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Reverse Vaccinology (RV) is a widely used approach to identify potential vaccine candidates (PVCs) by screening the proteome of a pathogen through computational analyses. Since its first application in Group B (MenB) vaccine in early 1990's, several software programs have been developed implementing different flavors of the first RV protocol. However, there has been no comprehensive review to date on these different RV tools. We have compared six of these applications designed for bacterial vaccines (NERVE, Vaxign, VaxiJen, Jenner-predict, Bowman-Heinson, and VacSol) against a set of 11 pathogens for which a curated list of known bacterial protective antigens (BPAs) was available. We present results on: (1) the comparison of criteria and programs used for the selection of PVCs (2) computational runtime and (3) performances in terms of fraction of proteome identified as PVC, fraction and enrichment of BPA identified in the set of PVCs. This review demonstrates that none of the programs was able to recall 100% of the tested set of BPAs and that the output lists of proteins are in poor agreement suggesting in the process of prioritize vaccine candidates not to rely on a single RV tool response. Singularly the best balance in terms of fraction of a proteome predicted as good candidate and recall of BPAs has been observed by the machine-learning approach proposed by Bowman (1) and enhanced by Heinson (2). Even though more performing than the other approaches it shows the disadvantage of limited accessibility to non-experts users and strong dependence between results and training dataset composition. In conclusion we believe that to significantly enhance the performances of next RV methods further studies should focus on the enhancement of accuracy of the existing protein annotation tools and should leverage on the assets of machine-learning techniques applied to biological datasets expanded also through the incorporation and curation of bacterial proteins characterized by negative experimental results.

Here, we hypothesize that dysbiotic gut microbiota might contribute to the development of Kawasaki disease (KD), a pediatric disease with unknown etiology. This is the second report on gut microbiota composition in KD patients. 16S amplicon sequencing was performed on fecal DNA samples and revealed predominance of bacterial pathogens, such as , ,  and , in the gut of KD patients, but absent or suppressed after immunoglobulin/antibiotics therapy. In addition, beneficial bacteria propagated after the therapy. We conclude that prevalence of Fusobacteria,  and might contribute to KD pathogenesis.

Inquilinus limosus belongs to the class of the Alphaproteobacteria and was first described in 2002. So far, the species has mainly been isolated from respiratory specimens of patients with cystic fibrosis. A main characteristic of Inquilinus limosus is the prolonged time until bacterial colony growth is detectable. As the defined incubation times in many laboratories are too short to detect the growth of Inquilinus limosus , it is likely that the species is less frequently detected in the clinical setting than it actually occurs. This also explains why there are currently only very few data on the incidence available. Furthermore, as an uncommon pathogen, Inquilinus limosus may be familiar to only a few specialised clinicians. Due to these reasons, only little research (e.g. case reports and research papers) have been published on this species to date. However, given that a clear human pathogenic significance can be deduced from the existing literature, we have decided to present the current state of knowledge in this review and to address further aspects for the future elucidation of the pathogenesis of Inquilinus limosus .

Objective: This work was conducted for the development of a 5-combi lateral flow immunochro¬matographic kit (LFK) for rapid and simultaneous identification of the common bacterial causes of bovine mastitis. The following pathogens are the identification targets of this kit: Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus agalactiae, and Streptococcus pyogenes in milk samples from suspected bovine mastitis cases. The conventional microbiological identification of these agents is not only time-consuming and requires a fully equipped laboratory but also requires experienced personnel. Materials and Methods: Rabbit polyclonal antibodies (PAbs) specific to the antigenic components of the selected pathogens were prepared, and the pathogen-specific IgG was separated, purified, and conjugated with nanogold that was laid on the conjugate pad. Guinea pig PAbs specific to the microbial antigens of the selected pathogens were prepared, and their IgG content was separated, purified, and used as a capture antibody in the test (T) line on the nitrocellulose (NC) strips. Goat anti-rabbit IgG antibodies were used to capture the rabbit antibodies in the control (C) line of NC strips. The kit was held in a device comprising five strip-holding channels for the above-mentioned five bacterial species antigens. The developed LFK was evaluated, and its sensitivity and specificity were determined. Results: The developed kits were applied for the examination of bovine milk samples from suspected mastitis cases, and the average sensitivity, specificity, and accuracy of 5-combi LFK for the detection of the five selected bacterial species compared to bacteriological examination (gold standard test) were 93.90%, 80.83%, and 90.53%, respectively. The minimal microbial count that gave positive results using the developed LFK was 103 colony forming unit/ml. Treatment of the milk samples with an application buffer and its pre-incubation in trypticase soy broth for 6 h at 37°C before testing significantly increased the sensitivity of the prepared LFK. The developed kit proved simple and convenient, and the results could be obtained in less than 10 min.

This paper highlights insights gained from fully sequenced bacterial pathogen genomes that are of particular relevance to plant biologists. It describes the range of bacterial phytopathogens and their lifestyles in plants, lessons gained from type III effector repertoires (a focus of much study during this period), major insights arising from each of the phytopathogen groups with completely sequenced genomes, and future challenges.

The Pseudomonas syringae-Arabidopsis (Arabidopsis thaliana) interaction is an extensively studied plant-pathogen system. Arabidopsis possesses approximately 150 putative resistance genes encoding nucleotide binding site (NBS) and leucine-rich repeat (LRR) domain-containing proteins. The majority of these belong to the Toll/Interleukin-1 receptor (TIR)-NBS-LRR (TNL) class. Comparative studies with the coiled-coil-NBS-LRR genes RPS2, RPM1, and RPS5 and isogenic P. syringae strains expressing single corresponding avirulence genes have been particularly fruitful in dissecting specific and common resistance signaling components. However, the major TNL class is represented by a single known P. syringae resistance gene, RPS4. We previously identified hopA1 from P. syringae pv syringae strain 61 as an avirulence gene that signals through ENHANCED DISEASE SUSCEPTIBILITY1, indicating that the corresponding resistance gene RPS6 belongs to the TNL class. Here we report the identification of RPS6 based on a forward-genetic screen and map-based cloning. Among resistance proteins of known function, the deduced amino acid sequence of RPS6 shows highest similarity to the TNL resistance protein RAC1 that determines resistance to the oomycete pathogen Albugo candida. Similar to RPS4 and other TNL genes, RPS6 generates alternatively spliced transcripts, although the alternative transcript structures are RPS6 specific. We previously characterized SRFR1 as a negative regulator of avrRps4-triggered immunity. Interestingly, mutations in SRFR1 also enhanced HopA1-triggered immunity in rps6 mutants. In conclusion, the cloning of RPS6 and comparisons with RPS4 will contribute to a closer dissection of the TNL resistance pathway in Arabidopsis.

Stable surface adhesion of cells is one of the early pivotal steps in bacterial biofilm formation, a prevalent adaptation strategy in response to changing environments. In Pseudomonas fluorescens, this process is regulated by the Lap system and the second messenger cyclic-di-GMP. High cytoplasmic levels of cyclic-di-GMP activate the transmembrane receptor LapD that in turn recruits the periplasmic protease LapG, preventing it from cleaving a cell surface-bound adhesin, thereby promoting cell adhesion. In this study, we elucidate the molecular basis of LapG regulation by LapD and reveal a remarkably sensitive switching mechanism that is controlled by LapD's HAMP domain. LapD appears to act as a coincidence detector, whereby a weak interaction of LapG with LapD transmits a transient outside-in signal that is reinforced only when cyclic-di-GMP levels increase. Given the conservation of key elements of this receptor system in many bacterial species, the results are broadly relevant for cyclic-di-GMP- and HAMP domain-regulated transmembrane signaling. While bacteria often live as unicellular microorganisms, many bacteria are capable of sticking together on a surface and forming a multicellular structure called a biofilm. Bacterial biofilms occur frequently in nature; for example, on the roots of plants and submerged rocks. While these biofilms are generally innocuous, others pose significant health threats to humans, causing tooth decay, gum disease, and—when they occur on implanted devices such as prosthetic heart valves—potentially serious infections. When in biofilms, many bacteria are tolerant to antibiotics; therefore, working out how to disrupt these films is crucial for developing new treatments. The microorganism Pseudomonas fluorescens is an example of a bacterium that can be found living in a complex biofilm. In response to certain environmental cues, free-swimming P. fluorescens cells adhere to a surface and produce a slime that encases them in a robust biofilm. The decision to shift between a free-swimming and a biofilm life-style is orchestrated by a signaling molecule found inside the bacteria called cyclic-di-GMP. In P. fluorescens, the availability of nutrients—in particular, phosphate—controls how much cyclic-di-GMP is produced inside the cell. If not enough phosphate is available, the level of cyclic-di-GMP falls and the biofilm disperses. Cyclic-di-GMP affects the stability of the biofilm via a group of proteins called the Lap system. When levels of cyclic-di-GMP are high, cyclic-di-GMP binds to a protein called LapD, which can then in turn bind to an enzyme known as LapG. When bound to LapD, LapG is unable to break apart the molecules that help P. fluorescens cells bind to a surface, and so a biofilm can form. If cyclic-di-GMP levels drop, fewer LapD molecules can bind to cyclic-di-GMP. As cyclic-di-GMP-unbound LapD proteins interact poorly with LapG, this leaves some LapG molecules able to destabilize the attachments between the cells and the surface, which disperses the biofilm. Here, Chatterjee et al. reveal the molecular mechanism by which LapD and LapG interact in P. fluorescens. When cyclic-di-GMP is bound to LapD, the shape of LapD changes to produce features that fit into the surface of LapG. It is this shape compatibility, more so than an increase in the number or quality of interactions between the chemical groups that make up the proteins, that enables LapD to bind to LapG. Chatterjee et al. also provide evidence that the LapD–LapG interaction can be disrupted, thereby raising the possibility that biofilm formation could be manipulated by targeting this system. Given that systems similar to the P. fluorescens Lap system exist in numerous other bacterial species, including important pathogens, the findings of Chatterjee et al. could assist efforts to develop medicines and products that eradicate bacterial biofilms. LapD also shares many structural elements with a large number of other signaling proteins; therefore, these findings could also improve the understanding of how other cell signaling systems work.

Garlic has long been known as the most effective plant species in treatment of bacterial infections. Considering the vast potential of garlic as a source of antimicrobial drugs, this study is aimed to evaluate the antibacterial activity of Allium sativum extracts and their interactions with selected antibiotics against drug-sensitive and multidrug-resistant isolates of emerging bacterial pathogens that are frequently found in healthcare settings. As shown by the in vitro data obtained in this study, the whole Allium sativum extract inhibited the growth of a broad range of bacteria, including multidrug-resistant strains with bactericidal or bacteriostatic effects. Depending on the organism, the susceptibility to fresh garlic extract was comparable to the conventional antibiotic gentamycin. Since the combinations of fresh garlic extract with gentamycin and ciprofloxacin inhibited both the drug sensitive and MDR bacteria, in most cases showing a synergistic or insignificant relationship, the potential use of such combinations may be beneficial, especially in inhibiting drug-resistant pathogens. The study results indicate the possibility of using garlic as e.g. a supplement used during antibiotic therapy, which may increase the effectiveness of gentamicin and ciprofloxacin.

One of the key mechanisms enabling bacterial cells to create biofilms and regulate crucial life functions in a global and highly synchronized way is a bacterial communication system called quorum sensing (QS). QS is a bacterial cell-to-cell communication process that depends on the bacterial population density and is mediated by small signalling molecules called autoinducers (AIs). In bacteria, QS controls the biofilm formation through the global regulation of gene expression involved in the extracellular polymeric matrix (EPS) synthesis, virulence factor production, stress tolerance and metabolic adaptation. Forming biofilm is one of the crucial mechanisms of bacterial antimicrobial resistance (AMR). A common feature of human pathogens is the ability to form biofilm, which poses a serious medical issue due to their high susceptibility to traditional antibiotics. Because QS is associated with virulence and biofilm formation, there is a belief that inhibition of QS activity called quorum quenching (QQ) may provide alternative therapeutic methods for treating microbial infections. This review summarises recent progress in biofilm research, focusing on the mechanisms by which biofilms, especially those formed by pathogenic bacteria, become resistant to antibiotic treatment. Subsequently, a potential alternative approach to QS inhibition highlighting innovative non-antibiotic strategies to control AMR and biofilm formation of pathogenic bacteria has been discussed.