Outer-Membrane Penetration Barriers as Components of Intrinsic Resistance to Beta-Lactam and Other Antibiotics in Escherichia coli K-12

Abstract

A new technique has been devised to investigate the penetration of antibiotics through the gram-negative outer membrane; the application here was to study intrinsic resistance of Escherichia coli K-12. Exponential cells in broth were briefly treated with 2.5 mM ethylenediaminetetraacetic acid at 5°C to disrupt the outer membrane penetration barrier, and the response of treated and untreated cells to antibiotics was compared by turbidimetry. A barrier index was derived to describe the ability of 7 beta-lactam and 10 other antibiotics to penetrate the outer membrane of strain Y10. There was correlation between the molecular weight and logio barrier index (r = 0.59, P = 0.01). The envelope mutant D22 (envA) had low barrier indexes for erythromycin, rifampin, ampicillin, and cloxacillin. For the beta-lactams, outer membrane penetration and affinity for inner membrane target site(s) triggering cell lysis were measured as independent components of the overall activity; although penetration and overall activity varied greatly, the affinities of most were within a narrow range.The limiting layers of the gram-negative cell envelope are the cytoplasmic or inner membrane (IM) and the outer membrane (OM) (29). Between the IM and OM is a murein sacculus within a tenuous space known as the periplasm, which contains a number of wall-associated enzymes (28).The OM is a barrier to molecules above a critical size; in Escherichia coli and Salmonella typhimurium, the penetration of oligopeptides (25) and oligosaccharides (8) declines sharply as the molecular weight exceeds 600, and they are essentially excluded at 1,000. Similarly, there is at least partial exclusion of some antibiotics by the OM contributing to "intrinsic" resistance (12), although in most cases the effect has not been well quantified. According to a popular model, solutes may penetrate the OM through narrow aqueous pores which restrict entry nonspecifically by size, and so provide a molecular sieving function (8). This function involves certain OM proteins which have been called "porins" (14,17,18,19,22).In this paper, we describe a technique which was used to measure the penetration of 17 antibiotics through the OM of E. coli K-12. A preliminary report has been made (30). The OM penetrability barrier was disrupted by brief treatment with ethylenediaminetetraacetic acid (EDTA), and the turbidimetric response of treated and normal cells to antibiotics was then compared over a short period of growth. (There was no adjustment of the external osmotic pressure). In support of the pore model, we found that antibiotic penetration through the OM was limited by molecular weight, and that the limits agreed well with those for oligosaccharides (8). However, it was clear that in some cases other variables must also control penetration.Beta-lactam antibiotics were differentiated with respect to penetration ofthe OM and affinity for the IM target site(s) promoting cell lysis. This gave an insight into the basis for the wide range of activities against E. coli which are encount...

High pH during Trisodium Phosphate Treatment Causes Membrane Damage and Destruction ofSalmonella entericaSerovar Enteritidis

“…As a consequence of EDTA treatment, the outer membrane becomes more permeable to agents that otherwise would not cross the cell membrane (15,23,24,29). Short-term EDTA treatments of bacteria have been used to introduce macromolecules and hydrophobic compounds through the outer membrane without affecting cell viability (16,23,33,36). In this study, short-term (30-min) treatment with EDTA alone did not affect cell viability or induce PI uptake (data not shown).…”

Differences in susceptibility to quinolones of outer membrane mutants of Salmonella typhimurium and Escherichia coli

“…The ability of drugs to pass through the bacterial outer membrane is a very important factor in their antibacterial activity and spectrum (2,10,13,14,22,23,26). There are many reports on the penetration into bacterial cells of various antimicrobial agents, especially beta-lactam antibiotics (4,8,15,22,23,26), but the penetration mechanisms of quinolones have not been studied in detail.…”

“…There are many reports on the penetration into bacterial cells of various antimicrobial agents, especially beta-lactam antibiotics (4,8,15,22,23,26), but the penetration mechanisms of quinolones have not been studied in detail.…”

Mutant Alleles of lptD Increase the Permeability of Pseudomonas aeruginosa and Define Determinants of Intrinsic Resistance to Antibiotics

“…For the most part, tolC is the major contributor to efflux in E. coli, and knockout of this gene is often used to assess whether novel compounds lack cellular activity due to efflux (16)(17)(18)(19)(20)(21)(22)(23)(24). Similarly, mutations in several genesincluding lpxC (25)(26)(27), lptD (28,29), and lptE (30, 31)-leading to increased permeability of E. coli have been described, and strains harboring such lesions are often used to assess the effect of increased cellular penetration on the bioactivity of molecules (32)(33)(34)(35)(36)(37). These three genes in particular are often targeted because of their essential role in biosynthesis and biogenesis of lipopolysaccharide (LPS), which comprises the outer leaflet of the outer membrane (OM) in Gram-negative organisms and greatly contributes to reduced permeability.…”