vibrios

The viable but non-culturable (VBNC) state in vibrios

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By: Ph.D. Stephen G. Newman*

It appears that many in the field that advocate the control of vibrios either in the environment or through the gut do not understand the concept of “viable but nonculturable” (VBNC). This is not a new idea although we continue to learn more about it.

The widespread dogmatic approach towards shrimp culture dictates that certain bacteria pose problems simply by being present. These are the vibrios. Vibrios are ubiquitous bacteria in most marine environments. They are highly evolved and can grow under a variety of conditions.

A few of them are opportunistic pathogens of fish and shrimp and a few are well known pathogens of humans (V. cholerae and V. parahaemolyticus). For shrimp most are what are termed opportunistic pathogens. The host must be weakened in some manner for the specific species/strains to cause pathology.

“There are many other bacteria that have been associated with disease in farmed shrimp that are largely ignored. Most are opportunistic although will cause disease in healthy animals, termed obligate pathogens. Many companies appear to be offering approaches to a problem that in reality is not a problem.”

Healthy strong animals in stress-free environments are not bothered by most vibrios. Only a few are obligate pathogens and there is some justification for attempting to control the levels of them. Notably the strains of V. parahaemolyticus that produces PIRa and PIRb and T6SS (type 6 secretory systems that allow the vibrio to kill other bacteria).

This strain is unique in many respects with the evidence strongly suggesting that it dominates in environments where efforts have been made to eliminate bacteria and other potential pathogens through the use of chlorine. Selective elimination of vibrios that are incapable of degrading the sugar, sucrose and thus appearing green on the selective media, TCBS is more than likely not helpful in the long run.

“There are species that are capable of degrading the sugar that are highly virulent obligate pathogens their colonies are yellow on TCBS.”

Farmers are much better off minimizing the stressors that are present via proactive management strategies including but not limited to adequate levels of aeration at all times, the use of automatic feeders to reduce waste and lessen competitive stress, and bioremediation to lessen the food available for potential pathogens than trying to selectively eliminate one species of bacteria.

Genetic selection can also play a role in reducing the overall susceptibility to common stressors. It appears that many in the field that advocate the control of vibrios either in the environment or through the gut do not understand the concept of VBNC. This is not a new idea although we continue to learn more about it.

“The acronym stands for “viable but nonculturable”. It means what it says; bacteria are viable but cannot be cultured. This is actually fairly common, and the vibrios are not the only taxon that this applies to. Conditions that appear to be sufficient to kill a specific group of bacteria turn out to be at best temporary.”

The bacteria are injured and cannot be cultured yet they are still metabolically active to some extent and may in some instances continue to produce toxins. Once the insult is no longer present, they can revert to their prior status, becoming metabolically active and being culturable.

vibrios

Any effort to culture the bacteria will be fruitless until they have recovered sufficiently. Strategies that are aimed at eliminating vibrios, whether it is through the use of chlorine, the use of antibiotics, whether man made or produced by competitive species of bacteria, and the use of compounds in the feed to inhibit bacteria in the gut that do not take this into account are prone to failure.

Elimination of large swatches of potentially “bad” bacteria is not necessarily the best approach to take. The data strongly suggests that with EMS or AHPNS that we are creating an environment that allows the etiologic agent to proliferate at the expense of other bacteria.

“By treating ponds with chlorine in a misguided attempt to kill off all possible pathogens it appears that we are making it easier for these strains to proliferate at the expense of whatever bacteria might still be present. When we look for them via culture, we may not find them yet there can be tell-tale signs that they are present, specifically the presence of the toxins that are pathognomic.”

Manipulation of the microbiome is a relatively recent concept and there are any numbers of publications that purport to demonstrate that certain disease states are a result of alterations in the microbiome and that these changes are predictive.

The tools used are based on the detection of specific genes. It does not make a distinction between living and VBNC bacteria. VBNC can still be actively synthesizing proteins. Even though we have known about this state for almost 40 years, there are still many unanswered questions.

“Many different factors have been identified for various species that induce the VNBC state and many more are likely to be found.”

The use of culture to validate the effectiveness of any number of feed additives should be viewed with caution. Often these damaged bacteria when removed from the immediate environment where they are declared dead because they cannot be grown up on or in bacteriological media will revert to being culturable.

They are also capable of producing toxins when they are in the VNBC state. This could explain why there are instances with the strain of VP that is responsible for EMS or AHPNS where the etiologic agent cannot be cultured. Enriching samples by adding them to culture media and incubating them for 18 to 24 hours can reveal that there are viable cells present.

“This should not be taken to mean that there are not instances when the application of disinfectants and/or compounds in the feed is in fact effective at killing them.”

There are instances where the data regarding the use of disinfectants or compounds in the feed (such as organic acids) should be viewed from the standpoint that the VNBC state is why they cannot be cultured on agar plates and not due to the treatment.

Care should be taken in experimental design to ensure that not only are there appropriate controls but specific properties, such as VNBC, are taken into account. Concluding that a given modality results in killing bacteria when in fact they have become VBNC can lead to misleading claims and the use of products that do not do the job that the end users believe that they need.

Stephen G. Newman

Stephen G. Newman has a bachelor’s degree from the University of Maryland in Conservation and Resource Management (ecology) and a Ph.D. from the University of Miami, in Marine Microbiology.
He has over 40 years of experience working within a range of topics and approaches on aquaculture such as water quality, animal health, biosecurity with special focus on shrimp and salmonids.
He founded Aquaintech in 1996 and continues to be CEO of this company to the present day.
It is heavily focused on providing consulting services around the world on microbial technologies and biosecurity issues.
sgnewm@aqua-in-tech.com
www.aqua-in-tech.com
www.bioremediationaquaculture.com
www.sustainablegreenaquaculture.com

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