Supplement bij veertiende jaargang, april 2006 - NVMM

75 mgr/liter aztreonam and 5 mg/liter ceftizoxim and
incubated for > 15 hours at 370°C. DNA was extracted
(Magna-Pure Roche) from both the saline and the broth and
subjected to a real-time multiplex PCR assay based on the
Huletsky protocol (Québec, Canada). DNA extracted from
the broth was also subjected to the MRSA-test from HAIN
(Germany). Two swabs obtained from two patients with a
MRSA infected wound were included as positive controls.
PCR positive samples were subjected to extensive culture
procedures.
Results: The 2 MRSA-positive swabs were positive in
all tests. None of the 152 swabs were MRSA-positive on
standard culture. However, the Huletski-PCR showed that
4/152 saline samples and 25/152 broth samples were clearly
MRSA-positive (Ct values < 40). By using the HAIN-test
on the broth samples, 7/152 samples proved to be MRSApositive,
these 7 samples were also positive in the Huletsky-
PCR. After extensive culturing from 17/25 Huletsky-PCR
positive samples a MRSA-strain could be recovered.
Conclusions: Molecular detection provides a very powerful
method for the detection of MRSA, especially when a preenrichment
broth is part of the procedure. Disturbing
however is the high MRSA prevalence when using these
strategies. Based on these result, the MRSA prevalence in
our region is between 5 and 15%, so at least 10 times higher
than expected.
05.09
novel variants of Staphylococcus Cassette Chromosomes
excised by ccrA/B type 2 recombinases in Staphylococcus
aureus
W.T.M. Jansen, M.M. Beitsma, C.J. Koeman, W.J.B. Wamel,
J. Verhoef, A.C. Fluit
UMCU, Medical Microbiology, Utrecht
Background: Staphylococcal Cassette Chromosome mec
(SCCmec) type IV is associated with virulent communityacquired
MRSA and frequent horizontal transfer among
Staphylococci. To gain insight into the mechanism of
transfer, we studied the by ccrA/B type 2 recombinase
mediated excision of SCCmec II and IV.
Methods: A ccrA/B type 2 plasmid was constructed, in
which the ccrA/B genes were placed under control of a
constitutive phage-repressor promoter. The plasmid was
transduced into different MRSA strains, including MR108,
MW2, Ca05 (JCSC1968), JCSC1978, Mu50 and N315, and
a set of strains that demonstrated the in vivo transfer of
SCCmec IV (MSSA wkz1, MRSA wkz2, and Staphylococcus
epidermidis O7.1 (Wielders et al. Lancet 2001:357:1674).
Excision of SCC elements was determined by PCR and
sequencing.
Results: In wild-type MRSA and S. epidermidis strains
containing SCCmec IV, but not in SCCmec II MRSA
Ned Tijdschr Med Microbiol 2006; 4:Supplement
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strains, excision of the cassette was observed. Introduction
of the ccrA/B type 2 plasmid in the different strains
yielded excision of SCCmec II and multiple excision
variants of SCCmec IV. Sequencing of the alternatively
excised products in several SCCmec IV wild-type strains
and clones identified a 100 bp shortened SCCmec variant
and novel excision products in MRSA wkz2 not present
in SCCmec donor O7.1. Sequencing showed a 5,877
bp, conserved SCC-like element that lacks mecA and
ccrA/B recombinases, which was also present in MR108.
Excision of the SCC-like element in wild type S. aureus
was dependent on the presence of SCCmec. The element
could be excised separately or as part of a novel composite
cassette together with SCCmec.
Conclusions: Four SCC excision variants were identified
in SCCmec IV strains, including the in vivo formation of a
new composite SCCmec cassette with a SCC-like element.
SCCmec II was only excised in the presence of the ccrA/B
2 plasmid. The variety in SCC excisions may increase
horizontal transfer and genetic plasticity in SCCmec IV
MRSA strains.
05.10
A specific secretion system mediates PPe protein transport
in Mycobacteria and is required for virulence
W. Bitter1 , A.M. Hannes1 , C.M.J.E. Vandenbroucke-Grauls 1 ,
T. Verboom1 , B.J. Appelmelk1 , J. Luirink3 , R. Musters2 ,
F. Abdallah1 1 2 VU medical centre, Medical Microbiology, Amsterdam, VU
medical centre, Physiology, 3VU medical centre, Molecular
Microbiology, Amsterdam
The Mycobacterium tuberculosis genome contains two
large gene families, the so-called PE and PPE genes, that
are unique to mycobacteria. Here we report that one of
the PPE proteins, i.e. PPE41, is secreted both in vitro and
in macrophages. Furthermore, we have identified that
PPE41 is secreted by a novel secretion system, which is
homologous to the mycobacterial ESAT-6 secretion system.
This secretion system, designated ESX-5, is conserved in
pathogenic mycobacteria, but is not present in the environmental
species Mycobacterium smegmatis. However, introduction
of the entire ESX-5 region in M. smegmatis resulted
in efficient secretion of heterologously expressed PPE41,
which shows that ESX-5 is necessary and sufficient for
PPE transport. ESX-5 secretion mutants showed reduced
spreading of mycobacteria to uninfected macrophages,
which shows that ESX-5 secreted substrates play an
important role in virulence.