*4.1. General*

#### 4.1.1. Screened Compounds

Fifteen AKGs previously synthesized in our laboratory in enantiomerically pure forms, such as the naturally occurring (Figure 1), were included in this study [42]: (2*S*)-3-*O*-hexyl-1,2-propanediol **1,** (2*S*)-3-*O*-octyl-1,2-propanediol **2**, (2*S*)-3-*O*-decyl-1,2-propanediol **3**, (2*S*)- 3-*O*-dodecyl-1,2-propanediol **4**, (2*S*)-3-*O*-tetradecyl-1,2-propanediol **5**, (2*S*)-3-*O*-hexadecyl-1,2-propanediol **6**, (2*S*)-3-*O*-(*cis*-9'-hexadecenyl)-1,2-propanediol **7,** (2*S*)-3-*O*-octadecyl-1,2-propanediol **8**, (2*S*)-3-*O*-(*cis*-9'-octadecenyl)-1,2-propanediol **9,** (2*S*)-3-*O*-(*cis*,*cis*-9',12' octadecadienyl)-1,2-propanediol **10**, (2*S*)-3-*O*-(*cis*,*cis*,*cis*-9',12',15'-octadecatrienyl)-1,2 propanediol **11**, (2*S*)-3-*O*-(eicosyl)-1,2-propanediol **12**, (2*S*)-3-*O*-(*cis*-11'-eicosenyl)-1,2 propanediol **13**, (2*S*)-3-*O*-(docosyl)-1,2-propanediol **14** and (2*S*)-3-*O*-(*cis*-13'-docosenyl)-1,2 propanediol **15**.

#### 4.1.2. Bacterial Strains and Culture Conditions

The following bacterial strains were used during our antibacterial, antibiofilm and QSI experiments. The Gram-negative clinical isolates included *Klebsiella pneumoniae* 792, *Enterobacter cloacae* 520, *Escherichia coli* 667, *Pseudomonas aeruginosa* 740 and *Proteus mirabilis* 26. The Gram-negative reference strain *Chromobacterium violaceum* 12472 was donated by Prof. Catalina Arevalo from the Universidad Nacional de Colombia. The Gram-positive clinical isolates include *Enterococcus faecalis* 12 and *Enterococcus gallinarum* 662 and reference strains *Staphylococcus epidermidis* ATCC 12228 and *Staphylococcus aureus* ATCC 6538. The clinical strains were previously isolated from hospital patients and provided by the Hospital of Neiva (Huila), Hospital of Tunal, Hospital of Engativá and Universidad del Bosque (Bogotá) [41] and together with the other reference strains, they were obtained from our microbiology laboratory collection [41,42].

The strains were grown for 24 h at 37 ◦C in yeast extract malt extract dextrose agar (YMD, Merck, Darmstadt, Germany) and the inoculum was prepared in Mueller-Hinton broth (MHB, Merck), supplemented with glycerol (15% *w*/*v*) and kept at −20 ◦C until use. In addition, *C. violaceum* 12472 was freshly cultured for 24 h at 22 ◦C in lysogeny broth (LB, Merck) supplemented with kanamycin 100 µg/mL (Merck) before use.

#### *4.2. Determination of the Minimum Inhibitory Concentration of Screened Compounds*

The MIC of AKGs was determined in a microbroth dilution assay according to the Clinical and Laboratory Standards Institute guidelines with slight modifications [42]. Briefly, the inoculum (100 <sup>µ</sup>L, 1 <sup>×</sup> <sup>10</sup><sup>4</sup> CFU/mL) was mixed with AKG dissolved in 3.6% dimethyl sulfoxide (DMSO, Merck) solution in water to final concentrations of 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 or 0.49 µg/mL in MHB, with up to 200 µL added to each well. All experiments were conducted using a maximum of 0.9% (*v*/*v*) DMSO in medium. The plates were incubated at 37 ◦C for 24 h under aerobic conditions, while *C. violaceum* 12472 was incubated at 22 ◦C. Ciprofloxacin (CPX, Merck) was used as the standard antibacterial agent (stock concentrations of 64 µg/mL). MIC was defined as the lowest concentration of AKGs to completely inhibit bacterial growth, which was indicated by a lack of visual turbidity. An inoculated well with solvent in culture medium (positive control) and a well containing only medium (negative control) were included. All experiments were performed in triplicate.

#### *4.3. Effectiveness of Alkylglycerols on the Inhibition of Biofilm Formation*

The ability of AKGs to prevent biofilm formation of bacterial strains included in this study was evaluated on 96-well polystyrene microplates (Techno Plastic Products, Trasadingen, Switzerland) according to the method described by Stepanovi´c et al. [64], with some modifications [42]. Briefly, 100 µL of the bacterial strain grown in MHB medium supplemented with 1% glucose was inoculated in each well to 10<sup>5</sup> CFU/mL in the presence of 100 µL subinhibitory concentrations of each AKG dissolved in aqueous solution of DMSO, equivalent to 0.50, 0.25 and 0.12 of MIC or of the highest concentration assayed during the MIC evaluation (250 µg/mL). The DMSO concentrations were not toxic to strains assayed and did not affect biofilm formation (data not shown). After incubation for 48 h at 37 ◦C, the wells were washed three times with 200 µL of water to remove planktonic bacteria and dried. The remaining bacteria that adhered to the surface of the wells were fixed with 200 µL of methanol for 15 min. Then, the wells were emptied, air dried and subsequently stained with 200 µL of 2% crystal violet solution (Merck) for 5 min, followed by washing with water, drying and solubilizing the stain using 200 µL of 33% acetic acid for 10 min. The absorbance of each well was measured at 570 nm on a microplate reader (xMark Bio-Rad, Hercules, CA, USA). Additionally, CPX was subject to the same essay. The values are expressed as the percent biofilm inhibited in comparison to the untreated control biofilm (positive control). All tests were performed in triplicate.

#### *4.4. Screening for Quorum Sensing Inhibition Activity*

The QSI ability of the AKGs was evaluated in vitro in 96-well microtiter plates by a *C. violaceum* ATCC 12472 biosensor bioassay [4], as previously described with some modifications [41]. Briefly, preinoculum cultured in LB supplemented with kanamycin was adjusted to 1 <sup>×</sup> <sup>10</sup><sup>9</sup> CFU/mL at 600 nm. Then, 100 <sup>µ</sup>L was added to each well, followed by 100 µL of AKGs dissolved in DMSO aqueous solution at subinhibitory concentrations of each compound or of the highest MIC assessed (250 µg/mL) and the plate was incubated for 48 h at 22 ◦C. Thus, the QSI activity was evaluated as the minimum quantity in µg/mL of sample required to inhibit violacein pigment and it was established by the appearance of a colorless and opaque well and did not have an effect on bacterial growth. The sample solvent that had no antibacterial activity served as the negative control and *C. violaceum* 12472 grown in the presence of the same amount of solvent without AKG was used as the positive control. To determine the effect of the presence of each AKG on the growth of the biosensor, a colony count was performed in each assay. All experiments were carried out with three replicates.

#### *4.5. Statistical Analysis*

All antibiofilm experiments were performed in triplicate and the results were expressed as the means ± standard deviation and calculated using statistical analyses of random uncertainties and rejection of data [65]. MIC and QSI data are representative of three independent experiments and the minimum concentration is expressed as the mean value.

#### **5. Conclusions**

AKGs are known bioactive compounds with weak to moderate antibacterial activity. Here, fifteen natural enantiopure AKGs were tested to determine their effect on biofilm development by seven clinical bacterial isolates and two reference strains as well their effect on the QSI activity in *C. violaceum* ATCC 12472 by microtiter plate assays. Ciprofloxacin as the standard antibiotic underwent the same antibiofilm assay. At subinhibitory concentrations, the highest biofilm inhibition rates (%) exhibited for all AKGs were influenced in a concentration-dependent manner and each AKG acted individually against the bacterial isolates, reaching rates up to 97.2%. In addition, AKGs displayed minimum QS inhibitory concentrations at different levels but did not affect the growth of *C. violaceum*. (2*S*)-3-*O*-(*cis*-13'-docosenyl)-1,2-propanediol **15** was the most effective AKG against QSI (20 µM), while (2*S*)-3-*O*-(*cis*-9'-hexadecenyl)-1,2-propanediol **7** was the least active (795 µM). Additional to the novelty of antibiofilm data, the results showed for the first time the QSI activity of this natural AKG series, which indeed, suggests that AKGs constitute a class of promising candidates for further studies on preventing antimicrobial resistance.

**Author Contributions:** K.E.C.D. performed the microbiological studies and interpreted the results. E.S.G. contributed to the experimental work supervision, data analysis and manuscript revision. B.D.M.M. reviewed the full paper. H.M.W. conducted the project supervision and manuscript preparation. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Universidad Nacional de Colombia, Vicerrectoría de Investigación, project number 46978.

**Acknowledgments:** The authors gratefully acknowledge financial support from the Universidad Nacional de Colombia. The authors express their gratitude to Catalina Arevalo Ferro from Universidad Nacional de Colombia, Grupo de Comunicación y Comunidades Bacterianas, Departamento de Biología, for her great assistance.

**Conflicts of Interest:** The authors declare that they had no conflict of interest.

## **References**

