… of SHV, TEM, CTX-M and extended-spectrum β-lactamase produced by Escherichia coli, Acinobacter baumannii and Klebsiella isolates in a Turkish hospital
ABSTRACT A total of 94 clinical isolates were collected from Gazi University Hospital, Turkey. Presence of ESBL positivity was detected using the double disk synergy test (DDST). ESBL isolates were further typed for the bla TEM , bla SHV , bla CTX-M and bla OXA using designed primers. ESBLs were found in 65 (69.14%) isolates using DDST. Plasmid DNAs of potentially ESBL positive strains were isolated. About 7.69% of the ESBL positive isolates did not harbour plasmid DNA. According to the PCR technique, only 2 additional isolates were found to be ESBL producers. blaTEM was the commonest genotype (73.43%), followed by blaSHV (21.87%) and blaCTX-M (17.18%), either alone or in combination. ESBL positive strains of Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa are increasingly found in hospital isolates. Because these strains become resistant to available antibiotics and they can pass the gene to other clinical strains, the quick detection of these strains in clinical laboratories is very important.
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ABSTRACT: The aim of the present study was to study the phenotypic and molecular characterization of 5 novel CTX-M-beta-1actamases carried by 5 Klebsiella pneumoniae isolates and 3 Escherichia coli isolates collected from 4 hospitals in Hefei, China. The purified PCR products were ligated with pGEM-Teasy vectors, expressed, and sequenced. The complete genes of the CTX-M-beta-lactamases were ligated with the pHSG398 vector to express prokaryotic recombinant proteins. Plasmids were extracted by rapid alkaline lysis protocol, and the PCR method was performed to determine whether the prokaryotic expression was successful or not. Antimicrobial susceptibility was tested and the phenotypes of transformants were determined according to criteria recommended by the Clinical and Laboratory Standards Institute. The kinetic parameters of enzymes were confirmed. The isoelectric points (pI) were determined by isoelectric focusing assay. Pulsed-field gel electrophoresis and plasmid profiling were performed. The PCR products had 1101 nucleotides and were determined as CTX-M-46, CTX-M-47, CTX-M-48, CTX-M-49, and CTX-M-50. All strains were resistant to cefotaxime, but most of them were susceptible or intermediate to ceftazidime. The phenotypes of novel enzymes were determined as extended-spectrum-beta-lactamases (ESBL). Penicillin G, cephalothin, cefuroxime, and cefotaxime were determined to good substrates, whereas ceftazidime hydrolysis was not detected. The pI of the 5 novel CTX-M-beta-lactamases were 8.0. CTX-M-derivatives could be the multiplex genesis in our area. This is the first report of these 5 novel plasmid-mediated CTX-M ESBL produced from China in the world. Molecular typing reveals notably different origin in genes encoding different CTX-M variants of 8 strains.Acta Pharmacologica Sinica 03/2008; 29(2):217-25. · 2.35 Impact Factor
African Journal of Microbiology Research Vol. 4 (8), pp. 650-654, 18 April, 2010
Available online http://www.academicjournals.org/ajmr
ISSN 1996-0808 © 2010 Academic Journals
Full Length Research Paper
Phenotypic and molecular characterization of SHV,
TEM, CTX-M and extended-spectrum ?-lactamase
produced by Escherichia coli, Acinobacter baumannii
and Klebsiella isolates in a Turkish hospital
Elif Burcu Bali1, Leyla Açık1* and Nedim Sultan2
1Department of Biology, Science and Arts Faculty, Gazi University, Ankara, 06500, Turkey.
2Department of Microbiology, Faculty of Medicine, Gazi University, Ankara, 06500, Turkey.
Accepted 2 April, 2010
A total of 94 clinical isolates were collected from Gazi University Hospital, Turkey. Presence of ESBL
positivity was detected using the double disk synergy test (DDST). ESBL isolates were further typed for
the blaTEM, blaSHV, blaCTX-M and blaOXA using designed primers. ESBLs were found in 65 (69.14%) isolates
using DDST. Plasmid DNAs of potentially ESBL positive strains were isolated. About 7.69% of the ESBL
positive isolates did not harbour plasmid DNA. According to the PCR technique, only 2 additional
isolates were found to be ESBL producers. blaTEM was the commonest genotype (73.43%), followed by
blaSHV (21.87%) and blaCTX-M (17.18%), either alone or in combination. ESBL positive strains of
Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii and Pseudomonas aeruginosa are
increasingly found in hospital isolates. Because these strains become resistant to available antibiotics
and they can pass the gene to other clinical strains, the quick detection of these strains in clinical
laboratories is very important.
Key words: ESBL, double disk synergy test, plasmid, PCR.
Extended-spectrum ?-lactamases (ESBLs) were first des-
cribed in the 1980s and they have been detected in
Klebsiella species, and later in Escherichia coli, Pseudo-
monas aeruginosa and Serratia marcescens and other
gram-negative bacilli (Kiratin et al., 2008; Cheng et al.,
2008; Morris et al., 2003). ESBLs are also able to
hydrolyze 3 and 4 generation cephalosporins and mono-
bactams. ESBL producing strains are inhibited by ?-
lactamase inhibitors (clavulanic acid, sulbactam and
tazobactam) (Bradford, 2001; Pitout et al., 2007; Giraud-
Morin and Fosse, 2003). ESBLs are a group of enzymes
encoded by genes described predominantly on plasmid
that are common among Enterobacteriaceae (Poole,
2004). ESBL are an increasingly important cause of
transferable multidrug resistance in gram-negative bac-
*Coreesponding author: E-mail: email@example.com. Tel:
0(312)2021185. Fax: 0(312)2122279.
teria throughout the world. These bacteria have spread
rapidly and have become a serious threat to human
health worldwide (Giraud-Morin and Fosse, 2003; Poole,
2004; Gupta, 2007).
ESBLs are undergoing continuous mutation, causing
the development of new enzymes showing expanded
substrate profiles. At present, there are more than 300
different ESBL variants, and these have been clustered
into nine different structural and evolutionary families
based on amino acid sequence. TEM and sulphydryl
variable SHV were the major types. However, CTX-M
type is more common in some countries (Paterson et al.,
2003). Determination of TEM and SHV genes by mole-
cular techniques in ESBL producing bacteria and their
pattern of antimicrobial resistance can supply useful data
about their epidemiology and risk factors associated with
these infections (Jain and Mondal, 2008). The aim of this
study was to isolate and identify the types of extended-
spectrum beta-lactamases (ESBL) produced by E. coli, A.
baumannii and Klebsiella spp. (K. pneumoniae, K. oxy-
Bali et al. 651
Table 1. Oligonucleotide primers used for detection of beta-lactamase genes.
Primers (° C) Nucleotide Sequences (5’-3’)
Expected amplicon size (bp)
toca, K. terrigena and K. ornithinolytica) isolated from
various specimens (urine, blood, sputum, wound, absces-
ses, catheter, peritoneum and cerebrospinal fluid) of
patients hospitalized in different units of Gazi University
Medical Faculty Hospital, Turkey.
MATERIALS AND METHODS
Bacterial isolates and detection of ESBL
Ninety-three clinical isolates of E. coli, A. baumannii, K. pneumo-
niae, K. oxytoca, K. terrigena and K. ornithinolytica from various
clinical specimens (urine, blood, sputum, wound, abscesses,
catheter, peritoneum and cerebrospinal fluid) were obtained in 2006
- 2007 and identified with API ID 32E (Bio Mérieux, Marcy l’Etoile,
France). E. coli, A. baumannii and Klebsiella isolates were
screened for ESBL production by DDST using ceftazidime (30 ?g),
cefotaxime (30 ?g), ceftriaxone (30 ?g) and cefpodoxime (10 ?g)
antibiotic on Mueller–Hinton agar as recommended by the Clinical
and Laboratory Standards Institute (CLSI guideline 2009). ESBL
production was confirmed by disk potentiation test using ceftazi-
dime (30 ?g) and cefotaxime (30 ?g) antibiotic disks with and
without clavunalic acid (10 ?g) and by DDST (Koneman et al.,
1997; Jarlier et al., 1988; Philippon et al., 1989).
Plasmid and chromosomal DNA analysis
Plasmid DNA was isolated from clinical isolates using the alkaline
lysis method (Stürenburg and Mack, 2003). All clinical isolates were
grown for 12 h on nutrient agar plates. A loopful of cells from a
single colony was transferred to 0.1 ml of H2O, and the mixture was
boiled for 10 min to lyse the cells. The resulting cell lysate was
centrifuged briefly (10 s at 10,000 rpm), and 15 µl of the
supernatant was used as the DNA sample for the PCR reaction
(Sambrook and Russel, 2001).
Detection of ESBL types by PCR
The specific oligonucleotide primers were designed according to
TEM, SHV, CTX-M-type and OXA ?-lactamase DNA sequences
deposited in the GenBank nucleotide sequence database with the
accession numbers AB282997, EF125011, DQ303459 and L07945
(Table 1). Multiple nucleotide sequence alignments were performed
with the CLC Workbench program. Coding sequences were detect-
ed with the Artemis program and primers were designed with the
Perl Primer program.
Amplification of the ESBL genes
ESBL producing isolates were amplified using bla TEM/SHVCTX-M, OXA
specific primers listed in Table 1. Amplification reactions were
carried out under the following conditions: initial denaturation at
94° C for 3 m, followed by 35 cycle of denaturation at 94° C for 45 s,
annealing at 60° C for TEM, SHV and CTX-M and at 62° C for OXA
for 30 s and extension at 72° C for 1 min, and a final extension at
72° C for 3 min. A molecular marker (Fermentase SM0241 effective
size range: 80 to 1000 kb) was used to assess PCR product size.
RESULTS AND DISCUSSION
During an 8-month period, a total of 94 clinical specimens
from Gazi University Hospital were identified, 50 as E.
coli, followed by A. baumannii (n = 19), K. pneumoniae (n
= 17), K. oxytoca (n = 4), K. terrigena (n = 2) and K.
ornithinolytica (n = 2). Sixty-five of the 94 isolates were
confirmed as potentially
ceftazidime/clavulanate and cefotaxime/clavulanate disks
(Table 2). Occurrence of ESBL in isolates was as follows:
42 (84%) of 50 E. coli, 1 (5.26%) of 19 A. baumannii, 15
(88.23%) of 17 K. pneumoniae, 4 (100%) of 4 K. oxytoca,
2 (100%) of 2 K. terrigena, and 1 (50%) of 2 K.
ornithinolytica (Table 2). Of the 65 isolates, 48 (73.84%)
were from urine, 5 (7.69%) from blood, 4 (6.15%) from
sputum, 1 (1.53%) from a wound, 1 (1.53%) from an
abscess, 2 (3.07%) from catheters, 2 (3.07%) from
peritoneums and 1 (1.53%) from cerebrospinal fluid
The plasmid DNAs were isolated from 58 isolates
(90.76%) of potentially ESBL positive E. coli, A.
baumannii, Klebsiella spp., potentially ESBL positive E.
coli, A. baumannii and Klebsiella species that harboured
plasmid varying from 1 to 10 (Figure 1 a, b and c). Five of
the ESBL positive E. coli and 2 of the K. pneumoniae
isolates had no plasmid DNA. All ESBL-producing isolat-
es were screened by PCR using blaTEM, blaSHV, blaCTX-M
and blaOXA specific primers (Figure 2). Some of the TEM,
SHV, and CTX-M PCR products were subsequently se-
ESBL producers using
652 Afr. J. Microbiol. Res.
Table 2. According to clinical material, distribution of ESBL positive isolates using disk diffusion (P*: Peritoneum, CSF*:
Positive for ESBL
by DDST / Source
34 2 2
8 2 2
4 - -
1 1 -
1 - -
- - 1
Total 48 5 5
Urine Blood Sputum Wound P* Catheter CSF* Abscess Total
a b c
Figure 1. Plasmid profiles of some Klebsiella (a), E. coli (b) and A. baumannii (c) isolates. M. ?-pUC mix.
Figure 2. PCR products of blaSHV (a: lines 1-4; b: lines 1, 4,5), blaTEM (a: Lines 5-7) and blaCTX-M (b: Lines 2, 3, 6,). M: 1000 bp DNA ladder.
sequenced and compared with DNA GenBank
sequences using BLAST search. ESBLs were found in 65
isolates (68.14%): 44 E. coli and 21 Klebsiella isolates.
Some of the isolates harboured two or more ESBL
genes. However, the A. baumannii isolate had none of
the ESBL genes.
TEM type ESBLs were found in 72.72% of E. coli,
73.33% of K. pneumoniae, 25% of K. oxytoca and 100%
of K. terrigena and K. ornithinolytica isolates (Table 3).
None of the isolates had blaOXA genes. Eighteen (19.2%)
out of 65 isolates carried more than one type of ?-
lactamase genes, with nine isolates harbouring blaTEM
and blaSHV genes and nine isolates harbouring blaTEM and
blaCTX-M genes. No isolates with blaTEM, blaSHV, and blaCTX-
M genes together were detected. BlaTEM genes were the
most common ESBLs detected in E. coli (72.72%).
In recent years, the problem of gradually increasing
resistance to antibiotics has threatened the entire world.
Production of beta-lactamase, which hydrolyses and
inactivates beta-lactam antibiotics, has been one of the
Bali et al. 653
Table 3. Distribution of ESBL positive isolates including blaTEM, blaSHV and blaCTX-M genes by PCR. Total ESBL
positive isolates number 64.
ESBL positive isolates / ESBL types
E. coli (n = 44)
K. pneumoniae (n = 15)
K. terrigena (n = 2)
K. ornithinolytica (n = 1)
K. oxytoca (n = 2)
most important resistance mechanisms of many bacterial
species, mainly in the family Enterobacteriace (Akcam et
al., 2004). Resistance to extended-spectrum ?-lactams
among gram-negative pathogens is increasingly associa-
ted with ESBLs (Kimura et al., 2007). ESBL positive
enterobacterial species are becoming
throughout the world (Timko, 2004).
In Turkey, several studies have revealed that the
distribution of ESBLs has been observed in different
rates. Bülüç et al. (2003) found that ESBL frequencies
were 48% for K. pneumoniae, 40% for K. oxytoca and
14% for E. coli isolates. However, Delialioglu et al. (2005)
stated that ESBL frequencies of K. pneumoniae, K.
oxytoca and E. coli isolates were 29.7, 4.2 and 18.3%,
respectively. The prevalence of confirmed ESBL-positive
isolates in the USA, Europe, Latin America, the Middle
East and Asia/Pacific was 3, 5, 10 and 13% for E. coli
and 17, 7, 11, 14, 20 and 18% for Klebsiella spp.
(Paterson et al., 2005). ESBL production in Acinetobacter
strains in India was 28% (Sinha et al., 2007).
In this study, ESBL positive isolates were detected by
DDST and PCR. Sixty-five isolates were positive in the
disk diffusion test and 67 isolates were positive using
PCR. The fact that only two additional isolates were
ESBL positive proves that the disk diffusion test is quite
accurate. One of the positive isolates with DDST had no
ESBL with PCR; these isolates may have different ESBL
types than the ESBL tested in this study. TEM type
ESBLs were found in 72.72% of E. coli, 73.33% of K.
pneumoniae, 25% of K. oxytoca and 100% of K. terrigena
and K. ornithinolytica isolates. ESBLs are mostly
encoded by large plasmids (up to 100 kb or even more)
that are transferable from strain to strain and between
bacterial species (Jarlier et al., 1988). However, in
different studies, different ESBL plasmid sizes have been
detected; one study in South Africa proved that plasmid
sizes of 25 isolates of K. pneumoniae varied from 5 to
186 kb in size (Essack et al., 2001). In another study, 48
E. coli isolates were investigated and it was found that
the size of plasmid DNA varied from 95 to 120 kb for
CTX-M type ESBL and from 60 to 160 kb for SHV type
ESBL (Velasco et al., 2007). In our study, we found that
ESBL positive E. coli, A. baumannii and Klebsiella
species harboured plasmid DNA from 1 to 10 of size 1 kb
to 19 kb. The other finding was that ESBL positive iso-
lates showed plasmid DNA more often than ESBL
negative isolates. This may be considered evidence that
most ESBL isolates depended on plasmids. These
finding support the result of relation between the number
of plasmids harbored by an isolate resistance to
antibiotic. However, there were seven ESBL positive
isolates with no plasmid, showing that some of the ESBL
genes were coded potentially by chromosomal DNA.
TEM and SHV beta lactamases are mainly found in E.
coli and Klebsiella pneumoniae, but can occur in other
members of the family Enterobacteriaceae and in
nonenteric organisms, such as Acinetobacter species
(Turner, 2005). In Turkey, SHV-2 (K. pneumoniae, E.
coli), SHV-5 (K. pneumoniae,Enterobacter aerogenes
and Enterobacter cloacae), SHV-12 (K. Pneumoniae and
E. coli), OXA-16 (Pseudomonas aeruginosa), PER-1
(Acinetobacter baumannii and P. aeruginosa), CTX-M-2,
CTX-M-15 and TEM-1 (K. pneumoniae) type ESBLs were
reported (Gupta, 2007; Velasco et al., 2007).
In summary, from a total of 94 isolates, we found 50%
(n = 47), 14.89% (n = 14) and 11.70% (n = 11) ESBL
rates for TEM, SHV and CTX-M type beta lactamases,
respectively. There were no strains harboring OXA type
beta lactamase. TEM and CTX-M type ESBL were
observed in 72.72 and 22.72% of E. coli isolates,
respectively. SHV type ESBL was frequently found in K.
pneumoniae (53.3%) isolates. ESBL positive strains of K.
pneumoniae, E. coli, A. baumannii and P. aeruginosa are
increasingly found in hospital isolates. These strains are
usually multi-drug resistant. Because these strains
become resistant to available antibiotics and they can
pass the gene to other clinical strains, the quick detection
of these strains in microbiology laboratories is very
important. Molecular typing would determine which types
of ESBL are present in each isolate. Molecular detection
and identification of beta lactamases would be essential
for a reliable epidemiological investigation of antimicrobial
resistance. Antimicrobial therapy has played an important
role in the treatment of human bacterial infections, but the
drug resistance that has emerged in the treatment of
bacterial infections due to ESBL enzymes degrades all
beta lactam antibiotics and thus bacteria become multi-
drug resistant (Gupta, 2007). These enzymes can be
chromosomal or plasmid mediated. The gene code for
the enzymes may be carried on integrons. Integrons help
in the dissemination of antimicrobial drug resistance in
health care settings (Gupta, 2007). Therefore, ESBL pro-
654 Afr. J. Microbiol. Res.
producing organisms should be identified quickly so that
appropriate antibiotic usage and infection control
measures can be implemented.
This study was supported by Gazi University Department
of Scientific Research Projects (project no: 05/2007-39).
The authors wish to thank Dr. Doruk Engin, Department
of Microbiology, Medical Faculty, University of Gazi.
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