Differential Virulence of Aggregatibacter actinomycetemcomitans Serotypes Explained by Exoproteome Heterogeneity.

Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative bacterial pathogen associated with periodontitis and nonoral diseases like rheumatoid arthritis and Alzheimer´s disease. Aa isolates with the serotypes a, b, and c are globally most prevalent. Importantly, isolates displaying these serotypes have different clinical presentations. While serotype b isolates are predominant in severe periodontitis, serotypes a and c are generally encountered in mild periodontitis or healthy individuals. It is currently unknown how these differences are reflected in the overall secretion of virulence factors. Therefore, this study was aimed at a comparative analysis of exoproteomes from different clinical Aa isolates with serotypes a, b, or c by mass spectrometry, and a subsequent correlation of the recorded exoproteome profiles with virulence. Overall, we identified 425 extracellular proteins. Significant differences in the exoproteome composition of isolates with different serotypes were observed in terms of protein identification and abundance. In particular, serotype a isolates presented more extracellular proteins than serotype b or c isolates. These differences are mirrored in their virulence in infection models based on human salivary gland epithelial cells and neutrophils. Remarkably, serotype a isolates displayed stronger adhesive capabilities and induced more lysis of epithelial cells and neutrophils than serotype b or c isolates. Conversely, serotype c isolates showed relatively low leukotoxicity, while provoking NETosis to similar extents as serotype a and b isolates. Altogether, we conclude that the differential virulence presentation by Aa isolates with the dominant serotypes a, b, or c can be explained by their exoproteome heterogeneity. IMPORTANCE Periodontitis is an inflammatory disease that causes progressive destruction of alveolar bone and supporting tissues around the teeth, ultimately resulting in tooth loss. The bacterium Aggregatibacter actinomycetemcomitans (Aa) is a prevalent causative agent of periodontitis, but this oral pathogen is also associated with serious extraoral diseases like rheumatoid arthritis and Alzheimer's disease. Clinical Aa isolates are usually distinguished by serotyping, because of known serotype-specific differences in virulence. Aa with serotype b is associated with aggressive forms of periodontitis, while isolates with serotypes a or c are usually encountered in cases of mild periodontitis or healthy individuals. The molecular basis for these differences in virulence was so far unknown. In the present study, we pinpoint serotype-specific differences in virulence factor production by clinical Aa isolates. We consider these findings important, because they provide new leads for future preventive or therapeutic approaches to fight periodontitis and associated morbidities.

Exploration of oxygen-mediated disinfection of medical devices reveals a high sensitivity of Pseudomonas aeruginosa to elevated oxygen levels.

The microbiological safety of medical devices is of paramount importance for patients and manufacturers alike. However, during usage medical devices will inevitably become contaminated with microorganisms, including opportunistic pathogens. This is a particular problem if these devices come in contact with body sites that carry high bacterial loads, such as the oral cavity. In the present study, we investigated whether high oxygen concentrations can be applied to disinfect surfaces contaminated with different Gram-positive and Gram-negative bacteria. We show that some opportunistic pathogens, exemplified by Pseudomonas aeruginosa, are particularly sensitive to oxygen concentrations above the atmospheric oxygen concentration of 21%. Our observations also show that high oxygen concentrations can be applied to reduce the load of P. aeruginosa on nebulizers that are used by cystic fibrosis patients, who are particularly susceptible to colonization and infection by this bacterium. We conclude that the efficacy of oxygen-mediated disinfection depends on the bacterial species, duration of oxygen exposure and the oxygen concentration. We consider these observations relevant, because gas mixtures with high oxygen content can be readily applied for microbial decontamination. However, the main challenge for oxygen-based disinfection approaches resides in a potentially incomplete elimination of microbial contaminants, which makes combined usage with other disinfectants like ethanol or hydrogen peroxide recommendable.

Fighting Acinetobacter baumannii infections with the acylase PvdQ.

Acinetobacter baumannii is an opportunistic Gram-negative bacterial pathogen that poses a threat for frail patients worldwide. The high ability to withstand environmental stresses as well as its resistance towards a broad range of antibiotics make A. baumannii an effective hard-to-eradicate pathogen. One of the key mechanisms mediating tolerance against antibiotic treatment is the formation of biofilms, a process that is controlled by a multitude of different regulatory mechanisms. A key factor with major impact on biofilm formation is cell-to-cell communication by quorum-sensing, which in A. baumannii is mediated by acyl homoserine lactone signaling molecules. Here we show that the Ntn-Hydrolase PvdQ from Pseudomonas aeruginosa can reduce biofilm formation by the A. baumannii ATCC 17978 type strain and several clinical isolates on abiotic surfaces. Further, our study shows that a combination treatment of PvdQ-mediated quorum-quenching with the antibiotic gentamicin has a synergistic effect on the clearance of A. baumannii biofilms and possible biofilm dispersal. Moreover, we demonstrate in a Galleria mellonella larval infection model that PvdQ administration significantly prolongs survival of the larvae. Altogether, we conclude that the acylase-mediated irreversible cleavage of quorum-sensing signaling molecules as exemplified with PvdQ can set a profound limit to the progression of A. baumannii infections.

Bdellovibrio bacteriovorus: a potential 'living antibiotic' to control bacterial pathogens.

Bdellovibrio bacteriovorus is a small Deltaproteobacterium which, since its discovery, has distinguished itself for the unique ability to prey on other Gram-negative bacteria. The studies on this particular "predatory bacterium", have gained momentum in response to the rising problem of antibiotic resistance, because it could be applied as a potential probiotic and antibiotic agent. Hereby, we present recent advances in the study of B. bacteriovorus, comprehending fundamental aspects of its biology, obligatory intracellular life cycle, predation resistance, and potential applications. Furthermore, we discuss studies that pave the road towards the use of B. bacteriovorus as a "living antibiotic" in human therapy, focussing on its interaction with biofilms, the host immune response, predation susceptibility and in vivo application models. The available data imply that it will be possible to upgrade this predator bacterium from a predominantly academic interest to an instrument that could confront antibiotic resistant infections.

Proteomic Charting of Imipenem Adaptive Responses in a Highly Carbapenem Resistant Clinical Enterobacter roggenkampii Isolate.

Gram-negative bacteria belonging to the Enterobacter cloacae complex are increasingly implicated in difficult-to-treat nosocomial infections, as exemplified by a recently characterized highly carbapenem-resistant clinical Enterobacter roggenkampii isolate with sequence type (ST) 232. While mechanisms of carbapenem resistance are well-understood, little is known about the responses of highly drug-resistant bacteria to these antibiotics. Our present study was therefore aimed at charting the responses of the E. roggenkampii ST232 isolate to the carbapenem imipenem, using a 'stable isotope labeling of amino acids in cell culture' approach for quantitative mass spectrometry. This unveiled diverse responses of E. roggenkampii ST232 to imipenem, especially altered levels of proteins for cell wall biogenesis, central carbon metabolism, respiration, iron-sulfur cluster synthesis, and metal homeostasis. These observations suggest a scenario where imipenem-challenged bacteria reduce metabolic activity to save resources otherwise used for cell wall biogenesis, and to limit formation of detrimental reactive oxygen species at the cytoplasmic membrane due to respiration and Fenton chemistry. We consider these observations important, because knowing the adaptive responses of a highly resistant bacterium of the E. cloacae complex to last-resort antibiotics, such as imipenem, provides a 'sneak preview' into the future development of antibiotic resistance in this emerging group of pathogens.

Determining the Virulence Properties of Escherichia coli ST131 Containing Bacteriocin-Encoding Plasmids Using Short- and Long-Read Sequencing and Comparing Them with Those of Other E. coli Lineages.

Escherichia coli ST131 is a clinical challenge due to its multidrug resistant profile and successful global spread. They are often associated with complicated infections, particularly urinary tract infections (UTIs). Bacteriocins play an important role to outcompete other microorganisms present in the human gut. Here, we characterized bacteriocin-encoding plasmids found in ST131 isolates of patients suffering from a UTI using both short- and long-read sequencing. Colicins Ia, Ib and E1, and microcin V, were identified among plasmids that also contained resistance and virulence genes. To investigate if the potential transmission range of the colicin E1 plasmid is influenced by the presence of a resistance gene, we constructed a strain containing a plasmid which had both the colicin E1 and blaCMY-2 genes. No difference in transmission range was found between transformant and wild-type strains. However, a statistically significantly difference was found in adhesion and invasion ability. Bacteriocin-producing isolates from both ST131 and non-ST131 lineages were able to inhibit the growth of other E. coli isolates, including other ST131. In summary, plasmids harboring bacteriocins give additional advantages for highly virulent and resistant ST131 isolates, improving the ability of these isolates to compete with other microbiota for a niche and thereby increasing the risk of infection.