2.2. Biofilm Development throughout Time of S. aureus Strains in the Absence and the Presence of ICs
As a preliminary step to evaluate activity of antimicrobial compounds against biofilm formation, we quantified biofilm mass production by all strains. Thus, the biofilm mass was measured during 40 h of incubation (
Figure 1).
Using this approach, the patterns of biofilm formation and biofilm thickness were evaluated for each strain at every time point. These patterns were characterized by the alternation of the assembly-dissasembly of the biofilm. CA-MRSA strain, SC-01 formed the thickest biofilm as measured at any growth time from 16 h onwards, reaching a biofilm mass corresponding to 8 absorbance units (AU) after 40 h (
Figure 1A).
Figure 1.
Inhibition of biofilm mass production in presence of NIC of carvacrol, citral and (+)-limonene. Biofilm development (expressed as optical absorbance units at 595 nm) at 37 °C of Staphylococcus aureus SC-01 (A); USA300 (B); UAMS-1 (C); or Newman (D) in the absence of antimicrobial compounds (●) or in the presence of the non-inhibitory concentration (NIC) of each compound: 100 µL/L of carvacrol (), 200 µL/L of citral (), or 2000 µL/L of (+)-limonene ().
Figure 1.
Inhibition of biofilm mass production in presence of NIC of carvacrol, citral and (+)-limonene. Biofilm development (expressed as optical absorbance units at 595 nm) at 37 °C of Staphylococcus aureus SC-01 (A); USA300 (B); UAMS-1 (C); or Newman (D) in the absence of antimicrobial compounds (●) or in the presence of the non-inhibitory concentration (NIC) of each compound: 100 µL/L of carvacrol (), 200 µL/L of citral (), or 2000 µL/L of (+)-limonene ().
Besides, it presented a different growth pattern from the other three strains, since it continued increasing its biofilm mass throughout the whole experiment. In contrast, the biofilms formed by UAMS-1 (MSSA) and USA300 (CA-MRSA) increased throughout the first 8 h of incubation (over 4 AU) and then biofilm mass decreased (
Figure 1B,C). Moreover, Newman strain (MSSA) presented the weakest biofilm forming ability: a growth plateau or declining phase started after peaking at 8 h, and the measured absorbance values remained under 3 AU (
Figure 1D).
The effect of the addition of the NIC of each IC on the biofilm development was evaluated by the representation of the absorbance values throughout time (
Figure 1). The most prominent effect observed was the decrease in the biofilm mass after 8 h of incubation in the presence of all ICs tested, which corresponded to the peak time point of biofilm mass production in the Newman and UAMS-1 strains (MSSA), and the USA300 strain (CA-MRSA). In addition to this, we detected an important reduction in the production of biofilm mass in
S. aureus SC-01 after 8 h onwards of incubation time in the presence of any of the ICs in the growth medium (
Figure 1A) (
p < 0.05).
S. aureus Newman was the least susceptible strain to the action of the ICs, since their addition seemed to cause a delay in the biofilm development rather than a reduction in the biofilm mass (
Figure 1D). Yet, Newman strain formed weak biofilms in all conditions tested, which prevented us from characterizing with precision the efficiency of the ICs inhibiting biofilm formation in this particular strain.
Although we detected similar anti-biofilm activity of the three ICs at their respective NICs, carvacrol was active at a lower concentration. Carvacrol decreases the biofilm mass production of USA300 and UAMS-1 at every time point tested (
Figure 1B,C). This contrasted to the anti-biofilm activity of the rest ICs, which showed an inhibitory effect that was dependable of the incubation period. For instance, the addition of 2000 µL/L of (+)-limonene reduced the production of biofilm mass in
S. aureus USA300 by 90% after 8 h of incubation, but increased it by 30% after 40 h of incubation (
Figure 1B). As a consequence, to compare the effect of each IC on
S. aureus strains in relation to the differences in biofilm development between strains and throughout time, we evaluated the decrease in the total biofilm mass production (expressed as the cumulative absorbance thickness measured over 40 h of incubation period) for each strain when adding each IC in comparison with the control (
Table 2).
Table 2.
Global inhibitory effect of ICs in biofilm formation by S. aureus. Percentage of decrease in the cumulative absorbance values in the presence of non-inhibitory concentration (NIC) of carvacrol, citral, or (+)-limonene against Staphylococcus aureus strains (SC-01, USA300, UAMS-1, and Newman), in comparison to each value in the absence of added compounds (control). Cumulative absorbance values indicating the biofilm thickness were measured throughout time for a total of 40 h. Values were obtained from the measurement of the area under the curve (AUC) and are expressed in optical absorbance units (at 595 nm)·h (mean ± standard deviation). Asterisks indicate statistically significant differences between each mean AUC value in the presence of each compound with that of the AUC value in its absence. Different superscript letters indicate statistically significant differences in the percentage of decrease in the AUC value among strains for the same compound. Different superscript numbers indicate statistically significant differences in the AUC values among compounds for the same strain. ANOVA tests with Bonferroni’s multiple comparison Post-tests were used (α = 0.05).
Table 2.
Global inhibitory effect of ICs in biofilm formation by S. aureus. Percentage of decrease in the cumulative absorbance values in the presence of non-inhibitory concentration (NIC) of carvacrol, citral, or (+)-limonene against Staphylococcus aureus strains (SC-01, USA300, UAMS-1, and Newman), in comparison to each value in the absence of added compounds (control). Cumulative absorbance values indicating the biofilm thickness were measured throughout time for a total of 40 h. Values were obtained from the measurement of the area under the curve (AUC) and are expressed in optical absorbance units (at 595 nm)·h (mean ± standard deviation). Asterisks indicate statistically significant differences between each mean AUC value in the presence of each compound with that of the AUC value in its absence. Different superscript letters indicate statistically significant differences in the percentage of decrease in the AUC value among strains for the same compound. Different superscript numbers indicate statistically significant differences in the AUC values among compounds for the same strain. ANOVA tests with Bonferroni’s multiple comparison Post-tests were used (α = 0.05).
| SC-01 | USA300 | UAMS-1 | Newman |
---|
Carvacrol | 81.51 *,a1 ± 11.16 | 74.62 *,a1 ± 12.23 | 74.66 *,a1 ± 2.79 | 33.28 *,b1 ± 17.34 |
Citral | 65.00 *,a1 ± 15.30 | 47.34 *,a1,2 ± 21.50 | 57.10 *,a2 ± 6.75 | 0.21 b1 ± 29.78 |
(+)-Limonene | 77.77 *,a1 ± 6.06 | 28.54 b2 ± 19.45 | 26.46 b2 ± 11.83 | 11.19 b1 ± 9.42 |
Thus,
Table 2 shows that SC-01 was the most sensitive strain to any IC, decreasing its total biofilm mass production to at least 65%; and that Newman experienced a significantly lower reduction in its biofilm production when compared to the other strains, regardless of the added compound (
p < 0.05). Carvacrol was the most effective IC regardless of the tested strain, decreasing more than 30% of biofilm mass production to all strains tested (
Table 2) and being statistically more effective than (+)-limonene (
p < 0.05).
Next, we further evaluated the potential of ICs at lower concentrations to inhibit biofilm mass production in the SC-01 strain (CA-MRSA).
Figure 2 was obtained by evaluating the evolution of the biofilm mass of SC-01 in the presence of 0.1-fold NIC of each IC. These conditions evidenced a reduction in the biofilm mass by approximately 40%, 55%, and 80% after 40 h of incubation using ICs concentrations as low as 10, 20, or 200 µL/L of carvacrol, citral, and (+)-limonene, respectively.
Figure 2.
Inhibition of biofilm mass production by S. aureus SC-01 in presence of 0.1× NIC of carvacrol, citral and (+)-limonene. Biofilm development (expressed as optical absorbance units at 595 nm) at 37 °C of Staphylococcus aureus SC-01 in the absence of antimicrobial compounds (continuous line) or in the presence of 1/10 the non-inhibitory concentration (NIC) of each compound: 10 µL/L of carvacrol (), 20 µL/L of citral (), or 200 µL/L of (+)-limonene ().
Figure 2.
Inhibition of biofilm mass production by S. aureus SC-01 in presence of 0.1× NIC of carvacrol, citral and (+)-limonene. Biofilm development (expressed as optical absorbance units at 595 nm) at 37 °C of Staphylococcus aureus SC-01 in the absence of antimicrobial compounds (continuous line) or in the presence of 1/10 the non-inhibitory concentration (NIC) of each compound: 10 µL/L of carvacrol (), 20 µL/L of citral (), or 200 µL/L of (+)-limonene ().
2.4. Discussion
Natural compounds with antimicrobial and anti-biofilm activity are being promoted for different purposes, such as food preservatives, disinfectants and chemotherapeutic agents to replace traditionally used chemical agents and antibiotics [
22,
23]. In addition, exposure to sub-MIC concentrations of some traditional antibiotics and disinfectants can act as an environmental signal triggering biofilm formation [
24,
25], supporting the research on new antimicrobial compounds. MIC values for ICs against
S. aureus strains showed in this investigation (
Table 1) were in agreement with previous studies showing the strong inhibitory activity of carvacrol, citral, and (+)-limonene against several strains of
S. aureus and other bacterial species [
21,
26,
27].
Figure 3.
Inactivation of planktonic cells at sub-MIC values of carvacrol, citral, and (+)-limonene. Log10 concentration of planktonic cells (CFU/mL) of Staphylococcus aureus Newman when grown at 37 °C on polystyrene surfaces in growth medium without added antimicrobial compounds (●), or in the presence of the non-inhibitory concentration (NIC) of each compound: 100 µL/L of carvacrol (), 200 µL/L of citral (), or 2000 µL/L of (+)-limonene ().
Figure 3.
Inactivation of planktonic cells at sub-MIC values of carvacrol, citral, and (+)-limonene. Log10 concentration of planktonic cells (CFU/mL) of Staphylococcus aureus Newman when grown at 37 °C on polystyrene surfaces in growth medium without added antimicrobial compounds (●), or in the presence of the non-inhibitory concentration (NIC) of each compound: 100 µL/L of carvacrol (), 200 µL/L of citral (), or 2000 µL/L of (+)-limonene ().
The study of the evolution of the biofilm mass production revealed that highly diverse patterns of adhesion and biofilm formation exist among the different assayed
S. aureus strains (
Figure 1). We found no correlation between each strain’s ability to form a biofilm and its original source of isolation and its susceptibility to methicillin. Yet, we detected that biofilm mass production is directly related to the incubation time interval, which varies from strain to strain. Therefore, incubation time should be considered when comparing biofilm formation ability among strains. For instance, the strains SC-01 and Newman exhibited increasing and decreasing biofilm thickness over 40 h of incubation period (
Figure 1A,D). Thus, after 24 h of incubation, which is the usual time to evaluate antimicrobial activity of EOs [
16,
17,
26,
28], UAMS-1 was a better biofilm forming strain than USA300 and Newman (
Figure 1). However, after 32 h of incubation, USA300 strain showed a thicker biofilm than Newman and UAMS-1 strains which is consistent with what has been previously reported [
3,
29,
30].
Addition of NIC of ICs to growth medium inhibited biofilm mass production by
S. aureus strains. Although most studies agree about the inhibitory effect of EOs and ICs on the biofilm formation [
31,
32,
33,
34,
35,
36,
37,
38,
39], different authors have detected an enhancement of the biofilm production by these compounds under some conditions [
40,
41,
42]. As described for biofilm formation in absence of ICs, the inhibitory effect of these compounds on biofilm mass production varied as a function of the time point tested. The biofilm formation is a dynamic and cyclical process that involves two initial steps; an initial attachment and a subsequent maturation phase. A final dispersal phase occurs when the biofilm reaches a nutrient-deprived critical mass or as a response to changing environmental conditions, which causes the detachment of bacteria from the outermost layers of the biofilm and become planktonic single individuals [
2,
18]. Therefore, dependence of the incubation period in the inhibition of biofilm mass production could be attributable to a possible delay in the cycles of biofilm assembly-disassembly caused by antimicrobial effect of ICs, emphasizing the importance of measuring the anti-biofilm properties of any compound of interest at different time points during the process of biofilm formation. Although the representation of the stages of biofilm development has been applied to the identification of particularities in mutants or the differentiation of strains [
43,
44], to the best of our knowledge, most studies describing the effect of EOs or ICs on the biofilm development only compare the biofilm produced after a certain period of incubation, usually 24 h [
16,
17,
26,
28]. Comparison of the area under the curve of biofilm for biofilm mass production in absence or presence of ICs (
Table 2) provided us with a useful parameter to describe inhibitory properties of these compounds in biofilm formation. This parameter revealed carvacrol as a potent inhibitor of biofilm mass production by all
S. aureus strains tested.
Although resistance to ICs (MIC values) was similar for the planktonic cells of all the strains (
Table 1), inhibition of biofilm mass production varied as a function of the assayed
S. aureus strain (
Figure 1). This differential behaviour could indicate that different or specific mechanisms of biofilm inhibition (other than those related to the delay in the initial attachment) are present depending on the
S. aureus strain and on the antimicrobial compound. Difference between the planktonic cell concentration of
S. aureus Newman in the absence and in the presence of the NICs of the ICs after 8 h of incubation (
Figure 3) could account for the difference observed in the biofilm production at this time point (
Figure 1D) by delaying the initial attachment and consequently the biofilm mass production. In this regard, it is known that the first step of the biofilm development (the initial bacterial adhesion to the surface) is conditioned by the growth stage of the bacterial cells [
11]. On the other hand, high cell density triggers
quorum sensing response, based on cell-to-cell communication that regulates genes involved in biofilm maturation and maintenance [
2,
23].
Quorum sensing response is activated when the concentration of autoinducers (small molecules secreted by bacteria) exceeds a requisite threshold. Therefore, lower initial bacterial counts would delay biofilm mass production [
9]. Moreover, these ICs can damage cell envelopes [
20,
21] which could result in a diminished ability to attach to the surface and form biofilms, as previously suggested by Kerekes
et al. [
17]. Likewise, inhibitory effects of ICs on planktonic cells could also explain the delay in biofilm formation by the other
S. aureus strains. However, while biofilm formation by
S. aureus Newman in presence of citral and (+)-limonene was only delayed, growth of the other strains in presence of ICs also reduced the highest biofilm mass (
Table 2). Therefore, in contrast to the results obtained with Newman strain, the effect of ICs on inhibiting biofilm formation by SC-01, USA300 and UAMS-1 strains would not only be due to the inhibitory effect of ICs on planktonic cells, indicating a different mechanism of action by ICs for these strains and Newman strain.
The control of
S. aureus in clinical settings has been traditionally performed by increasing concentrations of conventional antibiotics, such as β-lactams or glycopeptides. Unfortunately, the uncontrolled use of penicillins, like methicillin, to treat patients and livestock contributed in emergence of MRSA [
45]. MRSA infections show a mortality rate of 20%, and are the leading cause of death by a single infectious agent in the USA, high above HIV [
46]. In addition to this, a subset of CA-MRSA strains has emerged, which are no longer restricted to patients from hospitals or immuno-compromised high-risk citizens but in fact have the ability to cause severe and pandemic infections in healthy individuals [
47]. The reduced number of effective antimicrobial treatments that are available to eradicate multi-drug resistant pathogenic bacteria could lead to recurrent microbial contaminations in food industry, and to hard-to-treat infections frequently related to hospitals. In food processing, EOs and ICs are of special interest since their natural origin meets consumers’ current reluctance towards chemically-synthesized antimicrobials [
48]. In addition to their use as sanitizers and disinfectants of food equipment and environments, food packaging materials containing antimicrobial compounds have gained practical importance in the control of surface contamination [
49].
The potential of sub-MIC concentrations of carvacrol, citral, and (+)-limonene as inhibitors of the biofilm formation in multi-drug resistant strains, such as the CA-MRSA strain SC-01 (
Figure 2), whose biofilms can develop in the presence of many conventional antibiotics [
50], should be further considered as anti-biofilm compounds. Finally, another important reason that supports the use of ICs in food and clinical environments is the evidence that, unlike conventional antibiotics, these compounds would effectively kill bacteria while applying a less selective pressure for the development of resistance [
51].