| Objective:1.Analyzing the clinical characteristics,epidemiological status and molecular biological characteristics of hypervirulent Klebsiella pneumoniae from Jiujiang area.2.Analyzing the basic clinical data,drug resistance status and epidemiological status of carbapenem-resistant hypervirulent Klebsiella pneumoniae from Nanchang and Jiujiang,and their kinship were analyzed.3.Studing the molecular characteristics,plasmid replicon typing and virulence plasmid structure of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae.Methods:1.251 strains of Klebsiella pneumoniae isolated from November 2021 to June2022 from Jiujiang area were collected.Among them,118 strains of hvkp,which included 9 strains of CR-hvkp,were detected by LAMP.In this paper,we used the medical record system to query the clinical data of 118 patients infected with hvkp.Vitek-2 Compact was used for strain identification and drug sensitivity test.Polymerase chain reaction（PCR）was used to detect the common capsular serotypes and virulence genes of the strains.ST typing was detected by multilocus sequence typing（MLS）.2.In the same period,658 strains of Klebsiella pneumoniae isolated from Jiujiang area were collected.Among them,282 strains of hvkp,which included 36strains of CR-hvkp,were detected by LAMP.36 strains of CR-hvkp in Nanchang and9 strains of CR-hvkp from Jiujiang were analyzed.Polymerase chain reaction（PCR）was used to detect the common virulence genes and carbapenemase resistance genes,serum capsular typing and sequence typing（ST）.The genetic relationship between CR-hvkp in the two regions was analyzed by pulsed field gel electrophoresis（PFGE）.3.The virulence phenotypic characteristics,virulence gene spectrum and drug resistance gene spectrum of 15 ST11 CR-hvkp strains isolated in the previous chapter were analyzed.The virulence of ST11 CR-hvkp strains was detected by biofilm formation assay and Galleria mellonella larvae infection assay.The plasmid replicon type and virulence plasmid structure of ST11 CR-hvkp strain were detected by whole genome sequencing.Results:1.In this study,251 strains of Klebsiella pneumoniae were collected from Jiujiang area,and 118 strains of hypervirulent Klebsiella pneumoniae were isolated by LAMP,with a separation rate of 47.01%.Average years of patients with isolated hvkp strains was 65.37±13.46 years old,including 94 males（79.66%）and 24 females（20.34%）.The specimens included 98 strains of sputum（83.05%）,8 strains of blood（6.78%）and 6 strains of pus（5.08%）.There were 49 cases（41.53%）of individuals without a basic medical history among patients and 58 cases（49.15%）of underlying diseases in patients with hvkp infection.Among these patients with underlying diseases,7 cases had a history of diabetes or elevated blood glucose（31.36%）,45cases had a history of hypertension（38.14%）,and 7 patients had tumors（5.93%）;46cases（38.98%）received invasive treatment,Some patients had invasive infections outside the respiratory system,including 6 cases of hepatobiliary system infection（5.08%）,2 cases of renal infection（1.69%）,and 8 cases of bloodstream infection（6.78%）.33 cases（27.97%）had a history of ICU hospitalization.All of patients with isolated CR-hvkp strains were male patients,with an average age of 74.22±9.47years.Among the infected patients,there were 1 cases（11.11%）of individuals without a basic medical history among patients and 6 cases（66.67%）of underlying diseases.Among these patients with underlying diseases,5 cases had a history of diabetes or hyperglycemia（55.56%）,4 cases had a history of hypertension（44.44%）,All of the 9 patients infected with CR-hvkp received invasive treatment,including 8cases of sputum specimens（88.89%）and 1 case of alveolar lavage fluid（11.11%）.6cases had poor prognosis（66.67%）,and 5 cases had a history of ICU hospitalization（55.56%）.2.The main departments of 118 strains of hvkp from Jiujiang area were from respiratory medicine（25 strains,21.19%）,ICU（22 strains,18.64%）and neurosurgery（14 strains,11.86%）.Among the 118 strains,there was no high drug resistance phenotype to commonly used antibiotics in clinic,the resistance rate to tigecycline was the lowest（0.85%,1/118）,the resistance rate to imipenem and meropenem was7.63%（9/118）,the resistance rate to nitrofurantoin was 17.80%（21/118）,and the resistance rate to amoxicillin was the highest（100%,118/118）,It was highly sensitive to other commonly used antibiotics（>88%）.9 strains of imipenem and meropenem resistant bacteria were determined as CR-hvkp.CR-hvkp strains were mainly distributed in ICU（44.44%）,respiratory medicine（33.33%）,neurology（11.11%）and nephrology（11.11%）.It has high resistance to commonly used antibiotics in clinical practice.Among them,the resistance rates of cephalosporins,carbapenems,amoxicillin,ciprofloxacin and nitrofurantoin were 100%.The resistance rates of aztreonam and levofloxacin were 88.89%（8/9）,the resistance rates of gentamicin,amikacin and cotrimoxazole were 77.78%（7/9）,and the resistance rate of tigecycline was 11.11%（1/9）.3.The hvkp from Jiujiang is mainly K1-ST23.Using capsular serotypes,among them,there were 47 strains K1（39.83%）,22 strains K2（18.64%）,14 strains K57（11.86%）,6 strains K64（5.08%）,6 strains K20（5.08%）,3 strains K5（2.54%）,1strain K54（0.85%）,and no K47.Using MLST serotypes,among them,there were 48strains ST23（40.68%）,34 strains ST65（28.81%）,6 strains ST11（5.08%）,5 strains ST25（4.23%）,4 strains ST268（3.39%）,3 strains ST86（2.54%）,2 strains ST218（1.69%）,1 strain ST1265,ST1764,ST412,ST592 and ST818（0.85%）.43 strains K1（91.49%）belonged to ST23,and 22 strains K2（90.91%）belonged to ST65,ST86,ST25 and ST818.CR-hvkp strains,using capsular serotypes,included 6 strains K64（66.67%）and 1 strain K1（11.11%）.Using MLST serotypes,among them,there were included 6 strains ST11（66.67%）,2 strains of ST65（22.22%）,and 1 strain ST23（11.11%）.all K64 strains belonged to ST11.There were 5 strains（5/9,55.56%）of CR-hvkp strains with mucus drawing.4.The results of high mucus phenotype showed that that among the 118 hvkp strains,74 strains had mucus formation（62.71%）.In the virulence experiment of Galleria mellonella,9 strains showed high virulence.After 12 hours,the survival rate of Galleria mellonella larvae was less than 40%.After 48 hours,the survival rate of Galleria mellonella larvae was less than 20%.5.Comparing CR-hvkp strains from Nanchang and Jiujiang,the conclusion is that:blood（14/36,38.89%）and sputum（11/36,30.56%）were the main specimens in Nanchang area,,while,sputum specimens（8/9,88.89%）were the main specimens,in Jiujiang area,with significant difference（P<0.05）.The carrying rates of virulence genes rmp A and iuc A in Nanchang were significantly higher than those in Jiujiang（P<0.05）.6.The strains from Nanchang and Jiujiang were mainly from ICU,mainly middle-aged and elderly male over 50 years old.The average age of patients from Jiujiang（74.22±9.47）was higher than that of patients from Nanchang（53.02±9.34）.Although the resistance rates of CR-hvkp strains in the two regions were not significantly different（P>0.05）,they all showed high levels of resistance to commonly used clinical antibiotics,with resistance rates of at least 50%for each class of drugs and over 90%for cephalosporins.7.The main podosome serotypes of CRhvkp strains from Nanchang and Jiujiang were K64.Nanchang strains:K64（25/36,69.44%）,K1（6/36,16.67%）,K2（3/36,8.33%）,K5（1/36,2.78%）and K47（1/36,2.78%）;Jiujiang strains:K64（6/9,66.67%）,K1（1/9,11.11%）,unknown typing（2/9,22.22%）,and the statistical difference between strains in the two regions was not statistically significant in podoconjugate serotypes and MLST typing.The affinities of CRhvkp from the two regions were analyzed by PFGE experiments,and the strains were found to be well related in the two regions.The epidemiological survey also revealed that some patients from Jiujiang had a past history of inpatient visits to provincial hospitals in Nanchang.This suggested that there was a cross transmission between CRhvkp strains from Nanchang and Jiujiang.8.The 15 ST11 CR-hvkp strains isolated in the previous chapter were detected.The main sources of infection specimens were blood（9/15,60%）,urine（2/15,13.33%）,venous catheterization（1/15,6.67%）,sputum（1/15,6.67%）,wound secretions（1/15,6.67%）,and ascites（1/15,6.67%）.The main source of strains was the ICU of each department（11/15,73.33%）.Compared with non-ST11 CR-hvkp,ST11 was K64 in capsular serotype,and non-ST11 was mainly K1/K2（31/36）,with significant difference（P<0.05）.Among the carbapenemase resistance genes,the bla KPCgene carried by ST11（100%）was significantly higher than that of non-ST11（47.22%）（P<0.001）.In the distribution of virulence genes,there was no notable difference between the two types of CR-hvkp strains,and both carried a large number of virulence genes.The resistance rate of K64 to ciprofloxacin was significantly higher than that of non-ST11（P=0.020）,while the resistance rate to gentamicin and tobramycin was significantly lower than that of non-ST11（P<0.05）.There was no significant difference in resistance to other commonly used drugs;the virulence experiment of Galleria mellonella showed that ST11 CR-hvkp had strong lethal ability to Galleria mellonella,showing high virulence phenotype.All ST11 strains produced biofilms.In terms of drug resistance,it also showed multiple drug resistance.In addition to tigecycline,it showed high resistance to commonly used antibiotics.The resistance rate of cephalosporins was 100%,the resistance rate of cefoxitin was 100%,the resistance rate of imipenem,meropenem and ertapenem was 100%,the resistance rate of sulfamethoxazole was 73.33%,the resistance rate of gentamicin,tobramycin and amikacin was relatively low,and the resistance rate of tigecycline was the lowest.Only one strain showed resistance to tigecycline.All of these strains carried bla KPC,bla SHVand bla CTX-M-14,and most of them carried bla TEM-1.The PMQR gene was mainly qnr S1,followed by acc（6’）-lb-cr.Each strain carried at least 4 drug resistance genes,and the drug resistance mechanism was polymorphic.9.In the virulence gene phenotype,15 ST11 CR-hvkp strains carried 4 kinds of rmp A,rmp A2,iuc A and iut A in the virulence plasmid p LVPK virulence genes.By the whole genome sequencing,15 CR-hvkp strains were detected 5 types of replicons,namely Inc HI1B/Inc FIB,Inc FII,Inc R,Inc Q and Col RNAI.Each strain carried more than 3 plasmid replicons,indicating that these strains carried multiple plasmids.The virulence plasmid replicon Inc HI1B/Inc FIB was detected in 15 strains,and the drug-resistant plasmid replicon Inc FII was also common to all strains,and 14 strains carried the drug-resistant plasmid replicon Inc R.It showed that all strains carry virulence plasmids and drug-resistant plasmids,and through the analysis of the genome structure of virulence plasmids.It was found that the virulence plasmids carried by these 15 strains have the same skeleton structure,and there are also differences,showing the polymorphism of virulence plasmids.At the same time,they both lacked a fragment of about 25 kbp,which was the Salmonella encoding gene（iroBCDN）.Conclusion:1.The hvkp strains from Jiujiang are mainly from the elderly male patients in respiratory department and ICU,mainly sputum specimens,and have good sensitivity to commonly used antibiotics.The molecular typing was mainly K1-ST23 and carried a large number of virulence genes.CR-hvkp strains are mainly from ICU,mainly sputum specimens,with multiple drug resistance phenotypes.The molecular typing was mainly K64-ST11,which also carried a large number of virulence genes.The strains from Jiujiang were mainly transmitted and colonized by respiratory tract.2.The CR-hvkp was mainly K64-ST11,and the resistance gene was mainly bla KPC from Nanchang and Jiujiang.The mainly specimens are blood from Nanchang,The mainly specimens are sputum from Jiujiang,and the CR-hvkp from Nanchang is more prone to invasive infection.The risk factors for the formation of CR-hvkp in the two hospitals were the middle-aged and elderly men over 50 years old,the history of ICU admission,invasive treatment,and long-term and large-scale combination of drugs.The CR-hvkp strains had cross-transmission between the two regions.3.The ST11 CR-hvkp strain has high virulence phenotype,carrying most of the virulence genes of p LVPK plasmid,and the virulence level is high.Multiple drug resistance genes were detected,and the drug resistance mechanism was polymorphic.All of strains carried virulence plasmids and drug-resistant plasmids.The virulence plasmid p LVPK was polymorphic and the Salmonella coding gene（iroBCDN）was deleted. |
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