Nontraditional Roles of Magnesium Ions in Modulating Sav2152: Insight from a Haloacid Dehalogenase-like Superfamily Phosphatase from Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg2+ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg2+ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg2+ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg2+ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.

SaLTy: a novel Staphylococcus aureus Lineage Typer.

Staphylococcus aureus asymptomatically colonises 30 % of humans but can also cause a range of diseases, which can be fatal. In 2017 S. aureus was associated with 20 000 deaths in the USA alone. Dividing S. aureus isolates into smaller sub-groups can reveal the emergence of distinct sub-populations with varying potential to cause infections. Despite multiple molecular typing methods categorising such sub-groups, they do not take full advantage of S. aureus genome sequences when describing the fundamental population structure of the species. In this study, we developed Staphylococcus aureus Lineage Typing (SaLTy), which rapidly divides the species into 61 phylogenetically congruent lineages. Alleles of three core genes were identified that uniquely define the 61 lineages and were used for SaLTy typing. SaLTy was validated on 5000 genomes and 99.12 % (4956/5000) of isolates were assigned the correct lineage. We compared SaLTy lineages to previously calculated clonal complexes (CCs) from BIGSdb (n=21 173). SALTy improves on CCs by grouping isolates congruently with phylogenetic structure. SaLTy lineages were further used to describe the carriage of Staphylococcal chromosomal cassette containing mecA (SCCmec) which is carried by methicillin-resistant S. aureus (MRSA). Most lineages had isolates lacking SCCmec and the four largest lineages varied in SCCmec over time. Classifying isolates into SaLTy lineages, which were further SCCmec typed, allowed SaLTy to describe high-level MRSA epidemiology. We provide SaLTy as a simple typing method that defines phylogenetic lineages (https://github.com/LanLab/SaLTy). SaLTy is highly accurate and can quickly analyse large amounts of S. aureus genome data. SaLTy will aid the characterisation of S. aureus populations and ongoing surveillance of sub-groups that threaten human health.

The 3' UTR of vigR is required for virulence in Staphylococcus aureus and has expanded through STAR sequence repeat insertions.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are alarmingly common, and treatment is confined to last-line antibiotics. Vancomycin is the treatment of choice for MRSA bacteremia, and treatment failure is often associated with vancomycin-intermediate S. aureus isolates. The regulatory 3' UTR of the vigR mRNA contributes to vancomycin tolerance and upregulates the autolysin IsaA. Using MS2-affinity purification coupled with RNA sequencing, we find that the vigR 3' UTR also regulates dapE, a succinyl-diaminopimelate desuccinylase required for lysine and peptidoglycan synthesis, suggesting a broader role in controlling cell wall metabolism and vancomycin tolerance. Deletion of the 3' UTR increased virulence, while the isaA mutant is completely attenuated in a wax moth larvae model. Sequence and structural analyses of vigR indicated that the 3' UTR has expanded through the acquisition of Staphylococcus aureus repeat insertions that contribute sequence for the isaA interaction seed and may functionalize the 3' UTR.

Exploration of agr types, virulence-associated genes, and biofilm formation ability in Staphylococcus aureus isolates from hemodialysis patients with vascular access infections.

Introduction: Staphylococcus aureus, is a pathogen commonly encountered in both community and hospital settings. Patients receiving hemodialysis treatment face an elevated risk of vascular access infections (VAIs) particularly Staphylococcus aureus, infection. This heightened risk is attributed to the characteristics of Staphylococcus aureus, , enabling it to adhere to suitable surfaces and form biofilms, thereby rendering it resistant to external interventions and complicating treatment efforts. Methods: Therefore this study utilized PCR and microtiter dish biofilm formation assay to determine the difference in the virulence genes and biofilm formation among in our study collected of 103 Staphylococcus aureus, isolates from hemodialysis patients utilizing arteriovenous grafts (AVGs), tunneled cuffed catheters (TCCs), and arteriovenous fistulas (AVFs) during November 2013 to December 2021. Results: Our findings revealed that both MRSA and MSSA isolates exhibited strong biofilm production capabilities. Additionally, we confirmed the presence of agr types and virulence genes through PCR analysis. The majority of the collected isolates were identified as agr type I. However, agr type II isolates displayed a higher average number of virulence genes, with MRSA isolates exhibiting a variety of virulence genes. Notably, combinations of biofilm-associated genes, such as eno-clfA-clfB-fib-icaA-icaD and eno-clfA-clfB-fib-fnbB-icaA-icaD, were prevalent among Staphylococcus aureus, isolates obtained from vascular access infections. Discussion: These insights contribute to a better understanding of the molecular characteristics associated with Staphylococcus aureus, infections in hemodialysis patients and provided more targeted and effective treatment approaches.

Evaluating the impact of genomic epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) on hospital infection prevention and control decisions.

Genomic epidemiology enhances the ability to detect and refute methicillin-resistant Staphylococcus aureus (MRSA) outbreaks in healthcare settings, but its routine introduction requires further evidence of benefits for patients and resource utilization. We performed a 12 month prospective study at Cambridge University Hospitals NHS Foundation Trust in the UK to capture its impact on hospital infection prevention and control (IPC) decisions. MRSA-positive samples were identified via the hospital microbiology laboratory between November 2018 and November 2019. We included samples from in-patients, clinic out-patients, people reviewed in the Emergency Department and healthcare workers screened by Occupational Health. We sequenced the first MRSA isolate from 823 consecutive individuals, defined their pairwise genetic relatedness, and sought epidemiological links in the hospital and community. Genomic analysis of 823 MRSA isolates identified 72 genetic clusters of two or more isolates containing 339/823 (41 %) of the cases. Epidemiological links were identified between two or more cases for 190 (23 %) individuals in 34/72 clusters. Weekly genomic epidemiology updates were shared with the IPC team, culminating in 49 face-to-face meetings and 21 written communications. Seventeen clusters were identified that were consistent with hospital MRSA transmission, discussion of which led to additional IPC actions in 14 of these. Two outbreaks were also identified where transmission had occurred in the community prior to hospital presentation; these were escalated to relevant IPC teams. We identified 38 instances where two or more in-patients shared a ward location on overlapping dates but carried unrelated MRSA isolates (pseudo-outbreaks); research data led to de-escalation of investigations in six of these. Our findings provide further support for the routine use of genomic epidemiology to enhance and target IPC resources.

Unveilling genetic profiles and correlations of biofilm-associated genes, quorum sensing, and antibiotic resistance in Staphylococcus aureus isolated from a Malaysian Teaching Hospital.

BACKGROUND: Staphylococcus aureus is a notorious multidrug resistant pathogen prevalent in healthcare facilities worldwide. Unveiling the mechanisms underlying biofilm formation, quorum sensing and antibiotic resistance can help in developing more effective therapy for S. aureus infection. There is a scarcity of literature addressing the genetic profiles and correlations of biofilm-associated genes, quorum sensing, and antibiotic resistance among S. aureus isolates from Malaysia. METHODS: Biofilm and slime production of 68 methicillin-susceptible S. aureus (MSSA) and 54 methicillin-resistant (MRSA) isolates were determined using a a plate-based crystal violet assay and Congo Red agar method, respectively. The minimum inhibitory concentration values against 14 antibiotics were determined using VITEK® AST-GP67 cards and interpreted according to CLSI-M100 guidelines. Genetic profiling of 11 S. aureus biofilm-associated genes and agr/sar quorum sensing genes was performed using single or multiplex polymerase chain reaction (PCR) assays. RESULTS: In this study, 75.9% (n = 41) of MRSA and 83.8% (n = 57) of MSSA isolates showed strong biofilm-forming capabilities. Intermediate slime production was detected in approximately 70% of the isolates. Compared to MSSA, significantly higher resistance of clindamycin, erythromycin, and fluoroquinolones was noted among the MRSA isolates. The presence of intracellular adhesion A (icaA) gene was detected in all S. aureus isolates. All MSSA isolates harbored the laminin-binding protein (eno) gene, while all MRSA isolates harbored intracellular adhesion D (icaD), clumping factors A and B (clfA and clfB) genes. The presence of agrI and elastin-binding protein (ebpS) genes was significantly associated with biofilm production in MSSA and MRSA isolates, respectively. In addition, agrI gene was also significantly correlated with oxacillin, cefoxitin, and fluoroquinolone resistance. CONCLUSIONS: The high prevalence of biofilm and slime production among MSSA and MRSA isolates correlates well with the detection of a high prevalence of biofilm-associated genes and agr quorum sensing system. A significant association of agrI gene was found with cefoxitin, oxacillin, and fluoroquinolone resistance. A more focused approach targeting biofilm-associated and quorum sensing genes is important in developing new surveillance and treatment strategies against S. aureus biofilm infection.

Small regulatory RNA RSaX28 promotes virulence by reinforcing the stability of RNAIII in community-associated ST398 clonotype Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a notorious pathogen that cause metastatic or complicated infections. Hypervirulent ST398 clonotype strains, remarkably increased in recent years, dominated Community-associated S. aureus (CA-SA) infections in the past decade in China. Small RNAs like RNAIII have been demonstrated to play important roles in regulating the virulence of S. aureus, however, the regulatory roles played by many of these sRNAs in the ST398 clonotype strains are still unclear. Through transcriptome screening and combined with knockout phenotype analysis, we have identified a highly transcribed sRNA, RSaX28, in the ST398 clonotype strains. Sequence analysis revealed that RSaX28 is highly conserved in the most epidemic clonotypes of S. aureus, but its high transcription level is particularly prominent in the ST398 clonotype strains. Characterization of RSaX28 through RACE and Northern blot revealed its length to be 533nt. RSaX28 is capable of promoting the hemolytic ability, reducing biofilm formation capacity, and enhancing virulence of S. aureus in the in vivo murine infection model. Through IntaRNA prediction and EMSA validation, we found that RSaX28 can specifically interact with RNAIII, promoting its stability and positively regulating the translation of downstream alpha-toxin while inhibiting the translation of Sbi, thereby regulating the virulence and biofilm formation capacity of the ST398 clonotype strains. RSaX28 is an important virulence regulatory factor in the ST398 clonotype S. aureus and represents a potential important target for future treatment and immune intervention against S. aureus infections.

Occurrence, antimicrobial susceptibility, and resistance genes of Staphylococcus aureus in milk and milk products in the Arsi highlands of Ethiopia.

BACKGROUND: In Ethiopia, milk production and handling practices often lack proper hygiene measures, leading to the potential contamination of milk and milk products with Staphylococcus aureus (S. aureus), including methicillin-resistant strains, posing significant public health concerns. This study aimed to investigate the occurrence, antimicrobial susceptibility profiles and presence of resistance genes in S. aureus strains isolated from milk and milk products. METHODS: A cross-sectional study was conducted in the Arsi highlands, Oromia, Ethiopia from March 2022 to February 2023. A total of 503 milk and milk product samples were collected, comprising 259 raw milk, 219 cottage cheese, and 25 traditional yogurt samples. S. aureus isolation and coagulase-positive staphylococci enumeration were performed using Baird-Parker agar supplemented with tellurite and egg yolk. S. aureus was further characterized based on colony morphology, Gram stain, mannitol fermentation, catalase test, and coagulase test. Phenotypic antimicrobial resistance was assessed using the Kirby-Bauer disc diffusion method, while the polymerase chain reaction (PCR) was employed for confirming the presence of S. aureus and detecting antimicrobial resistance genes. RESULTS: S. aureus was detected in 24.9% of the milk and milk products, with the highest occurrence in raw milk (40.9%), followed by yogurt (20%), and cottage cheese (6.4%). The geometric mean for coagulase-positive staphylococci counts in raw milk, yogurt, and cottage cheese was 4.6, 3.8, and 3.2 log10 CFU/mL, respectively. Antimicrobial resistance analysis revealed high levels of resistance to ampicillin (89.7%) and penicillin G (87.2%), with 71.8% of the isolates demonstrating multidrug resistance. Of the 16 S. aureus isolates analyzed using PCR, all were found to carry the nuc gene, with the mecA and blaZ genes detected in 50% of these isolates each. CONCLUSION: This study revealed the widespread distribution of S. aureus in milk and milk products in the Arsi highlands of Ethiopia. The isolates displayed high resistance to ampicillin and penicillin, with a concerning level of multidrug resistance. The detection of the mecA and blaZ genes in selected isolates is of particular concern, highlighting a potential public health hazard and posing a challenge to effective antimicrobial treatment. These findings highlight the urgent need to enhance hygiene standards in milk and milk product handling and promote the rational use of antimicrobial drugs. Provision of adequate training for all individuals involved in the dairy sector can help minimize contamination. These measures are crucial in addressing the threats posed by S. aureus, including methicillin-resistant strains, and ensuring the safety of milk and its products for consumers.

A platform for predicting mechanism of action based on bacterial transcriptional responses identifies an unusual DNA gyrase inhibitor.

In the search for much-needed new antibacterial chemical matter, a myriad of compounds have been reported in academic and pharmaceutical screening endeavors. Only a small fraction of these, however, are characterized with respect to mechanism of action (MOA). Here, we describe a pipeline that categorizes transcriptional responses to antibiotics and provides hypotheses for MOA. 3D-printed imaging hardware PFIboxes) profiles responses of Escherichia coli promoter-GFP fusions to more than 100 antibiotics. Notably, metergoline, a semi-synthetic ergot alkaloid, mimics a DNA replication inhibitor. In vitro supercoiling assays confirm this prediction, and a potent analog thereof (MLEB-1934) inhibits growth at 0.25 µg/mL and is highly active against quinolone-resistant strains of methicillin-resistant Staphylococcus aureus. Spontaneous suppressor mutants map to a seldom explored allosteric binding pocket, suggesting a mechanism distinct from DNA gyrase inhibitors used in the clinic. In all, the work highlights the potential of this platform to rapidly assess MOA of new antibacterial compounds.

Relapsing bronchopneumonia due to community-associated methicillin-resistant Staphylococcus aureus: a case report.

BACKGROUND: The emergence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has increased the incidence of community-onset MRSA infection. Respiratory tract infections caused by MRSA has been noted for their severity; however, repeated relapses that require extended antibiotic therapy are rare. CASE PRESENTATION: We report a case of relapsing bronchopneumonia caused by CA-MRSA in a 56-year-old man. The patient responded to antibiotics, but repeatedly relapsed after stopping treatment. MRSA was consistently isolated from airway specimens during each relapse. Extended oral antibiotic treatment with trimethoprim/sulfamethoxazole (TMP/SMX) for 6 months achieved infection control. Whole-genome sequencing of the isolated strain revealed that the causative agent was sequence type (ST)1/staphylococcal cassette chromosome mec (SCCmec) type IVa, a clone that is rapidly increasing in Japan. DISCUSSION AND CONCLUSIONS: This patient had an unusual course of MRSA bronchopneumonia with repeated relapses. Although the choice of antibiotics for long-term use in MRSA respiratory tract infections has not been well established, TMP/SMX was effective and well tolerated for long-term therapy in this case. The clinical course of infections related to the rapid emerging clone, ST1/SCCmec type IVa warrants further attention.

Accessory Gene Regulator (agr) group polymorphisms in methicillin-resistant Staphylococcus aureus and its association with biofilm formation.

 Methicillin-resistant Staphylococcus aureus (MRSA) is one of the main causes of community- and hospital-acquired infections. The expression of virulence genes in S. aureus is arranged by regulators like the accessory gene regulator (agr). The present study aims to estimate phenotypic characteristics of S. aureus and investigate the occurrence of agr genes with their correlation to biofilm formation. In this study, 34 MRSA strains out of 100 S. aureus isolates were recovered in a variety of clinical samples. Phenotypic characterization and ability of biofilm formation were assessed. About 8(24%) of isolates were biofilm producers. The percentages of biofilm production among isolates were 3(37.5%), 2(25%), 3(37.5%) as strong, moderate, and weak, respectively. Furthermore, the resistance rates for all antibiotics were higher in biofilm producers and 76% of the isolates were staphyloxanthin producers, around 82% of the strains showed resistance to H2O2. Hemolytic activity was detected in 74% of the total isolates. The activity of the protease enzyme was 68%. The lipase enzyme was active in 79% of the tested S. aureus isolates. The majority of isolates were established to be agrI 84%, followed by agrII 53%, agrIII 32%, and 30% of the isolates have agr IV. Our study indicated that the majority of MRSA isolates were non-biofilm producers and the agr I is the most dominant type. Thus, agr I is not correlated with biofilm production.

Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to next-generation ß-lactams (NGBs) such as methicillin, nafcillin, and oxacillin. Resistance to NGBs, which is alternatively known as broad-spectrum ß-lactam resistance, is classically mediated by PBP2a, a penicillin-binding protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus spp. serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA-deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as methicillin-resistant lacking mec (MRLM), are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs, can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance toward NGBs at levels comparable to those of MRSAs. Our study provides a fresh new perspective about alternative mechanisms of NGB resistance, challenging our current overall understanding of high-level, broad-spectrum ß-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach toward diagnosis and treatment of ß-lactam-resistant S. aureus infections. IMPORTANCE: In Staphylococcus aureus, high-level, broad-spectrum resistance to ß-lactams such as methicillin, also referred to as methicillin resistance, is largely attributed to mecA. This study demonstrates that S. aureus strains that lack mecA but contain mutations that functionally alter PBP4 and GdpP can also mediate high-level, broad-spectrum resistance to ß-lactams. Resistance brought about by the synergistic action of functionally altered PBP4 and GdpP was phenotypically comparable to that displayed by mecA, as seen by increased bacterial survival in the presence of ß-lactams. An analysis of mutations detected in naturally isolated strains of S. aureus revealed that a significant proportion of them had similar pbp4 and GGDEF domain protein containing phosphodiesterase (gdpP) mutations, making this study clinically significant. This study not only identifies important players of non-classical mechanisms of ß-lactam resistance but also indicates reconsideration of current clinical diagnosis and treatment protocols of S. aureus infections.

The Staphylococcus aureus regulatory program in a human skin-like environment.

Staphylococcus aureus is a Gram-positive pathogen responsible for the majority of skin and soft tissue infections (SSTIs). S. aureus colonizes the anterior nares of approximately 20%-30% of the population and transiently colonizes the skin, thereby increasing the risk of developing SSTIs and more serious infections. Current laboratory models that mimic the skin surface environment are expensive, require substantial infrastructure, and limit the scope of bacterial physiology studies under human skin conditions. To overcome these limitations, we developed a cost-effective, open-source, chemically defined media recipe termed skin-like medium (SLM) that incorporates key aspects of the human skin surface environment and supports growth of several staphylococcal species. We utilized SLM to investigate the transcriptional response of methicillin-resistant Staphylococcus aureus (MRSA) following growth in SLM compared to a commonly used laboratory media. Through RNA-seq analysis, we observed the upregulation of several virulence factors, including genes encoding functions involved in adhesion, proteolysis, and cytotoxicity. To further explore these findings, we conducted quantitative reverse transcription-PCR (qRT-PCR) experiments to determine the influence of media composition, pH, and temperature on the transcriptional response of key factors involved in adhesion and virulence. We also demonstrated that MRSA primed in SLM adhered better to human corneocytes and demonstrated adhesin-specific phenotypes that previously required genetic manipulation. This improved adherence to corneocytes was dependent on both acidic pH and growth in SLM. These results support the potential utility of SLM as an in vitro model for assessing staphylococcal physiology and metabolism on human skin. IMPORTANCE: Staphylococcus aureus is the major cause of skin diseases, and its increased prevalence in skin colonization and infections present a need to understand its physiology in this environment. The work presented here outlines S. aureus upregulation of colonization and virulence factors using a newly developed medium that strives to replicate the human skin surface environment and demonstrates roles for adhesins clumping factor A (ClfA), serine-rich repeat glycoprotein adhesin (SraP), and the fibronectin binding proteins (Fnbps) in human corneocyte adherence.

An essential protease, FtsH, influences daptomycin resistance acquisition in Enterococcus faecalis.

Daptomycin is a last-line antibiotic commonly used to treat vancomycin-resistant Enterococci, but resistance evolves rapidly and further restricts already limited treatment options. While genetic determinants associated with clinical daptomycin resistance (DAPR) have been described, information on factors affecting the speed of DAPR acquisition is limited. The multiple peptide resistance factor (MprF), a phosphatidylglycerol-modifying enzyme involved in cationic antimicrobial resistance, is linked to DAPR in pathogens such as methicillin-resistant Staphylococcus aureus. Since Enterococcus faecalis encodes two paralogs of mprF and clinical DAPR mutations do not map to mprF, we hypothesized that functional redundancy between the paralogs prevents mprF-mediated resistance and masks other evolutionary pathways to DAPR. Here, we performed in vitro evolution to DAPR in mprF mutant background. We discovered that the absence of mprF results in slowed DAPR evolution and is associated with inactivating mutations in ftsH, resulting in the depletion of the chaperone repressor HrcA. We also report that ftsH is essential in the parental, but not in the ΔmprF, strain where FtsH depletion results in growth impairment in the parental strain, a phenotype associated with reduced extracellular acidification and reduced ability for metabolic reduction. This presents FtsH and HrcA as enticing targets for developing anti-resistance strategies.

Utility of Methicillin-Resistant Staphylococcus aureus Nasal PCR Testing in Pediatric Patients With Suspected Respiratory Infections.

Observational studies in adults suggest nasal methicillin-resistant Staphylococcus aureus (MRSA) swabs have a high negative predictive value (NPV) for ruling out MRSA pneumonia, however, pediatric data are limited. This retrospective study of 505 pediatric patients found a 99.8% NPV among children with suspected respiratory infections.

Characterization of Methicillin-resistant Staphylococcus aureus (MRSA) isolated in Santa Catarina (SC), Brazil.

The study investigated the characteristics of Methicillin-resistant Staphylococcus aureus (MRSA) isolated in Santa Catarina. Findings revealed prevalent SCCmecII and IV, multiresistance, Leucocidin ED genes, and one ST105 isolate. The results indicated that the in-state MRSA isolates showed the same characteristics as the out-of-state isolates among the investigated features.

Distribution of Staphylococcus aureus carriage among healthcare workers in a Japanese convalescent and rehabilitation hospital.

Earlobes, nasal cavities, and fingers of 145 healthcare workers in convalescent and rehabilitation hospital (60 nurses and 85 rehabilitation healthcare workers) were sampled. Of the 3 sites sampled, Staphylococcus aureus was detected in one or more sites in 25 nurses and 27 rehabilitation workers. S. aureus was detected in all 3 sites in 2 (8.0%) nurses and 2 (7.4%) rehabilitation workers, and the S. aureus isolates in each case showed related PFGE pattern. S. aureus was detected in both the fingers and nasal cavities of 5 (18.5%) of the rehabilitation healthcare workers; in all 5 cases, the PFGE patterns of the S. aureus isolates from each site belonged to same cluster based on PFGE. Of the 2 cases in which methicillinresistant S. aureus (MRSA) was recovered from earlobes, fingers, and nasal cavities, in both cases, MRSA isolates from the 3 sites were the same clone according to PFGE analysis and SCCmec type IV. As S. aureus was detected in pierced earlobes of nurses, hand hygiene must be practiced after touching pierced earlobes and before patient contact. The same S. aureus clone in the nasal cavity and earlobes indicates that the route of transmission is through the fingers.

Emergence and clonal expansion of a qacA-harbouring sequence type 45 lineage of methicillin-resistant Staphylococcus aureus.

The past decade has seen an increase in the prevalence of sequence type (ST) 45 methicillin-resistant Staphylococcus aureus (MRSA), yet the underlying drivers for its emergence and spread remain unclear. To better understand the worldwide dissemination of ST45 S. aureus, we performed phylogenetic analyses of Australian isolates, supplemented with a global population of ST45 S. aureus genomes. Our analyses revealed a distinct lineage of multidrug-resistant ST45 MRSA harbouring qacA, predominantly found in Australia and Singapore. Bayesian inference predicted that the acquisition of qacA occurred in the late 1990s. qacA was integrated into a structurally variable region of the chromosome containing Tn552 (carrying blaZ) and Tn4001 (carrying aac(6')-aph(2")) transposable elements. Using mutagenesis and in vitro assays, we provide phenotypic evidence that qacA confers tolerance to chlorhexidine. These findings collectively suggest both antimicrobial resistance and the carriage of qacA may play a role in the successful establishment of ST45 MRSA.

The functional small RNA interactome reveals targets for the vancomycin-responsive sRNA RsaOI in vancomycin-tolerant Staphylococcus aureus.

Small RNAs have been found to control a broad range of bacterial phenotypes including tolerance to antibiotics. Vancomycin tolerance in multidrug resistance Staphylococcus aureus is correlated with dysregulation of small RNAs although their contribution to antibiotic tolerance is poorly understood. RNA-RNA interactome profiling techniques are expanding our understanding of sRNA-mRNA interactions in bacteria; however, determining the function of these interactions for hundreds of sRNA-mRNA pairs is a major challenge. At steady-state, protein and mRNA abundances are often highly correlated and lower than expected protein abundance may indicate translational repression of an mRNA. To identify sRNA-mRNA interactions that regulate mRNA translation, we examined the correlation between gene transcript abundance, ribosome occupancy, and protein levels. We used the machine learning technique self-organizing maps (SOMs) to cluster genes with similar transcription and translation patterns and identified a cluster of mRNAs that appeared to be post-transcriptionally repressed. By integrating our clustering with sRNA-mRNA interactome data generated in vancomycin-tolerant S. aureus by RNase III-CLASH, we identified sRNAs that may be mediating translational repression. We have confirmed sRNA-dependant post-transcriptional repression of several mRNAs in this cluster. Two of these interactions are mediated by RsaOI, a sRNA that is highly upregulated by vancomycin. We demonstrate the regulation of HPr and the cell-wall autolysin Atl. These findings suggest that RsaOI coordinates carbon metabolism and cell wall turnover during vancomycin treatment. IMPORTANCE: The emergence of multidrug-resistant Staphylococcus aureus (MRSA) is a major public health concern. Current treatment is dependent on the efficacy of last-line antibiotics like vancomycin. The most common cause of vancomycin treatment failure is strains with intermediate resistance or tolerance that arise through the acqusition of a diverse repertoire of point mutations. These strains have been shown to altered small RNA (sRNA) expression in response to antibiotic treatment. Here, we have used a technique termed RNase III-CLASH to capture sRNA interactions with their target mRNAs. To understand the function of these interactions, we have looked at RNA and protein abundance for mRNAs targeted by sRNAs. Messenger RNA and protein levels are generally well correlated and we use deviations from this correlation to infer post-transcriptional regulation and the function of individual sRNA-mRNA interactions. Using this approach we identify mRNA targets of the vancomycin-induced sRNA, RsaOI, that are repressed at the translational level. We find that RsaOI represses the cell wall autolysis Atl and carbon transporter HPr suggestion a link between vancomycin treatment and suppression of cell wall turnover and carbon metabolism.

Allosteric Probe-Based Colorimetric Assay for Direct Identification and Sensitive Analysis of Methicillin Resistance of Staphylococcus aureus.

The accurate and rapid detection of methicillin-resistance of Staphylococcus aureus (SA) holds significant clinical importance. However, the methicillin-resistance detection strategies commonly require complicated cell lysis and gene extraction. Herein, we devised a novel colorimetric approach for the sensitive and accurate identification of methicillin-resistance of SA by combining allosteric probe-based target recognition with self-primer elongation-based target recycling. The PBP2a aptamer in the allosteric probe successfully identified the target MRSA, leading to the initiation of self-primer elongation based-cascade signal amplification. The peroxidase-like hemin/G-quadruplex undergo an isothermal autonomous process that effectively catalyzes the oxidation of ABTS2- and produces a distinct blue color, enabling the visual identification of MRSA at low concentrations. The method offers a shorter duration for bacteria cultivation compared to traditional susceptibility testing methods, as well as simplified manual procedures for gene analysis. The overall amplification time for this test is 60 min, and it has a detection limit of 3 CFU/ml. In addition, the approach has exceptional selectivity and reproducibility, demonstrating commendable performance when tested with real samples. Due to its advantages, this colorimetric assay exhibits considerable potential for integration into a sensor kit, thereby offering a viable and convenient alternative for the prompt and on-site detection of MRSA in patients with skin and soft tissue infections.