Coinfection of channel catfish (Ictalurus punctatus) with virulent Aeromonas hydrophila and Flavobacterium covae exacerbates mortality.

Flavobacterium covae and virulent Aeromonas hydrophila are prevalent bacterial pathogens within the US catfish industry that can cause high mortality in production ponds. An assessment of in vivo bacterial coinfection with virulent A. hydrophila (ML09-119) and F. covae (ALG-00-530) was conducted in juvenile channel catfish (Ictalurus punctatus). Catfish were divided into seven treatments: (1) mock control; (2) and (3) high and low doses of virulent A. hydrophila; (4) and (5) high and low doses of F. covae; (6) and (7) simultaneous challenge with high and low doses of virulent A. hydrophila and F. covae. In addition to the mortality assessment, anterior kidney and spleen were collected to evaluate immune gene expression, as well as quantify bacterial load by qPCR. At 96 h post-challenge (hpc), the high dose of virulent A. hydrophila infection (immersed in 2.3 × 107 CFU mL-1 ) resulted in cumulative percent mortality (CPM) of 28.3 ± 9.5%, while the high dose of F. covae (immersed in 5.2 × 106 CFU mL-1 ) yielded CPM of 23.3 ± 12.9%. When these pathogens were delivered in combination, CPM significantly increased for both the high- (98.3 ± 1.36%) and low-dose combinations (76.7 ± 17.05%) (p < .001). Lysozyme activity was found to be different at 24 and 48 hpc, with the high-dose vAh group demonstrating greater levels than unexposed control fish at each time point. Three proinflammatory cytokines (tnfα, il8, il1b) demonstrated increased expression levels at 48 hpc. These results demonstrate the additive effects on mortality when these two pathogens are combined. The synthesis of these mortality and health metrics advances our understanding of coinfections of these two important catfish pathogens and will aid fish health diagnosticians and channel catfish producers in developing therapeutants and prevention methods to control bacterial coinfections.

Probiotics enhance resistance to Streptococcus iniae in Nile tilapia (Oreochromis niloticus) reared in biofloc systems.

Biofloc technology is a rearing technique that maintains desired water quality by manipulating carbon and nitrogen and their inherent mixture of organic matter and microbes. Beneficial microorganisms in biofloc systems produce bioactive metabolites that may deter the growth of pathogenic microbes. As little is known about the interaction of biofloc systems and the addition of probiotics, this study focused on this integration to manipulate the microbial community and its interactions within biofloc systems. The present study evaluated two probiotics (B. velezensis AP193 and BiOWiSH FeedBuilder Syn 3) for use in Nile tilapia (Oreochromis niloticus) culture in a biofloc system. Nine independent 3785 L circular tanks were stocked with 120 juveniles (71.4 ± 4.4 g). Tilapia were fed for 16 weeks and randomly assigned three diets: a commercial control diet or a commercial diet top-coated with either AP193 or BiOWiSH FeedBuilder Syn3. At 14 weeks, the fish were challenged with a low dose of Streptococcus iniae (ARS-98-60, 7.2 × 107 CFU mL-1 , via intraperitoneal injection) in a common garden experimental design. At 16 weeks, the fish were challenged with a high dose of S. iniae (6.6 × 108 CFU mL-1 ) in the same manner. At the end of each challenge trial, cumulative per cent mortality, lysozyme activity and expression of 4 genes (il-1ß, il6, il8 and tnfα) from the spleen were measured. In both challenges, the mortalities of the probiotic-fed groups were significantly lower (p < .05) than in the control diet. Although there were some strong trends, probiotic applications did not result in significant immune gene expression changes related to diet during the pre-trial period and following exposure to S. iniae. Nonetheless, overall il6 expression was lower in fish challenged with a high dose of ARS-98-60, while tnfα expression was lower in fish subjected to a lower pathogen dose. Study findings demonstrate the applicability of probiotics as a dietary supplement for tilapia reared in biofloc systems.

Switchable inhibitory behavior of divalent magnesium ion in DNA hybridization-based gene quantification.

Contrary to the understanding that divalent cations only result in under-estimation of gene quantification via DNA hybridization-based assays, we have discovered that Mg2+ could cause either under or over-estimation at different concentrations. Its switchable inhibitory behavior is likely due to its rigid first solvation (hydrated) shell and hence it is inclined to form non-direct binding with DNA. At low concentrations, it caused under-estimation by occupying the hybridization sites. At high concentrations, it caused probe, signaling and target DNA to aggregate non-specifically via Coulomb forces. By quantifying target DNAs at a range of Mg2+ concentrations using a gene quantification assay (NanoGene assay), a Mg2+ inflection concentration of ∼10-3 M was observed for both target ssDNA and dsDNA. Field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray spectroscopy (EDS), and Fourier transform infrared spectroscopy (FT-IR) were employed to observe Mg2+-induced non-specific binding in the complexes that mimicked the presence of target DNA. Together with two other divalent cations Ca2+ and Cu2+, they were further examined via zeta potential measurements as well as NanoGene assay. This study revealed the importance of Mg2+ in achieving accurate gene quantification. Through a better mechanistic understanding of this phenomenon, it will be possible to develop strategies to mitigate the impact of Mg2+ on DNA hybridization-based gene quantification.

Dispersant Effects on Single-Walled Carbon Nanotube Antibacterial Activity.

There is significant interest in understanding whether nanomaterials with outstanding mechanical or electrical properties also possess antibacterial properties. However, assessment of antibacterial activity is a complex problem at the interface of chemistry and microbiology. Results can be affected by many factors including nanomaterial size, surface chemistry, concentration, and the dispersion media. The difficulty of dispersing nanomaterials such as single-walled carbon nanotubes (SWNTs) has resulted in many studies being conducted in the presence of dispersion aides which may themselves contribute to bacterial stress. The recent discovery that a standard microbial growth media, tryptic soy broth (TSB), is an effective SWNT dispersant provides a new opportunity to investigate the potential antibacterial activity of SWNTs using dispersants that range from antibacterial to growth-supporting. The five dispersants chosen for this work were Sodium dodecyl sulfate (SDS), pluronic, lysozyme, DNA, and tryptic soy broth. Staphylococcus aureus and Salmonella enterica were used as the model Gram-positive and Gram-negative bacteria. Activity was measured in terms of colony forming unit (CFU) and optical density measurements. None of the systems exhibited activity against Salmonella. SDS was fatal to Staph. aureus regardless of the presence of SWNTs. The activity of pluronic and lysozyme against Staph. aureus was enhanced by the presence of SWNTs. In contrast, the DNA and TSB dispersions did not have any activity regardless of the presence of SWNTs. These results highlight that the purported antibacterial activity of SWNTs may only be effective against bacteria that are sensitized by the dispersant and suggests the need for additional research on the mechanisms by which SWNT-dispersant interactions can result in antibacterial activity.

Differential production and secretion of potentially toxigenic extracellular proteins from hypervirulent Aeromonas hydrophila under biofilm and planktonic culture.

BACKGROUND: Hypervirulent Aeromonas hydrophila (vAh) is an emerging pathogen in freshwater aquaculture that results in the loss of over 3 million pounds of marketable channel catfish, Ictalurus punctatus, and channel catfish hybrids (I. punctatus, ♀ x blue catfish, I. furcatus, ♂) each year from freshwater catfish production systems in Alabama, U.S.A. vAh isolates are clonal in nature and are genetically unique from, and significantly more virulent than, traditional A. hydrophila isolates from fish. Even with the increased virulence, natural infections cannot be reproduced in aquaria challenges making it difficult to determine modes of infection and the pathophysiology behind the devastating mortalities that are commonly observed. Despite the intimate connection between environmental adaptation and plastic response, the role of environmental adaption on vAh pathogenicity and virulence has not been previously explored. In this study, secreted proteins of vAh cultured as free-living planktonic cells and within a biofilm were compared to elucidate the role of biofilm growth on virulence. RESULTS: Functional proteolytic assays found significantly increased degradative activity in biofilm secretomes; in contrast, planktonic secretomes had significantly increased hemolytic activity, suggesting higher toxigenic potential. Intramuscular injection challenges in a channel catfish model showed that in vitro degradative activity translated into in vivo tissue destruction. Identification of secreted proteins by HPLC-MS/MS revealed the presence of many putative virulence proteins under both growth conditions. Biofilm grown vAh produced higher levels of proteolytic enzymes and adhesins, whereas planktonically grown cells secreted higher levels of toxins, porins, and fimbrial proteins. CONCLUSIONS: This study is the first comparison of the secreted proteomes of vAh when grown in two distinct ecological niches. These data on the adaptive physiological response of vAh based on growth condition increase our understanding of how environmental niche partitioning could affect vAh pathogenicity and virulence. Increased secretion of colonization factors and degradative enzymes during biofilm growth and residency may increase bacterial attachment and host invasiveness, while increased secretion of hemolysins, porins, and other potential toxins under planktonic growth (or after host invasion) could result in increased host mortality. The results of this research underscore the need to use culture methods that more closely mimic natural ecological habitat growth to improve our understanding of vAh pathogenesis.

Enhanced biological control of root-knot nematode, Meloidogyne incognita, by combined inoculation of cotton or soybean seeds with a plant growth-promoting rhizobacterium and pectin-rich orange peel.

LC-MS analysis of plant growth-promoting rhizobacterium (PGPR) Bacillus velezensis AP203 supernatants indicated the presence of nematode-inhibiting compounds that increased in abundance when B. velezensis AP203 was grown on orange peel. Meloidogyne incognita J2 were incubated with B. velezensis AP203 spores and orange peel, spores alone, orange peel alone, or with a non-inoculated control, and the combination of B. velezensis AP203 with orange peel resulted in 94% mortality of M. incognita juveniles (p ≤ 0.05). The J2 mortality rate for B. velezensis alone was 53%, compared to 59% mortality with orange peel, and the non-inoculated control exhibited 7% mortality. When tested on soybeans raised in a greenhouse, it was observed that when grown in the presence of orange peel, B. velezensis AP203 culture broth, cell suspension or supernatant reduced the numbers of M. incognita eggs per g of root at 45 days after planting (DAP) compared to inoculated controls in soybean and cotton (p ≤ 0.05). Likewise, soybean root length and fresh root weight significantly increased after inoculation with B. velezensis AP203 amended with orange peel. In cotton, shoot and root length significantly increased after inoculation with cell pellets of B. velezensis AP203 amended with orange peel compared to the M. incognita inoculated control. These data indicate that B. velezensis AP203 responds to growth on pectin-rich orange peel by production of biologically active secondary metabolites that can promote plant growth and inhibit root-knot nematode viability.

A simple reagent-less approach using electrical discharge as a substitution for chelating agent in addressing genomic assay inhibition by divalent cations.

Electrical discharge treatment was shown to be a viable substitution for chelating agent in genomic assays. Divalent cation Mg2+ inhibits the performance of DNA hybridization based genomic assays by binding to the DNA and disrupting DNA hybridization. Until now, chelating agents such as ethylenediaminetetraacetic acid (EDTA) was the only option to address the presence of Mg2+ in samples. However, EDTA is a well-known environmental contaminant. In this work, we successfully employed electrical discharge instead of EDTA to render Mg2+ insipid. Its preliminary efficacy was first observed via circular dichroism (CD) and zeta potential analyses. After electrical discharge treatment, the reduction in CD shift at 280 nm was significant for samples with 10-3 and 10-8 M Mg2+. The zeta potential of Mg2+ laden samples were also restored from -4.71 ± 1.38 to -20.59 ± 6.37 mV after electrical discharge treatment. Both CD shift and change in zeta potential suggested that 2 min of electrical discharge treatment could prevent Mg2+ from binding to DNA. The complete efficacy of electrical discharge treatment was demonstrated with the performance recovery (within ∼15% of the control) of a genomic assay variant (NanoGene assay) while analyzing Mg2+ laden samples (10-5-10-3 M). Assuming 10 million samples are analyzed annually, the proposed electrical discharge treatment (∼50 mW per sample) would allow us to trade environmental contamination by ∼50 kg of hazardous EDTA with a single 250 W STC (standard test conditions) solar panel.

A genomic island in a plant beneficial rhizobacterium encodes novel antimicrobial fatty acids and a self-protection shield to enhance its competition.

Rhizobacteria devote a relatively large percentage of their genomes to encode bioactive natural products that are important for competition in the rhizosphere. In this study, a plant beneficial rhizobacterium Bacillus velezensis SQR9 was discovered to produce novel antibacterial fatty acids, Bacillunoic acids, which are encoded on a genomic island (GI). This GI contains a hybrid type I fatty acid synthase (FAS)-polyketide synthase (PKS) system and an ABC transporter. The FAS was predicted to synthesize a primer that was transferred to the PKS to synthesize Bacillunoic acids. The synthesized Bacillunoic acids inhibit the growth of diverse bacteria, with the strongest activity against closely related Bacillus strains, the ABC transporter exported the toxic Bacillunoic acids upon their induction for protecting the producing strain. The inhibition of other Bacillus strains by Bacillunoic acids extended the antimicrobial spectrum of SQR9 and enhanced its competition with closely related root-associated bacteria. So, through the obtaining of this GI by horizontal gene transfer, strain SQR9 not only acquired a competitive weapon but also acquired a self-protecting shield, which increased its competition with other rhizobacteria.

Aeromonas veronii and aerolysin are important for the pathogenesis of motile aeromonad septicemia in cyprinid fish.

Aeromonas species are ubiquitous inhabitants of freshwater environments, and are responsible for fish motile aeromonad septicemia (MAS). A. hydrophila is implicated as the primary etiologic agent of MAS. Here, we analysed MAS epidemiological data for cyprinid fish in southern China, and found that A. veronii infections dominated. Consistent with this observation, A. veronii isolates were generally more virulent than A. hydrophila isolates when infecting germ-free zebrafish larvae via continuous immersion challenge. Through in vivo screening of the transposon library of the A. veronii strain Hm091, aerolysin was identified as the key virulence factor. Further results indicated that A. veronii Hm091 aerolysin disrupts the intestinal barrier of zebrafish, enabling systematic invasion by not only A. veronii Hm091 in a mono-infection, but also A. hydrophila NJ-1 in a mixed infection. Moreover, the differences in aerolysin expression and activity were the major contributor to the observed differences between the A. veronii and A. hydrophila strains regarding invasion efficacy via intestine. Together, our results provide new insights into the aetiology and pathogenesis of Aeromonas infections, and highlight the importance of A. veronii-targeted treatments in future efforts against MAS.

Chloramphenicol Derivatives with Antibacterial Activity Identified by Functional Metagenomics.

A functional metagenomic approach identified novel and diverse soil-derived DNAs encoding inhibitors to methicillin-resistant Staphylococcus aureus (MRSA). A metagenomic DNA soil library containing 19 200 recombinant Escherichia coli BAC clones with 100 Kb average insert size was screened for antibiotic activity. Twenty-seven clones inhibited MRSA, seven of which were found by LC-MS to possess modified chloramphenicol ( Cm) derivatives, including three new compounds whose structures were established as 1-acetyl-3-propanoylchloramphenicol, 1-acetyl-3-butanoylchloramphenicol, and 3-butanoyl-1-propanoylchloramphenicol. Cm was used as the selectable antibiotic for cloning, suggesting that heterologously expressed enzymes resulted in derivatization of Cm into new chemical entities with biological activity. An esterase was found to be responsible for the enzymatic regeneration of Cm, and the gene trfA responsible for plasmid copy induction was found to be responsible for inducing antibacterial activity in some clones. Six additional acylchloramphenicols were synthesized for structure and antibacterial activity relationship studies, with 1- p-nitrobenzoylchloramphenicol the most active against Mycobacterium intracellulare and Mycobacterium tuberculosis, with MICs of 12.5 and 50.0 µg/mL, respectively.

Diverse roles of Hcp family proteins in the environmental fitness and pathogenicity of Aeromonas hydrophila Chinese epidemic strain NJ-35.

The type VI secretion system (T6SS) has been considered as a crucial factor in bacterial competition and virulence. The hemolysin co-regulated protein (Hcp) is the hallmark of T6SS. The secretion of Hcp in Aeromonas hydrophila Chinese epidemic strain NJ-35 indicated a functional T6SS. In this study, three copies of the hcp gene were identified in the genome of strain NJ-35. We targeted these Hcp family proteins for generating deletion mutants. These mutants showed varying levels in Hcp production, the interaction with other bacteria or eukaryotic cells, and bacterial virulence. Hcp1 was necessary for T6SS assembly and played a predominant role in the bacterial competition; Hcp2 negatively functioned in the biofilm formation and bacterial adhesion and was more involved in the A. hydrophila virulence in zebrafish and survival against the predation of Tetrahymena, and Hcp3 positively influenced the biofilm formation and bacterial adhesion. These findings illustrate that the T6SS of A. hydrophila NJ-35 is active, and the three Hcp family proteins take part in different processes in environmental adaptation and virulence of this bacterium. This study will provide valuable insights into our understanding of microbial interactions and thus contribute to a broader effort to manipulate these interactions for therapeutic or environmental benefit.

Chitin degradation and utilization by virulent Aeromonas hydrophila strain ML10-51K.

Virulent Aeromonas hydrophila (vAh) is one of the most important bacterial pathogens that causes persistent outbreaks of motile Aeromonas septicemia in warm-water fishes. The survivability of this pathogen in aquatic environments is of great concern. The aim of this study was to determine the capability of the vAh strain ML10-51K to degrade and utilize chitin. Genome-wide analysis revealed that ML10-51K encodes a suite of proteins for chitin metabolism. Assays in vitro showed that four chitinases, one chitobiase and one chitin-binding protein were secreted extracellularly and participated in chitin degradation. ML10-51K was shown to be able to use not only N-acetylglucosamine and colloidal chitin but also chitin flakes as sole carbon sources for growth. This study indicates that ML10-51K is a highly chitinolytic bacterium and suggests that the capability of effective chitin utilization could enable the bacterium to attain high densities when abundant chitin is available in aquatic niches.

Complete genome of Staphylococcus aureus Tager 104 provides evidence of its relation to modern systemic hospital-acquired strains.

BACKGROUND: Staphylococcus aureus (S. aureus) infections range in severity due to expression of certain virulence factors encoded on mobile genetic elements (MGE). As such, characterization of these MGE, as well as single nucleotide polymorphisms, is of high clinical and microbiological importance. To understand the evolution of these dangerous pathogens, it is paramount to define reference strains that may predate MGE acquisition. One such candidate is S. aureus Tager 104, a previously uncharacterized strain isolated from a patient with impetigo in 1947. RESULTS: We show here that S. aureus Tager 104 can survive in the bloodstream and infect naïve organs. We also demonstrate a procedure to construct and validate the assembly of S. aureus genomes, using Tager 104 as a proof-of-concept. In so doing, we bridged confounding gap regions that limited our initial attempts to close this 2.82 Mb genome, through integration of data from Illumina Nextera paired-end, PacBio RS, and Lucigen NxSeq mate-pair libraries. Furthermore, we provide independent confirmation of our segmental arrangement of the Tager 104 genome by the sole use of Lucigen NxSeq libraries filled by paired-end MiSeq reads and alignment with SPAdes software. Genomic analysis of Tager 104 revealed limited MGE, and a νSaß island configuration that is reminiscent of other hospital acquired S. aureus genomes. CONCLUSIONS: Tager 104 represents an early-branching ancestor of certain hospital-acquired strains. Combined with its earlier isolation date and limited content of MGE, Tager 104 can serve as a viable reference for future comparative genome studies.

Three novel virophage genomes discovered from Yellowstone Lake metagenomes.

UNLABELLED: Virophages are a unique group of circular double-stranded DNA viruses that are considered parasites of giant DNA viruses, which in turn are known to infect eukaryotic hosts. In this study, the genomes of three novel Yellowstone Lake virophages (YSLVs)--YSLV5, YSLV6, and YSLV7--were identified from Yellowstone Lake through metagenomic analyses. The relative abundance of these three novel virophages and previously identified Yellowstone Lake virophages YSLV1 to -4 were determined in different locations of the lake, revealing that most of the sampled locations in the lake, including both mesophilic and thermophilic habitats, had multiple virophage genotypes. This likely reflects the diverse habitats or diversity of the eukaryotic hosts and their associated giant viruses that serve as putative hosts for these virophages. YSLV5 has a 29,767-bp genome with 32 predicted open reading frames (ORFs), YSLV6 has a 24,837-bp genome with 29 predicted ORFs, and YSLV7 has a 23,193-bp genome with 26 predicted ORFs. Based on multilocus phylogenetic analysis, YSLV6 shows a close evolutionary relationship with YSLV1 to -4, whereas YSLV5 and YSLV7 are distantly related to the others, and YSLV7 represents the fourth novel virophage lineage. In addition, the genome of YSLV5 has a G+C content of 51.1% that is much higher than all other known virophages, indicating a unique host range for YSLV5. These results suggest that virophages are abundant and have diverse genotypes that likely mirror diverse giant viral and eukaryotic hosts within the Yellowstone Lake ecosystem. IMPORTANCE: This study discovered novel virophages present within the Yellowstone Lake ecosystem using a conserved major capsid protein as a phylogenetic anchor for assembly of sequence reads from Yellowstone Lake metagenomic samples. The three novel virophage genomes (YSLV5 to -7) were completed by identifying specific environmental samples containing these respective virophages, and closing gaps by targeted PCR and sequencing. Most of the YSLV genotypes were associated primarily with photic-zone and nonhydrothermal samples; however, YSLV5 had a unique distribution with an occurrence in vent samples similar to that in photic-zone samples and with a higher GC content that suggests a distinct host and habitat compared to other YSLVs. In addition, genome content and phylogenetic analyses indicate that YSLV5 and YSLV7 are distinct from known virophages and that additional as-yet-uncharacterized virophages are likely present within the Yellowstone Lake ecosystem.

Effect of Clostridium perfringens infection and antibiotic administration on microbiota in the small intestine of broiler chickens.

The etiological agent of necrotic enteritis (NE) is Clostridium perfringens (CP), which is an economically significant problem for broiler chicken producers worldwide. Traditional use of in-feed antibiotic growth promoters to control NE disease have resulted in the emergence of antibiotic resistance in CP strains. Identification of probiotic bacteria strains as an alternative to antibiotics for the control of intestinal CP colonization is crucial. Two experiments were conducted to determine changes in intestinal bacterial assemblages in response to CP infection and in-feed bacitracin methylene disalicylate (BMD) in broiler chickens. In each experiment conducted in battery-cage or floor-pen housing, chicks were randomly assigned to the following treatment groups: 1) BMD-supplemented diet with no CP challenge (CM), 2) BMD-free control diet with no CP challenge (CX), 3) BMD-supplemented diet with CP challenge (PCM), or 4) BMD-free control diet with CP challenge (PCX). The establishment of CP infection was confirmed, with the treatment groups exposed to CP having a 1.5- to 2-fold higher CP levels (P < 0.05) compared to the non-exposed groups. Next-generation sequencing of PCR amplified 16S rRNA genes, was used to perform intestinal bacterial diversity analyses pre-challenge, and at 1, 7, and 21 d post-challenge. The results indicated that the intestinal bacterial assemblage was dominated by members of the phylum Firmicutes in all treatments before and after CP challenge, especially the Lactobacillaceae and Clostridiales families. In addition, we observed post-challenge emergence of members of the Enterobacteriaceae and Streptococcaceae in the non-medicated PCX treatment, and emergence of the Enterococcaceae in the medicated PCM treatment. This study highlights the bacterial interactions that could be important in suppressing or eliminating CP infection within the chicken intestine. Future studies should explore the potential to use commensal strains of unknown Clostridiales, Lactobacillaceae, Enterobacteriaceae, Streptococcaceae, and Enterococcaceae in effective probiotic formulations for the control of CP and NE disease.

Bacillusin A, an Antibacterial Macrodiolide from Bacillus amyloliquefaciens AP183.

Bioassay-guided fractionation of the organic extracts of a Bacillus amyloliquefaciens strain (AP183) led to the discovery of a new macrocyclic polyene antibiotic, bacillusin A (1). Its structure was assigned by interpretation of NMR and MS spectroscopic data as a novel macrodiolide composed of dimeric 4-hydroxy-2-methoxy-6-alkenylbenzoic acid lactones with conjugated pentaene-hexahydroxy polyketide chains. Compound 1 showed potent antibacterial activities against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium with minimum inhibitory concentrations in a range of 0.6 to 1.2 µg/mL. The biosynthetic significance of this unique class of antibiotic compounds is briefly discussed.

Discovery and Derivatization of Tridecaptin Antibiotics with Altered Host Specificity and Enhanced Bioactivity.

The prevalence of multidrug-resistant (MDR) pathogens combined with a decline in antibiotic discovery presents a major challenge for health care. To refill the discovery pipeline, we need to find new ways to uncover new chemical entities. Here, we report the global genome mining-guided discovery of new lipopeptide antibiotics tridecaptin A5 and tridecaptin D, which exhibit unusual bioactivities within their class. The change in the antibacterial spectrum of Oct-TriA5 was explained solely by a Phe to Trp substitution as compared to Oct-TriA1, while Oct-TriD contained 6 substitutions. Metabolomic analysis of producer Paenibacillus sp. JJ-21 validated the predicted amino acid sequence of tridecaptin A5. Screening of tridecaptin analogues substituted at position 9 identified Oct-His9 as a potent congener with exceptional efficacy against Pseudomonas aeruginosa and reduced hemolytic and cytotoxic properties. Our work highlights the promise of tridecaptin analogues to combat MDR pathogens.

Secreted pyomelanin of Legionella pneumophila promotes bacterial iron uptake and growth under iron-limiting conditions.

Iron acquisition is critical to the growth and virulence of Legionella pneumophila. Previously, we found that L. pneumophila uses both a ferrisiderophore pathway and ferrous iron transport to obtain iron. We now report that two molecules secreted by L. pneumophila, homogentisic acid (HGA) and its polymerized variant (HGA-melanin, a pyomelanin), are able to directly mediate the reduction of various ferric iron salts. Furthermore, HGA, synthetic HGA-melanin, and HGA-melanin derived from bacterial supernatants enhanced the ability of L. pneumophila and other species of Legionella to take up radiolabeled iron. Enhanced iron uptake was not observed with a ferrous iron transport mutant. Thus, HGA and HGA-melanin mediate ferric iron reduction, with the resulting ferrous iron being available to the bacterium for uptake. Upon further testing of L. pneumophila culture supernatants, we found that significant amounts of ferric and ferrous iron were associated with secreted HGA-melanin. Importantly, a pyomelanin-containing fraction obtained from a wild-type culture supernatant was able to stimulate the growth of iron-starved legionellae. That the corresponding supernatant fraction obtained from a nonpigmented mutant culture did not stimulate growth demonstrated that HGA-melanin is able to both promote iron uptake and enhance growth under iron-limiting conditions. Indicative of a complementary role in iron acquisition, HGA-melanin levels were inversely related to the levels of siderophore activity. Compatible with a role in the ecology and pathogenesis of L. pneumophila, HGA and HGA-melanin were effective at reducing and releasing iron from both insoluble ferric hydroxide and the mammalian iron chelates ferritin and transferrin.

Genomic characterization of a novel, widely distributed Mycoplasma species "Candidatus Mycoplasma mahonii" associated with the brittlestar Gorgonocephalus chilensis.

Symbiotic relationships are ubiquitous throughout the world's oceans, yet for many marine organisms, including those in the high latitudes, little is understood about symbiotic associations and functional relationships. From a recently determined genome sequence of a filter-feeding basket star from Argentina, Gorgonocephalus chilensis, we discovered a novel Mycoplasma species with a 796Kb genome (CheckM completeness of 97.9%, G+C content = 30.1%). Similar to other Mycoplasma spp. within Mycoplasmatota, genomic analysis of the novel organism revealed reduced metabolic pathways including incomplete biosynthetic pathways, suggesting an obligate association with their basket star host. Results of 16S rRNA and multi-locus phylogenetic analyses revealed that this organism belonged to a recently characterized non-free-living lineage of Mycoplasma spp. specifically associated with marine invertebrate animals. Thus, the name "Candidatus Mycoplasma mahonii" is proposed for this novel species. Based on 16S rRNA PCR-screening, we found that Ca. M. mahonii also occurs in Gorgonocephalus eucnemis from the Northwest Pacific and other Gorgonocephalus chilensis from Argentinian waters. The level of sequence conservation within Ca. M. mahonii is considerable between widely disparate high-latitude Gorgonocephalus species, suggesting that oceanic dispersal of this microbe may be greater than excepted.