BACTERIOLOGICAL SAFETY OF FRESH FISH FROM URBANAND RURAL AREAS SOLD AT MANSOURA CITY

Assiut University web-site: www.aun.edu.eg

BACTERIOLOGICAL SAFETY OF FRESH FISH FROM URBANAND RURAL AREAS SOLD AT MANSOURA CITY

EL-DOSOKY, H.F.A. and SHAFIK, S.

Animal Health Research Institute, Mansoura Provential Lab., Department of Food Hygiene

Received: 12 January 2017;       Accepted: 2 March2017

INTRODUCTION

Fish can contribute to a higher level of food safety and security by providing protein of high quality, essential fatty acids, vitamins and minerals. Italso plays an important role in the economy of many countries by increasing employment opportunities. The whole world fish production reached 52.5 million tons in 2008, responsible for 45.5% of the world food fish consumption (FAO, 2012). Furthermore, fish is eaten in many ways including smoked, cooked and raw. However, it has been shown that fish may be a  source of food borne illness, causing outbreaks, this has made consumers more aware and has therefore become an important public health issue, which in many cases were neglected (EFSA, 2010).

Fish could be spoiled from both outer and inner surfaces as fish stomach and intestine. After fish is being caught and dying, the immune system collapses and bacteria allowed to proliferate freely on the skin surface and viscera, penetrating the intestinal walls to move into the flesh  through  the  muscle  fiber  where

Corresponding author:  Dr. EL-DOSOKY, H.F.A.

E-mail address: rafat552008@yahoo.com

Present address: Animal Health Research Institute, Mansoura Provential Lab., Department of Food Hygiene

the intestinal microflora is the main causative agent of fish spoilage (Kaneko, 1971), besides in 2004, Novotny et al. found many listed pathogenic bacteria in fresh water fish including Staph. aureus, E.coli, Salmonella, Cl. Botulinum, L. monocytogenes.

MATERIALS AND METHODS

A-Collection of samples: A total number of 200 freshwater fish samples including 100 Tilapia nilotica  fish (50 from rural and 50 from urban areas) and100 Mugil cephalus fish (50 from rural and 50 from urban areas) were collected from different fish markets at Mansoura city. The collected samples were kept in an insulated ice-box and transferred to the laboratory without delay, where they directly exposed to the following examination.

B-Sensory evaluation of the examined fish samples: Fish samples were washed using potable water and examined physically for general appearance of the skin, Consistency of flesh, odor and color of gills, color and condition of eyes and slime formation following the scheme provided by FAO (1995).

C-Preparation of fish samples for bacteriological examination according to APHA (2001): all the examined samples were apparently normal. The scales and fins were removed. The skin was sterilized by alcohol and flamed under complete aseptic conditions then25 g of fish flesh from each sample were dessicated in a sterile flask with 225 ml sterile peptone water (0.1%) which added and thoroughly mixed using sterile blender for 1-1.5 minutes, followed by six fold serial dilutions.

D-Bacteriological examination: the prepared samples were subjected to the following analysis:

1-Determination of total aerobic bacterial count: according to APHA (2001).

2-Determination of total Coliforms count: was carried out according to the procedures recommended by FDA (2001) using Violet Red Bile agar medium.

3-Determination of total anaerobic bacterial count according to Savvaidis et al. (2001) and identification of anaerobic bacteria according to Koneman et al. (1992): All samples were inoculated into cooked meat broth medium in duplicate tubes. One of the two tubes was heated at 80⁰C for 10 min. in a water bath to eliminate vegetative organisms while the other inoculated medium was kept without heating and both were anaerobically incubated at 37⁰C for 48 h. A loopful from the inoculated heated broth was streaked onto 10% sheep blood agar plates while that from unheated media was streaked onto the same media containing 75 ug/ ml neomycinsulphate blood agar and the inoculated plates were incubated anaerobically at 37⁰C for 48h. The growing surface colonies which showed catalase negative reaction were picked up inpure form and reinoculated into cooked meat broth for further identification.

4-Isolation of Staph. aureus: on Baird parker agar according to FDA (2001). The presumptive Staph. Aureus colonies were confirmed by Coagulase test.

5-Isolation of Salmonellae: according to FDA (2007) enrichment in rappaport vassiliades broth at 35⁰C for 24h., platting on XLD agar at 42⁰C for 24h. The presumptive colonies were confirmed biochemically and serologically.

6-Isolation and serotyping of E.coli: were carried out according to ICMSF (1996).

7- Isolation of Listeria monocytogenes: 25 g of each sample were homogenized separately in 225 ml of UVM I (University of Vermont Listeria enrichment broth Ryser et al. (1996). Aliquots of 1 ml of primary enrichments were transferred to 20 ml of UVM II (UVM I with 0.025 g. of acriflavine hydrochloride in 10 ml of sterile distilled water, pH 7.2) and incubated again at 37⁰C for 24-48 h Jorgensen and Huss (1998). Aliquots of 0.1 ml of secondary enrichments were plated in duplicate PALCAM Listeria selective agar (Merck) supplemented with PALCAM Listeria selective supplement (Merck). Suspected colonies were confirmed by Gram staining, motility test (hanging drop), catalase and B-haemolysis tests and sugar fermentation tests for rhamnose, xylose and mannitol APHA (2001) and Harrigan (1998). Serotypes were determined using Bacto-Listeria-O polyvalent antiserum and Bacto-Listeria-O antisera types 1 and 4 (Difco). The obtained results were statistically evaluated by using t test according to Feldman et al. (2003).

RESULTS

Table1: Statistical analytical results of bacteriological counts (log₁₀cfu/g) for the examined fresh Tilapia nilotica and Mugil cephalus fish samples (N=50 of each).

*=Egyptian Organization for Standardization and Quality Control,  **= not mentioned

Table 2: Number of isolated organisms in the examined fresh fish samples.

* = Egyptian Organization number of examined positive samples from rural areas or urban areas.

Table 3: Serological identification of isolated E.coli in the examined positive fresh fish samples.

Table4: Serological identification of isolated Salmonella serovars in the examined positive fresh fish samples.

DISCUSSION

Contamination of hands and surfaces during catching, cleaning and evisceration of fish is the common route of pathogen infection to other food (Buras, 1993) hence, Fish not only transmit diseases to man but received many diseases and capable of transmitting some of the established food borne microbial infections and intoxications (FAO/WHO, 1974).

The obtained results were calculated and analysed statistically as shown in Table (1) where the APC were 4.5±3,4.8±3.5,3.9±2 and 4.4± 3.2 log₁₀cfu/g for Tilapia nilotica from (rural and urban areas) and Mugil cephalus from (rural and urban areas) respectively, these results were nearly in accordance with Mahmoud (1999) who recorded that the APC were 3±0.12 x10³ and 1.86±0.10 x10³ cfu/g for Tilapia nilotica and Mugil cephalus, Vieira, et al. (2000) found that the APC were 0.3x10⁴cfu/g for frozen tilapia, Samaha et al. (2011) who reported that the APC were 4.38x10⁴±3.2x 10³ and 6.6x10³± 4.0x10²cfu /g for Tilapia nilotica and Mugil cephalusmusculature. Meanwhile, higher results were obtained by Abd El-Aziz, (2010) who recorded that the APC were 1.8x10⁷±4.0x10⁷cfu/g for Tilapia nilotica, Wang et al. (2011) found that the APC were 4.96-6.53 log₁₀ cfu/g with 15.8% which were unacceptable. As the recorded limit was (APC>7 log cfu/g) for seafood, Hafez and Megahed (2011) found that the APC were 4.2X10⁶ ±0.2x10³cfu/g for Tilapia nilotica and Budiati et al. (2015) who found that 5.77-9.12 log₁₀ cfu /g for Tilapia, the APC in rural areas were lower than in urban areas with significant difference (P<0.05) in the examined samples of Tilapia nilotica and Mugil cephalus. These results were in accordance with the Egyptian Organization for Standardization and Quality Control (EOS) (2007) for APC (<10⁶cfu/g).

The aforementioned results in Table (1) declared that all the examined samples were positive for Coliforms with variant mean counts 2.5±1.3, 2.7±1.7, 2.1±1.5 and 2.5±1.3log₁₀cfu/g for Tilapia nilotica and Mugil cephalus from (rural and urban areas) respectively, these results were nearlyin accordance with Abd ELShahid et al. (2009) who found that Coliform count were1.02x10³ and2.1×10²cfu/g.for O.niloticus and Mugil cephalus, Abd El-Aziz (2010) found Coliform count were 4.3x10²±8.4x10²cfu/g. for Tilapia nilotica, Hafez and Megahed (2011) showed that Coliform count were 2.8x10² ± 0.1x10²cfu/gfor Tilapia nilotica, El-Hakem et al. (2013) couldisolate Coliform by 2.4 x 10² ±1.4 x10²cfu/g from Tilapia musculaturealso, Eissa et al. (2014) found coliform count 9.5x10²±4.2x10² and 9.3x10²±4.3x10²cfu/g in raw Tilapia nilotica and Mugil cephalus, Junior, et al. (2014) obtained counts of coliforms were 3-1100 cfu/g in fish and Budiati et al. (2015) isolate 1.6 - 4.04 log₁₀cfu coliforms /g. for tilapia, while Samaha et al. (2011) recorded higher results in Tilapia nilotica and Mugil cephalus 1.69x10³±0.15x10³ and 1.98x10³±0.32x10³cfu/g which may be due to water pollution with sewage, improper handling during catching, storage and distribution in the markets. Coliforms in rural areas were lower than in urban areas with significant difference (P<0.05) in the examined samples of Tilapia nilotica and Mugil cephalus. The results which were higher than the EOS (2007) for Coliforms were unacceptable.

The anaerobic countresults in tables(1&2)were 2±1.8, 2.3±2, 2±1.6 and 2.3±1.5 log₁₀ cfu/g the counts were higher in urban  areas than rural areas, with incidence percent 6%,8%,4% and 4% for Tilapia nilotica from (rural and urban areas) and Mugil cephalus from (rural and urban areas) respectively. Meanwhile the vegetative form of Clostridium perfringens was detected in 2%, 4% and 2% of examined samples from Tilapia nilotica collected from (rural and urban areas) and Mugil cephalus from (urban areas) and the spore form of Clostridium perfringens were present in 4%, 4% 2% and 2% of examined samples from Tilapia nilotica collected from (rural and urban areas) and Mugil cephalus from (rural and urban areas). There was a significant difference (P<0.05) in anaerobic count between rural and urban areas in the examined samples of Tilapia nilotica and Mugil cephalus, these results were relatively in accordance with Voidarou et al. (2011) who isolate the vegetative and spore forms of Clostridium perfringens from 6% and 35% of the examined Mugil cephalus while, Novotny et al. (2004) found Cl. Botulinum in fresh water fish. The positive results were not in accordance with the EOS (2007) for anaerobic counts.

The obtained results of Staph.aureus in Tables (1&2) were 2.6±1.4, 2.7±1.8, 2.5±1.4 and 2.5±1.7 log₁₀cfu/g with incidence percent 4%,6%,0% and 2% for Tilapia nilotica from (rural and urban areas) and Mugil cephalus from (rural and urban areas)  respectively. There was a significant difference (P<0.05) in Staph. aureus count between rural and urban areas which were lower in rural areas than urban areas of the examined samples. These results were in accordance with Vieira et al. (2000) who found that the counts were 10.0 and 10.6x10²cfu/g in frozen Tilapia, Pacheco et al. (2000), Novotny et al. (2004) found coagulase-positive Staph. aureus counts ranged from 10-21x10³cfu/g.in frozen Tilapia and Voidarou et al. (2011) found 8% of Mugil cephalus contain Staph. aureus, Samaha et al. (2011) could isolate Staph. Aureus by 3.84x10²±0.46x10² and 3.56x10² ±0.41 x10²cfu/g from Tilapia niloticus and Mugil cephalus, Hafez and Megahed (2011) could isolate 5.1x10¹±0.114 cfu/g Staph. Aureus from 12% of the examined Tilapia nilotica, El-Hakem et al. (2013) detect 6.8x10²±2.9x10²cfu/g Staph. Aureus in Tilapia nilotica musculature and Eissa et al. (2014) could isolate 7.5X10±1.2 and 5.0x10±1.2cfu/g from raw Tilapia nilotica and Mugil cephilus fish. Meanwhile, higher results were obtained by Junior et al. (2014) who examined skin and muscles of Tilapia for Coagulase positive Staph. Aureus which were 1.0×10²–1.2×10⁶cfu/g the results were in accordance with the EOS (2007) for Staph. Aureus (<10³cfu/g), while coagulase positive Staph. Aureus were negative.

The results of E.coli incidence in (tables 2&3) were 10%,16%,6% and 8% in the examined Tilapia nilotica and Mugil cephalus samples collected from rural and urban areas respectively, with three serotypes of E. coli O111:H4 and two serotypes of E. coli O125:H21isolated from Tilapia nilotica samples collected from rural areas, five serotypes of E. coli O114:H21 and three serotypes of E. coli O127:H6 in Tilapia nilotica samples collected from urban areas, three serotypes of E. coliO44:H18 in Mugil cephalus collected from rural areas and two serotypes of E. coli O111:H4 and two serotypes of E. coli O127:H6 in Mugil cephalus collected from urban areas.

These results were nearly similar with those achieved by Mahmoud (1999), Novotny et al. (2004), Abd EL-Shahid et al. (2009) who found E. coli in 20% and 8% for Oreochromis niloticus and Mugil cephalus samples and so Voidarou et al. (2011) (6%) for Mugil cephalus and Tilapia nilotica respectively. Wang et al. (2011) could isolate E.coli by 9.4% from seafood, Elsherief et al. (2014) found E.coli in 12% and 4% of examined Tilapia nilotica and Mugilcephalus. Meanwhile, higher results were recorded by Hassan et al. (2012) 27% and 42.8% for Oreochromus niloticus and Mugilcapito respectively and Amr et al. (2012) 57.10% and 91.40% from Tilapia and Mugil cephalus. The presence of high counts and incidence of E. coli serotypes as shown in Table (3) in some samples indicates sewage pollution of fish inducing food poisoning and hemorrhagic enterocolitis in human due to eating improperly processed fish meals Galal, (2013). The positive results were not in accordance with the EOS (2007).

The results in (Table2) declared that the incidence of Salmonellae spp. were 2%,4%,0% and 2% in the examined Tilapia nilotica and Mugil cephalus samples collected from rural and urban areas respectively, Nearly similar or slightly higher results were obtained by Vieira et al. (2000) isolate 8.3% Salmonellae spp. from Tilapia nilotica, Abd EL-Shahid et al. (2009) detect Salmonellae spp. In 8% and 4% of examined Oreochromis niloticus and Mugil cephalus, Shinkafi and Ukwaja (2010) found Salmonellae spp. In 3.2% of examined Tilapia nilotica, Voidarou et al. (2011) isolate Salmonellae spp. from 2% of examined Mugil cephalus samples, Elsherief et al. (2014) detect Salmonellae spp. In Tilapia nilotica and Mugil cephalus by 8% and 16% and so Mahmoud (1999), Novotny et al. (2004). In contrary Pacheco et al. (2000) found higher results from Salmonellae spp. in the examined Tilapia (60%) and Wang et al. (2011) isolate Salmonellae spp. from 17.5% of examined seafood, Hassan et al. (2012) could isolate Salmonella arizonae from O. niloticus and Mugil capito which were 21.6% and 14.2%. Amr et al. (2012) could isolate Salmonellae spp. by 57.10% and 17.1% from Tilapia and Mugil cephalus, while Hafez and Megahed (2011), EL-Hakem et al. (2013) and Eissa et al. (2014) failed to detect Salmonellae spp. from the examined raw Tilapia nilotica and Mugil cephalus. The positive results were unacceptable and not in accordance with the EOS (2007). 

The data reported in (Table4) revealed that serotypes of Salmonellae spp. isolated from Tilapia nilotica were represented by Salmonella typhimurium and Salmonella anatu m in rural and urban areas While, in Mugil cephalus Salmonella enteritidis could be isolated. Some members of Salmonellae are pathogenic and may cause infection and food poisoning to human and unacceptable according to the EOS (2007). Salmonella typhimuium represents about 50 - 60 % of food poisoning and commonly isolated from cases of food poisoning. Meanwhile, presence of Salmonellae in fish reflect unsatisfactory hygienic conditions during catching, handling and marketing of fish WHO (1997).

The obtained results in Table (2) declared that L. monocytogenes could be isolated from Tilapia nilotica and Mugil cephalus collected from rural and urban areas by18%, 26%, 12% and 16% respectively, these results were in agreement with Novotny et al. (2004) and Abd El-Aziz, (2010) who could isolate L. monocytogenes from Tilapia nilotica by 26.7% while lower results were recorded by Wang et al. (2011) 4.1% and Shinkafi and Ukwaja (2010) 9.67 %. This may be attributed to fish species, methods of catching, handling sanitation level during transportation, distribution and storage conditions as well as marketing Wang et al. (1994) and the positive results were unacceptable according to the EOS (2007).

CONCLUSION AND RECOMMENDATIONS

From the obtained results, it could be concluded that raw fish had a high bacterial load. Some samples were free from Salmonella, E.coli and L. monocytogenes this may be attributed to the condition of fish itself or method of fish manipulation after catching. Staph. aureus and Coliform count which present in raw fish indicate pollution which must be taken in consideration, hence Fish should be chilled as quickly as possible to the lowest temperature from the harvesting point up to consumption. Containers used for fish transportation should be cleaned and disinfected periodically. Education of the fishermen and fish handlers about the hygienic methods for fish preservation, transportation and distribution.

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