采用RAPD分析臭鼻克雷伯菌临床菌株的基因型

臭鼻克雷伯菌是重要的条件致病菌 ,常引起老人及婴幼儿重症护理病房的交叉感染及爆发流行 ,对其进行流行病学监测有重要的医学意义。用随机引物聚合酶链式反应 (RAPD)对 37株臭鼻克雷伯菌临床菌株基因组DNA进行分型 ,分析扩增产物的指纹图谱。实验结果表明 ,37株臭鼻克雷伯菌RAPD后可出现不同的指纹图谱 ,根据特异泳带C ,J ,F ,Q的有无 ,可将其分 7型 (I～VII)。RAPD可用于检测臭鼻克雷伯菌基因组DNA的异质性 ,是医院内感染分子流行病学研究的好方法。     

Metabolism of the herbicide bromoxynil by Klebsiella pneumoniae subsp. ozaenae

Enrichment of soil samples for organisms able to utilize the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) as a nitrogen source yielded bacterial isolates capable of rapidly metabolizing this compound. One isolate, identified as Klebsiella pneumoniae subsp. ozaenae, could completely convert 0.05% bromoxynil to 3,5-dibromo-4-hydroxybenzoic acid and use the liberated ammonia as a sole nitrogen source. Assays of cell extracts of this organism for the ability to produce ammonia from bromoxynil revealed the presence of a nitrilase (EC 3.5.51) activity. The enzyme could not utilize 3,5-dibromo-4-hydroxybenzamide as a substrate, and no 3,5-dibromo-4-hydroxybenzamide could be detected as a product of bromoxynil transformation. Comparison of related aromatic nitriles as substrates demonstrated that the Klebsiella enzyme is highly specific for bromoxynil.     

Metabolism of the herbicide bromoxynil by Klebsiella pneumoniae subsp. ozaenae.

Enrichment of soil samples for organisms able to utilize the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) as a nitrogen source yielded bacterial isolates capable of rapidly metabolizing this compound. One isolate, identified as Klebsiella pneumoniae subsp. ozaenae, could completely convert 0.05% bromoxynil to 3,5-dibromo-4-hydroxybenzoic acid and use the liberated ammonia as a sole nitrogen source. Assays of cell extracts of this organism for the ability to produce ammonia from bromoxynil revealed the presence of a nitrilase (EC 3.5.51) activity. The enzyme could not utilize 3,5-dibromo-4-hydroxybenzamide as a substrate, and no 3,5-dibromo-4-hydroxybenzamide could be detected as a product of bromoxynil transformation. Comparison of related aromatic nitriles as substrates demonstrated that the Klebsiella enzyme is highly specific for bromoxynil.     

Draft Genome Sequence of Klebsiella pneumoniae subsp. pneumoniae DSM 30104T

Klebsiella pneumoniae is a Gram-negative, rod-shaped, nonmotile, and opportunistic pathogenic species with clinical importance. It is a part of natural flora of humans and animals. Here we report the draft genome sequence of the type strain of Klebsiella pneumoniae subsp. pneumoniae (DSM 30104^T) to provide taxonomic and functional insights into the species.     

Complete genome sequence of Klebsiella pneumoniae subsp. pneumoniae HS11286, a multidrug-resistant strain isolated from human sputum

Klebsiella pneumoniae is an important pathogen commonly associated with opportunistic infections. Here we report the genome sequence of a strain, HS11286, isolated from human sputum in 2011 in Shanghai, China. It contains one chromosome (5.3 Mb), three multidrug resistance plasmids (～110 kb), including a carbapenemase producer, and three small plasmids (～3 kb).     

Immunoprotective potential of polysaccharide-tetanus toxoid conjugate in Klebsiella pneumoniae induced lobar pneumonia in rats

The polysaccharide (PS) derived from K. pneumoniae NCTC 5055 lipopolysaccharide (LPS) was covalently linked to tetanus toxoid by using carbodimide with adipic acid dihydrazide as a spacer molecule. The conjugate was found to be non-toxic and non-pyrogenic at 100 microg dose level. At a similar dose, the conjugate did not elicit any local skin reaction on intradermal preparatory injection in rabbits. The conjugate was immunoprotective as was evident from the decrease in relative colonization of bacteria in lungs of immunized rats as compared to the control animals. Immunization with the conjugate resulted in alveolar macrophage activation in terms of their ability to phagocytose bacteria in vitro.     

PCR-based identification of Klebsiella pneumoniae subsp. rhinoscleromatis, the agent of rhinoscleroma

Rhinoscleroma is a chronic granulomatous infection of the upper airways caused by the bacterium Klebsiella pneumoniae subsp. rhinoscleromatis. The disease is endemic in tropical and subtropical areas, but its diagnosis remains difficult. As a consequence, and despite available antibiotherapy, some patients evolve advanced stages that can lead to disfiguration, severe respiratory impairment and death by anoxia. Because identification of the etiologic agent is crucial for the definitive diagnosis of the disease, the aim of this study was to develop two simple PCR assays. We took advantage of the fact that all Klebsiella pneumoniae subsp. rhinoscleromatis isolates are (i) of capsular serotype K3; and (ii) belong to a single clone with diagnostic single nucleotide polymorphisms (SNP). The complete sequence of the genomic region comprising the capsular polysaccharide synthesis (cps) gene cluster was determined. Putative functions of the 21 genes identified were consistent with the structure of the K3 antigen. The K3-specific sequence of gene Kr11509 (wzy) was exploited to set up a PCR test, which was positive for 40 K3 strains but negative when assayed on the 76 other Klebsiella capsular types. Further, to discriminate Klebsiella pneumoniae subsp. rhinoscleromatis from other K3 Klebsiella strains, a specific PCR assay was developed based on diagnostic SNPs in the phosphate porin gene phoE. This work provides rapid and simple molecular tools to confirm the diagnostic of rhinoscleroma, which should improve patient care as well as knowledge on the prevalence and epidemiology of rhinoscleroma.     

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.     

Synergistic effect of low-frequency ultrasound and antibiotics on the treatment of Klebsiella pneumoniae pneumonia in mice

The antibiotic-resistant Klebsiella pneumoniae (Kp) has become a significant crisis in treating pneumonia. Low-frequency ultrasound (LFU) is promising to overcome the obstacles. Mice were infected with bioluminescent Kp Xen39 by intratracheal injection to study the therapeutic effect of LFU in combination with antibiotics. The counts per second (CPS) were assessed with an animal biophoton imaging system. Bacterial clearance, histopathology, and the concentrations of cytokines were determined to evaluate the therapeutic effect. LC–MS/MS was used to detect the distribution of antibiotics in the lung and plasma. LFU in combination with meropenem (MEM) or amikacin (AMK) significantly improved the behavioural state of mice. The CPS of the LFU combination group were more significantly decreased compared with those of the antibiotic alone groups. The average colony-forming units of lung tissue in the LFU combination groups were also lower than those of the antibiotic groups. Although no significant changes of cytokines (IL-6 and TNF-α) in plasma and bronchoalveolar lavage fluid were observed, LFU in combination with antibiotics showed less inflammatory damage from histopathological results compared with the antibiotic-alone groups. Moreover, 10 min of LFU treatment promoted the distribution of MEM and AMK in mouse lung tissue at 60 and 30 min, respectively, after dosage. LFU could enhance the effectiveness of MEM and AMK in the treatment of Kp-induced pneumonia, which might be attributed to the fact that LFU could promote the distribution of antibiotics in lung tissue and reduce inflammatory injury.     

自然感染途径建立肺炎链球菌感染性肺炎小鼠模型

目的探索自然感染途径制备肺炎链球菌感染性肺炎小鼠模型的方法,为肺炎链球菌性肺炎的相关研究提供实验基础。方法肺炎链球菌标准菌株(CMCC 31203)经血平板培养18 h后配成2麦氏浊度。小鼠浅麻醉状态下,破损鼻黏膜,滴注一定量肺炎链球菌菌液。于3、7、14和21 d分别处死小鼠,取肺脏进行组织病理学观察,确定肺炎小鼠模型制备的最佳方式。结果鼻黏膜破损组小鼠3 d时肺泡间隔内毛细血管扩张,肺泡腔内以红细胞和纤维素渗出为主;7 d时病变以纤维素和中性粒细胞浸润为主;14 d时肺泡腔内渗出减少,炎症开始减轻;21 d时肺脏外观趋于正常,肺泡腔内渗出物质基本溶解吸收。结论以3×107CFU的肺炎链球菌菌液通过破损的鼻黏膜感染7 d时可以制备出比较典型、稳定的肺炎小鼠模型。     

A comparative study of induction of pneumonia in mice with planktonic and biofilm cells of Klebsiella pneumoniae

In the present study, the course of acute pneumonia in normal BALB/c mice infected by intranasal inoculation of planktonic and preformed biofilm cells (3 days old) of Klebsiella pneumoniae B5055 was studied and compared. With both cell forms the peak of infection was observed on the third post infection day, as assessed on the basis of lung bacterial load and corresponding pathology. There was an intense neutrophil infiltration in bronchoalveolar lavage fluid. Tissue damage was assessed on the basis of increased amounts of nitrite, malondialdehyde and lactate dehydrogenase in lung homogenates. The phagocytic potential of alveolar macrophages was lower in biofilm cell-induced infection than in that induced by planktonic cells. Biofilm cell induced infection generated significantly greater production of tumor necrosis factor-α and interleukin-1β on the third and fifth days of infection, respectively. Production of interleukin-10 was, however, variable. There was no significant difference in the ability of planktonic and biofilm cell forms of K. pneumoniae to induce acute pneumonia in mice in terms of bacterial counts and histopathological changes. However, biofilm cell-induced infection showed delayed clearance as compared to infection induced with the planktonic form.     

Infection of mice by aerosols of Klebsiella pneumoniae under hyperbaric conditions.

Both the physical behavior of aerosols and survival of airborne Serratia marcescens in hyperbaric chambers with a helium-air mixture at 20 atm of pressure was approximately the same as in the system at ambient pressures. Exposure of mice to aerosols of Klebsiella pneumoniae at 1-, 2-, and 17-atm (ca. 101-, 203-, and 1,722-kPa) pressures of helium-oxygen mixture showed that the number of viable organisms constituting a 50% lethal dose was not significantly affected by the hyperbaric conditions.     

Lactobacillus pentosus strain b240 suppresses pneumonia induced by Streptococcus pneumoniae in mice

Aims: Oral administration of probiotics has been known to improve inflammatory responses against infectious diseases. Here, we describe the inhibitory effect of oral intake of heat‐killed Lactobacillus pentosus strain b240 (b240) on pneumococcal pneumonia in a murine experimental model. Method and Results: The mice treated with oral b240 for 21 days before Streptococcus pneumoniae infection exhibited prolonged survival time and less body weight loss, compared with saline‐treated control mice. Mild pneumonia with significantly reduced secretion of inflammatory cytokines/chemokines according to related mitogen‐activated protein kinase signalling molecules (phosphorylated c‐Jun N‐terminal kinase) was found in b240‐treated mice, whereas severe pneumonia with hypercytokinemia was evident in control mice. Prominent reduction in the number of pneumococci and elevated expression of Toll‐like receptor 2 and 4 in the lung tissues was concomitantly noted in b240‐treated mice. Conclusions: These findings indicate that b240 has inhibitory effects on pneumococcal pneumonia induced by Strep. pneumoniae infection and improves inflammatory tissue responses, resulting in reduced damages to the respiratory tissues. Significance and Impact of the Study: These results demonstrate that oral administration of b240 might protect host animals from Strep. pneumoniae infection by augmentation of innate immune 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.     

Extracellular pullulanase of Klebsiella pneumoniae is a lipoprotein.

Pullulanase is a starch-debranching enzyme produced by the gram-negative bacterium Klebsiella pneumoniae. In this organism, the enzyme is first exported to the outer membrane and is subsequently released into the growth medium. Evidence reported here indicates that pullulanase is a lipoprotein. It is apparently synthesized as a precursor with a 19-residue-long signal sequence and modified by the covalent attachment of palmitate to the cysteine residue which becomes the amino terminus after cleavage of the signal sequence. In this respect, pullulanase is similar to some penicillinases produced by gram-positive bacteria which are initially exported to the cell surface and subsequently released into the medium. However, pullulanase and the penicillinases differ in one important aspect, namely, that the extracellular pullulanase still carries the covalently attached fatty acyls, whereas extracellular penicillinases lack the modified amino-terminal cysteine together with a limited number of other residues from the amino terminus.     

Magnetic resonance imaging of Klebsiella pneumoniae-induced pneumonia in mice

In vivo imaging of small animals is a rapidly developing field. However, the potential of global imaging of infectious processes in animal models remains poorly explored. We used magnetic resonance imaging (MRI) to follow the development and regression of inflammatory lesions caused by infection by Klebsiella pneumoniae in mouse lungs. A virulent strain caused an intense inflammation within 2 days in the whole lungs, while an avirulent strain did not show significant changes. Mice infected with the virulent strain and subsequently treated with antibiotics presented a severe inflammation localized mainly in the left lung that disappeared after a week. The lesions observed by MRI correlated with the damage seen by histological analysis and a 3D representation of the tissue allowed better visualization of the development and healing of inflammatory lesions. MRI thus represents a powerful technique to study in vivo the interactions between a pathogen and its host in real time.     

Discovery and investigation of a new, second triose phosphate isomerase in Klebsiella pneumoniae

In this study, a tpi1 gene encoding for the enzyme triose phosphate isomerase in Klebsiella pneumoniae DSM2026 was knocked out in an effort to metabolically engineer this strain as a model system for the production of 1,3-propanediol. Investigations of the tpi1 knockout mutant led to the discovery of a second tpi gene (tpi2) in this organism. The new tpi2 gene was cloned and sequenced. The coding region of the tpi2 gene contains 795bp (base pairs) and the deduced protein consists of 265 amino acids. Sequence comparison of TPI2 proteins in different organisms revealed the presence of a highly conserved signature A-Y-E-P-V-W-A-I-G-[EDVS]-[GKNASH], which is nearly the same as the reported TPI consensus signature. The tpi1 gene of K. pneumoniae DSM2026 shows a high sequence similarity to that of E. coli, whereas, the tpi2 gene resembles more its relatives in the alpha-proteobacteria, suggesting that they evolve from different ancestors. The overexpression of the tpi2 gene restores the growth deficiency of tpi1 knockout mutant on the minimal medium containing glucose or glycerol. Furthermore, the catalytic activity of this new triose phosphate isomerase was confirmed in both tpi1 knockout mutant and tpi2 over-expressing strain by enzyme assays. For the first time, the co-existence of two tpi genes in an enteric bacterium is experimentally confirmed.