Dynamics and diversity of Escherichia coli in animals and system management of the manure on a commercial farrow-to-finish pig farm

The objective of this study was to determine the dynamics and diversity of Escherichia coli populations in animal and environmental lines of a commercial farrow-to-finish pig farm in Spain along a full production cycle (July 2008 to July 2009), with special attention to antimicrobial resistance and the presence of integrons. In the animal line, a total of 256 isolates were collected from pregnant sows (10 samples and 20 isolates), 1-week-old piglets (20 samples and 40 isolates), unweaned piglets (20 samples and 38 isolates), growers (20 samples and 40 isolates), and the finishers' floor pen (6 samples and 118 isolates); from the underfloor pits and farm slurry tank environmental lines, 100 and 119 isolates, respectively, were collected. Our results showed that E. coli populations in the pig fecal microbiota and in the farm environment are highly dynamic and show high levels of diversity. These issues have been proven through DNA-based typing data (repetitive extragenic palindromic PCR [REP-PCR]) and phenotypic typing data (antimicrobial resistance profile comprising 19 antimicrobials). Clustering of the sampling groups based on their REP-PCR typing results showed that the spatial features (the line) had a stronger weight than the temporal features (sampling week) for the clustering of E. coli populations; this weight was less significant when clustering was performed based on resistotypes. Among animals, finishers harbored an E. coli population different from those of the remaining animal populations studied, considering REP-PCR fingerprints and resistotypes. This population, the most important from a public health perspective, demonstrated the lowest levels of antimicrobial resistance and integron presence.

VanB-type Enterococcus faecium clinical isolate successively inducibly resistant to, dependent on, and constitutively resistant to vancomycin

Three Enterococcus faecium strains isolated successively from the same patient, vancomycin-resistant strain BM4659, vancomycin-dependent strain BM4660, and vancomycin-revertant strain BM4661, were indistinguishable by pulsed-field gel electrophoresis and harbored plasmid pIP846, which confers VanB-type resistance. The vancomycin dependence of strain BM4660 was due to mutation P(175)L, which suppressed the activity of the host Ddl D-Ala:D-Ala ligase. Reversion to resistance in strain BM4661 was due to a G-to-C transversion in the transcription terminator of the vanRS(B) operon that lowered the free energy of pairing from -13.08 to -6.65 kcal/mol, leading to low-level constitutive expression of the resistance genes from the P(RB) promoter, as indicated by analysis of peptidoglycan precursors and of VanX(B) D,D-dipeptidase activity. Transcription of the resistance genes, studied by Northern hybridization and reverse transcription, initiated from the P(YB) resistance promoter, was inducible in strains BM4659 and BM4660, whereas it started from the P(RB) regulatory promoter in strain BM4661, where it was superinducible. Strain BM4661 provides the first example of reversion to vancomycin resistance of a VanB-type dependent strain not due to a compensatory mutation in the ddl or vanS(B) gene. Instead, a mutation in the transcription terminator of the regulatory genes resulted in transcriptional readthrough of the resistance genes from the P(RB) promoter in the absence of vancomycin.