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Epidemiologia de β-Lactamases adquiridas do tipo AmpC em isolados clínicos humanos de Enterobacteriaceae
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Advisor(s)
Abstract(s)
Os β-lactâmicos constituem antibióticos de primeira linha no tratamento de
infecções causadas por Enterobacteriaceae, mas a resistência a estes compostos tem
aumentado progressivamente, sendo uma das principais causas a produção de
β-lactamases. Nestas incluem-se as AmpC adquiridas (qAmpC), cujos genes
codificantes coexistem habitualmente com genes de resistência a outros antibióticos na
mesma plataforma genética, limitando as opções terapêuticas. O objectivo global do
presente trabalho consistiu em analisar a contribuição da expansão clonal e da
transferência horizontal de genes na disseminação de Enterobacteriaceae produtoras de
qAmpC (sem AmpC indutivel).
Foram analisados 675 isolados clínicos provenientes de 5 instituições de saúde
Portuguesas (Norte e Centro). A identificação bacteriana e a avaliação da
susceptibilidade aos antibióticos foram realizadas por métodos universalmente aceites.
A caracterização das qAmpC incluiu a realização do teste do ácido borónico, o teste do
duplo sinergismo entre cefoxitina e a ceftazidima ou a cefotaxima, e a identificação de
genes blaqAmpC por PCR e sequenciação. Efectuou-se também a análise da estrutura
populacional, da transferibilidade de blaqAmpC, e análise plasmídica. O ambiente
genético em torno dos genes blaqAmpC foi também pesquisado (PCR, sequenciação).
A expressão de AmpC foi observada em 59% (59/100) dos isolados
identificados como presumíveis produtores de qAmpC, detectando-se apenas entre os
isolados resistentes à cefoxitina (66%, 59/89). Em 50% (50/100) dos isolados
detectaram-se dois tipos distintos de genes blaqAmpC, identificados como blaDHA-1 (94%,
n=47/50) e blaCMY-2 (6%, n=3/50).
A co-produção de β-lactamases de espectro alargado (ESBLs), predominantemente
do tipo SHV, é comum entre os isolados produtores de qAmpC. Reportam-se
associações de genes blaqAmpC e blaESBL nunca antes descritas. Em todos os isolados
produtores de qAmpC se observou fenótipo de multirresistência, verificando-se
elevadas taxas de resistência à maioria dos antibióticos: canamicina (98%), tobramicina
(96%), sulfonamidas (96%), ceftazidima (94%), cefotaxima (88%), aztreonamo (84%),
ciprofloxacina (82%), trimetoprim (80%), cloranfenicol (78%) e tetraciclina (70%). Os
antibióticos com menores taxas de resistência foram: o meropenemo (0%), o imipenemo
(4%), a cefepima (8%), a tigeciclina (8%) e a amicacina (26%). Genes blaDHA-1 ocorreram em diferentes espécies, K. pneumoniae (n=41), K.
oxytoca (n=4) e E. coli (n=2), durante todo o período de estudo e na maioria das
instituições analisadas, verificando-se uma grande diversidade clonal (n=14). Entre os
isolados de K. pneumoniae produtores de DHA-1 foram identificados os clones ST11
(n=14, 2 tipos de PFGE) e ST443 (n=1), nos quais se confirmou a presença de blaDHA-1
em plasmídeos não transferíveis pertencentes ao grupo de incompatibilidade IncR (50kb
e 180 kb, respectivamente). Em K. oxytoca e E. coli blaDHA-1 detectou-se em plasmídeos
não transferíveis pertencentes ao grupo de incompatibilidade IncHI2 (300 e 350 kb,
respectivamente) Estas associações nunca antes foram descritas.
Genes blaCMY-2 ocorreram apenas em E. coli (n=3), durante o período 2006-2007
e apenas em duas das instituições analisadas, verificando-se a presença de distintos
clones (n=3) pertencentes ao grupo filogenético B1. Em dois clones, um dos quais da
linhagem clonal ST101, blaCMY-2 foi encontrado em plasmídeos conjugativos de
incompatibilidade IncK de 80 kb, enquanto no terceiro clone (B1-fumC65) blaCMY-2 se
detectou em plasmídeos não transferíveis de incompatibilidade IncA/C2 de 150 kb.
A epidemiologia de isolados produtores de qAmpCs em Portugal é complexa,
parecendo envolver a disseminação de clones e/ou plasmideos no caso de blaDHA-1, e
predominantemente plasmídeos, mas também sequências de inserção do tipo ISEcp1 no
caso de blaCMY-2.
β-lactam antibiotics are first-line treatment of infections caused by Enterobacteriaceae, but resistance to these compounds is increasing, being β-lactamase production one of the key causes. These include the acquired AmpCs (qAmpCs), which encoding genes usually coexist with other antibiotic resistance genes in the same genetic platform, thus limiting therapeutic options. The main goal of this study was to assess the contribution of clonal expansion and horizontal gene transfer in the dissemination of qAmpC-producing Enterobacteriaceae lacking inducible AmpC. We analyzed 675 clinical isolates from five Portuguese health institutions (North and Centre) during a seven year period (2002-2008). The identification and antimicrobial susceptibility tests were performed using standard procedures. qAmpC characterization included the boronic acid test, the double disk synergy test between cefoxitin and ceftazidime or cefotaxime, and identification of blaqAmpC genes by PCR and sequencing. Population structure analysis, blaqAmpC transfer capability and plasmid characterization was also done. The genetic environment surrounding blaqAmpC genes was also investigated (PCR, sequencing). qAmpC expression was observed in 59% (59/100) of the presumptive qAmpC producers, being detected only among cefoxitin resistant isolates (66%, 59/89). blaqAmpC genes were detected in 50% (50/100) of the selected isolates, of which 94% (n=47/50) were identified as blaDHA-1 and 6% (n=3/50) as blaCMY-2. ESBL co-production was frequently detected among qAmpC-producers, predominantly of the SHV-type. Here we report several new blaqAmpC and blaESBL associations. All qAmpC producers had a multidrug resistance phenotype, and high resistance rates to most antibiotics: kanamycin (98%), tobramycin (96%), sulfonamides (96%), ceftazidime (94%), cefotaxime (88%), aztreonam (84%), ciprofloxacin (82%), trimethoprim (80%), chloramphenicol (78%) and tetracycline (70%). The lower resistance rates were observed for: meropenem (0%) to imipenem (4%), cefepime (8%), tigecycline (8%) and amikacin (26%). blaDHA-1 genes occurred in different species, K. pneumoniae (n = 41), K. oxytoca (n = 4) and E. coli (n = 2) and in a multiclonal scenario (n=14), throughout all the study period and in most of the analyzed institutions. Among DHA-1-producing K. pneumoniae the ST11 (n=14) and ST443 (n=1) clones were identified, in which blaDHA-1 was carried within IncR non-transferable plasmids belonging to incompatibility group IncR (of 50 and 180 kb, respectively). In K. oxytoca and E. coli blaDHA-1 was carried within non-transferable plasmids belonging to incompatibility group IncHI2 (of 300 and 350 kb, respectively). These associations are described here for the first time. blaCMY-2 genes occurred only in E. coli (n=3) from three distinct clones belonging to phylogenetic group B1, during the 2006-2007 period, and only in two of the analyzed institutions. In two clones, one of the ST101 clone, blaCMY-2 was carried within IncK conjugative plasmids of 80 kb, while in the third clone (B1-fumC65) blaCMY-2 was carried within IncA/C2 non-transferable plasmid of 150 kb. In Portugal the epidemiology qAmpC producers is quite complex, and seems to involve clonal and / or plasmid dissemination in the case of blaDHA-1, and predominantly plasmids, but also insertion sequences like ISEcp1 in the case blaCMY-2.
β-lactam antibiotics are first-line treatment of infections caused by Enterobacteriaceae, but resistance to these compounds is increasing, being β-lactamase production one of the key causes. These include the acquired AmpCs (qAmpCs), which encoding genes usually coexist with other antibiotic resistance genes in the same genetic platform, thus limiting therapeutic options. The main goal of this study was to assess the contribution of clonal expansion and horizontal gene transfer in the dissemination of qAmpC-producing Enterobacteriaceae lacking inducible AmpC. We analyzed 675 clinical isolates from five Portuguese health institutions (North and Centre) during a seven year period (2002-2008). The identification and antimicrobial susceptibility tests were performed using standard procedures. qAmpC characterization included the boronic acid test, the double disk synergy test between cefoxitin and ceftazidime or cefotaxime, and identification of blaqAmpC genes by PCR and sequencing. Population structure analysis, blaqAmpC transfer capability and plasmid characterization was also done. The genetic environment surrounding blaqAmpC genes was also investigated (PCR, sequencing). qAmpC expression was observed in 59% (59/100) of the presumptive qAmpC producers, being detected only among cefoxitin resistant isolates (66%, 59/89). blaqAmpC genes were detected in 50% (50/100) of the selected isolates, of which 94% (n=47/50) were identified as blaDHA-1 and 6% (n=3/50) as blaCMY-2. ESBL co-production was frequently detected among qAmpC-producers, predominantly of the SHV-type. Here we report several new blaqAmpC and blaESBL associations. All qAmpC producers had a multidrug resistance phenotype, and high resistance rates to most antibiotics: kanamycin (98%), tobramycin (96%), sulfonamides (96%), ceftazidime (94%), cefotaxime (88%), aztreonam (84%), ciprofloxacin (82%), trimethoprim (80%), chloramphenicol (78%) and tetracycline (70%). The lower resistance rates were observed for: meropenem (0%) to imipenem (4%), cefepime (8%), tigecycline (8%) and amikacin (26%). blaDHA-1 genes occurred in different species, K. pneumoniae (n = 41), K. oxytoca (n = 4) and E. coli (n = 2) and in a multiclonal scenario (n=14), throughout all the study period and in most of the analyzed institutions. Among DHA-1-producing K. pneumoniae the ST11 (n=14) and ST443 (n=1) clones were identified, in which blaDHA-1 was carried within IncR non-transferable plasmids belonging to incompatibility group IncR (of 50 and 180 kb, respectively). In K. oxytoca and E. coli blaDHA-1 was carried within non-transferable plasmids belonging to incompatibility group IncHI2 (of 300 and 350 kb, respectively). These associations are described here for the first time. blaCMY-2 genes occurred only in E. coli (n=3) from three distinct clones belonging to phylogenetic group B1, during the 2006-2007 period, and only in two of the analyzed institutions. In two clones, one of the ST101 clone, blaCMY-2 was carried within IncK conjugative plasmids of 80 kb, while in the third clone (B1-fumC65) blaCMY-2 was carried within IncA/C2 non-transferable plasmid of 150 kb. In Portugal the epidemiology qAmpC producers is quite complex, and seems to involve clonal and / or plasmid dissemination in the case of blaDHA-1, and predominantly plasmids, but also insertion sequences like ISEcp1 in the case blaCMY-2.