Enterococci from Wild Magellanic Penguins (Spheniscus magellanicus) as an Indicator of Marine Ecosystem Health and Human Impact.

Enterococci are commensals that proliferated as animals crawled ashore hundreds of millions of years ago. They are also leading causes of multidrug-resistant hospital-acquired infections. While most studies are driven by clinical interest, comparatively little is known about enterococci in the wild or the effect of human activity on them. Pharmaceutical pollution and runoff from other human activities are encroaching widely into natural habitats. To assess their reach into remote habitats, we investigated the identity, genetic relatedness, and presence of specific traits among 172 enterococcal isolates from wild Magellanic penguins. Four enterococcal species, 18 lineage groups, and different colonization patterns were identified. One Enterococcus faecalis lineage, sequence type 475 (ST475), was isolated from three different penguins, making it of special interest. Its genome was compared to those of other E. faecalis sequence types (ST116 and ST242) recovered from Magellanic penguins, as well as to an existing phylogeny of E. faecalis isolated from diverse origins over the past 100 years. No penguin-derived E. faecalis strains were closely related to dominant clinical lineages. Most possessed intact CRISPR defenses, few mobile elements, and antibiotic resistances limited to those intrinsic to the species and lacked pathogenic features conveyed by mobile elements. Interestingly, plasmids were identified in penguin isolates that also had been reported for other marine mammals. Enterococci isolated from penguins showed limited anthropogenic impact, indicating that they are likely representative of those naturally circulating in the ecosystem inhabited by the penguins. These findings establish an important baseline for detecting the encroachment of human activity into remote planetary environments.IMPORTANCE Enterococci are host-associated microbes that have an unusually broad range, from the built hospital environment to the guts of insects and other animals in remote locations. Despite their occurrence in the guts of animals for hundreds of millions of years, we know little about the properties that confer this range or how anthropogenic activities may be introducing new selective forces. Magellanic penguins live at the periphery of human habitation. It was of interest to examine enterococci from these animals for the presence of antibiotic resistance and other markers reflective of anthropogenic selection. Diverse enterococcal lineages found discount the existence of a single well-adapted intrinsic penguin-specific species. Instead, they appear to be influenced by a carnivorous lifestyle and enterococci present in the coastal sea life consumed. These results indicate that currently, the penguin habitat remains relatively free of pollutants that select for adaptation to human-derived stressors.

Antimicrobial Resistance Profiles in Enterococcus spp. Isolates From Fecal Samples of Wild and Captive Black Capuchin Monkeys (Sapajus nigritus) in South Brazil.

The environment, human, and animals play an important role in the spread of antibiotic-resistant bacteria. Enterococci are members of the gastrointestinal tracts of humans and animals and represent important reservoirs of antibiotic resistance genes. Until today, few studies have examined antibiotic susceptibility in enterococci isolated from primates. Therefore, the present study investigated species distribution, antibiotic susceptibility, and resistance genes in enterococci isolated from wild and captive black capuchins monkeys (Sapajus nigritus) in Rio Grande do Sul, South Brazil. A total of 24 swabs/fecal samples were collected, including 19 from wild monkeys living in two forest fragments [São Sebastião do Caí (SSC) and Santa Cruz do Sul (SCS)], and five in captive [Parque Zoológico da Fundação Zoobotânica (ZOO)], between August 2016 and November 2017. Fifteen colonies were randomly selected from each sample. Enterococci were identified by MALDI-TOF, tested for susceptibility to 12 antibiotics; and screened for tet(S), tet(M), tet(L), msrC, and erm(B) genes by PCR. Two-hundred ninety-six enterococci were isolated (SSC n = 137; SCS n = 86; ZOO n = 73) and differences in Enterococcus species distribution were detected on three monkey groups, with low abundance in SCS (1 - D = 0.2), followed by ZOO (1 - D = 0.68), and SSC (1 - D = 0.73). The enterococci frequently recovered include the following: Enterococcus faecalis (42.6%), E. hirae (29.1%), and E. faecium (15.9%). Antibiotic-nonsusceptible was observed in 202 (67.9%) strains. The rate of non-susceptibility to rifampicin, tetracycline, erythromycin, nitrofurantoin, chloramphenicol, and ampicillin was 46%, 26%, 22% and 19%, 13%, 0.3%, and 0.3%, respectively. All strains were susceptible to vancomycin, streptomycin, gentamycin, and linezolid. Forty-three (14.52%) isolates were identified as multidrug resistant (MDR), and the highest number of MDR enterococci were E. faecium recovered from wild monkeys living close to a hospital and water treatment plant. Elevated rates of antibiotic resistance genes msrC and tet(L) were isolates from ZOO. In conclusion, differences in the frequency of enterococci species, antibiotic-nonsusceptible and antibiotic resistance genes in all groups of monkeys were identified. These data suggest that anthropogenic activities could have an impact in the resistome of primate gut enterococci communities.

Resistance to antimicrobial agents among enterococci isolated from fecal samples of wild marine species in the southern coast of Brazil.

The purpose of this study was to evaluate species distribution, antimicrobial resistance profiles, and presence of resistance genes in enterococci isolated from fecal samples of wild marine species, including seabirds (n=12), sea turtles (n=8), and mammals (n=3) found alive or dead in southern coast of Brazil. Enterococci were classified based on phenotypic and genotypic characteristics, tested for antibiotic susceptibility, and the presence of tet(S), tet(M), tet(L), mrsC, and erm(B) genes by PCR. Enterococcus faecalis and Enterococcus faecium were the most common species. Single (37.09%), double (25.80%), and multiple (16.12%) antibiotic resistance patterns were observed. Resistance to rifampicin occurred most frequently. The msrC, tet(M), and/or tet(L) genes were detected in 60.15%, 73.07%, and 23.07% of the resistant strains, respectively. In conclusion, the presence of antibiotic resistant strains in these species could be related to food web interactions and aquatic pollutants or linked to environmental resistome.

Complete Genome Sequence of Enterococcus faecalis Strain P8-1 Isolated from Wild Magellanic Penguin (Spheniscus magellanicus) Feces on the South Coast of Brazil.

Enterococcus faecalis strains have a ubiquitous nature that allows them to survive in different niches. Studies involving enterococci isolated from marine animals are scarce. Therefore, in this study, we report the complete genome sequence of E. faecalis strain P8-1 isolated from feces of a Magellanic penguin on the south coast of Brazil.

Antimicrobial resistance and virulence factor gene profiles of Enterococcus spp. isolates from wild Arctocephalus australis (South American fur seal) and Arctocephalus tropicalis (Subantarctic fur seal).

Enterococci are natural inhabitants of the gastrointestinal tracts in humans and animals. Epidemiological data suggest that enterococci are important reservoirs of antimicrobial resistant genes that may be transmitted from other bacterial species The aim of this study was to investigate the species composition, antimicrobial resistance and virulence genes in enterococci recovered from fecal samples of wild Arctocephalus australis and A. tropicalis found dead along the South Coast of Brazil. From a total of 43 wild fur seals, eleven were selected for this study. Phenotypic and genotypic characterizations were used to classify Enterococcus species. Strains were tested for susceptibility to 10 antibiotics, presence of ace, gelE, asa, cylA, tet(L), tet(M) and erm(B) genes by PCR, and genetic variability using RAPD-PCR. Among the 50 enterococci isolated, 40% were Enterococcus faecalis, 40% E. hirae, 12% E. casseliflavus and 8 % other enterococcal species. Resistance profiles were observed to erythromycin, nitrofurantoin, tetracycline, norfloxacin and ciprofloxacin. The prevalence of virulence genes was ace (68%), gelE (54%), asa (22%) and cylA (4%). In erythromycin- and tetracycline strains, erm(B) and tet(M) were detected, respectively. The RAPD-PCR demonstrated a close phylogenetic relationship between the enterococci isolated from A. australis and A. tropicalis. In conclusion, different enterococcus species showing antimicrobial resistance and virulence determinates were isolated from fecal samples of fur seals. Antibiotic resistant strains in these animals could be related within food chain and aquatic pollutants or linked to environmental resistome, and demonstrates the potential importance of these animals as reservoirs and disseminators of such determinants in marine environmental.

PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples.

INTRODUCTION: This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP. METHODS: Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5%) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus. RESULTS: Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species. CONCLUSIONS: A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.

PCR-RFLP of 16S ribosomal DNA to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples / PCR-RFLP do 16S DNA ribossomal para confirmar a identificação de Enterococcus gallinarum e Enterococcus casseliflavus isolados de amostras clínicas e alimentares

INTRODUCTION: This study aimed to confirm the identification of Enterococcus gallinarum and Enterococcus casseliflavus isolated from clinical and food samples by PCR-RFLP. METHODS: Fifty-two strains identified by conventional biochemical exams were submitted to PCR amplification and digested with HinfI. Only 20 (38.5 percent) of the 52 strains showed a DNA pattern expected for E. gallinarum and E. casseliflavus. RESULTS: Analysis of the results of this study showed that E. gallinarum and E. casseliflavus are occasionally erroneously identified and confirmed the potential application of 16S rDNA analysis for accurate identification of these species. CONCLUSIONS: A correct identification is important to distinguish between intrinsic and acquired vancomycin resistance.

INTRODUÇÃO: O objetivo deste estudo foi confirmar a identificação de amostras clínicas e alimentos de Enterococcus gallinarum e Enterococcus casseliflavus por PCR-RFLP. MÉTODOS: Cinquenta e duas cepas identificadas por exames bioquímicos convencionais foram submetidos a amplificação por PCR e digestão com HinfI. Apenas 20 (38,5 por cento) das 52 amostras apresentaram um padrão de DNA esperado E. gallinarum e E. casseliflavus. RESULTADOS: Analise dos resultados deste estudo demonstraram que, algumas vezes E. gallinarum e E. casseliflavus são erroneamente identificados e confirmaram a potencial aplicação da análise do 16S rDNA para identificação exata destas espécies. CONCLUSÕES: A correta identificação é importante a fim de distinguir entre resistência intrínseca e adquirida à vancomicina.