Strain-level bacterial typing directly from patient samples using optical DNA mapping

For bacterial infections, it is important to rapidly and accurately identify and characterize the type of bacteria involved so that optimal antibiotic treatment can be given quickly to the patient. However, current diagnostic methods are sometimes slow and cannot be used for mixtures of bacteria. We have, therefore, developed a method to identify bacteria directly from patient samples. The method was tested on two common species of disease-causing bacteria - Escherichia coli and Klebsiella pneumoniae - and it could correctly identify the bacterial strain or subtype in both urine samples and mixtures. Hence, the method has the potential to provide fast diagnostic information for choosing the most suited antibiotic, thereby reducing the risk of death and suffering. Nyblom, Johnning et al. develop an optical DNA mapping approach for bacterial strain typing of patient samples. They demonstrate rapid identification of clinically relevant E. coli and K. pneumoniae strains, without the need for cultivation. BackgroundIdentification of pathogens is crucial to efficiently treat and prevent bacterial infections. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions.MethodsIn this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation. For the typing, different taxonomical resolutions were examined and cultivated pure Escherichia coli and Klebsiella pneumoniae samples were used for parameter optimization. Finally, the method was applied to mixed bacterial samples and uncultured urine samples from patients with urinary tract infections.ResultsWe demonstrate that optical DNA mapping of single DNA molecules can identify Escherichia coli and Klebsiella pneumoniae at the strain level directly from patient samples. At a taxonomic resolution corresponding to E. coli sequence type 131 and K. pneumoniae clonal complex 258 forming distinct groups, the average true positive prediction rates are 94% and 89%, respectively. The single-molecule aspect of the method enables us to identify multiple E. coli strains in polymicrobial samples. Furthermore, by targeting plasmid-borne antibiotic resistance genes with Cas9 restriction, we simultaneously identify the strain or subtype and characterize the corresponding plasmids.ConclusionThe optical DNA mapping method is accurate and directly applicable to polymicrobial and clinical samples without cultivation. Hence, it has the potential to rapidly provide comprehensive diagnostics information, thereby optimizing early antibiotic treatment and opening up for future precision medicine management

Interplay between pleiotropy and secondary selection determines rise and fall of mutators in stress response

Dramatic rise of mutators has been found to accompany adaptation of bacteria in response to many kinds of stress. Two views on the evolutionary origin of this phenomenon emerged: the pleiotropic hypothesis positing that it is a byproduct of environmental stress or other specific stress response mechanisms and the second order selection which states that mutators hitchhike to fixation with unrelated beneficial alleles. Conventional population genetics models could not fully resolve this controversy because they are based on certain assumptions about fitness landscape. Here we address this problem using a microscopic multiscale model, which couples physically realistic molecular descriptions of proteins and their interactions with population genetics of carrier organisms without assuming any a priori fitness landscape. We found that both pleiotropy and second order selection play a crucial role at different stages of adaptation: the supply of mutators is provided through destabilization of error correction complexes or fluctuations of production levels of prototypic mismatch repair proteins (pleiotropic effects), while rise and fixation of mutators occur when there is a sufficient supply of beneficial mutations in replication-controlling genes. This general mechanism assures a robust and reliable adaptation of organisms to unforeseen challenges. This study highlights physical principles underlying physical biological mechanisms of stress response and adaptation

Spontaneous Emergence of Multiple Drug Resistance in Tuberculosis before and during Therapy

The emergence of drug resistance in M. tuberculosis undermines the efficacy of tuberculosis (TB) treatment in individuals and of TB control programs in populations. Multiple drug resistance is often attributed to sequential functional monotherapy, and standard initial treatment regimens have therefore been designed to include simultaneous use of four different antibiotics. Despite the widespread use of combination therapy, highly resistant M. tb strains have emerged in many settings. Here we use a stochastic birth-death model to estimate the probability of the emergence of multidrug resistance during the growth of a population of initially drug sensitive TB bacilli within an infected host. We find that the probability of the emergence of resistance to the two principal anti-TB drugs before initiation of therapy ranges from 10−5 to 10−4; while rare, this is several orders of magnitude higher than previous estimates. This finding suggests that multidrug resistant M. tb may not be an entirely “man-made” phenomenon and may help explain how highly drug resistant forms of TB have independently emerged in many settings

Acetate availability and utilization supports the growth of mutant sub-populations on aging bacterial colonies

When bacterial colonies age most cells enter a stationary phase, but sub-populations of mutant bacteria can continue to grow and accumulate. These sub-populations include bacteria with mutations in rpoB (RNA polymerase β-subunit) or rpoS (RNA polymerase stress-response sigma factor). Here we have identified acetate as a nutrient present in the aging colonies that is utilized by these mutant subpopulations to support their continued growth. Proteome analysis of aging colonies showed that several proteins involved in acetate conversion and utilization were upregulated during aging. Acetate is known to be excreted during the exponential growth phase but can be imported later during the transition to stationary phase and converted to acetyl-CoA. Acetyl-CoA is used in multiple processes, including feeding into the TCA cycle, generating ATP via the glyoxylate shunt, as a source of acetyl groups for protein modification, and to support fatty acid biosynthesis. We showed that deletion of acs (encodes acetyl-CoA synthetase; converts acetate into acetyl-CoA) significantly reduced the accumulation of rpoB and rpoS mutant subpopulations on aging colonies. Measurement of radioactive acetate uptake showed that the rate of conversion decreased in aging wild-type colonies, was maintained at a constant level in the rpoB mutant, and significantly increased in the aging rpoS mutant. Finally, we showed that the growth of subpopulations on aging colonies was greatly enhanced if the aging colony itself was unable to utilize acetate, leaving more acetate available for mutant subpopulations to use. Accordingly, the data show that the accumulation of subpopulations of rpoB and rpoS mutants on aging colonies is supported by the availability in the aging colony of acetate, and by the ability of the subpopulation cells to convert the acetate to acetyl-CoA

Influence of mutations affecting acetate metabolism on the growth advantage of <i>rpoB</i> P564L and Δ<i>rpoS</i> mutants.

<p>Fold increase in wild-type and mutant cells added to 24 h wild-type colonies and allowed to age for a further 7 days, as a function of acetate metabolism activity. <b>A</b>. Relative to the <i>rpoB</i> 564L mutant. <b>B</b>. Relative to the Δ<i>rpoS</i> mutant. The box plots show the first quartile, median, and third quartile values. Outliers are indicated as triangles. Δ<i>apa</i> indicates deletion of the three genes, <i>ackA-pta</i> and <i>acs</i>. Statistical significance of differences, compared to the <i>rpoB</i> or <i>rpoS</i> mutants, is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109255#pone.0109255.s002" target="_blank">Table S2</a> and is indicated in the figure by asterisks (**  = 99% confidence interval, ***  = 99.9% confidence interval).</p

Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies

<div><p>When bacterial colonies age most cells enter a stationary phase, but sub-populations of mutant bacteria can continue to grow and accumulate. These sub-populations include bacteria with mutations in <i>rpoB</i> (RNA polymerase β-subunit) or <i>rpoS</i> (RNA polymerase stress-response sigma factor). Here we have identified acetate as a nutrient present in the aging colonies that is utilized by these mutant subpopulations to support their continued growth. Proteome analysis of aging colonies showed that several proteins involved in acetate conversion and utilization were upregulated during aging. Acetate is known to be excreted during the exponential growth phase but can be imported later during the transition to stationary phase and converted to acetyl-CoA. Acetyl-CoA is used in multiple processes, including feeding into the TCA cycle, generating ATP via the glyoxylate shunt, as a source of acetyl groups for protein modification, and to support fatty acid biosynthesis. We showed that deletion of <i>acs</i> (encodes acetyl-CoA synthetase; converts acetate into acetyl-CoA) significantly reduced the accumulation of <i>rpoB</i> and <i>rpoS</i> mutant subpopulations on aging colonies. Measurement of radioactive acetate uptake showed that the rate of conversion decreased in aging wild-type colonies, was maintained at a constant level in the <i>rpoB</i> mutant, and significantly increased in the aging <i>rpoS</i> mutant. Finally, we showed that the growth of subpopulations on aging colonies was greatly enhanced if the aging colony itself was unable to utilize acetate, leaving more acetate available for mutant subpopulations to use. Accordingly, the data show that the accumulation of subpopulations of <i>rpoB</i> and <i>rpoS</i> mutants on aging colonies is supported by the availability in the aging colony of acetate, and by the ability of the subpopulation cells to convert the acetate to acetyl-CoA.</p></div

RpoB and RpoS mutants have a growth advantage on aging colonies.

<p>Fold increase in wild-type and mutant cells added to 24 h wild-type colonies and allowed to age for a further 7 days. The box plots show the first quartile, median, and third quartile values. Outlier indicated by a triangle. Statistical significance of differences in the distribution of values between strains, compared to the wild-type, is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109255#pone.0109255.s002" target="_blank">Table S2</a> and indicated in the figure by asterisks (*  = 95% confidence interval, ***  = 99.9% confidence interval).</p

Protein quantification in aging colonies.

<p>Total protein was prepared from colonies of either wild-type, <i>rpoB</i> P564L mutant or Δ<i>rpoS</i> mutant, all grown for 1, 3, 5 and 7 days. Concentrations of the proteins of interest were quantified by Single Ion Monitoring mass spectrometry. Bars represent averages of two or three peptides per protein, measured in biological duplicates, each measured twice, with error bars representing standard deviation between the runs. Concentration values are in arbitrary units, normalized to total protein in the samples, to enable direct comparison between different days and different samples. The day 5 sample from the Δ<i>rpoS</i> mutant could not be analyzed. Statistical significance of differences, compared to wild-type samples of the same age, are indicated in the figure (-  =  not significant, *  = 95% confidence interval, **  = 99% conficence interval, ***  = 99.9% confidence interval). <b>A</b>. Concentration of Acs, acetyl-CoA synthase. <b>B</b>. Concentration of AceA, isocitrate lyase. <b>C</b>. Concentration of AceB, malate synthase. <b>D</b>. Concentration of AckA, acetate kinase. <b>E</b>. Concentration of Pta, phosphotransacetylase.</p

Influence of the <i>acs</i> status of the background colony on the growth advantage of Rif^R and RpoS mutants.

<p>Fold increase in wild-type and mutant cells added to 24 h wild-type or Δ<i>acs</i> colonies and allowed to age for a further 7 days. The box plots show the first quartile, median, and third quartile values. Outliers are indicated as triangles. Statistical significance of differences between strains is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109255#pone.0109255.s002" target="_blank">Table S2</a> and is indicated in the figure by asterisks (**  = 99% confidence interval, ***  = 99.9% confidence interval).</p

Outline of central metabolism indicating acetate production and utilization pathways.

<p>Outline of central metabolism showing the acetate synthesis and utilization pathways <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109255#pone.0109255-Wolfe1" target="_blank">[24]</a>. Gene names are shown in italics, substrates and products are referred to in the text.</p