Unravelling Multiple Antibiotic Resistance in Salmonella Enterica

<p>Non-typhoidal Salmonella enterica (NTS) are major human pathogens. The morbidity of NTS infections is ~60% and the fatality rate of NTS ~1-4%. For the elderly and very young this rate is increased, especially in the developing world where the numbers of invasive strains of NTS are increasing; antibiotic resistance increases rates 2 fold. Chronic and extra-intestinal infections require antibiotic therapy, but the numbers of antibiotic resistant NTS are increasing making therapy problematic. Bacteria can be multiply antibiotic resistant (MAR) via the constitutive over-expression of chromosomally encoded efflux pumps. Several publications indicate that over expression of E. coli marA or soxS, and/or the efflux pump AcrAB-TolC system are associated with MAR, however our work shows that the same is not always true for NTS.</p>

<p>We will examine four hypotheses:</p>

<ol>
<li>MAR can be due to over expression of one or more efflux pumps possibly in synergy with an OMP involved with influx, mediated by over expression of a transcriptional activator;</li>
<li>ramA is the transcriptional activator that mediates MAR;</li>
<li>MAR can be chromosomally mediated by two mechanisms, only one of which also confers cyclohexane tolerance;</li>
<li>Efflux mediated MAR occurs in high numbers of naturally occurring antibiotic resistant NTS.</li>
</ol>

<p>We predict that there are two mechanisms of resistance of MAR: altered gene expression or mutation conferring a change in transporter substrate specificity.</p>

<p>Two strategies to identify the cause of MAR will be pursued, one using microarrays to determine the gene expression profile of stable constitutive isogenic MAR mutants compared with the parent strain, SL1344; the second strategy will clone MAR from two mutants. The regulon of ramA will also be explored. Genes that are differentially expressed specific to phenotype will be disrupted and then complemented to determine their role, if any, in MAR. Even if differential expression is observed, it may not lead to easily identifiable genes for disruption thereby necessitating cloning of MAR into pBLUESCRIPT and electroporation into E. coli DH5 alpha. Representative MAR clones will be transformed into SL1344 and MAR genes characterised. Finally, human and animal isolates of NTS will be screened to determine the prevalence of the identified mechanism(s) of MAR.</p>