Antibiotic resistance has been identified as one of the greatest threats to human health. Gram-negative bacteria in particular have been identified as the highest priority for new antibiotic development. Gram-negatives have an intrinsically low permeability to many antibiotics due to a combination of two cell membranes and active efflux. To rationally design new antibiotics capable of breaching this barrier, more information is required about how specific components of the Gram-negative cell envelope contribute to resistance to compounds with distinct physiochemical properties.
Ampicillin and benzylpenicillin are two β-lactam antibiotics with identical chemical structures, except for a primary amine group in ampicillin. Ampicillin has broad-spectrum activity, while benzylpenicillin is only clinically effective against Gram-positives. Primary amines have been found to promote compound accumulation in Gram-negatives, which is understood to occur through improved uptake through outer membrane porins such as OmpF in Escherichia coli. It is not known, however, how this feature may affect interaction with other cell envelope components. This study applied TraDIS to identify genes that affect ampicillin and benzylpenicillin accumulation in the model Gram-negative species E. coli, with a primary focus on cell envelope components.
Insertions that compromised the outer membrane, particularly the lipopolysaccharide layer, were found to decrease fitness under benzylpenicillin exposure, but had little effect on survival under ampicillin. These results align with expectations if benzylpenicillin is unable to pass through porins. Disruption of genes involved in the AcrAB efflux system was detrimental to survival under both antibiotics, but particularly ampicillin. Disruption of acrR, which encodes a negative regulator of acrAB expression, led to a greater increase in fitness under ampicillin than disruption of ompF, which is believed to be the main contributor to ampicillin sensitivity in E. coli. These results suggest that maintaining ampicillin efflux may be more significant to E. coli survival than inhibiting OmpF-mediated uptake.