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September 22, 2019

Genomics and host specialization of honey bee and bumble bee gut symbionts.

Gilliamella apicola and Snodgrassella alvi are dominant members of the honey bee (Apis spp.) and bumble bee (Bombus spp.) gut microbiota. We generated complete genomes of the type strains G. apicola wkB1(T) and S. alvi wkB2(T) (isolated from Apis), as well as draft genomes for four other strains from Bombus. G. apicola and S. alvi were found to occupy very different metabolic niches: The former is a saccharolytic fermenter, whereas the latter is an oxidizer of carboxylic acids. Together, they may form a syntrophic network for partitioning of metabolic resources. Both species possessed numerous genes [type 6 secretion systems, repeats in toxin (RTX) toxins, RHS proteins, adhesins, and type IV pili] that likely mediate cell-cell interactions and gut colonization. Variation in these genes could account for the host fidelity of strains observed in previous phylogenetic studies. Here, we also show the first experimental evidence, to our knowledge, for this specificity in vivo: Strains of S. alvi were able to colonize their native bee host but not bees of another genus. Consistent with specific, long-term host association, comparative genomic analysis revealed a deep divergence and little or no gene flow between Apis and Bombus gut symbionts. However, within a host type (Apis or Bombus), we detected signs of horizontal gene transfer between G. apicola and S. alvi, demonstrating the importance of the broader gut community in shaping the evolution of any one member. Our results show that host specificity is likely driven by multiple factors, including direct host-microbe interactions, microbe-microbe interactions, and social transmission.

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September 22, 2019

High-resolution phylogenetic microbial community profiling.

Over the past decade, high-throughput short-read 16S rRNA gene amplicon sequencing has eclipsed clone-dependent long-read Sanger sequencing for microbial community profiling. The transition to new technologies has provided more quantitative information at the expense of taxonomic resolution with implications for inferring metabolic traits in various ecosystems. We applied single-molecule real-time sequencing for microbial community profiling, generating full-length 16S rRNA gene sequences at high throughput, which we propose to name PhyloTags. We benchmarked and validated this approach using a defined microbial community. When further applied to samples from the water column of meromictic Sakinaw Lake, we show that while community structures at the phylum level are comparable between PhyloTags and Illumina V4 16S rRNA gene sequences (iTags), variance increases with community complexity at greater water depths. PhyloTags moreover allowed less ambiguous classification. Last, a platform-independent comparison of PhyloTags and in silico generated partial 16S rRNA gene sequences demonstrated significant differences in community structure and phylogenetic resolution across multiple taxonomic levels, including a severe underestimation in the abundance of specific microbial genera involved in nitrogen and methane cycling across the Lake’s water column. Thus, PhyloTags provide a reliable adjunct or alternative to cost-effective iTags, enabling more accurate phylogenetic resolution of microbial communities and predictions on their metabolic potential.

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September 22, 2019

Characteristics of ARG-carrying plasmidome in the cultivable microbial community from wastewater treatment system under high oxytetracycline concentration.

Studies on antibiotic production wastewater have shown that even a single antibiotic can select for multidrug resistant bacteria in aquatic environments. It is speculated that plasmids are an important mechanism of multidrug resistance (MDR) under high concentrations of antibiotics. Herein, two metagenomic libraries were constructed with plasmid DNA extracted from cultivable microbial communities in a biological wastewater treatment reactor supplemented with 0 (CONTROL) or 25 mg/L of oxytetracycline (OTC-25). The OTC-25 plasmidome reads were assigned to 72 antibiotic resistance genes (ARGs) conferring resistance to 13 types of antibiotics. Dominant ARGs, encoding resistance to tetracycline, aminoglycoside, sulfonamide, and multidrug resistance genes, were enriched in the plasmidome under 25 mg/L of oxytetracycline. Furthermore, 17 contiguous multiple-ARG carrying contigs (carrying =?2 ARGs) were discovered in the OTC-25 plasmidome, whereas only nine were found in the CONTROL. Mapping of the OTC-25 plasmidome reads to completely sequenced plasmids revealed that the conjugative IncU resistance plasmid pFBAOT6 of Aeromonas caviae, carrying multidrug resistance transporter (pecM), tetracycline resistance genes (tetA, tetR), and transposase genes, might be a potential prevalent resistant plasmid in the OTC-25 plasmidome. Additionally, two novel resistant plasmids (containing contig C301682 carrying multidrug resistant operon mexCD-oprJ and contig C301632 carrying the tet36 and transposases genes) might also be potential prevalent resistant plasmids in the OTC-25 plasmidome. This study will be helpful to better understand the role of plasmids in the development of MDR in water environments under high antibiotic concentrations.

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September 22, 2019

KIR3DL01 upregulation on gut natural killer cells in response to SIV infection of KIR- and MHC class I-defined rhesus macaques.

Natural killer cells provide an important early defense against viral pathogens and are regulated in part by interactions between highly polymorphic killer-cell immunoglobulin-like receptors (KIRs) on NK cells and their MHC class I ligands on target cells. We previously identified MHC class I ligands for two rhesus macaque KIRs: KIR3DL01 recognizes Mamu-Bw4 molecules and KIR3DL05 recognizes Mamu-A1*002. To determine how these interactions influence NK cell responses, we infected KIR3DL01+ and KIR3DL05+ macaques with and without defined ligands for these receptors with SIVmac239, and monitored NK cell responses in peripheral blood and lymphoid tissues. NK cell responses in blood were broadly stimulated, as indicated by rapid increases in the CD16+ population during acute infection and sustained increases in the CD16+ and CD16-CD56- populations during chronic infection. Markers of proliferation (Ki-67), activation (CD69 & HLA-DR) and antiviral activity (CD107a & TNFa) were also widely expressed, but began to diverge during chronic infection, as reflected by sustained CD107a and TNFa upregulation by KIR3DL01+, but not by KIR3DL05+ NK cells. Significant increases in the frequency of KIR3DL01+ (but not KIR3DL05+) NK cells were also observed in tissues, particularly in the gut-associated lymphoid tissues, where this receptor was preferentially upregulated on CD56+ and CD16-CD56- subsets. These results reveal broad NK cell activation and dynamic changes in the phenotypic properties of NK cells in response to SIV infection, including the enrichment of KIR3DL01+ NK cells in tissues that support high levels of virus replication.

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September 22, 2019

Diverse antibiotic resistance genes in dairy cow manure.

Application of manure from antibiotic-treated animals to crops facilitates the dissemination of antibiotic resistance determinants into the environment. However, our knowledge of the identity, diversity, and patterns of distribution of these antibiotic resistance determinants remains limited. We used a new combination of methods to examine the resistome of dairy cow manure, a common soil amendment. Metagenomic libraries constructed with DNA extracted from manure were screened for resistance to beta-lactams, phenicols, aminoglycosides, and tetracyclines. Functional screening of fosmid and small-insert libraries identified 80 different antibiotic resistance genes whose deduced protein sequences were on average 50 to 60% identical to sequences deposited in GenBank. The resistance genes were frequently found in clusters and originated from a taxonomically diverse set of species, suggesting that some microorganisms in manure harbor multiple resistance genes. Furthermore, amid the great genetic diversity in manure, we discovered a novel clade of chloramphenicol acetyltransferases. Our study combined functional metagenomics with third-generation PacBio sequencing to significantly extend the roster of functional antibiotic resistance genes found in animal gut bacteria, providing a particularly broad resource for understanding the origins and dispersal of antibiotic resistance genes in agriculture and clinical settings. IMPORTANCE The increasing prevalence of antibiotic resistance among bacteria is one of the most intractable challenges in 21st-century public health. The origins of resistance are complex, and a better understanding of the impacts of antibiotics used on farms would produce a more robust platform for public policy. Microbiomes of farm animals are reservoirs of antibiotic resistance genes, which may affect distribution of antibiotic resistance genes in human pathogens. Previous studies have focused on antibiotic resistance genes in manures of animals subjected to intensive antibiotic use, such as pigs and chickens. Cow manure has received less attention, although it is commonly used in crop production. Here, we report the discovery of novel and diverse antibiotic resistance genes in the cow microbiome, demonstrating that it is a significant reservoir of antibiotic resistance genes. The genomic resource presented here lays the groundwork for understanding the dispersal of antibiotic resistance from the agroecosystem to other settings.

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September 22, 2019

Application of PacBio Single Molecule Real-Time (SMRT) sequencing in bacterial source tracking analysis during milk powder production

This work developed a 16S rRNA-PacBio Single Molecule Real-Time (SMRT) sequencing-based method to identify and track the bacterial community of milk powder (MP) from two kinds of production settings, i.e., small-scale production contained within an in-house environment (minimal milk storage before pasteurization, milk concentration, and spray drying) and a large-scale factory production (prolonged milk storage before direct spray drying). A total of 18 samples were analyzed at the species level. Comparing with the large-scale factory production, only relatively little changes were observed in the bacterial community during the in-house production process, without significant loss in the levels of bioactive minor proteins (namely, lactoferrin, immunoglobulin G, lactoperoxidase, and lysozyme). The two most prevalent species in the in-house production, Bacillus cereus and Bacillus flexus, were likely originated from the raw milk with only small changes in their relative abundances (from 25.97% to 26.40%–28.89% and 27.40%, respectively) throughout the processing (from raw milk to MP). In contrast, large-scale factory production resulted in more obvious variation in the microbial content. This microbial tracking approach is valuable in identifying the contamination source and the specific stage when contamination happens; the implementation of such technique may also enhance food quality assurance systems that are currently used in the dairy industry.

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September 22, 2019

Prey range and genome evolution of Halobacteriovorax marinus predatory bacteria from an estuary

Halobacteriovorax strains are saltwater-adapted predatory bacteria that attack Gram-negative bacteria and may play an important role in shaping microbial communities. To understand how Halobacteriovorax strains impact ecosystems and develop them as biocontrol agents, it is important to characterize variation in predation phenotypes and investigate Halobacteriovorax genome evolution. We isolated Halobacteriovorax marinus BE01 from an estuary in Rhode Island using Vibrio from the same site as prey. Small, fast-moving, attack-phase BE01 cells attach to and invade prey cells, consistent with the intraperiplasmic predation strategy of the H. marinus type strain, SJ. BE01 is a prey generalist, forming plaques on Vibrio strains from the estuary, Pseudomonas from soil, and Escherichia coli. Genome analysis revealed extremely high conservation of gene order and amino acid sequences between BE01 and SJ, suggesting strong selective pressure to maintain the genome in this H. marinus lineage. Despite this, we identified two regions of gene content difference that likely resulted from horizontal gene transfer. Analysis of modal codon usage frequencies supports the hypothesis that these regions were acquired from bacteria with different codon usage biases than H. marinus. In one of these regions, BE01 and SJ carry different genes associated with mobile genetic elements. Acquired functions in BE01 include the dnd operon, which encodes a pathway for DNA modification, and a suite of genes involved in membrane synthesis and regulation of gene expression that was likely acquired from another Halobacteriovorax lineage. This analysis provides further evidence that horizontal gene transfer plays an important role in genome evolution in predatory bacteria. IMPORTANCE Predatory bacteria attack and digest other bacteria and therefore may play a role in shaping microbial communities. To investigate phenotypic and genotypic variation in saltwater-adapted predatory bacteria, we isolated Halobacteriovorax marinus BE01 from an estuary in Rhode Island, assayed whether it could attack different prey bacteria, and sequenced and analyzed its genome. We found that BE01 is a prey generalist, attacking bacteria from different phylogenetic groups and environments. Gene order and amino acid sequences are highly conserved between BE01 and the H. marinus type strain, SJ. By comparative genomics, we detected two regions of gene content difference that likely occurred via horizontal gene transfer events. Acquired genes encode functions such as modification of DNA, membrane synthesis and regulation of gene expression. Understanding genome evolution and variation in predation phenotypes among predatory bacteria will inform their development as biocontrol agents and clarify how they impact microbial communities.

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September 22, 2019

Comparative genome and phenotypic analysis of three Clostridioides difficile strains isolated from a single patient provide insight into multiple infection of C. difficile.

Clostridioides difficile infections (CDI) have emerged over the past decade causing symptoms that range from mild, antibiotic-associated diarrhea (AAD) to life-threatening toxic megacolon. In this study, we describe a multiple and isochronal (mixed) CDI caused by the isolates DSM 27638, DSM 27639 and DSM 27640 that already initially showed different morphotypes on solid media.The three isolates belonging to the ribotypes (RT) 012 (DSM 27639) and 027 (DSM 27638 and DSM 27640) were phenotypically characterized and high quality closed genome sequences were generated. The genomes were compared with seven reference strains including three strains of the RT 027, two of the RT 017, and one of the RT 078 as well as a multi-resistant RT 012 strain. The analysis of horizontal gene transfer events revealed gene acquisition incidents that sort the strains within the time line of the spread of their RTs within Germany. We could show as well that horizontal gene transfer between the members of different RTs occurred within this multiple infection. In addition, acquisition and exchange of virulence-related features including antibiotic resistance genes were observed. Analysis of the two genomes assigned to RT 027 revealed three single nucleotide polymorphisms (SNPs) and apparently a regional genome modification within the flagellar switch that regulates the fli operon.Our findings show that (i) evolutionary events based on horizontal gene transfer occur within an ongoing CDI and contribute to the adaptation of the species by the introduction of new genes into the genomes, (ii) within a multiple infection of a single patient the exchange of genetic material was responsible for a much higher genome variation than the observed SNPs.

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September 22, 2019

Functional characterization of the mucus barrier on the Xenopus tropicalis skin surface.

Mucosal surfaces represent critical routes for entry and exit of pathogens. As such, animals have evolved strategies to combat infection at these sites, in particular the production of mucus to prevent attachment and to promote subsequent movement of the mucus/microbe away from the underlying epithelial surface. Using biochemical, biophysical, and infection studies, we have investigated the host protective properties of the skin mucus barrier of the Xenopus tropicalis tadpole. Specifically, we have characterized the major structural component of the barrier and shown that it is a mucin glycoprotein (Otogelin-like or Otogl) with similar sequence, domain organization, and structural properties to human gel-forming mucins. This mucin forms the structural basis of a surface barrier (~6 µm thick), which is depleted through knockdown of Otogl. Crucially, Otogl knockdown leads to susceptibility to infection by the opportunistic pathogen Aeromonas hydrophila To more accurately reflect its structure, tissue localization, and function, we have renamed Otogl as Xenopus Skin Mucin, or MucXS. Our findings characterize an accessible and tractable model system to define mucus barrier function and host-microbe interactions. Copyright © 2018 the Author(s). Published by PNAS.

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September 22, 2019

Genomic diversity in the endosymbiotic bacterium Rhizobium leguminosarum.

Rhizobium leguminosarum bv. viciae is a soil a-proteobacterium that establishes a diazotrophic symbiosis with different legumes of the Fabeae tribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome of R. leguminosarum bv. viciae UPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains of R. leguminosarum bv. viciae, 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.

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September 22, 2019

Sooty mangabey genome sequence provides insight into AIDS resistance in a natural SIV host.

In contrast to infections with human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques, SIV infection of a natural host, sooty mangabeys (Cercocebus atys), is non-pathogenic despite high viraemia. Here we sequenced and assembled the genome of a captive sooty mangabey. We conducted genome-wide comparative analyses of transcript assemblies from C. atys and AIDS-susceptible species, such as humans and macaques, to identify candidates for host genetic factors that influence susceptibility. We identified several immune-related genes in the genome of C. atys that show substantial sequence divergence from macaques or humans. One of these sequence divergences, a C-terminal frameshift in the toll-like receptor-4 (TLR4) gene of C. atys, is associated with a blunted in vitro response to TLR-4 ligands. In addition, we found a major structural change in exons 3-4 of the immune-regulatory protein intercellular adhesion molecule 2 (ICAM-2); expression of this variant leads to reduced cell surface expression of ICAM-2. These data provide a resource for comparative genomic studies of HIV and/or SIV pathogenesis and may help to elucidate the mechanisms by which SIV-infected sooty mangabeys avoid AIDS.

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September 22, 2019

Whole-genome-sequencing characterization of bloodstream infection-causing hypervirulent Klebsiella pneumoniae of capsular serotype K2 and ST374.

Hypervirulent K. pneumoniae variants (hvKP) have been increasingly reported worldwide, causing metastasis of severe infections such as liver abscesses and bacteremia. The capsular serotype K2 hvKP strains show diverse multi-locus sequence types (MLSTs), but with limited genetics and virulence information. In this study, we report a hypermucoviscous K. pneumoniae strain, RJF293, isolated from a human bloodstream sample in a Chinese hospital. It caused a metastatic infection and fatal septic shock in a critical patient. The microbiological features and genetic background were investigated with multiple approaches. The Strain RJF293 was determined to be multilocis sequence type (ST) 374 and serotype K2, displayed a median lethal dose (LD50) of 1.5 × 102 CFU in BALB/c mice and was as virulent as the ST23 K1 serotype hvKP strain NTUH-K2044 in a mouse lethality assay. Whole genome sequencing revealed that the RJF293 genome codes for 32 putative virulence factors and exhibits a unique presence/absence pattern in comparison to the other 105 completely sequenced K. pneumoniae genomes. Whole genome SNP-based phylogenetic analysis revealed that strain RJF293 formed a single clade, distant from those containing either ST66 or ST86 hvKP. Compared to the other sequenced hvKP chromosomes, RJF293 contains several strain-variable regions, including one prophage, one ICEKp1 family integrative and conjugative element and six large genomic islands. The sequencing of the first complete genome of an ST374 K2 hvKP clinical strain should reinforce our understanding of the epidemiology and virulence mechanisms of this bloodstream infection-causing hvKP with clinical significance.

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September 22, 2019

Complete genome sequence of Lactobacillus pentosus SLC13, isolated from mustard pickles, a potential probiotic strain with antimicrobial activity against foodborne pathogenic microorganisms.

Lactobacillus pentosus SLC13 is a high exopolysaccharide (EPS)-producing strain with broad-spectrum antimicrobial activity and the ability to grow in simulated gastrointestinal conditions. SLC13 was isolated from mustard pickles in Taiwan for potential probiotic applications. To better understand the molecular base for its antimicrobial activity and high EPS production, entire genome of SLC13 was determined by PacBio SMRT sequencing.L. pentosus SLC13 contains a genome with a 3,520,510-bp chromosome and a 62,498-bp plasmid. GC content of the complete genome was 46.5% and that of plasmid pSLC13 was 41.3%. Sequences were annotated at the RAST prokaryotic genome annotation server, and the results showed that the genome contained 3172 coding sequences and 82 RNA genes. Seventy-six protein-coding sequences were identified on the plasmid pSLC13. A plantaricin gene cluster, which is responsible for bacteriosins biosynthesis and could be associated with its broad-spectrum antimicrobial activity, was identified based on comparative genomic analysis. Two gene clusters involved in EPS production were also identified.This genomic sequence might contribute to a future application of this strain as probiotic in productive livestock potentially inhibiting competing and pathogenic organisms.

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September 22, 2019

Magic pools: Parallel assessment of transposon delivery vectors in bacteria

Transposon mutagenesis coupled to next-generation sequencing (TnSeq) is a powerful approach for discovering the functions of bacterial genes. However, the development of a suitable TnSeq strategy for a given bacterium can be costly and time-consuming. To meet this challenge, we describe a part-based strategy for constructing libraries of hundreds of transposon delivery vectors, which we term “magic pools.” Within a magic pool, each transposon vector has a different combination of upstream sequences (promoters and ribosome binding sites) and antibiotic resistance markers as well as a random DNA barcode sequence, which allows the tracking of each vector during mutagenesis experiments. To identify an efficient vector for a given bacterium, we mutagenize it with a magic pool and sequence the resulting insertions; we then use this efficient vector to generate a large mutant library. We used the magic pool strategy to construct transposon mutant libraries in five genera of bacteria, including three genera of the phylum Bacteroidetes. IMPORTANCE Molecular genetics is indispensable for interrogating the physiology of bacteria. However, the development of a functional genetic system for any given bacterium can be time-consuming. Here, we present a streamlined approach for identifying an effective transposon mutagenesis system for a new bacterium. Our strategy first involves the construction of hundreds of different transposon vector variants, which we term a “magic pool.” The efficacy of each vector in a magic pool is monitored in parallel using a unique DNA barcode that is introduced into each vector design. Using archived DNA “parts,” we next reassemble an effective vector for making a whole-genome transposon mutant library that is suitable for large-scale interrogation of gene function using competitive growth assays. Here, we demonstrate the utility of the magic pool system to make mutant libraries in five genera of bacteria.

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September 22, 2019

Tn6450, a novel multidrug resistance transposon characterized in a Proteus mirabilis isolate from chicken in China.

A novel 65.8-kb multidrug resistance transposon, designated Tn6450, was characterized in a Proteus mirabilis isolate from chicken in China. Tn6450 contains 18 different antimicrobial resistance genes, including cephalosporinase gene blaDHA-1 and fluoroquinolone resistance genes qnrA1 and aac(6′)-Ib-cr It carries a class 1/2 hybrid integron composed of intI2 and a 3′ conserved segment of the class 1 integron. Tn6450 is derived from Tn7 via acquisition of new mobile elements and resistance genes. Copyright © 2018 American Society for Microbiology.

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