ArticlePDF AvailableLiterature Review

Bacterial proton motive force as an unprecedented target to control antimicrobial resistance

March 2023
Medicinal Research Reviews 43(10325)

March 2023
43(10325)

DOI:10.1002/med.21946

Authors:

Novel antibacterial therapies are urgently required to tackle the increasing number of multidrug-resistant pathogens. Identification of new antimicrobial targets is critical to avoid possible cross-resistance issues. Bacterial proton motive force (PMF), an energetic pathway located on the bacterial membrane, crucially regulates various biological possesses such as adenosine triphosphate synthesis, active transport of molecules, and rotation of bacterial flagella. Nevertheless, the potential of bacterial PMF as an antibacterial target remains largely unexplored. The PMF generally comprises electric potential (ΔΨ) and transmembrane proton gradient (ΔpH). In this review, we present an overview of bacterial PMF, including its functions and characterizations, highlighting the representative antimicrobial agents that specifically target either ΔΨ or ΔpH. At the same time, we also discuss the adjuvant potential of bacterial PMF-targeting compounds. Lastly, we highlight the value of PMF disruptors in preventing the transmission of antibiotic resistance genes. These findings suggest that bacterial PMF represents an unprecedented target, providing a comprehensive approach to controlling antimicrobial resistance.

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Received: 20 April 2022

Revised: 10 January 2023

Accepted: 26 February 2023

DOI: 10.1002/med.21946

REVIEW ARTICLE

Bacterial proton motive force as an

unprecedented target to control antimicrobial

resistance

Bingqing Yang

|Ziwen Tong

|Jingru Shi

Zhiqiang Wang

1,2

|Yuan Liu

1,2,3

Jiangsu Co‐innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of

Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China

Joint International Research Laboratory of Agriculture and Agri‐Product Safety, the Ministry of Education of China, Yangzhou

University, Yangzhou, Jiangsu, China

Institute of Comparative Medicine, Yangzhou University, Yangzhou, Jiangsu, China

Correspondence

Zhiqiang Wang and Yuan Liu, Jiangsu

Co‐innovation Center for Prevention and

Control of Important Animal Infectious Diseases

and Zoonoses, College of Veterinary Medicine,

Yangzhou University, Yangzhou, Jiangsu, China.

Email: zqwang@yzu.edu.cn and

liuyuan2018@yzu.edu.cn

Funding information

National Key Research and Development

Program of China, Grant/Award Numbers:

2021YFD1801000, 2018YFA0903400; Young

Elite Scientists Sponsorship Program by CAST,

Grant/Award Number: 2020QNRC001; Priority

Academic Program Development of Jiangsu

Higher Education Institutions (PAPD),

Grant/Award Number: 2019; Jiangsu

Agricultural Science and Technology Innovation

Fund, Grant/Award Numbers: CX(20)3091,

CX(21)2010; National Natural Science

Foundation of China, Grant/Award Numbers:

32222084, 32172907, 32002331; 111 Project,

Grant/Award Number: D18007

Abstract

Novel antibacterial therapies are urgently required

to tackle the increasing number of multidrug‐

resistant pathogens. Identification of new antimicro-

bial targets is critical to avoid possible cross‐

resistance issues. Bacterial proton motive force

(PMF), an energetic pathway located on the bacterial

membrane, crucially regulates various biological pos-

sesses such as adenosine triphosphate synthesis,

active transport of molecules, and rotation of bacte-

rial flagella. Nevertheless, the potential of bacterial

PMF as an antibacterial target remains largely

unexplored. The PMF generally comprises electric

potential (ΔΨ) and transmembrane proton gradient

(ΔpH). In this review, we present an overview of

bacterial PMF, including its functions and characteri-

zations, highlighting the representative antimicrobial

agents that specifically target either ΔΨ or ΔpH. At

the same time, we also discuss the adjuvant potential

of bacterial PMF‐targeting compounds. Lastly, we

highlight the value of PMF disruptors in preventing

Med Res Rev. 2023;43:1068–1090.wileyonlinelibrary.com/journal/med1068

the transmission of antibiotic resistance genes. These

findings suggest that bacterial PMF represents an

unprecedented target, providing a comprehensive

approach to controlling antimicrobial resistance.

KEYWORDS

antibiotic resistance, antimicrobial agents, bacteria, proton motive

force, target

1|INTRODUCTION

Antimicrobial resistance (AMR) is an increasingly severe problem threatening global health. It is indicated that nearly

1.27 million deaths directly result from antibiotic resistance and 4.95 million deaths are closely correlated to

resistant bacterial infections in 2019.

It is medically urgent to develop new antimicrobial strategies with traditional

antibiotics gradually losing their effectiveness.

Newly discovered lead compounds hold immense potential in this

battle. However, identifying compounds targeting the repetitious intracellular physiological processes, such as

membrane and cell wall disruption, inhibition of DNA/RNA, and protein synthesis, easily forms cross‐resistance

with the existing drug resistance determinants, greatly hampering their clinical translation.

Therefore, seeking

novel targets will be conducive to expanding the current antibacterial arsenals.

The cell membrane, an unexplored promising target for antimicrobial agents, has gained increasing attention

because of its importance in nongrowing bacteria and the success of emerging antibiotics.

5–7

Several novel

membrane targets, especially membrane phospholipids, have been successfully identified recently. For example,

daptomycin displays antimicrobial activities against Staphylococcus aureus by targeting phosphatidylglycerol (PG), a

central phospholipid component, and triggering severe membrane blebbing.

A linear undecapeptide, SLAP‐S25,

potentiates multiple antibiotic efficacies through simultaneously binding to lipopolysaccharide (LPS) and PG of

drug‐resistant Escherichia coli membrane.

Additionally, two other essential phospholipids, phosphatidylethanola-

mine (PE) and cardiolipin (CL), also inspire the development of new patterns of antibiotics. Specifically, by inhibiting

CL, a microbiota‐derived nonribosomal peptide termed telomycin, and its natural analogs, leads to the rapid

membrane lysis of S. aureus and Bacillus subtilis, causing a bactericidal effect.

Likewise, the methylation status of

the whole PE opens the premise for high‐level cinnamycin production in inducing antibacterial activities.

More interestingly, some unique previously unexplored elements exist on the membrane, such as menaquinone

(vitamin K2), the target of lysocin E,

and BamA, the target of novel antibiotic darobactin.

Specifically, the

binding of lysocin E with menaquinone leads to a series of membrane responses, such as potassium leakage and

membrane depolarization, thus exhibiting potent bactericidal activity against Gram‐positive bacteria. It is also

noteworthy that BamA, an emergent target in recent years, mainly comprises the β‐barrel folding complex (BAM),

which serves as the essential chaperone and translocator, folding the outer membrane proteins of Gram‐negative

bacteria.

The designed chimeric peptidomimetic antibiotics harboring a β‐hairpin peptide macrocycle, targeting

both LPS and BamA, also achieved considerable success.

Undoubtedly, the discoveries of these novel membrane targets and their ligands not only provide a paradigm

for the development of novel antimicrobial drugs but also reveal the feasibility and importance of bacterial cell

membranes as potential antibacterial targets. In bacteria, electron transfer and proton pumping via respiratory chain

on the cytoplasmic membrane generate a proton electrochemical gradient called proton motive force (PMF), which

is considered critical for various cell membrane functions.

The dysfunction of PMF directly hinders the synthesis

of adenosine triphosphate (ATP), a kind of unstable high‐energy compound that broadly acts as an activator,

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inhibitor, product, or substrate in the complex bacterial physiological network.

Generally, the dynamic synthesis

and consumption of ATP is carried out in parallel with many intracellular processes such as signal transduction,

stress response, active transport, polypeptide folding, subunit aggregation, and protein phosphorylation.

18–23

Despite the successive findings of potent antibacterial agents targeting PMF, including tridecaptin A1, bacaucin‐1,

halicin, WRK‐12, α‐mangostin (AMG), and isobavachalcone (IBC),

24–28

the potential roles of PMF in bacterial

resistance control remain poorly understood. Such a situation warrants a comprehensive investigation of the

function and measurement of PMF and its potential as a novel target in tackling AMR crisis.

This review presents an overview of the cellular functions and characterizations of PMF in bacteria. We

summarize recent advances in developing new antibacterial compounds targeting PMF. Additionally, the adjuvant

potential of bacterial PMF regulators in coordination with existing antibiotics against drug‐resistant bacteria is

highlighted. Moreover, we explore the potential of PMF disruptors in tackling the transmission of antibiotic

resistance genes (ARGs). Lastly, challenges and future perspectives for the translations and applications of

PMF‐targeting compounds are also discussed.

2|FUNCTIONS AND CHARACTERIZATIONS OF PMF IN BACTERIA

PMF, namely the electrochemical potential difference of proton across the cell membrane, comprises two

components: electric potential (ΔΨ) and transmembrane proton gradient (ΔpH).

PMF generation in eukaryotes

involves many intimate elements, including mitochondrion, respiratory chain, and ATPase. In the whole biological

system, most energy originated from numerous nutrient transfers from the substrates in the form of high‐energy

electrons by nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD). Subsequently, after

passing through a series of electric mediators in electron transfer chain (ETC), these electrons carried by NADH and

FADH

are finally transported to O

at the mitochondrial inner membrane (Figure 1A). Meanwhile, the considerable

energy produced along with the electron translocation is used for the transmembrane pumping of protons. More

specifically, protons are transported from the mitochondrial matrix to the intramembranous space, thus reversing

the concentration gradient and creating the electrochemical gradient (PMF) on both sides of the inner membrane.

Subsequently, the famous theory called the “chemiosmotic hypothesis”claims the coupling of PMF and ATP

synthesis. Driven by the concentration gradient, H

flows back to the mitochondrial matrix through specific pores

or channels in F1Fo‐ATPase, through which the released energy drives the ATPase to catalyze the interaction of

ADP and Pi to generate ATP.

While once the respiration process is interrupted, ATP would be hydrolyzed to

sustain PMF.

Lack of mitochondria in prokaryotes like bacteria leads to the harboring of the respiratory chain and ATPase on

the cytoplasmic membrane. Moreover, the flow of electrons and protons is dynamically changing across the

membrane. Generally, bacteria have a more dedicated and flexible network for electron transport,

including

branched pathways and various substitutable terminal electron receptors.

Additionally, the respiratory pathways

could be easily adjusted along with the external environment changes. For example, two terminal oxidases in the

aerobic respiratory chain of E. coli, cytochrome o oxidase under the command of cyoABCDE and cydAB‐encoding

cytochrome d oxidase, are converted to cater to the oxygen richness. Herein, only oxygen‐demanding growth

conditions express cytochrome o oxidase. In contrast, cytochrome d oxidase tends to be less restricted in

oxygen conditions, despite being moderately aerobic. It is also noteworthy that nitrate, fumarate, and

trimethylamine oxide are alternative terminal acceptors when the environment becomes anaerobic.

Despite the ability to provide the proximate energy for respiratory ATP synthesis by F1Fo‐ATPase, PMF plays

multifaced roles in bacterial bioprocesses, including the import of nutrients, the efflux of toxic products, and flagella

motility (Figure 1B).

Substance transport is significant in bacterial biological processes as bacteria undergoing

active metabolism have enormous requirements for a variety of small molecules like ions and macromolecules like

proteins. They primarily serve as energy supplements, raw materials for synthesizing new cellular substances, and

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YANG ET AL.

intermediate metabolites indispensable to maintaining essential survival and reproductive processes. Meanwhile,

the accumulation of metabolic waste and toxic products causes damage and burden to bacterial cells. Thus, PMF‐

dependent transmembrane processes with aid of the electrochemical gradient allow the active transport through

the inward and outward pumping of H

. Furthermore, H

homeostasis is the fundamental premise of all life

activities as enzymes playing catalytic roles require suitable pH conditions. Also, the rotation of bacterial flagella

relies heavily on the energy released from ATP hydrolysis and participates in many physiological activities such as

motion, surface adherence, biofilm formation, and host tissue colonization.

Altogether, stable PMF should be

ensured for maintaining healthy bacterial activities.

PMF is distributed in ΔΨ and ΔpH, with stored energy in ΔpH exploiting a persistent change in pH in one or

more cellular compartments. The physical distance between the redox factors and cofactors determines the

influence of ΔΨ on the charge‐separated state. In most cases, PMF remains stable as mutual compensation and

transformation exist between its two components.

Specifically, the dissipation of ΔΨ increases the ΔpH to

equilibrate and vice versa.

As a testing index, a hydrophobic fluorescent probe 3,3ʹ‐dipropylthiacarbocyanine

iodide (DiSC

(5)) is used to probe the dissipation of ΔΨ (Figure 2A).

DiSC

(5) is a kind of membrane‐labeling

fluorescence probe, which is sensitive to small changes of membrane potential (ΔΨ) or membrane configuration.

During cell membrane depolarization or permeabilization, the transmembrane electric gradient is disrupted.

Therefore, this probe is translocated to the outer membrane space with enhanced fluorescence units. In contrast,

hyperpolarization of the cell membrane or the loss of chemical gradient (ΔpH) enhances ΔΨ. DiSC

(5) uptake

activities will be more significant in this case, weakening the related fluorescence intensity. Together, the increase

of fluorescence suggests the dissipation of ΔΨ, while a decrease otherwise implies the disruption of ΔpH

(Figure 2B). Meanwhile, the investigation of H

and K

flow using specific fluorescent dyes provides more details for

understanding PMF perturbation (Figure 2C). For some classical PMF disrupters such as CCCP (carbonyl cyanide

FIGURE 1 Generation of proton motive force (PMF) and its functions in multiple bacterial intracellular

activities. (A) Elaborate depiction of how the electron transmitters in the respiratory chain complex pump H

from

bacterial cytoplasm to the outside. H

on the outer side of the membrane cannot return to the inner side freely

through the inner membrane. In this electron transfer process, the proton concentration gradient and membrane

potential difference are established on both sides of the inner membrane, constituting the transmembrane

electrochemical potential gradient of proton, the PMF. (B) Cellular functions of bacterial PMF. Bacterial PMF drives

many cellular processes, including ATP synthesis, transport of molecules by either symport or antiport (e.g., uptake

of nutrients and efflux of toxic drugs), and rotation of bacterial flagella. ΔΨ, electric potential; ΔpH, transmembrane

proton gradient; ATP, adenosine triphosphate; FAD, flavin adenine dinucleotide; NAD, nicotinamide adenine

di·nucleotide. [Color figure can be viewed at wileyonlinelibrary.com]

YANG ET AL.

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m‐chlorophenylhydrazone) and nigericin, the internal pH is decreased, with a drop in the intracellular potassium

levels in the meantime.

This process differs from the action of valinomycin, as only K

influx is observed.

Additionally, biocide triclosan (TCL) and antimalarial drug proguanil hydrochloride (PROG) display another

possibility of inducing the enhanced influx of both H

and K

3|PMF AS A PROMISING TARGET FOR NOVEL ANTIMICROBIAL

AGENTS

As increasing therapeutic drugs gradually lose their efficacies against drug‐resistant pathogens, developing novel

antimicrobial agents with distinct targets is urgently warranted. As mentioned above, PMF regulates many critical

physiological processes, such as ATP synthesis, signal transduction, macromolecule synthesis, and proteostasis in

bacterial cells.

As a result, those compounds with PMF‐disturbing activities are potential candidates for

suppressing bacterial infections, especially involving transmembrane transport of solutes or cytoplasmic pH

maintenance.

3.1 |Antimicrobial agents acting upon the Δψ component of PMF

The two components of PMF are closely linked as the positively charged protons generate Δψ and the chemical

separation across the membrane otherwise produces the ΔpH. More generally, Δψ contributes more to PMF

FIGURE 2 Measurement of bacterial PMF and ion changes using fluorescent probes. (A) Structural formula of

DiSC

(5), a fluorescence dye sensitive to membrane potential. (B) The relationship between the fluorescence

intensity of DiSC

(5) and the disruption of two components of the bacterial PMF. (C) Effect of some PMF disruptors

on the influx and efflux of H

and K

ions. Nigericin decreases intracellular pH and potassium levels, triclosan

decreases internal pH but enhances K

influx, while valinomycin only leads to the accumulation of intracellular

.ΔΨ, electric potential; ΔpH, transmembrane proton gradient; DiSC

(5), 3,3ʹ‐dipropylthiacarbocyanine

iodide; PMF, proton motive force. [Color figure can be viewed at wileyonlinelibrary.com]

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YANG ET AL.

generation than ΔpH due to the production of larger potential energy from charge separation.

Suitable membrane

potential is vital for bacterial survival and various activities; meanwhile, bacteria can harbor completely independent

space isolated from the external environment, especially in extreme conditions.

Hence, minor ionic disturbances

induced by capable compounds is prone to disturb PMF homeostasis in bacteria, and more severely, its survival.

Thus, the Δψ component of PMF can be regarded as a potential antibacterial target (Table 1and Figure 3A).

As a proof‐of‐concept, several FDA‐approved drugs display their antimicrobial capabilities by disrupting the ΔΨ

component. For example, daptomycin,

telavancin,

and HT61,

all work through potassium or calcium‐

dependent dissipation of ΔΨ, permeabilizing and depolarizing the cytoplasmic membrane. Moreover, pyrazinamide

(PZA), an effective first‐line drug against Mycobacterium tuberculosis, is hydrolyzed to pyrazinoic acid (POA) after

undergoing a series of intra‐and extracellular processes, which leads to the intracellular accumulation of protons

and the loss of membrane potential.

Additionally, some natural products derived from microorganisms or other

sources have been reported to dissipate bacterial ΔΨ. Examples include Lacticin 3147, a bacteriocin produced by

Lactococcus lactis subsp. lactis DPC3147 against a broad range of Gram‐positive bacteria.

At low concentrations,

PMF promotes the interaction between lacticin 3147 and the cytoplasmic membrane, producing the K

‐selective

pores and immediate membrane potential dissipation. Also, Farha et al. performed a high‐throughput screen to

identify molecules that specifically dissipate PMF in S. aureus.

As a consequence, molecules I1–I3 are proved to be

TABLE 1 Antimicrobials acting upon the ΔΨ component of bacterial proton motive force.

Compounds Antibacterial spectrum MIC values References

Daptomycin S. aureus 8μg/mL [41]

Telavancin Gram‐positive bacteria 0.06 μg/mL [42]

HT61 S. aureus 8μg/mL [43]

Pyrazinamide M. tuberculosis 50 μg/mL [44]

Lactincin 3147 Gram‐positive bacteria / [45]

Molecule I1–I3 S. aureus 2–8μg/mL [46]

JBC 1847 S. aureus 0.5–2μg/mL [47]

Oritavancin S. aureus 0.5–4μg/mL [48]

SC5005 S. aureus 0.5 mg/L [49]

Blestriacin S. aureus 2–8 µg/mL [50]

Pyrazolopyrimidinnons S. aureus 5–40 μM[51]

IMRG4 S. aureus 25–50 mg/L [52]

Paenipeptin C' P. aeruginosa 1–2μg/mL [53]

S. aureus 16–32 μg/mL

Pal‐α‐MSH (6–13) MRSA >45.45 μM[54]

Pal‐α‐MSH (11–13) 11.36 μM

WRK‐12 MDR Gram‐negative bacteria 2 μg/mL [24]

WW307 MDR bacteria 1–8μg/mL [55]

Bacaucin‐1 MRSA 4 μg/mL [27]

Abbreviations: ΔΨ, electric potential; MDR, multidrug‐resistant; MIC, minimum inhibitory concentration; MRSA, methicillin‐

resistant Staphylococcus aureus; M. tuberculosis, Mycobacterium tuberculosis; P. aeruginosa, Pseudomonas aeruginosa;

S. aureus, Staphylococcus aureus.

YANG ET AL.

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the potential modulators of ΔΨ, subsequently hindering electron transport and ATP synthesis. In particular,

molecule I1 is identified as a type II topoisomerase inhibitor in E. coli, and molecule I3 is a 7,9‐dialkylpurinium salt

naturally isolated from marine sponges as a secondary metabolite.

56,57

Furthermore, the emergence of some synthesized compounds aimed at disrupting ΔΨ is also exciting. JBC

1847, a phenothiazine derivative, is a positively charged cationic amphiphilic compound exhibiting significantly

improved antistaphylococcal activity through membrane depolarization.

Likewise, oritavancin is a semisynthetic

lipoglycopeptide effective in eliminating S. aureus cells in an exponential growth state.

The underlying

mechanisms include cell wall injury and membrane potential disruption. In our previous studies, some

synthetic antimicrobial peptides (AMPs) are also exploited to have antibacterial activities by disrupting ΔΨ.

FIGURE 3 Chemical structures of representative antibacterial compounds as (A) ΔΨ disruptors and

(B) ΔpH disruptors in fighting against Gram‐positive bacteria or Gram‐negative bacteria. ΔΨ,electric

potential; ΔpH, transmembrane proton gradient; AMG, α‐mangostin; IBC, isobavachalcone. [Color figure can

be viewed at wileyonlinelibrary.com]

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YANG ET AL.

Specifically, an amphiphilic peptide WRK‐12 is designed to cope with various multidrug‐resistant (MDR) bacteria,

including methicillin‐resistant S. aureus (MRSA), mcr‐1‐positive E. coli (MCRPEC), and tigecycline‐resistant E. coli

through membrane permeabilization, ΔΨ dissipation, and reactive oxygen species (ROS) accumulation.

Similarly,

in combating MDR pathogens, WW307 peptide damages the membrane by selectively targeting multiple

components such as LPS in the outer membrane and PG in the bacterial cytoplasmic membrane,

disrupting ΔΨ

and enhancing ROS. Additionally, the safer derivative of bacaucin, ring‐opened heptapeptide bacaucin‐1, shows

specific antibacterial activity against MRSA by dissipating the ΔΨ component,

which is similar to lysocin.

Recently, more compounds such as sorafenib derivative SC5005,

blestriacin,

pyrazolopyrimidinones,

IMRG4,

paenipeptin C’(C8‐Pat),

Pal‐α‐MSH (6–13), and Pal‐α‐MSH (11–13)

have been identified as

potential ΔΨ dissipators to fight against various notorious pathogenic bacteria.

3.2 |Antimicrobial agents acting upon the ΔpH component of PMF

As another component of PMF, ΔpH also plays an integral part in many membrane processes. The ΔpH directly

influences PMF. Generally, the antibacterial activity of the potential dissipaters of ΔΨ is intensely enhanced when

the pH becomes alkaline, decreasing the pH gradient. In contrast, a shift toward acidity increases ΔpH, reinforcing

the antibacterial ability of the ΔpH dissipators. The representative ΔpH dissipators are listed in Table 2and

Figure 3B.

In this context, some membrane‐targeting peptide compounds show tremendous potential. As a nonribosomal

peptide, tridecaptin A1 originated from Bacillus and Paenibacillus species is effective against various Gram‐negative

bacteria, including MDR Klebsiella pneumoniae and E. coli.

Independent of membrane lysis or depolarization,

tridecaptin A1 causes pore formation and the loss of proton gradient on the inner membrane by specifically binding

TABLE 2 Antimicrobials acting upon the ΔpH component of bacterial proton motive force.

Compounds Antibacterial spectrum MIC values References

Tridecaptin A1 Gram‐negative bacteria 3.13–50 μg/mL [28]

EcDBS1R4 E. coli 25 μg/mL [58]

Hepcidin 20 and 25 E. coli 25/50 μg/mL [59]

Bifidobacterium breve (YH68) C. difficile /[60]

Bile salts S. aureus 20/1/>200 mM [61]

Nisin L. monocytogenes /[62]

Halicin M. tuberculosis 2 µg/mL [26]

A. baumannii

Molecule D1–D3 S. aureus 8–128 μg/mL [46]

Niclosamide H. pylori 0.25 µg/mL [63]

α‐mangostin (AMG) Isobavachalcone (IBC) MRSA 0.5 µg/mL [25]

4 µg/mL

Nature flavones MRSA 1–8 µg/mL [64]

Abbreviations: ΔpH, transmembrane proton gradient; A. baumannii, Acinetobacter baumannii; C. difficile, Clostridioides

difficile; E. coli, Escherichia coli; H. pylori, Helicobacter pylori; L. monocytogenes, Listeria monocytogenes; MIC, minimum

inhibitory concentration; MRSA, methicillin‐resistant Staphylococcus aureus; M. tuberculosis, Mycobacterium tuberculosis; S.

aureus, Staphylococcus aureus.

YANG ET AL.

1075

with lipid II in Gram‐positive bacteria. Likewise, an AMP, EcDBS1R4, causes membrane hyperpolarization and the

increased assembly of inner membrane‐like (IML) vesicles in E. coli.

Herein, rearrangements of CL, a constitutive

phospholipid closely contacting with electron transport chain proteins, block the dissipation of proton potential

used for ATP synthesis, eventually causing hyperpolarization and the subsequent collapse of ΔpH. Additionally,

another research discusses the pH‐dependent interference of E. coli membranes by human AMPs hepcidin 20

and 25.

Biological agents such as probiotics and organic salts are widely used daily. Some of them harbor impressive

antibacterial properties by targeting ΔpH. For example, bifidobacterium breve (YH68) is common in food

fermentation. It can counter Clostridioides difficile infections by disrupting ΔΨ and ΔpH of the cytoplasmic

membrane, leading to pore formation and cell disintegration.

Bile salt supplements lower cholesterol levels by

emulsifying lipids, providing a new train of thought for controlling drug‐resistant bacteria.

It has been reported

that both conjugated and unconjugated bile salts at subinhibitory concentrations collapse the bioenergetic process

by reducing intracellular pH in S. aureus.

Moreover, food preservatives such as bacteriocin produced by

Lactobacillus spp. effectively eliminates Listeria monocytogenes cells.

With 1 μg/mL nisin, a ribosomally

synthesized peptide commonly used as a food preservative, entirely dissipates ΔpH in a time‐and

concentration‐dependent manner; however, ΔΨ is only slightly decreased. Also, the natural flavones isolated

from the Morus alba (white mulberry), such as kuwanon G, kuwanon H, mulberrin, and morusin, effectively kill some

MRSA isolates by collapsing ΔpH.

Likewise, AMG and IBC bind to the membrane phospholipids and dissipate

ΔpH, leading to metabolic perturbation.

25,66

In the face of the increasing emergence and dissemination of MDR

bacterial pathogens, these natural products have immense potential to provide new chemical scaffolds, thus

accelerating the discovery of antibacterial agents.

Recently, a deep neural network is trained to predict molecules with antimicrobial activities.

Growth

inhibition is an essential index in library screening. A novel antibiotic termed halicin stands ahead with

predicted antimicrobial activity, lower minimum inhibitory concentration (MIC), and lower structural similarity

to its nearest neighbor antibiotic. Most inspiringly, halicin effectively copes with antibiotic‐tolerant E. coli and

M. tuberculosis and inhibits the growth of numerous MDR Gram‐negative clinical isolates, demonstrating its

unusual antimicrobial effect. Moreover, whole‐transcriptome sequencing comprehensively explains how

halicin combines with iron to cause the dissipation of transmembrane ΔpH. It is an opportune time for

antibiotic discovery based on machine learning approaches. The expectation for narrow‐spectrum agents and

new scaffolds circumventing pre‐existing resistance determinants can be gradually achieved with decreasing

costs. Furthermore, considering the compensation mechanism between ΔΨ and ΔpH and their sensitivities to

a series of perturbations, a more targeted high‐throughput screen is performed and molecules D1–D3 with

capabilities to collapse transmembrane pH gradient of S. aureus are found.

Discovering new effective agents

is a stunning process, while repurposing conventional drugs achieves more productivity. Niclosamide

effectively treats cestodes (tapeworms) by inhibiting energy derived from anaerobic metabolism.

It is also an

anthelmintic drug and has been listed as an essential medicine by World Health Organization (WHO).

A recent study has further revealed its activity against Helicobacter pylori via disruption of transmembrane pH,

especially at lower MIC values.

For a long time, membrane‐active agents have been set aside during the process of developing novel

antimicrobials as their propensity to induce toxicity in mammals.

However, considering its performance in treating

drug‐resistant bacteria and the recent success of some antibiotic mechanisms, this initially apparent dismal

antibacterial target has regained increasing attention.

70–72

As the two components of PMF, ΔΨ and ΔpH, are

compensated mutually for either dissipation. This phenomenon enables PMF to be exquisitely susceptible to a

series of perturbances, rendering them fatal for bacteria.

Collectively, we highlight the significance of ΔΨ and

ΔpH in all bacterial activities, leading to the possibility of discovering previously unknown compounds as new

antimicrobial agents.

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4|ADJUVANT POTENTIAL OF BACTERIAL PMF‐ACTING COMPOUNDS

Although the discovery of novel antimicrobial agents is the most direct approach to killing these drug‐resistant

pathogens, the very low output‐to‐input ratio including substantial economic and time investment, coupled with the

rapid generation of resistant bacteria, limits the feasibility of this strategy. In contrast, restoring the efficacy of

initially effective antimicrobials using antibiotic adjuvants offers an innovative approach to minimizing the

emergence and impact of antibiotic resistance from a novel perspective.

Generally, the resistance mechanism has been comprehensively illustrated until now. Besides inherent

resistance, acquired resistance can be attributed to four main reasons: producing modifying enzymes, altering

targets, active efflux, or reduced uptake.

The latter two are closely related to the functions of bacterial

membrane, especially those of PMF. In the bacterial respiratory chain, PMF can serve as an acute energy responder

for bacterial metabolism and vitality.

Also, relying on the electrochemical gradient across the membrane, PMF

undoubtedly controls the influx and efflux, which is essential for intracellular antibiotic accumulation.

Altogether,

it is not hard to surmise the potential roles of PMF in developing resistance or even tolerance, which can be

attentively considered in the deployment of new antibiotic adjuvants. In Table 3, we provide a list of these

representative PMF‐targeting adjuvants.

4.1 |PMF correlates with bacterial resistance and tolerance

It is indisputable that we still get confronted with the intensified resistance crisis despite the numerous antibiotic

categories, warranting urgent novel antibacterial strategies from different angles. Recently, bacterial metabolism

has been implicated in the development of resistance.

Studies have thoroughly profiled drug resistance issues

across the whole metabolic state of bacteria, and explored ways to increase intracellular antibiotic concentrations

and reverse antibiotics resistance by establishing overall metabolism regulation.

Their efforts identified some key

metabolites that rearrange resistant metabolome to a sensitive, promoting the uptake of antibiotics and effectively

diminishing resistant bacteria.

77,87

Moreover, central carbon energy metabolism‐PMF is discovered as a new

resistance metabolism regulation pathway.

78,88

These findings not only highlight the critical reason behind the

decreased PMF in resistant bacteria, but also connect the tricarboxylic acid (TCA) cycle, glycolysis, and pyruvate

metabolism with the respiratory chain and PMF. This creates a new therapeutic method that combines antibiotics

and metabolism regulators. Furthermore, drug‐resistant bacteria have active efflux tactics to decrease intracellular

antibiotic concentration. Therefore, some compounds successfully perturb membrane PMF and damage the normal

operation of efflux pumps and ATP synthesis, ultimately restoring bacterial susceptibility to therapeutic drugs.

Tolerance describes how nongrowing or slow‐growing bacteria survive bactericidal antibiotics that target an

active metabolic state. The nongrowing phenotype subpopulation occupying a small fraction of the bacterial

population, such as that in biofilms, is called persisters.

Recent studies have shown that intermittent antibiotic

exposure promotes rapid evolution of tolerance and persistence.

90,91

Moreover, tolerance contributes to resistance

evolution by giving rise to numerous possible mutations in a bacterial population under periodic exposure to high

antibiotic concentrations.

As a consequence, blocking the development of tolerance makes a significant

difference in resistance evolution. Thus, the factors underlying tolerance phenotype are worthy of being studied

intensively. To investigate the physiological responses outside metabolism shutdown during tolerance evolvement,

Wang et al. showed that active efflux and the activities of some critical membrane proteins due to PMF contribute

to conferring the starvation‐induced tolerance phenotype in E. coli.

Meanwhile, disrupting PMF through

ionophore CCCP or inhibitors of ETC complex otherwise suspends this phenotype. It has been shown that the

preaddition of mannitol to lung infections of Pseudomonas aeruginosa persister cells during biofilm growth reverts

the persister phenotype in a PMF‐dependent manner.

Collectively, both tolerant and persistent phenotypes have

a close link with the state of PMF, suggesting a potential biological switch to block the pathway to resistance.

YANG ET AL.

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TABLE 3 Antibiotic adjuvants targeting bacterial proton motive force and their mechanisms of action.

Compounds Antibiotics Mechanisms References

Glycine, serine, and threonine Kanamycin ΔΨ↑[77]

Amino acids biosynthesis↑

TCA cycle↑

Fructose Kanamycin ΔΨ↑[78]

TCA cycle↑

L‐lysine Kanamycin ΔpH↑[79]

/Gentamicin Antibiotic uptake↑

/Amikacin

Pyruvate, succinate and glutamate Tobramycin ΔpH↑[75]

Biofilm inhibitory activity↑

Antibiotic uptake↑

ML‐7 Tigecycline ΔpH↑,ΔΨ↓[80]

Efflux pump↓

ATP synthesis↓

Carprofen Doxycycline ΔpH↓[81]

Efflux pump↓

Protein synthesis↓

Benzydamine Doxycycline ΔpH↑ΔΨ↓[76]

Antibiotic uptake↑

Protein synthesis↓

Efflux pump↓

Metformin Doxycycline/Minocycline ΔpH↑,ΔΨ↓[82]

Antibiotic uptake↑

Efflux pump↓

TetA protein synthesis↓

Citral Norfloxacin ΔΨ↓[83]

Efflux pump↓

IMRG4 Norfloxacin ΔΨ↓[52]

Membrane permeabilization↑

Efflux pump ↓(NorA)

Melatonin Colistin ΔΨ↓[84]

Efflux pumps↓

Protein synthesis↓

Abbreviations: ΔΨ,electricpotential;ΔpH, transmembrane proton gradient; ATP, adenosine triphosphate; TCA,

tricarboxylic acid.

1078

YANG ET AL.

Herein, we are about to elaborate on the adjuvant functions of PMF regulators in two main classes of antibiotics, as

their primary modes of action rely on different components.

4.2 |PMF in the development of aminoglycoside adjuvants

Aminoglycoside antibiotics exert solid antibacterial activities against the broad extension of notorious MDR bacteria

such as P. aeruginosa, S. aureus, and E. coli.

They are mainly composed of a 2‐deoxystreptamine (2‐DOS) ring and

several amino‐modified sugars, usually connected by glycosidic bonds to form glycosides.

Generally,

aminoglycosides interact with the bacterial outer membrane and arrive at the protein synthesis site in an

energy‐dependent way, irreversibly binding to the 30 S subunit of the 16 S ribosomal RNA (rRNA).

The following

consequences include codon misreading, interrupted peptide elongation, and even the production of malfunction-

ing protein, disrupting the structure of the cell membrane and increasing intracellular antibiotic accumulation.

Representative members like streptomycin, gentamicin, neomycin, and paromomycin are usually suggested in

treating clinical infections such as tuberculosis, brucellosis, tularemia, enterococcal endocarditis, and hepatic

encephalopathy.

Aminoglycoside antibiotics are gradually getting attentioninclinicaluse,withtheresistanceofβ‐lactam

antibiotics and fluoroquinolones in Gram‐negative bacteria becoming severe.

100

Despite this trend, controlling

toxicity and hindering resistance are always of foremost importance. In the treatment of MDR Edwardsiella

piscicida, the combinations of glycine, serine, and threonine present the best efficacy, leading to increased

pyruvate dehydrogenase, α‐ketoglutarate dehydrogenase, and succinate dehydrogenase activities in TCA cycle.

This line of treatment is also marked by enhanced membrane potential and intracellular kanamycin.

Similarly,

fructose effectively activates the TCA cycle to produce NADH, generating PMF to increase the uptake of

kanamycin in treating MDR Edwardsiella tarda.

Later, Webster et al. highlighted the relationship between

antibiotic toxicity, ROS production, and PMF, elaborating how the uncoupler 2,4‐dinitrophenol (DNP) weakens

the efficacy of gentamicin through ΔΨ dissipation in pathogenic E. coli.

This conversely indicates that PMF

actively underpins respiration‐mediated potentiation of aminoglycoside lethality. These findings strongly

suggest that the main adjuvants of aminoglycosides enhance antibiotic efficacy by boosting ΔΨ to regulate

metabolism and activate respiration (Figure 4A). Furthermore, the exogenous addition of L‐lysine potentiates

aminoglycoside activity against Acinetobacter baumannii by enhancing ΔpH and increasing antibiotic uptake.

Endogenous metabolites secreted from 3‐D lung cells also enhance the biofilm inhibitory activity of tobramycin

and promote antibiotic uptake against the opportunistic pathogen P. aeruginosa through the same

mechanism.

These two cases improve our understanding of the underlying modes of action of aminoglycoside

adjuvants on bacterial PMF.

4.3 |PMF in the development of tetracyclines adjuvants

Tetracycline antibiotics have broad‐spectrum antimicrobial activities against microorganisms and serve as the most

widely used antibiotic in veterinary clinics worldwide.

101,102

Numerous compounds are subsequently included in

this antibiotic category, such as the natural product chlortetracycline and second and third‐generation derivatives

with improved antibacterial potency and resistance coverage. Typical drugs are semisynthetic rolitetracycline,

doxycycline, minocycline, and, more importantly, tigecycline.

103

Nowadays, tigecycline is one of the last‐resort

antibiotics to treat complicated infections caused by MDR bacteria, including vancomycin‐resistant Enterococcus

faecium, MRSA and carbapenem‐resistant Enterobacteriaceae (CRE).

104

Unfortunately, due to increasing clinical

usage, tetracycline resistance is conferred in susceptible bacterial populations through horizontal gene transfer

(HGT), triggering overexpression of efflux pumps and ribosome protection.

105–107

Altogether, it is urgent to develop

YANG ET AL.

1079

potent adjuvants for tigecycline or other tetracyclines. Sun et al. found that tigecycline with ML‐7 entirely inhibits

bacterial growth with synergistic bactericidal activities against drug‐resistant isolates.

The underlying mechanisms

include the increased ΔpH and the subsequent inhibition of the efflux pump and ATP synthesis. Moreover,

coexposure to carprofen restores doxycycline susceptibility in methicillin‐resistant Staphylococcus pseudintermedius

(MRSP) ST71.

Herein, TetK‐mediated drug efflux is paralyzed by ΔpH dissipation and the intracellular protein

synthesis is then broken off. Our previous studies showed that nonantibiotic pharmaceutical benzydamine is also

effective against a series of clinically hazardous pathogens in combination with tetracyclines,

76,108

including

TMexCD‐TOprJ‐expressing bacteria that confer high‐level tigecycline resistance.

109

In‐depth studies indicate that

the potentiation of benzydamine to tigecycline correlates with PMF changes. On the one hand, benzydamine

addition disrupts ΔΨ and upregulates ΔpH to compensate, leading to the increased uptake of tetracyclines and the

inhibition of protein synthesis. On the other hand, dissipated PMF reduces efflux pump activity, contributing to the

increased intracellular drug accumulation, thus more effectively killing bacteria. Similarly, the adjuvant potential of

PMF‐acting drugs is also thoroughly clarified in the case of metformin, a first‐line antidiabetic drug, which could be

a potent adjuvant for tetracyclines in the fight against various MDR pathogens, including the notorious MRSA.

The increased ΔpH promotes antibiotic uptake, while the decrease of PMF inhibits the activity of the efflux pump

and the protein synthesis of TetA. Altogether, ΔpH critically regulates tetracyclines resistance and can serve as a

FIGURE 4 Adjuvants of aminoglycoside or tetracycline antibiotics act by targeting different components of

bacterial PMF. (A) Active bacterial metabolism induced by exogenously adding adjuvants makes aminoglycosides

more effective as they potentiate the tricarboxylic acid (TCA) cycle and produce more NADH, which together

boosts ΔΨ and PMF, thus promoting aminoglycoside uptake. (B) Tetracyclines adjuvants not only increase ΔpH to

enhance intracellular drug accumulation but also disrupt PMF to paralyze the function of the efflux pump, thereby

restoring the antibacterial activity of tetracyclines. ΔΨ, electric potential; ΔpH, transmembrane proton gradient;

ATP, adenosine triphosphate; PMF, proton motive force. [Color figure can be viewed at wileyonlinelibrary.com]

1080

YANG ET AL.

promising target for screening effective tetracycline adjuvants, significantly improving drug uptake and reducing

efflux activity (Figure 4B).

4.4 |PMF‐modulating compounds resensitize other antibiotic classes

Besides these two classical categories, PMF also restores the susceptibility of resistant bacteria to many other

antibiotics. Fluoroquinolones harbor a broader antibacterial spectrum and better efficacy than quinolones due to

the additional fluorine atom at the sixth position.

110

Owing to such properties, the current UK prescribing

guidelines once recommended fluoroquinolones as second‐line agents to maintain their effectiveness. However,

resistance development has never slowed, warranting suitable sensitizers.

111

Previous research has reported high‐

level fluoroquinolone resistance of Shigella dysenteriae induced by a PMF‐dependent efflux system.

112

Several

fluoroquinolone adjuvants are found to influence ΔΨ or ΔpH. For example, in the battle against MRSA, one

monoterpenoid aldehyde citral exerts a synergistic effect with norfloxacin through multiple mechanisms, including

efflux pump inhibition and membrane potential disruption.

Similarly, a novel bi‐functional chalcone IMRG4

effectively inhibits MRSA by potentiating the activity of fluoroquinolone through membrane depolarization and

permeabilization.

Additionally, colistin is a “last resort”antibiotic for treating infections caused by many dangerous Gram‐

negative pathogens. Some previous studies in Burkholderia thailandensis have demonstrated a DedA family protein

as a transmembrane transporter in the development of colistin resistance via covalent modification of lipid A in

LPS.

113

However, the activity of DbcA (DedA of Burkholderia required for colistin resistance) is proton‐dependent,

with PMF disruptors restoring colistin efficacy.

114

Additionally, our previous study illustrated how melatonin

reverses MCR‐mediated colistin resistance in Gram‐negative bacteria by disrupting membrane potential and

inhibiting efflux pumps and ABC transporter.

Meanwhile, the uptake of erythromycin and azithromycin,

representatives of macrolides in Haemophilus influenzae ATCC 19418, is evidently enhanced through a passive

diffusion upon ΔpH disruption.

115

Taken together, these successful examples encouragingly demonstrate the

universal applicability of bacterial PMF in regulating bacterial resistance. The recent decades have witnessed

the diminishing effectiveness of various originally potent antibiotics, posing a global threat to public health. In this

regard, exploiting new classes of antibiotics is time‐consuming and requires a long cycle while restoring the

susceptibility of existing antibiotics has more advantages. PMF modulators display their adjuvant potential against

resistance as sensitizers of antimicrobials and can reverse the resistance or tolerance phenotype. Such findings

immensely aid this branch of research in the present and the future, finding the optimal antibacterial strategies with

the lowest possibility of inducing resistance. The involvement of bacterial PMF is opening new insights toward

identifying reliable antibiotic adjuvants. We anticipate the development of similar combinations in treating

nosocomial infections.

5|PMF DISRUPTORS PREVENT THE TRANSMISSION OF ARGs

Compared with intrinsic resistance, acquired resistance is recognized as the leading cause of the rapid prevalence of

drug‐resistant bacteria worldwide.

74,116–118

ARGs on the chromosome are usually replicated during bacterial

division and proliferation. At the same time, plasmid‐mediated HGT is achieved through conjugation,

transformation, transduction, and vesiduction.

119

Direct transmission through conjugation and indirect transmission

through transformation have a subversive impact on the whole genetic environment in the bacterial population.

120

To effectively control the propagation of AMR, it is necessary to suppress these pathways through additional

interferences. Thus, preventing the plasmid‐mediated horizontal transfer of ARGs provides a feasible approach to

minimizing this global crisis.

YANG ET AL.

1081

HGT involves the transmembrane process and requires energy consumption, indicating the potential role of

PMF in its regulation. In agreement with this notion, increased PMF is reported to provide more power for

exogenous DNA uptake in many conjugation processes.

121,122

Interestingly, we recently found that melatonin

effectively inhibits the horizontal transfer of mcr‐harboring plasmids between intra‐and interspecies (Figure 5A).

123

We also highlight how melatonin disrupts bacterial PMF, which is intimately related to the energy supply and

conjugative transfer process. These findings shed new light on screening effective conjugation inhibitors by

perturbing bacterial PMF. Not merely, compounds blocking the competence can also interrupt transformation as

competence provides a physiological state to meet the requirements for the uptake and integration of the

exogenous DNA (Figure 5B).

124

Therefore, Domenech et al. identified COM‐blockers in Streptococcus pneumoniae

from 1380 drugs and further illustrated their mechanisms of action by disrupting the PMF‐dependent export

of quorum‐sensing peptide that activates competence.

The following studies confirm the efficacy of these

COM‐blockers in inhibiting transformation in human cell line colonization and in vivo models. Altogether, it would

be interesting in practice to develop strategies restricting resistance transmission through PMF disruption to reduce

the global burden of AMR.

6|CONCLUSION AND OUTLOOK

AMR is one of the most pressing issues faced by the healthcare system today. Such a situation demands the urgent

development of novel antibacterial agents with distinct scaffolds or targets to combat the resistance crisis in the

postantibiotic era. In the early stages, all kinds of antibiotics are subsequently discovered with targets in the cell

walls, on the membrane or during the synthesis of protein and genetic material.

Yet, in recent years, researchers

have tried to steer clear from discovering membrane‐active agents either damaging the membrane integrity or

FIGURE 5 The role of PMF in exogenous DNA uptake during bacterial conjugation and transformation. (A) The

potential mechanisms related to PMF in the donor strain during conjugation. For example, melatonin inhibits DNA

uptake, flagellar motility, and ATP synthesis through PMF collapse, especially disrupting ΔΨ. Changes in the

intracellular ion concentrations further support the PMF disruption. (B) The underlying mechanisms of competence

and transformation inhibition in S. pneumoniae. The comC‐encoded competence‐stimulating peptide (CSP) is

cleaved and transported outside the cell by ComAB with the support of PMF. Then, histidine‐kinase ComD is

autophosphorylated upon binding with CSP. The phosphate group is then transferred to the regulator ComE to

form phosphorylated ComE, which activates the former competence genes, including comAB and comCDE,

generating a positive feedback pathway. Meanwhile, comX is also activated to export a sigma factor (SigX),

contributing to the activation of genes responsible for transformation and DNA repair. ΔΨ, electric potential; ΔpH,

transmembrane proton gradient; ATP, adenosine triphosphate; PMF, proton motive force. [Color figure can be

viewed at wileyonlinelibrary.com]

1082

YANG ET AL.

targeting energetics, especially those with a propensity to induce mammalian toxicity. Meanwhile, the gradually

emerging combined consensus indicates that potential candidates acting on cell membranes should be excluded

from further studies in whole‐cell screenings. Analytical methods for detecting these undesirable compounds are

also available to ease this process.

125,126

But recently, PMF has been frequently reported to have indispensable

functions in various membrane processes during the safe administration of potent antimicrobials, vastly raising our

confidence and anticipation.

70,127

This phenomenon implies that this originally unpopular and unexplored drug

target related to the energetic pathways of bacterial membrane is being rejuvenated for further research.

From a comprehensive perspective, we conclude three main approaches to combating infections of pathogenic

bacteria, that is, exploiting direct bactericidal or bacteriostatic drugs, identifying novel antibiotic adjuvants, and

blocking the transmission of AMR. In this review, we have illustrated the roles of bacterial PMF modulators in

regulating these three aspects with multitudinous examples (Figure 6). Most importantly, PMF is closely linked with

the respiratory chain that participates in intracellular energy transformation and transmembrane substance

transport, satisfying the essential physiological requirements and supporting common bacterial activities. Also,

stable membrane potential allows the ion flows on both sides of the membrane to maintain a dynamic balance.

Herein, H

homeostasis is pivotal to the bacterial inner environment, with a suitable pH ensuring the normal

functions of various critical enzymes. Therefore, such important status and flexible changes render PMF a target

that can be regulated through external intervention. Indeed, we are excited to identify some potent agents showing

direct bactericidal or bacteriostatic effects via PMF interruption (Figure 6A). Moreover, a dedicated bacterial

internal regulation network crucially regulates bacterial survival. The pressure exerted by aminoglycoside antibiotics

FIGURE 6 Three strategies to cope with drug‐resistant pathogens by targeting bacterial PMF. (A) PMF is a

promising target for new antibacterial agents. (B) The supplementation of PMF regulators restores the antibacterial

activity of certain antibiotics against drug‐resistant bacteria. (C) PMF disruptors block the propagation of resistance

genes by preventing conjugation and transformation. PMF, proton motive force. [Color figure can be viewed at

wileyonlinelibrary.com]

YANG ET AL.

1083

causes the bacteria to survive at the cost of reduced metabolism.

Main energy circles may have defects in one or

several segments, causing downstream responses such as PMF dissipation and ATP synthesis inhibition. In this way,

the suitable addition of exogenous substances, especially those defective metabolites, accelerates the accumulation

and uptake of aminoglycosides by promoting PMF, primarily by regulating ΔΨ, thus resensitizing drug‐resistant

bacteria (Figure 6B). Meanwhile, some PMF disruptors serve as potent adjuvants for tetracycline antibiotics,

restoring their antibacterial activities via enhanced uptake due to an increased ΔpH and reduced efflux by inhibiting

PMF. Furthermore, comprehensive consideration reveals that suppressing bacterial resistance is warranted before

it becomes rampant, with more attention being paid to cutting off transmission pathways. Conjugation and

transformation are common ways of plasmid‐mediated horizontal transfer of ARGs.

120

Recent studies highlight that

disrupted PMF induced by some compounds cannot maintain the competence or sufficiently provide energy for

exogenous DNA uptake, thus enormously preventing the dissemination of ARGs (Figure 6C).

39,123

In summary, we highlight that bacterial PMF essentially regulates resistance emergence, evolution, and

transmission. PMF may be one of the promising targets for developing novel strategies to control the increasing

MDR bacterial infections. However, it is also noteworthy that both PMF disruptors and regulators still warrant a

series of preclinical studies. Many setbacks in using these promising compounds, such as nonspecific cytotoxicity on

mammalian cells or other multimodal activities, are undesirable for drug development. For example, despite being

typical ΔΨ disruptors, the applications of polymyxins (i.e., polymyxin B and colistin) are largely limited by safety

concerns such as acute toxicity and dose‐limiting nephrotoxicity. Likewise, the development of surotomycin

targeting C. difficile through cellular membrane depolarization is eventually suspended after Phase III studies for

unsatisfactory noninferiority criteria compared with vancomycin and a higher propensity to cause nausea and

headache.

128,129

Despite these obstacles, the introduction of new technologies, such as computer‐assisted design,

will undoubtedly contribute to the development of safer and more effective antibacterial drugs, as reflected in the

success of novel polymycin derivatives with lower nephrotoxicity and improved therapeutic indices.

130–132

Therefore, it is worth anticipating the extraordinary efficacy of PMF‐targeting compounds in treating MDR bacterial

infections by effectively avoiding their potential side effects.

ACKNOWLEDGMENTS

This work was supported by the National Key Research and Development Program of China (2021YFD1801000

and 2018YFA0903400), National Natural Science Foundation of China (32222084, 32172907 and 32002331),

Jiangsu Agricultural Science and Technology Innovation Fund (CX(21)2010), A Project Funded by the Priority

Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Young Elite Scientists

Sponsorship Program by CAST (2020QNRC001) and 111 Project D18007.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT

The data used to support the findings of this study are available from the corresponding authors upon request.

ORCID

Yuan Liu http://orcid.org/0000-0002-9622-6471

REFERENCES

1. Murray CJ, Ikuta KS, Sharara F, et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic

analysis. Lancet. 2022;399(10325):629‐655.

2. Theuretzbacher U, Outterson K, Engel A, Karlén A. The global preclinical antibacterial pipeline. Nat Rev Microbiol.

2020;18(5):275‐285.

1084

YANG ET AL.

3. Galarion LH, Mohamad M, Alzeyadi Z, Randall CP, O'Neill AJ. A platform for detecting cross‐resistance in

antibacterial drug discovery. J Antimicrob Chemother. 2021;76(6):1467‐1471.

4. Vila J, Moreno‐Morales J, Ballesté‐Delpierre C. Current landscape in the discovery of novel antibacterial agents. Clin

Microbiol Infect. 2020;26(5):596‐603.

5. MacNair CR, Farha MA, Serrano‐Wu MH, et al. Preclinical development of pentamidine analogs identifies a potent

and nontoxic antibiotic adjuvant. ACS Infect Dis. 2022;8(4):768‐777.

6. Zhang N, Ma S. Recent development of membrane‐active molecules as antibacterial agents. Eur J Med Chem.

2019;184:111743.

7. Zhou M, Zheng M, Cai J. Small molecules with membrane‐active antibacterial activity. ACS Appl Mater Interfaces.

2020;12(19):21292‐21299.

8. Pader V, Hakim S, Painter KL, Wigneshweraraj S, Clarke TB, Edwards AM. Staphylococcus aureus inactivates

daptomycin by releasing membrane phospholipids. Nat Microbiol. 2016;2:16194.

9. Song M, Liu Y, Huang X, et al. A broad‐spectrum antibiotic adjuvant reverses multidrug‐resistant Gram‐negative

pathogens. Nat Microbiol. 2020;5(8):1040‐1050.

10. Johnston CW, Skinnider MA, Dejong CA, et al. Assembly and clustering of natural antibiotics guides target

identification. Nat Chem Biol. 2016;12(4):233‐239.

11. O'Rourke S, Widdick D, Bibb M. A novel mechanism of immunity controls the onset of cinnamycin biosynthesis in

Streptomyces cinnamoneus DSM 40646. J Ind Microbiol Biotechnol. 2017;44(4‐5):563‐572.

12. Hamamoto H, Urai M, Ishii K, et al. Lysocin E is a new antibiotic that targets menaquinone in the bacterial membrane.

Nat Chem Biol. 2015;11(2):127‐133.

13. Kaur H, Jakob RP, Marzinek JK, et al. The antibiotic darobactin mimics a β‐strand to inhibit outer membrane

insertase. Nature. 2021;593(7857):125‐129.

14. Imai Y, Meyer KJ, Iinishi A, et al. A new antibiotic selectively kills Gram‐negative pathogens. Nature. 2019;576(7787):

459‐464.

15. Luther A, Urfer M, Zahn M, et al. Chimeric peptidomimetic antibiotics against Gram‐negative bacteria. Nature.

2019;576(7787):452‐458.

16. Le D, Krasnopeeva E, Sinjab F, Pilizota T, Kim M. Active efflux leads to heterogeneous dissipation of proton motive

force by protonophores in bacteria. mBio. 2021;12(4):e00676‐21.

17. Zhou J, Liu L, Shi Z, Du G, Chen J. ATP in current biotechnology: regulation, applications and perspectives. Biotech

Adv. 2009;27(1):94‐101.

18. Yuroff AS, Sabat G, Hickey WJ. Transporter‐mediated uptake of 2‐chloro‐and 2‐hydroxybenzoate by Pseudomonas

huttiensis strain D1. Appl Environ Microbiol. 2003;69(12):7401‐7408.

19. Klipp E, Nordlander B, Krüger R, Gennemark P, Hohmann S. Integrative model of the response of yeast to osmotic

shock. Nature Biotechnol. 2005;23(8):975‐982.

20. Watanabe Y, Oshima N, Tamai Y. Co‐expression of the Na

‐antiporterand H

‐ATPase genes of the salt‐tolerant

yeast Zygosaccharomyces rouxii in Saccharomycescerevisiae.FEMS Yeast Res. 2005;5(4‐5):411‐417.

21. Kragol G, Lovas S, Varadi G, Condie BA, Hoffmann R, Otvos L. The antibacterial peptide pyrrhocoricin inhibits the

ATPase actions of DnaK and prevents chaperone‐assisted protein folding. Biochemistry. 2001;40(10):3016‐3026.

22. Deutscher J, Francke C, Postma PW. How phosphotransferase system‐related protein phosphorylation regulates

carbohydrate metabolism in bacteria. Microbiol Mol Biol Rev. 2006;70(4):939‐1031.

23. Kipnis Y, Papo N, Haran G, Horovitz A. Concerted ATP‐induced allosteric transitions in GroEL facilitate release of

protein substrate domains in an all‐or‐none manner. Proc Natl Acad Sci USA. 2007;104(9):3119‐3124.

24. Liu Y, Shi J, Tong Z, Jia Y, Yang K, Wang Z. Potent broad‐spectrum antibacterial activity of amphiphilic peptides

against multidrug‐resistant bacteria. Microorganisms. 2020;8(9):1398.

25. Song M, Liu Y, Li T, et al. Plant natural flavonoids against multidrug resistant pathogens. Adv Sci. 2021;

8(15):2100749.

26. Stokes JM, Yang K, Swanson K, et al. A deep learning approach to antibiotic discovery. Cell. 2020;180(4):688‐702.

27. Liu Y, Ding S, Dietrich R, Märtlbauer E, Zhu K. A biosurfactant‐inspired heptapeptide with improved specificity to kill

MRSA. Angew Chem Int Ed. 2017;56(6):1486‐1490.

28. Cochrane SA, Findlay B, Bakhtiary A, et al. Antimicrobial lipopeptide tridecaptin A1 selectively binds to Gram‐

negative lipid II. Proc Natl Acad Sci USA. 2016;113(41):11561‐11566.

29. Mitchell P. Coupling of phosphorylation to electron and hydrogen transfer by a chemi‐osmotic type of mechanism.

Nature. 1961;191:144‐148.

30. Rieger B, Arroum T, Borowski MT, Villalta J, Busch KB. Mitochondrial F1Fo ATP synthase determines the local

proton motive force at cristae rims. EMBO Rep. 2021;22(12):e52727.

31. Berry BJ, Trewin AJ, Amitrano AM, Kim M, Wojtovich AP. Use the protonmotive force: mitochondrial uncoupling

and reactive oxygen species. J Mol Biol. 2018;430(21):3873‐3891.

YANG ET AL.

1085

32. Kaila VRI, Wikström M. Architecture of bacterial respiratory chains. Nat Rev Microbiol. 2021;19(5):319‐330.

33. Cotter PA, Chepuri V, Gennis RB, Gunsalus RP. Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene

expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product. JBacteriol. 1990;172(11):

6333‐6338.

34. Tseng CP, Albrecht J, Gunsalus RP. Effect of microaerophilic cell growth conditions on expression of the aerobic

(cyoABCDE and cydAB) and anaerobic (narGHJI, frdABCD, and dmsABC) respiratory pathway genes in Escherichia

coli.J Bacteriol. 1996;178(4):1094‐1098.

35. Abdulkadieva MM, Sysolyatina EV, Vasilieva EV, et al. Strain specific motility patterns and surface adhesion of

virulent and probiotic Escherichia coli.Sci Rep. 2022;12(1):614.

36. Bakker EP, Mangerich WE. Interconversion of components of the bacterial proton motive force by electrogenic

potassium transport. J Bacteriol. 1981;147(3):820‐826.

37. Mohiuddin SG, Ghosh S, Kavousi P, Orman MA. Proton motive force inhibitors are detrimental to Methicillin‐

Resistant Staphylococcus aureus strains. Microbiol Spectr. 2022;10(4):e0202422.

38. Te Winkel JD, Gray DA, Seistrup KH, Hamoen LW, Strahl H. Analysis of antimicrobial‐triggered membrane

depolarization using voltage sensitive dyes. Front Cell Dev Biol. 2016;4:29.

39. Domenech A, Brochado AR, Sender V, et al. Proton motive force disruptors block bacterial competence and

horizontal gene transfer. Cell Host Microbe. 2020;27(4):544‐555.

40. Benarroch JM, Asally M. The microbiologist's guide to membrane potential dynamics. Trends Microbiol. 2020;28(4):

304‐314.

41. Ma W, Zhang D, Li G, et al. Antibacterial mechanism of daptomycin antibiotic against Staphylococcus aureus based on

a quantitative bacterial proteome analysis. J Proteomics. 2017;150:242‐251.

42. Al Jalali V, Zeitlinger M. Clinical pharmacokinetics and pharmacodynamics of telavancin compared with the other

glycopeptides. Clin Pharmacokinet. 2018;57(7):797‐816.

43. Hubbard ATM, Barker R, Rehal R, Vandera KKA, Harvey RD, Coates ARM. Mechanism of action of a membrane‐

active quinoline‐based antimicrobial on natural and model bacterial membranes. Biochemistry. 2017;56(8):

1163‐1174.

44. Lamont EA, Dillon NA, Baughn AD. The bewildering antitubercular action of pyrazinamide. Microbiol Mol Biol Rev.

2020;84(2):e00070‐19.

45. Ryan A, Patel P, O'Connor PM, Ross RP, Hill C, Hudson SP. Pharmaceutical design of a delivery system for the

bacteriocin lacticin 3147. Drug Delivery Transl Res. 2021;11(4):1735‐1751.

46. Farha MA, Verschoor CP, Bowdish D, Brown ED. Collapsing the proton motive force to identify synergistic

combinations against Staphylococcus aureus.Chem Biol. 2013;20(9):1168‐1178.

47. Ronco T, Kappel LH, Aragao MF, et al. Insight into the anti‐staphylococcal activity of JBC 1847 at sub‐inhibitory

concentration. Front Microbiol. 2022;12:786173.

48. Belley A, Neesham‐Grenon E, McKay G, et al. Oritavancin kills stationary‐phase and biofilm Staphylococcus aureus

cells in vitro.Antimicrob Agents Chemother. 2009;53(3):918‐925.

49. Lu CH, Shiau CW, Chang YC, et al. SC5005 dissipates the membrane potential to kill Staphylococcus aureus persisters

without detectable resistance. J Antimicrob Chemother. 2021;76(8):2049‐2056.

50. Chen BC, Lin CX, Chen NP, Gao CX, Zhao YJ, Qian CD. Phenanthrene antibiotic targets bacterial membranes and

kills Staphylococcus aureus with a low propensity for resistance development. Front Microbiol. 2018;9:1593.

51. Crawford CL, Dalecki AG, Narmore WT, et al. Pyrazolopyrimidinones, a novel class of copper‐dependent bactericidal

antibiotics against multi‐drug resistant S. aureus.Metallomics. 2019;11(4):784‐798.

52. Gupta VK, Gaur R, Sharma A, et al. A novel bi‐functional chalcone inhibits multi‐drug resistant Staphylococcus aureus

and potentiates the activity of fluoroquinolones. Bioorg Chem. 2019;83:214‐225.

53. Moon SH, Huang E. Novel linear lipopeptide paenipeptin C’binds to lipopolysaccharides and lipoteichoic acid and

exerts bactericidal activity by the disruption of cytoplasmic membrane. BMC Microbiol. 2019;19(1):6.

54. Mumtaz S, Behera S, Mukhopadhyay K. Lipidated short analogue of α‐melanocyte stimulating hormone exerts

bactericidal activity against the stationary phase of methicillin‐resistant Staphylococcus aureus and inhibits biofilm

formation. ACS Omega. 2020;5(44):28425‐28440.

55. Shi J, Chen C, Wang D, Tong Z, Wang Z, Liu Y. Amphipathic peptide antibiotics with potent activity against

multidrug‐resistant pathogens. Pharmaceutics. 2021;13(4):438.

56. Oyamada Y, Ito H, Fujimoto‐Nakamura M, et al. Anucleate cell blue assay: a useful tool for identifying novel type II

topoisomerase inhibitors. Antimicrob Agents Chemother. 2006;50(1):348‐350.

57. Trachtenberg MC, Packey DJ, Sweeney T. In vivo functioning of the Na

‐activated ATPase. Curr Top Cell Regul.

1981;19:159‐217.

58. Makowski M, Felício MR, Fensterseifer ICM, Franco OL, Santos NC, Gonçalves S. EcDBS1R4, an antimicrobial

peptide effective against Escherichia coli with in vitro fusogenic ability. Int J Mol Sci. 2020;21(23):9104.

1086

YANG ET AL.

59. Maisetta G, Vitali A, Scorciapino MA, et al. pH‐dependent disruption of Escherichia coli ATCC 25922 and model

membranes by the human antimicrobial peptides hepcidin 20 and 25. FEBS J. 2013;280(12):2842‐2854.

60. Yang J, Yang H. Antibacterial activity of Bifidobacterium breve against Clostridioides difficile.Front Cell Infect Microbiol.

2019;9:288.

61. Sannasiddappa TH, Lund PA, Clarke SR. In vitro antibacterial activity of unconjugated and conjugated bile salts on

Staphylococcus aureus.Front Microbiol. 2017;8:1581.

62. Bruno ME, Kaiser A, Montville TJ. Depletion of proton motive force by nisin in Listeria monocytogenes cells. Appl

Environ Microbiol. 1992;58(7):2255‐2259.

63. Tharmalingam N, Port J, Castillo D, Mylonakis E. Repurposing the anthelmintic drug niclosamide to combat

Helicobacter pylori.Sci Rep. 2018;8(1):3701.

64. Wu SC, Han F, Song MR, et al. Natural flavones from Morus alba against methicillin‐resistant Staphylococcus

aureus via targeting the proton motive force and membrane permeability. J Agricult Food Chem. 2019;67(36):

10222‐10234.

65. Begley M, Gahan CGM, Hill C. The interaction between bacteria and bile. FEMS Microbiol Rev. 2005;29(4):625‐651.

66. Webster CM, Woody AM, Fusseini S, Holmes LG, Robinson GK, Shepherd M. Proton motive force underpins

respiration‐mediated potentiation of aminoglycoside lethality in pathogenic Escherichia coli.Arch Microbiol.

2022;204(1):120.

67. Kappagoda S, Singh U, Blackburn BG. Antiparasitic therapy. Mayo Clin Proc. 2011;86(6):561‐583.

68. Chen W, Mook RA Jr., Premont RT, Wang J. Niclosamide: beyond an antihelminthic drug. Cell Signal. 2018;41:89‐96.

69. Silver LL. Challenges of antibacterial discovery. Clin Microbiol Rev. 2011;24(1):71‐109.

70. Hurdle JG, O'Neill AJ, Chopra I, Lee RE. Targeting bacterial membrane function: an underexploited mechanism for

treating persistent infections. Nat Rev Microbiol. 2011;9(1):62‐75.

71. Kim W, Zou G, Pan W, et al. The neutrally charged diarylurea compound PQ401 kills antibiotic‐resistant and

antibiotic‐tolerant Staphylococcus aureus.mBio. 2020;11(3):e01140‐20.

72. Kubo S, Niina T, Takada S. Molecular dynamics simulation of proton‐transfer coupled rotations in ATP synthase Fo

motor. Sci Rep. 2020;10(1):8225.

73. Zhao X, Kuipers OP. BrevicidineB, a new member of the brevicidine family, displays an extended target specificity.

Front Microbiol. 2021;12:693117.

74. Munita JM, Arias CA. Mechanisms of antibiotic resistance. Microbiol Spectr. 2016;4(2):VMBF‐0016‐201.

75. Crabbé A, Ostyn L, Staelens S, et al. Host metabolites stimulate the bacterial proton motive force to enhance the

activity of aminoglycoside antibiotics. PLoS Pathog. 2019;15(4):e1007697.

76. Liu Y, Tong Z, Shi J, Jia Y, Deng T, Wang Z. Reversion of antibiotic resistance in multidrug‐resistant pathogens using

non‐antibiotic pharmaceutical benzydamine. Commun Biol. 2021;4(1):1328.

77. Ye J, Lin X, Cheng Z, et al. Identification and efficacy of glycine, serine and threonine metabolism in potentiating

kanamycin‐mediated killing of Edwardsiella piscicida.J Proteomics. 2018;183:34‐44.

78. Su Y, Peng B, Han Y, Li H, Peng X. Fructose restores susceptibility of multidrug‐resistant Edwardsiella tarda to

kanamycin. J Proteome Res. 2015;14(3):1612‐1620.

79. Deng W, Fu T, Zhang Z, et al. L‐lysine potentiates aminoglycosides against Acinetobacter baumannii via regulation of

proton motive force and antibiotics uptake. Emerg Microbes Infect. 2020;9(1):639‐650.

80. Sun L, Sun L, Li X, et al. A novel tigecycline adjuvant ML‐7 reverses the susceptibility of tigecycline‐resistant

Klebsiella pneumoniae.Front Cell Infect Microbiol. 2022;11:809542.

81. Magnowska Z, Jana B, Brochmann RP, et al. Carprofen‐induced depletion of proton motive force reverses TetK‐

mediated doxycycline resistance in methicillin‐resistant Staphylococcus pseudintermedius.Sci Rep. 2019;9(1):17834.

82. Liu Y, Jia Y, Yang K, et al. Metformin restores tetracyclines susceptibility against multidrug resistant bacteria. Adv Sci.

2020;7(12):1902227.

83. Gupta P, Patel DK, Gupta VK, Pal A, Tandon S, Darokar MP. Citral, a monoterpenoid aldehyde interacts

synergistically with norfloxacin against methicillin resistant Staphylococcus aureus.Phytomedicine. 2017;34:85‐96.

84. Liu Y, Jia Y, Yang K, et al. Melatonin overcomes MCR‐mediated colistin resistance in Gram‐negative pathogens.

Theranostics. 2020;10(23):10697‐10711.

85. Bhargava P, Collins JJ. Boosting bacterial metabolism to combat antibiotic resistance. Cell Metab. 2015;21(2):

154‐155.

86. Peng B, Li H, Peng XX. Functional metabolomics: from biomarker discovery to metabolome reprogramming. Protein

Cell. 2015;6(9):628‐637.

87. Yong Y, Zhou Y, Liu K, Liu G, Wu L, Fang B. Exogenous citrulline and glutamine contribute to reverse the resistance

of salmonella to apramycin. Front Microbiol. 2021;12:759170.

88. Ma Y, Guo C, Li H, Peng X. Low abundance of respiratory nitrate reductase is essential for Escherichia coli in

resistance to aminoglycoside and cephalosporin. J Proteomics. 2013;87:78‐88.

YANG ET AL.

1087

89. Maffei E, Fino C, Harms A. Antibiotic tolerance and persistence studied throughout bacterial growth phases.

Methods Mol Biol. 2021;2357:23‐40.

90. Van den Bergh B, Michiels JE, Wenseleers T, et al. Frequency of antibiotic application drives rapid evolutionary

adaptation of Escherichia coli persistence. Nat Microbiol. 2016;1:16020.

91. Mechler L, Herbig A, Paprotka K, Fraunholz M, Nieselt K, Bertram R. A novel point mutation promotes growth

phase‐dependent daptomycin tolerance in Staphylococcus aureus.Antimicrob Agents Chemother. 2015;59(9):

5366‐5376.

92. Levin‐Reisman I, Ronin I, Gefen O, Braniss I, Shoresh N, Balaban NQ. Antibiotic tolerance facilitates the evolution of

resistance. Science. 2017;355(6327):826‐830.

93. Wang M, Chan EWC, Wan Y, Wong MH, Chen S. Active maintenance of proton motive force mediates starvation‐

induced bacterial antibiotic tolerance in Escherichia coli.Commun Biol. 2021;4(1):1068.

94. Ciofu O, Tolker‐Nielsen T. Tolerance and resistance of Pseudomonas aeruginosa biofilms to antimicrobial agents‐how

P. aeruginosa can escape antibiotics. Front Microbiol. 2019;10:913.

95. Becker B, Cooper MA. Aminoglycoside antibiotics in the 21st century. ACS Chem Biol. 2013;8(1):105‐115.

96. Khan F, Pham DTN, Kim YM. Alternative strategies for the application of aminoglycoside antibiotics against the

biofilm‐forming human pathogenic bacteria. Appl Microbiol Biotechnol. 2020;104(5):1955‐1976.

97. Kotra LP, Haddad J, Mobashery S. Aminoglycosides: perspectives on mechanisms of action and resistance and

strategies to counter resistance. Antimicrob Agents Chemother. 2000;44(12):3249‐3256.

98. Walter F, Vicens Q, Westhof E. Aminoglycoside‐RNA interactions. Curr Opin Chem Biol. 1999;3(6):694‐704.

99. Barza M, Scheife RT. Drug therapy reviews: antimicrobial spectrum, pharmacology and therapeutic use of antibiotics,

part 4: aminoglycosides. Am J Health Syst Pharm. 1977;34(7):723‐737.

100. Eliopoulos GM, Drusano GL, Ambrose PG, et al. Back to the future: using aminoglycosides again and how to dose

them optimally. Clin Infect Dis. 2007;45(6):753‐760.

101. Chopra I, Roberts M. Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of

bacterial resistance. Microbiol Mol Biol Rev. 2001;65(2):232‐260.

102. Keßler DN, Fokuhl VK, Petri MS, Spielmeyer A. Abiotic transformation products of tetracycline and chlortetracycline

in salt solutions and manure. Chemosphere. 2019;224:487‐493.

103. Nguyen F, Starosta AL, Arenz S, Sohmen D, Dönhöfer A, Wilson DN. Tetracycline antibiotics and resistance

mechanisms. Biol Chem. 2014;395(5):559‐575.

104. Seifert H, Blondeau J, Dowzicky MJ. In vitro activity of tigecycline and comparators (2014‐2016) among key WHO

‘priority pathogens’and longitudinal assessment (2004‐2016) of antimicrobial resistance: a report from the T.E.S.T.

study. Int J Antimicro Ag. 2018;52(4):474‐484.

105. Gerson S, Nowak J, Zander E, et al. Diversity of mutations in regulatory genes of resistance‐nodulation‐cell division

efflux pumps in association with tigecycline resistance in Acinetobacter baumannii.J Antimicrob Chemother.

2018;73(6):1501‐1508.

106. He F, Shi Q, Fu Y, Xu J, Yu Y, Du X. Tigecycline resistance caused by rpsJ evolution in a 59‐year‐old male patient

infected with KPC‐producing Klebsiella pneumoniae during tigecycline treatment. Infect Genet Evol. 2018;66:

188‐191.

107. Sun J, Chen C, Cui CY, et al. Plasmid‐encoded tet(X) genes that confer high‐level tigecycline resistance in Escherichia

coli.Nat Microbiol. 2019;4(9):1457‐1464.

108. Tong Z, Xu T, Deng T, Shi J, Wang Z, Liu Y. Benzydamine reverses TMexCD‐TOprJ‐mediated high‐level tigecycline

resistance in Gram‐negative bacteria. Pharmaceuticals. 2021;14(9):907.

109. Lv L, Wan M, Wang C, et al. Emergence of a plasmid‐encoded resistance‐nodulation‐division efflux pump conferring

resistance to multiple drugs, including tigecycline, in Klebsiella pneumoniae.mBio. 2020;11(2):e02930‐19.

110. Redgrave LS, Sutton SB, Webber MA, Piddock LJV. Fluoroquinolone resistance: mechanisms, impact on bacteria, and

role in evolutionary success. Trends Microbiol. 2014;22(8):438‐445.

111. Azimi A, Rezaei F, Yaseri M, Jafari S, Rahbar M, Douraghi M. Emergence of fluoroquinolone resistance and possible

mechanisms in clinical isolates of Stenotrophomonas maltophilia from Iran. Sci Rep. 2021;11(1):9582.

112. Ghosh AS, Ahamed J, Chauhan KK, Kundu M. Involvement of an efflux system in high‐level fluoroquinolone

resistance of Shigella dysenteriae.Biochem Biophys Res Commun. 1998;242(1):54‐56.

113. Panta PR, Kumar S, Stafford CF, et al. A DedA family membrane protein is required for Burkholderia thailandensis

colistin resistance. Front Microbiol. 2019;10:2532.

114. Panta PR, Doerrler WT. A link between pH homeostasis and colistin resistance in bacteria. Sci Rep. 2021;

11(1):13230.

115. Capobianco JO, Goldman RC. Erythromycin and azithromycin transport into Haemophilus influenzae ATCC 19418

under conditions of depressed proton motive force (delta mu H). Antimicrob Agents Chemother. 1990;34(9):

1787‐1791.

1088

YANG ET AL.

116. Papkou A, Hedge J, Kapel N, Young B, MacLean RC. Efflux pump activity potentiates the evolution of antibiotic

resistance across S. aureus isolates. Nat Commun. 2020;11(1):3970.

117. Wang Y, Lu J, Mao L, et al. Antiepileptic drug carbamazepine promotes horizontal transfer of plasmid‐borne multi‐

antibiotic resistance genes within and across bacterial genera. ISME J. 2019;13(2):509‐522.

118. Zhang Y, Gu AZ, Cen T, Li X, Li D, Chen J. Petrol and diesel exhaust particles accelerate the horizontal transfer of

plasmid‐mediated antimicrobial resistance genes. Environ Int. 2018;114:280‐287.

119. Liu Y, Tong Z, Shi J, Jia Y, Yang K, Wang Z. Correlation between exogenous compounds and the horizontal transfer

of plasmid‐borne antibiotic resistance genes. Microorganisms. 2020;8(8):1211.

120. Lerminiaux NA, Cameron ADS. Horizontal transfer of antibiotic resistance genes in clinical environments. Can

J Microbiol. 2019;65(1):34‐44.

121. Li X, Wen C, Liu C, et al. Herbicide promotes the conjugative transfer of multi‐resistance genes by facilitating cellular

contact and plasmid transfer. J Environ Sci. 2022;115:363‐373.

122. Liao J, Huang H, Chen Y. CO

promotes the conjugative transfer of multiresistance genes by facilitating cellular

contact and plasmid transfer. Environ Int. 2019;129:333‐342.

123. Jia Y, Yang B, Shi J, Fang D, Wang Z, Liu Y. Melatonin prevents conjugative transfer of plasmid‐mediated antibiotic

resistance genes by disrupting proton motive force. Pharmacol Res. 2022;175:105978.

124. Johnston C, Martin B, Fichant G, Polard P, Claverys JP. Bacterial transformation: distribution, shared mechanisms

and divergent control. Nat Rev Microbiol. 2014;12(3):181‐196.

125. O'Neill AJ, Chopra I. Preclinical evaluation of novel antibacterial agents by microbiological and molecular techniques.

Expert Opin Invest Drugs. 2004;13(8):1045‐1063.

126. Gentry DR, Wilding I, Johnson JM, et al. A rapid microtiter plate assay for measuring the effect of compounds on

Staphylococcus aureus membrane potential. J Microbiol Meth. 2010;83(2):254‐256.

127. Zhanel GG, Calic D, Schweizer F, et al. New lipoglycopeptides: a comparative review of dalbavancin, oritavancin and

telavancin. Drugs. 2010;70(7):859‐886.

128. Boix V, Fedorak RN, Mullane KM, et al. Primary outcomes from a phase 3, randomized, double‐blind, active‐

controlled trial of surotomycin in subjects with Clostridium difficile infection. Open Forum Infect Dis.

2017;4(1):ofw275.

129. Lee CH, Patino H, Stevens C, et al. Surotomycin versus vancomycin for Clostridium difficile infection: phase 2,

randomized, controlled, double‐blind, non‐inferiority, multicentre trial. J Antimicrob Chemother. 2016;71(10):

2964‐2971.

130. Wang Z, Koirala B, Hernandez Y, et al. A naturally inspired antibiotic to target multidrug‐resistant pathogens. Nature.

2022;601(7894):606‐611.

131. Roberts KD, Zhu Y, Azad MAK, et al. A synthetic lipopeptide targeting top‐priority multidrug‐resistant Gram‐

negative pathogens. Nat Commun. 2022;13(1):1625.

132. Velkov T, Gallardo‐Godoy A, Swarbrick JD, et al. Structure, function, and biosynthetic origin of octapeptin antibiotics

active against extensively drug‐resistant Gram‐negative bacteria. Cell Chem Biol. 2018;25(4):380‐391.

AUTHOR BIOGRAPHIES

Bingqing Yang graduated in 2020 as a Bachelor in veterinary medicine at Yangzhou University. Currently, she is

a postgraduate under the supervision of Prof. Yuan Liu at Yangzhou University. She is investigating the

inhibitors of horizontal transfer of antibiotic resistance genes and their mechanisms of action.

Ziwen Tong graduated in 2019 as a Bachelor in veterinary medicine at Xinjiang Agricultural University.

Currently, she is a postgraduate of Prof. Yuan Liu at Yangzhou University. She is investigating the combined use

of antibiotic adjuvants and existing antibiotics in the treatment of drug‐resistant infections.

Jingru Shi graduated in 2019 with a Bachelor in veterinary pharmacology at Henan Institute of Science and

Technology. Currently, she is a postgraduate of Prof. Yuan Liu at Yangzhou University. She is investigating the

synergistic basis of antimicrobial peptides in combination with antibiotics.

Zhiqiang Wang is a Professor of Veterinary Pharmacology and dean of the College of Veterinary Medicine at

Yangzhou University. He received his PhD degree in veterinary pharmacology in 2000 from South China

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Agricultural University. His research focuses on the transmission mechanisms and control strategies of drug

resistance of clinically important pathogens. To date, he has published more than 100 articles in peer‐reviewed

journals.

Yuan Liu is a Professor of Veterinary Pharmacology at the College of Veterinary Medicine at Yangzhou

University. He obtained his Bachelor's degree in veterinary medicine in 2013 from Southwest University and

achieved PhD degree in veterinary pharmacology in 2018 from China Agricultural University. His research

focuses on the identification of novel strategies to combat multidrug‐resistant (MDR) pathogens both in

preclinical studies and in the clinical setting, including the discovery of novel antimicrobial agents and antibiotic

adjuvants. To date, he has published more than 80 origin articles in this area, including Nature Microbiology,

Advanced Science, Angewandte Chemie International Edition, and Natural Product Reports.

How to cite this article: Yang B, Tong Z, Shi J, Wang Z, Liu Y. Bacterial proton motive force as an

unprecedented target to control antimicrobial resistance. Med Res Rev. 2023;43:1068‐1090.

doi:10.1002/med.21946

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Host defense peptides mitigate the spread of antibiotic resistance in physiologically relevant condition

Article

Full-text available

Feb 2024
ANTIMICROB AGENTS CH

Antibiotic resistance represents a significant challenge to public health and human safety. The primary driver behind the dissemination of antibiotic resistance is the horizontal transfer of plasmids. Current conjugative transfer assay is generally performed in a standardized manner, ignoring the effect of the host environment. Host defense peptides (HDPs) possess a wide range of biological targets and play an essential role in the innate immune system. Herein, we reveal that sub-minimum inhibitory concentrations of HDPs facilitate the conjugative transfer of RP4-7 plasmid in the Luria Broth medium, and this observation is reversed in the RPMI medium, designed to simulate the host environment. Out of these HDPs, indolicidin (Ind), a cationic tridecapeptide from bovine neutrophils, significantly inhibits the conjugation of multidrug resistance plasmids in a dose-dependent manner, including blaNDM- and tet(X4)-bearing plasmids. We demonstrate that the addition of Ind to RPMI medium as the incubation substrate downregulates the expression of conjugation-related genes. In addition, Ind weakens the tricarboxylic acid cycle, impedes the electron transport chain, and disrupts the proton motive force, consequently diminishing the synthesis of adenosine triphosphate and limiting the energy supply. Our findings highlight the importance of the host-like environments for the development of horizontal transfer inhibitors and demonstrate the potential of HDPs in preventing the spread of resistance plasmids.

Boosting MRSA Infectious Osteoporosis Treatment: Mg‐Doped Nanofilm on Vacancy‐Enriched TiO2 Coating for Providing In Situ Sonodynamic Bacteria‐Killing and Osteogenic Alkaline Microenvironment

Article

Full-text available

Dec 2023
ADV FUNCT MATER

To effectively combat infectious osteoporosis, Ti‐based implants with enhanced antibacterial and osseointegrative properties are urgently required. Herein, a one‐step method involving Mg thermal‐reduction is employed to modify a hydroxyapatite (HA) array on Ti, which comprises an inner TiO2 layer and an outer nanorod‐like HA layer. This process introduces oxygen vacancies (OVs) into the TiO2 layer and deposits a Mg─O‐contained amorphous nanolayer on each HA nanorod. The introduced OVs enhance reactive oxygen species (ROS) yield by the array during ultrasound treatment via narrowing TiO2 bandgap and improving H2O molecules absorption. The produced ROS, combined with a weak alkaline microenvironment created by the degraded Mg─O nanolayer, endows the array with potent bacterial‐killing and biofilm‐eradicating efficacies within a 5 min ultrasound treatment via the combination of proton‐consumption and cell envelop‐detriment effects. Moreover, due to the alkaline microenvironment and the released Mg²⁺, the array hinders osteoclastogenesis by activating the inflammation‐related FAK‐PI3K‐AKT signaling pathway in macrophages, as revealed by transcriptomic analysis, resulting in robust osseointegration in rat femoral shaft with concurrent bacterial‐infection and osteoporosis. This work paves a new way for simultaneously endowing a sonosensitive coating on Ti with sonodynamic treatment‐derived high antibacterial ability and alkaline microenvironment‐mediated strong osseointegration for infectious osteoporosis treatment.

Enhanced Anti-Bacterial Activity of Arachidonic Acid against the Cariogenic Bacterium Streptococcus mutans in Combination with Triclosan and Fluoride

Article

Full-text available

Jun 2024

Dental caries is a global health problem that requires better prevention measures. One of the goals is to reduce the prevalence of the cariogenic Gram-positive bacterium Streptococcus mutans. We have recently shown that naturally occurring arachidonic acid (AA) has both anti-bacterial and anti-biofilm activities against this bacterium. An important question is how these activities are affected by other anti-bacterial compounds commonly used in mouthwashes. Here, we studied the combined treatment of AA with chlorhexidine (CHX), cetylpyridinium chloride (CPC), triclosan, and fluoride. Checkerboard microtiter assays were performed to determine the effects on bacterial growth and viability. Biofilms were quantified using the MTT metabolic assay, crystal violet (CV) staining, and live/dead staining with SYTO 9/propidium iodide (PI) visualized by spinning disk confocal microscopy (SDCM). The bacterial morphology and the topography of the biofilms were visualized by high-resolution scanning electron microscopy (HR-SEM). The effect of selected drug combinations on cell viability and membrane potential was investigated by flow cytometry using SYTO 9/PI staining and the potentiometric dye DiOC2(3), respectively. We found that CHX and CPC had an antagonistic effect on AA at certain concentrations, while an additive effect was observed with triclosan and fluoride. This prompted us to investigate the triple treatment of AA, triclosan, and fluoride, which was more effective than either compound alone or the double treatment. We observed an increase in the percentage of PI-positive bacteria, indicating increased bacterial cell death. Only AA caused significant membrane hyperpolarization, which was not significantly enhanced by either triclosan or fluoride. In conclusion, our data suggest that AA can be used together with triclosan and fluoride to improve the efficacy of oral health care.

A Broad-Spectrum Horizontal Transfer Inhibitor Prevents Transmission of Plasmids Carrying Multiple Antibiotic Resistance Genes

Article

Full-text available

May 2024
TRANSBOUND EMERG DIS

The dissemination of antimicrobial resistance (AMR) severely degrades the performance of antibiotics and constantly paralyzes the global health system. In particular, plasmid-mediated transfer of antibiotic resistance genes (ARGs) across bacteria is recognized as the primary driver. Therefore, antiplasmid transfer approaches are urgently warranted to resolve this intractable problem. Herein, we demonstrated the potential of azidothymidine (AZT), an FDA-approved anti-HIV drug, as a broad-spectrum horizontal transfer inhibitor to effectively prevent the transmission of multiple ARGs, including mcr-1, blaNDM−5, and tet(X4), both in vitro and in vivo. It was also noteworthy that the inhibitory effect of AZT was proved to be valid within and across bacterial genera under different mating conditions. Mechanistic studies revealed that AZT dissipated bacterial proton motive force, which was indispensable for ATP synthesis and flagellar motility. In addition, AZT downregulated bacterial secretion systems involving general and type IV secretion systems (T4SS). Furthermore, the thymidine kinase, which is associated with DNA synthesis, turned out to be the potential target of AZT. Collectively, our work demonstrates the broad inhibitory effect of AZT in preventing ARGs transmission, opening new horizons for controlling AMR.

Antibacterial activity of isopropoxy benzene guanidine against Riemerella anatipestifer

Article

Full-text available

Feb 2024

Introduction: Riemerella anatipestifer (R. anatipestifer) is an important pathogen in waterfowl, leading to substantial economic losses. In recent years, there has been a notable escalation in the drug resistance rate of R. anatipestifer. Consequently, there is an imperative need to expedite the development of novel antibacterial medications to effectively manage the infection caused by R. anatipestifer. Methods: This study investigated the in vitro and in vivo antibacterial activities of a novel substituted benzene guanidine analog, namely, isopropoxy benzene guanidine (IBG), against R. anatipestifer by using the microdilution method, time-killing curve, and a pericarditis model. The possible mechanisms of these activities were explored. Results and Discussion: The minimal inhibitory concentration (MIC) range of IBG for R. anatipestifer was 0.5–2 μg/mL. Time-killing curves showed a concentration-dependent antibacterial effect. IBG alone or in combination with gentamicin significantly reduced the bacterial load of R. anatipestifer in the pericarditis model. Serial-passage mutagenicity assays showed a low probability for developing IBG resistance. Mechanistic studies suggested that IBG induced membrane damage by binding to phosphatidylglycerol and cardiolipin, leading to an imbalance in membrane potential and the transmembrane proton gradient, as well as the decreased of intracellular adenosine triphosphate. In summary, IBG is a potential antibacterial for controlling R. anatipestifer infections.

Elevated Membrane Potential as a Tetracycline Resistance Mechanism in Escherichia coli

Article

Jun 2024

Evolution of triclosan resistance modulates bacterial permissiveness to multidrug resistance plasmids and phages

Article

Full-text available

Apr 2024

The horizontal transfer of plasmids has been recognized as one of the key drivers for the worldwide spread of antimicrobial resistance (AMR) across bacterial pathogens. However, knowledge remain limited about the contribution made by environmental stress on the evolution of bacterial AMR by modulating horizontal acquisition of AMR plasmids and other mobile genetic elements. Here we combined experimental evolution, whole genome sequencing, reverse genetic engineering, and transcriptomics to examine if the evolution of chromosomal AMR to triclosan (TCS) disinfectant has correlated effects on modulating bacterial pathogen (Klebsiella pneumoniae) permissiveness to AMR plasmids and phage susceptibility. Herein, we show that TCS exposure increases the evolvability of K. pneumoniae to evolve TCS-resistant mutants (TRMs) by acquiring mutations and altered expression of several genes previously associated with TCS and antibiotic resistance. Notably, nsrR deletion increases conjugation permissiveness of K. pneumoniae to four AMR plasmids, and enhances susceptibility to various Klebsiella-specific phages through the downregulation of several bacterial defense systems and changes in membrane potential with altered reactive oxygen species response. Our findings suggest that unrestricted use of TCS disinfectant imposes a dual impact on bacterial antibiotic resistance by augmenting both chromosomally and horizontally acquired AMR mechanisms.

Development of novel indole-quinoline hybrid molecules targeting bacterial proton motive force

Article

Apr 2024
J APPL MICROBIOL

Aims This study aimed to develop an editable structural scaffold for improving drug development, including pharmacokinetics and pharmacodynamics of antibiotics by using synthetic compounds derived from a (hetero)aryl-quinoline hybrid scaffold. Methods and results In this study, 18 CF3-substituted (hetero)aryl-quinoline hybrid molecules were examined for their potential antibacterial activity against Staphylococcus aureus by determining minimal inhibitory concentrations. These 18 synthetic compounds represent modifications to key regions of the quinoline N-oxide scaffold, enabling us to conduct a structure-activity relationship analysis for antibacterial potency. Among the compounds, 3 m exhibited potency against with both methicillin resistant S. aureus strains, as well as other Gram-positive bacteria, including Enterococcus faecalis and Bacillus subtilis. We demonstrated that 3 m disrupted the bacterial proton motive force (PMF) through monitoring the PMF and conducting the molecular dynamics simulations. Furthermore, we show that this mechanism of action, disrupting PMF, is challenging for S. aureus to overcome. We also validated this PMF inhibition mechanism of 3 m in an Acinetobacter baumannii strain with weaken lipopolysaccharides. Additionally, in Gram-negative bacteria, we demonstrated that 3 m exhibited a synergistic effect with colistin that disrupts the outer membrane of Gram-negative bacteria. Conclusions Our approach to developing editable synthetic novel antibacterials underscores the utility of CF3-substituted (hetero)aryl-quinoline scaffold for designing compounds targeting the bacterial proton motive force, and for further drug development, including pharmacokinetics and pharmacodynamics.

Regulation of metabolism and proton motive force generation during mixed carbon fermentation by an Escherichia coli strain lacking the FOF1-ATPase

Article

Feb 2024
BBA-BIOENERGETICS

Multitarget antibacterial drugs: An effective strategy to combat bacterial resistance

Article

Oct 2023
PHARMACOL THERAPEUT

Proton Motive Force Inhibitors Are Detrimental to Methicillin-Resistant Staphylococcus aureus Strains

Article

Full-text available

Aug 2022

Methicillin-resistant Staphylococcus aureus (MRSA) strains are tolerant of conventional antibiotics, making them extremely dangerous. Previous studies have shown the effectiveness of proton motive force (PMF) inhibitors at killing bacterial cells; however, whether these agents can launch a new treatment strategy to eliminate antibiotic-tolerant cells mandates further investigation. Here, using known PMF inhibitors and two different MRSA isolates, we showed that the bactericidal potency of PMF inhibitors seemed to correlate with their ability to disrupt PMF and permeabilize cell membranes. By screening a small chemical library to verify this correlation, we identified a subset of chemicals (including nordihydroguaiaretic acid, gossypol, trifluoperazine, and amitriptyline) that strongly disrupted PMF in MRSA cells by dissipating either the transmembrane electric potential (ΔΨ) or the proton gradient (ΔpH). These drugs robustly permeabilized cell membranes and reduced MRSA cell levels below the limit of detection. Overall, our study further highlights the importance of cellular PMF as a target for designing new bactericidal therapeutics for pathogens. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) emerged as a major hypervirulent pathogen that causes severe health care-acquired infections. These pathogens can be multidrug-tolerant cells, which can facilitate the recurrence of chronic infections and the emergence of diverse antibiotic-resistant mutants. In this study, we aimed to investigate whether proton motive force (PMF) inhibitors can launch a new treatment strategy to eliminate MRSA cells. Our in-depth analysis showed that PMF inhibitors that strongly dissipate either the transmembrane electric potential or the proton gradient can robustly permeabilize cell membranes and reduce MRSA cell levels below the limit of detection.

A synthetic lipopeptide targeting top-priority multidrug-resistant Gram-negative pathogens

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Mar 2022

The emergence of multidrug-resistant (MDR) Gram-negative pathogens is an urgent global medical challenge. The old polymyxin lipopeptide antibiotics (polymyxin B and colistin) are often the only therapeutic option due to resistance to all other classes of antibiotics and the lean antibiotic drug development pipeline. However, polymyxin B and colistin suffer from major issues in safety (dose-limiting nephrotoxicity, acute toxicity), pharmacokinetics (poor exposure in the lungs) and efficacy (negligible activity against pulmonary infections) that have severely limited their clinical utility. Here we employ chemical biology to systematically optimize multiple non-conserved positions in the polymyxin scaffold, and successfully disconnect the therapeutic efficacy from the toxicity to develop a new synthetic lipopeptide, structurally and pharmacologically distinct from polymyxin B and colistin. This resulted in the clinical candidate F365 (QPX9003) with superior safety and efficacy against lung infections caused by top-priority MDR pathogens Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae.

Preclinical Development of Pentamidine Analogs Identifies a Potent and Nontoxic Antibiotic Adjuvant

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Mar 2022

Mitochondrial F1FO ATP synthase determines the local proton motive force at cristae rims

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Feb 2022
BIOPHYS J

Strain specific motility patterns and surface adhesion of virulent and probiotic Escherichia coli

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Full-text available

Jan 2022

Bacterial motility provides the ability for bacterial dissemination and surface exploration, apart from a choice between surface colonisation and further motion. In this study, we characterised the movement trajectories of pathogenic and probiotic Escherichia coli strains (ATCC43890 and M17, respectively) at the landing stage (i.e., leaving the bulk and approaching the surface) and its correlation with adhesion patterns and efficiency. A poorly motile strain JM109 was used as a control. Using specially designed and manufactured microfluidic chambers, we found that the motion behaviour near surfaces drastically varied between the strains, correlating with adhesion patterns. We consider two bacterial strategies for effective surface colonisation: horizontal and vertical, based on the obtained results. The horizontal strategy demonstrated by the M17 strain is characterised by collective directed movements within the horizontal layer during a relatively long period and non-uniform adhesion patterns, suggesting co-dependence of bacteria in the course of adhesion. The vertical strategy demonstrated by the pathogenic ATCC43890 strain implies the individual movement of bacteria mainly in the vertical direction, a faster transition from bulk to near-surface swimming, and independent bacterial behaviour during adhesion, providing a uniform distribution over the surface.

Insight Into the Anti-staphylococcal Activity of JBC 1847 at Sub-Inhibitory Concentration

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Full-text available

Jan 2022

Multidrug-resistant pathogens constitute a serious global issue and, therefore, novel antimicrobials with new modes of action are urgently needed. Here, we investigated the effect of a phenothiazine derivative (JBC 1847) with high antimicrobial activity on Staphylococcus aureus , using a wide range of in vitro assays, flow cytometry, and RNA transcriptomics. The flow cytometry results showed that JBC 1847 rapidly caused depolarization of the cell membrane, while the macromolecule synthesis inhibition assay showed that the synthesis rates of DNA, RNA, cell wall, and proteins, respectively, were strongly decreased. Transcriptome analysis of S. aureus exposed to sub-inhibitory concentrations of JBC 1847 identified a total of 78 downregulated genes, whereas not a single gene was found to be significantly upregulated. Most importantly, there was downregulation of genes involved in adenosintrifosfat (ATP)-dependent pathways, including histidine biosynthesis, which is likely to correlate with the observed lower level of intracellular ATP in JBC 1847–treated cells. Furthermore, we showed that JBC 1847 is bactericidal against both exponentially growing cells and cells in a stationary growth phase. In conclusion, our results showed that the antimicrobial properties of JBC 1847 were primarily caused by depolarization of the cell membrane resulting in dissipation of the proton motive force (PMF), whereby many essential bacterial processes are affected. JBC 1847 resulted in lowered intracellular levels of ATP followed by decreased macromolecule synthesis rate and downregulation of genes essential for the amino acid metabolism in S. aureus . Bacterial compensatory mechanisms for this proposed multi-target activity of JBC 1847 seem to be limited based on the observed very low frequency of resistance toward the compound.

A Novel Tigecycline Adjuvant ML-7 Reverses the Susceptibility of Tigecycline-Resistant Klebsiella pneumoniae

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Full-text available

Jan 2022

The increasing incidence of tigecycline resistance undoubtedly constitutes a serious threat to global public health. The combination therapies had become the indispensable strategy against this threat. Herein, 11 clinical tigecycline-resistant Klebsiella pneumoniae which mainly has mutations in ramR, acrR, or macB were collected for tigecycline adjuvant screening. Interestingly, ML-7 hydrochloride (ML-7) dramatically potentiated tigecycline activity. We further picked up five analogs of ML-7 and evaluated their synergistic activities with tigecycline by using checkerboard assay. The results revealed that ML-7 showed certain synergy with tigecycline, while other analogs exerted attenuated synergistic effects among tigecycline-resistant isolates. Thus, ML-7 was selected for further investigation. The results from growth curves showed that ML-7 combined with tigecycline could completely inhibit the growth of bacteria, and the time-kill analysis revealed that the combination exhibited synergistic bactericidal activities for tigecycline-resistant isolates during 24 h. The ethidium bromide (EtBr) efflux assay demonstrated that ML-7 could inhibit the functions of efflux pump. Besides, ML-7 disrupted the proton motive force (PMF) via increasing ΔpH, which in turn lead to the inhibition of the functions of efflux pump, reduction of intracellular ATP levels, as well as accumulation of ROS. All of which promoted the death of bacteria. And further transcriptomic analysis revealed that genes related to the mechanism of ML-7 mainly enriched in ABC transporters. Taken together, these results revealed the potential of ML-7 as a novel tigecycline adjuvant to circumvent tigecycline-resistant Klebsiella pneumoniae.

Proton motive force underpins respiration-mediated potentiation of aminoglycoside lethality in pathogenic Escherichia coli

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Full-text available

Jan 2022
ARCH MICROBIOL

It is well known that loss of aerobic respiration in Gram-negative bacteria can diminish the efficacy of a variety of bactericidal antibiotics, which has lead to subsequent demonstrations that the formation of reactive oxygen species (ROS) and the proton motive force (PMF) can both play a role in antibiotic toxicity. The susceptibility of Gram-negative bacteria to aminoglycoside antibiotics, particularly gentamicin, has previously been linked to both the production of ROS and the rate of antibiotic uptake that is mediated by the PMF, although the relative contributions of ROS and PMF to aminoglycoside toxicity has remained poorly understood. Herein, gentamicin was shown to elicit a very modest increase in ROS levels in an aerobically grown Escherichia coli clinical isolate. The well-characterised uncoupler 2,4-dinitrophenol (DNP) was used to disrupt the PMF, which resulted in a significant decrease in gentamicin lethality towards E. coli . DNP did not significantly alter respiratory oxygen consumption, supporting the hypothesis that this uncoupler does not increase ROS production via elevated respiratory oxidase activity. These observations support the hypothesis that maintenance of PMF rather than induction of ROS production underpins the mechanism for how the respiratory chain potentiates the toxicity of aminoglycosides. This was further supported by the demonstration that the uncoupler DNP elicits a dramatic decrease in gentamicin lethality under anaerobic conditions. Together, these data strongly suggest that maintenance of the PMF is the dominant mechanism for the respiratory chain in potentiating the toxic effects of aminoglycosides.

A naturally inspired antibiotic to target multidrug-resistant pathogens

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Full-text available

Jan 2022
NATURE

Bimal Koirala

Gram-negative bacteria are responsible for an increasing number of deaths caused by antibiotic-resistant infections1,2. The bacterial natural product colistin is considered the last line of defence against a number of Gram-negative pathogens. The recent global spread of the plasmid-borne mobilized colistin-resistance gene mcr-1 (phosphoethanolamine transferase) threatens the usefulness of colistin³. Bacteria-derived antibiotics often appear in nature as collections of similar structures that are encoded by evolutionarily related biosynthetic gene clusters. This structural diversity is, at least in part, expected to be a response to the development of natural resistance, which often mechanistically mimics clinical resistance. Here we propose that a solution to mcr-1-mediated resistance might have evolved among naturally occurring colistin congeners. Bioinformatic analysis of sequenced bacterial genomes identified a biosynthetic gene cluster that was predicted to encode a structurally divergent colistin congener. Chemical synthesis of this structure produced macolacin, which is active against Gram-negative pathogens expressing mcr-1 and intrinsically resistant pathogens with chromosomally encoded phosphoethanolamine transferase genes. These Gram-negative bacteria include extensively drug-resistant Acinetobacter baumannii and intrinsically colistin-resistant Neisseria gonorrhoeae, which, owing to a lack of effective treatment options, are considered among the highest level threat pathogens⁴. In a mouse neutropenic infection model, a biphenyl analogue of macolacin proved to be effective against extensively drug-resistant A. baumannii with colistin-resistance, thus providing a naturally inspired and easily produced therapeutic lead for overcoming colistin-resistant pathogens.

The antibiotic darobactin mimics a β-strand to inhibit outer membrane insertase

Article

Aug 2022

Bacterial proton motive force as an unprecedented target to control antimicrobial resistance

Abstract

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