Mechanism of resistance to carbapenems in Ps. aeruginosa (Part 1)

1- Loss of OprD
The outer membrane of gram-negative bacteria constitutes an asymmetric bilayer, in which the inner monolayer is composed of phospholipid, whereas the outer monolayer contains the unique lipid species lipopolysaccharide (LPS) (fig. 6). Porins are group of proteins forming trans-outer-membrane, water filled channels. In general porins have monomer molecular weights in the range of 28 KD to 48 KD, are present in membrane as oligomers (usually trimers), are often strongly but non-covalentely associated with the underlying peptidoglycan and with LPS, and have a high content of β-sheet structure. In Ps. aeruginosa, OprB, OprC, OprD, OprE, OprF, OprP and OprO have been identified as porins (Hancock et al., 1990).
Porins are generally divided into two classes: non-specific (general) porins and specific porins (Nikaido and Vaara, 1985). General porins form water-filled channels that permit the passive diffusion of hydrophilic molecules below a certain size. Specific porins also produce water-filled channels, which contain stereospecific substrate-binding sites (Hancock, 1987). OprF is a major non-specific porin in Ps. aeruginosa, it allows the passage of saccharides with molecular weights of approximately 3,000. however, only 400 out of 200,000 OprF molecules per cell are proposed to form such large channels; the rest appear to form small channels that are predicted to be antibiotic impermeable. OprC and OprE are also general porins with small channel size. The above cited data explains the low outer membrane permeability of Ps. aeruginosa compared to E. coli. This, in turn, was proposed to be the major basis for the high intrinsic reistance of Ps. aeruginosa to hydrophilic antibiotics (Nikaido and Hancock, 1986).

Fig 6: Gram negative bacteria is surrounded by the outer membrane, which functions as an efficient permeability barrier because it contains lipopolysaccharide (LPS) and porins with narrow, restrictive channels (Nikaido, 1994).

To overcome the low permeability and to permit the effective uptake of essential nutrients available at low concentrations in the medium, several specific porins are present in Ps. aeruginosa outer membrane. OprB, which is induced by the presence of glucose (Hancock and Carey, 1980), form a channel that prefers D-glucose and D-xylose. OprP is induced by growth under phosphate starvation conditions (Hancock et al., 1982). Porin OprO, which is highly homologus to OprP, forms pyrophosphate-specific channels (Hancock et al., 1992). OprD was discovered due to its role in the facilitated uptake of imipenem. However the natural substrate for OprD is not imipenem, but its structural analogues, presumably basic amino acids and small peptides containing those amino acids. Lack of an outer membrane protein D2 (now called OprD) with molecular weight range 46 KD lead to mainly resistance to imipenem but only a low degree of resistance to meropenem (Naenna et al., 2010).
Genetic analysis shows that the elimination of OprD results from gene rearrangements in the OprD coding region or the upstream promoter region (Trias and Nikaido, 1990a). The loss of OprD expression occur at the levels of transcription and translation (Köhler et al., 1997); (Kolayli et al., 2004). Mutations (base transitions or deletions) in oprD structural gene generate a premature stop codon and early termination of translation. Deletions have also been shown to interfere with expression of oprD at the transcriptional level. (Yoneyama and Nakae, 1993) observed a large deletion encompassing the promoter, initiation codon and putative Shine-Dalgarno sequence of oprD preventing transcription initiation. In addition to the deletion of oprD gene, (Wolter et al., 2004) have shown the first report of carbapenem resistance occurring through insertional inactivation of the oprD gene by insertion sequence (IS) elements. It was suggested that elimination of OprD porin from most imipenem resistant Ps. aeruginosa isolates is due to efficient selection of oprD gene mutation. Thus, imipenem resistance mechanism of Ps. aeruginosa results from a loss-of function mutation, and detection of the mutated structural gene alone is not always possible to determine this type of resistance (Naenna et al., 2010).