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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]

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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

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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).

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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-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-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.


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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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