Prevalence and antimicrobial susceptibility of virulent and avirulent multidrug-resistant Escherichia coli isolated from diarrheic neonatal calves.

OBJECTIVE: To determine the prevalence of selected virulence genes and the antimicrobial susceptibility of multidrug-resistant (MDR) Escherichia coli isolated from diarrheic neonatal calves. SAMPLE: 97 E coli isolates from diarrheic neonatal calves. PROCEDURES: E coli isolates were tested via PCR assay for 6 virulence genes and susceptibility to 17 drugs belonging to 9 classes. A 2-sample test of proportions was used to make comparisons between proportions of virulent and avirulent MDR isolates. RESULTS: 23 of 97 (23.7%) isolates were virulent, and 74 (76.3%) were avirulent. Of the 23 virulent isolates, 15 (65.2%) were positive for K99, 14 (60.9%) for F41, 12 (52.2%) for STa, 9 (39.1%) for Stx1, 6 (26.1%) for intimin, and 0 (0%) for Stx2. Twenty of 23 (87.0%) virulent isolates expressed ≥ 2 virulence genes, and 3 of 23 (13.0%) were positive for 1 virulence factor. Eight of 23 (34.8%) virulent isolates expressed STa, K99, and F41, whereas 1 of 23 (4.4%) was positive for STa, F41, intimin, and Stx1. The second most frequent gene pattern was Stx1 and intimin. Twenty of 23 (87.0%) virulent isolates were MDR; the highest prevalence of resistance was recorded for the macrolide-lincosides, followed by the tetracyclines and penicillins. Also, 17 of 23 (74.0%) virulent isolates were resistant to sulfadimethoxine, and 10 of 23 (43.5%) were resistant to trimethoprim-sulfamethoxazole. Additionally, 60 of 74 (81.0%) avirulent isolates were MDR. CONCLUSIONS AND CLINICAL RELEVANCE: The prevalence of multidrug resistance was comparable for virulent and avirulent E coli isolated from diarrheic neonatal calves. Cephalosporins and aminoglycosides had reasonable susceptibility.

Proteomic analysis of multidrug resistant Escherichia coli strains from scouring calves.

A number of researchers have used chemical inhibitors that target membrane efflux pumps as an experimental treatment strategy for multidrug resistant (MDR) bacterial infections. However, most of these compounds are toxic in vertebrate animals. The present research was therefore done to describe expression dynamics of drug resistance-associated Escherichia coli proteins that could serve as novel drug targets. Proteomes of MDR and antimicrobial susceptible (AS) E. coli were studied in two dimensional (2-D) polyacrylamide gels and liquid chromatography-mass spectrometry (LC-MS) was performed on proteins of interest. The number of recovered peptides per protein was used to elucidate the amounts of target proteins expressed in MDR and AS E. coli strains. Eight proteins that may be potentially involved in mechanisms of drug resistance were analyzed and identified by LC-MS. These were grouped into membrane porins (TolC, OmpA, OmpC, Nmpc Precursor), proteins involved in microbial protein synthesis (EF-Ts, EF-Tu, RpsA) and Dps, a protein of unknown location and function. Experimental data demonstrated variability in the expression patterns and quantities of the four porins (TolC, OmpA, OmpC, Nmpc precursor), the three microbial protein synthesis associated proteins (EF-Ts, EF-Tu and RpsA), and Dps which has been previously associated with drug resistance. While variability was seen in quantities and expression patterns of some of the proteins of interest, the present data falls short of determining the suitability of these proteins as novel drug targets. Further studies are required to explore how these proteins could be targeted for drug development.

Molecular analysis of porin gene transcription in heterogenotypic multidrug-resistant Escherichia coli isolates from scouring calves.

BACKGROUND: Despite evidence that altered membrane porins may impair microbial drug uptake thereby potentially compounding efflux pump-mediated multidrug resistance, few studies have evaluated gene transcription to identify multidrug-resistance-associated porins and other potential drug targets. METHODS: Genes that encode six membrane porins (fadL, lamB, ompC, ompF, ompW and yiaT) and two membrane proteins (tolC and ompT) were assessed by PCR and by quantitative real-time PCR (qRT-PCR) analysis of 10 multidrug-resistant (MDR) and 10 antibiotic-susceptible (AS) Escherichia coli isolates. The mean DeltaDeltaCt values for the study E. coli genes were analysed by the Wilcoxon test (P = 0.05). RESULTS: All 20 E. coli isolates tested positive for tolC, lamB, ompC, ompF genes, while 10 MDR and 9/10 (90%) AS isolates were positive for the fadL gene. Seven out of 10 (70%) MDR and 7/10 (70%) AS isolates were positive for the yiaT gene, while 7/10 (70%) MDR and only 4/10 (40%) AS isolates were positive for the ompT gene. The mean DeltaDeltaCt values for the tolC and yiaT genes were significantly higher in MDR than in AS isolates (Wilcoxon test; P < 0.05). No significant difference was seen with respect to fadL, lamB, ompC, ompF, ompT and ompW gene transcription (Wilcoxon test; P > 0.05). CONCLUSIONS: Findings suggest up-regulated transcription of tolC and yiaT genes in the MDR E. coli isolates. These results indirectly suggest that TolC and YiaT proteins may play some role(s) in multidrug resistance, but proteomic studies are needed before the two proteins are considered potential drug targets.