On-Chip Isoniazid Exposure of Mycobacterium smegmatis Penicillin-Binding Protein (PBP) Mutant Using Time-Lapse Fluorescent Microscopy

Antibiotic resistance has been one of the biggest threats to global health. Despite the available prevention and control strategies and efforts in developing new antibiotics, the need remains for effective approaches against antibiotic resistance. Efficient strategies to cope with antimicrobial resistance require a quantitative and deeper understanding of microbial behavior, which can be obtained using different techniques to provide the missing pieces of the current antibiotic-resistance puzzle. Microfluidic-microscopy techniques are among the most promising methods that contribute modernization of traditional assays in microbiology. They provide monitoring and manipulation of cells at micro-scale volumes. Here, we combined population-level, culture-based assays with single-cell resolution, microfluidic-microscopy systems to investigate isoniazid response of Mycobacterium smegmatis penicillin-binding protein (PBP) mutant. This mutant exhibited normal growth in plain medium and sensitivity to stress responses when treated with thermal stress (45 °C), detergent stress (0.1% sodium dodecyl sulfate), acid stress (pH 4.5), and nutrient starvation (1XPBS). The impact of msm0031 transposon insertion on drug-mediated killing was determined for isoniazid (INH, 50 µg/mL), rifampicin (RIF, 200 µg/mL), ethionamide (ETH, 200 µg/mL), and ethambutol (EMB, 5 µg/mL). The PBP mutant demonstrated remarkable isoniazid-killing phenotype in batch culture. Therefore, we hypothesized that single-cell analysis will show increased lysis kinetics and fewer intact cells after drug treatment. However, the single-cell analysis data showed that upon isoniazid exposure, the percentage of the intact PBP mutant cells was 24%, while the percentage of the intact wild-type cells was 4.6%. The PBP mutant cells exhibited decreased cell-lysis profile. Therefore, the traditional culture-based assays were not sufficient to provide insights about the subpopulation of viable but non-culture cells. Consequently, we need more adequate tools to be able to comprehend and fight the antibiotic resistance of bacteria.

. mRNA expressions for M. smegmatis msm0031 transposon mutant. Bars represent the ratio of mRNA levels relative to sigA, a constitutive gene whose expression remains relatively unchanged. Number of transcript copies determined by qRT-PCR using gene-specific primers as listed below.
Complementation of the msm0031::Tn mutant. For complementation of the msm0031::Tn transposon mutant, the msm0031 gene was PCR-amplified from wild-type M. smegmatis genomic DNA ( Figure S1). The full-length gene was cloned into integrating plasmid as described below. Integrative complementation (pND200_Strep_msm0031). Complementation of the msm0031::Tn mutant with a single-copy attB-integrating plasmid (pND200_Strep) containing the intact msm0031 gene was performed. The INH-mediated killing response of the pND200_Strep_msm0031 strain followed wild-type kinetics only for the first 24 hours of drug exposure; thereafter, the killing kinetics were slower than wild-type ( Figure S2). The minimum inhibitory concentrations for INH of the integrating complemented and over-expression strains were higher than 100 µ g/mL (compared to 5 µ g/mL for wild-type bacteria).
The msm0031 deletion mutant: In-frame and unmarked deletion of the msm0031 gene in the WT background was made by allelic exchange, using the two-step counter-selection method (Pelicic et al., 1996). The RT-qPCR experiment confirmed that the msm0031 gene in the deletion strain was not expressed, Figure S3. Moreover, deletion of this gene might have affected the downstream genes (msm0028 and msm0030).  Figure S3. Confirmation for the msm0031 deletion mutant measuring the mRNA expression levels.
Bars represent the ratio of mRNA levels relative to sigA, a constitutive gene whose expression remains relatively unchanged. Number of transcript copies determined by qRT-PCR using gene specific primers as listed in materials and methods. The RT-qPCR experiments were performed at least 2 times.
Knockout of msm0031 gene resulted in hindered growth and defective cell separation in M. smegmatis ( Figure S4). The ability and time required to form colonies on standard LB plates was impaired relative to WT. This differs from the transposon mutant strain, which exhibited no growth phenotype and had higher transcript levels of msm0028, msm0030, and msm0031. Figure S4. Deletion of msm0031 gene altered morphology and growth rate. Deletion of msm0031 gene affected colony morphology (A), limited growth in standard 7H9 broth (B). Bright field images showed PBPA-inactivated cells were longer (C). Fluorescence microscopy of msm0031 deletion mutant transformed with Wag31-GFP showed defective septum formation in the presence of INH (D). Using time-lapse movies generation time was calculated as 3 hours for the msm0031 deletion cells (E). The measured cell area for the msm0031 deletion strain was 2 times larger than WT (F).  Complementation studies for the msm0031 and msm0031::Tn mutant strains.

Drug
Complementation studies for the transposon mutant and the deletion strain were performed in parallel with the same construct. The wild-type copy of the msm0031 gene was PCR-amplified from M. smegmatis genomic DNA and cloned into pND200_Strep integrating vector. Then, the pND200_Strep_msm0031 plasmid was delivered into the msm0031::Tn and msm0031 strains, Figure   S6. The integrating complementation improved the growth defect for the complemented msm0031 deletion strain. However, it was not possible to restore exact WT growth [25]. Delivery of the pND200_Strep_msm0031 plasmid did not alter the growth kinetics of the complemented transposon mutant, which were like WT, Figure S7.  Besides, the 1% SDS response for the complemented strains was similar to WT. The obtained results showed that there was no killing difference for 6 and 24 hours (data not shown).