- Volume 52, Issue 4, 2003

The nucleotide sequence of an ORF (vcfQ) within the type IV pilus gene cluster of Vibrio cholerae O34 strain NAGV14 was determined, thereby completing the sequence analysis of the structural operon. The vcfQ gene showed homology to the mshQ gene of the mannose-sensitive haemagglutinin pilus gene cluster. The vcfQ was 651 bp larger than mshQ, and the G+C content of the extra 651 bp portion (35.6 mol%) was lower than that of the overall vcfQ gene (42.5 mol%). Except for the first 270 aa residues, the deduced amino acid sequence of VcfQ showed high homology to the MshQ protein. There was immunological cross-reaction between VcfQ and MshQ by Western blotting. Cell fractionation studies showed that VcfQ is located in both the inner and the outer membranes. Mutational analysis showed that vcfQ-deficient mutant expressed detectable levels of major pilin (VcfA), but failed to assemble them into pili, indicating that VcfQ is essential for pilus assembly. Colony-blotting analyses showed that the N-terminal region of vcfQ is variable in V. cholerae strains.

All strains of Yersinia pestis examined have been found to lack an O-antigen. In other members of the Enterobacteriaceae, the rough phenotype often results in attenuation. However, Y. pestis is the aetiological agent of bubonic plague. In evolving from the ancestral enteropathogenic Yersinia pseudotuberculosis, and with the development of an arthropod-vectored systemic pathogenesis, smooth LPS production is not necessary for Y. pestis virulence and the metabolic burden has been alleviated by inactivation of the O-antigen biosynthetic operon. To investigate this, Y. pestis strain KIM D27 was transformed with a plasmid carrying the operon encoding the O-antigen of Yersinia enterocolitica O : 3. Expression of the O-antigen could be detected in silver-stained gels. The receptor for bacteriophage ϕYeO3-12 has been shown to be O-antigen, and infection by this bacteriophage results in lysis of Y. enterocolitica O : 3. Expression of the O-antigen in Y. pestis conferred sensitivity to lysis by ϕYeO3-12. The O-antigen-expressing clone was shown to be as virulent in mice by the intravenous route of challenge as the rough wild-type. Assays showed no alteration in the ability of Y. pestis to resist lysis by cationic antimicrobial peptides, serum or polymyxin.

Pseudomonas aeruginosa, an opportunistic human pathogen and ubiquitous environmental bacterium, is capable of forming specialized bacterial communities, referred to as biofilm. The results of this study demonstrate that the unique environment of the cystic fibrosis (CF) lung seems to select for a subgroup of autoaggregative and hyperpiliated P. aeruginosa small-colony variants (SCVs). These morphotypes showed increased fitness under stationary growth conditions in comparison with clonal wild-types and fast-growing revertants isolated from the SCV population in vitro. In accordance with the SCVs being hyperpiliated, they exhibited increased twitching motility and capacity for biofilm formation. In addition, the SCVs attached strongly to the pneumocytic cell line A549. The emergence of these highly adherent SCVs within the CF lung might play a key role in the pathogenesis of P. aeruginosa lung infection, where a biofilm mode of growth is thought to be responsible for persistent infection.

Cellular fatty acid analysis by GLC is widely used in the species identification of mycobacteria. Combining mycolic acid cleavage products with shorter cellular fatty acids increases the informative value of the analysis. A key has been created to aid in the identification of all currently known slowly growing environmental species. In this scheme, the species are classified into six categories, each characterized by a combination of fatty markers shared by those species. Within each category, individual species may be distinguished by the presence or absence of specific marker substances, such as methyl-branched fatty acids or secondary alcohols. This study also describes earlier unpublished GLC profiles of 14 rare, slowly growing, environmental mycobacteria, Mycobacterium asiaticum, Mycobacterium botniense, Mycobacterium branderi, Mycobacterium conspicuum, Mycobacterium cookii, Mycobacterium doricum, Mycobacterium heckeshornense, Mycobacterium heidelbergense, Mycobacterium hiberniae, Mycobacterium kubicae, Mycobacterium lentiflavum, Mycobacterium scrofulaceum, Mycobacterium triplex and Mycobacterium tusciae. Though no single identification technique alone, even sequencing of an entire single gene such as 16S rRNA, can identify all mycobacterial species accurately, GLC has proven to be both reliable and reproducible in the identification of slowly growing mycobacteria. In cases of earlier unknown species, it generates useful information that allows their further classification and may lead to the description of novel species.

A detection system for Legionella DNA in blood samples based on the PCR was developed and evaluated in A/J mice with experimentally induced Legionella pneumonia. Primers were designed to amplify a 106 bp DNA fragment of the 16S rRNA gene specific to Legionella species. The PCR system could detect clinically relevant Legionella species including Legionella pneumophila, Legionella micdadei, Legionella bozemanae, Legionella dumoffii, Legionella longbeachae, Legionella gormanii and Legionella jordanis. The sensitivity of the PCR system was 20 fg extracted DNA. In the mouse model, the blood PCR was compared with results obtained by PCR on bronchoalveolar lavage fluid (BALF) samples, cultures of blood and BALF and detection of Legionella urinary antigen. Blood PCR was positive until 8 days after infection, while BALF PCR became negative on day 4. These results indicate that PCR using blood samples may be a useful, convenient and non-invasive method for the diagnosis of Legionella pneumonia.

Rapid, sensitive and low-cost methods are needed urgently for the detection of Mycobacterium tuberculosis in clinical samples, especially in developing countries. To this end, the clinical performance of FASTPlaqueTBTM (a bacteriophage-based method) has been studied in parallel with microscopy, standard microbiological culture and in-house IS6110-based PCR methods. A total of 64 samples, including 42 sputum samples and 22 urine samples, were tested in this study. The sensitivity, specificity and overall accuracy values for the FASTPlaqueTB assay relative to that of culture were respectively 76.5, 95 and 90 %. The corresponding values for the in-house IS6110-based PCR assay were 88, 91 and 90 % and, for Ziehl–Neelsen staining, were 59, 95 and 85 %. FASTPlaqueTB gave better clinical performance with urine samples than with sputum samples (sensitivity, specificity and overall accuracy were 100 % with urine samples and 64, 93 and 84 % with sputum samples). The 100 % sensitivity of FASTPlaqueTB was higher than that of the corresponding values for PCR (67 %) with urine samples. In conclusion, FASTPlaqueTB proved to be sensitive, cheap relative to the PCR and rapid. It is able to detect M. tuberculosis in clinical samples within 1 day, reducing the time to diagnosis in comparison with culture.

This study forms part of the development of an integrated genotyping system for Streptococcus agalactiae (group B streptococcus, GBS) that can be used to study the population genetics of the organism and the pathogenesis and epidemiology of GBS disease. In recent previous studies, two sets of markers, the capsular polysaccharide synthesis (cps) gene cluster and surface protein antigen genes, have been used to assign molecular serotypes (MS) and protein-gene profiles (PGP) to more than 200 isolates. In the present study, five mobile genetic elements (MGE) have been used as a third set of markers, to characterize further 194 invasive isolates, recovered from blood or cerebrospinal fluid (CSF). Of these, 97 % contained one or more of the five MGE, the distribution of which was related to MS and PGP, as illustrated by MS III, which is divisible into four serosubtypes with different combinations of the MGE (or none). Fifty-six different genotypes and eight genetic clusters were identified, each with different combinations of the three sets of molecular markers. Five predominant genotypes (Ia-1, Ib-1, III-1, III-2 and V-1) contained 62 % of the isolates and five of the eight genetic clusters contained 92 % of the isolates. The 17 CSF isolates were relatively widely distributed between 10 genotypes and across seven of the eight clusters. Further study is needed to determine whether these genotypes or clusters share common markers of increased virulence. In future, comparison of invasive with colonizing strains of GBS may elucidate the significance of these findings.

Although HIV-infected individuals harbour multiple strains of oral Candida albicans, little is known of their micro-evolution over time. Therefore, a prospective study was conducted with 16 HIV-infected ethnic Chinese individuals with and without symptoms of oropharyngeal candidiasis to evaluate the genotype distribution of oral C. albicans isolates during HIV disease progression. Oral-rinse samples were obtained from all individuals and up to five C. albicans colonies were selected for each visit, over a 12 month period of multiple visits. After identification of isolates using standard mycological criteria, the genetic similarities of yeast isolates within and between sequential clones of C. albicans were assessed by DNA fingerprinting through random amplification of polymorphic DNA (RAPD). The results of RAPD gel profiles and the lineage of each isolate were further analysed using commercially available software. RAPD studies revealed the prevalence of up to 14 different genotypes per individual during the study period, with multiple genotypes isolated simultaneously from a single oral rinse. Computer analysis of RAPD profiles revealed that yeasts isolated over sequential visits from symptomatic individuals demonstrated a striking level of relatedness compared with isolates from asymptomatic individuals. Genetically identical C. albicans strains also formed ‘loosely’ connected subclusters that overlapped multiple visits, implying genetic ‘shuffling’ in these isolates during disease progression. These data point to varying evolutionary genetic trends in C. albicans associated with symptomatic oral candidiasis and asymptomatic carriage in HIV disease.