Identification of porins in outer membrane of Proteus, Morganella, and Providencia spp. and their role in outer membrane permeation of beta-lactams

Abstract

Proteus mirabiis, Proteus vulgaris, MorganeUa morganii, Providencia rettgeri, and Providencia alcalifaciens, which were once classified into the same genus, Proteus, were studied. Cefoxitin-resistant mutants from these species were isolated, and it was confirmed that the resistance was attributed to the lack of an outer membrane protein, resulting in a significant decrease in the penetration of hydrophilic cephalosporins through the outer membrane. Comparison of the mutant strains with their parental strains in the diffusion rates of six monoanionic cephalosporins, a zwitterionic cephalosporin (cephaloridine), and a divalent anionic cephalosporin (cephalosporin C) suggested that each species had only one kind of porin protein, with molecular weights of 40,000 (Proteus mirabilis) or 37,000 (the other four species) and that the porins formed channels with cation selectivity, except for Proteus vulgaris. Porin proteins were purified from all the bacterial species except Providencia alcalifaciens, and the radius of the pores formed by the purified porins was estimated by the use of the liposome swelling assay. The pore radii were estimated to be approximately 0.59 nm (Proteus mirabilis), 0.63 nm (Proteus vulgaris), 0.58 nm (Providencia rettgeri), and 0.60 nm (M. morganiO), similar to the size of the pore radius of Escherichia coli porins.Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia rettgeri, and Providencia alcalifaciens were once classified into the same genus, Proteus (7), and, together with Serratia marcescens and Pseudomonas aeruginosa, are known as opportunistic pathogens. Except for Proteus mirabilis, these species show low susceptibility to many antibiotics. One of the reasons for such an intrinsic resistance may be the barrier effect of the outer membrane on antibiotic permeation. In the case of Pseudomonas aeruginosa, there is evidence suggesting that intrinsic resistance involves the outer membrane (1, 4, 32). However, the characterization of the outer membrane as the permeation route of antibiotics in the former Proteus species is incomplete. In our preliminary work (24), Proteus mirabilis N-51 was found to produce only a single major porinlike protein, with a molecular weight of 40,000. This 40K protein contributed to the bacterial susceptibility to cephalosporins and tetracycline. The present investigation was undertaken as an extension of the previous study to identify the porin proteins produced by Proteus vulgaris, M. morganii, Providencia rettgeri, and Providencia alcalifaciens and to evaluate the porin pores of these four species and of Proteus mirabilis as permeation routes for ,-lactam antibiotics. MATERIALS AND METHODSBacterial strains and P-lactamase production. Proteus mirabilis N-51, Proteus vulgaris K22-2, Providencia rettgeri RE-18, and Providencia alcalifaciens IN-06 are clinical isolates which barely produce 1-lactamase activity under usual growth conditions. M. morganii 1510/9, which is a mutant strain with lower 1-lactamase activity, was isolated from strain 151...

Construction and characterization of Pseudomonas aeruginosa protein F-deficient mutants after in vitro and in vivo insertion mutagenesis of the cloned gene

“…(Tables 2 and 3). This was in contrast to results for E. coli and other members of the family Enterobacteriaceae in which porin-deficient mutants were 8-to 32-fold more resistant to certain antibiotics (4,7,17,20,23 (Table 3). This dichotomy in levels of increase in resistance to P-lactam antibiotics of porin-deficient E. coli strains (i.e., large versus small increases) has been previously noted by other researchers (7,17).…”

“…A role for some porins in antibiotic uptake has been proposed. This proposal is based largely on observations, in strains of the family Enterobacteriaceae, showing that cells lacking porin proteins in their outer membranes were more resistant than wild-type strains to hydrophilic antibiotics, particularly P-lactams (7,17,18,20,23). However, for some of the more recently introduced P-lactams, there is still some dispute, based on similar studies, as to the role of Escherichia coli porins in their uptake across the outer membrane (7,17)..…”

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

“…Biochemical studies of porins from Proteus vulgaris, Proteus mirabilis, Morganella morganii, Providencia rettgeri, and Providencia alcalifaciens suggested that each of these species produced predominantly a single porin with a subunit size of around 35 to 37 kDa (422). Proteoliposome swelling studies showed pore sizes similar to that in E. coli porins, and the channels favored more hydrophilic members among cephalosporins, just as in E. coli (462).…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…In an extensive study in 1987 (308), it was shown that two major OM proteins (presumably porins) are produced in Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia rettgeri, and Providencia alcalifaciens and that the major porin in every case had channel properties similar to those of E. coli OmpF. Furthermore, in vitro selection with cefoxitin led to the isolation of mutants deficient in the major porin in each species.…”