Bacterial Indicators Research Articles

Uncovering the potential of novel micromonosporae isolated from an extreme hyper-arid Atacama Desert soil

The taxonomic status, biotechnological and ecological potential of several Micromonospora strains isolated from an extreme hyper arid Atacama Desert soil were determined. Initially, a polyphasic study was undertaken to clarify the taxonomic status of five micromonosporae, strains LB4, LB19, LB32T, LB39T and LB41, isolated from an extreme hyper-arid soil collected from one of the driest regions of the Atacama Desert. All of the isolates were found to have chemotaxonomic, cultural and morphological properties consistent with their classification in the genus Micromonospora. Isolates LB32T and LB39T were distinguished from their nearest phylogenetic neighbours and proposed as new species, namely as Micromonospora arida sp. nov. and Micromonospora inaquosa sp. nov., respectively. Eluted methanol extracts of all of the isolates showed activity against a panel of bacterial and fungal indicator strains, notably against multi-drug resistant Klebsiella pneumoniae ATCC 700603 while isolates LB4 and LB41 showed pronounced anti-tumour activity against HepG2 cells. Draft genomes generated for the isolates revealed a rich source of novel biosynthetic gene clusters, some of which were unique to individual strains thereby opening up the prospect of selecting especially gifted micromonosporae for natural product discovery. Key stress-related genes detected in the genomes of all of the isolates provided an insight into how micromonosporae adapt to the harsh environmental conditions that prevail in extreme hyper-arid Atacama Desert soils.

Molecular Characterization of a Fungus Producing Membrane Active Metabolite and Analysis of the Produced Secondary Metabolite

Background:The majority of studies on soil Aspergillus concern the isolation and characterization of the antimicrobial compounds produced by this organism. Our previous studies indicated an isolated Aspergillus strain soil to be of interest, and this subject is further investigated here. Method: Soil samples of various locations in Iran were collected. Extract from Aspergillus sp. culture was obtained using ethyl acetate fractionation. Antimicrobial activity testing was performed using broth microdilution assay against Escherichia coli, Candida albicans, and Staphylococcus aureus microorganisms. One metabolite PA3-d10 was isolated from these active extracts and identified using thin layer chromatography, preparative thin-layer chromatography, HPLC, 1HNMR (proton nuclear magnetic resonance), 2D NMR, and LC-MS (liquid chromatography-mass spectrometry). Results:According to morphological and biochemical properties as well as ITS rDNA sequencing, we identified an isolate of Aspergillus flavus. The ethyl acetate fraction of the fermentation medium containing membrane active metabolites showed antimicrobial effects against different bacterial and yeast indicator strains. One metabolite from these active extracts was finally identified. Conclusion:Membrane active fraction produced by Aspergillus strain in this research demonstrated antimicrobial activities against bacteria and yeast strains. Therefore, this metabolite can be considered as a potential antimicrobial membrane active agent.

Molecular Characterization of a Fungus Producing Membrane Active Metabolite and Analysis of the Produced Secondary Metabolite

The majority of studies on soil Aspergillus concern the isolation and characterization of the antimicrobial compounds produced by this organism. Our previous studies indicated an isolated Aspergillus strain soil to be of interest, and this subject is further investigated here. Soil samples of various locations in Iran were collected. Extract from Aspergillus sp. culture was obtained using ethyl acetate fractionation. Antimicrobial activity testing was performed using broth microdilution assay against Escherichia coli, Candida albicans, and Staphylococcus aureus microorganisms. One metabolite PA3-d10 was isolated from these active extracts and identified using thin layer chromatography, preparative thin-layer chromatography, HPLC, 1HNMR (proton nuclear magnetic resonance), 2D NMR, and LC-MS (liquid chromatography-mass spectrometry). According to morphological and biochemical properties as well as ITS rDNA sequencing, we identified an isolate of Aspergillus flavus. The ethyl acetate fraction of the fermentation medium containing membrane active metabolites showed antimicrobial effects against different bacterial and yeast indicator strains. One metabolite from these active extracts was finally identified. Membrane active fraction produced by Aspergillus strain in this research demonstrated antimicrobial activities against bacteria and yeast strains. Therefore, this metabolite can be considered as a potential antimicrobial membrane active agent.

Bacterial indicator reduction in dairy manure using hybrid zero-valent iron (h-ZVI) system.

Novel and efficient animal wastewater treatment technologies of bacteria reduction are needed for preventing disease outbreak in animal herds and safeguarding environmental health. Zero-valent iron (ZVI) has been used to treat bacteria contaminated water for the past decades, but its passivation issue has been a major challenge. In this study, batch tests were performed to evaluate the effect of a hybrid zero-valent iron (h-ZVI) or a mixed ZVI/Fe3O4 media system on reduction of Escherichia coli (E. coli) levels. The h-ZVI media was created through a wet chemical process that uses nitrate to oxidize ZVI in the presence of externally added Fe2+ (aq.). Transforming ZVI into a h-ZVI system could overcome the passivation of ZVI and increase the reactivity of the media. The results demonstrated that E. coli cells in the bulk phase were removed rapidly by h-ZVI media. Majority of E. coli was attached (or adsorbed) to the surface of h-ZVI media within a few minutes, which suggested that adsorption was the dominant mechanism for bacterial removal in the initial phase. This adsorption was confirmed by fluorescence microscopy with CTC-DAPI double staining and transmission electron microscopy (TEM). Increasing contact time steadily inactivated E. coli; all cells were inactivated after 120min of contact. The TEM results indicated that h-ZVI inactivated E. coli by causing direct damage on bacterial cell membrane. The results of this study strongly suggest that h-ZVI treatment can be used in water treatment industry where bacterial contamination is concerned.

Antibacterial activity of the skin secretion of Phyllomedusa azurea (Anura: Hylidae) from the Central Brazil Cerrado

Phyllomedusa azurea is a frog species well distributed geographically in South America, including Brazilian biomes as Pantanal and Cerrado. Compared with other anurans from the Phyllomedusinae family, there are few reports on the bioactive potential of skin-derived molecules from this species. In this perspective, the aim of the present study was to evaluate the in vitro antibacterial activity of skin secretion of P. azurea by detection of Minimum Inhibitory Concentration (MIC) of the growth of bacterial indicator strains and to determine if occurs a changing in the bacterial cell envelope permeability. The MIC determination was carried out by the microdilution plate method. The absorbance was measured and analyzed statistically using the t-test to compare two groups (0.05 % of significance). The impact of the crude extract on cell envelope permeability of Staphylococcus aureus ATCC 25923 was conducted by the crystal violet assay, and the absorbance was measured spectrophotometry followed by the calculation of the crystal violet uptake percentage. The specific MIC for S. aureus ATCC 25923 and Escherichia coli ATCC 25922 was 31.25 µg/mL, while for Bacillus subtilis ATCC 6633 was 125 µg/mL and Pseudomonas aeruginosa ATCC 27853 was 250 µg/mL. The treatment with crescent concentrations of frog skin secretion increased the crystal violet uptake by S. aureus ATCC 25923 cells, suggesting an action on the cell plasma membrane. The results demonstrated that the skin secretion of P. azurea presents antibacterial activity and merit further investigations to characterize the bioactive molecules.

Assessment of Recommendation for the Containment and Disinfection of Human Excreta in Cholera Treatment Centers

Containment, safe handling and disinfection of human excreta in cholera treatment centers (CTC) are key to preventing the onward spread of the disease. This study compared the efficacy of three chlorine-based approaches at concentrations of 0.5%, 1%, and 2% and one hydrated lime-based (Ca(OH)2 at 30% w:v) approach. Experiments followed existing Médecins Sans Frontières (MSF) cholera guidelines. Three simulated human excreta matrices consisting of either raw municipal wastewater (4.5 liters), or raw municipal wastewater plus 1%, or 20% faecal sludge (w:v), were treated in 14 liter Oxfam® buckets containing 125 mL of chlorine solution or hydrated lime suspension. Bacterial indicators (faecal coliforms (FC) and intestinal enterococci (IE)) and viral indicator (somatic coliphages (SOMPH)) were used to determine treatment efficacy following contact times of 10, 30 and 60min. Results showed that efficacy improved as chlorine concentrations increased. No statistical differences were observed with respect to the various contact times. Overall median log removal for 0.5% chlorine were: FC (1.66), IE (1.41); SOMPH (1.28); for 1% chlorine: FC (1.98), IE (1.82); SOMPH (1.79); and for 2% chlorine: FC (2.88), IE (2.60), SOMPH (2.38). Hydrated lime (30%) provided the greatest overall log removal for bacterial indicators (FC (3.93) and IE (3.50), but not for the viral indicator, SOMPH (1.67)). These findings suggest that the use of 30% hydrated lime suspensions or 2% chlorine solutions may offer a simple public health protection measure for the containment, safe handling, and disinfection of human excreta during humanitarian emergencies.

Towards a Miniaturized Device to Evaluate the BOD Parameter of Wastwater

The concentration of organic pollutants present in wastewater is classically evaluated by measuring the quantity of dissolved oxygen during five days; it is quantified by the so called BOD5 parameter (Biological Oxygen Demand). This work constitutes the first step of an overall strategy targeting to improve the monitoring of this indicator of the water quality. We focus on the development of a microsystem approach allowing monitoring the O 2 consumption induced by the biodegradation process of organic matter. To evaluate the organic pollutants concentration, we use Escherichia coli as bacterial indicator, confined in a PDMS-glass chip. Their metabolic activity in presence of organic molecules is deduced from their oxygen consumption. These measurements are ensured by optical sensors present in each of the five instrumented chambers of the chip. The results show that the microsystem approach is suitable to measure simultaneously different concentrations of organic load, and that it is possible to reduce the analysis time. By examining the O 2 diffusion towards the walls of the device, we analyze the different part of the experimental results; it allows, on the basis of a theoretical model, to predict a precise evaluation of the BOD value within few hours.

Presence of bacteria and bacteriophages in full-scale trickling filters and an aerated constructed wetland

Aerated Constructed Wetlands are a state-of-the-art design that provides a different physical and chemical environment (compared to traditional passive wetland designs) for the wastewater treatment processes and, thus, may have different pathogen removal characteristics. In order to establish the fate of bacterial and viral indicators, a field study was carried out at a Sewage Treatment Works (STW) in the UK (serving 20,000 pe). The STW consists of primary and secondary sedimentation tanks and trickling filters (TF) as the biological stage. A large (1,160 m2) pilot aerated Vertical Flow Constructed Wetland (AVFCW) was constructed at the STW as tertiary stage receiving ¼ of the total flow rate, i.e., 1250 m3/day. Effluent quality of the AVFCW complied with national and international standards for environmental discharge and reuse. For the first time, two sets of bacterial (Faecal coliforms, E.coli and intestinal enterococci) and viral indicators (Somatic coliphages, F-RNA specific bacteriophages and human-specific B. fragilis GB124 phages) were simultaneously investigated in an AVFCW and TF. High elimination rates were detected (up to 3.7 and 2.2 log reduction for bacteria indicators and phages, respectively) and strong correlations between the two sets were found. The superior efficiency of the aerated Constructed Wetlands in microbiological contamination removal compared to passive wetland systems was established for the first time, which may have implications for process selection for wastewater reuse. This field study therefore provides new evidence on the fate of bacteriophages and a first indication of their potential use for performance evaluation in TF and aerated Constructed Wetlands. It also demonstrates that the combination of TF with aerated constructed wetlands could be a novel and effective treatment scheme for new STW or for the upgrade of existing STW.

Kū Hou Kuapā: Cultural Restoration Improves Water Budget and Water Quality Dynamics in Heʻeia Fishpond

In Hawaiʻi, the transition from customary subsistence flooded taro agroecosystems, which regulate stream discharge rate trapping sediment and nutrients, to a plantation-style economy (c. the 1840s) led to nearshore sediment deposition—smothering coral reefs and destroying adjacent coastal fisheries and customary fishpond mariculture. To mitigate sediment transport, Rhizophora mangle was introduced in estuaries across Hawaiʻi (c. 1902) further altering fishpond ecosystems. Here, we examine the impact of cultural restoration between 2012–2018 at Heʻeia Fishpond, a 600–800-year-old walled fishpond. Fishpond water quality was assessed by calculating water exchange rates, residence times, salinity distribution, and abundance of microbial indicators prior to and after restoration. We hypothesized that R. mangle removal and concomitant reconstruction of sluice gates would increase mixing and decrease bacterial indicator abundance in the fishpond. We find that Heʻeia Fishpond’s physical environment is primarily tidally driven; wind forcing and river water volume flux are secondary drivers. Post-restoration, two sluice gates in the northeastern region account for >80% of relative water volume flux in the fishpond. Increase in water volume flux exchange rates during spring and neap tide and shorter minimum water residence time corresponded with the reconstruction of a partially obstructed 56 m gap together with the installation of an additional sluice gate in the fishpond wall. Lower mean salinities post-restoration suggests that increased freshwater water volume influx due to R. mangle removal. Spatial distribution of microbial bio-indicator species was inversely correlated with salinity. Average abundance of Enterococcus and Bacteroidales did not significantly change after restoration efforts, however, average abundance of a biomarker specific to birds nesting in the mangroves decreased significantly after restoration. This study demonstrates the positive impact of biocultural restoration regimes on water volume flux into and out of the fishpond, as well as water quality parameters, encouraging the prospect of revitalizing this and other culturally and economically significant sites for sustainable aquaculture in the future.

Assessment of thin-film photocatalysis inactivation of different bacterial indicators and effect on their antibiotic resistance profile

The presence of bacterial pathogens in water bodies, alongside their growing antibiotic resistance, endanger access to freshwater sources and necessitate their successful inactivation with a proper disinfection technology. In the present study, a parallel plate reactor (PPL) with immobilized photocatalyst was used as a disinfection system for the inactivation of two bacterial indicators (Escherichia coli and Enterococcus faecalis) in aqueous samples. Experiments were carried out at parallel plate reactor configuration (PPL) operated in recycling batch mode. Titanium Tetraisopropoxide (TTIP) based thin-film coated photocatalyst surfaces were used and assessed operations parameters were; pH, initial bacteria concentration, source and type of bacteria. The effect of the photocatalytic process on antibiotic resistance profile of target bacteria was also investigated as it may serve as a pioneering step in the field of well-established and modern disinfection method development, without causing proliferation of antibiotic resistance. The observed courses of bacterial inactivation and the final disinfection rates point out diversity in the level of interaction between different type/source of bacteria and photocatalyst of concern. According to the results, a stationary phase of bacteria inactivation proceeded with a rapid and efficient one for the case of E. faecalis (99% removal after 180 min and 99.9% removal after 240 min), while the trend for E. coli is more likely to be described as extended along the process time. Among the tested antibiotics, E. coli Minimum inhibition concentration (MIC) values for beta-lactam, macrolide and aminoglycoside groups were considerably altered (namely Ampicillin, Cefaclor, Clarithromycin - Erythromycin and Amikacin, respectively). PC oxidation was approved to be efficient on bacterial inactivation and trigger alterations on resistance behaviour of E.coli and Enterococcus sp. strains.

Fate of bacterial indicators and Salmonella in biofilm developed on ultrafiltration membranes treating secondary effluents of domestic wastewater

The fate of representative indicator and pathogenic bacteria on ultrafiltration (UF)-membrane surfaces treating secondary wastewater effluent, as well as their reaction to common biofouling-removal techniques was investigated. Field-condition experiments showed that the number of heterotrophic bacteria, fecal coliforms, E. coli and Salmonella on membrane surface increased rapidly and continuously until the end of the experiment, reaching 9, 6.5, 6, and 2.4 logs, respectively. Similar results were obtained under controlled laboratory conditions. However, the increase in the bacterial numbers was dependent on the supply of fresh wastewater. Quantitative real-time PCR verified the behavior of attached E. coli cells, although the numbers were 1–2 logs higher compared to the standard culture-based method. The number of attached bacteria was positively correlated to increases in DNA and protein content and negatively correlated to the membrane flux. In-situ membrane cleaning using sodium hypochlorite significantly reduced the number of attached bacteria. However, the effect was temporary and affected bacterial cell cultivability rather than viability. Taken together, these findings suggest that, under the studied conditions, indicator and pathogenic bacteria can initiate rapid biofilm development, persist on UF membrane surfaces, and survive membrane cleaning with sodium hypochlorite.

Microbial Community Diversity as a Potential Bioindicator of AMD and Steel Plant Effluent in a Channelled Valley Bottom Wetland

Freshwater resources in semi-arid countries are under constant threat from pollution. One of the major pollutants is acid mine drainage (AMD), which not only lowers the pH of the water, but contains high sulphuric acid and high metal concentrations. Bacteria and algae are the first organisms to respond to stressors such as reduced pH and high metal concentrations. The bacterial community in a natural freshwater wetland impacted by AMD and steel plant effluent was identified, with the objective to include bacterial indicator communities in an ecotoxicological screening tool for wetland ecosystem health estimation. Five study sites at the Grootspruit canal valley bottom wetland in Mpumalanga, South Africa, were identified as case study areas which include a reference site and four AMD-impacted sites displaying various degrees of degradation. Physical, chemical and microbiological parameters were measured at each site. The bacterial community was sampled from both the water column and bottom sediment and subjected to next-generation sequencing for identification. The bacterial diversity was high, even at the most impacted sites. The phyla that were predominant in all the samples were the alpha-, beta- and gamma-Proteobacteria and Bacteriodetes. The bacterial based bio-assessment tool scored the reference site as mostly unaffected by anthropogenic impacts, while the AMD and steel plant effluent-impacted sites were classified as modified to severely modify. The outcome of the study showed that the proposed bacterial bioindicators can potentially be employed as part of the ecotoxicological screening tool to determine wetland ecosystem health.

ANTIFUNGAL AND ANTIBACTERIAL ACTIVITY OF SOME LACTOBACILLI ISOLATED FROM CAMEL’S MILK BIOTOPE IN THE SOUTH OF ALGERIA

Antifungal activity has been the subject of numerous recent works to fight the development of molds in food products and to reduce alteration. The objective of this study is the detection of new indigenous lactic acid bacteria (LAB) strains isolated from Algerian camel’s milk which have an antifungal activity. A number of 264 LAB were isolated from fermented camel’s milk where 80 rods isolates were identified by phenotypical, physiological and biochemical tests. All retained isolates belong to the genus Lactobacillus (Lb.). The antifungal activity was determined by using confrontation and overlay techniques. The screening of antifungal activity against Aspergillus niger, Aspergillus fumigatus and Penicillium sp. showed that 14 strains have an antifungal activity. These later isolates were identified as Lb. plantrarum, Lb. paracasei subsp. paracasei, Lb. brevis, Lb. rhamnosus and Lb. pentosus. These strains have shown an effect on mycelial growth and spore germination. Penicillium sp. is the most inhibited followed by Aspergillus ssp. No inhibitory activity could be detected against fungi with the supernatant. However, supernatant have an inhibitory activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Listeria ivanovii. P. aeruginosa is the only specie who has been affected by the neutralized supernatant. The redissolved dry supernatant of Lb. brevis revealed an inhibition of both fungal and bacterial indicator strains. Results suggest that camel’s milk LAB’s strains could be selected for application to control spoilage, fungal growth and pathogenic bacteria. The use of LAB producing antifungal metabolites represents a technological advantage for the food industry.

Occurrence and distribution of viruses and picoplankton in tropical freshwater bodies determined by flow cytometry

This study aimed to examine the drivers in shaping the occurrence and distribution of total viruses and picoplankton in tropical freshwater ecosystems. Flow cytometry was used to quantify the concentrations of total viruses, picoheterotrophs, picophytoplankton, and picocyanobacteria. Three land use patterns (urban-, agriculture- and parkland-dominated) were evaluated using ArcGIS. Significant correlations were observed between water-borne microbial targets and water quality parameters (0.175 ≤ |r| ≤ 0.441), nutrients (0.250 ≤ r ≤ 0.570) and land use factors (0.200 ≤ |r| ≤ 0.460). In particular, the concentrations of total viruses and picoheterotrophic cells were higher in catchments whereas the abundances of picophytoplankton and picocyanobacteria were higher in reservoirs. Total viruses and picoplankton had higher concentrations in urban- and agriculture-dominated areas, probably due to anthropogenic inputs and agricultural inputs, respectively. Although surface water is a complex matrix influenced by niche-based (i.e., physicochemical properties, nutrients, land use impact etc.) and neutral-based factors (i.e., ecological drift, dispersal and species), land use patterns could help to elucidate the occurrence and distribution of the total microbial community at the macroscopic level. Meanwhile, inter-correlations among viruses, picoplankton and picoheterotrophs (0.715 ≤ r ≤ 0.990) also substantiates their mutual interactions in influencing the microbial community. Furthermore, the relationships between total microbial cells and bacterial and viral indicators were also investigated. Concentrations of total viruses, picoplankton and picoheterotrophs were positively correlated with bacterial indicators (0.427 ≤ r ≤ 0.590) and viral indicators (0.201 ≤ r ≤ 0.563). These results indicated that faecal and viral contamination could contribute to the numbers of total viruses and bacteria.

The QuantiPhage assay: A novel method for the rapid colorimetric detection of coliphages using cellulose pad materials

Assessment of viral contamination is essential for monitoring the microbial quality of water and protection of public health, as human virus presence is not accurately determined using bacterial indicators. Currently, the time required for conventional viral testing means that water contaminated with human pathogens may be used (e.g. for drinking, recreation or irrigation) days before results are available. Here we report a new rapid method for coliphage enumeration, the QuantiPhage (QP) assay. The novelty of the assay is the use of cellulose absorbent pad materials to support coliphage growth and colorimetric detection, in place of agar that is used in the plaque assay. In addition to saving time associated with agar preparation and tempering, the QP assay enabled enumeration of somatic coliphages in 1.5–2 h and F+ coliphages in 2.5–3 h. The assays were highly sensitive, with a lower detection limit of 1 plaque forming unit (PFU) per mL where 1 mL sample volumes were analysed, and 1 PFU per 10 mL where 10 mL sample volumes were analysed. This is the first rapid culture assay to enable low numbers of coliphages to be reliably detected and to produce directly equivalent results to agar-based plaque assays. A novel gelatin-immobilisation method is also reported, that reduces time to prepare bacterial cells from ∼20 h to 40–60 min (depending on the assay format), and provides a ready to use form of cells, that is compatible with rapid detection and kit formats. When applied to analysis of somatic coliphages in wastewater samples and surface water samples, mean differences in results of the QP assay and the conventional plaque assay were not statistically significant (mean difference ≤ 0.15 log10 PFU/L and 0.5 PFU/10 mL respectively, P > 0.05). The QP is a valuable tool for assessing microbial water quality, which may assist in improving the management of water resources.

Temporal variability of parasites, bacterial indicators, and wastewater micropollutants in a water resource recovery facility under various weather conditions

Wastewater discharges lead to the deterioration of receiving waters through treated effluents and by-passes, combined and sanitary sewer overflows, and cross-connections to storm sewers. The influence of weather conditions on fecal indicator bacteria, pathogens and wastewater micropollutants on raw and treated sewage concentrations has not been extensively characterized. However, such data are needed to understand the effects of by-pass discharges and incomplete treatment on receiving waters. A water resource recovery facility was monitored for pathogenic parasites (Cryptosporidium oocysts, Giardia cysts), fecal indicator bacteria (Escherichia coli, Clostridium perfringens), and wastewater micropollutants (caffeine, carbamazepine, 2-hydroxycarbamazepine, acesulfame, sucralose, and aspartame) during 6 events under different weather conditions (snowmelt and trace to 32 mm 2-day cumulative precipitation). Greater intra- and inter-event variability was observed for Giardia, E. coli and C. perfringens than for studied WWMPs. Even with the addition of inflow and infiltration, daily variations dominated concentration trends. Thus, afternoon and early evening were identified as critical times with regards to high concentrations and flows for potential by-pass discharges. Peak concentrations of Giardia were observed during the June wet weather event (1010 cysts/L), with the highest flowrates relative to the mean monthly flowrate. Overall, Giardia, E. coli and C. perfringens concentrations were positively correlated with flowrate (R > 0.32, p < 0.05). In raw sewage samples collected under high precipitation conditions, caffeine, carbamazepine and its metabolite 2-OH-carbamazepine were significantly correlated (p < 0.05) with Giardia, E. coli, and C. perfringens demonstrating that they are useful markers for untreated sewage discharges. Data from the study are needed for estimating peak concentrations discharged from wastewater sources in relation to precipitation or snowmelt events.

Analysis of large deletion mutations induced by abasic site analog in human cells

BackgroundAbasic sites are formed spontaneously and by nucleobase chemical modifications and base excision repair. A chemically stable abasic site analog was site-specifically introduced into replicable plasmid DNAs, which were transfected into human U2OS cells. The amplified DNAs were recovered from the cells and used for the transformation of a bacterial indicator strain.ResultsLarge deletion mutations were induced by the analog, in addition to point mutations at the modified site. No apparent sequence homology at the deletion junctions was found.ConclusionThese results suggested that the large deletions induced by the abasic site analog are formed by homology-independent events.

Viral tools for detection of fecal contamination and microbial source tracking in wastewater from food industries and domestic sewage

Alternative indicators may be more suitable than thermotolerant coliform bacteria to assess enteric virus pollution in environmental waters and their removal from wastewaters. In this study, F-specific RNA bacteriophages (F-RNAPh) showed to be potential viral indicators of fecal contamination when they were quantified from domestic and food-industrial effluents containing human, chicken, swine or bovine wastes. In addition, they showed to be resistant to the primary and secondary treatments of the wastewater treatment plants. The viable F-RNAPh count showed correlation with viable thermotolerant coliforms but also with human polyomaviruses (HPyV) quantified by a new molecular method. In domestic effluents, F-RNAPh and HPyV indicators significantly correlated with a human viral pathogen, norovirus, while the bacterial indicator did not, being then better predictors of the behavior of enteric pathogenic viruses. In addition, we assessed human, bovine and fowl microbial source tracking markers, based on the molecular detections of human polyomavirus, bovine polyomavirus, and fowl adenovirus, respectively. The techniques implemented extend the range of viruses detected, since they target different viral types simultaneously. These markers could be applied when multiple source pollution is suspected, contributing to making decisions on public health interventions.

Assessing microbial contamination and antibiotic resistant bacteria using zebra mussels (Dreissena polymorpha)

Aquatic pollution with faecal bacteria and subsequent consumption of contaminated water or food is a worldwide issue that causes severe health effects (e.g. meningitis, salmonellosis, dysentery). In addition, the excessive use of antibiotics in animal husbandry and human medicine has enhanced the selective pressure on pathogenic bacteria, further increasing human health risks and detrimental effects on natural microbial communities. This urges the need to monitor faecal contamination using a time-integrated approach, as grab water samples can miss pathogen peaks. We tested the ability of zebra mussels (Dreissena polymorpha) to take up and depurate faecal indicator bacteria such as Escherichia coli and intestinal enterococci. Furthermore, we quantified the frequency of antibiotic resistant bacteria in water and mussels both in controlled laboratory tests and under in situ conditions downstream of a sewage treatment plant (STP). Laboratory results show that bacterial indicators in mussels were 132 times higher than their concentration in water, and that mussels retained bacteria up to 2 days after pulse exposure. Field results show decreasing bacterial concentrations in both water and mussels downstream the STP, with maximum E. coli concentrations ranging 173–9 cfu mL−1 in water and 2970–330 cfu g−1 in mussels. Similarly, enterococci ranged 59–4 cfu mL−1 and 1450–240 cfu g−1 in water and mussels, respectively. High proportions of antibiotic resistant E. coli were found in mussels (72%) and water (65%), and slightly lower proportion of resistant enterococci was found in mussels (47%) and in water (34%). Moreover, 33% of the bacteria isolated from mussels were resistant to multiple antibiotics, which emphasizes that resistance is a common feature in surface waters and highlights the need for safe water management. Our results show that zebra mussels provide an efficient, time-integrating tool for quantifying faecal indicators, including resistant and multidrug resistant bacteria.

Human adenoviruses as waterborne index pathogens and their use for Quantitative Microbial Risk Assessment

The current microbial water quality standards are based on the monitoring of fecal indicator organisms, which are mainly bacterial indicators (i.e., Escherichia coli, intestinal enterococci), however epidemiological data indicate that viruses are important etiological agents of waterborne illnesses. Among waterborne viruses, human adenovirus can be considered as an index pathogen, owing to its abundance in sewage and persistence in the environment, as well as its potential infectivity. In this study, data on human adenoviruses from different water matrices (the entrance and exit of a water treatment plant, rivers and seawaters) were analyzed, in parallel with traditional fecal bacterial indicators and somatic coliphages. The results showed a 64% frequency of positive adenovirus samples, decreasing from the sewage system (100% at the entrance and 94% at the exit) to rivers (92% and 72% for different rivers) and seawater (21%). Adenovirus concentrations showed a significant correlation with somatic coliphages in one river and seawater, thus supporting the recent inclusion of coliphages as viral indicators in water safety guidelines. The data collected were used to estimate adenovirus to indicator ratios, which could be used as input in Quantitative Microbial Risk Assessment (QMRA) studies.