Quantifying bacterial evolution in the wild: A birthday problem for Campylobacter lineages.

Measuring molecular evolution in bacteria typically requires estimation of the rate at which nucleotide changes accumulate in strains sampled at different times that share a common ancestor. This approach has been useful for dating ecological and evolutionary events that coincide with the emergence of important lineages, such as outbreak strains and obligate human pathogens. However, in multi-host (niche) transmission scenarios, where the pathogen is essentially an opportunistic environmental organism, sampling is often sporadic and rarely reflects the overall population, particularly when concentrated on clinical isolates. This means that approaches that assume recent common ancestry are not applicable. Here we present a new approach to estimate the molecular clock rate in Campylobacter that draws on the popular probability conundrum known as the 'birthday problem'. Using large genomic datasets and comparative genomic approaches, we use isolate pairs that share recent common ancestry to estimate the rate of nucleotide change for the population. Identifying synonymous and non-synonymous nucleotide changes, both within and outside of recombined regions of the genome, we quantify clock-like diversification to estimate synonymous rates of nucleotide change for the common pathogenic bacteria Campylobacter coli (2.4 x 10-6 s/s/y) and Campylobacter jejuni (3.4 x 10-6 s/s/y). Finally, using estimated total rates of nucleotide change, we infer the number of effective lineages within the sample time frame-analogous to a shared birthday-and assess the rate of turnover of lineages in our sample set over short evolutionary timescales. This provides a generalizable approach to calibrating rates in populations of environmental bacteria and shows that multiple lineages are maintained, implying that large-scale clonal sweeps may take hundreds of years or more in these species.

Co-evolutionary Signals Identify Burkholderia pseudomallei Survival Strategies in a Hostile Environment.

The soil bacterium Burkholderia pseudomallei is the causative agent of melioidosis and a significant cause of human morbidity and mortality in many tropical and subtropical countries. The species notoriously survives harsh environmental conditions but the genetic architecture for these adaptations remains unclear. Here we employed a powerful combination of genome-wide epistasis and co-selection studies (2,011 genomes), condition-wide transcriptome analyses (82 diverse conditions), and a gene knockout assay to uncover signals of "co-selection"-that is a combination of genetic markers that have been repeatedly selected together through B. pseudomallei evolution. These enabled us to identify 13,061 mutation pairs under co-selection in distinct genes and noncoding RNA. Genes under co-selection displayed marked expression correlation when B. pseudomallei was subjected to physical stress conditions, highlighting the conditions as one of the major evolutionary driving forces for this bacterium. We identified a putative adhesin (BPSL1661) as a hub of co-selection signals, experimentally confirmed a BPSL1661 role under nutrient deprivation, and explored the functional basis of co-selection gene network surrounding BPSL1661 in facilitating the bacterial survival under nutrient depletion. Our findings suggest that nutrient-limited conditions have been the common selection pressure acting on this species, and allelic variation of BPSL1661 may have promoted B. pseudomallei survival during harsh environmental conditions by facilitating bacterial adherence to different surfaces, cells, or living hosts.

Integrated chromosomal and plasmid sequence analyses reveal diverse modes of carbapenemase gene spread among Klebsiella pneumoniae.

Molecular and genomic surveillance systems for bacterial pathogens currently rely on tracking clonally evolving lineages. By contrast, plasmids are usually excluded or analyzed with low-resolution techniques, despite being the primary vectors of antibiotic resistance genes across many key pathogens. Here, we used a combination of long- and short-read sequence data of Klebsiella pneumoniae isolates (n = 1,717) from a European survey to perform an integrated, continent-wide study of chromosomal and plasmid diversity. This revealed three contrasting modes of dissemination used by carbapenemase genes, which confer resistance to last-line carbapenems. First, blaOXA-48-like genes have spread primarily via the single epidemic pOXA-48-like plasmid, which emerged recently in clinical settings and spread rapidly to numerous lineages. Second, blaVIM and blaNDM genes have spread via transient associations of many diverse plasmids with numerous lineages. Third, blaKPC genes have transmitted predominantly by stable association with one successful clonal lineage (ST258/512) yet have been mobilized among diverse plasmids within this lineage. We show that these plasmids, which include pKpQIL-like and IncX3 plasmids, have a long association (and are coevolving) with the lineage, although frequent recombination and rearrangement events between them have led to a complex array of mosaic plasmids carrying blaKPC Taken altogether, these results reveal the diverse trajectories of antibiotic resistance genes in clinical settings, summarized as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage. Our study provides a framework for the much needed incorporation of plasmid data into genomic surveillance systems, an essential step toward a more comprehensive understanding of resistance spread.

Stem Region of tRNA Genes Favors Transition Substitution Towards Keto Bases in Bacteria.

Transversion and transition mutations have variable effects on the stability of RNA secondary structure considering that the former destabilizes the double helix geometry to a greater extent by introducing purine:purine (R:R) or pyrimidine:pyrimidine (Y:Y) base pairs. Therefore, transversion frequency is likely to be lower than that of transition in the secondary structure regions of RNA genes. Here, we performed an analysis of transition and transversion frequencies in tRNA genes defined well with secondary structure and compared with the intergenic regions in five bacterial species namely Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Staphylococcus aureus and Streptococcus pneumoniae using a large genome sequence data set. In general, the transversion frequency was observed to be lower than that of transition in both tRNA genes and intergenic regions. The transition to transversion ratio was observed to be greater in tRNA genes than that in the intergenic regions in all the five bacteria that we studied. Interestingly, the intraspecies base substitution analysis in tRNA genes revealed that non-compensatory substitutions were more frequent than compensatory substitutions in the stem region. Further, transition to transversion ratio in the loop region was observed to be significantly lesser than that among the non-compensatory substitutions in the stem region. This indicated that the transversion is more deleterious than transition in the stem regions. In addition, substitutions from amino bases (A/C) to keto bases (G/T) were also observed to be more than the reverse substitutions in the stem region. Substitution from amino bases to keto bases are likely to facilitate the stable G:U pairing unlike the reverse substitution that facilitates the unstable A:C pairing in the stem region of tRNA. This work provides additional support that the secondary structure of tRNA molecule is what drives the different substitutions in its gene sequence.

Challenges in realising the potential of wastewater-based epidemiology to quantitatively monitor and predict the spread of disease.

Researchers around the world have demonstrated correlations between measurements of SARS-CoV-2 RNA in wastewater (WW) and case rates of COVID-19 derived from direct testing of individuals. This has raised concerns that wastewater-based epidemiology (WBE) methods might be used to quantify the spread of this and other diseases, perhaps faster than direct testing, and with less expense and intrusion. We illustrate, using data from Scotland and the USA, the issues regarding the construction of effective predictive models for disease case rates. We discuss the effects of variation in, and the problem of aligning, public health (PH) reporting and WW measurements. We investigate time-varying effects in PH-reported case rates and their relationship to WW measurements. We show the lack of proportionality of WW measurements to case rates with associated spatial heterogeneity. We illustrate how the precision of predictions is affected by the level of aggregation chosen. We determine whether PH or WW measurements are the leading indicators of disease and how they may be used in conjunction to produce predictive models. The prospects of using WW-based predictive models with or without ongoing PH data are discussed.

Power and Efficacy of Maternal Voice in Neonatal Intensive Care Units: Implicit Bias and Family-Centered Care.

INTRODUCTION: Implicit bias can lead medical professionals in Neonatal Intensive Care Units (NICUs) to disregard mothers who are Black and economically disadvantaged as they advocate for their infants' health. Disregard can weaken underlying communication principles within the Family-Centered Care (FCC) model of pediatric health in NICUs and increase maternal distress. This study is the first to address communication disregard by examining mothers' perceived power and efficacy of voice with NICU doctors and nurses. We hypothesized that mothers who are Black and economically disadvantaged would report lower efficacy of voice and higher levels of distress as compared to White mothers with higher income. METHODS: During pre-assessment within a small clinical trial of a parenting intervention, 33 racially and economically diverse mothers, from three Midwest NICUs serving the urban poor, responded to a 14-item measure of maternal power and efficacy of voice and measures of somatization, depression, anxiety and eating/sleeping disorders. Nonparametric examinations assessed the relation of power and efficacy of voice to maternal race, income, and distress. RESULTS: In contrast to White, higher-income mothers, Black, economically disadvantaged mothers reported lower perceived efficacy of voice with doctors (U = 74.5, d = 0.65) and nurses (U = 74.0; d = .0.66). These mothers with lower perceived efficacy with doctors and nurses, reported higher levels of somatization (U = 16.5, d = 1.14; U = 13.5, d = 1.38, respectively) and eating disorders (U = 14.0, d = 1.29; U = 12.0, d = 1.48, respectively). DISCUSSION: Study results are discussed within the framework of implicit bias in FCC in the NICU, expanding our understanding of effective communication with economically stressed, Black mothers.

Rapid Genomic Characterization and Global Surveillance of Klebsiella Using Pathogenwatch.

BACKGROUND: Klebsiella species, including the notable pathogen K. pneumoniae, are increasingly associated with antimicrobial resistance (AMR). Genome-based surveillance can inform interventions aimed at controlling AMR. However, its widespread implementation requires tools to streamline bioinformatic analyses and public health reporting. METHODS: We developed the web application Pathogenwatch, which implements analytics tailored to Klebsiella species for integration and visualization of genomic and epidemiological data. We populated Pathogenwatch with 16 537 public Klebsiella genomes to enable contextualization of user genomes. We demonstrated its features with 1636 genomes from 4 low- and middle-income countries (LMICs) participating in the NIHR Global Health Research Unit (GHRU) on AMR. RESULTS: Using Pathogenwatch, we found that GHRU genomes were dominated by a small number of epidemic drug-resistant clones of K. pneumoniae. However, differences in their distribution were observed (eg, ST258/512 dominated in Colombia, ST231 in India, ST307 in Nigeria, ST147 in the Philippines). Phylogenetic analyses including public genomes for contextualization enabled retrospective monitoring of their spread. In particular, we identified hospital outbreaks, detected introductions from abroad, and uncovered clonal expansions associated with resistance and virulence genes. Assessment of loci encoding O-antigens and capsule in K. pneumoniae, which represent possible vaccine candidates, showed that 3 O-types (O1-O3) represented 88.9% of all genomes, whereas capsule types were much more diverse. CONCLUSIONS: Pathogenwatch provides a free, accessible platform for real-time analysis of Klebsiella genomes to aid surveillance at local, national, and global levels. We have improved representation of genomes from GHRU participant countries, further facilitating ongoing surveillance.

Understanding the Steps Toward Mobile Early Intervention for Mothers and Their Infants Exiting the Neonatal Intensive Care Unit: Descriptive Examination.

BACKGROUND: Neonatal intensive care unit (NICU) history, combined with systemic inequities for mothers of nondominant cultures and mothers who are socioeconomically disadvantaged, places infants at an extraordinary risk for poor developmental outcomes throughout life. Although receipt of early intervention (EI) is the best single predictor of developmental outcomes among children with and at risk for early developmental delays, mothers and infants with the greatest needs are least likely to receive EI. Mobile internet-based interventions afford substantial advantages for overcoming logistical challenges that often prevent mothers who are economically disadvantaged from accessing EI. However, the bridge from the NICU to a mobile internet intervention has been virtually unexplored. OBJECTIVE: This study aims to examine progression flow from NICU exit referral to an early mobile internet intervention to increase EI access and promote parent mediation of infant social-emotional and communication development. METHODS: Three NICUs serving the urban poor in a Midwestern city were provided support in establishing an electronic NICU exit referral mechanism into a randomized controlled trial of a mobile internet intervention for mothers and their infants. Measurement domains to reflect the bridge to service included each crucial gateway required for navigating the path into Part C EI, including referral, screening, assessment, and intervention access. An iterative process was used and documented to facilitate each NICU in establishing an individualized accountability plan for sharing referral materials with mothers before their NICU exit. Subsequent to the referral, progression flow was documented on the basis of a real-time electronic recording of service receipt and contact records. Mother and infant risk characteristics were also assessed. Descriptive analyses were conducted to summarize and characterize each measurement domain. RESULTS: NICU referral rates for EI were 3 to 4 times higher for open-shared versus closed-single gatekeeper referral processes. Of 86 referred dyads, 67 (78%) were screened, and of those screened, 51 (76%) were eligible for assessment. Of the 51 assessment-eligible mothers and infants, 35 dyads (69%) completed the assessment and 31 (89%) went on to complete at least one remote coaching intervention session. The dyads who accessed and engaged in intervention were racially and ethnically diverse and experiencing substantial adversity. CONCLUSIONS: The transition from the NICU to home was fraught with missed opportunities for an EI referral. Beyond the referral, the most prominent reason for not participating in screening was that mothers could not be located after exiting the NICU. Stronger NICU referral mechanisms for EI are needed. It may be essential to initiate mobile interventions before exiting the NICU for maintaining post-NICU contact with some mothers. In contrast to a closed, single point of referral gatekeeper systems in NICUs, open, shared referral gating systems may be less stymied by individual service provider biases and disruptions.

Randomized control trial of an internet-based parenting intervention for mothers of infants.

Early parenting home-visiting interventions have been found to be highly effective in promoting child development. Yet, there are many obstacles in the implementation of home-visiting programs, including travel and access to trained providers. Internet-based interventions can reach many parents of infants to overcome these barriers. The objective of this randomized control trial was to evaluate the impact of the Internet-adaptation of the Play and Learning Strategies (PALS) program, a preventive intervention program to strengthen effective parenting practices that promote early language, cognitive, and social development. others in low-income environments (N = 164) of infants were randomized to either (a) an Internet-facilitated PALS parenting intervention or (b) an Internet-facilitated attention control condition. Measures included direct observations of maternal behavior with her infant, questionnaires about maternal functioning and parenting knowledge, and real-time program usage. Experimental participants demonstrated significantly greater increases in parenting knowledge and observed language-supportive parenting behaviors with a correlated positive change in infant language behaviors. Effects were pronounced when participants received a greater dosage of the intervention. Results suggest that the Internet-based translation of the PALS program is effective as a remotely delivered intervention for economically disadvantaged families to strengthen early parenting behaviors that promote infant social communication and child language development.

Real time monitoring of Aeromonas salmonicida evolution in response to successive antibiotic therapies in a commercial fish farm.

Our ability to predict evolutionary trajectories of pathogens in response to antibiotic pressure is one of the promising leverage to fight against the present antibiotic resistance worldwide crisis. Yet, few studies tackled this question in situ at the outbreak level, due to the difficulty to link a given pathogenic clone evolution with its precise antibiotic exposure over time. In this study, we monitored the real-time evolution of an Aeromonas salmonicida clone in response to successive antibiotic and vaccine therapies in a commercial fish farm. The clone was responsible for a four-year outbreak of furunculosis within a Recirculating Aquaculture System Salmo salar farm in China, and we reconstructed the precise tempo of mobile genetic elements (MGEs) acquisition events during this period. The resistance profile provided by the acquired MGEs closely mirrored the antibiotics used to treat the outbreak, and we evidenced that two subclonal groups developed similar resistances although unrelated MGE acquisitions. Finally, we also demonstrated the efficiency of vaccination in outbreak management and its positive effect on antibiotic resistance prevalence. Our study provides unprecedented knowledge critical to understand evolutionary trajectories of resistant pathogens outside the laboratory.

Building a genomic framework for prospective MRSA surveillance in the United Kingdom and the Republic of Ireland.

The correct interpretation of microbial sequencing data applied to surveillance and outbreak investigation depends on accessible genomic databases to provide vital genetic context. Our aim was to construct and describe a United Kingdom MRSA database containing over 1000 methicillin-resistant Staphylococcus aureus (MRSA) genomes drawn from England, Northern Ireland, Wales, Scotland, and the Republic of Ireland over a decade. We sequenced 1013 MRSA submitted to the British Society for Antimicrobial Chemotherapy by 46 laboratories between 2001 and 2010. Each isolate was assigned to a regional healthcare referral network in England and was otherwise grouped based on country of origin. Phylogenetic reconstructions were used to contextualize MRSA outbreak investigations and to detect the spread of resistance. The majority of isolates (n = 783, 77%) belonged to CC22, which contains the dominant United Kingdom epidemic clone (EMRSA-15). There was marked geographic structuring of EMRSA-15, consistent with widespread dissemination prior to the sampling decade followed by local diversification. The addition of MRSA genomes from two outbreaks and one pseudo-outbreak demonstrated the certainty with which outbreaks could be confirmed or refuted. We identified local and regional differences in antibiotic resistance profiles, with examples of local expansion, as well as widespread circulation of mobile genetic elements across the bacterial population. We have generated a resource for the future surveillance and outbreak investigation of MRSA in the United Kingdom and Ireland and have shown the value of this during outbreak investigation and tracking of antimicrobial resistance.

WGS of 1058 Enterococcus faecium from Copenhagen, Denmark, reveals rapid clonal expansion of vancomycin-resistant clone ST80 combined with widespread dissemination of a vanA-containing plasmid and acquisition of a heterogeneous accessory genome.

OBJECTIVES: From 2012 to 2015, a sudden significant increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) was observed in the Capital Region of Denmark. Clonal relatedness of VREfm and vancomycin-susceptible E. faecium (VSEfm) was investigated, transmission events between hospitals were identified and the pan-genome and plasmids from the largest VREfm clonal group were characterized. METHODS: WGS of 1058 E. faecium isolates was carried out on the Illumina platform to perform SNP analysis and to identify the pan-genome. One isolate was also sequenced on the PacBio platform to close the genome. Epidemiological data were collected from laboratory information systems. RESULTS: Phylogeny of 892 VREfm and 166 VSEfm revealed a polyclonal structure, with a single clonal group (ST80) accounting for 40% of the VREfm isolates. VREfm and VSEfm co-occurred within many clonal groups; however, no VSEfm were related to the dominant VREfm group. A similar vanA plasmid was identified in ≥99% of isolates belonging to the dominant group and 69% of the remaining VREfm. Ten plasmids were identified in the completed genome, and ∼29% of this genome consisted of dispensable accessory genes. The size of the pan-genome among isolates in the dominant group was 5905 genes. CONCLUSIONS: Most probably, VREfm emerged owing to importation of a successful VREfm clone which rapidly transmitted to the majority of hospitals in the region whilst simultaneously disseminating a vanA plasmid to pre-existing VSEfm. Acquisition of a heterogeneous accessory genome may account for the success of this clone by facilitating adaptation to new environmental challenges.

Closed genome sequences of Staphylococcus lloydii sp. nov. and Staphylococcus durrellii sp. nov. isolated from captive fruit bats (Pteropus livingstonii).

The increasing availability of whole genome sequencing of bacteria has accelerated the discovery of novel species which may not have been easy to discriminate using standard phenotypic or single gene methods. Phylogenomic analysis of genome sequences from a collection of coagulase-negative staphylococcal species isolated from captive fruit bats revealed two clusters which were close to Staphylococcus kloosii. To assess the relatedness of the strains we used digital DNA-DNA hybridization (dDDH) and two methods for average nucleotide identity (ANI) computation which predicted two novel species having dDDH less than 70 % and ANI less than 95%. We propose these species as Staphylococcus lloydii sp. nov. (type strain 23_2_7_LYT=NCTC 14453T=DSM 111639T) and Staphylococcus durrellii sp. nov (type strain 27_4_6_LYT=NCTC 14454T=DSM 111640T).

Comprehensive Cost Analysis of First Step Next for Preschoolers with Disruptive Behavior Disorder: Using Real-World Intervention Data to Estimate Costs at Scale.

While the long-term societal costs for youth with disruptive behavior disorders are well documented, there is a dearth of information about the comprehensive costs of implementing even the most well-regarded early intervention programs, and the costs of scaling effective interventions are even less well understood. This study estimated the costs of delivering and disseminating First Step Next (FSN), an established tier two school-based early intervention, in preschool and kindergarten settings, including the training and ongoing technical assistance that support sustained, high-quality implementation. Using the Ingredients Method, we estimated (a) the per student costs of implementation, (b) the incremental cost of offering FSN to an additional student, and (c) the cost to disseminate FSN to 40 preschool and kindergarten students, including a sensitivity analysis to examine potential areas of cost savings. The per child cost to implement the FSN intervention with 29 triads in two cohorts was $4330. The incremental cost per additional student was only $2970, highlighting efficiencies gained once intervention infrastructure had been established. The cost of disseminating the intervention to a single cohort of 40 students was $170,106, or $4253 per student. The range in sensitivity analysis was $3141-$7829 per student, with variability in personnel wages having the greatest impact on cost estimates. This research expands on existing literature by providing a more comprehensive understanding of the cost of effective disruptive behavior interventions based on real-world implementation data, using these data to estimate dissemination costs, and showing how dissemination costs are particularly sensitive to personnel wages.

Mediation analyses of Internet-facilitated cognitive behavioral intervention for maternal depression.

This study evaluated the putative mediating mechanisms of an Internet-facilitated cognitive-behavioral therapy (CBT) intervention for depression tailored to economically disadvantaged mothers of preschool-age children. The CBT mediators were tested across two previously published randomized controlled trials which included the same measures of behavioral activation, negative thinking, and savoring of positive events. Trial 1 included 70 mothers with elevated depressive symptoms who were randomized to either the eight-session, Internet-facilitated intervention (Mom-Net) or to treatment as usual. Trial 2 included 266 mothers with elevated depressive symptoms who were randomized to either Mom-Net or to a motivational interviewing and referral to services condition. Simple mediation models tested each putative mediator independently followed by tests of multiple mediation that simultaneously included all three mediators in the model to assess the salient contributions of each mediator. The pattern of results for the mediating effects were systematically replicated across the two trials and suggest that behavioral activation and negative thinking are salient mediators of the Mom-Net intervention; significant mediating effects for savoring were obtained only in the simple mediation models and were not obtained in the multiple mediation models.

The distribution of bacterial doubling times in the wild.

Generation time varies widely across organisms and is an important factor in the life cycle, life history and evolution of organisms. Although the doubling time (DT) has been estimated for many bacteria in the laboratory, it is nearly impossible to directly measure it in the natural environment. However, an estimate can be obtained by measuring the rate at which bacteria accumulate mutations per year in the wild and the rate at which they mutate per generation in the laboratory. If we assume the mutation rate per generation is the same in the wild and in the laboratory, and that all mutations in the wild are neutral, an assumption that we show is not very important, then an estimate of the DT can be obtained by dividing the latter by the former. We estimate the DT for five species of bacteria for which we have both an accumulation and a mutation rate estimate. We also infer the distribution of DTs across all bacteria from the distribution of the accumulation and mutation rates. Both analyses suggest that DTs for bacteria in the wild are substantially greater than those in the laboratory, that they vary by orders of magnitude between different species of bacteria and that a substantial fraction of bacteria double very slowly in the wild.

Evolutionary Trade-Offs Underlie the Multi-faceted Virulence of Staphylococcus aureus.

Bacterial virulence is a multifaceted trait where the interactions between pathogen and host factors affect the severity and outcome of the infection. Toxin secretion is central to the biology of many bacterial pathogens and is widely accepted as playing a crucial role in disease pathology. To understand the relationship between toxicity and bacterial virulence in greater depth, we studied two sequenced collections of the major human pathogen Staphylococcus aureus and found an unexpected inverse correlation between bacterial toxicity and disease severity. By applying a functional genomics approach, we identified several novel toxicity-affecting loci responsible for the wide range in toxic phenotypes observed within these collections. To understand the apparent higher propensity of low toxicity isolates to cause bacteraemia, we performed several functional assays, and our findings suggest that within-host fitness differences between high- and low-toxicity isolates in human serum is a contributing factor. As invasive infections, such as bacteraemia, limit the opportunities for onward transmission, highly toxic strains could gain an additional between-host fitness advantage, potentially contributing to the maintenance of toxicity at the population level. Our results clearly demonstrate how evolutionary trade-offs between toxicity, relative fitness, and transmissibility are critical for understanding the multifaceted nature of bacterial virulence.

A Randomized Study of a Mobile Behavioral Parent Training Application.

Background/Introduction: Never before have parents had such immediate access to parenting support. The extension of the Internet to smartphones, offers the opportunity to provide families with the highest-quality information at the time and place that it can be the most useful. However, there remain considerable barriers to getting the right information to the right people at the right time. MATERIALS AND METHODS: This study includes the initial feasibility testing of a smartphone application "ParentNet" that attempts to deliver on the potential of empirically supported therapy by connecting family members with specific behavioral goals and outcomes in real time. Participation was solicited from community parenting support groups and through online social media. Data were collected from 73 parents and 88 children on child behavior (adult only) and satisfaction. RESULTS: Data analyses showed positive satisfaction and utilization results: (1) users rated the ParentNet app very positively (i.e., 85% of caregivers and 88% of youth would recommend the app to others), and (2) parenting behavior was improved with a small/moderate effect-size. DISCUSSION AND CONCLUSIONS: Findings from this initial testing are reviewed along with future development possibilities to be considered. Limitations of small pilot sample and brief administration period could have reduced effects. Further study would include a more robust sample.

Predicting the virulence of MRSA from its genome sequence.

Microbial virulence is a complex and often multifactorial phenotype, intricately linked to a pathogen's evolutionary trajectory. Toxicity, the ability to destroy host cell membranes, and adhesion, the ability to adhere to human tissues, are the major virulence factors of many bacterial pathogens, including Staphylococcus aureus. Here, we assayed the toxicity and adhesiveness of 90 MRSA (methicillin resistant S. aureus) isolates and found that while there was remarkably little variation in adhesion, toxicity varied by over an order of magnitude between isolates, suggesting different evolutionary selection pressures acting on these two traits. We performed a genome-wide association study (GWAS) and identified a large number of loci, as well as a putative network of epistatically interacting loci, that significantly associated with toxicity. Despite this apparent complexity in toxicity regulation, a predictive model based on a set of significant single nucleotide polymorphisms (SNPs) and insertion and deletions events (indels) showed a high degree of accuracy in predicting an isolate's toxicity solely from the genetic signature at these sites. Our results thus highlight the potential of using sequence data to determine clinically relevant parameters and have further implications for understanding the microbial virulence of this opportunistic pathogen.

Genomic analysis of 495 vancomycin-resistant Enterococcus faecium reveals broad dissemination of a vanA plasmid in more than 19 clones from Copenhagen, Denmark.

OBJECTIVES: From 2012 to 2014, there has been a huge increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) in Copenhagen, Denmark, with 602 patients infected or colonized with VREfm in 2014 compared with just 22 in 2012. The objective of this study was to describe the genetic epidemiology of VREfm to assess the contribution of clonal spread and horizontal transfer of the vanA transposon (Tn1546) and plasmid in the dissemination of VREfm in hospitals. METHODS: VREfm from Copenhagen, Denmark (2012-14) were whole-genome sequenced. The clonal structure was determined and the structure of Tn1546-like transposons was characterized. One VREfm isolate belonging to the largest clonal group was sequenced using long-read technology to close a 37 kb vanA plasmid. RESULTS: Phylogeny revealed a polyclonal structure where 495 VREfm isolates were divided into 13 main groups and 7 small groups. The majority of the isolates were located in three groups (n = 44, 100 and 218) and clonal spread of VREfm between wards and hospitals was identified. Five Tn1546-like transposon types were identified. A dominant truncated transposon (type 4, 92%) was spread across all but one VREfm group. The closed vanA plasmid was highly covered by reads from isolates containing the type 4 transposon. CONCLUSIONS: This study suggests that it was the dissemination of the type 4 Tn1546-like transposon and plasmid via horizontal transfer to multiple populations of E. faecium, followed by clonal spread of new VREfm clones, that contributed to the increase in and diversity of VREfm in Danish hospitals.