Healing journey: Experiences of First Nations individuals with recovery from opioid use.

OBJECTIVE: To understand experiences of recovery from opioid use among First Nations individuals living in a small remote community. DESIGN: Qualitative phenomenologic study. SETTING: Northwestern Ontario. PARTICIPANTS: Sixteen First Nations individuals living in a remote community who had participated in or completed the community opioid agonist therapy program. METHODS: Extensive community consultation took place to ensure local acceptance of the study and permission for publication. Semistructured telephone interviews with consenting participants were audiorecorded between November and December 2021 and transcribed. Transcripts were reviewed and discussed in meetings with Indigenous and non-Indigenous research team members who conducted thematic analysis using immersion and crystallization. MAIN FINDINGS: Participants described their opioid use as a form of self-management of trauma. Their recovery processes were multifaceted and included developing cultural and self-awareness. Motivation for change often arose from concerns about family well-being and finances. Traditional cultural practices and time spent on the land were identified as important wellness experiences. Barriers to healing included limited clinical and holistic addiction services, particularly around dose weaning and opioid agonist therapy discontinuation. CONCLUSION: Community-based addiction programming for First Nations patients needs to be robust. It requires resources for trauma-informed clinical and addiction care, culturally appropriate addictions education, aftercare support, and land-based activities.

Molecular Epidemiology of Mycobacterium tuberculosis To Describe the Transmission Dynamics Among Inuit Residing in Iqaluit Nunavut Using Whole-Genome Sequencing.

BACKGROUND: In the last decade, tuberculosis (TB) incidence among Inuit in the Canadian Arctic has been rising. Our aim was to better understand the transmission dynamics of TB in this remote region of Canada using whole-genome sequencing. METHODS: Isolates from patients who had culture-positive pulmonary TB in Iqaluit, Nunavut, between 2009 and 2015 underwent whole-genome sequencing (WGS). The number of transmission events between cases within clusters was calculated using a threshold of a ≤3 single nucleotide polymorphism (SNP) difference between isolates and then combined with detailed epidemiological data using a reproducible novel algorithm. Social network analysis of epidemiological data was used to support the WGS data analysis. RESULTS: During the study period, 140 Mycobacterium tuberculosis isolates from 135 cases were sequenced. Four clusters were identified, all from Euro-American lineage. One cluster represented 62% of all cases that were sequenced over the entire study period. In this cluster, 2 large chains of transmission were associated with 3 superspreading events in a homeless shelter. One of the superspreading events was linked to a nonsanctioned gambling house that resulted in further transmission. Shelter to nonshelter transmission was also confirmed. An algorithm developed for the determination of transmission events demonstrated very good reproducibility (κ score .98, 95% confidence interval, .97-1.0). CONCLUSIONS: Our study suggests that socioeconomic factors, namely residing in a homeless shelter and spending time in a gambling house, combined with the superspreading event effect may have been significant factors explaining the rise in cases in this predominantly Inuit Arctic community.

The Legionella pneumophila collagen-like protein mediates sedimentation, autoaggregation, and pathogen-phagocyte interactions.

Although only partially understood, multicellular behavior is relatively common in bacterial pathogens. Bacterial aggregates can resist various host defenses and colonize their environment more efficiently than planktonic cells. For the waterborne pathogen Legionella pneumophila, little is known about the roles of autoaggregation or the parameters which allow cell-cell interactions to occur. Here, we determined the endogenous and exogenous factors sufficient to allow autoaggregation to take place in L. pneumophila. We show that isolates from Legionella species which do not produce the Legionella collagen-like protein (Lcl) are deficient in autoaggregation. Targeted deletion of the Lcl-encoding gene (lpg2644) and the addition of Lcl ligands impair the autoaggregation of L. pneumophila. In addition, Lcl-induced autoaggregation requires divalent cations. Escherichia coli producing surface-exposed Lcl is able to autoaggregate and shows increased biofilm production. We also demonstrate that L. pneumophila infection of Acanthamoeba castellanii and Hartmanella vermiformis is potentiated under conditions which promote Lcl dependent autoaggregation. Overall, this study shows that L. pneumophila is capable of autoaggregating in a process that is mediated by Lcl in a divalent-cation-dependent manner. It also reveals that Lcl potentiates the ability of L. pneumophila to come in contact, attach, and infect amoebae.

Mechanism of invasion of lung epithelial cells by filamentous Legionella pneumophila.

Legionella, the aetiological agent responsible for Legionellosis, is an opportunistic pathogen that infects humans upon the inhalation of contaminated aerosolized water droplets. Legionella is pleomorphic and its different morphotypes exhibit varying degrees of virulence. While the filamentous forms of Legionella pneumophila (Lp) have been reported in patient samples since the first description of legionellosis, their role in disease has not been studied. Our results show that both E-cadherin and ß1 integrin receptors mediate filamentous Lp (FLp) attachment to lung epithelial cells (LECs). The activation of these receptors induces the formation of actin enriched membrane surface structures that we designated 'hooks' and 'membrane wraps'. These structures entrap the filaments on the cell surface leading to their gradual internalization through a zipper mechanism of phagocytosis dependent on actomyosin activity. The supply of E-cadherin receptors from the recycling pathway and ß1 integrins released from focal adhesion turnover are required to sustain this process. Intracellular FLp inhabits a vacuolar compartment where filaments differentiate into short rods and replicate to produce infective progeny. Here we are reporting a first description of the invasion mechanism used by FLp to invade LECs. Therefore, filamentous morphotype of Lp can induce its own uptake by LECs and has the potential ability to cause disease.

Biofilms: the stronghold of Legionella pneumophila.

Legionellosis is mostly caused by Legionella pneumophila and is defined as a severe respiratory illness with a case fatality rate ranging from 5% to 80%. L. pneumophila is ubiquitous in natural and anthropogenic water systems. L. pneumophila is transmitted by inhalation of contaminated aerosols produced by a variety of devices. While L. pneumophila replicates within environmental protozoa, colonization and persistence in its natural environment are also mediated by biofilm formation and colonization within multispecies microbial communities. There is now evidence that some legionellosis outbreaks are correlated with the presence of biofilms. Thus, preventing biofilm formation appears as one of the strategies to reduce water system contamination. However, we lack information about the chemical and biophysical conditions, as well as the molecular mechanisms that allow the production of biofilms by L. pneumophila. Here, we discuss the molecular basis of biofilm formation by L. pneumophila and the roles of other microbial species in L. pneumophila biofilm colonization. In addition, we discuss the protective roles of biofilms against current L. pneumophila sanitation strategies along with the initial data available on the regulation of L. pneumophila biofilm formation.

Diagnostic and public health investigation of Mycobacterium tuberculosis infection in a dog in Ontario, Canada.

A 4-y-old, female mixed-breed dog was presented to the Ontario Veterinary College for further evaluation of multiple pulmonary and hepatic masses, intrathoracic lymphadenitis, and recent development of a pyogranulomatous pleural effusion. Along with other comprehensive tests, a thoracic lymph node biopsy was performed, and Mycobacterium tuberculosis complex infection was confirmed by real-time PCR. The dog's condition declined post-operatively, and euthanasia was elected. Postmortem examination confirmed severe granulomatous pneumonia, hepatitis, intrathoracic and intraabdominal lymphadenitis, omentitis, and nephritis. Line-probe assays performed on samples collected postmortem confirmed the species as M. tuberculosis. 24-loci MIRU-VNTR genotyping, spoligotyping, and whole-genome sequencing revealed relations to known human isolates, but no epidemiologic link to these cases was investigated. Given the concern for potential human exposure during this animal's disease course, a public health investigation was initiated; 45 individuals were tested for M. tuberculosis exposure, and no subsequent human infections related to this animal were identified. Our case highlights the need for more readily available, minimally invasive testing for the diagnosis of canine mycobacteriosis, and highlights the ability of canid species to act as potential contributors to the epidemiology of M. tuberculosis infections.

Lcl of Legionella pneumophila is an immunogenic GAG binding adhesin that promotes interactions with lung epithelial cells and plays a crucial role in biofilm formation.

Legionellosis is mostly caused by Legionella pneumophila and is defined by a severe respiratory illness with a case fatality rate ranging from 5 to 80%. In vitro and in vivo, interactions of L. pneumophila with lung epithelial cells are mediated by the sulfated glycosaminoglycans (GAGs) of the host extracellular matrix. In this study, we have identified several Legionella heparin binding proteins. We have shown that one of these proteins, designated Lcl, is a polymorphic adhesin of L. pneumophila that is produced during legionellosis. Homologues of Lcl are ubiquitous in L. pneumophila serogroups but are undetected in other Legionella species. Recombinant Lcl binds to GAGs, and a Δlpg2644 mutant demonstrated reduced binding to GAGs and human lung epithelial cells. Importantly, we showed that the Δlpg2644 strain is dramatically impaired in biofilm formation. These data delineate the role of Lcl in the GAG binding properties of L. pneumophila and provide molecular evidence regarding its role in L. pneumophila adherence and biofilm formation.

New endemic Legionella pneumophila serogroup I clones, Ontario, Canada.

The water-borne pathogen Legionella pneumophila serogroup 1 (Lp1) is the most commonly reported etiologic agent of legionellosis. To examine the genetic diversity, the long-term epidemiology, and the molecular evolution of Lp1 clinical isolates, we conducted sequence-based typing on a collection of clinical isolates representing 3 decades of culture-confirmed legionellosis in Ontario, Canada. Analysis showed that the population of Lp1 in Ontario is highly diverse and combines lineages identified worldwide with local strains. Identical types were identified in sporadic and outbreak-associated strains. In the past 15 years, the incidence of some lineages distributed worldwide has tended to decrease, and local endemic clones and lineages have emerged. Comparative geographic distribution analysis suggests that some lineages are specific to eastern North America. These findings have general clinical implications for the study of Lp1 molecular evolution and for the identification of Lp1 circulating strains in North America.

Polymorphisms of a Collagen-Like Adhesin Contributes to Legionella pneumophila Adhesion, Biofilm Formation Capacity and Clinical Prevalence.

Legionellosis is a severe respiratory illness caused by the inhalation of aerosolized water droplets contaminated with the opportunistic pathogen Legionella pneumophila. The ability of L. pneumophila to produce biofilms has been associated with its capacity to colonize and persist in human-made water reservoirs and distribution systems, which are the source of legionellosis outbreaks. Nevertheless, the factors that mediate L. pneumophila biofilm formation are largely unknown. In previous studies we reported that the adhesin Legionella collagen-like protein (Lcl), is required for auto-aggregation, attachment to multiple surfaces and the formation of biofilms. Lcl structure contains three distinguishable regions: An N-terminal region with a predicted signal sequence, a central region containing tandem collagen-like repeats (R-domain) and a C-terminal region (C-domain) with no significant homology to other known proteins. Lcl R-domain encodes tandem repeats of the collagenous tripeptide Gly-Xaa-Yaa (GXY), a motif that is key for the molecular organization of mammalian collagen and mediates the binding of collagenous proteins to different cellular and environmental ligands. Interestingly, Lcl is polymorphic in the number of GXY tandem repeats. In this study, we combined diverse biochemical, genetic, and cellular approaches to determine the role of Lcl domains and GXY repeats polymorphisms on the structural and functional properties of Lcl, as well as on bacterial attachment, aggregation and biofilm formation. Our results indicate that the R-domain is key for assembling Lcl collagenous triple-helices and has a more preponderate role over the C-domain in Lcl adhesin binding properties. We show that Lcl molecules oligomerize to form large supramolecular complexes to which both, R and C-domains are required. Furthermore, we found that the number of GXY tandem repeats encoded in Lcl R-domain correlates positively with the binding capabilities of Lcl and with the attachment and biofilm production capacity of L. pneumophila strains. Accordingly, the number of GXY tandem repeats in Lcl influences the clinical prevalence of L. pneumophila strains. Therefore, the number of Lcl tandem repeats could be considered as a potential predictor for virulence in L. pneumophila isolates.

Whole transcriptomic and proteomic analyses of an isogenic M. tuberculosis clinical strain with a naturally occurring 15 Kb genomic deletion.

Tuberculosis remains one of the most difficult to control infectious diseases in the world. Many different factors contribute to the complexity of this disease. These include the ability of the host to control the infection which may directly relate to nutritional status, presence of co-morbidities and genetic predisposition. Pathogen factors, in particular the ability of different Mycobacterium tuberculosis strains to respond to the harsh environment of the host granuloma, which includes low oxygen and nutrient availability and the presence of damaging radical oxygen and nitrogen species, also play an important role in the success of different strains to cause disease. In this study we evaluated the impact of a naturally occurring 12 gene 15 Kb genomic deletion on the physiology and virulence of M. tuberculosis. The strains denominated ON-A WT (wild type) and ON-A NM (natural mutant) were isolated from a previously reported TB outbreak in an inner city under-housed population in Toronto, Canada. Here we subjected these isogenic strains to transcriptomic (via RNA-seq) and proteomic analyses and identified several gene clusters with differential expression in the natural mutant, including the DosR regulon and the molybdenum cofactor biosynthesis genes, both of which were found in lower abundance in the natural mutant. We also demonstrated lesser virulence of the natural mutant in the guinea pig animal model. Overall, our findings suggest that the ON-A natural mutant is less fit to cause disease, but nevertheless has the potential to cause extended transmission in at-risk populations.

Preliminary evaluation of exome sequencing to identify genetic markers of susceptibility to tuberculosis disease.

BACKGROUND: Recent studies have shown that certain human genetic polymorphisms could be associated with susceptibility to tuberculosis (TB) infection and disease. Advances in next generation sequencing include the ability to rapidly sequence the entire human exome. These new technologies can be exploited to identify new associations of human genetic polymorphisms and TB infection and disease. In this preliminary study we compared two different strategies for sequencing of the human exome in a small sample set consisting of three individuals with a history of TB disease and two individuals with latent TB infection. FINDINGS: Sequencing of the entire exome of the five participants using Agilent SureSelect kit resulted in the identification of 1611 single nucleotide polymorphisms (SNPs) that were only present in the individuals with a history of active TB but not in the latent TB cases. Alternatively, sequencing of 4000 target genes available in the TruSight kit resulted in identification of 182 SNPs only present in the active TB cases and not in the latent TB participants. The overlap of the two kits was 112 SNPs. CONCLUSIONS: Even though this pilot study was restricted to a small number of participants, we demonstrated the feasibility of using exome sequencing technologies to mine potential genetic associations of susceptibility to TB disease and presented a number of potential targets that can be further explore in larger research trials.

Filamentous morphology of bacteria delays the timing of phagosome morphogenesis in macrophages.

Although filamentous morphology in bacteria has been associated with resistance to phagocytosis, our understanding of the cellular mechanisms behind this process is limited. To investigate this, we followed the phagocytosis of both viable and dead Legionella pneumophila filaments. The engulfment of these targets occurred gradually and along the longitudinal axis of the filament, therefore defining a long-lasting phagocytic cup stage that determined the outcome of phagocytosis. We found that these phagocytic cups fused with endosomes and lysosomes, events linked to the maturation of phagosomes according to the canonical pathway, and not with the remodeling of phagocytic cups. Nevertheless, despite acquiring phagolysosomal features these phagocytic cups failed to develop hydrolytic capacity before their sealing. This phenomenon hampered the microbicidal activity of the macrophage and enhanced the capacity of viable filamentous L. pneumophila to escape phagosomal killing in a length-dependent manner. Our results demonstrate that key aspects in phagocytic cup remodeling and phagosomal maturation could be influenced by target morphology.

Comparative Genomics Reveal That Host-Innate Immune Responses Influence the Clinical Prevalence of Legionella pneumophila Serogroups.

Legionella pneumophila is the primary etiologic agent of legionellosis, a potentially fatal respiratory illness. Amongst the sixteen described L. pneumophila serogroups, a majority of the clinical infections diagnosed using standard methods are serogroup 1 (Sg1). This high clinical prevalence of Sg1 is hypothesized to be linked to environmental specific advantages and/or to increased virulence of strains belonging to Sg1. The genetic determinants for this prevalence remain unknown primarily due to the limited genomic information available for non-Sg1 clinical strains. Through a systematic attempt to culture Legionella from patient respiratory samples, we have previously reported that 34% of all culture confirmed legionellosis cases in Ontario (n = 351) are caused by non-Sg1 Legionella. Phylogenetic analysis combining multiple-locus variable number tandem repeat analysis and sequence based typing profiles of all non-Sg1 identified that L. pneumophila clinical strains (n = 73) belonging to the two most prevalent molecular types were Sg6. We conducted whole genome sequencing of two strains representative of these sequence types and one distant neighbour. Comparative genomics of the three L. pneumophila Sg6 genomes reported here with published L. pneumophila serogroup 1 genomes identified genetic differences in the O-antigen biosynthetic cluster. Comparative optical mapping analysis between Sg6 and Sg1 further corroborated this finding. We confirmed an altered O-antigen profile of Sg6, and tested its possible effects on growth and replication in in vitro biological models and experimental murine infections. Our data indicates that while clinical Sg1 might not be better suited than Sg6 in colonizing environmental niches, increased bloodstream dissemination through resistance to the alternative pathway of complement mediated killing in the human host may explain its higher prevalence.

Essential roles and regulation of the Legionella pneumophila collagen-like adhesin during biofilm formation.

Legionellosis is mostly caused by Legionella pneumophila (Lp) and is defined by a severe respiratory illness with a case fatality rate ranging from 5 to 80%. In a previous study, we showed that a glycosaminoglycan (GAG)-binding adhesin of Lp, named Lcl, is produced during legionellosis and is unique to the L. pneumophila species. Importantly, a mutant depleted in Lcl (Δlpg2644) is impaired in adhesion to GAGs and epithelial cells and in biofilm formation. Here, we examine the molecular function(s) of Lcl and the transcriptional regulation of its encoding gene during different stages of the biofilm development. We show that the collagen repeats and the C-terminal domains of Lcl are crucial for the production of biofilm. We present evidence that Lcl is involved in the early step of surface attachment but also in intercellular interactions. Furthermore, we address the relationship between Lcl gene regulation during biofilm formation and quorum sensing (QS). In a static biofilm assay, we show that Lcl is differentially regulated during growth phases and biofilm formation. Moreover, we show that the transcriptional regulation of lpg2644, mediated by a prototype of QS signaling homoserine lactone (3OC12-HSL), may play a role during the biofilm development. Thus, transcriptional down-regulation of lpg2644 may facilitate the dispersion of Lp to reinitiate biofilm colonization on a distal surface.

Analytical and clinical validation of novel real-time reverse transcriptase-polymerase chain reaction assays for the clinical detection of swine-origin H1N1 influenza viruses.

During the early stages of the 2009/2010 swine-origin H1N1 influenza A (S-OIV H1N1 FluA) outbreak, the development and validation of sensitive and specific detection methods were a priority for rapid and accurate diagnosis. Between May and June 2009, 2 real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assays targeting the hemagglutinin and neuraminidase genes of the S-OIV H1N1 FluA virus were developed. These assays are highly specific, showing no cross-reactivity against a panel of respiratory viruses and can differentiate S-OIV H1N1 from seasonal FluA viruses. Analytical sensitivities of the 2 assays were found to be 10(-1) tissue culture infectious dose, 50%/ml. Clinical testing showed 99.2% sensitivity and 94.6-98.1% specificity. A large prospective analysis showed that 94.8-95.5% of S-OIV positive specimens were negative by seasonal H1/H3 subtyping. The large-scale validation data presented in this report indicate that these novel assays provide an accurate and efficient method for the rapid detection of S-OIV H1N1 FluA viruses.

Prevalence and characterization of invasive isolates of Streptococcus pyogenes with reduced susceptibility to fluoroquinolones.

Fluoroquinolone susceptibility testing was performed on invasive group A streptococcus isolates from 1992-1993 and 2003 from Ontario, Canada. None were nonsusceptible to levofloxacin. Two of 153 (1.3%) from 1992-1993 and 7 of 160 (4.4%) from 2003 had a levofloxacin MIC of 2 mug/ml; all nine had parC mutations, and eight were serotype M6.

Identification of a streptolysin S-associated gene cluster and its role in the pathogenesis of Streptococcus iniae disease.

Streptococcus iniae causes meningoencephalitis and death in cultured fish species and soft-tissue infection in humans. We recently reported that S. iniae is responsible for local tissue necrosis and bacteremia in a murine subcutaneous infection model. The ability to cause bacteremia in this model is associated with a genetic profile unique to strains responsible for disease in fish and humans (J. D. Fuller, D. J. Bast, V. Nizet, D. E. Low, and J. C. S. de Azavedo, Infect. Immun. 69:1994-2000, 2001). S. iniae produces a cytolysin that confers a hemolytic phenotype on blood agar media. In this study, we characterized the genomic region responsible for S. iniae cytolysin production and assessed its contribution to virulence. Transposon (Tn917) mutant libraries of commensal and disease-associated S. iniae strains were generated and screened for loss of hemolytic activity. Analysis of two nonhemolytic mutants identified a chromosomal locus comprising 9 genes with 73% homology to the group A streptococcus (GAS) sag operon for streptolysin S (SLS) biosynthesis. Confirmation that the S. iniae cytolysin is a functional homologue of SLS was achieved by PCR ligation mutagenesis, complementation of an SLS-negative GAS mutant, and use of the SLS inhibitor trypan blue. SLS-negative sagB mutants were compared to their wild-type S. iniae parent strains in the murine model and in human whole-blood killing assays. These studies demonstrated that S. iniae SLS expression is required for local tissue necrosis but does not contribute to the establishment of bacteremia or to resistance to phagocytic clearance.

Type II topoisomerase mutations in Bacillus anthracis associated with high-level fluoroquinolone resistance.

OBJECTIVES: To identify and characterize the mechanisms of high-level fluoroquinolone resistance in two strains of Bacillus anthracis following serial passage in increasing concentrations of fluoroquinolones. METHODS: Fluoroquinolone-resistant isolates of the Sterne and Russian Anthrax Vaccine STi strains were obtained following serial passage in the presence of increasing concentrations of four different fluoroquinolones. The quinolone-resistance-determining regions of the type II topoisomerase genes from the resistant strains were amplified by PCR and characterized by DNA sequence analysis. The MICs in the presence and absence of reserpine were determined using broth microdilution as a means of detecting active efflux. RESULTS: Single and double amino acid substitutions in the GyrA (Ser-85-Leu; Glu-89-Arg/Gly/Lys) and GrlA (Ser-81-Tyr; Val-96-Ala; Asn-70-Lys) were most common. A single amino acid substitution in GyrB (Asp-430-Asn) was also identified. Efflux only applied to isolates selected for by either levofloxacin or ofloxacin. CONCLUSIONS: Specific amino acid substitutions in the type II topoisomerase enzymes significantly contributed to the development of high-level fluoroquinolone resistance in B. anthracis. However, notable differences between the strains and the drugs tested were identified including the role of efflux and the numbers and types of mutations identified.

Emergence of macrolide resistance in throat culture isolates of group a streptococci in Ontario, Canada, in 2001.

Of 500 group A streptococci isolated from pharyngeal swabs, 72 (14.4%) were macrolide resistant, compared to 2.1% in 1997. Of these, 66 (92%) were of the M phenotype and 6 (8.3%) were of the MLS phenotype. Pulsed-field gel electrophoresis found that two clones, with patterns identical to those of serotypes M1 and M4, accounted for 19.4 and 68.1% of the macrolide-resistant isolates, respectively.