Vol. 24 No. 3
Article 117
Phenotypes and genotypes of ESBL-producing Klebsiella pneumoniae:- Tribuddharat C,Original
et al.
A Correlation between Phenotypes and Genotypes of
Extended-Spectrum Beta-Lactamase (ESBL)-Producing
Klebsiella pneumoniae in a University Hospital,
Thailand
Chanwit Tribuddharat, M.D., Ph.D.,
Somporn Srifuengfung, Ph.D.,
Wararat Chiangjong, M.Sc.
ABSTRACT
There are many extended-spectrum beta-lactamase (ESBL)-producing isolates of Klebsiella
pneumoniae in Thailand. In this study, we attempted to determine whether there was a correlation between
ESBL phenotypes and genotypes of ESBL-producing K. pneumoniae from 30 patients hospitalized at Siriraj
Hospital, Thailand. The ESBL phenotypes and genotypes were detected by using an antimicrobial susceptibility
test and a polymerase chain reaction, respectively. We found that the phenotypic resistance to cefotaxime,
ceftazidime, and ceftriaxone of ESBL-producing isolates may provide information concerning the correlation
with the presence of blaSHV, blaCTX-M, or blaVEB-1-like genes (p < 0.05), but not the blaTEM gene (p > 0.05). A
good correlation between the phenotypes and genotypes of ESBLs may provide the guidelines for selecting
appropriate antimicrobial agents for empirical treatment before obtaining the susceptibility results. (J Infect
Dis Antimicrob Agents 2007;24:117-23.)
INTRODUCTION
more than 157 TEM, 101 SHV, 65 CTX-M, and 5 VEB
Extended-spectrum beta-lactamases (ESBLs)
variants.6 ESBLs are most prevalent in Klebsiella
are the enzymes that hydrolyze a wide variety of beta-
pneumoniae. 7 They are frequently isolated from
lactam antibiotics including oxyimino-cephalosporins
clinical specimens from patients with nosocomial
and monobactams but not cephamycins, and are usually
septicemia, pneumonia, or urinary tract infection.8-10
inhibited by clavulanic acid. 1-4 These ESBLs are
At Siriraj Hospital which is the largest university
derivatives, predominantly, of class A (e.g., TEM, SHV,
hospital in Thailand, the number of patients who were
CTX-M, and VEB families).5 At present, there are
infected with ESBL-producing K. pneumoniae
Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.
Received for publication: October 29, 2007.
Reprint request: Somporn Srifuengfung, Ph.D., Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University,
Bangkok 10700, Thailand.
E-mail: [email protected]
Keywords: Klebsiella pneumoniae, Extended-spectrum beta-lactamase (ESBL), phenotype, genotype
117
J INFECT DIS ANTIMICROB AGENTS
118
Sep.-Dec. 2007
increased every year. A correlation between the
≤ 22 mm for ceftazidime, and ≤ 25 mm for ceftriaxone
phenotypes and genotypes of ESBLs may be useful in
were suspected of producing ESBL enzyme. The
providing the guidelines for clinicians in the selection
suspected isolates were further determined by using
of appropriate antimicrobial agents for empirical
the combination disk method of cefotaxime/clavulanate
treatment before obtaining the susceptibility results. The
and ceftazidime/clavulanate (BD BBL TM Sensi-
purpose of this study was to examine the correlation
DiskTM, MD, USA). A positive result was indicated
between the phenotypes and genotypes of ESBL-
by an inhibition zone size difference of ≥ 5 mm
producing K. pneumoniae isolates from patients
between the combination disk and the corresponding
hospitalized at Siriraj Hospital. The ESBL phenotypes
standard antimicrobial disk. The minimal inhibitory
were detected by an antimicrobial susceptibility test,
concentrations (MICs) of cefotaxime, ceftazidime,
and the ESBL genotypes were detected by using a
ceftriaxone, cefotaxime/clavulanate, and ceftazidime/
polymerase chain reaction (PCR)-based amplification
clavulanate were determined by the broth microdilution
of blaSHV, blaTEM, blaCTX-M, and blaVEB-1-like genes.
method in all 100 K. pneumoniae isolates. For quality
control, the positive control ESBL-producing K.
MATERIALS AND METHODS
pneumoniae ATCC 700603 and the negative control
Clinical isolates
E. coli ATCC 25922 were used.
One hundred isolates of ESBL-producing K.
pneumoniae from clinical specimens were studied.
PCR amplification
They were obtained from different patients hospitalized
The first 30 isolates of ESBL-producing K.
at Siriraj Hospital during the two periods from January
pneumoniae were extracted for DNA by a Puregene
to February 2004 and from July to August 2005. The
DNA Purification kit (Gentra Systems, USA). The
clinical specimens included the blood (11 specimens),
DNA primers13,14 were used to amplify bla gene (Table
the pus (16 specimens), the sputum from patients with
1). The reaction mixture contained 10 mM Tris-HCl
respiratory tract infections (20 specimens), and the
(pH 8.3), 50 mM KCl, 2 mM MgCl2, 0.5 µM each
urine from patients with urinary tract infections (53
primer, 250 µM each deoxynucleoside triphosphate, 1
specimens). Duplicate isolates from a single patient
U of i-TaqTM DNA polymerase (Intron Biotechnology,
were identified, and only one was kept.
K.
Korea), and 100-500 ng of total genomic DNA in a
pneumoniae was identified by standard micro-
final volume of 25 µl for blaSHV, blaCTX-M, and blaVEB-1
biological techniques.11
genes. For bla TEM gene, the reaction mixture
contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5
mM MgCl 2, 0.5 µM each primer, 400 µM each
Detection of ESBL production
The ESBL production was initially screened by
deoxynucleoside triphosphate, 1 U of DyNazymeTM
using the disk diffusion method of cefotaxime,
II DNA polymerase (Finzyme, Finland), and 100-500
ceftazidime, and ceftriaxone from Becton, Dickinson
ng of total genomic DNA in a final volume of 25 µl.
Sensi-Disk , MD, USA),
Amplification was performed in a PCR Sprint
according to the Clinical and Laboratory Standards
ThermoHybaid (Hausen Berstein, Thailand) for 5 min
The isolates which
at 95°C, followed by 30 consecutive cycles of 1 min
showed an inhibition zone of ≤ 27 mm for cefotaxime,
at 95°C, 1 min at 50°C for bla TEM gene, 55°C for
and Company (BD BBL
TM
Institute recommendation.
TM
12
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Phenotypes and genotypes of ESBL-producing Klebsiella pneumoniae:- Tribuddharat C, et al.
119
Table 1. Primers for extended-spectrum beta-lactamase (ESBL) genotypic detection.
Primer
TEM FTEM R
Tm (°°C)
Nucleotide sequences
5′-ATGAGTATTCAACATTTCCG-3′
54
5′-CTGACAGTTACCAATGCTTA-3′
56
5′-GGTTATGCGTTATATTCGCC-3′
58
5′-TTAGCGTTGCCAGTGCTC-3′
56
5′-CGACTTCCATTTCCCGATGC-3′
62
5′-GGACTCTGCAACAAATACGC-3′
60
CTX-M MA1
5′-S*CSATGTGCAGY**ACCAGTAA-3′
47.6
CTX-M MA2
5′-CCGCR***ATATGRTTGGTGGTG-3′
59.1
SHV FSHV R
VEB-1 FVEB-1 R
Product length
Reference
867 bp
13
865 bp
13
643 bp
This study
544 bp
14
*S = G or C, **Y = C or T, ***R = A or G.
Genotypic characteristics
bla SHV and bla VEB-1-like genes, 58°C for bla CTX-M
gene, and 1 min at 72°C, with a single final
The first 30 ESBL-producing K. pneumoniae
extension step of 10 min at 72°C. PCR products
isolates were detected by using a PCR of blaSHV, blaTEM,
were separated by electrophoresis in a 1.5 percent
bla CTX-M , and bla VEB-1-like genes. The results for
agarose gel and stained with ethidium bromide.
detection of blaSHV, b1aTEM, blaCTX-M, and blaVEB-1-like
gene were 30 (100%), 21 (70%), 15 (50%), and 11
(36.7%), respectively.
Statistical analysis
The correlation between antimicrobial susceptibility and genotypic characteristics was examined by
Correlation between ESBL phenotypes and
using the SPSS package. A P-value of < 0.05 was
genotypes
The first 30 ESBL-producing K. pneumoniae
considered significant.
isolates which were detected by antimicrobial
susceptibility (the MIC levels of cefotaxime, ceftazidime,
RESULTS
and ceftriaxone) and genotypic characteristics were
Antimicrobial susceptibility
Of 100 isolates of ESBL-producing K. pneumo-
studied for the correlation between them. A correlation
niae tested, on initial screening test , a positive
between the phenotypes and genotypes was observed
result was noted with 100 percent for cefotaxime
with the presence of blaSHV, blaCTX-M, or blaVEB-1-like
and ceftriaxone disks, and with 88 percent for
gene, but not the blaTEM gene (Table 2). The presence
ceftazidime disk. Of these 100 isolates, 100 percent
of blaCTX-M gene correlated well with the phenotypic
were positive by the confirmatory method (the
resistance to cefotaxime (p = 0.002) and ceftriaxone
combination disk) and the MIC methods. All
(p = 0.000), whereas it did not correlate with the
suspected ESBL-producing isolates showed the
phenotypic resistance to ceftazidime (p = 0.285). The
MICs of cefotaxime of ≥ 2 µg/ml, and of ceftazidime
presence of blaVEB-1 like gene correlated well with the
12
and ceftriaxone of ≥ 1 µg/ml.
119
phenotypic resistance to ceftazidime (p = 0.014),
J INFECT DIS ANTIMICROB AGENTS
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Sep.-Dec. 2007
whereas it did not correlate with the resistance to
groups of DNA sequences were found. They were
cefotaxime (p = 0.164) and ceftriaxone (p = 0.241).
SHV (pI 7.6), LEN (pI 7.1), and OKP (for other K.
The presence of blaTEM gene did not correlate with the
pneumoniae beta-lactamases) families. According
resistance to cefotaxime, ceftazidime, and ceftriaxone
to earlier studies, a gene related to blaSHV-1 gene in
(p > 0.05). In this study, there were only two isolates
most K. pneumoniae was found to be chromosomal
containing only blaSHV gene. The first isolate had MIC
and not plasmid borne.16,17 Because all first 30 ESBL-
of 16, 32, 256 µg/ml for ceftriaxone, cefotaxime, and
producing K. pneumoniae isolates contained blaSHV
ceftazidime, respectively. The second isolate had MIC
genes, the P-value of the correlation between the
of 32, 32, 128 µg/ml for ceftriaxone, cefotaxime, and
presence of blaSHV gene and the phenotypic resistance
ceftazidime, respectively. The presence of blaSHV gene
could not be calculated. However, 2 in 30 isolates
in these two isolates seemed to correlate with the
had only the bla SHV gene, so we found that the
resistance to ceftazidime better than to cefotaxime and
presence of bla SHV gene correlated well with the
ceftriaxone.
phenotypic resistance to ceftazidime better than to
cefotaxime and ceftriaxone. For the correlation
DISCUSSION
between phenotypic and genotypic characteristics, it
We used the first 30 isolates of ESBL-producing
was concluded that the presence of blaTEM gene was
K. pneumoniae to study the correlation between the
not significantly associated with the ESBL phenotype
phenotypes and genotypes, because 30 samples were
(Table 2). Our data was in accordance with the
usually the lowest number samples which could give a
previous study.18 For the blaCTX-M gene, it was reported
statistically significant value. Furthermore, the overall
that CTX-M-3 producers exhibited higher MICs to
properties of 30 isolates were not much different from
cefotaxime than cefta-zidime.19 In this study, the
the 100 isolates. The MIC ranges and MIC50 of these
correlations between the presence of blaCTX-M gene
30 isolates were almost identical to those from 100
and phenotypic resistance to cefotaxime and
isolates, with the MIC90 of the same values between
ceftriaxone were significant, whereas the resistance
both groups. Even though the genotypic study was
to ceftazidime was not significantly associated with
limited with the small number of isolates tested in this
the presence of blaCTX-M gene (Table 2). There may
study, the results may be useful for drawing a general
be some specific point mutations in the catalytic sites
conclusion between the broad characteristics of
in these ESBLs, which could confer an extraordinary
certain ESBL families and their preferred substrates.
substrate preference, such as the P167S mutation at
To analyze all 100 isolates for their actual genotypes of
the omega loop of CTX-M-19 or P167Q mutation in
each ESBL gene will have to deal with extensive
CTX-M-54 that make these enzymes hydrolyze
cloning and sequencing of the whole open reading
ceftazidime better than other variants of CTX-M
frames of each variant from each individual isolate,
ESBLs.20-22 For the blaVEB-1-like gene, it was reported
such study will have to deal with a very high cost, and
that MICs of ceftazidime for the blaVEB-1 gene carrier
the general findings and conclusion may have not
were higher than for cefotaxime and ceftriaxone.23-24
changed much from our results.
In this study, the correlation between blaVEB-1-like genes
Different chromosomal beta-lactamase variants
with the phenotypic resistance to of ceftazidime was
from K. pneumoniae have been reported.15 Three
significant, whereas the correlations with the
Vol. 24 No. 3
Phenotypes and genotypes of ESBL-producing Klebsiella pneumoniae:- Tribuddharat C, et al.
121
Table 2. P-values of the correlation between the presence of bla genes and the phenotypic
resistance to cefotaxime, ceftazidime, and ceftriaxone (determined by the minimal
inhibitory concentration) of the first 30 isolates of ESBL-producing Klebsiella
pneumoniae.
P-values (significant at 0.05 levels) of the correlation
bla gene
Cefotaxime
Ceftazidime
Ceftriaxone
TEM
0.089
0.084
0.093
CTX-M
0.002
0.285
0.000
VEB-1-like
0.164
0.014
0.241
resistance to cefotaxime and ceftriaxone were not
2.
significant.
Coudron PE, Moland ES, Sanders CC. Occurrence
and detection of extended-spectrum beta-lactamases
In conclusion, we found that the phenotypic
in members of the family Enterobacteriaceae at a
resistance of cefotaxime, ceftazidime, and cef-
veterans medical center: seek and you may find. J
triaxone of ESBL-producing K. pneumoniae isolates
Clin Microbiol 1997;35:2593-7.
may correlate well with the presence of bla SHV,
3.
Shahid M, Malik A, Agrawal M, Singhal S. Phenotypic
bla CTX-M, bla VEB-1-like genes, but not the blaTEM gene.
detection of extended-spectrum and AmpC beta-
Even though we did not study the enzyme kinetics
lactamases by a new spot-inoculation method and
of beta-lactamases from each bla gene, the
modified three-dimensional extract test: comparison
correlation in these thirty ESBL-producing K.
with the conventional three-dimensional extract test.
pneumoniae isolates from clinical specimens
J Antimicrob Chemother 2004;54:684-7.
concerning phenotypes and genotypes were in
4.
agreement with previous studies. 19-25
Weinbren MJ, Borthwick MA. Rapid detection of
extended-spectrum beta-lactamase (ESBL)-producing
organisms in blood culture. J Antimicrob Chemother
ACKNOWLEDGEMENT
2005;55:131-2.
This work was supported by a grant from the
5.
Siriraj Graduate Thesis Scholarship 2005. The
Poole K. Resistance to beta-lactam antibiotics. Cell
Mol Life Sci 2004;61:2200-23.
expertise of the technical staff at the Department of
6.
Lahey Clinic. Amino acid sequences for TEM, SHV
Microbiology, Faculty of Medicine Siriraj Hospital,
and OXA extended-spectrum and inhibitor resistant
Mahidol University is gratefully acknowledged.
beta-lactamases. 2007 [cited 2007 Mar 29]. Available
from: http://www.lacey.org/studies.
7.
References
1.
1999;115:3S-8S.
Cagatay AA, Kocagoz T, Eraksoy H. Dio-Sensimedia:
8.
a novel culture medium for rapid detection of
Nassif X, Sansonetti PJ. Correlation of the virulence
of Klebsiella pneumoniae K1 and K2 with the
extended spectrum beta-lactamases. BMC Infect Dis
2003;3:22.
File TM Jr. Overview of resistance in the 1990s. Chest
121
presence of a plasmid encoding aerobactin. Infect
J INFECT DIS ANTIMICROB AGENTS
122
9.
Immun 1986;54:603-8.
variant of the chromosomal gene for class A beta-
Podschun R, Ullmann U. Klebsiella spp. as nosocomial
lactamase K2, specific for Klebsiella pneumoniae,
pathogens: epidemiology, taxonomy, typing methods,
is the ancestor of SHV-1. Antimicrob Agents
and pathogenicity factors. Clin Microbiol Rev
Chemother 1997;41:2705-9.
1998;11:589-603.
10.
18.
12.
13.
Survey of plasmid-associated genetic markers in
Enterobacteriaceae. In: Winn W Jr, Allen S, Janda W,
Enterobacteriaceae with reduced susceptibilities to
et al, eds. Koneman’s Color Atlas and Textbook of
cephalosporins. Antimicrob Agents Chemother
Diagnostic Microbiology. 6 th ed. Philadelphia:
2003;47:2179-85.
15.
Serratia, Plesiomonas, and other Enterobacteriaceae.
beta-lactamase (ESBL) among cefotaxime-resistant
In: Murray PR, Baron EJ, Jorgensen JH, Pfaller MA,
Serratia marcescens at a medical center in middle
Yolken RH,eds. Manual of Clinical Microbiology. 8th
Taiwan. Diagn Microbiol Infect Dis 2004;49:
ed. Washington DC: ASM Press, 2003:684-97.
125-9.
Clinical and Laboratory Standards Institute. Perfor-
20.
Poirel L, Naas T, Le Thomas I, Karim A, Bingen E,
mance standards for antimicrobial susceptibility
Nordmann P. CTX-M-type extended-spectrum beta-
testing. Fifteenth informational supplement M100-
lactamase that hydrolyzes ceftazidime through a single
S15. Wayne, Pa: CLSI, 2005.
amino acid substitution in the omega loop. Antimicrob
Rasheed JK, Jay C, Metchock B, et al. Evolution of
Agents Chemother 2001;45:3355-61.
21.
Kimura S, Ishiguro M, Ishii Y, Alba J, Yamaguchi K.
a strain of Escherichia coli during multiple episodes
Role of a mutation at position 167 of CTX-M-19 in
of bacteremia. Antimicrob Agents Chemother
ceftazidime hydrolysis. Antimicrob Agents Chemother
1997;41:647-53.
2004;48:1454-60.
Saladin M, Cao VT, Lambert T, et al. Diversity of
22.
Bae IK, Lee BH, Hwang HY, et al. A novel ceftazidime-
CTX-M beta-lactamases and their promoter regions
hydrolysing extended-spectrum beta-lactamase,
from Enterobacteriaceae isolated in three Parisian
CTX-M-54, with a single amino acid substitution at
hospitals. FEMS Microbiol Lett 2002;209:161-8.
position 167 in the omega loop. J Antimicrob
Haeggman S, Lofdahl S, Paauw A, Verhoef J, Brisse S.
Chemother 2006;58:315-9.
23.
Naas T, Poirel L, Karim A, Nordmann P. Molecular
beta-lactamase gene in Klebsiella pneumoniae.
characterization of In50, a class 1 integron encoding
Antimicrob Agents Chemother 2004;48:2400-8.
the gene for the extended-spectrum beta-lactamase
Chaves J, Ladona MG, Segura C, Coira A, Reig R,
VEB-1 in Pseudomonas aeruginosa. FEMS Microbiol
Ampurdanes C. SHV-1 beta-lactamase is mainly a
Lett 1999;176:411-9.
chromosomally encoded species-specific enzyme
in Klebsiella pneumoniae.
17.
Wu LT, Tsou MF, Wu HJ, Chen HE, Chuang YC,
Yu WL. Survey of CTX-M-3 extended-spectrum
Diversity and evolution of the class A chromosomal
16.
19.
Abbott SL. Klebsiella, Enterobacter, Citrobacter,
extended-spectrum beta-lactam resistance (SHV-8) in
14.
Preston KE, Graffunder EM, Evans AM, Venezia RA.
The
Winn WC Jr, Allen S, Janda W, et al.
Lippincott William & Wilkins, 2006:261-3.
11.
Sep.-Dec. 2007
Antimicrob Agents
24.
Poirel L, Naas T, Guibert M, Chaibi EB, Labia R,
Nordmann P. Molecular and biochemical charac-
Chemother 2001;45:2856-61.
terization of VEB-1, a novel class A extended-spectrum
Haeggman S, Lofdahl S, Burman LG. An allelic
beta-lactamase encoded by an Escherichia coli
Vol. 24 No. 3
25.
Phenotypes and genotypes of ESBL-producing Klebsiella pneumoniae:- Tribuddharat C, et al.
123
integron gene. Antimicrob Agents Chemother
extended-spectrum beta-lactamase, SHV-57, that
1999;43:573-81.
confers resistance to ceftazidime but not cefazolin.
Ma L, Alba J, Chang FY, et al. Novel SHV-derived
Antimicrob Agents Chemother 2005;49: 600-5.
123