Prevalence of multidrug resistant ( MDR ) food-borne pathogens in raw chicken meat in Dhaka city , Bangladesh : an increasing food safety concern

The present study was performed to investigate the prevalence of multidrug resistant (MDR) foodborne pathogens in raw chicken meat in Dhaka city, Bangladesh: an increasing food safety concern. A total of 100 meat samples (05 from each market) were collected from randomly selected 20 retail markets of Dhaka city using sterile polythene bags in a view to prevent extraneous contamination. The collected meat samples were then processed and inoculated onto nutrient broth and nutrient agar plates for isolation. The isolated organisms were identified based on staining, motility, cultural and biochemical properties according to standard laboratory methods. The isolated bacteria were also subjected to characterize their antibiotic sensitivity. In the present study, it was revealed that 100% of samples were contaminated by at least one species of bacteria belonging to 5 genera such as Staphylococcus, Escherichia coli, Salmonella, Enterobacter and Bacillus. Out of total 100 meat samples, 56% were contaminated with Escherichia coli whereas 42% were contaminated with Coagulase positive Staphylococcus and 36% were Salmonella species followed by 20%, 14% and 10% were contaminated with Enterobacter species, Coagulase negative Staphylococcus and Bacillus species respectively. A total of 174 bacteria were isolated and identified from raw chicken meat samples inspected of which 29.89% were Escherichia coli, 24.14% were Coagulase positive Staphylococcus, 20.69% were Salmonella species, 11.49% were Enterobacter species, 8.05% were Coagulase negative Staphylococcus and 5.75% were Bacillus species. The antimicrobial sensitivity tests showed that 96.15% of Escherichia coli (50 out of 52), 95.24% of Coagulase positive Staphylococcus (40 out of 42) and 86.11% of Salmonella (31 out of 36) isolates displayed multidrug resistance phenotypes (resistant to more than two antimicrobial agents). All most all the isolates of E. coli, coagulase positive Staphylococcus and Salmonella were more resistant to tetracycline, amoxicillin, ampicillin and streptomycin whereas less resistant to Ceftriaxone and Cefotaxime. The resistance patterns against azithromycin, ciprofloxacin, chloramphenicol, gentamycin, nalidixic acid and kanamycin were fluctuated from 25% to 71.43% among the isolates. This increasing development of multidrug resistance is alarming for the poultry industry and an increasing food safety concern for human.


Introduction
Microbial food safety is an increasing public health concern worldwide.Food-borne illnesses occur following recent consumption of a particular food or drink contaminated with pathogens.Each year around one-third of the world population is affected by food-borne pathogens especially in developing countries.Even in developed nation like US, billions are spent in treatment of food-borne diseases caused by major pathogens.Each year 48 million people are affected in US with food-borne illness (Scallan et al., 2011;CDC, 2013).Salmonella, Campylobacter, Escherechia coli, Staphylococcus, Clostridium, Yersisnia, Listeria, Arcobacter, Mycobacterium, Taenia, Trichinella, Sarcocystis, Toxoplasma gondii and Cryptosporidium parvum are the important zoonotic food-borne pathogens of animal origin (Dhama et al., 2011;Dhama et al., 2013).Global use of antimicrobial agents in human and veterinary medicine, agriculture and aquaculture has promoted both the survival of resistant microorganisms and the elimination of susceptible ones in the resulting antibiotic containing environments (Levy and Marshall, 2004).In modern agriculture, production of meat, milk, and eggs has attained industrial dimensions, animals being kept on species-specific farms in large numbers for the various stages of production (breeding, raising, fattening, milk, and egg production) (FAO, 1995).Globally, about 48 billion animals (cattle, pigs, sheep, goats, chickens, and turkeys) are slaughtered and kept in stock annually, most of these animals live on species specific farms and are potential consumers of drugs and antibiotics (FAO, 1995).Since the early 1950s, antimicrobial agents have been used in livestock farming to treat infections and improve growth and feed efficiency.In 1997, the World Health Organization published its first report on the medical impact of the use of antimicrobials in food animals (WHO, 1997).The main threats identified were: (i) an increase in the prevalence of resistant bacteria in animals; (ii) the transfer of resistant pathogens to humans via direct contact with animals, or through the consumption of contaminated food or water; (iii) the transfer of resistance to human bacteria; (iv) an increase in the incidence of human infections caused by resistant pathogens; and (v) potential therapeutic failures in animals and humans (WHO, 1997).Poultry meat is a good source of animal protein, appealing to consumers very easily due to its sensorial attributes.Antibiotics are used for control and treatment of bacterial diseases in poultry.There is growing scientific evidence that the use of antibiotics in food animals leads to the development of resistant pathogenic bacteria that can reach humans through the food chain (Van Looveren et al., 2001).Recent reports have shown that different types of food and environmental sources harbor bacteria that are resistant to one or more antimicrobial drugs used in human or veterinary medicine and in food-producing animals (Anderson et al., 2003;Schroeder et al., 2004).Annual cost of treating infections caused by antibiotic-resistant bacteria is estimated to be $4 to $5 billion (McGowan, 2001).International and US public health agencies have targeted antibiotic resistance as an emerging public health concern (Barza and Travers, 2002) and one of the most pressing public health needs.Contaminated food of animal origin is one source of human bacterial infections; therefore, the presence of antibiotic-resistant strains in food animals such as poultry has raised concerns that the treatment of human infections will be compromised.So, considering the above facts the present study is designated as "Prevalence of multidrug resistant (MDR) food-borne pathogens in raw chicken meat in Dhaka city, Bangladesh: an increasing food safety concern".

Materials and Methods
The whole study was conducted in the laboratory of the department of Microbiology and Parasitology, Sher-e-Bangla Agricultural University (SAU), Dhaka-1207 during the period from August, 2016 to July, 2017.

Collection and transportation of samples
A total of 100 meat samples (05 from each market) were collected from randomly selected 20 retail markets of Dhaka city using sterile polythene bags in a view to prevent extraneous contamination.The collected meat samples were then transferred into the laboratory immediate after collection using ice box.

Isolation and identification of food-borne pathogens
At first, meat with hard pieces or bony samples was trimmed with sterile knife aseptically.Samples (25 g) were transferred to 225 mL of buffered peptone water (BPW) and macerated in a mechanical blender as per recommendation of International Organization for Standardization (ISO, 1995).The mixture samples were incubated at 37 °C for 18-24 h.Part of inoculated BPW was then transferred to nutrient agar plate for primary isolation.The subculture technique was followed up to obtaining pure culture (Cheesbrough, 2006).Stock cultures were maintained in both Agar slant and 20% sterile buffered glycerin (Merchant and Packer, 1967).The isolated organisms were identified based on gram's staining, motility by hanging drop techniques, cultural characteristics with colony morphology on different selective medias and available biochemical tests such as sugar fermentation test, Catalase test, Methyl red test, Voges-Proskauer test and Indole test according to standard laboratory methods (Cheesbrough, 2006).

Prevalence of food-borne pathogens in raw chicken meats
Contaminated raw chicken meat is one of the main sources of foodborne illnesses and a potential risk of the transmission of zoonotic infections.In the present study, it was revealed that 100% of samples were contaminated by at least one species of bacteria belonging to 5 genera such as Staphylococcus, Escherichia coli, Salmonella, Enterobacter and Bacillus.In the prevalence study, out of total 100 meat samples, 52% were contaminated with Escherichia coli whereas 42% were contaminated with Coagulase positive Staphylococcus and 36% were Salmonella species followed by 20%, 14% and 10% were contaminated with Enterobacter species, Coagulase negative Staphylococcus and Bacillus species respectively (Table 1 and Figure 1).A total of 174 bacteria were isolated and identified from raw chicken meat samples inspected based on morphological, cultural and biochemical characterization and their overall prevalence were presented in Table 2 and Figure 2. The present study is in close agreement with the findings of Bhaisare et al. (2014) who revealed that the Indian chicken meat contains pathogenic bacteria like Salmonella spp.(33.16%),Campylobacter spp.(95%), Escherichia coli (70.22%),Clostridium spp.(13.88%),Listeria monocytogenes (15%) and Staphylococcus aureus (11.25%).These findings also supported with some of the previous study (Javadi andSaeid, 2011 andAl-Salauddin et al., 2015).Javadi and Saeid (2011) were performed bacteriological analysis on 80 fresh chicken meat samples, marketed in Tabriz in Iran, results demonstrated the presence of S. aureus (65%), Cl. perfringens (83%), Streptococcus (100%) and Coliforms (100%) whereas Al-Salauddin et al. (2015) reported E. coli was isolated from 50 (83.33%)broiler meat samples and Salmonella spp.from 18 (31.66%)samples from some selected areas of Bangladesh.Owuna et al. (2015) observed that 72.5% (29 out of 40) of fresh poultry meat samples, obtained from different location in keffi metropolis of Nigeria, were contaminated with S. aureus.Osman et al. (2015) observed 100% chicken meat samples were contaminated with Staphylococcus species in Egypt where as other studies have reported that the rates varies from 6% to 100% in Spain (Alvarez-Astorga et al., 2002), Japan (Kitai et al., 2005), Italy (Normanno et al., 2007), Nigeria (Achi and Madubuike, 2007), Jordan (Al-Tarazi et al., 2009), Korea (Lim et al., 2010), United States (Waters et al., 2011), Turkey (Citak and Duman, 2011), India (Arul and Saravanan, 2011), China (Wang et al., 2013), EFSA (2013), Egypt (EI-Jakee et al., 2013), and Thailand (Akbar and Anil, 2013).In Crotia, bacteriological analysis was performed on 66 samples of fresh, retail-cut chicken meat (21 samples of chicken breasts without skin -"fillet", and 19 samples of chicken breasts with skin) and 26 samples of frozen ground chicken meat and found the presence of Salmonella spp.(10.60%), S. aureus (30.30%),L. monocytogenes (3.03%), Enterobacteria spp.(34.84%) and sulphite-reducing Clostridia (1.50%) (Kozacinski et al., 2006).The variation of prevalence is may be due to geographic location, management practices in poultry farms, slaughter house hygiene practices, etc.

Conclusions
Contaminated raw chicken meat is one of the main sources of foodborne illnesses and a potential risk of the transmission of zoonotic infections.In this study, it was revealed that 100% of samples were contaminated by at least one species of bacteria belonging to 5 genera such as Staphylococcus, Escherichia coli, Salmonella, Enterobacter and Bacillus.All most all isolates of Escherichia coli, Coagulase positive Staphylococcus and Salmonella species were developed multidrug resistance properties (resistant to more than two antimicrobial agents) which is alarming for the poultry industry and an increasing food safety concern for human.Further molecular characterization is prerequisite to detect multidrug resistant genes in order to find out the ways to prevent multidrug resistance properties of food borne pathogens.
Legends: % = Percentage; CP= Coagulase positive and CN= Coagulase negative Figure 1.Name and percentage of samples positive for bacterial isolates in raw chicken meats.

Figure 2 .
Figure 2. Overall prevalence of bacteria isolated from raw chicken meats.

Table 2 . Overall prevalence of bacteria isolated from raw chicken meats.
Legends: No. = Number and % = Percentage

Table 8 . Result of antibiotic sensitivity and resistance patterns of isolated Salmonella species. Name of antibiotics used No. of isolates tested Antibiotic sensitivity & resistant patterns
Legends: No. = Number and % = Percentage