Substituting Allose as the Primary Carbon Source During Enrichment Helps Improve Detection and Isolation of Lineage II Listeria monocytogenes From Food.

Testing of foods for low levels of the human pathogen, Listeria monocytogenes (Lm), involves a selective enrichment procedure. A nonpathogenic species of Listeria, L. innocua (Li), is often present in foods and food-manufacturing environments and is an interference organism for Lm detection due to competition during enrichment. The present study investigated whether a novel enrichment strategy incorporating the sugar allose into the secondary enrichment broth (allose method) could improve the detection of Lm from foods when Li is present. First, Canadian food isolates of Listeria spp. were tested to confirm recent reports that lineage II Lm (LII-Lm), but not Li, could metabolize allose. All LII-Lm isolates (n = 81), but not Li (n = 36), possessed the allose genes lmo0734-lmo0739, and could efficiently metabolize allose. Next, smoked salmon was contaminated with mixtures of LII-Lm and Li and tested using different enrichment procedures to compare the ability to recover Lm. Allose broth was more effective than Fraser Broth, with Lm detected in 87% (74 of 85) compared to 59% (50 of 85) of the samples (P < 0.05), following a common preenrichment. When evaluated against a current Health Canada method (MFLP-28), the allose method was more effective, with LII-Lm detected in 88% (57 of 65) compared to 69% (45 of 65) of the samples (P < 0.05). The allose method also remarkably increased the ratio of LII-Lm to Li postenrichment, which improved the ease of obtaining isolated Lm colonies for confirmation tests. Allose may therefore provide a tool for use when the presence of background flora interferes with Lm detection. As this tool is specifically applicable to a subset of Lm, the use of this method modification may provide a working example of tailoring methodology to target the known subtype of the pathogen of interest in an outbreak investigation, or for regular monitoring activities in conjunction with a PCR screen for allose genes on preenrichment cultures.

Real-Time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2 Associated with B.1.1.7 Variant of Concern.

The N501Y amino acid mutation caused by a single point substitution A23063T in the spike gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possessed by three variants of concern (VOCs), B.1.1.7, B.1.351, and P.1. A rapid screening tool using this mutation is important for surveillance during the coronavirus disease 2019 (COVID-19) pandemic. We developed and validated a single nucleotide polymorphism real-time reverse transcription PCR assay using allelic discrimination of the spike gene N501Y mutation to screen for potential variants of concern and differentiate them from SARS-CoV-2 lineages without the N501Y mutation. A total of 160 clinical specimens positive for SARS-CoV-2 were characterized as mutant (N501Y) or N501 wild type by Sanger sequencing and were subsequently tested with the N501Y single nucleotide polymorphism real-time reverse transcriptase PCR assay. Our assay, compared to Sanger sequencing for single nucleotide polymorphism detection, demonstrated positive percent agreement of 100% for all 57 specimens displaying the N501Y mutation, which were confirmed by Sanger sequencing to be typed as A23063T, including one specimen with mixed signal for wild type and mutant. Negative percent agreement was 100% in all 103 specimens typed as N501 wild type, with A23063 identified as wild type by Sanger sequencing. The identification of circulating SARS-CoV-2 lineages carrying an N501Y mutation is critical for surveillance purposes. Current identification methods rely primarily on Sanger sequencing or whole-genome sequencing, which are time consuming, labor intensive, and costly. The assay described herein is an efficient tool for high-volume specimen screening for SARS-CoV-2 VOCs and for selecting specimens for confirmatory Sanger or whole-genome sequencing. IMPORTANCE During the coronavirus disease 2019 (COVID-19) pandemic, several variants of concern (VOCs) have been detected, for example, B.1.1.7, B.1.351, P.1, and B.1.617.2. The VOCs pose a threat to public health efforts to control the spread of the virus. As such, surveillance and monitoring of these VOCs is of the utmost importance. Our real-time RT-PCR assay helps with surveillance by providing an easy method to quickly survey SARS-CoV-2 specimens for VOCs carrying the N501Y single nucleotide polymorphism (SNP). Samples that test positive for the N501Y mutation in the spike gene with our assay can be sequenced to identify the lineage. Thus, our assay helps to focus surveillance efforts and decrease turnaround times.

Impact of coronavirus disease 2019 (COVID-19) pre-test probability on positive predictive value of high cycle threshold severe acute respiratory coronavirus virus 2 (SARS-CoV-2) real-time reverse transcription polymerase chain reaction (RT-PCR) test results.

OBJECTIVES: Performance characteristics of SARS-CoV-2 nucleic acid detection assays are understudied within contexts of low pre-test probability, including screening asymptomatic persons without epidemiological links to confirmed cases, or asymptomatic surveillance testing. SARS-CoV-2 detection without symptoms may represent presymptomatic or asymptomatic infection, resolved infection with persistent RNA shedding, or a false-positive test. This study assessed the positive predictive value of SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) assays by retesting positive specimens from 5 pre-test probability groups ranging from high to low with an alternate assay. METHODS: In total, 122 rRT-PCR positive specimens collected from unique patients between March and July 2020 were retested using a laboratory-developed nested RT-PCR assay targeting the RNA-dependent RNA polymerase (RdRp) gene followed by Sanger sequencing. RESULTS: Significantly fewer (15.6%) positive results in the lowest pre-test probability group (facilities with institution-wide screening having ≤3 positive asymptomatic cases) were reproduced with the nested RdRp gene RT-PCR assay than in each of the 4 groups with higher pre-test probability (individual group range, 50.0%-85.0%). CONCLUSIONS: Large-scale SARS-CoV-2 screening testing initiatives among low pre-test probability populations should be evaluated thoroughly prior to implementation given the risk of false-positive results and consequent potential for harm at the individual and population level.

Characteristics of SARS-CoV-2 testing for rapid diagnosis of COVID-19 during the initial stages of a global pandemic.

Accurate SARS-CoV-2 diagnosis is essential to guide prevention and control of COVID-19. Here we examine SARS-CoV-2 molecular-based test performance characteristics and summarize case-level data related to COVID-19 diagnosis. From January 11 through April 22, 2020, Public Health Ontario conducted SARS-CoV-2 testing of 86,942 specimens collected from 80,354 individuals, primarily using real-time reverse-transcription polymerase chain reaction (rRT-PCR) methods. We analyzed test results across specimen types and for individuals with multiple same-day and multi-day collected specimens. Nasopharyngeal compared to throat swabs had a higher positivity (8.8% vs. 4.8%) and an adjusted estimate 2.9 Ct lower (SE = 0.5, p<0.001). Same-day specimens showed high concordance (98.8%), and the median Ct of multi-day specimens increased over time. Symptomatic cases had rRT-PCR results with an adjusted estimate 3.0 Ct (SE = 0.5, p<0.001) lower than asymptomatic/pre-symptomatic cases. Overall test sensitivity was 84.6%, with a negative predictive value of 95.5%. Molecular testing is the mainstay of SARS-CoV-2 diagnosis and testing protocols will continue to be dynamic and iteratively modified as more is learned about this emerging pathogen.

Genomic Epidemiology of Invasive Methicillin-Resistant Staphylococcus aureus Infections Among Hospitalized Individuals in Ontario, Canada.

BACKGROUND: Prevention and control of methicillin-resistant Staphylococcus aureus (MRSA) infections remain challenging. In-depth surveillance integrating patient and isolate data can provide evidence to better inform infection control and public health practice. METHODS: We analyzed MRSA cases diagnosed in 2010 (n = 212) and 2016 (n = 214) by hospitals in Ontario, Canada. Case-level clinical and demographic data were integrated with isolate characteristics, including antimicrobial resistance (AMR), classic genotyping, and whole-genome sequencing results. RESULTS: Community-associated MRSA (epidemiologically defined) increased significantly from 23.6% in 2010 to 43.0% in 2016 (P < .001). The MRSA population structure changed over time, with a 1.5× increase in clonal complex (CC)8 strains and a concomitant decrease in CC5. The clonal shift was reflected in AMR patterns, with a decrease in erythromycin (86.7% to 78.4%, P = .036) and clindamycin resistance (84.3% to 47.9%, P < .001) and a >2-fold increase in fusidic acid resistance (9.0% to 22.5%, P < .001). Isolates within both CC5 and CC8 were relatively genetically diverse. We identified 6 small genomic clusters-3 potentially related to transmission in healthcare settings. CONCLUSIONS: Community-associated MRSA is increasing among hospitalized individuals in Ontario. Clonal shifting from CC5 to CC8 has impacted AMR. We identified a relatively high genetic diversity and limited genomic clustering within these dominant CCs.

Potential Ad Hoc Markers of Persistence and Virulence in Canadian Listeria monocytogenes Food and Clinical Isolates.

The Listeria monocytogenes gene inlA, encoding a surface virulence protein, was examined for the presence of premature stop codon (PMSC) mutations in 82 isolates obtained by the Canadian Food Inspection Agency (CFIA) from foods and food contact surfaces. These mutations were coanalyzed for the presence of stress survival islet 1 (SSI-1) and for the abilities of the isolates to invade Caco-2 intestinal epithelial cells and form biofilms on polystyrene. PMSC mutations were present in one-third of the isolates (predominantly those of serogroup 1/2a), and their presence was correlated with a noninvasive phenotype. The presence of SSI-1 and the ability to form biofilms were also linked to the 1/2a serogroup. Serogroup 4b isolates lacked inlA PMSC mutations and were invasive, but neither formed biofilms nor carried SSI-1. To expand upon these experimental findings, an in silico analysis was performed on L. monocytogenes genomes from Canadian databases of 278 food isolates and 607 clinical isolates. The prevalence of inlA PMSC mutations in genomes of food isolates was significantly higher (P < 0.0001) than that in clinical isolates. Also, a three-codon deletion in inlA associated with a hyperinvasive phenotype was more prevalent in genomes from clinical isolates (primarily of clonal complex 6, serogroup 4b) than in those from food isolates (P < 0.001). In contrast, SSI-1 was significantly overrepresented (P < 0.001) in genomes from food isolates. We propose the hypothesis that SSI-1 and inlA play a role in the evolution of Canadian L. monocytogenes strains into either a virulent (represented by serogroup 4b clinical isolates) or an environmentally persistent (represented by serogroup 1/2a food isolates) phenotype. The combined presence of SSI-1 and inlA PMSC mutations have potential for use as genetic markers for risk assessment when L. monocytogenes is recovered from foods, indicating low potential for pathogenesis.

Draft Genome Sequences of Four Clinical Legionella pneumophila Isolates from Ontario, Canada.

Legionella pneumophila outbreak investigations require the development of reliable typing methods to better understand the genetic relationships of the isolates involved. Here, we report the draft genome sequences of four clinical Legionella pneumophila isolates obtained between 2000 and 2012 in Ontario, Canada.

Genome Sequence of Listeria monocytogenes Plasmid pLM-C-273 Carrying Genes Related to Stress Resistance.

Mobile genetic elements in bacteria, such as plasmids, act as important vectors for the transfer of antibiotic resistance, virulence, and metal resistance genes. Here, we report the genome sequence of a new plasmid pLM-C-273, identified in a Listeria monocytogenes strain isolated from a clinical sample in Ontario, Canada.

Genomic Recombination Leading to Decreased Virulence of Group B Streptococcus in a Mouse Model of Adult Invasive Disease.

Adult invasive disease caused by Group B Streptococcus (GBS) is increasing worldwide. Whole-genome sequencing (WGS) now permits rapid identification of recombination events, a phenomenon that occurs frequently in GBS. Using WGS, we described that strain NGBS375, a capsular serotype V GBS isolate of sequence type (ST)297, has an ST1 genomic background but has acquired approximately 300 kbp of genetic material likely from an ST17 strain. Here, we examined the virulence of this strain in an in vivo model of GBS adult invasive infection. The mosaic ST297 strain showed intermediate virulence, causing significantly less systemic infection and reduced mortality than a more virulent, serotype V ST1 isolate. Bacteremia induced by the ST297 strain was similar to that induced by a serotype III ST17 strain, which was the least virulent under the conditions tested. Yet, under normalized bacteremia levels, the in vivo intrinsic capacity to induce the production of pro-inflammatory cytokines was similar between the ST297 strain and the virulent ST1 strain. Thus, the diminished virulence of the mosaic strain may be due to reduced capacity to disseminate or multiply in blood during a systemic infection which could be mediated by regulatory factors contained in the recombined region.

Assessment of Listeria sp. Interference Using a Molecular Assay To Detect Listeria monocytogenes in Food.

Detection of Listeria monocytogenes in food is currently based on enrichment methods. When L. monocytogenes is present with other Listeria species in food, the species compete during the enrichment process. Overgrowth competition of the nonpathogenic Listeria species might result in false-negative results obtained with the current reference methods. This potential issue was noted when 50 food samples artificially spiked with L. monocytogenes were tested with a real-time PCR assay and Canada's current reference method, MFHPB-30. Eleven of the samples studied were from foods naturally contaminated with Listeria species other than those used for spiking. The real-time PCR assay detected L. monocytogenes in all 11 of these samples; however, only 6 of these samples were positive by the MFHPB-30 method. To determine whether L. monocytogenes detection can be affected by other species of the same genus due to competition, an L. monocytogenes strain and a Listeria innocua strain with a faster rate of growth in the enrichment broth were artificially coinoculated at different ratios into ground pork meat samples and cultured according to the MFHPB-30 method. L. monocytogenes was detected only by the MFHPB-30 method when L. monocytogenes/L. innocua ratios were 6.0 or higher. In contrast, using the same enrichments, the real-time PCR assay detected L. monocytogenes at ratios as low as 0.6. Taken together, these findings support the hypothesis that L. monocytogenes can be outcompeted by L. innocua during the MFHPB-30 enrichment phase. However, more reliable detection of L. monocytogenes in this situation can be achieved by a PCR-based method mainly because of its sensitivity.

Genome Sequence of Listeria monocytogenes Strain F6540 (Sequence Type 360) Collected from Food Samples in Ontario, Canada.

Comparative genomic analysis between pathogenic and nonpathogenic Listeria monocytogenes strains provides a good model for studying the virulence of this organism. Here, we report the genome sequence of the nonpathogenic L. monocytogenes strain F6540 (sequence type 360) identified specifically in food samples in Ontario, Canada, in 2010.

SopB promotes phosphatidylinositol 3-phosphate formation on Salmonella vacuoles by recruiting Rab5 and Vps34.

Salmonella colonizes a vacuolar niche in host cells during infection. Maturation of the Salmonella-containing vacuole (SCV) involves the formation of phosphatidylinositol 3-phosphate (PI(3)P) on its outer leaflet. SopB, a bacterial virulence factor with phosphoinositide phosphatase activity, was proposed to generate PI(3)P by dephosphorylating PI(3,4)P2, PI(3,5)P2, and PI(3,4,5)P3. Here, we examine the mechanism of PI(3)P formation during Salmonella infection. SopB is required to form PI(3,4)P2/PI(3,4,5)P3 at invasion ruffles and PI(3)P on nascent SCVs. However, we uncouple these events experimentally and reveal that SopB does not dephosphorylate PI(3,4)P2/PI(3,4,5)P3 to produce PI(3)P. Instead, the phosphatase activity of SopB is required for Rab5 recruitment to the SCV. Vps34, a PI3-kinase that associates with active Rab5, is responsible for PI(3)P formation on SCVs. Therefore, SopB mediates PI(3)P production on the SCV indirectly through recruitment of Rab5 and its effector Vps34. These findings reveal a link between phosphoinositide phosphatase activity and the recruitment of Rab5 to phagosomes.

Arrested maturation of Neisseria-containing phagosomes in the absence of the lysosome-associated membrane proteins, LAMP-1 and LAMP-2.

Mature, microbicidal phagosomes are rich in the lysosome-associated membrane proteins, LAMP-1 and LAMP-2, two highly glycosylated proteins presumed to form a protective barrier lining the phagosomal membrane. Pathogenic Neisseria secrete a protease that selectively cleaves LAMP-1, suggesting a critical role for LAMP proteins in the microbicidal competence of phagosomes. To determine the requirement for LAMP proteins in bacterial phagocytosis, we employed embryonic fibroblasts isolated from knockout mice lacking lamp-1, lamp-2 or both genes, as well as small interfering RNA (siRNA)-mediated knockdown of LAMP expression in a human epithelial cell line. Like wild-type cells, those lacking either LAMP-1 or LAMP-2 alone formed phagosomes that gradually acquired microbicidal activity and curtailed bacterial growth. In contrast, LAMP-1 and LAMP-2 double-deficient fibroblasts failed to kill engulfed Neisseria gonorrhoeae. In these cells, maturation was arrested prior to the acquisition of Rab7. As a result, the Rab7-interacting lysosomal protein (RILP, a Rab7 effector) was not recruited to the phagosomes, which were consequently unable to undergo dynein/dynactin-mediated centripetal displacement along microtubules and remained in a predominantly peripheral location. The inability of such phagosomes to migrate towards lysosomes likely contributed to their incomplete maturation and inability to eliminate bacteria. These findings suggest that neisserial degradation of LAMP-1 is not sufficient to affect its survival within the phagosome, and establish LAMP proteins as critical components in the process whereby phagosomes acquire microbicidal capabilities.

Alteration of epithelial structure and function associated with PtdIns(4,5)P2 degradation by a bacterial phosphatase.

Elucidation of the role of PtdIns(4,5)P(2) in epithelial function has been hampered by the inability to selectively manipulate the cellular content of this phosphoinositide. Here we report that SigD, a phosphatase derived from Salmonella, can effectively hydrolyze PtdIns(4,5)P(2), generating PtdIns(5)P. When expressed by microinjecting cDNA into epithelial cells forming confluent monolayers, wild-type SigD induced striking morphological and functional changes that were not mimicked by a phosphatase-deficient SigD mutant (C462S). Depletion of PtdIns(4,5)P(2) in intact SigD-injected cells was verified by detachment from the membrane of the pleckstrin homology domain of phospholipase Cdelta, used as a probe for the phosphoinositide by conjugation to green fluorescent protein. Single-cell measurements of cytosolic pH indicated that the Na(+)/H(+) exchange activity of epithelia was markedly inhibited by depletion of PtdIns(4,5)P(2). Similarly, anion permeability, measured using two different halide-sensitive probes, was depressed in cells expressing SigD. Depletion of PtdIns(4,5)P(2) was associated with marked alterations in the actin cytoskeleton and its association with the plasma membrane. The junctional complexes surrounding the injected cells gradually opened and the PtdIns(4,5)P(2)-depleted cells eventually detached from the monolayer, which underwent rapid restitution. Similar observations were made in intestinal and renal epithelial cultures. In addition to its effects on phosphoinositides, SigD has been shown to convert inositol 1,3,4,5,6-pentakisphosphate (IP(5)) into inositol 1,4,5,6-tetrakisphosphate (IP(4)), and the latter has been postulated to mediate the diarrhea caused by Salmonella. However, the effects of SigD on epithelial cells were not mimicked by microinjection of IP(4). In contrast, the cytoskeletal and ion transport effects were replicated by hydrolyzing PtdIns(4,5)P(2) with a membrane-targeted 5-phosphatase or by occluding the inositide using high-avidity tandem PH domain constructs. We therefore suggest that opening of the tight junctions and inhibition of Na(+)/H(+) exchange caused by PtdIns(4,5)P(2) hydrolysis combine to account, at least in part, for the fluid loss observed during Salmonella-induced diarrhea.

The amino-terminal non-catalytic region of Salmonella typhimurium SigD affects actin organization in yeast and mammalian cells.

The internalization of Salmonella into epithelial cells relies on the function of bacterial proteins which are injected into the cell by a specialized type III secretion system. Such bacterial effectors interfere with host cell signalling and induce local cytoskeletal rearrangements. One of such effectors is SigD/SopB, which shares homology with mammalian inositol phosphatases. We made use of the Saccharomyces cerevisiae model for elucidating new aspects of SigD function. Endogenous expression of SigD in yeast caused severe growth inhibition. Surprisingly, sigD alleles mutated in the catalytic site or even deleted for the whole C-terminal phosphatase domain still inhibited yeast growth by inducing loss of actin polarization and precluding the budding process. Accordingly, when expressed in HeLa cells, the same sigD alleles lost the ability of depleting phosphatidylinositol 4,5-bisphosphate from the plasma membrane, but still caused disappearance of actin fibres and loss of adherence. We delineate a region of 25 amino acids (residues 118-142) that is necessary for the effect of SigD on actin in HeLa cells. Our data indicate that SigD exerts a toxic effect linked to its N-terminal region and independent of its phosphatase activity.

Inducible antibacterial defense system in C. elegans.

The term innate immunity refers to a number of evolutionary ancient mechanisms that serve to defend animals and plants against infection. Genetically tractable model organisms, especially Drosophila, have contributed greatly to advances in our understanding of mammalian innate immunity. Essentially, nothing is known about immune responses in the nematode Caenorhabditis elegans. Using high-density cDNA microarrays, we show here that infection of C. elegans by the Gram-negative bacterium Serratia marcescens provokes a marked upregulation of the expression of many genes. Among the most robustly induced are genes encoding lectins and lysozymes, known to be involved in immune responses in other organisms. Certain infection-inducible genes are under the control of the DBL-1/TGFbeta pathway. We found that dbl-1 mutants exhibit increased susceptibility to infection. Conversely, overexpression of the lysozyme gene lys-1 augments the resistance of C. elegans to S. marcescens. These results constitute the first demonstration of inducible antibacterial defenses in C. elegans and open new avenues for the investigation of evolutionary conserved mechanisms of innate immunity.

The HMG-I/Y-related protein p8 binds to p300 and Pax2 trans-activation domain-interacting protein to regulate the trans-activation activity of the Pax2A and Pax2B transcription factors on the glucagon gene promoter.

p8 is a nuclear DNA-binding protein, which was identified because its expression is strongly activated in response to several stresses. Biochemical and biophysical studies revealed that despite a weak sequence homology p8 is an HMG-I/Y-like protein, suggesting that p8 may be involved in transcription regulation. Results reported here strongly support this hypothesis. Using a pull-down approach, we found that p8 interacts with the general co-activator p300. We also found that, similar to the HMG proteins, p300 was able to acetylate recombinant p8 in vitro, although the significance of such modification remains to be determined. Then a screening by the two-hybrid system, using p8 as bait, allowed us to identify the Pax2 trans-activation domain-interacting protein (PTIP) as another partner of p8. Transient transfection studies revealed that PTIP is a strong inhibitor of the trans-activation activities of Pax2A and Pax2B on the glucagon gene promoter, which was chosen as a model because it is a target of the Pax2A and Pax2B transcription factors. This effect is completely abolished by co-transfection of p8 in glucagon-producing InRIG9 cells, indicating that p8 binding to PTIP prevents inhibition of the glucagon gene promoter. This was not observed in NIH3T3 fibroblasts that do not express glucagon. Finally, expression of p8 enhances the effect of p300 on Pax2A and Pax2B trans-activation of the glucagon gene promoter. These observations suggest that in glucagon-producing cells p8 is a positive cofactor of the activation of the glucagon gene promoter by Pax2A and Pax2B, both by recruiting the p300 cofactor to increase the Pax2A and Pax2B activities and by binding the Pax2-interacting protein PTIP to suppress its inhibition.

Cloning and expression of the rat vacuole membrane protein 1 (VMP1), a new gene activated in pancreas with acute pancreatitis, which promotes vacuole formation.

To characterize the emergency program set up by pancreatic cells in response to pancreatitis, we established the phenotype of the pancreatitis-affected pancreas by characterizing a large number of its transcripts. In this report, we describe the cloning, sequencing, and expression pattern of a new gene, named VMP1 (vacuole membrane protein 1). The VMP1 mRNA codes for a putative protein of 406 amino acids. In situ hybridization studies revealed that pancreatic expression of VMP1 mRNAs was restricted to the acinar cells. Interestingly, VMP1 mRNA was also overexpressed in kidney after transient ischemic injury. However, many healthy tissues express VMP1 mRNA. Structure analysis suggested that VMP1 is a transmembrane protein with six hydrophobic regions. VMP1/EGFP fusion protein was located to the Golgi apparatus and the endoplasmic reticulum area. Expression of this protein promoted the formation of intracytoplasmatic vacuoles and VMP1/EGFP was located to the membranes of these vacuoles. Cells overexpressing this protein died after 48 h. In conclusion, we have identified a new stress-induced gene which codes for a transmembrane protein that, when overexpressed, promotes formation of intracellular vacuoles followed by cell death.