Structural basis of promoter recognition by Staphylococcus aureus RNA polymerase.

Bacterial RNAP needs to form holoenzyme with σ factors to initiate transcription. While Staphylococcus aureus σA controls housekeeping functions, S. aureus σB regulates virulence, biofilm formation, persistence, cell internalization, membrane transport, and antimicrobial resistance. Besides the sequence difference, the spacers between the -35 element and -10 element of σB regulated promoters are shorter than those of σA regulated promoters. Therefore, how σB recognizes and initiates transcription from target promoters can not be inferred from that of the well studied σ. Here, we report the cryo-EM structures of S. aureus RNAP-promoter open complexes comprising σA and σB, respectively. Structural analyses, in combination with biochemical experiments, reveal the structural basis for the promoter specificity of S. aureus transcription. Although the -10 element of σA regulated promoters is recognized by domain σA2 as single-stranded DNA, the -10 element of σB regulated promoters is co-recognized by domains σB2 and σB3 as double-stranded DNA, accounting for the short spacers of σB regulated promoters. S. aureus RNAP is a validated target of antibiotics, and our structures pave the way for rational drug design targeting S. aureus RNAP.

News sequences types of Staphylococcus aureus isolated from human pathologicals fluids in Burkina Faso.

Staphylococcus aureus is a pathogen with high epidemic potential frequently involved in nosocomials and communities infections. The pathogenicity of Staphylococcus aureus is due to both its ability to resist antibiotics and to Produce toxins. This work aims at studying the resistance and Molecular Epidemiology of Staphylococcus aureus. Antibiotic susceptibility of the 70 strains isolates of Staphylococcus aureus was determined by agar diffusion while Multiplex PCR and MLST were used to search toxin-coding genes and MRSA typing, respectively. 14.28% of isolates were multidrug resistant. Staphylococcus aureus showed high susceptibility to aminoglycoside and Macrolides familly. lukS-PV/lukF-PV and sea genes were detected in 45% and 3% of Staphylococcus aureus respectively. Ten (10) sequence types including ST5710, ST2430, ST5289, ST5786, ST6942, ST6943, ST6944, ST6945, ST6946, ST6947 have been reported. The study showed a diversity of antibiotic resistance phenotypes and a great diversity of MRSA clones causing infections.

Rule-based omics mining reveals antimicrobial macrocyclic peptides against drug-resistant clinical isolates.

Antimicrobial resistance remains a significant global threat, driving up mortality rates worldwide. Ribosomally synthesized and post-translationally modified peptides have emerged as a promising source of novel peptide antibiotics due to their diverse chemical structures. Here, we report the discovery of new aminovinyl-(methyl)cysteine (Avi(Me)Cys)-containing peptide antibiotics through a synergistic approach combining biosynthetic rule-based omics mining and heterologous expression. We first bioinformatically identify 1172 RiPP biosynthetic gene clusters (BGCs) responsible for Avi(Me)Cys-containing peptides formation from a vast pool of over 50,000 bacterial genomes. Subsequently, we successfully establish the connection between three identified BGCs and the biosynthesis of five peptide antibiotics via biosynthetic rule-guided metabolic analysis. Notably, we discover a class V lanthipeptide, massatide A, which displays excellent activity against gram-positive pathogens, including drug-resistant clinical isolates like linezolid-resistant S. aureus and methicillin-resistant S. aureus, with a minimum inhibitory concentration of 0.25 µg/mL. The remarkable performance of massatide A in an animal infection model, coupled with a relatively low risk of resistance and favorable safety profile, positions it as a promising candidate for antibiotic development. Our study highlights the potential of Avi(Me)Cys-containing peptides in expanding the arsenal of antibiotics against multi-drug-resistant bacteria, offering promising drug leads in the ongoing battle against infectious diseases.

Expression, purification and characterization of CTP synthase PyrG in Staphylococcusaureus.

Staphylococcus aureus (S. aureus) presents a significant challenge in both nosocomial and community settings due to its pathogenicity. The emergence of drug-resistant strains exacerbates S. aureus infections, leading to increased mortality rates. PyrG, a member of the cytidine triphosphate (CTP) synthase family, serves as a crucial therapeutic target against S. aureus due to the pivotal role of CTP in cellular metabolism. However, the structural and mechanistic details of S. aureus PyrG remains unknown. Here, we successfully expressed and purified monomeric PyrG. Mutational experiments were conducted based on the results of molecular docking. Based on the results of the molecular docking, we carried out mutation experiments and found that Q386A dramatically decreased the CTP synthase activity compared to the wild-type protein, while Y54A almost completely abolished the activity. Exposure of S. aureus to the kinase inhibitor crizotinib increased expression of gene pyrG. Our results identify the two key sites on PyrG for the CTP synthase activity, and present PyrG gene expression increased during the treatment of crizotinib, which may eventually provide valuable guidance for the development of new drugs against S. aureus infections.

Cell-wall-anchored proteins affect invasive host colonization and biofilm formation in Staphylococcus aureus.

As a major human and animal pathogen, Staphylococcus aureus can attach to medical implants (abiotic surface) or host tissues (biotic surface), and further establish robust biofilms which enhances resistance and persistence to host immune system and antibiotics. Cell-wall-anchored proteins (CWAPs) covalently link to peptidoglycan, and largely facilitate the colonization of S. aureus on various surfaces (including adhesion and biofilm formation) and invasion into host cells (including adhesion, immune evasion, iron acquisition and biofilm formation). During biofilm formation, CWAPs function in adhesion, aggregation, collagen-like fiber network formation, and consortia formation. In this review, we firstly focus on the structural features of CWAPs, including their intracellular function and interactions with host cells, as well as the functions and ligand binding of CWAPs in different stages of S. aureus biofilm formation. Then, the roles of CWAPs in different biofilm processes with regards in development of therapeutic approaches are clarified, followed by the association between CWAPs genes and clonal lineages. By touching upon these aspects, we hope to provide comprehensive knowledge and clearer understanding on the CWAPs of S. aureus and their roles in biofilm formation, which may further aid in prevention and treatment infection and vaccine development.

Superantigen Encoding Genes in Staphylococcus aureus Isolated from Lesional Skin, Non-Lesional Skin, and Nares of Patients with Atopic Dermatitis.

Patients with atopic dermatitis (AD) are more likely than healthy individuals to harbour Staphylococcus aureus on their skin. Superantigens (SAgs) produced by specific S. aureus strains may contribute to AD-associated skin inflammation. The present study compared the prevalence and types of SAg-encoding genes between S. aureus isolated from patients with AD and from  controls, and within the AD group between isolates from different sampling sites (lesional skin, non-lesional skin, and nares). This retrospective case-control study extracted data from 2 previous studies that examined S. aureus using whole-genome sequencing. The 138 S. aureus isolates obtained from 71 AD patients contained 349 SAg-encoding genes; 22 (6.3%) were found in isolates from nares (0.4 ± 0.6 genes per isolate), 99 (28.4%) in isolates from non-lesional skin (3.7 ± 3.9), and 228 (65.3%) in isolates from lesional skin (4.2 ± 4.5). S. aureus (n = 101) from the control group contained 594 SAg-encoding genes (5.9 ± 4.2). Of the S. aureus isolated from lesional AD skin, 69% carried at least 1 gene encoding SAg compared with 33% of AD nasal isolates. SAg could be a factor in the pathogenesis of a subset of AD patients.

Prevalence and molecular detection of Staphylococcus aureus resistance to antibiotics.

In Algeria, the issue of antibiotic resistance is on the rise, being the Staphylococcus aureus infection as a significant concern of hospital-acquired infections. The emergence of antibiotic resistance in this bacterium poses a worldwide challenge. The aim of this study aims to establish the incidence of S aureus strains in Algeria as well as identify phenotypic and genotypic resistance based on the "mecA" and "nuc" genes. From 2014 to 2017, a total of 185 S aureus strains were isolated from patients at a hospital in the city of Rouïba, Algiers the number of isolates was slightly higher in males at 58.06% compared to females at 41.94%, resulting in a sex ratio of 1.38. the Oxacillin and Cefoxitin DD test (1 µg oxacillin disk and 30 µg cefoxitin disk) identified 42 strains as resistant. The results indicated high resistance to lactam antibiotics, with penicillin having a 100% resistance rate. There was also significant resistance to oxacillin (51.25%) and cefoxitin (50%). This resistance was frequently associated with resistance to other antibiotic classes, such as aminoglycosides (50%) and Macrolides (28.29%). To confirm methicillin-resistant characteristics, a polymerase chain reaction (PCR) multiplex was conducted on 10 isolates (6 SARM; 4 MSSA) on a phenotypic level. Three isolates tested positive for "mecA," while 7 were negative. All strains carry the nuc gene, which is specific to S aureus. In Algeria, the incidence of S aureus resistance is slightly lower compared to other countries, but it is increasing over time. It is now more crucial than ever to restrict the proliferation of multidrug-resistant strains and reduce undue antibiotic prescriptions. To achieve this, it is vital to keep updated on the epidemiology of this bacterium and its antibiotic susceptibility. This will enable the formulation of appropriate preventive control measures to manage its progression.

Integrative omics analysis reveals insights into small colony variants of Staphylococcus aureus induced by sulfamethoxazole-trimethoprim.

BACKGROUND: Long-term treatment with trimethoprim-sulfamethoxazole (SXT) can lead to the formation of small-colony variants (SCVs) of Staphylococcus aureus. However, the mechanism behind SCVs formation remains poorly understood. In this study, we explored the phenotype and omics-based characterization of S. aureus SCVs induced by SXT and shed light on the potential causes of SCV formation. METHODS: Stable SCVs were obtained by continuously treating S. aureus isolates using 12/238 µg/ml of SXT, characterized by growth kinetics, antibiotic susceptibility testing, and auxotrophism test. Subsequently, a pair of representative strains (SCV and its parental strain) were selected for genomic, transcriptomic and metabolomic analysis. RESULTS: Three stable S. aureus SCVs were successfully screened and proven to be homologous to their corresponding parental strains. Phenotypic tests showed that all SCVs were non-classical mechanisms associated with impaired utilization of menadione, heme and thymine, and exhibited slower growth and higher antibiotic minimum inhibitory concentrations (MICs), compared to their corresponding parental strains. Genomic data revealed 15 missense mutations in 13 genes in the representative SCV, which were involved in adhesion, intramolecular phosphate transfer on ribose, transport pathways, and phage-encoded proteins. The combination analysis of transcriptome and metabolome identified 35 overlapping pathways possible associated with the phenotype switching of S. aureus. These pathways mainly included changes in metabolism, such as purine metabolism, pyruvate metabolism, amino acid metabolism, and ABC transporters, which could play a crucial role in promoting SCVs development by affecting nucleic acid synthesis and energy metabolism in bacteria. CONCLUSION: This study provides profound insights into the causes of S. aureus SCV formation induced by SXT. The findings may offer valuable clues for developing new strategies to combat S. aureus SCV infections.

Host stress drives tolerance and persistence: The bane of anti-microbial therapeutics.

Antibiotic resistance, typically associated with genetic changes within a bacterial population, is a frequent contributor to antibiotic treatment failures. Antibiotic persistence and tolerance, which we collectively term recalcitrance, represent transient phenotypic changes in the bacterial population that prolong survival in the presence of typically lethal concentrations of antibiotics. Antibiotic recalcitrance is challenging to detect and investigate-traditionally studied under in vitro conditions, our understanding during infection and its contribution to antibiotic failure is limited. Recently, significant progress has been made in the study of antibiotic-recalcitrant populations in pathogenic species, including Mycobacterium tuberculosis, Staphylococcus aureus, Salmonella enterica, and Yersiniae, in the context of the host environment. Despite the diversity of these pathogens and infection models, shared signals and responses promote recalcitrance, and common features and vulnerabilities of persisters and tolerant bacteria have emerged. These will be discussed here, along with progress toward developing therapeutic interventions to better treat recalcitrant pathogens.

An induced mutation of ABC-transporter component VraF(K84E) contributes to vancomycin resistance and virulence in Staphylococcus aureus strain MW2.

Staphylococcus aureus is a notorious pathogen responsible for various severe diseases. Due to the emergence of drug-resistant strains, the prevention and treatment of S. aureus infections have become increasingly challenging. Vancomycin is considered to be one of the last-resort drugs for treating most methicillin-resistant S. aureus (MRSA), so it is of great significance to further reveal the mechanism of vancomycin resistance. VraFG is one of the few important ABC (ATP-binding cassette) transporters in S. aureus that can form TCS (two-component systems)/ABC transporter modules. ABC transporters can couple the energy released from ATP hydrolysis to translocate solutes across the cell membrane. In this study, we obtained a strain with decreased vancomycin susceptibility after serial passaging and selection. Subsequently, whole-genome sequencing was performed on this laboratory-derived strain MWA2 and a novel single point mutation was discovered in vraF gene, leading to decreased sensitivity to vancomycin and daptomycin. Furthermore, the mutation reduces autolysis of S. aureus and downregulates the expression of lytM, isaA, and atlA. Additionally, we observed that the mutant has a less net negative surface charge than wild-type strain. We also noted an increase in the expression of the dlt operon and mprF gene, which are associated with cell surface charge and serve to hinder the binding of cationic peptides by promoting electrostatic repulsion. Moreover, this mutation has been shown to enhance hemolytic activity, expand subcutaneous abscesses, reflecting an increased virulence. This study confirms the impact of a point mutation of VraF on S. aureus antibiotic resistance and virulence, contributing to a broader understanding of ABC transporter function and providing new targets for treating S. aureus infections.

Nasal carriage of Staphylococcus aureus in healthy dairy cows in Algeria: antibiotic resistance, enterotoxin genes and biofilm formation.

BACKGROUND: Staphylococcus aureus can colonize and infect a variety of animal species. In dairy herds, it is one of the leading causes of mastitis cases. The objective of this study was to characterize the S. aureus isolates recovered from nasal swabs of 249 healthy cows and 21 breeders of 21 dairy farms located in two provinces of Algeria (Tizi Ouzou and Bouira). METHODS: The detection of enterotoxin genes was investigated by multiplex PCRs. Resistance of recovered isolates to 8 antimicrobial agents was determined by disc-diffusion method. The slime production and biofilm formation of S. aureus isolates were assessed using congo-red agar (CRA) and microtiter-plate assay. Molecular characterization of selected isolates was carried out by spa-typing and Multi-Locus-Sequence-Typing (MLST). RESULTS: S. aureus was detected in 30/249 (12%) and 6/13 (28.6%) of nasal swabs in cows and breeders, respectively, and a total of 72 isolates were recovered from positive samples (59 isolates from cows and 13 from breeders). Twenty-six of these isolates (36.1%) harbored genes encoding for staphylococcal enterotoxins, including 17/59 (28.8%) isolates from cows and 9/13 (69.2%) from breeders. Moreover, 49.1% and 92.3% of isolates from cows and breeders, respectively, showed penicillin resistance. All isolates were considered as methicillin-susceptible (MSSA). Forty-five (76.3%) of the isolates from cows were slime producers and 52 (88.1%) of them had the ability to form biofilm in microtiter plates. Evidence of a possible zoonotic transmission was observed in two farms, since S. aureus isolates recovered in these farms from cows and breeders belonged to the same clonal lineage (CC15-ST15-t084 or CC30-ST34-t2228). CONCLUSIONS: Although healthy cows in this study did not harbor methicillin-resistant S. aureus isolates, the nares of healthy cows could be a reservoir of enterotoxigenic and biofilm producing isolates which could have implications in human and animal health.

Exploring the virulence potential of Staphylococcus aureus CC121 and CC152 lineages related to paediatric community-acquired bacteraemia in Manhiça, Mozambique.

Staphylococcus aureus is a frequent agent of bacteraemia. This bacterium has a variety of virulence traits that allow the establishment and maintenance of infection. This study explored the virulence profile of S. aureus strains causing paediatric bacteraemia (SAB) in Manhiça district, Mozambique. We analysed 336 S. aureus strains isolated from blood cultures of children younger than 5 years admitted to the Manhiça District Hospital between 2001 and 2019, previously characterized for antibiotic susceptibility and clonality. The strains virulence potential was evaluated by PCR detection of the Panton-Valentine leucocidin (PVL) encoding genes, lukS-PV/lukF-PV, assessment of the capacity for biofilm formation and pathogenicity assays in Galleria mellonella. The overall carriage of PVL-encoding genes was over 40%, although reaching ~ 70 to 100% in the last years (2014 to 2019), potentially linked to the emergence of CC152 lineage. Strong biofilm production was a frequent trait of CC152 strains. Representative CC152 and CC121 strains showed higher virulence potential in the G. mellonella model when compared to reference strains, with variations within and between CCs. Our results highlight the importance of monitoring the emergent CC152-MSSA-PVL+ and other lineages, as they display important virulence traits that may negatively impact the management of SAB paediatric patients in Manhiça district, Mozambique.

Phage protein Gp11 blocks Staphylococcus aureus cell division by inhibiting peptidoglycan biosynthesis.

Phages and bacteria have a long history of co-evolution. However, these dynamics of phage-host interactions are still largely unknown; identification of phage inhibitors that remodel host metabolism will provide valuable information for target development for antimicrobials. Here, we perform a comprehensive screen for early-gene products of ΦNM1 that inhibit cell growth in Staphylococcus aureus. A small membrane protein, Gp11, with inhibitory effects on S. aureus cell division was identified. A bacterial two-hybrid library containing 345 essential S. aureus genes was constructed to screen for targets of Gp11, and Gp11 was found to interact with MurG and DivIC. Defects in cell growth and division caused by Gp11 were dependent on MurG and DivIC, which was further confirmed using CRISPRi hypersensitivity assay. Gp11 interacts with MurG, the protein essential for cell wall formation, by inhibiting the production of lipid II to regulate peptidoglycan (PG) biosynthesis on the cell membrane. Gp11 also interacts with cell division protein DivIC, an essential part of the division machinery necessary for septal cell wall assembly, to disrupt the recruitment of division protein FtsW. Mutations in Gp11 result in loss of its ability to cause growth defects, whereas infection with phage in which the gp11 gene has been deleted showed a significant increase in lipid II production in S. aureus. Together, our findings reveal that a phage early-gene product interacts with essential host proteins to disrupt PG biosynthesis and block S. aureus cell division, suggesting a potential pathway for the development of therapeutic approaches to treat pathogenic bacterial infections. IMPORTANCE: Understanding the interplay between phages and their hosts is important for the development of novel therapies against pathogenic bacteria. Although phages have been used to control methicillin-resistant Staphylococcus aureus infections, our knowledge related to the processes in the early stages of phage infection is still limited. Owing to the fact that most of the phage early proteins have been classified as hypothetical proteins with uncertain functions, we screened phage early-gene products that inhibit cell growth in S. aureus, and one protein, Gp11, selectively targets essential host genes to block the synthesis of the peptidoglycan component lipid II, ultimately leading to cell growth arrest in S. aureus. Our study provides a novel insight into the strategy by which Gp11 blocks essential host cellular metabolism to influence phage-host interaction. Importantly, dissecting the interactions between phages and host cells will contribute to the development of new and effective therapies to treat bacterial infections.

The function of CozE proteins is linked to lipoteichoic acid biosynthesis in Staphylococcus aureus.

Coordinated membrane and cell wall synthesis is vital for maintaining cell integrity and facilitating cell division in bacteria. However, the molecular mechanisms that underpin such coordination are poorly understood. Here we uncover the pivotal roles of the staphylococcal proteins CozEa and CozEb, members of a conserved family of membrane proteins previously implicated in bacterial cell division, in the biosynthesis of lipoteichoic acids (LTA) and maintenance of membrane homeostasis in Staphylococcus aureus. We establish that there is a synthetic lethal relationship between CozE and UgtP, the enzyme synthesizing the LTA glycolipid anchor Glc2DAG. By contrast, in cells lacking LtaA, the flippase of Glc2DAG, the essentiality of CozE proteins was alleviated, suggesting that the function of CozE proteins is linked to the synthesis and flipping of the glycolipid anchor. CozE proteins were indeed found to modulate the flipping activity of LtaA in vitro. Furthermore, CozEb was shown to control LTA polymer length and stability. Together, these findings establish CozE proteins as novel players in membrane homeostasis and LTA biosynthesis in S. aureus.IMPORTANCELipoteichoic acids are major constituents of the cell wall of Gram-positive bacteria. These anionic polymers are important virulence factors and modulators of antibiotic susceptibility in the important pathogen Staphylococcus aureus. They are also critical for maintaining cell integrity and facilitating proper cell division. In this work, we discover that a family of membrane proteins named CozE is involved in the biosynthesis of lipoteichoic acids (LTAs) in S. aureus. CozE proteins have previously been shown to affect bacterial cell division, but we here show that these proteins affect LTA length and stability, as well as the flipping of glycolipids between membrane leaflets. This new mechanism of LTA control may thus have implications for the virulence and antibiotic susceptibility of S. aureus.

Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing.

Whole-genome reconstruction of bacterial pathogens has become an important tool for tracking transmission and antimicrobial resistance gene spread, but highly accurate and complete assemblies have largely only historically been achievable using hybrid long- and short-read sequencing. We previously found the Oxford Nanopore Technologies (ONT) R10.4/kit12 flowcell/chemistry produced improved assemblies over the R9.4.1/kit10 combination, however long-read only assemblies contained more errors compared to Illumina-ONT hybrid assemblies. ONT have since released an R10.4.1/kit14 flowcell/chemistry upgrade and recommended the use of Bovine Serum Albumin (BSA) during library preparation, both of which reportedly increase accuracy and yield. They have also released updated basecallers trained using native bacterial DNA containing methylation sites intended to fix systematic basecalling errors, including common adenosine (A) to guanine (G) and cytosine (C) to thymine (T) substitutions. To evaluate these improvements, we successfully sequenced four bacterial reference strains, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, and nine genetically diverse E. coli bloodstream infection-associated isolates from different phylogroups and sequence types, both with and without BSA. These sequences were de novo assembled and compared against Illumina-corrected reference genomes. In this small evaluation of 13 isolates we found that nanopore long-read-only R10.4.1/kit 14 assemblies with updated basecallers trained using bacterial methylated DNA produce accurate assemblies with ≥40×depth, sufficient to be cost-effective compared with hybrid ONT/Illumina sequencing in our setting.

Molecular insights into expression and silencing of resistance determinants in Staphylococcus aureus.

OBJECTIVE: This study aimed to investigate the status of antimicrobial-resistant strains of Staphylococcus aureus in Pakistan, their association in terms of co-occurrence with the biofilm-forming genes, resistance profiling and associated discrepancies in diagnostic methods. METHODOLOGY: A total of 384 milk samples from bovine was collected by using convenient sampling technique and were initially screened for subclinical mastitis, further preceded by isolation and confirmation of S. aureus. The S. aureus isolates were subjected to evaluation of antimicrobial resistance by phenotypic identification using Kirby-Bauer disc diffusion method, while the genotypic estimation was done by polymerase chain reaction to declare isolates as methicillin, beta-lactam, vancomycin, tetracycline, and aminoglycoside resistant S. aureus (MRSA, BRSA, VRSA, TRSA, and ARSA), respectively. RESULTS: The current study revealed an overall prevalence of subclinical mastitis and S. aureus to be 59.11% and 46.69%, respectively. On a phenotypic basis, the prevalence of MRSA, BRSA, VRSA, TRSA, and ARSA was found to be 44.33%, 58.49%, 20.75%, 35.84%, and 30.18%, respectively. The results of PCR analysis showed that 46.80% of the tested isolates were declared as MRSA, 37.09% as BRSA, and 36.36% as VRSA, while the occurrence of TRSA and ARSA was observed in 26.31% and 18.75%, respectively. The current study also reported the existence of biofilm-producing genes (icaA and icaD) in 49.06% and 40.57% isolates, respectively. Lastly, this study also reported a high incidence of discrepancies for both genotypic and phenotypic identification methods of resistance evaluation, with the highest discrepancy ratio for the accA-aphD gene, followed by tetK, vanB, blaZ, and mecA genes. CONCLUSION: The study concluded that different antibiotic resistance strains of S. aureus are prevalent in study districts with high potential to transmit between human populations. The study also determined that there are multiple resistance determinants and mechanisms that are responsible for the silencing and expression of antibiotic resistance genes.

Sensitive and on-Site Detection of Staphylococcus aureus Based on CRISPR/Cas 13a-Assisted Chemiluminescence Resonance Energy Transfer.

Developing a specific, sensitive, rapid, and on-site method for detecting pathogenic bacteria in food samples is critical to ensuring public safety. This article demonstrates a CRISPR/Cas13a system and a chemiluminescence resonance energy transfer (CRET) (CRISPR/Cas 13a-assisted CRET)-based strategy for sensitive and on-site detection of pathogenic bacteria in real samples. Once the hybrid double strand of aptamerS. aureus-cRNA recognizes the target model bacteria of Staphylococcus aureus (S. aureus), the released cRNA would bind with CRISPR/Cas 13a to form a complex of cRNA-CRISPR/Cas 13a, which could cleave the RNA molecule in the detecting probe of horseradish peroxidase (HRP) modified-gold nanoparticles (AuNPs) linked by RNA (AuNPs-RNA-HRP), resulting in an enhanced chemiluminescence signal due to the CRET "OFF" phenomenon after introducing the chemiluminescence substrate of luminol. The CRISPR/Cas 13a-assisted CRET strategy successfully detected S. aureus in drinking water and milk with detection limits of 20 and 30 cfu/mL, respectively, within the recovery of 90.07-105.50%. Furthermore, after integrating with an immunochromatographic test strip (ICTS), the CRISPR/Cas 13a-assisted CRET strategy achieved the on-site detection of as low as 102 cfu/mL of S. aureus in drinking water and milk via a smartphone, which is about 10 times lower than that in the previously reported AuNPs-based colorimetric ICTS, demonstrating a convenient and sensitive detection method for S. aureus in real samples.

Substrate Analogues Entering the Lipoic Acid Salvage Pathway via Lipoate-Protein Ligase 2 Interfere with Staphylococcus aureus Virulence.

Lipoic acid (LA) is an essential cofactor in prokaryotic and eukaryotic organisms, required for the function of several multienzyme complexes such as oxoacid dehydrogenases. Prokaryotes either synthesize LA or salvage it from the environment. The salvage pathway in Staphylococcus aureus includes two lipoate-protein ligases, LplA1 and LplA2, as well as the amidotransferase LipL. In this study, we intended to hijack the salvage pathway by LA analogues that are transferred via LplA2 and LipL to the E2 subunits of various dehydrogenases, thereby resulting in nonfunctional enzymes that eventually impair viability of the bacterium. Initially, a virtual screening campaign was carried out to identify potential LA analogues that bind to LplA2. Three selected compounds affected S. aureus USA300 growth in minimal medium at concentrations ranging from 2.5 to 10 µg/mL. Further analysis of the most potent compound (Lpl-004) revealed its transfer to E2 subunits of dehydrogenase complexes and a negative impact on its functionality. Growth impairment caused by Lpl-004 treatment was restored by adding products of the lipoate-dependent enzyme complexes. In addition, Caenorhabditis elegans infected with LpL-004-treated USA300 demonstrated a significantly expanded lifespan compared to worms infected with untreated bacteria. Our results provide evidence that LA analogues exploiting the LA salvage pathway represent an innovative strategy for the development of novel antimicrobial substances.

The genes mgtE and spoVG are involved in zinc tolerance of Staphylococcus aureus.

Metals are essential for all living organisms, but the type of metal and its concentration determines its action. Even low concentrations of metals may have toxic effects on organisms and therefore exhibit antimicrobial activities. In this study, we investigate the evolutionary adaptation processes of Staphylococcus aureus to metals and common genes for metal tolerance. Laboratory and clinical isolates were treated with manganese, cobalt, zinc, or nickel metal salts to generate growth-adapted mutants. After growth in medium supplemented with zinc, whole-genome sequencing identified, among others, two genes, mgtE (SAUSA300_0910), a putative magnesium transporter and spoVG (SAUSA300_0475), a global transcriptional regulator, as hot spots for stress-induced single-nucleotide polymorphisms (SNPs). SNPs in mgtE were also detected in mutants treated with high levels of cobalt or nickel salts. To investigate the effect of these genes on metal tolerance, deletion mutants and complementation strains in an S. aureus USA300 LAC* laboratory strain were generated. Both, the mgtE and spoVG deletion strains were more tolerant to cobalt, manganese, and zinc. The mgtE mutant was also more tolerant to nickel exposure. Inductively coupled plasma mass spectrometry analysis demonstrated that the mgtE deletion mutant accumulated less intracellular zinc than the wild type, explaining increased tolerance. From these results, we conclude that mgtE gene inactivation increases zinc tolerance presumably due to reduced uptake of zinc. For the SpoVG mutant, no direct effect on the intracellular zinc concentration was detected, indicating toward different pathways to increase tolerance. Importantly, inactivation of these genes offers a growth advantage in environments containing certain metals, pointing toward a common tolerance mechanism. IMPORTANCE: Staphylococcus aureus is an opportunistic pathogen causing tremendous public health burden and high mortality in invasive infections. Treatment is becoming increasingly difficult due to antimicrobial resistances. The use of metals in animal husbandry and aquaculture to reduce bacterial growth and subsequent acquisition of metal resistances has been shown to co-select for antimicrobial resistance. Therefore, understanding adaptive mechanisms that help S. aureus to survive metal exposure is essential. Using a screening approach, we were able to identify two genes encoding the transporter MgtE and the transcriptional regulator SpoVG, which conferred increased tolerance to specific metals such as zinc when inactivated. Further testing showed that the deletion of mgtE leads to reduced intracellular zinc levels, suggesting a role in zinc uptake. The accumulation of mutations in these genes when exposed to other metals suggests that inactivation of these genes could be a common mechanism for intrinsic tolerance to certain metals.

Multidimensional calibration spaces in Staphylococcus Aureus detection using chitosan-based genosensors and electronic tongue.

Mastitis diagnosis can be made by detecting Staphylococcus aureus (S. aureus), which requires high sensitivity and selectivity. Here, we report on microfluidic genosensors and electronic tongues to detect S. aureus DNA using impedance spectroscopy with data analysis employing visual analytics and machine learning techniques. The genosensors were made with layer-by-layer films containing either 10 bilayers of chitosan/chondroitin sulfate or 8 bilayers of chitosan/sericin functionalized with an active layer of cpDNA S. aureus. The specific interactions leading to hybridization in these genosensors allowed for a low limit of detection of 5.90 × 10-19 mol/L. The electronic tongue had four sensing units made with 6-bilayer chitosan/chondroitin sulfate films, 10-bilayer chitosan/chondroitin sulfate, 8-bilayer chitosan/sericin, and 8-bilayer chitosan/gold nanoparticles modified with sericin. Despite the absence of specific interactions, various concentrations of DNA S. aureus could be distinguished when the impedance data were plotted using a dimensionality reduction technique. Selectivity of S. aureus DNA was confirmed using multidimensional calibration spaces, based on machine learning, with accuracy up to 89 % for the genosensors and 66 % for the electronic tongue. Hence, with these computational methods one may opt for the more expensive genosensors or the simpler and cheaper electronic tongue, depending on the sensitivity level required to diagnose mastitis.