Pseudomonas aeruginosa (P. aeruginosa) is the primary pathogenic bacterium causing nosocomial-acquired infections, particularly in immunocompromised individuals. Owing to the abuse of colistin (COL), a frontline drug in clinical settings, the resistance rate of P. aeruginosa to COL has been escalating year by year, exerting a severe impact on public health. To solve this clinical problem, new therapeutic approaches are urgently required. This study initially proposes a strategy of using carvone in combination with COL to combat colistin-resistant Pseudomonas aeruginosa (COL-R P. aeruginosa). The checkerboard assay and time-kill assay validated the in vitro antibacterial action of this combination against COL-R P. aeruginosa. Furthermore, crystalline violet staining and scanning electron microscopy (SEM) confirmed that the combined treatment effectively inhibited biofilm formation. Moreover, the thigh infection model in neutropenic mice showcased the in vivo effectiveness of the combined therapy. In addition, the erythrocyte hemolysis experiment and the in vivo safety assessment revealed that the combination of carvone and COL exhibited a certain level of safety at effective concentrations. The synergistic antibacterial and antibiofilm mechanisms of carvone combined with COL may be associated with the inhibition of quorum-sensing gene expression, enhancement of bacterial inner and outer membrane permeability, and increased generation of reactive oxygen species. This study offered a new therapeutic strategy for COL-R P. aeruginosa infections.IMPORTANCEThe increasing prevalence of colistin-resistant Pseudomonas aeruginosa (COL-R P. aeruginosa) poses a significant threat to global public health, as it limits treatment options for severe infections. This study investigates the synergistic effects of carvone, a natural compound, combined with colistin (COL) to combat COL-R P. aeruginosa both in vitro and in vivo. The findings reveal that carvone can restore the susceptibility of COL-R P. aeruginosa to COL. By exploring this novel combination, the research aims to provide a potential alternative therapeutic strategy for COL-R P. aeruginosa infections. The findings could pave the way for new approaches to treat infections caused by this resilient pathogen.

H-NS (histone-like nucleoid-structuring protein) is a global regulator affecting diverse bacterial processes. This study aimed to elucidate the regulatory role of H-NS in the virulence of Klebsiella pneumoniae (K. pneumoniae), particularly in relation to capsule synthesis and anchoring. A clinically isolated ST11-KL64 strain of K. pneumoniae FK6741 with low virulence was used. The role of H-NS was evaluated using colony morphology, the string test, viscosity measurement, capsule quantification, transmission electron microscopy, growth curve, biofilm assay, a mouse infection model, transcriptomic analysis, and RT-qPCR. Deletion of hns converted FK6741 into a hypermucoid phenotype in the positive string test; capsule quantification and transmission electron microscopy (TEM) showed increased polysaccharide chains but a reduced and tightly bound capsule. The mutant was initially found to grow slowly but formed stronger biofilms. In vivo, it displayed reduced virulence but induced stronger inflammation. Molecular assays revealed upregulation of capsule synthesis genes (galF, wzi, wcaJ, and wzc) and downregulation of wabG, which is involved in capsule anchoring. H-NS represses capsule synthesis genes, limiting capsule formation in K. pneumoniae. In contrast, loss of H-NS downregulates wabG, a key gene involved in GalA-mediated capsule anchoring, resulting in unstable surface attachment and loss of capsular polysaccharides. Consequently, these unanchored polysaccharides fail to confer effective protection, resulting in reduced bacterial virulence.

Purpose:The alarming rise of carbapenem-resistant Klebsiella pneumoniae (CRKP) has escalated into a formidable global health threat, because of its steadily increasing resistance rates to therapeutically important antimicrobial treatments. To address this challenge, we synthesized fluconazole-decorated gold nanoparticles (FCZ_Au NPs) and evaluated the antibacterial efficacy. Methods:FCZ_Au NPs were synthesized using a one-pot method. Its antimicrobial activity, anti-biofilm activity and antimicrobial mechanisms through antimicrobial susceptibility testing, growth curve analysis, murine model of acute intraperitoneal infection, crystal violet staining, reactive oxygen species (ROS) detection, membrane permeability assay. Results:As determined by our assays, the minimum inhibitory concentration of FCZ_Au NPs against CRKP was found to be between 4 and 16 μg/mL, indicating strong inhibitory effects on bacterial growth. Furthermore, at the experimental concentrations, FCZ_Au NPs exhibited excellent safety profiles toward red blood cells and mouse RAW264.7 cells. In an acute intra-abdominal infection paradigm, a notable rise in the mice's survival rate and a commensurate decrease in the bacterial burden in peritoneal lavage fluid revealed the in vivo efficiency of FCZ_Au NPs. Investigations into the antibacterial mechanisms revealed that FCZ_Au NPs act by disrupting bacterial cell membranes and enhancing reactive oxygen species production. The crystal violet assay revealed the great potential of FCZ_Au NPs in inhibiting biofilm formation and eradicating mature biofilms. Conclusion:In this study, we utilized the clinically antifungal drug FCZ to modify gold nanoparticles, synthesizing FCZ_Au NPs. Beyond their significant antibacterial activity against CRKP, these nanoparticles also demonstrated a strong ability to combat biofilms. Thus, this study provides a novel strategy for combating CRKP.

OBJECTIVES:The global spread of antimicrobial resistance represents a severe threat to public health, with infections caused by multidrug-resistant Gram-negative bacteria (MDR-GNB) emerging as a major clinical challenge. The clinical utility of traditional antimicrobial agents, such as sulfamethoxazole (SMZ), has become increasingly restricted by the emergence of bacterial resistance. Recent advances in nanomedicine have opened opportunities for 'old drugs, new uses' strategies to improve the antibacterial performance of existing agents. METHODS:We synthesized SMZ-modified gold nanoclusters (SMZ_Au NCs) via a green one-pot method and evaluated their antibacterial and anti-biofilm activities, underlying mechanisms, biocompatibility, and in vivo therapeutic efficacy against clinical MDR-GNB isolates using multiple in vitro assays and murine infection models. RESULTS:SMZ_Au NCs exhibited enhanced antibacterial activity with substantially reduced minimum inhibitory concentrations compared with SMZ alone, inhibited biofilm formation, and partially disrupted established biofilms. Mechanistic investigations suggested that the antibacterial effects were primarily associated with membrane perturbation and disruption of bacterial homeostasis, accompanied by moderate increases in intracellular reactive oxygen species. Initial in vitro biocompatibility assessments demonstrated that SMZ_Au NCs were well tolerated up to 32 µg/mL, and in vivo studies showed no acute toxicity at antibacterial-relevant doses. Moreover, SMZ_Au NCs demonstrated therapeutic efficacy in murine infection models, supporting their in vivo antibacterial potential at a proof-of-concept level. CONCLUSIONS:Nanocluster-based modification may provide a feasible approach to enhance sulphonamide antibacterial performance and support further investigation of SMZ_Au NCs against MDR-GNB infections.

OBJECTIVE:Evaluating the in vitro and in vivo antimicrobial activity of FPI-1523, a novel β-lactamase inhibitor, in combination with imipenem against carbapenemase-producing carbapenem-resistant Enterobacterales (CRE). METHODS:The in vitro and in vivo antibacterial effect of FPI-1523 combined with imipenem against CRE strains were evaluated using antimicrobial susceptibility test, time-kill assays, and Galleria mellonella infection model. Haemolysis assays and cytotoxicity experiments were performed to evaluate the safety. Modified carbapenem inactivation method and carbapenemase inhibition assay of crude periplasmic extract were conducted to explore the mechanistic basis of the antibacterial effect of the FPI-1523 and imipenem combination. RESULTS:Combined with FPI-1523, the majority of these strains (21/28, 75%) regained susceptibility to imipenem (MIC ≤ 1 mg/L), including those producing KPC, NDM, and IMP carbapenemases. Further analysis revealed that FPI-1523 exhibited intrinsic antimicrobial activity against Enterobacter cloacae and Escherichia coli, with MIC values ranging from 2 to 128 mg/L. These in vitro significant antibacterial effects were further confirmed by time-kill assays. The in vivo significant antibacterial effects were validated using a G. mellonella infection model. Haemolysis assays and cytotoxicity tests confirmed the safety of the drug combination. Modified carbapenem inactivation method and carbapenemase inhibition assay of crude periplasmic extract revealed that FPI-1523 effectively inhibited carbapenemase (excluding class B carbapenemases) activity. Based on the antimicrobial susceptibility results, for class B carbapenemase-producing strains, the antibacterial effect may be primarily mediated by FPI-1523 monotherapy. CONCLUSIONS:The combination of FPI-1523 and imipenem demonstrates significant antibacterial activity against diverse carbapenemase-producing CRE.

Introduction:Carbapenem-resistant gram-negative bacteria (CR-GNB) pose a significant threat to public health and require immediate attention. The development of novel antibacterial agents against CR-GNB has become an urgent priority, and nanomaterials offer promising solutions due to their unique properties. This study introduces 5-amino-2-mercaptobenzimidazole (5-A-2MBI) functionalized gold nanoparticles (5-A-2MBI_Au NPs) and evaluates their antibacterial activity against CR-GNB. Methods:The 5-A-2MBI_Au NPs was synthesized using a one-pot method. Its biocompatibility, bactericidal properties, and mechanisms of action were systematically characterized through in vivo and in vitro toxicity tests, antimicrobial susceptibility testing, live/dead staining, membrane permeability and reactive oxygen species (ROS) generation assays, as well as transcriptomic analysis. Results:The results of this study demonstrate that 5-A-2MBI_Au NPs exhibit excellent antibacterial efficacy against carbapenem-resistant gram-negative bacteria with various resistance mechanisms, with a minimum inhibitory concentration (MIC) of 2 μg/mL. In vivo experiments further confirmed that 5-A-2MBI_Au NPs not only possess effective bactericidal activity but also exhibit satisfactory biocompatibility. Mechanistic studies revealed that 5-A-2MBI_Au NPs enhance bacterial membrane permeability, increase the generation of reactive oxygen species, and disrupt intracellular oxidative stress and succinate synthesis, thereby conferring potent antibacterial activity. This study results demonstrate that 5-A-2MBI_Au NPs exhibit notable antibacterial efficacy against CR-GNB, with a minimum inhibitory concentration of 2 μg/mL. The antibacterial mechanism involves enhanced membrane permeability, increased reactive oxygen species production, and interference with intracellular oxidative stress and succinate synthesis. These mechanisms collectively contribute to the potent antibacterial activity of 5-A-2MBI_Au NPs against CR-GNB. Discussion:5-A-2MBI_Au NPs are a novel and highly effective antibacterial agent prepared through a simple process using benzimidazole and HAuCl4•3H2O. They efficiently eradicate the most challenging multidrug-resistant GNB both in vitro and in vivo while demonstrating excellent biocompatibility. This highlights their potential as a promising antibacterial agent to combat multidrug-resistant GNB.

Multidrug-resistant gram-negative bacteria (GNB) have posed a serious public health hazard worldwide. Colistin (COL), the ultimate therapeutic option for treating GNB infections, has had escalating resistance rates concomitant with increased utilization. In this study, we attempted to increase the sensitivity to COL using a plant extract, α-terpineol (α-TP). Checkerboard and time-kill assays indicated that the combination of COL and α-TP exhibited pronounced synergistic antimicrobial effects against colistin-resistant (COL-R) GNB in vitro. Biofilm assessments revealed that COL combined with α-TP effectively inhibited biofilm development and eliminated established biofilms. Scanning electron microscopy indicated substantial disruption of bacterial cell membranes after 6 h of combined treatment. Additionally, cytotoxicity and erythrocyte hemolysis assays confirmed that α-TP maintained a degree of safety at synergistic concentrations. Mechanistically, the combination of COL and α-TP promoted the accumulation of reactive oxygen species and enhanced the permeability of both the inner and outer bacterial membranes. This study demonstrated that α-TP can restore the sensitivity of COL-R GNB to COL when used in combination. IMPORTANCE:Colistin (COL) has emerged as a last-line therapeutic agent for gram-negative bacteria (GNB) infections. However, widespread antibiotic use has led to an alarming increase in the number of colistin-resistant (COL-R) strains. In this study, we demonstrate that combining COL with a plant-derived extract could effectively restore the susceptibility of COL-R GNB. A possible treatment plan for COL-R GNB is presented.

Introduction:Biofilms formed by Pseudomonas aeruginosa (P. aeruginosa) are a major challenge in clinical settings due to their resilience and contribution to persistent infections, especially in patients with indwelling medical devices. There is an urgent need for effective strategies to disrupt mature biofilms and control associated infections. Methods:This study investigated the combined antibacterial activity and mature biofilm eradication efficacy of slightly acidic electrolyzed water (SAEW) and thymol against P. aeruginosa PAO1 through mature biofilm removal assays. The underlying antibacterial mechanism was explored by measuring intracellular reactive oxygen species (ROS) levels. The impact of the combined treatment on the expression of PAO1 virulence genes was assessed using RT-qPCR. Additionally, the safety of the combination was evaluated through acute dermal toxicity and ocular irritation tests in mice. Results:The combination of thymol and SAEW effectively disrupted mature biofilms, significantly reduced bacterial load on medical catheters, and enhanced ROS production. Furthermore, the treatment downregulated key virulence genes, lasA and lasB, which are critical for elastin degradation and pathogenicity. Safety assessments confirmed no acute skin or ocular toxicity, indicating its suitability for clinical applications. Discussion:Thymol-enhanced SAEW shows great potential as a safe and effective strategy for biofilm eradication and infection control, paving the way for innovative approaches to combat antimicrobial-resistant pathogens in healthcare settings.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) and Escherichia coli (CREC) are frequently detected in clinical settings, restricting the use of carbapenems. Therefore, there is an urgent need for new antimicrobial strategies to address infections caused by CRKP and CREC. This study investigated the antibacterial, anti-biofilm, and anti-inflammatory effects of the cationic antimicrobial peptide Hs02, along with its potential antimicrobial mechanisms against CRKP and CREC. The results revealed that Hs02 had a low minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against CRKP and CREC, effectively eliminating the bacteria within 30 min. Moreover, Hs02 significantly prevents biofilm formation and disrupts the established biofilms. Further mechanistic studies demonstrated that Hs02 specifically targeted and bound to bacterial outer membrane lipopolysaccharides (LPS), disrupted membrane permeability and integrity, which led to intracellular reactive oxygen species (ROS) accumulation. Furthermore, Hs02 neutralized LPS, thereby suppressing the production of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in murine macrophage RAW 264.7 cells. In vitro, hemolysis and cytotoxicity assays confirmed Hs02’s safety at the tested concentrations and proved that Hs02 improved the survival rate of Galleria mellonella larvae. In conclusion, the findings suggest that Hs02’s interaction with LPS and the resulting disruption of membrane integrity may be key factors driving its rapid bactericidal and anti-inflammatory effects. IMPORTANCE Eukaryotic antimicrobial peptides are typically amphipathic peptides consisting of approximately 50 amino acids. Many macromolecular proteins in our body contain polypeptide sequences that show characteristics similar to those of antimicrobial peptides. The present research highlights a gap in the current literature regarding the mechanisms by which the intragenic antimicrobial peptide Hs02, derived from human proteins, exerts its rapid bactericidal and anti-inflammatory effects. The findings demonstrate that lipopolysaccharide (LPS) is a key target of Hs02’s antimicrobial activity and that its ability to neutralize LPS is crucial for its anti-inflammatory effects.

Disinfectants are commonly utilized by humans to combat microorganisms. However, residual disinfectants may promote environmental antimicrobial resistance by facilitating horizontal gene transfer (HGT) of antibiotic resistance genes. Bronopol is a routinely used disinfectant that persists in the environment, and previous studies have concentrated on its ecotoxicity rather than its implications on the propagation of resistance genes. This study aimed to establish an in vitro conjugation model to investigate whether bronopol promotes the transfer of antibiotic resistance genes (ARGs) via plasmid conjugation. Using Escherichia coli DH5α and DC8855 as donors harboring RP4-7 and blaNDM-4-positive IncFII(K) plasmids, respectively, and J53 as the recipient strain, we found that sub-inhibitory concentrations of bronopol (2 μg/L and 20 μg/L) significantly increased the conjugative transfer frequency (CTF) of both plasmids. Mechanistic analysis revealed that bronopol enhanced bacterial membrane permeability, as demonstrated by propidium iodide (PI) staining, 1-N-phenylnaphthylamine (NPN) fluorescent probes, transmission electron microscopy (TEM), and upregulation of the outer membrane protein gene ompC. Additionally, bronopol treatment upregulated RP4 plasmid-encoded genes involved in DNA transfer/replication (trfAp) and the global regulator of HGT (kilA/kilB). These findings highlight a previously unrecognized role of bronopol in facilitating the dissemination of antibiotic resistance genes, particularly those of clinical significance.

Acinetobacter baumannii is a major pathogen in hospitals, causing a notable rise in bloodstream infections among inpatients. Its growing resistance to multiple drugs limits treatment options. This study aims to examine the antibacterial effects of gallium nitrate [Ga(NO3)3] against A. baumannii and elucidate the underlying molecular mechanism. 40 strains of A. baumannii with different antimicrobials susceptibility patterns were isolated from bloodstream infections. The in vitro antibacterial activity of Ga(NO3)3 was analyzed by micro-dilution method and time-kill assay. The influence of ferric chloride/hemin on the antibacterial efficacy of Ga(NO3)3 was investigated. Transcriptome sequencing was performed to elucidate the antibacterial mechanism of Ga(NO3)3. A mouse infection model was conducted to assess its in vivo performance. Ga(NO3)3 exhibited a potent antibacterial effect in RPMI 1640 medium containing 10

Klebsiella pneumoniae is an important meningeal pathogen. Penetration of the blood-brain barrier (BBB) is a prerequisite for K. pneumoniae meningitis, although the underlying mechanisms remain unclear. Our study found that outer membrane protein A (OmpA), a virulence factor of K. pneumoniae, facilitates BBB penetration and induces K. pneumoniae meningitis. Experimental results revealed that the mucoviscosity, biofilm formation, capsular polysaccharide production, serum resistance, in vitro competitiveness, and motarlity rates in Galleria mellonella were markedly reduced in an ompA deletion strain (FK3907 ΔompA). In a mouse meningitis model, significant reductions in bacterial loads, mortality rate, clinical symptoms, and brain tissue damage were observed in mice infected with FK3907 ΔompA compared to FK3907 (wild-type) and FK3907 ΔompA+ompA strains. Furthermore, the wild-type strain demonstrated a markedly enhanced ability to disrupt the BBB both in vitro and in vivo compared to the FK3907 ΔompA strain. This enhancement involved not only the rearrangement of F-actin in bEnd.3 cells but also the activation of an inflammatory cytokine storm. Importantly, the wild-type strain exhibited significantly enhanced adhesion, invasion, and intracellular proliferation within RAW264.7 cells. A wound healing assay indicated that wild-type strain promoted RAW264.7 cell migration. Collectively, we identified OmpA as a required virulence factor and essential pathogenic factor for K. pneumoniae. It promotes K. pneumoniae penetration BBB via transcellular pathway, trojan horse pathway, and pro-inflammatory pathway. Our study improves an in-depth understanding for K. pneumoniae penetration BBB from the perspective of bacterial-host interactions, highlighting OmpA as a potential target for intervention in K. pneumoniae meningitis.

Introduction:This study investigates the negative regulatory role of the global transcriptional regulator H-NS (Histone-like Nucleoid Structuring Protein) on the Type VI secretion system (T6SS) in Acinetobacter baumannii (A. baumannii). We explored potential targets of H-NS mediated silencing or activation within the regulation of A. baumannii T6SS, along with the specific regulatory mechanisms involved, thereby providing a theoretical foundation for further research on A. baumannii invasive infections stemming from mixed infections and the development of therapeutic target. Methods:Using the plasmids pAT04 and pYMAb2-hyg, we constructed A. baumannii ATCC19606 strains with the hns gene knocked out (ABΔhns) and overexpressed (ABhns+). We measured the expression of the T6SS-related gene hcp in wild-type (AB WT), ABΔhns, and ABhns+ strains using RT-qPCR, combined with a mouse sepsis model featuring mixed infections. We assessed their serum resistance, competitive ability against Escherichia coli (E. coli), and blood invasion capability. Proteomic analysis identified differentially expressed proteins, and we further investigated the regulatory role of H-NS on A. baumannii T6SS using electrophoretic mobility shift assays (EMSA). Results:We successfully constructed both ABΔhns and ABhns+ strains of A. baumannii ATCC19606. RT-qPCR results indicated that H-NS functions as a negative regulator of the T6SS-related gene hcp in A. baumannii. Phenotypic assays for extracellular virulence revealed that the loss of hns enhanced both the competitive ability and serum resistance of ATCC19606. Results from the mouse sepsis infection model demonstrated that knockout of hns significantly increased the bacterium's blood invasion capability. Bioinformatics analysis of differentially expressed proteins identified elevated levels of T6SS-related proteins in the knockout strain. Furthermore, EMSAs confirmed that H-NS directly binds to multiple sites in the upstream region of hcp. Conclusion:H-NS inhibits the expression of T6SS-related proteins in A. baumannii by regulating relevant targets associated with the T6SS. This regulation influences the bacterium's pathogenicity, interspecies competitive ability, and serum resistance.

Hospital-acquired infections (HAIs) significantly increase morbidity and mortality worldwide, with Klebsiella pneumoniae (K. pneumoniae) being a leading HAI pathogen requiring targeted eradication in healthcare settings. The growing bacterial tolerance to chemical disinfectants, like chlorhexidine, highlights an urgent need for novel disinfection strategies. Bacteriophages, which employ unique mechanisms to lyse bacteria, offer a potential solution. Combining phages with disinfectants could reduce the use of chemical agents and delay the development of bacterial resistance. However, the use of phages for contamination control in clinical environments remains underexplored. ΦK2046 was isolated from hospital wastewater and characterized by transmission electron microscopy, one-step growth curve, optimal multiplicity of infection, and stability analysis. Whole-genome sequencing was performed to identify the genomic characteristics of ΦK2046. The antibacterial and antibiofilm effects of ΦK2046 combined with chlorhexidine were assessed through growth curves, time-kill assays, crystal violet staining, and scanning electron microscopy. A contaminated medical device model was established to assess the ΦK2046-chlorhexidine combination’s biofilm reduction efficacy, and different dosing sequences and timing intervals were evaluated for their impact on biofilms formed on urinary catheters. ΦK2046, characterized by a short latency period, strong environmental stability, safety, and tolerance to chlorhexidine, significantly enhanced the antibacterial and antibiofilm effects of chlorhexidine against FK2046, and reduce the emergence of resistant strains. In contaminated medical device models, the combination of ΦK2046 and chlorhexidine diminished bacterial load and biofilm formation on surfaces. A "phage-first" dosing sequence, particularly with a 90-min interval before chlorhexidine treatment, showed superior efficacy in biofilm reduction. This study, using ΦK2046 as an example, demonstrates the potential of phages to enhance the antibacterial and antibiofilm effects of chlorhexidine and their feasibility in medical device disinfection. This innovative approach not only improves chlorhexidine’s disinfecting power but also effectively tackles the issue of reduced susceptibility of K. pneumoniae to chlorhexidine. The research advances the development and application of phage-based disinfectants and lays a foundation for establishing a phage library with adjuvant properties for disinfectants.

Klebsiella quasipneumoniae is a potential pathogen that has not been studied comprehensively. The emergence of multidrug-resistant (MDR) K. quasipneumoniae, specifically strains resistant to tigecycline and carbapenem, presents a significant challenge to clinical treatment. This investigation aimed to characterize MDR K. quasipneumoniae strain FK8966, co-carrying tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 by plasmids. It was observed that FK8966's MDR was primarily because of the IncHI1B-like plasmid co-carrying tmexCD2-toprJ2 and blaIMP-4, and an IncFIB(K)/IncFII(K) plasmid harboring blaNDM-1. Furthermore, the phylogenetic analysis revealed that IncHI1B-like plasmids carrying tmexCD2-toprJ2 were disseminated among different bacteria, specifically in China. Additionally, according to the comparative genomic analysis, the MDR regions indicated that the tmexCD2-toprJ2 gene cluster was inserted into the umuC gene, while blaIMP-4 was present in transposon TnAs3 linked to the class 1 integron (IntI1). It was also observed that an ΔTn3000 insertion with blaNDM-1 made a novel blaNDM-1 harboring IncFIB(K)/IncFII(K) plasmid. The antimicrobial resistance prevalence and phylogenetic analyses of K. quasipneumoniae strains indicated that FK8966 is a distinct MDR branch of K. quasipneumoniae. Furthermore, CRISPR-Cas system analysis showed that many K. quasipneumoniae CRISPR-Cas systems lacked spacers matching the two aforementioned novel resistance plasmids, suggesting that these resistance plasmids have the potential to disseminate within K. quasipneumoniae. Therefore, the spread of MDR K. quasipneumoniae and plasmids warrants further attention.IMPORTANCEThe emergence of multidrug-resistant K. quasipneumoniae poses a great threat to clinical care, and the situation is exacerbated by the dissemination of tigecycline- and carbapenem-resistant genes. Therefore, monitoring these pathogens and their resistance plasmids is urgent and crucial. This study identified tigecycline- and carbapenem-resistant K. quasipneumoniae strain, FK8966. Furthermore, it is the first study to report the coexistence of tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 in K. quasipneumoniae. Moreover, the CRISPR-Cas system of many K. quasipneumoniae lacks spacers that match the plasmids carried by FK8966, which are crucial for mediating resistance against tigecycline and carbapenems, indicating their potential to disseminate within K. quasipneumoniae.

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.

Introduction:Antibiotic misuse and overuse have led to the emergence of carbapenem-resistant bacteria. The global spread of resistance to the novel antibiotic combination ceftazidime-avibactam (CZA) is becoming a severe problem. Antimicrobial peptide PAM-1 offers a novel approach for treating infections caused by antibiotic-resistant bacteria. This study explores its antibacterial and anti-biofilm activities and mechanisms against CZA-resistant Escherichia. Coli (E. coli), evaluating its stability and biosafety as well. Methods:The broth microdilution method, growth curve analysis, crystal violet staining, scanning electron microscopy, and propidium iodide staining/N-phenyl-1-naphthylamine uptake experiments were performed to explore the antibacterial action and potential mechanism of PAM-1 against CZA-resistant E. coli. The biosafety in diverse environments of PAM-1 was evaluated by red blood cell hemolysis, and cytotoxicity tests. Its stability was further assessed under different temperatures, serum concentrations, and ionic conditions using the broth microdilution method to determine its minimum inhibitory concentration (MIC). Galleria mellonella infection model and RT-qPCR were used to investigate the in vivo antibacterial and anti-inflammatory effects. Results and discussion:In vitro antibacterial experiments demonstrated that the MICs of PAM-1 ranged from 2 to 8 μg/mL, with its effectiveness sustained for a duration of 24 h. PAM-1 exhibited significant antibiofilm activities against CZA-resistant E. coli (p < 0.05). Furthermore, Membrane permeability test revealed that PAM-1 may exert its antibacterial effect by disrupting membrane integrity by forming transmembrane pores (p < 0.05). Red blood cell hemolysis and cytotoxicity tests revealed that PAM-1 exerts no adverse effects at experimental concentrations (p < 0.05). Moreover, stability tests revealed its effectiveness in serum and at room temperature. The Galleria mellonella infection model revealed that PAM-1 can significantly improve the survival rate of Galleria mellonella (>50%)for in vivo treatment. Lastly, RT-qPCR revealed that PAM-1 downregulates the expression of inflammatory cytokines (p < 0.05). Overall, our study findings highlight the potential of PAM-1 as a therapeutic agent for CZA-resistant E. coli infections, offering new avenues for research and alternative antimicrobial therapy strategies.

Carbapenem-resistant Enterobacteriaceae have become widely prevalent globally because of antibiotic misuse and the spread of drug-resistant plasmids, where carbapenem-resistant Escherichia coli (CREC) is one of the most common and prevalent pathogens. Furthermore, E. coli has been identified as a member of normal gut flora and does not cause disease under normal circumstances. However, certain strains of E. coli, due to the expression of virulence genes, can cause severe intestinal and extra-intestinal infections. Therefore, clinically, drug resistance and pathogenic E. coli strains are significantly challenging to treat. In this study, a novel CREC strain DC8855 was isolated from the ascites of a patient with intestinal perforation, identified as a novel sequence type 12531 (ST12531) and an unreported serotype O8:H7. It was revealed that the resistance of ST12531 CREC was predominantly conferred by an IncFII(K) plasmid carrying blaNDM-4. Furthermore, phylogenetic analysis indicated that this is the first discovery of such plasmids in China and the first identification in E. coli. Moreover, regarding virulence, the swimming assays, qRT-PCR, and in vitro intestinal barrier model indicated that DC8855 had significantly higher motility, flagella gene expression, and intestinal epithelial cell barrier migration ability than the other sequence types CREC strains (ST167 and ST410). In conclusion, this study identified novel CREC which was multidrug resistant as well as enteropathogenic and therefore requires continuous monitoring.

The hypervirulent Klebsiella pneumoniae (hvKp) with K1 and K2 capsular types causes liver abscess, pneumonia, sepsis, and invasive infections with high lethality. The presence of capsular polysaccharide (CPS) resists phagocytic engulfment and contributes to excessive inflammatory responses. Bacteriophage depolymerases can specifically target bacterial CPS, neutralizing its defense. Based on our previous research, we expressed and purified a bacteriophage depolymerase (Dep1979) targeting hvKp with capsule type K2. Interestingly, although Dep1979 lacked direct bactericidal activity in vitro, it exhibited potent antibacterial activity in vivo. Low-dose Dep1979 (0.1 mg/kg) improved the 7-day survival of immunocompetent mice to 100%. Even at 0.01 mg/kg, mice achieved 100% survival at 5 days, although efficacy sharply declined at doses as low as 0.001 mg/kg. Following Dep1979 treatment, reduced expression of inflammatory factors and no apparent tissue damage were observed. However, therapeutic efficacy significantly diminished in immunosuppressed mice. These findings underscore the critical role of Dep1979 in disarming CPS, which synergizes with host immunity to enhance antibacterial activity against hvKp.

OBJECTIVES:To investigate the characteristics and clonal dynamics of tigecycline-resistant Acinetobacter baumannii (TRAB) isolates from a Chinese hospital from 2016 to 2021. METHODS:A total of 64 TRAB isolates were screened and WGS was performed. Phylogenetic analysis and non-polymorphic mutation analysis were used to analyse their clonal dynamics and tigecycline resistance-related mutations. RT-PCR was used to analyse the expression of the resistance-nodulation cell-division (RND) efflux pump genes adeB and adeJ. Gene cloning was used to explore the effect of tet(39) variants on tigecycline resistance. RESULTS:Most TRAB isolates were found to be MDR, with 95% (61/64) of the isolates showing resistance to carbapenems. These TRAB isolates were classified into three primary genetic clusters based on core-genome SNPs. The KL2 cluster persisted throughout the study period, whereas the KL7 cluster emerged in 2019 and became the dominant clone. The KL7 cluster carried more antimicrobial resistance genes than the other two clusters. The predominant tigecycline resistance mechanism of the KL2 cluster and KL7 cluster was IS insertion in adeN (82.1%, 23/28) and genetic alterations in adeS (76.2%, 16/21), respectively. Eleven novel AdeS mutations were identified associated with elevated AdeB expression and tigecycline resistance. Moreover, we characterized a plasmid-borne tet(39) variant with an Ala-36-Thr substitution that synergizes with the RND efflux pump to confer high-level tigecycline resistance. CONCLUSIONS:This work provides important insights into the diverse mechanisms associated with tigecycline resistance in A. baumannii, highlighting a pressing need for further monitoring of ST2-KL7 A. baumannii in clinical settings.