Impact of Acinetobacter baumannii Superoxide Dismutase on Motility, Virulence, Oxidative Stress Resistance and Susceptibility to Antibiotics

Acinetobacter baumannii is a Gram-negative bacterium appearing as an opportunistic pathogen in hospital settings. Superoxide dismutase (SOD) contributes to virulence in several pathogenic bacteria by detoxifying reactive oxygen species released in the course of host defense reactions. However, the biological role of SODs in A. baumannii has not yet been elucidated. Here, we inactivated in A. baumannii ATCC 17978 gene A1S_2343, encoding a putative SOD of the Fe-Mn type by transposon insertion, resulting in mutant ATCC 17978 sod2343::Km. The mutation was also introduced in two naturally competent A. baumannii isolates by transformation with chromosomal DNA derived from mutant ATCC 17978 sod2343::Km. We demonstrate that inactivation of sod2343 leads to significant motility defects in all three A. baumannii strains. The mutant strains were more susceptible to oxidative stress compared to their parental strains. Susceptibility to colistin and tetracycline was increased in all mutant strains while susceptibility of the mutants to gentamicin, levofloxacin and imipenem was strain-dependent. In the Galleria mellonella infection model the mutant strains were significantly attenuated. In conclusion, sod2343 plays an important role in motility, resistance to oxidative stress, susceptibility to antibiotics and virulence in A. baumannii

Adding to Yersinia enterocolitica Gene Pool Diversity: Two Cryptic Plasmids from a Biotype 1A Isolate

We report the nucleotide sequence of two novel cryptic plasmids (4357 and 14 662 base pairs) carried by a Yersinia enterocolitica biotype 1A strain isolated from pork. As distinguished from most biotype 1A strains, this isolate, designated 07-04449, exhibited adherence to eukaryotic cells. The smaller plasmid pYe4449-1 carries five attributable open reading frames (ORFs) encoding the first CcdA/CcdB-like antitoxin/toxin system described for a Yersinia plasmid, a RepA-like replication initiation protein, and mobilizing factors MobA and MobC. The deduced amino acid sequences showed highest similarity to proteins described in Salmonella (CcdA/B), Klebsiella (RepA), and Plesiomonas (MobA/C) indicating genomic fluidity among members of the Enterobacteriaceae. One additional ORF with unknown function, termed ORF5, was identified with an ancestry distinct from the rest of the plasmid. While the C+G content of ORF5 is 38.3%, the rest of pYe4449-1 shows a C+G content of 55.7%. The C+G content of the larger plasmid pYe4449-2 (54.9%) was similar to that of pYe4449-1 (53.7%) and differed from that of the Y. enterocolitica genome (47.3%). Of the 14 ORFs identified on pYe4449-2, only six ORFs showed significant similarity to database entries. For three of these ORFs likely functions could be ascribed: a TnpR-like resolvase and a phage replication protein, localized each on a low C+G island, and DNA primase TraC. Two ORFs of pYe4449-2, ORF3 and ORF7, seem to encode secretable proteins. Epitope-tagging of ORF3 revealed protein expression at 4°C but not at or above 27°C suggesting adaptation to a habitat outside swine. The hypothetical protein encoded by ORF7 is the member of a novel repeat protein family sharing the DxxGN(x)nDxxGN motif. Our findings illustrate the exceptional gene pool diversity within the species Y. enterocolitica driven by horizontal gene transfer events

Complete Genome Sequencing of Acinetobacter sp. Strain LoGeW2-3, Isolated from the Pellet of a White Stork, Reveals a Novel Class D Beta-Lactamase Gene

Whole-genome sequencing of Acinetobacter sp. strain LoGeW2-3, isolated from the pellet of a white stork (Ciconia ciconia), reveals the presence of a plasmid of 179,399 bp encoding a CRISPR-Cas (clustered regularly interspaced short palindromic repeats and associated genes) system of the I-F type, and the chromosomally encoded novel class D beta-lactamase OXA-568

DNA Uptake by the Nosocomial Pathogen Acinetobacter baumannii Occurs during Movement along Wet Surfaces

The emergence of Acinetobacter baumannii as an increasingly multidrug-resistant nosocomial pathogen largely relies on acquisition of resistance genes via horizontal gene transfer. Here, we demonstrate that many clinical isolates of A. baumannii take up DNA while they move along wet surfaces. We show that both motility and DNA uptake are abolished after inactivation of pilT, which putatively encodes the type 4 pilus (T4P) retraction ATPase, and comEC, which putatively encodes the DNA uptake channel, respectively. Inactivation of pilT correlates with an increase in the number and length of pili with an average diameter of 7.2 nm. In the Galleria mellonella infection model, the comEC mutant is significantly attenuated, whereas the pilT mutant is not, dissecting biologically distinct roles of T4P and the DNA uptake channel. Collectively, these findings promote our understanding of the mechanisms of DNA uptake and resistance development in A. baumannii, which may also apply to other important pathogens

Orbus hercynius gen. nov., sp. nov., isolated from faeces of wild boar, is most related to Enterobacteriales and Pasteurellales

A novel gammaproteobacterium, strain CN3T, was isolated from faeces of wild boar. It is facultative anaerobic and appears coccoid or rod shaped. The determined partial 16S rRNA gene sequence of strain CN3T suggests a distant relationship to Enterobacteriales and Pasteurellales. The sequence shows highest similarity of 90.3% with Obesumbacterium proteus DSM 2777T, a member of the Enterobacteriaceae. The closest relatives outside the Enterobacteriales according to 16S rRNA gene sequence analysis are members of the Pasteurellales with 88.7% similarity (Mannheimia haemolytica NCTC 9380T and Actinobacillus lignieresii NCTC 4189T). In contrast to most members of the Enterobacteriales, strain CN3T is oxidase-positive. The pattern of fatty acids, in particular the high relative abundance of C18:1{omega}7c (38.5%), is clearly distinct from the conserved pattern of Pasteurellales. EcoRI ribotyping of strain CN3T yielded no significant similarity to database entries. Major ubiquinone of strain CN3T is Q-8. The DNA G+C content is 36.4 mol%. CN3T hosts a phage and secretes considerable amounts of three proteins into the culture supernatant. A spontaneous mutant of strain CN3T was isolated forming long filaments. Microscopic studies revealed the presence of a capsule which the mutant strain is unable to partition after cell division. CN3T (=DSM 22228T=CCUG 57622T) is considered as the type strain of a novel species within a new genus, for which the name Orbus hercynius gen. nov., sp. nov. is proposed. Its classification to family and order requires further investigation

Acinetobacter equi sp. nov., isolated from horse faeces

The taxonomic position of five strains isolated from horse faeces, and which shared identical 16S rRNA gene sequences, were studied. Cells of all isolates are Gram-stain-negative, obligately aerobic and have a rod-shaped appearance. The strains show highest 16S rRNA gene sequence similarities to Acinetobacter lwoffii (98.3 %), Acinetobacter haemolyticus (98.0 %), Acienetobacter johnsonii (97.9 %) and Acinetobacter brisouii (97.9 %). Whole-genome sequencing of strain 114T and phylogeny reconstruction based on a core set of 1061 Acinetobacter genes indicated that A. bouvetii CIP 107468T was the closest relative among species of the genus Acinetobacter for which whole genome sequences are available. The genomic DNA G+C content of strain 114T is 34.9 mol%, which is lower than any other value reported for the genus Acinetobacter. The predominant polyamine is 1,3-diaminopropane, which is typical for the genus Acinetobacter. The most abundant fatty acids are C16 : 1ω7c and/or iso-C15 : 0 2-OH (36 %) and C16 : 0 (28 %). The proportion of C18 : 1ω9c (7 %) is distinctively low compared to most species of the genus. The major ubiquinone of strain 114T is Q-9. Microscopic studies revealed the presence of pili and the absence of flagella. The capability of all five strains to utilize l-arabinose and gentisate as well as their lack of growth at temperatures of 41 °C and above provide sufficient criteria to distinguish the isolates from all species of the genus Acinetobacter with validly published names. Based on these combined data, the five isolates represent a novel species of the genus Acinetobacter, for which the name Acinetobacter equi sp. nov. is proposed. The type strain is 114T ( = DSM 27228T = CCUG 65204T)

<i>A. baumannii sod2343</i> mutants are attenuated in the <i>Galleria mellonella</i> infection model.

<p>(A) Survival of <i>Galleria</i> caterpillars injected with 3×10⁵ CFU of ATCC 17978 <i>sod2343::Km</i> (red), ATCC 17978 (green) and PBS (blue). (B) Infection with 1.5×10⁶ CFU of 07–095 and its <i>sod2343::Km</i> derivative. (C) Infection with 3×10⁵ CFU of 07–102 and its <i>sod2343::Km</i> mutant. Results represent means and standard deviations of at least three independent experiments of 16 larvae per group.</p

Confirmation of <i>sod2343::Km</i> mutants by immunoblotting.

<p>Overnight cultures as indicated were diluted 1∶20, grown for another 4 hours at 37°C, then adjusted to 1 OD (600 nm) and 0.5 ml of each was centrifuged and the pellet resuspended in 50 µl of loading buffer. 10 µl of each sample was loaded on an SDS-PAGE that was subsequently electro-blotted. A polyclonal antiserum raised against GST-SOD2343 fusion protein was diluted 1∶5000 for detection. The blots shown are representative of three independent replicates.</p

Growth defects of <i>sod2343</i> mutants depend on extent of aeration.

<p>Bacterial cultures as indicated were OD-adjusted from overnight cultures and grown at 37°C in LB medium under constant shaking for 8 hours either in baffled flasks (A) or in tubes (B) and the OD (600 nm) was determined. For each strain, data obtained from three independent cultures were averaged; error bars represent plus/minus one standard deviation. Growth of all <i>sod2343</i> mutants was significantly delayed compared to their parentals when grown in baffled flasks (A) (p<0.005, Student's <i>t</i> test,two-tailed, unpaired, for the last three time points). When grown in tubes (B), only the difference between ATCC 17978 and ATCC 17978 <i>sod2343</i> was significant (p<0.05).</p

Minimal inhibitory concentrations determined by Etest.

<p>Average MIC values in [µg/ml] determined from three (MIC³) and six (MIC⁶) independent experiments, respectively; MIC value given in <b>bold</b> indicates that MIC value of mutant is significantly different from MIC value of the corresponding parental strain (p<0.05; Student's t-test, two-tailed, unpaired;</p><p>* indicates p<0.005).</p