Draft Genome Sequence of JVAP01T, the Type Strain of the Novel Species Acinetobacter dijkshoorniae

Here, we report the draft genome sequence of the type strain of Acinetobacter dijkshoorniae, a novel human pathogen within the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex. Strain JVAP01T has an estimated genome size of 3.9 Mb, exhibits a 38.8% G+C content, and carries a plasmid with the blaNDM-1 carbapenemase gene

Pathogenic Acinetobacter species including the novel Acinetobacter dijkshoorniae recovered from market meat in Peru

Species of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex are important human pathogens which can be recovered from animals and food, potential sources for their dissemination. The aim of the present study was to characterise the Acinetobacter isolates recovered from market meat samples in Peru. From July through August 2012, 138 meat samples from six traditional markets in Lima were cultured in Lysogeny and Selenite broths followed by screening of Gram-negative bacteria in selective media. Bacterial isolates were identified by MALDI-TOF MS and DNA-based methods and assessed for their clonal relatedness and antimicrobial susceptibility. Twelve Acinetobacter isolates were recovered from calf samples. All but one strain were identified as members of the clinically-relevant Acinetobacter calcoaceticus-Acinetobacter baumannii complex: 9 strains as Acinetobacter pittii, 1 strain as A. baumannii, and 1 strain as the recently described novel species A. dijkshoorniae. The remaining strain could not be identified at the species level unambiguously but all studies suggested close relatedness to A. bereziniae. All isolates were well susceptible to antibiotics. Based on macrorestriction analysis, six isolates were further selected and some of them were associated with novel MLST profiles. The presence of pathogenic Acinetobacter species in human consumption meat might pose a risk to public health as potential reservoirs for their further spread into the human population. Nevertheless, the Acinetobacter isolates from meat found in this study were not multidrug resistant and their prevalence was low. To our knowledge, this is also the first time that the A. dijkshoorniae species is reported in Peru

MALDI-TOF/MS identification of species from the Acinetobacter baumannii (Ab) group revisited: inclusion of the novel A. seifertii and A. dijkshoorniae species

OBJECTIVES: Rapid identification of Acinetobacter species is critical since members of the A. baumannii (Ab) group differ in antibiotic susceptibility and clinical outcomes. A. baumannii, A. pittii and A. nosocomialis can be identified by MALDI-TOF/MS, while the novel species A. seifertii and A. dijkshoorniae cannot. Low identification rates for A. nosocomialis have also been reported. We evaluated the use of MALDI-TOF/MS to identify isolates of A. seifertii and A. dijkshoorniae and revisited the identification of A. nosocomialis to update the Bruker taxonomy database. METHODS: Species characterisation was performed by rpoB-clustering and MLSA. MALDI-TOF/MS spectra were recovered from formic acid/acetonitrile bacterial extracts overlaid with alpha-cyano-4-hydroxy-cinnamic acid matrix on a MicroflexLT in linear positive mode and 2,000-20,000 m/z range mass. Spectra were examined with the ClinProTools v2.2 software. Mean spectra (MSP) were created with the BioTyper software. RESULTS: Seventy-eight Acinetobacter isolates representative of the Ab group were used to calculate the average spectra/species and generate pattern recognition models. Species-specific peaks were identified for all species, and MSPs derived from 3 A. seifertii, 2 A. dijkshoorniae and 2 A. nosocomialis strains were added to the Bruker taxonomy database, allowing successful identification of all isolates using spectra from either bacterial extracts or direct colonies, resulting in a positive predictive value (PPV) of 99.6% (777/780) and 96.8% (302/312), respectively. CONCLUSIONS: The use of post-processing data software identified statistically significant species-specific peaks to generate reference signatures for rapid accurate identification of species within the Ab group, providing relevant information for the clinical management of Acinetobacter infections

In vitro and in vivo Virulence Potential of the Emergent Species of the Acinetobacter baumannii (Ab) Group

The increased use of molecular identification methods and mass spectrometry has revealed that Acinetobacter spp. of the A. baumannii (Ab) group other than A. baumannii are increasingly being recovered from human samples and may pose a health challenge if neglected. In this study 76 isolates of 5 species within the Ab group (A. baumannii n = 16, A. lactucae n = 12, A. nosocomialis n = 16, A. pittii n = 20, and A. seifertii n = 12), were compared in terms of antimicrobial susceptibility, carriage of intrinsic resistance genes, biofilm formation, and the ability to kill Caenorhabditis elegans in an infection assay. In agreement with previous studies, antimicrobial resistance was common among A. baumannii while all other species were generally more susceptible. Carriage of genes encoding different efflux pumps was frequent in all species and the presence of intrinsic class D β-lactamases was reported in A. baumannii, A. lactucae (heterotypic synonym of A. dijkshoorniae) and A. pittii but not in A. nosocomialis and A. seifertii. A. baumannii and A. nosocomialis presented weaker pathogenicity in our in vitro and in vivo models than A. seifertii, A. pittii and, especially, A. lactucae. Isolates from the former species showed decreased biofilm formation and required a longer time to kill C. elegans nematodes. These results suggest relevant differences in terms of antibiotic susceptibility patterns among the members of the Ab group as well as highlight a higher pathogenicity potential for the emerging species of the group in this particular model. Nevertheless, the impact of such potential in the human host still remains to be determined

Acinetobacter dijkshoorniae sp. nov., a new member of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex mainly recovered from clinical samples in different countries

The recent advances in bacterial species identification methods have led to the rapid taxonomic diversification of the genus Acinetobacter. In the present study, phenotypic and molecular methods have been used to determine the taxonomic position of a group of 12 genotypically distinct strains belonging to the Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) complex, initially described by Gerner-Smidt and Tjernberg in 1993, that are closely related to A. pittii. Strains characterized in this study originated mostly from human samples obtained in different countries over a period of 15 years. rpoB and MLST sequences were compared against those of 94 strains representing all species included in the ACB complex. Cluster analysis based on such sequences showed that all 12 strains grouped together in a distinct clade closest to A. pittii that was supported by bootstrap values of 99%. Values of average nucleotide identity based on BLAST between the genome sequence of strain JVAP01 (NCBI accession n masculine. LJPG00000000) and those of other species from the ACB were always < 91.2%, supporting the species status of the group. In addition, the metabolic characteristics of the group matched those of the ACB complex and the analysis of their protein signatures by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry identified some specific peaks. Our results support the designation of these strains as a novel species and we propose the name A. dijkshoorniae sp. nov. The type strain is JVAP01T (CECT 9134T, LMG 29605T)

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

[eng] 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.[spa] 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

Clonal Spread and Intra- and Inter-Species Plasmid Dissemination Associated With Klebsiella pneumoniae Carbapenemase-Producing Enterobacterales During a Hospital Outbreak in Barcelona, Spain

Objectives: The study aimed to characterize the clonal spread of resistant bacteria and dissemination of resistance plasmids among carbapenem-resistant Enterobacterales at a tertiary hospital in Catalonia, Spain. Methods: Isolates were recovered from surveillance rectal swabs and diagnostic samples. Species identification was by matrix-assisted laser desorption ionization-time time of flight mass spectrometry (MALDI-TOF MS). Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Antimicrobial susceptibility was assessed by gradient-diffusion and carriage of bla genes was detected by PCR. Plasmid typing, conjugation assays, S1-PFGE studies and long-read sequencing were used to characterize resistance plasmids. Results: From July 2018 to February 2019, 125 Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales were recovered from 101 inpatients from surveillance (74.4%) or clinical samples (25.6%), in a tertiary hospital in Barcelona. Clonality studies identified a major clone of Klebsiella pneumoniae belonging to sequence type ST15 and additional isolates of K. pneumoniae, Escherichia coli and Enterobacter sp. from different STs. All isolates but one carried the blaKPC-2 allelic variant. The blaKPC-2 gene was located in an IncFIIk plasmid of circa 106 Kb in a non-classical Tn4401 element designated NTEKPC-pMC-2-1. Whole-genome sequencing revealed different rearrangements of the 106 Kb plasmid while the NTEKPC-pMC-2-1 module was highly conserved. Conclusion: We report a hospital outbreak caused by the clonal dissemination of KPCproducing ST15 K. pneumoniae but also the intra- and inter-species transmission of the blaKPC-2 gene associated with plasmid conjugation and/or transposon dissemination. To our knowledge, this is the first report of an outbreak caused by KPC-producing Enterobacterales isolated from human patients in Catalonia and highlights the relevance of surveillance studies in the early detection and control of antibiotic resistant highrisk clones