Isolation of antibiotic resistant bacteria from tropical swimming pool sand filter backwash

YUNG, PUI YI MARIA; LOH, YING RU; TAY, MARTIN; GIVSKOV, MICHAEL; RAJAL, VERONICA BEATRIZ; YANG, LIANG

Viena

Simposio; 20th International Symposium on Health-Related Water Microbiology; 2019

The aim of the present work was to evaluate the presence of antibiotic resistant bacteria (ARB) in swimming pools from a tropical country. Sand filter backwash was collected from six swimming pools in Singapore and 2 milliliters of the concentrated backwash were plated in Nutrient Agar (NA), supplemented with antibiotics: carbenicillin (100 µg/mL), gentamycin (30 µg/mL), kanamycin (50 µg/mL), and tetracycline (15 µg/mL) to isolate ARB. After up to 120 hours at room temperature, colonies with distinct morphologies were isolated and sub-cultured until pure isolates were obtained. The minimum inhibitory concentrations of seven antibiotics (covering 5 classes) were determined in nutrient broth with serial dilutions of carbenicillin (300 µg/ml), chloroquine (300 µg/ml), kanamycin (200 µg/ml), erythromycin (200 µg/ml), gentamicin (100 µg/ml), tetracycline (50 µg/ml), and ciprofloxacin (4 µg/ml). Optical density at 600 nm was measured to evaluate growth.Genomic DNA from the isolates was extracted and amplified by PCR using universal 16S rRNA primers followed by Sanger sequencing. The sequences were searched against the NCBI-NR database, followed by phylogenetic tree construction. Taxonomic classification of the isolates was also performed using the RDP online portal?s Naïve Bayesian Classifier.A total of 41 isolates were obtained including 15 different species from 8 genera, with Aeromonas being the most abundant (10 isolates). Seventeen isolates were resistant to at least four antibiotics, with 11 of them resistant to the 7 antibiotics analyzed. Some of the isolates found, for example Chryseobacterium sp., Elizabethkingia sp., and Stenotrophomonas sp., had sequences closely related to known nosocomial pathogens.Sand filtration is an effective method to remove microorganisms from swimming pool water. However, as the filter retains organic matter and solids, as well as microorganisms, it could become an ideal place for them to grow forming biofilms, where horizontal gene transfer is facilitated.