Genomic analysis of two novel bacteriophages infecting Acinetobacter beijerinckii and halotolerans species

Bacteriophages are the most diverse genetic entities on Earth. In this study, two novel bacteriophages, nACB1 (Podoviridae morphotype) and nACB2 (Myoviridae morphotype), which infect Acinetobacter beijerinckii and Acinetobacter halotolerans, respectively, were isolated from sewage samples. The genome sequences of nACB1 and nACB2 revealed that their genome sizes were 80,310 bp and 136,560 bp, respectively. Comparative analysis showed that both genomes are novel members of the Schitoviridae and the Ackermannviridae families, sharing ≤ 40% overall nucleotide identities with any other phages. Interestingly, among other genetic features, nACB1 encoded a very large RNA polymerase, while nACB2 displayed three putative depolymerases (two capsular depolymerases and one capsular esterase) encoded in tandem. This is the first report of phages infecting A. halotolerans and beijerinckii human pathogenic species. The findings regarding these two phages will allow us to further explore phage—Acinetobacter interactions and the genetic evolution for this group of phages.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and by LABBELS—Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnical Systems, LA/P/0029/2020.info:eu-repo/semantics/publishedVersio

Characterization of novel Acinetobacter baumannii phage-derived depolymerases with anti-virulence properties

Dissertação de mestrado em BiothecnologyAcinetobacter baumannii is a pathogenic bacterium and is the major cause of nosocomial infections in hospital environment. Treatment of A. baumannii infections with antibiotics is extremely difficult due to their multi-resistance antibiotics. Moreover, most A. baumannii strains carry a capsular polysaccharide that is considered its main virulence factor, and a high variety of capsular types are produced by this bacterium. Phages are viruses that only infect bacteria, using their cellular machinery to propagate. Some phages produce polysaccharide depolymerases, that bind specifically to certain capsular types and degrade the polysaccharides present in the bacterial capsules, allowing phages to infect. Therefore, capsular polysaccharide depolymerases have demonstrated to be a very powerful anti-virulence weapon with the capability to degrade and strip bacterial cells from their capsule. Prior to this work, 17 distinct A. baumannii capsular-specific depolymerases were identified and characterized (K1-2, K9, K19, K27, K30, K32, K37, K44-45, K47-48, K87, K89, K91, K93 and K116). In this study, oxa-51 and oxa-23 β-lactamases-encoding genes were the most predominant found within carbapenem-resistant A. baumannii clinical isolates, followed by Imp-like and oxa-24 β-lactamases-encoding genes. Furthermore, 6 novel phage derived-CPS depolymerases were identified (F70, 3042, 3043, 3060, 3073 and 3082). They were cloned and expressed in E. coli alongside with previously identified CPS depolymerases (B1, B9, P1 and P2). The activity spectra of the capsular depolymerases B1, B3, B9, P1, P2, F70, 3042, 3043 and 3073 matched the lytic spectra of their parental phages (K9, K2/K19, K30/K45, K1, K67, K44, K38 and K32 respectively), expanding the collection of enzymes to infect 19 different K types. However, the capsular depolymerases belonging to phages 3060 and 3082 did not demonstrate any activity against any strain. Furthermore, these enzymes were characterized, demonstrating a high structural stability and degrading activity against the capsular polysaccharides, leaving bacteria more susceptible to be eliminated by the serum’s complement system. However, no synergy was found between the combination of these enzymes and different antibiotics in the disruption of biofilms and in planktonic cells.Acinetobacter baumannii é uma bactéria patogénica e a maior causa de infeções nosocomiais em ambiente hospitalar. O tratamento de infeções causadas por A. baumannii com antibióticos é extremamente difícil devido a multi-resistências. Adicionalmente, a maioria das estirpes de A. baumannii possui um polissacarídeo capsular, considerado o maior fator de virulência desta espécie, e uma grande variedade de tipos capsulares são produzidos por esta bactéria. Os fagos são vírus que apenas infetam bactérias, usando a sua maquinaria celular para propagar. Alguns fagos evoluíram para codificar depolimerases capsulares, que se ligam especificamente a certos tipos capsulares e degradam os polissacarídeos presentes nas cápsulas bacterianas, permitindo que o fago infete as bactérias. Desta forma, as depolimerases capsulares têm demonstrado ser uma poderosa arma antivirulenta, com a capacidade de degradar e remover as cápsulas das bactérias. Previamente a este trabalho, 17 diferentes depolimerases capsulares-específicas foram identificadas e caracterizadas (K1-2, K9, K19, K27, K30, K32, K37, K44-45, K47-48, K87, K89, K91, K93 e K116). Neste trabalho, os genes codificantes para as β-lactamases oxa-51 e oxa-23 foram os mais predominantes encontrados em isolados clínicos de A. baumannii resistentes a carbapenemos, seguidos pelos genes correspondentes às β-lactamases Imp-like e oxa-24. Adicionalmente, 6 novas depolimerases capsulares derivadas de novos fagos isolados foram identificadas (F70, 3042, 3043, 3060, 3073 e 3082). Estas foram clonadas e expressas recombinantemente em E. coli, juntamente com depolimerases capsulares previamente identificadas (B1, B3, B9, P1 e P2). Os espetros de atividade das depolimerases capsulares B1, B3, B9, P1, P2, F70, 3042, 3043 e 3073 corresponderam aos espetros líticos dos seus fagos parentais (K9, K2/K19, K30/K45, K1, K67, K44, K38 e K32 respetivamente). Desta forma, a coleção destas enzimas expandiu para 19 diferentes depolimerases capsulares-específicas. Posteriormente, estas enzimas foram caracterizadas, demonstrando possuir uma elevada estabilidade estrutural e capacidade de degradação dos polissacarídeos capsulares, deixando as bactérias mais suscetíveis a serem eliminadas pelo sistema complemento do soro. Porém, um efeito sinergético entre as enzimas e diferentes antibióticos na disrupção de biofilmes e em células planctónicas não foi observado