Capsular genotype and lipooligosaccharide locus class distribution in Campylobacter jejuni from young children with diarrhea and asymptomatic carriers in Bangladesh.

Campylobacter jejuni-related diarrheal diseases is one of the major health issues among young children (0-59 months old) in low-income countries. Monitoring of the capsular (capsule polysaccharide, CPS) types of virulent C. jejuni strains in regions where the disease is endemic is of great importance for the development of a customized capsule-based multivalent vaccine. Therefore, we aimed to determine the prevalence of CPS genotypes among C. jejuni strains isolated from young children with enteritis (n = 152) and asymptomatic carriers matched by age, sex, and residence defined as the control group (n = 215) in Bangladesh. CPS genotyping was performed using a newly established multiplex polymerase chain reaction (PCR) method and lipooligosaccharide (LOS) locus classes (A-E) were characterized using PCR as well. We identified 24 different CPS genotypes among the 367 isolates. Four prevalent capsular types, HS5/31 complex (n = 27, 18%), HS3 (n = 26, 17%), HS4A (n = 10, 7%), and HS8/17 (n = 10, 7%) covered almost 50% of the strains from enteritis patients and 43% of the isolates from controls. In combination, the CPS genotype and LOS class was not discriminative between cases and controls. Dominant capsular types previously identified in C. jejuni strains isolated from patients with Guillain-Barré syndrome in Bangladesh were rarely detected in strains isolated from the young children. A similar distribution was evident among enteritis- and control-related strains when comparison was done between CPS types and LOS classes. This is the first systematic study presenting the distribution of CPS genotypes of C. jejuni strains isolated in Bangladesh from children with diarrhea and controls, with capsular genotypes HS5/31 complex, HS3, HS4A, and HS8/17 being prevalent in both. In conclusion, systematic studies are required to develop a multivalent capsule-based vaccine for children in low-income countries.

Caught in the act: in vivo development of macrolide resistance to Campylobacter jejuni infection.

We report the first instance of macrolide resistance developing in vivo following appropriate antibiotic use in a healthy volunteer as a part of a controlled human infection with Campylobacter jejuni. In vivo development of macrolide resistance may be an important contributor to antibiotic resistance in C. jejuni.

Sequence of the immunodominant epitope for the surface protein on sporozoites of Plasmodium vivax.

Plasmodium vivax is one of the four malaria parasites that cause disease in humans. The structure of the immunodominant repeating peptide of the circumsporozoite (CS) protein of P. vivax was determined. A fragment of P. vivax DNA that encodes this tandemly repeating epitope was isolated by use of an oligonucleotide probe whose sequence is thought to be conserved in CS protein genes. DNA sequence analysis of the P. vivax clone indicates that the CS repeat is nine amino acids in length (Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala). The structure of the repeating region was confirmed with synthetic peptides and monoclonal antibodies directed against P. vivax sporozoites. This information should allow synthesis of a vaccine for P. vivax that is similar to the one being tested for P. falciparum.

Significance of duplicated flagellin genes in Campylobacter.

The complex flagellum of Campylobacter coli VC167 contains two highly related (98%) flagellin subunit proteins which are produced from two 92% homologous, tandemly orientated genes, flaA and flaB. Mutants expressing only flaA form a full-length flagellar filament that confers slightly less than wild-type motility to the bacterium. However, flagellin mutants expressing only flaB produce extremely short, truncated filaments, and are only slightly motile. We have shown that the presence of two essentially identical genes is advantageous, in that flaAflaB+ mutants become highly motile upon passage by an event which allows the production of a full length simple flagellar filament containing a single FlaA-FlaB chimeric flagellin protein. Furthermore, we have demonstrated that the reassortment of DNA that results in this chimeric protein can occur by two mechanisms: intragenomic recombination and transformation-mediated intergenomic recombination.

Campylobacter jejuni capsular genotypes are related to Guillain-Barré syndrome.

In about one in a thousand cases, a Campylobacter jejuni infection results in the severe polyneuropathy Guillain-Barré syndrome (GBS). It is established that sialylated lipo-oligosaccharides (LOS) of C. jejuni are a crucial virulence factor in GBS development. Frequent detection of C. jejuni with sialylated LOS in stools derived from patients with uncomplicated enteritis implies that additional bacterial factors should be involved. To assess whether the polysaccharide capsule is a marker for GBS, the capsular genotypes of two geographically distinct GBS-associated C. jejuni strain collections and an uncomplicated enteritis control collection were determined. Capsular genotyping of C. jejuni strains from the Netherlands revealed that three capsular genotypes, HS1/44c, HS2 and HS4c, were dominant in GBS-associated strains and capsular types HS1/44c and HS4c were significantly associated with GBS (p 0.05 and p 0.01, respectively) when compared with uncomplicated enteritis. In a GBS-associated strain collection from Bangladesh, capsular types HS23/36c, HS19 and HS41 were most prevalent and the capsular types HS19 and HS41 were associated with GBS (p 0.008 and p 0.02, respectively). Next, specific combinations of the LOS class and capsular genotypes were identified that were related to the occurrence of GBS. Multilocus sequence typing revealed restricted genetic diversity for strain populations with the capsular types HS2, HS19 and HS41. We conclude that capsular types HS1/44c, HS2, HS4c, HS19, HS23/36c and HS41 are markers for GBS. Besides a crucial role for sialylated LOS of C. jejuni in GBS pathogenesis, the identified capsules may contribute to GBS susceptibility.

Construction of new Campylobacter cloning vectors and a new mutational cat cassette.

We have developed new Campylobacter shuttle vectors which are 6.5-6.8-kb plasmids carrying Campylobacter and Escherichia coli replicons, a multiple cloning site (MCS), the lacZ alpha gene, oriT and either a kanamycin or chloramphenicol resistance-encoding gene (KmR or CmR) from Campylobacter which functions in both hosts. These vectors can be mobilized efficiently from E. coli into C. jejuni or C. coli, and stably maintained in these hosts. Plasmids pRY107 and pRY108 carry a KmR marker and 17 unique cloning sites in two different orientations in lacZ alpha, allowing easy blue/white color selection. Plasmids pRY111 and pRY112 contain a CmR gene and 17 unique sites in both orientations. In addition, MCS are flanked by T7 and T3 late promoters and M13 forward and reverse primer sites, facilitating expression in T7 or T3 expression systems and sequence analysis. A Campylobacter CmR gene cartridge, bracketed by six restriction sites, has been developed for use in site-specific mutagenesis of Campylobacter genes.

Synthesis and structure-activity relationship of (lactamylvinyl)cephalosporins exhibiting activity against staphylococci, pneumococci, and enterococci.

The synthesis and structure-activity relationships of a new class of vinylcephalosporins substituted with a lactamyl residue are described. These compounds show excellent activity against enterococci and retain the broad spectrum activity of third-generation cephalosporins such as ceftriaxone.

Evaluation of a synthetic oligonucleotide probe for diagnosis of Plasmodium falciparum infections.

A radiolabeled synthetic oligonucleotide was evaluated as a diagnostic probe specific for Plasmodium falciparum using blood samples lysed directly on nitrocellulose filters. The probe technique successfully diagnosed malaria in experimentally infected chimpanzees that had 0.001% parasitemias (50 parasites/microliter) as determined by blood smears, and in 1 chimpanzee whose blood smear was negative, but whose blood was culture-positive for P. falciparum. In a double blind study of 50 patient samples from the Philippines, the probe results correlated well with blood smear results when the autoradiographs were read after 4-8 hr exposure. The results indicate that the oligonucleotide probe may be useful in the rapid and specific diagnosis of P. falciparum infection.

[Psychodynamic understanding of anxiety]. / Compréhension psychodynamique de l'anxiété.

Anxiety as a state of arousal--Is it the same thing in pathology as in the ordinary experience of every human being? The outward show of anxiety is evidently the same, but what exactly do we mean by anxiety? Anxiety is one of the less welcome sides of man's superiority over the animals, stemming from a consciousness of one's own being, the freedom to make choices about the future and to plan ahead, the need to develop one's own personality, to create an identity for oneself in an environment where the means for such self-fulfillment appear always to be insufficient. This existential anxiety becomes pathological through symptoms; it manifests a specific weakness for each psychopathological structure. There are two different, but not irreconcilable approaches to understanding the mechanisms of anxiety: one biodynamic , the other psychodynamic. These two approaches should complement rather oppose one another. Anxiety is the cry of a self in the process of becoming, it is expressed through language. Administration of an anti-anxiety drug has a biochemical impact, but this should not be sufficient in itself. When a doctor prescribes an anti-anxiety drug, he should always encourage the patient to talk. Drug therapy thus becomes a means rather than an end in itself: It enables the patient to find the words to express his anxiety.

The structure of phosphate glass biomaterials from neutron diffraction and 31P nuclear magnetic resonance data.

Neutron diffraction and 31P nuclear magnetic resonance spectroscopy were used to probe the structure of phosphate glass biomaterials of general composition (CaO)0.5-x(Na2O)x(P2O5)0.5 (x = 0, 0.1 and 0.5). The results suggest that all three glasses have structures based on chains of Q2 phosphate groups. Clear structural differences are observed between the glasses containing Na2O and CaO. The P-O bonds to bridging and non-bridging oxygens are less well resolved in the neutron data from the samples containing CaO, suggesting a change in the nature of the bonding as the field strength of the cation increases [Formula: see text]. In the (CaO)0.5(P2O5)0.5 glass most of the Ca2+ ions are present in isolated CaOx polyhedra whereas in the (Na2O)0.5(P2O5)0.5 glass the NaOx polyhedra share edges leading to a Na-Na correlation. The results of the structural study are related to the properties of the (CaO)0.4(Na2O)0.1(P2O5)0.5 biomaterial.

Nonlipopolysaccharide surface antigens of Campylobacter species.

Among the protein antigens of Campylobacter species, flagellin, the subunit of the flagellar filament, is the best characterized. The motility imparted by this locomotory organelle is absolutely essential for Campylobacter organisms to colonize the gastrointestinal tract and to cause diarrheal disease. Flagellin is the immunodominant protein recognized during infection and has been suggested to be involved in the protective immune response. Campylobacter flagellins are glycosylated, which is an unusual posttranslational modification for prokaryotic proteins. Although the chemical structure of the glycosylated moiety is undetermined, the posttranslational modification includes sialic acid. The association of glycosylated flagellin with development of Guillain-Barré syndrome remains speculative, but the possibility of molecular mimicry between glycosylated flagellin and eukaryotic glycoproteins exists.

Polynucleotide sequence relationships among some bacterial plasmids.

The deoxyribonucleic acid of F-like plasmids appear to share a high degree of nucleotide similarity with each other but are not highly related to I-like plasmids.

Molecular cloning and site-specific mutagenesis of a gene involved in arylsulfatase production in Campylobacter jejuni.

The arylsulfatase gene from Campylobacter jejuni 81-176 encodes a predicted protein of 69,293 Da which shows no sequence similarity with other known arylsulfatases. The gene hybridizes to other Ast+ strains of C. jejuni and Campylobacter sputorum subsp. bubulus, as well as to many Ast- strains of C. jejuni.

R62, a naturally occurring hybrid R plasmid.

R62, a naturally occurring R factor, was shown to be a single deoxyribonucleic acid molecule composed of polynucleotide sequences typical of I group plasmids and also sequences typical of the N group. It determined I pili and belonged to the Ialpha compatibility group. Although compatible with plasmids of group N, R62 showed complex genetic reactions with N plasmids which are described and interpreted. It is concluded that R62 was the product of illegitimate recombination between an I group and an N group plasmid.

Isolation of cryptic plasmid deoxyribonucleic acid from Kanagawa-positive strains of Vibrio parahaemolyticus.

Twelve strains of Vibrio parahaemolyticus was examined for plasmid deoxyribonucleic acid (DNA) by dye-buoyant gradient centrifugation. Four Kanagawa-positive strains, all isolated from the same outbreak of gastroenteritis, contained multiple plasmid species of cryptic function. However, three Kanagawa-negative strains and five Kanagawa-positive strains were not found to contain demonstrable plasmid DNA. R-plasmids were successfully transferred from Escherichia coli to V. parahaemolyticus.

A pyrimido[1,6-a]benzimidazole that enhances DNA cleavage mediated by eukaryotic topoisomerase II: a novel class of topoisomerase II-targeted drugs with cytotoxic potential.

Recently, a number of novel quinolones with potent activity against topoisomerase II and eukaryotic cells have been described. Many of these compounds contain aromatic substituents in their C-7 ring positions. To determine whether pyrimido[1,6-a]benzimidazoles, a class of drugs modeled on quinolones, also display activity toward eukaryotic systems, the effects of Ro 46-7864 and Ro 47-3359 on Drosophila melanogaster topoisomerase II and Kc cells were characterized. While the former drug contains an aliphatic group (4-N-methylpiperazine) at the ring position equivalent to C-7 in quinolones, the latter compound contains an aromatic substituent (2,6-dimethylpyridine). Both pyrimido[1,6-a]benzimidazoles inhibited DNA relaxation catalyzed by the type II enzyme. However, only Ro 47-3359 enhanced topoisomerase II-mediated DNA cleavage and was toxic to Kc cells. At a concentration of 100 microM, this drug approximately doubled the levels of DNA breakage in vitro and killed > 50% of the initial cell population of cultures. These results strongly suggest that selected pyrimido[1,6-a]benzimidazoles may function as topoisomerase II-targeted drugs with cytotoxic potential.

Distribution and polymorphism of the flagellin genes from isolates of Campylobacter coli and Campylobacter jejuni.

The complex flagellar filaments of the LIO8 serogroup member Campylobacter coli VC167 are composed of two highly related subunit proteins encoded by the flaA and flaB genes which share 92% identity. Using oligonucleotide primers based on the known DNA sequence of both the flaA and flaB genes from C. coli VC167 in the polymerase chain reaction, we have shown conservation of both fla genes among isolates within the LIO8 heat-labile serogroup by digestion of the amplified product with PstI and EcoRI restriction endonucleases. Amplification and subsequent restriction analysis of the flaA flagellin gene from Campylobacter isolates belonging to 13 different LIO serogroups further identified 10 unique polymorphic groups. Within most of the serogroups examined, isolates appeared to contain flaA genes with conserved primary structures. Only in serogroups LIO11 and LIO29 did independent isolates possess flagellin genes with different primary structures. Furthermore, by employing primers specific for the flaB gene of C. coli VC167, all serogroups examined contained a second fla gene corresponding to flaB. In all serogroups except the LIO5 and LIO6 isolates which were identical to each other, the polymorphic pattern of this flaB gene was identical to that of the corresponding flaA gene. These data indicate that the presence of a second highly homologous flagellin gene is widespread throughout Campylobacter isolates and that in most instances, the primary structure of the two fla genes is conserved within isolates belonging to the same heat-labile LIO serogroup. This may represent the presence of clonal evolutionary groups in Campylobacter spp.

Unusual microtubule-dependent endocytosis mechanisms triggered by Campylobacter jejuni and Citrobacter freundii.

Bacterial invasion of six different human epithelial cell lines showed that some strains of the intestinal pathogen Campylobacter jejuni invaded intestinal cell lines at a level 10(2)-10(4) times higher than reported previously for other Campylobacter strains. Separately, urinary tract isolates of Citrobacter freundii triggered a high-efficiency invasion of bladder cells. Use of multiple inhibitors with known effects on eukaryotic cell structures/processes allowed us to define in these genetically distinct bacterial genera unusual bacterial invasion mechanisms that uniquely require microtubules but not microfilaments. Campylobacter jejuni strain 81-176 uptake into 407 intestinal cells and Citrobacter entry into T24 bladder cells was blocked by microtubule depolymerization and inhibitors of coated-pit formation but not by microfilament depolymerization. Inhibitors of endosome acidification had no significant impact on intracellular survival of Campylobacter jejuni or Citrobacter freundii, but monensin markedly reduced Citrobacter uptake. Epithelial cell invasion by both of these bacterial genera was dependent upon de novo bacterial protein synthesis but not upon de novo eukaryotic cell protein synthesis. In contrast to the T24 cell line-specific, strict microtubule-dependent uptake, Citrobacter entry into other cell lines was inhibited by both microtubule- and microfilament-depolymerization, suggesting that these bacteria encode two separate pathways for uptake (i, microtubule-dependent; ii, microfilament-dependent) that are cell line-specific and are recognized perhaps depending on the presence and abundance of appropriate eukaryotic receptors.