Antimicrobial activity of nature-inspired molecules against multidrug resistant bacteria

doi:10.21203/rs.3.rs-2719689/v1

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Antimicrobial activity of nature-inspired molecules against multidrug resistant bacteria

https://doi.org/10.21203/rs.3.rs-2719689/v1

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Multidrug-resistant bacterial infections present a serious challenge to global health. In addition to the spread of antibiotic resistance, some bacteria can form persister cells which are tolerant to most antibiotics and can lead to treatment failure or relapse. In the present work, we report the discovery of a new class of small molecules with potent antimicrobial activity against Gram-positive bacteria and moderate activity against Gram negative drug resistant bacterial pathogens. The lead compound SIMR 2404 had a minimal inhibitory concentration (MIC) of 2 µg/ml against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-intermediate Staphylococcus aureus (VISA). The MIC values against Gram negative bacteria such as Escherichia coli and Actinobacteria baumannii were between 8–32 µg/ml. Time kill experiments show that compound SIMR 2404 can rapidly kill tested bacteria. Compound SIMR 2404 was also found to rapidly kill MRSA persisters which display high levels of tolerance to conventional antibiotics. In antibiotic evolution experiments, MRSA quickly developed resistance to ciprofloxacin but failed to develop resistance to compound SIMR 2404 even after 24 serial passages. The rapid antibacterial activity against both active and persister MRSA and to a lesser extent against Gram negative bacteria, suggest the potential of these molecules for further evaluation as antibacterial agents.

Biological sciences/Drug discovery

Biological sciences/Microbiology

Antimicrobial resistance (AMR) among bacterial pathogens is a silent pandemic and a challenge to global public health ^1,2. A recent study estimates that AMR was behind 4.95 million deaths in 2019 ³. Around three quarters of these deaths were attributed to only six bacterial pathogens which included Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa³. These Multidrug-resistant (MDR) bacterial pathogens were also classified by the World Health Organization (WHO) as priority for antimicrobial development and discovery ^4,5. Along with surveillance and antibiotic stewardship, the discovery of new antimicrobial agents is urgently needed to mitigate the global challenge of AMR ⁶.

Methicillin-resistant Staphylococcus aureus (MRSA), is a Gram-positive bacterial pathogen that causes a wide array of infections which are often difficult to treat ^7,8. Antibiotics used for MRSA treatment include vancomycin, daptomycin, and linezolid; however, several MRSA strains resistant to these antibiotics have emerged ^7,9,10. MRSA can also form persister cells and biofilms which are tolerant to most clinically available antibiotics ¹¹. Persisters cells can arise during stress conditions such as stationary phase, anaerobic conditions, biofilm, antibiotic treatment, or infections and usually have low metabolic rates ^11–15. During such conditions, bacterial cells enter a low energy state, and as most antibiotic requires active metabolism for their antibacterial activity, the low metabolic rates of persisters can cause tolerance to most clinically available antibiotics ¹⁶.

We have recently reported the synthesis of small molecules with antimicrobial activity against MRSA ^17–19. While being only active against Gram positive bacteria, these molecules can rapidly kill actively growing MRSA as well as persister MRSA ¹⁸. In this work, we describe the antimicrobial activity of a new series of compounds with potent antimicrobial activity against MRSA including vancomycin-intermediate Staphylococcus aureus (VISA). These molecules were also active against Escherichia coli and Actinobacteria baumannii, albeit having higher minimal inhibitory concertation (MICs). A representative compound, SIMR 2404, showed rapid killing activity against tested Gram positive and Gram negative bacteria. Furthermore, this compound was also found to have rapid killing activity against persister MRSA. In antibiotic resistance evolution experiments, MRSA failed to develop resistance to SIMR 2404 unlike the antibiotic ciprofloxacin whereby resistance can readily develop. On the other hand, E. coli was able to develop resistance against SIMR 2404 after several passages. Our data demonstrate the antimicrobial activity of these new compounds against Gram-positive MRSA bacteria and suggest their potential for further studies as possible antimicrobials.

Identification of small molecules with antimicrobial activity

We evaluated the antimicrobial activity of first-in-class compounds developed in our laboratory ^20,21, for their ability to inhibit the growth of Gram positive and Gram negative MDR pathogens. For Gram positive bacteria we tested 3 clinical MRSA strains and 3 reference ATCC strains including MRSA-ATCC700699 (vancomycin intermediate S. aureus, VISA) (Supplementary Table S1). Tested Gram negative bacteria were ATCC reference strains of E. coli, K. pneumoniae, A. baumannii and P. aeruginosa. All six compounds showed growth inhibition against all tested MRSA strains with a minimum inhibitory concentration (MIC) ranging between 2–8 µg/ml (Supplementary Table S2). Against E. coli and A. baumannii, all six compounds showed growth inhibition at an MIC range between 8–64 µg/ml (Supplementary Table S3). None of the tested compounds showed activity against K. pneumoniae or P. aeruginosa (Data not shown, Table 1 for compound SIMR 2404). Based on these results, tested compounds were more active against Gram positive bacteria compared to Gram negative ones. We chose compound SIMR 2404 for further investigation. The chemical structure and MIC values for compound SIMR 2404 are shown in Table 1 and Fig. 1 (Table 1, Fig. 1).

Table 1

MICs (µg/ml) for compound SIMR 2404 against relevant multidrug resistant bacteria.
Bacteria	SIMR 2404 MIC (µg/ml)
MRSA-ATCC33591	2
MRSA-ATCC700699 (VISA)	2
Acinetobacter baumannii ATCC 19606	8
Acinetobacter baumannii ATCC BAA 1605	32
E. coli CDC-AR-0346	64
E. coli ATCC BAA 2469	16
Klebsiella Pneumonia ATCC BAA- 2146	Not Active
Pseudomonas aeruginosa ATCC 27853	Not Active

Compound SIMR 2404 can rapidly kill MDR bacteria

To evaluate the killing kinetics of compound SIMR 2404, time killing assay of exponentially growing (log phase) bacterial cultures were next performed. At 16×MIC of ciprofloxacin, which targets DNA replication, MRSA culture was eradicated within 4 hours (Fig. 2A). This is in shape contrast to compound SIMR 2404 which can rapidly eradicate MRSA cultures within 30 minutes at a lower concentration of 4×MIC (Fig. 2B). For E. coli, colistin which target bacterial membranes, was used as a control antibiotic and showed fast killing kinetics (Fig. 2C). Surprisingly, compound SIMR 2404 showed similar rapid killing kinetics as colistin suggest a similar mechanism of action (Fig. 2D). For A. baumanii, treatment with amikacin which target protein synthesis was able to eradicate cultures at 8×MIC after 4 hours (Fig. 2E). On the other hand, compound SIMR 2404 rapidly eradicates A. baumanii within only 30 minutes at a lower concentration of 2×MIC (Fig. 2F). We next examined the killing activity of compound SIMR 2404 against MRSA using live dead staining and fluorescent microscopy. SYTO-9 is a green, fluorescent stain that penetrate cells with intact membranes, whereas the red fluorescent stain, propidium iodide (PI), can only penetrate cells with damaged membranes. Therefore, live bacteria with intact membranes will fluoresce green while dead bacteria with compromised membranes will fluoresce red or yellow ²². Actively growing cultures of MRSA treated with compound SIMR 2404 showed ~ 84% dead cells after only 30 min of treatment (Fig. 3). On the other hand, the control antibiotic ciprofloxacin showed much slower killing activity and achieved a similar death rate of ~ 82% after prolonged treatment time of 24 hours (Fig. 3). Taken together, our data indicate that compared to conventional antibiotics, compound SIMR 2404 had a faster killing activity against MDR bacteria.

Compounds SIMR 2404 Eradicates MRSA Persisters

We next evaluated the ability of compound SIMR 2404 to kill MRSA persisters which are tolerant to conventional antibiotics. During stationary phase, Staphylococcus aureus develops high number of persister cells which can be readily isolated by treating antibiotic-susceptible bacteria with very high concentration of antibiotic for around 4 hours ^11,13,23. We used MRSA-ATCC 33591 for persister experiments since it can develop persisters during stationary phase ²⁴, and had low MIC to the antibiotic gentamicin (Table 2S). Stationary phase MRSA-ATCC 33591 treated with 100×MIC of gentamicin for 4 hours remained mostly viable (Fig. 4A), unlike actively growing (log phase) cultures which were eradicated upon the same treatment (Fig. 4B). Treatment of MRSA persisters prepared in (Fig. 4A) with 10×MIC of compound SIMR 2404, resulted in complete eradication of viable cells (Fig. 4C). On the other hand, treatment with 100×MIC of gentamicin, ciprofloxacin or vancomycin, showed virtually no antibacterial effects against persister MRSA (Fig. 4C). We next performed time-kill experiments to evaluate the anti persister activity of compound SIMR 2404. Within 2 hours of treatment at 5×MIC, compound SIMR 2404 was able to eradicate persister culture (Fig. 4D). At 10×MIC and 15×MIC, eradication of persisters occurred faster and was achieved within only 30 minutes (Fig. 4D).

Evolution of resistance against compound 2404

To evaluate the ability of bacteria to develop resistance to compound SIMR 2404, we performed multistep resistance evolution experiments. For MRSA, serial dilution at increasing concentrations of ciprofloxacin resulted in the evolution of resistant cells which appeared within 5 passages (Fig. 5A). However, no resistance was observed against compound SIMR 2404 even after 24 passages (Fig. 5A). On the other hand, E. coli was able to develop resistance for compound SIMR 2404 after 7 passages but failed to develop resistance to the control antibiotic colistin (Fig. 5B). This data indicate that MRSA lacks the ability to develop resistance to compound SIMR 2404, unlike E. coli which can become resistant following repeated exposures.

The use of “Complexity to diversity” schemes for chemical synthesis of nature-inspired molecules is a promising approach for the development of underrepresented chemical space with unique biological activities ^25–27. We have previously described the synthesis schemes of nature-inspired molecules using this approach and, in this work, we evaluated the antimicrobial activity of few of these molecules against MDR pathogens. Our results show that these molecules have potent antimicrobial activity against the Gram positive bacteria methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-intermediate Staphylococcus aureus (VISA). These molecules also show moderate antimicrobial activity against Gram negative MDR pathogens such as E. coli, A. baumannii but not against P. aeruginosa, K. pneumoniae. The reduced activity against Gram negative bacteria is likely due to the permeability barrier of outer membrane which is only present in Gram negative bacteria ²⁸. We have previously described another set of small heterocyclic molecules which were also active only against Gram-positive bacteria ¹⁸. The lack of activity against P. aeruginosa and K. pneumoniae could be a result of the differences in outer membrane permeability due to the lipopolysaccharide structures ^29,30, or could be due to the presence of multidrug efflux pumps ^31,32.

Compound SIMR 2404 showed fast killing rates against both Gram positive and Gram negative bacteria when compared with conventional antibiotics with the exception of colistin. Colistin is considered as a last resort drug against Gram negative bacteria, and its main mechanism of action involves binding to bacterial membranes leading to cell lysis ^33,34. The killing kinetics of both colistin and compound SIMR 2404 against E. coli were very similar which suggest that both can lead to rapid cell lysis. This is further supported by fluorescent microcopy using live/dead staining which showed that compound SIMR 2404 can induce rapid membrane damage in MRSA within only 30 minutes.

In addition to its resistance to antibiotics, MRSA can form persister cells which are tolerant to conventional antibiotics ^11,13. Eradicating drug-tolerant persisters, although difficult, have been achieved through metabolic activation ¹⁶, or targeting bacterial components which are independent of active metabolism such as bacterial membranes ^35–37 or DNA crosslinking ^38,39. Compound SIMR 2404 was found to kill persister MRSA within 4 hours unlike the conventional antibiotics gentamicin, ciprofloxacin, and vancomycin. Furthermore, in time kill experiments, increasing concentration of compound SIMR 2404 led to faster eradication of MRSA persisters. While the antimicrobial mechanism of action of compound SIMR 2404 remains to be elucidated, its ability to kill persisters in a concentration dependent manner, its fast-killing rates, and similar killing with the membrane targeting antibiotic colistin, all suggest that its mechanism of action is likely due to bacterial membrane disruption. Finally, when compound SIMR 2404 was studied for evolution of resistance, MRSA failed to develop resistance despite repeated exposure, however E. coli was able to develop resistance. While we currently do not know the mechanisms or mutations behind resistance development in E. coli, current work in our laboratory using whole genome sequencing and comparative genomics with the parent sensitive strain is underway to identify these mutations. In conclusion, our data indicate that compound SIMR 2404 is a promising candidate for future studies as a candidate antimicrobial against MRSA infections.

Bacterial strains

All bacterial strains used in this study are listed in Supplementary material (Table S1). Mueller-Hinton (MH) media (Biolife, Italy) was used as a growth media and was incubated at 37^oC. Antibiotic resistance profiles were performed using agar disk diffusion assay according to Clinical and Laboratory Standards Institute guidelines ⁴⁰. Bacteria resistant to at least three different classes of antibiotics were considered multidrug resistant ⁴¹.

Synthesis Of Compounds

The synthesis of tested compounds is detailed in our previous reported procedures ^20,21. Confirmation of structures was carried out using NMR and purity was greater than 99% as determined by HPLC ^20,21. The chemical structures of compounds used in this study are presented in Supplementary material ( Figure S1).

Determination Of Minimum Inhibitory Concentrations (MIC)

For compounds or antibiotics, the MICs were determined using two-fold dilutions in MH broth according to the microdilution method by the Clinical and Laboratory Standards Institute guidelines ⁴².

Time-kill Experiments

Time-kill assays were conducted using broth microdilution method and enumeration of viable cells following treatment. Exponentially grown bacteria were adjusted to 5 × 10⁵ CFU/ml and cultures were treated with two-fold serial dilutions of test compounds. Samples were removed at different time points, serially diluted in 0.85% NaCl solution, and 5-100 µl of each dilution were plated onto MH agar. Following 24 hours incubation at 37°C colonies were counted to enumerate of viable cells. All experiments were conducted in triplicate.

Preparation Of Mrsa Persisters And Killing Assays Of Persisters:

MRSA persister cells were prepared as previously described ¹⁸. Persister S. aureus were isolated by treating stationary phase bacteria with high antibiotic concentrations for 4 hours ^23,35. MRSA-ATCC 33591 strain was used for these experiments due to its susceptibility to the antibiotics gentamicin, ciprofloxacin, and vancomycin. Stationary phase MRSA-ATCC 33591 grown in 25 ml MH broth for 16 hours with shaking were washed three times with phosphate buffered saline (PBS). The cultures were then treated for 4 hours with 100 × MIC gentamicin (250 µg/ml). Following treatment, cells were washed 3 times with PBS, adjusted to 10⁷ CFU/ml and then treated with the test compound or control antibiotics at indicated concentrations. At specific time points a 50 µl sample was removed, serially diluted, and spot-plated on MH agar plates to determine viable cell counts ⁴³. In a control experiment, exponentially growing MRSA-ATCC 33591 was treated with gentamicin at 100 × MIC to confirm that this high antibiotic concentration eradicates all none persisters. Detection limit was 1:100 CFU/ml. Experiments were conducted on three biologically independent cultures.

Fluorescence Microscopy

MRSA-ATCC 33591 was grown in MH broth to ~ 2 × 10⁵ CFU/ml (early exponential phase), after which 1.5 ml cultures were treated with 2 × MIC of compound SIMR 2404 (4 µg/ml) or control antibiotic ciprofloxacin (0.6 µg/ml). Treated cultures were incubated at 37°C, and at different time points, cells were harvested by centrifugation at 3,000 × g for 5 minutes and washed two times with sterile water. Cells where then resuspended in 50 µl of water and stained with Live/Dead nucleic acid stains (Molecular Probes, USA): SYTO 9 (5 µM) and propidium iodide (15 µM) in the dark at room temperature for 15 minutes. 5 µl of the stained bacteria were then applied onto a glass slide, covered with a glass coverslip, and images were obtained using fluorescence microscope (IX73, Olympus, USA) at an excitation/emission wavelength of 480/500 nm for SYTO 9 (green) and 490/635 nm for propidium iodide (red) ^22,44. ImageJ software was used to quantify live cells (green) and dead cells (red or yellow) (National Institutes of Health, US).

Multi-step Resistance Evolution Experiment

MRSA-ATCC 33591 or E. coli ATCC BAA2469 were adjusted to 5 × 10⁵ CFU/ml then exposed to sub-MIC concentrations of SIMR 2404 or control antibiotics. Ciprofloxacin and colistin were used as control antibiotics for MRSA and E. coli respectively. Experiments were carried out in a 96 well-plate at a volume of 100 µl MH broth. Following static incubation at 37°C for 24 hours, wells with visible bacterial growth at highest concentration were used to inoculate the next passage at 1:100 dilution. At each sequential passage, 10% increments increase of the concentration of SIMR 2404 or antibiotic was performed (for up to 80% increase). This procedure was carried out three times for 24 consecutive days for each experiment ⁴⁵.

Contributions

M.H., F.M., and A.S., conducted the microbiology experiments. T.H.A., M.H., wrote the first draft of manuscript. V.S. synthesized the compounds and characterized the structures under the supervision of T.H.A.. T.H.A., conceived the idea and supervised all biological and chemical studies. M.H., F.M., and T.H.A. corrected and finalized the manuscript which is submitted with the consent of all authors.

ASSOCIATED CONTENT

Supplementary Information

Supplementary Information to this article can be found online in Scientific reports.

Data availability

The data supporting the findings in this study are available within the article and its Supplementary martial. Raw data are available from the corresponding author upon request.

Funding

This work was supported by generous grants from the IsDB-Transformers Fund and the Research Funding Department, and UOS competitive grant (2001050154).

Competing interests

The authors declare no competing interests.

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No competing interests reported.

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Antimicrobial activity of nature-inspired molecules against multidrug resistant bacteria

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Abstract

Figures

Introduction

Results

Discussion

Materials And Methods

Bacterial strains

Synthesis Of Compounds

Determination Of Minimum Inhibitory Concentrations (MIC)

Time-kill Experiments

Preparation Of Mrsa Persisters And Killing Assays Of Persisters:

Fluorescence Microscopy

Multi-step Resistance Evolution Experiment

Declarations

References

Additional Declarations

Supplementary Files

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