Publish Time: 2022-12-14 Origin: 迪飞医学
/ guide /
In July 2022, the team of Professor Zhang Min from the First People's Hospital Affiliated to Shanghai Jiaotong University School of Medicine, together with experts from Washington University and Fuzhou Pulmonary HospitalFrontiers in Cellular and Infection Microbiology(IF=6.073) magazine published a prospective study to evaluate the risk of chronic aeruginosa in patients with non-cystic fibrosis bronchiectasis (NCFB) through quorum sensing (QS) system evaluation, whole genome sequencing (WGS), and evolutionary experiments. Whether there will be drug resistance selection of lasR mutation during Pseudomonas lung infection and explain its mechanism.Di Fei Medical participated in the data analysis of the study as a co-author.
/ Research Background /
Pseudomonas aeruginosa is a common opportunistic pathogen that causes chronic infection and releases multiple virulence factors in patients with non-cystic fibrosis bronchiectasis (NCFB), resulting in increased morbidity, decreased quality of life, and treatment The burden increases.Eradication of Pseudomonas aeruginosa has become increasingly difficult due to its marked resistance to antibiotics.
The development of multidrug resistance (MDR) can influence the evolution and adaptation of Pseudomonas aeruginosa isolates and facilitate their becoming frequent colonizers in chronically infected patients.During this process, some physiological system functions of bacteria may be changed, and quorum sensing (QS) is one of them.Pseudomonas aeruginosa utilizes the QS system to regulate the expression of hundreds of genes in a cell density-dependent manner, many of which encode secreted virulence factors such as proteases, elastase, hydrogen cyanide, and phenazine, which makes the QS system group into potential targets for anti-drug therapy.The QS system is mediated by many small signaling molecules, including N-3-oxo-dodecyl homoserine lactone (3OC12-HSL) and N-butanol-homoserine lactone (C4-HSL), two key Signaling stratification activates the LasR-LasI system and the RhlR-RhlI system.3OC12-HSL is the product of LasI synthase, and when its environmental concentration increases to a threshold value, it can bind to the transcriptional activator LasR and activate a series of genes, including rhlI encoding C4-HSL signaling synthase and C4-HSL receptor encoding rhl R.
Pseudomonas aeruginosa isolates from patients with chronic lung infection often lack QS, but lasR mutant strains are the most common, with frequencies greater than 30% reported.The mechanism behind this phenomenon is still unknown.There may be a strong selective pressure such that lasR mutants may have a physiological advantage under certain growth conditions.In addition, the emergence of lasR mutants also benefited from the extracellular products produced by the QS wild-type strain.Previous studies have reported that lasR mutants evolved from wild-type ancestors during long-term culture under QS-requiring conditions and have a selective advantage when co-cultured with wild-type parents.Previous studies have shown that there is an interaction between the emergence of Pseudomonas aeruginosa MDR and QS function.On the one hand, QS promotes the occurrence of MDR by regulating the expression of multidrug efflux pump genes; on the other hand, the QS system itself is also affected by the expression of multidrug efflux pumps.Whether the influence of efflux pumps on QS creates a selective pressure on LasR activity in clinical P. aeruginosa strains is currently unknown.
In this study, respiratory isolates of P. aeruginosa from NCFB patients were collected and the QS phenotypes of MDR and non-MDR strains were compared.The influence of MDR mechanism on the evolution of lasR mutants was investigated by whole genome sequencing (WGS) and evolution experiments.
/ research content /
The study prospectively enrolled adult patients with chronic Pseudomonas aeruginosa pulmonary infection in two centers for a period of 2 years. Lasts more than 14 days.Patients co-colonized with other detected pathogens were excluded.MDR P. aeruginosa is defined as the acquisition of three or more antipseudomonal drugs (eg, carbapenems, fluoroquinolones, penicillins/cephalosporins, and aminoglycosides) Not sensitive.
Patient's clinical characteristics
A total of 97 patients with Pseudomonas aeruginosa chronic pulmonary infection were included in the analysis, including 63 non-MDR colonized strains and 34 MDR colonized strains.Univariate analysis found that,Patients with MDR had significantly longer duration of infection and more frequent previous exposure to carbapenems than non-MDR patients.Further multivariate analysis showed that,Only prior carbapenem exposure independently predicts isolation of MDR Pseudomonas aeruginosa strains(OR: 24.9, 95% CI: 5.2-156.0, p <0.0001).
MDR isolates lack the QS phenotype
One isolate was selected from each patient to determine whether it was MDR, and the QS phenotypes between MDR and non-MDR strains were compared, including signaling molecules (3OC12-HSL and C4-HSL), and virulence factors (pyocyanin and protease).It was found that the production of QS-controlled signaling molecules and virulence factors was significantly reduced in MDR isolates compared with non-MDR isolates.And these phenotypes showed more heterogeneity among MDR isolates than among non-MDR isolates.Overall, MDR isolates had a poorer QS phenotype than non-MDR isolates in all samples, supporting that QS deficiency during chronic lung infection is associated with the development of MDR in Pseudomonas aeruginosa.Furthermore, by measuring the QS-controlled virulence yield of MDR isolates grown with or without 3OC12-HSL for 18 h, it was found that,The reduced virulence yield could not be significantly complemented by exogenous addition of 3OC12-HSL, suggesting that the lack of QS phenotype in MDR isolates is related to the dysfunction of LasR, but not LasI.
To determine whether there is an evolutionary relationship between the emergence of the QS-deficient phenotype and MDR characteristics, we analyzed 11 pairs of isolates from different patients, 7 isolates switched from non-MDR to MDR, while the other 4 isolates remained non-MDR.We found that among isolates from patients with MDR metastases, the QS phenotype of relapsed isolates was significantly worse than that of initial isolates; however, in patients who maintained non-MDR, there was no significant difference in QS phenotype between relapsed and initial isolates.It was shown that the QS-deficient phenotype occurred after the MDR of Pseudomonas aeruginosa strains.
WGS identifies MDR determinants and QS gene mutations
To elucidate the mechanism of the QS-deficient phenotype of MDR isolates in the study population,The study screened 30 strains with similar in vitro growth rate to the laboratory strain PAO1 for WGS.These isolates included 24 isolates from different patients and 3 pairs of isolates from the same patient.Single-nucleotide variation analysis was used to determine the degree of genetic relatedness between sequenced isolates.The analysis results show that,The genetic diversity among these isolates was high, even within pairs of isolates from the same patient.
The results of mutations of drug resistance determinants showed that no isolates containing the carbapenemase gene were found; 14 (93.3%) MDR isolates and 11 (73.3%) non-MDR isolates contained non-synonymous mutations in oprD, possibly Resulting in loss of functional oprD expression.Comparing the frequency of non-synonymous mutations in oprD between MDR isolates and non-MDR isolates, there was no significant difference between the two groups.Although there are non-synonymous mutations in other drug resistance determinants such as ampC, ampR, ampD and dacB, almost all of them are missense mutations, and there are no frameshift mutations or premature terminator mutations, suggesting that these gene mutations are in the isolates. does not play a major role in the development of MDR.
Twenty-eight isolates (14 MDR isolates and 14 non-MDR isolates) had non-synonymous mutations in the lasR-I and rhlR-I systems, which were different from the laboratory strain PAO1.rhlI was the most frequently mutated gene in the QS system, and its non-synonymous mutations were present in 26 isolates.However, its mutation frequency was not significantly different between MDR isolates and non-MDR isolates.Mutations in the lasI and rhlR genes were present in only 2 isolates and were rare.Eight isolates had lasR non-synonymous mutations, almost all of which belonged to the MDR group, and only one was from a non-MDR isolate.These data suggest that in the lasR-I and rhlR-I systems, the frequency of non-synonymous mutations in the lasR gene is significantly higher in MDR isolates than in non-MDR isolates.
Comparison of synonymous and non-synonymous mutation frequencies in QS genes (lasR and RhlI) and efflux pump regulatory genes (mexR, nalC, nalD, mexZ, and nfxB) in MDR and non-MDR, and analysis of the correlation between these frequencies found , the frequency of synonymous mutations was not significantly different between the two groups, but the frequency of non-synonymous mutations in lasR, nalC and mexZ genes of MDR isolates was significantly higher than that of non-MDR isolates.The correlation analysis results show that,For synonymous mutation frequencies, lasR was significantly positively correlated with efflux pump regulatory genes (mexR, nalC, and mexZ), while for non-synonymous mutations, lasR was only associated with nalC and mexZ.The Ka/Ks ratios of these genes were calculated to estimate the selection pressure they experienced, and it was found that the Ka/Ks ratios of lasR, nalC, and mexZ were significantly higher in MDR isolates than in non-MDR isolates.The Ka/Ks ratio of lasR was significantly positively correlated with the Ka/Ks ratio of nalC and mexZ, supporting the existence of selection pressure for these gene mutations in MDR strains.
Phenotypic assessment and evolution experiments of efflux pump-induced QS dysfunction
Efflux pump activity and its effect on QS activity was evaluated using the efflux pump inhibitor PAbN.Whether PAbN was added or not had little effect on the growth curve.The QS phenotype of non-MDR isolates was not significantly different in media with and without PAbN supplementation.The MICs of ciprofloxacin, ceftazidime, and meropenem decreased by more than 50% when PAbN was added to MDR isolates; meanwhile, their QS signal and pyocyanin increased significantly.These data supportEfflux pump activity was involved in MDR development in these strains, resulting in low production of QS signaling molecules and virulence factors.
To determine the selective pressure of efflux pumpins on lasR mutations, three independent evolution experiments were performed on three QS-efflux pump regulator mutants (isolates 17, 20, and 24) possessing complete lasR sequences.In seven of the nine experiments, lasR mutants emerged at different times.To confirm that the population phenotype was due to upregulation of efflux pump activity, we repeated the experiment by adding the efflux pump inhibitor PAbN.A stable balance was maintained between the QS strain and the QS-deficient strain, and made the MDR strain phenotype similar to wild-type PAO1.
/ Summarize /
1. This study was designed to test the hypothesis that the development of MDR exerts selective pressure on lasR mutations in Pseudomonas aeruginosa colonizing NCFB patients.
2. Clinical MDR isolates exhibit a QS-deficient phenotype, including low production of QS signaling molecules and QS-controlled virulence factors.Exogenous addition of the signal 3OC12-HSL did not improve these phenotypes.
3. Paired clinical isolates obtained at different times showed that their QS phenotype defects occurred after MDR.WGS analysis showed that efflux pump activity was upregulated in clinical MDR Pseudomonas aeruginosa isolates, and lasR was positively correlated with the mutation frequency of efflux pump regulatory genes nalC and mexZ.
4. Evolution experiments showed that the upregulation of efflux pump activity promoted lasR mutation in MDR isolates.
5.In summary, it is suggested that in chronic lung infection, the efflux pump activity up-regulated by MDR Pseudomonas aeruginosa is prone to lasR mutation.Anti-QS combined with efflux pump inhibitors may be a potential therapeutic strategy for MDR Pseudomonas aeruginosa-infected NCFB patients.
6. Disadvantages: Only the population of two centers was included, which may limit the promotion of the research results to the patient population of other centers.Additionally, the possibility that other mechanisms are involved in the evolution of lasR mutants in MDR isolates cannot be ruled out and more studies are needed to clarify.
Original link:
https://www.frontiersin.org/articles/10.3389/fcimb.2022.934439/full
Ding F, Han L, Xue Y, Yang IT, Fan X, Tang R, Zhang C, Zhu M, Tian X, Shao P and Zhang M (2022) Multidrug-resistant Pseudomonas aeruginosa is predisposed to lasR mutation through up-regulated activity of efflux pumps in non-cystic fibrosis bronchiectasis patients. Front. Cell. Infect. Microbiol. 12:934439. doi: 10.3389/fcimb.2022.934439
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