Biology and Chemistry Research Antibiotic Resistance Dissemination Increased by High Frequency of Conjugating Bacteria in Escherichia coli Populations Jennifer Wu
ABSTRACT
According to the World Health Organization, the total societal cost of antibiotic resistance amounts to over $35 billion dollars per year in the United States alone when accounting for lost lives, wages, and extended hospital stays. Bacterial conjugation, a type of horizontal gene transfer, is one of the processes by which antibiotic resistance is disseminated throughout a bacterial population. In the search for methods to inhibit the spread of antibiotic resistance, preventing bacterial conjugation is considered a promising target. However, the degree to which conjugation affects the rise of antibiotic resistance is unclear. This study investigated the effect of different ratios of conjugatory donors to recipients of Escherichia coli on the population’s resistance to tetracycline. Strains BB4 and DH5α served as the donor and recipient cultures respectively and were allowed to conjugate before being plated in tetracycline containing agar; resistance was quantified by colony density. Results showed that the presence of conjugating bacteria had a greater relative effect on colony density at higher tetracycline concentration. A donor percentage of 5% (1:19) more than doubled the minimum inhibitory concentration of tetracycline required. At a donor percentage of 20% (1:4), colony density approached levels of an entirely resistant population. This experiment has revealed that even at low levels, bacterial conjugation has the potential to rapidly increase the resistance of a bacterial population and presents conjugation as a crucial target for slowing the spread of antibiotic resistance. Conjugation is the transfer of plasmid DNA from one Introduction bacterial cell to another and can only occur between a donor containing a conjugatory plasmid and a recipient Potent antibiotics are a double edged sword because that lacks one. After the recipient receives a plasmid, it their effectiveness against susceptible bacterial strains becomes a donor itself. Conjugation has even been known is also a strong selective force for developing resistant to occur between two different species of bacteria [5]. The strains. The spread of antibiotic resistance has become a components of conjugation machinery are encoded on pressing problem as evidenced by the fact that the number plasmids; basic components include a relaxase, a coupling of methicillin resistant Staphylococcus aureus infections has protein, and a type IV secretion system (T4SS) [6]. risen by 300% in ten years [1]. In a retrospective study In another study conducted on the EcoR collection spanning from 1950 – 2002, over 1,500 E. coli isolates were strains, it was found that 21% of the strains were capable taken from humans, cattle, chickens, and pigs and assessed of conjugation [7]. Experiments on the frequency of for their susceptibility to 15 different antimicrobial drugs plasmid transfer performed on E. coli strain BW27783 (a [2]. Around 54% of the strains tested were resistant to K-12 strain derivative) estimated the transfer frequency more than one antibiotic, with resistance most commonly of plasmid R388 [8]. It was also found that increasing the found against older drugs such as tetracycline, ampicillin, donor to recipient ratio from 2.5% to 50% led to an increase streptomycin, and sulfonamide [2]. Once a bacterial cell in maximum transfer rate of 0.2 to 0.7 transconjugant per acquires resistance to an antibiotic, the gene encoding recipient cell [8]. Conjugation frequency was shown to this ability can be rapidly disseminated throughout the increase logarithmically which is explained by the fact that population through forms of horizontal gene transfer such after conjugation occurs, recipient cells themselves become as bacterial transformation, transduction, and conjugation. donors and further propagate their plasmid [8]. Current methods of combatting antibiotic resistance The next step is to identify mechanisms of lateral are mainly focused upon the discovery of new drugs and gene transfer that contribute the most to the spread of avoidance of unnecessary or low level dosages of antibiotics antibiotic resistance. The inhibition of these mechanims, [3]. However, natural selective pressures often occur more such as conjugation, has the greatest potential to delay quickly than the discovery of new drugs and over-theresistance development allowing more time for researchers counter medications are difficult to regulate. Because to discover new treatment methods. Previous studies have horizontal gene transfer is a major contributor to antibiotic only examined either the phenomenon of rising antibiotic resistance but is nonessential for survivorship except resistance or the prevalence of conjugation individually. in the presence of antibiotics, disrupting gene transfer In contrast, this study looks at the rise of antibiotic will allow bacterial populations can remain susceptible resistance in combination with conjugation by varying to current treatments for a longer period of time and the proportion of the antibiotic susceptible population resistance to transfer inhibition will be slow to occur [4]. in the presence and absence of conjugating strains and Resistance inhibitors can then be used in conjunction with quantifying the resulting population’s resistance to the conventional drugs to create long term potent treatment antibiotic tetracycline. This will assess the impact of combinations which are especially important for patients bacterial conjugation on antibiotic in nosocomial settings [4]. Volume 3 | 2013-2014 | 27
Biology and Chemistry Research resistance dissemination and conjugation’s suitability as a target in halting the spread of resistance.
Materials and Methods E. coli Strains and Media The study organism was Escherichia coli strains DH5α, DH5α (Tetr), and BB4 (Table 1). E. coli was chosen because it is a widely used model organism in which conjugatory plasmids are naturally found. The strain recipient DH5α (Carolina Biological) had an F- genotype meaning it lacked a conjugatory plasmid. The donor strain was BB4 which contains an F plasmid that confers tetracycline resistance (Ward Scientific). DH5α (Tetr), was a nonconjugating F- tetracycline resistant strain that was used as a control. DH5α (Tetr) was created by transforming the DH5α strain with plasmid pBR322 which confers tetracycline resistance (Carolina Biological). DH5α was made competent through CaCl2 and glycerol treatment and transformed using heat shock [9]. All E. coli strains were cultured in sterile Mueller-Hinton (MH) broth that was prepared according to manufacturer’s instructions and incubated at 37ºC. For strains BB4 and DH5α (Tetr), the media was supplemented with 10 µg/mL of tetracycline. For both donor and recipient cultures used in experiments, cells were diluted to an OD600 of 0.300 as measured by a spectrophotometer. Testing protocols for MIC values followed guidelines established by the Clinical and Laboratory Standards Institute [10].
Conjugation Experimental Design In this experiment, cultures with starting BB4 population percentages (donor to recipient ratio) of 100% (1:0), 20% (1:4), 10% (1:9), 5% (1:19), 2.5% (1:39), and 0% (0:1) by volume were allowed to conjugate with DH5α. After cultures were allowed to conjugate, E. coli inoculum from each donor percentage setup were plated on MH agar containing tetracycline concentrations of 32 µg/mL, 16 µg/mL, 8 µg/mL, 4 µg/mL, 2 µg/mL, and 0 µg/mL. After 18 hours of incubation at 37 ºC, growth was assessed by determining the colony density of each 100 mm plate. Each antibiotic concentration and donor percentage combination was replicated five times. The Control groups were incubated under the same conditions as the experimental and composed of the non-conjugating tetracycline resistant strain DH5α (Tetr), cultured with the recipient strain DH5α in the same percentages as listed above and then plated on MH agar containing the same tetracycline concentrations as above. The control was used to determine the colony formations that can be attributed 28 | 2013-2014 | Volume 3
to the initial population of tetracycline resistant cells. Growth Curve Generation A preliminary experiment involving the growth rate of all three strains of E. coli was conducted in order to control for genotypic discrepancies between the three strains that may cause differences in growth. Each of the three strains was grown in both MH broth containing 0 µg/mL and 8 µg/mL of tetracycline. The OD600 of each culture over 24 hours was measured using a SpectroVis Plus Spectrophotometer blanked with a cuvette filled with sterile MH broth. Falcon 50 mL conical centrifuge tubes were filled with 40 mL of MH broth containing the appropriate amount of antibiotic (either 0 µg/mL or 8 µg/ mL) and inoculated with 100 µL of an overnight culture of one of the three E.coli strains grown in tetracycline free MH broth to an OD600 of 0.300. All cultures were grown in a shaking water bath at 37º C. Data were collected every hour for the first nine hours and then once again at the 24 hour mark. Each treatment was performed in triplicate. Conjugation Protocol Both donor and recipient strains were vortexed and diluted with MH broth as necessary until they reach an OD600 of 0.3. Each culture was then further diluted by a factor of 10-6 before being allowed to conjugate so that individual colonies would form once plated. When mating the two strains, donor percentages (donor to recipient ratio) of 100% (1:0), 20% (1:4), 10% (1:9), 5% (1:19), 2.5% (1:39), and 0% (0:1) by volume were set up in a 1.5 mL microcentrifuge tube and vortexed. The total volume of each mixture was 1 mL. The E. coli culture was then allowed to conjugate for 60 minutes in a 37ºC shaking water bath. At the end of this period, the tube was vortexed for 10 seconds to disrupt mating. As a control, DH5α (Tetr) and recipient (DH5α) ratios equivalent to ones listed above were also prepared in the same manner. This setup represented colony densities that could be expected under antibiotic selective pressures without the effect of plasmid transfer. After the conjugating period was over, 30 µL of each setup was pipetted onto MH agar plates containing the appropriate antibiotic concentration and spread with an L-shaped spreader and incubated for 18 hours at 37°C. Minimum Inhibitory Determination
Concentration
(MIC)
The MIC was determined using the agar serial dilution method [10]. Four serial dilutions of tetracycline by a factor of ½ with a starting concentration of 32 µg/ml were used to prepare agar of varying antibiotic concentration [11]. Tetracycline free MH agar plates were separately prepared. 25 mL of agar was poured into
Biology and Chemistry Research each 100mm x 25mm Corning culture dish. Bacteria to be inoculated were grown overnight to an OD600 of 0.300. After the plates were inoculated, they were incubated at 37º C for 18 hours. The MIC was taken as the lowest antibiotic concentration at which no growth was observed. Colony Formation Comparison After all petri dishes have been incubated for 18 hours, the number of colonies on each dish was counted using a colony counter (Figure 1). The surface of each plate was divided into 36 one cm2 squares that were numbered and four unique randomly chosen squares chosen by a random number generator were counted. Colonies growing on the border of a square were included inside the square. The colony density of each plate was then averaged. The increase in colony formation caused by conjugation was calculated by subtracting the average number of colonies in the control experiment from the average in the corresponding conjugation experiment in the same experimental conditions. The difference was then divided by the average in the control group to determine the percent change.
Figure 1. Colony counting. The above image represents the grid used to count colonies. The square outlined in red has dimensions of 1cm x 1cm. Four squares on each plate were chosen at random and the number of colonies present in each square was averaged for each dish. Source: author’s photo. LD50 Assessment Average colony densities at each donor percentage were plotted against antibiotic concentration. Total density used to calculate LD50 was taken to be the average colony density in plates containing 0 µg/mL of tetracycline. For each initial BB4 or DH5α (Tetr) percentage, the antibiotic concentration at the intercept with a line plotted at 50% of the average density in 0 µg/mL of tetracycline was determined to be the LD50 for that initial BB4 or DH5α (Tetr) percentage.
Statistical Analysis The software program JMP version 10.0.0 (SAS Institute, 2012) and Microsoft Excel were used for all statistical analyses. E. coli growth curve: Optical densities of each strain were averaged at each time point and an ANOVA was performed to determine significance between the maximum optical densities of each strain at 24 hours. Difference in Colony Formation: A threeway ANOVA was performed using JMP. The main effects in the model were antibiotic concentration, presence of conjugation, and initial Tetr population size. Crossed effects of antibiotic concentration by Tetr population percent, antibiotic concentration by presence of conjugating bacteria, Tetr population percent by presence of conjugating bacteria, and antibiotic concentration by Tetr population percent by presence of conjugating bacteria were also analyzed.
Results From the growth curves plotted at the start of experimentation (Figure 2), it was seen that all three strains of E. coli that were used in this study exhibited similar growth rates and carrying capacities. In broth containing no tetracycline, all three strains grew robustly as expected and reached a maximum OD600 at 24 hours that was not significantly different (ANOVA, df = 2, F-ratio = 3.5633, p < 0.2624) (Figure 2A). In the growth curve generated with broth containing a tetracycline concentration of 8 µg/ml, the susceptible strain, DH5α was greatly inhibited compared to other two trains with a maximum OD600 of 0.075± 0.001414 (ANOVA, df = 2, F-ratio = 3874.23, p <0.0001). BB4 reached an OD600 of 0.2403 ± 0.00144 and DH5α (Tetr) reached a maximum OD600 of 0.2363± 0.00072 showing they had the same carrying capacity (Tukey-Kramer, p < 0.53) (Figure 2B). This shows that DH5α (Tetr) would function as a suitable control for BB4 in determining differences in colony formation and changes in minimum inhibitory concentrations. To determine the effect of conjugation on the tetracycline resistance of an E. coli population, various population sizes of BB4 cells were cultured with susceptible DH5α cells and then grown in tetracycline concentrations ranging from 0 µg/ml to 32 µg/ml (Figure 3). Across all non-zero tetracycline concentrations, colony density of plates with conjugating cells was higher than those without when the initial Tetr population was between 5% and 20% inclusive (Figure 3). The difference in colony density between the control and conjugation treatments was magnified at high tetracycline concentrations (16 µg/ mL and 32 µg/mL). A 5% conjugating population (BB4) was able to raise the MIC of the population to greater than 32 µg/mL (Figure 3F). As antibiotic concentration, and thus selective pressures, increases, the adaptive Volume 3 | 2013-2014 | 29
Biology and Chemistry Research
Figure 2. E. coli growth curves A) Growth as measured by OD600 in 0µg/mL tetracycline. All three strains of E.coli exhibit similar growth patterns over 24 hours. ANOVA at the 24 hour time point suggests that genotypic differences among strains shows minimal effect (p <0.2624). B) Growth as measured by OD600 in 8µg/mL tetracycline. Strains BB4 and DH5α (Tetr) display similar growth rates under antibiotic stress (p <0.53) while DH5α shows very little resistance to tetracycline over 24 hours (p < 0.001). DH5α (Tetr) would serve as suitable control strain in colony formation assessment. 425% due to conjugation (Figure 4). value of conjugation increases as well. At all antibiotic In another representation, the LD50 of the population concentrations, there was no significant difference at each initial resistant Tetr percentage was determined between the colony density when the initial conjugating for treatments with and without conjugation. From BB4 population was 20% and 100% (Tukey-Kramer, p < this, we see that the difference in LD50 concentrations 0.80). between populations with and without BB4 donors is ANOVA values displayed in Table 2 shows that the size most noticeable at initial Tetr populations of 5%, 10%, of the intial Tetr population, antibiotic concentration, and and 20% (Figure 5). Since the LD50 -concentration especially the presence of conjugation all have a significant was calculated without fitting the dose response curve, it effect individually upon colony density even when variance cannot be ascertained that the LD50 dosages are indeed due to all other variables are not taken into consideration. representative values. However, given the significance When analyzing the cross effect of tetracycline of the difference between the responses of populations concentration by presence of conjugation, it was noted with and without conjugation shown previously, the that at every non zero tetracycline concentration, there LD50 values are appropriate estimates to represent the was a significant difference between the colony density of difference conjugation exerts on colony density. setups with and without conjugation.
Discussion
The increase in E. coli survival due to conjugation becomes most apparent when the percent increase in colony density is viewed at each experimental condition (antibiotic and donor percentage interaction) (Figure 4). The impact of BB4’s ability to transfer antibiotic resistance on colony formation was amplified at both high tetracycline concentrations and donor population sizes. General trends show that conjugation has a larger effect on colony density relative to controls at high antibiotic concentrations and donor percentages. The highest increase from control conditions was seen at 32 µg/mL and 20% BB4 population; colony density increased by 30 | 2013-2014 | Volume 3
In this study, it has been shown that bacterial conjugation can greatly raise a population’s antibiotic resistance and is especially effective at improving colony viability in high antibiotic concentrations. The results suggest that bacterial conjugation is an important factor in antibiotic resistance dissemination; consequently, inhibiting conjugation is important target for slowing resistance development. Previous works have found that lateral gene transfer has given rise to multi-drug resistant pathogens in hospital settings due to the presence of similar resistance genes among four different bacterial species [12]. However, in these works, the mechanism of horizontal gene transfer with the greatest contribution to resistance dissemination was not investigated. Comparisons between the rate of resistance developed through natural and artificial selection and through conjugation are needed.
Biology and Chemistry Research
Figure 3. Colony density with and without conjugation in tetracycline concentrations. A-F) Colony density on plates with and without conjugation in respective antibiotic concentrations as labeled (0 µg/mL – 32 µg/mL). Initial Tetr percentages of 0% and 100% are negative and positive controls. Growth in 0 µg/mL (A) also served as a control for the absence of selective pressure. Growth of the 100% Tetr pops shows that when the entire population is resistant, there is no relative cost or benefit from conjugation (with compared to without) across all antibiotic concentrations. Error bars denote SEM.
Figure 4. Percent increase in colony density due to conjugation. Percent increase was calculated by subtracting the average colony density of control plates at each experimental condition from the respective average density in the conjugating populations and then dividing by the average colony density of the control. Figure legend indicates size of donor BB4 by volume. Initial donor BB4 proportions of 0 % and 100% were not shown since they served as negative and positive controls, respectively.
Figure 5. Effect of conjugation on tetracycline LD50 and increase in colony density. E.coli tetracycline LD50 (µg/ml) with and without conjugation. LD50 concentration for 5-20% initial Tetr population exhibit the greatest effect of conjugation on LD50 concentration. 0% and 100% initial Tetr population serve as negative and positive controls, respectively.
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Biology and Chemistry Research E. coli with a minimum inhibitory concentration greater than 16 µg/ml tetracycline are considered resistant [10]. This project has shown that at a tetracycline concentration of 16 µg/ml and a donor population of 20%, resistant colonies increased by over 100% relative to the control (Figure 4E). This represents a startling rise in resistance due to conjugation since 21% of E. coli strains have been found to contain F-like plasmids capable of transferring itself [7]. It should be noted that at high tetracycline concentrations, there is a low basal colony density. Thus numerical increases in colony density reflect a large percentile increase in density. However, a BB4 population size of 20% does allows the colony density on all plates to come close to that of an entirely resistant starting culture in all tetracycline concentrations tested (Figure 3). The logistic relationship between BB4 population and resistant colonies is likely due to the logistic nature of bacterial conjugation itself. Since transconjugant cells become donors themselves, the number of resistant bacterial cells increases exponentially due to conjugation and then levels off when all cells have become resistant [13]. Population density is also limited through interactions with the carrying capacity since there is a biological toll associated with producing antibiotic neutralizing compounds. Consistent with inferences based on literature, Figure 3 shows a logistic increase in colony density with respect to donor number. This is notable since it shows that even at low levels, the presence of conjugating bacteria can exert a large influence on the antimicrobial susceptibility of a bacterial population. LD50 assessments also show that populations with conjugating bacteria require a higher tetracycline concentration to reach 50% of their carrying capacity which reflects how conjugation can greatly decrease the potency of antibiotics (Figure 5). The effect of having a conjugating population was also magnified at high tetracycline concentrations. The density plateaus once initial BB4 proportion reaches 20% since it is logical to assume that enough cells have acquired resistance to be limited by carrying capacity instead of tetracycline concentration. At a BB4 proportion of 20%, percent increase in resistance jumped from a 36% increase to a 190% increase when the antibiotic concentration changed from 8 µg/ml to 16 µg/ml. This may be due to the fact that at low sublethal antibiotic levels, more E. coli cells are able to cope with the stress and evolve resistance over time [14]. The high basal growth rate in low tetracycline concentrations thus diminishes the importance of the resistance gene carried on the plasmid because other genotypes are sufficient for survival albeit at a higher metabolic cost. However, the converse is also true: in the previously bactericidal dosage of 32 µg/ml tetracycline, conjugation enabled resistant E. coli colonies to gain a foothold and increase resistance by 4.25-fold. This suggests that conjugation alone may lead to bacterial infections that cannot be cured with a previously bactericidal dose of antibiotics. Another factor in the acquisition of antibiotic resistance 32 | 2013-2014 | Volume 3
is the formation of biofilms. Studies have found that in biofilms can lead to decreased susceptibility through activation of altered metabolic pathways, formation of persister cells, as well as the creation of a physical barrier against foreign compounds [15]. Biofilms require an initial cluster of cells that adhere to a solid surface and secrete biofilm determinants [16]. Conjugation during the planktonic (free floating) phase may play into this phenomenon by spreading resistance genes that allow for the formation of an initial cluster of resistant cells. These cells ultimately divide to form a biofilm colony that even protects susceptible cells from antibiotics. There is also a cyclic nature to this process since Lactococcus lacti biofilms have been observed to promote plasmid pAMβ1 transfer to more than 10,000 times compared to a non-biofilm forming strain [17]. These additional plasmid transfers aids in the creation of a greater number of resistant clusters that could later form biofilms themselves. This would explain how cultures with 5% donor could grow on agar that contained a previously bactericidal antibiotic concentration of 32 µg/ml tetracycline.
Conclusion and Future Work This study showed that bacterial conjugation between E. coli strains BB4 and DH5α led to higher rates of resistant colony formation especially at tetracycline concentrations at or above the MIC of 32 µg/ml tetracycline. In some cases, resistance can be seen to double or even triple with increasing donor composition. Therefore, bacterial conjugation can be seen as a major mode of antibiotic resistance transmission. In bacterial fauna found outside the laboratory, conjugatory plasmids may also have a bigger role than what was explored in this study since many plasmids and conjugatory transposons have a broad host range and can be transferred between different bacterial species [14]. This can rapidly lead to the development of drug resistance among multiple species of infectious agents. In this experiment, resistance was represented by colony density at 18 hrs. What can be considered in future works is that individual colonies can vary in size and morphology from plate to plate even though the composition of the initial inoculum was standardized. Larger colonies that may contain more cells were accounted for equivalently as smaller colonies. Future studies may aim to measure growth through wavelength absorbance in liquid cultures where cells are more homogenously suspended or develop methods of uniformly accounting for growth on agar plates. Although plasmid movement has been noted to be the most common form of horizontal gene transfer, further experimentation can be done to investigate other forms of horizontal gene transfer such as transduction and transformation and their role in antibiotic resistance dissemination [18]. Furthermore, mathematical and experimental models can be developed to determine
Biology and Chemistry Research differences in conjugation rate in liquid and solid media. Bacterial cultures suspended in liquid media have increased motility and thus increased likelihood to come into contact with recipient cells [8]. Yet in colony formations, neighboring cells are closer in proximity and contact is aided by an extracellular polymeric matrix [16]. What this study ultimately highlights is that bacterial conjugation is a key mechanism in the rapid rate of antibiotic resistance development. Although selective forces cannot be stopped, they can be hindered. Conjugation inhibition, whether through interactions with conjugatory components or the cell membrane [19] should prove to be a promising next step for drug development.
Acknowledgments I would like to thank Dr. Amy Sheck, for guidance and expertise in all aspects of the experimental process; Dr. Dan Teague for statistical analysis and data interpretation; Ms. Korah Wiley for advice and aid during experimental setup and data collection; Research in Biology colleagues of 2013-2014 and Glaxo fellows for constant encouragement, assistance throughout the research process, and peer review of paper; and Glaxo Endowment for funding and sponsorship of project.
References [1] Pray, L. 2008. Antibiotic resistance, mutation rates and MRSA. Nature Education 1: 34-36. [2] Tadesse, D., S. Zhao, E. Tong, S. Ayers, A. Singh, M. Bartholomew, P. McDermott. 2012. Antimicrobial drug resistance in Escherichi coli from humans and food animals, United States, 1950-2002. Emerging Infectious Diseases 18: 741-749. [3] Fernando B., T. M Coque, and F. de la Cruz. 2011. Ecology and evolution as targets: the need for novel ecoevo drugs and strategies to fight antibiotic resistance. Antimicrobial Agents and Chemotherapy 55: 3649-3660. [4] Smith, A. and F.E Romesberg. 2007. Combating bacteria and drug resistance by inhibiting mechanisms of persistence and adaption. Nature Chemical Biology 3: 549-556. [5] Dahlberg, C., M Bergstrom, M Andreasen, B. Christensen, S. Molin, and M Hermansson. 1998. Interspecies bacterial conjugation by plasmids from marine environments visualized by gfp expression. Molecular Biology and Evolution 15: 385-390. [6] Llosa, M., Gomis-Ruth, F.X., Coll, M., and De La Cruz, F. 2002. Bacterial conjugation: a two-step mechanism for DNA transport. Molecular Microbiology 45: 1–8. [7] Boyd, E., C. Hill, S. Rich, and D. Hartl. 1996. Mosaic structure of plasmids from natural populations of Escherichi coli. Genetics 3: 1091 -1100. [8] del Campo I., R. Ruiz, A. Cuevas, C. Revilla, L. Vielva, and F. de la Cruz. 2012. Determination of conjugation rates on solid surfaces. Plasmid 67: 174-182.
[9] Chung, C.T, S.L. Miemela, and R. H. Miller. 1989. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proceedings of the National Academy of Sciences 86: 2172-2175. [10] Wikler M., F. Cockerill, W. Craig, M.N. Dudley, G. Eliopoulos, D.W. Hecht, J.F. Hindler, D. Low, D. Sheehan, F. Tenover, J. Turnidge, M. Weinstein, B. Zimmer. M.J. Ferraro, and J.M. Swenson. 2006. Performance standards for antimicrobial susceptibility testing seventeenth informational supplement. Clinical and Laboratory Standards Institute 26: 1-49. [11] Andrews, J. M. 2001. Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48: 5-16. [12] Naiemi N. A., B. Duim,P. Savelkoul, L. Spanjaard, E. Jonge, A.Bart, C, Vandenbroucke-Grauls, and M. de Jong. 2005. Widespread transfer of resistance genes between bacterial species in an intensive care unit: implications for hospital epidemiology. Journal of Clinical Microbiology 43: 4862-4864. [13] Gehring, R., P. Schumm, M. Youssef, and C. Scoglio. 2010. A network-based approach for resistance transmission in bacterial populations. Journal of Theoretical Biology 262: 97-106. [14] Dzidic, S., and V. Bedekovic. 2003. Horizontal gene transfer-emerging multidrug resistance in hospital bacteria. Acta Pharmocologica Sinica 6: 519-526. [15] Anderson, G.G., and G.A. O’Toole. 2008. Innate and induced resistance mechanisms of bacterial biofilms. Current Topics in Microbiology and Immunology 322: 85-105. [16] Beloin, C., A. Roux, and J-M. Ghigo. 2008. Escherichia coli biofilms. Current Topics in Microbiology and Immunology 322: 249-289. [17] Luo, H., K. Wan, and HH. Wang. 2005. Highfrequency conjugation system facilitates biofilm formation and pAMβ1 transmission by Lactococcus lactis. Applied and Environmental Microbiology 71: 2970-2978. [18] Ochman, H., J.G. Lawrence, and E. A. Groisman. 2000. Lateral gene transfer and the nature of bacterial innovation. Nature 405: 299-304. [19] Baquero, F., T.M. Coque, and F. de la Cruz. 2011. Ecology and evolution as targets: the need for novel ecoevo drugs and strategies to fight antibiotic resistance. Antimicrobial Agents and Chemotherapy 55: 3649-3660. *Agilent Technologies. 2013. Escherichia coli host strains. http://www.chem-agilent.com/pdf/strata/200256.pdf. Accessed: 9/24/13 **Invitrogen. 2013. DH5alpha Genotype. http://www. lifetechnologies.com/us/en/home/life-science/cloning/ competent-cells-for-transformation/chemicallycompetent/dh5alpha-genotypes.html. Accessed: 9/22/13
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