Abstract
Objectives
Antibacterial monomers were developed to combat oral biofilm acids and caries; however, little is known on whether quaternary ammonium monomers (QAMs) would induce drug resistance in oral bacteria. The objective of this study was to investigate the effects of new antimicrobial monomers dimethylaminohexadecyl methacrylate (DMAHDM) and dimethylaminododecyl methacrylate (DMADDM) on the induction of drug resistance in eight species of cariogenic, endodontic and periodontal bacteria for the first time.
Methods
Streptococcus mutans ( S. mutans ), Streptococcus sanguis , Streptococcus gordonii , Enterococcus faecalis ( E. faecalis ), Aggregatibacter actinomycetemcomitans ( A. actinomycetemcomitans ), Fusobacterium nucleatum ( F. nucleatum ), Porphyromonas gingivalis ( P. gingivalis ), and Prevotella intermedia ( P. intermedia ) were tested. Minimum inhibitory concentration (MIC) was assessed using chlorhexidine (CHX) as control. Minimal bactericidal concentration (MBC), bacterial growth and membrane permeability properties were also investigated.
Results
CHX induced drug resistance in four species. DMAHDM did not induce any resistance. DMADDM induced drug resistance in only one benign species S. gordonii . The DMADDM-resistant and CHX-resistant S. gordonii had the same MIC and MBC values as S. gordonii parental strain against DMAHDM (p > 0.1), hence DMAHDM effectively inhibited the resistant strains. The resistant strains had slower growth metabolism than parental strain.
Significance
DMAHDM induced no drug resistance, and DMADDM had much less drug resistance than the commonly-used CHX in the eight common oral species. With its potent antimicrobial functions shown previously, the new DMAHDM is promising for applications in restorative, preventive, periodontal and endodontic treatments to combat cariogenic and pathological bacteria with no drug resistance in all tested species.
1
Introduction
Oral diseases such as caries, periapical disease and periodontal disease are directly associated with oral biofilm communities containing hundreds of species of bacteria . Pathological changes in the biofilm composition can lead to infections which affect oral health as well as systemic diseases . The emergence of certain bacterial strains becoming resistant to antibiotics could lead to severe and refractory infections. Indeed, antimicrobial resistance has become a global issue, which is not only a serious threat to global public health but also increases the cost of health care. Among various biofilms, oral biofilms are complex bacterial communities that tend to be highly resistant to antibiotics and the human immune defense . To date, little has been reported on the investigation of possible antimicrobial drug resistance in oral bacteria with the use of quaternary ammonium monomers (QAMs) in dentistry.
Quaternary ammonium salts (QAS) have been widely used in water treatments, the food processing industry, textiles and surface coatings since the mid-1930s . Due to their low toxicity and a broad spectrum of antimicrobial activity, QAS were first incorporated into mouth rinses to inhibit oral biofilms in the 1970s . Antibacterial dental resins have attracted increasing attention; chlorhexidine (CHX) , silver and several QAMs have been incorporated into resins to reduce biofilm growth, acid production and secondary caries . Pioneering work by Imazato et al. developed 12-methacryloyloxydodecyl-pyridinium bromide (MDPB) and incorporated it into dental resins, showing effective suppression of biofilm growth . Since then, several different antimicrobial compositions have been developed, including quaternary ammonium polyethylenimine (QPEI) , methacryloxylethyl cetyl dimethyl ammonium chloride (DMAE-CB) , dimethylaminododecyl methacrylate (DMADDM) , and dimethylaminohexadecyl methacrylate (DMAHDM) . The antibacterial mechanism of QAMs is suggested to be the action of causing bacterial cell membrane lysis via the binding to bacterial membranes .
Although the antimicrobial effects of QAMs have promising clinical benefits, their frequent use may lead to a selection of resistant strains or the emergence of acquired resistance to those sensitive strains . Indeed, bacterial drug resistance is an increasingly worrying phenomenon . For example, Staphylococcus aureus (a common cause of skin infections, respiratory infections and food poisoning), Serratia marcescens (involved in hospital-acquired infections such as urinary tract infections and wound infections) and Escherichia coli (occasionally responsible for product recalls due to food contamination) have been reported to have acquired resistance against QAS .
However, there are very few reports on the investigation of oral bacteria potentially developing resistance to QAMs used in antibacterial dental materials. A literature search revealed that, to date, there is only one publication on this important topic . That study showed that after serial exposures to cationic biocides, Streptococcus mutans ( S. mutans ) (commonly found in human oral cavity), and Enterococcus faecalis ( E. faecalis ) (closely associated with apical periodontitis) did not exhibit resistance to MDPB after repeated exposures, while E. faecalis developed drug resistance to chlorhexidine (CHX) . With the increasing efforts in developing a new generation of antibacterial dental resins, studies are urgently needed to investigate the possible antibacterial resistance of QAMs against oral bacteria.
Several oral streptococci strains are associated with dental caries. Acidogenic species, e.g., S. mutans , and aciduric species Streptococcus sanguis ( S. sanguis ) and Streptococcus gordonii ( S. gordonii ) play important roles in microecological balance regulations . In addition, E. faecalis is a microorganism commonly detected in persistent endodontic infections, often leading to root canal treatment failures due to its various virulence factors . Furthermore, several subgingival microbiota including Aggregatibacter actinomycetemcomitans ( A. actinomycetemcomitans ), Fusobacterium nucleatum ( F. nucleatum ), Porphyromonas gingivalis ( P. gingivalis ) and Prevotella intermedia ( P. intermedia ) are associated with aggressive chronic periodontitis or refractory periodontitis . Therefore, the three common caries-related species ( S. mutans , S. sanguis and S. gordonii ), one common periapical periodontitis-related species ( E. faecalis ), and four common periodontitis-related species ( A. actinomycetemcomitans , F. nucleatum , P. gingivalis and P. intermedia ) were selected for the present study. We recently developed new QAMs, including DMADDM (with alkyl chain length of 12) and DMAHDM (with alkyl chain length of 16), whose chemical structures are shown in Fig. 1 . They exhibited potent antimicrobial effects in suppressing biofilm growth and acid production . However, there has no report on the investigation of potential bacterial drug resistance induced by DMADDM and DMAHDM.
Therefore, the objective of this study was to determine the potential drug resistance induced by DMADDM and DMAHDM against the aforementioned eight species of oral bacteria for the first time. The commonly-used agent CHX served as control. It was hypothesized that: (1) The new antibacterial monomers DMADDM and DMAHDM would not induce drug resistance against the eight species of oral bacteria; (2) CHX would induce resistance in several species of the tested oral bacteria; (3) The impermeability of the bacterial membranes would contribute to the development of drug resistance in the bacteria.
2
Material and methods
2.1
Bacterial strains and growth conditions
The bacterial strains used in this study were approved by the University of Maryland Baltimore Institutional Review Board. The following eight species were obtained from American Type Culture Collection (ATCC, Manassas, VA): S. mutans (ATCC 700610, UA159); S. sanguis (ATCC 10556); S. gordonii (ATCC 10558); E. faecalis (ATCC 4083); A. actinomycetemcomitans (ATCC 43717); F. nucleatum (ATCC 25586); P. gingivalis (ATCC 33277); and P. intermedia (ATCC 25611). The three streptococci strains ( S. mutans , S. sanguis and S. gordonii ) were selected because they are involved in the initiation and progression of dental caries . E. faecalis was selected because it is a commonly-isolated species from persistent apical or intraradicular periodontitis . The anaerobic bacteria ( A. actinomycetemcomitans , F. nucleatum , P. gingivalis and P. intermedia ) were selected because they are closely associated with periodontitis .
Fifteen microliters of the first four stock bacteria was separately added to 15 mL of brain-heart infusion (BHI) broth (Sigma, St. Louis, MO, USA) and incubated overnight at 37 °C with 5% CO 2 , following previous studies . For the other four periodontitis-related species, each was grown in a tryptic soy broth (TSB) (Sigma) with yeast extract (5 g/L), l-cysteine hydrochloride (0.5 g/L), hemin (5 mg/L) and menadione (1 mg/L) at 37 °C anaerobically (90% N 2 , 5% CO 2 , 5% H 2 ).
2.2
Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) measurements
To evaluate the antibacterial properties of DMADDM and DMAHDM, a microtiter plate assay was used to determine the MIC and MBC for the eight species, following a previous method . The MIC was measured as the lowest concentration of an antimicrobial agent at which no visible bacterial growth appeared, where the bacterial growth was completely inhibited. MIC measurements were conducted according to the serial two-fold microdilution method using BHI broth ( Fig. 2 ). DMADDM and DMAHDM were dissolved in sterile distilled water at 200 μg/mL and diluted with culture medium to prepare the starting concentrations. CHX (Sigma) served as control with a starting concentration of 250 μg/mL. Since the antibacterial potency of DMAHDM was similar to CHX as determined in preliminary study, and that of DMADDM was lower than that of DMAHDM, different initial concentrations were used. For the monomer solutions, serial two-fold dilutions were made into 100 μL volumes of culture medium in the 96-well plate. Overnight cultures of bacteria in broth were diluted by fresh medium and used for the experiments. The bacterial culture was adjusted to 2 × 10 6 CFU/mL of suspension, and 100 μL was inoculated into each well in the 96-well plate with culture medium. After incubation at 37 °C with 5% CO 2 or anaerobically (90% N 2 , 5% CO 2 , 5% H 2 ) for 2 days, MIC was determined by visual examination: the lowest concentration at which no visible bacterial growth appeared was recorded as the MIC value. All tests were repeated in three replicates each time, and then repeated again three times on different days.
MBC was determined as the lowest concentration of the antimicrobial agent that killed 99.9% of the initial inoculum. Ten μL of aliquots bacterial suspensions from the wells without viable cells, where bacterial growth was inhibited, was inoculated onto the surface of BHI agar plates and incubated under anaerobic conditions for 48 h at 37 °C with 5% CO 2 . After calculating the colony-forming units (CFU), the MBC value was determined.
2.3
Bacterial drug resistance assays against antibacterial agents
To further examine whether oral bacteria could develop drug resistance against the antibacterial agents tested, MIC measurements following repeated serial passages were performed to analyze the antibiotic resistance, following a previous method . The MIC values of DMADDM, DMAHDM and CHX against the eight species were measured as described in Section 2.2 . A 100 μL portion of the bacterial suspensions in the sub-MIC well was taken and inoculated into 10 mL of fresh culture medium broth at 37 °C with 5% CO 2 or anaerobically (90% N 2 , 5% CO 2 , 5% H 2 ). The overnight bacterial suspensions were diluted to concentration of approximately 1 × 10 6 CFU/mL for the next MIC test. All of the MIC tests were repeatedly performed for 10 passages ( Fig. 2 ). The potential incremental changes in MIC value through repeated passages in antibacterial agents-containing medium were used to evaluate the development of drug resistance, following a previous study . After repeated exposures to one antibacterial agent, an increase in MIC value above the initial MIC would indicate the acquisition of drug resistance by the bacterial cells. The tests were repeated in three replicates each time, and then repeated again three times on different days.
2.4
Measurement of bacterial growth curves
To investigate the growth metabolism changes in bacteria after being challenged with antibacterial agents, the bacterial growth curves were performed following previous methods . Prior to measuring the growth curve, the bacteria strains ( S. gordonii parental-strain, DMADDM-resistant S. gordonii and CHX-resistant S. gordonii ) were cultivated overnight in BHI broth. Stationary phase cultures were diluted 1:20 in BHI broth and incubated at 37 °C until the OD 600nm reached 0.2. A volume of 20 μL diluted bacterial suspension and 180 μL of BHI broth were added to each well of a 96-well plate. The plate was sealed and bacteria were shaken at 100 rpm at 37 °C. The OD values of the bacteria culture were measured at 30-min intervals at 600 nm with the microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA). The bacterial growth was thus monitored for 16 h. The OD data were analyzed and the growth curves were modeled with four different phases: (1) Lag phase with the bacteria adapting themselves to the growth conditions; (2) log phase or exponential phase with the bacterial cells doubling; (3) stationary phase with the bacterial cell numbers not increasing and the growth rate being stabilized; and (4) death phase with the bacteria dying due to their nutrients being exhausted . The bacterial growth curves tend to vary with different organisms. The changes of the observed kinetic growth curve can reflect whether the bacterial cellular metabolism is accelerating or decelerating. A long log phase would indicate that the bacterium would take more time to adjust to the new environment, and the accelerated cellular metabolism would tend to undergo an advanced lag phase and shortened doubling time, even reaching a high stationary phase. The tests were conducted in three replicates each time and repeated for three times on different days.
2.5
Bacterial membrane permeability test
To further investigate the possible mechanisms on acquiring drug resistance, the bacterial cell membranes were examined which could affect drug intake and discharge. Sodium dodecyl sulfonate (SDS) (Sigma), an anionic surfactant, and guanidine hydrochloride (CH 5 N 3 HCl) (Sigma), one membrane-permeabilizing agent, were used to test their influence on resistance of S. gordonii against DMADDM. The presence of SDS and CH 5 N 3 HCl can cause an increase in the permeability of the plasma membrane . Various concentration of drug-containing BHI plates were prepared, and the solid BHI plates were incorporated with final concentrations of 20 μg/mL SDS and 100 μM guanidine hydrochloride, and/or 25 μg/mL DMADDM. The concentrations of SDS and CH 5 N 3 HCl were chosen according to a previous study, in which Saccharomyces cerevisiae developed resistance to quaternary ammonium compounds . The concentration of DMADDM was chosen in that S. gordonii could develop resistance against DMADDM at this concentration. This was based on our purpose to examine the influence of SDS and CH 5 N 3 HCl on the DMADDM-resistance of S. gordonii at this concentration. Overnight bacterial strains were diluted in sterile phosphate buffered saline (PBS) (Sigma) solution by 1:10 (100 μL culture in 900 μL diluent) to obtain a series of inoculants. An equal amount 10 μL droplets of the serial bacterial inoculum were spotted onto BHI plates and incorporated with DMADDM, SDS and hydrochloride, or their combinations, or none of them. After drying the droplets for 5–10 min, these BHI plates, which were pre-impregnated with a standard concentration of DMADDM, SDS and hydrochloride, or their combinations, or none of them, were incubated for 1–2 days at 37 °C with 5% CO 2 . Then the bacterial growth on each disk was examined. If the bacteria were not resistant to the additives in the agar plates, a clear “no-growth” phenomenon would occur on the culture plates. If the bacteria were resistant to the additives in the agar plates, a clear “growth” phenomenon of bacterial colony morphology would appear. The visible bacterial colony morphology was imaged by digital camera and processed by the software Adobe Photoshop CS6 to obtain the integration of bacterial colony morphology images.
2.6
Statistical analysis
Statistical analysis was performed by one-way analysis of variance followed by the Student-Newman-Keuls posttest. Differences were considered statistically significant at the 5% level ( P < 0.05). Statistical analysis was performed with the SPSS software, version 16.0 (SPSS Inc., Chicago, IL, USA).
2
Material and methods
2.1
Bacterial strains and growth conditions
The bacterial strains used in this study were approved by the University of Maryland Baltimore Institutional Review Board. The following eight species were obtained from American Type Culture Collection (ATCC, Manassas, VA): S. mutans (ATCC 700610, UA159); S. sanguis (ATCC 10556); S. gordonii (ATCC 10558); E. faecalis (ATCC 4083); A. actinomycetemcomitans (ATCC 43717); F. nucleatum (ATCC 25586); P. gingivalis (ATCC 33277); and P. intermedia (ATCC 25611). The three streptococci strains ( S. mutans , S. sanguis and S. gordonii ) were selected because they are involved in the initiation and progression of dental caries . E. faecalis was selected because it is a commonly-isolated species from persistent apical or intraradicular periodontitis . The anaerobic bacteria ( A. actinomycetemcomitans , F. nucleatum , P. gingivalis and P. intermedia ) were selected because they are closely associated with periodontitis .
Fifteen microliters of the first four stock bacteria was separately added to 15 mL of brain-heart infusion (BHI) broth (Sigma, St. Louis, MO, USA) and incubated overnight at 37 °C with 5% CO 2 , following previous studies . For the other four periodontitis-related species, each was grown in a tryptic soy broth (TSB) (Sigma) with yeast extract (5 g/L), l-cysteine hydrochloride (0.5 g/L), hemin (5 mg/L) and menadione (1 mg/L) at 37 °C anaerobically (90% N 2 , 5% CO 2 , 5% H 2 ).
2.2
Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) measurements
To evaluate the antibacterial properties of DMADDM and DMAHDM, a microtiter plate assay was used to determine the MIC and MBC for the eight species, following a previous method . The MIC was measured as the lowest concentration of an antimicrobial agent at which no visible bacterial growth appeared, where the bacterial growth was completely inhibited. MIC measurements were conducted according to the serial two-fold microdilution method using BHI broth ( Fig. 2 ). DMADDM and DMAHDM were dissolved in sterile distilled water at 200 μg/mL and diluted with culture medium to prepare the starting concentrations. CHX (Sigma) served as control with a starting concentration of 250 μg/mL. Since the antibacterial potency of DMAHDM was similar to CHX as determined in preliminary study, and that of DMADDM was lower than that of DMAHDM, different initial concentrations were used. For the monomer solutions, serial two-fold dilutions were made into 100 μL volumes of culture medium in the 96-well plate. Overnight cultures of bacteria in broth were diluted by fresh medium and used for the experiments. The bacterial culture was adjusted to 2 × 10 6 CFU/mL of suspension, and 100 μL was inoculated into each well in the 96-well plate with culture medium. After incubation at 37 °C with 5% CO 2 or anaerobically (90% N 2 , 5% CO 2 , 5% H 2 ) for 2 days, MIC was determined by visual examination: the lowest concentration at which no visible bacterial growth appeared was recorded as the MIC value. All tests were repeated in three replicates each time, and then repeated again three times on different days.
MBC was determined as the lowest concentration of the antimicrobial agent that killed 99.9% of the initial inoculum. Ten μL of aliquots bacterial suspensions from the wells without viable cells, where bacterial growth was inhibited, was inoculated onto the surface of BHI agar plates and incubated under anaerobic conditions for 48 h at 37 °C with 5% CO 2 . After calculating the colony-forming units (CFU), the MBC value was determined.
2.3
Bacterial drug resistance assays against antibacterial agents
To further examine whether oral bacteria could develop drug resistance against the antibacterial agents tested, MIC measurements following repeated serial passages were performed to analyze the antibiotic resistance, following a previous method . The MIC values of DMADDM, DMAHDM and CHX against the eight species were measured as described in Section 2.2 . A 100 μL portion of the bacterial suspensions in the sub-MIC well was taken and inoculated into 10 mL of fresh culture medium broth at 37 °C with 5% CO 2 or anaerobically (90% N 2 , 5% CO 2 , 5% H 2 ). The overnight bacterial suspensions were diluted to concentration of approximately 1 × 10 6 CFU/mL for the next MIC test. All of the MIC tests were repeatedly performed for 10 passages ( Fig. 2 ). The potential incremental changes in MIC value through repeated passages in antibacterial agents-containing medium were used to evaluate the development of drug resistance, following a previous study . After repeated exposures to one antibacterial agent, an increase in MIC value above the initial MIC would indicate the acquisition of drug resistance by the bacterial cells. The tests were repeated in three replicates each time, and then repeated again three times on different days.
2.4
Measurement of bacterial growth curves
To investigate the growth metabolism changes in bacteria after being challenged with antibacterial agents, the bacterial growth curves were performed following previous methods . Prior to measuring the growth curve, the bacteria strains ( S. gordonii parental-strain, DMADDM-resistant S. gordonii and CHX-resistant S. gordonii ) were cultivated overnight in BHI broth. Stationary phase cultures were diluted 1:20 in BHI broth and incubated at 37 °C until the OD 600nm reached 0.2. A volume of 20 μL diluted bacterial suspension and 180 μL of BHI broth were added to each well of a 96-well plate. The plate was sealed and bacteria were shaken at 100 rpm at 37 °C. The OD values of the bacteria culture were measured at 30-min intervals at 600 nm with the microplate reader (SpectraMax M5, Molecular Devices, Sunnyvale, CA, USA). The bacterial growth was thus monitored for 16 h. The OD data were analyzed and the growth curves were modeled with four different phases: (1) Lag phase with the bacteria adapting themselves to the growth conditions; (2) log phase or exponential phase with the bacterial cells doubling; (3) stationary phase with the bacterial cell numbers not increasing and the growth rate being stabilized; and (4) death phase with the bacteria dying due to their nutrients being exhausted . The bacterial growth curves tend to vary with different organisms. The changes of the observed kinetic growth curve can reflect whether the bacterial cellular metabolism is accelerating or decelerating. A long log phase would indicate that the bacterium would take more time to adjust to the new environment, and the accelerated cellular metabolism would tend to undergo an advanced lag phase and shortened doubling time, even reaching a high stationary phase. The tests were conducted in three replicates each time and repeated for three times on different days.
2.5
Bacterial membrane permeability test
To further investigate the possible mechanisms on acquiring drug resistance, the bacterial cell membranes were examined which could affect drug intake and discharge. Sodium dodecyl sulfonate (SDS) (Sigma), an anionic surfactant, and guanidine hydrochloride (CH 5 N 3 HCl) (Sigma), one membrane-permeabilizing agent, were used to test their influence on resistance of S. gordonii against DMADDM. The presence of SDS and CH 5 N 3 HCl can cause an increase in the permeability of the plasma membrane . Various concentration of drug-containing BHI plates were prepared, and the solid BHI plates were incorporated with final concentrations of 20 μg/mL SDS and 100 μM guanidine hydrochloride, and/or 25 μg/mL DMADDM. The concentrations of SDS and CH 5 N 3 HCl were chosen according to a previous study, in which Saccharomyces cerevisiae developed resistance to quaternary ammonium compounds . The concentration of DMADDM was chosen in that S. gordonii could develop resistance against DMADDM at this concentration. This was based on our purpose to examine the influence of SDS and CH 5 N 3 HCl on the DMADDM-resistance of S. gordonii at this concentration. Overnight bacterial strains were diluted in sterile phosphate buffered saline (PBS) (Sigma) solution by 1:10 (100 μL culture in 900 μL diluent) to obtain a series of inoculants. An equal amount 10 μL droplets of the serial bacterial inoculum were spotted onto BHI plates and incorporated with DMADDM, SDS and hydrochloride, or their combinations, or none of them. After drying the droplets for 5–10 min, these BHI plates, which were pre-impregnated with a standard concentration of DMADDM, SDS and hydrochloride, or their combinations, or none of them, were incubated for 1–2 days at 37 °C with 5% CO 2 . Then the bacterial growth on each disk was examined. If the bacteria were not resistant to the additives in the agar plates, a clear “no-growth” phenomenon would occur on the culture plates. If the bacteria were resistant to the additives in the agar plates, a clear “growth” phenomenon of bacterial colony morphology would appear. The visible bacterial colony morphology was imaged by digital camera and processed by the software Adobe Photoshop CS6 to obtain the integration of bacterial colony morphology images.
2.6
Statistical analysis
Statistical analysis was performed by one-way analysis of variance followed by the Student-Newman-Keuls posttest. Differences were considered statistically significant at the 5% level ( P < 0.05). Statistical analysis was performed with the SPSS software, version 16.0 (SPSS Inc., Chicago, IL, USA).
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