Multicenter evaluation of arbitrarily primed PCR for typing of Staphylococcus aureus strains

Belkum, Alex van; Kluytmans, Jan; Leeuwen, Willem van; Bax, R.; Quint, W. G. V.; Peters, E. D. J.; Fluit, A.C.; Vandenbroucke-Grauls, Christina M. J. E.; Brule, A. van den; Koeleman, Hatem Mohamed Saleh Bobby

doi:10.1128/jcm.33.6.1537-1547.1995

Cited by 240 publications

(135 citation statements)

References 29 publications

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“…However, it has been evident for some years that PFGE is a reliable technique for helping solve epidemiological problems. Lack of intercentre reproducibility of RAPD assays has also been iden-ti¢ed before, but in a single institution this methodology can be standardised to a satisfactory level [13]. This gives us the con¢dence that both the RAPD and the PFGE tests performed during the present study give an appropriate index of genetic diversity among the strains tested.…”

Section: Discussionsupporting

confidence: 51%

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

Braak

Power

Anthony

et al. 2000

FEMS Microbiology Letters

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Genetic typing of vancomycin-resistant enterococci (VRE) can be performed using a variety of methods, but comparative analyses of the quality of these methods are still relatively scarce. We here compare random amplification of polymorphic DNA (RAPD) analysis with pulsed field gel electrophoresis (PFGE) of DNA macrorestriction fragments as examples of two of the recent and well-accepted molecular typing methods. For the latter method, empirical guidelines for the interpretation of the DNA fingerprints have been proposed in the international literature. Based on our experimental analyses, we define similar criteria for RAPD fingerprinting. A collection of 100 strains of VRE, comprising Enterococcus faecium, Enterococcus faecalis, Enterococcus avium, Enterococcus gallinarum and Enterococcus casseliflavus, was assembled. Fifty isolates were Dutch, another 50 were isolated in the UK. Strains were selected on the basis of previously determined putative identity, close relatedness or uniqueness. The strains were analysed using well-standardised RAPD and PFGE protocols. Resulting fingerprints were interpreted with computerised methods involving band positioning and we show that typing of VRE by PFGE and RAPD generates highly congruent DNA fingerprint clustering. When the proposed international criteria for interpretation of PFGE fingerprints were applied in our case, 86% PFGE homology as discriminating value between close relatedness and uniqueness, a 75% homology cut-off for the comparison of the RAPD-generated DNA fingerprints revealed essentially identical strain clusters. As a spin-off it is revealed that strains from the different species can be efficiently discriminated, that strains from the UK and The Netherlands form separate clusters and that strains from veterinary origin can be identified separately as well.

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Section: Discussionsupporting

confidence: 51%

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

Braak

Power

Anthony

et al. 2000

FEMS Microbiology Letters

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“…The strains of S. aureus were characterized by arbitrarily primed PCR (AP-PCR) using the primers ERIC2 and 1 referred to in ref. [18], as previously described with minor changes [19]. Briefly, the DNA extraction was performed in a QIAsymphony Ò machine using a virus/bacteria mini kit (Qiagen, Courtaboeuf, France).…”

Section: Gentoyping Of S Aureus Isolatesmentioning

confidence: 99%

An algorithm based on one or two nasal samples is accurate to identify persistent nasal carriers of Staphylococcus aureus

Verhoeven

Grattard

Carricajo

et al. 2012

Clinical Microbiology and Infection

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Persistent Staphylococcus aureus nasal carriers are at high risk of S. aureus infection. The present study delineates a simple strategy aimed at identifying rapidly and accurately this subset of subjects for clinical or epidemiological purposes. Ninety healthy volunteers were each identified as persistent, intermittent or non-nasal carriers of S. aureus by using seven specimens sampled over a 5-week period. By reference to this so-called reference standard, six other strategies aimed at simplifying and speeding the identification of persistent carriers and based on the qualitative or quantitative detection of S. aureus in one to three nasal samples were evaluated by the measure of the area under the curve of receiver operating characteristic diagrams. Among strategies using qualitative results, there was no statistical difference between protocols using seven and three samples. A threshold of 10(3) CFU of S. aureus per swab was found capable of defining persistent nasal carriage with a sensitivity of 83.1% and a specificity of 95.6%. These figures reached 95.5% and 94.9%, respectively, by using an algorithm including one or two nasal specimens according to the threshold of 10(3) CFU of S. aureus in the first swab. The latter two strategies were shown to be costly equivalents. The proposed algorithm-based strategy proved to be relevant to identify properly and consistently persistent nasal carriers of S. aureus. However, as it was built from data of healthy volunteers, it needs to be confirmed prospectively on patients potentially at risk for S. aureus infection.

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“…Three different primers were used to amplify polymorphic regions of the S. aureus genome in three independent standard PCRs as described elsewhere (van Belkum et al 1995;van Leeuwen et al 1996): 5 ng (in the AP-1 and AP-7 reaction) or 25 ng (in the ERIC-2 reaction) of genomic DNA from overnight cultures were used as templates together with 1· PCR Gold buffer (Applied Biosystems), 2AE5 mmol l )1 MgCl 2 , 0AE2 units AmpliTaq Gold Ò DNA Polymerase (Applied Biosystems) 0AE2 mmol l )1 dNTPs and 1 lmol l )1 Primer (AP-1, AP-7 or ERIC-2) in a 50-ll reaction. Forty cycles of 1 min 94°C, 1 min 40°C, 2 min 72°C were run and the amplified fragments were separated on a ethidium bromidestained 1AE5% agarose gel and analysed under UV light.…”

Section: Randomly Amplified Polymorphic Dna-pcrmentioning

confidence: 99%

Contaminations of laboratory surfaces with Staphylococcus aureus are affected by the carrier status of laboratory staff

Schmidlin¹,

Alt²,

Vogel³

et al. 2010

Journal of Applied Microbiology

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Aim: As a biosafety laboratory, we take samples from surfaces in microbiological laboratories to survey the handling of micro-organisms. Whereas contaminations with other micro-organisms were rare, Staphylococcus aureus was found in the working environment of many laboratories. As 20-60% of the healthy population are carriers of S. aureus we wanted to asses the effect of carriers on our sampling results. Methods and Results: Nasal swabs of staff members in nonmicrobiological laboratories and offices as well as surface samples from their personal work environment were taken and analysed for S. aureus DNA. In addition S. aureus strains were isolated using S. aureus-specific agar plates and analysed by randomly amplified polymorphic DNA (RAPD)-PCR and multilocus sequence typing (MLST). Our data show that contaminations with S. aureus in nonmicrobiological environments are common with 29% of the surface samples containing S. aureus DNA. In the working environment of carriers, the number of contaminations was significantly increased compared to the environment of noncarriers. Conclusion: The carrier status of staff members significantly affects the number of contaminations on laboratory surfaces. Therefore, even in the absence of intentional handling of S. aureus, contaminations can be detected on a substantial amount of surfaces. Significance and Impact of the Study: Sampling procedures need to be adapted based on these results with respect to the locations where samples are taken and the threshold for significant contaminations. Because of its wide distribution, S. aureus can serve as a marker for hygienic standards in laboratories.

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Multicenter evaluation of arbitrarily primed PCR for typing of Staphylococcus aureus strains

Cited by 240 publications

References 29 publications

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

Random amplification of polymorphic DNA versus pulsed field gel electrophoresis of SmaI DNA macrorestriction fragments for typing strains of vancomycin-resistant enterococci

An algorithm based on one or two nasal samples is accurate to identify persistent nasal carriers of Staphylococcus aureus

Contaminations of laboratory surfaces with Staphylococcus aureus are affected by the carrier status of laboratory staff

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