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Vancomycin resistant enterococci (VRE) in Swedish sewage sludge

Article  in  Acta Veterinaria Scandinavica · February 2009

DOI: 10.1186/1751-0147-51-24 · Source: PubMed

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Acta Veterinaria Scandinavica BioMed Central

Research Open Access

Vancomycin resistant enterococci (VRE) in Swedish sewage sludge
Leena Sahlström*†1, Verena Rehbinder†2, Ann Albihn†2, Anna Aspan†2 and
Björn Bengtsson†2

Address: 1Finnish Food safety Authority, Evira, Mustialankatu 3, 00790 Helsinki, Finland and 2National Veterinary Institute, 75189 Uppsala,
Sweden
Email: Leena Sahlström* - [email protected]; Verena Rehbinder - [email protected]; Ann Albihn - [email protected];
Anna Aspan - [email protected]; Björn Bengtsson - [email protected]
* Corresponding author †Equal contributors

Published: 29 May 2009 Received: 28 November 2008

Accepted: 29 May 2009
Acta Veterinaria Scandinavica 2009, 51:24 doi:10.1186/1751-0147-51-24
This article is available from: http://www.actavetscand.com/content/51/1/24
© 2009 Sahlström et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Antimicrobial resistance is a serious threat in veterinary medicine and human
healthcare. Resistance genes can spread from animals, through the food-chain, and back to humans.
Sewage sludge may act as the link back from humans to animals. The main aims of this study were
to investigate the occurrence of vancomycin resistant enterococci (VRE) in treated sewage sludge,
in a Swedish waste water treatment plant (WWTP), and to compare VRE isolates from sewage
sludge with isolates from humans and chickens.
Methods: During a four month long study, sewage sludge was collected weekly and cultured for
VRE. The VRE isolates from sewage sludge were analysed and compared to each other and to
human and chicken VRE isolates by biochemical typing (PhenePlate), PFGE and antibiograms.
Results: Biochemical typing (PhenePlate-FS) and pulsed field gel electrophoresis (PFGE) revealed
prevalence of specific VRE strains in sewage sludge for up to 16 weeks. No connection was found
between the VRE strains isolated from sludge, chickens and humans, indicating that human VRE did
not originate from Swedish chicken.
Conclusion: This study demonstrated widespread occurrence of VRE in sewage sludge in the
studied WWTP. This implies a risk of antimicrobial resistance being spread to new farms and to
the society via the environment if the sewage sludge is used on arable land.

Background growing problem and vancomycin resistant enterococci

Enterococci are naturally occurring bacteria in the intesti- (VRE) in particular are considered a serious threat in hos-
nal tract of humans and animals, and are often used as pitals around the world [4]. Vancomycin is often used as
indicators of faecal contamination in water [1]. Entero- a last resort in treatment of antibiotic resistant gram-posi-
cocci are resistant to environmental stress and may persist tive bacterial infections caused by organisms such as
for a long time outside their hosts. They are not consid- multi-resistant enterococci and methicillin resistant sta-
ered severe pathogenic organisms, but some species, e.g. phylococci. In the USA, the prevalence of VRE is mainly
Enterococcus (E.) faecalis and E. faecium, are important documented as nosocomial infection in humans [5,6]. In
causes of nosocomial infections [2,3]. Antimicrobial Europe, nosocomial infections with VRE are less com-
resistance in strains causing nosocomial infections is a mon, but such bacteria are widespread among healthy

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livestock [5] as a consequence of previous use of tial risk of entering food producing animals and thereby
avoparcin (an analogue to vancomycin) as a feed additive the human food chain. Moreover, resistance genes could
in animal husbandry [7-9]. be transferred to other bacteria in the environment.
Novais et al., [20] and Iversen et al., [21] highlight the risk
Vancomycin resistance in nosocomial isolates of entero- of spreading antibiotic resistant bacteria in the environ-
cocci is usually mediated by the resistance genes vanA or ment and of building up environmental reservoirs of anti-
vanB [4]. High-level vancomycin resistance (MIC >64 mg/ microbial resistance genes that might re-enter the
L) is mediated by the vanA gene cluster, located on the ecosystem in human pathogenic bacteria.
transferable genetic element transposon Tn1546 [10]. Var-
iable levels of vancomycin resistance (MIC 4–1000 mg/L) The main aim of the present study was to investigate the
characterise the vanB genotype and the gene cluster is occurrence of VRE in treated sewage sludge from a Swed-
located on another mobile genetic element, Tn1547 [10]. ish sewage treatment plant and to study their prevalence
Some enterococci (including E. gallinarum) may posses in the WWTP over time. A secondary aim was to compare
intrinsic, but not transferable, resistance against vancomy- VRE isolates from sewage sludge with isolates from
cin, coded by vanC (MIC 2–32 mg/L) [10]. humans and chickens.

Vancomycin resistance may be spread either by clonal dis- Methods

semination of resistant isolates or by transfer of the resist- Sampling in the wastewater treatment plant
ance genes to other strains of the same bacterial species or Sewage sludge was sampled at the Kungsängen waste
to other species or genera (horizontal gene transfer). Inter- water treatment plant in Uppsala, Sweden, which serves
species transfer of transposable (Tn1546-like) genetic ele- 200 000 population equivalents. At the plant, sewage
ments between different species of enterococci [11] and sludge is treated with mesophilic anaerobic digestion and
from enterococci to Listeria monocytogenes [12] have been dewatered by centrifugation. The site for sampling of
demonstrated. The first documented case of horizontal dewatered sludge was at the sludge feeder after centrifuga-
transfer of the vanA gene to the most feared cause of noso- tion. Samplings started 24 February 2003 and were
comial infections, Staphylococcus aureus, was documented repeated once a week for a total of 17 times during a
in 2002 in the USA [13]. In addition, Marcineck et al. [14] period of more than four months, with the last sampling
demonstrated gene transfer between different strains of E. occasion on 30 June 2003. On every sampling occasion
faecalis in the natural environment of a waste water treat- five (5) samples of approximately 200 g were taken during
ment plant. Moreover, E. faecium vanA genetic elements of 1 hour with 12 minutes intervals from the sludge feeder
animal origin have recently been proven capable of trans- (screw) after the centrifugation of the sewage sludge. On
ferring to E. faecium strains of human origin in the intes- the first and fifth sampling occasions, only one sample
tine of human volunteers [15]. was obtained due to technical problems at the plant. In
total, 77 samples were collected and every sample was
Iversen et al. [16] demonstrated 60% prevalence of VRE in analysed separately. The treated and centrifuged sewage
samples of raw sewage from Swedish waste water treat- sludge had a dry matter content of 25–32% according to
ment plants (WWTP) and 19% prevalence in treated sew- an electronic moisture analyser (Sartorius MA-30) at the
age. These levels are surprisingly high, since nosocomial WWTP. Samples were collected in clean disposable vessels
VRE infections are uncommon in Sweden [17] and the and cultured within three hours at the National Veterinary
presence of VRE in healthy humans is considered rare Institute, Uppsala, Sweden.
[18]. Moreover, vancomycin is seldom used in routine
human healthcare in Sweden [17]. Likewise, VRE are rare Bacteriological culture
in Swedish livestock production and avoparcin has not Because of an expected low concentration of VRE in the
been used as a growth promoter since the early 1980s. sludge, samples were pre-enriched in enterococcosel
Since VRE occur in sewage [16], it is likely that the bacteria broth (BBL, Baltimore, USA). Ten (10) g wet weight of
can also be found in sewage sludge. sludge were mixed in a stomacher with 90 g of enterococ-
cosel broth containing 8 mg/L vancomycin and 0.25 mg/
Antimicrobial resistance is a serious threat in human L clindamycin. Clindamycin was added to improve the
healthcare. It is thus important to maintain the low level detection of VRE by reducing background flora. The con-
of resistance among the Nordic food producing animals, centration of clindamycin used (0.25 mg/L) is much
which could spread the resistance genes to humans. lower than the MIC of enterococci and should not imply
Chicken consumption increases, and VRE do occur in low selection within the population of enterococci. After incu-
numbers in Swedish broiler chickens [19]. If sewage bation at 37°C for 24 h, an aliquot of 0.1 mL from the
sludge is applied as a fertiliser to arable land, any VRE pre-enrichment broth was plated on Slanetz-Bartley agar
present could be spread in the environment, with a poten-

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(Oxoid, UK) with 8 mg/L vancomycin (Sigma, Germany) PCR for detection of vanA and vanB genes was performed
and incubated at 37°C for 48 h. according to Dutka-Malen et al., [24]. Enterococcus faecium
BM 4147 and E. faecalis V583 were used as positive con-
In addition to the pre-enrichment procedure, direct cul- trol strains for vanA and vanB genes, respectively. Both
ture was performed on the samples from sampling occa- strains were obtained from the Swedish Institute for Infec-
sions 10–17. For this, aliquots of 0.1 mL were taken from tious Disease Control, Solna, Sweden.
the pre-enrichment broth before incubation (see above),
plated on Slanetz-Bartley agar with 8 mg/L vancomycin PhenePlate
and incubated at 37°C for 48 h. A total of 8 weeks of sam- The 84 VRE isolated from sewage sludge were subtyped by
pling (including 5 samples/week) gave 40 samples for a rapid screening method for enterococci using PhP-FS
direct plating. microtitre plates (PhenePlate™, PhPlate Microplate Tech-
niques AB, Stockholm, Sweden) [25]. The subtyping of
Confirmation and identification of VRE isolates enterococci on PhP-FS plates is based on biochemical tests
Colonies with morphology consistent with enterococci with 24 different reagents, and was performed according
were sub-cultured on bile-esculin agar (Difco, Maryland, to the manufacturer's instructions. In short, a few colonies
USA) and horse blood agar (LabM, Lancashire, UK) were suspended in PhP-suspending media, inoculated
(37°C, 24 h). From each sample, 1–3 colonies with differ- onto the PhenePlates, and incubated at 37°C. The plates
ent morphology were chosen for further identification. were read with an optical microplate reader connected to
Isolates with a positive reaction on bile-esculin agar and a computer after 16, 40 and 64 h. For the cluster analysis
in PYR testing (pyrrolidonyl aminopeptidase) (Rosco, of the PhP-data, the unweighted-pair group method using
Denmark) were tested for antimicrobial susceptibility and average linkages (UPGMA) by the PhP-software (PhPlate
identified to species level according to Devriese et al. [22] Microplate Techniques AB) was used. Isolates with a sim-
by use of the following biochemical tests: mannitol, sorb- ilarity index greater than 0.975 were considered to be of
itol, arabinose, saccharose, ribose, raffinose and methyl- the same PhP-subtype. The subtypes were named α, β, γ, δ
α-D-glucopyranoside. and Φ (Additional file 1).

Three isolates (SL26, SL63 and SL68), untypeable by bio- In addition, using the PhenePlate analysis, VRE isolates
chemical tests, were 16S rRNA sequenced according to a from sewage sludge were compared to other isolates.
procedure described by Johansson et al. [23]. These were: three VRE isolated in 1998–1999 from sewage
from the same WWTP as the sewage sludge [16]), three
In total, 84 suspected VRE strains were stored at -70°C for VRE isolated from chickens in 2005 (SVA, National Veter-
further analysis. inary Institute, Uppsala, Sweden), two human isolates
from SMI isolated 1997 and 2001 (Swedish Institute for
Antimicrobial susceptibility testing Infectious Disease Control, Solna, Sweden), and five
Antimicrobial susceptibility was tested by a microdilution human VRE isolated in 2005 in Uppsala (UAS, The Uni-
method following the standards of the Clinical and Labo- versity Hospital, Uppsala, Sweden). The three isolates
ratory Standards Institute (CLSI) using VetMIC™ microdi- from chickens are of the same PhP and PFGE-type as the
lution panels (National Veterinary Institute, Uppsala, vast majority of VRE isolated from Swedish chicken
Sweden). Antimicrobials and ranges tested are given in within the framework of a monitoring program during
Additional file 1. In short, 4–5 colonies of a fresh over- years 2000 to 2005 [26]. The five human isolates from
night culture were diluted in 5 mL Muller-Hinton broth Uppsala were from one outbreak and considered identical
(Difco, Maryland, USA) and incubated at 37°C for 4 h. when typed by PFGE [Torell E: The University Hospital,
Ten microlitre of this suspension were diluted in 10 mL of Uppsala, Sweden, 2006, personal communication].
Muller-Hinton broth and 50 μL of the diluted sample
were inoculated into each well on a microtitre plate con- Pulsed Field Gel Electrophoresis (PFGE)
taining the dried antibacterial substances. The microtitre PFGE was performed according to a modified protocol
plate was then sealed with plastic tape and incubated at based on Turabelidze et al. [27] to verify relationships of
37°C for 16–18 h. To check the purity, a droplet of the the PhP-subtypes. Two isolates from each PhP-subtype,
suspension was also streaked on blood agar, and incu- most distant regarding time of isolation, were chosen for
bated together with the microtitre plates. Analysis of the the PFGE. In addition, eight single subtype isolates from
results was based on growth or no growth in the microtitre the pheneplate screening were analysed by PFGE.
well. The MIC (Minimum Inhibitory Concentration)
value was considered to be the lowest concentration of Electrophoresis was performed using a CHEF DRII appa-
antibiotic that prevented bacterial growth. ratus (BioRad Laboratories, Richmond Calif., USA). In the
first block the initial switch time was 3.5 s, the final switch

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time was 25 s, and the run time was 20 h, and in the sec- The PhenePlate analysis showed that phenotypically iden-
ond block the initial switch time was 1 s and the final tical strains were repeatedly isolated for up to 16 weeks.
switch time 5 s for 16 h, at 6 V/cm. Sma I was the enzyme This observation was strengthened by indistinguishable
used for cleaving the DNA. Lambda Ladder PFG Marker PFGE patterns of isolates, most distant regarding time of
(New England BioLabs Inc.) was used as a molecular size isolation, within each PhP-subtype (Figure 1).
marker. Isolates were considered indistinguishable if the
PFGE patterns were indistinguishable or differed by only Antibiograms
one band. Antibiograms of isolates within a PhP-subtype were
mostly similar and to some extent discriminatory between
Results subtypes (Additional file 1). However, in E. faecium sub-
VRE isolates in sludge type β, five (5) isolates (SL33, SL37, SL69, SL70 and SL99)
After enrichment, VRE carrying the vanA or vanB genes had divergent MIC values for gentamicin; MIC = 8 instead
were isolated from sludge samples on each of the 17 once- of MIC>512 as in the other 31 isolates. These five isolates
weekly sampling occasions. Sixty-one (61) of the 77 sam- were found in weeks IV to XII, i.e. over a nine-week
ples cultured (79%) tested positive for VRE (Table 1). In period. In addition, one isolate (SL37) in the same sub-
contrast, VRE were isolated from only three of 40 samples type (E. faecium subtype β) had a MIC<16 to neomycin
after direct plating. Subtyping with the PhenePlate (PhP) compared to the others, which had MIC values > 1024.
system identified 66 isolates (out of 84 analysed), each One of the isolates from direct culture (SL93), which also
representing a unique PhP-subtype from a sample (Figure belonged to the E. faecium subtype β, had a MIC value >
1). All three isolates from direct culture belonged to one 64 to tetracycline, whereas the other isolates had MIC val-
PhP-subtype (Figure 1). ues < 0.5–8.

E. faecium was the most prevalent species 85% (56/66) Discussion

followed by E. hirae 14% (9/66) and E. durans 1.5% (1/ VRE were isolated every week from sewage sludge during
66) (Table 1). Three isolates (SL26, SL63 and SL68) with the four-month study. This indicates that VRE as well as
uncertain species identity as judged from biochemical enterococci survive mesophilic anaerobic digestion, as
tests were identified as E. faecium by 16S rRNA gene earlier shown by Sahlström et al 2004 [28]. Treatment
sequencing. The sequence similarity of SL26 to E. faecium, with mesophilic digestion is a common way to treat sew-
strain DSM20477T (GenBank accession number age sludge in WWTPs in Sweden, which means that VRE
AJ276355) was 100% and the sequence identity to strain may occur in sewage sludge in other parts of the country
DSM20477T was 99.7%. The sequence similarity of SL63 as well. The majority of the VRE isolated were E. faecium
and SL68 to strain DSM20477T was 100% and sequence harbouring the vanB gene. This agrees with the fact that
identity to strain DSM20477T was 99.9%. However, the most VRE isolated from Swedish hospital patients are E.
three isolates may also represent Enterococcus lactis (Gen- faecium carrying the vanB gene [17,29] but contrasts with
Bank accession number DQ255948), because the the situation in other European countries, where E. fae-
sequence similarities to this species is also high (99.8– cium vanA is the most common cause of VRE infections
99.9%). [30].

PCR analysis revealed four isolates to harbour the vanA Isolation of VRE in Swedish healthcare is rare and as a
gene (one E. durans and three E. faecium), whereas the consequence, inflow of VRE of human origin to WWTPs
vanB gene was confirmed in the remaining isolates (Figure should be low [31]. The few human VRE strains analysed
1). In addition, E. gallinarum harbouring neither vanA nor in this study differed regarding their PhenePlate profiles
vanB genes was isolated from one sample (MIC for vanco- from the VRE isolates from sewage sludge. However, fur-
mycin = 8). The three isolates from direct plating were all ther comparisons of VRE from humans and sewage sludge
E. faecium vanB. would be interesting. The frequent isolation of VRE in this
study could be due to the ability of enterococci (including
PhenePlate analysis VRE) to persist in the WWTP environment. PhenePlate
The dendrogram in Figure 1 illustrates the results of analysis revealed that indistinguishable strains were
PhenePlate analysis of 69 sewage sludge(SL) samples repeatedly isolated from the sludge during a period of sev-
(including three from direct plating) and 13 other VRE eral weeks, a finding confirmed by PFGE analysis (figure
isolates: human(HU), sewage(SE) and chickens(C). None 2). The longest interval between isolations was 15 weeks.
of the 13 isolates of other origin belonged to the same
PhP-subtype as the isolates from sludge samples (Figure Bruinsma et al. [32] suggest a horizontal spread of the
1). enterococcal vanA gene rather from pigs than from chick-
ens to human strains. The source of human E. faecium

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Figure
isolated
Dendrogram
1from showing
sludge (S)UPGMA clustering of PhenePlate typing data for vancomycin resistant E. faecium, E. hirae and E. durans
Dendrogram showing UPGMA clustering of PhenePlate typing data for vancomycin resistant E. faecium, E.
hirae and E. durans isolated from sludge (S). For comparison, vancomycin resistant E. faecium from sewage, chickens and
humans are included. The horizontal axis of the dendrogram shows the similarities between isolates, and the dotted line indi-
cates the identity level of 0.975. PFGE types are indicated for isolates tested by pulsed field gel electrophoresis (PFGE). Filled
circles indicate isolates with vanA, the other isolates carried the vanB gene. Arrows indicate the isolates from direct culture.

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Table 1: Species (E. hirae, E. durans and E. faecium) and PhenePlate subtypes of enterococci isolated by enrichment culture during the
study period.

Sampling week (date) E. hirae E. durans E. faecium

subtype α single single subtype β subtype γ subtype δ sutype Φ single

I (24.2)* 1 1
II (4.3) 5 1 (SL19)
III (10.3) 1 1 1 (SL26)
IV (18.3) 1 2 2 (SL29, SL35) 1 (SL34)
V (24.3)* 1
VI (31.3) 1
VII (7.4) 4 1
VIII (14.4) 5
IX (22.4) 1 1 (SL63)
X (28.4) 3 1
XI (5.5) 4 1 (SL84)
XII (12.5) 5 1
XIII (19.5) 5 1
XIV (26.5) 2 3
XV (3.6) 1 2
XVI (24.6) 3
XVII (30.6) 1 2

The figures represent the number of positive samples (out of 5 samples) on the actual date of sampling. The subtypes were differentiated using
UPGMA clustering of PhP data. Identification of single subtype isolates in brackets.
*Only one sample obtained

vanB is not known, but could be another animal reservoir enterococci in sewage sludge. In sewage from Spanish
[33]. Our findings from sewage sludge, which can be WWTPs, VRE were detected in low proportions, i.e.
assumed to reflect the human population, give no indica- approximately 1:5000 (0.05%) of the total enterococci
tion that human VRE in Sweden derive from animals, count, using pre-enrichment in enterococcosel broth [35].
since we isolated mainly E. faecium vanB, whereas VRE in In addition, Ferreira da Silva et al. [36] reported no VRE
Swedish chickens are E. faecium vanA [26]. Moreover, VRE without pre-enrichment of sewage from a WWTP serving
of the dominant PhP-subtype isolated from Swedish 100 000 inhabitants in Portugal. However in another Por-
chickens were not found in sewage sludge (Figure 1), indi- tuguese study, 3.1% VRE were isolated from a larger
cating that human colonisation with VRE from Swedish WWTP in the district capital Coimbra by direct plating,
chickens is rare. but the authors expected that findings would have been
higher if an enrichment procedure had been used [37].
Our analysis illustrated a good correlation between the
PhenePlate analysis (dendrogram) and the PFGE analysis All of the vancomycin resistant E. faecium isolates carrying
(Figure 1). PhenePlate analysis has previously been found the vanB gene had high MICs to ampicillin (32->32 mg/
to be less discriminatory for E. faecium than for E. faecalis L). This is in accordance with a described genetic link
[34]. The E. faecium isolates, indistinguishable by UPGM between ampicillin resistance and vancomycin resistance
clustering of PhenePlate-data (identity level = 0. 975), type vanB2, which is a subtype of vanB [38-40]. In con-
were likewise indistinguishable by PFGE, whereas less trast, the three E. faecium vanA isolates from sewage sludge
similar isolates (similarity level = 0.969) differed on PFGE had substantially lower MICs to ampicillin (0.5–1 mg/L).
(e.g. SE345 vs. SL26). Likewise, the antibiograms also dis-
criminated PhP-subtypes from each other (Additional file In addition to E. faecium, other species of Enterococci iso-
1). lated from sewage sludge were found to have high MICs
to vancomycin. One isolate of E. durans from the first sam-
The direct platings revealed three samples positive for E. pling of sludge had high level vancomycin resistance
faecium out of 40 samples cultured (7.5%). This is in con- (MIC >128 μg/mL) and harboured the vanA gene. Torres
trast to the high isolation frequency after selective enrich- et al. [41] were the first in the world to report E. durans
ment (79.2%), which appears to be the preferred vanA in sewage, but such strains have also been reported
procedure for isolation of VRE from sewage sludge. The in sewage exposed to vancomycin waste [11]. Gambarotto
lower isolation frequency after direct plating was probably et al. [42] isolated one E. durans vanA from pork meat in
due to the fact that VRE constitute a small proportion of France, while Jenney et al. [43] reported isolation of E.

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Conclusion
In conclusion, sewage sludge contains vancomycin resist-
ant enterococci, implying a potential risk of spreading
VRE and resistance genes to the environment and possibly
to the human and animal population. The frequent occur-
rence of VRE in mesophilically digested sewage sludge
implies a need for more efficient hygienic treatment of
sewage sludge, in order to avoid possible spread of anti-
microbial resistance through use of sewage sludge on ara-
ble land. Usage of sewage sludge may contribute in
spreading resistant bacteria by building up environmental
reservoirs of antimicrobial resistance genes that enter the
ecosystem, if there is not efficient hygienic treatment of
sewage sludge before use [29,46].

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
The study was designed by all authors. LS did the field
work and the analysis was done by LS, VR and AAS. LS
drafted the manuscript and all authors revised, read and
approved the final manuscript.

Additional material

Additional file 1
Minimum inhibitory concentration (MIC) for VRE per PhP subtype.
Only isolates after enrichment and only one isolate of each subtype per
sample are shown.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1751-
λ,
λ
PFGE
Figure
SL6,patterns
SL19,
2 SL25, SL26, λ,
of sewage sludge
SL29,samples
SL34, SL35,
(fromSL36,
left to
SL47
right)
and
0147-51-24-S1.doc]
PFGE patterns of sewage sludge samples (from left
to right) λ, SL6, SL19, SL25, SL26, λ, SL29, SL34,
SL35, SL36, SL47 and λ. λ = Lambda Ladder PFG Marker,
DNA size standard (New England BioLabs Inc.) Isolates SL6
and SL36 represent PFGE type 1 and isolates SL29 and SL35 Acknowledgements
represent PFGE type 4. Thanks to prof Karl-Erik Johansson, Swedish University of Agricultural Sci-
ences for 16S rRNA sequencing of isolates; to personnel at Kungsängen
WWTP, Uppsala; to Aina Iverssen, Karolinska Institutet, Stockholm, for
kind supply of VRE isolates from sewage; to both Barbro Olsson-Liljekvist,
durans vanB from a human patient. Enterococcus galli- Swedish Institute for Infectious Disease Control, Solna, and Erik Torell,
narum, also isolated in our study, usually has an intrinsic Uppsala University Hospital, Uppsala for kind supply of VRE isolates from
low level resistance against vancomycin, mediated by humans; and to the Swedish University of Agricultural Sciences, Uppsala,
vanC [10]. However, there are a few reports of E. galli- for funding this study.
narum carrying vanA or vanB [44,45]. The isolation of E.
durans, E. gallinarum and E. hirae in addition to E. faecium References
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