RESUMO
BACKGROUND: Quorum sensing (QS) is a cell density-based intercellular communication system that controls virulence gene expression and biofilm formation. In Pseudomonas aeruginosa (P. aeruginosa), the LasR system sits at the top of the QS hierarchy and coordinates the expression of a series of important traits. However, the role of lasR in phage infection remains unclear. This study aims to investigate the role of lasR QS in phage infection. METHODS: The P. aeruginosa phage was isolated from sewage, and its biological characteristics and whole genome were analyzed. The adsorption receptor was identified via a phage adsorption assay. Following lasR gene knockout, the adsorption rate and bactericidal activity of phage were analyzed. Finally, real-time quantitative polymerase chain reaction (RT-qPCR) was conducted to explore how lasR promoting phage infection. RESULTS: The lytic phage vB_Pae_PLY was isolated and lipopolysaccharide (LPS) was identified as its adsorption receptor. The adsorption rate and bactericidal activity of vB_Pae_PLY were reduced after lasR knockout. RT-qPCR results showed that the expression of galU, a key gene involved in LPS synthesis, was down-regulated, and several genes related to type IV pili (T4P) were also down-regulated in the lasR mutant PaΔlasR. CONCLUSIONS: The study showed that QS lasR may promote phage vB_Pae_PLY infection by involving in the synthesis of LPS and T4P. This study provides an example of QS in promoting phage infection and deepens the understanding of phage-bacteria interactions.
Assuntos
Proteínas de Bactérias , Pseudomonas aeruginosa , Percepção de Quorum , Transativadores , Pseudomonas aeruginosa/virologia , Pseudomonas aeruginosa/genética , Percepção de Quorum/genética , Transativadores/genética , Transativadores/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fagos de Pseudomonas/genética , Fagos de Pseudomonas/fisiologia , Esgotos/virologia , Esgotos/microbiologia , Regulação Bacteriana da Expressão Gênica , Lipopolissacarídeos/metabolismo , Técnicas de Inativação de GenesRESUMO
BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) present substantial challenges to clinical intervention, necessitating the formulation of novel antimicrobial strategies to counteract them. Nanomaterials offer a distinctive avenue for eradicating bacteria by employing mechanisms divergent from traditional antibiotic resistance pathways and exhibiting reduced susceptibility to drug resistance development. Non-caloric artificial sweeteners, commonly utilized in the food sector, such as saccharin, sucralose, acesulfame, and aspartame, possess structures amenable to nanomaterial formation. In this investigation, we synthesized gold nanoparticles decorated with non-caloric artificial sweeteners and evaluated their antimicrobial efficacy against clinical CRE strains. RESULTS: Among these, gold nanoparticles decorated with aspartame (ASP_Au NPs) exhibited the most potent antimicrobial effect, displaying minimum inhibitory concentrations ranging from 4 to 16 µg/mL. As a result, ASP_Au NPs were chosen for further experimentation. Elucidation of the antimicrobial mechanism unveiled that ASP_Au NPs substantially elevated bacterial reactive oxygen species (ROS) levels, which dissipated upon ROS scavenger treatment, indicating ROS accumulation within bacteria as the fundamental antimicrobial modality. Furthermore, findings from membrane permeability assessments suggested that ASP_Au NPs may represent a secondary antimicrobial modality via enhancing inner membrane permeability. In addition, experiments involving crystal violet and confocal live/dead staining demonstrated effective suppression of bacterial biofilm formation by ASP_Au NPs. Moreover, ASP_Au NPs demonstrated notable efficacy in the treatment of Galleria mellonella bacterial infection and acute abdominal infection in mice, concurrently mitigating the organism's inflammatory response. Crucially, evaluation of in vivo safety and biocompatibility established that ASP_Au NPs exhibited negligible toxicity at bactericidal concentrations. CONCLUSIONS: Our results demonstrated that ASP_Au NPs exhibit promise as innovative antimicrobial agents against clinical CRE.
Assuntos
Anti-Infecciosos , Enterobacteriáceas Resistentes a Carbapenêmicos , Nanopartículas Metálicas , Animais , Camundongos , Ouro/química , Nanopartículas Metálicas/química , Edulcorantes , Aspartame , Espécies Reativas de Oxigênio , Antibacterianos/farmacologia , Bactérias , Testes de Sensibilidade MicrobianaRESUMO
With the widespread misuse of disinfectants, the clinical susceptibility of Klebsiella pneumoniae (K. pneumoniae) to chlorhexidine (CHX) has gradually diminished, posing significant challenges to clinical disinfection and infection control. K. pneumoniae employs overexpression of efflux pumps and the formation of thick biofilms to evade the lethal effects of CHX. Plumbagin (PLU) is a natural plant extract that enhances membrane permeability and reduces proton motive force. In this study, we elucidated the synergistic antimicrobial activity of PLU in combination with CHX, effectively reducing the MIC of CHX against K. pneumoniae to 1 µg/mL and below. Crucially, through crystal violet staining and confocal laser scanning microscopy live/dead staining, we discovered that PLU significantly enhances the anti-biofilm capability of CHX. Mechanistically, experiments involving membrane permeability, alkaline phosphatase leakage, reactive oxygen species, and RT-qPCR suggest that the combination of PLU and CHX improves the permeability of bacterial inner and outer membranes, promotes bacterial oxidative stress, and inhibits oqxA/B efflux pump expression. Furthermore, we conducted surface disinfection experiments on medical instruments to simulate clinical environments, demonstrating that the combination effectively reduces bacterial loads by more than 3 log10 CFU/mL. Additionally, results from resistance mutation frequency experiments indicate that combined treatment reduces the generation of resistant mutants within the bacterial population. In summary, PLU can serve as an adjuvant, enhancing the anti-biofilm capability of CHX and reducing the occurrence of resistance mutations, thereby extending the lifespan of CHX.IMPORTANCEAs disinfectants are extensively and excessively utilized worldwide, clinical pathogens are progressively acquiring resistance against these substances. However, high concentrations of disinfectants can lead to cross-resistance to antibiotics, and concurrent use of different disinfectants can promote bacterial resistance mutations and facilitate the horizontal transfer of resistance genes, which poses significant challenges for clinical treatment. Compared with the lengthy process of developing new disinfectants, enhancing the effectiveness of existing disinfectants with natural plant extracts is important and meaningful. CHX is particularly common and widely used compared with other disinfectants. Meanwhile, Klebsiella pneumoniae, as a clinically significant pathogen, exhibits high rates of resistance and pathogenicity. Previous studies and our data indicate a significant decrease in the sensitivity of clinical K. pneumoniae to CHX, highlighting the urgent need for novel strategies to address this issue. In light of this, our research is meaningful.
RESUMO
The continuous rise of multidrug-resistant (MDR) Gram-negative bacteria poses a severe threat to public health worldwide. Colistin(COL), employed as the last-line antibiotic against MDR pathogens, is now at risk due to the emergence of colistin-resistant (COL-R) bacteria, potentially leading to adverse patient outcomes. In this study, synergistic activity was observed when colistin and diclofenac sodium (DS) were combined and used against clinical COL-R strains of Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), Acinetobacter baumannii (A. baumannii), and Pseudomonas aeruginosa (P. aeruginosa) both in vitro and in vivo. The checkerboard method and time-killing assay showed that DS, when combined with COL, exhibited enhanced antibacterial activity compared to DS and COL monotherapies. Crystal violet staining and scanning electron microscopy showed that COL-DS inhibited biofilm formation compared with monotherapy. The in vivo experiment showed that the combination of DS and COL reduced bacterial loads in infected mouse thighs. Synergistic activity was observed when COL and DS were use in combination against clinical COL-R strains of E. coli, K. pneumoniae, A. baumannii and P. aeruginosa both in vitro and in vivo. The synergistic antibacterial effect of the COL-DS combination has been confirmed by performing various in vitro and in vivo experiments, which provides a new treatment strategy for infections caused by MDR bacteria.
Assuntos
Antibacterianos , Colistina , Diclofenaco , Sinergismo Farmacológico , Bactérias Gram-Negativas , Testes de Sensibilidade Microbiana , Colistina/farmacologia , Animais , Antibacterianos/farmacologia , Diclofenaco/farmacologia , Camundongos , Bactérias Gram-Negativas/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla , Acinetobacter baumannii/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Humanos , Quimioterapia Combinada , FemininoRESUMO
The bacteriophage is an effective adjunct to existing antibiotic therapy; however, in the course of bacteriophage therapy, host bacteria will develop resistance to bacteriophages, thus affecting the efficacy. Therefore, it is important to describe how bacteria evade bacteriophage attack and the consequences of the biological changes that accompany the development of bacteriophage resistance before the bacteriophage is applied. The specific bacteriophage vB3530 of Pseudomonas aeruginosa (P. aeruginosa) has stable biological characteristics, short incubation period, strong in vitro cleavage ability, and absence of virulence or resistance genes. Ten bacteriophage-resistant strains (TL3780-R) were induced using the secondary infection approach, and the plaque assay showed that vB3530 was less sensitive to TL3780-R. Identification of bacteriophage adsorption receptors showed that the bacterial surface polysaccharide was probably the adsorption receptor of vB3530. In contrast to the TL3780 parental strain, TL3780-R is characterized by the absence of long lipopolysaccharide chains, which may be caused by base insertion of wzy or deletion of galU. It is also intriguing to observe that, in comparison to the parent strain, the bacteriophage-resistant strains TL3780-R mostly exhibited a large cost of fitness (growth rate, biofilm formation, motility, and ability to produce enhanced pyocyanin). In addition, TL3780-R9 showed increased susceptibility to aminoglycosides and chlorhexidine, which may be connected to the loss and down-regulation of mexX expression. Consequently, these findings fully depicted the resistance mechanism of P. aeruginosa to vB3530 and the fitness cost of bacteriophage resistance, laying a foundation for further application of bacteriophage therapy.IMPORTANCEThe bacteriophage is an effective adjunct to existing antibiotic therapy; However, bacteria also develop defensive mechanisms against bacteriophage attack. Thus, there is an urgent need to deeply understand the resistance mechanism of bacteria to bacteriophages and the fitness cost of bacteriophage resistance so as to lay the foundation for subsequent application of the phage. In this study, a specific bacteriophage vB3530 of P. aeruginosa had stable biological characteristics, short incubation period, strong in vitro cleavage ability, and absence of virulence or resistance genes. In addition, we found that P. aeruginosa may lead to phage resistance due to the deletion of galU and the base insertion of wzy, involved in the synthesis of lipopolysaccharides. Simultaneously, we showed the association with the biological state of the bacteria after bacteria acquire bacteriophage resistance, which is extremely relevant to guide the future application of therapeutic bacteriophages.
Assuntos
Bacteriófagos , Infecções por Pseudomonas , Humanos , Bacteriófagos/genética , Pseudomonas aeruginosa , Infecções por Pseudomonas/terapia , Infecções por Pseudomonas/microbiologia , Antibacterianos/farmacologia , VirulênciaRESUMO
Today, colistin is considered a last-resort antibiotic for treating multidrug-resistant (MDR) Gram-negative bacteria (GNB). However, the increased and improper use of colistin has led to the emergence of colistin-resistant (Col-R) GNB. Thus, it is urgent to develop new drugs and therapies in response to the ongoing emergence of colistin resistance. In this study, we investigated the antibacterial and antibiofilm activities of the quaternary ammonium compound domiphen bromide (DB) in combination with colistin against clinical Col-R GNB both in vitro and in vivo. Checkerboard assay and time-kill analysis demonstrated significant synergistic antibacterial effects of the colistin/DB combination. The synergistic antibiofilm activity was confirmed through crystal violet staining and scanning electron microscopy (SEM). Furthermore, the colistin/DB combination exhibited increased survival rates in infected larvae and reduced bacterial loads in a mouse thigh infection model. The cytotoxicity measurement and hemolysis test showed that the combination did not adversely affect cell viability at synergistic concentrations. The alkaline phosphatase (ALP) leak test and propidium iodide (PI) staining analysis further revealed that the colistin/DB combination enhanced the therapeutic effect of colistin by altering bacterial membrane permeability. The ROS assays revealed that the combination induced the accumulation of bacterial ROS, leading to bacterial death. In conclusion, our study is the first to identify DB as a colistin potentiator, effectively restoring the sensitivity of bacteria to colistin. It provides a promising alternative approach for combating Col-R GNB infections.
Assuntos
Colistina , Compostos de Amônio Quaternário , Animais , Camundongos , Colistina/farmacologia , Colistina/uso terapêutico , Compostos de Amônio Quaternário/farmacologia , Espécies Reativas de Oxigênio/farmacologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Bactérias Gram-Negativas , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade MicrobianaRESUMO
Acinetobacter baumannii, which is predominantly responsible for hospital-acquired infections, presents a tremendous clinical challenge due to its increasing antibiotic resistance to colistin (COL), a last-line antibiotic. As a result, the combination of antimicrobial and non-antimicrobial agents is emerging as a more popular treatment approach against infections caused by COL-resistant A. baumannii. This study administered COL and verapamil (VER), that is an antihypertensive and antiarrhythmic agent. We found that the susceptibility of A. baumannii to COL was restored both in vitro and in vivo. Scanning electron microscope and Crystal violet staining showed inhibition of the VER/COL combination on bacterial biofilm formation. Cytotoxicity assay and haemolysis test were used to confirm in vitro safety evaluation. Further experiments using propidium iodide staining revealed that the VER/COL combination improved the therapeutic efficacy of COL by modifying the permeability of bacterial membranes. As demonstrated by reactive oxygen species experiments, the drug combination caused the accumulation of bacterial reactive oxygen species and their eventual death. Additionally, VER/COL treatment significantly reduced the efflux of Rhodamine 123 (Rh123). For the first time, this study identifies the anti-hypertensive drug VER as a COL potentiator against A. baumannii, providing a potential treatment approach against A. baumannii infections and improving patient outcomes.
Assuntos
Infecções por Acinetobacter , Acinetobacter baumannii , Antibacterianos , Biofilmes , Colistina , Testes de Sensibilidade Microbiana , Verapamil , Acinetobacter baumannii/efeitos dos fármacos , Colistina/farmacologia , Antibacterianos/farmacologia , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/microbiologia , Biofilmes/efeitos dos fármacos , Verapamil/farmacologia , Animais , Sinergismo Farmacológico , Espécies Reativas de Oxigênio/metabolismo , Humanos , Farmacorresistência Bacteriana/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacosRESUMO
The Enterobacter cloacae complex (ECC) is a group of nosocomial pathogens that pose a challenge in clinical treatment due to its intrinsic resistance and the ability to rapidly acquire resistance. Colistin was reconsidered as a last-resort antibiotic for combating multidrug-resistant ECC. However, the persistent emergence of colistin-resistant (COL-R) pathogens impedes its clinical efficacy, and novel treatment options are urgently needed. We propose that azomycin, in combination with colistin, restores the susceptibility of COL-R ECC to colistin in vivo and in vitro. Results from the checkerboard susceptibility, time-killing, and live/dead bacterial cell viability tests showed strong synergistic antibacterial activity in vitro. Animal infection models suggested that azomycin-colistin enhanced the survival rate of infected Galleria mellonella and reduced the bacterial load in the thighs of infected mice, highlighting its superior in vivo synergistic antibacterial activity. Crystal violet staining and scanning electron microscopy unveiled the in vitro synergistic antibiofilm effects of azomycin-colistin. The safety of azomycin and azomycin-colistin at experimental concentrations was confirmed through cytotoxicity tests and an erythrocyte hemolysis test. Azomycin-colistin stimulated the production of reactive oxygen species in COL-R ECC and inhibited the PhoPQ two-component system to combat bacterial growth. Thus, azomycin is feasible as a colistin adjuvant against COL-R ECC infection.