Molecular detection and characterization of Escherichia coli , Salmonella spp . and Campylobacter spp . isolated from broiler meat in Jamalpur , Tangail , Netrokona and Kishoreganj districts of Bangladesh

The study was conducted to isolate, identify and characterize bacterial samples from broiler meat collected from 20 different upazilla markets of Jamalpur, Tangail, Netrokona and Kishoreganj districts of Bangladesh. A total of 20 samples were subjected to bacteriological isolation and identification, and the isolated bacteria were subjected to antimicrobial susceptibility testing using disc diffusion method. Among the samples, 75% (n=15) were contaminated with Campylobacter spp., 70% (n=14) were with Salmonella species and 85% (n=17) were contaminated with Escherichia coli. The Campylobacter spp., Salmonella spp. and E. coli were isolated and identified by culturing on Blood agar, Xylose-Lysine Deoxycholate (XLD) agar, and MacConky and eosin methylene blue (EMB) agar respectively. Isolates of Campylobacter spp., Salmonella spp. and E. coli were biochemically analyzed. Campylobacter specific 16S rRNA genes were amplified from the isolates. Campylobacter spp. and E. coli isolates were positive to 16S rRNA gene based polymerase chain reaction (PCR). Almost all isolates of Campylobacter spp., Salmonella spp. and E. coli showed highest susceptibility to ciprofloxacin, norfloxacin and gentamicin. However, most isolates were resistant to amoxicillin and erythromycin. Some isolates showed susceptibility to tetracycline, streptomycin and azithromycin. The findings of this study revealed that there is presence of multidrug resistant isolates of Campylobacter spp., Salmonella spp. and E. coli in broiler meat. Results of this research project demonstrated the high level of microbial contamination and occurrence of pathogenic bacteria in broiler meat sold in markets of Bangladesh.


INTRODUCTION
Poultry industry which has started during 1980s is an excellent agribusiness (Haque, 2001) now in Bangladesh.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License vital sector for generating employment, creating additional income and improving the nutritional level of the country.Broiler meat is more popular to the consumers because of its easy digestibility and acceptance by the majority of people, although it could be contaminated with various potential food borne pathogens such as Salmonella, Campylobacter, Escherichia coli (Mulder et al., 1999).Broiler entering slaughter processing is highly contaminated by microorganisms such as Salmonella and Campylobacter and tends to be disseminated during processing (Mead et al., 1994).
Particular concern for human health is the inappropriate use of antibiotics in poultry production and the development of antibiotic resistant strains of bacteria (Sarker et al., 2018).Effective control systems are critical in ensuring product safety, and considerable information is available on how to minimize the risks (FAO, 2013).Various pathogenic microbes, such as E. coli, Salmonella spp.and Staphylococcus have been implicated to reduce the growth of poultry including duck.
Campylobacter is one of the most important pathogen and is regarded major bacterial cause of human gastroenteritis worldwide.Food animals, mainly poultry, cattle, sheep and pigs, may act as asymptomatic intestinal carriers of Campylobacter and animal food products can become contaminated by this pathogen during slaughter and carcass dressing (Berndtson et al., 1996).The abusive use of antimicrobials in food animals has resulted in the emergence and dissemination of antimicrobial resistant bacteria, including antimicrobial resistant Campylobacter, which has potentially serious impact on human health.Moreover, Campylobacter infections pose a serious public health problem for which many countries have monitored their infection and antimicrobial resistance patterns (Kabir et al., 2014).E. coli frequently cause bacterial infections including urinary tract infection, cholangitis, bacterimia and traveler's diarrhea.Enteropathogenic E. coli (EPEC) are an important cause of diarrhea in humans (Savkovic et al., 2005).Haemolytic uramic syndrome caused by Shiga toxin producing E. coli (STEC) is dependent on release of Shiga toxin during intestinal infection and subsequent absorption into the blood stream.Poultry meat can be contaminated with E. coli during processing.Any food that has been in contact with raw meat can also be contaminated.The bacteria also spread from person to person, usually when infected person does not wash his hands well after using a toilet.E. coli cause different types of public health hazards including cholangitis, bacteremia, traveler's diarrhea, Shiga toxicity etc. (Savkovic et al., 2005).
Salmonella spp. is potentially responsible for various pathogenic processes in man and animal including poultry (Freeman, 1985).It can cause diarrhea, vomition, fever, abdominal cramps in human.Sometimes severe diarrhea requires medical interventions such as intravenous fluid therapy.In cases, where bacteria enter into the bloodstream, symptoms include high fever, malaise, pain in the thorax and abdomen, chills and anorexia (Bell, 2002).
Antibiotics are extensively used in poultry industry either as growth promoters or to control infectious diseases (Sarker et al., 2018).Concern about antibiotic resistance and its transmission to human is important because these resistant bacteria may colonize the human gastrointestinal tract and may contribute to the development of resistance genes to human through Rfactor, conjugative plasmids or chromosomal elements as reviewed by Kabir (2010).Therefore, the disease causing microbes that have become resistant to antibiotic drug therapy are increasing public health importance.
Undoubtedly, the poultry slaughtered and dressed under Bangladesh conditions carry extremely high initial contamination loading from the point of slaughtering process to the point at which the consumers are offered the product.There occurs biomagnifications at all levels of handling, poor transport and retailing conditions.Therefore, considering the present perspective, the present research project was designed to detect and characterize the bacteria specifically Campylobacter spp.Salmonella spp.and E. coli and their antimicrobial resistance patterns in broiler meat.

Collection and transportation of samples
A total of 20 apparently healthy dressed broilers were collected randomly from 20 different upazilla live bird markets of Jamalpur, Tangail, Kishoreganj and Netrokona district of Bangladesh.Five upazilla were selected randomly from each district.After collection, samples were immediately brought to Bacteriology Laboratory of the Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh through maintaining cool chain using ice box.

Bacterial culture media
Solid and liquid culture media were used to isolate the bacteria.Blood agar (BA), MacConkey (MC), Salmonella-Shigella (SS), Eosin Methylene Blue (EMB), Xylose-Lysine Deoxycholate (XLD), Mueller Hinton agars were used as solid culture media for this study.The liquid media used in the study were nutrient broth (NB), peptone broth, methyl-red and voges-proskauer broth (MR-VP), and sugar media, 1% hippurate solution, 3.5% ninhydrin solution, oxidase solution and sugar media.

Isolation and identification of bacteria
Pure culture of E. coli and Salmonella spp.were obtained as per the methods described by Krieg et al. (1994).Briefly; 10 g of samples were homogenized with 90 ml of 0.1% peptone water and 50 µl of homogenized sample was poured on to selective agar media and spread with glass spreader and incubated at 37°C for 24 h.Isolation of Campylobacter spp. was carried out by filtration method (0.45 μm filter) according to Shiramaru et al. (2012).The collected samples were allowed to prepare meat homogenates and then 50 μl of meat homogenates were spread on the filter papers that were placed on the surface of Blood base agar no.2 and allowed to stand for 30 min at room temperature, after 30 min filter papers were removed from the BA and the plates were incubated at 37°C for 48 h in microaerobic condition (5% O2, 10% CO2 and 85% N2).The colonies of primary cultures were repeatedly sub-cultured by streak plate method (Cheesbrough, 1985) until the pure cultures with homogenous colonies appeared.The representative bacterial colonies were characterized morphologically using Gram's stain according to the method describe by Merchant and Packer (1967).Biochemical characterizations of the E. coli and Salmonella isolates were performed with Sugar fermentation test, Methyl Red test (MR), Voges-Proskauer test (V-P) and indole test (Cheesbrough, 1985).Differentiation of isolated Campylobacter spp. with supporting growth characteristics were subjected to various biochemical tests such as catalase, oxidase and hippurate hydrolysis test according to the procedures followed by Nachamkin (2003) and Foster et al. (2004).

Molecular identification by polymerase chain reaction (PCR)
DNA template was prepared by boiling method as described by Rawool et al. (2007).All the samples were examined by two pairs of primers (Table 1) to detect 16S rRNA gene of Campylobacter spp., E. coli and Histidine transport operon gene of Salmonella spp.For Campylobacter spp. the PCR reactions were carried out using a thermocycler (ASTEC, Japan) with the following programme: initial denaturation for 5 minutes at 94°C, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 47°C for 30 s and extension at 72°C for 1 min and 30 s.The final extension was conducted at 72°C for 10 min.For E. coli, the PCR reactions were carried out using a thermocycler (ASTEC, Japan) with the following programme: Initial denaturation for 5 min at 95°C followed by 30 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s and extension at 72°C for 1 min.The final extension was conducted at 72°C for 5 min.For Histidine transport operon gene identification in Salmonella spp.initial denaturation for 5 min at 94°C, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s and extension at 72°C for 45 s.The final extension was conducted at 72°C for 5 min. 1 and 2% agarose (Invitrogen, USA) gel was used for electrophoresis of the PCR products.

Isolation of bacteria from poultry meat
A total of 20 samples were collected for isolation of bacteria from broiler meat of different upazilla markets of four districts where three types of bacteria were isolated from the collected meat samples.The isolates were identified as E. coli, Salmonella spp.and Campylobacter spp. on the basis of their morphological, cultural properties, biochemical characteristics with standard reference organisms and molecular methods.Among the isolated bacteria, E. coli were detected in 85% (n=17), Salmonella and Campylobacter in 70% (n=14) and 75% (n=15), respectively of the samples.The highest percentage of E. coli (100%) isolates was observed in Jamalpur and Tangail district.Salmonella isolates were higher (80%) in samples of Jamalpur, Tangail and Kishoreganj.Furthermore, Campylobacter isolates were 100% in samples of Jamalpur District.

Molecular detection of bacteria
Screening of E. coli isolates from meat sample in this study was performed by genus specific (16S rRNA gene) polymerase chain reaction (PCR).The PCR assay was able to amplify 585 bp fragment of the targeted gene from the genomic DNA of E. coli successfully (Figure 1).Specific 496 bp fragment of targeted histidine transport operon gene of Salmonella was amplified successfully (Figure 2).Genus specific (16S rRNA gene) PCR was performed for Campylobacter spp.1530 bp fragment of  presented in Figure 3.  targeted gene was amplified successfully.The result of PCR is presented in Figure 3. sensitive to ciprofloxacin and 7 (63.63%)were susceptible to streptomycin.Most of the isolates were resistant to amoxicillin (81.81%) and 9 (81.81%)isolates were intermediate resistant to erythromycin.On the other hand, out of four C. coli isolates, all (100%) were sensitive to both ciprofloxacin and norfloxacin.3 (75%) were sensitive to gentamicin, and 4 (100%) were resistant to amoxicillin, 2 were resistant to streptomycin.

Antimicrobial resistance pattern of E. coli
The results of the antimicrobial resistance pattern by disk diffusion method with 8 chosen antimicrobial agents are presented in Table 3.Out of 17 isolates, 6 (35.29%) were resistant to 1 antimicrobial agent.Each 1 (5.88%) was resistant to each of 2 antibiotics.Moreover, 2 (11.76%) were resistant to 2 antibiotics.Furthermore, each 1 (5.88%) was resistant to each of 3 antibiotics.
On the other hand, 1 was resistant to 5 antimicrobial agents.

DISCUSSION
Meat products are important not only from nutritional

C. coli (n=4)
Resistant to 2 agent (AMX-TE) 1 (25%) Resistant to 2 agent (AMX-E) 1 (25%) Resistant to 3 agent (AMX-S-TE) 2 (50%) Total resistant isolates 4 (100%) point of view, but also as an item of international trade and foreign exchange for a number of countries.However, they can also function as carriers of several microbial and other health hazards.The greatest risk to human health is due to the consumption of raw or contaminated meat and meat products.In this study, E. coli, Salmonella and Campylobacter were found in the poultry meat, which was similar to the reports by Zhao et al. (2001), Ahmed et al. (2009), Awad-Alla et al. (2010), Torok et al. (2011), Voidarou et al. (2011), Sudershan et al. (2012), Malmuthuge et al. (2012) and Hossain et al. (2015).Several selective culture media were used simultaneously in this study to culture the organism.The media used in this study were selected considering the experience of the past researcher worked in various fields relevant to the present study by Kabir et al. (2014).The colony characteristics of Campylobacter spp.were gray color spreading colony which was supported by (Doyle, 1990;Rowe and Madden, 2000).In Gram's staining, the morphology of the isolated Campylobacter spp. was also supported by Doyle (1990).The colony characteristics of E. coli observed in Mac conkey, EMB agar were similar to the findings of Sharada et al. (1999).And in gram staining isolated bacteria showed pink color small rod shaped organism which was reported by others previously Sharada et al. (1999) and Merchant and Packer (1967).The colony morphology of Salmonella spp. was similar to that of Sarker et al. (2009) and Khan et al. (2005).In staining, the morphology of the isolated bacteria exhibited Gram negative small rod arranged in single or paired and motile which was supported by Sharada et al. (1999).
In catalase test, all the isolates (n = 15) produced bubbles those indicated positive for Campylobacter.In oxidase test, a purple color change was observed within ten seconds in all the isolates (n=15).In hippurate hydrolysis test, some of the isolates (n=4) develop a faint purple to no color change that indicated the isolates were C. coli and some of the test isolates (n=11) developed deep purple color that indicated the isolates were C. jejuni.These findings are similar to the findings of Kabir et al. (2014) and Jamshidi et al. (2008).
The E. coli isolates revealed a complete fermentation of 5 basic sugars by producing both acid and gas which was supported by Thomas (1998) and Beutin et al. (1991).The isolates also revealed positive reaction in MR test and Indole test but negative reaction in VP test (Honda et al., 1982).The antimicrobial susceptibility of most of the isolates was sensitive to ciprofloxacin; gentamicin and all the isolates were resistant to amoxycillin.Some of the isolates were multidrug resistant which was similar to the result of Kabir et al. (2014).In this study, it was observed that the E. coli isolates were sensitive to ciprofloxacin, norfloxacin and gentamicin.The results strengthen the earlier observations of Akond et al. (2009) and Islam et al. (2004).Resistance of E. coli was observed against erythromycin, amoxycillin.The result was supported by Akond et al. (2009).The cause of such resistance by the E. coli might be for the fact that the organisms might have gained the resistance property due to the indiscriminate use of antibiotics.
The occurrence of isolation of bacterial pathogens from broiler should be considered as hazardous to health and advocate the preventing risk factors.However, in the present study ciprofloxacin were proved to be the best antibiotics to treat E. coli infection since they were highly effective.The results agreed with those reported by several investigations of Islam et al. (2004) and Ozaki et al. (2011) who also obtained similar resistant patterns of E. coli isolated from broiler.It was revealed that Salmonella spp.were sensitive to ciprofloxacin, gentamicin and norfloxacin.This result was supported by Jahan et al. (2013) and Khan et al. (2005) where isolates were sensitive to ciprofloxacin and chloramphenicol.The result is also consistent with Wouafo et al. (2010).The isolates were resistant to erythromycin, and amoxycillin which is similar to report of Hyeon et al. (2011) and Khan et al. (2005).

Conclusion
In this study, bacteria were isolated from only 20 samples of 20 different upazila of Bangladesh.The findings of the present study revealed the presence of multidrug resistant bacteria in broiler meat sold in markets of Bangladesh.Most of the isolates showed resistance to amoxycillin but sensitive to ciprofloxacin and gentamicin.Some isolates showed multidrug resistance.

Figure 1 .
Figure 1.PCR targeting 16s rRNA gene for identification of E. coli.

Table 1 .
List of primers used in this study.

Table 2 .
Antimicrobial susceptibility pattern of E. coli, Salmonella spp., C. jejuni and C. coli identified by disk diffusion method.

Table 3 .
Results of antimicrobial resistance pattern of E. coli.

Table 4 .
Results of antimicrobial resistance pattern of Salmonella spp.

Table 5 .
Results of antimicrobial resistance pattern of Campylobacter spp.