Detecting waterborne illness pathogens with high precision

Waterborne pathogens and their survival strategies

When it comes to waterborne pathogens, bacteria like Vibrio cholerae, Salmonella enterica, Escherichia coli (E. coli) and Shigella spp. are among the most common culprits responsible for widespread diseases. These organisms have developed mechanisms to survive and thrive in both fresh and saltwater environments. Vibrio cholerae, for instance, uses quorum sensing to assess its surroundings and regulate the expression of virulence factors accordingly. This strategy allows the bacteria to survive in the absence of a suitable host and then become highly pathogenic when it makes the most impact.

Some bacteria produce toxins that can make us sick, even if the bacteria themselves are no longer present in significant numbers. For example, Clostridium botulinum can form resilient spores that survive in water until conditions are favorable for growth, at which point the bacteria produce the potent botulinum toxin one of the most toxic substances known to us. Similarly, E. coli O157, a pathogenic form of E. coli, produces Shiga toxin, which usually gets into our water systems via animal waste or agricultural runoff. Detecting whether bacteria carry the genes responsible for toxin production, as well as understanding the presence of spores, are important for assessing potential risks and addressing contamination effectively.

Viruses are also a problem. Norovirus, Hepatitis A and Enteroviruses are highly infectious viruses that can survive in water for long periods. Unlike bacteria, viruses need a host to be able to replicate. However, their transmission via water makes them a significant risk to public health. Take Norovirus, for example: it is viable in water for several weeks and is resistant to chlorine treatments. Plus, the small size of viral particles and their ability to form tough outer shells known as viral capsids lets them evade filtration systems and persist, even in treated water systems.

Transmission pathways and environmental survival

How do waterborne pathogens get into our water sources? One of the most significant pathways is contamination from animal feces, often via agricultural runoff. This runoff can carry pathogens like Salmonella, E. coli, Shigella and Campylobacter from animal waste into nearby rivers, lakes and aquifers. These pathogens are commonly transmitted to humans through the fecal-oral route, causing illness when we consume contaminated water. Infected animals also release zoonotic pathogens like Leptospira into water systems through their urine, compounding the problem. Improper waste disposal practices further exacerbate this issue, creating transmission hotspots where animal and human waste accumulate.

Biofilms further complicate the issue of waterborne illness. These complex microbial communities often form in water pipes, sediments and other aquatic environments, and they protect the organisms from antimicrobial agents and water treatment processes. For example, Legionella pneumophila – the bacteria that causes Legionnaires’ disease – colonizes and forms biofilms in plumbing and cooling systems. Vibrio cholerae also forms biofilms that allow it to survive in aquatic environments between outbreaks.

The ability of pathogens to survive prolonged periods in water, even in poor conditions, often gives them the upper hand. Robust survival mechanisms, like tolerance to high temperatures or pH or resistance to disinfectants, let the pathogens remain infectious for a long time after they enter a water source. For example, Vibrio vulnificus lives happily in warm, brackish water, and makes us sick when we are exposed to the water or eat contaminated shellfish from it. Understanding these mechanisms is a critical first step for improving our prevention and control strategies.

How water ecosystems harbor and sustain pathogens

In order to predict where and when waterborne disease outbreaks may occur, we need to understand how different pathogens interact with various water ecosystems. Freshwater rivers and lakes are natural reservoirs for bacteria like Campylobacter jejuni, Salmonella and Leptospira, who find their way there via stormwater runoff, animal waste and untreated sewage. Vibrio parahaemolyticus and Vibrio vulnificus, on the other hand, are more likely to occur in coastal marine environments with warm waters.

Other bacteria, such as Legionella and Mycobacterium marinum, prefer man-made water systems like plumbing and cooling systems or recreational bodies of water. Poor maintenance, along with the formation of biofilms, allows for sporadic disease outbreaks. Legionella is particularly tough to control because it tolerates low-nutrient conditions and resists common disinfectant treatments.

Climate change also influences the epidemiology of waterborne illnesses: higher temperatures and changing weather patterns produce ideal conditions for the growth of Vibrio species in coastal waterways. Furthermore, flooding and storm surges from extreme weather events spread fecal contamination and increase the transmission of pathogens like E. coli and Norovirus.

Campylobacter jejuni: These highly adaptable bacteria are able to survive in water and poultry products by forming biofilms or entering a dormant state. Once ingested, they use motility and surface proteins to penetrate the intestinal mucus layer, leading to infection. This results in gastroenteritis, with symptoms like abdominal pain and diarrhea due to inflammation and cell invasion.

Enteroviruses (including Human poliovirus 1, Enterovirus C): Enteroviruses, including poliovirus, are remarkably hardy in water and sewage, withstanding various environmental pressures. These viruses enter the body through the gastrointestinal tract, but they target and replicate in various tissues, including the nervous system. Depending on the virus, infections can range from mild respiratory illness to severe diseases like poliomyelitis, which can cause paralysis.

Escherichia coli (Pathogenic strains such as ETEC, EHEC and EPEC): Pathogenic E. coli strains survive in aquatic environments by adhering to surfaces and forming protective biofilms. Some strains produce potent toxins like Shiga toxin or enterotoxins, which disrupt cellular processes and cause fluid secretion. This leads to illnesses ranging from traveler’s diarrhea to more severe conditions like hemorrhagic colitis, depending on the strain.

Hepatoviruses (including Hepatitis A and E viruses): Hepatitis A and E viruses can persist in water and food, remaining viable for extended periods, particularly in unhygienic conditions. They are typically transmitted via the fecal-oral route and target the liver after an initial infection in the gut. The resulting hepatitis causes inflammation of the liver, leading to symptoms like jaundice, fatigue and abdominal pain.

Legionella spp. (including L. pneumophila): Legionella thrives in warm, aquatic environments like water systems and cooling towers, where it can resist chlorine and form biofilms for protection. It infects humans by invading lung macrophages, using a specialized secretion system to avoid immune destruction. This leads to Legionnaires' disease, a severe pneumonia, or Pontiac fever, a milder respiratory illness.

Leptospira spp.: These bacteria have adaptations that allow them to survive in both fresh and saltwater environments and can also persist in the kidneys of host animals. Infection typically occurs when contaminated water contacts broken skin or mucous membranes, allowing the bacteria to enter the bloodstream. Once inside, Leptospira can cause leptospirosis, a systemic illness that ranges from mild flu-like symptoms to severe liver and kidney damage.

Mycobacterium marinum: These bacteria are highly adapted to survive in water, especially in warm, slow-moving bodies like aquariums and natural waters. They invade the skin through abrasions or wounds and uses its thick, waxy cell wall to resist immune attacks. In humans, it typically causes skin infections that can become chronic if untreated.

Norovirus and Norwalk virus (including Norovirus GI and GII): Noroviruses are exceptionally stable in water and can remain infectious for long periods, even in treated water. Once ingested, they infect the cells lining the intestine, hijacking the cellular machinery to replicate rapidly. This results in acute gastroenteritis, marked by severe vomiting and diarrhea, which helps spread the virus to new hosts.

Polyomaviruses (including Betapolyomavirus hominis and JC polyomavirus): Polyomaviruses are small, resilient viruses that can survive in water or persist in the kidneys of hosts. They usually remain dormant in the body but can reactivate in immunocompromised individuals, leading to severe complications. JC virus, for instance, can cause a progressive brain infection called PML (progressive multifocal leukoencephalopathy) in such cases.

Salmonella spp. (including S. enterica serovars Typhi and Typhimurium): Salmonella thrives in various environments, including water, by forming resilient biofilms that protect it from harsh conditions. It cleverly manipulates host cells using a type III secretion system, injecting proteins that facilitate its invasion and help it evade the immune system. Once established, it wreaks havoc in the intestines, causing symptoms ranging from gastroenteritis to severe typhoid fever through cell damage and inflammation.

Shigella spp. (including S. dysenteriae, S. flexneri, S. sonnei): Shigella can persist in water, especially when contaminated, and remains highly infectious even at very low doses. It uses specialized proteins, called invasion plasmid antigens (Ipas), to bypass many of the host's defenses and directly invade the intestinal lining. This invasion results in severe dysentery, characterized by damage to the colon's epithelial cells and intense inflammation.

Vibrio spp. (including V. cholerae, V. alginolyticus, V. parahaemolyticus, V. vulnificus): Vibrio species can flourish in marine and estuarine environments, often associating with shellfish, which helps them persist in those environments. These bacteria employ toxins, such as cholera toxin, which disrupt cellular ion transport and lead to profuse watery diarrhea or, in more severe cases, sepsis. Infection typically occurs after consuming contaminated water or seafood, causing gastrointestinal or systemic infections.

Detection challenges in low-resource areas

Detecting waterborne pathogens in areas with low resources or in emergency situations is challenging. Culturing bacteria in the laboratory requires skilled workers, controlled settings and considerable time. This can result in a significant delay between the sampling phase and the availability of the results. These critical gaps can allow diseases like cholera and typhoid fever to escalate quickly.

The challenge is further amplified by erratic electricity supplies, lack of sterile laboratory settings and limited access to necessary reagents and equipment. Without timely detection, outbreaks can rapidly affect large portions of the population. That is why affordable, portable and effective real-time detection tools are critical in resource-limited settings.

Complexity of pathogen detection in diverse water environments

When detecting waterborne pathogens, the diversity of water environments also presents a unique challenge. From freshwater rivers and streams to coastal estuaries, each environment hosts a unique community of microbes, which behave differently depending on the water temperature, chemistry and presence of organic material. For example, detecting E. coli and Salmonella in fresh water is technically less complicated than identifying low numbers of Vibrio cholerae – still capable of causing outbreaks – in brackish water.

Biofilms also complicate detection, because their structures protect the pathogen communities within, allowing them to withstand water treatment processes and elude traditional culture or microscopy methods. Furthermore, biofilm pathogens can remain dormant for long periods and become active again when conditions turn favorable. This increases the risk of delayed disease outbreaks.

Waterborne pathogens also adapt their behavior in response to fluctuations in water salinity, pH and temperature. As seasons change, so do temperature and rainfall, which dilute or concentrate the organisms, further complicating their detection. Therefore, detection methods must be robust enough to ensure accurate pathogen identification despite ever-changing environments.

Detection sensitivity and specificity issues

Traditional pathogen detection methods, such as culture-based techniques, can fall short when it comes to sensitivity and specificity. Many waterborne pathogens, especially viruses and protozoa, exist at low concentrations in water, and conventional tests can overlook them.

Culture-based methods are highly specific but may fail to detect Viable But Non-Culturable (VBNC) organisms – those that have entered a dormant state due to unfavorable conditions or environmental stresses. Despite being viable and potentially infectious, such bacteria may not grow on standard laboratory media, posing a unique challenge for traditional detection methods.

Immunoassays are faster than cultures, but cross-reactivity can lead to false positives, which can trigger unnecessary public health scares. Quantitative PCR (qPCR) offers higher sensitivity, but the results may be influenced by the presence of inhibitors or low pathogen numbers, resulting in false negatives, undetected health risks and delayed responses to actual threats.

Newer technologies like NGS and dPCR can overcome these shortcomings and bridge the gaps associated with traditional detection methods. NGS makes it possible to analyze a sample's entire microbial community and identify known and novel pathogens. dPCR provides extremely high sensitivity, making it possible to reliably detect low-abundance pathogens.

Next-generation sequencing supports broad and comprehensive pathogen detection

With NGS, it’s possible to identify all of the microorganisms in a water sample, ideal for applications that require broad-spectrum detection, such as environmental monitoring or outbreak investigations. Plus, the ability to sequence entire microbial communities lets you not only detect known pathogens but also emerging threats and antibiotic resistance genes. It's the go-to solution to help you understand a water system’s entire microbial landscape and identify potential risks that are not otherwise obvious.

NGS gives a comprehensive snapshot of microbial diversity, which is ideal for monitoring large-scale water systems or investigating contamination events. And the detection of genetic variations that confer antibiotic resistance or increase the pathogen’s virulence also provides critical data to inform public health decisions.

Digital PCR provides targeted and highly sensitive detection

dPCR provides precise and targeted detection of specific pathogens – ideal for applications that require accurate quantification of known pathogens. These include ensuring regulatory compliance, guarding drinking water safety and monitoring wastewater. The detection of low-abundance microbes with high sensitivity makes it an invaluable tool for rapidly identifying contamination events and making sure our drinking water supplies are safe.

When accurate and specific pathogen detection is the priority, dPCR is the method of choice. Its consistency and high precision provide rapid, actionable insights for timely decision-making.

Combining NGS and dPCR for a comprehensive approach to pathogen detection

Better yet, an integrated approach that combines NGS and dPCR can be particularly valuable for public health surveillance and outbreak management programs, offering both broad detection and targeted monitoring. First, NGS reveals the full spectrum of pathogens in a water sample and then dPCR precisely quantifies the specific threats.

For example, in an outbreak situation, officials can use NGS to identify potential microbial threats, followed by dPCR to monitor the progression of those threats as they implement control measures. This combination gives the best of both worlds to ensure comprehensive waterborne pathogen detection and inform decisions that protect public health.