Complex population structure of Borrelia burgdorferi in southeastern and south central Canada as revealed by phylogeographic analysis.

Lyme disease, caused by the bacterium Borrelia burgdorferi sensu stricto, is an emerging zoonotic disease in Canada and is vectored by the blacklegged tick, Ixodes scapularis. Here we used Bayesian analyses of sequence types (STs), determined by multilocus sequence typing (MLST), to investigate the phylogeography of B. burgdorferi populations in southern Canada and the United States by analyzing MLST data from 564 B. burgdorferi-positive samples collected during surveillance. A total of 107 Canadian samples from field sites were characterized as part of this study, and these data were combined with existing MLST data for samples from the United States and Canada. Only 17% of STs were common between both countries, while 49% occurred only in the United States, and 34% occurred only in Canada. However, STs in southeastern Ontario and southwestern Quebec were typically identical to those in the northeastern United States, suggesting a recent introduction into this region from the United States. In contrast, STs in other locations in Canada (the Maritimes; Long Point, Ontario; and southeastern Manitoba) were frequently unique to those locations but were putative descendants of STs previously found in the United States. The picture in Canada is consistent with relatively recent introductions from multiple refugial populations in the United States. These data thus point to a geographic pattern of populations of B. burgdorferi in North America that may be more complex than simply comprising northeastern, midwestern, and Californian groups. We speculate that this reflects the complex ecology and spatial distribution of key reservoir hosts.

Evolutionary aspects of emerging Lyme disease in Canada.

In North America, Lyme disease (LD) is a tick-borne zoonosis caused by the spirochete bacterium Borrelia burgdorferi sensu stricto, which is maintained by wildlife. Tick vectors and bacteria are currently spreading into Canada and causing increasing numbers of cases of LD in humans and raising a pressing need for public health responses. There is no vaccine, and LD prevention depends on knowing who is at risk and informing them how to protect themselves from infection. Recently, it was found in the United States that some strains of B. burgdorferi sensu stricto cause severe disease, whereas others cause mild, self-limiting disease. While many strains occurring in the United States also occur in Canada, strains in some parts of Canada are different from those in the United States. We therefore recognize a need to identify which strains specific to Canada can cause severe disease and to characterize their geographic distribution to determine which Canadians are particularly at risk. In this review, we summarize the history of emergence of LD in North America, our current knowledge of B. burgdorferi sensu stricto diversity, its intriguing origins in the ecology and evolution of the bacterium, and its importance for the epidemiology and clinical and laboratory diagnosis of LD. We propose methods for investigating associations between B. burgdorferi sensu stricto diversity, ecology, and pathogenicity and for developing predictive tools to guide public health interventions. We also highlight the emergence of B. burgdorferi sensu stricto in Canada as a unique opportunity for exploring the evolutionary aspects of tick-borne pathogen emergence.

Investigation of the population structure of the tick vector of Lyme disease Ixodes scapularis (Acari: Ixodidae) in Canada using mitochondrial cytochrome C oxidase subunit I gene sequences.

Genotyping of Ixodes scapularis Say (Acari: Ixodidae) ticks could enhance understanding of the occurrence and genotypes of I. scapularis-borne pathogens. We investigated the utility of mitochondrial (mt) Cytochrome C Oxidase subunit I gene (cox1) sequences as a tool for understanding the population structure of I. scapularis collected in Canada, where we also investigated the geographic occurrence of different cox1 haplotypes. Sequences obtained from 414 ticks were one of 55 unique haplotypes, most of which grouped into one of six clades. Demographic analysis suggested that cox1 sequences have haplotype and nucleotide diversity comparable to other mt genes. All haplotypes were connected in a single minimum spanning network tree. Despite low fixation index values there were significant differences in the frequency of occurrence of haplotypes of different clades among four geographic regions: 1) Alberta to western Ontario, 2) eastern Ontario, 3) Quebec, and 4) Atlantic Provinces; suggesting that cox1 sequences could reveal population structure differences between I. scapularis in geographically separated populations of northeastern and midwestern North America. Spatial clusters of ticks of the same haplotype identified in regions of southern Quebec and southern Ontario where I. scapularis is invading were consistent with population bottlenecks associated with founder events. These findings suggest that cox1 sequences are useful for the study of I. scapularis population structure, are of sufficient diversity that spatial analyses of haplotypes can be used to identify where I. scapularis is emerging in southern Canada, and may be useful for exploring differences between northeastern and midwestern populations of I. scapularis.

Combining Genome Surveillance and Metadata To Characterize the Diversity of Staphylococcus aureus Circulating in an Italian Hospital over a 9-Year Period.

Staphylococcus aureus is an opportunistic pathogen and a leading cause of morbidity and mortality worldwide. Genomic-based surveillance has greatly improved our ability to track the emergence and spread of high-risk clones, but the full potential of genomic data is only reached when used in conjunction with detailed metadata. Here, we demonstrate the utility of an integrated approach by leveraging a curated collection of clinical and epidemiological metadata of S. aureus in the San Matteo Hospital (Italy) through a semisupervised clustering strategy. We sequenced 226 sepsis S. aureus samples, recovered over a period of 9 years. By using existing antibiotic profiling data, we selected strains that capture the full diversity of the population. Genome analysis revealed 49 sequence types, 16 of which are novel. Comparative genomic analyses of hospital- and community-acquired infection ruled out the existence of genomic features differentiating them, while evolutionary analyses of genes and traits of interest highlighted different dynamics of acquisition and loss between antibiotic resistance and virulence genes. Finally, highly resistant clones belonging to clonal complexes (CC) 8 and 22 were found to be responsible for abundant infections and deaths, while the highly virulent CC30 was responsible for rare but deadly episodes of infections. IMPORTANCE Genome sequencing is an important tool in clinical microbiology, as it allows in-depth characterization of isolates of interest and can propel genome-based surveillance studies. Such studies can benefit from ad hoc methods of sample selection to capture the genomic diversity present in a data set. Here, we present an approach based on clustering of antibiotic resistance profiles that allows optimal sample selection for bacterial genomic surveillance. We apply the method to a 9-year collection of Staphylococcus aureus from a large hospital in northern Italy. Our method allows us to sequence the genomes of a large variety of strains of this important pathogen, which we then leverage to characterize the epidemiology in the hospital and to perform evolutionary analyses on genes and traits of interest. These analyses highlight different dynamics of acquisition and loss between antibiotic resistance and virulence genes.

Wastewater-based epidemiology in hazard forecasting and early-warning systems for global health risks.

With the advent of the SARS-CoV-2 pandemic, Wastewater-Based Epidemiology (WBE) has been applied to track community infection in cities worldwide and has proven succesful as an early warning system for identification of hotspots and changingprevalence of infections (both symptomatic and asymptomatic) at a city or sub-city level. Wastewater is only one of environmental compartments that requires consideration. In this manuscript, we have critically evaluated the knowledge-base and preparedness for building early warning systems in a rapidly urbanising world, with particular attention to Africa, which experiences rapid population growth and urbanisation. We have proposed a Digital Urban Environment Fingerprinting Platform (DUEF) - a new approach in hazard forecasting and early-warning systems for global health risks and an extension to the existing concept of smart cities. The urban environment (especially wastewater) contains a complex mixture of substances including toxic chemicals, infectious biological agents and human excretion products. DUEF assumes that these specific endo- and exogenous residues, anonymously pooled by communities' wastewater, are indicative of community-wide exposure and the resulting effects. DUEF postulates that the measurement of the substances continuously and anonymously pooled by the receiving environment (sewage, surface water, soils and air), can provide near real-time dynamic information about the quantity and type of physical, biological or chemical stressors to which the surveyed systems are exposed, and can create a risk profile on the potential effects of these exposures. Successful development and utilisation of a DUEF globally requires a tiered approach including: Stage I: network building, capacity building, stakeholder engagement as well as a conceptual model, followed by Stage II: DUEF development, Stage III: implementation, and Stage IV: management and utilization. We have identified four key pillars required for the establishment of a DUEF framework: (1) Environmental fingerprints, (2) Socioeconomic fingerprints, (3) Statistics and modelling and (4) Information systems. This manuscript critically evaluates the current knowledge base within each pillar and provides recommendations for further developments with an aim of laying grounds for successful development of global DUEF platforms.

Investigation of genotypes of Borrelia burgdorferi in Ixodes scapularis ticks collected during surveillance in Canada.

The genetic diversity of Borrelia burgdorferi sensu stricto, the agent of Lyme disease in North America, has consequences for the performance of serological diagnostic tests and disease severity. To investigate B. burgdorferi diversity in Canada, where Lyme disease is emerging, bacterial DNA in 309 infected adult Ixodes scapularis ticks collected in surveillance was characterized by multilocus sequence typing (MLST) and analysis of outer surface protein C gene (ospC) alleles. Six ticks carried Borrelia miyamotoi, and one tick carried the novel species Borrelia kurtenbachii. 142 ticks carried B. burgdorferi sequence types (STs) previously described from the United States. Fifty-eight ticks carried B. burgdorferi of 1 of 19 novel or undescribed STs, which were single-, double-, or triple-locus variants of STs first described in the United States. Clonal complexes with founder STs from the United States were identified. Seventeen ospC alleles were identified in 309 B. burgdorferi-infected ticks. Positive and negative associations in the occurrence of different alleles in the same tick supported a hypothesis of multiple-niche polymorphism for B. burgdorferi in North America. Geographic analysis of STs and ospC alleles were consistent with south-to-north dispersion of infected ticks from U.S. sources on migratory birds. These observations suggest that the genetic diversity of B. burgdorferi in eastern and central Canada corresponds to that in the United States, but there was evidence for founder events skewing the diversity in emerging tick populations. Further studies are needed to investigate the significance of these observations for the performance of diagnostic tests and clinical presentation of Lyme disease in Canada.

A link between virulence and ecological abundance in natural populations of Staphylococcus aureus.

Staphylococcus aureus is a major cause of severe infection in humans and yet is carried without symptoms by a large proportion of the population. We used multilocus sequence typing to characterize isolates of S. aureus recovered from asymptomatic nasal carriage and from episodes of severe disease within a defined population. We identified a number of frequently carried genotypes that were disproportionately common as causes of disease, even taking into account their relative abundance among carriage isolates. The existence of these ecologically abundant hypervirulent clones suggests that factors promoting the ecological fitness of this important pathogen also increase its virulence.

Predominance of the Hungarian clone (ST 239-III) among hospital-acquired meticillin-resistant Staphylococcus aureus isolates recovered throughout mainland China.

Multilocus sequence typing (MLST) data have revealed many insights into the global epidemiology of Staphylococcus aureus but, with notable exceptions such as Japan, the evidence from most Asian countries is currently limited. Here we have applied MLST to 132 hospital-acquired meticillin-resistant Staphylococcus aureus (HA-MRSA) isolates collected in mainland China in 2002. In all, 102 isolates were recovered from a single tertiary hospital in Guangzhou, South China, and the remaining 30 isolates were recovered from six metropolitan tertiary hospitals from geographically diverse districts corresponding to a total area of more than 2 million km2. The data reveal a striking predominance throughout mainland China of a single clonal lineage, ST 239, which accounts for 97% of the 132 isolates. These data support more limited evidence from previous studies suggesting the widespread predominance of ST 239 throughout hospitals in China, a pattern which possibly extends to the whole of continental Asia. Staphylococcal chromosome cassette mec (SCCmec) typing confirmed the homogeneity of the ST 239 isolates, with the vast majority corresponding to the Hungarian clone (ST 239-III).

Guidelines for the validation and application of typing methods for use in bacterial epidemiology.

For bacterial typing to be useful, the development, validation and appropriate application of typing methods must follow unified criteria. Over a decade ago, ESGEM, the ESCMID (Europen Society for Clinical Microbiology and Infectious Diseases) Study Group on Epidemiological Markers, produced guidelines for optimal use and quality assessment of the then most frequently used typing procedures. We present here an update of these guidelines, taking into account the spectacular increase in the number and quality of typing methods made available over the past decade. Newer and older, phenotypic and genotypic methods for typing of all clinically relevant bacterial species are described according to their principles, advantages and disadvantages. Criteria for their evaluation and application and the interpretation of their results are proposed. Finally, the issues of reporting, standardisation, quality assessment and international networks are discussed. It must be emphasised that typing results can never stand alone and need to be interpreted in the context of all available epidemiological, clinical and demographical data relating to the infectious disease under investigation. A strategic effort on the part of all workers in the field is thus mandatory to combat emerging infectious diseases, as is financial support from national and international granting bodies and health authorities.

The relative contributions of recombination and point mutation to the diversification of bacterial clones.

Low levels of recombination in bacterial species have often been inferred from the presence of linkage disequilibrium between the alleles at different loci in the population. However, significant linkage disequilibrium is inevitable in organisms that divide by binary fission, and recombinational replacements must be very frequent, compared to point mutation, to dissipate disequilibrium. Recent studies using data from multilocus sequence typing indicate that, in many species, recombinational replacements contribute more greatly to clonal diversification than do point mutations and, in some species, recombination has been sufficient to eliminate any phylogenetic signal from gene trees. Recent efforts to improve understanding of the extent and impact of homologous recombination in the diversification of bacterial clones are discussed.

Estimating recombinational parameters in Streptococcus pneumoniae from multilocus sequence typing data.

Multilocus sequence typing (MLST) is a highly discriminatory molecular typing method that defines isolates of bacterial pathogens using the sequences of approximately 450-bp internal fragments of seven housekeeping genes. This technique has been applied to 575 isolates of Streptococcus pneumoniae and identifies a number of discrete clonal complexes. These clonal complexes are typically represented by a single group of isolates sharing identical alleles at all seven loci, plus single-locus variants that differ from this group at only one out of the seven loci. As MLST is highly discriminatory, the members of each clonal complex can be assumed to have a recent common ancestor, and the molecular events that give rise to the single-locus variants can be used to estimate the relative contributions of recombination and mutation to clonal divergence. By comparing the sequences of the variant alleles within each clonal complex with the allele typically found within that clonal complex, we estimate that recombination has generated new alleles at a frequency approximately 10-fold higher than mutation, and that a single nucleotide site is approximately 50 times more likely to change through recombination than mutation. We also demonstrate how to estimate the average length of recombinational replacements from MLST data.

Estimating the relative contributions of mutation and recombination to clonal diversification: a comparison between Neisseria meningitidis and Streptococcus pneumoniae.

Both Neisseria meningitidis and Streptococcus pneumoniae are naturally transformable species and are known to be freely recombining in the wild. Large multilocus sequence typing (MLST) datasets have been generated for these species. Here we outline an approach which exploits these data sets in order to quantify the extent of recombination, thus enabling meaningful comparisons between the two species. Two parameters are estimated; the rate at which recombination changes alleles, compared to point mutation, and the rate at which recombination changes individual nucleotide sites, compared to point mutation. Estimates for the former parameter are 4:1 in the meningococcus (i.e. alleles are changed four-fold more frequently by recombination than by mutation), and 10:1 in the pneumococcus. However, estimates for the latter parameter are at least 80:1 in the meningococcus (i.e. an individual nucleotide site is at least 80-fold more likely to change by recombination than by mutation) and 50:1 in the pneumococcus. These data imply that recombination events, compared to mutational events, may be more common in the pneumococcus than in the meningococcus. However, because it is a more diverse species, each recombinational exchange in the meningococcus results in more nucleotide changes on average.

Who participates in Internet-based self-management programs? A study among novice computer users in a primary care setting.

PURPOSE: The purpose of this study was to evaluate the participation rates and factors associated with nonparticipation among primary care patients who were invited to join an Internet-based self-management research program. METHODS: Primary care providers invited their patients with type 2 diabetes to participate in an Internet-based diabetes self-management support program. Research staff contacted these patients by phone to assess their eligibility and interest in participating. Reasons for declining were assessed and demographic/medical status information was collected. RESULTS: Of the eligible patients, 60% participated in the program. No significant differences were found between participants and decliners in gender, insulin use, computer familiarity, or computer ownership. There were significant differences in age and years since diagnosis. Participants were slightly younger and had diabetes for a fewer number of years than nonparticipants. Nonparticipation was not related to computer or Internet issues. CONCLUSIONS: Most older diabetes patients without previous Internet experience will take part in Internet-based self-management support programs if barriers to participation are addressed.

Feasibility and use of an Internet support service for diabetes self-management.

Diabetes self-management is a challenging task due to its complexity and ever-present barriers to self-care. A key issue for patients, providers, and healthcare systems is how to deliver personalized behavioral support in ways that are affordable and can reach most patients. The Internet offers several advantages in this regard because it is available 24 hours a day, low cost, and capable of reaching thousands of patients. Although Internet sites for diabetes support are popular, they have not been evaluated. For this study, a Web site for diabetes self-management that emphasized personalized goal setting, feedback, and social support was developed. Over a 10-week period, 111 different persons logged onto D-NET for a total of 21,046 accesses. Users included persons across a broad age range (up to age 77 years) and duration of diabetes. The most popular areas of the site were the Social Support Conference and the Diabetes Information Pages. User ratings revealed high satisfaction with the service. The Internet has great potential for providing needed support and services for patients with diabetes and other chronic illnesses.

Bridging the gap between science and practice in managing low back pain. A comprehensive spine care system in a health maintenance organization setting.

STUDY DESIGN: A case study of spine care system changes in a multispecialty group practice health maintenance organization setting. OBJECTIVES: To reduce unnecessary use of imaging and specialty referrals for low back pain in the primary care setting and to reduce spine surgery rates. SUMMARY OF BACKGROUND DATA: Results of previous research indicate that diagnostic and therapeutic procedures for low back pain are frequently used even though there is no scientific evidence of their efficacy. This indicates that low back pain care can be made more efficient by reducing the use of unproven diagnostic and therapeutic interventions for low back pain. METHODS: Rates of diagnostic imaging and specialty referral rates for low back pain were monitored for 9 months before and 9 months after primary care physician education regarding appropriate low back pain evaluation and management. Spine surgery rates were also monitored before and after implementation of a nonsurgical spine clinic. RESULTS: Large reductions in rates of imaging and specialty referrals for low back pain were achieved after primary care physician education. After spine clinic implementation, visits to spine surgeons dropped by approximately 50%, and spine surgery rates per thousand health plan members were reduced by 35%. CONCLUSIONS: Primary care physician education regarding low back pain management can reduce use of imaging and specialty referrals without reductions in patient satisfaction, and implementation of a nonsurgical spine clinic for complex or chronic spine patients can significantly reduce spine surgery consultations and spine surgery rates.

Recombination and the population structures of bacterial pathogens.

The population structures of bacterial species are complex and often controversial. To a large extent, this is due to uncertainty about the frequency and impact of recombination in bacteria. The existence of clones within bacterial populations, and of linkage disequilibrium between alleles at different loci, is often cited as evidence for low rates of recombination. However, clones and linkage disequilibrium are almost inevitable in species that divide by binary fission and can be present in populations where recombination is frequent. In recent years, it has become possible to directly compare rates of recombination in different species. These studies indicate that in many bacterial species, including Neisseria meningitidis, Streptococcus pneumoniae, and Staphylococcus aureus, evolutionary change at neutral (housekeeping) loci is more likely to occur by recombination than mutation and can result in the elimination of any deep-rooted phylogenetic signal. In such species, the long-term evolution of the population is dominated by recombination, but this does not occur at a sufficiently high frequency to prevent the emergence of adaptive clones, although these are relatively short-lived and rapidly diversify.