Growth as a biofilm facilitates the emergence of antibiotic resistance by mutation in Staphylococcus aureus . Here we demonstrate that biofilm growth of this species also dramatically increases horizontal transfer of plasmid-borne antibiotic resistance determinants by conjugation/mobilization and that standard laboratory practices to induce conjugation in staphylococci achieve optimal efficiency owing to the presence of a biofilm.

To investigate the development of mutational resistance to antibiotics in staphylococcal biofilms. Mutation frequencies to resistance against mupirocin and rifampicin were determined for planktonic cultures and for biofilms generated using either a novel static biofilm model or by continuous flow. DNA microarray analysis was performed to detect differences in transcriptional profiles between planktonic and biofilm cultures. The mutability of biofilm cultures increased up to 60-fold and 4-fold for S. aureus and S. epidermidis, respectively, compared with planktonic cultures. Incorporation of antioxidants into S. aureus biofilms reduced mutation frequencies, indicating that increased oxidative stress underlies the heightened mutability. Transcriptional profiling of early biofilm cultures revealed up-regulation of the superoxide dismutase gene, sodA, also suggestive of enhanced oxidative stress in these cultures. The addition of catalase to biofilms of S. aureus SH1000 reduced mutation frequencies, a finding which implicated hydrogen peroxide in increased biofilm mutability. However, catalase had no effect on biofilm mutability in S. aureus UAMS-1, suggesting that there is more than one mechanism by which the mutability of staphylococci may increase during the biofilm mode of growth. Our findings suggest that biofilms represent an enriched source of mutational resistance to antibiotics in the staphylococci.

The novel bacterial topoisomerase inhibitor class is an investigational type of antibacterial inhibitor of DNA gyrase and topoisomerase IV that does not have cross-resistance with the quinolones. Here, we report the evaluation of the in vitro properties of a new series of this type of small molecule. Exemplar compounds selectively and potently inhibited the catalytic activities of Escherichia coli DNA gyrase and topoisomerase IV but did not block the DNA breakage-reunion step. Compounds showed broad-spectrum inhibitory activity against a wide range of Gram-positive and Gram-negative pathogens, including biodefence microorganisms and Mycobacterium tuberculosis . No cross-resistance with fluoroquinolone-resistant Staphylococcus aureus and E. coli isolates was observed. Measured MIC ₉₀ values were 4 and 8 μg/ml against a panel of contemporary multidrug-resistant isolates of Acinetobacter baumannii and E. coli , respectively. In addition, representative compounds exhibited greater antibacterial potency than the quinolones against obligate anaerobic species. Spontaneous mutation rates were low, with frequencies of resistance typically <10 ⁻⁸ against E. coli and A. baumannii at concentrations equivalent to 4-fold the MIC. Compound-resistant E. coli mutants that were isolated following serial passage were characterized by whole-genome sequencing and carried a single Arg38Leu amino acid substitution in the GyrA subunit of DNA gyrase. Preliminary in vitro safety data indicate that the series shows a promising therapeutic index and potential for low human ether-a-go-go-related gene (hERG) inhibition (50% inhibitory concentration [IC ₅₀ ], >100 μM). In summary, the compounds' distinct mechanism of action relative to the fluoroquinolones, whole-cell potency, low potential for resistance development, and favorable in vitro safety profile warrant their continued investigation as potential broad-spectrum antibacterial agents.

The biofilm mode of growth can lead to diversification of the bacterial population by promoting the emergence of variants. Here we report the identification and characterization of two major subpopulations of morphological variants arising in biofilms of S. aureus . One of these lacked pigmentation (termed white variants; WVs), whilst the other formed colonies on agar that were larger and paler than the parental strain (termed large pale variants; LPVs). WVs were unable to form biofilms, and exhibited increased proteolysis and haemolysis; all phenotypes attributable to loss-of-function mutations identified in the gene encoding the alternative sigma factor, sigB. For LPVs, no differences in biofilm forming capacity or proteolysis were observed compared with the parental strain. Genetic analysis of LPVs revealed that they had undergone mutation in the accessory gene regulator system ( agrA ), and deficiency in agr was confirmed by demonstrating loss of both colony spreading and haemolytic activity. The observation that S. aureus biofilms elaborate large subpopulations of sigB and agr mutants, both genotypes that have independently been shown to be of importance in staphylococcal disease, has implications for our understanding of staphylococcal infections involving a biofilm component.

Objectives The objective of this study was to characterize the in vitro and in vivo biological properties of a novel series of small-molecule bacterial type IIA topoisomerase inhibitors. Methods Bacterial susceptibility testing was performed by broth microdilution. Resistance frequencies were determined by plating bacteria onto agar containing test compound and enumerating mutants. Bacteria were passaged using subinhibitory concentrations of antibacterials to generate resistance. Target enzyme inhibition was determined by exposure to antibacterials and DNA; topoisomers were visualized by gel electrophoresis. Oral and intravenous pharmacokinetic profiles were determined in mice. In vivo efficacy was determined using a mouse model of septicaemia and thigh infection with MSSA and MRSA, respectively. Results Representative compounds REDX04139, REDX05604 and REDX05931 demonstrated in vitro potency against a range of Gram-positive and fastidious Gram-negative pathogens. Clinical isolate testing revealed REDX04139 and REDX05931 had MIC₉₀ values of 0.25 and 0.5 mg/L, respectively, for MRSA and MIC₉₀ values of 2 mg/L for streptococci. REDX04139 was bactericidal in vitro against Staphylococcus aureus at 8× MIC over 6 h. Pharmacokinetic profiling of REDX04139 and REDX05604 in mice revealed low clearance and excellent bioavailability (≥71%). REDX04139 provided 100% survival against S. aureus in a mouse septicaemia model, while REDX05604 reduced bacterial load by up to 3.7 log units in the MRSA mouse thigh infection model. Conclusions Redx Pharma has discovered a novel series of topoisomerase inhibitors that are being further developed for drug-resistant bacteria.