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Comparison of extended-spectrum-b-lactamase (ESBL) carrying Escherichia colifrom sewage sludge and human urinary tract infection
G. Zarfel a,*, H. Galler a, G. Feierl a, D. Haas a, C. Kittinger a, E. Leitner a, A.J. Grisold a, F. Mascher a, J. Posch a,B. Pertschy b, E. Marth a, F.F. Reinthaler a
a Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 8010 Graz, Austriab Institute of Molecular Biosciences, Karl-Franzens University Graz, Austria
a r t i c l e i n f o
Article history:
Received 23 February 2012
Received in revised form
26 September 2012
Accepted 28 September 2012
Keywords:
ESBL
Sewage sludge
E. coli
UTI
CTX-M
SHV
Austria
a b s t r a c t
For many years, extended-spectrum-beta-lactamase (ESBL) producing bacteria were a problem mainly
located in medical facilities. Within the last decade however, ESBL-producing bacteria have started
spreading into the community and the environment. In this study, ESBL-producing Escherichia coli from
sewage sludge were collected, analysed and compared to ESBL-E. coli from human urinary tract infections
(UTIs). The dominant ESBL-gene-family in both sample groups was blaCTX-M, which is the most prevalent
ESBL-gene-family in human infection. Still, the distribution of ESBL genes and the frequency of additional
antibiotic resistances differed in the two sample sets. Nevertheless, phenotyping did not divide isolates
of the two sources into separate groups, suggesting similar strains in both sample sets. We speculate that
an exchange is taking place between the ESBL E. coli populations in infected humans and sewage sludge,
most likely by the entry of ESBL E. coli from UTIs into the sewage system.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Pathogens carrying Extended-spectrum-b-lactamases (ESBLs)
represent main challenges to antibiotic therapy, with growing
prevalence rates all over the world (Coque et al., 2008; Falagas and
Karageorgopoulos, 20 09).
ESBLs are dened as enzymes able to hydrolyse penicillins,rst-,
second-, and third-generation cephalosporins and aztreonam (but
not cephamycins or carbapenems). They are normally inhibited by
b-lactamase inhibitors such as clavulanic-acid. Although many
species of gram-negative bacteria can be hosts of ESBLs, ESBLs are
mainly found in Enterobacteriaceae, particularly in Escherichia coli
and Klebsiella spp. (Falagas and Karageorgopoulos, 2009). Up tonow, more than 200 different ESBL genes have been identied. All
of them encode b-lactamases of the groups A and D of the Ambler
schemeand group into several different ESBL gene families(Ambler
et al., 1991; Paterson and Bonomo, 2005).
Up to the mid-1990s, TEM and SHV ESBL were the dominant
ESBL gene families worldwide. Within the last 15 years however,
these groups have been replaced by CTX-M. Only in North America,
TEM and SHV mutants are still the predominant ESBL genes. Beside
the three above mentioned groups, there are still some other b-
lactamases with ESBL phenotype, like PER, VEB, GES and some
members of the big familyof OXAb-lactamases, although most OXA
enzymes do not match the common ESBL criteria (Paterson and
Bonomo, 2005; Eisner et al., 2006; Livermore et al., 2007).
ESBL resistance genes are genetically diverse and are highly
mobile. Mobile genetic elements like plasmids, transposons and
integrons are the most common carriers of ESBL genes. Conse-
quently, horizontal gene transfer plays an important role in
spreading resistances into many different strains, species and into
different reservoirs (Woodford and Livermore, 2009).
ESBL-producing bacteria can also be found outside of medicalinstitutions, e.g. in wastewater (not only from hospitals), in sewage
sludge (used in agriculture) and in faeces of farm animals. Beside
these reservoirs with assumed high antibiotic pressure, there are
also cumulating reports of the occurrence of ESBL-producing
bacteria in healthy humans with no direct connection to medical
institutions, in food and even in wild living animals (Henriques
et al., 2006; Mesa et al., 2006; Carattoli, 2008; Poeta et al., 2009;
Vinue et al., 2009; Slama et al., 2010; Reinthaler et al., 2010).
The distribution of ESBL genes isolated from non-human
reservoirs differs from the distribution of ESBL genes reported in
medical institutions. For example, TEM-52 and CTX-M-1 genes are* Corresponding author.
E-mail address: [email protected] (G. Zarfel).
Contents lists available at SciVerse ScienceDirect
Environmental Pollution
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m/ l o c a t e / e n v p o l
0269-7491/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.envpol.2012.09.019
Environmental Pollution 173 (2013) 192e199
mailto:[email protected]://www.sciencedirect.com/science/journal/02697491http://www.elsevier.com/locate/envpolhttp://dx.doi.org/10.1016/j.envpol.2012.09.019http://dx.doi.org/10.1016/j.envpol.2012.09.019http://dx.doi.org/10.1016/j.envpol.2012.09.019http://dx.doi.org/10.1016/j.envpol.2012.09.019http://dx.doi.org/10.1016/j.envpol.2012.09.019http://dx.doi.org/10.1016/j.envpol.2012.09.019http://www.elsevier.com/locate/envpolhttp://www.sciencedirect.com/science/journal/02697491mailto:[email protected]
8/20/2019 Zar Fel 2013
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dominant in farm animals, while CTX-M-15, which is the dominant
ESBL gene in isolates taken from humans, is rarely found in animals
(Livermore et al., 2007; Carattoli, 2008; Chong et al., 2011).
In this study, ESBL E. coli strains from sewage sludge were
analysed and directly compared to ESBL E. coli from human infec-
tions with the same geographic origin. The investigation of urban
wastewater and sewage sludge can be used as a tool to analyse the
presence of ESBLs in the human population and in the environment
affected by humans. Sewage sludge can additionally be considered
as a source for antibiotic resistances, as it is used as a fertilizer in
agriculture and is consequently a potential source of infection.
Since the treatment of wastewater does not suf ciently eliminate
infectious pathogens, they may re-enter the food chain via treated
wastewater and sewage sludge which is applied on arable land.
Hence, the analysis of such environmental samples is important to
understand the ways of transmission of antibiotic resistance to
humans (Czechowski and Marcinkowski, 2006; Arthurson, 2008;
Koczura et al., 2012).
As a source of ESBLs from human infections, we chose to analyse
ESBLs from urinary tract infections (UTIs). UTIs are the most
common types of community associated ESBL infections caused by
E. coli. Therefore, ESBL E. coli from UTIs are a feasible bacterial
population for a comparative study. Furthermore, UTIs are animportant source of ESBLs entering the sewage system and the
extent of their contribution to ESBL E. coli in the sewage system is
an important issue.
Isolates from both sources were analysed with respect to the
occurrence of different ESBL gene families, variations in their
antibiotic susceptibility, and plasmid replicon types of contained
plasmids. Furthermore, strain relationships were determined by
analysis of the utilization of different carbon sources.
2. Material and methods
2.1. Isolates
Between February and July 2009, sewage sludge samples were collected
monthly from ve different Austrian domestic sewage treatment plants in the area
of Graz (province Styria, Austria). The population equivalent of sewage treatment
plants ranged from 100,000 and sewage treatment plants had a ow
rate of 100e1200 L/min wastewater. Sludge samples were collected from activated
untreated sludge and 50 ESBL E. coli were isolated.
ESBL E. coli primary isolates from 50 patients (at the Medical University of Graz,
Austria) with urinary tract infection were collected in the same sampling period.
2.2. Sample collection, identi cation and susceptibility testing
Sewage sludge samples (activated sludge) were collected using sterile wide-
mouth bottles. Samples were transported to the laboratory in a cool box, where
they were immediately stored in a refrigerator at 4e8 C for up to 24 h until
processing.
For qualitative analysis, an amount of 100 mg sewage sludge was transferred
into 3 ml thioglycolate and incubated at 37 C for 24 h. The suspension was inoc-
ulated onto ESBL-screeningagar (37 C, 24 h). The identication of E. coli strains was
carried out using the ID-GN card on Vitek 2 (bioMérieux, Marcy-l ’Etoile, France).
Antibiotic resistance was determined with the AST-N020-card, and ESBL-positive
E. coli were conrmed by CLSI conrmatory tests (CLSI, 2008).
Identication and resistance testing of ESBL-E. coli from human urinary tract
infections were performed as described for the sewage sludge samples.
Susceptibility to 11 antibiotics was tested (amoxicillin/clavulanic acid, piper-
acillin/tazobactam, imipenem, meropenem, gentamicin, tobramycin, amikacin,
trimethoprim/sulfamethoxazole, nitrofurantoin, ooxacin and ciprooxacin) using
Vitek 2 (Testcard: AST-N020) (McFarland between 0.55 and 0.62.). Susceptibilities to
nalidixic-acid, tetracycline and chloramphenicol were determined by disc diffusion
testing according to CLSI criteria.
2.3. Determination of the b-lactamase families through PCR analysis of b-lactamase
(bla) genes
PCR detection and gene identication were performed for ve different b-lac-
tamase gene families, blaTEM, blaSHV , blaCTX-M, blaVEB and blaGES. PCR and sequencing
procedures were performed as described previously ( Eckert et al., 2004; Kiratisin
et al., 2008).
Standard PCR protocols and conditions were modied in the following way:
initial denaturation at 94 C for 5 min; 35 cycles at 95 C for 30 s, 52 C for 45 s, and
72 C for 60 s; and nal incubation for 10 min at 72 C using Taq DNA polymerase
and dNTPs from QIAGEN (Hilden, Germany).
2.4. Phenotyping
In contrast to studies investigating nosocomial outbreaks, which are usuallycaused by either one or only few dominant strains, we investigated a broad spec-
trum of ESBL samples from UTIs and sewage sludge, and hence expected a high
variation in the strain backgrounds of the investigated ESBL E. coli isolates. For this
reason, we decided to use the automated PhenePlate (PhP) phenotyping system for
biochemical ngerprinting for basic strain differentiation. ESBL-E. coli isolates were
typed with the PhP-system using the PhP-EC kit for E. coli Batch 21 (PhP-FS, PhPlate
Microplate Techniques, Stockholm, Sweden). This system utilizes an automated,
microtitre plate based method for typing of bacteria which is based on the evalua-
tion of the kinetics of biochemical reactions (Kuhn et al., 1991). In brief, a loop full of
freshbacterial culture was suspended in 300 mL growth medium containing 0.11% w/
v bromothymol blue. Aliquots (7 mL) of the suspensions were inoculated into 24
wells in the ready-made microtiter plates containing 24 different substrates which
had each been dissolved in 150 mL growth medium. The plates were incubated at
37 C in water saturated atmosphere. The absorption A620 of each reaction was
measured after 16 h using a microplate reader. E. coli ATCC 25922 served as the
control strain for the PhP ngerprinting system.
The similarities between the pair-wise comparisons of isolates were calculatedas correlation coef cients, yielding a similarity matrix from which a dendrogram
was built by the sequential clustering unweighted pair-group method using arith-
metic averages (UPGMA). An identity level of 0.95 was set. Strains showing simi-
larities higher than this value were regarded identical and assigned to the same
PhPtypes and those not identical to any other isolates were called single (Si)
PhPtypes (Ansaruzzaman et al., 2000).
2.5. Plasmid replicon typing
Identication of replicon types of the 18 major plasmid incompatibility (inc)
groups present in Enterobacteriaceae was performed by multiplex PCR. PCRs were
performed as described previously(Carattoli et al., 2005).
The protocol allows detection of the following inc groups: Hl1, Hl2, I1-Ig, X, L/M,
N, FIA, FIB, W, Y, P, FIC, A/C, T, FIIAs, F, K, B/O.
2.6. Statistical analyses
The statistical analyses were carried out using R Version 2.12, a free software
environment for statistical computing and graphics (www.r-project.org ). Group
specic proportions were tested on their equality by a two-sided binomial test.
Pearson’s Chi-squared test was used to evaluate counts of the observed gene
patterns.
3. Results
3.1. Genetic variation of ESBL genes
The rst aim of this study was to detect ESBL genes present in
E. coli isolates from domestic Austrian sewage sludge and to
investigate how the ESBL gene distribution differs compared to
isolates from UTI patients living in the region of the investigated
wastewater treatment plants. 100 ESBL E. coli isolates were testedfor the presence of ve different b-lactamase gene families.
95% of all ESBL E. coli isolates carried ESBL genes of the family
blaCTX-M. To determine the blaCTX-M subtypes present in our isolates,
PCR products of the blaCTX-M genes were sequenced. A diagram
summarizing the ESBL genes found either alone or in combination
with the non-ESBL b-lactamase TEM-1 in the two different sample
types is shown in Fig. 1. Furthermore, the ESBL genes detected in
each single isolate are listed in Table 1.
Themost commonESBLgenesin sewagesludgewere blaCTX-M-15,
which was present in 22 (44%) of the isolates and blaCTX-M-1, which
was found in 20 (40%) of the isolates. In addition, four isolates (8%)
harboured the blaCTX-M-3 gene. Only in one sewage sludge isolate
a non-CTX-M ESBL gene, blashv-15, wasdetected. In UTI isolates, only
two different ESBL genes were detected both belonging to the
G. Zarfel et al. / Environmental Pollution 173 (2013) 192e199 193
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CTX-M family. 38 isolates (76%) harboured the blaCTX-M-15 gene, the
other 11 (22%) harboured blaCTX-M-1.
For four of the ESBL E. coli isolates investigated in this study
(three from sewage sludge and one from UTI) no ESBL gene was
detected. However, three of these isolates (two from sewage sludge
and one from UTI) harboured the non-ESBL b-lactamase gene TEM-
1 which has been reported to be associated with ESBL phenotypes
in some cases (Beceiro et al., 2011). Furthermore, TEM-1 was
present in addition to a CTX-M gene in 28 (56%) of the sewage
sludge and 26 (52%) of the UTI isolates.
In summary, the prevalence of genes producing ESBL pheno-
types clearly varies between human urinary tract infections and
sewage sludge samples (P value 0.02).
3.2. Additional resistances of the ESBL E. coli isolates
Bacteria with ESBL phenotypes frequently carry additional
antibiotic resistances. For the purpose of phenotypic differentia-
tion, all isolated ESBL E. coli isolates were tested for their suscep-
tibility to 14 antibiotics. The antibiotic resistances of each of the
investigated isolates are listed in Table 1. Table 2 summarizes the
antibiotics tested and the percentages of resistant isolates in each of
the two sources.
The most frequently found resistances in ESBL E. coli isolates
from sewage sludge samples were against tetracycline (66%
resistant strains) and nalidixic-acid (66% resistant strains), fol-
lowed by ampicillin/clavulanic-acid (54% resistant strains). All
sewage sludge isolates were susceptible to amikacin, imipenemand meropenem.
For UTI ESBL E. coli isolates, the highest proportions of resistant
isolates were found for nalidixic-acid (88%), ampicillin/clavulanic-
acid (86%), as well as for the two other tested quinolones, cipro-
oxacin (82%) and ooxacin (80%). Just as the sewage sludge
isolates, all UTI isolates were susceptible to the tested carbape-
nems, imipenem and meropenem.
ESBL E. coli from UTIs had signicantly higher rates of resistance
against ampicillin/clavulanic acid, tobramycin, amikacin, trimeth-
oprim/sulfamethoxazole, nalidixic-acid, ciprooxacin and ooxacin
(see also the P values depicted in Table 2) than sewage sludge
isolates. Higher rates of resistance, which were however not
statistically signicant (P values > 0.5) were observed for piper-
acillin/tazobactam and gentamicin.
For nitrofurantoin, tetracycline and chloramphenicol, resistance
was observed slightly more often in sewage sludge isolates than in
UTI isolates, but the difference was not statistically signicant
(Table 2).
Next, we compared the antibiotic resistance spectra of the
isolates (Table 1). We found a broad diversity of resistance patterns
in both sample groups, with a total of 58 different patterns (34 in
UTI and 35 in sewage sludge isolates). 39 resistance patterns were
only represented by one isolate. The most frequently observed
resistance patterns were resistance against Ampicillin/clavulanic
acid only (in four sewage sludge and two UTI isolates), resistance
against Ampicillin/clavulanic acid, Tobramycin, Trimethoprim/sul-
famethoxazole, Ciprooxacin, Ooxacin and Nalidixic acid (one
sewage sludge and ve UTI samples) and resistance against Tetra-
cycline (four sewage sludge samples).
3.3. Phenotyping of ESBL E. coli isolates
Phenotypic differentiation of all isolates by evaluation of
metabolic reactions was performed using the PhenePlate (PhP)
system. PhP strain differentiation resulted in 17 PhP groups
(PhPtype 1e17) and 41 single isolates. The corresponding dendro-
gram is displayed in Fig. 2. Of the single isolates, 21 originated fromsewage sludge and 20 from UTIs.
Only three PhPtypes 7, 11 and 12 were represented by more than
three isolates. The largest cluster, PhPtype 11, was formed by ten
ESBL E. coli UTI isolates and only one isolate from sewage sludge.
Similarly, PhPtype 7 contained mainly UTI isolates (ve) and only
one sewage sludge isolate. PhPtype 12 contained two UTI isolates
and four isolates from sewage sludge, hence representing the
PhPtype with the highest number of sewage sludge isolates clus-
tering together. The remaining 14 PhPtypes split up into 8 PhPtypes
harbouring only isolates from sewage sludge, two types containing
only isolates from UTIs, and four containing isolates from both
sources.
The majority of PhPtypes was formed by isolates carrying the
same ESBL genes, while only ve PhPtypes (1, 2, 3, 15 and 16),contained isolates with different ESBL genes.
Within the dendrogram, there is no clear borderline between
the isolates from the two different sources, which even clustered
together in the same PhPtypes. The only remarkable difference is
the tendency of ESBL E. coli from UTI to form bigger clusters, sug-
gesting less phenotypic variation in the UTI isolates.
3.4. Plasmid replicon typing
Finally, we further differentiated the isolates on the basis of the
inc/rep groups of contained plasmids. All isolates were positive for
at least one of the tested inc/rep groups, with most strains har-
bouring plasmids from two up to four different inc/rep groups
(Table 1).The most dominant inc/rep groups were FIB, which tested
positive in 38 UTI and 43 sewage sludge isolates, F (37 isolates from
UTIs, 28 from sewage sludge) and FIA (38 isolates from UTIs, 23
from sewage sludge). In addition, we found group Y in 3 UTI and 8
sewage sludge isolates, N in 11 UTI and seven sewage sludge
isolates, and P in one UTI and one sewage sludge isolate. Further-
more, four inc/rep groups were only present in isolates from
sewage sludge, L/M (ve isolates), Hl1 (two isolates) A/C (one
isolate) and K (one isolate).
In general, the diversity of inc/rep groups was higher in sewage
sludge isolates than in UTI isolates. Notably, CTX-M-15 from both
sample groups was mainly associated with the presence of FIA and
FIB plasmids, while all other plasmid inc/rep groups previously
documented to carry CTX-M-15 (FII, L/M, I1 and N) were only rarely
Detected ß-Lactamases
6
14
2 21
21
8
3
0 0
15
23
01
0
1012
0
5
10
15
20
25
C T X
- M - 1
C T X
- M - 1 / T E M
- 1
C T X - M
- 3
C T X
- M - 3 / T E M
- 1
C T X
- M - 1 5
C T X
- M - 1 5 /
T E M
- 1
S H V -
1 5
T E M
- 1 N o n
N u m b e r o f E . c o l i i s o l a t e s
ESBL sewage
sludgeESBL urinary
tract infection
Fig. 1. Distribution of identied ESBL genes (CTX-M and SHV family), as well as the
non-ESBL gene TEM-1 in ESBL-E. coli isolates from UTIs (black bars) and sewage sludge
(striped bars).
G. Zarfel et al. / Environmental Pollution 173 (2013) 192e199194
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Table 1
Phenotypical and genotypical proles of tested ESBL-E. coli, including antibiotic resistances, ESBL genes and deduced plasmid rep/inc groups. The origins of the isolates are
listed in the last column.
Isolatea Antibiotic resistancesb ESBL gene Plasmid replicon types Originc
SeS1 NA; TE; C CTX-M-1 FIA, FIB, F STP-1
SeS2 CIP; OFL; NA; TE; C CTX-M-1 FIA, FIB, F STP-1
SeS3 AMC; TM; SXT; CIP; OFL; NA; FT; TE CTX-M-15 FIB, F STP-2
SeS4 CIP; OFL; NA; TE; C CTX-M-1 N, FIB, F STP-4
SeS5 AMC CTX-M-3 I1-Ig, FIB STP-3SeS6 NA; FT; TE CTX-M-1 FIB, F STP-1
SeS7 AMC; TM; SXT; CIP; OFL; NA; TE CTX-M-1 I1-Ig, FIB, FIA, FIB, F STP-2
SeS8 NA; TE CTX-M-1 I1-Ig, FIA, FIB, F STP-4
SeS9 AMC; CIP; OFL; NA CTX-M-15 FIA, Y STP-4
SeS10 TE CTX-M-1 FIA, FIB STP-3
SeS11 GM; TM; SXT; NA; TE CTX-M-3 FIB, Y, N STP-5
SeS12 AMC; SXT; TE CTX-M-15 FIB, F STP-4
SeS13 TE CTX-M-1 I1-Ig, FIB, P, F STP-2
SeS14 CIP; OFL; NA CTX-M-1 I1-Ig, FIA, FIB STP-3
SeS15 AMC; TM; SXT; TE L/M, FIB STP-5
SeS16 SXT; FT CTX-M-15 N, FIA, FIB, F STP-1
SeS17 AMC CTX-M-1 L/M, FIA, FIB STP-2
SeS18 TE SHV-15 Hl1, FIB STP-3
SeS19 AMC; GM; TM; CIP; OFL; NA; TE CTX-M-15 FIA, FIB, Y, F STP-3
SeS20 AMC; SXT; CIP; OFL; NA; TE CTX-M-1 I1-Ig, FIB, F STP-4
SeS21 AMC; SXT; CIP; OFL; NA; TE CTX-M-1 I1-Ig, FIA STP-1
SeS22 TE CTX-M-1 I1-Ig, FIB, F STP-2
SeS23 FT; TE F STP-2
SeS24 SXT; CIP; OFL; NA; TE CTX-M-15 FIA, FIB, F STP-5
SeS25 F STP-3
SeS26 TM; SXT; CIP; OFL; NA CTX-M-15 I1-Ig, FIB, Y, F STP-5
SeS27 AMC; P/TZP; SXT; CIP; OFL; NA; FT; TE CTX-M-1 N, FIB, P STP-2
SeS28 AMC; GM; TM CTX-M-15 FIA, FIB STP-3
SeS29 SXT CTX-M-1 L/M STP-5
SeS30 CIP; OFL; NA CTX-M-1 FIA, FIB, F STP-4
SeS31 AMC; SXT; NA; TE; C CTX-M-1 I1-Ig, FIB STP-3
SeS32 AMC; NA; TE; C CTX-M-1 Hl1, N, FIB STP-1
SeS33 AMC; SXT; TE CTX-M-15 FA, FB STP-2
SeS34 AMC; CIP; OFL; NA CTX-M-15 A/C STP-3
SeS35 AMC; SXT; CIP; OFL; NA; TE; C CTX-M-15 FIA, FIB STP-1
SeS36 AMC; GM; TM; CIP; OFL; NA; FT; TE CTX-M-15 FIA, FIB STP-4
SeS37 AMC; CIP; OFL; NA CTX-M-15 FIB, Y, F STP-5
SeS38 AMC; GM; TM; CIP; OFL; NA; TE CTX-M-15 N, FIA, FIB, F STP-3
SeS39 AMC; TM; SXT; CIP; OFL; NA CTX-M-15 FIA, FIB, Y, F STP-4
SeS40 CTX-M-15 FIB, F STP-2SeS41 AMC; P/TZP; SXT; NA; TE CTX-M-1 I1-Ig, FIB, K STP-4
SeS42 AMC; GM; TM; SXT; CIP; OFL; NA; TE CTX-M-15 FIA, FIB, Y, F STP-4
SeS43 NA CTX-M-15 I1-Ig, FIB, F STP-5
SeS44 CIP; OFL; NA; TE CTX-M-1 I1-Ig, N, FIA, FIB STP-5
SeS45 AMC; GM; TM; SXT; CIP; OFL; NA; FT; TE CTX-M-15 FIA, FIB STP-1
SeS46 AMC CTX-M-3 L/M, FIB STP-1
SeS47 SXT; NA; TE CTX-M-15 I1-Ig, FIB, Y, F STP-5
SeS48 SXT; CIP; OFL; NA; TE CTX-M-15 I1-Ig, FIA, FIB, F STP-2
SeS49 AMC CTX-M-3 L/M STP-1
SeS50 AMC; GM; TM; SXT; NA; TE CTX-M-15 FIA, FIB, Y STP-5
UTI-1 AMC; TM; SXT; CIP; OFL; NA CTX-M-15 FIA, FIB, Y, F Human
UTI-2 AMC; GM; TM; SXT; NA; TE CTX-M-15 FIB, F Human
UTI-3 AMC CTX-M-15 FIA, FIB Human
UTI-4 AMC; CIP; OFL; NA CTX-M-15 FIA, FIB Human
UTI-5 AMC; CIP; OFL; NA CTX-M-15 FIA, FIB Human
UTI-6 AMC; P/TZP; TM; AN; SXT; CIP; OFL; NA; TE CTX-M-1 I1-Ig, N, FIB, P Human
UTI-7 AMC; CIP; OFL; NA; TE CTX-M-1 I1-Ig, N, FIB, F HumanUTI-8 AMC; CIP; OFL; NA; TE CTX-M-1 N, FIB, F Human
UTI-9 AMC; P/TZP; TM; SXT; CIP; OFL; NA; TE CTX-M-1 FIB, F Human
UTI-10 AMC; TM; SXT; CIP; OFL; NA; TE CTX-M-15 FIA, F Human
UTI-11 SXT; CIP; OFL; NA CTX-M-1 I1-Ig, FIA Human
UTI-12 AMC; GM; TM; AN; SXT; CIP; OFL; NA; TE CTX-M-15 FIA, F Human
UTI-13 AMC; TM; SXT; CIP; OFL; NA; TE; C CTX-M-15 FIA, FIB, F Human
UTI-14 AMC; GM; TM; SXT; CIP; OFL; NA; TE CTX-M-15 FIA, FIB, F Human
UTI-15 AMC; TM; SXT; CIP; OFL; NA CTX-M-15 FIA, FIB, Y, F Human
UTI-16 AMC; SXT; CIP; OFL; NA CTX-M-15 FIA, F Human
UTI-17 AMC; TM; SXT; CIP; OFL; NA CTX-M-15 FIA, FIB, F Human
UTI-18 AMC; SXT; TE CTX-M-15 N, FIB, F Human
UTI-19 AMC CTX-M-15 FIB, F Human
UTI-20 AMC; TM; SXT; NA; TE CTX-M-15 FIA, F Human
UTI-21 AMC; P/TZP; GM; TM; AN; SXT; CIP; OFL; NA; TE CTX-M-15 FIA, FIB, F Human
UTI-22 AMC; TM; SXT; CIP; OFL; NA; TE CTX-M-15 FIA Human
(continued on next page)
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represented or totally absent. CTX-M-1 isolates showed also two
dominant inc/rep groups I1-Ig and N, while all other plasmid inc/
rep groups documented to carry CTX-M-1 (FII, L/M) were only
rarely represented or totally absent(Carattoli, 2009).
4. Discussion
Several studies report growing numbers of antibiotic resistant
bacteria in the environment, including surface water. These
bacteria are an additional burden for the human healthcare system,
which already has to ght resistant bacteria that arise in medical
institutions. (Goni-Urriza et al., 2000; Kummerer, 2004;
Luczkiewicz et al., 2010). There are reports of Enterobacteriaceae
harbouring ESBL genes (primarily of the CTX-M family) in waste-
water and sewage sludge from several countries. As we demon-
strated in a previous study, E. coli can survive some of the sewage
sludge treatment procedures applied in Austria (Reinthaler et al.,
2010). However, the inuence of these “wild living” ESBL-
producing bacteria on human health is in discussion. Sewagesludge used for agriculture may be one way for ESBL-producing
bacteria to enter the food chain (Livermore et al., 2007; Lu et al.,
2010; Dolejska et al., 2011; Dhanji et al., 2011).
To better understand howthe ESBL pool in the environment and
the ESBL pool in humans inuence each other, we drew a compar-
ison between the ESBL types found in sewage sludge and ESBL
types present in UTI infections. We are aware that the exclusive
consideration of UTI samples as a source of ESBL from human
infections limits the diversity of ESBL positive isolates studied. The
absence of some ESBL genes or plasmid inc/rep groups in the
isolates directly sampled from humans may be a result of this
limitation. Nevertheless, as UTIs represent a dominant type of ESBL
E. coli infection, especially outside the hospital, and UTIs are an
important source of ESBLs entering the sewage system, ESBL E. colifrom UTIs are a feasible bacterial population for a comparative
analysis.
In this study, ESBL E. coli isolates drawn from sewage sludge did
not show any special characteristics that would allow a clear
differentiation from isolates drawn from human beings. However,
the diversity of ESBL encoding genes, as well as the diversity of inc/
rep groups was higher in sewage sludge isolates than in UTI
isolates.
CTX-M-15, which is known to be one of the most important
ESBL enzymes in human infections, was found in 76% of the UTI
isolates and hence was the predominant ESBL E. coli type recovered
from UTIs. Most other human isolates (22%) contained CTX-M-1,
which is also known to be frequently found in human isolates. In
samples drawn from sewage sludge, the distribution was shifted
Table 1 (continued )
Isolatea Antibiotic resistancesb ESBL gene Plasmid replicon types Originc
UTI-23 AMC; GM; TM; SXT; CIP; OFL; NA; TE CTX-M-15 FIA, FIB Human
UTI-24 SXT; CIP; OFL; NA CTX-M-15 FIA Human
UTI-25 AMC; GM; TM; SXT; NA CTX-M-15 FIB, F Human
UTI-26 AMC; GM; TM; SXT; CIP; OFL; NA; TE CTX-M-15 FIB, F Human
UTI-27 AMC; GM; TM; SXT; CIP; OFL; NA; FT; TE CTX-M-15 FIA, FIB Human
UTI-28 AMC; TM; SXT; CIP; OFL; NA; TE CTX-M-15 FIA, F Human
UTI-29 GM; TM; SXT; CIP; OFL; NA; TE CTX-M-1 N, FIA, FIB HumanUTI-30 AMC; TM; SXT; OFL; NA; TE CTX-M-15 FIA, FIB Human
UTI-31 AMC; P/TZP; GM; TM; AN; SXT; CIP; OFL; NA; FT; TE CTX-M-15 N, FIA, FIB, F Human
UTI-32 AMC; TM; SXT; CIP; OFL; NA CTX-M-15 FIA, FIB, F Human
UTI-33 AMC; TM; CIP; OFL; NA CTX-M-1 I1-Ig, N, FIA, FIB Human
UTI-34 SXT; TE CTX-M-1 N, FIB, F Human
UTI-35 AMC; P/TZP; GM; TM; CIP; OFL; NA; TE CTX-M-15 N, FIA, FIB, F Human
UTI-36 AMC; GM; TM; SXT; CIP; OFL; NA; FT; TE CTX-M-15 FIA, F Human
UTI-37 AMC; P/TZP; SXT; CIP; TE CTX-M-15 FIA, F Human
UTI-38 AMC; TM; CIP; OFL; NA CTX-M-15 N, FIA, FIB Human
UTI-39 AMC; TM; CIP; OFL; NA CTX-M-15 FIA, FIB, F Human
UTI-40 AMC; TM; SXT; CIP; OFL; NA CTX-M-15 FIA, FIB, Y, F Human
UTI-41 TM; SXT; CIP; OFL; NA CTX-M-1 I1-Ig, FIA, FIB, F Human
UTI-42 AMC; TM; AN; CIP; OFL; NA; TE FIA, F Human
UTI-43 AMC; AN; OFL; NA; TE CTX-M-1 N, FIB, F Human
UTI-44 AMC; GM; SXT; CIP; NA CTX-M-15 FIA, FIB, F Human
UTI-45 AMC; GM; TM; SXT; CIP; OFL; NA CTX-M-15 FIA, F Human
UTI-46 AMC; GM; TM; AN; SXT; CIP; OFL; NA; FT; TE CTX-M-1 FIB, F Human
UTI-47 AMC; TM; SXT; CIP; OFL; NA; TE CTX-M-15 FIA, FIB, F Human
UTI-48 AMC; GM; TM; CIP; OFL; NA; TE; C CTX-M-15 FIA, FIB, F Human
UTI-49 CIP; FT; TE CTX-M-15 FIA, FIB, F Human
UTI-50 CIP; OFL; NA CTX-M-15 FIA, FIB, F Human
a UTI, ESBL-E. coli from human urinary tract infection; SeS, ESBL-E. coli from sewage sludge.b AMC, Ampicillin/Clavulanic acid; P/TZP, Piperacillin/Tazobactam; GM, Gentamicin; TM, Tobramycin; AM, Amikacin; SXT, Trimethoprim/sulfamethoxazole; CIP, Cipro-
oxacin; OFL, Ooxacin; NA, Nalidixic acid; FT, Nitrofurantoin; TE, Tetracycline; C, Chloramphenicol.c STP, sewage treatment plant.
Table 2
Antibiotic resistance of 50 ESBL-E. coli isolates from UTI patients and 50 ESBL-E. coli
isolates from sewage sludge. The percentages of resistant isolates, as well as the P
values for signicant differences between the two sources are listed. UTI: urinary
tract infection, SeS: sewage sludge.
Antibiotic % of resistant isolates P value
ESBL-E. colifrom SeS
ESBL-E. colifrom UTI
Ampicillin/Clavulanic acid 54% 86%
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Fig. 2. Phenotypic differentiation of 50 ESBL-E. coli isolates from sewage sludge (SeS) and 50 ESBL-E. coli isolates from UTIs. The E. coli strain ATCC 25922 was used as a control.
The line drawn at 0.95 marks the set level of similarity for assignment into the same PhP-group.
G. Zarfel et al. / Environmental Pollution 173 (2013) 192e199 197
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towards CTX-M-1, which was found to similar extents (40%) as CTX-
M-15 (44%). Interestingly, CTX-M-1 has been reported to be very
dominant in animal isolates, while CTX-M-15 is very uncommon in
animals. Therefore, the high number of CTX-M-15 in sewage sludge
isolates supports the idea that human infections are a major source
of ESBL genes in wastewater. The higher number of CTX-M-1
positive isolates in sewage sludge compared to UTIs might be an
indication for an additional entry of animal-borne ESBLs into the
sewage system. However, other ESBLs, which are frequently found
in animals, like CTX-M-9, CTX-M-14 and TEM-52, were totally
absent in the samples from this study. This is not necessarily
contradictory to the contribution of ESBL E. coli from animals to the
sewage sludge ESBL E. coli population, as reports from neighbouring
countries showed a high domination of CTX-M-1 in animals and an
absence of TEM-52, CTX-M-9 and CTX-M-14 (Carattoli, 2008;
Peirano and Pitout, 2010; Schink et al., 2011; Wasyl et al., 2012).
Another expected nding was the high frequency of resistance
of ESBL E. coli to other antibiotics. As the resistance patterns of the
investigated isolates showed a high diversity, also within the same
sample groups and even within the same PhP analysis groups, we
consider it unlikely that the over-representation of specic strains
might bias the comparison of resistances in the two sample groups.
Compared to the resistance rates of ESBLs from sewage sludgesamples investigated in our study, Lu et al. (2010) reported similar
or higher resistance rates for ESBL-E. coli in river sediments. An
exception is ciprooxacin, to which we found higher rates of
resistant strains (46%) than Lu et al. (29%). In general, we found high
resistance rates against quinolones in both sample types. This is
fully comprehensible, as ciprooxacin is among all antibiotics the
one with the highest increase in consumption in the last decade in
Austria (Metz-Gercek et al., 2009). Apart from that, ESBL mediated
by CTX-M has been reportedto be more often than other ESBL types
combined with additional resistances against different classes of
antibiotics, especially against quinolones (Livermore et al., 2007).
Studies in other countries investigating E. coli from sewage
sludge reported highest resistance rates for tetracycline, ampicillin/
clavulanic acid and trimethoprim/sulfamethoxazole (Luczkiewiczet al., 2010; Holzel et al., 2010). Consistent with these studies, we
found the highest numbers of resistances against the same anti-
biotics in ESBL E. coli from Austrian sewage sludge (with the
additional high abundance of quinolone resistance characteristic to
CTX-M ESBL).
Remarkably, compared with UTI E. coli, ESBL E. coli from sewage
sludge showed signicantly lower rates of resistance against anti-
biotics which are in common use in human medicine. An exception
to this is Tetracycline, which is frequently used in human and
veterinary medicine and in animal farming (Ungemach et al., 2006;
Metz-Gercek et al., 2009). Resistance against Tetracycline is
a commonly known phenomenon in excessive animal farming
(Machado et al., 2008; Grisold et al., 2010; Su et al., 2011) Hence, the
high proportion of tetracycline resistant strains in sewage sludgeisolates might be an additional indication for some contribution of
animal-borne ESBL E. coli to the ESBL E. coli population in sewage
sludge.
Phenotyping showed that the main population of ESBL
expressing E. coli isolates from sewage sludge had no direct rela-
tionship to the investigated ESBL E. coli UTI strains. As however,
some of the ESBL E. coli PhPtypes from UTI were also found in the
sewage sludge, it is probable that UTI strains are able to survive in
the environment.
5. Conclusions
Our results clearly demonstrate that ESBL E. coli from UTI and
from sewage sludge can not be separated into two different groups.
The occurrence of the same ESBL genes (albeit with different
frequencies), antibiotic resistances and other phenotypic markers
suggests that both groups have a strong impact on each other on
the level of strains and resistant genes. The most likely way of
exchange between these two pools to occur is the release of ESBL E. coli from UTIs into the sewage system. However, in case that
sewage sludge is applied onto farmland, these bacteria are also able
to enter the food chain and transfer their resistancesto the bacterial
population in humans. Therefore, the use of sewage sludge in
agriculture without effective treatment to eliminate ESBL E. coli
should be judged critically in order to reduce the introduction of
resistance bearing pathogens into the environment.
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