I would especially like to thank Nurul Sir, Niranjan Sir and Prarthana Ma'am for helping me whenever I needed it. I would like to thank the Department of Biosciences and Biotechnology for providing infrastructure facilities for my research work.
Basu Bhattacharjee
It is my pleasure to thank my current lab members Barlina, Shubhangi and former lab members Tabassum, Narahari, Dipanjali, Gopika, Bhagyashree, Sanket and Muskan for their support and unconditional gesture of assistance in times of need, especially during the pandemic. I also want to thank my father and especially his lessons on how to overcome obstacles and live life in general.
C ONTENTS
- Introduction and Literature Review
- Potential of Urea-based Ligands as an Efflux Pump Inhibitor to Counter Ciprofloxacin Resistance in MRSA
- CHAPTER 3: Development of C8-loaded Nanocarrier as an Adjuvant for Mitigation of MRSA in Combination Therapy
- Bactericidal Potential, Membrane-directed Activity and Antibiofilm Activity of Quinoxaline-based Ligands for Targeting MRSA
- CHAPTER 5: Potential of Quinoxaline-based Antimicrobial (C2) in
- Development of a Quinoxaline Ligand-loaded Nanocomposite for Anti-MRSA therapy and Alleviation of MRSA invasion in an
Effect of the combinatorial treatment of C2-HNC and CPX on adhesion of MRSA to collagen-coated titanium wire.
A BBREVIATIONS
L IST OF T ABLES
MTT assay to determine the cytotoxic effect of varying concentrations of C2 on cultured MG-63 cells.
L IST OF S CHEMES AND F IGURES
Schematic of the protocol followed for estimating MRSA cell adhesion to collagen in the presence of C8-PNC and CPX. White arrow in panel (ii) indicates disruption of characteristic cell morphology. aureus 4s cell adhesion on collagen in different treatment sets. aureus 4s estimated during collagen adhesion assay performed in different treatment sets. aureus 4s cells adhered to collagen in case of different treatment sets.
Introduction and Literature Review
Introduction
Based on the aforementioned principle, the present study is an effort to create rationally designed synthetic small molecules and explore their potential as adjuvants in combinatorial antibacterial chemotherapy. The current study is significant as the fundamental understanding emerging from the study is likely to provide a guideline for developing effective adjuvants for combinatorial antibacterial therapy, thereby addressing a very serious global health problem.
Literature Review
In addition, other antibiotics that have emerged for therapeutic intervention against MRSA include ceftaroline, telavancin, delafloxacin, tedizolid and others (Lee et al., 2018). 11 antagonistic potential of the lipopeptide daptomycin against MRSA (Smith et al., 2009; Arbeit et al., 2004).
MOTIVATION AND OBJECTIVES OF THE PRESENT INVESTIGATION
Based on the prospect of efflux pump inhibitors and membrane-acting antibacterial agents as adjuvants in combination therapy for the treatment of MRSA, the essential goals of the Ph.D. Assessment of the potential of quinoxaline-based ligand in combination therapy to reduce MRSA in an in vitro bone cell infection model.
Potential of Urea-based Ligands as an Efflux Pump Inhibitor to Counter Ciprofloxacin Resistance
ABSTRACT
Materials and Methods 1. Compounds and Reagents
In a separate control experiment, the target cells were cultured in the absence of the ligands or CPX. Growth of the MRSA strain was monitored by measuring absorbance at 600 nm in a microtiter plate reader (Infinite M200, TECAN, Switzerland).
Results and Discussion 1. Design Rational of Ligands
MIC of CPX was assigned as the lowest concentration of the antibiotic resulting in an A600 value of <0.1 in the checkerboard assay. With regard to reduction in the MIC of CPX in the presence of C8, a dose-response relationship was evident in the presence of 5.0 µM and 10 µM C8 (Table 2.3). In the combinatorial treatment regimen, C8 (10 µM) was essentially nonbactericidal as significant growth of MRSA cells could be observed (~73% growth) (Figure 2.7A).
Significant Findings
Development of C8-loaded Nanocarrier as an Adjuvant for Mitigation of MRSA in
Combination Therapy
Materials and Methods 1. Compounds and Reagents
Microscopic and spectroscopic techniques were used to characterize PNPs and C8-PNC as described in the next section. After incubation, the solution was subjected to centrifugation at 10,000 rpm for 10 minutes and C8-PNC was recovered as the pellet and resuspended in sterile MilliQ water. FESEM analysis was also performed to determine the effect of the combination treatment of C8-PNC and CPX on MRSA.
Results and Discussion
The degree of growth inhibition caused by the combination treatment of C8-PNC and CPX was similar to 32 µM CPX (Figure 3.4B). FESEM analysis of (i) untreated MRSA cells and (ii) MRSA cells treated with C8-PNC and CPX. Furthermore, the adhesion of MRSA cells to collagen in the presence of the combinatorial treatment with C8-PNC and CPX was significantly lower compared to the adhesion of cells treated with 2.0 µM or 32 µM CPX (Figure 3.5A).
Significant Findings
In the ongoing effort to overcome the lack of therapeutic approaches against MRSA infections, an interesting prospect is the development of membrane-targeting synthetic adjuvants. Further, the potential of combining such membrane-targeting adjuvants with therapeutic antibiotics for MRSA eradication is also worth exploring. To this end, in the following chapter, quinoxaline ligands are examined for their membrane-targeting bactericidal activity against MRSA planktonic cells and biofilm detection.
Bactericidal Potential, Membrane-directed Activity and Antibiofilm Activity of Quinoxaline-based
Ligands for Targeting MRSA
Materials and Methods 1. Materials
The anti-MRSA activity of the synthetic ligands was calculated from three independent experiments, each with three replicates. After incubation, cells were removed by centrifugation and leakage of carboxyfluorescein from treated MRSA cells was determined by measuring the fluorescence of the dye in the supernatant (Thiyagarajan et al., 2014). The fold change in the expression of target genes was evaluated using the ΔΔCT method (Livak, et al., 2001).
Results and Discussion
A similar effect was also captured in FETEM analysis of C2-treated MRSA cells (Figure 4.4, Panels iv-vi). It was also observed that the minimum biofilm inhibitory concentration (MBIC) of C2 against the tested MRSA strain S. A qRT-PCR analysis indicated that the expression of agrC gene (coding for histidine kinase element of the agr operon ) in MRSA was significantly downregulated after treatment with sub-MIC levels of C2 in a dose-dependent manner (Figure 4.8A).
Significant Findings
To this end, it may be mentioned that in the current study, solution-based crystal violet and MTT assay indeed revealed significant inhibition of MRSA biofilm formation when C2 was used at concentrations above 16 µM (Figure 4.6). Based on its effect on the expression levels of the adhesins fnbA and cnbA, it is also predicted that C2 may hold significant potential as an anti-MRSA coating agent on catheters and other implanted medical devices, as these adhesins are implicated in the colonization of MRSA on medical devices and other abiotic surfaces (Lee et al., 2018; Aricola et al., 2012). To this end, in the following chapter, the adjuvant potential of C2 to increase the effectiveness of CPX against planktonic cells as well as biofilms of MRSA is established.
Potential of Quinoxaline-based Antimicrobial (C2) in Combination Therapy for Mitigation of MRSA in
Materials and Methods 1. Materials
A checkerboard test was performed in a sterile 96-well microtiter plate to determine the combination effect of C2 and ciprofloxacin on MRSA cells. The potential of C2 to enhance the potency of CPX against MRSA cells was also established by FESEM analysis. The MG-63 cells were then washed twice with sterile PBS and overnight-grown cells of S.
Results and Discussion
Inhibition of MRSA biofilm in the presence of 10 μM C2 and 12 μM CPX was further substantiated by microscopic analysis. Consequently, there will be a significant reduction in the extent of MRSA cells adhered to MG-63 cells when exposed to the combinatorial treatment regimen. Previous results seem to indicate that the population of MRSA cells adhered to MG-63 cells in the combinatorial treatment (~17%) was significantly lower than the untreated sample (Figure 5.4A).
Significant Findings
Fluorescence microscopy analysis to determine MRSA cell invasion in cultured MG-63 cells in the presence of different treatment regimens. A similar trend was observed in the invasion assay, where the relative invasion of MRSA cells to MG-63 cells was only ~0.37% in the case of the combined treatment regimen. Double-labeled fluorescence microscopy analysis confirmed the efficacy of the combined treatment regimen in reducing the invasion of MRSA cells into MG-63 cells.
Development of a Quinoxaline Ligand-loaded Nanocomposite for Anti-MRSA therapy and
Alleviation of MRSA invasion in an Orthopaedic Implant
Materials and Methods 1. Materials
To generate C2-loaded HSA nanocarrier (C2-HNC), HNPs (1.0 mg/ml in sterile MilliQ water) were incubated in separate sets overnight under rocking condition at room temperature with C2 (25 μM - 1254 μM) . For estimation of LE, HNPs (1.0 mg/ml in sterile MilliQ water) were incubated with varying concentrations of C2 (100 μM - 500 μM) for 12 h on a rocker at room temperature. An MTT-based assay was performed to determine the cytotoxic potential of C2-HNC (loaded with 45 µM, 90 µM, 180 µM and 360 µM C2) against MG-63 cells (human osteosarcoma cells).
Results and Discussion 1. Generation of C2-HNC
Characterization of C2-loaded HSA nanocarrier (C2-HNC) by (A) FESEM, (B) TEM, (C) AFM analysis. D) Estimation of the hydrodynamic radius of C2-HNC by DLS. The antagonistic activity of C2-incorporated collagen-coated Ti wire (C2-Co-TW) was studied by FESEM. In the case of C2-coated Ti wire, a sparse population of MRSA cells was observed to adhere to the surface of the Ti wire (Figure 6.9, panels ii and v) after 6 hours of incubation.
Significant Findings
Colonization of MRSA on Ti wire was also evident in the case of treatment with 90 µM C2-HNC alone (Figure 6.11, panels ii and v). Interestingly, FESEM analysis clearly indicated that the combination treatment with 90 µM C2-HNC and 16 µM CPX was able to significantly inhibit MRSA cell adhesion on Ti wire, and the few adhered cells of the pathogen appeared quite distorted, indicating a significant loss of cell integrity. A combinatorial treatment with C2-HNC (loaded with 90 µM C2) and CPX (16 µM) could effectively counteract the adhesion of MRSA cells on collagen-coated Ti wire, indicating that the bactericidal and adjuvant activity of the payload nanocarrier could be exploited for to prevent MRSA colonization on a Ti wire.
SUMMARY AND FUTURE PERSPECTIVE
It would be worthwhile to exploit the evidence obtained from the current study and validate the potential of the combination therapy regimen in an in vivo bone cell infection model of MRSA. In the present study, the adjuvant potential of the ligand C2 was exploited to develop a biocompatible HSA nanocarrier, which in combination with CPX could prevent the invasion of MRSA on Ti wire, used as an orthopedic model implant. Scheme 1 provides a schematic representation of the significant findings emerging from the current study.
BIBLIOGRAPHY
Role of the accessory gene regulator agr in community-associated methicillin-resistant Staphylococcus aureus pathogenesis. Development of membrane-active honokiol/magnolol amphiphiles as potent antibacterial agents against methicillin-resistant Staphylococcus aureus (MRSA). Leucyl-tRNA synthetase inhibitor, d-norvaline, in combination with oxacillin, is effective against methicillin-resistant Staphylococcus aureus.
APPENDIX
A PPENDIX
Significant difference means p value < 0.001 based on analysis of variance (ANOVA) followed by all pairwise multiple comparisons (Holm-Sidak method) of relative endpoint fluorescence measured in the EtBr efflux assay. FESEM analysis of (i) untreated, (ii) C8-treated and (iii) CPX-treated S. Scale bar for images is 1.0 µm. MTT assay to ascertain the cytotoxic effect of different concentrations of C8 on cultured HEK 293 cells.
LIST OF PUBLICATIONS
A quinoxaline-based membrane-targeting therapeutic material: implications for antibiotic rejuvenation and containment of MRSA invasion in an in vitro bone cell infection model. A biocompatible nanocarrier enhanced with a membrane-targeting adjuvant enables effective combination therapy to prevent MRSA invasion of an orthopedic implant (Manuscript in preparation). Insight into the assembly perspective of Schiff base AIEgens enabling an efficient sensor for hydrazine in their aggregated state.