A key step in Escherichia coli K1 meningitis is the crossing of the blood-brain barrier by the bacteria in order to gain entry into the central nervous system (CNS). In this study, a novel ex vivo model to study E. coli K1 invasion of the CNS is described that uses the African migratory locust, Locusta migratoria. By injecting bacteria into isolated locust head capsules, it was demonstrated that E. coli K1 invade the locust brain within 2 h in numbers depending on the concentration of bacteria injected. Using several mutants derived from K1, it was shown that outer-membrane protein A is a critical bacterial determinant required for the E. coli K1 invasion. The isogenic gene-deletion mutants, DfimH, Dcnf1, DneuDB and a rough LPS mutant showed significantly reduced invasion of locust brain. This novel model for the study of E. coli K1 pathogenesis offers several advantages over existing mammalian models in relation to its relative ease of use, cost-effectiveness and ethical acceptability.
INTRODUCTIONNeonatal bacterial meningitis caused by Escherichia coli (more than 80 % due to E. coli strains possessing K1 antigen) contributes to more than 50 000 annual deaths worldwide, and it is of serious concern that these numbers remain significant, despite advances in antimicrobial chemotherapy (de Louvois et al., 1991;Unhanand et al., 1993;Kim, 2002Kim, , 2006. Although haematogenous spread is known to be a pre-requisite in E. coli meningitis, it is not clear how circulating bacteria cross the blood-brain barrier to gain entry into the central nervous system (CNS) to produce disease (reviewed by Kim, 2001Kim, , 2002Kim, , 2006. Over the last few decades only a handful of bacterial virulence determinants (such as outer-membrane protein A, FimH, cytotoxic necrotizing factor-1, Ibe proteins, Traj and AslA) have been identified as associated with E. coli K1 pathogenesis (Kim, 2006). The E. coli genome possesses approximately 5.3 Mbp, and approximately 500 kb of the E. coli K1 genome (RS218 strain) is not present in the genome of the E. coli K-12 strain MG1655, so it is reasonable to anticipate the existence of several as yet unknown virulence determinants involved in E. coli meningitis. Current studies have relied upon vertebrate models to study E. coli pathogenesis, but mammalian models are expensive, not routinely available in many laboratories, require labour-intensive management and have ethical implications. There are many parallels between the innate immune responses of mammals and insects, and it is envisaged that insects could make useful models for the study of disease pathogenesis (Scully & Bidochka, 2006), especially as insects possess a highly selective blood-brain barrier exhibiting functional properties comparable with that of vertebrates (Carlson et al., 2000). Here, Locusta migratoria, the African migratory locust, is used as a model to study E. coli pathogenesis.The aim of the present study was to develop an ex vivo locust model specifically to study invasion of the CNS by E. coli. To achieve this, ligated he...