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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  |  

Genomic erosion and extensive horizontal gene transfer in gut-associated Acetobacteraceae.

Symbiotic relationships between animals and bacteria have profound impacts on the evolutionary trajectories of each partner. Animals and gut bacteria engage in a variety of relationships, occasionally persisting over evolutionary timescales. Ants are a diverse group of animals that engage in many types of associations with taxonomically distinct groups of bacterial associates. Here, we bring into culture and characterize two closely-related strains of gut associated Acetobacteraceae (AAB) of the red carpenter ant, Camponotus chromaiodes.Genome sequencing, assembly, and annotation of both strains delineate stark patterns of genomic erosion and sequence divergence in gut associated AAB. We found widespread horizontal gene transfer (HGT) in these bacterial associates and report elevated gene acquisition associated with energy production and conversion, amino acid and coenzyme transport and metabolism, defense mechanisms, and lysine export. Both strains have acquired the complete NADH-quinone oxidoreductase complex, plausibly from an Enterobacteriaceae origin, likely facilitating energy production under diverse conditions. Conservation of several lysine biosynthetic and salvage pathways and accumulation of lysine export genes via HGT implicate L-lysine supplementation by both strains as a potential functional benefit for the host. These trends are contrasted by genome-wide erosion of several amino acid biosynthetic pathways and pathways in central metabolism. We perform phylogenomic analyses on both strains as well as several free living and host associated AAB. Based on their monophyly and deep divergence from other AAB, these C. chromaiodes gut associates may represent a novel genus. Together, our results demonstrate how extensive horizontal transfer between gut associates along with genome-wide deletions leads to mosaic metabolic pathways. More broadly, these patterns demonstrate that HGT and genomic erosion shape metabolic capabilities of persistent gut associates and influence their genomic evolution.Using comparative genomics, our study reveals substantial changes in genomic content in persistent associates of the insect gastrointestinal tract and provides evidence for the evolutionary pressures inherent to this environment. We describe patterns of genomic erosion and horizontal acquisition that result in mosaic metabolic pathways. Accordingly, the phylogenetic position of both strains of these associates form a divergent, monophyletic clade sister to gut associates of honey bees and more distantly to Gluconobacter.

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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  |  

Assignment of virus and antimicrobial resistance genes to microbial hosts in a complex microbial community by combined long-read assembly and proximity ligation.

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.

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April 21, 2020  |  

Adaptive Strategies in a Poly-Extreme Environment: Differentiation of Vegetative Cells in Serratia ureilytica and Resistance to Extreme Conditions.

Poly-extreme terrestrial habitats are often used as analogs to extra-terrestrial environments. Understanding the adaptive strategies allowing bacteria to thrive and survive under these conditions could help in our quest for extra-terrestrial planets suitable for life and understanding how life evolved in the harsh early earth conditions. A prime example of such a survival strategy is the modification of vegetative cells into resistant resting structures. These differentiated cells are often observed in response to harsh environmental conditions. The environmental strain (strain Lr5/4) belonging to Serratia ureilytica was isolated from a geothermal spring in Lirima, Atacama Desert, Chile. The Atacama Desert is the driest habitat on Earth and furthermore, due to its high altitude, it is exposed to an increased amount of UV radiation. The geothermal spring from which the strain was isolated is oligotrophic and the temperature of 54°C exceeds mesophilic conditions (15 to 45°C). Although the vegetative cells were tolerant to various environmental insults (desiccation, extreme pH, glycerol), a modified cell type was formed in response to nutrient deprivation, UV radiation and thermal shock. Scanning (SEM) and Transmission Electron Microscopy (TEM) analyses of vegetative cells and the modified cell structures were performed. In SEM, a change toward a circular shape with reduced size was observed. These circular cells possessed what appears as extra coating layers under TEM. The resistance of the modified cells was also investigated, they were resistant to wet heat, UV radiation and desiccation, while vegetative cells did not withstand any of those conditions. A phylogenomic analysis was undertaken to investigate the presence of known genes involved in dormancy in other bacterial clades. Genes related to spore-formation in Myxococcus and Firmicutes were found in S. ureilytica Lr5/4 genome; however, these genes were not enough for a full sporulation pathway that resembles either group. Although, the molecular pathway of cell differentiation in S. ureilytica Lr5/4 is not fully defined, the identified genes may contribute to the modified phenotype in the Serratia genus. Here, we show that a modified cell structure can occur as a response to extremity in a species that was previously not known to deploy this strategy. This strategy may be widely spread in bacteria, but only expressed under poly-extreme environmental conditions.

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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  |  

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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September 22, 2019  |  

Metagenomic binning and association of plasmids with bacterial host genomes using DNA methylation.

Shotgun metagenomics methods enable characterization of microbial communities in human microbiome and environmental samples. Assembly of metagenome sequences does not output whole genomes, so computational binning methods have been developed to cluster sequences into genome ‘bins’. These methods exploit sequence composition, species abundance, or chromosome organization but cannot fully distinguish closely related species and strains. We present a binning method that incorporates bacterial DNA methylation signatures, which are detected using single-molecule real-time sequencing. Our method takes advantage of these endogenous epigenetic barcodes to resolve individual reads and assembled contigs into species- and strain-level bins. We validate our method using synthetic and real microbiome sequences. In addition to genome binning, we show that our method links plasmids and other mobile genetic elements to their host species in a real microbiome sample. Incorporation of DNA methylation information into shotgun metagenomics analyses will complement existing methods to enable more accurate sequence binning.

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September 22, 2019  |  

A microbial clock provides an accurate estimate of the postmortem interval in a mouse model system.

Establishing the time since death is critical in every death investigation, yet existing techniques are susceptible to a range of errors and biases. For example, forensic entomology is widely used to assess the postmortem interval (PMI), but errors can range from days to months. Microbes may provide a novel method for estimating PMI that avoids many of these limitations. Here we show that postmortem microbial community changes are dramatic, measurable, and repeatable in a mouse model system, allowing PMI to be estimated within approximately 3 days over 48 days. Our results provide a detailed understanding of bacterial and microbial eukaryotic ecology within a decomposing corpse system and suggest that microbial community data can be developed into a forensic tool for estimating PMI. DOI:http://dx.doi.org/10.7554/eLife.01104.001.

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September 22, 2019  |  

Species-level bacterial community profiling of the healthy sinonasal microbiome using Pacific Biosciences sequencing of full-length 16S rRNA genes.

Pan-bacterial 16S rRNA microbiome surveys performed with massively parallel DNA sequencing technologies have transformed community microbiological studies. Current 16S profiling methods, however, fail to provide sufficient taxonomic resolution and accuracy to adequately perform species-level associative studies for specific conditions. This is due to the amplification and sequencing of only short 16S rRNA gene regions, typically providing for only family- or genus-level taxonomy. Moreover, sequencing errors often inflate the number of taxa present. Pacific Biosciences’ (PacBio’s) long-read technology in particular suffers from high error rates per base. Herein, we present a microbiome analysis pipeline that takes advantage of PacBio circular consensus sequencing (CCS) technology to sequence and error correct full-length bacterial 16S rRNA genes, which provides high-fidelity species-level microbiome data.Analysis of a mock community with 20 bacterial species demonstrated 100% specificity and sensitivity with regard to taxonomic classification. Examination of a 250-plus species mock community demonstrated correct species-level classification of >?90% of taxa, and relative abundances were accurately captured. The majority of the remaining taxa were demonstrated to be multiply, incorrectly, or incompletely classified. Using this methodology, we examined the microgeographic variation present among the microbiomes of six sinonasal sites, by both swab and biopsy, from the anterior nasal cavity to the sphenoid sinus from 12 subjects undergoing trans-sphenoidal hypophysectomy. We found greater variation among subjects than among sites within a subject, although significant within-individual differences were also observed. Propiniobacterium acnes (recently renamed Cutibacterium acnes) was the predominant species throughout, but was found at distinct relative abundances by site.Our microbial composition analysis pipeline for single-molecule real-time 16S rRNA gene sequencing (MCSMRT, https://github.com/jpearl01/mcsmrt ) overcomes deficits of standard marker gene-based microbiome analyses by using CCS of entire 16S rRNA genes to provide increased taxonomic and phylogenetic resolution. Extensions of this approach to other marker genes could help refine taxonomic assignments of microbial species and improve reference databases, as well as strengthen the specificity of associations between microbial communities and dysbiotic states.

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September 22, 2019  |  

Daily HIV pre-exposure prophylaxis (PrEP) with tenofovir disoproxil fumarate-emtricitabine reduced Streptococcus and increased Erysipelotrichaceae in rectal microbiota.

Daily PrEP is highly effective at preventing HIV-1 acquisition, but risks of long-term tenofovir disoproxil fumarate plus emtricitabine (TDF-FTC) include renal decline and bone mineral density decrease in addition to initial gastrointestinal side effects. We investigated the impact of TDF-FTC on the enteric microbiome using rectal swabs collected from healthy MSM before PrEP initiation and after 48 to 72 weeks of adherent PrEP use. The V4 region of the 16S ribosomal RNA gene sequencing showed that Streptococcus was significantly reduced from 12.0% to 1.2% (p?=?0.036) and Erysipelotrichaceae family was significantly increased from 0.79% to 3.3% (p?=?0.028) after 48-72 weeks of daily PrEP. Catenibacterium mitsuokai, Holdemanella biformis and Turicibacter sanguinis were increased within the Erysipelotrichaceae family and Streptococcus agalactiae, Streptococcus oralis, Streptococcus mitis were reduced. These changes were not associated with host factors including PrEP duration, age, race, tenofovir diphosphate blood level, any drug use and drug abuse, suggesting that the observed microbiome shifts were likely induced by daily PrEP use. Long-term PrEP resulted in increases of Catenibacterium mitsuokai and Holdemanella biformis, which have been associated with gut microbiome dysbiosis. Our observations can aid in characterizing PrEP’s side effects, which is likely to improve PrEP adherence, and thus HIV-1 prevention.

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September 22, 2019  |  

Next-generation sequencing for pathogen detection and identification

Over the past decade, the field of genomics has seen such drastic improvements in sequencing chemistries that high-throughput sequencing, or next-generation sequencing (NGS), is being applied to generate data across many disciplines. NGS instruments are becoming less expensive, faster, and smaller, and therefore are being adopted in an increasing number of laboratories, including clinical laboratories. Thus far, clinical use of NGS has been mostly focused on the human genome, for purposes such as characterizing the molecular basis of cancer or for diagnosing and understanding the basis of rare genetic disorders. There are, however, an increasing number of examples whereby NGS is employed to discover novel pathogens, and these cases provide precedent for the use of NGS in microbial diagnostics. NGS has many advantages over traditional microbial diagnostic methods, such as unbiased rather than pathogen-specific protocols, ability to detect fastidious or non-culturable organisms, and ability to detect co-infections. One of the most impressive advantages of NGS is that it requires little or no prior knowledge of the pathogen, unlike many other diagnostic assays; therefore for pathogen discovery, NGS is very valuable. However, despite these advantages, there are challenges involved in implementing NGS for routine clinical microbiological diagnosis. We discuss these advantages and challenges in the context of recently described research studies.

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September 22, 2019  |  

Effects of antibiotic on microflora in ileum and cecum for broilers by 16S rRNA sequence analysis.

An experiment was conducted to analyze and compare the microbial composition, abundance, dynamic distribution, and functions without and with antibiotic fed to broilers. A 16S rRNA-sequencing approach was used to evaluate the bacterial composition of the gut of male broilers under different groups. A total of 240 1-day old AA male broilers were randomly assigned to two groups, with 120 broilers per group. The treatment group was administered an antibiotic with their feed, while the control group was not administered antibiotic (control group). A total of 10 replicates were assessed per treatment. The control group was fed a basal diet containing corn, soybean meal, and cottonseed meal and met the nutritional requirement. The antibiotic group was fed 100 mg/kg aureomycin (based on the basal diet). The trial lasted 42 days. Operational taxonomic unit partition and classification, alpha diversity, taxonomic composition, beta diversity, and microflora comparative analyses along with key species screening were performed for all of the treatment groups. Our data indicate that aureomycin treatment in broilers is directly correlated with variations of the gut content of specific bacterial taxa, and herein provide insights into the impact of antibiotic on microbial communities in cecum and ileum of broiler chickens.© 2018 Japanese Society of Animal Science.

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September 22, 2019  |  

The features of mucosa-associated microbiota in primary sclerosing cholangitis.

Little is known about the role of the microbiome in primary sclerosing cholangitis.To explore the mucosa-associated microbiota in primary sclerosing cholangitis (PSC) patients across different locations in the gut, and to compare it with inflammatory bowel disease (IBD)-only patients and healthy controls.Biopsies from the terminal ileum, right colon, and left colon were collected from patients and healthy controls undergoing colonoscopy. Microbiota profiling using bacterial 16S rRNA sequencing was performed on all biopsies.Forty-four patients were recruited: 20 with PSC (19 with PSC-IBD and one with PSC-only), 15 with IBD-only and nine healthy controls. The overall microbiome profile was similar throughout different locations in the gut. No differences in the global microbiome profile were found. However, we observed significant PSC-associated enrichment in Barnesiellaceae at the family level, and in Blautia and an unidentified Barnesiellaceae at the genus level. At the operational taxa unit level, most shifts in PSC were observed in Clostridiales and Bacteroidales orders, with approximately 86% of shifts occurring within the former order.The overall microbiota profile was similar across multiple locations in the gut from the same individual regardless of disease status. In this study, the mucosa associated-microbiota of patients with primary sclerosing cholangitis was characterised by enrichment of Blautia and Barnesiellaceae and by major shifts in operational taxa units within Clostridiales order.© 2016 John Wiley & Sons Ltd.

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