Microbial diversities (16S and 18S rRNA gene pyrosequencing) and environmental pathogens within drinking water biofilms grown on the common premise plumbing materials unplasticized polyvinylchloride and copper

Drinking water (DW) biofilm communities influence the survival of opportunistic pathogens, yet knowledge about the microbial composition of DW biofilms developed on common in-premise plumbing material is limited. Utilizing 16S and 18S rRNA gene pyrosequencing, this study characterized the microbial community structure within DW biofilms established on unplasticized polyvinyl chloride (uPVC) and copper (Cu) surfaces and the impact of introducing Legionella pneumophila (Lp) and Acanthamoeba polyphaga. Mature (\u3e 1 year old) biofilms were developed before inoculation with sterilized DW (control, Con), Lp, or Lp and A. polyphaga (LpAp). Comparison of uPVC and Cu biofilms indicated significant differences between bacterial (P = 0.001) and eukaryotic (P \u3c 0.01) members attributable to the unique presence of several family taxa: Burkholderiaceae, Characeae, Epistylidae, Goniomonadaceae, Paramoebidae, Plasmodiophoridae, Plectidae, Sphenomonadidae, and Toxariaceae within uPVC biofilms; and Enterobacteriaceae, Erythrobacteraceae, Methylophilaceae, Acanthamoebidae, and Chlamydomonadaceae within Cu biofilms. Introduction of Lp alone or with A. polyphaga had no effect on bacterial community profiles (P \u3e 0.05) but did affect eukaryotic members (uPVC, P \u3c 0.01; Cu, P = 0.001). Thus, established DW biofilms host complex communities that may vary based on substratum matrix and maintain consistent bacterial communities despite introduction of Lp, an environmental pathogen

Molecular Detection of \u3ci\u3eCampylobacter\u3c/i\u3e spp. and Fecal Indicator Bacteria during the Northern Migration of Sandhill Cranes (\u3ci\u3eGrus canadensis\u3c/i\u3e) at the Central Platte River

The risk to human health of the annual sandhill crane (Grus canadensis) migration through Nebraska, which is thought to be a major source of fecal pollution of the central Platte River, is unknown. To better understand potential risks, the presence of Campylobacter species and three fecal indicator bacterial groups (Enterococcus spp., Escherichia coli, and Bacteroidetes) was assayed by PCR from crane excreta and water samples collected during their stopover at the Platte River, Nebraska, in 2010. Genus-specific PCR assays and sequence analyses identified Campylobacter jejuni as the predominant Campylobacter species in sandhill crane excreta. Campylobacter spp. were detected in 48% of crane excreta, 24% of water samples, and 11% of sediment samples. The estimated densities of Enterococcus spp. were highest in excreta samples (mean, 4.6 x108 cell equivalents [CE]/g), while water samples contained higher levels of Bacteroidetes (mean, 5.1 x 105 CE/100 ml). Enterococcus spp., E. coli, and Campylobacter spp. were significantly increased in river water and sediments during the crane migration period, with Enterococcus sp. densities (~3.3 x 105 CE/g) 2 to 4 orders of magnitude higher than those of Bacteroidetes (4.9 x 103 CE/g), E. coli (2.2 x 103 CE/ g), and Campylobacter spp. (37 CE/g). Sequencing data for the 16S rRNA gene and Campylobacter species-specific PCR assays indicated that C. jejuni was the major Campylobacter species present in water, sediments, and crane excreta. Overall, migration appeared to result in a significant, but temporary, change in water quality in spring, when there may be a C. jejuni health hazard associated with water and crops visited by the migrating birds

Microbial diversities (16S and 18S rRNA gene pyrosequencing) and environmental pathogens within drinking water biofilms grown on the common premise plumbing materials unplasticized polyvinylchloride and copper

Drinking water (DW) biofilm communities influence the survival of opportunistic pathogens, yet knowledge about the microbial composition of DW biofilms developed on common in-premise plumbing material is limited. Utilizing 16S and 18S rRNA gene pyrosequencing, this study characterized the microbial community structure within DW biofilms established on unplasticized polyvinyl chloride (uPVC) and copper (Cu) surfaces and the impact of introducing Legionella pneumophila (Lp) and Acanthamoeba polyphaga. Mature (\u3e 1 year old) biofilms were developed before inoculation with sterilized DW (control, Con), Lp, or Lp and A. polyphaga (LpAp). Comparison of uPVC and Cu biofilms indicated significant differences between bacterial (P = 0.001) and eukaryotic (P \u3c 0.01) members attributable to the unique presence of several family taxa: Burkholderiaceae, Characeae, Epistylidae, Goniomonadaceae, Paramoebidae, Plasmodiophoridae, Plectidae, Sphenomonadidae, and Toxariaceae within uPVC biofilms; and Enterobacteriaceae, Erythrobacteraceae, Methylophilaceae, Acanthamoebidae, and Chlamydomonadaceae within Cu biofilms. Introduction of Lp alone or with A. polyphaga had no effect on bacterial community profiles (P \u3e 0.05) but did affect eukaryotic members (uPVC, P \u3c 0.01; Cu, P = 0.001). Thus, established DW biofilms host complex communities that may vary based on substratum matrix and maintain consistent bacterial communities despite introduction of Lp, an environmental pathogen

Comparative Study on the Performance of Anaerobic and Aerobic Biotrickling Filter for Removal of Chloroform

Use of biotrickling filter (BTF) for gas phase treatment of volatile trihalomethanes (THMs) stripped from water treatment plants could be an attractive treatment option. The aim of this study is to use laboratory-scale anaerobic BTF to treat gaseous chloroform (recalcitrant to biological transformation) as a model THM and compare results with aerobic BTF. Additional investigations were conducted to determine the microbial diversity present within the BTFs. Chloroform is a hydrophobic volatile THM known to be difficult to biodegrade. To improve the degradation process, ethanol was used as a cometabolite at a different ratio to chloroform. The experimental plan was designed to operate one BTF under anaerobic condition and the other one under aerobic acidic condition. Higher elimination capacity (EC) of 0.23 ± 0.01 g/[m3·h] was observed with a removal efficiency of 80.9% ± 4% for the aerobic BTF operating at pH 4 for the concentration ratio of 1:40 chloroform to ethanol. For similar ratio, the anaerobic BTF supported lower removal efficiency of 59% ± 10% with corresponding lower EC of 0.16 ± 0.01 g/[m3·h]. Carbon recovery acquired for anaerobic and aerobic BTFs was 59% and 63%, respectively. The loading rate for chloroform on both BTFs was 0.27 g/[m3·h] (per m3 of filter bed volume). Variations of the microbial community were attributed to degradation of chloroform in each BTF. Azospira oryzae and Azospira restrica were the dominant bacteria and potential candidates for chloroform degradation for the anaerobic BTF, whereas Fusarium sp. and Fusarium solani were the dominant fungi and potential candidates for chloroform degradation in the aerobic BTF

The Trait Repertoire Enabling Cyanobacteria to Bloom Assessed through Comparative Genomic Complexity and Metatranscriptomics

Water bloom development due to eutrophication constitutes a case of niche specialization among planktonic cyanobacteria, but the genomic repertoire allowing bloom formation in only some species has not been fully characterized. We posited that the habitat relevance of a trait begets its underlying genomic complexity, so that traits within the repertoire would be differentially more complex in species successfully thriving in that habitat than in close species that cannot. To test this for the case of bloom-forming cyanobacteria, we curated 17 potentially relevant query metabolic pathways and five core pathways selected according to existing ecophysiological literature. The available 113 genomes were split into those of blooming (45) or nonblooming (68) strains, and an index of genomic complexity for each strain’s version of each pathway was derived. We show that strain versions of all query pathways were significantly more complex in bloomers, with complexity in fact correlating positively with strain blooming incidence in 14 of those pathways. Five core pathways, relevant everywhere, showed no differential complexity or correlations. Gas vesicle, toxin and fatty acid synthesis, amino acid uptake, and C, N, and S acquisition systems were most strikingly relevant in the blooming repertoire. Further, we validated our findings using metagenomic gene expression analyses of blooming and non- blooming cyanobacteria in natural settings, where pathways in the repertoire were differentially overexpressed according to their relative complexity in bloomers, but not in nonbloomers. We expect that this approach may find applications to other habitats and organismal groups

Antibiotic Resistance of E. coli Isolated From a Constructed Wetland Dominated by a Crow Roost, With Emphasis on ESBL and AmpC Containing E. coli

Information on the dissemination of antibiotic resistance mechanisms in the environment as well as wild life is needed in North America. A constructed wetland (where ∼15,000 American crows roost) was sampled on the University of Washington Bothell Campus for the presence of antibiotic resistant E. coli (ARE). Crow droppings from individual birds and grab samples of water were collected in 2014–2015. E. coli were isolated by selective agar plating. The most frequent antibiotic resistance (AR) of the fecal isolates was to ampicillin (AMP) (53%), followed by amoxicillin-clavulanic acid (AMC) (45%), streptomycin (S) (40%), and nalidixic acid (NA) (33%). Water isolates had similar AR pattern and ∼40% were multidrug resistant. Isolates from water samples collected during storm events showed higher resistance than isolates from no rain days to tetracycline, AMP, AMC, NA, and gentamycin. Extended spectrum beta lactamase (ESBL) containing E. coli with the blactx-M was found in three water and nine fecal isolates while blacmy-2 in 19 water and 16 fecal isolates. Multilocus Sequence Typing analysis (MLST) yielded 13 and 12 different sequence types (STs) amongst fecal and water isolates, many of which could be correlated to livestock, bird, and humans. MLST identified ESBL E. coli belonging to the clinically relevant ST131 clone in six fecal and one water isolate. Three STs found in feces could be found in water on the same dates of collection but not subsequently. Thus, the strains do not appear to survive for long in the wetland. Phylogenetic analysis revealed similar distribution of the water and fecal isolates among the different phylo-groups, with the majority belonging to the commensal B1 phylo-group, followed by the pathogenic B2 phylo-group. This study demonstrates that corvids can be reservoirs and vectors of ARE and pathogenic E. coli, posing a significant environmental threat

Development and evaluation of a quantitative PCR assay targeting sandhill crane (Grus canadensis) fecal pollution

While the microbial water quality in the Platte River is seasonally impacted by excreta from migrating cranes, there are no methods available to study crane fecal contamination. Here we characterized microbial populations in crane feces using phylogenetic analysis of 16S rRNA gene fecal clone libraries. Using these sequences, a novel crane quantitative PCR (Crane1) assay was developed, and its applicability as a microbial source tracking (MST) assay was evaluated by determining its host specificity and detection ability in environmental waters. Bacteria from crane excreta were dominated by bacilli and proteobacteria, with a notable paucity of sequences homologous to Bacteroidetes and Clostridia. The Crane1 marker targeted a dominant clade of unclassified Lactobacillales sequences closely related to Catellicoccus marimammalium. The host distribution of the Crane1 marker was relatively high, being positive for 69% (66/96) of the crane excreta samples tested. The assay also showed high host specificity, with 95% of the nontarget fecal samples (i.e., n=553; 20 different free-range hosts) being negative. Of the presumed crane-impacted water samples (n=16), 88% were positive for the Crane1 assay, whereas none of the water samples not impacted by cranes were positive (n=165). Bayesian statistical models of the Crane1 MST marker demonstrated high confidence in detecting true-positive signals and a low probability of false-negative signals from environmental water samples. Altogether, these data suggest that the newly developed marker could be used in environmental monitoring studies to study crane fecal pollution dynamics.Peer reviewedBiosystems and Agricultural Engineerin

Molecular detection of Campylobacter spp. and fecal indicator bacteria during the northern migration of sandhill cranes (Grus canadensis) at the central Platte River

The risk to human health of the annual sandhill crane (Grus canadensis) migration through Nebraska, which is thought to be a major source of fecal pollution of the central Platte River, is unknown. To better understand potential risks, the presence of Campylobacter species and three fecal indicator bacterial groups (Enterococcus spp., Escherichia coli, and Bacteroidetes) was assayed by PCR from crane excreta and water samples collected during their stopover at the Platte River, Nebraska, in 2010. Genus-specific PCR assays and sequence analyses identified Campylobacter jejuni as the predominant Campylobacter species in sandhill crane excreta. Campylobacter spp. were detected in 48% of crane excreta, 24% of water samples, and 11% of sediment samples. The estimated densities of Enterococcus spp. were highest in excreta samples (mean, 4.6 x 10^8 cell equivalents [CE]/g), while water samples contained higher levels of Bacteroidetes (mean, 5.1 x 10^5 CE/100 ml). Enterococcus spp., E. coli, and Campylobacter spp. were significantly increased in river water and sediments during the crane migration period, with Enterococcus sp. densities (~3.3 x 10^5 CE/g) 2 to 4 orders of magnitude higher than those of Bacteroidetes (4.9 x 10^3 CE/g), E. coli (2.2 x 10^3 CE/g), and Campylobacter spp. (37 CE/g). Sequencing data for the 16S rRNA gene and Campylobacter species-specific PCR assays indicated that C. jejuni was the major Campylobacter species present in water, sediments, and crane excreta. Overall, migration appeared to result in a significant, but temporary, change in water quality in spring, when there may be a C. jejuni health hazard associated with water and crops visited by the migrating birds.Peer reviewedBiosystems and Agricultural Engineerin

Identification of chicken-specific fecal microbial sequences using a metagenomic approach

In this study, we applied a genome fragment enrichment (GFE) method to select for genomic regions that differ among different fecal metagenomes. Competitive DNA hybridizations were performed between chicken fecal DNA and pig fecal DNA (CP) and between chicken fecal DNA and an avian DNA composite consisting of turkey, goose, and seagull fecal DNA extracts (CB) to enrich for chicken-specific DNA fragments. A total of 471 non-redundant chicken metagenomic sequences were retrieved and analyzed. All of the clone sequences were similar to prokaryotic genes, of which more than 60% could not be assigned to previously characterized functional roles. In general terms, sequences assigned characterized functional roles were associated with cellular processes (11.7%), metabolism (11.0%) and information storage and processing (13.4%). Approximately 53% of the nonredundant sequences are similar to genes present in intestinal bacteria belonging to Clostridia (20.9%), Bacteroidetes (15.0%), and Bacilli (17.3%). Twenty-five sequences from the CP and CB clone libraries were selected to develop chicken fecal-specific PCR assays. These assays were challenged against fecal DNA extracted from 21 different animal species, including mammals and birds. The results from the host-specificity studies showed that 12 of the assays had a high degree of specificity to chicken feces. In addition, three assays were specific to chicken and turkey while another four assays tested positive to more than two avian species, suggesting a broader distribution of some of the enriched gene fragments among different avian fecal microbial communities. Fecal pollution signals were detected using chicken-specific assays in contaminated water samples, although the PCR assays showed different detection limits. These results indicate the need for multiple assays to detect poultry fecal sources of pollution. The competitive DNA hybridization approach used in this study can rapidly select for numerous chicken fecal metagenomic regions that can be used as potential genetic markers for fecal source tracking