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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.

The Auto Microbic System (AMS) is an almost completely automated system, capable of identifying Enterobacteriaceae after 8 hours, and some glucose nonfermenters after 13 hours of incubation. The Autobac ID system is a mechanized, computer-assisted system capable of identifying Enterobacteriaceae and many nonfermentative gram-negative bacilli within 3-6 hours. The present report combines the results of two independent studies, both of which evaluated the AMS and the Autobac ID system compared with standard reference tests. Among the 1,510 isolates that were tested, both systems reported equivocal identifications (low confidence values) with 5-6% of the strains. AMS produced fewer erroneous identifications (3.8% vs. 4.9%) but more equivocal test results (5.7% vs. 4.9%). Reproducibility of the two systems was compared by triplicate testing of 88 selected strains in both laboratories. AMS was somewhat more reproducible than the Autobac ID system. The AMS was capable of identifying more species with greater accuracy and reproducibility, but the Autobac ID system was more rapid. Both systems demonstrated excellent accuracy and reproducibility and both could be used efficiently in the clinical laboratory.

The isolation and analysis of the Citrobacter O-serogroup Ci23Vi+ murein are described. The murein consists of alanine, glutamic acid, diaminopimelic acid (occurring in the molar ratio 1.5 : 1: 0.9), N-acetylmuramic acid and N-acetylglucosamine. Dialysable products resulting from the digestion of the Citrobacter O-serogroup Ci23Vi+ murein with egg white lysozyme resemble closely those obtained from the E. coli B murein.

Novel enterobacterial core oligosaccharides were isolated from Citrobacter O4 and O36 lipopolysaccharides, and their structures were determined by methylation analysis, Smith degradation and enzymatic degradations, gas chromatography/mass spectrometry, and two-dimensional phase-sensitive correlated, relayed coherence transfer, double-quantum, triple-quantum-filtered, and nuclear Overhauser effect (NOE) 1H NMR spectroscopy at 500 MHz. In the formulas, all hexose residues are D-hexopyranoses, and heptoses are L-glycero-D-manno-heptopyranoses; the alternative locations of the side-chain heptose and pyrophosphorylethanolamine (PPEtN) residues are marked by dashed lines; dOclA stands for 3-deoxy-D-manno-octulosonic acid. (formula; see text) Along with these complete cores, incomplete ones, lacking the hexosamine trisaccharides, occur in the lipopolysaccharides of both types. Qualitative NOE data were in good agreement with the minimum energy conformation of the above O36 oligosaccharide, calculated with the aid of the SUGAR program [Sundin, A., Carter, R. E., & Liljefors, T. (1988) J. Mol. Graphics (in press)].

Citrobacter freundii from an environmental source displayed resistance to a wide variety of antibacterials. Some of these resistances could be transferred by conjugation with frequencies as high as 10(-2). The complexity of plasmid involvement in the antibacterial resistance of this species was demonstrated by isolation of plasmid DNA from two isolates. In one strain, resistance to beta-lactam antibiotics was specified by a 55 x 10(6) molecular weight plasmid, whereas kanamycin and neomycin resistance was specified by a 7 x 10(6) molecular weight plasmid. Resistance to chloramphenicol and tetracycline was not plasmid mediated. The other strain contained plasmids of molecular weight 113 x 10(6) and 80 x 10(6), which specified resistance to chloramphenicol, neomycin, kanamycin, tetracycline, and mercury.

Chemical studies on carbohydrate part of lipopolysaccharides isolated from Citrobacter 87/57 and Citrobacter 1556 strains (serotype 023) were carried out. Lipopolysaccharides (LPS) were hydrolyzed with 1% acetic acid and the carbohydrate material was fractionated on Sephadex G-50 and Bio-Gel P-4 columns. All fractions obtained were subjected to chemical analysis by colorimetric methods and by gas-liquid chromatography. It was found that O-specific polysaccharide from LPS 87/57 is composed of mannose and N-acetylgalactosamine in the molar ratio of 3:1. Both lipopolysaccharides contain unsubstituted core oligosaccharides (built of heptose, glucose and galactose in molar ratio of 3:3:1) as well as core oligosaccharides substituted with shorter (LPS 87/57 and LPS 1556) and longer (LPS 87/57) O-specific oligosaccharides. Strain 87/57 is of S (smooth) type and strain 1556 lacking O-specific polysaccharide is of SR (semi-rough) type. The data of Sephadex G-100 chromatography showed that O-antigen from Citrobacter 87/57 is heterogeneous; it contains polysaccharide chains of different length but the same composition. The heterogeneity of Citrobacter 023 lipopolysaccharides has been also detected by SDS-polyacrylamide gel electrophoresis.

Culture medium content of free lipopolysaccharide (LPS) components spontaneously released from a Citrobacter freundii culture grown in minimum synthetic medium was determined during early (8-hr culture) and late (24-hr culture) phases of growth. As judged by Limulus-lysate test, free LPS occurred in the medium as early as after 8 hrs of incubation, i.e. at the beginning of log growth phase. As the culture continued to grow the LPS amount released into culture medium kept rising, reaching 30% of endotoxin present in 24-hr Citrobacter culture. The released LPS complex was isolated by separation and its physicochemical, immunochemical and biological properties were determined and compared with those of cell-bound endotoxin recovered from cells by phenol extraction. Comparisons revealed distinct differences in the chemical composition and the degree of heterogeneity; free LPS was less heterogeneous. Immunologically, free LPS differed from bound LPS in the structure of macromolecules, but was identical with it in some antigenic determinants. The biological activity of free LPS preparation was greater than that of cell-bound LPS.

The structure of Citrobacter 027 lipopolysaccharide core has been established using sugar and methylation analyses and 1H-NMR spectroscopy, and was shown to be identical to the core described recently in PCM 1487 strain which represents a separate serotype in Citrobacter genus.

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.

Structural studies on the O-specific polysaccharide of Citrobacter PCM 1487 lipopolysaccharide, using methylation analysis, Smith degradation and 1H-NMR spectroscopy, indicate that it consists of the trisaccharide repeating units (formula, see text) In this structure, 4-deoxy-D-araHex stands for 4-deoxy-D-arabino-hexose.

Lipopolysaccharides from three gram-negative bacteria isolated from bale cotton and piggery air were analyzed for their chemical composition, and their pulmonary toxicity for guinea pigs, lethal toxicity for mice, and pyrogenicity for rabbits were measured. Lipopolysaccharides from Enterobacter agglomerans and Citrobacter freundii had closely related chemical compositions; both were pyrogenic for rabbits and caused a dose-dependent influx of polymorphonuclear leukocytes into the airways of guinea pigs. The lethal toxicities of these lipopolysaccharides in mice were comparable to that of Salmonella typhimurium lipopolysaccharide, which was used as a reference. Lipopolysaccharide from Agrobacterium sp. was chemically different from those of E. agglomerans and C. freundii, did not induce any influx of polymorphonuclear leukocytes, and was only weakly toxic or pyrogenic. The low biological activity of the agrobacterial lipopolysaccharide may be due to its different chemical composition.

The structure of Citrobacter O23-specific polysaccharide has been shown by sugar and methylation analyses of the native and chemically degraded polysaccharide and by 1H- and 13C-n.m.r. spectroscopy to consist of the tetrasaccharide repeating-units: ----4)-alpha-D-Man-(1----2)-alpha-D-Man-(1----2)-beta-D-Man- (1----3)-alpha-D-GalNAc-(1----, 80% of which are substituted by O-acetyl groups.

We purified and sequenced the heat-stable enterotoxin produced by Citrobacter freundii. The toxin was detected during purification by reaction with monoclonal antibody to Escherichia coli heat-stable enterotoxin. The C. freundii toxin amino acid sequence was identical to that of the 18-amino-acid heat-stable enterotoxin (STa) produced by toxigenic E. coli.

The authors studied antigens obtained by Grasset's method from 13 strains of Citrobacter of the International collection. The strains possessed O- and H-antigens whose behaviur in the electric field differed. All the strains under study were divided into two groups (by the number of serologically-active components of their O-antigens); representatives of the second group had no cathode O-antigen component. Chemical composition of specific lipopolysaccharides (LPS) obtained by Westphal's method was determined. Fourteen different sugars were revealed. The strains under study were referred to the known chemotypes. Strain 16/52 (8a, 8c) was for the first time studied in respect to the monosaccharide composition of specific LPS, and was referred to chemotype designated as CC-L.

The method of Lowry as modified by Peterson (who had changed concentrations of reagents and added a new reagent, sodium dodecyl sulfate) has been tested. It has been shown that the modified method can be applied for rapid measurement of protein in the suspension of bacterial cells. The effect of the following compounds on the protein estimation by the modified method in bovine serum albumin has been studied: potassium chloride, potassium phosphate, sucrose, EDTA, tris, ammonium acetate, Triton X-100, tyrosine, phenol, glycerol, 2-mercaptoethanol.

Lipopolysaccharide preparations from R(rough) Escherichia coli O8-,SR(semirough) Salmonella typhimurium and S (smooth) strains E. coli O8 and Citrobacter 396 were disintegrated with sodium dodecylsulfate and subjected to polyacrylamide gel electrophoresis in the presence of 1% sodium dodecylsulfate. The results obtained were compared with those obtained from the same lipopolysaccharide preparations by degradation analysis. In dodecylsulfate gel electrophoresis the lipopolysaccharide preparation from the E. coli R mutant and the S. typhimurium SR mutant showed one band each (R-and SR-band, respectively) with different electrophoretic mobilities. The lipopolysaccharide preparations from the E. coli O8-strain exhibited two bands, one of which had the same electrophoretic mobility as the R-band and the other was identified as S-band. The lipopolysaccharide preparation from the Citrobacter 396-S-strain exhibited four bands: one R-band, one SR-band and two S-bands. The results showed that wild-type S strains contain more than one type of lipopolysaccharide. They differ in the length of their O-specific polysaccharide chains. The lipid A content of the different lipopolysaccharide was expressed in their electrophoretic mobilities.

Polynucleotide sequence relatedness studies were carried out to determine the extent of divergence present in Salmonella sub-genus IV strains, related strains of Salmonella of other sub-genera and the Citrobacter genus. Salmonella sub-genus IV were 91-97% related. The Salmonella of sub-genus I, II and III showed lower binding (79-87%) to Salmonella sub-genus IV. The change in thermal elution midpoint closely followed the reassociation data. Relatedness of Salmonella sub-genus IV and C. freundii ranged between 44 and 57%, which confirms that these organisms belong to different genera. The taxonomic position of Salmonella sub-genus IV is discussed according to the actual classification of the family Enterobacteriaceae.

Demethylmenaquinone and menaquinone mixtures from some species of enterobacteria were analysed by reverse-phase partition high-performance liquid chromatography. This method allowed clear separation and quantitative determination of these quinone components.

The interaction of the 20-kDa pore-forming domain of colicin A with phospholipid vesicles was investigated by gel permeation chromatography, analytical centrifugation, and electron microscopy. Under the experimental conditions of this study, this peptide was found to interact only with vesicles containing negatively charged phospholipids. It forms a well-defined disklike complex with phosphatidylglycerols with a preference for those containing 12-14 atoms of carbon in their fatty acid chain. This complex has a diameter of 120 A and is about one bilayer thick. It contains nine molecules of peptide and is formed both at acidic pH (pH 5.0) and at neutral pH (pH 7.2).

The structure of the O-specific polysaccharide moiety of the lipopolysaccharide from Citrobacter 396 was elucidated by composition, methylation, and periodate oxidation studies. The repeating unit consists of four 2-linked mannoses and one 3-linked N-acetylglucosamine. One of the mannose units is substituted at C3 with alpha-glucose, and one is substituted at C3 with alpha-(2-O-acetyl)-abequose. All the mannosyl linkages appear to have the beta-configuration; the N-acetylglucosaminyl linkage has the alpha-configuration. In bacterial agglutination and passive hemagglutination in some Salmonella antisera, Citrobacter 396 as well as its O-antigenic lipopolysaccharide expressed the serological factors 5 and 6. In corroboration of our structural studies, this showed the presence of alpha-(2-O-acetyl)-abequosyl-1,3-mannose (factor 5) and alpha-glucosyl-1,3-mannose (factor 6).

The deoxyribonculeic acid (DNA) of 106 strains of Enterobacteria was analysed for the guanine + cytosine (GC) content. These strains, whose origin and principal characters are described in the text, belong to the genera Citrobacter (C. freundii H2S-) and Levinea (L. malonatica and L. amalonatica). Four other groups or classes named C.D.E. and F. could not be classified on the base of the usual phenotypic criteria. DNA from the strains of Levinea has a GC% of 50.3 to 53.3, while DNA from the strains of C. freundii H2S- has a GC% of 48.6 to 51.7. The representative values from the new classes are C, 50.9%; D, 54%; E, 52.7%; F, 49.5%. For the latter a genomic heterogeneity was shown, expressing itself as two subpopulations whose average GC% are 51.7 and 48.6 respectively. Statistical analysis of the averages give a significant individuality to these new classes.

Investigations of nursery outbreaks of Citrobacter diversus sepsis and meningitis have been hampered by lack of adequate epidemiologic markers for the organism. We studied outer membrane protein profiles from clinical isolates of C. diversus by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine whether this method might be useful in the epidemiologic differentiation of strains. Paired cerebrospinal fluid isolates from each of three separate nursery outbreaks of C. diversus meningitis, paired isolates from the vagina of a postpartum woman and the cerebrospinal fluid of her newborn infant, one isolate from an infant with pneumonia and two from colonized nursery cohorts, and 30 epidemiologically unrelated clinical isolates were included. Eleven distinct profiles were differentiated by the presence or absence of five outer membrane proteins. Complete concordance of profiles was observed for epidemiologically related isolates. Unrelated epidemic strains had outer membrane protein profiles distinct from one another. Biotyping complemented determination of outer membrane protein profiles; the two markers differentiated each of the five epidemic strains from all but one of 30 unrelated nonepidemic isolates. Determination of outer membrane protein profiles is potentially useful in epidemiologic investigations of disease caused by C. diversus.