Emerging Biocide Resistance in Staphylococcus aureus: Cross-Source Insights and Public Health Implications

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This study aimed to investigate the susceptibility of 553 S. aureus isolates collected from patients, healthy carriers, retail foods, and wastewater in Ardabil, Iran, to common antimicrobial biocides, including triclosan (TCS), chlorhexidine digluconate (CHDG), benzalkonium chloride (BZC), and formaldehyde (FOR). Additionally, the presence of Biocide Tolerance-Associated (BTA) genes was examined. Methods. The Minimum Inhibitory Concentration (MIC) of biocides was determined using the agar dilution technique. Concurrently, a polymerase chain reaction (PCR) was used to detect the BTA genes. Results. In the current study, MIC 90 for TCS, CHDG, BZC, and FOR were 1 µg/ml, 1 µg/ml, 2 µg/ml, and 128 µg/ml, respectively, for total isolates. The MIC 90 for TCS, CHDG, and BZC in clinical isolates was double the MIC 90 of total isolates. The BTA genes, norA , mdeA , mepA , sepA , qacA/B , qacC , qacD , qacG , and qacJ , were detected in 24.05%, 21.33%, 30.22%, 8.49%, 18.08%, 10.12%, 10.48%, 12.29%, and 4.15% of isolates, respectively. Notably, the sepA, qacA/B, qacC , and qacJ genes were associated with higher MIC 90 values with CHDG. Additionally, the presence of nor A , mdeA , mepA , sepA , qacA/B , qacC , qacD , qacG , and qacJ genes was associated with increased MIC 90 levels for BZC compared to isolates devoid of these genes (P < 0.05). Conclusion. According to the study, S. aureus has elevated MICs for popular antimicrobial biocides. The most effective compounds are TCS and CHDG, and their presence in personal care products can help control these organisms. Staphylococcus aureus Biocide tolerance-associated genes Triclosan Chlorhexidine-digluconate Benzalkonium chloride formaldehyde Figures Figure 1 Background Staphylococcus aureus is a Gram-positive coccus bacterium found in the human microbiota, hospitals, communities, and wastewater ( 1 – 4 ). It adapts to diverse conditions and forms biofilms that protect it from environmental stresses and antibiotics. The bacterium transmits through direct and indirect contact with infected individuals and contaminated surfaces. It spreads via food, animals, and wastewater ( 2 , 5 ). S. aureus causes infections ranging from minor skin issues to severe diseases and is a significant source of hospital-acquired infections ( 6 ). It has developed resistance to many antibiotics, including methicillin, resulting in multidrug-resistant MRSA strains that complicate treatment ( 3 ). S. aureus can also develop resistance to antimicrobial biocides, further complicating treatment efforts and infection control measures ( 7 ). Despite tighter regulations in Europe and the United States, the global use of antimicrobial biocides is continuing to rise across various applications. In hospitals, businesses, and homes, antimicrobial biocides are extensively used as preservatives, antiseptics, and disinfectants against multiple pathogens ( 8 ). Antimicrobial biocides are frequently employed to eliminate bacteria from many surfaces, including medical and laboratory devices ( 9 ). Additionally, these compounds in the food industry play a vital role in protecting public health and ensuring the safety of food products. These processes help prevent microbial contamination, extend the shelf life of food items, and control cross-contamination ( 10 ). In slaughterhouses, disinfecting equipment and the environment are crucial for reducing the risk of transmitting zoonotic diseases ( 11 ). Triclosan (TCS), chlorhexidine digluconate (CHDG), benzalkonium chloride (BZC), and formaldehyde (FOR) are among the most widely used antimicrobial biocides worldwide ( 8 ). FOR, an aldehyde compound, is highly toxic to microorganisms and widely used as a sterilizing disinfectant. It exhibits strong reactivity with amines and thiols, which makes it particularly prone to damaging various compounds, such as proteins, nucleic acids, and metabolites ( 12 ). TCS, a phenol compound, has been utilized in consumer products since 1968 as an antiseptic, disinfectant, and preservative in clinical environments. It can be found in various consumer items, including cosmetics, household cleaning products, and toys. Additionally, TCS has been applied to the surfaces of medical devices, plastics, textiles, and kitchen utensils. It is included in a positive list of preservatives approved for cosmetic use and exhibits broad-spectrum antimicrobial activity by inhibiting the enoyl-acyl-carrier-protein (ACP) reductase enzyme, which plays a key role in the biosynthesis of bacterial fatty acids ( 13 ). CHDG, as a biguanide, is a potent antiseptic used in hospitals for hand hygiene, cleaning surgical areas, and sterilizing instruments in various medical fields. It is effective against bacteria, particularly Gram-positive agents. The active ingredient, chlorhexidine, is a cationic bisbiguanide that disrupts microbial cell membranes, leading to the loss of intracellular contents and osmotic imbalance, thereby exhibiting its antimicrobial action ( 14 ). BZC, a class of quaternary ammonium compounds, is commonly identified as an active ingredient in disinfectants utilized in residential, commercial, agricultural, and healthcare settings. Benzalkonium chloride interacts with cell membranes or transmembrane proteins by embedding its hydrophobic alkyl tail into the hydrophobic core of the membrane, leading to membrane destabilization ( 15 ). A significant concern regarding the widespread use of biocides is the progressive bacterial resistance to these products and unrelated antimicrobials, including antibiotics. This issue undermines their effectiveness as antibacterial agents ( 16 ). Moreover, several outbreaks of nosocomial infections linked to biocide contamination have been documented ( 17 ), underscoring the need for careful management and monitoring of biocide use ( 18 ). Repeated exposure to antimicrobial biocides exerts selection pressure on bacteria, leading to reduced sensitivity ( 19 – 21 ). A primary resistance strategy employed by bacteria is the use of efflux pumps, which reduce biocide concentrations to levels that are not harmful. Research has demonstrated that these efflux mechanisms play a significant role in conferring high-level resistance to various substances, including bisphenol compounds (such as TCS), especially in environments with extensive antimicrobial usage, like those containing biguanides (such as CHDG) and quaternary ammonium compounds (such as BZC) ( 22 , 23 ). Studies have identified numerous efflux genes in resistant bacterial isolates, including seven distinct efflux systems in S. aureus . Most of these efflux pumps are encoded on the chromosome (NorA, NorB, NorC, MdeA, SepA, MepA, and SdrM), while some resistance genes are located on plasmids ( qacA/B , qacC , qacD , qacG , and qacJ ). Efflux mechanisms are particularly effective in enabling bacteria to withstand exposure to biocides. Investigating these resistance mechanisms is essential for developing new strategies to address the declining effectiveness of antimicrobial biocides ( 22 ). This study aimed to evaluate the susceptibility of 553 S. aureus isolates from various sources, including clinical samples, healthy carriers, wastewater, and food sources in Ardabil, northwest Iran, to common antimicrobial biocides such as BZC, CHDG, TCS, and FOR. It highlights the limited information in Iran regarding biocide resistance in S. aureus from diverse sources and emphasizes the need for comprehensive research. The primary objective was to investigate resistance patterns and the prevalence of associated genes, such as norA , mdeA , mepA , sepA , qacA/B , qacC , qacD , qacG , and qacJ . The diverse sources and large sample size provide a thorough understanding of the bacterium's status in the region. The findings can inform policymakers and public health officials in designing effective infection control programs and raising awareness about hygiene practices. However, further research is needed to explore the effectiveness of current biocide treatment regimens in controlling microbial contamination across different settings. Results FOR MIC distribution The MIC for FOR in all S aureus isolates was established at 128 µg/ml. Consequently, the MIC 50 and MIC 90 values were also determined to be 128 µg/ml. TCS MIC distribution Table 1 displays the MIC results for TCS in S. aureus isolates. In this study, the MIC for TCS ranged from 0.25 to 8 µg/ml. The MIC 50 and MIC 90 values were 0.25 and 1 µg/ml, respectively. According to the MIC 90 value, isolates from clinical specimens exhibited the highest resistance to TCS, with a 2 µg/ml. CHDG MIC distribution Table 2 shows the MIC results for CHDG in S. aureus isolates, categorized by the sources of bacterial isolation. The MIC for CHDG in isolates ranged from 0.5 to 4 µg/ml. The MIC 50 and MIC 90 values were 0.5 and 1 µg/ml, respectively. Based on the MIC 90 value, isolates from clinical specimens exhibited the highest resistance to CHDG, with a value of 2 µg/ml. B Z C MIC distribution Table 3 displays the MIC results for BZC in S. aureus isolates, categorized by the source of bacterial isolation. The MIC for BZC isolates ranged from 0.25 to 8 µg/ml. The MIC 50 and MIC 90 values were 1 and 2 µg/ml, respectively. Based on the MIC 90 value, S. aureus isolates from clinical specimens exhibited the highest resistance to BZC, with a value of 4 µg/ml. Frequency distribution of BTA genes Table 4 illustrates the frequency distribution of BTA genes among S. aureus isolates. The results indicate that the prevalence of the norA, mdeA, mepA, sepA, qacA/B, qacC, qacD, qacG, and qacJ genes was 24.05%, 21.33%, 30.22%, 8.49%, 18.08%, 10.12%, 10.48%, 12.29%, and 4.15%, respectively. Association between BTA genes and biocide's MIC As illustrated in Fig. 1 , isolates harboring the sepA , qacA/B , qacC , and qacJ genes were associated with higher MIC 90 for CHDG than isolates without these genes. Overall, isolates carrying the norA , mdeA , mepA , sepA , qacA/B , qacC , qacD , qacG , and qacJ genes exhibited a higher MIC 90 for BZC than those lacking these genes. Table 1 Distribution of MIC values for TCS in S. aureus isolates Source MIC (µg/ml) 0.25 0.5 1 2 4 8 MIC 50 MIC 90 Clinical N = 93 n (%) 17 (18.27) 23 (24.73) 41 (44.08) 8 (8.6) 3 (3.22) 1 (1.07) 1 2 Healthy carriers N = 310 n (%) 304 (98.06) 0 (0.0) 3 (0.96) 3 (0.96) 0 (0) 0 (0) 0.25 0.25 Wastewaters N = 50 n (%) 50 (100) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0.25 0.25 Foodstuffs N = 100 n (%) 81 (81) 12 ( 12 ) 4 ( 4 ) 3 (0) 0 (0) 0 (0) 0.25 1 Total N = 553 n (%) 452 (81.73) 35 (6.32) 48 (8.67) 14 (2.53) 3 (0.54) 1 (0.18) 0.25 1 Table 2 CHDG MIC distribution among S. aureus isolates Source MIC (µg/ml) 0.5 1 2 4 MIC 50 MIC 90 Clinical N = 93 n (%) 40 (43.01) 43 (46.23) 6 (6.45) 4 (4.3) 1 2 Healthy carriers N = 310 n (%) 234 (75.48) 76 (24.51) 0 (0) 0 (0) 0. 5 1 Wastewater N = 50 n (%) 30 (60) 20 ( 40 ) 0 (0) 0 (0) 0. 5 1 Foodstuffs N = 100 n (%) 55 (55) 45 ( 45 ) 0 (0) 0 (0) 0. 5 1 Total N = 553 n (%) 359 (64.91) 184 (33.27) 6 (1.08) 4 (0.72) 0.5 1 Table 3 MIC values of BZC across S. aureus isolates Source MIC (µg/ml) 0.25 0.5 1 2 4 8 MIC 50 MIC 90 Clinical N = 93 n (%) 23 (24.73) 11 (11.82) 24 (25.8) 21 (22.58) 12 (12.9) 2 (2.15) 1 4 Healthy carriers N = 310 n (%) 3 (0.98) 19 (6.12) 252 (81.29) 30 (9.67) 6 (1.93) 0 (0) 1 2 wastewater N = 50 n (%) 6 ( 12 ) 5 ( 10 ) 39 (78) 0 (0) 0 (0) 0 (0) 1 1 Foodstuffs N = 100 n (%) 10 ( 10 ) 9 ( 9 ) 75 (75) 6 ( 6 ) 0 (0) 0 (0) 1 1 Total N = 553 n (%) 42 (7.59) 44 (7.95) 390 (70.52) 57 (10.3) 18 (3.25) 2 (0.36) 1 2 Table 4 Prevalence of BTA genes in S. aureus isolates Source BTA genes norA MepA MdeA SepA qacA/B qacC qacD qacG qacJ Clinical N = 93 n (%) 43 (46.23) 31 (33.33) 31 (33.33) 20 (21.5) 40 (43.01) 26 (27.95) 27 (29.03) 24 (25.8) 10 (10.75) Healthy carriers N = 310 n (%) 34 (10.96) 35 (11.29) 35 (11.29) 17 (5.48) 29 (9.35) 15 (4.83) 17 (5.48) 26 (8.38) 6 (1.93) Wastewater N = 50 n (%) 20 ( 40 ) 14 ( 28 ) 19 ( 38 ) 4 ( 8 ) 19 ( 38 ) 7 ( 14 ) 7 ( 14 ) 9 ( 18 ) 2 ( 4 ) Foodstuffs N = 100 n (%) 36 ( 36 ) 43 ( 43 ) 33 ( 33 ) 6 ( 6 ) 12 ( 12 ) 8 ( 8 ) 7 ( 7 ) 9 ( 9 ) 5 ( 5 ) Total N = 553 n (%) 133 (24.05) 123 (22.24) 118 (21.33) 47 (8.49) 100 (18.08) 56 (10.12) 58 (10.48) 68 (12.29) 23 (4.15) Discussion Biocides are utilized globally in hospital and non-hospital settings as antiseptics, disinfectants, and preservatives ( 8 ). Evidences suggest that continuous exposure to antimicrobial biocides, combined with their widespread use driven by selective pressure, contributes to increased resistance among microorganisms to these compounds ( 7 , 16 ). Unlike antibiotics, data on global bacterial sensitivity patterns to biocides are scarce. Our study offers a comprehensive understanding of biocide resistance in S. aureus isolates from clinical samples and various ecological niches in Iran. TCS is one of the most commonly used antimicrobial agents in personal hygiene products, including soaps, toothpaste, and particular dairy items ( 13 ). In our study, TCS MICs ranged from 0.25 to 8 µg/ml, with MIC 50 and MIC 90 values of 0.25 and 1 µg/ml, respectively. Clinical isolates demonstrated the highest resistance, exhibiting an MIC90 value of 2 µg/ml. In comparison, previous research reported MIC 50 and MIC 90 values of 0.12 µg/ml and 0.25 µg/ml, respectively, for S. aureus , suggesting a higher susceptibility than observed in our study ( 29 ). For Staphylococcus epidermidis , the MIC 50 was 0.12 µg/ml, and the MIC 90 was 8 µg/ml, indicating a broader range and higher resistance in specific isolates ( 29 ). For Enterococcus species, the MIC 90 values were 0.125 µg/ml for Enterococcus faecalis and 32 µg/ml for Enterococcus faecium , reflecting significant variability in susceptibility and resistance ( 21 , 30 ). Frequent exposure to biocides creates selective pressure on bacterial isolates, promoting the development of resistance. In studies involving Escherichia coli , clinical isolates exhibited a notable increase in MIC after adaptation to TCS, with some isolates reaching MICs as high as 32 µg/ml ( 19 , 21 ), suggesting the potential for enhanced resistance development. Similarly, research by Stickler et al . reported that Proteus mirabilis had initial MICs of 0.2 µg/ml, with mutants demonstrating MICs of up to 80 µg/ml after TCS exposure, highlighting substantial resistance evolution ( 20 ). These findings may explain the elevated MICs observed in our clinical isolates, as TCS is frequently used in hospital settings. The antibacterial activity of TCS, an anionic and lipophilic compound, is primarily derived from its inhibition of enoyl-acyl-carrier protein (ACP) reductase, a crucial enzyme involved in the elongation cycles of type II fatty acid synthase systems and an essential target for antibacterial agents ( 27 , 31 ). Resistance to TCS can arise from increased expression of the fabI gene or the acquisition of an additional fabI allele via horizontal gene transfer from Staphylococcus haemolyticus ( 32 ). In our study, the expression of fabI was not evaluated, and the presence of efflux pumps was not linked to the observed higher MIC 90 value. The MICs for CHDG in bacterial isolates can vary significantly across different studies and bacterial species. In our study, the MIC for CHDG ranged from 0.5 to 4 µg/ml, with MIC 50 and MIC 90 values of 0.5 and 1 µg/ml, respectively. The highest resistance observed was an MIC 90 value of 2 µg/ml in clinical specimens. Research on various Gram-negative and Gram-positive isolates showed a wide range of MIC values, with Gram-negative isolates generally exhibiting higher MICs (2–256 µg/ml) compared to Gram-positive isolates (2–32 µg/ml) ( 33 ). Multidrug-resistant organisms, including New Delhi metallo-beta-lactamase-1 positive Enterobacteriaceae spp., exhibited higher MICs for CHDG compared to their susceptible counterparts, with MIC values for commercial disinfectants being 20–600 times higher than standard preparations ( 34 ). Another study on Acinetobacter baumannii reported MIC 90 values of 64 µg/ml for CHDG, indicating a higher level of resistance than our findings ( 35 ). In a study of methicillin-susceptible and resistant S. aureus , it has been found that 10% of isolates had elevated MICs for CHDG, with some isolates showing higher MIC values, particularly ST22-MRSA-IV ( 7 ). Another study reported that some S. aureus isolates required higher concentrations or prolonged treatment times to achieve bactericidal effects, indicating variability in susceptibility ( 36 ). Our findings of CHDG MICs ranging from 0.5 to 4 µg/ml with MIC 50 and MIC 90 values of 0.5 and 1 µg/ml, respectively, align with some studies but show lower resistance levels than others, particularly those involving multidrug-resistant organisms. The variability in MIC values across different studies highlights the importance of continuous monitoring and tailored approaches to CHDG use in clinical settings. A survey on Klebsiella pneumoniae found that chlorhexidine-resistant strains had MIC values significantly reduced in the presence of an efflux pump inhibitor, suggesting that efflux mechanisms contribute to resistance ( 37 , 38 ). Consistently, in our study, the efflux pump encoding genes sepA, qacA/B, qacC , and qacJ were associated with higher MIC 90 for CHDG than isolates without these genes. BZC is the most widely employed active ingredient in disinfectants used across residential, commercial, agricultural, and clinical environments ( 39 ). Its extensive use—often leading to the release of residual biocides—can impose selective pressure on microbial populations, thereby facilitating the emergence of chemical resistance ( 40 ). In our investigation, the MICs of BZC against S. aureus isolates ranged from 0.25 to 8 µg/ml, with MIC₅₀ and MIC₉₀ values of 1 and 2 µg/ml, respectively. The highest resistance level observed was 4 µg/ml, identified in clinical S. aureus strains. These results are consistent with prior research. Taheri et al . (2016) reported BZC MICs ranging from 0.25 to 8 µg/ml for various Staphylococcus isolates, including both methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains ( 41 ). Similarly, Aykan et al . (2013) documented MICs ranging from 2 to 8 µg/ml for MRSA isolates from Turkey ( 42 ). Likewise, Shamsudin et al. (2012) observed lower MICs between 0.5 and 2 µg/ml in MRSA isolates from Malaysia ( 43 ). The highest resistance level in our study (4 µg/ml) is also comparable to findings by Narui et al. (2007), who noted that certain MRSA strains exhibited inhibitory concentrations exceeding typical BZC usage levels, indicating potential resistance ( 44 ). Previous studies have identified the qacA/B efflux pump genes, which are linked to reduced susceptibility to BZC, in a significant proportion of MRSA isolates. These genes were consistently associated with elevated MIC values ( 41 – 43 ). Our study supported these findings by revealing that isolates harboring efflux pump–encoding genes—including norA , mdeA , mepA , sepA , qacA/B , qacC , qacD , qacG , and qacJ —exhibited markedly higher MIC₉₀ values compared to isolates lacking these resistance determinants. The FOR is an organic electrophilic biocide whose mechanism of action involves the cross-linking of macromolecules (proteins, RNA, and DNA) ( 9 ). FOR has been historically and contemporarily employed in diverse settings, including hospital sterilization, laboratory decontamination, biological specimen preservation, and industrial and wastewater treatment. While its bactericidal properties are well established, precise inhibitory concentrations remain inconsistently reported in the literature ( 9 ). Our results indicated that the MIC 90 values of FOR equaled 128 µg/ml for all S. aureus isolates. Other studies reported the MIC 90 values as 512, 64, and 512 µg/ml for Enterococcus spp., E. coli , and Pseudomonas aeruginosa ( 27 , 45 , 46 ). Conclusion Overall, MIC levels increased for all antimicrobial biocides against S. aureus isolates. TCS was the most effective agent in this study. Additionally, BTA genes were identified among the isolates. To reduce the development and spread of biocide resistance, we recommend cautiously using biocides and continuously monitoring their antimicrobial activity. Materials and Methods Bacterial isolates A total of 553 S. aureus isolates were included in the study. The authors collected these isolates in previous research from various sources: clinical specimens (n = 93) ( 24 ), healthy carriers (n = 310) ( 25 ), wastewaters (n = 50) ( 25 ), and foodstuffs (n = 100) ( 5 ), between 2018 and 2022 in Ardabil City, Northwest Iran. The isolates were identified using a series of standard microbiological assays, including catalase, tube coagulase, DNase tests, and cultivation on mannitol salt agar. Confirmation was performed by amplifying the nuc gene through polymerase chain reaction (PCR), as described in a previous study ( 24 ). Subsequently, the isolates were preserved in tryptic soy broth (TSB) with 15% glycerol and stored at -70°C for future use. Biocide susceptibility testing The minimum inhibitory concentrations (MICs) of biocides, including FOR (Acros Organics USA), TCS (Bio Basic, Canada), CHDG (Sigma-Aldrich, USA), and BZC (Sigma-Aldrich, USA), were determined utilizing the agar dilution method. This procedure was conducted following the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) ( 26 ) and as elaborated upon in the authors’ preceding publication ( 27 ). Detection of BTA genes Genomic DNA was isolated using the thermal lysis method ( 28 ). PCR testing assessed the presence of BTA genes, including norA , mdeA , mepA , sepA , qacA/B , qacC , qacD , qacG , and qacJ , which contribute to antimicrobial compound resistance in S. aureus . This analysis employed gene-specific primers (designed in this study; Supplementary material, Table S1 ) and adhered to the cycling and temperature parameters outlined in Table S2 . Statistical analysis The findings were illustrated using tables, graphical representations, and quantitative metrics. Abbreviations MRSA Methicillin-resistant Staphylococcus aureus TCS Triclosan CHDG Chlorhexidine digluconate BZC Benzalkonium chloride FOR Formaldehyde ACP Acyl-carrier-protein MIC Minimum Inhibitory Concentration BTA Biocide Tolerance-Associated PCR Polymerase Chain Reaction CLSI Clinical and Laboratory Standards Institute TSB Tryptic Soy Broth Declarations Acknowledgments The authors would like to express their sincere gratitude to the Vice Chancellor for Research and Technology, Ardabil University of Medical Sciences for funding and support. Author Contributions MN: conducted the experiments and prepared the initial draft of the manuscript. EG, MM, MRO, AYJ: also contributed to the experimental work. KNK and MAV: made substantial contributions to the formal data analysis. MA: played a key role in the conception and design of the study and was actively involved in the critical review and refinement of the manuscript. KNK: was instrumental in conceptualizing and designing the study and providing overall leadership throughout the project. All authors have thoroughly reviewed and approved the final version of the manuscript. Funding This study was financially supported by Ardabil University of Medical Sciences, Iran (Grant No. 401000185). Data Availability Statement The authors confirm that the data supporting the findings of this study are available upon reasonable request from the corresponding author. Ethics approval and consent to participate The study was approved by the Ethics Committee of Ardabil University of Medical Sciences, Iran (Registration No. IR.ARUMS.REC.1401.223). All methods followed relevant guidelines and regulations under the Helsinki Declaration ( https://www.wma.net/policies-post/wma-declaration-of-helsinki/). Clinical isolates were collected from the hospital’s bacterial repository solely for research purposes, and no patient samples or patient data were used in this study. Consequently, the requirement for informed consent from participants was waived by the Regional Research Ethics Committee of Ardabil University of Medical Sciences. Samples from healthy carriers (12 - 14 years old) were collected after informed consent was received from their respective parents or legal guardians. Clinical trial number Not applicable. Consent for publication Not applicable. Conflict of interest The authors declared no conflict of interest. Consent for Publication Not applicable. Declaration of AI Use Artificial intelligence (AI) tools were used to assist with language refinement, editorial clarity, formatting, and literature review during manuscript preparation. Specifically, Microsoft Copilot was employed to enhance sentence structure, improve scientific phrasing, and ensure consistency in terminology. All intellectual content, data interpretation, and scientific conclusions were developed solely by the authors. 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Antimicrob Agents Chemother. 2004;48(4):1397–9. Gorski DB, Vlainić J, Škrlec I, Novak S, Novosel Ž, Biloglav Z, et al. Virulence Factors and Susceptibility to Ciprofloxacin, Vancomycin, Triclosan, and Chlorhexidine among Enterococci from Clinical Specimens, Food, and Wastewater. Microorganisms. 2024;12(9):1808. Heath RJ, Li J, Roland GE, Rock CO. Inhibition of the staphylococcus aureusNADPH-dependent enoyl-acyl carrier protein reductase by triclosan and hexachlorophene. J Biol Chem. 2000;275(7):4654–9. Dittmann K, Schmidt T, Müller G, Cuny C, Holtfreter S, Troitzsch D, et al. Susceptibility of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) to chlorhexidine digluconate, octenidine dihydrochloride, polyhexanide, PVP-iodine and triclosan in comparison to hospital-acquired MRSA (HA-MRSA) and community-aquired MRSA (CA-MRSA): a standardized comparison. Antimicrob Resist Infect Control. 2019;8(1):122. Marchi AP, Farrel Côrtes M, Vásconez Noguera S, Rossi F, Levin AS, Figueiredo Costa S, et al. Chlorhexidine susceptibility and Eagle effect in planktonic cells and biofilm of nosocomial isolates. Eur J Clin Microbiol Infect Dis. 2023;42(6):787–92. Mal P, Farooqi J, Irfan S, Hughes M, Khan E. Reduced susceptibility to chlorhexidine disinfectant among New Delhi metallo-beta-lactamase-1 positive Enterobacteriaceae and other multidrug-resistant organisms: Report from a tertiary care hospital in Karachi, Pakistan. Indian J Med Microbiol. 2016;34(3):346–9. Namaki Kheljan M, Hassanzadeh M, Srdari Jabedar M, Mohammadi Gollou A, Ashouri P, Teimourpour R, et al. Characterization of disinfectant susceptibility profiles among clinical isolates of Acinetobacter baumannii in Ardabil, Iran. Acta Microbiol Immunol Hung. 2023;70(4):311–7. Kanazawa K, Ueda Y. Bactericial activity of chlorhexidine gluconate against recent clinical isolates of various bacterial species in Japan. Jpn J Antibiot. 2004;57(5):449–64. ZHANG Y, ZHANG X, ZHANG S, ZHAO Y, DONG G, ZHOU X et al. Resistance mechanisms and molecular epidemiology of chlorhexidine acetate-resistant Klebsiella pneu-moniae clinical isolates. Chin J Microbiol Immunol. 2019:202–7. Kheljan MN, Hasanzadeh M, Neyestani Z, Jafarizare MA, Nejati-Koshki K, Vostakoli MA. Investigating the Frequency of Genotypic and Phenotypic Resistance to Plasmid-Mediated Ciprofloxacin in Clinical Isolates of Klebsiellapneumoniae in Ardabil. Arch Clin Infect Dis. 2025;20(3). Kampf G, Kampf G. Glutaraldehyde. Antiseptic Stewardship: Biocide Resistance and Clinical Implications. New York: Springer; 2018. Merchel Piovesan Pereira B, Tagkopoulos I. Benzalkonium chlorides: uses, regulatory status, and microbial resistance. Appl Environ Microbiol. 2019;85(13):e00377–19. Taheri N, Ardebili A, Amouzandeh-Nobaveh A, Ghaznavi-Rad E. Frequency of antiseptic resistance among Staphylococcus aureus and coagulase-negative staphylococci isolated from a university hospital in central Iran. Oman Med J. 2016;31(6):426. Aykan SB, Cağlar K, Engin ED, Sipahi AB, Sultan N, Yalınay Çırak M. Investigation of the presence of disinfectant resistance genes qacA/B in nosocomial methicillin-resistant Staphylococcus Aureus isolates and evaluation of their in vitro disinfectant susceptibilities. Mikrobiyol Bul. 2013;47(1):1–10. Shamsudin M, Alreshidi M, Hamat R, Alshrari A, Atshan S, Neela V. High prevalence of qacA/B carriage among clinical isolates of meticillin-resistant Staphylococcus aureus in Malaysia. J Hosp Infect. 2012;81(3):206–8. Narui K, Takano M, Noguchi N, Sasatsu M. Susceptibilities of methicillin-resistant Staphylococcus aureus isolates to seven biocides. Biol Pharm Bull. 2007;30(3):585–7. Kheljan MN, Jafarizare MA, Nejati-Koshki K, Arzanlou M, Akbarieh S. Phenotypic and Genotypic Resistance Patterns to Anti-microbial Biocides in Escherichia coli Isolates in Ardabil, Iran 2021–2023. Arch Clin Infect Dis. 2025;20(20). Namaki Kheljan M, Teymorpour R, Peeri Doghaheh H, Arzanlou M. Antimicrobial biocides susceptibility and tolerance-associated genes in Enterococcus faecalis and Enterococcus faecium isolates collected from human and environmental sources. Curr Microbiol. 2022;79(6):170. Additional Declarations No competing interests reported. Supplementary Files TableS1.docx TableS2.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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10:39:36","extension":"html","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":141398,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8057258/v1/3117503ab6fafa3d96df2824.html"},{"id":97151986,"identity":"a1385f7e-5a71-4971-b56c-da2c081c75ec","added_by":"auto","created_at":"2025-12-01 10:39:36","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":217443,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between MIC\u003csub\u003e90 \u003c/sub\u003eof antimicrobial biocides and the presence of BTA genes in \u003cem\u003eS. aureus\u003c/em\u003e isolates.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8057258/v1/10e3dbf701d64e66a3a0cffe.jpeg"},{"id":97664486,"identity":"3cdd8a91-5b86-49f0-bc5b-2cd1cfbe4e66","added_by":"auto","created_at":"2025-12-08 09:02:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1180291,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8057258/v1/1c6d353f-f767-4851-99a4-39f6af0bc5ff.pdf"},{"id":97249200,"identity":"673e427e-80fe-4aea-a283-cdf1d1e566c9","added_by":"auto","created_at":"2025-12-02 13:11:14","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16300,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8057258/v1/8996dd23892aae07d8cbeb62.docx"},{"id":97151990,"identity":"184390e7-6384-447e-9d2f-4d1ac8a1a0d4","added_by":"auto","created_at":"2025-12-01 10:39:36","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12577,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8057258/v1/69c179fe8f227efb82573270.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Emerging Biocide Resistance in Staphylococcus aureus: Cross-Source Insights and Public Health Implications","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e is a Gram-positive coccus bacterium found in the human microbiota, hospitals, communities, and wastewater (\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). It adapts to diverse conditions and forms biofilms that protect it from environmental stresses and antibiotics. The bacterium transmits through direct and indirect contact with infected individuals and contaminated surfaces. It spreads via food, animals, and wastewater (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). \u003cem\u003eS. aureus\u003c/em\u003e causes infections ranging from minor skin issues to severe diseases and is a significant source of hospital-acquired infections (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). It has developed resistance to many antibiotics, including methicillin, resulting in multidrug-resistant MRSA strains that complicate treatment (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). \u003cem\u003eS. aureus\u003c/em\u003e can also develop resistance to antimicrobial biocides, further complicating treatment efforts and infection control measures (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Despite tighter regulations in Europe and the United States, the global use of antimicrobial biocides is continuing to rise across various applications. In hospitals, businesses, and homes, antimicrobial biocides are extensively used as preservatives, antiseptics, and disinfectants against multiple pathogens (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Antimicrobial biocides are frequently employed to eliminate bacteria from many surfaces, including medical and laboratory devices (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Additionally, these compounds in the food industry play a vital role in protecting public health and ensuring the safety of food products. These processes help prevent microbial contamination, extend the shelf life of food items, and control cross-contamination (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In slaughterhouses, disinfecting equipment and the environment are crucial for reducing the risk of transmitting zoonotic diseases (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTriclosan (TCS), chlorhexidine digluconate (CHDG), benzalkonium chloride (BZC), and formaldehyde (FOR) are among the most widely used antimicrobial biocides worldwide (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). FOR, an aldehyde compound, is highly toxic to microorganisms and widely used as a sterilizing disinfectant. It exhibits strong reactivity with amines and thiols, which makes it particularly prone to damaging various compounds, such as proteins, nucleic acids, and metabolites (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). TCS, a phenol compound, has been utilized in consumer products since 1968 as an antiseptic, disinfectant, and preservative in clinical environments. It can be found in various consumer items, including cosmetics, household cleaning products, and toys. Additionally, TCS has been applied to the surfaces of medical devices, plastics, textiles, and kitchen utensils. It is included in a positive list of preservatives approved for cosmetic use and exhibits broad-spectrum antimicrobial activity by inhibiting the enoyl-acyl-carrier-protein (ACP) reductase enzyme, which plays a key role in the biosynthesis of bacterial fatty acids (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). CHDG, as a biguanide, is a potent antiseptic used in hospitals for hand hygiene, cleaning surgical areas, and sterilizing instruments in various medical fields. It is effective against bacteria, particularly Gram-positive agents. The active ingredient, chlorhexidine, is a cationic bisbiguanide that disrupts microbial cell membranes, leading to the loss of intracellular contents and osmotic imbalance, thereby exhibiting its antimicrobial action (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). BZC, a class of quaternary ammonium compounds, is commonly identified as an active ingredient in disinfectants utilized in residential, commercial, agricultural, and healthcare settings. Benzalkonium chloride interacts with cell membranes or transmembrane proteins by embedding its hydrophobic alkyl tail into the hydrophobic core of the membrane, leading to membrane destabilization (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA significant concern regarding the widespread use of biocides is the progressive bacterial resistance to these products and unrelated antimicrobials, including antibiotics. This issue undermines their effectiveness as antibacterial agents (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Moreover, several outbreaks of nosocomial infections linked to biocide contamination have been documented (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), underscoring the need for careful management and monitoring of biocide use (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). Repeated exposure to antimicrobial biocides exerts selection pressure on bacteria, leading to reduced sensitivity (\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA primary resistance strategy employed by bacteria is the use of efflux pumps, which reduce biocide concentrations to levels that are not harmful. Research has demonstrated that these efflux mechanisms play a significant role in conferring high-level resistance to various substances, including bisphenol compounds (such as TCS), especially in environments with extensive antimicrobial usage, like those containing biguanides (such as CHDG) and quaternary ammonium compounds (such as BZC) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Studies have identified numerous efflux genes in resistant bacterial isolates, including seven distinct efflux systems in \u003cem\u003eS. aureus\u003c/em\u003e. Most of these efflux pumps are encoded on the chromosome (NorA, NorB, NorC, MdeA, SepA, MepA, and SdrM), while some resistance genes are located on plasmids (\u003cem\u003eqacA/B\u003c/em\u003e, \u003cem\u003eqacC\u003c/em\u003e, \u003cem\u003eqacD\u003c/em\u003e, \u003cem\u003eqacG\u003c/em\u003e, and \u003cem\u003eqacJ\u003c/em\u003e). Efflux mechanisms are particularly effective in enabling bacteria to withstand exposure to biocides. Investigating these resistance mechanisms is essential for developing new strategies to address the declining effectiveness of antimicrobial biocides (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThis study aimed to evaluate the susceptibility of 553 \u003cem\u003eS. aureus\u003c/em\u003e isolates from various sources, including clinical samples, healthy carriers, wastewater, and food sources in Ardabil, northwest Iran, to common antimicrobial biocides such as BZC, CHDG, TCS, and FOR. It highlights the limited information in Iran regarding biocide resistance in \u003cem\u003eS. aureus\u003c/em\u003e from diverse sources and emphasizes the need for comprehensive research. The primary objective was to investigate resistance patterns and the prevalence of associated genes, such as \u003cem\u003enorA\u003c/em\u003e, \u003cem\u003emdeA\u003c/em\u003e, \u003cem\u003emepA\u003c/em\u003e, \u003cem\u003esepA\u003c/em\u003e, \u003cem\u003eqacA/B\u003c/em\u003e, \u003cem\u003eqacC\u003c/em\u003e, \u003cem\u003eqacD\u003c/em\u003e, \u003cem\u003eqacG\u003c/em\u003e, and \u003cem\u003eqacJ\u003c/em\u003e. The diverse sources and large sample size provide a thorough understanding of the bacterium's status in the region. The findings can inform policymakers and public health officials in designing effective infection control programs and raising awareness about hygiene practices. However, further research is needed to explore the effectiveness of current biocide treatment regimens in controlling microbial contamination across different settings.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eFOR MIC distribution\u003c/h2\u003e\u003cp\u003eThe MIC for FOR in all \u003cem\u003eS aureus\u003c/em\u003e isolates was established at 128 \u0026micro;g/ml. Consequently, the MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e values were also determined to be 128 \u0026micro;g/ml.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTCS MIC distribution\u003c/h3\u003e\n\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e displays the MIC results for TCS in \u003cem\u003eS. aureus\u003c/em\u003e isolates. In this study, the MIC for TCS ranged from 0.25 to 8 \u0026micro;g/ml. The MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e values were 0.25 and 1 \u0026micro;g/ml, respectively. According to the MIC\u003csub\u003e90\u003c/sub\u003e value, isolates from clinical specimens exhibited the highest resistance to TCS, with a 2 \u0026micro;g/ml.\u003c/p\u003e\n\u003ch3\u003eCHDG MIC distribution\u003c/h3\u003e\n\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the MIC results for CHDG in \u003cem\u003eS. aureus\u003c/em\u003e isolates, categorized by the sources of bacterial isolation. The MIC for CHDG in isolates ranged from 0.5 to 4 \u0026micro;g/ml. The MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e values were 0.5 and 1 \u0026micro;g/ml, respectively. Based on the MIC\u003csub\u003e90\u003c/sub\u003e value, isolates from clinical specimens exhibited the highest resistance to CHDG, with a value of 2 \u0026micro;g/ml.\u003c/p\u003e\u003cp\u003e\u003cb\u003eB\u003c/b\u003eZ\u003cb\u003eC MIC distribution\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e displays the MIC results for BZC in \u003cem\u003eS. aureus\u003c/em\u003e isolates, categorized by the source of bacterial isolation. The MIC for BZC isolates ranged from 0.25 to 8 \u0026micro;g/ml. The MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e values were 1 and 2 \u0026micro;g/ml, respectively. Based on the MIC\u003csub\u003e90\u003c/sub\u003e value, \u003cem\u003eS. aureus\u003c/em\u003e isolates from clinical specimens exhibited the highest resistance to BZC, with a value of 4 \u0026micro;g/ml.\u003c/p\u003e\n\u003ch3\u003eFrequency distribution of BTA genes\u003c/h3\u003e\n\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrates the frequency distribution of BTA genes among \u003cem\u003eS. aureus\u003c/em\u003e isolates. The results indicate that the prevalence of the \u003cem\u003enorA, mdeA, mepA, sepA, qacA/B, qacC, qacD, qacG, and qacJ\u003c/em\u003e genes was 24.05%, 21.33%, 30.22%, 8.49%, 18.08%, 10.12%, 10.48%, 12.29%, and 4.15%, respectively.\u003c/p\u003e\n\u003ch3\u003eAssociation between BTA genes and biocide's MIC\u003c/h3\u003e\n\u003cp\u003eAs illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, isolates harboring the \u003cem\u003esepA\u003c/em\u003e, \u003cem\u003eqacA/B\u003c/em\u003e, \u003cem\u003eqacC\u003c/em\u003e, and \u003cem\u003eqacJ\u003c/em\u003e genes were associated with higher MIC\u003csub\u003e90\u003c/sub\u003e for CHDG than isolates without these genes. Overall, isolates carrying the \u003cem\u003enorA\u003c/em\u003e, \u003cem\u003emdeA\u003c/em\u003e, \u003cem\u003emepA\u003c/em\u003e, \u003cem\u003esepA\u003c/em\u003e, \u003cem\u003eqacA/B\u003c/em\u003e, \u003cem\u003eqacC\u003c/em\u003e, \u003cem\u003eqacD\u003c/em\u003e, \u003cem\u003eqacG\u003c/em\u003e, and \u003cem\u003eqacJ\u003c/em\u003e genes exhibited a higher MIC\u003csub\u003e90\u003c/sub\u003e for BZC than those lacking these genes.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDistribution of MIC values for TCS in \u003cem\u003eS. aureus\u003c/em\u003e isolates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e\u003cp\u003eMIC (\u0026micro;g/ml)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eMIC\u003csub\u003e90\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinical\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;93 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e17 (18.27)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23 (24.73)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e41 (44.08)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8 (8.6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3 (3.22)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1 (1.07)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHealthy carriers\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;310 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e304 (98.06)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0 (0.0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3 (0.96)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3 (0.96)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWastewaters\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;50 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e50 (100)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFoodstuffs\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;100 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e81 (81)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12 (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e4 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;553 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e452 (81.73)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35 (6.32)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e48 (8.67)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e14 (2.53)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3 (0.54)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1 (0.18)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCHDG MIC distribution among \u003cem\u003eS. aureus\u003c/em\u003e isolates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"6\" nameend=\"c7\" namest=\"c2\"\u003e\u003cp\u003eMIC (\u0026micro;g/ml)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eMIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eMIC\u003csub\u003e90\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinical\u003c/p\u003e\u003cp\u003eN = 93 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e40 (43.01)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43 (46.23)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6 (6.45)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4 (4.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHealthy carriers\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;310 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e234 (75.48)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e76 (24.51)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0. 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWastewater\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;50 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e30 (60)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e20 (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0. 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFoodstuffs\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;100 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e55 (55)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e45 (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0. 5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;553 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e359 (64.91)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e184 (33.27)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6 (1.08)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4 (0.72)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMIC values of BZC across \u003cb\u003eS. aureus\u003c/b\u003e isolates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"9\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"8\" nameend=\"c9\" namest=\"c2\"\u003e\u003cp\u003eMIC (\u0026micro;g/ml)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.5\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eMIC\u003csub\u003e50\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eMIC\u003csub\u003e90\u003c/sub\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinical\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;93 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e23 (24.73)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11 (11.82)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e24 (25.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e21 (22.58)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12 (12.9)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2 (2.15)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHealthy carriers\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;310 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e3 (0.98)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19 (6.12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e252 (81.29)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30 (9.67)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e6 (1.93)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ewastewater\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;50 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e6 (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5 (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e39 (78)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFoodstuffs\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;100 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e10 (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e75 (75)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0 (0)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;553 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e42 (7.59)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e44 (7.95)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e390 (70.52)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e57 (10.3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e18 (3.25)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2 (0.36)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrevalence of BTA genes in \u003cem\u003eS. aureus\u003c/em\u003e isolates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eSource\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"9\" nameend=\"c10\" namest=\"c2\"\u003e\u003cp\u003eBTA genes\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003enorA\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eMepA\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eMdeA\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eSepA\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u003cem\u003eqacA/B\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e\u003cem\u003eqacC\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003e\u003cem\u003eqacD\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003e\u003cem\u003eqacG\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003e\u003cem\u003eqacJ\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eClinical\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;93 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e43 (46.23)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31 (33.33)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e31 (33.33)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e20 (21.5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e40 (43.01)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e26 (27.95)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e27 (29.03)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e24 (25.8)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e10 (10.75)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHealthy carriers\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;310 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e34 (10.96)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e35 (11.29)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e35 (11.29)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17 (5.48)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e29 (9.35)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15 (4.83)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e17 (5.48)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e26 (8.38)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e6 (1.93)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWastewater\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;50 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e20 (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14 (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19 (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e19 (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7 (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7 (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e9 (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e2 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFoodstuffs\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;100 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e36 (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43 (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e33 (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6 (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12 (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e8 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e7 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e9 (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e5 (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003cp\u003eN\u0026thinsp;=\u0026thinsp;553 n (%)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e133 (24.05)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e123 (22.24)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e118 (21.33)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e47 (8.49)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e100 (18.08)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e56 (10.12)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e58 (10.48)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e68 (12.29)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e23\u003c/p\u003e\u003cp\u003e(4.15)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eBiocides are utilized globally in hospital and non-hospital settings as antiseptics, disinfectants, and preservatives (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Evidences suggest that continuous exposure to antimicrobial biocides, combined with their widespread use driven by selective pressure, contributes to increased resistance among microorganisms to these compounds (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Unlike antibiotics, data on global bacterial sensitivity patterns to biocides are scarce. Our study offers a comprehensive understanding of biocide resistance in \u003cem\u003eS. aureus\u003c/em\u003e isolates from clinical samples and various ecological niches in Iran.\u003c/p\u003e\u003cp\u003eTCS is one of the most commonly used antimicrobial agents in personal hygiene products, including soaps, toothpaste, and particular dairy items (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). In our study, TCS MICs ranged from 0.25 to 8 \u0026micro;g/ml, with MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e values of 0.25 and 1 \u0026micro;g/ml, respectively. Clinical isolates demonstrated the highest resistance, exhibiting an MIC90 value of 2 \u0026micro;g/ml.\u003c/p\u003e\u003cp\u003eIn comparison, previous research reported MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e values of 0.12 \u0026micro;g/ml and 0.25 \u0026micro;g/ml, respectively, for \u003cem\u003eS. aureus\u003c/em\u003e, suggesting a higher susceptibility than observed in our study (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). For \u003cem\u003eStaphylococcus epidermidis\u003c/em\u003e, the MIC\u003csub\u003e50\u003c/sub\u003e was 0.12 \u0026micro;g/ml, and the MIC\u003csub\u003e90\u003c/sub\u003e was 8 \u0026micro;g/ml, indicating a broader range and higher resistance in specific isolates (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). For \u003cem\u003eEnterococcus\u003c/em\u003e species, the MIC\u003csub\u003e90\u003c/sub\u003e values were 0.125 \u0026micro;g/ml for \u003cem\u003eEnterococcus faecalis\u003c/em\u003e and 32 \u0026micro;g/ml for \u003cem\u003eEnterococcus faecium\u003c/em\u003e, reflecting significant variability in susceptibility and resistance (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFrequent exposure to biocides creates selective pressure on bacterial isolates, promoting the development of resistance. In studies involving \u003cem\u003eEscherichia coli\u003c/em\u003e, clinical isolates exhibited a notable increase in MIC after adaptation to TCS, with some isolates reaching MICs as high as 32 \u0026micro;g/ml (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), suggesting the potential for enhanced resistance development. Similarly, research by Stickler \u003cem\u003eet al\u003c/em\u003e. reported that \u003cem\u003eProteus mirabilis\u003c/em\u003e had initial MICs of 0.2 \u0026micro;g/ml, with mutants demonstrating MICs of up to 80 \u0026micro;g/ml after TCS exposure, highlighting substantial resistance evolution (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). These findings may explain the elevated MICs observed in our clinical isolates, as TCS is frequently used in hospital settings.\u003c/p\u003e\u003cp\u003eThe antibacterial activity of TCS, an anionic and lipophilic compound, is primarily derived from its inhibition of enoyl-acyl-carrier protein (ACP) reductase, a crucial enzyme involved in the elongation cycles of type II fatty acid synthase systems and an essential target for antibacterial agents (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Resistance to TCS can arise from increased expression of the \u003cem\u003efabI\u003c/em\u003e gene or the acquisition of an additional \u003cem\u003efabI\u003c/em\u003e allele via horizontal gene transfer from \u003cem\u003eStaphylococcus haemolyticus\u003c/em\u003e (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). In our study, the expression of \u003cem\u003efabI\u003c/em\u003e was not evaluated, and the presence of efflux pumps was not linked to the observed higher MIC\u003csub\u003e90\u003c/sub\u003e value.\u003c/p\u003e\u003cp\u003eThe MICs for CHDG in bacterial isolates can vary significantly across different studies and bacterial species. In our study, the MIC for CHDG ranged from 0.5 to 4 \u0026micro;g/ml, with MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e values of 0.5 and 1 \u0026micro;g/ml, respectively. The highest resistance observed was an MIC\u003csub\u003e90\u003c/sub\u003e value of 2 \u0026micro;g/ml in clinical specimens. Research on various Gram-negative and Gram-positive isolates showed a wide range of MIC values, with Gram-negative isolates generally exhibiting higher MICs (2\u0026ndash;256 \u0026micro;g/ml) compared to Gram-positive isolates (2\u0026ndash;32 \u0026micro;g/ml) (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Multidrug-resistant organisms, including New Delhi metallo-beta-lactamase-1 positive \u003cem\u003eEnterobacteriaceae\u003c/em\u003e spp., exhibited higher MICs for CHDG compared to their susceptible counterparts, with MIC values for commercial disinfectants being 20\u0026ndash;600 times higher than standard preparations (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Another study on \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e reported MIC\u003csub\u003e90\u003c/sub\u003e values of 64 \u0026micro;g/ml for CHDG, indicating a higher level of resistance than our findings (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). In a study of methicillin-susceptible and resistant \u003cem\u003eS. aureus\u003c/em\u003e, it has been found that 10% of isolates had elevated MICs for CHDG, with some isolates showing higher MIC values, particularly ST22-MRSA-IV (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Another study reported that some \u003cem\u003eS. aureus\u003c/em\u003e isolates required higher concentrations or prolonged treatment times to achieve bactericidal effects, indicating variability in susceptibility (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Our findings of CHDG MICs ranging from 0.5 to 4 \u0026micro;g/ml with MIC\u003csub\u003e50\u003c/sub\u003e and MIC\u003csub\u003e90\u003c/sub\u003e values of 0.5 and 1 \u0026micro;g/ml, respectively, align with some studies but show lower resistance levels than others, particularly those involving multidrug-resistant organisms. The variability in MIC values across different studies highlights the importance of continuous monitoring and tailored approaches to CHDG use in clinical settings. A survey on \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e found that chlorhexidine-resistant strains had MIC values significantly reduced in the presence of an efflux pump inhibitor, suggesting that efflux mechanisms contribute to resistance (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Consistently, in our study, the efflux pump encoding genes \u003cem\u003esepA, qacA/B, qacC\u003c/em\u003e, and \u003cem\u003eqacJ\u003c/em\u003e were associated with higher MIC\u003csub\u003e90\u003c/sub\u003e for CHDG than isolates without these genes.\u003c/p\u003e\u003cp\u003eBZC is the most widely employed active ingredient in disinfectants used across residential, commercial, agricultural, and clinical environments (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Its extensive use\u0026mdash;often leading to the release of residual biocides\u0026mdash;can impose selective pressure on microbial populations, thereby facilitating the emergence of chemical resistance (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). In our investigation, the MICs of BZC against \u003cem\u003eS. aureus\u003c/em\u003e isolates ranged from 0.25 to 8 \u0026micro;g/ml, with MIC₅₀ and MIC₉₀ values of 1 and 2 \u0026micro;g/ml, respectively. The highest resistance level observed was 4 \u0026micro;g/ml, identified in clinical \u003cem\u003eS. aureus\u003c/em\u003e strains. These results are consistent with prior research. Taheri \u003cem\u003eet al\u003c/em\u003e. (2016) reported BZC MICs ranging from 0.25 to 8 \u0026micro;g/ml for various \u003cem\u003eStaphylococcus\u003c/em\u003e isolates, including both methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) strains (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Similarly, Aykan \u003cem\u003eet al\u003c/em\u003e. (2013) documented MICs ranging from 2 to 8 \u0026micro;g/ml for MRSA isolates from Turkey (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Likewise, Shamsudin et al. (2012) observed lower MICs between 0.5 and 2 \u0026micro;g/ml in MRSA isolates from Malaysia (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). The highest resistance level in our study (4 \u0026micro;g/ml) is also comparable to findings by Narui et al. (2007), who noted that certain MRSA strains exhibited inhibitory concentrations exceeding typical BZC usage levels, indicating potential resistance (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePrevious studies have identified the \u003cem\u003eqacA/B\u003c/em\u003e efflux pump genes, which are linked to reduced susceptibility to BZC, in a significant proportion of MRSA isolates. These genes were consistently associated with elevated MIC values (\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Our study supported these findings by revealing that isolates harboring efflux pump\u0026ndash;encoding genes\u0026mdash;including \u003cem\u003enorA\u003c/em\u003e, \u003cem\u003emdeA\u003c/em\u003e, \u003cem\u003emepA\u003c/em\u003e, \u003cem\u003esepA\u003c/em\u003e, \u003cem\u003eqacA/B\u003c/em\u003e, \u003cem\u003eqacC\u003c/em\u003e, \u003cem\u003eqacD\u003c/em\u003e, \u003cem\u003eqacG\u003c/em\u003e, and \u003cem\u003eqacJ\u003c/em\u003e\u0026mdash;exhibited markedly higher MIC₉₀ values compared to isolates lacking these resistance determinants.\u003c/p\u003e\u003cp\u003eThe FOR is an organic electrophilic biocide whose mechanism of action involves the cross-linking of macromolecules (proteins, RNA, and DNA) (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). FOR has been historically and contemporarily employed in diverse settings, including hospital sterilization, laboratory decontamination, biological specimen preservation, and industrial and wastewater treatment. While its bactericidal properties are well established, precise inhibitory concentrations remain inconsistently reported in the literature (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Our results indicated that the MIC\u003csub\u003e90\u003c/sub\u003e values of FOR equaled 128 \u0026micro;g/ml for all \u003cem\u003eS. aureus\u003c/em\u003e isolates. Other studies reported the MIC\u003csub\u003e90\u003c/sub\u003e values as 512, 64, and 512 \u0026micro;g/ml for \u003cem\u003eEnterococcus\u003c/em\u003e spp., \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOverall, MIC levels increased for all antimicrobial biocides against \u003cem\u003eS. aureus\u003c/em\u003e isolates. TCS was the most effective agent in this study. Additionally, BTA genes were identified among the isolates. To reduce the development and spread of biocide resistance, we recommend cautiously using biocides and continuously monitoring their antimicrobial activity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eBacterial isolates\u003c/h2\u003e\u003cp\u003eA total of 553 \u003cem\u003eS. aureus\u003c/em\u003e isolates were included in the study. The authors collected these isolates in previous research from various sources: clinical specimens (n\u0026thinsp;=\u0026thinsp;93) (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e), healthy carriers (n\u0026thinsp;=\u0026thinsp;310) (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), wastewaters (n\u0026thinsp;=\u0026thinsp;50) (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), and foodstuffs (n\u0026thinsp;=\u0026thinsp;100) (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e), between 2018 and 2022 in Ardabil City, Northwest Iran. The isolates were identified using a series of standard microbiological assays, including catalase, tube coagulase, DNase tests, and cultivation on mannitol salt agar. Confirmation was performed by amplifying the \u003cem\u003enuc\u003c/em\u003e gene through polymerase chain reaction (PCR), as described in a previous study (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Subsequently, the isolates were preserved in tryptic soy broth (TSB) with 15% glycerol and stored at -70\u0026deg;C for future use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eBiocide susceptibility testing\u003c/h2\u003e\u003cp\u003eThe minimum inhibitory concentrations (MICs) of biocides, including FOR (Acros Organics USA), TCS (Bio Basic, Canada), CHDG (Sigma-Aldrich, USA), and BZC (Sigma-Aldrich, USA), were determined utilizing the agar dilution method. This procedure was conducted following the guidelines provided by the Clinical and Laboratory Standards Institute (CLSI) (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) and as elaborated upon in the authors\u0026rsquo; preceding publication (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eDetection of BTA genes\u003c/h2\u003e\u003cp\u003eGenomic DNA was isolated using the thermal lysis method (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). PCR testing assessed the presence of BTA genes, including \u003cem\u003enorA\u003c/em\u003e, \u003cem\u003emdeA\u003c/em\u003e, \u003cem\u003emepA\u003c/em\u003e, \u003cem\u003esepA\u003c/em\u003e, \u003cem\u003eqacA/B\u003c/em\u003e, \u003cem\u003eqacC\u003c/em\u003e, \u003cem\u003eqacD\u003c/em\u003e, \u003cem\u003eqacG\u003c/em\u003e, and \u003cem\u003eqacJ\u003c/em\u003e, which contribute to antimicrobial compound resistance in \u003cem\u003eS. aureus\u003c/em\u003e. This analysis employed gene-specific primers (designed in this study; Supplementary material, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and adhered to the cycling and temperature parameters outlined in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe findings were illustrated using tables, graphical representations, and quantitative metrics.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMRSA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Methicillin-resistant Staphylococcus aureus\u003c/p\u003e\n\u003cp\u003eTCS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Triclosan\u003c/p\u003e\n\u003cp\u003eCHDG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Chlorhexidine digluconate\u003c/p\u003e\n\u003cp\u003eBZC\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Benzalkonium chloride\u003c/p\u003e\n\u003cp\u003eFOR \u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Formaldehyde\u003c/p\u003e\n\u003cp\u003eACP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e Acyl-carrier-protein\u003c/p\u003e\n\u003cp\u003eMIC\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Minimum Inhibitory Concentration\u003c/p\u003e\n\u003cp\u003eBTA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Biocide Tolerance-Associated\u003c/p\u003e\n\u003cp\u003ePCR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003eCLSI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Clinical and Laboratory Standards Institute\u003c/p\u003e\n\u003cp\u003eTSB \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Tryptic Soy Broth\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to express their sincere gratitude to the Vice Chancellor for Research and Technology, Ardabil University of Medical Sciences for funding and support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMN: conducted the experiments and prepared the initial draft of the manuscript. EG, MM, MRO, AYJ: also contributed to the experimental work. KNK and MAV: made substantial contributions to the formal data analysis. MA: played a key role in the conception and design of the study and was actively involved in the critical review and refinement of the manuscript. KNK: was instrumental in conceptualizing and designing the study and providing overall leadership throughout the project. All authors have thoroughly reviewed and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by Ardabil University of Medical Sciences, Iran (Grant No. 401000185).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available upon reasonable request from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee of Ardabil University of Medical Sciences, Iran (Registration No. IR.ARUMS.REC.1401.223).\u0026nbsp;All methods followed relevant guidelines and regulations under the Helsinki Declaration ( https://www.wma.net/policies-post/wma-declaration-of-helsinki/). Clinical isolates were collected from the hospital\u0026rsquo;s bacterial repository solely for research purposes, and no patient samples or patient data were used in this study. Consequently, the requirement for informed consent from participants was waived by the Regional Research Ethics Committee of Ardabil University of Medical Sciences. Samples from healthy carriers (12 - 14 years old) were collected after informed consent was received from their respective parents or legal guardians.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declared no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of AI Use\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eArtificial intelligence (AI) tools were used to assist with language refinement, editorial clarity, formatting, and literature review during manuscript preparation. Specifically, Microsoft Copilot was employed to enhance sentence structure, improve scientific phrasing, and ensure consistency in terminology. All intellectual content, data interpretation, and scientific conclusions were developed solely by the authors. The authors take full responsibility for the integrity and originality of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOmid MR, Jamali H, Kafilzadeh F, Borjian A, Arzanlou M. Molecular epidemiology, virulence factors, antibiotic resistance and risk factors for nasal carriage of \u003cem\u003eStaphylococcus aureus\u003c/em\u003e in a teenage student population: High prevalence of oxacillin susceptible MRSA isolates. Jundishapur J Microbio. 2021;30(14):9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRanjbar Omid M, Jamali H, Kafilzadeh F, Borjian A, Arzanlou M. 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Reduced susceptibility to chlorhexidine disinfectant among New Delhi metallo-beta-lactamase-1 positive Enterobacteriaceae and other multidrug-resistant organisms: Report from a tertiary care hospital in Karachi, Pakistan. Indian J Med Microbiol. 2016;34(3):346\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNamaki Kheljan M, Hassanzadeh M, Srdari Jabedar M, Mohammadi Gollou A, Ashouri P, Teimourpour R, et al. Characterization of disinfectant susceptibility profiles among clinical isolates of Acinetobacter baumannii in Ardabil, Iran. Acta Microbiol Immunol Hung. 2023;70(4):311\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKanazawa K, Ueda Y. Bactericial activity of chlorhexidine gluconate against recent clinical isolates of various bacterial species in Japan. Jpn J Antibiot. 2004;57(5):449\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZHANG Y, ZHANG X, ZHANG S, ZHAO Y, DONG G, ZHOU X et al. Resistance mechanisms and molecular epidemiology of chlorhexidine acetate-resistant Klebsiella pneu-moniae clinical isolates. Chin J Microbiol Immunol. 2019:202\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKheljan MN, Hasanzadeh M, Neyestani Z, Jafarizare MA, Nejati-Koshki K, Vostakoli MA. Investigating the Frequency of Genotypic and Phenotypic Resistance to Plasmid-Mediated Ciprofloxacin in Clinical Isolates of Klebsiellapneumoniae in Ardabil. Arch Clin Infect Dis. 2025;20(3).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKampf G, Kampf G. Glutaraldehyde. Antiseptic Stewardship: Biocide Resistance and Clinical Implications. New York: Springer; 2018.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMerchel Piovesan Pereira B, Tagkopoulos I. Benzalkonium chlorides: uses, regulatory status, and microbial resistance. Appl Environ Microbiol. 2019;85(13):e00377\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTaheri N, Ardebili A, Amouzandeh-Nobaveh A, Ghaznavi-Rad E. Frequency of antiseptic resistance among Staphylococcus aureus and coagulase-negative staphylococci isolated from a university hospital in central Iran. Oman Med J. 2016;31(6):426.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAykan SB, Cağlar K, Engin ED, Sipahi AB, Sultan N, Yalınay \u0026Ccedil;ırak M. Investigation of the presence of disinfectant resistance genes qacA/B in nosocomial methicillin-resistant Staphylococcus Aureus isolates and evaluation of their in vitro disinfectant susceptibilities. Mikrobiyol Bul. 2013;47(1):1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShamsudin M, Alreshidi M, Hamat R, Alshrari A, Atshan S, Neela V. High prevalence of qacA/B carriage among clinical isolates of meticillin-resistant Staphylococcus aureus in Malaysia. J Hosp Infect. 2012;81(3):206\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNarui K, Takano M, Noguchi N, Sasatsu M. Susceptibilities of methicillin-resistant Staphylococcus aureus isolates to seven biocides. Biol Pharm Bull. 2007;30(3):585\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKheljan MN, Jafarizare MA, Nejati-Koshki K, Arzanlou M, Akbarieh S. Phenotypic and Genotypic Resistance Patterns to Anti-microbial Biocides in Escherichia coli Isolates in Ardabil, Iran 2021\u0026ndash;2023. Arch Clin Infect Dis. 2025;20(20).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eNamaki Kheljan M, Teymorpour R, Peeri Doghaheh H, Arzanlou M. Antimicrobial biocides susceptibility and tolerance-associated genes in Enterococcus faecalis and Enterococcus faecium isolates collected from human and environmental sources. Curr Microbiol. 2022;79(6):170.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Staphylococcus aureus, Biocide tolerance-associated genes, Triclosan, Chlorhexidine-digluconate, Benzalkonium chloride, formaldehyde","lastPublishedDoi":"10.21203/rs.3.rs-8057258/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8057258/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e causes numerous clinical infections. This study aimed to investigate the susceptibility of 553 \u003cem\u003eS. aureus\u003c/em\u003e isolates collected from patients, healthy carriers, retail foods, and wastewater in Ardabil, Iran, to common antimicrobial biocides, including triclosan (TCS), chlorhexidine digluconate (CHDG), benzalkonium chloride (BZC), and formaldehyde (FOR). Additionally, the presence of Biocide Tolerance-Associated (BTA) genes was examined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Minimum Inhibitory Concentration (MIC) of biocides was determined using the agar dilution technique. Concurrently, a polymerase chain reaction (PCR) was used to detect the BTA genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the current study, MIC\u003csub\u003e90\u003c/sub\u003e for TCS, CHDG, BZC, and FOR were 1 µg/ml, 1 µg/ml, 2 µg/ml, and 128 µg/ml, respectively, for total isolates. The MIC\u003csub\u003e90\u003c/sub\u003e for TCS, CHDG, and BZC in clinical isolates was double the MIC\u003csub\u003e90\u003c/sub\u003e of total isolates. The BTA genes, \u003cem\u003enorA\u003c/em\u003e, \u003cem\u003emdeA\u003c/em\u003e, \u003cem\u003emepA\u003c/em\u003e, \u003cem\u003esepA\u003c/em\u003e, \u003cem\u003eqacA/B\u003c/em\u003e, \u003cem\u003eqacC\u003c/em\u003e, \u003cem\u003eqacD\u003c/em\u003e, \u003cem\u003eqacG\u003c/em\u003e, and \u003cem\u003eqacJ\u003c/em\u003e, were detected in 24.05%, 21.33%, 30.22%, 8.49%, 18.08%, 10.12%, 10.48%, 12.29%, and 4.15% of isolates, respectively. Notably, the \u003cem\u003esepA, qacA/B, qacC\u003c/em\u003e, and \u003cem\u003eqacJ\u003c/em\u003e genes were associated with higher MIC\u003csub\u003e90\u003c/sub\u003e values with CHDG. Additionally, the presence of nor\u003cem\u003eA\u003c/em\u003e, \u003cem\u003emdeA\u003c/em\u003e, \u003cem\u003emepA\u003c/em\u003e, \u003cem\u003esepA\u003c/em\u003e, \u003cem\u003eqacA/B\u003c/em\u003e, \u003cem\u003eqacC\u003c/em\u003e, \u003cem\u003eqacD\u003c/em\u003e, \u003cem\u003eqacG\u003c/em\u003e, and \u003cem\u003eqacJ\u003c/em\u003e genes was associated with increased MIC\u003csub\u003e90\u003c/sub\u003e levels for BZC compared to isolates devoid of these genes (P \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the study, \u003cem\u003eS. aureus\u003c/em\u003e has elevated MICs for popular antimicrobial biocides. The most effective compounds are TCS and CHDG, and their presence in personal care products can help control these organisms.\u003c/p\u003e","manuscriptTitle":"Emerging Biocide Resistance in Staphylococcus aureus: Cross-Source Insights and Public Health Implications","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 10:39:31","doi":"10.21203/rs.3.rs-8057258/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"409a95cb-2411-4fc8-835b-27718d9faade","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T10:39:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 10:39:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8057258","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8057258","identity":"rs-8057258","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}