Assessment of pathogenic microbial contamination in fish and environmental samples in Ambon City, Indonesia: a preliminary study

. This preliminary study investigated pathogenic microbial contamination on fish landed in Ambon City, Indonesia, and evaluated the presence of microbial foodborne pathogens in both fish and environmental samples. The study used a rapid dry sheet media as an in-situ presumptive assay and a PCR method as a confirmatory assay. The results indicated the presence of various foodborne pathogenic bacteria, including V. parahaemolyticus , Salmonella spp., and E. coli, in the water and fish basket samples. Three out of thirty-six fish samples were also contaminated with these bacteria. Although the presumptive Listeria assay showed high positive results (36/81), none of them were confirmed as Listeria monocytogenes by PCR assay, indicating that a rapid dry sheet media may not be suitable as an in-situ rapid screening assay for fish and environmental samples. The study highlights the need for better sanitation and hygiene practices, especially at ERI Traditional Fish Landing in Ambon, and may help competent authorities and the seafood industry to implement effective control measures to ensure food safety and public health.


Introduction
Fish plays a significant role as a primary source of animal-based protein, providing essential macro-and micronutrients for human nutrition worldwide, including in Indonesia [1].Particularly in Ambon City, the fish demand reached 17,712 tons annually for its population of approximately 478,616 individuals in 2019.This represents the second-highest demand within the Maluku province, known as one of the highest fish consumption per capita in Indonesia, with a value reaching 72.67 kg/year/capita [2].The fish supply in Ambon City is predominantly supplied by captured fish species, such as Tuna and Tuna-like species (TTC).Scad, and Mackerel.In addition, the TTC species is also known as the dominant species captured in Maluku, including Ambon; hence, this commodity is frequently found in traditional dishes from Ambon [3].
Despite its nutritional benefit and cultural importance, fish and its processed products often pose risks to consumers, especially due to the presence of natural toxins (biotoxins), pathogenic microorganisms, or other chemical contaminants in its environment [4,5].Pathogenic microorganisms have become the major cause of foodborne illnesses in several countries, resulting in health and economic losses to the community [6][7][8].Among the various types of pathogenic microorganisms, Vibrio spp., Salmonella spp., and E. coli are common contaminants of fish products in Indonesia, particularly [4,9,10], either through primary contamination or secondary contamination (cross-contamination) resulting from mishandling during raw material handling, processing, storage, and distribution of fishery products.
To date, numerous studies and research have been conducted on the detection method, occurrence and prevalence, general profiles, or risk assessment of pathogenic microorganisms in Indonesia from various fisheries products such as multiplex PCR detection of E. coli, Salmonella spp and V, parahaemolyticus in fish [4], detection of pathogenic V. parahaemolyticus in fish, squid and shrimp/cultured shrimp [11,12]; Salmonella in marketed fresh fish, Tuna, and processed Tuna's like [13][14][15][16], and E. coli in Decapterus sp from Ambon's fish market -Indonesia [17].Some of these studies applied conventional culture methods and molecular-based assay (such as PCR assay as confirmatory assay), which produced a comparable result.The prevalence from those studies may not provide extensive information about multiple contaminations of microbial foodborne pathogens and their possible contamination-route routes on the captured fish; especially those landed in Ambon, Indonesia.The studies also suggested that the environment, including water, ice, and fish handling facilities, serves as the initial source of microbial contamination in freshly captured fish during post-harvest processing [18].The previously available database about microbial contamination on fish products from Ambon was mostly observed from the traditional market, while only limited information was collected from the fish landing place and its environment.Addressing these data gaps poses a significant challenge for local governments in formulating effective control measures to mitigate microbial contamination.One of the challenges is the availability of low-cost and rapid assays for pathogenic microbial screening that can be applied in situ during monitoring in the field.Previous studies on the rapid assay for microbial detection showed that dry sheet media (i.e., 3M® Petri film, SimPlate ® Compact Dry®) produced comparable results in detecting the pathogenic foodborne bacteria in food matrices and their environment [19][20][21].Moreover, the dry sheet or dehydrated media in a plate is cheaper and easier to handle compared to the conventional culture method [22]; hence, it has the potential to be applied in the field as an in situ assay for monitoring and screening purposes of pathogenic bacteria contaminations in food and its environment.
To ensure the safety of fisheries products for consumers, it is crucial to implement preventive measures through monitoring and surveillance programs overseen by the Indonesian competent authority.Accurate surveillance requires data on the microbial profile, potential contamination routes, and suitable detection methods for targeted bacteria in fish products.This information is essential for both the local government and competent authority in Ambon.Furthermore, this database can serve as the foundation for developing risk control guidelines by the Indonesian competent authority during fish handling and processing.
Therefore, in this preliminary study, our objective is to present the microbial contamination profile of captured fish in Ambon and propose a rapid, cost-effective, and sensitive method for in situ analysis of foodborne microbial pathogens.

Data collection
Data on captured fish production and fisheries export of Maluku (including Ambon), prevalence studies, existing regulations toward seafood quality and safety, and their implementation were collected by direct interviews with the local fisheries authority and FQIA of Ambon.The data were also extracted from the Annual Fisheries Report of Ambon City of the Year 2020 [23], the Monitoring Report on Fisheries Product Quality and Safety 2014 -2018 [24], and the Statistical Profile of Ambon City in 2020 [25].These data were then sorted and tabulated for further analysis in this study.

Sample collection
The fish and environmental samples were collected from two fish landing sites, i.e., PPI Eri (3°45'45.1"S128°07'25.4"E)and PPN Ambon (3°40'43.4"S128°11'17.7"E).Sampling activities were done twice a year during June (wet/rainy season) and November (dry season) of 2020.Thirty-six fish samples (i.e., TTC, sardines, mackerel, scad, rainbow runner) and a total of forty-five environmental samples, including water, ice, a swab of the fish basket, boat deck, and fish hold/Palka, were aseptically collected.The fish, water, and ice samples were directly collected from the fisherman during fish unloading and/or collected from the retailer in the fish landing sites, while some environmental samples (i.e., fish basket, boat deck, fish hold/palka) were collected by swabbing.All samples were immediately stored in a cool box with ice flake for further analysis.

In situ presumptive assay
In this study, the presumptive assay was performed in situ using rapid dry media/sheet methods such as Petrifilm® (for Listeria, Salmonella typhi, and E. coli/Coliform assay) and Compact dry® (for V. parahaemolyticus assay) following procedures from the manufacturer with minor modification.Briefly, ten grams of fish tissue were aseptically mixed into 90 mL Butterfield's Phosphate Buffer (BFP) + 1% NaCl.One milliliter of the mixture was then directly inoculated to each dry sheet and incubated at 35±2°C for 24 hours.The dry sheet with non-typical colonies was further incubated for another 24 hours.The interpretation of the positive result of each bacterium followed the manufacturer's procedure.The American Type Culture Collection (ATCC) of targeted bacteria such as Listeria monocytogenes (ATCC 13932), Salmonella typhimurium (ATCC 14028), Vibrio parahaemolyticus (ATCC 17802), and E. coli (ATCC 25922) from Oxoid -Indonesia were used as positive control, while Staphylococcus aureus (ATCC2593) were used as negative control.These bacteria were also used as controls on the confirmatory assay.The remaining mixture was stored at -20°C and then transported to the laboratory for further analysis.

Confirmatory assay
The confirmatory assay in this study was based on the molecular identification method using the PCR technique with a specific primer, as described in previous studies (Table 1).In this study, we performed a single PCR reaction to avoid misinterpretation of the visualized band in the gel electrophoresis.Briefly, a hundred microliter of the remaining mixture of each sample from the presumptive assay was extracted using the Bacteria Geneaid® DNA Extraction Kit (Taiwan) following the manufacturer's procedure.Two microliters of extracted DNA were then used as a template for further PCR assay following the protocol from previous studies [4,[19][20][21][22][23][24].The PCR products were then visualized at 1.5% TBE agarose gel with the electrophoresis condition of 100 volts for 40 mins.The interpretation of positive samples was determined by the presence of PCR products at targeted sizes (Table 1).To ensure the PCR process is working well, the DNA of the negative result (no band presence) was amplified with a specific primer targeting the 16S rDNA gene.

Result and discussion
Ambon City of Maluku, with an estimated annual fish production of approximately 45,000 tons in 2017, significantly contributes to the fish production of the Maluku region (which is considered one of the major fish suppliers in Indonesia).The city has two pivotal Fish Landing Sites (TPI): PPI Eri (3°45'45.1"S128°07'25.4"E) and PPN Ambon (3°40'43.4"S128°11'17.7"E).Based on the available data from local FQIA (as shown in Fig. 1), the export of fisheries commodities in Maluku, including Ambon, was primarily contributed by TTC species, with a total volume of 1,332 tons valued at US$ 10.9 million in 2019.This highlights the substantial economic importance of TTC in the capture fisheries sector of Maluku, including Ambon.Based on the information from the local FQIA in Ambon, the authority conducted a fish monitoring program from 2014 to 2018 to detect the presence of microbial contamination in the product.As shown in Table 2, the prevalence of E. coli contamination in fish products from the traditional market is the highest (13.04%, 3 of 23) among other bacteria (i.e., Salmonella spp.and V. cholerae).Moreover, no microbial contamination was observed during the monitoring program.Our observation in the field showed that PPN Ambon has a bigger infrastructure and better sanitation program compared to PPI ERI.The PPN Ambon has been selected as one of the national fish ports in the Eastern Indonesia region; hence, it is required to participate in the sanitation program by FQIA -Indonesia.Thus, it is likely that the sanitation and hygienic condition of PPN Ambon is better than that of PPI Eri, as shown by the presence of microbial contamination in the fish from PPI ERI (Table 2).The presence of E. coli or coliform in the samples can be indicated as the result of poor sanitation and hygiene.Coliform bacteria, including E. coli, are generally recognized as the indicator of fecal contamination during food processing and handling [32], along with Salmonella spp., V. cholerae, V. parahaemolyticus, and Listeria monocytogenes; these bacteria are included as microbiological criteria in food by CODEX or US-FDA or other national standards worldwide.Data were collected from monitoring report of FQIA Ambon.
The prevalence data presented in Table 2 were obtained using conventional-culture methods following BAM/US FDA, ISO, and the National Standard of Indonesia (SNI).These methods produce robust, specific, and sensitive results; hence, it has been applied as a gold standard assay by the competent authority in many countries for detecting foodborne microbial pathogens in food [33].However, this method is time-consuming, labour-intensive, and requires biosafety level 2 (BSL 2) and experienced laboratory staff [4].Based on our interviews with the local competent authorities, their laboratory was lack of these requirements.Hence, the rapid and reliable method could overcome this problem during their routine monitoring essay.In this study, we applied the dry sheet method as a rapid presumptive assay to detect the presence of foodborne microbial pathogens (i.e., Listeria monocytogenes, V. parahaemolyticus, Salmonella typhimurium, and E. coli) in the fish and environmental samples using Petrifilm® (3M -US) and compact dry® (Nissui -Japan).Our preliminary assay using ATTC as a positive and negative control in the laboratory showed that this method was sufficient to detect the ATCC as targeted bacteria (Figure 2).All the ATCC bacteria were grown overnight in BHI broth before being inoculated into the dry sheet.Thus, the assay produced robust results, as expected.
The result from the in-situ presumptive assay in this study (Table 3) showed that the majority of fish species were contaminated by V. parahaemolyticus.This microbe is known to be naturally present in marine and brackish water, including in tropical countries [9]; hence, it is very likely to isolate this species from marine fish or shellfish and its environment, especially in Indonesia.Our investigation using presumptive assay also showed some fish species (i.e., scad, skipjack tuna, and tuna) and the environmental samples (i.e., water, ice, fish basket, boat deck, and fish hold) were contaminated by Listeria species.It should be noted that the Listeria petrifilm® used in our study is only capable of detecting up to the genus level of Listeria; hence, without further confirmatory assay, it is difficult to conclude that the samples are contaminated by L. monocytogenes.Note: 1 The assay was performed using pertifilm® (3M -US) 2 The assay was performed using compact dry® (Nissui -Japan) The application of the rapid dry sheet method as a presumptive assay is promising to be applied for in-situ analysis by local staff of FQIA Ambon with less experience in the culturing method.This assay is able to provide fast results in overviewing the general seafood quality and sanitation conditions before the seafood product is distributed.However, the result without confirmatory assay may overestimate the real condition of microbial contamination in the samples.In the monitoring report, the negative result in this presumptive assay can be reported as no contamination, but the positive result should be followed by a confirmatory assay to ensure that no false positive is produced.
We performed a molecular identification method to replace the common biochemical test as the confirmatory assay.This assay was used to overcome the false positive or negative results from the presumptive assay.The molecular method is based on a targeted PCR assay that has been validated by previous studies [4,[26][27][28][29][30][31].In this study, we observed that the high prevalence of Listeria by presumptive assay is contrary to the result from confirmatory assay.No Listeria monocytogenes contaminated both fish and environmental samples, as shown in Table 3.The PCR assay for L. monocytogenes in this study utilized a specific target gene of Hyla at a size of 456 bp PCR product (as shown in Fig. 3) that are commonly used to identify L. monocytogenes species [30]; other Listeria species is unlikely being detected by this assay.Listeria spp.Including non-pathogenic species, i.e., L. innocua, L. seeligeri, and L. ivanovii, were frequently isolated from either fish or fish markets/landing site environments [34,35].Listeria species can naturally occur in the environment; hence, it is likely to isolate Listeria spp in the samples during our observation.The high prevalence of V. parahaemolyticus (33.33% (8/24) and E. coli (29.17% (7/24) in the environment samples was observed in samples from PPI ERI (Table 4).The occurrence of V. parahaemolyticus in these samples was possibly due to their natural inhabitant in marine and brackish water [9,36].In fish particularly, the contaminations of V. parahaemolyticus are commonly found in the fish surface, gills, and viscera due to washing by seawater [37] or direct contact with contaminated equipment during handling and processing [9].Meanwhile, the presence of E. coli in the environmental samples, including fish handling facilities (i.e., fish basket and boat deck), can be correlated with poor sanitation and hygiene practices in PPI ERI.E. coli and coliforms are bacteria that are naturally present in the intestines of warm-blooded animals, including humans.Their presence in seafood indicates fecal contamination, which can occur due to poor sanitation and hygiene practices during food processing or handling [32].PPI ERI is categorized as a traditional fish landing site with limited handling facilities and clean water supply; thus, sometimes, the fisherman directly sold the fish outside the landing site facilities (as shown in Fig. 4).Based on the current study and previous control systems performed by the local authorities, we suggested conducting more frequent fish sampling and including V. parahaemolyticus as one compulsory parameter of fish testing on fish landing sites or fish markets.Additionally, the implementation of Good Handling Practices (GHP), Good Manufacturing Practices (GMP), Standard Sanitation and Operational Procedure (SSOP), and Hazard Analysis Critical Control Points (HACCP) during handling and processing [9,38,39] should be carried out by the relevant parties.The government has established several regulations related to food safety and control systems, such as Government Regulation Number 57 of 2015 on "Quality Assurance System and Safety of Fisheries Products and Enhancement of Fisheries Product Value" [40], Government Regulation No. 86 of 2019 on "Food Safety," Indonesian Food and Drug Monitoring Agency Regulation Number 13 of 2019 on "Maximum Limits of Microbial Contamination in Processed Foods," and Minister of Marine Affairs and Fisheries Regulation Number PER.19/MEN/2010 on "Control of Quality Assurance System and Safety of Fisheries Products."These regulations can serve as the basis for all stakeholders to ensure the safety of fish products in Indonesia.
The implementation of these regulations also needs to be accompanied by the development of adequate fish-handling facilities and infrastructure.For example, the indication of poor sanitation and hygiene practices in PPI ERI (indicated by the high microbial prevalence of E. coli in the environmental sample) may be due to the lack of sufficient fish handling facilities.Insufficient facilities may result in improper cleaning of equipment, poor storage conditions, and inadequate waste management by seafood operators, among other issues.These factors can contribute to the contamination of foodborne pathogenic bacteria in seafood products and increase the risk of foodborne illnesses.

Conclusion
The finding of the current study highlights the importance of monitoring and surveillance programs to ensure the safety and quality of fish in landing sites, with the inclusion of V. parahaemolyticus and E. coli as compulsory parameters.Additionally, the development of adequate fish handling facilities and infrastructure is crucial to improve sanitation and hygiene practices in the fish landing site.The rapid assay can be used as an in situ presumptive assay for routine surveillance of V. parahaemolyticus, E. coli, and Salmonella typhimurium in fish and environmental samples.However, further confirmatory assay, such as the PCR method, is required to avoid overestimation of microbial contamination in the sample due to false positive results.

*Fig. 1 .
Fig. 1.The total volume and value of fish exported from Maluku in 2019

Fig. 3 .
Fig. 3.The interpretation of positive result on PCR assay of V. parahaemolyticus (A); L. monocytogenes and Salmonella typhimurium (B); and E. coli (C)

Table 1 .
Primer pairs used in the PCR assay targeting Listeria monocytogenes, Vibrio parahaemolyticus, Salmonella enterica typhimurium, and E. coli.

Table 2 .
The prevalence of microbial contamination on fish products from Ambon during monitoring

Table 3 .
The presumptive detection of targeted bacteria in fish and environmental samples using rapid dry sheet method

Table 4 .
Result of confirmatory assay using PCR method targeting specific bacteria.