Immunoelectrophoretic study of lipopolysaccharide antigens fromVibrio anguillarum strains,serogroup O2

Vibrio anguillarum isolates, derived from feral as well as cultured fish and recorded as serogroup O2 by slide agglutination, were selected for an immunoelectrophoretic study of lipopolysaccharide antigens. Antigenic preparations for the immunoelectrophoretic analyses were simple water extracts, heated to 100°C for 1 h. Two immunoelectrophoretic distinct lipopolysaccharide entities were detected. The analyses did not demonstrate serologic variations in lipopolysaccharide antigens among 16 O group 2 strains. The study also included an O1K1V. anguillarum strain. Antigenic extract from this strain was not precipitated by OK antiserum againstV. anguillarum serogroup O2.     

Isolation of K88 Antigen Variants (ab,ac, ad) from Porcine Enterotoxigenic Escherichia coli Belonging to Different Serotypes

Fimbriae isolation by means of thermal shock was applied to fifteen K88-positive (three K88ab, nine K88ac and three K88ad) Escherichia coli reference strains belonging to serotypes O8:K87, O32, O45, O138:K81, O141:K85, O147:K89, O149:K91, and O157, as well as to ten K88-positive enterotoxigenic strains isolated from porcine diarrhea in Spain, all of them belonging to the O149 serogroup. Fimbriae were removed from the bacterial cells by thermal shock at 60 C and then precipitated using ammonium sulfate. The final amount of K88 antigen and the purification degree were not related to the serogroup of the bacteria or to the antigen variant but were related to the buffer used for isolation. Phosphate buffer containing urea was shown to be more effective than Tris-HCl for isolation of K88 antigen. The molecular weights by SDS-PAGE for K88ab, K88ac, and K88ad were 28.5, 29.2, and 31.0 kDa, respectively. All enterotoxigenic E. coli strains isolated in Spain showed the K88ac variant.     

Evidence for two newVibrio anguillarum K antigens

Surface polysaccharides from five strains ofVibrio anguillarum were studied by means of immunoelectrophoretic procedures. The study suggested existence of two new K antigens, displaying cross-reactivity, in strains derived from diseased feral fish. The importance of a detailed serologic characterization of isolates for ecologic and epidemiologic studies ofV. anguillarum is considered.     

Exposure of cell surface structures inEscherichia coli as studied with ultrastructural immunohistochemistry

The surfaces of two smoothEscherichia coli O6 strains, one with K13 capsular antigen and one without demonstrable capsule, and one roughE. coli strain were analyzed by using a sandwich technique with ultrastructural immunohistochemistry. Peroxidase-conjugated anti-rabbit serum and antisera corresponding to the distal lipopolysaccharide ofE. coli (O antigen), to the capsule (K antigen), or to the lipid part of the lipopolysaccharide, lipid A, were used. Using the anti-capsule serum on the smooth-capsule-producing strain, we observed the capsule as loosely connected clusters covering less of the surface than the O antigen. No reactions were observed with the capsule-negative strain having the same O antigen. The O antigen was identified all over the outer membrane in a patchy pattern with knobs covering the whole surface on the capsule-negative strain. Fewer knobs, but more variable in size than on the capsule-negative strain, were observed on the capsule-producing strain. The rough strain showed no reactions with either the O or K antiserum. Lipid A was not available for anti-lipid A serum on the rough or smooth strains.     

Further antigenic analyses of the fish pathogenic bacteriumVibrio anguillarum

TenVibrio anguillarum strains were selected for an immunoelectrophoretic study. Evidence was provided for existence of two new K antigens which displayed cross-reactivity. The importance of an exact characterization of surface antigens inV. anguillarum is considered.     

Subgrouping of lipopolysaccharide O antigens fromVibrio anguillarum serogroup O2 by immunoelectrophoretic analyses

Lipopolysaccharide antigens from 40Vibrio anguillarum slide agglutination serogroup O2 strains, isolated from different species of diseased fish, were studied by means of immunoelectrophoretic techniques. The study divided the examined strains into two O antigenic subgroups, designated O2a and O2b. Subgroup O2a was implicated more frequently than O2b in vibriosis among salmonids as well as other species of fish. Subgroup O2b was primarily associated with disease in nonsalmonids. Bacterial virulence and host preference are considered in relation to O specificity.     

Characterization of Vibrio cholerae bacteriophage K139 and use of a novel mini-transposon to identify a phage-encoded virulence factor

Temperate bacteriophage K139 was isolated from a Vibrio cholerae O139 isolate and characterized in this study. The phage genome consists of a 35 kbp, double-stranded, linear DNA molecule that circularizes and integrates into the chromosome in a site-specific manner. DNA sequences that cross-hybridize with K139 phage DNA are present in all strains of V. cholerae serogroup O1 of the classical biotype examined and in some strains of the El Tor biotype. Phage K139 produces plaques on El Tor O1 strains that do not carry the K139-related sequences but does not plaque on O139 strains that lack detectable phage DNA. This results suggests that O139 strains arose in part by horizontal gene transfer of the O139 antigen genes into an El Tor O1 strain that harboured a K139 prophage. Consistent with this interpretation, the morphology of K139 phage particles is identical to that displayed by the widely distributed family of O1 phages referred to as ‘kappa’. In order to test whether K139 phage is involved in lysogenic conversion of V. cholerae, we constructed a novel mini-transposon, Tn10d-bla, which was designed to produce β-lactamase fusions to phage-encoded, exported proteins. All Tn 10d-bla insertions obtained were closely linked to one location on the K139 phage genome. DNA sequence determination of the fusion joints revealed an open reading frame (ORF1), encoding a gene product of 137 amino acids with a typical N-terminal hydrophobic signal sequence. ORF1 was designated the glo gene (G protein-like O RF) because its amino acid sequence shows similarity to eukaryotic Gs(α) protein (34.5% identity over an 81-amino-acid overlap) and its C-terminus displays the consensus motif (CAAX) which is found in many small eukaryotic GTP-binding proteins. LD₅₀ assays with isogenic Glo⁺ and Glo^? K139 lysogens suggest that glo encodes a secreted virulence determinant of V. cholerae     

Sequence of the E. coli O104 antigen gene cluster and identification of O104 specific genes

The Escherichia coli O104 polysaccharide is an important antigen, which contains sialic acid and is often associated with EHEC clones. Sialic acid is a component of many animal tissues, and its presence in bacterial polysaccharides may contribute to bacterial pathogenicity. We sequenced the genes responsible for O104 antigen synthesis and have found genes which from their sequences are identified as an O antigen polymerase gene, an O antigen flippase gene, three CMP-sialic acid synthesis genes, and three potential glycosyl transferase genes. The E. coli K9 group IB capsular antigen has the same structure as the O104 O antigen, and we find using gene by gene PCR that the K9 gene cluster is essentially the same as that for O104. It appears that the distinction between presence as group IB capsule or O antigen for this structure does not involve any difference in genes present in the O antigen gene cluster. By PCR testing against representative strains for the 166 E. coli O antigens and some randomly selected Gram-negative bacteria, we identified three O antigen genes which are highly specific to O104/K9. This work provides the basis for a sensitive test for rapid detection of O104 E. coli. This is important both for decisions on patient care as early treatment may reduce the risk of life-threatening complications and for a faster response in control of food borne outbreaks.     

Role of Capsule and O Antigen in the Virulence of Uropathogenic Escherichia coli

Urinary tract infection (UTI) is one of the most common bacterial infections in humans, with uropathogenic Escherichia coli (UPEC) the leading causative organism. UPEC has a number of virulence factors that enable it to overcome host defenses within the urinary tract and establish infection. The O antigen and the capsular polysaccharide are two such factors that provide a survival advantage to UPEC. Here we describe the application of the rpsL counter selection system to construct capsule (kpsD) and O antigen (waaL) mutants and complemented derivatives of three reference UPEC strains: CFT073 (O6:K2:H1), RS218 (O18:K1:H7) and 1177 (O1:K1:H7). We observed that while the O1, O6 and O18 antigens were required for survival in human serum, the role of the capsule was less clear and linked to O antigen type. In contrast, both the K1 and K2 capsular antigens provided a survival advantage to UPEC in whole blood. In the mouse urinary tract, mutation of the O6 antigen significantly attenuated CFT073 bladder colonization. Overall, this study contrasts the role of capsule and O antigen in three common UPEC serotypes using defined mutant and complemented strains. The combined mutagenesis-complementation strategy can be applied to study other virulence factors with complex functions both in vitro and in vivo.     

Aerobactin production and dulcitol fermentation in clonal groups of Escherichia coli O1: K1 strains from urinary tract infections

Abstract 70 urinary Escherichia coli O1:K1 strains were characterized for O1 antigen factors, mannose-resistant hemagglutination of human erythrocytes, flagellar and fimbrial antigens, dulcitol fermentation and aerobactin production. On the basis of their O1 and H antigens the strains could be assigned to 6 distinct groups. The most prevalent groups were: O1abcd: H⁻ :F9 (33 strains; pattern II), O1abc: H⁻ :F11 (9 strains; pattern IV), and O1abc: H7: F11 (19 strains; pattern V). Strains with patterns IV and V, both expressing fimbrial antigen F11, fermented dulcitol and produced aerobactin, whereas strains with pattern II were negative for both characteristics.     

Serological variety of flagellar antigen H1 in natural Escherichia coli population

Abstract Variation of the Escherichia coli flagellar antigen H1 was studied among 120 human isolates belonging to more than 25 O:K serovars. Factor-specific antisera were prepared and shown to be useful in the identification of serological subtypes of H1. The three subtypes found were defined as H1abc, H1acd and H1abe. The H1abc corresponded to the standard flagellar antigen H1 present in 84% of all strains. It was found in all O groups except O15, O17 and O83. Eight O15 and two O17 strains studied were of subtype H1abe, while the one O83 strain studied was H1acd. Both subtypes H1abc and H1acd were found among strains within O6:K5, O6:K13 or O-non-typeable:K5 serovars.     

Different mutations in the oafA gene lead to loss of O5-antigen expression in Salmonella enterica serovar Typhimurium

Aims: To analyse genetic changes in the oafA gene explaining the loss of O5‐antigen expression in Salmonella Typhimurium and Salm. 4,[5],12:i:‐. Methods and Results: The oafA gene in 52 O5‐antigen‐negative and 77 O5‐antigen‐positive Salm. Typhimurium (N = 47) and Salm. 4,[5],12:i:‐ (monophasic Salm. Typhimurium strains, N = 82) was investigated by a combination of PCR screening and DNA sequencing to identify mutations leading to the suppression of the O5‐antigen. Various DNA sequence changes within the open reading frame (ORF) of oafA in O5‐antigen‐negative strains could be identified. In 77% of the O5‐antigen‐negative strains, a 7‐bp deletion of a duplicated sequence within the functional oafA gene led to a frameshift in the ORF. In four strains, an IS4 element and in two, an IS1 element was inserted at different positions. Four other strains carried at different positions single base pair substitutions causing a premature stop codon. Finally, in two strains, a deletion of the oafA 3′end of undetermined size was responsible for the lack of O5‐antigen expression. In none of the strains investigated, the complete ORF of oafA was deleted. Primers were designed and used to detect the most prominent variants. Conclusions: O5‐antigen‐negative Salm. Typhimurium and Salm. 4,[5],12:i:‐ strains carry an oafA pseudogene caused by different genetic events indicating that there is a selection for oafA mutations leading to the loss of O5‐antigen expression. Significance and Impact of the Study: The loss of O5‐antigen expression may be an example of a common evolutionary mechanism to escape host defence or to adapt to environmental changes. The data are the basis for the development of diagnostic PCR assays for the differentiation of O5‐antigen‐positive and O5‐antigen‐negative Salm. Typhimurium and its monophasic (Salm. 4,[5],12:i‐) strains.     

Serogroups, K1 antigen, and antimicrobial resistance patterns of Aeromonas spp. strains isolated from different sources in Mexico

A total of 221 strains of Aeromonas species isolated in Mexico from clinical (161), environmental (40), and food (20) samples were identified using the automated system bioMérieux-Vitek. Antisera for serogroups O1 to 044 were tested using the Shimada and Sakazaki scheme. The K1 antigen was examined using as antiserum the O7:K1C of Escherichia coli. Besides, we studied the antimicrobial patterns according to Vitek AutoMicrobic system. Among the 161 clinical strains 60% were identified as A. hydrophila, 20.4% as A. caviae, and 19.25% as A. veronii biovar sobria. Only A. hydrophila and A. veronii biovar sobria were found in food (55 and 90% respectively) and environmental sources (45 and 10% respectively). Using "O" antisera, only 42.5% (94/221) of the strains were serologically identified, 55% (121/221) were non-typable, and 2.5% (6/221) were rough strains. Twenty-two different serogroups were found, O14, O16, O19, O22, and O34 represented 60% of the serotyped strains. More than 50% of Aeromonas strain examined (112/221) expressed K1 encapsulating antigen; this characteristic was predominant among Aeromonas strains of clinical origin. Resistance to ampicillin/sulbactam and cephazolin was detected in 100 and 67% of Aeromonas strain tested for their susceptibility to antibiotics. In conclusion, antibiotic-resistant Aeromonas species that possess the K1 encapsulating antigen and represent serogroups associated with clinical syndrome in man are not uncommon among Aeromonas strains isolated from clinical, food and environmental sources in Mexico.     

Adjuvant Action of Capsular Polysaccharide of Klebsiella pneumoniae on Antibody Response

A study was made to characterize the active substance for the extraordinarily strong adjuvant effect of the capsular polysaccharide of Klebsiella pneumoniae (CPS-K) type 1 Kasuya strain. CPS-K was fractionated into acidic and neutral CPS-K by the addition of cetyl-pyridinium chloride. Neutral CPS-K exhibited an extremely strong adjuvant effect. The active substance in neutral CPS-K was precipitable when mixed with a rabbit homologous antiserum. The neutral CPS-K antigen was serologically distinct from the O antigen and from the acidic CPS-K which was the type-specific capsular antigen. Among preparations of neutral CPS-K from eight different strains of K. pneumoniae tested, the preparation from only one strain (MH-2) exhibited a strong adjuvant effect comparable to that of the neutral CPS-K from the Kasuya strain. The neutral CPS-Ks from Kasuya and MH-2 strains were antigenically identical. This antigen was not found in all preparations of neutral CPS-Ks obtained from seven different strains. Preparations of acidic CPS-Ks from all strains of K. pneumoniae tested with various serologic types including Kasuya and MH-2 strains were found to exhibit only weak adjuvant effects. The active substance (neutral CPS-K antigen from Kasuya strain) was shown to form a single peak upon analyses by gel filtration (Sephadex G-100) and ultracentrifugation. Sedimentation coefficient of the substance was approximately 20 S at a concentration of 5 mg per ml in 0.1 M NaCl. The active substance finally purified by gel filtration contained 65% sugars (as glucose equivalents), 6.8% hexuronic acids, 2.6% hexosamine, 2.3% proteins, and very small amounts of lipids.     

Characterization of Aeromonas hydrophila Strains of Clinical,Animal, and Environmental Origin Expressing the O:34 Antigen

A collection of Aeromonas strains of different origins were characterized for isolates expressing the O:34 somatic antigen. Of over 200 strains tested, approximately 14% belonged to serogroup O:34 with >85% of these strains identified as A. hydrophila regardless of source. A subset of 14 A. hydrophila O:34 strains were further analyzed for a number of structural and pathogenic features. Most O:34 strains expressed similar whole-cell protein profiles with regards to minor bands, but major band differences were noted in outer membrane proteins (OMPs) migrating between the 31K and 58K region. OMP profiles could be subdivided into three distinct patterns. All O:34 strains expressed a heterogeneous O polysaccharide side chain profile in their lipopolysaccharide (LPS), although some variation in the electrophoretic migration of lower and higher molecular weight LPS bands was noted. Polyclonal antisera raised against a 45-K OMP-associated protein of one O:34 strain (AH-195) reacted in immunoblot assays with a major 43 to 46-K OMP in 11 of 14 (79%) O:34 strains tested. Most O:34 strains (69%) were found to be pathogenic in mice with LD-50 values (i.p.) of <1.0 × 10⁷ CFU; pathogenicity appeared to correlate best with elevated protease activity. The collective results suggest significant differences in both structural and pathogenic properties between some members of the O:34 group originating from human and nonhuman (fish, water) sources. Received: 14 December 1995 / Accepted: 22 January 1996     

Laboratory adapted Escherichia coli K‐12 becomes a pathogen of Caenorhabditis elegans upon restoration of O antigen biosynthesis

Escherichia coli has been the leading model organism for many decades. It is a fundamental player in modern biology, facilitating the molecular biology revolution of the last century. The acceptance of E. coli as model organism is predicated primarily on the study of one E. coli lineage; E. coli K‐12. However, the antecedents of today's laboratory strains have undergone extensive mutagenesis to create genetically tractable offspring but which resulted in loss of several genetic traits such as O antigen expression. Here we have repaired the wbbL locus, restoring the ability of E. coli K‐12 strain MG1655 to express the O antigen. We demonstrate that O antigen production results in drastic alterations of many phenotypes and the density of the O antigen is critical for the observed phenotypes. Importantly, O antigen production enables laboratory strains of E. coli to enter the gut of the Caenorhabditis elegans worm and to kill C. elegans at rates similar to pathogenic bacterial species. We demonstrate C. elegans killing is a feature of other commensal E. coli. We show killing is associated with bacterial resistance to mechanical shear and persistence in the C. elegans gut. These results suggest C. elegans is not an effective model of human‐pathogenic E. coli infectious disease.     

Virulence of Escherichia coli in acute pyelonephritis,acute cystitis and asymptomatic bacteriuria

E. coli strains were isolated from urine specimens of hospitalised patients with acute pyelonephritis, acute cystitis or asymptomatic bacteriuria (ABU), and tested for virulence in an experimental mouse model. Of 12 pyelonephritisstrains 11 were shown to be virulent and 1 avirulent; of 12 cystitis-strains 4 were virulent and 8 avirulent; of 12 ABU-strains 5 were virulent and 7 avirulent. It is concluded that, while no difference in virulence was found between cystitis-and ABU-strains, pyelonephritis-strains were more often virulent than cystitis-and ABU-strains.No associations could be shown between virulence of the isolated strains and the presence of antibody-coated bacteria in the urine. Common urinary O types were not more often virulent than other O types. No relationship was seen between virulence and the presence of K antigen or the presence of particular K types.     

Novel aerobactin receptor in Klebsiella pneumoniae

Several Klebsiella pneumoniae strains which produced enterochelin but not aerobactin were nevertheless sensitive to cloacin DF13. In contrast, a strain of serotype K1:O1 which produced both siderophores was cloacin-resistant. Loss by mutation of the O1 but not K1 antigen rendered this strain cloacin-sensitive, indicating that the O1 antigen prevented access of cloacin to the cloacin/aerobactin receptor. Unlike the K1:O1 strain, the aerobactin-negative strains failed to hybridize in a colony blot assay with an aerobactin receptor gene probe prepared from pColV-K30. However, antisera raised against the 74 kDa pColV-K30 aerobactin receptor cross-reacted with a 76 kDa outer-membrane protein in each K. pneumoniae strain. In addition to the 76 kDa protein, the K1:O1 strain also produced a strongly cross-reacting 74 kDa protein. To determine whether these aerobactin-negative strains could use aerobactin, mutants unable to synthesize siderophores were isolated. Aerobactin promoted the growth of these mutants in iron-deficient media. The evidence presented suggests that some K. pneumoniae strains produce an aerobactin iron-uptake system without apparent production of aerobactin and which is probably based on a 76 kDa receptor, the gene for which does not hybridize with aerobactin receptor gene encoded on pColV-K30.     

Lipopolysaccharide O1 antigen contributes to the virulence in Klebsiella pneumoniae causing pyogenic liver abscess

Klebsiella pneumoniae is the common cause of a global emerging infectious disease, community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) and lipopolysaccharide (LPS) are critical for this microorganism's ability to spread through the blood and to cause sepsis. While CPS type K1 is an important virulence factor in K. pneumoniae causing PLA, the role of LPS in PLA is not clear. Here, we characterize the role of LPS O antigen in the pathogenesis of K. pneumoniae causing PLA. NTUH-K2044 is a LPS O1 clinical strain; the presence of the O antigen was shown via the presence of 1,3-galactan in the LPS, and of sequences that align with the wb gene cluster, known to produce O-antigen. Serologic analysis of K. pneumoniae clinical isolates demonstrated that the O1 serotype was more prevalent in PLA strains than that in non-tissue-invasive strains (38/42 vs. 9/32, P<0.0001). O1 serotype isolates had a higher frequency of serum resistance, and mutation of the O1 antigen changed serum resistance in K. pneumoniae. A PLA-causing strain of CPS capsular type K2 and LPS serotype O1 (i.e., O1:K2 PLA strain) deleted for the O1 synthesizing genes was profoundly attenuated in virulence, as demonstrated in separate mouse models of septicemia and liver abscess. Immunization of mice with the K2044 magA-mutant (K(1) (-) O(1)) against LPS O1 provided protection against infection with an O1:K2 PLA strain, but not against infection with an O1:K1 PLA strain. Our findings indicate that the O1 antigen of PLA-associated K. pneumoniae contributes to virulence by conveying resistance to serum killing, promoting bacterial dissemination to and colonization of internal organs after the onset of bacteremia, and could be a useful vaccine candidate against infection by an O1:K2 PLA strain.     

Further studies of the polysaccharide of Klebsiella pneumoniae possessing strong adjuvanticity. II. Serological relationship between the adjuvant polysaccharide and O3 antigen of Klebsiella

The serological specificity of the neutral polysaccharide possessing extraordinarily strong adjuvanticity originally isolated from the culture supernatant of Klebsiella K1 strain Kasuya has been investigated. Among all of the reference strains (K1-K82) of Klebsiella obtained from the International Escherichia and Klebsiella Center, Statens Seruminstitut, Copenhagen, only 13 strains have been shown to produce the adjuvant polysaccharide by the passive hemagglutination inhibition test. All of these 13 strains belong to the O3 group, and the strains which belong to other O groups of which were not identifiable did not produce it. The gel precipitation test has demonstrated that the adjuvant polysaccharide is antigenically identical to O3 antigen isolated from the cells of the decapsulated mutant (strain LEN 1) of Klebsiella K1 strain Kasuya and to O9 antigen of Escherichia coli isolated from either the culture supernatant or the cells, which has already been shown to be antigenically and structurally identical to the O3 antigen of Klebsiella.