Development of Antibiotic-Inducible Reporter Systems as Tools for Study of Antibiotic Penetration through Biofilms

Edward Mabry, Jeremy Tzeng, Maya Elhage, Cedric Taylor, Maryam Saffarian

Abstract

Biofilm formations on medical implants are a major cause for infection and disrupt the function of medical devices. Modern antibiotics typically penetrate the surface but fail to effectively eradicate each layer of the biofilm, which in some cases can lead to the development of a drug-resistant biofilm. In this project, Bacillus subtilis served as a model organism for studying biofilm formation on implant surfaces. We designed a genetic construct with with inducible green fluorescent protein (GFP) under the control of a lac promoter and antibiotic-induced red fluorescent protein (RFP) which allows for the quantification of antibiotic penetrance in vitro. Expression of RFP is under the control of a vancomycin-inducible Plia1 promoter. We will validate the protocols we have developed for transformation of B. subtilis 1012wt strain. The transformed B. subtilis will then be utilized to validate the constructed vector, namely, pHT01-GFP-Plia1-RFP for detection of vancomycin and determine the detection limit. B. subtilis carrying pHT01 will then be used as a tool for development and valuation of methods that could facilitate penetration of antibiotics through biofilm.

Introduction

Biofilms are vital extracellular structures associated with  microorganisms that form communities in hostile environments and consist of a variety of biological molecules ranging from ions to nucleic acids. Biofilms usually consist of the extra polymeric matrices which, along with other bio-molecules, protect the microorganism community who take on new specialized functions themselves that are not performed by other non-biofilm forming or free floating organisms. Biofilms are not one simple mixture of slime, but quite layered and organized such that microbes in separate layers can have completely differing gene functions (1). These biofilms even can protect the microbial community from host immune factors, and the extra polymeric matrix is usually the first means of defense against such dangers to the pathogens. The presence of exopolysaccharide alginate and similar molecules prevents macrophages and neutrophils from utilizing phagocytosis on the microbial community within, in which the microbes would normally be engulfed and destroyed. As such, most infection-causing biofilms are rarely removed by the host own immune system alone (2). Biofilms increase the antibiotic resistance of the microbes within. This is due to the biofilms structure which can be a consequence of chemical interactions that occurs within the biofilm itself or even the perpetuation of anionic polysaccharides which can prevent or reduce antibiotic penetration (3). When combined with horizontal gene transfer that allows resistance of an antibiotic to be transferred easily if the biofilm is not completely wiped out, it leads to strains of antibiotic resistant microbial pathogens which are even harder to kill with antibiotics than before.

Unfortunately, it is not easy to visualize the antibiotic penetrance for biofilms with current tools. If such tools were to be developed, then study of the effects of drugs on biofilms either alone or with antibiotics could be possible. The goal of this project is to produce an antibiotic reporter-system in the microbes of the biofilm themselves. Not only would study of the antibiotics through layers be possible for new drugs, LSCM (laser scanning confocal microscopy) could be utilized to make a three dimensional map of the penetration. If these studies bear fruit, it could be more likely that more antibiotic treatments could be utilized as a replacement to invasive treatments of dealing with these microbes such as surgery to physically remove the biofilms. Antibiotic treatments are currently only mildly effective due to the resilience of most biofilms, which can be formed in the host from medical devices or implants. The current and most effective method is still surgery though to manually remove these biofilms, but they pose a high risk for the patient due to the nature of surgery itself.

Materials and Methods

Plasmid Construction and Extraction from Transformed E. coli DH10B

The plasmid components were obtained from the iGEM registry and E. coli DH10B was utilized as the host cells for both plasmid construction and amplification. BioBrick Standard Assembly method was utilized to assemble the individual gene components of the target plasmid and the BioBrick plasmids were utilized for the design of the final construct (4). The plasmids have two restriction sites upstream of the insert, Eco R1 and Xba I (known as the BioBrick Prefix), and two restriction sites downstream of the insert, Spe I and Pst I (known as the BioBrick Suffix), assuming a left to right orientation. The individual gene components were assembled in a stepwise manner. First, RFP was extracted from the BioBrick plasmid using restriction enzymes and ligated into pGEM, the target vector. The transformation was then confirmed via gel electrophoresis, and was repeated for the constructs in the other steps. In the second and third step, the TT (which stands for two transcriptional terminator sites in a row) and ribosome binding site (RBS) were ligated into pGEM the same way as RFP. The promoter, Plia1, was excised and a prefix insertion was performed at the beginning of the pGEM construct as to establish the vancomycin-induced RFP expression. The GFP portion was assembled similarly, but addition of RBS and a promoter was not necessary since the lac operon would be utilized for induction. GFP was excised using restriction enzymes and ligated to a TT part utilizing a prefix insertion. Final insertion into pGEM was completed using restriction enzymes BamHI and AatII.

Once the individual gene components were ligated, pGEM was inserted into competent E. coli DH10B cells. Transformation was then performed by electroporation of the cells. After incubation, plasmid extraction was performed using standard QIAGEN Spin Miniprep Kit protocol to confirm success of ligation and proper orientation of gene components (5). Extracted plasmids were digested with restriction enzymes and later screened using gel electrophoresis to ensure success of ligation. Subsequent DNA sequencing was then used to confirm proper gene orientation within the plasmid. Upon confirmation of successful gene component design in pGEM, gene components were separately excised and ligated into pHT01. Transformation and ligation were then confirmed as described above, and gene components were ligated into one pHT01 system to achieve the desired plasmid vector.

Transformation of pHT01-GFP-RFP into B. subtilis 1012

In order to induce competence in B. subtilis 1012, a large culture of B. subtilis 1012 in LB media is grown at 37℃ in a shaker for about 16 hours. Then some culture is then mixed with SM1 solution and incubated again until the OD reaches about 0.5 to 06, which is when the microbes have departed log phase are now in stationary phase. At that point, SM2 is added to the shaker, and after 90 more minutes of incubation, the cells should be considered competent.

In order to confirm transformation of B. subtilis 1012, some of the competent cells are combined with the pHT01-GFP-RFP plasmid vector and are incubated for 30 minutes on a rotator. Then LB broth is added to the solution and incubated for another 30 minutes. Then the solution is used for 5 separate plates: 1 TSA plate, 2 TSA + Cat plates, and 2 TSA + Cat + ITPG plates. One plate additional was used as a control, with untransformed B. subtilis 1012 on TSA.

Results

Figure 1: Final plasmid construct of pHT01-GFP-RFP shuttle vector with GFP under lac expression and RFP under Plia1 expression.

Plasmid sequence confirmed with DNA sequencing and realigned with plasmid shuttle vector sequence. BioBrick scar sites labeled for part insertion confirmation. The back bone pHT01 plasmid has the lac operon, ampicillin resistance gene (for E. coli), chloramphenicol resistance gene (for B. subtilis), and lacI gene.

Figure 2: Gel electrophoresis products of ligated products of antibiotic-inducible reporter system. (a) Gel product of Plia1 promoter. (b) Gel product of pGEM-RBS-RFP-TT ligation product. (c) Gel product of Plia1-RBS-RFP-TT-pGEM ligation product. (d) Gel product for template and GFP-TT ligation. (e) Gel product of digested pHT01 with BamH1 And AatII enzymes. (f) 2 log ladder (.1-10Kb).

The gel electrophoresis images obtained confirm ligation of the individual gene components. Figure 2c confirms ligation of the entire RFP construct from the pGEM plasmid, while figure 2d confirms the entire GFP construct assembly in pGEM. Figure 2e provides evidence that BamHI and AatII enzymes were functional and could be used to assemble the gene components in pHT01. Confirmation of insertion of the GFP and RFP gene constructs into pHT01 vector isn’t shown.

Figure 3. pHT01-GFP-RFP Transformed E. coli DH10B plates in a 25 grid format. (a) TSA plate with E. coli DH10B cells transformed with the pHT01-GFP-RFP vector via electroporation. (b) TSA + Ampicillin plate with E. coli DH10B cells transformed with the pHT01-GFP-RFP vector via electroporation. (c)  TSA + Chloramphenicol plate with E. coli DH10B cells transformed with the pHT01-GFP-RFP vector via electroporation.

Non-selective plates (not shown) were initially streaked to isolate the individual 25 colonies for each of the 25 grids utilizing the quadrant scoring method. The TSA plate and the TSA + Amp plates both show clear growth on most grids. The TSA + CAT plate shows no colony growth in any grids.

Figure 4: pHT01-GFP-RFP Transformed B. subtilis 1012 plates in a 25 grid format and GFP fluorescence comparison. (a) TSA plate with B. subtilis 1012 cells transformed with the pHT01-GFP-RFP vector via natural competence, colonies 1-25 scored. (b) TSA + Ampicillin plate with B. subtilis 1012 cells transformed with the pHT01-GFP-RFP vector via natural competence, colonies 1-25 scored. (c)  TSA + Chloramphenicol plate with B. subtilis 1012 cells transformed with the pHT01-GFP-RFP vector via natural competence, colonies 1-25 scored. (d)  TSA + Chloramphenicol + IPTG plate with B. subtilis 1012 cells transformed with the pHT01-GFP-RFP vector via natural competence, colonies 1-25 scored. (e) Untransformed control B. subtilis 1012 TSA plate (left) and pHT01-GFP-RFP transformed B. subtilis 1012 TSA + Chloramphenicol + IPTG plate (right) equally exposed with UV light, colonies 1-25 scored on transformed plate. (f) TSA plate with B. subtilis 1012 cells transformed with the pHT01-GFP-RFP vector via natural competence, colonies 26-50 scored. (g) TSA + Ampicillin plate with B. subtilis 1012 cells transformed with the pHT01-GFP-RFP vector via natural competence, colonies 26-50 scored. (h)  TSA + Chloramphenicol plate with B. subtilis 1012 cells transformed with the pHT01-GFP-RFP vector via natural competence, colonies 26-50 scored. (i)  TSA + Chloramphenicol + IPTG plate with B. subtilis 1012 cells transformed with the pHT01-GFP-RFP vector via natural competence, colonies 26-50 scored. (j) Untransformed control B. subtilis 1012 TSA plate (left) and pHT01-GFP-RFP transformed B. subtilis 1012 TSA + Chloramphenicol + IPTG plate (right) equally exposed with UV light, colonies 26-50 scored on transformed plate.

Non-selective plates (not shown) were initially streaked to isolate the 50 individual colonies for each of the 25 grids utilizing the quadrant scoring method (plates from figures f-j utilized only 1 streak on each grid square [not including final TSA streaks] except for squares 26 and 27 for the TSA plate). The TSA plates, the TSA + Cat plates, and the TSA + Cat + IPTG plates both show clear growth on most grids. The TSA + Amp plates shows no colony growth in any grids. When both the control TSA plates and the transformed B. subtilis 1012 TSA + Cat + IPTG plates are exposed to UV light, the transformed colonies show no significant fluorescence when compared to the untransformed control colonies.

Figure 5. Transformed pHT01-GFP-RFP E. coli DH10B and B. subtilis 1012 broth cultures and control broth cultures fluorescence comparison in UV light, in both glass and plastic tubes. (a) 4 tubes filled with TSB-Amp-IPTG broth cultures, including 2 uninoculated broth tubes (left most and right most tubes) and 2 inoculated with transformed  E. coli tubes (middle two tubes). (b)  4 tubes filled with TSB-Cat-IPTG broth cultures, including 2 uninoculated broth tubes (left most and right most tubes) and 2 inoculated with transformed  B. subtilis tubes (middle two tubes).

Broths were inoculated from previously transformed cells and incubated for 24 hours. The left most tubes are made of glass and the right most tubes are plastic centrifuge tubes, to confirm tube material would not negatively affect the passage of light through the tubes. When both the control broths and the transformed cell inoculated broths are exposed to UV light, the inoculated broth tubes for both E. coli and B. subtilis, in both plastic and glass tubes, show no significant fluorescence when compared to the uninoculated control broth tubes.

Conclusions

In Figure 1, the final plasmid construct is shown and confirmed to have the assembled insert for the antibiotic-inducible reporter system via sequencing of the final constructed plasmid and alignment to the reference sequence with the resulting fragments. In addition, each of the ligated products used in prefix and suffix insertions were confirmed to be present in the gel products as expected. Therefore, if can be safe to ascertain that, since all of the gels confirmed the ligation product constructs were present and the sequencing of the final plasmid shuttle vector confirmed the insertions were successful, the genotypic construct is validated. However, it is important to note that the GFP-TT insert did not include a RBS, which could be a reason phenotypic validation is not yet complete.

In Figure 3, the phenotypic expression is supported in the experiment, with E. coli lacking resistance to chloramphenicol but expressing resistance to ampicillin, which is the direct result of uptake of the constructed plasmid vector. Figure 4 shows very similar results, with B. subtilis lacking resistance for ampicillin as the gene could only be expressed in E. coli, but demonstrating resistance to chloramphenicol. However, when it seemed that IPTG did not enable B. subtilis to glow via expression of the GFP gene which should be controlled by the lac operon, another comparison was made in Figure 4 E and J, directly to a untransformed control group. It was determined that there was no significant fluorescence on the plates in comparison to the control and as such, another comparison experiment was performed in Figure 5. To answer whether it was a question of whether B. subtilis didn’t express GFP as brightly as E. coli or if the microbes only expressed GFP significantly in liquid media, the microbes were grown in media with IPTG and their resistant antibiotic which should enable GFP expression when exposed to UV light. They were also put in two separate containers to be exposed to UV light so that UV absorbance would be less of an issue. However, when compared to inoculated broth media of the same composition, there was no significant fluorescence from either E. coli or B. subtilis. When observed under fluorescence microscopy, neither of the individual cells visually expressed GFP as well.

One of the likely reasons that the GFP gene was not expressed could simply be the lack of an RBS site on the construct. Without that site, the gene would be unable to be expressed in any way. Another reason could be that the GFP gene was misaligned with a RBS, which means it could be subject to an early stop codon or make the entire protein not fluorescent. Another cause could be that the final construct did not have the final antibiotic-inducible reporter system, however, that was proven to exist via both sequencing and gel electrophoresis and could not be the issue. Future research will focus on phenotypic validation to accommodate the genotypic validation.

Research is to be continued on this project, as there are still so many factors to determine before the construct can be utilized as a tool for antibiotic penetration analyzation. Vancomycin sensitivity should be determined in the transformed B. subtilis 1012, as once it is confirmed by spectrometry analysis of RFP expression, the RFP expression can be used as a measure for the depth of penetration. Laser scanning confocal microscopy (LSCM) could be utilized as a method to three dimensionally map the antibiotic penetrance through the layers of the biofilm to help determine whether separate drugs can pierce separate layers.

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