Molecular typing of Klebsiella pneumoniae isolates from public hospitals in Recife, Brazil

Thirty nosocomial isolates of Klebsiella pneumoniae, collected from three public hospitals in Recife, Brazil, between 1999 and 2000, were analysed in order to determine their epidemiological relatedness and genetic characteristics. The isolates were characterised by biotyping, antibiotyping, protein analysis, plasmid profile and random amplified polymorphic DNA (RAPD). The majority of the clinical isolates were resistant to multiple antibiotics, in particular to beta-lactams, and 30% were found to be ESBLs producers. RAPD proved to be the most effective technique in discriminating unrelated K. pneumoniae isolates. It was confirmed by the highly genetic similarity found among related isolates from an hospital outbreak. We conclude that K. pneumoniae infections in Recife has been caused by a variety of bacterial genotypes. This is the first report that revealed the subtypes of K. pneumoniae in Brazil by plasmid analysis and RAPD.     

超广谱β-内酰胺酶肺炎克雷伯菌噬菌体F20的分离鉴定及其对小鼠败血症治疗效果

【背景】肺炎克雷伯菌是引起临床感染的重要条件致病菌之一,肺炎克雷伯菌中产超广谱β-内酰胺酶(Extended-spectrum beta-lactamases,ESBLs)的耐药菌株增多迫切需要找到一种新的治疗方法。【目的】自污水中分离超广谱β-内酰胺酶肺炎克雷伯菌噬菌体,并明确其生物学特性、观察其治疗小鼠产ESBLs肺炎克雷伯菌感染的疗效。【方法】电镜观察F20形态,调查其噬菌谱、生长曲线等生物学特性。建立小鼠败血症感染模型观察F20治疗小鼠肺炎克雷伯菌感染的疗效。【结果】F20在其宿主菌的菌苔上形成裂解性噬菌体所具有的完全透明的噬菌斑,电镜观察F20具典型的有尾噬菌体目长尾病毒科病毒的形态特征。一步生长曲线显示F20的潜伏期为18 min,裂解量为89 PFU/细胞。稳定性试验显示F20在pH 5.0-9.0及50°C环境均具良好稳定性。使用噬菌体F20对败血症小鼠治疗后,治疗组小鼠各外周血和各脏器(肺脏、肝脏、脾脏和肾脏)中的细菌数也显著小于对照组细菌数(P0.001),与对照组相比下降大约1–3数量级。F20治疗败血症小鼠存活率达到87.5%,无毒副作用,而对照组小鼠在1 d内全部死亡,可显著提高小鼠的存活率(P0.001)。【结论】新分离的裂解性噬菌体F20在小鼠体内能安全有效地治疗超广谱β-内酰胺酶肺炎克雷伯菌引起的败血症,可作为生物抗菌剂的有效成分。     

产ESBLs肺炎克雷伯菌的基因型和耐药性研究

超广谱β-内酰胺酶(extended spectrumβ-lactamase,ESBLs)与细菌耐药性密切相关,为了揭示产ESBLs肺炎克雷伯菌的基因型和耐药性,本研究在2017年1月至2018年9月期间,共检测了466例肺炎克雷伯菌感染的患者标本,并分析了产ESBLs肺炎克雷伯菌的基因型和耐药性。研究结果显示,466例患者中共检出562个肺炎克雷伯菌菌株。其中,呼吸内科的检出数最多(212株,37.72%),其次为重症监护科(106株,18.86%)。对于不同标本来源,痰液中检出菌数最多(331株),占总数的58.90%。562株肺炎克雷伯菌中共检测出237株产ESBLs(42.17%)。产ESBLs肺炎克雷伯菌对阿莫西林(89.03%)、替卡西林(87.34%)、氨苄西林(81.43%)的耐药率最高;而对替加环素(9.28%)、阿米卡星(6.75%)、亚胺培南(1.27%)和美罗培南(0.84%)的耐药率最低。237株产ESBLs的肺炎克雷伯菌菌株中,53.59%扩增出CTX-M型,48.95%扩增出TEM型,40.93%增出SHV型。另外,237株产ESBLs的肺炎克雷伯菌菌株中,产两个及以上的ESBLs的菌株共111株(46.84%)。     

Spreading and mechanisms of antimicrobial resistance in microorganisms,producing beta-lactamases. Molecular mechanisms of resistance to beta-lactam antibiotics of <Emphasis Type="Italic">Klebsiella</Emphasis> spp. strains,isolated in cases of nosocomial infections

Antibiotic sensitivity has been investigated in nosocomial bacterial Klebsiella spp. strains isolated from patients treated in 30 hospitals of 15 Russian regions. Among Klebsiella strains (n = 212) studied the following species were found: Klebsiella pneumoniae ss. pneumoniae—182 (85.8%), Klebsiella pneumoniae ss. ozaenae—1 (0.5%), Klebsiella oxytoca—29 (13.7%) strains. Their sensitivity to antibacterial preparations was estimated by the method of serial dilutions in microvolume (the microdilution method). Carbapenems (imipenem and meropenem) exhibited the highest antibacterial activity against the strains studied. Among third generation cephalosporins the lowest MIC (Minimum inhibitory concentration) were found in the inhibitor protected preparations: ceftazidime/clavulanic acid (MIC₅₀ of 0.25 μg/ml; MIC₉₀ of 64 μg/ml) and cefoperazone/sulbactam (MIC₅₀ of 16 μg/ml; MIC₉₀ of 64 μg/ml). Using the PCR method the detection of class A betalactamases genes (TEM, SHV, CTX) was carried out in 42 strains of Klebsiella pneumoniae ss. pneumoniae. TEM type beta-lactamases were found alone or in various combinations in 16 (38.1%) strains, SHV—in 29 (69%), and CTX—in 27 (64.3%). Combinations of 2 and 3 different resistance determinants were detected in 23.8 and 26.2% of strains, respectively. Screening of carbapenem-resistant Klebsiella strains for production of class B metallo-beta-lactamases did not reveal nosocomial strains with phenotypically documented production of these enzymes.     

Protection against Klebsiella pneumoniae induced lobar pneumonia in rats with lipopolysaccharide and related antigens

The immunoprotective role of lipopolysaccharide and related antigens from Klebsiella pneumoniae was studied in a lobar pneumonia model developed in rats. Various antigens were obtained by different chemical treatments of the lipopolysaccharide. All these antigens (purified lipopolysaccharide, reduced lipopolysaccharide, lipopolysaccharide--bovine serum albumin complex, and lipid A--bovine serum albumin complex were tested for pyrogenicity and the Shwartzman reaction. The lipopolysaccharide and the various related antigens were pyrogenic and elicited a positive Shwartzman reaction at high concentrations. However, at low concentrations, the same preparations did not show any side effects. All these antigens, on the other hand, were protective against bacterial challenge in Klebsiella pneumoniae induced lobar pneumonia in rats, as the bacterial colonization of lungs in the immunized animals was significantly lower when compared with the controls. The alveolar macrophages from these animals also showed significantly more uptake of Klebsiella pneumoniae as compared with those obtained from control animals.     

Inhibitors of bacterial virulence identified in a surrogate host model

Antibiotic resistance continues to reduce the number of available antibiotics, increasing the need for novel antibacterial drugs. Since the seminal work of Sir Alexander Fleming, antibiotic identification has been based exclusively on the inhibition of bacterial growth in vitro. Recently, inhibitors of bacterial virulence which interfere with bacterial pathogenesis mechanisms have been proposed as an alternative to antibiotics, and a few were discovered using assays targeting specific virulence mechanisms. Here we designed a simple surrogate host model for the measurement of virulence and systematic discovery of anti-virulence molecules, based on the interaction of Tetrahymena pyriformis and Klebsiella pneumoniae cells. We screened a library of small molecules and identified several inhibitors of virulence. In a mouse pneumonia model we confirmed that an anti-virulence molecule displayed antibacterial activity against Klebsiella pneumoniae and Pseudomonas aeruginosa, by reducing dramatically the bacterial load in the lungs. This molecule did not inhibit bacterial growth in vitro but prevented biosynthesis of the Klebsiella capsule and lipopolysaccharides, a key requirement for virulence. Our results demonstrate that anti-virulence molecules represent an alternative to antibiotics and those can be discovered using non-animal host models.     

Klebsiella pneumoniae secretes outer membrane vesicles that induce the innate immune response

Outer membrane vesicles (OMVs) derived from pathogenic Gram-negative bacteria are an important vehicle for delivery of effector molecules to host cells, but the production of OMVs from Klebsiella pneumoniae, an opportunistic pathogen of both nosocomial and community-acquired infections, and their role in bacterial pathogenesis have not yet been determined. In the present study, we examined the production of OMVs from K. pneumoniae and determined the induction of the innate immune response against K. pneumoniae OMVs. Klebsiella pneumoniae ATCC 13883 produced and secreted OMVs during in vitro culture. Proteomic analysis revealed that 159 different proteins were associated with K. pneumoniae OMVs. Klebsiella pneumoniae OMVs did not inhibit cell growth or induce cell death. However, these vesicles induced expression of proinflammatory cytokine genes such as interleukin (IL)-1β and IL-8 in epithelial cells. An intratracheal challenge of K. pneumoniae OMVs in neutropenic mice resulted in severe lung pathology similar to K. pneumoniae infection. In conclusion, K. pneumoniae produces OMVs like other pathogenic Gram-negative bacteria and K. pneumoniae OMVs are a molecular complex that induces the innate immune response.     

Expression,Purification, and Biophysical Characterization of Klebsiella Pneumoniae Nicotinate Nucleotide Adenylyltransferase

The Protein Journal - Patients in health-care settings develop nosocomial infections due to prolonged hospital stay. The Gram negative Klebsiella pneumoniae (K. pneumoniae), is a bacterial pathogen...     

致细菌性肝脓肿的肺炎克雷伯杆菌多位点序列分型及药敏分析

目的了解致细菌性肝脓肿的肺炎克雷伯杆菌多位点序列分型及药敏情况。方法收集2011年1月至2012年6月在浙江大学医学院附属第一医院感染科住院的细菌性肝脓肿患者,脓液培养为肺炎克雷伯杆菌的23株菌株,对23株菌株进行多位点序列分型;应用K—B纸片扩散法检测23株肺炎克雷伯杆菌菌株对10种抗菌药物的敏感性;应用超广谱β-内酰胺酶（ESBLs）确认试验了解23株菌株的产ESBLs情况。结果23株菌株经过多位点序列分型：ST23为最主要序列型,共有10株,ST25、ST30、ST65、ST86、ST163、ST367、ST375、ST380、ST660、ST700及ST806各1株,2株为新的ST型,未发现文献报道的产耐碳氢酶烯酶的常见sT型;23株菌株的药敏结果对哌拉西林他唑巴坦及头孢哌酮舒巴坦等8种抗菌药物的耐药率为0％,对头孢呋辛耐药率4．4％,而对氨苄西林的耐药率为100％;ESBLs确认试验其中22株为ESBL^-;1株为ESBL^＋。结论收治的致细菌性肝脓肿的肺炎克雷伯杆菌均为敏感菌株,可以经验性的选用青霉素（三代头孢）复合制剂,避免碳氢酶烯类抗生素的乱用及滥用。     

肺炎克雷伯菌生物被膜的体外模型建立

目的研究临床分离的肺炎克雷伯菌在体外形成生物被膜的情况,为进一步研究生物被膜肺炎克雷伯菌的耐药机制奠定基础。方法采用改良平板法建立肺炎克雷伯菌生物被膜模型,用喷金法和扫描电镜观察鉴定,并对生物被膜的形成进行定量分析。结果23株临床分离的肺炎克雷伯菌菌株生物被膜形成能力不同,以强阳性生物被膜形成能力者为最多数。结论绝大多数临床分离的肺炎克雷伯菌菌株具有较强的生物被膜形成能力。应用改良平板法能够较好的在体外建立其生物被膜模型。     

Decolourization and biodegradation of N,N'-dimethyl-p-phenylenediamine by Klebsiella pneumoniae RS-13 and Acetobacter liquefaciens S-1

Klebsiella pneumoniae RS-13 and Acetobacter liquefaciens S-1, both methyl red (MR)-degrading bacterial strains, degraded N,N'-dimethyl- p -phenylenediamine (DMPD) under aerobic conditions. DMPD, a toxic and mutagenic aromatic amine, is formed during the reductive cleavage of azo dyes such as MR. The effects of physical parameters, such as temperature and aeration, and chemical parameters, such as pH and concentrations of glucose, ethanol and ammonium sulphate in the culture medium, on the degradation of DMPD by these bacteria were determined. Klebsiella pneumoniae RS-13 degraded DMDP more efficiently than A. liquefaciens S-1 under all physicochemical conditions, except in the presence of ethanol as carbon and energy sources. In addition, Kl. pneumoniae RS-13 degraded DMDP at low levels of carbon and nitrogen at pH 6–8. These results indicate that it is feasible to use Kl. pneumoniae RS-13 to completely degrade the detoxify MR under aerobic conditions.     

The influence of the O-antigen and the capsular polysaccharide on the establishment of PlCmts lysogeny in Klebsiella pneumoniae

The O-antigen of the lipopolysaccharide in Klebsiella pneumoniae caused a significant reduction in the frequency of establishment of PlCmts lysogeny, while the capsular polysaccharide showed no effect on this frequency. The bacterial receptor for PlCmts are the lipopolysaccharide-core oligosaccharides, the results suggest that K. pneumoniae strains with an O-antigen in their lipopolysaccharide have a poorly accessible lipopolysaccharide-core (the PlCmts bacterial receptor), while K. pneumoniae strains lacking the O-antigen have a highly accessible lipopolysaccharide-core. The accessibility of the receptor is independent of the K antigen (capsular polysaccharide).     

肺炎克雷伯菌耐药性与I类整合子关系的研究

目的检测I类整合子在肺炎克雷伯菌临床分离株中的分布,分析整合子对细菌耐药性的影响。方法采用K—B纸片扩散法对127株肺炎克雷伯菌临床分离株进行药敏试验;并用WHONET5．6软件分析菌株药敏情况;采用聚合酶链反应（PCR）分析127株肺炎克雷伯菌株的I类整合子。并对I类整合子阳性株与阴性株的耐药性进行对比分析。结果127株菌中有53株（41．70％）含有I类整合子,I类整合子阳性菌株对氨基糖苷类、喹诺酮类及大多数B一内酰胺类的耐药率高于整合子阴性的菌株。结论I类整合子在肺炎克雷伯菌临床分离株存在较广,含有I类整合子的肺炎克雷伯菌更易获得耐药性。     

Examination of host defense factors responsible for experimental chronic respiratory tract infection caused by Klebsiella pneumoniae in mice.

We have studied the host defense factors that operate during the course of chronic respiratory tract infection caused by Klebsiella pneumoniae 27 in CBA/J mice. A large number of polymorphonuclear leukocytes (PMNs) rapidly infiltrated the alveolar spaces after infection. Treatment with cyclophosphamide (CY) before infection greatly reduced the infiltration of PMNs and caused an increase in bacterial counts. CY treatment of mice in the chronic phase also caused bacterial proliferation in the lungs. The administration of a high titer immune serum efficiently reduced the bacterial counts in the lungs during the early phase but not during the chronic phase. The proliferation of bacteria induced by CY treatment was not suppressed by the administration of the immune serum in either phase. When the mice were exposed to an aerosol containing Pseudomonas aeruginosa P9 in the chronic phase, the organisms from the secondary infection were eliminated from the lungs in the same manner as in the case of primary infection with P. aeruginosa. Thus, PMNs seem to play an important role in the suppression of bacterial proliferation in the early and chronic phases, and the specific antibody might have a supplementary effect on the defensive action of PMNs in the chronic phase. It is also presumed that the bacteria in the chronic phase of infection are sequestered at sites hardly accessible to PMNs.     

肺炎克雷伯菌噬菌体解聚酶的研究进展

肺炎克雷伯菌(Klebsiella pneumoniae)是在临床引起多种感染的常见条件致病菌之一。多重耐药肺炎克雷伯菌株的出现,给防控细菌感染带来了巨大阻力。肺炎克雷伯菌噬菌体编码的解聚酶是一种稳定性高、特异性强的生物酶,具有分解细菌胞外多糖、限制细菌生长等多种功能。解聚酶可为防控肺炎克雷伯菌感染提供新思路,在抗菌应用中具有广阔前景。本文就肺炎克雷伯菌噬菌体解聚酶的研究进展进行综述。     

痰液中产ESBL肺炎克雷伯菌Ⅰ、Ⅱ类整合子检测及基因分型研究

目的研究临床痰液分离的产ESBLs肺炎克雷伯菌Ⅰ、Ⅱ类整合子分布情况,并进行基因分型。方法分离临床痰液中100株产ESBLs的肺炎克雷伯菌,用WHONET 5.4分析菌株药敏情况,PCR检测整合酶Ⅰ、整合酶Ⅱ,ERIC-PCR进行基因分型。结果 100株菌对碳青霉烯类敏感率100%,对β-内酰胺类、氨基糖苷类、氟喹诺酮类多数耐药。整合酶Ⅰ检出率为60%,未检出整合酶Ⅱ。100株菌分为72个基因型。结论Ⅰ类整合子广泛存在于产ESBLS肺炎克雷伯菌中,与肺炎克雷伯菌的耐药相关,ERIC-PCR可用于临床分离肺炎克雷伯的基因分型。     

Chromosomal and plasmid encoded drug resistances of a <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> UTI 2 strain isolated from urine of a post-operative patient

The multi drug resistance Klebsiella pneumoniae in urinary tract infection is a common clinical problem in developing country like India. Use of random antibiotics, resulting multi drug resistance development, creates difficulties for treatment. In our present study, we investigated a strain of Klebsiella pneumoniae UTI 2 with multiple drug resistance, which was isolated from urine of a post operative woman patient (50 years) suffering from urinary tract infection with high fever. This strain is resistant to 36 antibiotics and sensitive to cefotaxime (Ce) and imipenem (I). After curing of plasmids, we observed that, 55% of drug resistant loci of K. pneumoniae UTI 2 are chromosomal and 40% are plasmid encoded. The organism is sensitive to 5% of drugs tested, i.e. Ce and I. This study contributes to understand the drug resistance of Klebsiella pneumoniae, which will enable better clinical management of catheter-associated urinary tract infections, a major health problem.     

Submicroscopical changes in Klebsiella pneumoniae cells treated with concentrated sucrose and polyethylene glycol 400 solutions

This study reports the extent and character of plasmolysis and other morphological changes as shown by electron microscopy in a strain of Klebsiella pneumoniae and with sucrose or polyethylene glycol 400 (PEG-400) as the plasmolysing agent at a water activity of 0.935.
Both solutes produced severe plasmolysis in K. pneumoniae cells; PEG-400 also caused some cell wall collapse and fingerlike extrusions to emerge from the bacterial cell.     

Submicroscopical changes in Klebsiella pneumoniae cells treated with concentrated sucrose and polyethylene glycol 400 solutions

This study reports the extent and character of plasmolysis and other morphological changes as shown by electron microscopy in a strain of Klebsiella pneumoniae and with sucrose or polyethylene glycol 400 (PEG-400) as the plasmolysing agent at a water activity of 0.935. Both solutes produced severe plasmolysis in K. pneumoniae cells; PEG-400 also caused some cell wall collapse and finger like extrusions to emerge from the bacterial cell.