# Effect of Sub-Inhibitory Concentrations of Quaternary Ammonium Compounds and Heavy Metals on Antibiotic Resistance and Expression of Virulence Factors Among Staphylococcus spp. from Dairy Products

## TL;DR

This study shows that low concentrations of disinfectants and heavy metals can increase antibiotic resistance and virulence in Staphylococcus bacteria from dairy products.

## Contribution

The novel finding is that sub-inhibitory concentrations of disinfectants and heavy metals can induce antibiotic resistance and virulence gene expression in Staphylococcus isolates.

## Key findings

- Sub-inhibitory concentrations of benzalkonium chloride and cadmium chloride increased antibiotic resistance gene expression in Staphylococcus isolates.
- Biofilm production and virulence gene (eno) expression were elevated in response to these stressors.
- Use of low disinfectant concentrations may lead to more resistant and virulent bacterial strains.

## Abstract

Antimicrobial resistance is spreading rapidly throughout the world. The food chain can be one of the routes of transmission for microorganisms containing drug-resistance genes and thus serve as a channel for their transmission. Environmental stress and methods of preventing the spread of microorganisms trigger adaptive responses in bacterial cells. The aim of the present study was to determine the effect of the stress induced by sub-inhibitory concentrations (SICs) of cadmium chloride and benzalkonium chloride on antibiotic resistance and the expression of selected virulence factors in Staphylococcus isolates from food. The study was conducted on strains of the species S. epidermidis, S. heamolyticus, S. saprophyticus, and S. aureus. The values of the minimum inhibitory concentration against erythromycin, tetracycline, and oxacillin were determined before and after the incubation of the tested strains under stress conditions. The ability to form biofilm and slime production was also investigated. The expression levels of the genes responsible for antibiotic resistance (blaZ, tetK, tetM, ermB, and mecA) and virulence (eno) were conducted using Real-Time PCR. The MIC values of the antibiotics tested against the strains analyzed were found to be elevated in the presence of SICs of benzalkonium chloride and cadmium chloride. Furthermore, the intensity of biofilm production was also increased. SICs of benzalkonium chloride induced the expression of the tetM, tetK, mecA, and blaZ genes in 75%, 66.6%, 33.3%, and 40% of the isolates tested, respectively. Similar treatment with cadmium chloride induced the expression of the same genes in 75%, 100%, 66.6%, and 40% of the strains. In both cases, the expression of the ermB gene was reduced in 100% of the isolates. The eno gene was found to be overexpressed in 66.6% of the strains following benzalkonium chloride stress, and in 100% of the strains following cadmium chloride stress. These findings suggest that in Staphylococcus spp. strains, changes in the expression of the genes encoding antibiotic resistance and virulence factors may occur in response to the applied stress factors. The results indicate the possibility of selecting more resistant and virulent strains due to the use of too low concentrations of disinfectants, which emphasizes the need to use appropriate inhibitory doses of disinfectants in the food industry.