Broiler meat production has been growing faster since the 1960s and it becomes the fastest growing sector in meat production through worldwide  . In Bangladesh, broiler meat is popular in the consumer market because of its easy digestibility and as a low-cost source of animal protein. The modern poultry industry can provide prepared broiler chickens in less than six weeks through genetic selection, improved feeding and keen health management practices. Nowadays antibiotics are used as therapeutic agents to treat bacterial diseases in intensive farming systems   because poultry meat offers an excellent medium for the multiplication of many bacteria even which are not inhibited by low temperatures. Storage of processed poultry meat is essential and considered only under circumstances which inhibit the multiplication of the initial load of bacteria  . Special attention should maintain because live animals are hosts to a large number of different microorganisms residing on their skin or feathers. During slaughter most of these microorganisms are eliminated. Contamination is possible at any stage of the production process, from feather plucking to freezing. Microorganisms from the environment, equipment and operators hands can contaminate the meat too   .
Poultry meat has significant contribution to the human diet  . The modernization of chicken farms and globalization of the bird breeding trade have played a role in infection  . During the slaughter of poultry birds there can be fecal contamination of the carcasses from the gut of these birds which means bacteria present in the spilled gut contents is passed on as contaminants  . Although due to short production time and low investment small scale commercial boiler farms are gradually rising but contamination of poultry meat with food borne pathogens remains an important health hazardous issue  . Foodborne diseases are great public health concerns of the modern world. Especially developing countries are largely affected by foodborne infections. These diseases affect people’s health and have an economic impact on the countries  . Bangladesh, as a developing country, have concerned about the foodborne diseases and economic impacts, because poultry industry is one of her significant source of income  . There are many regulatory agencies responsible for ensuring food safety and quality assurance. They are offered to the consumers that poultry chicken will be pure and healthful. Such agencies belonging to International forum include the Food and Agriculture Organization (FAO), World Health Organization (WHO), United Nations International Children’s Emergency Fund (UNICEF) and CAC  .
The significance of different foodborne diseases varies among countries depending on foods consumed, food processing, preparation, handling, storage techniques employed and sensitivity of the population. Microbiological food borne diseases are usually caused by bacteria or their metabolites, parasites, viruses or toxins  . The bacteria E. coli and Salmonella infections of poultry have been shown to be of critical importance in Bangladesh. Meat can be contaminated with E. coli during slaughter of the animals. E. coli from meat has mostly been associated with intestinal pathogenic E. coli. But many studies also proved that E. coli of animal origin has been shown to be associated with extra-intestinal infections, such as urinary tract infections  . Among the diseases caused by E. coli spp. some are often severe and sometimes causes lethal infections such as meningitis, endocarditis, septicemia, epidemic diarrhea of adults and children  . The health hazard from Salmonella spp. must be estimated because they have shown the enumeration of microbial indicators of fecal contamination. Salmonella was also detected in frozen samples from the supermarkets which indicates that the spread of infection is confined to apparently unhygienic environments  and the animal itself may be initially contaminated  . Most Salmonella found on poultry meat are non-host-specific. They are considered to capable of causing human food poisoning. Salmonellosis is the most common disease in human caused by Salmonella spp.  .Worldwide epidemiological reports incriminate poultry meat as a source of outbreaks of human foodborne disease. These outbreaks are caused by undercooking meat, cross-contamination of ready products to eat with microbial contaminants from the raw poultry. Poultry industry take aim to find ways to avoid contamination of live poultry and poultry products with potential pathogens  . Microbiological quality of processed carcasses mostly depends on a healthy condition and external micro flora of an animal  and the hygienic conditions during slaughtering and processing   . Foodborne diseases are not only associated with the consumption of poultry meat but also its processed products which have a great public health significance  . The relationship between the consumption of meat and health is multifaceted. The relevance of poultry meat for humans has been recognized by the United Nations (UN) Food and Agricultural Organization (FAO). These organizations have considered poultry meat as widely available, relatively inexpensive food to be particularly useful in developing countries, where it also can help to meet shortfalls in essential nutrients  .
Microorganisms have developed resistance against many antibiotics  and antibiotics are sometimes associated with side effects too  . Though there are some advantages of using antimicrobial compounds of medicinal plants  . In particular, Azadirachta indica (local name-neem) is one of the most promising medicinal plants which have several biological activities such as antioxidant, anti-inflammatory, antibacterial, antifungal, and antiulcer ones     . These biological activities are attributed to the presence of many bioactive compounds in its different parts. For example aqueous extract of Neem leaf extract has a good therapeutic potential as an antihyperglycaemic agent in insulin-dependent and non-insulin-dependent diabetes mellitus  . The phytochemicals like alkaloids, glycosides, flavanoids and saponins which are importance components of Azadirachta indica contain antibiotic principles of plants. They help in the defensive mechanism of the plants against different pathogen  . It is now considered as a precious source of unique natural products for development of medicines against various diseases  . The main objectives of our study was to investigate the microbial load of broiler meat, its impact on public health, antibiotic resistance pattern of these isolated bacteria and to seek a fruitful way for successful application of medicinal plants to minimize the hazards and risk related to bacterial contamination in poultry farms.
2. Method and Materials
2.1. Study Area
The study was conducted at different area of Nalitabari paurosova (Figure 1) in
Figure 1. Geographical location of the study area.
Sherpur district of Bangladesh during the periods of February 2015 to December 2018. Recently, huge number of poultry firm were established in this area that was an important reason for analyzing the poultry meat and all the samples were collected from poultry farms located in this region.
2.2. Collection of Samples
Fifteen samples were aseptically collected from study area. The slaughtered broilers at first immersed in a special tank containing hot water for some time. The immersed birds were de-feathered traditionally by hand plucking and subsequently evisceration was done using special tricks or techniques. The muscle of breast region were cut and put into a sterilized container. During transportation the sterile containers were kept cool in iceboxes containing fragments of ice.
2.3. Anthropometric Assessment
The anthropometric data were collected based on literature review of similar existing study  . In this study, randomly 400 respondents were selected by semi structured questionnaire based cross-sectional population selection system from study area. Among total respondents, 200 were females and 200 males. Age of the subjects under this study was determined by interrogation and confirmed with birth certificate or the health card. Measurements of weight were obtained by digital machine at three times and the average was calculated with minimal clothes and bare footed. The height was measured using a measuring tape without shoes and the average was calculated and recorded with standard error. Body Mass Index (BMI) was computed using the following standard equation: BMI = Weight (kg)/height (m2)  . Based on the interrelationships of height, weight, age, height for age, weight for age and height for weight have been calculated to determine their nutritional status  . Nutritional status (such as: thinness, malnourished, obesity, normal weight and overweight etc.) was evaluated following the recently published international BMI cut off points  .
2.4. Preparation of Sample for Bacteriological Studies
Each of the raw meat samples was macerated in a mechanical blender using a sterile diluent as per recommendation of International Organization for Standardization (ISO). Ten grams of the breast meat sample was taken aseptically with a sterile forceps and transferred into sterile containers containing 90 ml of 0.1% peptone water. A homogenized suspension was made in a sterile blender. Thus 1:10 dilution of the samples was obtained. Later on, different serial dilutions ranging from 10−2 to 10−9 were prepared according to the standard method  .
2.5. Enumeration of TVC and TCC
For the determination of TVC and TCC, 100 µl of each ten-fold dilution were transferred and spread on Plate Count Agar (PCA) and MacConkey agar using a fresh pipette for each dilution. The diluted samples were spread as quickly as possible on the surface of the plate with a sterile glass spreader. The plates were then kept in an incubator at 37˚C for 18 hours. The average number of colonies in a particular dilution was multiplied by the dilution factor to obtain the total viable count. The TVC and TCC were calculated according to ISO  . The results of the total bacterial count were expressed as the number of organism or colony forming units per gram (CFU/gm) of meat sample  .
2.6. Enumeration of Pathogenic Bacteria
For the identification of pathogenic bacteria, 100 µl of each sample were transferred into Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS) media and Shigella Salmonella Agar (SS) media with ten-fold dilution. The diluted samples were spread as quickly on the surface of the plate with a sterile glass spreader and incubated at 37˚C for overnight. The presence of pathogenic bacteria were observed and counted.
2.7. Statistical Analysis of Experimental Data
The data on TVC and TCC obtained from the bacteriological examination of meat samples of the poultry carcass collected from different area of Nalitabari. Data were analyzed with Microsoft Office Excel-2013  .
2.8. Cultural and Biochemical Examination of Samples
The cultural examination of chicken breast meat samples for bacteriological analysis was done according to the standard method by International Commission on Microbiological Specifications for Foods (ICMSF)  . The examination followed detail study of colony characteristics including the morphological and biochemical properties. In order to find out different types of microorganisms in chicken breast meat samples, different kinds of bacterial colonies were isolated in pure culture from the Plate Count Agar (PCA), MacConkey agar, SS agar and TCBS agar and subsequently identified according to the methods described by Krieg et al.  . Gram staining and biochemical reaction were performed for further confirmation of presumptively identified bacteria according to Bergey’s Manual Determinative Bacteriology  . Among these 15 samples 12 kinds of biochemical test such as Kligler Iron Agar (KIA), Motility-Indole-Urease (MIU), Citrate, Voges Proskauer (VP), Oxidase, Catalase, Mannitol, Starch, Methyl Red (MR), Glucose, Lactose, Eosin Methylene Blue (EMB) were performed.
2.9. Antibiotic Susceptibility Testing
The antibiotic susceptibility of the Escherichia coli and Salmonella spp. isolates was determined according to the standard disc-diffusion method  . Well isolated single colony obtained from overnight grown cultures, were used for making young culture for the test. The commercially available antibiotic discs (Oxoid, UK) used in this study were: Amoxicillin (30μg), Ampicillin (10μg), Tetracycline (30μg), Nalidixic Acid (30μg), Ciprofloxacin (5μg), Norfloxacin (10μg), Erythromycin (15μg) and Gentamicin (10μg).
2.10. Preparation of Plant Extracts
Healthy and disease free plant leaves of Azadirachta indica were collected from nearby area. The freshly collected leaves were washed with tap water, 70% ethanol and then distilled water to sterilize and remove the external impurities. These were further slice into small pieces and shade dried for two weeks and then blended into powder using mortar. The powdered leaves of A. indica were separately extracted with different solvents (30%, 40% of ethanol and methanol) by using Whatman No. 1 filter paper. The crude extracts were obtained by concentrating the mixture solution using a rotary evaporator and used for further tests.
Antibacterial Screening Test for Plant Extract
Young culture of bacterial aliquots of the test organisms in 4 ml sterile Mueller Hinton Broth (MHB) were made from well isolated single colony obtained from 24 h growth cultures. Each aliquots (10 μl) containing approximately 5 × 104 bacterial cells or colony forming units was transferred into Mueller-Hinton Agar (MHA) plates. The disc containing plant extract were diffused into the plate where, the extract allowed to stands for an hour for reaction to take place between the extracts and the bacterial organisms. The plate were then inoculated on separate and incubated at 37˚C for 18 hours.
3. Results and Discussion
3.1. Microbiological Analysis
3.1.1. Total Viable Count (TVC)
International Commission on Microbiological Specifications for Foods (ICMSF) recommended that the general viable count of fresh meat tissue at 35˚C should be less than 106 CFU per gram  . The average value of total viable count (TVC) of 15 samples was 4.3 × 106 CFU/gm. The lowest TVC was found 2.3 × 104 CFU/gm (sample 3) and highest was 3.6 × 107 CFU/gm (sample 13). Other studies reported the range of TVC from 6.1 × 106 to 6.5 × 105 CFU/gm  . Another study revealed that the mean value of total viable count of chicken meat was 5.0 × 105 to CFU/gm  and 1.3 × 106 CFU/gm  .
3.1.2. Total Coliform Count (TCC)
Total coliform count is the key indicator of the severity of bacterial contamination of broiler meat and its suitability to human consumption. In most cases of processed broiler meat, the high quantity of coliform bacteria causes severe food poisoning especially among children. In this study, the lowest value of TCC was found 1.6 × 103 CFU/gm (Sample 3) and the highest was 1.5 × 105 CFU/gm (Sample 8). The mean value was obtained as 3.6 × 104 CFU/gm. In another study, the average values of TCC at three different markets of Bangladesh were found as 4.7 × 104, 4.2 × 104 and 5.1 × 104 CFU/gm  and many other studies also showed the similar result regarding TCC. The isolation of coliform bacteria also indicates probable fecal contamination and the prevalence of fecal coliform was recorded as about 33% in this study where, fecal contamination were detected in five samples (2, 7, 8, 11 and 13). Presence of coliforms in broiler meat might be due to poor quality of water used for washing of meats, fecal contamination with own feces, inadequate light and air in poultry culture room, unhygienic places and personal unhygiene dinning meat processing.
3.1.3. Presence of Escherichia coli
E. coli can cause severe gastrointestinal tract-related complications like diarrhea, dysentery, urinary tract infections, pneumonia and even meningitis  . It has been recommended to be totally absent in poultry meat. Highly E. coli contaminated poultry meats is unfit for human consumption and considered as unhealthy. This study had revealed the presence of E coli in 53% samples which is a significant matter of concern. Some studies have reported the presence of higher level of E. coli in chicken meat samples like 76%  and 34.6%  . However, some studies also showed the prevalence in chicken meat samples in lower rate like 0.4%  , 5.6%  , 11.1%  and 16%  . The hygiene of sources, living places and environment, broiler feed and processing process with appropriate management has a great influence in minimizing the total E coli count in Table 1.
Table 1. Microbial load of broiler meat.
ND = Not Detected, +VE = Positive.
3.1.4. Total Salmonella spp. Count and Presence of Shigella spp., Vibrio spp. and Fungi.
In this study, Salmonella spp. was found 26.7% where other studies showed as 15.39%  and 7.41% in chicken meat  . The mean value of Salmonella spp. was 4.6 × 103 CFU/gm, the maximum was 5.6 × 103 CFU/gm and minimum was 3.1 × 103 CFU/gm. More than 25 g of Salmonella contaminated poultry meat is considered as unsafe for human consumption. Salmonellosis remains one of the most frequent food-borne diseases that constituting a worldwide major public health concern. The majority of food born infections may be occurred by consumption of contaminated poultry meat with Salmonella from any source in-spite of the success of Salmonella control measures implemented in food-animal production of industrialized countries  . The outbreaks of Shigellosis may have been associated with the consumption of several kinds of contaminated foods, milk, poultry, and some dairy products  . No Shigella spp. has been detected among 15 samples in this study. The result is similar with another research in which no Shigella spp. was found after evisceration  . Likely, no species of Vibrio has been detected in this present study. Earlier, a study has been reported a low prevalence of Vibrio spp. (0.3%)  . If the Vibrio spp. frequency found at a considerable high percentage, it will indicate the alarming situation of chicken farming and for public health as well  . There is lack of information about the acceptable limit for fungal contaminants could be of concern to the public health  . Any type of fungi is totally absent in all samples, although some other studies found the fungal contamination in some fresh chicken samples   .
3.2. Result of Biochemical Test
Three kinds of bacteria (E. coli, Salmonella spp. and Vibrio spp.) were isolated by observing distinct morphological characteristics on selective media and further confirmed with standard Biochemical test (Table 2).
Table 2. Reaction of biochemical test.
K = Alkaline, A = Acid, G = Gas, AG = Acid and Gas, + = Presence, − = Absence.
3.3. Antibiotic Resistance Pattern
Resistance against commercially available commonly used antibiotics has been observed in bacteria present in broiler since the introduction of these antimicrobial agents in poultry. The rise in antibiotic resistance has been reported in the past two decades in many countries including Bangladesh  . Based on the susceptibility to antibiotics, the bacteria were categorized into three groups—sensitive, intermediate, and resistance. Total 20 isolates (10 E. coli and 10 Salmonella spp.) were randomly selected for antibiotic susceptibility test.
In this study, E. coli showed highly resistance to Ampicillin (80%) and Amoxicillin (70%). Where, 40% were resistance to Nalidixic acid and Erythromycin. About 90% E. coli showed sensitivity to Ciprofloxacin, 80% to Gentamicin, 60% to Tetracycline and 50% to Norfloxacin (Figure 2). The results strengthen the earlier observations of some study where it was found that the E. coli isolated from broiler were sensitive to Azithromycin, Ciprofloxacin, Norfloxacin and Gentamicin and resistant to Amoxicillin and Erythromycin. The possessions of such factors by the E. coli isolates signify the fact that the organisms might have gained the resistance property due to the unsystematic use of antibiotics  . Salmonella spp. showed 100% resistance to Ampicillin, 90% resistance to Amoxicillin and highly sensitive to Ciprofloxacin (80%). About 50% were resistance to Nalidixic Acid and Norfloxacin whereas sensitive to Erythromycin (60%) and Gentamicin (50%) (Figure 3). Some others earlier study also revealed that
Figure 2. Antibiotic resistance pattern of Escherichia coli.
Figure 3. Antibiotic resistant pattern of Salmonella spp.
Salmonella spp. were sensitive to Ciprofloxacin, Gentamicin and Azithromycin   and resistant to Erythromycin and Amoxicillin   . Potential drug resistant pathogens in normal broilers may be a serious public health concern.
3.4. Evaluation of Antibacterial Activity of Azadirachta indica against Some Multidrug Resistance Escherichia coli and Salmonella spp.
The zone of inhibition of negative (30% methanol, 40% methanol, 30% ethanol, 40% ethanol, DMSO) control of plant extract and positive control (Ciprofloxacin) with and their comparison is presented in (Table 3). The above result specified the antimicrobial activity of extract against Escherichia coli and salmonella spp. depending on the nature of the active ingredients present in the extracts and their capacity of diffusion into agar medium. Antibacterial activities of methanolic and ethanolic plant extract (both 30% and 40%) were significant against E. coli. The average zone of inhibition of methanolic and ethanolic extract were 13 and 14.33 respectively, where 40% ethanolic plant extract was more potent than the 30% (Figure 4). No zone of inhibition was formed against isolated Salmonella spp.
Table 3. Antibacterial activity of Azadirachta indica plant extract.
N/A = No Activity.
Figure 4. Average zone of inhibition of methanolic and ethanolic plant extract with different concentrations against Escherichia coli.
A previous study found the average zone of inhibition of methanolic extract of Azadirachta indica against Escherichia coli was 7.5 mm  . The zone of inhibition against E. coli in both methanolic and ethanolic extract was not found by another research  . But Zone of inhibition of methanolic extract of same plants against Salmonella spp. was reported as 10 - 20 mm  . Another study evaluated the zone of inhibition with different concentrations of methanolic plant extract were 20 mm, 22 mm, 24 mm and 21 mm in diameter against E. coli, where 18 mm, 22 mm, 20 mm and 21 mm in diameter against Salmonella spp.  .
3.5. Assessment of Public Health Impact of Broiler Meat
The total respondents were divided into five groups based on age range—5 to 15, 16 to 30, 31 to 40, 40 to 50 and above 50 years old (Table 4). Each group had 40 male and 40 female respondents (Table 4). Total 300 (75%) people eat broiler meat among the total respondents, where 150 (50%) were females and 150 (50%) were males. The educational status of the respondent showed that, about 8% were illiterate, 10% completed primary level, 21% passed up to secondary level, 32% were higher secondary level and 35% were in graduation level (Table 5).
On the basis of family income, the respondents were divided into five socio-economic groups, like poor (15.75%), lower middle class (23.5%), middle class (49.25%), upper middle class (6.75%) and higher class (4.75%) (Table 6). Most of the female respondents in this study were domestic worker (34.5%). The major source of family income among these respondents were day laborer (18%) and businessman (18%), rest of them were private service holder (14.75%) and government service holder (14.75%) (Figure 5).
In this study, it was found that consumption of broiler meat largely varies with the socio-economic status of respondents (Table 7). Depending on economic status, most of the poor people (N = 31, 49.20%) ate broiler meat at least one day in a week where very few people of upper middle class (N = 9, 21.95%) and higher class (N = 2, 4.88%) ate occasionally, and majority of last two groups never eat broiler meat (N = 12, 44.44%) and (N = 17, 89.47% respectively).Large number of broiler meat consumer were middle classes and lower middle classes
Table 4. Frequency distribution of the respondents of this study.
Table 5. Educational status of subjected people.
Table 6. Socio-economic condition of the people.
Table 7. Broiler meat intake pattern.
where most of the mate broiler meat frequently (N = 85, 73.91%) and At least 2 - 4 days in a week (N = 44, 61.97%). Very few people of middle and lower middle classes ate broiler meat at least 5 days in a week (Figure 6).
BMI is an important consideration for evaluating the health condition of particular people. Various BMI had been observed among the responders who ate broiler meat or not. By considering various BMI group, it was found that, overweight (male 24.67%, female 22.67%) and obesity (male 32%, female 8%) of the responders who ate broiler meat were high in comparison with overweight (male 10%, female 16%) and obesity (male 8%, female 6%) of responders who never ate broiler meat (Figure 7).
Some common diseases such as food allergy, overweight, obesity, high blood pressure, and diabetes were considered to determine the possible correlation with consumption of broiler meat. Food allergy is allergic reaction of food includes fish, shellfish, peanuts, tree nuts, walnuts, etc., but not directly related to broiler meat. In this study, it was found that the occurrence of food allergy relatively high among the people who never ate broiler (male 32%, female 12%) compare to the people who ate broiler meat (male 23.3%, female 9.3%), whereas, in case of other diseases, it was vice versa. Among the broiler meat consumer, overweight, obesity and high blood pressure were found in elevated rate than any other diseases both in male and female. Male who eat broiler meat had high blood pressure, high cholesterol level, digestive disorders, constipation and diabetes in alarming percentage with 35.3%, 22.7%, 18.7%, 24% and 7.3% respectively whereas male who never eat broiler meat had this diseases in percentage with 16%, 10%, 14%, 6% and 4% in some respect. Surprisingly, it is a matter of great concern that, 3.3% male who eat broiler meat found having benign fat deposition like tiny tumor but this disease did not found in male and female who never eat broiler meat (Figure 8).
Figure 5. Source of family income of the respondents.
Figure 6. Broiler meat intake pattern.
Figure 7. Various health condition of broiler meat consumer according to BMI.
Figure 8. Comparison of disease frequency between who eat broiler meat and never eat broiler meat (male and female).
The present study demonstrated that the contamination of poultry and poultry products should be prevented during handling, slaughtering and processing to protect the public from infections and diseases. Present data also indicated that the viable count of microorganisms causing public hazards is also appropriate for analysis. Due to increasing density of poultry farms and infectious diseases in poultry caused by pathogenic bacteria, the healthy development of the poultry industry is facing serious threat. Therefore, application of hygienic measurements appears to be important to reduce the contamination of bacteria after processing of meat. The presence of E. coli and Salmonella demonstrates a potential health risk since the organisms are pathogenic and give warning signal for the possible occurrence of food borne intoxication. The need for microbial assessment of fresh meats for human consumption is emphasized and recommended to reduce possible hazard. Sensible use of antibiotics should be considered in broiler production since many strains get resistant to common antibiotics. The leaf extract of Azadirachta indica showed potent antibacterial activity against E. coli. It is recommended to isolate and separate the bioactive compounds responsible for this antibacterial activity and to apply such medicinal plants to minimize bacterial contamination in broiler meat.