Staphylococcus aureus : caractérisation moléculaire des isolats Afghans et étude du transfert conjugatif des gènes de résistance au linézolide

1. Molecular characterization of S. aureus isolates from Afghanistan: Staphylococcus aureus (SA) is a major cause of infections in human and plethora of animals. One of the main objective of this thesis was to investigate the molecular features and the antibiotic (ATB) resistance profile of infection and colonization strains of SA/methicillin-resistant SA (MRSA) isolated from human and livestock in Kabul. All strains were identified by conventional phenotypic methods. MRSA Strains were then extensively characterized using DNA microarray. Among SA isolates from human infection in the two hospitals of Kabul, from January to June 2017, MRSA prevalence was high at 66.3%. The 98 isolates (65 MRSA and 33 MSSA) were grouped into 12 clonal complexes (CCs) and 27 distinct clones. The prevalence of nasal carriage of the SA and MRSA among healthy population in Kabul was 33.3% and 12.7% respectively. Four CCs and 7 clones were identified. Around 11.8% (2/17) of SA strains caused bovine mastitis were MRSA, belonged to two distinct clones. We found a very low prevalence (1.05%) of SA nasal carriage in sheep in Kabul. Molecular characterization of the isolates, indicates a large genetic diversity and the presence of multiple virulence and ATB resistance genes among SA/MRSA strains circulating in the health-care facilities, community and livestock in Kabul. 2. Conjugative transfer of linezolid resistance gene: Linezolid is one of the last resort for the treatment of MRSA infections. The objective of this study was to investigate the risk of conjugative transfer of linezolid resistance from S. epidermidis (SE) strains to major SA clones. Five SE strains harboring different cfr-carrying plasmids (pSA737, p12-02300, p-cfr-PBR-A, p-cfr-PBR-B), and 9 major clones of SA circulating in France were used as donors and recipients respectively. The plasmid transfer rates were highly variable. The ST8-MRSA-IV USA300 was able to receive the cfr-carrying plasmids from all the donors tested. The SE ST5 strains was more able to transfer the plasmid p-cfr-PBR-B to different recipients, compared to SE ST2. The plasmids pSA737, and p12-02300 transferred to the most of the recipients tested. Plasmid fitness cost were highly variable. Under selective pressure, the linezolid resistance mediated by cfr vectors, can be efficiently transferred to major clones of SA circulating in France.1. Caractérisation moléculaire des isolats de S. aureus en provenance d’Afghanistan : Staphylococcus aureus (SA) est une cause majeure d'infections chez l'Homme et de nombreux animaux. L'un des principaux objectifs de cette thèse était d'étudier les caractéristiques moléculaires et le profil de résistance aux antibiotiques des souches d'infection et de colonisation de SA / SA résistante à la méticilline (SARM) isolées chez l'Homme et le bétail à Kaboul. Toutes les souches ont été identifiées par des méthodes phénotypiques conventionnelles. Les souches de SARM ont ensuite été caractérisées à l'aide d'une puce à ADN. Parmi les isolats de SA provenant d'infections humaines dans deux hôpitaux de Kaboul entre Janvier et Juin 2017, la prévalence des SARM était élevée (66,3 %). Les 98 isolats de SASM et SARM appartenaient à 12 complexes clonaux (CC) et 27 clones distincts. La prévalence du portage nasal de SA et du SARM dans la communauté, plus précisément chez des étudiants (hors médecine) de l’Université de Kaboul, était respectivement de 33,3% et 12,7%. Les 19 isolats de SARM appartenaient à 4 Enfin, les études réalisées chez l’animal ont montré qu’environ 11,8% des souches SA responsables de mammite bovine à Kabul étaient des SARM (2/17) et que la prévalence du portage nasal de SA chez les moutons était très faible (1.05 %). La caractérisation moléculaire des isolats a mis en évidence une grande diversité génétique et la présence de multiples gènes de virulence et de résistance parmi les souches SA/SARM circulant dans les établissements de soins, la communauté et le bétail à Kaboul. 2. Transfert conjugatif de gène de résistance au linézolide : Le linézolide est l'un des derniers recours pour le traitement des infections à SARM. L'objectif de cette étude était d'étudier le risque de transfert conjugatif de la résistance au linézolide des souches de S. epidermidis (SE) vers des souches SA de fonds génétiques différents. Cinq souches de SE portant différents plasmides possédant le gène cfr (pSA737, p12-02300, p-cfr-PBR-A, p-cfr-PBR-B), et 9 clones majeurs de SA circulant en France ont été respectivement utilisés comme donneuses et receveuses. Le clone ST8-MRSA-IV USA300 a pu recevoir les plasmides contenant le gène cfr de toutes les donneuses testées. Les souches SE ST5 avaient une plus grande capacité à transférer le plasmide p-cfr-PBR-B à différentes receveuses, par rapport aux souches du ST2. Les plasmides pSA737 et p12-02300 ont été transférés à la plupart des receveuses testées. Les coûts de fitness des plasmides testés étaient très variables. Les résultats ont au final montré que la résistance au linézolide, médiée par les vecteurs cfr, peut être transférée efficacement par conjugaison de SE aux clones pathogènes majeurs de SA circulant en France

Staphylococcus aureus : caractérisation moléculaire des isolats Afghans et étude du transfert conjugatif des gènes de résistance au linézolide

1. Caractérisation moléculaire des isolats de S. aureus en provenance d’Afghanistan : Staphylococcus aureus (SA) est une cause majeure d'infections chez l'Homme et de nombreux animaux. L'un des principaux objectifs de cette thèse était d'étudier les caractéristiques moléculaires et le profil de résistance aux antibiotiques des souches d'infection et de colonisation de SA / SA résistante à la méticilline (SARM) isolées chez l'Homme et le bétail à Kaboul. Toutes les souches ont été identifiées par des méthodes phénotypiques conventionnelles. Les souches de SARM ont ensuite été caractérisées à l'aide d'une puce à ADN. Parmi les isolats de SA provenant d'infections humaines dans deux hôpitaux de Kaboul entre Janvier et Juin 2017, la prévalence des SARM était élevée (66,3 %). Les 98 isolats de SASM et SARM appartenaient à 12 complexes clonaux (CC) et 27 clones distincts. La prévalence du portage nasal de SA et du SARM dans la communauté, plus précisément chez des étudiants (hors médecine) de l’Université de Kaboul, était respectivement de 33,3% et 12,7%. Les 19 isolats de SARM appartenaient à 4 Enfin, les études réalisées chez l’animal ont montré qu’environ 11,8% des souches SA responsables de mammite bovine à Kabul étaient des SARM (2/17) et que la prévalence du portage nasal de SA chez les moutons était très faible (1.05 %). La caractérisation moléculaire des isolats a mis en évidence une grande diversité génétique et la présence de multiples gènes de virulence et de résistance parmi les souches SA/SARM circulant dans les établissements de soins, la communauté et le bétail à Kaboul. 2. Transfert conjugatif de gène de résistance au linézolide : Le linézolide est l'un des derniers recours pour le traitement des infections à SARM. L'objectif de cette étude était d'étudier le risque de transfert conjugatif de la résistance au linézolide des souches de S. epidermidis (SE) vers des souches SA de fonds génétiques différents. Cinq souches de SE portant différents plasmides possédant le gène cfr (pSA737, p12-02300, p-cfr-PBR-A, p-cfr-PBR-B), et 9 clones majeurs de SA circulant en France ont été respectivement utilisés comme donneuses et receveuses. Le clone ST8-MRSA-IV USA300 a pu recevoir les plasmides contenant le gène cfr de toutes les donneuses testées. Les souches SE ST5 avaient une plus grande capacité à transférer le plasmide p-cfr-PBR-B à différentes receveuses, par rapport aux souches du ST2. Les plasmides pSA737 et p12-02300 ont été transférés à la plupart des receveuses testées. Les coûts de fitness des plasmides testés étaient très variables. Les résultats ont au final montré que la résistance au linézolide, médiée par les vecteurs cfr, peut être transférée efficacement par conjugaison de SE aux clones pathogènes majeurs de SA circulant en France.1. Molecular characterization of S. aureus isolates from Afghanistan: Staphylococcus aureus (SA) is a major cause of infections in human and plethora of animals. One of the main objective of this thesis was to investigate the molecular features and the antibiotic (ATB) resistance profile of infection and colonization strains of SA/methicillin-resistant SA (MRSA) isolated from human and livestock in Kabul. All strains were identified by conventional phenotypic methods. MRSA Strains were then extensively characterized using DNA microarray. Among SA isolates from human infection in the two hospitals of Kabul, from January to June 2017, MRSA prevalence was high at 66.3%. The 98 isolates (65 MRSA and 33 MSSA) were grouped into 12 clonal complexes (CCs) and 27 distinct clones. The prevalence of nasal carriage of the SA and MRSA among healthy population in Kabul was 33.3% and 12.7% respectively. Four CCs and 7 clones were identified. Around 11.8% (2/17) of SA strains caused bovine mastitis were MRSA, belonged to two distinct clones. We found a very low prevalence (1.05%) of SA nasal carriage in sheep in Kabul. Molecular characterization of the isolates, indicates a large genetic diversity and the presence of multiple virulence and ATB resistance genes among SA/MRSA strains circulating in the health-care facilities, community and livestock in Kabul. 2. Conjugative transfer of linezolid resistance gene: Linezolid is one of the last resort for the treatment of MRSA infections. The objective of this study was to investigate the risk of conjugative transfer of linezolid resistance from S. epidermidis (SE) strains to major SA clones. Five SE strains harboring different cfr-carrying plasmids (pSA737, p12-02300, p-cfr-PBR-A, p-cfr-PBR-B), and 9 major clones of SA circulating in France were used as donors and recipients respectively. The plasmid transfer rates were highly variable. The ST8-MRSA-IV USA300 was able to receive the cfr-carrying plasmids from all the donors tested. The SE ST5 strains was more able to transfer the plasmid p-cfr-PBR-B to different recipients, compared to SE ST2. The plasmids pSA737, and p12-02300 transferred to the most of the recipients tested. Plasmid fitness cost were highly variable. Under selective pressure, the linezolid resistance mediated by cfr vectors, can be efficiently transferred to major clones of SA circulating in France

Staphylococcus aureus : caractérisation moléculaire des isolats Afghans et étude du transfert conjugatif des gènes de résistance au linézolide

1. Molecular characterization of S. aureus isolates from Afghanistan: Staphylococcus aureus (SA) is a major cause of infections in human and plethora of animals. One of the main objective of this thesis was to investigate the molecular features and the antibiotic (ATB) resistance profile of infection and colonization strains of SA/methicillin-resistant SA (MRSA) isolated from human and livestock in Kabul. All strains were identified by conventional phenotypic methods. MRSA Strains were then extensively characterized using DNA microarray. Among SA isolates from human infection in the two hospitals of Kabul, from January to June 2017, MRSA prevalence was high at 66.3%. The 98 isolates (65 MRSA and 33 MSSA) were grouped into 12 clonal complexes (CCs) and 27 distinct clones. The prevalence of nasal carriage of the SA and MRSA among healthy population in Kabul was 33.3% and 12.7% respectively. Four CCs and 7 clones were identified. Around 11.8% (2/17) of SA strains caused bovine mastitis were MRSA, belonged to two distinct clones. We found a very low prevalence (1.05%) of SA nasal carriage in sheep in Kabul. Molecular characterization of the isolates, indicates a large genetic diversity and the presence of multiple virulence and ATB resistance genes among SA/MRSA strains circulating in the health-care facilities, community and livestock in Kabul. 2. Conjugative transfer of linezolid resistance gene: Linezolid is one of the last resort for the treatment of MRSA infections. The objective of this study was to investigate the risk of conjugative transfer of linezolid resistance from S. epidermidis (SE) strains to major SA clones. Five SE strains harboring different cfr-carrying plasmids (pSA737, p12-02300, p-cfr-PBR-A, p-cfr-PBR-B), and 9 major clones of SA circulating in France were used as donors and recipients respectively. The plasmid transfer rates were highly variable. The ST8-MRSA-IV USA300 was able to receive the cfr-carrying plasmids from all the donors tested. The SE ST5 strains was more able to transfer the plasmid p-cfr-PBR-B to different recipients, compared to SE ST2. The plasmids pSA737, and p12-02300 transferred to the most of the recipients tested. Plasmid fitness cost were highly variable. Under selective pressure, the linezolid resistance mediated by cfr vectors, can be efficiently transferred to major clones of SA circulating in France.1. Caractérisation moléculaire des isolats de S. aureus en provenance d’Afghanistan : Staphylococcus aureus (SA) est une cause majeure d'infections chez l'Homme et de nombreux animaux. L'un des principaux objectifs de cette thèse était d'étudier les caractéristiques moléculaires et le profil de résistance aux antibiotiques des souches d'infection et de colonisation de SA / SA résistante à la méticilline (SARM) isolées chez l'Homme et le bétail à Kaboul. Toutes les souches ont été identifiées par des méthodes phénotypiques conventionnelles. Les souches de SARM ont ensuite été caractérisées à l'aide d'une puce à ADN. Parmi les isolats de SA provenant d'infections humaines dans deux hôpitaux de Kaboul entre Janvier et Juin 2017, la prévalence des SARM était élevée (66,3 %). Les 98 isolats de SASM et SARM appartenaient à 12 complexes clonaux (CC) et 27 clones distincts. La prévalence du portage nasal de SA et du SARM dans la communauté, plus précisément chez des étudiants (hors médecine) de l’Université de Kaboul, était respectivement de 33,3% et 12,7%. Les 19 isolats de SARM appartenaient à 4 Enfin, les études réalisées chez l’animal ont montré qu’environ 11,8% des souches SA responsables de mammite bovine à Kabul étaient des SARM (2/17) et que la prévalence du portage nasal de SA chez les moutons était très faible (1.05 %). La caractérisation moléculaire des isolats a mis en évidence une grande diversité génétique et la présence de multiples gènes de virulence et de résistance parmi les souches SA/SARM circulant dans les établissements de soins, la communauté et le bétail à Kaboul. 2. Transfert conjugatif de gène de résistance au linézolide : Le linézolide est l'un des derniers recours pour le traitement des infections à SARM. L'objectif de cette étude était d'étudier le risque de transfert conjugatif de la résistance au linézolide des souches de S. epidermidis (SE) vers des souches SA de fonds génétiques différents. Cinq souches de SE portant différents plasmides possédant le gène cfr (pSA737, p12-02300, p-cfr-PBR-A, p-cfr-PBR-B), et 9 clones majeurs de SA circulant en France ont été respectivement utilisés comme donneuses et receveuses. Le clone ST8-MRSA-IV USA300 a pu recevoir les plasmides contenant le gène cfr de toutes les donneuses testées. Les souches SE ST5 avaient une plus grande capacité à transférer le plasmide p-cfr-PBR-B à différentes receveuses, par rapport aux souches du ST2. Les plasmides pSA737 et p12-02300 ont été transférés à la plupart des receveuses testées. Les coûts de fitness des plasmides testés étaient très variables. Les résultats ont au final montré que la résistance au linézolide, médiée par les vecteurs cfr, peut être transférée efficacement par conjugaison de SE aux clones pathogènes majeurs de SA circulant en France

Determination of antimicrobial susceptibility patterns in Staphylococcus aureus strains recovered from patients at two main health facilities in Kabul, Afghanistan

Abstract Background Staphylococcus aureus (S. aureus) is a major pathogen implicated in skin and soft tissue infections, abscess in deep organs, toxin mediated diseases, respiratory tract infections, urinary tract infections, post-surgical wound infections, meningitis and many other diseases. Irresponsible and over use of antibiotics has led to an increased presence of multidrug resistant organisms and especially methicillin resistant Staphylococcus aureus (MRSA) as a major public health concern in Afghanistan. As a result, there are many infections with many of them undiagnosed or improperly diagnosed. We aimed to establish a baseline of knowledge regarding the prevalence of MRSA in Kabul, Afghanistan, as well as S. aureus antimicrobial susceptibility to current available antimicrobials, while also determining those most effective to treat S. aureus infections. Methods Samples were collected from patients at two main Health facilities in Kabul between September 2016 and February 2017. Antibiotic susceptibility profiles were determined by the disc diffusion method and studied using standard CLSI protocols. Results Out of 105 strains of S. aureus isolated from pus, urine, tracheal secretions, and blood, almost half (46; 43.8%) were methicillin-sensitive Staphylococcus aureus (MSSA) while 59 (56.2%) were Methicillin-resistant Staphylococcus aureus (MRSA). All strains were susceptible to vancomycin. In total, 100 (95.2%) strains were susceptible to rifampicin, 96 (91.4%) susceptible to clindamycin, 94 (89.5%) susceptible to imipenem, 83 (79.0%) susceptible to gentamicin, 81(77.1%) susceptible to doxycycline, 77 (77.1%) susceptible to amoxicillin + clavulanic acid, 78 (74.3%) susceptible to cefazolin, 71 (67.6%) susceptible to tobramycin, 68 (64.8%) susceptible to chloramphenicol, 60 (57.1%) were susceptible to trimethoprim-sulfamethoxazole, 47 (44.8%) susceptible to ciprofloxacin, 38 (36.2%) susceptible to azithromycin and erythromycin, 37 (35.2%) susceptible to ceftriaxone and 11 (10.5%) were susceptible to cefixim. Almost all (104; 99.05%) were resistant to penicillin G and only 1 (0.95%) was intermediate to penicillin G. Interestingly, 74.6% of MRSA strains were azithromycin resistant with 8.5% of them clindamycin resistant. Ninety-six (91.4%) of the isolates were multi-drug resistant. Conclusions There was a high rate of Methicillin resistance (56.2%) among S. aureus strains in the samples collected and most (91.4%) were multidrug resistant. The most effective antibiotics to treat Staph infections were vancomycin, rifampicin, imipenem, clindamycin, amoxicillin-clavulanic acid, cefazolin, gentamicin and doxycycline. The least effective were azithromycin, ceftriaxone, cefixim and penicillin. We recommend that, where possible, in every case of S. aureus infection in Kabul, Afghanistan, Antibiotic susceptibility testing (AST) should be performed and responsible use of antibiotics should be considered

Characterizing the molecular epidemiology of Staphylococcus aureus across and within fitness facility types

Abstract Background Staphylococcus aureus is a common bacterium found in the nose and throat of healthy individuals, and presents risk factors for infection and death. We investigated environmental contamination of fitness facilities with S. aureus in order to determine molecular types and antibiotic susceptibility profiles of contaminates that may be transmitted to facility patrons. Methods Environmental swabs (n = 288) were obtained from several fitness facilities (n = 16) across Northeast Ohio including cross-fit type facilities (n = 4), traditional iron gyms (n = 4), community center-based facilities (n = 5), and hospital-associated facilities (n = 3). Samples were taken from 18 different surfaces at each facility and were processed within 24 h using typical bacteriological methods. Positive isolates were subjected to antibiotic susceptibility testing and molecular characterization (PVL and mecA PCR, and spa typing). Results The overall prevalence of S. aureus on environmental surfaces in the fitness facilities was 38.2% (110/288). The most commonly colonized surfaces were the weight ball (62.5%), cable driven curl bar, and CrossFit box (62.5%), as well as the weight plates (56.3%) and treadmill handle (50%). Interestingly, the bathroom levers and door handles were the least contaminated surfaces in both male and female restroom facilities (18.8%). Community gyms (40.0%) had the highest contamination prevalence among sampled surfaces with CrossFit (38.9%), traditional gyms (38.9%), and hospital associated (33.3%) contaminated less frequently, though the differences were not significant (p = 0.875). The top spa types found overall were t008 (12.7%), t267 (10.0%), t160, t282, t338 (all at 5.5%), t012 and t442 (4.5%), and t002 (3.6%). t008 and t002 was found in all fitness facility types accept Crossfit, with t267 (25%), t548, t377, t189 (all 10.7%) the top spa types found within crossfit. All samples were resistant to benzylpenicillin, with community centers having significantly more strains resistant to oxacillin (52.8%), erythromycin (47%), clindamycin (36%), and ciprofloxacin (19%). Overall, 36.3% of isolates were multidrug resistant. Conclusions Our pilot study indicates that all facility types were contaminated by S. aureus and MRSA, and that additional studies are needed to characterize the microbiome structure of surfaces at different fitness facility types and the patrons at these facilities

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