Treatment of Donor-derived Carbapenem-resistant Klebsiella pneumoniae Infection after Renal Transplantation with Tigecycline and Extended-infusion Meropenem

Objective Donor-derived carbapenem-resistant Klebsiella pneumoniae(CRKP)infection has recently emerged as a critical early complication after renal transplantation.Although CRKP is usually sensitive to tigecycline,monotherapy with this drug is often less than effective.We investigated the efficacy of a combined regimen of tigecycline with high-dose,extended-infusion meropenem in the treatment of donor-derived CRKP infection after kidney transplantation.Methods From Jan.2016 to Dec.2017,a total of 12 CRKP isolates were detected from cultures of the organ preservation solution used for soaking the donor kidneys at our institute.Probable or possible donor-derived infection(DDI)was identified in 8 transplant recipients.Clinical data were retrospectively analyzed.Results Klebsiella pneumoniae carbapenemase-2(KPC-2)-producing CRKP was reported to be positive in organ preservation solution cultures at 3.5±0.9 days after transplantation,leading to surgical site(n=3),urinary tract(n=4),and/or bloodstream(n=2)infections in 8 recipients.The drug susceptibility tests showed that CRKP was sensitive to tigecycline,but resistant to meropenem.In 7 patients who received tigecycline combined with high-dose extended-infusion meropenem,DDIs were successfully cured.The length of hospital stay was 31(18–129)days,and the serum creatinine at discharge was 105.8±16.7µmol/L.The one remaining patient who received tigecycline combined with intravenous-drip meropenem died of septic shock.A median follow-up of 43 months(33–55)showed no recurrence of new CRKP infection in the 7 surviving recipients.Conclusion It was suggested that a prompt and appropriate combination therapy using tigecycline with high-dose extended-infusion meropenem is effective in treating donor-derived KPC-2-producing CRKP infection after renal transplantation.

Prevalence of Carbapenem-Resistant Klebsiella Pneumoniae （CRKP） and the Distribution of Class 1 Integron in Their Strains Isolated from a Hospital in Central China

Objective The aim of our study is to investigate the prevalence of Carbapenem-resistant Klebsiella pneumoniae （CRKP） and the genetic characteristics of the class 1 integron in CRKP on multi-drug resistance. Methods Clinical Klebsiella pneumoniae strains were collected from multiple departments of a hospital in central China. CRKP strains were identified among the isolates, and antibiotics susceptibility of CRKP strains was analyzed. The polymerase chain reaction （PCR） was adopted to amplify the class 1 integron variable area. The integron genetic structure was analyzed with enzyme digestion and DNA sequencing technology. The relation between class 1 integron and drug resistance was analyzed statistically. Results Totally 955 strains of Klebsiella pneumoniae were isolated from varied sites of the hospital, and 117（12.3%） of them were identified as CRKP, with a separation rate of 8.9% （26/292） in 2013, 11.3% （38/336） in 2014 and 16.2% （53/327） in 2015, which shows an increasing trend by year. 44.4% （52/117） of CRKP strains were separated from specimen of ICU, and 61.5% （72/117） were from sputum. Over 95% CRKP strains were resistant to ampicillin/sulbactam, aztreonam, imipenem, meropenem Ceftazidme,Cefotaxime,Cefepime,and Piperacillin, while relatively low resistant rates were found in Tigecycline （12.8%） and colistin （35.9%）. The class 1 integron was detected in 77.8% （91/117） of CRKP strains. Class 1 integron of CRKP was significantly correlated with the antibiotic resistance to the tobramycin, gentamicin and amikacin （all P〈0.01）. The gene cassette analysis of variable area of class 1 integron showed that aadA2 accounts for 64.8% （59/91）, aacA4-catB8-aadA1 23.1% （21/91）, and aadA2-dfrA25 12.1% （11/91）. Conclusions CRKP has an increasing trend in a clinical setting in China, and most of them were resistant to multiple antibiotics. Class 1 integron in CRKP has strong ability to capture the genes resistant to aminoglycosides antibiotics from environment, with the aadA2 gene as the most popular one.

Molecular Epidemiology and Risk Factors of Carbapenem-Resistant Klebsiella Pneumoniae Bloodstream Infections in Wuhan,China

Objective:The clinical characteristics and microbiological data of patients with K.pneumoniae bloodstream infections(BSI)from January 2018 to December 2020 were retrospectively analyzed to study the molecular epidemiology of Carbapenem-resistant Klebsiella pneumoniae(CRKP).We also aimed to identify the risk factors for the development of CRKP BSI.Methods:This retrospective study was conducted at Renmin Hospital of Wuhan University from January 2018 to December 2020.The date of non-duplicate K.pneumoniae isolates isolated from blood samples was identified using the microbiology laboratory database.The data from patients diagnosed with K.pneumoniae BSI were collected and analyzed.

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.

Acquisition of a novel conjugative multidrug-resistant hypervirulent plasmid leads to hypervirulence in clinical carbapenem-resistant Klebsiella pneumoniae strains

The co-occurrence of plasmid-mediated multidrug resistance and hypervirulence in epidemic carbapenem-resistant Klebsiella pneumoniae has emerged as a global public health issue.In this study,an ST23 carbapenem-resistant hyper-virulent K.pneumoniae(CR-HvKP)strain VH1-2 was identified from cucumber in China and harbored a novel hybrid plasmid pVH1-2-VIR.The plasmid pVH1-2-VIR carrying both virulence and multidrug-resistance(MDR)genes was likely generated through the recombination of a virulence plasmid and an IncFIIK conjugative MDR plasmid in clinical ST2318622 isolated from a sputum sample.The plasmid pVH1-2-VIR exhibited the capacity for transfer to the clinical ST11 carbapenem-resistant K.pneumoniae(CRKP)strain via conjugation assay.Acquisition of pVH1-2-VIR plasmid directly converted a CRKP into CR-HvKP strain characterized by hypermucoviscosity,heightened virulence for Galleria mellonella larvae,and increased colonization ability in the mouse intestine.The emergence of such a hybrid plasmid may expedite the spread of CR-HvKP strains,posing a significant risk to human health.

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 Antibiotic Resistance of Urinary Tract Pathogens, with Molecular Identification of Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp., Using Multiplex Real-Time PCR

Urinary tract infections (UTIs) caused by uropathogens are a significant public health problem, and their treatment primarily relies on antibiotic therapy. However, the increasing global development of antibiotic resistance necessitates updating diagnostic techniques to ensure higher sensitivity and specificity, especially with advancements in science and medicine. This study aimed to evaluate the prevalence of UTIs and antibiotic resistance profiles through urine culture, as well as to identify Klebsiella pneumoniae, Klebsiella oxytoca, and Acinetobacter spp. in urine samples using a molecular approach with multiplex real-time PCR. From May 3 to July 25, 2023, at the Pietro Annigoni Biomolecular Research Center (CERBA) and Saint Camille Hospital of Ouagadougou (HOSCO), 209 urine samples collected from patients with suspected UTIs were analyzed using both urine culture and multiplex real-time PCR. Among the 209 patients, 52.15% were male and 47.85% female, with an average age of 46.87 ± 21.33 years. Urine cultures revealed an overall UTI prevalence of 23.44%, with a prevalence of 8.13% in men versus 15.31% in women (P = 0.023). The bacterial prevalence rates were as follows: Escherichia coli (12.92%), Klebsiella spp. (7.18%), Enterobacter cloacae (1.44%), Staphylococcus aureus (0.96%), and other bacteria. Klebsiella spp. demonstrated 100% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, while Escherichia coli showed 96.2% and 65.4% resistance to Amoxicillin and Amoxicillin/Clavulanic Acid, respectively. PCR analysis of the target bacteria revealed mono-infection prevalence rates of Klebsiella pneumoniae (10.39%), Klebsiella oxytoca (7.79%), and Acinetobacter spp. (7.79%), along with a co-infection prevalence rate of Klebsiella pneumoniae/Acinetobacter spp. (1.30%). This study demonstrated that PCR, with its high sensitivity and specificity, could effectively distinguish Klebsiella pneumoniae from Klebsiella oxytoca and detect Acinetobacter spp. in less than 24 hours—something urine culture alone could not achieve. The relative ease of automating urine PCR testing, combined with its diagnostic accuracy and rapid turnaround time, makes it a valuable addition to modern medical practice for the laboratory diagnosis of UTIs.

Impact of Genetic Diversity of Uropathogenic Escherichia coli and Klebsiella pneumoniae Strains on the Dissemination of Extended Spectrum Beta-Lactam Resistance Genes in Côte d’Ivoire

The increase and spread of bacterial resistance to extended-spectrum beta-lactam antibiotics are reported in many infections and are a real public health problem worldwide. Drug pressure is a factor that favors the emergence of a population of better adapted bacteria. However, there is no literature highlighting the genetic diversity and evolutionary structure of E. coli and K. pneumoniae in an environment with high selection pressure in Côte d’Ivoire. The objective of this study was to evaluate the genetic diversity of E. coli and K. pneumoniae strains circulating at the HKB Hospital in Abobo and at the Daloa Regional Hospital and its impact on the dissemination of extended spectrum beta-lactam resistance genes. A total of 39 strains isolated from the urinary tract of infected patients, including 30 strains of E. coli and 9 strains of K. pneumoniae were studied. A total of 39 strains isolated from the urinary tract of infected patients, including 30 strains of E. coli and 9 strains of K. pneumoniae were studied. From genomic DNA extracts, ESBL resistance genes were amplified by PCR and sequenced, in addition to genetic typing by ERIC-PCR. The data obtained were submitted to genetic and bioinformatics analyses. The results have shown a genetic diversity important in E. coli and K. pneumoniae with diversity indexs (SID) ranging from 0.5 to 0.77. The genetic structure of the bacterial species studied has shown a clonal distribution of strains with clones expressing TEM-9 and CTX-M-15 variants. Also, this clonal structure was correlated with the spread of resistance genes in E. coli and K. pneumoniae. The spread of resistant clones is a factor that might limit the fight against antibiotic resistance.

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.

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.

Risk Factors for Carbapenem-resistant Klebsiella pneumoniae Infection and Mortality of Klebsiella pneumoniae Infection

Background： Klebsiellapneumoniae （KP） is a pathogen commonly causing nosocomial infection. Carbapenem-resistant KP （CRKP） is more resistant to multiple antimicrobial drugs than carbapenem-susceptible KP （CSKP） isolates. The aim of the present study was to identify the risk factors for CRKP infection and the predictors of mortality among KP-infected adult patients. Methods： Patients with CRKP and CSKP infection were categorized as the case group and control group, respectively, and we conducted a 1：1 ratio case-control study on these groups. The CRKP isolates collected were tested tbr antimicrobial susceptibility and presence of KP carbapenemase （KPC） gene. Clinical data were collected to identity risk factors for CRKP infection and mortality of KP infection. Risk factors were analyzed under univariable and multivariable logistic regression model. Results： The independent risk factors for CRKP infection were admission to Intensive Care Unit （odds ratio [OR]： 15.486, 95% confidence interval [CI]： 3.175-75.541, P 〈 0.001）; use of I3-1actams and [3-1actamase inhibitor combination （OR： 4.765, 95% CI： 1.508-15.055, P = 0.008）： use of cephalosporins （OR： 8.033, 95% CI： 1.623-39.763, P = P= 0.019）： and indwelling of urethral catheter （OR： 6.164, 95% CI： 1.847 0.011 ）; fluoroquinolones （OR： 6.090, 95% CI： 1.343-27.613, -20.578, P = 0.003）. However, older age （OR： 1.079, 95% CI： 1.005-1.158, P= 0.036）, Charlson comorbidity index （OR： 4.690, 95% CI：2.094-10.504, P= 0.000）, and aminoglycoside use （OR： 670.252, 95% CI： 6.577-68,307.730, P = 0.006） were identified as independent risk factors for patient deaths with KP infection. The mortality of CRKP group was higher than that of the CSKP group. KPC gene did not play a role in the CRKP group. CRKP mortality was high. Conclusion： Implementation of infection control measures and protection of the immunefunction are crucial.

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。

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.

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.

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%。

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%。

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.