Ola Brønstad Brynildsrud

Rapid scoring of genes in microbial pan-genome-wide association studies with Scoary.

Genome-wide association studies (GWAS) have become indispensable in human medicine and genomics, but very few have been carried out on bacteria. Here we introduce Scoary, an ultra-fast, easy-to-use, and widely applicable software tool that scores the components of the pan-genome for associations to observed phenotypic traits while accounting for population stratification, with minimal assumptions about evolutionary processes. We call our approach pan-GWAS to distinguish it from traditional, single nucleotide polymorphism (SNP)-based GWAS. Scoary is implemented in Python and is available under an open source GPLv3 license at https://github.com/AdmiralenOla/Scoary.

Whole-Genome Characterization of Epidemic Neisseria meningitidis Serogroup C and Resurgence of Serogroup W, Niger, 2015

In 2015, Niger reported the largest epidemic of Neisseria meningitidis serogroup C (NmC) meningitis in sub-Saharan Africa. The NmC epidemic coincided with serogroup W (NmW) cases during the epidemic season, resulting in a total of 9,367 meningococcal cases through June 2015. To clarify the phylogenetic association, genetic evolution, and antibiotic determinants of the meningococcal strains in Niger, we sequenced the genomes of 102 isolates from this epidemic, comprising 81 NmC and 21 NmW isolates. The genomes of 82 isolates were completed, and all 102 were included in the analysis. All NmC isolates had sequence type 10217, which caused the outbreaks in Nigeria during 2013–2014 and for which a clonal complex has not yet been defined. The NmC isolates from Niger were substantially different from other NmC isolates collected globally. All NmW isolates belonged to clonal complex 11 and were closely related to the isolates causing recent outbreaks in Africa.

Microevolution of Renibacterium salmoninarum: evidence for intercontinental dissemination associated with fish movements.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, a major pathogen of salmonid fish species worldwide. Very low levels of intra-species genetic diversity have hampered efforts to understand the transmission dynamics and recent evolutionary history of this Gram-positive bacterium. We exploited recent advances in the next-generation sequencing technology to generate genome-wide single-nucleotide polymorphism (SNP) data from 68 diverse R. salmoninarum isolates representing broad geographical and temporal ranges and different host species. Phylogenetic analysis robustly delineated two lineages (lineage 1 and lineage 2); futhermore, dating analysis estimated that the time to the most recent ancestor of all the isolates is 1239 years ago (95% credible interval (CI) 444–2720 years ago). Our data reveal the intercontinental spread of lineage 1 over the last century, concurrent with anthropogenic movement of live fish, feed and ova for aquaculture purposes and stocking of recreational fisheries, whilst lineage 2 appears to have been endemic in wild Eastern Atlantic salmonid stocks before commercial activity. The high resolution of the SNP-based analyses allowed us to separate closely related isolates linked to neighboring fish farms, indicating that they formed part of single outbreaks. We were able to demonstrate that the main lineage 1 subgroup of R. salmoninarum isolated from Norway and the UK likely represent an introduction to these areas ∼40 years ago. This study demonstrates the promise of this technology for analysis of micro and medium scale evolutionary relationships in veterinary and environmental microorganisms, as well as human pathogens.

Armed conflict and population displacement as drivers of the evolution and dispersal of Mycobacterium tuberculosis

Significance We used population genomic analyses to reconstruct the recent history and dispersal of a major clade of Mycobacterium tuberculosis in central Asia and beyond. Our results indicate that the fall of the Soviet Union and the ensuing collapse of public health systems led to a rise in M. tuberculosis drug resistance. We also show that armed conflict and population displacement is likely to have aided the export of this clade from central Asia to war-torn Afghanistan and beyond. The “Beijing” Mycobacterium tuberculosis (Mtb) lineage 2 (L2) is spreading globally and has been associated with accelerated disease progression and increased antibiotic resistance. Here we performed a phylodynamic reconstruction of one of the L2 sublineages, the central Asian clade (CAC), which has recently spread to western Europe. We find that recent historical events have contributed to the evolution and dispersal of the CAC. Our timing estimates indicate that the clade was likely introduced to Afghanistan during the 1979–1989 Soviet–Afghan war and spread further after population displacement in the wake of the American invasion in 2001. We also find that drug resistance mutations accumulated on a massive scale in Mtb isolates from former Soviet republics after the fall of the Soviet Union, a pattern that was not observed in CAC isolates from Afghanistan. Our results underscore the detrimental effects of political instability and population displacement on tuberculosis control and demonstrate the power of phylodynamic methods in exploring bacterial evolution in space and time.

Brominated flame retardants (BFRs) in breast milk and associated health risks to nursing infants in Northern Tanzania.

The main aim of this study was to assess brominated flame retardants (BFRs) in breast milk in the Northern parts of Tanzania. Ninety-five colostrum samples from healthy, primiparous mothers at Mount Meru Regional Referral Hospital (MMRRH), Arusha Tanzania, were analyzed for polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCD), 1,2-bis(2,4,6-tribromophenoxy) ethane (BTBPE), hexabromobenzene (HBB), (2,3-dibromopropyl) (2,4,6-tribromophenyl) ether (DPTE), pentabromoethylbenzene (PBEB) and 2,3,4,5,6-pentabromotoluene (PBT). The Ʃ7PBDE (BDE 28, 47, 99, 100, 153, 154, 183) ranged from below level of detection (<LOD) to 785ng/g lipid weight (lw). BDE 47, 99, 100 and 153 were the dominating congeners, suggesting recent and ongoing exposure to banned, commercial PentaBDE mixture. A multiple linear regression model revealed that mothers eating clay soil/Pemba during pregnancy had significantly higher levels of BDE 47, 99, 100 and 153 in their breast milk than mothers who did not eat clay soil/Pemba. Infant birth weight and birth length were significantly correlated with the levels of BDE 47, 99, 100 and 153. The estimated daily intake (ng/kg body weight/day) of BDE 47 and 99 exceeded the US EPA Reference doses (RfD) in four and eight mothers, respectively, suggesting a potential health risk to the nursing infants.

Dioxins, PCBs, chlorinated pesticides and brominated flame retardants in free-range chicken eggs from peri-urban areas in Arusha, Tanzania: Levels and implications for human health.

The environment in the northern part of Tanzania is influenced by rapid population growth, and increased urbanization. Urban agriculture is common and of economic value for low income families. In Arusha, many households sell eggs from free-ranging backyard chicken. In 2011, 159 eggs from different households in five different locations in Arusha were collected, homogenized, pooled into 28 composite samples and analyzed for a wide selection of POPs. Levels of POPs varied widely within and between the locations. The levels of dieldrin and ΣDDT ranged between 2 and 98,791 and 2 and 324ng/g lipid weight (lw), respectively. EU MRLs of 0.02mg/kg dieldrin for eggs were exceeded in 4/28 samples. PCBs, HCHs, chlordanes, toxaphenes and endosulfanes were found at lower frequency and levels. Brominated flame retardants (BFRs), e.g polybrominated diphenylethers (PBDEs), hexabromocyclododecane (HBCD) and 1,2-bis(2,4,6-tribromphenoxy)ethane (BTBPE) were present in 100%, 60% and 46% of the composite samples, respectively. Octa-and deca-BDEs were the dominating PBDEs and BDE 209 levels ranged between <LOQ (limit of quantification) - 312ng/g lw. Dioxins were measured using the DR-LUC bio-assay and found in levels of <LOQ - 20pg bio-TEQs/g lw. Four samples (13%) exceeded the maximum level of 5pg/g total WHO-TEQs for hen eggs set by the Commission Regulation (EU) No 1259/2011. The daily/weekly intake was calculated and risk was characterized for all compounds comparing with available toxicity reference values (TRVs) such as the provisional tolerable intake (PTDI) or Reference Doses (RfDs). In one sample dieldrin exceeded the PTDI (100ng/kgbw/day). Correlation was found between bio-TEQs and lipid adjusted levels of ΣPBDEs, suggesting similar sources. Open fires in backyards may be one of the sources for contamination of eggs with BFRs and dioxins.

Amino acid usage is asymmetrically biased in AT- and GC-rich microbial genomes.

Introduction Genomic base composition ranges from less than 25% AT to more than 85% AT in prokaryotes. Since only a small fraction of prokaryotic genomes is not protein coding even a minor change in genomic base composition will induce profound protein changes. We examined how amino acid and codon frequencies were distributed in over 2000 microbial genomes and how these distributions were affected by base compositional changes. In addition, we wanted to know how genome-wide amino acid usage was biased in the different genomes and how changes to base composition and mutations affected this bias. To carry this out, we used a Generalized Additive Mixed-effects Model (GAMM) to explore non-linear associations and strong data dependences in closely related microbes; principal component analysis (PCA) was used to examine genomic amino acid- and codon frequencies, while the concept of relative entropy was used to analyze genomic mutation rates. Results We found that genomic amino acid frequencies carried a stronger phylogenetic signal than codon frequencies, but that this signal was weak compared to that of genomic %AT. Further, in contrast to codon usage bias (CUB), amino acid usage bias (AAUB) was differently distributed in AT- and GC-rich genomes in the sense that AT-rich genomes did not prefer specific amino acids over others to the same extent as GC-rich genomes. AAUB was also associated with relative entropy; genomes with low AAUB contained more random mutations as a consequence of relaxed purifying selection than genomes with higher AAUB. Conclusion Genomic base composition has a substantial effect on both amino acid- and codon frequencies in bacterial genomes. While phylogeny influenced amino acid usage more in GC-rich genomes, AT-content was driving amino acid usage in AT-rich genomes. We found the GAMM model to be an excellent tool to analyze the genomic data used in this study.

Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in human breast milk and associated health risks to nursing infants in Northern Tanzania

&NA; This is the first study to report organochlorines (OCs), including chlorinated pesticides (OCPs) and polychlorinated biphenyls (PCBs) in human milk from Tanzania. The main aims of this study were to assess the level of contamination and the possible health risks related to OC exposure in nursing infants from the Northern parts of Tanzania. Ninety‐five healthy mother‐infant couples attending Mount Meru Regional Referral Hospital (MMRRH), Arusha, Tanzania, were assessed for associations between maternal/infant characteristics, i.e. mothers age, BMI, gestational weight gain, occupation, residence and fetal growth parameters and breast milk levels of OCPs, such as dichlorodiphenyltrichloroethane (DDT) and its metabolites, dieldrin and PCBs. p,p′‐DDE and p,p′‐DDT were detected in 100% and 75% of the breast milk samples, respectively, and ranged between 24 and 2400 ng/g lipid weight (lw) and <LOD and 133 ng/g lw, respectively. Dieldrin was detected in 66% of the samples in levels up to 937 ng/g lw. Σ7PCBs ranged between <LOD and 157 ng/g lw. Other OCPs were detected in low levels. For assessment of health risks, the Hazard Quotient (HQ) was calculated by comparing estimated daily intakes of OCPs and PCBs with health based guidance values. The estimated daily intake (ng/kg body weight/day) of ΣDDTs, dieldrin and nondioxin‐like PCBs (Σ6PCBs) exceeded the provisional tolerable daily intake (PTDI) in two, six and forty‐eight of the nursing infants, respectively, suggesting potential health risks. In addition, head circumference were negatively associated with p,p′‐DDE in female infants, suggesting that OC exposure during pregnancy may influence fetal growth. Graphical abstract Figure. No caption available. Highlightsp,p′‐DDE the dominating OC in Tanzanian breast milk.Dieldrin detected in levels up to 937 ng/g lw.p,p′‐DDE negatively associated to head circumference in female infants.EDIs of ΣDDTs and dieldrin exceeded PTDIs in 2 and 6 of the infants, respectively.EDIs of Σ6PCBs exceeded PTDI in 48 of the infants.

Positive correlations between genomic %AT and genome size within strains of bacterial species

Genomic %AT has been found to correlate negatively with genome size in microbes. While microbes with large genomes are often GC rich and free living, AT-rich bacteria tend to be host associated with smaller genomes. With over 2000 fully sequenced and assembled microbial genomes available, we explored the relationship among genomic %AT, genome size, relative entropy (a measure associated with genetic drift) and fraction of genome islands (GIs) in microbial species with the genomes of more than 10 strains available. A negative correlation with genome size was found in six out of 12 phylogenetic groups and subphyla and a positive correlation in only two. At the species level, we found a trend of positive correlations between genomic %AT and genome size in eight out of 20 species, while only four showed a negative correlation. Estimated chromosomal fractions of GIs were found to correlate positively with genome size in the strains of 14 out of 18 species and genomic %AT in the strains of seven species (two correlated negatively). Although GIs explain most of the observed positive correlations between genomic %AT and size, Chlamydia trachomatis seem to be an exception; therefore, these findings needs to be further explored.

An evolutionary analysis of genome expansion and pathogenicity in Escherichia coli

BackgroundThere are several studies describing loss of genes through reductive evolution in microbes, but how selective forces are associated with genome expansion due to horizontal gene transfer (HGT) has not received similar attention. The aim of this study was therefore to examine how selective pressures influence genome expansion in 53 fully sequenced and assembled Escherichia coli strains. We also explored potential connections between genome expansion and the attainment of virulence factors. This was performed using estimations of several genomic parameters such as AT content, genomic drift (measured using relative entropy), genome size and estimated HGT size, which were subsequently compared to analogous parameters computed from the core genome consisting of 1729 genes common to the 53 E. coli strains. Moreover, we analyzed how selective pressures (quantified using relative entropy and dN/dS), acting on the E. coli core genome, influenced lineage and phylogroup formation.ResultsHierarchical clustering of dS and dN estimations from the E. coli core genome resulted in phylogenetic trees with topologies in agreement with known E. coli taxonomy and phylogroups. High values of dS, compared to dN, indicate that the E. coli core genome has been subjected to substantial purifying selection over time; significantly more than the non-core part of the genome (p<0.001). This is further supported by a linear association between strain-wise dS and dN values (β = 26.94 ± 0.44, R2~0.98, p<0.001). The non-core part of the genome was also significantly more AT-rich (p<0.001) than the core genome and E. coli genome size correlated with estimated HGT size (p<0.001). In addition, genome size (p<0.001), AT content (p<0.001) as well as estimated HGT size (p<0.005) were all associated with the presence of virulence factors, suggesting that pathogenicity traits in E. coli are largely attained through HGT. No associations were found between selective pressures operating on the E. coli core genome, as estimated using relative entropy, and genome size (p~0.98).ConclusionsOn a larger time frame, genome expansion in E. coli, which is significantly associated with the acquisition of virulence factors, appears to be independent of selective forces operating on the core genome.