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Applied and Environmental Microbiology, November 2006, p. 7383-7385, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01246-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

System for Determining the Relative Fitness of Multiple Bacterial Populations without Using Selective Markers

Hyo-Jin Ahn, Hyun-Joon La, and Larry J. Forney^*

Department of Biological Sciences, University of Idaho, Moscow, Idaho 83844-3051

Received 30 May 2006/ Accepted 28 August 2006

Top Abstract Introduction References

 
A device for simultaneously measuring the relative fitness of multiple bacterial populations was developed and evaluated. The new device eliminates the need to construct strains with selectively neutral markers so that strains can be readily distinguished, and it provides a means to perform multispecies competition experiments.

Top Abstract Introduction References

 
The relative fitness of bacterial populations can be measured by performing pairwise competition experiments in which two populations are cocultured and allowed to compete for the same resources (4-8). In population genetics, fitness can be defined as a measure of the reproductive success of a population (2), which can be expressed as the natural logarithm of the ratio of the final and initial cell densities of the culture. Differences in the fitness of the populations are typically estimated by determining the population sizes by plating diluted samples of the coculture onto selective growth media. In pairwise competition experiments, two kinds of media are usually employed, and each medium contains one or more antibiotics that permit the growth of one competitor while precluding the growth of the other (3). Alternatively, only one medium is used and the competing populations are distinguished based on differences in tetrazolium dye reduction due to differences in the abilities of the strains to metabolize a particular carbon source (5). Differences in antibiotic resistance or the ability to utilize a carbon source are often created by selecting spontaneous mutants of each strain. The use of mutagens or procedures that cause multiple mutations must be avoided because some mutations could affect fitness in unknown ways. Alternatively, mutations or selective markers can be introduced though genetic manipulation (transposon mutagenesis, for example). Obviously, for this approach the strains must be genetically tractable, and the genes introduced must be expressed in the new host. While these conditions might be easily met for commonly used laboratory strains of bacteria (e.g., Escherichia coli), studies of most bacterial species are more complicated. Obviously, the problems increase if more than two strains are used in a competition experiment. To circumvent these problems, we developed a device that permits investigators to conduct multispecies competition experiments without first constructing two or more marked strains.

The assembled device, which basically consisted of a medium reservoir into which cell culture inserts were placed as growth chambers, is shown in Fig. 1. The bottom sides of the cell culture inserts (Nunc, Rochester, NY) had hydrophilic Anapore membranes (pore size, 0.2 µm; 30 mm by 10 mm) that allowed free diffusion of medium between the interior of an insert and the medium reservoir. In addition, the membrane physically isolated bacterial cells inside the cell culture inserts; therefore, the inserts functioned as growth chambers for individual bacterial populations. The membranes were approximately 1.0 mm above the bottom of the medium reservoir as a result of legs on the bottom of the cell culture inserts. A lid from a sterile culture dish (35 mm by 10 mm; Corning, Acton, MA) was used to cover the cell culture inserts to prevent accidental migration of cells from the inserts to the medium reservoir during incubation. Empty boxes for pipette tips (11.8 cm by 8.2 cm) were used as medium reservoirs. The medium reservoir can be any kind of rectangular culture dish as long as it can be autoclaved (or manufactured sterile), has a lid, and fits in a microplate incubator. Pieces of plastic tubing with rectangular notches on either end (referred to as "bumpers" below) were used to maintain spaces between the cell culture insert lids and the cell culture inserts. The bumpers were made of perfluoroalkoxy tubing (inside diameter, 0.125 in.) by cutting rectangular notches in the ends to fit over the walls of the cell culture inserts. The use of bumpers prevented cross contamination of cultures and the bulk medium by capillary action of liquids into and out of the cell culture inserts. All the components were manufactured sterile or autoclaved before use, and the device was assembled in a laminar flow hood by using sterile gloves and forceps. Growth medium was added to the cell culture inserts (maximum, 3 ml) and the medium reservoir (maximum, 40 ml). After inoculation of the cell culture inserts, lids were placed on the inserts and medium reservoir, and the entire device was incubated in a microplate incubator (Jencons Scientific, Bridgeville, PA) with shaking at 108 rpm at 37°C for E. coli and at 35°C for other species. Samples from cell culture inserts were periodically taken after lids were removed from the medium reservoir and the inserts with sterile forceps, and the lids were put back in place after sampling.

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FIG. 1. Assembled device used in bacterial competition experiments. Cell culture inserts were placed in a medium reservoir which contained medium used for growth of bacterial populations in individual cell culture inserts.

Several replicate experiments were done to validate the use of the device in microbial competition experiments, and these experiments are described below.

First, we tested whether nutrients could freely diffuse between the interior of cell culture inserts and medium in the reservoir. Three cell culture inserts were filled with either Luria-Bertani (LB) broth, half-strength LB broth, or 0.9% NaCl and placed in a medium reservoir that contained half-strength LB broth. All three cell culture inserts were inoculated with the same density of E. coli MG1655 (= ATCC47076), and the assembled device was incubated at 37°C. Samples were periodically taken from each cell culture insert, and the numbers of CFU were determined by plating diluted samples on LB agar. The numbers of cells in the three cell culture inserts increased in parallel (Table 1) even though the cell culture inserts initially contained different levels of nutrients. This indicates that the medium in the reservoir readily equilibrated with the media in the cell culture inserts.

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TABLE 1. Resource partitioning among populations in different cell culture inserts

Second, we determined whether resources in the medium reservoir were equally partitioned among populations in different cell culture inserts. To evaluate this, three devices that had one, two, or three cell culture inserts were set up. All cell culture inserts were inoculated with the same number of E. coli MG1655 cells. M9 minimal medium supplemented with 0.05% glucose or LB medium diluted 1/20 was added to the inserts and the medium reservoir. After 24 h of incubation at 37°C, the final cell density in each cell culture insert was determined as described above. The results showed that the number of bacterial cells in each chamber decreased as the number of cell culture inserts per medium reservoir increased (Table 2). In addition, the total cell yield in a medium reservoir was approximately the same in both media regardless of the number of cell culture inserts used.

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TABLE 2. Growth of E. coli in cell culture inserts containing M9 medium supplemented with 0.05% glucose or LB medium diluted 1/20 with a 0.9% NaCl solution

Third, we conducted a pairwise competition experiment using the device and compared the results to the results obtained in a competition experiment done in a traditional manner with a coculture of two antibiotic-resistant bacterial strains. Two E. coli MG1655 strains were used (1); these strains were isogenic except that one was resistant to rifampin (E. coli K-12 Rif^r), while the other was resistant to kanamycin (E. coli K-12 Km^r). In one trial, two cell culture inserts were placed in a single medium reservoir that contained M9 minimal medium supplemented with 0.05% glucose. In a second trial, the two strains were cocultured in the same medium in Erlenmeyer flasks. Each trial was conducted in quadruplicate. At 0, 6, and 12 h, samples taken from cell culture inserts were diluted and plated onto LB agar that did not contain antibiotics to determine the number of each competitor. Likewise, samples that were taken from the cocultured bacterial populations were diluted and plated onto both LB agar with 100 µg/ml of rifampin and LB agar with 50 µg/ml of kanamycin. We calculated the specific growth rate from the slope of the line that resulted from plotting the natural logarithm of cell density against time. The results of trials done with our device showed that the specific growth rate of E. coli K-12 Rif^r was 26.5% ± 2.6% greater than that of E. coli K-12 Km^r, while the apparent difference in the growth rates when the strains were cocultured was 28.9% ± 3.6%. The differences in the specific growth rates of the strains in the two different competition systems were not statistically significant (P = 0.3175, as determined by an unpaired t test; P < 0.05), indicating that the outcome of competition experiments using this device was comparable to the outcome when traditional batch cocultures were used.

Finally, the new device was tested to determine whether it could be used for competition experiments involving multiple species that lacked selective markers. In these experiments three bacterial species were used: E. coli K-12, Pseudomonas putida UWC1, and Serratia marcescens. These organisms were chosen because their colonies could easily be distinguished on agar media based on color and morphology. Each species was inoculated (individually) into cell culture inserts that had been placed in a medium reservoir that contained LB broth. In parallel, the three strains were also cocultured in Erlenmeyer flasks that contained LB broth. The initial cell densities and final cell densities of each species after 12 h of incubation at 35°C were determined using the methods described above. The fitness of each strain (natural logarithm of the ratio of the final and initial densities) was calculated. The outcomes of competition in the two systems were not significantly different (Table 3). These findings suggest that the device can be used for multispecies competition experiments.

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TABLE 3. Comparison of E. coli, P. putida, and S. marcescens growth in cell culture inserts and mixed batch cultures

In conclusion, it was shown that the device described here could be used as an alternative to coculturing bacteria in pairwise competition experiments. This device obviates the need to construct or otherwise obtain strains that can be distinguished on the basis of differences in sensitivity to antibiotics or catabolism of a carbon source. Moreover, multispecies competition experiments could be done by increasing the number of cell culture inserts used.

 
We thank Leen De Gelder for providing Km^r and Rif^r strains of E. coli K-12 and for helpful discussions and Eva Top for critically reading the manuscript and providing useful advice.

 
* Corresponding author. Mailing address: Department of Biological Sciences, Life Sciences South, Rm. 252, University of Idaho, Moscow, ID 83844-3051. Phone: (208) 885-6280. Fax: (208) 885-7905. E-mail: lforney{at}uidaho.edu.

Published ahead of print on 8 September 2006.

Top Abstract Introduction References

 

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Applied and Environmental Microbiology, November 2006, p. 7383-7385, Vol. 72, No. 11
0099-2240/06/$08.00+0     doi:10.1128/AEM.01246-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Ahn, H.-J. Articles by Forney, L. J. Search for Related Content PubMed PubMed Citation Articles by Ahn, H.-J. Articles by Forney, L. J. Agricola Articles by Ahn, H.-J. Articles by Forney, L. J.