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Identification of major vancomycin resistant Enterococcus faecium reservoirs in livestock and the environment
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Os enterecocos são bactérias comensais do trato gastrointestinal de humanos e
animais, que em condições específicas se tornam agentes patogénicos oportunistas.
Enterococcus faecium resistentes à vancomicina (VRE) são uma das principais causas de
infeções nosocomiais mundialmente. Os VRE são endémicos nos Estados Unidos e
atingiram uma prevalência de 16,8% nos hospitais Europeus em 2020. Este estudo teve
como objetivo identificar reservatórios de VRE em amostras de animais de produção e
do meio ambiente, e identificar a sua relação epidemiológica com os VRE responsáveis
por infeções nos hospitais portugueses.
Foram rastreadas 294 amostras de animais de produção (porcos, n=80 e vacas,
n=55) e do ambiente (autocarros, n=89; mãos de passageiros, n=18; ambiente urbano
próximo de hospitais, n=52) para pesquisa de VRE. Especiação, deteção de genes de
resistência à vancomicina e de virulência foram realizados por PCR. A resistência aos
antibióticos foi estudada por difusão em disco, Etest® e diluição em agar. A análise
genómica de 23 E. faecium foi realizada por cgMLST (chewBBACA v2.5) e a deteção
de resistência aos antimicrobianos, virulência e plasmídeos por ResFinder_v3.1,
VirulenceFinder_v2.0 e PlasmidFinder_v2.0.
Um total de 470 isolados de Enterococcus spp. foram identificados em 90%
(266/294) das amostras, incluindo seis espécies diferentes: E. faecalis (30%), E.
casseliflavus (20%), E. gallinarum (14%), E. faecium (13%), E. hirae (5%) e E. durans
(< 1%). E. faecium foi mais prevalente nas amostras do ambiente [ambiente urbano
(27%), mãos dos passageiros (23%) e autocarros (19%)] comparativamente aos animais
[porcos (7%) e vacas (5%)]. Apesar de todos os isolados serem suscetíveis aos
glicopéptidos e de não terem sido detetados os genes vanA e vanB, 23% dos Enterococcus
continham o vanC2/3 e 17% o vanC1, incluindo 12 (40%) E. faecalis. Estes E. faecalis,
apresentaram um padrão de resistência associado principalmente a resistência intrínseca,
enquanto os E. faecium apresentaram elevada resistência aos aminoglicosídeos e
macrólidos (100%; genes aac(6’)-Ii e msrC ), à ampicilina (83%; mutações na pbp5),
tetraciclinas (39%; genes tetM and L) e elevada resistência à estreptomicina (35%; ant(6)-
Ia) e gentamicina (4%; aac(6')-aph(2'')). Foram identificados diferentes determinantes de
virulência em E. faecalis (asa1, gelE e ace) e E. faecium (ace, acm, scm, sgrA, efaAfm e
IS16).
A população de E. faecium mostrou uma elevada variabilidade genética, tendo
sido identificado um “sequence type” (ST) diferente por isolado, incluindo STs
pertencentes aos complexos clonais (CC) 17 (ST32) e CC94 (ST296, ST1205, ST800 e
ST2206), frequentemente associados a infeções hospitalares e a colonização. Embora não
tenha sido possível traçar uma ligação epidemiológica por cgMLST, entre os E. faecium
deste estudo e E. faecium isolados nos hospitais portugueses, foram identificados os
mesmos patrimónios clonais definidos por MLST em ambas as coleções.
Apesar de nenhuma das cinco coleções rastreadas neste estudo constituir um
reservatório de VRE em Portugal, foi detetada uma elevada prevalência de resistência aos
antibióticos e determinantes de virulência em isolados comensais de E. faecium e E.
faecalis. Estes resultados sugerem que uma vigilância ativa de reservatórios de Enterococcus, para além dos VRE, é fortemente recomendada na prevenção da
disseminação global da resistência aos antimicrobianos
Enterococci are commensals of the gastrointestinal tract of humans and animals, that under certain conditions, become opportunistic pathogens. Vancomycin-resistant Enterococcus faecium (VRE) is one of the leading causes of nosocomial infections worldwide. VRE are considered endemic in the United States and reached a prevalence of 16.8% in European hospitals in 2020. The present study aimed to identify VRE reservoirs among livestock and in the environment, and to determine the epidemiological relationship with VRE responsible for infections in Portuguese hospitals. A total of 294 samples from livestock (pigs, n=80 and cows, n=55) and from the environment (buses, n=89; hands of passengers, n=18; urban environment close to hospitals, n=52) were screened for VRE. Speciation, detection of vancomycin resistance and virulence genes were performed by PCR. Antimicrobial resistance was characterized by disk diffusion, Etest® and agar dilution. Genomic analysis of 23 selected E. faecium was performed by cgMLST (chewBBACA v2.5) and detection of antimicrobial resistance, virulence and plasmids by ResFinder_v3.1, VirulenceFinder_v2.0 and PlasmidFinder_v2.0. Overall, 470 Enterococcus spp. isolates were recovered from 90% (266/294) of the screened samples, and six species were identified: E. faecalis (30%), E. casseliflavus (20%), E. gallinarum (14%), E. faecium (13%), E. hirae (5%) and E. durans (< 1%). E. faecium was most prevalent among environmental samples [urban environment (27%), hands of bus passengers (23%) and buses (19%)] compared to livestock [pigs (7%) and cows (5%)]. Despite the fact that all isolates were susceptible to glycopeptides and no vanA or vanB genes were found, 23% of Enterococcus harbored the vanC2/3 and 17% carried the vanC1, including 12 (40%) E. faecalis. These E. faecalis, showed a resistance pattern mainly associated to intrinsic resistance, while E. faecium showed high resistance to aminoglycosides and macrolides (100%; aac(6’)-Ii and msrC genes), to ampicillin (83%; associated with mutations in pbp5), tetracyclines (39%; tetM and L genes) and high-level resistance to streptomycin (35%; ant(6)-Ia) and gentamicin (4%; aac(6')- aph(2'')). Different virulence determinants were identified among both E. faecalis (asa1, gelE and ace) and E. faecium (ace, acm, scm, sgrA, efaAfm and IS16). E. faecium population showed a high genetic variability, with a different sequence type (ST) per isolate, and including isolates from clonal complex (CC) 17 (ST32) and CC94 (ST296, ST1205, ST800 and ST2206), which are highly associated to hospital infections and human carriage. Although no epidemiological link could be drawn by cgMLST, between E. faecium isolates from this study and E. faecium isolated in Portuguese hospitals, the same clonal backgrounds defined by MLST were identified in both collections. Although none of the five settings screened in this study appear to be a VRE reservoir in Portugal, a high prevalence of antibiotic resistance and virulence determinants was identified among E. faecium and E. faecalis commensal isolates. Our results suggest that an active surveillance of Enterococcus reservoirs, beyond VRE, to prevent antimicrobial resistance spread is strongly recommended
Enterococci are commensals of the gastrointestinal tract of humans and animals, that under certain conditions, become opportunistic pathogens. Vancomycin-resistant Enterococcus faecium (VRE) is one of the leading causes of nosocomial infections worldwide. VRE are considered endemic in the United States and reached a prevalence of 16.8% in European hospitals in 2020. The present study aimed to identify VRE reservoirs among livestock and in the environment, and to determine the epidemiological relationship with VRE responsible for infections in Portuguese hospitals. A total of 294 samples from livestock (pigs, n=80 and cows, n=55) and from the environment (buses, n=89; hands of passengers, n=18; urban environment close to hospitals, n=52) were screened for VRE. Speciation, detection of vancomycin resistance and virulence genes were performed by PCR. Antimicrobial resistance was characterized by disk diffusion, Etest® and agar dilution. Genomic analysis of 23 selected E. faecium was performed by cgMLST (chewBBACA v2.5) and detection of antimicrobial resistance, virulence and plasmids by ResFinder_v3.1, VirulenceFinder_v2.0 and PlasmidFinder_v2.0. Overall, 470 Enterococcus spp. isolates were recovered from 90% (266/294) of the screened samples, and six species were identified: E. faecalis (30%), E. casseliflavus (20%), E. gallinarum (14%), E. faecium (13%), E. hirae (5%) and E. durans (< 1%). E. faecium was most prevalent among environmental samples [urban environment (27%), hands of bus passengers (23%) and buses (19%)] compared to livestock [pigs (7%) and cows (5%)]. Despite the fact that all isolates were susceptible to glycopeptides and no vanA or vanB genes were found, 23% of Enterococcus harbored the vanC2/3 and 17% carried the vanC1, including 12 (40%) E. faecalis. These E. faecalis, showed a resistance pattern mainly associated to intrinsic resistance, while E. faecium showed high resistance to aminoglycosides and macrolides (100%; aac(6’)-Ii and msrC genes), to ampicillin (83%; associated with mutations in pbp5), tetracyclines (39%; tetM and L genes) and high-level resistance to streptomycin (35%; ant(6)-Ia) and gentamicin (4%; aac(6')- aph(2'')). Different virulence determinants were identified among both E. faecalis (asa1, gelE and ace) and E. faecium (ace, acm, scm, sgrA, efaAfm and IS16). E. faecium population showed a high genetic variability, with a different sequence type (ST) per isolate, and including isolates from clonal complex (CC) 17 (ST32) and CC94 (ST296, ST1205, ST800 and ST2206), which are highly associated to hospital infections and human carriage. Although no epidemiological link could be drawn by cgMLST, between E. faecium isolates from this study and E. faecium isolated in Portuguese hospitals, the same clonal backgrounds defined by MLST were identified in both collections. Although none of the five settings screened in this study appear to be a VRE reservoir in Portugal, a high prevalence of antibiotic resistance and virulence determinants was identified among E. faecium and E. faecalis commensal isolates. Our results suggest that an active surveillance of Enterococcus reservoirs, beyond VRE, to prevent antimicrobial resistance spread is strongly recommended
Descrição
Palavras-chave
Microbiologia médica Enterococcus Animais de produção Meio ambiente VRE Resistência aos antimicrobianos