Rifampicin enhances activity of daptomycin and vancomycin against both a polysaccharide intercellular adhesin (PIA)-dependent and -independent Staphylococcus epidermidis biofilm.

OBJECTIVES AND METHODS: This study addressed the efficacy of daptomycin, vancomycin, rifampicin, daptomycin/rifampicin and vancomycin/rifampicin against a polysaccharide intercellular adhesin (PIA)-dependent and -independent Staphylococcus epidermidis biofilm using flow cell and guinea pig tissue cage models. RESULTS: The flow cell model of both PIA-dependent and -independent biofilms demonstrated that the viable cell count after treatment with daptomycin/rifampicin was significantly lower (P<0.05) than after treatment with vancomycin, vancomycin/rifampicin, daptomycin or rifampicin alone. To validate these observations, a guinea pig tissue cage model was used. The results demonstrated that the addition of rifampicin to daptomycin or vancomycin sterilized 5/6 tissues cages colonized with S. epidermidis 1457 (PIA producing). Similar results were noted with S. epidermidis 1457 icaADBC::dhfr (non-PIA producing), where daptomycin/rifampicin and vancomycin/rifampicin sterilized 5/6 and 6/6 tissue cages, respectively. There was no statistical difference in comparison with the no-treatment control when both 1457 and 1457 icaADBC::dhfr were treated with vancomycin and daptomycin alone. Furthermore, treatment with rifampicin alone sterilized 5/6 and 3/6 1457 and 1457 icaADBC::dhfr tissue cages, respectively. CONCLUSIONS: Interpretation of these data suggests that rifampicin is highly active against S. epidermidis biofilms and both vancomycin and daptomycin are effective at reducing the subpopulation of bacteria that develop rifampicin resistance.

A staphylococcal GGDEF domain protein regulates biofilm formation independently of cyclic dimeric GMP.

Cyclic dimeric GMP (c-di-GMP) is an important biofilm regulator that allosterically activates enzymes of exopolysaccharide biosynthesis. Proteobacterial genomes usually encode multiple GGDEF domain-containing diguanylate cyclases responsible for c-di-GMP synthesis. In contrast, only one conserved GGDEF domain protein, GdpS (for GGDEF domain protein from Staphylococcus), and a second protein with a highly modified GGDEF domain, GdpP, are present in the sequenced staphylococcal genomes. Here, we investigated the role of GdpS in biofilm formation in Staphylococcus epidermidis. Inactivation of gdpS impaired biofilm formation in medium supplemented with NaCl under static and flow-cell conditions, whereas gdpS overexpression complemented the mutation and enhanced wild-type biofilm development. GdpS increased production of the icaADBC-encoded exopolysaccharide, poly-N-acetyl-glucosamine, by elevating icaADBC mRNA levels. Unexpectedly, c-di-GMP synthesis was found to be irrelevant for the ability of GdpS to elevate icaADBC expression. Mutagenesis of the GGEEF motif essential for diguanylate cyclase activity did not impair GdpS, and the N-terminal fragment of GdpS lacking the GGDEF domain partially complemented the gdpS mutation. Furthermore, heterologous diguanylate cyclases expressed in trans failed to complement the gdpS mutation, and the purified GGDEF domain from GdpS possessed no diguanylate cyclase activity in vitro. The gdpS gene from Staphylococcus aureus exhibited similar characteristics to its S. epidermidis ortholog, suggesting that the GdpS-mediated signal transduction is conserved in staphylococci. Therefore, GdpS affects biofilm formation through a novel c-di-GMP-independent mechanism involving increased icaADBC mRNA levels and exopolysaccharide biosynthesis. Our data raise the possibility that staphylococci cannot synthesize c-di-GMP and have only remnants of a c-di-GMP signaling pathway.

Application of chromosomal DNA and protein targeting for the identification of Yersinia pestis.

PURPOSE: A comprehensive strategy was developed and validated for the identification of pathogens from closely related near neighbors using both chromosomal and protein biomarkers, with emphasis on distinguishing Yersinia pestis from the ancestral bacterium Yersinia pseudotuberculosis. EXPERIMENTAL DESIGN: Computational analysis was used to discover chromosomal targets unique to Y. pestis. Locus identifier YPO1670 was selected for further validation and PCR was used to confirm that this biomarker was exclusively present in Y. pestis strains, while absent in other Yersinia species. RT-PCR and Western blot analyses were utilized to evaluate YPO1670 expression and MRM MS was performed to identify the YPO1670 protein within cell lysates. RESULTS: The described study validated that YPO1670 was exclusive to Y. pestis. PCR confirmed the locus to be unique to Y. pestis. The associated transcript and protein were produced throughout growth with the highest abundance occurring in stationary phase and MRM MS conclusively identified the YPO1670 protein in cell extracts. CONCLUSIONS AND CLINICAL RELEVANCE: These findings validated YPO1670 as a reliable candidate biomarker for Y. pestis and that a dual DNA and protein targeting approach is feasible for the development of next-generation assays to accurately differentiate pathogens from near neighbors.