1. Introduction
Foods which contain essential nutrients such as fats, proteins, carbohydrates, vitamins and minerals, are the most indispensible part of human life as well as microorganisms. They are the basic necessity without which the survivals of all living things are not feasible. Consequently, milk is an ideal medium for the growth of many organisms [1] , having a high water content and abundant nutrients, and being nearly neutral pH (6.4 - 6.8) [2] [3] . It contains a plentiful of energy in the form of milk sugar or lactose, milk fat, citrate, and nitrogenous compounds (proteins, amino acids, ammonia, urea and other non-protein nitrogenous compounds) [3] [4] [5] . In addition, the electron potential (Eh) of milk is above 0.3 volts, thus enabling aerobic organisms to grow readily [5] .
The relative ease with which milk can be converted into a wide variety of products makes it an extremely useful base material. In some cases, milk undergoes relatively limited processing, consisting of heat treatment to increase the bacterial shelf life of the product [6] and homogenization to increase the physical shelf life through retarding fat separation. Other well-known processes involve the acid-induced coagulation of milk to produce yoghurt, or the enzymatic coagulation of milk to manufacture cheese. In addition, milk may be spray-dried or be used as a base from which constituents, e.g. proteins or fats, are isolated [7] . As a result of the widespread applications and use of milk, and products derived there from, in human nutrition, it has been the subject of scientific study for over a century [2] [3] [4] [8] [9] [10] .
Throughout the world, it has become established that fermented dairy products provide nutrients in the diet of both young and the elderly people. This occurs as a result of the fact that some of the microorganisms involved in the processing of these products are capable of producing several metabolites that not only add value in the form of taste, aroma, and firm consistency that are unique and attractive to the consumers during fermentation, but also have positive health implications [11] [12] [13] [14] .
Consumption of local spontaneously fermented milk drinks has increased over time, due in part, to the dietary benefits and also the cheap cost of the products; the ingestion of which is considered to be the main cause of several outbreaks of diseases posing a major public health concern [15] . According to Anyanwu [14] , locally fermented products offer a great alternative to mainstream food drinks. Raw cow milk forms the basis for most of the commonly sold local milk products in Nigeria and in order to extend the shelf life and add value, it is processed into various products like Nono, Kindirmo, Mayin Shanu and Wara [11] [15] . In order to prevent spoilage and disease transmission via these highly nutritious foods, milk should be adequately pasteurized [6] [12] . This is essential because at the level of processing of these products, they can be easily populated by a diverse array of both pathogenic and spoilage microorganisms, capable of imparting undesirable organoleptic and physicochemical characteristics that could be detrimental to health [13] [15] [16] [17] . Due to the spontaneous nature of the fermentation process associated with the dairy fermentation, it is exposed to massive contamination regarding the presence of undesirable microorganisms in the final products. The contamination may be as a result of a cow’s dirty exterior, the milking environment, and/or poorly cleaned equipment [18] . Especially remarkable is the incidence of spore-formers which are contaminants of public health significance in the food and dairy processing industries [7] [16] [19] . For instance, a case of food poisoning caused by Clostridium perfringes which resulted in the death of thirteen (13) individuals and ten (10) survivors was reported to have occurred in Saburi community, Gwagwa Ward of Abuja Municipal Area Council (AMAC), Nigeria in 2016 (Premium Times Online: https://www.premiumtimesng.com/regional/north-central/199091-mystery-deaths-abuja-community-caused-food-poisoning-health-board.html).
For these reasons, there is renewed interest in the possible presence of some Clostridia species in the raw milk and milk products, particularly traditionally manufactured products—including nono, in Nigeria. Isolation and molecular characterization of this group of microorganisms will offer knowledge of proper identification for effective control measures to be taken to ensure the wholesomeness and integrity of nono and other locally made dairy products. The main objective of this study therefore, was to determine the incidence of clostridium specie in nono (local yoghurt drink) vended in some parts of the FCT, Abuja, Nigeria.
2. Materials and Methods
2.1. The Study Area
Figure 1 shows the physical map of the Federal Capital Territory (FCT) Abuja, indicating the areas covered in this study.
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Figure 1. Physical map of the Federal Capital Territory (FCT) Abuja, indicating the areas covered in this study.
2.2. Collection of Samples
Thirty-two (32) samples of locally fermented cow milk, nono were randomly purchased in some markets within Abuja metropolis namely, Kuje (KJ03), Gosa (GS26A), Lokogoma (LK23), Giri (GR41), Lugbe (LG04), Garki (GK21) and Kubwa (KB06) respectively. The samples were collected in previously sterilzed capped bottles, labeled and transported to the microbiology laboratory of The Biotechnology Center, Abuja, for the analysis.
2.3. Isolation of Bacteria from the Nono Samples
The Isolation of Clostridium species was performed by serially diluting the nono samples up to 10−8 fold and inoculated on Reinforced Clostridial Agar media which is selective for Clostridium species. The plates were incubated anaerobically at 37˚C for 24 hours [20] .
2.4. Identification of the Clostridium Isolates
Cultural characteristics of the isolates were identified on the basis of different colony characteristics including color, shape and size of colony on the plates. The isolates were equally identified on the basis of their morphological and biochemical characteristics as described in [17] [21] . The morphological characteristics like gram staining of isolated bacteria was carried out to determine the gram status while the different biochemical tests such as Methyl Red (MR) test, Voges-Proskauer (VP), Oxidase, Indole, Catalase, Motility, Starch hydrolysis, Esculin Hydrolysis, and Lipase tests were equally performed on the pure overnight-grown cultures as described Allameh et al. [22] .
2.5. Molecular Confirmation of Isolates
2.5.1. Extraction of Bacterial DNA for 16S rRNA Gene Sequencing
The 16S rRNA gene sequencing is used as a tool to identify bacteria at the species level and assist in differentiating between closely related bacterial species [23] . It also enhances the analytical methods for detection and enumeration of Clostridia species. In this study, the process was carried out using the technique described by Kennedy et al. [24] . In this method, aliquots of 80 μl of NaOH (0.05 M) were added to 20 μl of bacterial cells suspended in distilled water and the mixture was incubated at 60˚C for 45 minutes, followed by the addition of 6 μl of Tris/HCl (pH 7.0), to achieve a final pH of 8.0. The resultant mixture was then diluted × 100 and 5 μl of the diluted extract was used for the Polymerase Chain Reaction (PCR).
2.5.2. PCR, Gel Electrophoresis, and 16S rRNA Gene Sequencing
The bacterial DNA extracts and control were amplified with 0.5 μM primers (LPW58, 5'-AGGCCCGGGAACGTATTCAC-3' and LPW81, 5'-TGGCGAAC GGGTGAGTAA-3'. After which, 10 μl aliquot of each amplified product was electrophoresed in 1.0% (wt/vol) agarose gel, with a molecular size marker (λ DNA AvaII digest; Boehringer Mannheim) in parallel. Electrophoresis in Tris/borate/EDTA buffer was performed at 100 V for 1.5 hours. The gel was stained with ethidium bromide (0.5 μg/ml) for 15 minutes, rinsed, and photographed under ultraviolet light illumination. The PCR products were gel purified using the QIAquick PCR purification kit and then sequences of the PCR products were compared with known 16S rRNA gene sequences in the GenBank (http://www.ncbi.nlm.nih.gov) by BLAST analysis on the NCBI [23] .
3. Results and Discussions
Milk is usually sterile when secreted into the alveoli of the udder. Microbial contamination occurs mainly during and after milking processes. Vissers and Driehuis [18] have summarized the main sources of microorganisms occurring in milk and associated spoilage and safety issues in the dairy products. The summary is provided here in Table 1 for more readerships. While the types of organisms present in raw milk and the products there from are influenced by many factors including temperatures and time of storage as well as the methods of handling during and after milking, the prevalence of these organisms vary considerably depending on geographical area, season, farm size, number of animals, on farm hygiene and farm management practices [5] . These factors, particularly the prevailing tropical temperatures, lack of adequate knowledge of milking, unhygienic handling and storage of raw and the processed milk products of the local milk producers, may also be regarded as potential factors for the proliferation of diverse forms of microorganisms in nono and other similar dairy
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Table 1. Main sources of microorganisms occurring in milk and associated spoilage and safety issues in dairy products*.
1For species having the environment as the major source of contamination and are the main microbial carriers indicated between brackets; 2Relevance for human health in unclear; * Vissers and Driehuis [18] .
products in Nigeria.
The results of this present study indicate positive growth for Clostridium species on the Reinforced Clostridial Agar media (Table 2). Out of the 32 samples collected and analyzed, the percentage occurrence of the target bacteria was: KJ03 (27%), GS26A (0%), LK24 (33%), GR41 (0%), LG04 (21%), GK21 (14%) and KB06 (5%). The occurrence ranged from 0 (None) at locations GS26A and GR41 to the highest in the products obtained from LK24 (33%) which were therefore found to be highly contaminated with Clostridium species as compared to samples from the other locations investigated; and the difference was found to be statistically significant (P < 0.05).
Also, the results of the biochemical characterization of the identified Clostridium isolates with 16S ribosomal RNA (rRNA) gene sequencing showed that the species had a percentage nucleotide identity of >99. Given the present trend towards the love for natural products by consumers, there has been an increased preference for yogurts and fermented yogurt-like products, due to their several perceived and proven health benefits; nono is one of such fermented yogurt-like milk products that is gaining increasing acceptance by many consumers, particularly in Nigeria and other parts of West Africa. Due to the fact that nono is produced using either pasteurized or unpasteurized cow's milk, the lack of pasteurization is presumed to be one of the reasons for the proliferation of diverse forms of microorganisms in the finished product; in addition to the factors mentioned earlier in this section and in Table 1.
This study has adjudged the traditional yoghurt drinks (nono) sold by the vendors in the locations investigated, to be of very poor quality owing to the high incidence of Clostridium sporogens in the finished products. Taylor et al. [25] , reported high level of contamination in a similar study on the conditions associated with Clostridium sporogens growth as a surrogate for Clostridium botulinum in non-thermally processed canned butter. In this study, five out of the seven locations investigated, had incidence of the spore-forming bacterial contaminants, which represents 71.4% of the study areas (Figure 2). The massive
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Table 2. Incidence of C. sporogens in Selected Nono Samples from the Seven Locations.
*KJO3 = Kuje03; GS26A = Gosa26A; LK24 = Lokogoma24; GR41 = Giri21; LG04 = Lugbe04; Gk21 = Garki; KB06 = Kubwa06.
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Figure 2. Pie-Chart Presentation of the occurrence of Clostridium sporogens in the studied locations.
occurrence of Clostridium species of this nature in milk and milk products is an indication of the potentially hazardous product which should be of food safety concern, due to the close relationship between Clostridium sporogenes and the dangerous and proteolytic strains of Clostridium botulinum [7] .
This observed incidence of these health-threatening organisms in nono and other similar products could be due to either inadequate pasteurization (boiling) of the milk or the post-processing contamination of the products as also indicated by Eluchie et al. [26] . Other human microbial pathogens have been reported in raw milk and/or products made from it include Listeria monocytogenes, Salmonella spp. and Campylobacter jejuni [27] . In addition to their significance for public health, a very good microbial quality of raw milk is also important to prevent production losses and to achieve an optimal shelf life of dairy products.
The findings of this study have underscored the importance of adequate adherence to basic hygienic practice during and after processing of dairy products. There is also need for further studies on good manufacturing procedures in order to completely eliminate the occurrence of toxins produced by Clostridium species and other harmful microorganisms in the human food supply chain. The use of molecular tool for the identification of bacteria was confirmed in this work to be simple, comprehensive and reliable which is in line with the findings of Lim et al. [28] in a similar study.
4. Conclusion
Knowledge of the microbiology of raw milk and of milk after different heat treatments is essential for ensuring the safety and quality of milk and it’s product at consumption. The preliminary handling, storage and treatment conditions of raw milk will have a major effect on the type of bacteria present and their effects on the final product. This study concludes that the naturally fermented milk products, nono, as presently vended in the sampled locations within and around Abuja Capital Territory (FCT), are obviously contaminated with the dangerous strains of Clostridium species. The study recommends adequate sensitization of all those involved: the producers, vendors and consumers on the best milk handling and processing practices to prevent adverse health consequences.
Acknowledgements
The authors highly acknowledge the management of the National Biotechnology Development Agency, Abuja, and that of all the Institutions that provided the suitable research environments for this study.