Vicente J. Benedí

Roles of β-Lactamases and Porins in Activities of Carbapenems and Cephalosporins against Klebsiella pneumoniae

ABSTRACT Two clinical isolates of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae were noted to be less susceptible than expected to imipenem. Both were missing outer membrane proteins that serve as channels for antibiotic entry. The role of β-lactamase in resistance was investigated by eliminating the original ESBL and introducing plasmids encoding various ESBLs and AmpC β-lactamase types, by studying the effect of an increased inoculum, and by evaluating interactions with β-lactamase inhibitors. The contribution of porin deficiency was investigated by restoring a functional ompK36 gene on a plasmid. Plasmids encoding AmpC-type β-lactamases provided resistance to imipenem (up to 64 μg/ml) and meropenem (up to 16 μg/ml) in strains deficient in porins. Carbapenem resistance showed little inoculum effect, was not affected by clavulanate but was blocked by BRL 42715, and was diminished if OmpK36 porin was restored. Plasmids encoding TEM- and SHV-type ESBLs conferred resistance to cefepime and cefpirome, as well as to earlier oxyimino-β-lactams. This resistance was magnified with an increased inoculum, was blocked by clavulanate, and was also lowered by OmpK36 porin restoration. In addition, SHV-2 β-lactamase had a small effect on carbapenem resistance (imipenem MIC, 4 μg/ml, increasing to 16 μg/ml with a higher inoculum) when porins were absent. In K. pneumoniae porin loss can thus augment resistance provided either by TEM- or SHV-type ESBLs or by plasmid-mediated AmpC enzymes to include the latest oxyimino-β-lactams and carbapenems.

Virulence Characteristics of Klebsiella and Clinical Manifestations of K. pneumoniae Bloodstream Infections

Differences in clinical manifestations are due to virulence factors expressed by the organism.

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 beta-lactamases express only porin OmpK36, and the OmpK35 porin is either very low or not expressed.

Role of the htrA Gene in Klebsiella pneumoniae Virulence

ABSTRACT We recently described the use of mini-Tn5 to generate complement-sensitive mutants derived from a complement-resistant Klebsiella pneumoniae clinical isolate deficient in the lipopolysaccharide O side chain. One mutant with a reduced capacity to survive in nonimmune human sera carried the transposon inserted in the htrA gene. We cloned and sequenced the gene and predicted from the deduced amino acid sequence that the putative HtrA homolog contains structural features similar to those of previously described HtrA proteins. To investigate the biological functions and the role of the htrA gene in the virulence of K. pneumoniae, we constructed an isogenic mutant by insertion-duplication mutagenesis. Characterization of the mutant showed that it had greater sensitivity to temperature (50°C) and oxidative stress (H2O2) than the parent strain. Furthermore, the htrA mutant produced less capsule, bound more molecules of complement component C3, and was more sensitive to complement and whole-blood killing than was the parent strain. Finally, disruption of the htrA gene in a virulent K. pneumoniae strain caused a reduction of its virulence in a mice model. Our results indicate that the htrA gene plays an important role in the virulence of K. pneumoniae.

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

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

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

ABSTRACT The relationships between porin deficiency, active efflux of fluoroquinolones, and extended-spectrum β-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.

Capsular polysaccharide is a major complement resistance factor in lipopolysaccharide O side chain-deficient Klebsiella pneumoniae clinical isolates

ABSTRACT We have previously demonstrated the existence of Klebsiella pneumoniae clinical isolates deficient in the lipopolysaccharide O side chain, the major factor for resistance to complement-mediated killing in this bacterial species. These isolates are complement resistant, and their mechanisms to resist complement were investigated by selecting transposon-generated complement-sensitive mutants. One mutant with a drastically reduced capacity to grow in nonimmune human serum carried the transposon inserted in an open reading frame of a gene cluster involved in capsule synthesis. This mutant produced less capsule, bound more molecules of the complement component C3, and was more sensitive to complement-mediated and opsonophagocytic killings than was the parent strain. Four additional clinical isolates representing four different K serotypes were studied, and results showed that capsular polysaccharide is a major complement resistance factor in these O side chain-deficient isolates.

Increase of Enterobacter in neonatal sepsis: a twenty-two-year study.

Background. Data on the incidence of Enterobacter infections in neonates over prolonged periods of time are scant. We determined the epidemiology of Enterobacter sepsis and/or meningitis and the trends of infection in a neonatal unit. Methods. Retrospective review of sepsis and/or meningitis in inborn neonates admitted to Son Dureta University Hospital during a 22-year period. Molecular study by ribotyping of the Enterobacter strains isolated from 1995 to 1997. Results. There were 513 cases of culture-proved sepsis and/or meningitis in neonates. In late onset infections Klebsiella pneumoniae and Staphylococcus epidermidis were the most frequent isolates in the period 1977 through 1991. Enterobacter was the most common isolate in the period 1992 through 1998. During this latter period Candida infections also increased, and the resistance rate of Enterobacter to cefotaxime was higher (59.2%). Decrease in early onset infections and increase in late onsets (4.6/1000 live births) were observed in the second period. From 1977 to 1998, 45 episodes of sepsis and/or meningitis by Enterobacter species were identified in 44 patients (8.7% of all neonatal bacteremias). Three patients with Enterobacter bacteremia died (6.6%, 0.03/1000 live births). During 1995 through 1997 5 different clones causing sepsis were identified and 3 were predominant. In 1997 there was an outbreak of Enterobacter disease. After cleaning, cohort nursing and hygiene reinforcement, Enterobacter was not isolated in the next 2 years. No change in the antibiotic policy was made. Conclusions. We observed a resurgence of Enterobacter infections in our neonatal intensive care unit. The sudden disappearance of this microorganism after reinforcement of hygienic measures, without withdrawing cefotaxime, confirms the importance of patient-to-patient transmission of this nosocomial infection. Further studies are needed to establish the role of antibiotics in the emergence of microorganisms in neonatal intensive care units.

A Gene, uge, Is Essential for Klebsiella pneumoniae Virulence

ABSTRACT Klebsiella pneumoniae strains typically express both smooth lipopolysaccharide (LPS) with O antigen molecules and capsule polysaccharide (K antigen) on the surface. A single mutation in a gene that codes for a UDP galacturonate 4-epimerase (uge) renders a strain with the O−:K− phenotype (lack of capsule and LPS without O antigen molecules and outer core oligosaccharide). The uge gene was present in all the K. pneumoniae strains tested. The K. pneumoniae uge mutants were unable to produce experimental urinary tract infections in rats and were completely avirulent in two different animal models (septicemia and pneumonia). Reintroduction of the single uge wild-type gene in the corresponding mutants completely restored the wild-type phenotype (presence of capsule and smooth LPS) independently of the O or K serotype of the wild type. Furthermore, complemented uge mutants recovered the ability to produce experimental urinary tract infections in rats and virulence in the septicemia and pneumonia animal models.

Cloning, Sequencing, and Role in Serum Susceptibility of Porin II from Mesophilic Aeromonas hydrophila

ABSTRACT We cloned and sequenced the structural gene for Aeromonas hydrophila porin II from strain AH-3 (serogroup O:34). The genetic position of this gene, like that of ompF inEscherichia coli, is adjacent to aspC and transcribed in the same direction. However, upstream of the porin II gene no similarities with E. coli were found. We obtained defined insertion mutants in porin II gene either in A. hydrophila (O:34) or A. veronii sobria (serogroup O:11) serum-resistant or -sensitive strains. Furthermore, we complemented these mutants with a plasmid harboring only the porin II gene, which allowed us to define the role of porin II as an important surface molecule involved in serum susceptibility and C1q binding in these strains.