The effect of recombination on the evolution of a population of Neisseria meningitidis.

Neisseria meningitidis (the meningococcus) is a major human pathogen with a history of high invasive disease burden, particularly in sub-Saharan Africa. Our current understanding of the evolution of meningococcal genomes is limited by the rarity of large-scale genomic population studies and lack of in-depth investigation of the genomic events associated with routine pathogen transmission. Here, we fill this knowledge gap by a detailed analysis of 2839 meningococcal genomes obtained through a carriage study of over 50,000 samples collected systematically in Burkina Faso, West Africa, before, during, and after the serogroup A vaccine rollout, 2009-2012. Our findings indicate that the meningococcal genome is highly dynamic, with highly recombinant loci and frequent gene sharing across deeply separated lineages in a structured population. Furthermore, our findings illustrate how population structure can correlate with genome flexibility, as some lineages in Burkina Faso are orders of magnitude more recombinant than others. We also examine the effect of selection on the population, in particular how it is correlated with recombination. We find that recombination principally acts to prevent the accumulation of deleterious mutations, although we do also find an example of recombination acting to speed the adaptation of a gene. In general, we show the importance of recombination in the evolution of a geographically expansive population with deep population structure in a short timescale. This has important consequences for our ability to both foresee the outcomes of vaccination programs and, using surveillance data, predict when lineages of the meningococcus are likely to become a public health concern.

Antibiotic Treatment Regimes as a Driver of the Global Population Dynamics of a Major Gonorrhea Lineage.

The Neisseria gonorrhoeae multilocus sequence type (ST) 1901 is among the lineages most commonly associated with treatment failure. Here, we analyze a global collection of ST-1901 genomes to shed light on the emergence and spread of alleles associated with reduced susceptibility to extended-spectrum cephalosporins (ESCs). The genetic diversity of ST-1901 falls into a minor and a major clade, both of which were inferred to have originated in East Asia. The dispersal of the major clade from Asia happened in two separate waves expanding from ∼1987 and 1996, respectively. Both waves first reached North America, and from there spread to Europe and Oceania, with multiple secondary reintroductions to Asia. The ancestor of the second wave acquired the penA 34.001 allele, which significantly reduces susceptibility to ESCs. Our results suggest that the acquisition of this allele granted the second wave a fitness advantage at a time when ESCs became the key drug class used to treat gonorrhea. Following its establishment globally, the lineage has served as a reservoir for the repeated emergence of clones fully resistant to the ESC ceftriaxone, an essential drug for effective treatment of gonorrhea. We infer that the effective population sizes of both clades went into decline as treatment schemes shifted from fluoroquinolones via ESC monotherapy to dual therapy with ceftriaxone and azithromycin in Europe and the United States. Despite the inferred recent population size decline, the short evolutionary path from the penA 34.001 allele to alleles providing full ceftriaxone resistance is a cause of concern.

Pre-detection history of extensively drug-resistant tuberculosis in KwaZulu-Natal, South Africa.

Antimicrobial-resistant (AMR) infections pose a major threat to global public health. Similar to other AMR pathogens, both historical and ongoing drug-resistant tuberculosis (TB) epidemics are characterized by transmission of a limited number of predominant Mycobacterium tuberculosis (Mtb) strains. Understanding how these predominant strains achieve sustained transmission, particularly during the critical period before they are detected via clinical or public health surveillance, can inform strategies for prevention and containment. In this study, we employ whole-genome sequence (WGS) data from TB clinical isolates collected in KwaZulu-Natal, South Africa to examine the pre-detection history of a successful strain of extensively drug-resistant (XDR) TB known as LAM4/KZN, first identified in a widely reported cluster of cases in 2005. We identify marked expansion of this strain concurrent with the onset of the generalized HIV epidemic 12 y prior to 2005, localize its geographic origin to a location in northeastern KwaZulu-Natal ∼400 km away from the site of the 2005 outbreak, and use protein structural modeling to propose a mechanism for how strain-specific rpoB mutations offset fitness costs associated with rifampin resistance in LAM4/KZN. Our findings highlight the importance of HIV coinfection, high preexisting rates of drug-resistant TB, human migration, and pathoadaptive evolution in the emergence and dispersal of this critical public health threat. We propose that integrating whole-genome sequencing into routine public health surveillance can enable the early detection and local containment of AMR pathogens before they achieve widespread dispersal.

Import and transmission of Mycobacterium orygis and Mycobacterium africanum, Norway.

BACKGROUND: The aim of the current study was to improve our understanding of the origins and transmission of Mycobacterium africanum (MAF) in Norway. METHODS: Whole-genome sequences (WGS) were generated for all (n = 29) available clinical isolates received at the Norwegian National Reference Laboratory for Mycobacteria (NRL) and identified as MAF in Norway, in the period 2010-2020. Phylogenetic analyses were performed. RESULTS: The analyses indicated several imports of MAF lineage 6 from both East and West African countries, whereas MAF lineage 5 was restricted to patients with West African connections. We also find evidence for transmission of MAF in Norway. Finally, our analyses revealed that a group of isolates from patients originating in South Asia, identified as MAF by means of a commercial line-probe assay, in fact belonged to Mycobacterium orygis. CONCLUSIONS: Most MAF cases in Norway are the result of import, but transmission is occurring within Norway.

Acquisition of virulence genes by a carrier strain gave rise to the ongoing epidemics of meningococcal disease in West Africa.

In the African meningitis belt, a region of sub-Saharan Africa comprising 22 countries from Senegal in the west to Ethiopia in the east, large epidemics of serogroup A meningococcal meningitis have occurred periodically. After gradual introduction from 2010 of mass vaccination with a monovalent meningococcal A conjugate vaccine, serogroup A epidemics have been eliminated. Starting in 2013, the northwestern part of Nigeria has been affected by yearly outbreaks of meningitis caused by a novel strain of serogroup C Neisseria meningitidis (NmC). In 2015, the strain spread to the neighboring country Niger, where it caused a severe epidemic. Following a relative calm in 2016, the largest ever recorded epidemic of NmC broke out in Nigeria in 2017. Here, we describe the recent evolution of this new outbreak strain and show how the acquisition of capsule genes and virulence factors by a strain previously circulating asymptomatically in the African population led to the emergence of a virulent pathogen. This study illustrates the power of long-read whole-genome sequencing, combined with Illumina sequencing, for high-resolution epidemiological investigations.

Lineage specific histories of Mycobacterium tuberculosis dispersal in Africa and Eurasia.

Mycobacterium tuberculosis (M.tb) is a globally distributed, obligate pathogen of humans that can be divided into seven clearly defined lineages. An emerging consensus places the origin and global dispersal of M.tb within the past 6,000 years: identifying how the ancestral clone of M.tb spread and differentiated within this timeframe is important for identifying the ecological drivers of the current pandemic. We used Bayesian phylogeographic inference to reconstruct the migratory history of M.tb in Africa and Eurasia and to investigate lineage specific patterns of spread from a geographically diverse sample of 552 M.tb genomes. Applying evolutionary rates inferred with ancient M.tb genome calibration, we estimated the timing of major events in the migratory history of the pathogen. Inferred timings contextualize M.tb dispersal within historical phenomena that altered patterns of connectivity throughout Africa and Eurasia: trans-Indian Ocean trade in spices and other goods, the Silk Road and its predecessors, the expansion of the Roman Empire, and the European Age of Exploration. We found that Eastern Africa and Southeast Asia have been critical in the dispersal of M.tb. Our results further reveal that M.tb populations have grown through range expansion, as well as in situ, and delineate the independent evolutionary trajectories of bacterial subpopulations underlying the current pandemic.

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

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.

Modeling of the GC content of the substituted bases in bacterial core genomes.

BACKGROUND: The purpose of the present study was to examine the GC content of substituted bases (sbGC) in the core genomes of 35 bacterial species. Each species, or core genome, constituted genomes from at least 10 strains. We also wanted to explore whether sbGC for each strain was associated with the corresponding species' core genome GC content (cgGC). We present a simple mathematical model that estimates sbGC from cgGC. The model assumes only that the estimated sbGC is a function of cgGC proportional to fixed AT→GC (α) and GC → AT (ß) mutation rates. Non-linear regression was used to estimate parameters α and ß from the empirical data described above. RESULTS: We found that sbGC for each strain showed a non-linear association with the corresponding cgGC with a bias towards higher GC content for most core genomes (66.3% of the strains), assuming as a null-hypothesis that sbGC should be approximately equal to cgGC. The most GC rich core genomes (i.e. approximately %GC > 60), on the other hand, exhibited slightly less GC-biased sbGC than expected. The best fitted regression model indicates that GC → AT mutation rates ß = (1.91 ± 0.13) p < 0.001 are approximately (1.91/0.79) = 2.42 times as high, on average, as AT→GC α = (- 0.79 ± 0.25) p < 0.001 mutation rates. Whether the observed sbGC GC-bias for all but the most GC-rich prokaryotic species is due to selection, compensating for the GC → AT mutation bias, and/or selective neutral processes is currently debated. Residual standard error was found to be σ = 0.076 indicating estimated errors of sbGC to be approximately within ±15.2% GC (95% confidence interval) for the strains of all species in the study. CONCLUSION: Not only did our mathematical model give reasonable estimates of sbGC it also provides further support to previous observations that mutation rates in prokaryotes exhibit a universal GC → AT bias that appears to be remarkably consistent between taxa.

The nucleotide composition of microbial genomes indicates differential patterns of selection on core and accessory genomes.

BACKGROUND: The core genome consists of genes shared by the vast majority of a species and is therefore assumed to have been subjected to substantially stronger purifying selection than the more mobile elements of the genome, also known as the accessory genome. Here we examine intragenic base composition differences in core genomes and corresponding accessory genomes in 36 species, represented by the genomes of 731 bacterial strains, to assess the impact of selective forces on base composition in microbes. We also explore, in turn, how these results compare with findings for whole genome intragenic regions. RESULTS: We found that GC content in coding regions is significantly higher in core genomes than accessory genomes and whole genomes. Likewise, GC content variation within coding regions was significantly lower in core genomes than in accessory genomes and whole genomes. Relative entropy in coding regions, measured as the difference between observed and expected trinucleotide frequencies estimated from mononucleotide frequencies, was significantly higher in the core genomes than in accessory and whole genomes. Relative entropy was positively associated with coding region GC content within the accessory genomes, but not within the corresponding coding regions of core or whole genomes. CONCLUSION: The higher intragenic GC content and relative entropy, as well as the lower GC content variation, observed in the core genomes is most likely associated with selective constraints. It is unclear whether the positive association between GC content and relative entropy in the more mobile accessory genomes constitutes signatures of selection or selective neutral processes.

Whole-Transcriptome and -Genome Analysis of Extensively Drug-Resistant Mycobacterium tuberculosis Clinical Isolates Identifies Downregulation of ethA as a Mechanism of Ethionamide Resistance.

Genetics-based drug susceptibility testing has improved the diagnosis of drug-resistant tuberculosis but is limited by our lack of knowledge of all resistance mechanisms. Next-generation sequencing has assisted in identifying the principal genetic mechanisms of resistance for many drugs, but a significant proportion of phenotypic drug resistance is unexplained genetically. Few studies have formally compared the transcriptomes of susceptible and resistant Mycobacterium tuberculosis strains. We carried out comparative whole-genome transcriptomics of extensively drug-resistant (XDR) clinical isolates using RNA sequencing (RNA-seq) to find novel transcription-mediated mechanisms of resistance. We identified a promoter mutation (t to c) at position -11 (t-11c) relative to the start codon of ethA that reduces the expression of a monooxygenase (EthA) that activates ethionamide. (In this article, nucleotide changes are lowercase and amino acid substitutions are uppercase.) Using a flow cytometry-based reporter assay, we show that the reduced transcription of ethA is not due to transcriptional repression by ethR Clinical strains harboring this mutation were resistant to ethionamide. Other ethA promoter mutations were identified in a global genomic survey of resistant M. tuberculosis strains. These results demonstrate a new mechanism of ethionamide resistance that can cause high-level resistance when it is combined with other ethionamide resistance-conferring mutations. Our study revealed many other genes which were highly up- or downregulated in XDR strains, including a toxin-antitoxin module (mazF5 mazE5) and tRNAs (leuX and thrU). This suggests that global transcriptional modifications could contribute to resistance or the maintenance of bacterial fitness have also occurred in XDR strains.

Tuberculosis Outbreak in an Educational Institution in Norway.

Within 1 week in April 2013, three cases of pulmonary tuberculosis (TB) were reported among students attending training sessions at an educational institution in Oslo, Norway. By the end of October 2013, a total of nine epidemiologically linked cases had been reported. The outbreak encompassed a total of 24 cases from 2009 to 2014, among which all of the 22 Mycobacterium tuberculosis isolates available had identical mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) profiles (MtbC15-9 code 10287-189) belonging to the Beijing lineage. Whole-genome sequencing (WGS) of the M. tuberculosis isolates revealed 20 variable nucleotide positions within the cluster, indicating a clonal outbreak. The most likely index case was identified and diagnosed in Norway in 2009 but was born in Asia. WGS analyses verified that all of the cases were indeed part of a single transmission chain. However, even when combining WGS and intensified contact tracing, we were unable to fully reconstruct the TB transmission events.

Cdc28 kinase activity regulates the basal transcription machinery at a subset of genes.

The cyclin-dependent kinase Cdc28 is the master regulator of the cell cycle in Saccharomyces cerevisiae. Cdc28 initiates the cell cycle by activating cell-cycle-specific transcription factors that switch on a transcriptional program during late G1 phase. Cdc28 also has a cell-cycle-independent, direct function in regulating basal transcription, which does not require its catalytic activity. However, the exact role of Cdc28 in basal transcription remains poorly understood, and a function for its kinase activity has not been fully explored. Here we show that the catalytic activity of Cdc28 is important for basal transcription. Using a chemical-genetic screen for mutants that specifically require the kinase activity of Cdc28 for viability, we identified a plethora of basal transcription factors. In particular, CDC28 interacts genetically with genes encoding kinases that phosphorylate the C-terminal domain of RNA polymerase II, such as KIN28. ChIP followed by high-throughput sequencing (ChIP-seq) revealed that Cdc28 localizes to at least 200 genes, primarily with functions in cellular homeostasis, such as the plasma membrane proton pump PMA1. Transcription of PMA1 peaks early in the cell cycle, even though the promoter sequences of PMA1 (as well as the other Cdc28-enriched ORFs) lack cell-cycle elements, and PMA1 does not recruit Swi4/6-dependent cell-cycle box-binding factor/MluI cell-cycle box binding factor complexes. Finally, we found that recruitment of Cdc28 and Kin28 to PMA1 is mutually dependent and that the activity of both kinases is required for full phosphorylation of C-terminal domain-Ser5, for efficient transcription, and for mRNA capping. Our results reveal a mechanism of cell-cycle-dependent regulation of basal transcription.

CTCF mediates the TERT enhancer-promoter interactions in lung cancer cells: identification of a novel enhancer region involved in the regulation of TERT gene.

Telomerase activation is a hallmark of cancer. Although the regulation of the telomerase reverse transcriptase catalytic subunit (TERT), the rate-limiting factor for telomerase activity, has been studied intensively it remains incompletely understood. In cells devoid of telomerase activity, TERT is embedded in a region of condensed chromatin and the chromatin remodeling protein CCCTC-binding factor (CTCF) has been implicated in the inhibition of TERT expression. The importance of TERT activation for cellular immortalization and carcinogenesis is attested by the fact that the gene is expressed in more than 90% of immortal cell lines and tumors and that gain of TERT is the most frequent amplification event in early stage lung cancer. This study was designed to study the mechanisms of regulation of the TERT gene expression by the CTCF transcription factor in three human lung cancer cell lines, A427, A549 and H838. Depletion of CTCF by siRNA resulted in reduced TERT mRNA levels in two (A427 and A549) of the three cell lines. A novel enhancer element was identified approximately 4.5 kb upstream of the TERT transcription start site. Chromatin immunoprecipitation experiments revealed recruitment of CTCF to this enhancer element. Chromosome conformation capture experiments demonstrated the presence of CTCF-dependent chromatin loops between this enhancer element and the TERT proximal promoter in A427 and A549 cell lines. In summary, the results show that CTCF plays an important role in maintaining TERT expression in a subset of human lung cancer cell lines. This role may be due to CTCF-dependent enhancer-promoter interactions.

A chemical-genetic screen to unravel the genetic network of CDC28/CDK1 links ubiquitin and Rad6-Bre1 to cell cycle progression.

Cyclin-dependent kinases (CDKs) control the eukaryotic cell cycle, and a single CDK, Cdc28 (also known as Cdk1), is necessary and sufficient for cell cycle regulation in the budding yeast Saccharomyces cerevisiae. Cdc28 regulates cell cycle-dependent processes such as transcription, DNA replication and repair, and chromosome segregation. To gain further insight into the functions of Cdc28, we performed a high-throughput chemical-genetic array (CGA) screen aimed at unraveling the genetic network of CDC28. We identified 107 genes that strongly genetically interact with CDC28. Although these genes serve multiple cellular functions, genes involved in cell cycle regulation, transcription, and chromosome metabolism were overrepresented. DOA1, which is involved in maintaining free ubiquitin levels, as well as the RAD6-BRE1 pathway, which is involved in transcription, displayed particularly strong genetic interactions with CDC28. We discovered that DOA1 is important for cell cycle entry by supplying ubiquitin. Furthermore, we found that the RAD6-BRE1 pathway functions downstream of DOA1/ubiquitin but upstream of CDC28, by promoting transcription of cyclins. These results link cellular ubiquitin levels and the Rad6-Bre1 pathway to cell cycle progression.

Tracing the adaptive evolution of SARS-CoV-2 during vaccine roll-out in Norway.

Vaccination against SARS-CoV-2 has greatly mitigated the impact of the COVID-19 pandemic. However, concerns have been raised about the degree to which vaccination might drive the emergence and selection of immune escape mutations that will hamper the efficacy of the vaccines. In this study, we investigate whether vaccination impacted the micro-scale adaptive evolution of SARS-CoV-2 in the Oslo region of Norway, during the first nine months of 2021, a period in which the population went from near-zero to almost 90 per cent vaccine coverage in the population over 50 years old. Weekly aggregated data stratified by age on vaccine uptake and number of SARS-CoV-2 cases in the area were obtained from the National Immunization Registry and the Norwegian Surveillance System for Communicable Diseases, respectively. A total of 6,438 virus sequences (7.5 per cent of the registered cases) along with metadata were available. We used a causal-driven approach to investigate the relationship between vaccination progress and changes in the frequency of 362 mutations present in at least ten samples, conditioned on the emergence of new lineages, time, and population vaccination coverage. After validating our approach, we identified 21 positive and 12 negative connections between vaccination progress and mutation prevalence, and most of them were outside the Spike protein. We observed a tendency for the mutations that we identified as positively connected with vaccination to decrease as the vaccinated population increased. After modelling the fitness of different competing mutations in a population, we found that our observations could be explained by a clonal interference phenomenon in which high fitness mutations would be outcompeted by the emergence or introduction of other high-fitness mutations.

Detection of a historic reservoir of bedaquiline/clofazimine resistance-associated variants in Mycobacterium tuberculosis.

BACKGROUND: Drug resistance in tuberculosis (TB) poses a major ongoing challenge to public health. The recent inclusion of bedaquiline into TB drug regimens has improved treatment outcomes, but this advance is threatened by the emergence of strains of Mycobacterium tuberculosis (Mtb) resistant to bedaquiline. Clinical bedaquiline resistance is most frequently conferred by off-target resistance-associated variants (RAVs) in the mmpR5 gene (Rv0678), the regulator of an efflux pump, which can also confer cross-resistance to clofazimine, another TB drug. METHODS: We compiled a dataset of 3682 Mtb genomes, including 180 carrying variants in mmpR5, and its immediate background (i.e. mmpR5 promoter and adjacent mmpL5 gene), that have been associated to borderline (henceforth intermediate) or confirmed resistance to bedaquiline. We characterised the occurrence of all nonsynonymous mutations in mmpR5 in this dataset and estimated, using time-resolved phylogenetic methods, the age of their emergence. RESULTS: We identified eight cases where RAVs were present in the genomes of strains collected prior to the use of bedaquiline in TB treatment regimes. Phylogenetic reconstruction points to multiple emergence events and circulation of RAVs in mmpR5, some estimated to predate the introduction of bedaquiline. However, epistatic interactions can complicate bedaquiline drug-susceptibility prediction from genetic sequence data. Indeed, in one clade, Ile67fs (a RAV when considered in isolation) was estimated to have emerged prior to the antibiotic era, together with a resistance reverting mmpL5 mutation. CONCLUSIONS: The presence of a pre-existing reservoir of Mtb strains carrying bedaquiline RAVs prior to its clinical use augments the need for rapid drug susceptibility testing and individualised regimen selection to safeguard the use of bedaquiline in TB care and control.

Genetic Characterization and Population Structure of Drug-Resistant Mycobacterium tuberculosis Isolated from Brazilian Patients Using Whole-Genome Sequencing.

The present study aimed to determine the genetic diversity of isolates of Mycobacterium tuberculosis (Mtb) from presumed drug-resistant tuberculosis patients from several states of Brazil. The isolates had been submitted to conventional drug susceptibility testing for first- and second-line drugs. Multidrug-resistant (MDR-TB) (54.8%) was the most frequent phenotypic resistance profile, in addition to an important high frequency of pre-extensive resistance (p-XDR-TB) (9.2%). Using whole-genome sequencing (WGS), we characterized 298 Mtb isolates from Brazil. Besides the analysis of genotype distribution and possible correlations between molecular and clinical data, we determined the performance of an in-house WGS pipeline with other online pipelines for Mtb lineages and drug resistance profile definitions. Sub-lineage 4.3 (52%) was the most frequent genotype, and the genomic approach revealed a p-XDR-TB level of 22.5%. We detected twenty novel mutations in three resistance genes, and six of these were observed in eight phenotypically resistant isolates. A cluster analysis of 170 isolates showed that 43.5% of the TB patients belonged to 24 genomic clusters, suggesting considerable ongoing transmission of DR-TB, including two interstate transmissions. The in-house WGS pipeline showed the best overall performance in drug resistance prediction, presenting the best accuracy values for five of the nine drugs tested. Significant associations were observed between suffering from fatal disease and genotypic p-XDR-TB (p = 0.03) and either phenotypic (p = 0.006) or genotypic (p = 0.0007) ethambutol resistance. The use of WGS analysis improved our understanding of the population structure of MTBC in Brazil and the genetic and clinical data correlations and demonstrated its utility for surveillance efforts regarding the spread of DR-TB, hopefully helping to avoid the emergence of even more resistant strains and to reduce TB incidence and mortality rates.

Naturally occurring Neisseria gonorrhoeae can have large deletions in housekeeping gene abcZ, making them untypable with multilocus sequence typing.

The abcZ gene is an essential housekeeping gene in all the Neisseria species. It is one of the seven genes used for multilocus sequence typing (MLST) this genus. It encodes the cytosolic component of an ATP-binding cassette (ABC) transporter complex of unknown function. We report here the finding of a strain of Neisseria gonorrhoeae with a 485 base pair deletion in the 5' region of the abcZ gene that truncates the protein product from 636 amino acids to 89 amino acids. A second open reading frame (ORF), encoding the latter 388 amino acids of the abcZ gene, was predicted downstream. The deletion will affect MLST profiling; interrogation of genomic sequences from PubMLST revealed that this isolate is not an anomaly. Deletions in abcZ were identified in 256 Neisseria genomes, roughly 0.6% of isolates. Furthermore, these deletions could leave the abcZ gene in a pseudogenized state. Our strain, isolated from a patient with symptoms of gonorrheal infection, nevertheless behaved normal in terms of growth and in vitro phenotypic properties.