Study of OmpK35 and OmpK36 Expression in Carbapenem Resistant ESBL Producing Clinical Isolates of Klebsiella pneumoniae

Background: Carbapenem resistant extended spectrum β-lactamase (ESBL) producing Klebsiella pneumoniae (K. pneumoniae) is increasing worldwide. Carbapenem resistance (CR) has been attributed not only to production of carbapenemases but also to permeability barriers due to outer membrane proteins (OmpK35 and OmpK36) disruption. Objective: Phenotypic detection of CR among ESBL producing K. pneumoniae isolates, followed by the evaluation of the role of ompK35 and ompK36 gene expression among carbapenem resistant K. pneumoniae (CR-KP) isolates. Methods: 100 ESBL producing K. pneumoniae isolates were included in this study. Minimum inhibitory concentration (MIC) of imipenem was performed for all isolates by broth microdilution method. For CR-KP isolates, phenotypic detection of K. pneumoniae carbapenemase (KPC), metallo-β-lactamase (MBL) and AmpC enzymes was performed followed by Realtime qRT-PCR to detect and quantify ompK35 and ompK36 gene expression. Results: 42% of our isolates were carbapenem resistant, and all of them were KPC producers either singly or in combination with MBL and/or AmpC production. Reduced expression of both ompK35 and ompK36 was detected in (52.38%) of CR-KP isolates, while reduced expression of ompK36 or ompK35 alone was found in (2.38%) and (33.33%) respectively. Twenty of 42 CR-KP isolates (47.62%), showing reduced ompK35 and ompK36 expression, exhibited high level resistance (HLR) (>32 μg/ml) to imipenem. There was a significant correlation between reduced expression of ompK36 and increase MIC values (p < 0.05). The combined production of MBL or AmpC together with reduced expression of ompK35 and/or ompK36 resulted in significant increase in imipenem MIC (p < 0.05). Conclusion: The combined OmpK35/OmpK36 loss resulted in HLR. However OmpK36 seems to play a major role in those strains. Imipenem MIC was markedly increased among K. pneumoniae showing carbapenemase and/or AmpC production together with loss of OmpK35 and/or OmpK36.

<i>In Vitro</i>Activity of Colistin and Vancomycin or Azithromycin Combinations on Extensively Drug Resistant <i>Acinetobacter baumannii</i>Clinical Isolates

Background: Extensively drug resistant Acinetobacter baumannii (XDR-AB) presents an increasing challenge to health care in Egypt as they are among the most common bacteria isolated in hospital setting. Treatment of such infections usually involves the use of antimicrobial agents in combination. Various combinations have been proposed, with colistin serving as the backbone in many of them even for colistin resistant isolates. Aim: The study was conducted in order to test the in vitro combined effects of colistin and vancomycin or azithromycin against (XDR-AB) causing infections at Alexandria Main University Hospital in Egypt, in an attempt to detect the possibility of a beneficial combination therapy. Material/Methods: Thirty XDR-AB clinical isolates were included in the study. Antibiotic susceptibility testing was performed using automated Vitek 2 compact system and disc diffusion method. Colistin antibiotic disc diffusion test was compared with broth microdilution method. Organisms were also tested against colistin and vancomycin or azithromycin in combination using checkerboard synergy test and FICI (Fractional Inhibitory Concentration Index) was calculated. Synergy was defined as a FICI of ≤0.5. Results: On comparing the two methods used to detect susceptibility to colistin to broth microdilution for MIC (minimum inhibitory concentration) determination, as a reference method, the Vitek showed 100% categorical agreement (CA), on the other hand, the disc diffusion showed CA of 93% with very major errors. Synergy was detected for all isolates (100%) when combining colistin with vancomycin (FICI mean = 0.08). As for azithromycin, 21 strains had FICI range from 0.7 to 1.001, denoting indifference;the remaining 9 strains showed synergy with FICI range from 0.06 to 0.241. The mean colistin/azithromycin FICI was 0.71 for the 30 isolates. Conclusion: These findings suggest that regimens containing vancomycin may confer therapeutic benefit for infection due to XDR-AB;however, other methods (time-kill assay) should be used to confirm such synergy. Furthermore, the optimal combination treatment for serious XDR-AB infection should be addressed in a prospective clinical trial.