Research Article | Open Access
Okoro Samuel Chukwu1 , 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. Ugwuocha1
1Department of Microbiology, Faculty of Biological Sciences, Alex Ekwueme Federal University, Ndufu-Alike, Ebonyi State, Nigeria.
2Department of Microbiology, Gregory University, Uturu, P.M.B. 1012, Achara Uturu, Abia State, Nigeria.
Article Number: 8342 | © The Author(s). 2023
J Pure Appl Microbiol. 2023;17(3):1620-1627. https://doi.org/10.22207/JPAM.17.3.23
Received: 16 December 2022 | Accepted: 21 March 2023 | Published online: 01 September 2023
Issue online: September 2023
Abstract

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 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.

Keywords

Isolation, Identification, Antibiotic Resistant Profile, Enteric Pathogens, Milk, Dairy Products

Introduction

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.

Materials and Methods

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.

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).

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).

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 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.

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 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.

Declarations

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.

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