Increase in antimicrobial resistance and emergence of major international high-risk clonal lineages in dogs and cats with urinary tract infection: 16 year retrospective study

Increase in antimicrobial resistance and emergence of major international high-risk clonal... Abstract Objectives To evaluate temporal trends in antimicrobial resistance, over 16 years, in bacteria isolated from dogs and cats with urinary tract infection (UTI) and the clonal lineages of bacteria harbouring critical antimicrobial resistance mechanisms. Methods Antimicrobial susceptibility testing was conducted for 948 bacteria isolated from dogs and cats with UTI (1999–2014). Resistance mechanisms were detected by PCR, namely ESBL/AmpC in third-generation cephalosporin (3GC)-resistant Escherichia coli and Proteus mirabilis, mecA in methicillin-resistant staphylococci, and aac(6′)-Ieaph(2″)-Ia and aph(2″)-1d in high-level gentamicin-resistant (HLGR) enterococci. Resistant bacteria were typed by MLST, and temporal trends in E. coli and Enterobacteriaceae antimicrobial resistance were determined by logistic regression. Results Enterobacteriaceae had a significant temporal increase in resistance to amoxicillin/clavulanate, 3GCs, trimethoprim/sulfamethoxazole, fluoroquinolones, gentamicin and tetracycline (P < 0.001). An increase in MDR was also detected (P < 0.0001). 3GC resistance was mainly caused by the presence of blaCTX-M-15 and blaCMY-2 in E. coli and the presence of blaCMY-2 in P. mirabilis. Two major 3GC-resistant E. coli clonal lineages were detected: O25b:H4-B2-ST131 and ST648. The mecA gene was detected in 9.2% (n = 11/119) of Staphylococcus spp., including MRSA clonal complex (CC) 5 (n = 2) and methicillin-resistant Staphylococcus epidermidis CC5 (n = 4). A temporal increase in MDR methicillin-resistant Staphylococcus pseudintermedius was detected (P = 0.0069). Some ampicillin-resistant and/or HLGR Enterococcus spp. were found to belong to hospital-adapted CCs, namely Enterococcus faecalis ST6-CC6 (n = 1) and Enterococcus faecium CC17 (n = 8). Conclusions The temporal increase in antimicrobial resistance and in MDR bacteria causing UTI in dogs and cats creates important therapeutic limitations in veterinary medicine. Furthermore, the detection of MDR high-risk clonal lineages raises public health concerns since companion animals with UTI may contribute to the spread of such bacteria. Introduction Urinary tract infections (UTIs) are frequently diagnosed in veterinary medicine1 and may require antimicrobial treatment.2 Since antimicrobial resistance is known to change geographically and over time,3 updated and long-term studies are critical to investigate the spread of antimicrobial resistance. Escherichia coli and Proteus mirabilis are the most frequently isolated Gram-negative bacteria from dogs and cats with UTI, while Staphylococcus spp. and Enterococcus spp. are the most common Gram-positive bacteria.1,4 These bacteria, isolated from dogs and cats, may harbour clinically and epidemiologically important resistance mechanisms of human and veterinary relevance such as ESBL,5,6 cephalosporinases (AmpC),7 PBP2a8 and high-level gentamicin resistance (HLGR) bifunctional enzyme.9 Moreover, the detection of MDR bacteria in dogs and cats is being increasingly reported,7,8 posing a difficult veterinary therapeutic challenge and often requiring the use of antimicrobials critically important to humans.10 With the growing contact between companion animals and humans, the risk of animal-to-human transfer of such bacteria is of concern.11 Additionally, several studies have shown that dogs and cats may share uropathogenic bacteria with the remaining household members.12 Therefore, the identification of the clonal lineages of bacteria isolated from dogs and cats with UTI, especially those harbouring important resistance mechanisms, is crucial to evaluate the extent to which dogs and cats with UTI may act as reservoirs for resistant bacteria. The goal of this study was to determine the temporal trends of antimicrobial resistance of bacteria isolated from dogs and cats with UTI over 16 years and to characterize their major antimicrobial resistance mechanisms, namely ESBL and AmpC in E. coli and P. mirabilis, methicillin resistance in Staphylococcus spp., and ampicillin and HLGR in Enterococcus spp. Furthermore, this study aimed to determine the clonal lineages of E. coli, Staphylococcus spp. and Enterococcus spp. harbouring such resistance genes and hence evaluate their potential public health relevance. Materials and methods Bacterial isolates A total of 948 consecutive positive bacterial isolates from dogs and cats with UTI (n = 869), collected from 1999 to 2014 at the Laboratory of Antimicrobial Resistance from the Veterinary Teaching Hospital of the Faculty of Veterinary Medicine/University of Lisbon and from several private practices in the Lisbon region were included in this study. Samples were collected by cystocentesis, catheterization or free catch as part of the routine care of dogs and cats with UTI. UTI was diagnosed based on clinical and urine cytological findings together with the detection of significant bacteriuria by quantitative urine culture. Bacteriological methods Quantitative urine culture was performed. Briefly, 10 μL of urine was inoculated onto 5% sheep blood (bioMérieux, Marcy-l’Étoile, France) and MacConkey (Biokar Diagnostics, Allonne, France) agar plates. After incubation at 37 °C for 24–48 h under atmospheric conditions, colonies were quantified and scored as having significant bacteriuria according to the urine sample collection method as defined elsewhere.13 Samples were included in the study regardless of the type of UTI. Species identification was conducted by phenotypic tests (API, bioMérieux and BD™ BBL™ Crystal Gram Positive ID Kit, Becton Dickinson, MD, USA). At the time of collection, isolated bacteria were stored in 20% glycerol (Sigma–Aldrich, St Louis, MO, USA) brain heart infusion broth (Biokar Diagnostics) at −80 °C for future studies. For the present study, stored isolates were recovered by streaking them onto 5% sheep blood agar. Whenever recovery was not possible, existing antimicrobial susceptibility results were used. E. coli,14,Klebsiella spp.,15,16,Proteus spp.,17,Enterococcus spp.18 and Staphylococcus spp.8 were confirmed by PCR and/or sequencing of 16S rRNA. Susceptibility testing Susceptibility testing was performed by the disc diffusion method according to CLSI guidelines, and E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used for quality control.19 The following antimicrobials were tested (Oxoid, Hampshire, UK): amoxicillin/clavulanate 30 μg, cefotaxime 30 μg, cefovecin 30 μg, cefoxitin 30 μg, ceftazidime 30 μg, ciprofloxacin 5 μg, enrofloxacin 5 μg, gentamicin 10 μg, high-level gentamicin 120 μg, oxacillin 1 μg, penicillin 10 U, tetracycline 30 μg and trimethoprim/sulfamethoxazole 25 μg. Veterinary CLSI breakpoints20 were used for amoxicillin/clavulanate, cefoxitin, enrofloxacin, gentamicin, high-level gentamicin, oxacillin, penicillin, tetracycline and trimethoprim/sulfamethoxazole; human CLSI breakpoints21 were used for cefotaxime, ceftazidime and ciprofloxacin. Finally, cefovecin results were interpreted according to the manufacturer’s breakpoints. Initial ESBL screening was conducted by a double-disc synergy test.19 Cefoxitin or oxacillin were used to predict methicillin resistance in Staphylococcus spp. according to CLSI guidelines.20,21 DNA extraction, sample purification and sequencing DNA extraction was conducted using a boiling method.5 For PCR amplicon sequencing, DNA purification was conducted using a NZYTech Gel Pure Kit (NZYTech—Genes and Enzymes, Lisbon, Portugal) and sequencing was performed by Stabvida (Caparica, Portugal). Sequences were analysed using Ugene Unipro software (Unipro, Novosibirsk, Russia) and the nucleotide basic local alignment search tool (http://blast.ncbi.nlm.nih.gov/). Antimicrobial resistance genes Third-generation cephalosporin (3GC)-resistant E. coli and P. mirabilis were screened for the presence of ESBL blaCTX-M-type and AmpC blaCIT-type,blaDHA-type,blaMOX-type,blaACT-type,blaFOX-type and blaMIR-type by PCR and sequencing.22–24 3GC-resistant Enterobacteriaceae without blaCTX-M-type or AmpC genes were further tested for the presence of blaTEM-type and blaSHV-type ESBL genes.5,Staphylococcus spp. were screened for the presence of the mecA gene8 and HLGR Enterococcus spp. for the presence of aac(6′)-Ieaph(2″)-Ia and aph(2″)-1d25 genes by PCR. Negative and previously sequenced positive controls were included in all PCR reactions. Clonal lineages of resistant isolates ESBL- or AmpC-producing E. coli were tested for the clonal lineage O25b:H4-B2-ST131 by PCR.14,26 3GC-resistant E. coli isolates not belonging to the O25b:H4-B2-ST131 clonal lineage were typed by MLST.27 Methicillin-resistant Staphylococcus spp. were previously characterized by MLST, SCCmec and spa typing elsewhere.8 Ampicillin-resistant and/or HLGR Enterococcus faecium and Enterococcus faecalis were also typed by MLST.28,29 eBURST v. 3 software (http://eburst.mlst.net/) was used to estimate the relationships between the isolate STs from this study and all MLST profiles known to date. Statistical analysis The SAS statistical software package for Windows v. 9.3 (SAS Institute Inc., Cary, NC, USA) was used for statistical analysis. Antimicrobial resistance frequencies were only calculated if at least 10 isolates were tested for a specific organism/antimicrobial combination and results were presented with the 95% CI. For statistical purposes, intermediate isolates were considered susceptible. An isolate was considered resistant to 3GCs when it was found to be resistant to at least one of the three 3GCs tested (cefotaxime, ceftazidime and cefovecin). Ciprofloxacin and enrofloxacin were used as markers of fluoroquinolone resistance. An isolate was considered MDR when it was found to be fully resistant to three or more antimicrobial categories. The antimicrobial categories were adapted from those proposed by other authors30 and varied according to the bacterial species considered (Table S1, available as Supplementary data at JAC Online). When at least 10 isolates were tested per year, temporal trends of antimicrobial resistance were determined using an SAS LOGISTIC regression model with the year as a continuous variable and an α value of 0.05. Thus, temporal trends in antimicrobial resistance were determined for E. coli and for Enterobacteriaceae (including E. coli, Proteus spp., Klebsiella spp. and Enterobacter spp. as a group). When determining Enterobacteriaceae temporal trends, intrinsic resistance data was excluded from analysis. Furthermore, P. mirabilis and Staphylococcus spp. antimicrobial resistance were compared between two time periods: 1999–2006 and 2007–14. The Fisher’s exact test was used for comparisons between groups with an α value of 0.05. Results From 1999 to 2014, 948 bacteria were isolated from 649 dogs and 220 cats with UTI. The majority of UTIs (91.1%, CI 89.2%–93.0%, n = 792/869) were caused by single organisms. Coinfections were most commonly caused by the combinations of E. coli/Enterococcus spp. (14.3%, CI 6.5%–22.1%, n = 11/77), E. coli/P. mirabilis (11.7%, CI 4.5%–18.9%, n = 9/77) and E. coli/Streptococcus spp. (10.4%, CI 3.6%–17.2%, n = 8/77). Although E. coli (43.5%) was the most frequently isolated bacterium, Proteus spp. (16.4%), Staphylococcus spp. (13.2%) and Enterococcus spp. (7.0%) were also common (Table S2). The frequency of infection by Proteus spp. was significantly higher (P < 0.0001) in dogs and Enterococcus spp. in cats, respectively (Table S2). Overall, Staphylococcus spp. had similar frequencies in cats and dogs. However, Staphylococcus pseudintermedius was significantly more common in dogs (P < 0.0001), while cats were infected by a higher diversity of staphylococcal species, with S. pseudintermedius, Staphylococcus felis and Staphylococcus epidermidis being the most frequent (Table S2). Enterobacteriaceae accounted for 66.1% (CI 63.1%–69.2%) of all isolated bacteria and showed a significant temporal increase in resistance to all tested antimicrobials (Table S3 and Figure 1). In 2012–14, resistance of Enterobacteriaceae to all the antimicrobials tested, except gentamicin, was >30% (Figure 1). Considering E. coli and Proteus spp. alone, no significant change over time was detected in trimethoprim/sulfamethoxazole resistance (Tables S3 and S4). Nevertheless, from 1999/2006 to 2007/2014, E. coli showed a 3-fold increase in amoxicillin/clavulanate resistance and a 4-fold increase in 3GC resistance. Proteus spp. had an even higher increase in resistance, showing a 5- and 9-fold increase in amoxicillin/clavulanate and 3GC resistance, respectively (Table S4). Proteus spp. and E. coli also had a significant increase in gentamicin resistance (Tables S3 and S4). Detection of MDR Enterobacteriaceae, E. coli and Proteus spp. increased significantly over time (Tables S3 and S4 and Figure 1). Most MDR Enterobacteriaceae (excluding Enterobacter spp.) were susceptible to at least one antimicrobial (Table S5). Gentamicin was the antimicrobial to which most MDR Enterobacteriaceae (excluding Enterobacter spp.) were susceptible (46.2%, CI 36.1%–56.4%). On the contrary, MDR Enterobacteriaceae were seldom susceptible to fluoroquinolones (9.7%, CI 3.7%–15.7%) (Table S5). Furthermore, among MDR E. coli, only 19.35% (CI 9.5%–29.2%) were susceptible to tetracycline. Figure 1. View largeDownload slide Enterobacteriaceae and E. coli resistance trends over the 16 years of the study. Depiction of the temporal trends in resistance obtained by logistic regression over 16 years. (a) Enterobacteriaceae. There was a significant increase (P < 0.05) in antimicrobial resistance to all antimicrobials. (b) E. coli. There was a significant increase (P < 0.05) in antimicrobial resistance to all antimicrobials, except trimethoprim/sulfamethoxazole. AMC, amoxicillin/clavulanate; SXT, trimethoprim/sulfamethoxazole; FQs, fluoroquinolones; GEN, gentamicin; TET, tetracycline. Figure 1. View largeDownload slide Enterobacteriaceae and E. coli resistance trends over the 16 years of the study. Depiction of the temporal trends in resistance obtained by logistic regression over 16 years. (a) Enterobacteriaceae. There was a significant increase (P < 0.05) in antimicrobial resistance to all antimicrobials. (b) E. coli. There was a significant increase (P < 0.05) in antimicrobial resistance to all antimicrobials, except trimethoprim/sulfamethoxazole. AMC, amoxicillin/clavulanate; SXT, trimethoprim/sulfamethoxazole; FQs, fluoroquinolones; GEN, gentamicin; TET, tetracycline. A total of 33 E. coli and 9 P. mirabilis that were 3GC-resistant were recovered and screened for the presence of ESBL and AmpC genes. The first 3GC-resistant E. coli isolate was detected in 1999, yet none of the tested genes were detected. Another resistance mechanism may be involved. In total, only seven 3GC-resistant E. coli were negative for all tested genes, including blaTEM-type and blaSHV-type ESBLs. E. coli 3GC resistance was mainly related to the presence of blaCTX-M-15 and blaCMY-2 (Tables 1 and 2). The first CTX-M-producing E. coli was detected in 2004 and belonged to the O25b:H4-B2-ST131 clonal lineage (Table 1). Besides O25b:H4-B2-ST131, CTX-M-producing E. coli were frequently found to belong to clonal complex (CC) 23, including a novel ST described here (Table 1 and Figure S1). From 2010 onwards, an increase in 3GC-resistant E. coli ST648 harbouring blaCMY-2 was observed (Table 2). 3GC-resistant P. mirabilis was first detected in 2004. All isolates with this phenotype were blaCMY-2 positive, except one P. mirabilis from 2007 possessing blaDHA-1 (Table S6). Table 1. CTX-M-producing E. coli clonal lineages Isolate  Year  β-Lactamase  Clonal lineage  CC  Animal  MDR  AMC  3GCs  SXT  FQs  GEN  TET  FMV5825/04  2004  CTX-M-15  ST131:H4-B2-O25b  CC131  dog  yes  R  R  R  R  R  R  FMV521/07  2007  CTX-M-32  ST224  —  cat  likely  I  R  S  R  S  R  FMV1630/07  2007  CTX-M-15  unassigned STb  CC23  dog  yes  I  R  R  R  S  R  FMV7261/07  2007  CTX-M-32  ST609  CC46  dog  yes  I  R  R  R  S  R  FMV635/08  2008  CTX-M-32  ST23  CC23  cat  yes  R  R  R  R  S  S  FMV2777/08  2008  CTX-M-15  ST131:H4-B2-O25b  CC131  cat  yes  R  R  S  R  R  R  FMV5827/08  2008  CTX-M-15  ST23  CC23  dog  yes  R  R  R  R  S  S  FMV1952/10a  2010  CTX-M-9  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV4479/13a  2013  CTX-M-15  ST533  —  dog  yes  R  R  R  R  S  S  FMV5338/13  2013  CTX-M-15  ST131:H4-B2-O25b  CC131  dog  yes  I  R  S  R  R  R  FMV58/2013  2013  CTX-M-1-type  ST131:H4-B2-O25b  CC131  cat  yes  S  R  R  R  S  R  FMV121/2014RE  2014  CTX-M-1-type  ST539  —  dog  yes  S  R  R  R  S  R  Isolate  Year  β-Lactamase  Clonal lineage  CC  Animal  MDR  AMC  3GCs  SXT  FQs  GEN  TET  FMV5825/04  2004  CTX-M-15  ST131:H4-B2-O25b  CC131  dog  yes  R  R  R  R  R  R  FMV521/07  2007  CTX-M-32  ST224  —  cat  likely  I  R  S  R  S  R  FMV1630/07  2007  CTX-M-15  unassigned STb  CC23  dog  yes  I  R  R  R  S  R  FMV7261/07  2007  CTX-M-32  ST609  CC46  dog  yes  I  R  R  R  S  R  FMV635/08  2008  CTX-M-32  ST23  CC23  cat  yes  R  R  R  R  S  S  FMV2777/08  2008  CTX-M-15  ST131:H4-B2-O25b  CC131  cat  yes  R  R  S  R  R  R  FMV5827/08  2008  CTX-M-15  ST23  CC23  dog  yes  R  R  R  R  S  S  FMV1952/10a  2010  CTX-M-9  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV4479/13a  2013  CTX-M-15  ST533  —  dog  yes  R  R  R  R  S  S  FMV5338/13  2013  CTX-M-15  ST131:H4-B2-O25b  CC131  dog  yes  I  R  S  R  R  R  FMV58/2013  2013  CTX-M-1-type  ST131:H4-B2-O25b  CC131  cat  yes  S  R  R  R  S  R  FMV121/2014RE  2014  CTX-M-1-type  ST539  —  dog  yes  S  R  R  R  S  R  AMC, amoxicillin/clavulanate; SXT, trimethoprim/sulfamethoxazole; FQs, fluoroquinolones; GEN, gentamicin; TET, tetracycline; R, resistant; I, intermediate; S, susceptible. a Also harbours blaCMY-2. b Refer to Figure S1 to see the new ST allelic profile. Table 2. AmpC-producing E. coli clonal lineages Isolate  Year  β-Lactamase  Clonal lineage  CC  Animal  MDR  AMC  3GCs  SXT  FQs  GEN  TET  FMV434/00  2000  CMY-2  ST1775  —  dog  yes  R  R  R  S  S  R  FMV1953/01  2001  CMY-2  ST57  CC350  dog  yes  R  R  R  I  I  R  FMV203/03  2003  CMY-2  ST405  CC405  dog  yes  R  R  S  R  S  R  FMV6346/05  2005  CMY-2  ST539  —  cat  yes  R  R  S  R  R  R  FMV3389/06  2006  CMY-2  ST354  CC354  dog  yes  R  R  R  R  R  R  FMV1952/10a  2010  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV25/2011  2011  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV29/2011  2011  CMY-2  ST648  CC648  dog  yes  R  R  S  R  R  R  FMV469/13  2013  CMY-2  ST648  CC648  dog  yes  R  R  R  R  R  R  FMV1389/13  2013  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV4479/13a  2013  CMY-2  ST533  —  dog  yes  R  R  R  R  S  S  FMV55/2013  2013  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV546/14  2014  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV966/14  2014  CMY-2  ST648  CC648  dog  yes  R  R  R  R  S  R  FMV1549/14  2014  CMY-2  ST648  CC648  cat  yes  R  R  S  R  S  R  FMV43/2014  2014  CMY-2  ST648  CC648  cat  yes  R  R  S  R  S  R  Isolate  Year  β-Lactamase  Clonal lineage  CC  Animal  MDR  AMC  3GCs  SXT  FQs  GEN  TET  FMV434/00  2000  CMY-2  ST1775  —  dog  yes  R  R  R  S  S  R  FMV1953/01  2001  CMY-2  ST57  CC350  dog  yes  R  R  R  I  I  R  FMV203/03  2003  CMY-2  ST405  CC405  dog  yes  R  R  S  R  S  R  FMV6346/05  2005  CMY-2  ST539  —  cat  yes  R  R  S  R  R  R  FMV3389/06  2006  CMY-2  ST354  CC354  dog  yes  R  R  R  R  R  R  FMV1952/10a  2010  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV25/2011  2011  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV29/2011  2011  CMY-2  ST648  CC648  dog  yes  R  R  S  R  R  R  FMV469/13  2013  CMY-2  ST648  CC648  dog  yes  R  R  R  R  R  R  FMV1389/13  2013  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV4479/13a  2013  CMY-2  ST533  —  dog  yes  R  R  R  R  S  S  FMV55/2013  2013  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV546/14  2014  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV966/14  2014  CMY-2  ST648  CC648  dog  yes  R  R  R  R  S  R  FMV1549/14  2014  CMY-2  ST648  CC648  cat  yes  R  R  S  R  S  R  FMV43/2014  2014  CMY-2  ST648  CC648  cat  yes  R  R  S  R  S  R  AMC, amoxicillin/clavulanate; SXT, trimethoprim/sulfamethoxazole; FQs, fluoroquinolones; GEN, gentamicin; TET, tetracycline; R, resistant; I, intermediate; S, susceptible. a Also harbours blaCTX-M. Regarding Staphylococcus spp., 9.2% (CI 4.0%–14.4%, n = 11/119) of Staphylococcus spp. were methicillin resistant and were found to harbour the mecA gene. Overall only resistance to fluoroquinolones was significantly higher in the second time period (2007–14; P = 0.0189) (Table S4). However, if S. pseudintermedius are analysed alone, a significant increase in methicillin (14.8%, CI 1.4%–28.2%, in 2007–14; P = 0.0069) and gentamicin (17.9%, CI 3.7%–32.0%, in 2007–14; P = 0.0099) resistance was also detected. All MDR Staphylococcus spp. were associated with the presence of the mecA gene. Methicillin-resistant Staphylococcus pseudintermedius (MRSP; n = 4) and methicillin-resistant S. epidermidis (MRSE; n = 4) were the most common methicillin-resistant Staphylococcus species, followed by MRSA (n = 2) and methicillin-resistant Staphylococcus lentus (n = 1). Although uncommon, S. epidermidis showed a high frequency of methicillin resistance (n = 4/6). MRSP showed resistance to all the tested antimicrobials, except two that were susceptible to tetracycline. Thus all MRSP were considered MDR. All methicillin-resistant Staphylococcus spp. but one were isolated from cats. The 11 methicillin-resistant Staphylococcus spp. found in this study were fully characterized elsewhere.8 Briefly, MRSP belonged to ST71-II-III (n = 3) and ST196-V (n = 1); MRSE belonged to CC 5 (ST2-nt, ST20-nt, ST23-IV, ST35-nt) and MRSA to CC5 (ST5-t311-VI, ST105-t002-II). Enterococcus spp. showed very high tetracycline (75.8%, CI 65.2%–86.5%, n = 47/62) and fluoroquinolone (56.4%, CI 44.1%–68.8%, n = 35/62) resistance. Ampicillin resistance in Enterococcus spp. (12.1%, CI 4.2%–20.0%, n = 8/66) was lower, though if only E. faecium were considered, almost all were ampicillin resistant (n = 8/9). HLGR was detected in 15.2% (CI 6.1%–24.4%, n = 9/59) of the tested Enterococcus spp. and was mostly found in E. faecalis harbouring aac(6′)-Ieaph(2″)-Ia (Table S7). HLGR E. faecalis belonged to known sequence types, including one isolate from ST6-CC6 (former CC2) (Table S7). All ampicillin-resistant and/or HLGR E. faecium belonged to CC17 (Table S7), including two novel STs (ST1282 and ST1283) identified in this study (Figure S2). Discussion As seen in other studies, E. coli was the most frequently isolated bacterium in dogs and cats with UTI.1,4 Overall, Enterobacteriaceae caused more than half of the UTIs in dogs and cats; therefore, given that β-lactams are among the most important antimicrobials nowadays,10 the great increase detected in this study in Enterobacteriaceae resistance to amoxicillin/clavulanate and 3GCs is worrisome (Figure 1 and Table S4). 3GCs are considered highest-priority critically important antimicrobials for humans,10 and resistance is frequently associated with the production of β-lactamases.3 The increase in 3GC resistance observed in this study was frequently associated with the presence of blaCTX-M-15 and blaCMY-2 in E. coli and blaCMY-2 in P. mirabilis (Tables 1 and 2 and Table S6). ESBL CTX-M-15 is distributed worldwide in E. coli.31 Moreover, E. coli O25b:H4-B2-ST131 is a widespread human uropathogenic clonal lineage that frequently harbours blaCTX-M-1532 and has been previously described in dog and cat faecal samples (as a commensal),12 and also causes UTI.6 Therefore the detection of E. coli O25b:H4-B2-ST131 in this study came as no surprise. Furthermore, E. coli CC23 has been described in humans with UTI in the community33 and has also been shown to be a common CC among CTX-M-producing E. coli.34 This study shows an increase in the detection of CMY-2-producing E. coli and Proteus spp. over time. Although less frequently reported than blaCTX-M in previously published data,6,35 this increase in blaCMY-2 should not be neglected since this enzyme shows stronger β-lactamase activity36 and may in the future become more prevalent. Furthermore, blaCMY-2-carrying Enterobacteriaceae may exhibit resistance to carbapenems in the absence of carbapenemases, owing to the presence of other resistance mechanisms such as porin defiency,37 which further highlights their clinical relevance. In the present study, the first CMY-2-producing E. coli was detected in 2000, yet it was from 2010 onwards that the E. coli CMY-2-producing ST648 clonal lineage was increasingly detected (Table 2). E. coli ST648 has been described in human infections harbouring several β-lactamases, such as ESBL and carbapenemases.31,38 Nevertheless, both in humans and companion animals, the ST648 E. coli clonal lineage has mostly been described harbouring blaCTX-M genes.38,39 The significant increase in ST648 CMY-2-producing E. coli observed in this study in companion animals with UTI may point to the possible expansion of a blaCMY-2-producing MDR ST648 clonal lineage, though more studies are needed to clarify the clonal relatedness between these isolates. However, a few studies have also found a high frequency of ST648 CMY-2-producing E. coli in faecal samples and specimens from infections of companion animals.40,41 Although only detected once in this study, E. coli ST405 also belongs to a highly successful clonal lineage that causes human infection.38 Dogs and cats with UTI are, therefore, shown in this study to be infected with 3GC-resistant E. coli belonging to clonal lineages of great importance to humans. Furthermore, CTX-M- and CMY-producing E. coli and P. mirabilis were also found to be MDR, thus increasing the relevance of these findings. Additionally, Enterobacteriaceae from this study showed a significant increase in resistance to all the antimicrobials commonly used in the treatment of dogs and cats with UTI (Figure 1). Together with the significant increase in detection of MDR Enterobacteriaceae over time, these results point to growing therapeutic limitations in veterinary medicine that in the future may lead to an increasing need to prescribe antimicrobials originally intended for human use. ESBL- and AmpC-producing E. coli presented MDR susceptibility phenotypes with limited therapeutic options (Tables 1 and 2). A study in humans showed that the use of amoxicillin/clavulanate for the treatment of UTIs caused by ESBL-producing E. coli may be suitable if the isolate is fully susceptible to this antimicrobial.42 Although most ESBL-producing E. coli from this study displayed intermediate resistance to amoxicillin/clavulanate, similar studies should be conducted in veterinary medicine to evaluate its effectiveness in the treatment of MDR ESBL-producing Enterobacteriaceae. MDR Enterobacteriaceae were more often susceptible to gentamicin; hence, although sometimes impractical, the use of this antimicrobial should be considered for the treatment of UTIs caused by MDR Enterobacteriaceae in dogs and cats (Table S5). Staphylococcus spp. were the second most frequently isolated bacteria; however, the identified Staphylococcus species varied significantly between dogs and cats (Table S2). The high frequency of S. felis in cats with UTI is in agreement with a previous study conducted in Australia, in which the authors associated the presence of S. felis with clinical signs of lower urinary tract disease in cats.4 The significant increase in the detection of MDR MRSP over time is a concerning finding since it creates major therapeutic limitations. The MRSP detected in this study belonged mainly to ST71-II-III, which is known to be one of the most disseminated clonal lineages in dogs and cats in Europe.43,44 Although rarely, human infection by MRSP ST71-II-III has already been described, thus highlighting its zoonotic potential.45 As seen in reports on humans, S. epidermidis showed a high frequency of methicillin resistance46 and all belonged to STs also found in humans.47 The two MRSA isolated in this study belonged to S. aureus CC5, which is frequently associated with human hospital-acquired MRSA.48 Furthermore, both MRSA STs have been reported in humans from Portugal.49,50 Interestingly, in this study, the mecA gene was mainly detected in Staphylococcus spp. isolated from cats. As this was a retrospective study, it was not possible to obtain information on the possible source of these infections. Nevertheless, the detection of MRSA and MRSE in dogs and cats with UTI is a public health issue since companion animals will have a role in dissemination of these bacteria to the household and public environment. Ampicillin resistance and HLGR in Enterococcus spp. strongly limit the therapeutic options against enterococcal infections.51 Although in this study ampicillin-resistant and/or HLGR Enterococcus spp. were uncommon, some belonged to high-risk CCs associated with hospital-acquired infections in humans, such as E. faecalis CC6 (formerly CC2) and E. faecium CC17 (Table S7).52,53 HLGR was mostly detected in E. faecalis and was caused by the presence of a bifunctional enzyme that is known to also confer resistance to a wide range of aminoglycosides.51,E. faecalis ST6 (CC6) has been previously described in hospitalized patients from Portugal but also in samples from pigs and from hospital waste waters.54 As in this study, the E. faecalis ST16 is a clonal lineage that has been found to frequently harbour the bifunctional enzyme.29,55 Also, E. faecalis ST16 has been previously detected in healthy and hospitalized humans as well as in animals.29,54,55 E. faecium isolates were less frequent than those of E. faecalis, but were more commonly ampicillin resistant (Table S7). A previous study has pointed to healthy dogs as reservoirs of ampicillin-resistant E. faecium CC17 including ST19.56 Besides belonging to CC17, the E. faecium ST19 and ST440 strains isolated in this study are also noteworthy for being simultaneously ampicillin resistant and HLGR. Enterobacteriaceae and Staphylococcus spp. together caused around 79% of the UTIs in dogs and cats. Thus, the increase in fluoroquinolone resistance in both groups of bacteria is of great relevance. Furthermore, Enterococcus spp. also showed high levels of resistance to this antimicrobial class. Bearing in mind fluoroquinolones are considered highest-priority critically important antimicrobials for humans,10 and of great importance in the treatment of pyelonephritis in companion animals,2 judicious use of these antimicrobials should be pursued. The fact that this study relied on samples submitted to the laboratory based on clinical judgement could be considered a bias towards resistance because urine cultures from complicated UTIs may be requested more frequently than from simple uncomplicated UTIs.1 However, if present, this bias was constant throughout the study time frame and therefore the increase in antimicrobial resistance is unequivocal. Only in recent years has the EMA started to publish data about antimicrobial sales for companion animals. Nevertheless, the high β-lactam and fluoroquinolone resistance frequencies detected in this study could be expected since these are the first and second most sold antimicrobials for companion animals in Portugal.57 The high frequency of tick-borne diseases in Portugal that require the use of doxycycline in companion animals could have contributed to the high tetracycline resistance seen in this study. Also, the increase in MDR Enterobacteriaceae may, to some extent, explain the increasing resistance trends observed for all the tested antimicrobials. The limited access to complete data about the epidemiological and clinical history of the dogs and cats infected with high-risk clonal lineages was also a limitation of this study. Nevertheless, since most of these animals were treated at home by their owners, the role of dogs and cats with UTI in the spread of resistant bacteria into the environment should be considered. Furthermore, it has been shown that companion animals may share uropathogenic bacteria with their household members,12 and therefore the role of dogs and cats as reservoirs of high-risk clonal lineages is a concern to veterinary professionals and owners. This study showed that bacteria causing UTI in dogs and cats, especially Enterobacteriaceae, are increasingly resistant to the antimicrobials most widely used in UTI treatment. These bacteria were found to harbour clinically relevant antimicrobial resistance mechanisms and simultaneously belong to high-risk clonal lineages, namely 3GC-resistant E. coli O25b:H4-B2-ST131, CC23 and ST648, MRSA CC5, MRSE CC5, HLGR E. faecalis CC6, and ampicillin/HLGR E. faecium CC17. Therefore, when such resistance mechanisms are suspected based on susceptibility testing, veterinary professionals and owners should be advised to take measures in order to reduce the spread of these bacteria, such as strict hand washing, cleaning of the animals’ living environment as well as adequate faecal disposal. Also, longitudinal studies on the faecal carriage of these resistant high-risk clonal lineages during and after UTI should be conducted to assess the time of carriage and evaluate the extent and duration of the infection control measures that should be taken. Acknowledgements We acknowledge the use of the E. faecium (http://pubmlst.org/efaecium/) and E. faecalis (http://pubmlst.org/efaecalis/) MLST websites hosted by the University of Oxford (Jolley KA, Maiden MC. BMC Bioinformatics 2010; 11: 595) and funded by the Wellcome Trust. Funding This work was supported by FEDER funds through the Programa Operacional Factores de Competitividade—COMPETE and by National funds through the FCT—Fundação para a Ciência e a Tecnologia—CIISA Project (UID/CVT/00276/2013) and PhD grants to A. B. (grant SFRH/BD/113142/2015) and to C. M. (grant SFRH/BD/77886/2011) Transparency declarations None to declare. Supplementary data Tables S1–S7 and Figures S1 and S2 are available as Supplementary data at JAC Online. References 1 Hall JL , Holmes MA, Baine SJ. Prevalence and antimicrobial resistance of canine urinary tract pathogens. Vet Rec  2013; 173: 549. Google Scholar CrossRef Search ADS   2 Weese S , Blondeau JM, Boothe D et al.   Antimicrobial use guidelines for treatment of urinary tract disease in dogs and cats: Antimicrobial Guidelines Working Group of the International Society for Companion Animal Infectious Diseases. Vet Med Int  2011; 2011: 263768. Google Scholar CrossRef Search ADS   3 ECDC. Antimicrobial Resistance Surveillance in Europe 2014. Annual Report of the European Antimicrobial Resistance Surveillance Network (EARS-Net) . Stockholm: ECDC, 2015. 4 Litster A , Moss SM, Honnery M et al.   Prevalence of bacterial species in cats with clinical signs of lower urinary tract disease: recognition of Staphylococcus felis as a possible feline urinary tract pathogen. Vet Microbiol  2007; 121: 182– 8. Google Scholar CrossRef Search ADS   5 Féria C , Ferreira E, Correia JD et al.   Patterns and mechanisms of resistance to β-lactams and β-lactamase inhibitors in uropathogenic Escherichia coli isolated from dogs in Portugal. J Antimicrob Chemother  2002; 49: 77– 85. Google Scholar CrossRef Search ADS   6 Ewers C , Grobbel M, Stamm I et al.   Emergence of human pandemic O25:H4-ST131 CTX-M-15 extended-spectrum-β-lactamase-producing Escherichia coli among companion animals. J Antimicrob Chemother  2010; 65: 651– 60. Google Scholar CrossRef Search ADS   7 Wagner S , Gally DL, Argyle SA. Multidrug-resistant Escherichia coli from canine urinary tract infections tend to have commensal phylotypes, lower prevalence of virulence determinants and ampC-replicons. Vet Microbiol  2014; 169: 171– 8. Google Scholar CrossRef Search ADS   8 Couto N , Monchique C, Belas A et al.   Trends and molecular mechanisms of antimicrobial resistance in clinical staphylococci isolated from companion animals over a 16 year period. J Antimicrob Chemother  2016; 71: 1479– 87. Google Scholar CrossRef Search ADS   9 Jackson CR , Fedorka-Cray PJ, Davis JA et al.   Mechanisms of antimicrobial resistance and genetic relatedness among enterococci isolated from dogs and cats in the United States. J Appl Microbiol  2010; 108: 2171– 9. 10 WHO. Critically Important Antimicrobials for Human Medicine—5th Revision. 2017. http://apps.who.int/iris/bitstream/10665/255027/1/9789241512220-eng.pdf. 11 Pomba C , Rantala M, Greko C et al.   Public health risk of antimicrobial resistance transfer from companion animals. J Antimicrob Chemother  2017; 72: 957– 68. 12 Johnson JR , Miller S, Johnston B et al.   Sharing of Escherichia coli sequence type ST131 and other multidrug-resistant and urovirulent E. coli strains among dogs and cats within a household. J Clin Microbiol  2009; 47: 3721– 5. Google Scholar CrossRef Search ADS   13 Bartges JW. Diagnosis of urinary tract infections. Vet Clin North Am Small Anim Pract  2004; 34: 923– 33. Google Scholar CrossRef Search ADS   14 Doumith M , Day MJ, Hope R et al.   Improved multiplex PCR strategy for rapid assignment of the four major Escherichia coli phylogenetic groups. J Clin Microbiol  2012; 50: 3108– 10. Google Scholar CrossRef Search ADS   15 Kovtunovych GL , Lytvynenko T, Negrutska VV et al.   A PCR-mediated method for discrimination of Klebsiella oxytoca between closely related bacteria in environmental and clinical specimens. Biopolym Cell  2003; 19: 520– 4. Google Scholar CrossRef Search ADS   16 Padmavathy B , Vinoth Kumar R, Patel A et al.   Rapid and sensitive detection of major uropathogens in a single-pot multiplex PCR assay. Curr Microbiol  2012; 65: 44– 53. Google Scholar CrossRef Search ADS   17 Stankowska D , Kwinkowski M, Kaca W. Quantification of Proteus mirabilis virulence factors and modulation by acylated homoserine lactones. J Microbiol Immunol Infect  2008; 41: 243– 53. 18 Woodford N , Egelton CM, Morrison D. Comparison of PCR with phenotypic methods for the speciation of enterococci. Adv Exp Med Biol  1997; 418: 405– 8. Google Scholar CrossRef Search ADS   19 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals—Fourth Edition: Approved Standard VET01-A4 . CLSI, Wayne, PA, USA, 2013. 20 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Second Informational Supplement VET01-S2 . CLSI, Wayne, PA, USA, 2013. 21 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Sixth Informational Supplement M100-S26 . CLSI, Wayne, PA, USA, 2016. 22 Pérez-Pérez FJ , Hanson ND. Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol  2002; 40: 2153– 62. Google Scholar CrossRef Search ADS   23 Guessennd N , Bremont S, Gbonon V et al.   Résistance aux quinolones de type qnr chez les entérobactéries productrices de bêta-lactamases à spectre élargi à Abidjan en Côte d’Ivoire. Pathol Biol (Paris)  2008; 56: 439– 46. Google Scholar CrossRef Search ADS   24 Belas A , Salazar AS, Telo da Gama L et al.   Risk factors for faecal colonisation with Escherichia coli producing extended-spectrum and plasmid-mediated AmpC β-lactamases in dogs. Vet Rec  2014; 175: 202. Google Scholar CrossRef Search ADS   25 Kao SJ , You I, Clewell DB et al.   Detection of the high-level aminoglycoside resistance gene aph(2")-Ib in Enterococcus faecium. Antimicrob Agents Chemother  2000; 44: 2876– 9. Google Scholar CrossRef Search ADS   26 Johnson J , Clermont O, Johnstone B et al.   Rapid and specific detection, molecular epidemiology, and experimental virulence of the O16 subgroup within Escherichia coli sequence type 131. J Clin Microbiol  2014; 52: 1358– 65. Google Scholar CrossRef Search ADS   27 Wirth T , Falush D, Lan R et al.   Sex and virulence in Escherichia coli: an evolutionary perspective. Mol Microbiol  2006; 60: 1136– 51. Google Scholar CrossRef Search ADS   28 Homan WL , Tribe D, Poznanski S et al.   Multilocus sequence typing scheme for Enterococcus faecium. J Clin Microbiol  2002; 40: 1963– 71. Google Scholar CrossRef Search ADS   29 Ruiz-Garbajosa P , Bonten MJ, Robinson DA et al.   Multilocus sequence typing scheme for Enterococcus faecalis reveals hospital-adapted genetic complexes in a background of high rates of recombination. J Clin Microbiol  2006; 44: 2220– 8. Google Scholar CrossRef Search ADS   30 Magiorakos AP , Srinivasan A, Carey RB et al.   Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect  2012; 18: 268– 81. Google Scholar CrossRef Search ADS   31 Ewers C , Bethe A, Semmler T et al.   Extended-spectrum β-lactamase-producing 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. Google Scholar CrossRef Search ADS   32 Nicolas-Chanoine MH , Blanco J, Leflon-Guibout V et al.   Intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15. J Antimicrob Chemother  2008; 61: 273– 81. Google Scholar CrossRef Search ADS   33 Lau SH , Reddy S, Cheesbrough J et al.   Major uropathogenic Escherichia coli strain isolated in the northwest of England identified by multilocus sequence typing. J Clin Microbiol  2008; 46: 1076– 80. Google Scholar CrossRef Search ADS   34 Brisse S , Diancourt L, Laouénan C et al.   Phylogenetic distribution of CTX-M- and non-extended-spectrum-β-lactamase-producing Escherichia coli isolates: group B2 isolates, except clone ST131, rarely produce CTX-M enzymes. J Clin Microbiol  2012; 50: 2974– 81. Google Scholar CrossRef Search ADS   35 Aragón LM , Mirelis B, Miró E et al.   Increase in β-lactam-resistant Proteus mirabilis strains due to CTX-M- and CMY-type as well as new VEB- and inhibitor-resistant TEM-type β-lactamases. J Antimicrob Chemother  2008; 61: 1029– 32. Google Scholar CrossRef Search ADS   36 Jacoby GA , Amp C. β-lactamases. Clin Microbiol Rev  2009; 22: 161– 82. Google Scholar CrossRef Search ADS   37 Chia JH , Siu LK, Su LH et al.   Emergence of carbapenem-resistant Escherichia coli in Taiwan: resistance due to combined CMY-2 production and porin deficiency. J Chemother  2009; 21: 621– 6. Google Scholar CrossRef Search ADS   38 Pitout JDD. Extraintestinal pathogenic Escherichia coli: a combination of virulence with antibiotic resistance. Front Microbiol  2012; 3: 9. Google Scholar CrossRef Search ADS   39 Ewers C , Bethe A, Stamm I et al.   CTX-M-15-D-ST648 Escherichia coli from companion animals and horses: another pandemic clone combining multiresistance and extraintestinal virulence? J Antimicrob Chemother  2014; 69: 1224– 30. Google Scholar CrossRef Search ADS   40 Tamang MD , Nam HM, Jang GC et al.   Molecular characterization of extended-spectrum-β-lactamase-producing and plasmid-mediated AmpC β-lactamase-producing Escherichia coli isolated from stray dogs in South Korea. Antimicrob Agents Chemother  2012; 56: 2705– 12. Google Scholar CrossRef Search ADS   41 Liu X , Thungrat K, Boothe DM. Occurrence of OXA-48 carbapenemase and other β-lactamase genes in ESBL-producing multidrug resistant Escherichia coli from dogs and cats in the United States, 2009–2013. Front Microbiol  2016; 7: 1057. 42 Beytur A , Yakupogullari Y, Oguz F et al.   Oral amoxicillin-clavulanic acid treatment in urinary tract infections caused by extended-spectrum β-lactamase-producing organisms. Jundishapur J Microbiol  2015; 8: e13792. 43 Kadlec K , Schwarz S, Perreten V et al.   Molecular analysis of methicillin-resistant Staphylococcus pseudintermedius of feline origin from different European countries and North America. J Antimicrob Chemother  2010; 65: 1826– 8. Google Scholar CrossRef Search ADS   44 Perreten V , Kadlec K, Schwarz S et al.   Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study. J Antimicrob Chemother  2010; 65: 1145– 54. Google Scholar CrossRef Search ADS   45 Stegmann R , Burnens A, Maranta CA et al.   Human infection associated with methicillin-resistant Staphylococcus pseudintermedius ST71. J Antimicrob Chemother  2010; 65: 2047– 8. Google Scholar CrossRef Search ADS   46 Becker K , Heilmann C, Peters G. Coagulase-negative staphylococci. Clin Microbiol Rev  2014; 27: 870– 926. Google Scholar CrossRef Search ADS   47 Rolo J , de Lencastre H, Miragaia M. Strategies of adaptation of Staphylococcus epidermidis to hospital and community: amplification and diversification of SCCmec. J Antimicrob Chemother  2012; 67: 1333– 41. Google Scholar CrossRef Search ADS   48 Aires-de-Sousa M. Methicillin-resistant Staphylococcus aureus among animals: current overview. Clin Microbiol Infect  2017; 23: 373– 80. Google Scholar CrossRef Search ADS   49 Conceição T , Tavares A, Miragaia M et al.   Prevalence and clonality of methicillin-resistant Staphylococcus aureus (MRSA) in the Atlantic Azores islands: predominance of SCCmec types IV, V and VI. Eur J Clin Microbiol Infect Dis  2010; 29: 543– 50. Google Scholar CrossRef Search ADS   50 Espadinha D , Faria NA, Miragaia M et al.   Extensive dissemination of methicillin-resistant Staphylococcus aureus (MRSA) between the hospital and the community in a country with a high prevalence of nosocomial MRSA. PLoS One  2013; 8: e59960. Google Scholar CrossRef Search ADS   51 Chow JW. Aminoglycoside resistance in enterococci. Clin Infect Dis  2000; 31: 586– 9. Google Scholar CrossRef Search ADS   52 Leavis HL , Bonten MJ, Willems RJ. Identification of high-risk enterococcal clonal complexes: global dispersion and antibiotic resistance. Curr Opin Microbiol  2006; 9: 454– 60. Google Scholar CrossRef Search ADS   53 Kuch A , Willems RJ, Werner G et al.   Insight into antimicrobial susceptibility and population structure of contemporary human Enterococcus faecalis isolates from Europe. J Antimicrob Chemother  2012; 67: 551– 8. Google Scholar CrossRef Search ADS   54 Freitas AR , Novais C, Ruiz-Garbajosa P et al.   Clonal expansion within clonal complex 2 and spread of vancomycin-resistant plasmids among different genetic lineages of Enterococcus faecalis from Portugal. J Antimicrob Chemother  2009; 63: 1104– 11. Google Scholar CrossRef Search ADS   55 Larsen J , Schønheyder HC, Lester CH et al.   Porcine-origin gentamicin-resistant Enterococcus faecalis in humans, Denmark. Emerg Infect Dis  2010; 16: 682– 4. Google Scholar CrossRef Search ADS   56 Damborg P , Top J, Hendrickx AP et al.   Dogs are a reservoir of ampicillin-resistant Enterococcus faecium lineages associated with human infections. Appl Environ Microbiol  2009; 75: 2360– 5. Google Scholar CrossRef Search ADS   57 EMA. Sales of Veterinary Antimicrobial Agents in 29 European Countries in 2014. 2016. http://www.ema.europa.eu/docs/en_GB/document_library/Report/2016/10/WC500214217.pdf. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Increase in antimicrobial resistance and emergence of major international high-risk clonal lineages in dogs and cats with urinary tract infection: 16 year retrospective study

Loading next page...
 
/lp/ou_press/increase-in-antimicrobial-resistance-and-emergence-of-major-BwpXeQsrNJ
Publisher
Oxford University Press
Copyright
© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
ISSN
0305-7453
eISSN
1460-2091
D.O.I.
10.1093/jac/dkx401
Publisher site
See Article on Publisher Site

Abstract

Abstract Objectives To evaluate temporal trends in antimicrobial resistance, over 16 years, in bacteria isolated from dogs and cats with urinary tract infection (UTI) and the clonal lineages of bacteria harbouring critical antimicrobial resistance mechanisms. Methods Antimicrobial susceptibility testing was conducted for 948 bacteria isolated from dogs and cats with UTI (1999–2014). Resistance mechanisms were detected by PCR, namely ESBL/AmpC in third-generation cephalosporin (3GC)-resistant Escherichia coli and Proteus mirabilis, mecA in methicillin-resistant staphylococci, and aac(6′)-Ieaph(2″)-Ia and aph(2″)-1d in high-level gentamicin-resistant (HLGR) enterococci. Resistant bacteria were typed by MLST, and temporal trends in E. coli and Enterobacteriaceae antimicrobial resistance were determined by logistic regression. Results Enterobacteriaceae had a significant temporal increase in resistance to amoxicillin/clavulanate, 3GCs, trimethoprim/sulfamethoxazole, fluoroquinolones, gentamicin and tetracycline (P < 0.001). An increase in MDR was also detected (P < 0.0001). 3GC resistance was mainly caused by the presence of blaCTX-M-15 and blaCMY-2 in E. coli and the presence of blaCMY-2 in P. mirabilis. Two major 3GC-resistant E. coli clonal lineages were detected: O25b:H4-B2-ST131 and ST648. The mecA gene was detected in 9.2% (n = 11/119) of Staphylococcus spp., including MRSA clonal complex (CC) 5 (n = 2) and methicillin-resistant Staphylococcus epidermidis CC5 (n = 4). A temporal increase in MDR methicillin-resistant Staphylococcus pseudintermedius was detected (P = 0.0069). Some ampicillin-resistant and/or HLGR Enterococcus spp. were found to belong to hospital-adapted CCs, namely Enterococcus faecalis ST6-CC6 (n = 1) and Enterococcus faecium CC17 (n = 8). Conclusions The temporal increase in antimicrobial resistance and in MDR bacteria causing UTI in dogs and cats creates important therapeutic limitations in veterinary medicine. Furthermore, the detection of MDR high-risk clonal lineages raises public health concerns since companion animals with UTI may contribute to the spread of such bacteria. Introduction Urinary tract infections (UTIs) are frequently diagnosed in veterinary medicine1 and may require antimicrobial treatment.2 Since antimicrobial resistance is known to change geographically and over time,3 updated and long-term studies are critical to investigate the spread of antimicrobial resistance. Escherichia coli and Proteus mirabilis are the most frequently isolated Gram-negative bacteria from dogs and cats with UTI, while Staphylococcus spp. and Enterococcus spp. are the most common Gram-positive bacteria.1,4 These bacteria, isolated from dogs and cats, may harbour clinically and epidemiologically important resistance mechanisms of human and veterinary relevance such as ESBL,5,6 cephalosporinases (AmpC),7 PBP2a8 and high-level gentamicin resistance (HLGR) bifunctional enzyme.9 Moreover, the detection of MDR bacteria in dogs and cats is being increasingly reported,7,8 posing a difficult veterinary therapeutic challenge and often requiring the use of antimicrobials critically important to humans.10 With the growing contact between companion animals and humans, the risk of animal-to-human transfer of such bacteria is of concern.11 Additionally, several studies have shown that dogs and cats may share uropathogenic bacteria with the remaining household members.12 Therefore, the identification of the clonal lineages of bacteria isolated from dogs and cats with UTI, especially those harbouring important resistance mechanisms, is crucial to evaluate the extent to which dogs and cats with UTI may act as reservoirs for resistant bacteria. The goal of this study was to determine the temporal trends of antimicrobial resistance of bacteria isolated from dogs and cats with UTI over 16 years and to characterize their major antimicrobial resistance mechanisms, namely ESBL and AmpC in E. coli and P. mirabilis, methicillin resistance in Staphylococcus spp., and ampicillin and HLGR in Enterococcus spp. Furthermore, this study aimed to determine the clonal lineages of E. coli, Staphylococcus spp. and Enterococcus spp. harbouring such resistance genes and hence evaluate their potential public health relevance. Materials and methods Bacterial isolates A total of 948 consecutive positive bacterial isolates from dogs and cats with UTI (n = 869), collected from 1999 to 2014 at the Laboratory of Antimicrobial Resistance from the Veterinary Teaching Hospital of the Faculty of Veterinary Medicine/University of Lisbon and from several private practices in the Lisbon region were included in this study. Samples were collected by cystocentesis, catheterization or free catch as part of the routine care of dogs and cats with UTI. UTI was diagnosed based on clinical and urine cytological findings together with the detection of significant bacteriuria by quantitative urine culture. Bacteriological methods Quantitative urine culture was performed. Briefly, 10 μL of urine was inoculated onto 5% sheep blood (bioMérieux, Marcy-l’Étoile, France) and MacConkey (Biokar Diagnostics, Allonne, France) agar plates. After incubation at 37 °C for 24–48 h under atmospheric conditions, colonies were quantified and scored as having significant bacteriuria according to the urine sample collection method as defined elsewhere.13 Samples were included in the study regardless of the type of UTI. Species identification was conducted by phenotypic tests (API, bioMérieux and BD™ BBL™ Crystal Gram Positive ID Kit, Becton Dickinson, MD, USA). At the time of collection, isolated bacteria were stored in 20% glycerol (Sigma–Aldrich, St Louis, MO, USA) brain heart infusion broth (Biokar Diagnostics) at −80 °C for future studies. For the present study, stored isolates were recovered by streaking them onto 5% sheep blood agar. Whenever recovery was not possible, existing antimicrobial susceptibility results were used. E. coli,14,Klebsiella spp.,15,16,Proteus spp.,17,Enterococcus spp.18 and Staphylococcus spp.8 were confirmed by PCR and/or sequencing of 16S rRNA. Susceptibility testing Susceptibility testing was performed by the disc diffusion method according to CLSI guidelines, and E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were used for quality control.19 The following antimicrobials were tested (Oxoid, Hampshire, UK): amoxicillin/clavulanate 30 μg, cefotaxime 30 μg, cefovecin 30 μg, cefoxitin 30 μg, ceftazidime 30 μg, ciprofloxacin 5 μg, enrofloxacin 5 μg, gentamicin 10 μg, high-level gentamicin 120 μg, oxacillin 1 μg, penicillin 10 U, tetracycline 30 μg and trimethoprim/sulfamethoxazole 25 μg. Veterinary CLSI breakpoints20 were used for amoxicillin/clavulanate, cefoxitin, enrofloxacin, gentamicin, high-level gentamicin, oxacillin, penicillin, tetracycline and trimethoprim/sulfamethoxazole; human CLSI breakpoints21 were used for cefotaxime, ceftazidime and ciprofloxacin. Finally, cefovecin results were interpreted according to the manufacturer’s breakpoints. Initial ESBL screening was conducted by a double-disc synergy test.19 Cefoxitin or oxacillin were used to predict methicillin resistance in Staphylococcus spp. according to CLSI guidelines.20,21 DNA extraction, sample purification and sequencing DNA extraction was conducted using a boiling method.5 For PCR amplicon sequencing, DNA purification was conducted using a NZYTech Gel Pure Kit (NZYTech—Genes and Enzymes, Lisbon, Portugal) and sequencing was performed by Stabvida (Caparica, Portugal). Sequences were analysed using Ugene Unipro software (Unipro, Novosibirsk, Russia) and the nucleotide basic local alignment search tool (http://blast.ncbi.nlm.nih.gov/). Antimicrobial resistance genes Third-generation cephalosporin (3GC)-resistant E. coli and P. mirabilis were screened for the presence of ESBL blaCTX-M-type and AmpC blaCIT-type,blaDHA-type,blaMOX-type,blaACT-type,blaFOX-type and blaMIR-type by PCR and sequencing.22–24 3GC-resistant Enterobacteriaceae without blaCTX-M-type or AmpC genes were further tested for the presence of blaTEM-type and blaSHV-type ESBL genes.5,Staphylococcus spp. were screened for the presence of the mecA gene8 and HLGR Enterococcus spp. for the presence of aac(6′)-Ieaph(2″)-Ia and aph(2″)-1d25 genes by PCR. Negative and previously sequenced positive controls were included in all PCR reactions. Clonal lineages of resistant isolates ESBL- or AmpC-producing E. coli were tested for the clonal lineage O25b:H4-B2-ST131 by PCR.14,26 3GC-resistant E. coli isolates not belonging to the O25b:H4-B2-ST131 clonal lineage were typed by MLST.27 Methicillin-resistant Staphylococcus spp. were previously characterized by MLST, SCCmec and spa typing elsewhere.8 Ampicillin-resistant and/or HLGR Enterococcus faecium and Enterococcus faecalis were also typed by MLST.28,29 eBURST v. 3 software (http://eburst.mlst.net/) was used to estimate the relationships between the isolate STs from this study and all MLST profiles known to date. Statistical analysis The SAS statistical software package for Windows v. 9.3 (SAS Institute Inc., Cary, NC, USA) was used for statistical analysis. Antimicrobial resistance frequencies were only calculated if at least 10 isolates were tested for a specific organism/antimicrobial combination and results were presented with the 95% CI. For statistical purposes, intermediate isolates were considered susceptible. An isolate was considered resistant to 3GCs when it was found to be resistant to at least one of the three 3GCs tested (cefotaxime, ceftazidime and cefovecin). Ciprofloxacin and enrofloxacin were used as markers of fluoroquinolone resistance. An isolate was considered MDR when it was found to be fully resistant to three or more antimicrobial categories. The antimicrobial categories were adapted from those proposed by other authors30 and varied according to the bacterial species considered (Table S1, available as Supplementary data at JAC Online). When at least 10 isolates were tested per year, temporal trends of antimicrobial resistance were determined using an SAS LOGISTIC regression model with the year as a continuous variable and an α value of 0.05. Thus, temporal trends in antimicrobial resistance were determined for E. coli and for Enterobacteriaceae (including E. coli, Proteus spp., Klebsiella spp. and Enterobacter spp. as a group). When determining Enterobacteriaceae temporal trends, intrinsic resistance data was excluded from analysis. Furthermore, P. mirabilis and Staphylococcus spp. antimicrobial resistance were compared between two time periods: 1999–2006 and 2007–14. The Fisher’s exact test was used for comparisons between groups with an α value of 0.05. Results From 1999 to 2014, 948 bacteria were isolated from 649 dogs and 220 cats with UTI. The majority of UTIs (91.1%, CI 89.2%–93.0%, n = 792/869) were caused by single organisms. Coinfections were most commonly caused by the combinations of E. coli/Enterococcus spp. (14.3%, CI 6.5%–22.1%, n = 11/77), E. coli/P. mirabilis (11.7%, CI 4.5%–18.9%, n = 9/77) and E. coli/Streptococcus spp. (10.4%, CI 3.6%–17.2%, n = 8/77). Although E. coli (43.5%) was the most frequently isolated bacterium, Proteus spp. (16.4%), Staphylococcus spp. (13.2%) and Enterococcus spp. (7.0%) were also common (Table S2). The frequency of infection by Proteus spp. was significantly higher (P < 0.0001) in dogs and Enterococcus spp. in cats, respectively (Table S2). Overall, Staphylococcus spp. had similar frequencies in cats and dogs. However, Staphylococcus pseudintermedius was significantly more common in dogs (P < 0.0001), while cats were infected by a higher diversity of staphylococcal species, with S. pseudintermedius, Staphylococcus felis and Staphylococcus epidermidis being the most frequent (Table S2). Enterobacteriaceae accounted for 66.1% (CI 63.1%–69.2%) of all isolated bacteria and showed a significant temporal increase in resistance to all tested antimicrobials (Table S3 and Figure 1). In 2012–14, resistance of Enterobacteriaceae to all the antimicrobials tested, except gentamicin, was >30% (Figure 1). Considering E. coli and Proteus spp. alone, no significant change over time was detected in trimethoprim/sulfamethoxazole resistance (Tables S3 and S4). Nevertheless, from 1999/2006 to 2007/2014, E. coli showed a 3-fold increase in amoxicillin/clavulanate resistance and a 4-fold increase in 3GC resistance. Proteus spp. had an even higher increase in resistance, showing a 5- and 9-fold increase in amoxicillin/clavulanate and 3GC resistance, respectively (Table S4). Proteus spp. and E. coli also had a significant increase in gentamicin resistance (Tables S3 and S4). Detection of MDR Enterobacteriaceae, E. coli and Proteus spp. increased significantly over time (Tables S3 and S4 and Figure 1). Most MDR Enterobacteriaceae (excluding Enterobacter spp.) were susceptible to at least one antimicrobial (Table S5). Gentamicin was the antimicrobial to which most MDR Enterobacteriaceae (excluding Enterobacter spp.) were susceptible (46.2%, CI 36.1%–56.4%). On the contrary, MDR Enterobacteriaceae were seldom susceptible to fluoroquinolones (9.7%, CI 3.7%–15.7%) (Table S5). Furthermore, among MDR E. coli, only 19.35% (CI 9.5%–29.2%) were susceptible to tetracycline. Figure 1. View largeDownload slide Enterobacteriaceae and E. coli resistance trends over the 16 years of the study. Depiction of the temporal trends in resistance obtained by logistic regression over 16 years. (a) Enterobacteriaceae. There was a significant increase (P < 0.05) in antimicrobial resistance to all antimicrobials. (b) E. coli. There was a significant increase (P < 0.05) in antimicrobial resistance to all antimicrobials, except trimethoprim/sulfamethoxazole. AMC, amoxicillin/clavulanate; SXT, trimethoprim/sulfamethoxazole; FQs, fluoroquinolones; GEN, gentamicin; TET, tetracycline. Figure 1. View largeDownload slide Enterobacteriaceae and E. coli resistance trends over the 16 years of the study. Depiction of the temporal trends in resistance obtained by logistic regression over 16 years. (a) Enterobacteriaceae. There was a significant increase (P < 0.05) in antimicrobial resistance to all antimicrobials. (b) E. coli. There was a significant increase (P < 0.05) in antimicrobial resistance to all antimicrobials, except trimethoprim/sulfamethoxazole. AMC, amoxicillin/clavulanate; SXT, trimethoprim/sulfamethoxazole; FQs, fluoroquinolones; GEN, gentamicin; TET, tetracycline. A total of 33 E. coli and 9 P. mirabilis that were 3GC-resistant were recovered and screened for the presence of ESBL and AmpC genes. The first 3GC-resistant E. coli isolate was detected in 1999, yet none of the tested genes were detected. Another resistance mechanism may be involved. In total, only seven 3GC-resistant E. coli were negative for all tested genes, including blaTEM-type and blaSHV-type ESBLs. E. coli 3GC resistance was mainly related to the presence of blaCTX-M-15 and blaCMY-2 (Tables 1 and 2). The first CTX-M-producing E. coli was detected in 2004 and belonged to the O25b:H4-B2-ST131 clonal lineage (Table 1). Besides O25b:H4-B2-ST131, CTX-M-producing E. coli were frequently found to belong to clonal complex (CC) 23, including a novel ST described here (Table 1 and Figure S1). From 2010 onwards, an increase in 3GC-resistant E. coli ST648 harbouring blaCMY-2 was observed (Table 2). 3GC-resistant P. mirabilis was first detected in 2004. All isolates with this phenotype were blaCMY-2 positive, except one P. mirabilis from 2007 possessing blaDHA-1 (Table S6). Table 1. CTX-M-producing E. coli clonal lineages Isolate  Year  β-Lactamase  Clonal lineage  CC  Animal  MDR  AMC  3GCs  SXT  FQs  GEN  TET  FMV5825/04  2004  CTX-M-15  ST131:H4-B2-O25b  CC131  dog  yes  R  R  R  R  R  R  FMV521/07  2007  CTX-M-32  ST224  —  cat  likely  I  R  S  R  S  R  FMV1630/07  2007  CTX-M-15  unassigned STb  CC23  dog  yes  I  R  R  R  S  R  FMV7261/07  2007  CTX-M-32  ST609  CC46  dog  yes  I  R  R  R  S  R  FMV635/08  2008  CTX-M-32  ST23  CC23  cat  yes  R  R  R  R  S  S  FMV2777/08  2008  CTX-M-15  ST131:H4-B2-O25b  CC131  cat  yes  R  R  S  R  R  R  FMV5827/08  2008  CTX-M-15  ST23  CC23  dog  yes  R  R  R  R  S  S  FMV1952/10a  2010  CTX-M-9  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV4479/13a  2013  CTX-M-15  ST533  —  dog  yes  R  R  R  R  S  S  FMV5338/13  2013  CTX-M-15  ST131:H4-B2-O25b  CC131  dog  yes  I  R  S  R  R  R  FMV58/2013  2013  CTX-M-1-type  ST131:H4-B2-O25b  CC131  cat  yes  S  R  R  R  S  R  FMV121/2014RE  2014  CTX-M-1-type  ST539  —  dog  yes  S  R  R  R  S  R  Isolate  Year  β-Lactamase  Clonal lineage  CC  Animal  MDR  AMC  3GCs  SXT  FQs  GEN  TET  FMV5825/04  2004  CTX-M-15  ST131:H4-B2-O25b  CC131  dog  yes  R  R  R  R  R  R  FMV521/07  2007  CTX-M-32  ST224  —  cat  likely  I  R  S  R  S  R  FMV1630/07  2007  CTX-M-15  unassigned STb  CC23  dog  yes  I  R  R  R  S  R  FMV7261/07  2007  CTX-M-32  ST609  CC46  dog  yes  I  R  R  R  S  R  FMV635/08  2008  CTX-M-32  ST23  CC23  cat  yes  R  R  R  R  S  S  FMV2777/08  2008  CTX-M-15  ST131:H4-B2-O25b  CC131  cat  yes  R  R  S  R  R  R  FMV5827/08  2008  CTX-M-15  ST23  CC23  dog  yes  R  R  R  R  S  S  FMV1952/10a  2010  CTX-M-9  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV4479/13a  2013  CTX-M-15  ST533  —  dog  yes  R  R  R  R  S  S  FMV5338/13  2013  CTX-M-15  ST131:H4-B2-O25b  CC131  dog  yes  I  R  S  R  R  R  FMV58/2013  2013  CTX-M-1-type  ST131:H4-B2-O25b  CC131  cat  yes  S  R  R  R  S  R  FMV121/2014RE  2014  CTX-M-1-type  ST539  —  dog  yes  S  R  R  R  S  R  AMC, amoxicillin/clavulanate; SXT, trimethoprim/sulfamethoxazole; FQs, fluoroquinolones; GEN, gentamicin; TET, tetracycline; R, resistant; I, intermediate; S, susceptible. a Also harbours blaCMY-2. b Refer to Figure S1 to see the new ST allelic profile. Table 2. AmpC-producing E. coli clonal lineages Isolate  Year  β-Lactamase  Clonal lineage  CC  Animal  MDR  AMC  3GCs  SXT  FQs  GEN  TET  FMV434/00  2000  CMY-2  ST1775  —  dog  yes  R  R  R  S  S  R  FMV1953/01  2001  CMY-2  ST57  CC350  dog  yes  R  R  R  I  I  R  FMV203/03  2003  CMY-2  ST405  CC405  dog  yes  R  R  S  R  S  R  FMV6346/05  2005  CMY-2  ST539  —  cat  yes  R  R  S  R  R  R  FMV3389/06  2006  CMY-2  ST354  CC354  dog  yes  R  R  R  R  R  R  FMV1952/10a  2010  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV25/2011  2011  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV29/2011  2011  CMY-2  ST648  CC648  dog  yes  R  R  S  R  R  R  FMV469/13  2013  CMY-2  ST648  CC648  dog  yes  R  R  R  R  R  R  FMV1389/13  2013  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV4479/13a  2013  CMY-2  ST533  —  dog  yes  R  R  R  R  S  S  FMV55/2013  2013  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV546/14  2014  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV966/14  2014  CMY-2  ST648  CC648  dog  yes  R  R  R  R  S  R  FMV1549/14  2014  CMY-2  ST648  CC648  cat  yes  R  R  S  R  S  R  FMV43/2014  2014  CMY-2  ST648  CC648  cat  yes  R  R  S  R  S  R  Isolate  Year  β-Lactamase  Clonal lineage  CC  Animal  MDR  AMC  3GCs  SXT  FQs  GEN  TET  FMV434/00  2000  CMY-2  ST1775  —  dog  yes  R  R  R  S  S  R  FMV1953/01  2001  CMY-2  ST57  CC350  dog  yes  R  R  R  I  I  R  FMV203/03  2003  CMY-2  ST405  CC405  dog  yes  R  R  S  R  S  R  FMV6346/05  2005  CMY-2  ST539  —  cat  yes  R  R  S  R  R  R  FMV3389/06  2006  CMY-2  ST354  CC354  dog  yes  R  R  R  R  R  R  FMV1952/10a  2010  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV25/2011  2011  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV29/2011  2011  CMY-2  ST648  CC648  dog  yes  R  R  S  R  R  R  FMV469/13  2013  CMY-2  ST648  CC648  dog  yes  R  R  R  R  R  R  FMV1389/13  2013  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV4479/13a  2013  CMY-2  ST533  —  dog  yes  R  R  R  R  S  S  FMV55/2013  2013  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV546/14  2014  CMY-2  ST648  CC648  cat  yes  R  R  R  R  R  R  FMV966/14  2014  CMY-2  ST648  CC648  dog  yes  R  R  R  R  S  R  FMV1549/14  2014  CMY-2  ST648  CC648  cat  yes  R  R  S  R  S  R  FMV43/2014  2014  CMY-2  ST648  CC648  cat  yes  R  R  S  R  S  R  AMC, amoxicillin/clavulanate; SXT, trimethoprim/sulfamethoxazole; FQs, fluoroquinolones; GEN, gentamicin; TET, tetracycline; R, resistant; I, intermediate; S, susceptible. a Also harbours blaCTX-M. Regarding Staphylococcus spp., 9.2% (CI 4.0%–14.4%, n = 11/119) of Staphylococcus spp. were methicillin resistant and were found to harbour the mecA gene. Overall only resistance to fluoroquinolones was significantly higher in the second time period (2007–14; P = 0.0189) (Table S4). However, if S. pseudintermedius are analysed alone, a significant increase in methicillin (14.8%, CI 1.4%–28.2%, in 2007–14; P = 0.0069) and gentamicin (17.9%, CI 3.7%–32.0%, in 2007–14; P = 0.0099) resistance was also detected. All MDR Staphylococcus spp. were associated with the presence of the mecA gene. Methicillin-resistant Staphylococcus pseudintermedius (MRSP; n = 4) and methicillin-resistant S. epidermidis (MRSE; n = 4) were the most common methicillin-resistant Staphylococcus species, followed by MRSA (n = 2) and methicillin-resistant Staphylococcus lentus (n = 1). Although uncommon, S. epidermidis showed a high frequency of methicillin resistance (n = 4/6). MRSP showed resistance to all the tested antimicrobials, except two that were susceptible to tetracycline. Thus all MRSP were considered MDR. All methicillin-resistant Staphylococcus spp. but one were isolated from cats. The 11 methicillin-resistant Staphylococcus spp. found in this study were fully characterized elsewhere.8 Briefly, MRSP belonged to ST71-II-III (n = 3) and ST196-V (n = 1); MRSE belonged to CC 5 (ST2-nt, ST20-nt, ST23-IV, ST35-nt) and MRSA to CC5 (ST5-t311-VI, ST105-t002-II). Enterococcus spp. showed very high tetracycline (75.8%, CI 65.2%–86.5%, n = 47/62) and fluoroquinolone (56.4%, CI 44.1%–68.8%, n = 35/62) resistance. Ampicillin resistance in Enterococcus spp. (12.1%, CI 4.2%–20.0%, n = 8/66) was lower, though if only E. faecium were considered, almost all were ampicillin resistant (n = 8/9). HLGR was detected in 15.2% (CI 6.1%–24.4%, n = 9/59) of the tested Enterococcus spp. and was mostly found in E. faecalis harbouring aac(6′)-Ieaph(2″)-Ia (Table S7). HLGR E. faecalis belonged to known sequence types, including one isolate from ST6-CC6 (former CC2) (Table S7). All ampicillin-resistant and/or HLGR E. faecium belonged to CC17 (Table S7), including two novel STs (ST1282 and ST1283) identified in this study (Figure S2). Discussion As seen in other studies, E. coli was the most frequently isolated bacterium in dogs and cats with UTI.1,4 Overall, Enterobacteriaceae caused more than half of the UTIs in dogs and cats; therefore, given that β-lactams are among the most important antimicrobials nowadays,10 the great increase detected in this study in Enterobacteriaceae resistance to amoxicillin/clavulanate and 3GCs is worrisome (Figure 1 and Table S4). 3GCs are considered highest-priority critically important antimicrobials for humans,10 and resistance is frequently associated with the production of β-lactamases.3 The increase in 3GC resistance observed in this study was frequently associated with the presence of blaCTX-M-15 and blaCMY-2 in E. coli and blaCMY-2 in P. mirabilis (Tables 1 and 2 and Table S6). ESBL CTX-M-15 is distributed worldwide in E. coli.31 Moreover, E. coli O25b:H4-B2-ST131 is a widespread human uropathogenic clonal lineage that frequently harbours blaCTX-M-1532 and has been previously described in dog and cat faecal samples (as a commensal),12 and also causes UTI.6 Therefore the detection of E. coli O25b:H4-B2-ST131 in this study came as no surprise. Furthermore, E. coli CC23 has been described in humans with UTI in the community33 and has also been shown to be a common CC among CTX-M-producing E. coli.34 This study shows an increase in the detection of CMY-2-producing E. coli and Proteus spp. over time. Although less frequently reported than blaCTX-M in previously published data,6,35 this increase in blaCMY-2 should not be neglected since this enzyme shows stronger β-lactamase activity36 and may in the future become more prevalent. Furthermore, blaCMY-2-carrying Enterobacteriaceae may exhibit resistance to carbapenems in the absence of carbapenemases, owing to the presence of other resistance mechanisms such as porin defiency,37 which further highlights their clinical relevance. In the present study, the first CMY-2-producing E. coli was detected in 2000, yet it was from 2010 onwards that the E. coli CMY-2-producing ST648 clonal lineage was increasingly detected (Table 2). E. coli ST648 has been described in human infections harbouring several β-lactamases, such as ESBL and carbapenemases.31,38 Nevertheless, both in humans and companion animals, the ST648 E. coli clonal lineage has mostly been described harbouring blaCTX-M genes.38,39 The significant increase in ST648 CMY-2-producing E. coli observed in this study in companion animals with UTI may point to the possible expansion of a blaCMY-2-producing MDR ST648 clonal lineage, though more studies are needed to clarify the clonal relatedness between these isolates. However, a few studies have also found a high frequency of ST648 CMY-2-producing E. coli in faecal samples and specimens from infections of companion animals.40,41 Although only detected once in this study, E. coli ST405 also belongs to a highly successful clonal lineage that causes human infection.38 Dogs and cats with UTI are, therefore, shown in this study to be infected with 3GC-resistant E. coli belonging to clonal lineages of great importance to humans. Furthermore, CTX-M- and CMY-producing E. coli and P. mirabilis were also found to be MDR, thus increasing the relevance of these findings. Additionally, Enterobacteriaceae from this study showed a significant increase in resistance to all the antimicrobials commonly used in the treatment of dogs and cats with UTI (Figure 1). Together with the significant increase in detection of MDR Enterobacteriaceae over time, these results point to growing therapeutic limitations in veterinary medicine that in the future may lead to an increasing need to prescribe antimicrobials originally intended for human use. ESBL- and AmpC-producing E. coli presented MDR susceptibility phenotypes with limited therapeutic options (Tables 1 and 2). A study in humans showed that the use of amoxicillin/clavulanate for the treatment of UTIs caused by ESBL-producing E. coli may be suitable if the isolate is fully susceptible to this antimicrobial.42 Although most ESBL-producing E. coli from this study displayed intermediate resistance to amoxicillin/clavulanate, similar studies should be conducted in veterinary medicine to evaluate its effectiveness in the treatment of MDR ESBL-producing Enterobacteriaceae. MDR Enterobacteriaceae were more often susceptible to gentamicin; hence, although sometimes impractical, the use of this antimicrobial should be considered for the treatment of UTIs caused by MDR Enterobacteriaceae in dogs and cats (Table S5). Staphylococcus spp. were the second most frequently isolated bacteria; however, the identified Staphylococcus species varied significantly between dogs and cats (Table S2). The high frequency of S. felis in cats with UTI is in agreement with a previous study conducted in Australia, in which the authors associated the presence of S. felis with clinical signs of lower urinary tract disease in cats.4 The significant increase in the detection of MDR MRSP over time is a concerning finding since it creates major therapeutic limitations. The MRSP detected in this study belonged mainly to ST71-II-III, which is known to be one of the most disseminated clonal lineages in dogs and cats in Europe.43,44 Although rarely, human infection by MRSP ST71-II-III has already been described, thus highlighting its zoonotic potential.45 As seen in reports on humans, S. epidermidis showed a high frequency of methicillin resistance46 and all belonged to STs also found in humans.47 The two MRSA isolated in this study belonged to S. aureus CC5, which is frequently associated with human hospital-acquired MRSA.48 Furthermore, both MRSA STs have been reported in humans from Portugal.49,50 Interestingly, in this study, the mecA gene was mainly detected in Staphylococcus spp. isolated from cats. As this was a retrospective study, it was not possible to obtain information on the possible source of these infections. Nevertheless, the detection of MRSA and MRSE in dogs and cats with UTI is a public health issue since companion animals will have a role in dissemination of these bacteria to the household and public environment. Ampicillin resistance and HLGR in Enterococcus spp. strongly limit the therapeutic options against enterococcal infections.51 Although in this study ampicillin-resistant and/or HLGR Enterococcus spp. were uncommon, some belonged to high-risk CCs associated with hospital-acquired infections in humans, such as E. faecalis CC6 (formerly CC2) and E. faecium CC17 (Table S7).52,53 HLGR was mostly detected in E. faecalis and was caused by the presence of a bifunctional enzyme that is known to also confer resistance to a wide range of aminoglycosides.51,E. faecalis ST6 (CC6) has been previously described in hospitalized patients from Portugal but also in samples from pigs and from hospital waste waters.54 As in this study, the E. faecalis ST16 is a clonal lineage that has been found to frequently harbour the bifunctional enzyme.29,55 Also, E. faecalis ST16 has been previously detected in healthy and hospitalized humans as well as in animals.29,54,55 E. faecium isolates were less frequent than those of E. faecalis, but were more commonly ampicillin resistant (Table S7). A previous study has pointed to healthy dogs as reservoirs of ampicillin-resistant E. faecium CC17 including ST19.56 Besides belonging to CC17, the E. faecium ST19 and ST440 strains isolated in this study are also noteworthy for being simultaneously ampicillin resistant and HLGR. Enterobacteriaceae and Staphylococcus spp. together caused around 79% of the UTIs in dogs and cats. Thus, the increase in fluoroquinolone resistance in both groups of bacteria is of great relevance. Furthermore, Enterococcus spp. also showed high levels of resistance to this antimicrobial class. Bearing in mind fluoroquinolones are considered highest-priority critically important antimicrobials for humans,10 and of great importance in the treatment of pyelonephritis in companion animals,2 judicious use of these antimicrobials should be pursued. The fact that this study relied on samples submitted to the laboratory based on clinical judgement could be considered a bias towards resistance because urine cultures from complicated UTIs may be requested more frequently than from simple uncomplicated UTIs.1 However, if present, this bias was constant throughout the study time frame and therefore the increase in antimicrobial resistance is unequivocal. Only in recent years has the EMA started to publish data about antimicrobial sales for companion animals. Nevertheless, the high β-lactam and fluoroquinolone resistance frequencies detected in this study could be expected since these are the first and second most sold antimicrobials for companion animals in Portugal.57 The high frequency of tick-borne diseases in Portugal that require the use of doxycycline in companion animals could have contributed to the high tetracycline resistance seen in this study. Also, the increase in MDR Enterobacteriaceae may, to some extent, explain the increasing resistance trends observed for all the tested antimicrobials. The limited access to complete data about the epidemiological and clinical history of the dogs and cats infected with high-risk clonal lineages was also a limitation of this study. Nevertheless, since most of these animals were treated at home by their owners, the role of dogs and cats with UTI in the spread of resistant bacteria into the environment should be considered. Furthermore, it has been shown that companion animals may share uropathogenic bacteria with their household members,12 and therefore the role of dogs and cats as reservoirs of high-risk clonal lineages is a concern to veterinary professionals and owners. This study showed that bacteria causing UTI in dogs and cats, especially Enterobacteriaceae, are increasingly resistant to the antimicrobials most widely used in UTI treatment. These bacteria were found to harbour clinically relevant antimicrobial resistance mechanisms and simultaneously belong to high-risk clonal lineages, namely 3GC-resistant E. coli O25b:H4-B2-ST131, CC23 and ST648, MRSA CC5, MRSE CC5, HLGR E. faecalis CC6, and ampicillin/HLGR E. faecium CC17. Therefore, when such resistance mechanisms are suspected based on susceptibility testing, veterinary professionals and owners should be advised to take measures in order to reduce the spread of these bacteria, such as strict hand washing, cleaning of the animals’ living environment as well as adequate faecal disposal. Also, longitudinal studies on the faecal carriage of these resistant high-risk clonal lineages during and after UTI should be conducted to assess the time of carriage and evaluate the extent and duration of the infection control measures that should be taken. Acknowledgements We acknowledge the use of the E. faecium (http://pubmlst.org/efaecium/) and E. faecalis (http://pubmlst.org/efaecalis/) MLST websites hosted by the University of Oxford (Jolley KA, Maiden MC. BMC Bioinformatics 2010; 11: 595) and funded by the Wellcome Trust. Funding This work was supported by FEDER funds through the Programa Operacional Factores de Competitividade—COMPETE and by National funds through the FCT—Fundação para a Ciência e a Tecnologia—CIISA Project (UID/CVT/00276/2013) and PhD grants to A. B. (grant SFRH/BD/113142/2015) and to C. M. (grant SFRH/BD/77886/2011) Transparency declarations None to declare. Supplementary data Tables S1–S7 and Figures S1 and S2 are available as Supplementary data at JAC Online. References 1 Hall JL , Holmes MA, Baine SJ. Prevalence and antimicrobial resistance of canine urinary tract pathogens. Vet Rec  2013; 173: 549. Google Scholar CrossRef Search ADS   2 Weese S , Blondeau JM, Boothe D et al.   Antimicrobial use guidelines for treatment of urinary tract disease in dogs and cats: Antimicrobial Guidelines Working Group of the International Society for Companion Animal Infectious Diseases. Vet Med Int  2011; 2011: 263768. Google Scholar CrossRef Search ADS   3 ECDC. Antimicrobial Resistance Surveillance in Europe 2014. Annual Report of the European Antimicrobial Resistance Surveillance Network (EARS-Net) . Stockholm: ECDC, 2015. 4 Litster A , Moss SM, Honnery M et al.   Prevalence of bacterial species in cats with clinical signs of lower urinary tract disease: recognition of Staphylococcus felis as a possible feline urinary tract pathogen. Vet Microbiol  2007; 121: 182– 8. Google Scholar CrossRef Search ADS   5 Féria C , Ferreira E, Correia JD et al.   Patterns and mechanisms of resistance to β-lactams and β-lactamase inhibitors in uropathogenic Escherichia coli isolated from dogs in Portugal. J Antimicrob Chemother  2002; 49: 77– 85. Google Scholar CrossRef Search ADS   6 Ewers C , Grobbel M, Stamm I et al.   Emergence of human pandemic O25:H4-ST131 CTX-M-15 extended-spectrum-β-lactamase-producing Escherichia coli among companion animals. J Antimicrob Chemother  2010; 65: 651– 60. Google Scholar CrossRef Search ADS   7 Wagner S , Gally DL, Argyle SA. Multidrug-resistant Escherichia coli from canine urinary tract infections tend to have commensal phylotypes, lower prevalence of virulence determinants and ampC-replicons. Vet Microbiol  2014; 169: 171– 8. Google Scholar CrossRef Search ADS   8 Couto N , Monchique C, Belas A et al.   Trends and molecular mechanisms of antimicrobial resistance in clinical staphylococci isolated from companion animals over a 16 year period. J Antimicrob Chemother  2016; 71: 1479– 87. Google Scholar CrossRef Search ADS   9 Jackson CR , Fedorka-Cray PJ, Davis JA et al.   Mechanisms of antimicrobial resistance and genetic relatedness among enterococci isolated from dogs and cats in the United States. J Appl Microbiol  2010; 108: 2171– 9. 10 WHO. Critically Important Antimicrobials for Human Medicine—5th Revision. 2017. http://apps.who.int/iris/bitstream/10665/255027/1/9789241512220-eng.pdf. 11 Pomba C , Rantala M, Greko C et al.   Public health risk of antimicrobial resistance transfer from companion animals. J Antimicrob Chemother  2017; 72: 957– 68. 12 Johnson JR , Miller S, Johnston B et al.   Sharing of Escherichia coli sequence type ST131 and other multidrug-resistant and urovirulent E. coli strains among dogs and cats within a household. J Clin Microbiol  2009; 47: 3721– 5. Google Scholar CrossRef Search ADS   13 Bartges JW. Diagnosis of urinary tract infections. Vet Clin North Am Small Anim Pract  2004; 34: 923– 33. Google Scholar CrossRef Search ADS   14 Doumith M , Day MJ, Hope R et al.   Improved multiplex PCR strategy for rapid assignment of the four major Escherichia coli phylogenetic groups. J Clin Microbiol  2012; 50: 3108– 10. Google Scholar CrossRef Search ADS   15 Kovtunovych GL , Lytvynenko T, Negrutska VV et al.   A PCR-mediated method for discrimination of Klebsiella oxytoca between closely related bacteria in environmental and clinical specimens. Biopolym Cell  2003; 19: 520– 4. Google Scholar CrossRef Search ADS   16 Padmavathy B , Vinoth Kumar R, Patel A et al.   Rapid and sensitive detection of major uropathogens in a single-pot multiplex PCR assay. Curr Microbiol  2012; 65: 44– 53. Google Scholar CrossRef Search ADS   17 Stankowska D , Kwinkowski M, Kaca W. Quantification of Proteus mirabilis virulence factors and modulation by acylated homoserine lactones. J Microbiol Immunol Infect  2008; 41: 243– 53. 18 Woodford N , Egelton CM, Morrison D. Comparison of PCR with phenotypic methods for the speciation of enterococci. Adv Exp Med Biol  1997; 418: 405– 8. Google Scholar CrossRef Search ADS   19 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals—Fourth Edition: Approved Standard VET01-A4 . CLSI, Wayne, PA, USA, 2013. 20 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Disk and Dilution Susceptibility Tests for Bacteria Isolated from Animals: Second Informational Supplement VET01-S2 . CLSI, Wayne, PA, USA, 2013. 21 Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Sixth Informational Supplement M100-S26 . CLSI, Wayne, PA, USA, 2016. 22 Pérez-Pérez FJ , Hanson ND. Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol  2002; 40: 2153– 62. Google Scholar CrossRef Search ADS   23 Guessennd N , Bremont S, Gbonon V et al.   Résistance aux quinolones de type qnr chez les entérobactéries productrices de bêta-lactamases à spectre élargi à Abidjan en Côte d’Ivoire. Pathol Biol (Paris)  2008; 56: 439– 46. Google Scholar CrossRef Search ADS   24 Belas A , Salazar AS, Telo da Gama L et al.   Risk factors for faecal colonisation with Escherichia coli producing extended-spectrum and plasmid-mediated AmpC β-lactamases in dogs. Vet Rec  2014; 175: 202. Google Scholar CrossRef Search ADS   25 Kao SJ , You I, Clewell DB et al.   Detection of the high-level aminoglycoside resistance gene aph(2")-Ib in Enterococcus faecium. Antimicrob Agents Chemother  2000; 44: 2876– 9. Google Scholar CrossRef Search ADS   26 Johnson J , Clermont O, Johnstone B et al.   Rapid and specific detection, molecular epidemiology, and experimental virulence of the O16 subgroup within Escherichia coli sequence type 131. J Clin Microbiol  2014; 52: 1358– 65. Google Scholar CrossRef Search ADS   27 Wirth T , Falush D, Lan R et al.   Sex and virulence in Escherichia coli: an evolutionary perspective. Mol Microbiol  2006; 60: 1136– 51. Google Scholar CrossRef Search ADS   28 Homan WL , Tribe D, Poznanski S et al.   Multilocus sequence typing scheme for Enterococcus faecium. J Clin Microbiol  2002; 40: 1963– 71. Google Scholar CrossRef Search ADS   29 Ruiz-Garbajosa P , Bonten MJ, Robinson DA et al.   Multilocus sequence typing scheme for Enterococcus faecalis reveals hospital-adapted genetic complexes in a background of high rates of recombination. J Clin Microbiol  2006; 44: 2220– 8. Google Scholar CrossRef Search ADS   30 Magiorakos AP , Srinivasan A, Carey RB et al.   Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect  2012; 18: 268– 81. Google Scholar CrossRef Search ADS   31 Ewers C , Bethe A, Semmler T et al.   Extended-spectrum β-lactamase-producing 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. Google Scholar CrossRef Search ADS   32 Nicolas-Chanoine MH , Blanco J, Leflon-Guibout V et al.   Intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15. J Antimicrob Chemother  2008; 61: 273– 81. Google Scholar CrossRef Search ADS   33 Lau SH , Reddy S, Cheesbrough J et al.   Major uropathogenic Escherichia coli strain isolated in the northwest of England identified by multilocus sequence typing. J Clin Microbiol  2008; 46: 1076– 80. Google Scholar CrossRef Search ADS   34 Brisse S , Diancourt L, Laouénan C et al.   Phylogenetic distribution of CTX-M- and non-extended-spectrum-β-lactamase-producing Escherichia coli isolates: group B2 isolates, except clone ST131, rarely produce CTX-M enzymes. J Clin Microbiol  2012; 50: 2974– 81. Google Scholar CrossRef Search ADS   35 Aragón LM , Mirelis B, Miró E et al.   Increase in β-lactam-resistant Proteus mirabilis strains due to CTX-M- and CMY-type as well as new VEB- and inhibitor-resistant TEM-type β-lactamases. J Antimicrob Chemother  2008; 61: 1029– 32. Google Scholar CrossRef Search ADS   36 Jacoby GA , Amp C. β-lactamases. Clin Microbiol Rev  2009; 22: 161– 82. Google Scholar CrossRef Search ADS   37 Chia JH , Siu LK, Su LH et al.   Emergence of carbapenem-resistant Escherichia coli in Taiwan: resistance due to combined CMY-2 production and porin deficiency. J Chemother  2009; 21: 621– 6. Google Scholar CrossRef Search ADS   38 Pitout JDD. Extraintestinal pathogenic Escherichia coli: a combination of virulence with antibiotic resistance. Front Microbiol  2012; 3: 9. Google Scholar CrossRef Search ADS   39 Ewers C , Bethe A, Stamm I et al.   CTX-M-15-D-ST648 Escherichia coli from companion animals and horses: another pandemic clone combining multiresistance and extraintestinal virulence? J Antimicrob Chemother  2014; 69: 1224– 30. Google Scholar CrossRef Search ADS   40 Tamang MD , Nam HM, Jang GC et al.   Molecular characterization of extended-spectrum-β-lactamase-producing and plasmid-mediated AmpC β-lactamase-producing Escherichia coli isolated from stray dogs in South Korea. Antimicrob Agents Chemother  2012; 56: 2705– 12. Google Scholar CrossRef Search ADS   41 Liu X , Thungrat K, Boothe DM. Occurrence of OXA-48 carbapenemase and other β-lactamase genes in ESBL-producing multidrug resistant Escherichia coli from dogs and cats in the United States, 2009–2013. Front Microbiol  2016; 7: 1057. 42 Beytur A , Yakupogullari Y, Oguz F et al.   Oral amoxicillin-clavulanic acid treatment in urinary tract infections caused by extended-spectrum β-lactamase-producing organisms. Jundishapur J Microbiol  2015; 8: e13792. 43 Kadlec K , Schwarz S, Perreten V et al.   Molecular analysis of methicillin-resistant Staphylococcus pseudintermedius of feline origin from different European countries and North America. J Antimicrob Chemother  2010; 65: 1826– 8. Google Scholar CrossRef Search ADS   44 Perreten V , Kadlec K, Schwarz S et al.   Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study. J Antimicrob Chemother  2010; 65: 1145– 54. Google Scholar CrossRef Search ADS   45 Stegmann R , Burnens A, Maranta CA et al.   Human infection associated with methicillin-resistant Staphylococcus pseudintermedius ST71. J Antimicrob Chemother  2010; 65: 2047– 8. Google Scholar CrossRef Search ADS   46 Becker K , Heilmann C, Peters G. Coagulase-negative staphylococci. Clin Microbiol Rev  2014; 27: 870– 926. Google Scholar CrossRef Search ADS   47 Rolo J , de Lencastre H, Miragaia M. Strategies of adaptation of Staphylococcus epidermidis to hospital and community: amplification and diversification of SCCmec. J Antimicrob Chemother  2012; 67: 1333– 41. Google Scholar CrossRef Search ADS   48 Aires-de-Sousa M. Methicillin-resistant Staphylococcus aureus among animals: current overview. Clin Microbiol Infect  2017; 23: 373– 80. Google Scholar CrossRef Search ADS   49 Conceição T , Tavares A, Miragaia M et al.   Prevalence and clonality of methicillin-resistant Staphylococcus aureus (MRSA) in the Atlantic Azores islands: predominance of SCCmec types IV, V and VI. Eur J Clin Microbiol Infect Dis  2010; 29: 543– 50. Google Scholar CrossRef Search ADS   50 Espadinha D , Faria NA, Miragaia M et al.   Extensive dissemination of methicillin-resistant Staphylococcus aureus (MRSA) between the hospital and the community in a country with a high prevalence of nosocomial MRSA. PLoS One  2013; 8: e59960. Google Scholar CrossRef Search ADS   51 Chow JW. Aminoglycoside resistance in enterococci. Clin Infect Dis  2000; 31: 586– 9. Google Scholar CrossRef Search ADS   52 Leavis HL , Bonten MJ, Willems RJ. Identification of high-risk enterococcal clonal complexes: global dispersion and antibiotic resistance. Curr Opin Microbiol  2006; 9: 454– 60. Google Scholar CrossRef Search ADS   53 Kuch A , Willems RJ, Werner G et al.   Insight into antimicrobial susceptibility and population structure of contemporary human Enterococcus faecalis isolates from Europe. J Antimicrob Chemother  2012; 67: 551– 8. Google Scholar CrossRef Search ADS   54 Freitas AR , Novais C, Ruiz-Garbajosa P et al.   Clonal expansion within clonal complex 2 and spread of vancomycin-resistant plasmids among different genetic lineages of Enterococcus faecalis from Portugal. J Antimicrob Chemother  2009; 63: 1104– 11. Google Scholar CrossRef Search ADS   55 Larsen J , Schønheyder HC, Lester CH et al.   Porcine-origin gentamicin-resistant Enterococcus faecalis in humans, Denmark. Emerg Infect Dis  2010; 16: 682– 4. Google Scholar CrossRef Search ADS   56 Damborg P , Top J, Hendrickx AP et al.   Dogs are a reservoir of ampicillin-resistant Enterococcus faecium lineages associated with human infections. Appl Environ Microbiol  2009; 75: 2360– 5. Google Scholar CrossRef Search ADS   57 EMA. Sales of Veterinary Antimicrobial Agents in 29 European Countries in 2014. 2016. http://www.ema.europa.eu/docs/en_GB/document_library/Report/2016/10/WC500214217.pdf. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Journal

Journal of Antimicrobial ChemotherapyOxford University Press

Published: Feb 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off