Recipient UvrD helicase is involved in single- to double-stranded DNA conversion during conjugative plasmid transfer.

Dissemination of antibiotic resistance, a current societal challenge, is often driven by horizontal gene transfer through bacterial conjugation. During conjugative plasmid transfer, single-stranded (ss) DNA is transferred from the donor to the recipient cell. Subsequently, a complete double-stranded (ds) plasmid molecule is generated and plasmid-encoded genes are expressed, allowing successful establishment of the transconjugant cell. Such dynamics of transmission can be modulated by host- or plasmid-encoded factors, either in the donor or in the recipient cell. We applied transposon insertion sequencing to identify host-encoded factors that affect conjugative transfer frequency in Escherichia coli. Disruption of the recipient uvrD gene decreased the acquisition frequency of conjugative plasmids belonging to different incompatibility groups. Results from various UvrD mutants suggested that dsDNA binding activity and interaction with RNA polymerase are dispensable, but ATPase activity is required for successful plasmid establishment of transconjugant cells. Live-cell microscopic imaging showed that the newly transferred ssDNA within a uvrD- recipient often failed to be converted to dsDNA. Our work suggested that in addition to its role in maintaining genome integrity, UvrD is also key for the establishment of horizontally acquired plasmid DNA that drives genome diversity and evolution.

L-arabinose induces the formation of viable non-proliferating spheroplasts in Vibrio cholerae.

Vibrio cholerae, the agent of the deadly human disease cholera, propagates as a curved rod-shaped bacterium in warm waters. It is sensitive to cold, but persists in cold waters under the form of viable but non-dividing coccoidal shaped cells. Additionally, V. cholerae is able to form non-proliferating spherical cells in response to cell wall damage. It was recently reported that L-arabinose, a component of the hemicellulose and pectin of terrestrial plants, stops the growth of V. cholerae. Here, we show that L-arabinose induces the formation of spheroplasts that lose the ability to divide and stop growing in volume over time. However, they remain viable and upon removal of L-arabinose they start expanding in volume, form branched structures and give rise to cells with a normal morphology after a few divisions. We further show that WigKR, a histidine kinase/response regulator pair implicated in the induction of a high expression of cell wall synthetic genes, prevents the lysis of the spheroplasts during growth restart. Finally, we show that the physiological perturbations result from the import and catabolic processing of L-arabinose by the V. cholerae homolog of the E. coli galactose transport and catabolic system. Taken together, our results suggest that the formation of non-growing spherical cells is a common response of Vibrios exposed to detrimental conditions. They also permit to define conditions preventing any physiological perturbation of V. cholerae when using L-arabinose to induce gene expression from the tightly regulated promoter of the Escherichia coli araBAD operon.Importance Vibrios among other bacteria form transient cell wall deficient forms as a response to different stresses and revert to proliferating rods when permissive conditions have been restored. Such cellular forms have been associated to antimicrobial tolerance, chronic infections and environmental dispersion.The effect of L-Ara on V. cholerae could provide an easily tractable model to study the ability of Vibrios to form viable reversible spheroplasts. Indeed, the quick transition to spheroplasts and reversion to proliferating rods by addition or removal of L-Ara is ideal to understand the genetic program governing this physiological state and the spatial rearrangements of the cellular machineries during cell shape transitions.

Vertical and Horizontal Transmission of ESBL Plasmid from Escherichia coli O104:H4.

Multidrug resistance (MDR) often results from the acquisition of mobile genetic elements (MGEs) that encode MDR gene(s), such as conjugative plasmids. The spread of MDR plasmids is founded on their ability of horizontal transference, as well as their faithful inheritance in progeny cells. Here, we investigated the genetic factors involved in the prevalence of the IncI conjugative plasmid pESBL, which was isolated from the Escherichia coli O104:H4 outbreak strain in Germany in 2011. Using transposon-insertion sequencing, we identified the pESBL partitioning locus (par). Genetic, biochemical and microscopic approaches allowed pESBL to be characterized as a new member of the Type Ib partitioning system. Inactivation of par caused mis-segregation of pESBL followed by post-segregational killing (PSK), resulting in a great fitness disadvantage but apparent plasmid stability in the population of viable cells. We constructed a variety of pESBL derivatives with different combinations of mutations in par, conjugational transfer (oriT) and pnd toxin-antitoxin (TA) genes. Only the triple mutant exhibited plasmid-free cells in viable cell populations. Time-lapse tracking of plasmid dynamics in microfluidics indicated that inactivation of pnd improved the survival of plasmid-free cells and allowed oriT-dependent re-acquisition of the plasmid. Altogether, the three factors-active partitioning, toxin-antitoxin and conjugational transfer-are all involved in the prevalence of pESBL in the E. coli population.

Using a Concept Inventory to Reveal Student Thinking Associated with Common Misconceptions about Antibiotic Resistance.

Misconceptions, also known as alternate conceptions, about key concepts often hinder the ability of students to learn new knowledge. Concept inventories (CIs) are designed to assess students' understanding of key concepts, especially those prone to misconceptions. Two-tiered CIs include prompts that ask students to explain the logic behind their answer choice. Such two-tiered CIs afford an opportunity for faculty to explore the student thinking behind the common misconceptions represented by their choice of a distractor. In this study, we specifically sought to probe the misconceptions that students hold prior to beginning an introductory microbiology course (i.e., preconceptions). Faculty-learning communities at two research-intensive universities used the validated Host-Pathogen Interaction Concept Inventory (HPI-CI) to reveal student preconceptions. Our method of deep analysis involved communal review and discussion of students' explanations for their CI answer choice. This approach provided insight valuable for curriculum development. Here the process is illustrated using one question from the HPI-CI related to the important topic of antibiotic resistance. The frequencies with which students chose particular multiple-choice responses for this question were highly correlated between institutions, implying common underlying misconceptions. Examination of student explanations using our analysis approach, coupled with group discussions within and between institutions, revealed patterns in student thinking to the participating faculty. Similar application of a two-tiered concept inventory by general microbiology instructors, either individually or in groups, at other institutions will allow them to better understand student thinking related to key concepts in their curriculum.

Decision-making processes shaping the home food environments of young adult women with and without children.

Although young adult women consume the majority of their total daily energy intake from home food sources, the decision-making processes that shape their home food environments have received limited attention. Further, how decision-making may be affected by the transformative experience of motherhood is unknown. In this study, we explore the factors that influence two key decision-making processes-food choices while grocery shopping and the use of non-home food sources-and whether there are differences by motherhood status. In-depth interviews were conducted with 40 women, aged 20-29, living in southwest Georgia. Thematic analysis was used to analyze qualitative data stratified by whether or not children were present in the home. Decision-making was affected by numerous factors, which differed across groups. In regard to grocery shopping, women with children more frequently discussed the influence of nutrition and the preferences of children, while women without children more frequently discussed the influence of taste and the preferences of other household members. Cost, convenience, weight control, and pre-planning meals emerged as salient in both groups. In regard to the use of non-home food sources, convenience and taste were discussed by both groups, while social factors were only discussed by women without children. The cost of eating out was the only reason cited for eating inside the home, and this factor only emerged among women with children. Motherhood may be an important contributor to the decision-making processes that shape young adult women's home food environments. Interventions may find success in framing messaging to emphasize factors identified as motivating healthy decisions, such as protecting the health of children, and practical strategies may be adapted from those already in use, such as pre-planning and budgeting for healthy meals.

No differences in clinical outcomes between fixed- and mobile-bearing computer-assisted total knee arthroplasties and no correlations between navigation data and clinical scores.

PURPOSE: The theoretical advantages of mobile-bearing (MB) designs over the conventional fixed bearings (FBs) for total knee arthroplasty (TKA) have not been proved yet through clinical studies. The aim of the study was to test whether the MB design has advantages in terms of better clinical outcomes when compared to FB. Furthermore, the relationships between intra-operative obtained implant positioning data and the clinical scores were analysed. METHODS: A total of 99 patients were randomized into the FB or the MB group. All patients received the same posterior cruciate retaining implants and were operated with the use of a computer-assisted navigation system. The clinical outcomes of both groups were compared pre-operatively, at 1 year, and at a mean follow-up time of 4 years after surgery. RESULTS: The MB implants showed no advantages over the FB when comparing the Knee Society Scores, the Oxford Score, the range of movement (ROM) and pain intensity of the patients in both groups at 1 and 4 years after surgery. There were no relationships between the computer navigation data and the clinical scores. CONCLUSIONS: In view of the 4-year results, there is no evidence to support the recommendation of one design over the other in terms of better clinical outcome scores, higher ROM or lower pain rates. Long-term follow-up results may be necessary, including survival rates. Further research comparing different TKA designs should also include standardized performance-based tests. LEVEL OF EVIDENCE: Prospective study (Randomized controlled trial with adequate statistical power to detect differences), Level I.

Research to reality: a process evaluation of a mini-grants program to disseminate evidence-based nutrition programs to rural churches and worksites.

OBJECTIVES: To describe a project that used mini-grants plus technical assistance to disseminate evidence-based programs, to understand how the project worked in different settings, and to generate recommendations for future programming and evaluation. DESIGN: Process evaluation using program records, activity forms completed by grantees, interviews, and focus groups. SETTING: Churches and worksites in rural, southwest Georgia. PARTICIPANTS: Site coordinators (n = 10), organizational leaders (n = 7), and project committee members (n = 25) involved in program implementation at 7 funded organizations. INTERVENTION: The Emory Cancer Prevention and Control Research Network solicited applications from churches and worksites to implement one of 2 evidence-based nutrition programs: Body & Soul for churches and Treatwell 5-a-Day for worksites. Successful applicants (n = 7) received funding and technical assistance from Emory and agreed to conduct all required elements of the evidence-based program. MAIN OUTCOME MEASURES: We assessed adoption, reach, implementation, and maintenance of specific programs and their core elements, as well as contextual influences and the resources required to implement the mini-grants program. RESULTS: Four of the 7 funded organizations conducted all programmatic core elements; all 7 sites conducted at least 6 of 8 core elements, including at least 1 food-related policy or environmental change as a result of the program. Program reach varied widely across sites and core elements. All site coordinators stated that they intend to continue at least some of the activities conducted under the project. Sites reported that contextual factors such as the program's fit with the organization's mission, leadership support, and leadership or staffing transitions influenced program implementation. Over 18 months, Emory staff spent 47.7 hours providing technical assistance to grantees. CONCLUSIONS: A mini-grants and technical assistance model has the potential to be an effective mechanism for disseminating evidence-based programs to community organizations, and further study of this method is warranted.

Smoke-free policies at home, church, and work: smoking levels and recent quit attempts among a southeastern rural population, 2007.

OBJECTIVE: The objective of this study was to examine the cumulative effect of smoke-free policies and social support for smoking cessation in the home, at church, and at work on smoking levels and quit attempts in the context of a community-based study of rural African Americans and whites in the Southeast. METHODS: We conducted a baseline survey to assess sociodemographics, smoking behavior, level of social support for smoking cessation, and smoke-free policies at home, church, and work. We created a variable for a weighted "dose" of smoking restrictions on the basis of the existence of policies in the 3 settings and a weighted score for social support and used bivariate analyses and multivariate regression to analyze data. RESULTS: Of 134 survey participants, 18.7% had complete restrictions at home. Among church attendees, 39.4% had complete restrictions at church, and among those employed outside the home, 15.4% had complete restrictions at work. After controlling for age, sex, race, and education, the weighted dose of smoking restrictions was significantly related to having made a quit attempt in the past 12 months (odds ratio, 2.2; 95% confidence interval, 1.1-4.3) but not number of cigarettes smoked per day. Social support for cessation had no effect on recent quit attempts or number of cigarettes smoked per day. CONCLUSION: Smoke-free policies have a cumulative effect on smoking behavior. These findings may inform interventions aimed at promoting comprehensive community-wide smoke-free policies.