April 21, 2020  |  

Rapid transcriptional responses to serum exposure are associated with sensitivity and resistance to antibody-mediated complement killing in invasive Salmonella Typhimurium ST313

Background: Salmonella Typhimurium ST313 exhibits signatures of adaptation to invasive human infection, including higher resistance to humoral immune responses than gastrointestinal isolates. Full resistance to antibody-mediated complement killing (serum resistance) among nontyphoidal Salmonellae is uncommon, but selection of highly resistant strains could compromise vaccine-induced antibody immunity. Here, we address the hypothesis that serum resistance is due to a distinct genotype or transcriptome response in S. Typhimurium ST313.


April 21, 2020  |  

Dual Role of gnaA in Antibiotic Resistance and Virulence in Acinetobacter baumannii.

Acinetobacter baumannii is an important Gram-negative pathogen in hospital-related infections. However, treatment options for A. baumannii infections have become limited due to multidrug resistance. Bacterial virulence is often associated with capsule genes found in the K locus, many of which are essential for biosynthesis of the bacterial envelope. However, the roles of other genes in the K locus remain largely unknown. From an in vitro evolution experiment, we obtained an isolate of the virulent and multidrug-resistant A. baumannii strain MDR-ZJ06, called MDR-ZJ06M, which has an insertion by the ISAba16 transposon in gnaA (encoding UDP-N-acetylglucosamine C-6 dehydrogenase), a gene found in the K locus. The isolate showed an increased resistance toward tigecycline, whereas the MIC decreased in the case of carbapenems, cephalosporins, colistin, and minocycline. By using knockout and complementation experiments, we demonstrated that gnaA is important for the synthesis of lipooligosaccharide and capsular polysaccharide and that disruption of the gene affects the morphology, drug susceptibility, and virulence of the pathogen.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Whole Genome Sequencing of the Giant Grouper (Epinephelus lanceolatus) and High-Throughput Screening of Putative Antimicrobial Peptide Genes.

Giant groupers, the largest grouper type in the world, are of economic importance in marine aquaculture for their rapid growth. At the same time, bacterial and viral diseases have become the main threats to the grouper industry. Here, we report a high-quality genome of a giant grouper sequenced by an Illumina HiSeq X-Ten and PacBio Bioscience Sequel platform. A total of 254 putative antimicrobial peptide (AMP) genes were identified, which can be divided into 34 classes according to the annotation of the Antimicrobial Peptides Database (APD3). Their locations in pseudochromosomes were also determined. Thrombin-, lectin-, and scolopendin-derived putative AMPs were the three largest parts. In addition, expressions of putative AMPs were measured by our transcriptome data. Two putative AMP genes (gapdh1 and gapdh2) were involved in glycolysis, which had extremely high expression levels in giant grouper muscle. As it has been reported that AMPs inhibit the growth of a broad spectrum of microbes and participate in regulating innate and adaptive immune responses, genome sequencing of this study provides a comprehensive cataloging of putative AMPs of groupers, supporting antimicrobial research and aquaculture therapy. These genomic resources will be beneficial to further molecular breeding of this economically important fish.


April 21, 2020  |  

Harnessing long-read amplicon sequencing to uncover NRPS and Type I PKS gene sequence diversity in polar desert soils.

The severity of environmental conditions at Earth’s frigid zones present attractive opportunities for microbial biomining due to their heightened potential as reservoirs for novel secondary metabolites. Arid soil microbiomes within the Antarctic and Arctic circles are remarkably rich in Actinobacteria and Proteobacteria, bacterial phyla known to be prolific producers of natural products. Yet the diversity of secondary metabolite genes within these cold, extreme environments remain largely unknown. Here, we employed amplicon sequencing using PacBio RS II, a third generation long-read platform, to survey over 200 soils spanning twelve east Antarctic and high Arctic sites for natural product-encoding genes, specifically targeting non-ribosomal peptides (NRPS) and Type I polyketides (PKS). NRPS-encoding genes were more widespread across the Antarctic, whereas PKS genes were only recoverable from a handful of sites. Many recovered sequences were deemed novel due to their low amino acid sequence similarity to known protein sequences, particularly throughout the east Antarctic sites. Phylogenetic analysis revealed that a high proportion were most similar to antifungal and biosurfactant-type clusters. Multivariate analysis showed that soil fertility factors of carbon, nitrogen and moisture displayed significant negative relationships with natural product gene richness. Our combined results suggest that secondary metabolite production is likely to play an important physiological component of survival for microorganisms inhabiting arid, nutrient-starved soils. © FEMS 2019.


April 21, 2020  |  

Sensitivity to the two peptide bacteriocin plantaricin EF is dependent on CorC, a membrane-bound, magnesium/cobalt efflux protein.

Lactic acid bacteria produce a variety of antimicrobial peptides known as bacteriocins. Most bacteriocins are understood to kill sensitive bacteria through receptor-mediated disruptions. Here, we report on the identification of the Lactobacillus plantarum plantaricin EF (PlnEF) receptor. Spontaneous PlnEF-resistant mutants of the PlnEF-indicator strain L. plantarum NCIMB 700965 (LP965) were isolated and confirmed to maintain cellular ATP levels in the presence of PlnEF. Genome comparisons resulted in the identification of a single mutated gene annotated as the membrane-bound, magnesium/cobalt efflux protein CorC. All isolates contained a valine (V) at position 334 instead of a glycine (G) in a cysteine-ß-synthase domain at the C-terminal region of CorC. In silico template-based modeling of this domain indicated that the mutation resides in a loop between two ß-strands. The relationship between PlnEF, CorC, and metal homeostasis was supported by the finding that PlnEF-resistance was lost when PlnEF was applied together with high concentrations of Mg2+ , Co2+ , Zn2+ , or Cu2+ . Lastly, PlnEF sensitivity was increased upon heterologous expression of LP965 corC but not the G334V CorC mutant in the PlnEF-resistant strain Lactobacillus casei BL23. These results show that PlnEF kills sensitive bacteria by targeting CorC. © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.


April 21, 2020  |  

Complete Genome Sequence of Lactic Acid Bacterium Pediococcus acidilactici Strain ATCC 8042, an Autolytic Anti-bacterial Peptidoglycan Hydrolase Producer

Pediococcus acidilactici is a probiotic bacterium that is industrially utilized in the food industry and antibiotics development. Here, we determine the complete nucleotide sequence of the genome of Pediococcus acidilactici ATCC 8042. The genome was sequenced by the PacBio RSII to generate a single contig consisting of circular chromosome sequence. Illumina MiniSeq sequencing platform and Sanger sequencing method were additionally utilized to correct errors resulting from the long-read sequencing platform. The sequence consists of 2,009,598 bp with a G + C content of 42.1% and contains 1,865 protein-coding sequences. Based on the sequence information, we could confirm and predict the presence of four peptidoglycan hydrolases by HyPe software. This work, therefore, provides the complete genomic information of P. acidilactici ATCC 8042 with a profitable potential of genome-scale comprehension of anti-pathogenic activity, which can be applied in nutraceutical and pharmaceutical biotechnology field.


April 21, 2020  |  

Phylogenetic barriers to horizontal transfer of antimicrobial peptide resistance genes in the human gut microbiota.

The human gut microbiota has adapted to the presence of antimicrobial peptides (AMPs), which are ancient components of immune defence. Despite its medical importance, it has remained unclear whether AMP resistance genes in the gut microbiome are available for genetic exchange between bacterial species. Here, we show that AMP resistance and antibiotic resistance genes differ in their mobilization patterns and functional compatibilities with new bacterial hosts. First, whereas AMP resistance genes are widespread in the gut microbiome, their rate of horizontal transfer is lower than that of antibiotic resistance genes. Second, gut microbiota culturing and functional metagenomics have revealed that AMP resistance genes originating from phylogenetically distant bacteria have only a limited potential to confer resistance in Escherichia coli, an intrinsically susceptible species. Taken together, functional compatibility with the new bacterial host emerges as a key factor limiting the genetic exchange of AMP resistance genes. Finally, our results suggest that AMPs induce highly specific changes in the composition of the human microbiota, with implications for disease risks.


April 21, 2020  |  

Full-length transcriptome analysis of Litopenaeus vannamei reveals transcript variants involved in the innate immune system.

To better understand the immune system of shrimp, this study combined PacBio isoform sequencing (Iso-Seq) and Illumina paired-end short reads sequencing methods to discover full-length immune-related molecules of the Pacific white shrimp, Litopenaeus vannamei. A total of 72,648 nonredundant full-length transcripts (unigenes) were generated with an average length of 2545 bp from five main tissues, including the hepatopancreas, cardiac stomach, heart, muscle, and pyloric stomach. These unigenes exhibited a high annotation rate (62,164, 85.57%) when compared against NR, NT, Swiss-Prot, Pfam, GO, KEGG and COG databases. A total of 7544 putative long noncoding RNAs (lncRNAs) were detected and 1164 nonredundant full-length transcripts (449 UniTransModels) participated in the alternative splicing (AS) events. Importantly, a total of 5279 nonredundant full-length unigenes were successfully identified, which were involved in the innate immune system, including 9 immune-related processes, 19 immune-related pathways and 10 other immune-related systems. We also found wide transcript variants, which increased the number and function complexity of immune molecules; for example, toll-like receptors (TLRs) and interferon regulatory factors (IRFs). The 480 differentially expressed genes (DEGs) were significantly higher or tissue-specific expression patterns in the hepatopancreas compared with that in other four tested tissues (FDR <0.05). Furthermore, the expression levels of six selected immune-related DEGs and putative IRFs were validated using real-time PCR technology, substantiating the reliability of the PacBio Iso-seq results. In conclusion, our results provide new genetic resources of long-read full-length transcripts data and information for identifying immune-related genes, which are an invaluable transcriptomic resource as genomic reference, especially for further exploration of the innate immune and defense mechanisms of shrimp. Copyright © 2019 Elsevier Ltd. All rights reserved.


April 21, 2020  |  

Heteroresistance to the model antimicrobial peptide polymyxin B in the emerging Neisseria meningitidis lineage 11.2 urethritis clade: mutations in the pilMNOPQ operon.

Clusters of Neisseria meningitidis (Nm) urethritis among primarily heterosexual males in multiple US cities have been attributed to a unique non-encapsulated meningococcal clade (the US Nm urethritis clade, US_NmUC) within the hypervirulent clonal complex 11. Resistance to antimicrobial peptides (AMPs) is a key feature of urogenital pathogenesis of the closely related species, Neisseria gonorrhoeae. The US_NmUC isolates were found to be highly resistant to the model AMP, polymyxin B (PmB, MICs 64-256 µg ml-1 ). The isolates also demonstrated stable subpopulations of heteroresistant colonies that showed near total resistant to PmB (MICs 384-1024 µg ml-1 ) and colistin (MIC 256 µg ml-1 ) as well as enhanced LL-37 resistance. This is the first observation of heteroresistance in N. meningitidis. Consistent with previous findings, overall PmB resistance in US_NmUC isolates was due to active Mtr efflux and LptA-mediated lipid A modification. However, whole genome sequencing, variant analyses and directed mutagenesis revealed that the heteroresistance phenotypes and very high-level AMP resistance were the result of point mutations and IS1655 element movement in the pilMNOPQ operon, encoding the type IV pilin biogenesis apparatus. Cross-resistance to other classes of antibiotics was also observed in the heteroresistant colonies. High-level resistance to AMPs may contribute to the pathogenesis of US_NmUC. © 2018 John Wiley & Sons Ltd.


April 21, 2020  |  

Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages.

The use of bacteriophages represents a valid alternative to conventional antimicrobial treatments, overcoming the widespread bacterial antibiotic resistance phenomenon. In this work, we evaluated whether biomimetic hydroxyapatite (HA) nanocrystals are able to enhance some properties of bacteriophages. The final goal of this study was to demonstrate that biomimetic HA nanocrystals can be used for bacteriophage delivery in the context of bacterial infections, and contribute – at the same time – to enhance some of the biological properties of the same bacteriophages such as stability, preservation, antimicrobial activity, and so on.Phage isolation and characterization were carried out by using Mitomycin C and following double-layer agar technique. The biomimetic HA water suspension was synthesized in order to obtain nanocrystals with plate-like morphology and nanometric dimensions. The interaction of phages with the HA was investigated by dynamic light scattering and Zeta potential analyses. The cytotoxicity and intracellular killing activities of the phage-HA complex were evaluated in human hepatocellular carcinoma HepG2 cells. The bacterial inhibition capacity of the complex was assessed on chicken minced meat samples infected with Salmonella Rissen.Our data highlighted that the biomimetic HA nanocrystal-bacteriophage complex was more stable and more effective than phages alone in all tested experimental conditions.Our results evidenced the important contribution of biomimetic HA nanocrystals: they act as an excellent carrier for bacteriophage delivery and enhance its biological characteristics. This study confirmed the significant role of the mineral HA when it is complexed with biological entities like bacteriophages, as it has been shown for molecules such as lactoferrin.


April 21, 2020  |  

Comparative genome analysis provides novel insight into the interaction of Aquimarina sp. AD1, BL5 and AD10 with their macroalgal host.

The Aquimarina genus is widely distributed throughout the marine environment, however little is understood regarding its ecological role, particularly when in association with eukaryotic hosts. Here, we examine the genomes of two opportunistic pathogens, Aquimarina sp. AD1 and BL5, and a non-pathogenic strain Aquimarina sp. AD10, that were isolated from diseased individuals of the red alga Delisea pulchra. Each strain encodes multiple genes for the degradation of marine carbohydrates and vitamin biosynthesis. These traits are hypothesised to promote nutrient exchange between the Aquimarina strains and their algal host, facilitating a close symbiotic relationship. Moreover, each strain harbours the necessary genes for the assembly of a Type 9 Secretion System (T9SS) and the associated gliding motility apparatus. In addition to these common features, pathogenic strains AD1 and BL5, encode genes for the production of flexirubin type pigments and a number of unique non-ribosomal peptide synthesis (NRPS) gene clusters, suggesting a role for these uncharacterised traits in virulence. This study provides valuable insight into the potential ecological role of Aquimarina in the marine environment and the complex factors driving pathogenesis and symbiosis in this genus.Copyright © 2019 Elsevier B.V. All rights reserved.


April 21, 2020  |  

Mobilome of Brevibacterium aurantiacum Sheds Light on Its Genetic Diversity and Its Adaptation to Smear-Ripened Cheeses.

Brevibacterium aurantiacum is an actinobacterium that confers key organoleptic properties to washed-rind cheeses during the ripening process. Although this industrially relevant species has been gaining an increasing attention in the past years, its genome plasticity is still understudied due to the unavailability of complete genomic sequences. To add insights on the mobilome of this group, we sequenced the complete genomes of five dairy Brevibacterium strains and one non-dairy strain using PacBio RSII. We performed phylogenetic and pan-genome analyses, including comparisons with other publicly available Brevibacterium genomic sequences. Our phylogenetic analysis revealed that these five dairy strains, previously identified as Brevibacterium linens, belong instead to the B. aurantiacum species. A high number of transposases and integrases were observed in the Brevibacterium spp. strains. In addition, we identified 14 and 12 new insertion sequences (IS) in B. aurantiacum and B. linens genomes, respectively. Several stretches of homologous DNA sequences were also found between B. aurantiacum and other cheese rind actinobacteria, suggesting horizontal gene transfer (HGT). A HGT region from an iRon Uptake/Siderophore Transport Island (RUSTI) and an iron uptake composite transposon were found in five B. aurantiacum genomes. These findings suggest that low iron availability in milk is a driving force in the adaptation of this bacterial species to this niche. Moreover, the exchange of iron uptake systems suggests cooperative evolution between cheese rind actinobacteria. We also demonstrated that the integrative and conjugative element BreLI (Brevibacterium Lanthipeptide Island) can excise from B. aurantiacum SMQ-1417 chromosome. Our comparative genomic analysis suggests that mobile genetic elements played an important role into the adaptation of B. aurantiacum to cheese ecosystems.


April 21, 2020  |  

Whole Genome Analysis of Lactobacillus plantarum Strains Isolated From Kimchi and Determination of Probiotic Properties to Treat Mucosal Infections by Candida albicans and Gardnerella vaginalis.

Three Lactobacillus plantarum strains ATG-K2, ATG-K6, and ATG-K8 were isolated from Kimchi, a Korean traditional fermented food, and their probiotic potentials were examined. All three strains were free of antibiotic resistance, hemolysis, and biogenic amine production and therefore assumed to be safe, as supported by whole genome analyses. These strains demonstrated several basic probiotic functions including a wide range of antibacterial activity, bile salt hydrolase activity, hydrogen peroxide production, and heat resistance at 70°C for 60 s. Further studies of antimicrobial activities against Candida albicans and Gardnerella vaginalis revealed growth inhibitory effects from culture supernatants, coaggregation effects, and killing effects of the three probiotic strains, with better efficacy toward C. albicans. In vitro treatment of bacterial lysates of the probiotic strains to the RAW264.7 murine macrophage cell line resulted in innate immunity enhancement via IL-6 and TNF-a production without lipopolysaccharide (LPS) treatment and anti-inflammatory effects via significantly increased production of IL-10 when co-treated with LPS. However, the degree of probiotic effect was different for each strain as the highest TNF-a and the lowest IL-10 production by the RAW264.7 cell were observed in the K8 lysate treated group compared to the K2 and K6 lysate treated groups, which may be related to genomic differences such as chromosome size (K2: 3,034,884 bp, K6: 3,205,672 bp, K8: 3,221,272 bp), plasmid numbers (K2: 3, K6 and K8: 1), or total gene numbers (K2: 3,114, K6: 3,178, K8: 3,186). Although more correlative inspections to connect genomic information and biological functions are needed, genomic analyses of the three strains revealed distinct genomic compositions of each strain. Also, this finding suggests genome level analysis may be required to accurately identify microorganisms. Nevertheless, L. plantarum ATG-K2, ATG-K6, and ATG-K8 demonstrated their potential as probiotics for mucosal health improvement in both microbial and immunological contexts.


April 21, 2020  |  

Comparative Genomic Analyses Reveal Core-Genome-Wide Genes Under Positive Selection and Major Regulatory Hubs in Outlier Strains of Pseudomonas aeruginosa.

Genomic information for outlier strains of Pseudomonas aeruginosa is exiguous when compared with classical strains. We sequenced and constructed the complete genome of an environmental strain CR1 of P. aeruginosa and performed the comparative genomic analysis. It clustered with the outlier group, hence we scaled up the analyses to understand the differences in environmental and clinical outlier strains. We identified eight new regions of genomic plasticity and a plasmid pCR1 with a VirB/D4 complex followed by trimeric auto-transporter that can induce virulence phenotype in the genome of strain CR1. Virulence genotype analysis revealed that strain CR1 lacked hemolytic phospholipase C and D, three genes for LPS biosynthesis and had reduced antibiotic resistance genes when compared with clinical strains. Genes belonging to proteases, bacterial exporters and DNA stabilization were found to be under strong positive selection, thus facilitating pathogenicity and survival of the outliers. The outliers had the complete operon for the production of vibrioferrin, a siderophore present in plant growth promoting bacteria. The competence to acquire multidrug resistance and new virulence factors makes these strains a potential threat. However, we identified major regulatory hubs that can be used as drug targets against both the classical and outlier groups.


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