SitA contributes to the virulence of Klebsiella pneumoniae in a mouse infection model

Klebsiella pneumoniae is an opportunistic pathogen, which causes a wide range of nosocomial infections. Recently, antibiotic resistance makes K. pneumoniae infection difficult to deal with. Investigation on virulence determinants of K. pneumoniae can provide more information about pathogenesis and unveil new targets for treatment or vaccine development. In this study, SitA, a Fur-regulated divalent cation transporter, was found significantly increased when K. pneumoniae was cultured in a nutrient-limited condition. A sitA-deletion strain (ΔsitA) was created to characterize the importance of SitA in virulence. ΔsitA showed higher sensitivity toward hydroperoxide than its parental strain. In a mouse intraperitoneal infection model, the survival rate of mice infected with ΔsitA strain increased greatly when compared with that of mice infected with the parental strain, suggesting that sitA deletion attenuates the bacterial virulence in vivo. To test whether ΔsitA strain is a potential vaccine candidate, mice were immunized with inactivated bacteria and then challenged with the wild-type strain. The results showed that using ΔsitA mutant protected mice better than using the wild-type strain or the capsule-negative congenic bacteria. In summary, SitA was found being important for the growth of K. pneumoniae in vivo and deleting sitA might be a potential approach to generate vaccines against K. pneumoniae.     

血流感染产ESBLs肺炎克雷伯菌毒力基因及分子分型研究

目的 研究血流感染产ESBLs肺炎克雷伯菌的毒力基因和基因分型特点。方法 采用PCR检测菌株中高毒力因子、荚膜血清型以及ST分型;采用微量肉汤稀释法对菌株进行药敏试验;采用加克拉维酸的复合药（头孢他啶/克拉维酸或头孢噻肟/克拉维酸）与单药（头孢噻肟或头孢他啶）的药敏纸片组合进行肺炎克雷伯菌产ESBLs的表型确证试验。结果 128株血流感染肺炎克雷伯菌中,有23株产ESBLs（产ESBLs组）,占17.97%（23/128）;105株不产ESBLs（非产ESBLs组）,占82.03%（105/125）。本地区血流感染肺炎克雷伯菌主要流行ST型别为ST23、ST65、ST37和ST29,其中ST23、ST29、ST65为非产ESBLs的优势ST型别菌株,而在产ESBLs菌株中无优势型别。两组菌在高黏液表型、荚膜血清型和毒力基因分布上差异均无统计学意义（P>0.05）。产EBSLs组中发现8株高毒力产EBSLs肺炎克雷伯菌。结论 临床诊疗中需在肺炎克雷伯菌耐药株中识别出高毒力肺炎克雷伯菌并给与及时的治疗,避免其并发症的发生。     

Protease activity of Klebsiella pneumoniae of different virulence

The study of substrate specificity and activity of proteolytic enzymes secreted by K. pneumoniae strains with different virulence was carried out. The strains were cultivated in a liquid semi-synthetic medium. The biomass was inactivated, and the supernatant fluid was separated from microbial cells by centrifuging. In the supernatant thus obtained and in the fractions isolated by gel filtration with the subsequent purification on DEAE Sepharose elastase-like, trypsin-like and chemotrypsin-like proteolytic activity was determined. In K. pneumoniae strains with different virulence only a single proteolytic enzyme--elastase with a mol. wt. of 21 kD--was detected. The protease activity of the supernatant culture fluid did not depend on the virulence of the strain and was equal to 5,416-7,476 I.U./ml. The activity of the purified enzyme was 100% of the elastase-like activity of the supernatant culture fluid. The most virulent K. pneumoniae strain K2, whose LD50 for white mice was less than 10 microbial cells, was characterized by lower elastase-like activity. The absence of correlation between protease activity and K. pneumoniae virulence may be explained by the fact that surface glycoproteins of eukaryotic cells are glycosilated and thus slightly accessible for proteases.     

肺炎克雷伯氏菌特异性抗原的筛选与鉴定

目的寻找肺炎克雷伯氏菌（Klebsiella pneumoniae）血清学检测用特异性抗原。方法双向电泳分离K.pneumoniae总蛋白,通过免疫印迹（Western blotting）与常见病原菌的多抗反应筛选特异性抗原蛋白,原核表达该蛋白并用ELISA法验证。结果获得K.pneumoniae的双向电泳图谱。寻找Western blotting中与K.pneumoniae自身多抗反应而不与与其它病原菌多抗反应的蛋白,质谱鉴定为酸性磷酸酶（Acid phosphatase,GI：238894261）。经表达纯化并以酶联免疫吸附试验（ELISA）验证,证明该蛋白作为包被抗原的灵敏度高,特异性强。结论 K.pneumoniae酸性磷酸酶是适用于该菌感染检测特异性抗原蛋白。     

肺炎克雷伯菌血流感染的临床回顾性分析

目的 探讨肺炎克雷伯菌血流感染的临床特点、治疗方案和预后。方法 对诸暨市人民医院2014年3月-2015年9月诊断为肺炎克雷伯菌血流感染的住院患者临床资料进行回顾性分析。结果 66例肺炎克雷伯菌血流感染患者平均年龄64.6岁,检出前平均住院5.7 d。13例（19.7%）合并有其他细菌感染,49例（74.2%）合并其他系统感染。肺炎克雷伯菌血流感染多见于糖尿病（33.3%）、实体肿瘤（30.3%）等免疫力低下疾病,最常见于肝胆外科（24.2%）、内分泌科（16.7%）等科室。患者预后好转47例（71.2%）,APACHEⅡ评分为13.78±4.33;死亡19例（28.8%）,APACHEⅡ评分为21.10±7.45,两组评分差异有统计学意义（P<0.05）。合并其他感染状况与合并神经系统疾病状况病死率更高（P<0.05）。结论 肺炎克雷伯菌血流感染多见于糖尿病、实体肿瘤等免疫力低下的危重疾病患者,多发生在医院内环境的外科手术操作的科室,容易合并其他系统感染。APACHEⅡ评分可作为判断肺炎克雷伯菌血流感染预后的重要指标。控制其他感染情况可有助于临床防控肺炎克雷伯菌血流感染,改善其预后。     

Application of S-thanatin,an antimicrobial peptide derived from thanatin,in mouse model of Klebsiella pneumoniae infection

Thanatin was first discovered from the hemipteran insect Podisus maculiventris and showed a promising antimicrobial activity. Multidrug-resistant (MDR) clinical isolates of Klebsiella pneumoniae have developed resistance to current therapies. As an attempt to resolve this problem, the efficacy of thanatin and its analogues against clinical isolates of K. pneumoniae was studied in vitro and in vivo. S-thanatin showed an improved antimicrobial activity with the tested MIC values was 2–8-fold lower than those of other thanatin analogs. Antimicrobial assay indicated a high activity of S-thanatin against K. pneumoniae in vitro with MIC between 4 and 8 μg/ml. Its in vivo activity was evaluated using a K. pneumoniae-infected mice model. Adult male ICR mice were randomly grouped and given an intraperitoneal (i.p.) administration of 2 × 10¹⁰ colony-forming units of K. pneumoniae (CI 120204205). Afterwards, mouse groups were subjected to i.p. administration of saline or S-thanatin (5, 10, or 15 mg/kg). After an inspection of 72 h, the mice were finally sacrificed for analysis of in vivo bacterial growth and plasma endotoxin level. The results showed that S-thanatin administration apparently improved the survival rate and reduced the bacterial CFU from intra-abdominal fluid in mice. The plasma endotoxin level was improved as well. All above implied that S-thanatin, as an alternative, may provide a novel strategy for treating K. pneumoniae infection and other infections due to multidrug-resistant bacteria.     

洋葱伯克氏菌基因型的鉴定及其在苜蓿模型上的毒力分析

[目的]证实来自我国农业和医院环境中部分洋葱伯克氏菌的基因型并通过苜蓿植物模型探测不同基因型对人体的可能毒力.[方法]采用洋葱伯克氏菌基因型的PCR特异性扩增技术对来源于根围、土壤和医院中的57株洋葱伯克氏菌进行了基因型的鉴定,并利用苜蓿植物模型对这些基因型菌株进行了毒力探测.[结果]获得4种不同的基因型,包括基因型Ⅰ、ⅢA、ⅢB、Ⅴ和Ⅸ.来源于医院的基因型Ⅰ和ⅢA菌株以及根围的基因型ⅢB菌株均对苜蓿幼苗有较强的毒力,其对苜蓿幼苗的平均发病率分别达到69%、68%和55%,与农田环境中基因型Ⅴ和Ⅸ对苜蓿幼苗的发病率相比,表现出显著的差异性.[结论]农田环境中洋葱伯克菌的基因型在苜蓿模型上的毒力差异大,根围的基因型ⅢB菌株对苜蓿幼苗具有强毒力,其毒力程度接近于医院致病基因型ⅢA菌.     

Extracellular polysaccharide production by Klebsiella pneumoniae and its relationship to virulence

Klebsiella pneumoniae serotype 1 and serotype 2 and their capsular variants were examined for production of cell-associated capsular polysaccharides and extracellular capsular polysaccharides. The virulence of these organisms in experimental animals was examined via intraperitoneal injection in mice and transtracheal inoculation into the lungs of rats. It was found that the production of either polysaccharide component correlated with the observed virulence. The extracellular polysaccharides were purified by ethanol precipitation, electrodialysis, extraction with quaternary ammonium salts, and gel filtration. These purification steps allowed for the separation and purification of both the extracellular lipopolysaccharide and the extracellular capsular polysaccharide. Purified extracellular capsular polysaccharide and extracellular lipopolysaccharide were co-injected with K. pneumoniae intraperitoneally into mice to determine if either of these substances would produce an effect on the natural course of infection in these animals. These studies showed that only purified extracellular lipopolysaccharide enhanced the virulence of K. pneumoniae when co-injected into mice, and this virulence enhancement correlated with the content of extracellular lipopolysaccharide, but not extracellular capsular polysaccharide in mixtures of these polysaccharides. Saponification of K. pneumoniae serotype 1 extracellular polysaccharides significantly decreased their virulence-enhancing capabilities in mice, further suggesting that extracellular lipopolysaccharide may play a role in these infections.     

The efficacy of Klebsiella pneumoniae bacteriophage in the therapy of experimental Klebsiella infection

The effectiveness of specific phage therapy was studied on Klebsiella experimental sepsis in noninbred white mice, caused by the intraperitoneal injection of K. pneumoniae highly virulent strain K2 5055 into the animals. For treatment, Klebsiella polyvalent bacteriophage administered on day 2 after the infection of the animals with Klebsiella was used. The study revealed that bacteriophage could be detected in the blood and internal organs of the animals within 24 hours irrespective of the route of its administration: intraperitoneal, intravenous or intranasal. The bacteriophage preparation, introduced intraperitoneally, was shown to be effective in the treatment of generalized Klebsiella infection. One daily intraperitoneal injection of Klebsiella bacteriophage for 15-20 days proved to be the optimum scheme of treatment. In contrast to chemotherapeutic preparations, bacteriophages had no effect on normal microflora and did not aggravate dysbiotic disturbances. For this reason, bacteriophages may become one of alternative antimicrobial remedies, selectively affecting infective agents.     

Contribution of fucose-containing capsules in Klebsiella pneumoniae to bacterial virulence in mice

Bacterium Klebsiella pneumoniae (KP) contains a prominent capsule. Clinical infections usually are associated with pneumonia or urinary tract infection (UTI). Emerging evidence implicates KP in severe liver abscess especially in diabetic patients. The goal of this study was to investigate the capsular polysaccharides from KP of liver abscess (hepatic-KP) and of UTI-KP. The composition of capsular polysaccharides was analyzed by capillary high-performance liquid chromatography (HPLC, Dionex system). The terminal sugars were assayed by binding ability to lectins. The results showed that the capsule of a hepatic KP (KpL1) from a diabetic patient contained fucose, while the capsule from UTI-KP (KpU1) did not. The absence of fucose was verified by the absence of detectable polymerase chain reaction (PCR) fragment for fucose synthesis genes, gmd and wcaG in KpU1. Mice infected with the KpL1 showed high fatality, whereas those infected with the KpU1 showed high survival rate. The KpL1 capsule was reactive to lectins AAA and AAL, which detect fucose, while the KpU1 capsule was reactive to lectin GNA, which detects mannose. Phagocytosis experiment in mouse peritoneal cavity indicated that the peritoneal macrophages could interact with KpU1, while rare association of KpL1 with macrophages was observed. This study revealed that different polysaccharides were displayed on the bacterial capsules of virulent KpL1 as compared with the less virulent KpU1. Interaction of KpU1 with mice peritoneal macrophages was more prominent than that of KpL1. The possession of fucose might contribute to KpL1 virulence by avoiding phagocytosis since fucose on bacteria had been implicated in immune evasion.     

Pilot-scale production of 1,3-propanediol using Klebsiella pneumoniae

The conversion of glycerol to 1,3-propanediol (PDO) using Klebsiella pneumoniae M5al under anaerobic condition was scaled up from scale 5 to 5000 l in series. A simple strategy for scale-up was to transfer the optimized conditions of a lab scale bioreactor to pilot-scale fermentation. Multistage inocula were developed and their fermentation abilities were assessed in a small-scale fermenter. The experimental results showed that inoculum development in the early steps of a scale-up process could influence the outcomes of a large scale fermentation. Through three-stage liquid inoculum development and a pulse addition of (NH₄)₂SO₄ and yeast extract at 30 h of fermentation, the best results in a 5000 l fermentation were achieved leading to 58.8 g l⁻¹ 1,3-propanediol with a yield of 0.53 mol mol⁻¹ glycerol and productivity of 0.92 g l⁻¹ h⁻¹. This is the first report on pilot-scale 1,3-propanediol production using K. pneumoniae.     

Modification of susceptibility to Klebsiella pneumoniae during murine cytomegalovirus infection

The effect of murine cytomegalovirus (MCMV) infection on susceptibility to bacterial infection was studied in mice by a combination of intraperitoneal (ip) inoculation of a sublethal dose of MCMV with subsequent ip challenge of 2 X 10(3) cfu of a strain of Klebsiella pneumoniae (KP). When given alone, KP produced a mortality of 30-40%. Mortality was increased when KP was given 1 to 7 days after MCMV injection with the peak increase at the 4th to 5th day when 100% mortality occurred. Virus levels in various organs of mice infected with MCMV alone, or superinfected with KP did not differ. Bacterial counts on the other hand, showed that increased mortality in mixed MCMV and KP infected mice was due to an uncontrolled growth of bacteria at the site of primary lodgment, i.e., the peritoneum, and severe systemic infection. Neutrophil response to growth of KP during the first 3 days of bacterial infection was defective in MCMV infected mice. While the initial clearance of KP from the blood was more efficient in MCMV infected mice, probably due to activated reticuloendothelial function, it did not protect the mice against KP infection. Using the in vivo model, it was shown that poor neutrophil response and possibly other defective neutrophil functions were responsible for increased mortality to KP infection in MCMV infected mice.     

Molecular epidemiology of nosocomial infection by extended-spectrum beta-lactamases-producing Klebsiella pneumoniae

Molecular epidemiology applied to the study of nosocomial infection has been fundamental in formulating and evaluating control methods. From patients in a level 3 Bogota hospital, Klebsiella pneumoniae samples were isolated that produced extended-spectrum beta-lactamases (ESBL). Each of 15 isolates was characterized microbiologically and by molecular characters realized by pulsed field gel electrophoresis (PFGE) and by repetitive-DNA sequences amplification (REP-PCR). Antimicrobial susceptibility and ESBL production was determined in accordance with NCCLS guidelines. The beta-lactamases were evaluated by isoelectric-focusing and PCR. Twelve (80%) of the isolates were associated with nosocomial infection; 11 of them were from intensive care units. The antibiotic susceptibility displayed 13 resistance patterns--87% presented co-resistance to amikacin, 53% to gentamicin, 33% to ciprofloxacin, 40% to cefepime, 67% to piperacillin/tazobactam, 60% to trimethoprim/sulfamethoxazole and 47% to chloranphenicol. All were sensitive to imipenem. Production of TEM and SHV beta-lactamases was detected simultaneously in most isolates by isoelectric focusing and 93.3% produced a ceftazidimase of pl 8.2 of the SHV-5 type. The 15 isolates were grouped into 11 and 12 electrophoretic patterns by PFGE and REP-PCR, respectively. The degree of genetic variability indicated an endogenous origin of the nosocomial infections.     

Crystallization of an R-Form Lipopolysaccharide from Klebsiella pneumoniae

An R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3^–:K1^–) formed crystals, whose shapes were elongated hexagonal plates, trapezoid plates, and rhomboid plates, and whose greatest dimensions were 3.1 × 0.8 μm, when it was suspended in 50 mM Tris buffer at pH 8.5 containing 5 mM MgCl₂ and kept at 4 C for as long as 870 days. K. pneumoniae LEN-111 synthesized LPS molecules possessing incomplete repeating units of the O-antigenic polysaccharide portion besides the R-form LPS because of a leaky characteristic, but crystals consisted exclusively of the R-form LPS. Although the size of crystals was not large enough for X-ray analysis and limited crystallographic information was available, it was suggested that the crystals consist of hexagonal lattices with an a axis of 4.62 Å and c axis of 79.8 ±2.6 Å. The present results showed that R-form LPS lacking the O-antigenic polysaccharide portion tends to form crystals during long-term incubation in Tris buffer at pH 8.5 containing MgCl₂ at 4 C.     

Experimental chronic pulmonary infection in mice caused by Klebsiella pneumoniae

Examination of mouse strain differences in susceptibility to experimental respiratory tract infection with Klebsiella pneumoniae 27 revealed that a chronic pulmonary infection model could be established using CBA/J mice. After 6 X 10(5) colony-forming units of K. pneumoniae 27 were inoculated into the lung, the bacterial counts in the lungs changed with time showing four different phases: initial decrease, regrowth, steady-state, and final increase leading to death. Throughout the course of the infection, the challenge bacteria were isolated mainly from the respiratory organs. Pulmonary gross lesions appeared on day 2 after infection and persisted thereafter. Lobar consolidation was the primary lesion and occurred mainly in the anterior and middle lobes of the right lung, and the median lobe. Mice began to die from 4 weeks after aerosol exposure. This model may be useful for investigating the pathogenesis of chronic pulmonary infection by Klebsiella and its therapy.     

Identification of vaccine candidate antigens of an ESBL producing Klebsiella pneumoniae clinical strain by immunoproteome analysis

Klebsiella pneumoniae is an opportunistic pathogen which causes pneumoniae, urinary tract infections and septicemia in immunocompromised patients. Hospital outbreaks of multidrug-resistant K. pneumoniae, especially those in neonatal wards, are often caused by strains producing the extended-spectrum-beta-lactamases (ESBLs). An immunoproteome based approach was developed to identify candidate antigens of K. pneumoniae for vaccine development. Sera from patients with acute K. pneumoniae infections (n = 55) and a control group of sera from healthy individuals (n = 15) were analyzed for reactivity by Western blot against ESBL K. pneumoniae outer membrane proteins separated by 2-DE. Twenty highly immunogenic protein spots were identified by immunoproteomic analysis. The immunogenic proteins that are most frequently recognized by positive K. pneumoniae sera were OmpA, OmpK36, FepA, OmpK17, OmpW, Colicin I receptor protein and three novel proteins. Two of the vaccine candidate genes, OmpA (Struve et al. Microbiology 2003, 149, 167-176) and FepA (Lai, Y. C. et al.. Infect Immun 2001, 69, 7140-7145), have recently been shown to be essential in colonization and infection in an in vivo mouse model. Hence, these two immunogenic proteins could serve as potential vaccine candidates.     

Strain-specific virulence phenotypes of Streptococcus pneumoniae assessed using the Chinchilla laniger model of otitis media

Background

Streptococcus pneumoniae [Sp] infection is associated with local and systemic disease. Our current understanding of the differential contributions of genetic strain variation, serotype, and host response to disease phenotype is incomplete. Using the chinchilla model of otitis media [OM] we investigated the disease phenotype generated by the laboratory strain TIGR4 and each of thirteen clinical strains (BS68-75, BS290, BS291, BS293, BS436 and BS437); eleven of the thirteen strains have been genomically sequenced.

Methodology/Principal Findings

For each strain 100 colony forming units were injected bilaterally into the tympanic bullae of 6 young adult chinchillas under general anesthesia. All animals were examined daily for local and systemic disease by a blinded observer. Pneumatic otoscopy was used to evaluate local disease, and behavioral assessments served as the measure of systemic disease. Virulence scoring was performed using a 4-point scale to assess four clinical parameters [severity and rapidity of local disease onset; and severity and rapidity of systemic disease onset] during a 10-day evaluation period. Highly significant variation was observed among the strains in their ability to cause disease and moribundity.

Conclusions/Significance

As expected, there was a significant correlation between the rapidity of systemic disease onset and severity of systemic disease; however, there was little correlation between the severity of otoscopic changes and severity of systemic disease. Importantly, it was observed that different strains of the same serotype produced as broad an array of disease phenotypes as did strains of different serotypes. We attribute these phenotypic differences among the strains to the high degree of genomic plasticity that we have previously documented.