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“We have long been looking for a good viral surrogate, and coliphages are one of the groups we are most interested in”

Apr 5, 2023

Bluephage interviews Dr. John F. Griffith, head of the SCCWRP Microbiology Department and Molecular Technology Coordinator at SCCWRP, to learn more about his work in developing and evaluating methods for detecting and counting fecal indicators and pathogens in fresh and marine waters and the relationships between water quality indicators and swimming-related diseases.

Do you consider that fecal bacterial indicators can provide the same information as fecal indicator viruses, and how does this relate to related recreational aquatic diseases?

Viruses and bacteria provide different information. Viruses cause most illnesses in bathers and people exposed to marine waters and rarely do bacteria, although there are indications of some wound infections due to bathing. We know that most gastrointestinal illnesses, the leading causes of disease in U.S. recreational waters, are not caused by bacteria, so we have long been looking for a good viral surrogate, and coliphages are one of the groups we are most interested in.

Does the U.S. Recreational Water Act not require virus testing? 

Since 1986, the EPA has recommended Enterococcus as an indicator for marine waters based on epidemiological studies. It replaced fecal coliforms, which are a broader group of non-gram-positive bacteria. Enterococcus is more resistant to degradation because this gram-positive has a tough cell wall that allows it to survive easily in the marine environment and in more saline waters.

It is, therefore, longer-lived and has proven to be a better indicator of fecal contamination, but it is a relatively nonspecific indicator present in most warm-blooded animals.

A major concern is the exclusive use of bacterial indicators in determining water quality, especially in California, where the way sewage is treated by collecting it in large plants that either discharge miles offshore into the ocean so that treated sewage does not return to shore or, if it does inland, receives tertiary treatment.

Especially when it flows into the ocean, where most recreational exposures occur – because our rivers have never had sufficient flow for recreational bathing- Enterococcus is not specific enough. Still, it is adequate if there is a sewage discharge, although it does not tell us, for example, whether the treated sewage is sufficiently treated.

Modern sewage treatment plants are designed to remove bacteria and then, when discharged to the ocean, mix the water with a diffuser so that what is discharged into the environment is within bacterial standards. So, what is recommended by US EPA for marine water is Enterococcus, and what is recommended for fresh water is E Coli.

Do you believe that the U.S. Recreational Water Directive is representative of all recreational water risks in the United States?

It is Federal Regulations that determine that determines bacterial standards based on risk and epidemiological studies. But we have done much work in California with recreational waters because we felt that the national beach studies were not representative. You don’t have the same circumstances at beaches in California, the Great Lakes in Ohio, or the East Coast, where the water is warm, and it rains in the summer. Rainfall is one of the main drivers of fecal pollution episodes, as it washes everything in from the watershed.

Also, on the Gulf Coast, the southeast coast, in Florida, and the Gulf of Mexico, which is very different from our cold water climate, with temperatures around 16 degrees Celsius, and the maximum they can reach is 30 degrees Celsius.

The other main feature is that we have a relatively deep ocean near the beach, and discharges are usually up to one or more miles offshore and at least 50 meters deep. On the east coast, it’s different, and even if it’s discharged quite a distance offshore, it comes to the surface.

Our biggest problem in California in dry weather is urban runoff from car washes, pool drains, and lawn watering. The local governments have intercepted the flows before they reach the beach to treat them as sewage because we have found sewage organisms such as Enterococci, which would otherwise end up in bathing waters.

Enterococci tested with selected media grow as if they were associated with sewage, regardless of the source. And that causes problems at the beach. That’s why we have a separate sewer, sanitary and stormwater system. the most typical way a diversión is accomplished is to build a weir, a low wall that crosses the drain channel; as long as the water doesn’t get over it, it doesn’t end up on the beach. There’s a pump on it, so every time water gets near the top of that wall, it’s pumped into the nearby sewer and never hits the beach.

There are beaches where birds are a problem. In our experience, we often don’t see other sewage markers, but we do see genetic markers of birds in abundance, and even the levels are correlated with fecal indicator bacteria. So there is no problem with human waste, but the beach may still violate the federal Clean Water Act for exceeding bacterial standards.

Are you working on rapid tests for analyzing beach water quality?

Rapid testing is something we’ve been working on, and I, in particular, for 25 years. The US EPA was ordered by federal legislation as part of the Beaches Act of 1999 to develop and validate a rapid test measuring water quality in one day. They were supposed to have done that in 2005 but did not meet the deadline. Because we wanted to make sure that any rapid method developed by EPA would also work in California, we worked with them to look at all possible methods. Many teams tried to develop rapid tests to measure fecal indicator bacteria, but the sensitivity was about a thousand cells or targets per hundred millimeters in all these methods but qPCR for Enterococcus.

The epidemiological study conducted in 1995 around storm drains in Los Angeles, the results of which eventually prompted the construction of the infrastructure to pump sewage from storm drains to the sanitary sewer, showed that there was an increased risk of GI illness that became greater the closer bathers were to flowing storm drains. This led to legislation in California that requires all beaches with > 50K visitors/year to be monitored at least weekly for FIB. If water quality does not meet standards for any of 3 indicators (enterococci, fecal coliform, total coliform), by law, the beach must be posted with a water quality advisory warning that exposure could cause illness.

Water quality testing takes time. Usually, the samples are collected first thing in the morning, and the warning sign arrives the next day in the afternoon. So in the meantime, swimmers have been exposed to polluted water. Ironically, we know from our work that the next day when a sign is posted, the water quality problem has already abated. So, because of the delay from sampling to results, warnings cannot be issued quickly enough to protect bathers from exposure to dirty water, and when the sign does go up, most of the time, the water is safe.

We have not been able to use qPCR, even though there was an EPA-validated method, because it was too laborious and required standard reference material. You have to dilute the reference material and create a standard curve to be able to plot what you get from your unknown sample on that line. The qPCR software calculates for you and tells you where your sample is on the curve so you can extrapolate to the original concentration, but when the samples are low, and especially with the EPA method, because of interferences, you have to dilute one to five, showing a limit of quantification of about 250 cell equivalents, well above the 104 we discussed earlier. So we needed a different method.

In California, we only dilute when we have an inhibition problem. Also, we were able to demonstrate that we can get results before noon by sending someone out at seven or eight o’clock in the morning and taking the sample directly to the lab without doing culture-based work and qPCR.

However, local laboratories have not adopted qPCR because it is too expensive, requires more staff in the lab, and in the case of the wastewater agencies, does not relieve them from the requirement to also monitor the use of culture-based methods required by their discharge permits.

How could coliphages be applied in bathing water testing in California? 

There is a potential beach monitoring market for coliphages in the U.S., but likely not for Southern California because they have done an excellent job of keeping sewage away from the beaches. But there is a market for places like the Great Lakes and in rivers, where there are sewage discharges, and at the sewage treatment plants themselves, as a surrogate for enteric viruses, which are thought to behave very similar to coliphages, like Noroviruses, which are a problem. And, of course, we can only measure them by molecular methods because we need a good culture host.

What is the most common source of fecal contamination in bathing waters, and what measures are in place in the United States or, in particular, in California to control it? 

In California, sewage typically only reaches the ocean beaches after a spill. Spills at the beach most often result from a pump station failure. When one of these pumps fails, which happens occasionally, and the redundant systems designed to prevent a spill also fail, then potentially millions or even hundreds of millions of gallons of sewage reach beaches. In those circumstances, the reaction of the health department is to close large parts of the coastline so that people are not exposed to fecal contamination. But they also want to reopen the beach as soon as possible, and this is a situation where a rapid test would be decisive.

Because of these rapid diagnostic needs, some municipalities are turning to droplet digital PCR to look for Enterococci and human sewage indicators, such as the HF183 gene, a well-known and validated human sewage marker.

What is the reopening time of a beach when a sewage spill has occurred? 

Typically, if you test a beach’s water quality with culture-based methods, it takes at least 24 hours plus the time it takes for the administrative process. Therefore, from an operational point of view, the time needed to reopen a beach to bathers is likely to be more than 36 to 48 hours, which is too long.

For really rapid beach surveillance, the best scenario would be a test that could be initiated by the lifeguard first thing in the morning. They would go down to the water and collect a water sample, put it into a machine connected to a lab, and the device would analyze it there and send the result to the health department within hours.

Could climate change increase the risk of fecal contamination in recreational waters? And if so, to what extent and how will this relate to increases in waterborne diseases? 

To the extent that it interferes with infrastructure. If the sea level rises above where sewage has been diverted, it could pose a problem. Walls are already being built to reinforce that infrastructure because the sea level is rising.

If it interferes with the capacity of the treatment infrastructure or the collection system, climate change may affect the increased risk of fecal contamination in recreational waters from possible sewage spills on beaches.

Another concern is the increase in water temperature, especially here in California, which could lead to more Vibrio infections, which only rarely seen now and are warm water phenomena.

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