Enterococci and Their Interactions with the Intestinal Microbiome

Background: We investigated the virulence factors, genes, antibiotic resistance patterns, and genotypes (VRE and ESBL/AmpC) production in Enterococci and Enterobacteriaceae strains isolated from fecal samples of humans, dogs, and cats. Methods: A total of 100 fecal samples from 50 humans, 25 dogs, and 25 cats were used in the study. MICs of nine antimicrobials were determined using the broth microdilution method. Polymerase chain reaction was used for the detection of genes responsible for antibiotic resistance (VRE and ESBL/AmpC) and virulence genes both in Enterococcus species, such as cytolysin (cylA, cylB, cylM), aggregation substance (agg), gelatinase (gelE), enterococcal surface protein (esp), cell wall adhesins (efaAfs and efaAfm), and in Enterobacteriaceae, such as cytolysin (hemolysin) and gelatinase production (afa, cdt, cnf1, hlyA, iutA, papC, sfa). Results: Enterococcus faecium was the most prevalent species in humans and cats, whereas Enterococcus faecalis was the species isolated in the remaining samples. A total of 200 Enterobacteriaceae strains were also detected, mainly from humans, and Escherichia coli was the most frequently isolated species in all types of samples. In the Enterococcus spp, the highest percentages of resistance for ampicillin, amoxicillin/clavulanate, erythromycin, tetracycline, ciprofloxacin, teicoplanin, and vancomycin were detected in cat isolates (41.6%, 52.8%, 38.9%, 23.6%, 62.5%, 20.8%, and 23.6% respectively), and in E. coli, a higher rate of resistance to cefotaxime and ceftazidime emerged in cat and dog samples, if compared with humans (75.4% and 66.0%, 80.0% and 71.4%, and 32.0% and 27.2%, respectively). Regarding the total number of enterococci, 5% and 3.4% of the strains were vancomycin and teicoplanin resistant, and the vancomycin resistance (van A) gene has been detected in all samples by PCR amplification. All the Enterobacteriaceae strains were confirmed as ESBL producers by PCR and sequencing, and the most frequent ESBL genes in E. coli strains from humans and pet samples were blaCTX-M-1 and blaCTX-M-15. Conclusions: Our results provide evidence that one or more virulence factors were present in both genera, underlining again the ability of pet strains to act as pathogens.

The objective of this study was to investigate the distribution and persistence of antimicrobial resistance genotypes of enterococci from broilers fed bambermycin (BAM), penicillin (PEN), salinomycin (SAL), bacitracin (BAC), or a salinomycin/bacitracin combination (SALBAC) for 35 days. A total of 95 enterococci from cloacal (n = 40), cecal (n = 38), and litter samples collected on day 36 (n = 17) were isolated weekly from day 7 to 36. All isolates were identified by API-20 Strep and their antimicrobial susceptibilities were evaluated using the Sensititre system with the plates for Gram positive bacteria. Whole genome sequencing (WGS) was used to assess their intra-and inter-genetic variability, with a focus on virulence and antibiotic resistance characteristics. All isolates were further characterized for hemolysin production (HEM), bile salt hydrolysis (BSH), and gelatinase (GEL) activities. Of the 95 isolates, Enterococcus faecium (n = 58) and Enterococcus faecalis (n = 24) were the most common Enterococcus species identified. Significant differences in the level of resistance for the E. faecium isolates to ciprofloxacin, macrolide, penicillin and tetracycline were observed among treatments. The bcrR, mefA, and aac(6) genes were higher in BAM treatment than the other groups whereas bcrR, ermA, ermB, aphA(3), and tetL were more prevalent in PEN and BAC treatments. Overall, E. faecium isolates showed a higher prevalence of antimicrobial resistance, but E. faecalis from litter also exhibited a significant level of resistance. A range of 4-15 different virulence genes was detected in E. faecalis. All isolates from litter but one (94.1%) showed BSH activities while 52.9% of them produced GEL. HEM activity was observed only in isolates collected on Day 7 (n = 9) and Day 14 (n = 1). This study confirmed that genetically diverse AMR enterococci harboring virulence factors can be promoted by the use of certain antimicrobials in feed. Such enterococci could persist in broiler chickens and their litter, which can potentially contaminate the soil upon land application.

26Bacterial pathogens encounter a variety of nutritional environments in the human host, 27 including nutrient metal restriction and overload. Uptake of manganese (Mn) is essential for 28 Enterococcus faecalis growth and virulence; however, it is not known how this organism 29 prevents Mn toxicity. In this study, we examine the role of the highly conserved MntE 30 transporter in E. faecalis Mn homeostasis and virulence. We show that inactivation of mntE 31 results in growth restriction in the presence of excess Mn, but not other metals, demonstrating 32 its specific role in Mn detoxification. Upon growth in the presence of excess Mn, an mntE 33 mutant accumulates intracellular Mn, iron (Fe), and magnesium (Mg), supporting a role for 34 MntE in Mn and Fe export, and a role for Mg in offsetting Mn toxicity. Growth of the mntE 35 mutant in excess Fe also results in increased levels of intracellular Fe, but not Mn or Mg, 36 providing further support for MntE in Fe efflux. Inactivation of mntE in the presence of 37 excess iron also results in the upregulation of glycerol catabolic genes and enhanced biofilm 38 growth, and addition of glycerol is sufficient to augment biofilm growth for both the mntE 39 mutant and its wild type parental strain, demonstrating that glycerol availability significantly 40 enhances biofilm formation. Finally, we show that mntE contributes to infection of the 41 antibiotic-treated mouse gastrointestinal (GI) tract, suggesting that E. faecalis encounters 42 excess Mn in this niche. Collectively, these findings demonstrate that the manganese exporter 43 MntE plays a crucial role in E. faecalis metal homeostasis and virulence. 44 45 nutritional immunity (1, 4-6). To counteract these host-mediated defences, many bacterial 51 pathogens including Enterococci encode dedicated systems to acquire Mn. 52 53Bacteria encode conserved ABC and NRAMP-family transporters for manganese uptake (1, 54 2). In Enterococcus faecalis, three manganese uptake systems have been described: the ABC-55 type transporter encoded by efaCBA and two NRAMP transporters encoded by mntH1 and 56 mntH2 (7). These three Mn transport systems are functionally redundant since deletion of all 57 three transporter systems (efaCBA, mntH1, mntH2) is required to abrogate intracellular Mn 58 accumulation, rendering the triple mutant severely impaired in growth (7). Furthermore, 59 deletion of both efaCBA and mntH2, or all three transporters together results in attenuated 60 colonization in rabbit endocarditis and mouse catheter-associated urinary tract infection 61 (CAUTI) models (7). Together these observations demonstrate that the ability to acquire Mn 62 is essential for E. faecalis virulence. 64In contrast to Mn influx mechanisms that have been characterized in E. faecalis, Mn export 65 pathways have not been described. The contribution of Mn export to bacterial pathogenesis 66 and virulence has been demonstrated for some bacterial species (8-15) and is dependent on 67 two widely characterized classes of exporters -MntE, a cat...