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Paper title:
Multiplex PCR for TEM, OXA and SHV β-Lactamases of Escherichia coli and Klebsiella
spp from tertiary care hospital, Lahore
Huma Liaqat1, Shahida Hussain2, Farhan Rasheed3, Saba Riaz4
1. M. Phil Microbiology and Molecular Genetics
Email: [email protected]
2. M. Phil Biotechnology
Email: [email protected]
3. MD. M. Phil Microbiology
Email: [email protected]
4. Ph. D Microbiology and Molecular Genetics
Email: [email protected]
Disclaimers: All authors declared no conflict of interest. No funding from any agency.
Short running title: Multiplex PCR system and detection of β-lactamases
Number of Authors: 4
Number of Words: 2430
Number of Tables: 2
Number of Figures: 2
Number of References: 21
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1
Department of Microbiology and Molecular Genetics, University of the Punjab,
Lahore, Pakistan
2
Citi Lab and Research Center, Faisal Town Lahore, Pakistan
3
Allama Iqbal Medical College Lahore.
* Corresponding author: Saba Riaz, PhD.
Assistant Professor
Department of Microbiology and Molecular Genetics, University of the Punjab, Lahore, Pakistan
Citi Lab and Research Center, Faisal Town Lahore, Pakistan
Tel: 00923364208516
Email: [email protected]
3
Abstract
Background: The emergence of extended-spectrum-β-lactamase producing pathogens has
important clinical consequence in community and especially hospital settings.
Objective: In this study the distribution of OXA, TEM and SHV ESBLs producing Escherichia
coli and Klebsiella spp were investigated using phenotypic and molecular techniques.
Methods: This cross-sectional study was conducted in Jinnah Hospital, Lahore during June 2015
to November 2015. Antimicrobial Sensitivity Testing (AST), Double Disc Synergy Testing
(DDST), Combination Drug Synergy Testing (CDST) and Epsilometer Testing (E-test) were
used for the phenotypic detection. Multiplex PCR assays were used for the detection of OXA,
TEM and SHV genes in ESBL positive strains. Sequencing results were aligned and matched to
the reference sequences from NCBI BLAST.
Results: A total of 85 strains of Enterobacteriaceae were selected on the basis of AST
screening tests, out of which 45 strains were Klebsiella spp and 40 strains were Escherichia coli.
Among the phenotypic techniques used, CDST was proved to be the most sensitive as compared
to DDST and E-test. PCR results revealed that 15.5 % Klebsiella spp and 7.5 % E. coli were
positive for all OXA/TEM/SHV genes. ATCC no KX789530 and KX789531 were obtained for
two Klebsiella spp and ATCC KX 789532 were obtained for E. coli. Eight TEM and six OXA
amplicons were sequenced for further analysis.
Conclusion: We report a useful multiplex PCR composed of blaSHV, blaTEM and blaOXA genes
most frequently isolated from our hospitals. There is an urgent need to employ effective methods
for the detection of ESBL infections in our diagnostic laboratories.
Key words: Multiplex PCR, ESBLs, β-lactams
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INTRODUCTION:
Extended-spectrum-β-lactamase-producing-Enterobacteriaceae (ESBL-E) is multi-drugresistant Gram-negative bacteria. They have important clinical consequence in hospital settings
and especially among immunocompromised people in the community (1). In Enterobacteriaceae
the main reason behind the development of resistance for third generation cephalosporin is ESBL
enzymes of Ambler class A. The most significance ESBL genes are SHV-1, TEM-1 and TEM-2
which are generated due to mutations, and broaden the substrate specificity against broad
spectrum cephalosporin’s, commonly to ceftazidime, cefotaxime and ceftriaxone. These βlactamases, show evidence of a broad range of amino acid substitutions which leads to
enhancement of substrate specificity. The mutations influence the identification of substrates and
acyl–enzyme complex formation and hydrolysis (2). Presently, many clinical pathogens harbour
more than one β-lactam gene. Due to the high diversity of these enzymes, multiplex PCR based
detection methods have become a widely used tool for epidemiological surveys (3).
The tendency of ESBL-producing pathogens to be concurrently resistant to other groups of
antibiotics significantly limits the selection of antibiotics for treatment of infections. In Pakistan,
an increase in the number of ESBL associated infections has been observed. Antimicrobial
resistance is evolving as a global concern exacerbated by under reporting in some regions of the
world. Lack of regular surveillance programs at national or international levels, inadequate
infection control agencies, lack of facilities and inappropriate diagnostic approaches contribute to
the emergence of the antibiotic resistance in bacteria (4). Infections due to carbapenem-resistant
ESBLs strains are very common in Asian countries. Moreover, dissemination of MDR- strains or
the conjugative transmission of resistance-mediating plasmids are of alarming concern, and
therapeutic options should be restricted to control the increasing threat of lethal outcomes. This
demands the urgent call for surveillance of resistance and molecular characterization for Gramnegative ESBLs.
Objectives:
The purpose of this research was to evaluate the diagnostic accuracy of a multiplex PCR system
for the rapid detection of commonly encountered β-lactamase genes in clinical isolates of a
tertiary care hospital in Pakistan.
METHODS:
This cross sectional study was conducted at the department of Microbiology and Molecular
Genetics, University of the Punjab, Lahore in collaboration with the Pathology department
Allama Iqbal Medical College/ Jinnah Hospital, Lahore. A total of 500, non-duplicated clinical
isolates from various sources were collected by random sampling technique form June 2015November 2015 for culture and sensitivity testing. Repeated and Non ESBL producing isolates
were excluded.
Ethical Approval:
This study was approved by the ethical review board of the Citilab and Research, Lahore.
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All specimens were subjected to culturing according to the standard CLSI protocol (5).
The bacterial pathogens were identified on the basis of Gram staining, colony characteristics,
motility, biochemical tests and biochemical profiles. Analytical profile index (API) was used
where necessary. Isolated organisms were subjected to antibiotic susceptibility testing (AST).
The standard antibiotic discs (HiMEDIA, India) were used.
Quality controlling:
ESBL producing E. coli ATCC 25922 and non ESBL producing K. pneumoniae ATCC 700603
strains were used as a positive control and negative control respectively for quality controlling
purpose (6). For multiplex PCR, E. coli strains ATCC (GU594301, GU594304 and GU59302)
were used as positive controls.
ESBL screening and confirmation:
Screening of ESBLs was initially done on the basis of Antibiotic Susceptibility Tests
(AST) and Double Disk Synergism Tests (DDST). For confirmation of EBSLs, Combination
Disk Tests (CDT) and Epsilometer Tests (E-test) was performed according to Clinical and
Laboratory Standards Institute (CLSI) guidelines (7). In DDST, augmentin (AMC 20/10μg) and
discs of cefpodoxime (30μg), ceftazidime (CAZ 30μg) and cefotaxime (CTX 30μg) were
applied. For this test, suitable distances were required to detect the synergy (8) . All the strains
which were resistant to CAZ (30μg) and CTX (30μg) were subjected to ESBL confirmatory
testing by CDT. In CDT discs with only CAZ (30μg) and CTX (30μg) and in combination with
Clavulanic acid (CTC (40μg) and CZC (40μg) were used. A zone of inhibition ≥ 5mm with the
addition of Clavulanic acid was reported as an ESBL positive strain (7). For the E-test
(HIMEDIA, India), two strips, one having CTX on one side and cefotaxime /clavulanic acid on
the other side and another strip having CAZ on one side and ceftazidime /clavulanic acid on the
other side of the Muller Hinton (MH) plate. Interpretation of results was performed with the help
of the user manual (9).
Extractions of DNA and Plasmid Isolation:
The DNA used for Multiplex PCR was extracted by the heat lysis method (10). Briefly, a 24
hour old bacterial colony was mixed in the 50 µl low TE buffer solution and heated at 1000C for
10 minutes. This step was followed by a centrifugation step at maximum speed for 5 minutes.
The supernatant was separated and stored as template DNA for multiplex PCR.
Multiplex PCR:
In Multiplex PCR 2 µl DNA was used in 25 µl PCR master mixture containing 10x PCR buffer,
1.5 Mm MgCl2, 200 µM of each deoxynucleotide triphosphate (dNTPs), 20 pmol of three primer
sets and 2.5 U of Taq polymerase (Thermo scientific, Germany) (11) . PCR amplification
conditions were: Initial step of denaturation at 95°C for 5 min followed by 35 cycles of
denaturation at 95o C for 1 min then annealing at 56o C for 1:30 min, extension at 72 C for 1 min
and the final extension was done at 72o C for 10 min.
Sequencing and phylogenetic analysis:
Selected strains were ribotyped and amplicon sequences for TEM and OXA were obtained.
Phylogenetic analysis of sequenced strains was done by using MEGA 6.0 software. We used the
sequence service of Advance Bioscience International, Pakistan.
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Statistical analysis:
The descriptive and statistical analysis of all collected data was done after putting into Microsoft
Office Excel. The frequency, mean values and percentages were determined and presented in the
form of tables and bar charts.
RESULTS:
Out of 500 clinical samples, only 240 were identified as Enterobacteriaceae. We
selected only E. coli and Klebsiella spp isolates for ESBL screening. Finally 85 bacteria strains
were identified as ESBL producers based on phenotypic test. On the basis of biochemical tests
and API 20 E- strip tests 52.9 % of isolates were identified as Klebsiella spp and 47.1 % were
identified as Escherichia coli. These 85 isolates were obtained from urine (n=26), pus (n=18),
sputum (n=12), wound swab (n=12), tracheal secretion (n=7), blood (n=5), HVS (n=2), Foley’s
tip (n=2) and bile secretions (n=1) respectively. Sampling covered all the age groups from 1-80
years. According to gender distribution of the study population, males were more prone to
ESBLs positivity with 55.2 % (47/85) and females showed a 44 % (38/85) ESBL positivity rate.
Our results showed that infectivity rate was more confined to >20-60 years of age.
Resistance trends of different antibiotics were observed as follows: Amoxicillin (100
%) > Aztreonam(100 %) > Cefotaxime (100 %) > Ceftazidime(100 %) > Cefuroxime(100%) >
Cefepime(100 %) > Norfloxacin (100 %) > Levofloxacin(98 %) > Tazocin (97.8 %) >
Gentamycin (90.5%) > Ciprofloxacin (89.4 %) > Amikacin (82 %) > Sulzone(77.6 %) >
Tobramycin (75%) > and Meropenem (28.2 %). Generally E. coli shows higher resistance than
Klebsiella spp. Here, same trends were observed (Table 1).
Table 1: Antimicrobial susceptibility trends towards fifteen drugs
Sr.
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Antimicrobial Agent
Amoxicillin
Aztreonam
Cefotaxime
Ceftazidime
Cefuroxime
Cefepime
Meropenem
Gentamycin
Tobramycin
Amikacin
Ciproxin
Levofaxacin
Norfloxacin
Tazocin
Sulzone
Abb. and
Conc.
(μg/ml)
AMC(30/10)
ATM(30)
CTX(30)
CAZ(30)
CRO(30)
CXM(30)
MEM(10)
CN(10)
TOB(30)
AK(30)
CIP(5)
LEV(5)
NOR(10)
TAZ(100/10)
SUL(105)
Profile of Antibiotic Resistance (AST)(%)
E. coli (n=40)
Klebsiella spp (n=45)
Resistance
Sensitive
Resistance
Sensitive
100
0
100
0
100
0
100
0
100
0
100
0
100
0
100
0
100
0
100
0
100
0
100
0
20
80
37.78
62.22
87.5
12.5
95.0
4.4
60
40
91.2
8.8
85
15
82.3
17.7
92.5
7.5
86.7
13.33
98
2.5
100
0
100
0
100
0
75
2.5
74.8
2.2
77.5
22.5
77.8
22.2
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Selection of β-lactamase producing strains was confirmed by using three phenotypic
techniques. In comparison among phenotypic tests, DDST showed 21.5 % (18/85), CDST
showed (85/85) 100 % and E-test showed 43.5 % efficiency (Figure 1).
percentage efficiency
Determined
100
80
60
40
20
0
Non-determined
100
78.9
56.4
43.5
21.1
0
DDST
CDST
E-Test
Figure 1: Comparative Efficiency of applied ESBLs phenotypic detection techniques
After screening, all ESBL positive strains were processed for the detection of OXA, TEM and
SHV gene by PCR. Singleplex PCR results showed that OXA genes were predominant with a
frequency of 48.23 % followed by TEM at 45.8 %. The SHV genes were found in only 21.1 %
of strains (Figure 2).
Percentage Efficiency
Determined
Non Determined
100
78.9
80
60
48.2351.77
45.8
54.2
40
21.1
20
0
OXA
TEM
SHV
β-Lactamases
Figure 2: Detection of OXA/TEM/SHV Genes in ESBLs positive strains by singleplex PCR
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Multiplex PCR showed that OXA/TEM/SHV genes were present in 15.5% (7/45) and 7.5%
(3/40) of Klebsiella spp and E. coli respectively. 6 (13.3%) Klebsiella spp and only 1 E. coli (2.5
%) were positive for TEM/SHV combination. 4 (8.8 %) Klebsiella spp and 9 (22.5%) E. coli
were positive for OXA/TEM. Neither Klebsiella spp nor E. coli was positive for only the SHV
gene. Among 85 ESBL strains detected by phenotypic methods only 27 strains were negative
(TEM, SHV and TEM) by multiplex PCR (Table 2). This might be due to presence of ESBL
genes other OXA, TEM and SHV. ATCC numbers of two Klebsiella sp were KX789530 and
KX789531 and ATCC KX 789532 for E. coli.
Table 2: Multiplex PCR for TEM, SHV and OXA in E. coli and Klebsiella spp
Strain (n=85)
E. coli (40)
Klebsiella Spp (45)
TEM
7.5
13.3
OXA
15
28.8
SHV
-
TEM/OXA
22.5
8.8
TEM/SHV
2.5
13.3
SHV/OXA
2.2
TEM/SHV/OXA
7.5
15.55
Others
19
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DISCUSSION:
Antimicrobial resistance is a global problem that has gained the attention of scientists
worldwide. It affects countries all over the world but is most prominent in developing countries.
The control and prevention of this antimicrobial resistance is severely compromised in
underdeveloped countries. Clinical microbiologists strongly believe that a multidrug resistant
Gram-negative bacterium is a danger to public health (12). This research was an attempt to
understand the prevalence of ESBL producing Escherichia coli and Klebsiella spp with a view to
introducing the multiplex PCR in our health setting. The study was conducted at the Department
of Microbiology and Molecular Genetics (MMG) University of the Punjab from June 2015 to
November 2015.
Out of 85 strains, 52.9 % of the selected strains were Klebsiella spp and 47 % were E.
coli. Generally, 40 % isolates were ESBLs positive, these results are comparable to work done in
Tanzania where 45% ESBLs producers have been reported (13). As far as the studies from other
regions of the world are concerned, similar results were reported in previous studies (14, 15).
ESBL infectivity rate varied in accordance to the gender and age. Here, 55.2% patients were
male and 44.7 % were females. This rate is quite similar to the rate reported by Afirdi et al.,
2011(16). ESBL infections were significantly higher in the mean age group of 41-60 years.
Similar high rates of infection have been reported at the mean age of 60 years (17). According to
many reports males have significantly higher rates of nosocomial infection compared to females.
Community acquired infections are more prevalent in females(17).
We used AST for screening and three phenotypic techniques were used for the ESBLs
detection. CDST was found to be more sensitive than DDST. DDST determined only 21.1%
strains as positive ESBLs while CDT determined 100% strains as positive ESBLs. A study
reported the same combination disc test results as obtained in our result (99.5 %)(18). In India
similar results were observed, where 90 % sensitivity for the detection of ESBLs by combination
disc test were reported(19). E-test revealed that 43.5 % strains were s ESBLs producers while
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56.4 % remained non determined by this technique. Mohanty et al., (2009) also reported similar
findings i.e. 61 % positive strains for ESBLs producers by E-test technique (20).
ESBL producers showed high resistance to all cephalosproins, but results for other β-lactams
were less straight forward. This phenomenon is a hindrance in the diagnosis of ESBL in clinical
laboratories. ESBLs mostly failed to reach detectable levels by the routinely used phenotypic
method of DDST which could result in failure of treatment and sub optimal management of
infected patients. The lack of correlation between ESBL production and DDST results is clear
from the current study. Such discrepancies between susceptibility data and phenotypic test
results have increased the demand for more sensitive methods of ESBL detection for
implementation into routine susceptibility testing procedures.
For the detection of β-lactumases, we selected 3 sets of primers for TEM, OXA and SHV genes.
Amplification was carried out on DNA of Klebsiella pneumoniae and E. coli isolates by
multiplex PCR. With multiplex PCR, 65% ESBL positive strains of Klebsiella pneumonia and E.
coli with OXA, TEM and SHV primers were detected. A study conducted by Sharma et al
reported that PCR is the most rapid and reliable method of ESBL detection (21).
Early detection of ESBLs is a major concern especially for life threatening infections.
Combination disc along with PCR is a good choice for the detection of ESBLs. Good hygienic
practices can reduce the incidence of ESBL infection. Antibiotic treatment regimens should be
followed properly. In Pakistan there should be a proper diagnostic laboratory for ESBLs.
Conclusion:
In conclusion, TEM/SHV/OXA type ESBLs are common types of ESBLs which are now
commonly isolated from health care settings. The strains of E. coli and Klebsiella spp studied in
this research have high resistance towards cephalosporins. Carbapenems are a good choice for
the treatment of ESBLs. In Pakistan there are no efficient diagnostic tools for the detection of
TEM, SHV and OXA ESBLs and such genes at laboratory level. This is one of the major reasons
why ESBLs infections are increasing so rapidly. Multiplex PCR, incorporating the primers for
commonly prevalent ESBLs genes may be a valuable clinical and research tool for the
characterization of ESBLs. Therefore it is strongly recommended that we have ESBLs diagnostic
reference labs with more efficient and advance singleplex as well as multiplex PCR techniques.
Limitations:
The other classes of β-lactams were not determined in this present study due to time constrains
but they are in progress.
Recommendations
It is necessary to do more research to evaluate the existing therapeutic agents and recognize new
ones; this will help the development of more appropriate strategies to control the emergence of
these organisms in hospital and community.
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Acknowledgement:
We acknowledged the laboratory personnel of the Microbiology section of Allama Iqbal Medical
College/Jinnah Hospital, Lahore in the collection of bacterial strains. The whole research work
was fully funded by the University of the Punjab, Lahore. Pakistan.
REFERENCES:
1.
O'Connell N, Keating D, Kavanagh J, Schaffer K. Detection and characterization of
extended-spectrum beta-lactamase-producing Enterobacteriaceae in high-risk patients in an Irish
tertiary care hospital. Journal of Hospital Infection. 2015;90(2):102-7. doi:
10.1016/j.jhin.2015.01.018.
2.
Pimenta A, Fernandes R, Moreira I. Evolution of drug resistance: insight on TEM βlactamases structure and activity and β-lactam antibiotics. Mini reviews in medicinal chemistry.
2014;14(2):111-22.
3.
Dallenne C, Da Costa A, Decré D, Favier C, Arlet G. Development of a set of multiplex
PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae.
Journal of Antimicrobial Chemotherapy. 2010: doi: 10.1093/jac/dkp498
4.
Vernet G, Mary C, Altmann DM, Doumbo O, Morpeth S, Bhutta ZA, et al. Surveillance
for antimicrobial drug resistance in under-resourced countries. Emerg Infect Dis.
2014;20(3):434-41. doi: 10.3201/EID2003.121157.
5.
Standard A, Edition E. CLSI Document M02-A11. Clinical and Laboratory Standards
Institute, Wayne, PA, USA. 2012.
6.
Alfaresi M, Elkoush A. Real-time polymerase chain reaction for rapid detection of genes
encoding SHV extended-spectrum β-lactamases. Indian journal of medical microbiology.
2010;28(4):332. doi: 10.4103/0255-0857.71827.
7.
Wayne P. Performance standards for antimicrobial susceptibility testing. Ninth
informational supplement NCCLS document M100-S9 National Committee for Clinical
Laboratory Standards. 2008:120-6.
8.
Oğünç D, Ongüt G, Ozen NS, Baysan BO, Günseren F, Dağlar D, et al. [Shall we report
the carbapenem resistance in Pseudomonas aeruginosa and Acinetobacter baumannii strains
detected by BD Phoenix system?]. Mikrobiyoloji bulteni. 2010;44(2):197-202.
9.
Edition AS-N. CLSI document M07-A9. Clinical and Laboratory Standards Institute.
2012;950.
10.
Shahzadi I, Ahmed R, Hassan A, Shah M. Optimization of DNA extraction from seeds
and fresh leaf tissues of wild marigold (Tagetes minuta) for polymerase chain reaction analysis.
Genetics and Molecular Research. 2010;9(1):386-93. doi: 10.4238/vol9-1gmr747.
11.
Bhat MA, Sageerabanoo S, Kowsalya R, Sarkar G. The occurrence of CTX-M3 type
extended spectrum beta lactamases among Escherichia coli causing urinary tract infections in a
tertiary care hospital in puducherry. Journal of Clinical and Diagnostic Research.
2012;6(7):1203-6.
12.
Carlet J, Jarlier V, Harbarth S, Voss A, Goossens H, Pittet D. Ready for a world without
antibiotics? The pensières antibiotic resistance call to action. Antimicrobial resistance and
infection control. 2012;1(1):1.
13.
Moyo SJ, Aboud S, Kasubi M, Lyamuya EF, Maselle SY. Antimicrobial resistance
among producers and non-producers of extended spectrum beta-lactamases in urinary isolates at
a tertiary Hospital in Tanzania. BMC research notes. 2010;3(1):1.
11
14.
Bindayna K, Khanfar HS, Senok AC, Botta GA. Predominance of CTX-M genotype
among extended spectrum beta lactamase isolates in a tertiary hospital in Saudi Arabia. Saudi
medical journal. 2010;31(8):859-63.
15.
Maina D, Revathi G, Kariuki S, Ozwara H. Genotypes and cephalosporin susceptibility in
extended-spectrum beta-lactamase producing enterobacteriaceae in the community. The Journal
of Infection in Developing Countries. 2011;6(06):470-7.
16.
Afridi FI, Farooqi BJ, Hussain A. Frequency of extended spectrum beta lactamase
producing enterobacteriaceae among urinary pathogen isolates. J Coll Physicians Surg Pak.
2011;21(12):741-4.
17.
Ben-Ami R, Rodríguez-Baño J, Arslan H, Pitout JD, Quentin C, Calbo ES, et al. A
multinational survey of risk factors for infection with extended-spectrum β-lactamase-producing
Enterobacteriaceae in nonhospitalized patients. Clinical Infectious Diseases. 2009;49(5):682-90.
doi: 10.1086/604713.
18.
Ejaz H. Detection of extended-spectrum β-lactamases in Klebsiella pneumoniae:
Comparison of phenotypic characterization methods. Pakistan journal of medical sciences.
2013;29(3):768.
19.
Dalela G. Prevalence of extended spectrum beta lactamase (ESBL) producers among
gram negative bacilli from various clinical isolates in a tertiary care hospital at Jhalawar,
Rajasthan, India. J Clin Diagn Res. 2012;6(2):182-7.
20.
Mohanty S, Gaind R, Ranjan R, Deb M. Use of the cefepime-clavulanate ESBL Etest for
detection of extended-spectrum beta-lactamases in AmpC co-producing bacteria. The Journal of
Infection in Developing Countries. 2009;4(01):024-9.
21.
Sharma J, Sharma M, Ray P. Detection of TEM & SHV genes in Escherichia coli &
Klebsiella pneumoniae isolates in a tertiary care hospital from India. 2010.