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“Using coliphages as a treatment efficacy indicator on a larger scale would allow better preparation for future unknown pandemic viruses and a better understanding of actual viruses transmitted through food and water.”

Feb 20, 2023

Christophe GANTZER, Directeur, Laboratoire de Chimie Physique et Microbiologie pour les matériaux etl’Environnement (LCPME)

Interview with Christophe Gantzer,  Director of the Laboratory of Physical Chemistry and Microbiology of Materials and Environment (LCPME) belonging to the University of Lorraine and CNRS in Nancy (France). This laboratory is a multidisciplinary unit that studies material and biological interfaces in aqueous media. 

Could you briefly describe how coliphages interact with the aquatic environment at different temperatures and exposure times and if their behavior is suitable to be considered a conservative indicator of fecal contamination in water? 

We can consider two types of coliphages that infect E. coli and are excreted in human or animal feces. On the one hand, somatic coliphages infect E. coli using a receptor located on the bacterium’s surface (outer membrane). They belong to several phage families, consisting mainly of a head containing the DNA genome and a tail. On the other hand, F-specific RNA phages are the most homogeneous structural group having an icosaheadral capsid with an RNA genome. They infect their host through the F-pilus, expressed by bacteria for gene transfer. Their structure is similar to that of the most frequent and important pathogenic enteric viruses, as explained below.

Human enteric viruses cannot multiply outside their human host, and coliphages of both groups also have little chance to multiply due to the low concentration of host bacteria in the medium and their low metabolic activity in such media. Furthermore, the possibility of multiplication is even lower in the case of F-specific RNA phages because F-pili are poorly expressed in the medium. Therefore, the presence in feces and the low probability of multiplication make coliphages suitable indicators of fecal contamination.

After excretion into the environment via feces, coliphages are recognized to have higher survival in the environment and during treatments than bacteria, also used as fecal indicators. Therefore, it may be better to compare their survival with that of pathogenic enteric viruses. However, due to the diversity of pathogenic enteric viruses and bacteriophages, it is pretty complex to give a direct answer on the behavior of each species in the environment. Nevertheless, temperature and ultraviolet light are the most critical environmental factors in decreasing the survival of viruses or phages.

Globally, we know that F-specific RNA phages are conservative indicators compared to enteric viruses when UV light is the most crucial inactivation factor. The action of UV is mainly localized to the genome. The genome of these F-specific RNA phages is between 3.5 and 4.3 kilobases (kb), compared to 7.5 kb for the main pathogenic enteric viruses. Thus, there are statistically fewer reactive sites in F-specific RNA phages than in viruses, and therefore, these phages have more prolonged survival than pathogenic enteric viruses by UV inactivation.

However, F-specific RNA phages express less stability with increasing temperature, especially above 25°C. Somatic coliphages then represent much higher survival than F-specific RNA phages, especially to enteric viruses.

Adhesion to solids can significantly modify survival in the medium. The ability to adhere changes considerably from one virus or phage species to another. This adds high complexity when comparing the survival of one particular viral species to another in complex environmental media.

Globally we may retain that in many situations, somatic coliphages have a higher survival compared to pathogenic viruses but with a different structural organization, whereas F-specific RNA phages have a similar structure but a lower heat resistance capacity.

What are the main viruses causing waterborne diseases in Europe, what are the most common sources of viral contamination, and what methodology do you suggest to prevent possible infections better? 

According to the European Food Safety Agency (EFSA), noroviruses are between the second and the fourth leading cause of foodborne outbreaks related to microbes in terms of number of cases since 2016. More globally, the most critical fecal-oral transmitted viruses worldwide may be gastroenteritis viruses, including noroviruses, but also others such as hepatitis A and E viruses and enteroviruses.

Human fecal contamination is the leading cause of transmission of these viruses, with hands being a primary source of infection. However, waterborne transmission is significant, for example, in oyster farming, sludge spreading on land, or inadequate drinking water consumption.

“Limiting fecal contamination through effective treatment is the main objective of reducing transmission. Such treatment should be evaluated using viral treatment efficacy indicators in addition to bacterial indicators due to the significant differences in behavior between the two microorganisms (viruses versus bacteria). In environmental media, viral contamination should also be assessed using viral in addition to bacterial indicators, although it is assumed that for a high content of bacterial indicators, there is also high viral contamination. Better prevention of viral transmission is based on the broader use of viral indicators.”

Could you tell us more about the replication process of these viruses and the causes that influence their lower infectivity?  

Two different strategies can be used to evaluate viral pollution in the environment and food, each with its advantages and disadvantages.

1. The first strategy can detect the main pathogenic viruses directly by molecular techniques, usually by reverse transcription-polymerase chain reaction (RT-PCR). Such detection answers the question: what type of viruses are present? Detection of the viral genome confirms the presence of the virus but does not provide information on its infectivity. There is no simple relationship between the number of viral genomes and the number of infectious viruses. The genome, even RNA, has a much greater persistence than infectivity.

An excellent example is what happened during the last SARS-CoV-2 pandemic, where 107 genome copies (gc)/L were detected at the entrance of the scrubber, and it could be confirmed that no virus was infectious. This can be applied at different scales for all viral particles present in the environment. The difference between infectivity and genome quantity is more significant when the number or level of inactivating factors is high. This is often the case in old contaminations and after water or food treatments. Thus, the molecular technique cannot be used to estimate an infectious risk.

2. The second strategy uses a fecal infectious indicator of viral origins, such as coliphages. This detection answers the question: is there an infectious viral risk? If the viral indicator coming from feces is present in its virulent form in a particular environment, a pathogenic virus could have followed the same pathway and remained infectious because its survival is similar to that of the indicator. Demonstration of coliphage infectivity is easy, whereas that of pathogenic enteric viruses is complicated and time-consuming.

Infectious risk can only be assessed by measuring infectious viruses.

What are the advantages of working with coliphages, non-infectious human viruses, as indicators of infectious activity or inactivation of other viruses, particularly human waterborne viruses?  

As indicated in the previous question, working with coliphages as indicators allow us to easily and quickly estimate virucidal treatment efficacy using infectious criteria. We applied this strategy in France during the pandemic to demonstrate that more frequent sludge treatments based on temperature or pH effectively inactivate SARS-CoV-2 (if it was present). Treatment efficacy was estimated in the field with the help of coliphages. Inactivation by temperature and pH was determined in both viruses (SARS-CoV-2 and coliphages) in the laboratory. Inactivation was so different between the two viruses that there was no doubt about the conservative use of coliphages as an indicator of SARS-CoV-2. The use of coliphages as an indicator of treatment efficacy on a larger scale would allow better preparation for future unknown pandemic viruses and better knowledge about the dissemination of the actual viruses transmitted through food and the environment.

What do you think of the JO Legifrance decree 0121 of 05/27/2021 specifying methods for spreading sludge from urban wastewater treatment? Is it not too restrictive (feasible? and limited to some sludge sanitization treatments with high costs?) The previous French regulation required infectious human enteroviruses to be < 3 in 10 g of dehydrated sludge. This value corresponds to ≈ 1.2 x 10E4 somatic coliphages /gr of dewatered sludge per 1 enterovirus according to the ratio determined in sludge for somatic coliphages and enteroviruses. What is your opinion on this level of sludge sanitization?

In my opinion, looking only at the level of infectious enteroviruses after sludge treatment before spreading is very insufficient. Its absence does not provide any information on the viral risk because other types of viruses may be present (see above), and, above all, it does not give any information on the effectiveness of the treatment because the level of infectious enteroviruses before treatment is unknown. Knowing the efficacy of viral treatment is especially important during a pandemic or even during other viral epidemics that increase the level of the virus in wastewater. In addition, the search for infectious enteroviruses is especially long and complex in sludge matrices.

Therefore, coliphages are valuable indicators for assessing the effectiveness of sludge treatment. Asking for the removal of at least 4 logs seems to me justified without any other criteria because the level of viruses during an epidemic or pandemic can be high and even more so in the concentration of coliphages.

What can you tell us about the French transposition of the new European Drinking Water Directive and whether you consider it sufficient to prevent waterborne diseases? 

The French transposition refers to the level of somatic coliphages in water resources at the point of collection (< 50 PFU/100mL), and if present, the absence must be verified after overall treatment (0/100mL). I believe that monitoring water resources with a viral indicator is positive. In the final product, the probability of detecting coliphages is low; therefore, it is better to control the process carefully and, in parallel, define virus removal by this process.

In the context of wastewater reuse, a 6-log removal of coliphages is required in France.

It is perhaps also important to note that, in a research context, LCPME participates in a Joint Technological Unit (UMT) Actia VIROcontrol, which is approved by the French Ministry in charge of food. Since 2016, we have promoted the use of coliphages for the evaluation of food treatments. Currently, shellfish industries are validating the use of infectious F-specific RNA phages to evaluate the performances of depuration processes of contaminated oysters before reléase the products to the market. We have obtained very promising results in France for a few years, showing that these coliphages (especially F-specific RNA phages) can be conservative indicators of norovirus contamination of oysters.

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