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.

Community Engaged Cumulative Risk Assessment of Exposure to Inorganic Well Water Contaminants, Crow Reservation, Montana.

An estimated 11 million people in the US have home wells with unsafe levels of hazardous metals and nitrate. The national scope of the health risk from consuming this water has not been assessed as home wells are largely unregulated and data on well water treatment and consumption are lacking. Here, we assessed health risks from consumption of contaminated well water on the Crow Reservation by conducting a community-engaged, cumulative risk assessment. Well water testing, surveys and interviews were used to collect data on contaminant concentrations, water treatment methods, well water consumption, and well and septic system protection and maintenance practices. Additive Hazard Index calculations show that the water in more than 39% of wells is unsafe due to uranium, manganese, nitrate, zinc and/or arsenic. Most families' financial resources are limited, and 95% of participants do not employ water treatment technologies. Despite widespread high total dissolved solids, poor taste and odor, 80% of families consume their well water. Lack of environmental health literacy about well water safety, pre-existing health conditions and limited environmental enforcement also contribute to vulnerability. Ensuring access to safe drinking water and providing accompanying education are urgent public health priorities for Crow and other rural US families with low environmental health literacy and limited financial resources.

Detection of Pathogenic and Non-pathogenic Bacteria in Drinking Water and Associated Biofilms on the Crow Reservation, Montana, USA.

Private residences in rural areas with water systems that are not adequately regulated, monitored, and updated could have drinking water that poses a health risk. To investigate water quality on the Crow Reservation in Montana, water and biofilm samples were collected from 57 public buildings and private residences served by either treated municipal or individual groundwater well systems. Bacteriological quality was assessed including detection of fecal coliform bacteria and heterotrophic plate count (HPC) as well as three potentially pathogenic bacterial genera, Mycobacterium, Legionella, and Helicobacter. All three target genera were detected in drinking water systems on the Crow Reservation. Species detected included the opportunistic and frank pathogens Mycobacterium avium, Mycobacterium gordonae, Mycobacterium flavescens, Legionella pneumophila, and Helicobacter pylori. Additionally, there was an association between HPC bacteria and the presence of Mycobacterium and Legionella but not the presence of Helicobacter. This research has shown that groundwater and municipal drinking water systems on the Crow Reservation can harbor potential bacterial pathogens.

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.

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.

Increase in antimicrobial resistance in bacteria isolated from stranded marine mammals of the Northwest Atlantic.

Studies on marine mammals can inform our understanding of the environmental health of the ocean. To evaluate the potential for changes in antimicrobial resistance, we analyzed a database spanning 2004-2010 that consisted of bacterial isolate identity and antimicrobial sensitivity for stranded pinnipeds in the Northwest Atlantic. Samples (n = 170) from treated animals yielded 310 bacterial isolates representing 24 taxa. We evaluated changes in antimicrobial class resistance from 2004 to 2010 for eight taxa. Escherichia coli displayed a significant increase in resistance to several antimicrobial classes. Other taxa displayed significant increases in resistance to aminoglycosides, and/or fluoroquinolones. In addition, we observed a significant increase in multiple antimicrobial resistance in cultures from untreated animals. These results demonstrate an increase in resistance among common bacterial pathogens of marine mammals over a time span of 6 years.

Optimizing the growth of stressed Helicobacter pylori.

Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach and is responsible for causing gastric ulcers. H. pylori is known to become stressed and nonculturable after exposure to unfavorable conditions. In this study, we enhanced previously published resuscitation procedures, characterized conditions under which stressed H. pylori can be recovered, and formulated a selective and differential resuscitation medium. Results showed that a specialized broth supplemented with trace minerals and lysed human erythrocytes and serum is required for the recovery of nonculturable H. pylori. The type of stress was an important factor in the efficacy of resuscitation, with cells exposed to atmospheric oxygen more readily resuscitated than nutrient deprived cells. After resuscitation, culturable cells were recovered from previously nonculturable oxygen stressed cells (24 and 72 h of exposure) and nonculturable nutrient deprived cells (24 h of exposure). The length of time the cells were exposed to the stress was also an important factor in the recovery of stressed H. pylori. RNA levels were quantified and transcription of the cell division related gene, cdrA (HP0066), was assessed by qRT-PCR. The low levels of RNA detected in stressed cells, after resuscitation, support the idea that a small population of viable cells may be responsible for the colonies recovered on solid agar. The modification of the resuscitation broth into a selective and differential slant culture medium also allowed the recovery of stressed H. pylori. The methods presented here highlight the benefits and limitations of using human blood products for recovering nonculturable H. pylori.

Temporal bacterial diversity associated with metal-contaminated river sediments.

The temporal activity, abundance and diversity of microbial communities were evaluated across a metal-contamination gradient around a Superfund site in Montana. In order to analyze short-term variability, samples were collected from six sites on four occasions over 12 months. Measurements of community activity, diversity and richness, quantified by dehydrogenase activity and through denaturant gradient gel electrophoresis (DGGE), respectively, were higher at contaminated sites adjacent to the smelter, relative to reference sites. 16S rRNA gene copy numbers, measured by quantitative PCR, showed seasonal variability, yet were generally higher within polluted sediments. Jaccard similarity coefficients of DGGE profiles, found sites to cluster based primarily on geographical proximity rather than geochemical similarities. Intra-site clustering of the most polluted sites also suggests a stable metal-tolerant community. Sequences from DGGE-extracted bands were predominantly Beta and Gammaproteobacteria, although the communities at all sites generally maintained a diverse phylogeny changing in composition throughout the sampling period. Spearman's rank correlations analysis found statistically significant relationships between community composition and organic carbon (r-value = 0.786) and metals (r-values As = 0.65; Cu = 0.63; Zn = 0.62). A diverse and abundant community at the most polluted site indicates that historical contamination selects for a metal-resistant microbial community, a finding that must be accounted for when using the microbial community within ecosystem monitoring studies. This study highlights the importance of using multiple time-points to draw conclusions on the affect of metal contamination.

Using satellite images of environmental changes to predict infectious disease outbreaks.

Recent events clearly illustrate a continued vulnerability of large populations to infectious diseases, which is related to our changing human-constructed and natural environments. A single person with multidrug-resistant tuberculosis in 2007 provided a wake-up call to the United States and global public health infrastructure, as the health professionals and the public realized that today's ease of airline travel can potentially expose hundreds of persons to an untreatable disease associated with an infectious agent. Ease of travel, population increase, population displacement, pollution, agricultural activity, changing socioeconomic structures, and international conflicts worldwide have each contributed to infectious disease events. Today, however, nothing is larger in scale, has more potential for long-term effects, and is more uncertain than the effects of climate change on infectious disease outbreaks, epidemics, and pandemics. We discuss advances in our ability to predict these events and, in particular, the critical role that satellite imaging could play in mounting an effective response.

Quantifying nonspecific TEM beta-lactamase (blaTEM) genes in a wastewater stream.

To control the antibiotic resistance epidemic, it is necessary to understand the distribution of genetic material encoding antibiotic resistance in the environment and how anthropogenic inputs, such as wastewater, affect this distribution. Approximately two-thirds of antibiotics administered to humans are beta-lactams, for which the predominant bacterial resistance mechanism is hydrolysis by beta-lactamases. Of the beta-lactamases, the TEM family is of overriding significance with regard to diversity, prevalence, and distribution. This paper describes the design of DNA probes universal for all known TEM beta-lactamase genes and the application of a quantitative PCR assay (also known as Taqman) to quantify these genes in environmental samples. The primer set was used to study whether sewage, both treated and untreated, contributes to the spread of these genes in receiving waters. It was found that while modern sewage treatment technologies reduce the concentrations of these antibiotic resistance genes, the ratio of bla(TEM) genes to 16S rRNA genes increases with treatment, suggesting that bacteria harboring bla(TEM) are more likely to survive the treatment process. Thus, beta-lactamase genes are being introduced into the environment in significantly higher concentrations than occur naturally, creating reservoirs of increased resistance potential.

Autoinducer-2 triggers the oxidative stress response in Mycobacterium avium, leading to biofilm formation.

Mycobacterium avium is an environmental organism and opportunistic pathogen with inherent resistance to drugs, environmental stresses, and the host immune response. To adapt to these disparate conditions, M. avium must control its transcriptional response to environmental cues. M. avium forms biofilms in various environmental settings, including drinking water pipes and potable water reservoirs. In this study, we investigated the role of the universal signaling molecule autoinducer-2 (AI-2) in biofilm formation by M. avium. The addition of the compound to planktonic M. avium cultures resulted in increased biofilm formation. Microarray and reverse transcriptase PCR studies revealed an upregulation of the oxidative stress response upon addition of AI-2. This suggests that the response to AI-2 might be related to oxidative stress, rather than quorum sensing. Consistent with this model, addition of hydrogen peroxide, a known stimulus of the oxidative stress response, to M. avium cultures resulted in elevated biofilm formation. These results suggest that AI-2 does not act as a quorum-sensing signal in M. avium. Instead, biofilm formation is triggered by environmental stresses of biotic and abiotic origins and AI-2 may exert effects on that level.

Quantification of changing Pseudomonas aeruginosa sodA, htpX and mt gene abundance in response to trace metal toxicity: a potential in situ biomarker of environmental health.

Sediment-dwelling prokaryotes play a vital role in determining the fate and speciation of metals, yet are also susceptible to the biological effects of trace metals. In this article, optimized DNA extraction and purification techniques and species-specific primers are used to assess the genetic incidence and abundance of metal detoxification and general stress genes of Pseudomonas aeruginosa to complement chemical analysis in inferring the severity of metal-contaminated sites along the Clark Fork River, Montana. Results show the highest incidence of candidate genes related to bacterial stress at the most polluted site, while multiple regression analysis demonstrated significant correlations (P<0.05, r(2)=0.9) between in situ metal concentrations (As, Cu and Zn), total gene incidence, and the incidence of metal detoxification genes. Furthermore, principal components plotting the incidence of genes related to metal resistance show clear separation of sites giving clear clusters on the basis of contamination. Quantification of three genes (sodA, htpX and mt) from surveyed sites found significantly higher (anova, P<0.05) copy numbers at the more contaminated sites compared with reference sites. The development of rapid microbial biomarker tools represents a significant advance in the field of environmental biomonitoring and the prediction of metal bioavailability.

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.

Roles for cell wall glycopeptidolipid in surface adherence and planktonic dispersal of Mycobacterium avium.

The opportunistic pathogen Mycobacterium avium is a significant inhabitant of biofilms in drinking water distribution systems. M. avium expresses on its cell surface serovar-specific glycopeptidolipids (ssGPLs). Studies have implicated the core GPL in biofilm formation by M. avium and by other Mycobacterium species. In order to test this hypothesis in a directed fashion, three model systems were used to examine biofilm formation by mutants of M. avium with transposon insertions into pstAB (also known as nrp and mps). pstAB encodes the nonribosomal peptide synthetase that catalyzes the synthesis of the core GPL. The mutants did not adhere to polyvinyl chloride plates; however, they adhered well to plastic and glass chamber slide surfaces, albeit with different morphologies from the parent strain. In a model that quantified surface adherence under recirculating water, wild-type and pstAB mutant cells accumulated on stainless steel surfaces in equal numbers. Unexpectedly, pstAB mutant cells were >10-fold less abundant in the recirculating-water phase than parent strain cells. These observations show that GPLs are directly or indirectly required for colonization of some, but by no means all, surfaces. Under some conditions, GPLs may play an entirely different role by facilitating the survival or dispersal of nonadherent M. avium cells in circulating water. Such a function could contribute to waterborne M. avium infection.

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.

Body weight and water ingestion estimates for women in two communities in the Philippines: the importance of collecting site-specific data.

In this paper, we report data on exposure factors relevant to assessing health risks of contaminant exposures to women of childbearing age (age 15-49) in two communities in the Philippines. We collected exposure factor data through an interview survey of 182 women conducted between January and May 2002 and we present distributions of self-reported body weight and water ingestion rates. A simple comparison of our results to those from large studies of US women suggests that these small subpopulations may both weigh less and consume more water than might be expected based on the extensive national US data, and it suggests that exposure analysts focused on a particular geographic area should consider the value of obtaining site-specific data to characterize exposure and risk. The lack of a comparable large study of water ingestion rates and body weights of Filipino women makes it difficult to determine whether the communities studied are typical of the population of the Philippines or represent unique subgroups. We suggest that Philippine health authorities consider the possibility of including questions about water ingestion rates in future national health or nutrition surveys.

Concentrations of lead and mercury in multimedia samples from homes near the former Clark Air Base, Philippines.

We measured lead and mercury in samples collected from 31 homes in communities near the former Clark Air Base, Philippines during May and October 2002. Sample media included water used for drinking and cooking, house dust and entryway soil. Composite samples of 15 food items purchased at local markets were also collected. Samples were analyzed for total lead (Pb) and total mercury (Hg) to evaluate the relative importance of each media to residential exposure concentrations in the community adjacent to Clark (Community A) versus a control community 5 km away (Community B). In general, we measured low (e.g. background) to undetectable levels of the target analytes in all media sampled with two important exceptions. First, the Hg concentrations we measured in canned mackerel composites, which were within the range reported for mackerel from other locations worldwide, may pose a risk to pregnant women who are frequent consumers (e.g. one or more cans per day). Second, we measured Pb above the USEPA residential screening concentration (400 mug/g) in dust and soil from two homes, illustrating the need for periodic residential lead monitoring in these and other communities in the Philippines. We found no significant difference between Communities A and B with respect to Pb and Hg concentrations in water or food, although we were not able to detect very low levels of Pb in most of the foods we sampled because of trace Pb contamination added during sample homogenization. Although the Pb levels we measured in dust and soil from Community A homes were higher on average than Community B homes, the levels in both communities were low (e.g. background) thus we did not investigate the difference further. To our knowledge, these are the first reported measurements of Pb in house dust in the Philippines. The concentrations of Pb we measured in house dust were significantly higher than those in entryway soil from both communities, adding empirical support to the assertion that yard soil should not be considered a proxy for house dust in exposure studies in the Philippines or elsewhere.