Multiple-Locus Variable Number Tandem Repeat Analysis of Staphylococcus Aureus: Comparison with Pulsed-Field Gel Electrophoresis and spa-Typing

(MRSA) is required to study the routes and rates of transmission of this pathogen. Currently available typing techniques are either resource-intensive or have limited discriminatory ability. Multiple-locus variable number tandem repeat analysis (MLVA) may provide an alternative high throughput molecular typing tool with high epidemiological resolution.-sequence typing and PFGE, at the MLVA complex level with group separation values of 95.1% and 89.2%. MLVA could not discriminate between pig-related MRSA strains isolated from humans and pigs, corroborating the high degree of relationship. MLVA was also superior in the grouping of MRSA isolates previously assigned to temporal-spatial clusters with indistinguishable SpaTypes, demonstrating its enhanced epidemiological usefulness. that yields discrete and unambiguous data that can be used to assign biological meaningful genotypes and complexes and can be used for interlaboratory comparisons in network accessible databases. Results suggest that MLVA offsets the disadvantages of other high discriminatory typing approaches and represents a promising tool for hospital, national and international molecular epidemiology

A MLST typing scheme for Enterococcus faecium

C1 - Journal Articles Referee

Widespread Dissemination in The Netherlands of the Epidemic Berlin Methicillin-Resistant Staphylococcus aureus Clone with Low-Level Resistance to Oxacillin

Methicillin-resistant Staphylococcus aureus (MRSA) is an important human pathogen and represents a growing public health burden due to the emergence and spread of epidemic strains, particularly within the hospital environment. An epidemic MRSA clone, with characteristic low-level resistance to oxacillin, emerged in the year 2000 and became endemic in the Netherlands. Multilocus sequence typing characterized the strain as sequence type 45, which was previously designated the Berlin epidemic MRSA clone. In 2 years, this strain has become the predominant MRSA clone in the Netherlands

Multiple Cases of Familial Transmission of Community-Acquired Methicillin-Resistant Staphylococcus aureus

The worldwide emergence of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) can have severe public health implications. Familial transmissions of CA-MRSA in The Netherlands were investigated. Among the families studied, two clusters of CA-MRSA could be identified. This report demonstrates that family members can serve as reservoirs of CA-MRSA which may become a serious problem in containing the spread of MRSA

High prevalence of methicillin resistant Staphylococcus aureus in pigs. Vet. Microbiol

Abstract Recently methicillin resistant Staphylococcus aureus (MRSA) was isolated from pigs and pig farmers in The Netherlands. In order to assess the dissemination of MRSA in the Dutch pig population, we screened 540 pigs in 9 slaughterhouses, where a representative portion of Dutch pigs (63%) was slaughtered in 2005. We found 209 (39%) of the pigs to carry MRSA in their nares. Forty-four of 54 groups of 10 consecutive pigs (81%), each group from a different farm, and all slaughterhouses were affected. All MRSA isolates belonged to 1 clonal group, showing Multi-Locus Sequence Type 398 and closely related spa types (mainly t011, t108 and t1254). Three types of the Staphylococcal Chromosome Cassette (SCCmec) were found: III (3%), IVa (39%) and V (57%). All 44 tested isolates (1 isolate per group) were resistant to tetracycline, reflecting the high and predominant use of tetracyclines in pig husbandry. Twenty-three percent of the isolates were resistant to both erythromycin and clindamycin and 36% to kanamycin, gentamicin and tobramycin but only a single isolate was resistant to co-trimoxazole and none to ciprofloxacin and several other antibiotics. The percentage of MRSA positive pigs was significantly different among slaughterhouses and among groups within slaughterhouses, indicating a high prevalence of MRSA in pigs delivered from the farms as well as cross contamination in the slaughterhouses.

Minimum spanning tree of the 1681 <i>S. aureus</i> isolates typed by MLVA.

<p>Clustering of MLVA profiles was done using a categorical coefficient. In the minimum spanning tree the MLVA types are displayed as circles. The size of each circle indicates the number of isolates with this particular type. Thick solid lines connect types that differ in a single VNTR locus and a thin solid connects types that differ in 2 VNTR loci. The color of the halo surrounding the MLVA types denote types that belong to the same complex. MLVA complexes were assigned if 2 neighboring types did not differ in more than 1 VNTR locus and if at least 5 types fulfilled this criterion. MLVA complexes are also indicated in characters e.g. MC8 denotes MLVA complex 8.</p

Schematic map of the genome of <i>S. aureus</i> strain N315, indicating the positions of the VNTR loci used in MLVA.

<p>Schematic map of the genome of <i>S. aureus</i> strain N315, indicating the positions of the VNTR loci used in MLVA.</p

List of primers used for MLVA of <i>S. aureus</i>.

1<p>Fluorescent dyes are indicated by a character. F, FAM; N, NED; V, VIC; P, PET.</p>2<p>Coordinates in NC_002745.</p>3<p>Unlabeled version of the primer VNTR61_02, used to reduce the fluorescence signal in the MLVA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005082#s2" target="_blank">Materials and Methods</a>).</p

Minimum spanning tree of 1304 <i>S. aureus</i> isolates typed by PFGE.

<p>The PFGE types are displayed as circles. Clustering was performed using the Pearson correlation coefficient and a 2% optimization setting. The resulting similarity matrix was used to construct a minimum spanning tree using a 10% similarity bin size. Related PFGE grouped and this is denoted by the double circle (compacted complexes). The MLVA complex is denoted as the color of the circles (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005082#pone-0005082-g002" target="_blank">figure 2</a>) and is also indicated in characters.</p

Space-time SpaType clusters found in isolates collected from 15 hospitals during the time period January–October 2008.

1<p>MT, MLVA type.</p