Contribution of the Klebsiella pneumoniae capsule to bacterial aggregate and biofilm microstructures

We studied the interaction between capsule production and hydrodynamic growth conditions on the internal and macroscopic structure of biofilms and spontaneously formed aggregates of Klebsiella pneumoniae. Wild-type and capsule-deficient strains were studied as biofilms and under strong and mild hydrodynamic conditions. Internal organization of multicellular structures was determined with a novel image-processing algorithm for feature extraction from high-resolution confocal microscopy. Measures included interbacterial spacing and local angular alignment of individual bacteria. Macroscopic organization was measured via the size distribution of aggregate populations forming under various conditions. Compared with wild-type organisms, unencapsulated mutant organisms formed more organized aggregates with less variability in interbacterial spacing and greater interbacterial angular alignment. Internal aggregate structure was not detectably affected by the severity of hydrodynamic growth conditions. However, hydrodynamic conditions affected both wild-type and mutant aggregate size distributions. Bacteria grown under high-speed shaking conditions (i.e., at Reynolds' numbers beyond the laminar-turbulent transition) formed few multicellular aggregates while clumpy growth was common in bacteria grown under milder conditions. Our results indicate that both capsule and environment contribute to the structure of communities of K. pneumoniae, with capsule exerting influence at an interbacterial length scale and fluid dynamic forces affecting overall particle size.     

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.     

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.     

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.     

Identification of Klebsiella pneumoniae virulence determinants using an intranasal infection model

Klebsiella pneumoniae is a Gram-negative enterobacterium that has historically been, and currently remains, a significant cause of human disease. It is a frequent cause of urinary tract infections and pneumonia, and subsequent systemic infections can have mortality rates as high as 60%. Despite its clinical significance, few virulence factors of K. pneumoniae have been identified or characterized. In this study we present a mouse model of acute K. pneumoniae respiratory infection using an intranasal inoculation method, and examine the progression of both pulmonary and systemic disease. Wild-type infection recapitulates many aspects of clinical disease, including significant bacterial growth in both the trachea and lungs, an inflammatory immune response characterized by dramatic neutrophil influx, and a steady progression to systemic disease with ensuing mortality. These observations are contrasted with an infection by an isogenic capsule-deficient strain that shows an inability to cause disease in either pulmonary or systemic tissues. The consistency and clinical accuracy of the intranasal mouse model proved to be a useful tool as we conducted a genetic screen to identify novel virulence factors of K. pneumoniae. A total of 4800 independent insertional mutants were evaluated using a signature-tagged mutagenesis protocol. A total of 106 independent mutants failed to be recovered from either the lungs or spleens of infected mice. Small scale independent infections proved to be helpful as a secondary screening method, as opposed to the more traditional competitive index assay. Those mutants showing verified attenuation contained insertions in loci with a variety of putative functions, including a large number of hypothetical open reading frames. Subsequent experiments support the premise that the central mechanism of K. pneumoniae pathogenesis is the production of a polysaccharide-rich cell surface that provides protection from the inflammatory response.     

Infectivity of hepatic strain Klebsiella pneumoniae in diabetic mice

Besides urinary tract infection (UTI) and pneumonia, increased severe liver abscesses caused by Klebsiella pneumoniae (KP), especially in diabetic patients, have been observed in infections acquired in hospitals. This indicates that different KP strains with higher virulence have emerged in recent years. Our goal was to investigate the infectivity of KP isolates in mice from liver abscess or UTI patients. Mice were injected with streptozotocin to induce diabetes. Male ICR mice were infected with KpU1 (UTI strain CG3 for survival experiment only) and KpL1 (liver abscess strain CG5) by tail-vein injection of 5 x 10(4) colony-forming units (CFU) bacterial suspension. The mice survival rates, cytokine level by enzyme-linked immunosorbent assay (ELISA), and bacterial presence in liver tissue by Giemsa stain were examined. The survival rates for the KpL1-infected animals were 28% and 0% in normal and diabetic groups, respectively, whereas, for the KpU1-infected mice, the rates were 100% and 75% during a 30-day observation. Nonsurviving KpL1-infected mice showed > 10(5) bacteria/ml blood and the bacteria appeared in the liver sinus area and inside liver cells. The KpL1-infected mice showed a tendency to increase the blood interleukin 1beta (IL-1beta) level in both nondiabetic and diabetic groups, whereas the tumor necrosis factor-alpha (TNF-alpha) level was significantly decreased in the KpL1-infected diabetic mice (P = 0.002). In conclusion, the KP strain from liver abscess showed a greater virulence in mice than the KP from UTI and was more virulent in diabetic than in nondiabetic mice. The infection with KP from liver abscess significantly decreased the blood TNF-alpha level in diabetes mellitus (DM) mice and the blood IL-1beta level tended to increase in both infected nondiabetic and diabetic groups. High blood bacterial count and appearance of bacteria in liver sinus and cells usually contribute to death of the animals.     

Lipopolysaccharide O1 antigen contributes to the virulence in Klebsiella pneumoniae causing pyogenic liver abscess

Klebsiella pneumoniae is the common cause of a global emerging infectious disease, community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) and lipopolysaccharide (LPS) are critical for this microorganism's ability to spread through the blood and to cause sepsis. While CPS type K1 is an important virulence factor in K. pneumoniae causing PLA, the role of LPS in PLA is not clear. Here, we characterize the role of LPS O antigen in the pathogenesis of K. pneumoniae causing PLA. NTUH-K2044 is a LPS O1 clinical strain; the presence of the O antigen was shown via the presence of 1,3-galactan in the LPS, and of sequences that align with the wb gene cluster, known to produce O-antigen. Serologic analysis of K. pneumoniae clinical isolates demonstrated that the O1 serotype was more prevalent in PLA strains than that in non-tissue-invasive strains (38/42 vs. 9/32, P<0.0001). O1 serotype isolates had a higher frequency of serum resistance, and mutation of the O1 antigen changed serum resistance in K. pneumoniae. A PLA-causing strain of CPS capsular type K2 and LPS serotype O1 (i.e., O1:K2 PLA strain) deleted for the O1 synthesizing genes was profoundly attenuated in virulence, as demonstrated in separate mouse models of septicemia and liver abscess. Immunization of mice with the K2044 magA-mutant (K(1) (-) O(1)) against LPS O1 provided protection against infection with an O1:K2 PLA strain, but not against infection with an O1:K1 PLA strain. Our findings indicate that the O1 antigen of PLA-associated K. pneumoniae contributes to virulence by conveying resistance to serum killing, promoting bacterial dissemination to and colonization of internal organs after the onset of bacteremia, and could be a useful vaccine candidate against infection by an O1:K2 PLA strain.     

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。     

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。     

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.     

Fine structures of the capsules of Klebsiella pneumoniae and Escherichia coli K1.

The fine structures of the capsules of Klebsiella pneumoniae and Escherichia coli were determined by the rapid-freezing technique. The capsular layer was seen as a densely packed accumulation of fine fibers. The thickness of the capsule was approximately 160 nm in K. pneumoniae and less than 10 nm in E. coli K1. Two layers were observed in the Klebsiella capsule in which the arrangements of the fibers were different. The inner layer of the capsule was formed by a palisade of thick and dense bundles of the fibers standing at right angles on the surface of the outer membrane. In the outer layer these thick bundles of fibers loosened into fine fibers which spread over the bacterial surface, forming a fine network structure.     

Complete genome sequence of the N2-fixing broad host range endophyte Klebsiella pneumoniae 342 and virulence predictions verified in mice

We report here the sequencing and analysis of the genome of the nitrogen-fixing endophyte, Klebsiella pneumoniae 342. Although K. pneumoniae 342 is a member of the enteric bacteria, it serves as a model for studies of endophytic, plant-bacterial associations due to its efficient colonization of plant tissues (including maize and wheat, two of the most important crops in the world), while maintaining a mutualistic relationship that encompasses supplying organic nitrogen to the host plant. Genomic analysis examined K. pneumoniae 342 for the presence of previously identified genes from other bacteria involved in colonization of, or growth in, plants. From this set, approximately one-third were identified in K. pneumoniae 342, suggesting additional factors most likely contribute to its endophytic lifestyle. Comparative genome analyses were used to provide new insights into this question. Results included the identification of metabolic pathways and other features devoted to processing plant-derived cellulosic and aromatic compounds, and a robust complement of transport genes (15.4%), one of the highest percentages in bacterial genomes sequenced. Although virulence and antibiotic resistance genes were predicted, experiments conducted using mouse models showed pathogenicity to be attenuated in this strain. Comparative genomic analyses with the presumed human pathogen K. pneumoniae MGH78578 revealed that MGH78578 apparently cannot fix nitrogen, and the distribution of genes essential to surface attachment, secretion, transport, and regulation and signaling varied between each genome, which may indicate critical divergences between the strains that influence their preferred host ranges and lifestyles (endophytic plant associations for K. pneumoniae 342 and presumably human pathogenesis for MGH78578). Little genome information is available concerning endophytic bacteria. The K. pneumoniae 342 genome will drive new research into this less-understood, but important category of bacterial-plant host relationships, which could ultimately enhance growth and nutrition of important agricultural crops and development of plant-derived products and biofuels.     

Effect of capsular polysaccharide of Klebsiella pneumoniae on host resistance to bacterial infections. I. Induction of increased susceptibility to infections in mice.

When Klebsiella pneumoniae capsular polysaccharide (CPS-K) from type 1, Kasuya strain, was injected intraperitoneally (i.p.) immediately before i.p. bacterial challenge, the survival time of mice infected with Salmonella enteritidis NUB 1 (virulent strain) was shortened and the mortality rate for mice infected with S. enteritidis NUB 31 (avirulent strain) was enhanced. The promotion of infection with S. enteritidis NUB 1 by CPS-K depended upon its dose, the effect of CPS-K being demonstrable up to as little as 0.2 mug per mouse. In the case of S. enteritidis NUB 31, the effect of CPS-K was detectable only when more than 20 mug per mouse was injected. As a result of enumeration of bacterial populations in the peritoneal washing, blood, liver and spleen, it was revealed that CPS-K promoted in vivo growth of S. enteritidis NUB 1 and NUB 31. In addition, CPS-K enhanced the mortality rate in mice infected with Streptococcus pyogenes or Streptococcus pneumoniae. The peak CPS-K effect on infection with S. enteritidis NUB 1 was seen when given immediately before bacterial challenge. The active substance responsible for the infection-promoting effect of CPS-K was neutral CPS-K, which is distinct from the O antigen and from acidic CPS-K (the type-specific capsular antigen). Preparations of neutral CPS-K isolated from the other three strains of K. pneumoniae exhibited a marked infection-promoting effect comparable with that of preparations from the Kasuya strain. Neutral CPS-K, with identical antigenicity to that from the Kasuya strain, has already been found to exert a strong adjuvant effect on antibody responses to various antigens in mice. No parallelism exists between infection-promoting activity and adjuvant activity of neutral CPS-K.     

肺炎克雷伯菌研究进展

近年来由于各种抗菌药物的广泛使用,导致肺炎克雷伯菌多重耐药现象普遍存在,给临床治疗带来极大的困扰,造成医院获得性肺炎感染严重的现状。着重论述了肺炎克雷伯菌流行现状、耐药机制、致病因子及防治措施。     

The Klebsiella pneumoniae wabG gene: role in biosynthesis of the core lipopolysaccharide and virulence

To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LPS) of Klebsiella pneumoniae, we constructed wabG nonpolar mutants. Data obtained from the comparative chemical and structural analysis of LPS samples obtained from the wild type, the mutant strain, and the complemented mutant demonstrated that the wabG gene is involved in attachment to alpha-L-glycero-D-manno-heptopyranose II (L,D-HeppII) at the O-3 position of an alpha-D-galactopyranosyluronic acid (alpha-D-GalAp) residue. K. pneumoniae nonpolar wabG mutants were devoid of the cell-attached capsular polysaccharide but were still able to produce capsular polysaccharide. Similar results were obtained with K. pneumoniae nonpolar waaC and waaF mutants, which produce shorter LPS core molecules than do wabG mutants. Other outer core K. pneumoniae nonpolar mutants in the waa gene cluster were encapsulated. K. pneumoniae waaC, waaF, and wabG mutants were avirulent when tested in different animal models. Furthermore, these mutants were more sensitive to some hydrophobic compounds than the wild-type strains. All these characteristics were rescued by reintroduction of the waaC, waaF, and wabG genes from K. pneumoniae.     

Apolipoprotein E knock-out mice are highly susceptible to endotoxemia and Klebsiella pneumoniae infection

Lipoproteins are able to neutralize bacterial lipopolysaccharide (LPS) and thereby inhibit the proinflammatory cytokine response. In a previous study, we demonstrated that hypercholesterolemic low density lipoprotein receptor knock-out (LDLr-/-) mice are protected against lethal endotoxemia and gram-negative infection. In the present study we investigated the susceptibility of apolipoprotein E knock-out mice (apoE-/-) to LPS and to Klebsiella pneumoniae. These mice have increased plasma lipoprotein concentrations in the very low density lipoprotein (VLDL)-sized fraction. Despite 8 -fold higher plasma cholesterol levels compared to controls, and in contrast to LDLr-/- mice, apoE-/- mice were significantly more susceptible to endotoxemia and to K. pneumoniae infection. Circulating TNFalpha concentrations after intravenously injected LPS were 4 - to 5-fold higher in apoE-/- mice, whereas IL-1alpha, IL-1beta, and IL-6 did not differ. This TNF response was not due to an increased cytokine production capacity of cells from apoE-/- mice, as ex vivo cytokine production in response to LPS did not differ between apoE-/- and control mice. The LPS-neutralizing capacity of apoE-/- plasma was significantly less than that of controls. Most likely, the absence of apoE itself in the knock-out mice explains the failure to neutralize LPS, despite the very high cholesterol concentrations.