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. 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Journal of Antimicrobial Chemotherapy – Oxford University Press
Published: Feb 1, 2018
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