A Global Survey of Hypervirulent Aeromonas hydrophila (vAh) Identified vAh Strains in the Lower Mekong River Basin and Diverse Opportunistic Pathogens from Farmed Fish and Other Environmental Sources.

Hypervirulent Aeromonas hydrophila (vAh) has emerged as the etiologic agent of epidemic outbreaks of motile Aeromonas septicemia (MAS) in high-density aquaculture of farmed carp in China and catfish in the United States, which has caused millions of tons of lost fish. We conducted a global survey to better understand the evolution, geographical distribution, and phylogeny of vAh. Aeromonas isolates were isolated from fish that showed clinical symptoms of MAS, and pure cultures were screened for the ability to utilize myo-inositol as the sole carbon source. A total of 113 myo-inositol-utilizing bacterial strains were included in this study, including additional strains obtained from previously published culture collections. Based on a gyrB phylogeny, this collection included 66 A. hydrophila isolates, 48 of which were vAh. This collection also included five new vAh isolates from diseased Pangas catfish (Pangasius pangasius) and striped catfish (Pangasianodon hypophthalmus) obtained in Cambodia and Vietnam, respectively. Genome sequences were generated from representative vAh and non-vAh isolates to evaluate the potential for lateral genetic transfer of the myo-inositol catabolism pathway. Phylogenetic analyses of each of the nine genes required for myo-inositol utilization revealed the close affiliation of vAh strains regardless of geographic origin and suggested lateral genetic transfer of this catabolic pathway from an Enterobacter species. Prediction of virulence factors was conducted to determine differences between vAh and non-vAh strains in terms of virulence and secretion systems. Core genome phylogenetic analyses on vAh isolates and Aeromonas spp. disease isolates (55 in total) were conducted to evaluate the evolutionary relationships among vAh and other Aeromonas sp. isolates, which supported the clonal nature of vAh isolates. IMPORTANCE This global survey of vAh brought together scientists that study fish disease to evaluate the evolution, geographical distribution, phylogeny, and hosts of vAh and other Aeromonas sp. isolates. In addition to vAh isolates from China and the United States, four new vAh isolates were isolated from the lower Mekong River basin in Cambodia and Vietnam, indicating the significant threat of vAh to modern aquaculture and the need for improved biosecurity to prevent vAh spread.

Bacillus velezensis AP183 Inhibits Staphylococcus aureus Biofilm Formation and Proliferation in Murine and Bovine Disease Models.

The increasing frequency of S. aureus antimicrobial resistance has spurred interest in identifying alternative therapeutants. We investigated the S. aureus-inhibitory capacity of B. velezensis strains in mouse and bovine models. Among multiple B. velezensis strains that inhibited S. aureus growth in vitro, B. velezensis AP183 provided the most potent inhibition of S. aureus proliferation and bioluminescence in a mouse cutaneous wound (P = 0.02). Histology revealed abundant Gram-positive cocci in control wounds that were reduced in B. velezensis AP183-treated tissues. Experiments were then conducted to evaluate the ability of B. velezensis AP183 to prevent S. aureus biofilm formation on a tracheostomy tube substrate. B. velezensis AP183 could form a biofilm on a tracheostomy tube inner cannula substrate, and that this biofilm was antagonistic to S. aureus colonization. B. velezensis AP183 was also observed to inhibit the growth of S. aureus isolates originated from bovine mastitis cases. To evaluate the inflammatory response of mammary tissue to intramammary inoculation with B. velezensis AP183, we used high dose and low dose inocula in dairy cows. At the high dose, a significant increase in somatic cell count (SCC) and clinical mastitis was observed at all post-inoculation time points (P < 0.01), which resolved quickly compared to S. aureus-induced mastitis; in contrast, the lower dose of B. velezensis AP183 resulted in a slight increase of SCC and no clinical mastitis. In a subsequent experiment, all mammary quarters in four cows were induced to have grade 1 clinical mastitis by intramammary inoculation of a S. aureus mastitis isolate; following mastitis induction, eight quarters were treated with B. velezensis AP183 and milk samples were collected from pretreatment and post-treatment samples for 9 days. In groups treated with B. velezensis AP183, SCC and abundance of S. aureus decreased with significant reductions in S. aureus after 3 days post-inoculation with AP183 (P = 0.04). A milk microbiome analysis revealed significant reductions in S. aureus relative abundance in the AP183-treated group by 8 days post-inoculation (P = 0.02). These data indicate that B. velezensis AP183 can inhibit S. aureus biofilm formation and its proliferation in murine and bovine disease models.

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.

Draft Genome Sequence of Pseudomonas mosselii Gil3, Isolated from Catfish and Antagonistic against Hypervirulent Aeromonas hydrophila.

Pseudomonas mosselii Gil3 was isolated from a catfish that survived from lethal challenge with hypervirulent Aeromonas hydrophila (vAh). When assayed in vitro, the bacterium showed antagonism against vAh. Sequence analysis revealed that the genome of P. mosselii Gil3 encodes numerous aromatic metabolism pathways and proteins for biosynthesis of antimicrobial compounds.

Classification of a Hypervirulent Aeromonas hydrophila Pathotype Responsible for Epidemic Outbreaks in Warm-Water Fishes.

Lineages of hypervirulent Aeromonas hydrophila (vAh) are the cause of persistent outbreaks of motile Aeromonas septicemia in warm-water fishes worldwide. Over the last decade, this virulent lineage of A. hydrophila has resulted in annual losses of millions of tons of farmed carp and catfish in the People's Republic of China and the United States (US). Multiple lines of evidence indicate US catfish and Asian carp isolates of A. hydrophila affiliated with sequence type 251 (ST251) share a recent common ancestor. To address the genomic context for the putative intercontinental transfer and subsequent geographic spread of this pathogen, we conducted a core genome phylogenetic analysis on 61 Aeromonas spp. genomes, of which 40 were affiliated with A. hydrophila, with 26 identified as epidemic strains. Phylogenetic analyses indicate all ST251 strains form a coherent lineage affiliated with A. hydrophila. Within this lineage, conserved genetic loci unique to A. hydrophila were identified, with some genes present in consistently higher copy numbers than in non-epidemic A. hydrophila isolates. In addition, results from analyses of representative ST251 isolates support the conclusion that multiple lineages are present within US vAh isolated from Mississippi, whereas vAh isolated from Alabama appear clonal. This is the first report of genomic heterogeneity within US vAh isolates, with some Mississippi isolates showing closer affiliation with the Asian grass carp isolate ZC1 than other vAh isolated in the US. To evaluate the biological significance of the identified heterogeneity, comparative disease challenges were conducted with representatives of different vAh genotypes. These studies revealed that isolate ZC1 yielded significantly lower mortality in channel catfish, relative to Alabama and Mississippi vAh isolates. Like other Asian vAh isolates, the ZC1 lineage contains all core genes for a complete type VI secretion system (T6SS). In contrast, more virulent US isolates retain only remnants of the T6SS (clpB, hcp, vgrG, and vasH) which may have functional implications. Collectively, these results characterize a hypervirulent A. hydrophila pathotype that affects farmed fish on multiple continents.

Virulence Factors of Aeromonas hydrophila: In the Wake of Reclassification.

The ubiquitous "jack-of-all-trades," Aeromonas hydrophila, is a freshwater, Gram-negative bacterial pathogen under revision in regard to its phylogenetic and functional affiliation with other aeromonads. While virulence factors are expectedly diverse across A. hydrophila strains and closely related species, our mechanistic knowledge of the vast majority of these factors is based on the molecular characterization of the strains A. hydrophila AH-3 and SSU, which were reclassified as A. piscicola AH-3 in 2009 and A. dhakensis SSU in 2013. Individually, these reclassifications raise important questions involving the applicability of previous research on A. hydrophila virulence mechanisms; however, this issue is exacerbated by a lack of genomic data on other research strains. Collectively, these changes represent a fundamental gap in the literature on A. hydrophila and confirm the necessity of biochemical, molecular, and morphological techniques in the classification of research strains that are used as a foundation for future research. This review revisits what is known about virulence in A. hydrophila and the feasibility of using comparative genomics in light of this phylogenetic revision. Conflicting data between virulence factors, secretion systems, quorum sensing, and their effect on A. hydrophila pathogenicity appears to be an artifact of inappropriate taxonomic comparisons and/or be due to the fact that these properties are strain-specific. This review audits emerging data on dominant virulence factors that are present in both A. dhakensis and A. hydrophila in order to synthesize existing data with the aim of locating where future research is needed.

Deciphering the conserved genetic loci implicated in plant disease control through comparative genomics of Bacillus amyloliquefaciens subsp. plantarum.

To understand the growth-promoting and disease-inhibiting activities of plant growth-promoting rhizobacteria (PGPR) strains, the genomes of 12 Bacillus subtilis group strains with PGPR activity were sequenced and analyzed. These B. subtilis strains exhibited high genomic diversity, whereas the genomes of B. amyloliquefaciens strains (a member of the B. subtilis group) are highly conserved. A pairwise BLASTp matrix revealed that gene family similarity among Bacillus genomes ranges from 32 to 90%, with 2839 genes within the core genome of B. amyloliquefaciens subsp. plantarum. Comparative genomic analyses of B. amyloliquefaciens strains identified genes that are linked with biological control and colonization of roots and/or leaves, including 73 genes uniquely associated with subsp. plantarum strains that have predicted functions related to signaling, transportation, secondary metabolite production, and carbon source utilization. Although B. amyloliquefaciens subsp. plantarum strains contain gene clusters that encode many different secondary metabolites, only polyketide biosynthetic clusters that encode difficidin and macrolactin are conserved within this subspecies. To evaluate their role in plant pathogen biocontrol, genes involved in secondary metabolite biosynthesis were deleted in a B. amyloliquefaciens subsp. plantarum strain, revealing that difficidin expression is critical in reducing the severity of disease, caused by Xanthomonas axonopodis pv. vesicatoria in tomato plants. This study defines genomic features of PGPR strains and links them with biocontrol activity and with host colonization.