Publicação
Characterization of streptococcus pyogenes associated with tonsillo-pharyngitis in Portugal with particular emphasis on macrolide resistance
| Resumo: | There are large geographical and temporal variations in the frequency of macrolide-resistant Streptococcus pyogenes, as well in the associated resistance phenotypes. Although decreases in macrolide resistance were often associated with low macrolide consumption, natural fluctuations of macrolide resistant clones were also suggested to play an important role in the prevalence of resistant isolates and of resistance phenotypes. The recent report of heterogeneity found among group A streptococcus (GAS) isolates recovered from the same patient raised the question of whether the strategy used in the microbiology laboratory, the study of microorganisms from a single colony, was leading to the underestimation of macrolide resistance rate. For that purpose, 321 GAS isolates, recovered from 35 pharyngitis patients from one hospital were screened for potential differences in antimicrobial resistance profiles and emm types of up to 10 isolates recovered from the same sample. In our collection, all the isolates recovered from the same patient presented an identical antimicrobial resistance profile and emm type, indicating that the single colony approach was suitable for a correct identification of macrolide resistance rates and other epidemiological studies. In this thesis, the epidemiology of macrolide resistant GAS in Portugal is divided in 3 periods - 1998 to 2003, 2004 to 2006 and 2007 to 2011. In all periods, macrolide resistance rates and macrolide resistance phenotypes and genotypes were determined; molecular characterization of the resistant population was accomplished by T typing, emm typing, pulsed field gel electrophoresis (PFGE) profiling and multilocus sequence typing (MLST). In the first period, macrolide resistance was high (27%) and stable. However, this stability was accompanied by a variation in the prevalence of the resistance phenotypes. In just 6 years, there was a complete inversion in the prevalence of macrolide resistance phenotypes. The molecular characterization of 325 resistant isolates revealed a diverse population, and the genetic lineages identified in Portugal were also identified in other European countries; the most common were T12-emm22-ST46, T28-emm28-ST52, T4- emm4-ST39, T12-emm12-ST36 and T1-emm1-ST28. This unusual situation motivated continuing the surveillance studies. Results from the analysis of the isolates recovered in 2004-2006 allowed the identification of a decreasing trend in macrolide resistance that had started in 1999. The diversity in the clonal composition was still evident among the 156 isolates studied and, with minor exceptions, the same genetic lineages were identified, although differences in their prevalence were noted. Among the most prevalent lineages were T28-emm28-ST52, T4-emm4-ST39, T11-emm11-ST403, and T12-emm22-ST46. In order to determine if this decline in macrolide resistance was sustained, the susceptibility of isolates recovered in 2007-2011 was determined and the clonal composition of the resistant population (n=139) was analyzed. The results showed that macrolide resistance continued to decline, reaching in 2011 the lowest value ever recorded in Portugal (2%). Again, macrolide resistant genetic lineages had been previously described in Portugal and other countries, with some changes in their prevalence. The lineage defined by T11-emm11-ST403 was now the most prevalent, followed by T12-emm22-ST46, T12-emm12-ST36 and T28-emm28-ST52. One of the most intriguing observations was that this situation was paralleled by high macrolide consumption, emphasizing the critical role of the fluctuations in the resistant clones for the prevalence of resistance. Although a small group of resistant lineages has been shown to be widely disseminated, little is known about the reasons for their success and their origin - ongoing acquisition of macrolide resistance genes followed by local spread or simple geographic dissemination. In order to see if the macrolide resistant GAS population was mirroring the general pharyngeal population or if their dynamics were independent, a collection of 803 isolates, representing 50% of all isolates recovered from tonsillo-pharyngitis patients in Portugal from 2000 to 2005 was analyzed by all the typing methods mentioned above. This comparison allowed the identification of specific emm types and PFGE clones associated with each of the macrolide resistant phenotypes. Among the emm types found in this collection, emm4, emm22 and emm28 were associated with macrolide resistance and emm3, emm6, emm87 and emm89 with macrolide susceptibility. Furthermore, the usefulness of PFGE in typing S. pyogenes was reinforced with this work since it was the only method capable of differentiating macrolide resistant and susceptible isolates of the same emm types. Examples of this could be found in emm4 and emm1. Additionally, both populations presented different diversities indicating that macrolide resistant GAS has its own dynamics, independent of the changing patterns of the general population. The production of the exotoxins SpeA and SpeC was for a long time believed to be responsible for scarlet fever, one of the infections caused by S. pyogenes. However, the literature is contradictory and some studies failed to detect such associations. In order to find out any potential markers of this disease, we characterized a collection of 101 scarlet fever isolates and compared it with pharyngitis isolates (n=202) (these not associated with scarlet fever). To achieve this, the superantigen gene profile was determined for all isolates, in addition to the typing methods already mentioned. The results showed that ssa, speA and speC were associated with scarlet fever and that scarlet fever isolates are less diverse than the pharyngitis isolates, indicating that a restricted number of lineages have a higher propensity to cause this presentation. |
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| Autores principais: | Silva-Costa, Catarina |
| Assunto: | Streptococcus pyogenes Faringite Escarlatina Portugal Teses de doutoramento - 2014 |
| Ano: | 2014 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade de Lisboa |
| Idioma: | inglês |
| Origem: | Repositório da Universidade de Lisboa |
| Resumo: | There are large geographical and temporal variations in the frequency of macrolide-resistant Streptococcus pyogenes, as well in the associated resistance phenotypes. Although decreases in macrolide resistance were often associated with low macrolide consumption, natural fluctuations of macrolide resistant clones were also suggested to play an important role in the prevalence of resistant isolates and of resistance phenotypes. The recent report of heterogeneity found among group A streptococcus (GAS) isolates recovered from the same patient raised the question of whether the strategy used in the microbiology laboratory, the study of microorganisms from a single colony, was leading to the underestimation of macrolide resistance rate. For that purpose, 321 GAS isolates, recovered from 35 pharyngitis patients from one hospital were screened for potential differences in antimicrobial resistance profiles and emm types of up to 10 isolates recovered from the same sample. In our collection, all the isolates recovered from the same patient presented an identical antimicrobial resistance profile and emm type, indicating that the single colony approach was suitable for a correct identification of macrolide resistance rates and other epidemiological studies. In this thesis, the epidemiology of macrolide resistant GAS in Portugal is divided in 3 periods - 1998 to 2003, 2004 to 2006 and 2007 to 2011. In all periods, macrolide resistance rates and macrolide resistance phenotypes and genotypes were determined; molecular characterization of the resistant population was accomplished by T typing, emm typing, pulsed field gel electrophoresis (PFGE) profiling and multilocus sequence typing (MLST). In the first period, macrolide resistance was high (27%) and stable. However, this stability was accompanied by a variation in the prevalence of the resistance phenotypes. In just 6 years, there was a complete inversion in the prevalence of macrolide resistance phenotypes. The molecular characterization of 325 resistant isolates revealed a diverse population, and the genetic lineages identified in Portugal were also identified in other European countries; the most common were T12-emm22-ST46, T28-emm28-ST52, T4- emm4-ST39, T12-emm12-ST36 and T1-emm1-ST28. This unusual situation motivated continuing the surveillance studies. Results from the analysis of the isolates recovered in 2004-2006 allowed the identification of a decreasing trend in macrolide resistance that had started in 1999. The diversity in the clonal composition was still evident among the 156 isolates studied and, with minor exceptions, the same genetic lineages were identified, although differences in their prevalence were noted. Among the most prevalent lineages were T28-emm28-ST52, T4-emm4-ST39, T11-emm11-ST403, and T12-emm22-ST46. In order to determine if this decline in macrolide resistance was sustained, the susceptibility of isolates recovered in 2007-2011 was determined and the clonal composition of the resistant population (n=139) was analyzed. The results showed that macrolide resistance continued to decline, reaching in 2011 the lowest value ever recorded in Portugal (2%). Again, macrolide resistant genetic lineages had been previously described in Portugal and other countries, with some changes in their prevalence. The lineage defined by T11-emm11-ST403 was now the most prevalent, followed by T12-emm22-ST46, T12-emm12-ST36 and T28-emm28-ST52. One of the most intriguing observations was that this situation was paralleled by high macrolide consumption, emphasizing the critical role of the fluctuations in the resistant clones for the prevalence of resistance. Although a small group of resistant lineages has been shown to be widely disseminated, little is known about the reasons for their success and their origin - ongoing acquisition of macrolide resistance genes followed by local spread or simple geographic dissemination. In order to see if the macrolide resistant GAS population was mirroring the general pharyngeal population or if their dynamics were independent, a collection of 803 isolates, representing 50% of all isolates recovered from tonsillo-pharyngitis patients in Portugal from 2000 to 2005 was analyzed by all the typing methods mentioned above. This comparison allowed the identification of specific emm types and PFGE clones associated with each of the macrolide resistant phenotypes. Among the emm types found in this collection, emm4, emm22 and emm28 were associated with macrolide resistance and emm3, emm6, emm87 and emm89 with macrolide susceptibility. Furthermore, the usefulness of PFGE in typing S. pyogenes was reinforced with this work since it was the only method capable of differentiating macrolide resistant and susceptible isolates of the same emm types. Examples of this could be found in emm4 and emm1. Additionally, both populations presented different diversities indicating that macrolide resistant GAS has its own dynamics, independent of the changing patterns of the general population. The production of the exotoxins SpeA and SpeC was for a long time believed to be responsible for scarlet fever, one of the infections caused by S. pyogenes. However, the literature is contradictory and some studies failed to detect such associations. In order to find out any potential markers of this disease, we characterized a collection of 101 scarlet fever isolates and compared it with pharyngitis isolates (n=202) (these not associated with scarlet fever). To achieve this, the superantigen gene profile was determined for all isolates, in addition to the typing methods already mentioned. The results showed that ssa, speA and speC were associated with scarlet fever and that scarlet fever isolates are less diverse than the pharyngitis isolates, indicating that a restricted number of lineages have a higher propensity to cause this presentation. |
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