Álvaro San Millán Cruz defended the PhD Thesis at the Faculty of Veterinary Medicine of the Complutense University of Madrid

May 27^th, 2010

Antibiotics are one of the main tools to fight against infectious diseases. Since the introduction of antibiotics in clinical practice, the development and progressive dispersion of antimicrobial resistance mechanisms among bacteria have dramatically increased. Acquisition of antimicrobial resistance determinants by microorganisms of clinical relevance is a concerning phenomenon both for animal medicine and public health. Research on the mechanisms responsible for decreased susceptibility to antibiotics in bacteria, together with the study of the genetic platforms responsible for the spread of these determinants among bacteria are crucial to understand and prevent this threat. In this work, the antimicrobial resistance mechanisms of bacteria from the Pasteurellaceae family have been studied. This family includes pathogens of great relevance for animals and human beings.

Initially, we analyzed the molecular mechanisms of ß-lactam resistance in the swine pathogen [Haemophilus parasuis], the causative agent of Glasser disease. In this species, the clonal dissemination of a strain bearing the new mobilizable plasmid pB1000, belonging to the MOBHEN family (ColE1 Superfamily), has been described. This replicon of 4613 bp encodes the ROB-1 ß-lactamase, and it is responsible for the ß-lactam resistance phenotype in [H. parasuis]. Further, antimicrobial resistance in Pasteurella multocida swine clinical isolates has been characterized. In P. multocida, ß-lactam resistance is also due to the presence of pB1000, or the new plasmid pB1002, identical to the former, but with the insertion of the transposable element ISApl1 downstream blaROB-1. In addition to ß-lactam resistance, these P. multocida strains present resistance to tetracyclines and/or streptomycin. This phenotype is mediated by tet(H), tet(B) and tet(O) genes, for tetracycline resistance, and by strA, for streptomycin resistance. These genes are located in small plasmids, and each strain bears two or three plasmids. Complete nucleotide sequences of seven plasmids have been determined, encoding one, or maximum two, antimicrobial resistance determinants each (pB1000, pB1001, pB1002, pB1003, pB1005, pB1006, and p9956), and six out of seven belong to the MOBHEN family of mobilizable plasmids.
In most bacteria, acquired multiresistance is conferred by large plasmids compiling numerous antimicrobial resistance genes; notwithstanding, in P. multocida the coexistence of two or three small plasmids (between 4 and 6 kb) in a single strain is responsible for the multiresistance phenotype. Hence, this is a new evolutionary strategy to achieve multiresistance in the Pasteurellaceae family.

Finally, presence of plasmid pB1000, and its derivative pB1000’, is reported from Haemophilus influenzae clinical strains isolated in different hospitals from Spain. This is an important episode for public health, as this plasmid, previously described from animal Pasteurellaceae, is reported for the first time in a strict human pathogen. Spread of pB1000 within Spain has been analyzed, as well as the mechanisms of transfer of this plasmid among Pasteurellaceae members from animal and human origin, showing that pB1000 can be acquired by transformation and conjugation by P. multocida and H. influenzae. In addition, the fitness cost that pB1000 entails to H. influenzae has also been determined.

We propose P. multocida as the possible vehicle of transmission of antimicrobial resistance mechanisms, as pB1000, between Pasteurellaceae family animal pathogens and H. influenzae. This hypothesis is support by the data from our study and by the fact that P. multocida is the sole species from this family able to colonize both humans and animals.

In conclusion, in this work we characterize the molecular mechanisms of resistance to antibiotics of clinical relevance in the swine pathogens [H. parasuis] and P. multocida, discovering a new strategy to acquire multiresistance to antibiotics. Moreover, the dispersion of these resistance determinants to the human pathogen H. influenzae has been shown.