Characterization and Molecular Identification of Bacteria Isolated from Raw Camel’s Milk in Al-Ahsa, Saudi Arabia

Camels play a pivotal role in the social culture of Saudi Arabia. Additionally, they own unique qualities that make them superior to other domesticated animals under harsh desert conditions. According to the FAO estimates, the global camel population is about 35 million heads and the camel population in the Kingdom of Saudi Arabia is about 1.6 million heads (FAO, 2019).
       
Camel’s milk  contains proteins, fats and considerably high amounts of minerals and vitamins; for this reason, it can stand as a complete diet (Aqib et al., 2019), for nomads for weeks. Camel’s milk has multiple benefits of on human health. It has been reported that drinking camel’s milk improves liver function in hepatitis patients and helps treat juvenile diabetes and improves histopathological parameters in cardiovascular and hepatorenal patients (Hassani et al., 2022). In addition, camel’s milk has antimicrobial activity against some bacterial pathogens such as Staphylococcus aureus and Escherichia coli (Ayyash et al., 2020). Additionally, it has been reported that raw camel’s milk is inhabited by diverse bacterial species such as probiotic strains Enterococcus, Streptococcus, Lactobacillus and Bifidobacterium (Suez et al., 2020). Such benefits contribute in increasing camel’s milk demand (Mohan et al., 2020).
 
Despite the presence of beneficial bacteria, potentially pathogenic species can contaminate camel’s milk. It has been reported that raw camel’s milk in Taif, Saudi Arabia, harbors potentially pathogenic species, such as Serratia nematodiphila (Samy et al., 2017), Brucella (Hirad et al., 2018) posing a serious health concern. Consumption of untreated camel’s milk has been reported as a mode of transmission of the Middle East respiratory syndrome-related coronavirus (Nooh et al., 2020) and hence poses a potential health concern for both humans and animals. Desert camping is a common tradition among Saudis, where they drink fresh untreated camel’s milk. Few reports address camel’s milk from the bacteriological viewpoint in Al-Ahsa. Therefore, this study aimed to assess the bacterial biodiversity of camel’s milk in Al-Ahsa, Saudi Arabia. The specific aim of the project was to identify isolated strains using genotypic techniques.

Collection of milk samples
 
Three samples of raw camel’s milk (~200 ml each) were collected under aseptic conditions in sterile bottles directly from the udder of apparently healthy camels, in 9^th March, 2019, in Al-Ahsa. To avoid microbial contamination, the first three streams of the milk were discarded. Samples were transferred immediately to an icebox and transported to the laboratory for analysis. The experimental work was conducted in the laboratories of Biological Sciences Department, College of Science, King Faisal University, Al-Ahsa, Saudi Arabia.
 
Bacterial isolation and enumeration
 
Briefly, 10 mL of fresh camel’s milk was diluted with 90 mL of sterilized saline solution (8.5 g/L, NaCl w/v). The suspension was shaken for 5 min before it was subjected to serial dilutions up to 10-8 by transferring 1 mL into 9 mL of the sterile saline solution in sterile tubes. One milliliter from each dilution was pour-plated on M17 agar medium (Frantzen et al., 2016). The dried plates were incubated at 37°C for 48 h. After the incubation period, the formed colonies were counted and reported as CFU per liter of milk. Discreet colonies were picked and restreaked onto fresh plates containing the M17 medium to obtain pure isolates.
 
Phenotypic characterization of bacterial isolates
 
The bacterial isolates were screened visually for colour, shape, margin and elevation of the formed colonies. To avoid redundancy, five different bacterial isolates representing different morphological colonies were selected for further characterization.
 
Biochemical characterization of bacterial isolates using the API 20E kit
 
The ability of the five bacterial isolates to metabolize 20 different biochemical substrates was investigated using the commercially available API 20E strips (bioMérieux, Marcy-l’Etoile, France) according to the manufacturer’s instructions. After inoculation, the strips were incubated at 30°C and the results were recorded after 24 h.
 
Hemolytic activity
 
The isolates were checked for hemolysis using the blood agar lysis technique. The isolates were inoculated into blood agar medium, supplemented with 5% sheep blood and incubated at 30°C for 48 h. After incubation, the plates were checked for formation of a hemolytic zone around the bacterial colonies.
 
Catalase activity assay
 
The ability of the isolates to produce catalase was determined as previously described (Khalifa and Almalki, 2015). Aliquots of the actively growing bacterial cultures were were flooded with hydrogen peroxide (5%). Positive results were recorded when gas bubbles evolved within a few seconds after the addition of the reagent.
 
Genotypic identification of bacterial isolates using 16S ribosomal RNA gene sequencing
 
Extraction of genomic DNA
 
Total DNA of the bacterial isolates at the mid-exponential growth phase was extracted by boiling the bacterial suspension in the presence of InstaGene Matrix (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions.
 
PCR amplification of 16S rRNA gene
 
PCR amplification of the 16S rRNA gene was carried out in a 20 mL reaction using the universal primers 27F 5' -AGA GTT TGA TCM TGG CTC AG-3' and 1492R 5'-TACGGYTACCTTGTTACGACTT-3' as outlined by Khalifa et al., (2015). PCR products were purified using the Clean-up kit (Millipore, ThermoFisher Scientific, Loughborough, UK) according to the manufacturer’s protocol.
 
16S rRNA sequencing
 
The purified 16S rRNA gene of the bacterial isolates was sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied BioSystems, Foster City, CA) on an Applied Biosystems 3730xl DNA Analyzer. The obtained sequences were deposited in the NCBI database.
 
Phylogenetic analysis
 
The 16S rRNA sequences of the bacterial isolates were identified by the BLASTn alignment against the GenBank nr/nt database (BLASTN) on the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/). Phylogenetic relationships were inferred using the maximum likelihood method based on the Tamura-Nei model suing MEGA5.02 (Tamura et al., 2011).

Camel’s milk is the main source of food for nomads in Saudi Arabia. A popular habit among Saudi people is to drink fresh untreated camel milk, which could be contaminated with virulent microbes. Therefore, the current study aimed to provide in-depth insights into the bacterial species inhabiting raw camel milk and the quality of milk from a bacteriological viewpoint.
 
Bacterial isolation and enumeration
 
The average total bacterial count of raw camel’s milk from Al-Ahsa was 3×10⁷ CFU mL^-1 and thus exceeded the allowed upper limit recommended by the Saudi Food and Drug Authority (Saudi Arabian Standards 2019).  The finding that the total bacterial count of raw camel’s milk from Al-Ahsa exceeded the allowed upper limit recommended by the Saudi Food and Drug Authority was in agreement with previous studies in Taif (Samy et al., 2017) studies in Qassim (El-Ziney and Al-Turki, 2007) where high bacterial loads were found in raw camel’s milk. The total viable counts of bacteria reported in this study were also higher than those reported in raw camel’s milk in Taif (Samy et al., 2017). Additionally, a high incidence of pathogenic Brucella has been reported in raw camel’s milk in Riyadh (Hirad et al., 2018). This result suggests poor hygiene practices during milking that can be explained by inadequate handwashing and improper udder preparation. This finding also uncovers potential health and economic concerns.
 
Phenotypic characterization of bacterial isolates
 
Five morphologically different bacterial isolates, called CMK1 to CMK5, were selected for further characterization. The colonial appearance of the isolates is presented in Table 1. The isolates formed colonies that were rounded, entire, convex and elevated from the surface of the agar. The diameter of the colonies ranged from 1 to 4 mm after a 48-h incubation at 30°C on the M17 medium. The colour of the colonies varied among the bacterial isolates: CMK2, milky; CMK1, yellowish white; CMK4, white; CMK3, faint orange; and CMK5, yellow (Table 1). All isolates except CKM4 were gram-positive cells. While three isolates (CKM1, CKM2 and CKM3) were rod-shaped, two (CMK3 and CMK5) were spherical-shaped. The morphological differences among the bacterial isolates were remarkably similar to those described for the relevant strains (Bacillus, Pseudomonas and Kocuria), as will be discussed later.
 

 
Genotypic identification of bacterial isolates using 16S ribosomal RNA gene sequencing
 
A milestone tool for the identification and classification of bacterial and archaeal taxa is 16S rRNA gene sequencing. The widespread presence and high variability of the 16S rRNA gene in prokaryotes enable efficient discrimination of the assigned taxa. The 16S rRNA sequences of the bacterial isolates were obtained and deposited in the NCBI database under the following accession numbers: CMK1 (MT084027), CMK2 (MT084031), CMK3 (MT084030), CMK4 (MT084032) and CMK5 (MT084034). BLAST analysis of the 16S rRNA sequences of the five bacterial isolates revealed that they were closely related to various known bacterial genera (e.g., Bacillus, Kocuria and Pseudomonas). CMK1 and CMK2 displayed a 97.8% and 99.1% homology with Bacillus sp. and B. zanthoxyli, respectively, while CMK3 and CMK5 shared a 99.8% and 99.5% homology with K. tytonicola and K. marina, respectively (Table 1 and Fig 1), indicating the composition of the microflora in raw camel’s milk. Indeed, many reports have documented the existence of Bacillus and Kocuria (Wang et al., 2018) and Pseudomonas (Hirad et al., 2018) in in raw camel’s milk.
 

       
Inferring phylogenetic relationships and evolutionary history among bacterial and archaeal taxa relies on the 16S rRNA genes. Therefore, 16S rRNA gene-based phylogenetic tree among the bacterial isolates from camel’s milk and the related genera was generated (Fig 1). Bacterial isolates were clustered within three distinctive clades inferred by the maximum likelihood method27. CMK1 and CMK2 were clustered within the Bacillus clade, while CMK3 and CMK5 clustered within the Kocuria clade. CMK4 formed a monophyletic group within the Pseudomonas clade. Phylogenetic trees generated with the neighbor-joining method using the MEGA7 software were similar to the trees constructed with the maximum likelihood method, thus confirming the robust positioning of the bacterial isolates within the clades (data not shown). CMK2 and CMK5 clustered with the closest bacterial species, B. aryabhattai B8W22 (EF114313) and K. marina KMM 3905 (AY211385), respectively. Interestingly, CMK1, CMK3 and CMK4 formed outgroups within the Bacillus and Pseudomonas clades, respectively. This could be because 16S rRNA genes contain highly conserved domains that unable distinction between closely related taxa. Alternatively, the three isolates could be three novel species or subspecies within the bacterial genera. Further studies are hence needed to determine the exact taxonomic position of these isolates; for instance, techniques such as DNA–DNA hybridization and sequencing of other housekeeping genes.
 
Hemolytic activity
 
Unlike the other four bacterial isolates, a clear zone was noted around the growing CMK1colonies highlighting a remarkable degradation of the hemoglobin in the erythrocytes (Fig 2). The finding highlighted potential existence of virulence determinants. Similar findings have been reported in Bacillus sp., (Klichko et al., 2003).  Additionally, six potential membrane-damaging proteins, were existed in the genome of as Bacillus sp. Such proteins were highly similar to hemolysins and phospholipases C from B. cereus (Klichkoet_al2003). More investigations are needed to verify the potential virulence of the strain CMK1.
 

 
Biochemical characterization of bacterial isolates using the API 20E kit
 
To further characterize the isolates, a biochemical analysis with the API E20 test was performed. As shown in Table 1, the strains were able to metabolize 12 (60%) to 15 (75%) out of 20 different compounds as growth substrates present in the API 20E test system. All isolates consumed arginine, ornithine, citrate, glucose, mannitol, rhamnose, sucrose, melibiose, amygdalin and arabinose as growth substrates. No isolate was able to consume all compounds (Table 1). None of the isolates metabolized urea. The biochemical diversity among the bacterial isolates as revealed by the API E20 test, indicating the existence of the enzymatic machinery that enables the isolates to metabolize these substrates. For example, arginine dihydrolase and ornithine decarboxylase are enzymes responsible for the first step in the utilization of arginine and ornithine, respectively. None of the isolates metabolized urea, suggesting the possible lack of urease. These observations are in agreement with those reported for Bacillus, Kocuria and Pseudomonas (Khalifa and Almalki 2015). Unlike K. marina, CMK5 was able to metabolize certain carbohydrates such as glucose, sucrose, melibiose and arabinose as the only carbon source indicating preferences toward different substrates at the species level. Similar phenotypic variations have been reported in Pseudomonas aeruginosa  (Chandler et al., 2019).
 
Catalase activity assay
 
All isolates formed air bubbles upon addition of drops of H₂O₂ indicating the presence of the catalase enzyme. This enzyme splits the toxic hydrogen peroxide into water and oxygen, indicating the protective role of catalase enzyme in preventing the damage to the vital macromolecules existed inside bacterial cells.

Collectively, the total bacterial count of raw camel’s milk in Al-Ahsa exceeded the allowed upper limit recommended by the Saudi Food and Drug Authority. This finding indicates a lack of proper hygiene practices during the milking process and, consequently, poses potential health and economic concerns. Camel milk is inhabited by morphologically, biochemically and genetically diverse bacteria.Taken together, this study sheds light on the bacterial composition of raw camel’s milk that has a direct impact on public health and the economy.

This work was supported by the Deanship of Scientific Research, Vice Presidency for Graduate Studies and Scientific Research, King Faisal University, Al-Ahsa, Saudi Arabia [Project No. GRANT26 (180115)].

There are no conflicts of interest to declare.