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Complex Interplay of Physiology and Selection in the Emergence of Antibiotic Resistance.

ABSTRACT: Emergence of antibiotic resistance, an evolutionary process of major importance for human health [1], often occurs under changing levels of antibiotics. Selective sweeps, in which resistant cells become dominant in the population, are a critical step in this process [2]. While resistance emergence has been studied in laboratory experiments [3-8], the full progression of selective sweeps under fluctuating stress, from stochastic events in single cells to fixation in populations, has not been characterized. Here, we study fluctuating selection using Escherichia coli populations engineered with a stochastic switch controlling tetracycline resistance. Using microfluidics and live-cell imaging, we treat multiple E. coli populations with the same total amount of tetracycline but administered in different temporal patterns. We find that populations exposed to either short or long antibiotic pulses are likely to develop resistance through selective sweeps, whereas intermediate pulses allow higher growth rates but suppress selective sweeps. On the basis of single-cell measurements and a dynamic growth model, we identify the major determinants of population growth and show that both physiological memory and environmental durations can strongly modulate the emergence of resistance. Our detailed quantification in a model synthetic system provides key lessons on the interaction between single-cell physiology and selection that should inform the design of treatment regimens [9-12] and the analysis of phenotypically diverse populations adapting under fluctuating selection [13-17].

SUBMITTER: Lin WH

PROVIDER: S-EPMC4899102 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Complex Interplay of Physiology and Selection in the Emergence of Antibiotic Resistance.

Lin Wei-Hsiang WH Kussell Edo E

Current biology : CB 20160519 11

Emergence of antibiotic resistance, an evolutionary process of major importance for human health [1], often occurs under changing levels of antibiotics. Selective sweeps, in which resistant cells become dominant in the population, are a critical step in this process [2]. While resistance emergence has been studied in laboratory experiments [3-8], the full progression of selective sweeps under fluctuating stress, from stochastic events in single cells to fixation in populations, has not been char ...[more]

PMID: 27212408

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Project description:The origin of novel genes and beneficial functions is of fundamental interest in evolutionary biology. New genes can originate from different mechanisms, including horizontal gene transfer, duplication-divergence, and de novo from noncoding DNA sequences. Comparative genomics has generated strong evidence for de novo emergence of genes in various organisms, but experimental demonstration of this process has been limited to localized randomization in preexisting structural scaffolds. This bypasses the basic requirement of de novo gene emergence, i.e., lack of an ancestral gene. We constructed highly diverse plasmid libraries encoding randomly generated open reading frames and expressed them in Escherichia coli to identify short peptides that could confer a beneficial and selectable phenotype in vivo (in a living cell). Selections on antibiotic-containing agar plates resulted in the identification of three peptides that increased aminoglycoside resistance up to 48-fold. Combining genetic and functional analyses, we show that the peptides are highly hydrophobic, and by inserting into the membrane, they reduce membrane potential, decrease aminoglycoside uptake, and thereby confer high-level resistance. This study demonstrates that randomized DNA sequences can encode peptides that confer selective benefits and illustrates how expression of random sequences could spark the origination of new genes. In addition, our results also show that this question can be addressed experimentally by expression of highly diverse sequence libraries and subsequent selection for specific functions, such as resistance to toxic compounds, the ability to rescue auxotrophic/temperature-sensitive mutants, and growth on normally nonused carbon sources, allowing the exploration of many different phenotypes.IMPORTANCEDe novo gene origination from nonfunctional DNA sequences was long assumed to be implausible. However, recent studies have shown that large fractions of genomic noncoding DNA are transcribed and translated, potentially generating new genes. Experimental validation of this process so far has been limited to comparative genomics, in vitro selections, or partial randomizations. Here, we describe selection of novel peptides in vivo using fully random synthetic expression libraries. The peptides confer aminoglycoside resistance by inserting into the bacterial membrane and thereby partly reducing membrane potential and decreasing drug uptake. Our results show that beneficial peptides can be selected from random sequence pools in vivo and support the idea that expression of noncoding sequences could spark the origination of new genes.

Project description:Biocide use is essential and ubiquitous, exposing microbes to sub-inhibitory concentrations of antiseptics, disinfectants, and preservatives. This can lead to the emergence of biocide resistance, and more importantly, potential cross-resistance to antibiotics, although the degree, frequency, and mechanisms that give rise to this phenomenon are still unclear. Here, we systematically performed adaptive laboratory evolution of the gut bacteria Escherichia coli in the presence of sub-inhibitory, constant concentrations of ten widespread biocides. Our results show that 17 out of 40 evolved strains (43%) also decreased the susceptibility to medically relevant antibiotics. Through whole-genome sequencing, we identified mutations related to multidrug efflux proteins (mdfA and acrR), porins (envZ and ompR), and RNA polymerase (rpoA and rpoBC), as mechanisms behind the resulting (cross)resistance. We also report an association of several genes (yeaW, pyrE, yqhC, aes, pgpA, and yeeP-isrC) and specific mutations that induce cross-resistance, verified through mutation repairs. A greater capacity for biofilm formation with respect to the parent strain was also a common feature in 11 out of 17 (65%) cross-resistant strains. Evolution in the biocides chlorophene, benzalkonium chloride, glutaraldehyde, and chlorhexidine had the most impact in antibiotic susceptibility, while hydrogen peroxide and povidone-iodine the least. No cross-resistance to antibiotics was observed for isopropanol, ethanol, sodium hypochlorite, and peracetic acid. This work reinforces the link between exposure to biocides and the potential for cross-resistance to antibiotics, presents evidence on the underlying mechanisms of action, and provides a prioritized list of biocides that are of greater concern for public safety from the perspective of antibiotic resistance.Significance statementBacterial resistance and decreased susceptibility to antimicrobials is of utmost concern. There is evidence that improper biocide (antiseptic and disinfectant) use and discard may select for bacteria cross-resistant to antibiotics. Understanding the cross-resistance emergence and the risks associated with each of those chemicals is relevant for proper applications and recommendations. Our work establishes that not all biocides are equal when it comes to their risk of inducing antibiotic resistance; it provides evidence on the mechanisms of cross-resistance and a risk assessment of the biocides concerning antibiotic resistance under residual sub-inhibitory concentrations.

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Complex Interplay of Physiology and Selection in the Emergence of Antibiotic Resistance.

Publications

Complex Interplay of Physiology and Selection in the Emergence of Antibiotic Resistance.

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