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.

The objective of this study was to compare chlorhexidine digluconate and other antibiotics susceptibility of four species of the Acinetobacter baumannii complex, and further investigate the chlorhexidine digluconate (CHG) tolerance mechanisms and molecular epidemic characteristics. Of 889 A. baumannii complex isolates, A. baumannii, A. nosocomialis, A. pittii, and A. seifertii accounted for 84.2%, 10.9%, 3.4%, and 1.5%. Acinetobacter baumannii was generally resistant to all tested antibiotics, while other three species were commonly more susceptible; 92.1% (313/340) CHG-tolerant A. baumannii, 19.6% (19/97) CHG-tolerant A. nosocomialis, 3.3% (1/30) CHG-tolerant A. pittii, and 15.4% (2/13) CHG-tolerant A. seifertii were identified. Furthermore, compared to A. baumannii ATCC 19606, upregulated expression was found in qacEΔ1, fabI, and efflux pump encoding genes in CHG-tolerant A. baumannii, but the expression level of oprD was reduced. Additionally, only the expression level of fabI was increased in the CHG-tolerant A. nosocomialis, and the expression level of adeG was increased in the CHG-tolerant A. pittii and A. seifertii. Furthermore, CHG-tolerant A. baumannii may have a relatively high clonal correlation, the predominant sequence type of which was ST208 (90%, 36/40). It is rather necessary to identify specific species members among the A. baumannii complex for clinical treatment options and antibiotics resistance monitoring.

Purpose:The global emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) represent a major threat to effective clinical antimicrobial therapy, highlighting the urgent demand for alternative treatment strategies. This study aims to develop dichlorophen-functionalized gold nanoparticles (DDM_Au NPs) as a novel approach to combat CRE and their associated biofilms. Methods:Two structurally related antiparasitic compounds, bithionol and dichlorophen, were functionalized with Au NPs using a one-pot synthesis technique and thoroughly characterized. Their antibacterial activity was assessed through standard antimicrobial susceptibility testing and bacterial growth curve analysis. Antibiofilm properties were evaluated using crystal violet staining, scanning electron microscopy, and confocal laser scanning microscopy. The underlying mechanism of action was investigated by measuring reactive oxygen species production and assessing bacterial membrane permeability. Biocompatibility was evaluated via hemolysis assays, in vivo murine studies, and Galleria mellonella infection models. A urinary catheter model contaminated with biofilms, along with murine models of abdominal and pulmonary infection, was employed to assess device-associated applicability and therapeutic efficacy in vivo. Results:DDM_Au NPs demonstrated potent antibacterial activity against CRE, with minimum inhibitory concentrations ranging from 4 to 16 μg/mL. These nanoparticles effectively inhibited biofilm formation and promoted the disruption of mature biofilms, resulting in bacterial load reductions of 2-6  log10 CFU/mL on infected urinary catheters. Mechanistic studies revealed that their antimicrobial activity was primarily driven by disruption of bacterial membrane integrity and induction of intracellular oxidative stress through elevated reactive oxygen species production. Notably, DDM_Au NPs exhibited favorable biocompatibility and significantly reduced bacterial burdens at infection sites by 4-5 log10 CFU/mL, while also alleviating inflammatory responses and limiting tissue damage across multiple animal infection models. Conclusion:This study introduces a streamlined and effective strategy for achieving both antibacterial and antibiofilm effects using antiparasitic drug-functionalized Au NPs. DDM_Au NPs show strong promise as innovative antimicrobial agents for treating clinical CRE infections and reducing environmental contamination in healthcare environments.

Hypervirulent Klebsiella pneumoniae (hvKp) presents challenges in infection management due to antibiotic resistance associated with its intracellular persistence. This study investigates the efficacy of phage therapy against intracellular hvKp using a two-stage murine model. We assessed changes in virulence, host survival, and immune responses through phagocytosis assays, transmission electron microscopy, and Western blotting, complemented by transcriptomic and proteomic analyses. Results indicate that phage therapy reduces mortality and modulates bacterial virulence by downregulating capsule production. Following phage exposure, hvKp adapts by enhancing its oxidative stress resistance. Crucially, these adaptations weaken host inflammatory and autophagy responses, enabling better survival within host cells. These adaptations suggest that while phage therapy can mitigate infection severity, the capacity of hvKp to modulate host pathways underscores the complexity of treating intracellular infections and highlights the importance of targeting both bacterial and cellular responses.

ObjectivesThe emergence and spread of carbapenem-resistant Morganella morganii (M. morganii) pose a serious global challenge. This study aimed to investigate the clinical characteristics, resistance patterns, and molecular mechanisms of carbapenem-resistant M. morganii.MethodsA total of 170 M. morganii clinical isolates were collected from the First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China) between January 2016 and December 2017. Carbapenem MICs were determined by antimicrobial susceptibility testing. Carbapenem resistance determinants, including carbapenemase genes (blaKPC-2, blaVIM, blaIMP, blaNDM, and blaOXA-48) and extended-spectrum β-lactamase (ESBL) genes (blaTEM, blaCTX-M-1, and blaSHV), were analyzed by polymerase chain reaction (PCR). PCR and sequencing assays were performed to detect penicillin-binding protein (PBP) mutations. Efflux pump activity was also assessed in carbapenem-resistant isolates. Quantitative real-time PCR (qRT-PCR) was used to determine the relative mRNA expression levels of outer membrane porin-encoding gene ompC and PBP activator-encoding genes lpoA and lpoB.ResultsTwenty-six imipenem-resistant and 108 imipenem-intermediate M. morganii isolates were identified, accounting for 15.29% and 63.53% of cases, respectively. No isolates were resistant to meropenem or ertapenem. Among the 26 carbapenem-resistant isolates, the prevalence of ESBL genes blaTEM and blaCTX-M-1 was 30.77% and 11.54%, respectively, while carbapenemase genes were not detected. Resistant isolates carried more specific PBP mutations than carbapenem-susceptible and carbapenem-intermediate isolates. Efflux pump phenotypes were associated with reduced imipenem susceptibility in 13 carbapenem-resistant isolates. qRT-PCR revealed no significant differences in ompC expression among the resistant, intermediate, and susceptible groups; however, significant differences were observed in lpoA and lpoB expression. Isolates in the imipenem-resistant group carried more PBP mutations.ConclusionM. morganii isolates were commonly non-susceptible to imipenem but remained susceptible to meropenem and ertapenem. Low expression of PBP activator genes (lpoA and lpoB), along with the presence of specific PBP mutations, appeared to be the primary mechanisms of resistance. In addition, efflux pump overexpression may contribute to imipenem resistance in M. morganii.

Bacteriophages (phages), viruses capable of infecting and lysing bacteria, are a promising alternative for treating infections from hypervirulent, antibiotic-resistant pathogens like Klebsiella pneumoniae, though narrow host range and phage resistance remain challenges. In this study, the hypervirulent K. pneumoniae NTUH-K2044 was used to purify phage Phi K2044, while two Phi K2044-resistant strains were used to purify two further phages: Phi KR1, and Phi KR8 from hospital sewage. A detailed characterization showed that Phi K2044 specifically killed KL1 capsule-type K. pneumoniae, while Phi KR1 and Phi KR8 targeted 13 different capsular serotypes. The phage cocktail (Phi K2044 + Phi KR1 + Phi KR8) effectively killed K. pneumoniae in biofilms, pre-treatment biofilm formation, and delayed phage-resistance. The phage cocktail improved 7-day survival in Galleria mellonella and mouse models and showed therapeutic potential in a catheter biofilm model. In summary, this proof-of-principle phage cocktail has a broad host range, including hypervirulent and highly drug-resistant K. pneumoniae, and serves as a promising starting point for optimizing phage therapy.

ABSTRACT Antimicrobial resistance has become a growing public health threat in recent years. Klebsiella pneumoniae is one of the priority pathogens listed by the World Health Organization. Antimicrobial peptides are considered promising alternatives to antibiotics due to their broad-spectrum antibacterial activity and low resistance. In this study, we investigated the antibacterial activity of antimicrobial peptide A20L against K. pneumoniae. In vitro antibacterial activity of A20L against K. pneumoniae was demonstrated by broth microdilution method. We confirmed the in vivo efficacy of A20L by Galleria mellonella infection model. In addition, we found that A20L also had certain antibiofilm activity by crystal violet staining. We also evaluated the safety and stability of A20L, and the results revealed that at a concentration of ≤128 µg/mL, A20L exhibited negligible toxicity to RAW264.7 cells and no substantial toxicity to G. mellonella . A20L was stable at different temperatures and with low concentration of serum [5% fetal bovine serum (FBS)]; however, Ca 2+ , Mg 2+ , and high serum concentrations reduced the antibacterial activity of A20L. Scanning electron microscope (SEM) and membrane permeability tests revealed that A20L may exhibit antibacterial action by damaging bacterial cell membranes and increasing the permeability of outer membrane. Taken together, our results suggest that A20L has significant development potential as a therapeutic antibiotic alternative, which provides ideas for the treatment of K. pneumoniae infection. IMPORTANCE A20L showed antibacterial and anti-infective efficacy in vitro and in vivo against Klebsiella pneumoniae . It can have an antibacterial effect by disrupting the integrity of cell membranes. A20L displayed anti-biofilm and anti-inflammatory activity against carbapenem-resistant K. pneumoniae and certain application potential in vivo , which provides a new idea for the clinical treatment of biofilm-associated infections.

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.

Combining pentamidine with Gram-positive-targeting antibiotics has been proven to be a promising strategy for treating infections from Gram-negative bacteria (GNB). However, which antibiotics pentamidine can and cannot synergize with and the reasons for the differences are unclear. This study aimed to identify the possible mechanisms for the differences in the synergy of pentamidine with rifampicin, linezolid, tetracycline, erythromycin, and vancomycin against GNB. Checkerboard assays were used to detect the synergy of pentamidine and the different antibiotics. To determine the mechanism of pentamidine, fluorescent labeling assays were used to measure membrane permeability, membrane potential, efflux pump activity, and reactive oxygen species (ROS); the LPS neutralization assay was used to evaluate the target site; and quantitative PCR was used to measure changes in efflux pump gene expression. Our results revealed that pentamidine strongly synergized with rifampicin, linezolid, and tetracycline and moderately synergized with erythromycin, but did not synergize with vancomycin against E. coli, K. pneumoniae, E. cloacae, and A. baumannii. Pentamidine increased the outer membrane permeability but did not demolish the outer and inner membranes, which exclusively permits the passage of hydrophobic, small-molecule antibiotics while hindering the entry of hydrophilic, large-molecule vancomycin. It dissipated the membrane proton motive force and inactivated the efflux pump, allowing the intracellular accumulation of antimicrobials that function as substrates of the efflux pump, such as linezolid. These processes resulted in metabolic perturbation and ROS production which ultimately was able to destroy the bacteria. These mechanisms of action of pentamidine on GNB indicate that it is prone to potentiating hydrophobic, small-molecule antibiotics, such as rifampicin, linezolid, and tetracycline, but not hydrophilic, large-molecule antibiotics like vancomycin against GNB. Collectively, our results highlight the importance of the physicochemical properties of antibiotics and the specific mechanisms of action of pentamidine for the synergy of pentamidine–antibiotic combinations. Pentamidine engages in various pathways in its interactions with GNB, but these mechanisms determine its specific synergistic effects with certain antibiotics against GNB. Pentamidine is a promising adjuvant, and we can optimize drug compatibility by considering its functional mechanisms.

目的 通过研究庆大霉素诱导粪肠球菌耐药突变株对氯霉素协同敏感(collateral sensitivity,CS)的相关特性,为基于协同敏感治疗策略的临床应用提供依据.方法 采用庆大霉素通过梯度浓度平板对3株庆大霉素敏感的粪肠球菌临床分离株和1株粪肠球菌标准菌株ATCC29212进行体外实验诱导耐药(experimental evolution);通过微量肉汤稀释法测定庆大霉素诱导后耐药突变株对常见抗菌药物的最低抑菌浓度(minimum inhibitory concentrations,MICs),分析抗菌药物间交叉敏感性;PCR检测诱导耐药菌株氨基糖苷类修饰酶编码基因携带情况;通过琼脂平板稀释法测定庆大霉素诱导耐药(evolved gentamicin-resistant)菌株和亲本菌株对其协同敏感药物(氯霉素)的防耐药突变浓度(mutant prevention concentration,MPC),最后,测定氯霉素对庆大霉素诱导耐药菌株和亲本菌株的时间杀菌曲线.结果 结果显示4株粪肠球菌亲本菌株经过庆大霉素诱导后,对庆大霉素MICs升高了 16-64倍,并对阿米卡星表现出交叉耐药;诱导耐药菌株及其亲本菌株均未检出aac(6′)-Ie-aph(2″)-Ia.此外,研究发现诱导后菌株对氯霉素表现出协同敏感.氯霉素对庆大霉素诱导耐药粪肠球菌及其亲本菌株的MPC均大于1024 μg/mL,突变选择窗较宽;时间杀菌曲线显示当氯霉素浓度为亲本菌株的1/2×MIC或1/4×MIC时,庆大霉素诱导耐药菌株的生长被有效抑制,而亲本菌株则不能被抑制.结论 基于协同敏感性制定抗菌药物治疗策略可能有助于有效清除耐药病原菌并延长抗菌药物的使用寿命.

目的 分析脑脓肿的临床、微生物学特征及其预后相关因素.方法 回顾性分析2012年1月至2019年12月温州医科大学附属第一医院脑脓肿患者的临床资料,采用MALDI-TOF-MS微生物鉴定系统和VITEK?2?Compact全自动微生物分析系统对脑脓肿患者各类送检标本中培养出的病原菌进行鉴定和药敏试验,并通过分析比较脑脓肿患者的临床结局以发现其预后不良的危险因素.结果 共纳入患者96例,主要见于>50岁的中老年患者,平均年龄为54.47岁,以男性患者居多,临床表现以局灶性症状多见,74.0％的患者为隐源性感染来源,37.5％的患者为多发性脑脓肿,85.4％的患者预后良好.Logistic多因素分析结果显示,GCS评分下降和脓肿临近脑室是脑脓肿患者出院时预后不良的危险因素.共有15株病原菌分离自38例经手术治疗的脑脓肿患者脓液标本,其中大多数分离菌株对临床常用抗菌药物具有较好的敏感性.结论 脑脓肿常见于中老年男性患者,其中隐源性脑脓肿更为常见,临床表现具有不典型性,所分离菌株对临床常用抗菌药物的耐药率较低,GCS评分下降和脓肿临近脑室是脑脓肿患者预后不良的危险因素.

Cysteiniphilum is a newly discovered genus in 2017 and is phylogenetically closely related to highly pathogenic Francisella tularensis. Recently, it has become an emerging pathogen in humans. However, the complete genome sequence of genus Cysteiniphilum is lacking, and the genomic characteristics of genetic diversity, evolutionary dynamics, and pathogenicity have not been characterized. In this study, the complete genome of the first reported clinical isolate QT6929 of genus Cysteiniphilum was sequenced, and comparative genomics analyses to Francisella genus were conducted to unveil the genomic landscape and diversity of the genus Cysteiniphilum. Our results showed that the complete genome of QT6929 consists of one 2.61 Mb chromosome and a 76,819 bp plasmid. The calculated average nucleotide identity and DNA–DNA hybridization values revealed that two clinical isolates QT6929 and JM-1 should be reclassified as two novel species in genus Cysteiniphilum. Pan-genome analysis revealed genomic diversity within the genus Cysteiniphilum and an open pan-genome state. Genomic plasticity analysis exhibited abundant mobile genetic elements including genome islands, insertion sequences, prophages, and plasmids on Cysteiniphilum genomes, which facilitated the broad exchange of genetic material between Cysteiniphilum and other genera like Francisella and Legionella. Several potential virulence genes associated with lipopolysaccharide/lipooligosaccharide, capsule, and haem biosynthesis specific to clinical isolates were predicted and might contribute to their pathogenicity in humans. Incomplete Francisella pathogenicity island was identified in most Cysteiniphilum genomes. Overall, our study provides an updated phylogenomic relationship of members of the genus Cysteiniphilum and comprehensive genomic insights into this rare emerging pathogen.

Objective Acinetobacter baumannii is a hazardous bacterium that causes hospital-acquired nosocomial infections, and the advent of multidrug-resistant A. baumannii (MDR-AB) strains is concerning. Novel antibacterial therapeutic strategies must be developed. The biological effects of glabridin on MDR-AB were investigated in this study. Methods The minimum inhibitory concentrations (MICs) of glabridin against eight clinical MDR-AB strains were determined using the broth microdilution technique. Crystal violet staining was used to assess biofilm development, which has significant contribution to bacterial resistance. Swarming motility was measured according to surface growth zone of MDR-AB on LB agar medium. qRT-PCR was used to evaluate the expression of quorum sensing genes abaI and abaR. Glabridin and routinely used therapeutic antimicrobial agents were tested for synergistic action using the checkerboard method. Results According to our findings, glabridin suppressed MDR-AB growth at high doses (512–1024 μg/mL). The 1/4 MIC of glabridin significantly decreased MDR-AB biofilm formation by 19.98% (P < 0.05), inhibited MDR-AB motility by 44.27% (P < 0.05), whereas the 1/2 MIC of glabridin dramatically reduced MDR-AB biofilm development by 27.43% (P < 0.01), suppressed MDR-AB motility by 50.64% (P < 0.05). Mechanistically, glabridin substantially downregulated the expression of quorum sensing-related genes abaI and abaR by up to 39.12% (P < 0.001) and 25.19% (P < 0.01), respectively. However, no synergistic effect between glabridin and antibacterial drugs was found. Conclusion Glabridin might be a quorum sensing inhibitor that inhibits MDR-AB biofilm development and swarming motility.

Objectives: The emergence of carbapenem-resistant Escherichia coli (CREC) is a global concern as its prevalence restricts treatment options and poses a considerable threat to public health. In this study, in vitro and in vivo activity of the antimicrobial peptide Octominin against CREC was investigated to reveal possible mechanisms of action. Furthermore, its safety and factors influencing its antibacterial effect were assessed. Additionally, the anti-inflammatory effects of Octominin were examined. Methods: The antimicrobial activity of Octominin against 11 strains of CREC was determined using the broth microdilution method, growth curve, and time-kill assay. Its possible mechanism of action was unraveled using the propidium iodide and N-phenyl-1-naphthylamine fluorochrome and lipopolysaccharidebinding assays. To understand the safety and stability of Octominin, its cytotoxicity, hemolysis, and antibacterial activity under various conditions (i.e, temperature, ions) were estimated. Additionally, a Galleria mellonella infection model was utilized to evaluate the efficacy of Octominin in vivo, and qRT-PCR was performed to assess its effect on the expression of proinflammatory cytokines. Results: Octominin displayed a significant antibacterial effect, with MICs of 4-8 mu g/mL and MBCs of 8- 16 mu g/mL. Octominin exerted its antibacterial effect by disrupting bacterial membranes. Cytotoxicity and hemolysis tests demonstrated the potential application of Octominin in vivo. The G. mellonella infection model asserted the in vivo efficacy of Octominin. Furthermore, Octominin inhibited the expression of proinflammatory cytokines. Although the temperature had little effect on its the activity, serum and ions reduced activity. Conclusion: Octominin is a promising alternative agent with remarkable antibacterial and antiinflammatory effects for treating infections caused by CREC . (c) 2023 Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ )

Cooccurrence of multidrug resistant (MDR) and hypervirulence phenotypes in liver abscess-causing Klebsiella pneumoniae (LAKp) would pose a major threat to public health. However, relatively little information is available on the genomic and phenotypic characteristics of this pathogen. This study aimed to investigate the virulence and resistance phenotype and genotype of MDR LAKp strains from 2016 to 2020. We collected 18 MDR LAKp strains from 395 liver abscess samples and characterized these strains using antimicrobial susceptibility test, string test, mucoviscosity assay, biofilm formation assay, Galleria mellonella killing assay, and whole-genome sequencing. Besides, phylogenetic and comparative genomic analyses were performed on these MDR LAKp, along with 94 LAKp genomes from global sources. Most of these MDR LAKp strains exhibited resistance to cephalosporins, quinolones, and chloramphenicol. Virulence assays revealed that only half of MDR LAKp strains exhibited higher virulence than classical MDR strain K. pneumoniae MGH78578. Importantly, we identified three ST11 KL64 hypervirulence carbapenem-resistant strains carrying blaKPC-2 and one colistin-resistant strain carrying mcr-1. Phylogenetic analysis revealed that 112 LAKp genomes were divided into two clades, and most of MDR LAKp strains in this study belonged to clade 1 (83.33%, 15/18). We also detected the loss of mucoviscosity mediated by mutations and ISKpn14 insertion in rmpA, and the latter representing a novel mechanism by which bacteria regulate RmpA system. This study provides novel insights into MDR LAKp and highlights the necessity for measures to prevent further spread of such organisms in hospital settings and the community. IMPORTANCE Pyogenic liver abscess is a potentially life-threatening suppurative infection of hepatic parenchyma. K. pneumoniae has emerged as a predominant pathogen of pyogenic liver abscess. Liver abscess-causing K. pneumoniae is generally considered hypervirulent K. pneumoniae and is susceptible to most antibiotics. Recently, convergence of multidrug resistant and hypervirulence phenotypes in liver abscess-causing K. pneumoniae was emerging and poses a major threat to public health. However, relatively little information is available on liver abscess-causing multidrug-resistant hypervirulent K. pneumoniae. In this study, we characterized phenotype and genotype of virulence and resistance of 18 multidrug-resistant hypervirulent liver abscess-causing K. pneumoniae strains collected from 395 pyogenic liver abscess cases in a tertiary teaching hospital over a 5-year period to enable in-depth understanding of this pathogen.

Aims Quorum sensing (QS) is the intercellular communication used by bacteria to regulate collective behaviour. QS regulates the production of virulence factors in many bacterial species and is considered to be an attractive target for reducing bacterial pathogenicity. Chlorogenic acid (CA) is abundant in vegetables, fruits, and traditional Chinese medicine, and has multiple activities. This study aimed to investigate the QS quenching activity of CA against clinically isolated multidrug-resistant Pseudomonas aeruginosa. Methods and Results The results showed that CA inhibited the mobility of bacteria, reduced the production of pyocyanin, and inhibited the activity of elastase. Furthermore, crystal violet staining and scanning electron microscope experiments showed that CA inhibited the formation of multidrug-resistant P. aeruginosa biofilm. CA at or below the concentration of 2560 mu g/mL exerted negligible cytotoxicity to RAW264.7 cells. The study also examined the expression of QS-related genes, including lasI, lasR, rhlI, rhlR, pqsA, and pqsR in P. aeruginosa and found that the expression of these genes was down-regulated under CA treatment. Conclusions The study showed that CA could be used as an anti-virulence factor for treating clinical P. aeruginosa infection. Significance and Impact of Study For the first time, this study took clinically isolated multidrug-resistant P. aeruginosa as the experimental object, and suggested that CA might be an effective antimicrobial compound targeting QS in treating P. aeruginosa infection, thus providing a new therapeutic direction for treating bacterial infection and effectively alleviating bacterial resistance.

Objectives: This study investigated the underlying mechanism of the evolution of tigecycline resistance during treatment in a patient infected with Klebsiella pneumoniae harbouring bla(KPC-2). Methods: A total of seven clonal K. pneumoniae strains were continuously isolated from a patient during hospitalisation. Antimicrobial resistance in the strains was determined by antimicrobial susceptibility testing. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed to explore the homology of the isolates. Whole-genome shotgun (WGS) analysis and cloning experiments were used to investigate the underlying mechanism of the evolution of tigecycline resistance. Results: All of the isolates had a minimum inhibitory concentration (MIC) for tigecycline of 4 mu g/mL, except strain FK6768 that had a MIC of 32 mu g/mL. Carbapenem-resistant K. pneumoniae strains (FK6614, FK6768 and FK6809) were consecutively isolated from faeces at different times. Antimicrobial susceptibility testing indicated that tigecycline resistance increased in FK6768 and subsequently decreased in FK6809, which attracted our attention. WGS and further bioinformatics analysis showed a homology for the three faecal isolates of >99%. The bla(KPC-2) carbapenemase gene and a tet(A) mutation were found in tigecycline-resistant isolate FK6768. Subsequent cloning experiments confirmed the contribution of a tet(A) variant to reduced tigecycline susceptibility. Conclusion: Here we report a K. pneumoniae isolate carrying both tet(A) mutation and the bla(KPC-2) gene, which led to increased tigecycline resistance during tigecycline treatment. This is the first report describing tigecycline resistance of K. pneumoniae first increasing and subsequently decreasing in vivo. (C) 2022 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy.

Klebsiella pneumoniae (K. pneumoniae) is one of the most common causes of bacterial meningitis worldwide. The purpose of this study was to investigate the clinical and microbiological characteristics of K. pneumoniae meningitis, as well as the association of antimicrobial resistance, virulence, and patient prognosis. The clinical data of patients with K. pneumoniae meningitis from 2014 to 2020 in a tertiary teaching hospital were retrospectively evaluated. Antimicrobial susceptibility profiles were performed by the agar dilution method and broth microdilution method. The isolates were detected for virulence-related genes, resistance genes, capsular serotypes, and molecular subtypes. A total of 36 individuals with K. pneumoniae meningitis were included in the study, accounting for 11.3% (36/318) of all cases of bacterial meningitis. Of the 36 available isolates, K1, K47, and K64 were tied for the most frequent serotype (7/36, 19.4%). MLST analysis classified the isolates into 14 distinct STs, with ST11 being the most common (14/36, 38.9%). Carbapenem resistance was found in 44.4% (16/36) of the isolates, while hypervirulent K. pneumoniae (HvKP) was found in 66.7% (24/36) of the isolates. The isolates of hypervirulent carbapenem-resistant K. pneumoniae (Hv-CRKP) were then confirmed to be 36.1% (13/36). Importantly, individuals with meningitis caused by Hv-CRKP had a statistically significant higher mortality than the other patients (92.3%, 12/13 vs. 56.5%, 13/23; P < 0.05). The high percentage and fatality of K. pneumoniae-caused meningitis, particularly in Hv-CRKP strains, should be of significant concern. More effective surveillance and treatment solutions will be required in future to avoid the spread of these life-threatening infections over the world.

Colistin is used as the “last line of defense” against multidrug-resistant (MDR) Gram-negative bacteria (GNB). However, improper use of colistin may further lead to an increasing number of colistin-resistant (Col-R) strains worldwide, which greatly limits antibiotic treatment options. In this study, we investigated the antibacterial and antibiofilm activities of naringenin (NG) combined with colistin against Col-R GNB in vitro and in vivo. The checkerboard method and time-kill test showed that NG combined with colistin has better antibacterial activity (FICI < 0.5) compared with NG and colistin alone. Biofilm formation inhibition tests demonstrated that combining the two drugs could inhibit biofilm formation; scanning electron microscopy (SEM) confirmed that the combination of the two significantly reduces the number of cells in the biofilm compared with the drug alone. The in vivo experiment showed that the combination of NG and colistin can improve the survival rate of the Galleria mellonella (G. mellonella) and reduce the microbial load in the mouse thigh infection model. Mechanistically, the combination of NG and colistin synergistically enhances the antibacterial activity and changes the permeability of the bacterial outer membrane. More importantly, cytotoxicity tests showed no cell cytotoxicity of NG in combination with colistin. In conclusion, our data revealed that NG combined with colistin exhibited good synergistic effects in vivo and in vitro, thus providing a new therapeutic option for clinical Col-R GNB infections.

This study aimed to analyze the influence of the main aerobactin-encoding gene iucB and the regulator of mucoid phenotype rmpA on the virulence of Klebsiella pneumoniae causing liver abscess. In addition, the possible regulatory effects of the main encoding gene iucB on the regulator of mucoid phenotype rmpA were explored, thus providing novel strategies for the prevention and control of hypervirulent K. pneumoniae (hvKp) causing liver abscess. The virulence-related genes iucB and rmpA of K. pneumoniae were detected by PCR. iucB and rmpA were cloned into K. pneumoniae strain by using plasmid pET28b as vector. Quantitative real-time PCR (RT-qPCR) was employed to detect the relative expression of rmpA gene in K. pneumoniae. We investigated the potential effects of aerobactin coding gene iucB and regulator of mucoid phenotype rmpA on the virulence of K. pneumoniae by establishing the Galleria mellonella infection model. Capsule quantitative experiment was conducted to investigate the impact of aerobactin-encoding gene iucB on the modulation of regulator of mucoid phenotype rmpA. The results of the G. mellonella infection model indicated that iucB gene could significantly enhance the virulence of K. pneumoniae, but the presence of rmpA gene did not markedly affect the virulence of K. pneumoniae. RT-qPCR showed that iucB inhibited the expression of rmpA gene. Quantitative capsulation experiments showed that the presence of rmpA gene could not increase the capsulation production of K. pneumoniae. The main encoding gene of aerobactin, namely iucB, could substantially enhance the virulence of K. pneumoniae. The gene iucB might be involved in the biosynthesis of the capsular polysaccharide through an unknown mechanism instead of the gene rmpA. Overall, these findings provide important theoretical support for the treatment of infections caused by hvKp.