Haemophilus influenzae Clinical Isolates with Plasmid pB1000 Bearing bla _ROB-1 : Fitness Cost and Interspecies Dissemination

Abstract

Plasmid pB1000 is a mobilizable replicon bearing the bla ROB-1 ␤-lactamase gene that we have recently described in Haemophilus parasuis and Pasteurella multocida animal isolates. Here we report the presence of pB1000 and a derivative plasmid, pB1000, in four Haemophilus influenzae clinical isolates of human origin. Pulsed-field gel electrophoresis showed unrelated patterns in all strains, indicating that the existence of pB1000 in H. influenzae isolates is not the consequence of clonal dissemination. The replicon can be transferred both by transformation and by conjugation into H. influenzae, giving rise to recipients resistant to ampicillin and cefaclor (MICs, >64 g/ml). Stability experiments showed that pB1000 is stable in H. influenzae without antimicrobial pressure for at least 60 generations. Competition experiments between isogenic H. influenzae strains with and without pB1000 revealed a competitive disadvantage of 9% per 10 generations for the transformant versus the recipient. The complete nucleotide sequences of nine pB1000 plasmids from human and animal isolates, as well as the epidemiological data, suggest that animal isolates belonging to the Pasteurellaceae act as an antimicrobial resistance reservoir for H. influenzae. Further, since P. multocida is the only member of this family that can colonize both humans and animals, we propose that P. multocida is the vehicle for the transport of pB1000 between animal-and human-adapted members of the Pasteurellaceae.

TEM-1-encoding small plasmids impose dissimilar fitness costs on Haemophilus influenzae and Haemophilus parainfluenzae

“…2h). The competitive disadvantage of plasmid pB1000 on H. influenzae strain Rd found in this study is in agreement with that reported by San Millan et al (2010). In contrast, the three TEM-1-bearing plasmids were differentially tolerated by the two bacterial hosts: pPN223 and pA1606 did not impose a significant fitness cost to H. influenzae strain Rd, and pA1209 did not impose a significant fitness cost to H. parainfluenzae strain HK 23, while the reverse combinations resulted in significant disadvantages (Fig.…”

“…Small beta-lactamase bearing plasmids were isolated from strains of H. influenzae (pPN223, pA1209 and pA1606) (Søndergaard et al, 2012) or H. parainfluenzae (pSF3) (Tristram et al, 2008). Plasmid pB1000 was kindly donated by Professor Bruno Gonzales-Zorn (San Millan et al, 2010). The bla TEM-1 plasmids used in this study represent each of the three TEM-1-bearing small plasmid types hitherto characterized in H. influenzae.…”

“…The plasmid encoding ROB-1 in H. influenzae is designated pB1000 and is 4.6 kb in size (San Millan et al, 2010). pB1000 has been detected in other Pasteurellaceae species of animal origin (San Millan et al, 2007, 2009Tristram et al, 2010), but not in H. parainfluenzae.…”

“…A plasmid bearing TEM-34 was not available for this investigation; the reported plasmid (p72322) is identical to pLFH64, except for the single amino acid substitution in TEM that renders the beta-lactamase resistant to clavulanic acid (García-Cobos et al, 2013). pB1000 has a competitive disadvantage of approximately 9 % per ten generations relative to the parental strain when transformed into H. influenzae strain Rd (San Millan et al, 2010). Fitness cost analyses of bla TEM -bearing plasmids have not, to our knowledge, been performed.…”

Pasteurella multocida: from Zoonosis to Cellular Microbiology

“…In addition to the antibiotic resistance genes, a few of these plasmids have also been found to carry genes with other functions, such as plasmid mobilization, segregation, or replication genes (522) and putative cation transporter genes (80,532). Many of the plasmids carrying antibiotic resistance determinants are transferrable among the Pasteurellaceae members, as well as other Gram-negative bacteria (517,518,522,529,(533)(534)(535)(536).…”