Oxidative stress response plays a role in antibiotic tolerance of Streptococcus mutans biofilms

Antibiotic mode-of-action classification is based upon drug-target interaction and whether the resultant inhibition of cellular function is lethal to bacteria. Here we show that the three major classes of bactericidal antibiotics, regardless of drug-target interaction, stimulate the production of highly deleterious hydroxyl radicals in Gram-negative and Gram-positive bacteria, which ultimately contribute to cell death. We also show, in contrast, that bacteriostatic drugs do not produce hydroxyl radicals. We demonstrate that the mechanism of hydroxyl radical formation induced by bactericidal antibiotics is the end product of an oxidative damage cellular death pathway involving the tricarboxylic acid cycle, a transient depletion of NADH, destabilization of iron-sulfur clusters, and stimulation of the Fenton reaction. Our results suggest that all three major classes of bactericidal drugs can be potentiated by targeting bacterial systems that remediate hydroxyl radical damage, including proteins involved in triggering the DNA damage response, e.g., RecA.

Biofilms are surface-attached microbial communities with characteristic architecture and phenotypic and biochemical properties distinct from their free-swimming, planktonic counterparts. One of the best-known of these biofilm-specific properties is the development of antibiotic resistance that can be up to 1,000-fold greater than planktonic cells. We report a genetic determinant of this high-level resistance in the Gram-negative opportunistic pathogen, Pseudomonas aeruginosa. We have identified a mutant of P. aeruginosa that, while still capable of forming biofilms with the characteristic P. aeruginosa architecture, does not develop high-level biofilm-specific resistance to three different classes of antibiotics. The locus identified in our screen, ndvB, is required for the synthesis of periplasmic glucans. Our discovery that these periplasmic glucans interact physically with tobramycin suggests that these glucose polymers may prevent antibiotics from reaching their sites of action by sequestering these antimicrobial agents in the periplasm. Our results indicate that biofilms themselves are not simply a diffusion barrier to these antibiotics, but rather that bacteria within these microbial communities employ distinct mechanisms to resist the action of antimicrobial agents.

Streptococcus mutans is the leading cause of dental caries (tooth decay) worldwide and is considered to be the most cariogenic of all of the oral streptococci. The genome of S. mutans UA159, a serotype c strain, has been completely sequenced and is composed of 2,030,936 base pairs. It contains 1,963 ORFs, 63% of which have been assigned putative functions. The genome analysis provides further insight into how S. mutans has adapted to surviving the oral environment through resource acquisition, defense against host factors, and use of gene products that maintain its niche against microbial competitors. S. mutans metabolizes a wide variety of carbohydrates via nonoxidative pathways, and all of these pathways have been identified, along with the associated transport systems whose genes account for almost 15% of the genome. Virulence genes associated with extracellular adherent glucan production, adhesins, acid tolerance, proteases, and putative hemolysins have been identified. Strain UA159 is naturally competent and contains all of the genes essential for competence and quorum sensing. Mobile genetic elements in the form of IS elements and transposons are prominent in the genome and include a previously uncharacterized conjugative transposon and a composite transposon containing genes for the synthesis of antibiotics of the gramicidin/bacitracin family; however, no bacteriophage genomes are present.