Development of a qPCR assay for the detection of naturalized wastewater E. coli strains.

We recently demonstrated the presence of naturalized populations of Escherichia coli in municipal sewage. We wanted to develop a quantitative polymerase chain reaction (qPCR) assay targeting the uspC-IS30-flhDC marker of naturalized wastewater E. coli and assess the prevalence of these naturalized strains in wastewater. The limit of detection for the qPCR assay was 3.0 × 10-8 ng of plasmid DNA template with 100% specificity. This strain was detected throughout the wastewater treatment process, including treated effluents. We evaluated the potential of this marker for detecting municipal sewage/wastewater contamination in water by comparing it to other human and animal markers of fecal pollution. Strong correlations were observed between the uspC-IS30-flhDC marker and the human fecal markers Bacteroides HF183 and HumM2, but not animal fecal markers, in surface and stormwater samples. The uspC-IS30-flhDC marker appears to be a potential E. coli-based marker for human wastewater contamination.

A review of the taxonomy, genetics, and biology of the genus Escherichia and the type species Escherichia coli.

Historically, bacteriologists have relied heavily on biochemical and structural phenotypes for bacterial taxonomic classification. However, advances in comparative genomics have led to greater insights into the remarkable genetic diversity within the microbial world, and even within well-accepted species such as Escherichia coli. The extraordinary genetic diversity in E. coli recapitulates the evolutionary radiation of this species in exploiting a wide range of niches (i.e., ecotypes), including the gastrointestinal system of diverse vertebrate hosts as well as non-host natural environments (soil, natural waters, wastewater), which drives the adaptation, natural selection, and evolution of intragenotypic conspecific specialism as a strategy for survival. Over the last few years, there has been increasing evidence that many E. coli strains are very host (or niche)-specific. While biochemical and phylogenetic evidence support the classification of E. coli as a distinct species, the vast genomic (diverse pan-genome and intragenotypic variability), phenotypic (e.g., metabolic pathways), and ecotypic (host-/niche-specificity) diversity, comparable to the diversity observed in known species complexes, suggest that E. coli is better represented as a complex. Herein we review the taxonomic classification of the genus Escherichia and discuss how phenotype, genotype, and ecotype recapitulate our understanding of the biology of this remarkable bacterium.

Role of amoebae for survival and recovery of 'non-culturable' Helicobacter pylori cells in aquatic environments.

Helicobacter pylori is a fastidious Gram-negative bacterium that infects over half of the world's population, causing chronic gastritis and is a risk factor for stomach cancer. In developing and rural regions where prevalence rate exceeds 60%, persistence and waterborne transmission are often linked to poor sanitation conditions. Here we demonstrate that H. pylori not only survives but also replicates within acidified free-living amoebal phagosomes. Bacterial counts of the clinical isolate H. pylori G27 increased over 50-fold after three days in co-culture with amoebae. In contrast, a H. pylori mutant deficient in a cagPAI gene (cagE) showed little growth within amoebae, demonstrating the likely importance of a type IV secretion system in H. pylori for amoebal infection. We also demonstrate that H. pylori can be packaged by amoebae and released in extracellular vesicles. Furthermore, and for the first time, we successfully demonstrate the ability of two free-living amoebae to revert and recover viable but non-cultivable coccoid (VBNC)-H. pylori to a culturable state. Our studies provide evidence to support the hypothesis that amoebae and perhaps other free-living protozoa contribute to the replication and persistence of human-pathogenic H. pylori by providing a protected intracellular microenvironment for this pathogen to persist in natural aquatic environments and engineered water systems, thereby H. pylori potentially uses amoeba as a carrier and a vector of transmission.

Characterization of water treatment-resistant and multidrug-resistant urinary pathogenic Escherichia coli in treated wastewater.

A growing body of evidence has demonstrated that extraintestinal pathogenic E. coli (ExPEC), such as the urinary pathogenic E. coli (UPEC), are common constituents of treated wastewater, and therefore represent a potential public health risk. However, no single virulence gene, or set of virulence genes, can be used to conclusively identify this genetically diverse pathotype. As such we sought to identify and characterize the public health relevance of potential UPEC found in treated sewage/wastewater using a comparative genomics approach. Presumptive wastewater UPEC (W-UPEC) were initially identified by virulence gene screening against 5 virulence genes, and for which isolates containing ≥3 virulence genes were whole genome sequenced (n = 24). Single nucleotide polymorphic (SNP) spanning tree analysis demonstrated that many of these wastewater UPEC (WUPEC) were virtually identical at the core genome (0.4 Mbp) when compared to clinical UPEC (C-UPEC) sequences obtained from NCBI, varying by as little as 1 SNP. Remarkably, at the whole genome level, W-UPEC isolates displayed >96% whole genome similarity to C-UPEC counterparts in NCBI, with one strain demonstrating 99.5% genome similarity to a particular C-UPEC strain. The W-UPEC populations were represented by sequence types (ST) known to be clinically important, including ST131, ST95, ST127 and ST640. Many of the W-UPEC carried the exact same complement of virulence genes as their most closely related C-UPEC strains. For example, O25b-ST131 W-UPEC strains possessed the same 80 virulence genes as their most closely related C-UPEC counterparts. Concerningly, W-UPEC strains also carried a plethora of antibiotic resistance genes, and O25b-ST131strains were designated as extended spectrum beta-lactamase (ESBL) producing E. coli by both genome profiling and phenotypic resistance testing. W-UPEC ST131 strains were found in the effluents of a single treatment plant at different times, as well as different wastewater treatment plants, suggesting a differentially ability to survive wastewater treatment. Indeed, in sewage samples treated with chlorine doses sufficient for inducing a ∼99.99% reduction in total E. coli levels, UPEC represented a significant proportion of the chlorine-resistant population. By contrast, no Shiga toxin-producing E. coli were observed in these chlorinated sewage libraries. Our results suggest that clinically-relevant UPEC exist in treated wastewater effluents and that they appear to be specifically adapted to survive wastewater treatment processes.

Molecular characterization of Giardia spp. and Cryptosporidium spp. from dogs and coyotes in an urban landscape suggests infrequent occurrence of zoonotic genotypes.

Giardia spp. and Cryptosporidium spp. are common gastrointestinal parasites with the potential for zoonotic transmission. This study aimed to (1) determine the genotypes occurring in dogs and coyotes occupying a similar urban area; (2) determine if these hosts were infected with potentially zoonotic genotypes; (3) provide baseline molecular data. In August and September 2012, 860 dog owners living in neighborhoods bordering six urban parks in Calgary, Alberta, Canada, provided faecal samples from their dogs. From March 2012 through July 2013, 193 coyote faeces were also collected from five of six of the same parks. Direct immunofluorescence microscopy (DFA) indicated that Giardia spp. and Cryptosporidium spp. infected a total of 64 (7.4%) and 21 (2.4%) dogs, as well as 15 (7.8%) and three (1.6%) coyotes, respectively. Semi-nested, polymerase chain reactions targeting the 16S small-subunit ribosomal ribonucleic acid (SSU rRNA) and 18S SSU rRNA genes of Giardia spp. and Cryptosporidium spp., respectively, were conducted on samples that screened positive by DFA, and products were sequenced and genotyped. Dogs were infected with Giardia intestinalis canid-associated assemblages C (n = 14), D (n = 13), and Cryptosporidium canis (n = 3). Similarly, G. intestinalis assemblages C (n = 1), D (n = 1) and C. canis (n = 1), were detected in coyotes, as well as G. intestinalis assemblage A (n = 1) and Cryptosporidium vole genotype (n = 1). Dogs and coyotes were predominantly infected with host-specific genotypes and few potentially zoonotic genotypes, suggesting that they may not represent a significant risk for zoonotic transmission of these parasites in urban areas where these hosts are sympatric.

Evidence for the evolution, clonal expansion and global dissemination of water treatment-resistant naturalized strains of Escherichia coli in wastewater.

We previously demonstrated the existence of naturalized strains of E. coli in wastewater and herein perform an in-depth comparative whole genome analysis of these strains (n = 17). Fourteen of the Canadian E. coli strains, isolated from geographically separated wastewater treatment plants, were virtually identical at the core genome and were ≥96% similar at the whole genome level, suggesting clonal-relatedness among these isolates. Remarkably, these strains were shown to be extremely similar to the genome of an E. coli isolated from wastewater in Switzerland, suggesting a global distribution of these strains. The genomes of three other Canadian wastewater strains were more diverse but very similar to the genomes of E. coli isolates collected from U.S. wastewater samples. Based on maximum likelihood phylogenetic analysis, wastewater strains from Canada, the U.S. and Switzerland formed a clade separate from other known enteric phylogroups (i.e., A, B1, B2, D, E) and the cryptic clades. All Canadian, Swiss and U.S. wastewater strains possessed a common SNP biomarker pattern across their genomes, and a sub-population (i.e., 14 Canadian and 1 Swiss strain) also possessed a previously identified wastewater-specific marker known as uspC-IS30-flhDC element. Biochemical heat mapping of 518 categories of genes recapitulated phylogeny, with wastewater strains phenotypically clustering separately from enteric and cryptic clades. Wastewater strains were enriched for stress-response genes (i.e., nutrient acquisition/deprivation, DNA repair, oxidative stress, and UV resistance) - elements reflective of their environmental survival challenges. Wastewater strains were shown to carry a plethora of known antibiotic resistance (AR) genes, the patterns of which were remarkably similar among all Canadian, U.S. and Swiss wastewater strains. Virulence gene composition was also similar among all the wastewater strains, with an abundant representation of virulence genes commonly associated with urinary pathogenic E. coli (UPEC) as well as enterohemorrhagic (EHEC) E. coli. The remarkable degree of similarity between all wastewater strains from Canada, Switzerland and the U.S. suggests the evolution and global-dissemination of water treatment-resistant clone of E. coli. These finding, along with others, raise some important concerns about the potential for emergence of E. coli pathotypes resistant to water-treatment.

An Improved Culture Method for Selective Isolation of Campylobacter jejuni from Wastewater.

Campylobacter jejuni is one of the leading foodborne pathogens worldwide. C. jejuni is isolated from a wide range of foods, domestic animals, wildlife, and environmental sources. The currently available culture-based isolation methods are not highly effective for wastewater samples due to the low number of C. jejuni in the midst of competing bacteria. To detect and isolate C. jejuni from wastewater samples, in this study, we evaluated a few different enrichment conditions using five different antibiotics (i.e., cefoperazone, vancomycin, trimethoprim, polymyxin B, and rifampicin), to which C. jejuni is intrinsically resistant. The selectivity of each enrichment condition was measured with C t value using quantitative real-time PCR, and multiplex PCR to determine Campylobacter species. In addition, the efficacy of Campylobacter isolation on different culture media after selective enrichment was examined by growing on Bolton and Preston agar plates. The addition of polymyxin B, rifampicin, or both to the Bolton selective supplements enhanced the selective isolation of C. jejuni. The results of 16S rDNA sequencing also revealed that Enterococcus spp. and Pseudomonas aeruginosa are major competing bacteria in the enrichment conditions. Although it is known to be difficult to isolate Campylobacter from samples with heavy contamination, this study well exhibited that the manipulation of antibiotic selective pressure improves the isolation efficiency of fastidious Campylobacter from wastewater.

An evaluation of logic regression-based biomarker discovery across multiple intergenic regions for predicting host specificity in Escherichia coli.

Several studies have demonstrated that E. coli appears to display some level of host adaptation and specificity. Recent studies in our laboratory support these findings as determined by logic regression modeling of single nucleotide polymorphisms (SNP) in intergenic regions (ITGRs). We sought to determine the degree of host-specific information encoded in various ITGRs across a library of animal E. coli isolates using both whole genome analysis and a targeted ITGR sequencing approach. Our findings demonstrated that ITGRs across the genome encode various degrees of host-specific information. Incorporating multiple ITGRs (i.e., concatenation) into logic regression model building resulted in greater host-specificity and sensitivity outcomes in biomarkers, but the overall level of polymorphism in an ITGR did not correlate with the degree of host-specificity encoded in the ITGR. This suggests that distinct SNPs in ITGRs may be more important in defining host-specificity than overall sequence variation, explaining why traditional unsupervised learning phylogenetic approaches may be less informative in terms of revealing host-specific information encoded in DNA sequence. In silico analysis of 80 candidate ITGRs from publically available E. coli genomes was performed as a tool for discovering highly host-specific ITGRs. In one ITGR (ydeR-yedS) we identified a SNP biomarker that was 98% specific for cattle and for which 92% of all E. coli isolates originating from cattle carried this unique biomarker. In the case of humans, a host-specific biomarker (98% specificity) was identified in the concatenated ITGR sequences of rcsD-ompC, ydeR-yedS, and rclR-ykgE, and for which 78% of E. coli originating from humans carried this biomarker. Interestingly, human-specific biomarkers were dominant in ITGRs regulating antibiotic resistance, whereas in cattle host-specific biomarkers were found in ITGRs involved in stress regulation. These data suggest that evolution towards host specificity may be driven by different natural selection pressures on the regulome of E. coli among different animal hosts.

Evidence of Naturalized Stress-Tolerant Strains of Escherichia coli in Municipal Wastewater Treatment Plants.

UNLABELLED: Escherichia coli has been proposed to have two habitats-the intestines of mammals/birds and the nonhost environment. Our goal was to assess whether certain strains of E. coli have evolved toward adaptation and survival in wastewater. Raw sewage samples from different treatment plants were subjected to chlorine stress, and ∼59% of the surviving E. coli strains were found to contain a genetic insertion element (IS30) located within the uspC-flhDC intergenic region. The positional location of the IS30 element was not observed across a library of 845 E. coli isolates collected from various animal hosts or within GenBank or whole-genome reference databases for human and animal E. coli isolates (n = 1,177). Phylogenetics clustered the IS30 element-containing wastewater E. coli isolates into a distinct clade, and biomarker analysis revealed that these wastewater isolates contained a single nucleotide polymorphism (SNP) biomarker pattern that was specific for wastewater. These isolates belonged to phylogroup A, possessed generalized stress response (RpoS) activity, and carried the locus of heat resistance, features likely relevant to nonhost environmental survival. Isolates were screened for 28 virulence genes but carried only the fimH marker. Our data suggest that wastewater contains a naturalized resident population of E. coli We developed an endpoint PCR targeting the IS30 element within the uspC-flhDC intergenic region, and all raw sewage samples (n = 21) were positive for this marker. Conversely, the prevalence of this marker in E. coli-positive surface and groundwater samples was low (≤5%). This simple PCR assay may represent a convenient microbial source-tracking tool for identification of water samples affected by municipal wastewater. IMPORTANCE: The results of this study demonstrate that some strains of E. coli appear to have evolved to become naturalized populations in the wastewater environment and possess a number of stress-related genetic elements likely important for survival in this nonhost environment. The presence of non-host-adapted strains in wastewater challenges our understanding of using E. coli as a microbial indicator of wastewater treatment performance, suggesting that the E. coli strains present in human and animal feces may be very different from those found in treated wastewater.

Evaluation of Various Campylobacter-Specific Quantitative PCR (qPCR) Assays for Detection and Enumeration of Campylobacteraceae in Irrigation Water and Wastewater via a Miniaturized Most-Probable-Number-qPCR Assay.

UNLABELLED: Campylobacter spp. are the leading cause of bacterial gastroenteritis worldwide, and water is increasingly seen as a risk factor in transmission. Here we describe a most-probable-number (MPN)-quantitative PCR (qPCR) assay in which water samples are centrifuged and aliquoted into microtiter plates and the bacteria are enumerated by qPCR. We observed that commonly used Campylobacter molecular assays produced vastly different detection rates. In irrigation water samples, detection rates varied depending upon the PCR assay and culture method used, as follows: 0% by the de Boer Lv1-16S qPCR assay, 2.5% by the Van Dyke 16S and Jensen glyA qPCR assays, and 75% by the Linton 16S endpoint PCR when cultured at 37°C. Primer/probe specificity was the major confounder, with Arcobacter spp. routinely yielding false-positive results. The primers and PCR conditions described by Van Dyke et al. (M. I. Van Dyke, V. K. Morton, N. L. McLellan, and P. M. Huck, J Appl Microbiol 109:1053-1066, 2010, http://dx.doi.org/10.1111/j.1365-2672.2010.04730.x) proved to be the most sensitive and specific for Campylobacter detection in water. Campylobacter occurrence in irrigation water was found to be very low (<2 MPN/300 ml) when this Campylobacter-specific qPCR was used, with the most commonly detected species being C. jejuni, C. coli, and C. lari Campylobacters in raw sewage were present at ∼10(2)/100 ml, with incubation at 42°C required for reducing microbial growth competition from arcobacters. Overall, when Campylobacter prevalence and/or concentration in water is reported using molecular methods, considerable validation is recommended when adapting methods largely developed for clinical applications. Furthermore, combining MPN methods with molecular biology-based detection algorithms allows for the detection and quantification of Campylobacter spp. in environmental samples and is potentially suited to quantitative microbial risk assessment for improved public health disease prevention related to food and water exposures. IMPORTANCE: The results of this study demonstrate the importance of assay validation upon data interpretation of environmental monitoring for Campylobacter when using molecular biology-based assays. Previous studies describing Campylobacter prevalence in Canada utilized primers that we have determined to be nonspecific due to their cross-amplification of Arcobacter spp. As such, Campylobacter prevalence may have been vastly overestimated in other studies. Additionally, the development of a quantitative assay described in this study will allow accurate determination of Campylobacter concentrations in environmental water samples, allowing more informed decisions to be made about water usage based on quantitative microbial risk assessment.

Phage receptor binding protein-based magnetic enrichment method as an aid for real time PCR detection of foodborne bacteria.

We present a novel phage receptor binding protein-based magnetic separation and pre-enrichment method as an alternative to the immunomagnetic separation methods by replacing antibodies with bacteriophage receptor binding proteins (RBPs). We couple the proposed RBP-based magnetic separation with real time PCR for rapid, sensitive and specific detection of Campylobacter jejuni cells in artificially contaminated skim milk, milk with 2% fat and chicken broth. Recovery rates, assessed by real time PCR, were greater than 80% for the samples spiked with as low as 100 cfu mL(-1) of C. jejuni cells. The specificity of capture was confirmed using Salmonella Typhimurium as a negative control where no bacteria were captured on the RBP-derivatized magnetic beads. The combination of RBP-based magnetic separation and real time PCR improved PCR sensitivity and allowed the detection of C. jejuni cells in milk and chicken broth samples without a time consuming pre-enrichment step through culturing. The total sample preparation and analysis time in the proposed RBP-based enrichment method coupled with real time PCR was less than 3 h.

Characterization of the peroxide sensitivity of COX-deficient yeast strains reveals unexpected relationships between COX assembly proteins.

A number of distinct cuproproteins of the mitochondrial inner membrane are required for the assembly of cytochrome oxidase (COX), thought to function in a "bucket brigade" fashion to provide copper to the Cu(A) and Cu(B) sites. In yeast, the loss of two these proteins, Sco1p and Cox11p, leads to respiratory deficiency and a specific inability to survive exposure to hydrogen peroxide (H(2)O(2)). Using a quantitative assay, we have identified subtle differences in the peroxide-sensitive phenotypes between sco1 and cox11 mutant strains. Interestingly, the peroxide sensitivity of the sco1 null strain can be suppressed by overexpressing either SCO2 or COX11, although overexpression of neither SCO1 nor SCO2 can rescue the cox11 null strain. We also find that overexpression of either CTT1, encoding the cytosolic catalase T, or CTA1, encoding the mitochondrial matrix catalase, suppresses the peroxide sensitivity in both the sco1 and the cox11 null mutants. Direct measurement of peroxide metabolism shows that sco1 and cox11 null strains fail to degrade a significant amount of exogenously provided H(2)O(2). Taken together, our data demonstrate that although Cox11p and Sco1p play distinct roles in COX assembly, they seem to play overlapping or related roles in peroxide metabolism that require further investigation.

Mutational analysis of the Saccharomyces cerevisiae cytochrome c oxidase assembly protein Cox11p.

Cox11p is an integral protein of the inner mitochondrial membrane that is essential for cytochrome c oxidase assembly. The bulk of the protein is located in the intermembrane space and displays high levels of evolutionary conservation. We have analyzed a collection of site-directed and random cox11 mutants in an effort to further define essential portions of the molecule. Of the alleles studied, more than half had no apparent effect on Cox11p function. Among the respiration deficiency-encoding alleles, we identified three distinct phenotypes, which included a set of mutants with a misassembled or partially assembled cytochrome oxidase, as indicated by a blue-shifted cytochrome aa(3) peak. In addition to the shifted spectral signal, these mutants also display a specific reduction in the levels of subunit 1 (Cox1p). Two of these mutations are likely to occlude a surface pocket behind the copper-binding domain in Cox11p, based on analogy with the Sinorhizobium meliloti Cox11 solution structure, thereby suggesting that this pocket is crucial for Cox11p function. Sequential deletions of the matrix portion of Cox11p suggest that this domain is not functional beyond the residues involved in mitochondrial targeting and membrane insertion. In addition, our studies indicate that Deltacox11, like Deltasco1, displays a specific hypersensitivity to hydrogen peroxide. Our studies provide the first evidence at the level of the cytochrome oxidase holoenzyme that Cox1p is the in vivo target for Cox11p and suggest that Cox11p may also have a role in the response to hydrogen peroxide exposure.

Crystal structure of human SCO1: implications for redox signaling by a mitochondrial cytochrome c oxidase "assembly" protein.

Human SCO1 and SCO2 are copper-binding proteins involved in the assembly of mitochondrial cytochrome c oxidase (COX). We have determined the crystal structure of the conserved, intermembrane space core portion of apo-hSCO1 to 2.8 A. It is similar to redox active proteins, including thioredoxins (Trx) and peroxiredoxins (Prx), with putative copper-binding ligands located at the same positions as the conserved catalytic residues in Trx and Prx. SCO1 does not have disulfide isomerization or peroxidase activity, but both hSCO1 and a sco1 null in yeast show extreme sensitivity to hydrogen peroxide. Of the six missense mutations in SCO1 and SCO2 associated with fatal mitochondrial disorders, one lies in a highly conserved exposed surface away from the copper-binding region, suggesting that this region is involved in protein-protein interactions. These data suggests that SCO functions not as a COX copper chaperone, but rather as a mitochondrial redox signaling molecule.

CECR2, a protein involved in neurulation, forms a novel chromatin remodeling complex with SNF2L.

Chromatin remodeling complexes play critical roles in development. Here we describe a transcription factor, CECR2, which is involved in neurulation and chromatin remodeling. CECR2 shows complex alternative splicing, but all variants contain DDT and bromodomain motifs. A mutant mouse line was generated from an embryonic stem cell line containing a genetrap within Cecr2. Reporter gene expression demonstrated Cecr2 expression to be predominantly neural in the embryo. Mice homozygous for the Cecr2 genetrap-induced mutation show a high penetrance of the neural tube defect exencephaly, the human equivalent of anencephaly, in a strain-dependent fashion. Biochemical isolation of CECR2 revealed the presence of this protein as a component of a novel heterodimeric complex termed CECR2-containing remodeling factor (CERF). CERF comprises CECR2 and the ATP-dependent chromatin remodeler SNF2L, a mammalian ISWI ortholog expressed predominantly in the central nervous system. CERF is capable of remodeling chromatin in vitro and displays an ATP hydrolyzing activity that is stimulated by nucleosomes. Together, these data identify a novel chromatin remodeling complex with a critical role in neurulation.