A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana.

The sensing of microbe-associated molecular patterns (MAMPs) triggers innate immunity in animals and plants. Lipopolysaccharide (LPS) from Gram-negative bacteria is a potent MAMP for mammals, with the lipid A moiety activating proinflammatory responses via Toll-like receptor 4 (TLR4). Here we found that the plant Arabidopsis thaliana specifically sensed LPS of Pseudomonas and Xanthomonas. We isolated LPS-insensitive mutants defective in the bulb-type lectin S-domain-1 receptor-like kinase LORE (SD1-29), which were hypersusceptible to infection with Pseudomonas syringae. Targeted chemical degradation of LPS from Pseudomonas species suggested that LORE detected mainly the lipid A moiety of LPS. LORE conferred sensitivity to LPS onto tobacco after transient expression, which demonstrated a key function in LPS sensing and indicated the possibility of engineering resistance to bacteria in crop species.

New Obolenskvirus Phages Brutus and Scipio: Biology, Evolution, and Phage-Host Interaction.

Two novel virulent phages of the genus Obolenskvirus infecting Acinetobacter baumannii, a significant nosocomial pathogen, have been isolated and studied. Phages Brutus and Scipio were able to infect A. baumannii strains belonging to the K116 and K82 capsular types, respectively. The biological properties and genomic organization of the phages were characterized. Comparative genomic, phylogenetic, and pangenomic analyses were performed to investigate the relationship of Brutus and Scipio to other bacterial viruses and to trace the possible origin and evolutionary history of these phages and other representatives of the genus Obolenskvirus. The investigation of enzymatic activity of the tailspike depolymerase encoded in the genome of phage Scipio, the first reported virus infecting A. baumannii of the K82 capsular type, was performed. The study of new representatives of the genus Obolenskvirus and mechanisms of action of depolymerases encoded in their genomes expands knowledge about the diversity of viruses within this taxonomic group and strategies of Obolenskvirus-host bacteria interaction.

5,7-Diamino-3,5,7,9-tetradeoxynon-2-ulosonic Acids in the Capsular Polysaccharides of Acinetobacter baumannii.

The polysaccharide capsule surrounding bacterial cell plays an important role in pathogenesis of infections caused by the opportunistic pathogen Acinetobacter baumannii by providing protection from external factors. The structures of the capsular polysaccharide (CPS) produced by A. baumannii isolates and the corresponding CPS biosynthesis gene clusters are highly diverse, although many of them are related. Many types of A. baumannii CPSs contain isomers of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acid (DTNA). Three of these isomers, namely acinetaminic acid (l-glycero-l-altro isomer), 8-epiacinetaminic acid (d-glycero-l-altro isomer), and 8-epipseudaminic acid (d-glycero-l-manno isomer), have not been found so far in naturally occurring carbohydrates from other species. In A. baumannii CPSs, DTNAs carry N-acyl substituents at positions 5 and 7; in some CPSs, both N-acetyl and N-(3-hydroxybutanoyl) groups are present. Remarkably, pseudaminic acid carries the (R)-isomer and legionaminic acid carries the (S)-isomer of the 3-hydroxybutanoyl group. The review addresses the structure and genetics of biosynthesis of A. baumannii CPSs containing di-N-acyl derivatives of DTNA.

Structural and Serological Characterization of the O Antigen of Proteus mirabilis Clinical Isolates Classified into a New Proteus Serogroup, O84.

Two closely related Proteus mirabilis smooth strains, Kr1 and Ks20, were isolated from wound and skin samples, respectively, of two infected patients in central Poland. Serological tests, using the rabbit Kr1-specific antiserum, revealed that both strains presented the same O serotype. Their O antigens are unique among the Proteus O serotypes, which had been described earlier, as they were not recognized in an enzyme-linked immunosorbent assay (ELISA) by a set of Proteus O1-O83 antisera. Additionally, the Kr1 antiserum did not react with O1-O83 lipopolysaccharides (LPSs). The O-specific polysaccharide (OPS, O antigen) of P. mirabilis Kr1 was obtained via the mild acid degradation of the LPSs, and its structure was established via a chemical analysis and one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy applied to both initial and O-deacetylated polysaccharides, where most ß-2-acetamido-2-deoxyglucose (N-acetylglucosamine) (GlcNAc) residues are non-stoichiometrically O-acetylated at positions 3, 4, and 6 or 3 and 6, and a minority of α-GlcNAc residues are 6-O-acetylated. Based on the serological features and chemical data, P. mirabilis Kr1 and Ks20 were proposed as candidates to a new successive O-serogroup in the genus Proteus, O84, which is another example of new Proteus O serotypes identified lately among serologically differentiated Proteus bacilli infecting patients in central Poland.

Depolymerisation of the Klebsiella pneumoniae Capsular Polysaccharide K21 by Klebsiella Phage K5.

Klebsiella pneumoniae is a pathogen associated with various infection types, which often exhibits multiple antibiotic resistance. Phages, or bacterial viruses, have an ability to specifically target and destroy K. pneumoniae, offering a potential means of combatting multidrug-resistant infections. Phage enzymes are another promising therapeutic agent that can break down bacterial capsular polysaccharide, which shields K. pneumoniae from the immune response and external factors. In this study, Klebsiella phage K5 was isolated; this phage is active against Klebsiella pneumoniae with the capsular type K21. It was demonstrated that the phage can effectively lyse the host culture. The adsorption apparatus of the phage has revealed two receptor-binding proteins (RBPs) with predicted polysaccharide depolymerising activity. A recombinant form of both RBPs was obtained and experiments showed that one of them depolymerised the capsular polysaccharide K21. The structure of this polysaccharide and its degradation fragments were analysed. The second receptor-binding protein showed no activity on capsular polysaccharide of any of the 31 capsule types tested, so the substrate for this enzyme remains to be determined in the future. Klebsiella phage K5 may be considered a useful agent against Klebsiella infections.

Friunavirus Phage-Encoded Depolymerases Specific to Different Capsular Types of Acinetobacter baumannii.

Acinetobacter baumannii is a critical priority nosocomial pathogen that produces a variety of capsular polysaccharides (CPSs), the primary receptors for specific depolymerase-carrying phages. In this study, the tailspike depolymerases (TSDs) encoded in genomes of six novel Friunaviruses, APK09, APK14, APK16, APK86, APK127v, APK128, and one previously described Friunavirus phage, APK37.1, were characterized. For all TSDs, the mechanism of specific cleavage of corresponding A. baumannii capsular polysaccharides (CPSs) was established. The structures of oligosaccharide fragments derived from K9, K14, K16, K37/K3-v1, K86, K127, and K128 CPSs degradation by the recombinant depolymerases have been determined. The crystal structures of three of the studied TSDs were obtained. A significant reduction in mortality of Galleria mellonella larvae infected with A. baumannii of K9 capsular type was shown in the example of recombinant TSD APK09_gp48. The data obtained will provide a better understanding of the interaction of phage-bacterial host systems and will contribute to the formation of principles of rational usage of lytic phages and phage-derived enzymes as antibacterial agents.

Capsule-Targeting Depolymerases Derived from Acinetobacter baumannii Prophage Regions.

In this study, several different depolymerases encoded in the prophage regions of Acinetobacter baumannii genomes have been bioinformatically predicted and recombinantly produced. The identified depolymerases possessed multi-domain structures and were identical or closely homologous to various proteins encoded in other A. baumannii genomes. This means that prophage-derived depolymerases are widespread, and different bacterial genomes can be the source of proteins with polysaccharide-degrading activities. For two depolymerases, the specificity to capsular polysaccharides (CPSs) of A. baumannii belonging to K1 and K92 capsular types (K types) was determined. The data obtained showed that the prophage-derived depolymerases were glycosidases that cleaved the A. baumannii CPSs by the hydrolytic mechanism to yield monomers and oligomers of the K units. The recombinant proteins with established enzymatic activity significantly reduced the mortality of Galleria mellonella larvae infected with A. baumannii of K1 and K92 capsular types. Therefore, these enzymes can be considered as suitable candidates for the development of new antibacterials against corresponding A. baumannii K types.

Pectobacterium versatile Bacteriophage Possum: A Complex Polysaccharide-Deacetylating Tail Fiber as a Tool for Host Recognition in Pectobacterial Schitoviridae.

Novel, closely related phages Possum and Horatius infect Pectobacterium versatile, a phytopathogen causing soft rot in potatoes and other essential plants. Their properties and genomic composition define them as N4-like bacteriophages of the genus Cbunavirus, a part of a recently formed family Schitoviridae. It is proposed that the adsorption apparatus of these phages consists of tail fibers connected to the virion through an adapter protein. Tail fibers possess an enzymatic domain. Phage Possum uses it to deacetylate O-polysaccharide on the surface of the host strain to provide viral attachment. Such an infection mechanism is supposed to be common for all Cbunavirus phages and this feature should be considered when designing cocktails for phage control of soft rot.

Equine Intestinal O-Seroconverting Temperate Coliphage Hf4s: Genomic and Biological Characterization.

Tailed bacteriophages constitute the bulk of the intestinal viromes of vertebrate animals. However, the relationships between lytic and lysogenic lifestyles of phages in these ecosystems are not always clear and may vary between the species or even between the individuals. The human intestinal (fecal) viromes are dominated mostly by temperate phages, while in horse feces virulent phages are more prevalent. To our knowledge, all the previously reported isolates of horse fecal coliphages are virulent. Temperate coliphage Hf4s was isolated from horse feces, from the indigenous equine Escherichia coli 4s strain. It is a podovirus related to the Lederbergvirus genus (including the well-characterized Salmonella bacteriophage P22). Hf4s recognizes the host O antigen as its primary receptor and possesses a functional O antigen seroconversion cluster that renders the lysogens protected from superinfection by the same bacteriophage and also abolishes the adsorption of some indigenous equine virulent coliphages, such as DT57C, while other phages, such as G7C or phiKT, retain the ability to infect E. coli 4s (Hf4s) lysogens. IMPORTANCE The relationships between virulent and temperate bacteriophages and their impact on high-density symbiotic microbial ecosystems of animals are not always clear and may vary between species or even between individuals. The horse intestinal virome is dominated by virulent phages, and Hf4s is the first temperate equine intestinal coliphage characterized. It recognizes the host O antigen as its primary receptor and possesses a functional O antigen seroconversion cluster that renders the lysogens protected from superinfection by some indigenous equine virulent coliphages, such as DT57C, while other phages, such as G7C or phiKT, retain the ability to infect E. coli 4s (Hf4s) lysogens. These findings raise questions on the significance of bacteriophage-bacteriophage interactions within the ecology of microbial viruses in mammal intestinal ecosystems.

Synthesis of bodipy-labeled bacterial polysaccharides and their interaction with human dendritic cells.

In this report, we describe the fluorescent labeling of bacterial polysaccharides (Escherichia coli O86:B7, Escherichia coli O19ab, Pseudomonas aeruginosa O10a10b, and Shigella flexneri 2b) at the "natural" amino group of their phosphoethanolamine moiety. Two protocols for labeling are compared: 1) on a scale of a few mg of the polysaccharide, with a dialysis procedure for purification from excessive reagents; and 2) on a scale of 0.1 mg of the polysaccharide, with a simple precipitation procedure instead of dialysis. The microscale version is sufficient for comfortable cytofluorometric analysis. The resulting probes were found to specifically bind to human dendritic cells in a dose-dependent manner. The used limited set of polysaccharides did not allow us even to get close to understanding which dendritic cell-associated lectins and which cognate polysaccharide epitopes are involved in recognition, but the proposed microscale protocol allows to generate a library of fluorescent probes for further mapping of the polysaccharide specificity of the dendritic cells.

Acinetobacter baumannii K106 and K112: Two Structurally and Genetically Related 6-Deoxy-l-talose-Containing Capsular Polysaccharides.

Whole genome sequences of two Acinetobacter baumannii clinical isolates, 48-1789 and MAR24, revealed that they carry the KL106 and KL112 capsular polysaccharide (CPS) biosynthesis gene clusters, respectively, at the chromosomal K locus. The KL106 and KL112 gene clusters are related to the previously described KL11 and KL83 gene clusters, sharing genes for the synthesis of l-rhamnose (l-Rhap) and 6-deoxy-l-talose (l-6dTalp). CPS material isolated from 48-1789 and MAR24 was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy. The structures of K106 and K112 oligosaccharide repeats (K units) l-6dTalp-(1→3)-D-GlcpNAc tetrasaccharide fragment share the responsible genes in the respective gene clusters. The K106 and K83 CPSs also have the same linkage between K units. The KL112 cluster includes an additional glycosyltransferase gene, Gtr183, and the K112 unit includes α l-Rhap side chain that is not found in the K106 structure. K112 further differs in the linkage between K units formed by the Wzy polymerase, and a different wzy gene is found in KL112. However, though both KL106 and KL112 share the atr8 acetyltransferase gene with KL83, only K83 is acetylated.

Autographivirinae Bacteriophage Arno 160 Infects Pectobacterium carotovorum via Depolymerization of the Bacterial O-Polysaccharide.

Phytopathogenic bacteria belonging to the Pectobacterium and Dickeya genera (soft-rot Pectobacteriaceae) are in the focus of agriculture-related microbiology because of their diversity, their substantial negative impact on the production of potatoes and vegetables, and the prospects of bacteriophage applications for disease control. Because of numerous amendments in the taxonomy of P. carotovorum, there are still a few studied sequenced strains among this species. The present work reports on the isolation and characterization of the phage infectious to the type strain of P. carotovorum. The phage Arno 160 is a lytic Podovirus representing a potential new genus of the subfamily Autographivirinae. It recognizes O-polysaccahride of the host strain and depolymerizes it in the process of infection using a rhamnosidase hydrolytic mechanism. Despite the narrow host range of this phage, it is suitable for phage control application.

Expanding CSDB_GT glycosyltransferase database with Escherichia coli.

In 2017, we reported a new database on glycosyltransferase (GT) activities, CSDB_GT (http://csdb.glycoscience.ru/gt.html), which was built at the platform of the Carbohydrate Structure Database (CSDB, http://csdb.glycoscience.ru/database/index.html) and contained data on experimentally confirmed GT activities from Arabidopsis thaliana. All entries in CSDB_GT are curated manually upon the analysis of scientific publications, and the key features of the database are accurate structural, genetic, protein and bibliographic references and close-to-complete coverage on experimentally proven GT activities in selected species. In 2018, CSDB_GT was supplemented with data on Escherichia coli GT activities. Now it contains ca. 800 entries on E. coli GTs, including ca. 550 entries with functions predicted in silico. This information was extracted from research papers published up to the year 2018 or was obtained by the authors' efforts on GT annotation. Thus, CSDB_GT was extended to provide not only experimentally confirmed GT activities, but also those predicted on the basis of gene or protein sequence homology that could carry valuable information. Accordingly, a new confirmation status-predicted in silico-was introduced. In addition, the coverage on A. thaliana was extended up to ca. 900 entries, all of which had experimental confirmation. Currently, CSDB_GT provides close-to-complete coverage on experimentally confirmed GT activities from A. thaliana and E. coli presented up to the year 2018.

Acinetobacter baumannii K20 and K21 capsular polysaccharide structures establish roles for UDP-glucose dehydrogenase Ugd2, pyruvyl transferase Ptr2 and two glycosyltransferases.

Infections caused by Acinetobacter baumannii isolates from the major global clones, GC1 and GC2, are difficult to treat with antibiotics, and phage therapy, which requires extensive knowledge of the variation in the surface polysaccharides, is an option under consideration. The gene clusters directing the synthesis of capsular polysaccharide (CPS) in A. baumannii GC1 isolate A388 and GC2 isolate G21 differ by a single glycosyltransferase (gtr) gene. They include genes encoding a novel UDP-glucose dehydrogenase (Ugd2) and a putative pyruvyl transferase (Ptr2). The composition and structures of the linear K20 and K21 tetrasaccharide repeats (K units) of the CPSs isolated from A338 and G21, respectively, were established by sugar analyses and Smith degradation along with 1D and 2D 1H and 13C NMR spectroscopy. The K20 and K21 CPSs are the first known to include GlcpA produced by Ugd2 and d-galactose with an (R)-configured 4,6-pyruvic acid acetal added by Prt2. The first sugar in the tetrasaccharide K units is 2-acetamido-4-amino-2,4,6-trideoxy-d-glucose (d-QuipNAc4N) that carries a 4-N-[(S)-3-hydroxybutanoyl] group in some K units and a 4-N-acetyl group in the others. Accordingly, K unit polymerases WzyK20 and WzyK21 form a ß-d-QuipNAc4NR-(1→2)-d-Galp bond. The K20 and K21 units differ only in the configuration of the glycosidic linkages of d-GlcpNAc allowing the unique inverting glycosyltransferases Gtr43 and the retaining glycosyltransferase Gtr45 to be assigned to the formation of the ß-d-GlcpNAc-(1→4)-d-GlcpA and α-d-GlcpNAc-(1→4)-d-GlcpA linkages, respectively.

Function of bacteriophage G7C esterase tailspike in host cell adsorption.

Bacteriophages recognize and bind to their hosts with the help of receptor-binding proteins (RBPs) that emanate from the phage particle in the form of fibers or tailspikes. RBPs show a great variability in their shapes, sizes, and location on the particle. Some RBPs are known to depolymerize surface polysaccharides of the host while others show no enzymatic activity. Here we report that both RBPs of podovirus G7C - tailspikes gp63.1 and gp66 - are essential for infection of its natural host bacterium E. coli 4s that populates the equine intestinal tract. We characterize the structure and function of gp63.1 and show that unlike any previously described RPB, gp63.1 deacetylates surface polysaccharides of E. coli 4s leaving the backbone of the polysaccharide intact. We demonstrate that gp63.1 and gp66 form a stable complex, in which the N-terminal part of gp66 serves as an attachment site for gp63.1 and anchors the gp63.1-gp66 complex to the G7C tail. The esterase domain of gp63.1 as well as domains mediating the gp63.1-gp66 interaction is widespread among all three families of tailed bacteriophages.

Acinetobacter baumannii isolate BAL_212 from Vietnam produces the K57 capsular polysaccharide containing a rarely occurring amino sugar N-acetylviosamine.

The structures of capsular polysaccharides (CPSs) produced by different Acinetobacter baumannii strains have proven to be invaluable in confirming the role of specific genes in the synthesis of rare sugars through the correlation of genetic content at the CPS biosynthesis locus with sugars found in corresponding CPS structures. A module of four genes (rmlA, rmlB, vioA and vioB) was identified in the KL57 capsule biosynthesis gene cluster of A. baumannii isolate BAL_212 from Vietnam. These genes were predicted to direct the synthesis of 4-acetamido-4,6-dideoxy-d-glucose (N-acetylviosamine, d-Qui4NAc) and the K57 CPS was found to contain this monosaccharide. The K57 structure was determined and, in addition to d-Qui4NAc, included three N-acetylgalactosamine residues in the main chain, with a single glucose side branch. The KL57 gene cluster has not been found in any other A. baumannii genomes, but the rmlA-rmlB-vioA-vioB module is present in the KL119 gene cluster that would likely produce a d-Qui4NAc-containing CPS.

Genetics of biosynthesis and structure of the K53 capsular polysaccharide of Acinetobacter baumannii D23 made up of a disaccharide K unit.

The KL53 capsular polysaccharide (CPS) gene cluster of Acinetobacter baumannii D23 was sequenced, and includes a single gtr gene encoding the glycosyltransferase Gtr2, and the itrA1 gene for ItrA1 that is known to initiate CPS biosynthesis with d-QuiNAc4NAc. The K53 CPS was isolated and studied by one- and two-dimensional 1H and 13C nuclear magnetic resonance (NMR) spectroscopy before and after O-deacetylation. The disaccharide K unit of the CPS was established as →3)-α-d-GalpNAcA4Ac-(1→3)-ß-d-QuipNAc4NAc-(1→, where GalNAcA and QuiNAc4NAc indicate 2-acetamido-2-deoxygalacturonic acid and 2,4-diacetamido-2,4,6-trideoxyglucose, respectively. This established the linkage formed by Gtr2. The degree of 4-O-acetylation of d-GalNAcA by Atr18, encoded at the KL53 locus, is ~55 %.

Changes in the lipopolysaccharide of Proteus mirabilis 9B-m (O11a) clinical strain in response to planktonic or biofilm type of growth.

The impact of planktonic and biofilm lifestyles of the clinical isolate Proteus mirabilis 9B-m on its lipopolysaccharide (O-polysaccharide, core region, and lipid A) was evaluated. Proteus mirabilis bacteria are able to form biofilm and lipopolysaccharide is one of the factors involved in the biofilm formation. Lipopolysaccharide was isolated from planktonic and biofilm cells of the investigated strain and analyzed by SDS-PAGE with silver staining, Western blotting and ELISA, as well as NMR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry techniques. Chemical and NMR spectroscopic analyses revealed that the structure of the O-polysaccharide of P. mirabilis 9B-m strain did not depend on the form of cell growth, but the full-length chains of the O-antigen were reduced when bacteria grew in biofilm. The study also revealed structural modifications of the core region in the lipopolysaccharide of biofilm-associated cells-peaks assigned to compounds absent in cells from the planktonic culture and not previously detected in any of the known Proteus core oligosaccharides. No differences in the lipid A structure were observed. In summary, our study demonstrated for the first time that changes in the lifestyle of P. mirabilis bacteria leads to the modifications of their important virulence factor-lipopolysaccharide.

Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells.

LPS consists of a relatively conserved region of lipid A and core oligosaccharide and a highly variable region of O-antigen polysaccharide. Whereas lipid A is known to bind to the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex, the role of the O-antigen remains unclear. Here we report a novel molecular interaction between dendritic cell-associated C-type lectin-2 (Dectin-2) and mannosylated O-antigen found in a human opportunistic pathogen, Hafnia alvei PCM 1223, which has a repeating unit of [-Man-α1,3-Man-α1,2-Man-α1,2-Man-α1,2-Man-α1,3-]. H. alvei LPS induced higher levels of TNFα and IL-10 from mouse bone marrow-derived dendritic cells (BM-DCs), when compared with Salmonella enterica O66 LPS, which has a repeat of [-Gal-α1,6-Gal-α1,4-[Glc-ß1,3]GalNAc-α1,3-GalNAc-ß1,3-]. In a cell-based reporter assay, Dectin-2 was shown to recognize H. alvei LPS. This binding was inhibited by mannosidase treatment of H. alvei LPS and by mutations in the carbohydrate-binding domain of Dectin-2, demonstrating that H. alvei LPS is a novel glycan ligand of Dectin-2. The enhanced cytokine production by H. alvei LPS was Dectin-2-dependent, because Dectin-2 knock-out BM-DCs failed to do so. This receptor cross-talk between Dectin-2 and TLR4 involved events including spleen tyrosine kinase (Syk) activation and receptor juxtaposition. Furthermore, another mannosylated LPS from Escherichia coli O9a also bound to Dectin-2 and augmented TLR4 activation of BM-DCs. Taken together, these data indicate that mannosylated O-antigens from several Gram-negative bacteria augment TLR4 responses through interaction with Dectin-2.

A gene cluster at an unusual chromosomal location responsible for the novel O-antigen synthesis in Escherichia coli O62 by the ABC transporter-dependent pathway.

The O-antigen is a part of the outer membrane of Gram-negative bacteria and is related to bacterial virulence. It is one of the most variable cell constituents, and its structural diversity is almost entirely due to genetic variation of the O-antigen gene cluster. In this study, the O-antigen structure of Escherichia coli O62 was elucidated by chemical analysis and nuclear magnetic resonance spectroscopy, but showing not consistent with the O-antigen gene cluster between conserved genes galF and gnd reported earlier. The complete genome of E. coli O62 was then sequenced and analyzed, and another O-antigen gene cluster was found and characterized that correlated perfectly with the established O-antigen structure. A deletion and complementation experiment confirmed the functionality of the novel gene cluster and demonstrated that the O62-antigen is synthesized by the ABC transporter-dependent system. To our knowledge, this is the first report that the O-antigen gene cluster is positioned at a novel locus in E. coli. Comparative analysis indicated that E. coli O62 likely originated from E. coli O68 via an IS event resulting in the repression of the O68-antigen synthesis, followed by the acquisition of a novel O-antigen gene cluster from Enterobacter aerogenes.