A Study on the Epidemiological-Molecular Role of Staphylococcus aureus Strains in the Development of Ventilator-Associated Pneumonia in a Tertiary Hospital in Brazil.

This study aimed to explore the molecular epidemiology of Staphylococcus aureus isolated from patients on mechanical ventilation and the participation of virulence factors in the development of ventilator-associated pneumonia (VAP). A prospective cohort study was conducted on patients under mechanical ventilation, with periodic visits for the collection of tracheal aspirates and clinical data. The S. aureus isolates were analyzed regarding resistance profile, virulence, expression of protein A and alpha-toxin using Western blot, clonal profile using PFGE, sequence type using MLST, and characterization and quantification of phenol-soluble modulins. Among the 270 patients in the study, 51 S. aureus strains were isolated from 47 patients. The incidence density of S. aureus and MRSA VAP was 2.35/1000 and 1.96/1000 ventilator days, respectively; of these, 45% (n = 5) were resistant to oxacillin, with 100% (n = 5) harboring SCCmec types II and IV. The most frequent among the tested virulence factors were icaA, hla, and hld. The clonal profile showed a predominance of sequence types originating from the community. Risk factors for VAP were the presence of solid tumors and the sea gene. In conclusion, patient-related risk factors, together with microbiological factors, are involved in the development of S. aureus VAP, which is caused by the patient's own strains.

Acidonemycins A-C, Glycosylated Angucyclines with Antivirulence Activity Produced by the Acidic Culture of Streptomyces indonesiensis.

The genome of Streptomyces indonesiensis is highly enriched with cryptic biosynthetic gene clusters (BGCs). The majority of these cryptic BGCs are transcriptionally silent in normal laboratory culture conditions as determined by transcriptome analysis. When cultured in acidic pH (pH 5.4), this strain has been shown to produce a set of new metabolites that were not observed in cultures of neutral pH (pH 7.4). The organic extract of the acidic culture displayed an antivirulence activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we report the structures of new glycosylated aromatic polyketides, named acidonemycins A-C (1-3), belonging to the family of angucyclines. Type II polyketide synthase BGC responsible for the production of 1-3 was identified by a transcriptome comparison between acidic (pH 5.4) and neutral (pH 7.4) cultures and further confirmed by heterologous expression in Streptomyces albus J1074. Of the three new compounds, acidonemycins A and B (1 and 2) displayed antivirulence activity against MRSA. The simultaneous identification of both antivirulent compounds and their BGC provides a starting point for the future effort of combinatorial biosynthesis.

New meroterpenoids from a soil-derived fungus Penicillium sp. SSW03M2 GY and their anti-virulence activity.

Two new berkeley meroterpenoids (1 and 2), along with seven known compounds (3‒9) were isolated from a fungus, Penicillium sp. SSW03M2 GY derived from a sediment at Seosan bay, South Korea. Chemical structures of the isolated compounds were elucidated on the basis of 1D, 2D NMR, HRESIMS, and optical rotation. All the isolated compounds, 1 showed anti-virulence activity by significantly inhibiting α-toxin (Hla) secreted by methicillin-resistant Staphylococcus aureus without its growth inhibition.

Study of SarA by DNA Affinity Capture Assay (DACA) Employing Three Promoters of Key Virulence and Resistance Genes in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA), one of the most well-known human pathogens, houses many virulence factors and regulatory proteins that confer resistance to diverse antibiotics. Although they have been investigated intensively, the correlations among virulence factors, regulatory proteins and antibiotic resistance are still elusive. We aimed to identify the most significant global MRSA regulator by concurrently analyzing protein-binding and several promoters under same conditions and at the same time point. DNA affinity capture assay (DACA) was performed with the promoters of mecA, sarA, and sarR, all of which significantly impact survival of MRSA. Here, we show that SarA protein binds to all three promoters. Consistent with the previous reports, ΔsarA mutant exhibited weakened antibiotic resistance to oxacillin and reduced biofilm formation. Additionally, production and activity of many virulence factors such as phenol-soluble modulins (PSM), α-hemolysin, motility, staphyloxanthin, and other related proteins were decreased. Comparing the sequence of SarA with that of clinical strains of various lineages showed that all sequences were highly conserved, in contrast to that observed for AgrA, another major regulator of virulence and resistance in MRSA. We have demonstrated that SarA regulates antibiotic resistance and the expression of various virulence factors. Our results warrant that SarA could be a leading target for developing therapeutic agents against MRSA infections.

Identification and characterization of the pathogenic potential of phenol-soluble modulin toxins in the mouse commensal Staphylococcus xylosus.

In contrast to the virulent human skin commensal Staphylococcus aureus, which secretes a plethora of toxins, other staphylococci have much reduced virulence. In these species, commonly the only toxins are those of the phenol-soluble modulin (PSM) family. PSMs are species-specific and have only been characterized in a limited number of species. S. xylosus is a usually innocuous commensal on the skin of mice and other mammals. Prompted by reports on the involvement of PSMs in atopic dermatitis (AD) and the isolation of S. xylosus from mice with AD-like symptoms, we here identified and characterized PSMs of S. xylosus with a focus on a potential involvement in AD phenotypes. We found that most clinical S. xylosus strains produce two PSMs, one of the shorter α- and one of the longer ß-type, which were responsible for almost the entire lytic and pro-inflammatory capacities of S. xylosus. Importantly, PSMα of S. xylosus caused lysis and degranulation of mast cells at degrees higher than that of S. aureus δ-toxin, the main PSM previously associated with AD. However, S. xylosus did not produce significant AD symptoms in wild-type mice as opposed to S. aureus, indicating that promotion of AD by S. xylosus likely requires a predisposed host. Our study indicates that non-specific cytolytic potency rather than specific interaction underlies PSM-mediated mast cell degranulation and suggest that the previously reported exceptional potency of δ-toxin of S. aureus is due to its high-level production. Furthermore, they suggest that species that produce cytolytic PSMs, such as S. xylosus, all have the capacity to promote AD, but a high combined level of PSM cytolytic potency is required to cause AD in a non-predisposed host.

Molecular Basis of Non-ß-Lactam Antibiotics Resistance in Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most successful human pathogens with the potential to cause significant morbidity and mortality. MRSA has acquired resistance to almost all ß-lactam antibiotics, including the new-generation cephalosporins, and is often also resistant to multiple other antibiotic classes. The expression of penicillin-binding protein 2a (PBP2a) is the primary basis for ß-lactams resistance by MRSA, but it is coupled with other resistance mechanisms, conferring resistance to non-ß-lactam antibiotics. The multiplicity of resistance mechanisms includes target modification, enzymatic drug inactivation, and decreased antibiotic uptake or efflux. This review highlights the molecular basis of resistance to non-ß-lactam antibiotics recommended to treat MRSA infections such as macrolides, lincosamides, aminoglycosides, glycopeptides, oxazolidinones, lipopeptides, and others. A thorough understanding of the molecular and biochemical basis of antibiotic resistance in clinical isolates could help in developing promising therapies and molecular detection methods of antibiotic resistance.

Arctic Psychrotolerant Pseudomonas sp. B14-6 Exhibits Temperature-Dependent Susceptibility to Aminoglycosides.

Bacteria can evade antibiotics by acquiring resistance genes, as well as switching to a non-growing dormant state without accompanying genetic modification. Bacteria in this quiescent state are called persisters, and this non-inheritable ability to withstand multiple antibiotics is referred to as antibiotic tolerance. Although all bacteria are considered to be able to form antibiotic-tolerant persisters, the antibiotic tolerance of extremophilic bacteria is poorly understood. Previously, we identified the psychrotolerant bacterium Pseudomonas sp. B14-6 from the glacier foreland of Midtre Lovénbreen in High Arctic Svalbard. Herein, we investigated the resistance and tolerance of Pseudomonas sp. B14-6 against aminoglycosides at various temperatures. This bacterium was resistant to streptomycin and susceptible to apramycin, gentamicin, kanamycin, and tobramycin. The two putative aminoglycoside phosphotransferase genes aph1 and aph2 were the most likely contributors to streptomycin resistance. Notably, unlike the mesophilic Pseudomonas aeruginosa PA14, this cold-adapted bacterium demonstrated reduced susceptibility to all tested aminoglycosides in a temperature-dependent manner. Pseudomonas sp. B14-6 at a lower temperature formed the persister cells that shows tolerance to the 100-fold minimum inhibitory concentration (MIC) of gentamicin, as well as the partially tolerant cells that withstand 25-fold MIC gentamicin. The temperature-dependent gentamicin tolerance appears to result from reduced metabolic activity. Lastly, the partially tolerant Pseudomonas sp. B14-6 cells could slowly proliferate under the bactericidal concentrations of aminoglycosides. Our results demonstrate that Pseudomonas sp. B14-6 has a characteristic ability to form cells with a range of tolerance, which appears to be inversely proportional to its growth rate.

Simultaneous monitoring of each component on degradation of blended bioplastic using gas chromatography-mass spectrometry.

The increasing interest in bioplastics, with regard to future environmental issues, has rendered research on bioplastic biodegradation highly important. However, only a few tools directly monitor the degradation of bioplastics without measuring the levels of gaseous products, such as carbon dioxide. Classical nonquantitative methods, such as clear zone tests on solid plates, and less-sensitive weight-loss experiments in liquid media measured using a precision scale, are still employed to screen the microbial players associated with bioplastic degradation and monitor the biodegradation rates. However, the simultaneous monitoring of the degradation of each component of blended bioplastics has not been previously reported. In the present study, to provide information regarding the degradation rates and compositional changes of different bioplastics in a blend in a time-dependent manner, we simultaneously monitored and quantified the degradation of four bioplastics, polyhydroxybutyrate (PHB), polybutylene succinate (PBS), polycaprolactone (PCL), and poly(butylene adipate-co-terephthalate) (PBAT), by Bacillus sp. JY36 using gas chromatography-mass spectrometry (GC-MS) analysis after fatty acid methyl ester (FAME) derivatization. Our results demonstrate the feasibility of using the GC-MS-based method described here to obtain comprehensive data regarding blended bioplastics and their degradation. Moreover, our findings indicate that this method may support classical analytic tools for assessing bioplastic biodegradation.

Genotypes of Staphylococcus aureus Clinical Isolates Are Associated with Phenol-Soluble Modulin (PSM) Production.

Phenol-soluble modulins (PSMs) are important S. aureus virulence factors that cause cytolysis, mast cell degranulation, and stimulate inflammatory responses. In this study, PSM production by S. aureus clinical isolates was measured by liquid chromatography/mass spectrometry (LC-MS) and correlated with staphylococcal protein A (spa) type and staphylococcal cassette chromosome mec (SCCmec) type. Of 106 S. aureus clinical isolates, 50 (47.2%) corresponded to methicillin-susceptible S. aureus (MSSA) and 56 (52.8%) to methicillin-resistant S. aureus (MRSA). LC-MS analysis revealed no significant difference in average PSMα3, PSMα4, PSMß2, and δ-toxin production between MSSA and MRSA isolates, but PSMα1, PSMα2, and PSMß1 production were higher in MSSA than MRSA. This study demonstrated that average PSMα1-α4, PSMß1-ß2, and δ-toxin production by SCCmec type II strains was significantly lower than the IV, IVA, and V strains. Most of the SCCmec type II strains (n = 17/25; 68.0%) did not produce δ-toxin, suggesting a dysfunctional Agr system. The spa type t111 (except one strain) and t2460 (except one strain producing PSM α1-α4) did not produce PSMα1-α4 and δ-toxin, while average PSM production was higher among the t126 and t1784 strains. This study showed that the genotype of S. aureus, specifically the spa and SCCmec types, is important in characterizing the production of PSMs.

Leucyl-tRNA Synthetase Inhibitor, D-Norvaline, in Combination with Oxacillin, Is Effective against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogenic bacterium that causes severe diseases in humans. For decades, MRSA has acquired substantial resistance against conventional antibiotics through regulatory adaptation, thereby posing a challenge for treating MRSA infection. One of the emerging strategies to combat MRSA is the combinatory use of antibacterial agents. Based on the dramatic change in phospholipid fatty acid (PLFA) composition of MRSA in previous results, this study investigated branched-chain amino acid derivatives (precursors of fatty acid synthesis of cell membrane) and discovered the antimicrobial potency of D-norvaline. The compound, which can act synergistically with oxacillin, is among the three leucine-tRNA synthetase inhibitors with high potency to inhibit MRSA cell growth and biofilm formation. PLFA analysis and membrane properties revealed that D-norvaline decreased the overall amount of PLFA, increasing the fluidity and decreasing the hydrophobicity of the bacterial cell membrane. Additionally, we observed genetic differences to explore the response to D-norvaline. Furthermore, deletion mutants and clinically isolated MRSA strains were treated with D-norvaline. The study revealed that D-norvaline, with low concentrations of oxacillin, was effective in killing several MRSA strains. In summary, our findings provide a new combination of aminoacyl-tRNA synthetase inhibitor D-norvaline and oxacillin, which is effective against MRSA.

Thymol Reduces agr-Mediated Virulence Factor Phenol-Soluble Modulin Production in Staphylococcus aureus.

Staphylococcus aureus is a major human bacterial pathogen that carries a large number of virulence factors. Many virulence factors of S. aureus are regulated by the accessory gene regulator (agr) quorum-sensing system. Phenol-soluble modulins (PSMs) are one of the agr-mediated virulence determinants known to play a significant role in S. aureus pathogenesis. In the present study, the efficacy of thymol to inhibit PSM production including δ-toxin in S. aureus was explored. We employed liquid chromatography-mass spectrometry (LC-MS) to quantify the PSMsα1-PSMα4, PSMß1 and PSMß2, and δ-toxin production from culture supernatants. We found that thymol at 0.5 MIC (128 µg/mL) significantly reduced the PSMα and δ-toxin production in S. aureus WKZ-1, WKZ-2, LAC USA300, and ATCC29213. Downregulation in transcription by quantitative real-time (qRT) PCR analysis of response regulator agrA and receptor histidine kinase agrC upon 0.5 MIC thymol treatment affirmed the results of LC-MS quantification of PSMs. In silico molecular docking analysis demonstrated the binding affinity of thymol with receptors AgrA and AgrC. Transmission electron microscopy images revealed no ultrastructural alterations (cell wall and membrane) in thymol-treated WKZ-1 and WKZ-2 S. aureus strains. Here, we demonstrated that thymol reduces various PSM production in S. aureus clinical isolates and reference strains with mass spectrometry.

4-Chloro-2-Isopropyl-5-Methylphenol Exhibits Antimicrobial and Adjuvant Activity against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) causes severe infections and poses a global healthcare challenge. The utilization of novel molecules which confer synergistical effects to existing MRSA-directed antibiotics is one of the well-accepted strategies in lieu of de novo development of new antibiotics. Thymol is a key component of the essential oil of plants in the Thymus and Origanum genera. Despite the absence of antimicrobial potency, thymol is known to inhibit MRSA biofilm formation. However, the anti-MRSA activity of thymol analogs is not well characterized. Here, we assessed the antimicrobial activity of several thymol derivatives and found that 4-chloro-2-isopropyl-5-methylphenol (chlorothymol) has antimicrobial activity against MRSA and in addition it also prevents biofilm formation. Chlorothymol inhibited staphyloxanthin production, slowed MRSA motility, and altered bacterial cell density and size. This compound also showed a synergistic antimicrobial activity with oxacillin against highly resistant S. aureus clinical isolates and biofilms associated with these isolates. Our results demonstrate that chlorinated thymol derivatives should be considered as a new lead compound in anti-MRSA therapeutics.

Comparative Genomics Reveals a Remarkable Biosynthetic Potential of the Streptomyces Phylogenetic Lineage Associated with Rugose-Ornamented Spores.

The genus Streptomyces is one of the richest sources of secondary metabolite biosynthetic gene clusters (BGCs). Sequencing of a large number of genomes has provided evidence that this well-known bacterial genus still harbors a large number of cryptic BGCs, and their metabolites are yet to be discovered. When taking a gene-first approach for new natural product discovery, BGC prioritization would be the most crucial step for the discovery of novel chemotypes. We hypothesized that strains with a greater number of BGCs would also contain a greater number of silent unique BGCs due to the presence of complex regulatory systems. Based on this hypothesis, we employed a comparative genomics approach to identify a specific Streptomyces phylogenetic lineage with the highest and yet-uncharacterized biosynthetic potential. A comparison of BGC abundance and genome size across 158 phylogenetically diverse Streptomyces type strains identified that members of the phylogenetic group characterized by the formation of rugose-ornamented spores possess the greatest number of BGCs (average, 50 BGCs) and also the largest genomes (average, 11.5 Mb). The study of genetic and biosynthetic diversities using comparative genomics of 11 sequenced genomes and a genetic similarity network analysis of BGCs suggested that members of this group carry a large number of unique BGCs, the majority of which are cryptic and not associated with any known natural product. We believe that members of this Streptomyces phylogenetic group possess a remarkable biosynthetic potential and thus would be a good target for a metabolite characterization study that could lead to the discovery of novel chemotypes. IMPORTANCE It is now well recognized that members of the genus Streptomyces still harbor a large number of cryptic BGCs in their genomes, which are mostly silent under laboratory culture conditions. Activation of transcriptionally silent BGCs is technically challenging and thus forms a bottleneck when taking a gene-first approach for the discovery of new natural products. Thus, it is important to focus activation efforts on strains with BGCs that have the potential to produce novel metabolites. The clade-level analysis of biosynthetic diversity could provide insights into the relationship between phylogenetic lineage and biosynthetic diversity. By exploring BGC abundance in relation to Streptomyces phylogeny, we identified a specific monophyletic lineage associated with the highest BGC abundance. Then, using a combined analysis of comparative genomics and a genetic network, we demonstrated that members of this lineage are genetically and biosynthetically diverse, contain a large number of cryptic BGCs with novel genotypes, and thus would be a good target for metabolite characterization studies.

Comparative Study of the Difference in Behavior of the Accessory Gene Regulator (Agr) in USA300 and USA400 Community-Associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA).

Community-associated Methicillin-Resistant Staphylococcus aureus (CA-MRSA) is notorious as a leading cause of soft tissue infections. Despite several studies on the Agr regulator, the mechanisms of action of Agr on the virulence factors in different strains are still unknown. To reveal the role of Agr in different CA-MRSA, we investigated the LACΔagr mutant and the MW2Δagr mutant by comparing LAC (USA300), MW2 (USA400), and Δagr mutants. The changes of Δagr mutants in sensitivity to oxacillin and several virulence factors such as biofilm formation, pigmentation, motility, and membrane properties were monitored. LACΔagr and MW2Δagr mutants showed different oxacillin sensitivity and biofilm formation compared to the LAC and MW2 strains. Regardless of the strain, the motility was reduced in Δagr mutants. And there was an increase in the long chain fatty acid in phospholipid fatty acid composition of Δagr mutants. Other properties such as biofilm formation, pigmentation, motility, and membrane properties were different in both Δagr mutants. The Agr regulator may have a common role like the control of motility and straindependent roles such as antibiotic resistance, biofilm formation, change of membrane, and pigment production. It does not seem easy to control all MRSA by targeting the Agr regulator only as it showed strain-dependent behaviors.

Increased Antibiotic Resistance of Methicillin-Resistant Staphylococcus aureus USA300 Δpsm Mutants and a Complementation Study of Δpsm Mutants Using Synthetic Phenol-Soluble Modulins.

Phenol-soluble modulins (PSMs) are responsible for regulating biofilm formation, persister cell formation, pmtR expression, host cell lysis, and anti-bacterial effects. To determine the effect of psm deletion on methicillin-resistant Staphylococcus aureus, we investigated psm deletion mutants including Δpsmα, Δpsmß, and Δpsmαß;. These mutants exhibited increased ß-lactam antibiotic resistance to ampicillin and oxacillin that was shown to be caused by increased Nacetylmannosamine kinase (nanK) mRNA expression, which regulates persister cell formation, leading to changes in the pattern of phospholipid fatty acids resulting in increased anteiso-C15:0, and increased membrane hydrophobicity with the deletion of PSMs. When synthetic PSMs were applied to Δpsmα and Δpsmß mutants, treatment of Δpsmα with PSMα1-4 and Δpsmß with PSMß1-2 restored the sensitivity to oxacillin and slightly reduced the biofilm formation. Addition of a single fragment showed that α1, α2, α3, and ß2 had an inhibiting effect on biofilms in Δpsmα; however, ß1 showed an enhancing effect on biofilms in Δpsmß. This study demonstrates a possible reason for the increased antibiotic resistance in psm mutants and the effect of PSMs on biofilm formation.

Unusual bridged angucyclinones and potent anticancer compounds from Streptomyces bulli GJA1.

Two new unique angucyclinones (1 and 2) with unprecedented ether bridges connecting carbons 5 and 7 were isolated from the cultures of Streptomyces bulli GJA1, an endophyte of Gardenia jasminoides, together with two known ones (3 and 4). The MS2-based molecular networking system facilitated the isolation of compounds with target functionalities. The stereochemistry of 1 was completely established by ROESY and ECD experiments. Compound 3 showed antivirulence activities by inhibiting the peptide toxin (PSMs) production and the biofilm formation of MRSA. Compounds 3 and 4 showed very potent anti-proliferative effects against OV1 and ES2 ovarian cancer cells.

Increased resistance of a methicillin-resistant Staphylococcus aureus Δagr mutant with modified control in fatty acid metabolism.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are distinct from general Staphylococcus strains with respect to the composition of the membrane, ability to form a thicker biofilm, and, importantly, ability to modify the target of antibiotics to evade their activity. The agr gene is an accessory global regulator of gram-positive bacteria that governs virulence or resistant mechanisms and therefore an important target for the control of resistant strains. However, the mechanism by which agr impacts resistance to ß-lactam antibiotics remains unclear. In the present study, we found the Δagr mutant strain having higher resistance to high concentrations of ß-lactam antibiotics such as oxacillin and ampicillin. To determine the influence of variation in the microenvironment of cells between the parental and mutant strains, fatty acid analysis of the supernatant, total lipids, and phospholipid fatty acids were compared. The Δagr mutant strain tended to produce fewer fatty acids and retained lower amounts of C16, C18 fatty acids in the supernatant. Phospholipid analysis showed a dramatic increase in the hydrophobic longer-chain fatty acids in the membrane. To target membrane, we applied several surfactants and found that sorbitan monolaurate (Span20) had a synergistic effect with oxacillin by decreasing biofilm formation and growth. These findings indicate that agr deletion allows for MRSA to resist antibiotics via several changes including constant expression of mecA, fatty acid metabolism, and biofilm thickening.

Phenol-Soluble Modulin-Mediated Aggregation of Community-Associated Methicillin-Resistant Staphylococcus Aureus in Human Cerebrospinal Fluid.

Phenol-soluble modulins (PSMs) are major determinants of Staphylococcus aureus virulence and their increased production in community-associated methicillin-resistant S. aureus (CA-MRSA) likely contributes to the enhanced virulence of MRSA strains. Here, we analyzed the differences in bacterial cell aggregation according to PSM presence in the specific human cerebrospinal fluid (CSF) environment. CSF samples from the intraventricular or lumbar intrathecal area of each patient and tryptic soy broth media were mixed at a 1:1 ratio, inoculated with WT and PSM-deleted mutants (Δpsm) of the CA-MRSA strain, USA300 LAC, and incubated overnight. Cell aggregation images were acquired after culture and image analysis was performed. The cell aggregation ratio in WT samples differed significantly between the two sampling sites (intraventricular: 0.2% vs. lumbar intrathecal: 6.7%, p < 0.001). The cell aggregation ratio in Δpsm samples also differed significantly between the two sampling sites (intraventricular: 0.0% vs. lumbar intrathecal: 1.2%, p < 0.001). Division of the study cases into two groups according to the aggregated area ratio (WT/Δpsm; group A: ratio of ≥ 2, group B: ratio of < 2) showed that the median aggregation ratio value differed significantly between groups A and B (5.5 and 0, respectively, p < 0.001). The differences in CSF distribution and PSM presence within the specific CSF environment are significant factors affecting bacterial cell aggregation.

Multi-omics based characterization of antibiotic response in clinical isogenic isolates of methicillin-susceptible/-resistant Staphylococcus aureus.

As demands for new antibiotics and strategies to control methicillin-resistant Staphylococcus aureus (MRSA) increase, there have been efforts to obtain more accurate and abundant information about the mechanism of the bacterial responses to antibiotics. However, most of the previous studies have investigated responses to antibiotics without considering the genetic differences between MRSA and methicillin-susceptible S. aureus (MSSA). Here, we initially applied a multi-omics approach into the clinical isolates (i.e., S. aureus WKZ-1 (MSSA) and S. aureus WKZ-2 (MRSA)) that are isogenic except for the mobile genetic element called staphylococcal cassette chromosome mec (SCCmec) type IV to explore the response to ß-lactam antibiotics (oxacillin). First, the isogenic pair showed a similar metabolism without oxacillin treatment. The quantitative proteomics demonstrated that proteins involved in peptidoglycan biosynthesis (MurZ, PBP2, SgtB, PrsA), two-component systems (VrsSR, WalR, SaeSR, AgrA), oxidative stress (MsrA1, MsrB), and stringent response (RelQ) were differentially regulated after the oxacillin treatment of the isogenic isolates. In addition, targeted metabolic profiling showed that metabolites belonging to the building blocks (lysine, glutamine, acetyl-CoA, UTP) of peptidoglycan biosynthesis machinery were specifically decreased in the oxacillin-treated MRSA. These results indicate that the difference in metabolism of this isogenic pair with oxacillin treatment could be caused only by SCCmec type IV. Understanding and investigating the antibiotic response at the molecular level can, therefore, provide insight into drug resistance mechanisms and new opportunities for antibiotics development.

Biofilm Formation by Staphylococcus aureus Clinical Isolates is Differentially Affected by Glucose and Sodium Chloride Supplemented Culture Media.

Staphylococcus aureus (S. aureus) causes persistent biofilm-related infections. Biofilm formation by S. aureus is affected by the culture conditions and is associated with certain genotypic characteristics. Here, we show that glucose and sodium chloride (NaCl) supplementation of culture media, a common practice in studies of biofilms in vitro, influences both biofilm formation by 40 S. aureus clinical isolates (methicillin-resistant and methicillin-sensitive S. aureus) and causes variations in biofilm quantification. Methicillin-resistant strains formed more robust biofilms than methicillin-sensitive strains in tryptic soy broth (TSB). However, glucose supplementation in TSB greatly promoted and stabilized biofilm formation of all strains, while additional NaCl was less efficient in this respect and resulted in significant variation in biofilm measurements. In addition, we observed that the ST239-SCCmec (Staphylococcal Cassette Chromosome mec) type III lineage formed strong biofilms in TSB supplemented with glucose and NaCl. Links between biofilm formation and accessory gene regulator (agr) status, as assessed by δ-toxin production, and with mannitol fermentation were not found. Our results show that TSB supplemented with 1.0% glucose supports robust biofilm production and reproducible quantification of S. aureus biofilm formation in vitro, whereas additional NaCl results in major variations in measurements of biofilm formation.