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Our study investigates the effectiveness of Oxford Nanopore Technologies for accurate outbreak tracing by resequencing 33 isolates of a 3-year-long Klebsiella pneumoniae outbreak with Illumina short-read sequencing data as the point of reference. We detect considerable base errors through cgMLST and phylogenetic analysis of genomes sequenced with Oxford Nanopore Technologies, leading to the false exclusion of some outbreak-related strains from the outbreak cluster. Nearby methylation sites cause these errors and can also be found in other species besides K. pneumoniae Based on these data, we explore PCR-based sequencing and a masking strategy, which both successfully address these inaccuracies and ensure accurate outbreak tracing. We offer our masking strategy as a bioinformatic workflow (MPOA) to identify and mask problematic genome positions in a reference-free manner. Our research highlights limitations in using Oxford Nanopore Technologies for sequencing prokaryotic organisms, especially for investigating outbreaks. For time-critical projects that cannot wait for further technological developments by Oxford Nanopore Technologies, our study recommends either using PCR-based sequencing or using our provided bioinformatic workflow. We advise that read mapping-based quality control of genomes should be provided when publishing results., (© 2024 Lohde et al.; Published by Cold Spring Harbor Laboratory Press.)

Background: Antimicrobial resistance (AMR) in Enterobacterales constitutes a significant threat to the health of both humans and animals and a socioeconomic problem. Enterobacterales, mainly Escherichia coli, carrying β-lactamases has become one of the main indicators to estimate the burden of AMR in animals within "One Health" approach., Objectives: To assess the presence of extended-spectrum cephalosporin-resistant Enterobacterales associated with ruminants (cattle, sheep, goats) habituated in all five provinces of Rwanda and to perform in depth characterization of isolates., Methods: We screened 454 rectal swabs from 203 cows, 170 goats, and 81 sheep and selective isolation of extended-spectrum cephalosporin-resistant Enterobacterales was conducted. Isolates were identified as a members of the order Enterobacterales by MALDI-TOF MS and further characterized by susceptibility testing and by whole-genome sequencing., Results: Out of the 454 samples, 64 extended-spectrum cephalosporin-resistant Enterobacterales were isolated from 58 animals. Isolates belonged to seven bacterial species and were identified as Escherichia coli (n = 54), Enterobacter bugandensis (n = 4), Enterobacter mori (n = 2), Klebsiella pneumoniae (n = 2), Enterobacter dykesii (n = 1), and Citrobacter freundii (n = 1). All isolates displayed an Extended-spectrum β-lactamases (ESBL) phenotype, with exception of Citrobacter freundii isolate displayed both an ESBL and AmpC phenotype. In addition, all Enterobacter isolates were identified as stably de-repressed AmpC-producers. ESBLs genes, bla CTX-M-15 was predominant. Resistance to tetracycline and tet(A) was most frequently observed among non-β-lactam resistance. Forty-eight isolates displayed multidrug-resistance phenotypes. A shiga toxin-producing E. coli and an enterotoxigenic E. coli isolate were observed. Genome comparisons revealed thirty-five E. coli sequence types (ST) (ST10, ST307 being predominate)., Conclusions: Considering the high proximity between ruminants and humans in Rwanda, the dissemination of antimicrobial drug resistance highlights the public health threats and requires the joint and multisectoral action of human and veterinary medicine, at human-animal-environment interfaces. Therefore, it is important to establish national and global "One Health" surveillance programs of AMR to tackle the antibiotic-resistant crisis in human and veterinary medicine., Competing Interests: Declarations Ethics approval and consent to participate The study was discussed, and the rectal swabbing was approved by the Research Screening and Ethical Clearance Committee of the College of Agriculture, Animal Sciences and Veterinary Medicine, University of Rwanda (003/2021/DRI). The study was performed in compliance with institutional guidelines for research on animals and an “informed consent named “Animal Health Survey Informed Consent Form” was obtained and signed from animal owners. All methods were performed in accordance with the relevant intitutaion guidelines and regulations. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

In the present study, the presence of the Enterobacterales, Staphylococcus spp., Mammaliicoccus spp., and Enterococcus spp. in cloacal samples of nestling ospreys (Pandion haliaetus), a fish-eating specialist, from Mono Lake, California, USA was examined by a multiphasic approach, including antimicrobial and biocide susceptibility testing, genotyping, and whole genome sequencing of selected isolates. The most commonly detected species was Escherichia coli, followed by Mammaliicoccus sciuri, Staphylococcus delphini, Enterococcus faecalis, Enterococcus faecium, Hafnia alvei, Klebsiella pneumoniae, Citrobacter braakii and single isolates of Edwardsiella tarda, Edwardsiella albertii, Klebsiella aerogenes, Plesiomonas shigelloides and Staphylococcus pseudintermedius. Multi-drug resistance (MDR) was observed in two E. coli isolates and in an Enterococcus faecium isolate. The MDR blaCTX-M-55-positive E. coli belonged to the pandemic clone ST58. The results of the present study suggest that nestling ospreys are exposed to MDR bacteria, possibly through the ingestion of contaminated fish. Ospreys may be good biosentinels for the presence of these microorganisms and antibiotic resistance in the local environment and the risk for other wildlife, livestock and humans., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Loncaric et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Staphylococcus aureus CC239-MRSA-III is an ancient pandemic strain of hospital-associated, methicillin-resistant S. aureus that spread globally for decades and that still can be found in some parts of the world. In Kuwait, microarray-based surveillance identified from 2019 to 2022 a series of isolates of a hitherto unknown variant of this strain that carried a second set of recombinase genes, ccrA/B-2. To elucidate the structure of its SCCmec element, two isolates were subjected to nanopore sequencing. This revealed, in addition to ccrA/B-2, several SCC-associated genes including speG (spermidine N acetyltransferase) and a gene encoding a large "E-domain containing protein" (dubbed as edcP-SCC). This gene contained three regions consisting of multiple repeating units. In terms of sequence and structure it was similar but not identical to the biofilm-related aap gene from S. epidermidis. A review of published sequences identified edcP-SCC in eighteen genome sequences of S. aureus, S. epidermidis and S. capitis, and frequently it appears in a similar cluster of genes as in the strains sequenced herein. Isolates also carried a prophage with the adhesion factor sasX/sesI and aminoglycoside resistance genes. This is consistent with an affiliation to the "South-East Asian" Clade of CC239. The emergence of edcP-SCC and sasX-positive CC239 strain shows that, against a global trend towards community-associated MRSA, the ancient pandemic CC239 hospital strain still continues to evolve and to cause outbreaks., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Staphylococcus argenteus is a recently described staphylococcal species that is related to Staphylococcus aureus but lacks the staphyloxanthin operon. It is able to acquire both resistance markers such as the SCC mec elements and mobile genetic elements carrying virulence-associated genes from S. aureus . This includes those encoding the Panton-Valentine leukocidin (PVL), which is associated mainly with severe and/or recurrent staphylococcal skin and soft tissue infections. Here, we describe the genome sequences of two PVL-positive, mecA -negative S. argenteus sequence type (ST) 2250 isolates from the United Arab Emirates in detail. The isolates were found in a dental clinic in the United Arab Emirates (UAE). Both were sequenced using Oxford Nanopore Technology (ONT). This demonstrated the presence of temperate bacteriophages in the staphylococcal genomes, including a PVL prophage. It was essentially identical to the published sequence of phiSa2wa_st78 (GenBank NC_055048), a PVL phage from an Australian S. aureus clonal complex (CC) 88 isolate. Besides the PVL prophage, one isolate carried another prophage and the second isolate carried two additional prophages, whereby the region between these two prophages was inverted. This "flipped" region comprised about 1,083,000 bp, or more than a third of the strain's genome, and it included the PVL prophage. Prophages were induced by Mitomycin C treatment and subjected to transmission electron microscopy (TEM). This yielded, in accordance to the sequencing results, one or, respectively, two distinct populations of icosahedral phages. It also showed prolate phages which presumptively might be identified as the PVL phage. This observation highlights the significance bacteriophages have as agents of horizontal gene transfer as well as the need for monitoring emerging staphylococcal strains, especially in cosmopolitan settings such as the UAE.

Salmonella enterica , a bacterium causing foodborne illnesses like salmonellosis, is prevalent in Europe and globally. It is found in food, water, and soil, leading to symptoms like diarrhea and fever. Annually, it results in about 95 million cases worldwide, with increasing antibiotic resistance posing a public health challenge. Therefore, it is necessary to detect and serotype Salmonella for several reasons. The identification of the serovars of Salmonella enterica isolates is crucial to detect and trace outbreaks and to implement effective control measures. Our work presents a protein-based microarray for the rapid and accurate determination of Salmonella serovars. The microarray carries a set of antibodies that can detect different Salmonella O- and H-antigens, allowing for the identification of multiple serovars, including Typhimurium and Enteritidis, in a single miniaturized assay. The system is fast, economical, accurate, and requires only small sample volumes. Also, it is not required to maintain an extensive collection of sera for the serotyping of Salmonella enterica serovars and can be easily expanded and adapted to new serovars and sera. The scientific state of the art in Salmonella serotyping involves the comparison of traditional, molecular, and in silico methods, with a focus on economy, multiplexing, accuracy, rapidity, and adaptability to new serovars and sera. The development of protein-based microarrays, such as the one presented in our work, contributes to the ongoing advancements in this field.

Objectives: The objective of the present study was to examine the diversity of Staphylococcus aureus from mastitis milk samples of cows in Rwanda., Methods: A total of 1080 quarter milk samples from 279 dairy cows were collected in 80 different farms from all five provinces of Rwanda. In total, 135 S. aureus isolates were obtained and subjected to genotyping (spa typing, DNA microarray, whole-genome sequencing (WGS)), antimicrobial susceptibility testing (AST) and phenotypic profiling by Fourier Transform Infrared (FTIR) spectroscopy (including capsular serotyping)., Results: Resistance to penicillin and/or tetracycline was most frequently observed. Ten sequence types (STs) (ST1, ST151, ST152, ST5477, ST700, ST7110, ST7983, ST7984, ST8320, ST97) belonging to seven clonal complexes (CCs) (CC1, CC130, CC152, CC3591, CC3666, CC705, CC97) were detected. The Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83) and the human and bovine toxic shock syndrome toxin gene tst-1 variants were detected. FTIR-based capsular serotyping showed CC-specific differences. Most CC97 (cap5 allele) isolates were primarily nonencapsulated (82%), whereas isolates of CC3591 and CC3666 (cap8 allele) were mostly encapsulated (86.4% and 57.8%, respectively). Our results underline the widespread global distribution of cattle-adapted CC97., Conclusion: The presence of CC3591 and CC3666 in bovine mastitis suggests an important role in cattle health and dairy production in Rwanda. The results of the present study support the need for a rigorous One-Health Surveillance program of the bovine-human interface., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

One of the greatest challenges to the use of molecular methods for diagnostic purposes is the detection of target DNA that is present only in low concentrations. One major factor that negatively impacts accuracy, diagnostic sensitivity, and specificity is the sample matrix, which hinders the attainment of the required detection limit due to the presence of residual background DNA. To address this issue, various methods have been developed to enhance sensitivity through targeted pre-amplification of marker sequences. Diagnostic sensitivity to the single molecular level is critical, particularly when identifying bloodstream infections. In cases of clinically manifest sepsis, the concentration of bacteria in the blood may reach as low as one bacterial cell/CFU per mL of blood. Therefore, it is crucial to achieve the highest level of sensitivity for accurate detection. In the present study, we have established a method that fills the analytical gap between low concentrations of molecular markers and the minimum requirements for molecular testing. For this purpose, a sample preparation of whole blood samples with a directly downstream pre-amplification was developed, which amplifies specific species and resistance markers in a multiplex procedure. When applying pre-amplification techniques, the sensitivity of the pathogen detection in whole blood samples was up to 100 times higher than in non-pre-amplified samples. The method was tested with blood samples that were spiked with several Gram-positive and Gram-negative bacterial pathogens. By applying this method to artificial spiked blood samples, it was possible to demonstrate a sensitivity of 1 colony-forming unit (CFU) per millilitre of blood for S. aureus and E. faecium . A detection limit of 28 and 383 CFU per ml of blood was achieved for E. coli and K. pneumoniae , respectively. If the sensitivity is also confirmed for real clinical blood samples from septic patients, the novel technique can be used for pathogen detection without cultivation, which might help to accelerate diagnostics and, thus, to decrease sepsis mortality rates.

Staphylococcus aureus is a versatile pathogen that does not only occur in humans but also in various wild and domestic animals, including several avian species. When characterizing S. aureus isolates from waterfowl, isolates were identified as atypical CC133 by DNA microarray analysis. They differed from previously sequenced CC133 strains in the presence of the collagen adhesin gene cna ; some also showed a different capsule type and a deviant spa type. Thus, they were subjected to whole-genome sequencing. This revealed multiple insertions of large regions of DNA from other S. aureus lineages into a CC133-derived backbone genome. Three distinct strains were identified based on the size and extent of these inserts. One strain comprised two small inserts of foreign DNA up- and downstream of oriC ; one of about 7000 nt or 0.25% originated from CC692 and the other, at ca. 38,000 nt or 1.3% slightly larger one was of CC522 provenance. The second strain carried a larger CC692 insert (nearly 257,000 nt or 10% of the strain's genome), and its CC522-derived insert was also larger, at about 53,500 nt or 2% of the genome). The third strain carried an identical CC692-derived region (in which the same mutations were observed as in the second strain), but it had a considerably larger CC522-like insertion of about 167,000 nt or 5.9% of the genome. Both isolates of the first, and two out of four isolates of the second strain also harbored a hemolysin-beta-integrating prophage carrying "bird-specific" virulence factors, ornithine cyclodeaminase D0K6J8 and a putative protease D0K6J9. Furthermore, isolates had two different variants of SCC elements that lacked mecA/mecC genes. These findings highlight the role of horizontal gene transfer in the evolution of S. aureus facilitated by SCC elements, by phages, and by a yet undescribed mechanism for large-scale exchange of core genomic DNA.

Aims: To examine the diversity of Staphylococcus aureus isolated from nasal swabs of ruminants in Rwanda., Methods and Results: A total of 454 nasal swabs from 203 cows, 170 goats, and 81 sheep were examined for the presence of S. aureus, and 30 S. aureus isolates were detected and characterized pheno- and genotypically. Resistance to penicillin and/or tetracycline was observed. The isolates were assigned to eight different spa types (t21057 (novel), t10103, t18853, t20842, t318, t355, t458, and t9432) belonging to six clonal complexes (CCs) (CC152, CC30, CC3591, CC3666, CC522, and CC97). Panton-Valentine leukocidin (PVL) genes (lukF-PV/lukS-PV), the bovine leukocidin genes (lukM/lukF-P83), and the human and bovine variants of the toxic shock syndrome toxin gene tst-1 variants were detected., Conclusion: These findings demonstrate that the nares of ruminants in Rwanda are colonized with mastitis-associated S. aureus, including lineages that are also carried by humans, underscoring the zoonotic risk, especially for livestock keepers. These results highlight the crucial importance of hygiene measures when handling livestock., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

The aim of this study was to comprehensively characterise S. aureus from the Caribbean Islands of Trinidad and Tobago, and Jamaica. A total of 101 S. aureus / argenteus isolates were collected in 2020, mainly from patients with skin and soft tissue infections. They were characterised by DNA microarray allowing the detection of ca. 170 target genes and assignment to clonal complexes (CC)s and strains. In addition, the in vitro production of Panton-Valentine leukocidin (PVL) was examined by an experimental lateral flow assay. Two isolates were identified as S. argenteus , CC2596. The remaining S. aureus isolates were assigned to 21 CCs. The PVL rate among methicillin-susceptible S. aureus (MSSA) isolates was high (38/101), and 37 of the 38 genotypically positive isolates also yielded positive lateral flow results. The isolate that did not produce PVL was genome-sequenced, and it was shown to have a frameshift mutation in agrC . The high rate of PVL genes can be attributed to the presence of a known local CC8-MSSA clone in Trinidad and Tobago (n = 12) and to CC152-MSSA (n = 15). In contrast to earlier surveys, the USA300 clone was not found, although one MSSA isolate carried the ACME element, probably being a mecA -deficient derivative of this strain. Ten isolates, all from Trinidad and Tobago, were identified as MRSA. The pandemic ST239-MRSA-III strain was still common (n = 7), but five isolates showed a composite SCC mec element not observed elsewhere. Three isolates were sequenced. That showed a group of genes (among others, speG , crzC, and ccrA / B -4) to be linked to its SCC element, as previously found in some CC5- and CC8-MRSA, as well as in S. epidermidis . The other three MRSA belonged to CC22, CC72, and CC88, indicating epidemiological connections to Africa and the Middle East.

Staphylococcus ( S. ) aureus colonizes up to 30% of all humans and can occasionally cause serious infections. It is not restricted to humans as it can also often be found in livestock and wildlife. Recent studies have shown that wildlife strains of S. aureus usually belong to other clonal complexes than human strains and that they might differ significantly with regard to the prevalence of genes encoding antimicrobial resistance properties and virulence factors. Here, we describe a strain of S. aureus isolated from a European badger ( Meles meles ). For molecular characterisation, DNA microarray-based technology was combined with various next-generation sequencing (NGS) methods. Bacteriophages from this isolate were induced with Mitomycin C and characterized in detail by transmission electron microscopy (TEM) and NGS. The S. aureus isolate belonged to ST425 and had a novel spa repeat sequence (t20845). It did not carry any resistance genes. The uncommon enterotoxin gene see was detected in one of its three temperate bacteriophages. It was possible to demonstrate the induction of all three prophages, although only one of them was expected to be capable of excision based on its carriage of the excisionase gene xis . All three bacteriophages belonged to the family Siphoviridae . Minor differences in size and shape of their heads were noted in TEM images. The results highlight the ability of S. aureus to colonize or infect different host species successfully, which can be attributed to a variety of virulence factors on mobile genetic elements, such as bacteriophages. As shown in the strain described herein, temperate bacteriophages not only contribute to the fitness of their staphylococcal host by transferring virulence factors, but also increase mobility among themselves by sharing genes for excision and mobilization with other prophages.

The present study aims to characterise clinical MRSA isolates from a tertiary care centre in Egypt's second-largest city, Alexandria. Thirty isolates collected in 2020 were genotypically characterised by microarray to detect their resistance and virulence genes and assign them to clonal complexes (CC) and strains. Isolates belonged to 11 different CCs and 14 different strains. CC15-MRSA-[V+ fus ] (n = 6), CC1-MRSA-[V+ fus+tir+ccrA/B-1 ] (PVL+) (n = 5) as well as CC1-MRSA-[V+ fus+tir+ccrA/B-1 ] and CC1153-MRSA-[V+ fus ] (PVL+) (both with n = 3) were the most common strains. Most isolates (83%) harboured variant or composite SCC mec V or VI elements that included the fusidic acid resistance gene fusC . The SCC mec [V+ fus+tir+ccrA/B -1] element of one of the CC1 isolates was sequenced, revealing a presence not only of fusC but also of blaZ , aacA-aphD and other resistance genes. PVL genes were also common (40%). The hospital-acquired MRSA CC239-III strain was only found twice. A comparison to data from a study on strains collected in 2015 (Montelongo et al., 2022) showed an increase in fusC and PVL carriage and a decreasing prevalence of the CC239 strain. These observations indicate a diffusion of community-acquired strains into hospital settings. The beta-lactam use in hospitals and the widespread fusidic acid consumption in the community might pose a selective pressure that favours MRSA strains with composite SCC mec elements comprising mecA and fusC . This is an unsettling trend, but more MRSA typing data from Egypt are required.

Staphylococcus aureus is a widespread and common opportunistic bacterium that can colonise or infect humans as well as a wide range of animals. There are a few studies of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolated from monkeys, apes, and lemurs, indicating a presence of a number of poorly or unknown lineages of the pathogen. In order to obtain insight into staphylococcal diversity, we sequenced strains from wild and captive individuals of three macaque species ( Macaca mulatta , M. assamensis , and M. sylvanus ) using Nanopore and Illumina technologies. These strains were previously identified by microarray as poorly or unknown strains. Isolates of novel lineages ST4168, ST7687, ST7688, ST7689, ST7690, ST7691, ST7692, ST7693, ST7694, ST7695, ST7745, ST7746, ST7747, ST7748, ST7749, ST7750, ST7751, ST7752, ST7753, and ST7754 were sequenced and characterised for the first time. In addition, isolates belonging to ST2990, a lineage also observed in humans, and ST3268, a MRSA strain already known from macaques, were also included into the study. Mobile genetic elements, genomic islands, and carriage of prophages were analysed. There was no evidence for novel host-specific virulence factors. However, a conspicuously high rate of carriage of a pathogenicity island harbouring edinB and etD2 / etE as well as a higher number of repeat units within the gene sasG (encoding an adhesion factor) than in human isolates were observed. None of the strains harboured the genes encoding Panton-Valentine leukocidin. In conclusion, wildlife including macaques may harbour an unappreciated diversity of S. aureus lineages that may be of clinical relevance for humans, livestock, or for wildlife conservation, given the declining state of many wildlife populations.

Poultry is one of the most important reservoirs for zoonotic multidrug-resistant pathogens. The indiscriminate use of antimicrobials in poultry production is a leading factor for development and dissemination of antimicrobial resistance. This study aimed to describe the prevalence and antimicrobial resistance of E. coli isolated from healthy turkey flocks of different ages in Nile delta region, Egypt. In the current investigation, 250 cloacal swabs were collected from 12 turkey farms in five governorates in the northern Egypt. Collected samples were cultivated on Brilliance TM ESBL agar media supplemented with cefotaxime (100 mg/L). The E. coli isolates were identified using MALDI-TOF-MS and confirmed by a conventional PCR assay targeting 16S rRNA-DNA. The phenotypic antibiogram against 14 antimicrobial agents was determined using the broth micro-dilution method. DNA-microarray-based assay was applied for genotyping and determination of both, virulence and resistance-associated gene markers. Multiplex real-time PCR was additionally applied for all isolates for detection of the actual most relevant Carbapenemase genes. The phenotypic identification of colistin resistance was carried out using E-test. A total of 26 E. coli isolates were recovered from the cloacal samples. All isolates were defined as multidrug-resistant. Interestingly, two different E. coli strains were isolated from one sample. Both strains had different phenotypic and genotypic profiles. All isolates were phenotypically susceptible to imipenem, while resistant to penicillin, rifampicin, streptomycin, and erythromycin. None of the examined carbapenem resistance genes was detected among isolates. At least one beta-lactamase gene was identified in most of isolates, where bla TEM was the most commonly identified determinant (80.8%), in addition to bla CTX-M9 (23.1%), bla SHV (19.2%) and bla OXA-10 (15.4%). Genes associated with chloramphenicol resistance were flo R (65.4%) and cml A1 (46.2%). Tetracycline- and quinolone-resistance-associated genes tet A and qnr S were detected in (57.7%) and (50.0%) of isolates, respectively. The aminoglycoside resistance associated genes aad A1 (65.4%), aad A2 (53.8%), aph A (50.0%), str A (69.2%), and str B (65.4%), were detected among isolates. Macrolide resistance associated genes mph and mrx were also detected in (53.8%) and (34.6%). Moreover, colistin resistance associated gene mcr -9 was identified in one isolate (3.8%). The class 1 integron integrase int I1 (84.6%), transposase for the transposon tnp ISEcp1 (34.6%) and OqxB -integral membrane and component of RND-type multidrug efflux pump oqx B (7.7%) were identified among the isolates. The existing high incidence of ESBL/colistin-producing E. coli identified in healthy turkeys is a major concern that demands prompt control; otherwise, such strains and their resistance determinants could be transmitted to other bacteria and, eventually, to people via the food chain.

This study aimed to estimate the prevalence of extended-spectrum β-lactamase-producing (ESBL) bacteria in swine. Thus, 214 fecal samples were collected from suckling and weaned piglets from 34 farms in Greece (out of an overall population of about 14,300 sows). A subset of 78 (36.5%) ESBL producers were identified as E. coli (69/78, 88.5%), K. pneumoniae spp. pneumoniae (3.8%), P. mirabilis (5.1%), E. cloacae complex (1.3%) and S. enterica spp. diarizonae (1.3%). Resistance to at least one class of non-β-lactam antibiotics was detected in 78 isolates. Among the E. coli strains, resistance was identified with regard to aminoglycosides ( n = 31), fluoroquinolones ( n = 49), tetracycline ( n = 26) and trimethoprim/sulfamethoxazole ( n = 46). Of the three K. pneumoniae spp. pneumoniae, two displayed resistances to aminoglycosides and all were resistant to fluoroquinolones, tetracyclines and trimethoprim/sulfamethoxazole. As for the four P. mirabilis isolates, three had a resistant phenotype for aminoglycosides and all were resistant to imipenem, fluoroquinolones, tetracyclines and trimethoprim/sulfamethoxazole. Molecular characterization of the isolates revealed the presence of CTX-M, SHV and TEM genes, as well as of genes conferring resistance to fluoroquinolones, aminoglycosides, sulfonamides, trimethoprim, macrolides and colistin. High levels of antimicrobial resistance (AMR) were demonstrated in Greek swine herds posing a concern for the efficacy of treatments at the farm level as well as for public health.

Wild and feral birds are known to be involved in the maintenance and dissemination of clinically-important antimicrobial-resistant pathogens, such as extended-spectrum β-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae. The aim of our study was to evaluate the presence of ESBL- and carbapenemase-producing Escherichia coli among wild and feral birds from Greece and to describe their antimicrobial resistance characteristics. In this context, fecal samples of 362 birds were collected and cultured. Subsequently, the antimicrobial resistance pheno- and geno-type of all the obtained E. coli isolates were determined. A total of 12 multidrug-resistant (MDR), ESBL-producing E. coli were recovered from eight different wild bird species. Eleven of these isolates carried a bla CTX-M-1 group gene alone or in combination with bla TEM and one carried only bla TEM . AmpC, fluoroquinolone, trimethoprim/sulfamethoxazole, aminoglycoside and macrolide resistance genes were also detected. Additionally, one carbapenemase-producing E. coli was identified, harboring bla NDM along with a combination of additional resistance genes. This report describes the occurrence of ESBL- and carbapenemase-producing E. coli among wild avian species in Greece, emphasizing the importance of incorporating wild birds in the assessment of AMR circulation in non-clinical settings.

Strategies to contain the current SARS-CoV-2 pandemic rely, beside vaccinations, also on molecular and serological testing. For any kind of assay development, screening for the optimal antigen is essential. Here we describe the verification of a new protein microarray with different commercially available preparations significant antigens of SARS-CoV-2 that can be used for the evaluation of the performance of these antigens in serological assays and for antibody screening in serum samples. Antigens of other pathogens that are addressed by widely used vaccinations were also included. To evaluate the accuracy of 21 different antigens or antigen preparations on the microarray, receiver operating characteristics (ROC) curve analysis using ELISA results as reference were performed. Except for a single concentration, a diagnostic sensitivity of 1 was determined for all antigen preparations. A diagnostic specificity, as well as an area under the curve (AUC) of 1 was obtained for 16 of 21 antigen preparations. For the remaining five, the diagnostic specificity ranged from 0.942 to 0.981 and AUC from 0.974 to 0.999. The optimized assay was subsequently also applied to determine the immune status of previously tested individuals and/or to detect the immunization status after COVID-19 vaccination. Microarray evaluation of the antibody profiles of COVID-19 convalescent and post vaccination sera showed that the IgG response differed between these groups, and that the choice of the test antigen is crucial for the assay performance. Furthermore, the results showed that the immune response is highly individualized, depended on several factors (e.g., age or sex), and was not directly related to the severity of disease. The new protein microarray provides an ideal method for the parallel screening of many different antigens of vaccine-preventable diseases in a single sample and for reliable and meaningful diagnostic tests, as well as for the development of safe and specific vaccines., (© 2022. The Author(s).)

The phylogenetic tree of the Staphylococcus aureus complex consists of several distinct clades and the majority of human and veterinary S. aureus isolates form one large clade. In addition, two divergent clades have recently been described as separate species. One was named Staphylococcus argenteus , due to the lack of the "golden" pigment staphyloxanthin. The second one is S. schweitzeri , found in humans and animals from Central and West Africa. In late 2021, two additional species, S. roterodami and S. singaporensis , have been described from clinical samples from Southeast Asia. In the present study, isolates and their genome sequences from wild Straw-coloured fruit bats ( Eidolon helvum ) and a Diamond firetail ( Stagonopleura guttata , an estrildid finch) kept in a German aviary are described. The isolates possessed staphyloxanthin genes and were closer related to S. argenteus and S. schweitzeri than to S. aureus . Phylogenetic analysis revealed that they were nearly identical to both, S. roterodami and S. singaporensis . We propose considering the study isolates, the recently described S. roterodami and S. singaporensis as well as some Chinese strains with MLST profiles stored in the PubMLST database as different clonal complexes within one new species. According to the principle of priority we propose it should be named S. roterodami . This species is more widespread than previously believed, being observed in West Africa, Southeast Asia and Southern China. It has a zoonotic connection to bats and has been shown to be capable of causing skin and soft tissue infections in humans. It is positive for staphyloxanthin, and it could be mis-identified as S. aureus (or S. argenteus ) using routine procedures. However, it can be identified based on distinct MLST alleles, and " S. aureus " sequence types ST2470, ST3135, ST3952, ST3960, ST3961, ST3963, ST3965, ST3980, ST4014, ST4075, ST4076, ST4185, ST4326, ST4569, ST6105, ST6106, ST6107, ST6108, ST6109, ST6999 and ST7342 belong to this species., Competing Interests: DG is employed by a company, Illumina, but he performed experiments for this study before commencing this employment. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Monecke, Schaumburg, Shittu, Schwarz, Mühldorfer, Brandt, Braun, Collatz, Diezel, Gawlik, Hanke, Hotzel, Müller, Reinicke, Feßler and Ehricht.)

Carbapenem-resistant Gram-negative bacteria are a public health threat that requires urgent action. The fact that these pathogens commonly also harbor resistance mechanisms for several other antimicrobial classes further reduces patient treatment options. The present study aimed to provide information regarding the multidrug resistance genetic background of carbapenem-resistant Gram-negative bacteria in Central Greece. Strains from a tertiary care hospital, collected during routine practice, were characterized using a DNA microarray-based assay. Various different resistance determinants for carbapenems, other beta-lactams, aminoglycosides, quinolones, trimethoprim, sulfonamides and macrolides were detected among isolates of the same sequence type. Eighteen different multidrug resistance genomic profiles were identified among the twenty-four K. pneumoniae ST258, seven different profiles among the eight K. pneumoniae ST11, four profiles among the six A. baumannii ST409 and two among the three K. oxytoca . This report describes the multidrug resistance genomic background of carbapenem-resistant Gram-negative bacteria from a tertiary care hospital in Central Greece, providing evidence of their continuous genetic evolution.

Staphylococcus aureus can be a harmless coloniser, but it can also cause severe infections in humans, livestock and wildlife. Regarding the latter, only few studies have been performed and knowledge on virulence factors is insufficient. The aim of the present study was to study S. aureus isolates from deceased wild beavers (Castor fiber). Seventeen isolates from eleven beavers, found in Germany and Austria, were investigated. Antimicrobial and biocide susceptibility tests were performed. Isolates were characterised using S. aureus-specific DNA microarrays, spa typing and whole-genome sequencing. From two isolates, prophages were induced by mitomycin C and studied by transmission electron microscopy. Four isolates belonged to clonal complex (CC) 8, CC12, and CC398. Twelve isolates belonged to CC1956 and one isolate was CC49. The CC49 and CC1956 isolates carried distinct lukF/S genes related to the Panton-Valentine leukocidin (PVL) from human isolates of S. aureus. These genes were located on related, but not identical, Siphovirus prophages. The beavers, from which those isolates originated, suffered from abscesses, purulent organ lesions and necrotising pneumonia, i.e., clinical manifestations resembling symptoms of severe PVL-associated disease in humans. It might thus be assumed that the "Beaver Leukocidin (BVL, lukF/S-BV)"-positive strains are beaver-specific pathogens, and further studies on their clinical role as well as on a possible transmissibility to other species, including humans, are warranted., (© 2021. The Author(s).)

In the context of microarray-based epidemiological typing of the clonal organism Staphylococcus aureus /MRSA , a strain was identified that did not belong to known clonal complexes. The molecular analysis by microarray-based typing yielded signals suggesting that it was a mosaic or hybrid strain of two lineages. To verify this result, the isolate was sequenced with both, short-read Illumina and long-read Nanopore technologies and analysed in detail. This supported the hypothesis that the genome of this strain, ST6610-MRSA-IVg comprised of segments originating from two different clonal complexes (CC). While the backbone of the strain's genome, i.e., roughly 2 megabases, belongs to CC8, a continuous insert of 894 kb (approx. 30% of the genome) originated from CC140. Beside core genomic markers in the normal succession and orientation, this insert also included the mecA gene, coding for PbP2a and causing methicillin resistance, localised on an SCC mec IVg element. This particular SCC mec type was also previously observed in CC140 MRSA from African countries. A second conspicuous observation was the presence of the trimethoprim resistance gene dfrG within on a prophage that occupied an attachment site normally used by Panton-Valentine Leucocidin phages. This observation could indicate a role of large-scale chromosomal recombination in the evolution of S. aureus as well as a role of phages in the dissemination of antibiotic resistance genes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Burgold-Voigt, Monecke, Simbeck, Holzmann, Kieninger, Liebler-Tenorio, Braun, Collatz, Diezel, Müller, Schneider-Brachert and Ehricht.)

Background: Pulmonary diseases have considerable prognostic relevance for all-cause mortality. Most patients with lung diseases such as chronic obstructive pulmonary disease are treated by general practitioners. Understanding the clinical consequences such as pulmonary hyperinflation or reduced diffusion capacity is important for the management and prognosis of patients with chronic respiratory disorders. Therefore, the interpretation of pulmonary function testing (PFT) results needs to see more emphasis in the medical education curriculum., Objective: To develop PFT training for final-year medical students and to compare the efficacy of instructional training to self-reliant textbook study., Methods: A two-armed randomized control trial compares learning outcomes in PFT interpretation. A total of 25 final-year medical students were selected at random into the 1 ) instructional training group or 2 ) self-reliant textbook study group on PFT interpretation. The learning time for both groups was 2 hours. The duration of the written pre- and post-training examinations was 60 minutes each. Both exams had a knowledge section (30 questions, maximum 120 points) and a skills section (11 case studies, maximum 75 points)., Results: The instructional training group acquired significantly more knowledge and, in particular, higher skill levels when compared with the self-reliant reading group. In the reading group, knowledge scores increased from 48 to 60% (12%) and skills scores increased from 14 to 22% (8%), whereas in the instructional group, knowledge increased from 47 to 71% (24%) and skills from 18 to 58% (40%). A multivariate analysis (Pillai's Trace: 0.633; P  < 0.001) as well as follow-up univariate analyses reveal that these differences are statistically significant (knowledge: F = 8.811, df = 1, P  = 0.007; skills F = 33.965, df = 1, P  < 0.001). Interestingly, there was no significant group effect in the pure knowledge gain about respiratory disorders per se., Conclusion: The self-reliant study group was less able to translate their newly acquired knowledge into interpretation of comprehensive PFT reports. A mandatory 2-hour instructional training greatly enhances the students' knowledge and skills about PFT interpretation. Obligatory PFT instructional training should therefore be included in the students' curriculum., (Copyright © 2021 by the American Thoracic Society.)

The aim of this study was continuous monitoring of the presence of mcr-1 to mcr-5 genes in Enterobacterales isolated from cattle, pigs, and domestic poultry at intensive breeding facilities in Northern Vojvodina, Serbia, from 1 January 1 to 1 October 2020. Out of 2167 examined samples, mcr-1 was observed in five E. coli isolates originating from healthy turkeys. Four isolates belonged to the phylogenetic group B1, and one isolate to the phylogenetic group A. Detected E. coli serogenotypes (somatic O and flagellar H antigens) were O8:H25 and O29:H25. Core-genome multi-locus sequence typing (cgMLST) revealed three ST58 isolates clustering together in Clonal Complex (CC) 155 and two singletons of ST641-CC86 and ST410-CC23, respectively. Clonotyping revealed CH4-32 ( n = 3), CH6-53 ( n = 1) and CH4-24 ( n = 1). In all isolates, the mcr-1 gene was located on a large IncX4 replicon type plasmid. Eight virulence-associated genes (VAGs) typical of avian pathogenic E. coli (APEC) ( fyuA, fimH , hlyF , iss , ompT , sitA , traT , iroN ) were detected in four isolates. These isolates were investigated for susceptibility to four biocides and revealed MIC values of 0.125% for glutardialdehyde, of 0.00003-0.00006% for chlorohexidine, of 4-6% for isopropanol and of 0.001-0.002% for benzalkonium chloride. All obtained MIC values of the tested biocides were comparable to the reference strain, with no indication of possible resistance. This is the first report of mcr-1.1 -carrying E. coli from Serbia. Although only samples from turkeys were mcr -positive in this study, continuous monitoring of livestock samples is advised to prevent a spill-over from animals to humans.

Escherichia ( E. ) coli is the main causative pathogen of neonatal and post-weaning diarrhea and edema disease in swine production. There is a significant health concern due to an increasing number of human infections associated with food and/or environmental-borne pathogenic and multidrug-resistant E. coli worldwide. Monitoring the presence of pathogenic and antimicrobial-resistant E. coli isolates is essential for sustainable disease management in livestock and human medicine. A total of 102 E. coli isolates of diseased pigs were characterized by antimicrobial and biocide susceptibility testing. Antimicrobial resistance genes, including mobile colistin resistance genes, were analyzed by PCR and DNA sequencing. The quinolone resistance-determining regions of gyrA and parC in ciprofloxacin-resistant isolates were analyzed. Clonal relatedness was investigated by two-locus sequence typing (CH clonotyping). Phylotyping was performed by the Clermont multiplex PCR method. Virulence determinants were analyzed by customized DNA-based microarray technology developed in this study for fast and economic molecular multiplex typing. Thirty-five isolates were selected for whole-genome sequence-based analysis. Most isolates were resistant to ampicillin and tetracycline. Twenty-one isolates displayed an ESBL phenotype and one isolate an AmpC β-lactamase-producing phenotype. Three isolates had elevated colistin minimal inhibitory concentrations and carried the mcr-1 gene. Thirty-seven isolates displayed a multi-drug resistance phenotype. The most predominant β-lactamase gene classes were bla TEM-1 (56%) and bla CTX-M-1 (13.71%). Mutations in QRDR were observed in 14 ciprofloxacin-resistant isolates. CH clonotyping divided all isolates into 51 CH clonotypes. The majority of isolates belonged to phylogroup A. Sixty-four isolates could be assigned to defined pathotypes wherefrom UPEC was predominant. WGS revealed that the most predominant sequence type was ST100, followed by ST10. ST131 was detected twice in our analysis. This study highlights the importance of monitoring antimicrobial resistance and virulence properties of porcine E. coli isolates. This can be achieved by applying reliable, fast, economic and easy to perform technologies such as DNA-based microarray typing. The presence of high-risk pathogenic multi-drug resistant zoonotic clones, as well as those that are resistant to critically important antibiotics for humans, can pose a risk to public health. Improved protocols may be developed in swine farms for preventing infections, as well as the maintenance and distribution of the causative isolates.

Marine mammals have been described as sentinels of the health of marine ecosystems. Therefore, the aim of this study was to investigate (i) the presence of extended-spectrum β-lactamase (ESBL)- and AmpC-producing Enterobacterales, which comprise several bacterial families important to the healthcare sector, as well as (ii) the presence of Salmonella in these coastal animals. The antimicrobial resistance pheno- and genotypes, as well as biocide susceptibility of Enterobacterales isolated from stranded marine mammals, were determined prior to their rehabilitation. All E. coli isolates ( n = 27) were screened for virulence genes via DNA-based microarray, and twelve selected E. coli isolates were analyzed by whole-genome sequencing. Seventy-one percent of the Enterobacterales isolates exhibited a multidrug-resistant (MDR) pheno- and genotype. The gene bla CMY ( n = 51) was the predominant β-lactamase gene. In addition, bla TEM-1 ( n = 38), bla SHV-33 ( n = 8), bla CTX-M-15 ( n = 7), bla OXA-1 ( n = 7), bla SHV-11 ( n = 3), and bla DHA-1 ( n = 2) were detected. The most prevalent non-β-lactamase genes were sul2 ( n = 38), strA ( n = 34), strB ( n = 34), and tet (A) ( n = 34). Escherichia coli isolates belonging to the pandemic sequence types (STs) ST38, ST167, and ST648 were identified. Among Salmonella isolates ( n = 18), S . Havana was the most prevalent serotype. The present study revealed a high prevalence of MDR bacteria and the presence of pandemic high-risk clones, both of which are indicators of anthropogenic antimicrobial pollution, in marine mammals.

While many data on molecular epidemiology of MRSA are available for North America, Western Europe and Australia, much less is known on the distribution of MRSA clones elsewhere. Here, we describe a poorly known lineage from the Middle East, CC1153, to which several strains from humans and livestock belong. Isolates were characterised using DNA microarrays and one isolate from the United Arab Emirates was sequenced using Nanopore technology. CC1153 carries agr II and capsule type 5 genes. Enterotoxin genes are rarely present, but PVL is common. Associated spa types include t504, t903 and t13507. PVL-positive CC1153-MSSA were found in Egyptian cattle suffering from mastitis. It was also identified among humans with skin and soft tissue infections in Saudi Arabia, France and Germany. CC1153-MRSA were mainly observed in Arabian Gulf countries. Some isolates presented with a previously unknown SCCmec/SCCfus chimeric element in which a mec B complex was found together with the fusidic acid resistance gene fusC and accompanying genes including ccrA/B-1 recombinase genes. Other isolates carried SCCmec V elements that usually also included fusC. Distribution and emergence of CC1153-MRSA show the necessity of molecular characterization of MRSA that are resistant to fusidic acid. These strains pose a public health threat as they combine resistance to beta-lactams used in hospitals as well as to fusidic acid used in the community. Because of the high prevalence of fusC-positive MRSA in the Middle East, sequences and descriptions of SCC elements harbouring fusC and/or mecA are reviewed. When comparing fusC and its surrounding regions from the CC1153 strain to available published sequences, it became obvious that there are four fusC alleles and five distinct types of fusC gene complexes reminiscent to the mec complexes in SCCmec elements. Likewise, they are associated with different sets of ccrA/B recombinase genes and additional payload that might include entire mec complexes or SCCmec elements.

The prevalence of multidrug resistant, extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae is increasing worldwide. The present study aimed to provide an overview of the multidrug resistance phenotype and genotype of ESBL-producing Escherichia coli ( E. coli ) isolates of livestock and wild bird origin in Greece. Nineteen phenotypically confirmed ESBL-producing E. coli strains isolated from fecal samples of cattle ( n = 7), pigs ( n = 11) and a Eurasian magpie that presented resistance to at least one class of non β-lactam antibiotics, were selected and genotypically characterized. A DNA-microarray based assay was used, which allows the detection of various genes associated with antimicrobial resistance. All isolates harbored bla CTX-M-1/15 , while bla TEM was co-detected in 13 of them. The AmpC gene bla MIR was additionally detected in one strain. Resistance genes were also reported for aminoglycosides in all 19 isolates, for quinolones in 6, for sulfonamides in 17, for trimethoprim in 14, and for macrolides in 8. The intI1 and/or tnpISE cp1 genes, associated with mobile genetic elements, were identified in all but two isolates. This report describes the first detection of multidrug resistance genes among ESBL-producing E. coli strains retrieved from feces of cattle, pigs, and a wild bird in Greece, underlining their dissemination in diverse ecosystems and emphasizing the need for a One-Health approach when addressing the issue of antimicrobial resistance.

Background: Antimicrobial resistance (AMR) is an increasing global health concern reducing options for therapy of infections and also for perioperative prophylaxis. Many Enterobacteriaceae cannot be treated anymore with third generation cephalosporins (3GC) due to the production of certain 3GC hydrolysing enzymes (extended spectrum beta-lactamases, ESBLs). The role of animals as carriers and vectors of multi-resistant bacteria in different geographical regions is poorly understood. Therefore, we investigated the occurrence and molecular characteristics of ESBL-producing Escherichia coli (E. coli) in wild birds and slaughtered cattle in Ibadan, Nigeria. Cattle faecal samples (n = 250) and wild bird pooled faecal samples (cattle egrets, Bubulcus ibis, n = 28; white-faced whistling duck, Dendrocygna viduata, n = 24) were collected and cultured on cefotaxime-eosin methylene blue agar. Antimicrobial susceptibility was determined by agar diffusion assays and all 3GC resistant isolates were genotypically characterised for AMR genes, virulence associated genes (VAGs) and serotypes using DNA microarray-based assays., Results: All 3GC resistant isolates were E. coli: cattle (n = 53), egrets (n = 87) and whistling duck (n = 4); cultured from 32/250 (12.8%), 26/28 (92.9%), 2/24(8.3%), cattle, egrets and whistling duck faecal samples, respectively. blaCTX-M gene family was prevalent; blaCTX-M15 (83.3%) predominated over blaCTX-M9 (11.8%). All were susceptible to carbapenems. The majority of isolates were resistant to at least one of the other tested antimicrobials; multidrug resistance was highest in the isolates recovered from egrets. The isolates harboured diverse repositories of other AMR genes (including strB and sul2), integrons (predominantly class 1) and VAGs. The isolates recovered from egrets harboured more AMR genes; eight were unique to these isolates including tetG, gepA, and floR. The prevalent VAGs included hemL and iss; while 14 (including sepA) were unique to certain animal isolates. E. coli serotypes O9:H9, O9:H30 and O9:H4 predominated. An identical phenotypic microarray profile was detected in three isolates from egrets and cattle, indicative of a clonal relationship amongst these isolates., Conclusion: Wild birds and cattle harbour diverse ESBL-producing E. coli populations with potential of inter-species dissemination and virulence. Recommended guidelines to balance public health and habitat conservation should be implemented with continuous surveillance.

A PVL-positive, methicillin-susceptible Staphylococcus aureus was cultured from pus from cervical lymphadenitis of a patient of East-African origin. Microarray hybridisation assigned the isolate to clonal complex (CC) 80 but revealed unusual features, including the presence of the ORF-CM14 enterotoxin homologue and of an ACME-III element as well as the absence of etD and edinB. The isolate was subjected to both, Illumina and Nanopore sequencing allowing characterisation of deviating regions within the strain´s genome. Atypical features of this strain were attributable to the presence of two genomic regions that originated from other S. aureus lineages and that comprised, respectively, 3% and 1.4% of the genome. One deviating region extended from walJ to sirB. It comprised ORF-CM14 and the ACME-III element. A homologous but larger fragment was also found in an atypical S. aureus CC1/ST567 strain whose lineage might have served as donor of this genomic region. This region itself is a chimera comprising fragments from CC1 as well as fragments of unknown origin. The other deviating region comprised the region from htsB to ecfA2, i.e., another 3% of the genome. It was very similar to CC1 sequences. Either this suggests an incorporation of CC1 DNA into the study strain, or alternatively a recombination event affecting "canonical" CC80. Thus, the study strain bears witness of several recombination events affecting supposedly core genomic genes. Although the exact mechanism is not yet clear, such chimerism seems to be an additional pathway in the evolution of S. aureus. This could facilitate also a transmission of virulence and resistance factors and therefore offer an additional evolutionary advantage., Competing Interests: DG is employee of PTC - Phage Technology Center GmbH, Bönen, Germany; AZC is employee of T-Systems Multimedia Solutions GmbH, Dresden, Germany. In both cases, work on this project was performed before the respective employments started. Thus, employers of the authors did not have any role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The other authors declare that no competing interests exist. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Staphylococcus aureus is a ubiquitous pathogen and colonizer in humans and animals. There are few studies on the molecular epidemiology of S. aureus in wild monkeys and apes. S. aureus carriage in rhesus macaques ( Macaca mulatta ) and Assam macaques ( Macaca assamensis ) is a species that has not previously been sampled and lives in remote environments with limited human contact. Forty Staphylococcus aureus isolates including 33 methicillin-susceptible S. aureus (MSSA) and seven methicillin-resistant S. aureus (MRSA) were characterized. Thirty-four isolates were from rhesus macaques and six isolates (five MSSA, one MRSA) were from Assam macaques. Isolates were characterized using StaphyType DNA microarrays. Five of the MRSA including one from Assam macaque were CC22 MRSA-IV (PVL+/ tst +), which is a strain previously identified in Nepalese rhesus. One MRSA each were CC6 MRSA-IV and CC772 MRSA-V (PVL+). One MSSA each belonged to CC15, CC96, and CC2990. Six MRSA isolates carried the blaZ , while ten known CC isolates (seven MRSA, three MSSA) carried a variety of genes including aacA-aphD , aphA3, erm (C), mph (C), dfrA , msrA , and/or sat genes. The other 30 MSSA isolates belonged to 17 novel clonal complexes, carried no antibiotic resistance genes, lacked Panton-Valentine Leukocidin (PVL), and most examined exotoxin genes. Four clonal complexes carried egc enterotoxin genes, and four harbored edinB , which is an exfoliative toxin homologue.

Introduction . Identification of typhoidal Salmonella (TS) serovars and their discrimination from non-typhoidal Salmonella (NTS) is conventionally performed by seroagglutination. This method is labour-intensive, requires technical experience and can be inconclusive in some cases. Molecular assays may be reliable alternative diagnostic tools. Aim . This study was designed to evaluate the eazyplex TyphiTyper based on loop-mediated isothermal amplification (LAMP) for fast identification of TS and S . Choleraesuis in culture. Methodology . A total of 121 Salmonella strains and 33 isolates of other Enterobacterales species were tested by the eazyplex TyphiTyper. Simulated and clinical blood cultures (BCs) were used to examine the performance of the assay for diagnosis of systemic infection. Sample preparation took about 5 min and the test running time was 20 min. Amplification was measured by real-time fluorescence detection. Results . All TS and S . Choleraesuis strains were correctly identified. The most common NTS S . Typhimurium ( n =34) and S . Enteritidis ( n =15) were detected as Salmonella species without any false positive result for TS targets. Cross-reactions of NTS with TS were only rarely observed. Direct testing of positive BCs gave correct results. Sensitivities and specificities of the assay were as follows: 100 and 99.3 % for S . Typhi, 100 and 98.7 % for S . Paratyphi A, 100 and 97.3 % for S . Paratyphi B, 100 and 100 % for S . Paratyphi C, 100 and 100 % for S . Choleraesuis, and 100 and 100 % for Salmonella species, respectively. Conclusion . The eazyplex TyphiTyper is very easy to perform and allows the rapid identification of TS and S . Choleraesuis isolates.

In order to obtain more information on the MRSA population structure in the border region of Afghanistan and Pakistan, we collected and genotyped MRSA causing bloodstream infections from a tertiary care hospital in Peshawar, Pakistan, that serves the local population as well as Afghan immigrants and refugees. Thirty-one MRSA isolates from 30 patients were included and characterized by microarray hybridisation. For 25 patients, serum samples were tested using protein microarrays in order to detect antibodies against staphylococcal virulence factors. The most conspicuous result was the high rate of PVL-positive MRSA. Twenty-two isolates (71%) harboured lukF/S-PV genes. The most common lineage was CC772-MRSA-V/VT (PVL+) to which eleven isolates were assigned. The second most common strain was, surprisingly, CC8-MRSA-[IV+ACME] (PVL+), "USA300" (9 isolates). Two isolates were tst1 positive CC22-MRSA-IV, matching the Middle Eastern "Gaza Epidemic Strain". Another two were PVL-positive CC30-MRSA-IV. The remaining isolates belonged to, possibly locally emerging, CC1, CC5, and CC8 strains with SCC mec IV elements. Twenty-three patient sera were positive for anti-PVL-IgG antibodies. Several questions arise from the present study. It can be assumed that MRSA and high rates of PVL-positive S. aureus/MRSA are a public health issue in the Afghanistan/Pakistan border region. A possible emergence of the "USA300" clone as well as of the CC772 lineage warrants further investigation.