ISSN: 0973-7510
E-ISSN: 2581-690X
, Chukwuma Victor Uzoh1, Isaiah Yusuf1,K.E. Aroh1, O.J. Owolabi1, A.C. Kalu2, M.M. Egwu-Ikechukwu1, C.C. Nnabugwu1, O.C. Azuama1, D.C. Ilang1, B. Ugwu1, D.O. Okata-Nwali1 and C.S. Ugwuocha1Milk 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 used for isolation of enteric bacteria from retail dairy products and 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. Salmonella spp 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.
Isolation, Identification, Antibiotic Resistant Profile, Enteric Pathogens, Milk, Dairy Products
Foodborne diseases result from the consumption of food contaminated or infected by pathogens.1 These diseases cause serious and continued challenges to human health globally.2 The epidemiology of foodborne diseases has changed rapidly due to changes in the environment and the ability of pathogens to adapt to a new environment.3,4 Foodborne pathogens cause illness and death especially in developing countries, leading to a loss of labour force that could have contributed to economic growth.5,6 Poor 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.7 Annually, millions of cases of foodborne illness and thousands of associated deaths occur especially in developing countries.8,9 The 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.10 The nutrient composition of milk and dairy products ensures a favourable environment for different microorganisms to grow and multiple.11,12 Drug-resistant microorganisms are widely in circulation in the environment with a significant rise in their negative effects over the past few years as a result of empirical antibiotic use and knowledge deficiency.13-16 Public health threat pathogenic bacteria of the Enterobacteriaceae family are mostly implicated in foodborne infections globally.17,18 Salmonella spp., Shigella spp., Listeria spp., Escherichia coli, Enterobacter spp., Campylobacter spp. and Yersinia spp. are among public health threat pathogenic microorganisms commonly implicated in foodborne diseases.19-21 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 was conducted in Abakaliki the capital of Ebonyi State, South-East Nigeria. Abakaliki is located at Latitude 6.32485 and Longitude 8.11368. It is part of West Africa and in the Northern hemisphere. The study was conducted from August to December 2022 at the Microbiology laboratory of Alex Ekwueme Federal University Ndufu Alike, Ebonyi State, Nigeria.
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 (H2S) or (Blackened), while Shigella spp. and Escherichia coli don’t.
Antimicrobial susceptibility test of enteric bacteria
The antimicrobial susceptibility profile for all enteric bacteria of concern (Salmonella spp., Escherichia coli, and Shigella spp.) was carried out using the disc diffusion method on Mueller Hinton agar. The following antibiotics was used; ciprofloxacin (5µg), ampicillin (10 µg), augmentin (30 µg), gentamycin (10µg), perfloxacin (5µg), ofloxacin (5µg), streptomycin (10 µg), trimethoprim-sulfamethoxazole (1.25 µg /23.75 µg), chloramphenicol (30 µg) and spectinomycin (10µg). Within 30 minutes of applying the discs, the plates were inverted and incubated aerobically at 35°C for 16–18 hrs. After incubation, the control and test plates were examined to ensure the growth is confluent or near confluent. The zone of inhibition was measured in millimetres and interpreted as described in Haile et al.22
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.
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).
Figure 1. Percentage prevalence of bacteria in dairy products
Figure 2. Percentage distribution of Escherichia coli in dairy products
Figure 3. Percentage distribution of Salmonella spp. in dairy products
Figure 4. Percentage distribution of Shigella spp. in dairy products
Figure 5. Percentage susceptibility profile of antibiotics among enteric bacteria from dairy products
KEY: CPX: Ciprofloxacin (5µg), AM: Ampicillin (10µg), AU: Augmentin (30µg), CN: Gentamycin (10µg), PEF: Perfloxacin (5µg), OFX: Ofloxacin (5µg), S: Streptomycin (10µg), SXT: Trimethoprim-Sulfamethaxazole (1.25/23.75), CH: Chloramphenicol (30µg), SP: Spectinomycin (10µg)
Figure 6. Percentage resistance and susceptibility of pathogenic bacteria from dairy food
Table (1):
Distribution of pathogenic bacteria in dairy products
Dairy foods |
Number of samples |
Escherichia coli |
Salmonella spp. |
Shigella spp. |
No growth |
|---|---|---|---|---|---|
Milk |
20 |
16 |
8 |
13 |
0 |
Yoghurt |
20 |
13 |
11 |
10 |
0 |
Ice-cream |
20 |
10 |
10 |
8 |
2 |
Cheese |
20 |
14 |
7 |
9 |
1 |
Butter |
20 |
18 |
6 |
8 |
2 |
Total |
100 |
71 |
42 |
48 |
5 |
Table (2):
Antibiotic susceptibility profile of bacteria isolates from dairy products
Isolates |
CPX |
AM |
AU |
CN |
PEF |
OFX |
S |
SXT |
CH |
SP |
|---|---|---|---|---|---|---|---|---|---|---|
Escherichia coli |
S |
R |
R |
S |
R |
S |
S |
S |
R |
R |
Shigella spp. |
S |
R |
R |
S |
S |
R |
I |
R |
R |
R |
Salmonella spp. |
S |
R |
R |
S |
R |
S |
I |
R |
R |
R |
Key: CPX: Ciprofloxacin (5µg), AM: Ampicillin (10µg), AU: Augmentin (30µg), CN: Gentamycin (10µg), PEF: Perfloxacin (5µg), OFX: Ofloxacin (5µg), S: Streptomycin (10µg), SXT: Trimethoprim-Sulfamethaxazole (1.25/23.75), CH: Chloramphenicol (30µg), SP: Spectinomycin (10µg).
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 suggests ciprofloxacin 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.
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 pathogens from milk and dairy products. The present research work is of unique significance since no existing literature has any work on the microbiological quality of dairy products retail and consume in Abakaliki, Nigeria. The research work informs society of the poor hygiene standard during storage and retail of milk and dairy products, leading to cross-contamination of the dairy products and milk by pathogenic microorganism of public health threat (Salmonella, Escherichia coli and Shigella); therefore, close monitoring of milk and dairy products is important to avoid outbreak of foodborne disease caused by milk and dairy. There is therefore the need to improve the quality of materials (water and milk) used for production of dairy, as well as storage conditions, and environmental and personnel hygiene to meet food safety standards.
ACKNOWLEDGMENTS
The authors would like to thank the Laboratory Technologist in Microbiology Department, Alex Ekwueme Federal University, Ndufu Alike, and Mr. Kayode Akindele for his Technical Assistance and constant encouragement throughout the duration of the research work.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
AUTHORS’ CONTRIBUTION
OSC conceptualized the study. CVU, IY, KEA, and OJO performed culture and identification. ACK, MME, CCN, OCA, and DCI performed antibiogram of isolates. DOO and CSU performed isolation and characterization of the isolates. BU performed data analysis. OSC supervised the study and wrote the manuscript. All authors read and approved the final manuscript for publication.
FUNDING
None.
DATA AVAILABILITY
All datasets generated or analyzed during this study are included in the manuscript.
ETHICS STATEMENT
This article does not contain any studies on human participants or animals performed by any of the authors.
- Getie M, Abebe W, Tessema B. Prevalence of Enteric Bacteria and their Antimicrobial Susceptibility Patterns Among Food Handlers in Gondar Town, Northwest Ethiopia. Antimicrob Resist Infect Control. 2019;8:111.
Crossref - Fahim KM, Ismael E, Khalifa HS, Farag HS, Hamzad DA. Isolation and Characterization of E.coli strains Causing Intramammary Infections from Dairy Animals and Wild Birds. Inter J Veter Sci Med. 2019;7(1):61-70.
Crossref - Zhang L, Wei Q, Han Q, et al. Detection of Shigella in Milk and Clinical Samples by Magnetic Immunocaptured-Loop-Mediated Isothermal Amplification Assay. Front Microbiol. 2018;9(94):1-7.
Crossref - Ababu A, Endashaw D, Fesseha H. Isolation and Antimicrobial Susceptibility Profile of Escherichia coli O157: H7 from Raw Milk of Dairy Cattle in Holeta District, Central Ethiopia. Inter J Microbiol. 2020;6626488.
Crossref - El-Baz AH, El-Sherbini M, Abdelkhalek A, Al-Ashmawy MA. Prevalence and Molecular Characterization of Salmonella serovars in Milk and Cheese in Mansoura city, Egypt. J Adv Vet Ani Res. 2017;4(1):45-51.
Crossref - Fadihl SJ, Mohammad S Q, Al-qrtani YM. Microbiological Evaluation of Locally Produced Ice Cream in Baquba City Iraq. J Physics: Conference Series. 2019;1294(6): 062057.
Crossref - Wallace MJ, Fishbein SRS, Dantas G. Antimicrobial Resistance in Enteric Bacteria: Current State and Next-generation Solutions. Gut Microbes. 2020;12(1):1-21.
Crossref - Reta MA, Bereda TW, Alemu AN. Bacterial Contaminations of Raw Cow’s Milk Consumed at Jigjiga City of Somali Regional State, Eastern Ethiopia. Inter J Food Contamination. 2016;3:4.
Crossref - Sobeih AMK, Al-Hawary I I, Khalifa EM, Ebied NA. Prevalence of Enterobacteriaceae in Raw Milk and Some Dairy Products. Kafrelsheikh Veter Med J. 2020;18(2):9-13.
Crossref - Lobacz A, Zulewska J. Fate of Salmonella spp. in the Fresh Soft Raw Milk Cheese during Storage at Different Temperatures. MDPI-Microorganisms. 2021;9(5):938.
Crossref - Musa JA, Kwoji ID, Lawan F, Diyo D, Meshack MM, Jauro S. Prevalence and Antibiotic Sensitivity Pattern of Salmonella isolates from Milk Products and Water Reservoirs in Maiduguri, North-Eastern Nigeria. ISOR J Agric Vet Sci. 2017;10(2):87-92.
Crossref - Rahman MA, Rahman AKMA, Islam MA, Alam MM. Detection of Multi-drug Resistant Salmonella from Milk and Meat in Bangladesh. Bangladesh J Vet Med. 2018;16(1): P115-120.
Crossref - Muayad SB. Isolation and Characterization of Bacteria Isolated from Ice Cream Samples in Hyderabad, India. J Pure Appl Microbiol. 2018;12(4):2275-2282.
Crossref - Tan CW, Noor Hazirah MZ, Shu’aibu I, et al. Occurence and Antibiotic Resistance of Salmonella spp. in Raw Beef from Wet Market and Hypermarket in Malaysia. Food Res. 2019;3(1):21-27.
Crossref - Manishimwe R, Moncada PM, Bugarel M, Scott HM, Loneragan GH. Antibiotic Resistance among Escherichia coli and Salmonella isolated from Dairy Cattle Feces in Texas. PLoS ONE. 2021;16(5):e0242390.
Crossref - Sheet I, Sakr S, Hallak AN, Khatib AA. Microbial Quality Assessment of Selected Lebanese Dairy Products and the Antimicrobial Resistance of Bacterial Isolates. Inter J Clin Studies and Medical Case Reports. 2020;2(2):1-7.
Crossref - Younis GAM, Elkenany RM, Abd-Elmoati WS. Advanced Studies on Virulence Genes of Salmonella and Shigella species Isolated from Milk and Dairy Products. Ann Res Rev Biol. 2018;29(3):1-11.
Crossref - Mariam SH. Isolation and Characterization of Gram-Negative Bacterial Species from Pasteurized Dairy Products: Potential Risk to Consumer Health. Journal of Food Quality. 2021;8876926.
Crossref - Haliu D, Gelaw A, Molla W, Garedew L, Cole L, Johnson R. Prevalence and Antibiotic Resistance Patterns of Salmonella isolates from Lactating cows and in-contact humans in diary farms, Northwest Ethiopia. J Environ Occupat Sci. 2015;4(4):171-178.
Crossref - Tanzin T, Nazir KHM, Zahan MN, Md-Parvej S, Zesmin K, Rahman MT. Antibiotic Resistance Profile of Bacteria Isolated from Raw milk Samples of Cattle and Buffaloes. J Adv Vet Anim Res. 2016;3(1):62-67.
Crossref - Haile AF, Alonso S, Berhe N, Atoma TB, Boyaka PN, Grace D. Prevalence, Antibiogram, and Multidrug-Resistant Profile of E. coli O157:H7 in Retail Raw Beef in Addis Ababa, Ethiopia. Front Vet Sci. 2022;9:734896.
Crossref - Bedasa S, Shiferaw D, Abraha A, Moges T. Occurrence and Antimicrobial Susceptibility Profile of Escherichia coli O157: H7 from Food of Animal Origin in Bishoftu Town, Central Ethiopia. Inter J Food Contam. 2018;5:2-8.
Crossref - Tadesse HA, Gidey NB, Workelule K, et al. Antimicrobial Resistance Profile of E. coli Isolated from Raw Cow Milk and Fresh Fruit Juice in Mekelle, Tigray, Ethiopia. Vet Med Inter. 2018; 8903142.
Crossref - Dadi S, Lakew M, Seid M, et al. Isolation of Salmonella and E. coli (E. coli O157:H7) and its Antimicrobial Resistance Pattern from Bulk Tank Raw Milk in Sebeta Town, Ethiopia. HSOA J Anim Res Vet Sci. 2020;4(1):021.
Crossref - Abunna F, Ashenafi D, Beyene T, Ayana MB, Duguma R. Isolation, Identification and Antimicrobial Susceptibility Profile of Salmonella Isolates from Dairy Farms in and Around Modjo Town, Ethiopian. Ethiopian Veter J. 2017;21:2.
Crossref - Frederick A, Courage KS, Gabriel AT. Antibiotic Susceptibility of Escherichia coli Isolated from Milk and Hands of Milkers in Nyankpala community of Ghana. Curr Res Dairy Sci. 2016;8(1):6-11.
Crossref - Demirci M, Yioin A, Altun SK, Uysal HK, Sarybaþ S, Kocazeybek BS. Salmonella spp. and Shigella spp. Detection via Multiplex Real-Time PCR and Discrimination via MALDI-TOF MS in Different Animal Raw Milk Samples. Nig J Clin Practice. 2019;22(8):1083-1090.
Crossref - Qamar A, Ismail T, Akhtar S. Prevalence and Antibiotic Resistance of Salmonella spp. in South Punjab-Pakistan. PLoS ONE. 2020;15(11):e0232382.
Crossref
© The Author(s) 2023. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.