Download Genetic relatedness of ESBL-producing E. coli from pig

Genetic relatedness of ESBL-producing E. coli from pig holdings and humans in the Dutch-German border region by whole
genome sequencing.
S. García-Cobos1, R. Köck2, J. Frenzel1, S. Surie1, A. W. Friedrich1, J. W. A. Rossen1.
1Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, The Netherlands2Institute of Hygiene, University Hospital
Münster, Robert-Koch-Str. 41, 48149 Münster, Germany
Objectives
The globally rising incidence of extended-spectrum β-lactamase
(ESBL)-producing Enterobacteriaceae in livestock and the
possible transmission of these bacteria from animals to humans
is an important health concern that needs to be addressed. The
aim of this study was to investigate the genetic relatedness of
ESBL-Escherichia coli from pig holdings and hospitalized
patients in the Dutch-German border region by high-throughput
sequencing technologies.
Conclusions
Human and animal isolates, which clustered
together by DL were analyzed by whole
genome sequencing (WGS) (Illumina MiSeq)
and assembled using CLC Bio Genomics
Wokbench software. Ridom SeqSephere+
software was used to determine an MLST+
scheme using E. coli K12 as a reference.
Twenty additional E. coli genomes of pigs,
farm workers and Dutch patients were added
during target definition to develop a more
stable MLST scheme plus 37 plasmid
sequences for exclusion of genes located on
plasmids. This resulted in a scheme
consisting of 2,653 target genes used for
genotypic analyses. In addition, the
resistome to β-lactams was analyzed by
ResFinder 2.1.
1
2
ESBL-producing E.coli from pig holdings and
humans that initially clustered together using
DL or MLST showed more than 162 different
alleles of 2,600 genes using MLST+.
Therefore,
to
determine
possible
transmission of resistant bacteria from
animals to humans, is required high
discriminatory power typing methods in
combination with epidemiological metadata to
obtain reliable data.
ST58
3
4
5
Cluster 5
ST539
Cluster 3
2077
Results
Figure 1. Distribution of hospitals and farms included in the study.
Red circles and yellow squares indicate hospital and farm origin,
respectively.
Material and Methods
A total of 305 ESBL- producing E. coli isolates were initially typed
by Diversilab® (DL): 199 clinical E. coli isolates from patients of
four hospitals in the Dutch-German border region (Groningen,
Enschede, Oldenburg, and Münster) collected from January to
June 2011 and 106 E. coli isolates collected from 47 different pig
farms (dust and manure samples) on the German side of the
border between February and September 2013 (Figure 1).
Analysis was done using the Pearson correlation. Isolates with a
similarity >95% were considered indistinguishable. Then, typing
results of one representative isolate of every cluster obtained
from the hospital and animal DL previous dendrograms were
selected (in total 95 E. coli clinical isolates and 61 E. coli pig
isolates) and analyzed together by the DL software.
The molecular typing by DL of clinical and
pig-associated E. coli isolates (n=154)
showed a total of six clusters containing
isolates from both sources (Figure 2).
Isolates belonging to these clusters were
selected for WGS. One isolate from cluster 4
showed low sequencing quality and was not
further analyzed and an additional isolate for
cluster 3 was included. Four pairs of isolates
combining porcine and German clinical
isolates showed the same MLST pattern
(ST38, ST167, ST410 and ST58). However,
using the MLST+ scheme showed 19
(ST410), 162 (ST38), 316 (ST167) and 328
(ST58) different alleles between isolates with
the same MLST type (Figure 3). In addition,
most isolates had a different resistome profile
for β-lactams, being CTX-M-15 and CTX-M14 the predominant ESBL genes. Only the
two isolates of ST410 had the same β-lactam
resistome profile (CTX-M-15 gene) (Figure
3).
Cluster 3
ST162
ST410
Cluster 1
Cluster 4
Cluster 1
ST10
ST410
Cluster 6
ST167
6
Cluster 2
ST38
Figure 3.
Figure 2. Dendrogram for E. coli
Diversilab© (DL) patterns. Type
designations for DL clusters are
given as numbers on the right.
Minimum Spanning Tree for 12
isolates based on 2,653 targets, pairwise ignoring
missing values. Between branches is indicated the
number of different alleles. The same colour
indicates isolates that clustered together by DL.