Spacer oligotyping of Mycobacterium bovis isolates compared to typing by restriction fragment length polymorphism using PGRS, DR and IS6110 probes.

Ninety-two Mycobacterium bovis isolates from cattle, deer and badgers in Northern Ireland and the Republic of Ireland were genotyped by spacer-oligotyping (spoligotyping) and 67 of these were analysed by restriction fragment length polymorphism (RFLP). RFLP analysis was performed using three DNA probes, PGRS, DR and IS6110. Forty-seven of the M. bovis isolates were from 45 different sources; these were typed using both RFLP and spoligotyping. These 47 isolates could be differentiated into 24 different RFLP types and 15 distinct spoligotypes. Although RFLP was found to be more discriminatory compared to the present spoligotyping technique, spoligotyping was able to differentiate 21 RFLP type 'ACA' isolates into three different patterns. The remaining 45 M. bovis isolates were from a small case study, involving infected cattle, deer and badgers from the same geographic region. All these isolates were analysed by spoligotyping and a selection of 20 isolates were RFLP typed. All the isolates in the case study had the same spoligotype pattern with the exception of one cervine isolate. Similarly all the isolates typed by RFLP had the same pattern. Consequently, the predominant strain in the case study was not host restricted. The consistency between the results obtained using the two techniques indicates the potential value of both techniques for epidemiological studies. Spoligotyping was found to be a much more rapid technique and easier to perform, requiring less sophisticated computer software for strain typing. Spoligotyping results were more readily documented and analysed and the technique was also more suitable than RFLP analysis for large-scale screening studies.

Spacer oligonucleotide typing of Mycobacterium bovis strains from cattle and other animals: a tool for studying epidemiology of tuberculosis.

The spacer oligonucleotide typing (spoligotyping) method was evaluated for its ability to differentiate Mycobacterium bovis strains. This method detects the presence or absence of spacers of the direct repeat locus of the M. bovis genome. The spacers in the direct repeat locus are amplified by PCR and are detected by hybridization of the biotin-labelled PCR product with a membrane containing oligonucleotides derived from spacer sequences that have previously been bound to a membrane. One hundred eighty-two M. bovis isolates from domestic animals (cattle, goat, sheep, and cats) and wild animals (deer and wild boar) were spoligotyped, and the results were compared with those obtained by IS6110 restriction fragment length polymorphism analysis. Two rather homogeneous clusters of isolates containing 20 and 4 types, respectively, were identified by spoligotyping. The first cluster included isolates from cattle, cats, and feral animals. By spoligotyping, isolates from the Spanish wild boar and deer had the same pattern as some bovine isolates, suggesting transmission between these animals and cattle and highlighting the importance of the study of these reservoirs. The second cluster included all the caprine and ovine isolates. Within each cluster, the patterns of the different strains differed only slightly, suggesting that the spoligotypes may be characteristic of strains from particular animal species. Spoligotyping proved to be useful for studying the epidemiology of bovine M. bovis isolates, especially of those isolates containing only a single copy of IS6110. In view of our results, we suggest fingerprinting all M. bovis strains by the spoligotyping method initially and then by IS6110 restriction fragment length polymorphism typing of the strains belonging to the most common spoligotypes.

Rapid species identification of "Streptococcus milleri" strains by line blot hybridization: identification of a distinct 16S rRNA population closely related to Streptococcus constellatus.

A collection of 399 "Streptococcus milleri" strains were identified to the species level by the use of a line blot assay. Their PCR-amplified partial 16S rRNA gene sequences were hybridized with species-specific 5'-biotinylated oligonucleotide probes homologous to the bp 213 to 231 regions of the 16S rRNA gene sequences of the type strains Streptococcus anginosus ATCC 33397, Streptococcus constellatus ATCC 27823, and Streptococcus intermedius ATCC 27335. The hybridization results were compared with the reference phenotypic identification method data (R. A. Whiley, H. Fraser, J. M. Hardie, and D. Beighton, J. Clin. Microbiol. 28:1497-1501, 1990). Most strains (357 of 399 [89.5%]) reacted unambiguously with only one probe. However, 42 of the 399 strains (10.5%) reacted with both the S. constellatus- and S. intermedius-specific probes; 41 of them were phenotypically identified as S. constellatus. These dually reactive strains hybridized with a 5'-biotinylated probe based on the bp 213 to 231 region of the 16S rRNA gene sequence of one of two species. Analysis of the 5' ends of the 16S rRNA gene sequences (487 bp) demonstrated that the dually reactive strains represent a distinct rRNA population sharing 98.1% sequence similarity with S. constellatus. Phenotypic consistency between the dually reactive strains and the S. constellatus strains was not demonstrated. Line blot hybridization proved to be a simple and inexpensive method to screen large numbers of strains for genetic relatedness, and it allowed the detection of a distinct 16S rRNA type within the "S. milleri" group.

Genetic organization and functional analysis of the otn DNA essential for cell-wall polysaccharide synthesis in Vibrio cholerae O139.

In 1992 a new Vibrio cholerae strain, designated V. cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh. Previously, we have shown that this strain arose from a V. cholerae O1 strain by the acquisition of novel DNA. Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains. The mosaic structure of rfaDVCO139 indicated that it was one of the regions involved in recombination between donor and acceptor DNA. However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second recombination site between donor and O1-acceptor DNA is probably located downstream of rfbDVCO139. The DNA region between rfaDVCO139 and rfbQRSVCO139, designated otn, contained seven open reading frames (ORFs). Two ORFs, otnA and otnB, showed homology with genes involved in cell-wall polysaccharide synthesis. Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis. The otn DNA is also found in V. cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain. However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains. No antigenic relationship was found between the different V. cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA. The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide-synthetic genes in several bacterial species. Furthermore, a repeat motif was observed in extragenic regions. A number of observations suggest that these sequences may facilitate gene flow between V. cholerae strains and the assembly of clusters of functionally related genes.

Molecular typing of methicillin-resistant Staphylococcus aureus on the basis of protein A gene polymorphism.

The polymorphic X-region of the protein A gene (spa) was used for molecular typing of methicillin-resistant Staphylococcus aureus (MRSA) strains. The X-region is characterized by a variable number (between 3 and 15) of small repeats. DNA sequencing of MRSA strains revealed 25 distinct repeats. Analysis of MRSA strains grown in vitro and in vivo revealed that the X-region was sufficiently stable for epidemiologic typing of MRSA strains. Spa typing of MRSA strains was compared to phage typing and, in general, concordance was found between the two methods. However, spa typing was more sensitive, allowing differentiation of strains within a particular phage type. Results obtained with spa typing suggest that hospital outbreaks may be caused by two or more MRSA strains. Spa typing may be an important tool in unravelling the spread of MRSA strains within and between hospitals.

Genesis of the novel epidemic Vibrio cholerae O139 strain: evidence for horizontal transfer of genes involved in polysaccharide synthesis.

Only Vibrio cholerae strains of serotype O1 are known to cause epidemics, while non-O1 strains are associated with sporadic cases of cholera. It was therefore unexpected that the recent cholera epidemic in Asia was caused by a non-O1 strain with the serotype O139. We provide evidence that O139 arose from a strain closely related to the causative agent of the present cholera pandemic, V. cholerae O1 El Tor, by acquisition of novel DNA which was inserted into, and replaced part of, the O antigen gene cluster of the recipient strain. Part of the novel DNA was sequenced and two open reading frames (otnA and otnB) were observed, the products of which showed homology to proteins involved in capsule and O antigen synthesis, respectively. This suggests that the otnAB DNA determines the distinct antigenic properties of the O139 cell surface. The otnAB DNA was not detected in O1 strains, but was present in two non-O1 V. cholerae strains with serotypes O69 and O141. In the O69 and O139 strains the otnAB genes were located proximate to the putative insertion sequence (IS) element rfbQRS, which is associated with O antigen synthesis genes in O1 strains, and may have played a role in the insertion of the otnAB DNA in the recipient chromosome. Our results suggest that the O139 strain arose by horizontal gene transfer between a non-O1 and an O1 strain. The acquired DNA has altered the antigenic properties of the recipient O1 strain, providing a selective advantage in a region where a large part of the population is immune to O1 strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method.

Mycobacterium tuberculosis complex strains contain a unique chromosomal region, which consists of multiple 36bp direct repeats (DRs), which are interspersed by unique spacers 35 to 41 bp in length. In this study we investigated the nature of the DNA polymorphism of this DR cluster by sequencing part of this region in a large number of M. tuberculosis complex strains. Two types of genetic rearrangements were observed. One type consists of the variation in one or a few discrete, contiguous DRs plus spacer sequences. This variation is probably driven by homologous recombination between adjacent or distant DRs. The other type of polymorphism is probably driven by transpositional events of the insertion sequence, IS6110, which is almost invariably present in the DR cluster of M. tuberculosis complex strains. Based on the nature of the DNA polymorphism in the DR cluster, we developed a novel method of strain differentiation, direct variable repeat polymer chain reaction (DVR-PCR), which enables typing of individual M. tuberculosis strains in a single PCR. The method allows an excellent differentiation of epidemiologically unrelated isolates and, in principle, the DVR-PCR allows the detection of M. tuberculosis and strain differentiation at the same time.