| ObjectivePatients with chronic obstructive pulmonary disease(COPD)are mainly elderly and prone to recurrent acute exacerbations,mainly due to air pollution and bacterial infections.The air pollution in Shenyang is more severe in winter than in summer.Air pollution can exacerbate the deterioration of patients’ conditions and lead to abnormal changes in the diversity and abundance of pulmonary microbiota.The disorder of respiratory microbiota in COPD patients can easily lead to increased colonization of pathogenic bacteria such as P.aeruginosa in the respiratory tract,inducing respiratory infections and exacerbating the condition.At present,there is limited research on the upper respiratory tract microbiota of COPD patients both domestically and internationally,while the pulmonary microbiota mainly comes from the upper respiratory tract microbiota such as the oropharynx.Therefore,combining the characteristics of air pollution in Shenyang area,analyzing the composition and differences of oropharyngeal microbiota in elderly COPD patients in winter and summer can help clarify the characteristics of oropharyngeal microbiota disorder in COPD patients in this area,and provide a basis for controlling COPD from the perspective of adjusting the balance of microbiota.P.aeruginosa is the most common pathogenic bacterium in COPD patients,and P.aeruginosa infection can accelerate the deterioration of COPD patients’ condition.Intervening in P.aeruginosa colonization and growth can help reduce P.aeruginosa respiratory infections and reduce the frequency of acute exacerbation in COPD patients.At present,the effectiveness of conventional antibiotics in the treatment of P.aeruginosa infection is not ideal,while some dominant bacteria in respiratory symbiotic bacteria have an antagonistic effect on the colonization of pathogenic bacteria.In the early stage of this study,a symbiotic bacterium D-19 was isolated from the oropharyngeal tissues of healthy children,which can inhibit the growth of several pathogenic bacteria,including P.aeruginosa,in vitro.Therefore,on the basis of clarifying the characteristics of microbial community disorder in COPD patients,the effect of symbiotic bacteria D-19 on P.aeruginosa growth and biofilm formation is analyzed,providing a research basis for effectively utilizing symbiotic bacterial preparations to intervene in P.aeruginosa respiratory colonization and prevent and treat COPD.Methods(1)From January to March(winter)and June to August(summer)2018,COPD patients aged from 65 to 85 years as the patient group and healthy physical examiners of the same age group as the control group of a hospital in Shenyang were selected to collect oropharyngeal swabs for bacterial 16 S r RNA high-throughput sequencing.Through alpha diversity,beta diversity analysis,species composition analysis and species difference analysis,the diversity,community structure and species abundance of oropharyngeal microbiota between COPD patients and healthy people,as well as between COPD patients in summer and winter were compared.(2)The effect of D-19 cell-free supernatant collected at different culture time on the growth of P.aeruginosa,the effect of D-19 sup after heating,protease treatment and p H regulation on the bacteriostatic effect of P.aeruginosa,and whether D-19 sup combined with antibiotics has synergistic effect on the inhibition of P.aeruginosa growth were comprehensively evaluated by Oxford Cup antagonistic test and growth inhibition test.(3)Crystal violet staining was used to detect whether D-19 sup could inhibit the biofilm formation of P.aeruginosa and destroy the preformed biofilm.The biofilm morphology of P.aeruginosa co-cultured with D-19 sup was observed by optical microscope and scanning electron microscope.And the metabolic activity of viable P.aeruginosa biofilm cells treated with D-19 sup was evaluated by MTT reduction assay.(4)The effects of D-19 sup on extracellular polysaccharides of P.aeruginosa biofilm were detected by FITC-ConA staining assay,Congo red staining assay and RT-qPCR.Results(1)Alpha diversity analysis showed that in oropharyngeal flora abundance,COPD patients were significantly higher in summer than healthy people(P<0.01),no significant difference between COPD patients in winter and healthy population(P>0.05),and COPD patients in winter were significantly higher than COPD patients in summer(P<0.001);In terms of species diversity,COPD patients in summer and winter were lower than healthy people,and COPD patients in winter higher than COPD patients in summer.Unweighted Unifrac distance PCo A analysis showed that there were differences in the structure of oropharyngeal flora between COPD patients and healthy people,as well as between COPD patients in summer and COPD patients in winter.At the phylum level,the relative abundance of Actinobacteria and Cyanobacteria in the oropharyngeal flora of COPD patients in summer was significantly higher than that of healthy people(P < 0.001),while the relative abundance of Bacteroides,Patescibacteria,Spirochaetes and Tenericutes was significantly lower(P<0.05);There was no significant difference in the relative abundance of oropharyngeal flora between COPD patients and healthy people in winter(P > 0.05);In the oropharynx of patients with COPD in winter,the relative abundance of Proteobacteria,Planctomycetes,Verrucomicrobia,Epsilonbacteraeota,and Patescibacteria is higher(P<0.05),while the relative abundance of Firmicutes and Actinobacteria is higher in the oropharynx of COPD patients in summer(P<0.01).At the genus level,compared with the healthy people,the abundance of Rothia and Actinomyces in the oropharyngeal flora of COPD patients in summer was significantly higher(P<0.01),while the abundance of Neisseria,Alloprevotella and Prevotella decreased(P<0.05);The abundance of Actinomyces in the oropharyngeal flora of COPD patients in winter increased(P<0.05),while the abundance of Prevotella decreased(P < 0.05);Compared with COPD patients in summer,the relative abundance of Neisseria and Porphyromonas in the oropharynx of COPD patients in winter is higher(P < 0.05),while the relative abundance of Actinomyces and Veillonella is higher in the oropharynx of COPD patients in summer(P<0.05).Through species difference analysis,it was found that no matter COPD patients in summer or in winter,the relative abundance of Actinomyces graevenitzii F0530 in the oropharyngeal flora increased significantly,the relative abundance of Prevotella nigrescens CC14 M decreased significantly compared with healthy people(P<0.05).(2)The Oxford Cup antagonistic test and growth inhibition test showed that D-19 thallus could not inhibit the growth of P.aeruginosa,while D-19 sup could inhibit the growth of P.aeruginosa(P < 0.001);Compared with the control group,the bacteriostatic properties of D-19 sup after treatment at 40℃ did not change significantly,but gradually weakened after treatment at 60℃,80℃ and 100℃(P<0.01);The bacteriostatic activity of D-19 sup to P.aeruginosa was significantly reduced after treatment with protease K,and the higher the concentration of enzyme,the lower the bacteriostatic activity(P<0.01);The bacteriostatic activity of D-19 sup after trypsin(1mg/ml)treatment was not significantly different from that of the control group(P>0.05),but the bacteriostatic activity was significantly reduced after trypsin(2 mg/ml)treatment(P < 0.01);It was detected that D-19 sup was weakly acidic.When p H=3.0~6.0,the bacteriostatic property of the supernatant was significantly improved by lowering the p H value(P<0.05).When p H=6.0~8.0,the bacteriostatic property of the supernatant was not significantly changed by increasing the p H value;The combination of D-19 sup and cefoperazone sodium has a stronger antibacterial effect on P.aeruginosa than that of cefoperazone sodium alone(P<0.001).(3)D-19 sup can not only inhibit the formation of P.aeruginosa biofilm,but also destroy the mature biofilm that has been formed,and the cell-free supernatant collected from D-19 culture for 12 hours has the strongest inhibition and destruction effect on P.aeruginosa biofilm(P<0.001),with the inhibition rate of 53.55% and the destruction rate of 38.64%;Light microscope and scanning electron microscope showed that P.aeruginosa co-culture with D-19 sup for 24 hours,the cell mass of biofilm decreased,the density decreased,and the structure was loose;Compared with the control group,the number of active cells in the P.aeruginosa biofilm after D-19 sup treatment decreased by about 32.33%(P<0.05).(4)Fluorescence microscope observation showed that the biofilm exopolysaccharide complexes were significantly reduced,thin and scattered in the D-19 sup treatment group compared with the control group;Congo red binding test showed that the binding amount of P.aeruginosa biofilm and Congo red after D-19sup treatment decreased from 100% to 66.62% compared with the control group(P<0.01);Compared with the control group,the expression of extracellular polysaccharide related genes pelA,pslA and algD of P.aeruginosa biofilm treated with D-19 sup decreased(P<0.01).Conclusions(1)Compared with the healthy population,the composition of the normal flora in the oropharyngeal areas of elderly COPD patients in Shenyang during summer and winter has changed.The relative abundance of the normal flora has decreased,while the relative abundance of some opportunistic pathogenic bacteria has significantly increased.This suggests that the imbalance of the oropharyngeal flora in elderly COPD patients may be beneficial for the colonization of respiratory pathogenic bacteria,leading to infection and acute exacerbation of the disease;Compared with patients in summer,patients in winter have higher diversity of oropharyngeal flora,and there are differences in dominant species,which may be related to the higher degree of air pollution in Shenyang in winter than in summer.(2)P.aeruginosa is the main pathogenic bacteria of COPD,and D-19 cell-free supernatant can inhibit the growth of P.aeruginosa and enhance the sensitivity of P.aeruginosa to cefoperazone sodium.It is preliminarily believed that there are protein antibacterial substances in the supernatant,heat-resistant,acid-resistant and weak alkali-resistant.(3)The colonization of P.aeruginosa through biofilm is the main cause of acute exacerbation of COPD.D-19 sup can effectively inhibit the formation of P.aeruginosa biofilm,destroy the mature biofilm formed by P.aeruginosa,and reduce the metabolic activity of cells in the biofilm;It can reduce the aggregation of extracellular polysaccharides in P.aeruginosa biofilm and reduce the expression of extracellular polysaccharides related genes in biofilm. |
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