Περίληψη σε άλλη γλώσσα
During the last two decades vancomycin-resistant enterococci (VRE), have emerged as important nosocomial pathogens, due to the widespread use of glycopeptides, mainly vancomycin, in hospitals. Meanwhile, the use of glycopeptide avoparcin as growth promoter in food-producing animals resulted also in the emergence of VRE in livestock worldwide indicating that they might be a potential reservoir for vancomycin resistance determinants. The use of avoparcin has been banned from 1997 in the European Union (Commission Directive 97/6 EC).According to updated epidemiological data, Greek reports showed higher rates for clinical vancomycin-resistant Enterococcus faecium (VREF) compared to the majority of European countries. The aim of this study was to investigate the prevalence of VRE isolated from farm animals (broilers, pigs and calves) and humans in close association to them (slaughterers), the determination of their van genotype as well as the evaluation of their possible epidemiological rel ...
During the last two decades vancomycin-resistant enterococci (VRE), have emerged as important nosocomial pathogens, due to the widespread use of glycopeptides, mainly vancomycin, in hospitals. Meanwhile, the use of glycopeptide avoparcin as growth promoter in food-producing animals resulted also in the emergence of VRE in livestock worldwide indicating that they might be a potential reservoir for vancomycin resistance determinants. The use of avoparcin has been banned from 1997 in the European Union (Commission Directive 97/6 EC).According to updated epidemiological data, Greek reports showed higher rates for clinical vancomycin-resistant Enterococcus faecium (VREF) compared to the majority of European countries. The aim of this study was to investigate the prevalence of VRE isolated from farm animals (broilers, pigs and calves) and humans in close association to them (slaughterers), the determination of their van genotype as well as the evaluation of their possible epidemiological relationships with clinical VRE sharing the same geographical regions in Greece and at the same period of time.A total of 500 caecal samples of broilers were collected in two poultry slaughterhouses. The samples originated from 8 broiler farms in Northern Greece. During the same time period, cecal samples of sixty (60) fecal samples of healthy calves originated from seven (7) farms in northern Greece and 250 cecal samples of healthy fattening pigs from six (6) farms in northern and central Greece, were collected in two animal slaughterhouses. Moreover, during the same period, stool samples were collected from 50 poultry slaughterers and 50 slaughterers working at the corresponding slaughterhouses where broilers and pigs and calves were slaughtered. The isolation of VRE was performed using selective enrichment with media supplemented with vancomycin (6 mg/l).In addition, 63 human clinical VREF, isolated at the same period of time, were included in this study of which 48 originated from adult (n=13) and neonate (n=35) samples from Hippokratio University hospital whereas 15 originated from adult samples from Larissa University hospital.All presumptive VRE were tested by the Kirby-Bauer method for their susceptibilities to vancomycin and teicoplanin. The Minimun Inhibitory Concentrations (MICs) of vancomycin and teicoplanin were furthermore determined by the broth microdilution method.To detect the presence of van genes, multiplex PCR analyses were performed to all presumptive VRE exhibiting MIC to vancomycin ≥2 μg/ml using primers specific for the vanA, vanB, vanC1-C2, vanD, vanE, vanG, ddl-E. faecium and ddl-E. faecalis genes.A total of 120 VRE were recovered from 112 (22,4%) broiler samples. VREF carrying the vanA gene were predominant recovered from 74 (14,8%) samples from 5 out of 8 (62,5%) broiler farms, while E. gallinarum harboring the vanC gene were recovered from 39 (7,8%) samples obtained also from 5 farms. VRE were recovered from 14 (28%) poultry slaughtetrer samples consisted of 10 (20%) and 4 (8%) positive samples for VREF and E. gallinarum, respectively. Furthermore, one VREF isolate, carrying the vanA gene, was recovered from 1 (1,7%) calve sample. No VRE were recovered from pig samples as well as their slaughterers. The prevalence of broiler VREF indicates their remarkable persistence inbroiler production environment even more than a decade after the avoparcin ban and without any apparent glycopeptide selection. Additionally to their susceptibility testing to vancomycin and teicoplanin, all presumptive VRE isolates, were tested for their susceptibilities to 12 antimicrobials. Broiler VREF were constantly resistant to tetracycline (100%) whereas the most common trait among clinical VREF was resistant to ampicillin (93,7%) and penicillin (93,7%). In contrast, low resistance rates to tetracycline (9,5%) and ampicillin (16,5%) were observed among clinical and broiler VREF, respectively. Concerning poultry slaughterers VREF isolates, they exhibited low resistance to ampicillin (10%), similarly to those observed in broilers and moderate resistance to tetracycline (40%). Another antimicrobial that broiler and clinical VREF alsoexhibited high resistance rates, was erythromycin (54,4% and 57,1%, respectively). Analysis of Antimicrobial Resistance Profiles (ARPs) revealed 53 distinct types, with only 5 of them shared among isolates from the different sources. Meanwhile, 54,4% of broiler and 92,1 % of clinical VREF were multiresistant (resistant to 5 antimicrobial classes) whereas only 10% of poultry slaughterer VREF were multiresistant.The different resistance rates in combination with the existence of variant ARPs among isolates from the different sources (broilers, calves, poultry slaughterers, hospitalized patients) may reflect the broad (different) spectrum of selection pressure of antimicrobial agents used in each source.The relationship of antimicrobial resistance profiles (ARPs) among VREF isolates from the different sources, was determined using discriminant analysis (DA), based on all 14 antimicrobials tested. Broiler and human clinical VREF were classified to their corresponding source at a rate of 100% and 85.7%, respectively, showing a clear discrimination and association with their source. In contrast, the classification rate of poultry slaughterer VREF was just 50% indicating their low source specificity, with 40% of them classified closely to broiler source due to their sharing common ARPs.PFGE (Pulsed Field Gel Electrophoresis) analysis was performed to investigate the epidemiological relationship of VREF isolates due to its high discriminatory power. PFGE analysis revealed patterns clearly related to their source. Meanwhile, a high genetic diversity within each source (broilers, poultry slaughterers, hospitalized patients) demonstrated by the existence of more than one clusters, was observed. Specifically, 71 out of 79 broiler VREF were grouped into 4 distinct clusters, designated C1-C4 while 37 out of 63 human clinical isolates appeared to belong also to 4 distinct clusters, designated H1 - H4, exhibiting less than 60% similarity to the C clusters. The remaining broiler, human clinical, poultry slaughterer and the calf VREF isolate, exhibited distinct patterns unrelated to each other or C or H clusters.The comparison of PFGE patterns showed that clinical and broiler VREF isolates created clearly unrelated populations which strongly indicates no clonal spread among the two sources. Moreover, PFGE analysis in this study did not reveal any sharing of clones between broilers and poultry slaughterers despite the close association exhibited by DA, indicating no clonal spread among the two sources. However, the existence of genetically indistinguishable PFGE patterns of VREF isolates from farms of different geographical regions as well as the fact that H1 and H2 clusters shared patterns of isolates with common ARP types from different hospitals, indicate that clonal spread might have occurred inside ech source.The information above may serve as a basis to further clarify whether “specific” vanA genetic elements circulate in broiler production environment or in human environment as well as the mechanisms of VREF long-term persistence in Greece.
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