Molecular Characteristics of ESBL-Producing Escherichia coli Isolated From Clinical
Specimens at Vajira Hospital, Navamindradhiraj University
Thunwalai Hongsom1,*, Pornphan Diraphat2, Somchai Uaratanawong3, Kanokrat
Siripanichgon2,#
1
MS program in Infectious disease and Epidemiology, Faculty of Graduate Studies, Mahidol
University, Bangkok, Thailand.
2
Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok,
Thailand.
3
Faculty of Medicine Vajira Hospital, Navamindradhiraj University, Bangkok, Thailand
*e-mail: pophuenga@gmail.com, #e-mail: kanokrat.sir@mahidol.ac.th
Abstract
Increasing multidrug resistant E. coli infection in the hospital resulted in limited
choice of therapy and high mortality rate. The objective of this study was to determine
prevalence and genetic determinants of multidrug resistance in ESBL-producing E. coli
isolated from clinical specimens submitted to the Microbiology Laboratory of Vajira hospital.
ESBL-producing E. coli isolates were identified by using automated Microscan WalkAway40 SI system. Prevalence of ESBL-producing E. coli was 54.3% (252/464). Majority of the
ESBL isolates were from urine (48.4%, 122/252), and sputum (18.7%, 47/252). All ESBL
isolates (100%) were resistant to ampicillin, cefotaxime, ceftazidime, ceftriaxone, cefepime
but susceptible to ertapenem, imipenem, meropenem. The percentage of resistance to
cefoxitin, gentamicin, amikacin, ciprofloxacin, levofloxacin, moxifloxacin were 6.3%, 63%,
1.1%, 79.4%, 77.2%, 78.8%, respectively. Thirty ESBL producers were selected to
determine bla and int genes by PCR and multiplex PCR method, respectively. The
percentage of ESBL-producing E. coli positive for blaCTX-M, blaTEM, blaVEB, blaSHV, blaOXA-10like on chromosome were 97%, 47%, 27%, 3%, 13%, and on plasmid were 97%, 47%, 17%,
3%, 3%, respectively. None of isolates were positive for blaOXA-2-like and blaOXA-40-like. The
intl1 were detected on chromosome 77% and plasmid 73%. None of isolates were positive
for intI2 and intI3. The blaCTX-M, was predominantly found on both chromosome and plasmid
(97%). The results indicated more than 54% of clinical E. coli isolates were ESBL producers
that still susceptible to carbapenems. High percentages of blaCTX-M and intI1 on both
chromosome and plasmid suggested that horizontal transfer of ESBL-encoding genes would
be readily in this setting.
Key words: extended spectrum β-lactamase (ESBL), Escherichia coli, bla genes
Introduction
The healthcare-associated infections (HAIs) have increased severity in recent years,
which is due to increasing numbers of multidrug-resistant pathogens, especially among the
gram-negative bacteria. Inactivation of antibiotics by producing β-lactamases is the most
common mechanism of resistance. At present, extended-spectrum β-lactamases (ESBLs) was
the highest clinical impact due to they are able to hydrolyze and cause resistance to various
types of the newer β-lactam antibiotics, including third-generation cephalosporins such as,
cefotaxime, ceftriaxone, ceftazidime and monobactams (1). Infections due to ESBLproducing organisms are associated with a delay in initiation of appropriate antibacterial
therapy, which consequently prolongs hospital stays, increases hospital costs and high
mortality (2).
ESBL have been identified in various member of Enterobacteriaceae family as well as
non-fermentors such as Pseudomonas aeruginosa and Acinetobacter baumannii (3, 4). Since
2000 E. coli has emerged as an important organism responsible for producing ESBLs(2).
1
ESBL-producing E. coli is the most common cause of urinary tract infection (UTIs) in both
nosocomial and community acquired infections worldwide (5, 6). Furthermore, ESBLproducing E. coli have been involved in other sites of infections including bloodstream
infections, pneumonias, peritonitis, cholangitis, and other intra-abdominal infections (7, 8).
The prevalence of ESBL-producing E. coli from phenotypic detection was varied by
geographic region. The Study for Monitoring Antimicrobial Resistance Trends (SMART)
program found that 17.9% of all E. coli isolates in 2009-2010 from urinary tract specimens of
hospitalized patients in countries worldwide were extended-spectrum β-lactamases (ESBL)
producers. The highest ESBL rate was from the Asia/Pacific region (27.7%), followed by
Latin America (23.3%), Europe (18.8%), Middle East/Africa (16.2%), and North America
(7.4%) (5). In Thailand, data from National Antimicrobial Resistance Surveillance Thailand
(NARST) showed a trend towards increasing incidence rates of ESBL-producing E. coli from
2000 to 2005. Rates of ESBL-producing E. coli detected by cefotaxime screening were
ranging from 20.8% to 69.3% (9, 10). In 2008, Thailand reported the highest incidence rate
of ESBL-producing E. coli (53%) in the Asia–Pacific region (11).
Phenotypic methods of ESBL detection are not able to distinguish between the
specific genes responsible for ESBL production (SHV, TEM, and CTX-M types). Molecular
methods are widely used for confirmation and determination of ESBL genes (12). There are
many types or variants of ESBL genes which effect on enzyme hydrolytic activity. The most
common ESBL genes identified in the past were the TEM and SHV types. Currently, the
most important group is the CTX-M enzymes which have disseminated rapidly worldwide
(1). CTX-M enzymes are more active against cefotaxime and ceftriaxone than against
ceftazidime, and they are inhibited more by tazobactam than by clavulanic acid (4).
Although, point mutations at the active site of this types enzymes including CTX-M-1 and
CTX-M-9 groups have increased their ability to hydrolyze ceftazidime significantly (13).
Survival of a microorganism depends on its capacity to adapt to changing
environmental conditions. Antimicrobial agents commonly used in the hospital exert strong
selective pressures on bacterial populations, favoring organisms that are capable of resisting
them. Genetic variability occurs through a variety of mechanisms aside from mutation,
acquisition of drug resistant genes is common among gram negative bacteria (14) Most
resistance genes found in pathogens are acquired through horizontal gene transfer via mobile
genetic elements such as plasmids, insert sequence elements etc.(15). Many ESBL genes
have been often found on plasmids. Being plasmid mediated, they are easily transferred
among members of Enterobacteriaceae. Moreover, ESBL genes acquisition are also related
to others genetic elements including insertion sequences (ISs), transposons, class 1 integrons,
and sul1-type integrons containing the ISCR1 element. This diversity of mobile elements
enhances the spread of ESBLs (16).
Prevalence of ESBL-producing E. coli causing HAIs at Vajira Hospital showed
increasing trend recently in spite of infection control measures. Objectives of this study was
to determine prevalence and genetic determinants of multidrug resistance in ESBL-producing
E. coli isolated from clinical specimens submitted to the Microbiology Laboratory of Vajira
hospital. We were able to identify variety of bla genes as well as class 1 integron among
these clinical ESBL-producing E. coli.
Methodology
All clinical specimens submitted to the Microbiology Laboratory from August to
September in 2012 were identified by routinely conventional method and antimicrobial
susceptibility test by using 96 well Microscan® Neg Breakpoint Combo Panel type 34 in the
automated MicroScan system (WalkAway-40 SI, Siemens Healthcare Diagnostics, New
York). Antimicrobial susceptibility testing in this automated system is based on broth
2
microdilution method. ESBL producers were interpreted from MICs obtained for
cefotaxime, ceftazidime, ceftriaxone and aztreonam according to 2010 guidelines established
by Clinical and Laboratory Standards Institute (17). Additional patient information was
obtained from laboratory request form. Thirty isolates of ESBL-producing E. coli were
selected by types of specimen and antibiograms for study on molecular characteristics of
ESBL genes. This study was approved by the ethical committee of Faculty of Medicine
Vajira Hospital, Navamindradhiraj University.
Chromosome and plasmid DNA of ESBL-producing E. coli isolates were extracted by
using NucleoSpin® Tissue kit and NucleoSpin® Plasmid DNA purification kit by following
the manufacturer’s instruction. The bla genes and integron were detected by using PCR and
Multiplex PCR method, respectively. PCR reactions were performed in a volume of 20 µl.
Each reaction mixture contained 1X Thermopol® buffer (200 mM Tris-HCl pH 8.8, 100 mM
(NH4)2SO4, 100 mM KCl, 20 mM Mg2SO4, 1.0% Triton X-100; New England Biolabs,
USA), 1 unit of Taq DNA polymerase (New England Biolabs, USA), 200 µM each dNTP,
250 nM concentrations of each primer, approximately 200 ng of chromosome and 20 ng of
plasmid DNA template, and deionized water to a final volume of 20 µl. PCR amplification
were performed by using the following conditions; an initial denaturation at 95ºC for 2
minutes and followed by 35 cycles of denaturation at 95ºC for 15 seconds, annealing for 30
seconds, and extension at 68ºC for 1 minutes/1 Kb of PCR product, and final extension at
68ºC for 5 minutes.
Multiplex PCR for integron detection was performed in a volume of 50 µl. Each
reaction mixture contained 1X Thermopol® buffer, 1 unit of Taq DNA polymerase (New
England Biolabs, USA), 200 µM each dNTP, 250 nM concentrations of each primer,
approximately 200 ng of chromosome and 20 ng of plasmid DNA template, and deionized
water to a final volume of 50 µl. Multiplex PCR were performed by using the following
steps; an initial denaturation at 95ºC for 2 minutes and followed by 35 cycles of denaturation
at 94ºC for 1 minute, annealing at 59ºC for 1 minute, and extension at 68ºC for 1 minutes,
and final extension at 68ºC for 5 minutes. Table1 shows primer sets, annealing temperature
and expected PCR amplicon sizes used in this study. PCR products were analyzed by
electrophoresis in 1% agarose gel in 1X Tris-borated-EDTA and visualized by UV
transilluninator (BIS 303 PC, Jerusalem).
3
Table 1 Primer set used for PCR amplification of bla genes.
Primer name
Sequence (5'-3' direction)
Amplicon
size (bp)
Annealing
temperature
(ºC)
References
SHV-SE
SHV-AS
ATGCGTTATATTCGCCTGTG
TGCTTTGTTATTCGGGCCAA
747
56
(18)
TEM-164-F
TEM-165-R
TCGCCGCATACACTATTCTCAGAATGA
ACGCTCACCGGCTCCAGATTTAT
445
50
(19)
CTX-M-U1
CTX-M-U2
ATGTGCAGYACCAGTAARGTKATGGC
TGGGTRAARTARGTSACCAGAAYCAGCGG
593
58
(20)
VEB-FN
VEB-RN
GGCAAAAAATGCCAGAATAGG
CGTATTTGTTGCAGAGTCC
712
45
Diraphat P.
OXA-2-A
OXA-2-B
ATGGCAATCCGAATCTTCG
TTATCGCGCAGCGTCCG
828
50
(21)
OXA-10-F
OXA-10-B
TCTTTCGAGTACGGCATTAGC
CCAATGATGCCCTCACTTTCC
759
50
(22)
501
52
(23)
OXA-23-F
OXA-23-R
GATCGGATTGGAGAACCAGA
ATTTCTGACCGCATTTCCAT
OXA-40-F
OXA-40-R
GGTTAGTTGGCCCCCTTAAA
AGTTGAGCGAAAAGGGGATT
246
48
(23)
OXA-51-F
OXA-51-R
TAATGCTTTGATCGGCCTTG
TGGATTGCACTTCATCTTGG
353
47
(23)
IntF
IntR
CAGTGGACATAAGCCTGTTC
CCCGAGGCATAGACTGTA
160
59
(24)
Int2.F
Int2.R
CACGGATATGCGACAAAAAGGT
GTAGCAAACGAGTGACGAAATG
788
59
(25)
Int3.F
Int3.R
GCCTCCGGCAGCGACTTTCAG
ACGGATCTGCCAAACCTGACT
979
59
(25)
Results
There were 464 E. coli isolates from clinical specimens submitted to the Microbiology
Laboratory at the Faculty of Medicine Vajira Hospital, Navamindradhiraj University during
August to September, 2012. Two hundred and fifty two isolates were identified as ESBLproducing E. coli. Therefore, prevalence of ESBL-producing E. coli was 54.3%. Among
them, 122 isolates (48.4%) were isolated from urine, 47 isolates (18.7%) from sputum, 29
isolates (11.5%) from blood, 27 isolates (10.7%) from pus, 9 isolates (3.6%) from wound, 7
isolates (2.8%) from tissue, 7 isolates (2.8%) from abdominal fluid, 3 isolates (1.2%) from
cervix swab, and 1 isolate (0.4%) from catheter tip samples. One hundred and eighty nine
ESBL-producing E. coli isolates from clinical specimens of 137 patients were further
analyzed in this study. Other 63 isolates were lost and contaminated during the collection.
Over 60% of all patients used antibiotics drug during the time of specimen collection and 3rd
generation cephalosporin, such as ceftriaxone, was extensively use in this patient group.
The antimicrobial susceptibility of 189 ESBL-producing E. coli isolates was
summarized in Table 2. All of ESBL-producing isolates (100%) were resistant to ampicillin,
cefotaxime, ceftazidime, ceftriaxone, cefepime. Twelve isolates (6.3%) were resistant to
cefoxitin. In addition, most ESBL producers were also resistant to fluoroquinolones. There
were 150 isolates (79.4%) resistant to ciprofloxacin, 146 isolates (77.2%) resistant to
levofloxacin, and 149 isolates (78.8%) resistant to moxifloxacin. For aminoglycosides, 2
4
isolates (1.1%) were resistant to amikacin, 119 isolates (63.0%) were resistant to gentamicin.
As of penicillin combination group, 61.9% of all isolates were resistant to
ampicillin/sulbactam but 51.3% remained susceptible to amoxycillin/clavulanate. Most
isolates (91.5%) were susceptible to piperacillin/tazobactam. All isolates (100%) were
susceptible to ertapenem, imipenem, meropenem.
The results of bla and intI genes detection on chromosome and plasmid of the 30
ESBL-producing E. coli were shown in Table 3. There was 97% of ESBL producers carried
blaCTX-M, followed by blaTEM 47% on their chromosomes and plasmids. The blaVEB was
positive on chromosome 27% and 17% on plasmids. Other β-lactamase chromosomal
encoding genes of ESBL producers included blaOXA-10-like 13%, blaOXA-23-like 10%, blaOXA-51like and blaSHV 3% each. While plasmid-encoding genes were blaOXA-10-like and blaSHV 3%
each. The blaOXA-2-like and blaOXA-40-like were not detected in chromosome of all isolates. 77%
and 73% of the isolates were positive for intI1 on chromosome and plasmid, respectively.
None of them were positive for intI2 and intI3.
Table 2 Antimicrobial susceptibility of 189 isolates of ESBL-producing E. coli
Antimicrobial Drug
Sensitive
Intermediate
No (%)
No (%)
Ampicillin
0
0
Cefoxitin
Resistant
No (%)
189(100)
150 (79.4)
27(14.3)
12(6.3)
Cefotaxime
0
0
189(100)
Ceftazidime
0
0
189(100)
Ceftriaxone
0
0
189(100)
Cefepime
0
0
189(100)
Gentamicin
70(37.0)
0
119(63.0)
Amikacin
182(96.3)
5(2.6)
2(1.1)
Ciprofloxacin
36(19.0)
3(1.6)
161(80.5)
Levofloxacin
40(20)
3(1.6)
150(79.4)
Moxifloxacin
39(20.6)
1(0.5)
149(78.8)
Amoxicillin/Clavulanate
97(51.3)
65(34.4)
27(14.3)
Ampicillin/Sulbactam
35(18.5)
37(19.6)
117(61.9)
Piperacillin/Tazobactam
173(91.5)
11(5.8)
5(2.6)
Trimethoprim/Sulfamethoxazole
58(30.7)
0
131(69.3)
Ertapenem
189(100)
0
0
Imipenem
189(100)
0
0
Meropenem
189(100)
0
0
5
Table 3 Percentage of ESBL-producing E. coli with positive bla and intI gene
Gene
Chromosome
Plasmid
No(%)
No(%)
blaSHV
blaTEM
blaCTX-M
blaVEB
blaOXA-2-like
blaOXA-10-like
blaOXA-23-like
blaOXA-40-like
blaOXA-51-like
IntI1
IntI2
IntI3
1(3)
14(47)
29(97)
8(27)
0
4(13)
3(10)
0
1(3)
23(77)
0
0
1(3)
14(47)
29(97)
5(17)
0
1(3)
0
0
0
22(73)
0
0
Discussion and Conclusion
The prevalence of ESBL-producing E. coli was varied by geographic region.
However, the prevalence was high nearly in all countries across Asia including China (26).
The high prevalence of ESBL-producing E. coli corresponded with the high rates of 3rd
generation cephalosporins consumption (27). The prevalence of ESBL-producing E. coli in
this study was 54.3% of all identified E. coli isolates during the study period in 2012. The
prevalence was obviously higher than those of previous study 13.2% of HAIs in 2005 at two
university hospitals of Thailand (28). The increasing number could be due to the clinical
samples of the present study were come from the in-patients (75.9%) as well as out-patients
(24.1%). Interestingly, more than 60% of the patients infected with ESBL-producing E. coli
already took antibiotics, especially ceftriaxone, at the time of sample collection. All ESBLproducing E. coli in this study shows resistant to ceftriaxone (100%). Hence, the antibiogram
profile of the hospital ESBL-producing E. coli strain should be aware among the physicians
and pharmacists for the sake of effective treatment, cost and risk of emerging more MDR
organisms.
The blaCTX-M was predominant in clinical isolates of ESBL-producing E. coli which
similar to several studies in many countries worldwide such as USA (29), French (30), Brazil
(31), and Japan (32). CTX-M β- lactamses consists of a group of plasmid – mediated
enzymes that confer high resistance to cefotaxime but low level against ceftazidime (4).
CTX-M β- lactamses were the most prevalent among ESBL-producing E. coli since 2000 and
spread in both hospital and community settings as also observed in this study. Antimicrobial
susceptibility of most ESBLs producers showed a board resistance, including resistance to
quinolone and aminoglycosides. The blaCTX-M-15 gene is prevalent among ESBL-positive E.
coli strains that is frequently found together with aac(6')- Ib-cr, and aac(3)-II genes (33).
However all isolates in this study remained susceptible to carbapenem. Increased using of
this antibiotics group may create selective pressure and increase expression of already exist
ESBL genes (oxacillnases) which were found on both chromosome and plasmid. The results
indicated that ESBL-producing E. coli have potential to spread ESBL genes to both the same
and the next generations. Furthermore most isolates carried class 1 integron. Therefore,
these MDROs may acquire other resistant genes or exchange their resistant genes to other
bacteria and resulted in increasing prevalence of this MDR strains at the rate faster than
expected.
6
Acknowledgements
Thank staff of Microbiology Laboratory at the Faculty of Medicine Vajira Hospital,
Navamindradhiraj University for technical support in bacterial isolation. This study was
partially supported by Department of Microbiology, Faculty of Public Health, Mahidol
University.
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