Abstract
Quaternary ammonium compounds (QACs) are the most significant disinfectants utilised to control the contamination of Staphylococcus aureus in food establishments. S. aureus is a significant pathogen that carries genes responsible for resistance to QACs, which pose a risk to public health and food safety. The objective of the study was to investigate the prevalence of QAC genes (qacA/B, qacC, qacG, qacH, qacJ, and smr) and benzalkonium minimal inhibitory concentration (MIC) values in S. aureus strains isolated from food products and food production facilities (n = 200). The analysis results indicated that the qacC gene was the most frequently detected, with a prevalence of 12%. The qacA/B, qacG, qacH, qacJ, and smr genes were identified at frequencies of 2%, 3%, 1%, 4.5%, and 5%, respectively. The highest MIC level was identified in the surface sample, which carried the qacG gene, at a concentration of 6.25 μg mL−1. The study's results highlight the potential risks associated with disinfectant resistance in food establishments. To prevent the transfer of disinfectant resistance genes, which have become a global risk, it is imperative that the rules of disinfectant usage are observed rigorously and that scientific research in this field is diversified.
1 Introduction
Staphylococcus aureus (S. aureus) is a foodborne pathogen of global concern, classified by the World Health Organization as a high-level priority II pathogen (Gatadi et al., 2019). Since S. aureus is commonly found on the hands, nose, and mucosal membranes, personnel working in the food industry play an essential role as a source of contamination. It has been demonstrated that food handlers who carry S. aureus can contaminate food through hand contact or respiratory secretion (Bencardino and Vitali, 2019).
Quaternary ammonium compounds (QACs) are the most preferred disinfectants in the food industry, particularly in meat and dairy production facilities (Sidhu et al., 2001). The prolonged and high-level use of disinfectants in food production facilities has been observed to result in the development of resistance to these disinfectants amongst many microorganisms. Many studies have demonstrated that the inappropriate use of disinfectants can result in the emergence of cross-resistance to antibiotic compounds, which can subsequently facilitate the spread of antibiotic-resistance genes among microorganisms (Wu et al., 2023). The transfer of resistance genes via a unit transposon can lead to the spread of resistance development among microorganisms, which can present serious public health problems (Tong et al., 2021).
Gram-positive bacteria such as S. aureus are more susceptible to QACs than Gram-negative bacteria. This is because Gram-negative bacteria possess an outer membrane that precludes access of biocides to their cytoplasmic membrane, which is the target site (Boyce, 2023). S. aureus has been observed to exhibit acquired resistance, mainly against QACs. The qac and smr genes are responsible for resistance to QACs, one of the main disinfectants. A total of 6 different plasmids encoded as qac efflux pumps have been identified in Staphylococcus spp., belonging to two main protein families (Tong et al., 2021). The qacA and qacB belong to the Major Facilitator superfamily, while qacC, qacG, qacH, and qacJ belong to the Small Multidrug Resistance (SMR) family (Heir et al., 1999).
This study aimed to investigate the prevalence of qac and smr genes in 120 S. aureus strains isolated from various food products and 80 S. aureus strains isolated from food processing facilities. Furthermore, the efficacy and minimum inhibitory concentration (MIC) values of benzalkonium chloride (BAC), frequently used as disinfectant in food establishments, are among the objectives of this study.
2 Materials and methods
2.1 Sample collection and bacterial isolation
A total of 120 S. aureus strains were isolated from foods (meat and meat products [n = 18], milk and milk products [n = 96], bakery products [n = 3], ready-to-eat foods [n = 3]) collected around Istanbul. In addition to the isolates above, samples were taken from the surfaces in direct contact with food in food processing facilities, personnel, instruments and equipment, and some food in the facilities, and transported to the laboratory in thermobox at +4 °C. The isolation of S. aureus from surface samples was achieved using a swab-rinse technique (Legnani et al., 2004). To achieve this objective, a 5 × 5 cm2 area was sampled using a swab (Lp, Italy).
The isolation of S. aureus from food samples was conducted by the procedures outlined in the International Organization for Standardization (ISO) standard EN ISO 6881-1 (ISO 6888-1:1999). The typical colonies on Baird Parker Agar (BPA, Oxoid, CM275, Basingstoke, UK) were subcultured and identified biochemically by Gram staining, catalase test, latex agglutination test, growth on mannitol (Mannitol Salt Agar; Oxoid CM0085B), and DNase activity (DNase agar; Oxoid, CM0321). The prescribed methodology conducted the DNA isolation procedure (Sudagidan and Aydin, 2009). This was achieved by screening for the presence of three genes (nuc, coa, and spa) by PCR for identification. The previously described PCR conditions and primer sequences were utilised (Hookey et al., 1998; Aires-De-Sousa et al., 2006; Sudagidan and Aydin, 2009).
2.2 Detection of disinfectant resistance genes in S. aureus
The presence of disinfectant resistance genes qacA/B-1, qacA/B-2 (Noguchi et al., 2006), qacA/B-3, qacA/B-4 (Opacic et al., 2010), qacC-1, qacC-2 (Sidhu et al., 2002), qacG-1, qacG-2, qacH-1, qacH- 2, qacJ-F, qacJ-R (Smith et al., 2008), smr-1, smr-2 (Noguchi et al., 2006) and smr-3, smr-4 (Bjorland et al., 2001) was determined by PCR. The PCR products were electrophoresed in a horizontal electrophoresis system using 1xTris-acetate-EDTA (TAE) buffer containing 1.5% (w/v) agarose gel and 5% (v/v) fluorescent DNA dye (SafeView Classic, Applied Biological Materials Inc., Richmond, Canada). Visualisation of the gels was performed by the Infinity Gel Imaging System (Vilber Lourmat, Marne-la-Vallée, France). A total of two independent PCR experiments were performed for each isolate.
2.3 Determination of minimum inhibitory concentration (MIC) values of disinfectants
The efficacy and MIC values of BAC (Zefiran® Forte solution, 10g/100cc, Sandoz, Basel, Switzerland) QAC used as a disinfectant in the food industry, were determined by microdilution testing in 96-well microplates (Corning Costar, 3599, Corning, NY, USA).
Increasing concentrations of disinfectants were prepared as serial dilutions in Cation adjusted-Mueller Hinton Broth (CA-MHB, BD L007475) (0; 0.78; 1.56; 3.125; 6.25; 12.5; 25; 50; 100; 200, and 400 μg mL−1). The overnight growth of S. aureus strains at 37 °C in Tryptone Soya Agar (TSA, Oxoid, CM0131) was adjusted to McFarland 0.5 in 0.9% NaCl using a densitometer (Den-1B, Biosan, Latvia). In duplicate, two hundred microlitres per well and twenty microlitres per well of the McFarland 0.5 bacterial suspension of each concentration were added to the wells. At a concentration of 0%, only bacteria and CA-MHB were utilised as a negative control. The 96-well microplates were incubated at 37 °C for 24 h, after which the absorbance values were measured at 600 nm using a microplate reader (EPOCH, Bio-Tek, USA). The calculations were performed by subtracting the negative control values from the obtained absorbance values (blank subtraction). S. aureus ATCC 25923 strains were used as a reference in this study.
3 Results and discussion
3.1 Detection of disinfectant resistance genes
S. aureus represents a significant pathogen for those engaged in the food industry. A total of 200 isolates identified from foods (n = 120) and food facilities (n = 80) as S. aureus within the study's scope were examined by PCR to determine the presence of genes responsible for disinfectant resistance. In addition to the findings of our study, it has been reported that S. aureus is prevalent in food and food-contact surface samples (including personnel and food production facilities) (Marino et al., 2010; Bencardino and Vitali, 2019). In cases where hygiene and sanitation are inadequate, S. aureus contaminates food and significant public health problems occur because of the consumption of contaminated food. At the same time, the waste of contaminated food that cannot be consumed or the costs associated with its consumption cause significant economic problems.
The results demonstrated that the qacJ gene was the most frequently detected in S. aureus strains isolated from food samples (n = 9). Furthermore, the smr gene and all other studied genes were also positive in S. aureus strains (Table 1). Kroning et al. (2020) detected 9.6% and 3.2% qacA and smr genes in 31 S. aureus samples isolated from milk. In a study conducted with sheep milk, smr/qacC genes were detected at the highest rate in coagulase-negative staphylococci isolates, and the qacA/B gene was not detected (Turchi et al., 2020).
The distribution of disinfectant resistance genes in S. aureus strains (n = 200)
| Genes | Number of food samples with positive strains (n = 120) | Number of positive strains sampled from food production facilities (n = 80) | Total number (n = 200) of positive strains (%) |
| qacA/B-1/2 | 2 | – | 2 (1%) |
| qacA/B-3/4 | 2 | – | 2 (1%) |
| qacC | 3 | 21 | 24 (12%) |
| qacG | 5 | 1 | 6 (3%) |
| qacH | 2 | – | 2 (1%) |
| qacJ | 9 | – | 9 (4.5%) |
| smr-1/2 | 4 | 4 | 8 (4%) |
| smr-3/4 | 4 | 4 | 8 (4%) |
The studies in Türkiye were mainly carried out in milk samples and for the detection of qacA/B and qacC genes. Bayrakal and Aydin (2024) detected qacA/B (7.1%), qacC (7.1%), qacJ (17.8%), and smr (21.4%) genes, and Akin et al. (2020) detected qacA/B (18.8%) and qacC (2.2%) genes in Staphylococcus spp. isolates from milk samples.
The prevalence of disinfectant-resistant genes varies depending on the geographical location (Zaki et al., 2019). Studies for the detection of antimicrobial resistance genes in S. aureus strains have generally detected the qacA/B and qacC genes but not the qacG, qacH, and qacJ genes (Zaki et al., 2019; Kroning et al., 2020). Similarly, in another study, the qacC gene was detected in 3 isolates out of 38 S. aureus samples isolated from dairy products. On the other hand, the qacA/qacB gene was not detected in any isolate (da Silva Abreu et al., 2021). In this study, genes were also detected that had not been detected in the above-mentioned studies, the number of qacA/B, qacC, qacG, qacH, and qacJ positive samples were 4, 24, 6, 2, and 9, respectively. The observed differences in the rates of disinfectant resistance genes may be attributed to the location of the QAC resistance genes in different S. aureus isolates. These genes are typically carried on plasmids, facilitating rapid transmission, whereas in some S. aureus strains, different genes controlling QAC resistance are carried on the chromosome (Zaki et al., 2019). The differing prevalence of the genes identified in our study in comparison to those identified in other studies indicates that the development of resistance is an ongoing process, with geographical variations in the patterns of resistance.
A total of 21 (26.25%) S. aureus strains were found to contain the qacC gene, which originated from food facilities. Furthermore, the presence of smr genes was identified in 10% of the S. aureus strains isolated from contact surfaces. qacA/B, qacH, and qacJ were not identified in any of the samples. Inadequate sanitation procedures for equipment in the food industry contribute to the transmission of microorganisms between personnel and equipment. The transmission of bacteria from personnel to food and from food to consumers represents a significant threat to public health. An investigation of studies conducted in the food industry revealed a high prevalence of contamination by S. aureus (Ye et al., 2012; da Silva Abreu et al., 2021). In a study conducted by Marino et al. (2010), S. aureus isolates were obtained from food processing surfaces (21.1 %) and gloves (46.3 %). Ye et al. (2012) observed that the prevalence rates of qacG, qacH, and qacJ genes in human carriage isolates were low and that they were rarely transferred to food from humans. They reported that staphylococci colonising food animals play a more significant role in the food industry than human strains.
3.2 Determination of minimum inhibitory concentration values of disinfectants
The MIC levels of disinfectants were analysed in S. aureus strains with the detected disinfectant gene. The present study investigated the MIC levels of 15 foodborne and 3 S. aureus strains isolated from food production facilities (Table 2). The benzalkonium MIC level was determined as 3.125 μg mL−1 in 75% (9/12) of the foodborne S. aureus isolates.
The distribution of QAC resistance genes and MIC values of benzalkonium chloride among S. aureus strains
| S. aureus codes | Isolated food types/surfaces | Genes | Benzalkonium chloride MIC (μg mL−1) | |
| Type of food | PY-5 | Local cheese | qacG | 3.125 |
| PY-100C | White cheese | qacA/B, qacG | 1.56 | |
| PY-128D | Local cheese | qacJ | 3.125 | |
| PY-358 | Curd cheese | smr, qacC | 3.125 | |
| PY-368A | Sheep-goat cheese | qacA/B | 1.56 | |
| PY-417A | Curd cheese | qacH, qacJ | 3.125 | |
| S-176A | Raw milk | smr | 3.125 | |
| S-267 | Raw milk | qacH | 1.56 | |
| SE-21D | Beef meat | qacJ | 3.125 | |
| TE-2 | Chicken skin | qacG | 3.125 | |
| Y-5 | Cake | qacC | 3.125 | |
| Y-41 | Semolina halva | smr, qacC | 3.125 | |
| Surfaces | Y-6 | Hand swab | qacG | 6.25 |
| Y-22 | Hand swab | smr, qacC | 3.125 | |
| Y-70 | Surface swab | smr | 3.125 |
The highest benzalkonium MIC level was identified in one surface sample as 6.25 μg mL−1, in which the qacG gene was also detected. In addition, a high level (3.125 μg mL−1) of benzalkonium was identified in two additional surface samples, in which the smr gene was also present.
The efficacy of BAC on S. aureus was evaluated by a disinfectant tolerance test, and MIC values were determined. As there is no standard for MIC values, the data were interpreted by comparison with other studies or by comparison of isolates. Turchi et al. (2020) found that 12/20 (60%) of the isolates had a MIC value ≥2 μg mL−1, and the highest MIC value for BAC was in the qac gene negative isolate. Another study identified a 30% reduction in susceptibility among isolates exhibiting MIC higher than 8 mg L−1 for BAC and documented a strong positive correlation between the presence of qac genes (qacA/B, qacJ, and smr) and elevated BAC MIC levels (Zaki et al., 2019). Our results were similar to the aforementioned studies and showed high MIC sensitivity in samples carrying the disinfectant gene. The data obtained shows the risk in the facilities, and the highest value obtained from the hand swab shows the importance of personnel in contamination in food establishments. As a result of not using appropriate hand sanitisers, not paying attention to their duration of effect, and not cleaning hands properly, sensitivity to sanitisers decreases and resistance increases.
4 Conclusions
The most significant future risk is horizontal gene transfer, which demonstrates that resistance genes can be transmitted between bacteria and humans, animals, and food. The findings of our study indicate that the detection of disinfectant-resistant genes in S. aureus strains isolated from food businesses and foods suggests a potential risk associated with these foods. To prevent the formation and transfer of resistance, it would be prudent to consider the expansion of disinfectants that are sensitive to the environment and living organisms as an alternative to the current disinfectants that are widely used. It is recommended that disinfectants with varying effects be used in combination. It is imperative to prevent the formation of biofilms. Furthermore, it is essential to ensure the appropriate selection of a disinfectant, along with the correct duration and concentration of the chosen agent. It is imperative to conduct comprehensive molecular investigations into gene transfer and resistance mechanisms to avert the advent of more substantial risks in the future.
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