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.

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.

能量驱动对Klebsiella pneumoniae发酵甘油合成1,3-丙二醇的影响

考察了外加能量对Klebsiella pneumoniae合成1,3-丙二醇的影响,结果表明,外加0.6～0.8 g/L三磷酸腺苷)(ATP)可以有效促进Klebsiella pneumoniae发酵甘油的还原代谢,1,3-丙二醇产量提高了50%～70%,得率在发酵后期仍能维持在较高水平. 利用休止细胞研究了甘油脱水酶催化失活后能量刺激复活的情况,结果表明外加三磷酸腺苷(ATP)对休止细胞中甘油脱水酶的复活有明显的驱动作用,经多次失活/驱动复活后甘油脱水酶活性可维持不变.

代谢副产物对Klebsiella pneumoniae生长及合成1,3-丙二醇的影响

研究了Klebsiellapneumoniae在厌氧摇瓶中的生长代谢特性和基质消耗情况,发现主要副产物乙醇是抑制菌体持续生长及1,3-丙二醇合成的主要因素,外源添加实验表明,8g/L乙醇可使K.pneumoniae比生长速率、1,3-丙二醇比合成速率、最大菌体浓度及1,3-丙二醇终浓度分别下降21.6%,22.1%,59.6%及33.5%;指数生长期加入乙醇对菌体生长代谢的抑制作用更加明显.其他代谢副产物乙酸、乳酸、2,3-丁二醇对K.pneumoniae生长代谢也有不同程度影响,乙酸浓度仅2g/L即可对菌体生长产生抑制,乙酸浓度达到5g/L以上时对菌体生长有显著的抑制作用,而2,3-丁二醇与乳酸浓度需达到10g/L时才会对菌体生长产生显著影响.

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.

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.

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%.

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%。

Klebsiella pneumoniae发酵稻草纸浆水解液生产2,3-丁二醇工艺的研究

对Klebsiella pneumoniae发酵稻草纸浆水解液生产2,3-丁二醇的工艺进行了初步的实验研究。考察了温度、时间、底物浓度、pH等不同因素对稻草纸浆酶水解和2,3-丁二醇发酵的影响。结果表明,在纸浆的酶用量为135IU/g、底物浓度为20g/L、50℃、pH4.8的条件下反应20h,还原糖得率最高为68.15%;2,3-丁二醇的最佳发酵条件为pH6.0、葡萄糖初始浓度100g/L、30℃、接种量15%、150r/min、反应72h,2,3-丁二醇的最高转化率为17.92%。

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。

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%.

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.

两种消除Klebsiella pneumoniae重组型质粒的方法

对于工业发酵菌种肺炎克雷伯氏菌(Klebsiella pneumoniae),研究发现有两种消除其重组型质粒的有效方法,一种是连续传代培养,另一种是使用消除剂十二烷基硫酸钠(SDS)。对K.pneumoniae重组菌连续20代传代培养后,发现其质粒具有较高的消除率;而以0.2%SDS复合Ca2+处理K.pneumoniae重组菌,也能有效消除其重组型质粒,且该方法省却了反复的传代培养,能快速得到质粒消除菌,更具简便易操作性。消除了质粒的K.pneumoniae能再次接纳新的质粒,有效避免了因质粒不相容性带来的转化不成功,进而可用作宿主菌积累更多的生理性状。

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

我们的学习的目的是调查 Carbapenem 抵抗的 Klebsiella pneumoniae (CRKP ) 和班的基因特征的流行的目的在临床的 Klebsiella pneumoniae 拉紧的多药 resistance.Methods 上的 CRKP 的 1 integron 在华中从一所医院的多重部门被收集。CRKP 紧张在 isolates 之中被识别，并且 CRKP 紧张的抗菌素危险性被分析。聚合酶链反应(PCR ) 被采用放大类 1 个 integron 变量区域。integron 基因结构与酶消化和 DNA 定序技术被分析。在班 1 integron 和药抵抗之间的关系被分析完全， 955 Klebsiella pneumoniae 拉紧的 statistically.Results 从医院的改变的地点被孤立，并且(12.3%) 117 作为 CRKP 他们被识别，与在 2013 的8.9%的分离率( 26/292 )，11.3%( 38/336 )在 2014 和16.2%( 53/327 )在 2015 ，哪个表演到年的一个增加的趋势。(52/117 )44.4% 与 ICU 的标本 CRKP 紧张被分开，并且(72/117 )61.5% 从唾沫。超过 95% CRKP 紧张对 ampicillin/sulbactam， aztreonam， imipenem， meropenem， ceftazidme， cefotaxime， cefepime，和 piperacillin 抵抗，当相对低的抵抗的率在 tigecycline (12.8%) 和 colistin (35.9%) 被发现时。班 1 integron 在 77.8% 被检测(91/117 ) CRKP 紧张。CRKP 的班 1 integron 显著地与抗菌素抵抗被相关到 tobramycin， gentamicin 和 amikacin (所有 P < 0.01 ) 。班 1 integron 的可变区域的基因盒子分析证明 aadA2 占 64.8%(59/91 ) ， aacA4-catB8-aadA1 23.1%(21/91 ) ，并且 aadA2-dfrA25 12.1%(11/91 ).Conclusions CRKP 在他们的中国，和大多数在临床的设置有一个增加的趋势对多重抗菌素抵抗。在 CRKP 的班 1 integron 有强壮的能力从环境捕获对 aminoglycosides 抗菌素抵抗的基因，与象最流行的 aadA2 基因。

Patients with culture negative pyogenic liver abscess have the same outcomes compared to those withKlebsiella pneumoniae pyogenic liver abscess

BACKGROUND:Etiologic organism is not frequently isolated despite multiple blood and fluid cultures during management of pyogenic liver abscess(PLA).Such culture negative pyogenic liver abscess(CNPLA) is routinely managed by antibiotics targeted to Klebsiella pneumoniae. In this study, we evaluated the outcomes of such clinical practice. METHODS: All the patients with CNPLA and Klebsiella pneumoniae PLA(KPPLA) admitted from January 2003 to December 2011 were included in the study. A retrospective review of medical records was performed and demographic, clinical and outcome data were collected.RESULTS: A total of 528 patients were treated as CNPLA or KPPLA over the study period. CNPLA presented more commonly with abdominal pain(P=0.024). KPPLA was more common in older age(P=0.029) and was associated with thrombocytopenia(P=0.001), elevated creatinine(P=0.002), bilirubin(P=0.001), alanine aminotransferase(P=0.006) and C-reactive protein level(P=0.036). CNPLA patients tend to have anemia(P=0.015) and smaller abscess(P=0.008). There was no difference in hospital stay(15.7 vs 16.8 days) or mortality(14.0% vs 11.0%). No patients required surgical drainage after initiation of medical therapy.CONCLUSION: Despite demographic and clinical differences between CNPLA and KPPLA, overall outcomes are not different.

Prostatic abscess of Klebsiella pneumoniae complicating septic pulmonary emboli and meningitis: A case report and brief review

Prostatic abscess is a rare entity with an incidence of 0.5% to 2.5% in all prostate diseases and usually occurs in the 5th and 6th decades of life with immunocompromised status,Prostatic abscess might be a process of evolution from acute prostatitis,Klebsiella pneumoniae is the leading microorganism in the diabetic patients of prostatic abscess in Taiwan,A 60-year-old diabetic man,with a one-week history of acute bacterial prostatitis was reported in this study,presenting to the emergency department with sudden altered mental status,The abdominal computed tomographic scan demonstrated lobulated prostatic abscess and multiple septic pulmonary emboli with lung abscesses,Analysis of cerebrospinal fluid showed white blood cells of 10 771 counts/mm3 with segmented neutrophils of 99%,Cultures of blood,cerebrospinal fluid and sputum yielded Klebsiella pneumoniae,We concluded that computed tomographic scan can make a definite diagnosis of prostatic abscess associated with complications and management with empiric antibiotics and adequate drainage is suggested.

一株环境Klebsiella pneumoniae硝化与反硝化研究

文章对分离于自然环境中的一株异养硝化-好氧反硝化菌的氮代谢特性进行研究,以期为解释异养硝化和好氧反硝化的机制和为生物脱氮工艺提供理论依据。用16S r DNA基因序列分析法对分离菌株进行鉴定。以铵盐、硝酸盐、亚硝酸盐作为细菌氮源,每隔3 h连续取样,检测培养基中氮和细菌浓度的变化。经鉴定该菌株属于肺炎克雷伯氏杆菌(Klebsiella pneumoniae)。该菌株具有降解铵盐、硝酸盐和亚硝酸盐的作用,24 h内的降解率分别为96%、100%、100%,氮去除速率分别为4.8、2.2、3.3 mg/(L·h)。该研究为中国内首次报道环境肺炎克雷伯氏杆菌具异养硝化与好氧反硝化特性,提示该类细菌具重要生态作用,广泛参与环境氮循环,也是污水中去除氮的一类可选微生物资源。

遗传改造Klebsiella pneumoniae提高1,3-丙二醇产量的研究进展

1,3-丙二醇是合成聚对苯二甲酸丙二醇酯(PTT)的重要原料。与化学法相比,微生物发酵生产1,3-丙二醇因对环境友好而成为当今研究热点。本文作者以肺炎克雷伯菌(Klebsiella pneumoniae)为表达宿主,从代谢途径、表达宿主、载体构建和关键酶基因等方面,综述了发酵菌种遗传改造的研究进展,并对一些新的菌种筛选和改造方法进行了讨论。