Metagenomics of Wastewater Influent from Wastewater Treatment Facilities across Ontario in the Era of Emerging SARS-CoV-2 Variants of Concern.

We report metagenomic sequencing analyses of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in composite wastewater influent from 10 regions in Ontario, Canada, during the transition between Delta and Omicron variants of concern. The Delta and Omicron BA.1/BA.1.1 and BA.2-defining mutations occurring in various frequencies were reported in the consensus and subconsensus sequences of the composite samples.

Epigenetic age is associated with baseline and 3-year change in frailty in the Canadian Longitudinal Study on Aging.

BACKGROUND: The trajectory of frailty in older adults is important to public health; therefore, markers that may help predict this and other important outcomes could be beneficial. Epigenetic clocks have been developed and are associated with various health-related outcomes and sociodemographic factors, but associations with frailty are poorly described. Further, it is uncertain whether newer generations of epigenetic clocks, trained on variables other than chronological age, would be more strongly associated with frailty than earlier developed clocks. Using data from the Canadian Longitudinal Study on Aging (CLSA), we tested the hypothesis that clocks trained on phenotypic markers of health or mortality (i.e., Dunedin PoAm, GrimAge, PhenoAge and Zhang in Nat Commun 8:14617, 2017) would best predict changes in a 76-item frailty index (FI) over a 3-year interval, as compared to clocks trained on chronological age (i.e., Hannum in Mol Cell 49:359-367, 2013, Horvath in Genome Biol 14:R115, 2013, Lin in Aging 8:394-401, 2016, and Yang Genome Biol 17:205, 2016). RESULTS: We show that in 1446 participants, phenotype/mortality-trained clocks outperformed age-trained clocks with regard to the association with baseline frailty (mean = 0.141, SD = 0.075), the greatest of which is GrimAge, where a 1-SD increase in ΔGrimAge (i.e., the difference from chronological age) was associated with a 0.020 increase in frailty (95% CI 0.016, 0.024), or ~ 27% relative to the SD in frailty. Only GrimAge and Hannum (Mol Cell 49:359-367, 2013) were significantly associated with change in frailty over time, where a 1-SD increase in ΔGrimAge and ΔHannum 2013 was associated with a 0.0030 (95% CI 0.0007, 0.0050) and 0.0028 (95% CI 0.0007, 0.0050) increase over 3 years, respectively, or ~ 7% relative to the SD in frailty change. CONCLUSION: Both prevalence and change in frailty are associated with increased epigenetic age. However, not all clocks are equally sensitive to these outcomes and depend on their underlying relationship with chronological age, healthspan and lifespan. Certain clocks were significantly associated with relatively short-term changes in frailty, thereby supporting their utility in initiatives and interventions to promote healthy aging.

Complete Genome Sequences of a Diverse Group of 13 Propionibacterium acnes Bacteriophages Isolated from Urban Raw Sewage.

We present complete genome sequences of 13 Propionibacterium acnes phages isolated from urban raw sewage. They belong to the family Siphoviridae, have genome sizes of 29,450.6 ± 256.5 nucleotides and G+C contents of 54.14% ± 0.22% and contain 42 to 45 coding DNA sequences (CDS). Genomic sequences of 9 of 13 phages were divergent by 6 to 10%, distinguishing them as species.

Complete Genome Sequence of Enterococcus thailandicus Strain a523 Isolated from Urban Raw Sewage.

We report here the first complete circularly closed genome sequence of Enterococcus thailandicus strain a523 isolated from raw urban sewage. This genome contains 2,646,250 bp with a G+C content of 36.8%, 2,499 genes, 2,370 protein-coding sequences, 6 rRNA operons, 65 tRNAs, and 6 clustered regularly interspaced short palindromic repeat arrays.

Splicing arrays reveal novel RBM10 targets, including SMN2 pre-mRNA.

BACKGROUND: RBM10 is an RNA binding protein involved in message stabilization and alternative splicing regulation. The objective of the research described herein was to identify novel targets of RBM10-regulated splicing. To accomplish this, we downregulated RBM10 in human cell lines, using small interfering RNAs, then monitored alternative splicing, using a reverse transcription-PCR screening platform. RESULTS: RBM10 knockdown (KD) provoked alterations in splicing events in 10-20% of the pre-mRNAs, most of which had not been previously identified as RBM10 targets. Hierarchical clustering of the genes affected by RBM10 KD revealed good conservation of alternative exon inclusion or exclusion across cell lines. Pathway annotation showed RAS signaling to be most affected by RBM10 KD. Of particular interest was the finding that splicing of SMN pre-mRNA, encoding the survival of motor neuron (SMN) protein, was influenced by RBM10 KD. Inhibition of RBM10 resulted in preferential expression of the full-length, exon 7 retaining, SMN transcript in four cancer cell lines and one normal skin fibroblast cell line. SMN protein is expressed from two genes, SMN1 and SMN2, but the SMN1 gene is homozygously disrupted in people with spinal muscular atrophy; as a consequence, all of the SMN that is expressed in people with this disease is from the SMN2 gene. Expression analyses using primary fibroblasts from control, carrier and spinal muscle atrophy donors demonstrated that RBM10 KD resulted in preferential expression of the full-length, exon 7 retaining, SMN2 transcript. At the protein level, upregulation of the full-length SMN2 was also observed. Re-expression of RBM10, in a stable RBM10 KD cancer cell line, correlated with a reversion of the KD effect, demonstrating specificity. CONCLUSION: Our work has not only expanded the number of pre-mRNA targets for RBM10, but identified RBM10 as a novel regulator of SMN2 alternative inclusion.

False Negative Results in Clostridium difficile Testing.

BACKGROUND: Accurate diagnosis of Clostridium difficile infection (CDI) is paramount for patient management. The wrong diagnosis places patients at risk, delays treatment, and/ or contributes to transmission of infection in the healthcare setting. Although amplification of the toxin B gene by polymerase chain reaction (PCR) is a sensitive method for detecting toxigenic C. difficile, false negative results still occur and could impact the diagnosis and treatment of this infection. METHODS: This study investigated 48 patients that tested negative for toxigenic C. difficile via GeneXpert C. difficile epi test, while simultaneously testing positive for toxigenic C. difficile via stool culture. Fifty discrepant samples were collected over a 15-month period and all C. difficile isolates were characterized by ribotype. Patient charts were reviewed to assess whether discrepant results impacted the treatment course or clinical outcome of affected patients. RESULTS: Fifty samples of a total of 2308 samples tested in an acute healthcare facility over a 15-month period had negative PCR and positive stool culture for toxigenic C. difficile. C. difficile isolated from the discrepant samples resulted in diverse ribotyping patterns suggesting they were derived from different strains. The samples belonged to patients who were distributed evenly between age groups and wards in the hospital. In the majority of cases, the false negative C. difficile test results did not seem to impact the clinical outcome in these patients. CONCLUSIONS: The PCR limit of detection may impact the results of molecular methods for C. difficile detection. Both clinical and analytical sensitivity of C. difficile tests should be considered when deciding which diagnostic assay to use, and clinical correlates should be examined carefully before excluding CDI as a cause of disease.

Antimicrobial susceptibility of Canadian isolates of Helicobacter pylori in Northeastern Ontario.

BACKGROUND: Helicobacter pylori plays a significant role in gastritis and ulcers. It is a carcinogen as defined by the WHO, and infection can result in adenocarcinomas and mucosa-associated lymphoid tissue lymphomas. In Canada, rates of antimicrobial resistance are relatively unknown, with very few studies conducted in the past 15 years. OBJECTIVE: To examine rates of resistance in Sudbury, Ontario, compare antimicrobial susceptibility methods and attempt to determine the molecular basis of antibiotic resistance. METHODS: Patients attending scheduled visits at Health Sciences North (Sudbury, Ontario) provided gastric biopsy samples on a volunteer basis. In total, 20 H pylori isolates were collected, and antimicrobial susceptibility testing (on amoxicillin, tetracycline, metronidazole, ciprofloxacin, levofloxacin and clarithromycin) was conducted using disk diffusion and E-test methods. Subsequently, genomic DNA from these isolates was sequenced to detect mutations associated with antimicrobial resistance. RESULTS: Sixty-five percent of the isolates were found to be resistant to at least one of the listed antibiotics according to E-test. Three isolates were found to be resistant to ≥3 of the above-mentioned antibiotics. Notably, 25% of the isolates were found to be resistant to both metronidazole and clarithromycin, two antibiotics that are normally prescribed as part of first-line regimens in the treatment of H pylori infections in Canada and most of the world. Among the resistant strains, the sequences of 23S ribosomal RNA and gyrA, which are linked to clarithromycin and ciprofloxacin/levofloxacin resistance, respectively, revealed the presence of known point mutations associated with antimicrobial resistance. CONCLUSIONS: In general, resistance to metronidazole, ciprofloxacin/levofloxacin and clarithromycin has increased since the studies in the early 2000s. These results suggest that surveillance programs of H pylori antibiotic resistance may need to be revisited or improved to prevent antimicrobial therapy failure.

HISTORIQUE: L'Helicobacter pylori contribue énormément à la gastrite et aux ulcères. L'OMS le définit comme un cancérigène, et l'infection peut provoquer l'apparition d'adénocarcinomes et de lymphomes de tissus lymphoïdes associés aux muqueuses. Au Canada, on connaît relativement peu les taux de résistance antimicrobienne, car très peu d'études ont été réalisées sur le sujet depuis 15 ans. OBJECTIF: Examiner les taux de résistance à Sudbury, en Ontario, comparer les méthodes de susceptibilité antimicrobienne et tenter de déterminer le fondement biologique de la résistance antibiotique. MÉTHODOLOGIE: Les patients qui allaient à un rendez-vous prévu au Health Sciences North de Sudbury ont remis les résultats de biopsies gastriques sur une base volontaire. Au total, 20 isolats de H pylori ont été recueillis, et les tests de susceptibilité antimicrobienne (à l'amoxicilline, à la tétracycline, au métronidazole, à la ciprofloxacine, à la lévofloxacine et à la clarithromycine) ont été effectués au moyen de la diffusion sur disque et de l'essai E. L'ADN génomique de ces isolats a ensuite été séquencé pour déceler les mutations associées à la résistance antimicrobienne. RÉSULTATS: Selon l'essai E, 65 % des isolats étaient résistants à au moins un des antibiotiques énumérés. Notamment, 25 % des isolats étaient résistants à la fois au métronidazole et à la clarithromycine, tous deux normalement prescrits en première ligne pour traiter les infections à H pylori au Canada et dans la plupart des régions du monde. Parmi les souches résistantes, les séquences d'ARN ribosomique 23S et de gyrA, qui sont liées à la résistance à la clarithromycine et à la ciprofloxacine-lévofloxacine, respectivement, révélaient la présence de mutations de points connus associés à une résistance antimicrobienne. CONCLUSIONS: En général, la résistance au métronidazole, à la ciprofloxacine-lévofloxacine et à la clarithromycine a augmenté depuis les études réalisées au début des années 2000. D'après ces résultats, il faudra peut-être revoir ou améliorer les programmes de surveillance de l'antibiorésistance au H pylori pour prévenir l'échec du traitement antimicrobien.

Impact of polymerase chain reaction testing on Clostridium difficile infection rates in an acute health care facility.

Two rapid methods of Clostridium difficile infection (CDI) diagnosis were compared between June 2012 and March 2013: a GeneXpert (Cepheid, Sunnyvale, Calif) polymerase chain reaction (PCR) test and an enzyme immunoassay (EIA). The influence of these methods on the detection of hospital-acquired CDI and identification of CDI outbreaks was evaluated. We tested 1,592 stool samples for C difficile. The GeneXpert PCR test identified 211 positive samples (68 determined to be hospital-acquired infection), whereas EIA identified 105 positive samples (36 determined to be hospital-acquired infection). The GeneXpert PCR method in contrast to the EIA method increased the detection rates of nosocomial CDI cases and contributed to the declaration of CDI outbreaks.

Geographic and haplotype structure of candidate type 2 diabetes susceptibility variants at the calpain-10 locus.

Recently, a positional cloning study proposed that haplotypes at the calpain-10 locus (CAPN10) are associated with increased risk of type 2 diabetes, or non-insulin-dependent diabetes mellitus, in Mexican Americans, Finns, and Germans. To inform the interpretation of the original mapping results and to look for evidence for the action of natural selection on CAPN10, we undertook a population-based genotyping survey of the candidate susceptibility variants. First, we genotyped sites 43, 19, and 63 (the haplotype-defining variants previously proposed) and four closely linked SNPs, in 561 individuals from 11 populations from five continents, and we examined the linkage disequilibrium among them. We then examined the ancestral state of these sites by sequencing orthologous portions of CAPN10 in chimpanzee and orangutan (the identity of sites 43 and 19 was further investigated in a limited sample of other great apes and Old World and New World monkeys). Our survey suggests larger-than-expected differences in the distribution of CAPN10 susceptibility variants between African and non-African populations, with common, derived haplotypes in European and Asian samples (including one of two proposed risk haplotypes) being rare or absent in African samples. These results suggest a history of positive natural selection at the locus, resulting in significant geographic differences in polymorphism frequencies. The relationship of these differences to disease risk is discussed.