Antibiotic resistance (AR) in the environment is a growing and global concern for public health, and intrinsic AR from pristine sites untouched by pharmaceutical antibiotics is not commonly studied. Forty aerobic bacteria were isolated from water and sediment samples of hot springs in South Africa. Resistance against ten antibiotics (carbenicillin, gentamicin, kanamycin, streptomycin, tetracycline, chloramphenicol, ceftriaxone, co-trimoxazole, nalidixic acid and norfloxacin) was tested using a standard disk diffusion assay. Resistance to one or two antibiotics were equally found in 37.5%, while the remaining 22% showed complete sensitivity. Intermediate resistance was found for ceftriaxone (52.5%), nalidixic acid (37.5%) and carbenicillin (22.5%), while low levels of resistance were observed for streptomycin (5%) and kanamycin (2.5%), and total sensitivity towards the other antibiotics. Twenty-nine isolates were also tested against eight different heavy-metal salts (Al, Cr, Cu, Fe, Hg, Mn, Ni and Pb) at 10 and 40 mM. All isolates were tolerant and able to grow on ≥2 heavy-metal salts at both concentrations. No association was observed between AR and heavy metal tolerance (HMT). Based on the relatively low AR levels, hot spring sites are pristine environments reflecting baseline levels for comparison to other potentially contaminated groundwater sites.

Heavy metal contamination of source water frequently occurred in developing countries as a result of accidents. To address the problems, most of the previous studies have focused on engineering countermeasures. In this study, we investigated the effects of heavy metals, particularly copper, on the development of antibiotic resistance by establishing a copper shock loading test. Results revealed that co-selection occurred rapidly within 6 h. Copper, at the levels of 10 and 100 mg/L, significantly increased bacterial resistance to the antibiotics tested, including rifampin, erythromycin, kanamycin, and a few others. A total of 117 antimicrobial-resistance genes were detected from 12 types of genes, and the relative abundance of most genes (particularly mobile genetic elements intⅠand transposons) was markedly enriched by at least one fold. Furthermore, the copper shock loading altered the bacterial community. Numerous heavy metal and antibiotic resistant strains were screened out and enriched. These strains are expected to enhance the overall level of resistance. More noticeably, the majority of the co-selected antibiotic resistance could sustain for at least 20 h in the absence of copper and antimicrobial drugs. Resistance to vancomycin, erythromycin and lincomycin even could remain for 7 days. The prominent selection pressure by the copper shock loading implies that a real accident most likely poses similar impacts on the water environment. An accidental release of heavy metals would not only cause harm to the ecological environment, but also contribute to the development of bacterial antibiotic resistance. Broader concerns should be raised about the biological risks caused by sudden releases of pollutants by accidents.

Vibrio parahaemolyticus can cause severe gastroenteritis, septicaemia and even death in humans. Continuous monitoring of V. parahaemolyticus contamination in aquatic products is imperative for ensuring food safety. In this study, we isolated and characterized 561 V. parahaemolyticus strains recovered from 23 species of commonly consumed shellfish, crustaceans, and fish collected in July and August of 2017 in Shanghai, China. The bacterium was not isolated from two fish species Carassius auratus and Parabramis pekinensis. The results revealed a very low occurrence of pathogenic V. parahaemolyticus carrying the toxin genes trh (0.2%) and tdh (0.0%). However, high percentages of resistance to the antimicrobial agents ampicillin (93.0%), rifampin (82.9%), streptomycin (75.4%) and kanamycin (50.1%) were found. A high incidence of tolerance to the heavy metals Hg²⁺ (74.7%) and Zn²⁺ (56.2%) was also observed in the isolates. ERIC-PCR-based fingerprinting of MDR isolates (77.5%) revealed 428 ERIC-genotypes, demonstrating remarkable genetic variation among the isolates. The results of this study support the urgent need for food safety risk assessment of aquatic products.

Vibrio parahaemolyticus is a major gastroenteritis-causing pathogen in Korea. Recent studies have reported that heavy metal and antimicrobial resistance in bacteria are related. In this study, we investigated heavy metal and antimicrobial resistance in wild strains of V. parahaemolyticus. First, we isolated and characterized 38 V. parahaemolyticus strains (toxR-positive) from shellfish collected from the West Sea of Korea between May and November 2018. Antibiotic and heavy metal resistance in the 38 strains were tested by disk diffusion assay and broth dilution assay, respectively. Then, we selected seven strains that showed resistance to cobalt (Co²⁺) and copper (Cu²⁺), to examine the relationship between heavy metal resistance and antimicrobial resistance. After heavy metal (Co²⁺ and Cu²⁺) pretreatment, the seven strains exhibited increased resistance to kanamycin, streptomycin, tetracycline, and gentamycin. Likewise, antimicrobial pretreatment resulted in increased heavy metal tolerance.

546 Vibrio isolates from rearing seawater (292 strains) and intestines of abalone (254 strains) were tested to ten antibiotics using Kirby–Bauer diffusion method. Resistant rates of abalone-derived Vibrio isolates to chloramphenicol (C), enrofloxacin (ENX) and norfloxacin (NOR) were <28%, whereas those from seawater showed large fluctuations in resistance to each of the tested antibiotics. Many strains showed higher resistant rates (>40%) to kanamycin (KNA), furazolidone (F), tetracycline (TE), gentamicin (GM) and rifampin (RA). 332 isolates from seawater (n=258) and abalone (n=74) were resistant to more than three antibiotics. Peaked resistant rates of seawater-derived isolates to multiple antibiotics were overlapped in May and August. Statistical analysis showed that pH had an important effect on resistant rates of abalone-derived Vibrio isolates to RA, NOR, and ENX. Salinity and dissolved oxygen were negatively correlated with resistant rates of seawater-derived Vibrio isolates to KNA, RA, and PG.

Antibiotic resistance in bacteria is a growing problem that is not only restricted to the clinical setting but also to other environments such as marine species that harbor antibiotic resistant bacteria and therefore may serve as reservoirs for antibiotic-resistance genetic determinants. The aim of this study was to evaluate antibiotic resistance phenotypes in enterococci isolated from fecal samples of gilthead seabream and the associated mechanisms of resistance. A collection of 118 samples were analyzed and 73 enterococci were recovered. The strains showed high percentages of resistance to erythromycin and tetracycline (58.9% and 17.8%, respectively). Lower level of resistance (<13%) was detected for quinupristin–dalfopristin, ampicillin, high-level-gentamicin, high-level-streptomycin, high-level-kanamycin, ciprofloxacin and chloramphenicol. The erm(B), tet(L) or tet(M), aac(6′)-aph(2″) and aph(3′)-IIIa genes were shown in isolates resistant to erythromycin, tetracycline, high-level gentamicin and high-level kanamycin, respectively. Antibiotic resistance in natural microbiota is becoming a concern of human and environmental health.

The Rozália Mine, with its long mining history, could represent an environmental threat connected with metal contamination and associated antibiotic tolerance. Metal and antibiotic tolerance profiles of heterotrophic, cultivable bacteria isolated from the Rozália Gold Mine in Hodruša-Hámre, Slovakia, and the surrounding area were analysed. Subsurface samples were collected from different mine levels or an ore storage dump. As expected, heterotrophic cultivable bacteria showed high minimum inhibitory concentrations for metals (up to 1000 mg/l for zinc and nickel, 2000 mg/l for lead and 500 mg/l for copper). Surprisingly, very high minimum inhibitory concentrations of selected antibiotics were observed, e.g. > 10,000 μg/ml for ampicillin, up to 4800 μg/ml for kanamycin, 800 μg/ml for chloramphenicol and 50 μg/ml for tetracycline. Correlation analysis revealed a linkage between increased tolerance to the antibiotics ampicillin and chloramphenicol and metal tolerance to nickel and copper. A correlation was also observed between tetracycline-kanamycin tolerance and zinc-lead tolerance. Our data indicate that high levels of antibiotic tolerance occur in deep subsurface microbiota, which is probably connected with the increased level of metal concentrations in the mine environment.

Marine fish species were analyzed for culturable and total metagenomic microbial diversity, antibiotic resistance (AR) pattern, and horizontal gene transfer in culturable microorganisms. We observed a high AR microbial load of 3 to 4 log CFU g⁻¹. Many fish pathogens like Providencia, Staphylococcus, Klebsiella pneumoniae, Enterobacter, Vagococcus, and Aeromonas veronii were isolated. Photobacterium and Vibrio were two major fish and human pathogens which were identified in the fish metagenome. Other pathogens that were identified were Shewanella, Acinetobacter, Psychrobacter, and Flavobacterium. Most of these pathogens were resistant to multiple antibiotics such as erythromycin, kanamycin, neomycin, streptomycin, penicillin, cefotaxime, bacitracin, rifampicin, trimethoprim, ciprofloxacin, and doxycycline with a high multiple antibiotic resistance index of 0.54–0.77. The fish microflora showed high prevalence of AR genes like bla TEM, Class I integron, tetA, aph(3′)-IIIa, ermB, aadA, and sul1. Nineteen of 26 AR isolates harbored Class I integrons showing high co-resistance to trimethoprim, kanamycin, doxycycline, and cefotaxime. Mobile R-plasmids from 6 of the 12 AR pathogens were transferred to recipient E. coli after conjugation. The transconjugants harbored the same R-plasmid carrying bla CTX₋M, dfr1, tetA, bla TEM, and cat genes. This study confirms that fish is a potential carrier of AR pathogens which can enter the human gut via food chain. To the best of our knowledge, this is the first study in the Indian subcontinent reporting a direct evidence of spread of AR pathogens to humans from specific marine fish consumption.

Several studies have suggested the existence of a close relationship between antibiotic-resistant phenotypes and resistance to other toxic compounds such as heavy metals, which involve co-resistance or cross-resistance mechanisms. A metagenomic library was previously constructed in Escherichia coli with DNA extracted from the bacterial community inhabiting an acid mine drainage (AMD) site highly contaminated with heavy metals. Here, we conducted a search for genes involved in antibiotic resistance using this previously constructed library. In particular, resistance to antibiotics was observed among five clones carrying four different loci originating from CARN5 and CARN2, two genomes reconstructed from the metagenomic data. Among the three CARN2 loci, two carry genes homologous to those previously proposed to be involved in antibiotic resistance. The third CARN2 locus carries a gene encoding a membrane transporter with an unknown function and was found to confer bacterial resistance to rifampicin, gentamycin, and kanamycin. The genome of Thiomonas delicata DSM 16361 and Thiomonas sp. X19 were sequenced in this study. Homologs of genes carried on these three CARN2 loci were found in these genomes, two of these loci were found in genomic islands. Together, these findings confirm that AMD environments contaminated with several toxic metals also constitute habitats for bacteria that function as reservoirs for antibiotic resistance genes.

Veterinary manure is an important pollution reservoir of antibiotics and antibiotic-resistant bacteria (ARB). However, little is known of the distribution of ARB in plant endophytic bacteria and the number/types of ARB in chicken manure. In this study, 454-pyrosequencing was used to investigate the distribution and composition of ARBs in chicken manure and fertilized vegetables. The prevalence of ARB in the samples of the chicken manure compost recovered from farms on which amoxicillin, kanamycin, gentamicin, and cephalexin were used was 20.91–65.9 % for ARBs and 8.24–20.63 % simultaneously resistant to two or more antibiotics (multiple antibiotic resistant bacteria (MARB)). Antibiotic-resistant endophytic bacteria were widely detected in celery, pakchoi, and cucumber with the highest rate of resistance to cephalexin. The pyrosequencing indicated that the chicken manure dominantly harbored Firmicutes, Bacteroidetes, Synergistetes, and Proteobacteria and that Bacteroidetes was significantly enhanced in farms utilizing antibiotics. In the total cultivable colonies, 62.58–89.43 % ARBs and 95.29 % MARB were clustered in Bacteroidetes with the dominant species (Myroides ordoratimimus and Spningobacterium spp., respectively) related to human clinical opportunistic pathogens.