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April 21, 2020  |  

Strain-level metagenomic assignment and compositional estimation for long reads with MetaMaps.

Metagenomic sequence classification should be fast, accurate and information-rich. Emerging long-read sequencing technologies promise to improve the balance between these factors but most existing methods were designed for short reads. MetaMaps is a new method, specifically developed for long reads, capable of mapping a long-read metagenome to a comprehensive RefSeq database with >12,000 genomes in <16?GB or RAM on a laptop computer. Integrating approximate mapping with probabilistic scoring and EM-based estimation of sample composition, MetaMaps achieves >94% accuracy for species-level read assignment and r2?>?0.97 for the estimation of sample composition on both simulated and real data when the sample genomes or close relatives are present in the classification database. To address novel species and genera, which are comparatively harder to predict, MetaMaps outputs mapping locations and qualities for all classified reads, enabling functional studies (e.g. gene presence/absence) and detection of incongruities between sample and reference genomes.

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April 21, 2020  |  

The Not-so-Sterile Womb: Evidence That the Human Fetus Is Exposed to Bacteria Prior to Birth.

The human microbiome includes trillions of bacteria, many of which play a vital role in host physiology. Numerous studies have now detected bacterial DNA in first-pass meconium and amniotic fluid samples, suggesting that the human microbiome may commence in utero. However, these data have remained contentious due to underlying contamination issues. Here, we have used a previously described method for reducing contamination in microbiome workflows to determine if there is a fetal bacterial microbiome beyond the level of background contamination. We recruited 50 women undergoing non-emergency cesarean section deliveries with no evidence of intra-uterine infection and collected first-pass meconium and amniotic fluid samples. Full-length 16S rRNA gene sequencing was performed using PacBio SMRT cell technology, to allow high resolution profiling of the fetal gut and amniotic fluid bacterial microbiomes. Levels of inflammatory cytokines were measured in amniotic fluid, and levels of immunomodulatory short chain fatty acids (SCFAs) were quantified in meconium. All meconium samples and most amniotic fluid samples (36/43) contained bacterial DNA. The meconium microbiome was dominated by reads that mapped to Pelomonas puraquae. Aside from this species, the meconium microbiome was remarkably heterogeneous between patients. The amniotic fluid microbiome was more diverse and contained mainly reads that mapped to typical skin commensals, including Propionibacterium acnes and Staphylococcus spp. All meconium samples contained acetate and propionate, at ratios similar to those previously reported in infants. P. puraquae reads were inversely correlated with meconium propionate levels. Amniotic fluid cytokine levels were associated with the amniotic fluid microbiome. Our results demonstrate that bacterial DNA and SCFAs are present in utero, and have the potential to influence the developing fetal immune system.

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April 21, 2020  |  

A Phage-Like Plasmid Carrying blaKPC-2 Gene in Carbapenem-Resistant Pseudomonas aeruginosa.

Background: Lateral gene transfer plays a central role in the dissemination of carbapenem resistance in bacterial pathogens associated with nosocomial infections, mainly Enterobacteriaceae and Pseudomonas aeruginosa. Despite their clinical significance, there is little information regarding the mobile genetic elements and mechanism of acquisition and propagation of lateral genes in P. aeruginosa, and they remain largely unknown. Objectives: The present study characterized the genetic context of blaKPC-2 in carbapenem-resistant P. aeruginosa strain BH9. Methods:Pseudomonas aeruginosa BH9 sequencing was performed using the long-read PacBio SMRT platform and the Ion Proton System. De novo assembly was carried out using the SMRT pipeline and Canu, and gene prediction and annotation were performed using Prokka and RAST. Results:Pseudomonas aeruginosa BH9 exhibited a 7.1 Mb circular chromosome. However, the blaKPC-2 gene is located in an additional contig composed by a small plasmid pBH6 from P. aeruginosa strain BH6 and several phage-related genes. Further analysis revealed that the beginning and end of the contig contain identical sequences, supporting a circular plasmid structure. This structure spans 41,087 bp, exhibiting all the Mu-like phage landmarks. In addition, 5-bp direct repeats (GGATG) flanking the pBH6 ends were found, strongly indicating integration of the Mu-like phage into the pBH6 plasmid. Mu phages are commonly found in P. aeruginosa. However, for the first time showing a potential impact in shaping the vehicles of the dissemination of antimicrobial (e.g., plasmid pBH6) resistance genes in the Pseudomonas genus. Conclusion: pBH6 captured the Mu-like Phage BH9, creating a co-integrate pBH6::Phage BH9, and this phage-plasmid complex may represent novel case of a phage-like plasmid.

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April 21, 2020  |  

Characterization of mcr-1-Harboring Plasmids from Pan Drug-Resistant Escherichia coli Strains Isolated from Retail Raw Chicken in South Korea

A number of studies from different countries have characterized mcr-1-harboring plasmids isolated from food; however, nothing has been reported about it in South Korea. In this study, we report the characterization of mcr-1 plasmids from pan drug-resistant (PDR) Escherichia coli strains isolated from retail food in the country. Colistin-resistant E. coli strains were isolated from retail raw chicken, and PCR was carried out to detect the mcr-1 gene. Whole genome sequencing of the mcr-1-positive strains was performed for further characterization. The results of whole genome sequencing revealed that all mcr-1 plasmids belonged to the IncI2 type. In addition to the mcr-1 plasmids, all of the isolates also carried additional plasmids possessing multiple antibiotic resistance genes, and the PDR was mediated by resistant plasmids except for fluoroquinolone resistance resulting from mutations in gyrA and parC. Interestingly, the mcr-1 plasmids were transferred by conjugation to other pathogenic strains including enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), Salmonella, and Klebsiella at the frequencies of 10−3−10−6, 10−2−10−5, 10−4−10−5, 10−4−10−6, and 10−5−10−6, respectively. The results showed that mcr-1 plasmids can be easily transmitted to pathogenic bacteria by conjugation.

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April 21, 2020  |  

ICESsuHN105, a Novel Multiple Antibiotic Resistant ICE in Streptococcus suis Serotype 5 Strain HN105.

Streptococcussuis serotype 5, an emerging zoonosis bacterial pathogen, has been isolated from infections in both pigs and humans. In this study, we sequenced the first complete genome of a virulent, multidrug-resistant SS5 strain HN105. The strain HN105 displayed enhanced pathogenicity in zebrafish and BABL/c mouse infection models. Comparative genome analysis identified a novel 80K integrative conjugative element (ICE), ICESsuHN105, as required for the multidrug resistance phenotype. Six corresponding antibiotic resistance genes in this ICE were identified, namely tet (O), tet (M), erm (two copies), aph, and spc. Phylogenetic analysis classified the element as a homolog of the ICESa2603 family, containing the typical family backbone and insertion DNA. DNA hybrids mediated by natural transformation between HN105 and ZY05719 verified the antibiotic resistant genes of ICESsuHN105 that could be transferred successfully, while they were dispersedly inserted with a single gene in different genomic locations of ZY05719(HN105) transformants. To further identify the horizontal transfer of ICESsuHN105 as a whole mobile genetic element, a circular intermediate form of ICESsuHN105 was detected by PCR. However, the effective conjugation using serotype 2 S. suis as recipients was not observed in current assays in vitro. Further studies confirmed the presence of the complete lantibiotic locus encoded in ICESsuHN105 that effectively inhibits the growth of other streptococci. In summary, this study demonstrated the presence of antibiotic resistance genes in ICE that are able to transfer between different clinical isolates and adapt to a broader range of Streptococcus serotype or species.

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April 21, 2020  |  

Comparative genomics reveals structural and functional features specific to the genome of a foodborne Escherichia coli O157:H7.

Escherichia coli O157:H7 (O157) has been linked to numerous foodborne disease outbreaks. The ability to rapidly sequence and analyze genomes is important for understanding epidemiology, virulence, survival, and evolution of outbreak strains. In the current study, we performed comparative genomics to determine structural and functional features of the genome of a foodborne O157 isolate NADC 6564 and infer its evolutionary relationship to other O157 strains.The chromosome of NADC 6564 contained 5466?kb compared to reference strains Sakai (5498?kb) and EDL933 (5547?kb) and shared 41 of its 43 Linear Conserved Blocks (LCB) with the reference strains. However, 18 of 41 LCB had inverse orientation in NADC 6564 compared to the reference strains. NADC 6564 shared 18 of 19 bacteriophages with reference strains except that the chromosomal positioning of some of the phages differed among these strains. The additional phage (P19) of NADC 6564 was located on a 39-kb insertion element (IE) encoding several hypothetical proteins, an integrase, transposases, transcriptional regulators, an adhesin, and a phosphoethanolamine transferase (PEA). The complete homologs of the 39-kb?IE were found in E. coli PCN061 of porcine origin. The IE-encoded PEA showed low homology (32-33%) to four other PEA in NADC 6564 and PEA linked to mobilizable colistin resistance in E. coli but was highly homologous (95%) to a PEA of uropathogenic, avian pathogenic, and enteroaggregative E. coli. NADC 6564 showed slightly higher minimum inhibitory concentration of colistin compared to the reference strains. The 39-kb?IE also contained dndBCDE and dptFGH operons encoding DNA S-modification and a restriction pathway, linked to oxidative stress tolerance and self-defense against foreign DNA, respectively. Evolutionary tree analysis grouped NADC 6564 with lineage I O157 strains.These results indicated that differential phage counts and different chromosomal positioning of many bacteriophages and genomic islands might have resulted in recombination events causing altered chromosomal organization in NADC 6564. Evolutionary analysis grouped NADC 6564 with lineage I strains and suggested its earlier divergence from these strains. The ability to perform S-DNA modification might affect tolerance of NADC 6564 to various stressors.

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April 21, 2020  |  

Long-read based de novo assembly of low-complexity metagenome samples results in finished genomes and reveals insights into strain diversity and an active phage system.

Complete and contiguous genome assemblies greatly improve the quality of subsequent systems-wide functional profiling studies and the ability to gain novel biological insights. While a de novo genome assembly of an isolated bacterial strain is in most cases straightforward, more informative data about co-existing bacteria as well as synergistic and antagonistic effects can be obtained from a direct analysis of microbial communities. However, the complexity of metagenomic samples represents a major challenge. While third generation sequencing technologies have been suggested to enable finished metagenome-assembled genomes, to our knowledge, the complete genome assembly of all dominant strains in a microbiome sample has not been demonstrated. Natural whey starter cultures (NWCs) are used in cheese production and represent low-complexity microbiomes. Previous studies of Swiss Gruyère and selected Italian hard cheeses, mostly based on amplicon metagenomics, concurred that three species generally pre-dominate: Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii.Two NWCs from Swiss Gruyère producers were subjected to whole metagenome shotgun sequencing using the Pacific Biosciences Sequel and Illumina MiSeq platforms. In addition, longer Oxford Nanopore Technologies MinION reads had to be generated for one to resolve repeat regions. Thereby, we achieved the complete assembly of all dominant bacterial genomes from these low-complexity NWCs, which was corroborated by a 16S rRNA amplicon survey. Moreover, two distinct L. helveticus strains were successfully co-assembled from the same sample. Besides bacterial chromosomes, we could also assemble several bacterial plasmids and phages and a corresponding prophage. Biologically relevant insights were uncovered by linking the plasmids and phages to their respective host genomes using DNA methylation motifs on the plasmids and by matching prokaryotic CRISPR spacers with the corresponding protospacers on the phages. These results could only be achieved by employing long-read sequencing data able to span intragenomic as well as intergenomic repeats.Here, we demonstrate the feasibility of complete de novo genome assembly of all dominant strains from low-complexity NWCs based on whole metagenomics shotgun sequencing data. This allowed to gain novel biological insights and is a fundamental basis for subsequent systems-wide omics analyses, functional profiling and phenotype to genotype analysis of specific microbial communities.

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April 21, 2020  |  

Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii.

Clostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways.We have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (>?5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication repL and repH. We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the C. ljungdahlii genome, resulting in the detection of four sequence motifs with N6-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a Himar1 transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by C. ljungdahlii cultures utilizing syngas as a carbon and energy source.The developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism C. ljungdahlii. The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well.

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April 21, 2020  |  

Characterization of an NDM-5 carbapenemase-producing Escherichia coli ST156 isolate from a poultry farm in Zhejiang, China.

The emergence of carbapenem-resistant Enterobacteriaceae strains has posed a severe threat to public health in recent years. The mobile elements carrying the New Delhi metallo-ß-lactqtamase (NDM) gene have been regarded as the major mechanism leading to the rapid increase of carbapenem-resistant Enterobacteriaceae strains isolated from clinics and animals.We describe an NDM-5-producing Escherichia coli strain, ECCRA-119 (sequence type 156 [ST156]), isolated from a poultry farm in Zhejiang, China. ECCRA-119 is a multidrug-resistant (MDR) isolate that exhibited resistance to 27 antimicrobial compounds, including imipenem and meropenem, as detected by antimicrobial susceptibility testing (AST). The complete genome sequence of the ECCRA-119 isolate was also obtained using the PacBio RS II platform. Eleven acquired resistance genes were identified in the chromosome; four were detected in plasmid pTB201, while six were detected in plasmid pTB202. Importantly, the carbapenem-resistant gene blaNDM-5 was detected in the IncX3 plasmid pTB203. In addition, seven virulence genes and one metal-resistance gene were also detected. The results of conjugation experiments and the transfer regions identification indicated that the blaNDM-5-harboring plasmid pTB203 could be transferred between E. coli strains.The results reflected the severe bacterial resistance in a poultry farm in Zhejiang province and increased our understanding of the presence and transmission of the blaNDM-5 gene.

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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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April 21, 2020  |  

Genome plasticity favours double chromosomal Tn4401b-blaKPC-2 transposon insertion in the Pseudomonas aeruginosa ST235 clone.

Pseudomonas aeruginosa Sequence Type 235 is a clone that possesses an extraordinary ability to acquire mobile genetic elements and has been associated with the spread of resistance genes, including genes that encode for carbapenemases. Here, we aim to characterize the genetic platforms involved in resistance dissemination in blaKPC-2-positive P. aeruginosa ST235 in Colombia.In a prospective surveillance study of infections in adult patients attended in five ICUs in five distant cities in Colombia, 58 isolates of P. aeruginosa were recovered, of which, 27 (46.6%) were resistant to carbapenems. The molecular analysis showed that 6 (22.2%) and 4 (14.8%) isolates harboured the blaVIM and blaKPC-2 genes, respectively. The four blaKPC-2-positive isolates showed a similar PFGE pulsotype and belonged to ST235. Complete genome sequencing of a representative ST235 isolate shows a unique chromosomal contig of 7097.241?bp with eight different resistance genes identified and five transposons: a Tn6162-like with ant(2?)-Ia, two Tn402-like with ant(3?)-Ia and blaOXA-2 and two Tn4401b with blaKPC-2. All transposons were inserted into the genomic islands. Interestingly, the two Tn4401b copies harbouring blaKPC-2 were adjacently inserted into a new genomic island (PAGI-17) with traces of a replicative transposition process. This double insertion was probably driven by several structural changes within the chromosomal region containing PAGI-17 in the ST235 background.This is the first report of a double Tn4401b chromosomal insertion in P. aeruginosa, just within a new genomic island (PAGI-17). This finding indicates once again the great genomic plasticity of this microorganism.

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