Cloning and nucleotide sequence of the aroA gene of Bordetella pertussis.

The aroA locus of Bordetella pertussis, encoding 5-enolpyruvylshikimate 3-phosphate synthase, has been cloned into Escherichia coli by using a cosmid vector. The gene is expressed in E. coli and complemented an E. coli aroA mutant. The nucleotide sequence of the B. pertussis aroA gene was determined and contains an open reading frame encoding 442 amino acids, with a calculated molecular weight for 5-enolpyruvylshikimate 3-phosphate synthase of 46,688. The amino acid sequence derived from the nucleotide sequence shows homology with the published amino acid sequences of aroA gene products of other microorganisms.     

Study of urinary tract infection in children in one health district.

OBJECTIVES--To determine the number of children who had urine specimens sent for culture, who had infections or sterile pyuria, and who were investigated further. To relate the laboratory findings to the results of imaging. DESIGN--One year survey of urine specimens submitted to a laboratory; review of previous and subsequent laboratory reports; review of the findings of imaging of the urinary tract. SETTING--Portsmouth and South East Hampshire health district. SUBJECTS--An estimated population of 89,086 children aged 12 years or under. MAIN OUTCOME MEASURES--Urine bacterial count and results of imaging. RESULTS--12,551 urine specimens were submitted from 7450 children, 3138 boys and 4312 girls. 2238 children had infection or sterile pyuria at least once during the study (13.9/1000 boys, 37/1000 girls). 996 (45%) of the children with infection or sterile pyuria underwent some form of imaging. 128 children who had infection or sterile pyuria were already known to have urinary tract abnormalities and 114 children had newly identified abnormalities (1.0/1000 boys, 1.5/1000 girls). 50 (44%) of the children with newly detected abnormalities had no pyuria and 48 (42%) had bacterial counts below 10(8)/l. Eight children who had sterile pyuria on presentation were found to have abnormalities on imaging. CONCLUSIONS--Urinary tract infection is much commoner in children than is widely believed. Low bacterial counts, the absence of pyuria, or a finding of sterile pyuria should not be disregarded.     

Cloning and molecular analysis of the galE gene of Neisseria meningitidls and its role in lipopolysaccharide biosynthesis

The galE gene from Haemophilus influenzae was used as a hybridization probe for the galE gene of Neisseria meningitidis Group B, identifying two different homologous loci. Each of the loci was cloned and nucleotide sequence analysis revealed that both loci contained sequences similar to galE. One contained a functional galE gene and mapped to the capsule biosynthetic locus. The second contained only a partial galE-coding sequence, which did not express a functional gene product. A galE mutant meningococcal strain was constructed by transformation with an inactivated galE gene. Analysis of the LPS from the galE mutant strain revealed an apparent reduction in molecular weight and a loss of reactivity with monoclonal antibodies specific for structures known to contain galactose. These results are consistent with an essential role for galE in the incorporation of galactose into meningococcal lipopolysaccharide.     

Gene Content and Diversity of the Loci Encoding Biosynthesis of Capsular Polysaccharides of the 15 Serovar Reference Strains of Haemophilus parasuis

Haemophilus parasuis is the causative agent of Glässer''s disease, a systemic disease of pigs, and is also associated with pneumonia. H. parasuis can be classified into 15 different serovars. Here we report, from the 15 serotyping reference strains, the DNA sequences of the loci containing genes for the biosynthesis of the group 1 capsular polysaccharides, which are potential virulence factors of this bacterium. We contend that these loci contain genes for polysaccharide capsule structures, and not a lipopolysaccharide O antigen, supported by the fact that they contain genes such as wza, wzb, and wzc, which are associated with the export of polysaccharide capsules in the current capsule classification system. A conserved region at the 3′ end of the locus, containing the wza, ptp, wzs, and iscR genes, is consistent with the characteristic export region 1 of the model group 1 capsule locus. A potential serovar-specific region (region 2) has been found by comparing the predicted coding sequences (CDSs) in all 15 loci for synteny and homology. The region is unique to each reference strain with the exception of those in serovars 5 and 12, which are identical in terms of gene content. The identification and characterization of this locus among the 15 serovars is the first step in understanding the genetic, molecular, and structural bases of serovar specificity in this poorly studied but important pathogen and opens up the possibility of developing an improved molecular serotyping system, which would greatly assist diagnosis and control of Glässer''s disease.     

Two msbB genes encoding maximal acylation of lipid A are required for invasive Shigella flexneri to mediate inflammatory rupture and destruction of the intestinal epithelium

Shigella flexneri is a Gram-negative pathogen that invades and causes inflammatory destruction of the human colonic epithelium, thus leading to bloody diarrhea and dysentery. A type III secretion system that delivers effector proteins into target eukaryotic cells is largely responsible for cell and tissue invasion. However, the respective role of this invasive phenotype and of lipid A, the endotoxin of the Shigella LPS, in eliciting the inflammatory cascade that leads to rupture and destruction of the epithelial barrier, was unknown. We investigated whether genetic detoxification of lipid A would cause significant alteration in pathogenicity. We showed that S. flexneri has two functional msbB genes, one carried by the chromosome (msbB1) and the other by the virulence plasmid (msbB2), the products of which act in complement to produce full acyl-oxy-acylation of the myristate at the 3' position of the lipid A glucosamine disaccharide. A mutant in which both the msbB1 and msbB2 genes have been inactivated was impaired in its capacity to cause TNF-alpha production by human monocytes and to cause rupture and inflammatory destruction of the epithelial barrier in the rabbit ligated intestinal loop model of shigellosis, indicating that lipid A plays a significant role in aggravating inflammation that eventually destroys the intestinal barrier. In addition, neutralization of TNF-alpha during invasion by the wild-type strain strongly impaired its ability to cause rupture and inflammatory destruction of the epithelial lining, thus indicating that TNF-alpha is a major effector of epithelial destruction by Shigella.     

Dynamics of bacterial growth and distribution within the liver during Salmonella infection

Salmonella enterica causes severe systemic diseases in humans and animals and grows intracellularly within discrete tissue foci that become pathological lesions. Because of its lifestyle Salmonella is a superb model for studying the in vivo dynamics of bacterial distribution. Using multicolour fluorescence microscopy in the mouse typhoid model we have studied the interaction between different bacterial populations in the same host as well as the dynamic evolution of foci of infection in relation to bacterial growth and localization. We showed that the growth of Salmonella in the liver results in the spread of the microorganisms to new foci of infection rather than simply in the expansion of the initial ones. These foci were associated with independently segregating bacterial populations and with low numbers of bacteria in each infected phagocyte. Using fast-growing and slow-growing bacteria we also showed that the increase in the number of infected phagocytes parallels the net rate of bacterial growth of the microorganisms in the tissues. These findings suggest a novel mechanism underlying growth of salmonellae in vivo with important consequences for understanding mechanisms of resistance and immunity.