Objectives Osteomyelitis is a severe bone infection that was frequently fatal before the introduction of antibiotics and remains a significant healthcare burden today. Staphylococcus aureus is the most common cause, alongside other hospital-acquired pathogens. Despite their clinical importance, the evolutionary history of these bacteria remains poorly understood. We investigated historical osteomyelitis specimens to identify causative pathogens and characterise their genomes, virulence and antimicrobial resistance (AMR) Methods Seven osteomyelitis-affected bones from adults dating to 19th-20th century Germany were analysed using ancient DNA (aDNA) approaches. Following sequencing and screening, candidate pathogens were prioritised based on authentic aDNA damage patterns, established association with osteomyelitis and exclusion as environmental contaminants. Identified species were characterised by phylogenetics, multilocus sequence typing and virulence/AMR profiling. Results In four patients, we detected authentic aDNA from Acinetobacter baumannii, Staphylococcus aureus, or Streptococcus pyogenes. Detected taxa in the remaining three patients did not fulfil the criteria for further analysis. Two patients carried A. baumannii genomes clustering closely with modern avian and freshwater isolates. Both harboured virulence genes, alongside intrinsic efflux pumps and β-lactamases. One patient carried an S. aureus strain belonging to the globally disseminated clonal complex 30, responsible for outbreaks since the 1950s. Molecular dating indicated that this strain diverged from the wider lineage around 1800, placing it among the earliest members of this group. It encoded multiple virulence genes, but no methicillin resistance genes. The fourth patient carried an S. pyogenes strain related to modern epidemic lineages from North America, encoding conserved virulence factors, but no AMR genes. Conclusions These specimens provide a window into the evolution of osteomyelitis pathogens. While modern developments such as widespread antibiotic use have intensified the global resistance crisis, our findings indicate that the genetic foundations for pathogenicity and resistance were already present more than 100 years ago.

IntroductionMethicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen of global public health importance. A key element of MRSA decolonization strategies is the administration of mupirocin, an antibiotic also used to treat superficial skin infections. However, the emergence and global increase of mupirocin-resistant (mupR)-MRSA highlight the need for active surveillance to support evidence-based MRSA control measures. This study investigated the antibiotic resistance, genetic diversity, and plasmid profiles of mupR-MRSA from Germany and South Africa.MethodsPhenotypic identification of mupR-MRSA strains was verified by molecular methods. Characterization of all strains included PCR detection of Panton-Valentine leucocidin, immune evasion cluster genes, and staphylococcal protein A typing. Representative strains from each spa type were selected for whole-genome sequencing to determine their clonal lineages and relationships, including antibiotic and virulence gene content. Comparative analysis, phylogeny and classification of mupA-carrying plasmids were determined.ResultsEighty-two mupR-MRSA were characterized, comprising 32 strains that exhibited high-level mupirocin resistance (HmupR) and 50 strains with low-level mupirocin resistance (LmupR). The plasmid-mediated mupA gene and the V588F mutation in the bacterial isoleucyl-tRNA synthetase gene were identified in strains that demonstrated HmupR and LmupR, respectively. However, mupA and the V588F mutation were detected in two strains from Germany. The mupR-MRSA from Germany were assigned to CC1, CC5, CC8, CC22, CC30, and ST59, while those from South Africa were grouped into CC8, CC22, and CC30. The strains harboured different SCCmec types (IIa, III, IVa, IVj, and V). Notably, mupR CC22-MRSA-IVj and tst-positive CC30-MRSA-IIa strains were identified in both countries. The mupA-positive plasmids formed four distinct communities, including two singletons. The mupA-positive plasmids from the strains in Germany often carried the aminoglycoside (aac(6')-Ie/aph(2'')-Ia) resistance genes. However, the β-lactam (blaZ), arsenic (arsB, arsC, arsR), and copper (mco) resistance genes were primarily identified on mupA-positive plasmids from strains obtained in South Africa.DiscussionThis study highlights the genetic diversity and potential for horizontal gene transfer among mupA-positive MRSA lineages in the two countries, further supported by the regional clustering of mupA plasmids. Integrating genomic surveillance of chromosomal- and plasmid-mediated resistance with antimicrobial use and clinical data is needed for targeted MRSA infection prevention and control.

Staphylococcal enterotoxins (SEs) are major contributors to foodborne intoxications. Reliable detection methods for SEs are essential to maintain food safety and protect public health. Since the heat-stable toxins also exert their toxic effect in the absence of the bacterium, reliance on DNA detection alone can be misleading: it does not allow for determining which specific toxins encoded by a given strain are produced and epidemiologically linked with a given outbreak. Commercially available diagnostic assays for SE detection are so far limited in sensitivity and specificity as well as in the range of targeted toxins (SEA-SEE), thus non-targeted SEs linked to foodborne illness remain undetected at the protein level. This study aimed to develop a highly sensitive and specific multiplex suspension immunoassay (SIA) for SEA to SEI. To this end, high-affinity monoclonal antibodies (mAbs) for the specific detection of the individual SEs were generated. When implemented in sandwich ELISAs and multiplex SIA, these mAbs demonstrated exceptional sensitivity with detection limits in the low picogram per millilitre range. When applied for the analysis of SE production in liquid cultures of a panel of 145 whole-genome sequenced strains of Staphylococcus spp. and Enterococcus faecalis, the novel multiplex SIA detected and differentiated the eight SEs with assay accuracies of 86.9-100%. Notably, the multiplex SIA covered one to four sequence variants for each of the individual SEs. Validation confirmed high recovery rates and reliable performance in three representative complex food matrices. The implementation of the novel mAbs in a multiplex SIA enabled, for the first time, simultaneous detection, differentiation, and quantification of multiple SEs from minimal sample volumes using Luminex® technology. As a result, the multiplex SIA will help strengthen food safety protocols and public health response capabilities.

ABSTRACT Metals are essential for all living organisms, but the type of metal and its concentration determines its action. Even low concentrations of metals may have toxic effects on organisms and therefore exhibit antimicrobial activities. In this study, we investigate the evolutionary adaptation processes of Staphylococcus aureus to metals and common genes for metal tolerance. Laboratory and clinical isolates were treated with manganese, cobalt, zinc, or nickel metal salts to generate growth-adapted mutants. After growth in medium supplemented with zinc, whole-genome sequencing identified, among others, two genes, mgtE (SAUSA300_0910) , a putative magnesium transporter and spoVG (SAUSA300_0475), a global transcriptional regulator, as hot spots for stress-induced single-nucleotide polymorphisms (SNPs). SNPs in mgtE were also detected in mutants treated with high levels of cobalt or nickel salts. To investigate the effect of these genes on metal tolerance, deletion mutants and complementation strains in an S. aureus USA300 LAC* laboratory strain were generated. Both, the mgtE and spoVG deletion strains were more tolerant to cobalt, manganese, and zinc. The mgtE mutant was also more tolerant to nickel exposure. Inductively coupled plasma mass spectrometry analysis demonstrated that the mgtE deletion mutant accumulated less intracellular zinc than the wild type, explaining increased tolerance. From these results, we conclude that mgtE gene inactivation increases zinc tolerance presumably due to reduced uptake of zinc. For the SpoVG mutant, no direct effect on the intracellular zinc concentration was detected, indicating toward different pathways to increase tolerance. Importantly, inactivation of these genes offers a growth advantage in environments containing certain metals, pointing toward a common tolerance mechanism. IMPORTANCE Staphylococcus aureus is an opportunistic pathogen causing tremendous public health burden and high mortality in invasive infections. Treatment is becoming increasingly difficult due to antimicrobial resistances. The use of metals in animal husbandry and aquaculture to reduce bacterial growth and subsequent acquisition of metal resistances has been shown to co-select for antimicrobial resistance. Therefore, understanding adaptive mechanisms that help S. aureus to survive metal exposure is essential. Using a screening approach, we were able to identify two genes encoding the transporter MgtE and the transcriptional regulator SpoVG, which conferred increased tolerance to specific metals such as zinc when inactivated. Further testing showed that the deletion of mgtE leads to reduced intracellular zinc levels, suggesting a role in zinc uptake. The accumulation of mutations in these genes when exposed to other metals suggests that inactivation of these genes could be a common mechanism for intrinsic tolerance to certain metals.

OBJECTIVES:Because of a steady increase in the detection of daptomycin-resistant (DAP-R) Staphylococcus aureus at three medical centres in Cologne, Germany, molecular surveillance was established from June 2016 to June 2018 to investigate the causes of the emergence and spread of respective isolates. Seventy-five S. aureus isolates, both DAP-R and DAP-susceptible, were collected from 42 patients for further analysis. METHODS:Broth microdilution was used to determine the MICs for DAP and polyhexamethylene biguanide/polyhexanide (PHMB). To investigate the effect of PHMB on the development of DAP resistance, we performed selection experiments with PHMB. All isolates studied were subjected to whole-genome sequencing. Epidemiological, clinical, microbiological and molecular data were analysed comparatively. RESULTS:Acquisition of DAP resistance was mainly observed in patients with acute and chronic wounds (40/42, 96.2%) treated with antiseptic (32/42, 76.2%) rather than systemic antibiotic therapy using DAP or vancomycin (7/42, 16.7%). DAP-R S. aureus had a diverse genetic background; however, within individual patients, isolates were closely related. At least three potential transmission events were detected. Most DAP-R isolates had concomitant elevated MICs for PHMB (50/54, 92.6%), and in vitro selection experiments confirmed that PHMB treatment is capable of generating DAP resistance. DAP resistance could be linked to 12 different polymorphisms in the mprF gene in the majority of clinical isolates (52/54, 96.3%) as well as in all in vitro selected strains. DISCUSSION:DAP resistance in S. aureus can occur independently of prior antibiotic therapy and can be selected by PHMB. Therefore, wound treatment with PHMB may trigger individual resistance development associated with gain-of-function mutations in the mprF gene.

Global spread of multidrug-resistant, hospital-adapted Staphylococcus epidermidis lineages underscores the need for new therapeutic strategies. Here we show that many S . epidermidis isolates belonging to these lineages display cryptic susceptibility to penicillin/β-lactamase inhibitor combinations under in vitro conditions, despite carrying the methicillin resistance gene mecA . Using a mouse thigh model of S . epidermidis infection, we demonstrate that single-dose treatment with amoxicillin/clavulanic acid significantly reduces methicillin-resistant S . epidermidis loads without leading to detectable resistance development. On the other hand, we also show that methicillin-resistant S . epidermidis is capable of developing increased resistance to amoxicillin/clavulanic acid during long-term in vitro exposure to these drugs. These findings suggest that penicillin/β-lactamase inhibitor combinations could be a promising therapeutic candidate for treatment of a high proportion of methicillin-resistant S . epidermidis infections, although the in vivo risk of resistance development needs to be further addressed before they can be incorporated into clinical trials.

Abstract Background A diversity of risk factors for ca-MRSA manifestations has been described so far. Up to date toxic contact dermatitis induced by plants has not been identified as one. Patients and Methods After intense skin contact with poison ivy in the US a 24-year-old Afro-American showed pronounced bullous contact dermatitis on the back of the neck and subsequently massive ca-MRSA furunculitis with proof of Panton-Valentine-leucocidin (PVL). After travelling to Germany, his German girlfriend developed a subacute ca-MRSA, PVL-positive superinfection of a mosquito bite at her lower leg. Both infections required surgical intervention. Results While the male patient displayed contact dermatitis by poison ivy, the female patient demonstrated two risk factors for ca-MRSA: contact with a ca-MRSA positive person and a predisposing skin lesion. Both cases underpin the role of ca-MRSA transmission and the potential severeness of wound infections in young and immunocompetent persons, just to be resolved by invasive intervention. Conclusions Marked and recalcitrant skin or soft tissue infections in otherwise healthy young patients require instant microbiological analysis and surgical intervention flanked by adequate antibiotic therapy. Contact dermatitis induced by plant toxins should be taken into consideration as possible risk factor for the acquisition of ca-MRSA.

Hintergrund Für die Behandlung von Infektionen mit multiresistenten Erregern stehen nur noch wenige Reserveantibiotika zur Verfügung. Bei schweren MRSA-Infektionen, insb. mit Beteiligung der Knochen, wird i.d.R. Linezolid eingesetzt. Eine Zunahme der Linezolid-Resistenzen wird befürchtet.

We describe the identification of a methicillin-resistant, high-level mupirocin-resistant Staphylococcus argenteus. The isolate (1801221) was characterized as t6675-ST2250-SCCmecIVc, and whole-genome sequencing revealed that the isolate possessed two plasmids. One plasmid (34,870 bp), designated p1_1801221 with rep23, harboured the mupirocin resistance (mupA) gene. The second plasmid (20,644 bp), assigned as p2_1801221 with rep5a and rep16, carried the resistance determinants for penicillin (blaZ) and cadmium (cadD). Phylogenetic analysis revealed that the isolate clustered with the European ST2250 lineage. The overall high similarity of both plasmids in S. argenteus with published DNA sequences of Staphylococcus aureus plasmids strongly suggests an interspecies transfer. The pathogenic potential, community and nosocomial spread, and acquisition of antibiotic resistance gene determinants, including the mupA gene by S. argenteus, highlight its clinical significance and the need for its correct identification.

Hintergrund Für die Behandlung von Infektionen mit multiresistenten Erregern (MRE) stehen nur noch wenige Reserveantibiotika zur Verfügung. Bei schweren MRSA-Infektionen, insbesondere mit Beteiligung der Knochen, wirkt i.d.R. noch Linezolid, für die Kombinationsbehandlung schwerer Infektionen mit MRGN wird in der Intensivtherapie Fosfomycin benötigt. Fosfomycin wird in der S3-Leitlinie bei unkomplizierten Harnwegsinfektionen (HWI) empfohlen und in großem Umfang eingesetzt, sodass die Gefahr besteht, dass hierdurch Resistenzen entstehen.

In Staphylococcus aureus, resistance to β-lactamase stable β-lactam antibiotics is mediated by the penicillinbinding protein 2a, encoded by mecA or by its homologues mecB or mecC. However, a substantial number of meticillin-resistant isolates lack known mec genes and, thus, are called meticillin resistant lacking mec (MRLM). This study aims to identify the genetic mechanisms underlying the MRLM phenotype. A total of 141 MRLM isolates and 142 meticillin-susceptible controls were included in this study. Oxacillin and cefoxitin minimum inhibitory concentrations were determined by broth microdilution and the presence of mec genes was excluded by PCR. Comparative genomics and a genome-wide association study (GWAS) approach were applied to identify genetic polymorphisms associated with the MRLM phenotype. The potential impact of such mutations on the expression of PBP4, as well as on cell morphology and biofilm formation, was investigated. GWAS revealed that mutations in gdpP were significantly associated with the MRLM phenotype. GdpP is a phosphodiesterase enzyme involved in the degradation of the second messenger cyclic-di-AMP in S. aureus. A total of 131 MRLM isolates carried truncations, insertions or deletions as well as amino acid substitutions, mainly located in the functional DHH-domain of GdpP. We experimentally verified the contribution of these gdpP mutations to the MRLM phenotype by heterologous complementation experiments. The mutations in gdpP had no effect on transcription levels of pbp4; however, cell sizes of MRLM strains were reduced. The impact on biofilm formation was highly strain dependent. We report mutations in gdpP as a clinically relevant mechanism for β-lactam resistance in MRLM isolates. This observation is of particular clinical relevance, since MRLM are easily misclassified as MSSA (meticillin-susceptible S. aureus), which may lead to unnoticed spread of β-lactam-resistant isolates and subsequent treatment failure.

Here, we present the circular and complete genome sequences of the Nosocomiicoccus ampullae isolate 19-00310 and type strain DSM 19163. To our knowledge, these represent the first complete, circular chromosomes in the entire genus. Sequencing of a growth-adapted mutant suggests iron availability as a factor for growth improvement.

BACKGROUND:As next generation sequencing (NGS) technologies have experienced a rapid development over the last decade, the investigation of the bacterial genetic architecture reveals a high potential to dissect causal loci of antibiotic resistance phenotypes. Although genome-wide association studies (GWAS) have been successfully applied for investigating the basis of resistance traits, complex resistance phenotypes have been omitted so far. For S. aureus this especially refers to antibiotics of last resort like daptomycin and ceftaroline. Therefore, we aimed to perform GWAS for the identification of genetic variants associated with DAP and CPT resistance in clinical S. aureus isolates.MATERIALS/METHODS:To conduct microbial GWAS, we selected cases and controls according to their clonal background, date of isolation, and geographical origin. Association testing was performed with PLINK and SEER analysis. By using in silico analysis, we also searched for rare genetic variants in candidate loci that have previously been described to be involved in the development of corresponding resistance phenotypes.RESULTS:GWAS revealed MprF P314L and L826F to be significantly associated with DAP resistance. These mutations were found to be homogenously distributed among clonal lineages suggesting convergent evolution. Additionally, rare and yet undescribed single nucleotide polymorphisms could be identified within mprF and putative candidate genes. Finally, we could show that each DAP resistant isolate exhibited at least one amino acid substitution within the open reading frame of mprF. Due to the presence of strong population stratification, no genetic variants could be associated with CPT resistance. However, the investigation of the staphylococcal cassette chromosome mec (SCCmec) revealed various mecA SNPs to be putatively linked with CPT resistance. Additionally, some CPT resistant isolates revealed no mecA mutations, supporting the hypothesis that further and still unknown resistance determinants are crucial for the development of CPT resistance in S. aureus.CONCLUSION:We hereby confirmed the potential of GWAS to identify genetic variants that are associated with antibiotic resistance traits in S. aureus. However, precautions need to be taken to prevent the detection of spurious associations. In addition, the implementation of different approaches is still essential to detect multiple forms of variations and mutations that occur with a low frequency.

BACKGROUND:Methicillin-resistant Staphylococcus aureus (MRSA) is a common healthcare-associated pathogen that remains a major public health concern. Sequence type 228 (ST228) was first described in Germany and spread to become a successful MRSA clone in several European countries. In 2000, ST228 emerged in Lausanne and has subsequently caused several large outbreaks. Here, we describe the evolutionary history of this clone and identify the genetic changes underlying its expansion in Switzerland.MATERIALS AND METHODS:We aimed to understand the phylogeographic and demographic dynamics of MRSA ST228/ST111 by sequencing 530 representative isolates of this clone that were collected from 14 European countries between 1997 and 2012.RESULTS:The phylogenetic analysis revealed distinct lineages of ST228 isolates associated with specific geographic origins. In contrast, isolates of ST111, which is a single locus variant of ST228 sharing the same spa type t041, formed a monophyletic cluster associated with multiple countries. The evidence points to a German origin of the sampled population, with the basal German lineage being characterized by spa type t001. The highly successful Swiss ST228 lineage diverged from this progenitor clone through the loss of the aminoglycoside-streptothricin resistance gene cluster and the gain of mupirocin resistance. This lineage was introduced first in Geneva and was subsequently introduced into Lausanne.CONCLUSION:Our results reveal the radiation of distinct lineages of MRSA ST228 from a German progenitor, as the clone spread into different European countries. In Switzerland, ST228 was introduced first in Geneva and was subsequently introduced into Lausanne.

Rats are a reservoir of human- and livestock-associated methicillin-resistant Staphylococcus aureus (MRSA). However, the composition of the natural S. aureus population in wild and laboratory rats is largely unknown. Here, 144 nasal S. aureus isolates from free-living wild rats, captive wild rats and laboratory rats were genotyped and profiled for antibiotic resistances and human-specific virulence genes. The nasal S. aureus carriage rate was higher among wild rats (23.4%) than laboratory rats (12.3%). Free-living wild rats were primarily colonized with isolates of clonal complex (CC) 49 and CC130 and maintained these strains even in husbandry. Moreover, upon livestock contact, CC398 isolates were acquired. In contrast, laboratory rats were colonized with many different S.aureus lineages-many of which are commonly found in humans. Five captive wild rats were colonized with CC398-MRSA. Moreover, a single CC30-MRSA and two CC130-MRSA were detected in free-living or captive wild rats. Rat-derived S. aureus isolates rarely harbored the phage-carried immune evasion gene cluster or superantigen genes, suggesting long-term adaptation to their host. Taken together, our study revealed a natural S. aureus population in wild rats, as well as a colonization pressure on wild and laboratory rats by exposure to livestock- and human-associated S.aureus, respectively.

The increasing number of nosocomial pathogens with resistances against last resort antibiotics like linezolid leads to a pressing need for the reliable detection of these drug-resistant bacteria. National guidelines on infection prevention, e.g., in Germany, have already recommend screening for linezolid-resistant bacteria, although a corresponding screening agar medium has not been provided. In this study we analyzed the performance and reliability of a commercial, chromogenic linezolid screening agar. The medium was capable to predict more than a hundred linezolid-resistant isolates of E. faecium, E. faecalis, S. aureus, S. epidermidis, and S. hominis with excellent sensitivity and specificity. All isolates were collected at the National Reference Centre between 2010 and 2020.