Browsing by Author "Hanski, Kirsi"

Antibiotic resistance is a growing threat to global health due to overuse and misuse of antibiotics leading to untreatable or difficult to treat infections. Natural environments are an important reservoir of antibiotic resistance. The release of antibiotics into the environment promotes the development of antibiotic resistant bacteria and environmental occurrence of antibiotic resistance genes (ARGs). ARGs are common in nature and prevalent in aquatic environments such as surface waters and effluent. Cyanobacteria are widely found in marine, freshwater, and terrestrial environments. Since their ubiquitous presence in water environments cyanobacteria are exposed to antibiotic pollution and are in contact with resistant bacteria. The role of cyanobacteria in the antimicrobial resistome and dissemination of ARGs has only been studied recently. This work aimed to evaluate the antibiotic susceptibilities of 51 cyanobacterial strains against different classes of antibiotics, using liquid batch cultures, antibiotic discs, and bioinformatics approaches. Cyanobacterial strains used in this work were sensitive to most of the tested antibiotics. However, majority of the strains also showed resistance against trimethoprim and novobiocin. Overall, there was little variation in the antibiotic resistances observed between strains but differences in sensitivity to different antibiotics was observed between species and strains with most differences seen with Nostoc spp. According to bioinformatic tools used (CARD database and BLASTp) FosA protein was found only in strains showing resistance against fosfomycin but not in any sensitive phenotypes and therefore fosA gene was selected as the most promising putative resistance gene for subsequent assays. To determine whether the fosA from cyanobacteria could confer resistance to fosfomycin, the fosA gene from Nostoc sp. XPORK 5A was cloned into pET28a(+) expression vector under the control of T7 promoter and subsequently native cyanobacterial promoter. The ability of Escherichia coli BL21 (DE3) carrying each plasmid constructs to grow in the presence of fosfomycin was determined with agar plates and growth curve assay. E. coli transformants containing the fosA gene and T7 promoter conferred high-level resistance to fosfomycin showing ability to grow at the highest concentrations tested (1mg/ml) on agar plates and (500 µg/ml) in growth curve assay. FosA protein expression from the native cyanobacterial promoter appeared to be weaker and conferred lower-level resistance to fosfomycin (≥ 10 µg/ml). The results of this study provide more information about the antibiotic susceptibility of cyanobacteria. In addition, replicating a horizontal transfer of the fosA gene from cyanobacteria to proteobacteria conferred resistance to fosfomycin and these results may indicate that also nonpathogenic cyanobacteria could act as a source of fosA antibiotic resistance genes.