Lactobacillus rhamnosus and Staphylococcus epidermidis in gut microbiota: in vitro antimicrobial resistance

Antimicrobial assays for planktonic bacteria

The inhibitory concentration IC50 was performed, and the optical density (OD600) was measured after 24 h of incubation. The obtained OD600 was converted into a percentage of viable cells. To note that different concentrations of EDTA varying from 1 mM to 2.5 mM were tested without observing any significant difference in the bacterial inhibition as compared to the control (data not shown).

Effect of increasing lysozyme concentration on Lactobacillus rhamnosus GG

Figure 1 illustrates the impact of lysozyme with and without 1 mM EDTA on planktonic L. rhamnosus GG at different pH.

Fig. 1figure 1

Effect of lysozyme with and without EDTA (1 mM) on Lactobacillus rhamnosus GG at different pH: a pH 2; b pH 6; c pH 7.5 and d pH 8.5. The results are mean values of three replicates. OD600 of the positive controls: C+(a) = 0.38; C+(b) = 1.36; C+(c) = 1.33;C+(d) = 1.32. (*) indicates a significant difference (P < 0.05) between each test and the positive control (without lysozyme). The lowercase letter a indicates a significant difference (P < 0.05) between each test and its preceding. Error bars represent the SD (standard deviation)

At pH 2, the addition of lysozyme addition did not have a wide effect on inhibiting viable cells (Fig. 1a). Thus, at a lysozyme concentration of 80 mg/mL, the percentage of viable cells decreased (P < 0.05) respectively from 100% (positive control) to 78% and 82% with and without adding EDTA. At this pH value, the growth of L. rhamnosus GG was limited compared to other pH values where the OD600 of the positive control increased from 0.38 at pH 2 to 1.33 at pH 7.5. At the same lysozyme concentration, a better inhibition effect was observed at pH 6 (Fig. 1b). Hence, an inhibition of 43% of the living cells with EDTA (57% viable cells) was reached. On the other hand, a higher percentage of cells (72%) remained viable without EDTA. However, lysozyme with EDTA exhibited its maximum antimicrobial activity at pH 7.5 (Fig. 1c). This gradual decrease occurred at a 1.25 mg/mL lysozyme concentration and beyond. At this concentration, a considerable drop (P < 0.05) of viable cells was observed to finally reach 35% at a concentration of 80 mg/mL. In contrast to other tested pH, a MIC was observed at 80 mg/mL of lysozyme (with EDTA) where an IC50 was detected at 30 mg/mL of lysozyme with EDTA. At a pH of 8.5 (Fig. 1d), a progressive decrease of cells viability was observed till reaching a 32% of bacterial growth inhibition (P < 0.05) at a lysozyme concentration of 80 mg/mL with EDTA. It is quite noticeable that increasing lysozyme concentration had a less significant effect at pH 8.5 compared to 7.5.

Effect of increasing lysozyme concentration on Staphylococcus epidermidis 444

The antimicrobial activity of the lysozyme with and without 1 mM EDTA on planktonic S. epidermidis 444 at different pH is highlighted in Fig. 2.

Fig. 2figure 2

Effect of lysozyme with and without EDTA (1 mM) on Staphylococcus epidermidis 444 at different pH: a pH 2; b pH 6; c pH 7.5 and d pH 8.5. The results are mean values of three replicates. OD600 of the positive controls: C+(a) = 0.45; C+(b) = 1.03; C+(c) = 1.30;C+(d) = 1.34. (*) indicates a significant difference (P < 0.05) between each test and the positive control (without lysozyme). The lowercase letter a indicates a significant difference (P < 0.05) between each test and its preceding. Error bars represent the SD (standard deviation)

At pH 2, S. epidermidis 444 underwent a decrease (P < 0.05) in viable cells respectively from 100 to 74% (with EDTA) and to 81% (without EDTA) at a lysozyme concentration of 80 mg/mL (Fig. 2a). A better effect of lysozyme was observed at pH 6 (Fig. 2b). Accordingly, significant inhibition of viable cells growth (P < 0.05) was progressively expressed to finally reach 40% (with EDTA) and 71% (without EDTA) respectively at the same lysozyme concentration (80 mg/mL). Thus, a MIC was observed at a concentration of 40 mg/mL of lysozyme (with EDTA), and the IC50 was detected at 22 mg/mL of lysozyme (with EDTA). Although, lysozyme’s optimal inhibition effect was reported at a pH of 7.5 (Fig. 2c). Therefore, at a concentration of 10 mg/mL of lysozyme (with EDTA) a highly significant drop (P < 0.05) occurred. Thus, the percentage of viable cells decreased from 100 to 60% and kept decreasing till reaching 25% (with EDTA) and 46% (without EDTA) at a lysozyme concentration of 80 mg/mL. However, the MIC was visually observed at a lysozyme concentration of 40 mg/mL (with EDTA). The IC50 was determined at 18 mg/mL (with EDTA) and 26 mg/mL (without EDTA) of lysozyme (Table 1). Moreover, a progressive drop in the density of viable cells was observed at a pH of 8.5 (Fig. 2d), where a significant decrease (P < 0.05) by 51% was detected at 80 mg/mL of lysozyme with EDTA. A MIC was observed at 80 mg/mL of lysozyme with EDTA, and the IC50 at 50 mg/mL. However, with the same lysozyme concentration and without EDTA, 75% of the cells remained viable.

Table 1 Comparative table between the different MIC, IC50 and MBC of Lactobacillus rhamnosus GG, Staphylococcus epidermidis 444 and the co-culture of both strainsEffect of increasing lysozyme on the co-culture of planktonic Lactobacillus rhamnosus GG and Staphylococcus epidermidis 444

The antimicrobial activity of the lysozyme with and without 1 mM EDTA on co-culture at different pH is highlighted in Fig. 3.

Fig. 3figure 3

Effect of lysozyme with and without EDTA (1 mM) on the co-culture of Lactobacillus rhamnosus GG and Staphylococcus epidermidis 444 at different pH: a pH 2; b pH 6; c pH 7.5 and d pH 8.5. The results are mean values of three replicates. OD600 of the positive controls: C+(a) = 0.37; C+(b) = 1.15; C+(c) = 1.19;C+(d) = 1.40. (*) indicates a significant difference (P < 0.05) between each test and the positive control (without lysozyme). The lowercase letter a indicates a significant difference (P < 0.05) between each test and its preceding. Error bars represent the SD (standard deviation)

According to Fig. 3a, at pH 2, results were in accordance with when testing each strain singly. At 80 mg/mL of lysozyme, the percentages of cells viability were 87% (with EDTA) and 84% (without EDTA). Figure 3b showed a significant decrease (P < 0.05) from 100% (positive control) to 73% (with EDTA) and 75% (without EDTA) of the viable cells at a lysozyme concentration of 80 mg/mL and a pH of 6. However, at a pH of 7.5 (Fig. 3c), an optimal antimicrobial effect was detected. Thus, at 80 mg/mL of lysozyme, a gradual decrease of viable cells occurred till reaching respectively 38% (with EDTA) and 59% (without EDTA). In contrast to the other tested pH levels, a MIC was observed at 80 mg/mL and the IC50 was at 26 mg/mL of lysozyme (with EDTA). The obtained results were quite similar to those of lysozyme performed with L. rhamnosus GG. At a pH of 8.5 (Fig. 3d), the effect of the lysozyme has become weak, thus a slower decrease (P < 0.05) in viable cells was observed and a reduction to 70% (with EDTA) and 86% (without EDTA) was observed at a lysozyme concentration of 80 mg/mL.

Despite the significant results obtained by increasing lysozyme concentration on the inhibition of the tested strains, the absence of minimum bactericidal concentrations (MBC) was remarkable at all pH levels.

Effect of oxytetracycline hydrochloride with and without lysozyme-EDTA mixture on planktonic Lactobacillus rhamnosus GG

The effect of using singly oxytetracycline hydrochloride or mixing it with lysozyme-EDTA (at IC50) on L. rhamnosus GG at pH 7.5 is shown in Fig. 4.

Fig. 4figure 4

Effect of oxytetracycline hydrochloride with and without lysozyme-EDTA (at IC50) on planktonic Lactobacillus rhamnosus GG at pH 7.5. OD600 of negative control (oxytetracycline hydrochloride) = 0.32, OD600 of 700 μg/mL of oxytetracycline hydrochloride with lysozyme-EDTA (at IC50) = 0.38. The results are mean values of three replicates. (*) indicates a significant difference (P < 0.05) between each test and the positive control (oxytetracycline hydrochloride). The lowercase letter a indicates a significant difference (P < 0.05) between each test and its preceding. Error bars represent the SD (standard deviation)

The oxytetracycline hydrochloride significant effect (P < 0.05) started to be observed at a concentration of 21.87 µg/mL. The antibiotic effect kept increasing while its concentration increased, where 69% of viable L. rhamnosus GG were inhibited at a concentration of 700 µg/mL and a MIC was observed. Nevertheless, after adding 30 mg/mL of lysozyme with EDTA (at IC50), the percentage of cell viability started to drop down from lower antibiotic concentrations (0.17 µg/mL). Hence, at this concentration a significant reduction (P < 0.05) in the percentage of viable cells (up to 71%) was observed. This reduction continued gradually till reaching 23% at an antibiotic concentration of 700 μg/mL after the addition of lysozyme and EDTA. The IC50 was reduced from 152 μg/mL when singly using oxytetracycline hydrochloride to 90 μg/mL after lysozyme addition.

After performing the MBC test, it was noted that in the absence of the lysozyme, oxytetracycline hydrochloride could not inhibit L. rhamnosus GG at 700 μg/mL. When plating the same concentration of antibiotic with lysozyme-EDTA (at IC50) no colonies of L. rhamnosus GG were observed and an MBC was detected (Additional file 1: Fig. S3). The OD600 at 700 μg/mL of oxytetracycline hydrochloride with lysozyme-EDTA (at IC50) was of 0.38, which was similar to that of the negative control (OD600 oxytetracycline hydrochloride).

Effect of oxytetracycline hydrochloride with and without lysozyme-EDTA mixture on planktonic Staphylococcus epidermidis 444

The effect of using singly oxytetracycline hydrochloride or mixing it with lysozyme-EDTA (at IC50) on S. epidermidis 444 at pH 7.5 is shown in Fig. 5.

Fig. 5figure 5

Effect of oxytetracycline hydrochloride with and without lysozyme-EDTA (at IC50) on planktonic Staphylococcus epidermidis 444 at pH 7.5. OD600 negative control (oxytetracycline hydrochloride) = 0.32, OD600 of 700 μg/mL of oxytetracycline hydrochloride with lysozyme-EDTA (at IC50) = 0.4. The results are mean values of three replicates. (*) indicates a significant difference (P < 0.05) between each test and the positive control (oxytetracycline hydrochloride). The lowercase letter (a) indicates a significant difference (P < 0.05) between each test and its preceding. Error bars represent the SD (standard deviation)

As seen in Fig. 5, at a concentration of 700 µg/mL, oxytetracycline hydrochloride significantly reduced the bacterial growth of viable S. epidermidis 444 (P < 0.05) up to 29% without being able to totally inhibit their growth. A MIC was visually observed at this concentration (700 µg/mL). One the other hand, the addition of 18 mg/mL of lysozyme-EDTA (at IC50) to this antibiotic revealed a significant drop (P < 0.05) in the percentage of viability from 100 to 26% when using 700 µg/mL of antibiotic (with lysozyme). The IC50 was observed at 130 μg/mL (without lysozyme) and at 1.22 μg/mL (with lysozyme) of oxytetracycline hydrochloride. Therefore, we hypothesize that the lysozyme has made S. epidermidis 444 more vulnerable to oxytetracycline hydrochloride, even with its broad-spectrum antibiotic profile. Consequently, lysozyme-EDTA mixture increased antibiotic killing of S. epidermidis 444 even after showing a certain resistance to high antibiotic concentrations (Additional file 1: Fig. S4). Similarly, an MBC was observed when lysozyme-EDTA mixture (at IC50) was added to 700 µg/mL of oxytetracycline hydrochloride.

Effect of oxytetracycline hydrochloride with and without lysozyme-EDTA mixture on a co-culture of planktonic Lactobacillus rhamnosus GG and Staphylococcus epidermidis 444

Figure 6 shows the effect of oxytetracycline hydrochloride on a co-culture of L. rhamnosus GG and S. epidermidis 444 planktonic strains after 24 h of incubation. The percentage of cells viability decreased progressively to 66% at an antibiotic concentration of 700 μg/mL at which a MIC was observed. A higher resistance to antibiotic was observed when the two strains have been co-cultured compared to when singly tested. When 26 mg/mL of lysozyme-EDTA (at IC50) were added to oxytetracycline hydrochloride, a significant and gradual reduction of the viable cells (P < 0.05) toward reaching 39% was observed. Thus, an IC50 was detected at an antibiotic concentration of 10.93 μg/mL. The positive effect of lysozyme combined to EDTA on the co-cultured strains is well noticeable. Interestingly, the minimum bactericidal concentrations (MBC) were absent at 700 µg/mL of oxytetracycline hydrochloride when lysozyme and EDTA were not added (Additional file 1: Fig. S5).

Fig. 6figure 6

Effect of oxytetracycline hydrochloride with and without lysozyme-EDTA (at IC50) on the planktonic co-culture of Lactobacillus rhamnosus GG and Staphylococcus epidermidis 444. OD600 negative control (oxytetracycline hydrochloride) = 0.32. OD600 of 700 μg/mL of oxytetracycline hydrochloride with lysozyme-EDTA (at IC50) = 0.30. The results are mean values of three replicates. (*) indicates a significant difference (P < 0.05) between each test and the positive control (oxytetracycline hydrochloride). The lowercase letter a indicates a significant difference (P < 0.05) between each test and its preceding. Error bars represent the SD (standard deviation)

Antimicrobial assays for bacterial biofilms

The minimal concentration to eradicate the formed biofilm by 50% (MCBEC50) was assayed where the optical density (OD570) was measured after 24 h of incubation. The obtained OD570 was converted into a percentage of biofilm formation.

Effect of increasing lysozyme concentration on Lactobacillus rhamnosus GG, Staphylococcus epidermidis 444, and co-culture biofilm

Figure 7 shows the effect of lysozyme (with and without EDTA) on different biofilms at pH 7.5.

Fig. 7figure 7

Effect of Lysozyme with and without EDTA (1 mM) on different biofilms at pH7.5. a Lactobacillus rhamnosus GG, b Staphylococcus epidermidis 444, and c co-culture of both strains. The results are mean values of three replicates. (*) indicates a significant difference (P < 0.05) between each test and the positive control (without lysozyme). The lowercase letter a indicates a significant difference (P < 0.05) between each test and its preceding. Error bars represent the SD (standard deviation)

When adding lysozyme with EDTA to L. rhamnosus GG biofilm (Fig. 7a), a gradual increase in the biofilm eradication was observed. The biofilm formation significantly decreased (P < 0.05) and reached 32% (with EDTA) and 40% (without EDTA) at a lysozyme concentration of 80 mg/mL. On the other hand, a gradual decreased by around 50% in the formed biofilm of S. epidermidis 444 was observed when using 20 mg/mL of lysozyme (with EDTA). Similarly, as shown in Fig. 7b, an eradication of 74% of the same biofilm was observed at 80 mg/mL of lysozyme (with EDTA). In the absence of EDTA, a less significant eradication (P < 0.05) of the formed biofilm was observed. Thus, at a concentration of 80 mg/mL of lysozyme, the biofilm formation by S. epidermidis 444 was of 39%. The co-culture biofilm of both strains shown in Fig. 7c, revealed a gradual decrease when increasing the lysozyme concentration. This gradual decrease in biofilm formation reached up to 35% (with EDTA) and 57% (without EDTA) at 80 mg/mL of lysozyme. The minimal complete biofilm eradication concentrations MCBEC50 were obtained by following a series of serial dilutions. 50, 26, and 30 mg/mL of lysozyme (with EDTA) were respectively MCBEC50 of L. rhamnosus GG, S. epidermidis 444, and the co-culture of both strains. The obtained lysozyme concentrations (with EDTA) will be added to different antibiotic concentrations to evaluate their effect on increasing biofilm eradication.

Effect of oxytetracycline hydrochloride with and without lysozyme-EDTA mixture on Lactobacillus rhamnosus GG, Staphylococcus epidermidis 444, and co-culture biofilm

The effect of oxytetracycline hydrochloride on L. rhamnosus GG biofilm eradication with and without the addition of lysozyme and EDTA at pH 7.5 is shown in Fig. 8a.

Fig. 8figure 8

Effect of oxytetracycline hydrochloride with and without lysozyme-EDTA (at MCBEC50) on different biofilms at pH7.5. a Lactobacillus rhamnosus GG, b Staphylococcus epidermidis 444, and c co-culture of both strains. The results are mean values of three replicates. (*) indicates a significant difference (P < 0.05) between each test and the positive control (oxytetracycline hydrochloride). The lowercase letter a indicates a significant difference (P < 0.05) between each test and its preceding. Error bars represent the SD (standard deviation)

The biofilm of L. rhamnosus GG showed a high resistance against the used antibiotic. Thus, the percentage of biofilm formation was constant until a significant decrease (P < 0.05) started at an antibiotic concentration of 350 μg/mL. Notably, when 50 mg/mL of lysozyme with EDTA (at MCBEC50) had been added to the antibiotic, the biofilm was eradicated by 12% starting from the lowest concentration (0.17 μg/mL). This eradication continued gradually until reaching 38% and 35% in the presence of 1400 μg/mL and 2800 μg/mL of oxytetracycline hydrochloride. A MCBEC50 was observed after adding 1144 μg/mL of oxytetracycline hydrochloride to lysozyme-EDTA mixture. However, lysozyme with EDTA induced higher eradication when added to the antibiotic.

The effect of oxytetracycline hydrochloride on S. epidermidis 444 biofilm eradication with and without the addition of lysozyme and EDTA at pH 7.5 is shown in Fig. 8b.

Hence, oxytetracycline hydrochloride weakly affected the biofilm eradication of S. epidermidis 444 up to a concentration of 350 μg/mL, where a significant (P < 0.05) drop of 28% was observed. The increase in the biofilm eradication continued gradually to reach its optimum of 43% at 2800 μg/mL oxytetracycline hydrochloride. On the other hand, when the antibiotic was coupled with 26 mg/mL of lysozyme with EDTA (at MCBEC50), a much greater eradication was observed even with lower antibiotic concentrations. Thus, at an antibiotic concentration of 2.73 μg/mL, the percentage of biofilm eradication was of 2% compared to 48% after the addition of lysozyme-EDTA mixture. Hence, a 21-fold of eradication increase was noticed. Significantly, the decrease in biofilm formation when increasing the antibiotic concentration (P < 0.05) caused a reduction of the formed S. epidermidis biofilm by 63% at an antibiotic concentration of 2800 μg/mL with the addition of 26 mg/mL of lysozyme mixed with EDTA. Accordingly, a new MCBEC50 was observed at 24 μg/mL of oxytetracycline hydrochloride after adding lysozyme and EDTA.

The effect of oxytetracycline hydrochloride on co-culture of L. rhamnosus GG and S. epidermidis 444 biofilm eradication with and without the addition of lysozyme and EDTA at pH 7.5 is shown in Fig. 8c.

The biofilm formed of the two strains showed high resistance at low concentrations of oxytetracycline hydrochloride. Thus, oxytetracycline hydrochloride weakly affected the co-culture biofilm eradication up to a concentration of 350 μg/mL, where a significant (P < 0.05) drop of 22% was observed. This eradication increased gradually to finally reach 33% (P < 0.05) at an antibiotic concentration of 2800 μg/mL. However, when the antibiotic was combined to 30 mg/mL of lysozyme with EDTA (at MCBEC50), a substantially higher level of eradication was observed starting at an antibiotic concentration of 10.93 μg/mL. The biofilm was eradicated by 22% starting with an antibiotic concentration of 10.93 μg/mL. This eradication continued to gradually increase until reaching 49% and 54% in the presence of 1400 μg/mL and 2800 μg/mL of oxytetracycline hydrochloride respectively. Accordingly, a new MCBEC50 was observed at a concentration of 1464 μg/mL of oxytetracycline hydrochloride after the addition of lysozyme and EDTA.

Comparison of IC50, MIC, MBC and MCBEC50 values

The IC50, MIC, MBC and MCBEC50 values obtained from the different tests performed are shown in Table 1. From the above results it may be noticed that S. epidermidis 444 was more sensitive to lysozyme than L. rhamnosus GG. Indeed, the addition of EDTA to the lysozyme induced a significant improvement in its effect. In addition, the activity of the lysozyme varied widely between the different tested pH levels. However, the lysozyme activity was almost absent at a pH of 2 and quite variable between 6 and 8.5. Indeed, lysozyme’s activity seems to be more effective at a pH of 7.5. Similarly, when lysozyme-EDTA mixture was combined to oxytetracycline hydrochloride a better biofilm eradication of the tested strains was observed.

Comparison of FIC50 values

Table 2 shows the effect of combining lysozyme-EDTA to oxytetracycline hydrochloride on the tested bacterial strains.

Table 2 FIC50 and ƩFIC50 for combinations of lysozyme-EDTA and oxytetracycline hydrochloride at pH 7.5 against planktonic Lactobacillus rhamnosus GG, Staphylococcus epidermidis 444 and co-culture strains

For L. rhamnosus GG, an indifferent effect when combining lysozyme-EDTA and oxytetracycline hydrochloride was observed where ΣFIC50 (= 3.59) was between 1 and 4. Thus, this indifference is mainly due to lysozyme-EDTA mixture that didn’t affect much L. rhamnosus GG (FIC50 = 3) as compared to oxytetracycline (FIC50 = 0.59). However, when combining antibiotic to lysozyme-EDTA mixture a high synergic effect (ΣFIC50 ≤ 0.5) was observed on S. epidermidis 444 indicating a remarkable increase in bacterial inhibition. Furthermore, same synergic results but with a lower rate were observed when the two strains where co-cultured.

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