Role of endogenous soil microorganisms in controlling antimicrobial resistance after the exposure to treated wastewater.

The reuse of treated wastewater (TWW) for irrigation appears to be a relevant solution to the challenges of growing water demand and scarcity. However, TWW contains not only micro-pollutants including pharmaceutical residues but also antibiotic resistant bacteria. The reuse of TWW could contribute to the dissemination of antimicrobial resistance in the environment. The purpose of this study was to assess if exogenous bacteria from irrigation waters (TWW or tap water-TP) affect endogenous soil microbial communities (from 2 soils with distinct irrigation history) and key antibiotic resistance gene sul1 and mobile genetic elements intl1 and IS613. Experiments were conducted in microcosms, irrigated in one-shot, and monitored for three months. Results showed that TP or TWW exposure induced a dynamic response of soil microbial communities but with no significant increase of resistance and mobile gene abundances. However, no significant differences were observed between the two water types in the current experimental design. Despite this, the 16S rDNA analysis of the two soils irrigated for two years either with tap water or TWW resulted in soil microbial community differentiation and the identification of biomarkers from Xanthomonadaceae and Planctomycetes families for soils irrigated with TWW. Low-diversity soils were more sensitive to the addition of TWW. Indeed, TWW exposure stimulated the growth of bacterial genera known to be pathogenic, correlating with a sharp increase in the copy number of selected resistance genes (up to 3 logs). These low-diversity soils could thus enable the establishment of exogenous bacteria from TWW which was not observed with native soils. In particular, the emergence of Planctomyces, previously suggested as a biomarker of soil irrigated by TWW, was here demonstrated. Finally, this study showed that water input frequency, initial soil microbial diversity and soil history drive changes within soil endogenous communities and the antibiotic resistance gene pool.

Effects of the chlorination and pressure flushing of drippers fed by reclaimed wastewater on biofouling.

Milli-channel baffle labyrinths are widely used in drip irrigation systems. They induce a pressure drop enabling drip irrigation. However, with a section thickness that is measured in mm2, they are sensitive to clogging, which reduces the performance and service life of a drip irrigation system. The impact of chlorination (1.5 ppm of free chlorine during 1 h application) and pressure flushing (0.18 MPa) on the biofouling of non-pressure-compensating drippers, fed by real reclaimed wastewater, was studied at lab scale using optical coherence tomography. The effect of these treatments on microbial composition (bacteria and eukaryotes) was also investigated by High-throughput DNA sequencing. Biofouling was mainly observed in the inlet, outlet and return areas of the milli-labyrinth channel from drippers. Chlorination reduced biofilm development, particularly in the mainstream of the milli-labyrinth channel, and it was more efficient when combined with pressure flushing. Moreover, chlorination was more efficient in maintaining water distribution uniformity (CU < 95% compared to less than 85% for unchlorinated lines). It reduced more efficiently the bacterial concentration (≈1 log) and the diversity of the bacterial community in the dripper biofilms compared to the pressure flushing method. Chlorination significantly modified the microbial communities, promoting chlorine-resistant bacteria such as Comamonadaceae or Azospira. Inversely, several bacterial groups were identified as sensitive to chlorination such as Chloroflexi and Planctomycetes. Nevertheless, one month after stopping the treatments bacterial diversity recovered and the chlorine-sensitive bacteria such as Chloroflexi phylum and the Saprospiraceae, Spirochaetaceae, Christensenellaceae and Hydrogenophilaceae families re-emerged in conjunction with the growth of biofouling, highlighting the resilience of the bacteria originating from drippers. Based on PCoA analyses, the structure of the bacterial communities still clustered separately from non-chlorinated drippers, showing that the effect of chlorination was still detectable one month after stopping the treatment.

Hydrodynamic effect on biofouling of milli-labyrinth channel and bacterial communities in drip irrigation systems fed with reclaimed wastewater.

The clogging of drippers due to the development of biofilms reduces the benefits and is an obstacle to the implementation of drip irrigation technology in a reclaimed water context. The narrow section and labyrinth geometry of the dripper channel results the development of a heterogeneous flow behaviours with the vortex zones which it enhance the fouling mechanisms. The objective of this study was to analyse the influence of the three dripper types, defined by their geometric and hydraulic parameters, fed with reclaimed wastewater, on the biofouling kinetics and the bacterial communities. Using optical coherence tomography, we demonstrated that the inlet of the drippers (mainly the first baffle) and vortex zones are the most sensitive area for biofouling. Drippers with the lowest Reynolds number and average cross-section velocity v (1 l·h-1) were the most sensible to biofouling, even if detachment events seemed more frequent in this dripper type. Therefore, dripper flow path with larger v should be consider to improve the anti-clogging performance. In addition, the dripper type and the geometry of the flow path influenced the structure of the bacterial communities from dripper biofilms. Relative abundancy of filamentous bacteria belonging to Chloroflexi phylum was higher in 1 l·h-1 drippers, which presented a higher level of biofouling. However, further research on the role of this phylum in dripper biofouling is required.

Drip irrigation biofouling with treated wastewater: bacterial selection revealed by high-throughput sequencing.

Clogging of drippers due to the development of biofilms weakens the advantages and impedes the implementation of drip irrigation technology. The objective of this study was to characterise the bacterial community of biofilms that develop in a drip irrigation system supplied with treated wastewater. High-throughput sequencing of 16S rRNA gene amplicons indicated that the bacterial community composition differed between drippers and pipes, mainly due to changes in the abundance of the genus Aquabacterium. Cyanobacteria were found to be involved in the biological fouling of drippers. Moreover, bacterial genera including opportunistic pathogenic bacteria such as Legionella and Pseudomonas were more abundant in dripper and pipe biofilms than in the incoming water. Some genera such as Pseudomonas were mostly recovered from drippers, while others (ie Bacillus, Brevundimonas) mainly occurred in pipes. Variations in the hydraulic conditions and properties of the materials likely explain the shift in bacterial communities observed between pipes and drippers.

Characterisation of the biodegradability of post-treated digestates via the chemical accessibility and complexity of organic matter.

The stability of digestate organic matter is a key parameter for its use in agriculture. Here, the organic matter stability was compared between 14 post-treated digestates and the relationship between organic matter complexity and biodegradability was highlighted. Respirometric activity and CH4 yields in batch tests showed a positive linear correlation between both types of biodegradability (R2=0.8). The accessibility and complexity of organic matter were assessed using chemical extractions combined with fluorescence spectroscopy, and biodegradability was mostly anti-correlated with complexity of organic matter. Post-treatments presented a significant effect on the biodegradability and complexity of organic matter. Biodegradability was low for composted digestates which comprised slowly accessible complex molecules. Inversely, solid fractions obtained after phase separation contained a substantial part of remaining biodegradable organic matter with a significant easily accessible fraction comprising simpler molecules. Understanding the effect of post-treatment on the biodegradability of digestates should help to optimize their valorization.

Persistence and Potential Viable but Non-culturable State of Pathogenic Bacteria during Storage of Digestates from Agricultural Biogas Plants.

Despite the development of on-farm anaerobic digestion as a process for making profitable use of animal by-products, factors leading to the inactivation of pathogenic bacteria during storage of digestates remain poorly described. Here, a microcosm approach was used to evaluate the persistence of three pathogenic bacteria (Salmonella enterica Derby, Campylobacter coli and Listeria monocytogenes) in digestates from farms, stored for later land spreading. Nine samples, including raw digestates, liquid fractions of digestate and composted digestates, were inoculated with each pathogen and maintained for 40 days at 24°C. Concentrations of pathogens were monitored using culture and qPCR methods. The persistence of L. monocytogenes, detected up to 20 days after inoculation, was higher than that of Salmonella Derby, detected for 7-20 days, and of C. coli (not detected after 7 days). In some digestates, the concentration of the pathogens by qPCR assay was several orders of magnitude higher than the concentration of culturable cells, suggesting a potential loss of culturability and induction of Viable but Non-Culturable (VBNC) state. The potential VBNC state which was generally not observed in the same digestate for the three pathogens, occurred more frequently for C. coli and L. monocytogenes than for Salmonella Derby. Composting a digestate reduced the persistence of seeded L. monocytogenes but promoted the maintenance of Salmonella Derby. The effect of NH[Formula: see text]/NH3 on the culturability of C. coli and Salmonella Derby was also shown. The loss of culturability may be the underlying mechanism for the regrowth of pathogens. We have also demonstrated the importance of using molecular tools to monitor pathogens in environmental samples since culture methods may underestimate cell concentration. Our results underline the importance of considering VBNC cells when evaluating the sanitary effect of an anaerobic digestion process and the persistence of pathogens during the storage of digestates and subsequent land spreading.

Virus removal and integrity in aged RO membranes.

Membrane ageing reduces the quality of the filtered water. Therefore, in order to warrant public health, monitoring membrane performances are of utmost importance. Reverse osmosis (RO) membranes are generally used to remove viruses and salt. However, there is no detailed study demonstrating the impact of aged membrane on the rejection of viruses and of membrane integrity indicators. In this paper, the impact of hypochlorite induced RO ageing on the rejection of a virus surrogate (MS2 phage) and four membrane integrity indicators (salt, dissolved organic matter, rhodamine WT and sulphate) was evaluated. Hypochlorite exposure was either active (under filtration) or passive (soaking), and the changes of the membrane surface chemistry were characterised using several autopsy techniques. Under this accelerated ageing condition, the introduction of chlorine in the membrane chemistry and the breakage of amide bonds caused an increase of the water permeability and a decrease of the virus surrogate's and indicators' rejection. Ageing resulted in a more negatively charged membrane and also in a higher hydrophobicity, which lead to the adsorption of MS2 phage. Despite severe physical membrane damage leading to a reduction of salt rejection to 1.2 log (94%), the minimum rejection of MS2 phage stayed on or above 4 log.

Bioaerosol emissions from open microalgal processes and their potential environmental impacts: what can be learned from natural and anthropogenic aquatic environments?

Open processes for microalgae mass cultivation and/or wastewater treatment present an air-water interface. Similarly to other open air-aquatic environments, they are subject to contamination, but as such, they also represent a source of bioaerosols. Indeed, meteorological, physico-chemical and biological factors cause aerial dispersion of the planktonic community. Operating conditions like liquid mixing or gas injection tend to both enhance microbial activity, as well as intensify aerosolization. Bacteria, virus particles, fungi and protozoa, in addition to microalgae, are all transient or permanent members of the planktonic community and can thus be emitted as aerosols. If they should remain viable, subsequent deposition on various habitats could instigate their colonization of other environments and the potential expression of their ecological function.

Needles of Pinus halepensis as biomonitors of bioaerosol emissions.

We propose using the surface of pine trees needles to biomonitor the bioaerosol emissions at a composting plant. Measurements were based on 16S rRNA gene copy numbers of Saccharopolyspora rectivirgula, a bioindicator of composting plant emissions. A sampling plan was established based on 29 samples around the emission source. The abundance of 16S rRNA gene copies of S. rectivirgula per gram of Pinus halepensis needles varied from 104 to 102 as a function of the distance. The signal reached the background level at distances around the composting plant ranging from 2 km to more than 5.4 km, depending on the local topography and average wind directions. From these values, the impacted area around the source of bioaerosols was mapped.

Bioaerosols from composting facilities--a review.

Bioaerosols generated at composting plants are released during processes that involve the vigorous movement of material such as shredding, compost pile turning, or compost screening. Such bioaerosols are a cause of concern because of their potential impact on both occupational health and the public living in close proximity to such facilities. The biological hazards potentially associated with bioaerosol emissions from composting activities include fungi, bacteria, endotoxin, and 1-3 ß-glucans. There is a major lack of knowledge concerning the dispersal of airborne microorganisms emitted by composting plants as well as the potential exposure of nearby residents. This is due in part to the difficulty of tracing specifically these microorganisms in air. In recent years, molecular tools have been used to develop new tracers which should help in risk assessments. This review summarizes current knowledge of microbial diversity in composting aerosols and of the associated risks to health. It also considers methodologies introduced recently to enhance understanding of bioaerosol dispersal, including new molecular indicators and modeling.

Diversity of bacterial communities that colonize the filter units used for controlling plant pathogens in soilless cultures.

In recent years, increasing the level of suppressiveness by the addition of antagonistic bacteria in slow filters has become a promising strategy to control plant pathogens in the recycled solutions used in soilless cultures. However, knowledge about the microflora that colonize the filtering columns is still limited. In order to get information on this issue, the present study was carried out over a 4-year period and includes filters inoculated or not with suppressive bacteria at the start of the filtering process (two or three filters were used each year). After 9 months of filtration, polymerase chain reaction (PCR)-single strand conformation polymorphism analyses point out that, for the same year of experiment, the bacterial communities from control filters were relatively similar but that they were significantly different between the bacteria-amended and control filters. To characterize the changes in bacterial communities within the filters, this microflora was studied by quantitative PCR, community-level physiological profiles, and sequencing 16SrRNA clone libraries (filters used in year 1). Quantitative PCR evidenced a denser bacterial colonization of the P-filter (amended with Pseudomonas putida strains) than control and B-filter (amended with Bacillus cereus strains). Functional analysis focused on the cultivable bacterial communities pointed out that bacteria from the control filter metabolized more carbohydrates than those from the amended filters whose trophic behaviors were more targeted towards carboxylic acids and amino acids. The bacterial communities in P- and B-filters both exhibited significantly more phylotype diversity and markedly distinct phylogenetic compositions than those in the C-filter. Although there were far fewer Proteobacteria in B- and P-filters than in the C-filter (22% and 22% rather than 69% of sequences, respectively), the percentages of Firmicutes was much higher (44% and 55% against 9%, respectively). Many Pseudomonas species were also found in the bacterial communities of the control filter. The persistence of the amended suppressive-bacteria in the filters is discussed with regards to the management of suppressive microflora in soilless culture.

Selective microbial aerosolization in biogas demonstrated by quantitative PCR.

Aerosolization of Bacteria, Archaea, Synergistes, Staphylococcus spp. and Propionibacterium acnes was investigated in situ with quantitative real-time PCR of DNA isolated from sludge and biogases of anaerobic digesters. The data revealed that in biogas, Staphylococcus spp. and P. acnes were, respectively, aerosolized 30 and 220 times more and Archaea and Synergistes, respectively, 8 and 20 times less aerosolized than Bacteria. This is the first demonstration of selective microbial aerosolization for anaerobic digestors microorganisms. This study illustrates the fact that some microbial groups, such as opportunistic pathogens, are more susceptible to be aerosolized, since they use air as a dissemination vector, and that this has to be taken in account when up-grading biogas into natural gas networks.

Human-specific fecal bacteria in wastewater treatment plant effluents.

The objective of this study was to identify fecal bacteria able to persist after wastewater treatment and that could be used as indicators of human fecal contamination. In a first step, the diversity of Bacteroidales, Clostridiaceae, Bifidobacterium, and Bacillus-Streptococcus-Lactobacillus cluster (BSL) was analysed using a fingerprint technique (CE-SSCP) and 16S rDNA libraries in waters collected at the end of the treatment process in different urban wastewater treatment plants. For each group, dominant bacteria present in most effluents were identified. Their origin (human feces, animal feces, non-fecal) was then analysed based on data of their closest relatives in public 16S rDNA databases. Among fecal bacteria recovered in the treated effluents analysed, phylotypes close to Bifidobacterium adolescentis and Bacteroides caccae seem to be specific to human beings. Phylotypes gathering only sequences of human fecal origin were also identified among the BSL and Clostridiaceae, two bacterial groups which have been poorly investigated for bacterial source-tracking purpose. Since these bacteria were detected post-treatment in most wastewater treatment plants, they may constitute potential new indicators of fecal contamination specific to humans that could be used to track fecal contamination of surface water by sewage.

Diversity of bacteria and fungi in aerosols during screening in a green waste composting plant.

This article outlines a comprehensive analysis of the microbial diversity of aerosols produced during screening in a green waste composting plant using both culture and molecular techniques. Bacteria, thermophilic actinomycetes and fungi were quantified in the aerosols. The structure of the microbial community was examined using a fingerprint technique and DNA libraries. The results show: (i) the very high diversity of bacteria and fungi in aerosols produced during the composting screening stage, (ii) the low percentage of cultivability for bacteria in aerosols, (iii) the abundance of Thermoactinomyces spp. and Aspergillus spp. in compost aerosols.

Dynamics of Legionella spp. and bacterial populations during the proliferation of L. pneumophila in a cooling tower facility.

The dynamics of Legionella spp. and of dominant bacteria were investigated in water from a cooling tower plant over a 9-month period which included several weeks when Legionella pneumophila proliferated. The structural diversity of both the bacteria and the Legionella spp. was monitored by a fingerprint technique, single-strand conformation polymorphism, and Legionella spp. and L. pneumophila were quantified by real-time quantitative PCR. The structure of the bacterial community did not change over time, but it was perturbed periodically by chemical treatment or biofilm detachment. In contrast, the structure of the Legionella sp. population changed in different periods, its dynamics at times showing stability but also a rapid major shift during the proliferation of L. pneumophila in July. The dynamics of the Legionella spp. and of dominant bacteria were not correlated. In particular, no change in the bacterial community structure was observed during the proliferation of L. pneumophila. Legionella spp. present in the cooling tower system were identified by cloning and sequencing of 16S rRNA genes. A high diversity of Legionella spp. was observed before proliferation, including L. lytica, L. fallonii, and other Legionella-like amoebal pathogen types, along with as-yet-undescribed species. During the proliferation of L. pneumophila, Legionella sp. diversity decreased significantly, L. fallonii and L. pneumophila being the main species recovered.

Microbial characteristics of biogas.

The microbial diversity of biogas was analyzed in order to examine the aerosolization behavior of microorganisms. Six biogas samples were analyzed: five from mesophilic and thermophilic anaerobic digestors treating different wastes, and one from landfill. Epifluorescent microscopic counts revealed 10(6) prokarya m(-3). To assess the difference occuring in aerosolization, 498 biogas-borne 16S ribosomal DNA were analyzed and compared to published anaerobic digestor microbial diversity. Results show a large microbial diversity and strong discrepancy with digestor microbial diversity. Three different aerosolisation behaviour patterns can be identified: (i) that of non-aerosolized microorganisms, Deltaproteobacteria, Spirochaetes, Thermotogae, Chloroflexi phyla and sulfate-reducing groups, (ii) that of passively aerosolized microorganisms, including Actinobacteria, Firmicutes and Bacteroidetes phyla and (iii) that of preferentially aerosolized microorganisms, including Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, as well as strictly aerobic and occasionally pathogenic species, presented high levels of aerosolization.

Behaviour of pathogenic and indicator bacteria during urban wastewater treatment and sludge composting, as revealed by quantitative PCR.

Two enteric pathogens, Salmonella spp. and Campylobacter jejuni, and two bacteria commonly used as indicators, Escherichia coli and Clostridium perfringens, were monitored using quantitative real-time PCR during municipal wastewater treatment and sludge composting. The results were compared with those obtained using standard culture methods. A reduction of all bacteria was observed during wastewater treatment and during the thermophilic phase of composting. However, the bacterial groups studied behaved differently during the process, and the main differences were observed during biological treatment in activated sludge basins. In particular, Salmonella spp. and C. jejuni survived better during activated sludge treatment than E. coli. C. jejuni was the most resistant to wastewater treatment among the four bacterial groups. Overall, differences in survival were observed for all bacteria studied, when submitted to the same environmental pressure. This holds both for differences between indicators and pathogenic bacteria and between pathogenic bacteria. These results show the difficulty in defining reliable indicators.

Vertebral malformations induced by sodium salicylate correlate with shifts in expression domains of Hox genes.

Several embryotoxic agents, which includes sodium salicylate, were reported to induce vertebral variations in the form of supernumerary ribs (SNR) when administered to pregnant rodents. Because the biological significance of SNR in toxicological studies is still a matter of debate, we investigated the molecular basis of this defect by analyzing the possible involvement of Hox genes, known to specify vertebrae identity. Sodium salicylate (300mg/kg) was administered to pregnant rats on gestational day 9 (GD 9). On GD 13, the expression of several Hox genes, selected according to the position of their anterior limit of expression, namely upstream (Hoxa9), at the level (Hoxa10) and downstream (Hoxd9) to the morphological alteration, were analyzed. Posterior shifts in the anterior limit of expression of Hoxa10 and Hoxd9 were observed following exposure to salicylate, which could explain an effect at the level of the axial skeleton. This finding suggests that the appearance of ectopic ribs can be attributed to an anterior transformation of lumbar vertebrae identity into thoracic vertebrae identity. Whether this transformation occurs with all compounds inducing SNR in rats remains to be determined.

Defects in cervical vertebrae in boric acid-exposed rat embryos are associated with anterior shifts of hox gene expression domains.

BACKGROUND: Previously, we showed that prenatal exposure to boric acid (BA), an industrial agent with large production, causes alterations of the axial skeleton in rat embryos, reminiscent of homeotic transformations. Indeed, Sprague-Dawley rats exposed in utero to BA on gestation day 9 (GD 9) had only six, rather than the normal seven, cervical vertebrae. This finding, observed in 91% of GD 21 fetuses, suggests posterior transformations of vertebrae. The present study attempts to determine if these skeletal alterations could be explained by modifications of the hox code, involved in the establishment of positional information along the craniocaudal axis of the embryo. METHODS: Pregnant rats were treated by gavage with BA (500 mg/kg, twice) on GD 9. Embryos were collected on GD 11 or GD 13.5 and processed for in situ hybridization. Several hox genes were selected according to the position of their cranial limit of expression in the cervical and thoracic region. RESULTS: At GD 13.5, we detected a cranial shift of the anterior limit of expression of hoxc6 and hoxa6. We observed no difference between control and treated embryos in the location of the cranial limit of expression of the other genes: hoxd4, hoxa4, hoxc5, and hoxa5. CONCLUSIONS: Our results demonstrate that following in utero exposure to BA on GD 9, a disturbance of the expression of hox genes involved inthe specification of most anterior vertebrae is observed at GD 13.5. Based on their expression domain and on their implication in the definition of the cervicothoracic vertebral boundary, it is likely that the anteriorization of hoxc6 and hoxa6 reported here is correlated to the morphological phenotype observed in BA-exposed fetuses at GD 21.

Use of a packed-column bioreactor for isolation of diverse protease-producing bacteria from antarctic soil.

Seventy-five aerobic heterotrophs have been isolated from a packed-column bioreactor inoculated with soil from Antarctica. The column was maintained at 10 degrees C and continuously fed with a casein-containing medium to enrich protease producers. Twenty-eight isolates were selected for further characterization on the basis of morphology and production of clearing zones on skim milk plates. Phenotypic tests indicated that the strains were mainly psychrotrophs and presented a high morphological and metabolical diversity. The extracellular protease activities tested were optimal at neutral pH and between 30 and 45 degrees C. 16S ribosomal DNA sequence analyses showed that the bioreactor was colonized by a wide variety of taxons, belonging to various bacterial divisions: alpha-, beta-, and gamma-Proteobacteria; the Flexibacter-Cytophaga-Bacteroides group; and high G+C gram-positive bacteria and low G+C gram-positive bacteria. Some strains represent candidates for new species of the genera Chryseobacterium and Massilia. This diversity demonstrates that the bioreactor is an efficient enrichment tool compared to traditional isolation strategies.