SummaryThe 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 5 220), 8% of geese (n 5 40) and 25% of white stork nestlings (n 5 661). 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.

The acute and chronic bacterial toxicity of 34 organic compounds comprising 19 baseline narcotics and 15 epoxides has been determined with regard to 30-min bioluminescence and 24-h growth inhibition in terms of EC50 (effective concentration 50%) values employing Vibrio fischeri. For the narcotics, linear regression of log EC50 on log Kow (octanol/water partition coefficient) yields r2 (squared correlation coefficient) and rms (root-mean-square error) values of 0.95 and 0.44 (30-min), and 0.94 and 0.34 (24-h), respectively. Employing the resultant baseline narcosis models, toxicity enhancement (Te) values were derived as a ratio of narcosis-predicted over experimental EC50 for the epoxides. For seven aliphatic epoxides, log Te was below 1 in both assays, indicating narcosis-range toxicity with regard to 30-min bioluminescence and 24-h growth inhibition. Concerning eight nonaliphatic epoxides, log Te values up to 2.4 were observed, reflecting excess toxicity through an enhanced electrophilic reactivity of the compounds. Here, however, the intercorrelation between both assays was very low (r2 = 0.09). The results are discussed in terms of electronic substituent effects activating an SN2-type epoxide reaction with nucleophilic protein sites and side-chain activation offering alternative electrophile-nucleophile reaction routes at side-chain sites, leading to respective structural alerts as indicators of excess toxicity. Surprisingly, 30-min bioluminescence appears to be slightly more sensitive to chemical stress than 24-h growth, which holds both for baseline narcotics and for most of the epoxides. This is also reflected by effective narcosis doses 50%, ED50, of 7.1 mmol/kg (30-min) and 7.7 mmol/kg (24-h) estimated from narcosis theory. Keeping in mind the different end points (bioluminescence vs growth) involved, this finding demonstrates that chronic toxicity is not always more sensitive than acute toxicity, calling for analyses with regard to further respective cases and associated mechanistic causes.

For eight acrylates, three methacrylates, and three propiolates as three subclasses of α,β-unsaturated esters, short-term and long-term bacterial toxicity with Vibrio fischeri was determined in terms of EC(50) (effective concentration 50%) values for the 30-min bioluminescence and 24-h growth inhibition. To this end, experimental exposure concentrations were corrected for volatilization through a thermodynamic model based on Henry's law constant of the compounds. Moreover, toxicity enhancements T(e) as the ratio of narcosis-predicted over actual EC(50) were determined and discussed in terms of underlying mechanisms of reaction of the electrophiles with endogenous nucleophiles such as glutathione (GSH) and proteins. Overall, log EC(50) [M] ranges from -2.28 to -3.70 (30 min) and from -2.80 to -5.28 (24 h), respectively, indicating a significantly larger sensitivity of the growth inhibition bioassay for the reactive toxicity of these Michael acceptors. The latter is also reflected in the observed toxicity enhancements, where log T(e) > 1 was obtained for only 5 of 14 30-min EC(50) values but for 11 of 13 24-h EC(50) values. Moreover, the average long-term to short-term difference in log T(e) is 1 unit for the acrylates and 0.7 units for both methacrylates and propiolates. Methacrylates exert narcosis-level toxicity except for the methyl derivative in the long-term assay. The log EC(50) (24 h) of a subset of 10 mostly excess-toxic acrylates and a propiolate correlates with their logarithmic rate constants of reaction with GSH, log k(GSH), significantly more than with log K(ow) (r(2) 0.76 vs 0.47), yielding a respective regression rms of 0.34 log units. For allyl and propargyl acrylate as well as propargyl methacrylate, the observed excess toxicity is likely caused by initial enzymatic hydrolysis and subsequent oxidation of the α,β-unsaturated alcohols to the respective carbonyls. The latter shows that in the context of nonanimal testing schemes such as for REACH, the metabolic capacity of in vitro screens requires attention.

Prostaglandin E₂ (PGE₂), an arachidonic acid metabolite regulating a broad range of physiological activities, is an important modulator of the severity of infection caused by Streptococcus pyogenes. Here, we investigated the role of streptococcal cytolysin S (SLS) and streptococcal cytolysin O (SLO) in the induction of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of prostaglandins, in in vitro cultured macrophages and during in vivo infection. Macrophages were infected with S. pyogenes wild type or with the isogenic mutant strains deficient in SLS (ΔSLS), SLO (ΔSLO), or both (ΔSLS/ΔSLO), and the expression of COX-2 was determined at the transcriptional and the protein level. The results indicated that S. pyogenes induced expression of COX-2 and concomitant synthesis of PGE₂ in macrophages mediated by the synergistic activity of both SLS and SLO, and involved calcium and the PKC/JNK signaling pathway. These results were validated using recombinant cytolysins. In a murine skin infection model, COX-2-positive cells were found more abundant at the site of S. pyogenes wild-type infection than at the site of infection with ΔSLS/ΔSLO mutant strain. These findings suggest that inhibitory targeting of SLS and SLO could ameliorate the adverse effects of high levels of prostaglandins during S. pyogenes infection.

Acinetobacter baumannii appears as an often multidrug-resistant nosocomial pathogen in hospitals worldwide. Its remarkable persistence in the hospital environment is probably due to intrinsic and acquired resistance to disinfectants and antibiotics, tolerance to desiccation stress, capability to form biofilms, and is possibly facilitated by surface-associated motility. Our attempts to elucidate surface-associated motility in A. baumannii revealed a mutant inactivated in a putative DNA-(adenine N6)-methyltransferase, designated A1S_0222 in strain ATCC 17978. We recombinantly produced A1S_0222 as a glutathione S-transferase (GST) fusion protein and purified it to near homogeneity through a combination of GST affinity chromatography, cation exchange chromatography and PD-10 desalting column. Furthermore we demonstrate A1S_0222-dependent adenine methylation at a GAATTC site. We propose the name AamA (Acinetobacteradenine methyltransferase A) in addition to the formal names M.AbaBGORF222P/M.Aba17978ORF8565P. Small angle X-ray scattering (SAXS) revealed that the protein is monomeric and has an extended and likely two-domain shape in solution.