Activity of Sodium Lauryl Sulfate,Rhamnolipids,and N -Acetylcysteine Against Biofilms of Five Common Pathogens

Abstract

Bacteria in biofilms are more resistant to antibacterial agents than bacteria in planktonic form. Hence, antibacterial agents should be able to eradicate biofilms to ensure the best outcomes. Little is known about how well many antibacterial agents can disrupt biofilms. In this study, we compared sodium lauryl sulfate (SDS), rhamnolipids (RHL), and N-acetylcysteine (NAC) for their ability to eradicate mature biofilms and inhibit new biofilm formation against Helicobacter pylori, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus mutans. SDS and RHL effectively inhibited formation of five bacterial biofilms in a dose-dependent manner, even at concentrations below the minimal inhibitory concentrations (MICs), suggesting that their antibiofilm activities are unrelated to their antibacterial activities. In contrast, NAC at certain concentrations promoted biofilm formation by all bacteria except P. aeruginosa, whereas at supra-MIC concentrations, it inhibited biofilm formation against the four bacteria, suggesting that its antibiofilm activity depends on its antibacterial activity. NAC was ineffective at eradicating mature H. pylori biofilms, and it actually promoted their formation at concentrations >10 mg/mL. Our results suggest that RHL is superior at eradicating biofilms of H. pylori, E. coli, and S. mutans; SDS is more effective against S. aureus biofilms; and NAC is more effective against P. aeruginosa biofilms. Our results may help determine which antibiofilm agents are effective against certain bacterial strains and develop agents effective against specific bacterial threats.

Introduction

Bacterial biofilms consist of a group of bacteria that adhere to surfaces and embed in an extracellular matrix made of extracellular polysaccharide substances extracellular polymeric substances (EPS), including nucleic acids, polysaccharides, proteins, and lipids.¹ Biofilms are considered distinct from planktonic bacteria, as they have the mushroom-like three-dimensional structure that allow the bacteria to resist disinfectants and antibacterial agents.¹ Eighty percent of bacterial infections are related to biofilms, such as lung cystic fibrosis, chronic bronchopneumonia, endocarditis, and prosthetic joint infection.²

Bacteria in biofilms are more resistant to antibiotics for a number of reasons. First, EPS impede the penetration of antibiotics¹ and bacteria in biofilms have lower metabolic activity and therefore reduce sensitivity to most antibacterial agents that target bacterial proliferation.³ Second, the high cell density in biofilms promotes efficient horizontal gene transfer,⁴ which is the primary mechanism for the spread of antibiotic resistance. Finally, multidrug efflux pumps are more effective at removing antibiotics in biofilms,³ such that antibiotic resistance can be 100- to 1000-fold higher in biofilms compared with their planktonic form.² This high antibiotic resistance makes it difficult to treat biofilm-related infections.

Recent studies have shown that quorum sensing inhibitors (QSIs) and antimicrobial peptides (AMPs) have been identified as important antibiofilm agents. QSIs are capable of decreasing biofilm formation in various bacteria by disrupting quorum sensing pathways.⁵ AMP is an alternative of conventional antibiotics and its antibiofilm activity has also been reported.⁶ The efficacy of AMPs can be reduced by the fact that most substances in EPS are negatively charged, so they interact with positively charged AMPs.⁷ Besides, many QSIs and AMPs are cytotoxic, so it is crucial to develop QSIs and AMPs with higher activity and lower toxicity.

Surfactants, which contain hydrophilic and hydrophobic groups, are also one of the most important antibiofilm agents. Surfactants, including chemical surfactants and biosurfactants, can anchor themselves to surfaces and effectively inhibit biofilm formation and eradicate mature biofilms.^8,9 Sodium lauryl sulfate (SDS) and rhamnolipids (RHL), which belong to the chemical surfactants and biosurfactants, respectively, are the effective antibiofilm agents with anti-adhesion and antibiofilm activities.^10,11 In addition, biosurfactants often exhibit lower toxicity and higher efficiency than chemical surfactants^12,13 because they are metabolites originating from microorganisms. Another compound that may be effective to treat biofilm-related infection is N-acetylcysteine (NAC).^14,15 NAC, a mucolytic and antioxidant agent,¹⁶ has been approved by the US Food and Drug Administration (FDA) for treating chronic bronchitis. Its ability to break down disulfide bonds may be help for its antibiofilm activity.¹⁵

The pathogenic bacteria Helicobacter pylori, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus mutans can develop firm biofilms on biotic and abiotic surfaces. Eradication of biofilms formed by all these bacteria has been studied, but much less is known about the eradication of H. pylori biofilms. H. pylori, a spiral Gram-negative bacterium, can form biofilms on the gastric mucosal epithelium and cause gastritis and peptic ulcers,¹⁷ which have been linked to resistance to triple and quadruple antibiotic therapy.¹⁸ In addition, our previous study demonstrated that lipid polymer nanoparticles containing RHL can effectively eradicate H. pylori biofilms.¹⁷

There are few studies that have compared the efficacy of antibiofilm agents across a panel of bacteria in parallel. Therefore, we compared the antibiofilm activities of SDS, RHL, and NAC against H. pylori, E. coli, P. aeruginosa, S. aureus, and S. mutans. The results can help guide the selection of antibiofilm agents against strains based on strain characteristics.

Materials and Methods

Chemicals

Luria–Bertani (LB) broth and brain–heart infusion (BHI) broth were purchased from Hopebio (Qingdao, China). Columbia blood plates were obtained from Huan kai Guangzhou Microbial (Guangzhou, China). NAC and crystal violet were supplied by Shanghai Macklin Biochemical (Shanghai, China); RHL, by Sigma-Aldrich (St. Louis, MO); and SDS, by Fu Chen Chemical Reagents Factory (Tianjin, China). LIVE/DEAD dye kit was provided by Molecular Probes, Inc. (Eugene, OR).

Bacterial strains

H. pylori SS1 was kindly provided by Guangdong General Hospital (Guangdong, China). E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), S. aureus (ATCC 25923), and S. mutans (UA 159) were purchased from Huankai Guangzhou Microbial.

Medium and culture conditions

All the microorganisms were stored in a freezer at −80°C. After E. coli, P. aeruginosa, S. aureus, and S. mutans were grown on LB agar for 24 h at 37°C, single colonies were selected and immediately transferred to LB medium (E. coli and P. aeruginosa) or BHI medium (S. aureus and S. mutans). Then, all bacterial suspensions were incubated for 12 h at 37°C with shaking at 200 rpm/min. As for H. pylori, 200 μL of bacterial culture was spread plated on blood agar plate supplemented with fetal bovine serum and incubated in the Genbox microaer under microaerophilic condition (5% O₂, 10% CO₂, and 85% N₂) at 37°C for 72 h. A piece of moist sterile filter paper was put into the device to provide and keep appropriate humidity.

Determination of minimum inhibitory concentrations

Minimum inhibitory concentrations (MICs) of the three antibiofilm agents were determined using the broth microdilution assay in 96-well microtiter plates (Corning, New York, NY). In brief, 100 μL of bacterial suspension (1.5 × 10⁶ cells/mL) was added to each well, then 50 μL of SDS and RHL with same concentrations (10, 5, 2.5, 1.25, 0.62, 0.31, 0.16, 0.08, 0.04, and 0.02 mg/mL), and 50 μL of NAC with concentrations (80, 40, 20, 10, 5, 2.5, 1.25, 0.62, 0.31, and 0.16 mg/mL) were added. Wells without antibacterial agents served as controls. After 24 h of incubation at 37°C, bacterial growth was measured based on optical density at 600 nm using a microtiter plate reader (BioTek, Winooski, VT).

Inhibition of biofilm formation

Cultured cells (250 μL) at concentrations of 1.5 × 10⁷ cells/mL (S. aureus), 5 × 10⁶ cells/mL (E. coli), or 1.5 × 10⁸ cells/mL (H. pylori, P. aeruginosa, and S. mutans) were incubated with 250 μL of serial dilutions of SDS, RHL, and NAC for 24 h at 37°C in 48-well microtiter plates. The medium in H. pylori was supplemented with 2% serum (ScienCell) to induce biofilm formation. Wells containing only bacteria without antibacterial agents served as controls, and wells containing only medium served as blanks. After incubation, the culture medium was carefully removed, wells were washed three times with sterile phosphate-buffered saline (PBS) to remove nonadherent cells, then wells were stained for 15 min with 500 μL of 1% crystal violet. Plates were then washed with water three times and dried at 37°C. Finally, 500 μL of 95% (v/v) ethanol was added, and optical density at 570 nm was measured using a microtiter plate reader. Tests were carried out in six replicates. Biofilm formation was calculated according to the following equation: $P e r c e n t o f b i o f i l m (%) = \frac{O D t - O D b}{O D c - O D b} \times 100,$ (1)

where ODt is the optical density (570 nm) in the treatment group; ODb, blank group; and ODc, control group.

Eradication of mature biofilms

The crystal violet assay was used to evaluate the effect of antibiofilm agents on mature biofilms. Mature biofilms were formed by incubating P. aeruginosa, S. aureus, and S. mutans for 24 h; H. pylori for 72 h; and E. coli for 48 h in 48-well microtiter plates. Culture medium of E. coli was replaced every day during incubation. After biofilms had formed, culture medium was discarded and the well was rinsed twice with fresh medium. The culture plate was then incubated with SDS and RHL at same concentrations of 10, 5, 2.5, 1.25, 0.62, 0.31, and 0.16 mg/mL and NAC at the concentrations of 80, 40, 20, 10, 5, 2.5, and 1.25 mg/mL for 24 h at 37°C. Wells containing only bacteria without antibacterial agents served as controls. Wells containing only medium served as blanks. Residual biofilm was quantified using crystal violet assay as described previously. Percentage of biofilm was calculated according to Equation (1). Tests were carried out in six replicates.

Confocal laser scanning microscopy

Biofilms were cultured as described previously in confocal culture plates. After incubation, medium was discarded and wells were rinsed twice with fresh medium. SDS, RHL, or NAC at 4 × MICs were added to the plates (RHL was incubated with mature E. coli and P. aeruginosa biofilms at a concentration of 2.5 mg/mL). Wells containing only bacteria served as controls. Plates were incubated for 24 h at 37°C, then medium was removed and wells were rinsed three times with sterile PBS. Cells were stained with 600 μL LIVE/DEAD dye (Molecular Probes, Inc.) for 15 min in the dark. Extra dye was removed by washing in PBS. Biofilms were observed under a confocal laser scanning microscope (CLSM) (Olympus, Japan) at an excitation wavelength of 488 nm for SYTO9 and 543 nm for propidium iodide. Three-dimensional images of biofilm were reconstructed by taking images in 2 μm steps along the z plane.

Scanning electron microscopy

Sterile microscopic glass slides were placed in sterile 24-well plates, and cells were added to the wells. After biofilms had matured, medium was discarded and wells were rinsed twice with fresh medium. SDS, RHL, or NAC at 4 × MICs were then added (RHL was incubated with mature E. coli and P. aeruginosa biofilms at a concentration of 2.5 mg/mL). Plates were incubated for 24 h at 37°C. Cells on glass slides were fixed overnight with 2.5% glutaraldehyde. On the following day, the glutaraldehyde was removed, and each well was rinsed three times with 1 mL PBS. Dehydration was performed by immersing slides in a gradient of ethanol solutions (30%, 50%, 70%, 80%, and 95%) for 10 min at each concentration. Then slides were immersed three times in pure ethanol, dried with flowing CO₂, and coated with gold. Biofilms were observed under a scanning electron microscope (SEM) (JSM-6330F; JEOL, Japan).

Statistical analysis

Statistical analysis was performed using a one-way analysis of variance of IBM SPSS package (version 21.0; SPSS, Inc., IBM, New York, NY). Values of p < 0.05 were considered statistically significant.

Results

Antibacterial activity of SDS, RHL, and NAC on planktonic bacteria

Antibacterial effects of SDS, RHL, and NAC were first tested on the five pathogens in planktonic form (Table 1). SDS and NAC exhibited antibacterial effects against all five bacteria, whereas RHL showed activity against only H. pylori, S. aureus, and S. mutans. SDS had the strongest antibacterial activity among the three agents with MIC as low as 0.08 mg/mL. RHL showed no activity against E. coli and P. aeruginosa even at concentrations as high as 10 mg/mL. In contrast, NAC showed quite weak activity against all bacteria, with MICs ranging from 5 to 10 mg/mL.

Table 1.

Minimum Inhibitory Concentrations of Three Antibiofilm Agents Against Planktonic Helicobacter pylori, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus mutans (mg/mL)

	Helicobacter pylori	Escherichia coli	Pseudomonas aeruginosa	Staphylococcus aureus	Streptococcus mutans
SDS	0.16	0.62	0.62	0.08	0.08
RHL	0.31	—	—	0.31	0.16
NAC	5	5	10	10	10

—, no antibacterial activity at 10 mg/mL.

SDS, sodium lauryl sulfate; RHL, rhamnolipids; NAC, N-acetylcysteine.

Inhibition of biofilm formation

Crystal violet colorimetric assay, a quantifiable method to examine both bacteria and their external EPS, was conducted to evaluate biofilm biomass.

SDS and RHL inhibited formation of all five bacterial biofilms, whereas NAC showed an unexpected biphasic effect on bacteria except P. aeruginosa at lower concentrations. It actually promoted biofilm formation: H. pylori, 10 mg/mL; E. coli, 2.5 mg/mL; S. aureus, 5 mg/mL; and S. mutans, 5 mg/mL (Fig. 1). SDS and RHL had similar biofilm inhibition pattern against H. pylori and E. coli. Both SDS and RHL even inhibited H. pylori biofilm formation at extremely low concentrations (0.005 and 0.02 mg/mL, respectively), and biofilm formation could be reduced up to 80% at tested concentrations (Fig. 1A, B). SDS at 5 mg/mL reduced E. coli biofilm biomass by ∼60% similar to RHL (Fig. 1D, E). However, RHL was more effective than SDS in inhibiting biofilm formation against P. aeruginosa, S. aureus, and S. mutans. RHL inhibited P. aeruginosa, S. aureus, and S. mutans biofilm formation by 80% at 0.31, 0.08 (1/4 × MIC), and 0.08 mg/mL (1/2 × MIC), respectively, whereas SDS failed to inhibit 80% of P. aeruginosa biofilm formation at the highest test concentration (5 mg/mL) and inhibited 80% of S. aureus and S. mutans biofilm formation at 0.08 mg/mL (MIC) and 0.62 (8 × MIC), respectively (Fig. 1).

FIG. 1.

Biofilm inhibition effectiveness of SDS, RHL, and NAC under or above MIC concentrations against Helicobacter pylori (A–C), Escherichia coli (D–F), Pseudomonas aeruginosa (G–I), Staphylococcus aureus (J–L), and Streptococcus mutans (M–O). Biofilms were quantified as absorption by crystal violet assay at 570 nm. Error bars indicate the standard deviation (SD). *Significant at p < 0.05, **Significant at p < 0.01, ***Significant at p < 0.001. SDS, sodium lauryl sulfate; RHL, rhamnolipids; NAC, N-acetylcysteine; MIC, minimum inhibitory concentration.

Eradication of mature biofilms

The biofilm eradicative activities of agents were studied by crystal violet assay first. The three agents could destroy mature biofilms formed by all five organisms in a dose-dependent manner. The only exception to this was NAC, which at concentrations >10 mg/mL was found to promote biofilm formation in H. pylori (Fig. 2). RHL was superior to SDS at eradicating biofilms of H. pylori and E. coli. It eradicated 80% of H. pylori and E. coli biofilms at 5 and 0.31 mg/mL, respectively (Fig. 2A–F). NAC was more effective against P. aeruginosa than SDS and RHL as >80% of biofilms were destroyed at 5 mg/mL (Fig. 2G–I). For S. aureus biofilms, SDS showed the strongest eradicative activity with 80% eradication of S. aureus biofilms at 0.62 mg/mL (Fig. 2J–L). Both SDS and RHL showed high activity against S. mutans biofilms: SDS at 0.31 mg/mL disrupted 80% of S. mutans biofilms, and RHL effectively eradicated S. mutans biofilms even at the lowest tested concentration of 0.16 mg/mL (Fig. 2M–O).

FIG. 2.

Eradication of mature biofilms by SDS, RHL, and NAC against H. pylori (A–C), E. coli (D–F), P. aeruginosa (G–I), S. aureus (J–L), and S. mutans (M–O). Biofilms were quantified as absorption by crystal violet assay at 570 nm. *Significant at p < 0.05, **significant at p < 0.01, ***significant at p < 0.001.

SEM results showed that RHL removed mature H. pylori biofilms more effectively than SDS and NAC and left fewer microcolonies. NAC promoted the growth of H. pylori biofilms at 4 × MIC (Fig. 3A). CLSM, which could observe biofilm architecture and distinguish live and dead cells, showed that after treatment with any of the three antibiofilm agents, the residual H. pylori biofilms contained mostly living bacteria (Fig. 4A). All three agents efficiently removed E. coli biofilms, with SDS and NAC causing more death of bacteria in mature E. coli biofilms than RHL (Figs. 3B and 4B). NAC was more effective against P. aeruginosa biofilms than RHL or SDS (Figs. 3C and 4C). SDS and NAC were significantly better than RHL in destroying S. aureus biofilms (Figs. 3D and 4D). All three antibiofilm agents were effective at eradicating S. mutans biofilms, and most of the remaining biofilms were dead bacteria (Figs. 3E and 4E). SEM and CLSM results were consistent with those from the crystal violet assay.

FIG. 3.

Scanning electron micrographs of mature H. pylori (A), E. coli (B), P. aeruginosa (C), S. aureus (D), and S. mutans (E) biofilms after treatment of three antibiofilm agents at their 4 × MICs (RHL was incubated with mature E. coli and P. aeruginosa biofilms at a concentration of 2.5 mg/mL as it did not have antibacterial activity against the two bacteria). Scale bars are 2 μm (A) and 5 μm (B–E), respectively.

FIG. 4.

Confocal laser microscopic visualization of the eradication effect of three antibiofilm agents at their 4 × MICs on mature H. pylori (A), E. coli (B), P. aeruginosa (C), S. aureus (D), and S. mutans (E) (RHL was incubated with mature E. coli and P. aeruginosa biofilms at a concentration of 2.5 mg/mL as it did not have antibacterial activity against the two bacteria). Biofilms were grown in confocal plates and stained with the LIVE/DEAD BacLight. Green fluorescence indicated living bacteria and red represented dead bacteria. Scale bars are 20 μm. Color images are available online.

Discussion

Antibiotic resistance caused by bacterial biofilms significantly lowers the cure rates of infectious disease. Bacterial biofilm eradication may decrease antibiotic resistance by directly exposing bacteria to antibiotics, and thus reducing both dosage and side effects of antibiotics in biofilm-related infections. To explore novel bacterial biofilm eradication strategies, a large number of compounds with potential antibiofilm activities have been studied.^19,20 However, studies that compare antibiofilm agents in multiple bacterial strains are rare. In this study, for the first time, we compared the antibiofilm activity of SDS, RHL, and NAC against five common pathogens: H. pylori, E. coli, P. aeruginosa, S. aureus, and S. mutans.

We found that SDS showed the strongest activity against the bacteria in their planktonic form. RHL also exhibited antibacterial activity against H. pylori, S. aureus, and S. mutans but not against E. coli or P. aeruginosa. NAC had the weakest antibacterial activity. SDS and RHL kill bacteria by damaging cell membranes, which allows leakage of cytoplasmic components.²¹ Gram-negative bacteria have thicker cell walls and thus are less susceptible to the membrane damage effect caused by surfactants.²² As expected, RHL and SDS showed weaker activity against the Gram-negative H. pylori, E. coli, and P. aeruginosa than against Gram-positive S. aureus and S. mutans. The mechanism of NAC antibacterial activity is unknown. It is possible that NAC kills bacteria by decreasing cysteine utilization or interacting with sulfhydryl groups in cellular proteins.¹⁵ Therefore, the antibacterial activity of NAC may not depend on Gram types or cell wall thickness.

In this study, we selected a static biofilm model to evaluate the activities of biofilm inhibition and biofilm eradication of three agents. Static and dynamic models are two commonly used models for evaluating antibiofilm activities. Although the dynamic model of biofilm formation can closely approximate in vivo conditions, the static biofilm cost-effectively applies batch and static growth conditions in such a way that smaller drug amounts are needed, allowing numerous tests to be performed at the same time.¹⁹

Biofilm formation likely involves several intermolecular forces, including Lifshitz-van der Waals forces, surface hydrophobicity, and Lewis acid–base interactions. The most important of these forces is likely to be surface hydrophobicity.²³ SDS and RHL, as surfactants, position themselves between the bacteria and surface, inhibiting bacterial adhesion.²⁴ Thus, the efficacy of antibiofilm agents should depend on the characteristics of the bacteria, including surface hydrophobicity and zeta potential. As bacteria have diversity in these characteristics,²⁵ the effect of the same antibiofilm agent may vary among different bacteria, as our results demonstrate.

Our results showed that SDS and RHL inhibited bacterial biofilm formation at concentrations below their MICs, suggesting that they worked by blocking adherence of bacteria to polystyrene surfaces rather than by interfering with bacterial growth. NAC, in contrast, did not significantly inhibit biofilm formation until concentrations were one to four times the MIC except P. aeruginosa. This implies that NAC inhibits biofilm formation of these four bacteria by interfering with bacterial growth. Surprisingly, NAC promoted biofilm formation at certain concentrations, in all bacteria except P. aeruginosa. This may be analogous to the way in which some antibiotics at certain concentrations strongly induce biofilm development in vitro by modulating levels of cyclic diguanosine monophosphate.²⁶ In contrast, the ability of lower concentrations of NAC to inhibit P. aeruginosa biofilm development is probably because of NAC-mediated inhibition of EPS production.²⁷

We found that the three antibiofilm agents effectively eradicated five bacterial mature biofilms in a dose-dependent manner except that NAC promoted H. pylori biofilm at high concentrations (Fig. 2). Besides the efficacy of agents assessed here, toxicity should also be considered for the choice of agents. RHL was reported to possess much lower toxicity, higher efficiency, and better biocompatibility than SDS.¹² NAC, an FDA-approved drug for chronic bronchitis treatment, also shows low toxicity. Eighty percent of eradication of H. pylori, E. coli, and S. mutans mature biofilms by RHL was observed at 5, 0.31, and 5 mg/mL, respectively. Therefore, RHL was a more proper agent to eradicate H. pylori, E. coli, and S. mutans mature biofilms after balancing the safety and efficiency. The ability of RHL to eradicate mature biofilms is probably because of the removal of EPS. Earlier study by Al-Tahhan reports that RHL can remove lipopolysaccharides by forming micelle.²⁸ The hydrophobic group of RHL inserts into EPS, and its hydrophilic group extends out into the aqueous phase, leading to EPS desorption from the substrate surface. In addition, RHL can destabilize EPS by binding metal ions, further disrupting biofilms.²⁹ It may also exert these effects by disturbing interbacterial signaling by the bacterial membrane.³⁰ RHL did not effectively destroy S. aureus biofilms in our study, which may reflect differences in EPS composition between S. aureus and the other bacteria.³¹

SDS, a synthetic anionic surfactant, performed differently in eradicating mature biofilms of different bacteria (Fig. 2). Unlike RHL, SDS had the best effect on S. aureus mature biofilms with an eradication rate of 80% at 0.62 mg/mL. In addition, at concentrations >0.62 mg/mL, SDS's eradication rate of mature biofilms is three to five times higher than that of RHL. Other studies have also concluded that SDS is more effective than RHL on S. enterica and Salmonella biofilms.^10,32 These differences may reflect different mechanisms of action: SDS may work primarily by denaturing the proteinaceous matrix,³³ whereas RHL may work primarily by interacting with lipopolysaccharides.³³

NAC, a powerful sulfhydryl antioxidant and mucolytic agent, was found to completely clear P. aeruginosa biofilms at a concentration of 10 mg/mL. Eradication of P. aeruginosa biofilms is extremely difficult because they locate under the mucus layers of the lung, and drugs become trapped in the mucus before reaching the biofilms. We suggest that NAC has a promising antibiofilm activity against P. aeruginosa biofilms than SDS and RHL because its mucolytic properties mean that it can overcome the mucus barrier in the lung. However, NAC did not work well against mature H. pylori biofilms at concentrations <5 mg/mL, and it actually stimulated formation of H. pylori biofilms at concentrations >5 mg/mL. To cultivate H. pylori biofilms, we found that 2% fetal bovine serum was essential. Yin et al. found that the combination of NAC and serum increased biomass of seven bacterial strains, and they attributed this effect to interaction between NAC and transferrin in the serum.³⁴ This interaction may explain why we observed NAC to improve growth of H. pylori biofilms.

Conclusion

To our knowledge, this is the first time that the antibiofilm abilities of SDS, RHL, and NAC were compared in antibiofilm activities on five common pathogens H. pylori, E. coli, P. aeruginosa, S. aureus, and S. mutans.

Our results showed that the efficacy of antibiofilm agents against specific pathogens cannot be predicted based on their MICs against planktonic bacteria. The abilities of SDS and RHL to inhibit biofilm formation were unrelated to their antibacterial activities. In fact, RHL showed strong antibiofilm activity against E. coli and P. aeruginosa although no antibacterial activities were observed against these two bacteria. We also found that the ability of these agents to inhibit biofilm formation did not always correlate with their ability to eradicate mature biofilms. These results highlight the need for further investigations into how antibiofilm agents interact with early and mature biofilms. Our results indicate that the same antibiofilm agent may not be suitable against all kinds of bacterial biofilms. For example, NAC was only suitable for P. aeruginosa biofilms, whereas it promoted the other four bacteria's biofilm development at certain concentrations. Taking into account the efficacy and safety of three substances, we suggest that RHL might be a suitable to eradicate H. pylori, E. coli, and S. mutans biofilms, whereas SDS and NAC may be preferable for eliminating S. aureus and P. aeruginosa biofilms.

Footnotes

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grants No. 81473154 and 81773659) and the Fundamental Research Funds for central universities (Grant No. 18ykzd08). Besides, the authors are also grateful to Dr. Liyan Zhang for kindly providing H. pylori SS1.

Disclosure Statement

The authors declare no competing conflicts of interest in this work.

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