Beta-lactam resistance in Haemophilus parasuis Is mediated by plasmid pB1000 bearing blaROB-1

Abstract

beta-Lactam resistance in Haemophilus parasuis is an emerging phenomenon that has not yet been characterized from a molecular perspective. Clinical high-level beta-lactam-resistant isolates from Spain bore a novel plasmid, pB1000, expressing a functionally active ROB-1 beta-lactamase. Pulsed-field gel electrophoresis was applied for the first time to H. parasuis and showed that beta-lactam resistance is due to clonal spread of a resistant strain, BB1018, bearing pB1000.

FIG. 1.

Location of bla_ROB-1 in H. parasuis. (A) GPS-PCR. Agarose gel electrophoresis of PCRs from plasmid extraction and total DNA. Note that plasmid extractions resulted in a positive PCR signal for bla_ROB-1 (upper left), whereas no amplification is observed using the chromosomally encoded 16S rRNA primers (upper right). Total DNA extraction serves as a PCR control for both reactions. The positive control is DNA from an A. pleuropneumoniae isolate bearing plasmid-encoded bla_ROB-1. Plasmid DNA extraction was carried out using the Plasmid Midi kit and QIAprep Spin Miniprep kit (QIAGEN, Inc., Chatworth, CA). PCR fragments were purified with QIAGEN PCR purification or gel extraction kits (QIAGEN, Inc., Chatworth, CA), following the manufacturer's instructions. (B) Southern blot of DNA extracted from a representative β-lactam-resistant H. parasuis isolate using bla_ROB-1 as a probe. The signal confirms that the bla_ROB-1 gene is located on a plasmid. Southern blotting was performed with DNA electrophoresed in 1% agarose gel and transferred onto Hybond N+ positively charged nylon membranes (Amersham Hybond; GE Healthcare). The bla_ROB-1 probe was obtained with primers MAP-4 and rob-1F (Table 1) prepared with the Nona primer kit (Q-BIO gene; MP Biomedicals).

FIG. 2.

Genetic structure of pB1000. (A) Comparison of the genetic structures of pB1000, pHS-Tet, and pAB2. pB1000 contains the transcriptional terminator and a conserved direct repeat (white box) from pHS-Tet (13). Analogously, the transcriptional terminator of bla_ROB-1 is present in pHS-Tet. These data indicate that pB1000 and pHS-Tet might have evolved from a common ancestor bearing together the tet(B) and bla_ROB-1 genes. pAB2 has an inverted copy of bla_ROB-1. The empty triangle indicates the position of the GACTT sequence. (B) Potential recombination sites of bla_ROB-1. pHS-Tet has a single copy of the GACTT sequence. Further, in pB1000 and pAB2, bla_ROB-1 is embedded between the GACTT repeated sequences, indicating strongly that these sequences mediate mobilization of bla_ROB-1 via an intermediary hairpin structure and duplication of the insertion site.

FIG. 3.

PFGE fingerprint patterns of H. parasuis. Lanes 1 to 8, β-lactam-susceptible clinical isolates; lanes 9 to 16, β-lactam-resistant isolates. M stands for molecular marker and ATCC for β-lactam-susceptible H. parasuis strain ATCC 19417. Lane 1, BB1027; lane 2, BB1028; lane 3, BB1029; lane 4, BB1026; lane 5, BB1030; lane 6, BB1031; lane 7, BB1032; lane 8, BB1033; lane 9, BB1018; lane 10, BB1019; lane 11, BB1020; lane 12, BB1021; lane 13, BB1022; lane 14, BB1023; lane 15, BB1024; lane 16, BB1025.