Caracterização dos mecanismos de resistência à ampicilina em estirpes clínicas de Haemophilus influenzae isoladas em Portugal entre o período de 2009 a 2012

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2013O Haemophilus influenzae (H. Influenzae) e uma bacteria Gram-negativa restrita ao aparelho respiratorio humano, normalmente responsavel por infecoes respiratorias adquiridas na comunidade. A gravidade destas infecoes resulta inicialmente num diagnostico presuntivo, sendo posteriormente estabelecido um tratamento antibiotico empirico. A resistencia desta bacteria aos antibioticos β-lactamicos pode constituir serias implicacoes a nivel clinico, sendo que, o uso inapropriado destes antibioticos e a utilizacao de antibioticos de largo espectro tem contribuido grandemente para a emergencia da resistencia a esta classe de antibioticos. A resistencia a ampicilina pode ser mediada por dois mecanismos: a producao de enzima β-lactamase, responsavel pela inativacao enzimatica dos antibioticos β- lactamicos, e pela presenca de proteinas de ligacao a penicilina alteradas que se traduz numa diminuicao da afinidade aos mesmos. A amostra de 248 estirpes clinicas de H. influenzae foi selecionada com base na concentracao inibitoria minima (CIM) a ampicilina, tendo como objetivo a caracterizacao dos mecanismos de resistencia aos antibioticos β- lactamicos em estirpes clinicas isoladas em Portugal. Foram estudadas 140 estirpes nao produtoras de β-lactamase com CIM a ampicilina ≥1mg/L, 66 estirpes produtoras de β- lactamase com CIM≥8mg/L, e ainda um grupo controlo de 42 estirpes suscetiveis com CIM1mg/L. A sequencia do gene ftsI foi amplificada e sequenciada para todas as estirpes. Da totalidade, 204 estirpes apresentavam mutacoes na regiao da transpeptidase do gene ftsI, nomeadamente 136 gBLNAR de 140 estirpes BLNAR, 24 gBLNAR de 42 estirpes BLNAS e 44 gBLPACR de 66 estirpes BLPAR. Foram identificados 40 padroes mutacionais que foram incluidos em grupos e subgrupos previamente descritos (I, IIa, IIb, IIc, IId e III-like). A analise destes padroes permitiu a classificacao da maioria das estirpes no grupo mutacional II (78,5%), e as substituicoes aminoacidicas mais frequentes situavam-se proximo do dominio KTG: Val547Ile (88,7%), Asn526Lys (76,9%) e Asn569Ser (72,6%). A relacao entre as mutacoes identificadas e os niveis de suscetibilidade a ampicilina permitiu constatar que 8,8% das estirpes eram suscetiveis, 8,8% eram resistentes devido a producao de β-lactamase (BLPAR), 64,5% eram resistentes pela alteracao das PBPs, e 17,7% apresentavam ambos os mecanismos de resistencia (BLPACR). Comparando estes resultados com os resultados obtido no periodo 2001-2008, verificou-se um aumento do numero de estirpes suscetiveis a ampicilina, e tambem do numero de estirpes resistentes com mutacoes no gene ftsI. Pela caracterizacao das estirpes e relacao clonal entre as estirpes BLNAR e BLPACR por Multilocus Sequence Typing (MLST), verificou-se que as estirpes BLNAR apresentam uma grande heterogeneidade genetica e que as estirpes BLPACR tambem nao aparentam ter uma grande proximidade filogenetica entre si.Haemophilus influenzae are Gram-negative bacilli restricted to the human respiratory tract and responsible for community-acquired respiratory tract infections. Due to the severity of these infections, it is standard procedure to perform a presumptive diagnostic test followed by an empirical antibiotic treatment. The developed resistance of these bacteria to the β-lactam antibiotics may constitute serious implications at the clinical level, and the inappropriate use of antibiotics or the use of broad-spectrum antibiotics has contributed greatly to the emergence of β-lactam antibiotic resistance. H. influenzae β-lactam antibiotic resistance develops via two mechanisms: β-lactamase production, which is responsible for the enzymatic inactivation of these antibiotics, especially β-lactams, and a change in the penicillin-binding proteins, which means decreased affinity to β-lactams. A sample of 248 clinical isolates of H. influenzae were selected according to their Minimum Inhibitory Concentration (MIC) to Ampicillin, with the purpose of characterizing β-lactam antibiotics resistance mechanisms in clinical strains isolated in Portugal. In total, this study analyzed 140 β-lactamase-non-producing strains (BLNAR) with ampicillin MIC≥1mg/L, 66 β- lactamase producing strains (BLPAR) with MIC≥8mg/L, and a control group of 42 β- lactamase-non-producing ampicillin susceptible strains (BLNAS) with MIC1mg/L. The ftsI gene encoding PBP3 was amplified and sequenced to all strains. Of the 248 H. influenzae strains, 204 had mutations in the ftsI transpeptidase domain as follows: 136 gBLNAR out of 140 BLNAR strains, 24 gBLNAR out of 42 BLNAS strains and 44 gBLPACR out of 66 BLPAR strains. Among gBLNAR and gBLPACR strains there were 40 different mutation patterns, that were included in the six previously described groups and subgroups (I, IIa, IIb, IIc, IId, III-like). The analysis of these patterns included the majority of the strains in group II (78,5%) and the most common amino acid substitutions were located near KTG motif: Val547Ile (88,7%), Asn526Lys (76,9%) and Asn569Ser (72,6%). The relation between these mutations and ampicillin susceptibility values showed that 8,8% of these strains were susceptible, 8,8% were resistant to ampicillin due to β-lactamase producing (BLPAR), 64,5% were resistant due to changes on the PBPs (BLNAR), and 17,7% had both resistant mechanisms (BLPACR). Comparing these results with previous ones obtained between 2001 and 2008, it was possible to verify that there was a rising of the strains susceptible to ampicillin (BLNAS), as well as resistant strains (BLNAR) with mutations in the ftsI gene. Through Multilocus Sequence Typing (MLST) it was possible to characterize the strains and study the clonal relationship between BLNAR and BLPACR. The BLNAR strains have a diverse genetic heterogeneity and the BLPACR strains did not seem to present a close phylogenetic relationship between them