A European-wide dataset to uncover adaptive traits of Listeria monocytogenes to diverse ecological niches.

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes listeriosis, a serious foodborne illness. In the nature-to-human transmission route, Lm can prosper in various ecological niches. Soil and decaying organic matter are its primary reservoirs. Certain clonal complexes (CCs) are over-represented in food production and represent a challenge to food safety. To gain new understanding of Lm adaptation mechanisms in food, the genetic background of strains found in animals and environment should be investigated in comparison to that of food strains. Twenty-one partners, including food, environment, veterinary and public health laboratories, constructed a dataset of 1484 genomes originating from Lm strains collected in 19 European countries. This dataset encompasses a large number of CCs occurring worldwide, covers many diverse habitats and is balanced between ecological compartments and geographic regions. The dataset presented here will contribute to improve our understanding of Lm ecology and should aid in the surveillance of Lm. This dataset provides a basis for the discovery of the genetic traits underlying Lm adaptation to different ecological niches.

Brewer's spent grain as a no-cost substrate for polyhydroxyalkanoates production: Assessment of pretreatment strategies and different bacterial strains.

Polyhydroxyalkanoates (PHAs) are polyesters of significant interest due to their biodegradability and properties similar to petroleum-derived plastics, as well as the fact that they can be produced from renewable sources such as by-product streams. In this study, brewer's spent grain (BSG), the main by-product of the brewing industry, was subjected to a set of physicochemical pretreatments and their effect on the release of reducing sugars (RS) was evaluated. The RS obtained were used as a substrate for further PHA production in Burkholderia cepacia, Bacillus cereus, and Cupriavidus necator in liquid cultures. Although some pretreatments proved efficient in releasing RS (acid-thermal pretreatment up to 42.1 gRS L-1 and 0.77 gRS g-1 dried BSG), the generation of inhibitors in such scenarios likely affected PHA production compared with the process run without pretreatment (direct enzymatic hydrolysis of BSG). Thus, the maximum PHA accumulation from BSG hydrolysates was found in the reference case with 0.31 ±â€¯0.02 g PHA per g cell dried weight, corresponding to 1.13 ±â€¯0.06 g L-1 and a PHA yield of 23 ±â€¯1 mg g-1 BSG. It was also found that C. necator presented the highest PHA accumulation of the tested strains followed closely by B. cepacia, reaching their maxima at 48 h. Although BSG has been used as a source for other bioproducts, these results show the potential of this by-product as a no-cost raw material for producing PHAs in a waste valorization and circular economy scheme.

Pharmaceuticals removal in an on-farm pig slurry treatment plant based on solid-liquid separation and nitrification-denitrification systems.

The fate and degradation of 28 multiple-class veterinary pharmaceuticals in an on-farm pig slurry treatment plant based on solid-liquid separation and a nitrification-denitrification (NDN) sequence batch reactor (SBR) were evaluated for the first time. The pharmaceuticals detected at the highest concentrations in raw pig slurries belonged to the group of tetracycline antibiotics. Fluoroquinolone, lincosamide and pleuromutilin antibiotics and other drugs such as flubendazole and flunixin were also frequently detected. After solid-liquid separation, target compounds were distributed in an average of 64% onto the liquid fraction. Pharmaceuticals distributed in this fraction were removed in an average of almost 50% after being treated in NDN-SBR. Lincomycin was the compound with the highest removal percentage, reaching 100% reduction, while tetracyclines and fluoroquinolones showed moderate removal percentages (50 and 40%, respectively). Regarding nitrogen removal, NDN-SBR reduced a 77% of the content of this nutrient in the liquid slurry fraction.

Fate of pharmaceuticals and antibiotic resistance genes in a full-scale on-farm livestock waste treatment plant.

This study investigated, for the first time, the distribution and fate of 28 multiple-class veterinary pharmaceuticals and antibiotics (PhACs), and their corresponding antibiotic resistance genes (ARGs), in a full-scale on-farm livestock waste treatment plant. The plant relies on several technologies, including: anaerobic digestion (AD), solid-liquid separation, and two stages reverse osmosis (RO) of the liquid digestate. Tetracycline, fluoroquinolone, lincosamide and pleuromutilin antibiotics, together with anti-helmintic (flubendazole) and anti-inflammatory (flunixin) drugs were the most frequently detected compounds in livestock waste and in slaughterhouse sludge. This last fraction is used as co-substrate in the AD process and showed to be an important input source of PhACs and ARGs. In terms of treatment performance, AD exhibited moderate to low PhACs and ARGs reduction, while a large fraction (<50%) of the PhACs present in the digestate were distributed onto the solid fraction, after solid-liquid separation. Both solid and liquid digestates had relatively high copy numbers of ARGs. Finally, RO showed high rejection percentages for all PhACs (<90%), with concentrations in the low ng L-1 range in permeates, for most target PhACs. Nevertheless, moderate copy numbers of ARGs were detected in permeates.

Abundance of antibiotic resistance genes and bacterial community composition in wild freshwater fish species.

This study was aimed to determine the abundance of four antibiotic resistance genes (blaTEM, ermB, qnrS and sulI), as well as bacterial community composition associated with the intestinal mucus of wild freshwater fish species collected from the Foix and La Llosa del Cavall reservoirs, which represent ecosystems with high and low anthropogenic disturbance, respectively. Water and sediments from these reservoirs were also collected and analyzed to determine the pollution level by antibiotics. The blaTEM gene was only detected in brown trout and Ebro barbel, which were collected from La Llosa del Cavall reservoir. In contrast, the sulI and qnrS genes were only detected in common carp, which were collected from the Foix reservoir. Although the ermB gene was also detected in common carp, the values were below the limit of quantification. Likewise, water and sediment samples from the Foix reservoir had higher concentrations and more classes of antibiotics than those from La Llosa del Cavall. Pyrosequencing analysis of 16S rRNA genes revealed significant differences in bacterial communities associated with the intestinal mucus of fish species. Therefore, these findings suggest that anthropogenic activities are not only increasing the pollution of aquatic environments, but also contributing to the emergence and spread of antibiotic resistance in organisms that inhabit such environments.

Detection of Potential Infectious Enteric Viruses in Fresh Produce by (RT)-qPCR Preceded by Nuclease Treatment.

Foodborne illnesses associated with contaminated fresh produce are a common public health problem and there is an upward trend of outbreaks caused by enteric viruses, especially human noroviruses (HNoVs) and hepatitis A virus (HAV). This study aimed to assess the use of DNase and RNase coupled to qPCR and RT-qPCR, respectively, to detect intact particles of human adenoviruses (HAdVs), HNoV GI and GII and HAV in fresh produce. Different concentrations of DNase and RNase were tested to optimize the degradation of free DNA and RNA from inactivated HAdV and murine norovirus (MNV), respectively. Results indicated that 10 µg/ml of RNase was able to degrade more than 4 log10 (99.99%) of free RNA, and 1 U of DNase degraded the range of 0.84-2.5 log10 of free DNA depending on the fresh produce analysed. The treatment with nucleases coupled to (RT)-qPCR was applied to detect potential infectious virus in organic lettuce, green onions and strawberries collected in different seasons. As a result, no intact particles of HNoV GI and GII were detected in the 36 samples analysed, HAdV was found in one sample and HAV was present in 33.3% of the samples, without any reasonable distribution pattern among seasons. In conclusion, RT-qPCR preceded by RNase treatment of eluted samples from fresh produce is a good alternative to detect undamaged RNA viruses and therefore, potential infectious viruses. Moreover, this study provides data about the prevalence of enteric viruses in organic fresh produce from Brazil.

Effects of subinhibitory ciprofloxacin concentrations on the abundance of qnrS and composition of bacterial communities from water supply reservoirs.

We used a short-term microcosm approach to investigate the influence of two different subinhibitory concentrations of ciprofloxacin (0.01 and 0.1 µg/ml) on both the abundance of a plasmid-mediated quinolone resistance determinant (qnrS) and the structure and composition of bacterial communities from impaired and pristine water supply reservoirs. The results showed that the abundance of the qnrS gene increases in water samples exposed to both subinhibitory concentrations of ciprofloxacin, especially in water samples from La Llosa del Cavall, which represents the pristine system. Subinhibitory ciprofloxacin concentrations also induced changes in bacterial community composition as indicated by the relative abundances of each operational taxonomic unit (OTU) across treatments. Therefore, our findings may be of significant importance because subinhibitory ciprofloxacin concentrations may promote antibiotic resistance and affect bacterial community composition in environmental settings.

Detection of human adenoviruses in organic fresh produce using molecular and cell culture-based methods.

The consumption of organic fresh produce has increased in recent years due to consumer demand for healthy foods without chemical additives. However, the number of foodborne outbreaks associated with fresh produce has also increased. Contamination of food with enteric viruses is a major concern because the viruses have a low infectious dose and high persistence in the environment. Human adenovirus (HAdV) has been proposed as a good marker of faecal contamination. Therefore, the aim of this study was to evaluate the efficiency of the plaque assay (PA), real time PCR (qPCR) and integrated cell culture-RT-qPCR (ICC-RT-qPCR) for the recovery of HAdV from artificially and naturally contaminated fresh produce. Organic lettuce, strawberries and green onions were selected because these fresh products are frequently associated with foodborne outbreaks. The virus extraction efficiencies from artificially contaminated samples varied from 2.8% to 32.8% depending on the food matrix and the quantification method used. Although the HAdV recoveries determined by qPCR were higher than those determined by PA and ICC-RT-qPCR, PA was defined as the most reproducible method. The qPCR assays were more sensitive than the PA and ICC-RT-qPCR assays; however, this technique alone did not provide information about the viability of the pathogen. ICC-RT-qPCR was more sensitive than PA for detecting infectious particles in fresh produce samples. HAdV genome copies were detected in 93.3% of the analysed naturally contaminated samples, attesting to the common faecal contamination of the fresh produce tested. However, only 33.3% of the total samples were positive for infectious HAdV particles based on ICC-RT-qPCR. In conclusion, this study reported that HAdV can be an efficient viral marker for fresh produce contamination. Good detection of infectious HAdV was obtained with the ICC-RT-qPCR and PA assays. Thus, we suggest that the ICC-RT-qPCR and PA assays should be considered when quantitative microbial risk assessment (QMRA) studies are required and to establish reliable food safety guidelines.

Aeromonas rivipollensis sp. nov., a novel species isolated from aquatic samples.

Two gram-negative, facultatively anaerobic, motile and rod-shaped bacteria, strains P2G1(T) and P1A11, were isolated from the Ter River in Ripoll, Spain. Phylogenetic analysis based on 16S rRNA gene sequences showed that both strains are closely related to each other and that their closest relatives were Aeromonas media ATCC 33907(T) (99.4%) and Aeromonas hydrophila ATCC 7966(T) (99.3%). Multilocus sequence analysis (MLSA) based on the partial sequences of gyrA, gyrB, rpoD, recA, and dnaJ genes suggested that these two strains represent a novel species that clustered with A. media ATCC 33907(T). This was further supported by DNA-DNA hybridization analysis between P2G1(T) and A. media LMG 9073(T). Phenotypic features also allowed their differentiation from closely related species. These two strains should, therefore, be considered to represent a novel species within the genus Aeromonas, for which the name Aeromonas rivipollensis sp. nov. is proposed.

Occurrence of antibiotics and antibiotic resistance genes in hospital and urban wastewaters and their impact on the receiving river.

Antibiotic resistance has become a major health concern; thus, there is a growing interest in exploring the occurrence of antibiotic resistance genes (ARGs) in the environment as well as the factors that contribute to their emergence. Aquatic ecosystems provide an ideal setting for the acquisition and spread of ARGs due to the continuous pollution by antimicrobial compounds derived from anthropogenic activities. We investigated, therefore, the pollution level of a broad range of antibiotics and ARGs released from hospital and urban wastewaters, their removal through a wastewater treatment plant (WWTP) and their presence in the receiving river. Several antimicrobial compounds were detected in all water samples collected. Among antibiotic families, fluoroquinolones were detected at the highest concentration, especially in hospital effluent samples. Although good removal efficiency by treatment processes was observed for several antimicrobial compounds, most antibiotics were still present in WWTP effluents. The results also revealed that copy numbers of ARGs, such as blaTEM (resistance to ß-lactams), qnrS (reduced susceptibility to fluoroquinolones), ermB (resistance to macrolides), sulI (resistance to sulfonamides) and tetW (resistance to tetracyclines), were detected at the highest concentrations in hospital effluent and WWTP influent samples. Although there was a significant reduction in copy numbers of these ARGs in WWTP effluent samples, this reduction was not uniform across analyzed ARGs. Relative concentration of ermB and tetW genes decreased as a result of wastewater treatment, whereas increased in the case of blaTEM, sulI and qnrS genes. The incomplete removal of antibiotics and ARGs in WWTP severely affected the receiving river, where both types of emerging pollutants were found at higher concentration in downstream waters than in samples collected upstream from the discharge point. Taken together, our findings demonstrate a widespread occurrence of antibiotics and ARGs in urban and hospital wastewater and how these effluents, even after treatment, contribute to the spread of these emerging pollutants in the aquatic environment.

Characterization of ciprofloxacin-resistant isolates from a wastewater treatment plant and its receiving river.

In this study we characterised the ciprofloxacin-resistant strains isolated in biofilm and sediments from a wastewater treatment plant (WWTP) discharge point and its receiving river. We also examined the prevalence of qnrA, qnrB, qnrS and aac(6')-Ib-cr genes in these isolates and determined whether they harbour plasmid-encoded ß-lactamases such as TEM, SHV and CTX-M. Moreover, antibiotic concentrations were also measured to evaluate the level of contamination of these pharmaceuticals in the sampling area. Antibiotics were found in the range of ng L(-1) in WWTP effluents, but most of them were no longer found in downstream river. However, some fluoroquinolones were detected in sediment downstream demonstrating their high persistence and their capacity to be retained in the river sediments. Most of the ciprofloxacin-resistant isolates belonged to the Gammaproteobacteria class and 17 of them, 8 (7.6%) from the first sampling and 9 (6.1%) from the second sampling, carried a qnr gene. In particular, 15 isolates carried the qnrS gene and 2 carried the qnrB gene. Among the qnr-positive isolates, 12 harboured the aac(6')-lb-cr gene and 2 of them also carried a ß-lactamase on the same plasmid, indicating that they may be transferred simultaneously. It is also noteworthy that all qnr-positive isolates identified as Aeromonas species harboured the same qnrS allele, namely the qnrS2. This study reinforces the importance of environmental bacteria as vehicles for dissemination of antibiotic resistance genes.

Prevalence of antibiotic-resistant fecal bacteria in a river impacted by both an antibiotic production plant and urban treated discharges.

In this study, the abundance and spatial dynamics of antibiotic-resistant fecal bacteria (Escherichia coli, total coliforms and Enterococcus spp.) were determined in water and sediment samples from a river impacted by both antibiotic production plant (APP) and urban wastewater treatment plant (WWTP) discharges. Agar dilution and disk diffusion methods were also used for antimicrobial susceptibility testing. Two antimicrobial agents, cephalexin (25 µg/ml) and amoxicillin (50 µg/ml), were evaluated using the agar dilution method for E. coli, total coliforms (TC) and Enterococcus spp., whereas the degree of sensitivity or resistance of E. coli isolates to penicillin (10 U), ampicillin (10 µg), doxycycline (30 µg), tetracycline (30 µg), erythromycin (15 µg), azithromycin (15 µg) and streptomycin (10 µg) was performed using the disk diffusion method. Real-time PCR assays were used to determine the prevalence of three antibiotic-resistance genes (ARGs). The agar dilution method showed that most E. coli isolates and TC were resistant to amoxicillin, especially after receiving the APP discharges. Antibiotic resistances to amoxicillin and cephalexin were higher after the APP discharge point than after the WWTP effluent. The disk diffusion method revealed that 100% of bacterial isolates were resistant to penicillin and erythromycin. Multidrug-resistant bacteria were detected and showed a higher proportion at the WWTP discharge point than those in the APP. Highly multidrug-resistant bacteria (resistance to more than 4 antibiotics) were also detected, reaching mean values of 41.6% in water samples and 50.1% in sediments. The relative abundance of the blaTEM, blaCTX-M and blaSHV genes was higher in samples from the treatment plants than in those collected upstream from the discharges, especially for water samples collected at the APP discharge point. These results clearly demonstrate that both the APP and the WWTP contribute to the emergence and spread of antibiotic resistance in the environment.

Use of pyrosequencing to explore the benthic bacterial community structure in a river impacted by wastewater treatment plant discharges.

In this study, we determined the diversity and composition of benthic bacterial communities collected in river sediments upstream and downstream from a wastewater treatment plant (WWTP). Pyrosequencing of bacterial 16S rRNA genes revealed notable differences between the communities from upstream and downstream sites. In particular, a higher relative abundance of Acidobacteria, Chloroflexi, Deltaproteobacteria and Firmicutes and a lower proportion of Gammaproteobacteria and Verrucomicrobia sequences were detected at the downstream site compared to the upstream site. These findings represent a first approximation of the impact of WWTP discharges on environmental microbial communities.

The role of aquatic ecosystems as reservoirs of antibiotic resistance.

Although antibiotic resistance has become a major threat to human health worldwide, this phenomenon has been largely overlooked in studies in environmental settings. Aquatic environments may provide an ideal setting for the acquisition and dissemination of antibiotic resistance, because they are frequently impacted by anthropogenic activities. This review focuses primarily on the emergence and dissemination of antibiotic resistance in the aquatic environment, with a special emphasis on the role of antibiotic resistance genes.

Prevalence of antibiotic resistance genes and bacterial community composition in a river influenced by a wastewater treatment plant.

Antibiotic resistance represents a global health problem, requiring better understanding of the ecology of antibiotic resistance genes (ARGs), their selection and their spread in the environment. Antibiotics are constantly released to the environment through wastewater treatment plant (WWTP) effluents. We investigated, therefore, the effect of these discharges on the prevalence of ARGs and bacterial community composition in biofilm and sediment samples of a receiving river. We used culture-independent approaches such as quantitative PCR to determine the prevalence of eleven ARGs and 16S rRNA gene-based pyrosequencing to examine the composition of bacterial communities. Concentration of antibiotics in WWTP influent and effluent were also determined. ARGs such as qnrS, bla TEM, bla CTX-M, bla SHV, erm(B), sul(I), sul(II), tet(O) and tet(W) were detected in all biofilm and sediment samples analyzed. Moreover, we observed a significant increase in the relative abundance of ARGs in biofilm samples collected downstream of the WWTP discharge. We also found significant differences with respect to community structure and composition between upstream and downstream samples. Therefore, our results indicate that WWTP discharges may contribute to the spread of ARGs into the environment and may also impact on the bacterial communities of the receiving river.

Exploring the links between antibiotic occurrence, antibiotic resistance, and bacterial communities in water supply reservoirs.

Antibiotic resistance represents a growing global health concern due to the overuse and misuse of antibiotics. There is, however, little information about how the selective pressure of clinical antibiotic usage can affect environmental communities in aquatic ecosystems and which bacterial groups might be responsible for dissemination of antibiotic resistance genes (ARGs) into the environment. In this study, chemical and biological characterization of water and sediments from three water supply reservoirs subjected to a wide pollution gradient allowed to draw an accurate picture of the concentration of antibiotics and prevalence of ARGs, in order to evaluate the potential role of ARGs in shaping bacterial communities, and to identify the bacterial groups most probably carrying and disseminating ARGs. Results showed significant correlation between the presence of ARG conferring resistance to macrolides and the composition of bacterial communities, suggesting that antibiotic pollution and the spreading of ARG might play a role in the conformation of bacterial communities in reservoirs. Results also pointed out the bacterial groups Actinobacteria and Firmicutes as the ones probably carrying and disseminating ARGs. The potential effect of antibiotic pollution and the presence of ARGs on the composition of bacterial communities in lacustrine ecosystems prompt the fundamental question about potential effects on bacterial-related ecosystem services supplied by lakes and reservoirs.

Real-Time PCR assays for quantification of qnr genes in environmental water samples and chicken feces.

Real-time PCR assays were developed for the enumeration of plasmid-mediated quinolone resistance (PMQR) determinants, such as the qnrA, qnrB, and qnrS genes, in different water samples and chicken feces. The results indicate that the developed assays are specific and sensitive for the quantification of qnr genes in complex samples.

Removal of microbial indicators from municipal wastewater by a membrane bioreactor (MBR).

The impact of removable and irremovable fouling on the retention of viral and bacterial indicators by the submerged microfiltration membrane in an MBR pilot plant was evaluated. Escherichia coli, sulphite-reducing Clostridium spores, somatic coliphages and F-specific RNA bacteriophages were used as indicators. The membrane demonstrated almost complete removal of E. coli and sulphite-reducing Clostridium spores. However, there was no correlation with membrane fouling. The phage removal varied in accordance with the irremovable fouling, rising from 2.6 to 5.6 log(10) units as the irremovable fouling increased (measured by the change in the transmembrane pressure). In contrast, removable fouling did not have any effect on the retention of viruses by the membrane. These results indicate that irremovable membrane fouling may affect the removal efficiency of MBRs and, therefore, their capacity to ensure the required microbiological standards for the permeate achieved.