Today, the emergence of antibiotic resistance in pathogenic bacteria is considered an important problem for society. Excessive consumption of antibiotics, long‐term treatments, and inappropriate prescriptions continually increase the severity of the problem. Improving antibiotic stewardship requires improved diagnostic testing, and, therefore, in vitro antibiotic susceptibility testing is becoming increasingly important. This research details the development of an antibiotic susceptibility test for Mycobacterium smegmatis using streptomycin as antibiotics. This strain was selected because it is a member of the slow growing Mycobacterium genus and serves as a useful surrogate organism for M. tuberculosis. A commercially available and low‐cost screen‐printed gold electrode in combination with a specifically developed nucleic acid probe sequence for the 16SrRNA region of the mycobacterial genome was employed to monitor M. smegmatis nucleic acid sequences using the techniques of square‐wave voltammetry and electrochemical impedance spectroscopy. The results show that it was possible to detect M. smegmatis sequences and distinguish antibiotic‐treated cells from untreated cells with a label‐free molecular detection. As a result, the in vitro antibiotic susceptibility test revealed that M. smegmatis showed sensitivity to streptomycin after a 24‐H incubation, with the developed protocol representing a potential approach to determining antibiotic susceptibility more quickly and economically than current methods.

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Heat shock proteins (HSPs) are produced when organisms are exposed to various environmental stress conditions such as extreme temperatures, light, and toxins. It is a known fact that in bacteria which have the HSP gene (hsp), antibiotics can trigger the expression of these proteins. However, the response of HSP genes to antibiotics has not been fully clarified in the literature, with studies still ongoing. In this work, a novel method of detecting heat shock protein 65 (HSP65) was investigated using electrochemical impedance spectroscopy (EIS) due to its sensitivity and selectivity. To do so, a specific HSP65 probe and target were designed and their hybridization behaviour was studied using low-cost screen-printed electrodes (SPEs). Cyclic voltammetry was performed to analyse surface characteristics of the SPEs and the performance of the electrodes was tested using EIS, by measuring changes in the charge transfer resistance upon probe binding and target hybridization. Increases in charge transfer resistance were measured and observed to be in-line with literature. Based on these results, the designed HSP65 probe was confirmed to bind the target sequence and proved that the EIS can be effectively used to detect HSP65 label-free. Results presented here could lead to development of antibiotic susceptibility assays based on hsp genes in future and provide a quicker test for the detection of many slow-growing bacteria.

Successful lysis of cells/microorganisms is a key step in the sample preparation in fields like molecular biology, bioengineering, and biomedical engineering. This study therefore aims to investigate the lysis of bacteria on-chip and its dependence on both microfluidic channel structure and flow rate. Effects of temperature on lysis on-chip were also investigated. To perform these investigations, three different microfluidic chips were designed and produced (straight, zigzag and circular configurations), while the length of the channels were kept constant. As an exemplary case, Mycobacterium smegmatis was chosen to represent the acid-fast bacteria. Bacterial suspensions of 1.5 McFarland were injected into the chips at various flow rates (0.6-8 µl/min) either at room temperature or 50° C. In order to understand the on-chip lysis performance fully, off-chip experiments were carried out at durations which are equal to those bacteria spent in the channel from inlet to the outlet at different flow rates. We also performed COMSOL multiphysics program simulations to evaluate further the effect of the applied parameters. As a result, we found that the structure and the flow rate do not affect lysis over all in all investigated channel types, however on-chip experiments at room temperature produced more effective lysis compared to the on-chip and the off-chip samples performed at higher temperatures. Interestingly on-chip experiments at higher tempratures do not result in effective lysis. Index Terms-Microfluidic chip, Thermal lysis, On-chip bacterial lysis, Mycobacterium smegmatis I. INTRODUCTION acterial infections have continued to be a major cause of human morbidity and mortality [1]. Among them, tuberculosis and leprosy are categorized as acid-fast bacteria diseases since they could resist decolorization in acid, and these bacteria are responsible for millions of deaths each year and a great effort has been made by health institutions to control these This study was conducted in the frame of a Newton Katip Celebi Fund between Turkey and UK and supported by Turkish Scientific and Technological Council under the grant number of 217S793.

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