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1 Monitoring pathogenic bacteria in fish processing Wednesday, February 25, :00 IST S Visnuvinayagam, Binsi P K, Viji P and Thriveni Adiga Introduction Fish meat spoils the quickest compared to other animal s meat. The quality of fish meat also varies based on difference - species, age and sex. Moreover, the autolytic and microbial enzymes present in fish play a huge role in variation of the fish quality. Hence, controlling the quality of the fish is difficult. Next to spoilage, fish is a potential source of pathogens, unless monitored properly. Pathogens enter the food either pre- or post-processing, and its existence in fish is hazardous. So, it is difficult to provide safe and wholesome food without a stringent hygienic and sanitary practice. Microbiological techniques are the tools for estimation of bacterial species present in the fish. Consideration of few bacterial species is adequate to evaluate the condition of the microbial load. These bacterial species are called as indicator species, which directly measure the total microbiological status in food materials. Quality assurance systems Codex Alimentarius Commission (CAC) stated that the following three practices were mandatory to achieve good hygienic and sanitary conditions: Good manufacturing practices (GMP); Good hygiene practice (GHP); and Hazard analysis and critical control point (HACCP) GMP is an overall management activity to reduce the contamination and provides a safe product to consumers. GHP is a part of GMP concerned with hygiene, microbial safety and product spoilage. Although GHP and GMP furnish the detailed procedure, obtaining a zero risk-free product is questionable. Hence, to ensure 100 per cent safety, we need to follow the HACCP. It is a systematic approach to identify the hazards in every stage of the product, and thus, starts from the raw material to the final packing section, and possible control over the hazards towards elimination. Possible chance of contamination Personnel: Inadequate, or the absence of, hand-washing is the most common cause for the transmittance of diseases, especially by ill persons. For instance, low doses of Shigella and pathogenic Escherichia coli may spread from infected handlers and cause severe infection. Equipment: The equipment that regularly come into contact with the fish have to be cleaned adequately. Unclean or inadequately-cleaned containers, pumps and transport material are major sources of contamination. Pests: It has been recorded that the wandering of rodents and birds leads to infection, because they are the carriers of most of the pathogens. The presence of insects in the environment allows small birds to occupy the processing plants. Water: Water is potential source of contamination. Numerous outbreaks are recorded worldwide due to contaminated water. Hence, clean potable water should be used to clean the equipment and fish, and the preparation of ice. Monitoring Swabs have to be collected from the surface of all equipment in each section, starting from the raw material receiving section to the final product. Likewise the fish handlers hand swabs also have to be collected and tested for the presence of bacteria. Simultaneously, fish samples must also be collected to find source of contamination. All the swab and fish samples have to be assessed as per the standard procedure.

2 Interpretation results Based on the microbiological results, we can identify the probable source of origin of pathogens. Some pathogenic bacteria are always found in the fishes, even before catching. These are called as indigenous bacteria. Other types of bacteria usually arise as contaminants either after catch or while processing. These are called non-indigenous bacteria. Table 1 provides the details for the product exposure to temperature, and the cumulative value is needed for toxin production, which helps to prevent the product to develop such toxins. Indigenous bacteria These are commonly present in the skin, muscles, intestine of the fish or the marine environment, and most of them are commensal in nature; remaining bacteria are weaker spoilage. A few of these bacteria, such as Vibrio parahaemolyticus, V vulnificus, Clostridium botulinum type E and Aeromonas, are pathogenic. C botulinum: It is widely distributed in the aquatic environment and fish. Its occurrence is more common in a colder environment. Based on toxin production, it is classified into five toxin types (A-F); among the five toxins, C botulinum type E is most commonly present in fish, which is non-proteolytic, sensitive to heat and less salt tolerant. But, non-proteolytic toxin is dangerous; since, it is non-proteolytic, no foul smell could be observed from the product. Hence, the consumer usually consumes the food without notification. C. botulism is a severe problem in canning industry; but very meagre in other food items (Huss, 1980). Vibrio species: These are the common bacterial species associated food poisoning, most of the Vibrio species are non pathogenic and few of them are potential pathogens such as V. parahaemolyticus, V. alginolyticus; moreover, most of the Vibrios species are heat sensitive and destroyed by cooking temperature except V. cholera; which is stable in boiling temperature upto 8 min, 25 min in steaming. In addition it also possess very shortest generation time i.e., 8 9 min. Aeromonas: These species are important food spoilage bacteria belonging to vibrioceae family which are ubiquitous in marine environment. A. hydrophilia is an important pathogen in this genus and it is able to grow even in 0o C. Even though it is sensitive to acid and salt, most of the A. hydrophilia food poisoning outbreaks are reported, when the ph of the food is more than 6.5 with less than 3% salt. Plesiomonas sp: These are ubiquitous in marine environment and belong to the vibrioceae family. Since, it is mesophilic in nature, most of the outbreak occurs in the summer season only; especially, shell fishes like clam and oysters are playing a major role for the outbreak. Listeria monocytogenes: It is widely spread in soil, milk, fish and other food items, particularly common in cold smoked fish products. Owing to its robust nature, it can grow even in low temperature (4oC) with wide range of ph as well as salt concentration (10% NaCl). Moreover, nitrite is not inhibitory to L. monocytogenes at permitted levels, unless there is an interaction with other inhibiting agents (Shahamat et al. 1980). 1. Non-indigenous bacteria These are not inhabitant of marine environment. So, arrival of these bacteria happens mostly as contaminants by following practices such as storage of fish in uncleaned storage tank in fishing vessels, involvement of unhygienic fish handlers, cleaning of fish in polluted water, improper icing, prolonged storage and improper handling of the fish. Examples of such nonindigenous bacteria are S. aureus and faecal indicators such as E. coli, faecal streptococci (FS) and sulphite reducing clostridia (SRC). Staphylococcus aureus: It is the most common food poisoning bacteria present as contaminants in fish. Most of the unhygienic food handlers and their uncleaned vessels play a major role for transmission of S. aureus. In the world, 60% of fisherman population harbours S. aureus in its skin; about 50% and 20 % of the human population harbours S. aureus in the skin and intestine respectively (Doyle et al, 2001). Faecal indicator organisms: Faecal indicator bacteria are commonly present as commensal bacteria in warm blooded animal s intestine viz., E. coli, faecal streptococci (FS) and sulphite reducing clostridia (SRC). Hence, presence of these bacteria in the water or food materials indicates the faecal contamination. E. coli are primary indicators for faecal contamination; but exist in environment for shortest period, rapidly destroyed while freezing; moreover the possibilities of E. coli may come from nonfaecal origin. So, thermostable E. coli is another suitable indicator than E. coli for the assessment of the faecal contamination. Since, it arrives from intestine of the warm blooded animal, it can be able to produce the gas at 44.5oC in 24 hrs, which is called as Eijkman s test. Faecal streptococci are another indicator for the faecal contamination, it can present in the

3 environment little longer period in environment than E. coli and also it will not be destroyed rapidly while freezing; possessing of these characters, faecal streptococci considered as a good indicator for water and food. Sulphite reducing clostridia (SRC) and its spore are ubiquitous in aquatic sediments, existences of these SRC indicates the remote or non-point faecal pollution. It is also used to evaluate the virus and cyst inactivation in the drinking water disinfection processes. The SRC is a suitable indicator for assessment of faecal pollution especially a remote origin; the following characters make the SRC act as best faecal indicators (i) Abundantly present in all warm blood animal faeces (ii) Highly stable in water (iii) Highly resistance to the disinfection processes and (iv) Easy to isolate from the sample. So, SRC is generally used in the assessment of public health risks associated with faecal contamination in or of freshwater ecosystems. Control The growth of bacteria is mostly based on the suitable ph and temperature; other factors like water activity and salt concentration also a limiting factor for the growth of bacteria. Even though, presence of non-indigenous bacteria in fish or instruments in the meagre level, chance of growth while processing the fish due to suitable temperature and ph. The table 1 & 2 prepared by the USFDA clearly describes various temperatures at which these bacteria can be killed. Conclusion Pathogenic bacteria exist in the fish processing unit can be identified by sampling at different sites with and without food materials. Presence of microbial indicators would suggest the possible source of contamination. Once the processing instruments are contaminated with certain types of pathogens like Listeria monocytogenes, it needs thorough cleaning to eliminate the pathogens. Sampling after cleaning and sanitation also provides the data for the efficiency of cleaning; hence, modification in cleaning and sanitation protocol can be tailored as per the existence of the microbial indicators. Table: 1 Control of the bacterial growth and their toxin formation: Potentially Hazardous Condition C. botulinum type a, and proteolytic C. botulinum type e, and non-proteolytic Product Temperature C Above 21 C C 6-10 C 21 C C. Perfringens C C C Maximum Cumulative Exposure Time 11 hours 7 days 11 hours 1 day 1 Escherichia coli Listeria monocytogenes C >21 C C 6-10 C 5 hours 7 days 1 day

4 Salmonella species Shigella species Staphylococcus aureus C >30 C C C 7-10 C 7 hours 3 hours 1 hour 5 hours 5 hours 14 days 1 Vibrio cholerae 10 C ( C >27 C V. parahaemolyticus 5-10 C C 3 hours 1 hour2 Vibrio vulnificus Yersinia enterocolitica >27 C 8-10 C C > 27 C C >21 C 1 hour2 1 hour2 1 day 2.5 hours

5 Table: 2 Factors limiting the growth of bacteria and their toxin PATHOGEN Min aw (using salt) Min ph Max ph Water phase Min temp Max temp Bacillus cereus C 55 C Campylobacter jejuni C 45 C C. botulinum, type a, & proteolytic C 48 C C. otulinum, type e, & nonproteolytic C 45 C Clostridium perfringens C 52 C Pathogenic strains of Escherichia coli C 49.4 C Listeria monocytogenes C 0.4 C Salmonella spp C 46.2 C Shigella spp C 47.1 C Staphylococcus aureus growth C 50 C Staphylococcus aureus toxin formation C 48 C Vibrio cholerae C 43 C Vibrioparahaemolyticus C 45.3 C Vibrio vulnificus C 43 C Yersinia enterocolitica C 1.3 C References APHA (American Public Health Association), Compendium of Methods for the Microbiological Examination of Foods, 3 Edition. Washington, DC CAC (Codex Alimentarius Commission), Hazard Analysis and Critical Control Point (HACCP)-System and Guidelines for its Application. Codex Alimentarius Commission CAC/RCP , Rev. 3, Rome Doyle, M E., Faye, A, Hartmann, Amy C and Lee Wong, White Paper on Sources of Methicillin-Resistant Staphylococcus aureus (MRSA) and Other Methicillin-Resistant Staphylococci: Implications for Our Food Supply? Fri food safety reviews. Food Research Institute, UW Madison United States FDA, 2011: US Food and drug administration, Bacterial Pathogen Growth and Inactivation, Fish and Fishery Products Hazards and Controls Guidance /guidanceregulation/ guidancedocumentsregulatoryinformation/seafood/ucm htm Fish and fishery products hazardous and control guidance, 4th edition. April 2011 by Department of health and human science, Food and drug administration and Control for food safety and applied nutrition office of food safety Jay, J M, Modern Food Microbiology. Microbiological Indicators of food safety and quality, Principles and Quality control, and microbiological criteria. New York: Van Nostrand Reinhold Samakupa, A P, Hygiene indicators in a fish processing establishment-a case study in a white fish processing establishment. University of Namibia, Department of Natural Resources, P/Bag 13301, Windhoek, Namibia WHO, Guidelines for drinking-water quality. World Health Organisation; Geneva Slanetz, L W and Bartley, C H Numbers of enterococci in water, sewage, and feces determined by the membrane filtration technique with an improved medium. J. of Bact. 57:

6 (Visnuvinayagam and Binsi are scientists, and Adiga is assistant chief technical officer, Mumbai research centre, Central Institute of Fisheries Technology [CIFT], Vashi, Navi Mumbai. They can be contacted at