Immunobiological properties and therapeutic effectiveness of preparations from Klebsiella bacteriophages]

The purified preparations of Klebsiella bacteriophages, viz. the monovalent preparation of K. pneumoniae bacteriophage and the polyvalent bacteriophage preparation for the treatment of infections caused by K. ozaenae, K. rhinoscleromatis scleromatis and K. pneumoniae sensu lato, have been obtained. The bacteriophage preparations have proved to be nontoxic and safe for laboratory animals after the intraperitoneal injection of these preparations followed by the pathomorphological study of the internal organs of the animals. The clinical study of the newly developed bacteriophage preparations in the course of the treatment of purulent inflammatory diseases in 109 patients has revealed that the preparations are not reactogenic and exhibit sufficient effectiveness in the therapy of ozena, rhinoscleroma and Klebsiella infections with different localization of the infectious process.     

Genome of Klebsiella sp.-infecting bacteriophage vB_KleM_RaK2

Despite the fact that multidrug-resistant Klebsiella sp. strains emerge rapidly (Xu J, et al., Adv. Mater. Res. 268-270:1954-1956, 2011) and bacteriophages have been reported to be useful in controlling these bacteria (Kumari S, Harjai K, Chhibber S, J. Med. Microbiol. 60:205-210, 2011), the complete genome sequences of only five Klebsiella phages (four siphoviruses and one myovirus) can be found in databases. In this paper, we report on the complete genome sequence of Klebsiella sp.-infecting bacteriophage vB_KleM_RaK2. With a genome size of 345,809 bp, this is the second largest myovirus and the largest Klebsiella phage sequenced to date. This phage differs substantially from other myoviruses since 411 out of 534 vB_KleM_RaK2 open reading frames have no known functions and lack any reliable database matches. Comparative analysis of the genome sequence of vB_KleM_RaK2 suggests that this phage forms a distinct phylogenetic branch within the family Myoviridae of tailed bacteriophages.     

Characterization of a T7-Like Lytic Bacteriophage of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> B5055: A Potential Therapeutic Agent

Characterization of bacteriophages to be used prophylactically or therapeutically is mandatory, as use of uncharacterized bacteriophages is considered as one of the major reasons of failure of phage therapy in preantibiotic era. In the present study, one lytic bacteriophage, KPO1K2, specific for Klebsiella pneumoniae B5055, with broad host range was selected for characterization. As shown by TEM, morphologically KPO1K2 possessed icosahedral head with pentagonal nature with apex to apex head diameter of about 39 nm. Presence of short noncontractile tail (10 nm) suggested its inclusion into family Podoviridae with a designation of T7-like lytic bacteriophage. The phage growth cycle with a latent period of 15 min and a burst size of approximately 140 plaque forming units per infected cell as well as a genome of 42 kbps and structural protein pattern of this bacteriophage further confirmed its T7-like characteristics. Phage was stable over a wide pH range of 4–11 and demonstrated maximum activity at 37°C. After injection into mice, at 6 h, a high phage titer was seen in blood as well as in kidney and urinary bladder, though titers in kidney and urinary bladder were higher as compared to blood. Phage got cleared completely in 36 h from blood while from kidneys and urinary bladder its clearance was delayed. We propose the use of this characterized phage, KPO1K2, as a prophylactic/therapeutic agent especially for the treatment of catheter associated UTI caused by Klebsiella pneumoniae.     

Lipopolysaccharide-specific bacteriophage for Klebsiella pneumoniae C3.

Bacteriophage FC3-1 is one of several specific bacteriophages of Klebsiella pneumoniae C3 isolated in our laboratory. Unlike receptors for other Klebsiella phages, the bacteriophage FC3-1 receptor was shown to be lipopolysaccharide, specifically the polysaccharide fraction (O-antigen and core region). We concluded that capsular polysaccharide, outer membrane proteins, and lipid A were not involved in phage binding. Mutants resistant to this phage were isolated and were found to be devoid of lipopolysaccharide O-antigen by several criteria but to contain capsular material serologically identical to that of the wild type. The polysaccharide fraction was concluded to be the primary phage receptor, indicating that it is available to the phage.     

Complete Genome Sequence of Klebsiella pneumoniae Phage JD001

Klebsiella pneumoniae is a member of the family Enterobacteriaceae, opportunistic pathogens that are among the eight most prevalent infectious agents in hospitals. The emergence of multidrug-resistant strains of K. pneumoniae has became a public health problem globally. To develop an effective antimicrobial agent, we isolated a bacteriophage, named JD001, from seawater and sequenced its genome. Comparative genome analysis of phage JD001 with other K. pneumoniae bacteriophages revealed that phage JD001 has little similarity to previously published K. pneumoniae phages KP15, KP32, KP34, and phiKO2. Here we announce the complete genome sequence of JD001 and report major findings from the genomic analysis.     

Use of bacteriophage Mu to isolate deletions in the his-nif region of Klebsiella pneumoniae.

Klebsiella pneumoniae M5a1 is naturally resistant to infection by bacteriophage Mu. Mutants of K. pneumoniae sensitive to Mu infection were isolated and found to support both lytic and lysogenic development of Mu. K. pneumoniae lysogens containing a heat-inducible Mu prophage integrated in his were isolated. Strains carrying deletions extending from his into nif were obtained after heat treatment of these lysogens. Such deletions should be useful for determining the map order and cistronic organization of the nif genes.     

Interferon type I in protective body reactions in an experimental Klebsiella infection]

The study on mice with experimental generalized Klebsiella infection, carried out with the use of microbiologic, immunologic and pathomorphologic methods, revealed that the intraperitoneal injection of type I interferon into the animals prevented their death and led to the rapid elimination of the infective agent from their body, enhanced the phagocytic and metabolic activity of polymorphonuclear lymphocytes of their peritoneal exudate, decreased the manifestation of microcirculatory and dystrophic changes in the parenchyma of their internal organs.     

Influence of environmental conditions on infection of Klebsiella pneumoniae by two different types of bacteriophages

The adsorption and efficiency of plating of bacteriophages FC3-1 and FC3-9 on Klebsiella pneumoniae C3 (serotype O1:K66) cells grown at different pHs and temperatures were quantitated. Bacteriophage FC3-1, with lipopolysaccharide as its bacterial receptor, showed a large decrease in efficiency of plating on bacteria grown at low pH or low temperature. Under the same conditions, no significant decrease in efficiency of plating was found for bacteriophage FC3-9, a phage requiring capsule and lipopolysaccharide for its adsorption and carrying capsule-depolymerizing activity. We demonstrate that K. pneumoniae C3 cells grown at low pH or low temperature have less lipopolysaccharide exposed on their surface. We conclude that this is why lipopolysaccharide-specific phage FC3-1 less efficiently infects bacterial cells grown under those conditions. We propose that bacteriophage FC3-9 efficiently infects bacterial cells grown at low pH or low temperature because its enzymatic activity on the capsule makes lipopolysaccharide available to this phage.     

Isolation and characterization of bacteriophage FC3-10 from Klebsiella spp.

FC3-10 is a Klebsiella spp. specific bacteriophage isolated on a rough mutant (strain KT707, chemotype Rd) of K. pneumoniae C3. The bacteriophage receptor for this phage was shown to be the low-molecular mass lipopolysaccharide (LPS) fraction (LPS-core oligosaccharides), specifically the heptose content of the LPS inner-core. This is the first phage isolated on Klebsiella, the receptor for which is the LPS-core. This phage was unable to plate on Salmonella typhimurium LPS mutants with chemotypes Rd2 or Re showing incomplete or no heptose content on their LPS-core, respectively. Spontaneous phage-resistant mutants from different Klebsiella strains were deep-rough LPS mutants or encapsulated revertants from unencapsulated mutant strains.     

噬菌体治疗肺炎克雷伯菌感染的研究进展

肺炎克雷伯菌是肠杆菌科家族中的一员,在各种环境中广泛存在,可导致诸如奶牛乳房炎在内的多种动物疫病,引起人类的肺炎、尿路感染、菌血症、伤口性感染和化脓性脓肿在内的多种临床感染。该菌对抗生素的耐受日趋严重,而且高毒力菌株不断出现,给该菌的防控带来了巨大挑战。噬菌体是一种裂解细菌的病毒,因其具有治疗耐药细菌感染的潜力而备受关注,世界各地均有使用噬菌体成功治疗耐药细菌感染的案例。本文基于国内外对肺炎克雷伯菌及其噬菌体的研究数据,综述了肺炎克雷伯菌的流行病学调查情况和噬菌体在治疗肺炎克雷伯菌感染方面的应用,以期为基于肺炎克雷伯菌噬菌体的抗菌研究和临床应用提供参考。     

Antibacterial effects of gamma-interferon in experimental Klebsiella infection]

The results of the experimental study on the effect of the natural and recombinant gamma-interferons (gamma-IFs) of mice on the process of the infection caused by Klebsiella sp. are presented. The infection was reproduced by intraperitoneal contamination of mice with a virulent culture of Klebsiella pneumoniae 5055, line SHK. The gamma-IFs were administered to the animals in a dose of 250 units per mouse on days 1 and 3 after the contamination. Survival of the animals, clearance of the pathogen from the blood and liver and functional activity of the phagocytes in the contaminated mice were investigated. It was shown that both the natural and recombinant gamma-IF stimulated the phagocytic activity and oxidative metabolism of the phagocytes in the contaminated mice. Activation of these functions after the use of the natural gamma-IF correlated with its marked protective effect and accelerated elimination of the pathogen from the host which was not observed after the use of the recombinant gamma-IF.     

工业微生物的噬菌体感染与防治策略

以细菌为基础的生物技术在蓬勃发展的同时也不断受到噬菌体感染的威胁,噬菌体感染已成为微生物发酵过程中的一个顽疾,其实质是噬菌体与细菌之间复杂的共进化关系。在漫长的进化过程中,噬菌体已经形成了多种针对细菌抗性系统的逃逸机制。合理的工厂设计、菌株的轮换策略和传统的基因工程方法能在一定程度上降低噬菌体感染的风险,但仍然无法避免。基于CRISPR-Cas系统的防治策略仅需噬菌体的序列信息就可以理性设计噬菌体抗性菌株,且可以通过叠加效应不断增强菌种抗性,从而避免噬菌体的逃逸;群体感应信号分子则可以从整体水平上调节细菌的噬菌体抗性。这些新发现为噬菌体感染问题的解决带了新的希望,而噬菌体基因组编辑技术和合成生物学的快速发展则将进一步加深人们对噬菌体感染防治领域的认识。     

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.     

一株新型牛源肺炎克雷伯菌噬菌体的分离鉴定

【背景】肺炎克雷伯菌(Klebsiella pneumoniae)是一种广泛分布于环境中的重要致病菌,该菌较高的耐药性致其在养殖业中治疗较为困难。【目的】分离一株裂解性肺炎克雷伯菌噬菌体,对分离株进行生物学特性鉴定和基因组学分析。【方法】使用双层平板法从四川省某奶牛场中分离、纯化出一株裂解性噬菌体,测定其裂解谱、热稳定性、酸碱耐受度、最佳感染复数及一步生长曲线等生物学特性,并进行全基因组的测序及注释分析。【结果】得到一株裂解性肺炎克雷伯菌噬菌体vB_Kpn_B01,该噬菌体拥有透明且无晕环的噬菌斑,热稳定性较高,在极酸或极碱环境下不进行裂解活动,特异性较强,属长尾噬菌体科(Siphoviridae)。vB_Kpn_B01全基因组大小为113 227 bp,GC含量为47.97%。注释结果显示噬菌体拥有149个编码序列和25个tRNAs,不含耐药基因及毒力基因。通过噬菌体的进化树分析发现,该噬菌体为Sugarlandvirus。【结论】vB_Kpn_B01拥有高效的生长特性和对不利环境的低耐受性,拥有裂解宿主菌的必备基因,并不含耐药基因及毒力基因,具有应用于畜牧业中防治多重耐药细菌的潜力。     

噬菌体治疗肺炎克雷伯菌感染的研究进展

随着细菌的进化以及部分抗生素的滥用，耐药细菌的感染已成为21世纪主要的公共卫生挑战之一。其中，耐药肺炎克雷伯菌(Klebsiella pneumoniae)问题尤为突出。噬菌体在治疗耐药细菌感染引起的疾病方面展现出一定的潜力及独特优势，但目前噬菌体治疗尚缺乏统一的临床指导规范。虽然临床上有少数将噬菌体用于治疗肺炎克雷伯菌感染的成功案例，但多数情况下是采用噬菌体配合抗生素疗法，噬菌体在其中的作用仍不明确。本文综合评述国内外研究数据，回顾与噬菌体治疗肺炎克雷伯菌感染相关的数个重点问题，包括噬菌体的特性以及影响其疗效的因素，旨在为肺炎克雷伯菌和其他耐药细菌的噬菌体治疗提供参考。     

Structural changes induced by a lytic bacteriophage make ciprofloxacin effective against older biofilm of Klebsiella pneumoniae

Bacteria have evolved multiple mechanisms, such as biofilm formation, to thwart antibiotic action. Yet antibiotics remain the drug of choice against clinical infections. It has been documented that young biofilm of Klebsiella pneumoniae could be eradicated significantly by ciprofloxacin treatment alone. Since age of biofilm is a decisive factor in determining the outcome of antibiotic treatment, in the present study biofilm of K. pneumoniae, grown for extended periods was treated with ciprofloxacin and/or depolymerase producing lytic bacteriophage (KPO1K2). The reduction in bacterial numbers of older biofilm was greater after application of the two agents in combination as ciprofloxacin alone could not reduce bacterial biomass significantly in older biofilms (P > 0.05). Confocal microscopy suggested the induction of structural changes in the biofilm matrix and a decrease in micro-colony size after KPO1K2 treatment. The role of phage associated depolymerase was emphasized by the insignificant eradication of biofilm by a non-depolymerase producing bacteriophage that, however, eradicated the biofilm when applied concomitantly with purified depolymerase. These findings demonstrate that a lytic bacteriophage alone can eradicate older biofilms significantly and its action is primarily depolymerase mediated. However, application of phage and antibiotic in combination resulted in slightly increased biofilm eradication confirming the speculation that antibiotic efficacy can be augmented by bacteriophage.     

Mutational alteration of a nitrogen-fixing bacterium to sensitivity to infection by bacteriophage Mu: isolation of nif mutations of Klebsiella pneumoniae M5al induced by Mu.

The nitrogen-fixing bacterium Klebsiella pneumoniae M5al is not sensitive to infection by bacteriophage Mu. A mutant of K. pneumoniae that is sensitive to Mu infection was isolated. Several Mu-induced auxotrophic mutations of K. pneumoniae including nif, trp, and rtl were isolated and genetically characterized. Evidence is presented that the Mu-induced mutations of nif arise as the result of insertion of Mu within (or near) the nif operon(s). The rtl locus, which determines the ability to utilize ribitol as a carbon source, was found to be linked to nif loci.     

Preparation of branched hexasaccharides by the action of a viral lyase on Klebsiella K14 polysaccharide

Klebsiella K14 capsular polysaccharide was degraded by a bacteriophage-borne enzyme to afford oligosaccharides A-C which were studied by one- and two-dimensional n.m.r. spectroscopy. A and B were the repeating-unit hexasaccharide and pyruvylated hexasaccharide, respectively, while C was a dodecasaccharide. Each oligomer was terminated by a reducing mannose and a non-reducing 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid residue, indicating that the phage enzyme had cleaved the beta-D-Manp-(1----4)-beta-D-GlcpA linkages in the polysaccharide by a lyase, rather than the more common glycosidase, activity found with other Klebsiella bacteriophages. In this respect, the depolymerisation resembles those reported for the capsular polysaccharides of Klebsiella K5 and K64     

Involvement of colicin in the limited protection of the colicin producing cells against bacteriophage

The restriction/modification system is considered to be the most common machinery of microorganisms for protection against bacteriophage infection. However, we found that mitomycin C induced Escherichia coli containing ColE7-K317 can confer limited protection against bacteriophage M13K07 and lambda infection. Our study showed that degree of protection is correlated with the expression level of the ColE7 operon, indicating that colicin E7 alone or the colicin E7-immunity protein complex is directly involved in this protection mechanism. It was also noted that the degree of protection is greater against the single-strand DNA bacteriophage M13K07 than the double-strand bacteriophage(lambda). Coincidently, the K(A) value of ColE7-Im either interacting with single-strand DNA (2.94x10(5)M(-1)) or double-strand DNA (1.75x10(5)M(-1)) reveals that the binding affinity of ColE7-Im with ssDNA is 1.68-fold stronger than that of the protein complex interacting with dsDNA. Interaction between colicin and the DNA may play a central role in this limited protection of the colicin-producing cell against bacteriophages. Based on these observations, we suggest that the colicin exporting pathway may interact to some extent with the bacteriophage infection pathway leading to a limited selective advantage for and limited protection of colicin-producing cells against different bacteriophages.     

Protective effect of the aerosol immunization of mice with the polycomponent vaccine Immunovac VP-4 after the challenge of the animals with Klebsiella pneumoniae virulent strain

Used four schemes of the administration of the preparation with different time of the exposition of the animals in an aerosol chamber were tested with their subsequent intraperitoneal challenge with K. pneumoniae virulent strain K16. Irrespective of the number of immunization courses, the administration of the preparation made at intervals of 1 day, or daily, did not ensure any protective effect, but only led to an insignificant increase in their survival time in comparison with nonimmunized animals. After intervals between immunizations were increased to 3 days the protective effect of aerosol immumization was obtained (the survival rate was 65-80 % and considerably differed from that of the controls). The protective effect of aerosol immunization thus obtained was comparable with the effectiveness immunization made in a single subcutaneous injection. Aerosol immunization resulted in low antibody titers to the antigens contained in the vaccine, while after a single subcutaneous injection high antibody titers to Klebsiella and Proteus antigens were detected. The antigen-stimulated blast transformation of spleen lymphocytes in mice subjected to aerosol immunizations in 5 exposures was high. After subcutaneous immunization significant changes in such characteristics were detected on day 15. The data thus obtained were indicative of good prospects in the development Immunovac VP-4 as the medicinal form intended for use in aerosols.