Irrigation with wastewater releases pharmaceuticals, pathogenic bacteria, and resistance genes, but little is known about the accumulation of these contaminants in the environment when wastewater is applied for decades. We sampled a chronosequence of soils that were variously irrigated with wastewater from zero up to 100 years in the Mezquital Valley, Mexico, and investigated the accumulation of ciprofloxacin, enrofloxacin, sulfamethoxazole, trimethoprim, clarithromycin, carbamazepine, bezafibrate, naproxen, diclofenac, as well as the occurrence of Enterococcus spp., and sul and qnr resistance genes. Total concentrations of ciprofloxacin, sulfamethoxazole, and carbamazepine increased with irrigation duration reaching 95% of their upper limit of 1.4 µg/kg (ciprofloxacin), 4.3 µg/kg (sulfamethoxazole), and 5.4 µg/kg (carbamazepine) in soils irrigated for 19–28 years. Accumulation was soil-type-specific, with largest accumulation rates in Leptosols and no time-trend in Vertisols. Acidic pharmaceuticals (diclofenac, naproxen, bezafibrate) were not retained and thus did not accumulate in soils. We did not detect qnrA genes, but qnrS and qnrB genes were found in two of the irrigated soils. Relative concentrations of sul1 genes in irrigated soils were two orders of magnitude larger (3.15×10−3±0.22×10−3 copies/16S rDNA) than in non-irrigated soils (4.35×10−5±1.00×10−5 copies/16S rDNA), while those of sul2 exceeded the ones in non-irrigated soils still by a factor of 22 (6.61×10–4±0.59×10−4 versus 2.99×10−5±0.26×10−5 copies/16S rDNA). Absolute numbers of sul genes continued to increase with prolonging irrigation together with Enterococcus spp. 23S rDNA and total 16S rDNA contents. Increasing total concentrations of antibiotics in soil are not accompanied by increasing relative abundances of resistance genes. Nevertheless, wastewater irrigation enlarges the absolute concentration of resistance genes in soils due to a long-term increase in total microbial biomass.

Background Enterococcus faecalis is one of the leading causes of nosocomial infections. Due to its innate and acquired resistance to most antibiotics, identification of new targets for antimicrobial treatment of E. faecalis is a high priority. The multiple peptide resistance factor MprF, which was first described in Staphylococcus aureus, modifies phosphatidylglycerol with lysin and reduces the negative charge of the membrane, thus increasing resistance to cationic antimicrobial peptides. We studied the effect of mprF in E. faecalis regarding influence on bacterial physiology and virulence. Results Two putative mprF paralogs ( mprF1 and mprF2 ) were identified in E. faecalis by BLAST search using the well-described S. aureus gene as a lead. Two deletion mutants in E. faecalis 12030 were created by homologous recombination. Analysis of both mutants by thin-layer chromatography showed that inactivation of mprF 2 abolishes the synthesis of three distinct amino-phosphatidylglycerols (PGs). In contrast, deletion of mprF1 did not interfere with the biosynthesis of amino-PG. Inactivation of mprF 2 increased susceptibility against several antimicrobial peptides and resulted in a 42% increased biofilm formation compared to wild-type mprF . However, resistance to opsonic killing was increased in the mutant, while virulence in a mouse bacteremia model was unchanged. Conclusion Our data suggest that only mprF2 is involved in the aminoacylation of PG in enterococci, and is probably responsible for synthesis of Lys-PG, Ala-PG, and Arg-PG, while mprF1 does not seem to have a role in aminoacylation. As in other Gram-positive pathogens, aminoacylation through MprF2 increases resistance against cationic antimicrobial peptides. Unlike mprF found in other bacteria, mprF 2 does not seem to be a major virulence factor in enterococci.

d pIP501 is a conjugative broad-host-range plasmid frequently present in nosocomial Enterococcus faecalis and Enterococcus faecium isolates. We focus here on the functional analysis of the type IV secretion gene traG, which was found to be essential for pIP501 conjugative transfer between Gram-positive bacteria. The TraG protein, which localizes to the cell envelope of E. faecalis harboring pIP501, was expressed and purified without its N-terminal transmembrane helix (TraG⌬TMH) and shown to possess peptidoglycan-degrading activity. TraG⌬TMH was inhibited by specific lytic transglycosylase inhibitors hexa-N-acetylchitohexaose and bulgecin A. Analysis of the TraG sequence suggested the presence of two domains which both could contribute to the observed cell wall-degrading activity: an N-terminal soluble lytic transglycosylase domain (SLT) and a C-terminal cysteine-, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The protein domains were expressed separately, and both degraded peptidoglycan. A change of the conserved glutamate residue in the putative catalytic center of the SLT domain (E87) to glycine resulted in almost complete inactivity, which is consistent with this part of TraG being a predicted lytic transglycosylase. Based on our findings, we propose that TraG locally opens the peptidoglycan to facilitate insertion of the Gram-positive bacterial type IV secretion machinery into the cell envelope.

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Background:Little is known about the complement evasion strategies of Enterococcus faecalis. Results: Inactivation of tagB in E. faecalis V583 resulted in the loss of two wall teichoic acids (WTAs) associated with a strongly increased complement deposition by the lectin pathway. Conclusion: WTA is critical for complement evasion in E. faecalis. Significance: WTA biosynthesis may be a valuable target for novel antimicrobial agents.