Probing Genomic Aspects of the Multi-Host Pathogen Clostridium perfringens Reveals Significant Pangenome Diversity, and a Diverse Array of Virulence Factors

Clostridium perfringens is an important cause of animal and human infections, however information about the genetic makeup of this pathogenic bacterium is currently limited. In this study, we sought to understand and characterise the genomic variation, pangenomic diversity, and key virulence traits of 56 C. perfringens strains which included 51 public, and 5 newly sequenced and annotated genomes using Whole Genome Sequencing. Our investigation revealed that C. perfringens has an “open” pangenome comprising 11667 genes and 12.6% of core genes, identified as the most divergent single-species Gram-positive bacterial pangenome currently reported. Our computational analyses also defined C. perfringens phylogeny (16S rRNA gene) in relation to some 25 Clostridium species, with C. baratii and C. sardiniense determined to be the closest relatives. Profiling virulence-associated factors confirmed presence of well-characterised C. perfringens-associated exotoxins genes including α-toxin (plc), enterotoxin (cpe), and Perfringolysin O (pfo or pfoA), although interestingly there did not appear to be a close correlation with encoded toxin type and disease phenotype. Furthermore, genomic analysis indicated significant horizontal gene transfer events as defined by presence of prophage genomes, and notably absence of CRISPR defence systems in >70% (40/56) of the strains. In relation to antimicrobial resistance mechanisms, tetracycline resistance genes (tet) and anti-defensins genes (mprF) were consistently detected in silico (tet: 75%; mprF: 100%). However, pre-antibiotic era strain genomes did not encode for tet, thus implying antimicrobial selective pressures in C. perfringens evolutionary history over the past 80 years. This study provides new genomic understanding of this genetically divergent multi-host bacterium, and further expands our knowledge on this medically and veterinary important pathogen

Response: Commentary: Probing Genomic Aspects of the Multi-Host Pathogen Clostridium perfringens Reveals Significant Pangenome Diversity, and a Diverse Array of Virulence Factors

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The role of Clostridium perfringens in intestinal diseases

Clostridium perfringens, a spore-forming bacterium that produces >20 toxins, is known to cause both human and animal intestinal diseases including; foodborne diarrhoea, preterm necrotising enterocolitis (NEC) and necrotic enteritis (NE) in chickens. Currently, there is very limited information on genomic virulence determinants, and the phylogenic and epidemiology landscape of this enteric pathogen, thus in this thesis I sought to comprehensively explore intestinal-associated C. perfringens strains using both genomic and phenotypic methodologies. Whole Genome Sequencing (WGS) and bioinformatic approaches was used to examine a novel collection of C. perfringens isolates and publically available genomes (n=552, including 109 public genomes) from a diverse range of hosts and disease states including; NEC-associated preterm infants, foodborne diarrhoea patients, NEassociated broilers, and healthy humans and animals. These genomes were analysed in combination or as discreet disease subsets to determine infection-linked virulence features, genomic epidemiology and hyper-virulent genotypes, and indicated a highly diverse pangenome (7.4% core genome), toxin-specific and novel virulence factors, widespread distribution of conjugative plasmids, long-term persistence, and interregional transmission events during disease outbreaks. This work represents the largest WGS-based phylogenetic and comparative genomics on C. perfringens to date. Furthermore, a sub-set of C. perfringens isolates were characterised phenotypically using microbiological assays to determine hyper-virulent phenotypes, which linked to genomic analysis. These hyper-virulent strains were then used to establish a novel C. perfringens oral-challenge mouse model. This enteric infection model will allow further mechanistic work in understanding the role of C. perfringens in relevant intestinal diseases and may be used to facilitate therapy or vaccine development. Overall, this multidisciplinary work provides important novel insights into the intestinal pathogen C. perfringens at both a genomic and phenotypic level and provides a platform for subsequent translational studies into efforts to reduce C. perfringens-associated disease burden in both humans and animals

Preterm Infant-Associated Clostridium tertium, Clostridium cadaveris, and Clostridium paraputrificum Strains: Genomic and Evolutionary Insights.

Clostridium species (particularly Clostridium difficile, Clostridium botulinum, Clostridium tetani and Clostridium perfringens) are associated with a range of human and animal diseases. Several other species including Clostridium tertium, Clostridium cadaveris, and Clostridium paraputrificum have also been linked with sporadic human infections, however there is very limited, or in some cases, no genomic information publicly available. Thus, we isolated one C. tertium strain, one C. cadaveris strain and three C. paraputrificum strains from preterm infants residing within neonatal intensive care units and performed Whole Genome Sequencing (WGS) using Illumina HiSeq. In this report, we announce the open availability of the draft genomes: C. tertium LH009, C. cadaveris LH052, C. paraputrificum LH025, C. paraputrificum LH058, and C. paraputrificum LH141. These genomes were checked for contamination in silico to ensure purity, and we confirmed species identity and phylogeny using both 16S rRNA gene sequences (from PCR and in silico) and WGS-based approaches. Average Nucleotide Identity (ANI) was used to differentiate genomes from their closest relatives to further confirm speciation boundaries. We also analysed the genomes for virulence-related factors and antimicrobial resistance genes, and detected presence of tetracycline and methicillin resistance, and potentially harmful enzymes, including multiple phospholipases and toxins. The availability of genomic data in open databases, in tandem with our initial insights into the genomic content and virulence traits of these pathogenic Clostridium species, should enable the scientific community to further investigate the disease-causing mechanisms of these bacteria with a view to enhancing clinical diagnosis and treatment

Phylogenomic analysis of gastroenteritis-associated Clostridium perfringens in England and Wales over a 7-year period indicates distribution of clonal toxigenic strains in multiple outbreaks and extensive involvement of enterotoxin-encoding (CPE) plasmids

Clostridium perfringens is a major enteric pathogen known to cause gastroenteritis in human adults. Although major outbreak cases are frequently reported, only limited whole-genome sequencing (WGS) based studies have been performed to understand the genomic epidemiology and virulence gene content of outbreak-associated C. perfringens strains. We performed phylogenomic analysis on 109 C. perfringens isolates (human and food) obtained from disease cases in England and Wales between 2011 and 2017. Initial findings highlighted the enhanced discriminatory power of WGS in profiling outbreak C. perfringens strains, when compared to the current Public Health England referencing laboratory technique of fluorescent amplified fragment length polymorphism analysis. Further analysis identified that isogenic C. perfringens strains were associated with nine distinct care-home-associated outbreaks over the course of a 5-year interval, indicating a potential common source linked to these outbreaks or transmission over time and space. As expected, the enterotoxin cpe gene was encoded in all but 4 isolates (96.3 %; 105/109), with virulence plasmids encoding cpe (particularly pCPF5603 and pCPF4969 plasmids) extensively distributed (82.6 %; 90/109). Genes encoding accessory virulence factors, such as beta-2 toxin, were commonly detected (46.7 %; 51/109), and genes encoding phage proteins were also frequently identified. Overall, this large-scale genomic study of gastroenteritis-associated C. perfringens suggested that three major cpe-encoding (toxinotype F) genotypes underlie these outbreaks: strains carrying (1) pCPF5603 plasmid, (2) pCPF4969 plasmid and (3) chromosomal-cpe strains. Our findings substantially expanded our knowledge on type F C. perfringens involved in human-associated gastroenteritis, with further studies required to fully probe the dissemination and regional reservoirs of this enteric pathogen, which may help devise effective prevention strategies to reduce the food-poisoning disease burden in vulnerable patients, such as the elderly

Genomic analysis on broiler-associated Clostridium perfringens strains and exploratory caecal microbiome investigation reveals key factors linked to poultry necrotic enteritis.

BACKGROUND: Clostridium perfringens is a key pathogen in poultry-associated necrotic enteritis (NE). To date there are limited Whole Genome Sequencing based studies describing broiler-associated C. perfringens in healthy and diseased birds. Moreover, changes in the caecal microbiome during NE is currently not well characterised. Thus, the aim of this present study was to investigate C. perfringens virulence factors linked to health and diseased chickens, including identifying putative caecal microbiota signatures associated with NE. RESULTS: We analysed 88 broiler chicken C. perfringens genomes (representing 66 publicly available genomes and 22 newly sequenced genomes) using different phylogenomics approaches and identified a potential hypervirulent and globally-distributed clone spanning 20-year time-frame (1993-2013). These isolates harbored a greater number of virulence genes (including toxin and collagen adhesin genes) when compared to other isolates. Further genomic analysis indicated exclusive and overabundant presence of important NE-linked toxin genes including netB and tpeL in NE-associated broiler isolates. Secondary virulence genes including pfoA, cpb2, and collagen adhesin genes cna, cnaA and cnaD were also enriched in the NE-linked C. perfringens genomes. Moreover, an environmental isolate obtained from farm animal feeds was found to encode netB, suggesting potential reservoirs of NetB-positive C. perfringens strains (toxinotype G). We also analysed caecal samples from a small sub-set of 11 diseased and healthy broilers for exploratory microbiome investigation using 16S rRNA amplicon sequencing, which indicated a significant and positive correlation in genus Clostridium within the wider microbiota of those broilers diagnosed with NE, alongside reductions in beneficial microbiota members. CONCLUSIONS: These data indicate a positive association of virulence genes including netB, pfoA, cpb2, tpeL and cna variants linked to NE-linked isolates. Potential global dissemination of specific hypervirulent lineage, coupled with distinctive microbiome profiles, highlights the need for further investigations, which will require a large worldwide sample collection from healthy and NE-associated birds

Enterococcus innesii sp. nov., isolated from the wax moth Galleria mellonella

Four bacterial strains were isolated from two different colony sources of the wax moth Galleria mellonella. They were characterized by a polyphasic approach including 16S rRNA gene sequence analysis, core-genome analysis, average nucleotide identity (ANI) analysis, digital DNA–DNA hybridization (dDDH), determination of G+C content, screening of antibiotic resistance genes, and various phenotypic analyses. Initial analysis of 16S rRNA gene sequence identities indicated that strain GAL7T was potentially very closely related to Enterococcus casseliflavus and Enterococcus gallinarum, having 99.5–99.9 % sequence similarity. However, further analysis of whole genome sequences revealed a genome size of 3.69 Mb, DNA G+C content of 42.35 mol%, and low dDDH and ANI values between the genomes of strain GAL7T and closest phylogenetic relative E. casseliflavus NBRC 100478T of 59.0 and 94.5 %, respectively, indicating identification of a putative new Enterococcus species. In addition, all novel strains encoded the atypical vancomycin-resistance gene vanC-4. Results of phylogenomic, physiological and phenotypic characterization confirmed that strain GAL7T represented a novel species within the genus Enterococcus, for which the name Enterococcus innesii sp. nov. is proposed. The type strain is GAL7T (=DSM 112306T=NCTC 14608T)

Allocoprobacillus halotolerans gen. nov., sp. nov and Coprobacter tertius sp. nov., isolated from human gut microbiota

Two novel bacterial isolates were cultured from faecal samples of patients attending the Breast Care clinic at the Norwich and Norfolk University Hospital. Strain LH1062T was isolated from a 58-year-old female diagnosed with invasive adenocarcinoma with ductal carcinoma in situ. Strain LH1063T was isolated from a healthy 51-year-old female. Isolate LH1062T was predicted to be a potential novel genus most closely related to Coprobacillus , whilst LH1063T was predicted to be a novel species belonging to Coprobacter . Both strains were characterized by polyphasic approaches including 16S rRNA gene analysis, core-genome analysis, average nucleotide identity (ANI) comparisons and phenotypic analysis. Initial screening of the 16S rRNA gene of LH1062T returned a nucleotide identity of 93.4 % to Longibaculum muris . For LH1063T, nucleotide identity was a 92.6 % to Coprobacter secundus . Further investigations showed that LH1062T had a genome size of 2.9 Mb and G+C content of 31.3 mol %. LH1063T had a genome size of 3.3Mb and G+C content of 39.2 mol %. Digital DNA-DNA hybridization (dDDH) and ANI values of LH1062T with its closest relative, Coprobacillus cateniformis JCM 10604T, were 20.9 and 79.54 %, respectively. For LH1063T, the dDDH and ANI values with its closest relative, Coprobacter secundus 177T, were 19.3 and 77.81 %, respectively. Phenotypic testing confirmed that LH1062T could not be matched to a known validly published isolate in any database; thereby indicating a novel genus for which the name Allocoprobacillus gen. nov. is now proposed with LH1062T (=DSM 114537T=NCTC 14686T) being the type strain of the proposed novel species Allocoprobacillus halotolerans sp. nov. Strain LH1063T (=DSM 114538T=NCTC 14698T) fits within the genus Coprobacter and, it being the third species within this genus, the name Coprobacter tertius sp. nov. is proposed

Microbiota supplementation with Bifidobacterium and Lactobacillus modifies the preterm infant gut microbiota and metabolome: An observational study

Supplementation with members of the early-life microbiota as “probiotics” is increasingly used in attempts to beneficially manipulate the preterm infant gut microbiota. We performed a large observational longitudinal study comprising two preterm groups: 101 infants orally supplemented with Bifidobacterium and Lactobacillus (Bif/Lacto) and 133 infants non-supplemented (control) matched by age, sex, and delivery method. 16S rRNA gene profiling on fecal samples (n = 592) showed a predominance of Bifidobacterium and a lower abundance of pathobionts in the Bif/Lacto group. Metabolomic analysis showed higher fecal acetate and lactate and a lower fecal pH in the Bif/Lacto group compared to the control group. Fecal acetate positively correlated with relative abundance of Bifidobacterium, consistent with the ability of the supplemented Bifidobacterium strain to metabolize human milk oligosaccharides into acetate. This study demonstrates that microbiota supplementation is associated with a Bifidobacterium-dominated preterm microbiota and gastrointestinal environment more closely resembling that of full-term infants

An efficient method for high molecular weight bacterial DNA extraction suitable for shotgun metagenomics from skin swabs

The human skin microbiome represents a variety of complex microbial ecosystems that play a key role in host health. Molecular methods to study these communities have been developed but have been largely limited to low-throughput quantification and short amplicon-based sequencing, providing limited functional information about the communities present. Shotgun metagenomic sequencing has emerged as a preferred method for microbiome studies as it provides more comprehensive information about the species/strains present in a niche and the genes they encode. However, the relatively low bacterial biomass of skin, in comparison to other areas such as the gut microbiome, makes obtaining sufficient DNA for shotgun metagenomic sequencing challenging. Here we describe an optimised high-throughput method for extraction of high molecular weight DNA suitable for shotgun metagenomic sequencing. We validated the performance of the extraction method, and analysis pipeline on skin swabs collected from both adults and babies. The pipeline effectively characterised the bacterial skin microbiota with a cost and throughput suitable for larger longitudinal sets of samples. Application of this method will allow greater insights into community compositions and functional capabilities of the skin microbiome