Biochemical and Molecular Identification of Escherichia coli and Salmonella spp. Isolated from Milk and Fermented Milk Products Vended in Sabon-Gari and Zaria Local Government Area, Kaduna State, Nigeria

Salmonella spp. and Escherichia coli are two of the most prevalent food-poisoning organisms. As part of this investigation, Escherichia coli and Salmonella spp. were isolated from some fresh milk and fermented milk products in Zaria. This study aimed to investigate the prevalence and molecular characterisation of Salmonella spp. and Escherichia coli in fresh milk and fermented milk products vended Sabon-Gari and Zaria Local Government Area, Kaduna State, Nigeria . Four hundred (400) samples were used consisting of one hundred (100) samples of milk, 100 samples of Kindirimo, 100 samples of Nono, and 100 samples of yoghurt. In addition, a routine microbiological isolation detected 11% of the samples positive for Escherichia coli and 16% for Salmonella spp. All isolates were biochemically characterised and identified; however, none of the selected isolates of Escherichia coli or Salmonella spp. possessed the virulence genes rfbE or invA. Thus, 16S ribotyping was utilised to authenticate the isolates' identities as Escherichia coli strain NCCP 14540, Escherichia coli strain E57, Salmonella bongori and Salmonella enterica subsp. enterica serovar Typhimurium. However, more molecular research is required to determine which other Salmonella and Escherichia coli serotypes were present in the study region.


Introduction
The following terms have been defined by the National Agency for Food and Drug Administration and Control (NAFDAC) in Milk and Dairy Products Regulations (2021): "Milk" means a natural, unprocessed mammary secretion of an animal, whether it is collected from a single milking or multiple and is either consumed as a liquid or used for further processing.In this context, "milk product" means the result of processing milk, which may include added ingredients for food or other components necessary for processing.´Yoghurt" is a milk product pasteurized and fermented by Lactobacillus bulgaricus and Streptococcus thermophilus.
However, Indigenous fermented milk products (FMPs), such as Nono and Kindirimo, are not defined by NAFDAC.Oyeyinka et al. (2023) reported that Nigeria's Hausa people's beloved fermented milk products are made by the Fulani people, who often make cultured milk products for subsistence.This milk can be either full-fat (Kindirimo) or partially cultured skimmed (Nono).It is made by collecting unpasteurised cow milk in a kwarya (calabash) container and letting it ferment spontaneously for one day at room temperature.
The Enterobacteriaceae family includes the facultative anaerobic Garm-negative bacterium E. coli, characterised by its rodshaped, non-spore-forming, and flagellated bacteria (Sarba et al., 2023).Pathogenic E. coli is serotyped using surface antigen profiles of K [capsular], H [flagellar], and O [somatic] (Denamur et al., 2021).While other animals, such as sheep and goats, could harbour E. coli O157, cattle are the most common hosts.While E. coli O157 is a severe foodborne disease, most E. coli strains are innocuous (Sarba et al., 2023).
Salmonella species are members of the facultative intracellular bacteria, are gramnegative and rod-shaped, and may infect many hosts.Salmonellae are classified into two species: Salmonella enterica and Salmonella bongori.(Gebeyehu et al., 2022).By analyzing the bacterial outer membrane's flagellar 'H' antigen and the somatic 'O' antigen, more than 2700 unique Salmonella serovars have been found (Chatterjee et al., 2023).Abey et al. (2024) found that out of 2700 serovars, roughly 1500 are Salmonella enterica subsp.enterica, making it the most frequent zoonotic pathogen causing salmonellosis in humans and other animals.The finding of Salmonella spp.and E. coli O157 in milk and FMPs thus suggests a potential public health issue.
Ribotyping is a technique for identifying and characterising organisms from distinct species, such as bacteria with 16S ribotyping and fungi with 23S ribotyping (Qurban and Ameen, 2020).However, this method often finds microbes in every part of the body (Hatzenpichler et al., 2020).According to Regueira-Iglesias et al. ( 2023), the 16S ribosomal RNA gene is a popular tool in bacterial phylogenetic study because of its large size (about 1500 base pairs), prevalence in bacteria, and consistent activity.
The significance of this research lies in its potential to improve public health and food safety by identifying and characterising E. coli and Salmonella spp.gotten from milk, Nono, Kindirimo, and yoghurt.

Sample collection
The study utilised four hundred (400) samples-100 of each kind of milk and FMPsthat were marketed in Sabon Gari and Zaria.To isolate E. coli and Salmonella spp., 50 mL of milk and FMPs were collected from various sampling points, placed in a sterile, transparent zip-lock bag, labelled, and quickly sent to the Industrial Microbiology Laboratory at Ahmadu Bello University for analysis.

Isolation of Escherichia coli
The isolation followed the method employed by Naratama and Santoso (2020) with modifications.After mixing 25 mL of the sample with buffered peptone water 225 mL, the mixture was grown for 24 h at 37°C.A loopful was streaked onto Chromocult Coliform Agar Enhanced Selectivity (CCAES) plates, then incubated for 24 h at 37°C.Afterwards, positive colonies (purple) were subcultured onto Eosin Methylene Blue (EMB) Agar and incubated for 24 h at 37°C.The Green-metallic Sheen growth will be subcultured and stored on nutrient agar slants for Biochemical Identification.
Isolation of Salmonella spp.
The isolation procedure followed the method described by Fathuddin and Obidah (2024).In the pre-enrichment culture, 25 mL of the sample and 225 mL of buffered peptone water were mixed and grown for 24 h at 37 °C.Next, the pre-enrichment culture underwent centrifugation at 5000 rpm for 5 minutes to separate the pellets.The sediment was placed into 9.0 mL of a selective enrichment medium Rappaport-Vassiliadis Broth (RV) and then incubated at 42°C for seven days.After incubation, subculture onto Xylose Lysine Deoxycholate (XLD) agar was incubated at 37°C for 48 h.Plates were observed for growth.
Positive growth was then transferred to nutrient agar slants for biochemical identification.

Biochemical Identification of Isolates
The isolates were revived from the Nutrient Agar Slant by subculturing them on nutrient agar and incubating them for 24 h at 37°C.Then, the biochemical test carried out was Gram's reaction, Morphology, Indole, Methyl Red, Voges-Proskauer, Citrate, Triple Sugar Iron, Catalase, Motility, Blood Agar (βhaemolysis), DNase Production, Glucose, Lactose, Mannitol, Oxidase, Sucrose, Xylose, and Urease as per the protocol described Chauhan and Jindal (2020).

Detection of Virulence Gene in the Isolates
To detect the distinctive rfbE and invA genes in five (E.coli) and four (Salmonella spp.) biochemically identified isolates, they were evaluated using a Polymerase Chain Reaction (PCR) technique.The isolate was subjected to identification using 16S rRNA.At the Africa Centre of Excellence for Neglected Tropical Diseases and Forensic Biotechnology, the genome DNA was amplified using universal primers 27F (Table 2).Later, at the Inqaba Biotec West Africa, the samples were further amplified using universal primers 907R and 1492R (Table 2).The nucleotide sequences obtained were subjected to a BLAST search in the NCBI database to identify similarities with known sequences.

Results and Discussion
Isolation of Escherichia coli and Salmonella spp.
Forty-four suspected E. coli were isolated from the four hundred samples.Meanwhile, sixtyfive suspected Salmonella spp.were isolated.

Biochemical Characterisation of Isolates
The biochemical characterisation, as presented in

Detection of rfbE Virulence Gene
Plate 1 displays the PCR amplification product for E. coli's rfbE gene on an Agarose gel.The negative control was only normal Nuclease-free water.There was no amplification among the chosen isolates, indicating that the selected virulence gene does not exist but does not rule out the presence of other virulence genes.
Plate 3 shows the amplified 16S rRNA at 1500 bp.
Key: M -Molecular Ladder, (13,25,193,191,239,201,206,63,110)   Based on biochemical characterisation, the total number of E. coli and Salmonella spp.isolated were forty-four and sixty-five, respectively, which consisted of 11% and 16% of the sample collected, which was low considering the total samples collected.Furthermore, the rfbE and invA genes were not detected; however, the 16S ribotyping did confirm the isolates to be E.
coli and Salmonella spp. with a similarity of 92.14-98.50%.Additional molecular studies must determine the serotypes of E. coli and Salmonella spp.circulating in the research area.
Our results highlight the need to promote hygienic food handling and proper cooking practices to reduce or eliminate the risk of pathogenic bacteria from these foods.
-Wells containing samples, NC -Negative control (Nuclease-Free Water) Plate 01: Agarose gel post-electrophoresis showing PCR amplification product of rfbE gene for Escherichia coli at 601 bp.Barbour et al. (2015) reported that 0.0% of E. coli had the rfbE gene, which was isolated from milk from five provinces of Lebanon.Elafify et al. (2020) showed that in Egypt, out of thirtysix (36) isolates were only three (3) E. coli isolates which harboured the rfbE gene, which indicates the rarity of the E. coli O157: H7 in nature.Sobeih et al. (2023) studied milk and FMPs from Kafr El-Sheikh governorate in Egypt; out of twenty-one (21) E. coli isolates, thirteen (13) had the rfbE genes, which indicates an increase in the E. coli O157:H7 population.Detection of invA Virulence Gene Plate 2 shows the post-electrophoresis agarose gel showing the PCR amplification product for Salmonella spp.'s invA gene.Normal Nucleasefree water was the negative control.Key: M -Molecular Ladder, (201, 206, 63, 110) -Wells containing samples, NC -Negative control (Nuclease-Free Water) Plate 02: Agarose gel post-electrophoresis showing PCR amplification product of invA gene at 284 bp for Salmonella spp.Akinyemi et al. (2021) detected the invA gene among 75% of the isolated Salmonella spp. in Lagos.Almas et al. (2021) reported that 70% of isolated Salmonella spp. in Lahore had confirmed the presence of invA genes.Kanteh et al. (2021) reported that in the Gambia, invasive Salmonella spp.constituted about 10% of the total Salmonella spp.isolates.
-Wells containing samples, NC -Negative control (Nuclease-Free Water) Plate 03: DNA band from 16S rRNA gene amplification As shown in Tale 4, the sequencing results confirm the selected organisms to be Escherichia coli (193 and 239) and Salmonella spp.(110 and 201).
Table 1 gives details of the primers used.The study was carried out at the Africa Centre of Excellence for Neglected Tropical Diseases and Forensic Biotechnology and the Department of Veterinary Public Health at Ahmadu Bello University.

Table 01 :
Sequences and parameters of primers

Table 02 :
Sequences of 16S rRNA primers Target gene

Table 3
They could also utilise Triple Sugar Iron and produce acid and gas and ferment sugars such as glucose, lactose and mannitol.And , shows that both organisms were gram-negative rods, with E. coli being positive for indole, methyl-red, catalase, motility, and urease.

Table 04 :
16S Ribotyping of the selected E. coli isolates Conclusion This study detected and characterised E. coli and Salmonella spp.gotten from milk and FMPs vended around Sabon-gari and Zaria Local government areas.The current study shows that E. coli and Salmonella spp.are influential milk contaminations regardless of the locations sampled, year's season and source of milk.