In vitro effects of sub-minimum inhibitory concentrations of azithromycin on Bordetella pertussis tohama-I

Bordetella pertussis is the primary causative agent of whooping cough (pertussis) and is known to infect only humans. This bacterium colonizes the lungs and respiratory tract of the infected host and exhibits high contagiousness. The lack of oxygen caused by the disease can lead to brain damage that can be fatal, especially in infants under one year of age. Although whooping cough is primarily known as a childhood disease, it can also occur in adolescents and adults with mild symptoms such as a persistent cough. According to the World Health Organization (WHO), there were over 151,000 reported cases of pertussis worldwide in 2018. Despite widespread vaccination efforts, recent studies indicate a resurgence in pertussis cases. Antibiotics, particularly macrolide antibiotics, are commonly used in the clinic to control pertussis. This thesis study focused on azithromycin, a macrolide antibiotic, and aimed to determine the minimum inhibitory concentration (MIC) dose against the B. pertussis Tohama-I strain. Additionally, the study investigated the effects of the sub-MIC doses on bacterial growth rate, biofilm formation capacity, and expression of two critical virulence factors: ptxS1 and prn genes. The minimum inhibitory concentration (MIC) of azithromycin was determined to be 0.08 μg/ml using the broth dilution method. Subsequently, the effects of MIC ratios of 1/2, 1/4, 1/8, 1/16, 1/32, and 1/64 on bacterial growth rate were investigated. However, due to insufficent cell growth, the experiments could not be performed for sub-MIC doses of 1/2, 1/4, and 1/8. Therefore, the sub-MIC doses of azithromycin against the Tohama-I strain were set at 1/16, 1/32, and 1/64. To evaluate the effects of these doses on bacterial biofilm formation, a 96-well cell culture plate was used. In addition, gene expression was analyzed using real-time polymerase chain reaction (RT-PCR). The ability of bacteria to form biofilms decreased proportionally with increasing antibiotic dose. However, the expression levels of ptxS1 and prn genes did not exhibit a direct correlation with the escalating antibiotic dose. Specifically, it was observed that the expression levels of both genes increased at azithromycin concentration of MIC/32 compared with control. Conversely, at MIC/16 and MIC/64, the expression of ptxS1 decreased, whereas the expression of prn increased compared with the control. Nevertheless, the effects of antibiotic doses on gene expression were statistically insignificant compared to control (p>0.05). There are not adequate and widespread studies on the effects of sub-MIC antibiotic doses on B. pertussis in the literature. The present study is the first to specifically address the effects of sub-MIC antibiotic doses using azithromycin against B. pertussis. Considering the widespread use of azithromycin for pertussis treatment, the results of this study will provide valuable insight into the effects of the antibiotic on bacterial cells and improve our understanding of the relationship between bacteria and host cells.