Genetically distinct genogroup IV norovirus strains identified in wastewater.

We investigated the prevalence and genetic diversity of genogroup IV norovirus (GIV NoV) strains in wastewater in Arizona, United States, over a 13-month period. Among 50 wastewater samples tested, GIV NoVs were identified in 13 (26 %) of the samples. A total of 47 different GIV NoV strains were identified, which were classified into two genetically distinct clusters: the GIV.1 human cluster and a unique genetic cluster closely related to strains previously identified in Japanese wastewater. The results provide additional evidence of the considerable genetic diversity among GIV NoV strains through the analysis of wastewater containing virus strains shed from all populations.

Occurrence and genetic diversity of human cosavirus in influent and effluent of wastewater treatment plants in Arizona, United States.

Human cosavirus (HCoSV) is a novel member of the family Picornaviridae. We investigated the prevalence and genetic diversity of HCoSV in influent and effluent wastewater in Arizona over a 12-month period, from August 2011 to July 2012. HCoSV sequences were identified in six (25%) influent samples and one (4%) effluent sample, with the highest concentration of 3.24 × 10(5) and 1.54 × 10(3) copies/liter in influent and effluent, respectively. The strains were characterized based on their 5' untranslated region and classified into species A and D, demonstrating that genetically heterogeneous HCoSV were circulating with a clear temporal shift of predominant strains in the study area.

Norovirus Outbreaks Among Colorado River Rafters in the Grand Canyon, Summer 2012.

OBJECTIVE: To investigate the incidence and causative agent of the recurrent outbreaks of acute gastrointestinal illness (AGI) among different rafting groups on the Colorado River in the Grand Canyon National Park during the 2012 summer season. METHODS: Confidential illness reports were completed by all individuals with symptoms of AGI, and samples of fecal matter and vomitus, surface swabs of rafting equipment, and environmental swabs at stops along the hiking corridor were collected and tested for the presence of norovirus using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). RESULTS: During the active outbreak period between May 9 and July 9, 2012, 97 rafters (1.4%) from 10 trips (2.9% of all trips) declared AGI symptoms. AGI incidence within the 10 infected trips varied from 6% to 88%. Outbreaks occurred in 3 distinct temporal clusters that involved 2 different genogroups of norovirus. All available toilet fecal samples (5 samples) were positive for norovirus RNA: 1 with genogroup I (GI) and 4 with GII. The vomitus sample tested positive for GI. None of the fomite samples from rafting equipment or from the hiking corridors were confirmed for norovirus. CONCLUSIONS: The results suggest that norovirus may have been introduced by ill or asymptomatic individuals actively shedding the virus in their vomitus or feces, and spread within, or between, river trips by different modes of transmission. This study reinforces the importance of appropriate guidance and practice regarding norovirus prevention and the necessity of postoutbreak containment in relatively isolated groups of individuals.

Molecular survey of concrete sewer biofilm microbial communities.

The microbial composition of concrete biofilms within wastewater collection systems was studied using molecular assays. SSU rDNA clone libraries were generated from 16 concrete surfaces of manholes, a combined sewer overflow, and sections of a corroded sewer pipe. Of the 2457 sequences analyzed, α-, ß-, γ-, and δ-Proteobacteria represented 15%, 22%, 11%, and 4% of the clones, respectively. ß-Proteobacteria (47%) sequences were more abundant in the pipe crown than any of the other concrete surfaces. While 178 to 493 Operational Taxonomic Units (OTUs) were associated with the different concrete samples, only four sequences were shared among the different clone libraries. Bacteria implicated in concrete corrosion were found in the clone libraries while archaea, fungi, and several bacterial groups were also detected using group-specific assays. The results showed that concrete sewer biofilms are more diverse than previously reported. A more comprehensive molecular database will be needed to better study the dynamics of concrete biofilms.

Microbial atrazine breakdown in a karst groundwater system and its effect on ecosystem energetics.

In the absence of sunlight energy, microbial community survival in subterranean aquifers depends on integrated mechanisms of energy and nutrient scavenging. Because karst aquifers are particularly sensitive to agricultural land use impacts due to rapid and direct hydrologic connections for pollutants to enter the groundwater, we examined the fate of an exogenous pesticide (atrazine) into such an aquifer and its impact on microbial ecosystem function. Atrazine and its degradation product deethylatrazine (DEA) were detected in a fast-flowing karst aquifer underlying atrazine-impacted agricultural land. By establishing microbial cultures with sediments from a cave conduit within this aquifer, we observed two distinct pathways of microbial atrazine degradation: (i) in cave sediments previously affected by atrazine, apparent surface-derived catabolic genes allowed the microbial communities to rapidly degrade atrazine via hydroxyatrazine, to cyanuric acid, and (ii) in low-impact sediments not previously exposed to this pesticide, atrazine was also degraded by microbial activity at a much slower rate, with DEA as the primary degradation product. In sediments from both locations, atrazine affected nitrogen cycling by altering the abundance of nitrogen dissimulatory species able to use nitrogenous compounds for energy. The sum of these effects was that the presence of atrazine altered the natural microbial processes in these cave sediments, leading to an accumulation of nitrate. Such changes in microbial ecosystem dynamics can alter the ability of DEA to serve as a proxy for atrazine contamination and can negatively affect ecosystem health and water quality in karst aquifers.

New Insights into the Intrinsic and Extrinsic Factors That Shape the Human Skin Microbiome.

Despite recognition that biogeography and individuality shape the function and composition of the human skin microbiome, we know little about how extrinsic and intrinsic host factors influence its composition. To explore the contributions of these factors to skin microbiome variation, we profiled the bacterial microbiomes of 495 North American subjects (ages, 9 to 78 years) at four skin surfaces plus the oral epithelium using 16S rRNA gene amplicon sequencing. We collected subject metadata, including host physiological parameters, through standardized questionnaires and noninvasive biophysical methods. Using a combination of statistical modeling tools, we found that demographic, lifestyle, and physiological factors collectively explained 12 to 20% of the variability in microbiome composition. The influence of health factors was strongest on the oral microbiome. Associations between host factors and the skin microbiome were generally dominated by operational taxonomic units (OTUs) affiliated with the Clostridiales and Prevotella A subset of the correlations between microbial features and host attributes were site specific. To further explore the relationship between age and the skin microbiome of the forehead, we trained a Random Forest regression model to predict chronological age from microbial features. Age was associated mostly with two mutually coexcluding Corynebacterium OTUs. Furthermore, skin aging variables (wrinkles and hyperpigmented spots) were independently correlated to these taxa.IMPORTANCE Many studies have highlighted the importance of body site and individuality in shaping the composition of the human skin microbiome, but we still have a poor understanding of how extrinsic (e.g., lifestyle) and intrinsic (e.g., age) factors influence its composition. We characterized the bacterial microbiomes of North American volunteers at four skin sites and the mouth. We also collected extensive subject metadata and measured several host physiological parameters. Integration of host and microbial features showed that the skin microbiome was predominantly associated with demographic, lifestyle, and physiological factors. Furthermore, we uncovered reproducible associations between chronological age, skin aging, and members of the genus Corynebacterium Our work provides new understanding of the role of host selection and lifestyle in shaping skin microbiome composition. It also contributes to a more comprehensive appreciation of the factors that drive interindividual skin microbiome variation.

Genetic Analysis of Norovirus Strains that Caused Gastroenteritis Outbreaks Among River Rafters in the Grand Canyon, Arizona.

Toilet solid waste samples collected from five outbreaks among rafters in the Grand Canyon were subjected to sequencing analysis of norovirus partial capsid gene. The results revealed that a GI.3 strain was associated with one outbreak, whereas the other outbreaks were caused by GII.5 whose sequences shared >98.9% homology.

Occurrence of Cryptosporidium, Giardia, and Cyclospora in influent and effluent water at wastewater treatment plants in Arizona.

We investigated the occurrence of Cryptosporidium, Giardia, and Cyclospora at two wastewater treatment plants (WWTPs) in Arizona over a 12-month period, from August 2011 to July 2012. Influent and effluent wastewater samples were collected monthly, and protozoan (oo)cysts were concentrated using an electronegative filter, followed by the detection of protozoa using fluorescent microscopy (Cryptosporidium oocysts and Giardia cysts) and PCR-based methods (Cryptosporidium spp., Giardia intestinalis, and Cyclospora cayetanensis). The concentration of Giardia cysts in the influent was always higher than that of Cryptosporidium oocysts (mean concentration of 4.8-6.4×10(3) versus 7.4×10(1)-1.0×10(2)(oo)cysts/l) with no clear seasonality, and log10 reduction of Giardia cysts was significantly higher than that of Cryptosporidium oocysts for both WWTPs (P<0.05). Log10 reduction of Giardia cysts at the WWTP utilizing activated sludge was significantly higher than the other WWTP using trickling filter (P=0.014), while no statistically significant difference between the two WWTPs was observed for the log10 reduction of Cryptosporidium oocysts (P=0.207). Phylogenetic analysis revealed that G. intestinalis strains identified in wastewater belonged to two assemblages, AII and B, which are potentially infectious to humans. C. cayetanensis was also detected from both influent and effluent using a newly developed quantitative PCR, with the highest influent concentration of 1.2×10(4)copies/l. Our results demonstrated that these protozoan pathogens are prevalent in the study area and that efficacy of the conventional wastewater treatment processes at physically removing (oo)cysts is limited.

Relative abundance and treatment reduction of viruses during wastewater treatment processes--identification of potential viral indicators.

Waterborne pathogenic viruses discharged from wastewater treatment plants (WWTPs) pose potential public health risks. In the present study, we investigated the relative abundance, occurrence, and reduction of eleven different viruses at two WWTPs in southern Arizona over a 12-month period, from August 2011 to July 2012. Influent and effluent samples from the two WWTPs were collected monthly. Viruses were concentrated using an electronegative filter method and quantified using TaqMan-based quantitative PCR (qPCR) assays for each of the virus types (i.e., genogroup I, II and IV noroviruses, sapovirus, enterovirus, group A rotavirus, Aichi virus, pepper mild mottle virus, adenovirus, and JC and BK polyomaviruses), with murine norovirus internal control for the monitoring of extraction-RT-qPCR efficiencies. The pepper mild mottle virus, a plant virus, was found to be the most prevalent virus in both influent and effluent wastewater (annual mean concentration of 3.7-4.4×10(6) copies/L and 4.6-6.3×10(5) copies/L in influent and effluent wastewater, respectively), showing a low reduction by the treatment processes (0.76-0.99 annual mean log10 reduction), and no significant seasonal change in concentration. Aichi virus, a human enteric virus, was also found in greater abundance, and showed lower reduction during wastewater treatment than other human enteric viruses. Our results suggest that these viruses could be used as potential indicators of wastewater reclamation system performance, with respect to virus occurrence and removal.

Quantification and genetic analysis of salivirus/klassevirus in wastewater in Arizona, USA.

Salivirus/klassevirus sequences were identified in 7 (15%) wastewater samples collected in Arizona monthly for a year, with the highest concentration of 2.28 × 10(5) and 2.46 × 10(4) copies/L in influent and effluent, respectively. This is the first report of quantification and genetic analysis of salivirus/klassevirus in water samples in the United States.

Evaluation of commercial kits for the extraction and purification of viral nucleic acids from environmental and fecal samples.

The extraction and purification of nucleic acids is a critical step in the molecular detection of enteric viruses from environmental or fecal samples. In the present study, the performance of three commercially available kits was assessed: the MO BIO PowerViral Environmental DNA/RNA Isolation kit, the Qiagen QIAamp Viral RNA Mini kit, and the Zymo ZR Virus DNA/RNA Extraction kit. Viral particles of adenovirus 2 (AdV), murine norovirus (MNV), and poliovirus type 1 (PV1) were spiked in molecular grade water and three different types of sample matrices (i.e., biosolids, feces, and surface water concentrates), extracted with the kits, and the yields of the nucleic acids were determined by quantitative PCR (qPCR). The MO BIO kit performed the best with the biosolids, which were considered to contain the highest level of inhibitors and provided the most consistent detection of spiked virus from all of the samples. A qPCR inhibition test using an internal control plasmid DNA and a nucleic acid purity test using an absorbance at 230 nm for the nucleic acid extracts demonstrated that the MO BIO kit was able to remove qPCR inhibitors more effectively than the Qiagen and Zymo kits. These results suggest that the MO BIO kit is appropriate for the extraction and purification of viral nucleic acids from environmental and clinical samples that contain high levels of inhibitors.

Application of leftover sample material from waterborne protozoa monitoring for the molecular detection of Bacteroidales and fecal source tracking markers.

In this study, we examined the potential for detecting fecal bacteria and microbial source tracking markers in samples discarded during the concentration of Cryptosporidium and Giardia using USEPA Method 1623. Recovery rates for different fecal bacteria were determined in sewage spiked samples and environmental waters using different group-specific and host-specific PCR assays. Bacteroidales DNA recovery ranged from 59 to 71% for aliquots of supernatant collected after the elution step. The recovery of human-specific Bacteroidales DNA from sewage spiked samples was 54% in the elution step. An additional 1-7% Bacteroidales DNA was recovered after the immunomagnetic separation step, while recovery from the pellet left after the immunomagnetic separation of protozoa parasites was substantially lower. Comparison of Bacteroidales 16S rRNA gene sequences from elution and immunomagnetic separation discarded samples indicated that the distribution of clones was not statistically different, suggesting that there were no recovery biases introduced by these steps. Human- and cow-specific Bacteroidales and fecal indicator bacteria (i.e., enterococci,) were also detected in the discarded fractions of environmental samples collected from different geographic locations. Overall, the results of this study demonstrated the potential application of leftover sample fractions that are currently discarded for the PCR detection of fecal bacterial indicators and molecular source tracking.

Identification of bacterial populations in drinking water using 16S rRNA-based sequence analyses.

Intracellular RNA is rapidly degraded in stressed cells and is more unstable outside of the cell than DNA. As a result, RNA-based methods have been suggested to study the active microbial fraction in environmental matrices. The aim of this study was to identify bacterial populations in drinking water by analyzing 16S rRNA-based clone libraries. Hollow-fiber ultrafiltration was used to concentrate bacterial communities from 40l of tap water collected at 12 different times during three different summer months from a single point-of-use. Total RNA was extracted from the microbial concentrates and used to develop 16S rRNA-based clone libraries. Phylogenetic analyses of 1231 partial 16S rRNA gene sequences showed that difficult-to-classify bacterial sequences were the most predominant clones, representing 57.6% of the sequences analyzed. Within these unclassified clades, most sequences were closely related to sequences retrieved from previous DNA- and RNA-based drinking water studies. Other bacterial groups represented in this study included Proteobacteria, cyanobacteria, Actinobacteria, Bacteroidetes, and Planctomycetes. Overall, the results suggest that these bacterial groups are amongst potentially active bacteria in drinking water. Diversity analyses of clones generated show that while overall diversity is similar amongst the different months, membership changes with respect to time. The results from this study further improve our understanding of the molecular diversity and bacterial population dynamics of drinking water microbial communities. Moreover, these results provide the sequence foundation for the development of molecular assays that target active drinking water bacteria.