Frequency and antibiotic sensitivity of gram negative bacteria isolated from raw fish sold in Karachi, Pakistan

Fish serve as an important source of nutrition and also acts as a reservoir for different pathogenic organisms. Sewage pollution in the water bodies is the major source of pathogenic bacteria in seafood. In this study, the presence of Gram negative bacteria in raw fish samples was detected and the antibiotic susceptibility pattern of the isolates was determined.A total of 25 raw fish samples were collected at random from different local markets of Karachi in sterile zip-lock plastic bags. From these samples, a total of 73 Gram negative bacterial strains were isolated. Of these 73 Gram negative isolates, 6 were E. coli, 14 K. pneumoniae, 7 E. aerogenes, 4 E. cloacae, 5 C. freundii, 1 C. koseri, 2 P. vulgaris, 3 P. mirabilis, 7 Salmonella spp., 6 Shigella spp., 2 Serratia spp., 2 Pseudomonas spp., 7 Yersinia spp., 3 Aeromonas spp. and 4 were Vibrio spp. The most abundant species among all the isolated Gram negative bacteria was Klebsiellapneumoniae(19.17%) followed by Enterobacter aerogenes(9.59%), Salmonella spp. (9.59%) and Yersinia spp. (9.59%). Antibiotic susceptibility pattern of all the Gram negative isolates was determined by Kirby-Bauer disc diffusion method in which a panel of eight antibiotics was tested. The incidence of resistance was highest against Cefazolin (93.2%) and lowest resistance was observed against Gentamicin (1.4%). Of the 73 Gram negative isolates, 24 were found to be multi-drug resistant (MDR). In conclusion, the bacteriological analysis of raw fish samples showed that the quality of raw fish was poor as high bacterial load was obtained and enteric bacteria were isolated from most of the samples indicating fecal pollution. This study provides valuable information about the presence of MDR Gram negative pathogens in raw fish sold locally which could be useful in the selection of antibiotics for empirical treatment.


Introduction
Fish are consumed as a major source of protein and 30 % of fish comes from aquaculture for human consumer [1]. Fish is more readily available food having low fat content and is taken into consideration because it is inexpensive or low cost source of protein in comparison with beef and goat meat so, easy to access by all social classes' individuals [2,3]. Fish are enriched with micronutrients such as Vitamin D, minerals including Iodine, Magnesium, Selenium and Zinc than animal meat or other food (e.g. plants) and its consumption is helpful in reducing hunger or minimizing the deficiency of micronutrients thereby preventing disease in under developed countries [2]. The easy availability and high nutritional value of fresh fish is responsible for its preferable consumption by coastal people [4]. Karachi being a coastal city has a well-developed fishery industry. However, very little attention is given to the microbiological quality of fresh fish sold at local markets. Proper storage and handling practices are not followed by local vendors mainly due to ignorance, lack of facilities and professional training. There are many sources of contamination of seafood which are often difficult to identify [5]. The sources of contamination of fish include improper storage conditions i.e. maintaining temperature and moisture, allowing customers to deal with fish by their own, use of ice prepared from contaminated water, keeping fish on contaminated surfaces such as weighing balance and unhygienic practices [6], the intake of fecally contaminated water by fish and consumption of those fish can cause intoxication in consumers as they may be harbouring enteric pathogens [7]. All these factors can facilitate the contamination or spoilage of fish thereby resulting in foodborne illnesses. Due to increase in the cases of food intoxication or poisoning, it is a major concern now to consider presence of pathogens in food and its control [8]. Seafood or fish acts as a reservoir or vehicle for bacteria which are pathogenic for human and capable of causing infections [5,9]. Enteric bacteria present in seafood indicate fecal pollution of the aquatic environment [5]. A major source of food contamination is fecal contamination indicated by the presence of members of family Enterobacteriaceae such as Salmonella spp., Proteus spp., Escherichia coli and Klebsiella spp. To improve the quality of food and to reduce the occurrence of food borne illnesses proper screening of zoonotic bacterial pathogens in food is needed [10]. Most of the foodborne diseases in large proportion are caused by bacterial pathogens (76%) than by viruses (21%) followed by parasites (2.6%). Main pathogenic bacteria carried by seafood are E.coli, Salmonella enterica, Listeria monocytogenes and Vibrio spp. [11,12]. The contaminated raw or partially cooked food consumption poses a higher risk to the human health [13]. There is very little data available regarding the prevalence of pathogenic bacteria and the antibiotic resistance in locally available fish. Therefore, this study was aimed to detect the presence of Gram negative bacteria from raw fish samples sold at local markets in Karachi, Pakistan and to evaluate the antibiotic susceptibility pattern of the Gram negative bacteria thereby assessing the quality of fish sold locally.

Materials and methods Sample collection
A total of 25 raw fish samples were randomly collected in a sterile zip lock plastic bags separately from different shops of local markets in Karachi, Pakistan. The fish species collected for this study were Tilapia sp., Pangasius sp., Lutjanus argentimaculatus (Red snapper), Labeorohita (Rohu) and Scomberomorus guttatus (King Mackerel/Surmai).

Sample processing Total viable count/ Total plate count
The sample was serially diluted and 0.1ml of the 10 -3 and 10 -4 dilution was transferred to Plate Count Agar (PCA) media plates separately to make lawn and plates were incubated at 37°C for 24-48 hours. Pre-enrichment of sample 10 gm of fish sample was weighed and inoculated into 100 ml of Buffered Peptone Water in a flask. The flask containing sample was then incubated at 37°C for 3 hours.

Isolation of bacteria Streaking on media plates
A loopful inoculum of pre-enriched sample was streaked on MacConkey's Agar and on Bismuth Sulfide Agar media plates with the help of sterile wire loop to obtain isolated colonies. All the media plates were incubated at 37°C for 24 hours.

Subculture
The different types of isolated colonies were picked from different media plates and streaked on Nutrient agar media plates for the purpose of sub-culturing. All the media plates were incubated at 37°C for 24 hours.
Purification and maintenance of culture The isolated colonies with different morphology after sub-culturing were picked and maintained on Nutrient Agar slants. These cultures were further characterized and identified biochemically.

Results
Total aerobic count was determined and a very high bacterial load was observed on Plate Count Agar from all 25 raw fish samples which was interpreted as too numerous to count (TNTC). A Total of 73 Gram negative bacterial strains were isolated.
Among 73 Gram negative bacterial strains, 64 belonged to different genera of the family Enterobacteriaceae (i.e. Escherichia, Klebsiella, Enterobacter, Salmonella, Shigella, Citrobacter, Serratia, Proteus and Yersinia), while 9 belonged to non-Enterobacteriaceae genera (i.e. Pseudomonas, Aeromonas and Vibrio). The most frequently isolated Gram negative bacteria was found to be Klebsiella pneumoniae (19.17%) followed by Enterobacter aerogenes, Salmonella spp., and Yersinia spp. (9.6% each) ( Table 1). Antibiotic susceptibility pattern of all the Gram negative isolates was determined against Ofloxacin (5μg), Gentamycin (10μg), Trimethoprim-Sulfamethoxazole or Co-trimoxazole (25μg), Chloramphenicol (30μg), Cefazolin (30μg), Ceftriaxone (30μg), Tetracycline (30μg) and Oxacillin (1μg). The resistance pattern of the isolated Gram negative bacteria against different antibiotics is given in ( Table 2). The highest number of organisms were resistant to Cefazolin (93.15%) followed by Oxacillin (89.04%) and Ceftriaxone (30%) while maximum number of organisms were susceptible to Gentamicin (98.63%) followed by Ofloxacin (93.15%) and Chloramphenicol (93.15%). Of the 73 Gram negative isolates obtained from fish, 24 were resistant to multiple drugs, however none of the isolates of genus Proteus showed multidrug resistance. Highest frequency of multidrug resistance was observed among Yersinia spp. and Salmonella spp. followed by Serratia marcescens, Pseudomonas, Escherichia coli, Enterobacter aerogenes and Citrobacter freundii ( Table 3). The resistance of bacteria with respect to the number of antibiotics is given in ( Figure  1). The highest number of isolates were resistant to a combination of two antibiotics and none of the isolates were resistant to all the antibiotics tested. Few strains were sensitive to all the eight antibiotics tested and organisms were found to be tolerant to a maximum of six antibiotics simultaneously. The occurrence of multidrug resistance (MDR) was also noted among the different species of Gram negative bacteria isolated. Salmonella (57%) and Yersenia (57%) species exhibited the highest rate of MDR strains, followed by E. coli (50%) ( Table 4).

Discussion
The prevalence of antibiotic resistant bacteria in fish is a public health concern.
In the present study, total aerobic count was performed to assess the hygienic quality of fish. The results represented that the fish were of poor quality and unsafe to consume without proper washing and cooking as high bacterial load was observed on Plate Count Agar in form of uncountable colonies which was interpreted as too numerous to count (TNTC). Ghosh [25]. In this study, the most frequently isolated Gram negative bacteria was found to be Klebsiella pneumoniae (19.17%) which could be due to the unhygienic processing of fish and fecal contamination, as Klebsiella are not only found in the mucosal lining of mammals where they colonize and contribute towards the source of fecal contamination of food but are also common in the environment i.e. water, sewage, soil and plants [26]. Antibiotic susceptibility pattern of the Gram negative bacteria isolated from raw fish was also determined against eight commonly used antibiotics representing different classes of antibiotics. Most of the isolates were found to be resistant to Cefazolin (93.2%) and Oxacillin (85%)  [37] in bacteria including E. coli, K. pneumoniae and Enterobacter isolates. Tetracycline is a bacteriostatic drug and inhibits bacterial protein synthesis. In this study, only 6.8% resistance was observed to tetracycline among the isolated Gram negative bacteria which is much lower than the 52% reported by Newaj-Fyzul et al. [36]. In this study, only 5.5% isolates were resistant to Chloramphenicol. Verner-Jeffreys et al. [35] reported a high resistance (56%) to chlormaphenicol in their fish isolates. Ofloxacin, a broad spectrum second generation fluoroquinolone drug acts by inhibiting DNA gyrase or topoisomerase IV to inhibit bacterial cell division. In this study, 4.1% resistance was observed by the isolated Gram negative bacteria. Similar findings of low resistance to ofloxacin was also reported in a previous study where out of 94 clinical isolates from fish, 78% to 80% of isolates (members of family Enterobacteriaceae and Staphylococcus) were susceptible to Ofloxacin [32]. Gentamicin, a member of aminoglycoside drug whose mode of action is to inhibit the protein synthesis by binding with 30s ribosomal subunit was found to be the most effective antibiotic. In this study, only a single isolate belonging to Vibrio spp. was resistant to Gentamicin. Similar findings were reported by Kathleen et al. [38] where 1.1% isolates were also resistant to gentamicin. In another study, 50% to 90% of bacterial isolates among which most were Gram negative and member of family Enterobacteriaceae showed susceptibility to Gentamicin [32]. The differences in the antibiotic resistance pattern of these isolates from other researches could be due to the fact that fish sold in the local market are the catch from natural marine waters while most of the reported studies are based on the samples obtained from aquacultures and also bacterial flora differs among various geographical regions. The quality of fish can be maintained or food products can be saved not only from pathogenic bacterial species but also from other micro-organisms by following the guidelines provided by Centre for Disease Control and Prevention (CDC), The International Commission on Microbiological Specifications for Foods (ICMSF) and Hazard Analysis Critical Control Point (HACCP). Fish should be microbiologically examined for the presence of pathogens especially enteric pathogens (belonging to family Enterobacteriaceae), hygienic practices should be followed during collection, processing, transportation and distribution of food. Fish processing environment, equipment and storage conditions such as appropriate cold temperature and humidity should also be maintained and fish should be properly cooked before it is consumed in order to prevent foodborne illnesses or food poisoning.

Conclusion
In this study a number of Gram negative food-borne pathogens were isolated from raw fish samples including E. coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Proteus spp., Salmonella spp., Shigella spp., Serratia spp., Pseudomonas spp., Yersinia spp., Aeromonas spp. and Vibrio spp. Resistance against different antibiotics was also observed by most of the isolates and some isolates also showed multidrug resistance. So, it is important to assess the quality of fish before it is being consumed and to consider the emergence of resistance among Gram negative bacteria by the observed isolates because they are a threat to public health worldwide and difficult to treat. Efforts should be directed to improve the quality of fish sold at local markets and proper storage facilities should be provided to the vendors/merchants.