Discovery of potential anti-infectives against Staphylococcus aureus using a Caenorhabditis elegans infection model

Bacterial and nematode strains

The methicillin-susceptible S. aureus strain NCTC8325-4 [19] and methicillin-resistant S. aureus (MRSA) strain ATCC33591 were grown with aeration in Trypticase Soy (TS) media (Oxoid/Pronadisa) at 37°C while Escherichia coli strain OP50 was grown in Luria Bertani (LB) broth supplemented with streptomycin (100 μg/mL). The wild type C. elegans Bristol N2 hermaphrodite strain was propagated on nematode growth medium (NGM) and fed on the standard laboratory food source, E. coli OP50. The animals were age-synchronized by bleaching with alkaline hypochlorite and sodium hydroxide to release embryos. Embryos were placed on plates containing concentrated E. coli DH5α at 25°C until the worms reached the young adult stage. To eliminate the confounding effects of reproduction on the scoring of surviving worms, wild type C. elegans were made sterile by RNAi knockdown of the pos-1 gene which resulted in worms laying unhatched eggs [23]. The pos-1 RNAi treated worms were grown for ~45 hrs at 25ºC until they reached young adult stage and were ready to be used in the screen.

Preparation of extracts and compounds

A total of 18 plant species and one marine sample were the source of the natural extracts. Extracts UE-01-1 to UE-08 were extracted according to the previously described method [24]. Briefly, the plant materials and animal sample were collected from several locations in Malaysia. The samples were authenticated, deposited and catalogued with specimen voucher numbers (Additional file 1). All samples were then air-dried, powdered and extracted with polar and non-polar solvents. Solvents were then removed by evaporation using a rotary evaporator. Extracts UE-09 to UE-20 (except UE-15) were prepared by Herbal Science Pte. Ltd., a local pharmaceutical company. In brief, the raw materials were collected and quality assessed through a series of stringent microbial tests as well as heavy metals test using an Atomic Absorption Spectrophotometer (AAS). They were then dried, comminuted or ground into fine powder before water-based extraction was performed. The microbial and trace metal element tests were repeated again on the yield obtained. The finished products were examined for the presence of certain functional groups/molecules using Fourier transform infrared spectroscopy (FTIR) and High-performance liquid chromatography (HPLC). UE-15 was purchased from Merck, Germany.

Four different series of butylated hydroxytoluene (BHT) derivatives (Figure 1) were designed and synthesized by the Chemistry Department, University of Malaya, Malaysia. Thiosemicarbazides were synthesized according to the method of Yehye et al. [25,26]. 1,3,4-thiadiazoles and 1,2,4-triazole bearing the free radical scavenger BHT were synthesized using acid-(base-) catalyzed intramolecular dehydrative cyclization reaction of the 1-acylthiosemicarbazides whilst hydrazones were synthesized from the reaction between free amino groups and properative aldehydes (Yehye et al., in preparation). All extracts and compounds were dissolved in dimethyl sulfoxide (DMSO), filtered through a 0.2 μm membrane filter and stored at −20°C. Detailed information on all natural extracts and synthetic compounds used in this study is provided in Additional file 2.

Figure 1

Chemical structure of butylated hydroxytoluene (BHT) and stilbenes. (A) BHT, with its IUPAC name 2,6-bis(1,1-dimethylethyl)-4-methylphenol, is chemical derivative of phenol (C₆H₅OH) [25]. (B) and (C) refer to the two isomers of stilbene (IUPAC name: 1,2-diphenylethene). (B) (Z)-stilbene or cis-stilbene and (C) (E)-stilbene or trans-stilbene.

C. elegans survival assay

For the agar-based assay, 10 μL of a S. aureus overnight culture in TS broth was spread on 3.5 cm TS agar plates and incubated at 37°C for 24 hrs. The plates were then allowed to equilibrate to room temperature for at least 1 hour before use. A total of 40 young adult worms were transferred to the agar plate and incubated at 25°C. Worm mortality was monitored at 24-hour intervals. E. coli OP50 on NGM was used as the negative control. Three independent experiments were performed.

For the liquid-based assay, a 24-well plate was filled with S. aureus/MRSA in a liquid medium (80% M9 buffer, 20% S. aureus overnight culture and 10 μg/mL cholesterol) and 20 synchronized young adult nematodes were transferred into each well. Wells containing C. elegans, E. coli OP50 and M9 buffer served as the control. The total number of worms for each treatment was 120 in six wells representing six technical replicates. The plate was incubated at 25°C and survival was monitored every 24 hrs following exposure to the pathogen.

Anti-infective screening

Screening was performed in liquid medium in a 24-well plate. The liquid screening medium consisted of worm M9 buffer and overnight culture of S. aureus/MRSA in TS broth with 10 μg/mL of cholesterol at a ratio of 4:1 (v/v). The medium was supplemented with either natural extracts or synthetic compounds to a final concentration of 200 μg/mL (natural extracts) and 200 μM (synthetic compounds). All components of the assay were mixed evenly and 500 μL of medium was transferred into each well in a 24-well plate. In control wells, the extract or synthetic compound was replaced with 1% DMSO or S. aureus was replaced by E. coli OP50 as the nematode food source. Each extract or compound was tested in triplicate wells. Approximately 10–15 age-synchronized pos-1 treated young adult stage N2 nematodes were transferred into each well and the plate was incubated at 25°C. Worms treated with pos-1 RNAi produced unhatched eggs thereby eliminating potential complications during the scoring of worms. Worm survival was scored manually every 24 hrs for 5 days. In addition, turbidity of the extract/compound containing media was also observed and recorded. An extract or compound was considered a positive hit if >50% of the infected worms survived compared to ≤ 20% survival in the untreated control worms in least two replicates. All positive hits from the primary screen were subjected to a secondary screen for confirmation. A summary of the one-step anti-infective liquid screen approach used in this study is presented in Additional file 3 and is compared to the previously reported two-step agar-liquid protocol used by Moy et al. [8] for screening of antimicrobials against Enterococcus faecalis and high throughput liquid-based chemical screen to screen for molecules that attenuate Pseudomonas aeruginosa virulence and rescue C. elegans from infection [11].

Determination of antimicrobial property by disc diffusion assay

The disc diffusion method [27] was used to determine if the extracts and compounds also promoted antimicrobial features. Sterile filter paper discs (Whatman No. 1, 6 mm) were impregnated with each of the extracts (20 mg/mL) or compounds (20 mM) and left to dry under sterile conditions overnight. S. aureus was spread evenly onto the surface of TS agar plates using glass beads before the discs were positioned on the inoculated agar surface. The bacterial inoculum size was standardized at 10⁶ cfu/mL by adjusting the optical density of the bacterial suspension. DMSO served as the negative control with gentamicin (200 μg/mL) as the positive control. All plates were incubated for 18 hrs at 37°C. Antibacterial activity was confirmed by the presence of a clear zone of inhibition around the disc.

Determination of MIC and MBC

The bacteriostatic and bactericidal effects of all positive extracts and compounds were evaluated using the broth microdilution minimum inhibitory concentration (MIC) assay [28]. A range of different concentrations of extract/compound was prepared in a 48-well plate, followed by inoculation with 10⁶ cfu/mL S. aureus. The plate was then incubated at 37°C for 18 hrs. Gentamicin, the standard antibiotic for S. aureus infections, was used as a control. All extracts/compounds were assayed in triplicate. The MIC endpoint recorded was the lowest concentration of the extract/compound used in wells that showed no turbidity after 18 hrs incubation, indicating an absence of bacterial growth. The presence or absence of turbidity was confirmed by absorption readings at 600 nm. The minimum bactericidal concentration (MBC) was determined by spreading the culture from the respective wells on sterile agar plates. The MBC value was the lowest concentration of extract/compound used where no apparent growth of bacteria was observed on the agar plate.

Enumeration of bacterial colony forming units (CFU) within the C. elegans gut

After 24 hours exposure to S. aureus in liquid medium in the presence and absence of extracts/compounds, 10–12 live worms were randomly picked and briefly anesthetized in 25 mM Levamisole. The worms were washed at least twice in 200 μL antibiotic cocktail comprising 25 mM Lev and gentamicin 10 μg/mL followed by incubation for 45 min to 1 hour to completely kill bacterial cells associated with the worm cuticle. Then, the worms were washed three times with 200 μL of 25 mM Lev to eliminate the killed bacteria and residual antibiotic. The number of worms were recorded followed by mechanical disruption in 50 μL of 1% Triton X (Sigma-Aldrich, USA; X100) using a motorized pestle. Serial dilutions were performed on the worm lysates and 10 μL of each dilution was spotted on TS agar supplemented with 5 μg/mL nalidixic acid. Colonies were counted after incubating the plates at 37°C overnight. Bacterial CFU per worm was calculated using the formula reported by Ooi et al. [29].

Statistical analysis

All data are presented as mean ± standard deviation (SD) of at least two independent experiments. All screening assays, antibacterial tests and in vivo CFU assay were replicated in a comparable manner. Data from the killing assays were analyzed with StatView® 5.0.1 (SAS Institute, Inc) and plotted using the Kaplan-Meier Cumulative Survival Plot for Time (nonparametric survival analysis). The pair wise comparison was analyzed using the Log-rank (Mantel-Cox) significance test.

Liquid-based screen was able to detect more hits

Most studies on C. elegans infections by S. aureus are routinely performed on agar plates. In this study, we observed significant reduction in the lifespan of C. elegans infected by S. aureus in both the agar-based and liquid-based assays (Figure 2A and B). Specifically, the mean time to death (TD_mean) for S. aureus-infected animals assayed on agar was 73 ± 1.5 hrs which was consistent with previous studies [20]. Similarly, in the liquid-based assay, worms in M9 buffer and S. aureus culture (4:1, v/v) had a TD_mean of 72 ± 5 hrs. S. aureus required 4 to 5 days for complete killing of C. elegans. In liquid medium, the worms infected by S. aureus started to die after 24 hrs exposure to the pathogen. Mobility, pharyngeal pumping and foraging rates of worms exposed to pathogens progressively decreased with time until all worms became immobile and died. The results confirmed that in liquid medium, S. aureus is able to kill the nematodes as efficiently as the standard agar-based assay system.

Figure 2

S. aureus kills C. elegans in both liquid and agar medium. Representative survival curves of C. elegans following infection by S. aureus strain NCTC8325-4 in (A) liquid medium consisting of 80% M9 buffer, 20% S. aureus overnight culture and cholesterol and (B) TS agar medium. Graph (A) shows the mean ± SD of six replicates (20 nematodes/replicate) and graph (B) presents the mean ± SD of three replicates (40 nematodes/replicate) from a representative of three independent assays.

To establish the utility of the liquid assay for anti-infective screening, we screened a subset of 18 natural extracts using both liquid- and agar-based assays. In the agar-based assay, extracts were added into the TS agar whilst the prepared agar was in a molten state. Nematode survival on extract-added plates was compared to the control (agar without extract). Only 1/18 demonstrated potential anti-infective properties when compared to controls in the absence of extract on solid agar (Figure 3). By contrast, when the assay was performed in the liquid-based assay, we were able to detect 7/18 potential hits that reproducibly extended the lifespan of S. aureus-infected worms in at least two independent experiments (Figure 3). These results indicated that screening using a liquid-based assay was able to detect more hits than the conventional agar-based assay. Thus, we carried out the anti-infective screen on the rest of the extracts and compounds using the liquid-based screen.

Figure 3

Liquid-based assay identified more hits than the agar-based assay. Comparison of hits from the preliminary screen of 18 natural extracts on both agar-based and liquid-based assays. All natural extracts were tested at same concentration (200 μg/mL) in both agar and liquid assays. The graph represents the mean percentage of survival ± SD at 72 hours post-infection of three replicates from a representative of two individual experiments. The survival of extract-treated worms is compared to the control (no extract). (*) indicates positive in agar-based screens while (**) indicates positive in liquid-based screens.

Direct comparison of the hits identified from both the liquid and agar based screens (Figure 3) confirmed that only UE-08 promoted the survival of infected worms in both agar-based and liquid based screens. UE-01-1, UE-01-2, UE-01-4, UE-01-6, UE-01-7 and UE-03-5 were able to improve the lifespan of infected worms in liquid medium but not in agar and this could be due to thermal degradation of certain active molecules (saponins, flavonoids) during the media preparation. Further comparisons between the solid agar and liquid-based screens are elaborated in Table 1.

Selected extracts and compounds enhanced the survival of S. aureus-infected nematodes

We performed a small scale screen in 24-well plates using the S. aureus – C. elegans liquid infection assay to identify more potential anti-infectives that improved the survival of infected worms. Screening over 37 natural plant and marine invertebrate extracts and 29 synthetic compounds revealed a total of 14 natural extracts and 14 synthetic compounds that conferred increased survival of S. aureus-infected nematodes (Figure 4). The criteria adopted to classify an extract or compound as a positive hit was the survival of >50% of the infected worms compared to ≤ 20% survival in untreated wells over at least two replicates. All positive hits contributed to more than 60% C. elegans survival following infection and treatment for 72 hrs compared to about 20% survival in untreated controls (Table 2). All positive natural extracts increased nematode survival by 2.8-fold or more while all positive synthetic compounds exhibited a more significant increase in survival of at least 4-fold. The worms fed on E. coli OP50 remained alive throughout the assay. Detailed information on all positive hits is shown in Table 2.

Figure 4

Extracts and compounds rescued the worms from a S. aureus infection. Percentage of survival for S. aureus-infected nematodes upon exposure to extracts (200 μg/mL) and compounds (200 μM) in the primary anti-infective screen. The graph depicts the percentage of survival at 72 hours post-infection when the assay was carried out in the absence of presence of different extracts/compound. Results are expressed as mean ± SD from a representative of at least two individual assays. Dashed lines show the percentage of survival for untreated worms (no extract/no compound) while straight lines demarcate the positive hits. Extracts and compounds that contributed to > 50% infected worm survival were considered as positive in the primary screen.

The striking differences in the appearance of live worms and worms that succumbed to infection facilitated the screening process. The infected worms were generally smaller in size as compared to healthy worms fed on E. coli OP50. Dead worms did not move or exhibit muscle tone and did not respond to external stimuli. They appeared as straight rigid rods in the medium with no observable pharyngeal pumping (Figure 5A). In contrast, worms exposed to E. coli OP50 maintained a sinusoidal ‘S’ shape and moved actively. These worms grew in size and developed into gravid adults which produced eggs (Figure 5B). With the addition of extract/compound that exhibited potential anti-infective properties, the worms were rescued from the S. aureus infection as demonstrated by their ability to overcome the debilitating effects of the infection. As indicated in Figure 5C, the surviving infected nematodes appear to be smaller in size, similar to that of the untreated infected worms. Nevertheless, the live worms were able to maintain the sinusoidal ‘S’ shape posture and respond to external stimuli when touched. In addition, the worms continued to move actively even after 72 hrs exposure to S. aureus. Although the worms were able to survive upon S. aureus infection in the presence of hit extracts/compounds, most of the live worms appeared to experience delayed development as compared to healthy worms fed on E. coli OP50.

Figure 5

Distinct appearances of dead and alive C. elegans. Representative micrographs of C. elegans exposed either to S. aureus (A), E. coli OP50 (B) or S. aureus in the presence of extracts/compounds (C). The worms were examined under a stereomicroscope (Nikon SMZ645) at a magnification of x40. White arrows denote the worms.

Among 18 plant species tested, extracts from six plants (N. fruticans, S. macrophylla, C. longa, E. longifolia, O. stamineus and S. eburneum) were able to extend the lifespan of S. aureus-infected animals. The marine sample (F. ater) also possessed potential anti-infective activity. N. fruticans, S. macrophylla, C. longa, E. longifolia and O. stamineus are commonly found in Malaysia and used widely by the local community as folk medicine. Hence, local traditional herbs are a promising source of largely unexplored classes of anti-infectives.

We tested seven samples from N. fruticans obtained from different parts of the plant through the use of different extraction solvents. Our screen identified 5/7 N. fruticans samples that increased the survival of infected nematodes by 2.9 to 3.7 fold. Different parts of the plant, for example, leaves, roots and husks, may contain different sets of bioactive compounds whilst the different extraction solvents used may also separate different bioactive constituents. Alternatively, the differences may be due to variations in the quantity of the individual active components in these tissues. Both C. longa and curcumin showed an equally strong effect by enhancing the survival of S. aureus-infected nematodes by at least 4-fold (Table 2). Curcumin is the main active ingredient in C. longa suggesting that curcumin might be the contributing bioactive component of C. longa. Methanol, butanol and ethyl acetate extracts of S. macrophylla were also able to prolong the lifespan of S. aureus-infected worms with the butanol extract showing the most enhanced effect, whereby worm survival increased by 4.2-fold. Of all the extracts tested, the S. macrophylla butanol extract, curcumin and S. eburneum seed extract demonstrated very promising anti-infective activity by promoting the survival of S. aureus-infected worms by more than 4-fold.

With the synthetic compounds, all 14 hits promoted the survival of infected worms by at least 4-fold with UC-15, UC-16, UC-27 and UC-28 demonstrating the most prominent effect, whereby worm survival increased by 5-fold. Compounds UC-01, UC-02 and UC-03 are variations of thiosemicarbazide, UC-07, UC-08, UC-09, UC-10, UC-11 and UC-12 were synthesized based on of 1,2,4-triazole-5-thiones, UC-15 and UC-16 are derived from hydrazones, UC-27 and UC-28 are stilbene derivatives while UC-06 is a variant of 1,3,4-thiadiazole. With the exception of stilbene, all other compounds were fused to a BHT moiety. The structures of all 14 positive synthetic compounds are shown in Figure 6.

Figure 6

Structure of the compounds that promoted the survival of S. aureus infected nematodes. Compounds UC-01, UC-02 and UC-03 are thiosemicarbazides, UC-06 is 1,3,4-thiadiazoles, UC-07, UC-08 and UC-09 are 1,2,4-triazole-5-thiones, UC-10, UC-11 and UC-12 are 4-(substituted benzylideneamino-1,2,4-triazole-5-thiones, UC-15 and UC-16 are hydrazones and UC-27 and UC-28 are stilbene derivatives. All compounds, with the exception of the stilbene derivatives, are fused to a BHT moiety.

As illustrated in Figure 7, different extracts/compounds exhibited different degrees of protection in extending the lifespan of nematodes following S. aureus infection. During the screening process, we also took note of changes in media turbidity indicative of bacterial presence and growth. Upon addition of selected extracts and compounds, for example UE-08 and compound UC-15, into the medium, a significant clearing of the medium was noted. When we correlated this observation to the MIC and MBC results of all positive hits as shown in Table 3, we noted that extract UE-08 and compound UC-15 possessed strong antibacterial activity against S. aureus. The survival curves presented in Figure 7A and B reflect that both these hits exerted a strong protective effect on the worms and ~100% of worms were rescued from the lethal infection throughout the experiment (over 5 days). Based on this, we propose that UE-08 and UC-15 extend the lifespan of worms during a S. aureus infection through their antimicrobial activity against S. aureus.

Figure 7

Survival of C. elegans is improved over time upon supplementation of extracts and compounds. (A and B) The survival curves of S. aureus-infected C. elegans upon exposure to selected hit extracts (200 μg/mL) and compounds (200 μM). Enhanced survival of infected worms can be seen in the presence of extracts UE-01-2, UE-03-5, UE-08 (A) and compounds UC-10, UC-12 and UC-15 (B). Data at each time point are the average of three wells with each well containing approximately 10–15 animals per well. In a pair-wise comparison to no extract using log-rank tests, the difference between all of the treatments was significant (p < 0.0001).

In contrast, upon supplementation of the other selected extracts and compounds into the screening media, we observed a cloudy appearance (similar to the untreated control) of the media indicating continued bacterial growth. We confirmed our observations by measuring the turbidity at 600 nm. Nevertheless, addition of these extracts and compounds significantly delayed the killing of worms by S. aureus. Data for lifespan extension by representative hits (extracts UE-01-2 & UE-03-5 and compounds UC-10 & UC-12) that exhibited this phenomenon are presented in Figure 7. All these hits either did not kill S. aureus or only inhibited S. aureus growth at very high concentrations (Table 3). As they did not affect S. aureus viability at the tested concentrations, we propose that extracts UE-01-2 & UE-03-5 and compounds UC-10 & UC-12 enhance the survival of S. aureus-infected worms using other distinct mechanisms such as reducing bacterial virulence and/or by enhancing host immunity.

Positive hits exhibited different anti-S. aureus activity

The disc diffusion assay and MIC test were used to evaluate the positive hits obtained from the anti-infective screen for their ability to inhibit S. aureus growth. The disc diffusion assay detected two natural extracts (UE-08 & UE-15) and four synthetic compounds (UC-01, UC-15, UC-27 and UC-28) out of 28 positive hits, with antibacterial effect on S. aureus (Figure 8). To further validate the hits, we performed the broth microdilution MIC test to obtain both MIC and MBC values.

Figure 8

Inhibition of S. aureus growth by extracts and compounds. The clear zone formed around the discs indicated that S. aureus was susceptible to extract UE-08 (A) and compounds UC-27 & UC-28 (B). The negative control (DMSO) showed no clear zone around the disc while the positive control (200 μg/mL gentamicin) suppressed the growth of S. aureus and formed a 20 mm zone of inhibition around the disc.

The respective MIC and MBC values of all 28 positive hits in standard medium (TS broth) and screening medium (20% TS broth + 80% M9 medium) are presented in Table 3A and B. The negative control (DMSO) and positive control (gentamicin) were run in parallel with a recorded MIC for gentamicin towards S. aureus at 1.2 μg/mL and MBC of 2.4 μg/mL. As the clearing of the liquid medium has been used as a criterion during the anti-infective screening, we also performed the MIC microdilution test using the screening medium. We found relatively similar values for both media tested. Of the positive hits screened, two natural extracts (UE-01-1 and UE-08) were found to inhibit S. aureus growth at a concentration of < 200 μg/mL while six compounds (UC-01, UC-07, UC-11, UC-15, UC-27 and UC-28) exhibited bacteriostatic effects at a concentration of ≤ 200 μM. Of note is compound UC-15, the most potent antimicrobial agent which suppressed S. aureus growth at concentrations less than 62.5 μM in both standard medium and screening medium. In the anti-infective screens discussed above, both 200 μg/mL natural extracts and 200 μM synthetic compounds were the working concentrations used. A further five natural extracts (UE-01-2, UE-01-6, UE-01-7, UE-15 and UE-18) and seven synthetic compounds (UC-02, UC-03, UC-06, UC-08, UC-09, UC-12 and UC-16) affected S. aureus growth in vitro but at ~ 2-fold higher concentrations than that used in the screen. Interestingly, seven natural extracts (UE-01-4, UE-02-4, UE-03-3, UE-03-5, UE-03-6, UE-11 and UE-12) and one synthetic compound (UC-10) which enhanced the survival of S. aureus-infected nematodes, did not exhibit any bacteriostatic or bactericidal activity on S. aureus growth in vitro. Collectively, the C. elegans model-based screen identified eight hits (29%) that inhibited S. aureus growth at concentrations lower than that used in the screens, 12 (42%) hits that suppressed S. aureus growth at concentrations at least two-fold higher than the screen concentrations and eight hits (29%) that had no effect on S. aureus growth in vitro even though they were effective in curing the worms from infection.

Both C. longa and curcumin demonstrated anti-S. aureus properties at different concentrations with C. longa inhibiting S. aureus growth at a lower concentration (<125 μg/mL) than curcumin (500 μg/mL). This suggests that the C. longa extract may contain multiple bioactive compounds that target S. aureus. Out of five N. fruticans positive samples, 4 of 5 were capable of inhibiting S. aureus growth with the husk methanol extract exhibiting the strongest activity.

All synthetic compounds, with the exception of UC-10, demonstrated antibacterial activity towards S. aureus at different concentrations. A comparison between all 14 hits and antibiotics currently used in a clinical setting did not reveal any structural relationship. However, some of the variants from these groups of compounds have previously been reported as potential antibacterials. UC-01, UC-02 and UC-03 are thiosemicarbazides with different elements attached to different positions of the second benzene ring and thiosemicarbazide derivatives have known antimicrobial and antifungal properties [30,31]. Derivatives of thiadiazoles and triazole-thiones (UC-06, UC-07, UC-08, UC-09, UC-11 and UC-12) also showed an inhibitory effect towards S. aureus at concentrations ranging from 200 – 2000 μM. Compounds containing a 1,3,4-thiadiazole nucleus and 1,2,4-trizole-thiones have a wide range of pharmacological activities including potential antimicrobial properties [32,33]. On the other hand, hydrazone derivatives are also active against S. aureus and P. aeruginosa[34]. Natural stilbenes are typical plant metabolites (e.g. resveratrol in grapes) which play a role in defending the host against microbial attack [35]. Stilbene variants such as hydroxystilbene and isopropylstilbene are potential antimicrobials and show a synergistic effect when applied together with antibiotics to inhibit and kill bacteria [36]. Our data consistently demonstrated that both stilbene derivatives (UC-27 and UC-28) showed bactericidal effect towards S. aureus at 200 μM. Despite ample evidence indicating the antimicrobial activity of the derivatives of these compounds, the exact mechanism on how these molecules inhibit/kill bacteria remains unknown.

Positive hits also rescued worms from MRSA infection

From the anti-infective screen and antimicrobial susceptibility test, we identified seven natural extracts (UE-01-4, UE-02-4, UE-03-3, UE-03-5, UE-03-6, UE-11 and UE-12) and one synthetic compound (UC-10) which significantly increased survival of infected nematodes without affecting S. aureus replication. The S. aureus strain NCTC8325-4 used in the screen is a methicillin-sensitive S. aureus (MSSA) [37]. To further explore the potency of these eight candidates, we extended our study by determining their ability in promoting the survival of MRSA-infected worms.

As the susceptibility of C. elegans towards MRSA strain ATCC33591 has not been studied before, we performed the survival assay in liquid medium to assess the virulence of this MRSA strain towards wild-type C. elegans. Our results showed that MRSA ATCC33591 killed all nematodes after 5 days post-infection with a TD_mean of 68 ± 3.2 hrs which is comparable to the MSSA strain NCTC8325-4 (Figure 9A). Interestingly, when we evaluated the eight hits from the MSSA screen, 5/8 were also able to rescue the worms from MRSA infection. The five natural extracts that significantly promoted the survival of MRSA-infected nematodes after 72 hours infection were UE-01-4, UE-03-3, UE-03-5, UE-03-6 and UE-12 (p < 0.005). All five extracts contributed to more than 70% C. elegans survival upon MRSA infection compared to almost 80% death of untreated worms (Figure 9B). Specifically, the five extracts that protected the worms from both antibiotic-sensitive and antibiotic-resistant S. aureus infection were extracted from N. fruticans roots, S. macrophylla seeds and O. stamineus leaves.

Figure 9

Nematodes infected by MRSA are rescued by natural extracts. (A) Kinetics of the killing of C. elegans by MRSA ATCC33591, S. aureus NCTC-8325-4 and E. coli OP50 in liquid medium. Graph shows the mean ± SD of six replicates (20 worms / replicate) from a representative of three independent experiments. (B) Survival rate of MRSA-infected nematodes upon exposure to selected extracts (200 μg/mL)/compound (200 μM). Graph shows the percentage of survival at 72 hours post-infection when the assay was carried out without any treatment or treatment with different extracts/compound. Results are expressed as mean ± SD from a representative of two individual assays. (**) denotes significant difference in percentage of survival between untreated (no extract) and treated animals (p < 0.005).

The survival of MRSA-infected nematodes exposed to UE-02-4, UE-11 and UC-10 was less than 40% (Figure 9B) indicating that these three hits were only efficacious for MSSA but not MRSA infections. Based on this, we suggest that these compounds may be able to render the antibiotic-sensitive strain less virulent but not the antibiotic-resistant strain. It was possible that these hits function as a specific inhibitor of virulence determinants that are complex and strain specific.

Two natural extracts caused reduction in intestinal bacterial loads

The killing of C. elegans by S. aureus is correlated with the ability of the bacteria to colonize the worm intestine [20]. S. aureus do not persist in the host, yet when the host has been in contact with the bacteria for an adequate period of time, death is observed. Similarly, Pseudomonas aeruginosa PA14 also colonize and distend the worm intestinal lumen but do not persist within the host [38]. To address the question of whether the level of live S. aureus in the worm intestine remained the same in the presence of extracts, we measured the number of bacteria by performing a CFU assay. In vivo CFU counts were enumerated at 12 and 24 hours when >70% of the infected non-treated population remained alive. For this assay, we tested the eight hits that showed no interference with S. aureus viability in the antimicrobial test.

In the absence of extract, S. aureus grossly colonized the worm intestine and the in vivo CFU counts for S. aureus-infected worms was 6.25 × 10⁴ bacteria per worm after 24 hours exposure to the pathogen. This result is consistent with the data reported for worms exposed to PA14, where the bacterial CFU counts can reach up to 10³-10⁵ bacteria per worm [29]. Upon supplementation with extract UE-01-4 and UE-12, we observed a significant reduction in the number of bacteria harvested from the C. elegans intestine, 2.5 × 10² and 2.3 × 10² CFU/worm respectively (p < 0.0001) (Figure 10). One possible explanation for this is that the ingestion of bacteria is abated as a consequence of reduced feeding following exposure to a specific bioactive molecule. We tested this hypothesis by counting the pharyngeal pumping rate of the worms in the presence of UE-01-4, as an indirect measure of the intake of bacteria. In the presence of UE-01-4, pharyngeal pumping rates were similar to untreated worms (Additional file 4). Hence, suffice to say, the low number of intestinal bacteria is not a consequence of a decrease in bacteria consumption, but rather, due to other in vivo factor/s that suppress the number of bacteria in the host intestine. For the other five extracts (UE-02-4, UE-03-3, UE-03-5, UE-03-6, UE-11) and compound UC-10, we did not observe any significant difference in the intestinal bacterial loads compared to the untreated animals.

Figure 10

Extracts suppressed the number of in vivo S. aureus in infected animals. Bacterial CFU in the intestine of S. aureus-infected worms were enumerated at 24 hours post-infection in the presence and absence of extracts/compound. All extracts were tested at 200 μg/mL and compound was tested at 200 μM. The bars correspond to mean ± SD of the number of CFU per worm from one representative of two independent replicates. (**) marks the significant difference of in vivo CFU counts between untreated (no extract) and treated worms (p < 0.0001).

The extracts from N. fruticans root (UE-01-4) and O. stamineus leaves (UE-12) limited the number of S. aureus in the host intestine, suggesting that the mode of action of these extracts likely involved activation of host immunity to reduce the bacterial numbers and thus clear the infection. For extracts UE-02-4, UE-03-3, UE-03-5, UE-03-6, UE-11 and compound UC-10, the manifestation of a healthy worm population that remained colonized by S. aureus proposed that these extracts/compound might have rendered the bacteria less virulent and therefore harmless. The less virulent bacteria continued to colonize the host intestine but were unable to bring about death of the host. Alternatively, they may have also modulated the host response to tolerate chronic innocuous intestinal colonization. Both protective outcomes (healthy worms with harmless infection or healthy worms with cleared infection) may arise from either impairing the bacterial virulence or upregulating the host defenses.