Engineered lactobacilli display anti-biofilm and growth suppressing activities againstPseudomonas aeruginosa

Abstract

17Biofilms are an emerging target for new therapeutics in the effort to address the continued 18 increase in resistance and tolerance to traditional antimicrobials. In particular, the distinct nature 19 of the biofilm growth state often means that traditional antimicrobials, developed to combat 20 planktonic cells, are ineffective. Biofilm treatments are designed to both reduce pathogen load 21 at an infection site and decrease the development of resistance by rendering the embedded 22 organisms more susceptible to treatment at lower antimicrobial concentrations. In this work, we 23 developed a new antimicrobial treatment modality by characterizing the natural capacity of two 24 lactobacilli, L. plantarum and L. rhamnosus, to inhibit P. aeruginosa growth, biofilm formation, 25 and biofilm viability. We further engineered these lactic acid bacteria (LAB) to secrete enzymes 26 known to degrade P. aeruginosa biofilms and show that our best performing engineered LAB, 27 secreting a pathogen-derived enzyme (PelAhyd), degrades up to 85 % of P. aeruginosa biofilm. 28 29 30 31 . Materials and Methods 97 98 2.1. Bacterial growth and transformation 99All strains used in this study are listed in Table S1. E. coli strains were grown in LB broth and plated 100 on LB agar, unless stated otherwise. Erythromycin and ampicillin were added to E. coli cultures 101 at 200 or 100 µg/mL, respectively. Lactobacilli were grown in De Man, Rogosa and Sharpe (MRS; 102 RPI Corp) broth and plated on MRS agar (1.5 % w/v) plates unless stated otherwise. Erythromycin 103 was added lactobacillus cultures at 5 µg/mL when necessary. All cultures were grown at 37 °C; 104 E. coli and L. plantarum cultures were grown shaking (250 rpm) and LGG was grown statically, 105 unless stated otherwise. E. coli transformation was performed using MES or TSS competent cells. 106 L. plantarum WCFS1 transformation was performed using a method derived from Aukrust and 107 Blom 31 . LGG transformation was performed using the method described in De Keersmaecker et 108 al. 32 . 109 110 2.2. LAB antimicrobial plate assay 111Overnight cultures of LAB were diluted 1000× into 10 mL fresh media and 1 mL aliquots were 112 removed from the culture at the designated times. Following aliquot removal from LAB culture, 113 cells were pelleted at 4000 ×g for 15 min and the resulting supernatant was filtered through 0.22 114 µm PES filter and frozen at ─20 °C until the following day. The following day, overnight cultures 115 of P. aeruginosa were diluted 100× in LB broth and 100 µL was plated on the surface of LB agar. 116Agar wells were excised from the agar plate and 200 µL of fresh lactobacilli culture or filtered 117 culture supernatant was added to each well. Plates were incubated at 37 °C overnight and 118 inhibition was evaluated qualitatively by inhibition of pathogen growth. 119 120 Plasmid construction 121All vectors used and constructed in this study are listed in Table S2. E. coli TG1 was modified by 122 knockout of endA to improve transformation efficiency and plasmid ...