Klebsiella pneumoniae infections after liver transplantation:Drug resistance and distribution of pathogens,risk factors,and influence on outcomes

BACKGROUND Liver transplantation(LT)is the only curative treatment for end-stage liver disease.However,LT recipients are susceptible to infection,which is the leading cause of early mortality after LT.Klebsiella pneumoniae infections(KPIs)in the bloodstream are common in LT recipients.We hypothesized that KPIs and carbapenemresistant Klebsiella pneumoniae(CRKP)infections may affect the outcomes of LT recipients.AIM To assess KPI incidence,timing,distribution,drug resistance,and risk factors following LT and its association with outcomes.METHODS This retrospective study included 406 patients undergoing LT at The Third Xiangya Hospital of Central South University,a tertiary hospital,from January 2015 to January 2023.We investigated the risk factors for KPIs and assessed the impact of KPIs and CRKP infections on the prognosis of LT recipients using logistic regression analysis.RESULTS KPI incidence was 7.9%(n=32),with lung/thoracic cavity the most frequent site of infection;the median time from LT to KPI onset was 7.5 d.Of 44 Klebsiella pneumoniae isolates,43(97.7%)and 34(77.3%)were susceptible to polymyxin B or ceftazidime/avibactam and tigecycline,respectively;>70%were resistant to piperacillin/tazobactam,ceftazidime,cefepime,aztreonam,meropenem,and levofloxacin.Female sex[odds ratio(OR)=2.827,95%confidence interval(CI):1.256-6.364;P=0.012],pre-LT diabetes(OR=2.794,95%CI:1.070-7.294;P=0.036),day 1 post-LT alanine aminotransferase(ALT)levels≥1500 U/L(OR=3.645,95%CI:1.671-7.950;P=0.001),and post-LT urethral catheter duration over 4 d(OR=2.266,95%CI:1.016-5.054;P=0.046)were risk factors for KPI.CRKP infections,but not KPIs,were risk factors for 6-month all-cause mortality post-LT.CONCLUSION KPIs occur frequently and rapidly after LT.Risk factors include female sex,pre-LT diabetes,increased post-LT ALT levels,and urethral catheter duration.CRKP infections,and not KPIs,affect mortality.

Occurrence of K1 and K2 serotypes and genotypic characteristics of extended spectrumβ-lactamases-producing Klebsiella pneumoniae isolated from selected hospitals in Malaysia

Objective:To determine the distribution,phenotypic and genetic background of extended spectrumβ-lactamases(ESBL)-producing Klebsiella(K.)pneumoniae clinical isolates associated with K1 and K2 serotypes in two selected hospitals in Malaysia.Methods:A total of 192 K.pneumoniae isolates were collected and subjected to antibiotic susceptibility,hypermucoviscosity test and multiplex PCR to detect the presence of K1-and K2-serotype associated genes.Multilocus sequence typing(MLST)was performed on ESBL-producing K.pneumoniae isolates presented with K1 and K2 serotypes,followed by phylogenetic analysis.Results:A total of 87 out of 192(45.3%)of the K.pneumoniae isolates collected were ESBL producers.However,only 8.3%(16/192)and 10.9%(21/192)of the total isolates were detected to carry K1-and K2-serotype associated genes,respectively.Statistical analysis showed that K1 and K2 capsular serotypes were not significantly associated with ESBL phenotype(P=0.196).However,they were significantly associated with hypervirulent,as demonstrated by the positive string test(P<0.001).MLST analysis revealed that ST23 as the predominant sequence type(ST)in the K1 serotype,while the ST in the K2 serotype is more diverse.Conclusions:Although the occurrence of ESBL-producing isolates among the hypervirulent strains was low,their coexistence warrants the need for continuous surveillance.MLST showed that these isolates were genetically heterogeneous.

Prevalence and Study of the Clonality of Extended-Spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Strains in Neonatology at the University Hospitals of Abidjan by the Pulsed Field Gel Electrophoresis and the Quantitative Antibiogram

Background: ESBL-producing strains of Klebsiella pneumoniae, one of the main causes of nosocomial and hospital-acquired infections, are commonly associated with therapeutic impasses. Surveillance of these multidrug-resistant pathogens is a crucial tool for controlling and preventing infections. This surveillance involves the use of appropriate molecular and phenotypic typing techniques. The choice of techniques is based on criteria such as discriminatory power, intra- and inter-laboratory reproducibility, epidemiological concordance, ease of use and cost. The aim of our study was to identify clusters of Extended-Spectrum Beta-Lactamase-producing Klebsiella pneumoniae (ESBL-K. pneumoniae) strains circulating in neonatology using quantitative antibiogram (QA) and Pulsed Field Gel Electrophoresis (PFGE). Materials and Methods: This cross-sectional study included 55 K. pneumoniae strains isolated from a total of 513 samples. These various samples are taken from newborns, healthcare personnel, and the environment. K. pneumoniae identification followed standard bacteriological procedures and was confirmed using the Vitek® 2 (bioMérieux). The detection of the ESBL phenotype was performed using the synergy test. QA and PFGE were used to identify clonal relationships between the various strains isolated. Concordance between these two methods was assessed by calculating Cohen’s KAPPA coefficient and Simpson’s diversity index. Results: Among the 55 K. pneumoniae strains included in this study, 58.2% (32/55) were found to be Extended-Spectrum Beta-Lactamase (ESBL) producers. Most of these strains were isolated from neonatal samples (blood samples and rectal swabs). The quantitative antibiogram method applied to 28 out of the 32 ESBL-producing strains revealed that the isolates were grouped into 5 clusters. Pulsed Field Gel Electrophoresis performed on a total of 16 ESBL-producing strains showed the existence of four profiles. A perfect concordance was observed between the two methods. Conclusion: The results of this study highlighted the existence of clonal strains of various origins within neonatology units.

一株大口黑鲈源ST-873型肺炎克雷伯菌(Klebsiella pneumoniae)的分离鉴定及生物学特性研究

从患腹水病大口黑鲈(Micropterus salmoides)的头肾、脾脏、肝脏组织中分离到一株优势菌,生长出圆形、边缘整齐的灰白色黏液状菌落;染色镜检可见短粗、卵圆形、有荚膜的革兰氏阴性杆菌。结合形态、生理生化特征、16S rRNA基因序列分析鉴定为肺炎克雷伯菌(Klebsiella pneumoniae),MLST分析进一步确定其为ST-873型,与ST-65型聚为一支;该菌株携带aere、alls、wca和ybt四种毒力基因,具有溶血活性;人工感染大口黑鲈,发现患病鱼呈现腹水等与自然发病类似的症状,且病鱼内脏的分离菌株与攻毒菌株相同;经统计,其LD50为3.4×10^(7) CFU/mL,具有中等生物被膜形成能力。耐药性分析结果显示,该菌株携带bla-shv、sul2、aadA和tetB四种耐药基因,对β-内酰胺类、氨基糖苷类、喹诺酮类、黏菌素类药物敏感,对四环素类、磺胺类、林可酰胺类、利福霉素类耐药;中药三七、款冬花对分离菌株有明显抑制作用。研究探明了大口黑鲈腹水病的主要病原,可为鱼源肺炎克雷伯菌的诊断和防治提供科学依据和数据参考。

Newly Detected Transmission of bla_(KPC-2) by Outer Membrane Vesicles in Klebsiella Pneumoniae

Objective The prevalence of carbapenem-resistant Klebsiella pneumoniae(CR-KP)is a global public health problem.It is mainly caused by the plasmid-carried carbapenemase gene.Outer membrane vesicles(OMVs)contain toxins and other factors involved in various biological processes,includingβ-lactamase and antibiotic-resistance genes.This study aimed to reveal the transmission mechanism of OMV-mediated drug resistance of Klebsiella(K.)pneumoniae.Methods We selected CR-KP producing K.pneumoniae carbapenemase-2(KPC-2)to study whether they can transfer resistance genes through OMVs.The OMVs of CR-KP were obtained by ultracentrifugation,and incubated with carbapenem-sensitive K.pneumoniae for 4 h.Finally,the carbapenem-sensitive K.pneumoniae was tested for the presence of bla_(KPC-2)resistance gene and its sensitivity to carbapenem antibiotics.Results The existence of OMVs was observed by the electron microscopy.The extracted OMVs had bla_(KPC-2)resistance gene.After incubation with OMVs,bla_(KPC-2)resistance gene was detected in sensitive K.pneumoniae,and it became resistant to imipenem and meropenem.Conclusion This study demonstrated that OMVs isolated from KPC-2-producing CR-KP could deliver bla_(KPC-2)to sensitive K.pneumoniae,allowing the bacteria to produce carbapenemase,which may provide a novel target for innovative therapies in combination with conventional antibiotics for treating carbapenem-resistant Enterobacteriaceae.

Isolation and Identification Methods for Oral Klebsiella pneumoniae Involved in Onset of Inflammatory Bowel Disease

Purpose: Recently, it was reported that Klebsiella pneumoniae is related to the onset of inflammatory bowel disease including the Crohn disease. It was frequently reported that K. pneumoniae was detected in human oral cavities. Regrettably, it currently remains unclear whether K. pneumoniae is part of the normal oral flora. The aim of this study was to establish the isolation and identification methods for K. pneumoniae from human oral cavities, and investigate its transmission pattern. Methods: A selective medium, OKPSM, for the isolation of K. pneumoniae from oral cavities was developed in this study. Also, PCR primer for the identification and detection at subspecies level of K. pneumoniae was designed. Results: OKPSM and PCR method using the primers designed in this study were useful for the isolation and identification of K. pneumoniae from human oral cavities. K. pneumoniae subsp. pneumoniae was detected at 10.0% in 30 saliva samples. On the other hand, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis were detected from no sample. Moreover, K. pneumoniae subsp. pneumoniae isolates from same subject at 0 month and after 3 months showed same genotypes on AP-PCR using OPA-07 primer. Conclusion: These results indicated that human oral cavities were not suitable for the habitat of K. pneumoniae.

甲酸脱氢酶在Klebsiella pneumoniae中的表达和功能分析

在甘油厌氧发酵生产1,3-丙二醇的过程中,需要消耗还原当量NADH,NADH的有效供给决定了1,3-丙二醇的产量和得率。采用PCR方法从Candidaboidinii基因组中克隆编码甲酸脱氢酶基因fdh,将fdh基因片段插入载体pMALTM-p2X中,构建表达载体pMALTM-p2X-fdh,并转入1,3-丙二醇生产菌Klebsiella pneumoniae YMU2,获得重组菌Klebsiella pneumoniae F-1。研究了重组质粒的稳定性和IPTG诱导fdh基因过量表达的条件。结果表明,重组质粒具有良好的稳定性;fdh基因表达的蛋白分子量为40.2kDa;IPTG诱导表达研究表明,在IPTG浓度为0.5mmol/L时,诱导4h后甲酸脱氢酶表达明显;发酵过程中甲酸脱氢酶比酶活达到5.47U/mg;与出发菌株K.pneumoniae YMU2相比,重组菌F-1合成1,3-丙二醇的浓度提高了12.5%。

Klebsiella pneumoniae合成1,3-丙二醇过程中的生长与催化耦联

This paper focuses on the research of the bioconversion of 1,3-propanediol by Klebsiella pneumoniae. The linear correlation of cell growth and 1,3-propanediol synthesis was found. An equation of the relationship between cell growth and biocatalysis was given.With the analysis of metabolism, it was discovered that the cell regulated the NADH production by cell growth in order to supply enough reductive equivalent for enzyme catalysis. A conclusion was drawn that the cell growth was coupled with the reactivation of a key-enzyme which catalyzes 1,3-propanediol production in Klebsiella pneumoniae.

维生素C和E对Klebsiella pneumoniae合成1,3-丙二醇的调控

通过外源添加还原剂的方式调控细胞内NADH/NAD再生系统的状态,研究了40～150 mg/LVC及20～100mg/LVE对Klebsiella penumoniae合成1,3-丙二醇的影响,发现外源添加150 mg/L VC或30 mg/L VE均可使1,3-PD合成浓度提高20%～30%;但同时也提高了某些副产物的合成浓度,对代谢流分布的调控作用不明显;1,3-丙二醇得率稍有提高但不显著.提高1,3-pD得率宜从代谢节点(丙酮酸)通量调节方面考虑.

3-羟丙醛对Klebsiella pneumoniae发酵产1,3-丙二醇的影响及其调控

中间产物3-羟丙醛在发酵液中的积累对Klebsiella pneumoniae细胞生长及1,3-丙二醇的合成有显著的抑制作用,而调节发酵的起始甘油浓度及控制发酵pH值可调控发酵液中3-羟丙醛的积累.当起始甘油质量浓度分别为20、30、50、70g/L的批式发酵中,发酵液中3-羟丙醛的积累的高峰分别为4.31、6.87、11.48及13.49mmol/L,当起始甘油质量浓度大于50g/L时,3-羟丙醛在到达积累高峰后不能被菌体有效转化,在发酵后期维持较高浓度,抑制了细胞生长及1,3-丙二醇的合成,发酵不能继续进行.控制发酵pH值为7.75～8.0可促进发酵液堆积的3-羟丙醛被迅速转化.在流加发酵中起始甘油质量浓度采用30g/L,发酵pH值控制为7.75条件下,发酵32 h,1,3-丙二醇质量浓度可达37.16g/L,1,3-丙二醇的生产强度和质量得率分别达到1.16g/(L·h)和52.66%.

利用氧化还原电位调控基因工程菌株Klebsiella pneumoniae F-1合成1,3-丙二醇

基因工程菌株Klebsiella pneumoniae厌氧发酵过程中,氧化还原电位(ORP)是一个监控发酵过程的重要指标.考察了K.pneumoniae F-1在不同ORP(-190,-210,-240和-290mV)下菌体生长和代谢产物合成的情况.在ORP为-240mV时,1,3-丙二醇的发酵终浓度和得率分别为81.5g/L和0.423mol/mol,分别为4种ORP条件下的最高值.这说明ORP=-240mV是菌株K.pneumoniae F-1的最适ORP.此ORP既不同于野生菌株K.pneumoniae M5aL的最适ORP(-190mV),也不同于经过诱变的菌株K.pneumoniae YMU2的最适ORP(-280mV).另外,通过研究发酵体系ORP对甘油代谢流的影响,发现还原性氛围较有利于甘油向1,3-PD、乙醇、2,3-丁二醇等还原性较高的代谢物转化.首次报道了ORP调控发酵过程在基因工程改造菌株中的应用,这对于进一步研究ORP调控厌氧发酵过程具有一定的意义.

在Klebsiella pneumoniae醛脱氢酶失活菌中构建NADH再生系统

生物法生产1,3-丙二醇(1,3-Propanediol,1,3-PD)是当前工业生物技术研究的热点之一,生产过程中,需要消耗还原当量NADH,NADH的有效供给决定了1,3-PD的产量和得率。采用PCR的方法从Candidaboidinii基因组中克隆编码fdh的基因,将该基因片段插入载体pMALTM-p2X,构建表达载体pMALTM-p2X-fdh,并转入醛脱氢酶失活菌KlebsiellapneumoniaeDA-1HB,获得重组菌KlebsiellapneumoniaeDAF-1。在IPTG浓度0.5mmol/L时,诱导3h后甲酸脱氢酶表达明显;发酵过程中甲酸脱氢酶比酶活达到4.82U/mg;与出发菌株K.pneumoniaeDA-1HB相比,重组菌DAF-1合成1,3-丙二醇的浓度提高了19.2%。

分段通气对Klebsiella pneumoniae生产1,3-丙二醇关键酶和辅酶的影响

通过考察1,3-丙二醇合成中关键酶、辅酶的变化,研究了分段通入空气对Klebsiellapneumoniae厌氧发酵生产1,3-丙二醇的影响.与对照组相比,在发酵前期(12h)通入空气4h后,甘油脱氢酶酶活提高1.5倍,1,3-丙二醇氧化还原酶酶活提高18%,1,3-丙二醇浓度提高16%;发酵后期(28和48h)通入空气后,甘油脱氢酶酶活不变,1,3-丙二醇氧化还原酶酶活下降,1,3-丙二醇浓度降低.发酵前期,通气对辅酶NADH和NAD的浓度无影响;发酵后期,菌体生长停滞,辅酶的浓度也随之下降.

有氧条件下Klebsiella pneumoniae发酵生产1,3-丙二醇的研究

探讨了有氧条件下利用Klebsiella pneumoniae发酵生产1,3-丙二醇的可能性,研究了通气量、初始底物浓度、pH和3-羟基丙醛等因素对发酵过程的影响.当空气通量为0.25vvm时,1,3-丙二醇的得率和生产强度与厌氧时相近.有氧条件下,当初始甘油质量浓度大于50g/L时,发酵中后期出现3-羟基丙醛的长期积累,甘油不能消耗完全.控制pH为7.75～8.00可以促进3-羟基丙醛的转化使甘油完全消耗.

葡萄糖作辅助碳源对Klebsiella pneumoniae发酵甘油生产1,3-丙二醇的影响

在Klebsiella pneumoniae发酵甘油生产1,3-丙二醇的种子培养及发酵实验中,考察了葡萄糖作辅助碳源对菌体生长及1,3-丙二醇生成的影响. 结果表明,在种子培养期,以葡萄糖和甘油为混合碳源可缩短种子培养周期;在批次发酵和流加发酵中,葡萄糖作辅助碳源可使1,3-丙二醇产率及得率明显提高,但不同的葡萄糖加入方式对产率及得率促进的效果不同. 在初始发酵培养基中添加5 g/L葡萄糖、并在4～40 h进行葡萄糖与甘油混合液连续流加的条件下,1,3-丙二醇浓度、产率及得率较单一甘油为底物的流加发酵结果分别提高41.2%, 38.6%和8.3%.

氧对Klebsiella pneumoniae产1,3-丙二醇代谢的影响

考察了氧对Klebsiellapneumoniae发酵产1,3?丙二醇(PDO)代谢的影响.研究结果表明,在厌氧或供氧条件下,K.pneumoniae都能利用甘油产PDO.起始甘油浓度20g/L,发酵时间4h,在充分供氧条件下,PDO产量仅为1.1mmol/L;但在微量供氧条件下,PDO产量为16mmol/L,是厌氧发酵时的1.28倍.在微量供氧条件下,调控PDO合成的关键酶甘油脱氢酶、甘油脱水酶及1,3?丙二醇氧化还原酶的活性分别为7.28,1.14,0.52U/mg,高于厌氧或充分供氧条件下的相应酶活性.氧对细胞内NADH的合成也有影响,厌氧及微量供氧条件下菌体内NADH含量分别为3.78及3.72μmol/g(DCW),高于充分供氧发酵时的0.85μmol/g(DCW).

基因敲除弱化产1,3-丙二醇Klebsiella pneumoniae荚膜多糖的合成

1,3-丙二醇(1,3-PDO)是重要的平台化合物,在高性能纤维合成等领域具有广泛应用。Klebsiella pneumoniae是优良的1,3-PDO生物合成细胞工厂,但该菌代谢甘油合成1,3-PDO时积累大量荚膜多糖,影响底物与产物的高效转运,导致发酵液黏度增大,限制发酵法生产1,3-丙二醇的下游提取及产业化。本研究基于已报道的K.pneumoniae荚膜合成途径,利用Red重组技术对K.pneumoniae荚膜多糖合成途径中的起始糖基转移酶wec A基因及参与荚膜多糖后期组装的跨膜蛋白wzi基因进行敲除,并考察上述基因缺失对菌株荚膜含量、细胞形态、菌体生长、发酵液黏度及产物合成的影响。研究发现,同时缺失wec A和wzi基因对于弱化荚膜多糖效果显著,荚膜含量降低38.5%,细胞基本丧失菌毛合成及相互粘连能力,1,3-PDO产量提高23.0%,为菌株改造提供了一种新思路。

甘油耐受菌Klebsiella pneumoniae XJPD-Li合成1，3-丙二醇的研究

利用自行筛选的1,3-丙二醇(1,3-PD)合成菌株Klebsiella pneumoniae XJPD-Li进行发酵,考察了甘油浓度对菌株生长及合成1,3-PD的影响。经多次甘油耐受后菌株XJPD-Li在摇瓶培养48 h,菌体生物量(OD_(650))和1,3-PD产量分别达到1.52和11.16 g/L,相比耐受前分别提高48%和159%。在5 L发酵罐批式培养中,甘油耐受后XJPD-Li菌体生物量并未显著增长,但合成1,3-PD的能力却提高了77%,达到53.13 g/L。

不同碳源对Klebsiella pneumoniae WA-1孔雀石绿脱色和降解的影响

利用变色圈法从孔雀石绿污染的养殖池塘中筛选到1株具有较强孔雀石绿脱色和降解能力的细菌,经过16S rDNA基因序列比对及系统进化树分析,结合VITEK 2微生物鉴定系统,鉴定该菌为Klebsiella pneumoniae,重命名为K.pneumoniae WA-1。采用分光光度法和硼氢化钾还原-高效液相色谱法研究不同碳源对孔雀石绿脱色和降解的影响,发现菌株在盐培养基中表现出很强的孔雀石绿脱色和降解能力,添加葡萄糖、半乳糖、蔗糖等碳源时孔雀石绿(5~40 mg/L)的脱色率有所提高,均接近100%,但是孔雀石绿(5 mg/L)降解率显著降低。明确了孔雀石绿首先在细胞外由孔雀石绿还原酶还原成隐性孔雀石绿,然后通过主动运输进入到细胞被进一步降解的脱色和降解过程,并揭示了不同碳源对菌株孔雀石绿脱色和降解的影响机制。本研究为K.pneumoniae WA-1的孔雀石绿降解机制研究提供重要思路,也为后期孔雀石绿降解的应用提供重要理论基础。

A Five-year Surveillance of Carbapenemase-producing Klebsiella pneumoniae in a Pediatric Hospital in China Reveals Increased Predominance of NDM-1

Objective To characterize carbapenem （CPM）-non-susceptible Klebsiella pneumoniae （K. pneumoniae） and carbape-nemase produced by these strains isolated from Beijing Children＇s Hospital based on a five-year surveillance. Methods The Minimal Inhibition Concentration values for 15 antibiotics were assessed using the Phonixl00 compact system. PCR amplification and DNA sequencing were used to detect genes encoding carbapenemases. WHONET 5.6 was finally used for resistance analysis. Results In total, 179 strains of CPM-non-susceptible K. pneumoniae were isolated from January, 2010 to December, 2014. The rates of non-susceptible to imipenem and meropenem were 95.0% and 95.6%, respectively. In the 179 strains, 95 （53.1%） strains carried the blalMP gene, and IMP-4 and IMP-8 were detected in 92 （96.8%） and 3 （3.2%） IMP-producing isolates, respectively. 65 （36.3%） strains carried the blaNDM_1 gene. 6 （3.4%） strains carried the blaKpc gene, and KPC-2 were detected in 6 KPC-producing isolates. In addition, New Delhi-Metallo-1 （NDM-1） producing isolates increased from 7.1% to 63.0% in five years and IMP-4 producing isolates decreased from 75.0% to 28.3%. Conclusion High frequencies of multiple resistances to antibiotics were observed in the CPM-non-susceptible K. pneumoniae strains isolated from Beijing Children＇s Hospital. The production of IMP-4 and NDM-1 metallo-13-1actamases appears to be an important mechanism for CPM-non- susceptible in K. pneumoniae.