Biocide Resistance in Populations of E.coli

The subject of bacterial resistance to antibiotics has not only inundated the commercial market, but also provided powerful evolutionary insight into larger studies of evolution. Genetic resistance using bacterial models is highly regarded for their rapid proliferation, easy maintenance, and simple manipulation of variables. Despite their widespread use, these studies have yet to signify the presence of evolution and note residual costs on competition in strains of E.coli. To observe both aspects, six repeated rounds of three various treatments were subjected to cultured bacteria with each plate measured for zone of inhibition. Two control groups of sterile water and ethanol provided the basis for evolutionary explanation, while Triclosan acted as the antimicrobial agent from which the trend was analyzed. Control groups revealed no distinct pattern of evolution and little to no zones of inhibition, as expected. Triclosan samples showed a relatively steady decrease in zone measurements from first to final round. Competition cultures detect heightened presence of ancestral colonies over evolved in all three conditions. However, results of a one-way ANOVA analysis through Dunn’s multiple comparisons test disclose significant difference between Triclosan and each control group (P=0.0009), while both controls do not vary significantly from each other. Despite mixed results, the distinct portrayal of evolution is evident and the cost associated with resistance has yet to exhibit in a distinct manner. The truncated timeline of this evolutionary study provides insight, yet is strengthened when extrapolated to include more, subsequent generations of resistant bacteria. Introduction The dynamics of adaptation and variation have long baffled those in search of powerful evidence to the phenomenon of evolution. Finding this evidence has, in many cases, come to explaining the genetic variation and inheritance from generation to generation. Such transgenerational influence is portrayed best in those models that reproduce and proliferate over a short time period, namely bacteria. In particular, countless evolutionary studies have resorted to using bacterial models to show antibiotic resistance over time (Levy, S.B., 1998). This resistance is associated with genetic variation, yet is greatly affected by the environmental stimuli within a bacterial culture. These short-term studies provide potent evidence to the adaptive qualities of bacteria that, in turn, can be extrapolated to larger systems. Determining the source of variation behind this antibiotic resistance is complex, and typically varies between bacterial subjects and their antibiotic counterpart of choice (Lenski, 1994). Among the most widely used antibiotics in these studies, Triclosan is one that is considered an effective antimicrobial ingredient of many health care and consumer products (Schweizer, 2001). This agent has been deemed potent in its ability to suppress harmful bacteria and other microorganisms through its supposed “multicellular target attacks” (Schweizer, 2001). However, it has been recently found that Triclosan instead targets a particular artificial fatty acid enzyme, enoyl-[acyl-carrier protein] reductase (Lenski, C.W. 1998). Many have found this enzyme to be particularly useful in determining the sources of variation that cause antibiotic resistance. With Triclosan, increased prevalence of target expression and mutation along with enzymatic inactivation/degradation leads to similar mechanisms involved in antibiotic resistance (Schweizer, 2001). This correlation between genetic variation and qualities of resistance has begun to explain the worry associated with communicable bacteria in spite of antimicrobial agents. The links between this agent and various bacteria are endless, yet confirmed links between antimicrobials in clinical isolates have been seen with Escherichia coli subjects. The aim of this study is to exhibit the potential resistance of E.coli bacterial cultures to repeated exposures of Triclosan. Significance of results comes from notable portrayal of increased resistance through observation of zones of inhibition. Triclosan has been shown to act as a site-directed inhibitor of the enoyl-[acyl carrier protein] reductase by imitating its substrate (Lenski, C.W. 1998). From here, Triclosan affects a number of bacterial cell properties. In particular, studies have found both Triclosan and antibiotics cause expression of efflux pumps (used for detoxification) through selection of similar mutations in respective regulatory loci (Schweiser, 2001). With this theory, turning on additional efflux pumps within the bacteria, further resistance to the antibiotic could be achieved. Therefore, the experimental procedures of this study can base its findings off of substantiated evolutionary perspective. Similarly, the resistance mechanisms associated with Triclosan come with a series of trade-offs with other cell properties and functions. The cost of resistance against antibiotics is notable, especially noted in previous clinical tests using E.coli. The mutations associated with Triclosan confer resistance by disturbing normal physiological cell processes that would otherwise be used for cell regulation (Lenski, 1998). The energy given toward producing additional nucleic acids and proteins toward resistance place a great burden on the cell, making the cell inferior in a case of competition (DaSilva, 1986). Assuming the resistance of bacterial subjects is evident upon continual exposure to Triclosan, the inferiority of the resistant population to the ancestral generation should also be evident. With these methodologies in mind, a set of hypotheses are propagated for this particular experiment: i. A negative relationship between generation and size of inhibition zones exists, suggesting evolved antibiotic resistance over six generations. ii. The evolved population of bacteria is inferior to that of the ancestral line under the course of competition. Methods A series of six selection events take the form of this evolutionary simulation, using three treatments exposed to each culture growth of E.coli. To create the beginnings of the ancestral line of E.coli cultures, a stock culture was created using a green fluorescent protein (GFP). This culture was incubated at 35° C for 24 hours. 48 petri dishes were prepared with a standard agarose gel medium, and divided into three groupings of 16 dishes each, one for each of the treatments: Ethanol, Sterile water, Triclosan. Each of the 16 preliminary trials were swabbed with a complete lawn covering of the ancestral E.coli growth culture. For each group, a paper disk was placed into the middle of each dish, each soaked in one of the three treatments. Petri dishes were sealed with parafilm and incubated at 35° C for 24 hours. After incubation, zones of inhibition of each dish were measured (in mm) in two different positions, averaging the recordings. The most resistant strain of the dish was isolated by swabbing the inner margin of the zone of inhibition. In the absence of inhibition, swabs were taken from the perimeter of the disk. These swabbings were transferred to a new tube of sterile tryptic soy broth and swirled to ensure mixture of the two. Tubes were capped and placed into the incubation chamber for another 24 hours. After 24 hours, the process was repeated from the point of swabbing new cultures onto new petri dishes of ancestral lawn coverings. After the last round of treatments, the turbidity of each GFP E.coli culture was measured at 600nm using the Nanodrop spectrophotometer. Each culture tube volume was compensated for by adding more tryptic soy broth so it’s absorption at 600nm would be close to the stock culture of BFP E.coli. The platform for determining the outcome of competition was created using 1 ul of GFP E.coli culture and 1 ul of BFP E.coli culture to 1 ml of tryptic soy broth using 1x IPTG fluorescent inducer. Cultures were placed into a 35-37° C incubation for 24 hours and held at 2.2° C until measurements were taken. Measuring the outcome of competition was dependent upon the interaction of the E.coli with the kanamycin sulfate (50 ug.ml) placed in all tryptic soy broth and soy agar throughout the experiment. The bacteria contained a plasmid for fluorescent protein as well as kanalycin resistance. The kanamycin resistance selected those E.coli that were successfully transformed. Kanamycin was included throughout the remainder of the experiment to ensure any back mutations were negated from the equation. For measurement, 100 uL of each culture was pipetted into a 96-well microtiter plate, including eight wells of tryptic soy broths for use as blank readings. Two scans of the Walla Victor 2 mictoplate reader were conducted to determine relative amounts of BFP and GFP E.coli. BFP bacteria were agitated at a reading of 355nm and absorbance was recorded at 460nm. GFP bacteria were agitated at a reading of 485nm and absorbance was recorded at 530nm. To account the difference in wavelength readings through microplate reader filters, the ratio between absorbances is instead used for calculations and analysis.