Multi-drug-resistant (MDR) pathogens are an increasing problem worldwide. Annually, 700 000 deaths are attributed to MDR and antimicrobial-resistant (AMR) strains of common bacterial infections. This number, if current trends in the use of antibiotics continue, is projected to increase beyond 10 million annual deaths by 2050. (1) MDR infections create an increasingly large burden in healthcare and preventative practices. (2) In their 2013 antibiotic-resistant threat report, the Centers for Disease Control and Prevention (CDC) listed 18 MDR and AMR pathogens that require immediate attention. Carbapenem-resistant Enterobacteriaceae (CRE) were identified as one of three pathogens at the highest threat level, demanding urgent action. (3) Recognizing the global impact of MDR and AMR pathogens on patient care, the World Health Organization (WHO) put forth a Global Action Plan (GAP) in 2015 to ensure continued success in effective treatment and prevention of these infectious diseases. (4) In 2017, the WHO also identified CRE as one of three carbapenem-resistant pathogens in their highest priority category (Priority 1: Critical) for research and development of new antibiotics, again highlighting the urgent need for solutions to counter pathogens resistant to last resort antibiotics. (5)

Klebsiella pneumoniae belongs to the family of Enterobacteriaceae and is one of the most important causes of nosocomial infections worldwide.K. pneumoniae causes various healthcare-associated infections, including pneumonia, bloodstream infections, urinary tract infections, wound or surgical site infections, and meningitis.K. pneumoniae infections have rapidly increased in hospital settings, making first-line antibiotics vastly ineffective. The emergence of carbapenem-resistant strains of K. pneumoniae as a major nosocomial infection has raised many concerns as antibiotic treatment options available against this pathogen are very limited. belongs to the family of Enterobacteriaceae and is one of the most important causes of nosocomial infections worldwide. (6) This Gram-negative opportunistic pathogen colonizes the human intestine and is of great clinical importance, especially among very sick patients. (7) causes various healthcare-associated infections, including pneumonia, bloodstream infections, urinary tract infections, wound or surgical site infections, and meningitis. (8−10) Over the last few decades, MDRinfections have rapidly increased in hospital settings, making first-line antibiotics vastly ineffective. The emergence of carbapenem-resistant strains ofas a major nosocomial infection has raised many concerns as antibiotic treatment options available against this pathogen are very limited. (11−13) With the rapid emergence of resistance to conventional antibiotics that were once considered wonder drugs, there is an emergent need for the development of new unconventional antibiotic agents that can effectively counter MDR pathogens.

1 (1 rotates ∼2–3 million revolutions per second and is considered a fast motor. Light-activated MNM 2 is a slow motor, rotating only ∼1.8 revolutions per hour and is a nanomechanical control for MNM 1. MNM 3 is similar to MNM 1 but with a triphenylphosphonium (TPP) cation attached to its stator portion. TPP targets eukaryotic mitochondria, causing MNM 3 to accumulate within the mitochondria.1 can disrupt bacterial cell walls and act as a potent nanomechanical antibacterial agent either alone or facilitating the action of conventional antimicrobials. Molecular nanomachines (MNMs) are synthetic organic nanomolecules that have a rotor component with light-induced actuation (motorization) that rotates unidirectionally relative to a stator ( Figure 1 a). (14−16) These MNMs can disrupt synthetic lipid bilayers and cell membranes with their rapid rotational movement. In the absence of light, MNMs diffuse into bilayers and display little or no toxicity. (16−18) The mechanism for light-induced unidirectional rotation of MNMs can be explained as follows: (1) upon photoexcitation of the central twisted double bond, it will move into an excited state, wherein the rotor and stator are orthogonal to each other; (2) normally relaxation could occur in either direction, but since there is a neighboring stereogenic center at the methyl group, the two ways that it can rotate are diastereomeric and therefore different in energy, so relaxation occurs through the preferred lower energy twist direction that completes a half turn; (3) the molecule finds itself in a sterically encumbered twist state, and the rotor and stator will thermally slide past each other to a lower energy state; (4) then a second photoexcitation followed by (5) a thermally induced twist puts the molecule back in the original state. (16−18) Recently, ultraviolet-light-activated MNMs were shown to use nanomechanical action to drill into cell membranes, creating pores in targeted cancer cells and causing cell death. (17) Light-activated fast motor, MNM 1 Figure b), was shown to cause cell necrosis in human prostate adenocarcinoma cells (PC-3) and mouse embryonic fibroblast cells (NIH 3T3). MNMs have various properties dependent on their steric structure and attached functional groups. They can be modified to give them specific properties and functions. Light-activated MNMrotates ∼2–3 million revolutions per second and is considered a fast motor. Light-activated MNMis a slow motor, rotating only ∼1.8 revolutions per hour and is a nanomechanical control for MNM. MNMis similar to MNMbut with a triphenylphosphonium (TPP) cation attached to its stator portion. TPP targets eukaryotic mitochondria, causing MNMto accumulate within the mitochondria. (19) MNMs can also have peptide appendages for specific cell adhesion. Nanomechanical action of fast motor MNMs makes them potential broad-spectrum antibacterials. We hypothesized that MNMcan disrupt bacterial cell walls and act as a potent nanomechanical antibacterial agent either alone or facilitating the action of conventional antimicrobials.

Figure 1 Figure 1. Molecular nanomachine structures. (a) Representative MNMs, illustrating the rotor portions (red), which rotate upon light activation relative to the stator portion (blue). R groups (green) are functional molecules that can be added to provide increased solubility, fluorophores for tracking, or serve as recognition sites for cellular targeting. (b) MNM 1 is a fast motor with a unidirectional rotor activated by 365 nm light. (c) MNM 2 is the corresponding slow motor that serves as a control. (d) MNM 3 is a fast motor similar to MNM 1 but with a triphenylphosphonium (TPP) cation attached to the stator portion. TPP targets eukaryotic mitochondria, causing MNM 3 to accumulate within mitochondria. This served as a control to demonstrate eukaryotic cell targeting of MNMs.

K. pneumoniae to resist carbapenems, the loss of cell wall outer membrane porins and production of K. pneumoniae carbapenemase (KPC) confer the highest levels of carbapenem resistance.1 nanomechanical properties to drill pores and disrupt the cell wall in MDR K. pneumoniae to allow carbapenem to traverse the cell wall OM and cause bacterial cell death. One of the effective ways to kill AMR and MDR bacteria is to increase the intracellular concentration of antibiotics. Biological and chemical approaches have been shown to modulate metabolic pathways to help increase the effective concentrations of antibiotics within bacteria.K. pneumoniae cell wall and increase the effective concentrations of antibiotics. Among various AMR mechanisms used by MDRto resist carbapenems, the loss of cell wall outer membrane porins and production ofcarbapenemase (KPC) confer the highest levels of carbapenem resistance. (20−23) The cell wall outer membrane (OM) lacking porins acts as a mechanical barrier that prevents carbapenem to permeate the OM and reach its target site, penicillin-binding proteins (PBP) in the periplasmic space. (24) We explore the use of light-activated MNMnanomechanical properties to drill pores and disrupt the cell wall in MDRto allow carbapenem to traverse the cell wall OM and cause bacterial cell death. One of the effective ways to kill AMR and MDR bacteria is to increase the intracellular concentration of antibiotics. Biological and chemical approaches have been shown to modulate metabolic pathways to help increase the effective concentrations of antibiotics within bacteria. (25,26) In this study, we assay the use of a nanomechnical approach to disrupt thecell wall and increase the effective concentrations of antibiotics.

Here, we use an extensively drug-resistant (ψkp6) and an antibiotic-sensitive (ψkp7) strain of K. pneumoniae to first show that light-activated MNM 1 using its nanomechanical action can display antibacterial properties irrespective of pathogen antibiotic susceptibility profiles. Then we show that light-activated MNM 1 in combination with Meropenem has the ability to make an extensively drug-resistant K. pneumoniae susceptible to Meropenem at subtherapeutic concentrations. Our results indicate that light-activated MNM 1 uses its nanomechanical action to assist in bypassing the cell wall OM-induced antibacterial resistance posed by K. pneumoniae. Thus, MNM 1, together with antibiotics like Meropenem, is shown as a potent antibacterial agent with the potential to effectively counter the increasing problem of multidrug resistance not only in K. pneumoniae but also in many other MDR pathogens.