Determination of the bacterial microbiome of free-living amoebae isolated from wastewater by 16S rRNA amplicon-based sequencing.

Free-living amoebae (FLA) are ubiquitous protozoa commonly found in water. FLA are well-established hosts for amoeba-resistant bacteria, most of which are pathogenic, and offer them shelter from adverse environmental conditions or water treatments. Since there is very little knowledge about the complete bacterial microbiome of FLA, in this work the bacterial microbiome of FLA isolated from wastewater both after secondary and tertiary treatments was studied by amplicon-based sequencing. FLA were detected in 87.5% and 50.0% of wastewater samples taken after secondary and tertiary disinfection treatments, respectively. The most abundant bacterial phyla were Proteobacteria, Planctomycetes, Bacteroidetes and Firmicutes, which represented 83.77% of the total bacterial FLA microbiome. The most abundant class of bacteria was Gammaproteobacteria, which contains an important number of relevant pathogenic bacteria. The bacteria of public health concern Aeromonas, Arcobacter, Campylobacter, Helicobacter, Klebsiella, Legionella, Mycobacterium, Pseudomonas and Salmonella were detected as part of the FLA microbiome. Although different microbial communities were identified in each sample, there is no correlation between the microbiome of FLA and the extent of wastewater treatment. To our knowledge, this is the first work in which the bacterial microbiome of FLA isolated from wastewater is studied. Obtained results indicate that FLA are hosts of potentially pathogenic bacteria in treated wastewater used for irrigation, which may pose a public health threat.

Detection of viable Helicobacter pylori inside free-living amoebae in wastewater and drinking water samples from Eastern Spain.

Helicobacter pylori is one of the most concerning emerging waterborne pathogens. It has been suggested that it could survive in water inside free-living amoebae (FLA), but nobody has studied this relationship in the environment yet. Thus, we aimed to detect viable H. pylori cells from inside FLA in water samples. Sixty-nine wastewater and 31 drinking water samples were collected. FLA were purified and identified by PCR and sequencing. For exclusively detecting H. pylori inside FLA, samples were exposed to sodium hypochlorite and assayed by specific PMA-qPCR, DVC-FISH and culture. FLA were detected in 38.7% of drinking water and 79.7% of wastewater samples, even after disinfection. In wastewater, Acanthamoeba spp. and members of the family Vahlkampfiidae were identified. In drinking water, Acanthamoeba spp. and Echinamoeba and/or Vermamoeba were present. In 39 (58.2%) FLA-positive samples, H. pylori was detected by PMA-qPCR. After DVC-FISH, 21 (31.3%) samples harboured viable H. pylori internalized cells. H. pylori was cultured from 10 wastewater samples. To our knowledge, this is the first report that demonstrates that H. pylori can survive inside FLA in drinking water and wastewater, strongly supporting the hypothesis that FLA could play an important role in the transmission of H. pylori to humans.

Influence of drinking water biofilm microbiome on water quality: Insights from a real-scale distribution system.

Biofilms, constituting over 95 % of the biomass in drinking water distribution systems, form an ecosystem impacting both the aesthetic and microbiological quality of water. This study investigates the microbiome of biofilms within a real-scale drinking water distribution system in eastern Spain, utilizing amplicon-based metagenomics. Forty-one biofilm samples underwent processing and sequencing to analyze both bacterial and eukaryotic microbiomes, with an assessment of active biomass. Genus-level analysis revealed considerable heterogeneity, with Desulfovibrio, Ralstonia, Bradyrhizobium, Methylocystis, and Bacillus identified as predominant genera. Notably, bacteria associated with corrosion processes, including Desulfovibrio, Sulfuricella, Hyphomicrobium, and Methylobacterium, were prevalent. Potentially pathogenic bacteria such as Helicobacter, Pseudomonas, and Legionella were also detected. Among protozoa, Opisthokonta and Archaeplastida were the most abundant groups in biofilm samples, with potential pathogenic eukaryotes (Acanthamoeba, Naegleria, Blastocystis) identified. Interestingly, no direct correlation between microbiota composition and pipe materials was observed. The study suggests that the usual concentration of free chlorine in bulk water proved insufficient to prevent the presence of undesirable bacteria and protozoa in biofilms, which exhibited a high concentration of active biomass.

Microbiome of Free-Living Amoebae (FLA) Isolated from Fresh Organic Produce: Potential Risk to Consumers?

In response to growing global interest in organic agriculture, this study delves into the microbial landscape of organically grown raw produce with a focus on food safety. Vegetables that are consumed raw are potential vehicles for the transmission of any type of microorganism capable of causing human disease. Free-living amoebae (FLA) are ubiquitous protozoa found in many ecosystems and can serve as hosts to pathogenic bacteria. So far, data regarding the FLA bacterial microbiome in fresh produce remain scarce and are non-existent for those of organic origin. Thus, the aim of this preliminary work is to characterize the microbiome of FLA in commonly consumed raw vegetables to know their possible implications for consumers. A total of 40 organic cabbage, lettuce, spinach, and strawberry samples were analyzed. FLA were found in all samples, and their bacterial microbiome was obtained via amplicon sequencing using the Illumina MiSeq platform and pair-end protocol. Acanthamoeba spp. and Vermamoeba vermiformis were identified via qPCR in 65.0% and 25.0% of the samples, respectively. Regarding the bacterial microbiome of FLA, the most abundant genera were Pseudomonas (1.8-17.8%) and Flavobacterium (1.7-12.6%). Bacteria not previously related to FLA, such as Prosthecobacter or Cellvibrio, are described in this work. Importantly, several bacterial genera found within the FLA microbiome were identified as potential human pathogens, including Pseudomonas, Flavobacterium, Arcobacter, Klebsiella, Mycobacterium, Salmonella and Legionella. This is the first work in which FLA microbiome isolated from organic products has been characterized, underscoring the significance of understanding FLA's role as carriers of pathogenic bacteria in the context of organic food safety.

Characterization of eukaryotic microbiome and associated bacteria communities in a drinking water treatment plant.

The effectiveness of drinking water treatment is critical to achieve an optimal and safe drinking water. Disinfection is one of the most important steps to eliminate the health concern caused by the microbial population in this type of water. However, no study has evaluated the changes in its microbiome, specially the eukaryotic microbiome, and the fates of opportunistic pathogens generated by UV disinfection with medium-pressure mercury lamps in drinking water treatment plants (DWTPs). In this work, the eukaryotic community composition of a DWTP with UV disinfection was evaluated before and after a UV disinfection treatment by means of Illumina 18S rRNA amplicon-based sequencing. Among the physicochemical parameters analysed, flow and nitrate appeared to be related with the changes in the eukaryotic microbiome shape. Public health concern eukaryotic organisms such as Blastocystis, Entamoeba, Acanthamoeba, Hartmannella, Naegleria, Microsporidium or Caenorhabditis were identified. Additionally, the relation between the occurrence of some human bacterial pathogens and the presence of some eukaryotic organisms has been studied. The presence of some human bacterial pathogens such as Arcobacter, Mycobacterium, Pseudomonas and Parachlamydia were statistically correlated with the presence of some eukaryotic carriers showing the public health risk due to the bacterial pathogens they could shelter.

Deep-amplicon sequencing (DAS) analysis to determine the presence of pathogenic Helicobacter species in wastewater reused for irrigation.

Wastewater has become one of the most important and least expensive water for the agriculture sector, as well as an alternative to the overexploitation of water resources. However, inappropriate treatment before its reuse can result in a negative impact on the environment, such as the presence of pathogens. This poses an increased risk for environmental safety, which can subsequently lead to an increased risk for human health. Among all the emerging wastewater pathogens, bacteria of the genus Helicobacter are some of the most disturbing ones, since they are directly related to gastric illness and hepatobiliary and gastric cancer. Therefore, the aim of this study was to determine the presence of potentially pathogenic Helicobacter spp. in treated wastewater intended for irrigation. We used a next generation sequencing approach, based on Illumina sequencing in combination with culture and other molecular techniques (qPCR, FISH and DVC-FISH), to analyze 16 wastewater samples, with and without an enrichment step. By culture, one of the direct samples was positive for H. pylori. FISH and DVC-FISH techniques allowed for detecting viable Helicobacter spp., including H. pylori, in seven out of eight samples of wastewater from the tertiary effluents, while qPCR analysis yielded only three positive results. When wastewater microbiome was analyzed, Helicobacter genus was detected in 7 samples. The different molecular techniques used in the present study provided evidence, for the first time, of the presence of species belonging to the genus Helicobacter such as H. pylori, H. hepaticus, H. pullorum and H. suis in wastewater samples, even after disinfection treatment.

Evidence of viable Helicobacter pylori and other bacteria of public health interest associated with free-living amoebae in lettuce samples by next generation sequencing and other molecular techniques.

Vegetables are one of the sources from which Helicobacter pylori can be acquired. This bacterium infects >50% of the global population and is a recognized type I human carcinogen. H. pylori enters into the viable but non-culturable state when it is in the environment, and therefore the use of molecular techniques is much convenient for its detection. Free-living amoebae (FLA) are protozoans found in vegetables. They are transmission vehicles for amoeba-resistant bacteria, among which H. pylori is included. The aim of this study is to study the occurrence and viability of H. pylori from lettuce samples, H. pylori internalized into FLA and the microbiome of FLA isolated from these samples. Special focus was pointed to human pathogenic bacteria. H. pylori was not directly detected in any lettuce sample by means of molecular techniques and neither by culture. However, intra-amoebic H. pylori DNA was detected by means of PMA-qPCR in 55% of the samples and viable intra-amoebic H. pylori cells in 25% of the samples by means of DVC-FISH technique. When FLA microbiome was studied, 21 bacterial genera were part of FLA microbiome in all samples. Helicobacter genus was detected as part of the FLA microbiome in two samples. Other bacteria of public health interest such as Aeromonas sp., Arcobacter sp., Legionella sp., Mycobacterium sp., Pseudomonas sp. and Salmonella sp. were detected as part of FLA microbiome along the analysed samples. This study demonstrates for the first time that H. pylori is internalized as well as alive inside FLA isolated from vegetables. Moreover, this study shows that FLA promote H. pylori detection in environmental samples. In addition, as far as we are aware, this is the first study which studies the microbiome of FLA isolated from vegetables. Among the FLA microbiome, bacteria of public health interest were detected, pointing out that FLA are carriers of these pathogens which can reach humans and cause a public health concern.

Microbiological contamination of conventional and reclaimed irrigation water: Evaluation and management measures.

The wide diversity of irrigation water sources (i.e., drinking water, groundwater, reservoir water, river water) includes reclaimed water as a requested measure for increasing water availability, but it is also a challenge as pathogen exposure may increase. This study evaluates the level of microbial contamination in different irrigation waters to improve the knowledge and analyses management measures for safety irrigation. Over a one-year period, the occurrence of a set of viruses, bacteria and protozoa, was quantified and the performance of a wetland system, producing reclaimed water intended for irrigation, was characterized. Human fecal pollution (HAdV) was found in most of the irrigation water types analysed. Hepatitis E virus (HEV), an emerging zoonotic pathogen, was present in groundwater where porcine contamination was identified (PAdV). The skin-carcinoma associated Merkel cell polyomavirus (MCPyV), was found occasionally in river water. Noroviruses were detected, as expected, in winter, in river water and reclaimed water. Groundwater, river water and reservoir water also harboured potential bacterial pathogens, like Helicobacter pylori, Legionella spp. and Aeromonas spp. that could be internalized and viable inside amoebas like Acanthamoeba castellanii, which was also detected. Neither Giardia cysts, nor any Cryptosporidium oocysts were detected. The wetland system removed 3 Log10 of viruses and 5 Log10 of bacteria, which resembled the river water quality. Irrigation waters were prone to variable contamination levels and according to the European guidance documents, the E. coli (EC) levels were not always acceptable. Sporadic detection of viral pathogens as NoV GII and HAdV was identified in water samples presenting lower EC than the established limit (100MNP/100 mL). When dealing with reclaimed water as a source of irrigation the analysis of some viral parameters, like HAdV during the peak irrigation period (summer and spring) or NoV during the coldest months, could complement existing water management tools based on bacterial indicators.

Metagenomic analysis of viruses, bacteria and protozoa in irrigation water.

Viruses (e.g., noroviruses and hepatitis A and E virus), bacteria (e.g., Salmonella spp. and pathogenic Escherichia coli) and protozoa (e.g., Cryptosporidium parvum and Giardia intestinalis) are well-known contributors to food-borne illnesses linked to contaminated fresh produce. As agricultural irrigation increases the total amount of water used annually, reclaimed water is a good alternative to reduce dependency on conventional irrigation water sources. European guidelines have established acceptable concentrations of certain pathogens and/or indicators in irrigation water, depending on the irrigation system used and the irrigated crop. However, the incidences of food-borne infections are known to be underestimated and all the different pathogens contributing to these infections are not known. Next-generation sequencing (NGS) enables the determination of the viral, bacterial and protozoan populations present in a water sample, providing an opportunity to detect emerging pathogens and develop improved tools for monitoring the quality of irrigation water. This is a descriptive study of the virome, bacteriome and parasitome present in different irrigation water sources. We applied the same concentration method for all the studied samples and specific metagenomic approaches to characterize both DNA and RNA viruses, bacteria and protozoa. In general, most of the known viral species corresponded to plant viruses and bacteriophages. Viral diversity in river water varied over the year, with higher bacteriophage prevalences during the autumn and winter. Reservoir water contained Enterobacter cloacae, an opportunistic human pathogen and an indicator of fecal contamination, as well as Naegleria australiensis and Naegleria clarki. Hepatitis E virus and Naegleria fowleri, emerging human pathogens, were detected in groundwater. Reclaimed water produced in a constructed wetland system presented a virome and bacteriome that resembled those of freshwater samples (river and reservoir water). Viral, bacterial and protozoan pathogens were occasionally detected in the different irrigation water sources included in this study, justifying the use of improved NGS techniques to get a comprehensive evaluation of microbial species and potential environmental health hazards associated to irrigation water.

Characterization of the efficiency and uncertainty of skimmed milk flocculation for the simultaneous concentration and quantification of water-borne viruses, bacteria and protozoa.

In this study, the use of skimmed milk flocculation (SMF) to simultaneously concentrate viruses, bacteria and protozoa was evaluated. We selected strains of faecal indicator bacteria and pathogens, such as Escherichia coli and Helicobacter pylori. The viruses selected were adenovirus (HAdV 35), rotavirus (RoV SA-11), the bacteriophage MS2 and bovine viral diarrhoea virus (BVDV). The protozoa tested were Acanthamoeba, Giardia and Cryptosporidium. The mean recoveries with q(RT)PCR were 66% (HAdV 35), 24% (MS2), 28% (RoV SA-11), 15% (BVDV), 60% (E. coli), 30% (H. pylori) and 21% (Acanthamoeba castellanii). When testing the infectivity, the mean recoveries were 59% (HAdV 35), 12% (MS2), 26% (RoV SA-11) and 0.7% (BVDV). The protozoa Giardia lamblia and Cryptosporidium parvum were studied by immunofluorescence with recoveries of 18% and 13%, respectively. Although q(RT)PCR consistently showed higher quantification values (as expected), q(RT)PCR and the infectivity assays showed similar recoveries for HAdV 35 and RoV SA-11. Additionally, we investigated modelling the variability and uncertainty of the recovery with this method to extrapolate the quantification obtained by q(RT)PCR and estimate the real concentration. The 95% prediction intervals of the real concentration of the microorganisms inoculated were calculated using a general non-parametric bootstrap procedure adapted in our context to estimate the technical error of the measurements. SMF shows recoveries with a low variability that permits the use of a mathematical approximation to predict the concentration of the pathogen and indicator with acceptable low intervals. The values of uncertainty may be used for a quantitative microbial risk analysis or diagnostic purposes.

Prevalence of Cryptosporidium oocysts and Giardia cysts in raw and treated sewage sludges.

Treated sludge from wastewater treatment plants (WWTPs) is commonly used in agriculture as fertilizers and to amend soils. The most significant health hazard for sewage sludge relates to the wide range of pathogenic microorganisms such as protozoa parasites.The objective of this study was to collect quantitative data on Cryptosporidium oocysts and Giardia cysts in the treated sludge in wastewater treatment facilities in Spain. Sludge from five WWTPs with different stabilization processes has been analysed for the presence of Cryptosporidium and Giardia in the raw sludge and after the sludge treatment. A composting plant (CP) has also been assessed. After a sedimentation step, sludge samples were processed and (oo)cysts were isolated by immunomagnetic separation (IMS) and detected by immunofluorescence assay (IFA). Results obtained in this study showed that Cryptosporidium oocysts and Giardia cysts were present in 26 of the 30 samples (86.6%) of raw sludge samples. In treated sludge samples, (oo)cysts have been observed in all WWTP's analysed (25 samples) with different stabilization treatment (83.3%). Only in samples from the CP no (oo)cysts were detected. This study provides evidence that (oo)cysts are present in sewage sludge-end products from wastewater treatment processes with the negative consequences for public health.

DVC-FISH and PMA-qPCR techniques to assess the survival of Helicobacter pylori inside Acanthamoeba castellanii.

Free-living amoebae (FLA) are ubiquitous microorganisms commonly found in water. They can act as Trojan Horses for some amoeba-resistant bacteria (ARB). Helicobacter pylori is a pathogenic bacteria, suggested to be transmitted through water, which could belong to the ARB group. In this work, a co-culture assay of H. pylori and Acanthamoeba castellanii, one of the most common FLA, was carried out to identify the presence and survival of viable and potentially infective forms of the bacteria internalized by the amoeba. Molecular techniques including FISH, DVC-FISH, qPCR and PMA-qPCR were used to detect the presence of internalized and viable H. pylori. After 24 h in co-culture and disinfection treatment to kill extra-amoebic bacteria, viable H. pylori cells were observed inside A. castellanii. When PMA-qPCR was applied to the co-culture samples, only DNA from internalized H. pylori cells was detected, whereas qPCR amplified total DNA from the sample. By the combined DVC-FISH method, the viability of H. pylori cells in A. castellanii was observed. Both specific techniques provided evidence, for the first time, that the pathogen is able to survive chlorination treatment in occurrence with A. castellanii and could be very useful methods for performing further studies in environmental samples.