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.

Identification, antimicrobial resistance profiles, and virulence of members from the family Enterobacteriaceae from the feces of yellow-headed blackbirds (Xanthocephalus xanthocephalus) in North Dakota.

Public pressure to reduce or eliminate antimicrobials as ingredients of feed for poultry and other agricultural animals is mounting, primarily due to the fear of multidrug-resistant bacteria in clinical infections in both animals and humans. Exploration of the occurrence of antibiotic resistance in the gut flora of wildlife avian flocks that presumptively do not receive antimicrobials will determine the rate of resistance in a naïve population. Fecal samples collected from a healthy population of the yellow-headed blackbirds (YHB) (Xanthocephalus xanthocephalus) in North Dakota were cultured to determine what genera and species of gram-negative facultative anaerobic bacteria these wild birds carry in their intestinal flora and to evaluate the antimicrobial susceptibility profiles. Isolates of Escherichia coli were further characterized for the presence of putative virulence factors and for pathogenic potential using the chicken embryo lethality assay (ELA). The ELA was performed in chicken embryos with challenges at both 12 days and 16 days of incubation to determine whether the 16-day-old embryos were better able to fight the infection and subsequent disease and also to determine whether the ELA could distinguish between primary and secondary avian Escherichia coli pathogens. After screening 33 isolates from the 21 fecal samples, only two E. coli isolates were identified. The predominant genus and species of bacterium identified was Pantoea agglomerans. Collectively, 12 of the 33 isolates (36%) exhibited no resistance to any antimicrobial tested. However, several multidrug-resistant isolates of varying genera were identified. Among the antimicrobial resistances observed, the most common was to ampicillin (60%), followed by cephalothin (33%). Neither E. coli isolate belonged to serogroups that are notorious for causing major outbreaks of colibacillosis in poultry, and only one E. coli isolate retained resistance to any antibiotics; nevertheless, the ELA results indicate that at least one of these E. coli may be a primary pathogen of chickens. This study demonstrates that antibiotic resistance occurs in the gut flora of natural populations of YHB despite the absence of antibiotic pressure. In addition, these results indicate that YHB will harbor E. coli isolates that are potentially pathogenic in poultry. However, these E. coli isolates are not a significant reservoir for multiple antibiotic resistances nor are they widespread in the population of YHB surveyed in North Dakota.