Isolation, Identification and Antibiotic Resistance Profile of Public Health Threat Enteric Bacteria from Milk and Dairy Products Retail in Abakaliki, South-East, Nigeria

Milk and foods made from milk is manufactured into more stable dairy products of worldwide value, such as butter, cheese, ice cream, and yoghurt. Consumption of contaminated milk or dairy products by pathogens causes human gastrointestinal infection, which leads to diarrheal disease in human and hospitalization or death in severe cases especially among elderly and children. An assessment of milk and dairy products was designed to determine the microbiological quality of milk and dairy products consumed in Abakaliki, Nigeria. Culture techniques were usedfor isolation of enteric bacteria from retail dairy productsand disk diffusion method were used to determine the Antibiotic Resistance profile of isolates. Bacteria pathogens isolated were characterized and identified using morphological and biochemical techniques. SPSS and Chi-square test were used for the analysis of the study,P-value of 0.02 indicates a significant difference between the bacteria pathogens counts. A total of 161 pathogenic bacteria were isolated from 100 dairy products. Salmonellaspp heard (26.1%), Escherichia coli (44.1%) and Shigella spp. (29.8%). All identified isolates were found to be 100% susceptible to ciprofloxacin and gentamycin, with 66.7% for ofloxacin. Augmentin, ampicillin, chloramphenicol and spectinomycin was 100% resistant. Data obtained confirm that milk and dairy products retailed in Abakaliki pose a serious public health threat to consumers due to the presence of pathogenic bacteria. Standard and good storage conditions, as well as environmental and personnel hygiene should be practiced to prevent contamination of milk and dairy products for the safety of consumers.


INTRODUCTION
Foodborne diseases result from the consumption of food contaminated or infected by pathogens. 1These diseases cause serious and continued challenges to human health globally. 2The epidemiology of foodborne diseases has changed rapidly due to changes in the environment and the ability of pathogens to adapt to a new environment. 3,4Foodborne pathogens cause illness and death especially in developing countries, leading to a loss of labour force that could have contributed to economic growth. 5,6Poor environmental sanitation, changes in eating habits, and lack of hygienic practice during production, packaging, transportation, storage and retail of food are contributing factors that leads to contamination of dairy products. 7nnually, millions of cases of foodborne illness and thousands of associated deaths occur especially in developing countries. 8,9The primary materials for the production of dairy products such as ice cream, yoghurt, butter and cheese are water, condensed and dried milk.Contamination of the primary materials affects the quality of the dairy products.Pathogenic microorganisms contaminate dairy products through impure air in the processing environment or through poor personnel hygiene during processing, as well as the use of contaminated water during preparation and poor storage condition. 10The nutrient composition of milk and dairy products ensures a favourable environment for different microorganisms to grow and multiple. 11,12][15][16] Public health threat pathogenic bacteria of the Enterobacteriaceae family are mostly implicated in foodborne infections globally. 17,18Salmonella spp., Shigella spp., Listeria spp., Escherichia coli, Enterobacter spp., Campylobacter spp.and Yersinia spp.0][21] Thus, this study aimed to determine the microbiological quality of various dairy products consumed in Abakaliki, and the antibiogram of the pathogenic enteric bacteria isolates from milk and different dairy products.

Study area and duration
The

Study design and samples collection
A total of 100 samples (ice cream, yoghurt, butter, cheese, dried milk) of dairy products were randomly collected from dairy shops and stores in Abakaliki metropolis, Ebonyi State, Nigeria.Twenty of each of the samples were transmitted to the microbiology laboratory in an insulating icebox as soon as possible for examination.

Bacteriological examination
The study was conducted using cultural techniques for the detection of enteric bacteria following the international standard guideline from ISO, 20976-1:2019(E).

Biochemical tests
All isolates were gram stained to detect if isolates were gram-negative or positive.Suspected bacteria pathogens stored on nutrient agar were identified by inoculation unto tubes of different biochemical test mediums such as; Motility Indole Urea (MIU) agar, Simmon Citrate Agar (SCA), Triple sugar iron (TSI), Methyl Red Voges Proskauer (MR-VP) broth, incubated for 24hr at 37°C.Escherichia coli and Salmonella spp.isolates are motile, Shigella spp. was not.All isolates are urease and citrate negative.Escherichia coli was indole positive while Salmonella spp.and Shigella spp.are indole negative.TSI agar test showed yellow slop and yellow butt for Escherichia coli while it showed red slop and yellow butt for Salmonella spp.and pink for both slop and butt for Shigella spp.Escherichia coli produced gas while Salmonella spp.and Shigella spp.doesn't.Salmonella spp.produces Hydrogen Sulphide (H 2 S) or (Blackened), while Shigella spp.and Escherichia coli don't.

Data analysis
Data was analyzed using the statistical software programme SPSS version 20.0.(SPSS 20.0 Command Syntax Reference, SPSS Inc. Chicago, 2013).The Chi-square test was utilized to assess significant differences between each pathogen from different dairy products.

RESULTS
Bacteriological examination of dairy products through culture techniques and identification of isolates through morphological and biochemical methods implicated Escherichia coli (44.10%),Salmonella spp.(26.10%) and Shigella spp.(29.80%) as the prevalent bacteria.Escherichia coli was most prevalent and Salmonella spp.least prevalent (Figure 1).Among the dairy products, butter had the highest Escherichia coli, while ice cream had the least (Figure 2).The percentage distribution of Salmonella spp.heard yoghurt with the highest percentage and butter with the least (Figure 3).The highest percentage of Shigella spp. was detected in dried milk, while ice cream had the least (Figure 4).The antibiogram of the isolates confirmed that ciprofloxacin and gentamycin were completely susceptible while ampicillin, augmentin, chloramphenicol and spectinomycin were completely resisted by the isolates (Figure 5).The percentage resistance and susceptibility confirmed that Escherichia coli was 50% resistance and 50% susceptibility.Shigella spp.and Salmonella spp. was 60% resistance and 30% susceptible, respectively, with 10% intermediate (Figure 6).Out of 100 dairy products sampled, a total of 161 bacteria was isolated (Table 1).The first line of antibiotics for the treatment of dairy foodborne disease are ciprofloxacin and gentamycin (Table 2).

DISCUSSION
In the present study, out of 100 dairy products sampled, a total of 161 isolates were isolated from all the samples.Statistical analysis showed a significant difference between bacteria species isolated across the sampling period (P<0.05).Escherichia coli had the highest distribution of bacteria isolates of 71 (44.10%), while Salmonella spp.had the least number of 42 (26.10%).The isolation rate of Escherichia coli agreed with a report by Tadesse et al. 23 which reported 44.57%.The results also agreed with Dadi et al. 24 that Escherichia coli was the most prevalent bacteria in dairy products while Salmonella spp. was the least.In this study, the highest distribution of Escherichia coli was discovered in butter (25.4%) and dried milk (22.50%), while the least was in ice cream (14.10%).Salmonella spp. was found to have the highest distribution in yoghurt (26.25%) and least in butter (14.30%).Shigella spp. was found to have the highest distribution in milk (27.10%), this agreed with a report by Abunna et al. 25 Above all, the water used for cleaning milking equipment and the air in the environment are the likely sources of contamination by enteric bacteria.However, the high percentage distribution of Escherichia coli may be due to poor hygiene practices in dairy products firms.The antibiotic susceptibility testing for Escherichia coli had an overall susceptibility of 50% and resistance of 50%, this slightly agreed with Fredrick et al. 26 who reported overall susceptibility of 64.35%.All isolates were 100% resistant to ampicillin, augmentin, chloramphenicol, and spectinomycin, the findings agreed with Federick et al. 26 The highest resistance (60%) and least susceptibility (30%) were reported for Shigella spp.and Salmonella spp. in the present study, this agreed with Demirci et al. 27 ; Qamar et al. 28 Furthermore, Escherichia coli, Salmonella spp., and Shigella spp.had 100% susceptible to ciprofloxacin and gentamycin, 66.7% for ofloxacin, 33.3% for pefloxacin.These findings agreed with Qamar et al. 28 who reported ciprofloxacin (100%).The present study suggestsciprofloxacin as the first drug of choice in the treatment of food-borne illnesses, followed by gentamycin.Moreover, hygiene practices should be encouraged among dairy food handlers to avoid contamination.

CONCLUSION
Pathogenic microorganisms are highly resistant to so many conventional antibiotics/ drugs, therefore regular antibiotic surveillance is necessary from time to time to determine antibiotics that are susceptible to a particular pathogen.Ciprofloxacin and gentamycin antibiotics were confirmed as the antibiotics/drugs for the treatment of illness caused by foodborne

Table 1 .
Distribution of pathogenic bacteria in dairy products