A Decade-Long Commitment to Antimicrobial Resistance Surveillance in Portugal

Abstract

Antimicrobial resistance (AMR) is a worldwide problem with serious health and economic repercussions. Since the 1940s, underuse, overuse, and misuse of antibiotics have had a significant environmental downside. Large amounts of antibiotics not fully metabolized after use in human and veterinary medicine, and other applications, are annually released into the environment. The result has been the development and dissemination of antibiotic-resistant bacteria due to many years of selective pressure. Surveillance of AMR provides important information that helps in monitoring and understanding how resistance mechanisms develop and disseminate within different environments. Surveillance data is needed to inform clinical therapy decisions, to guide policy proposals, and to assess the impact of action plans to fight AMR. The Functional Genomics and Proteomics Unit, based at the University of Trás-os-Montes and Alto Douro in Vila Real, Portugal, has recently completed 10 years of research surveying AMR in bacteria, mainly commensal indicator bacteria such as enterococci and Escherichia coli from the microbiota of different animals. Samples from more than 75 different sources have been accessed, from humans to food-producing animals, pets, and wild animals. The typical microbiological workflow involved phenotypic studies followed by molecular approaches. Throughout the decade, 4,017 samples were collected and over 5,000 bacterial isolates obtained. High levels of AMR to several antimicrobial classes have been reported, including to β-lactams, glycopeptides, tetracyclines, aminoglycosides, sulphonamides, and quinolones. Multi-resistant strains, some relevant to human and veterinary medicine like extended-spectrum β-lactamase-producing E. coli and vancomycin-resistant enterococci, have been repeatedly isolated even in non-synanthropic animal species. Of particular relevance are reports of AMR bacteria in wildlife from natural reserves and endangered species. Future work awaits as this threatening yet unsolved problem persists. GRAPHICAL ABSTRACTSummary diagram of the antimicrobial resistance surveillance work developed by the UTAD Functional Genomics and Proteomics Unit.

Keywords: Escherichia coli; antimicrobial resistance; enterococci; molecular microbiology; surveillance; wildlife.

GRAPHICAL ABSTRACT

Summary diagram of the antimicrobial resistance surveillance work developed by the UTAD Functional Genomics and Proteomics Unit.

FIGURE 1

Geographical distribution in Portugal showing the sources of samples containing bacterial isolates. The number and provenance of samples containing enterococci (orange) and Escherichia coli (blue) are shown. Both types of bacteria were frequently isolated from several samples from humans, pets, food-producing animals, and wild animals.

FIGURE 2

Percentage of phenotypic antibiotic resistance detected in enterococci and E. coli isolates from each of the four sampling groups. The resistance profile of enterococci from food-producing animals is distinct from those of the other sampling groups, showing the highest resistance to tetracycline, streptomycin, trimethoprim-sulfamethoxazole, and chloramphenicol. Enterococci from pets displayed the lowest resistance profile to all tested antibiotics. TET, tetracycline; STR, streptomycin; GEN, gentamicin; NAL, nalidixic acid; CIP, ciproflaxin; SXT, trimethoprim-sulfamethoxazole; CHL, chloramphenicol; AMP, ampicillin.

FIGURE 3

Percentage of antimicrobial resistance (AMR) genes detected in E. coli resistant isolates from all sources over a decade.

FIGURE 4

Number of ESBL-producing E. coli and resistance genes present in all isolates from different groups. blaCTX-M15 was the most frequent ESBL gene recovered from E. coli, followed by blaCTX-M-1 and blaTEM-52. Otherwise, blaTEM-20, blaOXA-30, and blaCTX-M-9 genes were found in a few ESBL-producing E. coli.

FIGURE 5

Percentages of enterococci species isolated from samples. Enterococcus faecium is the most frequently isolated enterococci species among all recovered samples and Enterococcus gallinarum the least.

FIGURE 6

Percentage of phenotypic antibiotic resistance detected in Enterococcus spp. isolates from each of the four sampling groups. Enterococci isolates showed high resistance levels to tetracycline and erythromycin, regardless of the source. However, food-producing animals and pets stand out from the other sampling groups. Streptomycin, kanamycin, and chloramphenicol-resistant enterococci are more frequent in samples from humans than from other sources. TET, tetracycline; ERY, erythromycin; STR, streptomycin; GEN, gentamicin, KAN, kanamycin; CHL, chloramphenicol.

FIGURE 7

Percentage of AMR genes detected in resistant Enterococci isolates from all sources over a decade. Resistance genes tet(M)/tet(L), erm(B), ant(6)-Ia, aac(6′)-aph(2′), aph(3′)-IIIA, and catA were present in enterococci isolates resistant to tetracycline, erythromycin, streptomycin, gentamicin, kanamycin, and chloramphenicol, respectively. TET, tetracycline; ERY, erythromycin; STR, streptomycin; GEN, gentamicin; KAN, kanamycin, CHL, chloramphenicol.

FIGURE 8

Number of vancomycin-resistant enterococci species, and resistance genes present in all isolates from different groups. E. gallinarum and E. casseliflavus are intrinsically resistant to vancomycin, and harbor the genes vanC1 and vanC2, respectively, in their genome.