The characterisation of nickel resistance in sewage-borne enterobacteriaceae

Abstract

This thesis is a continuation of experimental work conducted by Pickup el al. (1997) analysing nickel resistant bacteria isolated from effluent discharged from a sewage outfall in the Lake District, Cumbria. Its aim is to further characterise the nickel resistance determinant ftom Enterobacter cloacae FBA30. FBA30 was found to harbour a 55-60 kb plasmid designated pFBA30 which confers nickel resistance on its host and in laboratory strains of E. coh. pFBA30 is transferable to enteric relatives, but not to non-enteric species and is considered to have a narrow host range. A 10.2kb Sad restriction fi7agment was cloned fi7om pFBA30, which exhibited constitutive nickel resistance both in its original host and in laboratory strains of Eschericia coli.
Further work on the 10.2 kb Sacl fragment localised the nickel resistance locus to an internal 2.3 kb PvuII restriction fragment exhibiting extensive homology to the nre determinant of plasmid pTOM9 (Schmidt and Schlegel, 1994). This fragment and a further 1.75 kb Smal fragment derived from it, were used as DNA probes to examine other bacterial strains isolated from the same site.
Six strains showing extensive homology were retained for further study (Pickup et al., 1997). The nickel resistance of the six strains appears to be chromosomally encoded. The strains were sorted into groups based upon their sampling date and size of restriction fragment homologous to the probe developed from pFBA30. Group A isolates carry a homologous Null fragment approximately 6.5 kb in size whereas group B isolates carry a 2.5 kb Null fragment. All strains display constitutive nickel resistance and are capable of expressing resistance under both anaerobic and aerobic growth conditions. Furthermore, each strain was discovered to be resistant to a spectrum of heavy metals and antimicrobial agents. Each group consists of a range of enteric bacterial species including Klebsiella, Citrobacter and Enterobacter species.