In vivo evolution of antimicrobial resistance in a biofilm model of Pseudomonas aeruginosa lung infection.

The evolution of antimicrobial resistance (AMR) in biofilms has been repeatedly studied by experimental evolution in vitro, but rarely in vivo. The complex microenvironment at the infection site imposes selective pressures on the bacterial biofilms, potentially influencing the development of AMR. We report here the development of AMR in an in vivo mouse model of Pseudomonas aeruginosa biofilm lung infection. The P. aeruginosa embedded in seaweed alginate beads underwent four successive lung infection passages with or without ciprofloxacin (CIP) exposure. The development of CIP resistance was assessed at each passage by population analysis of the bacterial populations recovered from the lungs of CIP-treated and control mice, with subsequent whole-genome sequencing of selected isolates. As inflammation plays a crucial role in shaping the microenvironment at the infection site, its impact was explored through the measurement of cytokine levels in the lung homogenate. A rapid development of AMR was observed starting from the second passage in the CIP-treated mice. Genetic analysis revealed mutations in nfxB, efflux pumps (mexZ), and two-component systems (parS) contribution to CIP resistance. The control group isolates exhibited mutations in the dipA gene, likely associated with biofilm dispersion. In the initial two passages, the CIP-treated group exhibited an elevated inflammatory response compared to the control group. This increase may potentially contribute to the release of mutagenic reactive oxygen species and the development of AMR. In conclusion, this study illustrates the complex relationship between infection, antibiotic treatment, and immune response.

Long-term evolution of antibiotic tolerance in Pseudomonas aeruginosa lung infections.

Pathogenic bacteria respond to antibiotic pressure with the evolution of resistance but survival can also depend on their ability to tolerate antibiotic treatment, known as tolerance. While a variety of resistance mechanisms and underlying genetics are well characterized in vitro and in vivo, an understanding of the evolution of tolerance, and how it interacts with resistance in situ is lacking. We assayed for tolerance and resistance in isolates of Pseudomonas aeruginosa from chronic cystic fibrosis lung infections spanning up to 40 years of evolution, with 3 clinically relevant antibiotics: meropenem, ciprofloxacin, and tobramycin. We present evidence that tolerance is under positive selection in the lung and that it can act as an evolutionary stepping stone to resistance. However, by examining evolutionary patterns across multiple patients in different clone types, a key result is that the potential for an association between the evolution of resistance and tolerance is not inevitable, and difficult to predict.

Comparative whole-genome analysis of China and global epidemic Pseudomonas aeruginosa high-risk clones.

OBJECTIVES: The various sequence types (STs) of Pseudomonas aeruginosa (P. aeruginosa) high-risk clones (HiRiCs) have been sporadically reported in China, but the systematic analysis of genomes for these STs remains limited. This study aimed to address the evolutionary pathways underlying the emergence of HiRiCs and their routes of dissemination from Chinese and global perspectives. METHODS: The phylogenetic analysis was performed based on 416 newly sequenced clinical P. aeruginosa strains from Guangdong (GD), published genome sequences of 282 Chinese isolates, and 868 HiRiCs isolates from other countries. The genomic comparison study of global HiRiC ST244 was conducted to detect the model of global dissemination and local separation driven by association regional-specific antibiotic resistance genes. Furthermore, the evolutionary route of the emerging, China-specific HiRiC ST1971 was explored using Most Recent Common Ancestor (MRCA) analysis. RESULTS: Based on comparative genomics analysis, we found a clear geographical separation of ST244 isolates, yet with an association between ST244 isolates from GD and America. We identified a set of 38 AMR genes that contribute to the geographical separation in ST244, and we identified genetic determinants either positively (MexB) and negatively (opmD) associated with GD ST244. For the China-unique HiRiC ST1971, its evolutionary history across different continents before emerging as ST1971 in China was also deduced. CONCLUSION: This study provides insight into the specific genetics underlying regional differences among globally disseminated P. aeruginosa HiRiCs (ST244) as well as new understanding of the dissemination and evolution of a regional HiRiC (ST1971). Understanding the genetics of these and other HiRiCs may assist in controlling their emergence and further spread.

The effects of antibiotic use on the dynamics of the microbiome and resistome in pigs.

Antibiotics are widely used in pig farming across the world which has led to concerns about the potential impact on human health through the selection of antibiotic resistant pathogenic bacteria. This worry has resulted in the development of a production scheme known as pigs Raised Without Antibiotics (RWA), in which pigs are produced in commercial farms, but are ear-tagged as RWA until slaughter unless they receive treatment, thus allowing the farmer to sell the pigs either as premium priced RWA or as conventional meat. Development of antibiotic resistance in pig farming has been studied in national surveys of antibiotic usage and resistance, as well as in experimental studies of groups of pigs, but not in individual pigs followed longitudinally in a commercial pig farm. In this study, a cohort of RWA designated pigs were sampled at 10 time points from birth until slaughter along with pen-mates treated with antibiotics at the same farm. From these samples, the microbiome, determined using 16S sequencing, and the resistome, as determined using qPCR for 82 resistance genes, was investigated, allowing us to examine the difference between RWA pigs and antibiotic treated pigs. We furthermore included 176 additional pigs from six different RWA farms which were sampled at the slaughterhouse as an endpoint to substantiate the cohort as well as for evaluation of intra-farm variability. The results showed a clear effect of age in both the microbiome and resistome composition from early life up until slaughter. As a function of antibiotic treatment, however, we observed a small but significant divergence between treated and untreated animals in their microbiome composition immediately following treatment, which disappeared before 8 weeks of age. The effect on the resistome was evident and an effect of treatment could still be detected at week 8. In animals sampled at the slaughterhouse, we observed no difference in the microbiome or the resistome as a result of treatment status but did see a strong effect of farm origin. Network analysis of co-occurrence of microbiome and resistome data suggested that some resistance genes may be transferred through mobile genetic elements, so we used Hi-C metagenomics on a subset of samples to investigate this. We conclude that antibiotic treatment has a differential effect on the microbiome vs. the resistome and that although resistance gene load is increased by antibiotic treatment load, this effect disappears before slaughter. More studies are needed to elucidate the optimal way to rear pigs without antibiotics.

Whole-Genome Sequencing of 11 High-Risk Clone ST111 Pseudomonas aeruginosa Isolates from French Hospitals.

This study reports high-quality genomes of 11 sequence type 111 (ST111) isolates of Pseudomonas aeruginosa. This ST is known for its worldwide dissemination and high capacity to acquire antibiotic resistance mechanisms. This study used long- and short-read sequencing to provide high-quality closed genomes for most of the isolates.

Whole-genome sequencing reveals high-risk clones of Pseudomonas aeruginosa in Guangdong, China.

The ever-increasing prevalence of infections produced by multidrug-resistant or extensively drug-resistant Pseudomonas aeruginosa is commonly linked to a limited number of aptly-named epidemical 'high-risk clones' that are widespread among and within hospitals worldwide. The emergence of new potential high-risk clone strains in hospitals highlights the need to better and further understand the underlying genetic mechanisms for their emergence and success. P. aeruginosa related high-risk clones have been sporadically found in China, their genome sequences have rarely been described. Therefore, the large-scale sequencing of multidrug-resistance high-risk clone strains will help us to understand the emergence and transmission of antibiotic resistances in P. aeruginosa high-risk clones. In this study, 212 P. aeruginosa strains were isolated from 2 tertiary hospitals within 3 years (2018-2020) in Guangdong Province, China. Whole-genome sequencing, multi-locus sequence typing (MLST) and antimicrobial susceptibility testing were applied to analyze the genomic epidemiology of P. aeruginosa in this region. We found that up to 130 (61.32%) of the isolates were shown to be multidrug resistant, and 196 (92.45%) isolates were Carbapenem-Resistant Pseudomonas aeruginosa. MLST analysis demonstrated high diversity of sequence types, and 18 reported international high-risk clones were identified. Furthermore, we discovered the co-presence of exoU and exoS genes in 5 collected strains. This study enhances insight into the regional research of molecular epidemiology and antimicrobial resistance of P. aeruginosa in China. The high diversity of clone types and regional genome characteristics can serve as a theoretical reference for public health policies and help guide measures for the prevention and control of P. aeruginosa resistance.

Epidemiological and Genetic Characteristics of Clinical Carbapenem-Resistant Pseudomonas aeruginosa Strains in Guangdong Province, China.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a bacterial pathogen that may cause serious drug-resistant infections that are potentially fatal. To investigate the genetic characteristics of these organisms, we tested 416 P. aeruginosa strains recovered from 12 types of clinical samples collected in 29 different hospital wards in 10 hospitals in Guangdong Province, China, from 2017 to 2020. These strains were found to belong to 149 known sequence types (STs) and 72 novel STs, indicating that transmission of these strains involved multiple routes. A high rate of resistance to imipenem (89.4%) and meropenem (79.4%) and a high prevalence of pathogenic serotypes (76.4%) were observed among these strains. Six STs of global high-risk clones (HiRiCs) and a novel HiRiC strains, ST1971, which exhibited extensive drug resistance, were identified. Importantly, ST1971 HiRiC, which was unique in China, also exhibited high virulence, which alarmed the further surveillance on this highly virulent and highly resistant clone. Inactivation of the oprD gene and overexpression of efflux systems were found to be mainly responsible for carbapenem resistance in these strains; carriage of metallo-ß-lactamase (MBL)-encoding genes was less common. Interestingly, frameshift mutations (49.0%) and introduction of a stop codon (22.4%) into the oprD genes were the major mechanisms of imipenem resistance. On the other hand, expression of the MexAB-OprM efflux pump and MBL-encoding genes were mechanisms of resistance in >70% of meropenem-resistant strains. The findings presented here provide insights into the development of effective strategies for control of worldwide dissemination of CRPA. IMPORTANCE Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a major concern in clinical settings worldwide, yet few genetic and epidemiological studies on CRPA strains have been performed in China. Here, we sequence and analyze the genomes of 416 P. aeruginosa strains from hospitals in China to elucidate the genetic, phenotypic, and transmission characteristics of CRPA strains and to identify the molecular signatures responsible for the observed increase in the prevalence of CRPA infections in China. These findings may provide new insight into the development of effective strategies for worldwide control of CRPA and minimize the occurrence of untreatable infections in clinical settings.

A bittersweet fate: detection of serotype switching in Pseudomonas aeruginosa.

High-risk clone types in Pseudomonas aeruginosa are problematic global multidrug-resistant clones. However, apart from their ability to resist antimicrobial treatment, not much is known about what sets these clones apart from the multitude of other clones. In high-risk clone ST111, it has previously been shown that replacement of the native serotype biosynthetic gene cluster (O4) by a different gene cluster (O12) by horizontal gene transfer and recombination may have contributed to the global success of this clone. However, the extent to which isolates undergo this type of serotype switching has not been adequately explored in P. aeruginosa. In the present study, a bioinformatics tool has been developed and utilized to provide a first estimate of serotype switching in groups of multidrug resistant (MDR) clinical isolates. The tool detects serotype switching by analysis of core-genome phylogeny and in silico serotype. Analysis of a national survey of MDR isolates found a prevalence of 3.9 % of serotype-switched isolates in high-risk clone types ST111, ST244 and ST253. A global survey of MDR isolates was additionally analysed, and it was found that 2.3 % of isolates had undergone a serotype switch. To further understand this process, we determined the exact boundaries of the horizontally transferred serotype O12 island. We found that the size of the serotype island correlates with the clone type of the receiving isolate and additionally we found intra-clone type variations in size and boundaries. This suggests multiple serotype switch events. Moreover, we found that the housekeeping gene gyrA is co-transferred with the O12 serotype island, which prompted us to analyse this allele for all serotype O12 isolates. We found that 95 % of ST111 O12 isolates had a resistant gyrA allele and 86 % of all O12 isolates had a resistant gyrA allele. The rates of resistant gyrA alleles in isolates with other prevalent serotypes are all lower. Together, these results show that the transfer and acquisition of serotype O12 in high-risk clone ST111 has happened multiple times and may be facilitated by multiple donors, which clearly suggests a strong selection pressure for this process. However, gyrA-mediated antibiotic resistance may not be the only evolutionary driver.

Sequential interspecies interactions affect production of antimicrobial secondary metabolites in Pseudomonas protegens DTU9.1.

Soil and rhizosphere microbiomes play important roles in suppression of plant pathogens through production of antagonistic secondary metabolites, yet mechanisms that determine the strength of pathogen control are not well understood. Many Pseudomonas species are associated with soil and rhizosphere microbiomes, and their ability to suppress pathogens is well documented. Here, we investigate how interactions within the Pseudomonas genus affect their production of antimicrobial metabolites. From a biosensor-based screen, we identify P. capeferrum species as capable of modulating secondary metabolite production in P. protegens. We show that P. capeferrum alters production of pyoluteorin and 2,4-diacetylphloroglucinol (DAPG) in P. protegens via two distinct and sequential mechanisms that depends on spatial proximity of the two species. Specifically, P. capeferrum secretes a diffusible signal that induce pyoluteorin production up to 100-fold in neighboring P. protegens colonies. In contrast, the interaction results in reduced DAPG production, but only within mixed-species colonies. Additionally, we found that increased pyoluteorin production and cell lysis of P. capeferrum is required for inhibition of DAPG production, suggesting that pyoluteorin-facilitated antibiosis of P. protegens on P. capeferrum leads to release of cell-associated metabolites and subsequent inhibition of DAPG production in P. protegens. As the interaction modulates in vitro bioactivity of the species, genus-specific interactions may assist in improving efficacy of biocontrol strains and consortia.

Orally active bivalent VHH construct prevents proliferation of F4+ enterotoxigenic Escherichia coli in weaned piglets.

A major challenge in industrial pig production is the prevalence of post-weaning diarrhea (PWD) in piglets, often caused by enterotoxigenic Escherichia coli (ETEC). The increased use of antibiotics and zinc oxide to treat PWD has raised global concerns regarding antimicrobial resistance development and environmental pollution. Still, alternative treatments targeting ETEC and counteracting PWD are largely lacking. Here, we report the design of a pH, temperature, and protease-stable bivalent VHH-based protein BL1.2 that cross-links a F4+ ETEC model strain by selectively binding to its fimbriae. This protein inhibits F4+ ETEC adhesion to porcine epithelial cells ex vivo and decreases F4+ ETEC proliferation when administrated as a feed additive to weaned F4+ ETEC challenged piglets. These findings highlight the potential of a highly specific bivalent VHH-based feed additive in effectively delimiting pathogenic F4+ ETEC bacteria proliferation in piglets and may represent a sustainable solution for managing PWD while circumventing antimicrobial resistance development.

Identification and Differentiation of Pseudomonas Species in Field Samples Using an rpoD Amplicon Sequencing Methodology.

Species of the genus Pseudomonas are used for several biotechnological purposes, including plant biocontrol and bioremediation. To exploit the Pseudomonas genus in environmental, agricultural, or industrial settings, the organisms must be profiled at the species level as their bioactivity potential differs markedly between species. Standard 16S rRNA gene amplicon profiling does not allow for accurate species differentiation. Thus, the purpose of this study was to develop an amplicon-based high-resolution method targeting a 760-nucleotide (nt) region of the rpoD gene enabling taxonomic differentiation of Pseudomonas species in soil samples. The method was benchmarked on a 16-member Pseudomonas species mock community. All 16 species were correctly and semiquantitatively identified using rpoD gene amplicons, whereas 16S rRNA V3-V4 amplicon sequencing only correctly identified one species. We analyzed the Pseudomonas profiles in 13 soil samples in northern Zealand, Denmark, where samples were collected from grassland (3 samples) and agriculture soil (10 samples). Pseudomonas species represented up to 0.7% of the 16S rRNA gene abundance, of which each sampling site contained a unique Pseudomonas composition. Thirty culturable Pseudomonas strains were isolated from each grassland site and 10 from each agriculture site and identified by Sanger sequencing of the rpoD gene. In all cases, the rpoD amplicon approach identified more species than were found by cultivation, including hard-to-culture nonfluorescent pseudomonads, as well as more than were found by 16S rRNA V3-V4 amplicon sequencing. Thus, rpoD profiling can be used for species profiling of Pseudomonas, and large-scale prospecting of bioactive Pseudomonas may be guided by initial screening using this method. IMPORTANCE A high-throughput sequencing-based method for profiling of Pseudomonas species in soil microbiomes was developed and identified more species than 16S rRNA gene sequencing or cultivation. Pseudomonas species are used as biocontrol organisms and plant growth-promoting agents, and the method will allow tracing of specific species of Pseudomonas as well as enable screening of environmental samples for further isolation and exploitation.

The plasmidome of multidrug-resistant emergent Salmonella serovars isolated from poultry.

The rapid emergence of resistant bacteria is occurring worldwide. The understanding of the dissemination of antimicrobial resistance using high-throughput sequencing and bioinformatics approaches is providing valuable insights into the genetic basis of the horizontal gene transfer and the emergence of the antibiotic resistance threat. This ultimately can offer vital clues to the development of coordinated efforts to implement new policies to continue fighting against bacterial infections. The poultry microbiota is characterized as a potential reservoir of resistance genes, mostly derived from the Enterobacteriaceae which have become increasingly important in human and animal infections. In this work, complete genome sequences were achieved for four multidrug-resistant Salmonella spp. isolated from poultry from different farms in Brazil. We identified highly similar IncHI2-ST2 megaplasmids (larger than 275.000 bp) in all Salmonella isolates studied. These megaplasmids carry a resistome comprised of eleven different resistance genes (aac(6')-Iaa, aadA1b, aph(4)-Ia, aph(6)-Id, aph(3″)-Ib, aph(3')-Ia, aac(3)-Iva, sul1, tetA, tetB and dfrA1b) and four heavy metal tolerance operons (telluride, mercury, silver and copper). In conclusion, the multidrug-resistant plasmids identified in S. enterica serovar Schwarzengrund and Newport isolated from poultry show a variety of antibiotic resistance and heavy metal tolerance genes, providing advantages for the bacteria to survive under extremely unfavorable conditions.

A Whole-Cell Biosensor for Detection of 2,4-Diacetylphloroglucinol (DAPG)-Producing Bacteria from Grassland Soil.

Fluorescent Pseudomonas spp. producing the antibiotic 2,4-diacetylphloroglucinol (DAPG) are ecologically important in the rhizosphere, as they can control phytopathogens and contribute to disease suppression. DAPG can also trigger a systemic resistance response in plants and stimulate root exudation and branching as well as induce plant-beneficial activities in other rhizobacteria. While studies of DAPG-producing Pseudomonas have predominantly focused on rhizosphere niches, the ecological role of DAPG as well as the distribution and dynamics of DAPG-producing bacteria remains less well understood for other environments, such as bulk soil and grassland, where the level of DAPG producers are predicted to be low. In this study, we constructed a whole-cell biosensor for detection of DAPG and DAPG-producing bacteria from environmental samples. The constructed biosensor contains a phlF response module and either lacZ or lux genes as output modules assembled on a pSEVA plasmid backbone for easy transfer to different host species and to enable easy future genetic modifications. We show that the sensor is highly specific toward DAPG, with a sensitivity in the low nanomolar range (>20 nM). This sensitivity is comparable to the DAPG levels identified in rhizosphere samples by chemical analysis. The biosensor enables guided isolation of DAPG-producing Pseudomonas Using the biosensor, we probed the same grassland soil sampling site to isolate genetically related DAPG-producing Pseudomonas kilonensis strains over a period of 12 months. Next, we used the biosensor to determine the frequency of DAPG-producing pseudomonads within three different grassland soil sites and showed that DAPG producers can constitute part of the Pseudomonas population in the range of 0.35 to 17% at these sites. Finally, we showed that the biosensor enables detection of DAPG produced by non-Pseudomonas species. Our study shows that a whole-cell biosensor for DAPG detection can facilitate isolation of bacteria that produce this important secondary metabolite and provide insight into the population dynamics of DAPG producers in natural grassland soil.IMPORTANCE The interest in bacterial biocontrol agents as biosustainable alternatives to pesticides to increase crop yields has grown. To date, we have a broad knowledge of antimicrobial compounds, such as DAPG, produced by bacteria growing in the rhizosphere surrounding plant roots. However, compared to the rhizosphere niches, the ecological role of DAPG as well as the distribution and dynamics of DAPG-producing bacteria remains less well understood for other environments, such as bulk and grassland soil. Currently, we are restricted to chemical methods with detection limits and time-consuming PCR-based and probe hybridization approaches to detect DAPG and its respective producer. In this study, we developed a whole-cell biosensor, which can circumvent the labor-intensive screening process as well as increase the sensitivity at which DAPG can be detected. This enables quantification of relative amounts of DAPG producers, which, in turn, increases our understanding of the dynamics and ecology of these producers in natural soil environments.

Complete Genome Sequence of Pseudomonas sp. Strain SK, Isolated from Organic Wheat Rhizosphere.

Here, we report the annotated whole-genome sequence of Pseudomonas sp. strain SK, isolated in India from organic wheat rhizosphere. This strain has proved to be a species with potential biocontrol activity against soilborne plant pathogens based on antiSMASH analysis.

Recurrent tuberculosis in patients infected with the predominant Mycobacterium tuberculosis outbreak strain in Denmark. New insights gained through whole genome sequencing.

OBJECTIVES: Recurrent tuberculosis (TB) is defined by more than one TB episode per patient and is caused by reinfection with a new M. tuberculosis (Mtb) strain or relapse with the previous strain. In Denmark, a major TB outbreak caused by one specific Mtb genotype "DKC2" is ongoing. Of the 892 patients infected with DKC2 between 1992 and 2014, 32 had recurrent TB with 67 TB episodes in total. METHODS: The 32 cases were evaluated in terms of number of single-nucleotide polymorphism (SNP) differences and time between episodes derived from whole-genome sequencing data. RESULTS: For four TB cases, the subsequent episodes could be confirmed as relapse and for one case as reinfection. Eight cases with SNP differences <6, theoretically indicating relapse, could be classified as likely reinfections based on phylogenetic analysis in combination with geographical data. Subsequent TB episodes for the remaining 19 cases could not be classified as relapse or reinfection even though they all had a SNP difference of <6 SNPs. CONCLUSIONS: In newer studies, investigating recurrent TB with the use of WGS, the number of SNPs has been used to distinguish between relapse and reinfection. The algorithm proposed for this is not valid in the Danish TB outbreak setting as our findings challenge the interpretation of few SNP differences as representing relapse. However, when including phylogenetic analysis and geographical data in the analysis, classification of 13 of the 32 cases were possible.

A Rare Thioquinolobactin Siderophore Present in a Bioactive Pseudomonas sp. DTU12.1.

Many of the soil-dwelling Pseudomonas species are known to produce secondary metabolite compounds, which can have antagonistic activity against other microorganisms, including important plant pathogens. It is thus of importance to isolate new strains of Pseudomonas and discover novel or rare gene clusters encoding bioactive products. In an effort to accomplish this, we have isolated a bioactive Pseudomonas strain DTU12.1 from leaf-covered soil in Denmark. Following genome sequencing with Illumina and Oxford Nanopore technologies, we generated a complete genome sequence with the length of 5,943,629 base pairs. The DTU12.1 strain contained a complete gene cluster for a rare thioquinolobactin siderophore, which was previously described as possessing bioactivity against oomycetes and several fungal species. We placed the DTU12.1 strain within Pseudomonas gessardii subgroup of fluorescent pseudomonads, where it formed a distinct clade with other Pseudomonas strains, most of which also contained a complete thioquinolobactin gene cluster. Only two other Pseudomonas strains were found to contain the gene cluster, though they were present in a different phylogenetic clade and were missing a transcriptional regulator of the whole cluster. We show that having the complete genome sequence and establishing phylogenetic relationships with other strains can enable us to start evaluating the distribution and evolutionary origins of secondary metabolite clusters.

Complete Genome Sequence of a Bioactive Pseudomonas sp. Strain, DTU12.3, Isolated from Soil in Denmark.

Here, we report the complete annotated genome sequence of a Pseudomonas sp. strain, DTU12.3. It was isolated from leaf-covered soil in Denmark and potentially has bioactivity against certain plant pathogens.

Within-Host Adaptation Mediated by Intergenic Evolution in Pseudomonas aeruginosa.

Bacterial pathogens evolve during the course of infection as they adapt to the selective pressures that confront them inside the host. Identification of adaptive mutations and their contributions to pathogen fitness remains a central challenge. Although mutations can either target intergenic or coding regions in the pathogen genome, studies of host adaptation have focused predominantly on molecular evolution within coding regions, whereas the role of intergenic mutations remains unclear. Here, we address this issue and investigate the extent to which intergenic mutations contribute to the evolutionary response of a clinically important bacterial pathogen, Pseudomonas aeruginosa, to the host environment, and whether intergenic mutations have distinct roles in host adaptation. We characterize intergenic evolution in 44 clonal lineages of P. aeruginosa and identify 77 intergenic regions in which parallel evolution occurs. At the genetic level, we find that mutations in regions under selection are located primarily within regulatory elements upstream of transcriptional start sites. At the functional level, we show that some of these mutations both increase or decrease transcription of genes and are directly responsible for evolution of important pathogenic phenotypes including antibiotic sensitivity. Importantly, we find that intergenic mutations facilitate essential genes to become targets of evolution. In summary, our results highlight the evolutionary significance of intergenic mutations in creating host-adapted strains, and that intergenic and coding regions have different qualitative contributions to this process.

A Major Mycobacterium tuberculosis outbreak caused by one specific genotype in a low-incidence country: Exploring gene profile virulence explanations.

Denmark, a tuberculosis low burden country, still experiences significant active Mycobacterium tuberculosis (Mtb) transmission, especially with one specific genotype named Cluster 2/1112-15 (C2), the most prevalent lineage in Scandinavia. In addition to environmental factors, antibiotic resistance, and human genetics, there is increasing evidence that Mtb strain variation plays a role for the outcome of infection and disease. In this study, we explore the reasons for the success of the C2 genotype by analysing strain specific polymorphisms identified through whole genome sequencing of all C2 isolates identified in Denmark between 1992 and 2014 (n = 952), and the demographic distribution of C2. Of 234 non-synonymous (NS) monomorphic SNPs found in C2 in comparison with Mtb reference strain H37Rv, 23 were in genes previously reported to be involved in Mtb virulence. Of these 23 SNPs, three were specific for C2 including a NS mutation in a gene associated with hyper-virulence. We show that the genotype is readily transmitted to different ethnicities and is also found outside Denmark. Our data suggest that strain specific virulence factor variations are important for the success of the C2 genotype. These factors, likely in combination with poor TB control, seem to be the main drivers of C2 success.