Based On Nanopore Gene Sequencing Technology,Drug Resistancegene Detection And Risk Factors Analysis Of Alveolar Lavage Fluid In Patients With Bacterial Pneumonia Were Carried Out

Background and objectives:The incidence rate of pneumonia is very high at home and abroad,and Bacterial pneumonia occupies an absolute advantage in CAP,HAP and VAP.Before the advent of antibiotics,the treatment of bacterial infections was ineffective and had a high mortality rate.With the advent of antibiotics,many patients with bacterial infections received better treatment.While killing or inhibiting the growth of bacteria,these antibiotics bring selective pressure to the survival of bacteria.Under the influence of multiple factors such as the pressure of antibiotic selection and Antibiotic misuse,bacteria gradually become resistant,which brings difficulties and challenges to the treatment of patients with Bacterial pneumonia.At present,the situation of antibiotic resistance in Bacterial pneumonia in China is grim.A variety of common bacteria in Bacterial pneumonia,such as Acinetobacter baumannii,Streptococcus pneumoniae,Staphylococcus aureus,Klebsiella pneumoniae,etc.,all show different degrees of resistance and are not optimistic.It is urgent to reduce the drug resistance rate of Bacterial pneumonia.Antibiotic resistance in Bacterial pneumonia is closely related to bacterial resistance genes,At present,there is a lack of research on the risk factors of antibiotic resistance genes in patients with Bacterial pneumonia.Nowadays,the main methods for detecting bacterial drug resistance genes include NGS and nanopore sequencing.The reading length of NGS is generally about 150 bp,and there are high repetition regions and high GC regions in bacterial genes,and there are often many missing sequences in the assembled genome.The disadvantage of reading length brings difficult problems to the assembly of bacterial genes,which is difficult to obtain complete sequences of pathogenic microorganisms and identify antibiotic resistance genes,However,nanopore sequencing overcomes the problems faced by NGS due to its ability to directly and completely detect all bacterial genes and the advantage of not requiring gene amplification during the detection process.Compare the results of nanopore sequencing for detecting bacteria with those of bacterial culture,and then compare the results of nanopore sequencing for detecting drug-resistant genes with those of drug sensitivity tests to explore whether nanopore sequencing technology has a stronger ability in detecting bacteria and detecting antibiotic resistance compared to bacterial culture and drug sensitivity experiments.In this study,nanopore sequencing technology was used to detect drug resistance Genetic testing in patients with Bacterial pneumonia in alveolar lavage fluid,and the samples were divided into drug resistant genomes and non drug resistant genomes,to compare whether there were statistical differences between the two groups in multiple factors such as gender and age of patients in the two groups,and to find out the independent risk factors of antibiotic resistance genes in bacteria in alveolar lavage fluid of patients with Bacterial pneumonia.By combining the results of drug sensitivity experiments and nanopore sequencing,explore the antibiotics that may be more sensitive to several bacteria with high detection rates in this study,in order to guide drug use.Research methods:From June 2022 to March 2023,patients with CAP/HAP/VAP in Ganzhou People’s Hospital and Gannan First Affiliated Hospital who were considered to have pneumonia infection by imaging methods were collec ted,and their bronchoalveolar lavage fluid was collected for nanopore technology sequencing and bacterial culture.The bacteria detected by the two methods of the patients were recorded.The samples of bacteria detected by any of the two methods were included in the study.The samples included in the study were divided into drug resistance genome and non-drug resistance genome according to the presence or absence of drug resistance genes,and the data of patients were collected.Including gender,age,consciousness,history of smoking,drinking,diabetes,coronary heart disease,hypertension,albumin,PCT,WBC,CRP,length of hospital stay,glucocorticoid use,immunosuppressant use,days of antibiotic use,types of antibiotic use,antibiotic carbapenem use,postoperative surgery,invasive operation(except surgery).SPSS 22.0 statistical software was used for statistical operation to compare the above factors between patients with and without drug resistance genes.Logistic analysis was used to find out the independent risk factors of bacterial pneumonia patients with drug resistance genes.By combining the results of drug sensitivity test and nanopore sequencing,the four bacteria with high detection rate in this study were explored to guide drug use.Results:1.Fifty-five samples included in the study were subjected to bacterial culture and nanopore sequencing.Bacteria were detected in 33 samples during bacterial culture,while 40 samples were detected during nanopore sequencing.The ability of nanopore sequencing to detect bacteria was stronger than that of bacterial culture(P<0.05).2.Drug sensitivity tests and nanopore sequencing were conducted on 40 samples of bacteria,respectively.Antibiotic resistance was detected in 24 samples in the drug sensitivity test,and drug resistance genes were detected in 24 samples by nanopore sequencing.The ability of nanopore sequencing to detect drug resistance genes was stronger than that of drug sensitivity tests(P<0.05).3.There were significant statistical differences between patients with bacterial pneumonia who had antibiotic resistant genomes and patients without antibiotic resistant genomes in terms of long hospital stay,long days of antibiotic use,consciousness,invasive operations,and procalcitonin(P<0.05).The number of days of antibiotic use(OR=1.801,95% CI: 1.001-3.238,P=0.049)and patients’ unconsciousness(OR=30.39,95% CI: 1.971-51.49,P=0.016)were independent risk factors for patients with bacterial pneumonia.The optimal value for predicting antibiotic resistance genes in patients with bacterial pneumonia is the duration of antibiotic use of 6.5 days(AUC=0.848,95% CI: 0.731-0.966,P=0.001),with sensitivity and specificity of 70% and 100%,respectively.4.A total of 20 samples were detected for Acinetobacter baumannii,0 samples had peptide antibiotic resistance genes,and 3 samples showed positive peptide antibiotic susceptibility tests.Combining the two detection methods,Acinetobacter baumannii was more sensitive to peptide antibiotics;The number of samples with resistance genes for macrolide antibiotics and penicillin antibiotics in Acinetobacter baumannii is 12 and 1,and the number of samples with positive drug sensitivity tests for macrolide antibiotics and penicillin antibiotics is 0 and 10,respectively.The results of resistance tests for macrolide antibiotics and penicillin antibiotics in Acinetobacter baumannii differ significantly.14 samples detected Pseudomonas aeruginosa,3 samples had genes for resistance to glycylcycline antibiotics,and 2samples showed resistance to glycylcycline antibiotics through drug sensitivity tests.Combined with the 2-hour detection method,Pseudomonas aeruginosa is more likely to be sensitive to glycylcycline antibiotics;Pseudomonas aeruginosa has resistance genes to macrolide antibiotics,and the number of samples tested for macrolide antibiotic resistance in drug sensitivity experiments is 6 and 0,respectively.The results of the two detection methods for detecting macrolide antibiotic resistance differ significantly.Eleven samples were detected for Klebsiella pneumoniae.The number of Klebsiella pneumoniae samples with cephalosporin antibiotic resistance genes and the number of cephalosporin antibiotic resistance samples detected in drug sensitivity experiments were 2 and 3,respectively.Klebsiella pneumoniae may be more sensitive to cephalosporin antibiotics;Klebsiella pneumoniae has aminoglycoside antibiotic resistance genes and drug sensitivity experiments suggest that the number of samples with cephalosporin antibiotic resistance is 6 and 0,respectively.There is a significant difference in the results of the two detection methods for detecting aminoglycoside antibiotic resistance.Among the 12 samples,Maltotrophomonas maltophilia was detected.There were peptide antibiotic resistance genes in Maltotrophomonas maltophilia,and drug sensitivity experiments suggested that the number of samples with peptide antibiotic resistance was 2 and 3,respectively.Maltotrophomonas maltophilia may be more sensitive to peptide antibiotics.Conclusion:1.Nanopore sequencing is more effective than traditional bacterial culture in detecting bacteria in alveolar lavage fluid of patients with Bacterial pneumonia.2.The ability of nanopore sequencing to detect bacterial antibiotic resistance in alveolar lavage fluid of patients with Bacterial pneumonia is stronger than that of traditional drug sensitivity test.3.The independent risk factors for bacterial resistance genes in the alveolar lavage fluid of patients with bacterial pneumonia are the number of days of antibiotic use and mental state.4.For patients with high suspicion or pathogenic evidence suggesting infection with Acinetobacter baumannii,Pseudomonas aeruginosa,and Klebsiella pneumoniae,the use of peptide antibiotics,glycylcyclines,and cephalosporins may be effective;Maltotrophomonas maltophilia is often infected by its resistant bacteria,and multiple sensitive drugs such as peptide antibiotics can be chosen for the resistance of the bacteria.More research may be needed on the resistance genes of Acinetobacter baumannii macrolide antibiotics and penicillin antibiotics,Pseudomonas aeruginosa macrolide antibiotic resistance genes,and Klebsiella pneumoniae macrolide and aminoglycoside antibiotic resistance genes.