Class 1 integrons and tetracycline resistance genes in alcaligenes, arthrobacter, and Pseudomonas spp. isolated from pigsties and manured soil

Abstract

The presence of tetracycline resistance (Tc(r)) genes and class I integrons (in-1), and their ability to cotransfer were investigated in Tc(r) gram-negative (185 strains) and gram-positive (72 strains) bacteria from Danish farmland and pigsties. The isolates belonged to the groups or species Escherichia coli, Enterobacter spp., Arthrobacter spp., Alcaligenes spp., Pseudomonas spp., and Corynebacterium glutamicum. The 257 isolates were screened for in-1. Eighty-one of the gram-negative isolates were also screened for the Tc(r) genes tet(A), tet(B), and tet(C), and all (n = 72) gram-positive isolates were screened for tet(33). Fourteen (7%) of the soil isolates and eleven (25%) of the pigsty isolates contained in-1. All isolates that contained tet genes also contained in-1, except one gram-negative isolate from a pigsty that contained tet(B). All gram-positive isolates with in-1 also contained tet(33). No isolates contained more than one tet gene. The in-1-positive isolates were tested for resistance to selected antimicrobial agents and showed resistance to three to nine drugs. Filter-mating experiments showed cotransfer of Tc(r) and class I integrons from soil isolates to Escherichia coli and/or Pseudomonas putida. We conclude that soil bacteria in close contact to manure or pigsty environment may thus have an important role in horizontal spread of resistance. Use of tetracyclines in food animal production may increase not only Tc(r) but also multidrug resistance (caused by the presence tet genes and in-1) in bacteria.

FIG. 1.

Southern blot of plasmid extractions. (A) Hybridized with class 1 integron probe (qacEΔ1). Lane 1, HindIII-cut λ marker; lane 2, A. faecalis R194; lane 3, transconjugant R194-P; lane 4, transconjugant R194-E; lane 5, A. faecalis R214; lane 6, transconjugant R214-P; lane 7, transconjugant R214-E; lane 8, recipient P. putida; lane 9, recipient E. coli. (B) Hybridized with Tc^r tet(A) probe. Lane 1, HindIII-cut λ marker; lane 2, A. faecalis R194; lane 3, transconjugant R194-P; lane 4, transconjugant R194-E; lane 5, A. faecalis R214; lane 6, transconjugant R214-P; lane 7, transconjugant R214-E; lane 8, recipient P. putida; lane 9, recipient E. coli. (C) BamHI-restricted 36-kb plasmids hybridized with tet(A). Lane 1, strain R194; lane 2, transconjugant R194-P; lane 3, transconjugant R194-E; lane 4, recipient P. putida; lane 5, E. coli; lane 6, strain R214; lane 7, transconjugant R214-P; lane 8, transconjugant R214-E; lane 9, HindIII-digested λ marker. The size markers were based on HindIII-digested λ marker and plasmids extracted from E. coli R39.