Bacteriological study of fish samples collected from different markets in some Egyptian governorates and antimicrobial sensitivity of isolates

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A total of (80) samples of living diseased cultured Tilapia fish (Oreochromusniloticus), were collected from different fish farms in Egypt (Behera and Kafr El-Sheikh) which showed the clinical signs of loss of scales from some areas of the skin, excessive mucus all over the body surfaces with petechial haemorrhages over the dorsal musculature, large necrotic lesions extending all over the body and darkness of skin.

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Nội dung Text: Bacteriological study of fish samples collected from different markets in some Egyptian governorates and antimicrobial sensitivity of isolates

Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 2765-2776 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 5 (2017) pp. 2765-2776 Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2017.605.310 Bacteriological Study of Fish Samples Collected from Different Markets in Some Egyptian Governorates and Antimicrobial Sensitivity of Isolates Elham I. Atwa* Hafr Al Batin University, College of Science and Arts - Al Khafji, Biology Department *Corresponding author ABSTRACT A total of (80) samples of living diseased cultured Tilapia fish (Oreochromusniloticus), were collected from different fish farms in Egypt (Behera and Kafr El-Sheikh) which showed the clinical signs of loss of scales from some areas of the skin, excessive mucus all Keywords over the body surfaces with petechial haemorrhages over the dorsal musculature, large necrotic lesions extending all over the body and darkness of skin. Differentiation and Haemorrhages, characterization of various isolates was based on their growth characteristics on specific Fluorescens, culture media (biochemical and gram staining reactions). The following human pathogenic Norfloxacin, bacteria were isolated Staph. aureus, Staph. epidermidis, Staph. Saprophytics, Bacteria, Streptococcus spp., E. coli, Salmonella, P. aeruginosa, P. fluorescens, and Fish, Enterobacteriaceae from skin with the incidence of 12.5%, 23.8%, 31.3%, 10%, 25%, Epidermidis 7.5%, 22.5%, 20%, and 18.8% respectively, but from muscle with the incidence of 7.5%, Article Info 7.5%, 12.5%, 8.8%, 22.5%, 5%, 20%, 18.8%, and 16.3% respectively. On the other hand these bacteria isolated from intestine with the incidence of8.8%, 7.5%, 12.5%, 13.8%, Accepted: 25%, 8.8%, 17.5%, 15%, and 16.3% respectively, while from liver with incidence of 15%, 26 April 2017 12.5%, 16.3%, 15%, 35%, 6.3%, 25%, 23.8%, and 15% respectively. In vitro sensitivity Available Online: test indicated that, the most prevalent bacteria isolated from examined fish samples were 10 May 2017 sensitive to enrofloxacin, norfloxacin, ciprofloxacin and kanamycin. Most of these strains were highly resistant to erythromycin and a moxycillin. PCR panel could help for rapid diagnoses to determine the causative agents from fish samples, Staph. aureus coagulase gene and variable fragments for 16SrRNA genes from the extracted DNA at 228bp. Introduction Fish and fish products have long been used as before it begins to spoil. Freezing does not a major food component for humans and prevent spoilage of fish because of autolytic animals. Fishes are known to be enriched by activities and chemical changes occurring in high nutritional components and concentrated fish after harvest (Huss et al., 1974, and Jay source of energy, in addition to their high Jm modern food microbiology, 1992). palatability and good digestibility (Mead et al., 1986; Mol et al., 2007; Dinakaran et al., The degeneration of fish is accelerated by 2010; Kawarazuka, 2010). microorganism associated with aquatic environment as well as contaminated during Fish is a perishable protein food, when fish is post –harvest handling, when fish dies stored at
Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 2765-2776 and gills begins to utilize the fish protein and associated with particular fish depends on its food nutrient resulting in loss of nutritional habitat (Claucas and Ward, 1996). The value (Ames,1992). Microbial activities bacterial pathogens associated with fish have create undesirable changes like off-flavors, been classified as indigenous and non- texture and appearance (Jhonstone et al., indigenous (Kvenberg, 1991). The non- 1994). indigenous contaminate the fish or the habitat one way or the other and examples include Fish disease due to bacterial infection are Escherichia coli, Clostridium botulinum, considered one of the major problems in Shigella dysenteriae, Staphylococcus aureus, aquaculture which lead to heavy losses Listeria monocytogens and Salmonella. The (Austin and Allen Austin 1985 and Austin indigenous bacterial pathogens are found and Austin, 1999), causing a great drop in fish naturally living in the fish s habitat for production and industry. Most of the bacteria example Vibrio species and Aeromonas associated with these diseases are naturally species (Rodricks, 1991). The bacteria from saprophytic organism and widely distributed fish only become pathogens when fish are in the aquatic environment (Frerichs and physiologically unbalanced, nutritionally Handrie, 1985). deficient, or there are other stressors, i.e., poor water quality, overstocking, which allow A variety of fishes consumed regularly are opportunistic bacterial infections to prevail prone to pathogenic spoilage especially by (Austin, 2011). Pathogenic and potentially Vibrio spp., Shigella spp, Salmonella spp., pathogenic bacteria associated with fish and streptococci, Staphylococci, Coliforms, shellfish include Mycobacteium, Listeria spp., Clostridium spp. (Rahman et al., Streptococcus spp., Vibrio spp., Aeromonas 2012) which may get entry into the fish from spp., Salmonella spp. and others (Lipp and their habitat or during the fish transportation Ross, 1997). and storage (Frazier and Westhoff, 1995; Eze et al., 2010). A number of reports suggested Staphylococcus, Escherichia coli, that the consumption of the microbiologically Pseudomonas, Shigella and Salmonella, were spoiled seafoods might be responsible for the common pathogenic bacteria found food-borne diseases like diarrhea, associated with fish from the ponds associated salmonellosis, shigellosis, cholera and even with integrated farming systems. Their some neurological diseases by an array of presence was attributed to the contamination viruses, bacteria, fungi and parasites of the fish ponds by animal waste (Snowdon et al., 1989; Starutch, 1991; (Abdelhamid et al., 2006). The isolation of Karunasagar et al., 1994; Cray and Moon, Salmonella, Shigella and E. coli from the fish 1995; Wallace et al., 1999; WHO, 2012). samples indicates faecal contamination of the ponds resulting from the livestock manure However fish are susceptible to a wide variety that they add to the fish ponds as feed. The of bacterial pathogens, most of which are isolation of Salmonella, Shigella, and E. coli capable of causing disease and are considered indicate faecal and environmental pollution by some to be saprophytic in nature (Lipp and (Yagoub, 2009). Coliforms such as E. coli are Ross, 1997). The microbiological diversity of usually present where there has been faecal fresh fish muscle depends on the fishing contamination from warm blooded animals grounds and environmental factors around it (Chao et al., 2003). E. coli is recognized as (Cahill, 1990). It has been suggested that the the reliable indicator of faecal contamination type of micro-organisms that are found in small numbers and in large numbers, it is 2766
Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 2765-2776 an indicator of mishandling (Eze et al., 2011). Materials and Methods E. coli is the only species in the coliform group that is found in the human intestinal Samples tract and in the other warm blooded animals as a commensal and is subsequently excreted A total of (80) samples of living diseased in large quantities in faeces (Geldreich, 1983). Tilapia fish (Oreochromus niloticus), were collected from different fish farms in Egypt One of the risks involved in livestock (Behera and Kafr El-Sheikh), during the integrated fish farming is possible transfer of period from May 2014 till February 2015. pathogens between livestock and humans. The collected fish subjected to gross clinical Previous research has shown that, different examination according to the method kinds of livestock manure are contaminated described by (Amlacher 1970),showed the with pathogenic bacteria such as Salmonella, clinical signs of Loss of scales from some Shigella, Pseudomonas, Vibrio, areas of the skin, excessive mucus all over the Streptococcus, and E. coli species body surfaces with petechial haemorrhages (Abdelhamid et al., 2006). Rate of bacterial over the dorsal musculature. Large necrotic spoilage is dependent on the initial microbial lesions extending all over the body with tail load, ambient temperature and improper rot. Skin showed superficial ulcers, and handling. Therefore, proper storage critical in darkness of skin (Fig. 1). The fish samples maintaining a high standard of safety when were transferred a aseptically to laboratory of processing fish. (Jay Jm modern food microbiology without delay in large sterile microbiology, 1992). jars containing water. The transmission of these pathogens to people The surface of fish bodies were disinfected by can be through improperly cooked food or the alcohol (70%), then dissected under antiseptic handling of the fish. There have been great conditions. The freshly dead collected economic losses reported due to food borne samples of fishes were subjected to post illness such as dysentery and diarrhea mortem examination (PM) before the resulting from consumption of contaminated bacteriological examination according to fish and such can be a problem to the immune (Lucky 1977, and Conroy and Herman compromised, children and elderly people. 1981).The post mortem examination revealed of generalized septicemia and enlargement in The aim of the present study is to prove the most internal organs especially kidney, liver, demonstration, isolation and identification of intestinal tract, spleen and severe distension of human pathogenic bacteria from the skin, of gall bladder with bile secretion, congested muscle, intestines and liver of living diseased gills and gall bladder (Fig. 2). fish samples. Bacteriological examination Besides, susceptibility of most predominant isolates to chemotherapeutic agents as an aid Specimens of fish obtained from skin, muscle, to overcome this problem and reduce losses. intestine and liver were inoculated directly in Also, using polymerase chain reaction (PCR) nutrient broth and incubated at 37ºC for 6 test to substitute the conventional cultural hours. Loopful from each broth culture were methods and rapid diagnosis of Staph. aureus inoculated directly onto nutrient agar, blood directly from fish samples. agar, Macconkey's agar, bile salt lactose agar, Salmonella. Shegilla agar, Mannitol Salt agar, 2767
Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 2765-2776 Baird parker’s agar media for isolation of S. extraction kit (Qiagen, Germany) according to aureus,, Eosin Methylene blue agar media for manufacturer’s protocol for gram-positive isolation of E. coli and Enterobacteriacae bacteria. The extracted DNA from samples family. The plates were incubated at 37ºC for was dissolved in 25 μl sterile distilled water 24-48 hours. Suspected colonies onto the and stored at −20°C until further use. surface of these media were identified by studying characters of the colonies as well as Multiplex PCR was performed on the Gram’s stain, then identified morphologically extracted DNA from samples (part C) to according to the method described by (Kloss detect coagulase (coa) and 16SrRNA genes and Schleifer1986, Barrow and Feltham 1993 (Hookey et al., 1998 and Løvseth and and Austin and Austin 1999). BerdalK 2004). One single colony showed typical colonial Primers for Staph. aureus coagulase (coa) appearance and morphological characters was and 16SrRNA genes picked up and streaked into semisolid agar media and incubated at 37ºC for 24 hours to Specific oligonuclotide multiplex primer obtain pure culture, for further identification. assay (synthesized by MWG-Biotech AG, The pure colonies were biochemically Holle & Huttner GmbH, Germany), for rapid identified according to (Cruickshank et al., diagnosis of Staph. aureus coagulase (coa) 1975, Koneman et al., 1992, Quinn et al., and 16SrRNA genes. The forward primer for 2002). The Gram negative bacteria included coagulase (coa) was 5'- Enterobacteriacae family were biochemically ATAGAGATGCTGGT -3', while the reverse identified according to (Morrison et al., 1981 primer was 5`-GCTTCCGATTGTTCG -3` and Krieg and Holt 1984). (Hookey et al., 1998). While the forward primer for 16SrRNA gene was 5'- Antibiotic sensitivity tests (Antibiogram) GTAGGTGGCAAGCG -3', while the reverse primer was 5`-CGCACATCAGCGTC-3` The sensitivity of bacterial isolates to (Løvseth and BerdalK 2004). different antimicrobial agents were investigated using the disc diffusion method Staph. aureus DNA amplification by PCR as described by (Lennette et al.,1980, and Finegold and Martin 1982) to detect the drug The PCR was performed (Hookey et al., 1998 of choice against different isolated bacteria and Løvseth and BerdalK 2004) in a for trials of treatment. The results were touchdown thermocycler in a total reaction interpretated according to (Koneman et al., volume of 30 ul containing 2.5 µl of extracted 1992). DNA, 1 µl of each primer (10 pmol/µl), 0.6 µl of deoxynucleoside triphosphate (10 Extraction of Staph. aureus DNA (Løvseth mmol/L), 3 µl of 10 X thermophilic buffer and BerdalK 2004) (Promega), 1.8 µl of MgCl2 (25 mmol/L), 0.1 µl of Taq DNA polymerase (5 U/µl), and Isolated Staph. aureus strains were incubated complete the reaction volume using distilled overnight in 10 ml brain heart infusion broth water in 0.2-ml reaction tube. The presence of (Oxoid), centrifuged at (5000 rpm, for 15 PCR products was determined by min) and resuspended in 0.5 ml TE buffer (10 electrophoresis of 10 µl of the DNA product mMTris, 1 mM EDTA - pH 8). Total cellular in a 1.5 % agarose gel with 1 X TAE buffer DNA was extracted using Qiagen DNA (40 mMTris-HCl, 1 mM EDTA/L, 1.14 ml/L 2768
Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 2765-2776 glacial acetic acid, pH 7.8) at a voltage of 4 chemotherapeutic agents. Most tested strains volts /cm and stained with 0.5 mg/ml of Staph. aureus, Staph. epidermidis, Staph. ethidium bromide and the Fluorescent bands Saprophytics, Streptococcus spp., E. coli, were visualized with a UV transilluminator Salmonella, P. aeruginosa, P. fluorescens, and photographed. A 100-bp DNA ladder and Enterobacteriaceae were sensitive to (Gibco BRL) was used as a molecular marker. enrofloxacin, norfloxacin, ciprofloxacin and Amplification was obtained with 35 cycles. kanamycin. Most of these strains were highly Each cycle involved initial denaturation at resistant to erythromycin and amoxicillin. 93ºC for 3 minutes, denaturation at 92ºC for 1 minutes, annealing at 52ºC for 1 minutes, and Figure 3 showed three diseased fish samples extension at 72ºC for 1 minutes. The final representative for positive Staph. aureus extension was performed at 72ºC for 7 isolates, were selected and subjected to PCR minutes. analysis. The specificity of the oligonucleotide primer was confrimed by the The presence of PCR products was positive amplification of 228bp fragments for determined by electrophoresis of 10 µl of the Staph. aureus coagulase (coa) and variable DNA product in a 1.5 % agarose gel with 1 X fragments for 16SrRNA genes from the TAE buffer (40 mMTris-HCl, 1 mM extracted DNA of Staph. aureus. EDTA/L, 1.14 ml/L glacial acetic acid, pH 7.8) at a voltage of 4 volts /cm and stained Aquaculture products can harbor pathogenic with 0.5 mg/ml ethidium bromide and the bacteria which are part of the natural Fluorescent bands were visualized with a UV microflora of the environment. Bacterial transilluminator and photographed. A 100-bp pathogens associated with fish can be DNA ladder (Gibco BRL) was used as a transmitted to human beings from fish used as molecular marker. food or by handling the fish causing human diseases. The role of bacteria varies from their Results and Discussion effect as primary pathogen to that of secondary invader in the presence of other Results in table 1 shows the total bacterial disease agents; they may also serve as a stress isolates from different site of (80 skin, 80 factor and predispose fish to other diseases muscle, 80 intestine and 80 liver) of examined (Badran and Eissa, 1991). naturally infected fishes which were (30.4%, 21.1%, 22.2 % and 26.2 % respectively). The The isolation of enteric bacteria in fish serves prevalence of bacterial isolates from the as indicator organisms of faecal different examined naturally infected fishes is contamination and or water pollution. Their illustrated in table 1 also. The result revealed presence also represents a potential hazard to that isolation rate was (10.9) for Staph. humans. Generally, the presence of coliform aureus, (12.8) for Staph. epidermidis, (18.1) and faecal coliform is not the normal flora of for Staph. Saprophytics, (11.9) for bacteria in fish (Mandal et al., 2009). This is Streptococcus spp., (22.8) for E. coli, (6.9) for reflecting the contamination of fish habitat Salmonella, (21.3) for P. aeruginosa, (19.4) with the human and animal faeces. for P. fluorescens and (16.6) for Enterobacteriaceae. Staphylococcus spp. It is associated with food poisoning, produced toxin, which makes man Table 2 showed the in vitro sensitivity of the sick, usually associated with the nausea, most prevalent bacteria isolated from vomiting and diarrhea after eating the collected fish samples were done against (14) staphylococci infected food (O'connell, 2002). 2769
Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 2765-2776 In the present work, we spot light on the niloticus fish. In regard to the results of clinical picture and PM lesions of the most clinical signs and PM lesions of bacterial predominant bacterial pathogens affecting naturally infected fishes, our results were Oreochromus niloticus fishes. Moreover, similar to the findings recorded by (Post isolation and identification of these bacterial 1987, Badran and Eissa, 1991and Austin and infections by both biochemical traditional Austin, 1999), where they mentioned that methods serological as well as by PCR, bacterial infection causes generalized Concerning the clinical signs and Postmortem septicemia and enlargement in most internal (PM) lesions of examined Oreochromus organs. Table.1 Prevalence of bacteria isolated from skin, muscle, intestine and liver of collected fish samples Site of isolation Skin (80) Muscle(80) Intestine(80 Liver(80) Total Bacterial isolates ) No. %* No. % * No. %* No %* No %** Staph. aureus 10 12.5 6 7.5 7 8.8 12 15 35 10.9 Staph. epidermidis 19 23.8 6 7.5 6 7.5 10 12.5 41 12.8 Staph. saprophytics 25 31.3 10 12.5 10 12.5 13 16.3 58 18.1 Streptococcus spp. 8 10 7 8.8 11 13.8 12 15 38 11.9 E. coli 20 25 18 22.5 20 25 15 35 73 22.8 Salmonella 6 7.5 4 5 7 8.8 5 6.3 22 6.9 P. aeruginosa 18 22.5 16 20 14 17.5 20 25 68 21.3 P. fluorescens 16 20 15 18.8 12 15 19 23.8 62 19.4 Enterobacteriaceae 15 18.8 13 16.3 13 16.3 12 15 53 16.6 Total bacterial 137 30.4 95 21.1 100 22.2 11 26.2 45 100 isolates 8 0 %* was calculated according to total number of samples (80) %**was calculated according to the total number of samples (320) Fig.1 Oreochromus niloticus naturally infected with bacteria showing petechial haemorrhages over the dorsal musculature. Large necrotic lesions extending all over the body 2770
Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 2765-2776 Table.2 Results of antibiogram pattern of the most prevalent bacteria Isolated from collected fish samples Staph. P. Staph. P. Enterobacter Antibacterial agents Staph. Sapro Streptococcus Salmonella aeruginosa epidermidis E.coli fluorescens iaceae aureus phyticus S. % S. % S. % S. % S. % S. % S. % S. % S. % Amoxycillin (25ug) 6/15 40 5/15 33.3 2/15 13.3 6/15 40 0/15 0 2/15 13.3 1/15 6.7 2/15 13.3 1/15 6.7 Chloramphenicol 6/15 40 5/15 33.3 7/15 46.7 11/15 73.3 0/15 0 9/15 60 9/15 60 10/15 66.7 2/15 13.3 (30ug) Ciprofloxacin (5ug) 14/15 93.3 13/15 86.7 14/15 93.3 13/15 86.7 14/15 93.3 14/15 93.3 15/15 100 15/15 100 13/15 86.7 Erythromycin (10ug) 1/15 6.7 1/15 6.7 5/15 33.3 3/15 20 2/15 13.3 2/15 13.3 0/15 0 1/15 6.7 3/15 20 Flumequine (30ug) 11/15 73.3 8/15 53.3 14/15 93.3 11/15 73.3 1/15 6.7 1/15 6.7 0/15 0 1/15 6.7 14/15 93.3 Gentamicin (10ug) 10/15 66.7 15/15 100 14/15 93.3 14/15 93.3 13/15 86.7 13/15 86.7 12/15 80 13/15 86.7 9/15 60 Norfloxacin (10ug) 15/15 100 13/15 86.7 12/15 80 15/15 100 15/15 100 15/15 100 8/15 53.3 2/15 13.3 13/15 86.7 Polymyxin 9/15 60 12/15 80 6/15 40 8/15 53.3 1/15 6.7 1/15 6.7 0/15 0 2/15 13.3 6/15 40 (10ug) Streptomycin 12/15 80 10/15 66.7 8/15 53.3 5/15 33.3 14/15 93.3 14/15 93.3 9/15 60 10/15 66.7 5/15 33.3 (10ug) Penicillin G 0/15 0 13/15 86.7 7/15 46.7 9/15 60 1/15 6.7 1/15 6.7 0/15 0 1/15 6.7 5/15 33.3 (10ug) Kanamycin (30ug) 13/15 86.7 12/15 80 13/15 86.7 13/15 86.7 15/15 100 15/15 13/15 86.7 100 14/15 93.3 15/15 100 S: Sensitive; %: Percentage of sensitive isolates in relation to total isolates. Fig.2 Oreochromus niloticus naturally infected with bacteria showing generalized septicemia and enlargement in most internal organs 2771
Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 2765-2776 Fig.3 Electrophoresis analysis of PCR product of amplified Staph. aureus coagulase (coa) and 16SrRNA genes M: 100bp marker. Lane 1, 2 and 3 indicate a positive amplification Staph. aureus coagulase (coa) at the 228bp and variable for 16SrRNA genes: C1: Control positive for Staph. aureus coagulase (coa) and 16SrRNA genes In regards to the Incidence of the bacterial (35%), P. aeruginosa (25%), P. fluorescens isolates among naturally examined fishes as (23.8%), Staph. saprophytics (16.3%), Staph. shown in table 1, the most predominant isolates aureus (15%), Streptococcus spp. (15%), from skin were Staph. Saprophytics (31.3%), E. Enterobacteriaceae (15%), Staph. epidermidis coli (25%), Staph. Epidermidis (23.8%), P. (12.5%) and Salmonella (6.3%). aeruginosa (22.5%), P. fluorescens (20%), Enterobacteriaceae (18.8%), Staph. aureus, In similar studies, Escherichia coli, (12.5%), Streptococcus spp. (10%) and Pseudomonas aueriginosa, Staphylococcus Salmonella (7.5%), while the most predominant aureus and Salmonella typhi were isolated from isolates from muscle were E. coli (22.5%), P. the gills, intestines, muscle and skin of aeruginosa (20%), P. fluorescens (18.8%), Megalaspiscordyla and muscles of Enterobacteriaceae (16.3%), Staph. Priacanthushamrur from Royapuram waters in Saprophytics (12.5%), Streptococcus spp India by (Sujatha et al., 2011). This was (8.8%), Staph. aureus (7.5%), Staph. attributed to the heavy load of sewage disposal Epidermidis (7.5%) and Salmonella (5%). On into the seas which could act as a suitable the other hand the most predominant isolates environment for the growth and survival of the from intestine were E. coli (25%), P. human pathogens. Members of the genus aeruginosa (17.5%), Enterobacteriaceae Pseudomonas are found in the soil and natural (16.3%), P. fluorescens (15%), Streptococcus sources of water and are important spp. (13.8%), Staph. saprophytics (12.5%), phytopathogens and agents of human infections Staph. aureus (8.8%), Salmonella (8.8%) and being considered opportunistic pathogens Staph. epidermidis (7.5%), but the most (Sujatha et al., 2011). predominant isolates from liver were E. coli 2772
Int.J.Curr.Microbiol.App.Sci (2017) 6(5): 2765-2776 This also confirms the findings of simultaneous detection of Staph. aureus in fish (Koutsoumanisand Nychas, 2000; Gonzalez- samples. Our results were in agreement with Podriguez et al., 2001; Herrera et al., 2006), those of other studies (Coton and Coton, 2005; who isolated similar organisms from fish and Janda and Abbott, 2007; Olivares et al., 2008). fish products. The presence of these bacterial Also, these results are agreement with the species in fish has been reported to be of public results of (Phuektes and Browning, 2001; health significance, because of their primary Gillespie and Oliver, 2005; Cremonesi et al., role as occupational hazard to fish handlers 2006). (Ibiwoye et al., 2001). In conclusion, fish are susceptible to all Table 2 showed the in vitro sensitivity of the contaminant organisms that may be found in most prevalent bacteria isolated from collected water, post harvesting, marketing, dealing, fish fish samples were done against (11) handlers, this processing will result in chemotherapeutic agents. Most tested strains of microbiological activities leading to loss of fish Staph. aureus, Staph. epidermidis, Staph. meat quality so the basic principles for saprophytics Streptococcus spp., E. coli, prevention of food borne disease and sanitation Salmonella, P. aeruginosa, P. fluorescens and should be followed to protect the consumers Enterobacteriaceae were sensitive to against the public health hazard, the widespread enrofloxacin, norfloxacin, ciprofloxacin and presence of antibiotic resistance kanamycin. Most of these strains were highly microorganisms should be a priority to reinforce resistant to erythromycin and amoxicillin. The the importance of basic hygiene for fish. data generated from this experiment are essential in the choice of most effective References antimicrobial agents against fish pathogenic bacteria of fish. This results nearly agreed with Abdelhamid, A.M., Gawish, M.M., Soryal, those of (Zaky 2009) who arranged the K.A. (2006): Comparative study between antibiotic according its effect on E. coli isolated desert cultivated and natural fisheries of from fish, the least to high were mullet fish in Egypt. IIMicrobiological chloramphenicol (30 mcg), ampicillin (10 mcg), concern. J. Agric. Scie., Mansoura Univ., penicillin G (10 mcg), streptomycin (10 mcg) 31: 5681 5687. and Gentamycin (10 mcg). Ames GR (1992): The kinds and levels of post- harvest losses in Africa in land fisheries. Antimicrobial agents are widely used by Food agriculture organization of the farmers especially in the intensive culture united nations. Rome Italy, (1992). CIFA system. Misuse of drugs and non-compliance technical paper 19. with treatment regiments among users can cause Amlacher, E. (1970): Textbook of fish diseases, treatment to be less effective thereby prolonging T.E.S. Publication, NewJersey. USA, P. the duration of disease (Muniruzzaman and 117-135. Choudhury, 2004). Austin, B. (2011): Taxonomy of bacterial fish pathogens. Vet. Res., 42 (1): 20. The use of polymerase chain reaction (PCR), as Austin, B. and Allen- Austin, D. (1985): A shown in figure 3, revealed positive review. Bacterial pathogens of fish. J. amplification of Staph. aureus on 228 bp Appl. Bact., 58: 483 -506. fragments for Staph. aureus coagulase (coa) and Austin, B. and Austin, D. A. (1999): Bacterial variable fragments for 16SrRNA genes from the fish pathogens: disease of farmed and extracted DNA of Staph. aureus on lane 1-3. wild fish, 3rd edn (revised). Springer- These results suggest that the PCR assay could Praxis series in aquaculture and fisheries. be used as an alternative method in routine Springer-Verlag, London. diagnosis for rapid, sensitive, and specific 2773
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