Corinne Jay

Multilocus Sequence Typing of Staphylococcus aureus with DNA Array Technology

ABSTRACT A newly developed oligonucleotide array suited for multilocus sequence typing (MLST) of Staphylococcus aureus strains was analyzed with two strain collections in a two-center study. MLST allele identification for the first strain collection fully agreed with conventional strain typing. Analysis of strains from the second collection revealed that chip-defined MLST was concordant with conventional MLST. Array-mediated MLST data were reproducible, exchangeable, and epidemiologically concordant.

Species differentiation and antibiotic susceptibility testing with DNA microarrays

1. SUMMARYThere is a need for accurate and rapid species identification,strain typing and antibiotic susceptibility testing for severalbacterial genera that contain some of the major humanpathogens like Staphylococcus aureus or Mycobacterium tuber-culosis. Assays based on molecular biology technologies havebeen introduced in recent years and their clinical value is nowwell established, especially for the identification of thedifficult-to-grow bacteria, for the epidemiological character-ization of strains involved in nosocomial infections and for thedetectionofmutationsassociatedwithresistancetoantibiotics.Analysis with high density microarrays following multi-plex nucleic acid amplification allows for simultaneoustesting of specimens for suspected pathogens. Feasibilitystudies have been conducted on two assays for the Myco-bacterium and Staphylococcus genera, based on the photoli-thography DNA-Chip technology developed by Affymetrix.The Mycobacterium assay combines 16S ribosomal DNAidentification of 80 species, strain typing of species inside theM. tuberculosis (MTB) complex and detection of mutationslinked to resistance to rifampin. Spoligotyping can furtherdiscriminate between species inside the TB complex and, fora given species, perform strain typing. A total of 104rifampin mutations were detected in the rpoB gene ofM. tuberculosis. The assay is performed within a working dayon a single smear-positive sputum sample.The Staphylococcus assay combines 16S ribosomal DNAidentification of 34 species, typing of S. aureus strains bymulti-locus sequence typing and detection of genes ormutations linked to resistance to methicillin, fluoroquinolo-nes and aminoglycosides. The objective of this assay was toperform the multiplex analysis of these different targetswithin a working day starting from a single colony.Results obtained with these two prototype assays, andwith other similar techniques in the fields of clinical virologyor food industry testing, show that the microarray technol-ogy is very promising and will soon be part of the laboratorytools available to microbiologists.2. INTRODUCTIONDNA microarrays are expected to take an important place inthe microbiology laboratory. To date, reagents using DNAmicroarrays are mostly used in research laboratories, forexample to look at up- and down-regulated genes. Thisfundamental work yields a set of gene targets that can beused later on as diagnostic tools to routinely serve clinicianneeds. Only few if any DNA microarray are commercializedfor routine in vitro diagnosis of infectious diseases. DNAmicroarrays offer the possibility of multiplex parallelhybridization on DNA probes of nucleic acids eitherdirectly extracted from a specimen or, more often, generatedby an amplification step. They can be used for the initialdiagnosis of an infection when several pathogens can be thecause of a disease (panel diagnostic) or for the precisecharacterization of this pathogen, when it is required for amore accurate treatment management. Panel diagnosis can1. Summary, 592. Introduction, 593. Applications, 603.1 Species identification, 603.2 Strain typing, 623.3 Antibiotic resistance, 634. Procedures, 654.1 Tiling strategies using high-density DNA micro-arrays, 654.1.1 4L-tiling, 654.1.2 2L-tiling, 654.2 Spoligotyping of MTB species, 654.2.1 Reference spoligotyping, 654.2.2 Comparison of spoligotypes obtained by dot-blot and by the microarray with a strain ofM. bovis,654.3 Complete Mycobacterium assay, 665. Conclusions, 666. References, 67

Efficient amplification with NASBA® of hepatitis B virus, herpes simplex virus and methicillin resistant Staphylococcus aureus DNA

A new mechanism is described for DNA amplification using nucleic acid sequence-based amplification (NASBA) including a restriction enzyme digestion and P1 primer binding directly upstream of the digestion. For hepatitis B virus (HBV), herpes simplex virus (HSV) and methicillin resistant Staphylococcus aureus (MRSA) DNA, which all show very poor amplification with normal NASBA, assay sensitivity was improved by a factor 100-1000 when restriction enzyme digestion was performed prior to amplification. For the quantitative HBV assay, in combination with the NucliSENS Extractor (bioMérieux, Boxtel, The Netherlands), a 95% target detection rate of 242 WHO IU/ml and 50% detection rate of 35 WHO IU/ml was achieved. The lowest detectable HBV concentration was 10 WHO IU/ml. HBV DNA could be quantified with an algorithm comparable to that used for RNA quantitation and by using a two step approach a dynamic range of 10(2)-10(9)WHO IU/ml (>6 log) was shown to be quantifiable. For the qualitative HSV assay, in combination with the NucliSENS miniMAG (bioMérieux, Boxtel, The Netherlands), the 95% detection rate was determined to be 84 and 138 copies/isolation for HSV 1 and HSV 2, respectively, which corresponds to approximately 10 copies per amplification for both targets. For MRSA, the limit of detection was <10 equivalent CFU per amplification.

Characterization of 26 isolates of Staphylococcus aureus, predominantly from dairy sheep, using four different techniques of molecular epidemiology.

Little information is available regarding the molecular epidemiology of Staphylococcus aureus–induced mastitis in dairy sheep. In this study, 4 different typing techniques were compared in typing 26 S. aureus isolates, predominantly from cases of subclinical mastitis in dairy ewes. The 4 techniques were pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP) on 2 genes (coagulase and clumping factor B), randomly amplified polymorphic DNA–polymerase chain reaction (PCR) (RAPD-PCR), and multilocus sequence typing (MLST). On the basis of discriminatory power as the key parameter of typing systems, MLST and PFGE were found to be the most powerful techniques. The MLST and PFGE could contribute to epidemiological surveillance and evaluation of mastitis control programs, by documenting prevalence and dissemination of endemic clones in infected populations. The results of this study show that a single clone of S. aureus is widely distributed in infected ewe mammary glands.