Molecular detection and phylogenetic diversity of Staphylococcus aureus isolated from goat subclinical mastitis in Nineveh governorate

1. Isolation and identification of aureus using the classical methods.
2. Detection of the same genes encoding virulence factors such as (nuc, mecA, clfA, clfB, and coa genes) using the PCR assay.
3. Make the phylogenetic analysis tree to find the relationship between aureus isolates in this study with S. aureus isolated from other studies.

Introduction

Staphylococcus (S.) aureus is one major pathogen identified in dairy goats responsible for both preclinical and symptomatic intramammary infections (1). S. aureus has been determined to cause 4.1% to 18.0% of all bacterial intramammary infections (2). Additionally, these microbes are commonly found in milk and its products (3). From mild-local, superficial skin lesions to significant invasive infections, S. aureus causes many clinical symptoms and may be fatal (4). There is a correlation between virulence factors and the severity and signs of S. aureus infections (5). The nuc gene encodes the virulence factor heat-resistant nuclease gene, strongly associated with enterotoxin production. At the same time, the pathogen attachment mechanism is carried out by fibrinogen-binding proteins termed clumping factors (clf) A and B, also a penicillin-binding protein (PBP 2a) with low affinity for β-lactam antibiotics that is encoded by the mecA gene, and staphylococcal enterotoxins (SEs) are the primary cause of foodborne poisoning among them. Food poisoning from S. aureus bearing SEs can occur quickly, develop quickly, and be extremely dangerous to human health (6). In addition, S. aureus possesses various types of toxics such as exfoliative toxins A and B (eta and etb), toxic shock syndrome toxin-1 (tst), staphylococcal enterotoxins (SEs), and toxic shock syndrome toxin-like (SEls) are expressed by certain S. aureus strains. It is commonly known that SEs, or SEls, are a primary reason for illness caused by food (7). Reports state that 95% of staphylococcal food poisoning (SFP) cases are caused by these five traditional enterotoxins, with newly identified SEs responsible for the remaining 5% of infections (8). When the mammary glands udder tissue becomes inflamed due to trauma or infectious bacteria, it results in mastitis in ruminants (9). It is the most widespread illness that is economically harmful to dairy company operations because it reduces milk production by nearly 10% to 20% and has a negative impact on milk ingredients, making them less nutritious (low quality) and unfit to undergo processing and consumption (10,11). The direct and indirect mastitis costs result in a large financial loss (12). Bovine mastitis directly costs money for veterinary care and increases labour needs (13). The significant financial losses caused by mastitis-related losses in milk availability and quality are the cost of various forms of bovine mastitis (14). The mammary glands contain ruminants of more than 100 different species of bacteria, yet only a tiny percentage of these bacteria have been shown to cause mastitis (15). The cause of mastitis microorganisms may be categorized into two groups based on their reservoir, source, and transmission mechanism: the first category is an infectious bacterium. In contrast, the second category is an environmental bacterium (16). Mastitis risks public health because it can spread zoonoses and diseases associated with contaminated food (17). Because of the significant risk of harmful microorganisms from cattle, automated milking machines, and milk containers contaminating raw milk, it is not recommended to eat raw milk directly (18). Goat milk production is a burgeoning industry that produces a significant amount of mainstream dairy milk in hard-climate regions where large ruminants are either impossible to rear or very difficult to raise (19). Goat milk offers several advantages over human and cow milk, including superior digestion, alkalinity, buffer capacity, and therapeutic value (20). Goat milk plays a significant role in several countries' economies and dairy industries. Asia and Africa are home to more than 80% of the worldwide goats (21). Goat milk output is estimated to be 12 million tons worldwide (including 2 million tons in Europe), or 2% of all animal milk produced worldwide (22). Product consumption derived from goat milk has risen, which can be attributed to the health advantages and nutritional benefits associated with goat milk (23). The cleanliness of the milking system, the conditions during transportation, and storage all affect raw milk quality. On the other hand, it is also intimately linked to several animal illnesses, including mastitis, which could cause pathogenic bacteria in milk (24).

Because pathogenic S. aureus is important in goat subclinical mastitis, this research aims to determine the amount of damage caused by pathogenic S. aureus in milk samples collected during the governorate of Nineveh's goat subclinical mastitis, identify the methicillin-resistant strain of S. aureus, and identify the genes encoding virulence factors in S. aureus isolates.

Materials and methods

Ethical approval

With the approved ID of UM. Vet. 2024.003, all samples were obtained with the owners' consent and implemented in compliance with the ethical guidelines provided by the Institutional Animal Care and Use Committee at Mosul University's College of Veterinary Medicine.

Samples collection

Sixty goat milk samples were obtained from various governorate areas from October 2023 to February 2024. These regions included Ali Rash, Brtala, Kukagly, and Hama Al Alil. The goat milk samples were collected in sanitary containers and sent straight to the laboratory of the Dept. Veterinary Public Health/College of Veterinary Medicine/Mosul University. All containers containing peptone water were placed in an incubator and underwent pre-enrichment for 18 to 24 hours at 37°C. After streaking milk samples across Blood and 7.5% Mannitol salt media plates, they were incubated at 37°C for 24 hr.

1. aureus isolation and characterization

Morphological assessment, catalase and coagulase activity tests, and gram staining were used to study the characteristic S. aureus colonies (25).

Extraction of DNA

To extract the S. aureus genomic DNA, the positive isolates of S. aureus were cultivated for 24 hours at 37°C on a mannitol salt medium. The DNeasy Blood and Tissue Kit (Qiagen, Germany) was used to extract DNA, following the Gram-positive bacteria-specific protocol provided by the producer. Following Nanodrop (Biodrop, UK) measurement, the separated DNA was maintained at -20°C.

PCR Reaction

The PCR method detected the nuc, mecA, clfA, clfB, and coa genes of S. aureus. The molecular weight of the nuc gene is 166 bp (26), the mecA gene is 147 bp (27), the clfA gene is 288 bp (28), the clfB gene is 203 bp (28), and the coa gene is 674 bp (29) are shown in table 1. A total of 30 μl was used for the PCR reaction, and the mixture was prepared in a 200 μl tube (Biozym, Oldenhorf, Germany). The resultant amplicons were analyzed using gel electrophoresis on a 2% agarose gel (Peqlab, Erlangen, Germany) using a 100 bp ladder as a reference. 15 μl of Promega Corporation's (2×) GoTaq Green Mix Master, 1 μl of primer 1, 1 μl of primer 2, 9 μl of Promega Corporation's (USA) DNeasy-free water, and 4 μl of the S. aureus DNA template made up the reaction mixture.

Table 1: PCR techniques and primers for identifying different S. aureus genes

PCR program: I: 35 times (94°C - 30s, 55°C - 30s, 72°C - 30s), II: 35 times (94°C - 30s, 54°C - 30s, 72°C - 30s), III: 35 times (94°C - 30s, 60°C - 30s, 72°C - 30s)

DNA sequencing

The samples were sent to Macrogen, a South Korean commercial sequencing company, to purify and sequence six PCR amplicons acquired from milk goat isolates previously found to be S. aureus-positive by classical PCR. The target genes for sequencing were the clfA, clfB, and coa genes. The obtained clfA, clfB, and coa genes sequences were then compared to previously published S. aureus sequences that are available on GenBank using the NCBI BLASTn program, which is accessible at http://www.ncbi.nlm.nih.gov. The alignment and comparability of these sequences were further examined using the online multiple sequence alignment program CLUSTALW from GenomeNet (available at http://www.genome.jp/tools/clustalw/). The Neighbor-Joining (NJ) program and the same Genome Net tool, CLUSTALW, were used to generate phylogenetic trees. Five hundred duplicates of the S. aureus clfA, clfB, and coa gene sequences were utilized as an outgroup while creating the phylogenetic tree to increase robustness. This comprehensive approach aimed to shed light on the genetic relationships between the S. aureus isolates by using purification, sequencing, and subsequent bioinformatics analysis to better understand the phylogenetic context of the isolates from milk goats.

Results

The colonies of S. aureus that yielded good results had a golden-yellowish tint based on how they appeared on Mannitol salt agar. In addition, positive results were obtained for particular biochemical evaluations, including coagulase and catalase, which verified the existence of S. aureus isolates. This study showed that the S. aureus isolates in this current research had a rate of occurrence of 5% (3/60). In the Ali Rash region, the highest percentage of S. aureus found in goat sub-clinical mastitis was 20% (3/15), whereas no S. aureus has been isolated from any other regions (Tables 2-4). According to Table 3, the results of the PCR assay agreed with those of the traditional methods, showing that all isolates of S. aureus had the nuc gene (Figure 1). According to Table 3, 100% (3/3) of the S. aureus possessed the mecA gene (Figure 2). Furthermore, our findings showed that just two S. aureus contained the clfA gene 66.7% (2/3) (Figure 3). Additionally, from this study, 100% (3/3) of S. aureus had the clfB gene (Figure 4). Furthermore, 100% (3/3) of S. aureus had the coa gene (Figure 5).  The current study's findings additionally revealed that the S. aureus isolates were hooked on two separate gene profiles based on the presence of unique genes in each isolate (Table 4). Gene profile I (nuc + mecA + clfA + clfB + coa) was most frequently present in 2 (66.7%) S. aureus isolates, while gene profile II (nuc + mecA + clfB + coa) was reported in 1 (33.3%) isolate. Single genes were absent from all S. aureus isolates.

Table 2: The number of samples and percentage of positive S. aureus isolates

Table 3: The number and percentage of the genes found in S. aureus isolates

Table 4: Genetic characterization of three S. aureus isolates obtained from milk samples of sheep with sub-clinical mastitis

Figure 1: Electrophoretic analysis of PCR products for S. aureus nuc gene detection. The DNA amplification appears as a ladder-like pattern. Lane 1 contains a negative control, Lane 2 shows the positive control, and Lanes marked as "M" are DNA markers using a 100 bp ladder from Biozym Diagnostic. Lanes 3 to 5 display positive isolates, while Lanes 6 and 7 show negative isolates.

Figure 2: Electrophoretic analysis of PCR products for S. aureus mecA gene detection. The DNA amplification appears as a ladder-like pattern. Lane 1 contains a non-template control, Lane 2 shows the positive control, Lanes marked as "M" are DNA markers using a 100 bp ladder from Biozym Diagnostic, and Lanes 3 to 5 display positive isolates, while Lanes 6 and 7 show negative isolates.

Figure 3: Electrophoretic analysis of PCR products for S. aureus clfA gene detection. The amplification of DNA appears as a ladder-like pattern. Lane 1 contains a non-template control, Lane 2 shows the positive control, Lanes marked as "M" are DNA markers using a 100 bp ladder from Biozym Diagnostic, and Lanes 3 and 4 display positive isolates, while Lanes 5 to7 show negative isolates.

Figure 4: Electrophoretic analysis of PCR products for S. aureus clfB gene detection. The DNA amplification appears as a ladder-like pattern. Lane 1 contains a non-template control, Lane 2 shows the positive control, Lanes marked as "M" are DNA markers using a 100 bp ladder from Biozym Diagnostic, and Lanes 3 to 5 display positive isolates, while Lanes 6 and 7 show negative isolates.

Figure 5: Electrophoretic analysis of PCR products for S. aureus coa gene detection. The DNA amplification appears as a ladder-like pattern. Lane 1 contains a non-template control, Lane 2 shows the positive control, Lanes marked as "M" are DNA markers using a 100 bp ladder from Biozym Diagnostic, and Lanes 3 to 5 display positive isolates, while Lanes 6 and 7 show negative isolates.

The sequencing results of this research showed that the individual sequencing analysis (BLASTn) was carried out on nine novel gene sequences (three novel clfA gene sequences, three novel clfB gene sequences, and three novel coa gene sequences) obtained from the subclinical mastitis milk of goats. As indicated in table 5, the S. aureus sequences that are provided in the NCBI Genbank are indexed under the following accession numbers:( PP958240, PP958241, PP958242, PP973841, PP973842, PP973843, PP951509, PP951510, and PP951511). A phylogenetic tree analysis in MegAlign software applying the maximum likelihood technique showed that local difference gene sequences displayed a distinct relationship from those previously described and available in the GenBank database. Furthermore, the S. aureus sequence types based on the clfA gene (PP958240, PP958241, and PP958242) and the United Kingdom's CP001996.1 and France CP025395 sequence types have a tight link was 100%. Whereas the sequence types (PP958240, PP958241, and PP958242) of this research have 99.6% and 98.3% similarity with LT615218.1 Australia and FR821779.1 United Kingdom, respectively. Furthermore, the S. aureus sequence types based on the clfB gene (PP973841, PP973842, and PP973843) and HQ424333.1 Germany and HQ424333.1 USA appeared to have the closest relationship of 100%. Meanwhile, the sequence types (PP973841, PP973842, and PP973843) have a 99.4% similarity with CP134882.1 South Korea. The S. aureus sequence types based on the clfA and clfB did not compare with those of S. aureus isolated from Iraq. Moreover, a strong correlation was seen between the S. aureus sequence types depending on the coa gene (PP951509, PP951510, and PP951511) and the sequence types of CP042650.1 China and CP085320.1 South Korea achieved 100%. In addition, the sequence types (PP951509, PP951510, and PP951511) were similar to CP041634.1 China, CP127671.1 Australia, CP075511.1 Thailand, and CP020354.1 Republic of Korea were 97.2%, 95.3%, 95%, and 94.2%, respectively. Moreover, the sequence types (PP951509, PP951510, and PP951511) appeared similar with LC425081.1 Al Basra, Iraq and ON631029.1 Al Diyala, Iraq was 95% and 94.5%, respectively (Figures 6 and 7).

Table 5: The NCBI GenBank accession numbers for the clfA, clfB, and coa genes of S. aureus sequences in goat^’s milk

Figure 6: Clustering analysis of the clfA, clfB, and coa gene sequences of S. aureus and other various gene sequences of S. aureus isolates retrieved from NCBI GenBank. The designation in the parentheses indicates the NCBI accession number.

Figure 7: clfa, clfB, and coa gene sequences similarity and divergence of each pair for S. aureus calculated by DNASTAR

Discussion

A microorganism infection is typically the cause of mastitis, a significant mammary gland illness that can be either subclinical (mild) or clinical (severe). To increase our knowledge of the spread of S. aureus in Nineveh goat herds, we characterized S. aureus detected in cases of goat subclinical mastitis collected in several herds and various areas of Nineveh governorate/Iraq. Several reports of S. aureus isolated from bovine subclinical mastitis (30) and camel mastitis (31) in Nineveh governorate/Iraq. The present investigation revealed that 5% (3/60) of goat milk samples with subclinical mastitis had S. aureus isolated from them. Several studies showed that clinical and subclinical goat mastitis caused by S. aureus varies from 5.6% to 37% in different counties (32-36). The rate of occurrence of S. aureus in goat's milk has been reported in many previous studies to be greater than the findings of our research; it was 46% (47/104) in the United States (37), 66% in Spain (38), and 76.9% (60/78) in Italy (39). The rate of occurrence of S. aureus in this research was near to the findings of other investigations, at 6.3% in Iran (40) and 6.2% in Norway (41). While several additional studies have revealed a lower rate of occurrence of S. aureus in goat's milk than the current research results, it was 1.4% in the USA (42) and 1.5% in China (43). The variations in S. aureus occurrence rates are based on the varying geographic distributions and sanitary conditions in milk production facilities and farms. Numerous earlier investigations revealed that S. aureus was found in udder skin, bedding, labourers' hands, insects, and dust, significant in transmitting S. aureus among mammary glands of animals and tainted milk (44).

Furthermore, the isolation of S. aureus from various animals’ organs, including the vagina, muzzle, and skin wounds, causes the spread of S. aureus from body sites to environments on the one hand and from dairy herds to their calves on the other, via ventilation or by providing infants milk from animals that has S. aureus (45). In addition, often pointed-out issues included dirty milking utensils, insufficient cleanliness of milking staff, inadequate udder preparation, unhygienic farms, carrying milk without cold chains, and not realizing enough about foodborne illnesses (46). All of the investigated farms had refrigerated milk storage facilities in place. Therefore, seasonal management methods (i.e., grazing in the summer and confinement in the winter) are more likely to be to blame for this effect than seasonal variations in the temperature (47).

Additionally, all S. aureus isolates had 100% (3/3) of the nuc, mecA, clfB, and coa genes; only two isolates had 66.7% (2/3) of the clfA gene. Many previous investigations discovered that the nuc gene was identified in all coagulase-positive S. aureus (48,49). Moreover, the mecA gene was present for each isolate of S. aureus, indicating that the organism was methicillin-resistant S. aureus (MRSA). According to earlier investigations, the S. aureus isolated from goat milk in Spain lacked the mecA gene (47). People in close proximity to animals are exposed to colonization and subsequent infection in areas where the occurrence of livestock-associated clones is significant. The MRSA outbreak may be prevented by systematically surveilling MRSA in animals produced for food (50). Numerous studies concluded that there was no MRSA isolated from goat's milk (51-53). While the occurrence of MRSA in Greece was 5.5% (9/162) (54), in China it was 51.6% (55), and in Uganda it was 56.1% (56). Encoding adhesion factors are the clfA and clfB genes. Numerous studies have reported revealing these genes in S. aureus in goat milk throughout the globe (57-59). The results of our research concurred with those of prior investigations, which showed that all strains of S. aureus possessed the clfA and clfB genes (60). In Iran, 84 and 65.3% of S. aureus in bovine milk carried the clfA and clfB genes (61). Numerous investigations found clfA in 19%-100% of isolates with bovine mastitis and clfB in 91.8%-92.9% (62-65). Although every isolate displayed a coagulase-positive reaction on mannitol salt agar, all S. aureus isolates carried the coa gene when the primers mentioned above were used. After agarose gel investigation of the amplified products, PCR amplification of the coa gene revealed a single amplicon for the 674 bp. Our results are consistent with previous research that the coa gene existed in all S. aureus, and the coa gene produced five distinct PCR products with a range of around 500, 580, 660, 740, and 820 bp (66,49). A different study revealed that all S. aureus isolates had the coa gene, which produced 580 bp amplicons (67-70).

Conclusion

The presence of S. aureus, and mainly MRSA in subclinical mastitis goat milk, raises serious concerns for the health of the animals because S. aureus is capable of resisting β-lactams, one of the most effective antimicrobials for treating mastitis. Raw milk has S. aureus and MRSA, which can spread throughout the community via the dairy supply chain. Therefore, from their existence in raw milk, there is a significant health danger to people of all ages. In low-input goat farms, the high incidence rate of S. aureus is justified by bad husbandry techniques, a disregard for basic hygiene precautions, and the haphazard use of antibiotics. Numerous genes encode various virulence factors in S. aureus isolated from goat milk at various geographical locations worldwide.

Acknowledgment

We thank the College of Veterinary Medicine / Mosul University for furnishing the essential resources needed to complete this study.

Conflict of interest

The writer of the manuscript has affirmed that neither the writing nor the information analysis phases included any conflicts of interest.