Rapid Identification And Epidemiological Analysis Of Urinary Tract Infection Microorganisms

Objective:At present,the treatment of clinical urinary tract infections mainly has the problems of long-term urine culture and multi-drug resistance of pathogenic bacteria.For this reason,this article intends to carry out research on rapid identification of urinary tract infection microorganisms and molecular epidemiology of major pathogens,with a view to providing a theoretical basis for the rapid clinical diagnosis of urinary tract infections,rational drug use and avoidance of outbreaks.Method:1.Application of MALDI-TOF MS combined with UF1000i and short-term culture method in rapid identification of urinary tract infection microorganisms(1)Specimen pretreatment:differential centrifugation to concentrate bacteria.(2)Direct MALDI-TOF MS detection:the pre-treated precipitate was directly subjected to mass spectrometry and the identification rate was calculated.(3)MALDI-TOF MS identification after short-term culture:the remaining precipitates after direct mass spectrometry identification were mixed and inoculated on Columbia blood agar plates.After incubation for 5 hours,the membrane growth was detected by mass spectrometry,and the results were recorded.(4)ROC curve count bacteria threshold:when the direct mass spectrometry detection result and the short-term culture post-mass spectrometry identification result are consistent with the mid-stage urine culture results,calculation of UF-1000i counting bacterial threshold using ROC curve.2.Epidemiological analysis of urinary tract infection with Escherichia coli and Klebsiella pneumoniae(1)Microbial identification and drug susceptibility:microbial and drug susceptibility tests were identified using the Phoenix System-100BD automated microbiology system and the disk diffusion method(K-B method).(2)Drug resistance phenotype confirmation experiment:modified Hodge test and improved carbapenemase inactivation test to detect carbapenemase.(3)Detection of drug resistance genes:the carbapenemase-encoding gene and the extended-spectrum β-lactamase gene were detected by PCR,and the positive strains were sequenced.(4)Molecular epidemiological analysis:the homology of the strains was analyzed by pulsed-field gel electrophoresis PFGE.(5)Whole genome sequencing:second generation sequencer sequencing.(6)ST classification,serum capsule typing and virulence gene detection:all collected carbapenemase-producing Escherichia coli and Klebsiella pneumoniae were detected by multiplex PCR technique.Result:1.Application of MALDI-TOF MS combined with UF1000i and short-term culture method in rapid identification of urinary tract infection microorganisms(1)Identification rate of mass spectrometry:after 5 hours of short-term culture,the identification rate of the membrane mass spectrometry was 87.2%,which was higher than the 77.1%of the direct mass spectrometry detection rate.(2)The colony count of the specimen corresponds to the mass spectrometry detection rate:the more the colony count of the specimen,the higher the direct mass spectrometry detection rate and the mass spectrometry detection rate after short-term culture.(3)Mass spectrometry identification score:this experiment can find that the number of strains with mass spectrometry score≥1.7(Escherichia coli≥1.5)after short-term culture is significantly more than the direct mass spectrometry score≥1.7(Escherichia coli≥1.5)the number of strains.(4)Mixed infection:whether it is direct mass spectrometry detection,or short-term culture after mass spectrometry detection,in most cases can only identify a higher proportion of strains,and the credibility is higher.(5)ROC curve count bacteria threshold:when UF1000i detects bacterial value≥ 1100/μl,direct mass spectrometry detection is easy to identify the species.Similarly,when UF1000i detects bacterial values≥470/μ1,mass spectrometry detection in the short-term culture is easy to identify the species.2.Epidemiological analysis of urinary tract infection with Escherichia coli and Klebsiella pneumoniae(1)The detection rate of cefotaxime-resistant Escherichia coli(CTX-R-ECO)in urine samples was 55.2%,slightly higher than the national bacterial resistance monitoring network(CARSS)monitoring data,and the detection rate of cefotaxime-resistant Klebsiella pneumoniae(CTX-R-KPN)was 46.0%,slightly lower than the national bacterial resistance monitoring network monitoring data.(2)In this study,there were 2 and 11 strains of carbapenemase-resistant Escherichia coli and Klebsiella pneumoniae isolated from urinary tract infections.(3)From the perspective of drug resistance genes,carbapenemase-producing Escherichia coli and Klebsiella pneumoniae in our hospital,carbapenemase-producing NDM and KPC are the main resistance mechanisms.ESBLs-producing Escherichia coli and Klebsiella pneumoniae are mainly strains producing CTX,TEM,and SHV.(4)In the homology analysis,the results of PFGE study showed that the urinary tract infection of Escherichia coli in our hospital was non-clonal transmission.(5)Whole genome sequencing showed:4117 functional genes,multiple drug resistance genes,and a complete virulent island.(6)The results of ST typing,serum capsule typing and virulence-related genes showed that:Klebsiella pneumoniae of urinary tract infections were all ST11 types,and most of the serum capsule typing were KL64 types.The virulence-related genes showed diverse.Conclusion:1.Compared with traditional urine culture,in this study,urine culture specimens were differentially centrifuged to enrich bacteria,and MALDI-TOF MS combined with UF1000i and short-term culture method were used to rapidly identify urinary tract infection microorganisms,which was simple,rapid and low in cost.The high accuracy rate can greatly improve the clinical application value of MALDI-TOF MS technology for rapid identification of urinary tract infection microorganisms.It has important clinical significance for rapid clinical diagnosis of urinary tract infection,rational drug use and timely control of urinary tract infection.2.Our hospital urinary tract infection Escherichia coli and Klebsiella pneumoniae:resistance to third-generation cephalosporins,monocyclic β-lactam antibiotics and even carbapenem antibiotics,especially the emergence of ESBLs strains and carbon-resistant enzymes,which have brought great difficulties to clinical treatment;Like most other cities,the CRE strain of urinary tract infection in our hospital is mainly Klebsiella pneumoniae;Urinary tract infection Klebsiella pneumoniae mainly consists of cloned ST11 carrying blaKPC;Non-clonal transmission is the main mode of transmission of urinary tract infection Escherichia coli.