Serology Unit, Animal Health Research Institute,

Dokki, Giza.

Contamination of some local fish with Listeria monocytogenes and studying its characterization and control

(With 7 Tables)

By

Hala S. Ibrahim and  Hala F. Hassan*

*Dept. of Food Hygiene, Animal Health Research Institute, Dokki, Giza.

(Received at 12/12/2005)    

مدى تلوث الاسماک المحلية بميکروب الليستيريا مونوسيتوجين

ودراسة خصائصها والتحکم فيها

هالة سلطان إبراهيم ، هالة فريد حسن

تم عزل عشرون عترة ليستريا مونوسيتوجين من بعض الاسماک المحلية من المحلات والسوبر مارکت فى خمس مدن مصرية وقد وجد أن نسبة العزل الکلية (9.3%) کانت أکثر نسبة عزل فى القرموط  (4.19%) والمکرونه (2.79%) والسردين (2.33%). وقد صنفت هذه العترات على حسب ثمانية خصائص ظاهرية منها تخمر السکريات ، وجد ان هذه العترات تختلف فى قدرتها على تخمر الدالسيت ولاکتوز ومالتوز ومانينول والنشا والزيلوز بينما لها القدرة على تخمر کل من الجلوکوز والسالسين وسکروز وتريهالوز والرامينوز ولها المقدرة على تحمل الترکيزات العالية من کلوريد الصوديوم من (8-20%) وقد وجد ان الاحماض العضوية ليس لها المقدرة على تثبيط نمو الليستيريا مونوسيتوجين خلال 5-10 دقائق ولکنها لها المقدرة بعد 24 ساعة برغم استخدام ترکيزات مختلفة (0.5، 1، 2و 5%) ونسبة مقدرتها على الاتحاد بصبغة الکونجو الحمراء وانتاج الليثيثين هى 90 و85% على التوالى. قد وضحت اختبارات الحساسية للمضادات الحيوية ان کل العترات المعزولة کانت مقاومه للکولستين سلفات وانروفلوکساسين وناليدکسک اسد واکرولينک اسد بنسبة 100% ولکنها حساسة 100% للکلورامفينيکول ثم يليه الاريثرومايسين (80%) ، اموکسيسللين     (75%) ثم اوکسىتتراسيکلين (70%) وسبروفلوکساسين (65%). وقد وجد ان العترات المعزولة احدثت التهاب الملتحمة فى خنازير غينيا و16 عترة فقط احدثت نفوق الفئران خلال 7 أيام وجميع العترات ادت الى تحلل کرات الدم الحمراء للحصان والخروف وخنازير غينيا والانسان ولکنها تختلف عند اضافة المانوز حيث کانت النسبة 85، 75، 50، 50 % على التوالى. قد وجد ان العد البکتيرى لميکروب الليستيريا المحقون فى السردين قد تاثر بالتعرض لدرجات الحرارة المختلفة فزيادة درجة الحرارة ووقت التعرض لها تؤثر على العدد فعند درجة 100º م لمدة 20دقيقة يؤدى الى القضاء عليها تماماَ اما عند 70º م لمدة 20 دقيقه يقل العدد فقط بينما عند 50º م حتى مع زيادة الوقت لم يؤثر على العدد بينما المعالجة بالاحماض العضوية اوضح ان زيادة ترکيز الحمض يقلل معدل النمو وکانت علاقة تثبيط النمو باستخدام الاحماض العضوية کالاتى (حمض الاکتيک ثم الاسيتک ثم الستريک ) ومن الملاحظ فى هذا البحث ان عند استخدام ترکيز 0.05% من الاحماض العضوية شجع نمو الميکروب، وفى حالة استخدام انتى ميکروبيل ايس على السردين لم يقضى تماماَ على الميکروب بل قل عده من 1×10 ⁸ الى 1.5 × 10º و2 × 10 ³ بعد 60 و 120 دقيقة على التوالى.

SUMMARY

A total of 20 L. monocytogenes  strains isolated from some local fish from different shops and markets located in 5 towns in Egypt. The total incidence of L. monocytogenes in raw fish was 9.3%, it was predominated in Clarias (4.19%) followed by Saurus (2.79%) then Sardines (2.33%). They were phenotypic characterized with respect to 8 characters, carbohydrate fermentation profiles showed variation in utilization of dalcite, lactose, maltose, mannitol, starch and xylose  while glucose, salicin, sucrose, trehalose and rhaminose yielded positive in all strains. They were tolerant to NaCl at high salt concentrations ranged from 8% to 20%. The growth inhibition of L. monocytogenes in presence of organic acids not occurred within 5 and 10 minutes but occurred after 24 h incubation at 37°C even with different concentrations (0.5, 1, 2 and 5%). The positive percent of Congo red binding activity and Lecithinase production were 90% and 85% respectively. The drug susceptibility characterization of L. monocytogenes cleared that all isolates were resistant (100%) to colistin sulphate, enrofloxacin, nalidixic acid and oxolinic acid, but were susceptible to chloramphenicol (100%) followed by erythromycin (80%), amoxycillin (75%) then oxytetracycline (70%)and ciprofloxacin (65%). Pathogenicity in laboratory animals showed that 20 strains  caused keratoconjunctivities in G. pig and only 16 strains killed the mice within 7 days. All strains were haemolytic to RBCs of horse, sheep, G. pig and human and only showed change in haemolysis with addition of D-mannose as follows 85, 75, 50 and 50% in horse, sheep, G. pig and human RBCs respectively. The count of       L. monocytogenes inoculated in Sardine was affected by heat treatment where increasing time of exposure and degree of temperature reduced count as 100°C for 20 minutes completely eliminated L. monocytogenes , while at 70°C for 20 minutes only reduced count and having no effect at 50°C  even with increasing time. Whereas treatment with organic acids revealed that when the acid concentration increased, the growth rate of L. monocytogenes decreased and the relative inhibition effect was generally  lactic > acetic > citric acid , form interest of this work that low concentration of organic acids (0.5%) enhanced growth of                     L. monocytogenes. Application of antimicrobial ice on Sardine not completely eliminated but reduced L. monocytogenes count from 1 X 10⁸ to 1.5 X 10⁵ and 2 X 10³ after 60 and 120 minutes exposure respectively.

Key words: Fish, Clarias spp., saurus spp., sardine, L. monocytogenes.

INTRODUCTION

            Fish is considered a very important diet for human due to its protein content and high level of calcium and phosphorus. Fish may be subjected to various forms of contamination by different microorganism including L. monocytogenes which is an opportunistic bacterial pathogen of man and animal. It has the ability to survive under extreme environmental conditions causing illness to animals and man called listeriosis, specially in pregnant woman, new born infants, children and adults whose immune system are weakened (Rocourt et al.,2000).

             More recently a new form of the disease has been recognized,it is characterized by mild disorders of the gastrointestinal system and short incubation period. L. monocytogenes has been involved in a food borne outbreaks of gastroenteritis (FAO report,1999).Due to ubiquitous nature of Listeria spp. and its ability to grow in refrigerated temperature and low oxygen level, its isolation from fish is recorded by Kwiatek, (2004) who isolated it from raw fish.

           The recent concern about decontamination of L. monocytogenes by physical and chemical methods that inhibit this bacteria to prevent future outbreaks as heat treatment or addition of certain substance that remove or minimize the Listeria load in fish and fish products included acetic and citric acid (Naser, 2003), antimicrobial ice containing chlorine dioxide (ClO₂) (Shin et al.,2004) or addition of lactic acid alone or with hot water (Koutsoumanis et al.,2004).

The investigation dealt with:

- The prevalence of L. monocytogenes in some local fish including Saurus, Sardines and Clarias Spp

- Studying some phenotypic characterization with respect to carbohydrate fermentation profiles, haemolytic activity, growth inhibition by sodium chloride, and organic acids (acetic, citric and lactic acid) Congo red binding activity, lecithinase production test, drug susceptibility and pathogenicity in laboratory animals (G. pig and mice).

- In addition to studying the effect of heat treatment, addition of acetic, citric and lactic acid and also antimicrobial ice on contaminated samples with L. monocytogenes was investigated.

MATERIALS and METHODS

Fish samples:

A total of 215 fish samples including 80 Saurus, 70 Clariasand 65 Sardines were purchased from different shops and markets of various size located in 5 towns in Egypt, collected in sterile polyethylene bags and transferred to the laboratory in an ice box then kept at 0- 4ºC until tested within 24 h.

Bacterial strains:

         Rhodococcus equi and Staphylococcus aureus for CAMP test were obtained from Animal Health Research Institute. L. monocytogenes microorganism was isolated during this study. The stock culture was maintained at -20°C in trypticase soy broth containing 10% glycerol.

Blood:

 Blood was collected from horse, sheep, G. pig and human into tubes containing sodium citrate in distilled water per 9.0 ml of blood. Blood was diluted 1:4 with phosphate buffered saline (PBS) of pH 7.2 to test for haemagglutination (HA) and 1:4 with 1% D mannose in PBS test for mannose resistant haemagglutination MRHA.

Laboratory animals:

1- Mice:

A total of 100 albino white mice with average weight of about 18- 20 gm and aged 30- 35 days were used for detection of pathogenicity of L. monocytogenes.

2- G. pig:       

A total of 20 G pigs were used for interaconjunctival infection for detection of conjunctivitis caused by L. monocytogenes.

Disinfectants :

- Acetic, citric and lactic acids, each was used in a concentration of 0.5, 1, 2 and 5%.

- Antimicrobial ice was obtained from some factories containing chlorine dioxide.

 Isolation of Listeria monocytogenes from fish:

            According to FAO (1992)25 gms from each fish sample were added to 225 ml of Listeria enrichment broth (L.E.B.), then incubated at 30ºC for 48 h. The enrichment culture was streaked on PALCAM selective agar and incubated at 30ºC for 24- 48 h.

 Identification and characterization of L. monocytogenes isolated from fish:

            Colonies suspected to be L. monocytogenes were identified according to Koneman et al. (1996) and Quinn et al. (2002) and characterized according to Margolles et al. (2000) by Gram stain, tumbling motility, V.P, catalase, oxidase, haemolysis on horse blood agar and CAMP tests.

CAMP technique:

            B haemolytic Stayphlococcus aureus and Rhodococcus equi cultures in parallel and diametrically opposite each other were  streaked on sheep blood agar plate, where several test cultures were streaked parallel to one another, but at right angles to and between S. aureus and R. equi streaks. The plates were incubated at 35°C for 24- 48h then examined for haemolysis.

All L. monocytogenes give haemolytic reactions which are enhanced in the zone influenced by S. aureus streak.

characterization of L. monocytogenes

1. Carbohydrate fermentation profiles:

This test was performed by addition of single colony to 5 ml of basal liquid medium (meat extract 1%, sodium chloride 0.5% and bromocresol purple 0.02 g pH 6.8) containing 1% of the appropriate carbohydrate including glucose, dalcite, lactose, maltose mannitol, salicin, starch, sucrose trechalose, xylose, and rhamnase. Development of yellow colour after 24- 48 h of incubation at 37ºC was considered a positive result.

2. Growth inhibition by sodium chloride:

Over night cultures on tryptic soya broth (T.S.B) were used to inoculate fresh T.S.B supplemented with NaCl in a range from 8 to 20% at 1% intervals then 200 ml of each inoculated media were dispensed into wells of microtitre plates and were incubated at 37ºC for 48 h. To ensure the growth of L. monocytogenes a loopfull from each well was cultured on PALCAM agar as solid media and was incubated 30ºC for 24h.

3. Growth inhibition by organic acids:

  Over night cultures on T.S.B were inoculated to fresh T.S.B supplemented with one of each of the 3 organic acids (acetic, citric and lactic acid) at mentioned concentrations, then 200 ml of each suspension was dispensed into wells of microtitre plates that were incubated at 37ºC for 48 h and ensured growth by cultivation on PALCAM.

4. Congo red (C.R) binding activity: Delgado-da-Silva et al. (2001).

Listeria monocytogenes strains were cultured into Congo red media, the reaction was best seen after 24 h of incubation at 37ºC and then left at room temperature for additional 2 days. CR⁺ was indicated by development of red colonies, CR^- colonies appeared white as did not bind to the dye.

5. Lecithinase production test: Delgado-da-Silva et al. (2001)                                                               Listeria monocytogenes isolates were inoculated on the surface of egg yolk agar media and incubated at 37ºC for 24 h. Positive result was indicated by appearance of colonies surrounded by zone of opacity.

6. Drug susceptibility testing:  Finegold and Martin (1982) and Quinn et al. (2002).

On Muelleur Hinton medium the standard disk diffusion assay was used to determine susceptibility of the isolated L. monocytogenes to 12 antimicrobial agents (Oxoid), using the following discs, amikacin (30µg), amoxicillin (25µg), cefadroxil (30µg), chloramphenicol (30µg), ciprofloxacin (5µg), colistin sulphate (50µg), enrofloxacin (5µg), erythromycin (15µg), nalidixic acid (30µg), oxolinic acid (2µg) oxytetracycline (30µg) and streptomycin (10µg). The results were interpretated according to the manual supplied by the manufacture company.

7. Pathogenicity in laboratory animals:

All laboratory animals were examined bacteriologically and proved to be free from pathogens.

- Mouse lethality test: Stelma et al. (1987).

 The mice were divided into groups (5 in each) according to number of isolates. Last group was kept as control and each mouse was inoculated with 0.1 ml of 10¹⁰ C F U of test strain of L. monocytogenes  I/ P, each group was kept separately. All mice were kept under observation for 7 days. Number of deaths were recorded daily and reisolation of L. monocytogenes was done from dead mice. Strain that killed 3 or more mice was considered pathogenic.

- Anton test:

 1 G. pig was used for each isolate. Instillation of G. pig eye with early pure culture causing conjunctivitis within 24- 48 h.

8. Haemolytic activity: Dominguez et al. (1986)

Two well developed colonies from sheep blood agar were suspended in 0.5 ml of saline solution. Two fold dilutions were made in microtitre plate with U form wells containing 50ml of saline solution then  50 ml of bacterial suspension was added to the first well. 50ml from well were transferred to another containing  saline solution and so on. 100 ml of 3% of washed sheep RBCs were added to each bacterial dilution. Negative control well contained 100m of washed sheep RBCs and 50ml of saline solution. The microtitre plates were incubated at 37ºC for 16 h. The haemolytic activity was expressed as complete haemolysis unite C.H.U. (the inverse of highest dilution at which 100% haemolysis occur) and minimal haemolysis unite M.H.U. (the inverse of highest dilution at which haemolysis was detected). The test was repeated using washed G pig then human RBCs.

Preparation of Listeria  inocula and fish inoculation: Koutsoumanis et al.,( 2004).

A mixture of six L. monocytogenes strains showed various phenotypic characterization isolated from fish in this work, were grew on tryptic soya broth at 30ºC for 24 h, then centrifuged at 2000 rpm for 15 minutes. The harvested cells were washed in 10 ml phosphate buffer saline, centrifuged as previously then the washed cells were diluted with P.B.S till obtain 10⁸ CFU/ ml. Then 1 ml was inoculated to each fish sample (previously examined to ensure it is free from L. monocytogenes, and left to dry off for 15 minutes at 4ºC. Listeria was counted using PALCAM agar.

Treatments used to control L. monocytogenes:

1-      Heat treatment:

Fish were experimentally contaminated with L. monocytogenes and subjected to various degrees of temperature with different (increasing) durations in hot air oven that was considered as grilling in following temperatures 50, 70 and 100ºC each with the following time 10, 15 and 20 minutes.

 2- Addition of organic acids:

            Each of acetic, citric and lactic acids was added in mentioned concentrations to fish contaminated with L. monocytogenes in polyethylene bags for 30 seconds, 1 and 5 minutes.

3- Application of antimicrobial ice:

            Contaminated fish were put in plastic bag with antimicrobial ice               and tested for time intervals of 60-120 minutes

RESULTS

Table 1: Occurrence of L. monocytogenes in some local raw fish.

* Percent was calculated according to the number of each species of the examined fish.

** Percent was calculated according to the total number of the examined fish (215).

Table 2: Characterization of L. monocytogenes isolated from local fish.

The percent was calculated according to the total number of strains (20).

Glucose, salicin, sucrose, trehalose and rhaminose were +ve in all isolates.

Sodium chloride was used in range from 8 to 20 % at 1% intervals for each strain in growth inhibition test.

Colistin sulphate, enrofloxacin, nalidixic acid, oxolinic acid were–ve in all strains.

Inoculation dose: 0.1 ml of 10¹⁰ CFU/mice I.P.

Table 3: Haemolytic characterization of L. monocytogenes isolated from local raw fish.

The percent was calculated according to the total number of samples (20).

 Table 4: Haemolytic titre of L. monocytogenes isolated form local raw fish.

S:  sheep                    G: G pig                      H:  human                           

Table 5: Efficacy of heat treatment on count of L. monocytogenes in Sardines.

Table 6: Efficacy of organic acids treatment on count of L. monocytogenes in Sardines.

S = seconds                                           m =minutes

Table 7: Efficacy of antimicrobial ice on count of L. monocytogenes in Sardines.

DISCUSSION

            Listeria monocytogenes is of greatest concern from the public health point of view, consistently associated with human illness and linked to many outbreaks due to consumption of food. Dalton et al. (2004) in their studies about foodborne diseases outbreaks, found that the most frequently implicated vehicles in the 173 outbreaks were seafood and L. monocytogenes caused 40% of deaths.

           The present study dealt with the occurrence of L. monocytogenes in some local fish including Saurus, Clarias and Sardines. The total incidence of L. monocytogenes in raw examined fish was 9.3%.             L. monocytogenes  was predominated in Clarias (4.19%) followed by Saurus (2.79%) then Sardines (2.33%). In relation to the total incidence, the occurrence of L. monocytogenes differed in the examined fish species as it was higher (12.86%) in Clarias and lower incidence was observed in Sardines (7.69%) then Saurus (7.5%) as shown in Table (1).

           Many investigators recorded the incidence of L. monocytogenes in raw fish. Higher values were recorded by Hartemink and Georgsson (1991) who isolated L. monocytogenes  from 25.7% of raw fish samples while Bianchini et al. (1999) and Hoffman et al. (2003) recorded 34% and 14.6%. On the other hand, lower values were recorded by kwiatek (2004), Thimothe et al. (2004) and Markkula et al. (2005) as the incidence in raw fish was 1.26%, 3.8% and 4%, respectively. In contrast,  Baek et al. (2000) reported that no L. monocytogenes was found in salt water fish and Handa et al. (2005) never isolate L. monocytogenes from raw fish. Our results are in accordance to large extent with Erdenling et al. (2000) who found certain type of L. monocytogenes associated with cat fish fillets might be a risk of listeriosis for human and  Naser (2003) who recorded the presence of L. monocytogenes  (8%) in Saurus spp. This variation in results may be attributed to fish species, transport, purchased places, country variation and hygienic measures or seasons.

             Only limited data are available on the growth characteristics of   L. monocytogenes in naturally contaminated raw fish, so Table (2) deals with characterization of 20 strains of L. monocytogenes isolated from some local raw fish. Carbohydrate profiles showed variation in sugar utilization of dalcite, lactose, maltose, mannitol, starch and xylose, while glucose, salicin, sucrose, trehalose and rhaminose yielded positive in all strains. This result  is in accordance to Margolles et al. (2000) who found that all isolates of L. monocytogenes fermented L. rhammose and mannoside, while Hitchins (2003) recoded that L. monocytogenes fail to utilize xylose and was positive to rhaminose utilization. Van Coillie et al. (2004) depended on haemolycin and mannitol, rhaminose and xylose utilization test in identification and characterization of                            L. monocytogenes.

          Also, tolerance of L. monocytogenes to NaCI that grew at high salt concentrations ranged from 8% to 20% was observed. The results in the present study are in accordance with Guyer and Jemmi (1991) and Margolles et al. (2000) who found that the minimal inhibition concentration was between 9% and 12% at 37°C for L. monocytogenes growth. Liu et al. (2005)  recorded that L. monocytogenes strains tested were resistant to saturated NaCl (40 W/V) for a long time. It is clear from this result that high light  salt treatment is not sufficient to eliminate L. monocytogenes from salted food products.

It is obvious form Table (2) that the growth inhibition of            L. monocytogenes in presence of organic acids did not occur within 5 and 10 minutes but occurred after 24 h incubation at 37°C (even with different concentrations 0.5, 1, 2 and 5%). There were no differences in the effect among organic acids used for growth inhibition of                   L. monocytogenes. Many authors concerned with the effect of organic acids on growth of L. monocytogenes as Young and Foegeding (1993) who found that when the initial pH values of acetic and lactic acids decreased or the total acid concentration increased the growth rates of    L. monocytogenes decreased, the relative inhibition effect was generally acetic > lactic > citric. Margolles et al. (2000) recorded that the inhibitory capability of organic acids followed the order citric> lactic>acetic high level and high concentration being more inhibitory than low concentration. While Gravesen et al. (2004) said that all Listeria strains were sensitive to D -lactic acid which gave 0.6-2.2 log unit greater reduction than L- lactic acid midway in the activation curves

Positive Congo red binding activity and Lecithinase production were 90% and 85%, respectively. Many authors concerned with Congo red and Lecithinase production as Nunes and Hofer (1994) who evaluated Lecithinase production and capacity for Congo red absorption, presented 51.8% and 88.8% positively rates respectively. Whereas Delgado-da-Silva et al. (2001) recorded that the percents were 27.5% and 12.6%  respectively.

The drug susceptibility is one of the important factors of characterization of L. monocytogenes. It is clear that all isolates were resistant (100%) to colistin sulphate, enrofloxacin, nalidixic acid and oxolinic acid, but all strains were susceptible to chloramphenicol (100%) so is considered the antibiotic of choice, followed by erythromycin (80%), amoxycillin (75%) then oxytetracycline (70) and ciprofloxacin (65%).

Lowest number of isolates were sensitive to the remaining studied antibiotics. These results indicated an increasing incidence of antibiotic resistant of L. monocytogenes. This could be attributed to the fact that Listeria may have plasmid encoding antimicrobial resistance. Also, Vaz-Velho et al. (2001) recorded that 190 isolates of                    L. monocytogenes isolated from fresh and cold smoked fish were susceptible to tetracycline. Poyart- Salmeron et al. (1990) and Hadorn et al. (1993) concluded that some strains of L. monocytogenes contain plasmid encoding resistance to choramphenicol, erythromycin tetracycline and streptomycin, while McLauchlin et al. (1997) suggested that resistance to tetracycline may be useful as an easily recognized epidemiological marker for L. monocytogenes .

Table (2) clears that all 20 strains  could cause keratoconjunctivities in G. pig and only 16 strains killed the mice within 7 days. The obtained resultscoincided to large extent with Nunes and Hofer (1994) who recorded the ability of L. monocytogenes to produce keratoconjunctivitis in G. pigs and Hitchins (2003)whofound that 10⁴ CFU pathogenic  L. monocytogenes caused death usually within 3 days in mice. The obtained results disagree with Takeuchi et al. (2003) who observed no deaths of mice inoculated with 10⁸ CFU ,but (20%) to         (40%) of mice died when inoculated with 10⁹ CFU of L. monocytogenes.

           Concerning Table (3), it is clear that all strains were haemolytic (100%) to RBCs of horse, sheep, G. pig and human. Only 85, 75, 50 and 50% showed change in haemolysis with addition of D-mannose in horse, sheep, G. pig and human respectively. Also, there were variations in C.H.U. and M.H.U. between strains and species of used RBCs as high concentration (titre) occurred in sheep followed by G. pigs then human RBCs as shown in Table (4). Our results are in accordance with Margolles et al. (2000) who found that L. monocytogenes displayed  haemolytic activity to a variable extent depending on the strain C.H.U between 0 and 6 while M.H.U between 12 and 96. The synthesis of haemolysin was correlated with the virulence due to  its cytotoxic for leucocytes.

All strains of L. monocytogenes recovered from fish appeared to contain most pathoegnic and virulence factor. The trails to control          L. monocytogenes in raw fish includingheat treatment were shown in Table (5). There was direct relation between time of exposure and degree of temperature on count of L. monocytogenes in Sardines. It was clear that increasing time of exposure and degree of temperature reduced count as 100°C for 20 minutes completely eliminated L. monocytogenes, while at 70°C for 20 minutes only reduced count. On the other hand, 50°C had no effect on count even with increasing time. Our results  agreed to some extent with Autio et al. (1999) who recorded that use of hot steam, hot air and hot water seemed to be useful in eliminating L. monocytogenes, while Thimothe et al. (2002) found that heat treatment significantly reduced L. monocytogenes contamination of RTE craw fish meat. Naser (2003) recorded that fish fillets samples that subjected to grilling for 6 and 8 minutes gave 4 X 10⁴ and 1.3 X 10³ CFU/ g respectively.

The counts of L. monocytogenes (Table 6) inoculated in Sardine were affected by treatment with organic acids in used concentrations as the acid concentration increased the growth rates of L. monocytogenes decreased. The relative inhibition effect was generally  lactic > acetic > citric acid.The obtained results coincidedwith that of Zeitoun and Debevere (1991) who found that the antimicrobial effect of lactic acid buffer systems pH 3.0 increased with increasing concentration of lactic acid in the buffer system on chicken leg. Also Pothuri et al. (1996) studied the effect of lactic acid on growth and survival of                       L. monocytogenes in cry fish at concentration of 0.5, 1, 1.5 and 2% and found that count declined steadily with 2% lactic acid. Naser (2003) said that citric acid was less effective than acetic acid in controlling  L.monocytogenes in fish fillet samples

It is of interest in this work that low concentration of organic acids (0.5%) enhanced growth of L. monocytogenes and this is in accordance with Pothuri et al. (1996) who found that stimulations of growth by 0.5% lactic acid may be due to utilization of lactic acid as a carbon and energy sources. Our results indicated the importance of choosing an adequate concentration of organic acids used for the decontamination of fish because low concentration could favour the growth of L. monocytogenes.

          The inhibitory action of acetic acid is produced through utilization of electrochemical ingredient of the cell membrane as well as denatutrating protein inside the cell but the microbial effect of citric acid is partially due to its ability to chelate divalent cations, while lactic acid  penetrate the cell membrane and act by releasing a proton and acidifying the cytoplasm of the cell.

           It is clear from Table (7) that application of antimicrobial ice in commercial concentration on fish not completely eliminated but reduced L. monocytogenes count from 1 X 10⁸ to 1.5 X 10⁵ and 2 X 10³ after 60 and 120 min exposure respectively.

          This is in agreement with Kim et al. (1999) who reported that treatment with antimicrobial ice up to 200 ppm slightly reduced the number of mesophilic bacteria. Also Shin et al. (2004) recorded that the use of antimicrobial ice (100 ppm) on fish skin for 120 min, only reduced count of L. monocytogenes. There is difference with the study conducted by Huang et al. (1997) who found that the amount of antimicrobial ice required to destroy (95%) of gram +ve and gram –ve bacteria was 1.3 and 1.18 ppm respectively. This may be attributed to nature of strains and country difference. Also antimicrobial ice can be easily used on seafood products as generally stored and displayed on ice, also maintain a constant level of ClO₂ gas during melting.

From the results of the present work, it can be concluded the importance of regular L. monocytogenes testing of raw fish and improve microbiological quality, good hygienic measure and continuous disinfection of utensil, shops and markets. We suggest the inactivation treatments that effectively eliminate L. monocytogenes present in        raw fish by addition of lactic or acetic or citric acid to fish during eating and / or good grilling of fish. A need of further studies of sensory quality (appearance odour freshness- texture and freshness of taste) must be taken in consideration.

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