Autores
Ulises
Garza-Ramos, M en C,(1) Esperanza
Martínez-Romero, PhD,(2) Jesús Silva-Sánchez,
PhD.(1)
(1)
Departamento de Resistencia Bacteriana, Centro de Investigaciones Sobre
Enfermedades Infecciosas. Instituto Nacional de Salud Pública.
(2) Centro
de Ciencias Genómicas, Universidad Nacional Autónoma de México.
Cuernavaca, México.
Resumen
Objetivo:
En este trabajo se reporta la caracterización molecular de la
resistencia a antibiótico ß-lactámicos conferida por genes contenidos
en plásmidos de enterobacterias productoras de ß-lactamasas de espectro
extendido (BLEEs). Material
y métodos: Catorce aislamientos clínicos de
enterobacterias fueron seleccionados por conveniencia de un banco de
cepas obtenidas de siete diferentes hospitales de México durante los
periodos 1990-1992 y 1996-1998 y fueron procesados en el Laboratorio de
Resistencia Bacteriana (Instituto Nacional de Salud Pública,
Cuernavaca). En la caracterización se empleó PFGE, IEF para
ß-lactamasas, conjugación bacteriana, amplificación por PCR y
secuenciación de DNA, extracción y restricción de plásmidos. Resultados: Las 14
cepas fueron no relacionadas genéticamente. Se identificaron BLEEs tipo
SHV-2 (5/14) y SHV-5 (9/14). La resistencia a cefalosporinas fue
transferida por conjugación en 9 de 14 (64%) aislamientos clínicos
mediante un plásmido que mostró un patrón de restricción similar entre
ellos. Conclusión:
Se sugiere que la diseminación de la resistencia a cefalosporinas fue
debida a plásmidos relacionados que contienen los genes que codifican
BLEEs.
Palabras clave: enterobacterias, plásmido, BLEE, SHV-5, SHV-2; México
Abstract
Objective: In this
work we report the molecular characterization of ß-lactam antibiotics
resistance conferred by genes contained in plasmids
from enterobacteria producing extended-spectrum ß-lactamases
(ESBL). Material and Methods: Fourteen
enterobacterial clinical isolates selected from a group of strains
obtained from seven different hospitals in Mexico during 1990-1992 and
1996-1998 were analyzed at the Bacterial Resistance
Laboratory (National Institute Public Health, Cuernavaca).
Molecular characterization included PFGE, IEF of
ß-lactamases, bacterial conjugation, PCR amplification and DNA
sequencing, plasmid extraction and restriction. Results: Isolates
were genetically unrelated. ESBL identified were SHV-2
(5/14) and SHV-5 (9/14) type. Cephalosporin-resistance was
transferable in 9 of 14 (64%) clinical isolates with only
one conjugative plasmid, DNA finger printing showed a similar band
pattern in plasmids. Conclusions:
The
dissemination of cephalosporin resistance was due to related plasmids
carrying the ESBL genes.
Key words:
enterobacteria, plasmid, ESBL, SHV-5, SHV-2; Mexico
Address reprint requests to:
Dr. Jesús Silva-Sánchez. Instituto Nacional de Salud Pública. Av.
Universidad 655, col.
Santa María Ahuacatitlán, 62508 Cuernavaca, Morelos, México. E-mail: jsilva@correo.insp.mx.
Introducción
There is a need to study the epidemiology of
extended-spectrum ß-lactamase-producing enterobacteria (ESBL-PE) as
antibiotic resistance is an increasing problem in healthcare
institutions.1 Novel ESBLs gene variants have
emerged.2,3 Most ESBLs are variants of the
classical TEM-1 and SHV-1 ß-lactamases, with one or more amino acid
substitutions that confer resistance to broad-spectrum cephalosporins
and aztreonam.3 These changes alter the
catalytic site allowing the hydrolysis of oxyimino cephalosporins and
monobactams.4 Almost all SHV coding ESBL genes
have G/A mutations, which specify glycine/serine and glutamate/lysine
substitutions at amino acids 238 and 240, respectively.5
In general, the substitution at position 238 (SHV-2) confers a large
increase in resistance to cefotaxime, while the additional substitution
at position 240 (SHV-5) confers a large increase in resistance to
ceftazidime.6 These mutations have been
documented in clinical isolates of Klebsiella
pneumoniae from hospitals in Mexico and have been
implicated in outbreaks with high mortality.7-9.Meanwhile,
101 different SHV mutants have been recognized worldwide
(http://www.lahey.org/Studies/).
Because the genes coding for these enzymes are located on plasmids,8-10
we wondered if some of the plasmids expressing shv-derived
enzymes from different clinical isolates in Mexico could be related,
thus we used restriction enzymes to analyze the plasmids expressing
these enzymes isolated from different geographically distant hospitals
over several years.
The approach that we followed was to characterize ESBL-producers by the
isoelectric point of the ß-lactamases, PCR analyses, sequence of the
genes involved
and restriction patterns of the DNA of the plasmids encoding these
genes.
Material
y Métodos
Bacterial
strains. One-hundred and fifty-seven clinical isolates of
ESBL-PE causing nosocomial infections were each collected from a
different patient during a six-year period (1990-1992 and 1996-1998) in
seven hospitals in Mexico. Hospitals 1, 2, 3 and 6 are located in
Mexico City, hospitals 5 and 7 are 70 km from Mexico City, and hospital
4 is 785 km from Mexico City (table I). All clinical isolates were
identified with the API 20E system (BioMerieux, Merck. Germany) and
confirmed as ESBL-producers. In order to perform further molecular
characterization, 14 strains were selected to include one strain from
each hospital. In cases corresponding to an outbreak or endemic clone
in the hospital, the strain represents from 6 to 94 independent
clinical isolates: this was the case of strains K806-4 (6 isolates),11
K910-5 (21 isolates),12 K1333-2 and K1335-2 (12 isolates),13 K1509-6
(94 isolates)9 and C1177-7 (16 isolates) (this work) as indicated in
table I. The species included were: nine of K. pneumoniae, two K. variicola,
two E. coli, and one E. cloacae. Strains were named first
with the first letter of the corresponding specie, followed by the
number of the strain and finally number of the hospital where it was
obtained.
Susceptibility
testing. Bacterial antimicrobial susceptibility was
initially determined with the MicroScan (Dade, Behring, USA) system
using the combo 20 panel. Subsequently, MICs for cefotaxime and
ceftazidime were determined by Etest strips on Mueller-Hinton agar
following the recommendations of Clinical and Laboratory Standards
Institute (CLSI). ESBL production was confirmed by the double disk
diffusion method with cefotaxime and ceftazidime alone and in
combination with clavulanic acid, interpretation criteria was as
indicated in the CLSI.14
Genomic DNA
typing. For pulse-field gel electrophoresis (PFGE) typing, whole cell
DNA was obtained according to the method described by Kaufmann.15
DNA was digested with XbaI
(Gibco, BRL, UK.) and separated in 2% agarose gels (Pulsed
Field-Certified; Pronadisa, Madrid) with a Gene-Path System (Bio-Rad,
Hercules, USA). Gels were stained with ethidium bromide and analysed
according to the criteria of Tenover et al.16
Plasmid
profile and mating experiments. Plasmid DNA was extracted
from clinical isolates and the respective transconjugant according to
the method described by Kieser.17 DNA was
visualized after vertical electrophoresis in 0.7% agarose gels stained
with ethidium bromide. Plasmids R6K (40 kb), RP4 (54 kb), R1 (205 kb)
and pUA21 (300 kb) were used as molecular weight markers. Matings were
performed on filters placed on solid LB-Luria medium according to
Miller,18 using Escherichia coli J53-2
(F-, pro, met,
Rifr) as the receptor strain. In all cases, transconjugants were
selected on Luria agar supplemented with rifampin (100μg/ml) in
combination with cefotaxime (1μg/ml), or ampicillin (50μg/ml).
Transconjugants were denoted with an X after the donor strain
designation. IEF of ß-lactamases and bioassay. Crude cell protein
preparations from clinical isolates and transconjugants were obtained
from sonicated extracts prepared in 0.05M phosphate buffer (pH 7.0).
Crude extracts were subjected to isoelectric focusing (IEF) by the
procedure described previously,19 using a Phast
system minigel with a pH range from 3 to 9 (Amersham, Biosciences, UK).
Following IEF, ß-lactamase bands were visualized with nitrocefin
(Oxoid, Hampshire, UK). After IEF, the cefotaxidimase activities of
separated ß-lactamases were detected by the bioassay described
previously.20 Extracts from TEM-1, SHV-1 and
SHV-5-producing strains were used as the standards for pIs of 5.4, 7.6
and 8.2, respectively.
TEM and SHV-specific PCR and DNA sequencing.
The oligonucleotide primers OT1 and OT2 which amplify a region of TEM
genes were used.21 Only ESBL genes were
considered for further molecular characterization using the DNA from
some ESBL strains and resulting transconjugants. To amplify SHV-related
genes the oligonucleotide primers SE5 and SB3 were used for PCR
amplification as described by Silva et al.12
Reactions were carried out in a 50 μl volume containing 1 X PCR
amplification buffer, 3 X Enhancer buffer (PCRx Enhancer System; Gibco,
BRL, USA), amplification conditions were: 5 min of denaturation at 94°
C; 30 cycles of 30 sec at 94° C, 30 sec at 58° C and 2 min at 72° C;
and a final extension for 15 min at 72° C. The resulting PCR products
were analysed in 1.5% agarose gels; samples producing one sharp band
were purified with a column kit (High PureTM PCR
Purification Kit, Boheringer, USA) and used for sequencing reactions
with the dideoxy chain termination using an automatic sequencer (ABI
PRISM 377-18, kit EL:Taq FS Dye Terminator Cycle Sequencing
Fluorescence-Based Sequencing). Primers SEC5´
(5´-TCAGGAGGTTGACTATGCGT-3´) (this study) and P1
(5´-ATCGAATGAGGCGCTTCC-3´),22 were used for
sequencing the amplified PCR products.
Sequence
Analysis. Amino acid sequences were obtained using
Translate tool, available at ExPASy (http://www. expasy.ch/tools/dna.
html). Multiple-alignment of nucleotide and amino acid sequences was
done with ClustalW (http://clustalw.genome.jp/) and compared with the
SHV-1 gene (GENBANK Accession number AF148850).
Plasmid DNA fingerprinting. Plasmid DNA purification from
transconjugants was performed with QIAGEN Plasmid Midi Kit (QIAGEN,
Hilden, Germany) ionic interchange columns, according to the
manufacturer’s procedure. Fingerprinting analysis was performed with
DraI restriction enzyme (Promega. USA and Gibco, BRL, USA). The
resulting DNA fragments were analysed in 2% agarose gels. The
dendrogram of DraI
restrict pattern was achieved using the NTSYSpc 2.0 program.
Resultados
Clinical
isolates and susceptibility test. Isolation dates spanned
a period of six years, from 1990 to 1992 and 1996 to 1998, collected
from seven hospitals and three different cities in Mexico. General
characteristics of all 14 clinical isolates are described in table I.
All strains were susceptible to cefoxitin, imipenem and ciprofloxacine,
they were confirmed as ESBL producers by the double disc method. In
general the MICs for ceftazidime and cefotaxime were heterogeneous.
Five of these clinical isolates represent outbreaks or endemic clones
at each hospital, some of them reported previously: K806-4, K910-5,
K1332-2, K1335-2, K1509-6 and C1177-7, representing 6, 21,
12, 94 and 16 endemic clones, respectively (see methods and table).
Typing by
genomic DNA PFGE
analysis. In this work, PFGE genomic DNA analysis from
each species group was carried out with the nine clinical isolates of
K. pneumoniae, two K.
variicola and two E. coli. The XbaI restriction profiles
showed different DNA patterns between these three groups (data not
shown), indicating no genetic relation within the species.
Plasmid
profile and mating experiments. The 14 clinical isolates
harboured 1-4 plasmids with sizes of 40-290 kb. To define if the
resistance genes were plasmid borne, bacterial conjugation was
performed and resistant recipients were selected. Only 9 of 14 clinical
isolates were capable of transferring the resistance to the susceptible
E. coli J53-2
strain, and corresponded to five of the nine K. pneumoniae
clinical isolates tested, one of the two K. variicola tested, the two E. coli and the E. cloacae clinical
isolates included in this study. In all cases the transconjugants
received one plasmid originally contained in the respective isolate;
these plasmids are underlined in table I.
ß-Lactamase profiles and
detection of ESBL. All 14 clinical isolates expressed 1
to 3 ß-lactamases with different isoelectric points (pI). Three main
bands were identified with pI’s of 5.4, 7.6 and 8.2. In addition to
these bands, two more bands were detected with pI’s of 7.4 (strain
E86-2) and 8.1 (strain K65-1), (table I). The ß-lactamase with pI 5.4
was identified in 12/14 isolates, and the enzyme with pI 7.6 was
expressed in 10/14 isolates. The ß-lactamase with pI 8.2 was present in
9/14 strains. In order to identify the ESBL encoded in each clinical
isolate and the respective transconjugant, the bioassay (as mentioned
in Material and Methods) was performed on the IEF gels. Two bands (one
from each clinical isolate and transconjugant) were detected as ESBL.
The enzyme with pI 7.6 was identified in 5/14 clinical isolates and was
obtained from three K.
pneumoniae, one K. variicola, and one E. coli.
The ESBL with pI 8.2 was detected in 9/14 clinical isolates which
included six K.
pneumoniae, one K.
variicola, one E. coli, and one E. cloacae. Five of
these strains produced an enzyme with pI 7.6 with no cefotaximase
activity (non-ESBL). Considering the ß-lactamase profile and the ESBL
encoded in transconjugants, two major groups (including four strains
each) were identified with pI 5.4, 7.6; and 5.4, 8.2. Data
corresponding to transconjugants are underlined indicated in table. The
detection of TEM-encoding ß-lactamase by PCR amplification for blaTEM
allele was carried out with all clinical isolates. A fragment of 503-pb
was obtained in 12 of 14 bacterial strains and corresponded to the
isolates that expressed the ß-lactamase with pI 5.4, which does not
have cefotaximase activity in the bioassay and does not correspond to
an ESBL (table I).
Identification
of SHV-type ESBL by PCR amplification and sequencing. Only
ESBL genes were considered for the molecular characterization,
according to the pI values (7.6 and 8.2) these enzymes could correspond
to SHVderived ESBL. PCR amplification of SHV genes was performed with
14 isolates; all of them produced a DNA fragment corresponding to the
expected size of 0.9 kb (representing the complete blaSHV
gene and the signal peptide region). In order to identify mutations in
this gene, the 14 PCR products obtained were sequenced and the deduced
amino acid sequence compared with the wild type SHV-1 enzyme.23
In five cases, a Gli238Ser (G697A) substitution was detected indicating
the presence of the previously described SHV- 2.25 Nine clinical
isolates showed Gli238Ser (G697A) and Glu240Lys (G680/A) substitutions,
which corresponded to SHV-5.25 (table I).
Amplicons from clinical isolates K910-5, K1333-2, K1335-2 and K1319-2
yielded sequences with clear double G/A peak nucleotides at the first
position of codons 238 and 240. In addition to this mutation, the PCR
sequence of strain K1335-2 included a double peak nucleotide at the
third position of codon 240. The double nucleotide peak at the first
position of codon 238 corresponded to adenine (A) and guanine (G)
nucleotides, corresponding to serine (AGC) and
glycine (GGC)
codons, respectively. A similar case was identified at the first and
third positions of codon 240 in strain K1335-2, where the base
combinations corresponded to glutamic acid (GAG, GAA) and
lysine (AAG, AAA) codons.
These results suggest the amplification of a mixture of PCR products,
one of them corresponding to SHV-1 (non ESBL) and the other to SHV-5
(ESBL).
Plasmid
profile.
With the goal of identifying similar DNA regions in the
conjugative plasmids expressing SHVderived ESBL, the plasmidic DNA from
transconjugants (which contain only one plasmid) was digested with DraI
enzyme. The restriction pattern showed at least three common bands in
all plasmids with sizes of 6.5, 2.1 and 1.0 kb (figure 1), with
additional bands shared between several plasmids. Interestingly, the
plasmid from the transconjugants of clinical isolates K96-1 and K97-2
had identical DNA restriction pattern, even though they were collected
during different years (1990 and 1991, respectively) and hospitals
settings (figure 1, slots 4 and 5). The dendrogram generated with the
DraI restriction pattern of conjugative plasmids showed three main
groups of plasmids excluding the XK55-1 and XC1177-7 strains; in
general the correlation coefficient was up to 0.55 (figure 2). Group I
included XK96-1 and XK97-2, group II included XK102-2 and XE128-3 and
group III
included XK65-1 and XE86-2 transconjugants.
Discusión
The present study includes the molecular
characterization of cephalosporin resistance in 14 clinical isolates of
ESBL-EP (including endemic clones from different hospitals) and the
respective plasmids patterns encoding the SHV-derived enzyme. The ESBL
identified in all clinical isolates analysed were SHV-2 and SHV-5.
There was a relatedness of plasmids coding these enzymes from isolates
collected during six years from seven different hospitals and three
different locations in Mexico.
Three groups were identified according to the results of IEF, sequence
analysis of PCR product and mating experiments: Group 1:
Transconjugants XK55-1, XK65-1, XK96-1 XE128-3 and XK806-44 contained
only one plasmid from the clinical isolate, they expressed the SHV-2
ESBL with a pI of 7.6; Group 2: Transconjugants XE86-2, XK97-2, XK102-2
and XC1177-7 harbour only one plasmid, they expressed the SHV-5 ESBL
with a pI of 8.2; Finally, Group 3: The clinical isolates K910-5,
K1319-2, K1333-2, K1335-2 and K1509-6 that did not have the ability to
transfer the resistance by conjugation and they expressed the SHV-5
ESBL with pI 8.2 and SHV-1 not ESBL with pI 7.6. The interpretation of
these results was based on the sequence analysis of the PCR products
including double C/A peaks at the first position of codons 238 and 240,
indicating a mixture of PCR products. These amino acid combinations
suggested that one enzyme corresponded to SHV-1 (glycine 238,
glutamic acid 240) with a pI of 7.6, and the second to the SHV-5
(serine 238, lysine 240) with pI of 8.2. These possible combinations of
amino acids are deduced based on the fact that a mutant of SHV
including only the lysine at position 240 has not been previously
reported (http://www.lahey.org/Studies/). Similar results have been
described in 13 ESBL-producer strains, in which at least four of them
carried blaSHV-11 (non-ESBL) genes in addition
to the blaSHV-2a or blaSHV-2 (ESBLs).26
It is known that the blaSHV-1 gene has a
chromosomal K.
pneumoniae origin,27 this
observation must be in the clinical isolates of third group.
Interestingly, the gene coding for the plasmidic SHV-5 enzyme was
located on a compound transposon which originated from the K.
pneumoniae chromosome.10 Coexistence of two
different ß-lactamases of SHV (SHV-11 and SHV-2a, or SHV-11 and SHV-12)
in the same clinical isolate has been reported previously.26
The horizontal transfer of a plasmid carrying ESBL SHV genes between
non-related strains has been reported.28 Also,
the clonal and horizontal spread of SHV genes in clinical isolates of
K. pneumoniae has been documented.9 In general,
transconjugants harboured the conjugative plasmid corresponded to the
largest one, with exception of XK55-1 and XC1177-7. Five clinical
isolates (K910-5, K1319-2, K1333-2, K1335-2 and K1509-6) did not have
the ability to transfer the resistance by conjugation under the
conditions tested; however, the K1333-2 and K1319-2 isolates belong to
different species which were collected in different years with
identical number and size plasmids and ESBLs.
According to the plasmid restriction pattern from transconjugants,
group I have identical restriction profile but encode different
SHV-type ESBL: this group included K96-1 and K97-2 isolates which were
collected in different years and hospitals. This pattern had a
correlation coefficient of >0.55 with the plasmid pattern IV
that represents the plasmid from isolate K55-1 collected in the same
hospital and year as isolate K96-1, both plasmids encode SHV-2 ESBL.
Plasmid pattern II includes theK102-2 and E128-3 isolates that
corresponded to two different species and encode SHV-5 and SHV-2
enzymes respectively, these isolates were collected in different
hospitals. This situation is similar to isolates included in plasmid
pattern III (K65-1 and E86-2 isolates), both groups have a high
similarity (> 0.65 correlation coefficient). In general there is
relatedness between plasmids encoding different SHV-type ESBLs. This
situation could be explained first as plasmids are promiscuous
mobile enetic elements and can be transferred horizontally
within different enterobacterial species.29 Secondly,
one mutation distinguishes SHV-2 from SHV-5 enzymes (E240K), this
mutation could occur in an independent event from SHV-2 and selected by
the antibiotic exposure converting the enzyme to SHV-5.4,30 The
dissemination of cephalosporin resistance among nosocomial
enterobacteria strains in different hospitals in Mexico, suggests that
it could be due to horizontal plasmid transmission among clonally
unrelated strains (PFGE).
This situation highlights the need to establish a study for molecular
epidemiology of ESBLs and plasmids harboured in this kind of clinical
isolates. We hope that this study will contribute to a more complete
understanding of the spread and evolution of the SHV genes in hospital
settings.
Acknowledgments
This work was supported by grants 30938 and SALUD 2003-C01-009 from
CONACyT. Ulises Garza-Ramos was a fellow from CONACyT. We thank Bertha
Carrillo and Teresa Rojas for excellent technical assistance.
Authors want to thank Dr. Jose Sifuentes (INCMNSZ), Dra. Luz
Elena Espinosa de los Monteros (HIM), Dr. Rodolfo Gatica (HCC), Q.
Eduardo Escamilla (HNT), Dra. Guadalupe Miranda y Dr. Fortino Solórzano
(HP CMN, IMSS), MC Veronica Andrade (HNM), for providing the clinical
isolates for this study, also to Dr. Michael Dunn (Centro de Ciencias
Genómicas, UNAM), Cuernavaca, Morelos, Mexico, for reviewing the
manuscript.
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