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Selected Abstracts

Abstract 10

Antimicrobial Agents and Chemotherapy (2003) 47(10):3332-3335

Role of Klebsiella pneumoniae OmpK35 porin in antimicrobial resistance.

Domenech-Sanchez A, Martinez-Martinez L, Hernandez-Alles S, del Carmen Conejo M, Pascual A, Tomas JM, Alberti S, Benedi VJ.
 

OmpK35 from Klebsiella pneumoniae is the homologue of Escherichia coli OmpF porin. Expression of OmpK35 in K. pneumoniae strain CSUB10R (lacking both OmpK35 and OmpK36) decreased the MICs of cephalosporins and meropenem > or = 128-fold and decreased the MICs of imipenem, ciprofloxacin, and chloramphenicol > or = 8-fold. MIC reductions by OmpK35 were 4 times (cefepime), 8 times (cefotetan, cefotaxime, and cefpirome), or 128 times (ceftazidime) higher than those caused by OmpK36, but the MICs were similar or 1 dilution lower for other evaluated agents.

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Abstract 9

Microbial Drug Resistance 2002 Winter;8(4):245-51

Mutations in gyrA and parC QRDRs are not relevant for quinolone resistance in

epidemiological unrelated Stenotrophomonas maltophilia clinical isolates.

Ribera A, Domenech-Sanchez A, Ruiz J, Benedi VJ, Jimenez de Anta MT, Vila J.

Clinical strains of Stenotrophomonas maltophilia are often highly resistant to multiple antibiotics and this resistance is steadily rising. Quinolones are included in the group of antimicrobial agents to which this microorganism is developing resistance. Therefore, the aim of this study was to analyze the epidemiological relationship among 22 clinical isolates of S. maltophilia as well as the molecular mechanisms responsible for the acquisition of quinolone-resistance in these strains. The results of the pulsed-field gel electrophoresis (PFGE) showed an heterogenicity of 82% among the strains used in the study. On the other hand, no amino acid changes were found in the quinolone resistance-determining region (QRDR) of either gyrA and parC genes among quinolone-susceptible and -resistant S. maltophilia strains. Besides, the amino acid of the GyrA found in the position equivalent to Ser-83 of E. coli was Gln instead of a Ser or Thr, the amino acids usually encountered in this position among Gram-negative bacteria. The results suggest that there is not a relationship between the presence of this Gln and the resistance to quinolones in S. maltophilia. We can conclude that, contrary to what has been described in other microorganisms, in these S. maltophilia isolates, the development of resistance to quinolones was not related to mutations in the QRDR of gyrA and parC genes. Thus, to our knowledge,  this is the first report describing this phenomenon.

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Abstract 8

Antimicrobial Agents and Chemotherapy (2002) 46(12):3926-3932

Energy-Dependent Accumulation of Norfloxacin and Porin Expression in Clinical Isolates of Klebsiella pneumoniae and Relationship to Extended-Spectrum ß-Lactamase Production

Luis Martínez-Martínez, Alvaro Pascual, María del Carmen Conejo, Isabel García, Providencia Joyanes, Antonio Doménech-Sánchez, and Vicente Javier Benedí

The relationships between porin deficiency, active efflux of fluoroquinolones, and extended-spectrum ß-lactamase (ESBL) production were determined for 53 clinical isolates of Klebsiella pneumoniae. Thirty-two ESBL-positive strains (including 22 strains expressing porins and 10 strains lacking porins) and 21 ESBL-negative strains were evaluated. Active efflux of norfloxacin was defined as a 50% increase in the accumulation of norfloxacin in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) in comparison with the corresponding basal value in the absence of CCCP. The quinolone resistance-determining regions of both gyrA and parC from 13 strains, representing all isolates with different porin profiles and with or without active efflux, were determined. Porin loss was significantly more common among ESBL-positive strains (10 of 32 [31.2%]) than among ESBL-negative strains (0 of 2 [0%]) (P < 0.01). Active efflux was observed in 7 of 10 (70%) strains lacking porins and in 4 of 43 (9.3%) strains producing porins (P < 0.001). The 11 strains showing active efflux corresponded to 3 of 21 (14.3%) ESBL-negative strains and 8 of 32 (25.5%) ESBL-positive strains (P > 0.05). Basal values of norfloxacin accumulation were higher in strains lacking active efflux than in those that had this mechanism (P < 0.05). In the absence of topoisomerase changes, the contribution of either porin loss or active efflux to
fluoroquinolone resistance in K. pneumoniae was negligible. It is concluded that among K. pneumoniae strains of clinical origin, porin loss was observed only in those producing ESBL, and that a significant number of porin-deficient strains also expressed active efflux of norfloxacin. In terms of fluoroquinolone resistance, both mechanisms are significant only in the presence of topoisomerase modifications.

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Abstract 7

Antimicrobial Agents and Chemotherapy, 2002, 46(11):3679-3682

Expression of SHV-2 ß-Lactamase and of Reduced Amounts of OmpK36 Porin in Klebsiella pneumoniae Results in Increased Resistance to Cephalosporins and Carbapenems

Brendan Crowley, Vicente J. Benedí, and Antonio Doménech-Sánchez

A Klebsiella pneumoniae clinical isolate was resistant to cefoxitin, cefotaxime, ceftazidime, ceftazidime-clavulanate, piperacillin-tazobactam (MICs, >256 ?g/ml in all cases), and meropenem (MIC, 16 µg/ml) and was intermediate to imipenem (MIC, 8 ?g/ml). Decreased expression of the OmpK36 porin and expression of an SHV-2 ß-lactamase contributed to the observed resistance to these ß-lactam-containing agents.  [pdf]

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Abstract 6

Antimicrobial Agents and Chemotherapy, 2001, 45(4):1151-1161

Novel Carbapenem-Hydrolyzing beta-Lactamase, KPC-1, from a Carbapenem-Resistant Strain of Klebsiella pneumoniae

Hesna Yigit, Anne Marie Queenan, Gregory J. Anderson, Antonio Domenech-Sanchez, James W. Biddle, Christine D. Steward, Sebastian Alberti, Karen Bush, and Fred C. Tenover

A Klebsiella pneumoniae isolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 µg/ml. The beta-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three beta-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence of blaSHV and blaTEM genes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the beta-lactamase with a pI of 6.7, KPC-1 (K.pneumoniae carbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene, blaKPC-1, was cloned in E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing beta-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing beta-lactamase, Sme-1, from Serratia marcescens S6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parent K. pneumoniae strain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance in K. pneumoniae strain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing beta-lactamase, KPC-1, although alterations in porin expression may also play a role. [pdf]

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Abstract 5

Journal of Antimicrobial Chemotherapy , 2000 46(5): 858-859.

Activity of nine antimicrobial agents against clinical isolates of Klebsiella pneumoniae producing extended-spectrum beta-lactamases and deficient or not in porins.

A. Domenech-Sanchez, A. Pascual, A.I. Suarez, D. Alvarez, V.J. Benedi,  and L. Martinez-Martinez

The activities of nine antimicrobial agents against 65 clinical isolates of Klebsiella pneumoniae producing extended-spectrum beta-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 beta-lactamase-producing Klebsiella pneumoniae. [pdf]

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Abstract 4

Antimicrobial Agents and Chemotherapy,  2000, 44(9):2382-2388

Characterization of the Extended-Spectrum beta-Lactamase Reference Strain, Klebsiella pneumoniae K6 (ATCC 700603), Which Produces the Novel Enzyme SHV-18

Klebsiella pneumoniae K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and other oxyimino-beta-lactams. A consistent reduction in the MICs of oxyimino-beta-lactams by at least 3 twofolddilutions in the presence of clavulanic acid confirmed the utilityof K. pneumoniae K6 as a quality control strain for extended-spectrum beta-lactamase (ESBL) detection. Isoelectric-focusing analysis of crude lysates of K6 demonstrated a single beta-lactamase with a pI of 7.8 and a substrate profile showing preferential hydrolysis of cefotaxime compared to ceftazidime. PCR analysis of total bacterialDNA from K6 identified the presence of a bla_SHV gene. K6 contained two large plasmids with molecular sizes of approximately 160 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 nine nucleotides, five of which resulted in amino acid substitutions:Ile to Phe at position 8, Arg to Ser at position 43, Gly to Alaat position 238, and Glu to Lys at position 240. In addition tothe production of this novel ESBL, designated SHV-18, analysisof the outer membrane proteins of K6 revealed the loss of theOmpK35 and OmpK37 porins. [pdf]

Identification and characterization of a new porin gene of Klebsiella pneumoniae:its role in beta-lactam antibiotics resistance.

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. [pdf]

Porin expression in clinical isolates of Klebsiella pneumoniae.

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 beta-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. [pdf]

Klebsiella pneumoniae lipopolysaccharide O-typing: revision of prototype strains and O-group distribution among clinical isolates of different sources and countries.

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. [pdf]