PREVALENCE OF HYDROGEN SULFIDE PRODUCING PSYCHROPHILIC BACTERIA IN CHILLED MUGIL CEPHALUS “MULLET” FISH AND THEIR PUBLIC HEALTH SIGNIFICANCE.

Dept. Food Hygiene, Port-Said Lab.,

Animal Health Research Institute, Dokki, Giza, Egypt.

PREVALENCE OF HYDROGEN SULFIDE PRODUCING PSYCHROPHILIC BACTERIA IN CHILLED MUGIL CEPHALUS “MULLET” FISH AND THEIR PUBLIC HEALTH SIGNIFICANCE.

(With 4 Tables)

By

H.E.M. FARAG and NAHLA T. KORASHY*

* Dept. of Microbiology, Port-Said Lab., Animal Health Research Institute, Dokki, Giza, Egypt.

(Received at 22/4/2009)

مدي تواجد البکتيريا المحبة للبرودة المنتجة لکبريتيد الهيدروجين في أسماک البوري المبرد وتاثيرها علي الصحة العامة

حسن السيد محمد فرج ، نهلة طه عبد الجواد قرشي

في دراسة لتحديد مدي تواجد البکتيريا المحبة للبرودة المنتجة لکبريتيد الهيدروجين تم فحص خمسون عينة صالحة ظاهريا للاستهلاک الادمي من أسماک البوري المبرد  والتي تم جمعها من أسواقِ مدينة بورسعيد بهدف عد وعزل وتصنيف البکتيريا المحبة للبرودة المنتجة لکبريتيد الهيدروجين. اظهرت النتائج ان نسبة العينات الايجابية للبکتيريا المحبة للبرودة المنتجة لکبريتيد الهيدروجين کانت 100 % (50) وکان متوسط العد الکلي للبکتيريا المحبة للبرودة والعد الکلي للبکتيريا المحبة للبرودة المنتجة لکبريتيد الهيدروجين 2,1X  ⁵10 ± 2,9  X⁴10 و 1,8X ³10 ± 3,3X  ²10 خلية/ جرام في العينات موضع الدراسة على التوالي. وکان متوسط نسبة البکتيريا المحبة للبرودة المنتجة لکبريتيد الهيدروجين الي نسبة  البکتيريا الکلية المحبة للبرودة 0,86 %. تم تصنيف عترات البکتيريا المحبة للبرودة المنتجة لکبريتيد الهيدروجين المعزولة من أسماک البوري المبرد الي انواع من سودوموناس,  سودوموناس فلوريسنس وشيوانيلا بيتروفيکانس وکانت بنسبة 77,78% , 14,44% و 7,78% علي التوالي. تم مناقشة تاثير البکتيريا المحبة للبرودة والمنتجة لکبريتيد الهيدروجين علي الصحة العامة.

SUMMARY

Fifty samples of chilled Mugil cephalus “Mullet” were randomly purchased from Port-Said markets. The samples were examined for enumeration and isolation of hydrogen sulfide producing psychrophilic bacteria. The incidence of positive samples for hydrogen sulfide producing psychrophilic bacteria was 100% (50). The mean values of the total psychrophilic bacterial counts and hydrogen sulfide producing psychrophilic bacterial counts were 2.1 X 10⁵ ± 2.9 X 10⁴ and 1.8 X 10³ ± 3.3 X 10² CFU/g of chilled Mugil cephalus “Mullet” respectively. The incidence of hydrogen sulfide producing psychrophilic bacterial colony in compared to the total psychrophilic bacteria was 0.86%. The hydrogen sulfide producing psychrophilic bacterial isolates in the examined samples were identified as Pseudomonas spp., Pseudomonas fluorescens and Shewanella putrefaciens with an incidence of 77.78%, 14.44% and 7.78% respectively. The effect of the hydrogen sulfide producing psychrophilic bacteria on the public health was discussed.

Key words: Fish, Mugil cephalus, psychrophiles

INTRODUCTION

Fish is a very perishable, high-protein food that typically contains a high level of free amino acids and volatile nitrogen bases which are essential for human consumption, in addition to the high levels of hydrosoluble and liposoluble vitamins, minerals and polyunsaturated fatty acids (Ashie et al., 1996; Haugen and Undeland, 2003; Gonzalez-Fandos, et al., 2005).

Although ice storage has been the most widely used methods for the preservation of fresh fish (Chai and Levin, 1975) to keep it fresh during marketing, bacterial activity is generally accepted as the primary cause of fish spoilage (Shewan, 1961) depends on specific organisms, climates, storage conditions, the type of fish and even the place of fish was harvested (Gram and Huss, 1996).

Microbes can metabolize and degrade these compounds resulting in undesirable metabolites and spoilage of the fish (Haugen and Undeland, 2003; Olafsdottir et al., 2005). The main spoilage agents of chilled stored fresh fish are the bacteria that produce hydrogen sulfide (Satomi, et al., 2006).

Hydrogen sulfide producing psychrophilic bacteriais a group of bacteria including Shewanella and Pseudomonas specieswhich are the predominant species found as specific spoilage organisms in chilled fresh fish under aerobic conditions (Miller III, et al., 1973; Fonnesbech Vogel, et al., 2005; Hozbor, et al., 2006). Also some species of Aeromonaceae, Vibrionaceae and Enterobacteriaceae were able to produce hydrogen sulfide (NMKL, 2006).     

These types of psychrophilic bacteria are widely distributed in marine and freshwater environments. They are mostly Gram negative, motile rods, facultative anaerobic, non spore forming and catalase positive but oxidase and sugar fermentation were variable. The fresh isolates on iron media show black colonies due to the precipitation of the insoluble iron sulfide (Allen and Geldreich, 1975; Baumann and Schubert, 1984; Krieg, 1984; Popoff, 1984; Greenberg and Hunt, 1985; Gennari and Dragotto, 1992 Ziemke et al., 1999; Satomi, et al., 2006)

In spite of the hydrogen sulfide producing (HSP) bacteria constitute a minor fraction in the initial microflora of the newly caught fish (Jorgensen et al., 1988), they can cause organoleptic changes such as offensive, fishy, rotten H₂S off odors in the spoiled aerobically and cold stored fish (Adams, et al., 1964; Skjerdal et al., 2004).

Different illness in the consumer has been recorded by the hydrogen sulfide producing (HSP) bacteria. Chen et al., 1997recorded that Shewanella putrefaciens and Shewanella algae have been associated with septicemia, cellulitis, skin, and soft tissue infections. Other Shewanella spp., especially S. baltica, is commonly associated with food spoilage (Gram and Huss 1996). Patients with liver disease (Otsuka       et al., 2007) and the immunocompromised individuals appear to be at higher risk for shewanellosisis (Krsnik et al., 2002). Some Pseudomonas species has been transmitted by food affecting primarily immunocompromised people and those suffering from cystic fibrosis (Morais, et al., 1997) leads to several outbreaks (Pererra et al., 1977). Food borne Aeromonas causing food poisoning and traveler’s diarrhea (Cholera-like illness and dysentery like illness). Septicemia and peritonitis can be produced specially in malignancies and immunocompromised host (Palumbo, et al., 1992; FDA, 2001). Non-cholera Vibrio infections cause self-limiting gastroenteritis lasting 2-3 days and characterized by diarrhea, sometimes bloody stools, abdominal cramps, nausea, vomiting, headache and fever. While necrotizing fascitis and septicemia were associated with chronic liver diseases, adrenal insufficiency, portal hypertension and immunocompromised hosts (Morris, 2003; Yeung and Boor, 2004). The enterotoxigenic strains of the Enterobactericae have been isolated from infants and children with acute gastroenteritis while the endotoxins show a bacteraemia in human (Guentzel, 1982; APHA, 1984).

The objective of the present study was to determine safety of chilled Mugil cephalus “Mullet” fish for human consumption through the enumeration and identification of the psychrophilic and hydrogen sulfide producing psychrophilic bacteria and their public health significance.

MATERIALS and METHODS

1- Samples collection:

A total of 50 random samples of chilled Mugil cephalus “Mullet” were randomly purchased from Port-Said markets. Each individual sample was placed separately into sealed sterile plastic bag on ice, thoroughly identified and delivered to the laboratory to be examined bacteriologically for enumeration, isolation and biochemical identification of the isolated psychrophilic and hydrogen sulfide producing psychrophilic bacteria. All specimens were processed within 4 hours of collection.

2- Preparation, homogenation and enrichment of the samples:

A representative twenty five grams of fish sample were transferred aseptically to a stomacher bag (Seward medical, London, UK) containing 225  ml of sterile 0.1% (W/V) peptone saline (0.1% peptone + 0.85% NaCl) and homogenized for 60 second with stomacher (Lab. Blender 400, Seward Medical, London UK) at room temperature. Serial tenfold dilutions were made in the same dilution up to 10⁶ according to the BSI (1996) and ISO/DIS 6887-1 (1997).

3- Isolation and enumeration of psychrophilic bacteria:

0.1 ml of each serial dilution was spread thoroughly and uniformly onto duplicate marked Petri plates of plate count agar (PCA) using surface plate technique. All plates were inverted and incubated at 7^°C for 10 days. The psychrophilic bacterial count was calculated and expressed as colony forming units per gram (CFU/g) of fish (Cousin      et al. (1992).

4- Isolation and enumeration of hydrogen sulfide producing psychrophilic bacteria:

Another 0.1ml of the appropriate dilutions was spread onto duplicate marked petri plates of iron peptone agar (IPA). After solidification the plates were covered with a thin layer of the same growth media. All plates were inverted and incubated at 25^°C for 3 days. Plates containing 25-250 black colonies were counted. The hydrogen sulfide producing psychrophilic bacterial count was calculated and expressed as colony forming units per gram (CFU/g) of fish (Gram       et al., 1987; Gennari and Campanini 1991).

5- Purification and keeping the working isolates:

            Approximately 15 typical colonies were picked randomly each from plate count agar and peptone iron agar plates for each sample (Leroi et al., 1998). The picked colonies from plate count agar were purified by reinoculation in nutrient broth and incubated at 25^°C for 18-24 hr. A loopful of the inoculated nutrient broth was streaked onto nutrient agar slope (Murray et al., 2003). Meanwhile the black colonies from peptone iron agar plates were further purified in brain heart infusion broth supplemented with NaCl (0.5%) and streaked onto peptone iron agar slant (IAS) and incubated at 15^°C for 18-24 hr (Gibson and Khoury, 1986; Leroi et al., 1998). All purified isolates were maintained at 4^°C and subculture periodically each 3 weeks to ensure viability, till the morphological and biochemical identification of the isolates (Harrigan and McCance, 1966).

6- Morphological and biochemical identification of the Isolation:

            All isolates were morphologically and biochemically identified at 25^°C for Gram stain, cell morphology, oxidase and catalase reactions, motility, oxidation fermentation test (O/F test) and other biochemical tests according to Skerman (1967), Sneath et al. (1986) and Gram et al. (1987)

7- Statistical methods

Minimum, maximum, mean, standard deviation and standard error of mean as well as frequency distribution were used to describe data. T-test was used to evaluate the relationship between the number of psychrophilic bacteria and hydrogen sulfide producing psychrophilic bacteria in chilled Mugil cephalus “Mullet”. P value was considered significant if less than 0.05 and 0.01 at 95% and 99% respectively. These tests were analyzed using the Statistical Package for Social Scientists (SPSS) for windows 16.0 (SPSS Inc., Chicago, IL, and USA).

RESULTS

Table 1: Statistical analytical results of the psychrophilic and hydrogen sulfide producing psychrophilic bacterial count (CFU/g) recovered from chilled Mugil cephalus “Mullet” (n=50).

Min. = Minimum.  Max. = Maximum.  SE = Standard Error   SD = Standard Deviation.

Table 2: Frequency distribution of the examined Mugil cephalus based on their psychrophilic and hydrogen sulfide producing psychrophilic bacterial count.

Table 3: Relationship between the counts of psychrophilic and hydrogen sulfide producing psychrophilic bacteria recovered from chilled Mugil cephalus According to Chai, et al. (1968)

 ^(**) = Highly significant correlation at p <0.01 (99% confidence) by using t-test (Paired            system test).

Table 4: Incidence of psychrophilic and hydrogen sulfide producing psychrophilic bacterial isolates recovered from chilled Mugil cephalus

DISCUSSION

Hydrogen sulfide gram negative bacteria are commonly associated with the spoilage of foods. These bacteria usually constitute only a small fraction of the initial flora on newly caught fish but constitute a significant, sometimes dominant part of the microbiota during chilled storage and their numbers determine the shelf life of the product (Herbert et al., 1971; Jorgensen et al., 1988; Gram and Fonnesbech Vogel, 2000).

The obtained results in Table 1 showed that the mean values of the psychrophilic and hydrogen sulfide producing psychrophilic bacterial count of the chilled Mugil cephalus “Mullet” samples were 2.1 X 10⁵ ± 2.9 X 10⁴ and 1.8 X 10³ ± 3.3 X 10² CFU/g fish respectively. These results agree with the results recorded by Hobbs (1983) and Hayes, (1992) but lower than the results recorded by Chai, et al. (1968) and higher than the results recorded by Fonnesbech Vogel, et al. (2005). The variation between our results and the results recorded by other authors may be attributed to the variation between the species of fish and the season where the counts increased in summer (Chen and Chai; 1982 and Gram and Huss; 1996) the type of the specific spoilage organisms, the storage conditions and the place in which the fish was harvested (Gram and Huss; 1996).

Regarding frequency distribution of the examined samples presented in Table 2 it is evident that most of the examined chilled Mugil cephalus “Mullet” (62%)had hydrogen sulfide producing psychrophilic bacteria within the range of 2 X 10² - <10³ CFU/g  while 54% had psychrophilic bacterial counts within the range of 10⁵ - <10⁶ CFU/g. On the other hand, 38% and 46% of the examined samples had hydrogen sulfide producing psychrophilic bacteria and psychrophilic bacteria within the range of 10³- <10⁴ CFU/g and 10⁴- <10⁵ CFU/g respectively. The lower range of most of the examined samples (62%) for hydrogen sulfide producing psychrophilic bacteria may be attributed to the lower range of the psychrophilic bacteria than the level (10⁷-10⁹ CFU/g) at which typically grow of hydrogen sulfide producing psychrophilic bacteria occur (Shewan 1977). Also these levels are required to produce off-odors of the food sample (Jorgensen and Huss, 1989).

The results given in Table 3 reveal that the number of H₂S producing psychrophilic bacterial colonies per gram of fish was 0.86% of the total psychrophilic flora. This result was lower than the results recorded by Chai, et al. (1968). The lowering of this percentage (0.86%) may be attributed to the lower of hydrogen sulfide producing psychrophilic bacterial count of our results than the results recoded by the other authors.

Statistically by using t-test (Paired system test), a highly significant relationship between the counts of each of the psychrophilic and hydrogen sulfide producing psychrophilic bacteria of the examined chilled Mugil cephalus “Mullet” samples (Table 3). This means that the counts of H₂S producing psychrophilic bacterial could be used as indicators of spoilage of iced fish (Kyrana and Lougovois, 2002).

Table 4 showed that the isolated strains from the examined samples were identified as Pseudomonas spp, Pseudomonas fluorescens and Shewanella putrefaciens with an incidence of 77.78, 14.44 and 7.78% respectively.

Although Shewanella putrefaciens play a prominent role as spoilage organisms of fish and other food products (Gram and Dalgaard, 2002), it was found in low incidence (7.78%). This may be attributed to the inhibitory activity of the isolated siderophore producing Pseudomonas species (Gram, 1993), through the competition inhibition of iron (Henry et al., 1991) or due to the antibiotic activity of the siderophore (Neilands 1981). On the other hand the high incidence of the isolated Pseudomonas spp. (77.78 %,) may be due to their faster growth rates at chill temperature (Miller III, et al., 1973), their greater affinity for oxygen and as a consequence their catabolism of glucose and lactose (Miller III, et al., 1973; Gill and Molin 1991).

 In conclusion, to lower the number and activity of the psychrophilic and hydrogen sulfide producing psychrophilic bacterial count and prevent their spoilage effect, a good fish handling practices include: icing or rapid immersion of the catch in water chilled to -1^°C, good time/temperature storage besides prevention the cross and secondary contamination, strictly hygienic measurement for prevention and removal the source of pollution from the harvested and storage sites.

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