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Molecular Biology of the Staphylococci

30 Nov 1990-
TL;DR: Introduction genome organization and evolution gene transfer and transposons plasmids exoproteins and pathogenicity factors resistance and its spread resistance and epidemiology.
Abstract: Introduction genome organization and evolution gene transfer and transposons plasmids exoproteins and pathogenicity factors resistance and its spread resistance and epidemiology.
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Journal ArticleDOI
TL;DR: In the NYC collection, spa typing provided a clonal assignment for 185 of 195 strains within the five major groups previously described, and spa sequencing appears to be a highly effective rapid typing tool for S. aureus that, despite some expense of specificity, has significant advantages in terms of speed, ease of use, easy of interpretation, and standardization among laboratories.
Abstract: Three hundred and twenty isolates of Staphylococcus aureus were typed by DNA sequence analysis of the X region of the protein A gene (spa). spa typing was compared to both phenotypic and molecular techniques for the ability to differentiate and categorize S. aureus strains into groups that correlate with epidemiological information. Two previously characterized study populations were examined. A collection of 59 isolates (F. C. Tenover, R. Arbeit, G. Archer, J. Biddle, S. Byrne, R. Goering, G. Hancock, G. A. Hebert, B. Hill, R. Hollis, W. R. Jarvis, B. Kreiswirth, W. Eisner, J. Maslow, L. K. McDougal, J. M. Miller, M. Mulligan, and M. A. Pfaller, J. Clin. Microbiol. 32:407-415, 1994) from the Centers for Disease Control and Prevention (CDC) was used to test for the ability to discriminate outbreak from epidemiologically unrelated strains. A separate collection of 261 isolates form a multicenter study (R. B. Roberts, A. de Lencastre, W. Eisner, E. P. Severina, B. Shopsin, B. N. Kreiswirth, and A. Tomasz, J. Infect. Dis. 178:164-171, 1998) of methicillin-resistant S. aureus in New York City (NYC) was used to compare the ability of spa typing to group strains along clonal lines to that of the combination of pulsed-field gel electrophoresis and Southern hybridization. In the 320 isolates studied, spa typing identified 24 distinct repeat types and 33 different strain types. spa typing distinguished 27 of 29 related strains and did not provide a unique fingerprint for 4 unrelated strains from the four outbreaks of the CDC collection. In the NYC collection, spa typing provided a clonal assignment for 185 of 195 strains within the five major groups previously described. spa sequencing appears to be a highly effective rapid typing tool for S. aureus that, despite some expense of specificity, has significant advantages in terms of speed, ease of use, ease of interpretation, and standardization among laboratories.

993 citations

Journal ArticleDOI
TL;DR: Using techniques of molecular epidemiology, the authors demonstrate that an organism isolated from the vitreous was genetically indistinguishable from an isolate recovered from the patient's eyelid, conjunctiva, or nose in 14 (82%) of 17 cases of endophthalmitis.

618 citations

Journal ArticleDOI
24 Jul 1992-Cell
TL;DR: This work proposes the existence of a hitherto undescribed sorting mechanism that positions proteins on the surface of gram-positive bacteria, which consists of an LPXTGX motif, a C-terminal hydrophobic domain, and a charged tail.

565 citations

Journal ArticleDOI
TL;DR: Cl cloning and determination of the structure of the entire mec DNA sequence from a Japanese S. aureus strain found no ORF that might encode mecDNA-specific transposase or integrase proteins, indicating that themec DNA is not a transposon or a bacteriophage in nature.
Abstract: In methicillin-resistant Staphylococcus aureus, the methicillin resistance gene mecA is localized within a large chromosomal region which is absent in the methicillin-susceptible S. aureus chromosome. The region, designated mec DNA, is speculated to have originated from the genome of another bacterial species and become integrated into the chromosome of the S. aureus cell in the past. We report here cloning and determination of the structure of the entire mec DNA sequence from a Japanese S. aureus strain, N315. The mec DNA was found to be 51,669 bp long, including terminal inverted repeats of 27 bp and a characteristic pair of direct repeat sequences of 15 bp each: one is situated in the right extremity of mec DNA, and the other is situated outside the mec DNA and abuts the left boundary of mec DNA. The integration site of mec DNA was found to be located in an open reading frame (ORF) of unknown function, designated orfX. Clusters of antibiotic resistance genes were noted in mec DNA carried by transposon Tn554 and an integrated copy of plasmid pUB110. Both the transposon and plasmid were integrated in the proximity of the mecA gene, the latter being flanked by a pair of insertion sequence IS431 elements. Many ORFs other than those encoding antibiotic resistance were considered nonfunctional because of the acquired mutations or partial deletions found in the ORFs. Two ORFs potentially encoding novel site-specific recombinases were found in mec DNA. However, there was no ORF that might encode mec DNA-specific transposase or integrase proteins, indicating that the mec DNA is not a transposon or a bacteriophage in nature.

498 citations

Journal ArticleDOI
TL;DR: A cell‐based, drug‐screening assay is described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product, thereby greatly facilitating the search for new antibiotics.
Abstract: Summary To address the need for new approaches to antibiotic drug development, we have identified a large number of essential genes for the bacterial pathogen, Staphylococcus aureus, using a rapid shotgun antisense RNA method. Staphylococcus aureus chromosomal DNA fragments were cloned into a xylose-inducible expression plasmid and transformed into S. aureus. Homology comparisons between 658 S. aureus genes identified in this particular antisense screen and the Mycoplasma genitalium genome, which contains 517 genes in total, yielded 168 conserved genes, many of which appear to be essential in M. genitalium and other bacteria. Examples are presented in which expression of an antisense RNA specifically reduces its cognate mRNA. A cell-based, drug-screening assay is also described, wherein expression of an antisense RNA confers specific sensitivity to compounds targeting that gene product. This approach enables facile assay development for high throughput screening for any essential gene, independent of its biochemical function, thereby greatly facilitating the search for new antibiotics.

481 citations


Cites methods from "Molecular Biology of the Staphyloco..."

  • ...RN4220 carrying either an rplQ antisense clone (S1–760 A) or a lig antisense clone (S1–396 A) were grown to mid-log phase and diluted 1:10 into prewarmed Luria–Bertani (LB) broth....

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  • ...The genomic library was amplified by passage through Escherichia coli to provide sufficient amounts of DNA, and DNA from the pooled library was then transformed into S. aureus strain RN4220....

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  • ...Cell-based assay for target-specific inhibitors An overnight culture of RN4220 carrying plasmid S1- © 2002 Blackwell Science Ltd, Molecular Microbiology, 43, 1387–1400 1941 expressing antisense to the fab gene encoding bketoacyl-acyl carrier protein synthase was inoculated into fresh LB plus 34 mg ml–1 of chloramphenicol, and incubated with shaking at 37∞C. Exponential cultures were diluted to a final OD600 of 0.002 into two flasks of the same medium containing 0 and 12 mM xylose....

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  • ...Staphylococcus aureus strains used include RN450 and RN4220 (Novick, 1990) and E. coli strains, DH5a and XL1Blue, which were obtained from Gibco-BRL and Stratagene respectively....

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  • ...This plasmid library was electroporated into RN4220 to generate approximately 315 000 transformants that were recovered on LBG + 15 mg ml–1 chloramphenicol agar plates....

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