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Identification of Enterococcus faecium and

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Enterococcus faecalis as vanC-type Vancomycin-
Resistant Enterococci (VRE) from sewage and river
water in the provincial city of Miyazaki, Japan
a b a
Masateru Nishiyama , Atsushi Iguchi & Yoshihiro Suzuki
a
Department of Civil and Environmental Engineering, Faculty of Engineering, University of
Miyazaki, Miyazaki, Japan
b
Department of Animal and Grassland Sciences, Faculty of Agriculture, University of
Miyazaki, Miyazaki, Japan
Published online: 01 Dec 2014.

To cite this article: Masateru Nishiyama, Atsushi Iguchi & Yoshihiro Suzuki (2015) Identification of Enterococcus faecium and
Enterococcus faecalis as vanC-type Vancomycin-Resistant Enterococci (VRE) from sewage and river water in the provincial
city of Miyazaki, Japan, Journal of Environmental Science and Health, Part A: Toxic/Hazardous Substances and Environmental
Engineering, 50:1, 16-25, DOI: 10.1080/10934529.2015.964599

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 Journal of Environmental Science and Health, Part A (2015) 50, 16–25
Copyright © Taylor & Francis Group, LLC
ISSN: 1093-4529 (Print); 1532-4117 (Online)
DOI: 10.1080/10934529.2015.964599

Identification of Enterococcus faecium and Enterococcus

faecalis as vanC-type Vancomycin-Resistant Enterococci
(VRE) from sewage and river water in the provincial city
of Miyazaki, Japan

MASATERU NISHIYAMA1, ATSUSHI IGUCHI2 and YOSHIHIRO SUZUKI1

1
Department of Civil and Environmental Engineering, Faculty of Engineering, University of Miyazaki, Miyazaki, Japan
2
Department of Animal and Grassland Sciences, Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan
Downloaded by [George Mason University] at 09:59 01 January 2015

As a first step for assessing the risk to human health posed by vancomycin-resistant enterococci (VRE) in the aquatic environment,
we screened sewage and urban river water samples from Miyazaki, Japan for VRE. Because vancomycin-resistant organisms are not
as prevalent in sewage and river water as vancomycin-susceptible organisms, the samples were screened by minimum inhibitory
concentration test using the vancomycin-supplemented membrane-Enterococcus indoxyl-b-d-glucoside (mEI) agar. The isolates,
presumed to be enterococci, were identified using 16S rRNA sequencing analysis. The percentages of VRE isolates screened using
4 mg mL¡1 vancomycin-supplemented mEI agar from sewage and urban river water samples were 12% and 24%, respectively. The
vancomycin-resistant genes vanC1 and vanC2/3 were detected in the isolates from both samples by PCR analysis. All enterococci
isolates containing vanC1, which is a specific gene for vanC-type of VRE, were identified as Enterococcus casseliflavus/gallinarum.
Further, 92% enterococci isolates containing vanC2/3 were identified as E. casseliflavus/gallinarum, the remaining isolates
containing vanC2/3 were E. faecium (4%) and E. faecalis (4%). Thereafter, the distribution of E. faecium and E. faecalis, which are
the major types of enterococci in humans containing vanC2/3, was observed in the water samples collected.
Keywords: Antibiotic-resistance, minimum inhibitory concentration, Vancomycin-resistant enterococci, vancomycin-resistant genes,
water environment.

Introduction Fecal pollution of recreational beaches in the United

Enterococci are gram-positive bacteria present in the intes-
tinal microflora of humans and animal gastrointestinal
tracts. Because of their abundance in the feces of warm-
blooded animals and their long-term survival in the envi-
ronment, they have been traditionally used as indicators of
fecal contamination in the aquatic environment, including
sewage, rivers, and coastal areas.[1–3] Enterococci are ubiq-
uitously detected in the aquatic environments of both
developing and developed countries [4,5] and are able to
survive in aquatic environments for longer periods of time
than other intestinal microorganisms.[6,7]
Address correspondence to Yoshihiro Suzuki, Department of
Civil and Environmental Engineering, Faculty of Engineering,
University of Miyazaki, Miyazaki 889-2192, Japan, E-mail:
suzuki@civil.miyazaki-u.ac.jp
Received April 30, 2014.
States has been aggravating because enterococci count lev-
els occasionally exceed the acceptable standard safety lev-
els in recreational coastal waters and sands.[8,9] In
European countries, enterococci have been detected at
concentrations of 101–103 CFU 100 mL¡1 collected from
principal rivers, such as the Danube River.[10] Even in
Japan, where the building of advanced infrastructure has
been completed, enterococci have been detected in urban
river water or coastal waters at relatively high concentra-
tions (101–103 CFU 100 mL¡1 level).[11,12] Enterococci
closely and ubiquitously exist in the water surrounding our
living areas.
Unfortunately, some enterococci are opportunistic
pathogens that infect compromised hosts such as infants,
hospital patients, and elderly.[13–15] With the development
of new antimicrobial agents and use of increased dosage of
antimicrobials in the medical treatment, drug-resistant
enterococci that cause serious problems in the treatment of
nosocomial infections have arisen.[16,17] Furthermore, the
most serious drug-resistant enterococci, with high-level
 Vancomycin-Resistant Enterococci (VRE) from sewage and river water in Japan
resistance to the glycopeptide antibiotic vancomycin, have
emerged in medical institutions. Vancomycin is the ulti-
mate antibiotic, prescribed when other antibiotics are
ineffective.
Therefore, vancomycin-resistant enterococci (VRE) are
the most troublesome bacteria causing nosocomial infec-
tions.[18,19] In recent years, VRE occurrence has been glob-
ally increasing, particularly in countries such as US,
Europe, and South Asia.[20–23] Although the prevalence of
VRE in Japan is much lower than that in US, Europe, and
Asia, the Japanese medical establishment is apprehensive
regarding the expansion of nosocomial infections caused
by VRE.[8,24,25]
However, there are few data concerning the presence of
VRE in the aquatic environment and little information on
the relationship between the resistance to vancomycin and
Downloaded by [George Mason University] at 09:59 01 January 2015
resistance to other antibiotics. When antibiotic-resistant
enterococci exist in the aquatic environment surrounding
our communities, there is a risk of opportunistic infection
in not only the medical institutions but also our communi-
ties. As a first step to reduce the risk, it is important to
accumulate information regarding the prevalence of VRE
and enterococci resistant to other antibiotics in our aquatic
environment. In this study, the resistance of enterococci
isolates to vancomycin and other various antibiotics was
investigated in sewage and urban river water. Because
VRE are not as prevalent as susceptible enterococci in
sewage and river water, they were screened using vanco-
mycin-supplemented membrane-Enterococcus indoxyl-
b-d-glucoside (mEI) agar as an enterococci-selective
media. The isolates collected were also quantitatively eval-
uated for susceptibility to vancomycin and other antibiot-
ics using antibiotic susceptibility testing.
Materials and methods
Sampling
Samples were collected from two sites, a sewage and river.
These sampling sites were located in Miyazaki City, which
was considered a model provincial city in this study. Miya-
zaki City has an approximate population of 400,000 and a
developed infrastructure with sewer systems or septic tanks
for wastewater treatment. The sewage sample was col-
lected from raw wastewater from an urban treatment plant
that treats wastewater from a population of approximately
14,000 people.
Its average daily flow is 6,300 m3 per day. The river
water sample was collected from the Yae River, which
flows through Miyazaki City. The main water supply for
the Yae River is domestic wastewater and/or treated sew-
age water. The collected water samples were stored in 1-L
sterile polyethylene bottles that are immediately trans-
ported to the laboratory for microbial analysis and water
quality tests. Microbial and water quality analyses were
17
started within 4 h of sampling. Electrical conductivity,
pH, and turbidity were also determined using a conductiv-
ity meter (CM30S; TOA DKK, Tokyo, Japan), pH meter
(HM-30G; TOA DKK), and turbidity meter (SEP-PT-
706D; Mitsubishi Kagaku, Tokyo, Japan), respectively.
Enumeration and isolation
The water samples were tested for total coliform, Escheri-
chia coli, and enterococci. Total coliform and E. coli were
analyzed using the Colilert-18 kit (IDEXX, Westbrook,
ME, USA) , which is based on defined-substrate technol-
ogy. This kit provides a most probable number value
based on of the presence or absence of fluorescence in a
Quanti-Tray. Enterococci were isolated using membrane
filtration followed by culture on mEI agar plates.[26] Sam-
ples consisting of 1, 10, or 100 mL of sewage or river water
were diluted with 10 mL of sterile distilled water, filtered
through a membrane filter (0.45-mm pore, 47-mm diame-
ter, sterile, mixed cellulose ester; Advantec, Tokyo,
Japan), and then incubated on mEI agar plates for 24 h at
41
C § 1.0
C.
To screen for vancomycin-resistant strains in sewage
and river water, the mEI agar was supplemented with the
following four different vancomycin concentrations
(Wako Pure Chemical Industries, Ltd., Japan): 0, 2, 4, and
32 mg mL¡1. After incubation, colonies on the filter that
had blue halos were considered putative enterococci. The
number of enterococci isolated by membrane filtration
was expressed as CFU 100 mL¡1 of water. A representa-
tive bacterial count for each of the samples isolated using
membrane filtration was determined from the mean CFU
of three replicate mEI agar plates.
For further analysis of isolated bacteria, 11–27 single
colonies with blue halos were randomly isolated from each
mEI agar plate and streaked on a Todd–Hewitt agar plate
(1.5% agar; Bacto; Becton, Dickinson, NJ, USA). These
plates were then incubated for 24 h at 37
C.
Identification of enterococci by 16S rRNA sequencing
Presumptive enterococci were selected for identification
using 16S rRNA gene sequence analysis. For DNA extrac-
tion, isolates were grown overnight at 37
C in Todd–
Hewitt broth. Cells were harvested by centrifugation
(8,000 g) for 1 min. Genomic DNA was extracted using
the Insta Gene Matrix (Bio-Rad Laboratories, Hercules,
CA, USA) according to the manufacturer’s instructions.
The primers used for 16S rRNA amplification, which were
designed to produce a 1498-bp fragment from the gene
encoding 16S rRNA, were 16S-27F (50 -AGAGTTT-
GATCCTGGCTCAG-30 ) and 16S-1497R (50 -AAAG-
GAGGTGATCCAGCC-30 ).[27]
The enzyme KAPA taq Extra (Kapa Biosystems, Nip-
pon Genetics Co, Ltd., Japan) was used for the PCR
 18
amplification of the 16S rRNA gene. PCR was performed
in a 15 mL of reaction containing 3.0 mL of 5£ KAPA
Extra Buffer, 1.5 mL of 25 mM MgCl2, 0.45 mL of
0.3 mM dNTP Mix, 0.75 mL of 0.5 mM forward and
reverse primers, 0.08 mL of 1.25 U KAPA Taq Extra
DNA polymerase, 7.48 mL of sterile distilled water, and
1.0 mL of template DNA extracted from a putative entero-
coccal strain. The PCR amplification program used was as
follows: 25 cycles of denaturation at 94
C for 20 s, anneal-
ing at 62
C for 20 s, and elongation at 72
C for 1 min.
Amplification was confirmed by electrophoretic analysis
of a 5 mL aliquot of the reaction mixture on a 1% agarose
gel.
The amplified PCR products were sequenced using an
ABI 3170 automated sequencer (Applied Biosystems, San
Mateo, CA, USA) and BigDye Terminator v3.1 Cycle
Downloaded by [George Mason University] at 09:59 01 January 2015
Sequencing Kits (Applied Biosystems). Primers 16S-27F,
16S-1497R, Ent-16S-2ndF (50 -ATGGACGAAAGTCT-
GACCGA-30 ; this study), and Ent-16S-3rdF (50 -TAGA-
TACCCTGGTAGTCCAC-30 ; this study) were used as
sequencing primers. Sequencing data were edited using a
Sequencher software, ver. 4.9 (Gene Code Corporation,
Ann Arbor, MI, USA), and enterococci were identified by
BLAST searching the GenBank database.
Antimicrobial agents
The antibiotics used in this study included the penicillins
such as ampicillin (Wako Pure Chemical Industries, Ltd.,
Japan), piperacillin (MP Biomedicals, LLC; France), and
penicillin G (Wako Pure Chemical Industries, Ltd.); tetra-
cycline (Wako Pure Chemical Industries, Ltd.); the carba-
penem imipenem (LKT Laboratories, Inc., USA); the
macrolide erythromycin (Wako Pure Chemical Industries,
Ltd.), and the glycopeptide vancomycin. All the tested
agents were dissolved in distilled water or other appropri-
ate solvents according to the recommendations of the Clin-
ical and Laboratory Standards Institute.[28]
Antimicrobial susceptibility testing
Susceptibility testing was conducted on the Mueller–Hin-
ton agar using the agar dilution method according to
CLSI guidelines.[29] Enterococcal strains were cultured for
24 h in the Mueller–Hinton broth (Becton Dickinson,
Spark, MD, USA) and then diluted to a final
Table 1. Water quality of samples collected from the survey point.
pH Water temp Conductivity
Sampling point Sampling date (¡) (
C) (mS cm¡1)
Sewage 2012/1/25 7.72 15.8 1780
River water 7.76 15.8 490
Nishiyama et al.
concentration of approximately 107 CFU 100 mL¡1 with
fresh Mueller–Hinton broth. Inoculums were then applied
to the surface of the Mueller–Hinton agar (1.5% agar)
plates containing graded concentrations (0.25–128 mg mL
¡1
) of each antibiotic using a microplanter (Sakuma Co.,
Japan). The plates were incubated at 37
C for 20 h, and
then minimum inhibitory concentrations (MICs) were
determined. MIC breakpoints for intermediate resistance
and complete resistance were based on CLSI criteria.[28]
The percentage susceptibility toward each antimicrobial
agent used was calculated by dividing the number of sus-
ceptible isolates by the total number of isolates tested.
Resistance to 3 chemical groups of antimicrobial agents
was considered multidrug resistance.
Identification of vancomycin-resistant genes
PCR was performed to identify vancomycin-resistant
genes in the enterococcal strains. All the isolates were
screened for the presence of vancomycin-resistant genes
vanA, vanB, vanC1, and vanC2/C3 using the conditions
described by Dutka-Malen et al.[30] The PCR amplifica-
tion program used during the screening was as follows: 30
cycles of denaturation at 94
C for 1 min, annealing at
54
C for 1 min, and elongation at 72
C for 1 min. Ampli-
fication was followed by a final extension at 72
C for
10 min. Amplification was confirmed by electrophoretic
analysis of a 5-mL aliquot of each reaction mixture on a
1% agarose gel.
Results
Bacterial counts of sewage and urban river water
Water quality indicators, total coliform counts, and E. coli
counts of sewage and river water are shown in Table 1.
The coliform and E. coli counts of the wastewater are typi-
cal of those of municipal wastewater in Japan.[31] The data
demonstrate that the E. coli count for the river water is
approximately 1/100 that of the wastewater.
The bacterial counts derived from the number of blue
colonies on the mEI agar that were identified as entero-
cocci are shown in Figure 1. The data from the normal
mEI agar plate show that, similar to the E. coli count, the
enterococci count of the river water corresponded to
approximately1/100 of that of sewage. The bacterial
Turbidity Total coliform Escherichia coli
(kaolin unit) (MPN 100 mL¡1) (MPN 100 mL¡1)
75.2 2.9 £ 107 8.6 £ 106
1.91 3.6 £ 105 6.5 £ 104
 Vancomycin-Resistant Enterococci (VRE) from sewage and river water in Japan 19

Table 2. Identification of presumptive enterococcal isolates from

sewage and river water using each vancomycin-supplemented
mEI agar.
Sampling point Sewage River water
Vancomycin concentration
(mg mL¡1) 0 2 4 32 0 2 4 32

Enterococcus
faecium 17 17 3 1
faecalis 8 6 1 1 2 2
casseliflavus / gallinarum 5 24 1 18
hirae 2 9 4
Leuconostoc 4
Pediococcus 2 18 1 6 27
Weissella 5
No. of isolates 27 11 27 27 27 11 27 27
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Fig. 1. Bacterial counts (CFU 100 mL¡1) on supplemented the
mEI agar, which was prepared with four different vancomycin
concentrations: 0, 2, 4, and 32 mg mL¡1. The black bar indicates
counts from sewage and the shaded bar indicates counts from
river water.
counts of both sewage and river water decreased as the
vancomycin concentration in the supplemented mEI agar
increased. On the mEI agar supplemented with 2 mg mL¡1
vancomycin, which is 1/16 of its standard MIC, entero-
coccal growth was limited to 1/10 of the growth observed
on the mEI agar without vancomycin.
On the mEI agar supplemented with vancomycin at
4 mg mL¡1, the growth of the enterococci was further
restricted. The number of enterococcal colonies formed on
the agar supplemented with vancomycin at the highest
concentration, 32 mg mL¡1, was 100 times lower than that
formed on the normal mEI agar without vancomycin.
Because the fraction of the total bacterial count in sewage
that was tolerant to 32 mg mL¡1 vancomycin was
100 times higher than that in river water (compare bars at
32 mg mL¡1 vancomycin to those at 0 mg mL¡1 vancomy-
cin in Fig. 1) sewage and river water contained enterococci
with different tolerance for vancomycin.
Identification of Enterococcus spp.
Blue colonies (11–27) were randomly picked from each
mEI agar plate (without vancomycin or with 2–32 mg
mL¡1 of vancomycin) as putative enterococcal strains.
Each of the 184 isolates was then identified by the analysis
of the sequence of their 16S rRNA genes. The number of
isolates from each species is shown in Table 2. Of the 184
isolates, 121 (66%) were identified as Enterococcus spp.
These isolates, which were isolated from the normal
mEI agar (54 isolates) and the mEI agar supplemented
with 2 mg mL¡1 (21 isolates) or 4 mg mL¡1 (46 isolates)
vancomycin, included isolates belonging to the species
Enterococcus faecium (38 isolates), Enterococcus faecalis
(20 isolates), Enterococcus casseliflavus/gallinarum (48 iso-
lates), and Enterococcus hirae (15 isolates). The nonentero-
cocci (63 isolates, 34%) were identified as belonging to the
genera Pediococcus (54 isolates), Weissella (5 isolates), or
Leuconostoc (4 isolates).
All the presumptive enterococci isolated from sewage or
river water using the normal mEI agar were identified as
enterococci. The predominant species isolated from the
normal mEI agar was E. faecium (63%), whereas the
remaining isolates belonged to E. hirae (20%) or E. faecalis
(17%). However, the distribution of bacterial species
changed as the concentration of vancomycin in the mEI
agar increased.
The predominant species from the normal mEI agar
inoculated with sewage, identified as E. faecium, was not
detected among the sewage isolates on the mEI agar sup-
plemented with 2 or 4 mg mL¡1 vancomycin. In addition,
the percentage of Enterococcus spp. in the river water
decreased to <27% when the samples were cultured on the
mEI media containing 2 or 4 mg mL¡1 vancomycin. On
the mEI agar supplemented with 4 mg mL¡1 vancomycin,
the percentages of the 27 isolates from sewage and river
water that were identified as E. casseliflavus or E. gallina-
rum were 89% and 67%, respectively. The dominant spe-
cies of enterococci changed from E. faecium to E.
casseliflavus/gallinarum with the increase in vancomycin
concentration from 0 mg mL¡1 to 4 mg mL¡1 in the mEI
agar.
Although its percentage was small (<15%), Pediococcus
was identified from the mEI agar supplemented with 4 mg
mL¡1 vancomycin. In spite of the positive blue halo, which
suggested that the colonies picked from the mEI agar
plates were presumptive enterococci, none of the 54 iso-
lates from the mEI agar supplemented with 32 mg mL¡1
vancomycin were identified as Enterococcus spp. They
were classified as Leuconostoc (4 isolates), Pediococcus (45
isolates), and Weissella (5 isolates). The genera Pediococ-
cus, Leuconostoc, and Weissella belong to the lactic acid
 20
bacteria, which are part of the commensal intestinal flora
of humans and animals, and these strains are intrinsically
resistant to vancomycin.[32,33] Microbial substitution arose
on the mEI agar that contained vancomycin at high con-
centration, and different types of bacteria, including Ped-
iococcus, Leuconostoc, and Weissella, formed false-
positive colonies.
Antimicrobial susceptibility of enterococci isolates
from sewage and river water
MIC50 and MIC90 values for vancomycin displayed by the
enterococci isolates from the normal mEI agar were lower
than those displayed by enterococci isolates from the van-
comycin-supplemented mEI agar. Resistance to vancomy-
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cin tended to be enhanced with increasing vancomycin
concentration in the mEI agar used to isolate the strains
(Fig. 2A). Both the percentage of vancomycin-resistant
strains and their MICs for vancomycin increased using the
vancomycin-supplemented mEI agar.
High levels of resistance to piperacillin and tetracy-
cline were also detected among the 121enterococcal iso-
lates from sewage and river water. In contrast, many
isolates from sewage and river water were susceptible
to imipenem and ampicillin. The percentage of isolates
that showed complete resistance to penicillin G in each
sample was <11% (Table 3). Although enterococci with
high levels of resistance to erythromycin were isolated
from sewage water, erythromycin-resistant enterococci
were not detected in the river water. Because the curve
representing the cumulative percentage of enterococcal
isolates from sewage displaying MICs for tetracycline
lies above that of the enterococcal isolates from the
river water, the sewage isolates were more tolerant to
tetracycline than the river water isolates (Fig. 2B).
Enterococci resistant to vancomycin and to other anti-
biotics were detected among the isolates collected from
the sewage and river water. Multidrug-resistant strains
were also observed.
(A) 100
Cumulative percentage (%)
80
60
normal mEI: S
normal mEI: R
40 -1
2 µg mL mEI: S
2 µg mL-1 mEI: R
20 4 µg mL-1 mEI: S
-1
4 µg mL mEI: R
0 0
0 5 10 15 20 25 30
MIC (µg mL-1)
Fig. 2. Susceptibility distribution of isolates from sewage (S) and river water (R). (A) Vancomycin. (B) Tetracycline.
Nishiyama et al.
Determination of vancomycin-resistant genes
The vancomycin-resistant genes (vanA, vanB, vanC1, and
vanC2/C3) of all of the enterococcal strains collected from
sewage and river water were identified using PCR. The
gene identities and antibiotic susceptibility profiles of all
isolates are summarized in Figure 3. The vanA and vanB
were not detected among all enterococcal strains. On the
other hand, enterococcal strains possessing vanC were
detected among isolates collected from the vancomycin-
supplemented mEI agar.
Strains possessing vanC1 (8 isolates) and vanC2/3 (9 iso-
lates) were isolated from the sewage using the mEI agar
supplemented with 2 and 4 mg mL¡1 vancomycin. Fifteen
more isolates possessing vanC2/3 were isolated from the
river water using the mEI agar supplemented with 4 mg
mL¡1 vancomycin.
All of the enterococcal isolates that contained vanC1
were identified as E. casseliflavus/gallinarum. In contrast,
92% of the enterococcal isolates possessing vanC2/3
belonged to E. casseliflavus/gallinarum, while the remain-
ing belonged to E. faecalis (4%) and E. faecium (4%).
There is a no significant difference between vanC1-positive
isolates (100%, 8/8) and vanC2/3-positive isolates (92%,
22/24), which are E. casseliflavus/gallinarum (P > 0.05).
Although vanA and vanB, including the mobilized genes,
were not detected, vanC1 and vanC2/3 are present in the
aquatic environment in Japan.
Discussion
Enterococci, which are found in many aquatic compart-
ments, have an advantage in terms of persistence and
multiplication because of their tolerance to various environ-
mental factors, such as alkaline pH, increased temperature,
and sodium chloride concentration.[6,7] Because long-term
survival is possible in the aquatic environment, antibiotic-
resistant enterococci present a higher risk of spreading anti-
biotic genes than other bacteria. The presence of VRE,
(B) 100
Cumulative percentage (%)
80
60
normal mEI: S
normal mEI: R
40
2 µg mL-1 mEI: S
2 µg mL-1 mEI: R
20 4 µg mL-1 mEI: S
4 µg mL-1 mEI: R
0 20 40 60 80 100 120
MIC (µg mL-1)
 Downloaded by [George Mason University] at 09:59 01 January 2015

21
Table 3. Antibiotic susceptibility of enterococci total isolates (121 isolates) from sewage and river water.
Sewage River water

Breakpoint Vancomycin concentration MIC range MIC50* MIC90** Resistant MIC range MIC50 MIC90 Resistant
¡1 ¡1 ¡1 ¡1 ¡1 ¡1 ¡1
Group Antimicrobial agent (mg mL ) (mg mL ) (mg mL ) (mg mL ) (mg mL ) (%) (mg mL ) (mg mL ) (mg mL¡1) (%)
Penicillins Ampicillin 16 0 0.25–128 1 4 7.4 0.25–4 0.25 4 0
2 0.25–2 1 2 0 0.5–2 1 1 0
4 0.25–2 0.5 1 0 0.25–16 0.25 2 9.5
Penicillins Piperacillin 16 0 2–128 16 64 52 8–128 16 64 67
2 16–64 16 64 100 16–32 16 16 100
4 4–64 8 64 44 8–128 8 16 48
Penicillins Penicillin G 16 0 0.25–128 2 4 7.4 0.25–16 0.5 16 11
2 0.25–4 2 4 0 1–8 2 4 0
4 0.25–4 0.5 2 0 0.25–128 0.25 0.5 9.5
Tetracyclines Tetracycline 16 0 0.5–128 1 128 41 0.5–16 1 1 3.7
2 4–128 64 64 82 1–64 1 64 20
4 1–128 4 128 40 2–32 2 32 19
Carbapenems Imipenem 16 0 0.25–128 2 8 7.4 0.25–16 1 16 11
2 0.5–2 1 2 0 0.5–2 1 1 0
4 0.25–1 0.25 0.5 0 0.25–16 0.25 0.25 9.5
Macrolides Erythromycin 8 0 0.25–128 2 4 7.4 0.25–4 2 2 0
2 0.25–128 128 128 55 0.25–1 1 1 0
4 0.25–128 0.5 128 24 0.25–1 0.5 1 0
Glycopeptides Vancomycin 32 0 0.5–2 1 2 0 0.25–1 0.5 1 0
2 1–8 4 8 0 1–4 2 4 0
　 4 2–256 4 32 12 4–256 8 256 24
MIC: minimum inhibitory concentration.
*MIC50: Minimum concentration at which growth of 50% the isolates is inhibited.
**MIC90: Minimum concentration at which growth of 90% the isolates is inhibited.
 22 Nishiyama et al.
concentration

concentration
vanC2/C3

vanC2/C3
Vancomycin

Vancomycin
No. isolates

No. isolates
ABPC

ABPC
(% isolates)

(% isolates)
VCM

VCM
vanC1

vanC1
MDR

MDR
PIPC

PIPC
PCG

PCG
vanB

vanB
IMP

IMP
vanA

vanA
EM

EM
TC

TC
( g mL )

(µ g mL )
-1

-1
Species

Species
Genus

Genus
2 - - - - 6 - - - -
6 - - - - 3 - - - -
17 17
E. faecium 2 - - - - E. faecium 6 - - - -
(63%) (63%)
5 * - - - - 1 * - - - -
0 2 * - - - - 1 * - - - -
0
E. hirae 2 (7%) - - - - E. faecalis 1 (4%) * - - - -
2 - - - - 1 - - - -
8
E. faecalis 4 - - - - 9 2 - - - -
(30%) E. hirae
2 - - - - (33%) 3 - - - -
6 1 - - - - 3 - - - -
E. faecalis
(55%) 5 * - - - - 1 - - - -
3
1 * - - - + E. faecium 1 - - - -
(27%)
2 1 - - + - 1 * - - - -
E. casseliflavus / 5
1 * - - + - 2 1 - - - -
E. gallinarum (45%) E. faecalis
1 * - - + - 2 (18%) 1 * - - - -
1 * - - + - 4 2 - - - -
E. hirae
E. faecalis 1 (4%) - - - + (36%) 2 - - - -
2 - - - - E. c / g* 1 (9%) - - - -
Downloaded by [George Mason University] at 09:59 01 January 2015

1 - - + - Pediococcus 1 (9%) - - - -
1 - - - - E. faecium 1 (4%) - - - +
2 - - - - 2 1 * - - - -
E. faecalis
1 - - + - (7%) 1 * - - - -
2 - - - + 6 - - - +
2 - - - - 2 - - - +
E. casseliflavus / 24 2 - - + - 3 - - - -
4 4 E. casseliflavus / 18
E. gallinarum (89%) 1 - - - + 3 - - - +
E. gallinarum (67%)
2 - - - - 1 * - - - -
1 - - - + 2 - - - +
1 - - - - 1 * - - - +
1 - - - - 6 4 - - - -
Pediococcus
3 * - - - + (22%) 2 - - - -
1 * - - - - 32 Pediococcus 32 (100%) - - - -
1 * - - - -
Pediococcus 2 (7%) - - - -
Leuconostoc 4 (15%) - - - -
32 Pediococcus 18 (67%) - - - -
Weissella 5 (18%) - - - -

Fig. 3. Antibiotic susceptibility profiles and genotypes for total isolates from sewage and river water. For antibiotic resistance pheno-
types, black indicates resistance, gray indicates intermediate resistance, and white indicates susceptibility. ABPC, Ampicillin; PIPC,
Piperacillin; PCG, Penicillin G; TC, Tetracycline; IMP, Imipenem; EM, Erythromycin; VCM, Vancomycin, MDR, multi-drug resis-
tance; E.c/g, E. casseliflavus/E. gallinarum.

particularly in the aquatic environment is crucially impor- This study confirms the presence of VRE in the commu-
tant because VRE are recognized as a major cause of noso- nity of Miyazaki, a provincial city in Japan. VRE has been
comial infections. Therefore, isolating VRE from aquatic detected in wastewater from hospitals and the water treat-
environments, such as sewage and rivers, is important. ment process, including effluent samples.[34–37] In US,
These data may help identify the source of antibiotic-resis- where VRE cause serious nosocomial infections, VRE
tant bacteria, clarify resistance mechanisms, and reveal bac- constituted 3% of enterococci isolated from municipal
terial dynamics in aquatic environments. wastewaters.[37] VRE has also been detected in municipal
All vancomycin-resistant and intermediate-resistant wastewaters in Europe at percentages ranging from 0.6%
strains isolated using the mEI agar supplemented with 2 or to 60%.[25,34,35,38] VRE concentrations in the three princi-
4 mg mL¡1 vancomycin were identified as Enterococcus pal rivers of Korea were found to range from 1 to 23 CFU
spp. In contrast, although all strains isolated from sewage 100 mL¡1.[39] In addition, vancomycin-resistant entero-
and river water using the mEI agar supplemented with cocci have been isolated from coastal waters in Northern
32 mg mL¡1 vancomycin showed resistance to vancomycin Greece.[13] The percentages of VRE in sewage and river
(MIC, >256 mg mL¡1); none of the 54 isolates were water indicate that Japan has a lower proportion of VRE
Enterococcus spp. They were identified as species of Pedio- than other countries.
coccus, Leuconostoc, or Weissella. The mEI agar contain- Strains possessing vanA and vanB, which are the most
ing vancomycin at an excessively high concentration allows troublesome in nosocomial infections, were not detected in
the emergence of false-positive colonies. Therefore, the the sewage and river water tested in this study. However,
results presented here suggest that 4 mg mL¡1 is a suitable isolates that retained vanC1 and vanC2/3 were detected in
concentration of vancomycin for screening sewage and the aquatic environment in Japan. The vanC genes, which
river water for the presence of VRE using the mEI agar. confer resistance to low levels of glycopeptide, are
 Vancomycin-Resistant Enterococci (VRE) from sewage and river water in Japan
chromosomally encoded by vanC1 and vanC2/3. These
genes are intrinsic to E. casseliflavus and E. gallinarum and
can be used for species identification.[30,40]
Previous studies have demonstrated that E. faecalis
acquired vanC by horizontal gene transfer from E. casseli-
flavus/gallinarum in the poultry gut.[41–43] The fact that
vanC2/3 was confirmed in E. faecalis and E. faecium strains
that were isolated along with E. casseliflavus/gallinarum
from the aquatic environment suggests that the E. faecalis
and E. faecium strains observed in this study may have
acquired vanC2/3 by horizontal gene transfer in the aque-
ous environment. However, they may also have acquired
vanC2/3 through horizontal gene transfer prior to entering
the aquatic environment. Because there is little information
on the acquisition or transfer of antibiotic resistance in
enterococci that are discharged into the aquatic environ-
Downloaded by [George Mason University] at 09:59 01 January 2015
ment, future studies are required to address this issue.
Resistance to piperacillin and tetracycline were common
among the 54 enterococcal strains isolated from sewage
and river water using normal mEI agar plates. Numerous
isolates from sewage also showed resistance to piperacillin,
tetracycline, and erythromycin. These findings are similar
to those obtained by the surveillance of antimicrobial-
resistant enterococci at wastewater treatment plants in
Poland and Portugal.[25,34] The results of national surveil-
lance studies of antimicrobial consumption conducted by
WHO in a Japanese hospital were reported on the basis of
the defined daily dose (DDD).
The use of penicillins was the highest among the antibiot-
ics, with a consumption rate of 4.27 DDDs per 100 bed
days.[44] The high erythromycin resistance rate observed
among enterococci in this study is consistent with the exten-
sive therapeutic use of macrolides in Portugal.[45] In con-
trast, the percentage of enterococcal strains resistant to the
universally applicable tetracycline, which is a classic antibi-
otic, is 41% in sewage but only 3.7% in river water. Tetra-
cycline is not used to treat enterococcal infections, yet
resistance to this antimicrobial agent is still common
among clinical [45] and sewage isolates.[25,34] When treating
other infectious diseases by administering doses of tetracy-
cline, enterococci in the human gastrointestinal tract are
strongly influenced, and this leads to a higher percentage of
tetracycline-resistant strains in sewage than in river water.
Resistance to 3 chemical classes of antimicrobial
agents is defined as multidrug resistance. Multidrug-resis-
tant enterococci were observed in both sewage and river
water. Two E. faecium isolates from sewage were suscepti-
ble to vancomycin, but showed resistance to six antimicro-
bial agents (ampicillin, piperacillin, penicillin G,
tetracycline, imipenem, and erythromycin), representing
four chemical classes (Fig. 3). Multidrug-resistant entero-
cocci that showed resistance to four antimicrobial agents
(piperacillin, penicillin G, tetracycline, and imipenem)
were isolated from river water. It has been reported that
VRE, which has become a problematic cause of nosoco-
mial infectious diseases, shows strong tolerance to almost
23
all other antibiotics. However, the VRE strains collected
form sewage and river water in this study were susceptible
to other antibiotics, including ampicillin, piperacillin, and
imipenem.
Although VRE isolates were collected from sewage and
river water, vanA and vanB, which confer high-level resis-
tance to vancomycin, were not detected. VRE possessing
vanA and vanB have been isolated from hospitals in Japan
[23,24,46,47]
and VRE possessing vanA were detected not
only in the clinic but also in imported and domestic chick-
ens.[48–50] Therefore, drainage water from houses and retail
outlets that sells food products made from chicken is
potentially contaminated with VRE. Wastewater treat-
ment processes that treat miscellaneous drainage from
human and hospital effluents use biological processes
based on activated sludge. Because of the high cell density
within these sludges, wastewater treatment processes are
regarded as factors promoting the dissemination of bacte-
rial resistance.
Conclusion
Antibiotic-resistant Enterococcus spp, including VRE,
were isolated from samples taken from sewage and river
water in the provincial city of Miyazaki, Japan. The isola-
tion of VRE strains required the supplementation of the
isolation medium with vancomycin; the mEI agar supple-
mented with 4 mg mL¡1 vancomycin is suitable for the iso-
lation of VRE from sewage and river water. The bacterial
strains isolated from sewage and river water were 12% and
24% VRE, respectively. Vancomycin-resistant genes
vanC1 and vanC2/3 were detected in VRE isolates
obtained from both sources.
All the enterococcal isolates that contained vanC1 were
identified as E. casseliflavus/gallinarum. In contrast, 92%
of the enterococcal isolates possessing vanC2/3 were E.
casseliflavus/ gallinarum, wheras the remaining were E.
faecalis (4%) or E. faecium (4%). Therefore, E. faecium
and E. faecalis, which are the major types of enterococci in
humans, containing vanC2/3 were observed in sewage and
river water. The pollution of the aquatic environment with
VRE containing vancomycin-resistant genes is a signifi-
cant concern. Accurate information concerning the preva-
lence of VRE in aquatic water environments is important
for the improvement of public hygiene. Further informa-
tion on the presence of VRE and antibiotic-resistant
enterococci in the aquatic environment should be gathered
as soon as possible.
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