Evaluation of a fluorescence resonance energy transfer quantitative polymerase chain reaction assay for identification of gyrA mutations conferring enrofloxacin resistance in canine urinary Escherichia coli isolates and canine urine specimens.

OBJECTIVE To evaluate a fluorescence resonance energy transfer quantitative PCR (FRET-qPCR) assay for detection of gyrA mutations conferring fluoroquinolone resistance in canine urinary Escherichia coli isolates and canine urine specimens. SAMPLE 264 canine urinary E coli isolates and 283 clinical canine urine specimens. PROCEDURES The E coli isolates were used to validate the FRET-qPCR assay. Urine specimens were evaluated by bacterial culture and identification, isolate enrofloxacin susceptibility testing, and FRET-qPCR assay. Sensitivity and specificity of the FRET-qPCR assay for detection of gyrA mutations in urine specimens and in E coli isolated from urine specimens were computed, with results of enrofloxacin susceptibility testing used as the reference standard. RESULTS The validated FRET-qPCR assay discriminated between enrofloxacin-resistant and enrofloxacin-susceptible E coli isolates with an area under the receiver operating characteristic curve of 0.92. The assay accurately identified 25 of 40 urine specimens as containing enrofloxacin-resistant isolates (sensitivity, 62.5%) and 226 of 243 urine specimens as containing enrofloxacin-susceptible isolates (specificity, 93.0%). When the same assay was performed on E coli isolates recovered from these specimens, sensitivity (77.8%) and specificity (94.8%) increased. Moderate agreement was achieved between results of the FRET-qPCR assay and enrofloxacin susceptibility testing for E coli isolates recovered from urine specimens. CONCLUSIONS AND CLINICAL RELEVANCE The FRET-qPCR assay was able to rapidly distinguish between enrofloxacin-resistant and enrofloxacin-susceptible E coli in canine clinical urine specimens through detection of gyrA mutations. Therefore, the assay may be useful in clinical settings to screen such specimens for enrofloxacin-resistant E coli to avoid inappropriate use of enrofloxacin and contributing to antimicrobial resistance.

Quality assessment of fluconazole capsules and oral suspensions compounded by pharmacies located in the United States.

OBJECTIVE To evaluate pharmaceutical characteristics (strength or concentration, accuracy, and precision), physical properties, and bacterial contamination of fluconazole compounded products. SAMPLE Fluconazole compounded products (30- and 240-mg capsules; 30- and 100-mg/mL oral suspensions) from 4 US veterinary compounding pharmacies. PROCEDURES Fluconazole compounded products were ordered 3 times from each of 4 pharmacies at 7- or 10-day intervals. Generic fluconazole products (50- and 200-mg tablets; 10- and 40-mg/mL oral suspensions) served as references. Compounded products were evaluated at the time of receipt; suspensions also were evaluated 3 months later and at beyond-use dates. Evaluations included assessments of strength (concentration), accuracy, precision, physical properties, and bacterial contamination. Acceptable accuracy was defined as within ± 10% of the labeled strength (concentration) and acceptable precision as within ± 10%. Fluconazole was quantified by use of high-performance liquid chromatography. RESULTS Physical characteristics of compounded products differed among pharmacies. Aerobic bacterial cultures yielded negative results. Capsules (30 and 240 mg) had acceptable accuracy (median, 96.3%; range, 87.3% to 135.2%) and precision (mean ± SD, 7.4 ± 6.0%). Suspensions (30 and 100 mg/mL) had poor accuracy (median, 73.8%; range, 53.9% to 95.2%) and precision (mean ± SD, 15.0 ± 6.9%). Accuracy and precision were significantly better for capsules than for suspensions. CONCLUSIONS AND CLINICAL RELEVANCE Fluconazole compounded products, particularly suspensions, differed in pharmaceutical and physical qualities. Studies to evaluate the impact of inconsistent quality on bioavailability or clinical efficacy of compounded fluconazole products are indicated, and each study should include data on the quality of the compounded product evaluated.

Occurrence of OXA-48 Carbapenemase and Other ß-Lactamase Genes in ESBL-Producing Multidrug Resistant Escherichia coli from Dogs and Cats in the United States, 2009-2013.

OBJECTIVE: The aim of this study was to explore the occurrence and molecular characterization of extended-spectrum ß-lactamases (ESBL), plasmid-mediated AmpC ß-lactamase (pAmpC) and carbapenemases among ESBL-producing multidrug resistant (MDR) Escherichia coli from dogs and cats in the United States. METHODS: Of 2443 E.coli isolated from dogs and cats collected between August 2009 and January 2013, 68 isolates were confirmed as ESBL-producing MDR ones. PCR and sequencing were performed to identify ß-lactamases and plasmid-mediated quinolone resistance (PMQR) genes, and shed light on the virulence gene profiles, phylogenetic groups and ST types. RESULTS: Phylogenic group D and B2 accounted for 69.1% of the isolates. 50 (73.5%) isolates carried CTX-M ESBL gene, and the most predominant specific CTX-M subtype identified was bla CTX-M-15 (n = 33), followed by bla CTX-M-1 (n = 32), bla CTX-M-123 (n = 27), bla CTX-M-9 (n = 19) and bla CTX-M-14 (n = 19), and bla CTX-M-123 was firstly reported in E. coli isolates in the United States alone or in association. Other ß-lactamase genes bla TEM, bla SHV, bla OXA-48, and bla CMY-2 were detected in 41.2, 29.4, 19.1, and 17.6% of 68 ESBL-producing MDR isolates, respectively. The bla TEM and bla SHV genes were classfied as ESBLs with the exception of the bla TEM-1 gene. Additionally, 42.6% (29/68) of isolates co-expressed bla CTX-M-15 and PMQR gene aac(6')-Ib-c. The overall occurrence of virulence genes ranged from 11.8 (ireA) to 88.2% (malX), and most of virulence genes were less frequent among CTX-M-producing isolates than non-CTX-M isolates with the exception of malX and iutA. The 68 isolates analyzed were assigned to 31 STs with six being novel. Three pandemic clonal lineages ST131 (n = 10), ST648 (n = 9), and ST405 (n = 9) accounted for more than 41% of the investigated isolates, and ST648 and ST405 of phylogenetic D were firstly reported in E. coli from dogs and cats in the United States. CONCLUSION: bla CTX-M-123 of ESBLs and carbapenemase bla OXA-48 were firstly reported in ESBL-producing MDR E.coli from dogs and cats in the United States, and ST131, ST648, and ST405 were the predominant clonal groups.

Multilocus Sequence Typing and Virulence Profiles in Uropathogenic Escherichia coli Isolated from Cats in the United States.

The population structure, virulence, and antimicrobial resistance of uropathogenic E. coli (UPEC) from cats are rarely characterized. The aim of this study was to compare and characterize the UPEC isolated from cats in four geographic regions of USA in terms of their multilocus sequence typing (MLST), virulence profiles, clinical signs, antimicrobial resistance and phylogenetic grouping. The results showed that a total of 74 E. coli isolates were typed to 40 sequence types with 10 being novel. The most frequent phylogenetic group was B2 (n = 57). The most frequent sequence types were ST73 (n = 12) and ST83 (n = 6), ST73 was represented by four multidrug resistant (MDR) and eight non-multidrug resistant (SDR) isolates, and ST83 were significantly more likely to exhibit no drug resistant (NDR) isolates carrying the highest number of virulence genes. Additionally, MDR isolates were more diverse, and followed by SDR and NDR isolates in regards to the distribution of the STs. afa/draBC was the most prevalent among the 29 virulence-associated genes. Linking virulence profile and antimicrobial resistance, the majority of virulence-associated genes tested were more prevalent in NDR isolates, and followed by SDR and MDR isolates. Twenty (50%) MLST types in this study have previously been associated with human isolates, suggesting that these STs are potentially zoonotic. Our data enhanced the understanding of E. coli population structure and virulence association from cats. The diverse and various combinations of virulence-associated genes implied that the infection control may be challenging.

Antimicrobial susceptibility patterns of clinical Escherichia coli isolates from dogs and cats in the United States: January 2008 through January 2013.

Escherichia coli is among the most common bacterial pathogens in dogs and cats. The lack of a national monitoring program limits evidence-based empirical antimicrobial choices in the United States. This study describes antimicrobial susceptibility patterns for presumed clinical E. coli isolates from dogs (n=2392) or cats (n=780) collected from six geographic regions in the United States between May 2008 and January 2013. Minimum inhibitory concentrations (MIC) were determined for 17 drugs representing 6 drug classes. Urinary tract isolates were most common (71%). Population MIC distributions were generally bimodal with the second mode above the resistant breakpoint for all drugs except gentamicin, amikacin, and meropenem. The MIC90 exceeded the susceptible breakpoint for ampicillin, amoxicillin-clavulanic acid, cephalothin (surrogate drug for cephalexin), and doxycycline but was below the susceptible breakpoint for all others. None of isolates was susceptible or resistant to all drug tested; 46% were resistant to 1 or 2 antimicrobial categories, and 52% to more than three categories. The resistance percentages were as follows: doxycycline (100%), cephalothin (98%)>ampicillin (48%)>amoxicillin-clavulanic acid (40%)>ticarcillin-clavulanic acid (18%)>cefpodoxime (13%), cefotaxime (12%), cefoxitin (11%), cefazolin (11%), enrofloxacin (10%), chloramphenicol (9.6%)>ciprofloxacin (9.2%), ceftazidime (8.7%), trimethoprim-sulfamethoxazole (7.9%), gentamicin (7.9%)>meropenem (1.5%), amikacin (0.7%) (P<0.05). Resistance to ampicillin and amoxicillin-clavulanic acid was greatest in the South-Central region (P<0.05). E. coli resistance may preclude empirical treatment with doxycycline, cephalexin, ampicillin, or amoxicillin-clavulanic acid. Based on susceptibility patterns, trimethoprim-sulfonamides may be the preferred empirical oral treatment.

In vitro selection of resistance to pradofloxacin and ciprofloxacin in canine uropathogenic Escherichia coli isolates.

This study explored and compared the mechanisms and selective concentration of resistance between a 3rd (pradofloxacin) and 2nd (ciprofloxacin) generation fluoroquinolone. Pradofloxacin- and ciprofloxacin-resistant mutants were selected by stepwise exposure of Escherichia coli (E. coli) to escalating concentrations of pradofloxacin and ciprofloxacin. The sequence of the quinolone resistance determining region (QRDR) and the transcriptional regulator soxS were analyzed, and efflux pump AcrAB-TolC activity was measured by quantitative real-time reverse transcription-PCR (qRT-PCR). First-step mutants reduced the fluoroquinolone sensitivity and one mutant bore a single substitution in gyrA. Four of six second-step mutants expressed ciprofloxacin resistance, and displayed additional mutations in gyrA and/or parC, while these mutants retained susceptibility to pradofloxacin. All the third-step mutants were fluoroquinolone resistant, and each expressed multidrug resistance (MDR) phenotypes. Further, they displayed resistance to all antibacterials tested except cefotaxime, ceftazidime and meropenem. The number of mutations in QRDR of gyrA and parC correlated with fluoroquinolone MICs. Mutations in parC were not common in pradofloxacin-associated mutants. Moreover, one second- and one third-step ciprofloxacin-associated mutants bore both mutations at position 12 (Ala12Ser) and 78 (Met78Leu) in the soxS gene, yet no mutations in the soxS gene were detected in the pradofloxacin-selected mutants. Altogether, these results demonstrated that resistance emerged relatively more rapidly in 2nd compared to 3rd generation fluoroquinolones. Point mutations in gyrA were a key mechanism of resistance to pradofloxacin, and overexpression of efflux pump gene acrB played a potential role in the emergence of MDR phenotypes identified in this study.

Uterine bacterial isolates from mares and their resistance to antimicrobials: 8,296 cases (2003-2008).

OBJECTIVE: To describe bacteria isolated from reproductive tracts of mares and to examine the extent and patterns of resistance to antimicrobials commonly used for treatment of endometritis. DESIGN: Retrospective case series. SAMPLE: 8,296 uterine swab, lavage, or biopsy samples obtained between January 2003 and December 2008 from 7,665 horses in central Florida. PROCEDURES: Results of bacterial culture and antimicrobial susceptibility testing were obtained for uterine swab, lavage, and biopsy samples collected from mares undergoing a routine breeding examination or examined because of a reproductive disorder. Bacterial organisms were identified by means of standard techniques, and proportions of samples resistant to various antimicrobials were determined. RESULTS: At least 95% of samples (n = 1,451) were collected with uterine swabs. Potentially pathogenic organisms were cultured from 2,576 (31%) samples, with Escherichia coli (n = 729 [29%]) and ß-hemolytic Streptococcus equi subsp zooepidemicus (733 [28%]) being most common. Resistance to antimicrobials changed over time for E coli, S equi subsp zooepidemicus, and Enterobacteriaceae isolates. Overall, E coli was most resistant to trimethoprim-sulfonamide and ampicillin and least to amikacin and enrofloxacin. For S equi subsp zooepidemicus, resistance was greatest to oxytetracycline and enrofloxacin and least to ceftiofur and ticarcillin with or without clavulanic acid. Inflammatory response was greater for S equi subsp zooepidemicus. CONCLUSIONS AND CLINICAL RELEVANCE: E coli and S equi subsp zooepidemicus were the most common pathogens recovered from uterine samples, with S equi subsp zooepidemicus more commonly associated with inflammation. Antimicrobials most commonly used empirically to treat endometritis are appropriate on the basis of these data. However, as antimicrobial resistance changes over time, susceptibility assays should aid antimicrobial selection.

In vitro potency and efficacy favor later generation fluoroquinolones for treatment of canine and feline Escherichia coli uropathogens in the United States.

Information regarding in vitro activity of newer fluoroquinolones (FQs) is limited despite increasing resistance in canine or feline pathogenic Escherichia coli (E. coli). This study describes in vitro potency and efficacy toward E. coli of seven FQs grouped according to similarities in chemical structure: enrofloxacin, ciprofloxacin, orbifloxacin (first-group), levofloxacin, marbofloxacin (second-group) and pradofloxacin, moxifloxacin (third-group; latest S, S-pyrrolidino-piperidine at C-7). Potency measures included minimum inhibitory concentration (MIC) (geometric mean MIC, MIC(50), MIC(90)); and mutant prevention concentration (MPC) for FQ susceptible isolates only. In vitro efficacy measures included relative susceptibility (MIC(BP-S):MIC) or resistance (MIC:MIC(BP-R)) and mutant selection window (MSW) (MPC:MIC). For enrofloxacin susceptible isolates, mean MIC (µg/ml) was least for each third-group drug and ciprofloxacin and greatest for enrofloxacin and orbifloxacin (P = 0.006). For enrofloxacin susceptible isolates, MPC were below MIC:MIC(BP-R) and least for pradofloxacin (0.29 ± 0.16 µg/ml) and greatest for enrofloxacin (1.55 ± 0.55 µg/ml) (P = 0.006). MSW was least for pradofloxacin (55 ± 30) and greatest for ciprofloxacin (152 ± 76) (P = 0.0024). MIC(BP-S):MIC was greatest (P = 0.025) for pradofloxacin (190.1 ± 0.61) and least for enrofloxacin (23.53 ± 0.83). For FQ susceptible isolates, FQs MIC:MIC(BP-R) may serve as a surrogate for MPC. Because in vitro efficacy was greatest for pradofloxacin; it might be preferred for treatment of urinary tract infections (UTIs) associated with FQ susceptible E. coli uropathogens.

Mechanisms accounting for fluoroquinolone multidrug resistance Escherichia coli isolated from companion animals.

Multidrug resistance (MDR) is associated with fluoroquinolone (FQ) resistance in companion animal Escherichia coli (E. coli). In this study, gyrA, gyrB, parC, and parE quinolone resistance determining regions (QRDR) were sequenced among uropathogenic E. coli isolates with different resistant phenotypes. Also determined were porin, efflux pump and regulatory gene expression based on quantitative real-time reverse transcriptase PCR (qRT-PCR), the impact of efflux pump inhibition (Phe-Arg-ß-naphthylamide) and the presence of plasmid-mediated quinolone resistance (PMQR). Using enrofloxacin as the prototypic FQ, we found that (i) the number of mutations in target genes correlate well with minimum inhibitory concentrations (MICs). A single mutation (Ser83Leu) in gyrA increases FQ MIC in susceptible isolates; subsequent mutations result in resistance that increases from low (enrofloxacin MICs 4-16 µg/ml) to high level (enrofloxacin MICs≥128 µg/ml) with each progressive mutation. (ii) as MIC increase, acrB activity and the number of drug classes contributing to the MDR phenotype increases; (iii) a consistent relationship between regulatory gene expression and MIC could not be identified; and (iv) qnrS and aac(6')-Ib-cr gene were detected in 14 and 5 ENR(R)-MDR isolates containing the target mutation, respectively. Of 13 isolates expressing PDR isolates, 10 (77%) were positive for qnrS gene, and 4 (40%) carried both qnrS and aac(6')-Ib-cr gene. These findings demonstrated that MDR-associated FQ resistance in canine and feline uropathogenic E. coli reflects a combination of point mutations, enhanced efflux pump activities, and PMQR mechanisms. Point mutations in DNA gyrase, however, are necessary to achieve a clinical level of FQ resistance.