The Biological Burden of Antibiotic Resistance in Staphylococcus aureus

Antibiotic resistance is a major problem. Resistance is often mediated by genes on bacterial plasmids and dissemination of resistance is commonly achieved by plasmid transfer. The mechanisms of resistance that are presently understood suggest that resistance is energetically costly. If this is generally true, resistance would reduce the fitness of cells in the absence of antibiotics. We examined the hypothesis that resistance reduces the fitness of microorganism and requires continuous selection to be maintained. To test this hypothesis, we measured the effects of plasmid-borne resistance genes on cell growth by comparing the growth rates of Staphylococcus aureus with and without 10 plasmids. Plasmid-ffee cells were mixed with equal numbers of plasmid-containing cells and propagated serially through 10 cultures for 5 days without antibiotics. The ratio of sensitive to resistant cells was measured at regular intervals by plating. The results show that plasmids reduce the growth rates of a sensitive laboratory strain, 8325-4 and also the native MRSA strain from which the plasmids were obtained. Because of its growth reducing effect we selected plasmid pS A2 to test our secondary hypothesis that the resistance function rather than the biological cost of maintaining plasmids causes most of the growth rate reduction. Transposon mutagenesis was used to create randomly distributed mutations saturating the plasmid. We analyzed a major portion of the plasmid by enrichment experiments where 113 mutated plasmids were mixed in equal proportions and grown serially for 28 days, through 56 transfers. Using restriction analysis and DNA sequencing, we found that the final population was highly enriched for mutations in a 3 kb region of the plasmid corresponding to the plactamase operon and two ORFs with homology to the abortive infection protein AbiK of Lactococcus lactis. Pairwise competition experiments show that 8325-4 grows considerably faster carrying pS A2 with blaZ mutations than it does with unmutated pSA2 and almost as rapidly as 8325-4 with no plasmid at all. We conclude that the biological function of blaZ impedes the growth of cells. This is significant since p-lactamase, unlike most resistance genes, is not known to affect the cellular apparatus for DNA, RNA, or protein production