- Volume 50, Issue 3, 2001

A pool of heparan sulphate-binding proteins (HSBPs) from Helicobacter pylori culture supernates was obtained by sequential ammonium sulphate precipitation and affinity chromatography on heparin-Sepharose. The chromatographic procedure yielded one major fraction that contained proteins with heparan sulphate affinity as revealed by inhibition studies of heparan sulphate binding to H. pylori cells. Preparative iso-electric focusing, SDS-PAGE and blotting experiments, with peroxidase(POD)-labelled heparan sulphate as a probe, indicated the presence of two major extracellular proteins with POD-heparan sulphate affinity. One protein had a molecular mass of 66.2 kDa and a pI of 5.4, whilst the second protein had a molecular mass of 71.5 kDa and a pI of 5.0. The N-terminal amino acid sequence of the 71.5-kDa HSBP did not show homology to any other heparin-binding protein, nor to known proteins of H. pylori , whereas the 66.2-kDa HSBP showed a high homology to an Escherichia coli chaperon protein and equine haemoglobin. A third HSBP was isolated from an outer-membrane protein (OMP) fraction of H. pylori cells with a molecular mass of 47.2 kDa. The amino acid sequence of an internal peptide of the OMP-HSBP did not show homology to the extracellular HSBP of H. pylori , or to another microbial HSBP.

Iron is an essential metabolite for pathogenic bacteria, and the specificity exhibited by bacteria for host-iron chelates may be correlated with host and tissue tropism. The effect of iron restriction on Chlamydia pneumoniae and C. trachomatis was studied by use of the iron-chelating compound deferoxamine. Growth of C. pneumoniae was inhibited much more than that of C. trachomatis and the effect of iron restriction largely depended on the cell line used for propagation. This might reflect differences in tissue tropism of the two chlamydial species. As iron levels are usually higher in men than in women, this might also be connected with the higher prevalence rate of C. pneumoniae antibodies in males, observed in all populations studied so far.

Samples of atherosclerotic tissue from 58 patients undergoing carotid surgery were analysed by tissue culture and PCR for Chlamydia pneumoniae ; PCR was performed to detect Omp1, 16S rRNA and HSP-70 genes. To understand the active pathogenic role of C. pneumoniae , a reverse transcriptase-PCR (RT-PCR) assay was applied to detect the specific RNAs expressed either in the replicative form, or in the cryptic form found in chronic infection. The C. pneumoniae omp1 gene, encoding the major outer-membrane protein (MOMP), was detected in 13 of 58 samples. Among these, the result was confirmed in 11 samples after amplification of a further target, the 16S rRNA, and the presence of the HSP-70 gene, encoding heat-shock protein 70, was revealed in only five cases. All the samples were negative for evidence of specific RNAs by RT-PCR. The presence of genomic DNA and absence of specific RNAs in atherosclerotic tissue samples suggests a lack of an active metabolic or persistent infective role for C. pneumoniae . Thus, traces of C. pneumoniae DNA in these samples could be due to a degradative pathway of the host defensive cellular and biochemical mechanisms.

The effect of endotoxin on the regulation of vascular endothelial growth factor (VEGF) mRNA expression in human monocytic (THP-1) cells was examined. Endotoxic lipopolysaccharide (LPS) from Escherichia coli and synthetic E. coli -type lipid A (LA-15-PP) enhanced VEGF mRNA expression. LPS-induced VEGF mRNA accumulation was regulated, at least in part, at the transcriptional level. Enhancement of VEGF gene expression by LPS was shown by gel shift analysis and use of transcription factor inhibitors to be mediated via the activation of SP-1.

Peptostreptococcus micros is a gram-positive anaerobic coccus which, although considered to be a natural commensal of the human oral cavity, is associated with periodontal, endodontal and peritonsillar infections. Identification of the organism has to date relied upon conventional culture methods and biochemical analyses. The purpose of this study was to develop a PCR method for rapid and specific identification of this organism in clinical samples. A pair of primers was selected, each of which was specific at the 3′ end for P. micros DNA; they were used in the PCR assay, resulting in a 1074-bp product. The primers were shown to be specific for P. micros DNA as no PCR products were obtained when genomic DNA extracts from a wide range of other Peptostreptococcus species and other oral bacteria were used as templates. The PCR assay was then applied to the identification of P. micros DNA in subgingival plaque samples from adult periodontitis patients and pus samples from subjects with acute dento-alveolar abscesses. Confirmation of specific amplification of P. micros DNA was obtained by digestion of PCR products with the restriction endonuclease Rsa I, which gives a unique restriction profile for P. micros , and DNA sequencing. Sixty-eight subgingival plaque samples from 18 patients were analysed, of which 19 (28%) were positive for P. micros DNA; the proportion of patients carrying P. micros DNA in at least one sampled site was 11 (61%) of 18. Twenty (71%) of 28 pus samples analysed by PCR contained P. micros DNA. These results confirm that P. micros may be involved in the aetiology of acute dento-alveolar abscesses and adult periodontitis. The PCR assay provides a more rapid and reliable alternative to conventional methods for identification of P. micros in clinical samples.

Chronic respiratory infection by Pseudomonas aeruginosa is a significant determinant in the prognosis of cystic fibrosis patients. Cross-infection between cystic fibrosis patients and the prevalence of P. aeruginosa among them were investigated by microbiological surveillance and RAPD typing of the isolates. A total of 748 samples was cultured, including specimens from the respiratory tract (sputum or throat swabs) and hands of patients and medical staff, resulting in the collection of 86 isolates of P. aeruginosa from 65 samples. Prevalence of P. aeruginosa was 39.3% in respiratory samples, 0.2% on patients’ hands and none in the medical staff's hand samples. RAPD typing characterised 51 genotypes and clonal persistence was observed in the majority of patients. These results suggest that cross-infection is not common in the outpatient clinic studied and a common source of acquisition is unlikely.

Seven rough isolates of Vibrio cholerae isolated as the sole infecting agent from patients with cholera-like diarrhoea were examined for the presence of the regulatory element toxR and certain virulence-associated genes of the CTX genetic element and V. cholerae pathogenicity island (VPI). Multiplex PCR analysis with wb -specific genes of either O1 or O139 origin showed that six of the seven isolates produced an O1 wb -specific amplicon and the remaining isolate produced an O139-specific amplicon. Analysis of lipopolysaccharide profiles of smooth variants of V. cholerae revealed the presence of long repeated units of ‘O’ polysaccharide side chains but all the rough variants appeared to be devoid of the latter and possessed only core oligosaccharide. PCR amplification with primers specific to the ctxA, ctxB, tcpA, tagA, int, aldA, toxT, LJ, RJ and toxR genes revealed that six of the seven rough isolates were positive for these genes. One isolate was found to be negative for tagA and RJ , indicating the presence of an altered VPI. Each of these isolates showed media-dependent expression of cholera toxin (CT) and produced more toxin than the reference V. cholerae O1 El Tor strain VC20 or O139 strain SG24 under comparable conditions. Studies on the clonality of these isolates by the analysis of rRNA genes indicated their relatedness to strains of V. cholerae O1 El Tor or O139, isolated during the same time period.

A total of 87 invasive isolates of Haemophilus influenzae isolated throughout Taiwan from 1994 to 1998 was collected; 57 were from children <14 years old. In all, 60.9% of isolates were resistant to ampicillin and produced β -lactamase. Ribotyping revealed six different profiles in 55 isolates of type b, nine profiles in 10 isolates of non-type b and 12 profiles in 22 isolates of non-typable H. influenzae . Among isolates from 35 cases of meningitis, 30 (86%) were in ribogroups 1, 2 and 3 with >90% genetic similarity. Compared with all the other ribogroups, ribogroups 1, 2 and 3, which encompassed all H. influenzae type b, were significantly more prevalent as a cause of meningitis in children <14 years old. Further subtyping of the predominant ribogroup by pulsed-field gel electrophoresis (PFGE) identified differences of 0–6 bands among these isolates of ribogroup 1, which indicated distant relatedness. Automated ribotyping was found to be a useful method and was less time-consuming for molecular epidemiology studies of H. influenzae . PFGE is suggested as an addition to ribotyping to improve discrimination if H. influenzae type b is involved. Differentiating ribogroups between type b and non-type b H. influenzae by genotyping may help to understand the molecular characteristics of outbreaks, endemicity and value of vaccination. According to the results of ribotyping and PFGE, it seems possible that spread of invasive H. influenzae type b had occurred and ribotyping confirmed that there was no clonal spread of non-type b H. influenzae in Taiwan.