Investigating the Diversity of Spoilage and Food Intoxicating Bacteria from Chicken Meat of Biratnagar, Nepal

Research Article

J Bacteriol Mycol. 2020; 7(5): 1141.

Investigating the Diversity of Spoilage and Food Intoxicating Bacteria from Chicken Meat of Biratnagar, Nepal

Mahato A1,2, Mahato S1,3*, Dhakal K3 and Dhakal A3

¹AASRA Research and Education Academy Counsel, Biratnagar-6, 56613, Nepal, India

²Department of Military and Veterans Affairs, New Jersey Veterans Memorial Home, NJ, USA

³Department of Microbiology, Mahendra Morang Adarsh Multiple Campus, Tribhuvan University, Biratnagar, Nepal, India

*Corresponding author: Sanjay Mahato, Department of Microbiology, Mahendra Morang Adarsh Multiple Campus, Tribhuvan University, Biratnagar, Keshaliya Road, Janpariya Tole, Biratnagar-06, Nepal, India

Received: June 05, 2020; Accepted: July 02, 2020; Published: July 09, 2020


Background: Foodborne diseases are global human health problems, especially in developing countries where substandard hygiene of food like meat and unsafe water supplies prevail which are aggravated by multidrug resistance.

Objectives: The study was designed for investigating the diversity of microbial population like E. coli, Staphylococcus spp, Vibrio spp, Salmonella, Shigella and Pseudomonas from raw chicken meat and their drug resistance.

Results: The major bacterial pathogens isolated were Shigella spp (60%) followed by Salmonella typhi (53.3%), Pseudomonas aeruginosa (46.7%) and E. coli (46.7%), Staphylococcus aureus (40%), Staphylococcus epidermidis (33.3%) and Vibrio spp (13.3%) were isolated. With four antimicrobial drugs, 57.1% isolates of E. coli were sensitive to cefotaxime and levofloxacin while 65% resistant to amoxicillin. S. aureus isolates were 100% sensitive to cefotaxime and amoxicillin while 50% were sensitive to erythromycin. All isolates of Vibrio were found to be 100% sensitive to levofloxacin and amoxicillin. The isolates of Shigella were 37.5% resistant to cefotaxime and levofloxacin; while 12.5% resistant to amoxicillin. Salmonella typhi showed 33.3% resistant to cefotaxime and 22.2% resistant to levofloxacin and amoxicillin. Pseudomonas aeruginosa is 28.6% resistant to amoxicillin; while 14.3% resistant to levofloxacin and cefotaxime.

Conclusion: The meat in retail shops of Biratnagar is highly contaminated so this could bring foodborne infections in the city. In this light, it is recommended that microbial assessment of fresh meats and other meat products for human consumption should be performed and proper cleanliness should be adopted to reduce a possible hazard.

Keywords: Staphylococcus; Pseudomonas; Spoilage; Chicken; Multidrug-Resistant


spp: Species; MDR: Multi-Drug Resistant; g: Gram; MHA: Muller- Hinton agar; NA: Nutrient Agar; XLDA: Xylose Lysine Deoxycholate Agar; TCBS: Thiosulfate-Citrate-Bile Salts-Sucrose; MSA: Mannitol Salt Agar; mL: Milliliter; CLSI: Clinical and Laboratory Standards Institute; CTX: Cefotaxime; LE: Levofloxacin; AMX: Amoxicillin; E: Erythromycin; S: Sensitive; I: Intermediate; R: Resistance


Spoilage is the procedure by which food is deteriorated and becomes unacceptable for humans or its quality is reduced making it inappropriate for sale or consumption [1]. Chicken is one of the most consumed meats which contains enough nutrition needed to support the growth of microorganisms [2]. Nepal produces 16662 metric ton of chicken meat [3]. Since meat contains fat, protein, minerals, carbohydrate and water, the quality of meat and meat products degrade because of digestive enzymes, microbial spoilage and lipid oxidation [4, 5]. Fat oxidation, protein degradation and the loss of other biomolecules are the results of meat spoilage process.

The intestinal tract and the skin of the animal are the main sources of these microorganisms. Chicken meat can be contaminated at several points throughout the processing operations like stunning, bleeding, skinning, evisceration and carcass splitting [6]. Moreover, cutting of meat at retail outlets could result in greater microbial growth owing to a large amount of exposed surface area, more readily available water, nutrient and greater oxygen penetration which leads to spoilage of meat [7]. Microorganisms reach the meat via butcher’s hands, utensils, and tools, clothing and apron, water, etc. The number can be multiplied during cutting and distribution [4].

Spoilage bacteria include Bacillus spp, Shigella spp, Streptococcus pyogenes, Proteus, Leuconostoc, Lactobacillus spp, Pseudomonas, Micrococcus, Streptococcus, Lactobacillus, Salmonella, Escherichia, Clostridium and Bacillus [8, 9]. The most important pathogens associated with meat include Salmonella, Staphylococcus aureus, Escherichia coli, Clostridium perfringens, Campylobacter jejuni, Listeria monocytogenes, Yersinia spp, and Aeromonas hydrophila, Acrobacter, Mycobacterium, Vibrio spp, etc [10,11].

Staphylococcal food intoxication generally occurs within one to six hours after the ingestion of contaminated water or food and symptoms are nausea, vomiting, abdominal cramps and diarrhea [12]. Vibrio cholerae produces cholera enterotoxin and responsible for the life-threatening secretory diarrhea. It is strongly aerobic, growth being scanty and slow anaerobically [13]. Since poultry meat is usually not consumed raw, these outbreaks are caused by undercooking or cross-contamination of ready-to-eat products with microbial contaminants from the raw poultry or others introduced during preparation of the food [14].

Widely using antibiotic in the poultry as treatment prophylaxis or growth promoters in livestock lead to widely spread antibioticresistant pathogens that cause the problem in the humans [15]. The prevalence of Multi-Drug Resistant (MDR) foodborne pathogens is increased by consumption of contaminated food because they are responsible for more serious disease than susceptible bacteria [16].

The main aim of this study was to assess the microbial diversity of spoilage and food intoxicating bacteria of raw meat from outlets of Biratnagar and to understand its possible role in spoilage and foodborne illnesses.

Materials and Methods

Sample Collection

Fifteen samples of raw chicken meat (25 g) were collected aseptically in a sterile plastic container from different meat shops in Biratnagar from July 2017 to August 2017. After collection, the samples were transported to the laboratory for further processing i.e., Isolation, identification.

Sample Processing

Twenty-five gram of meat sample was weighed and grinded with the help of motor and pestle. The initial dilution was prepared by adding 25 g of the sample into 225 mL of dilution blank (10-1), thoroughly mixing, and thereby performing serial dilution up to 10-7.

Isolation and Identification of Bacteria

For isolation of E. coli and Pseudomonas spp., Eosin Methylene Blue (EMB) agar (HiMedia, Mumbai, India) plate and Muller-Hinton Agar (MHA) (HiMedia, Mumbai, India) plate were spread with 0.1 mL inoculum from several dilutions respectively and incubated at 37°C for 24 hr and consequently sub-cultured onto Nutrient Agar (NA) (HiMedia, Mumbai, India) plate to get pure culture for further identification. For Salmonella typhi and Shigella spp, bacterial suspension was spread into Xylose Lysine Deoxycholate Agar (XLDA) (HiMedia, Mumbai, India) and incubated at 37°C for 24 hr and consequently sub-cultured onto NA to get pure culture for further identification. Similarly, Thiosulfate-Citrate-Bile salts- Sucrose (TCBS) Agar plate (HiMedia, Mumbai, India) and Mannitol Salt Agar (MSA) plate (HiMedia, Mumbai, India) were used for cultivation of Vibrio spp and Staphylococcus spp respectively. After aerobic incubation at 37°C for 24 hr, consequently, the characteristic S. aureus and S. epidermidis colonies were yellow and cream color respectively. Selected colonies were sub-cultured onto NA plates to get pure culture. The isolation of the bacteria was done by using spread plate technique. 0.1 mL of bacterial suspension from 10-3 and 10-4 was taken.

Microscopic and Biochemical Identification of the Isolates

Characterization and identification of the colony isolates were achieved by initial morphological examination of the colonies in the plate (macroscopically) for colonial appearance, size, elevation, form, edge, consistency, color, odor, opacity, and pigmentation, and the results were recorded. Gram’s staining, capsule and spore staining were done to the selected colonies for preliminary identification of the bacteria. The bacterial isolates were identified by cultural, physiological, morphological and biochemical tests as per Bergey’s manual of determinative bacteriology [17]. Biochemical identification of the isolated bacteria was done by particular tests such as catalase, oxidase, TSI, Indole, Methyl red, Voges-Proskauer and citrate tests, carbohydrate fermentation tests, coagulase, O/F tests and urease test [18].

Antibiotic Sensitivity Testing for the Isolates

The identified isolates were submitted to antimicrobial susceptibility testing according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [19]. Antibiotic sensitivity testing by disc diffusion method was performed for the isolates using commercially available antibiotic discs (HiMedia, Mumbai, India) on Muller-Hinton agar (MHA) (HiMedia, Mumbai, India). The organism was diluted to obtain a turbidity equivalent to the 0.5 McFarland test standard. Immediately a sterile cotton swab was dipped into bacterial suspension and lawn culture was performed on the surface of MHA plate. The antibiotic discs like Cefotaxime (CTX, 30 μg), Levofloxacin (LE, 5 μg), Amoxicillin (AMX, 10 μg), and Erythromycin (E, 15 μg) were placed on the plates. Then the plates were incubated at 37°C for 18 hours. After incubation, the zone diameter was measured and compared to the standard chart. Thereby the zone of inhibition was interpreted as Sensitive (S), Intermediate (I) or Resistance (R).


A total number of 15 chicken meat samples were examined for the presence of bacteria. E. coli isolates appeared as greenish metallic sheen (Figure 1) on EMB agar plate. The large yellow colored colony on TCBS (Figure 2) represents Vibrio spp. Staphylococcus aureus and Staphylococcus epidermidis showed golden yellow and cream-colored colony respectively (Figure 3) on MSA agar plate and showed gelatin hydrolyzing activity (Figure 4). The bacterial pathogen isolated from chicken meat sample was Shigella (n=9, 60%) (Figure 5) followed by Salmonella (n=8, 53.3%), Pseudomonas (n=7, 46.7%) (Figure 6 and Table 1), E. coli (n=7, 46.7%); S. aureus (n=6, 40%); S. epidermidis (n=5, 33.3%) and V. cholera (n=2, 13.3%) and were identified by cultural and biochemical properties.