Prerpints.org logo
Preprint
Article

Investigation of Various Toxigenic Genes, Antibiotic and Disinfectant Resistance Profiles in Staphylococcus aureus Originating from Raw Milk

Altmetrics

Downloads

67

Views

38

Comments

0

A peer-reviewed article of this preprint also exists.

This version is not peer-reviewed

Submitted:

27 September 2024

Posted:

29 September 2024

You are already at the latest version

Alerts
Abstract
This study investigated the toxigenic genes and antimicrobial resistance profiles Staphylococcus aureus strains isolated from 260 raw milk samples collected from dairy farms in Türkiye. Results indicated that 60.7% of staphylococcal enterotoxin genes (sea, seb, sed, seg, sei, sej, sek, seq, sem, seo, and seu) and 21.4% of the tst and eta genes were positive, with most enterotoxin-positive samples carrying more than one gene. The genes sec, see, seh, sel, sen, sep, and etb were not identified in any samples. The prevalence of antibiotic resistance genes (mecA, blaR, blaI, blaZ, vanA, ermT, tetK, aac/aph, ant, dfrA, tcaR, IS256, and IS257) was high at 89.2%, with bla being the most frequently detected gene (75%). The mecA gene was present in 14.2% of samples, while tcaR was detected in 78.5%. Nevertheless, the mecC was not identified. Disinfectant resistance genes (qacA/B, qacC, qacJ, smr) were detected in 21.4% of the samples. The results of the disc diffusion test showed that 64% of strains were resistant to penicillin G and ampicillin, with additional resistance found for cefoxitin, teicoplanin, levofloxacin, norfloxacin, and other antibiotics. These findings highlight a significant public health and food safety risk associated with raw milk due to the presence of S. aureus strains with toxigenic genes and high antimicrobial resistance.
Keywords: 
Subject: Biology and Life Sciences  -   Food Science and Technology

1. Introduction

Milk is a nutritionally complete food, providing all the nutrients required for growth and healthy development in humans [1]. Nevertheless, raw milk is also an optimal source of nutrition for the cultivation of pathogens and spoilage organisms [2]. As the demand for raw milk, perceived to be more nutritionally beneficial by the public, continues to rise, a corresponding increase in health concerns has been observed. Staphylococcus aureus (S. aureus) is one of the most significant pathogenic microorganisms found in milk and is responsible for several serious diseases in humans [3,4]. S. aureus is a significant pathogen for dairy farms, due to its virulence and antimicrobial resistance properties.
S. aureus is the most frequently identified causative agent of clinical and subclinical mastitis in dairy cattle [4,5]. It is worth noteworthy that, in addition to mastitis in animals, S. aureus, which can be transmitted from farm personnel to milk, has been identified as a causative agent of food poisoning in humans [6,7]. In contrast to other toxins secreted by S. aureus, a relatively small quantity is sufficient for enterotoxins to exert their toxic effects. Staphylococcal food poisoning represents a significant cause of foodborne illness globally, attributed to the presence of enterotoxins with enhanced tolerance to environmental agents [8,9].
S. aureus possesses a variety of virulence factors that contribute to its pathogenicity [3]. The known secretory virulence factors include toxins (staphylococcal enterotoxins, toxic shock syndrome toxin-1 (tst), haemolysins, and exfoliative toxins A and B (eta and etb), as well as enzymes (coagulase, staphylokinase, DNAase, phosphatase, lipase, and phospholipase) [3,10]. Due to its virulence properties, S. aureus can overcome the host defense system, thereby facilitating the occurrence of disease, and prolonging the course of treatment.
Food represents a significant vector for the transmission of antimicrobial resistance [7]. The term "antimicrobial resistance" encompasses both disinfectant and antibiotic resistance. In recent years, there has been an alarming increase in the severity of infections caused by S. aureus, largely due to the emergence of antibiotic resistance [11]. The emergence of penicillin, vancomycin, and methicillin resistance is frequently observed because of excessive antibiotic treatment [12]. Methicillin-resistant S. aureus (MRSA) represents a significant global public health threat on a global scale, particularly in humans and animals, and is a common cause of severe infection [13,14]. The primary cause of hospital-acquired infections, MRSA, exhibits resistance to numerous known antibiotics, rendering the fight against the disease even more challenging [15].
Quaternary ammonium compounds (QACs) are a commonly used disinfectant in the dairy industry, employed for the disinfection of milking equipment and udder disinfection, particularly for the prevention of mastitis [16]. In the absence of the requisite conditions, including the correct selection of disinfectant, appropriate dosage, and pre-cleaning, the efficacy of disinfectants is diminished, leading to the emergence of resistant strains [17]. This situation represents a significant public health concern that requires urgent attention.
The objective of this study was to investigate the virulence characteristics and antimicrobial resistance profile of S. aureus strains isolated from raw milk. The antimicrobial resistance profiles of S. aureus strains isolated from raw milk were investigated by analyzing the presence of antibiotic and disinfectant resistance genes. Moreover, agar diffusion assays were performed to evaluate the antibiotic susceptibility of the samples against nine distinct antibiotic groups. To identify the virulence properties, an investigation was conducted into the presence of SEs genes responsible for enterotoxin production, exfoliative toxin genes (eta and etb), and the toxic shock syndrome toxin-1 gene (tst). Furthermore, the milk to be analyzed in the study was collected from the Thrace region, which represents a border area with Europe. The objective was to examine a region that is significant in terms of disease control.

2. Materials and Methods

2.1. Isolation and Strain Identification

A total of 260 raw milk samples were collected from various dairy farms in the Thrace region and plated on Baird Parker agar (BPA, Oxoid, CM275, Basingstoke, UK) and S. aureus was isolated. The isolation of S. aureus from food samples was conducted by the EN ISO 6881-1 standardized procedures documented by the International Organization for Standardization [18]. The strains were identified by biochemical tests, including gram staining, catalase testing, latex agglutination testing, growth on mannitol fermentation (using Mannitol Salt Agar, Oxoid CM0085B), and DNase activity testing (using DNase agar, Oxoid CM0321). The identifications were subsequently confirmed by PCR following DNA extraction [7]. To confirm the identity of the S. aureus strains, an analysis was conducted to determine the presence of the thermonuclease gene (nuc), the coagulase gene (coa), and the production of the surface protein A gene (spa) [7,19,20].

2.2. Detection of Toxigenic Genes in S. aureus Isolates

The presence of staphylococcal enterotoxin genes sea, seb, sec, sed, see [21], seg, seh, sei, sej, sek, sel, sep, seq [22], sem, sen, seo [23], and seu [24] were detected using multiplex and monoplex PCR. Moreover, the presence of the exfoliative toxins’ genes (eta, etb) [21], and the toxic shock syndrome toxin-1 gene (tst) [25] were investigated.

2.3. Determination of Antimicrobial Susceptibility of S. aureus Isolates

The antimicrobial susceptibility of the isolates was determined by the agar disk diffusion method, in accordance with the guidelines set forth by the EUCAST (European Committee on Antimicrobial Susceptibility Testing) [26]. The following antibiotic disks were used: penicillin G (Oxoid-CT0043, 10 U), ampicillin (Oxoid-CT0003, 10 μg), gentamicin (Oxoid-CT0024,10 μg), tobramycin (Oxoid-CT0056, 10 µg), teicoplanin (Oxoid-CT0647, 30 μg), ceftaroline (Oxoid-CT1942, 5 μg), cefoxitin (Oxoid-CT0119, 30 μg), tetracycline (Oxoid-CT0054, 30 µg), erythromycin (Oxoid-CT0020, 15 μg), levofloxacin (Oxoid-CT1587, 5 µg), ofloxacin (Oxoid-CT0446 5 μg), norfloxacin (Oxoid-CT0434, 10 µg), fusidic acid (Oxoid-CT0023, 10 μg), trimethoprim-sulfamethoxazole (Oxoid-CT0052, 1.25 μg/23.75 μg), linezolid (Oxoid-CT1649, 10 μg). The antibiotics were selected from nine preferred antibiotic groups, namely penicillins, aminoglycosides, glycopeptides and lipoglycopeptides, cephalosporins, tetracyclines, macrolides, lincosamides and streptogramins, fluoroquinolones, miscellaneous agents, and oxazolidinones. Antimicrobial discs were placed on Mueller-Hinton agar (MHA, CM 337 Oxoid) containing S. aureus. Following the incubation period, the diameter of the inhibition zone was measured and evaluated. MDR isolates were defined as those exhibiting resistance to at least three distinct antimicrobial classes.

2.4. Detection of Antimicrobial Resistance Genes in S. aureus Isolates

Multiplex and monoplex PCR was used to detect the presence of antibiotic resistance gene; methicillin resistance genes (mecA [27] and mecC [28]), penicilin resistance genes (blaR, blaI, blaZ [blaZF-R]) [29], and (blaZ1-2) [30], aminoglycoside resistance genes (aac/aph, aph, ant) [31], teicoplanin associated locus (tcaR) [32], vancomycin resistance gene (vanA) [33], trimethoprim resistance gene (dfrA) [29], erythromycin resistance gene (ermT) [34], tetracycline resistance gene (tetK) [29], and insertion sequence (IS) elements (IS256, IS257) [29]. Isolates were classified as multidrug-resistant when they exhibited an inability to respond to at least three distinct drug classes. To detect disinfectant resistance, which is the other responsible factor for the formation of antimicrobial resistance, the presence of the QAC resistance gene qacAB and smr [35], qacC [29], and qacJ [36] was determined by monoplex PCR.
The PCR products were analyzed by horizontal electrophoresis in a system containing 1xTris-acetate-EDTA (TAE) buffer, 1.5% (w/v) agarose gel, and 5% (v/v) fluorescent DNA dye (SafeView Classic, Applied Biological Materials Inc. Richmond, Canada). The gels were imaged using the Infinity Gel Imaging System (Vilber Lourmat, Marne-la-Vallée, France). All the PCR experiments were done twice for each strain.

3. Results

3.1. Toxigenic Genes (Virulence Genes)

In this study, 260 raw milk samples were collected, and 28 strains (10.7%) of S. aureus were confirmed by PCR following microbiological analysis. The investigation of the presence of virulence and antimicrobial resistance genes in these strains is presented in Table 1, Table 2 and Table 3 respectively.
The research findings indicated that at least one gene encoding for enterotoxin was present in 60.7% of the S. aureus samples obtained from raw milk. Fourteen of the 17 enterotoxin-positive samples demonstrated the presence of two or more distinct enterotoxin genes. The most prevalent gene observed in the samples was the seo gene (35.7%), and the gene observed in three samples carrying a single gene was also the seo gene. The percentages of the sea, seb, sed, seg, sei, sej, sek, sem, seo, seu and seq enterotoxin genes that were isolated from the 28 samples were found to be 7.1%, 17.8%, 17.8%, 25%, 28.5%, 3.5%, 7.1%, 25%, 35.7%, 32.1% and 17.8%, respectively. The genes sec, see, seh, sel, sen, and sep were not identified in any samples.
Regarding the virulence genes examined, the tst gene was identified in 21.4% of the samples. Among the exfoliative toxin genes, the eta gene was identified in a single sample (3.5%), while the etb gene was not detected.

3.2. Antimicrobial Susceptibility

The antimicrobial susceptibility of the isolates was determined by the agar disk diffusion method. The results of the disc diffusion test indicated that the highest resistance was observed against penicillin G and ampicillin (64%), followed by cefoxitin (28%), teicoplanin (21%), levofloxacin (21%), and norfloxacin (17%). The prevalence of resistance to ofloxacin and fucidic acid was observed to be 14%, while the prevalence of resistance to tobramycin, tetracycline, and erythromycin was observed to be 7.1%. The lowest resistance was observed in the gentamicin treatment group. All isolates demonstrated susceptibility to ceftaroline, trimethoprim-sulfamethoxazole, and linezolid.

3.3. Antimicrobial Resistance Genes

The present study demonstrated a high prevalence (89.2%) of antimicrobial resistance genes in S. aureus strains isolated from raw milk. The results of the analysis of antibiotic resistance genes revealed the presence of the mecA gene, responsible for resistance to the methicillin antibiotic, at a rate of 14.2%. In contrast, the mecC gene was not detected in any of the examined strains. The most frequently identified antibiotic resistance gene was the bla gene, which is responsible for resistance to beta-lactam antibiotics. This gene was identified in 75% of the samples analyzed. In the present study, the aac/aph and ant genes, which are responsible for the development of resistance to the aminoglycoside antibiotic group, were identified to be present in 3.5% and 7.1% of the strains, respectively. The aph gene was not detected in any of the strains. The tcaR gene, which is responsible for resistance to the teicoplanin antibiotic, was identified in 78% of the isolates. The vanA gene, which is responsible for vancomycin resistance, was analyzed at a rate of 17%, while the dfrA gene, which is responsible for resistance to trimethoprim antibiotics, was analyzed at a rate of 7.1%. The ermT gene, which is responsible for resistance to the erythromycin antibiotic from the lincosamide group, and the tetK gene, which is responsible for tetracycline resistance, were identified in a single sample each. The insertion sequence (IS) elements IS256 were identified in 53.5% of the cases, while IS257 was analyzed in 3.5%.
The QAC genes responsible for QAC disinfectant resistance were identified in 21.4% of the samples. The prevalence of disinfectant resistance genes qacAB, qacC, qacJ, and smr was determined to be 7.1%, 7.1%, 17.8%, and 21.4%, respectively.

4. Discussions

4.1. Toxigenic Genes

Epidemiological studies have demonstrated that S. aureus strain agents in milk produce a group of virulence factors and have indicated a correlation between the severity of infection and the virulence factors produced by S. aureus [3]. The most significant virulence factor of S. aureus is the production of enterotoxin. SEs are regarded as a significant public health concern [37]. A number of studies have investigated the occurrence of enterotoxin genes in strains of S. aureus isolated from food sources, reporting a range of prevalence rates for different genes. In their study, Zhang et al. (2022, a) reported the detection of the sea (17.45%), seb (16.44%), sec (7.38%), and sed (1.68%) enterotoxin genes, while the see gene was not detected. In their 2020 study, Titouche et al. reported that 62.5% of the samples tested positive for at least one gene encoding SEs. The most prevalent genes were sei and seg (47.69%), followed by seb (23.08%). It is noteworthy that, none of the isolates carried the sed and see genes. In another study, the seg, sei and sec genes were identified as positive in 35% of raw milk samples, while the sea and sei genes were found to be negative [39]. In a study conducted by Khemiri et al. (2019), at least one enterotoxin gene was identified in 87.5% of S. aureus strains isolated from raw milk samples. Of these, the sed gene was the most frequently analyzed. Consistent with previous studies [3,38,39], our analysis revealed the presence of at least one enterotoxin gene in the strains under investigation, while the sec and see genes were not identified. In contrast with the findings of the present study, Fursova et al. (2018) examined the sea gene in 53.30% and the sec gene in 50% of S. aureus strains isolated from food sources. Furthermore, the present study identified the presence of the seb, sed (17.8%), and sei (28.5%) genes, which were not detected in the studies conducted by Adame-Gómez et al. (2020) and Pereira et al. (2009). One of the primary causes for the disparate outcomes observed in the study was attributed to the distinct characteristics exhibited by the strains in varying geographical regions. As observed in our study, the see gene was not detected, while the sed gene was among the most prevalent in a similar study conducted in the Thrace region by Papadopoulos et al. (2019). The findings of our study are consistent with those of a similar investigation conducted in our neighboring country, in the Thrace region. In the study, the see gene was not detected, while the sed gene was identified with high frequency [43].
In addition to food poisoning, S. aureus is a causative agent of a range of acute and chronic diseases, including septicemia, pneumonia, endocarditis, and respiratory tract illnesses, as well as autoimmune diseases. These diseases have a high morbidity rate in a variety of hosts. One of these diseases is toxic shock syndrome [44]. In their study, Zhang et al. (2022, a) identified the presence of the tst gene at a rate of 23.50%, while Dan et al. (2019) reported a rate of 26.5%. As in the aforementioned studies, the tst gene was identified in 21.4% of cases in our own investigation. In contrast with the findings of our study, the tst gene was not identified in other studies conducted on milk [3,42,46].
Exfoliative toxins (eta, etb) are virulence factors secreted by staphylococci. These toxins are responsible for the degradation of keratinocytes in both human and animal skin, which can result in the development of skin infections [47]. In the present study, the eta gene was identified as positive, whereas the etb gene was identified as negative. In other studies, Tegegne et al. (2021) analyzed the eta gene in 22.05% of S. aureus strains isolated from milk. Similarly, Kot et al. (2016) analyzed the eta gene in 5.6% of S. aureus strains isolated from milk. The studies conducted by Dan et al. (2019), Gharsa et al. (2019) and Chenouf (2021) did not identify the presence of the eta and etb genes in any of the samples. The findings demonstrate that S. aureus strains may encode a range of virulence factors and that the distribution of these factors varies between different genotypes. These results indicate that S. aureus virulence is not exclusive to the strain level but is also influenced by factors such as the origin and genetic background of the strain [45].

4.2. Antimicrobial Susceptibility

Upon examination of the antibiotic resistance profiles, it was observed that the results of the disc diffusion and resistance genes assays were consistent for the beta lactam group. In the course of our investigation, it was found that 75% of the isolates encoding the bla gene and 64% of the isolates were resistant to penicillin G. The aac/aph and ant genes responsible for resistance to the aminoglycoside group were detected in 3.5% and 7.1% of isolates, while resistance to gentamicin and tobramycin was 3.5% and 7.1%, respectively. The findings of our study are in accordance with those of other researchers indicating that the highest resistance was observed in the penicillin group [39,45,51]. As observed by Dan et al. (2019), the current study revealed that the isolates did not demonstrate resistance to linezolid. However, in contrast to the findings of this study, the isolates exhibited high resistance to teicoplanin. As previously observed by Pereira et al. (2009), a minor percentage of isolates exhibited resistance to gentamicin, erythromycin, and tetracycline. The current study reports a lower prevalence of norfloxacin-resistant isolates than that observed by Kotb and Gafer (2020).
The data presented here are similar to those reported by Dan et al. (2019), in that all MRSA isolates demonstrated resistance to penicillin G and ampicillin. Of the analyzed samples, 78% showed resistance to more than one antibiotic. The findings of our study indicate that some isolates encoding resistance genes (tcaR and dfrA) did not develop resistance to the antibiotic. These findings indicate that, despite the presence of antimicrobial resistance genes, their expression may be insufficient to confer a resistant phenotype. Nevertheless, these strains have the potential to transform into resistant strains under certain conditions and may therefore have epidemiological importance in the transmission of antibiotic resistance [45].

4.3. Antimicrobial Resistance Genes

The increasing prevalence of antimicrobial resistance in S. aureus represents a significant global public health concern [4]. As a consequence of the excessive and inappropriate usage of antimicrobials, microorganisms develop resistance to these substances, thereby transferring this resistance to other microorganisms through the transfer of genes [53]. This situation gives cause for concern about both food safety and public health. The range of treatment options for life-threatening infections caused by MDR strains of S. aureus is limited. Considering these considerations, the World Health Organization (WHO) classified S. aureus as the most significant bacterial pathogen exhibiting antibiotic resistance in 2017 [54].
Methicillin-resistant S. aureus is one of the most significant antibiotic-resistant bacteria. Isolates of MRSA are frequently multidrug resistant, which results in increased costs, longer treatment durations, and higher rates of hospitalization and comorbidity. The mecA gene, which encodes the production of penicillin-binding proteins, is responsible for methicillin resistance in the MRSA chromosome. It is therefore considered the most reliable method for the detection of methicillin resistance [4,55]. The present study revealed the presence of the mecA gene in 14.2% of the samples. A review of the literature on the detection of the meC gene in milk revealed that the findings of Dan et al. (2019) were consistent with those of the present study. The results of the mecA analysis in the present study were found to be lower than those reported by Ganai et al. (2016), Riva et al. (2015), and our neighboring country [43], but higher than those observed by Mahanti et al. (2020), Chenouf et al. (2021), and Pereira et al. (2009). In studies conducted in Türkiye, the prevalence of strains carrying the mecA gene was found to be 6.3% [59] and 1.70% [60], which is lower than the data obtained in the present study. In numerous additional studies conducted in Türkiye, the presence of the mecA gene was not detected in S. aureus strains isolated from food sources [51,61,62]. It is also noteworthy that, as with the present study, the mecC gene was not detected in studies conducted in Brazil [63] and Greece [43].
The primary mechanism of resistance to penicillin and penicillin derivatives is the blaZ gene, which encodes the production of beta-lactamases that hydrolytically break down beta-lactams [4,55]. Elevated levels of the blaZ gene have been reported in milk [63 (74.07%), 64 (94.6%), 65 (95.7%)]. In this study, the blaI and blaR genes, which are responsible for the production of beta-lactamase, were also subjected to analysis. A review of the literature revealed no previous analysis of these genes in raw milk in Türkiye. The blaI and blaR genes are responsible for the regulation of the expression of the blaZ gene [[66]. In a study conducted by Kreausukon et al. (2012), the presence of the blaI, blaR, and blaZ genes was identified in all MRSA strains. Additionally, the study demonstrated the presence of elevated levels of the blaI and blaR genes. Furthermore, the investigation revealed that the blaI and blaZ genes were present in all isolates that also carried the blaR gene.
The bifunctional enzyme AAC/APH encoded by the aac/aph gene, the APH enzyme encoded by the aph gene, and the ANT enzyme encoded by the ant gene are responsible for resistance to aminoglycosides [68]. The present study identified the presence of the aac/aph and ant genes in various strains, while the aph gene was not detected in any strains. In other studies, the presence of these genes in milk was confirmed [68,69]. The Teicoplanin-associated locus (tcaR) gene, which is associated with teicoplanin resistance, was analyzed for the first time in Türkiye and the results demonstrated that it was present in 78.5% of the samples examined. In contrast, the tcaR gene was identified in clinical isolates in a study conducted in India [70].
Vancomycin, a glycopeptide antibiotic, has been employed in the treatment of MRSA for several decades. This has resulted in the emergence of vancomycin-resistant S. aureus strains [71]. In the present study, the prevalence of the vanA resistance gene was determined to be 17%, which is consistent with the findings of Kou et al. (2021). The dihydrofolate reductase protein, which confers resistance to trimethoprim (dfrA), was identified at a prevalence of 7.1% in our study. Nevertheless, the dfrA gene was not detected in raw milk in Egypt [73] and Mozambique [74], nor in chicken, fish, and red meat in Türkiye [75].
The resistance to lincosamides, including erythromycin and clindamycin, is a consequence of the methylation of the receptor-binding site on the ribosome. Methylation is a process that is mediated by an enzyme called methylase and is encoded by erm genes. During our investigation, the ermT gene was isolated from a single isolate. Nevertheless, other studies have indicated that the ermT gene was not identified [50]. The efflux pump system encoded by the tetK gene and carried by plasmids represents one of the resistance pathways to tetracyclines, a broad-spectrum antibiotic [71]. The prevalence of the tetK gene, which was identified in a single isolate in our study, was found to vary significantly in other studies [50 (30%), 45 (3.2%), 4 (48.66%)].
The insertion sequence (IS) elements IS256 and IS257, which are associated with multiple antibiotic resistance, were identified as being present in the study. As observed in our data, studies by Zhang et al. (2022, b) and Miao et al. (2018) similarly identified the presence of IS257 and IS256, respectively. Araújo et al. (2017) did not identify the presence of IS256 and IS257 in their samples. The present study revealed that 64.2% of S. aureus strains demonstrated multidrug resistance (MDR), predominantly to beta-lactams, methicillin, and teicoplanin (Table 3). The findings indicated a high prevalence of MDR S. aureus in raw milk, which represents a significant public health concern. Absent the implementation of necessary preventive measures, the probability of encountering far more significant difficulties in the future is considerable. To prevent the development and transmission of antibiotic-resistant strains, it is recommended that different groups of antibiotics be preferred over this resistance group of antibiotics, particularly when antibiotics are used on dairy farms.
A further component of antimicrobial resistance is disinfectant resistance. To investigate the potential for disinfectant resistance, this study examined the presence of the efflux pump gene (qac and smr), which is responsible for resistance to QACs. The qac and smr genes resulted in 21.4% positive results. The findings of Kroning et al. (2020), Kotb and Gafer (2020) and Ergun et al. (2017) indicate a lower prevalence of the qac gene than that observed in our data. Furthermore, the qacJ gene, identified as 17% positive in our study, was found to be negative by Kroning et al. (2020). Upon analysis of the results of the study, it was observed that all samples exhibiting disinfectant resistance genes also carried at least one antibiotic resistance gene. Three of the four MRSA strains were found to carry a disinfectant resistance gene. This was regarded as an additional risk associated with MRSA strains. Furthermore, previous research has indicated a genetic correlation between disinfectant and antibiotic resistance genes [80,81]. The co-detection of genes responsible for both disinfectant and antibiotic resistance in strains is indicative of the presence of antimicrobial-resistant strains. Despite the disparate mechanisms of action exhibited by the two groups of genes, genetic studies have revealed striking similarities in their genetic systems and the locations of their genes on the same mobile genetic elements [80]. Moreover, research has indicated that the probability of the pathogen developing antibiotic resistance increases with prolonged exposure to the disinfectant [82].

5. Conclusions

This study represents the first comprehensive investigation into the antimicrobial resistance levels and virulence characteristics of raw milk in Türkiye. The high prevalence of virulence and antimicrobial genes in S. aureus in our study indicates a potential risk associated with raw milk. The necessary measures must be taken to eliminate this situation, which presents a significant risk to both food safety and public health. To prevent the development of MDR bacteria, which represents an important potential health threat in the future, it is essential to focus on the selection of antibiotics and the utilization of novel, advanced-generation antibiotics. To prevent the development of enterotoxins and disinfectant resistance, it is essential to implement hygiene and sanitation procedures, as well as to ensure the correct selection of disinfectants. Further studies are necessary to determine the risks associated with food products and to identify the precautions to be taken before these risks occur.

Author Contributions

Conceptualization, G.M.B. and A.A..; design and methodology, G.M.B. and A.A.; sampling and investigation, literature searching, G.M.B.; resources, A.A.; writing – original draft preparation, G.M.B; writing – review & editing, G.M.B. and A.A. supervision, A.A. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data presented in this study are available on request from the corresponding authors.

Acknowledgments

The authors would like to acknowledge to Goknur Yetimler for her technical assistance.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Zakary, E.M.; Nassif, M.Z.; Mohammed, G.M. Detection of Staphylococcus aureus in bovine milk and its product by real time PCR assay. GJBB. 2011, 6, 171–177. [Google Scholar]
  2. Bayrakal, G.M.; Aydin, A. Sütteki Kontaminantlar. Süt ve Süt Ürünleri, Editor Atasever, M. Turkiye Klinikleri; Ankara, Türkiye, 2019: Volume 5, pp. 114-119.
  3. Tegegne, D.T.; Mamo, G.; Waktole, H.; Messele, Y.E. Molecular characterization of virulence factors in Staphylococcus aureus isolated from bovine subclinical mastitis in central Ethiopia. Ann. Microbiol., 2021, 71, 1–8. [Google Scholar] [CrossRef]
  4. a Zhang, Z.; Chen, Y.; Li, X.; Wang, X.; Li, H. Detection of antibiotic resistance, virulence gene, and drug resistance gene of Staphylococcus aureus isolates from bovine mastitis. Microbiol. Spectr. 2022, 10, e00471–22. [Google Scholar] [CrossRef] [PubMed]
  5. Kadariya, J.; Smith, T.C.; Thapaliya, D. Staphylococcus aureus and staphylococcal food-borne disease: an ongoing challenge in public health. BioMed Res. Int. 2014, 2014, 827965. [Google Scholar] [CrossRef] [PubMed]
  6. Hennekinne, J.A.; De Buyser, M.L.; Dragacci, S. Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation. FEMS Microbiol. Rev. 2012, 36, 815–836. [Google Scholar] [CrossRef]
  7. Sudagıdan, M.; Aydın, A. Screening virulence properties of staphylococci isolated from meat and meat products. Vet. Med. Austria / Wien. Tierärztl. Mschr. 2008, 95, 128–134. [Google Scholar]
  8. Antoszewska, A.; Maćkiw, E.; Kowalska, J.; Patoleta, M.; Ławrynowicz-Paciorek, M.; Postupolski, J. Microbiological risks of traditional raw cow’s milk cheese (Koryciński Cheeses). Foods. 2024, 13, 1364. [Google Scholar] [CrossRef]
  9. Pérez-Boto, D.; D’Arrigo, M.; García-Lafuente, A.; Bravo, D.; Pérez-Baltar, A.; Gaya, P.; Medina, M.; Arqués, J.L. Staphylococcus aureus in the processing environment of cured meat products. Foods. 2023, 12, 2161. [Google Scholar] [CrossRef]
  10. Abril, A.G.; Villa, T.G.; Barros-Velázquez, J.; Cañas, B.; Sánchez-Pérez, A.; Calo-Mata, P.; Carrera, M. Staphylococcus aureus exotoxins and their detection in the dairy industry and mastitis. Toxins. 2020, 12, 537. [Google Scholar] [CrossRef]
  11. Neyra, R.C.; Frisancho, J.A.; Rinsky, J.L.; Resnick, C.; Carroll, K.C.; Rule, A.M.; Ross, T.; You, Y.; Price, L.B.; Silbergeld, E.K. Multidrug-resistant and methicillin-resistant Staphylococcus aureus (MRSA) in hog slaughter and processing plant workers and their community in North Carolina (USA). Environ. Health Perspect. 2014, 122, 471–477. [Google Scholar] [CrossRef]
  12. Chambers, H.F. The changing epidemiology of Staphylococcus aureus? Emerg. Infect. Dis. 2001, 7, 178. [Google Scholar] [CrossRef] [PubMed]
  13. Caruso, M.; Latorre, L.; Santagada, G.; Fraccalvieri, R.; Miccolupo, A.; Sottili, R.; Palazzo, L.; Parisi, A. Methicillin-resistant Staphylococcus aureus (MRSA) in sheep and goat bulk tank milk from Southern Italy. Small Rumin. Res. 2016, 135, 26–31. [Google Scholar] [CrossRef]
  14. González-Machado, C.; Alonso-Calleja, C.; Capita, R. Methicillin-Resistant Staphylococcus aureus (MRSA) in different food groups and drinking water. Foods. 2024, 13, 2686. [Google Scholar] [CrossRef] [PubMed]
  15. Enright, M.C.; Robinson, D.A.; Randle, G.; Feil, E.J.; Grundmann, H.; Spratt, B.G. The evolutionary history of methicillin-resistant Staphylococcus aureus (MRSA). PNAS. 2002, 99, 7687–7692. [Google Scholar] [CrossRef]
  16. Bjorland, J.; Sunde, M.; Waage, S. Plasmid-borne smr gene causes resistance to quaternary ammonium compounds in bovine Staphylococcus aureus. J. Clin. Microbiol. 2001, 39, 3999–4004. [Google Scholar] [CrossRef]
  17. Kroning, I.S.; Haubert, L.; Kleinubing, N.R.; Jaskulski, I.B.; Scheik, L.K.; Ramires, T.; da Silva, W.P. New spa types, resistance to sanitisers and presence of efflux pump genes in Staphylococcus aureus from milk. Int. Dairy J. 2020, 109, 104712. [Google Scholar] [CrossRef]
  18. ISO 6881-1; Microbiology of food and animal feeding stuffs: horizontal method for the enumeration of coagulase positive staphylococci (Staphylococcus aureus and other species). Part 1: technique using Baird Parker agar medium. International Organization for Standardization (ISO): Geneva, Switzerland, 1999.
  19. Hookey, J.V.; Richardson, J.F.; Cookson, B.D. Molecular typing of Staphylococcus aureus based on PCR restriction fragment length polymorphism and DNA sequence analysis of the coagulase gene. J. Clin. Microbiol. 1998, 36, 1083–1089. [Google Scholar] [CrossRef]
  20. Aires-de-Sousa, M.; Boye, K.; de Lencastre, H.; Deplano, A.; Enright, M.C.; Etienne, J.; Friedrich, A.; Harmsen, D.; Holmes, A.; Huijsdens, X.W.; Kearns, A.M.; Mellmann, A.; Meugnier, H.; Rasheed, J.K.; Spalburg, E.; Strommenger, B.; Struelens, M.J.; Tenover, F.C.; Thomas, J.; Vogel, U.; Westh, H.; Xu, J.; Witte, W. High interlaboratory reproducibility of DNA sequence-based typing of bacteria in a multicenter study. J. Clin. Microbiol. 2006, 44, 619–621. [Google Scholar] [CrossRef] [PubMed]
  21. Johnson, W.M; Tyler, S.D.; Ewan, E.P.; Ashton, F.E.; Pollard, D.R.; Rozee, K.R. Detection of genes for enterotoxins, exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction. J. Clin. Microbiol. 1991, 29, 426–430. [Google Scholar] [CrossRef]
  22. Bania, J.; Dabrowska, A.; Bystron, J.; Korzekwa, K.; Chrzanowska, J.; Molenda, J. Distribution of newly described enterotoxin-like genes in Staphylococcus aureus from food. Int. J. Food Microbiol. 2006, 108, 36–41. [Google Scholar] [CrossRef]
  23. Jarraud, S.; Mougel, C.; Thioulouse, J.; Lina, G.; Meugnier, H.; Forey, F.; Nesme, X.; Etienne, J.; Vandenesch, F. Relationships between Staphylococcus aureus genetic background, virulence factors, agr groups (alleles), and human disease. Infect. Immun. 2002, 70, 631–641. [Google Scholar] [CrossRef] [PubMed]
  24. Nashev, D.; Toshkova, K.; Bizeva, L.; Akineden, Ö.; Lämmler, C.; Zschöck, M. Distribution of enterotoxin genes among carriage-and infection-associated isolates of Staphylococcus aureus. Lett. Appl. Microbiol. 2007, 45, 681–685. [Google Scholar] [CrossRef] [PubMed]
  25. Booth, M.C.; Pence, L.M.; Mahasreshti, P.; Callegan, M.C.; Gilmore, M.S. Clonal associations among Staphylococcus aureus isolates from various sites of infection. Infect. Immun. 2001, 69, 345–352. [Google Scholar] [CrossRef] [PubMed]
  26. EUCAST (European Committee on Antimicrobial Susceptibility Testing). Antimicrobial Susceptibility Testing EUCAST Disk Diffusion Method. Version 10.0. 2022. Available online: Manual_v_10.0_EUCAST_Disk_Test_2022.pdf (accessed on 10 April 2022).
  27. Lem, P.; Spiegelman, J.; Toye, B.; Ramotar, K. Direct detection of mecA, nuc and 16S rRNA genes in BacT/Alert blood culture bottles☆. Diagn. Microbiol. Infect. Dis. 2001, 41, 165–168. [Google Scholar] [CrossRef]
  28. Paterson, G.K.; Larsen, A.R.; Robb, A.; Edwards, G.E.; Pennycott, T.W.; Foster, G.; Mot, D.; Hermans, K.; Baert, K.; Peacock, S.J.; Parkhill, J.; Zadoks, R.N.; Holmes, M.A. The newly described mecA homologue, mecA LGA251, is present in methicillin-resistant Staphylococcus aureus isolates from a diverse range of host species. J. Antimicrob. Chemother. 2012, 67, 2809–2813. [Google Scholar] [CrossRef]
  29. Sidhu, M.S.; Heir, E.; Leegaard, T.; Wiger, K.; Holck, A. Frequency of disinfectant resistance genes and genetic linkage with β-lactamase transposon Tn 552 among clinical staphylococci. Antimicrob. Agents Chemother. 2002, 46, 2797–2803. [Google Scholar] [CrossRef]
  30. Bjorland, J.; Steinum, T.; Kvitle, B.; Waage, S.; Sunde, M.; Heir, E. Widespread distribution of disinfectant resistance genes among staphylococci of bovine and caprine origin in Norway. J. Clin. Microbiol. 2005, 43, 4363–4368. [Google Scholar] [CrossRef]
  31. Ardic, N.; Sareyyupoglu, B.; Ozyurt, M.; Haznedaroglu, T.; Ilga, U. Investigation of aminoglycoside modifying enzyme genes in methicillin-resistant staphylococci. Microbiol. Res. 2006, 161, 49–54. [Google Scholar] [CrossRef]
  32. Jefferson, K.K.; Pier, D.B.; Goldmann, D.A.; Pier, G.B. The teicoplanin-associated locus regulator (TcaR) and the intercellular adhesin locus regulator (IcaR) are transcriptional inhibitors of the ica locus in Staphylococcus aureus. J. Bacteriol. 2004, 186, 2449–2456. [Google Scholar] [CrossRef]
  33. Clark, N.C.; Cooksey, R.C.; Hill, B.C.; Swenson, J.M.; Tenover, F.C. Characterization of glycopeptide-resistant enterococci from U.S. hospitals. Antimicrob. Agents Chemother. 1993, 37, 2311–2317. [Google Scholar] [CrossRef]
  34. Feßler, A.; Scott, C.; Kadlec, K.; Ehricht, R.; Monecke, S.; Shwarz, S. Characterization of methicillin-resistant Staphylococcus aureus ST398 from cases of bovine mastitis. J. Antimicrob. Chemother. 2010, 65, 619–25. [Google Scholar] [CrossRef] [PubMed]
  35. Noguchi, N.; Nakaminami, H.; Nishijima, S.; Kurokawa, I.; So, H.; Sasatsu, M. Antimicrobial agent of susceptibilities and antiseptic resistance gene distribution among methicillin-resistant Staphylococcus aureus isolates from patients with impetigo and staphylococcal scalded skin syndrome. J. Clin. Microbiol. 2006, 44, 2119–2125. [Google Scholar] [CrossRef] [PubMed]
  36. Smith, K.; Gemmell, C.G.; Hunter, I.S. The association between biocide tolerance and the presence or absence of qac genes among hospital-acquired and community-acquired MRSA isolates. J. Antimicrob. Chemother. 2008, 61, 78–84. [Google Scholar] [CrossRef] [PubMed]
  37. Grispoldi, L.; Karama, M.; Armani, A.; Hadjicharalambous, C.; Cenci-Goga, B.T. Staphylococcus aureus enterotoxin in food of animal origin and staphylococcal food poisoning risk assessment from farm to table. Ital. J. Anim. Sci. 2021, 20, 677–690. [Google Scholar] [CrossRef]
  38. Titouche, Y.; Houali, K.; Ruiz-Ripa, L.; Vingadassalon, N.; Nia, Y.; Fatihi, A.; Cauquil, A.; Bouchez, P.; Bouhier, L.; Torres, C.; Hennekinne, J.A. Enterotoxin genes and antimicrobial resistance in Staphylococcus aureus isolated from food products in Algeria. J. Appl. Microbiol. 2020, 129, 1043–1052. [Google Scholar] [CrossRef]
  39. Pereira, V.; Lopes, C.; Castro, A.; Silva, J.; Gibbs, P.; Teixeira, P. Characterization for enterotoxin production, virulence factors, and antibiotic susceptibility of Staphylococcus aureus isolates from various foods in Portugal. Food Microbiol. 2009, 26, 278–282. [Google Scholar] [CrossRef]
  40. Khemiri, M.; Abbassi, M.S.; Elghaieb, H.; Zouari, M.; Dhahri, R.; Pomba, C.; Hammami, S. High occurrence of enterotoxigenic isolates and low antibiotic resistance rates of Staphylococcus aureus isolated from raw milk from cows and ewes. Lett. Appl. Microbiol. 2019, 68, 573–579. [Google Scholar] [CrossRef]
  41. Fursova, K.K.; Shchannikova, M.P.; Loskutova, I.V.; Shepelyakovskaya, A.O.; Laman, A.G.; Boutanaev, A.M. , Sokolov, S.L.; Artem'eva, O.A.; Nikanova, D.A.; Zinovieva, N.A.; Brovko, F.A. Exotoxin diversity of Staphylococcus aureus isolated from milk of cows with subclinical mastitis in Central Russia. J. Dairy Sci. 2018, 101, 4325–4331. [Google Scholar] [CrossRef]
  42. Adame-Gómez, R.; Castro-Alarcón, N.; Vences-Velázquez, A.; Toribio-Jiménez, J.; Pérez-Valdespino, A.; Leyva-Vázquez, M.A.; Ramírez-Peralta, A. Genetic diversity and virulence factors of S. aureus isolated from food, humans, and animals. Int. J. Microbiol. 2020, 2020, 1048097. [Google Scholar] [CrossRef]
  43. Papadopoulos, P.; Papadopoulos, T.; Angelidis, A.S.; Kotzamanidis, C.; Zdragas, A.; Papa, A.; Filioussis, G.; Sergelidis, D. Prevalence, antimicrobial susceptibility and characterization of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus isolated from dairy industries in north-central and north-eastern Greece. Int. J. Food Microbiol. 2019, 291, 35–41. [Google Scholar] [CrossRef]
  44. Fisher, E.L.; Otto, M.; Cheung, G.Y. Basis of virulence in enterotoxin-mediated staphylococcal food poisoning. Front. Microbiol. 2018, 9, 436. [Google Scholar] [CrossRef] [PubMed]
  45. Dan, M.; Yehui, W.; Qingling, M.; Jun, Q.; Xingxing, Z.; Shuai, M.; Kuojun, C.; Jinsheng, Z.; Zibing, C.; Zaichao, Z.; Xuepeng, C. Antimicrobial resistance, virulence gene profile and molecular typing of Staphylococcus aureus isolates from dairy cows in Xinjiang Province, northwest China. J. Glob. Antimicrobe. Resist. 2019, 16, 98–104. [Google Scholar] [CrossRef] [PubMed]
  46. Mello, P.L.; Moraes Riboli, D.F.; Pinheiro, L.; de Almeida Martins, L.; Vasconcelos Paiva Brito, M.A.; Ribeiro de Souza da Cunha, M.D.L. Detection of enterotoxigenic potential and determination of clonal profile in Staphylococcus aureus and coagulase-negative staphylococci isolated from bovine subclinical mastitis in different Brazilian states. Toxins. 2016, 8, 104. [Google Scholar] [CrossRef] [PubMed]
  47. Imanishi, I.; Nicolas, A.; Caetano, A.C.B.; Castro, T.L.D.P.; Tartaglia, N.R.; Mariutti, R.; Guédon, E.; Even, S.; Berkova, N.; Arni, R.K.; Seyffert, N.; Azevedo, V.; Nishifuji, K.; Le Loir, Y. Exfoliative toxin E, a new Staphylococcus aureus virulence factor with host-specific activity. Sci. Rep. 2019, 9, 16336. [Google Scholar] [CrossRef]
  48. Kot, B.; Szweda, P.; Frankowska-Maciejewska, A.; Piechota, M.; Wolska, K. Virulence gene profiles in Staphylococcus aureus isolated from cows with subclinical mastitis in eastern Poland. J. Dairy Res. 2016, 83, 228–235. [Google Scholar] [CrossRef]
  49. Gharsa, H.; Chairat, S.; Chaouachi, M.; Ben Yahia, H.; Boudabous, A.; Ben Slama, K. High diversity of genetic lineages and virulence genes of Staphylococcus aureus isolated from dairy products in Tunisia. Ann. Microbiol. 2019, 69, 73–78. [Google Scholar] [CrossRef]
  50. Chenouf, N.S.; Mama, O.M.; Messaï, C.R.; Ruiz-Ripa, L.; Fernández-Fernández, R.; Carvalho, I.; Zitouni, A.; Hakem, A.; Torres, C. Detection of methicillin-resistant coagulase-negative staphylococci and PVL/mecA genes in cefoxitin-susceptible Staphylococcus aureus (t044/ST80) from unpasteurized milk sold in stores in Djelfa, Algeria. J. Dairy Sci. 2021, 104, 2684–2692. [Google Scholar] [CrossRef]
  51. Aydin, A.; Muratoglu, K.; Sudagidan, M.; Bostan, K.; Okuklu, B.; Harsa, S. Prevalence and antibiotic resistance of foodborne Staphylococcus aureus isolates in Turkey. Foodborne Pathog. Dis. 2011, 8, 63–69. [Google Scholar] [CrossRef] [PubMed]
  52. Kotb, E.; Gafer, J. Molecular detection of toxins and disinfectant resistance genes among Staphylococcus aureus isolated from dairy cattle in Egypt. J. Appl. Vet. Sci. 2020, 5, 35–45. [Google Scholar] [CrossRef]
  53. Al-Mebairik, N.F.; El-Kersh, T.A.; Al-Sheikh, Y.A.; Marie, M.A.M. A review of virulence factors, pathogenesis, and antibiotic resistance in Staphylococcus aureus. Rev. Med. Microbil. 2016, 27, 50–56. [Google Scholar] [CrossRef]
  54. World Health Organization (WHO) Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. Feb 27, 2017. Available online: http://www.who.int/medicines/publications/global-priority-list-antibiotic-resistant-bacteria/en/.
  55. Algammal, A.M.; Hetta, H.F.; Elkelish, A.; Alkhalifah, D.H.H.; Hozzein, W.N.; Batiha, G.E.S.; Nahhas, N.E.; Mabrok, M.A. Methicillin-Resistant Staphylococcus aureus (MRSA): one health perspective approach to the bacterium epidemiology, virulence factors, antibiotic-resistance, and zoonotic impact. Infect. Drug Resist. 2020, 3255–3265. [Google Scholar] [CrossRef] [PubMed]
  56. Ganai, A.W.; Kotwal, S.K.; Wani, N.; Malik, M.A.; Jeelani, R.I.Z.W.A.N.; Kour, S.; Zargar, R. Detection of mecA gene of methicillin resistant Staphylococcus aureus by PCR assay from raw milk. Indian J. Anim. Sci. 2016, 86, 508–511. [Google Scholar] [CrossRef]
  57. Riva, A.; Borghi, E.; Cirasola, D.; Colmegna, S.; Borgo, F.; Amato, E.; Pontello, M.M.; Morace, G. Methicillin-resistant Staphylococcus aureus in raw milk: prevalence, SCCmec typing, enterotoxin characterization, and antimicrobial resistance patterns. J. Food Pro. 2015, 78, 1142–1146. [Google Scholar] [CrossRef] [PubMed]
  58. Mahanti, A.; Joardar, S.N.; Bandyopadhyay, S.; Banerjee, J.; Ghosh, S.; Batabyal, K.; Sar, T.K.; Dutta, T.K.; Samanta, I. Characterization of methicillin-resistant and enterotoxins producing Staphylococcus aureus in bovine milk in India. J. Agric. Food Res. 2020, 2, 100017. [Google Scholar] [CrossRef]
  59. Tavsanli, H.; Cibik, R. The prevalence, genetic diversity and antibiotic resistance of Staphylococcus aureus associated with subclinical bovine mastitis in Balikesir, Turkey. Vet. Arh. 2022, 92, 17–25. [Google Scholar] [CrossRef]
  60. Gokmen, M.; Ektik, N.; Cibik, R. The prevalence and antibiotic resistance of methicillin-resistant Staphylococcus aureus (MRSA) in milk and dairy products in Balikesir, Turkey. J. Hellenic Vet. Med.Soc. 2017, 68, 613–620. [Google Scholar] [CrossRef]
  61. Mus, T.E.; Cetinkaya, F.; Soyutemiz, G.E.; Erten, B. Toxigenic genes of coagulase-negative staphylococci and Staphylococcus aureus from milk and dairy. J. Agric. Sci. 2023, 29, 924–932. [Google Scholar] [CrossRef]
  62. Gucukoglu, A.; Cadirci, O.; Gulel, G.T.; Uyanik, T.; Abdullahi, A. Enterotoxin gene content and antibiotic resistance profiles of Staphylococcus aureus isolated from traditional Turkish dairy-based desserts. Fresenius Environ. Bull. 2020, 29, 2073–2080. [Google Scholar]
  63. Silva, A.T.; da Silva, J.G.; Aragão, B.B.; Peixoto, R.M.; Mota, R.A. Occurrence of β-lactam-resistant Staphylococcus aureus in milk from primiparous dairy cows in the northeastern region of Brazil. Trop. Anim. Health Prod. 2020, 52, 2303–2307. [Google Scholar] [CrossRef]
  64. Yang, F.; Wang, Q.; Wang, X.; Wang, L.; Xiao, M.; Li, X.; Luo, J.; Zhang, S.; Li, H. Prevalence of blaZ gene and other virulence genes in penicillin-resistant Staphylococcus aureus isolated from bovine mastitis cases in Gansu, China. Turk. J. Vet. Anim. Sci. 2015, 39, 634–636. [Google Scholar] [CrossRef]
  65. Lucas, A.P.; de Farias, A.R.B.; da Silva, E.C.; Santoro, K.R.; Mendonça, M.; da Silva, E.R. Detection of β-lactamase, blaZ and mecA in penicillin-resistant Staphylococcus aureus isolated from bovine mastitis in Garanhuns, Brazil. Acta Vet. Bras. 2021, 15, 140–145. [Google Scholar] [CrossRef]
  66. Rocha, G.D.; Nogueira, J.F.; Dos Santos, M.V.G.; Boaventura, J.A.; Soares, R.A.N.; de Simoni Gouveia, J.J.; da Costa, M.M.; Gouveia, G.V. Impact of polymorphisms in blaZ, blaR1 and blaI genes and their relationship with β-lactam resistance in S. aureus strains isolated from bovine mastitis. Microb. Pathog. 2022, 165, 105453. [Google Scholar] [CrossRef] [PubMed]
  67. Kreausukon, K.; Fetsch, A.; Kraushaar, B.; Alt, K.; Müller, K.; Krömker, V.; Zessin, K.H.; Käsbohrer, A.; Tenhagen, B.A. Prevalence, antimicrobial resistance, and molecular characterization of methicillin-resistant Staphylococcus aureus from bulk tank milk of dairy herds. J. Dairy Sci. 2012, 95, 4382–4388. [Google Scholar] [CrossRef] [PubMed]
  68. Szczuka, E.; Porada, K.; Wesołowska, M.; Łęska, B. Occurrence and characteristics of Staphylococcus aureus isolated from dairy products. Molecules. 2022, 27, 4649. [Google Scholar] [CrossRef]
  69. Patel, K.; Godden, S.M.; Royster, E.E.; Crooker, B.A.; Johnson, T.J.; Smith, E.A.; Sreevatsan, S. Prevalence, antibiotic resistance, virulence, and genetic diversity of Staphylococcus aureus isolated from bulk tank milk samples of US dairy herds. BMC Genomics. 2021, 22, 367. [Google Scholar] [CrossRef]
  70. Bakthavatchalam, Y.D.; Babu, P.; Munusamy, E.; Dwarakanathan, H.T.; Rupali, P.; Zervos, M.; Victor, P.J.; Veeraraghavan, B. Genomic insights on heterogeneous resistance to vancomycin and teicoplanin in Methicillin-resistant Staphylococcus aureus: A first report from South India. PLoS One. 2019, 14, e0227009. [Google Scholar] [CrossRef]
  71. Rasheed, N.A.; Hussein, N.R. Staphylococcus aureus: an overview of discovery, characteristics, epidemiology, virulence factors and antimicrobial sensitivity. Eur. J. Mol. Clin. Med. 2021, 8, 1160–1183. [Google Scholar]
  72. Kou, X.; Cai, H.; Huang, S.; Ni, Y.; Luo, B.; Qian, H.; Ji, H.; Wang, X. Prevalence and characteristics of Staphylococcus aureus isolated from retail raw milk in Northern Xinjiang, China. Front. Microbiol. 2021, 12, 705947. [Google Scholar] [CrossRef]
  73. Emam, A.; El-Diasty, M.; Abdelkhalek, A. Prevalence of Staphyloсoссus aureus and Streptococcus agalaсtiae isolated from raw milk in Dakahlia Governorate, Egypt. Zagazig Vet. J. 2021, 49, 67–77. [Google Scholar] [CrossRef]
  74. Nhatsave, N.; Garrine, M.; Messa, A., Jr.; Massinga, A.J.; Cossa, A.; Vaz, R.; Ombi, A.; Zimba, T.F.; Alfredo, H.; Mandomando, I.; Tchamo, C. Molecular characterization of Staphylococcus aureus isolated from raw milk samples of dairy cows in Manhiça District, Southern Mozambique. Microorganisms. 2021, 9, 1684. [Google Scholar] [CrossRef]
  75. Ozdemir, F. Antimicrobial resistance, multilocus sequence, and spa typing of Staphylococcus aureus isolated from retail raw meat products. BioMed Res. Int. 2022, 2022, 6035987. [Google Scholar] [CrossRef] [PubMed]
  76. b Zhang, F.; Wu, S.; Lei, T.; Wu, Q.; Zhang, J.; Huang, J.; Dai, J.; Chen, M.; Ding, Y.; Wang, J.; Wei, X.; Zhang, Y. Presence and characterization of methicillin-resistant Staphylococcus aureus co-carrying the multidrug resistance genes cfr and lsa (E) in retail food in China. Int. J. Food Microbiol. 2022, 363, 109512. [Google Scholar] [CrossRef] [PubMed]
  77. Miao, J.; Wang, W.; Xu, W.; Su, J.; Li, L.; Li, B.; Zhang, X.; Xu, Z. The fingerprint mapping and genotyping systems application on methicillin-resistant Staphylococcus aureus. Microb. Pathog. 2018, 125, 246–251. [Google Scholar] [CrossRef] [PubMed]
  78. Araújo, R.D.M.P.; de Moraes Peixoto, R.; Gouveia, G.V.; da Costa, M.M. Virulence factors in Staphylococcus aureus and quality of raw milk from dairy cows in a semiarid region of northeastern Brazil. Acta Sci. Vet. 2017, 45, 1–8. [Google Scholar] [CrossRef]
  79. Ergun, Y.; Cantekin, Z.; Gurturk, K.; Solmaz, H.; Ekin, I.H.; Ozturk, D. Distribution of antiseptic resistance genes in Staphylococcus spp. from bovine mastitis. Vet. Med. 2017, 62, 200–203. [Google Scholar] [CrossRef]
  80. Allaion, J.R.; Barrionuevo, K.G.; Grande Burgos, M.J.; Gálvez, A.; Franco, B.D.G.D.M. Staphylococcus aureus from Minas Artisanal Cheeses: Biocide tolerance, antibiotic resistance and enterotoxin genes. Appl. Sci. 2022, 12, 1019. [Google Scholar] [CrossRef]
  81. Turchi, B.; Bertelloni, F.; Marzoli, F.; Cerri, D.; Tola, S.; Azara, E.; Longheu, C.M.; Tassi, R.; Schiavo, M.; Cilia, G.; Fratini, F. Coagulase negative staphylococci from ovine milk: Genotypic and phenotypic characterization of susceptibility to antibiotics, disinfectants and biofilm production. Small Rumin. Res. 2020, 183, 106030. [Google Scholar] [CrossRef]
  82. Chen, B.; Han, J.; Dai, H.; Jia, P. Biocide-tolerance and antibiotic-resistance in community environments and risk of direct transfers to humans: Unintended consequences of community-wide surface disinfecting during COVID-19. Environ. Pollut. 2021, 283, 117074. [Google Scholar] [CrossRef]
Table 1. The results of the virulence gene profile analysis.
Table 1. The results of the virulence gene profile analysis.
Strain Number (n=28) Virulence Gene
Enterotoxin Producing Gene Toxic Shock Syndrome Toxin-1 Gene and Exfoliative Toxin Genes
2 tst
4
10
18 seo tst
22
23 seo, seu, seq
24 sed, sei, sej, sek, seo, seq
25 seu, seq
26 sei, seo tst
29 sei, seu
43
44 seo
46 sea, sed, sek, sem, seq tst
47 sea, sed, seq
97
100
101 seb, sed, seg, sei, sem, seu
102
112 seb, seg, sei, sem, seo, seu
114 seb, seg, sei, sem, seu,
123 seb, seg, sei, sem, seo, seu
124 seb, seg, sei, sem, seu
131 tst
167 seo
168
190
194 seg, seo tst, eta
218 sed, seg, sem, seo, seu
Total Strain Number 17/28 6/28
Table 2. Distribution of antibiotic-resistant strains of S. aureus.
Table 2. Distribution of antibiotic-resistant strains of S. aureus.
Antibiotic Groups Name of Antibiotics Distribution of S. aureus Strains According to EUCAST
R        S
(%)       (%)
Penicillins Penicillin G 10 µg
Ampicillin 10 µg		 18        10
(%64)     (%35)
18        10
(%64)     (%35)
Aminoglikozid Gentamicin 10µg 1         27
(%3.5)     (%96)
Tobramycin 10µg 2         26
(%7.1)     (%92)
Glycopeptides and Lipoglycopeptides Teicoplanin 30 µg 6         22
(%21)     (%78)
Cephalosporins Ceftaroline 5 µg
Cefoxitin 30 µg		 0        28
(%0)      (%100)
8         20
(%28)     (%71)
Tetracycline Tetracycline 30µg 2        26
(%7.1)    (%92)
Macrolides, lincosamides and streptogramins Erythromycin 15µg 2        26
(%7.1)     (%92)
Fluoroquinolones Levofloxacin 5 µg
Ofloxacin 5 µg
Norfloxacin 10 µg
 6        22
(%21)     (%78)
4         24
(%14)     (%85)
5         23
(%17)     (%82)
Miscellaneous Agents Fusidic Acid 10 µg
Trimethoprim-Sulfamethoxazole (1.25 μg/23.75 μg)		 4        24
(%14)     (%85)
0        28
(%0)     (%100)
Oxazolidinones Linezolid 10 µg 0        28
(%0)     (%100)
Table 3. The results of the antimicrobial resistance gene and antimicrobial susceptibility test.
Table 3. The results of the antimicrobial resistance gene and antimicrobial susceptibility test.
Strain Number (n=28) Antimicrobial Resistance Gene Antimicrobial Susceptibility Test
Disinfectant Resistance Gene Antibiotic Resistance gene
2 BlaI, BlaZ1-2, tcaR, IS256 Penicillin G, Ampilcillin
4 BlaI, BlaZ1-2, tcaR, IS256 Penicillin G, Ampilcillin
10 BlaR, BlaI, BlaZ1-2, BlaZF-R, tcaR, IS256 Ampilcillin,
Cefoxitin, Fusidic Acid
18
22 BlaI, BlaZ1-2, BlaZF-R, tcaR, vanA Penicillin G, Ampilcillin, Levofloxacin, Norfloxacin
23 qacC, qacJ, smr BlaR, BlaI, BlaZ1-2, BlaZF-R, tcaR, vanA, dfrA, ermT, IS256 Penicillin G, Ampilcillin,
Cefoxitin, Erythromycin, Levofloxacin
24 qacJ, smr BlaR, BlaI, BlaZ1-2, BlaZF-R, tcaR, IS256 Penicillin G, Levofloxacin,
Ofloxacin, Norfloxacin
25 BlaI, BlaZ1-2, BlaZF-R, tcaR, dfrA Penicillin G, Levofloxacin,
Ofloxacin, Norfloxacin
26 BlaI, BlaZ1-2, BlaZF-R, ant, tcaR, IS256 Cefoxitin, Fusidic Acid
29 BlaR, BlaI, BlaZ1-2, BlaZF-R, tcaR, IS256 Penicillin G, Ampilcillin, Levofloxacin, Norfloxacin
43 BlaI, BlaZ1-2, BlaZF-R, aac/aph, tcaR, IS256 Penicillin G, Ampilcillin
44 qacJ, smr BlaI, BlaZ1-2, vanA, IS256 Penicillin G, Ampilcillin
46 qacAB, qacJ, smr mecA, BlaR, BlaI, BlaZ1-2, BlaZF-R, tcaR Penicillin G, Ampilcillin, Levofloxacin,
Ofloxacin, Norfloxacin
47 BlaI, BlaZ1-2, BlaZF-R, tcaR Penicillin G, Ampilcillin,
Cefoxitin
97 BlaI, BlaZ1-2, ant, tcaR, IS256 Cefoxitin, Fusidic Acid
100 BlaI, BlaZ1-2, BlaZF-R, tcaR, IS256 Tetracycline
101 qacAB, smr mecA, BlaI, BlaZ1-2, BlaZF-R, tcaR Penicillin G, Ampilcillin, Teicoplanin
102 mecA, BlaR, BlaI, BlaZ1-2, BlaZF-R, tcaR, vanA, tetK, IS256 Penicillin G, Ampilcillin, Teicoplanin, Tetracycline, Erythromycin
112 BlaI, BlaZ1-2, BlaZF-R, tcaR Penicillin G, Ampilcillin
114 BlaI, BlaZ1-2, BlaZF-R, tcaR Penicillin G, Ampilcillin
123 BlaI, BlaZ1-2, BlaZF-R, tcaR Penicillin G, Ampilcillin
124 BlaI, BlaZ1-2, BlaZF-R, tcaR Penicillin G, Ampilcillin, Gentamicin, Tobramycin,
Cefoxitin, Ofloxacin, Fusidic Acid
131 IS257 Teicoplanin, Cefoxitin
167 tcaR, IS256 Teicoplanin
168 IS256 Cefoxitin
190 Teicoplanin
194 qacC, qacJ, smr mecA, tcaR, vanA, IS256 Penicillin G, Ampilcillin, Tobramycin, Teicoplanin
218 Ampilcillin
Total Strain Number 6/28 25/28 27/28
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.
Copyright: This open access article is published under a Creative Commons CC BY 4.0 license, which permit the free download, distribution, and reuse, provided that the author and preprint are cited in any reuse.
Prerpints.org logo

Preprints.org is a free preprint server supported by MDPI in Basel, Switzerland.

facebook logotwitter logolinkedin logo
MDPI Initiatives
Important Links
Subscribe

Disclaimer

Terms of Use

Privacy Policy

© 2024 MDPI (Basel, Switzerland) unless otherwise stated