Supplement bij veertiende jaargang, april 2006 - NVMM

15.01
Molecular typing of bacterial pathogens reveals a spectrum
from clonal to panmictic population structures
L.M. Schouls
National institute of Public Health, Laboratory for Vaccine-
Preventable Diseases, Bilthoven
Introduction: Strain typing is an integral part of epidemiological
investigations of bacterial infections. However, strain
typing may also serve to study the composition of bacterial
populations and the impact of human interference on these
populations. Methods to distinguish bacterial strains have
improved dramatically over the last decade, mainly due to
the introduction of molecular typing technology.
Methods: Many studies have been performed to assess
source and contacts in outbreaks of community acquired or
nosocomial bacterial infections. Mostly these studies rely
on rapid, easy to perform band based typing methods like
pulsed-field gel electrophoresis and restriction fragment
length polymorphism. However, more recent and reliable
typing technology, such as multi-locus sequence typing
(MLST), relies on DNA sequence information. This
portable technique seems to be better suited to create
(inter)national databases required for population studies.
Furthermore, the availability of whole genome sequences
over a large number of bacterial species has enabled
the development of microarrays to perform comparative
genome hybridizations.
Results: MLST has yielded a number of publicly available
databases with typing results that have been used to
study bacterial populations. Some of the best studied
bacterial species are Neisseria meningitidis and Streptococcus
pneumoniae. Analysis of the MLST databases showed that
species like Helicobacter pylori are extremely diverse due
to constant lateral transfer and genome reshuffeling. In
other species like N. meningitidis where DNA is exchanged
and recombined to a lesser extend MLST has proven to be
extremely useful for population studies. However, some
species like Bordetella pertussis hardly exchange DNA
and have a nearly uniform population often referred to
as clonal. For highly variable and clonal species MLST is
unsuitable and other typing methods are required.
Conclusion: Molecular typing techniques, particularly
MLST, have revealed that bacterial species may have
population structures that vary from panmictic to clonal.
15.02
Phylogenomic Analysis of Enterococcus faecium (efm) Using
Mixed Whole genome Microarray Technology Discerns a
globally Dispersed Hospital Clade
H.L. Leavis1,2 , R.J.L. Willems1 , W.J.B. van Wamel1 , F.H.
Schuren2 , A.C. Fluit1 , M.J.M. Bonten1 1 2 UMC Utrecht, Eijkman-Winkler Institute, Utrecht, TNO,
Quality of Life, Zeist
Ned Tijdschr Med Microbiol 2006; 4:Supplement
S49
Introduction: Efm, ubiquous colonizers of humans and
animals, have emerged as nosocomial pathogens in the last
decade. Comparative phylogenomic analysis (CPA) using a
mixed whole genome microarray was used to elucidate the
population structure of Efm, using 97 Efm isolates from
different backgrounds (hospital outbreaks, hospitalized
patients, healthy subjects and animals) typed by multilocus
sequence typing (MLST).
Methods: A shotgun library constructed of DNA from
9 Efm strains and additional PCR products (virulence
genes and resistance markers) were spotted. Mixtures of
Cy5 labeled DNA of the 97 Efm isolates and Cy3 labeled
DNA of the library strains were hybridized. 3474 inserts
met quality criteria and were analyzed. 151 hospital clade
associated inserts were amplified, sequenced and blasted
in GenBank. Genomic mosaicism was visualized using
split decomposition analysis (SDA) and a Bayesian-based
algorithm on binary data was used to study phylogeny.
Character evolution was studied with maximum likelihoodbased
models.
Results: 1183 inserts (34 %) were conserved among all Efm
strains. CPA identified a clade, supported by Bayesian
probabilities (p=1), containing all outbreak-associated
strains and closely resembling the previously described
MLST-based Clonal Complex-17. 447 inserts (13%) were
associated with this clade. The predicted most prominent
inserts of this clade encoded a mutator type transposase
and a predicted metal-dependent hydrolase (98% sensitive,
100% specific). Clade-specific inserts (specificity > 78%,
sensitivity 40-94%) included membrane proteins (n=8),
hypothetical (n=26), resistance and (n=3), regulatory genes
(n=7), mobile elements (n=35), phage (n=6) and plasmid
genes (n=6). The genes were mainly located on 2 contigs
of the unfinished Efm DO genome. In SDA presence
and absence of genes on these contigs was inconsistent
with a branched phylogeny, indicating a highly mosaic
structure.
Conclusion: These findings demonstrate the evolution
of a specific Efm subpopulation, associated with hospital
outbreaks worldwide, and characterized by mobile
elements, regulatory and potential virulence genes and a
highly mosaic structure.
15.03
non-typeable methicillin-resistant Staphylococcus aureus
form a clonal cluster which seems to be related to pig
farmers and pigs
X.W. Huijsdens 1 , E. Spalburg 1 , M.G. van Santen-Verheuvel 1 ,
M.E.O.C. Heck1 , G.N. Pluister1 , B.J. van Dijke2 , A. Voss3 ,
W.J.B. Wannet1 , A.J. de Neeling1 1 2 RIVM, LIS-BBT, Bilthoven, St.Jansgasthuis, Weert,
3Canisius-Wilhelmina Hospital, Medical Microbiology,
Nijmegen