Persistence and Diffusion of mecC-Positive CC130 MRSA Isolates in Dairy Farms in Meurthe-et-Moselle County (France)

Background: Methicillin resistance in Staphylococcus aureus (MRSA) is classically conferred by the acquisition of the mecA gene encoding an additional penicillin binding protein with low affinity for beta-lactams. A mecA variant, named mecC, was described in 2011. MRSA isolates harboring mecC of both animal and human origin have since been collected in different European countries. In France, animal cases were reported in 4 dairy farms between 2008 and 2013 in the Meurthe-et-Moselle county, all located in a 30 km perimeter, suggesting a possible dissemination of mecC-positive MRSA strains. We performed a prospective study to evaluate the local epidemiology of such strains in terms of (i) dissemination among animals, humans and in the environment, and (ii) persistence in Meurthe-et-Moselle dairy cattle farms.Methods: The 4 French dairy farms with previous reports of mecC-positive MRSA strains and 14 farms in the same perimeter were included in this study. In each farm, nasal swabs, rectal swabs and milk samples were collected from 10 randomly selected cows, as well as nasal samples from family pets, volunteer farmers and veterinarians. One farm (E0), in which mecC-MRSA isolates were detected, was selected to study more deeply the dissemination of mecC-positive strains within the farm. After pre-enrichment of swabs and milk, they were subcultured on MSSA/MRSA chromogenic selective agar plates. S. aureus colonies were tested with a multiplex PCR to detect the mecA and mecC genes. The mecC-positive strains were characterized using DNA microarray.Results:mecC-positive strains were recovered in four farms, corresponding to the ones with previous reports of mecC-positive MRSA strains, and originated only from dairy cow samples. The screening in the E0 farm showed that 22% of the dairy cows carried mecC-positive MRSA. Three strains were also isolated from the environmental samples. All mecC-positive strains belonged to the clonal complex CC130 and harbored the same spa-type t1736.Conclusion: This study found that mecC-positive MRSA isolates are able to persist within the same farms for several years after being introduced in this setting and are able to widely disseminate but only among dairy cows suggesting that milking machines might be a key player

Pathogénie intracellulaire des staphylococcus aureus hypervirulents (rôle des phénol-soluble modulins, de l'alpha-toxine et de la beta-toxine dans l'échappement phagosomal et l'induction de l'autophagie)

Staphylococcus aureus est un pathogène intracellulaire facultatif capable d'envahir les cellules eucaryotes et d'induire leur mort. Cette virulence intracellulaire ou cytotoxicité a été associée à deux phénomènes, l'échappement phagosomal (destruction de la vacuole d'internalisation et relargage de bactéries libres dans le cytoplasme) et le détournement de l'autophagie (séquestration de S. aureus dans un autophagosome, blocage de la fusion lysosomale et réplication bactérienne intra-autophagosomale ). Les connaissances sur ces phénomènes intracellulaires sont limitées et comportent plusieurs résultats contradictoires: (i) l'expression d'alpha-toxine par S. aureus semble associée au détournement de l'autophagie mais pas à la cytotoxicité, (ii) l'expression des phenol-soluble modulins (PSM) est associée à la cytotoxicité mais pas à l'échappement phagosomal et (iii) l'échappement phagosomal, prérequis à la cytotoxicité, semble nécessiter l'expression de bêtatoxine alors que cette toxine est inactivée par l'insertion d'un phage dans le gène hlb chez la majorité des S. Aureus rencontrés en clinique. Dans le but de résoudre ces contradictions apparentes, nous avons investigué de façon intégrée les impacts respectifs de l'alpha-toxine et des PSM sur l'échappement phagosomal et l'induction de l'autophagie dans un modèle ex vivo d'infection intracellulaire sur ostéoblastes humains; nous avons également testé l'hypothèse d'une excision du phage convertissant hlb (avec restauration concomitante de l'expression de bêta-toxine) induite par le stress oxydatif intraphagosomal. Des cellules MG-63 (ostéosarcome) ont été infectées par la souche SF8300 (S. Aureus hypervirulent) et ses mutants isogéniques délétés pour l'alpha-toxine (dhla), les PSMa (D.psma) ou le système régulateur agr (D.agr) contrôlant l'expression d'hla et psma. Après 6h d'infection, les cellules ont été observées en microscopie électronique à transmission (MET) pour quantifier les S. aureus libres (échappement phagosomal) ou inclus dans un phagosome ou un autophagosome. Une méthode de microscopie confocale permettant d'évaluer le taux de perméabilisation phagosomale chez une lignée MG-63 exprimant un marqueur d'intégrité phagosomale a également été développée. Enfin, les fréquences d'excision du phage convertissant hlb ont été mesurées par méthode phénotypique (modification de l'hémolyse sur gélose au sang de mouton) et génotypique (confirmation par PCR de la restauration d' hlb) en l'absence de stress, en présence de stress oxydatif in vitro (H20 2 0,9 mM) et après infection intracellulaire ex vivo. La délétion d'agr était associée à une diminution de l'échappement phagosomal (22,9% vs. 30,9%; p<0,0001, test de Fisher) et de l'induction de l'autophagie (4,3% vs. 7,6%; p<O,OOOI). La délétion depsma était associée à une diminution de l'induction de l'autophagie (3,0% vs. 7,6%; p<0,0001) mais pas de l'échappement phagosomal. La délétion d'hia n'impactait ni l'échappement phagosomal ni l'induction de l'autophagie. L'excision du phage convertissant hlb présentait une fréquence basale faible (< 1%), augmentée modestement par le stress oxydatif mais pas par le passage intracellulaire.Ce travail permet de préciser les rôles respectifs des toxines staphylococciques majeures dans la cytotoxicité des S. aureus hypervirulents: (i) le rôle cytotoxique des PSMa. est associé à l'induction de l'autophagie plutôt qu'à l'échappement phagosomal; (ii) l'alpha-toxine ne joue aucun rôle dans ce contexte chez ces souches ; et (iii) la restauration du gène hlb par excision du phage n'est ni induite ni sélectionnée après passage intracellulaire, éliminant un rôle de la bêta-toxine. Le développement d'un outil de mesure de l'intégrité phagosomale permettra de déterminer prochainement si l'induction de l'autophagie est liée à une perméabilisation de la membrane phagosomale par les PSM. Ces résultats soulignent l'importance des PSM comme acteurs majeurs de la virulence intracellulaire de S. aureus et comme cibles thérapeutiques potentiellesLYON1-BU Santé (693882101) / SudocSudocFranceF

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in Horses, Cats, and Dogs Over a 5-Year Period in France

Methicillin-resistant Staphylococcus aureus (MRSA) has been reported as a worldwide pathogen in humans and animals including companion animals, i.e., cats, dogs, and horses. France lacked a comprehensive nationwide study describing the molecular features of MRSA circulating among companion animals over a large period of time. Here is reported the characterization of 130 non-duplicate clinical MRSA isolates collected from those three animal species from 2010 to 2015 through the French national Resapath network. Characterization of isolates was performed using phenotypic (antimicrobial susceptibility tests) and molecular (DNA arrays, spa-typing) methods. A horse-specific epidemiology was observed in France with the large dissemination of a unique clone, the CC398 clone harboring a Staphylococcal chromosomal cassette mec (SCCmec) type IV and spa-type t011. It was even the unique clone collected in 2015 whereas the clone CC8 USA500 (SCCmec type IV), classically described in horses, was present until 2014. Contrarily, cats and dogs were mainly infected by human-related MRSA isolates, i.e., clones usually reported in human infections, thus mirroring the human epidemiology in hospitals in France. Isolates belonging to the CC398 clone (SCCmec type IV or V) were also identified in 21.4% of dogs’ and 26.5% of cats’ MRSA isolates. In order to differentiate human-related from CC398 MRSA, tetracycline-resistance [or tet(M) detection] could be useful since this resistance is scarce in human-related strains but constant in CC398 MRSA isolates. In all, our data give a nationwide epidemiological picture of MRSA in companion animals over a 5-year period in France, adding further epidemiological information on the contribution of those animal species to a major public health issue. Considering the wide dissemination of CC398 MRSA isolates and the fact that 11/64 (17.2%) of them presented the Immune Evasion Cluster which enhances CC398 capacities to colonize humans, a specific attention should be paid in the coming years to determine the risk associated to the transmission in people in frequent contacts with companion animals. Our data also show that the prevalence of MRSA has likely decreased in cats, dogs, and horses between 2012 and 2015 in France. This trend should be monitored in the years to come

Methicillin-resistant Staphylococcus aureus in the environment of public transport: data from the metropolitan network in Lyon, France

International audienceMethicillin-resistant Staphylococcus aureus (MRSA) is involved in community-acquired and nosocomial diseases. The means of MRSA transmission and dissemination in the community remain uncertain. Studies have shown that public transport systems could be a source of MRSA and may serve as a potential source for community-acquired MRSA infections. This study aimed to investigate MRSA contamination on Lyon's metropolitan network (Métro) in France. Hand-touched surfaces were sampled with sterile swabs (Transystem(\textregistered)) during a 1-day transversal study by collecting 50 samples in seven hub stations and two trains for each of the four Métro lines. Then, during a longitudinal study, one sample was collected twice daily for 30 consecutive days in the busiest and most congested hub station. All swabs were incubated in enrichment medium for 24 h and then each suspension was plated onto a chromogenic selective medium for MRSA. After 24 h at 36 °C, all presumptive MRSA colonies were tested using VITEK(\textregistered) MS to confirm identification as S. aureus as well as by Alere\texttrademark PBP2a Culture Colony Test and mecA/mecC PCR to check methicillin resistance. Of the 110 swabs tested, 24 presumptive MRSA colonies were isolated, of which 2 were confirmed as S. aureus by VITEK(\textregistered) MS. These two isolates were tested negative using the PBP2a Culture Colony Test and PCR. Unlike other foreign cities such as Lisbon, the current data suggest a low level of MRSA contamination of hand-touched surfaces on Lyon's Métro. This should be put in perspective with the low level of MRSA colonisation in the French community

Does β-toxin production contribute to the cytotoxicity of hypervirulent Staphylococcus aureus?

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Implant-associated ESBL-Klebsiella pneumonia producing small colony variant bone and joint infection in a healthy 40-year-old man

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Molecular characterization and antimicrobial resistance of nasal Staphylococcus aureus in the community of Kabul

ABSTRACT: Objectives: This study aimed to investigate the prevalence and molecular characteristics of community methicillin-resistant Staphylococcus aureus (MRSA) nasal carriage among students at Kabul University. Methods: Nasal swabs were collected from anterior nares of 150 healthy non-medical students at Kabul University. Antimicrobial susceptibility testing was performed on all S. aureus isolates, and all detected MRSA isolates were then confirmed by mecA/mecC polymerase chain reaction and characterized using DNA microarray. Results: A total of 50 S. aureus strains were isolated from the anterior nares of the 150 participants. The prevalence of S. aureus and MRSA nasal carriage among Kabul students was 33.3% and 12.7%, respectively. Seven (36.8%) MRSA isolates and 8 (25.8%) methicillin-susceptible S. aureus (MSSA) isolates were multidrug-resistant (i.e. resistant to at least three different antimicrobials tested). All MRSA isolates (n = 19) were susceptible to linezolid, rifampicin, and fusidic acid. Seven MRSA clones, belonging to four clonal complexes (CCs), were identified. The most commonly identified clone was CC22-MRSA-IV TSST-1-positive, which accounted for 63.2% (12/19) of MRSA isolates. SCCmec typing showed that most MRSA strains harboured SCCmec type IV (94.7%). Thirteen (68.4%) MRSA isolates carried the TSST-1 and 5 (26.3%) PVL genes. Conclusion: Our findings revealed the relatively high prevalence of MRSA nasal carriers in the community in Kabul, with the predominance of the CC22-MRSA-IV TSST-1-positive clone and frequent multidrug resistance among these isolates

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in Horses, Cats, and Dogs Over a 5-Year Period in France

International audienceMethicillin-resistant Staphylococcus aureus (MRSA) has been reported as a worldwide pathogen in humans and animals including companion animals, i.e., cats, dogs, and horses. France lacked a comprehensive nationwide study describing the molecular features of MRSA circulating among companion animals over a large period of time. Here is reported the characterization of 130 non-duplicate clinical MRSA isolates collected from those three animal species from 2010 to 2015 through the French national Resapath network. Characterization of isolates was performed using phenotypic (antimicrobial susceptibility tests) and molecular (DNA arrays, spa-typing) methods. A horse-specific epidemiology was observed in France with the large dissemination of a unique clone, the CC398 clone harboring a Staphylococcal chromosomal cassette mec (SCCmec) type IV and spa-type t011. It was even the unique clone collected in 2015 whereas the clone CC8 USA500 (SCCmec type IV), classically described in horses, was present until 2014. Contrarily, cats and dogs were mainly infected by human-related MRSA isolates, i.e., clones usually reported in human infections, thus mirroring the human epidemiology in hospitals in France. Isolates belonging to the CC398 clone (SCCmec type IV or V) were also identified in 21.4% of dogs' and 26.5% of cats' MRSA isolates. In order to differentiate human-related from CC398 MRSA, tetracycline-resistance [or tet(M) detection] could be useful since this resistance is scarce in human-related strains but constant in CC398 MRSA isolates. In all, our data give a nationwide epidemiological picture of MRSA in companion animals over a 5-year period in France, adding further epidemiological information on the contribution of those animal species to a major public health issue. Considering the wide dissemination of CC398 MRSA isolates and the fact that 11/64 (17.2%) of them presented the Immune Evasion Cluster which enhances CC398 capacities to colonize humans, a specific attention should be paid in the coming years to determine the risk associated to the transmission in people in frequent contacts with companion animals. Our data also show that the prevalence of MRSA has likely decreased in cats, dogs, and horses between 2012 and 2015 in France. This trend should be monitored in the years to come

mecC-positive MRSA in horses

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