BACKGROUND:Escherichia (E.) coli is the primary cause of urinary tract infections in companion animals. OBJECTIVES:Therefore, the present study aimed to characterise antimicrobial resistance profiles, virulence-associated genes, and phylogenetic diversity of E. coli with haemolytic colony morphology isolated from urine samples of cats and dogs during routine diagnostic activities, which have not been characterised previously in Austria. METHODS:A total of 51 isolates were included from dogs (n = 32) and cats (n = 19) and characterised by a polyphasic approach, including susceptibility testing and DNA microarray-based assays. In addition, all isolates were analysed using two-locus fumC and fimH sequence typing (CH-clonotyping) and E. coli phylotyping. RESULTS:The majority of E. coli isolates belonged to phylogenetic group B2, with one isolate assigned to phylogenetic group B1. Thirty-four CH-clonotypes were detected using two-locus sequence typing. All isolates detected could be classified as ExPEC. CONCLUSIONS:The identification of clonal complexes common to humans indicates interspecies transmission, thereby emphasising the critical need for surveillance in companion animal populations.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important public health hazard at the human-food-environment interface. Poultry meat has been increasingly implicated as a potential source of MRSA exposure, yet molecular data from the Middle East remain scarce. This study aimed to characterize MRSA isolated from retail chicken meat in the United Arab Emirates (UAE) using a DNA microarray platform and to compare these profiles with recent UAE clinical MRSA data. A total of 34 non-duplicate MRSA isolates recovered from retail chicken meat were analyzed using a high-resolution DNA microarray targeting clonal complexes, SCCmec elements, antimicrobial resistance genes and virulence determinants. Isolates were assigned to six clonal complexes, with CC5 (18/34, 52.9%) and CC6 (12/34, 35.3%) predominating, while CC1, CC22, CC88 and CC97 each occurred at low frequency. Most isolates carried community-associated SCCmec types IV or V/VT. All (100%) harbored the mecA gene, whereas resistance genes associated with fusidic acid (fusC), macrolide-lincosamide resistance (ermC), tetracycline resistance (tetM and/or tetK), phenicol resistance (fexA) and fosfomycin resistance (fosB) were detected in 14/34 (41.2%), 15/34 (44.1%), 20/34 (58.8%), 18/34 (52.9%) and 30/34 (88.2%) isolates, respectively, while no vanA or vanB genes were identified. Virulence profiling revealed widespread carriage of immune evasion cluster genes, including sak (32/34, 94.1%) and scn (30/34, 88.2%), together with universal detection of major adhesion and biofilm-associated genes. Panton-Valentine leukocidin genes (lukF-PV/lukS-PV) were present in 2/34 (5.9%) isolates and the toxic shock syndrome toxin gene (tst1) in 1/34 (2.9%). A diverse repertoire of staphylococcal enterotoxin genes was observed, with enterotoxin gene cluster components (e.g., seg, selm, selu) detected in up to 19/34 (55.9%) isolates and the classical enterotoxin gene sea in 12/34 (35.3%) isolates. Overall, these findings indicate that MRSA contaminating retail chicken meat in the UAE is dominated by human-associated, community-type lineages with extensive antimicrobial resistance and virulence gene content, paralleling recent clinical MRSA clonal structures in the country. This study provides the first microarray-based molecular characterization of MRSA from retail chicken meat in the UAE and supports integration of food-chain surveillance into national One Health MRSA monitoring efforts.

Syndromic PCR panels are becoming a pivotal diagnostic tool in precision medicine. Yet, the design and validation of such test panels is cumbersome, and the integration of new markers typically necessitates complete panel revalidation, a challenge that becomes even more complex for quantitative assays. Here, we introduce a novel multiplex digital PCR strategy utilizing fluorescence-encoded magnetic Nanoreactor Beads (femNRB), enabling cross-reactivity-free detection and absolute quantification of multiple genetic markers within a single sample. These uniquely encoded nanoreactor beads are reversibly conjugated to target-specific primers and probes, allowing the formation of highly adaptable panels composed of multiple PCR assays. As proof of concept, we assembled and validated a 10-assay panel targeting species-specific markers for common nosocomial pathogens. We subsequently expanded this panel with six additional markers for antibiotic resistance and virulence, systematically evaluating both configurations. Remarkably, without further optimization, both panels demonstrated equivalent analytical performance, underscoring the robustness and scalability of this method. This approach operates without complex microfluidic hardware and represents a diagnostic multiplex digital PCR platform that enables seamless assay integration while maintaining uncompromised quantification across variable sample concentrations. By eliminating the need for recalibration, this method substantially simplifies assay expansion, providing a powerful, modular solution for developing and upgrading diagnostic test panels with minimal effort.

Chronic rhinosinusitis (CRS) is a persistent inflammatory condition frequently associated with Staphylococcus aureus. The bacterium's ability to evade immune clearance and establish long-term infection complicates treatment. In our previous study, we demonstrated that S. aureus isolates obtained from patients with CRS (CRS-S. aureus isolates; CSS) exhibit reduced glycolytic activity and cytotoxicity, which is consistent with a persistence-associated phenotype. Here, we present transcriptomic evidence that supports this shift. Comparative RNA sequencing of CSS and control (S. aureus isolates from healthy carriers, MIN) isolates from healthy individuals revealed significantly lower expression of genes involved in canonical virulence pathways in CSS isolates, particularly during the early growth phase. These profiles suggest reduced acute virulence in favour of metabolic changes that aid survival in the chronically inflamed sinus. The distinct transcriptional state of CSS isolates might reflect the influence of the CRS host milieu in shaping bacterial behaviour. Host factors such as sustained inflammation or altered nutrient availability may select for persistence-associated phenotypes. Together, these findings advance our understanding of chronic S. aureus infection and may aid/guide the development of therapies aimed at disrupting persistence programmes or enhancing host resilience.

Genome annotation is an important step in deriving functional meaning from prokaryotic sequencing data, yet systematic evaluations guiding tool selection are lacking. We present the first large-scale investigation of four prominent open-source annotation tools (Prokka, Bakta, EggNOG-mapper, and PGAP) across 156,033 diverse genomes. This includes Escherichia coli strains for baseline performance, thousands of archaea and bacteria genomes, as well as frameshifted and metagenome-assembled genomes. Bakta excels in annotating high-quality bacterial genomes, while PGAP was better for archaeal genomes and challenging bacterial assemblies, including metagenome-assembled, fragmented, or contaminated samples. For Gene Ontology annotation, PGAP consistently provides broader term coverage, whereas EggNOG-mapper offers more terms per feature. Our findings highlight tool-specific strengths crucial for selecting optimal solutions based on genome quality, taxonomy, and origin (e.g. MAGs). This study provides an evidence-based guide for users and informs future tool development.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major public health concern due to its resistance and pathogenic potential. This study investigated the prevalence and genetic characterization of MRSA and methicillin-sensitive S. aureus (MSSA) in aquaculture farms in Damietta Governorate, Egypt. Sampling was conducted across three commercial farms cultivating shrimp, marine fish, and freshwater fish. A total of 509 specimens were collected, including 150 shrimp, 75 each of sea bass, sea bream, catfish, and tilapia, 15 farm water samples, and 44 samples from farm workers. S. aureus was isolated using classical techniques and characterized by MALDI-TOF MS, a PbP2a lateral flow assay, and DNA microarrays. Sixty isolates (11.8%) were identified, comprising 46 MRSA and 14 MSSA strains. The carriage rates of S. aureus and MRSA were 6.7% and 4.7% in shrimp, 11.3% and 8.6% in marine fish, 8% and 6.6% in freshwater fish, respectively and were notably higher among farm workers (71.5% and 42.9% in shrimp farms; 50% and 42.9% in marine farms). Four MRSA clonal complexes (CC88, CC361, CC15, CC152) and two MSSA complexes (CC1, CC361) were identified. MRSA CC15 was restricted to shrimp and freshwater fish, while CC152 was found only in workers. Several MRSA clones exhibited multidrug resistance and carried virulence genes encoding leukocidins, hemolysins, proteases, and adhesion & biofilm factors. This is the first report of these clonal complexes in Egyptian aquaculture. The presence of MRSA clones in aquatic ecosystems could represent a significant route for the transmission and spread of multiple pathogenic factors to human populations and emphasize the need to expand monitoring of antimicrobial resistance (AMR) in the study regions.

BACKGROUND: Staphylococcus aureus is a Gram-positive coccus that colonises or infects humans and a wide range of domestic and wild animals. Various lineages of S. aureus are adapted to ruminants, including cattle, where they commonly cause mastitis. This is of significant animal welfare and economic relevance. Clonal complex (CC) 97 is one of the S. aureus lineages found predominantly in bovines, but also in other animals and humans. Sequence Type (ST) 71 is a related bovine-associated lineage with part of its genome identical to CC97. Thus, it has been assumed that ST71 was a chimera consisting of a fragment of DNA from an unknown donor strain inserted into a CC97 backbone genome. RESULTS: In this study, S. aureus ST3042 was identified as a putative donor of this fragment based on core genome multilocus sequence typing and alignment of core and genomic island markers. We also identified a second ST71 lineage from Sardinia that differs from previously known ST71 from Ireland, the United Kingdom and continental Europe, both in size and demarcations of the ST3042-derived insert. Sardinian ST71 sequences also differed from other ST71 in the orientation of a large part of the genome. As the inversion affected the ST3042-derived region, this event must have taken place later in time than the recombination of CC97 and ST3042. CONCLUSIONS: The observation that ST71 emerged twice adds to the body of evidence that recombination involving large segments of the genome is another, highly effective, mode of horizontal gene transfer in S. aureus and likely contributes to the extraordinary success of this pathogen.

This study investigated the carriage of Staphylococcus (S.) intermedius group (SIG) isolates in marine mammals in the Pacific coastal region of Southern California, USA. We screened 452 samples from the oral cavities of California sea lions (Zalophus californianus), northern elephant seals (Mirounga angustirostris), harbor seals (Phoca vitulina), northern fur seals (Callorhinus ursinus), Guadalupe fur seals (Arctocephalus townsendi), long beaked common dolphin (Delphinus delphis bairdii), Pacific white-sided dolphins (Sagmatias obliquidens), and pygmy sperm whales (Kogia breviceps). Forty-two S. pseudintermedius and four S. delphini were obtained. Isolates were characterized by a polyphasic approach, which included antimicrobial susceptibility testing, molecular identification and characterization by rpoB/nuc sequencing, PCRs, DNA-based microarray, genotyping via spa and MLVA, and whole-genome sequencing of selected isolates. Resistance to penicillin, penicillin/tetracycline, and tetracycline alone was observed. Spa typing resulted either in novel types or in isolates that were non‑typeable. Twenty-nine MLVA types were detected. Multi-locus sequence typing revealed four STs: ST453 (previously described) and three novel STs: ST3046-8) among S. pseudintermedius and various virulence-associated genes were detected. Long-term monitoring remains essential to fully map its epidemiology, pathogenicity, and evolution of SIG in marine mammals.

Biofilms formed by multidrug-resistant (MDR) Klebsiella spp. present a significant clinical challenge due to elevated antibiotic tolerance. Bacteriophages (phages) represent a promising alternative, particularly in combination with antibiotics, where phage–antibiotic synergy (PAS) can increase antibiofilm activity. Evaluating treatment efficacy in these complex structures requires real-time, noninvasive viability analysis. To address this, we used light-sheet fluorescence microscopy (LSFM), a high-resolution, minimally invasive approach, for dynamic tracking of PAS in intact biofilms. To our knowledge, this is the first in vitro application of LSFM for investigating PAS. We studied the combined activity of a virulent phage (vB_KpUKJ_2) and ceftazidime (CAZ) against an extended-spectrum β-lactamase-producing Klebsiella quasipneumoniae. In planktonic cultures, PAS was strongly affected in a dose-dependent manner. In mature biofilms, LSFM imaging revealed that high-dose phages (10⁸ PFU/mL) combined with CAZ at a 0.25 × minimum inhibitory concentration (MIC) induced a rapid and sustained reduction in viability over 24 h. This regimen significantly outperformed mono-treatments (p < 0.01), demonstrating that phage coadministration can reduce the required antibiotic dose. Mechanistically, treatment resulted in phage-mediated degradation of α- and β-polysaccharides within the extracellular polymeric substance (EPS). Crucially, while phage mono-treatment led to the emergence of resistant mutants, the combination treatment fully suppressed resistance. Whole-genome sequencing revealed mutations in genes such as fhuA, purA, and rpoC, suggesting diverse resistance mechanisms linked to fitness trade-offs such as impaired biofilm formation. Our findings highlight a precision-guided strategy with translational potential for device-associated infections, providing a mechanistic and methodological foundation for optimizing PAS-based therapies.

Staphylococcus (S.) aureus can harbour many virulence factors and these include leukocidins, which are bi-component toxins encoded by two co-expressed genes. Some of these toxin genes are localised on prophages and thus they are potentially transmissible. In many cases, they determine the host specificity of the strain they reside in. One set of leukocidin genes , lukM/lukF-P83 , is known to occur in isolates from bovids, small ruminants and rodents. It has also been shown to occur in two lineages of S. aureus from red deer ( Cervus elaphus ) in Italy. Three deer isolates from two lineages, clonal complexes (CC) 350 and 425, were subjected to genome sequencing applying nanopore technology. In addition, phages were induced by mitomycin C treatment, sequenced and imaged by transmission electron microscopy (TEM). The lukM/lukF-P83 leukocidin genes were situated on a Peeveelvirus prophage that integrated into the A5IU43 = yfkAB gene and that was inducible by mitomycin C. The prophage sequences of CC350 and CC425 were nearly identical. They differed from other, previously published lukM/lukF-P83- prophage sequences with most of which, however, they shared the integration site. In addition, the CC350 isolate harboured another, apparently hybrid prophage which was not inducible. Both CC425 isolates also carried an inducible Triavirus prophage. While various S. aureus lineages have been observed in deer, data on their virulence factors remain insufficient. Our observations should prompt further investigations on cervids, both wild or captive. Red deer in zoo or game park populations, together with post-mortem examinations of free-ranging deer found dead, culled for management or harvested by hunters, should provide ample opportunities to observe animals for skin and soft tissue infections including mastitis which are known to be associated with lukM/lukF-P83 in other animals.

Introduction:This study investigated the epidemiology and distribution of carbapenem resistance and virulence genes in Klebsiella pneumoniae strains isolated from the oral cavity of hospitalized patients, highlighting their role as reservoirs in non-epidemic contexts. Methods:Carbapenem-resistant Klebsiella spp. were isolated from the oral cavity of 180 hospitalized patients in medical wards at two hospitals in Bejaia, Algeria. Screening for carbapenem resistance was performed on oral mucosa and saliva using Carba-MTL broth. Antibiotic susceptibility was assessed with the Vitek2 system and interpreted according to EUCAST guidelines. Whole genome sequencing (WGS) was carried out using Oxford Nanopore Technologies, with ABRicate used for resistance/virulence gene detection and Kleborate for hypervirulence assessment. Whole-genome sequences were further examined to identify single-nucleotide polymorphisms (SNPs) and to reconstruct a SNP-based phylogenetic tree in order to assess the genetic relatedness among the isolates. Results:Twenty Klebsiella strains were identified as K. pneumoniae. Among these, 85% were carbapenem-resistant, carrying OXA-48 (80%) or NDM-5 (5%), and all harbored blaCTX-M-15. WGS of the 20 K. pneumoniae strains revealed a broad resistome, including β-lactamases (CTX-M-15, CMY-4, OXA-1, TEM-1), sulfonamide (sul1, sul2), aminoglycoside (aac(3)-IIa, aadA2, aph(3')-VI, armA, strA, strB), trimethoprim (dfrA12, dfrA5, dfrA14), and tetracycline (tetA). Quinolone resistance was linked to QRDR mutations (gyrA S83I, parC S80I) and plasmid-mediated genes (qnrS1, qnrB10, qnrS10, aac(6')-Ib-cr). Five distinct sequence types (STs) were identified, including high-risk clones ST13 and ST48. Virulence profiling revealed yersiniabactin (85%), frequently linked to ICEKp elements (ICEKp4, ICEKp10), and colibactin (40%) among OXA-48 isolates. Notably, a single K. pneumoniae isolate harboring NDM-5 (K21) carried both hypervirulence markers (ybt9/ICEKp3, iuc1, rmp1/kpvp-1) and carbapenem resistance, documenting, for the first time in Algeria, the convergence of these traits in oral isolates. ICEKp was identified as the key vehicle for dissemination of yersiniabactin and colibactin, and a novel association between ICEKp and kpvp-1 was observed. Capsular typing showed predominance of K57-O1/O2v2 among OXA-48 producers and K27/O4 among NDM-5 strains. Conclusion:This study provides the first evidence in Algeria of OXA-48- and NDM-5-producing K. pneumoniae in the oral cavity of hospitalized patients. The coexistence of carbapenem resistance and hypervirulence underscores the oral cavity as a critical reservoir, potentially fueling nosocomial infections and the dissemination of high-risk clones within hospitals and the wider community.

Accurate in silico evaluation of primers and probes is essential for the rational design of molecular multi-parameter assays. We present AssayBLAST v2 to automate and simplify this process for extensive assay designs. A newly integrated strand and proximity check enables precise validation of corresponding oligonucleotides, ensuring correct orientation and spacing required for amplification. Based on predicted oligonucleotide interactions, AssayBLAST v2 determines the theoretical amplification outcomes, offering a computational benchmark for downstream wet-lab validation and performance correlation. Additionally, the updated software integrates an adaptive BLAST parameter optimization that dynamically scales with database size, thereby improving both analytical sensitivity and computational performance. These improvements are supported by a comparative evaluation against the previous version of AssayBLAST. Collectively, these enhancements streamline the assay development workflow, reduce costs associated with suboptimal primer and probe synthesis, and increase the robustness and reliability of molecular diagnostics and research applications.

Abstract Staphylococcus aureus is a major human commensal and pathogen, with the Panton-Valentine leukocidin (PVL) genes being associated with increased virulence. Rapid detection and molecular typing of such isolates are essential for effective epidemiological surveillance and infection control. Studies from Lithuania indicate that PVL is relatively common among S. aureus isolates, but detailed molecular typing data are still lacking. Clinical S. aureus isolates were collected from two hospitals in Vilnius in 2018–2019 and 2024, and from healthy volunteers between 2012 and 2020. Isolates were screened for PVL genes using real-time PCR. In addition, positive isolates harvested directly from the agar plate were tested for PVL production using an experimental lateral flow assay (LFA). Positive isolates were characterised using DNA-microarrays that facilitated the detection of resistance markers and virulence genes including PVL as well as an assignment to clonal complexes, strains and SCC mec types. Epidemiologically relevant isolates were subjected to whole-genome sequencing using Oxford nanopore technology. Out of 1296 S. aureus isolates, 124 yielded PVL-positive PCR results. 100 isolates were available for genotyping. Two PCR-positive isolates were negative by array and LFA, but PVL detection by DNA-microarray and lateral flow test (LF) showed complete concordance. Among PVL-positive isolates, 61.2% were methicillin-resistant S. aureus (MRSA). The most common PVL-MRSA strain ( n = 43) was a Clonal Complex (CC) 8 MRSA that resembled the North American “USA300” strain but that lacked the arginine catabolic mobile element (ACME). This suggested an outbreak in one of the participating hospitals. Other common strains were PVL-positive CC30-MSSA, CC121-MSSA and CC8-MRSA-[IV+ACME] “USA300”, while other lineages were represented by single isolates only. Whole-genome sequencing of two ACME-negative CC8-MRSA-IV isolates and of one local “USA300” isolate, as well as a comparison to international reference sequences, showed a very high degree of similarity in core genome and prophage content. The dominant PVL-MRSA strains were “USA300”, indicating a possible importation from North America, and a locally emerged variant of “USA300” that lost the ACME-associated genes of its SCC mec element. These findings suggest that real-time PVL detection is essential for outbreak prevention. Given its clinical relevance, routine PVL screening —via PCR or lateral flow assays—in routine diagnostics should be seriously considered and surveillance of PVL-MRSA is urgently recommended.

Antimicrobial resistance poses a significant challenge for infection control, requiring the development of accurate and high-throughput diagnostic techniques. We expanded and optimized an existing DNA microarray platform for the molecular characterization of vancomycin-resistant Enterococcus (VRE) by incorporating resistance, virulence, species-specific, and typing markers. The enhanced microarray allows for the simultaneous analysis of up to 96 strains, providing detailed genetic profiles of clinical isolates. VRE strains from Romania and Bavaria, Germany, were analyzed, and the results were compared to those obtained using traditional typing methods, such as multilocus sequence typing (MLST). Next-generation sequencing (NGS) was used in parallel to validate the microarray findings and explore genomic relationships. The microarray revealed considerable genetic diversity and potential epidemiological linkages among isolates. A novel hexadecimal-based nomenclature system was introduced for standardized and scalable strain classification. Comparative analysis demonstrated that the array profiles provided greater discriminatory power and practical resolution than MLST. Receiver operating characteristic (ROC) curve analysis of 187 target genes in 220 isolates gave diagnostic sensitivity and specificity of 100%. This integrated approach offers a cost-effective, rapid, and adaptable global VRE surveillance and infection control tool. It provides a practical alternative to conventional typing systems and facilitates early detection of outbreaks and emerging clones.

Despite advances in dairy husbandry, mastitis-associated Escherichia (E.) coli (MAEC) remains a primary causative agent of bovine mastitis, contributing significantly to economic losses in the cattle industry. This study characterized 50 E. coli isolates from severe, moderate, mild, and subclinical mastitis by antimicrobial susceptibility testing and genotypic analysis, including PCR, DNA microarray, E. coli phylotyping and whole-genome sequencing of selected strains. Antimicrobial resistance rates were highest for ampicillin (30%), tetracycline (20%), amoxicillin-clavulanate (20%), and trimethoprim-sulfamethoxazole (14%); The majority of isolates (66%) was susceptible to antibiotics. One isolate displayed an ESBL phenotype, carrying blaCTX-M-3 and blaTEM-1 genes. Phylogenetic analysis indicated that members of phylogroups B1 (50%) and A (16%) are predominant among the isolates, followed by group C (12%), with the remaining isolates belonging to groups D, E, and F, and two unassigned. The most prevalent virulence-associated genes detected were fimH (100%), and genes of the ferric dicitrate uptake locus (fecIRABCDE). Overall, MAEC isolates exhibited diverse and heterogeneous genetic profiles. The proliferation of ESBL-producing E. coli poses a threat within a One Health framework, given the risk of multidrug-resistant strains at the human-animal-environment interface, compromising critical antimicrobials in both veterinary and human medicine.

BackgroundStaphylococcal mastitis is a common disease of small ruminants causing major economic losses. The problem is particularly significant in the rural areas of the Mediterranean region, where almost two thirds of the global sheep and a quarter of the global goat milk are produced. This study aimed to gain insight into the genotypes, antimicrobial resistance (AMR) profiles and virulence factors of Staphylococcus aureus isolated from clinical mastitis in small ruminants’ farms from different 25 dairy herds in six different provinces in Sardinia, Italy between December 2021 and May 2022.MethodsThirty- two S. aureus were phenotypically identified and confirmed by real-time PCR. Whole-genome sequencing (WGS) was conducted and the sequence data were analyzed to reveal the genetic diversity, AMR markers and virulence genes to draw a conclusion for a current situation of small ruminants’ clinical mastitis in dairy herds in the region and the potential public health risk. Furthermore, the phylogenetic relations between S. aureus strains within one farm and from various farms was analyzed.ResultsAll isolates proved to be phenotypically Methicillin-susceptible S. aureus (MSSA), and none of them harbored mecA/C genes. The antimicrobial resistance against Tetracycline and Erythromycin were 15.62% and 3.12%, respectively. The isolates were assigned to ten sequence types in addition to five different clonal complexes using multilocus sequence typing (MLST). Sequence types ST133 (46.9%) and ST700 (21.9%) were the dominant types, and the majority of isolates were assigned to either CC133 (56.25%) or CC130 (34.4%). Twelve different spa-types were identified among isolates, while six isolates were not assigned to known spa-types. The dominant spa-type was t1773 (18.75%) which is known to be associated with CC130. All Tetracycline-resistant isolates harbored tet genes. The only Erythromycin-resistant isolate carried the erm(T) gene. The leucocidin genes lukF-PV (P83)/lukM were detected in 20 isolates (62.5%), while one isolate (ST522) carried a chimeric leukocidin.ConclusionIn conclusion, this study showed a considerable genetic diversity of S. aureus isolated from sheep and goat mastitis in Sardinia, Italy, and the prominent sensitivity to most of antimicrobial agents relevant for mastitis treatment. These findings inform about the local mastitis control strategies and highlight a low immediate public health risk from antimicrobial resistance in this setting.

Panton–Valentine leukocidin (PVL) is a staphylococcal toxin associated with chronic/recurrent skin and soft tissue infections (SSTIs) and necrotizing pneumonia. Its detection in clinical isolates of Staphylococcus aureus warrants aggressive therapy and infection control measures. However, PVL detection relies on molecular methods of limited use, especially in outpatient or resource-poor settings. In order to aid the development of a lateral flow (LF) test for PVL, clinical isolates from SSTIs were collected in 2020/21 at three laboratories in two cities in the Eastern part of Germany. After the exclusion of duplicate and serial isolates, 83 isolates were eligible. These were tested using an experimental LF test for PVL production. They were also characterized using DNA microarrays, facilitating the detection of virulence and resistance markers as well as the assignment to clonal complexes and epidemic/pandemic strains. Thirty-nine isolates (47%) were PVL-positive, and the LF results were in 81 cases (97.6%) concordant with genotyping. One false-positive and one false-negative case were observed. This translated into a diagnostic sensitivity of 0.974 and a diagnostic specificity of 0.977. The most common PVL-positive MSSA lineages were CC152 (n = 6), CC121 (n = 4), and CC5 and CC30 (each n = 2). Thirty isolates (36%) were mecA-positive. The MRSA rate among PVL-negatives was 20% (nine isolates), but among the PVL-positives, it was as high as 54% (n = 21). The most common PVL-MRSA strains were CC398-MRSA-VT (n = 5), CC5-MRSA-IV “Sri Lanka Clone” (n = 4), CC8-MRSA-[mec IV+Hg] “Latin American USA300” (n = 4), and CC22-MRSA-IV (PVL+/tst+) (n = 2). While the PVL rate was similar just like the German isolates from a previous study a decade before, the MRSA rate among PVL-positives was clearly higher. All PVL-MRSA strains detected, as well as the most common methicillin-susceptible lineage (CC152), are known to be common locally in other parts of the world, and might, thus, be regarded as travel-associated. Therefore, patients with suspected PVL-associated disease should be asked for their history of travel or migration, and, in case of hospitalization, they should be treated as MRSA cases until proven otherwise.

Methicillin-resistant Staphylococcus aureus (MRSA) has moved beyond healthcare settings and is increasingly documented in food, yet genomic data from the Arabian Peninsula are scarce. This study aimed to characterize the genomic diversity, antimicrobial-resistance (AMR), and virulence repertoire of MRSA in retail red meat sold in Dubai, United Arab Emirates (UAE), and to evaluate evidence for potential human-to-food transmission. In a cross-sectional survey (September 2024 - February 2025), 140 red-meat samples (beef, mutton, camel) were collected from supermarket chains. Fifty-one MRSA isolates were confirmed by multiplex PCR and phenotypic testing. Short-read whole-genome sequencing was followed by bioinformatic characterization of the isolates for clonal complex (CC) multilocus sequence typing (ST), spa typing, SCCmec assignment, AMR and virulence genes identification, plasmid-replicon detection, and core-genome phylogeny. Genotype, meat commodity, and products' origin associations were statistically assessed. Eight sequence types were detected; CC5-ST6 (27.5 %) and CC8-ST789 (23.5 %) predominated. SCCmec IV and V accounted for 82.3 % of genomes, and no livestock-associated CC398 was found. CC5/ST6-t304 and CC8-t091 comprised 47 % of isolates but showed no association with meat type (p = 0.451). Core-genome analysis split the collection into six clusters with shallow branch lengths, signaling recent clonal expansion across local and imported meat products. All genomes carried the methicillin resistance mecA gene plus a median of five additional resistance genes; 41 % carried multidrug-resistant determinants. Classical enterotoxin genes occurred in 80.4 % of isolates, and Panton-Valentine leukocidin genes in 27.5 %, mainly within CC8 and CC22. Distinct plasmid backbones-RepA_N-RepL-Rep_trans in CC5-ST6 and Inc18-Rep3 in CC8-ST789-were associated with lineage-specific AMR profiles. Retail red meat in the UAE is contaminated by clinically important, community-associated MRSA clones, likely introduced via human handling rather than animal reservoirs. Genomic surveillance at the human-food interface can guide regional One Health policies and help curb foodborne antimicrobial resistance dissemination.

Wastewater-based surveillance (WBS) is valuable for monitoring antimicrobial resistance (AMR). Staphylococci are key targets, as wastewater can facilitate gene transfer and resistance emergence. Data on WBS for population-level AMR in the Arabian-Gulf remain limited. This study assessed Staphylococcus diversity and resistance in Dubai wastewater. Samples were collected over eight months from nine community sites, two hospital nodes, and two wastewater treatment plants (WWTPs) and were analysed by culture-based method. Ninety-six Staphylococcus isolates were recovered from community, hospital, and WWTP influent, with no growth in effluent. Most isolates (n/N = 88/96) were coagulase-negative Staphylococcus (CoNS), spanning 15 species, dominated by S. saprophyticus, S. cohnii and S. sciuri. The only coagulase-positive Staphylococcus was S. aureus (n = 8) and the only species detected across all wastewater sources. Resistance was highest to benzylpenicillin (88%) and fusidic acid (82%), whereas all isolates remained susceptible to glycopeptides, tigecycline, and linezolid. Fusidic acid resistance was higher in community-wastewater isolates, whereas β-lactam resistance predominated in hospital-wastewater isolates. Sixty percent of CoNS were multidrug-resistant; methicillin resistance occurred in 37.5% of CoNS and 50% of S. aureus. Wastewater is a reservoir of diverse multidrug-resistant staphylococci, underscoring One Health relevance reflecting the potential for circulation between humans, animals, and the shared environment. WBS can support population-level AMR monitoring to inform public health and veterinary interventions.