Detection and quantification of Shiga toxin-encoding genes in sheep faeces by real-time PCR.

Sheep faeces may be an important source of Shiga toxin (Stx)-producing Escherichia coli. We have, therefore, established and evaluated a real-time 5'-nuclease PCR assay to quantify the stx(1) and stx(2) genes in sheep faeces. The detection limit of our assay for both stx(1) and stx(2) in spiked samples corresponded to 10(2)--10(3)CFU/g, which is lower than for other assays for measuring these genes in faecal samples. Quantification values for our assay ranged from 10(2) to 10(7)CFU/g faeces. The assay was evaluated on native, un-spiked faeces. All sheep tested (n=7) shed stx(1), and the quantitative results corresponded to the gene copies in 10(3)--10(4)CFU/g. The level of stx(2), however, was below the quantitative detection limit in all the samples analyzed. This quantitative stx(1) and stx(2) assay may be important in assessing whether sheep harbouring Shiga toxin-producing bacteria represent a potential hazard to human health.

Integron-containing IncU R plasmids pRAS1 and pAr-32 from the fish pathogen Aeromonas salmonicida.

A 45-kb R plasmid, pRAS1, that confers resistance to tetracyclines, trimethoprim, and sulfonamides was isolated in 1989 from an atypical strain of the fish pathogen Aeromonas salmonicida. This plasmid could be transferred by conjugation to Escherichia coli with a high degree of efficiency (frequency, 0.48). The following year pRAS1 was isolated from A. salmonicida subsp. salmonicida in the same area. Incompatibility group U plasmid pRAS1 contained a drug resistance-determining region of 12 kb consisting of a class 1 integron similar to In4 of Tn1696 but with a dfrA16 gene cassette inserted. Close to IS6100 at the right end of Tn4 was a truncated Tn1721. Restriction enzyme analysis showed that R plasmid pAr-32, isolated from A. salmonicida in Japan in 1970, had the same backbone structure as pRAS1, while the drug resistance-determining region contained a complex class 1 integron with an aadA2 cassette; the chloramphenicol resistance gene catA2, as in In6 of pSa; and a duplicate of the 3' conserved segment of the integron.