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Objectives: The aim of this analysis was to characterize transmitted drug resistance (TDR) in Strategic Timing of Antiretroviral Treatment (START) study participants by next‐generation sequencing (NGS), a sensitive assay capable of detecting low‐frequency variants. Methods: Stored plasma from participants with entry HIV RNA > 1000 copies/mL were analysed by NGS (Illumina MiSeq). TDR was based on the WHO 2009 surveillance definition with the addition of reverse transcriptase (RT) mutations T215N and E138K, and integrase strand transfer inhibitor (INSTI) surveillance mutations (Stanford HIVdb). Drug resistance mutations (DRMs) detected at three thresholds are reported: > 2%, 5% and 20% of the viral population. Results: Between 2009 and 2013, START enrolled 4684 antiretroviral therapy (ART)‐naïve individuals in 35 countries. Baseline NGS data at study entry were available for 2902 participants. Overall prevalence rates of TDR using a detection threshold of 2%/5%/20% were 9.2%/5.6%/3.2% for nucleoside reverse transcriptase inhibitors (NRTIs), 9.2%/6.6%/4.9% for non‐NRTIs, 11.4%/5.5%/2.4% for protease inhibitors (PIs) and 3.5%/1.6%/0.1% for INSTI DRMs and varied by geographic region. Using the 2% detection threshold, individual DRMs with the highest prevalence were: PI M46IL (5.5%), RT K103NS (3.5%), RT G190ASE (3.1%), T215ISCDVEN (2.5%), RT M41L (2.2%), RT K219QENR (1.7%) and PI D30N (1.6%). INSTI DRMs were detected almost exclusively below the 20% detection threshold, most commonly Y143H (0.4%), Q148R (0.4%) and T66I (0.4%). Conclusions: Use of NGS in this study population resulted in the detection of a large proportion of low‐level variants which would not have been detected by traditional Sanger sequencing. Global surveillance studies utilizing NGS should provide a more comprehensive assessment of TDR prevalence in different regions of the world. [ABSTRACT FROM AUTHOR]

Objectives: The main objective of this analysis was to evaluate the impact of pre-existing drug resistance by next-generation sequencing (NGS) on the risk of treatment failure (TF) of first-line regimens in participants enrolled in the START study., Methods: Stored plasma from participants with entry HIV RNA >1000 copies/mL were analysed using NGS (llumina MiSeq). Pre-existing drug resistance was defined using the mutations considered by the Stanford HIV Drug Resistance Database (HIVDB v8.6) to calculate the genotypic susceptibility score (GSS, estimating the number of active drugs) for the first-line regimen at the detection threshold windows of >20%, >5%, and >2% of the viral population. Survival analysis was conducted to evaluate the association between the GSS and risk of TF (viral load >200 copies/mL plus treatment change)., Results: Baseline NGS data were available for 1380 antiretroviral therapy (ART)-naïve participants enrolled over 2009-2013. First-line ART included a non-nucleoside reverse transcriptase inhibitor (NNRTI) in 976 (71%), a boosted protease inhibitor in 297 (22%), or an integrase strand transfer inhibitor in 107 (8%). The proportions of participants with GSS <3 were 7% for >20%, 10% for >5%, and 17% for the >2% thresholds, respectively. The adjusted hazard ratio of TF associated with a GSS of 0-2.75 versus 3 in the subset of participants with mutations detected at the >2% threshold was 1.66 (95% confidence interval 1.01-2.74; p = 0.05) and 2.32 (95% confidence interval 1.32-4.09; p = 0.003) after restricting the analysis to participants who started an NNRTI-based regimen., Conclusions: Up to 17% of participants initiated ART with a GSS <3 on the basis of NGS data. Minority variants were predictive of TF, especially for participants starting NNRTI-based regimens., (© 2023 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Society for the Study of Evolution (SSE) and European Society for Evolutionary Biology (ESEN).)

In 2020, the novel coronavirus, SARS-CoV-2, caused a pandemic, which is still raging at the time of writing this. Here, we present results from SpikeSeq, the first published Sanger sequencing-based method for the detection of Variants of Concern (VOC) and key mutations, using a 1 kb amplicon from the recognized ARTIC Network primers. The proposed setup relies entirely on materials and methods already in use in diagnostic RT-qPCR labs and on existing commercial infrastructure offering sequencing services. For data analysis, we provide an automated, open source, and browser-based mutation calling software (https://github.com/kblin/covid-spike-classification, https://ssi.biolib.com/covid-spike-classification). We validated the setup on 195 SARS-CoV-2 positive samples, and we were able to profile 85% of RT-qPCR positive samples, where the last 15% largely stemmed from samples with low viral count. We compared the SpikeSeq results to WGS results. SpikeSeq has been used as the primary variant identification tool on > 10.000 SARS-CoV-2 positive clinical samples during 2021. At approximately 4€ per sample in material cost, minimal hands-on time, little data handling, and a short turnaround time, the setup is simple enough to be implemented in any SARS-CoV-2 RT-qPCR diagnostic lab. Our protocol provides results that can be used to choose antibodies in a clinical setting and for the tracking and surveillance of all positive samples for new variants and known ones such as Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) Delta (B.1.617.2), Omicron BA.1(B.1.1.529), BA.2, BA.4/5, BA.2.75.x, and many more, as of October 2022., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Infection with human immunodeficiency virus type 1 (HIV) typically results from transmission of a small and genetically uniform viral population. Following transmission, the virus population becomes more diverse because of recombination and acquired mutations through genetic drift and selection. Viral intrahost genetic diversity remains a major obstacle to the cure of HIV; however, the association between intrahost diversity and disease progression markers has not been investigated in large and diverse cohorts for which the majority of the genome has been deep-sequenced. Viral load (VL) is a key progression marker and understanding of its relationship to viral intrahost genetic diversity could help design future strategies for HIV monitoring and treatment., Methods: We analysed deep-sequenced viral genomes from 2,650 treatment-naive HIV-infected persons to measure the intrahost genetic diversity of 2,447 genomic codon positions as calculated by Shannon entropy. We tested for associations between VL and amino acid (AA) entropy accounting for sex, age, race, duration of infection, and HIV population structure., Results: We confirmed that the intrahost genetic diversity is highest in the env gene. Furthermore, we showed that mean Shannon entropy is significantly associated with VL, especially in infections of >24 months duration. We identified 16 significant associations between VL (p-value<2.0x10-5) and Shannon entropy at AA positions which in our association analysis explained 13% of the variance in VL. Finally, equivalent analysis based on variation in HIV consensus sequences explained only 2% of VL variance., Conclusions: Our results elucidate that viral intrahost genetic diversity is associated with VL and could be used as a better disease progression marker than HIV consensus sequence variants, especially in infections of longer duration. We emphasize that viral intrahost diversity should be considered when studying viral genomes and infection outcomes., Trial Registration: Samples included in this study were derived from participants who consented in the clinical trial, START (NCT00867048) (23), run by the International Network for Strategic Initiatives in Global HIV Trials (INSIGHT). All the participant sites are listed here: http://www.insight-trials.org/start/my&#95;phpscript/participating.php?by=site., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Gabrielaite et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Second primary cancer (SPC) is the second most common cause of death among patients diagnosed with head and neck cancer. This study examined the risk of SPC following oropharyngeal squamous cell carcinoma (OPSCC) and the impact of human papillomavirus (HPV) on survival following SPC. The study was a population-based, retrospective study including all patients diagnosed with OPSCC in eastern Denmark from 2000-2020 who received curative intended treatment. The incidence rate ratio (IRR), age-adjusted incidence rates (AAIR), and hazard ratios (HR) were calculated. A total of 2584 patients with primary OPSCC were included (median follow-up time: 3.1 years), with 317 patients (12.3%) diagnosed with SPC. The risk of SPC was approximately five times the occurrence of cancer in the general population (IRR: 4.96). The median time to SPC after a primary OPSCC was 2.0 years (interquartile range (IQR) = 0.6-4.2 years). HPV-positive (HPV+) patients had a significantly longer median time to SPC, and a significant better survival compared to HPV-negative (HPV-) patients. SPC was most frequently found in lungs, head, and neck (LHN) for HPV- OPSCC patients and lungs followed by gender-specific (prostate, ovaries, or endometrium) for HPV+ OPSCC. There was a significant difference between the two groups when distributed between "within" or "outside" LHN. Patients with SPC outside LHN had a significant better overall survival. This knowledge should be considered during post-treatment surveillance and might guide targeted imaging.

Objectives: It is unclear whether recurrent sputum culture with Pseudomonas aeruginosa from patients with chronic obstructive pulmonary disease (COPD) is caused by intermittent airway carriage by different P. aeruginosa lineages or persistent carriage by the same lineage, and whether lineages genetically adapt during carriage., Methods: Whole-genome sequencing was performed for P. aeruginosa isolates sampled longitudinally from sputum cultures in patients with COPD who were enrolled in an ongoing randomized controlled trial (clinicaltrials.gov: NCT03262142)., Results: A total of 153 P. aeruginosa isolates were sequenced for 23 patients during 365 days of follow-up. Recurrent presence of P. aeruginosa was seen in 19 patients (83%) and was caused by persistence of the same clonal lineage in all but one patient. We identified 38 genes mutated in parallel in two or more lineages, suggesting positive selection for adaptive mutations. Mutational enrichment analysis revealed genes important in antibiotic resistance and chronic infections to be more frequently mutated., Discussion: Recurrent P. aeruginosa was common and carried for a prolonged time after initial detection in the airways of patients with COPD. Recurrence was caused by persistence of the same clonal lineage and was associated with genetic adaptation. Trial data on possible clinical benefits of attempting antibiotic eradication of P. aeruginosa in COPD are warranted., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bacteria with increased mutation rates (mutators) are common in chronic infections and are associated with poorer clinical outcomes, especially in the case of Pseudomonas aeruginosa infecting cystic fibrosis (CF) patients. There is, however, considerable between-patient variation in both P. aeruginosa mutator frequency and the composition of co-infecting pathogen communities. We investigated whether community context might affect selection of mutators. Using an in vitro CF model community, we show that P. aeruginosa mutators were favoured in the absence of other species but not in their presence. This was because there were trade-offs between adaptation to the biotic and abiotic environments (for example, loss of quorum sensing and associated toxin production was beneficial in the latter but not the former in our in vitro model community) limiting the evolvability advantage of an elevated mutation rate. Consistent with a role of co-infecting pathogens selecting against P. aeruginosa mutators in vivo, we show that the mutation frequency of P. aeruginosa population was negatively correlated with the frequency and diversity of co-infecting bacteria in CF infections. Our results suggest that co-infecting taxa can select against P. aeruginosa mutators, which may have potentially beneficial clinical consequences., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Knowledge of the genetic variation underlying Primary Immune Deficiency (PID) is increasing. Reanalysis of genome-wide sequencing data from undiagnosed patients with suspected PID may improve the diagnostic rate., Methods: We included patients monitored at the Department of Infectious Diseases or the Child and Adolescent Department, Rigshospitalet, Denmark, for a suspected PID, who had been analysed previously using a targeted PID gene panel (457 PID-related genes) on whole exome- (WES) or whole genome sequencing (WGS) data. A literature review was performed to extend the PID gene panel used for reanalysis of single nucleotide variation (SNV) and small indels. Structural variant (SV) calling was added on WGS data., Results: Genetic data from 94 patients (86 adults) including 36 WES and 58 WGS was reanalysed a median of 23 months after the initial analysis. The extended gene panel included 208 additional PID-related genes. Genetic reanalysis led to a small increase in the proportion of patients with new suspicious PID related variants of uncertain significance (VUS). The proportion of patients with a causal genetic diagnosis was constant. In total, five patients (5%, including three WES and two WGS) had a new suspicious PID VUS identified due to reanalysis. Among these, two patients had a variant added due to the expansion of the PID gene panel, and three patients had a variant reclassified to a VUS in a gene included in the initial PID gene panel. The total proportion of patients with PID related VUS, likely pathogenic, and pathogenic variants increased from 43 (46%) to 47 (50%), as one patient had a VUS detected in both initial- and reanalysis. In addition, we detected new suspicious SNVs and SVs of uncertain significance in PID candidate genes with unknown inheritance and/or as heterozygous variants in genes with autosomal recessive inheritance in 8 patients., Conclusion: These data indicate a possible diagnostic gain of reassessing WES/WGS data from patients with suspected PID. Reasons for the possible gain included improved knowledge of genotype-phenotype correlation, expanding the gene panel, and adding SV analyses. Future studies of genotype-phenotype correlations may provide additional knowledge on the impact of the new suspicious VUSs., Competing Interests: TK has participated in advisory boards for Gilead Sciences, GlaxoSmithKline/ViiV and MSD. TK has received fees for teaching from Takeda and CSL Behring, and a research grant from Gilead Sciences. MH has participated in advisory boards for AstraZeneca, Gilead Sciences, GlaxoSmithKline/ViiV, MSD, Roche, and Sobi. MH has received fees for teaching from Gilead Sciences and GSK. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mørup, Nazaryan-Petersen, Gabrielaite, Reekie, Marquart, Hartling, Marvig, Katzenstein, Masmas, Lundgren, Murray, Helleberg and Borgwardt.)

Background: In early 2021, the SARS-CoV-2 lineage B.1.1.7 (Alpha variant) became dominant across large parts of the world. In Denmark, comprehensive and real-time test, contact-tracing, and sequencing efforts were applied to sustain epidemic control. Here, we use these data to investigate the transmissibility, introduction, and onward transmission of B.1.1.7 in Denmark., Methods: We analyzed a comprehensive set of 60,178 SARS-CoV-2 genomes generated from high-throughput sequencing by the Danish COVID-19 Genome Consortium, representing 34% of all positive cases in the period 14 November 2020 to 7 February 2021. We calculated the transmissibility of B.1.1.7 relative to other lineages using Poisson regression. Including all 1976 high-quality B.1.1.7 genomes collected in the study period, we constructed a time-scaled phylogeny, which was coupled with detailed travel history and register data to outline the introduction and onward transmission of B.1.1.7 in Denmark., Results: In a period with unchanged restrictions, we estimated an increased B.1.1.7 transmissibility of 58% (95% CI: [56%, 60%]) relative to other lineages. Epidemiological and phylogenetic analyses revealed that 37% of B.1.1.7 cases were related to the initial introduction in November 2020. The relative number of cases directly linked to introductions varied between 10 and 50% throughout the study period., Conclusions: Our findings corroborate early estimates of increased transmissibility of B.1.1.7. Both substantial early expansion when B.1.1.7 was still unmonitored and continuous foreign introductions contributed considerably to case numbers. Finally, our study highlights the benefit of balanced travel restrictions and self-isolation procedures coupled with comprehensive surveillance efforts, to sustain epidemic control in the face of emerging variants., (© 2022. The Author(s).)

The increases observed in incidence and survival of oropharyngeal squamous cell carcinoma (OPSCC) have been attributed to human papillomavirus (HPV) infection, but the survival-impact of specific genotypes is poorly understood. We investigated the potential influence of HPV genotypes on survival in HPV-positive (HPV+) OPSCC. All patients with HPV+/p16+ OPSCC and available genotype data within the period 2011 to 2017 in Eastern Denmark were included. Descriptive statistics on clinical and tumor data, as well as overall survival (OS) and recurrence-free survival (RFS) with Cox hazard models and Kaplan-Meier plots were performed. Overall, 769 HPV+/p16+ OPSCC patients were included of which genotype HPV16 accounted for 86% (n = 662). Compared to high-risk non-HPV16 genotypes (HR non-HPV16), HPV16 patients were younger at diagnosis (median years, 60 vs 64), had a higher male to female ratio (3.7:1 vs 2.1:1), and lower performance scores of ≤1 (90%, n = 559, vs 81%, n = 74). Regarding 5-year OS and RFS, no difference was observed between HPV16 and HR non-HPV16 patients. Subgrouping the HR non-HPV16 group into HPV33 (n = 57), HPV35 (n = 26) and "other genotypes" (n = 24) a significantly worse OS in the "other genotypes" group (hazard rate: 2.33, P = .027) was shown. With similar survival results between HPV16 and non-HPV16 genotypes, genotyping in OPSCC is interesting from an epidemiological point of view as well as in vaccination programs, but not a necessary addition in prognostication of HPV+/p16+ OPSCC., (© 2021 UICC.)

Following emergence of the SARS-CoV-2 variant Omicron in November 2021, the dominant BA.1 sub-lineage was replaced by the BA.2 sub-lineage in Denmark. We analysed the first 2,623 BA.2 cases from 29 November 2021 to 2 January 2022. No epidemiological or clinical differences were found between individuals infected with BA.1 versus BA.2. Phylogenetic analyses showed a geographic east-to-west transmission of BA.2 from the Capital Region with clusters expanding after the Christmas holidays. Mutational analysis shows distinct differences between BA.1 and BA.2.

Introduction: Extended-spectrum beta-lactamase (ESBL) producing Escherichia coli have become prevalent worldwide, with E. coli of sequence type 131 (ST131) as the dominant genotype. E. coli ST131 predominantly exhibits the serotype O25, is associated with the ESBL CTX-M-15 and belongs to a well-defined subclade within the Fim H 30-R clade, Fim H 30-Rx/C2. Multidrug resistance may have fitness costs for the bacteria. The aim of the current study was to investigate the fitness burden compared to a susceptible ST131 isolate without resistance genes in vitro and in vivo and describe genetic differences between fit and less fit isolates., Materials and Methods: From a collection of clinical ESBL and non-ESBL E. coli isolates from urinary tract infection, we selected 16 bla CTX-M-15 -positive isolates of ST131. The in vitro fitness was examined, and relative bacterial fitness (fit t ) was determined by direct competition with a fully susceptible ST131 isolate and illustrated in percent, with <100% resulting in a lower fitness, compared to the susceptible reference isolate. The isolates were subjected to whole-genome sequencing and analyzed for resistance markers, plasmids, phage content, and serotype. In vivo competition was tested in a mouse colonization model., Results: The majority (12 out of 16) of the CTX-M-15-producing isolates had a slightly lower relative fitness compared to the susceptible ST131 isolate (mean, 97.6%; range, 82.6-108%) in vitro . Three isolates had a better fitness than the susceptible ST131 isolate, and one isolate had an identical fitness to the susceptible ST131 isolate. The in vitro fitness showed no correlation to the number of plasmids, number of phages, number of resistances, or genome size. For the in vivo competition assays, all three ESBL-producing isolates showed better colonization of the ESBL-resistant ST131 isolates compared to the susceptible ST131 isolate., Conclusion: This study shows that ESBL-producing ST131/ H 30-Rx are not necessarily burdened by multidrug resistance, however, have a better in vitro fitness than the susceptible isolate. These data contribute to the understanding of the success of ST131/ H 30-Rx, although they do not indicate ways to overcome this highly fit, virulent, and antimicrobial-resistant clone., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hertz, Marvig, Frimodt-Møller and Nielsen.)

Background: Single-nucleotide polymorphisms (SNPs) of the glucocorticoid receptor (GR) gene NR3C1 have been associated with an altered sensitivity to glucocorticoids, and thus may alter the therapeutic effects of glucocorticoids. We investigated the prevalence of adrenal suppression after treatment with glucocorticoids and evaluated whether GR SNPs were associated with altered risks of adrenal suppression and metabolic disorders in patients with chronic obstructive pulmonary disease (COPD)., Methods: In an observational prospective cohort study, we recruited 78 patients with severe COPD receiving 5 days glucocorticoid treatment for an exacerbation of COPD. In total, 55% of these patients were also receiving regular inhaled corticosteroids (ICS). Adrenal function was evaluated with a corticotropin test 30 days after the exacerbation. Patients were genotyped for Bcl1, N363S, ER22/23EK, and 9β SNPs., Results: The prevalence of adrenal suppression (corticotropin-stimulated plasma-cortisol ≤ 420 nmol/L) 30 days after glucocorticoid treatment was 4/78 (5%). There was no difference between carriers and non-carriers of the polymorphisms (Bcl1, 9β, ER22/23K, and N363S) in corticotropin stimulated plasma-cortisol concentrations. In the haplotype analyses, we included the 50 patients who had a high-sensitivity (76%), a low-sensitivity (4%), or a wild-type (20%) GR haplotype. There was no difference in the frequency of adrenal suppression or metabolic disorders between the two stratified groups: (a) high-sensitivity (Bcl1 and/or N363S) haplotypes vs. (b) low-sensitivity (9β and/or ER22/23K) plus wild-type haplotypes (p > 0.05). Carriers of the high-sensitivity GR gene haplotype exhibited a steeper decline in stimulated P-cortisol with increased ICS dose (slope, -1.35 vs. 0.94; p = 0.17), compared to the group with low-sensitivity or wild-type haplotypes, respectively., Conclusions: In total, 5% of patients exhibited insufficient adrenal function. The Bcl1 and N363S polymorphisms did not seem to increase the risk of glucocorticoid suppression or metabolic disorders in adults treated with glucocorticoids for COPD exacerbations., Competing Interests: The authors have declared that no competing interests exist.

Background: Understanding the genetic interplay between human hosts and infectious pathogens is crucial for how we interpret virulence factors. Here, we tested for associations between HIV and host genetics, and interactive genetic effects on viral load (VL) in HIV-positive antiretroviral treatment-naive clinical trial participants., Methods: HIV genomes were sequenced and the encoded amino acid (AA) variants were associated with VL, human single nucleotide polymorphisms (SNPs), and imputed HLA alleles using generalized linear models with Bonferroni correction., Results: Human (388 501 SNPs) and HIV (3010 variants) genetic data were available for 2122 persons. Four HIV variants were associated with VL (P < 1.66 × 10-5). Twelve HIV variants were associated with a range of 1-512 human SNPs (P < 4.28 × 10-11). We found 46 associations between HLA alleles and HIV variants (P < 1.29 × 10-7). HIV variants and immunotypes when analyzed separately were associated with lower VL, whereas the opposite was true when analyzed in concert. Epitope binding predictions supported our observations., Conclusions: Our results show the importance of immunotype specificity on viral antigenic determinants, and the identified genetic interplay emphasizes that viral and human genetics should be studied in the context of each other.Clinical Trials Registration: NCT00867048., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Copy-number variations (CNVs) have important clinical implications for several diseases and cancers. Relevant CNVs are hard to detect because common structural variations define large parts of the human genome. CNV calling from short-read sequencing would allow single protocol full genomic profiling. We reviewed 50 popular CNV calling tools and included 11 tools for benchmarking in a reference cohort encompassing 39 whole genome sequencing (WGS) samples paired current clinical standard-SNP-array based CNV calling. Additionally, for nine samples we also performed whole exome sequencing (WES), to address the effect of sequencing protocol on CNV calling. Furthermore, we included Gold Standard reference sample NA12878, and tested 12 samples with CNVs confirmed by multiplex ligation-dependent probe amplification (MLPA). Tool performance varied greatly in the number of called CNVs and bias for CNV lengths. Some tools had near-perfect recall of CNVs from arrays for some samples, but poor precision. Several tools had better performance for NA12878, which could be a result of overfitting. We suggest combining the best tools also based on different methodologies: GATK gCNV, Lumpy, DELLY, and cn.MOPS. Reducing the total number of called variants could potentially be assisted by the use of background panels for filtering of frequently called variants.

Hospitalization and treatment with antibiotics increase the risk of acquiring multidrug-resistant bacteria due to antibiotic-mediated changes in patient microbiota. This study aimed to investigate how broad- and narrow-spectrum antibiotics affect the gut microbiome and the resistome in antibiotic naïve patients during neurointensive care. Patients admitted to the neurointensive care unit were treated with broad-spectrum (meropenem or piperacillin/tazobactam) or narrow-spectrum antibiotic treatment (including ciprofloxacin, cefuroxime, vancomycin and dicloxacillin) according to clinical indications. A rectal swab was collected from each patient before and after 5-7 days of antibiotic therapy ( N = 34), respectively. Shotgun metagenomic sequencing was performed and the composition of metagenomic species (MGS) was determined. The resistome was characterized with CARD RGI software and the CARD database. As a measure for selection pressure in the patient, we used the sum of the number of days with each antibiotic (antibiotic days). We observed a significant increase in richness and a tendency for an increase in the Shannon index after narrow-spectrum treatment. For broad-spectrum treatment the effect was more diverse, with some patients increasing and some decreasing in richness and Shannon index. This was studied further by comparison of patients who had gained or lost >10 MGS, respectively. Selection pressure was significantly higher in patients with decreased richness and a decreased Shannon index who received the broad treatment. A decrease in MGS richness was significantly correlated to the number of drugs administered and the selection pressure in the patient. Bray-Curtis dissimilarities were significant between the pre- and post-treatment of samples in the narrow group, indicating that the longer the narrow-spectrum treatment, the higher the differences between the pre- and the post-treatment microbial composition. We did not find significant differences between pre- and post-treatment for both antibiotic spectrum treatments; however, we observed that most of the antibiotic class resistance genes were higher in abundance in post-treatment after broad-spectrum treatment.

Introduction SARS-CoV-2 outbreaks at care homes are associated with a high morbidity and mortality. We aimed to study the molecular epidemiology of a major care home outbreak in Denmark. Methods After a staff member had been tested positive on 16 November 2020, a bundle approach programme was initiated including frequent surveillance screenings of residents and staff, isolation and cohorting procedures. This approach also involved limiting the number of visitors and enhancing the use of personal protective equipment, hand hygiene, and environmental cleaning. Naso/oropharyngeal swabs were tested for SARS-CoV-2 by polymerase chain reaction. Available positive samples were sequenced and phylogenetic relationships between the outbreak and local circulating strains were reconstructed. Results In all, 50% (56/114) of residents and 26% (49/190) of staff members became infected during the 46-day outbreak period. Altogether 16% of the infected residents died within 30 days after becoming infected. A total of 44% (46/105) of the samples with SARS-CoV-2 were sequenced. and phylogenetic analysis demonstrated a dominant outbreak lineage belonging to Global Lineage B.1.1.29 containing the mutation I233V in the S gene. The outbreak lineage was detected in the community 28 days before its introduction into the care home. Conclusions Introduction of SARS-CoV-2 to care homes is associated with severe outbreaks. Initiation of a bundle approach infection control programme in addition to measures ensuring enhanced herd immunity were successful in controlling the outbreak. Genome sequencing proved to be a powerful tool to describe the relatedness of the various clones and may help focusing outbreak interventions. Funding The study was funded in part by The Poul Due Jensen Foundation and The Danish Ministry of Higher Education and Science. The authors have no conflicts of interest to report. Trial registration not relevant., (Articles published in the DMJ are “open access”. This means that the articles are distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits any non-commercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.)

Achromobacter spp. are emerging pathogens in patients with cystic fibrosis (CF) and Achromobacter spp. caused infections are associated with more severe disease outcomes and high intrinsic antibiotic resistance. While conventional CF pathogens are studied extensively, little is known about the genetic determinants leading to antibiotic resistance and the genetic adaptation in Achromobacter spp. infections. Here, we analysed 101 Achromobacter spp. genomes from 51 patients with CF isolated during the course of up to 20 years of infection to identify within-host adaptation, mutational signatures and genetic variation associated with increased antibiotic resistance. We found that the same regulatory and inorganic ion transport genes were frequently mutated in persisting clone types within and between Achromobacter species, indicating convergent genetic adaptation. Genome-wide association study of six antibiotic resistance phenotypes revealed the enrichment of associated genes involved in inorganic ion transport, transcription gene enrichment in β-lactams, and energy production and translation gene enrichment in the trimethoprim/sulfonamide group. Overall, we provide insights into the pathogenomics of Achromobacter spp. infections in patients with CF airways. Since emerging pathogens are increasingly recognized as an important healthcare issue, our findings on evolution of antibiotic resistance and genetic adaptation can facilitate better understanding of disease progression and how mutational changes have implications for patients with CF.

Bacterial pathogens evolve during chronic colonization of the human host by selection for pathoadaptive mutations. One of the emerging and understudied bacterial species causing chronic airway infections in patients with cystic fibrosis (CF) is Achromobacter xylosoxidans. It can establish chronic infections in patients with CF, but the genetic and phenotypic changes associated with adaptation during these infections are not completely understood. In this study, we analyzed the whole-genome sequences of 55 clinical A. xylosoxidans isolates longitudinally collected from the sputum of 6 patients with CF. Four genes encoding regulatory proteins and two intergenic regions showed convergent evolution, likely driven by positive selection for pathoadaptive mutations, across the different clones of A. xylosoxidans. Most of the evolved isolates had lower swimming motility and were resistant to multiple classes of antibiotics, while fewer of the evolved isolates had slower growth or higher biofilm production than the first isolates. Using a genome-wide association study method, we identified several putative genetic determinants of biofilm formation, motility and β-lactam resistance in this pathogen. With respect to antibiotic resistance, we discovered that a combination of mutations in pathoadaptive genes ( phoQ and bigR ) and two other genes encoding regulatory proteins ( spoT and cpxA ) were associated with increased resistance to meropenem and ceftazidime. Altogether, our results suggest that genetic changes within regulatory loci facilitate within-host adaptation of A. xylosoxidans and the emergence of adaptive phenotypes, such as antibiotic resistance or biofilm formation. IMPORTANCE A thorough understanding of bacterial pathogen adaptation is essential for the treatment of chronic bacterial infections. One unique challenge in the analysis and interpretation of genomics data is identifying the functional impact of mutations accumulated in the bacterial genome during colonization in the human host. Here, we investigated the genomic and phenotypic evolution of A. xylosoxidans in chronic airway infections of patients with CF and identified several mutations associated with the phenotypic evolution of this pathogen using genome-wide associations. Identification of phenotypes under positive selection and the associated mutations can enlighten the adaptive processes of this emerging pathogen in human infections and pave the way for novel therapeutic interventions.

Understanding of pandemics depends on the characterization of pathogen collections from well-defined and demographically diverse cohorts. Since its emergence in Congo almost a century ago, Human Immunodeficiency Virus Type 1 (HIV-1) has geographically spread and genetically diversified into distinct viral subtypes. Phylogenetic analysis can be used to reconstruct the ancestry of the virus to better understand the origin and distribution of subtypes. We sequenced two 3.6-kb amplicons of HIV-1 genomes from 3,197 participants in a clinical trial with consistent and uniform sampling at sites across 35 countries and analyzed our data with another 2,632 genomes that comprehensively reflect the HIV-1 genetic diversity. We used maximum likelihood phylogenetic analysis coupled with geographical information to infer the state of ancestors. The majority of our sequenced genomes ( n  = 2,501) were either pure subtypes (A-D, F, and G) or CRF01&#95;AE. The diversity and distribution of subtypes across geographical regions differed; USA showed the most homogenous subtype population, whereas African samples were most diverse. We delineated transmission of the four most prevalent subtypes in our dataset (A, B, C, and CRF01&#95;AE), and our results suggest both continuous and frequent transmission of HIV-1 over country borders, as well as single transmission events being the seed of endemic population expansions. Overall, we show that coupling of genetic and geographical information of HIV-1 can be used to understand the origin and spread of pandemic pathogens., (© The Author(s) 2021. Published by Oxford University Press.)

Intense genome sequencing of Pseudomonas aeruginosa isolates from cystic fibrosis (CF) airways has shown inefficient eradication of the infecting bacteria, as well as previously undocumented patient-to-patient transmission of adapted clones. However, genome sequencing has limited potential as a predictor of chronic infection and of the adaptive state during infection, and thus there is increasing interest in linking phenotypic traits to the genome sequences. Phenotypic information ranges from genome-wide transcriptomic analysis of patient samples to determination of more specific traits associated with metabolic changes, stress responses, antibiotic resistance and tolerance, biofilm formation and slow growth. Environmental conditions in the CF lung shape both genetic and phenotypic changes of P. aeruginosa during infection. In this Review, we discuss the adaptive and evolutionary trajectories that lead to early diversification and late convergence, which enable P. aeruginosa to succeed in this niche, and we point out how knowledge of these biological features may be used to guide diagnosis and therapy.

Whenever Pseudomonas aeruginosa is cultured from cystic fibrosis (CF) patient airways, the primary goal is eradication by antibiotic therapy. Success is defined by ≥6 months of negative bacterial airway cultures. However, we suspect that P. aeruginosa persists in airways without clinical detection for long periods.Out of 298 P. aeruginosa -infected Copenhagen CF patients, we identified 80 with complete P. aeruginosa monitoring records and measured their maximum P. aeruginosa -free eradication periods (MEP). Isolates from 72 patients were whole-genome sequenced (n=567) and clone typed. Select isolate relatedness was examined through phylogenetic analysis and phenotypic multivariate modelling.69 (86%) patients exhibited eradication in the monitoring period (2002-2018). Sequenced isolates bridged the MEP of 42 patients, and the same clone type persisted over the MEP in 18 (43%) patients. Patients with failed eradication were on average treated more intensively with antibiotics, but this may be linked to their more severe pre-MEP infection trajectories. Of the 42 patients, 26 also had sinus surgery; the majority (n=15) showed MEPs adjacent to surgery, and only five had persisting clone types. Importantly, combined phylogenetic-phenomic evaluation suggests that persisting clone types are a result of re-emergence of the same strain rather than re-infection from the environment, and similar relatedness is exhibited by paired lower and upper airway samples and in transmission cases.In conclusion, nearly half of CF patients with supposed eradication may not truly be cleared of their original bacteria according to omics-based monitoring. This distinct cohort that is persistently infected would probably benefit from tailored antibiotic therapy., Competing Interests: Conflict of interest: J.A. Bartell reports grants from Cystic Fibrosis Foundation (grant number BARTEL18F0), during the conduct of the study. Conflict of interest: L.M. Sommer has nothing to disclose. Conflict of interest: R.L. Marvig reports grants from Danish National Research Foundation (grant number 126), during the conduct of the study. Conflict of interest: M. Skov has nothing to disclose. Conflict of interest: T. Pressler has nothing to disclose. Conflict of interest: S. Molin reports grants from Cystic Fibrosis Foundation (grant MOLIN18G0), during the conduct of the study. Conflict of interest: H.K. Johansen has nothing to disclose., (Copyright ©ERS 2021.)

Achromobacter species are increasingly being detected in patients with cystic fibrosis (CF), and this emerging pathogen is associated with antibiotic resistance and more-severe disease outcomes. Nonetheless, little is known about the extent of transmission and antibiotic resistance development in Achromobacter infections. We sequenced the genomes of 101 Achromobacter clinical isolates (identified as Achromobacter xylosoxidans based on matrix-assister laser desorption ionization-time of flight [MALDI-TOF] or API N20 typing) collected from 51 patients with CF-the largest longitudinal data set to date. We performed phylogenetic analysis on the genomes and combined this with epidemiological and antibiotic resistance data to identify patient-to-patient transmission and the development of antibiotic resistance. We confirmed that the MALDI-TOF or API N20 method was not sufficient for Achromobacter species-level typing and that the population of Achromobacter isolates was composed of five different species, among which A. xylosoxidans accounted for 52% of infections. Most patients were infected by unique Achromobacter clone types; nonetheless, suspected patient-to-patient transmission cases identified by shared clone types were observed in 35% ( n  = 18) of patients. In 15 of 16 cases, the suspected transmissions were further supported by genome- or clinic visit-based epidemiological analysis. Finally, we found that resistance developed over time. We show that whole-genome sequencing (WGS) is essential for Achromobacter species typing and identification of patient-to-patient transmission, which was revealed for Achromobacter ruhlandii , A. xylosoxidans , and, for the first time, Achromobacter insuavis Furthermore, we show that the development of antibiotic resistance is associated with chronic Achromobacter infections. Our findings emphasize that transmission and antibiotic resistance should be considered in future treatment strategies., (Copyright © 2021 Gabrielaite et al.)

Purpose: Historically, cancer predisposition syndromes (CPSs) were rarely established for children with cancer. This nationwide, population-based study investigated how frequently children with cancer had or were likely to have a CPS., Methods: Children (0-17 years) in Denmark with newly diagnosed cancer were invited to participate in whole-genome sequencing of germline DNA. Suspicion of CPS was assessed according to Jongmans'/McGill Interactive Pediatric OncoGenetic Guidelines (MIPOGG) criteria and familial cancer diagnoses were verified using population-based registries., Results: 198 of 235 (84.3%) eligible patients participated, of whom 94/198 (47.5%) carried pathogenic variants (PVs) in a CPS gene or had clinical features indicating CPS. Twenty-nine of 198 (14.6%) patients harbored a CPS, of whom 21/198 (10.6%) harbored a childhood-onset and 9/198 (4.5%) an adult-onset CPS. In addition, 23/198 (11.6%) patients carried a PV associated with biallelic CPS. Seven of the 54 (12.9%) patients carried two or more variants in different CPS genes. Seventy of 198 (35.4%) patients fulfilled the Jongmans' and/or MIPOGG criteria indicating an underlying CPS, including two of the 9 (22.2%) patients with an adult-onset CPS versus 18 of the 21 (85.7%) patients with a childhood-onset CPS (p = 0.0022), eight of the additional 23 (34.8%) patients with a heterozygous PV associated with biallelic CPS, and 42 patients without PVs. Children with a central nervous system (CNS) tumor had family members with CNS tumors more frequently than patients with other cancers (11/44, p = 0.04), but 42 of 44 (95.5%) cases did not have a PV in a CPS gene., Conclusion: These results demonstrate the value of systematically screening pediatric cancer patients for CPSs and indicate that a higher proportion of childhood cancers may be linked to predisposing germline variants than previously supposed., Competing Interests: The authors have declared that no competing interests exist.

Genome analyses have documented that there are differences in gene repertoire between evolutionary distant lineages of the same bacterial species; however, less is known about microevolutionary dynamics of gene loss and acquisition within bacterial lineages as they evolve over years. Here, we analyzed the genomes of 45 Pseudomonas aeruginosa lineages evolving in the lungs of cystic fibrosis (CF) patients to identify genes that are lost or acquired during the first years of infection. On average, lineage genome content changed by 88 genes (range, 0 to 473). Genes were more often lost than acquired, and prophage genes were more variable than bacterial genes. We identified convergent loss or acquisition of the same genes across lineages, suggesting selection for loss and acquisition of certain genes in the host environment. We found that a notable proportion of such genes are associated with virulence; a trait previously shown to be important for adaptation. Furthermore, we also compared the genomes across lineages to show that the within-lineage variable genes (i.e., genes that had been lost or acquired during the infection) often belonged to genomic content not shared across all lineages. In sum, our analysis adds to the knowledge on the pace and drivers of gene loss and acquisition in bacteria evolving over years in a human host environment and provides a basis to further understand how gene loss and acquisition play roles in lineage differentiation and host adaptation. IMPORTANCE Bacterial airway infections, predominantly caused by P. aeruginosa , are a major cause of mortality and morbidity of CF patients. While short insertions and deletions as well as point mutations occurring during infection are well studied, there is a lack of understanding of how gene loss and acquisition play roles in bacterial adaptation to the human airways. Here, we investigated P. aeruginosa within-host evolution with regard to gene loss and acquisition. We show that during long-term infection P. aeruginosa genomes tend to lose genes, in particular, genes related to virulence. This adaptive strategy allows reduction of the genome size and evasion of the host's immune response. This knowledge is crucial to understand the basic mutational steps that, on the timescale of years, diversify lineages and adds to the identification of bacterial genetic determinants that have implications for CF disease., (Copyright © 2020 Gabrielaite et al.)

A previously healthy 19-year-old Syrian man presented with atypical and severe mucosal leishmaniasis caused by Leishmania tropica . During a 2-year period, he had three severe relapses despite various treatment strategies, including liposomal amphotericin B and Miltefosine. Because of the unusual clinical presentation, potential underlying immunodeficiency was investigated. Normal T and NK cell counts were found. The B cell count was slightly elevated at 0.7 × 10 9 cells/L (0.09 × 10 9 to 0.57 × 10 9 cells/L), but the proportions of memory and isotype switched memory B cells were severely diminished IgG levels were low, at 309 mg/dL (610-1490 mg/dL). The initial IgM and IgA levels were within normal range, but the IgA levels decreased to 57 mg/dL (70-430 mg/dL) during follow up. Common variable immunodeficiency (CVID) was initially suspected, because the immunological results of low IgG and IgA, low switched memory B cells, no profound T cell deficiency found and absence of secondary cause of hypogammaglobulinemia were compatible with this diagnosis (ESID 2019). However, the highly unusual and severe clinical presentation of L. tropica is not suggestive of B-cell deficiency or CVID. Eventually a pathogenic nonsense variant in the CD40 ligand gene [p.(Arg11 ∗ )] was identified by whole genome sequencing, thus enabling the diagnosis of X-linked hyper IgM syndrome. This case illustrates and supports the potential for the use of whole genome sequencing in accurate diagnosis of primary immunodeficiencies., (Copyright © 2020 Drabe, Marvig, Borgwardt, Lundgren, Maquart, Katzenstein and Helleberg.)

We sequenced 29 carbapenemase-producing Klebsiella pneumoniae isolates from a neonatal intensive care unit in Ghana. Twenty-eight isolates were sequence type 17 with bla OXA-181 and differed by 0-32 single-nucleotide polymorphisms. Improved surveillance and infection control are needed to characterize and curb the spread of multidrug-resistant organisms in sub-Saharan Africa.

Background: Bacterial gene loss and acquisition is a well-known phenomenon which contributes to bacterial adaptation through changes in important phenotypes such as virulence, antibiotic resistance and metabolic capability. While advances in DNA sequencing have accelerated our ability to generate short genome sequence reads to disentangle phenotypic changes caused by gene loss and acquisition, the short-read genome sequencing often results in fragmented genome assemblies as a basis for identification of gene loss and acquisition events. However, sensitive and precise determination of gene content change for fragmented genome assemblies remains challenging as analysis needs to account for cases when only a fragment of the gene is assembled or when the gene assembly is split in more than one contig., Results: We developed GenAPI, a command-line tool that is designed to compare the gene content of bacterial genomes for which only fragmented genome assemblies are available. GenAPI, unlike other available tools of similar purpose, accounts for imperfections in sequencing and assembly, and aims to compensate for them. We tested the performance of GenAPI on three different datasets to show that GenAPI has a high sensitivity while it maintains precision when dealing with partly assembled genes in both simulated and real datasets. Furthermore, we benchmarked the performance of GenAPI with six popular tools for gene presence-absence identification., Conclusions: Our developed bioinformatics tool, called GenAPI, has the same precision and recall rates when analyzing complete genome sequences as the other tools of the same purpose; however, GenAPI's performance is markedly better on fragmented genome assemblies.

Pseudomonas aeruginosa exploits intrinsic and acquired resistance mechanisms to resist almost every antibiotic used in chemotherapy. Antimicrobial resistance in P. aeruginosa isolates recovered from cystic fibrosis (CF) patients is further enhanced by the occurrence of hypermutator strains, a hallmark of chronic infections in CF patients. However, the within-patient genetic diversity of P. aeruginosa populations related to antibiotic resistance remains unexplored. Here, we show the evolution of the mutational resistome profile of a P. aeruginosa hypermutator lineage by performing longitudinal and transversal analyses of isolates collected from a CF patient throughout 20 years of chronic infection. Our results show the accumulation of thousands of mutations, with an overall evolutionary history characterized by purifying selection. However, mutations in antibiotic resistance genes appear to have been positively selected, driven by antibiotic treatment. Antibiotic resistance increased as infection progressed toward the establishment of a population constituted by genotypically diversified coexisting sublineages, all of which converged to multidrug resistance. These sublineages emerged by parallel evolution through distinct evolutionary pathways, which affected genes of the same functional categories. Interestingly, ampC and ftsI , encoding the β-lactamase and penicillin-binding protein 3, respectively, were found to be among the most frequently mutated genes. In fact, both genes were targeted by multiple independent mutational events, which led to a wide diversity of coexisting alleles underlying β-lactam resistance. Our findings indicate that hypermutators, apart from boosting antibiotic resistance evolution by simultaneously targeting several genes, favor the emergence of adaptive innovative alleles by clustering beneficial/compensatory mutations in the same gene, hence expanding P. aeruginosa strategies for persistence., (Copyright © 2020 American Society for Microbiology.)

Whole-genome sequencing has enabled detailed studies on bacterial evolution during infection, but there is limited knowledge on intraclonal variation. In this study, we sought to provide a snapshot of the intraclonal diversity of Escherichia coli as both commensal in the faecal environment and pathogen during urinary tract infection, respectively. This was performed by whole-genome sequencing and analyses of single nucleotide polymorphisms (SNPs) and gene-content variation in ten isolates belonging to the same clone and isolated from rectal swabs or urine samples. We identified only one clone in eight of the nine urines sampled (89 %). In both the commensal and pathogenic state, the within-host diversity was limited with intraclonal SNP diversity of 0-2 non-synonymous SNPs for each clone. The genetic diversity showed variation in gene content in a range of 2-15 genes in total for all clones, including genes positioned on plasmids, and in the K- and O-antigen cluster. The observed SNP- and gene variation shows that sampling of one colony would be enough for surveillance, outbreak investigations and clonal evolution. However, for studies of adaptation during or between colonization and infection, this variation is relevant to consider., Competing Interests: Declaration of Competing Interest None to declare., (Copyright © 2020 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Carriage of multidrug resistant (MDR) Gram-negative bacteria (GN) in hospitalized neonates may increase the risk of difficult-to-treat invasive infections at neonatal intensive care units (NICUs). Data on MDRGN carriage among hospitalized newborns in Africa are limited., Methods: We conducted a cross-sectional study at the NICUs of 2 tertiary hospitals in Ghana. Swabs from the axilla, groin, perianal region, and the environment were cultured, GN were identified, and antibiotic susceptibility was tested. We obtained blood culture isolates from neonates with sepsis. Whole-genome sequencing was used to characterize carbapenemase-producing Klebsiella pneumoniae . Typing was done by multilocus sequence typing (MLST) and single nucleotide polymorphism (SNP) analysis., Results: A total of 276 GN were isolated from 228 screened neonates. Pathogenic GN were cultured in 76.8% (175 of 228) of neonates. Klebsiella spp (41.7%; 115 of 276) and Escherichia coli (26.4%; 73 of 276) were the commonest organisms. Carriage rates of MDRGN and third-generation cephalosporin resistant organisms were 49.6% (113 of 228) and 46.1% (105 of 228), respectively. Among Klebsiella spp, 75.6% (87 of 115) phenotypically expressed extended-spectrum β-lactamase activity, whereas 15.6% expressed carbapenemase and harbored bla- OXA-181 and bla- CTX-M-15 . Overall, 7.0% (16 of 228) of neonates developed GN bloodstream infection. In 2 of 11 neonates, sequencing showed the same identity between carriage and the bloodstream isolate. Length of stay before specimen collection and antibiotic use were independently associated with carriage rates, which increased from 13% at admission to 42% by day 2 and reached a plateau at 91% by day 15., Conclusions: High carriage rates of MDRGN, including carbapenemase-producing enterobacterales may be an emerging problem in NICUs in Africa., (© The Author(s) 2020. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: In low- and middle-income countries (LMICs) the rate of surgical site infections (SSI) is high, leading to negative patient outcomes and excess healthcare costs. A causal relationship between airborne bacteria in the operating room and SSI has not been established, at a molecular or genetic level. We studied the relationship between intraoperative airborne bacteria and bacteria causing SSI in an LMIC., Methods: Active air sampling using a portable impactor was performed during clean or clean-contaminated elective surgical procedures. Active patient follow-up consisting of phone calls and clinical examinations was performed 3, 14 and 30 days after surgery. Bacterial isolates recovered from SSI and air samples were compared by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) identification, ribotyping, whole genome sequencing (WGS), and metagenomic analysis., Results: Of 128 included patients, 116 (91%) completed follow-up and 11 (9%) developed SSI. Known pathogenic bacteria were isolated from intraoperative air samples in all cases with SSI. A match between air and SSI isolates was found by MALDI-TOF in eight cases. Matching ribotypes were found in six cases and in one case both WGS and metagenomic analysis showed identity between air- and SSI-isolates., Conclusion: The study showed high levels of intraoperative airborne bacteria, an SSI-rate of 9% and a genetic link between intraoperative airborne bacteria and bacteria isolated from SSIs. This indicates the need for awareness of intraoperative air quality in LMICs., (Copyright © 2019 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Behçet disease (BD) is an immune-mediated disease. The cause of BD remains unknown, but the existence of multiple pathological pathways is suspected, including different genetic factors. Polymorphisms in ERAP1 gene have been associated with an increased risk of BD. However, while current BD-associated ERAP1 variants are suggested to contribute to disease by altering the activity of the encoded protein, there is no knowledge of variants that alter the expression level of ERAP1, despite previous associations between ERAP1 expression and BD. Here, we used whole-exome sequencing of a patient with a Behçet-like MHC-I-opathy to identify that the patient, unlike its healthy parents, was homozygous for a rare 1-bp deletion, rs140416843, in the promoter region of ERAP1. rs140416843 has not previously been associated with disease, but is linked to ERAP1 haplotype Hap10 which is associated with BD. The expression of ERAP1 by both RT-qPCR and RNA sequencing showed that ERAP1 mRNA expression correlated with the zygosity for the identified deletion and was decreased in comparison to a healthy cohort. In conclusion, we diagnosed the patient as having BD, and hypothesize that rs140416843-mediated changes in ERAP1 expression play a causative role in BD and that this risk factor is contributing to the association between Hap10 and BD. This is the first report to identify a variant that may cause BD by altering the expression of ERAP1, and our findings suggest that downregulation of ERAP1 expression can serve as a diagnostic marker for BD.

Filamentous bacteriophage (Pf phage) contribute to the virulence of Pseudomonas aeruginosa infections in animal models, but their relevance to human disease is unclear. We sought to interrogate the prevalence and clinical relevance of Pf phage in patients with cystic fibrosis (CF) using sputum samples from two well-characterized patient cohorts. Bacterial genomic analysis in a Danish longitudinal cohort of 34 patients with CF revealed that 26.5% ( n = 9) were consistently Pf phage positive. In the second cohort, a prospective cross-sectional cohort of 58 patients with CF at Stanford, sputum qPCR analysis showed that 36.2% ( n = 21) of patients were Pf phage positive. In both cohorts, patients positive for Pf phage were older, and in the Stanford CF cohort, patients positive for Pf phage were more likely to have chronic P. aeruginosa infection and had greater declines in pulmonary function during exacerbations than patients negative for Pf phage presence in the sputum. Last, P. aeruginosa strains carrying Pf phage exhibited increased resistance to antipseudomonal antibiotics. Mechanistically, in vitro analysis showed that Pf phage sequesters these same antibiotics, suggesting that this mechanism may thereby contribute to the selection of antibiotic resistance over time. These data provide evidence that Pf phage may contribute to clinical outcomes in P. aeruginosa infection in CF., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

A single cheating mutant can lead to the invasion and eventual eradication of cooperation from a population. Consequently, cheat invasion is often considered equal to extinction in empirical and theoretical studies of cooperator-cheat dynamics. But does cheat invasion necessarily equate extinction in nature? By following the social dynamics of iron metabolism in Pseudomonas aeruginosa during cystic fibrosis lung infection, we observed that individuals evolved to replace cooperation with a 'private' behaviour. Phenotypic assays showed that cooperative iron acquisition frequently was upregulated early in infection, which, however, increased the risk of cheat invasion. With whole-genome sequencing we showed that if, and only if, cooperative iron acquisition is lost from the population, a private system was upregulated. The benefit of upregulation depended on iron availability. These findings highlight the importance of social dynamics of natural populations and emphasizes the potential impact of past social interaction on the evolution of private traits., Competing Interests: SA, MG, RM, ZL, SM, HJ, AG No competing interests declared, (© 2018, Andersen et al.)

Bacterial opportunistic pathogens are feared for their difficult-to-treat nosocomial infections and for causing morbidity in immunocompromised patients. Here, we study how such a versatile opportunist, Pseudomonas aeruginosa, adapts to conditions inside and outside its model host Caenorhabditis elegans, and use phenotypic and genotypic screens to identify the mechanistic basis of virulence evolution. We found that virulence significantly dropped in unstructured environments both in the presence and absence of the host, but remained unchanged in spatially structured environments. Reduction of virulence was either driven by a substantial decline in the production of siderophores (in treatments without hosts) or toxins and proteases (in treatments with hosts). Whole-genome sequencing of evolved clones revealed positive selection and parallel evolution across replicates, and showed an accumulation of mutations in regulator genes controlling virulence factor expression. Our study identifies the spatial structure of the non-host environment as a key driver of virulence evolution in an opportunistic pathogen.

Whole genome sequencing is increasing used in epidemiology, e.g. for tracing outbreaks of food-borne diseases. This requires in-depth understanding of pathogen emergence, persistence and genomic diversity along the food production chain including in food processing plants. We sequenced the genomes of 80 isolates of Listeria monocytogenes sampled from Danish food processing plants over a time-period of 20 years, and analysed the sequences together with 10 public available reference genomes to advance our understanding of interplant and intraplant genomic diversity of L. monocytogenes. Except for three persisting sequence types (ST) based on Multi Locus Sequence Typing being ST7, ST8 and ST121, long-term persistence of clonal groups was limited, and new clones were introduced continuously, potentially from raw materials. No particular gene could be linked to the persistence phenotype. Using time-based phylogenetic analyses of the persistent STs, we estimate the L. monocytogenes evolutionary rate to be 0.18-0.35 single nucleotide polymorphisms/year, suggesting that the persistent STs emerged approximately 100 years ago, which correlates with the onset of industrialization and globalization of the food market., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The genome of Mycobacterium tuberculosis (Mtb) of latently infected individuals may hold the key to understanding the processes that lead to reactivation and progression to clinical disease. We report here analysis of pairs of Mtb isolates from putative prolonged latent TB cases. We identified two confirmed cases, and used whole genome sequencing to investigate the mutational processes that occur over decades in latent Mtb. We found an estimated mutation rate between 0.2 and 0.3 over 33 years, suggesting that latent Mtb accumulates mutations at rates similar to observations from cases of active disease., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Colistin is an antimicrobial peptide that has become the only remaining alternative for the treatment of multidrug-resistant Gram-negative bacterial infections, but little is known of how clinical levels of colistin resistance evolve. We use in vitro experimental evolution and whole-genome sequencing of colistin-resistant Pseudomonas aeruginosa isolates from cystic fibrosis patients to reconstruct the molecular evolutionary pathways open for high-level colistin resistance. We show that the evolution of resistance is a complex, multistep process that requires mutation in at least five independent loci that synergistically create the phenotype. Strong intergenic epistasis limits the number of possible evolutionary pathways to resistance. Mutations in transcriptional regulators are essential for resistance evolution and function as nodes that potentiate further evolution towards higher resistance by functionalizing and increasing the effect of the other mutations. These results add to our understanding of clinical antimicrobial peptide resistance and the prediction of resistance evolution.

Background: The primary cause of morbidity and mortality in cystic fibrosis (CF) patients is lung infection by Pseudomonas aeruginosa. Therefore much work has been done to understand the adaptation and evolution of P. aeruginosa in the CF lung. However, many of these studies have focused on longitudinally collected single isolates, and only few have included cross-sectional analyses of entire P. aeruginosa populations in sputum samples. To date only few studies have used the approach of metagenomic analysis for the purpose of investigating P. aeruginosa populations in CF airways., Results: We analysed five metagenomes together with longitudinally collected single isolates from four recently chronically infected CF patients. With this approach we were able to link the clone type and the majority of SNP profiles of the single isolates to that of the metagenome(s) for each individual patient., Conclusion: Based on our analysis we find that when having access to comprehensive collections of longitudinal single isolates it is possible to rediscover the genotypes of the single isolates in the metagenomic samples. This suggests that information gained from genome sequencing of comprehensive collections of single isolates is satisfactory for many investigations of adaptation and evolution of P. aeruginosa to the CF airways.

Infections with Pseudomonas aeruginosa increase morbidity in primary ciliary dyskinesia (PCD) and cystic fibrosis (CF) patients. Both diseases are associated with a defect of the mucociliary clearance; in PCD caused by non-functional cilia, in CF by changed mucus. Whole genome sequencing of P. aeruginosa isolates from CF patients has shown that persistence of clonal lineages in the airways is facilitated by genetic adaptation. It is unknown whether this also applies to P. aeruginosa airway infections in PCD. We compared within-host evolution of P. aeruginosa in PCD and CF patients. P. aeruginosa isolates from 12 PCD patients were whole genome sequenced and phenotypically characterised. Ten out of 12 PCD patients were infected with persisting clone types. We identified convergent evolution in eight genes, which are also important for persistent infections in CF airways: genes related to antibiotic resistance, quorum sensing, motility, type III secretion and mucoidity. We document phenotypic and genotypic parallelism in the evolution of P. aeruginosa across infected patients with different genetic disorders. The parallel changes and convergent adaptation and evolution may be caused by similar selective forces such as the intensive antibiotic treatment and the inflammatory response, which drive the evolutionary processes.

Combination therapy with several antibiotics is one strategy that has been applied in order to limit the spread of antimicrobial resistance. We compared the de novo evolution of resistance during combination therapy with the β-lactam ceftazidime and the fluoroquinolone ciprofloxacin with the resistance evolved after single-drug exposure. Combination therapy selected for mutants that displayed broad-spectrum resistance, and a major resistance mechanism was mutational inactivation of the repressor gene mexR that regulates the multidrug efflux operon mexAB-oprM. Deregulation of this operon led to a broad-spectrum resistance phenotype that decreased susceptibility to the combination of drugs applied during selection as well as to unrelated antibiotic classes. Mutants isolated after single-drug exposure displayed narrow-spectrum resistance and carried mutations in the MexCD-OprJ efflux pump regulator gene nfxB conferring ciprofloxacin resistance, or in the gene encoding the non-essential penicillin-binding protein DacB conferring ceftazidime resistance. Reconstruction of resistance mutations by allelic replacement and in vitro fitness assays revealed that in contrast to single antibiotic use, combination therapy consistently selected for mutants with enhanced fitness expressing broad-spectrum resistance mechanisms., (Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

Background: Chronic infection with Pseudomonas aeruginosa is a major cause of morbidity and mortality in cystic fibrosis (CF) patients, and a more complete understanding of P. aeruginosa within-host genomic evolution, transmission, and population genomics may provide a basis for improving intervention strategies. Here, we report the first genomic analysis of P. aeruginosa isolates sampled from Italian CF patients., Results: By genome sequencing of 26 isolates sampled over 19 years from four patients, we elucidated the within-host evolution of clonal lineages in each individual patient. Many of the identified mutations were located in pathoadaptive genes previously associated with host adaptation, and we correlated mutations with changes in CF-relevant phenotypes such as antibiotic resistance. In addition, the genomic analysis revealed that three patients shared the same clone. Furthermore, we compared the genomes of the Italian CF isolates to a panel of genome sequenced strains of P. aeruginosa from other countries. Isolates from two of the Italian lineages belonged to clonal complexes of P. aeruginosa that have previously been identified in Danish CF patients, and our genomic comparison showed that clonal isolates from the same country may be more distantly related than clonal isolates from different countries., Conclusions: This is the first whole-genome analysis of P. aeruginosa isolated from Italian CF patients, and together with both phenotypic and clinical information this dataset facilitates a more detailed understanding of P. aeruginosa within-host genomic evolution, transmission, and population genomics. We conclude that the evolution of the Italian lineages resembles what has been found in other countries.

Laboratory experiments show that social interactions between bacterial cells can drive evolutionary change at the population level, but significant challenges limit attempts to assess the relevance of these findings to natural populations, where selection pressures are unknown. We have increasingly sophisticated methods for monitoring phenotypic and genotypic dynamics in bacteria causing infectious disease, but in contrast, we lack evidence-based adaptive explanations for those changes. Evolutionary change during infection is often interpreted as host adaptation, but this assumption neglects to consider social dynamics shown to drive evolutionary change in vitro. We provide evidence to show that long-term behavioral dynamics observed in a pathogen are driven by selection to outcompete neighboring conspecific cells through social interactions. We find that Pseudomonas aeruginosa bacteria, causing lung infections in patients with cystic fibrosis, lose cooperative iron acquisition by siderophore production during infection. This loss could be caused by changes in iron availability in the lung, but surprisingly, we find that cells retain the ability to take up siderophores produced by conspecifics, even after they have lost the ability to synthesize siderophores. Only when cooperative producers are lost from the population is the receptor for uptake lost. This finding highlights the potential pitfalls of interpreting loss of function in pathogenic bacterial populations as evidence for trait redundancy in the host environment. More generally, we provide an example of how sequence analysis can be used to generate testable hypotheses about selection driving long-term phenotypic changes of pathogenic bacteria in situ.