CXCR2 perturbation promotes Staphylococcus aureus implant-associated infection.

Introduction. Staphylococcus aureus is the leading cause of acute medical implant infections, representing a significant modern medical concern. The success of S. aureus as a pathogen in these cases resides in its arsenal of virulence factors, resistance to multiple antimicrobials, mechanisms of immune modulation, and ability to rapidly form biofilms associated with implant surfaces. S. aureus device-associated, biofilm-mediated infections are often persistent and notoriously difficult to treat, skewing innate immune responses to promote chronic reoccurring infections. While relatively little is known of the role neutrophils play in response to acute S. aureus biofilm infections, these effector cells must be efficiently recruited to sites of infection via directed chemotaxis. Here we investigate the effects of modulating CXC chemokine receptor 2 (CXCR2) activity, predominantly expressed on neutrophils, during S. aureus implant-associated infection.Hypothesis. We hypothesize that modulation of CXCR2 expression and/or signalling activities during S. aureus infection, and thus neutrophil recruitment, extravasation and antimicrobial activity, will affect infection control and bacterial burdens in a mouse model of implant-associated infection.Aim. This investigation aims to elucidate the impact of altered CXCR2 activity during S. aureus biofilm-mediated infection that may help develop a framework for an effective novel strategy to prevent morbidity and mortality associated with implant infections.Methodology. To examine the role of CXCR2 during S. aureus implant infection, we employed a mouse model of indwelling subcutaneous catheter infection using a community-associated methicillin-resistant S. aureus (MRSA) strain. To assess the role of CXCR2 induction or inhibition during infection, treatment groups received daily intraperitoneal doses of either Lipocalin-2 (Lcn2) or AZD5069, respectively. At the end of the study, catheters and surrounding soft tissues were analysed for bacterial burdens and dissemination, and Cxcr2 transcription within the implant-associated tissues was quantified.Results. Mice treated with Lcn2 developed higher bacterial burdens within the soft tissue surrounding the implant site, which was associated with increased Cxcr2 expression. AZD5069 treatment also resulted in increased implant- and tissues-associated bacterial titres, as well as enhanced Cxcr2 expression.Conclusion. Our results demonstrate that CXCR2 plays an essential role in regulating the severity of S. aureus implant-associated infections. Interestingly, however, perturbation of CXCR2 expression or signalling both resulted in enhanced Cxcr2 transcription and elevated implant-associated bacterial burdens. Thus, CXCR2 appears finely tuned to efficiently recruit effector cells and mediate control of S. aureus biofilm-mediated infection.

Identification of kinase modulators as host-directed therapeutics against intracellular methicillin-resistant Staphylococcus aureus.

The increasing prevalence of antimicrobial-resistant Staphylococcus aureus strains, especially methicillin-resistant S. aureus (MRSA), poses a threat to successful antibiotic treatment. Unsuccessful attempts to develop a vaccine and rising resistance to last-resort antibiotics urge the need for alternative treatments. Host-directed therapy (HDT) targeting critical intracellular stages of S. aureus emerges as a promising alternative, potentially acting synergistically with antibiotics and reducing the risk of de novo drug resistance. We assessed 201 ATP-competitive kinase inhibitors from Published Kinase Inhibitor Sets (PKIS1 and PKIS2) against intracellular MRSA. Seventeen hit compounds were identified, of which the two most effective and well-tolerated hit compounds (i.e., GW633459A and GW296115X) were selected for further analysis. The compounds did not affect planktonic bacterial cultures, while they were active in a range of human cell lines of cervical, skin, lung, breast and monocyte origin, confirming their host-directed mechanisms. GW633459A, structurally related to lapatinib, exhibited an HDT effect on intracellular MRSA independently of its known human epidermal growth factor receptor (EGFR)/(HER) kinase family targets. GW296115X activated adenosine monophosphate-activated protein kinase (AMPK), thereby enhancing bacterial degradation via autophagy. Finally, GW296115X not only reduced MRSA growth in human cells but also improved the survival rates of MRSA-infected zebrafish embryos, highlighting its potential as HDT.

Staphylococcus aureus foldase PrsA contributes to the folding and secretion of protein A.

BACKGROUND: Staphylococcus aureus secretes a variety of proteins including virulence factors that cause diseases. PrsA, encoded by many Gram-positive bacteria, is a membrane-anchored lipoprotein that functions as a foldase to assist in post-translocational folding and helps maintain the stability of secreted proteins. Our earlier proteomic studies found that PrsA is required for the secretion of protein A, an immunoglobulin-binding protein that contributes to host immune evasion. This study aims to investigate how PrsA influences protein A secretion. RESULTS: We found that in comparison with the parental strain HG001, the prsA-deletion mutant HG001ΔprsA secreted less protein A. Deleting prsA also decreased the stability of exported protein A. Pulldown assays indicated that PrsA interacts with protein A in vivo. The domains in PrsA that interact with protein A are mapped to both the N- and C-terminal regions (NC domains). Additionally, the NC domains are essential for promoting PrsA dimerization. Furthermore, an immunoglobulin-binding assay revealed that, compared to the parental strain HG001, fewer immunoglobulins bound to the surface of the mutant strain HG001ΔprsA. CONCLUSIONS: This study demonstrates that PrsA is critical for the folding and secretion of protein A. The information derived from this study provides a better understanding of virulent protein export pathways that are crucial to the pathogenicity of S. aureus.

Identification of small molecules affecting the interaction between human hemoglobin and Staphylococcus aureus IsdB hemophore.

Human hemoglobin (Hb) is the preferred iron source of Staphylococcus aureus. This pathogenic bacterium exploits a sophisticated protein machinery called Iron-regulated surface determinant (Isd) system to bind Hb, extract and internalize heme, and finally degrade it to complete iron acquisition. IsdB, the surface exposed Hb receptor, is a proven virulence factor of S. aureus and the inhibition of its interaction with Hb can be pursued as a strategy to develop new classes of antimicrobials. To identify small molecules able to disrupt IsdB:Hb protein-protein interactions (PPIs), we carried out a structure-based virtual screening campaign and developed an ad hoc immunoassay to screen the retrieved set of commercially available compounds. Saturation-transfer difference (STD) NMR was applied to verify specific interactions of a sub-set of molecules, chosen based on their efficacy in reducing the amount of Hb bound to IsdB. Among molecules for which direct binding was verified, the best hit was submitted to ITC analysis to measure the binding affinity to Hb, which was found to be in the low micromolar range. The results demonstrate the viability of the proposed in silico/in vitro experimental pipeline to discover and test IsdB:Hb PPI inhibitors. The identified lead compound will be the starting point for future SAR and molecule optimization campaigns.

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.

Antibacterial effect of Moringa oleifera on Staphylococcus aureus and Pseudomonas aeruginosa isolated from raw milk and some dairy products with special reference to biofilm gene expression.

Background: Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) are well defined as food poisoning pathogens that are highly resistant and need continuous studies. Aim: The purpose of the work was to examine phenotypic and genotypic characteristics of both P. aeruginosa and S. aureus, and treatment trials with medicinal plants. Methods: Samples were examined for isolation of P. aeruginosa and S. aureus on selective media followed by biochemical confirmation, biofilm formation, genes detection, and expression of P. aeruginosa pslA biofilm gene was performed by quantitative real-time polymerase chain reaction after treatment with 0.312 mg/ml Moringa oleifera aqueous extract as a minimum inhibitory concentration. Results: The highest isolation rate of P. aeruginosa was 20% from both raw milk and Kariesh cheese, followed by 16% and 12% from ice cream and processed cheese, respectively, while the highest isolation rate of S. aureus was 36% from raw milk followed by 28% in ice cream and 16% in both Kariesh cheese and processed cheese. 30% of P. aeruginosa isolates were biofilm producers, while only 21% of S. aureus isolates were able to produce biofilm. The P. aeruginosa isolates harbor virulence-associated genes nan1, exoS, toxA, and pslA at 100%, 80%, 40%, and 40%, respectively. Staphylococcus aureus SEs genes were examined in S. aureus strains, where SEA and SEB genes were detected with 60%, but no isolate harbored SEC, SED, or SEE. The significant fold change of P. aeruginosa pslA expression was 0.40332 after treatment with M. oleifera aqueous extract. Conclusion: Pseudomonas aeruginosa and S. aureus harbor dangerous virulence genes that cause food poisoning, but M. oleifera extract could minimize their action.

Screening of antibiogram, virulence factors, and biofilm production of Staphylococcus aureus and the bio-control role of some probiotics as alternative antibiotics.

Background: Food safety is a serious challenge in the face of increasing population and diminishing resources. Staphylococcus aureus is a critical foodborne pathogen characterized by its capability to secret a diverse range of heat-resistant enterotoxins. Antibiotic usage in dairy herds resulted in the occurrence of antimicrobial resistance (AMR) patterns among bacterial species, which were consequently transmitted to humans via dairy products. Lactic acid bacteria (LAB) produce bacteriocins, which provide an excellent source of natural antimicrobials with the further advantage of being environmentally friendly and safe. Aim: Detection of multidrug resistance (MDR) S. aureus isolates in concerned samples, molecular characteristics, biofilm production, and the inhibitory role of LAB against it. Methods: Random samples of raw milk and other dairy products were analyzed for S. aureus isolation. Phenotypic and genotypic assessment of AMR was performed, in addition to detection of classical enterotoxin genes of S. aureus. Finally, evaluation of the antimicrobial action of some Lactobacillus strains against S. aureus. Results: Incidence rates of presumptive S. aureus in raw milk, Kariesh cheese, and yogurt samples were 50%, 40%, and 60%, respectively. The highest resistance of S. aureus was to Kanamycin (100%) and Nalidixic acid (89.3%), respectively. (78.66%) of S. aureus were MDR. 11.1% of S. aureus carried mecA gene. In concern with enterotoxins genes, PCR showed that examined isolates harbored sea with a percentage of (22.2%), while sed was found in (11.1%) of isolates. Regarding biofilm production, (88.88%) of S. aureus were biofilm producers. Finally, agar well diffusion showed that Lactobacillus acidophilus had the strongest antimicrobial action against S. aureus with inhibition zone diameter ranging from 18 to 22 mm. Conclusion: There is a widespread prevalence of MDR S. aureus in raw milk and dairy products. Production of staphylococcal enterotoxins, as well as biofilm production are responsible for public health risks. Therefore, installing proper hygienic routines and harsh food safety policies at food chain levels is substantial.

Expression of norA, norB and norC efflux pump genes mediating fluoroquinolones resistance in MRSA isolates.

INTRODUCTION: Although fluoroquinolones are used to treat methicillin-resistant Staphylococcus aureus (MRSA)-induced infections, acquisition of antibiotic resistance by bacteria has impaired their clinical relevance. We aimed to evaluate the frequency of norA, norB, and norC efflux pump genes-mediating fluoroquinolones resistance and measure their expression levels in MRSA isolates. METHODOLOGY: 126 S. aureus isolates were collected from different clinical samples of adult hospitalized patients and identified by conventional microbiological methods. MRSA was diagnosed by cefoxitin disc diffusion method and minimum inhibitory concentration (MIC) of ciprofloxacin by broth microdilution method. The expression levels of efflux pump genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: 80 (63.5%) MRSA isolates were identified and showed high level of resistance to erythromycin (80%), gentamicin (75%), clindamycin (65%) and ciprofloxacin (60 %). norA, norB and norC were detected in 75%, 35% and 55% of the MRSA isolates respectively. norC was the most commonly overexpressed gene measured by qRT-PCR, occurring in 40% of MRSA isolates, followed by norA (35%) and norB (30%). The expression of these genes was significantly higher in ciprofloxacin-resistant than quantitative real-time PCR ciprofloxacin-sensitive MRSA isolates. CONCLUSIONS: This study showed high prevalence and overexpression of efflux pump genes among MRSA isolates which indicates the significant role of these genes in the development of multidrug resistance against antibiotics including fluoroquinolones.

Pathogen group-specific risk factors for intramammary infection in water buffalo.

A cross-sectional study was conducted to estimate the prevalence of intramammary infection (IMI) associated bacteria and to identify risk factors for pathogen group-specific IMI in water buffalo in Bangladesh. A California Mastitis Test (CMT) and bacteriological cultures were performed on 1,374 quarter milk samples collected from 763 water buffalo from 244 buffalo farms in nine districts in Bangladesh. Quarter, buffalo, and farm-related data were obtained through questionnaires and visual observations. A total of 618 quarter samples were found to be culture positive. Non-aureus staphylococci were the predominant IMI-associated bacterial species, and Staphylococcus (S.) chromogenes, S. hyicus, and S. epidermidis were the most common bacteria found. The proportion of non-aureus staphylococci or Mammaliicoccus sciuri (NASM), S. aureus, and other bacterial species identified in the buffalo quarter samples varied between buffalo farms. Therefore, different management practices, buffalo breeding factors, and nutrition were considered and further analyzed when estimating the IMI odds ratio (OR). The odds of IMI by any pathogen (OR: 1.8) or by NASM (OR: 2.2) was high in buffalo herds with poor milking hygiene. Poor cleanliness of the hind quarters had a high odds of IMI caused by any pathogen (OR: 2.0) or NASM (OR: 1.9). Twice daily milking (OR: 3.1) and farms with buffalo purchased from another herd (OR: 2.0) were associated with IMI by any pathogen. Asymmetrical udders were associated with IMI-caused by any bacteria (OR: 1.7). A poor body condition score showed higher odds of IMI by any pathogen (OR: 1.4) or by NASM (OR: 1.7). This study shows that the prevalence of IMI in water buffalo was high and varied between farms. In accordance with the literature, our data highlight that IMI can be partly controlled through better farm management, primarily by improving hygiene, milking management, breeding, and nutrition.

Staphylococcus haemolyticus: An updated review on nosocomial infections, antimicrobial resistance, virulence, genetic traits, and strategies for combating this emerging opportunistic pathogen.

Staphylococcus haemolyticus, a key species of the Staphylococcus genus, holds significant importance in healthcare-associated infections, due to its notable resistance to antimicrobials, like methicillin, and proficient biofilms-forming capabilities. This coagulase-negative bacterium poses a substantial challenge in the battle against nosocomial infections. Recent research has shed light on Staph. haemolyticus genomic plasticity, unveiling genetic elements responsible for antibiotic resistance and their widespread dissemination within the genus. This review presents an updated and comprehensive overview of the clinical significance and prevalence of Staph. haemolyticus, underscores its zoonotic potential and relevance in the one health framework, explores crucial virulence factors, and examines genetics features contributing to its success in causing emergent and challenging infections. Additionally, we scrutinize ongoing studies aimed at controlling spread and alternative approaches for combating it.

Bacteriophage vB_SepP_134 and Endolysin LysSte_134_1 as Potential Staphylococcus-Biofilm-Removing Biological Agents.

Bacteria of the genus Staphylococcus are significant challenge for medicine, as many species are resistant to multiple antibiotics and some are even to all of the antibiotics we use. One of the approaches to developing new therapeutics to treat staphylococcal infections is the use of bacteriophages specific to these bacteria or the lytic enzymes of such bacteriophages, which are capable of hydrolyzing the cell walls of these bacteria. In this study, a new bacteriophage vB_SepP_134 (St 134) specific to Staphylococcus epidermidis was described. This podophage, with a genome of 18,275 bp, belongs to the Andhravirus genus. St 134 was able to infect various strains of 12 of the 21 tested coagulase-negative Staphylococcus species and one clinical strain from the Staphylococcus aureus complex. The genes encoding endolysin (LysSte134_1) and tail tip lysin (LysSte134_2) were identified in the St 134 genome. Both enzymes were cloned and produced in Escherichia coli cells. The endolysin LysSte134_1 demonstrated catalytic activity against peptidoglycans isolated from S. aureus, S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus warneri. LysSte134_1 was active against S. aureus and S. epidermidis planktonic cells and destroyed the biofilms formed by clinical strains of S. aureus and S. epidermidis.

Injectable Phage-Loaded Microparticles Effectively Release Phages to Kill Methicillin-Resistant Staphylococcus aureus.

The increasing prevalence of bacterial multidrug antibiotic resistance has led to a serious threat to public health, emphasizing the urgent need for alternative antibacterial therapeutics. Lytic phages, a class of viruses that selectively infect and kill bacteria, offer promising potential as alternatives to antibiotics. However, injectable carriers with a desired release profile remain to be developed to deliver them to infection sites. To address this challenge, phage-loaded microparticles (Phage-MPs) have been developed to deliver phages to the infection site and release phages for an optimal therapeutic effect. The Phage-MPs are synthesized by allowing phages to be electrostatically attached onto the porous polyethylenimine-modified silk fibroin microparticles (SF-MPs). The high specific surface area of SF-MPs allows them to efficiently load phages, reaching about 1.25 × 1010 pfu per mg of microparticles. The Phage-MPs could release phages in a controlled manner to achieve potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Unlike the diffuse biodistribution of free phages post-intraperitoneal injection, Phage-MPs could continuously release phages to effectively boost the local phage concentration at the bacterial infection site after they are intraperitoneally injected into an abdominal MRSA-infected mouse model. In a mouse abdominal MRSA infection model, Phage-MPs significantly reduce the bacterial load in major organs, achieving an efficient therapeutic effect. Furthermore, Phage-MPs demonstrate outstanding biocompatibility both in vitro and in vivo. Overall, our research lays the foundation for a new generation of phage-based therapies to combat antibiotic-resistant bacterial infections.

Contribution of staphylococcal virulence factors in the pathogenesis of thrombosis.

Staphylococci are responsible for many infections in humans, starting with skin and soft tissue infections and finishing with invasive diseases such as endocarditis, sepsis and pneumonia, which lead to high mortality. Patients with sepsis often demonstrate activated clotting pathways, decreased levels of anticoagulants, decreased fibrinolysis, activated endothelial surfaces and activated platelets. This results in disseminated intravascular coagulation and formation of a microthrombus, which can lead to a multiorgan failure. This review describes various staphylococcal virulence factors that contribute to vascular thrombosis, including deep vein thrombosis in infected patients. The article presents mechanisms of action of different factors released by bacteria in various host defense lines, which in turn can lead to formation of blood clots in the vessels.

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.

Prevalence of Staphylococcus argenteus among food handlers, kitchen utensils, and food samples in Japan.

Staphylococcus argenteus has received increased attention from an aspect of food safety since several food poisoning outbreaks caused by the bacterium were reported in Japan. However, S. argenteus prevalence among food handlers and utensils has not yet been investigated. In this study, we investigated S. argenteus prevalence among a collection of coagulase-positive staphylococci (CPS) that were isolated during food sanitary inspections in Japan. Out of a total of 191 CPS isolates, 14 were identified as S. argenteus. One was isolated from shelled shrimp, nine were isolated from food handlers' hand swabs, and four were isolated from kitchen utensils. Whole-genome sequencing revealed that transmission of S. argenteus from human hands to utensils was possible. Though all 14 isolates were negative for the pvl and tst-1 genes, 6 harbored the seb gene. Only 21.4% of S. argenteus isolates were resistant to antibiotics, while 62.1% of the S. aureus isolates from the same sources were confirmed to be resistant. To the best of our knowledge, this is the first report to demonstrate possible transmission of S. argenteus from food handlers to utensils in food-processing environments.

Detection of Staphylococcus aureus virulence gene pvl based on CRISPR strip.

Introduction: Staphylococcus aureus (S. aureus) is a prominent pathogen responsible for both hospital-acquired and community-acquired infections. Among its arsenal of virulence factors, Panton-Valentine Leucocidin (PVL) is closely associated with severe diseases such as profound skin infections and necrotizing pneumonia. Patients infected with pvl-positive S. aureus often exhibit more severe symptoms and carry a substantially higher mortality risk. Therefore, it is crucial to promptly and accurately detect pvl-positive S. aureus before initiating protective measures and providing effective antibacterial treatment. Methods: In this study, we propose a precise identification and highly sensitive detection method for pvl-positive S. aureus based on recombinase-assisted amplification and the CRISPR-ERASE strip which we previously developed. Results: The results revealed that this method achieved a detection limit of 1 copy/µL for pvl-positive plasmids within 1 hour. The method successfully identified all 25 pvl-positive and 51 pvl-negative strains among the tested 76 isolated S. aureus samples, demonstrating its concordance with qPCR. Discussion: These results show that the CRISPR-ERASE detection method for pvl-positive S. aureus has the advantages of high sensitivity and specificity, this method combines the characteristics of recombinase-assisted amplification at room temperature and the advantages of ERASE test strip visualization, which can greatly reduce the dependence on professional laboratories. It is more suitable for on-site detection than PCR and qPCR, thereby providing important value for rapid on-site detection of pvl.

Insights from a decade of surveillance: Molecular epidemiology of methicillin-resistant Staphylococcus aureus in Norway from 2008 to 2017.

AIM: Norway has a low prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and reporting of all MRSA cases has been mandatory, including infections and carriage, since 1995 and 2005 accordingly. This provides a unique window to study the spread of MRSA in Norway over time. The aim of this study was to analyze the nationwide trends in the molecular epidemiology of MRSA in Norway over a period of 10 years. METHODS: Clinical and epidemiological data as well as bacterial genotype (spa-type and PVL) were analyzed for all reported MRSA cases in Norway in the period 2008-2017. RESULTS: During the study period, there were 15,200 MRSA cases reported in Norway, from 14,386 patients. The notification rate per 100,000 population increased by 15% annually, rising from 14.2 in 2007 to 48.6 in 2017. This increase was primarily driven by MRSA carriage and community-associated MRSA cases. The incidence of invasive infections remained stable and low, at less than 0.5. The incidence of healthcare-associated MRSA showed an increasing trend, while the number of outbreak-related cases, particularly those associated with nursing homes, decreased. Overall, there were significantly more MRSA infections in males than females. Interestingly, there was a significantly higher prevalence of MRSA infections in female young adolescents compared to males. spa-typing revealed a very heterogeneous MRSA population (D = 0.97), predominantly impacted by international travel and migration patterns, and less by domestic spread in the community. CONCLUSIONS: This study highlights that Norway, while still classified as a low-prevalence country, has experienced a significant increase in the incidence of MRSA between 2008 and 2017, which can predominantly be attributed to CA-MRSA and MRSA carriage.

Antimicrobial resistance and virulence profiling of Staphylococcus pseudintermedius isolated from cats, Bangladesh.

Staphylococcus pseudintermedius is a significant bacterial pathogen that frequently colonizes different body sites and mucous membranes of pets. The objectives of the cross-sectional study were to estimate the prevalence, antimicrobial resistance pattern, and detection of diverse resistance as well as virulence genes of S. pseudintermedius in cats. A standard bacteriological method, species-specific gene and different antimicrobial resistance as well as virulence genes were confirmed by PCR assay. A total of 233 swab samples were collected from different body sites of 102 cats, among them 146 swabs from 73 healthy cats, and 87 from 29 diseased cats. Overall, prevalence of S. pseudintermedius in cats was 12.01%, while dermatitis and otitis affected cats were 26.08% and 33.33%, respectively. The highest antimicrobial resistance was observed against penicillin (96.42%) followed by streptomycin (85.71%) and erythromycin (78.57%). Moreover, 89.28% of S. pseudintermedius isolates exhibit multi-drug resistance (MDR) (≥ 3 classes' antimicrobial resistant). In addition, 17.86% isolates harbored the mecA gene; thus, were classified as methicillin-resistant S. pseudintermedius (MRSP). Furthermore, the erythromycin resistance genes ermA and ermB were harbored by 25% and 10.71% of isolates, while 42.86% and 17.86% of isolates carried tetK and tetL (tetracycline resistance) genes, respectively. In virulence profiling, 32.14% (sea) and 10.71% (seb) of isolates were found positive for enterotoxin genes, whereas, the toxic shock syndrome toxin-1 (tst-1) gene and the Panton-Valentine leukocidin gene (pvl) were detected in 25% and 14.29% of isolates, respectively. To our knowledge, this is the first report of cats in Bangladesh for MDR S. pseudintermedius, MRSP, and their virulence profiling.

LysSYL: a broad-spectrum phage endolysin targeting Staphylococcus species and eradicating S. aureus biofilms.

BACKGROUND: Staphylococcus aureus and its single or mixed biofilm infections seriously threaten global public health. Phage therapy, which uses active phage particles or phage-derived endolysins, has emerged as a promising alternative strategy to antibiotic treatment. However, high-efficient phage therapeutic regimens have yet to be established. RESULTS: In this study, we used an enrichment procedure to isolate phages against methicillin-resistant S. aureus (MRSA) XN108. We characterized phage SYL, a new member of the Kayvirus genus, Herelleviridae family. The phage endolysin LysSYL was expressed. LysSYL demonstrated stability under various conditions and exhibited a broader range of efficacy against staphylococcal strains than its parent phage (100% vs. 41.7%). Moreover, dynamic live/dead bacterial observation demonstrated that LysSYL could completely lyse MRSA USA300 within 10 min. Scan and transmission electron microscopy revealed evident bacterial cell perforation and deformation. In addition, LysSYL displayed strong eradication activity against single- and mixed-species biofilms associated with S. aureus. It also had the ability to kill bacterial persisters, and proved highly effective in eliminating persistent S. aureus when combined with vancomycin. Furthermore, LysSYL protected BALB/c mice from lethal S. aureus infections. A single-dose treatment with 50 mg/kg of LysSYL resulted in a dramatic reduction in bacterial loads in the blood, liver, spleen, lungs, and kidneys of a peritonitis mouse model, which resulted in rescuing 100% of mice challenged with 108 colony forming units of S. aureus USA300. CONCLUSIONS: Overall, the data provided in this study highlight the strong therapeutic potential of endolysin LysSYL in combating staphylococcal infections, including mono- and mixed-species biofilms related to S. aureus.

Exploring the potential of bis(thiazol-5-yl)phenylmethane derivatives as novel candidates against genetically defined multidrug-resistant Staphylococcus aureus.

Antimicrobial resistance (AMR) represents an alarming global challenge to public health. Infections caused by multidrug-resistant Staphylococcus aureus (S. aureus) pose an emerging global threat. Therefore, it is crucial to develop novel compounds with promising antimicrobial activity against S. aureus especially those with challenging resistance mechanisms and biofilm formation. Series of bis(thiazol-5-yl)phenylmethane derivatives were evaluated against drug-resistant Gram-positive bacteria. The screening revealed an S. aureus-selective mechanism of bis(thiazol-5-yl)phenylmethane derivatives (MIC 2-64 µg/mL), while significantly lower activity was observed with vancomycin-resistant Enterococcus faecalis (MIC 64 µg/mL) (p<0.05). The most active phenylmethane-based (p-tolyl) derivative, 23a, containing nitro and dimethylamine substituents, and the naphthalene-based derivative, 28b, harboring fluorine and nitro substituents, exhibited strong, near MIC bactericidal activity against S. aureus with genetically defined resistance phenotypes such as MSSA, MRSA, and VRSA and their biofilms. The in silico modeling revealed that most promising compounds 23a and 28b were predicted to bind S. aureus MurC ligase. The 23a and 28b formed bonds with MurC residues at binding site, specifically Ser12 and Arg375, indicating consequential interactions essential for complex stability. The in vitro antimicrobial activity of compound 28b was not affected by the addition of 50% serum. Finally, all tested bis(thiazol-5-yl)phenylmethane derivatives showed favorable cytotoxicity profiles in A549 and THP-1-derived macrophage models. These results demonstrated that bis(thiazol-5-yl)phenylmethane derivatives 23a and 28b could be potentially explored as scaffolds for the development of novel candidates targeting drug-resistant S. aureus. Further studies are also warranted to understand in vivo safety, efficacy, and pharmacological bioavailability of bis(thiazol-5-yl)phenylmethane derivatives.