Exploring the mechanisms by which camel lactoferrin can kill Salmonella enterica serovar typhimurium and Shigella sonnei

Lactoferrins

In the present study, hLf (15% iron saturation) and bLf (10% iron saturation) purchased from Sigma-Aldrich (St. Louis, MO, USA) were comparatively evaluated to camel lactoferrins regarding their in vitro antimicrobial mechanisms (bacteriostatic and bactericidal activities) against S. typhimurium and S. sonnei. Protocols of milk collection from four different breeds of Saudi camels, milk processing, cLfs purification, and Lfs labeling with activated NHS-biotin/gold nanoparticles (AuNPs) were reported in earlier studies (Redwan et al., 2016; Almehdar et al., 2019; Almehdar et al., 2020).

Cultivation conditions

The seed stock cultures of enteric bacterial strains Salmonella typhimurium LT2 and Shigella sonnei ATCC 25931 were inoculated into tryptic soy broth (TSB). After overnight incubation, a 200 µl inoculum of each enteric bacterial strain was plated on tryptic soy agar (TSA) and incubated overnight. Finally, TSB was inoculated with one of the colonies of each enteric bacterial strain and incubated at 37 °C for 16 h.

Antimicrobial activity examination using agar disc-diffusion test and monitoring bacterial growth

Standard discs of 17 antibiotics were obtained from Mast Diagnostics (Merseyside, Liverpool, UK) to be used in this assay; amikacin (30 µg), ampicillin (10 µg), augmentin (30 µg), aztreonam (30 µg), cefoxitin (30 µg), cefepime (30 µg), ceftazidime (30 µg), cephalothin (30 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), cotrimoxazole (25 µg), fucidic acid (10 µg), gentamicin (10 µg), imipenem (10 µg), oxacillin (1 µg), piperacillin (100 µg), and vancomycin (30 µg).

Antimicrobial activity of four cLfs isolated from milk of four different breeds of Saudi camels (cLf1, cLf2, cLf3, and cLf4), bLf, hLf, and the standard antibiotics against S. typhimurium and S. sonnei was tested by agar disc-diffusion technique. Plates of Mueller-Hinton (MH) agar were overlaid with 2 × 10⁶ CFU/ml of Salmonella typhimurium LT2 and Shigella sonnei. Wells of about 5 mm diameter were made on inoculated plates then different concentrations of lactoferrins (0.00, 0.125, 0.250, 0.50, 0.750, 1, 1.5, 2, 2.5, and 3 mg/ml) were added, and left to diffuse in agar at 4 °C for 2 h before plates finally incubated overnight at 37 °C. The diameters of clear inhibition zones were measured in millimeters. Antibacterial effects of different lactoferrins on growth of S. typhimurium and S. sonnei after 1, 3, 6, 12, and 24 h of incubation at 37 °C were monitored spectrophotometrically (measuring OD at 620 nm).

Determination of minimum inhibitory concentrations (MICs) by broth microdilution assay

Cation adjusted Mueller-Hinton (CAMH) broth containing test antibacterial agents in serial dilutions was inoculated with 2 × 10⁶ CFU/ml of Salmonella typhimurium LT2 and Shigella sonnei then added to 96-well microtiter plates. Plates were incubated overnight at 37 °C and then absorbance was recorded at 620 nm (monitoring bacterial growth). The lowest concentration of each antibacterial agent at which bacterial growth was completely inhibited was recorded as MIC. Bacteria in CAMH broth was the growth control. To confirm the presence or absence of a ferrochelating-dependent antibacterial mechanism of Lfs, the MICs for all Lfs were evaluated once more after iron supplementation (adding ferrinitrilotriacetate (FeNTA) at a concentration of 50 µM) at the time of inoculation (Redwan et al., 2016; Almehdar et al., 2019; Almehdar et al., 2020).

Checkerboard assay

To study synergistic interaction between the different lactoferrins and antibiotics regarding their antibacterial activity against Salmonella typhimurium LT2 and Shigella sonnei, combinations of cLf-chloramphenicol, cLf-cefepime, cLf-imipenem, hLf-chloramphenicol, hLf-cefepime, hLf-imipenem, bLf-chloramphenicol, bLf-cefepime, and bLf-imipenem were examined and compared with their individual antibacterial activities via microdilution checkerboard method as described previously (Redwan et al., 2016; Almehdar et al., 2019; Almehdar et al., 2020). The combined antibacterial effects of cLf, hLf, and bLf with each studied antibiotic were analyzed by calculation of fractional inhibitory concentration index (FICI) (Redwan et al., 2016; Almehdar et al., 2019; Almehdar et al., 2020). Firstly, we calculated the fractional inhibitory concentration (FIC) of each lactoferrin or antibiotic by dividing MIC value of the agent in combination by MIC value of the agent alone. Afterward, FIC value of each lactoferrin was divided by FIC of the antibiotic to estimate FICI. The FICI values of 0.5 indicate that the MIC values of both antibiotic and Lf in the combination treatment were reduced to $\frac{1}{4}$ MIC. The FICI values of 0.37 indicate that MICs of antibiotics were reduced to $\frac{1}{4}$ MIC and MICs of Lfs were reduced to 1/8 MIC. The antibacterial combination against S. typhimurium and S. sonnei was considered indifferent at 0.5 < FICI < 4, antagonistic at FICI ≥ 4, and synergistic at FICI ≤ 0.5.

Time-kill assay

Assays for the killing rate of S. typhimurium and S. sonnei by the different lactoferrins either alone or combined with antibiotics were performed as previously reported (Redwan et al., 2016; Almehdar et al., 2019; Almehdar et al., 2020). Each lactoferrin at concentrations of $\frac{1}{4}$ MIC or 1 × MIC was combined with chloramphenicol, imipenem, or cefepime at concentration of $\frac{1}{4}$ MIC. Lactoferrins alone were added at concentrations of $\frac{1}{2}\times$, 1 ×, and 2 × MIC in LB broth. Antimicrobials were added to S. typhimurium and S. sonnei (2 ×10⁶ CFU/ml) cultures followed by incubation at 37 °C and 150 rpm. Viable colony counts were done at 0, 4, and 8 h. Experiments were conducted three times and the obtained results were demonstrated as mean and the standard error of the mean (SEM). Bactericidal activity was defined as ≥ 3 log₁₀ CFU/ml reduction (99.9% kill) from the count of the control culture (without addition of any antimicrobial).

Calculation of minimum bactericidal concentration

All MIC wells from broth microdilution assay with no bacterial growth were subcultured on plates of MH agar that did not contain test lactoferrins and incubated at 37 °C for 24 h. Bacterial viability was measured by the count of colonies. Bacterial growth resumption means that test lactoferrins did not cause bacterial death, thus identified as bacteriostatic, while absence of bacterial proliferation means that test lactoferrins caused bacterial death, thus identified as bactericidal. The lowest concentration of test lactoferrins resulting in a ≥ 99.9% decrease in viable cells (bacterial death) was recorded as minimum bactericidal concentration (MBC). Generally, antibacterial compounds are considered bactericidal if their MBC value is at most four times their MIC value (French, 2006).

Analysis of interaction between the biotinylated lactoferrins and S. typhimurium/S. sonnei LPS

Reactivity of Lfs with LPS of S. typhimurium or S. sonnei obtained from Sigma Aldrich (St. Louis, MO, USA) was estimated using ELISA as previously described (Almehdar et al., 2019; Almehdar et al., 2020).

Effects of the different antibacterial agents (antibiotics, cLf, and their combination) on the integrity of S. typhimurium/S. sonnei cell and its protein profile

Cell pellets of 5 ml S. typhimurium and S. sonnei cultures were collected then suspended in 5 ml phosphate buffered saline (PBS) buffer, pH 7.4. Suspended bacterial cells were treated with either carbenicillin, chloramphenicol, or imipenem alone, or combined with cLf, or cLf alone, and incubated at 37 °C. Samples (200 µl) were withdrawn from each treatment at different time intervals (0, 30, 60, and 150 min). These samples were split into two halves, the first one was centrifuged and its corresponding supernatant protein content was evaluated by Bradford assay kit at 595 nm (Thermo Fisher Scientific, Waltham, MA, USA). The second half was used for SDS-PAGE protein profiling (Almehdar et al., 2019; Almehdar et al., 2020).

Separation of bacterial membrane proteins fraction and analysis of their interactions with cLf-biotin

S. typhimurium and S. sonnei cells were pelleted and then washed with PBS, pH 7.4. The membrane proteins fraction of bacterial cells were then separated and run on 12.5% reducing SDS-PAGE as described previously (Redwan et al., 2016; Almehdar et al., 2019; Almehdar et al., 2020). The resolved bacterial membrane proteins (BMP) of S. typhimurium or S. sonnei on SDS-PAGE were transferred into poly-vinylidene fluoride (PVDF) membrane by western blotting then incubated with cLf-biotin. Afterwards, the membrane was incubated with streptavidin-peroxidase and developed with peroxidase substrate. The interaction between separated bacterial membrane proteins and cLf was analyzed by ELISA as described in previous studies (Redwan et al., 2016; Almehdar et al., 2019; Almehdar et al., 2020). After coating of 96-well ELISA microtiter plate with the blank buffer of carbonate/bicarbonate pH 9.6, free camel lactoferrin and BMP extract of S. typhimurium/S. sonnei were added. Subsequently, the plate was incubated with biotinylated cLf then streptavidin-peroxidase, and developed with TMP; peroxidase substrate. The plate OD was recorded at 450 nm, the results were demonstrated as mean ± SD of eight replicates.

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM)

To track morphological changes both extracellularly and intracellularly after S. typhimurium and S. sonnei treatment by an antibiotic (carbenicillin) or cLf alone (labeled with AuNPs) and in combination, scanning and transmission electron microscopy were used as previously reported (Almehdar et al., 2019; Almehdar et al., 2020).

Protein content evaluation

The content of test proteins (lactoferrins either labeled or free) was estimated by Bradford assay. Determination of the remaining proteins present in the supernatant solution from combining washes was used to indirectly evaluate the labeled proteins. The amount of bound or labeled protein was determined by difference spectrophotometry.

Statistical analysis

The experiments were carried out in triplicate and the obtained results were demonstrated as mean ± SD of triplicate. Data analysis was done by using Student’s t-test and McNemar’s test. A P-value of less than 0.01 was regarded as statistically significant.

Antimicrobial activity examination

Salmonella typhimurium LT2 was susceptible to 13 of the 17 tested antibiotics, but showed resistance to vancomycin, fucidic acid, oxacillin, and ampicillin (Table 1). While S. sonnei ATCC 25931 was susceptible to 10 of the 17 tested antibiotics and showed resistance to oxacillin, ampicillin, augmentin, cefoxitin, ceftazidime, aztreonam, and piperacillin (Table 1). The corresponding experiments showing the microbial resistance to antibiotics were conducted to confirm that we are carrying out our study on the pathogenic multiple antibiotic resistant strains of S. typhimurium and S. sonnei. The in vitro antibacterial activity of cLf subtypes isolated from different clans of Saudi camels (cLf1, cLf2, cLf3, and cLf4), bLf, or hLf against S. typhimurium and S. sonnei was evaluated by measuring their inhibition zones at different Lf concentrations in agar disc-diffusion assay. All lactoferrins efficiently inhibited S. typhimurium and S. sonnei growth, producing concentration-dependent inhibition zones (Table 2). We noticed that bLf and hLf were able to inhibit S. typhimurium growth at concentrations ranging from 0.75 to 3 mg/ml and S. sonnei growth at concentrations ranging from 1 to 3 mg/ml, whereas all camel lactoferrins could inhibit the growth of both bacterial strains at concentrations ranging from 0.125 to 3 mg/ml (Table 2). This suggests the superiority (six and eight folds) of cLf antibacterial activity against S. typhimurium and S. sonnei, respectively over that of both hLf and bLf. No significant differences in antimicrobial activity were observed between cLf subtypes.

Inhibitory effects of lactoferrins on S. typhimurium and S. sonnei

After incubation of S. typhimurium and S. sonnei with various concentrations of bLf, hLf, and different cLfs, it was observed that all cLfs inhibited the growth of both bacterial strains completely after incubation for 3–24 h at concentrations of 1–3 mg/ml, 6–24 h at a concentration of 0.75 mg/ml, 12–24 h at a concentration of 0.5 mg/ml, and 24 h at concentrations of 0.125−0.25 mg/ml (Table S1). On the other hand, bLf and hLf could inhibit S. typhimurium growth completely after incubation for 3–24 h at concentrations of 1.5–3 mg/ml, 6–24 h at a concentration of 1 mg/ml, and 12–24 h at a concentration of 0.75 mg/ml. Both bLf and hLf also inhibited S. sonnei growth completely after incubation for 3–24 h at concentrations of 2–3 mg/ml, 6–24 h at a concentration of 1.5 mg/ml, and 12–24 h at a concentration of 1 mg/ml. No antibacterial effects were noticed after incubation with hLf or bLf at concentrations of 0.125−0.5 mg/ml in the case of S. typhimurium and at concentrations of 0.125−0.75 mg/ml in case of S. sonnei, suggesting superior activity of camel lactoferrins (Table S1). Furthermore, the data presented in Table S1 demonstrate that the culture OD decreases over time in the presence of lactoferrins before reaching complete inhibition of growth suggesting that Lfs not only inhibit bacterial growth but also induce cell death via lysis. Since no difference was found between the different types of camel lactoferrin in their inhibitory effectiveness, we used only cLf1 in the remaining parts of this work.

Determination of MIC values of antibacterial agents by broth microdilution assay

The MIC values of cLf, and hLf or bLf against S. typhimurium were calculated to be 125 and 750 µg/ml, respectively. While MIC values of cLf, and hLf or bLf against S. sonnei were calculated to be 0.125 and 1 mg/ml, respectively. These values confirm the superior (six and eight folds against S. typhimurium and S. sonnei, respectively) antibacterial effect of cLf. MIC values determined for chloramphenicol, cefepime, and imipenem against S. typhimurium and S. sonnei were 1.3, 1.7, and 1.9 µg/ml, respectively. We also found that the inhibitory activity of lactoferrins was reversed after addition of iron as FeNTA to bacterial cultures. The MIC values obtained for cLf and hLf or bLf against S. typhimurium after iron addition were 1 and 3 mg/ml, respectively, and against S. sonnei were 1 and 4 mg/ml, respectively. The reported here reverse of the inhibitory activity means that Lf antimicrobial mechanisms include exhibiting bacteriostatic effect based on the Lf ability to bind free iron (at concentration lower than 2 × MIC), and this bacteriostatic effect can be reversed by iron supplementation as observed in this study and our previous work (Redwan et al., 2016; Almehdar et al., 2019; Almehdar et al., 2020).

Checkerboard assay

The synergy between the studied antibiotics and cLf, hLf, or bLf against S. typhimurium and S. sonnei was evident from the corresponding FICI values ranging from 0.37 to 0.5 (Table 3). This was further explored by examining the changes induced in the morphology of bacterial cells caused by these agents alone or combined under electron microscopy.

Time-kill assay

Figure 1 demonstrates the time-kill study data as changes in the log₁₀ CFU/ml of S. typhimurium and S. sonnei after either 4 h or 8 h of incubation with each Lf alone, and combined with different antibiotics. The reduction in viable cell count was greatest after 8 h of incubation with cLf combined with chloramphenicol (−4.334 log₁₀ and −4.230 log₁₀) in the case of S. typhimurium and S. sonnei, respectively. Also, it was observed that all combinations were significantly more effective at reducing viable bacteria count than Lf alone even at a concentration of 2 × MIC (Fig. 1).

Calculation of minimum bactericidal concentration

All tested Lfs can be considered as bacteriostatic agents against S. typhimurium and S. sonnei after resuming bacterial growth of MIC wells with no growing bacteria. However, cLf and hLf/bLf achieved bactericidal activity against S. typhimurium and S. sonnei only at a concentration of 2 × MIC; 0.25 mg/ml and 1.5 mg/ml, respectively in the case of S. typhimurium and 0.25 mg/ml and 2 mg/ml, respectively in case of S. sonnei. Since the ratio of MBC/MIC for all test lactoferrins was equal to two, they were labeled as bactericidal agents. These data also confirm the superior antibacterial activity of cLf.

Analysis of interaction between the biotinylated lactoferrins and S. typhimurium/S. sonnei LPS

We failed many times in running LPS of S. typhimurium or S. sonnei on SDS-PAGE then transferring them to nitrocellulose blots, and only ELISA could detect LPS possibly because of the interference of SDS with the bacterial LPS as we mentioned in previous studies (Almehdar et al., 2019; Almehdar et al., 2020). Nevertheless, silver nitrate could stain LPS of S. typhimurium and S. sonnei on SDS-PAGE (data not shown).

ELISA results of lactoferrins interaction with S. typhimurium or S. sonnei LPS demonstrated a significant (p < 0.01) interaction between all tested Lfs and S. typhimurium or S. sonnei LPS (Fig. 2). Camel lactoferrin was more reactive (p < 0.001) than bLf and hLf against S. typhimurium or S. sonnei LPS, with no evident differences between the cLf sub-types. These observations reveal that the cLf sub-types (cLf1, cLf2, cLf3, and cLf4) isolated from milk of four different breeds of Saudi camels have possibly no differences in their glycosylation moieties that are involved in the interaction between the LPS and lactoferrin, which needs further comprehensive investigation.

Effect of the different antibacterial agents on the integrity of the S. typhimurium/S. sonnei cells and their protein profile

Table 4 and Fig. 3 reveal the effect of antibiotics (carbenicillin, chloramphenicol, or imipenem), cLf, and cLf-antibiotics combinations on the integrity of S. typhimurium or S. sonnei cell (protein released from treated cells was measured by Bradford assay at 595 nm). Furthermore, Table 5 described the effect of antibiotics, cLf, and their combinations on protein profile of S. typhimurium or S. sonnei cell (protein appearance, disappearance, and/or high expression). Protein profile of S. typhimurium or S. sonnei cell clearly changed on SDS-PAGE over time (from 30 min to 150 min) depending on the used antibacterial agents in treatment. These results obviously indicate that cLf and antibiotics either alone or in combination exert significant (p < 0.01, and p < 0.001) effect on S. typhimurium or S. sonnei cell integrity (impairing the integrity of the bacterial cells and their membranes, which lead to release of cell protein content) and change bacterial protein profile. Additionally, these effects increase over time. The presence of synergism between cLf and antibiotics with regard to their effect on the cell integrity and protein profile of the two bacterial strains was also observed.

Separation of bacterial membrane proteins fraction and analysis of their interactions with cLf-biotin

The BMP extract of S. typhimurium or S. sonnei was resolved on SDS-PAGE (Fig. 4). The profile of membrane proteins was found to be different from the profile of whole-cell lysate (Fig. 4). Results of western blotting confirmed the interaction of cLf-biotin with multiple protein receptors of S. typhimurium or S. sonnei, and Table 6 summarizes the bacterial membrane proteins of S. typhimurium or S. sonnei that could interact with cLf, and showed that each strain has its distinctive binding membrane proteins for cLf.

ELISA plate coated with BMP crude extract of S. typhimurium or S. sonnei exposed to cLf-biotin showed significant signals (p < 0.001) (Fig. 5). From these data and those of Lf-LPS interaction analysis (Fig. 2), we propose that cLf could kill S. typhimurium and S. sonnei via a bactericidal mechanism that involves binding to bacterial LPS and membrane proteins.

SEM and TEM analyses

The SEM examination of S. typhimurium in Figs. 6A–6D and S. sonnei in Figs. 7A–7D demonstrated that treated bacterial cells showed irregular and wrinkled outer surfaces, adhesion and aggregation of damaged cells, fragmentation, or the presence of crushed cells and cellular debris. These observations were more evident with cLf-carbenicillin treated cells indicating a clear synergistic effect of both agents on S. typhimurium and S. sonnei. On the other hand, TEM experiments (Figs. 6E–6H and Figs. 7E–7H) showed deformation of cell membrane and wall, and the gold nanoparticles (black dots) can be easily observed both on and inside the treated cells. The cLf-carbenicillin combination treatment showed the clearest deformation signs; disintegration of cell membrane and wall, bigger cell size, loss of regular cellular shape, and separation of the cell wall (manifested as zigzag or erosion) from the cell.