Open Veterinary Journal, (2025), Vol. 15(10): 5175-5182

Research Article

10.5455/OVJ.2025.v15.i10.34

The molecular evidence of toxic shock syndrome toxin-1 on methicillin-resistant Staphylococcus aureus isolated from cats in Surabaya, Indonesia

Daniah Ashri Afnani^1,2, Aswin Rafif Khairullah³, Mustofa Helmi Effendi^4,5*, Wiwiek Tyasningsih⁶, John Yew Huat Tang⁵, Budiastuti Budiastuti⁷, Saifur Rehman⁸, Ikechukwu Benjamin Moses⁹, Dea Anita Ariani Kurniasih¹⁰, Sheila Marty Yanestria¹¹, Riza Zainuddin Ahmad², Siti Hamidatul Aliyah¹², Alfiana Laili Dwi Agustin¹³ and Katty Hendriana Priscilia Riwu¹³

¹Doctoral Program of Veterinary Science, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

²Department of Microbiology and Parasitology, Faculty of Veterinary Medicine, Universitas Pendidikan Mandalika, Mataram, Indonesia

³Research Center for Veterinary Science, National Research and Innovation Agency (BRIN), Bogor, Indonesia

⁴Division of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

⁵School of Food Industry, Faculty of Bioresources, and Food Industry, Universiti Sultan Zainal Abidin, Besut, Malaysia

⁶Division of Veterinary Microbiology, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia

⁷Study Program of Pharmacy Science, Faculty of Health Science, Universitas Muhammadiyah Surabaya, Surabaya, Indonesia

⁸Department of Pathobiology, Faculty of Veterinary and Animal Sciences, Gomal University, Dera Ismail Khan, Pakistan

⁹Department of Applied Microbiology, Faculty of Science, Ebonyi State University, Abakaliki, Nigeria

¹⁰Research Center for Public Health and Nutrition, National Research and Innovation Agency (BRIN), Bogor, Indonesia

¹¹Department of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Wijaya Kusuma Surabaya, Surabaya, Indonesia

¹²Center for Biomedical Research, National Research and Innovation Agency (BRIN), Bogor, Indonesia

¹³Department of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Pendidikan Mandalika, Mataram, Indonesia

*Corresponding Author: Mustofa Helmi Effendi. Division of Veterinary Public Health, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia. Email: mhelmieffendi [at] gmail.com

Submitted: 10/04/2025 Revised: 14/09/2025 Accepted: 21/09/2025 Published: 31/10/2025

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Abstract

Background: Staphylococcus aureus is a significant microorganism in both human and veterinary medicine due to its ability to colonize mucosal surfaces, particularly the nasal cavity, and its potential to cause opportunistic infections in domestic cats. Methicillin-resistant S. aureus (MRSA) is a new strain of S. aureus with multidrug-resistance (MDR) characteristics and is resistant to β-lactam antibiotics. Companion animals are classified as potential reservoirs of MRSA. In this study, 150 cats were brought to veterinary clinics and animal hospitals in Surabaya for routine check-ups or medical treatment. Cats were not selected based on the presence of clinical symptoms, allowing for the identification of both symptomatic and asymptomatic carriers. Swab samples were collected from the nasal cavities using sterile cotton swabs under aseptic conditions. This approach enabled the assessment of MRSA colonization.

Aim: This study aimed to detect the tst gene that produces toxic shock syndrome toxin-1 (TSST-1) from MRSA isolates in cats.

Methods: A total of 150 nose swab samples were collected from cats visiting veterinary clinics and hospitals as pet patients spread over Surabaya, Indonesia. Amies Medium Transport was used to swab the noses of the samples, and the microbiological standard procedure was used for identification. Staphylococcus aureus isolates were subjected to an antibiotic resistance test using the Kirby-Bauer diffusion method by streaking a bacterial suspension according to a 0.5 McFarland standard, then five different antibiotic discs on Mueller-Hinton Agar. Cefoxitin-resistant S. aureus isolates grew on Oxacillin Resistance Screening Agar Base (ORSAB) as a confirmatory test for MRSA.

Results: There were 18 (12%) S. aureus isolates identified and isolated. The antibiotic resistance test results revealed 7 (38.88%) MDR isolates; there were 3 (16.66%) MDR of S. aureus isolates and 4 (22.22%) MDR of S. aureus isolates that were ORSAB positive; there were MDR and MRSA isolates. 4 MRSA isolates (22.22%) were subjected to molecular detection.

Conclusion: Polymerase chain reaction 1 (5.55%) of the MRSA isolate was test gene positive at 143 bp. This indicates that cats may be a potential source of transmission to humans and endanger public health.

Keywords: Antibiotic, Cats, MRSA, Public health, TSST-1.

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Introduction

The popularity of cats as pets is rapidly rising, particularly in metropolitan areas, and domestication and globalization have increased the risk of bacterial transmission between humans and animals (Afnani et al., 2022). The skin and mucous membrane flora of mammals and birds naturally contain Staphylococcus aureus, a common opportunistic infection in humans and veterinary medicine (Khairullah et al., 2019; Khairullah et al., 2024a). Resistance by mutation is the term used to describe how S. aureus develops drug resistance as a result of genetic changes that change the target DNA gyrase or decrease outer membrane proteins (Ramandinianto et al., 2020a; Pinchuk et al., 2010). Bacteria can become drug-resistant by producing high levels of α-lactamase through plasmid-mediated transduction, transformation, and insertion of drug-resistance genes. This is a type of plasmid-mediated resistance (Ramandinianto et al., 2020b).

The primary source of methicillin-resistant S. aureus (MRSA) is drug-resistant gene transmission mediated by plasmids, which can expand the genome and enable the transfer of resistance genes between S. aureus and other bacteria (Waruwu et al., 2023). Human, animal, and environmental antimicrobial resistance (AMR) is increased via intricate interactions between bacterial species from various "environments" (Yunita et al., 2020; Kumar et al., 2016). MRSA is a methicillin-resistant strain of the bacteria that is currently posing a threat to the entire world (Putra et al., 2023). It has spread to the public among people without risk factors for contracting MRSA, posing a fresh danger after becoming well-established in the health care setting (Kaben et al., 2024). The subsequent finding that MRSA can colonize or infect animals and food derived from animals was highly alarming, and more MRSA reservoirs were identified (Tyasningsih et al., 2019).

Close contact between pets and their owners fosters an environment that is conducive to MRSA transmission, and pet owners are more likely to become colonized by MRSA than the general population, suggesting that dogs also operate as reservoirs (Ramadhani et al., 2024). However, several studies found that the majority of MRSA in pets comes from humans, and that close contact with people is associated with a higher risk of S. aureus colonization in pets (Khairullah et al., 2023). High population density, the potential for nosocomial transmission from humans, poor cleaning and disinfection practices, the unknown carrier status of multiple animals, or a stressful environment are some potential reasons for this exposure (Khairullah et al., 2024b).

Staphylococcal infections from animals can be transmitted to humans, and the most common staphylococcal strain in cats is S. aureus because these germs can occasionally flourish on household animals as opportunistic pathogens (Pantosti, 2012; Haag et al., 2019). The virulence factors that drive the host's innate and adaptive immune responses are the main pathophysiology of S. aureus as a pathogen (Cheung et al., 2021; Fazal et al., 2023). The test gene encodes the toxin known as toxic shock syndrome toxin 1 (TSST-1), which is one of the most significant virulence factors of S. aureus (Zheng et al., 2020). A diverse range of virulence factors determines the pathogenicity of S. aureus, with secreted toxins playing a major role. Numerous toxins produced by S. aureus harm biological membranes, causing cell death (Zhang et al., 2017; Rehab et al., 2016). The most well-known S. aureus superantigen, the 22-kD TSST-1, causes toxic shock syndrome (TSS), a disease that causes the release of cytokines such as TNF-, IL-1, and IL-2 (Ortega et al., 2010). Although TSST does not cause emesis, unlike enterotoxin superantigens, TSS is a dangerous and occasionally fatal condition (Otto, 2014; Chen et al., 2023).

MRSA strains have been linked to no instances in Europe, a considerable percentage of TSS cases in Japan, and occasional cases in the United States (Jamart et al., 2005). Historically, TSS was typically linked to menstruation and high-absorbency tampons; however, TSS is more commonly caused by other staphylococcal infections, especially those affecting the skin and soft tissues (Sharma et al., 2019). Staphylococcus aureus toxin-induced fever, rash, desquamation, hypotension, and multiple organ failure are the hallmarks of TSS in both clinical and laboratory settings (Atchade et al., 2024). Because TSST-1-carrying MRSA is difficult to treat, it poses a risk to public health. This study aimed to detect the tst gene that produces TSST-1 in MRSA isolates from cats.

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Materials and Methods

Study design and sample collection

This research was conducted using a cross-sectional study design, which aims to analyze the correlational relationship between potential causes and their associated outcomes. A random sampling technique was employed, in which nasal swabs were collected using Amies transport medium. A total of 150 nasal swabs from both healthy and diseased cats were taken from five regions (North, East, West, Central, and South) of Surabaya, East Java, Indonesia, between May 2022 and July 2023. The required sample size was calculated using the disease prevalence formula described by Martin et al. (1987), expressed as N=4PQ/L². Where N represents the sample size, P is the assumed prevalence, q equals 100% minus P, and L denotes the desired margin of error. Samples were obtained from several laboratories, namely, the Department of Veterinary Microbiology and Mycology, Faculty of Veterinary, Airlangga University, and the Central for Health Laboratory Surabaya.

Bacterial isolates

Amies was used as a medium to collect cat nasal swab samples, which were then kept in an icebox at 4°C. Staphylococcus aureus was isolated using a sterilized Amies cotton swab and streaked on MSA. The bacterial inoculum was cultured on MSA medium at 37°C for 24 hours (Effendi et al., 2019). Gram staining, positive catalase, coagulase, and Voges-Proskauer test results, as well as yellow colonies with yellow zones on MSA media, were used to identify S. aureus (Effendi et al., 2018).

Antibiotic sensitivity and MRSA confirmation tests

Antibiotic sensitivity tests were performed using five different antibiotics disk using Kirby-Bauer diffusion method, there were cefoxitin 5 µg, erythromycin 15 µg, tetracycline 30 µg, ciprofloxacin 5 µg, and chloramphenicol 30 µg. A McFarland equivalent of 0.5 was used to prepare purified cultures in bacterial suspension. The Mueller Hinton Agar (MHA) (Oxoid-CM00337) was streaked with a sterile cotton swab to create the inoculum. Five distinct antibiotic disks were then added, and the mixture was incubated at 35°C for 24 hours. The width of the inhibition zone of the clear region surrounding the antibiotic disk, as specified by the (Clinical and Laboratory Standards Institute, 2020), was measured in millimeters (mm) to determine the results of antibiotic sensitivity tests.

The MRSA confirmation test was performed using several cefoxitin-resistant S. aureus isolates from MHA (Oxoid-CM00337), and the outcomes were then streaked on Oxacillin Resistance Screening Agar Base (ORSAB) (HiMedia M1415) and Oxacillin Resistance Selective Supplement (Supplement, HiMedia FD191) (Decline et al., 2020).

Detection of the tst gene

DNA was extracted from the MRSA isolate using a QIAamp DNA Mini Kit 50 (Qiagen, Germany) according to the manufacturer’s instructions. MRSA isolates were added with 180 µl lysozyme (20 mg/ml) and incubated for 45 minutes at 37°C. Then, the extracted DNA was diluted to 20 µl Proteinase K and 200 µl Buffer kit. 3 µl of DNA solution was used for polymerase chain reaction (PCR) amplification. Pure S. aureus genomic DNA was used as a template for amplification using a Promega GoTag Master Mix in accordance with the manufacturer’s guidelines. PCR amplification was performed using test (F) and test (R) as described in Table 1. The amplification process included an initial denaturation at 95°C for 4 minutes of 1 cycle, denaturation at 95°C for 30 seconds, followed by annealing at 54°C for 30 seconds in 40 cycles, then an extension at 72°C for 1 minutes, and a final extension at 72°C for 1 minute. Following amplification reactions, the PCR products were electrophoretically separated on a 2% agarose gel using Ultrapore Agarose Gel (Invitrogen, USA) and stained with Safe DNA gel stain (Invitrogen, USA) at 100 V for 45 minutes in 10X UltrapureTM TBE buffer (Invitrogen, USA) before being illuminated by a UV transilluminator and visualized.

Table 1. The primers used in this study.

Ethical approval

This research received ethical approval from the Animal Ethics Committee, Faculty of Veterinary Medicine, Wijaya Kusuma University. The approval was issued through the Animal Care and Use Committee (ACUC) with certificate number 217-KKE.

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Results

Isolation and identification of S. aureus strains

A total of 150 nasal swabs from cat samples were analyzed, and 18 (12%) were positive for S. aureus based on morphological culture by the shape, color, and edges of the bacterial colonies (Fig. 1). Gram staining was used as the identification method, and biochemical tests including catalase, coagulase, and Vogues Proskauer were used.

Fig. 1. Staphylococcus aureus colonies in the MSA.

Antibiotic sensitivity and MRSA confirmation tests

From 150 samples, 18 (12%) S. aureus isolates were identified: one resistant to tetracycline, two to tetracycline and erythromycin, and seven classified as multidrug-resistant (MDR). The main MDR patterns were cefoxitin–ciprofloxacin–tetracycline–erythromycin–chloramphenicol (four isolates) and erythromycin–tetracycline–chloramphenicol (three isolates). Seven isolates showed no resistance (Tables 2 and 3).

Table 2. Prevalence results from a regional MRSA screening and the presence of the tst gene in MRSA isolates.

Table 3. Ratio of antibiotic resistance test and region-based MRSA screening from cat nasal swabs (%).

Four S. aureus isolates that were resistant to cefoxitin were classified as presumptive MRSA isolates and then continued to streaked on ORSAB that contained oxacillin resistance selective supplement as a confirmation test of MRSA. The results of the ORSA test showed that the blue color indicated positive and the white color indicated negative confirmation results, as shown in Figure 2. Four presumptive MRSA isolates that tested with ORSA showed all positive results; it can be concluded that they were phenotypically confirmed as MRSA isolates.

Fig. 2. ORSAB as a confirmation test for MRSA. The blue color indicates positive confirmation results, and the white color indicates negative confirmation results.

Detection of the tst gene in MRSA isolates

Four MRSA isolates were further examined molecularly using conventional PCR, and one of four MRSA isolates harbored the tst gene (Fig. 3).

Fig. 3. Gel electrophoresis demonstrating the presence of gen test at a nucleotide length of 143 bp in one of the four MRSA isolates from nasal swabs in cats. Note: K- stands for S. aureus ATCC 25923 as the negative control, K+ stands for positive control, and J7, M7, and E38 are MRSA isolates.

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Discussion

Based on the prevalence results of multidrug resistant S. aureus isolates were found seven (38.88%) in South Surabaya, four of them were MRSA isolates confirmed with ORSAB phenotypically and there was found one MRSA isolate harbored the test gene. Among the many virulence traits of MRSA are toxins called TSST-1, which are encoded by the test gene (Zarei Koosha et al., 2016). These results are consistent with those of prior research conducted in multiple countries, which similarly identified MRSA isolates harboring the test gene. The detection of the test gene in our MRSA isolate is consistent with the findings of several international studies. For example, Zhao et al. (2019) reported that 198 of 208 MRSA isolates (29.88%) carried the test gene. Similarly, Motamedifar et al. (2015) found that 11.66% of MRSA isolates in Iran were positive for tst. In contrast, a study conducted in Japan reported a significantly higher prevalence, with 75% of MRSA isolates carrying the test gene (Nagao et al., 2009). These variations suggest geographical and epidemiological differences in the distribution of tst-positive MRSA (Fisher et al., 2018).

Furthermore, Al Laham et al. (2015) reported a high prevalence of MRSA isolates carrying the test gene in the Gaza region, especially within the CC22 clone ("Gaza strain"), indicating the potential for widespread carriage in community settings. In contrast, our study showed a relatively low prevalence (one isolate) of the test gene among MRSA strains, which may be attributed to differences in sample size, population, or local practices.

Numerous studies have also reported the detection of the test gene in MRSA from animal sources. For instance, Farahmand-Azar et al. (2013) detected test in 19.2% of bovine S. aureus isolates, whereas Bruce et al. (2022) identified it in 2 of 7 MRSA isolates from pets (dogs and cats). These findings, along with the report by Crespo-Piazuelo and Lawlor (2021), support the idea that close human–animal contact can facilitate the zoonotic transmission of MRSA strains harboring virulence genes such as test.

The widespread resistance of S. aureus strains to multiple antibiotics, particularly in hospital settings, remains a major global health concern. Due to its resistance to methicillin and other commonly used antibiotics, MRSA is categorized as a superbug Khairullah et al., 2022). The high rates of AMR observed in our isolates may reflect the overuse or misuse of antibiotics, which has been reported to reduce the effectiveness of treatments and enhance the survival of resistant strains (Mustapha et al., 2014).

Moreover, AMR may influence the expression of virulence factors. The co-occurrence of multidrug resistance and test gene expression, although low in our study, may have serious clinical implications, as TSST-1 is associated with higher mortality rates (Zarei Koosha et al., 2016). TSST-1 acts as a superantigen, leading to excessive T-cell activation and cytokine release, which may result in TSS characterized by rapid fever onset, organ failure, and potentially death (Spaulding et al., 2013; Juwita et al., 2021).

Interestingly, while a high percentage of tst-positive isolates have been reported in some studies, the clinical incidence of TSS remains low. Environmental and host factors significantly influence the expression of tst (Deurenberg et al., 2005; Mrochen et al., 2020). TSST-1 production is affected by temperature, pH, oxygen levels, magnesium concentration, and bacterial density (Yarwood and Schlievert, 2000; Khojasteh et al., 2016; Kulhankova et al., 2018). The optimal conditions for TSST-1 expression include a temperature range of 37°C–40°C, pH 6.5–7, and aerobic conditions (Zschöck et al., 2000).

Historically, TSS was often associated with tampon use in menstruating women, but recent studies have linked it to nonmenstrual cases, particularly skin and soft tissue infections (Schlievert and Davis, 2020). The potential of tampons to absorb magnesium and create an environment conducive to test gene expression highlights the importance of understanding the host-environment interactions that modulate virulence factor production (Tong et al., 2015).

Lastly, the detection of tst-positive MRSA in both humans and animals underscores the importance of the One Health approach to infection control (Nandhini et al., 2022). Close contact with domestic animals, such as cats and dogs, may contribute to the transmission of these strains to humans (Crespo-Piazuelo and Lawlor, 2021; Krakauer and Stiles, 2013). Therefore, proper sanitation and hygiene practices, including routine disinfection, pet hygiene, waste management, and limiting unnecessary contact between animals, are crucial for preventing the spread of MRSA (Haag et al., 2019).

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Conclusion

In conclusion, this study demonstrated that MRSA is a serious worldwide problem; its occurrences are not limited to human health alone, as it can spread from humans to animals in numerous ways. Among the virulence factors carried by MRSA are toxins known as TSST-1. At trace doses, TSST-1 is hazardous to humans, in contrast to most other secreted toxins produced by S. aureus. TSST-1 is impervious to proteolysis, heat, and acid as a superantigen. The TSST-1 superantigen is essential for controlling the host and preserving a colonizing environment. The frequency of TSST-1 from MRSA isolates can be used as a guide to prevent and control MRSA infections and raise public awareness, particularly among high-risk groups including pet owners, paramedics, and veterinarians. More investigation is required to identify the additional MRSA virulence factors than TSST-1.

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Acknowledgments

The authors would like to thank the Faculty of Veterinary Medicine, Universitas Airlangga.

Conflict of interest

The authors declare no conflict of interest.

Funding

This study was partly supported by The International Research Consortium, Lembaga Penelitian dan Pengabdian Masyarakat, Universitas Airlangga, Surabaya, Indonesia, in 2024 (Grant number: 171/UN3.LPPM/PT.01.03/2024).

Author’s contributions

DAA, WT, and MHE: Conceived, designed, and coordinated the study. JYHT, SMY, and BB: Designed data collection tools, supervised the field sample and data collection, and performed laboratory work and data entry. ALDA, IBM, and KHPR: Validation, supervision, and formal analysis. RZA and DAAK: Contributed reagents, materials, and analysis tools. SHA, SR, and ARK: Carried out the statistical analysis and interpretation and participated in the preparation of the manuscript. All authors have read, reviewed, and approved the final version of the manuscript.

Data availability

All data are available in the revised manuscript.

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