The quantification and identification of new plasmid-acquiring bacteria in representative mating conditions is critical to characterize the risk of horizontal gene transfer in the environment. This study aimed to quantify conjugation events resulting from manure application to soils and identify the transconjugants resulting from these events. Conjugation was quantified at multiple time points by plating and flow cytometry, and the transconjugants were recovered by fluorescence-activated cell sorting and identified by 16S rRNA sequencing. Overall, transconjugants were only observed within the first 4 days after manure application and at values close to the detection limits of this experimental system (1.00-2.49 log CFU/g of manured soil, ranging between 10-5 and 10-4 transconjugants-to-donor ratios). In the pool of recovered transconjugants, we found amplicon sequence variants (ASVs) of genera whose origin was traced to soils (Bacillus and Nocardioides) and manure (Comamonas and Rahnella). This work showed that gene transfer from fecal to soil bacteria occurred despite the less-than-optimal conditions faced by manure bacteria when transferred to soils, but these events were rare, mainly happened shortly after manure application, and the plasmid did not colonize the soil community. This study provides important information to determine the risks of AMR spread via manure application.

Plasmid-mediated dissemination of antibiotic resistance among fecal Enterobacteriaceae in natural ecosystems may contribute to the persistence of antibiotic resistance genes in anthropogenically impacted environments. Plasmid transfer frequencies measured under laboratory conditions might lead to overestimation of plasmid transfer potential in natural ecosystems. This study assessed differences in the conjugative transfer of an IncP-1 (pKJK5) plasmid to three natural Escherichia coli strains carrying extended-spectrum beta-lactamases, by filter mating. Matings were performed under optimal laboratory conditions (rich LB medium and 37°C) and environmentally relevant temperatures (25, 15 and 9°C) or nutrient regimes mimicking environmental conditions and limitations (synthetic wastewater and soil extract). Under optimal nutrient conditions and temperature, two recipients yielded high transfer frequencies (5 × 10^–1) while the conjugation frequency of the third strain was 1000-fold lower. Decreasing mating temperatures to psychrophilic ranges led to lower transfer frequencies, albeit all three strains conjugated under all the tested temperatures. Low nutritive media caused significant decreases in transconjugants (−3 logs for synthetic wastewater; −6 logs for soil extract), where only one of the strains was able to produce detectable transconjugants. Collectively, this study highlights that despite less-than-optimal conditions, fecal organisms may transfer plasmids in the environment, but the transfer of pKJK5 between microorganisms is limited mainly by low nutrient conditions.

Application of animal manure to soils results in the introduction of manure-derived bacteria and their antimicrobial resistance genes (ARGs) into soils. ResCap is a novel targeted-metagenomic approach that allows the detection of minority components of the resistome gene pool without the cost-prohibitive coverage depths and can provide a valuable tool to study the spread of antimicrobial resistance (AMR) in the environment. We used high-throughput sequencing and qPCR for 16S rRNA gene fragments as well as ResCap to explore the dynamics of bacteria, and ARGs introduced to soils and adjacent water ditches, both at community and individual scale, over a period of three weeks. The soil bacteriome and resistome showed strong resilience to the input of manure, as manuring did not impact the overall structure of the bacteriome, and its effects on the resistome were transient. Initially, manure application resulted in a substantial increase of ARGs in soils and adjacent waters, while not affecting the overall bacterial community composition. Still, specific families increased after manure application, either through the input of manure (e.g., Dysgonomonadaceae) or through enrichment after manuring (e.g., Pseudomonadaceae). Depending on the type of ARG, manure application resulted mostly in an increase (e.g., aph(6)-Id), but occasionally also in a decrease (e.g., dfrB3) of the absolute abundance of ARG clusters (FPKM/kg or L). This study shows that the structures of the bacteriome and resistome are shaped by different factors, where the bacterial community composition could not explain the changes in ARG diversity or abundances. Also, it highlights the potential of applying targeted metagenomic techniques, such as ResCap, to study the fate of AMR in the environment.

Manure application can spread antimicrobial resistance (AMR) from manure to soil and surface water. This study evaluated the role of the soil texture on the dynamics of antimicrobial resistance genes (ARGs) in soils and surrounding surface waters. Six dairy farms with distinct soil textures (clay, sand, and peat) were sampled at different time points after the application of manure, and three representative ARGs sul1, erm(B), and tet(W) were quantified with qPCR. Manuring initially increased levels of erm(B) by 1.5 ± 0.5 log copies/kg of soil and tet(W) by 0.8 ± 0.4 log copies/kg across soil textures, after which levels gradually declined. In surface waters from clay environments, regardless of the ARG, the gene levels initially increased by 2.6 ± 1.6 log copies/L, after which levels gradually declined. The gene decay in soils was strongly dependent on the type of ARG (erm(B) < tet(W) < sul1; half-lives of 7, 11, and 75 days, respectively), while in water, the decay was primarily dependent on the soil texture adjacent to the sampled surface water (clay < peat < sand; half-lives of 2, 6, and 10 days, respectively). Finally, recovery of ARG levels was predicted after 29–42 days. The results thus showed that there was not a complete restoration of ARGs in soils between rounds of manure application. In conclusion, this study demonstrates that rather than showing similar dynamics of decay, factors such as the type of ARG and soil texture drive the ARG persistence in the environment.

Conventional wastewater treatment has a limited capacity to reduce antibiotic resistant bacteria and genes (ARB&ARG). Tertiary treatment processes are promising solutions, although the transitory inactivation of bacteria may select ARB&ARG. This study aimed at assessing the potential of ozonation and UV_{254 nm} radiation to inactivate cultivable fungal and bacterial populations, and the selected genes 16S rRNA (common to all bacteria), intI1 (common in Gram-negative bacteria) and the ARG vanA, bla_TEM, sul1 and qnrS. The abundance of the different microbiological parameters per volume of wastewater was reduced by ∼ 2 log units for cultivable fungi and 16S rRNA and intI1 genes, by ∼3–4 log units, for total heterotrophs, enterobacteria and enterococci, and to values close or below the limits of quantification for ARG, for both processes, after a contact time of 30 min. Yet, most of the cultivable populations, the 16S rRNA and intI1 genes as well as the ARG, except qnrS after ozonation, reached pre-treatment levels after 3 days storage, suggesting a transitory rather than permanent microbial inactivation. Noticeably, normalization per 16S rRNA gene evidenced an increase of the ARG and intI1 prevalence, mainly after UV_{254 nm} treatment. The results suggest that these tertiary treatments may be selecting for ARB&ARG populations.

Photocatalytic ozonation was employed for the first time in continuous mode with TiO₂-coated glass Raschig rings and light emitting diodes (LEDs) to treat urban wastewater as well as surface water collected from the supply area of a drinking water treatment plant (DWTP). Different levels of contamination and types of contaminants were considered in this work, including chemical priority substances (PSs) and contaminants of emerging concern (CECs), as well as potential human opportunistic antibiotic resistant bacteria and their genes (ARB&ARG). Photocatalytic ozonation was more effective than single ozonation (or even than TiO₂ catalytic ozonation) in the degradation of typical reaction by-products (such as oxalic acid), and more effective than photocatalysis to remove the parent micropollutants determined in urban wastewater. In fact, only fluoxetine, clarithromycin, erythromycin and 17-alpha-ethinylestradiol (EE2) were detected after photocatalytic ozonation, by using solid-phase extraction (SPE) pre-concentration and LC-MS/MS analysis. In surface water, this treatment allowed the removal of all determined micropollutants to levels below the limit of detection (0.01-0.20 ng L^-1). The efficiency of this process was then assessed based on the capacity to remove different groups of cultivable microorganisms and housekeeping (16S rRNA) and antibiotic resistance or related genes (intI1, blaTEM, qnrS, sul1). Photocatalytic ozonation was observed to efficiently remove microorganisms and ARGs. Although after storage total heterotrophic and ARB (to ciprofloxacin, gentamicin, meropenem), fungi, and the genes 16S rRNA and intI1, increased to values close to the pre-treatment levels, the ARGs (blaTEM, qnrS and sul1) were reduced to levels below/close to the quantification limit even after 3-days storage of treated surface water or wastewater. Yeast estrogen screen (YES), thiazolyl blue tetrazolium reduction (MTT) and lactate dehydrogenase (LDH) assays were also performed before and after photocatalytic ozonation to evaluate the potential estrogenic activity, the cellular metabolic activity and the cell viability. Compounds with estrogenic effects and significant differences concerning cell viability were not observed in any case. A slight cytotoxicity was only detected for Caco-2 and hCMEC/D3 cell lines after treatment of the urban wastewater, but not for L929 fibroblasts.

Disinfection processes aim at reducing the number of viable cells through the generation of damages in different cellular structures and molecules. Since disinfection involves unspecific mechanisms, some microbial populations may be selected due to resilience to treatment and/or to high post-treatment fitness. In this study, the bacterial community composition of secondarily treated urban wastewater and of surface water collected in the intake area of a drinking water treatment plant was compared before and 3-days after disinfection with ultraviolet radiation, ozonation or photocatalytic ozonation. The aim was to assess the dynamics of the bacterial communities during regrowth after disinfection. In all the freshly collected samples, Proteobacteria and Bacteroidetes were the predominant phyla (40–50% and 20–30% of the reads, respectively). Surface water differed from wastewater mainly in the relative abundance of Actinobacteria (17% and < 5% of the reads, respectively). After 3-days storage at light and room temperature, disinfected samples presented a shift of Gammaproteobacteria (from 8 to 10% to 33–65% of the reads) and Betaproteobacteria (from 14 to 20% to 31–37% of the reads), irrespective of the type of water and disinfection process used. Genera such as Pseudomonas, Acinetobacter or Rheinheimera presented a selective advantage after water disinfection. These variations were not observed in the non-disinfected controls. Given the ubiquity and genome plasticity of these bacteria, the results obtained suggest that disinfection processes may have implications on the microbiological quality of the disinfected water.

Escherichia coli with reduced susceptibility to ciprofloxacin, isolated from urban streams, wastewater treatment plants and hospital effluent between 2004 and 2012, were compared based on multilocus sequence typing (MLST), quinolone and beta-lactam resistance determinants and plasmid replicon type. Isolates from the different types of water and isolation dates clustered together, suggesting the persistence and capacity to propagate across distinct aquatic environments. The most prevalent MLST groups were ST10 complex and ST131. Almost all isolates (98%) carried mutations in the chromosomal genes gyrA and/or parC, and 10% possessed the genes qepA, aac(6')-Ib-cr and/or qnrS1. Over 80% of the isolates were resistant to three or more classes of antibiotics (MDR ≤3). The most prevalent beta-lactamase encoding gene was bla_TEM, followed by bla_CTX-M-15, co-existing with plasmid mediated quinolone resistance. The plasmid replicon types of the group IncF were the most prevalent and distributed by different MLST groups. The genes aac(6')-Ib-cr and/or qnrS1 could be transferred by conjugation in combination with the genes bla_TEM,bla_SHV-12 or bla_OXA-1 and the plasmid replicon types I1-Iγ, K, HI2 and/or B/O. The potential of multidrug resistant E. coli with reduced susceptibility to ciprofloxacin, harboring mobile genetic elements and with ability to conjugate and transfer resistance genes, to spread and persist across different aquatic environments was demonstrated.

Mycobacterium avium subsp. paratuberculosis (MAP) and adherent-invasive Escherichia coli (AIEC) have been implicated as primary triggers in Crohn’s disease (CD). In this study, we evaluated the prevalence of MAP and E. coli (EC) DNA in peripheral blood from 202 inflammatory bowel disease (IBD) patients at various disease periods and compared against 24 cirrhotic patients with ascites (CIR) (non-IBD controls) and 29 healthy controls (HC). MAP DNA was detected by IS900-specific nested PCR, EC DNA by malB-specific nested PCR and AIEC identity, in selected samples, by sequencing of fimH gene. CD patients with active disease showed the highest MAP DNA prevalence among IBD patients (68 %). Infliximab treatment resulted in decreased MAP detection. CIR patients had high individual and coinfection rates (75 % MAP, 88 % EC and 67 % MAP and EC), whilst HC controls had lower MAP prevalence (38 %) and EC was undetectable in this control group. EC DNA prevalence in IBD patients was highly associated with CD, and 80 % of EC from the selected samples of CD patients analyzed carried the fimH30 allele, with a mutation strongly associated with AIEC. Our results show that coinfection with MAP and AIEC is common and persistent in CD, although the high MAP and EC detection in CIR patients suggested that colonization is, at least, partially dependent on increased gut permeability. Nevertheless, facilitative mechanisms between a susceptible host and these two potential human pathogens may allow their implication in CD pathogenesis.

Over the last decade, numerous evidences have contributed to establish a link between the natural and human-impacted environments and the growing public health threat that is the antimicrobial resistance. In the environment, in particular in areas subjected to strong anthropogenic pressures, water plays a major role on the transformation and transport of contaminants including antibiotic residues, antibiotic-resistant bacteria, and antibiotic resistance genes. Therefore, the urban water cycle, comprising water abstraction, disinfection, and distribution for human consumption, and the collection, treatment, and delivery of wastewater to the environment, is a particularly interesting loop to track the fate of antibiotic resistance in the environment and to assess the risks of its transmission back to humans. In this article, the relevance of different transepts of the urban water cycle on the potential enrichment and spread of antibiotic resistance is reviewed. According to this analysis, some gaps of knowledge, research needs, and control measures are suggested. The critical rationale behind the measures suggested and the desirable involvement of some key action players is also discussed.

Background: Inflammatory bowel disease (IBD) is associated with a higher prevalence of opportunistic infections. Epstein-Barr virus (EBV) is a ubiquitous virus related to several malignancies, namely lymphoma; its prevalence in patients with IBD and its relation with different therapeutic regimens are not well studied. Methods: Patients followed in our IBD outpatient clinic were consecutively enrolled for participation in a prospective study, and healthy volunteers were recruited as controls. EBV DNA was measured at least 1 time in each patient. Results: Three hundred and seventy-nine individuals were enrolled in the study (93 treated with 5-aminosalicylates, 91 with azathioprine, 70 with infliximab, 43 with combined treatment with infliximab and azathioprine, and 82 controls). More than 90% of the patients had previous EBV exposure. EBV DNA was found in 132 samples (35%); its prevalence was significantly higher in every group of patients with IBD, comparing to controls. Among patients with IBD, infliximab with or without azathioprine was related to higher prevalence of EBV comparing to azathioprine alone or 5-aminosalicylates (P < 0.05). Age above 60 years was related to EBV DNA positivity with a specificity of 92%. Concerning treated groups, ulcerative colitis was the only risk factor identified for high levels of EBV DNA (>1000 and 2500 copies per milliliter). No relationship was found between EBV and C-reactive protein. Conclusions: IBD is a risk factor for the presence of EBV DNA in blood, particularly in older patients and in those taking infliximab. C-reactive protein was not related to EBV DNA prevalence.