The porin and the permeating antibiotic: A selective diffusion barrier in gram-negative bacteria

Gram-negative bacteria are responsible for a large proportion of antibiotic resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channel

In vivo selection of porin-deficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expanded-spectrum-cephalosporins.

Four Klebsiella pneumoniae isolates (LB1, LB2, LB3, and LB4) with increased antimicrobial resistance were obtained from the same patient. The four isolates were indistinguishable in biotype, plasmid content, lipopolysaccharide, and DNA analysis by pulse-field gel electrophoresis. Isolate LB1 made TEM-1 and SHV-1 beta-lactamases. Isolates LB2, LB3, and LB4 produced SHV-5 in addition to TEM-1 and SHV-1. MICs of cefoxitin, ceftazidime, and cefotaxime against LB1 were 4, 1, and 0.06 micrograms/ml, respectively. MICs of ceftazidime against K. pneumoniae LB2, LB3, and LB4 were > 256 micrograms/ml, and those of cefotaxime were 2, 4, and 64 micrograms/ml, respectively. MICs of cefoxitin against K. pneumoniae LB2 and LB3 were 4 micrograms/ml, but that against K. pneumoniae LB4 was 128 micrgrams/ml. K. pneumoniae LB4 could transfer resistance to ceftazidime and cefotaxime, but not that to cefoxitin, to Escherichia coli. Isolate LB4 and cefoxitin-resistant laboratory mutants lacked an outer membrane protein of about 35 kDa whose molecular mass, mode of isolation, resistance to proteases, and reaction with a porin-specific antiserum suggested that it was a porin. MICs of cefoxitin and cefotaxime reverted to 4 and 2 micrograms/ml, respectively, when isolate LB4 was transformed with a gene coding for the K. pneumoniae porin OmpK36. We conclude that the increased resistance to cefoxitin and expanded-spectrum cephalosporins of isolate LB4 was due to loss of a porin channel for antibiotic uptake

VIM-2 beta-lactamase in Pseudomonas aeruginosa isolates from Zagreb, Croatia

The aim of this investigation was to characterize metallo-beta-lactamases (MBLs) in Pseudomonas aeruginosa isolates from Zagreb, Croatia. One hundred P. aeruginosa isolates with reduced susceptibility to either imipenem or meropenem were tested for the production of MBLs by MBL-Etest. The susceptibility to a wide range of antibiotics was determined by broth microdilution method. The presence of bla(MBL) genes was detected by polymerase chain reaction (PCR). Hydrolysis of 0.1 mM imipenem by crude enzyme preparations of beta-lactamases was monitored by UV spectrophotometer. Outer membrane proteins were prepared and analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Six out of 100 isolates were positive for MBLs by Etest. All strains were resistant to gentamicin, ceftazidime and cefotaxime, and all except 1 were resistant to imipenem. Six strains positive for MBLs by Etest were identified as VIM MBL-producers by PCR. Sequencing of bla(VIM) genes revealed the production of VIM-2 beta-lactamase in all 6 strains. This investigation proved the occurrence of VIM-2 beta-lactamase among P. aeruginosa strains from Zagreb, Croatia. VIM-2 beta-lactamase with similar properties has previously been described in another region of Croatia and in Italy, France, Spain, Greece, Taiwan and South Korea, suggesting that this type of enzyme is widespread in the Mediterranean region of Europe and in the Far East