Elucidation of the structure of the lipopolysaccharide core and the linkage between the core and the O-antigen in Pseudomonas aeruginosa immunotype 5 using strong alkaline degradation of the lipopolysaccharide

The products of the strong alkaline degradation of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa immunotype 5 were separated by anion-exchange HPLC and studied by electrospray ionization mass spectrometry and NMR spectroscopy.It was found that two major products have the same inner core region and lipid A carbohydrate backbone but different outer core regions.The difference is in the position of a rhamnose residue,which is substituted with either an additional glucose residue or a disaccharide remainder of the degraded O-polysaccharide.The site and the configuration of the linkage between the O-polysaccharide and the core were determined and,together with published data,the structure of the so-called biological repeating unit of the O-antigen was defined.The glycosidic linkage of the 2-acetamido-2,6-dideoxy D-glucose (N-acetyl-D-quinovosamine) residue is when it links the O-polysaccharide to the core and when it connects the interior repeating units of the O-polysaccharide to each other.     

Full Structure of the Lipopolysaccharide of Pseudomonas aeruginosa Immunotype 5

The lipopolysaccharide (LPS) of the opportunistic human pathogen Pseudomonas aeruginosa immunotype 5 was delipidated by mild acid hydrolysis, and the products were separated by high-performance anion-exchange chromatography and analyzed by ESI MS and NMR spectroscopy. LPS species of three types were found, including those with an unsubstituted core and the core substituted with one O-polysaccharide repeating unit or with an O-polysaccharide of a variable number of repeating units. The core region is highly phosphorylated, the major species containing two monophosphate groups and one ethanolamine diphosphate group. Based on these and published data on the O-polysaccharide structure, the full structure of the LPS of P. aeruginosa immunotype 5 was established.     

Specific and non-specific mouse protection induced by different chemotypes of the Pseudomonas aeruginosa lipopolysaccharides

Abstract Lipopolysaccharides (LPS) of Pseudomonas aeruginosa were studied by the mouse active, cross-protection test. The primary structure of O-specific polysaccharides (O-repeating units) of different chemotypes was determined and their cross-protective activity demonstrated. Low doses of LPS (0.1–1 μg) stimulated chemotype-specific protection against P. aeruginosa in mice. This immunity was associated with the primary structure of the LPS and it lasted for 14 days after the first or second immunization. High doses of LPS (10–100 μg) induced cross-protection against P. aeruginosa in mice. The cross-protective capacity was caused evidently by the secondary structure or conformation of LPS molecule, i.e. by the common conformational protective determinant. This cross-protection lasted for only 5 days after the first or second immunization.     

Monoclonal antibodies to the surface antigens of Pseudomonas aeruginosa

Abstract A panel of 48 monoclonal antibodies was prepared against 8 O-serotype strains of Pseudomonas aeruginosa , and 43 of the antibodies reacted specifically with whole cells of the vaccine strain in an enzyme-linked immunosorbent assay (ELISA). 4 antibodies showed varying degrees of reactivity for more than one of the serotype strains, and one antibody bound to all of the serotype strains as well as strains of Pseudomonas putida and Pseudomonas fluorescens . The epitopes recognised by these antibodies were characterised by immunoblotting and the serotype-specific antibodies reacted only with lipopolysaccharide (LPS) of the vaccine strain. The antibodies that bound to more than one serotype strain were specific for outer-membrane proteins common to the serotype strains. The antibody that cross-reacted with all strains of P. aeruginosa apparently recognised an antigen associated with the core or lipid A components of LPS.     

301株铜绿假单胞菌的分布及耐药性分析

目的 通过对临床中分离的铜绿假单胞菌的分布及对临床常用13种抗生素的耐药性进行分析指导临床合理用药.方法 收集大连医科大学附属第二医院2010年1月至12月临床送检的标本,采用全自动细菌和药敏分析仪分离铜绿假单胞菌同时进行药敏试验.结果 铜绿假单胞菌在痰液标本中分离率高达79.06％;对头孢噻肟、头孢西丁、头孢唑啉3种药物的耐药率均大于50％;对头孢他啶,哌拉西林/他唑巴坦耐药率低于20％.结论 铜绿假单胞菌易产生多源耐药,加强耐药性监测,控制医院内感染,对临床医生选用有效的抗生素具有十分重要的意义.     

Chemical and biological characterization of lipopolysaccharides from the Pseudomonas syringae pv. maculicola IMV 381 collection culture and its dissociants

Lipopolysaccharides (LPS) were isolated from the crude bacterial mass of the Pseudomonas syringae pv. maculicola IMV 381 collection culture and its virulent and avirulent subcultures isolated earlier from the heterogeneous collection culture due to its natural variability during long-term storage. The composition, immunochemical properties, and certain parameters of the biological activity of the LPS preparations obtained were studied. The structural parts of the LPS macromolecule—lipid A, the core oligosaccharide, and O-specific polysaccharide (OPS)—were isolated and characterized. The following fatty acids were identified in the lipid A composition of all cultures: 3-OH-C_10:0, C_12:0, 2-OH-C_12:0, 3-OH-C_12:0, C_16:1, C_16:0, C_18:1, and C_18:0. Glucosamine (GlcN), ethanolamine (EtN), phosphoethanolamine (EtN-P), and phosphorus (P) were revealed in the hydrophilic portion of the macromolecule. In the core portion of the LPS macromolecule, glucose (Glc), rhamnose (Rha), GlcN, galactosamine (GalN), 2-keto-3-deoxyoctulosonic acid (KDO), alanine (Ala), and P were found. The peculiarities of the structure of LPS isolated from the stable collection culture (LPS_stab) and its virulent (LPS_vir) and avirulent (LPS_avir) subcultures were studied. LPS_vir and LPS_avir were identical in the monosaccharide composition and contained as the main components L-rhamnose (L-Rha) and 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc), like LPS_stab, studied earlier. The NMR spectra of LPS_vir were identical to the spectra of LPS_stab, whose O-chain repeating unit structure was studied by us earlier, whereas LPS_avir differed from LPS_vir in the NMR spectrum and was identified by us as the SR form. LPS_avir was serologically identical to LPS_stab and LPS_vir. Hence, the degree of polymerism of the LPS O-chain of P. Syringae pv. maculicola IMV 381 is the main virulence factor in infected model plants. Serological relationships were studied between P. Syringae pv. maculicola IMV 381 and the strains of other pathovars with structurally similar LPS.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 790–801.Original Russian Text Copyright © 2004 by G. Zdorovenko, Varbanets, E. Zdorovenko, Vinarskaya, Yakovleva.     

126株铜绿假单胞菌的临床分布及耐药性分析

目的调查分析象山县中医医院铜绿假单胞菌的临床分布及药敏情况,为临床合理选用抗生素提供可靠的依据。方法采集疑似患者的标本,进行分离、培养与鉴定。采用自动微生物鉴定／药敏分析仪进行鉴定及药敏试验,对2012年7月至2013年10月分离出的126株铜绿假单胞菌（包括21株黏液型铜绿假单胞菌）的分布及耐药性进行回顾性分析。结果126株铜绿假单胞菌临床主要分布情况：痰占80．2％,尿液占11．1％,脓液占7．1％,以呼吸道感染为主。对铜绿假单胞菌保持活性较强同时耐药率〈20％的抗生素有阿米卡星、妥布霉素、亚胺培南、哌拉西林／他唑巴坦,其中碳青霉烯类耐药率升至5％,原来被认为抗铜绿假单胞菌较为有效的喹诺酮类抗生素的耐药率也有了很大提升,左氧氟沙星耐药率升至33％。黏液型铜绿假单胞菌的体外抗菌药物敏感试验耐药性较弱,且明显弱于非黏液型铜绿假单胞菌的耐药性。结论铜绿假单胞菌为医院呼吸道感染的常见致病菌,对多种抗菌药物呈不同程度耐药。加强动态监测,合理使用抗菌药物,对铜绿假单胞菌感染的预防和药物治疗具有重要指导意义。     

哈尔滨市112株绿脓杆菌的血清学分型和耐药性测定

对从临床分离的112株绿脓杆菌进行系统鉴定后,血清学分型表明:6、2和3型分别占32.14％、15.18％、15.18％,为主要流行型,共占总分离株的62.50％。耐药性测定结果为:对10种抗生素5耐以上者占69.6％。其中对多粘菌素、妥布霉素、丁胺卡那霉素三种抗生素最为敏感,敏感率分别为100％、70.6％、86.5％。     

Structural relationship between the polyagglutinable antigen and the core polysaccharide of Pseudomonas aeruginosa

Abstract It has been observed that each strain of the Pseudomonas aeruginosa species harbours the so-called polyagglutinable antigen (PA). Some strains may produce it in a form which is linked to the core moiety of lipopolysaccharide (LPS) and this type of PA can thus be detected by passive haemagglutination using the isolated LPS as coating antigen. Other strains synthesize PA exclusively in a free form, which is also coextractable with LPS, its presence can, however, be demonstrated by the haemagglutination inhibition test. From a polyagglutinable strain of P. aeruginosa an R-type LPS was isolated having the core-linked PA. This LPS preparation was highly immunogenic with regard to its PA moiety. The core-bound PA seems to exert an immunosuppression on the core region, hence, the polyagglutinable strains isolated from cystic fibrosis patients only engender anti-PA antibodies, whereas antibodies against both, side chain and core region of LPS, are not engendered. The mucoid exopolysacharides also contains the PA which could possibly play an important role in the patient by protecting P. aeruginosa cells against anti-PA antibodies.     

Saccharides of seven Pseudomonas aeruginosa immunotypes

Abstract The structures of O-specific polysaccharides obtained by mild acid degredation of lipopolysaccharides (LPS) from seven Pseudomonas aeruginosa Fisher's immunotypes have been studied. The polysaccharides consist mainly of monoamino and diamino sugars, frequently also carrying acidic functions. Some of the sugars were detected in nature for the O-specific polysaccharides of the immunotypes 2, 3, 4, 5 and 6 are identical to those of the polysaccharides of the 011; 0(2a)2c; 01; 010a, 10b and 07a, 7d Lányi-Bergan serological subgroups respectively, whereas no analogues have been found for the immunotypes 1 and 7. Some cross-reactions between the LPS of different immunotypes were observed in passive haemagglutination tests; the results of inhibition of passive haemagglutination and agar gel immunoprecipitation point, however, to a specificity of the LPS. Many of the LPS of the seven Pseudomonas aeruginosa immunotypes manifest rather a high cross-protective activity in active immunization tests in mice. The nature of the cross-protective activity of the LPS is discussed.     

铜绿假单胞菌lasI,rhlI基因功能缺陷株的构建和生物学功能验证

构建铜绿假单胞菌lasI,rhlI基因功能缺陷株,为进一步阐明氦氧饱和高气压暴露条件诱导lasI,rhlI基因介导铜绿假单胞菌毒力调节的分子机制研究奠定基础。用双亲株接合转移法删除lasI,rhlI基因ORF编码区,通过RT-PCR方法验证目标基因编码序列mRNA的缺失;通过对细菌生长增殖能力、弹性蛋白酶代谢活性和细菌绿脓菌素分泌能力等表型的测定,验证目标基因编码序列缺失后的基因调节功能的缺陷。结果表明成功构建铜绿假单胞菌lasI,rhlI基因功能缺陷株,可作为进一步研究的基因工程菌。     

Evidence for the location of OprM in the Pseudomonas aeruginosa outer membrane

Abstract OprM with a M _r of 49 K is associated with the multidrug resistance of Pseudomonas aeruginosa . Detergent fractionation of bacterial cells has demonstrated that OprM is located in the outer membrane from which it sediments with the other major outer membrane proteins. In this study we have determined the location of OprM as the P. aeruginosa outer membrane. Western immunoblots of cell fractions, obtained by sucrose density gradient centrifugation of whole cell lysates, were probed with an OprM-specific murine polyclonal antiserum.     

Copper uptake and its compartmentalization in Pseudomonas aeruginosa strains: Chemical nature of cellular metal

Copper-sensitive (Cu^s) and copper-resistant (Cu^r) strains of Pseudomonas aeruginosa were characterized in terms of Cu²⁺ sensitivity, uptake and its compartmentalization in the possible cell sectors. Minimum inhibitory concentrations (MICs) of Cu²⁺ for the Cu^r strain (3.2 mM and 0.12 mM in enriched- and in minimal-medium, respectively) were almost 5-fold higher over that of its sensitive counterpart. While Cu^s strain accumulated Cu²⁺ to a maximum of 1.8 mol mg^–1 protein, Cu^r strain increased it to 2.37 mol mg^–1 protein. Both the strains also demonstrated energy- and pH-dependent Cu²⁺ uptake through the broad-substrate range divalent cation (Zn²⁺, Mg²⁺, Co²⁺) uptake system as well as through the system specific for Cu²⁺. Cell-fractionation study revealed that in Cu^r strain, periplasm and membrane are the main Cu²⁺ binding sites, whereas, in case of Cu^s strain, it is the cytoplasm. The overall observations indicate that the Cu^r strain restricted Cu²⁺ sequestration exterior to the cytoplasm as the possible strategy for Cu-resistance. The chemical nature of Cu²⁺ deposition in the respective strains was also ascertained by X-ray powder diffraction analysis.     

Arbitrary primed PCR fingerprinting and serotyping of clinical Pseudomonas aeruginosa strains

Arbitrary primed PCR (AP-PCR) analysis was compared with serotyping as a means of high-resolution typing of Pseudomonas aeruginosa. Seventy-four isolates from 3 different hospitals and 18 reference strains were studied. Serotyping provided good index of discrimination, although eleven isolates could not be serotyped. Genomic DNA was amplified with a single 10 nucleotide primer (sequence 5′-AGG GGT CTT G-3′). The strains were genetically diverse and 61 different AP-PCR profiles of 2–7 bands between 0.3 and 2.4 kb were obtained. AP-PCR profiles were not consistently associated with serotypes, but they clearly subtyped strains of the same serotype. Numerical analysis of AP-PCR patterns defined 7 groups at the 55% similarity level, and identified predominant strains in each hospital. The results show that AP-PCR analysis provides a simple and practical approach to typing P. aeruginosa that is more discriminatory than traditional serotyping scheme. We suggest that maximum discrimination can be achieved by a combination of both methods.     

The structure of the lipopolysaccharide from a galU mutant of Pseudomonas aeruginosa serogroup-O11

The lipopolysaccharide (LPS) of a galU mutant of Pseudomonas aeruginosa PA103, a serogroup O11 strain, was sequentially extracted with phenol–chloroform–petroleum ether (PCP) followed by hot phenol–water extraction of the bacterial pellet remaining after PCP extraction. LPS was found in both the PCP extract as well as in the water phase of the hot phenol–water extract. Analysis of the carbohydrate portion released by mild acid hydrolysis of both LPS preparations, both before and after removal of all phosphate groups by treatment with aqueous HF, was performed by glycosyl composition and linkage analyses as well as by NMR and mass spectrometric analyses. The results showed that the carbohydrate portion of these two LPS extracts contained the same structure: namely, -GalN(Ala)-(1→3)--(7-Cm)HepII-(1→3)--HepI-(1→5)--Kdo-(2→. The oligosaccharide preparation from PCP-extracted LPS consisted of a variety of structures containing up to six phosphate groups present as mono-, pyro-, and possibly triphosphate, primarily located on the HepI residue with some molecules having a monophosphate on HepII. The oligosaccharide preparation from the hot phenol–water-extracted LPS contained a similar variety of structures, but with an additional structure in which HepI contained a PPEA group at O-2. In addition, PAGE immunoblot analysis of the crude cellular extract with anti-A-antibodies revealed the presence of A-band material in both PA103 and the galU mutant. The A-band material was purified and characterized by glycosyl composition and linkage analyses, as well as by NMR spectroscopy, which confirmed that the A-band rhamnan polysaccharide was present but not as typical LPS since lipid-A or LPS core oligosaccharide components were not detected.     

Auxotrophy of Pseudomonas aeruginosa in cystic fibrosis

Seventy-four of 403 (18.4%) sputum isolates of Pseudomonas aeruginosa from 49 of 136 (36.0%) adults with cystic fibrosis (CF) were auxotrophic mutants. Two of 11 (18.2%) isolates of P. aeruginosa taken from patients with non-CF bronchiectasis were also auxotrophic. All 99 strains taken from non-bronchiectatic sources were prototrophic. Forty-six of 55 (83.6%) CF auxotrophs required one or more of 36 growth factors tested; the requirements for the remaining 9 isolates were not identified. Methionine was the sole factor required by 17 of 22 (77.3%) isolated which depended on a single factor. We conclude that auxotrophy is a feature of P. aeruginosa infection in cystic fibrosis.     

Carbons from choline present in the phospholipids of Pseudomonas aeruginosa

The phospholipid composition of Pseudomonas aeruginosa grown in a mineral medium with choline as the carbon source was: phosphatidylethanolamine, 71.6±1.4%; phosphatidylglycerol, 11.8±0.4%; diphosphatidylglycerol, 0.8±0.4%; phosphatidic acid, 2.4±0.6%; lysophosphatidylethanolamine, 1.6±0.3%; phosphatidylcholine 7.9±0.3%; lysophosphatidylcholine, 3.9±0.7%. The molar ratio between the acidic and the neutral phospholipids was 0.18. Radiolabeling experiments with [methyl-¹⁴C]choline or [1,2-¹⁴C]choline carried out in cell suspension from bacteria that were grown in the presence of choline as the sole carbon source demonstrated that the carbons of the N-methyl groups of choline contributed to the synthesis of fatty acids while the carbons comprising the backbone of choline were used for the synthesis of glycerol.