The Isolation And Cloning of Chromosomal Dna Specific for a Clonal Population of Staphylococcus Aureus By Subtractive Hybridisation

Abstract

Subtractive hybridisation was used to screen for and identify conserved DNA sequences associated with clinical, clonal populations of Staphylococcus aureus. DNA from S. aureus strain ISPS (a clinical isolate) was digested with TaqI and hybridised with randomly fragmented pooled DNA from 10 non-clinical (community) isolates of S. aureus. The mixture of DNA fragments was then ligated to AccI-digested and dephosphorylated pTZl9U. One recombinant plasmid (pWASA) was identified as containing specific DNA from strain ISP8; DNA sequencing revealed a 40-bp TaqI DNA fragment (WASA). PCR amplification and hybridisation analysis, with pWASA as a probe, showed that 84% of clinical isolates from a clonal line present in hospitals in major eastern Australian cities carried sequences homologous to WASA, compared with only 10% of community isolates. The isolates that hybridised were closely related by RFLP analysis to strain ISPS. The plasmid pWASA was used to identify, isolate and clone a 3.5-kb DraI fragment (DSA) from strain ISP8 total DNA. Sequence analysis of the DSA fragment identified two open reading frames (ORFs) of 2475 and 576bp, respectively; the larger O W 1 contained a series of six tandem repeats, each consisting of 384 nucleotides. The nucleotide sequence of the repeats was 96% identical, and no significant sequence homologies to previously described protein sequences were identified. However, tandem repeats of amino acids are structural motifs characteristic of a number of gram-positive surface proteins, and are thought to play a role in generating genetic and phenotypic diversity in these and other bacteria.

Spot the difference: applications of subtractive hybridisation to the study of bacterial pathogens

“…SH has been used to identify genomic regions as diagnostic targets in various animal and human pathogens [17,[31][32][33][34][35][36][37]. Strains of Chlamydia psittaci that can invade and colonise the placenta of sheep cause enzootic abortion in ewes (EAE).…”

“…El Adhami et al [34] [36] used SH to identify nine short III-3-specific sequences as a first step to identifying genes that might account for differences in virulence.…”

Antimicrobial resistance in Salmonella isolated from animals and their environment in England and Wales from 1988 to 1999

“…The 1994 to 1996, respectively. 199119921994199719981999 Annual numbers of Salmonella Dublin and Salmonella Enteritidis isolates tested for antimicrobial sensitivity between 1988 and 1999…”

“…This phage type was first detected in 1979, when it showed resistance to chloramphenicol, streptomycin, sulphonamides, chlortetracycline and sulphamethoxazole/trimethoprim (Threlfall and others 1980); it subsequently became widely disseminated . I 1988I 1991I 1992I 1993I 1994I 1995I 1996I 1997I 1998 Annual numbers of Salmonella Typhimunum and STyphimurium DT1O4 isolates tested for antimicrobial sensitivity between 1988 and 1999 in the cattle population and was the most common phage type received at this laboratory. Its dedining prevalence during the late 1980s and early 1990s is indicated by the increased numbers of susceptible S Typhimurium isolated in this period (Table 3).…”

Application of suppressive subtractive hybridization to the identification of genetic differences between two Lactococcus garvieae strains showing distinct differences in virulence for rainbow trout and mouse

“…This technique is especially useful for the identification of genetic differences between two closely related bacteria whose genomes have not been sequenced (Winstanley, 2002). It has been applied successfully on several occasions to the identification of virulence-related factors, host-specific determinants and epidemiological markers (Bohnsack et al, 2001;el-Adhami et al, 1997;Juíz-Río et al, 2005;Lai et al, 2000;Olivares-Fuster & Arias, 2008;Reckseidler et al, 2001;Soule et al, 2005;Wang et al, 2009;Zhang et al, 2000).The objective of this work was to study the virulence of two L. garvieae strains, one isolated from an outbreak in rainbow trout (L. garvieae 074) and the other from a human patient affected by endocarditis provoked by the bacterium (L. garvieae HF), in rainbow trout and mouse models. Based on their different virulence, the SSH approach was applied to the identification of genetic variation between the two strains.…”