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Lost in plasmids: next generation sequencing and the complex genome of the tick-borne pathogen Borrelia burgdorferi
Background
Borrelia
(
B.
)
burgdorferi
sensu lato, including the tick-transmitted agents of human Lyme borreliosis, have particularly complex genomes, consisting of a linear main chromosome and numerous linear and circular plasmids. The number and structure of plasmids is variable even in strains within a single genospecies. Genes on these plasmids are known to play essential roles in virulence and pathogenicity as well as host and vector associations. For this reason, it is essential to explore methods for rapid and reliable characterisation of molecular level changes on plasmids.
In this study we used three strains: a low passage isolate of
B. burgdorferi
sensu stricto strain B31(−NRZ) and two closely related strains (PAli and PAbe) that were isolated from human patients. Sequences of these strains were compared to the previously sequenced reference strain B31 (available in GenBank) to obtain proof-of-principle information on the suitability of next generation sequencing (NGS) library construction and sequencing methods on the assembly of bacterial plasmids. We tested the effectiveness of different short read assemblers on Illumina sequences, and of long read generation methods on sequence data from Pacific Bioscience single-molecule real-time (SMRT) and nanopore (Oxford Nanopore Technologies) sequencing technology.
Results
Inclusion of mate pair library reads improved the assembly in some plasmids as did prior enrichment of plasmids. While cp32 plasmids remained refractory to assembly using only short reads they were effectively assembled by long read sequencing methods. The long read SMRT and nanopore sequences came, however, at the cost of indels (insertions or deletions) appearing in an unpredictable manner. Using long and short read technologies together allowed us to show that the three
B. burgdorferi
s.s. strains investigated here, whilst having similar plasmid structures to each other (apart from fusion of cp32 plasmids), differed significantly from the reference strain B31-GB, especially in the case of cp32 plasmids.
Conclusion
Short read methods are sufficient to assemble the main chromosome and many of the plasmids in
B. burgdorferi
. However, a combination of short and long read sequencing methods is essential for proper assembly of all plasmids including cp32 and thus, for gaining an understanding of host- or vector adaptations. An important conclusion from our work is that the evolution of
Borrelia
plasmids appears to be dynamic. This has important implications for the development of useful research strategies to monitor the risk of Lyme disease occurrence and how to medically manage it.
Journal Article
I know an old lady who swallowed a fly
Collects the author's folk songs, including \"I Know an Old Lady Who Swallowed a Fly,\" \"Heel, Toe, and Away We Go,\" and \"Sailing Over the Sea.\"
BOOK
Clinical symptoms cannot predict influenza infection during the 2013 influenza season in Bavaria, Germany
For influenza surveillance and diagnosis typical clinical symptoms are traditionally used to discriminate influenza virus infections from infections by other pathogens. During the 2013 influenza season we performed a multiplex assay for 16 different viruses in 665 swabs from patients with acute respiratory infections (ARIs) to display the variety of different pathogens causing ARI and to test the diagnostic value of both the commonly used case definitions [ARI, and influenza like illness (ILI)] as well as the clinical judgement of physicians, respectively, to achieve a laboratory-confirmed influenza diagnosis. Fourteen different viruses were identified as causing ARI/ILI. Influenza diagnosis based on clinical signs overestimated the number of laboratory-confirmed influenza cases and misclassified cases. Furthermore, ILI case definition and physicians agreed in only 287/651 (44%) cases with laboratory confirmation. Influenza case management has to be supported by laboratory confirmation to allow evidence-based decisions. Epidemiological syndromic surveillance data should be supported by laboratory confirmation for reasonable interpretation.
Journal Article
Epidemiology and transmission characteristics of early COVID-19 cases, 20 January–19 March 2020, in Bavaria, Germany
Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) led to a significant disease burden and disruptions in health systems. We describe the epidemiology and transmission characteristics of early coronavirus disease 2019 (COVID-19) cases in Bavaria, Germany. Cases were reverse transcription polymerase chain reaction (RT-PCR)-confirmed SARS-CoV-2 infections, reported from 20 January−19 March 2020. The incubation period was estimated using travel history and date of symptom onset. To estimate the serial interval, we identified pairs of index and secondary cases. By 19 March, 3546 cases were reported. A large proportion was exposed abroad (38%), causing further local transmission. Median incubation period of 256 cases with exposure abroad was 3.8 days (95%CI: 3.5–4.2). For 95% of infected individuals, symptom onset occurred within 10.3 days (95%CI: 9.1–11.8) after exposure. The median serial interval, using 53 pairs, was 3.5 days (95%CI: 3.0–4.2; mean: 3.9, s.d.: 2.2). Travellers returning to Germany had an important influence on the spread of SARS-CoV-2 infections in Bavaria in early 2020. Especially in times of low incidence, public health agencies should identify holiday destinations, and areas with ongoing local transmission, to monitor potential importation of SARS-CoV-2 infections. Travellers returning from areas with ongoing community transmission should be advised to quarantine to prevent re-introductions of COVID-19.
Journal Article
The rosetta stone of successful ageing: does oral health have a role?
Ageing is an inevitable aspect of life and thus successful ageing is an important focus of recent scientific efforts. The biological process of ageing is mediated through the interaction of genes with environmental factors, increasing the body’s susceptibility to insults. Elucidating this process will increase our ability to prevent and treat age-related disease and consequently extend life expectancy. Notably, centenarians offer a unique perspective on the phenomenon of ageing. Current research highlights several age-associated alterations on the genetic, epigenetic and proteomic level. Consequently, nutrient sensing and mitochondrial function are altered, resulting in inflammation and exhaustion of regenerative ability.
Oral health, an important contributor to overall health, remains underexplored in the context of extreme longevity. Good masticatory function ensures sufficient nutrient uptake, reducing morbidity and mortality in old age. The relationship between periodontal disease and systemic inflammatory pathologies is well established. Diabetes, rheumatoid arthritis and cardiovascular disease are among the most significant disease burdens influenced by inflammatory oral health conditions. Evidence suggests that the interaction is bi-directional, impacting progression, severity and mortality. Current models of ageing and longevity neglect an important factor in overall health and well-being, a gap that this review intends to illustrate and inspire avenues for future research.
Journal Article
A novel duplex real-time PCR permits simultaneous detection and differentiation of Borrelia miyamotoi and Borrelia burgdorferi sensu lato
For simultaneous detection of Borrelia miyamotoi (relapsing fever spirochete) and Borrelia burgdorferi sensu lato, we have developed a duplex real-time PCR targeting the flagellin gene (flaB; p41), a locus frequently used in routine diagnostic PCR for B. burgdorferi s.l. detection.
Primers and probes were designed using multiple alignments of flaB sequences of B. miyamotoi and B. burgdorferi s.l. species. The sensitivity and specificity of primers and probes were determined using serial dilutions (ranging from 10(4) to 10(-1)) of B. miyamotoi and B. burgdorferi s.l. DNA and of several species of relapsing fever spirochetes. Conventional PCR on recG and glpQ and sequencing of p41 PCR products were used to confirm the species assignment.
The detection limit of both singleplex and duplex PCR was 10 genome equivalents except for B. spielmanii and two B. garinii genotypes which showed a detection limit of 10(2) genome equivalents. There was no cross reactivity of the B. miyamotoi primers/probes with B. burgdorferi s.l. DNA, while the B. burgdorferi s.l. primer/probe generated a signal with B. hermsii DNA. Out of 2341 Ixodes ricinus ticks from Germany and Slovakia that were screened simultaneously for the presence of B. miyamotoi and B. burgdorferi s.l., 52 were positive for B. miyamotoi and 276 for B. burgdorferi s.l., denoting an average prevalence of 2.2% for B. miyamotoi and 11.8% for B. burgdorferi s.l., and B. miyamotoi DNA was also detectable by PCR using artificial clinical samples.
The duplex real-time PCR developed here represents a method that permits simultaneous detection and differentiation of B. burgdorferi s.l. and B. miyamotoi in environmental and potentially clinical samples.
Journal Article
Trans-Atlantic exchanges have shaped the population structure of the Lyme disease agent Borrelia burgdorferi sensu stricto
The origin and population structure of
Borrelia burgdorferi
sensu stricto (s.s.), the agent of Lyme disease, remain obscure. This tick-transmitted bacterial species occurs in both North America and Europe. We sequenced 17 European isolates (representing the most frequently found sequence types in Europe) and compared these with 17 North American strains. We show that trans-Atlantic exchanges have occurred in the evolutionary history of this species and that a European origin of
B. burgdorferi
s.s. is marginally more likely than a USA origin. The data further suggest that some European human patients may have acquired their infection in North America. We found three distinct genetically differentiated groups: i) the outgroup species
Borrelia bissettii
, ii) two divergent strains from Europe, and iii) a group composed of strains from both the USA and Europe. Phylogenetic analysis indicated that different genotypes were likely to have been introduced several times into the same area. Our results demonstrate that irrespective of whether
B. burgdorferi
s.s. originated in Europe or the USA, later trans-Atlantic exchange(s) have occurred and have shaped the population structure of this genospecies. This study clearly shows the utility of next generation sequencing to obtain a better understanding of the phylogeography of this bacterial species.
Journal Article
Prevention of smoking in adolescents with lower education: a school based intervention study
Objective: To assess the effect of an antismoking intervention focusing on adolescents in lower education. Students with lower education smoke more often and perceive more positive norms, and social pressure to smoke, than higher educated students. An intervention based on peer group pressure and social influence may therefore be useful to prevent smoking among these students. Design: Group randomised controlled trial. Setting: 26 Dutch schools that provided junior secondary education. Subjects: 1444 students in the intervention and 1118 students in the control group, all in the first grade, average age 13 years. Intervention: Three lessons on knowledge, attitudes, and social influence, followed by a class agreement not to start or to stop smoking for five months and a class based competition. Main outcome measures: Comparison of smoking status before and immediately after and one year after the intervention, using multilevel analysis. Results: In the intervention group, 9.6% of non-smokers started to smoke, in the control group 14.2%. This leads to an odds ratio of 0.61 (95% CI= 0.41 to 0.90) to uptake smoking in the intervention group compared with the control group. One year after the intervention, the effect was no longer significant. Conclusions: In the short-term, an intervention based on peer pressure decreases the proportion of adolescents with lower education who start smoking. Influencing social norms and peer pressure would therefore be a promising strategy in terms of preventing smoking among adolescents. The results also suggest that additional interventions in later years are needed to maintain the effect.
Journal Article
