Epidemiología molecular y factores de virulencia de Staphylococcus aureus resistente a meticilina : emergencia en la comunidad e impacto a nivel intrahospitalario

Tesis (Doctora en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2017.Staphylcoccus aureus Resistente a Meticilina (MRSA) es un exitoso patógeno que se presenta asociado tanto al ambiente hospitalario (HA-MRSA) como a la comunidad (CA-MRSA), ha demostrado tener alta virulencia, capacidad para adquirir resistencia a diferentes antimicrobianos y adaptabilidad para transmitirse y diseminarse a través de la colonización de seres humanos y de la supervivencia en superficies inertes comportándose, estas, como reservorios ambientales para el microorganismo. A partir de 1990 CA-MRSA emergió y se diseminó en diferentes países del mundo y en aquellos de mayores prevalencia (> 50%) se ha descripto que estas cepas CA-MRSA comenzaron a introducirse en el ambiente hospitalario. Sin embargo, el conocimiento de su impacto en los hospitales es limitado, particularmente en América Latina. En este trabajo de tesis se realizó un estudio prospectivo de corte transversal y multicéntrico (nov-2009) de las infecciones por S. aureus mediante el análisis de 591 aislamientos clínicos de 66 hospitales de Argentina. Los casos se clasificaron considerando el tipo de inicio de la infección y la presencia o ausencia de factores de riesgo relacionados a la atención sanitaria (HRF) Se consideró que una infección es de inicio hospitalario (HO) si el cultivo positivo para S. aureus se obtuvo 48 horas después de la admisión en el hospital y el paciente no tenía evidencia de infección en el momento de la admisión Todos los demás casos se definieron como infección de inicio en la comunidad (CO). Las infecciones asociadas a la comunidad (CA) fueron definidas como casos de inicio en la comunidad de pacientes sin HRF durante el año anterior (CACO), según los criterios del CDC. Las infecciones asociadas al hospital o a la atención sanitaria (HA) incluyen: (i) infecciones de inicio hospitalario independientemente de la presencia de otros HRF (HAHO) e (ii) infecciones de inicio en la comunidad que ocurren en pacientes con al menos un HRF (HACO). Las cepas de MRSA fueron genéticamente tipificadas como genotipos CAMRSA y HA-MRSA (CA MRSAG y HA-MRSAG) por tipificación de SCCmec y spa, PFGE, MLST y perfil de genes de virulencia por PCR. Teniendo en cuenta todos los aislamientos, el 63% fueron de infecciones de inicio en la comunidad (CO) y de ellas el 55% fueron MRSA [39% CA-MRSAG y 16% HA-MRSAG].Detectó la proporción significativamente mayor de MRSA entre las infecciones de inicio en la comunidad CO vs las de inicio en el hospital HAHO (58% vs 49% respectivamente); Esta diferencia se notó principalmente en pediatría (48 hospitalization hours) and community-onset (CO) infections [including both, infections (HACO) in patients with healthcare-associated risk-factors (HRFs) and infections (CACO) in those without HRFs]. MRSA strains were genetically typed as CA-MRSA and HA-MRSA genotypes (CA-MRSAG and HA-MRSAG) by SCCmecand spa-typing, PFGE, MLST and virulence genes profile by PCR. Considering all isolates, 63% were from CO-infections and 55% were MRSA [39% CAMRSAG and 16% HAMRSAG]. A significantly higher MRSA proportion among CO- than HAHO-S. aureus infections was detected (58% vs 49%); mainly in children (62% vs 43%). The CAMRSAG/ HA-MRSAG have accounted for 16%/33% of HAHO-, 39%/13% of HACO- and 60.5%/0% of CACO-infections. In addition trends over time in MRSA were studied based on the national annual antimicrobial-susceptibility reports [WHONET-database, 2001- 2011, 49.909 S. aureus infections] using Phenotypic-definitions. These ones were validated by genotyping of isolates from the prospective cross-sectional multicenter study. Hence, the phenotypic definitions were significantly associated with genotypic definitions (p<0.0001) and it has correctly identified 94% of all MRSA isolates, with 94.7%, 92.4%, 96.9% and 87.8% of sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), respectively. The CA-MRSAP proportion, increased from 6.4% to 38.9% whereas HA-MRSAP decreased from 33.3% to 12.4% (P<0.0001). All these results, demonstrate that in Argentina, CA-MRSAG appears to be replacing HA-MRSAG strains in healthcare setting, along with an increasing reservoir in the community. Regarding the epidemiological associations identified in multivariate models for patients with healthcare-onset CA-MRSAG infections, CA-MRSAG behave like HA-MRSAG within hospitals but children were the highest risk group for healthcare-onset CA-MRSAG infections. Most CA-MRSAG belonged to two major clones: PFGE-type N-ST30-IVc-t019- PVL+ and PFGE-type I-ST5-IVa-t311 PVL+ (45% each). The ST5-IV-PVL+/ST30-IV-PVL+ clones have caused 31%/33% of all infections, 20%/4% of HAHO-, 43%/23% of HACOand 35%/60% of CACO- infections, with significant differences by age groups (children/adults) and geographical regions. Importantly, an isolate belonging to USA300- (ST8-SCCmecIVa-spat008- PVL+-ACME+) was detected for the first time in Argentina. Most of HA-MRSAG (66%) were related to the Cordobes/Chilean clone-(PFGE-type AST5- SCCmecI-t149) causing 18% of all infections (47% of HAHO and 13% of HACOinfections). Regarding to the antimicrobial susceptibility of the different MRSA clones and the possibility of using the new anti-MRSA β-lactam antibiotic for the treatment of MRSA infections in the country, the sensitivity of these strains to ceftaroline (CPT) was investigated. The prevalence of intermediate resistance (MIC = 2ug/mL) to CPT was 6.4% for all tested S. aureus isolates, with MIC50/90 of 0.25/1 ug/ml. All MSSA and CA-MRSA clones were 100% susceptible to CPT with MIC50/90 of 0.25/0.25 ug/ml for MSSA and 0.5/0.5 ug/ml for CA-MRSA. CPT susceptibility was strongly associated with clonal type in HA-MRSA isolates, being ST5-I (Cordobes/Chilean) and ST239-IIIA (Brazilian) the main clones with intermediate susceptibility. This result implies its molecular surveillance over time In addition, for the purpose of to describe the population structure of MSSA and to shed light on the origin of these CA-MRSA clones a total of 132 MSSA clinical isolates were analyzed by the same methodology as MRSA. Showing a reverse distribution to that detected for MRSA, with a north predominance of the MSSA [Is ST5-t311-PVL +] clone whereas MSSA [Ns-ST30-t012-PVL-] prevails in the south. spa types analysis showed that MRSA and MSSA isolates belonging to CC5 shared related t (t311 and t002), suggesting that these were closely related. Contrary, for MRSA (t019) and MSSA (mainly t012) isolates from ST30 no common t was found. These results suggest that: 1) CA-MRSA CC5-ST5-IV-t311-PVL(+) clone have emerged from MSSA CC5-t311-PVL(+) ancestor, already established in this country and 2) CA-MRSA CC30-ST30-IV-t019-PVL(+) clone was probably imported from neighboring countries. In addition, international CA-MRSA clones (ST72-IV, ST97-IV, ST8-IV) and closely related MSSA genetic lines were identified, supporting the hypothesis of local and continuous emergence of new CA-MRSA clones from successful MSSA lineages. In order to investigate the relationship between the epidemiological success of epidemic MRSA clones and their capacity to persist on an environmental inert surface, the survival and genes expression level (spa, fnbpA, fnbpB, hla, psmA1A2, IsdD, agrA, hld) during the persistence on melamine surface and biofilm form were analyzed by qRT-PCR. All MRSA clones showed a similar survival tendency, decreasing progressively towards 40 day, being higher (P<0,05) in [A-ST5-I-t149] and [C-ST100-IVnv-t002] clones than the others. The genes expression levels by each clone at 10 day, were higher than those in its initial condition 24h (p<0,001). IsdD transcription was increased about 40 and 35 fold of change in [A-ST5-I-t149] and [C-ST100-IVnv-t002] clones, respectively. These results suggest that the isdD could be involved in the persistence onto inert surfaces in the hospital setting of both epidemic MRSA clones. Regarding biofilm, the multiwell plate formation and the genes level of expression of the fnbpA, isdD, hla, spa, agrA were evaluated and showed that t: i) the clone CAMRSA [I1-ST5-IVa-t311] had a strong-moderate ability to form biofilm with high protein content, likely related to spa type t311 (ii) hla gene would be involved in the different capacity of biofilm formation in clinical strains (iii) the agr regulator could be involved with the different ability to form the biofilm, a through different pathways in several clones, including hla and/or fnbpA (clone USA300), iv) Different patterns of expression of the same genes were detected among the various clones in the biofilm phenotype, which implies a great variability between the different clonal complexes and the strategies used to survive in biofilm. Therefore, this study contributes to the existing literature, information about the factors associated with the dissemination of CA-MRSA clones in the community in addition to entry and transferability in hospitals. These contributions will be especially useful in children to evaluate strategies for the control and prevention of MRSA infections, both in the community and in hospitals. In a second instance, this work provides data on the possible characteristics (related to the survival capacity in inert materials) that the described clones could have for dissemination and establishment in the country both in the hospital and in the community that will help to address measures for control of transmission considering the differential capacities of these MRSA clones.Fil: Egea, Ana Lía. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Sola, Claudia Del Valle. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.Fil: Sola, Claudia Del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigación en Bioquímica Clínica e Inmunología; Argentina.Fil: Bocco, José Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Bocco, José Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Smania, Andrea María. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.Fil: Smania, Andrea María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina.Fil: Argaraña, Carlos Enrique. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.Fil: Argaraña, Carlos Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina.Fil: Gomez, Sonia Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Administración Nacional de Laboratorios e Institutos de Salud Dr. Carlos G. Malbran. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina

Molecular epidemiology of hospital-onset methicillin-resistant Staphylococcus aureus infections in Southern Chile

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen of public health importance. In Chile, the Cordobes/Chilean clone was the predominant healthcareassociated MRSA (HA-MRSA) clone in 1998. Since then, the molecular epidemiological surveillance of MRSA has not been performed in Southern Chile. We aimed to investigate the molecular epidemiology of HA-MRSA infections in Southern Chile to identify the MRSA clones involved, and their evolutionary relationships with epidemic international MRSA lineages. A total of 303 single inpatient isolates of S. aureus were collected in the Valdivia County Hospital (2007–2008), revealing 33 % (100 MRSA/303) prevalence for HA-MRSA infections. The SCCmec types I and IV were identified in 97 % and 3 % of HA-MRSA, respectively. All isolates lacked the pvl genes. A random sample (n = 29) of all MRSA was studied by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), SCCmec subtyping, agr and spa typing, and virulence genes profiling. PFGE analysis revealed the predominance (89 %, 26/29) of pulsotype A and three additional pulsotypes, designated H1, I33, and G1. Pulsotype A (ST5-SCCmecI-spa-t149) is clonally related to the Cordobes/Chilean clone. Pulsotype H1 (ST5-SCC mecIVNT-spa-t002) is genetically related to the Pediatric clone (ST5-SCCmecIV). Pulsotype I33 (ST5-SCCmecIVc-spa-t002) is clonally related by PFGE to the community-associated MRSA (CA-MRSA) clone spread in Argentina, I-ST5-IVa-PVL+ . The G1 pulsotype (ST8-SCCmecIVc-spa-t024) is clonally related to the epidemic USA300 CA-MRSA. Here, we demonstrate the stability of the Cordobes/Chilean clone over time as the major HA-MRSA clone in Southern Chile. The identification of two CA-MRSA clones might suggest that these clones have entered into the healthcare setting from the community. These results emphasize the importance of the local surveillance of MRSA infections in the community and hospital settings.Fil: Medina, G.. Universidad Austral de Chile. Facultad de Medicina. Instituto de Microbiología Clínica; ChileFil: Egea, Ana Lía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Otth, C.. Universidad Austral de Chile. Facultad de Medicina. Instituto de Microbiología Clínica; ChileFil: Otth, L.. Universidad Austral de Chile. Facultad de Medicina. Instituto de Microbiología Clínica; ChileFil: Fernández, H.. Universidad Austral de Chile. Facultad de Medicina. Instituto de Microbiología Clínica; ChileFil: Bocco, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Wilson, M.. Universidad Austral de Chile. Facultad de Medicina. Instituto de Microbiología Clínica; ChileFil: Sola, Claudia del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

New patterns of methicillin-resistant Staphylococcus aureus (MRSA) clones, community-associated MRSA genotypes behave like healthcare-associated MRSA genotypes within hospitals, Argentina

Methicillin-resistant Staphylococcus aureus (MRSA) burden is increasing worldwide in hospitals [healthcare-associated (HA)-MRSA] and in communities [community-associated (CA)-MRSA]. However, the impact of CA-MRSA within hospitals remains limited, particularly in Latin America. A countrywide representative survey of S. aureus infections was performed in Argentina by analyzing 591 clinical isolates from 66 hospitals in a prospective cross-sectional, multicenter study (Nov-2009). This work involved healthcare-onset infections-(HAHO, >48 hospitalization hours) and community-onset (CO) infections [including both, infections (HACO) in patients with healthcare-associated risk-factors (HRFs) and infections (CACO) in those without HRFs]. MRSA strains were genetically typed as CA-MRSA and HA-MRSA genotypes (CA-MRSAG and HA-MRSAG) by SCCmec- and spa-typing, PFGE, MLST and virulence genes profile by PCR. Considering all isolates, 63% were from CO-infections and 55% were MRSA [39% CA-MRSAG and 16% HA-MRSAG]. A significantly higher MRSA proportion among CO- than HAHO-S. aureus infections was detected (58% vs 49%); mainly in children (62% vs 43%). The CA-MRSAG/HA-MRSAG have accounted for 16%/33% of HAHO-, 39%/13% of HACO- and 60.5%/0% of CACO-infections. Regarding the epidemiological associations identified in multivariate models for patients with healthcare-onset CA-MRSAG infections, CA-MRSAG behave like HA-MRSAG within hospitals but children were the highest risk group for healthcare-onset CA-MRSAG infections. Most CA-MRSAG belonged to two major clones: PFGE-type N-ST30-SCCmecIVc-t019-PVL+ and PFGE-type I-ST5-IV-SCCmecIVa-t311-PVL+ (45% each). The ST5-IV-PVL+/ST30-IV-PVL+ clones have caused 31%/33% of all infections, 20%/4% of HAHO-, 43%/23% of HACO- and 35%/60% of CACO- infections, with significant differences by age groups (children/adults) and geographical regions. Importantly, an isolate belonging to USA300-0114-(ST8-SCCmecIVa-spat008-PVL+-ACME+) was detected for the first time in Argentina. Most of HA-MRSAG (66%) were related to the Cordobes/Chilean clone-(PFGE-type A-ST5-SCCmecI-t149) causing 18% of all infections (47% of HAHO- and 13% of HACO-infections). Results strongly suggest that the CA-MRSA clone ST5-IV-PVL+ has begun to spread within hospitals, replacing the traditional Cordobes/Chilean-HA-MRSA clone ST5-I-PVL−, mainly in children. Importantly, a growing MRSA reservoir in the community was associated with spreading of two CA-MRSA clones: ST5-IV-PVL+, mainly in children with HRFs, and ST30-IV-PVL+ in adults without HRFs. This is the first nationwide study in Argentina providing information about the molecular and clinical epidemiology of CA-MRSA, particularly within hospitals, which is essential for designing effective control measures in this country and worldwide.Fil: Egea, Ana Lía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Gagetti, Paula Silvana. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Área de Antimicrobianos; ArgentinaFil: Lamberghini, Ricardo. Universidad Nacional de Córdoba. Facultad de Ciencias Médicas; ArgentinaFil: Faccone, Diego Francisco. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Área de Antimicrobianos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lucero, Celeste. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Área de Antimicrobianos; ArgentinaFil: Vindel, Ana. Universidad Carlos III de Madrid. Instituto de Salud; EspañaFil: Tosoroni, Dario. Universidad Catolica de Córdoba. Facultad de Medicina; ArgentinaFil: Garnero, Analía. Hospital de Ninos de la Santísima Trinidad de Córdoba; ArgentinaFil: Saka, Hector Alex. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Galas, Marcelo Fabián. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorio e Instituto de Salud ; ArgentinaFil: Study Group of S. aureus in Argentina. No especifica;Fil: Bocco, Jose Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Corso, Alejandra. Dirección Nacional de Institutos de Investigación. Administración Nacional de Laboratorios e Institutos de Salud. Instituto Nacional de Enfermedades Infecciosas. Área de Antimicrobianos; ArgentinaFil: Sola, Claudia del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin