Pitfalls associated with evaluating enzymatic quorum quenching activity: the case of MomL and its effect on Pseudomonas aeruginosa and Acinetobacter baumannii biofilms

Bacterial strains, culture conditions and chemicals

P. aeruginosa PAO1, A. calcoaceticus LMG 10517, A. nosocomialis M2 and A. baumannii LMG 10520, LMG 10531 and AB5075 were cultured on tryptic soy agar (TSA) or in Mueller-Hinton broth (MH) at 37 °C. Escherichia coli BL21(DE3) harboring MomL expression plasmid pET24a(+)-momL-(−SP) (Tang et al., 2015) was cultured on Luria-Bertani (LB) agar supplemented with kanamycin (50 μg/mL) at 37 °C. The AHL biosensor Agrobacterium tumefaciens A136 (pCF218) (pCF372) (Zhu et al., 1998) was maintained on LB agar supplemented with spectinomycin (50 μg/mL) and tetracycline (4.5 μg/mL), and grown in AT minimal medium (Tempé et al., 1977) containing 0.5% (wt/vol) glucose for detecting AHLs in the liquid X-Gal (5-bromo-4-chloro-3-indolyl- β-D-galactopyranoside) assay. 3-OH-C12-HSL was purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO) as stock solution (100 mM). Caenorhabditis elegans N2 (glp-4; sek-1) was propagated under standard conditions, synchronized by hypochlorite bleaching, and cultured on nematode growth medium using E. coli OP50 as a food source (Stiernagle, 1999).

MomL expression and purification

MomL was expressed and purified according to Tang et al. (2015). In brief, protein expression was induced by 0.5 mM IPTG (isopropyl- β-D-thiogalactopyranoside) when E. coli cells in LB reaching an optical density at 600 nm (OD600) of 0.5–0.7. The induction was carried out at 16 °C with moderate shaking (150 rpm) for 12 h. Cells were harvested and sonicated, and the obtained supernatant was loaded on NTA-Ni (Qiagen, Hilden, Germany) columns for purification according to the manufacturer’s instruction. Desalting of the protein solution was accomplished by Amicon Ultra-15 centrifugal filter devices, and the purified MomL was stored at −20 °C in Tris–HCl buffer (50 mM, pH 6.5) with 25% glycerol.

Detection for degradation of 3-OH-C12-HSL

The amount of 3-OH-C12-HSL was quantified using A. tumefaciens A136 liquid X-gal assay and expressed as the normalized β-galactosidase activity as previously described (Tang et al., 2013). The correction factor a and b were obtained and calculated for our experimental conditions, and the final formula to calculate the normalized β-galactosidase activity is $\frac{0.716\times OD492-OD620}{0.205\times OD620-OD492}$. To test the degradation of 3-OH-C12-HSL by MomL, 3-OH-C12-HSL (10 μM) was mixed with MomL in different concentrations (0.05–5 μg/mL) and incubated at 37 °C for 1 h. No MomL was added to the control. Afterwards the residual 3-OH-C12-HSL was quantified by adding 10 μL solution to the A136 biosensor, as described previously (Tang et al., 2013).

Biofilm formation assays

Overnight cultures of P. aeruginosa and Acinetobacter strains in MH broth were diluted to contain approximately 5 × 10⁷ CFU/mL. 90 μL of this diluted bacterial suspension was transferred to the wells of a round-bottomed 96-well microtiter plate. Uninoculated MH broth served as blank control. To test the effect of MomL on biofilms, 10 μL purified enzyme (in different concentrations) was added to the wells, while 10 μL Tris–HCl buffer (50 mM, pH 6.5) with 25% glycerol was added to the control. The plate was incubated at 37 °C for 4 h before the supernatant was removed. The wells were rinsed once with sterile physiological saline (PS) and re-filled with fresh medium (90 μL) and MomL (10 μL). The plate was incubated at 37 °C for an additional 20 h. The biofilm biomass was quantified by crystal violet (CV) staining as described previously (Peeters, Nelis & Coenye, 2008). After rinsing the wells with sterile PS, the biofilm was fixed with 100 μL 99% methanol for 15 min and stained with 100 μL 0.1% CV for 20 min. The excess CV was removed by washing the plates under running tap water and bound CV was released by adding 150 μl of 33% acetic acid. The absorbance was measured at 590 nm.

Biofilm susceptibility assays

After a 24 h-biofilm of P. aeruginosa or A. baumannii strains was formed as described above either in presence of MomL or not, the plate was emptied and biofilm cells were rinsed with sterile PS. Antibiotics were dissolved in PS and 90 μL of these solutions were added to treat the biofilm for another 24 h, either with or without 10 μl MomL. Tobramycin (TOB; 4 μg/mL as final concentration), ciprofloxacin (CIP; 0.5 μg/mL), meropenem (MEM; 16 μg/mL) and colistin (CST; 16 μg/mL) were used to treat the biofilm of P. aeruginosa PAO1; TOB (6 μg/mL), CIP (4 μg/mL), MEM (8 μg/mL) and CST (16 μg/mL) were used to treat the biofilm of the A. baumannii strains. The supernatant was removed and the wells were washed once with sterile PS. To release bacterial cells from biofilm, two cycles of vortex (5 min) and sonication (5 min) were performed, and the number of CFU/biofilm was determined by plating the resulting suspensions on TSA.

Fluorescence microscopy

Biofilms of P. aeruginosa PAO1 or A. baumannii strains were formed in the absence or presence of MomL and treated with antibiotics as described above using a flat-bottomed 96-well microtiter plates. A total of 3 μL SYTO9 and 3 μL propidium iodide were diluted to 1mL in sterile PS, and 100 μL of this staining solution was transferred to each well. The plate was incubated for 15 min at room temperature and fluorescence microscopy was performed with EVOS FL Auto Imaging System (Life Technologies, Carlsbad, CA, USA). The red fluorescent signal was detected with 531/40 nm excitation filter cube and 593/40 nm emission filter cube and the green fluorescent signal was detected with 470/22 nm excitation filter cube and 510/42 nm emission filter cube.

Dual-species biofilm formation

Overnight cultures of P. aeruginosa and A. baumannii strains in MH broth were diluted to contain approximately 5 × 10⁵ CFU/mL and 5 × 10⁷ CFU/mL, respectively, and equal volume of suspensions of P. aeruginosa and A. baumannii were mixed. MomL (200 μg/mL) and tobramycin (6 μg/mL) were added as described above. To quantify CFU in the dual-species biofilm, Pseudomonas Isolation Agar (Difco) and TSA supplemented with 5 μg/mL cefsulodin were used as selective media for P. aeruginosa and A. baumannii respectively.

Biofilm formation in wound model

Artificial dermis composed of hyaluronic acid and collagen was used in our wound model, as described before (Brackman et al., 2016). Each disk of artificial dermis was placed in 24-well microtiter plate. One mL medium containing Bolton Broth, heparinized bovine plasma and freeze-thaw laked horse blood cells was added on and around the dermis. Suspensions (10 μL) of P. aeruginosa or A. baumannii containing 10⁴ bacterial cells were added on the top of dermis. Final concentrations of MomL added were 200 μg/mL and 10 μg/mL for P. aeruginosa and A. baumannii, respectively. Tobramycin (10 μg/mL) was added after 8 h incubation at 37 °C. After 24 h, the infected dermis was washed with 1 mL PS and was transferred into 10 ml PS. Biofilm cells on the dermis were loosen and collected by three cycles of vortex (30 s) and sonication (30 s). The number of CFU/dermis was quantified by standard plating techniques.

C. elegans survival assay

The C. elegans survival assay was carried out as described before with minor modification (Brackman et al., 2011). Synchronized worms (L4 stage) were suspended in medium containing 95% M9 buffer (3 g of KH₂PO₄, 6 g of Na₂HPO₄, 5 g of NaCl, and 1 ml of 1 M MgSO₄⋅7H₂O in 1 l of water) and 5% brain heart infusion broth (Oxoid), and 25 μL of this nematode suspension was transferred to the wells of a 96-well microtiter plate. Overnight culture of A. baumannii was suspended in the assay medium and added in a final concentration of 2.5 × 10⁷ CFU/ml. MomL was added in a final concentration of 10 μg/mL. The plates were incubated at 25 °C for 24 h. The fraction of dead worms was determined by counting the number of dead worms and the total number of worms in each well.

Statistics

The normal distribution of the data was checked by the D’Agostino–Pearson normality test. Normally distributed data were analyzed by one-way ANOVA, and non-normally distributed data were analyzed by the Kruskal–Wallis test or the Mann–Whitney test. All statistical analyses were carried out using GraphPad Prism 6.0.

Degradation of 3-OH-C12-HSL by purified MomL

MomL was produced in E. coli and subsequently successfully purified (Fig. 1). Although MomL had been shown to degrade various AHLs (Tang et al., 2015), its activity on AHLs with hydroxyl substituent at the C3 position was not tested yet. We could demonstrate that MomL, in a concentration of 1 μg/mL or higher, can degrade almost all 3-OH-C12-HSLs (10 μM) under the experimental conditions used in the present study (Fig. 2).

Effect of MomL on biofilm formation by P. aeruginosa and A. baumannii

Following biofilm formation in 96-well microtiter plates and quantification by crystal violet staining, a significant difference was observed between P. aeruginosa PAO1 control biofilms and biofilms grown in the presence of MomL (concentration > 50 μg/mL) (Fig. 3A). When grown with 150 μg/mL MomL, an average decrease of approximately 35% was observed. MomL inhibited A. baumannii LMG 10531 biofilm formation at concentrations as low as 0.1 μg/mL, and the biofilm biomass was reduced by approximately 42% when exposed to 5 μg/mL MomL (Fig. 3B). No further decrease was observed when A. baumannii LMG 10531 biofilms were grown in the presence of higher concentration of MomL.

Effect of MomL on biofilm susceptibility to antibiotics

Application of MomL alone (200 μg/mL for P. aeruginosa PAO1 and 10 μg/mL for A. baumannii LMG 10531) reduced the number of cultivable biofilm cells by approximately 50% in both P. aeruginosa PAO1 and A. baumannii LMG 10531. For P. aeruginosa PAO1, combining CIP or MEM with MomL led to >70% more reduction compared to treatment with CIP or MEM alone (Fig. 4A). For A. baumannii LMG 10531, MomL also increased killing of biofilm cells when antibiotics were used together with MomL (Fig. 4B). In case of TOB, cell number was reduced by 80% when used in combination with MomL compared to TOB alone. Consistent with results obtained by plating, fewer living cells were observed in fluorescence microscope images of biofilms treated with MomL, TOB, or a combination of both, compared to control biofilms (Fig. 5).

Effect of MomL on dual-species biofilm formed by P. aeruginosa and A. baumannii

We also evaluated the effect of MomL on dual-species biofilm formed by P. aeruginosa PAO1 and A. baumannii LMG 10531. We found that P. aeruginosa PAO1 inhibited growth of A. baumannii LMG 10531 in dual-species biofilm, and most A. baumannii LMG 10531 cells were killed by P. aeruginosa PAO1 after 48 h (Fig. 6). When MomL was added, there was a reduction in A. baumannii LMG 10531 cell numbers; however no difference was observed in either total cell numbers or number of surviving P. aeruginosa PAO1 cells (Fig. 6A). MomL in combination of TOB was also tested, but no change in susceptibility to TOB was observed in the dual-species biofilm (Fig. 6B).

Effect of MomL on other Acinetobacter strains

We also tested MomL on four other Acinetobacter strains. However, only A. baumannii LMG 10520 showed reduction in biofilm biomass when treated with MomL at 50 μg/mL (Fig. 7). No significant difference was observed for A. calcoaceticus LMG 10517, A. nosocomialis M2 and A. baumannii AB5075. The effect of MomL on susceptibility of A. baumannii LMG 10520 and A. calcoaceticus LMG 10517 biofilms was also tested. For A. baumannii LMG 10520, significant differences were detected when MomL was added alone or in combination with several antibiotics (Fig. 8). For A. calcoaceticus LMG 10517, no difference was observed between biofilms receiving MomL treatment and biofilms receiving the control treatment, either by plating or fluorescence microscope.

Effect of MomL in a biofilm wound model system and in the C. elegans model

An in vitro wound model was used to mimic the conditions in an infected wound. For both P. aeruginosa PAO1 and A. baumannii LMG 10531, MomL had no effect on biofilm formation in this wound model (Fig. 9).

The C. elegans model was used to further evaluate whether MomL can increase survival of nematodes infected with A. baumannii. However, no significant increase of C. elegans survival was found after treating nematodes infected with A. baumannii LMG 10520 or A. baumannii LMG 10531 with MomL (Fig. 10), although AHL-degrading activity was maintained under these test conditions (Fig. S2).

Conclusion

The results of the present study highlight that there are considerable hurdles to be cleared before QQ enzymes could potentially be used to combat infections. Our data indicate that demonstrating AHL degrading activity in vitro and/or anti-biofilm activity in simple in vitro biofilm model systems is not sufficient to predict an anti-biofilm effect in more complex systems.