Inactivation of the Response Regulator AgrA Has a Pleiotropic Effect on Biofilm Formation, Pathogenesis and Stress Response in Staphylococcus lugdunensis.

Staphylococcus lugdunensis is a coagulase-negative Staphylococcus that emerges as an important opportunistic pathogen. However, little is known about the regulation underlying the transition from commensal to virulent state. Based on knowledge of S. aureus virulence, we suspected that the agr quorum sensing system may be an important determinant for the pathogenicity of S. lugdunensis. We investigated the functions of the transcriptional regulator AgrA using the agrA deletion mutant. AgrA played a role in cell pigmentation: ΔargA mutant colonies were white while the parental strains were slightly yellow. Compared with the wild-type strain, the ΔargA mutant was affected in its ability to form biofilm and was less able to survive in mice macrophages. Moreover, the growth of ΔagrA was significantly reduced by the addition of 10% NaCl or 0.4 mM H2O2 and its survival after 2 h in the presence of 1 mM H2O2 was more than 10-fold reduced. To explore the mechanisms involved beyond these phenotypes, the ΔagrA proteome and transcriptome were characterized by mass spectrometry and RNA-Seq. We found that AgrA controlled several virulence factors as well as stress-response factors, which are well correlated with the reduced resistance of the ΔagrA mutant to osmotic and oxidative stresses. These results were not the consequence of the deregulation of RNAIII of the agr system, since no phenotype or alteration of the proteomic profile has been observed for the ΔRNAIII mutant. Altogether, our results highlighted that the AgrA regulator of S. lugdunensis played a key role in its ability to become pathogenic. IMPORTANCE Although belonging to the natural human skin flora, Staphylococcus lugdunensis is recognized as a particularly aggressive and destructive pathogen. This study aimed to characterize the role of the response regulator AgrA, which is a component of the quorum-sensing agr system and known to be a major element in the regulation of pathogenicity and biofilm formation in Staphylococcus aureus. In the present study, we showed that, contrary to S. aureus, the agrA deletion mutant produced less biofilm. Inactivation of agrA conferred a white colony phenotype and impacted S. lugdunensis in its ability to survive in mice macrophages and to cope with osmotic and oxidative stresses. By global proteomic and transcriptomic approaches, we identified the AgrA regulon, bringing molecular bases underlying the observed phenotypes. Together, our data showed the importance of AgrA in the opportunistic pathogenic behavior of S. lugdunensis allowing it to be considered as an interesting therapeutic target.

Identification of the iron-limitation stimulon in Staphylococcus lugdunensis.

During the infectious process, pathogens such as Staphylococcus lugdunensis have to cope with the condition of host-induced iron-limitation. Using the RNAseq approach, we performed the first global transcriptomic analysis of S. lugdunensis cells incubated in the absence and presence of iron chelator. One hundred and seventy-five genes were identified as members of the iron-limitation stimulon (127 up- and 48 downregulated). Six gene clusters known or likely required for the acquisition of iron have been identified. Among them, a novel Energy-Coupling Factor type transporter (ECF), homologous to the lhaSTA operon, has been found into a 13-gene putative operon and strongly overexpressed under iron-limitation condition. Moreover, the transcription of genes involved in resistance to oxidative stress (including catalase), virulence, transcriptional regulation, and hemin detoxification were also modified. These data provide some answers on the cellular response to the iron-limitation stress that is important for the opportunistic behavior of this pathogen.

Phenotypic and proteomic approaches of the response to iron-limited condition in Staphylococcus lugdunensis.

BACKGROUND: Staphylococcus lugdunensis is a coagulase-negative Staphylococcus part of the commensal skin flora but emerge as an important opportunistic pathogen. Because iron limitation is a crucial stress during infectious process, we performed phenotypic study and compared proteomic profiles of this species incubated in absence and in presence of the iron chelator 2,2'-dipyridyl (DIP). RESULTS: No modification of cell morphology nor cell wall thickness were observed in presence of DIP. However iron-limitation condition promoted biofilm formation and reduced the ability to cope with oxidative stress (1 mM H2O2). In addition, S. lugdunensis N920143 cultured with DIP was significantly less virulent in the larvae of Galleria mellonella model of infection than that grown under standard conditions. We verified that these phenotypes were due to an iron limitation by complementation experiments with FeSO4. By mass spectrometry after trypsin digestion, we characterized the first iron-limitation stress proteome in S. lugdunensis. Among 1426 proteins identified, 349 polypeptides were differentially expressed. 222 were more and 127 less abundant in S. lugdunensis incubated in iron-limitation condition, and by RT-qPCR, some of the corresponding genes have been shown to be transcriptionally regulated. Our data revealed that proteins involved in iron metabolism and carriers were over-expressed, as well as several ABC transporters and polypeptides linked to cell wall metabolism. Conversely, enzymes playing a role in the oxidative stress response (especially catalase) were repressed. CONCLUSIONS: This phenotypic and global proteomic study allowed characterization of the response of S. lugdunensis to iron-limitation. We showed that iron-limitation promoted biofilm formation, but decrease the oxidative stress resistance that may, at least in part, explained the reduced virulence of S. lugdunensis observed under low iron condition.

The Transcriptional Repressor SmvR Is Important for Decreased Chlorhexidine Susceptibility in Enterobacter cloacae Complex.

Major facilitator superfamily (MFS) efflux pumps have been shown to be important for bacterial cells to cope with biocides such as chlorhexidine (CHX), a widely used molecule in hospital settings. In this work, we evaluated the role of two genes, smvA and smvR, in CHX resistance in Enterobacter cloacae complex (ECC). smvA encodes an MFS pump whereas smvR, located upstream of smvA, codes for a TetR-type transcriptional repressor. To this aim, we constructed corresponding deletion mutants from the ATCC 13047 strain (CHX MIC, 2 mg/liter) as well as strains overexpressing smvA or smvR in both ATCC 13047 and three clinical isolates exhibiting elevated CHX MICs (16 to 32 mg/liter). Determination of MICs revealed that smvA played a modest role in CHX resistance, in contrast to smvR that modulated the ability of ECC to survive in the presence of CHX. In clinical isolates, the overexpression of smvR significantly reduced MICs of CHX (2 to 8 mg/liter). Sequence analyses of smvR and promoter regions pointed out substitutions in conserved regions. Moreover, transcriptional studies revealed that SmvR acted as a repressor of smvA expression even if no quantitative correlation between the level of smvA mRNA and MICs of CHX could be observed. On the other hand, overproduction of smvA was able to complement the lack of the major resistance-nodulation-cell division (RND) superfamily efflux pump AcrB and restored resistance to ethidium bromide and acriflavine. Although SmvA could expel biocides such as CHX, other actors, whose expression is under SmvR control, should play a critical role in ECC.

Comparative Genome Analysis of Staphylococcus lugdunensis Shows Clonal Complex-Dependent Diversity of the Putative Virulence Factor, ess/Type VII Locus.

Staphylococcus lugdunensis is a commensal bacterium of human skin that has emerged as a virulent Coagulase-Negative Staphylococcus in both community-acquired and healthcare associated infections. Genotyping methods have shown a clonal population structure of this pathogen but failed to identify hypervirulent lineages. Here, complete genomes of three pathogenic and three carriage S. lugdunensis strains were obtained by Single-Molecule sequencing (PacBio) and compared to 15 complete genomes available in GenBank database. The aim was to identify (i) genetic determinants specific to pathogenic or carriage strains or specific to clonal complexes (CCs) defined by MultiLocus Sequence Typing, and (ii) antibiotic resistance genes and new putative virulence factors encoded or not by mobile genetic elements (MGE). Comparative genomic analysis did not show a strict correlation between gene content and the ability of the six strains to cause infections in humans and in a Galleria mellonella infection model. However, this study identified new MGEs (five prophages, two genomic islands and one plasmid) and genetic variations of some putative virulence-associated loci, especially in CC3 strains. For a clonal population, high variability and eight CC-dependent genetic organizations were observed for the ess locus, which encodes a putative type VII secretion system (T7SS) homologous to that of S. aureus. Further phenotypic and functional studies are needed to characterize this particular CC3 and to evaluate the role of T7SS in the virulence of S. lugdunensis.

Characterization of the gen locus involved in ß-1,6-oligosaccharide utilization by Enterococcus faecalis.

The bicistronic genBA operon (formerly named celBA) of the opportunistic pathogen Enterococcus faecalis, encodes a 6-phospho-ß-glucosidase (GenA) and a phosphotransferase system permease EIIC (GenB). It resembles the cel operon of Streptococcus pyogenes, which is implicated in the metabolism of cellobiose. However, genBA mutants grew normally on cellobiose, but not (genA) or only slowly (genB) on gentiobiose and amygdalin. The two glucosides were also found to be the main inducers of the operon, confirming that the encoded proteins are involved in the utilization of ß-1,6- rather than ß-1,4-linked oligosaccharides. Expression of the genBA operon is regulated by the transcriptional activator GenR, which is encoded by the gene upstream from genB. Thermal shift analysis showed that it binds gentiobiose-6'-P with a Kd of 0.04 mM and with lower affinity also other phospho-sugars. The GenR/gentiobiose-6'-P complex binds to the promoter region upstream from genB. The genBA promoter region contains a cre box and gel-shift experiments demonstrated that the operon is under negative control of the global carbon catabolite regulator CcpA. We also show that the orphan EIIC (GenB) protein needs the EIIA component of the putative OG1RF_10750-OG1RF_10755 operon situated elsewhere on the chromosome to form a functional PTS transporter.

Exercise transcutaneous oximetry significantly modifies the diagnostic hypotheses and impacts scheduled investigations or treatments of patients with exertional limb pain.

INTRODUCTION: In lower extremity peripheral artery disease (PAD), transcutaneous oximetry at exercise (Ex-TcpO2) has been largely validated in research practice, but evidence of routine practice in various vascular laboratories is missing. We hypothesized that Ex-TcPO2 would change the diagnosis hypotheses, investigations and treatments for patients referred for exertional limb pain. MATERIAL & METHODS: A multicenter prospective trial was conducted in nine different referral centers. Investigators performed Ex-TcpO2 and recorded investigations and treatments already scheduled for the patient. We encoded referral physician's diagnostic hypothesis. Before Ex-TcpO2, vascular physicians were asked to give their diagnosis hypotheses. A minimal decrease from rest of oxygen pressure (DROP)

[Vascular rehabilitation in patients with peripheral arterial disease]. / Rééducation des patients ayant une artériopathie oblitérante des membres inférieurs avec une claudication.

Lower limb peripheral arterial disease (PAD) is a frequent debilitating disease associated with a high morbidity and mortality rate. The benefit of rehabilitation in PAD patients has been largely demonstrated, both for patients that undergo amputation, and for patients with claudication. In these latter patients, rehabilitation programs rely on a variety of additional techniques or tools, among which: stretching, specific muscle proprioception, walking and a variety of other physical activities, exercise or situations adapted to community life, lower limb and respiratory physiotherapy, patient's education, support for smoking cessation and healthy nutrition, social support, etc. Whether rehabilitation is performed in specialised integrated structures or performed on a home-based basis, various clinicians are involved. Despite evidence-based proof of efficacy, rehabilitation of PAD patients with claudication is still under-used.

Development and evaluation of the Walking Estimated-Limitation Calculated by History questionnaire in patients with claudication.

BACKGROUND: The Walking Impairment Questionnaire (WIQ) is used to estimate walking impairment in patients with peripheral artery disease; however, it faces frequent errors when self-completed and is complex to score. We aimed to validate an alternative, easily scored four-item tool, the Walking Estimated-Limitation Calculated by History (WELCH) questionnaire. METHODS: The WIQ and WELCH were prospectively tested in five centers. We studied 434 patients, among which 298 had a treadmill test (3.2 km/h; 10% slope) to determine their maximum walking time (MWT), and 30 were seen twice during the study period. RESULTS: After self-completion, we found at least one error in 177 WIQ (40.8%; 95% confidence interval [CI], 36.3%-45.5%) vs 56 WELCH (12.9%; 95% CI, 10.1%-16.4%) questionnaires (P < .0001). When scoring only questionnaires without missing or duplicate answers, 267 WIQ (61.5%; 95% CI, 56.9%-66.0%) vs 393 WELCH (90.6%; 95% CI, 87.4%-93.0%) questionnaires could be scored (P < .001). The median MWT was 233 seconds (interquartile range, 133-654 seconds) for the 298 patients who had a treadmill test. When the 296 patients who had both questionnaire scores available were studied, no difference was found between the Pearson r coefficient of correlation of the WIQ (r = 0.615) and the WELCH (r = 0.653) with MWT (P = .211). In the 30 patients who completed the WELCH twice, correlation was r = 0.839 (P < .001) between the two scores in 22 nonrevascularized patients, and the area under the receiver-operating characteristic curve was 0.830 ± 0.105 (P < .01) to discriminate the eight revascularized from the 22 nonrevascularized patients. CONCLUSIONS: The WELCH questionnaire is a simple tool to estimate walking limitation in patients with suspected peripheral artery disease. It is easily scored by mental calculation. It may help to standardize the estimation of walking limitation in routine clinical practice.