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

Charakterisierung einer Sulfit-Reduktase von Acinetobacter baumannii

Acinetobacter baumannii ist ein gram-negativer Erreger nosokomialer Infektionen, der zunehmend multiresistent ist, so dass die therapeutischen Möglichkeiten immer begrenzter werden. Kürzlich konnte gezeigt werden, dass Schwefelwasserstoff (H2S) in verschiedenen Bakterien als Signalmolekül wirkt, welches zur Resistenzausprägung gegenüber antimikrobiellen Substanzen beiträgt. Nach neueren Erkenntnissen lösen bakterizid wirkende Antibiotika oxidativen Stress durch die Entstehung reaktiver Sauerstoffspezies (ROS) aus. H2S kann dabei offensichtlich ROS direkt inaktivieren und zusätzlich die Aktivierung von Entgiftungsenzymen wie Superoxid-Dismutase (SOD) stimulieren. Vor diesem Hintergrund wurde die Sulfit-Reduktase (SR), die aus Sulfit H2S generiert und aus α- und β-Untereinheit besteht, in A. baumannii näher charakterisiert. Dazu wurde die ß-Untereinheit der SR zunächst in unterschiedlichen klinischen Isolaten von A. baumannii inaktiviert. Die SR-Mutanten waren auf Minimalmedium mit Sulfat als einziger Schwefelquelle nicht mehr kultivierbar. Sowohl durch Zugabe des H2S-Donors Natriumhydrogensulfid, als auch von Cystein sowie durch genetische Komplementierung des inaktivierten Genlocus konnte dieser Wachstumsdefekt kompensiert werden. Damit wurde hier gezeigt, dass die SR die einzige H2S-produzierende Reaktion in A. baumannii katalysiert. Die Inaktivierung der SR führte außerdem zu einer Virulenzabschwächung im Infektionsmodell der Wachsmottenlarve (Galleria mellonella), einer reduzierten Biofilmbildung und einer erhöhten Empfindlichkeit gegenüber verschiedenen Antibiotika-Wirkstoffklassen. Hemmstoffe gegen die SR, die in Säugern nicht vorkommt, könnten damit die Wirksamkeit gängiger Antibiotika unterstützen und gleichzeitig die Virulenz der Erreger schwächen. Die ß-Untereinheit der SR konnte in dieser Arbeit bereits erfolgreich rekombinant hergestellt werden und steht nun für strukturbiologische Arbeiten und für die weitere Wirkstoffsuche zur Verfügung. Aus Säugern ist bekannt, dass H2S als Signalmolekül über die Modifikation von Aminosäuren (Sulfhydrierung von Cystein) wirkt und so die Aktivität von Enzymen beeinflusst. Die Möglichkeit der Sulfhydrierung von Proteinen ist in Bakterien bislang jedoch noch nicht beschrieben wurden

<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

Recovery of <i>A. baumannii sod2343</i> mutant from <i>Galleria mellonella</i> larvae is minor compared to parental strain.

<p>Groups of 10 larvae were injected with 1.5×10⁶ CFU of ATCC 17978, ATCC 17978 <i>sod2343::Km</i>, and ATCC 17978 <i>sod2343::Km</i> pWH<i>sod2343</i>, respectively. Directly after injection of the 10 larvae within a group, the larvae were frozen at −80°C, homogenized and serial dilutions were plated on CHROMagar Acinetobacter to determine CFUs. Boxes indicate the 25–75% region, the median is indicated by a line in the box, the average is indicated by a small square, whiskers indicate maximum and minimum values, respectively.</p

Relatedness of wildlife and livestock avian isolates of the nosocomial pathogen Acinetobacter baumannii to lineages spread in hospitals worldwide

The natural habitats and potential reservoirs of the nosocomial pathogen Acinetobacter baumannii are poorly defined. Here, we put forth and tested the hypothesis of avian reservoirs of A. baumannii. We screened tracheal and rectal swab samples from livestock (chicken, geese) and wild birds (white stork nestlings) and isolated A. baumannii from 3% of sampled chicken (n = 220), 8% of geese (n = 40) and 25% of white stork nestlings (n = 661). Virulence of selected avian A. baumannii isolates was comparable to that of clinical isolates in the Galleria mellonella infection model. Whole genome sequencing revealed the close relationship of an antibiotic-susceptible chicken isolate from Germany with a multidrug-resistant human clinical isolate from China and additional linkages between livestock isolates and human clinical isolates related to international clonal lineages. Moreover, we identified stork isolates related to human clinical isolates from the United States. Multilocus sequence typing disclosed further kinship between avian and human isolates. Avian isolates do not form a distinct clade within the phylogeny of A. baumannii, instead they diverge into different lineages. Further, we provide evidence that A. baumannii is constantly present in the habitats occupied by storks. Collectively, our study suggests A. baumannii could be a zoonotic organism that may disseminate into livestock