Cellular impermeability and uptake of biocides and antibiotics in Gram-negative bacteria S.P. Denyer and J.-Y. Maillard School of Pharmacy and Biomolecular Sciences, University of Brighton, UK 1. SUMMARY The principal targets for antibacterial agents reside at the cytoplasm and cytoplasmic membrane, damage to other structures often arising from initial events at these loci. The Gram-negative bacteria offer a complex barrier system to biocides and antibiotics, regulating, and sometimes prevent- ing, their passage to target regions. Routes of entry differ between hydrophobic and hydrophilic agents, often with a structure dependency; specialized uptake mechanisms are exploited and portage transport can occur for pro-drug antibacterials. Uptake isotherms offer insight into the sorption process and can sometimes shed light on biocide mechanisms of action. The multi-component barrier system of Gram-negative bacteria offers opportunities for pheno- typic resistance development where partitioning or exclusion minimizes the delivery of an antibacterial agent to the target site. Active efflux processes are recognized as increasingly relevant mechanisms for resistance, potentially offering routes to biocide:antibiotic cross-resistance. These mecha- nisms may be targeted directly in an attempt to compromise their role in microbial survival. 2. INTRODUCTION Biocides, antimicrobial agents with a disinfectant, antiseptic or preservative activity, have been used for centuries, originally in the preservation of water and foodstuffs, and later for antisepsis. Since the beginning of the 20th century, there has been an explosion in the number of biocides available, finding broad applications from simple cleaning to sterilization. The balance of antimicrobial effectiveness against adverse effects (e.g. corrosion, toxicity) is of prime importance in determining the application to which a biocidal formulation is used. In terms of antimicrobial activity, biocides have been classified according to the type of micro-organism inactivated (Favero and Bond 1991), prions and spores being the least susceptible to biocides. Among vegetative bacteria, mycobacteria are deemed to be the least sensitive to biocides, followed by Gram-negative and then Gram-positive bacteria. This classification is primarily based on the difference in permeability/imperme- ability of the micro-organism to an active agent. The phenomenon of cellular impermeability is partic- ularly important in the resistance of mycobacteria and Gram-negative bacteria (Hancock 1997), since target sites for biocides (Denyer and Stewart 1998; Maillard 2002) are often situated within the cell and particularly, at the cytoplasmic membrane level or within the cytoplasm. The 1. Summary, 35S 2. Introduction, 35S 3. The structure of the Gram-negative bacterial cell envelope, 36S 3.1. Structural organization and composition of the outer membrane, 36S 3.1.1. Lipopolysaccharides (LPS), 36S 3.1.2. Porins, 37S 3.1.3. Periplasmic space, 37S 3.1.4. Peptidoglycan, 37S 3.2. Environmental influences (plasticity), 38S 3.3. Barrier function rather than target site, 38S 4. Biocide interactions, 38S 5. Molecular characteristics required for the penetration of biocides, 39S 6. Strategies adopted by Gram-negative bacteria to reduce penetration, 39S 6.1. Outer membrane changes, 40S 6.2. Loss of porin proteins, 40S 6.3. Other mechanisms, 41S 7. Strategies to overcome penetration constraints, 41S 7.1. Compromising the permeability barrier, 41S 7.2. Intracellular delivery of biocides, 41S 7.3. Ultrasonic enhancement of action, 42S 7.4. Self-promoted uptake for cationic antimicrobial peptides, 42S 7.5. Other strategies, 42S 8. Conclusion, 42S 9. References, 43S Correspondence to: Prof. S.P. Denyer, School of Pharmacy and Biomolecular Sciences, University of Brighton, Cockcroft Building, Moulsecoomb, Brighton BN2 4GJ, UK. ª 2002 The Society for Applied Microbiology Journal of Applied Microbiology Symposium Supplement 2002, 92, 35S–45S