# Antibiotic Resistance in Filters from Car Air Conditioning Systems
## 2 Materials and Methods  
The samples tested were cabin filters from vehicles of five different brands: Toyota, Subaru, Suzuki, Hyundai, and Volkswagen. The vehicles were mainly operated in the area of Central Europe. The filters were obtained during their periodic replacement at authorized service stations of the brands in Warsaw. Among the 20 filters tested were both filters with activated carbon and those equipped with an additional layer providing antibacterial properties. The pieces of filter material taken from several separate places of each filter were stored in sterile containers and then transported to the laboratory where they were subjected to microbiological testing. Sample preparation for quantitative studies consisted of placing fragments of filter material with a total weight of 2 g in 100 mL of sterile sodium pyrophosphate and then shaking for 30 min at 120 rpm to flush out the microorganisms contained in the filter. The obtained microbial suspension was inoculated in parallel onto two culture media: nutrient agar (Biocorp Ltd., Warsaw, Poland), for the determination of the total number of bacteria, and Rose Bengal Chloramphenicol medium (Biocorp Ltd., Warsaw, Poland), for the quantitative analysis of molds. Bacteria were cultured for 48 h, simultaneously at 26 \({}^{\circ}\)C and 37 \({}^{\circ}\)C, while fungi were cultured for 5 days at 26 \({}^{\circ}\)C. The number of microorganisms was calculated as the number of colony forming units--CFU/g of the filter. In parallel with the determination of the total number of microorganisms, surface culture of the test suspension was performed on nutrient agar medium with the addition of individual antibiotics: penicillin, nitrofurantoin, doxycycline, rifampicin, and gentamicin, at concentrations of 100 mg/L. The cultures were incubated at 26 \({}^{\circ}\)C for 48 h and then the number of bacterial colonies capable of growth in the presence of antibiotics was determined. The result was reported as the number of CFU/g of the filter. The percentage of antibiotic-resistant bacteria was determined by comparing bacterial counts on individual selective media with antibiotics with the total number of bacteria in the sample. In order to obtain pure isolates of antibiotic-resistant bacteria, the colonies of bacteria were selected from among those obtained on media with the addition of individual antibiotics, and purified by passaging using a streak plate method. Identification of the isolates was carried out using API (Bio-Merieux Poland, Ltd., Warsaw, Poland) biochemical tests and sequencing methods. Samples for sequencing were prepared at A&A Biotechnology, Gdansk, Poland. Genomic DNA was isolated by a method based on the Genomic Mini AX Bacteria+ kit (A&A Biotechnology) with additional mechanical lysis of the sample in a FastPrep-24-type device using zirconia beads. The PCR reaction mixture contained 25 \(\upmu\)L of PCR Mix Plus (A&A Biotechnology), 0.2 \(\upmu\)L each of FOR and REV primers and 1 \(\upmu\)L of test DNA. Pre-denaturation was carried out at 94 \({}^{\circ}\)C for 120 s, followed by 30 cycles of denaturation, primer connection, and extension, at 94 \({}^{\circ}\)C, 58 \({}^{\circ}\)C, and 72 \({}^{\circ}\)C, respectively, and a final extension phase for 300 s. PCR products were then purified using Gel-Out Kit (A&A Biotechnology), resuspended in 10 mM Tris-HCl buffer pH 8.0, and sent for sequencing to Macrogen (Amsterdam, The Netherlands). The resulting sequences posted were analyzed in CLC Main Workbench 22 and in BLAST software ([[http://blast.ncbi.nlm.nih.gov](http://blast.ncbi.nlm.nih.gov)]([http://blast.ncbi.nlm.nih.gov](http://blast.ncbi.nlm.nih.gov)): rRNA/ITS databases, accessed on 17 June 2025). Determination of inhibitory concentrations of individual antibiotics against selected bacterial isolates was carried out by the dilution method. A series of solutions of individual antibiotics in twice-diluted nutrient broth (Biocorp Ltd., Warsaw, Poland) were prepared to obtain test media for microbial culture. Two test series were carried out.