Unravelling multiple antibiotic resistance in Salmonella enterica

We are performing experiments that will allow us to understand how salmonella bacteria become multi-drug (antibiotic) resistant (MDR) after exposure to antibiotics.
Every year people are exposed to salmonella and some people get ill. The very old and young are particularly vulnerable to this infection and they may be so sick that they need antibiotics to help them recover. Treatment of antibiotic resistant salmonella can be difficult.
One way in which bacteria can resist antibiotic action is to pump the drug out of the cell. This is why some bacteria are always resistant to some drugs. Some MDR salmonella produce even more of these pumps than normal strains, making these MDR strains additionally resistant to antibiotics. Whilst we know the identity of some of these pumps, we do not know what controls when they are produced, or what makes the bacterium produce more.
New methods allow us to look at what happens to bacteria when we have removed or ‘knocked out’ the pump. Other methods allow us to both inactivate, and label, the antibiotic resistance. By doing this the DNA sequence, and hence the gene/s involved, are found.
Understanding how MDR is caused will allow us to develop screening tests to quickly identify this type of resistance. Inhibitors of the pumps or genes that control their production can also be developed.

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. We will examine four hypotheses (i) 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; (ii) ramA is the transcriptional activator that mediates MAR; (iii) MAR can be chromosomally mediated by two mechanisms, only one of which also confers cyclohexane tolerance; and (iv) Efflux mediated MAR occurs in high numbers of naturally occurring antibiotic resistant NTS. We predict that there are two mechanisms of resistance of MAR: altered gene expression or mutation conferring a change in transporter substrate specificity. 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.