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Resistance to Industrial Sanitizers due to the Presence of HorA Morgan Kirkpatrick, Christopher Parker, PhD Texas Wesleyan University, Department of Biology Abstract The mul(drug efflux pump HorA confers resistance to hops in certain beer-‐spoilage organisms. Because of HorA’s similarity to other mul(drug efflux pumps, HorA might also confer resistance to industrial sani(zers. The model organism E. coli DH5α’s lack of Figure 1: Clustal Omega Alignment of HorA protein sequence in Lactobacillus and Pediococcus spp. suscep(bility in a disk diffusion assay suggests a need • Figure 2: Amplifica(on of 16s Disk Diffusion Assay cont. to increase compound volumes. Ubiquitous growth in a nd h orA f rom J ester K ing • Diffused g rowth a round H O m ade a n a ccurate 2 2 a minimum inhibitory concentra(on assay suggests M RS b roth s ample h ad n o zone o f c learing m easurement n ot p ossible. the iso-‐α-‐acids were structurally altered when band at the expected Minimum Inhibitory Concentration extracted. An alignment of horA sequences a mplicon l ength o f 2 158bp. -‐1 to 2-‐12 in LB Broth • Iso-‐α-‐acids w ere d iluted f rom 2 determined that the gene is highly conserved. Thus, and inoculated with E. coli DH5α to find the the nega(ve PCR results on several cultures suggests a minimum inhibitory concentra(on of iso-‐α-‐acids. possible need to adjust the PCR protocol. • E. c oli D H5α g rew u biquitously a cross t he s erial Background Conclusions dilu(on. • Iso-‐α-‐acids in hops preserve beer by disrup(ng the • Disk volumes might be too low on diffusion assay. HorA Alignment and Culture Methods • Iso-‐α-‐acids might be chemically altered. proton mo(ve force (Sakamoto, 2001). • Due to limited access to L. brevis BSO 464, HorA • Sani(zers prevent spoilage from hop-‐resistant • horA is highly conserved in lac(c acid bacteria. amino acid sequences of lac(c acid bacteria were organisms (Palmer 1999). • Either no sample contained horA, or the PCR aligned using Clustal Omega to study conserva(on. • Lactobacillus brevis strain BSO 464 contains a hop-‐ primers or temperatures need to be adjusted. • Lac(c acid bacteria were cultured from a Wyeast resistance gene horA on the plasmid pLb464 Future Directions Lactobacillus and Pediococcus samples and a (Bergsveinson, 2012). • Culture L. brevis BSO 464. Jester King sour beer. • HorA pumps iso-‐α-‐acids out of the cell and shows • Clone horA into E. coli with TOPO TA Kit. • Samples were cultured in MRS broth and 85% similarity to other ABC mul(drug efflux pumps • Determine survivorship with increasing Lager 15% 2X MRS broth. (Sakamoto, 2001). concentra(ons of sani(zers with cloned cells. • MRS+Lager broth cultures were then plated on • Hypothesis: Organisms expressing horA are more • Construct Dose Response Curves, t-‐test IC50 values MRS agar. resistant to industrial sani(zers than those without. Disk Diffusion Assay • E. coli DH5α was chosen as the model organism. • E. coli DH5α were tested for suscep(bility to: Betadine, 10% Bleach, 70% EtOH, H2O2, 100% Isopropanol, and iso-‐α-‐acids (5 IBUs, 10 IBUs, and 20 IBUs) on LB agar. • E. coli DH5α did not demonstrate suscep(bility to Betadine, 10% Bleach, 70% EtOH, 100% Isopropanol, or iso-‐α-‐acids. PCR of Culture Samples • To test for the presence of horA, PCR was performed on cultures from: • Wyeast Pediococcus MRS broth • Jester King MRS broth • 3 colonies from Jester King MRS agar • 16s amplified as posi(ve control • Gel electrophoresis performed. • Primers are expected to amplify 2158bp. References Bergsveinson J, Pihet V, Ziola B. 2012. RT-‐qPCR analysis of puta(ve beer-‐ spoilage gene expression during growth of Lactobacillus brevis BSO 464 and Pediococcus claussenii ATCC BAA-‐344(T) in beer. Appl Microbiol Biotechnol 96(2):461-‐70. Palmer J. 1999. How to Brew: Everything You Need to Know to Brew Beer Right the First Time [Internet]. First Edi(on. Boulder(CO):Brewers Publica(ons; [cited 2014 Nov 20] Available from: hhp://www.realbeer.com/jjpalmer/sitemap.html Sakamoto K, Margolles A, van Veen HW, Konings WN. 2001. Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-‐binding cassehe mul(drug transporter HorA. J Bacteriol
