Optimizing phage-based mutant recovery and minimizing heat effect in the construction of transposon libraries in Staphylococcus aureus.

Staphylococcus aureus (S. aureus), particularly Methicillin-resistant S. aureus (MRSA), poses a significant global public health threat, necessitating advanced methodologies to enhance our understanding of this organism at the omics levels. This study introduces a refined protocol for constructing and curing high-density transposon mutant (tn-mutant) libraries in S. aureus, addressing the challenges associated with low transductant yields, and the complex genetic manipulation mechanism in Gram-positive bacteria. Our methodology employs a Himar1 transposon based on a two-plasmid system, leveraging Himar1's high insertional efficiency in AT-rich organisms. Enhanced transduction efficiency was achieved through chloramphenicol pre-treatment and the use of modified enriched media. Complementing this, an optimized plasmid curing procedure ensured a representative and stable tn-mutant library. The protocol was successfully applied to multiple S. aureus strains, demonstrating an increase in mutant recovery and reduced post-curing impact. The method offers a robust approach for Transposon Insertion Sequencing (TIS) applications in S. aureus, enabling deeper insights into survival, resistance, and pathogenicity mechanisms. This protocol holds a significant potential for accelerating the construction of tn-mutant libraries in various S. aureus strains.

Occurrence, genetic diversity, and antimicrobial resistance of methicillin-resistant Staphylococcus spp. in hospitalized and non-hospitalized cats in Brazil.

Methicillin-resistant Staphylococci (MRS) cause infections at various sites and exhibit multidrug resistance. Despite their importance in veterinary medicine, only little is known about Staphylococcus spp. colonizing and infecting cats. Therefore, in this study, we aimed to isolate and identify Staphylococcus spp. colonizing hospitalized and non-hospitalized domestic cats and analyze their antimicrobial resistance profiles, genetic diversity, and risk factors associated with MRS colonization. A total of 218 oral and axillary swabs were obtained from 109 cats, including 77 non-hospitalized and 32 hospitalized cats. After plating on selective media, the isolates were identified via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and rpoB and 16S rRNA gene sequencing. Subsequently, antimicrobial sensitivity of the strains was assessed, and they were screened for mecA gene. Methicillin-resistant S. haemolyticus (MRSH) isolates were subjected to multilocus sequence typing, whereas methicillin-resistant S. pseudintermedius (MRSP) and S. felis isolates were subjected to whole genome sequencing. S. felis was most commonly isolated from non-hospitalized cats (28.1%), whereas S. pseudintermedius and MRS were commonly isolated from hospitalized cats (25%). MRSH isolates from hospitalized animals were classified as ST3. The identified MRSP strains belonged to two well-known sequence types, ST551 and ST71. Moreover, antimicrobial use (p = 0.0001), hospitalization (p = 0.0141), and comorbidities (p = 0.002) were associated with increased MRS prevalence in cats.

The roles of cell wall inhibition responsive protein CwrA in the pathogenicity of Staphylococcus aureus.

The ability to form robust biofilms and secrete a diverse array of virulence factors are key pathogenic determinants of Staphylococcus aureus, causing a wide range of infectious diseases. Here, we characterized cwrA as a VraR-regulated gene encoding a cell wall inhibition-responsive protein (CwrA) using electrophoretic mobility shift assays. We constructed cwrA deletion mutants in the genetic background of methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains. Phenotypic analyses indicated that deletion of cwrA led to impaired biofilm formation, which was correlated with polysaccharide intercellular adhesin (PIA). Besides, the results of real-time quantitative PCR (RT-qPCR) and ß-galactosidase activity assay revealed that CwrA promoted biofilm formation by influence the ica operon activity in S. aureus. Furthermore, cwrA deletion mutants released less extracellular DNA (eDNA) in the biofilm because of their reduced autolytic activity compared to the wild-type (WT) strains. We also found that cwrA deletion mutant more virulence than the parental strain because of its enhanced hemolytic activity. Mechanistically, this phenotypic alteration is related to activation of the SaeRS two-component system, which positively regulates the transcriptional levels of genes encoding membrane-damaging toxins. Overall, our results suggest that CwrA plays an important role in modulating biofilm formation and hemolytic activity in S. aureus.

Role of Xpert PCR kit in assessing MRSA colonization in medical and surgical units of a tertiary care teaching hospital.

BACKGROUND AND RATIONALE: Methicillin resistant Staphylococcus aureus (MRSA) colonization increases the risk of MRSA infection. Detecting MRSA colonization can influence postoperative outcomes and prolong hospital stay. The conventional standard culture method for detecting MRSA colonization has limitations in terms of sensitivity and turnaround time. Hence, we sought out use of Xpert PCR kit for prompt evaluation of MRSA colonization to support MRSA prevention in a tertiary care hospital in Karachi, Pakistan. MATERIALS AND METHODS: During 1st April-31st December 2022, 290 nasal and skin swab samples were collected from 257 patients and processed using routine culture (as gold standard method) and PCR-based MRSA detection assay (MRSA Xpert). RESULTS: A total of two hundred and ninety (290) swab samples from 257 patients were obtained, 33 of which were paired. The overall prevalence of MRSA colonization was 12% by both methods, with 90% of cases classified as community-associated (CA-MRSA) whereas 10% as hospital-acquired (HA-MRSA). The colonized group showed a higher subsequent MRSA infection rate (11% vs. 3.5%) compared to the noncolonized group. Culture identified 11% of screening samples as MRSA positive, Xpert MRSA assay showed 100% sensitivity and 95% specificity. The cost of a single MRSA Xpert assay was $50 while MRSA culture cost around $7.50. CONCLUSION: Our study findings suggest that the presence of MRSA colonization in our cohort of patients is consistent with the existing trends in hospital epidemiology. Both conventional culture and Xpert MRSA methods showed comparable efficacy for detection of MRSA colonization. Larger-scale studies are recommended to validate these findings conclusively.

Phagemid-based capsid system for CRISPR-Cas13a antimicrobials targeting methicillin-resistant Staphylococcus aureus.

In response to the escalating antibiotic resistance in multidrug-resistant pathogens, we propose an innovative phagemid-based capsid system to generate CRISPR-Cas13a-loaded antibacterial capsids (AB-capsids) for targeted therapy against multidrug-resistant Staphylococcus aureus. Our optimized phagemid system maximizes AB-capsid yield and purity, showing a positive correlation with phagemid copy number. Notably, an 8.65-fold increase in copy number results in a 2.54-fold rise in AB-capsid generation. Phagemids carrying terL-terS-rinA-rinB (prophage-encoded packaging site genes) consistently exhibit high packaging efficiency, and the generation of AB-capsids using lysogenized hosts with terL-terS deletion resulted in comparatively lower level of wild-type phage contamination, with minimal compromise on AB-capsid yield. These generated AB-capsids selectively eliminate S. aureus strains carrying the target gene while sparing non-target strains. In conclusion, our phagemid-based capsid system stands as a promising avenue for developing sequence-specific bactericidal agents, offering a streamlined approach to combat antibiotic-resistant pathogens within the constraints of efficient production and targeted efficacy.

The genetic relationship between human and pet isolates: a core genome multilocus sequence analysis of multidrug-resistant bacteria.

INTRODUCTION: The global increase of multidrug-resistant organisms (MDROs) is one of the most urgent public health threats affecting both humans and animals. The One Health concept emphasizes the interconnectedness of human, animal and environmental health and highlights the need for integrated approaches to combat antimicrobial resistance (AMR). Although the sharing of environments and antimicrobial agents between companion animals and humans poses a risk for MDRO transmission, companion animals have been studied to a lesser extent than livestock animals. This study therefore used core genome multilocus sequence typing (cgMLST) to investigate the genetic relationships and putative transmission of MDROs between humans and pets. METHODS: This descriptive integrated typing study included 252 human isolates, 53 dog isolates and 10 cat isolates collected from 2019 to 2022 at the Charité University Hospital in Berlin, Germany. CgMLST was performed to characterize methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and multidrug-resistant gram-negative bacteria. The genetic diversity of the MDROs of the different host populations was determined and compared based on sequence type and core genome complex type. RESULTS: Within this study the majority of samples from pets and humans was genetically distinct. However, for some isolates, the number of allelic differences identified by cgMLST was low. Two cases of putative household transmission or shared source of VR E. faecium and MDR E. coli between humans and pets were documented. CONCLUSIONS: The interaction between humans and their pets appears to play a minor role in the spread of the MDROs studied. However, further research is needed. This study emphasizes the importance of comprehensive molecular surveillance and a multidisciplinary One Health approach to understand and contain the spread of MDROs in human and animal populations. TRIAL REGISTRATION: The study is registered with the German Clinical Trials Register (DRKS00030009).

Genomic portraits of methicillin-resistant staphylococci (MRS) from food fish unveiled the genes associated with staphylococcal food poisoning (SFP), virulence and antimicrobial resistance.

BACKGROUND: Characteristics of non-clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) especially from fishery environment are poorly understood. This research, in addition to comprehensive characterisation, sought to delineate the genetic relatedness between the MRSA strains originating from clinical as well as non-clinical settings. Out of 39 methicillin-resistant staphylococcal isolates from 197 fish samples, 6 (Three each of methicillin-resistant S. haemolyticus (MRSH) and MRSA) with distinct resistance profiles were selected for whole-genome sequencing. Using respective bioinformatics tools, MRSA genomes were comprehensively characterized for resistome, virulomes, molecular epidemiology and phylogenetic analysis. Simultaneously, MRSH genomes were specifically examined to characterize antimicrobial resistance genes (ARGs), owing to the fact that MRSH is often recognized as a reservoir for resistance determinants. RESULTS: Three MRSA clones identified in this study include ST672-IVd/t13599 (sequence type-SCCmec type/spa type), ST88-V/t2526, and ST672-IVa/t1309. Though, the isolates were phenotypically vancomycin-sensitive, five of the six genomes carried vancomycin resistance genes including the VanT (VanG cluster) or VanY (VanM cluster). Among the three MRSA, only one harbored the gene encoding Panton-Valentine Leukocidin (PVL) toxin, while staphylococcal enterotoxin (SEs) genes such as sea and seb, associated with staphylococcal food poisoning were identified in two other MRSA. Genomes of MRSH carried a composite of type V staphylococcal cassette chromosome mec (SCCmec) elements (5C2 & 5). This finding may be explained by the inversion and recombination events that may facilitate the integration of type V elements to the SCC elements of S. aureus with a methicillin-susceptible phenotype. Phylogenetically, MRSA from a non-clinical setting displayed a considerable relatedness to that from clinical settings. CONCLUSION: This study highlights the genetic diversity and resistance profiles of MRSA and MRSH, with non-clinical MRSA showing notable relatedness to clinical strains. Future research should explore resistance gene transfer mechanisms and environmental reservoirs to better manage MRSA spread.

Inducible clindamycin-resistant and biofilm formation in the Staphylococcus aureus isolated from healthcare worker's anterior nasal carriage.

OBJECTIVE: The purpose of this study is a new update on the resistance profile, Macrolide-Lincosamide-Streptogramin B resistance mechanisms and biofilm formation in the Staphylococcus aureus isolated from health care workers (HCWs) nasal carriage at a children's teaching hospital in Babol (Northern Iran). RESULTS: A total of 143 non-repetitive nasal swab samples were collected from volunteers, where 53.8% (n; 77/143) were HCWs, 33.6% (n; 48/143) medical students, and 12.6% (n; 18/143) resident students. The prevalence of nasal carriers of S. aureus was 22.4% (n; 32/143), among them, 40.6% (n; 13/32) were identified as methicillin-resistant Staphylococcus aureus (MRSA( carriers. Antimicrobial susceptibility testing showed that erythromycin (68.8%, n; 22/32) and ciprofloxacin (15.6%, n; 5/32) had the highest and lowest resistance rate, respectively. The frequency of resistance genes in the strains was as follows; ermC (n; 17/32, 53.1%), ermA (n; 11/32, 34.4%), ermB (n; 6/32, 18.7%), ereA (n; 3/32, 9.4%). Moreover, 50.0% (n; 16/32), 28.1% (n; 9/32) and 21.8% (n; 7/32) of isolates were strongly, weakly and moderately biofilm producer, respectively. Macrolides-lincosamides-streptogramins B (MLSB) antibiotic resistance among S. aureus isolates from HCWs nasal carriage have found significant prevalence rates throughout the globe. It is crucial to remember that the development of biofilms and MLS B antibiotic resistance are both dynamic processes.

Proven anti-virulence therapies in combating methicillin- and vancomycin-resistant Staphylococcus aureus infections.

Introduction: Despite years of efforts to develop new antibiotics for eradicating multidrug-resistant (MDR) and multi-virulent Methicillin-Resistant Staphylococcus aureus (MRSA) and Vancomycin-Resistant Staphylococcus aureus (VRSA) infections, treatment failures and poor prognoses in most cases have been common. Therefore, there is an urgent need for new therapeutic approaches targeting virulence arrays. Our aim is to discover new anti-virulence therapies targeting MRSA and VRSA virulence arrays. Methodology: We employed phenotypic, molecular docking, and genetic studies to screen for anti-virulence activities among selected promising compounds: Coumarin, Simvastatin, and Ibuprofen. Results: We found that nearly all detected MRSA and VRSA strains exhibited MDR and multi-virulent profiles. The molecular docking results aligned with the phenotypic and genetic assessments of virulence production. Biofilm and hemolysin productions were inhibited, and all virulence genes were downregulated upon treatment with sub-minimum inhibitory concentration (sub-MIC) of these promising compounds. Ibuprofen was the most active compound, exhibiting the highest inhibition and downregulation of virulence gene products. Moreover, in vivo and histopathological studies confirmed these results. Interestingly, we observed a significant decrease in wound area and improvements in re-epithelialization and tissue organization in the Ibuprofen and antimicrobial treated group compared with the group treated with antimicrobial alone. These findings support the idea that a combination of Ibuprofen and antimicrobial drugs may offer a promising new therapy for MRSA and VRSA infections. Conclusion: We hope that our findings can be implemented in clinical practice to assist physicians in making the most suitable treatment decisions.

Evaluation of virulence determinants and cell surface properties associated with biofilm formation in methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrum beta-lactamase (ESBL) Escherichia coli from livestock and poultry origin.

Antibiotic resistance poses a persistent threat to modern medicine due to the emergence of novel antibiotic-resistant strains. Therefore, a timely understanding of antibiotic resistance and the virulence biology of pathogenic bacteria, particularly those of public health significance, is crucial for implementing effective mitigation strategies. This study aimed to investigate the virulence profiles of ten S. aureus isolates (NDa to NDj) and ten E. coli isolates (ND1 to ND10) originating from livestock and poultry, and to assess how various cell surface properties and biofilm formation abilities influence antibiotic resistance phenotypes. Antibiotic resistance profiling through phenotypic (AST) and genotypic methods (PCR) confirmed that NDa to NDe were methicillin-resistant S. aureus (MRSA) and ND1 to ND5 were extended-spectrum ß-lactamase (ESBL) producing E. coli isolates. Virulence properties such as hemolytic activity, coagulase activity, and nuclease activity were found to be independent of the antibiotic resistance phenotype in S. aureus. In contrast, biofilm formation phenotype was observed to influence antibiotic resistance phenotypes, with MRSA and ESBL E. coli isolates demonstrating higher biofilm formation potency. Chemical and enzymatic analysis of S. aureus and E. coli biofilms revealed proteins and polysaccharides as major components, followed by nucleic acids. Furthermore, cell surface properties such as auto-aggregation and hydrophobicity were notably higher in isolates with strong to medium biofilm-forming capabilities (ESBL and MRSA isolates), corroborated by genomic confirmation of various genes associated with biofilm, adhesion, and colonization. In conclusion, this study highlights that surface hydrophobicity and biofilm formation ability of MRSA (NDa to NDe) and ESBL E. coli (ND1 to ND5) isolates may influence antibiotic resistance phenotypes.

Methicillin resistant Staphylococcus aureus mazEF expression promotes infections by influencing cellular growth, antibiotic sensitivity, and formation of biofilms.

Staphylococcus aureus infections are hard to treat due to the emergence of antibiotic resistant strains, as well as their ability to form biofilms. The MazEF toxin-antitoxin system is thought play a role in bacterial biofilm phenotype as well as antibiotic resistance. In S. aureus, the physiologic function of the mazEF gene in the disease transition from acute to chronic infection is not well understood. In methicillin resistant S. aureus (MRSA), loss of mazF expression results in loss of resistance to first generation cephalosporins. mazF::tn displayed sensitivity while the isogenic wild type (WT) remained resistant. mazF::tn displayed significantly increased growth of biofilms on metal implants over 48 h compared to WT and the complemented transposon mutant. mazF::tn biofilms displayed significantly decreased antibiotic tolerance to vancomycin and cefazolin in comparison to WT and complement biofilms. Mice given mazF::tn in a sepsis model displayed less abscess burden and increased survival (100%) when treated with cefazolin compared to WT bacteremia treated with cefazolin (20%). mazF::tn periprosthetic joint infections displayed increased biofilm burden at acute time points and decreased biofilm burden at chronic time points. Our data suggests MazEF in MRSA is responsible for controlling growth of biofilms, antibiotic tolerance, and influence chronic infections in vivo.

Unraveling novel mutation patterns and morphological variations in two dalbavancin-resistant MRSA strains in Austria using whole genome sequencing and transmission electron microscopy.

BACKGROUND: The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) strains resistant to non-beta-lactam antimicrobials poses a significant challenge in treating severe MRSA bloodstream infections. This study explores resistance development and mechanisms in MRSA isolates, especially after the first dalbavancin-resistant MRSA strain in our hospital in 2016. METHODS: This study investigated 55 MRSA bloodstream isolates (02/2015-02/2021) from the University Hospital of the Medical University of Vienna, Austria. The MICs of dalbavancin, linezolid, and daptomycin were assessed. Two isolates (16-33 and 19-362) resistant to dalbavancin were analyzed via whole-genome sequencing, with morphology evaluated using transmission electron microscopy (TEM). RESULTS: S.aureus BSI strain 19-362 had two novel missense mutations (p.I515M and p.A606D) in the pbp2 gene. Isolate 16-33 had a 534 bp deletion in the DHH domain of GdpP and a SNV in pbp2 (p.G146R). Both strains had mutations in the rpoB gene, but at different positions. TEM revealed significantly thicker cell walls in 16-33 (p < 0.05) compared to 19-362 and dalbavancin-susceptible strains. None of the MRSA isolates showed resistance to linezolid or daptomycin. CONCLUSION: In light of increasing vancomycin resistance reports, continuous surveillance is essential to comprehend the molecular mechanisms of resistance in alternative MRSA treatment options. In this work, two novel missense mutations (p.I515M and p.A606D) in the pbp2 gene were newly identified as possible causes of dalbavancin resistance.

Clinical, demographic characteristics and antimicrobial resistance profile of Staphylococcus aureus isolated in clinical samples from pediatric patients in a tertiary hospital in Rio de Janeiro: 7-year longitudinal study.

BACKGROUND: In the pediatric population, Staphylococcus aureus infections are responsible for increased morbidity and mortality, length of hospitalization and the cost of inpatient treatment. The aim of this study is to describe the antimicrobial resistance profile of S. aureus isolated in clinical specimens from pediatric patients admitted to a tertiary hospital in Rio de Janeiro, Brazil. METHODS: Culture reports and medical records of hospitalized patients under 18 years of age with S. aureus infections between January 2015 and December 2022 were retrospectively analyzed. Information was collected on recent antibiotic use, previous hospital admission, inpatient unit, clinical specimen, time of infection (community or nosocomial), classification according to susceptibility to methicillin (methicillin sensitive - MSSA or methicillin resistant - MRSA) and sensitivity to other antimicrobials. We analyzed the distribution of the sensitivity profile of S. aureus infections over the 7 years evaluated in the study. RESULTS: Were included 255 unique clinical episodes, among which the frequencies of MSSA and MRSA were 164 (64%) and 91 (36%), respectively. Over the 7 years evaluated, there was stability in the prevalence percentage, with a predominance of MSSA in the range of 60 to 73.3%, except in 2020, when there was a drop in the prevalence of MSSA (from 73.3% in 2019 to 52.5%) with an increase in MRSA (from 26.7% in 2019 to 47.5%). Ninety-seven (38%) infections were community-acquired and 158 (62%) were healthcare-associated. The main clinical specimens collected were blood cultures (35.2%) and wound secretions (17%). The MRSA isolates presented percentages of sensitivity to trimethoprim-sulfamethoxazole from 90.4 to 100%, and to clindamycin from 77 to 89.8% in MRSA healthcare associated and MRSA community respectively. CONCLUSION: There was a constant predominance in the prevalence of MSSA with percentage values ​​maintained from 2015 to 2022, except in 2020, in which there was a specific drop in the prevalence of MSSA with an increase in MRSA. MSSA infections are still predominant in the pediatric population, but MRSA rates also present significant values, including in community infections, and should be considered in initial empiric therapy.

TMT-Based Quantitative Proteomics Revealed the Antibacterial Mechanism of Cinnamaldehyde against MRSA.

Natural plant extracts have demonstrated significant potential in alternative antibiotic therapies. Cinnamaldehyde (CA) has garnered considerable attention as a natural antibacterial agent. In this study, Tandem mass tag (TMT) quantitative proteomics combined with Western blot and RT-qPCR methods were employed to explore the antibacterial mechanism of CA against Methicillin-Resistant Staphylococcus aureus (MRSA) at the protein level. The results showed that a total of 254 differentially expressed proteins (DEPs) were identified in the control group and CA treatment group, of which 161 were significantly upregulated and 93 were significantly downregulated. DEPs related to nucleotide synthesis, homeostasis of the internal environment, and protein biosynthesis were significantly upregulated, while DEPs involved in the cell wall, cell membrane, and virulence factors were significantly downregulated. The results of GO and KEGG enrichment analyses demonstrated that CA could exert its antibacterial effects by influencing pyruvate metabolism, the tricarboxylic acid (TCA) cycle, teichoic acid biosynthesis, and the Staphylococcus aureus (S. aureus) infection pathway in MRSA. CA significantly inhibited the expression of recombinant protein MgrA (p < 0.05), significantly reduced the mRNA transcription levels of mgrA, hla, and sdrD genes (p < 0.05), and thermostability migration assays demonstrated that CA can directly interact with MgrA protein, thereby inhibiting its activity. These findings suggest that CA exerts its antibacterial mechanism by regulating the expression of related proteins, providing a theoretical basis for further development of clinical applications of antimicrobial agents derived from natural plant essential oils in the treatment of dairy cow mastitis.

CRISPR-Responsive RCA-Based DNA Hydrogel Biosensing Platform with Customizable Signal Output for Rapid and Sensitive Nucleic Acid Detection.

Current nucleic acid-responsive DNA hydrogels face significant challenges, such as the requirement for high target concentrations, frequent redesigns, and increased costs, which limit their practical applications in biosensing. To address these issues, we developed a novel biosensing platform integrating a CRISPR/Cas12a system into an RCA-based DNA hydrogel. The hydrogel used in the platform could preencapsulate diverse signal molecules comprising GelRed, methylene blue, and gold nanoparticles, which were released upon Cas12a-mediated cleavage. This design enabled customizable signal output, including fluorescence, electrochemistry, and colorimetry, thereby ensuring the platform's adaptability to various detection scenarios. Our platform was highly specific for methicillin-resistant Staphylococcus aureus, with a mecA gene detection limit of 10 copies/µL, and provided fast and accurate results within 2 h for clinical samples. Hence, based on these advantages, the proposed biosensing platform exhibits promising application prospects in the field of nucleic acid detection.

The transmission risk of multidrug-resistant organisms between hospital patients and their pets - a case-control study, Germany, 2019 to 2022.

BackgroundCarriage of multidrug-resistant organisms (MDROs) in humans constitutes an important public health concern. Cross-transmission of bacteria between animals and humans has been demonstrated before.AimOur aim was to quantify the risk factor 'pet ownership' for MDRO colonisation in hospital patients.MethodsWe performed a matched case-control study from 2019 to 2022 in Berlin, Germany and compared MDRO-positive and MDRO-negative patients in terms of contact with pets and other risk factors for MDRO acquisition. Patients completed a questionnaire-based interview and provided nasal and rectal swabs. Pet owners provided swab samples from the throat and stool of their pets (dogs and cats). Phenotypically matching samples of owners and pets were analysed via whole genome sequencing.ResultsThe analyses included 2,891 patients. Reported pet ownership was 17.7% in MDRO-positives (154/871) and 23.4% in MDRO-negatives (472/2,020). Among 397 owner-pet pairs, we identified one pair sharing genotypically indistinguishable pathogens (0.3%). A risk factor analysis of pet ownership was performed for carriers of meticillin-resistant Staphylococcus aureus (MRSA) (OR = 0.662; 95% CI: 0.343-1.277), vancomycin-resistant enterococci (VRE) (OR = 0.764; 95% CI: 0.522-1.118) and multidrug-resistant Gram-negative bacteria (MDR-GNB) (OR = 0.819; 95% CI: 0.620-1.082). Colonisation with MDRO was rare in pets, and dogs were more often colonised than cats (MRSA: 0% vs 0%, VRE: 1.5% vs 1.0%, MDR-GNB: 17.2% vs 3.6%).ConclusionTransmission of MDROs between humans and pets is possible though rare. In an urban living space, neither cat nor dog ownership appears as a relevant risk factor for MDRO carriage in hospital patients.

Functional and Proteomic Dissection of the Contributions of CodY, SigB and the Hibernation Promoting Factor HPF to Interactions of Staphylococcus aureus USA300 with Human Lung Epithelial Cells.

Staphylococcus aureus is a leading cause of severe pneumonia. Our recent proteomic investigations into S. aureus invasion of human lung epithelial cells revealed three key adaptive responses: activation of the SigB and CodY regulons and upregulation of the hibernation-promoting factor SaHPF. Therefore, our present study aimed at a functional and proteomic dissection of the contributions of CodY, SigB and SaHPF to host invasion using transposon mutants of the methicillin-resistant S. aureus USA300. Interestingly, disruption of codY resulted in a "small colony variant" phenotype and redirected the bacteria from (phago)lysosomes into the host cell cytoplasm. Furthermore, we show that CodY, SigB and SaHPF contribute differentially to host cell adhesion, invasion, intracellular survival and cytotoxicity. CodY- or SigB-deficient bacteria experienced faster intracellular clearance than the parental strain, underscoring the importance of these regulators for intracellular persistence. We also show an unprecedented role of SaHPF in host cell adhesion and invasion. Proteomic analysis of the different mutants focuses attention on the CodY-perceived metabolic state of the bacteria and the SigB-perceived environmental cues in bacterial decision-making prior and during infection. Additionally, it underscores the impact of the nutritional status and bacterial stress on the initiation and progression of staphylococcal lung infections.

Penicillin susceptibility among Staphylococcus aureus skin and soft tissue infections at a children's hospital.

Shortly after its introduction into clinical practice, Staphylococcus aureus isolates gained resistance to penicillin via the acquisition of ß-lactamases. A number of centers have recently described an increase in the proportion of invasive methicillin-susceptible S. aureus (MSSA), which are also susceptible to penicillin (PSSA). Little data are available regarding the prevalence or impact of PSSA in skin and soft tissue infections (SSTI). Community-acquired MSSA SSTI isolates were obtained through a surveillance study at Texas Children's Hospital from January 2017 to December 2021. A total of 200 random isolates underwent PCR for blaZ ß-lactamase; blaZ-negative isolates then underwent penicillin susceptibility testing using macrobroth dilution. Isolates which were blaZ negative and had a penicillin MIC ≤0.125 µg/mL were regarded as PSSA with the remainder regarded as penicillin-resistant MSSA (PR-MSSA). All PSSA underwent multilocus sequence typing. Medical records were reviewed. The median age of subjects was 4.2 years (IQR: 1.6-10.5). PSSA accounted for 9% of isolates during the study period. PSSA and PR-MSSA cases were similar with respect to age, demographics, and rates of prior antibiotic exposure. PSSA isolates less often had vancomycin MIC ≥1.5 µg/mL. Furthermore, 39% of PSSA were variants of sequence type 1. In multivariable analyses, penicillin susceptibility was independently associated with both hospital admission and surgical intervention. PSSA account for a small but significant proportion of MSSA SSTI in children. Clinically distinguishing patients with PSSA and PR-MSSA SSTI is challenging. However, PSSA SSTI were independently associated with higher rates of hospital admission as well as the need for surgical intervention suggesting a significant clinical impact.IMPORTANCEThe vast majority of Staphylococcus aureus in the US are penicillin resistant with most clinical labs no longer reporting penicillin susceptibility for this organism. A number of centers, however, have reported increasing penicillin susceptibility among invasive S. aureus infections. Skin and soft tissue infections (SSTI) are far more common than invasive infections, yet the frequency and impact of penicillin-susceptible S. aureus (PSSA) in this population are uncertain. Through active surveillance at a children's hospital, we found that 9% of methicillin-susceptible S. aureus SSTI isolates were PSSA. PSSA were independently associated with hospital admission for the management of SSTI as well as the need for debridement in the operating room. Given that most SSTI are managed in the outpatient setting, these findings suggest a clinical impact of this phenotype and the need for a reassessment of the value in susceptibility testing and potentially even treatment with penicillin.

YjbH contributes to Staphylococcus aureus skin pathology and immune response through Agr-mediated α-toxin regulation.

Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) with Methicillin-Resistant S. aureus (MRSA) strains being a major contributor in both community and hospital settings. S. aureus relies on metabolic diversity and a large repertoire of virulence factors to cause disease. This includes α-hemolysin (Hla), an integral player in tissue damage found in various models, including SSTIs. Previously, we identified a role for the Spx adapter protein, YjbH, in the regulation of several virulence factors and as an inhibitor of pathogenesis in a sepsis model. In this study, we found that YjbH is critical for tissue damage during SSTI, and its absence leads to decreased proinflammatory chemokines and cytokines in the skin. We identified no contribution of YjbI, encoded on the same transcript as YjbH. Using a combination of reporters and quantitative hemolysis assays, we demonstrated that YjbH impacts Hla expression and activity both in vitro and in vivo. Additionally, expression of Hla from a non-native promoter reversed the tissue damage phenotype of the ΔyjbIH mutant. Lastly, we identified reduced Agr activity as the likely cause for reduced Hla production in the ΔyjbH mutant. This work continues to define the importance of YjbH in the pathogenesis of S. aureus infection as well as identify a new pathway important for Hla production.