K.pneumoniae XJPD-Li甘油脱水酶基因的定点突变及其性能研究

针对菌株K.pneumoniae XJPD-Li高效转化生产1,3-丙二醇的特性,将其甘油脱水酶与已知报道的甘油脱水酶(U60992)的序列比对分析,根据差异位点设计了Nβ47I的定点突变,利用反向PCR定点突变技术构建了甘油脱水酶(dhaBCE)的突变体质粒M1821,并在E.coli BL21(DE3)中高效表达。SDS-PAGE结果显示,诱导表达后在相对分子质量66、24、16KD出现三条特征条带,与预期结果一致。突变体M1821甘油脱水酶的比酶活为38.7U/mg,催化反应最适pH为8.0,最适温度为40℃,与未突变重组甘油脱水酶比较,最适温度降低了约5℃。

Class Ⅰ integron with a novel cassette array in an ESBL-producing multidrug-resistant Klebsiella pneumoniae isolate

Objective： To analyze the molecular mechanism of integron mediated mulfi-resistanc, e in an ESBL-producing K. pneumoniae NJ 12 isolate. Methods： Susceptibility test was carried out by Kirby-Bauer method. Class Ⅰ, Ⅱ and Ⅲ integrons were detected by integrase gene PCR with primers that annealed to conserved regions of integron-encoded integrase genes intll, intl2 and intl3. The variable region of integron was amplified by integron PCR with primers that targeted the conserved flanking regions, and the product was sequenced. Six aminoglycoside modifying-enzyme genes, including ant（2＂）-Ⅰ, ant（3＂）- Ⅰ, aac（3）- Ⅰ, aac（3）-Ⅱ, aac （6＇）-Ⅰ, and aac（6＇）-Ⅱ , were detected. Results： K. pneumoniae NJ 12 was resistant to nine antibiotics, including piperacillin, ampicillin, cefuroxime, ceftazidime, cefotaxime, aztreonam, streptomycin, gentamicin and amikacin. This isolate was shown that there was positive with class Ⅰ integron, ant（2＂）- Ⅰ , ant（3＂）- Ⅰ , aac（3）-Ⅱ and aac（6＇）- Ⅰ modifying-enzyme genes. Neither class Ⅱ nor Ⅲ integron was detected; DNA sequencing of the fragment amplified by integron PCR revealed a novel cassette array aadR-cat-blaoxa-10/ aadA1. Conclusion： Class I integron with a novel cassette array in an ESBL-producing multidrug-resistant K. pneumon/ae NJ 12 isolate was reported from Nanjing area of China, with the GenBank accession number DQ141319.

Study of OmpK35 and OmpK36 Expression in Carbapenem Resistant ESBL Producing Clinical Isolates of Klebsiella pneumoniae

Background: Carbapenem resistant extended spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae (K. pneumoniae) is increasing worldwide. Carbapenem resistance (CR) has been attributed not only to production of carbapenemases but also to permeability barriers due to outer membrane proteins (OmpK35 and OmpK36) disruption. Objective: Phenotypic detection of CR among ESBL producing K. pneumoniae isolates, followed by the evaluation of the role of ompK35 and ompK36 gene expression among carbapenem resistant K. pneumoniae (CR-KP) isolates. Methods: 100 ESBL producing K. pneumoniae isolates were included in this study. Minimum inhibitory concentration (MIC) of imipenem was performed for all isolates by broth microdilution method. For CR-KP isolates, phenotypic detection of K. pneumoniae carbapenemase (KPC), metallo-β-lactamase (MBL) and AmpC enzymes was performed followed by Realtime qRT-PCR to detect and quantify ompK35 and ompK36 gene expression. Results: 42% of our isolates were carbapenem resistant, and all of them were KPC producers either singly or in combination with MBL and/or AmpC production. Reduced expression of both ompK35 and ompK36 was detected in (52.38%) of CR-KP isolates, while reduced expression of ompK36 or ompK35 alone was found in (2.38%) and (33.33%) respectively. Twenty of 42 CR-KP isolates (47.62%), showing reduced ompK35 and ompK36 expression, exhibited high level resistance (HLR) (>32 μg/ml) to imipenem. There was a significant correlation between reduced expression of ompK36 and increase MIC values (p < 0.05). The combined production of MBL or AmpC together with reduced expression of ompK35 and/or ompK36 resulted in significant increase in imipenem MIC (p < 0.05). Conclusion: The combined OmpK35/OmpK36 loss resulted in HLR. However OmpK36 seems to play a major role in those strains. Imipenem MIC was markedly increased among K. pneumoniae showing carbapenemase and/or AmpC production together with loss of OmpK35 and/or OmpK36.

Incidence of Extended-Spectrum Be-ta-Lactamase-Producing Klebsiella pneumoniae among Patients and in the Environment of Hassan II Hospital, Settat, Morocco

Aim: The aim of the current study is to determine: (1) the prevalence of extended-spectrum β-lactamase-producing K. pneumoniae (ESBL-Kp) isolated from clinical samples and a hospital environment in Hassan II Hospital (Settat, Morocco);(2) the associated risk factors of ESBL-Kp infections;(3) the link between clinical and environmental isolates. Methods: During the study period (April 2010 to March 2011), all patients infected and hospital environment sites contaminated by K. pneumoniae were considered as the potential study population and environmental site. The clinical data were collected to identify risk factors for ESBL carriage of K. pneumoniae infection. Screening of ESBL-and carbapenemase-producing isolates was performed by using a double-disk synergy test and the modified Hodge test, respectively. ESBL-Kp isolates were tested for the presence of genes encoding β-lactamases and were investigated by PCR. The clonal relationship between ESBL-producing isolates was analysed by ERIC- and REP-PCR method. Results: The overall prevalence of ESBL-Kp among clinical and environmental K. pneumoniae isolates was 35.13% (13/37) and 4.04% (4/99), respectively. The main risk factors for carrying ESBL-Kp were renal disease (46.15%), recent surgery (53.84%), previous hospitalisation (76.92%), and the presence of many invasive devices (53.84%). All ESBL isolates were multidrug resistant. The blaCTX-M group1and blaSHV (70.58% for each) were the most prevalent followed by blaTEM (52.94%). Thirteen strains expressed at least two bla genes. One isolate was positive in the modified Hodge test and was a blaOXA-48 producer. ERIC and Rep-PCR methods revealed an epidemic clonal dissemination of these isolates. Conclusion: The emergence of OXA-48 carbapenemase, endemic clonal dissemination and multi-drug resistance of ESBL-Kp isolates in our institution is highly alarming.

Characteristics of β-Lactamase Synthesis in E. coli and K. pneumanie Strains in Nosocomial Infections

Background: Recently micro-organisms that synthesize extended-spectrum β-lactamase (ESBLs) were increased. The peculiarities of ESBL synthesis of Escherichia coli and Klebsiella pneumoniae strains that cause nosocomial urinary tract infections, surgical site infections and pneumonia in surgical clinic were studied. ESBL synthesis were observed 38.9% of E. coli strains obtained from urine, 92.3% of strains obtained from surgical site infections, and 50% of strains obtained from sputum. ESBL synthesis were observed 37.5% of K. pneumoniae strains obtained from urine, 85.7% of strains obtained from surgical site infections, and 60% of strains obtained from sputum. Different levels of ESBL synthesize of E. coli and K. pneumoniae strains isolated from different pattern is discussed. Conclusion. ESBL synthesis is common in E. coli and K. pneumoniae strains, which cause nosocomial infections. The frequency of occurrence of ESBL s synthesis among of these strains depends on clinical forms of nosocomial infections.

Isolation and Identification of Alkali-Resistant 1,3-Propanediol Producing Strain

1,3-Propanediol is a promising renewable resource produced by microbial production. It is mainly used in many synthetic reactions, particularly applied to the polymer synthesis and cosmetics industry. We described here the isolation of strain ZH-1, which has the ability of high production with 1,3-propanediol, from Fenhe River in China. It was classified as a member of K. pneumoniae after the study of phenotypic, physio-logical, biochemical and phylogenetic (16S rDNA). The initial glycerol concentration, fermentation time and pH value of strain ZH-1 were determined to be 50 g·L-1, 36 h and 8.0. Under these conditions, the practical yield of 1,3-PD was 18.53 g·L-1 and a molar yield (mol1,3-PD molGlycerol-1 of 1,3-propanediol to glycerol of 0.497. In addition, we found that for the strain ZH-1, the optimum grown pH was 9.0, so we can deter-mine that it is a new member of alkali-resistant strains.

Epidemiology and genetic characterization of tet(X4)-positive Klebsiella pneumoniae and Klebsiella quasipneumoniae isolated from raw meat in Chengdu City, China

The rapid spread of mobile tigecycline resistance presents a significant public health threat,particularly with the increasing prevalence of tet(X4)-positive Enterobacterales across various species.This study aimed to inves-tigate the epidemic features and transmission dynamics of tet(X4)-positive Klebsiella pneumoniae(K.pneumo-niae)through the analysis of 206 raw meats,including pork(n=182),beef(n=16),duck(n=5),and chicken(n=3).These samples were collected from schools,markets,and restaurants in Chengdu City,China.A total of 25 isolates were obtained from 13 administrative regions.All isolates exhibited resistance to tetracycline,tigecycline,ampicillin,chloramphenicol,and florfenicol.Over half of the isolates also demon-strated resistance to streptomycin(80%),sulfamethoxazole/trimethoprim(72%),ciprofloxacin(64%),and ampicillin/sulbactam(56%).Among these strains,14 distinct sequence types(STs)were identified,revealing evidence of inter-regional clonal spread,notably among 9 K.pneumoniae ST3393.Phylogenetic analysis revealed the presence of two K.pneumoniae ST5 closely resembling hypervirulent K.pneumoniae from Jiangsu.Importantly,12 isolates were capable of transferring tigecycline resistance to Escherichia coli J53.Further plasmid analysis showed that the tet(X4)-harboring plasmids in K.pneumoniae could be classified into four types,primarily belonging to the IncFIA(HI1)/HI1A/HI1B hybrid plasmid(n=16)and IncFII plasmid(n=7),which significantly contributed to the cross-species dissemination of tet(X4).In summary,this study highlights the prevalence of MDR tet(X4)-positive K.pneumoniae in Chengdu,driven predominantly by clonal expansion and plasmid-mediated horizontal gene transfer.These findings emphasize the importance of contin-uous surveillance of tet(X4)-positive K.pneumoniae in raw meat and the implementation of effective measures to control their spread.

晚期内体/溶酶体适配蛋白2(LAMTOR2)基因缺失加重肺炎克雷伯菌引起的小鼠脓毒症

目的建立肺炎克雷伯菌(K.pneumoniae)感染肝组织晚期内体/溶酶体适配蛋白2(LAMTOR2)基因条件敲除小鼠模型,初步探究LAMTOR2基因在K.pneumoniae感染引起肝脓毒症过程中的作用及机制。方法繁育获得肝脏LAMTOR2基因条件性敲除小鼠(LAMTOR2^(flox/flox);Alb⁃Cre^(+))和同窝阴性对照组小鼠(LAMTOR2^(flox/flox))。提取实验组和对照组小鼠的肝组织,利用实时荧光定量PCR和Western blot法检测LAMTOR2的mRNA和蛋白表达水平确定LAMTOR2在肝组织的敲除效果。实验组和对照组小鼠分别感染K.pneumoniae,观察24、36、48、72 h的小鼠生存率,HE染色观察肝组织病变情况;利用实时荧光定量PCR检测实验组和对照组小鼠感染K.pneumoniae 24 h后肝组织肿瘤坏死因子α(TNF⁃α)、白细胞介素1β(IL⁃1β)和CXC趋化因子配体1(CXCL1)的mRNA的表达水平。结果成功获得肝组织LAMTOR2基因条件性敲除小鼠;与对照组小鼠相比,实验组小鼠在感染K.pneumoniae后生存率降低,肝组织出现更严重损伤;实验组小鼠肝组织TNF⁃α、IL⁃1β和CXCL1的mRNA表达水平显著降低,小鼠免疫应答能力降低。结论LAMTOR2在K.pneumoniae感染诱导脓毒症的过程中起保护作用。

Current status and trends of antimicrobial resistance among clinical isolates in China:a retrospective study of CHINET from 2018 to 2022

Antimicrobial resistance(AMR)is a pressing issue in China,with antibiotic therapy becoming less effective against bacterial infections.To address this challenge,the China Antimicrobial Surveillance Network(CHINET)was established in 2005 to monitor antimicrobial resistance in the country.This study analyzed the CHINET data from teaching hospi-tals and evaluated the trends of AMR in China from 2018 to 2022.A range of 163,636 to 301,917 isolates was obtained per year,with the majority being Gram-negative bacilli(69.0%to 71.8%).The proportion of important multidrug-resistant pathogens remained stable over the years.While the analysis showed diverse AMR profiles for different bac-terial species.Over the five years,generally decreased resistance rates were observed from the majority of the tested species.For example,resistance to ceftriaxone decreased in Escherichia coli and Klebsiella pneumoniae,while resistance to imipenem and meropenem decreased in Pseudomonas aeruginosa.Moreover,resistance to methicillin,gentamicin,fosfomycin,and clindamycin also decreased in clinical Staphylococcus aureus isolates.On the other hand,resistance levels of Acinetobacter baumannii remained stable.Our study provides a comprehensive overview of the AMR profiles of common bacterial species in China and highlights the ongoing efforts to address this challenge.