No abstract

In this study, we investigated the antimicrobial susceptibility profiles and the distribution of some well known genetic determinants of virulence in clinical isolates of Salmonella enterica from New Mexico. The minimum inhibitory concentrations (MICs) for various antimicrobials were determined by using the E-test strip method according to CLSI guidelines. Virulence genotyping was performed by polymerase chain reaction (PCR) using primers specific for known virulence genes of Salmonella enterica. Of 15 isolates belonging to 11 different serovars analyzed, one isolate of Salmonella Typhimurium was resistant to multiple drugs namely ampicillin, amoxicillin / clavulanic acid, chloramphenicol and tetracycline, that also harbored class 1 intergron, bla TEM encoding genes for β-lactamase, chloramphenicol acetyl transferase (cat1), plus floR, tet(C) and tet(G). This strain was phage typed as DT104. PCR analysis revealed the presence of invA, hilA, stn, agfA and spvR virulence genes in all the isolates tested. The plasmid-borne pefA gene was absent in 11 isolates, while 5 isolates lacked sopE. One isolate belonging to serogroup E4 (Salmonella Sombre) was devoid of multiple virulence genes pefA, iroB, shdA and sopE. These results demonstrate that clinical Salmonella serotypes from New Mexico used here are predominantly sensitive to multiple antimicrobial agents, but vary in their virulence genotypes. Information on antimicrobial sensitivity and virulence genotypes will help in understanding the evolution and spread of epidemic strains of Salmonella enterica in the region of study.

MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) is a key enzyme involved in bacterial cell wall peptidoglycan synthesis and a target for the antimicrobial agent fosfomycin, a structural analog of the MurA substrate phosphoenol pyruvate. In this study, we identified, cloned and sequenced a novel murA gene from an environmental isolate of Vibrio fischeri that is naturally resistant to fosfomycin. The fosfomycin resistance gene was isolated from a genomic DNA library of V. fischeri. An antimicrobial agent hypersensitive strain of Escherichia coli harbouring murA from V. fischeri exhibited a high fosfomycin resistance phenotype, with an MIC of 3000 µg/ml. The cloned murA gene was 1269 bp long encoding a 422 amino acid polypeptide with an estimated pI of 5.0. The deduced amino acid sequence of the putative protein was identified as UDP-Nacetylglucosamine enolpyruvyl transferase by homology comparison. The MurA protein with an estimated molecular weight of 44.7 kDa was expressed in E. coli and purified by affinity chromatography. MurA of V. fischeri will be a useful target to identify potential inhibitors of fosfomycin resistance in pharmacological studies.

The purpose of this study was to examine the sugar recognition and transport properties of the sucrose permease (CscB), a secondary active transporter from Escherichia coli. We tested the hypothesis that maltose transport is conferred by the wild-type CscB transporter. Cells of E. coli HS4006 harboring pSP72/cscB were red on maltose MacConkey agar indicator plates. We were able to measure "downhill" maltose transport and establish definitive kinetic behavior for maltose entry in such cells. Maltose was an effective competitor of sucrose transport in cells with CscB, suggesting that the respective maltose and sucrose binding sites and translocation pathways through the CscB channel overlap. Accumulation ("uphill" transport) of maltose by cells with CscB was profound, demonstrating active transport of maltose by CscB. Sequencing of cscB encoded on plasmid pSP72/ cscB used in cells for transport studies indicate an unaltered primary CscB structure, ruling out the possibility that mutation conferred maltose transport by CscB. We conclude that maltose is a bona fide substrate for the sucrose permease of E. coli. Thus, future studies of sugar binding, transport and permease structure should consider maltose, as well as sucrose.

SUMMARYWe inoculated pasteurized whole milk with E. coli strains GC4468 (intact marRAB locus), JHC1096 (Δ marRAB), or AG112 (Δ marR), and incubated each overnight at 37°C. All strains were then recovered from the milk cultures, and susceptibilities to antimicrobial agents were determined by the E-test strip method (CLSI). Cells of strain GC4468, prior to culturing in milk, were susceptible to trimethoprim, gatifloxacin, cefotaxime and tetracycline. After culturing GC4468 in pasteurized milk, however, the MICs increased 1.4-fold for trimethoprim (P ≤ 0.05), 1.5-fold for gatifloxacin (P ≤ 0.05), 2.0-fold for cefotaxime (P = 0.008), and 1.4-fold for tetracycline (P ≥ 0.05). After culturing GC4468 on milk count agar the MICs were enhanced 3.4-fold for trimethoprim (P ≤ 0.05), 10-fold for gatifloxacin (P = 0.001), 7.1-fold for cefotaxime (P = 0.011), and 40.5-fold for tetracycline (P = 0.074), but exhibiting tetracycline resistance with a mean MIC of 74.7 ± 18.47 µg/ml (CLSI). The MICs of the antimicrobial agents for JHC1096 cells after culturing in pasteurized whole milk were indistinguishable (P ≥ 0.05) from baseline MICs measured before culturing in the same type of milk. Thus, E. coli cells harboring the marRAB locus exhibit reduced susceptibilities to multiple antimicrobial agents after culturing in pasteurized whole milk.Infectious diseases caused by bacteria that are resistant to antimicrobial agents are a serious public health concern (Levy & Marshall, 2004;Barbosa & Levy, 2000b;Neu, 1992). Members of the Enterobacteriaceae family of bacteria are causative agents of infectious disease, and their resistance to antimicrobial agents compromises chemotherapeutic efforts (Paterson, 2002). Antimicrobial agents are used in agriculture for the treatment of infection, prophylaxis and growth promotion (Levy & Marshall, 2004;Levy, 2002).Bacterial resistance to antimicrobial agents may be of clinical significance due to dissemination of pathogenic bacteria through a population of food animals (McDermott et al., 2002;Angulo et al., 2004;Silbergeld et al., 2008). In isolated instances, outbreaks of food-borne infectious disease from contaminated milk and occurrences of milk-derived isolates of members from the Enterobacteriaceae family of bacteria have been studied (Gillespie et al., 2003). Although relatively well-studied within dairy cattle (Hershberger et al., 2005), bacteria that are resistant to antimicrobial agents in other dairy farm environments, such as soil (Burgos et al., 2005), water (Biyela et al., 2004) and milk (Makovec & Ruegg, 2003) are less well-characterized. Dissemination mechanisms for drug resistant bacterial pathogens within dairy farms are also poorly understood (Hershberger et al., 2005 NIH-PA Author ManuscriptThe marRAB locus is a well studied genetic element in Escherichia coli and mediates bacterial resistance to multiple antimicrobial agents such as β-lactams, chloramphenicol, quinolones, and tetracycline (Randall & Woodward, 2002). Several organic compounds are known to modulate resistance to...