Significance of Cross-contamination on Bacteriological Quality of Black Tiger Shrimp (Penaeus monodon Fabricius 1798) for Export Trade Produced in Fish Processing Plant

Shrimp is the major value added exported fishery product of India [1]. Southeast Asia continued to be the largest buyer of Indian marine products with a share of 25.75% in US$ realization and 39.58% in quantity. Exports to Southeast Asia have shown a growth of 43.19% in quantity and 95.99% in US$ realization. European Union ranks second in terms of frozen shrimp export with a share of 22.02%, followed by USA 19.17%, Japan 14.09%, China 7.06%, Middle East 4.39% and other countries 7.51% [2]. In Indian shrimp culture scenario, West Bengal is the second dominant shrimp producing state across its 158 km coastline [1]. The dominant species of shrimp, Penaeus monodon Fabricius 1798 (black tiger) are extensively cultured in the coastal area of West Bengal due to its unique taste, high unit value and everexpanding export demand in the global market.


Introduction
Shrimp is the major value added exported fishery product of India [1]. Southeast Asia continued to be the largest buyer of Indian marine products with a share of 25.75% in US$ realization and 39.58% in quantity. Exports to Southeast Asia have shown a growth of 43.19% in quantity and 95.99% in US$ realization. European Union ranks second in terms of frozen shrimp export with a share of 22.02%, followed by USA 19.17%, Japan 14.09%, China 7.06%, Middle East 4.39% and other countries 7.51% [2]. In Indian shrimp culture scenario, West Bengal is the second dominant shrimp producing state across its 158 km coastline [1]. The dominant species of shrimp, Penaeus monodon Fabricius 1798 (black tiger) are extensively cultured in the coastal area of West Bengal due to its unique taste, high unit value and everexpanding export demand in the global market.
Most of the farm-raised products of shrimps are being processed as either block frozen or individually quick frozen (IQF) for export to various countries in Middle East Asia, Europe, Japan, and USA [3]. Frozen shrimp in India continued to be the major export item accounting for 51.35% of the total foreign exchange earnings. Shrimp exports from India during 2010-2011 increased by 17.67%, 34.77% and 32.98% in quantity, Indian rupee value and US$ value respectively than the previous year [2]. In past decades, the frozen shrimp exported from India had faced many problems such as high bacteriological count in the cooked and peeled frozen shrimps, as well as the presence of other pathogenic bacteria such as Salmonella [3]. Food security is a complex issue, where animal proteins such as fish and fishery products are generally regarded as high risk commodity with respect to pathogen contamination, presence of natural toxins and adulterants [4]. Moreover coliforms, especially Escherichia coli are the most alarming concern in all types of food products as their presence reflects the unhygienic handling during production process, improper storage conditions and post-process contamination [5][6][7]. It is important to note that cross-contamination of pathogenic bacteria in the finished shrimp products is generally introduced by human contact in the various steps of the process flow. The problem of cross-contamination in relation to potential transmission of pathogens to food is a serious issue in food processing and allied industries. The possible route of pathogenic bacterial contamination in shrimp products takes place either by direct contact with contaminated containers or through airborne particles. Some potential pathogenic bacteria are attached to the surfaces of the products as their predominant form of survival in nature and man-made ecosystems [8]. Several studies indicated that various bacteria including Escherichia coli, Staphylococcus aureus and Salmonella spp., survive on hands, cloths, utensils etc. after initial contact with the microorganisms [9][10][11].
Augmented import of seafood from developing countries resulted in the acceptance of international guidelines for food processing

Bacteriological procedures
The frozen samples were thawed before analysis by overnight storage at 5°C in a refrigerator. After that total plate count (TPC), detection of food borne pathogenic bacteria such as V. cholerae, E. coli, S. aureus, Salmonella spp and L. monocytogenes were undertaken as part of this study as per the instruction mentioned in Bacteriological Analytical Manual of Food and Drug Administration [22]. For swab test, solutions of peptone (1 g L -1 ) and sodium chloride (8.5 g L -1 ) were prepared. The solutions were stored in bottles and were sterilized for 15 minutes at 121°C.  [22]. For Salmonella detection, Rappaport-Vassilliadis (RV) broth (HiMedia, Mumbai) and tetrathionate broth (TTB) (HiMedia, Mumbai) were used for selective enrichment after pre-enrichment in lactose broth. Selective media used were xylose lysine deoxycholate (XLD) agar, bismuth sulphite agar (BSA), and hektoen enteric agar (HEA) (HiMedia, Mumbai). For detection of L. monocytogens, Listeria enrichment broth (BLEB) (HiMedia, Mumbai) was used after pre-enriched at 30°C for 4 h [22].

Statistical analysis
The data obtained in the present investigations were subjected to one-way Analysis of Variance (ANOVA) using statistical software Medcalc® version 12.7.0. (MedCalc Software bvba, Ostend, Belgium). Tukey's (HSD) test was used to determine the differences among the means. Significant differences are stated at P<0.01 level.

Microbial analysis to determine the bacterial load in receiving table, panning crate and workers' hands before and after cross-contamination
Total plate count values of all cases were found to be significantly much higher (P<0.01) in cross-contaminated samples than in preprocessing and processing areas. After cross-contamination, the TPC value was found to be highest (P<0.01) in panning crates (394.66 ± 90.38 cfu sq cm -1 ) followed by worker's hands (273.33 ± 154.38 cfu sq cm -1 ) and receiving tables (80.56 ± 5.98 cfu sq cm -1 ) respectively. In pre-processing areas, the TPC was highest in panning crates (61.33 ± 11.23 cfu sq cm -1 ) followed by receiving tables (60.66 ± 8.50 cfu sq cm -1 ) and workers' hands (48.33 ± 9.29 cfu sq cm -1 ) respectively. In processing areas the lowest TPC was observed in receiving tables (4.33 ± 2.51 cfu sq cm -1 ) followed by workers' hands (9.66 ± 2.88 cfu sq cm -1 ) and panning crates (11.00 ± 3.60 cfu sq cm -1 ) respectively.

Microbial analysis of water and ice samples before and after cross contamination
V. cholerae was not detected in all the samples and E. coli were positive in cross-contaminated samples. High TPC values were observed (P<0.01) in cross-contaminated water and ice samples than such as Hazard Analysis Critical Control Points (HACCP) and European Union (EU) guidelines. In an attempt to control the microbial contamination of foods, various international agencies have recommended microbiological criteria for assessing the effectiveness of HACCP program [12]. If coliforms were effectively destroyed at the pasteurizing step, still there was a possibility to detect their presence in the cooked products. These cases were also noticed when Good Management Practice (GMP) and HACCP programs are implemented to access best hygienic conditions for human consumption. As the bacterial contamination come from various sources in the processing work flow, it is extremely important to identify the possible route of cross-contamination to establish effective control process of the bacterial contamination and strengthens the GMP and HACCP programs [13]. There are some reports on the bacteriological assessment of frozen raw, processed IQF and ready to eat shrimps produced from processing plant in India [3,[14][15][16]. Rao et al. [17] studied the seasonal variations in the supply of raw materials at processing plants located in Cochin, Veraval and Kakinada in India. Iyer et al. [18] observed that bacterial content and handling of raw materials influence the bacteriological quality of frozen shrimps. Reilly et al. [19] reported microbiological changes of insufficiently iced and improperly stored shrimps at higher temperatures. Zuberi and Qadri [20] showed the important role of microorganisms in the deterioration of shrimp quality. Jeyasekaran et al. [21] reported the incidence of Listeria monocytogenes in seafood landed at the Mangalore coast of India. However, there are no such studies on differences of bacteriological load in the shrimp products before and after the food processing work flow. In continuation to the fact, the main objective of this study is designed to access the bacteriological quality of various processing steps, raw and processed black tiger shrimp in a processing plant following the HACCP guidelines.

Study area and sampling design
The experimental work was carried out in a processing plant located at East Kolkata Township (22° 30 ' 48.07 '' N and 88° 24 ' 06.43 '' E), West Bengal, India with an approved HACCP plan. The processing plant has been regularly monitored and verified by respective competent authority.
Samples (N=24; three samples from eight treatments) were taken for microbiological evaluation such as total plate count (TPC), presence of V. cholerae, E. coli, S. aureus, Salmonella spp and L. monocytogenes in order to access the bacteriological quality of crosscontaminated condition and also the effectiveness of HACCP program. All samples were collected aseptically from pre-processing and processing areas according to the recommendation of USFDA [22]. Cross-contaminations of equipments were established by transferring them from processing to pre-processing areas at considerable distance and in direct contact with contaminated surfaces. Water and ice were also moved from processing to pre-processing areas and were placed in contaminated surface for long duration. The distance from preprocessing and processing area of the study design was about 115 meter. Bacteriological analysis was undertaken from three important steps of the process flow viz., i) receiving table, panning crate (a metal container or tray) and workers' hands ii) water and ice sample; and iii) head on black tiger shrimp (HOBT; shrimp with whole body parts), headless black tiger shrimp (HLBT; shrimp without head) and processed shrimp product from pre-processing and processing areas after cross-contamination. samples from pre-processing and processing areas, such as 122.50 ± 25.35 cfu ml -1 and 271.66 ± 53.79 cfu ml -1 respectively. Bacterial load in water samples from pre-processing and processing areas was 15.33 ± 6.02 cfu ml -1 and 7.66 ± 5.50 cfu ml -1 respectively. In ice sample, TPC was lowest in processing area (12.33 ± 6.02 cfu ml -1 ) followed by preprocessing area (16.33 ± 3.78 cfu ml -1 ) but did not differ significantly (P>0.01). E. coli in cross contaminated water and ice sample was 17.0 cfu ml -1 and 19.0 cfu ml -1 respectively. However, E. coli were not detected in samples collected from pre-processing and processing area.

Discussion
Pérez-Rodríguez et al. [23] defined cross-contamination as "a general term which refers to the transfer, direct or indirect, of bacteria or virus from a contaminated product to a non-contaminated product". According to the World Health Organization [24], 25% of foodborne outbreaks are closely associated with cross-contamination events involving scarce hygiene practices, contaminated equipment, and contamination via food handlers, processing, or inadequate storage. Chen et al. [25] demonstrated that bacterial transfer rates varied by more than five orders of magnitude depending on the nature of the surfaces involved in the cross-contamination. According to him a transfer rate of 100% was identified as the maximum value, although in some cases it was found that the recipients demonstrated higher numbers of microbial load than the contributor of contamination. Our study clearly indicated that maximum microbial load in crosscontaminated condition due to transfer or shifting of the place, equipments and products from the concerned place to another places.
The total bacterial load in all the evaluated samples was high in cross-contaminated condition. In pre-processing, processing and cross-contaminated samples, the value was not exceeding 10 4 cfu g -1 , which is lower than the standard limits of 5 × 10 5 cfu g -1 as per International Commission on Microbiological Specifications for Foods Standards [26,27] and Export Inspection Council, India [28,29]. Most of the previous studies indicated that bacterial load in fresh tropical shrimps ranged from 10 3 to 10 5 cfu g -1 [20,[30][31][32][33][34].
The presence of E. coli in ready-to-eat foods is undesirable because it indicates poor hygienic conditions during processing. In the present study, E. coli were positive in cross-contaminated samples. In water and ice samples E. coli levels exceed ICMSF standards but within marginally acceptable range, however in HOBT, HLBT and processed shrimp products E. coli was within ICMSF acceptable limits i.e., 11 cfu g -1 . Ideally E. coli should not be detected and as such a level of <3 g -1 has been given as the satisfactory criteria for this organism. Levels exceeding 100 g -1 are unacceptable and indicate a level of contamination [27]. This finding substantiates with the study of Iyer and Shrivastava [14] and Antony et al. [35]. Jeyasekaran et al. [36] reported that E. coli in shrimp ranged from 0.6 to 240 cfu g -1 . Iyer et al. [18] also stated that season plays a role in controlling the bacterial quality of raw shrimps and observed that the bacterial counts were higher in certain specific seasons.
V. cholerae, S. aureus, Salmonella spp and L. monocytogenes were below detection limit in all the samples. Varma et al. [37] documented that out of the 7,238 tested shrimps, V. cholerae was present in only one raw shrimp. Antony et al. [35] also found similar results. ICMSF acceptable range of S. aureus was <1.0 × 10 3 cfu g -1 , however, levels of ≥10 4 cfu g -1 are considered hazardous as foods with this level of contamination may result in food borne illness if consumed. Hatha et al. [3] detected Staphylococci less than 1% of the IQF shrimp samples. Ready-to-eat foods should be free of Salmonella as consumption of food containing this pathogen indicates poor food preparation and handling practices or cross-contamination which may result in food borne illness. Their presence in high numbers (>10 4 g -1 ) in ready-toeat foods indicates that an unacceptable level of contamination has occurred. Consistent with the present study, Chen et al. [38] while analyzing bacteriological quality of shrimp, did not find Salmonella and Antony et al. [35] also observed similar findings. In Thailand, Dalsgaard et al. [39] also reported that Salmonella was not detected from shrimps. However, there were very few reports on the incidence of Salmonella in fish and fishery products in India [40]. Bhaskar et al. [41] reported the incidence of Salmonella from aquacultured shrimps. Food under refrigeration should be free from L. monocytogenes. However levels of 10 2 cfu g -1 indicate a failure with food handling controls and are considered a public health risk [42]. The present study is well supported by earlier studies of Manoj et al. [43], Kamat and Nair [44], Antony et al. [35] and Prabakaran et al. [16]. However, Jeyasekaran et al. [21] reported the incidence of L. monocytogenes in raw shrimps. As humans are the major source of contamination in the processing environment [45], good private hygiene and adherence to GMPs are a must to control contamination. Wearing of nose-mouth cover and periodical dipping of workers' hands in chlorinated water must be strictly followed in the processing unit [3].
The present study indicated that microbial analyses of all the samples obtained from the processing areas were within the safe limits suggested in the guidelines by competent authority [26][27][28] and these values were far lower than pre-processing areas. This finding reveals that the adherence to Current Good Manufacturing Practice (CGMPs) and HACCP plan greatly enhances the quality of food products in the processing plants. The sanitary conditions of seafood processing plant are also associated with the microbial quality of finished products [46,47].
Though raw shrimps were procured exclusively from farms, the chances of natural contamination were expected in case of samples from polluted coastal waters [48]. However, the raw material could pick up microbial load during transportation and storage. Excellent temperature control of the raw material (less than 5°C) during preprocessing might be able to control the multiplication of mesophilic microflora present in the raw material [3].

Conclusion
The risk of food-borne infection associated with crosscontamination depends on two factors: the level of contamination on the surfaces and the probability of its transfer to the foods being consumed [49]. The present microbiological survey revealed that there is an urgent need for considerable improvement in the bacteriological quality of products and it should be free from cross-contamination. Strict adherence to CGMPs and HACCP plan can greatly augment the product quality and minimize the risk of cross-contamination. The processing plants should take paramount care while collecting raw specimens to produce uniformly high quality product. Prior to food preparation, all workers' hands should be free of all test microorganisms and changing of equipments from low risk zone to high risk zone should be avoided. Regular training programs should be organized for processing workers for effective implementation of HACCP programme and also to meet the requirements of European Commission and the other international food regulating bodies. Further studies on quantifying the cross-contamination risk associated with the various steps in the food preparation process can provide a scientific basis for risk management efforts in shrimp processing industries.