A Novel Nickel Resistance Determinant Found in Sewage-Associated Bacteria

Abstract

Nickel-resistant bacteria were isolated from effluent discharged from a sewage treatment outfall over an 18-month period. One of these strains, Enterobacter cloacae FBA30, was found to harbor a narrow host range conjugative plasmid, designated pFBA30, which confers nickel resistance on its host. A 10.2-kb SstI restriction fragment was cloned from pFBA30 and was shown to specify inducible nickel resistance, both in its original host and in laboratory strains of Escherichia coli. This DNA fragment, and a 1.75-kb SmaI fragment derived from it, were used as probes to examine other bacterial strains isolated during the study. Homologous nickel resistance genes were detected in enteric bacteria emerging directly from the treatment plant, but not in strains isolated from sediments downstream of the outfall, or in strains harboring well-characterized determinants such as ncc/nre, cnr, or Klebsiella oxytoca type. Thus, this element constitutes a new nickel resistance determinant and was named nrf.

Nickel-resistant strains were sorted into two groups, based on the sampling date and the size of restriction fragments homologous to probes developed from pFBA30. Group A isolates all carried a homologous 6.5-kb PvuII restriction fragment and expressed nickel resistance constitutively. Group B strains, isolated independently from the same outfall, were different from group A in that the nickel resistance was associated with a 4.7-kb PvuII fragment, and group B strains displayed a slower growth rate on nickel salts media. Each group contained a range of enteric bacterial species, including K. oxytoca, Citrobacter freundii, and Enterobacter spp. Localized genetic exchange probably occurs within wastewater treatment effluents, and, once effluents are discharged into the environment, enteric bacteria carrying pFBA30-like determinants do not persist downstream.