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New quantitative methods for measuring plasmid loss rates reveal unexpected stability.

ABSTRACT: Plasmid loss rate measurements are standard in microbiology and key to understanding plasmid stabilization mechanisms. The conventional assays eliminate selection for plasmids at the beginning of the experiment and screen for the appearance of plasmid-free cells over long-term population growth. However, it has been long appreciated in plasmid biology that the growth rate differential between plasmid-free and plasmid-containing cells at some point overshadows the effect of primary loss events, such that the assays can greatly over-estimate inherent loss rates. The standard solutions to this problem are to either consider the very early phase of loss where the fraction of plasmid-free cells increases linearly, or to measure the growth rate difference either by following the population for longer time or by measuring growth rates separately. Here we mathematically show that in all these cases, seemingly small experimental errors in the growth rate estimates can overshadow the estimates of the loss rates. For many plasmids, loss rates may thus be much lower than previously thought, and for some plasmids, the estimated loss rate may have nothing to do with actual loss rates. We further modify two independent experimental methods to separate inherent losses from growth differences and apply them to the same plasmids. First we use a high-throughput microscopy-based approach to screen for plasmid-free cells at extremely short time scales--tens of minutes rather than tens of generations--and apply it to a par? version of mini-R1. Second we modify a counterselection-based plasmid loss assay inspired by the Luria-Delbrück fluctuation test that completely separates losses from growth, and apply it to various R1 and pSC101 derivatives. Concordant results from the two assays suggest that plasmids are lost at a lower frequency than previously believed. In fact, for par? mini-R1 the observed loss rate of about 10?³ per cell and generation seems to be so low as to be inconsistent with what we know about the R1 stabilization mechanisms, suggesting these well characterized plasmids may have some additional and so far unknown stabilization mechanisms, for example improving copy number control or partitioning at cell division.

SUBMITTER: Lau BT

PROVIDER: S-EPMC3966108 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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New quantitative methods for measuring plasmid loss rates reveal unexpected stability.

Lau Billy T C BT Malkus Per P Paulsson Johan J

Plasmid 20130913 3

Plasmid loss rate measurements are standard in microbiology and key to understanding plasmid stabilization mechanisms. The conventional assays eliminate selection for plasmids at the beginning of the experiment and screen for the appearance of plasmid-free cells over long-term population growth. However, it has been long appreciated in plasmid biology that the growth rate differential between plasmid-free and plasmid-containing cells at some point overshadows the effect of primary loss events, s ...[more]

PMID: 24042048

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Project description:There has been increasing interest in measuring under-five mortality as a health indicator and as a critical measure of human development. In countries with complete vital registration systems that capture all births and deaths, under-five mortality can be directly calculated. In the absence of a complete vital registration system, however, child mortality must be estimated using surveys that ask women to report the births and deaths of their children. Two survey methods exist for capturing this information: summary birth histories and complete birth histories. A summary birth history requires a minimum of only two questions: how many live births has each mother had and how many of them have survived. Indirect methods are then applied using the information from these two questions and the age of the mother to estimate under-five mortality going back in time prior to the survey. Estimates generated from complete birth histories are viewed as the most accurate when surveys are required to estimate under-five mortality, especially for the most recent time periods. However, it is much more costly and labor intensive to collect these detailed data, especially for the purpose of generating small area estimates. As a result, there is a demand for improvement of the methods employing summary birth history data to produce more accurate as well as subnational estimates of child mortality.We used data from 166 Demographic and Health Surveys (DHS) to develop new empirically based methods of estimating under-five mortality using children ever born and children dead data. We then validated them using both in- and out-of-sample analyses. We developed a range of methods on the basis of three dimensions of the problem: (1) approximating the average length of exposure to mortality from a mother's set of children using either maternal age or time since first birth; (2) using cohort and period measures of the fraction of children ever born that are dead; and (3) capturing country and regional variation in the age pattern of fertility and mortality. We focused on improving estimates in the most recent time periods prior to a survey where the traditional indirect methods fail. In addition, all of our methods incorporated uncertainty. Validated against under-five estimates generated from complete birth histories, our methods outperformed the standard indirect method by an average of 43.7% (95% confidence interval [CI] 41.2-45.2). In the 5 y prior to the survey, the new methods resulted in a 53.3% (95% CI 51.3-55.2) improvement. To illustrate the value of this method for local area estimation, we applied our new methods to an analysis of summary birth histories in the 1990, 2000, and 2005 Mexican censuses, generating subnational estimates of under-five mortality for each of 233 jurisdictions.The new methods significantly improve the estimation of under-five mortality using summary birth history data. In areas without vital registration data, summary birth histories can provide accurate estimates of child mortality. Because only two questions are required of a female respondent to generate these data, they can easily be included in existing survey programs as well as routine censuses of the population. With the wider application of these methods to census data, countries now have the means to generate estimates for subnational areas and population subgroups, important for measuring and addressing health inequalities and developing local policy to improve child survival. Please see later in the article for the Editors' Summary.

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New quantitative methods for measuring plasmid loss rates reveal unexpected stability.

Publications

New quantitative methods for measuring plasmid loss rates reveal unexpected stability.

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