Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of β-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of β-lactam antibiotics induce the RpoS regulon. RpoS-regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies.

Triggering receptors expressed on myeloid cell (TREM) proteins are a family of cell surface receptors that participate in diverse cellular processes such as inflammation, coagulation, and bone homeostasis. TREM-1, in particular, is expressed on neutrophils and monocytes and is a potent amplifier of inflammatory responses. LPS and other microbial products induce up-regulation of cell surface-localized TREM-1 and the release of its soluble form, sTREM-1. Two hypotheses have been advanced to explain the origin of sTREM-1: alternative splicing of TREM-1 mRNA and proteolytic cleavage(s) of mature, membrane-anchored TREM-1. In this report, we present conclusive evidence in favor of the proteolytic mechanism of sTREM-1 generation. No alternative splicing forms of TREM-1 were detected in monocytes/macrophages. Besides, metalloproteinase inhibitors increased the stability of TREM-1 at the cell surface while significantly reducing sTREM-1 release in cultures of LPS-challenged human monocytes and neutrophils. We conclude that metalloproteinases are responsible for shedding of the TREM-1 ectodomain through proteolytic cleavage of its long juxtamembrane linker.

Clinical isolates of Pseudomonas aeruginosa that hyperproduce a dark-brown pigment are quite often found in the lungs of chronically infected patients, suggesting that they may have an adaptive advantage in chronic infections. We have screened a library of random transposon insertions in P. aeruginosa. Transposon insertions resulting in the hyperproduction of a darkbrown pigment were found to be located in the hmgA gene, which putatively encodes the enzyme homogentisate-1,2-dioxygenase. Complementation studies indicate that hmgA disruption is responsible for the hyperproduction of pyomelanin in both laboratory and clinical isolates. A relationship between hmgA disruption and adaptation to chronic infection was explored and our results show that the inactivation of hmgA produces a slight reduction of killing ability in an acute murine model of lung infection. On the other hand, it also confers decreased clearance and increased persistence in chronic lung infections. Whether pyomelanin production is the cause of the increased adaptation to chronicity or just a side effect of hmgA inactivation is a question to be studied in future; however, this adaptation is consistent with the higher resistance to oxidative stress conferred in vitro by the pyomelanin pigment. Our results clearly demonstrate that hmgA inactivation leads to a better adaptation to chronic infection, and strongly suggest that this mechanism may be exploited in naturally occurring P. aeruginosa strains.

The fact that inactivation of recA reduces mutagenicity and/or increases the activity of a large number of antimicrobials supports the hypothesis that RecA inhibition might have favourable effects on antibiotic therapy.

Bacterial infections caused by antibiotic-resistant isolates have become a major health problem in recent years, since they are very difficult to treat, leading to an increase in morbidity and mortality. Fosfomycin is a broad-spectrum bactericidal antibiotic that inhibits cell wall biosynthesis in both Gram-negative and Gram-positive bacteria. This antibiotic has a unique mechanism of action and inhibits the initial step in peptidoglycan biosynthesis by blocking the enzyme, MurA. Fosfomycin has been used successfully for the treatment of urinary tract infections for a long time, but the increased emergence of antibiotic resistance has made fosfomycin a suitable candidate for the treatment of infections caused by multidrug-resistant pathogens, especially in combination with other therapeutic partners. The acquisition of fosfomycin resistance could threaten the reintroduction of this antibiotic for the treatment of bacterial infection. Here, we analyse the mechanism of action and molecular mechanisms for the development of fosfomycin resistance, including the modification of the antibiotic target, reduced antibiotic uptake and antibiotic inactivation. In addition, we describe the role of each pathway in clinical isolates.

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Fosfomycin is transported into Escherichia coli via both glycerol-3-phosphate (GlpT) and a hexose phosphate transporter (UhpT). Consequently, the inactivation of either glpT or uhpT confers increased fosfomycin resistance in this species. The inactivation of other genes, including ptsI and cyaA, also confers significant fosfomycin resistance. It has been assumed that identical mechanisms are responsible for fosfomycin transport into Pseudomonas aeruginosa cells. The study of an ordered library of insertion mutants in P. aeruginosa PA14 demonstrated that only insertions in glpT confer significant resistance. To explore the uniqueness of this resistance target in P. aeruginosa, the linkage between fosfomycin resistance and the use of glycerol-3-phosphate was tested. Fosfomycin-resistant (Fos-R) mutants were obtained in LB and minimal medium containing glycerol as the sole carbon source at a frequency of 10 ؊6 . However, no Fos-R mutants grew on plates containing fosfomycin and glycerol-3-phosphate instead of glycerol (mutant frequency, <5 ؋ 10 ؊11 ). In addition, 10 out of 10 independent spontaneous Fos-R mutants, obtained on LB-fosfomycin, harbored mutations in glpT, and in all cases the sensitivity to fosfomycin was recovered upon complementation with the wild-type glpT gene. The analysis of these mutants provides additional insights into the structure-function relationship of glycerol-3-phosphate the transporter in P. aeruginosa. Studies with glucose-6-phosphate and different mutant derivatives strongly suggest that P. aeruginosa lacks a specific transport system for this sugar. Thus, glpT seems to be the only fosfomycin resistance mutational target in P. aeruginosa. The high frequency of Fos-R mutations and their apparent lack of fitness cost suggest that Fos-R variants will be obtained easily in vivo upon the fosfomycin treatment of P. aeruginosa infections.