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Enterobacterial outer membrane
proteins and porins: isolation and polyacrylamide gel electrophoretic analysis.
A. Doménech-Sánchez
y V.J. Benedí
En Antimicrobial resistance,
a laboratory approach. Ed. V.J. Benedí, R. Cantón, M.A. Domínguez,
L. Martínez y J. Vila
Coimpress S.A., Madrid.
2000. ISBN: 84-607-1399-7 [pdf]
Activity of nine antimicrobial
agents against clinical isolates of Klebsiella pneumoniae producing extended-spectrum
ß-lactamases and deficient or not in porins.
A. Doménech-Sánchez,
A. Pascual, A.I. Suárez, D. Álvarez, V.J. Benedí,
L. Martínez-Martínez
Journal of Antimicrobial
Chemotherapy 2000 46(5): 858-859. [pdf]
ABSTRACT: The activities
of nine antimicrobial agents against 65 clinical isolates of Klebsiella
pneumoniae producing extended-spectrum ß-lactamases and expressing
(50 strains) or not expressing (15 strains) porins was evaluated. Meropenem
and imipenem were the most active agents against these strains, being meropenem
slightly affected in three strains defficient in porins. Among the cephalosporins,
cefepime and cefpirome were the most active agents. All the cephalosporins
were higly affected by porin loss. Ciprofloxacin and amikacin showed variable
activity against extended-spectrum ß-lactamase-producing K. pneumoniae.
Characterization of the
extended-spectrum ß-lactamase (ESBL) reference strain, Klebsiella
pneumoniae K6 (ATCC 700603), which produces the novel enzyme SHV-18.
J.K. Rasheed, J.M. Swenson,
H. Yigit, G.J. Anderson, J.W. Biddle, A.M. Queenan, A.
Doménech-Sánchez, M.J. Ferraro,
G.A. Jacoby, F.C. Tenover
Antimicrobial Agents and
Chemotherapy 2000. 44(9):2382-2388.[pdf]
ABSTRACT: Klebsiella pneumoniae
K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and the
oxyimino-ß-lactams. A consistent reduction in the MICs of oxyimino-ß-lactams
by at least 3 two-fold dilutions in the presence of clavulanic acid confirmed
the utility of K. pneumoniae K6 as a quality control strain for extended-spectrumß-lactamase
(ESBL) production. Isoelectric focusing analysis of crude lysates
of K6 demonstrated a single ß-lactamase with a pI of 7.8. PCR
analysis of total bacterial DNA from K6 identified the presence of a bla-SHV
gene. Plasmid profile analysis showed that K6 contained two large
plasmids with molecular sizes of approximately 160 kb and 80 kb.
Hybridization of plasmid DNA with a bla-SHV -specific probe indicated that
a bla-SHV gene was encoded on the 80 kb plasmid, which was shown to transfer
resistance to ceftazidime in conjugal mating experiments with Escherichia
coli HB101. DNA sequencing of this bla-SHV-related gene revealed
that it differs from bla-SHV-1 at eight nucleotides, five of which resulted
in amino acid substitutions: Ile-to-Phe at position 8, Arg-to-Ser
at position 43, Gly-to-Ala at position 238, and Glu-to-Lys at position
240. In addition to the production of this novel ESBL, designated
SHV-18, analysis of the outer membrane proteins of K6 revealed the
loss of the OmpK35 and OmpK37 porins.
Identification and characterization
of a new porin gene of Klebsiella pneumoniae:its role in ß-lactam
antibiotics resistance.
A. Doménech-Sánchez,
S. Hernández-Allés, L. Martínez-Martínez, V.J.
Benedí, S. Albertí
Journal of Bacteriology
1999. 181:2726-2732. [pdf]
ABSTRACT: Klebsiella pneumoniae
porin genes were analyzed to detect mutations accounting for the porin
deficiency observed in many Beta-lactam-resistant strains. PCR and Southern
blot analysis revealed the existence of a third porin gene in addition
to the OmpK36 and OmpK35 porin genes previously described. This new porin
gene was designated ompK37 and is present in all of the clinical isolates
tested. The OmpK37 porin gene was cloned, sequenced, and overexpressed
in Escherichia coli. In contrast to that of the major porins, OmpK37 porin
expression was only detectable by Western blot analysis in porin-deficient
Beta-lactam-resistant strains, suggesting strong down regulation under
standard laboratory conditions. Functional characterization suggested a
narrower pore for the OmpK37 porin than for K. pneumoniae porins OmpK36
and OmpK35. This correlated with the susceptibility to certain Beta-lactam
antibiotics, since a K. pneumoniae strain expressing porin OmpK37, but
not porin OmpK36 or OmpK35, was less susceptible to Beta-lactam antibiotics
than the same strain expressing either porin OmpK36 or OmpK35.
Porin expression in clinical
isolates of Klebsiella pneumoniae.
S. Hernández-Allés,
S. Albertí, D. Álvarez, A. Doménech-Sánchez,
L. Martínez-Martínez, J. Gil, J.M. Tomás, V.J. Benedí.
Microbiology 1999. 145:673-679.
[pdf]
ABSTRACT: Two porins, OmpK36
and OmpK35, have been described previously in Klebsiella pneumoniae,and
they are homologous to the Escherichia coli porins OmpC and OmpF, respectively,
at both the DNA and amino acid levels. Optimal resolution of the two K.
pneumoniae porins by electrophoresis on polyacrylamide gels is not achieved
using gel systems already described for E. coli and requires modifications
of the bisacrylamide content of the resolving gels. Once resolved, identification
of porins OmpK36 and OmpK35 cannot be based solely on their apparent molecular
masses since in some strains the OmpK36 porin migrates faster than the
OmpK35 porin, whilst in other strains OmpK35 is the faster-migrating porin.
Expression of OmpK35 porin is increased in low-osmolarity medium and, combined
with Western blot analysis, this allows for the identification of both
porins. Application of this identification system showed that most isolates
lacking expression of extended-spectrum ß-lactamases express the
two porins, whereas most isolates producing these -lactamases express only
porin OmpK36, and the OmpK35 porin is either very low or not expressed.
Klebsiella pneumoniae lipopolysaccharide
O-typing: revision of prototype strains and O-group distribution among
clinical isolates of different sources and countries.
D. S. Hansen, F. Mestre,
S. Albertí, S. Hernández-Allés, D. Alvarez, A.
Doménech-Sánchez, J. Gil, S.
Merino, J.M. Tomás, and V.J. Benedí.
Journal of Clinical Microbiology
1999. 37:56-62 [pdf]
ABSTRACT: We have previously
described an inhibition enzyme-linked immunosorbent assay method for the
O typing of O1 lipopolysaccharide from Klebsiella pneumoniae which overcomes
the technical problems and limitations of the classical O-typing method.
In this study, we have extended the method to all of thecurrently recognized
O types. The method was validated by studying the prototype strains that
have defined the O groups by the classical tube agglutinatination O-typing
method. Based on these results, we confirmed the O types of 60 of 64 typeable
strains, and we propose a revised O-antigenic scheme, with minor but necessary
changes, consisting of serogroups or serotypes O1, O2, O2ac, O3, O4, O5,
O7, O8, and O12. Application of this typing method to 638 K. pneumoniae
clinical isolates from Denmark, Spain, and the United States from different
sources (blood, urine, and others) showed that up to 80% of these isolates
belong to serotypes or serogroups O1, O2, O3, and O5, independently of
the source of isolation, and that a major group of nontypeable isolates,
representing about 17% of the total, consists of half O+ and half O- strains.
Differences were observed, however, in the prevalence of the lipopolysaccharide
O types or groups, depending on the country and isolation source.
AUTORES: A. Doménech-Sánchez,
S. Hernández-Allés, L. Martínez-Martínez, V.J.
Benedí
TITULO: "Cloning of porin OmpK35 and the roles
of the three porins of Klebsiella pneumoniae in the activity of antimicrobial
agents"
TIPO DE PARTICIPACIÓN: Presentación
de póster.
CONGRESO: 39th Interscience Conference on
Antimicrobial Agents and Chemotherapy.
LUGAR DE CELEBRACIÓN: San Francisco
(USA) AÑO: 1999
AUTORES: A. Doménech-Sánchez,
S. Hernández-Allés, L. Martínez-Martínez, V.J.
Benedí
TITULO: "Porin expression and frequency of
selection of antimicrobial-resistant mutants of Klebsiella pneumoniae strains
producing or not extended-spectrum ß-lactamase"
TIPO DE PARTICIPACIÓN: Sesión
oral ("slide session").
CONGRESO: 38th Interscience Conference on
Antimicrobial Agents and Chemotherapy.
LUGAR DE CELEBRACIÓN: San Diego
(USA) AÑO: 1998
AUTORES: S. Hernández-Allés,
A.
Doménech-Sánchez, S. Albertí,
L. Martínez-Martínez, T. Schirmer, V.J. Benedí
TITULO: "Identification and characterization
of a novel porin from Klebsiella pneumoniae""
TIPO DE PARTICIPACIÓN: Presentación
de póster
CONGRESO: 8th International Congress on Infectious
Diseases
LUGAR DE CELEBRACIÓN: Boston
(USA) AÑO: 1998
AUTORES: A.
Doménech-Sánchez, S. Hernández-Allés
y V.J.Benedí
TITULO: "Clonaje y caracterización
de porinas de Klebsiella pneumoniae"
TIPO DE PARTICIPACIÓN: Presentación
de póster
CONGRESO: "XVI Congreso de la Sociedad Española
de Microbiología"
LUGAR DE CELEBRACIÓN: Barcelona (España)
AÑO: 1997
AUTORES: -
TITULO: -
TIPO DE PARTICIPACIÓN: Oyente.
CONGRESO: "I Congreso de Investigación
para estudiantes de Medicina y Ciencias de la Salud"
LUGAR DE CELEBRACIÓN: Reus (España)
AÑO: 1996
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