Investigating the Human Intestinal DNA Virome and Predicting Disease-Associated Virus-Host Interactions in Severe Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

Understanding how the human virome, and which of its constituents, contributes to health or disease states is reliant on obtaining comprehensive virome profiles. By combining DNA viromes from isolated virus-like particles (VLPs) and whole metagenomes from the same faecal sample of a small cohort of healthy individuals and patients with severe myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), we have obtained a more inclusive profile of the human intestinal DNA virome. Key features are the identification of a core virome comprising tailed phages of the class Caudoviricetes, and a greater diversity of DNA viruses including extracellular phages and integrated prophages. Using an in silico approach, we predicted interactions between members of the Anaerotruncus genus and unique viruses present in ME/CFS microbiomes. This study therefore provides a framework and rationale for studies of larger cohorts of patients to further investigate disease-associated interactions between the intestinal virome and the bacteriome.

Investigating Antibody Reactivity to the Intestinal Microbiome in Severe Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS): A Feasibility Study.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a multisystemic disease of unknown aetiology that is characterised by disabling chronic fatigue and involves both the immune and gastrointestinal (GI) systems. Patients display alterations in GI microbiome with a significant proportion experiencing GI discomfort and pain and elevated blood biomarkers for altered intestinal permeability compared with healthy individuals. To investigate a possible GI origin of ME/CFS we designed a feasibility study to test the hypothesis that ME/CFS pathogenesis is a consequence of increased intestinal permeability that results in microbial translocation and a breakdown in immune tolerance leading to generation of antibodies reactive to indigenous intestinal microbes. Secretory immunoglobulin (Ig) A and serum IgG levels and reactivity to intestinal microbes were assessed in five pairs of severe ME/CFS patients and matched same-household healthy controls. For profiling serum IgG, we developed IgG-Seq which combines flow-cytometry based bacterial cell sorting and metagenomics to detect mucosal IgG reactivity to the microbiome. We uncovered evidence for immune dysfunction in severe ME/CFS patients that was characterised by reduced capacity and reactivity of serum IgG to stool microbes, irrespective of their source. This study provides the rationale for additional studies in larger cohorts of ME/CFS patients to further explore immune-microbiome interactions.

A hybrid and poly-polish workflow for the complete and accurate assembly of phage genomes: a case study of ten przondoviruses.

Bacteriophages (phages) within the genus Przondovirus are T7-like podoviruses belonging to the subfamily Studiervirinae, within the family Autographiviridae, and have a highly conserved genome organisation. The genomes of these phages range from 37 to 42 kb in size, encode 50-60 genes and are characterised by the presence of direct terminal repeats (DTRs) flanking the linear chromosome. These DTRs are often deleted during short-read-only and hybrid assemblies. Moreover, long-read-only assemblies are often littered with sequencing and/or assembly errors and require additional curation. Here, we present the isolation and characterisation of ten novel przondoviruses targeting Klebsiella spp. We describe HYPPA, a HYbrid and Poly-polish Phage Assembly workflow, which utilises long-read assemblies in combination with short-read sequencing to resolve phage DTRs and correcting errors, negating the need for laborious primer walking and Sanger sequencing validation. Our assembly workflow utilised Oxford Nanopore Technologies for long-read sequencing for its accessibility, making it the more relevant long-read sequencing technology at this time, and Illumina DNA Prep for short-read sequencing, representing the most commonly used technologies globally. Our data demonstrate the importance of careful curation of phage assemblies before publication, and prior to using them for comparative genomics.

Genome-wide association reveals host-specific genomic traits in Escherichia coli.

BACKGROUND: Escherichia coli is an opportunistic pathogen which colonizes various host species. However, to what extent genetic lineages of E. coli are adapted or restricted to specific hosts and the genomic determinants of such adaptation or restriction is poorly understood. RESULTS: We randomly sampled E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, to construct a collection of 1198 whole-genome sequenced E. coli isolates. We identified associations between specific E. coli lineages and the host from which they were isolated. A genome-wide association study (GWAS) identified several E. coli genes that were associated with human, cattle, or chicken hosts, whereas no genes associated with the pig host could be found. In silico characterization of nine contiguous genes (collectively designated as nan-9) associated with the human host indicated that these genes are involved in the metabolism of sialic acids (Sia). In contrast, the previously described sialic acid regulon known as sialoregulon (i.e. nanRATEK-yhcH, nanXY, and nanCMS) was not associated with any host species. In vitro growth experiments with a Δnan-9 E. coli mutant strain, using the sialic acids 5-N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as sole carbon source, showed impaired growth behaviour compared to the wild-type. CONCLUSIONS: This study provides an extensive analysis of genetic determinants which may contribute to host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.

Using unique ORFan genes as strain-specific identifiers for Escherichia coli.

BACKGROUND: Bacterial identification at the strain level is a much-needed, but arduous and challenging task. This study aimed to develop a method for identifying and differentiating individual strains among multiple strains of the same bacterial species. The set used for testing the method consisted of 17 Escherichia coli strains picked from a collection of strains isolated in Germany, Spain, the United Kingdom and Vietnam from humans, cattle, swine, wild boars, and chickens. We targeted unique or rare ORFan genes to address the problem of selective and specific strain identification. These ORFan genes, exclusive to each strain, served as templates for developing strain-specific primers. RESULTS: Most of the experimental strains (14 out of 17) possessed unique ORFan genes that were used to develop strain-specific primers. The remaining three strains were identified by combining a PCR for a rare gene with a selection step for isolating the experimental strains. Multiplex PCR allowed the successful identification of the strains both in vitro in spiked faecal material in addition to in vivo after experimental infections of pigs and recovery of bacteria from faecal material. In addition, primers for qPCR were also developed and quantitative readout from faecal samples after experimental infection was also possible. CONCLUSIONS: The method described in this manuscript using strain-specific unique genes to identify single strains in a mixture of strains proved itself efficient and reliable in detecting and following individual strains both in vitro and in vivo, representing a fast and inexpensive alternative to more costly methods.

Genomic Diversity and Virulence Potential of ESBL- and AmpC-ß-Lactamase-Producing Escherichia coli Strains From Healthy Food Animals Across Europe.

The role of livestock animals as a putative source of ESBL/pAmpC E. coli for humans is a central issue of research. In a large-scale pan-European surveillance, 2,993 commensal Escherichia spp. isolates were recovered from randomly collected fecal samples of healthy cattle, pigs and chickens in various abattoirs. One-hundred Escherichia spp. isolates (0.5% from cattle, 1.3% pigs, 8.0% chickens) fulfilled the criteria for cefotaxime and ceftazidime non-wildtype (EUCAST). In silico screening of WGS data of 99 isolates (98 E. coli and 1 E. fergusonii) revealed bla SHV - 12 (32.3%), bla CTX - M - 1 (24.2%), and bla CMY - 2 (22.2%) as predominant ESBL/pAmpC types. Other types were bla SHV - 2 (1.0%), bla CTX - M - 2 / - 14 / - 15 (1.0/6.1/1.0%), and bla TEM - 52 (5.1%). Six isolates revealed AmpC-promoter mutations (position -42 (C > T) and one carried mcr-1. The majority (91.3%) of ESBL/pAmpC genes were located on plasmids. SHV-12 was mainly (50%) encoded on IncI1α plasmids (pST-3/-26/-95), followed by IncX3 (12.5%) and IncK2 (3.1%). The bla TEM - 52 genes were located on IncI1α-pST-36 (60%) and IncX1 plasmids (20%). The dominant plasmid lineage among CTX-M-1 isolates was IncI1α (pST-3/-295/-317) (87.5%), followed by IncN-pST-1 (8.3%). CMY-2 was mostly identified on IncI1α (pST-12/-2) (54.5%) and IncK2 (31.8%) plasmids. Several plasmids revealed high similarity to published plasmids from human and animal Enterobacteriaceae. The isolates were assigned to phylogroups A/C (34.7/7.1%), B1 (27.6%), B2 (3.1%), D/F (9.2/10.2%), E (5.1%), and to E. clades (3.0%). With 51 known and 2 novel MLST types, a wide variety of STs was found, including STs previously observed in human isolates (ST10/38/117/131/648). ESBL/AmpC types or STs were rarely correlated with the geographic origin of the isolates or animal species. Virulence gene typing identified extraintestinal pathogenic E. coli (ExPEC; 2.0%), avian pathogenic E. coli (APEC; 51.5%), and atypical enteropathogenic E. coli (EPEC; 6.1%). In conclusion, the high diversity of STs and phylogenetic groups provides hardly any hint for clonal spread of single lineages but hints toward the dissemination of cephalosporin resistance genes in livestock via distinct, globally successful plasmid lineages. Even though a number of isolates could not be assigned to a distinct pathotype, our finding of combined multidrug-resistance and virulence in this facultative pathogen should be considered an additional threat to public health.

Risk of Transmission of Antimicrobial Resistant Escherichia coli from Commercial Broiler and Free-Range Retail Chicken in India.

Multidrug-resistant Escherichia coli infections are a growing public health concern. This study analyzed the possibility of contamination of commercial poultry meat (broiler and free-range) with pathogenic and or multi-resistant E. coli in retail chain poultry meat markets in India. We analyzed 168 E. coli isolates from broiler and free-range retail poultry (meat/ceca) sampled over a wide geographical area, for their antimicrobial sensitivity, phylogenetic groupings, virulence determinants, extended-spectrum-ß-lactamase (ESBL) genotypes, fingerprinting by Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR and genetic relatedness to human pathogenic E. coli using whole genome sequencing (WGS). The prevalence rates of ESBL producing E. coli among broiler chicken were: meat 46%; ceca 40%. Whereas, those for free range chicken were: meat 15%; ceca 30%. E. coli from broiler and free-range chicken exhibited varied prevalence rates for multi-drug resistance (meat 68%; ceca 64% and meat 8%; ceca 26%, respectively) and extraintestinal pathogenic E. coli (ExPEC) contamination (5 and 0%, respectively). WGS analysis confirmed two globally emergent human pathogenic lineages of E. coli, namely the ST131 (H30-Rx subclone) and ST117 among our poultry E. coli isolates. These results suggest that commercial poultry meat is not only an indirect public health risk by being a possible carrier of non-pathogenic multi-drug resistant (MDR)-E. coli, but could as well be the carrier of human E. coli pathotypes. Further, the free-range chicken appears to carry low risk of contamination with antimicrobial resistant and extraintestinal pathogenic E. coli (ExPEC). Overall, these observations reinforce the understanding that poultry meat in the retail chain could possibly be contaminated by MDR and/or pathogenic E. coli.

Comparative Genomic Analysis of Globally Dominant ST131 Clone with Other Epidemiologically Successful Extraintestinal Pathogenic Escherichia coli (ExPEC) Lineages.

Escherichia coli sequence type 131 (ST131), a pandemic clone responsible for the high incidence of extraintestinal pathogenic E. coli (ExPEC) infections, has been known widely for its contribution to the worldwide dissemination of multidrug resistance. Although other ExPEC-associated and extended-spectrum-ß-lactamase (ESBL)-producing E. coli clones, such as ST38, ST405, and ST648 have been studied widely, no comparative genomic data with respect to other genotypes exist for ST131. In this study, comparative genomic analysis was performed for 99 ST131 E. coli strains with 40 genomes from three other STs, including ST38 (n = 12), ST405 (n = 10), and ST648 (n = 18), and functional studies were performed on five in-house strains corresponding to the four STs. Phylogenomic analysis results from this study corroborated with the sequence type-specific clonality. Results from the genome-wide resistance profiling confirmed that all strains were inherently multidrug resistant. ST131 genomes showed unique virulence profiles, and analysis of mobile genetic elements and their associated methyltransferases (MTases) has revealed that several of them were missing from the majority of the non-ST131 strains. Despite the fact that non-ST131 strains lacked few essential genes belonging to the serum resistome, the in-house strains representing all four STs demonstrated similar resistance levels to serum antibactericidal activity. Core genome analysis data revealed that non-ST131 strains usually lacked several ST131-defined genomic coordinates, and a significant number of genes were missing from the core of the ST131 genomes. Data from this study reinforce adaptive diversification of E. coli strains belonging to the ST131 lineage and provide new insights into the molecular mechanisms underlying clonal diversification of the ST131 lineage.IMPORTANCEE. coli, particularly the ST131 extraintestinal pathogenic E. coli (ExPEC) lineage, is an important cause of community- and hospital-acquired infections, such as urinary tract infections, surgical site infections, bloodstream infections, and sepsis. The treatment of infections caused by ExPEC has become very challenging due to the emergence of resistance to the first-line as well as the last-resort antibiotics. This study analyzes E. coli ST131 against three other important and globally distributed ExPEC lineages (ST38, ST405, and ST648) that also produced extended-spectrum ß-lactamase (ESBL). This is perhaps the first study that employs the high-throughput whole-genome sequence-based approach to compare and study the genomic features of these four ExPEC lineages in relation to their functional properties. Findings from this study highlight the differences in the genomic coordinates of ST131 with respect to the other STs considered here. Results from this comparative genomics study can help in advancing the understanding of ST131 evolution and also offer a framework towards future developments in pathogen identification and targeted therapeutics to prevent diseases caused by this pandemic E. coli ST131 clone.

Comparative Genomics of Escherichia coli Isolated from Skin and Soft Tissue and Other Extraintestinal Infections.

Escherichia coli, an intestinal Gram-negative bacterium, has been shown to be associated with a variety of diseases in addition to intestinal infections, such as urinary tract infections (UTIs), meningitis in neonates, septicemia, skin and soft tissue infections (SSTIs), and colisepticemia. Thus, for nonintestinal infections, it is categorized as extraintestinal pathogenic E. coli (ExPEC). It is also an opportunistic pathogen, causing cross infections, notably as an agent of zoonotic diseases. However, comparative genomic data providing functional and genetic coordinates for ExPEC strains associated with these different types of infections have not proven conclusive. In the study reported here, ExPEC E. coli isolated from SSTIs was characterized, including virulence and drug resistance profiles, and compared with isolates from patients suffering either pyelonephritis or septicemia. Results revealed that the majority of the isolates belonged to two pathogenic phylogroups, B2 and D. Approximately 67% of the isolates were multidrug resistant (MDR), with 85% producing extended-spectrum beta-lactamase (ESBL) and 6% producing metallo-beta-lactamase (MBL). The blaCTX-M-15 genotype was observed in at least 70% of the E. coli isolates in each category, conferring resistance to an extended range of beta-lactam antibiotics. Whole-genome sequencing and comparative genomics of the ExPEC isolates revealed that two of the four isolates from SSTIs, NA633 and NA643, belong to pandemic sequence type ST131, whereas functional characteristics of three of the ExPEC pathotypes revealed that they had equal capabilities to form biofilm and were resistant to human serum. Overall, the isolates from a variety of ExPEC infections demonstrated similar resistomes and virulomes and did not display any disease-specific functional or genetic coordinates.IMPORTANCE Infections caused by extraintestinal pathogenic E. coli (ExPEC) are of global concern as they result in significant costs to health care facilities management. The recent emergence of a multidrug-resistant pandemic clone, Escherichia coli ST131, is of primary concern as a global threat. In developing countries, such as India, skin and soft tissue infections (SSTIs) associated with E. coli are marginally addressed. In this study, we employed both genomic analysis and phenotypic assays to determine relationships, if any, among the ExPEC pathotypes. Similarity between antibiotic resistance and virulence profiles was observed, ST131 isolates from SSTIs were reported, and genomic similarities among strains isolated from different disease conditions were detected. This study provides functional molecular infection epidemiology insight into SSTI-associated E. coli compared with ExPEC pathotypes.

Genome Dynamics and Molecular Infection Epidemiology of Multidrug-Resistant Helicobacter pullorum Isolates Obtained from Broiler and Free-Range Chickens in India.

Some life-threatening, foodborne, and zoonotic infections are transmitted through poultry birds. Inappropriate and indiscriminate use of antimicrobials in the livestock industry has led to an increased prevalence of multidrug-resistant bacteria with epidemic potential. Here, we present a functional molecular epidemiological analysis entailing the phenotypic and whole-genome sequence-based characterization of 11 H. pullorum isolates from broiler and free-range chickens sampled from retail wet markets in Hyderabad City, India. Antimicrobial susceptibility tests revealed all of the isolates to be resistant to multiple antibiotic classes such as fluoroquinolones, cephalosporins, sulfonamides, and macrolides. The isolates were also found to be extended-spectrum ß-lactamase producers and were even resistant to clavulanic acid. Whole-genome sequencing and comparative genomic analysis of these isolates revealed the presence of five or six well-characterized antimicrobial resistance genes, including those encoding a resistance-nodulation-division efflux pump(s). Phylogenetic analysis combined with pan-genome analysis revealed a remarkable degree of genetic diversity among the isolates from free-range chickens; in contrast, a high degree of genetic similarity was observed among broiler chicken isolates. Comparative genomic analysis of all publicly available H. pullorum genomes, including our isolates (n = 16), together with the genomes of 17 other Helicobacter species, revealed a high number (8,560) of H. pullorum-specific protein-encoding genes, with an average of 535 such genes per isolate. In silico virulence screening identified 182 important virulence genes and also revealed high strain-specific gene content in isolates from free-range chickens (average, 34) compared to broiler chicken isolates. A significant prevalence of prophages (ranging from 1 to 9) and a significant presence of genomic islands (0 to 4) were observed in free-range and broiler chicken isolates. Taken together, these observations provide significant baseline data for functional molecular infection epidemiology of nonpyloric Helicobacter species such as H. pullorum by unraveling their evolution in chickens and their possible zoonotic transmission to humans. IMPORTANCE: Globally, the poultry industry is expanding with an ever-growing consumer base for chicken meat. Given this, food-associated transmission of multidrug-resistant bacteria represents an important health care issue. Our study involves a critical baseline approach directed at genome sequence-based epidemiology and transmission dynamics of H. pullorum, a poultry pathogen having established zoonotic potential. We believe our studies would facilitate the development of surveillance systems that ensure the safety of food for humans and guide public health policies related to the use of antibiotics in animal feed in countries such as India. We sequenced 11 new genomes of H. pullorum as a part of this study. These genomes would provide much value in addition to the ongoing comparative genomic studies of helicobacters.

Molecular Epidemiology and Genome Dynamics of New Delhi Metallo-ß-Lactamase-Producing Extraintestinal Pathogenic Escherichia coli Strains from India.

The global dissemination and increasing incidence of carbapenem-resistant, Gram-negative organisms have resulted in acute public health concerns. Here, we present a retrospective multicenter study on molecular characterization of metallo-ß-lactamase (MBL)-producing clinical Escherichia coli isolates recovered from extraintestinal infections in two hospitals in Pune, India. We screened a large sample size of 510 E. coli isolates for MBL production wherein we profiled their molecular determinants, antimicrobial resistance phenotypes, functional virulence properties, genomic features, and transmission dynamics. Approximately 8% of these isolates were MBL producers, the majority of which were of the NDM-1 (69%) type, followed by NDM-5 (19%), NDM-4 (5.5%), and NDM-7 (5.5%). MBL producers were resistant to all antibiotics tested except for colistin, fosfomycin, and chloramphenicol, which were effective to various extents. Plasmids were found to be an effective means of dissemination of NDM genes and other resistance traits. All MBL producers adhered to and invaded bladder epithelial (T24) cells and demonstrated significant serum resistance. Genomic analysis of MBL-producing E. coli isolates revealed higher resistance but a moderate virulence gene repertoire. A subset of NDM-1-positive E. coli isolates was identified as dominant sequence type 101 (ST101) while two strains belonging to ST167 and ST405 harbored NDM-5. A majority of MBL-producing E. coli strains revealed unique genotypes, suggesting that they were clonally unrelated. Overall, the coexistence of virulence and carbapenem resistance in clinical E. coli isolates is of serious concern. Moreover, the emergence of NDM-1 among the globally dominant E. coli ST101 isolates warrants stringent surveillance and control measures.

Contig-Layout-Authenticator (CLA): A Combinatorial Approach to Ordering and Scaffolding of Bacterial Contigs for Comparative Genomics and Molecular Epidemiology.

A wide variety of genome sequencing platforms have emerged in the recent past. High-throughput platforms like Illumina and 454 are essentially adaptations of the shotgun approach generating millions of fragmented single or paired sequencing reads. To reconstruct whole genomes, the reads have to be assembled into contigs, which often require further downstream processing. The contigs can be directly ordered according to a reference, scaffolded based on paired read information, or assembled using a combination of the two approaches. While the reference-based approach appears to mask strain-specific information, scaffolding based on paired-end information suffers when repetitive elements longer than the size of the sequencing reads are present in the genome. Sequencing technologies that produce long reads can solve the problems associated with repetitive elements but are not necessarily easily available to researchers. The most common high-throughput technology currently used is the Illumina short read platform. To improve upon the shortcomings associated with the construction of draft genomes with Illumina paired-end sequencing, we developed Contig-Layout-Authenticator (CLA). The CLA pipeline can scaffold reference-sorted contigs based on paired reads, resulting in better assembled genomes. Moreover, CLA also hints at probable misassemblies and contaminations, for the users to cross-check before constructing the consensus draft. The CLA pipeline was designed and trained extensively on various bacterial genome datasets for the ordering and scaffolding of large repetitive contigs. The tool has been validated and compared favorably with other widely-used scaffolding and ordering tools using both simulated and real sequence datasets. CLA is a user friendly tool that requires a single command line input to generate ordered scaffolds.