The role of two-component regulatory systems in environmental sensing and virulence in Salmonella.

Adaptation to environments with constant fluctuations imposes challenges that are only overcome with sophisticated strategies that allow bacteria to perceive environmental conditions and develop an appropriate response. The gastrointestinal environment is a complex ecosystem that is home to trillions of microorganisms. Termed microbiota, this microbial ensemble plays important roles in host health and provides colonization resistance against pathogens, although pathogens have evolved strategies to circumvent this barrier. Among the strategies used by bacteria to monitor their environment, one of the most important are the sensing and signalling machineries of two-component systems (TCSs), which play relevant roles in the behaviour of all bacteria. Salmonella enterica is no exception, and here we present our current understanding of how this important human pathogen uses TCSs as an integral part of its lifestyle. We describe important aspects of these systems, such as the stimuli and responses involved, the processes regulated, and their roles in virulence. We also dissect the genomic organization of histidine kinases and response regulators, as well as the input and output domains for each TCS. Lastly, we explore how these systems may be promising targets for the development of antivirulence therapeutics to combat antibiotic-resistant infections.

Small Molecules Produced by Commensal Staphylococcus epidermidis Disrupt Formation of Biofilms by Staphylococcus aureus.

The microbiota influences host health through several mechanisms, including protecting it from pathogen colonization. Staphylococcus epidermidis is one of the most frequently found species in the skin microbiota, and its presence can limit the development of pathogens such as Staphylococcus aureusS. aureus causes diverse types of infections ranging from skin abscesses to bloodstream infections. Given the increasing prevalence of S. aureus drug-resistant strains, it is imperative to search for new strategies for treatment and prevention. Thus, we investigated the activity of molecules produced by a commensal S. epidermidis isolate against S. aureus biofilms. We showed that molecules present in S. epidermidis cell-free conditioned media (CFCM) caused a significant reduction in biofilm formation in most S. aureus clinical isolates, including all 4 agr types and agr-defective strains, without any impact on growth. S. epidermidis molecules also disrupted established S. aureus biofilms and reduced the antibiotic concentration required to eliminate them. Preliminary characterization of the active compound showed that its activity is resistant to heat, protease inhibitors, trypsin, proteinase K, and sodium periodate treatments, suggesting that it is not proteinaceous. RNA sequencing revealed that S. epidermidis-secreted molecules modulate the expression of hundreds of S. aureus genes, some of which are associated with biofilm production. Biofilm formation is one of the main virulence factors of S. aureus and has been associated with chronic infections and antimicrobial resistance. Therefore, molecules that can counteract this virulence factor may be promising alternatives as novel therapeutic agents to control S. aureus infections.IMPORTANCES. aureus is a leading agent of infections worldwide, and its main virulence characteristic is the ability to produce biofilms on surfaces such as medical devices. Biofilms are known to confer increased resistance to antimicrobials and to the host immune responses, requiring aggressive antibiotic treatment and removal of the infected surface. Here, we investigated a new source of antibiofilm compounds, the skin microbiome. Specifically, we found that a commensal strain of S. epidermidis produces molecules with antibiofilm activity, leading to a significant decrease of S. aureus biofilm formation and to a reduction of previously established biofilms. The molecules potentiated the activity of antibiotics and affected the expression of hundreds of S. aureus genes, including those associated with biofilm formation. Our research highlights the search for compounds that can aid us in the fight against S. aureus infections.

Differential proteomic analysis of outer membrane enriched extracts of Bacteroides fragilis grown under bile salts stress.

Bacteroides fragilis is the most commonly isolated anaerobic bacteria from infectious processes. Several virulence traits contribute to the pathogenic nature of this bacterium, including the ability to tolerate the high concentrations of bile found in the gastrointestinal tract (GIT). The activity of bile salts is similar to detergents and may lead to membrane permeabilization and cell death. Modulation of outer membrane proteins (OMPs) is considered a crucial event to bile salts resistance. The primary objective of the current work was to identify B. fragilis proteins associated with the stress induced by high concentration of bile salts. The outer membrane of B. fragilis strain 638R was isolated after growth either in the presence of 2% conjugated bile salts or without bile salts. The membrane fractions were separated on SDS-PAGE and analyzed by ESI-Q/TOF tandem mass spectrometry. A total of 37 proteins were identified; among them nine were found to be expressed exclusively in the absence of bile salts whereas eight proteins were expressed only in the presence of bile salts. These proteins are related to cellular functions such as transport through membrane, nutrient uptake, and protein-protein interactions. This study demonstrates the alteration of OMPs composition in B. fragilis during bile salts stress resistance and adaptation to environmental changes. Proteomics of OMPs was also shown to be a useful approach in the identification of new targets for functional analyses.

Microbe Interactions within the Skin Microbiome.

The skin is the largest human organ and is responsible for many important functions, such as temperature regulation, water transport, and protection from external insults. It is colonized by several microorganisms that interact with each other and with the host, shaping the microbial structure and community dynamics. Through these interactions, the skin microbiota can inhibit pathogens through several mechanisms such as the production of bacteriocins, proteases, phenol soluble modulins (PSMs), and fermentation. Furthermore, these commensals can produce molecules with antivirulence activity, reducing the potential of these pathogens to adhere to and invade human tissues. Microorganisms of the skin microbiota are also able to sense molecules from the environment and shape their behavior in response to these signals through the modulation of gene expression. Additionally, microbiota-derived compounds can affect pathogen gene expression, including the expression of virulence determinants. Although most studies related to microbial interactions in the skin have been directed towards elucidating competition mechanisms, microorganisms can also use the products of other species to their benefit. In this review, we will discuss several mechanisms through which microorganisms interact in the skin and the biotechnological applications of products originating from the skin microbiota that have already been reported in the literature.

Influence of Penicillin on Biofilm Formation by Streptococcus agalactiae Serotype Ia/CC23.

Streptococcus agalactiae (Group B Streptococcus , GBS) is a major agent of perinatal infections. Biofilms have been associated with GBS colonization and disease, as well as with infection persistence and recurrence. Although GBS remains susceptible to beta-lactams, it is still unknown how sessile cells respond to these antibiotics. Here, we evaluated the effect of different concentrations of penicillin (3-48 mg/L) on in vitro biofilm formation by four GBS strains belonging to serotype Ia/clonal complexes23 that were recovered from the oropharynx or urine of pregnant women and were previously characterized as strong biofilm producers. All four GBS strains were fully susceptible to penicillin (minimum inhibitory concentration = 0.023 mg/L), but penicillin was not able to fully prevent biofilm formation by these GBS strains. Biofilms formed in the presence of penicillin had reduced biomasses and thickness, but they were still classified as strong. Penicillin significantly reduced the density of live cells, but higher penicillin concentrations did not lead to improved prevention of biofilm formation. Biofilms formed in the presence of penicillin had no channels or long cocci chains observed in penicillin-free biofilms. Overall, results highlight the concerning possible impacts of biofilm formation in penicillin-based treatment and preventive strategies of GBS infections, even when the bacterial strain involved is fully antibiotic-susceptible.

Antibiofilm activity of Cutibacterium acnes cell-free conditioned media against Staphylococcus spp.

Staphylococcus spp. and Cutibacterium acnes are members of the skin microbiome but can also act as pathogens. Particularly, Staphylococcus species are known to cause medical devices-associated infections, and biofilm production is one of their main virulence factors. Biofilms allow bacteria to adhere and persist on surfaces, protecting them from antimicrobials and host defenses. Since both bacteria are found in the human skin, potentially competing for niches, we aimed to investigate if C. acnes produces molecules that affect Staphylococcus spp. biofilm formation and dispersal. Thus, we evaluated the impact of C. acnes cell-free conditioned media (CFCM) on S. aureus, S. epidermidis, S. hominis, and S. lugdunensis biofilm formation. S. lugdunensis and S. hominis biofilm formation was significantly reduced with C. acnes CFCM without impact on their planktonic growth. C. acnes CFCM also significantly disrupted S. hominis established biofilms. The active molecules against S. lugdunensis and S. hominis biofilms appeared to be distinct since initial characterization points to different sizes and sensitivity to sodium metaperiodate, although the activity is highly resistant to heat in both cases. Mass spectrometry analysis of the fractions active against S. hominis revealed several potential candidates. Investigating how species present in the same environment interact, affecting the dynamics of biofilm formation, may reveal clinically useful compounds as well as molecular aspects of interspecies interactions.

Panton-Valentine leukocidin (PVL) isoforms among Staphylococcus aureus lineages isolated from hospitals in Rio de Janeiro, Brazil.

Panton-Valentine leukocidin (PVL) is a Staphylococcus aureus virulence factor codified by lukSF-PV genes. Single-nucleotide polymorphisms (SNPs) at lukSF-PV genes can lead to two PVL sequence variants (R and H) generating different PVL isoforms. This study analyzed lukSF-PV genes SNPs among four different clonal lineages (STs/CC 1, 5, 8, and 30) of nine S. aureus isolated at Brazilian hospitals. The sequenced products showed SNPs at seven sites (positions 121, 470, 527, 663, 856, 1396, and 1729), leading to non-synonymous substitutions in all isolates investigated. Our findings showed new R and H isoforms variants in S. aureus isolated in Brazil and suggest a possible relationship between H2b isoform and the ST30/CC30 lineage.

Genetic mutations in the quinolone resistance-determining region are related to changes in the epidemiological profile of methicillin-resistant Staphylococcus aureus isolates.

OBJECTIVES: Methicillin-resistant Staphylococcus aureus (MRSA) is an important causative agent of nosocomial infections. Mutations in the quinolone resistance-determining regions (QRDRs) of the gyr and par genes have been described. This study aimed to characterise phenotypic and genotypic fluoroquinolone resistance in 69 MRSA isolates of different clonal lineages from hospitals in Rio de Janeiro, Brazil. METHODS: QRDR mutations in the gyrA, gyrB, parC and parE genes were detected by DNA sequencing. Minimum inhibitory concentrations (MICs) for ciprofloxacin and moxifloxacin were determined by broth microdilution. The occurrence of associations between mutations and MICs among the different clonal lineages of MRSA isolates was then verified. RESULTS: Most isolates from the USA400/ST1/SCCmec IV lineage, but mainly USA100/ST5/SCCmec II isolates, which have been more recently found in Rio de Janeiro hospitals, showed different patterns of mutations, including double mutation in the QRDR of parC (Ser-80â¿¿â¿¿â¿¿Tyr and Glu-84â¿¿â¿¿â¿¿Lys/Gly) and/or gyrA (Ser-84â¿¿â¿¿â¿¿Leu and/or Glu-88â¿¿â¿¿â¿¿Lys) associated with higher moxifloxacin and ciprofloxacin MICs (MIC90, â¿¥8â¿¿mg/L and 256â¿¿mg/L, respectively). On the other hand, all USA800/ST5/SCCmec IV and the BEC/ST239/SCCmec III isolates, which have disappeared from hospitals, showed single mutations in parC (Ser-80â¿¿â¿¿â¿¿Phe) and gyrA (Ser-84â¿¿â¿¿â¿¿Leu or Glu-88â¿¿â¿¿â¿¿Gly) and lower fluoroquinolones MICs (MIC90, â¿¥2â¿¿mg/L and â¿¥16â¿¿mg/L). CONCLUSION: This study highlights an increase in the number and types of mutations in the QRDRs ofgyrA and parC associated with high fluoroquinolones MICs that may be related to changes in the epidemiological profile of MRSA isolates from hospitals in Rio de Janeiro.

Simultaneous detection of the mecA and ileS-2 genes in coagulase-negative staphylococci isolated from Brazilian hospitals by multiplex PCR.

Coagulase-negative Staphylococcus spp. (CNS) has been associated with primary bloodstream infections and implanted medical devices. Its importance is increasing due to the acquisition of resistance to oxacillin (Oxa) and, recently, resistance to mupirocin (Mup). Mupirocin, a topical antimicrobial, has been used in the prevention of staphylococci catheter colonization. Susceptibility to Oxa and Mup was analyzed by different testing methods in clinical CNS isolates. Among 112 CNS strains, 69 (61.6%) were Oxa(R) by the disk diffusion (DD) method and 72 (64.2%) grew on the oxacillin agar screen plate. S. epidermidis and S. haemolyticus presented high rates of oxacillin resistance, 75.4% and 96.1%, respectively. Twenty four (21.4%) strains were Mup(R) by the DD test and 21 of them (87.5%) were identified as S. epidermidis. The detection of the mecA and ileS-2 genes, determined by multiplex-PCR, showed that 72 (64.2%) CNS strains possessed the mecA gene, while 16 (14.3%) possessed the ileS-2 gene. Fifteen of these strains presented the two resistance genes simultaneously. The isolates containing the ileS-2 gene presented a minimum inhibitory concentration (MIC) >1024 microg/mL in the E-test, while low-level mupirocin resistance (MICs of 12-16 microg/mL) was observed in those strains without ileS-2. The resistances to high and low levels of mupirocin could not be distinguished when the DD test was used. The analysis of the Mup(R) S. epidermidis strains by Pulsed Field Gel Electrophoresis showed that 17 (80.9%) strains belonged to one of two patterns (A and B), which have been shown to be prevalent in hospitals in Rio de Janeiro. This report showed that the PCR method for detection of oxacillin and mupirocin resistance in CNS is necessary to determine accurate rates of these resistance, and will can help in the staphylococcal infections prevention and control policies in Brazil.

Simplified and reliable scheme for species-level identification of Staphylococcus clinical isolates.

Reliable and rapid identification of staphylococcal strains continues to be a problem faced by many microbiology laboratories. This study evaluates a simplified method that uses a flowchart to assist in the identification of 12 clinical species of Staphylococcus, including eight subspecies. A total of 198 isolates and 11 control strains were identified by the reference method, which employed 22 tests. The results were compared with those obtained by two other methods: an automated system (MicroScan WalkAway) and a simplified method composed of nine tests. The simplified scheme showed an accuracy of 98.5%, while the automated method showed an accuracy of 79.3% (P < 0.001), in identifying staphylococcal species. Atypical phenotypic profiles were detected by both the reference (55.6%) and the simplified (19.7%) methods. The simplified method proposed here was shown to be reliable, with the advantage of being more practical and economic than the reference method.