FULL PAPER Bacteriology
Antimicrobial resistance of Escherichia coli isolates from canine urinary tract infections Shao-Kuang CHANG1), Dan-Yuan LO2), Hen-Wei WEI3) and Hung-Chih KUO2)* 1)Graduate
Institute of Veterinary Medicine, National Taiwan University, Taipei 106, Taiwan, ROC of Veterinary Medicine, National Chiayi University, Chiayi, 600, Taiwan, ROC 3)Department of Animal Science and Technology, National Taiwan University, Taipei 106, Taiwan, ROC 2)Department
(Received 1 June 2013/Accepted 17 September 2014/Published online in J-STAGE 28 October 2014) ABSTRACT. This study determined the antimicrobial resistance profiles of Escherichia coli isolates from dogs with a presumptive diagnosis of urinary tract infection (UTI). Urine samples from 201 dogs with UTI diagnosed through clinical examination and urinalysis were processed for isolation of Escherichia coli. Colonies from pure cultures were identified by biochemical reactions (n=114) and were tested for susceptibility to 18 antimicrobials. The two most frequent antimicrobials showing resistance in Urinary E. coli isolates were oxytetracycline and ampicillin. Among the resistant isolates, 17 resistance patterns were observed, with 12 patterns involving multidrug resistance (MDR). Of the 69 tetracycline-resistant E. coli isolates, tet(B) was the predominant resistance determinant and was detected in 50.9% of the isolates, whereas the remaining 25.5% isolates carried the tet(A) determinant. Most ampicillin and/or amoxicillin-resistant E. coli isolates carried blaTEM-1 genes. Class 1 integrons were prevalent (28.9%) and contained previously described gene cassettes that are implicated primarily in resistance to aminoglycosides and trimethoprim (dfrA1, dfrA17-aadA5). Of the 44 quinolone-resistant E. coli isolates, 38 were resistant to nalidixic acid, and 6 were resistant to nalidixic acid, ciprofloxacin and enrofloxacin. Chromosomal point mutations were found in the GyrA (Ser83Leu) and ParC (Ser80Ile) genes. Furthermore, the aminoglycoside resistance gene aacC2, the chloramphenicol resistant gene cmlA and the florfenicol resistant gene floR were also identified. This study revealed an alarming rate of antimicrobial resistance among E. coli isolates from dogs with UTIs. KEY WORDS: antimicrobial resistance, class 1 integrons, urinary Escherichia coli
doi: 10.1292/jvms.13-0281; J. Vet. Med. Sci. 77(1): 59–65, 2015
Escherichia coli (E. coli) is the most important causative bacterium of urinary tract infections (UTIs) in both humans and dogs, and strains of these species are often abundant in the gastrointestinal tract at the time of infection [7, 24, 26, 29]. In past studies of canine UTIs, the majority of bacterial isolates were E. coli (33–56%) [2, 12, 20, 29, 34, 39]. Historically, a range of antimicrobial agents has been used to treat UTIs in veterinary medicine, including penicillins, cephalosporins, tetracyclines, chloramphenicols, aminoglycosides, fluoroquinolones and potentiated sulfonamides. The use of antimicrobial drugs has been associated with an increasing trend of antimicrobial resistance among canine E. coli isolates over the last decade [33]. Thus, the management of UTIs in dogs has become more complicated, as the prevalence of antibiotic-resistant strains of E. coli has increased. In addition, the frequent close physical contact between dogs and humans increases the potential for transmission of resistant bacteria between companion animals and humans, as well as the potential for exchange or transfer of antimicrobialresistant genes to human pathogens [22]. The objectives of this study were to determine (i) the frequency of canine uro*Correspondence to: Kuo, H. J., Department of Veterinary Medicine, National Chiayi University, No. 580, Xinmin Rd., Chiayi City 60054, Taiwan.e-mail:
[email protected] ©2015 The Japanese Society of Veterinary Science
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License .
pathogenic E. coli at the National Taiwan University Veterinary Hospital (NTUVH) and the National Chiayi University Veterinary Teaching Hospital (NCYUVTH), (ii) the percentage of antimicrobial resistance and (iii) the distribution and mechanisms of bacterial resistance to β-lactams, chloramphenicols, tetracyclines, quinolones, aminoglycosides and sulfamethoxazole-trimethoprim. The results should provide useful information for veterinarians in the management of persistent or recurrent UTI in dogs. MATERIALS AND METHODS Sampling methods: Urine samples were obtained from 201 non-medicated adult dogs (aged >1 y) of both sexes with a presumptive diagnosis of UTI from July 2010 to June 2011. The inclusion criteria included clinical signs of UTI, such as hematuria and dysuria, urinalysis results that included red blood cell counts >5 under a high-power field (HPF) and proteinuria, and >103 colony-forming units (CFU) of bacteria per milliliter of urine at the first plating. Urine samples were obtained by catheterization after thorough cleansing of the genital area. Bacterial isolation, culture conditions and identification: The fresh urine samples were refrigerated temporarily and were cultured using a calibrated pipette to deliver 10 µl and 100 µl of samples onto Columbia agar supplemented with 5% sheep blood and onto MacConkey agar (Becton Dickinson Microbiology Systems, Cockeysville, MD, U.S.A.) upon receipt. The blood agar plates were incubated with
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5% CO2, and the MacConkey agar plates were incubated aerobically. All samples were incubated at 37°C for 18 to 24 hr until adequate growth was present. Primary plates were carefully inspected for colonies of E. coli, which were plated onto sheep blood agar plates; these plates were incubated at 37°C for 24 hr. Suspected colonies were identified as E. coli using standard techniques, including indole, Methyl red– Voges-Proskauer (MR-VP) and citrate biochemical testing and analysis with an API-20E system (bioMérieux, Marcy l’Etoile, France) [17]. Antimicrobial susceptibility testing: Susceptibility was tested quantitatively by broth microdilution with cationadjusted Mueller-Hinton broth, according to Clinical and Laboratory Standards Institute (CLSI) guidelines [9]. Eight drug classes were included in this study. These classes represented the most frequently used antibiotics for cases of UTI in our community. Tetracyclines were represented by oxytetracycline and doxycycline. Aminoglycosides were represented by gentamicin and amikacin. Quinolones were represented by nalidixic acid, enrofloxacin and ciprofloxacin. Penicillins were represented by the amoxicillin-clavulanic acid combination, amoxicillin and ampicillin. Lastly, cephalosporins were represented by cefazolin, ceftiofur, ceftazidime and cefotaxime. Sulfamethoxazole-trimethoprim, colistin, florfenicol and chloramphenicol were also tested. E. coli ATCC 25922, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 were used as control strains. Bacterial DNA preparation, PCR assays and DNA sequencing: PCR analyses for preselected resistant genes were performed for all of the isolates. The total DNA of E. coli isolates was extracted by using an InstaGene DNA Purification Matrix kit (Bio-Rad, Hercules, CA, U.S.A.) according to the manufacturer’s instructions. PCR primers specific to these resistant genes were designed based on the gene sequence information from previously published studies [5, 28, 32, 42]. PCR was performed in a 20 µl mixture containing 1 µl of template DNA, 0.5 µl of each primer (10 nmol l−1), 10 µl of 2× PCR Mix (Fermentas, MBI) and 8 µl of ddH2O. PCR was performed using 30 cycles of denaturation at 94°C and amplification at 72°C for 10 min. Amplicons were visualized by electrophoresis at 100 V in a 2% agarose gel. Amplified PCR products were purified with a QIAquick PCR purification Kit (Qiagen, Valencia, CA, U.S.A.) and sequenced by ABI 3730 × l capillary sequencers (Applied Biosystems, Foster City, CA, U.S.A.). The DNA sequences were compared using the BLAST online search engine from GenBank at the National Center for Biotechnology Information website (http://www. ncbi.nlm.nih.gov/blast) [1]. The gyrA, gyrB, parC and parE genes were amplified by PCR using the primers and PCR conditions described previously [16]. Both strands of the purified PCR products were sequenced, and the results were compared with the sequences of wild-type E. coli gyrA (NCBI X06373), gyrB (P06982), parC (P20082) and parE (P20083). Comparisons were performed using NCBI BLAST, the ClustalW Multiple Sequence Alignment program and the Lasergene sequence analysis software package (version 4.0, DNASTAR, Madison, WI, U.S.A.) [44].
Statistical analysis: The statistical tests used were the chisquare test and Fisher’s exact test, which were performed using the Mixed Procedure in SAS (version 8.2; SAS Institute, Inc., Cary, NC, U.S.A.). P1024
0 8.7 0 0 0 0 60.9 56.5 0 43.5 34.8 0 0 0 43.5 43.5
4 1 2 1 1 0.5 16 8 8/4 8 4 1 1024 >1024
0 13.3 0 0 0 0 33.3 26.7 6.7 13.3 26.7 0 13.3 13.3 33.3 20
2 1 2 1 2 0.5 16 16 8/4 8 4 1 0.125 0.125 8 2/38
8 64 8 2 2 1 >1024 >1024 16/8 512 16 2 0.25 1 1024 >1024
0 10.5 0 0 0 0 50 44.7 2.6 31.6 31.6 0 5.3 5.3 38.6 34.2
8 256
128 1024
39.1 73.9
2 8
32 1024
26.7 40
4 8
128 1024
28.9 60.5
a) MIC breakpoints according to CLSI guidelines. b) Percentage of resistant isolates. c) Because the MIC breakpoints for UTIs were not provided in the 2007 CLSI guidelines, the values for bovine respiratory pathogens were used [10].
Table 2. Distribution of antimicrobial drug resistance patterns among the 114 uropathogenic E. coli strains Resistance pattern
No. of isolates
AMP AUG FLO NA CHL, FLO AMP, NA, SUL CHL, NA, OTC AMO, AMP, CHL, OTC AMO, AMP, GEN, SUL DOX, GEN, NA, OTC AMO, AMP, CHL, FLO, OTC AMO, AMP, CIP, EN, NA AMO, AMP, DOX, NA, OTC, SUL AMO, AMP, CHL, DOX, GEN, OTC, SUL AMO, AMP, DOX, FLO, NA, OTC, SUL AMO, AMP, CHL, DOX, FLO, NA, OTC, SUL AMO, AMP, CHL, CIP, DOX, EN, FLO, NA, OTC, SUL
3 3 9 5 6 3 3 9 6 3 3 3 9 3 6 9 3
AMO, amoxicillin; AMP, ampicillin; AUG, amoxicillin-clavulanic acid; CHL, chloramphenicol; CIP, ciprofloxacin; DOX, doxycycline; EN, enrofloxacin; FLO, florfenicol; GEN, gentamicin; NA, nalidixic acid; OTC, oxytetracycline; SUL, sulfamethoxazole-trimethoprim.
The fluoroquinolone-resistant isolates (n=6) had 2 point mutations, one in GyrA (Ser-83 to Leu) and one in ParC (Ser-80 to Ile) (Table 3). No mutations were detected in the known resistance regions of GyrB or ParE. Nalidixic acid-resistant isolates (n=44) with a low level of MICs for ciprofloxacin (
