Research Article

Molecular Detection of mecA Gene in Staphylococcus sp. Associated with Ruminant Mastitis

Hanan Yousif Jasim1*, Rasha Munther Othman2, Wasan Moaed Shaker3

1Department of Internal and Preventive Medicine, College of Veterinary Medicine, Basrah University, Iraq; 2Department of Microbiology, College of Veterinary Medicine, Basrah University, Iraq; 3Department of Public Health, College of Veterinary Medicine, Basrah University, Iraq.

Received | March 25, 2024; Accepted | April 11, 2024; Published | May 27, 2024

*Correspondence | Hanan Yousif Jasim, Department of Internal and Preventive Medicine, College of Veterinary Medicine, Basrah University, Iraq; Email: hanan.yousef@uobasrah.edu.iq

Citation | Jasim HY, Othman RM, Shaker WM (2024). Molecular detection of mecA gene in Staphylococcus sp. associated with ruminant mastitis. Adv. Anim. Vet. Sci., 12(7):1348-1355.

DOI | https://dx.doi.org/10.17582/journal.aavs/2024/12.7.1348.1355

ISSN (Online) | 2307-8316

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Mastitis is an important global disease for the dairy industry, it is related with economic losses and decreasing milk production and quality, since it affects animal wellbeing (Sharun et al., 2021; Yang et al., 2020). Mastitis is an inflammation of the mammary gland caused mainly by a bacterial infection. It is characterized by a variety of local and systemic symptoms. It is a significant concern for both meat and milk producers as the infection can lead to considerable economic losses. This is due to the reduction in milk yield, decreased quality of milk, and treatment costs Sawant et al. (2009).

Many bacterial agents are causing animals mastitis. Among these bacteria are Staphylococcus sp. The genus Staphylococcus is a group of Gram-positive bacteria, and S. aureus in particular is one of the most harmful species of Staphylococcus. According to a report by Miles et al. (1992), Staphylococcus aureus is likely the most dangerous agent, as it causes persistent and severe infections in mammary glands which are challenging to treat. Staphylococcus cause damage to the tissues lining the teat and gland cisterns then, move to the duct system and form deep– seated pockets of infection which are difficult to treat and may result in abscess formation. The body’s defense mechanism walls-off the bacteria with scar tissue, which helps to isolate the infection but also lowers the effectiveness of antibiotics. As a result, the cure rates for this type of infection are generally poor (Philpot and Nickerson, 2000).

The virulence of S. aureus is believed to be multifactorial as it is caused by the combined effect of various virulence factors (Bien et al., 2011). It is a worldwide bacterium that causes subclinical and clinical mastitis infection in mammary glands of the domestic animals, that usually spreading through milking processing and leading to chronic illness, especially with antibiotics bacterial strain like Methicillin Resistant Staphylococcus aureus (MRSA) (Kumar et al., 2010; Zigo et al., 2021).

Additionally, methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogenic bacterium in veterinary medicine and public health, in addition to resistance to methicillin, most strains are also resistant to other β-lactam antibiotics (Nandhini et al., 2022). The methicillin resistance in Staphylococcus sp. is associated by mecA gene, that encodes the protein PBP 2A which is a low-affinity penicillin-binding (Stapleton and Taylor, 2002). The mecA gene is a 21-60 kb structural component that integrates into the staphylococcal large chromosome cassette (SCCmec). It is a mobile genetic element acquired by horizontal transfer from coagulase-negative staphylococcal species and may contain genetic constructs (Tn554, pUB110, pT181) that also encode resistance to non-lactam antibiotics (Becker et al., 2014; Ito and Hiramatsu, 1998).

At least five different types of SCCmec differing in gene sequence and size, have been reported. The types I to III of MRSA strains found in healthcare are usually large and multi-drug resistant. Types IV and V associated with community-acquired MRSA strains tend to be smaller and more sensitive to antibiotics other than β-lactams (Ahmad et al., 2009). However, the identification of the mecA gene as the primary mechanism behind MRSA isolates cause bovine mastitis led to the creation of quick molecular assays, first using DNA probes and later PCR, to differentiate between MRSA and methicillin-susceptible S. aureus (MSSA) colonies (Tokue et al., 1992). However, it proved more challenging to directly identify MRSA in clinical cases, since mecA can also be exist in methicillin-resistant strains of coagulase-negative staphylococci, which are also prevalent in a variety of clinical cases (Wang et al., 2014).

Nevertheless, the interest on methicillin-susceptible S. aureus (MSSA) has increased in recent years, given that they can also be implicated in important in human or animal infections This helps explain the onset and progression of infections and the different and successful MRSA lineages. Few data exist in the circulating genetic lineages of MSSA in food producing animals or in derived food products, as in the case of milk (Silva et al., 2013). Coagulase-positive and coagulase-negative staphylococci were recovered in a previous study from milk of local dairy farms with mastitis in Basrah government (de Freitas Guimarães et al., 2013; Hashosh et al., 2022a, b). Therefore, the recent study was aimed to the determine the genetic diversity of mecA gene among Staphylococcus sp. isolated from mastitis cows in Basrah/ Iraq.

MATERIALS AND METHODS

Samples collection and isolation of Staphylococcus sp.

A total of 70 milk samples were taken randomly from the following animals with clear signs of mastitis: 34 from cows, 19 from sheep, and 17 from goats in Basrah governorate. The examined animals were shown moderate to severe clinical signs that can included: Abnormalities and swelling the udder also heat, hardness, redness, or pain; and milk have a watery appearance, clots, or pus.

Before collecting the milk samples, the udder of the affected animals was cleaned from the dust, mud and dirt with water, then wipe with a cotton pad soaked in 70% alcohol and dried with a clean cloth. 10ml of milk were collected in sterile tubes and preservative in an icebox and transported as fast as possible to the microbiology laboratory. All milk samples were immediately plated on mannitol salt agar (MSA) and incubated at 37 °C for 24 h to identify Staphylococcus sp. Mannitol-fermenting colonies were subcultured on MSA medium and incubation at 37oC for 24-48h as performed previously by (Talan et al., 1989). Subsequently gram stain and the coagulase tube were further used according to (Cowan, 1993; Macfaddin, 2000).

Positive isolates were identified presumptively as Staphylococcus spp. DNA extraction and PCR analysis for confirmation.

DNA extraction and PCR analysis

Total genomic DNA was extracted using the Genomic DNA Purification Wizard® Kit according to the manufacturer’s instructions (Promega, USA). Two sets of primer were used: The 16Sup primers used for amplification and detection of 16S rDNA gene of Staphylococcus sp., and mecup primer for detection of mecA gene in the Staphylococcus sp. isolates (Poulsen et al., 2003). The oligonucleotides primers used for the 16S rDNA and mecA genes were (forward primer 5-GTG CCA GCA GCC GCG GTA A-3), (reverse primer 5-AGA CCC GGG AAC GTA TTC AC-3) and (forward primer 5-GGG ATC ATA GCG TCA TTA TTC-3), (reverse primer 5- AAC GAT TGT GAC ACG ATA GCC-3).

The PCR reaction was performed in 25 µl reaction mixtures with 1 µl (10 pmol/µl) for forward primer and reverse primer, 12.5 µl ready to use GoTaq® Hot Start Master Mixes (Promega, USA) and 5.5 µl nuclease-free water. Finally, the 5 µl DNA template was added to each reaction tube. For genes amplification, the PCR cycling and conditions was started with initial denaturation step for 5 min at 94°C, following by 30 cycles of 30 s at 94C, 30 s at 55C for annealing step and 30 s at 72C for elongation. The final elongation for 2 min at 72°C. The PCR products were then running on 1% agarose gel staining using ethidium bromide in order to assess the successful of amplification. Agarose gel was visualized using a gel documentation system. The DNA ladder from (SolGent™ 1 Kb Plus DNA Ladder) was used as a molecular mass marker and the fragment size of approximately 886 bp and 527bp was verified as positive for 16SrDNA gene of Staphylococcus sp., and mecup gene, respectively (Sambrook et al., 1989).

Genes sequencing and phylogenetic analysis

The PCR products of 16S rDNA and mecA genes were sequenced in (Macrogen Inc./ Seoul, South Korea Korea) by using the 16Sup and mecup primers for partial nucleotide sequence of the 16S rDNA and mecA genes respectively. The results of sequencing were analysis useing BLAST (Basic Local Alignment Search Tool) to compare and search for similarities in the National Center for Biological Information (NCBI) sequence database. The phylogenetic tree was constructed using the Neighbor Join method using the software MEGA-X (Kumar et al., 2018).

RESULTS AND DISCUSSION

Incidence of isolation and identification of Staphylococcus sp.

The emerging of mecA gene in Staphylococcus sp. bacteria pose a serious threat to human and animal health also stimulating the interest of researchers, as it could help bacteria to be resistant to antibiotics such as methicillin, penicillin and penicillin-like antibiotics. Based on previous studies, the mecA gene has been found to be present in all strains of MRSA (Berger-Bächi, 1999; Al-Deewan and Adlan, 2017). Methicillin resistance in Staphylococcus aureus occurs primarily due to the production of a unique penicillin-binding protein (PBP)-2a. This protein has a lower binding affinity for β-lactam antibiotics than other PBPs.

 

Table 1: A prevalence of mastitis milk samples taken from dairy animals with clear signs of mastitis.

Type of animals	Positive No. and (%) of isolates based on
	Clinical signs	MSA and Gram stain	PCR and sequence of 16S rDNA	PCR and sequence of mecA
Cows	34	13	7	7
Sheep	19	8	4	4
Goats	17	5	2	2
Total	70	26 (37.14%)	13(50%)	13(50%)

 

PBP-2a requires a higher concentration of penicillin (2-10 times more than PBP-2 and 20 times more than PBP-1) to be neutralized (Kaszanyitzky et al., 2003). The aim of this study was to examine the distribution of mecA in Staphylococcus aureus and other CON populations of dairy animals. Two molecular methods were used to detect the mecA gene in bacterial isolates, PCR and sequences analysis. Therefore, the initial target step in this study was the detection and isolation of Staphylococcus sp. Seventy milk samples were collected from cows, sheep, and goats with clear signs of mastitis (Figure 1). In this study, it was found that animals with clinical mastitis have discolored milk and abnormal consistency ranging from watery to viscous. This variation is due to the presence of pus, blood, and milk clots (Biggs, 2009). Table 1 shows the results of bacterial culturing on MSA and examined by Gram stain in which 26 (37.14%) isolates out of 70 tested samples were suspected Staphylococcus sp. Additionally, all suspected colonies were appeared as a round, golden-yellow clusters on mannitol salt media (Figure 2). A study conducted in Iraq by Aboud and Khudaier (2018) found that 39.47 % of milk samples were positive for Staphylococcus, which is consistent with the results observed in the present study. In contrast, previous studies by Dehkordi et al. (2015) and Tibebu et al. (2021), reported lower isolation rates, where 25.53% and 24.8% of the milk samples tested positive for Staphylococcus, respectively. However, in Turkey, a higher isolation rate of Staphylococcus was observed, where 56% of raw milk and dairy products were positive to Staphylococcus (Gundogan and Avci, 2014). This highlights the importance of implementing effective mastitis control programs and limiting the use of antibiotics in dairy projects. Staphylococcus is a common cause of mastitis, which can lead to persistent and recurring infections. Additionally, the cure rate after antibiotic therapy is usually low (Haag et al., 2019). Furthermore, the PCR results demonstrated that 16S rDNA and mecA genes were detected in 13 (50%) of isolates (Table 1). The PCR-amplified DNA products with expected size of 16S rDNA (527bp) and mecA gene (886 bp) are shown in Figures 3 and 4.

 

 

 

In this study Staphylococcus sp. was identified by PCR and sequence analysis methods through used specific primers for the 16S rDNA gene that is located as a tandem repeat on the bacterial chromosome and shows significant variation among Staphylococci groups, confirmed by PCR and sequence analysis using the specific primers used. enabling for quick, highly sensitive, and specific identification of these species (Poulsen et al., 2003).

 

Table 2: The A prevalence results of 13 isolates of Staphylococcus sp. based on 16S rDNA gene sequences.

Staphylococcus sp.	Positive No. based on sequence of 16S rDNA	(%) of isolates based on sequence of 16S rDNA
S. aureus	9	69.24%
Other Staphylococcus sp.	4	30.76%
Total	13	100%

 

Table 3: The Nucleotides BLAST Analysis results of 13 isolates of Staphylococcus sp. based on 16S rDNA gene sequences.

Sample number	Source	Identities	GenBank ID
1	Staphylococcus aureus	96.31%	MN871924.1
2	Staphylococcus sciuri	97.96%	AB795259.1
3	Staphylococcus aureus	99.08%	KP295472.1
4	Staphylococcus aureus	98.57%	MN629267.1
5	Staphylococcus succinus	99.71%	LC216396.1
6	Staphylococcus aureus	99.85%	OR054191.1
7	Staphylococcus aureus	97.48%	LR134087.1
8	Staphylococcus aureus	99.85%	OR098500.1
9	Staphylococcus equorum	100.00%	MT409914.1
10	Staphylococcus epidermidis	97.29%	JF784023.1
11	Staphylococcus aureus	97.87%	MT628394.1
12	Staphylococcus aureus	99.86%	KP295472.1
13	Staphylococcus aureus	99.69%	MH429612.1

 

Furthermore, and in order to complete the genetic analysis of Staphylococcus sp., the PCR product result that exposed an amplicon size (527bp) of 16S rDNA gene was send for sequences analysis and all sequencing results were analyzed using the Basic Local Alignment Search Tool (BLAST) to compare the sequences to NCBI information and identify any discrepancies, as shown in Tables 2, 3, according to the sequencing results in Table 2 the prevalence results of 13 isolates of Staphylococcus sp. based on 16S rDNA gene sequences. only 9 (69.24%) local samples had similarity ranged between (96.31%-99.86%) to the S. aureus strains that previously recorded in the GenBank. However, the remaining of the local analysis samples 4 (30.76%) were diagnosed as Staphylococcus sciuri, Staphylococcus succinus, Staphylococcus equorum and Staphylococcus epidermidis at a homology ranged between 100%-96%. This percentage was lower than the research conducted by Khairullah et al. (2022), who isolated 80 (53.33%) of S. aureus from 150 milk samples, but agree with the result of another study that conducted by Jahan et al. (2015) who isolated 12 (25.53%) of S. aureus from 47 milk samples regardless the number of S. aureus that could be isolated from milk samples the differences that occur could be attributed to variations in research design, such as residents and geographic distribution of samples, infection control procedures, the kind of antibiotic used for treatment, and poor hygiene procedure during milking, all of which can raise the risk of bacterial contamination in cow’s milk (Tohoyessou et al., 2020; Omrani et al., 2020).

 

 

On the other hand, the phylogenetic tree based on 16SrDNA gene was made by maximum likelihood method as in Figures 5, 6. In Figure 5 phylogeny of the Staphylococcus aureus was divided into two groups: The first group contained the S13, S6, S12, S8 and S7. In which S13 that shows extreme similarity to the samples from Colombia and Iraq while S6 show more relation to the samples from Japan, Pakistan and Russia.

Although S12 shares the same ancestor with S13 and S6 but it has the special branch. Additionally, both S7 and S8 arise from the same ancestor of groups one, but that had its evolution path. In contrast, groups two comprising S11, S1, S4 and S3. S11 shares the same ancestor with those from China and Turkey while S1 shows similarity with national strains from India and USA. Subsequently, both S3 and S4 were not national species, but shared a common ancestor with species from Iraq and China.

Alternatively, Figure 6 reveals the phylogeny of Iraqi isolates of other Staphylococcus sp., in this Figure 6, S9 shows extreme similarity with strain from China than those from India although it shares the same ancestor. While both S10 and S2 were lacks the similarity to the national isolates. Finally, S5 was shown more similarity with those strain from Japan than those came from Peru.

This indicates that Staphylococcus sp. Iraqi isolates are very similar to isolates from other countries and share the same 16s rDNA gene sequence. Because this gene is stable, has little mutation, and takes a long time to change, it is used to classify bacteria around the world, and lead to concluded that using of 16s rDNA in the clinical laboratory by sequence instead of phenotype can improve clinical microbiology technique for identifying bacteria and conducting taxonomic research (Clarridge, 2004).

 

Table 4: The nucleotides sequencing analysis results of mecA gene in 13 isolates of Staphylococcus sp.

Sample number	Source	Identities	GenBank ID
1	Staphylococcus aureus	100.00%	LC727174.1
2	Staphylococcus aureus	100.00%	CP127807.1
3	Staphylococcus epidermidis	100.00%	CP121525.1
4	Staphylococcus aureus	100.00%	LC727174.1
5	Staphylococcus aureus	100.00%	LC727174.1
6	Staphylococcus aureus	100.00%	LC727174.1
7	Staphylococcus aureus	100.00%	LC727174.1
8	Staphylococcus aureus	100.00%	CP127807.1
9	Staphylococcus aureus	100.00%	LC727174.1
10	Staphylococcus aureus	100.00%	CP127807.1
11	Staphylococcus aureus	100.00%	LC727174.1
12	Staphylococcus aureus	100.00%	CP121525.1
13	Staphylococcus aureus	100.00%	CP127807.1

 

On the other hand, the sequence analysis of mecA gene was also investigate using Blast algorithmic tool and listed in Table 4. In this Table 4 the sequencing results of mecA gene was revealed 100% homology to the Staphylococcus aureus S286 DNA, Staphylococcal Cassette Chromosome mec with ID number LC727174.1 and Staphylococcus aureus strain C249 chromosome with ID number CP127807.1. While one isolate was displayed 100% homology to the mecA gene that previously detected in Staphylococcus epidermidis strain 1FSE05 plasmid with ID: CP121525.1.

 

The phylogeny tree was also constructed in order to investigated the genetic distribution of mecA gene in local isolates and those listed and in GenBank as shown in Figure 7. The results of this Figure 7 give an adopt that the sources of mecA gene is mainly from Staphylococcus aureus which bring and share their Cassette Chromosome mec with other staphylococcal populations by horizontal gene transfer (Wielders et al., 2002). Additionally, it was recently discovered that using antibiotic treatment and or the abuse of antibiotics caused the gene to be transfer from S. epidermidis to S. aureus (Wielders et al., 2001), other similar investigations suggested that CoNS could also act as donors and source for the transmission of the mecA gene crossways the Staphylococcaceae family (Otto, 2013; John et al., 2019). On the other hand, it was recently discovered that using antibiotic in of treatment and or the abuse of antibiotics caused the gene to be transfer from S. epidermidis to S. aureus (Wielders et al., 2001). Moreover, the detection of mecA gene in the milk samples of mastitis cases have a significantly impact that there was a high degree of correlation between the presence of the mecA gene in Staphylococcus isolates and resistance to some common antibiotics such as cefoxitin and associated oxacillin and methicillin resistance (Wolk et al., 2009).

In this context, it is significantly notable that (70 to 75%) of all CoNS are currently methicillin-resistant (Diekema et al., 2001), creating a great potential reservoir of resistance.

CONCLUSION AND RECOMMENDATIONS

Regardless if the S. aureus or other CoNS is responsible for sharing and distribution of mecA gene, we concluded that all these information may offer crucial understanding of the origin and persistence of current and possibly even future of mecA in Staphylococcus strains. Additionally, it is advised to put control measures in place to prevent the establishment of resistance phenomena, such as a regular cleanliness regimen, routine MRSA screening, the use of antibiotic susceptibility testing before to treatment, or the random selection of antibiotics in field cases.

ACKNOWLEDGMENT

We thank to the Veterinary Medicine faculty, Basrah University, Iraq, for providing the laboratory facilities.

NOVELTY STATEMENT

According to a recent study, it has been found that the majority of staphylococci strains, which cause mastitis in lactating animals, possess mecA gene. This gene plays a crucial role in the survival and persistence of Staphylococcus sp. as well as in their resistance to antibiotics. The study highlights the importance of following correct diagnostic procedures to identify the pathogen and using antimicrobials appropriately to prevent the development of antibiotic resistance.

AUTHOR’S CONTRIBUTION

HYJ: Clinical examination, sample collection and presumptive laboratory diagnosis. RMO: Molecular diagnosis and wrote the article. WMS: Microbiological diagnosis.

Conflict of interest

The authors have declared no conflict of interest.

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