This work was supported by the Instituto de Salud Carlos III (Plan Estatal de I+D+i 2013-2016): grants CP15-00012, PI16-00860, and CIBER (CB06/02/0053), co-financed by the European Development Regional Fund &#39;&#39;A way to achieve Europe&#39;&#39; (ERDF). JAE is supported by the Atracci&#243;n de talento program of the government of the region of Madrid (2016-T1/BIO-1105) and the I+D Excelencia of the Spanish Ministerio de Econom&#237;a, Industria y Competitividad (BIO2017-85056-P). ASM is supported by a Miguel Servet Fellowship from the Instituto de Salud Carlos III (MS15/00012) co-financed by The European Social Fund &#34;Investing in your future&#34; (ESF) and ERDF.

1. Construction of the Experimental System Encoding Antibiotic Resistance Gene
Note: Here E. coli MG1655 was used as the recipient strain of the plasmid- or chromosome-encoded antibiotic resistance gene. The antibiotic resistance gene is encoded in the chromosome or a multicopy plasmid in an otherwise isogenic strain (Figure 1).
<ol>
	<li>Insertion of the antibiotic resistance gene in the &#955; phage integration site (attB)20 of the chromosome of MG1655.
	<ol>
		<li>Amplify 500 bp-long regions at both sides of the chromosomal attB site by PCR. Use oligonucleotides YbhC-F (5&#39;- CCTGTACCGTACAGAGTAAT-3&#39;) and attB-R (5&#39;- GCCCGCCACCCTCCGGGCCGGTATAAAAAAGCAGGCTTCA-3&#39;) for the left homology region and attB-F (5&#39;- AGCGCCCTAGCGCCCGCTCCTTATACTAACTTGAGCGAAA-3&#39;) and YbhB/R (5&#39;- TGGCGATAATATTTCACCGC-3&#39;) for the right one. Amplify the blaTEM-1 resistance gene using primers Tem1-pBAD-F (5&#39;- TGAAGCCTGCTTTTTTATACCGGCCCGGAGGGTGGCGGGC-3&#39;) and Tem1-pBAD-R (5&#39;- TTTCGCTCAAGTTAGTATAAGGAGCGGGCGCTAGGGCGCT-3&#39;). Design primers with approximately 20 bp (in the 5&#39; end) of sequence showing complementarity to the fragment that is going to be fused to.</li>
		<li>Fuse homology regions to the antibiotic resistance gene PCR product (including its promoter region) using isothermal assembly21, at 50 &#176;C for 30 min.</li>
		<li>Electroporate MG1655 cells containing plasmid pKOBEG. 
		Note: pKOBEG is a thermosensitive vector that contains the &#955; Red machinery, promoting homology-based allelic exchanges between the chromosome and PCR products22.
		<ol>
			<li>Use 1 &#181;L of the product from step 1.1.223 into 40 &#181;L of electrocompetent cells in 2 mm cuvettes at 4 &#176;C and 2.5 kV. Resuspend cells in 1 mL of LB broth + 0.2% arabinose to maintain the expression of the &#955; Red machinery.</li>
			<li>Transfer the total volume to a microfuge tube and incubate 2 h at 30 &#176;C (permissive temperature for pKOBEG) with intense shaking (200 rpm in an orbital shaker) to allow for the phenotypic expression of the resistance markers inserted in the chromosome.</li>
			<li>Plate the cells on LB agar containing the appropriate antibiotic to select for the antibiotic resistance gene (ampicillin or carbenicillin at 100 mg/l for blaTEM-1).</li>
		</ol>
		</li>
		<li>Plate 100 &#181;L on one Petri dish, and spin down the rest of the volume, resuspend it in 100 &#181;L of fresh LB broth and plate it on another Petri dish. Incubate overnight at 42 &#176;C. 
		Note: This temperature is non-permissive for the replication of pKOBEG. The colonies able to grow in these conditions will have lost pKOBEG and will present the resistance gene integrated into the attB site (for simplicity this strain will be called MG1655::resA from here on).</li>
		<li>Test for the loss of pKOBEG by replicating the colonies of interest in plates with chloramphenicol (the antibiotic against which pKOBEG contains a resistance marker). 
		Note: Lack of growth in chloramphenicol plates at the permissive temperature of 30 &#186;C is indicative of the absence of the plasmid.</li>
		<li>In order to verify clones, PCR amplify the construction using external primers YbhC-Extern (5&#39;-TTTGTGACCAGAAGACCGCA-3&#39;) and YbhB-Extern (5&#39;-CTCATCAGTAACGATCTGCG-3&#39;), verify through gel electrophoresis the correct size of the amplicon and sequence the purified product to ensure that the sequence of the inserted gene is correct.</li>
	</ol>
	</li>
	<li>Insert the antibiotic resistance gene in a multicopy plasmid. 
	Note: Because plasmids with very-high copy number tend to impose a large reduction in bacterial host fitness, we recommend the use of multicopy plasmids with a natural origin of replication, such as p3655 (pSU18T-pBADgfp2, ColE1-type origin of replication24). These plasmids usually present a moderate copy number of about 15-20 copies per cell.
	<ol>
		<li>PCR amplify the antibiotic resistance gene of choice (including promoter region), phosphorylate the PCR product and ligate it to the PCR-amplified backbone of the plasmid:
		<ol>
			<li>PCR amplify the antibiotic resistance gene; for blaTEM-1 use primers Tem1-pBAD-F (5&#39;- TGAAGCCTGCTTTTTTATACCGGCCCGGAGGGTGGCGGGC-3&#39;) and Tem1-pBAD-R (5&#39;- TTTCGCTCAAGTTAGTATAAGGAGCGGGCGCTAGGGCGCT-3&#39;).</li>
			<li>Phosphorylate the purified product using T4 polynucleotide kinase, following manufacturer&#39;s instructions.</li>
			<li>PCR amplify the plasmid backbone using a high-fidelity polymerase and oligonucleotides pBAD-F (5&#39;-CGTTGATCGGCACGTAAGAG-3&#39;) and pBAD-R (5&#39;-AAACGACGGCCAGTGCCAAG-3&#39;).</li>
			<li>Ligate both PCR fragments using T4-ligase following manufacturer&#39;s guidelines.</li>
		</ol>
		</li>
		<li>Electroporate, as described previously, 40 &#181;L of electrocompetent E. coli DH5-&#945; cells with a maximum of 1 &#181;L of the ligation product and select on the appropriate antibiotics for the antibiotic resistance gene (ampicillin or carbenicillin at 100 mg/L for blaTEM-1) and the plasmid backbone. 
		Note: Supplementing LB with arabinose here is unnecessary.</li>
		<li>Extract the plasmid using the commercial mini-prep kit of the reader&#39;s choice:
		<ol>
			<li>Harvest 5 mL of an overnight culture by centrifuging at 12,000 x g at room temperature. Resuspend the cells in 250 &#181;L of resuspension solution and mix well. Add 250 &#181;L of lysis solution and mix well. Add 350 &#181;L of neutralization and mix well.</li>
			<li>Transfer the supernatant to the DNA binding column provided with the kit, centrifuge for 1 min and discard the flow through. Wash twice the column with 500 &#181;L of washing solution, centrifuging for one minute and discarding the flowthrough at each wash. Centrifuge the empty column for 1 additional min to remove residual washing buffer.</li>
			<li>Place the column in a clean tube and add 50 &#181;L of ultra-pure water to the membrane to elute the purified DNA. Centrifuge for 1-2 min and collect the flow through that contains the purified plasmid. Sanger sequence the gene of interest in the plasmid using primers from outside the inserted sequence to confirm that the sequence is correct and that no mutations are present in the resistance gene prior to the evolution experiments.</li>
		</ol>
		</li>
		<li>Once the sequence is verified, electroporate the plasmid in the E. coli MG1655 strain, as described above. 
		Note: This yields strain MG1655/pRESA.</li>
	</ol>
	</li>
</ol>
2. Evolutionary Rescue Approach to Experimentally Evolve Antibiotic Resistance (Figure 1)
<ol>
	<li>Streak out the strains under study on LB plates: MG1655::resA and MG1655/pRESA plus the susceptible parental strain, MG1655. 
	Note: Plasmid pRESA should be stable in MG1655, so there is no need to add antibiotics to the LB plates. Mutation rates in E. coli are low enough so once the construction is verified it is not necessary to verify plasmid sequence at each step.</li>
	<li>Prepare 96-well plates with 200 &#181;L of LB in each well and inoculate 48 isolated colonies of each strain in independent wells (one plate per strain). Incubate the plates overnight at 37 &#176;C and 200 rpm. Keep a frozen stock of these founder populations. 
	Note: To prevent and control for culture cross-contamination in the 96-well plates, use a checkerboard plate design by intercalating inoculated wells with bacteria-free medium throughout the 96-well plate. Use this plate design during the entire experimental evolution.</li>
	<li>Start the evolutionary rescue experiment by inoculating 2 &#181;L from each well of the plates with the founder populations in new 96-well plates with 198 &#181;L of LB in each well with a sub-inhibitory concentration of the antibiotic under study. For the evolutionary rescue approach start with &#188; or 1/8 of the minimal inhibitory concentration (MIC, determined previously25) of the antibiotic in LB for each of the three strains and double the concentration of the antibiotic daily. Use this approach to maximize the chances of populations to acquire resistance mutations26. Incubate the plates at 37 &#176;C and 200 rpm for 20 h. 
	Note: Measure the overnight OD of the founder populations. If there are significant differences in OD among strains correct the initial inoculum to start the experiment with the same number of cells per well.</li>
	<li>Every day, measure the OD of each population after overnight culture and perform a transfer of the cultures, as described in 2.3, to new 96-well plates with double the concentration of the antibiotic than the day before. Incubate the plates 20 h at 37 &#176;C and 200 rpm. 
	Note: The 1:100 dilution factor produces approximately 6-7 generations per day.</li>
	<li>In parallel, propagate control populations of each strain in the same conditions as described in 2.3 and 2.4, but in the absence of antibiotics. 
	Note: Control populations will help discriminate between mutations arising due to the presence of the antibiotics and general mutations helping bacteria to adapt to the experimental conditions. Parallel mutations arising in the absence of antibiotics are likely to be helping bacteria to adapt to the experimental conditions and not related to antibiotic resistance.</li>
	<li>Track the number of surviving populations every day by measuring the absorbance at a wavelength of 600 nm (OD) of the cultures using a plate reader. 
	Note: Optical density values lower than 0.1 indicate the extinction of the population. See Figure 2 for an example of survival curves.</li>
	<li>Keep a frozen stock of all of the populations periodically (every 3-5 days).</li>
	<li>Use log-rank tests [package &#34;survival&#34; in RStudio (Version 0.99.486)] to determine statistical differences in the survival of populations of the different strains over time under increasing concentration of antibiotics12. 
	Note: This experiment will determine if multicopy plasmids potentiate the evolution of antibiotic resistance for the particular antibiotic and gene under study.</li>
</ol>
3. Molecular Basis of the Evolution of Antibiotic Resistance (Figure 1)
<ol>
	<li>Perform total DNA extractions from a representative number of populations and clones across strains and treatments. Include the parental strains (MG1655, MG1655::resA and MG1655/pRESA) to be able to detect the mutations accumulating during the experiment. 
	Note: For examples of DNA extraction kits see the table of materials. Each kit has different protocols; follow manufacturers instructions.</li>
	<li>Quantify DNA quality and concentration. There are different methods to determine DNA quality and concentration. Determine the quality measuring the ratios of absorbance 260 nm/280 nm and 260 nm/230 nm. Use a fluorescent, DNA-binding dye to measure DNA concentration following manufacturer&#39;s instructions, and agarose gel electrophoresis to confirm that there is no DNA degradation or RNA contamination.</li>
	<li>Use deep DNA sequencing from samples of whole populations and individual clones to investigate the genetic basis of antibiotic resistance. 
	Note: We conducted all sequencing at the Wellcome Trust Centre for Human Genetics, Oxford, UK. For more details see San Millan et al. 201612.</li>
	<li>Use the breseq 0.26.1 pipeline27,28 to detect the mutations, using polymorphism mode to estimate the frequency of mutations in populations. Compare the different evolved genomes to the parental genome to detect mutations that have accumulated during the experiment.</li>
	<li>Compare the mutations accumulated in parallel in the control populations with those of the populations evolved under increasing concentration of antibiotics to differentiate between mutations helping bacteria to adapt to the general experimental conditions (those found in the control populations) and those involved in antibiotic resistance (those found exclusively in populations subjected to antibiotic pressure). 
	Note: The number of parallel mutations is usually low, so different parallel mutations between treatments can be easily assessed. Apart from the mutations in the antibiotic resistance gene under study it is quite likely to find other mutations associated with antibiotic resistance in the chromosome, such mutations in porins or efflux systems12.</li>
	<li>Reconstruct the mutations in the antibiotic resistance gene in the parental strain using the same approach as in section 1 of this protocol. Follow points 1.1 and 1.2 of the protocol to introduce the new mutations in the parental background (using the evolved genes as a PCR template). Analyze the antibiotic resistance phenotypes of these new constructions to confirm the role of the mutations.</li>
</ol>

<ol>
	<li>Neill, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TACKLING+DRUG-RESISTANT+INFECTIONS+GLOBALLY:+FINAL+REPORT+AND+RECOMMENDATIONS.">TACKLING DRUG-RESISTANT INFECTIONS GLOBALLY: FINAL REPORT AND RECOMMENDATIONS.</a> Review on Antimicrobal Resistance. , (2016).</li><li>Palmer, A. C., Kishony, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding,+predicting+and+manipulating+the+genotypic+evolution+of+antibiotic+resistance.">Understanding, predicting and manipulating the genotypic evolution of antibiotic resistance.</a> Nat Rev Genet. 14 (4), 243-248 (2013).</li><li>MacLean, R. C., Hall, A. R., Perron, G. G., Buckling, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+population+genetics+of+antibiotic+resistance:+integrating+molecular+mechanisms+and+treatment+contexts.">The population genetics of antibiotic resistance: integrating molecular mechanisms and treatment contexts.</a> Nat Rev Genet. 11 (6), 405-414 (2010).</li><li>Buckling, A., Maclean, R. C., Brockhurst, M. A., Colegrave, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Beagle+in+a+bottle.">The Beagle in a bottle.</a> Nature. 457 (7231), 824-829 (2009).</li><li>Barrick, J. E., Lenski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+dynamics+during+experimental+evolution.">Genome dynamics during experimental evolution.</a> Nature Reviews Genetics. 14 (12), 827-839 (2013).</li><li>Elena, S. F., Lenski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+experiments+with+microorganisms:+The+dynamics+and+genetic+bases+of+adaptation.">Evolution experiments with microorganisms: The dynamics and genetic bases of adaptation.</a> Nature Reviews Genetics. 4 (6), 457-469 (2003).</li><li>Lenski, R. E., Rose, M. R., Simpson, S. C., Tadler, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+Experimental+Evolution+in+Escherichia+coli.+I.+Adaptation+and+Divergence+During+2,000+Generations.">Long-Term Experimental Evolution in Escherichia coli. I. Adaptation and Divergence During 2,000 Generations.</a> The American Naturalist. 138 (6), 1315-1341 (1991).</li><li>Bennett, A. F., Dao, K. M., Lenski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+evolution+in+response+to+high-temperature+selection.">Rapid evolution in response to high-temperature selection.</a> Nature. 346 (6279), 79-81 (1990).</li><li>Bell, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+rescue+and+the+limits+of+adaptation.">Evolutionary rescue and the limits of adaptation.</a> Philosophical Transactions of the Royal Society B-Biological Sciences. 368 (1610), (2013).</li><li>Bell, G., Gonzalez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+and+Evolutionary+Rescue+in+Metapopulations+Experiencing+Environmental+Deterioration.">Adaptation and Evolutionary Rescue in Metapopulations Experiencing Environmental Deterioration.</a> Science. 332 (6035), 1327-1330 (2011).</li><li>Bell, G., Gonzalez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+rescue+can+prevent+extinction+following+environmental+change.">Evolutionary rescue can prevent extinction following environmental change.</a> Ecology Letters. 12 (9), 942-948 (2009).</li><li>San Millan, A., Escudero, J. A., Gifford, D. R., Mazel, D., MacLean, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicopy+plasmids+potentiate+the+evolution+of+antibiotic+resistance+in+bacteria.">Multicopy plasmids potentiate the evolution of antibiotic resistance in bacteria.</a> Nature Ecology &amp; Evolution. 1, 0010 (2016).</li><li>Alekshun, M. N., Levy, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+antibacterial+multidrug+resistance.">Molecular mechanisms of antibacterial multidrug resistance.</a> Cell. 128 (6), 1037-1050 (2007).</li><li>Ochman, H., Lawrence, J. G., Groisman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+gene+transfer+and+the+nature+of+bacterial+innovation.">Lateral gene transfer and the nature of bacterial innovation.</a> Nature. 405 (6784), 299-304 (2000).</li><li>Carattoli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmids+and+the+spread+of+resistance.">Plasmids and the spread of resistance.</a> Int J Med Microbiol. 303 (6-7), 298-304 (2013).</li><li>Smillie, C., Garcill&#225;n-Barcia, M. P., Francia, M. V., Rocha, E. P., de la Cruz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobility+of+plasmids.">Mobility of plasmids.</a> Microbiol Mol Biol Rev. 74 (3), 434-452 (2010).</li><li>San Millan, A., Heilbron, K., Maclean, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+epistasis+between+co-infecting+plasmids+promotes+plasmid+survival+in+bacterial+populations.">Positive epistasis between co-infecting plasmids promotes plasmid survival in bacterial populations.</a> ISME J. , (2013).</li><li>Stoesser, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+History+of+the+Global+Emergence+of+the+Escherichia+coli+Epidemic+Clone+ST131.">Evolutionary History of the Global Emergence of the Escherichia coli Epidemic Clone ST131.</a> MBio. 7 (2), (2016).</li><li>San Millan, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiresistance+in+Pasteurella+multocida+is+mediated+by+coexistence+of+small+plasmids.">Multiresistance in Pasteurella multocida is mediated by coexistence of small plasmids.</a> Antimicrob Agents Chemother. 53 (8), 3399-3404 (2009).</li><li>Escudero, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unmasking+the+ancestral+activity+of+integron+integrases+reveals+a+smooth+evolutionary+transition+during+functional+innovation.">Unmasking the ancestral activity of integron integrases reveals a smooth evolutionary transition during functional innovation.</a> Nat Commun. 7, 10937 (2016).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proc Natl Acad Sci U S A. 97 (12), 6640-6645 (2000).</li><li>Chaveroche, M. K., Ghigo, J. M., d'Enfert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+efficient+gene+replacement+in+the+filamentous+fungus+Aspergillus+nidulans.">A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans.</a> Nucleic Acids Res. 28 (22), 97 (2000).</li><li>Le Roux, F., Binesse, J., Saulnier, D., Mazel, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+Vibrio+splendidus+mutant+lacking+the+metalloprotease+gene+vsm+by+use+of+a+novel+counterselectable+suicide+vector.">Construction of a Vibrio splendidus mutant lacking the metalloprotease gene vsm by use of a novel counterselectable suicide vector.</a> Appl Environ Microbiol. 73 (3), 777-784 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Performance standards for antimicrobial susceptibility testing; 19th ed. Approved standard M100-S19. , (2009).</li><li>Lindsey, H. A., Gallie, J., Taylor, S., Kerr, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+rescue+from+extinction+is+contingent+on+a+lower+rate+of+environmental+change.">Evolutionary rescue from extinction is contingent on a lower rate of environmental change.</a> Nature. 494 (7438), 463-467 (2013).</li><li>Deatherage, D. E., Barrick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mutations+in+laboratory-evolved+microbes+from+next-generation+sequencing+data+using+breseq.">Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq.</a> Methods Mol Biol. 1151, 165-188 (2014).</li><li>Barrick, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+structural+variation+in+haploid+microbial+genomes+from+short-read+resequencing+data+using+breseq.">Identifying structural variation in haploid microbial genomes from short-read resequencing data using breseq.</a> BMC Genomics. 15, 1039 (2014).</li><li>Salverda, M. L., De Visser, J. A., Barlow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+evolution+of+TEM-1+&#946;-lactamase:+experimental+reconstruction+and+clinical+relevance.">Natural evolution of TEM-1 &#946;-lactamase: experimental reconstruction and clinical relevance.</a> FEMS Microbiol Rev. 34 (6), 1015-1036 (2010).</li><li>Ramirez, M. S., Tolmasky, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminoglycoside+modifying+enzymes.">Aminoglycoside modifying enzymes.</a> Drug Resist Updat. 13 (6), 151-171 (2010).</li><li>Jerison, E. R., Desai, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+investigations+of+evolutionary+dynamics+and+epistasis+in+microbial+evolution+experiments.">Genomic investigations of evolutionary dynamics and epistasis in microbial evolution experiments.</a> Current Opinion in Genetics &amp; Development. 35, 33-39 (2015).</li><li>Toll-Riera, M., San Millan, A., Wagner, A., MacLean, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Genomic+Basis+of+Evolutionary+Innovation+in+Pseudomonas+aeruginosa.">The Genomic Basis of Evolutionary Innovation in Pseudomonas aeruginosa.</a> PLoS Genet. 12 (5), 1006005 (2016).</li></ol>

The authors have nothing to disclose.

We present a new protocol combining molecular biology, experimental evolution and deep DNA sequencing designed to investigate the role of multicopy plasmids in the evolution of antibiotic resistance in bacteria. Although this protocol combines techniques from different fields, all the methods required to develop it are simple, and can be performed in a regular microbiology laboratory. The most critical steps in the protocol probably are the construction of the model system strains and the reconstruction of the mutations ...

Antibiotic resistance in bacteria is a major health problem1. At a fundamental level, the spread of antibiotic resistance in pathogenic bacteria is a simple example of evolution by natural selection2,3. Put simply, the use of antibiotics generates selection for resistant strains. A key problem in evolutionary biology, therefore, is to understand the factors that influence the ability of bacterial populations to evolve resistance to antibiotics. Selection experiments have emerged as a very powerful tool to investigate the evolutionary biology of bacteria, and this field has produced incredible insights into a wide range of evolutionary problems4,5,6. In experimental evolution, bacterial populations initiated from a single parental strain are serially passaged under defined and tightly controlled conditions. Some of the mutations that occur during the growth of these cultures increase bacterial fitness, and these spread through the cultures by natural selection. During the experiment, samples of the populations are periodically cryogenically preserved to create a non-evolving frozen fossil record. A wide number of approaches can be used to characterize evolving bacterial populations, but the two most common methods are fitness assays, that measure the ability of evolved bacteria to compete against their distant ancestors, and whole genome sequencing, that is used to identify the genetic changes that drive adaptation. Following pioneering work by Richard Lenski and colleagues7,8, the standard approach in experimental evolution has been to challenge a relatively small number of replicate populations (typically &#60;10) with adapting to a new environmental challenge, such as new carbon sources, temperature, or a predatory phage.
Infections caused by antibiotic resistant bacteria become a big problem when resistance is high enough that it is not possible to increase antibiotic concentrations to lethal levels in patient tissues. Clinicians are therefore interested in what allows bacteria to evolve resistance to high doses of antibiotic that are above this threshold antibiotic concentration, the clinical breakpoint. How to study this experimentally? If a small number of bacterial populations are challenged with a high dose of antibiotic, as in a Lenski-style experiment, then the most likely outcome is that the antibiotic will drive all of the populations to extinction. At the same time, if the dose of antibiotic that is used is low, below the minimal inhibitory concentration (MIC) of the parental strain, then it is unlikely that the bacterial populations will evolve clinically relevant levels of resistance, especially if resistance carries a large cost. One compromise between these two scenarios is to use an &#34;evolutionary rescue&#34; experiment9,10,11. In this approach, a very large number of cultures (typically &#62;40) is challenged with doses of antibiotics that increase over time, typically by doubling antibiotic concentration every day12. The hallmark of this experiment is that any population that does not evolve increased resistance will be driven to extinction. Most populations that are challenged in this way will be driven extinct, but a small minority will persist by evolving high levels of resistance. In this paper, we show how this experimental design can be used to investigate multicopy plasmid contribution to the evolution of resistance.
Bacteria acquire resistance to antibiotics through two principal routes, chromosomal mutations, and acquisition of mobile genetic elements, mostly plasmids13. Plasmids play a key role in the evolution of antibiotic resistance because they are able to transfer resistance genes between bacteria by conjugation14,15. Plasmids can be divided into two groups according to their size and biology: &#34;small&#34;, with high copy number per bacterial cell and &#34;large&#34;, with low copy number16,17. The role of large plasmids in the evolution of antibiotic resistance has been extensively documented because they include conjugative plasmids, which are key drivers of the dissemination of resistance and multi resistance among bacteria15. Small multicopy plasmids are also extremely common in bacteria17,18, and they often code for antibiotic resistance genes19. However, the role of small multicopy plasmids in the evolution of antibiotic resistance has been studied to a lesser extent.
In a recent work, we proposed that multicopy plasmids could accelerate the evolution of the genes they carry by increasing gene mutation rates due to the higher gene copy number per cell12. Using an experimental model with E. coli strain MG1655 and the &#946;-lactamase gene blaTEM-1 it was shown that multicopy plasmids accelerated the rate of appearance of TEM-1 mutations conferring resistance to the third-generation cephalosporin ceftazidime. These results indicated that multicopy plasmids might play an important role in the evolution of antibiotic resistance.
Here, we present a detailed description of the method we have developed to investigate the multicopy plasmid-mediated evolution of antibiotic resistance. This method has three different steps: first, insertion of the gene under study either in a multicopy plasmid or the chromosome of the host bacteria. Second, use of experimental evolution (evolutionary rescue) to assess the potential of the different strains to adapt to the selective pressure. And third, determining the molecular basis underlying plasmid-mediated evolution using DNA sequencing and reconstructing the suspected mutations individually in the parental genotype.
Finally, although the protocol described here was designed to investigate the evolution of antibiotic resistance, one can argue that this method could be generally useful to analyze the evolution of innovations acquired by mutations in any multicopy plasmid-encoded gene.

<table><tbody><tr><td>Thermocycler</td><td>BioRad</td><td>C1000</td><td></td></tr><tr><td>Electroporator</td><td>BiorRad</td><td>1652660</td><td></td></tr><tr><td>Electroporation cuvettes</td><td>Sigma-Aldrich</td><td>Z706078</td><td></td></tr><tr><td>NanoDrop 2000/2000c</td><td>Thermo Fisher Scientific</td><td>ND-2000</td><td>Determine DNA quality measuring the ratios of absorbance 260nm/280nm and 260nm/230nm</td></tr><tr><td>Incubator</td><td>Memmert</td><td>UF1060</td><td></td></tr><tr><td>Incubator (shaker)</td><td>Cole-Parmer Ltd</td><td>SI500</td><td></td></tr><tr><td>Electrophoresis power supply</td><td>BioRad</td><td>1645070</td><td>Agarose gel electrophoresis</td></tr><tr><td>Electrophoresis chamber</td><td>BioRad</td><td>1704405</td><td>Agarose gel electrophoresis</td></tr><tr><td>Pippettes</td><td>Biohit</td><td>725020, 725050, 725060, 725070</td><td></td></tr><tr><td>Multi-channel pippetes</td><td>Biohit</td><td>728220, 728230,&lt;br/&gt; 728240</td><td></td></tr><tr><td>Plate reader Synergy HTX</td><td>BioTek</td><td>BTS1LF</td><td></td></tr><tr><td>Inoculating loops</td><td>Sigma-Aldrich</td><td>I8388</td><td></td></tr><tr><td>96-well plates</td><td>Falcon</td><td>351172</td><td></td></tr><tr><td>LB</td><td>BD Difco</td><td>DF0446-17-3</td><td></td></tr><tr><td>LB agar</td><td>Fisher scientific</td><td>BP1425-500</td><td></td></tr><tr><td>Phusion Polymerase</td><td>Thermo Fisher Scientific</td><td>F533S</td><td></td></tr><tr><td>Gibson Assembly</td><td>New England Biolabs</td><td>E2611S</td><td></td></tr><tr><td>Resctriction enzymes</td><td>Fermentas FastDigest</td><td></td><td></td></tr><tr><td>Antibiotics</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>27104</td><td>Plasmid extraction kit</td></tr><tr><td>Wizard Genomic DNA Purification Kit</td><td>Promega</td><td>A1120</td><td>gDNA extraction kit</td></tr><tr><td>DNeasy Blood &amp;amp; Tissue Kits</td><td>Qiagen</td><td>69506</td><td>gDNA extraction kit</td></tr><tr><td>Electroporation cuvettes</td><td>Sigma-Aldrich</td><td>Z706078</td><td></td></tr><tr><td>Petri dishes</td><td>Sigma-Aldrich</td><td>D9054</td><td></td></tr><tr><td>Cryotubes</td><td>ClearLine</td><td>390701</td><td></td></tr><tr><td>96-well plates (-80&amp;ordm;C storage)</td><td>Thermo Fisher Scientific</td><td>249945</td><td></td></tr><tr><td>QuantiFluor dsDNA System</td><td>Promega</td><td>E2670</td><td>Quantification of DNA concentartion</td></tr><tr><td>Agarose</td><td>BioRad</td><td>1613100</td><td>Agarose gel electrophoresis</td></tr><tr><td>50x TAE buffer</td><td>BioRad</td><td>1610743</td><td>Agarose gel electrophoresis</td></tr><tr><td>T4 Polynucleotide Kinase</td><td>Thermo Fisher Scientific</td><td>EK0031</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>Thermo Fisher Scientific</td><td>EL0014</td><td></td></tr></tbody></table>

testing the role of multicopy plasmids in the evolution of antibiotic resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

In our previous work, the evolution the &#946;-lactamase gene blaTEM-1 towards conferring resistance to the third generation cephalosporin ceftazidime12 was investigated. This gene was selected because, although TEM-1 does not confer resistance to ceftazidime, mutations in blaTEM-1 can expand the range of activity of TEM-1 to hydrolyze cephalosporins such as ceftazidime29. Mutations in antibiotic resis...

Watch this Scientific Journal Video about Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance at JoVE.com

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance | Genetics | Video

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

testing-role-multicopy-plasmids-evolution-antibiotic

Universidad San Sebastian

Research

Education

JoVE Journal

JoVE Core

Biology

Genetics

Evolution

Natural Selection

SCIENTISTS IN ACTION

11.5K Views.  Universidad Complutense de Madrid. Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

Video: Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major factor in the spread of diverse resistance mechanisms. Amongst the DNA elements facilitating lateral transfer, the class 1 integrons have largely been responsible for spreading antibiotic resistance determinants amongst Gram negative pathogens. In total, these integrons have acquired and disseminated over 130 different antibiotic resistance genes. With continued antibiotic use, class 1 integrons have become ubiquitous in commensals and pathogens of humans and their domesticated animals. As a consequence, they can now be found in all human waste streams, where they continue to acquire new genes, and have the potential to cycle back into humans via the food chain. This protocol details a streamlined approach for detecting class 1 integrons and their associated resistance gene cassettes in foodstuffs, using culturing and PCR. Using this protocol, researchers should be able to: collect and prepare samples to make enriched cultures and screen for class 1 integrons; isolate single bacterial colonies to identify integron-positive isolates; identify bacterial species that contain class 1 integrons; and characterize these integrons and their associated gene cassettes.

This protocol describes the detection of class 1 integrons and their associated gene cassettes in foodstuffs.

Antibiotic resistance is one of the greatest threats to health in the 21st century. Acquisition of resistance genes via lateral gene transfer is a major ...

Environment

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Our experimental approach provides a strategy to follow plasmid abundance and antibiotic resistance over time in bacterial populations.

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the study of the mobilization of naturally occurring conjugative plasmids, using two naturally-occurring plasmids as an example. These protocols rely on the differential presence of selectable markers in donor, recipient, and conjugative plasmid. Specifically, the methods described include 1) the identification of natural conjugative plasmids, 2) the quantification of conjugation rates in solid culture, and 3) the diagnostic detection of the antibiotic resistance genes and plasmid replicon types in transconjugant recipients by polymerase chain reaction (PCR). The protocols described here have been developed in the context of studying the evolutionary ecology of horizontal gene transfer, to screen for the presence of conjugative plasmids carrying antibiotic-resistance genes in bacteria found in the environment. The efficient transfer of conjugative plasmids observed in these experiments in culture highlights the biological relevance of conjugation as a mechanism promoting horizontal gene transfer in general and the spread of antibiotic resistance in particular.

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation mediates horizontal gene transfer by mobilizing plasmid DNA across two different cells, facilitating the spread of beneficial genes. This work describes a widely-used method for the efficient detection of conjugative plasmid transfer, based on the use of differential markers in the conjugative plasmid, donor, and recipient to detect transconjugation.

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the ...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Immunology and Infection

Antibiotic Dereplication Using the Antibiotic Resistance Platform

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this challenge, dereplication, which is the process of identifying known compounds, is performed on samples of interest. Methods for dereplication such as analytical separation followed by mass spectrometry are time-consuming and resource-intensive. To improve the dereplication process, we have developed the antibiotic resistance platform (ARP). The ARP is a library of approximately 100 antibiotic resistance genes that have been individually cloned into Escherichia coli. This strain collection has many applications, including a cost-effective and facile method for antibiotic dereplication. The process involves the fermentation of antibiotic-producing microbes on the surface of rectangular Petri dishes containing solid medium, thereby allowing for the secretion and diffusion of secondary metabolites through the medium. After a 6 day fermentation period, the microbial biomass is removed, and a thin agar-overlay is added to the Petri dish to create a smooth surface and enable the growth of the E. coli indicator strains. Our collection of ARP strains is then pinned onto the surface of the antibiotic-containing Petri dish. The plate is next incubated overnight to allow for E. coli growth on the surface of the overlay. Only strains containing resistance to a specific antibiotic (or class) grow on this surface enabling rapid identification of the produced compound. This method has been successfully used for the identification of producers of known antibiotics and as a means to identify those producing novel compounds.

We describe a platform that utilizes a library of isogenic antibiotic resistant Escherichia coli for the dereplication of antibiotics. The identity of an antibiotic produced by bacteria or fungi can be deduced by the growth of E. coli expressing its respective resistance gene. This platform is economically effective and time-efficient.

One of the main challenges in the search for new antibiotics from natural product extracts is the re-discovery of common compounds. To address this ...

Single&#45;Copy Gene Expression for Characterizing Multidrug Efflux Systems

Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux&#45;Deficient Bacterial Strain and a Single&#45;Copy Gene Expression System

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend the mechanisms behind this resistance to design new and effective therapeutic options. Unfortunately, our ability to investigate these mechanisms in A. baumannii is hindered by the paucity of suitable genetic manipulation tools. Here, we describe methods for utilizing a chromosomal mini-Tn7-based system to achieve single-copy gene expression in an A. baumannii strain that lacks functional RND-type efflux mechanisms. Single-copy insertion and inducible efflux pump expression are quite advantageous, as the presence of RND efflux operons on high-copy number plasmids is often poorly tolerated by bacterial cells. Moreover, incorporating recombinant mini-Tn7 expression vectors into the chromosome of a surrogate A. baumannii host with increased efflux sensitivity helps circumvent interference from other efflux pumps. This system is valuable not only for investigating uncharacterized bacterial efflux pumps but also for assessing the effectiveness of potential inhibitors targeting these pumps.

Author Spotlight: Advancing Antibiotic Resistance Research Using an Efflux&#45;Deficient Bacterial Strain and a Single&#45;Copy Gene Expression System

We describe a facile procedure for the single-copy chromosomal complementation of an efflux pump gene using a mini-Tn7-based expression system into an engineered efflux-deficient strain of Acinetobacter baumannii. This precise genetic tool allows for controlled gene expression, which is key for the characterization of efflux pumps in multidrug resistant pathogens.

Acinetobacter baumannii is recognized as a challenging Gram-negative pathogen due to its widespread resistance to antibiotics. It is crucial to comprehend ...

Bacterial Transformation

<h4>Background</h4>

In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccine against pneumonia, Frederick Griffith set out to study two different strains of the Streptococcus pneumoniae: a non-virulent strain with a rough appearance (R-strain) and a virulent strain with a smooth appearance (S-strain) due to an outer polysaccharide capsule2. This outer layer of the S-strain bacteria enabled them to withstand the host immune system, eventually leading to a life-threatening illness. When Griffith separately injected mice with heat-killed bacteria of either strain, the mice lived. However, when he injected mice with a combination of the heat-killed S-strain with live R-strain, the mice died2. When he analyzed the samples obtained from dead mice injected with the combination, he observed live S-strain bacteria to be present. In 1928, Griffith noted that a &#8220;transformation&#8221; process must have occurred to change the nonvirulent bacteria into the virulent strain. As the first known discovery of bacterial transformation, his discovery paved the way for the development of an essential tool in genetic engineering - transformation3.

Transformation is the genetic change in a cell due to the intake of DNA from the environment. In Griffith&#8217;s experiment, the DNA that encodes the protective polysaccharide coating of the S-strain bacteria did not break down from the heat shock and was introduced into the R-strain, allowing the latter to bypass the immune system of the mouse. While this process constantly occurs in the wild between organisms and even different species, scientists transform bacteria in laboratory settings for research purposes4.

<h4>Bacterial Transformation using Plasmids</h4>

Bacteria are the ideal organisms for transformation as they can easily take in exogenous genetic material into their genome and quickly amplify it3,5. They have one circular chromosome and multiple small circular pieces of double-stranded DNA called plasmids within the cytoplasm. These plasmids can replicate independently from the chromosomal DNA and generally provide certain functional benefits, such as resistance to antibiotics6,7. In their natural environment, bacteria undergo &#8220;bacterial transformation&#8221; by taking in plasmids from other bacteria in a process called conjugation8. Moreover, when they multiply, each of their progeny receives a copy of the new plasmid.

Plasmids that are used for experimental purposes are called plasmid vectors. In a laboratory setting, scientists can artificially create &#8220;recombinant plasmids&#8221; that are approximately 5,000-10,000 base pairs in length by inserting DNA fragments into a plasmid vector. These recombinant plasmids usually have certain components: an origin of replication (ORI), an antibiotic resistance gene, a multiple cloning site, a promoter, a selection marker, and the gene of interest. The origin of replication is where replication begins. The antibiotic resistance gene allows the bacteria that take in the plasmid to survive on plates in the presence of a certain antibiotic drug. Although plasmids are relatively small pieces of DNA, scientists need to treat the host cells to enable the plasmid penetration through the cell membrane. Hence, the efficiency of the transformation is directly related to porosity of the host membrane. One common approach is to heat-shock the bacteria that have been treated with a calcium chloride solution9. The bacteria that do not incorporate the plasmid will not transform, and therefore have no resistance to survive on the plate and be visible. The multiple cloning sites aid in DNA insertion by containing sites for restriction enzymes to cut the plasmid where the gene of interest can be inserted and ligated. The promoter drives transcription of the gene of interest. It is tagged by a marker, usually a fluorescent protein such as a green fluorescent protein (GFP), or can be an additional antibiotic resistance gene. The antibiotic resistance gene and the other selection markers help us determine whether the collected bacteria contain the plasmid of interest.

<h4>Applications</h4>

Effective transformation methods enabled scientists to isolate and profile genes and gene products and led to many advancements in life sciences and medicine, such as development of effective drugs, generation of genetically-modified crops, and advanced diagnostic tools10. In addition, with technological advancements, new methods of transformation have emerged. For instance, Gateway Cloning allows insertion of multiple DNA fragments into different vectors as well as transfer of DNA sequences between plasmids11. Furthermore, clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is a gene-editing technique that directly modifies nucleotides in the genome and does not require the use of plasmids12. After transformation, researchers often isolate and profile the gene of interest and its products. Subsequently, the whole process of genetic cloning has opened up a new field of genetic manipulation. Thanks to genetic cloning, researchers can manipulate bacteria to produce large amounts of specific human proteins, such as insulin to treat diabetes patients10. Cloning also has served a major importance in modern day agriculture. Genetically modified organisms (GMO) are a direct result of genetic cloning and bacterial transformation10. For instance, scientists are working to generate genetically modified crops with nitrogen-fixing genes incorporated into their genome to boost food production and reduce fertilizer use, thus, diminishing the economic and environmental impact of fertilizers13. In sum, bacterial transformation is the first step of modern-day biotechnology and the foundation of future research discoveries.

<h4>References</h4>

<ol class="references">
	<li>GL, Armstrong, LA, Conn and RW, Pinner. Trends in Infectious Disease Mortality in the United States During the 20th Century. JAMA. 1999, Vol. 281, 1 (61-66).</li>
	<li>F, Griffith. The Significance of Pneumococcal Types. J Hyg (Lond). . 1928 , 2 (113-59).</li>
	<li>Lorenz, MG and Wackernagel, F. Bacterial gene transfer by natural genetic transformation in the environment. Microbiol Rev. . 1994 Sep;, Vol. 58, 3: (563-602).</li>
	<li>S, Domingues, et al. Natural Transformation Facilitates Transfer of Transposons, Integrons and Gene Cassettes between Bacterial Species. PLOS Pathogens. 2012, 8(8): e1002837.</li>
	<li>Dubnau, D. DNA uptake in bacteria. Annu Rev Microbiol. . 1999, 53: (217-44).</li>
	<li>Solar, G del, et al. Replication and control of circular bacterial plasmids. Microbiol Mol Biol Rev. . 1998 , Vol. 62, 2: (434-64).</li>
	<li>Bennett, PM. Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria. Br J Pharmacol. . 2008 , Vol. 153, S1: (S347-S357).</li>
	<li>MLlosa, et al. Bacterial conjugation: a two-step mechanism for DNA transport. Mol Microbiol. 2002 Jul;, Vol. 45, 1: (1-8).</li>
	<li>Roychoudhury, A, Basu, S and Sengupta, DN. Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors. Indian J Biochem Biophys. . 2009 , Vol. 46, 5: (395-400).</li>
	<li>Khan, S, et al. Role of Recombinant DNA Technology to Improve Life. I. nt J Genomics. . 2016, 2016:2405954.</li>
	<li>Marsischky, G and LaBaer, J. Many Paths to Many Clones: A Comparative Look at High-Throughput Cloning Methods. Genome Res. 2004, Vol. 14, 2020-28.</li>
	<li>Doudna, JA and Charpentie, E. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014, Vol. 346, 6213-1258096.</li>
	<li>Good, A. Toward nitrogen-fixing plants. Science. 2018, Vol. 359, 6378: 869-70.</li>
</ol>

Bacterial Transformation using Plasmids - Concept

Background

In early 20th century, pneumonia was accountable for a large portion of infectious disease deaths1. In order to develop an effective vaccine ...

JoVE Lab Manual

Phage Transduction: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

Source: Alexander S. Gold1, Tonya M. Colpitts1 
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases Laboratories, Boston, MA
Transduction is a form of genetic exchange between bacteria that utilizes bacteriophages, or phages, a class of virus that infects exclusively prokaryotic organisms. This form of DNA transfer, from one bacterium to another by way of a phage, was discovered in 1951 by Norton Zinder and Joshua Ledererg, who termed the process &#34;transduction&#34; (1). Bacteriophages were first discovered in 1915 by British bacteriologist Frederick Twort, then independently discovered again in 1917 by French-Canadian microbiologist Felix d&#39;Herelle (2). Since then, the structure and function of these phages have been widely characterized (3), dividing these phages into two classes. The first of these classes are the lytic phages which upon infection multiply within the host bacterium, disrupting the bacterial metabolism, lysing the cell, and releasing progeny phage (4). As a result of this anti-bacterial activity and the increasing prevalence of antibiotic-resistant bacteria, these lytic phages have recently proved useful as a substitute treatment for antibiotics. The second of these classes are the lysogenic phages which can either multiply within the host via the lytic cycle or enter a quiescent state in which their genome is integrated into that of the host (Figure 1), a process known as lysogeny, with the ability for phage production to be induced in multiple later generations (4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/10517/10517fig1.jpg" /> 
Figure 1: Infection of host cell by bacteriophage. Adsorption by the phage to the bacterial cell wall via interactions between the tail fibers and receptor (purple). Once on the cell surface, the phage is irreversibly attached to the bacterial cell using the base plate (black) which is moved to the cell wall by the contractile sheath (yellow). Phage genome (red) then enters the cell and integrates into the host cell genome.
While bacterial transduction is a naturally occurring process, using modern technology it has been manipulated for the transfer of genes into bacteria in the laboratory setting. By inserting genes of interest into the genome of a lysogenic phage, such as phage, one is able to transfer these genes into the genomes of bacteria and consequently express them within these cells. While other methods of gene transfer, such as transformation, use a plasmid for gene transfer and expression, the insertion of the phage genome into that of the recipient bacterium not only has the potential to confer new traits to this bacterium, but also allows for naturally occurring mutations and other factors of the cellular environment to alter the function of the transferred gene.
Compared to other methods of horizontal gene transfer, such as conjugation, transduction is fairly flexible in the criteria required for donor and recipient cells. Any genetic element that can fit inside the genome of the phage being used can be transferred from any strain of donor bacteria to any strain of recipient bacteria as long as both are permissive to the phage, requiring the expression of necessary phage receptors on the cell surfaces. Once this gene is moved out of the donor genome and packaged into the phage, it can be transferred to the recipient. Following transduction, it is necessary to select for recipient bacteria that contain the gene of interest have to be selected for. This could be done by use of a genetic marker, such as a FLAG-tag or polyhistidine-tag, to mark the gene of interest, or the intrinsic function of the gene, in the case of genes that encode for antibiotic resistance. Additionally, PCR could be used to further confirm successful transduction. By using primers for a region within the gene of interest and comparing signal to a positive control, bacteria that has the gene of interest, and a negative control, bacteria that underwent the same steps as the transduction reaction with no phage. While bacterial transduction is a useful tool in molecular biology, it has and continues to play an important role in the evolution of bacteria, particularly with regard to the recent rise of antibiotic resistance.
In this experiment, bacterial transduction was used to transfer the gene encoding for resistance to the antibiotic ampicillin from the W3110 strain of E.coli to the J53 strain via the P1 bacteriophage (5). This experiment consisted of two main steps. First, the preparation of P1 phage containing the ampicillin resistance gene from the donor strain. Second, the transfer of this gene to the recipient strain by transduction with the P1 phage (Figure 1). Once carried out, the successful transfer of the ampicillin resistance gene could be determined by qPCR (Figure 2). If transduction was successful, the J53 strain of E. coli would be resistant to ampicillin, and the gene conferring this resistance detectable by qPCR. If unsuccessful, there would be no detection of the ampicillin resistance gene and ampicillin would still function as an effective antibiotic against the J53 strain.

<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/10517/10517fig2.jpg" /> 
Figure 2: The confirmation of successful transduction by qPCR. By comparing the Cq values detected for the gene of interest from the transduction reaction and the negative control reaction, and normalizing these values against a housekeeping gene, one was able to confirm that bacterial transduction was successful.

Phage Transduction: Transferring Ampicillin Resistance in E. coli

Source: Alexander S. Gold1, Tonya M. Colpitts1
1 Department of Microbiology, Boston University School of Medicine, National Emerging Infections Diseases ...

JoVE Science Education

Advanced Biology

Microbiology

Antibiotic Selection

<h4>Overview</h4>

Researchers use antibiotic resistance genes to identify bacteria that possess a plasmid containing their gene of interest. Antibiotic resistance naturally occurs when a spontaneous DNA mutation creates changes in bacterial genes that eliminate antibiotic activity. Bacteria can share these new resistance genes with their offspring and other bacteria. The overuse and misuse of antibiotics have created a public health crisis, as resistant and multi-resistant bacteria continue to develop.

<h4>Antibiotic Resistance is an Essential Tool in Genetic Engineering</h4>

Antibiotics, such as penicillin, are drugs that kill or stop bacterial growth. Bacteria that naturally or artificially acquired antibiotic resistance genes do not respond to antibiotics. Scientists exploit this by designing plasmids&mdash;small, self-replicating pieces of DNA&mdash;that carry both an antibiotic resistance gene and a gene of interest. Antibiotic resistance is an integral part of DNA cloning that allows a researcher to identify cells that absorbed a DNA of interest.

The researcher&rsquo;s DNA of interest is introduced into bacterial cells using a process called transformation. Bacterial transformation involves temporarily creating small holes in the bacterial cell wall to permit the uptake of external DNA such as a plasmid. Only some bacterial cells absorb new DNA. Since the plasmid includes both the DNA of interest and a gene that confers resistance to a specific antibiotic, applying the antibiotic to the bacterial cells (i.e., antibiotic selection) can help determine which cells were genetically modified.

The researcher spreads the bacterial cells onto a culture plate containing a chosen antibiotic. Only bacteria containing the antibiotic resistance gene survive and grow on the plate. After a few days, the researcher can select a bacterial colony to culture for further experiments&mdash;such as gene expression studies. Following antibiotic selection, the researcher will further test the bacteria using other methods (e.g., PCR) to confirm that DNA of interest is correct. Errors often occur such as the plasmid not containing the gene of interest at all.

<h4>Bacteria Can Acquire Antibiotic Resistance Naturally</h4>

Bacteria can acquire antibiotic resistance through spontaneous DNA mutations that alter the proteins produced by the cell. Resistant bacteria may produce proteins that cause the antibiotic to either be degraded, pumped out of the cell, or prevented from interacting with its target. For example, the antibiotic vancomycin inhibits synthesis of the bacterial cell wall. Some bacteria have developed resistance to this antibiotic by changing the types of protein subunits&mdash;amino acids&mdash;used in the assembly of their cell wall to ones that are unaffected by vancomycin.

Once antibiotic resistance genes emerge, bacteria can pass them on to their offspring. Bacteria can also acquire antibiotic resistance genes from other bacteria of the same or different species through a process called horizontal gene transfer (HGT). There are three mechanisms of HGT: transformation, conjugation, and transduction. Antibiotic resistance genes are often found in plasmids or transposons&mdash;pieces of DNA that are easily transferred between bacteria&mdash;that are exchanged during HGT. As a result, new types of antibiotic resistance can rapidly spread to multiple types of infectious bacteria.

<h4>Clinical Overuse and Misuse of Antibiotics Produces &ldquo;Superbugs&rdquo;</h4>

Antibiotics are a critical treatment for bacterial infections. However, their use can cause bacteria to become resistant and render the antibiotic ineffective, leading to untreatable and potentially deadly infections. Antibiotic overuse and misuse&mdash;for instance, using antibiotics to treat viral (rather than bacterial) infections or to increase livestock growth&mdash;is problematic because it promotes resistance.

Antibiotics cause resistance to evolve because they kill susceptible bacteria and leave only the resistant individuals. The surviving bacteria divide rapidly, producing offspring with the same antibiotic resistance. When antibiotics are overused, this selection pressure causes the number of resistant bacteria in the population to rise quickly. This is a major public health concern because it increases antibiotic resistance and creates &ldquo;superbugs&rdquo; that are resistant to multiple antibiotics. Continued overuse and misuse of antibiotics may eventually exhaust treatment options for bacterial infections.

Antibiotic Selection of Plasmids

Overview

Researchers use antibiotic resistance genes to identify bacteria that possess a plasmid containing their gene of interest. Antibiotic resistance ...

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Summary

Explore More Videos

Chapters in this video

Screening Foodstuffs for Class 1 Integrons and Gene Cassettes

High-Resolution Comparison of Bacterial Conjugation Frequencies

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Antibiotic Dereplication Using the Antibiotic Resistance Platform

Characterizing Multidrug Efflux Systems in Acinetobacter baumannii Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System

Bacterial Transformation

Phage Transduction: A Method to Transfer Ampicillin Resistance from Donor to Recipient E. coli

Antibiotic Selection