Displacement of Hospital-Acquired, Methicillin-Resistant Staphylococcus aureus Clones by Heterogeneous Community Strains in Kenya over a 13-Year Period.

We determined antibiotic susceptibility and employed Oxford Nanopore whole-genome sequencing to explore strain diversity, resistance, and virulence gene carriage among methicillin-resistant Staphylococcus aureus (MRSA) strains from different infection sites and timepoints in a tertiary Kenyan hospital. Ninety-six nonduplicate clinical isolates recovered between 2010 and 2023, identified and tested for antibiotic susceptibility on the VITEK ID/AST platform, were sequenced. Molecular typing, antibiotic resistance, and virulence determinant screening were performed using the relevant bioinformatics tools. The strains, alongside those from previous studies, were stratified into two periods covering 2010-2017 and 2018-2023 and comparisons were made. Mirroring phenotypic profiles, aac(6')-aph(2″) [aminoglycosides]; gyrA (S84L) and grlA (S80Y) [fluoroquinolones]; dfrG [anti-folates]; and tet(K) [tetracycline] resistance determinants dominated the collection. While the proportion of ST239/241-t037-SCCmec III among MRSA reduced from 37.7% to 0% over the investigated period, ST4803-t1476-SCCmec IV and ST152-t355-SCCmec IV were pre-eminent. The prevalence of Panton-Valentine leucocidin (PVL) and arginine catabolic mobile element (ACME) genes was 38% (33/87) and 6.8% (6/87), respectively. We observed the displacement of HA-MRSA ST239/241-t037-SCCmec III with the emergence of ST152-t355-SCCmec IV and a greater clonal heterogeneity. The occurrence of PVL+/ACME+ CA-MRSA in recent years warrants further investigations into their role in the CA-MRSA virulence landscape, in a setting of high PVL prevalence.

Targeting S. aureus Extracellular Vesicles: A New Putative Strategy to Counteract Their Pathogenic Potential.

Long-term inflammatory skin disease atopic dermatitis is characterized by dry skin, itching, and eczematous lesions. During inflammation skin barrier protein impairment promotes S. aureus colonisation in the inflamed skin, worsening AD patient's clinical condition. Proteomic analysis revealed the presence of several immune evasion proteins and virulence factors in S. aureus extracellular vesicles (EVs), suggesting a possible role for these proteins in the pathophysiology of atopic dermatitis. The objective of this study is to assess the efficacy of a wall fragment obtained from a patented strain of C. acnes DSM28251 (c40) and its combination with a mucopolysaccharide carrier (HAc40) in counteract the pathogenic potential of EVs produced by S. aureus ATCC 14458. Results obtained from in vitro studies on HaCaT keratinocyte cells showed that HAc40 and c40 treatment significantly altered the size and pathogenicity of S. aureus EVs. Specifically, EVs grew larger, potentially reducing their ability to interact with the target cells and decreasing cytotoxicity. Additionally, the overexpression of the tight junctions mRNA zona occludens 1 (ZO1) and claudin 1 (CLDN1) following EVs exposure was decreased by HAc40 and c40 treatment, indicating a protective effect on the epidermal barrier's function. These findings demonstrate how Hac40 and c40 may mitigate the harmful effects of S. aureus EVs. Further investigation is needed to elucidate the exact mechanisms underlying this interaction and explore the potential clinical utility of c40 and its mucopolysaccharide carrier conjugate HAc40 in managing atopic dermatitis.

Strain Differences in Bloodstream and Skin Infection: Methicillin-Resistant Staphylococcus aureus Isolated in 2018-2021 in a Single Health System.

Staphylococcus aureus is a common cause of skin and soft-tissue infections (SSTIs) and has become the most common cause of bloodstream infections (BSIs) in recent years, but whether the strains causing these two clinical syndromes overlap has not been studied adequately. USA300/500 (clonal complex [CC] 8-sequence type [ST] 8) and USA100 (CC5-ST5) have dominated among methicillin-resistant S aureus (MRSA) strains in the United States since the early 2000s. We compared the genomes of unselected MRSA isolates from 131 SSTIs with those from 145 BSIs at a single US center in overlapping periods in 2018-2021. CC8 MRSA was more common among SSTIs, and CC5 was more common among BSIs, consistent with prior literature. Based on clustering genomes with a threshold of 15 single-nucleotide polymorphisms, we identified clusters limited to patients with SSTI and separate clusters exclusively comprising patients with BSIs. However, we also identified eight clusters that included at least one SSTI and one BSI isolate. This suggests that virulent MRSA strains are transmitted from person to person locally in the healthcare setting or the community and that single lineages are often capable of causing both SSTIs and BSIs.

Pharmacotherapies for multidrug-resistant gram-positive infections: current options and beyond.

INTRODUCTION: Infections due to multidrug-resistant organisms (MDRO) are a serious concern for public health with high morbidity and mortality. Though many antibiotics have been introduced to manage these infections, there are remaining concerns regarding the optimal management of Gram-positive MDROs. AREAS COVERED: A literature search on the PubMed/Medline database was conducted. We applied no language and time limits for the search strategy. In this narrative review, we discuss the current options for managing Gram-positive MDROs as well as non-traditional antibacterial agents in development. EXPERT OPINION: Despite their introduction more than 70 years ago, glycopeptides are still the cornerstone in treating Gram-positive infections: all registrative studies of new antibiotics have glycopeptides as control; these studies are designed as not inferior studies, therefore it is almost impossible to give recommendations other than the use of glycopeptides in the treatment of Gram-positive infections. The best evidence on treatments different from glycopeptides comes from post-hoc analysis and meta-analysis. Non-traditional antibacterial agents are being studied to aid in short and effective antibiotic therapies. The use of non-traditional antibacterial agents is not restricted to replacing traditional antibacterial agents with alternative therapies; instead, they should be used in combination with antibiotic therapies.

Design, Synthesis and Bioactivity Evaluation of Ag(I)-, Au(I)- and Au(III)-Quinoxaline-Wingtip N-Heterocyclic Carbene Complexes Against Antibiotic Resistant Bacterial Pathogens.

Intending to homogenize the biological activities of both quinoxaline and imidazole moieties, the proligand, 1-methyl-3-quinoxaline-imidazolium hexaflurophosphate (1.HPF6), and [Ag(1)2][PF6], (2); [Au(1)2][PF6], (3); and [Au(1)Cl3], (4) NHC complexes were synthesized. All the synthesized compounds were characterized by elemental analysis, NMR, and UV-Vis spectroscopy. Finally, single crystal X-ray structures revealed a linear geometry for complex 2 whereas a square planar geometry for complex 4. The formation of complex 3 was confirmed and supported by its MS spectra. The antibacterial activities of all the synthesized complexes were investigated against gram-positive bacteria and gram-negative bacteria. The Au(III)-NHC complex, 4 showed the highest antibacterial activity with extremely low MIC values against both the bacterial strains (0.24 µg.mL-1).  Monitoring of zeta potential supports the higher activity of complex 4 compared to 2 and 3. ROS production by complex 4 has also been measured in vitro in the CT26 cancer cell lines, which is directly responsible for targetting and killing the bacterial pathogens. Cell cytotoxicity assay using 293T cell lines has been performed to investigate the biocompatibility nature of complex 4. Also, an excellent hemocompatibility was assigned to it from its hemolytic studies,   which provide valuable insights into the design of novel antibacterial agents.

Type III interferon drives pathogenicity to Staphylococcus aureus via the airway epithelium.

Type III interferon signaling contributes to the pathogenesis of the important human pathogen Staphylococcus aureus in the airway. Little is known of the cellular factors important in this response. Using Ifnl2-green fluorescent protein reporter mice combined with flow cytometry and cellular depletion strategies, we demonstrate that the alveolar macrophage is the primary producer of interferon lambda (IFN-λ) in response to S. aureus in the airway. Bone marrow chimeras showed reduced bacterial burden in IFN-λ receptor (IFNLR1)-deficient recipient mice, indicative that non-hematopoietic cells were important for pathogenesis, in addition to significant reductions in pulmonary inflammation. These observations were confirmed through the use of an airway epithelial-specific IFNLR knockout mouse. Our data suggest that upon entry to the airway, S. aureus activates alveolar macrophages to produce type III IFN that is subsequently sensed by the airway epithelium. Future steps will determine how signaling from the epithelium then exerts its influence on bacterial clearance. These results highlight the important, yet sometimes detrimental, role of type III IFN signaling during infection and the impact the airway epithelium plays during host-pathogen interactions.IMPORTANCEThe contribution of type III interferon signaling to the control of bacterial infections is largely unknown. We have previously demonstrated that it contributes to the pathogenesis of acute Staphylococcus aureus respiratory infection. In this report, we document the importance of two cell types that underpin this pathogenesis. We demonstrate that the alveolar macrophage is the cell that is responsible for the production of type III interferon and that this molecule is sensed by airway epithelial cells, which impacts both bacterial clearance and induction of inflammation. This work sheds light on the first two aspects of this important pathogenic cascade.

Naringin exerts antibacterial and anti-inflammatory effects on mice with Staphylococcus aureus-induced osteomyelitis.

Osteomyelitis is an invasive bone infection that can lead to severe pain and even disability, posing a challenge for orthopedic surgery. Naringin can reduce bone-related inflammatory conditions. This study aimed to elucidate the function and mechanism of naringin in a Staphylococcus aureus-induced mouse model of osteomyelitis. Femurs of S. aureus-infected mice were collected after naringin administration and subjected to microcomputed tomography to analyze cortical bone destruction and bone loss. Bacterial growth in femurs was also assessed. Proinflammatory cytokine levels in mouse femurs were measured using enzyme-linked immunosorbent assays. Pathological changes and bone resorption were analyzed using hematoxylin and eosin staining and tartrate-resistant acid phosphatase staining, respectively. Quantitative reverse transcription polymerase chain reaction and western blot analysis were used to quantify the messenger RNA and protein expression of osteogenic differentiation-associated genes in the femurs. The viability of human bone marrow-derived stem cells (hBMSCs) was determined using cell counting kit-8. Alizarin Red S staining and alkaline phosphatase staining were performed to assess the formation of mineralization nodules and bone formation in vitro. Notch signaling-related protein levels in femur tissues and hBMSCs were assessed using western blot analysis. Experimental results revealed that naringin alleviated S. aureus-induced cortical bone destruction and bone loss in mice by increasing the bone volume/total volume ratio. Naringin suppressed S. aureus-induced bacterial growth and inflammation in femurs. Moreover, it alleviated histopathological changes, inhibited bone resorption, and increased the expression of osteogenic markers in osteomyelitic mice. It increased the viability of hBMSCs and promoted their differentiation and bone mineralization in vitro. Furthermore, naringin activated Notch signaling by upregulating the protein levels of Notch1, Jagged1, and Hes1 in the femurs of model mice and S. aureus-stimulated hBMSCs. In conclusion, naringin reduces bacterial growth, inflammation, and bone resorption while upregulating the expression of osteogenic markers in S. aureus-infected mice and hBMSCs by activating Notch signaling.

Induction of acute silkworm hemolymph melanization by Staphylococcus aureus treated with peptidoglycan-degrading enzymes.

Staphylococcus aureus, a Gram-positive bacterium, causes inflammatory skin diseases, such as atopic dermatitis, and serious systemic diseases, such as sepsis. In the skin and nasal environment, peptidoglycan (PGN)-degrading enzymes, including lysozyme and lysostaphin, affects S. aureus PGN. However, the effects of PGN-degrading enzymes on the acute innate immune-inducing activity of S. aureus have not yet been investigated. In this study, we demonstrated that PGN-degrading enzymes induce acute silkworm hemolymph melanization by S. aureus. Insoluble fractions of S. aureus treated with lysozyme, lysostaphin, or both enzymes, were prepared. Melanization of the silkworm hemolymph caused by the injection of these insoluble fractions was higher than that of S. aureus without enzyme treatment. These results suggest that structural changes in S. aureus PGN caused by PGN-degrading enzymes affect the acute innate immune response in silkworms.

Antibacterial activity and mechanisms of D-3263 against Staphylococcus aureus.

Multi-drug-resistant Staphylococcus aureus infections necessitate novel antibiotic development. D-3263, a transient receptor potential melastatin member 8 (TRPM8) agonist, has potential antineoplastic properties. Here, we reported the antibacterial and antibiofilm activities of D-3263. Minimum inhibitory concentrations (MICs) against S. aureus, Enterococcus faecalis and E. faecium were ≤ 50 µM. D-3263 exhibited bactericidal effects against clinical methicillin-resistant S. aureus (MRSA) and E. faecalis strains at 4× MIC. Subinhibitory D-3263 concentrations effectively inhibited S. aureus and E. faecalis biofilms, with higher concentrations also clearing mature biofilms. Proteomic analysis revealed differential expression of 29 proteins under 1/2 × MIC D-3263, influencing amino acid biosynthesis and carbohydrate metabolism. Additionally, D-3263 enhanced membrane permeability of S. aureus and E. faecalis. Bacterial membrane phospholipids phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL) dose-dependently increased D-3263 MICs. Overall, our data suggested that D-3263 exhibited potent antibacterial and antibiofilm activities against S. aureus by targeting the cell membrane.

Virulence and resistance profiling of Staphylococcus aureus isolated from subclinical bovine mastitis in the Pakistani Pothohar region.

Mastitis is considered one of the most widespread infectious disease of cattle and buffaloes, affecting dairy herds. The current study aimed to characterize the Staphylococcus aureus isolates recovered from subclinical mastitis animals in Pothohar region of the country. A total of 278 milk samples from 17 different dairy farms around two districts of the Pothohar region, Islamabad and Rawalpindi, were collected and screened for sub clinical mastitis using California Mastitis Test. Positive milk samples were processed for isolation of Staphylococcus aureus using mannitol salt agar. The recovered isolates were analyzed for their antimicrobial susceptibility and virulence genes using disc diffusion and PCR respectively. 62.2% samples were positive for subclinical mastitis and in total 70 Staphylococcus aureus isolates were recovered. 21% of these isolates were determined to be methicillin resistant, carrying the mecA gene. S. aureus isolates recovered during the study were resistant to all first line therapeutic antibiotics and in total 52% isolates were multidrug resistant. SCCmec typing revealed MRSA SCCmec types IV and V, indicating potential community-acquired MRSA (CA-MRSA) transmission. Virulence profiling revealed high prevalence of key genes associated with adhesion, toxin production, and immune evasion, such as hla, hlb, clfA, clfB and cap5. Furthermore, the Panton-Valentine leukocidin (PVL) toxin, that is often associated with recurrent skin and soft tissue infections, was present in 5.7% of isolates. In conclusion, the increased prevalence of MRSA in bovine mastitis is highlighted by this study, which also reveals a variety of virulence factors in S. aureus and emphasizes the significance of appropriate antibiotic therapy in combating this economically burdensome disease.

A Rare and Dangerous Combination of COVID-19, Lemierre Syndrome, and Carotid Pseudoaneurysm: A Case Report.

Lemierre syndrome is a rare complication of oropharyngeal infection that causes septic thrombophlebitis in the internal jugular vein. Since the onset of the COVID-19 pandemic, this condition has been dangerously overlooked and poses an even greater threat when complicated by vascular pathologies. A case is presented where the patient required emergency endovascular exclusion of a right internal carotid artery pseudoaneurysm due to Lemierre syndrome. The treatment included stent graft placement and drainage of a neck abscess, along with appropriate antibiotic treatment during hospitalization. Recognizing this diagnosis requires a high index of suspicion, particularly during the COVID-19 pandemic. The complexity of the disease necessitates extensive multidisciplinary collaboration for effective treatment.

Polyclonal but not monoclonal circulating memory CD4+ T cells attenuate the severity of Staphylococcus aureus bacteremia.

Staphylococcus aureus bacteremia causes significant morbidity and mortality. Treatment of staphylococcal infections is hindered by widespread antibiotic resistance, and attempts to develop an S. aureus vaccine have failed. Improved S. aureus treatment and infection prevention options require a deeper understanding of the correlates of protective immunity. CD4+ T cells have been identified as key orchestrators in the defense against S. aureus, but uncertainties persist regarding the subset, polarity, and breadth of the memory CD4+ T-cell pool required for protection. Here, using a mouse model of systemic S. aureus infection, we discovered that the breadth of bacterium-specific memory CD4+ T-cell pool is a critical factor for protective immunity against invasive S. aureus infections. Seeding mice with a monoclonal bacterium-specific circulating memory CD4+ T-cell population failed to protect against systemic S. aureus infection; however, the introduction of a polyclonal and polyfunctional memory CD4+ T-cell pool significantly reduced the bacterial burden. Our findings support the development of a multi-epitope T-cell-based S. aureus vaccine, as a strategy to mitigate the severity of S. aureus bacteremia.

Fighting fibrin with fibrin: Vancomycin delivery into coagulase-mediated Staphylococcus aureus biofilms via fibrin-based nanoparticle binding.

Staphylococcus aureus skin and soft tissue infection is a common ailment placing a large burden upon global healthcare infrastructure. These bacteria are growing increasingly recalcitrant to frontline antimicrobial therapeutics like vancomycin due to the prevalence of variant populations such as methicillin-resistant and vancomycin-resistant strains, and there is currently a dearth of novel antibiotics in production. Additionally, S. aureus has the capacity to hijack the host clotting machinery to generate fibrin-based biofilms that confer protection from host antimicrobial mechanisms and antibiotic-based therapies, enabling immune system evasion and significantly reducing antimicrobial efficacy. Emphasis is being placed on improving the effectiveness of therapeutics that are already commercially available through various means. Fibrin-based nanoparticles (FBNs) were developed and found to interact with S. aureus through the clumping factor A (ClfA) fibrinogen receptor and directly integrate into the biofilm matrix. FBNs loaded with antimicrobials such as vancomycin enabled a targeted and sustained release of antibiotic that increased drug contact time and reduced the therapeutic dose required for eradicating the bacteria, both in vitro and in vivo. Collectively, these findings suggest that FBN-antibiotic delivery may be a novel and potent therapeutic tool for the treatment of S. aureus biofilm infections.

Multiplexed bacterial pathogen detection and clinical characteristics of orthopedic infection in hospitalized patients.

Introduction: Accurate identification of the etiology of orthopedic infection is very important for correct and timely clinical management, but it has been poorly studied. In the current study we explored the association of multiple bacterial pathogens with orthopedic infection. Methods: Hospitalized orthopedic patients were enrolled in a rural hospital in Qingdao, China. Wound or exudate swab samples were collected and tested for twelve bacterial pathogens with both culture and multiplex real time PCR. Results and discussion: A total of 349 hospitalized orthopedic patients were enrolled including 193 cases presenting infection manifestations upon admission and 156 with no sign of infection. Orthopedic infection patients were mainly male (72.5%) with more lengthy hospital stay (median 15 days). At least one pathogen was detected in 42.5% (82/193) of patients with infection while 7.1% (11/156) in the patients without infection (P < 0.001). S. aureus was the most prevalent causative pathogen (15.5%). Quantity dependent pathogen association with infection was observed, particularly for P. aeruginosa and K. pneumoniae, possibly indicating subclinical infection. Most of the patients with detected pathogens had a previous history of orthopedic surgery (odds ratio 2.8, P = 0.038). Pathogen specific clinical manifestations were characterized. Multiplex qPCR, because of its high sensitivity, superior specificity, and powerful quantification could be utilized in combination with culture to guide antimicrobial therapy and track the progression of orthopedic infection during treatment.

Evaluation of the mechanistic basis for the antibacterial activity of ursolic acid against Staphylococcus aureus.

The antibiotics are generally regarded as the first choice approach to treat dairy mastitis, targeting the public health problems associated with the food safety and the emergence of antibioticresistant bacteria. The objective of the study was to evaluate the antibacterial efficacy of ursolic acid (UA) when used to treat Staphylococcus aureus and other isolates associated with bovine mastitis and to clarify the mechanistic basis for these effects. The bacteriostatic properties of UA extracted from Rosmarinus officinalis L. at four different purity levels were assessed by calculating minimum inhibitory concentration (MIC) values, while the synergistic effects of combining 98% UA with antibiotics were evaluated by measuring the fractional inhibitory concentration index (FICI). Changes in biofilm formation and the growth curves of the clinical isolates were assessed to clarify the bacteriostatic effect of UA. Furthermore, the cell wall integrity, protein synthesis, and reactive oxygen species (ROS) production were assessed to determine the antibacterial mechanism of UA treatment. Ultimately, UA was revealed to exhibit robust activity against Gram-positive bacteria including S. aureus (ATCC 25923), Streptococcus dysgalactiae (ATCC27957), Streptococcus agalactiae (ATCC13813), Enterococcus faecalis (ATCC29212), and Streptococcus mutans (ATCC25175). However, it did not affect Escherichia coli (ATCC 25922). The MIC values of UA preparations that were 98, 50, 30, and 10% pure against S. aureus were 39, 312, 625, and 625 µg/mL, respectively, whereas the corresponding MIC for E. coli was >5,000 µg/mL. The minimum bactericidal concentrations of 98% UA when used to treat three clinical S. aureus isolates (S4, S5, and S6) were 78, 78, and 156 µg/mL, respectively. Levels of biofilm formation for clinical S. aureus isolates decreased with increasing 98% UA concentrations. Above the MIC dose, UA treatment resulted in the dissolution of bacterial cell walls and membranes, with cells becoming irregularly shaped and exhibiting markedly impaired intracellular protein synthesis. S. aureus treated with 98% UA was able to rapidly promote intracellular ROS biogenesis. Together, these data highlight the promising utility of UA as a compound that can be used together with other antibiotics for the treatment of infections caused by S. aureus.

A membrane-targeting magnolol derivative for the treatment of methicillin-resistant Staphylococcus aureus infections.

Multidrug-resistant bacterial infections are a major global health challenge, especially the emergence and rapid spread of methicillin-resistant Staphylococcus aureus (MRSA) urgently require alternative treatment options. Our study has identified that a magnolol derivative 6i as a promising agent with significant antibacterial activity against S. aureus and clinical MRSA isolates (MIC = 2-8 µg/mL), showing high membrane selectivity. Unlike traditional antibiotics, 6i demonstrated rapid bactericidal efficiency and a lower propensity for inducing bacterial resistance. Compound 6i also could inhibit biofilm formation and eradicate bacteria within biofilms. Mechanistic studies further revealed that 6i could target bacterial cell membranes, disrupting the integrity of the cell membrane and leading to increased DNA leakage, resulting in potent antibacterial effects. Meanwhile, 6i also showed good plasma stability and excellent biosafety. Notably, 6i displayed good in vivo antibacterial activity in a mouse skin abscess model of MRSA-16 infection, which was comparable to the positive control vancomycin. These findings indicated that the magnolol derivative 6i possessed the potential to be a novel anti-MRSA infection agent.

Meta-meta-analysis of the mortality risk associated with MRSA compared to MSSA bacteraemia.

Staphylococcus aureus bacteraemia (SAB) is a bloodstream infection that carries a high risk of exacerbating a diseased state and may result in an increased death rate. The aim of this study was to assess mortality risk in Methicillin Resistant Staphylococcus aureus (MRSA) bacteraemia compared to Methicillin Susceptible Staphylococcus aureus (MSSA) bacteraemia through meta-meta-analyses. The study followed PRISMA guidelines, conducting a comprehensive search in Scopus, PubMed, and Google Scholar. It included full-text systematic reviews and meta-analyses comparing MRSA vs. MSSA bacteraemia, excluding reviews without data pooling and unclear selection criteria. Validity was assessed using QUOROM and AMSTAR. Edwards' Venn diagrams were used to visualized overlaps between primary studies. Aggregated odds ratio (OR) and risk ratios with 95% confidence intervals were calculated using the random-effect model. Heterogeneity was evaluated using the Higgins I2 statistic. The study included 3 meta-analysis studies, a total of 38,159 patients, with 9,056 having MRSA bacteraemia and 29,103 having MSSA bacteraemia. Data were collected from 46 different outcome studies published between 2001 and 2022. The meta-analyses used 7 to 33 primary studies from 1990 to 2020, with no overlap. Odds ratios (ORs) ranged from 1.78 to 2.92, while relative risks (RR) ranged from 1.57 to 2.37 for the included meta-anlysis. The pooled analysis confirmed a higher risk of mortality in patients with MRSA bacteraemia (OR: 2.35, RR: 2.01, HR: 1.61) compared to MSSA bacteraemia. Heterogeneity among the studies was considerable (I2: 90-91%). The study strongly supports that most patient deaths from SAB are linked to MRSA rather than MSSA. This highlights the significant public health problem posed by SAB, with difficult and often unsuccessful treatment leading to increased mortality and high healthcare costs.

The mobilome of Staphylococcus aureus from wild ungulates reveals epidemiological links at the animal-human interface.

Staphylococcus aureus thrives at animal-human-environment interfaces. A large-scale work from our group indicated that antimicrobial resistance (AMR) in commensal S. aureus strains from wild ungulates is associated with agricultural land cover and livestock farming, raising the hypothesis that AMR genes in wildlife strains may originate from different hosts, namely via exchange of mobile genetic elements (MGE). In this work, we generate the largest available dataset of S. aureus draft genomes from wild ungulates in Portugal and explore their mobilome, which can determine important traits such as AMR, virulence, and host specificity, to understand MGE exchange. Core genome multi-locus sequence typing based on 98 newly generated draft genomes and 101 publicly available genomes from Portugal demonstrated that the genomic relatedness of S. aureus from wild ungulates assigned to livestock-associated sequence types (ST) is greater compared to wild ungulate isolates assigned to human-associated STs. Screening of host specificity determinants disclosed the unexpected presence in wildlife of the immune evasion cluster encoded in φSa3 prophage, described as a human-specific virulence determinant. Additionally, two plasmids, pAVX and pETB, previously associated with avian species and humans, respectively, and the Tn553 transposon were detected. Both pETB and Tn553 encode penicillin resistance through blaZ. Pangenome analysis of wild ungulate isolates shows a core genome fraction of 2133 genes, with isolates assigned to ST72 and ST3224 being distinguished from the remaining by MGEs, although there is no reported role of these in adaptation to wildlife. AMR related gene clusters found in the shell genome are directly linked to resistance against penicillin, macrolides, fosfomycin, and aminoglycosides, and they represent mobile ARGs. Altogether, our findings support epidemiological interactions of human and non-human hosts at interfaces, with MGE exchange, including AMR determinants, associated with putative indirect movements of S. aureus among human and wildlife hosts that might be bridged by livestock.

Genomic investigation and clinical correlates of the in vitro ß-lactam: NaHCO3 responsiveness phenotype among methicillin-resistant Staphylococcus aureus isolates from a randomized clinical trial.

NaHCO3 responsiveness is a novel phenotype where some methicillin-resistant Staphylococcus aureus (MRSA) isolates exhibit significantly lower minimal inhibitory concentrations (MIC) to oxacillin and/or cefazolin in the presence of NaHCO3. NaHCO3 responsiveness correlated with treatment response to ß-lactams in an endocarditis animal model. We investigated whether treatment of NaHCO3-responsive strains with ß-lactams was associated with faster clearance of bacteremia. The CAMERA2 trial (Combination Antibiotics for Methicillin-Resistant Staphylococcus aureus) randomly assigned participants with MRSA bloodstream infections to standard therapy, or to standard therapy plus an anti-staphylococcal ß-lactam (combination therapy). For 117 CAMERA2 MRSA isolates, we determined by broth microdilution the MIC of cefazolin and oxacillin, with and without 44 mM of NaHCO3. Isolates exhibiting ≥4-fold decrease in the MIC to cefazolin or oxacillin in the presence of NaHCO3 were considered "NaHCO3-responsive" to that agent. We compared the rate of persistent bacteremia among participants who had infections caused by NaHCO3-responsive and non-responsive strains, and that were assigned to combination treatment with a ß-lactam. Thirty-one percent (36/117) and 25% (21/85) of MRSA isolates were NaHCO3-responsive to cefazolin and oxacillin, respectively. The NaHCO3-responsive phenotype was significantly associated with sequence type 93, SCCmec type IVa, and mecA alleles with substitutions in positions -7 and -38 in the regulatory region. Among participants treated with a ß-lactam, there was no association between the NaHCO3-responsive phenotype and persistent bacteremia (cefazolin, P = 0.82; oxacillin, P = 0.81). In patients from a randomized clinical trial with MRSA bloodstream infection, isolates with an in vitro ß-lactam-NaHCO3-responsive phenotype were associated with distinctive genetic signatures, but not with a shorter duration of bacteremia among those treated with a ß-lactam.