations directly associated with ␤-lactam specificity and catalysis. Alternatively, this mutation could facilitate required structural changes at other positions. Other enzymes which display Ala-to-Val substitutions at 184 along with a mutation(s) at a different position(s) have been reported elsewhere (http://www.lahey.org/Studies/temtable .asp). Although the OXA-1 type ␤-lactamase is not regarded as an ESBL and does not hydrolyze ESCs, this enzyme hydrolyzes ␤-lactamase inhibitors (e.g., clavulanic acid), compromising effective treatment during combined use of these drugs (21). In our study, ⬃28% of E. coli strains carried this variant, compared to those found in canine uropathogenic E. coli strains in Portugal (0.8%) (18). The blaOXA-1 gene is widely disseminated among human E. coli strains of phylogroup B2 and/or plasmids containing blaCTX-M-15, blaTEM-1, and aac(6⬘)-Ib-cr resistance genes (30, 39). The increased incidence of the OXAtype lactamase could be linked to clinical use of ␤-lactams with a ␤-lactamase inhibitor for treatment of clinical infections in companion animals (26). Among plasmid-borne class C ␤-lactamase genes, blaCMY-2 was detected in 89% of the isolates. All isolates positive for the CMY-2 variant were resistant to broad-spectrum cephalosporins. The presence of a CMY-2 ␤-lactamase in E. coli strains from companion animals has been described from the United States (42) and Italy (7, 28). The blaCMY-2 gene can be trans-

ferred between different bacterial species and between animals and humans (49). The high prevalence of class C ␤-lactamases, particularly blaCMY-2, is of concern, since these ␤-lactamases not only confer resistance to a wide range of ESCs used for treatment of serious infections in humans or animals but also are not affected by ␤-lactamase inhibitors (2, 26). In the presence of CMY-2 in clinical E. coli strains, the MICs of ceftazidime and cefotaxime in the presence of clavulanic acid were increased ⱖ8-fold compared to that for E. coli J53 AZr, while for select transconjugants, increases in the MICs varied from ⱖ16-fold (e.g., NCTR-918) to ⱖ130-fold (e.g., NCTR-926) (Table 1). Such variation in the expression of CMY-2 ␤-lactamase in E. coli isolates, as displayed by diverse transconjugant MICs, possibly reflects different copy numbers of the cmy genes associated with their relationship to plasmid size and copy number. This also might reflect differences in the putative promoter sequence on blaCMY-2 transcription, as the promoter is found within an ISEcp1-like element located upstream of blaCMY-2 (19). The presence of CMY in E. coli isolates would mask the detection of ESBLs, which requires an innovative approach. In our study, we found that both IncI and IncFIA (96%), followed by IncFIB (87%), were the most prevalent plasmid replicon types in E. coli. Similarly, a recent study has identified IncI, IncFIA, and IncFIB as common replicon types in CTXM-15-type-ESBL-producing E. coli strains from companion animals in Europe (17). Given the high incidence of CTX-M15-type ESBLs in our isolates (Fig. 1), the data suggest a common plasmid backbone on which the blaCTX-M-15 gene is likely to be located. In contrast to a previous study (17), we observed a wide diversity of plasmids from different incompatible groups (Fig. 3). However, none of the isolates was positive for the FII replicon, previously identified in E. coli from humans (20). Multiple plasmid replicons (ⱖ4 replicon types) were observed in 20 E. coli strains (Fig. 3). The presence of multiple replicons in Salmonella and E. coli isolates from humans has been described previously (24, 32); however, their clinical significance is still unknown. Conjugation experiments demonstrated that ␤-lactam resis-

5674

SHAHEEN ET AL.

tance was transferred from nine clinical E. coli isolates among the 27 tested (Table 1). The transconjugants expressed resistance to aminopenicillins and narrow-, expanded-, and broadspectrum cephalosporins. PCR and Southern blotting revealed the presence of blaCMY, blaCTX-M, and blaTEM genes in donor strains and their respective transconjugants (Table 1; Fig. 4 and 5). Due to the presence of a CMY-type ␤-lactamase, the MICs of ceftazidime or cefotaxime combined with clavulanic acid for transconjugants were increased ⱖ16-fold relative to the recipient, E. coli J53 AZr. The blaCMY and blaCTX-M genes were located on the same plasmids (e.g., 93-kbp plasmids of NCTR-920 and -927) in the donors and were cotransferred to the recipients (Fig. 4). The blaTEM gene was located on different size plasmids (e.g., 55-kbp plasmids of NCTR-920 and -927) (Fig. 5). PCR detected blaTEM in all donors and only two transconjugants (NCTR-926 and -927) (Table 1). The blaTEM hybridization signal was absent from two donors and all transconjugants, which may indicate that blaTEM can be present in other, potentially transferrable genetic elements that were not readily detected by Southern hybridization of the plasmids. All transconjugants were susceptible to other drug classes (data not shown). These results suggest that MDR genes could be carried on integron-associated plasmids and that selective pressure of certain antimicrobials may be necessary to mobilize these plasmids. Mutation at position ⫺32 of ampC, which is necessary for the overexpression of this gene (5, 8), was found in only one isolate (NCTR-940) (Fig. 1) along with a mutation at ⫹58. In E. coli, the primary mechanism of hyperproduction of a chromosomal ampC gene is mediated through mutations in the promoter and an attenuator of ampC, which confers resistance to ampicillin, cefoxitin, and ESCs (47). A previous study has shown that mutations at ⫺32 overexpressed the ampC gene 124-fold (47). Other important mutations, at positions ⫺42 and ⫺1 (5, 8), were not found in this study. Mutations at ⫺18, which play an important role in over expression of the ampC gene by creating a new ⫺10 box (8), were found in 18% of the isolates. Mutation at ⫺28 with an uncertain function related to the expression of ampC (35) were observed in one-third of our isolates, and there was one novel insertion of adenine between ⫺28 and ⫺29 in E. coli NCTR-929. This insertion created an alternate displaced promoter with new ⫺35 and ⫺10 boxes, separated by 17 bp, and could be responsible for overexpression of ampC (47). In our study, the effect of the important mutations and insertion in the ampC promoter region on the resistance phenotype was not evaluated. Their clinical impact on the resistance phenotype could be masked by the presence of plasmid-mediated ␤-lactam resistance found in our isolates. To the best of our knowledge, this is the first report to describe mutations associated with chromosomal AmpC ␤-lactamases in E. coli strains from companion animals. In summary, ⬃6% of clinical E. coli isolates from companion animals had reduced susceptibility to ESCs. The resistant phenotypes harbored different variants of ESBL genes. The genes, which are located on conjugative plasmids, conferred resistance to ESCs in recipient cells. The emergence and dissemination of conjugative plasmid-mediated ESC resistance in canine and feline E. coli isolates can serve as a potential reservoir of ESBL genes. Hence, prudent use of ESCs by veterinarians in treating companion animals is warranted.

ANTIMICROB. AGENTS CHEMOTHER. ACKNOWLEDGMENTS We thank Shaohua Zhao (Center for Veterinary Medicine, U.S. Food and Drug Administration) for kindly providing ESBL-positive control strains, George A. Jacoby and Quynh T. Tran for providing E. coli J53 AZr, and Alessandra Carattoli for providing replicon typingpositive control strains for this study. We thank George A. Jacoby for helpful discussions and John B. Sutherland, Mark Hart, and Carl E. Cerniglia for their critical review of the manuscript. Special thanks go to Carl E. Cerniglia for his encouragement and support of this work. Bashar W. Shaheen is supported by the Oak Ridge Institute for Science and Education. Views presented in this article do not necessarily reflect those of the U.S. Food and Drug Administration. REFERENCES 1. Bonnet, R. 2004. Growing group of extended-spectrum ␤-lactamases: the CTX-M enzymes. Antimicrob. Agents Chemother. 48:1–14. 2. Bradford, P. A. 2001. 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