The identity of the O-specific polysaccharide structure of Citrobacter strains from serogroups O2, O20 and O25 and immunochemical characterisation of C. youngae PCM 1507 (O2a,1b) and related strains

Serological studies using SDS-PAGE and immunoblotting revealed that from five strains that are ascribed to Citrobacter serogroup O2, four strains, PCM 1494, PCM 1495, PCM 1496 and PCM 1507, are reactive with specific anti-Citrobacter O2 serum. In contrast, strain PCM 1573 did not react with anti-Citrobacter O2 serum and, hence, does not belong to serogroup O2. The LPS of Citrobacter youngae O2a,1b (strain PCM 1507) was degraded under mild acidic conditions and the O-specific polysaccharide (OPS) released was isolated by gel chromatography. Sugar and methylation analyses along with (1)H- and (13)C-NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, NOESY and (1)H,(13)C HSQC experiments, showed that the repeating unit of the OPS has the following structure: [structure: see text]. NMR spectroscopic studies demonstrated that Citrobacter werkmanii O20 and C. youngae O25 have the same OPS structure as C. youngae O2. Sugar and methylation analyses of the core oligosaccharide fractions demonstrated structural differences in the lipopolysaccharide core regions of these strains, which may substantiate their classification in different serogroups.     

Core region of Citrobacter O23 lipopolysaccharide. Structure elucidation by chemical methods, gas chromatography/mass spectrometry and NMR spectroscopy at 500 MHz

A novel enterobacterial core region in Citrobacter O23 lipopolysaccharide is described. Its structure was determined by methylation analysis/mass spectrometry, chemical degradation and one- and two-dimensional 1H-NMR spectroscopy: [formula; see text] where PPEtN stands for diphosphorylethanolamine, and dOclA for 3-deoxy-D-manno-octulosonic acid.     

Core region of Citrobacter lipopolysaccharide from strain PCM 1487. Structure elucidation by two-dimensional 1H-NMR spectroscopy at 500 MHz and methylation analysis/mass spectrometry

The core structure of Citrobacter PCM 1487 lipopolysaccharide has been established using methylation analysis/mass spectrometry, chemical degradations and one- and two-dimensional 1H-NMR spectroscopy at 500 MHz. 1H-NMR assignments are given for all sugar components of the core oligosaccharide. In the formula shown below, the alternative locations of branch terminal heptose (LDHep) and diphosphorylethanolamine (PPEtN) residues are marked by dashed lines; dOclA stands for 3-deoxy-D-manno-octulosonic acid. (Formula: see text). The sample of the core oligosaccharide showed some microheterogeneity due to a slightly incomplete substitution by terminal N-acetylgalactosamine and a partial splitting of diphosphorylethanolamine residues.     

Structure and serology of the lipopolysaccharides: Cell components of citrobacter

The genus Citrobacter consists of enteric rods that utilize citrate as a sole carbon source. Citrobacter serotypes are normal intestinal inhabitants, but some of them have been reported in gastroenteritis and in extraintestinal infections. Our immumochemical studies on Citrobacter lipopolysaccharides of chemotype G comprised serotypes: 04, 027 and 036. As we found the serotypes mentioned above are distinguished in having O-specific polysaccharides that are homopolymers of β [1–2] linked 4-deoxy-D-arabinohexopyranose. In accordance with the structural indentity, the Citrobacter lipopolysaccharides showed strong serological cross-reactivity. Nevertheless, serotypes 04, 027, and 036 form separate entities, as observed in early studies of serologists. To find the reason for the immunological diversity the structural studies on lipopolysaccharide core regions of these Citrobacter serotypes were carried out. Three novel enterobacterial cores were identified. The complete cores 04 and 036 are decasaccharides differing in one structural element only: side-chain glucose is present in 04 oligosaccharide in the place of galactose in the 036. The core 027 is octasaccharide. In conclusion, our structural studies have shown hitherto that in Citrobacter chemotype G group only one O-specific polysaccharide, but three core regions, exist.     

Frequency of detecting bacteria of the genera Citrobacter and Hafnia in different contingents of examined adults]

A study of the incidence of bacteria of the Citrobacter and Hafnia genus in adults permitted to establish a greater occurrence of the mentioned microbes in the patients with various acute intestinal diseases in comparison with the healthy ones. Bacteria of the Citrobacter and Hafnia genus were revealed in the patients with acute intestinal diseases of obscure etiology, which often were diagnosed as gastroenteritis, enteritis, etc. Bacteria of the Citrobacter and Hafnia genus were revealed in the dysentery patients. Results of studies carried out among various healthy groups of the population indicated no significant differences in respect to the carrier state of the Citrobacter and Hafnia bacteria both within each of the groups of the persons examined and between them. Further studies directed to the investigation of the etiological role of bacteria of the Citrobacter and Hafnia genus in the pathology of enteric disturbances are necessary.     

The occurrence of glycine in bacterial lipopolysaccharides

Abstract The aminoacyl analysis of endotoxic lipopolysaccharides (LPS) isolated from several bacteria revealed essential amounts of glycine, among the inherent LPS components. Significant amounts of the glycine was detected in lipopolysaccharides isolated from over 30 strains of Escherichia, Salmonella, Hafnia, Citrobacter and Shigella species. Glycine as a single amino acid was found only in a core part of LPS. Molar ratio of glycine in core oligosaccharide fraction ranged from 0.2 to 0.6 per 3 heptoses. The oligosaccharide enriched in glycine was isolated using the HPLC. The amino acid appeared to be terminally located in a core oligosaccharide. The labelling of the lipopolysaccharide cores was achieved when the bacteria were cultivated in the presence of radioactive [¹⁴C]glycine. The labelled core oligosaccharide released the radioactivity during treatment with mild alkali or acid (0.1 M NaOH or HCl, 100°C, 4 h). The radioactivity in SDS-polyacrylamide gel electrophoresis migrated exclusively with LPS. The results indicate that amino acid is an integral constituent of core oligosaccharide in lipopolysaccharide.     

Prediction of two- and three-amino-acid sequences of Citrobacter Freundii beta-lactamase from its amino acid composition

The repeated amino-acid sequences in Citrobacter Freundii beta-lactamase may be indispensable for its function, because such repetitions cannot be simply attributed to a chance. In order to fully explore the functional units in Citrobacter Freundii beta-lactamase, it may need to analyse all the amino acid pairs, triplets, etc. along Citrobacter Freundii beta-lactamase from one terminal to the other terminal, to count their frequencies and calculate their probabilities. The amino-acid sequence of Citrobacter Freundii beta-lactamase was counted according to two-, three- and four-amino-acid sequences. The counted frequency and probability were compared with the predicted frequency and probability. The amino acid sequences, which appear in Citrobacter Freundii beta-lactamase and can be predicted from its amino acid composition according to a purely random mechanism, should not be deliberately evolved and conserved. By contrast, the amino acid sequences, which appear in Citrobacter Freundii beta-lactamase but cannot be predicted from its amino acid composition according to a purely random mechanism, should be deliberately evolved and conversed. Accordingly 99 (26.053%) and 33 (8.684%) of 380 two-amino-acid sequences can be predicted by the frequency and probability according to a purely random mechanism. Some kinds of amino acid sequences, which absent in Citrobacter Freundii beta-lactamase and can be predicted from its amino acid composition according to a purely random mechanism, should not be deliberately excluded from Citrobacter Freundii beta-lactamase. By contrast, some kinds of amino acid sequences, which absent in Citrobacter Freundii beta-lactamase and cannot be predicted from its amino acid composition according to a purely random mechanism, should be deliberately excluded from Citrobacter Freundii beta-lactamase. Accordingly 89 (48.370%) and 41 (22.283%) of 184 kinds of absent two-amino-acid sequences can be predicted by the frequency and probability according to a purely random mechanism, and 7236 (99.848%) of 7247 kinds of absent three-amino-acid sequences can be predicted by the frequency according to a purely random mechanism. The amino acids, whose probabilities in following certain preceding amino acids can be predicted from Citrobacter Freundii beta-lactamase amino acid composition according to a purely random mechanism, should not be deliberately evolved and conversed, accordingly 2 (0.526%) of 380 counted first order Markov transition probabilities for the second amino acid in two-amino-acid sequences match the predicted conditional probabilities.     

The characteristic and clinical importance of bacteria of the genus Citrobacter isolated from patients with acute intestinal infections on the territory of Volgograd

A total of 309 Citrobacter strains isolated from patients with acute intestinal infections of obscure aetiology and from healthy subjects were investigated. Species specificity of the bacterial cultures was determined by biochemical tests recommended by the International Enterobacteriaceae Subcommittee. Citrobacter strains were titrated serologically in the reaction of agglutination on glass with a living culture and adsorbed 0-antisera. Eighteen serological 0 groups were identified; 0 groups 3, 9, 23, 1 and 8 were found most frequently. Citrobacter was isolated more frequently from patients with acute intestinal infections than from healthy subjects. Seasonal circulation of Citrobacter strains in spring and summer was established. The number of culture findings of Citrobacter in the patients increased against the background of reduction in the number of cases of bacteriologically confirmed dysentery.     

小龙虾肠道细菌群落多样性及产蛋白酶细菌的筛选

【背景】养殖动物对饲料的消化利用往往与肠道菌群密切相关。【目的】揭示小龙虾肠道细菌群落组成,并从小龙虾肠道筛选产蛋白酶细菌。【方法】在Illumina MiSeq PE300平台对细菌16S rRNA基因V3-V4区进行高通量测序,分析小龙虾肠道细菌群落多样性;通过酪蛋白平板法筛选产蛋白酶细菌并进行分子生物学鉴定。【结果】小龙虾肠道细菌优势门包括变形菌门、软壁菌门、厚壁菌门、拟杆菌门等4个门,累计占比为98.53%;优势属包括柠檬酸杆菌属、支原体科Candidatus_Bacilloplasma暂定属、哈夫尼菌属、肠球菌属、拟杆菌属、梭菌科未分类属、希瓦氏菌属等7个属,累计占比为91.67%。通过酪蛋白平板法筛选的产蛋白酶细菌来自哈夫尼菌属、柠檬酸杆菌属、克雷伯氏菌属和芽孢杆菌属。【结论】小龙虾肠道细菌核心类群在蛋白质消化利用中发挥了重要作用。     

Differentiation of Citrobacter strains using chromosomal DNA restriction patterns

The aim of this study was checking of the usefulness of chromosomal DNA restriction patterns in differentiation of Citrobacter strains. Molecular characterization of total 56 isolates of Citrobacter from Poland and Czech Republic, was performed by pulsed-field gel electrophoresis after digestion of chromosomal DNA with restriction endonuclease Xba I (5'-TCTAGA-3'). Chromosomal DNA of all tested Citrobacter strains gave after electrophoresis 12 to 21 bands and patterns consisting of 12 to 21 fragments ranging in size from 790 kb to 48.5 kb and smaller, which where not distinguishable. Pulsed-field gel electrophoresis patterns were useful for comparing Citrobacter strains. Identical restriction patterns generated by PFGE were observed in the case of selected strains e.g. strains C. sedlakii studied in this study, coming from an outbreak, having the some phenotype. In addition, PFGE patterns can be used to evaluate the clonal relatedness among bacterial isolates. PFGE can be helpful for assessing genetic relatedness among strains epidemiologicaly unrelated e.g. C. werkmanii strains tested in this study. The sum of DNA fragments after Xba I digestion indicates the genome size of Citrobacter strains. This suggests that PFGE should be useful for epidemiological investigations of Citrobacter strains.     

Isolation and properties of Citrobacter bacteriophages

Four phages differing in their antigenic properties, thermosensitivity and spectrum of action have been obtained from Citrobacter lysogenic cultures. To prepare the suspension of Citrobacter phages, phagolysates may be treated with chloroform 1:10 for 30 minutes. The isolated phages have proved to be highly specific. The possibility of distinguishing Citrobacter cultures by means of the phages under study has been shown.     

Biochemical and Antibiotic Susceptibility Studies of H2S-Negative Citrobacter

Ninety-four strains of H(2)S-negative Citrobacter were biochemically characterized and their antibiograms were determined. The antibiograms demonstrated not only a difference from Enterobacter cloacae but also a difference within the Citrobacter group between the indole-negative and indole-positive strains. These differences were statistically significant and emphasize the importance of the indole reaction as an aid to speciation of the H(2)S-negative Citrobacter.     

Differentiation of Citrobacter strains using electrophoretic protein patterns

The aim of this study was checking of the usefulness of electrophoretic protein patterns in differentiation of Citrobacter strains. For analysis of whole-cell proteins 181 Citrobacter strains were prepared. Electrophoresis was performed in Mini Protean Duall Slab Cell (Bio-Rad) apparatus. Electrophoresis was carried out in 10% polyacrylamide gel according to the SDS-PAGE method of Laemmli. Whole-cell proteins of all tested Citrobacter strains gave after electrophoresis 12 to 20 bands. Patterns consisting of 12 to 20 fragments ranging in size from 70,000 to 14,000 and smaller, were not distinguishable. There were no significant differences between electrophorograms of Citrobacter strains belonging to the different species, useful for species differentiation. Identical protein band patterns were observed in the case of selected strains e.g. strains C. sedlakii studied in this study, coming from an outbreak, having the some phenotype.     

Persistense of mono- and associated cultures of conditionally pathogenic enterobacteria

The comparative study of adhesive, hemolytic, DNA-ase, lecithinase, antilysozymic, anticomplementary activities of mono- and associated cultures of 57 Enterobacter spp., 61 Citrobacter spp. and 55 Serratia spp. strains, isolated from patients with pyoinflammatory, intestinal and urological diseases is carried out. Different variations of cocultivated bacteria including Enterobacter and Citrobacter, Enterobacter and Serratia, Citrobacter and Serratia are used. It was shown, that cocultivated Enterobacter, Citrobacter and Serratia bacteria increased the persistent properties of mixt cultures.     

Diminished reproduction, failure to thrive, and altered immunologic function in a colony of T-cell receptor transgenic mice: possible role of Citrobacter rodentium

Citrobacter rodentium from an undetermined source was detected in a breeding colony of T-cell receptor transgenic mice housed in a conventional mouse facility in which murine hepatitis virus had been endemic and Helicobacter spp. had been detected. Citrobacter rodentium, isolated from blood, spleen, and colon, correlated with a significant increase in mortality and morbidity in this breeding colony. Transgenic mice of all ages were affected by chronic debilitation, loss in reproductive efficiency, rectal prolapse, and acute death, resulting in the near loss of these noncommercially available strains. Several alterations in immunologic parameters were observed, including outgrowth of an unusual population of cells in the spleen and blood, reduction in ascites production, loss of the capacity of peritoneal exudate cells to serve as feeders for the cloning of long-term T-cell lines, and inhibition of antigen-specific cytotoxic T-cell activity. These altered immune functions also were apparent in commercially-derived nontransgenic mice cohoused with the infected colony and in overtly healthy transgenic and nontransgenic littermates. Citrobacter rodentium and murine hepatitis virus were eliminated ultimately on rederivation of the affected strains by embryo transfer. However, the rapid decrease in the health of the colony necessitated more immediate action. To reduce mortality and allow breeding to continue during rederivation of the transgenic lines, animals were treated with enrofloxacin and moved to a barrier facility. Antibiotic therapy significantly reduced morbidity and mortality, markedly increased litter size and frequency, and resulted in the normalization of many of the immunologic assays. The involvement of C. rodentium in altering viability of the colony and perturbing immunologic assays is suggested by correlation of the onset of the syndrome with the appearance of Citrobacter sp. and its resolution with the elimination of Citrobacter sp. from the colony. Whether infection with Citrobacter alone is causative or whether superinfection of murine hepatitis virus- and Helicobacter-infected mice is required remains to be determined.     

Metabolism of dicarboxylic amino acids and their amides in bacteria of the genus Citrobacter]

In 58 Citrobacter strains the pathways of the utilization of dicarbonic amino acids and their amides were studied. These organisms were found to be incapable of decarboxylating glutaminic and asparaginic acids, as well as their amides. All the strains could actively desamidizate asparagine. Not all of these strains showed glutaminase activity. Aspartate-aminotransferase occurred twice as often as alanine-aminotransferase, the level of activity being approximately the same. The Citrobacter strains desamidizated asparaginic acid with great constancy, but only in 1/3 of them this reaction occurred via an aspartase route. The desamidization of asparaginic acid in Citrobacter seemed to proceed in different ways. The desamidization of glutaminic acid was observed only in a part of the strains, and the reaction proceeded less actively.     

Western-style diet impedes colonization and clearance of Citrobacter rodentium

Western-style diet (WSD), which is high in fat and low in fiber, lacks nutrients to support gut microbiota. Consequently, WSD reduces microbiota density and promotes microbiota encroachment, potentially influencing colonization resistance, immune system readiness, and thus host defense against pathogenic bacteria. Here we examined the impact of WSD on infection and colitis in response to Citrobacter rodentium. We observed that, relative to mice consuming standard rodent grain-based chow (GBC), feeding WSD starkly altered the dynamics of Citrobacter infection, reducing initial colonization and inflammation but frequently resulting in persistent infection that associated with low-grade inflammation and insulin resistance. WSD’s reduction in initial Citrobacter virulence appeared to reflect that colons of GBC-fed mice contain microbiota metabolites, including short-chain fatty acids, especially acetate, that drive Citrobacter growth and virulence. Citrobacter persistence in WSD-fed mice reflected inability of resident microbiota to out-compete it from the gut lumen, likely reflecting the profound impacts of WSD on microbiota composition. These studies demonstrate potential of altering microbiota and their metabolites by diet to impact the course and consequence of infection following exposure to a gut pathogen.     

Flagellar antigens of E. coli serologically interrlelated to H40, 41 and H41, 42, 43 flagellar antigens of Citrobacter. New H-antigeny E. coli i Citrobacter.]

The authors confirmed the reference of the test strains H13 (P6c) and H22 (A231a) of the international collection of E. coli to Citrobacter; their antigenic formula was established. As shown, strains P6c possessed a variety of the H-antigen which was not described in Citrobacter earlier, designated as H41a, 97. Three types of flagellar antigens characterized by the presence of an interrelationship with the partial factor H41 of the flagellar Citrobacter antigens were revealed in E. coli; the partial composition of H-antigenic components common for E. coli and Citrobacter was studied. Two of three new varieties of the E. coli H-antigen revealed was characterized by a cross correlation and a relation to the standard H19 E. coli antigen. The strain with the third variety of the H-antigen was capable of forming the H-antigenic mutants which acquired the antigenic component identical to the standard H16 E. coli antigen. E. coli strain is recommended for the replacement of the strain P6c in the International collection of E. coli.     

Resolution of high-molecular-weight components in lipopolysaccharides of Escherichia coli, Morganella morganii, Citrobacter freundii and Citrobacter diversus strains with sodium dodecyl sulfate polyacrylamide gels

The use of 0.5% sodium dodecyl sulfate in polyacrylamide separation gels allowed the resolution in several bands of high-molecular-mass components in smooth lipopolysaccharide of bacterial outer membrane from Escherichia coli, Morganella morganii, Citrobacter freundii and Citrobacter diversus. With or without 0.1% SDS, however, such a result was not possible.