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Methicillin-resistant Staphylococcus aureus (MRSA) of the ST1-SCCmecIV lineage has been associated with community-acquired (CA) infections in North America and Australia. In Brazil, multi-drug resistant ST1-SCCmecIV MRSA has emerged in hospital-associated (HA) diseases in Rio de Janeiro. To understand these epidemiological differences, genomic and phylogenetic analyses were performed. In addition, virulence assays were done for representative CA – and HA-MRSA strains. Despite the conservation of the virulence repertoire, some genes were missing in Brazilian ST1-SCCmecIV including lukSF-PV, fnbB, and several superantigen-encoded genes. Additionally, CA-MRSA lost the splDE while HA-MRSA strains conserved the complete operon. Most of these variable genes were located in mobile genetic elements (MGE). However, conservation and maintenance of MGEs were often observed despite the absence of their associated virulence markers. A Bayesian phylogenetic tree revealed the occurrence of more than one entrance of ST1 strains in Rio de Janeiro. The tree shape and chronology allowed us to infer that the hospital-associated ST1-SCCmecIV from Brazil and the community-acquired USA400 from North America are not closely related and that they might have originated from different MSSA strains that independently acquired SCCmecIV cassettes. As expected, representatives of ST1 strains from Brazil showed lower cytotoxicity and a greater ability to survive inside human host cells. We suggest that Brazilian ST1-SCCmecIV strains have adapted to the hospital setting by reducing virulence and gaining the ability to persist and survive inside host cells. Possibly, these evolutionary strategies may balance the biologic cost of retaining multiple antibiotic resistance genes.

Abstract Staphylococcus capitis is a coagulase-negative staphylococcus that has been described primarily as causing bloodstream infections in neonatal intensive care units (NICUs), but has also recently been described in prosthetic joint infections (PJIs). The multidrug-resistant S. capitis subsp. urealyticus clone NRCS-A, comprising three sublineages, is prevalent in NICUs across the world, but its impact on other patient groups such as those suffering from PJIs or among adults planned for arthroplasty is unknown. Genome sequencing and subsequent analysis were performed on a Swedish collection of PJI isolates (n = 21), nasal commensals from patients planned to undergo arthroplasty (n = 20), NICU blood isolates (n = 9), operating theatre air isolates (n = 4), and reference strains (n = 2), in conjunction with an international strain collection (n = 248). The NRCS-A Outbreak sublineage containing the composite type V SCCmec-SCCcad/ars/cop element was present in PJIs across three Swedish hospitals. However, it was not found among nasal carrier strains, where the less virulent S. capitis subsp. capitis was most prevalent. The presence of the NRCS-A Outbreak clone in adult patients with PJIs demonstrates that dissemination occurs beyond NICUs. As this clone has several properties which facilitate invasive infections in patients with medical implants or immunosuppression, such as biofilm forming ability and multidrug resistance including heterogeneous glycopeptide-intermediate susceptibility, further research is needed to understand the reservoirs and distribution of this hospital-associated pathogen.

Background: Phage therapy a promising antimicrobial strategy to address antimicrobial resistance for infections caused by the major human pathogen Staphylococcus aureus. Development of therapeutic phages for human use should follow pharmaceutical standards, including selection of strictly lytic bacteriophages with high therapeutic potential and optimization of their production process. Results: Here, we describe three novel Silviavirus phages active against 82% of a large collection of strains (n = 150) representative of various methicillin-susceptible and -resistant S. aureus clones circulating worldwide. We also investigated the optimization of the efficiency and safety of phage amplification protocols. To do so, we selected a well-characterized bacterial strain in order to (i) maximize phage production yields, reaching phage titres of 1011 PFU/mL in only 4 h; and (ii) facilitate phage purity while minimizing the risk of the presence of contaminants originating from the bacterial host; i.e., secreted virulence factors or induced temperate phages. Conclusions: In sum, we propose a quality-by-design approach for the amplification of broad-spectrum anti-S. aureus phages, facilitating the subsequent steps of the manufacturing process; namely, purification and quality control.

Abstract Background The methicillin-resistant clone Staphylococcus capitis NRCS-A, involved in sepsis in neonatal intensive care units (NICUs) worldwide, is able to persist and spread in NICUs, suggesting the presence of reservoirs inside each setting. The purpose of the present study was to identify these reservoirs and to investigate the cycle of transmission of NRCS-A in one NICU. Methods In a single institution study, NRCS-A was sought in 106 consecutive vaginal samples of pregnant women to identify a potential source of NRCS-A importation into the NICU. Additionally NICU caregivers and environmental including incubators were tested to identify putative secondary reservoirs. Finally, the efficacy of disinfection procedure in the elimination of NRCS-A from incubators was evaluated. Results No S. capitis was isolated from vaginal samples of pregnant women. Three of the 21 tested caregivers (14%) carried S. capitis on their hands, but none remain positive after a five-day wash-out period outside NICU. Moreover, the clone NRCS-A persisted during six consecutive weeks in the NICU environment, but none of the sampled sites was constantly contaminated. Finally in our before/after disinfection study, all of 16 incubators were colonized before disinfection and 10 (62%) incubators remained colonized with NRCS-A after the disinfection procedure. Conclusions The partial ineffectiveness of incubators’ disinfection procedures is responsible for persistence of NRCS-A inside a NICU, and the passive hand contamination of caregivers could be involved in the inter-patient transmission of S. capitis.

Staphylococcus pseudintermedius is a colonizer as well as an important pathogen of dogs where it is responsible for skin, ear and post-operative infections. The emergence of methicillin-resistant S. pseudintermedius (MRSP) in the early 2000s, which were additionally resistant to most veterinary-licensed antibiotics, drew specific attention to these pathogens due to the limitations created in veterinary therapeutic options. Multiple studies showed that the sequence type (ST)71 was the most frequently identified clone in Europe. A few years ago, several publications have suggested a decline of the ST71 clone and the emergence of the ST258 lineage in Northern Europe. In this study, we show that ST71 is also decreasing over time in France and that the non-ST71 population is highly heterogeneous. Globally, the non-ST71 clones are more susceptible to antibiotics, which might be good news for veterinarians. Two other lineages, ST258 and ST496, seem to be successful in France. These isolates, as well as representatives of the ST71 clone, underwent whole-genome sequence. This study shows that the ST71 and ST496 clusters are highly homogenous while the ST258 cluster is more diverse. Each ST possesses a specific pattern of resistance and virulence genes. The reasons for the apparent and simultaneous success of the ST258 and ST496 clones remain unclear. But the emergence of the ST496 clone will require monitoring given its multi-resistant genotype and threat to canine health.

We report here on the taxonomic and molecular diversity of 10 929 terrestrial arthropod specimens, collected on four islands of the Society Archipelago, French Polynesia. The survey was part of the ‘SymbioCode Project’ that aims to establish the Society Islands as a natural laboratory in which to investigate the flux of bacterial symbionts (e.g., Wolbachia) and other genetic material among branches of the arthropod tree. The sample includes an estimated 1127 species, of which 1098 included at least one DNA-barcoded specimen and 29 were identified to species level using morphological traits only. Species counts based on molecular data emphasize that some groups have been understudied in this region and deserve more focused taxonomic effort, notably Diptera, Lepidoptera and Hymenoptera. Some taxa that were also subjected to morphological scrutiny reveal a consistent match between DNA and morphology-based species boundaries in 90% of the cases, with a larger than expected genetic diversity in the remaining 10%. Many species from this sample are new to this region or are undescribed. Some are under description, but many await inspection by motivated experts, who can use the online images or request access to ethanol-stored specimens.

• Staphylococcus aureus bacteraemia (SAB) can lead to infective endocarditis (IE). • Specific traits of IE-causing strains versus SAB-only are questioned. • GWAS from IE and SAB-only strains does not discriminate the 2 groups. • In murine and rabbit IE models, IE and SAB-only strains behave in the same way. We aimed at determining whether specific S. aureus strains cause infective endocarditis (IE) in the course of Staphylococcus aureus bacteraemia (SAB). A genome-wide association study (GWAS) including 924 S. aureus genomes from IE (274) and non-IE (650) SAB patients from international cohorts was conducted, and a subset of strains was tested with two experimental animal models of IE, one investigating the early step of bacterial adhesion to inflamed mice valves, the second evaluating the local and systemic developmental process of IE on mechanically-damaged rabbit valves. The genetic profile of S. aureus IE and non-IE SAB strains did not differ when considering single nucleotide polymorphisms, coding sequences, and k-mers analysed in GWAS. In the murine inflammation-induced IE model, no difference was observed between IE and non-IE SAB strains both in terms of adhesion to the cardiac valves and in the propensity to cause IE; in the mechanical IE-induced rabbit model, there was no difference between IE and non-IE SAB strains regarding the vegetation size and CFU. All strains of S. aureus isolated from SAB patients must be considered as capable of causing this common and lethal infection once they have accessed the bloodstream. [ABSTRACT FROM AUTHOR]

Implicated in more than 60% of bone and joint infections (BJIs), Staphylococci have a particular tropism for osteoarticular tissue and lead to difficult-to-treat clinical infections. To date, Staphylococcus aureus internalization in non-professional phagocytic cells (NPPCs) is a well-explored virulence mechanism involved in BJI chronicity. Conversely, the pathophysiological pathways associated with Staphylococcus non-aureus (SNA) BJIs have scarcely been studied despite their high prevalence. In this study, fifteen reference strains from 15 different SNA species were compared in terms of (i) adhesion to human fibronectin based on adhesion microplate assays and (ii) internalization ability, intracellular persistence and cytotoxicity based on an in vitro infection model using human osteoblasts. Compared to S. aureus, Staphylococcus pseudintermedius was the only species that significantly adhered to human fibronectin. This species was also associated with high (even superior to S. aureus) internalization ability, intracellular persistence and cytotoxicity. These findings were confirmed using a panel of 17 different S. pseudintermedius isolates. Additionally, S. pseudintermedius internalization by osteoblasts was completely abolished in β1 integrin-deficient murine osteoblasts. These results suggest the involvement of β1 integrin in the invasion process, although this mechanism was previously restricted to S. aureus. In summary, our results suggest that internalization into NPPCs is not a classical pathophysiologic mechanism of SNA BJIs. S. pseudintermedius appears to be an exception, and its ability to invade and subsequently induce cytotoxicity in NPPCs could explain its severe and necrotic forms of infection, notably in dogs, which exhibit a high prevalence of S. pseudintermedius infection.

Phage therapy appears to be a promising approach to tackle multidrug-resistant bacteria, including staphylococci. However, most anti-staphylococcal phages have been characterized in Staphylococcus aureus , while a limited number of studies investigated phage activity against S. epidermidis . We studied the potential of phage training to extend the host range of two types of anti- S . aureus phages against S. epidermidis isolates. The Appelmans protocol was applied to a mixture of Kayvirus and a mixture of Silviavirus phages repeatedly exposed to seven S . epidermidis strains representative of nosocomial-associated sequence types (ST), including the world-wide disseminated ST2. We observed increased activity only for the Kayvirus mixture against two of these strains (ST2 or ST35). Phage subpopulations isolated from the training mixture using these two strains (five/strain) exhibited different evolved phenotypes, active only against their isolation strain or strains of the same ST. Of note, 16/47 ST2 strains were susceptible to one of the groups of trained phages. A comparative genomic analysis of ancestral and trained phage genomes, conducted to identify potential bacterial determinants of such specific activity, found numerous recombination events between two of the three ancestors. However, a small number of trained phage genes had nucleotide sequence modifications impacting the corresponding protein compared to ancestral phages, two to four of them per phage genome being specific of each group of phage subpopulations exhibiting different host range. The results suggest that anti- S . aureus phages can be adapted to S. epidermidis isolates but with inter- and intra-ST specificity.Importance S. epidermidis is increasingly recognized as a threat for public health. Its clinical importance is notably related to multidrug resistance. Phage therapy is one of the most promising alternative therapeutic strategies to antibiotics. Nonetheless, only very few phages active against this bacterial species have been described. In the present study, we showed that phage training can be used to extend the host range of polyvalent Kayvirus phages within the Staphylococcus genera to include S. epidermidis species. In the context of rapid development of phage therapy, in vitro forced adaptation of previously characterized phages could be an appealing alternative to fastidious repeated isolation of new phages to improve the therapeutic potential of a phage collection.

Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis. Nucleic acid extraction was optimized on a first group of 30 FTs with Aspergillus fumigatus or Mucorales infection by macrodissecting the microscopically identified fungal-rich area and comparing Qiagen and Promega extraction methods through DNA amplification by A. fumigatus and Mucorales primers. Targeted NGS was developed on a second group of 74 FTs using three primer pairs (ITS-3/ITS-4, MITS-2A/MITS-2B, and 28S-12-F/28S-13-R) and two databases (UNITE and RefSeq). A prior fungal identification of this group was established on fresh tissues. Targeted NGS and Sanger sequencing results on FTs were compared. To be valid, the molecular identifications had to be compatible with the histopathological analysis. In the first group, the Qiagen method yielded a better extraction efficiency than the Promega method (100% and 86.7% of positive PCRs, respectively). In the second group, targeted NGS allowed fungal identification in 82.4% (61/74) of FTs using all primer pairs, in 73% (54/74) using ITS-3/ITS-4, in 68.9% (51/74) using MITS-2A/MITS-2B, and in 23% (17/74) using 28S-12-F/28S-13-R. The sensitivity varied according to the database used (81% [60/74] using UNITE compared to 50% [37/74] using RefSeq [ P = 0.000002]). The sensitivity of targeted NGS (82.4%) was higher than that of Sanger sequencing (45.9%; P < 0.00001). To conclude, fungal integrated histomolecular diagnosis using targeted NGS is suitable on FTs and improves fungal detection and identification.

Objectives: To describe two linezolid-resistant MRSA strains carrying the cfr(B) gene detected in the French National Reference Centre for staphylococci., Methods: Two linezolid-resistant MRSA strains isolated from cystic fibrosis patients in two different French hospitals in 2017 and 2019 were examined to explore the mechanisms of linezolid resistance. Antimicrobial susceptibility was tested using broth microdilution and gradient strips. The genetic determinants of linezolid resistance were assessed by a multiplex PCR targeting cfr/cfr(B), optrA and poxtA genes, by amplification and sequencing of individual 23S rRNA genes and by WGS using both Illumina and Nanopore technologies., Results: The two MRSA strains were resistant to linezolid but susceptible to tedizolid, and PCR-positive for cfr/cfr(B). The WGS analysis indicated that they belonged to two different STs (ST8-MRSA-IV and ST5382-MRSA-IV) and that they both harboured the cfr(B) gene on the same 9.7 kb Tn6218-like chromosomal transposon, a finding only previously reported in Enterococcus sp. and Clostridioides difficile., Conclusions: To the best of our knowledge, this is the first description of the presence of cfr(B) in staphylococci, more specifically in linezolid-resistant MRSA strains. This finding illustrates the risk of horizontal intergenus transfer of oxazolidinone resistance genes in Staphylococcus aureus and highlights the need to monitor such emergence in this species., (© The Author(s) 2022. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Phage therapy a promising antimicrobial strategy to address antimicrobial resistance for infections caused by the major human pathogen Staphylococcus aureus . Development of therapeutic phages for human use should follow pharmaceutical standards, including selection of strictly lytic bacteriophages with high therapeutic potential and optimization of their production process. Results: Here, we describe three novel Silviavirus phages active against 82% of a large collection of strains (n = 150) representative of various methicillin-susceptible and -resistant S. aureus clones circulating worldwide. We also investigated the optimization of the efficiency and safety of phage amplification protocols. To do so, we selected a well-characterized bacterial strain in order to (i) maximize phage production yields, reaching phage titres of 10 11 PFU/mL in only 4 h; and (ii) facilitate phage purity while minimizing the risk of the presence of contaminants originating from the bacterial host; i.e., secreted virulence factors or induced temperate phages. Conclusions: In sum, we propose a quality-by-design approach for the amplification of broad-spectrum anti- S. aureus phages, facilitating the subsequent steps of the manufacturing process; namely, purification and quality control.

Methicillin-resistant Staphylococcus aureus (MRSA) of the ST1-SCC mec IV lineage has been associated with community-acquired (CA) infections in North America and Australia. In Brazil, multi-drug resistant ST1-SCC mec IV MRSA has emerged in hospital-associated (HA) diseases in Rio de Janeiro. To understand these epidemiological differences, genomic and phylogenetic analyses were performed. In addition, virulence assays were done for representative CA - and HA-MRSA strains. Despite the conservation of the virulence repertoire, some genes were missing in Brazilian ST1-SCC mec IV including lukSF-PV, fnbB , and several superantigen-encoded genes. Additionally, CA-MRSA lost the splDE while HA-MRSA strains conserved the complete operon. Most of these variable genes were located in mobile genetic elements (MGE). However, conservation and maintenance of MGEs were often observed despite the absence of their associated virulence markers. A Bayesian phylogenetic tree revealed the occurrence of more than one entrance of ST1 strains in Rio de Janeiro. The tree shape and chronology allowed us to infer that the hospital-associated ST1-SCC mec IV from Brazil and the community-acquired USA400 from North America are not closely related and that they might have originated from different MSSA strains that independently acquired SCC mec IV cassettes. As expected, representatives of ST1 strains from Brazil showed lower cytotoxicity and a greater ability to survive inside human host cells. We suggest that Brazilian ST1-SCC mec IV strains have adapted to the hospital setting by reducing virulence and gaining the ability to persist and survive inside host cells. Possibly, these evolutionary strategies may balance the biologic cost of retaining multiple antibiotic resistance genes.

An emergent kanamycin-susceptible ST5 methicillin-resistant Staphylococcus aureus (MRSA) lineage has been identified in France. Whole-genome sequencing revealed a 40-kb staphylococcal cassette chromosome (SCC) composite island with a mosaic structure including three SCC elements: a ΨSCC cop/ars , a SCC Lim88A with a ccrC recombinase, and a novel subtype of SCCmec type VI (VIb). This mosaic structure suggests a high recombination rate of SCC elements from distinct staphylococci species., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Staphylococcus pseudintermedius is a colonizer as well as an important pathogen of dogs where it is responsible for skin, ear and post-operative infections. The emergence of methicillin-resistant S. pseudintermedius (MRSP) in the early 2000s, which were additionally resistant to most veterinary-licensed antibiotics, drew specific attention to these pathogens due to the limitations created in veterinary therapeutic options. Multiple studies showed that the sequence type (ST)71 was the most frequently identified clone in Europe. A few years ago, several publications have suggested a decline of the ST71 clone and the emergence of the ST258 lineage in Northern Europe. In this study, we show that ST71 is also decreasing over time in France and that the non-ST71 population is highly heterogeneous. Globally, the non-ST71 clones are more susceptible to antibiotics, which might be good news for veterinarians. Two other lineages, ST258 and ST496, seem to be successful in France. These isolates, as well as representatives of the ST71 clone, underwent whole-genome sequence. This study shows that the ST71 and ST496 clusters are highly homogenous while the ST258 cluster is more diverse. Each ST possesses a specific pattern of resistance and virulence genes. The reasons for the apparent and simultaneous success of the ST258 and ST496 clones remain unclear. But the emergence of the ST496 clone will require monitoring given its multi-resistant genotype and threat to canine health.

Staphylococcus aureus is a commensal and pathogen of both humans and bovines. While the epidemiology of both groups has been extensively studied individually, little is known about the potential zoonotic transfer from animal strains to human being and vice versa. To determine the S. aureus prevalence of bovine mastitis in Algeria and the zoonotic transfer of strains to human beings, mastitis milk samples were collected, and professionals in a close contact with bovines were nasal swabbed. S. aureus isolates were all characterized by methicillin resistance and spa-typing. DNA microarrays analysis was performed on a subset of strains in order to detect other virulence factors, including toxins, and to assign the isolates to theirs MLST clonal complexes. Overall, 116/222 (52.3%) cows suffered from mastitis, whose 38.8% (45/116) infected with S. aureus. Human nasal carriage was of 38% (49/129), with only 4 MRSA carriers (3.1%). A higher diversity of spa-types was observed in human (35/50) than in bovine (18/67) isolates, with a predominance of clonal complexes CC97 and CC22 in bovines. The typical animal clone CC97 was occasionally detected in human beings. Conversely, the CC22 S. aureus clone largely switched from humans to bovines. Our study highlights the potential dynamics of animal and human S. aureus strains in the farm environment in Algeria, which may represent a health threat in both populations., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The multiresistant Staphylococcus capitis clone NRCS-A is a major pathogen in neonates worldwide. We show that NRCS-A grows as mauve colonies with a cream-color halo after a 5-day incubation on MRSA Brilliance 2 agar (Oxoid®). This innovative protocol will ease the screening of clinical and environmental niches of this clone., (Copyright © 2017 Elsevier Inc. All rights reserved.)