Nanopore-based metagenomics analysis reveals prevalence of mobile antibiotic and heavy metal resistome in wastewater.

In-depth studies of the microbiome and mobile resistome profile of different environments is central to understanding the role of the environment in antimicrobial resistance (AMR), which is one of the urgent threats to global public health. In this study, we demonstrated the use of a rapid (and easily portable) sequencing approach coupled with user-friendly bioinformatics tools, the MinION (Oxford Nanopore Technologies), on the evaluation of the microbial as well as mobile metal and antibiotic resistome profile of semi-rural wastewater. A total of 20 unique phyla, 43 classes, 227 genera, and 469 species were identified in samples collected from the Amherst Wastewater Treatment Plant, both from primary and secondary treated wastewater. Alpha diversity indices indicated that primary samples were significantly richer and more microbially diverse than secondary samples. A total of 1041 ARGs, 68 MRGs, and 17 MGEs were detected in this study. There were more classes of AMR genes in primary than secondary wastewater, but in both cases multidrug, beta-lactam and peptide AMR predominated. Of note, OXA ß-lactamases, some of which are also carbapenemases, were enriched in secondary samples. Metal resistance genes against arsenic, copper, zinc and molybdenum were the dominant MRGs in the majority of the samples. A larger proportion of resistome genes were located in chromosome-derived sequences except for mobilome genes, which were predominantly located in plasmid-derived sequences. Genetic elements related to transposase were the most common MGEs in all samples. Mobile or MGE/plasmid-associated resistome genes that confer resistance to last resort antimicrobials such as carbapenems and colistin were detected in most samples. Worryingly, several of these potentially transferable genes were found to be carried by clinically-relevant hosts including pathogenic bacterial species in the orders Aeromonadales, Clostridiales, Enterobacterales and Pseudomonadales. This study demonstrated that the MinION can be used as a metagenomics approach to evaluate the microbiome, resistome, and mobilome profile of primary and secondary wastewater.

A Perspective on the Global Pandemic of Waterborne Disease.

Waterborne diseases continue to take a heavy toll on the global community, with developing nations, and particularly young children carrying most of the burden of morbidity and mortality. Starting with the historical context, this article explores some of the reasons why this burden continues today, despite our advances in public health over the past century or so. While molecular biology has revolutionized our abilities to define the ecosystems and etiologies of waterborne pathogens, control remains elusive. Lack of basic hygiene and sanitation, and failing infrastructure, remain two of the greatest challenges in the global fight against waterborne disease. Emerging risks continue to be the specter of multiple drug resistance and the ease with which determinants of virulence appear to be transmitted between strains of pathogens, both within and outside the human host.

Detection and source tracking of Escherichia coli, harboring intimin and Shiga toxin genes, isolated from the Little Bighorn River, Montana.

The Little Bighorn River flows through the Crow Indian Reservation in Montana. In 2008, Escherichia coli concentrations as high as 7179 MPN/100 ml were detected in the river at the Crow Agency Water Treatment Plant intake site. During 2008, 2009, and 2012, 10 different serotypes of E. coli, including O157:H7, harboring both intimin and Shiga toxin genes were isolated from a popular swim site of the Little Bighorn River in Crow Agency. As part of a microbial source tracking study, E. coli strains were isolated from river samples as well as from manure collected from a large cattle feeding operation in the upper Little Bighorn River watershed; 23% of 167 isolates of E. coli obtained from the manure tested positive for the intimin gene. Among these manure isolates, 19 were identified as O156:H8, matching the serotype of an isolate collected from a river sampling site close to the cattle feeding area.

Community-based participatory research in Indian country: improving health through water quality research and awareness.

Water has always been held in high respect by the Apsaálooke (Crow) people of Montana. Tribal members questioned the health of the rivers and well water because of visible water quality deterioration and potential connections to illnesses in the community. Community members initiated collaboration among local organizations, the tribe, and academic partners, resulting in genuine community-based participatory research. The article shares what we have learned as tribal members and researchers about working together to examine surface and groundwater contaminants, assess routes of exposure, and use our data to bring about improved health of our people and our waters.

Metagenomic Profiling of Microbial Pathogens in the Little Bighorn River, Montana.

The Little Bighorn River is the primary source of water for water treatment plants serving the local Crow Agency population, and has special significance in the spiritual and ceremonial life of the Crow tribe. Unfortunately, the watershed suffers from impaired water quality, with high counts of fecal coliform bacteria routinely measured during run-off events. A metagenomic analysis was carried out to identify potential pathogens in the river water. The Oxford Nanopore MinION platform was used to sequence DNA in near real time to identify both uncultured and a coliform-enriched culture of microbes collected from a popular summer swimming area of the Little Bighorn River. Sequences were analyzed using CosmosID bioinformatics and, in agreement with previous studies, enterohemorrhagic and enteropathogenic Escherichia coli and other E. coli pathotypes were identified. Noteworthy was detection and identification of enteroaggregative E. coli O104:H4 and Vibrio cholerae serotype O1 El Tor, however, cholera toxin genes were not identified. Other pathogenic microbes, as well as virulence genes and antimicrobial resistance markers, were also identified and characterized by metagenomic analyses. It is concluded that metagenomics provides a useful and potentially routine tool for identifying in an in-depth manner microbial contamination of waterways and, thereby, protecting public health.

Unexpected Prevalence of eae-Positive Escherichia coli in the Animas River, Durango, Colorado.

Since 2014, biology students at Fort Lewis College have studied the water quality of the Animas River in Durango, Colorado. Environmental microbiology and molecular biology techniques have been employed to study Escherichia coli isolates from the river and to define characteristics of the bacteria related to public health. E. coli was found in the river, as well as in culverts and tributary creeks that drain into the river within the Durango city limits. Concentrations of E. coli in the river occasionally exceeded the US EPA guideline of 126 CFU per 100 mL for recreational water use. Many of the E. coli isolates were able to be grown at 45 °C, an indication of mammalian origin. Unexpectedly, 8% of the isolates contained the intimin (eae) gene, a virulence gene characteristic of two pathotypes of E. coli, the enterohemorrhagic and enteropathogenic E. coli. Several isolates tested were resistant to multiple antibiotics commonly used in animal and human medicine. Further study is warranted to determine the source of these bacteria entering the Animas River, and to further characterize the possible disease potential of multi-antibiotic resistant and virulence gene-containing isolates found in a semi-rural/urban setting.

A Metagenomic Approach to Evaluating Surface Water Quality in Haiti.

The cholera epidemic that occurred in Haiti post-earthquake in 2010 has resulted in over 9000 deaths during the past eight years. Currently, morbidity and mortality rates for cholera have declined, but cholera cases still occur on a daily basis. One continuing issue is an inability to accurately predict and identify when cholera outbreaks might occur. To explore this surveillance gap, a metagenomic approach employing environmental samples was taken. In this study, surface water samples were collected at two time points from several sites near the original epicenter of the cholera outbreak in the Central Plateau of Haiti. These samples underwent whole genome sequencing and subsequent metagenomic analysis to characterize the microbial community of bacteria, fungi, protists, and viruses, and to identify antibiotic resistance and virulence associated genes. Replicates from sites were analyzed by principle components analysis, and distinct genomic profiles were obtained for each site. Cholera toxin converting phage was detected at one site, and Shiga toxin converting phages at several sites. Members of the Acinetobacter family were frequently detected in samples, including members implicated in waterborne diseases. These results indicate a metagenomic approach to evaluating water samples can be useful for source tracking and the surveillance of pathogens such as Vibrio cholerae over time, as well as for monitoring virulence factors such as cholera toxin.

Bile salts affect expression of Escherichia coli O157:H7 genes for virulence and iron acquisition, and promote growth under iron limiting conditions.

Bile salts exhibit potent antibacterial properties, acting as detergents to disrupt cell membranes and as DNA-damaging agents. Although bacteria inhabiting the intestinal tract are able to resist bile's antimicrobial effects, relatively little is known about how bile influences virulence of enteric pathogens. Escherichia coli O157:H7 is an important pathogen of humans, capable of causing severe diarrhea and more serious sequelae. In this study, the transcriptome response of E. coli O157:H7 to bile was determined. Bile exposure induced significant changes in mRNA levels of genes related to virulence potential, including a reduction of mRNA for the 41 genes making up the locus of enterocyte effacement (LEE) pathogenicity island. Bile treatment had an unusual effect on mRNA levels for the entire flagella-chemotaxis regulon, resulting in two- to four-fold increases in mRNA levels for genes associated with the flagella hook-basal body structure, but a two-fold decrease for "late" flagella genes associated with the flagella filament, stator motor, and chemotaxis. Bile salts also caused increased mRNA levels for seventeen genes associated with iron scavenging and metabolism, and counteracted the inhibitory effect of the iron chelating agent 2,2'-dipyridyl on growth of E. coli O157:H7. These findings suggest that E. coli O157:H7 may use bile as an environmental signal to adapt to changing conditions associated with the small intestine, including adaptation to an iron-scarce environment.

Isolation of potentially pathogenic Escherichia coli O157:H7 from the Ganges River.

Escherichia coli serotype O157:H7 was detected among bacteria collected from the Ganges River. O157:H7 isolates tested positive for stx(1), stx(2), and eae gene sequences. Identification of potentially pathogenic isolates from extensively used source water indicates that O157:H7 may be a significant but as yet underacknowledged public health concern in India.

The role of water use patterns and sewage pollution in incidence of water-borne/enteric diseases along the Ganges river in Varanasi, India.

In Varanasi, India, an estimated 200 million liters daily or more of untreated human sewage is discharged into the Ganges River. River water monitoring over the past 12 years has demonstrated faecal coliform counts up to 10(8) MPN (most probable number) per 100 ml and biological oxygen demand levels averaging over 40 mg/l in the most polluted part of the river in Varanasi. A questionnaire-based survey was used to estimate water-borne and enteric disease incidence and study river use among resident users of the Ganges River in Varanasi. The overall rate of water-borne/enteric disease incidence, including acute gastrointestinal disease, cholera, dysentery, hepatitis-A, and typhoid, was estimated to be about 66% during the one-year period prior to the survey. Logistic regression analysis revealed significant associations between water-borne/enteric disease occurrence and the use of the river for bathing, laundry, washing eating utensils, and brushing teeth. Thirty-three cases of cholera were identified among families exposed to washing clothing or bathing in the Ganges while no cholera cases occurred in unexposed families. Other exposure factors such as lack of sewerage and toilets at residence, children defecating outdoors, poor sanitation, low income and low education levels also showed significant associations with enteric disease outcome. This study provides an estimate of water-borne/enteric disease incidence and identifies possible risk factors for residents who live by and use the Ganges River in Varanasi.