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Int.J.Curr.Microbiol.App.Sci
(2015)
4(10
):
179
-
189
179
Original Research Article
Characterization of Extended Spectrum -
Lactamase Producing and Non
-
Producing
Escherichia coli
Isolated from Hospital and Fish Processing Plant
Untreated Wastewaters
M.
Divyashree
1, Malathi Shekar2
*,
K.
Shama Prakash
3
, M
.
N
.
Venugopal
2,
I.
Karunasagar
4
and
A.
Veena Shetty
5
1
Nitte University Centre for Science Education and Research, Nitte University, Deralakatte,
Mangalore, Karnataka
, India
2
Department of Microbiology, UNESCO Center for Marine Biotechnology, KVAFSU Coll
ege of
Fisheries, Mangalore, Karnataka, India
3
Department of General Medicine, K.S. Hegde Medical Academy, Nitte University, Deralakatte,
Mangalore, Karnataka
, India
4
Director, Nitte University
,
Centre for Science Education and Research, Nitte University,
Deralakatte, Mangalore, Karnataka
, India
5
Department of Microbiology, K.S.
Hegde Medical Academy,
Nitte University, Deralakatte,
Mangalore, Karnataka
, India
*Corresponding author
A B S T R A C T
ISSN: 2319
-7706
Volume
4
Number 10
(201
5
) pp.
179
-
189
http://
www.ijcmas.com
This study aimed to detect and characterize Extend
ed
-Spectrum -
lactamase
(ESBL) and non-ESBL producing Escherichia coli from untreated effluents let out
from hospitals and fish processing plants in Mangalore, India. Isolates were
phenotypically tested for ESBL production, resistance to ACCoT (ampicillin,
chloramphenicol, co-
trimoxazole,
tetracyclines) and for the presence of
their
resistance encoded genes.
ESBL
-
producing E.
coli
was seen to be dominant in
hospital wastewaters (HWW) as compared to Fish processing wastewaters
(FPWW). Among HWW ESBL positive strains 82% harbored the bla
CTX
-
M
gene,
while bla
TEM
accounted for only 36%. These strains also showed resistance to
ampicillin (100%), co-trimaxazole (59%), tetracycline (96%) and
chloramphenicol(18%). Similarly non-ESBL HWW E. coli isolates showed
resis
tance to ampicillin (92%), co-trimoxazole (64%), tetracycline (45%) and
chloramphenicol (22%). The HWW ESBL and non-ESBL isolates were observed
to be multi drug resistant with several encoding more than one gene determinant
corresponding to their antibiotic resistance phenotype. RAPD-PCR for ESBL
positive and negative E coli isolates from HWW and FPWW showed the existence
of several genotypes among ESBL positive strains. No correlation existed between
the ESBL phenotypes to antibiotic resistance genes harbo
red.
Key word s
E coli
,
ESBL,
bla
CTX
-M,
Antibiotic
resistance
genes,
Hospital
waste
water,
RAPD
Int.J.Curr.Microbiol.App.Sci
(2015)
4(10
):
179
-
189
180
I
ntroduction
The widespread use of antimicrobial agents
in human, aquaculture and veterinary
medicine has led to the emergence of
multidrug resistant bacteria which is now a
worldwide public health concern.
Wastewaters discharged into the
environment constitute a way for the
introduction of antibiotic residues as well as
antibiotic resistant bacteria into the
environment
(Harris
et al., 2012).
E.
coli
a
gram negative bacterium and an important
microflora of the human and animal
gastrointestinal tract has been frequently
isolated from effluent waters fed into aquatic
environments (Prado et al., 2008; Chagas
et
al.,
2011).
Beta
-lactam antibiotics constitute
the main therapeutic choice for treating
human infections caused by
Enterobacteriaceae
bacteria.
In
recent years,
extended
-spectrum -lactamase (ESBL)
producing
E.
coli
has gained recognition as a
major clinical problem worldwide due to
their increased resistance to most of the -
lactam antibiotics including penicillins,
carbapenems and third generation
cephalosporins (Banno et al., 2004; Canton
et al., 2007). The increased resistance to -
lactams is due to the ability of bacteria to
produce -lactamase enzymes capable of
hydrolyzing -lactams, rendering the
antibiotic inactive (
Nordmann
et al.,
2012).
Although many studies have reported
the
prevalence of ESBL producing
E.
coli
in
hospital settings
(Pitout
et al., 2005;
Korzeniewska and Harnisz, 2013), studies
on its prevalence in hospital effluents and
other non-clinical environments has been
limited
(W
atkinson
et al., 2009).The aim of
this study was to detect
E.
coli
from
wastewaters discharged from hospitals and
fish processing plants and characterize them
based on their ESBL production. The
rationale for including fish processing
wastewater was based
on the common use of
human intended drugs such as
chloramphenicol, sulphonamide and
tetracyclines in aquaculture practices,
which
has influenced the fecal coliforms to acquire
resistance to the antibiotics used
(Akinbowale
et al., 2007). ESBL and non-
ESBL
E.
coli
show simultaneous resistance
to other non- -lactam class of drugs
(Bradford, 2001; Qin et al.,
2008)
and
therefore the
E.
coli strains in this study
were tested for their antibiotic resistance to
selected antibiotics and PCR detected
further for the presence of implicated
antibiotic resistant gene(s). The
E.
coli
in
this study w
as
also genotyped by RAPD-
PCR to determine their genetic relatedness
and association to ESBL production and the
antibiotic resistance genes harbored.
Materials and Methods
Bacteria
l strains
E.
coli
isolated from untreated wastewater
let out from two hospitals (H1 and H2) and
two fish processing plants (FP1 and FP2)
were used in this study. The isolates were
identified as
E.
coli by standard biochemical
tests. Molecular identification of the strains
using PCR primer was based on targeting a
146-bp fragment of the
uid
A gene (Table 1)
by polymerase chain reaction (Asim et al.,
1991). Briefly, genomic DNA was extracted
according to the method of Ausubel et al.
(1995). The bacterial cell pellet was
suspended in 567 l of 1
×
TE buffer (10
mM
Tris
-Cl; 1 mM EDTA; pH 8.0), 30 l of
10%
sodium dodecyl sulphate and 3 l of
proteinase K (20 mg/ml) and incubated at
45oC for 1 hour. After incubation, 100 l of
5M NaCl and 80 l of CTAB/NaCl solutio
n
were added and the mixture incubated for a
further 10 min at 65oC. The solution was
centrifuged at 10000 rpm for 10 min with an
equal volume of chloroform/isoamyl
Int.J.Curr.Microbiol.App.Sci
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alcohol. The aqueous phase was transferred
to a fresh tube and 0.6 volume of
isopropanol added to precipitate DNA. The
mixture was centrifuged and the pellet
obtained was washed with 70% ethanol and
re
-centrifuged at 10,000 rpm for 5 min. The
DNA pellet was vacuum dried and re-
suspended in 100 l of sterile 1X TE buffer
(pH 8.0). The DNA concentration and purity
was estimated using a Nano Drop
spectrophotometer (ND-1000, V3.3.0,
Wilmington, DE, USA).
PCR was carried out in a programmable
thermocycler (MJ Research, USA) using 30
µl reaction mixture containing 10X buffer
(100mM of Tris- HCl, pH 8.3, 20mM 0f
MgCl2,
500mM of KCL and 0.1% gelatin)
200mM of deoxyribonucleotide triphosphate
(dATP, dTTP, dGTP and dCTP), 10
picomoles of each primer and 1 U of
Taq
polymerase (Bangalore Genei, Bangalore),
with 2.0 l of template DNA. The optimized
PCR programme consisted of an initial
denaturation at 94°C for 5 min followed by
30 cycles with each cycle consisting of 94
°
C
for 30 sec, Tm (annealing temperature) 60°C
fo
r 30 sec and extension for 72° C for 30
sec. The final extension was performed at
72°C for 10 min. The amplified products
were resolved by 1.5 % (w/v) agarose gel
electrophoresis.
Antibiotic susceptibility and ESBL
production test
E.coli
isolates were subjected to antibiotic
susceptibility test on
Mueller
-
Hinton
agarby
the Kirby-Bauer disk diffusion method
(CLSI, 2012). Antibiotic discs (HI Media,
Mumbai) tested were ampicillin (Amp,
10µg); cefotaxime (CTX,
30µg),
ceftazidime, (CAZ, 30µg) chloramphenicol
(C, 30µg), cotrimoxazole (CO,
25µg),
tetracycline (TET,
30µg)
and the resistance
estimated as per CLSI guidelines
(CLSI,
2012).
E coli ATCC strain 25922 was used
as a reference strain.
Strains resistant to cefotaxime and
ceftazidime were tested further for the
production of ESBL by double disc
diffusion assay (DDDT). To a young lawn
culture of the isolate on Muller-Hinton agar
cephalosporin/clavulanate combination discs
namely Ceftazidime (CAZ-30µg) and
Ceftazidime + Clavulanic acid (CAC,
30/10µg), Cefotaxime (CTX, 30µg) and
Cefotaxime + Clavulanic acid (CEC,
30/10µg) were placed. The plates were
incubated for 24hrs at 37°C. An increase in
zone diameter of equal or >5 mm around
both CAC and CEC discs as compared to
cefotaxime or ceftazidime tested alone we
re
interpreted as positive for ESBL (Figure 1).
E. coli ATCC 25922 w
as
used as a standard
strain.
Detection of antibiotic resistance genes
The
E.
coli
strains were characterized for
genes encoding -lactamases (blaCTX-M
,
bla
TEM
-1), and for resistant genes that
conferred resistance to chloramphenicol
(
cat1, cat2, cat
3), sulfonamides (
sul1, sul
2,
s
ul
3) and tetracyclines (tet
A, tet
B,
tet
C,
tet
D,
tet
E,
tet
G) by PCR assays. PCR
reaction was carried out in a 30 l reaction
mixture as described above using specific
primers as mentioned in t
able
1. The
amplicons were resolved in 1.5 % agarose
gel, stained with ethidium bromide and
visualized in a Gel Documentation system
(Bio
-
Rad, USA).
RAPD
-
PCR assay
RAPD
-PCR was performed initially using
six randomly designed 10-mer
oligonucleotide primers PM3 and PM5
(Tassanakajon
et al., 1997), CRA22,
CRA26, OPA11 and OPA18 (Neilan 1995)
Int.J.Curr.Microbiol.App.Sci
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to evaluate and check for the repeatability of
the fingerprints generated. PM5 was selected
for further analysis as it yielded well
resolved and better banding patterns. The
RAPD
-
PCR
was carried out in 30 L
reaction mixture consisting of 3 L of 10
×
PCR buffer (100 mM
Tris
-Cl pH 8.3, 20
mM MgCl2, 500 mM KCl, 0.1% gelatin),
250 M of each deoxynucleotide
triphosphates,
50 pmol of primer, 1.5 U of
Taq
polymerase (Bangalore
Genie,
Bangalo
re, India). Standardized PCR thermo
cycling
conditions for the RAPD included
initial denaturationat 95°C for 5 min and
final delay at 72oC for 30s followed by 35
cycles, 94oC for 50s, and 36oC for 45sand
72°C for 30s. The amplification was carried
out in a thermocycler
(MJResearch,
Watertown, USA) and the products resolved
in 1.0% agarose gel, stained with
ethidium
bromide and the results photograph captured
by Gel Documentation system (Bio-
Rad,
USA).
RAPD data analysis
Comparison of RAPD-PCR patterns
gen
erated for different gels was analysed
using Gel compare II version 2.5 (Applied
Maths, St Martens-Latem, Belgium). All
visible electrophoretic bands were included
in analysis. Similarities between profiles
was based on Pearson s correlation
coefficient and clustered using the
unweighted pair group method with
arithmetic mean (UPGMA). The similarity
is expressed as percentage similarity and
presented as a dendrogram. The numerical
discriminatory index value was calculated
(Hunter and Gatson 1988).
Results
and Discussion
Characterization of -
lactamase
-
producing
E.
coli
The incidence of
E.
coli
from FPWW in this
study was lower than those isolated from
HWW.
E.
coli
isolated from hospital
(n=150)
and fish processing
(n=32)
wastewaters were screened for thei
r
antibiotic resistance patterns and ESBL
production. A total of 114 (76%) and 12
(37.7%)
E.
coli
isolated
from HWW and
FPWW respectively, showed resistance to
one or more antibiotics tested.
The
remaining was sensitive to all the antibiotics
tested. The antibiotic resistance strains were
further characterized and grouped as ESBL
and non-ESBL producing phenotypes. Of
the 114 HWW isolates, 39 isolates were
classified as ESBL producers by phenotypic
assay. Analysis of the ESBL
-
encoding genes
indicated that majority of the strains
harbored CTX-M (82%) followed by bla
TEM
(36%). Similarly for
E.
coli
isolated from
FPWW, only one isolate was positive for
ESBL production and harbored the blaCTX-
M
gene, while 2 among the 11 ESBL negative
strains harbored bla
TEM
gene
(Table 2). All
non-ESBL HWW isolates were negative for
the presence of blaCTX-M gene, while 19
(26%) isolates confirmed positive for the
bla
TEM
gene.
Antibiotic resistance and distribution of
resistance genes
The HWW ESBL producing strains revealed
comp
lete resistance to ampicillin (100%),
followed by resistance to cotrimaxazole
(59%), tetracycline (46%) and
chloramphenicol (18%).Afew of these
strains also showed the simultaneous
harboring of other antimicrobial resistance
genes, the highest being for
su
l (41%),
followed by
tet
(26%) and
cat
(3%) genes.
The 74 non-ESBL HWW isolates showed
resistance to ampicillin (92%), co-
trimoxazole (64%), tetracyclines (45%) and
chloramphenicol (22%). Among the non-
ESBL 53%, 27% and 8% were observed to
encode for
sul,
tet
and
cat
genes
Int.J.Curr.Microbiol.App.Sci
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respectively. The encoding of more than one
gene corresponding to an antibiotic resistant
phenotype was also observed. For example,
strains phenotypically resistant to co-
trimoxazole were seen to encode either one
or two of the
sul
genes, but not all gene
determinants tested. This was also observed
for strains resistant to tetracycline, wherein
except for one strain which harbored four
tet
genes, the rest were seen to encode two, one
or no tet genes (Table 2). The details of
antibiotic resistance patterns and the
distribution of antibiotic resistance genes for
the
E.
coli
included in this study are
presented in
t
able
2.
RAPD PCR
Twenty eight
E.
coli
strains representative of
ESBL positive and negative strains from
both HWW and FPWW were used in RAPD
analysis. RAPD analysis generated seven
different banding patterns indicating the
prevalence of different genotypes (Figure 2).
The RAPD generated 6-12 bands with band
sizes ranging from 0.3 kb to 1.2 kb. The
number of bands generated for
E.
c
oli HWW
isolates
was observed to be higher in
comparison to FPWW isolates (Figure
2). At
a similarity of 70% all
E.
coli
strains in this
study grouped into eight clusters (C1-
C8).
All the non-ESBLs confirmed negative by
phenotypic assay were seen to group
into
one large cluster C2 (Figure
2).
However,
the ESBL positive HWW isolates grouped
themselves into seven clusters, with cluster
C1 grouping 7 isolates, followed by cluster
C5 (2 isolates) and the remaining (C3-
C4,
C6
-C8) having a single isolate
(Figure
2).
Further, at 79% percent similarity value the
ESBL producing HWW isolates were seen
to be statistically discriminatory (DI=0.92),
wherein the isolates in cluster C1 could be
further sub grouped into C1a (4 isolates),
C1b (2 isolates) and C1c (1 isolate). This
indicates the prevalence of several
genotypes among ESBL producing
E.
coli
strains in hospital waster waters. The RAPD
fingerprints generated showed no correlation
between ESBL phenotype to antibiotic
resistance genes harbored.
In this study the E. coli isolated from
HPWW was higher as compared to FPWW.
Almost all of the
E.
coli
isolated from fish
processing plant effluents were also
observed to be sensitive to antibiotics tested.
The low levels of resistance observed for
FPWW
E.
coli
in this study could be due to
the low numbers identified or due to the
non-use of these antibiotics following ban
on the use of these antibiotics in aquaculture
practice
(Aquaculture News, 2003).
However, the harboring of plasmid
associated resistance genes such as bla
TEM
,
sul
and tetseen in few of the FPWW isolates
is a matter of concern. I
n contrast to FPWW,
E.
coli
isolated from hospital effluents
showed high resistance to antibiotics tested
exhibiting several phenotypic resistance
patterns.
The higher multidrug resi
stant
phenotypes could be due to the extensive
usage of these drugs in hospitals for treating
patients, which eventually gets released into
hospital sewage. Majority (80%) of the -
lactamase producing ESBL phenotypes from
the hospital sewage in this study encodedthe
blaCTX
-M gene, which is in accordance to
observations made by other investigators
(Amaya
et al., 2012; Galvin et al.
2010).
The CTX-M type of ESBL strains has been
resp
onsible for outbreaks in hospitals (Rezai
et al., 2015) and environments
(Pitout
et al.,
2005; Rossolini et. al., 2008) throughout the
world. A few strains positive for the bla
CTM
-
M
gene also co
-
harbored the bla
TEM
gene. The
presence of more than one
bla
-
ge
ne has
been previously documented for
E.
coli
isolated from sewage (Korzeniewska and
Harnisz, 2013, Galvin et al., 2010) and the
reason suggested for the high-levels of -
lactamase resistance
(Kiratisin
et al.,
2008).
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Table.1
Primers used in this study
Primer
Primer sequence (5 -3 )
Product
size (bp)
Reference
bla
CTX
-M
F:ACGTTAAACACCGCCATTCC
R:TCGGTGACGATTTTAGCCGC
356 Colomer-
Lluch Marta et al.,
2
2011
bla
TEM
F:C
TCACCCAGAAACGCTGGTG
R:ATCCGCCTCCATCCAGTCTA
569 Colomer-
Lluch Marta et al.,
2011
cat1
F:AACCAGACCGTTCAGCTGGAT
R:CCTGCCACTCATCGCAGTAC
549
Ma et al.,
2007
cat2
F:AACGGCATGATGAACCTGAA
R:ATCCCAATGGCATCGTAAAG
547
Ma et al.,
2007
cat3
F:ATCGGCATCGGTTACCATGT
R:ATCCCCTTCTTGCTGATATT
531
Ma et al.,
2007
sul 1
F:TTTCCTGACCCTGCGCTCTAT
R:GTGCGGACGTAGTCAGCGCCA
425
Ma et al.,
2007
sul 2
R:CCTGTTTCGTCCGACACAGA
R:GAAGCGCAGCCGCAATTCAT
435
Ma et al.,
2007
sul 3
F:ATGAGCAAGATTTTTGGAATCGT
R:CTAACCTAGGGCTTTGGATATTT
792
Ma et al.,
2007
tetA
F:TTGGCATTCTGCATTCACTC
R:GTATAGCTTGCCGGAAGTCG
494
Ma et al.,
2007
tetB
F:CAGTGCTGTTGTTGTCATTAA
R:GCTTGGAATACTGAGTGTTAA
571
Ma et al.,
2007
tetC
F:CTTGAGAGCCTTCAACCCAG
R:ATGGTCGTCATCTACCTGCC
418
Ma et al.,
2007
tetD
F:GCAAACCAT
TACGGCATTCT
R:GATAAGCTGCGCGGTAAAAA
546
Ma et al.,
2007
tetE
F:TATTAACGGGCTGGCATTTC
R:AGCTGTCAGGTGGGTCAAAC
544
Ma et al.,
2007
tetG
F:GCTCGGTGGTATCTCTGCTC
R:CAAAGCCCCTTGCTTGTTAC
550
Ma et al.,
2007
PM5
CGACGCCCTG
-
Tassanakajon
et al.,
1997
uid
A
F:AA
AACGGCAAGAAAAAGCAG
R:ACGCGTGGTTACAGTCTTGCG
146
Asim
et al., 1991
Int.J.Curr.Microbiol.App.Sci
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Table.2 Antibiotic resistance profile and the distribution of resistance genes among ESBL and non
-ESBL producing
E.
coli
Antibiotic resistance profile
No. of isolates PCR positive for t
he antibiotic resistant gene corresponding to
ESBL
(
bla
CTX
-M
) (
bla
TEM
)
Chloramphenicol
(
cat1, cat2, cat3
)
Cotrimoxazole
(
sul1, sul2, sul3
)
Tetracycline
(
tetA,tetB, tetC, tetD, tetE
)
HWW ESBL+veE.coli
A, (
n
=9)
+ (8)
+ (4)
- - -
A,C (
n
=2)
+ (1)
- - - -
A,C,Co (
n
=2)
+ (2)
+ (1)
-
sul2
(1)
-
A,Co (
n
=8)
+ (6)
+ (4)
-
sul1
(1);
sul2
(1);
sul3
(1);
sul1,2
(1);
sul2,3
(1)*
-
A,Co,T (
n
=10)
+ (9)
+ (3)
-
sul1
(1);
sul
3(1);
sul
1,2(4);
sul1,3
(1)
tetE
(1);
tetC
(2);
tetA,E
(1);
tetA,D
(1)
A,C,Co,T (
n
=3)
+ (1)
+ (1)
cat1(1)
sul3
(1);
sul1,2,3
(2)
tetA
(1);
tetA,C
(1)
A,T (
n
=5)
+ (5)
+ (1)
- -
tetA
(1);
tetD
(1);
tetA,D
(1)
HWW ESBL
-
veE.coli
A (
n
=11)
- - - - -
C (
n
=1)
- - - - -
Co (
n=3
) - - -
sul1
(1
); sul1,2
(1)
-
A,C (
n
=3)
-
+ (2)
cat1,2
(2)
A, Co(
n
=18)
-
+ (6)
-
sul
1,2
(3)
; sul1
(6)
; sul2
(2); sul2,3(1); sul1,3(1)
; sul3
(2)
-
A,C, Co (
n
=5)
-
+ (2)
cat2(1)
sul1
(1);
sul2
(3);
sul1,2
(1)
-
A, C, Co, T (
n
=6)
-
+ (2)
cat1,2
(2);
cat2
(1)
sul1
(3)
; sul2
(2)
tetA
(1)
; tetD
(1);
tet B,D
(1)
A, C, T (
n
=1)
- - - - -
A,Co,T (
n
=15)
-
+ (
3)
-
sul1
(7)
; sul1,2
(4)
; sul2
(1)
tetB,E
(1)
; tetA
(2)
; tetB
(1);
tetD
(1)
;
tetB,C,D,E
(1)
; tetC(1)
A, T (
n
=9)
-
+ (4)
- -
tetE
(1)
; tetC
(1);
tetA,B
(1);
tetA
(4);
tetA,E
(1)
T (
n
=2)
- - - -
tetA
(2)
FPWW ESBL +veE.coli
CAZ, CTX, T(
n
=1)
+
(1)
- - - -
FPWW ESBL
-
veE
.coli
A (
n
=3)
- - - - -
T (
n
=3)
- - - - -
A, T (
n
=3)
-
+ (2)
- -
tet
D(1)
A, Co (
n
=2)
- - -
sul
1(1)
-
A, ampicillin; C, chloramphenicol; Co, co-trimoxazole; T, tetracycline; HWW: Hospital wastewater; FPWW: Fish processing plant wastewater, The HWW
ESBL
isolates were resistant to ceftazidime and cefotaxime. n indicates the number of isolates and no. in brackets indicates the no of isolates positive for the
gene; *presence of more than one gene coding for the same resistant antibiotic is separated by a
comma
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Figure.
1
ESBL detection by
double disk diffusion test
CAZ: ceftazidime; CAC: ceftazidime + clavulanic acid; CTX: cefotaxime; CEC: cefotaxime + clavulanic acid
Figure.2
Dendrogram generated based on RAPD PCR profiles for ESBL and non
-
ESBL E.co
li
isolates. H1 and H2 represents hospital wastewater isolates and FP1 and FP2 fish processing
wastewater isolates. C1-
C8 are clusters generated at 70% similarity
Int.J.Curr.Microbiol.App.Sci
(2015)
4(10
):
179
-
189
187
The TEM gene has been associated with
90% of ampicillin resistance in
E.
coli
(Livermore,
1995). In this study, a low
association of 36% and 26% was observed
for HWW ESBLs and non-
ESBL
respectively. A small percentage of the
E.
coli
positive for the production of -
lactamase but negative for the presence of
the
bla
genes could be harboring othe
r
variant genes such as SHV, OXA, PER
types implicated in ESBL production which
was not included in this study.
Several of the
E.
coli strains in this study
were observed to simultaneously harbor
antibiotic genes associated with non-
lactam class of antibiotics (Table 2). It was
interesting to note that many of the ESBL
and non-ESBL isolates that displayed
resistance to co-trimoxazole and tetracycline
showed the presence of multiple
determinants of
sul
and
tet
genes
respectively.
Genotyping of
E.
coli
by RAPD-
PCR
showed that there exists a genetically
diverse and heterogeneous group among
ESBL and non-ESBL producers in hospital
wastewaters, which maybe a reflection of
genotypes circulating within the hospitals.
The prevalence of several genotypes in
hosp
ital waste waters thus emphasizes the
need for appropriate control measures to be
taken before letting out untreated water into
the environment. Further, the isolates are
multidrug resistant and carry plasmid
mediated antibiotic resistance genes which
coul
d transfer these genes to other non-
resistant bacterial strains through exchange
of genetic material
(Rahube
, 2010).
Therefore molecular detection and
identification of ESBL producing
E.
coli
in
hospital untreated waste water becomes
important for implementation of appropriate
control measures. Our studies with RAPD
show
although RAPD-PCR cannot be used
in typing strains based on -lactam or non -
lactams genes they carry, this tool could be
used as a technique for epidemiologically
discriminating
ESBL producers from non-
ESBL producing
E.
coli
strains.
Acknowledgements
The molecular work was supported by
UNESCO MIRCEN for Marine
Biotechnology,
Department of
Microbiology, Karnataka Veterinary,
Animal and Fisheries Science University,
College of Fisheries Ma
ngalore.
Authors
acknowledge the DBT-
Bioinformatics
Centre, KVAFSU, College of Fisheries
Mangalore for carrying out the analysis
work.
Conflict of Interest: The authors declare
that they have no conflict of interest.
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