P seud omon as aeruginosa is a common opportunistic pathogen with growing resistance and presents heightened treatment challenges. Quorum sensing (QS) is a cell-to-cell communication system that contributes to the production of a variety of virulence factors and is also related to biofilm formation of P. aeruginosa . Compared to traditional antibiotics which kill bacteria directly, the anti-virulence strategy by targeting QS is a promising strategy for combating pseudomonal infections. In this study, the QS inhibition potential of the compounds derived from the Traditional Chinese Medicines was evaluated by using in silico, in vitro, and in vivo analyses. The results showed that psoralen, a natural furocoumarin compound derived from Psoralea corylifolia L., was capable of simultaneously inhibiting the three main QS regulators, LasR, RhlR, and PqsR of P . aeruginosa. Psoralen had no bactericidal activity but could widely inhibit the production of extracellular proteases, pyocyanin, and biofilm, and the cell motilities of the model and clinical P . aeruginosa strains. RNA-sequencing and quantitative PCR analyses further demonstrated that a majority of QS-activated genes in P . aeruginosa were suppressed by psoralen. The supplementation of psoralen could protect Caenorhabditis elegans from P . aeruginosa challenge, especially for the hypervirulent strain PA14. Moreover, psoralen showed synergistic antibacterial effects with polymyxin B, levofloxacin, and kanamycin. In conclusions, this study identifies the anti-QS and antibiofilm effects of psoralen against P. aeruginosa strains and sheds light on the discovery of anti-pseudomonal drugs among Traditional Chinese Medicines. Key points • Psoralen derived from Psoralea corylifolia L. inhibits the virulence-related phenotypes of P. aeruginosa. • Psoralen simultaneously targets the three core regulators of P. aeruginosa QS system and inhibits the expression of a large part of downstream genes. • Psoralen protects C. elegans from P. aeruginosa challenge and enhances the susceptibility of P. aeruginosa to antibiotics.

Objectives: The aim of this study was to characterize the co-occurrence of the blaSHV-182, blaKPC-2, blaCTX-M65, and blaTEM-1B genes in a multi-resistant carbapenem carbapenem-resistant K. pneumoniae (CRKP) strain. Methods: The multi-resistant CRKP strain was isolated from the bronchoalveolar lavage fluid (BAL) of a patient with acute exacerbation of chronic obstructive pulmonary disease (AECOPD), after cultured in lysogeny broth, the colonies with apparent phenotypic differences (size, shape, color, and surface states) were picked out for species identification by sequencing their 16S rDNA and BLASTN in the NCBI database, antimicrobial susceptibility was determined, Genomic DNA of overnight cultured K. pneumoniae D1a was extracted by using the Bacterial DNA Isolation Kit, the library was constructed by using NEBNext (R) UltraTM DNA Library Prep Kit for Illumina, and the whole-genome sequencing was performed on the Illumina HiSeq PE150 platform. The pair-end reads were assembled by SOAP denovo v2.04, SPAdes, and ABySS. Genome characteristics were analyzed by using bioinformatics methods. Results: The WGS of the multi-resistant CRKP strain D1a (CRKP D1a) yielded 84 scaffolds of genomic DNA (5, 492, 035 bp) and two circular plasmids, pD1a1 (173, 413 bp) and pD1a2 (57, 686 bp). CRKP D1a was classified as the sequence type 11, and harbored 16 antibiotic resistance genes and 30 multidrug efflux pump-encoding genes associated with the resistance to almost all kinds of commonly used antibiotics. The plasmid pD1a1 belonged to the IncFII/IncR hybrid plasmid family with blaSHV-182, blaKPC-2, blaCTX-M-65, and blaTEM-1B genes flanked by the IS6-like element. The plasmid pD1a2 was comprised of 95 phage-related genes but had no lysis activity on the tested K. pneumoniae strains. Conclusion: This study reports the isolation of a multidrug-resistant CRKP strain co-carrying IncFII/IncR-KPC2 plasmid and phage-like plasmid, the findings hold significant implications for further investigating the resistance mechanism and molecular diversity of CRKP.

The quorum-sensing (QS) system of Pseudomonas aeruginosa dominates the pathogenicity of the acute or chronic infection process. Hence, curbing the pathogenicity of P. aeruginosa by targeting QS system is an ideal strategy. This study aims to identify potential anti-virulence compounds that can effectively disrupt the QS system of P. aeruginosa using a combination of virtual screening and experimental validation techniques. We explored inhibitory effect of isovanillin obtained by virtual screening on P. aeruginosa QS regulated virulence factors extracellular protease, biofilm, and pyocyanin. Results displayed that isovanillin could inhibit the virulence phenotypes regulated by the las- and pqs-QS systems of P. aeruginosa. The synthesis of extracellular proteases, pyocyanin, and biofilm formation by P. aeruginosa were dramatically inhibited by sub-MICs doses of isovanillin. The results of RNA sequencing and quantitative PCR revealed that the QS-activated genes down-regulated by subinhibitory isovanillin in the transcriptional evels. Furthermore, the presence of isovanillin increased the susceptibility of drug-resistant P. aeruginosa to kanamycin, meropenem, and polymyxin B. Treatment of isovanillin as a monotherapy significantly decreased the mortality of C. elegans in P. aeruginosa PAO1 or UCBPP-PA14 (PA14) infection. Our study reported the anti-virulence activity of isovanillin against P. aeruginosa, and provided a structural foundation for developing anti-virulence drugs targeting the QS system of P. aeruginosa.

Colonization of Pseudomonas aeruginosa in the lung environments frequently leads to the enrichment of strains displaying enhanced antibiotic resistance and reduced production of quorum-sensing (QS) controlled products. However, the relationship between the emergence of QS deficient variants and antibiotic resistance remains less understood. In this study, 67 P. aeruginosa strains were isolated from the lungs of 14 patients with chronic obstructive pulmonary disease, followed by determining their genetic relationship, QS-related phenotypes and resistance to commonly used antibiotics. The integrity of P. aeruginosa QS system was checked by DNA sequencing. The relationship between the QS system and antibiotic resistance was then assessed by correlation analyses. The function of the LasR protein and bacterial virulence were evaluated through homology modeling and nematode-infection assay. The influence of antibiotic on the development of extracellular protease production ability of P. aeruginosa was tested by an evolutionary experiment. The results showed that P. aeruginosa clinical strains displayed abundant diversity in phenotype and genotype. The production of extracellular proteases was significantly negatively correlated with antibiotic resistance. The strains with enhanced antibiotic resistance also showed a notable overlap with the mutation of lasR gene, which is the core regulatory gene of P. aeruginosa QS system. Molecular docking and Caenorhabditis elegans infection assays further suggested that P. aeruginosa with impaired LasR protein could also have varying pathogenicity. Moreover, in vitro evolution experiments demonstrated that antibiotic-mediated selective pressure, particularly from Levofloxacin contributed to the emergence of extracellular protease-negative strains. Therefore, this study provides evidence for the connection of P. aeruginosa QS system and antibiotic resistance, and holds significance for developing targeted strategies to address antibiotic resistance and improving the management of antibiotic-resistant infections in chronic respiratory diseases.

Mycobacterium tuberculosis, the ancient master of causing tuberculosis, is one of the most successful pathogens capable of persistently colonizing host lungs. The EsxB (CFP-10) of ESX-1 system and PPE68 of the PPE family contribute to the virulence of M. tuberculosis. However, the virulence potential and pathogenetic characteristics of these two proteins during M. tuberculosis infection remain unclear. In this study, two prokaryotic expression plasmids for EsxB or PPE68 of M. tuberculosis were constructed and the recombinant proteins His-EsxB or His-PPE68 were purified. The proteome and transcriptome of MH-S cells treated with His-EsxB or His-PPE68 were explored, followed by validating the expression of the identified differentially expressed genes (DEGs) using quantitative PCR. A total of 159/439 specific proteins or 633/1117 DEGs were obtained between control and His-EsxB or His-PPE68 treated groups in the MH-S proteomes and transcriptomes. Additionally, 37/60 signal pathways were predicted in the His-EsxB or His-PPE68 treated groups and “Cytokine-cytokine receptor interaction” was the most represented pathway. Furthermore, the expression of the DEGs (IL-1β, IL-6, and TNF-α) was significantly upregulated, suggesting that these DEGs contributed to the host response during EsxB or PPE68 treatment. These findings provide detailed information on developing an effective intervention strategy to control M. tuberculosis infection.

Chronic infection of the common bacterial pathogen Pseudomonas aeruginosa frequently leads to the coexistence of heterogeneous individuals to engage in several group behaviors. However, further evolution of the polymorphic P. aeruginosa population, including the dynamic change of social cooperation and its impact on host immune system, still remain elusive. We show that the evolution of P. aeruginosa in the patients with chronic obstructive pulmonary disease frequently selects the isolates deficient in producing the costly and sharable extracellular products for nutrient acquisition. The evolution of polymorphic P. aeruginosa population is mainly concentrated on modifying the adaptability of lasR mutants, which are typical cheaters in the competition of quorum-sensing-controlled extracellular proteases. Importantly, lasR mutants with varying degrees of evolution interact with wild-type P. aeruginosa in a framework termed cascaded public goods game to compete for extracellular proteases and siderophores, and thus perpetuate social cooperation under different conditions. Finally, we find that a polymorphic population comprised of lasR- intact P. aeruginosa and evolved lasR- mutant can minimize the host immune fluctuation for persistent colonization. This study demonstrates the multistage evolution and complex interaction of P. aeruginosa in adaptation to host lungs, and provides an explanation for the success of cooperation in public goods game.### Competing Interest StatementThe authors have declared no competing interest.

Chronic infection with the bacterial pathogen Pseudomonas aeruginosa often leads to coexistence of heterogeneous populations carrying diverse mutations. In particular, loss-of-function mutations affecting the quorum-sensing regulator LasR are often found in bacteria isolated from patients with lung chronic infection and cystic fibrosis. Here, we study the evolutionary dynamics of polymorphic P. aeruginosa populations using isolates longitudinally collected from patients with chronic obstructive pulmonary disease (COPD). We find that isolates deficient in production of different sharable extracellular products are sequentially selected in COPD airways, and lasR mutants appear to be selected first due to their quorum-sensing defects. Polymorphic populations including lasR mutants display survival advantages in animal models of infection and modulate immune responses. Our study sheds light on the multistage evolution of P. aeruginosa populations during their adaptation to host lungs.

Dear Editor, Pseudomonas aeruginosa is a ubiquitous Gram-negative oppor-tunistic bacterium that notoriously causes infections with a high mortality rate in hospitalized patients,especially those with compromised immune systems.1 P.aeruginosa can cause acute infections that are typically associated with the cytotoxins secreted by the type Ⅲ secretion system(T3SS),2 as well as chronic persistence that relies on type Ⅵ secretion system(T6SS)and biofilm formation,including those in cystic fibrosis patients.3 An intercellular communication network based on cell density,quorum sensing(QS),regulates numerous gene expressions including those related to both acute and chronic virulence of P.aeruginosa.4 The global regulatory protein of the repressor of secondary metabolites(Rsm)system in bacteria,RsmA,is found to predominantly regulate these virulence entities in P.aeruginosa by modulating related gene expressions at transcriptional and posttranscriptional levels.5

Pseudomonas aeruginosa is a notorious Gram-negative opportunistic pathogen that normally causes acute and chronic infections in a wide range of hosts. In this study, a multi-resistant P. aeruginosa isolate L1a harboring an infrequent plasmid with red fluorescence was obtained from the bronchoalveolar lavage fluid of a patient with chronic obstructive pulmonary disease. The results of susceptibility testing and virulence-related phenotypic identification revealed that P. aeruginosa L1a was resistant to levofloxacin, cefepime, aztreonam, and imipenem and showed significantly stronger capacities for swimming and pyocyanin production than the reference strain PAO1. The genome of P. aeruginosa L1a was assembled into one circular chromosome (6,216,913 bp) and one circular plasmid (9111 bp). P. aeruginosa L1a was found to belong to the multilocus sequence type ST549, and serotype O5, and carried 8 drug resistance genes and 18 multidrug efflux pump-related genes in the chromosomal DNA. The plasmid pL1a harbored a tetracycline resistant gene tetA and a functional red fluorescence protein. This study reports a multidrug resistant P. aeruginosa clinical isolate harboring an infrequent red fluorescence plasmid for the first time.

Pseudomonas aeruginosa relies on its complex cellular regulatory network to produce a series of virulence factors and to cause various acute and chronic infections in a wide range of hosts. Compared with traditional antibiotics which frequently accompany with widespread antibiotic resistance, crippling the virulence system of bacteria is expected to be a promising anti-infective strategy. In this study, Dimetridazole and Ribavirin, which had poor antibacterial activities on P. aeruginosa reference isolate PAO1 in nutrient medium but significantly inhibited the growth of P. aeruginosa PAO1 in M9-adenosine, were selected from 40 marketed compounds with similar core structure (furan, benzofuran, or flavonoids) to the acyl-homoserine lactone signals of P. aeruginosa quorum sensing (QS) system. The production of QS-controlled proteases, pyocyanin, and biofilm formation of P. aeruginosa PAO1 and the clinical isolates were significantly decreased by the presence of Dimetridazole or Ribavirin. Correspondingly, the majority of QS-activated genes in P. aeruginosa, including the key regulatory genes lasR, rhlR, and pqsR and their downstream genes, were significantly inhibited by Ribavirin or Dimetridazole, as determined by RNA-sequencing and quantitative PCR. Furthermore, the susceptibilities of drug-resistant P. aeruginosa isolates to polymyxin B, meropenem, and kanamycin were remarkably promoted by the synergistic application of Dimetridazole or Ribavirin. Finally, the treatment of Ribavirin or Dimetridazole effectively protected Caenorhabditis elegans and mice from P. aeruginosa infection. In conclusion, this study reports the antivirulence potentials of Dimetridazole and Ribavirin on P. aeruginosa and provides structural basis and methodological reference for the development of anti-pseudomonal drugs.

AbstractPseudomonas aeruginosais an opportunistic human pathogen that poses threats to hospitalized immunocompromised patients1. A non-coding small RNA (sRNA) from the repressor of secondary metabolites (Rsm) system, RsmZ, sequesters the global repressor protein RsmA to regulate downstream gene expressions that reprogram virulence repertoires associated with acute and chronicP. aeruginosainfections2,3. Molecular insights into the full-length RsmZ architecture remain elusive, leading to the lack of understanding of RsmZ binding to RsmA and subsequent modulations of gene expressions. Here we use cryo-electron microscopy (cryo-EM) to resolve structures of the full-length RsmZ in complexes with RsmA, in which five stem-loops (SLs) and one single-stranded junction carrying the GGA binding sites in RsmZ form three pairs of clamps, each binding to a RsmA homodimer. Disruptions of the base-pairings in all stems of RsmZ significantly reduced the binding affinity to RsmA by 17-fold, which resulted in enhanced RsmA downregulation of gene expressions and phenotypes associated to both acute and chronic virulence ofP. aeruginosa. Double mutations that rescued these stems of RsmZ restored the binding to RsmA by more than 5-fold, and recovered the corresponding phenotypes. Our results reveal the molecular mechanism of RsmZ regulation ofP. aeruginosavirulence and suggest RsmZ as a potential target for the development of new antimicrobial agents.

Increasing evidence has demonstrated the polymicrobial characteristics of most chronic infections, and the frequent communications among bacterial pathogens result in many difficulties for clinical therapy. Exploring bacterial interspecific interaction during antibiotic treatment is an emerging endeavor that may facilitate the understanding of polymicrobial infections and the optimization of clinical therapies.