**1. Introduction**

Antibiotics were discovered in the early 20th century and have been widely used in the treatment of bacterial infections. However, the indiscriminate and excessive usage of antibiotics has led to the development of antimicrobial resistance, which has steadily decreased the effectiveness of current antibacterial therapies [1,2]. Resistance mechanisms occur in almost all clinical isolates of bacteria and recurrent infections caused by persistent bacteria hamper the successful treatment of infections [3]. This situation highlights the urgency of identifying new therapeutic candidates and less toxic treatment targets. Consequently, considerable research has focused on developing novel strategies to control bacterial diseases [4]. Infectious diseases cause the death of more than 16 million people annually and at least 65% of these cases are linked to bacterial communities that proliferate by forming biofilms [5,6]. Biofilms consist of microorganisms that are attached to

**Citation:** Chaverra Daza, K.E.; Silva Gómez, E.; Moreno Murillo, B.D.; Mayorga Wandurraga, H. Natural and Enantiopure Alkylglycerols as Antibiofilms Against Clinical Bacterial Isolates and Quorum Sensing Inhibitors of *Chromobacterium violaceum* ATCC 12472. *Antibiotics* **2021**, *10*, 430. https://doi.org/ 10.3390/antibiotics10040430

Academic Editors: Luís Melo and Andreia Azevedo

Received: 10 February 2021 Accepted: 2 April 2021 Published: 13 April 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

a substratum and embedded in a matrix of extracellular polymers that protects the cells from harsh environmental conditions, including disinfectant treatments and antibiotics [7]. Bacterial biofilms are developed by a cell communication process called quorum sensing (QS), through which Gram-negative and Gram-positive bacteria synchronize their behaviors in a cell density-dependent mode by a series of signal molecules termed autoinducers, thereby mediating the production and secretion of virulence traits. In general, QS is a regulatory mechanism that promotes the establishment of infection, expression of virulence factors, formation of biofilm and development of resistance [8,9]. Biofilms are involved in many clinical infections and during biofilm infection, simultaneous activation of both innate and acquired host immune responses may occur; however, these responses do not eliminate the biofilm pathogen but rather accelerate collateral tissue damage [10]. A strategy for controlling microbial infections involves the use of agents that inhibit bacterial pathogenicity (e.g., production of virulence factors and biofilm formation) rather than targeting growth-dependent mechanisms that will inevitably lead to the development of microbial resistance [11,12]. QS inhibitors do not kill bacteria or inhibit bacterial growth; rather, they quench QS-regulated pathogenic behaviors, which inhibits bacterial pathogenesis and does not easily induce antibiotic-resistant mutations [13,14]. The communication of individual cells is essential for the formation of biofilms; therefore, blocking this QS process is an important goal for the control of biofilm infections and represents a new method of combating antimicrobial resistance [15].

Efforts have been made to disrupt biofilms by inhibiting the QS system and many natural and synthetic molecules exhibiting quorum sensing inhibition (QSI) have been identified with potential therapeutic approaches [2]. However, natural products continue to be a prolific source of drugs [13,16] and the rich biodiversity in the marine environment suggests that it represents an enormous resource of novel therapeutic candidates with antibiofilm and QSI activities [17,18]. Marine sources, such as shark liver oil, contain high levels of alkylglycerols (AKGs), which are minor constituents of bacteria, protozoa, fungi, higher plants, animals and humans [19,20]. Natural AKGs called 1-*O*-alkyl-sn-glycerols are structurally glycerol ether lipids that occur as a mixture of alkyl chains of varying lengths and unsaturation levels and as pure enantiomers with an *S* configuration at the asymmetric carbon [21,22]. AKGs generally occur in their diacylated form as 1-*O*-alkyl-2,3-diacyl-snglycerols and they are also natural precursors of their derivatives ether phospholipids, which participate in the structure and function of cell membranes [20,23]. AKGs display a broad range of beneficial effects on human health and can be used to treat gastric ulcers and colon inflammation; stimulate hematopoiesis, immunological defense and vaccination efficiency; reduce tumor growth, metastasis, radiotherapy side effects, obesity and oxidative stress; increase therapeutic molecule permeability through the hematoencephalic barrier and sperm motility; and regulate cell differentiation and neuropathic pain [19,20,23–28]. Their activity spectrum also includes antifungal [29] and antibacterial properties [30–35].

In preliminary works to identify bioactive compounds from marine invertebrates [36], extracts from two soft coral *Eunicea* species and an AKG purified from them, which is known as batyl alcohol **8**, showed antibiofilm capacity against bacteria isolated from marine fouled surfaces [37,38]. Two more AKGs demonstrated growth inhibition of marine biofilm-forming bacteria, which are regarded as potential antifouling compounds [39,40]. In addition, a mixture of AKGs from *Eunicea* together with the AKG (2*S*)-3-*O*-dodecyl-1,2-propanediol **4** and (2*S*)-3-*O*-tetradecyl-1,2-propanediol **5** also showed QSI activity in *C. violaceum* ATCC 31532 [41]. Fifteen natural enantiopure AKGs were synthesized and they depicted promising ability against two biofilm-forming bacteria, clinical isolate *Staphylococcus aureus* 91 and *Pseudomonas aeruginosa* ATCC 15442 [42]. Thus, in the current study, we selected this natural AKG series to evaluate their antibiofilm activity against other clinical bacterial isolates, other reference strains and assess their QSI ability using *C. violaceum* ATCC 12472.

## **2. Results**

#### *2.1. Structures of Natural and Enantiopure Alkylglycerols*

The AKGs included in this study, **1–6**, **8**, **12** and **14**, are of saturated aliphatic chains, while AKGs **7**, **9**, **13** and **15** are mono-unsaturated, **10** is di-unsaturated and **11** is a triunsaturated AKG. They have a chain from 6 to 22 carbons atoms and between 176 to 400 amu (Figure 1).

**Figure 1.** Structures of alkylglycerols used in this study.

#### *2.2. Minimal Inhibitory Concentration (MIC) of Alkylglycerols*

An initial in vitro screening was performed to evaluate the MIC of all AKGs (Figure 1) against the bacterial strains included in this study and the results are summarized in Table 1.

**Table 1.** Minimal inhibitory concentration values (µM) of the examined alkylglycerols.


<sup>a</sup> Alkylglycerols **1**, **8**, **12**, **13**, **14** and **15** had a MIC values >1418, >726, >671, >675, >624 and >627 µM respectively for all tested bacterial strains. All experiments were performed with a maximum of 0.9% (*v*/*v*) DMSO in MHB medium and it did not interfere with bacterial growth. <sup>b</sup> CPX = ciprofloxacin. CPX was the antibacterial agent used as the standard. Values are the means of triplicate determinations.

#### *2.3. Alkylglycerols Ability to Inhibit of Biofilm Formation*

The clinical bacterial isolates and the reference strains were selected because of their biofilm-forming ability evidenced in this study. All examined AKGs were able to inhibit biofilm formation of most clinical isolates and reference strains and showed different capacities against each microorganism assayed. However, the inhibition intensity varied depending on the compound concentration (Figure 2a,b). The highest values of the biofilm inhibitory percentage were observed at AKGs concentrations corresponding to 0.50 MIC. Remarkable biofilm inhibition ratios of several AKGs above 50% were found for *E. cloacae* 250, *P. aeruginosa* 740 and *S. aureus* ATCC 6538, while a low response below 27% was found

for *S. epidermidis* ATCC 12228. *K. pneumoniae* 792 and *S. aureus* ATCC 6538 were the only strains active against all AKGs.

8

**Figure 2.** (**a**) Biofilm inhibition represented as percentages of enantiopure alkylglycerols against clinical bacterial isolates: *K. pneumoniae* 792, *E. cloacae* 250, *E. coli* 667 and *P. aeruginosa* 740 at different subinhibitory concentrations. (**b**) Biofilm inhibition represented as percentages of enantiopure alkylglycerols against clinical bacterial isolates: *P. mirabilis* 26, *E. faecalis* 12, *E. gallinarum* 662 and reference strains *S. epidermidis* ATCC 12228 and *S. aureus* ATCC 6538 at different subinhibitory concentrations. CPX was subject to the same assays. The results are expressed as the mean of three independent experiments and error bars represent the standard deviation. Biofilm formation was significantly reduced compared to untreated control biofilms of each of these bacteria at the tested concentrations of the compounds.

> AKG **9** showed a reduction in biofilm formation of all strains exhibiting the best antibiofilm activity and the highest inhibition ratios were observed for the Gram-negative clinical isolates, *E. cloacae* 250 (97.2%), *E. coli* 667 (82.6%) at 0.50 MIC and *P. aeruginosa* 740 (63.7%) at 0.25 MIC. AKG **11** showed great biofilm reduction for *E. cloacae* 250 (81.6%) and *P. aeruginosa* 740 (78.2%) at 0.50 MIC. The other AKGs were less effective.

> *K. pneumoniae* 792 was inhibited by AKG **3** up to 62.5% and AKGs **7**, **8** and **10** by close to 46.5% at 0.50 MIC. In *E. cloacae* 250 in addition to AKGs **9** and **11**, AKGs **1**, **3**, **4** and **14** showed effectiveness and reached inhibition rates of approximately 76% at 0.50 MIC. Other compounds, such as **1** and **6**, decreased biofilm formation by *E. coli* 667 by up to 56.9% and AKGs **2**, **3**, **8**, **12**, **14** and **15** reached almost 45% at 0.50 MIC.

> In *P. aeruginosa* 740, biofilm inhibition was also shown by AKGs **3** and **5** at up to 72.6% at 0.50 MIC and a lower response was observed with other compounds. At 0.50 MIC compounds **10** (47.3%), **3** and **8** were the most active against *P. mirabilis* 26. In the case of Gram-positive strains, *E. faecalis* 12 was most susceptible to AKGs **10**, **5** and **1** and an inhibition rate of 83.9% was found for **10**. In addition, for *E. gallinarum* 662 moderate inhibition by AKGs **13** (52.9%), **10** (52.4%) and **8** (40.7%) was observed at 0.50 MIC. For the reference strain *S. epidermidis* ATCC 12228, compounds **7** (27.0%) and **4** (20.3%) at 0.50 MIC exhibited low activity, whereas for *S. aureus* ATCC 6538 showed inhibition up to 70.6% for

AKG **8** at 0.05 MIC and up to 66.9% for compounds **12** and **14**. In comparison, CPX, which is the antibacterial agent and subjected to same treatment, only inhibited biofilm formation of *E. faecalis* 12 up to 90.7%, *E. coli* 667 up to 37.2% and *K. pneumoniae* 792 up to 28.2% at 0.50 MIC.

#### *2.4. Quorum Sensing Inhibition Activity of Natural Alkylglycerols*

To determine the effect on the QSI activity using *C. violaceum* ATCC 12472 without interference from the antibacterial activity of the tested compounds, the AKGs were analyzed at subinhibitory concentrations. All AKGs were capable of inhibiting violacein production with varying degrees of action depending on the aliphatic chains on their structures (Table 2). Notably, the highest QSI activity was exhibited by AKG **15**, with a minimum concentration of 20 µM. To a lesser extent, compounds **3**, **4**, **5** and **10** showed relevant activity and reached a reduction in QS up to an inhibitory concentration of 135, 120, 109 and 92 µM respectivelly, followed by AKG **6** at a minimum concentration of 197 µM. In regard to the majority of AKGs, the potential of QSI was lower for **1**, **2**, **8**, **9**, **11**, **12**, **13** and **14** at a concentration varied from 312 to 709 µM, while *C. violaceum* ATCC 12472 was less sensitive for the inhibition of QS to AKG **7** (795 µM).

**Table 2.** Minimum quorum sensing inhibitory concentration (µM) of alkylglycerols against *C. violaceum* ATCC 12472.


<sup>a</sup> Minimum quantity µM of compound required to inhibit violacein pigment. Values represent the means from three independent experiments.

The bacterial viability of *C. violaceum* ATCC 12472 was also assessed for both the positive control and AKG-treated samples and the results in terms of colony-forming units/mL (CFU/mL) did not show significant differences between the groups. Therefore, the reduction in violacein production was attributed to the effect of the AKGs.

#### **3. Discussion**

Bacteria have developed multiple mechanisms to adapt to changes in the environment, especially the widespread use of antibiotics and these mechanics allow them to avoid adverse conditions and maintain their pathogenesis. One of the mechanisms is QS, a cell-to-cell communication method that depends on the cell density to control collective behavior [1,9]. With the regulation of QS, bacteria can form virulence factors such as toxins, motility, enzymes, proteins and biofilm development to survive in disadvantageous circumstances [2,8]. Biofilms are another critical reason for microbial resistance and they consist of bacteria embedded in a self-produced extracellular matrix of polysaccharides, fibrins, lipids, proteins and DNA that can reduce the effect of antibiotics [7,10,43]. Consequently, there is an urgent search for new candidates and less toxic treatments to control pathogenic resistant bacteria [3,4].

To identify the inhibitors of biofilm formation from bioactive extracts of two Caribbean soft corals of *Eunicea* genus, we isolated an AKG from these species called batyl alcohol **8**, which was the most active inhibitor [37,38]. This class of natural AKGs that had not been previously studied for antibiofilm activity was selected and great variety of their compounds were evaluated. AKGs are bioactive compounds found in low concentrations in marine sources, such as fishes, sponges and corals and at trace levels in plants and mammal, including humans [19,20,23–28]. In nature, they are enantiomerically pure constituents with configuration *S* at the stereogenic center [21,22], which motivated us to obtain several of their naturally occurring structures by enantiomeric synthesis [41].

In this study, fifteen previously synthesized enantiopure AKGs [42] were tested to determine their effect on the biofilm formation of seven clinical bacterial isolates and two reference strains and evaluate their QSI ability using *C. violaceum* ATCC 12472 at

subinhibitory concentrations. These AKGs displayed weak antibacterial activity compared with CPX (MIC from 0.09 to 48.2 µM) against all strains at MIC values in the range of 27 to >1418 µM in a microtiter plate assay (Table 1, Figure 1). In this way, only saturated AKGs with an intermediate length of their aliphatic chain such as compounds **3**, **4** and **5** showed in vitro moderate antibacterial activity toward some Gram-positive strains, of them AKGs **4** and **5** were the most active against *S. aureus* ATCC 6538 (MIC of 30 and 27 µM). With less effectiveness against the most bacterial strains resulted the AKGs **2**, **6**, **7**, **9**, **10** and **11** between 99 and 1224 µM, while the AKGs, **1**, **8**, including **12**, **13**, **14** and **15**, the latter which have 20 or more carbon atoms at the aliphatic chain were much less active against all bacterial strains with a MIC from 624 to >1418 µM. Furthermore, the AKGs showed low bacteriostatic effects on the clinical isolates and reference strains because evaluation of the viability by seeding in fresh culture medium led to the recovery of each bacterial species (data not shown). The above is in accordance with other authors and our preliminary works [41,42]. In a previous study, the authors showed that racemic **4** (*rac*-**4)**, known as *rac*-1-*O*-dodecylglycerol, inhibited the growth of *Enterococcus faecium* and *Streptococcus mutans* primarily by stimulating autolysin activity and interfering with cell wall synthesis [30–32]. Racemic-**4** inhibits *S. aureus* and toxic shock syndrome toxin 1 production, although the mechanisms of action have not been characterized [35] and produces growth inhibition of members of two yeast genera, *Candida* and *Cryptococcus* [29]. AKGs **1** and **2** are able to prevent the growth of *Chlamydia trachomatis* [33], while AKG **8** show antimicrobial activity toward some marine bacterial species [34,38].

We found that all AKGs at subinhibitory amounts of 0.50, 0.25 and 0.12 MICs were capable of significantly inhibiting biofilm formation by up to 97.2% in both Gram-negative and Gram-positive clinical isolates and in the reference strains, although they had different efficiencies against each strain. (Figure 2a,b). This compound concentration-dependent antibiofilm activity is also dependent on the bacterial susceptibility of each strain to the respective structure. Consistent with the abovementioned findings, we previously observed that most of these AKGs reduced biofilm formation with different capacities against other two strains and the rates were 23.5–99.8% for the clinical isolate *S. aureus* 91 and 14.1–64.0% for *P. aeruginosa* ATCC 15442 [42]. Our additional results against these nine bacterial species considerably broaden the understanding of the antibiofilm abilities of AKGs.

The QSI activity of the AKGs in this work was explored by employing *C. violaceum* ATCC 12472 as a biological model and subinhibitory concentrations. All enantiopure AKGs significantly inhibited violacein production at minimum QS inhibitory concentrations in the range of 20 to 795 µM (Table 2). Surprisingly, among the tested compounds, the most potent QSI activity was noted for the highest molecular weight and unsaturated AKG, namely, (2*S*)-3-*O*-(*cis*-13'-docosenyl)-1,2-propanediol **15**, with a minimum concentration of 20 µM, while (2*S*)-3-*O*-(*cis*-9'-hexadecenyl)-1,2-propanediol **7** turned out to have the lowest QSI capacity (795 µM). In addition, AKGs **3**, **4**, **5** and **10** reached a reduction in QS at a minimum concentration of 135, 120, 109 and 92 µM, respectively. Neither AKG affected the growth of *C. violaceum* ATCC 12472. A compound that can to interfere with QS without affecting cell growth is considered a promising inhibitor [11,13,14]. The QSI effects reported in the current study support our preliminary results, where the disc diffusion assay in other strain, *C. violaceum* ATCC 31532, showed that AKG **4** exhibited the same minimum inhibition halo at 20 µg/disc as kojic acid, which is a positive control used as known inhibitor of QS systems, moreover, AKG **5** produced a minimum inhibitory halo at 50 µg/disc [41].

Although it was not possible to deduce a common relationship pattern between the structure and antibiofilm activity or between the structure and QSI activity, each strain positively responded with the aliphatic chain nature of the corresponding AKG. Some related natural molecules, such as fatty acids, are antibacterial agents at high concentrations and have great potential to attenuate microbial biofilm formation and virulence at low concentrations by modulating the QS systems without generating drug resistance [44]. Many studies have been performed to discover natural compounds that could combat

pathogenic bacteria. Many natural products with antibiofilm activity have been identified from plants, microbes and marine life and they include various compounds classes, such as alkaloids, fatty acids, organosulfurs, cyclic compounds, phenolics, steroids, terpenoids and other aliphatic compounds. These compounds include: elligic acid glycosides, hamamelitannin, carolacton, skyllamycins, promysalin, phenazines, bromoageliferin, flustramine C, meridianin D and brominated furanones [18,45–50]. The most significant groups of plant-derived QS inhibitors belong to terpenes, terpenoids, phenylpropanoids, acid derivatives, diarylheptanoids, coumarins, flavonoids and tannins [46,51,52]. Microorganisms and marine organisms produce a range of molecules that showing remarkable QSI activity. These molecules belong to organic compounds classes: derived alkaloids, terpenes, peptides, polyketides, derived carbohydrates, sesterterpenes, halogenated furanones, cembranoids, halogenated alkaloids and phenolics and they include compounds, such as kojic acid, phenethylamides, aculenes, meleagrin, malyngamides, manoalides, hymenialdisin, betonicines, floridoside and cembrane diterpenoids [18,53,54].

Among the Gram-negative bacteria susceptible to AKGs in the present study, *K. pneumoniae* is a pathogenic bacterium that shows high rates of antibiotic resistance through biofilm formation [55]; *E. cloacae* is an emerging pathogen responsible for various diseases, including respiratory tract infections associated with biofilm formation [56]; *E. coli* pathogenic strains cause problematic biofilm infections and employ a furanosyl borate diester (AI-2)-based QS system to regulate two of its most studied phenotypes, virulence and biofilm formation [57]; another pathogen that has been well studied and is frequently associated with a wide range of severe infections is *P. aeruginosa*, which includes cystic fibrosis pneumonia and chronic obstructive pulmonary disease-related infections. The high level of *P. aeruginosa* pathogenicity is mostly controlled by QS, which regulates genetic expression, with 30% encoding virulence factor production and biofilm formation based on the release and sensing of the signal molecules homoserine lactones [6,8,9,11,57]; *P. mirabilis* is another opportunistic pathogen implicated in various human diseases of the respiratory tract and gastrointestinal tract and such infections are complicated by the unique ability of this bacterium to form crystalline biofilms that protect it from antibiotics and the host immune response [58]; among the most known Gram-negative bacteria, *C. violaceum* is a biosensor strain used for QS research that produces the pigment violacein in response to QS-regulated gene expression and it has been widely used to study the inhibition of QS [4,59]. Among Gram-positive bacteria, *E. faecalis* has become one of the most prevalent multidrug-resistant hospital pathogens and its QS system is closely related to biofilm development and virulence production [60]; *E. gallinarum* is less commonly identified in humans but is responsible for bloodstream, urinary tract and surgical wound infections and is resistant to many antibiotics [61]; the most important virulence factor of *S. epidermidis* is considered to be its ability to form biofilms on implanted biomaterials, these films are difficult to treat because of the increased resistance to both antibiotics and the human immune system [62]; in some cases, *S. aureus* causes a wide range of human infections on skin and soft tissues, as well as life-threatening pneumonia, bacteremia, osteomyelitis and toxic shock syndrome. *S. aureus* uses an autoinducing peptide signal to mediate QS and its pathogenicity depends on its ability to produce biofilms and virulence factors, such as different toxins, that contribute to invasion of the host and bacterial spread, [35,63].

In the current research, the differences in susceptibility among the bacteria tested to each AKG suggested that a large range of these structures with different molecular sizes, unsaturation and polarities have both antibiofilm and QSI activities, which result in much more effectiveness than their respective antibacterial effects. However, the most noteworthy information obtained from the present work revealed for the first time that this variety of natural AKGs are promising QS inhibitors. Since the target of QSI is bacterial virulence factors and bacterial biofilms, not viability, there is less chance that the bacteria will develop resistance to QS inhibitors [5,12,63]. For the above reasons, AKGs could be considered potential candidates to control pathogenic bacterial diseases and thus warrant further experiments involving other model systems to establish their action mechanisms and the extent of their efficacy against antimicrobial resistance.

## **4. Materials and Methods**
