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Patients in the pediatric intensive care unit are exposed to multiple medications and are at high risk for adverse drug reactions. Pharmacogenomic (PGx) testing could help decrease their risk of adverse reactions. Although whole blood is preferred for PGx testing, blood volume in this population is often limited. However, for patients on mechanical ventilation, tracheal secretions are abundant, frequently suctioned, and discarded. Thus, the aim of this pilot study was to determine if tracheal aspirates could be used as a source of human genomic DNA for PGx testing. We successfully extracted DNA from tracheal secretions of all 23 patients in the study. The samples were successfully genotyped for 10 clinically actionable single nucleotide variants across 3 cytochrome P450 genes (CYP2D6, CYP2C19, and CYP3A5). Using DNA from whole blood samples in 11 of the patients, we confirmed the accuracy of the genotyping with 100% concordance. Therefore, our results support the use of tracheal aspirates from mechanically ventilated children as an adequate biospecimen for clinical genetic testing.

Abstract Single nucleotide variants (SNVs) in intronic regions have yet to be systematically investigated for their disease-causing potential. Using known pathogenic and neutral intronic SNVs (iSNVs) as training data, we develop the RegSNPs-intron algorithm based on a random forest classifier that integrates RNA splicing, protein structure, and evolutionary conservation features. RegSNPs-intron showed excellent performance in evaluating the pathogenic impacts of iSNVs. Using a high-throughput functional reporter assay called ASSET-seq (ASsay for Splicing using ExonTrap and sequencing), we evaluate the impact of RegSNPs-intron predictions on splicing outcome. Together, RegSNPs-intron and ASSET-seq enable effective prioritization of iSNVs for disease pathogenesis.

Currently, only three phages that infect the medically important bacterium Clostridium difficile have been discussed by the International Committee of Viral Taxonomy (ICTV). They are all myoviruses, and have been assigned to the genus “phicd119likevirus”. An additional nine phages have since been described in the literature with their genome data available. The Phicd119likevirus is named after the type species: the myovirus ΦCD119 which was the first C. difficile phage to be sequenced. The two additional myoviruses, ϕCD27 and φC2, also fall into this genus based on the similarity of their genome and morphological characteristics. The other nine phages have not been assigned to this genus, and four of them do not fit the criteria for the current taxonomic grouping. We have applied protein clustering analysis to determine their phylogenetic relationships. From these results we propose an additional myoviridae genus, that we term “phiMMP04likevirus”.

Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however developing suitable phages is challenging. In this review we summarise the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics.Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage-host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution.No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using whole-phages are such that they remain a major research focus. Whilst we don’t envisage working with C. difficile phages will be problem free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen.

The transfer of novel genetic material into the genomes of bacterial viruses (phages) has been widely documented in several host-phage systems. Bacterial genes are incorporated into the phage genome and, if retained, subsequently evolve within them. The expression of these phage genes can subvert or bolster bacterial processes, including altering bacterial pathogenicity. The phage phiCDHM1 infects Clostridium difficile, a pathogenic bacterium that causes nosocomial infections and is associated with antibiotic treatment. Genome sequencing and annotation of phiCDHM1 shows that despite being closely related to other C. difficile myoviruses, it has several genes that have not been previously reported in any phage genomes. Notably, these include three homologs of bacterial genes from the accessory gene regulator (agr) quorum sensing (QS) system. These are; a pre-peptide (AgrD) of an autoinducing peptide (AIP), an enzyme which processes the pre-peptide (AgrB) and a histidine kinase (AgrC) that detects the AIP to activate a response regulator. Phylogenetic analysis of the phage and C. difficile agr genes revealed that there are three types of agr loci in this species. We propose that the phage genes belonging to a third type, agr3, and have been horizontally transferred from the host. AgrB and AgrC are transcribed during the infection of two different strains. In addition, the phage agrC appears not to be confined to the phiCDHM1 genome as it was detected in genetically distinct C. difficile strains. The discovery of QS gene homologs in a phage genome presents a novel way in which phages could influence their bacterial hosts, or neighbouring bacterial populations. This is the first time that these QS genes have been reported in a phage genome and their distribution both in C. difficile and phage genomes suggests that the agr3 locus undergoes horizontal gene transfer within this species.

Bacteriophages (phages) are increasingly being explored as therapeutic agents to combat bacterial diseases, including Clostridium difficile infections. Therapeutic phages need to be able to efficiently target and kill a wide range of clinically relevant strains. While many phage groups have yet to be investigated in detail, those with new and useful properties can potentially be identified when phages from newly studied geographies are characterised. Here, we report the isolation of C. difficile phages from soil samples from the north of Iraq. Two myoviruses, CDKM15 and CDKM9, were selected for detailed sequence analysis on the basis of their broad and potentially useful host range. CDKM9 infects 25/80 strains from 12/20 C. difficile ribotypes, and CDKM15 infects 20/80 strains from 9/20 ribotypes. Both phages can infect the clinically relevant ribotypes R027 and R001. Phylogenetic analysis based on whole genome sequencing revealed that the phages are genetically distinct from each other but closely related to other long-tailed myoviruses. A comparative genomic analysis revealed key differences in the genes predicted to encode for proteins involved in bacterial infection. Notably, CDKM15 carries a clustered regularly interspaced short palindromic repeat (CRISPR) array with spacers that are homologous to sequences in the CDKM9 genome and of phages from diverse localities. The findings presented suggest a possible shared evolutionary past for these phages and provides evidence of their widespread dispersal.

ABSTRACT Clostridium difficile is an important human-pathogenic bacterium causing antibiotic-associated nosocomial infections worldwide. Mobile genetic elements and bacteriophages have helped shape C. difficile genome evolution. In many bacteria, phage infection may be controlled by a form of bacterial immunity called the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system. This uses acquired short nucleotide sequences (spacers) to target homologous sequences (protospacers) in phage genomes. C. difficile carries multiple CRISPR arrays, and in this paper we examine the relationships between the host- and phage-carried elements of the system. We detected multiple matches between spacers and regions in 31 C. difficile phage and prophage genomes. A subset of the spacers was located in prophage-carried CRISPR arrays. The CRISPR spacer profiles generated suggest that related phages would have similar host ranges. Furthermore, we show that C. difficile strains of the same ribotype could either have similar or divergent CRISPR contents. Both synonymous and nonsynonymous mutations in the protospacer sequences were identified, as well as differences in the protospacer adjacent motif (PAM), which could explain how phages escape this system. This paper illustrates how the distribution and diversity of CRISPR spacers in C. difficile, and its prophages, could modulate phage predation for this pathogen and impact upon its evolution and pathogenicity. IMPORTANCE Clostridium difficile is a significant bacterial human pathogen which undergoes continual genome evolution, resulting in the emergence of new virulent strains. Phages are major facilitators of genome evolution in other bacterial species, and we use sequence analysis-based approaches in order to examine whether the CRISPR/Cas system could control these interactions across divergent C. difficile strains. The presence of spacer sequences in prophages that are homologous to phage genomes raises an extra level of complexity in this predator-prey microbial system. Our results demonstrate that the impact of phage infection in this system is widespread and that the CRISPR/Cas system is likely to be an important aspect of the evolutionary dynamics in C. difficile.