w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 5 0 8 9 e5 0 9 7

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/watres

Antimicrobial resistance of fecal indicators in municipal

wastewater treatment plant

_
A. quczkiewicz*, K. Jankowska, S. Fudala-Ksia˛zek,  czuk-Neyman
K. Olan
 sk University of Technology,
Department of Water and Wastewater Technology, Faculty of Civil and Environmental Engineering, Gdan
 sk, Poland
G. Narutowicza 11/12, 80-233 Gdan

article info abstract

Article history:
Received 1 April 2010
Received in revised form
22 July 2010
Accepted 8 August 2010
Available online 14 August 2010
Keywords:
Fecal indicators
Antimicrobial resistance
Wastewater treatment processes
Antimicrobial resistance of fecal coliforms (n ¼ 153) and enterococci (n ¼ 199) isolates was
investigated in municipal wastewater treatment plant (WWTP) based on activated sludge
system. The number of fecal indicators (in influent and effluent as well as in the aeration
chamber and in return activated sludge mixture) was determined using selective media.
Susceptibility of selected strains was tested against 19 (aminoglycosides, aztreonam, car-
bapenems, cephalosporins, b-lactam/b-lactamase inhibitors, fluoroquinolones, penicillines,
tetracycline and trimethoprim/sulfamethoxazole) and 17 (high-level aminoglycosides,
ampicillin, chloramphenicol, erythromycin, fluoroquinolones, glycopeptides, linezolid, lin-
cosamides, nitrofuration, streptogramins, tetracycline) antimicrobial agents respectively.
Among enterococci the predominant species were Enterococcus faecium (60.8%) and Entero-
coccus faecalis (22.1%), while remaining isolates belonged to Enterococcus hirae (12.1%),
Enterococcus casseliflavus/gallinarum (4.5%), and Enterococcus durans (0.5%). Resistance to
nitrofuration and erythromycin was common among enterococci (53% and 44%, respec-
tively), and followed by resistance to ciprofloxacin (29%) and tetracycline (20%). The resis-
tance phenotypes related to glycopeptides (up to 3.2%) and high-level aminoglycosides (up to
5.4%) were also observed. Most frequently, among Escherichia coli isolates the resistance
patterns were found for ampicillin (34%), piperacillin (24%) and tetracycline (23%). Extended-
spectrum b-lactamase producing E. coli was detected once, in the aeration chamber. In the
study the applied wastewater treatment processes considerably reduced the number of fecal
indicators. Nevertheless their number in the WWTP effluent was higher than 104 CFU per
100 ml and periodically contained 90% of bacteria with antimicrobial resistance patterns. The
positive selection of isolates with antimicrobial resistance patterns was observed during the
treatment processes. Substantial concern should be paid to the isolates resistant to 3 or more
chemical classes of antimicrobials (MAR). In treated wastewater MAR E. coli and MAR
enterococci constituted respectively 9% and 29% of tested isolates.
ª 2010 Elsevier Ltd. All rights reserved.

* Corresponding author.
_
E-mail addresses: ansob@pg.gda.pl (A. quczkiewicz), kjank@pg.gda.pl (K. Jankowska), sksiazek@pg.gda.pl (S. Fudala-Ksia˛zek),
kola@pg.gda.pl (K. Olan czuk-Neyman).
0043-1354/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2010.08.007
5090 w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 5 0 8 9 e5 0 9 7
1. Introduction
In Poland the antimicrobial agents’ consumption varies from
year to year, with the increasing trend observed especially in
outpatient therapy. Daily identified doses per 1000 inhabitants
per day (DID) increased from 16.56 DID in 1997 to 24.77 DID in
2001, and in 2001 this number was sixth in Europe (Ferech
et al., 2006). According to the data collected in ESAC project
(European Study on Antibiotic Consumption), the increase
was observed in the classes of penicillin and fluo-
roquinolones, while the use of other antimicrobial agents
stagnated or decreased (Elseviers et al., 2007; Ferech et al.,
2006; Muller et al., 2007). Antimicrobial agents used in
human therapy as well as their residues reach the sewage
systems via urine and feces. Their concentrations in waste-
water, although significantly lower than therapeutic dosages,
are suspected to affect the susceptible bacteria and select
resistant strains (Kim et al., 2007; Al-Ahmad et al., 1999;
Backhaus and Grimme, 1999). The wastewater treatment
processes remove the antimicrobial agents by sorption and/or
by biodegradation (Kümmerer, 2009; Golet et al., 2003;
Lindberg et al., 2006), thus their antimicrobial activity and
fate in final effluents depends on their chemical properties.
Growing evidences have additionally supported the thesis of
horizontal genes transfer under wastewater treatment
conditions (Soda et al., 2008; Marcinek et al., 1998). Biological
processes based on the activated sludge, due to the high
density of cells, are regarded as factors promoting the
dissemination of resistance among bacteria (Soda et al., 2008;
Marcinek et al., 1998). In the environmental risk assessment,
the importance of continuous input of resistant bacteria
versus continuous input of antimicrobial agents themselves
requires investigations (Kümmerer, 2009).
The aim of the present study was to evaluate the effects of
wastewater treatment on the presence of antimicrobial
resistance patterns among the indicator bacteria of waste-
water origin. In the environmental studies Escherichia coli and
enterococci are taken as the indicators of fecal contamination.
Those bacteria are present in the intestinal tract of humans;
however, they have been also frequently related to the human
and animal diseases. Among humans some strains of E. coli
are for instance one of the most important food-borne path-
ogens and the most frequent agent of urinary tract infections
INF
internal recycle
influent grit primary
screens removal settling DP
sludge
Fig. 1 e Scheme of the WWTP (DP e dephosphatation, DN e denitrification, N e nitrification) and the sampling points
location (INF e influent, BR e aerobic chamber, RAS e return activated sludge, EFF e effluent).
(Gupta et al., 2001). The emergence of pathogenic enterococci
is connected with their high tenacious and intrinsic resistance
to a broad range of antimicrobial agents. The resistance
mechanisms, however, differ among different species
(Murray, 1998). Besides of the exposition of pathogenic E. coli
and Enterococcus spp. to antimicrobial agents, the selection of
resistant strains may also occur among commensals as
a result of bystander effect (Karami et al., 2006). In this study
the prevalence of antimicrobial resistant fecal indicators was
studied in the wastewater treatment plant (WWTP), which
discharges the treated wastewater into the recreationally
important coastal water of Gdan  sk Bay.
2. Methods
The samples of raw and treated wastewater as well as waste-
water from the aeration chamber and returned activated
sludge were collected from WWTP ‘Gdan  skeWschód’ (Northen
Poland) working in modified UCT (University of Cape Town) e
type system. The WWTP treats wastewater for the population
of about 570,000 people in the area of Gdan  sk, Sopot, Kolbudy,
Pruszcz Gdan _
 ski, Zukowo, and some local industry (5%) as well
as it treats undisinfected (0.17%) hospital wastewater. Pop-
ulation equivalent estimated for the WWTP is equal to 700,000,
the average daily flow is 96,000 m3 per day with 24 h of reten-
tion time. The schematic diagram of the WWTP and sampling
points are indicated in Fig. 1. During the study the average
values of chemical and biological oxygen demand, total
nitrogen and phosphorus as well as total suspended solids
observed in treated wastewater were lower than those legally
established for the examined WWTP ‘Gdan  skeWschód’
(125 mgO2 l1, 15 mgO2 l1, 10 mgN l1, 1 mgP l1, 35 mg l1,
respectively). Treated wastewater is pumped by the 2300 m
 sk Bay.
collector into the coastal water of Gdan
2.1. Samples collection
The 24 h flow-proportional wastewater samples (5 l) were
taken from the influent (INF) and effluent (EFF) as well as the
mixed liquor from the aerobic chamber (BR) and return acti-
vated sludge (RAS) in the three following days of January and
June (Fig. 1). The BR, RAS and EFF samples were taken
BR EFF
biological process
internal recycle
secondary effluent
DN I DN II N settling
return activated sludge
sludge
RAS
 w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 5 0 8 9 e5 0 9 7 5091

considering the retention time of WWTP (one day after the
inflow sampling). All together 6 series of sampling were con-
ducted. The microbiological analyses were performed within
6h after collection.
2.2. Enumeration and isolation of fecal bacteria
Detection and enumeration of E. coli and Enterococcus spp. were
carried out by means of membrane filtration according to ISO
9308-1:2000 and ISO 7899-2:2000, respectively. Wastewater
samples were diluted and filtered through 0.45 mm cellulose
acetate filters in triplicate. Next, in order to detect fecal coli-
forms filters were placed on mFC agar (Merck) and incubated at
44.5  C for 24 h. Blue colonies, regarded as presumptive E. coli
(n ¼ 153) were selected and subcultured onto the nutrient agar,
then kept in 4  C for further investigation. Enterococci were
determined using Enterococcus selective agar (Merck)
according to Slanetz-Bartley at 37  C for 48 h. Representative
colonies, dark red or maroon (n ¼ 199), were taken and kept on
nutrient agar in 4  C for further investigations.
2.3. Species identification (ID) and antimicrobial
susceptibility tests (AST)
The identification and drug susceptibility of presumptive
E. coli and Enterococcus spp. isolates were tested by the Phoenix
Automated Microbiology System (Phoenix AMS, BD).
Commercially available panels (BD Phoenix) were applied for
ID and AST tests. The susceptibility analyses, based on the
microdilution tests, were carried out against the antimicrobial
agents representative for drugs important in treating human
E. coli and enterococcal infection (Table 1).
Pure cultures of bacterial isolates (18e24 h old), were Gram
stained and then the appropriate commercially available
Phoenix panels were used according to the manufacturer’s
recommendations. ID broth was inoculated and bacterial
concentration was adjusted to a 0.5 McFarland standard using
BBLTM Crystal Spec TM Nephelometer (BD Diagnostics). Then
AST broth was inoculated with 25 ml of ID suspension. Next ID
and AST sections were filled and panel was placed into the
instrument, and incubated at 35  C. The quality control was
performed according to the manufacturer’s instructions.

Table 1 e The list of antimicrobial agents used in the BD Phoenix panel.

Antimicrobial agent

Class Name Code Range mg/cm3

E. coli enterococci

Aminoglycosides Amikacin AN 4e32 e

Gentamicin GM 1e8 e
Gentamicin-Synergy GMS e 500
Streptomycin-Synergy STS e 1000
Tobramycin NN 1e4 e
b-lactam penicillin Ampicillin AM 2e16 0.5e32
Piperacillin PIP 4e64 e
b-lactam/b-lactamase inhibitors Ampicillin/Clavulanate AMC 4/2e16/8 e
Piperacillin/Tazobactam TZP 4/4e64/4 e
Cephalosporin Cefazolin CZ 4e16 e
Cefuroxime CXM 4e16 e
Ceftazidime CAZ 4e16 e
Cefotaxime CTX 1e32 e
Cefepime FEP 2e16 e
Carbapenems Imipenem IPM 1e4 e
Meropenem MEM 1e8 e
Fluoroquinolones Ciprofloxacin CIP 0.5e2 0.5e4
Levofloxacin LVX 1e4 0.25e4
Moxifloxacin MXF e 0.5e4
Folate antagonist Trimethoprim/Sulphamethoxazole SXT 0.5/9.5e2/38
Glycopeptides Teicoplanin TEC e 0.5e32
Vancomycin VA e 0.5e32
Lincosamides Clindamycin CC e 0.25e4
Lincomycin L e 1e8
Macrolides Erythromycin E e 0.125e4
Monobactam Aztreonam ATM 2e16 e
Oxazolidinones Linezolid LZD e 0.5e4
Streptogramins Pristinamycin PR e 0.25e2
QuinupristineDalfopristin SYN e 0.5e2
Tetracyclines Tetracycline TE 1e8 0.5e8
Other Chloramphenicol C e 1e16
Nitrofurantoin FM e 32e256
ESBL ESR þ e
5092 w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 5 0 8 9 e5 0 9 7
2.4. Data analyses
In this study the confidence level of 90% was required as the
lowest limit of acceptability for ID tests. The bacterial
susceptibility data was analysed using the CLSI (Clinical and
Laboratory Standards Institute) standards for antimicrobial
susceptibility testing (CLSI, 2006). The presence of extended-
spectrum b-lactamase (ESBL) e producing E. coli isolate (n ¼ 1),
detected by Phoenix AMS, was additionally confirmed by the
disk diffusion method (CLSI, 2006). Ceftazidime (30 mg) and
ceftazidime e clavulanic acid (30/10 mg) as well as cefotaxime
(30 mg) and cefotaxime e clavulanic acid (30/10 mg) (Oxoid)
were placed on Mueller-Hinton agar plate, inoculated with the
tested organism. After 16 h of incubation in 35  C, enhance-
ment of the inhibition zone around the discs in combination
with clavulanic acid towards the antimicrobial agents tested
alone, indicated the presence of ESBL-producing strains.
Since the susceptibility criteria for lincomycin (L) and clin-
damycin (CC) (lincosamides) as well as for pristinamycin (PR)
(streptogramin) are not available in CLSI recommendations
(2006), the obtained data was evaluated according to the
recommendation of Société Française de Microbiologie
(CASFM, 2010). Enterococci showing minimum inhibitory
concentrations (MICs) > 2 mg ml1 for clindamycin and for
pristinamycin, as well as MIC > 8 mg ml1 for lincomycin, were
regarded as CC, PR and L e resistant, respectively.
Antibiotic resistance of the tested isolates was categorized
as sensitive and resistant (as an ‘intermediate resistant’ and
‘resistant’ behaviour). Resistance to 3 or more chemical
classes of antimicrobial agents was taken as the multiple
antibiotic resistance (MAR). The resistance rate was defined as
the ratio of isolates with antimicrobial resistance patterns to
all tested isolates. In estimation of resistance rate among
Enterococcus spp. data not taken into consideration was AST
data obtained for CC, L, PR e due to lack of CLSI (2006)
recommendations as well as data obtained for SYN e due to
intrinsic resistance of Enterococcus faecalis to streptogramin
reported for clinical strains.
Significant differences between antibiotic resistance rate
in influent and effluent were determined for fecal indicators
using a two-sided z-test for two proportions ( p < 0.05). For
statistical computations ‘Scilab environment’ was used.
3. Results
The number of fecal coliforms and fecal enterococci detected
in the influent reach up to 5.7  106 CFU ml1 and up to
3.5  106 CFU ml1, respectively. In the effluent this number
was reduced to the average values of 2.9  104 CFU 100 ml1
and 1.8  104 CFU 100 ml1.
Among presumptive E. coli (n ¼ 153) all isolates were
identified as E. coli. In case of enterococci among 199 tested
isolates 60.8% were identified as Enterococcus faecium, 22.1% as
E. faecalis, 12.1% as E. hirae, 4.5% as Enterococcus casseliflavus/
gallinarum and one strain as Enterococcus durans (0.5%).
Considering the sampling point the composition of Entero-
coccus species differ. During wastewater treatment processes,
the relative proportion of E. faecium in raw and treated
wastewater remained unchanged (about 60%), whereas this
proportion was reduced in E. hirae (from 17% to 7%) and
increased in E. faecalis (from 19% to 25%).
In the current study the susceptibility results were
obtained for all tested E. coli (n ¼ 153) and 93% (n ¼ 185) of
tested enterococci. Grow limitation, caused probably by the
physical stresses (Colwell and Grimes, 2000), was observed
mainly among E. casseliflavus/gallinarum (5 of 9 isolates).
In general, the antimicrobial susceptibility tests showed the
presence of resistance patterns among 48% of E. coli and 78%
enterococci isolates. Among E. coli most commonly resistance
to penicillins (ampicillin e 34% and piperacillin e 24%) as well
as to tetracycline (23%) was observed, and no resistance was
noted to amikacin and tobramycin as well as to imipenem and
meropenem (Table 2). As much as 9% (n ¼ 14) of E. coli isolates,
expressed the phenotype of resistance to three or more classes
of antimicrobial agents (MAR) and were detected in all ana-
lysed sampling points. The ESBL-producing isolate was
detected once in sample taken from the bioreactor.
In the case of enterococci two interpretive criteria for
susceptibility testing were used: CLSI (2006) and CASFM (2010).
According to CLSI (2006) E. durans (n ¼ 1) was sensitive to all
tested antimicrobial agents (Table 2). Resistance to quinupristin
e dalfopristin was observed only among E. faecalis (72.7%), what
is in agreement with clinical data (CLSI, 2006). Among other
species the predominance of nitrofurantoin (53%) and eryth-
romycin (44%) resistance patterns were noted. Isolates of
E. casseliflavus/gallinarum were resistant only to erythromycin,
nitrofurantoin and tetracycline, while E. hirae was resistant to
erythromycin, nitrofurantoin, linezolid and moxifloxacin
(Table 2). All species showed no or relatively low rate of resis-
tance to chloramphenicol (<3%). Resistance to high-level ami-
noglycosides (GMS and STS) as well as to glycopeptides (TEC
and VA), antimicrobials agents of clinical concern, was detected
only among E. faecium (4.5%, 6.3% and 2.7%, 2.7%, respectively)
and E. faecalis (2.3%, 6.8% and 2.3%, 6.8%, respectively). MAR
resistance patterns were detected among 29% of isolates (AST
data of CC, L, and PR were not taken under consideration).
Among the tested antimicrobial agents CLSI (2006) inter-
pretive criteria are also not given for clindamycin and linco-
mycin (lincosamides) as well as for pristinamycin
(streptogramin), thus the obtained MICs are showed in
Table 3. In general, according to the CASFM criteria (2010) CC-
resistant enterococci constitute 42%, L e resistant 38%, PR e
resistant 7% of the tested isolates, but the resistance level
varied between enterococcal species (Table 3).
On the basis of the obtained data, the daily load of antimi-
crobial resistant bacteria discharged in the final WWTP effluent
into the costal waters of Gdan  sk Bay, was estimated. It was
expressed in CFU of antimicrobial resistant bacteria per day
and was calculated, taking into consideration the total number
of fecal bacteria in treated the wastewater samples, the
detected rate of antimicrobial resistance and the average daily
flow (96000 m3 per day). Thus, the number of colony forming
units calculated for resistant isolates reached up to
3.7  104 CFU 100 ml1 for enterococci and up to 3.2  104 CFU
100 ml1 for E. coli in treated wastewater. The load of antimi-
crobial resistant E. coli in the final effluent reached from
5.2  1012 to 2.0  1013 CFU per day. In the case of enterococci the
estimated load varied from 9.8  1012 to 3.0  1013 CFU per day.
 w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 5 0 8 9 e5 0 9 7 5093

Table 2 e Resistance detected among indicator bacteria to tested antimicrobial agents according to CLSI (2006).
Resistance (%)

Enterococcus spp.

E. coli N ¼ 153 in general E. faecium E. faecalis E. hirae E. c/ga E. durans

n ¼ 185 N ¼ 112 N ¼ 44 n ¼ 24 n¼4 n¼1

AN NDb ec e e e e e
GM 2 e e e e e e
GMS e 3.2 4.5 2.3 ND ND ND
STS e 5.4 6.3 6.8 ND ND ND
NN ND e e e e e e
AM 34 7 10.7 2.3 ND ND ND
PIP 24.2 e e e e e e
AMC 7.8 e e e e e e
TZP 0.7 e e e e e e
CZ 2.6 e e e e e e
CXM 2.6 e e e e e e
CAZ 1.3 e e e e e e
CTX 0.7 e e e e e e
FEP 0.7 e e e e e e
IPM ND e e e e e e
MEM ND e e e e e e
CIP 10.5 28.6 38.4 22.7 ND ND ND
LVX 9.8/15 9.7 11.6 11.4 ND ND ND
MXF e 15.7 21.4 9.1 4.2 ND ND
SXT 11.1 e e e e e e
TEC e 2.2 2.7 2.3 ND ND ND
VA e 3.2 2.7 6.8 ND ND ND
E e 44.3 44.6 61.4 4.2 100 ND
ATM 0.7 e e e e e e
LZD e 5.9 6.3 4.5 8.3 ND ND
SYN e ND 72.7 ND ND ND
TE 23.5 20.0 18.8 29.5 ND 75 ND
C e 2.2 2.7 2.3 ND ND ND
FM e 53.0 63.4 29.5 45.8 75 ND
ESBL 0.7 e e e e e e
MARd 9.2 28.6 34.8 22.7 4.2 75 ND

a E. c/g, E. casseliflavus/gallinarum.
b ND, not detected.
c e, Not analysed.
d MAR, isolates with MAR patterns; AST data of CC, L, PR, and SYN were not taken under consideration.

Manero et al., 2002), however, the quantitative species

4. Discussion
The applied processes of wastewater treatment caused 99.8%
reduction of fecal coliforms and 99.6% reduction of fecal
enterococci what is in agreement with other studies carried
out in the WWTP Gdan  skeWschód (Olan czuk-Neyman et al.,
2003). Such stability of fecal indicators removal during
wastewater processes facilitates predicting the potential final
effluent quality in terms of the total and antimicrobial resis-
tant bacteria.
Since the sensitivity to antimicrobial agents varied among
the bacteria isolated from wastewater, the species informa-
tion is required to recognize the resistance level. Among the
studied presumptive thermotolerant fecal coliforms (n ¼ 153)
only E. coli was identified. Identification tests of enterococci
showed the following predominance of species: E. faecium, E.
faecalis and E. hirae, which together made up to 95% of isolates.
Similar species distribution of enterococci was observed
elsewhere (Ferreira da Silva et al., 2006; Blanch et al., 2003;
enterococcal composition differed. Knowledge about the
enterococcal species composition in human microflora and in
wastewater, with regard to the difference in diet and climate
etc., is scared. In general, the most specific for intestinal
humane flora are E. faecium and E. faecalis, also considered as
main agents of enterococcal infections, with predominance of
E. faecalis (90e95%) (Teixeira and Facklam, 2003). Similar
results of E. faecium predominance were received in waste-
water in UK and Spain (Blanch et al., 2003; Manero et al., 2002),
while in Portugal and in the United States the most abundant
was E. hirae (Ferreira da Silva et al., 2006; Bonilla et al., 2006). E.
hirae isolates as well as isolates of E. casseliflavus/gallinarum
and E. durans, detected in studied wastewater, are mainly
found in the gastrointestinal tract of animals (poultry, pigs,
rats, cats, horses). In minority they occur in human intestinal
flora and rarely are associated with human infections (Rice
et al., 2003; Devriese et al., 2002).
In this study the susceptibility tests were carried out
against antimicrobials important in treating human E. coli and
5094 w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 5 0 8 9 e5 0 9 7

strains presented in other studies (Ferreira da Silva et al., 2006;

Table 3 e Susceptibility of Enterococcus spp. to
clindamycin (CC), lincomycin (L) and pristinamycin (PR) Martins da Costa et al., 2006).
according to Société CASFM criteria (2010). All Enterococcus species, except E. durans, showed the
highest resistance rate to erythromycin (E) and nitrofurantoin
%
(FM). Prevalence of E  resistant enterococci in wastewater
MICv E. faecium E. faecalis E. hirae E.c/ga E. durans and in other environmental compartments, has been widely
mg/cm3 n ¼ 112 n ¼ 44 n ¼ 24 n ¼ 4 n¼1 reported, with resistance rate even up to 82% (Blanch et al.,
CC <0.25 S 45.5 9.1 16.7 0.0 0.0 2003; Ferreira da Silva et al., 2006; Martins da Costa et al.,
0.5 S 5.4 2.3 8.3 0.0 0.0 2006); in this study 44% (61.4% for E. faecalis)(Table 2)
2 S 14.3 15.9 16.7 25.0 0.0 Surprisingly high was the FM e resistance rate and amounted
3 R 8.0 0.0 12.5 0.0 0.0
to 53% (for E. faecium even 63.4%), while other studies reported
4 R 2.7 2.3 12.5 0.0 0.0
by Martins da Costa et al. (2006) showed 22.5% of wastewater
>4 R 24.1 70.5 33.3 75.0 100.0
L <1 S 45.5 11.4 20.8 0.0 0.0 enterococci. The high prevalence of nitrofurantoin-resistant
2 S 1.8 0.0 4.2 0.0 0.0 strains as well as the cross-resistance of nitrofurantoin with
4 S 13.4 2.3 29.2 0.0 0.0 furazolidone was noted among Gram-negative isolates of
8 S 15.2 4.5 29.2 25.0 0.0 poultry origin (Rampling et al., 1990). In the human therapy
>8 R 24.1 81.8 16.7 75.0 100.0 nitrofurantoin, together with trimethoprim/sulphamethox-
PR <0.25 S 45.5 11.4 45.8 0.0 0.0
azole, ciprofloxacin, ampicillin and others antimicrobial
0.5 S 42.9 2.3 50.0 0.0 0.0
agents, are used in treatment of urinary tract infections. Since
1 S 8.9 29.5 4.2 100.0 100.0
2 S 1.8 22.7 0.0 0.0 0.0 some strains of E. coli are regarded as the causative agent of
>2 R 0.9 34.1 0.0 0.0 0.0 the majority of urinary tract infections, the resistance to
nitrofurantoin is rarely tested among enterococci, also due to
a E. c/g, E. casseliflavus/gallinarum, S e sensitive, R e resistant.
the limited usage of this antimicrobial agent in treatment of
enterococcal infections. The results obtained in the present
enterococcal infection, although it should be stressed that the study indicate the need to monitor this phenomenon and/or
selection of resistance strains among commensal E. coli and concentration of nitrofurantoin also in municipal wastewater.
Enterococcus spp. can not be excluded (Karami et al., 2006). For For other antimicrobial agents, frequently used to treat
instance tetracycline (TE) is not used to treat E. coli and urinary tract infections (SXT, CIP and LVX) similar resistance
enterococcal infection but resistance to this antimicrobial rate (about 10%) was observed among tested E. coli (Table 2).
agent is still common among clinical (Kawalec et al., 2007; For enterococci, the resistance rate to fluoroquinolones (CIP,
Karami et al., 2006) and environmental strains (quczkiewicz LVX, MXF) was even higher (29%, 10% and 16%, respectively)
et al., in press, 2010a,b; Martins da Costa et al., 2006; and detected mainly among E. faecium and E. faecalis. It should
Reinthaler et al., 2003). In this study TE-resistant isolates of be stressed that resistance to fluoroquinolones is widely
the tested fecal indicators were present in all sampling points reported among isolates from different environmental
of WWTP (Figs. 2e4). Among E. coli the resistance rate reached compartments (Martins da Costa et al., 2006; Moore et al.,
23.5% and was close to that found among enterococci (20%). 2008). The resistance rate found in this study was compa-
The highest prevalence of resistance among E. coli was rable with the resistance of E. coli and enterococci isolated
observed for penicillines, the most commonly used antibiotics from the local surface water (quczkiewicz et al., in press,
in Poland, and it reached 34% for AM and 24.2% for PIP (Table 2010a) as well as from the clinical material in Poland (EARSS,
2), whereas among studied enterococci the resistance to AM 2008 e data only for E. coli). The findings are in agreement
was relatively low (2% of E. faecalis and 11% of E. faecium) in with previous reports showing similar level of fluo-
comparison to clinical data reported in Poland (up to 12% and roquinolones resistance, namely ciprofloxacin, among
up to 95%, respectively) (EARSS, 2008). These results confirm isolates of environmental and clinical origin (Ferreira da Silva
high susceptibility to penicillin of wastewater enterococcal et al., 2006).

a 100 b
100
%

50 50

0 0
R=11
R=10

MAR
R=1

R=2
R=3
R=4

R=6

R=7
R=5

R=8

R=9
FEP

PIP

CIP
TZP

TE
CZ

SXT
GM

CTX

LVX
CAZ
CXM

AMC
ATM
AM

INF (n = 19) BR (n = 23) RAS (n = 26) EFF(n = 85)

Fig. 2 e Resistance detected among E. coli isolates, (a) to the single antimicrobial agent, (b) to all tested antimicrobial agents
(S e sensitive, R e resistant).
 w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 5 0 8 9 e5 0 9 7 5095

a 100 b 100
%

50 50

0 0

MAR
R=2
R=1

R=4

R=7

R=9
R=3

R=5

R=6

R=8
MXF
LVX
LZD
TEC
STS

CIP

TE
C
AM

FM
GMS

VA

S
INF (n = 7) BR (n = 5) RAS (n = 4) EFF(n = 28)

Fig. 3 e Resistance detected among E. faecalis isolates, (a) to the single antimicrobial agent, (b) to all tested antimicrobial
agents (S e sensitive, R e resistant); AST data of CC, L, PR, and SYN was not taken into consideration.

Among the tested fecal indicators isolated from waste-
water, the isolates of clinical concern like: extended-spectrum
beta-lactamases (ESBL) e producing E. coli as well as entero-
cocci resistant to vancomycin (VRE) and to high-level amino-
glycosides (HLAR) were observed. The ESBL e producing E. coli
isolate was detected once, in the aerobic chamber, and
exhibited the resistance to cefepime e cephalosporin of 4th
generation used only in hospital practice in Poland. It is an
indirect confirmation of the reported association between co-
treatment of hospital and municipal wastewater and resis-
tance patterns found in wastewater (Reinthaler et al., 2003).
The ESBL e producing E. coli detected in this study was also
multiple resistant, like majority of clinical ESBL producers
(Livermore et al., 2007). Other studies present higher rate of
ESBL e producing E. coli in environmental samples, especially
in sewage sludge (Reinthaler et al., 2010), probably due to the
bacteria accumulation after dewatering.
In this study, clinically relevant isolates of enterococci,
were found only among E. faecalis and E. faecium. The emer-
gence of HLAR patterns among clinical isolates is of concern
due to their usual multiple-resistance (Kawalec et al., 2007).
Also HLAR enterococci isolated from wastewater in this and
other studies in majority were multiple resistant (Talebi et al.,
2008; Rice et al., 1995). High-level gentamicin resistance
(HLGR) was detected in 4.5% of tested E. faecium and in 2.3% of
tested E. faecalis. Additionally, both species showed higher
resistance rate to high-level streptomycin (HLSR), 6.3% and
6.8% respectively, what is in agreement with other studies
(Rice et al., 1995). Resistance to both tested glycopeptides (VA
and TEC) was noted in three isolates of E. faecium harbouring
vanA gene, whereas one isolate of E. faecalis was vanA and two
were vanB. Also in the clinical studies, among five identified
phenotypes of glycopeptide resistance (VanA to VanE) the
prevalence of VanA and VanB phenotypes was noted (EARSS,
2008; Kawalec et al., 2000). The VanC phenotypes the charac-
teristic feature of the E. casseliflavus and E. gallinarum, is con-
nected with low-level vancomycin intrinsic resistance, what
is in agreement with data obtained in the present study. All
E. casseliflavus/gallinarum isolates (n ¼ 4) showed resistance to
VA MICs ¼ 4 mg ml1 and TEC MICs  1 mg ml1. VA-resistance
rate detected among studied enterococci did not exceed 3%,
which is in agreement with previous studies (Martins da Costa
et al., 2006). In order to obtain better recovery of VA e resistant
enterococci the enrichment of Slanetz and Bartley medium
with vancomycin is, however, suggested (Novais et al., 2005).
In Poland, among clinical isolates, HLARs occur with
medium to high frequency (from 29% of E. faecalis to 100% of
E. faecium), while resistance to glycopeptides is relatively low
(up to 2% and up to 5%, respectively) in comparison with other
European countries (EARSS, 2008). In this study, both clinically
relevant patterns were mainly associated with E. faecium,
what is in agreement with other studies (Martins da Costa

a b
100 100
%

50 50

0 0
MAR
R=1

R=2

R=3

R=6

R=8
R=4

R=5

R=7

R=9
TEC

LZD

LVX
MXF
STS

CIP
GMS

TE
C

FM
AM

VA
E

INF (n = 20) BR (n = 11) RAS (n = 15) EFF (n = 66)

Fig. 4 e Resistance detected among E. faecium isolates, (a) to the single antimicrobial agent, (b) to all tested antimicrobial
agents (S e sensitive, R e resistant); AST data of CC, L, PR, and SYN was not taken into consideration.
5096 w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 5 0 8 9 e5 0 9 7
et al., 2006). Interestingly, the first reported VRE in Poland
occurred in Gdan  sk hospital in the end of 1996 (E. faecium of
the phenotype VanA), due to horizontal transfer of different
Tn1546-like transposon variants (Kawalec et al., 2000).
Even through wastewater has been considered to be
a reservoirs of antimicrobial resistant bacteria, resistance
plasmids and sub-therapeutic concentration of antimicrobial
agents and their metabolites (Kümmerer, 2009; Kim et al.,
2007), the role of wastewater treatment processes in selec-
tion of resistant isolates as well as in exchange and distribu-
tion of resistance determinants has not been fully explained
yet. Data obtained in the present study support the observa-
tion, reported elsewhere (quczkiewicz et al., 2010b; Zhang
et al., 2009; Kim et al., 2007; Ferreira da Silva et al., 2006;
Reinthaler et al., 2003) of positive selection of antimicrobial
isolates in course of wastewater treatment processes (Figs.
2e4). In general antimicrobial resistance rate noted for
enterococci isolated from raw and treated wastewater was
73% and 78%, respectively, while for E. coli 42% and 47%,
respectively. Significant increase in resistance prevalence
( p < 0.05) after treatment was observed for both E. faecalis and
E. faecium resistant to fluoroquinolones (CIP, LVX, and MXF).
Additionally treatment processes favour E and TE e resistant
E. faecalis. In the case of E. coli its resistance to penicillines (AM
and PIP), fluoroquinolones (CIP and LVX) as well as to SXT and
TE is more prevalent in treated wastewater.
According to the obtained results isolates of both fecal
indicators exhibiting MAR patterns (resistance to at least three
different classes of antimicrobial agents) were positively
selected during the treatment processes. Different environ-
mental factors can influence positive selection of wastewater
isolates with resistance patterns. The sewage system of
WWTP Gdansk-Wschod receives the mixture of various
wastewaters, due to the catchment’s characteristic. Bacterial
strains, which were previously exposed to antimicrobials and
may poses resistance determinants, are suspected to reach
the WWTP mainly via households wastewater as well as via
undisinfected wastewater from hospitals and community
homes. Hospital wastewater is additionally regarded as
contaminated by antimicrobial agents, which even in sub-
inhibitory concentrations, may promote resistance and
survival of resistant strains (Kim et al., 2007; Al-Ahmad et al.,
1999). Also the horizontal gene transfer is regarded as the
main mechanism of resistance dissemination (Soda et al.,
2008; Marcinek et al., 1998), since the presence of antimicro-
bial resistance genes on mobile genetic elements (plasmids,
transposable elements or integron-specific gene cassettes)
has been reported among wastewater isolates (Schlüter et al.,
2007; Tennstedt et al., 2003). In wastewater processes based on
activated sludge resistance dissemination may be enhanced
by high cell density (Soda et al., 2008; Marcinek et al., 1998) as
well as by the prolonged retention time of biomass in the
system (quczkiewicz et al., 2010b; Novo and Manaia, 2010).
In this study, fecal bacteria released with treated wastewater
to the receiving costal water of Gdansk Bay contained periodi-
cally even 90% of isolates with antimicrobial resistance
patterns. Considering the average volume of wastewater
treated per day by the WWTP ‘Gdan  skeWschód’ (96,000 m3 per
day), for both fecal indicators the estimated daily load of isolates
with resistance patterns can reach even 1013 CFU per day.
5. Conclusion
Indicator bacteria with resistance patterns were positively
selected by the wastewater treatment processes based on
activated sludge. Their number in treated wastewater peri-
odically reached even 90% of the total number of fecal bacteria.
Special attention should be paid to the selection of isolates
with MAR patterns. Due to the introduction and/or selection of
resistant bacteria in water environment, changes in natural
ecosystems of receivers can be expected. Thus to prevent the
receivers from the dissemination of resistance strains the
disinfection of final WWTP effluents is recommended.
Acknowledgement
The authors would like to acknowledge Wojciech Artichowicz
for data analysis and the engineers of WWTP “Wschód” for
their assistance in obtaining the wastewater samples. This
project was founded by the Minister of Science and Higher
Education Grant nr N N523 493134
references
Al-Ahmad, A., Daschner, F.D., Kümmerer, K., 1999. Biodegradability
of cefotiam, ciprofloxacin, meropenem, penicillin G, and
sulfamethoxazole and inhibition of wastewater bacteria. Arch.
Environ. Contam. Toxicol. 37, 158e163.
Backhaus, T., Grimme, L.H., 1999. The toxicity of antibiotic agents
to the luminescent bacterium Vibrio fischeri. Chemosphere 38
(14), 3291e3301.
Blanch, A.R., Caplin, J.L., Iversen, A., Kühn, I., Manero, A.,
Taylor, H.D., Vilanova, X., 2003. Comparison of enterococcal
populations related to urban and hospital wastewater in
various climatic and geographic European regions. J. Appl.
Microbiol. 94 (6), 994e1002.
Bonilla, T.D., Nowosielski, K., Esiobu, N., McCorquodale, D.S.,
Rogerson, A., 2006. Species assemblages of Enterococcus
indicate potential sources of fecal bacteria at a south Florida
recreational beach. Mar. Pollut. Bull. 52 (7), 807e810.
CASFM, 2010. Société Française de Microbiologie, 2010.
Recommendations 2010, last accessed March 2010. Comite De
L’antibiogramme De La Societe Francaise De Microbiologie.
http://www.sfm.asso.fr/page1/page1.php?la¼2&we¼1.
CLSI (Clinical and Laboratory Standards Institute), 2006.
Performance Standards for Antimicrobial Susceptibility Testing;
Sixteenth Informational Supplement, 26. CLSI. M100eS16, 3.
Colwell, R.R., Grimes, D.J. (Eds.), 2000. Non-culturable
Microorganisms in the Environment. American Society for
Microbiology, Washington, D.C., USA.
Devriese, L.A., Vancanneyt, M., Descheemaeker, P., Baele, M., Van
Landuyt, H.W., Gordts, B., Butaye, P., Swings, J.,
Haesebrouck, F., 2002. Differentiation and identification of
Enterococcus durans, E. hirae and E. villorum. J. Appl. Microbiol.
92 (5), 821e827.
EARSS, 2008 (European Antimicrobial Resistance Surveillance
System) Annual Report, 2008, last accessed July 2010. http://
www.rivm.nl/earss/result/Monitoring_reports/.
Elseviers, M.M., Ferech, M., Vander Stichele, R.H., Goossens, H., ESAC
project group, 2007. Antibiotic use in ambulatory care in Europe
(ESAC data 1997e2002): trends, regional differences and seasonal
fluctuations. Pharmacoepidemiol. Drug Saf. 16 (1), 115e123.
 w a t e r r e s e a r c h 4 4 ( 2 0 1 0 ) 5 0 8 9 e5 0 9 7
Ferech, M., Coenen, S., Dvorakova, K., Hendrickx, E., Suetens, C.,
Goossens, H.on behalf of the ESAC Project Group, 2006.
European surveillance of antimicrobial consumption (ESAC)
outpatient penicillin use in Europe. J. Antimicrob. Chemother.
58 (2), 408e412.
Ferreira da Silva, M., Tiago, I., Verı́ssimo, A., Boaventura, R.A.,
Nunes, O.C., Manaia, C.M., 2006. Antibiotic resistance of
enterococci and related bacteria in an urban wastewater
treatment plant. FEMS Microb. Ecol. 55, 322e329.
Golet, E.M., Xifra, I., Siegrist, H., Alder, A.C., Giger, W., 2003.
Environmental exposure assessment of fluoroquinolone
antibacterial agents from sewage to soil. Environ. Sci. Technol.
37 (15), 3243e3249.
Gupta, K., Hooton, T.M., Stamm, W.E., 2001. Increasing
antimicrobial resistance and the management of
uncomplicated community-acquired urinary tract infections.
Ann. Intern. Med. 135 (1), 41e50.
Karami, N., Nowrouzian, F., Adlerberth, I., Wold, A.E., 2006.
Tetracycline resistance in Escherichia coli and persistence in
the infantile colonic microbiota. Antimicrob. Agents
Chemother. 50 (1), 156e161.
Kawalec, M., Gniadkowski, M., Hryniewicz, W., 2000. Outbreak of
vancomycin-resistant enterococci in a hospital in Gdan  sk,
Poland, due to horizontal transfer of different Tn 1546-like
transposon variants and clonal spread of several strains. J. Clin.
Microbiol. 38, 3317e3322.
Kawalec, M., Pietras, Z., Dani1owicz, E., Jakubczak, A.,
Gniadkowski, M., Hryniewicz, W., Willems, R.J.L., 2007. Clonal
structure of Enterococcus faecalis isolated from Polish hospitals: the
characterization of epidemic clones. J. Clin. Microbiol. 45, 147e153.
Kim, S., Jensen, J.N., Aga, D.S., Weber, A.S., 2007. Tetracycline as
a selector for resistant bacteria in activated sludge.
Chemosphere 66 (9), 1643e1651.
Kümmerer, K., 2009. Antibiotics in the aquatic environmentdA
reviewePart I and Part II. Chemosphere 75, 417e441.
Lindberg, R.H., Olofsson, U.L., Rendahl, P., Johansson, M.I.,
Tysklind, M., Andersson, B.A.V., 2006. Behavior of
fluoroquinolones and trimethoprim during mechanical,
chemical, and active sludge treatment of sewage water and
digestion of sludge. Environ. Sci. Technol. 40 (3), 1042e1048.
Livermore, D.M., Canton, R., Gniadkowski, M., Nordmann, P.,
Rossolini, G.M., Arlet, G., Ayala, J., Coque, T.M., Kern-
Zdanowicz, I., Luzzaro, F., Poirel, L., Woodford, N., 2007. CTX-
M: changing the face of ESBLs in Europe. J. Antimicrob.
Chemother. 59 (2), 165e174.
quczkiewicz, A., Jankowska, K., Kurlenda, J., Olan  czuk-Neyman, K.
Identification and antimicrobial susceptibility of fecal coliforms
isolated from surface water. Polish J. Environ. Stud, in press.
quczkiewicz, A., Jankowska, K., Kurlenda, J., Olan  czuk-Neyman,
K., 2010a. Identification and antimicrobial resistance of
Enterococcus spp. isolated from surface water. Water Sci.
Technol. 62 (2), 466e473.
quczkiewicz, A., Fudala-Ksia˛zek, _ S., Jankowska, K., Quant, B.,
Olan czuk-Neyman, K., 2010b. Diversity of fecal coliforms and
their antimicrobial resistance patterns in wastewater
treatment model plant. Water Sci. Technol. 61 (6), 1383e1392.
Manero, A., Vilanova, X., Cerdà-Cuéllar, M., Blanch, A.R., 2002.
Characterization of sewage waters by biochemical
fingerprinting of Enterococci. Water Res. 36 (11), 2831e2835.
Marcinek, H., Wirth, R., Muscholl-Silberhorn, A., Gauer, M., 1998.
Enterococcus faecalis gene transfer under natural conditions in
municipal sewage water treatment plants. Appl. Environ.
Microbiol. 64 (2), 626e632.
Martins da Costa, M.P., Vaz-Pires, P., Bernardo, F., 2006.
Antimicrobial resistance in Enterococcus spp. isolated in inflow,
effluent and sludge from municipal sewage water treatment
plants. Water Res. 40, 1735e1740.
5097
Moore, D.F., Guzman, J.A., McGee, C., 2008. Species distribution
and antimicrobial resistance of enterococci isolated from
surface and ocean water. J. Appl. Microbiol. 105 (4), 1017e1025.
Muller, A., Coenen, S., Monnet, D.L., Goossens, H., 2007. European
surveillance of antimicrobial consumption (ESAC): outpatient
antibiotic use in Europe, 1998e2005. Eurosurveillance 12 (41)
Available online, last accessed March 2010. http://www.
eurosurveillance.org/ViewArticle.aspx?ArticleId¼3284.
Murray, B.E., 1998. Diversity among multidrug-resistant
enterococci. Emerg. Infect. Dis. 4, 37e47.
Novais, C., Coque, T.M., Ferreira, H., Sousa, J.C., Peixe, L., 2005.
Environmental contamination with vancomycin-resistant
enterococci from hospital sewage in Portugal. Appl. Environ.
Microbiol. 71 (6), 3364e3368.
Novo, A., Manaia, C.M., 2010. Factors influencing antibiotic
resistance burden in municipal wastewater treatment plants.
Appl. Microbiol. Biotechnol. 87, 1157e1166.
 czuk-Neyman, K., Geneja, M., Quant, B., Dembin
Olan  ska, M.,
Kruczalak, K., Kulbat, E., Kulik-Kuziemska, I., Miko1ajski, S.,
Gielert, M., 2003. Microbiological and biological aspects of the
wastewater treatment plant “Wschód” in Gdan  sk. Polish J.
Environ. Stud. 12 (6), 747e757.
Rampling, A., Upson, R., Brown, D.F.J., 1990. Nitrofurantoin
resistance in isolates of Salmonella enteritidis phage type 4 from
poultry and humans. J. Antimicrob. Chemother. 25, 285e290.
Reinthaler, F.F., Posch, J., Feierl, G., Wust, G., Haas, D.,
Ruckenbauer, G., Mascher, F., Marth, E., 2003. Antibiotic
resistance of E. coli in sewage and sludge. Water Res. 37 (8),
1685e1690.
Reinthaler, F.F., Feierl, G., Galler, H., Haas, D., Leitner, E.,
Mascher, F., Melkes, A., Posch, J., Winter, I., Yarfel, G.,
Marth, E., 2010. ESBL-producing E. coli in Austrian sewage
sludge. Water Res. 44, 1981e1985.
Rice, E.W., Messer, J.W., Johnson, C.H., Reasoner, D.J., 1995.
Occurrence of high-level aminoglycoside resistance in
environmentalisolates of enterococci. Appl. Environ.
Microbiol. 61 (1), 374e376.
Rice, E.W., Boczek, L.A., Johnson, C.H., Messer, J.W., 2003. Detection
of intrinsic vancomycin resistant enterococci in animal and
human feces. Diagn. Microbiol. Infect. Dis. 46 (2), 155e158.
Schlüter, A., Szczepanowski, R., Pühler, A., Top, E.M., 2007.
Genomics of IncP-1 antibiotic resistance plasmids isolated
from wastewater treatment plants provides evidence for
a widely accessible drug resistance gene pool. FEMS Microbiol.
Ecol. Rev. 31 (4), 449e477.
Soda, S., Otsuki, H., Inoue, D., Tsutsui, H., Sei, K., Ike, M., 2008.
Transfer of antibiotic multiresistant plasmid RP4 from
Escherichia coli to activated sludge bacteria. J. Biosci. Bioeng.
106 (3), 292e296.
Talebi, M., Pourshafie, M.R., Katouli, M., Mollby, R., 2008.
Molecular structure and transferability of Tn1546-Like
elements in Enterococcus faecium isolates from clinical, sewage,
and surface sater samples in Iran. Appl. Environ. Microbiol. 74,
1350e1356.
Teixeira, L.M., Facklam, R.R., 2003. Enterococcus. In: Murray, P.R.,
Baron, E.J., Jorgensen, J.H., Pfaller, M.A., Yolken, R.H. (Eds.),
Manual of Clinical Microbiology, eighth ed. American Society
for Microbiology, Washington, DC, pp. 422e433.
Tennstedt, T., Szczepanowski, R., Braun, S., Pühler, A.,
Schlüter, A., 2003. Occurrence of integron-associated
resistance gene cassettes located on antibiotic resistance
plasmids isolated from a wastewater treatment plant. FEMS
Microbiol. Ecol. 45, 239e252.
Zhang, Y., Marrs, C.F., Simon, C., Xi, C., 2009. Wastewater
treatment contributes to selective increase of antibiotic
resistance among Acinetobacter spp. Sci. Total Environ. 407 (12),
3702e3706.