The increasing resistance of gram negative bacteria is of worldwide concern.
Its rapid evolution to become resistant is an immense challenge to clinicians,
scientists and researchers worldwide. Despite many new β-lactam antibiotics
being developed over the last 20 years, new β-lactamases emerged,
causing them to be resistant to these antibiotics (Bradford, 2001). This
is presumably due to overuse of new antibiotics resulting in selective
pressure to select new variants of β-lactamases. Its infection has
now become even more difficult to treat as its resistance level has increased
over the years. Now with the threat of the so-called ESBL-producing pathogens,
it looks like we are fighting a losing battle.
Extended spectrum β-lactamase or ESBL is a β-lactamase enzyme
with increased spectrum of activity especially against the oxyimino-cephalosporins
such as cefotaxime and ceftazidime. Most ESBLs are derivatives of TEM
or SHV enzymes (Bush et al., 1995). There are now >150 TEM-type β-lactamases and >100 SHV-type enzymes (www.lahey.org/studies/webt.htm).
They are easily transmissible from one organism to another. Hence, SHV-and
TEM-types which have been recognized since the 1980s are now a major cause
of nosocomial infections all over the world.
In Malaysia, a national surveillance on antibiotic resistance was conducted
in 2005 and found that ceftazidime and cefotaxime resistance in E.
coli was 8.9% (0.9% from the community) and 10.1% (1.0% from the community)
respectively (unpublished data). Tests for ESBL production was unfortunately
not performed routinely in this survey. Another study done in a university
hospital in Malaysia reported blaSHV-5 to be dominant
in ESBL-producing E. coli and was postulated to have been acquired
from ESBL-producing Klebsiella pneumoniae via plasmid transfer
(Subramaniam et al., 2006).
Another family of plasmid-mediated ESBLs which are non-TEM and non-SHV
derivatives, called CTX-M has emerged. They preferentially hydrolyze cefotaxime
and have mainly found in wide range of bacteria, especially members of
the Enterobacteriaceae family (Bonnet, 2004). Strains expressing
CTX-M-type β-lactamases have been isolated from many parts of the
world. Based on communications with various experts in many local centres,
CTX-M-type is also available in Malaysia and it poses a potential threat
in healthcare management. Realizing the lack of published local research
on this issue, the aim of this study is to demonstrate the emergence of
CTX-M-type in ESBL-producing E. coli in a tertiary hospital in
MATERIALS AND METHODS
Sixteen non-duplicate isolates of validated ESBL-producing E. coli
from multiple types of specimens collected from a tertiary hospital in
Malaysia in 2006 were analyzed in this study.
β-Lactamase Gene Identification
Chromosomal DNA was prepared from an overnight bacterial culture (18-24
h) on a MacConkey agar plate. Approximately ten colonies were re-suspended
into 50μL sterile distilled water and the cells were lysed by boiling
for 15 min, then immediately cooled on ice for another 2 min. Cellular
debris were removed by centrifugation at 10,000 g for 1 min and the supernatant
was used as a source of template DNA for amplification of blaTEM
and blaSHV genes.
Plasmid DNA was prepared by using a commercial kit (Invitrogen, BioDiagnostic,
USA). The plasmid DNA was used as a source of template DNA for amplification
of blaCTX-M gene.
PCR primers used for amplification of CTX-M types were universal primers
of CTX-MA1 (5`-SCS ATG TGC AGY ACC AGT AA-3`) and CTX-MA2 (5`-CCG CRA
TAT GRT TGG TG-3`) described by Saladin et al. (2002) yielding
a 544 bp amplicon (S = G or C; Y = C or T; R = A or G). The primers used
for PCR amplification of blaTEM and blaSHV
were mentioned previously by Perilli et al. (2002)
yielding amplicons of 1080 and 1050 bp in size, respectively.
The cycling condition for the amplification of blaTEM
and blaSHV were described previously by Perilli et
al. (2002) and the DNA amplification steps for blaCTX-M
were as follows: initial denaturation at 94°C for 4 min followed
by 30 cycles of denaturation at 95°C for 30 sec, annealing at 57.6°C
for 1 min and elongation at 72°C for 1 min with final extension at
72°C for 4 min. All reactions were performed in a 25μL volume
using 6 units of Taq DNA polymerase (Bioron, Germany), 2.5μL of
10x PCR buffer, 2 mM MgCl2, 0.2 mM deoxynucleoside triphosphates,
0.3μL of 10 pmol mL-1 primers each and 1μL DNA
template. The PCR reactions were performed using PCR Thermal Cycler (Biometra-TPersonal,
Germany). All negative reactions were repeated twice.
All purified PCR product were sent for commercial sequencing (1st
Base Laboratories Sdn. Bhd., Malaysia). The sequences were edited and
analyzed by using DNAsis for Windows 2.6 (Hitachi Software Engineering,
USA). The nucleotide and the deduced protein sequences were analyzed by
using the software available online at the National Center for Biotechnology
Information website (http://www.ncbi.nlm.nih.gov).
The quality control strains used were E. coli ATCC 25922 (sensitive
strain), K. pneumoniae ATCC 700603 (blaSHV-18),
E. coli 13-00 (blaCTX-M-14), E. coli 78-01(blaCTX-M-15)
and E. coli 188-04 (blaCTX-M-2).
RESULTS AND DISCUSSION
Sixteen clinical isolates of ESBLs-producing E. coli carried at
least one of the blaCTX-M, blaTEM,
or blaSHV ESBL genes (Table 1). A
vast majority of the strains carried blaCTX-M (81.3%)
and blaTEM genes (75%). Only two strains (12.5%) carried
the blaSHV gene. There were at least three circulating
strains of which CTX-M-15 being the most dominant (61%), followed by CTX-M-14
(31%) and CTX-M-3 (8%).
Almost all strains demonstrated resistance to the second, third and fourth
generation of cephalosporins (Table 2). There were more
resistance to cefotaxime than ceftazidime among CTX-M-types, phenotypically
supporting the presence of CTX-M enzymes.
It is clearly shown that at least in this study, CTX-M-types have been
the dominant strains. This is in parallel to the worldwide trend where
CTX-M-types have overtaken dominance of TEM-and SHV-types globally. Though
prior data on the prevalence of TEM-types and SHV-types are not known
in Malaysia to demonstrate this fact, we believe similar trend is valid.
It is difficult to determine the proportion of our strains which originated
from the community as these patients were admitted to the hospital where
medical records access is restricted owing to confidentiality and security
In our study, we found at least three circulating strains of which CTX-M-15
being the most dominant (61%), followed by CTX-M-14 (31%) and CTX-M-3
(8%). CTX-M-15 and CTX-M-3 belong to the CTX-M-1 group while CTX-M-14
to the CTX-M-9 group (Bush et al., 1995). Based on this finding,
we agree to the hypothesis that states the current spread of CTX-M enzymes,
is allodemic in nature, rather than an epidemic pattern, reflecting that
the increase of CTX-M enzymes is a result of both multiple specific clones
and/ or mobile genetic elements and not dissemination of a particular
clone (Baquero et al., 2002). Owing to the dynamic nature of these
clones, it is not surprising if the dominant strains would change over
time (Bonnet, 2004).
||The β-lactamase types of ESBL-producing Escherichia
|+: Present; -: Absent
||Antibiotic resistant pattern of sixteen ESBL-producing
|*CXM: Cefuroxime; CFP: Ceforperazone; CAZ: Ceftazidime;
CTX: Cefotaxime; FEP: Cefepime; GN: Gentamicin; SXT: Trimethoprim-sulfamethoxazole;
CIP: Ciprofloxacine; IMP: Imipenem; MEM: Meropenem; S: Susceptible;
R: Resistance; I: Intermediate; -: Absent
Several studies have shown an increasing association with resistance
to other classes of antimicrobial agents among ESBL-producing organisms
isolated form the community (Pitout et al., 2005). This includes
co-resistant with trimethoprim-sulfamethoxazole, tetracycline, gentamicin
and ciprofloxacin. This association may be evident in this study as 46%
of the strain was resistant to gentamicin, 69% resistant to trimethoprim-sulfamethoxazole
and 46% resistant to ciprofloxacin.
In order to further understand the role of ESBL to healthcare, more extensive
studies will be conducted. These include linking CTX-M-types with E. coli
phylogenetic group. We certainly would like to know this association with
our strains, especially the dominant strains. CTX-M-types have been reported
to be associated with groups D and B2, both groups of which are mainly associated
with extraintestinal infections (Cantun and Conque, 2006). Virulence
markers in our local CTX-M-producing E. coli would be investigated. It
was reported that there were significant differences from individual virulence
factors among CTX-M producers and non-producers; however, aggregate virulence
factor scores were similar (Pitout et al., 2007).
A comprehensive surveillance study is in the pipeline which, in our opinion
will provide a good overview and more accurate picture of the problem
of ESBL in our country.
The increasing evidence of CTX-M-producing bacteria in food-producing
bacteria such as cattle and poultry and pets, namely, dogs and cats is
worrying (Pitout et al., 2005). This may acts as reservoir for
the acquisition of resistant organism which will make the quest for winning
this battle an uphill task. It is therefore, necessary for
us to study this situation in our region.
In conclusion, ESBL is indeed a significant problem in our clinical settings
in this tertiary hospital and therefore, it is not incorrect to postulate
this problem as also a national issue. We have succeeded in determining
the group type of the enzymes and in this preliminary study, the epidemiology
of the enzymes seem to give a slightly clearer picture of the impact of
ESBL in Malaysia. However, it is still premature to draw conclusions based
on the existing data.
This study was funded by Intensified Research in Priority Areas Grant
No. 54436 and 5523248 from the Ministry of Science, Technology and Innovation
of Malaysia. Our sincere appreciation to the Ministry of Science, Technology
and Innovation of Malaysia for the research grants and Dr. Johann Pitout
for the control strains E. coli 13-00 (blaCTX-M-14),
E. coli 78-01(blaCTX-M-15) and E. coli