Vibrio parahaemolyticus: an Australian perspective

Keywords: Australia, foodborne diseases, food safety, microbiology, oysters, qPCR, Vibrio parahaemolyticus.

Vibrios are ubiquitous in marine and estuarine environments and the genus encompasses over 140 species, with many involved in symbiotic relationships with marine life.¹ The comma-shaped, Gram-negative bacteria with polar flagella generally present no risk to humans; however, within the genus there are a small proportion, at least 12 species, that are human pathogens.² These pathogens are capable of causing infections ranging from ear or wound infections to foodborne illness and sepsis.² The most notable of these is Vibrio cholerae, a water and foodborne pathogen that is the causative agent of cholera. Presently, however, it is non-choleragenic vibrios causing concerns for the Australian seafood industry. The species of greatest concern in Australia currently is Vibrio parahaemolyticus. Some strains of V. parahaemolyticus are pathogenic and capable of causing gastroenteritis that is primarily associated with consumption of raw seafoods, particularly oysters.³ In Australia, oysters are a significant commodity and in 2019–2020 it is estimated that over 11.2 million retail dozens were sold, to a total of over $100 million for the industry.⁴

In many major seafood producing and consuming countries such as the United States, China, Japan and Korea, V. parahaemolyticus is recognised as a leading cause of seafood-associated illness.^5–7 Concerningly, the number of infections in many areas appears to be increasing.⁷

Like other species within the genus, there is a high degree of genetic diversity among V. parahaemolyticus strains and importantly, not all strains are pathogenic. The molecular markers typically utilised to indicate pathogenicity are the thermostable direct haemolysin (tdh) gene and the TDH-related hemolysin (trh) gene or a combination of both.⁸ Clinical isolates have been found to contain these markers approximately 90% of the time whereas environmental or food isolates carry a low likelihood of possessing these genes with only 1–10% of isolates carrying the markers.⁹ In terms of distribution in seawater, Vibrio species have an increased presence in coastal tropical areas due to their preference for warmer water.¹⁰ There are also indications that changing salinity levels and algal blooms can contribute to a greater risk of pathogenic vibrio species being present in certain areas.¹

It is this predilection of vibrios for warm water which has prompted concerns about changing climates and warming sea temperatures.¹¹ In recent decades, incidences of vibrio-associated illnesses have been occurring in cooler climates where there had previously been no known cases.¹² Studies have revealed an upward trend in case numbers in already affected populations and many have positively correlated large outbreaks to warm weather events.¹⁰ The persistence of this pathogen overseas has prompted surveys in many marketplaces and oyster farming regions to understand its prevalence.^13–15 Models have also been developed, such as the surface sea-water temperature monitoring system by the European Centre for Disease Prevention and Control (ECDC) to enhance risk prediction.¹⁶

In Australia, the prevalence of vibrio infections historically has been intermittent, with many of the reported infections linked to overseas travel or the consumption of imported foods.^17,18 However, in the recent decade, outbreaks of V. parahaemolyticus infections associated with locally produced seafood appear to have been increasing.¹⁹ The most recent of these outbreaks occurred over November–December 2021, where reports from this time period indicated that over 250 people had fallen ill across multiple states after consuming oysters harvested in Coffin Bay, South Australia.²⁰ In 2016, there were two separate outbreaks of V. parahaemolyticus infections in Australia, one in Tasmania associated with consumption of Tasmanian grown oysters involving 11 cases and one in Western Australia associated with consumption of South Australian oysters involving nine cases.¹⁹ This increase in infections could be correlated to warming sea surface temperatures and weather events, with Australia not being spared effects of climate change.²¹ There are other factors that might be contributing to an apparent increase in prevalence and it has been noted there is potential that infections are being under reported due to the bacteria previously not being included in clinical panels.¹⁹ There are also varying State and Territory requirements as to whether Vibrio spp. infections are notifiable illnesses which can leave gaps in epidemiological data collection.¹⁹

V. parahaemolyticus is not a new pathogen; however, its increasing prevalence with many unknown contributing factors does complicate risk management practices. There are few guidelines available for safe levels of V. parahaemolyticus in food as the virulence is unclear. Food Standards Australia New Zealand (FSANZ), the Australian statutory authority which develops and maintains the Australian Food Standards Code, provides recommendation for levels of the bacteria in Ready to Eat (RTE) foods.²² This serves as a guideline only and does not provide impetus for regular screening of seafood products. There are also some state guidelines in place to mitigate risks, such as control plans implemented by Tasmanian state authorities.²³ Despite these controls, there is still much to learn about the distribution of pathogenic strains of V. parahaemolyticus in Australian oyster growing areas. This prompts the need for further research to understand variations in the Vibrio populations during weather events and as sea water temperatures rise. Improvements in detection and identification methodologies would also assist in routine monitoring of the bacteria levels during varying weather conditions and assist in the development of appropriate risk management procedures.

Questions have also been raised about the suitability of currently recommended methodologies for the detection and enumeration of both total and pathogenic V. parahaemolyticus in food. To adopt routine or surveillance testing and ensure testing results are consistent across multiple areas, access to robust, validated methods that are easily adopted by laboratories with a range of capabilities are required. Available methods for use by laboratories are described in a joint document by the Food and Agriculture Organisation (FAO) and the World Health Organization (WHO).²⁴ The traditional culture-based method of detection for vibrios is laid out in the Bacteriological Analysis Manual (BAM) by the United States Food and Drug Administration (FDA) as well as in the International Standards Organisation (ISO) standard ISO21872-1.^25,26 Briefly, 10–12 oyster specimens are homogenised and weighed into a selective broth for enrichment and then plated onto selective agar. Suspect colonies are then analysed using probes or transferred to non-selective agar for biochemical testing to identify bacterial species. Enumeration can be achieved by combining this method with a Most Probable Number (MPN) protocol to determine counts per gram. Many MPN methods for detection of V. parahaemolyticus, including the current Australian Standard AS 5013.18, utilise a three tube technique.²⁷ This provides a limit of detection (LOD) of 3 MPN/g, which may not be sensitive enough in some applications. There are alternative methods available that can provide far greater sensitivity, such as the US FDA BAM method that utilises a polymerase chain reaction (PCR) MPN method that can quantify down to 0.3 MPN/g. The increased sensitivity confers many benefits and can improve low level detections of pathogenic bacteria.

The MPN technique for quantification is the most accessible for laboratories but carries limitations, including lengthy result turnarounds and numerous confirmation tests for suspect colonies. Confirmation testing is vital for characterising the pathogenicity and virulence of V. parahaemolyticus isolates and can include detection of genes which indicate pathogenicity, sequence typing and serotyping. The time to detection and identification is a key aspect for the oyster industry and regulators who require rapid information in order to deliver safe products to the marketplace. Conversely, this method provides the best opportunity for genomic investigation of colonies and for epidemiological tracing. Also of concern with culture-based methods is the potential for bacterial cells to be missed. Some research has indicated difficulties in detecting and identifying vibrio spp. in samples, which can be partially attributed to their ability to enter a viable but not culturable (VBNC) state.²⁸

The preferred alternative to culture-based methods in terms of rapidity and sensitivity is quantitative PCR (qPCR). For qPCR applications, there are multiple options available in terms of methodologies or commercial kits to use, many using different gene targets and having varying sensitivities. For detection and enumeration of total V. parahaemolyticus numbers within a sample, the ISO standard, ISO-21872, utilises VpToxR for species level detection and trh and tdh as molecular markers of pathogenicity.²⁶ Recent research has indicated the potential for false negative results using the trh gene as a marker, due to high sequence variability among strains and suggests an alternative target, a urease gene (UreR) which is located directly upstream to trh and highly conserved.²⁹ With such rich genetic diversity among V. parahaemolyticus strains and high homology to closely related species, it is important to investigate the most appropriate gene targets for PCR testing. Recent advances in technology have also provided accessibility to previously out of reach techniques, such as whole genome sequencing (WGS) and next generation sequencing (NGS). Utilising these tools to understand more about the genetic diversity of isolates and their pathogenicity will confer great benefits, particularly during outbreaks of illness. As V. parahaemolyticus infections increase, so too does the data available, and further work is warranted to determine the most appropriate analyses for detection and identification and to ensure there are standardised methods available to allow for optimal sensitivity and accuracy of testing.

As the climate continues to change around the world and microbial communities shift with it, further research is required to protect our industries and consumers. Improvement in methods of detection and identification of V. parahaemolyticus as well as increased surveillance with epidemiological tracing can ensure Australia’s safe enjoyment of oysters for years to come.

Data sharing is not applicable as no new data were generated or analysed during this study.

The author declares no conflicts of interest.

This work was supported by RMIT University and the National Measurement Institute (NMI).