Not the usual suspects: membrane translocation, pathogenic potential and bacterial species of the Acinetobacter baumannii group

Abstract

The most clinically relevant species of the Acinetobacter genus are comprised within the Acinetobacter baumannii group (Ab group, i. e. A. baumannii, Acinetobacter nosocomialis, Acinetobacter pittii, Acinetobacter seifertii and A. pittii-like/A. dijskhoorniae). Among these species A. baumannii is the most prevalent and usually shows multidrug resistance. This, and the fact that the Ab group species cannot be distinguished by phenotypic methods, has led to the misidentification of the species of the Ab group as A. baumannii in the clinical settings. Nevertheless, in the last years the incidence of the other species of the Ab group has risen, partly due to the use of molecular techniques and mass spectrometry tools. This thesis has characterised, using both genotypic (rpoB-based and MLSA phylogenetic analyses, and whole genome sequence analysis) and phenotypic (carbon utilisation assays and MALDI-TOF MS) techniques, a group of strains mainly recovered from human samples that represents a new bacterial species within the Ab group for which the name of Acinetobacter dijkshoorniae has been proposed. The genome of the type strain of the novel species has been sequenced and the identification of the species of the Ab group by MALDI-TOF MS has been optimised, since the novel species (A. dijkshoorniae and A. seifertii) could not be identified by this technique prior to this study. MALDI-TOF MS was shown to be rapid and accurate in the discrimination of all the species of the Ab group, indicating its suitability for the implementation of this technique in the clinical settings. In addition, the use of MALDI- TOF MS allowed the identification of members of the Ab group in market meat from Peru, leading to the first identification of A. dijkshoorniae from food and in South America. We also evaluated the differences among the species of the Ab group beyond those at the taxonomic level and found that while A. baumannii still presents the higher rates of antimicrobial resistance, this species and A. nosocomialis, which usually are more prevalent in our hospitals and have worst outcomes, showed a minor pathogenicity in terms of biofilm formation and virulence in the Caenorhabditis elegans infection model. In contrast, A. pittii, A. seifertii and A. dijkshoorniae presented a higher pathogenicity in the phenotypes studied. This data suggests the emergence of the non-baumannii species of the Ab group as well as different degrees of adaptation to the human host and the need of studying them as distinct entities. The rise on the antimicrobial resistance of A. baumannii has drastically reduced the therapeutic options left to treat infections caused by this pathogen. In view of the lack of effective antimicrobial drugs there is an urgent need for novel therapeutic approaches. In order to find novel targets for the development of antimicrobial drugs against A. baumannii, we evaluated the dual role of transport-related proteins in antimicrobial resistance and virulence using a transposon mutant strain collection derived from the A. baumannii AB5075 strain. The antimicrobial susceptibility of the mutant strains was compared against the wild- type strain and led to the identification of novel antimicrobial substrates for known efflux pumps and uncharacterised transport-related proteins which seem to participate in the transport of antibiotics across membranes. The evaluation of the virulence in the Galleria mellonella infection model also identified transport-related proteins putatively involved in the virulence of A. baumannii. This results are still preliminary but show the dual role of transport- related proteins in the antimicrobial resistance and pathogenicity of A. baumannii and open a

new line of research that might help to gain further knowledge about the virulence of this pathogen while finding novel targets for the development of new antimicrobial drugs against A. baumannii.

Dentro del género Acinetobacter, las especies del grupo Acinetobacter baumannii (Ab) (i. e. A. baumannii, Acinetobacter nosocomialis, Acinetobacter pittii y Acinetobacter seifertii y A. pittii-like/A. dijskhoorniae) destacan por su gran relevancia clínica, siendo A. baumannii el patógeno del grupo Ab de mayor importancia debido a su frecuente aislamiento y su usual fenotipo de multirresistencia. Esto, sumado a que las especies del grupo Ab son indistinguibles a nivel fenotípico, ha conllevado que habitualmente hayan sido erróneamente identificadas en el ámbito clínico como A. baumannii. Sin embargo, en los últimos años se ha observado un aumento en la incidencia de las otras especies del grupo Ab, en parte gracias a las técnicas moleculares, que han revolucionado la taxonomía de este género, y también al uso de la espectrometría de masas MALDI-ToF (EM MALDI-TOF). Este estudio proporciona las pruebas fenotípicas y genotípicas que respaldan la delineación de una nueva especie dentro del grupo Ab, para la cual se ha propuesto el nombre de Acinetobacter dijkshoorniae. El genoma de la cepa tipo de esta nueva especie ha sido secuenciado, y se ha puesto a punto la identificación mediante EM MALDI-TOF de los nuevos miembros del grupo Ab (A. dijkshoorniae y A. seifertii) así como ha sido optimizado para el resto de especies del grupo. Rasgos como la formación de biofilm o la virulencia en el modelo animal de Caenhorabditis elegans muestran que A. baumannii, junto con A. nosocomialis, presentan menor potencial patogénico que las otras especies del grupo Ab, y que A. dijkshoorniae es especialmente virulenta. Estos datos sugieren diferentes niveles de adaptación al ámbito hospitalario, y remarca la necesidad de identificar y estudiar las especies del grupo Ab individualmente. Además, a nivel de susceptibilidad antimicrobiana, A. baumannii sigue siendo la especie que presenta mayores tasas de resistencia. Finalmente, hemos hallado nuevas proteínas implicadas en el transporte de membrana que, de manera preliminar, parecen participar en la resistencia a antibióticos y virulencia de A. baumannii, y cuya caracterización podría aportar nuevas dianas para el desarrollo de fármacos antimicrobianos para tratar las infecciones causadas por A. baumannii multirresistente.