Evidence for a bimodal distribution of Escherichia coli doubling times below a threshold initial cell concentration

Abstract Background In the process of developing a microplate-based growth assay, we discovered that our test organism, a native E. coli isolate, displayed very uniform doubling times (τ) only up to a certain threshold cell density. Below this cell concentration (≤ 100 -1,000 CFU mL-1 ; ≤ 27-270 CFU well-1) we observed an obvious increase in the τ scatter. Results Working with a food-borne E. coli isolate we found that τ values derived from two different microtiter platereader-based techniques (i.e., optical density with growth time {=OD[t]} fit to the sigmoidal Boltzmann equation or time to calculated 1/2-maximal OD {=tm} as a function of initial cell density {=tm[CI]}) were in excellent agreement with the same parameter acquired from total aerobic plate counting. Thus, using either Luria-Bertani (LB) or defined (MM) media at 37°C, τ ranged between 17-18 (LB) or 51-54 (MM) min. Making use of such OD[t] data we collected many observations of τ as a function of manifold initial or starting cell concentrations (CI). We noticed that τ appeared to be distributed in two populations (bimodal) at low CI. When CI ≤100 CFU mL-1 (stationary phase cells in LB), we found that about 48% of the observed τ values were normally distributed around a mean (μτ1) of 18 ± 0.68 min (± στ1) and 52% with μτ2 = 20 ± 2.5 min (n = 479). However, at higher starting cell densities (CI>100 CFU mL-1), the τ values were distributed unimodally (μτ = 18 ± 0.71 min; n = 174). Inclusion of a small amount of ethyl acetate to the LB caused a collapse of the bimodal to a unimodal form. Comparable bimodal τ distribution results were also observed using E. coli cells diluted from mid-log phase cultures. Similar results were also obtained when using either an E. coli O157:H7 or a Citrobacter strain. When sterile-filtered LB supernatants, which formerly contained relatively low concentrations of bacteria(1,000-10,000 CFU mL-1), were employed as a diluent, there was an evident shift of the two populations towards each other but the bimodal effect was still apparent using either stationary or log phase cells. Conclusion These data argue that there is a dependence of growth rate on starting cell density.</p

The potential effect of improved provision of rabies post-exposure prophylaxis in Gavi-eligible countries: a modelling study

Background: Tens of thousands of people die from dog-mediated rabies annually. Deaths can be prevented through post-exposure prophylaxis for people who have been bitten, and the disease eliminated through dog vaccination. Current post-exposure prophylaxis use saves many lives, but availability remains poor in many rabies-endemic countries due to high costs, poor access, and supply. Methods: We developed epidemiological and economic models to investigate the effect of an investment in post-exposure prophylaxis by Gavi, the Vaccine Alliance. We modelled post-exposure prophylaxis use according to the status quo, with improved access using WHO-recommended intradermal vaccination, with and without rabies immunoglobulin, and with and without dog vaccination. We took the health provider perspective, including only direct costs. Findings: We predict more than 1 million deaths will occur in the 67 rabies-endemic countries considered from 2020 to 2035, under the status quo. Current post-exposure prophylaxis use prevents approximately 56 000 deaths annually. Expanded access to, and free provision of, post-exposure prophylaxis would prevent an additional 489 000 deaths between 2020 and 2035. Under this switch to efficient intradermal post-exposure prophylaxis regimens, total projected vaccine needs remain similar (about 73 million vials) yet 17·4 million more people are vaccinated, making this an extremely cost-effective method, with costs of US$635 per death averted and$33 per disability-adjusted life-years averted. Scaling up dog vaccination programmes could eliminate dog-mediated rabies over this time period; improved post-exposure prophylaxis access remains cost-effective under this scenario, especially in combination with patient risk assessments to reduce unnecessary post-exposure prophylaxis use. Interpretation: Investing in post-exposure vaccines would be an extremely cost-effective intervention that could substantially reduce disease burden and catalyse dog vaccination efforts to eliminate dog-mediated rabies. Funding: World Health Organization

International experiences on the formulation and implementation of transboundary climate change adaptation strategies

The Mekong River Commission’s (MRC) Climate Change and Adaptation Initiative (CCAI) aims at formulating the Climate Change Adaptation Strategy for the Lower Mekong Basin. An important first step in developing this strategy is to review international experiences of existing strategies, to learn from their process and their implementation, and to derive recommendations for a Mekong Adaptation Strategy and Action Plan (MASAP). This report provides a review of existing transboundary/basin-wide adaptation strategies. Relevant international basins and regions worldwide were first identified for the review. At the basin level, adaptation strategies of four basins were selected for review, including the Nile, Danube, Rhine and Neman. At the regional level, adaptation strategies of four regional cooperation organisations were reviewed – the Association of Southeast Asian Nations (ASEAN) adaptation strategy, the Common Market for Eastern and Southern Africa-East African Community-Southern African Development Community (COMESA-EAC-SADC) Climate Change Adaptation Strategy for the water sector, the European Adaptation Strategy, and the Amazonian Strategic Cooperation Agenda. The analysis in this report is based on the framework and steps for the development of an adaptation strategy, which is derived from the United Nations Economic Commission for Europe (UNECE) Guidance on Water and Adaptation to Climate Change. A comparison was made between the UNECE framework and the CCAI framework. The review was based on the following aspects: • the overall formulation process, which examines, inter alia, how the strategy was developed, the parties involved, the duration of the process and the time horizon of the strategy; • the enabling environment, which looks at hindrances to adaptation. The enabling environment includes the policy, legal and institutional frameworks within which the strategy is developed; • the information used and required, including the information management, the impact assessment and the vulnerability assessment; • the goals, objectives and measures of the strategy; • the implementation, monitoring and evaluation of the strategy. The review finds that initiatives for developing an adaptation strategy generally rely on the collaboration between the involved ministers, often ministers responsible for water management, and the transboundary institution responsible for coordination. Through the mandate, the parties 2 International experiences on the formulation and implementation of transboundary climate change adaptation strategies can ensure that their interests are included in the process. The first step in developing the strategy is usually a study on the climate change vulnerability of the basin or region. This is mainly achieved by compiling the available studies and drawing generic conclusions from them. Based on the vulnerability assessment, adaptation measures are identified, usually in broad terms, to enable adjusting these measures to the local conditions. The process of developing the adaptation strategy is generally carried out in close cooperation between the countries including the joint body, and representatives and experts of the individual countries at the national level. Usually also a broad range of stakeholders are involved through meetings and workshops. The process of developing an adaptation strategy is considered continuous as changing circumstances, due to climate change, socioeconomic and demographic developments, and changes in vulnerability and the relevant measures may change. In practice, this implies that an adaptation strategy once developed is reviewed every five to ten years to account for the changing environment. To make sure that both the process and the resulting strategy are relevant, efficient and effective, and have the expected impact, the process should be continuously evaluated. The time needed to elaborate the strategy, the time between the adoption of the mandate to develop a strategy and the adoption of the strategy by the parties, varies between one to six years. This depends, among others, on the available information and the extent of the mandate (e.g. a detailed versus a more generic strategy). In general, a period of two to three years is needed, but can be shortened when much of the preparatory work has been finalized, for instance on the vulnerability assessment. The climate change outlook to be included in the strategy usually extends to 2050. A longer time horizon is, however, useful when the strategy includes measures that imply long-term investments, for example, for infrastructures. The infrastructures should therefore be built so as to account for the longerterm projections of climate and other changes. Moreover, since climate change projections become more uncertain as they extend further in time, a time horizon of 2050 with some outlook until 2100 seems an appropriate approach. An important issue is to consider an integrated approach where the strategy is mainstreamed in the relevant policies. This will avoid contradictory policies and hindrances to implement specific measures. Benefits of transboundary cooperation are generally ignored or taken for granted. It is recommended to explicitly clarify the expected benefits of the joint strategy to ensure better implementation of the strategy. International experiences on the formulation and implementation of 3 transboundary climate change adaptation strategies Financing of the measures comes from the national budgets and from donors, depending on the economic situation in the countries. The Danube river basin strategy, for instance, has incorporated the implementation of the strategy in the process of implementing European Union (EU) directives, where the funding largely comes from national budgets. The EU strategy is aiming at directing funds to adaptation. The COMESA-EAC-SADC strategy aims at improving the access to international funds. The mandate adopted as well as the involvement of government representatives in the process of developing the strategy should ensure that the strategy is in line with the respective national policies and legislation, including international agreements. If it appears that the existing policy and/or legislation contradicts the goals and objectives of the mandate or the strategy, reconsideration of these policies and legislation should be included as part of the measures of the strategy. Also, adapting the institutional arrangements may be part of the strategy. The COMESA-EAC-SADC strategy, for instance, explicitly aims at capacity building and improvement of the institutions. An important element in developing the strategy is that there is agreement on the impacts and related vulnerabilities. This entails exchange of all relevant information and extended communication about scenarios and projections between the parties. The generic objective of the strategies is to reduce the vulnerability of society and ecosystems to climate change and improve resilience. The specific goals and objectives for each strategy reflect the priorities of the parties in the different strategies and are rooted in the differences in historical, geographical, political, and cultural contexts of the respective countries. Measures specified in the various strategy documents include both structural (e.g. protective dams) and non-structural (e.g. regulatory and economic instruments) measures. An important development in defining measures is the recognition of ecosystems as an adaptation option, either restoration and preservation or as a green infrastructural measure. The strategy should include an implementation plan to guide the implementation of the strategy and a monitoring and evaluation (M&E) plan to be able to keep track of the implementation. The M&E plan is also needed to ensure that if the climate change effects differ from the initial projections, the planned measures can be adapted to the changes. For this reason, it is also recommended to regularly revise the strategy. The strategy should therefore be valid for a period of five to ten years. The report ends with an overview of conclusions and recommendations for the development of the Mekong Adaptation Strategy and Action Plan (MASAP)

Sequence analysis and plasmid mobilization of a 6.6-kb kanamycin resistance plasmid, pSNC3-Kan, from a Salmonella enterica serotype Newport isolate.

Research on the transfer of antibiotic resistance plasmids has been mainly focused on the large multi-drug resistance conjugative plasmids, while the transmission of small mobilizable plasmids remains under-investigated. A series of diverse ColE-like kanamycin resistance plasmids ("KanR plasmids") from Salmonella enterica were characterized previously. In this study, the 6.6-kb pSNC3-Kan from a Salmonella enterica serotype Newport isolate was investigated. It possessed highly conserved RNA I/II and Tn602 (IS903-aph-IS903) regions to two other KanR plasmids pSe-Kan and pSBardo-Kan, but carried a mobC-mobA/BD operon. The mobilization proteins encoded by the mob operon of pSNC3-Kan showed high sequence identity (~95%) to those of an E. coli plasmid pEC34B, except that MobE was not present; and were much less conserved to those of another KanR plasmid pSN11/00Kan (43% - 86% identity). Four structurally different KanR plasmids were investigated for their ability to be mobilized by the conjugal transfer (tra) genes from F and IncP plasmids. Transfer genes derived from IncP plasmids can efficiently mobilize KanR plasmids possessing the mob operons (mobC-mobA/BD), such as pSNC3-Kan and pSN11/00Kan, in bi-parental mating experiments. On the other hand, F tra genes were able to mobilize pU302S, pSNC3-Kan and pSe-Kan, but not pSN11/00Kan. A plasmid-borne mob operon was not required for mobilization of the oriT(F)-bearing pSe-Kan by the F tra genes. This study underscores the complexity of plasmid interaction and the importance of how small mobilizable plasmids may contribute to the spread of antibiotic resistance genes

Genomic Comparison of Conjugative Plasmids from <i>Salmonella enterica</i> and <i>Escherichia coli</i> Encoding Beta-Lactamases and Capable of Mobilizing Kanamycin Resistance Col-like Plasmids

Salmonella enterica and Escherichia coli are important human pathogens that frequently contain plasmids, both large and small, carrying antibiotic resistance genes. Large conjugative plasmids are known to mobilize small Col plasmids, but less is known about the specificity of mobilization. In the current study, six S. enterica and four E. coli strains containing large plasmids were tested for their ability to mobilize three different kanamycin resistance Col plasmids (KanR plasmids). Large conjugative plasmids from five isolates, four S. enterica and one E. coli, were able to mobilize KanR plasmids of various types. Plasmids capable of mobilizing the KanR plasmids were either IncI1 or IncX, while IncI1 and IncX plasmids with no evidence of conjugation had disrupted transfer regions. Conjugative plasmids of similar types mobilized similar KanR plasmids, but not all conjugative plasmid types were capable of mobilizing all of the KanR plasmids. These data describe some of the complexities and specificities of individual small plasmid mobilization

Sequence alignment of the RNA I/II regions between CC7059F and CC7062R primer binding sites.

<p>A. Alignment of representative X and Y plasmids to pU302S (group A), pSN11/00Kan (group B), and pSe-Kan (group C). B. X plasmid against top 4 BLAST hits (pJJ1886_3, pColD-157, pO111_5, and pUB2380). Non-identical nucleotides are highlighted in colors.</p

Isolation and characterization of two novel groups of kanamycin-resistance ColE1-like plasmids in <i>Salmonella enterica</i> serotypes from food animals

<div><p>While antimicrobial resistance in <i>Salmonella enterica</i> is mainly attributed to large plasmids, small plasmids may also harbor antimicrobial resistance genes. Previously, three major groups of ColE1-like plasmids conferring kanamycin-resistance (Kan^R) in various <i>S</i>. <i>enterica</i> serotypes from diagnostic samples of human or animals were reported. In this study, over 200 Kan^R <i>S</i>. <i>enterica</i> isolates from slaughter samples, collected in 2010 and 2011 as a part of the animal arm of the National Antimicrobial Resistance Monitoring System, were screened for the presence of ColE1-like plasmids. Twenty-three Kan^R ColE1-like plasmids were successfully isolated. Restriction fragment mapping revealed five major plasmid groups with subgroups, including two new groups, X (n = 3) and Y/Y2/Y3 (n = 4), in addition to the previously identified groups A (n = 7), B (n = 6), and C/C3 (n = 3). Nearly 75% of the plasmid-carrying isolates were from turkey and included all the isolates carrying X and Y plasmids. All group X plasmids were from serotype Hadar. Serotype Senftenberg carried all the group Y plasmids and one group B plasmid. All Typhimurium isolates (n = 4) carried group A plasmids, while Newport isolates (n = 3) each carried a different plasmid group (A, B, or C). The presence of the selection bias in the NARMS strain collection prevents interpretation of findings at the population level. However, this study demonstrated that Kan^R ColE1-like plasmids are widely distributed among different <i>S</i>. <i>enterica</i> serotypes in the NARMS isolates and may play a role in dissemination of antimicrobial resistance genes.</p></div

Restriction digests of representative Kan^R ColE1-like plasmids.

<p>A. <i>Nla</i> III digests of representative plasmid groups: A (from BEAR099953), B (from BEAR097733), C (from BEAR101192), C3 (from BEAR097719), X (from BEAR099509), Y (from BEAR097612), Y2 (from BEAR099972) and Y3 (from BEAR101182). M1, 100 bp ladder (Invitrogen). B. <i>Xho</i> I+<i>Eco</i>R V and <i>Xho</i> I+<i>Bgl</i> II digests of plasmid groups X, Y, Y2, and Y3; M2, 1kb Extension ladder (Invitrogen).</p

Application of Machine Learning for eutrophication analysis and algal bloom prediction in an urban river: A 10-year study of the Han River, South Korea

The increasing release of nutrients to aquatic environments has led to great concern regarding eutrophication and the risk of unwanted algal blooms. Based on observational data of 20 water quality parameters measured on a monthly basis at 40 stations from 2011 to 2020, this study applied different Machine Learning (ML) algorithms to suggest the best option for algal bloom prediction in the Han River, a large river in South Korea. Eight different ML algorithms were categorized into several groups of statistical learning, regression family, and deep learning, and were then compared for their suitability to predict the chlorophyll-derived trophic index (TSI-Chla). ML algorithms helped identify the most important water quality parameters contributing to algal bloom prediction. The ML results confirmed that eutrophication and algal proliferation were governed by the complex interplay between nutrients (nitrogen and phosphorus), organic contaminants, and environmental factors. Of the models tested, the adaptive neuro-fuzzy inference system (ANFIS) exhibited the best performance owing to its consistent and outperforming prediction both quantitatively (i.e., via regression) and qualitatively (i.e., via classification), which was evidenced by the lowest value of mean absolute error (MAE) of 0.09, and the highest F1-score, Recall and Precision of 0.97, 0.98 and 0.96, respectively. In a further step, a representative web application was constructed to assist common users to predict the trophic status of the Han River. This study demonstrated that ML techniques are not only promising for highly accurate water quality modeling of urban rivers, but also reduce time and labor intensity for experiments, which decreases the number of monitored water quality parameters, providing further insights into the driving factors of water quality deterioration. They ultimately help devise proactive strategies for sustainable water management. (c) 2021 Elsevier B.V. All rights reserved