Production of capsular polysaccharide does not influence Staphylococcus aureus vancomycin susceptibility.

BACKGROUND: Diverse mechanisms (increased cell wall thickness, low cross linking, decreased autolysis, etc.) have been reported for Staphylococcus aureus strains with intermediate vancomycin susceptibility (VISA). This study was conducted to identify common mechanisms responsible for decreased vancomycin susceptibility in a VISA strain pair. RESULTS: Transcriptional profiling of the clinical heterogeneous VISA isolate SA137/93A and its spontaneous homogeneous mutant strain SA137/93G pointed to an increased capsule production in the strain pair compared to a susceptible control. Furthermore, transcript quantification of the gene cap5E, which is essential for capsule biosynthesis, revealed elevated levels in the VISA strains SA137/93A, SA137/93G and Mu50 in comparison with susceptible strains Reynolds, Newman and SA1450/94. The increased expression was observed in bacteria from exponential as well as stationary growth phase. However, suppression of type 5 capsule formation by expression of antisense RNA did not increase vancomycin susceptibility in the VISA strain SA137/93G. Likewise, construction of inducible mutants of S. aureus Newman or repair of capsule biosynthesis of S. aureus HG001 and S. aureus 1450/94 did not influence resistance to vancomycin. Furthermore, purified type 5 polysaccharide did not protect indicator strains from the action of vancomycin. CONCLUSIONS: The VISA strain tested in this study displayed an increased production of type 5 capsular polysaccharide. However, the production of capsule material did not protect strain SA137/93G and three vancomycin sensitive strains in the presence of vancomycin and thus is not part of the resistance mechanism; however it may represent a by-product of VISA life style that is often characterized by a high sigma factor B activity.

Genome sequence of Staphylococcus aureus VC40, a vancomycin- and daptomycin-resistant strain, to study the genetics of development of resistance to currently applied last-resort antibiotics.

The increasing emergence of multidrug-resistant Staphylococcus aureus is a problem of global importance. Here, we report the genome of S. aureus VC40, which is resistant to the last-resort antibiotics vancomycin and daptomycin. Its genome sequence will allow insights into the mechanisms that convey full resistance to these compounds.

Influence of exercise order on repetition performance during low-intensity resistance exercise.

The purpose of this study was to compare repetition maximum performance and ratings of perceived exertion during resistance exercise sessions conducted at a low intensity (i.e., 20 RM) and in different exercise orders. Twenty-one recreationally trained men performed two total body resistance exercise sessions in opposite orders; each exercise was performed for three sets with 2 minutes passive rest between sets and exercises. The results indicated significantly greater total repetitions for each exercise when performed near the beginning of a sequence and for the first set of each exercise, irrespective of the sequence. The ratings of perceived exertion, however, were not significantly different between sequences. In conclusion, repetition maximum performance for resistance exercise sessions conducted at a low intensity is significantly different based on exercise order. Therefore, when performing high repetitions with relatively low intensity loads, exercises should be prioritized based on individual needs and sports-specific movement patterns for greater volume and potential for the desired neuromuscular adaptations.

Influence of ciprofloxacin and vancomycin on mutation rate and transposition of IS256 in Staphylococcus aureus.

In Staphylococcus aureus, the development of intermediate resistance to vancomycin is due to an accumulation of mutations. To elucidate the mechanisms involved here, a standard laboratory strain (S. aureus HG001) and a clinical MRSA mutator strain (S. aureus SA1450/94, which is characterized by a spontaneous insertion of IS256 into the gene of the mismatch repair enzyme MutS) were incubated at subinhibitory concentrations of ciprofloxacin and vancomycin. Ciprofloxacin increased the mutation rates of both strains, but this effect was inhibited when the SOS response was blocked by the presence of a non-cleavable variant of the LexA repressor. In the presence of vancomycin, the mutation rate was slightly elevated in the mutator strain, and this increase also depended on the strain's ability to induce the SOS response. Furthermore, treatment with subinhibitory concentrations of both antibiotics resulted in an activation of transposition frequency of the insertion element IS256 in S. aureus HG001. Transposition was dependent on the presence of a functional transposase, and the activation of transposition depended on the presence of the functional phosphatase RsbU, which activates SigB transcription activity. An in silico analysis indicated a putative antisense sigma B promoter sequence within the transposase gene. Scrambling of this promoter resulted in an about 20-fold activation of transposition activity of IS256. These data indicate that sigma B is involved in the regulation of IS256 activity by generation of an antisense RNA.

The lantibiotic mersacidin is a strong inducer of the cell wall stress response of Staphylococcus aureus.

BACKGROUND: The lantibiotic mersacidin is an antimicrobial peptide of 20 amino acids that is ribosomally produced by Bacillus sp. strain HIL Y-85,54728. Mersacidin acts by complexing the sugar phosphate head group of the peptidoglycan precursor lipid II, thereby inhibiting the transglycosylation reaction of peptidoglycan biosynthesis. RESULTS: Here, we studied the growth of Staphylococcus aureus in the presence of subinhibitory concentrations of mersacidin. Transcriptional data revealed an extensive induction of the cell wall stress response, which is partly controlled by the two-component regulatory system VraSR. In contrast to other cell wall-active antibiotics such as vancomycin, very low concentrations of mersacidin (0.15xMIC) were sufficient for induction. Interestingly, the cell wall stress response was equally induced in vancomycin intermediately resistant S. aureus (VISA) and in a highly susceptible strain. Since the transcription of the VraDE ABC transporter genes was induced up to 1700-fold in our experiments, we analyzed the role of VraDE in the response to mersacidin. However, the deletion of the vraE gene did not result in an increased susceptibility to mersacidin compared to the wild type strain. Moreover, the efficacy of mersacidin was not affected by an increased cell wall thickness, which is part of the VISA-type resistance mechanism and functions by trapping the vancomycin molecules in the cell wall before they reach lipid II. Therefore, the relatively higher concentration of mersacidin at the membrane might explain why mersacidin is such a strong inducer of VraSR compared to vancomycin. CONCLUSION: In conclusion, mersacidin appears to be a strong inducer of the cell wall stress response of S. aureus at very low concentrations, which reflects its general mode of action as a cell wall-active peptide as well as its use of a unique target site on lipid II. Additionally, mersacidin does not seem to be a substrate for the resistance transporter VraDE.

Tying the knot: making of lasso peptides.

In this issue of Chemistry & Biology, Duquesne and colleagues [1] describe the biosynthetic machinery of microcin J25, an antibacterial peptide that adopts an exceptional three-dimensional structure resembling a knot. The dedicated enzymes, McjB and McjC, were identified and employed to produce this modification in vitro.

Living with an imperfect cell wall: compensation of femAB inactivation in Staphylococcus aureus.

BACKGROUND: Synthesis of the Staphylococcus aureus peptidoglycan pentaglycine interpeptide bridge is catalyzed by the nonribosomal peptidyl transferases FemX, FemA and FemB. Inactivation of the femAB operon reduces the interpeptide to a monoglycine, leading to a poorly crosslinked peptidoglycan. femAB mutants show a reduced growth rate and are hypersusceptible to virtually all antibiotics, including methicillin, making FemAB a potential target to restore beta-lactam susceptibility in methicillin-resistant S. aureus (MRSA). Cis-complementation with wild type femAB only restores synthesis of the pentaglycine interpeptide and methicillin resistance, but the growth rate remains low. This study characterizes the adaptations that ensured survival of the cells after femAB inactivation. RESULTS: In addition to slow growth, the cis-complemented femAB mutant showed temperature sensitivity and a higher methicillin resistance than the wild type. Transcriptional profiling paired with reporter metabolite analysis revealed multiple changes in the global transcriptome. A number of transporters for sugars, glycerol, and glycine betaine, some of which could serve as osmoprotectants, were upregulated. Striking differences were found in the transcription of several genes involved in nitrogen metabolism and the arginine-deiminase pathway, an alternative for ATP production. In addition, microarray data indicated enhanced expression of virulence factors that correlated with premature expression of the global regulators sae, sarA, and agr. CONCLUSION: Survival under conditions preventing normal cell wall formation triggered complex adaptations that incurred a fitness cost, showing the remarkable flexibility of S. aureus to circumvent cell wall damage. Potential FemAB inhibitors would have to be used in combination with other antibiotics to prevent selection of resistant survivors.

Expression of the lantibiotic mersacidin in Bacillus amyloliquefaciens FZB42.

UNLABELLED: Lantibiotics are small peptide antibiotics that contain the characteristic thioether amino acids lanthionine and methyllanthionine. As ribosomally synthesized peptides, lantibiotics possess biosynthetic gene clusters which contain the structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG). The lantibiotic mersacidin is produced by Bacillus sp. HIL Y-85,54728, which is not naturally competent. METHODOLOGY/PRINCIPAL FINDINGS: The aim of these studies was to test if the production of mersacidin could be transferred to a naturally competent Bacillus strain employing genomic DNA of the producer strain. Bacillus amyloliquefaciens FZB42 was chosen for these experiments because it already harbors the mersacidin immunity genes. After transfer of the biosynthetic part of the gene cluster by competence transformation, production of active mersacidin was obtained from a plasmid in trans. Furthermore, comparison of several DNA sequences and biochemical testing of B. amyloliquefaciens FZB42 and B. sp. HIL Y-85,54728 showed that the producer strain of mersacidin is a member of the species B. amyloliquefaciens. CONCLUSIONS/SIGNIFICANCE: The lantibiotic mersacidin can be produced in B. amyloliquefaciens FZB42, which is closely related to the wild type producer strain of mersacidin. The new mersacidin producer strain enables us to use the full potential of the biosynthetic gene cluster for genetic manipulation and downstream modification approaches.

Plectasin, a fungal defensin, targets the bacterial cell wall precursor Lipid II.

Host defense peptides such as defensins are components of innate immunity and have retained antibiotic activity throughout evolution. Their activity is thought to be due to amphipathic structures, which enable binding and disruption of microbial cytoplasmic membranes. Contrary to this, we show that plectasin, a fungal defensin, acts by directly binding the bacterial cell-wall precursor Lipid II. A wide range of genetic and biochemical approaches identify cell-wall biosynthesis as the pathway targeted by plectasin. In vitro assays for cell-wall synthesis identified Lipid II as the specific cellular target. Consistently, binding studies confirmed the formation of an equimolar stoichiometric complex between Lipid II and plectasin. Furthermore, key residues in plectasin involved in complex formation were identified using nuclear magnetic resonance spectroscopy and computational modeling.

The Shikani Optical Stylet: a useful adjunct to airway management in a neonate with popliteal pterygium syndrome.

The craniofacial abnormalities of popliteal pterygium syndrome present unique challenges to the pediatric anesthetist. Congenital fibrous bands between the maxilla and mandible limit mouth opening and the tongue may be fused to the palate. We describe the use of the Shikani Optical Stylet, a novel fiberoptic endoscope, in the airway management of a neonate with popliteal pterygium syndrome and syngnathia.

Role of insertion elements and yycFG in the development of decreased susceptibility to vancomycin in Staphylococcus aureus.

The glycopeptide antibiotic vancomycin acts by binding to the D-alanyl-D-alanine terminus of the cell wall precursor lipid II in the cytoplasmic membrane. The purpose of this study was the identification of genes that might be involved in the vancomycin resistance mechanism. To this end, the expression profiles of two vancomycin intermediately resistant Staphylococcus aureus (VISA) strains, the clinical isolate S. aureus SA137/93A (Etest: 8 microg/ml) and its laboratory mutant S. aureus SA137/93G (Etest: 12 microg/ml) were analyzed using an S. aureus full-genome chip. The results indicated that an essential two-component regulatory system, yycF (vicR) and yycG (vicK) was drastically up-regulated in strain SA137/93A. Sequencing of the yycFG promoter region of strain SA137/93A revealed an insertion of IS256 in the predicted promoter region creating a potentially stronger hybrid promoter. In strain SA137/93G, IS256 was not integrated in the yycFG promoter region but, in previous studies, a copy of IS256 had been found to inactivate the tcaA gene (Maki et al. Antimicrob. Agents and Chemother. 48, 1953-1959 (2004)). Detailed population analyses showed that, in addition to the loss of SCCmec, the inactivation of tcaA seems to cause at least part of the increase in teicoplanin and vancomycin resistance in strain SA137/93G.