The aim of this study was to map the spectrum of microorganisms belonging to the genus Acinetobacter in domestic animals with a specific focus on the prevalence of Acinetobacter pseudolwoffii. Additionally, the susceptibility of isolates to antimicrobial agents was determined. In the period from January 1, 2014, to August 31, 2015, a total of 9 544 samples originating from gross lesions and pathological processes of animals exhibiting clinical symptoms of the disease were examined across 41 districts in the Czech Republic. The examinations were carried out using culture methods involving meat-peptone blood agar and Endo agar under aerobic conditions at a temperature of 37 ± 1 °C for 18-24 hours. Isolates were confirmed using molecular phenotypic method MALDI-TOF MS with the MBT Compass Library Revision L 2020 covering 3 239 species/entries (9 607 MSP) from Bruker Daltonics company. Out of the 108 isolates (prevalence 1.13%), 14 species of Acinetobacter spp. were identified, with 5 isolates remaining unclassified as species. A. pseudolwoffii was the predominant species isolated in 25 cases (prevalence 0.26%). Antimicrobial susceptibility was determined for 12 antimicrobials by the disc diffusion method, with A. pseudolwoffii isolates exhibiting the lowest susceptibility to ceftazidime (32%) and co-trimoxazole (60%).

• Keywords
• organ, pathogenicity, prevalence, species, susceptibility, veterinary,
• Publication type
• Journal Article MeSH

The amikacin resistance gene aphA6 was first detected in the nosocomial pathogen Acinetobacter baumannii and subsequently in other genera. Analysis of 133 whole-genome sequences covering the taxonomic diversity of Acinetobacter spp. detected aphA6 in the chromosome of 2 isolates of A. guillouiae, which is an environmental species, 1 of 8 A. parvus isolates, and 5 of 34 A. baumannii isolates. The gene was also present in 29 out of 36 A. guillouiae isolates screened by PCR, indicating that it is ancestral to this species. The Pnative promoter for aphA6 in A. guillouiae and A. parvus was replaced in A. baumannii by PaphA6, which was generated by use of the insertion sequence ISAba125, which brought a -35 sequence. Study of promoter strength in Escherichia coli and A. baumannii indicated that PaphA6 was four times more potent than Pnative. There was a good correlation between aminoglycoside MICs and aphA6 transcription in A. guillouiae isolates that remained susceptible to amikacin. The marked topology differences of the phylogenetic trees of aphA6 and of the hosts strongly support its recent direct transfer within Acinetobacter spp. and also to evolutionarily remote bacterial genera. Concomitant expression of aphA6 must have occurred because, contrary to the donors, it can confer resistance to the new hosts. Mobilization and expression of aphA6 via composite transposons and the upstream IS-generating hybrid PaphA6, followed by conjugation, seems the most plausible mechanism. This is in agreement with the observation that, in the recipients, aphA6 is carried by conjugative plasmids and flanked by IS that are common in Acinetobacter spp. Our data indicate that resistance genes can also be found in susceptible environmental bacteria. Importance: We speculated that the aphA6 gene for an enzyme that confers resistance to amikacin, the most active aminoglycoside for the treatment of nosocomial infections due to Acinetobacter spp., originated in this genus before disseminating to phylogenetically distant genera pathogenic for humans. Using a combination of whole-genome sequencing of a collection of Acinetobacter spp. covering the breadth of the known taxonomic diversity of the genus, gene cloning, detailed promoter analysis, study of heterologous gene expression, and comparative analysis of the phylogenetic trees of aphA6 and of the bacterial hosts, we found that aphA6 originated in Acinetobacter guillouiae, an amikacin-susceptible environmental species. The gene conferred, upon mobilization, high-level resistance to the new hosts. This work stresses that nonpathogenic bacteria can act as reservoirs of resistance determinants, and it provides an example of the use of a genomic library to study the origin and dissemination of an antibiotic resistance gene to human pathogens.

• MeSH
• Acinetobacter drug effects   enzymology   genetics   isolation & purification   MeSH
• Aminoglycosides pharmacology   MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Drug Resistance, Bacterial MeSH
• Escherichia coli enzymology   genetics   MeSH
• Phylogeny MeSH
• Kanamycin Kinase genetics   MeSH
• Conjugation, Genetic MeSH
• Microbial Sensitivity Tests MeSH
• Environmental Microbiology MeSH
• Evolution, Molecular MeSH
• Molecular Sequence Data MeSH
• Gene Transfer, Horizontal MeSH
• Promoter Regions, Genetic MeSH
• Interspersed Repetitive Sequences MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Homology MeSH
• Cluster Analysis MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Aminoglycosides MeSH
• Anti-Bacterial Agents MeSH
• Kanamycin Kinase MeSH

Bacterial genomics has greatly expanded our understanding of microdiversification patterns within a species, but analyses at higher taxonomical levels are necessary to understand and predict the independent rise of pathogens in a genus. We have sampled, sequenced, and assessed the diversity of genomes of validly named and tentative species of the Acinetobacter genus, a clade including major nosocomial pathogens and biotechnologically important species. We inferred a robust global phylogeny and delimited several new putative species. The genus is very ancient and extremely diverse: Genomes of highly divergent species share more orthologs than certain strains within a species. We systematically characterized elements and mechanisms driving genome diversification, such as conjugative elements, insertion sequences, and natural transformation. We found many error-prone polymerases that may play a role in resistance to toxins, antibiotics, and in the generation of genetic variation. Surprisingly, temperate phages, poorly studied in Acinetobacter, were found to account for a significant fraction of most genomes. Accordingly, many genomes encode clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems with some of the largest CRISPR-arrays found so far in bacteria. Integrons are strongly overrepresented in Acinetobacter baumannii, which correlates with its frequent resistance to antibiotics. Our data suggest that A. baumannii arose from an ancient population bottleneck followed by population expansion under strong purifying selection. The outstanding diversification of the species occurred largely by horizontal transfer, including some allelic recombination, at specific hotspots preferentially located close to the replication terminus. Our work sets a quantitative basis to understand the diversification of Acinetobacter into emerging resistant and versatile pathogens.

• Keywords
• bacterial genus, comparative genomics, evolution, mobile genetic elements, nosocomial pathogens,
• MeSH
• Acinetobacter genetics   MeSH
• Phylogeny MeSH
• Genome, Bacterial genetics   MeSH
• Genomics methods   MeSH
• Interspersed Repetitive Sequences genetics   MeSH
• Clustered Regularly Interspaced Short Palindromic Repeats genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH