Zinc effects on bacteria: insights from Escherichia coli by multi-omics approach.

IMPORTANCE: A long-term exposure of bacteria to zinc oxide and zinc oxide nanoparticles leads to major alterations in bacterial morphology and physiology. These included biochemical and physiological processes promoting the emergence of strains with multi-drug resistance and virulence traits. After the removal of zinc pressure, bacterial phenotype reversed back to the original state; however, certain changes at the genomic, transcriptomic, and proteomic level remained. Why is this important? The extensive and intensive use of supplements in animal feed effects the intestinal microbiota of livestock and this may negatively impact the health of animals and people. Therefore, it is crucial to understand and monitor the impact of feed supplements on intestinal microorganisms in order to adequately assess and prevent potential health risks.

Zinc phosphate-based nanoparticles as alternatives to zinc oxide in diet of weaned piglets.

BACKGROUND: The high doses of zinc oxide (ZnO) administered orally to piglets for the prevention of diarrhea and increase of growth rate can contaminate pig farms and the surrounding environment. Therefore, there is a need to find a replacement of high doses of dietary ZnO with an equally effective alternative. In the present study, the effect of two formulations of zinc phosphate-based nanoparticles (ZnA and ZnC NPs) on growth performance, intestinal microbiota, antioxidant status, and intestinal and liver morphology was evaluated. A total of 100 weaned piglets were randomly divided into 10 equal groups with the base diet (control) or the base diet supplemented with ZnA, ZnC, or ZnO at concentrations 500, 1000, and 2000 mg Zn per kilogram of diet. Supplements were given to animals for 10 days. Fecal samples were collected on day 0, 5, 10 and 20. At the end of the treatment (day 10), three piglets from each group were sacrificed and analyzed. RESULTS: Comparing to that of control, the significantly higher piglet weight gain was observed in all piglet groups fed with ZnA (P < 0.05). Differences in the total aerobic bacteria and coliform counts in piglet feces after NPs supplementation compared to that of control and ZnO groups were also found (P < 0.05). The majority of aerobic culturable bacteria from the feces represented Escherichia (28.57-47.62%), Enterococcus (3.85-35.71%), and Streptococcus (3.70-42.31%) spp. A total of 542 Escherichia coli isolates were screened for the virulence genes STa, STb, Stx2, F4, and F18. The substantial occurrence of E. coli virulence factors was found on day 5, mainly in fimbrillary antigen and thermostable toxins, except for piglets fed by ZnC. Zn treatment decreased Zn blood levels in piglets fed with ZnO and ZnA (500 mg/kg) and increased in ZnC (2000 mg/kg) compared to that of control (P < 0.05). The antioxidant status of piglets was affected only by ZnA. While some changes in the liver and the intestinal morphology of piglets with NPs were observed, none were serious as reflected by the normal health status and increased weigh gain performance. CONCLUSIONS: Our results indicate that ZnA NPs have a positive effect on the piglet growth performance even at the lowest concentration. The prevalence of E. coli virulence factors was lowest in pigs supplemented with ZnC. Zinc phosphate-based nanoparticles may be an effective alternative to ZnO.

Correction to: Bacterial Community of the Digestive Tract of the European Medicinal Leech (Hirudo verbana) from the Danube River.

The original published version of this article had mistakes in figure legends. Correct figure legends are presented below.

Bacterial Community of the Digestive Tract of the European Medicinal Leech (Hirudo verbana) from the Danube River.

The digestive tract of medicinal leeches from commercial suppliers has been investigated previously and comprises of a relatively simple bacterial community. However, the microbiome of medicinal leeches collected directly from the natural habitat has not been examined. In this study, we characterized the bacterial community in the digestive tract (anterior crop, posterior crop, and intestine) of the European medicinal leech, Hirudo verbana, collected from the Danube river using culture-independent and culture-dependent approaches. Culture-independent approach confirmed that the digestive tract of H. verbana carries a relatively simple bacterial community with species richness in the individual samples ranging from 43 to164. The dominant bacterial taxon was Mucinivorans sp. (49.7% of total reads), followed by Aeromonas sp. (18.7% of total reads). Several low abundance taxa, new for H. verbana, such as Phreatobacter, Taibaiella, Fluviicola, Aquabacterium, Burkholderia, Hydrogenophaga, Wolinella, and unidentified Chitinophagia, were also detected. The aerobic culturing approach showed Aeromonas veronii (Proteobacteria), the known leech symbiont, as the most dominant taxon followed by several Pseudomonas and Acidovorax spp. No significant differences in the bacterial community composition were detected among different parts of the digestive tract of individual leeches. However, the overall composition of the bacterial community among individual specimen varied significantly and this is possibly due to differences in leech age, feeding status, and blood source. Our results showed that the core bacterial community of H. verbana collected from the natural habitat is similar to that reported from the digestive tract of commercially supplied leeches maintained in the laboratory.

Occurrence of tcrB, a transferable copper resistance gene, in fecal enterococci of swine.

High concentration of copper, fed as copper sulfate, is often used to increase growth rates in swine. Bacteria exposed to copper may acquire resistance, and in Enterococcus faecium and Enterococcus faecalis, a plasmid-borne transferable copper resistance (tcrB) gene that confers copper resistance has been reported. Our objectives were to determine the occurrence of tcrB in fecal enterococci from weaned piglets fed diets with a normal supplemental level (16.5 ppm) or an elevated supplemental level (125 ppm) of copper and to determine the association of tcrB with copper, erythromycin, and vancomycin resistance. A total of 323 enterococcal isolates were examined and 15 (4.6%) isolates (14 E. faecium and 1 E. faecalis) were positive for tcrB. Fifteen tcrB-positive and 15 randomly chosen tcrB-negative isolates from piglets fed the normal supplemental level of copper were tested for erm(B), tet(M), vanA, and vanB genes and susceptibilities to copper, erythromycin, tetracyclines, and vancomycin. All tcrB-positive and -negative isolates contained erm(B) and tet(M), but not vanA and vanB. The mean minimum inhibitory concentration of copper for tcrB-positive (21.1 mM) was higher (p < 0.001) compared with tcrB-negative isolates (6.1 mM). All isolates were resistant to erythromycin and tetracyclines and susceptible to vancomycin. The transferability of the tcrB gene from tcrB-positive strains to tcrB-negative strains was demonstrated by conjugation. The potential link between tcrB and antibiotic resistance genes and the propensity of enterococci to transfer tcrB to other strains raises the possibility that copper supplementation may exert selection pressure for antibiotic-resistant enterococci. This study is the first report on the occurrence of the tcrB gene in enterococci isolated from swine in the United States.

Occurrence of ciprofloxacin-, trimethoprim-sulfamethoxazole-, and vancomycin-resistant bacteria in a municipal wastewater treatment plant.

The occurrence of antibiotic-resistant bacteria was evaluated in aqueous samples obtained from a municipal wastewater treatment plant. Samples collected from the influent, clarifier effluent, and disinfected effluent were assayed for fecal coliforms, E. coli, and enterococci exhibiting resistance to ciprofloxacin, trimethoprim-sulfamethoxazole, and vancomycin. Membrane filtration of samples was followed by plating on growth media containing various concentrations of antibiotic. Bacterial colonies on plates with antibiotic exposures greater than the clinical minimum inhibitory concentrations were counted and considered resistant. The numbers of drug-resistant organisms in influent ranged from nondetectable to 7 x 10(5) colony-forming units (CFU)/100 mL for fecal coliforms, nondetectable to 5 x 10(4) CFU/100 mL for E. coli, and nondetectable to 6 x 10(5) CFU/100 mL for enterococci. Fecal coliforms, E. coli, and enterococci with reduced susceptibility to antibiotics were also detected in influent and clarifier effluent; however, the disinfected effluent did not contain resistant bacteria. Species-level identification of enterococci revealed that resistant enterococci were predominantly E. faecalis.