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The effects of d -Tyrosine combined with amikacin on the biofilms of Pseudomonas aeruginosa
Microbial Pathogenesis 86 (2015) 38e44
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The effects of D-Tyrosine combined with amikacin on the biofilms of Pseudomonas aeruginosa Pengfei She 1, Lihua Chen 1, Hongbo Liu, Yaru Zou, Zhen Luo, Asmaa Koronfel, Yong Wu* Department of Medicine Clinical Laboratory, The Third Xiangya Hospital of Central South University, Tong Zipo Road, Changsha 410013, Hunan, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 February 2015 Received in revised form 9 July 2015 Accepted 13 July 2015 Available online 15 July 2015
The biofilm formation of microorganisms causes persistent tissue infections resistant to treatment with antimicrobial agents. Pseudomonas aeruginosa is commonly isolated from the airways of patients with chronic fibrosis (CF) and often forms biofilms, which are extremely hard to eradicate and a major cause of mortality and morbidity. Recent studies have shown that D-amino acids (D-AAs) inhibited and disrupted biofilm formation by causing the release of the protein component of the polymeric matrix. However, the effects of D-AAs combined with common antibiotics on biofilms have rarely been studied. The current study first determined whether D-AAs disrupted the biofilms of PAO1 and the clinical airway isolates of P. aeruginosa. It was then determined whether combinations of D-Tyr (the most effective one) and the antibiotic amikacin (AMK) enhanced the activity against these biofilms. The results of the current study showed that D-Tyr is the most effective among those that disassemble the D-amino acids (D-leucine, Dmethionine, D-Tyrptophan, and D-tryptophan), and D-Tyr at concentrations higher than 5 mM significantly reduced the biofilm biomass of P. aeruginosa (p < 0.05) without influencing bacterial growth. It was also revealed that D-Tyr improved the efficacy of AMK to combat P. aeruginosa biofilms, as indicated by a reduction in the minimal biofilm-inhibiting concentration (MBIC50 and MBIC90) without a change in the minimal inhibitory concentration (MIC) of planktonic bacteria. Thus, the findings indicated that D-Tyr supplementation overcame the resistance of P. aeruginosa biofilms to AMK, which might be helpful for preventing AMK overuse when this specific D-Tyr is recommended for combatting these biofilms. Also, toxicity of the liver and kidney from AMK could be potentially mitigated by co-delivery with D-Tyr. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Pseudomonas aeruginosa Biofilm D-amino acid D-Tyrosine Amikacin Combination
1. Introduction Bacteria are able to live either as independent planktonic cells or as members of organized surface-attached microbial communities called biofilms, which are composed of microorganisms and the extracellular matrix-forming polymers they produce [1]. In contrast to their planktonic counterparts, biofilm-derived bacteria have a distinctive phenotype regarding metabolic activity and gene expression, conferring an inherent resistance to antimicrobial agents as well as mechanisms of host clearance and making the treatment of biofilm-associated infections extremely difficult [2]. Biofilms persist and are hard to eradicate because of mechanisms that involve the restricted penetration of antimicrobials, differential physiological activity, and the presence of phenotypic variants
* Corresponding author. E-mail address: [email protected] (Y. Wu). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.micpath.2015.07.009 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
and persisters, efflux systems, and enhanced repair systems [3]. Resistance is often genetically inherited and therefore transmitted progeny of bacterial colonies, or it can be acquired through horizontal gene transfer [4]. Pseudomonas aeruginosa is commonly isolated from the airways of patients with cystic fibrosis (CF). Colonization can occur both in the paranasal sinuses and lungs, where it most often establishes chronic infections that usually persist for the rest of the patients' lives [5], eventually leading to respiratory failure and lung transplantation or death [6]. Despite improvement in therapies and a considerable increase in longevity over the past decades, the prevalence of P. aeruginosa in respiratory cultures increases with age from approximately 30% at ages 0e5 years to 80% at 18 years and older [7,8], so it still represents a therapeutic challenge. Clinical data suggest that P. aeruginosa forms antibiotic-resistant biofilms in the CF lung, which hinders the efficacy of currently available antibiotics and precludes the eradication of P. aeruginosa [9]. Chronic CF is characterized by the continuous growth of P. aeruginosa in airway
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secretions and by the development of P. aeruginosa-specific antibodies. It is also correlated with a higher degree of inflammation, a higher number of neutrophils, and a greater amount of released serine proteinases than are found in intermittently colonized individuals. Together, these factors cause increased lung obstruction and destruction [10,5]. The continuous presence of P. aeruginosa in the chronically-infected lung leads to immune complex-mediated chronic inflammation, which is dominated by PMNs and represents a major cause of lung tissue damage and decreased lung function along with the damage actively caused by the bacteria [11]. Since D-amino acids are synthesized and released by many bacterial species, including the opportunistic human pathogen P. aeruginosa, and have been shown to lack significant toxicity, the idea of using them to combat biofilm-associated infections is highly attractive [3]. Peptidoglycan (PG) is the major component of the bacterial cell wall and the most commonly cited source of D-amino acids in bacteria. PG is comprised of long glycan chains linked by short peptide stems. It is a plastic structure that provides a protective barrier for the cell, enabling cells to survive under variable physicochemical conditions [12]. Stems from adjacent glycan strands can be linked either directly or by an interpeptide bridge, which is itself comprised of amino acids. D-amino acids can be incorporated into both the peptide stem and the interpeptide bridge [13]. Incorporation of a D-amino acid into PG was also first reported to induce biofilm disassembly in Bacillus subtilis (B. subtilis) [14,15]. In addition to their incorporation into PG, free Damino acids may directly modulate the activity of periplasmic transpeptidase enzymes [16], and certain D-amino acids may serve as signaling compounds to mediate biofilm dispersal. On the basis of these observations, we hypothesized that combining dispersal agents with antimicrobials may be an effective therapeutic strategy for biofilms, functionally restoring the susceptibility of biofilms to antimicrobials through the release of bacteria from the biofilm. The dispersal activity of D-AAs on the biofilms of standard strain (PAO1) and clinical isolates was evaluated, and it was also investigated whether the combination of D-Tyr with antibiotics enhanced the activity against biofilm-producing bacteria in vitro. 2. Materials and methods 2.1. Strains and growth conditions P. aeruginosa PAO1 Strain (ATCC 15692) was used in this study. Six biofilm-forming clinical isolates, (designated stains JYK-0001, JYK-0002, JYK-0016, JYK-0004, JYK-0047 and JYK-0071) were chosen from a collection of 72 clinical isolates of P. aeruginosa during Jan 2014 to Dec 2014, from the Third Xiangya Hospital of Central South University, Changsha, China. No special ethical permit was required for this study according to the Chinese law. Cultures were stored at 80 C, and all isolates were routinely grown in LuriaeBertani broth (LB) (10 g tryptone per liter, 5 g yeast extract per liter, 5 g NaCl per liter) at 37 C with constant shaking (160 rpm). 2.2. Antibiotics and D-AAs Amikacin (AMK) was purchased from SigmaeAldrich (St. Louis, MO). D-AAs were purchased from SigmaeAldrich and prepared as a concentrated stock solution in distilled water or 1.0 N HCl, followed by filter sterilization. From the prepared stock solutions, D-AAs was diluted into Mueller-Hinton (MH) broth to a final concentration of 50 mM and neutralized when necessary with NaOH (1 M) (pH 7 to 7.4). All subsequent working concentrations of D-AAs were prepared by diluting the neutralized 50 mM stock into MH broth to yield a final working concentration. Because D-Tyr is the most
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effective among those D-AAs (D-leucine, D-methionine, D-Tyrptophan, and D-Tryptophan) that disassemble the biofilms (Fig. 2), it was selected for the following investigation. 2.3. Biofilm development of PAO1 To determine the kinetics of biofilm formation, an overnight culture was diluted at a ratio of 1:200 with fresh media, and 200 ml was deposited in a 6-well plate with 4 ml LB broth and a 9 9 mm glass slide inside. The plate was incubated at 37 C without agitation, and the biofilm formation was determined by crystal violet (CV) staining after removing the culture and washing the slides vigorously with 1 PBS every 6 h for a total of 24 h. The biofilm was observed using an optical microscope at 10 100 magnification. 2.4. Minimal inhibitory concentration (MIC) and minimal biofilminhibiting concentration (MBIC) MICs were determined by a microtitre broth dilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). The MIC was defined as the lowest antibiotic concentration that yielded no visible growth. The test medium was MH broth, and the density of bacteria was 5 105 colony-forming units (CFU)/ mL. Cell suspensions (100 mL) were inoculated into the wells of 96well microtitre plates in the presence of AMK with different final concentrations (0, 1, 2, 4, 6, 8, 16, 32, 64, 128, 256, 512, and 1024 mg/ ml). The inoculated microplates were incubated at 37 C for 24 h before being read. Antimicrobial susceptibility assays were performed in triplicate. The MBICs was calculated as the concentration of the antibiotic in which biofilms were eradicated (i.e. killed and/or dispersed) to a level 50% for MBIC50 or 90% for MBIC90 in comparison to control wells only treated with LB broth for 24 h [17]. 2.5. Assessment of the effects of D-Try alone or D-Tyr combined with AMK on biofilms in 96-well plates The semi-quantitative determination of biofilm formation was performed in 96-well sterile tissue culture plates (Corning, NY, America) based on the modified method reported by Christensen et al. [18]. Briefly, 1:200 diluted overnight bacterial culture was added to a 96-well plate, and bacteria were grown at 37 C in LB medium. After 24 h of growth, the plates were washed with 1 phosphate-buffered saline (PBS), and 200 ml of media without or supplemented with D-Tyr or D-Tyr combined with AMK at the designated concentrations in MH broth was added to each well for an additional 8, 16, or 24 h, respectively. Following exposure, the cells were washed as described above, and the biomass was determined by measuring the A490nm of 0.5% (w/v) crystal violet solubilized in ethanol. Experimental assays were performed in triplicate. 2.6. Timeekill assay The 24 h biofilms formed in 96-well plates were washed with 1 PBS to remove the planktonic cells. After 8, 16, and 24 h of incubation in AMK alone or combined with D-Tyr at the designated concentrations, the cells that adhered to the wells were mixed with the planktonic cells by thoroughly rubbing the surfaces with two moistened swabs, then mixing vigorously with a 1 ml pipette. The mixture was serially diluted (101e105) in saline, and aliquots of 100 mL were plated onto sterile NA plates. The plates were incubated for 24 h at 37 C. After the incubation period, the number of viable cells were counted, and the results were expressed in Log10 cfu/ml.
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Fig. 1. The biofilm development of PAO1 on glass covers is shown. Representative images (magnification, 10 100) of PAO1 biofilm grown on glass covers for 6 h (A), 12 h (B), 18 h (C), and 24 h (D), respectively, are shown and were stained with crystal violet to assess biofilm formation.
temperature. Cells were washed twice in 1 PBS. Samples were then removed from the PBS rinse solution and inverted onto a slide glass. Biofilms or adhered cells were imaged through the cover glass using the following excitation and emission wavelengths: 488 and 500 nm for SYTO9 and 490 and 635 nm for PI, respectively. Only SYTO9 staining was used to detect the dispersal ability of DTyr on JYK-0047 at the designated concentration of 5 mM.
2.8. Statistical analysis The normal distribution of the data was checked using the ShapiroeWilk test. Differences between groups were examined for statistical significance using analysis of variance (ANOVA). Statistics were determined using SPSS software, version 19.0. P < 0.05 denoted a statistically significant difference. All experiments were performed in triplicate and repeated three times.
Fig. 2. The effects of D-amino acids against PAO1 biofilms are shown. 24 h mature biofilms were cultured overnight in 96-well plates with individual D-amino acids at concentrations ranging from 0.01 to 50 mM and the quantification of biofilm formation determined by crystal violet staining. Biofilm was significantly impaired by D-Tyrosine at concentrations of 5 mM *, P < 0.05 versus untreated control. Error bars represent SD.
2.7. Confocal laser scanning microscopy (CLSM) Confocal microscopy images were obtained by a Leica TCS SP8 spectral confocal inverted microscope. The 24 h biofilms of PAO1 formed in 6-well plates with 18 18 mm glass cover slides inside were washed with 1 PBS to remove the planktonic cells, transferred into new wells, and submerged in AMK alone or AMK combined with D-Tyr at a designated final concentration. After 8, 16, and 24 h of incubation, the planktonic cells were removed with 1 PBS. Pre-mixed dyes SYTO9 (green) and PI (red) [LIVE/DEAD BacLight Bacterial Viability Kit (L7012), USA] were added to the slides and incubated without shaking for 10 min at room
3. Results 3.1. Biofilm development of PAO1 PAO1 formed biofilms on glass covers. These biofilms were detected by staining the adhered cells with crystal violet. The development of a bacterial biofilm could be divided into three phases, which involved specific molecular factors: attachment, maturation, and detachment [19]. Fig. 1 shows the biofilm development of PAO1 from the stage of attachment to the surface to the growth of the cells into a sessile biofilm (Fig. 1AeD). Individual planktonic cells formed cell-to-surface and cell-to-cell contacts, resulting in the formation of microcolonies. The development involved the synthesis of an extracellular polymeric matrix, which held the bacterial cells together in a mass and firmly attached the bacterial mass to the underlying surface (Fig. 1AeB). Continued growth of bacterial cells on surfaces led to the development of mature biofilm colonies containing millions of tightly packed cells gathered into a three-dimensional structure (Fig. 1CeD).
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3.2. D-AAs causes disassembly of existing PAO1 biofilm To evaluate the potential clinical applications of D-AAs, it was tested whether D-AAs effectively dispersed the preformed biofilm of PAO1 at various concentration (0.01e50 mM). The results showed that the dispersal activity of D-AAs were significant (p < 0.05) at the concentration of 5, 1, 25 and 25 mM, respectively (Fig. 2). But D-Tyr is the most effective among those D-AAs that disassemble the biofilms at the same effective concentration. The 24-h treatment with 5 mM D-Tyr resulted in a reduction of the biomass by nearly 50%. Based on the initial screening of D-AAs dispersal activity, D-Tyr was chosen at the concentration of 5 mM for use in all subsequent in vitro assays. 3.3. The dispersive effects of D-Tyr on the 24 h mature biofilms of clinical airway isolates When tested against biofilms of PAO1, D-Tyr was observed to have the strongest significant dispersal activity compared with other D-AAs at the same concentration of 5 mM, as indicated by the reductions in biofilm biomass determined by crystal violet assay (Fig. 2). Then, when we tested against biofilms of six clinical isolates of P. aeruginosa, D-Tyr at 5 mM was also observed to have significant dispersal activity (Fig. 3A). Consistent with the result, the confocal microscopy analysis of biofilms of JYK-0047 treated with D-Tyr (5 mM) demonstrated a significant reduction of the biomass compared to that of the untreated controls (Fig. 3B). 3.4. D-AAs had no significant effects on the growth of P. aeruginosa The reduction in the biomass led to further investigation on whether this was due to cell death or detachment. Overnight culture of PAO1 was inoculated into LB broth in the presence or
Fig. 4. D-amino acids do not inhibit cell growth. Overnight culture of PAO1 was inoculated into 25 mL of LB broth and grown at 37 C with agitation in the presence or absence of individual D-amino acids (D-Tyr, D-Trp, D-Met and D-Leu) at 5 or 10 mM respectively. Absorbance at 600 nm was measured every 2 h up to 14 h. The average of two replicates is shown.
absence of individual D-AAs at 5 or 10 mM, respectively. Absorbance at 600 nm was measured every 2 h up to 14 h. The results showed that D-Tyr had no significant effects on the growth of PAO1, indicating that biofilm dispersive activity was not associated with growth inhibition (Fig. 4).
3.5. D-Tyr enhanced the activity of antimicrobials against P. aeruginosa Antimicrobial susceptibility assays with and without D-Tyr were performed on both the planktonic cells and biofilms of PAO1 and clinical isolates. The MICs of AMK for planktonic cells were determined for PAO1 (0.5 mg/ml), JYK-0001 (>256 mg/ml), JYK-0002 (1 mg/ml), JYK-0016 (1 mg/ml), JYK-0045 (0.5 mg/ml), JYK-0047
Fig. 3. The biofilm dispersive activity of D-Tyrosine (5 mM) on clinical isolates are shown. (A) Biofilm dispersal was assessed by measuring the absorbance of solubilized crystal violet from stained biofilms following treatment with D-Tyrosine at 490 nm *, P < 0.05 versus untreated control. Error bars represent SD. (B) Representative CLSM images of biofilms of JYK-0047 treated with D-Tyrosine for 24 h are shown. Biofilms were stained with SYTO9 (green).
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Table 1 MIC, MBIC50 and MBIC90 of AMK alone or combined with D-Tyr. Antimicrobial agent
PAO1 JYK-0001 JYK-0002 JYK-0004 JYK-0016 JYK-0047 JYK-0071
MIC (mg/ml)
MBIC50 (mg/ml)
MBIC90 (mg/ml)
AMK
AMK þ D-Tyr
AMK
AMK þ D-Tyr
AMK
AMK þ D-Tyr
0.5 >256 1 1 0.5 0.5 0.5
0.5 >256 1 1 0.5 0.5 0.5
32 256 512 128 32 32 >1024
8 64 64 32 16 16 512
128 >1024 >1024 512 256 512 >1024
32 1024 512 128 128 256 >1024
MIC, minimal inhibitory concentration, for planktonic cells; MBIC, minimal biofilm-inhibiting concentration, for biofilm cells; AMK, amikacin; D-Tyr, D-Tyrosine.
(0.5 mg/ml) and JYK-0071 (0.5 mg/ml) (Table 1). Moreover, the results showed that the combination of D-Tyr (5 mM) with AMK did not alter the susceptibilities of planktonic cells (MICs). Conversely, the MBIC50 and MBIC90 for the P. aeruginosa stains, respectively, were determined for PAO1 (32 and 128 mg/ml), JYK-0001 (256 and > 1024 mg/ml), JYK-0002 (512 and > 1024 mg/ml), JYK-0004 (128 and 512 mg/ml), JYK-0016 (32 and 256 mg/ml), JYK-0047 (32 and 512 mg/ml) and JYK-0071 (>1024 and > 1024 mg/ml) when used AMK alone, which exceeded the range of the MICs for planktonic cells. As anticipated, exposure to AMK combined with D-Tyr (5 mM) enhanced the activity of AMK by reducing the concentration of the MBIC50 and MBIC90 to 8 and 32 mg/ml for PAO1, 64 and 1024 mg/ml for JYK-0001, 64 and 512 mg/ml for JYK-0002, 32 and 128 mg/ml for JYK-0004, 16 and 128 mg/ml for JYK-0016, 16 and 256 mg/ml for JYK0047, 512 and > 1024 mg/ml for JYK-0071, respectively (Table 1). The results indicated that the combined exposure resulted in a reduction of the observed MBIC50 and MBIC90 from 16 to 512 mg/ml lower than with AMK used alone. The related fluorescent images showed that the exposure of PAO1 to the combined treatment at the experimentally determined MBIC50 described above for AMK (8 mg/ ml) and D-Tyr (5 mM) resulted in a greater reduction of the biomass (Fig. 5 A, C) as well as viable bacteria (Fig. 5 B,C) from the biofilm than treatment with AMK alone. 4. Discussion The differentiation process that transformed small groups of adherent bacteria into a thick matrix-enclosed biofilm community on a colonized surface was observed as a series of morphological changes (Fig. 1AeD). Stage 1 showed the initial attachment of cells to the surface (Fig. 1A). The individual adherent cells that initiated biofilm formation on a surface were surrounded only by small amounts of exopolymeric material, and many were capable of independent movement [20]. However, these adherent cells sometimes actually left the surface to resume the planktonic lifestyle. Before they exuded enough expolysaccharide and adhered irreversibly (Fig. 1B), the bacteria exhibited several behaviors including rolling, creeping, aggregate formation, and “windrow” formation [21]. As biofilms matured, they developed the basic threedimensional structure containing water channels, marking the early development of biofilm architecture (Fig. 1C). Also, many cells altered their physiological processes in response to conditions in their particular niches. During the period of maturation and dispersion for PAO1 (Fig. 1D), biofilm architecture improved, and individual microcolonies sometimes detached from the surface or increased in size as planktonic cells, leaving empty spaces that became part of the water channels [21]. P. aeruginosa is the most relevant pathogen colonizing the respiratory tract of CF patients and causes an important decline in pulmonary function [22]. In some of the stages of the process (planktonic dispersal), the microorganisms are frequently
susceptible to antibiotics, but compared to their planktonic counterparts, biofilm-derived bacteria have distinctive phenotypes in regards to growth, gene expression, and protein production that confer resistance to antimicrobial agents as well as host mechanisms of clearance [23,24]. Above all, the resistance is mainly because bacteria are encased in an exopolysaccharide (EPS) matrix that can act as a barrier, preventing antibiotics from penetrating the biofilm [25]. Finally, a small population (i.e., 0.1e10%) of “persister cells” survives antimicrobial therapy and rapidly grows after the cessation of antibiotic therapy, potentially resulting in recurrent infections [26]. Recent studies have shown that the D-amino acids can disperse biofilms formed by a diverse range of bacterial species, including S. aureus and P. aeruginosa. In contrast to other biofilm dispersal agents, D-AAs have minimal cellular toxicity [14,27,28]. Given the importance of the biofilms in disease and the limitations of conventional antimicrobials against this phenotype, it was herein assessed whether the use of D-AAs, the potential biofilm dispersal signaling molecules, could enhance the activity of antimicrobials against biofilms. Consistent with these studies, it was observed that D-AAs had significant dispersive activity on 24 h biofilms of P. aeruginosa at different concentrations (Fig. 1). Importantly, and in contrast to previous studies with B. subtilis, significant effects on either cell viability or bacterial growth were not observed at the effective concentrations (Fig. 4). Therefore, the effects of D-AAs seem to be specific for biofilm dispersal. The inhibition of biofilm formation by D-AAs is largely, if not entirely, mediated by misincorporation into protein, presumably resulting in proteotoxicity. It has been proposed that D-AAs affect the union of the TasA protein to the cell wall in B. subtilis NCBI3610 biofilms [29]. The protein TasA is polymerized to form amyloid-like fibers, which are considered to be the fundamental structural units in Bacillus biofilms that bind to the bacterial cell wall. Recently, scientists found that TapA, linking the TasA fibers to the cell wall, was separated from the cell wall in a similar fashion as the amyloid TasA fibers when treated with D-AAs [30]. Conversely, D-AAs inhibited the expressions of biofilm matrix genes epsA and tapA at concentrations that inhibited biofilm formation [29], but Kolodkin-Gal I found that D-AAs did not inhibit the expressions of the matrix operons epsA-O and yqxM-sipW-tasA [14]. We will rectify these contradictory results in our future study. AMK, a semisynthetic analog of kanamycin, is very active against most gram-negative bacteria, including gentamicin- and tobramycin-resistant strains. The effectiveness of AMK in the treatment of serious gram-negative bacillary infections is welldocumented [31]. As anticipated, the antimicrobial agent tested against biofilms of P. aeruginosa was ineffective at the tested concentration against the planktonic phenotype. However, the combination of D-Tyr (5 mM) significantly enhanced the antimicrobial activity of AMK and reduced the MBIC50 and MBIC90 (Table 1). AMK (10 mg/ml) combined with D-Tyr (5 mM) demonstrated the enhancement of bactericidal activity with 1.25 Log10 CFU/ml, 0.75
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Fig. 5. The effects of the combination of D-Tyrosine (5 mM) and AMK (8 mg/ml) against the biofilms of PAO1. (A) The kinetic dispersing effects of AMK alone or combined with DTyrosine on PAO1 biofilms are shown. Biofilm dispersal was assessed by measuring the absorbance of solubilized crystal violet at 490 nm. (B) The kinetic bactericidal effects of DTyrosine and AMK against biofilms of PAO1 are shown. The biofilm of PAO1 was developed in a 96-well plate for 24 h, followed by exposure to AMK in the absence or presence of DTyrosine for 8, 16, and 24 h, respectively. Viable bacteria formed biofilms were determined by plating serial dilutions, followed by the removal of adherent bacteria by swabbing from wells. (C) Representative CLSM images about the effects of D-Tyrosine and AMK on PAO1 biofilm morphology are shown. The biofilms were developed on glass covers for 24 h, followed by exposure to AMK in the absence or presence of D-Tyrosine for 8, 16, and 24 h, respectively. The cells were fixed and stained for fluorescence microscopy imaging.
Log10 CFU/ml, and 1.68 Log10 CFU/ml decreases compared to AMK (10 mg/ml) alone at 8, 16, and 24 h, respectively. The timeekill curve and timeedisperse curve were consistent with the results (Fig. 5AeB). Confocal microscopy gave a more detailed picture of biofilm dispersal during exposure to AMK (10 mg) alone or combined with D-Tyr (5 mM) for 8, 16, and 24 h, respectively [LIVE/ DEAD BacLight Bacterial Viability Kit (L7012), Invitrogen, USA] (Fig. 5C). Since the antimicrobial concentrations required for biofilm eradication may be reduced by co-delivery with D-Tyr, the toxicity of the liver and kidney due to AMK can potentially be mitigated. In summary, this work demonstrated the ability of D-Tyr to disperse biofilms in P. aeruginosa. The data suggest that combining AMK with D-Tyr may increase the success of a treatment by increasing the efficacy of AMK against biofilm-related infections. Because D-AAs are not bactericidal, they are anticipated to be most
effective as an adjuvant therapy to conventional treatment with systemic antibiotics. Most studies of biofilms have taken a reductionist approach, where single-species biofilms have been extensively investigated. However, biofilms in human beings can sometimes comprise multiple species. For example, about 100e200 bacterial species were found to colonize and form biofilms on hard and soft tissues of the oral cavity, where the interspecies interactions play important roles in determining the development, structure, and function of these biofilms [32]. Thus, we will continue studying the effects of D-amino acids combined with antibiotics on multi-species biofilms. References [1] R.D. Monds, G.A. O'Toole, The developmental model of microbial biofilms: ten years of a paradigm up for review, Trends Microbiol. 17 (2009) 73e87.
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The effects of D-Tyrosine combined with amikacin on the biofilms of Pseudomonas aeruginosa Pengfei She 1, Lihua Chen 1, Hongbo Liu, Yaru Zou, Zhen Luo, Asmaa Koronfel, Yong Wu* Department of Medicine Clinical Laboratory, The Third Xiangya Hospital of Central South University, Tong Zipo Road, Changsha 410013, Hunan, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 February 2015 Received in revised form 9 July 2015 Accepted 13 July 2015 Available online 15 July 2015
The biofilm formation of microorganisms causes persistent tissue infections resistant to treatment with antimicrobial agents. Pseudomonas aeruginosa is commonly isolated from the airways of patients with chronic fibrosis (CF) and often forms biofilms, which are extremely hard to eradicate and a major cause of mortality and morbidity. Recent studies have shown that D-amino acids (D-AAs) inhibited and disrupted biofilm formation by causing the release of the protein component of the polymeric matrix. However, the effects of D-AAs combined with common antibiotics on biofilms have rarely been studied. The current study first determined whether D-AAs disrupted the biofilms of PAO1 and the clinical airway isolates of P. aeruginosa. It was then determined whether combinations of D-Tyr (the most effective one) and the antibiotic amikacin (AMK) enhanced the activity against these biofilms. The results of the current study showed that D-Tyr is the most effective among those that disassemble the D-amino acids (D-leucine, Dmethionine, D-Tyrptophan, and D-tryptophan), and D-Tyr at concentrations higher than 5 mM significantly reduced the biofilm biomass of P. aeruginosa (p < 0.05) without influencing bacterial growth. It was also revealed that D-Tyr improved the efficacy of AMK to combat P. aeruginosa biofilms, as indicated by a reduction in the minimal biofilm-inhibiting concentration (MBIC50 and MBIC90) without a change in the minimal inhibitory concentration (MIC) of planktonic bacteria. Thus, the findings indicated that D-Tyr supplementation overcame the resistance of P. aeruginosa biofilms to AMK, which might be helpful for preventing AMK overuse when this specific D-Tyr is recommended for combatting these biofilms. Also, toxicity of the liver and kidney from AMK could be potentially mitigated by co-delivery with D-Tyr. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Pseudomonas aeruginosa Biofilm D-amino acid D-Tyrosine Amikacin Combination
1. Introduction Bacteria are able to live either as independent planktonic cells or as members of organized surface-attached microbial communities called biofilms, which are composed of microorganisms and the extracellular matrix-forming polymers they produce [1]. In contrast to their planktonic counterparts, biofilm-derived bacteria have a distinctive phenotype regarding metabolic activity and gene expression, conferring an inherent resistance to antimicrobial agents as well as mechanisms of host clearance and making the treatment of biofilm-associated infections extremely difficult [2]. Biofilms persist and are hard to eradicate because of mechanisms that involve the restricted penetration of antimicrobials, differential physiological activity, and the presence of phenotypic variants
* Corresponding author. E-mail address: [email protected] (Y. Wu). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.micpath.2015.07.009 0882-4010/© 2015 Elsevier Ltd. All rights reserved.
and persisters, efflux systems, and enhanced repair systems [3]. Resistance is often genetically inherited and therefore transmitted progeny of bacterial colonies, or it can be acquired through horizontal gene transfer [4]. Pseudomonas aeruginosa is commonly isolated from the airways of patients with cystic fibrosis (CF). Colonization can occur both in the paranasal sinuses and lungs, where it most often establishes chronic infections that usually persist for the rest of the patients' lives [5], eventually leading to respiratory failure and lung transplantation or death [6]. Despite improvement in therapies and a considerable increase in longevity over the past decades, the prevalence of P. aeruginosa in respiratory cultures increases with age from approximately 30% at ages 0e5 years to 80% at 18 years and older [7,8], so it still represents a therapeutic challenge. Clinical data suggest that P. aeruginosa forms antibiotic-resistant biofilms in the CF lung, which hinders the efficacy of currently available antibiotics and precludes the eradication of P. aeruginosa [9]. Chronic CF is characterized by the continuous growth of P. aeruginosa in airway
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secretions and by the development of P. aeruginosa-specific antibodies. It is also correlated with a higher degree of inflammation, a higher number of neutrophils, and a greater amount of released serine proteinases than are found in intermittently colonized individuals. Together, these factors cause increased lung obstruction and destruction [10,5]. The continuous presence of P. aeruginosa in the chronically-infected lung leads to immune complex-mediated chronic inflammation, which is dominated by PMNs and represents a major cause of lung tissue damage and decreased lung function along with the damage actively caused by the bacteria [11]. Since D-amino acids are synthesized and released by many bacterial species, including the opportunistic human pathogen P. aeruginosa, and have been shown to lack significant toxicity, the idea of using them to combat biofilm-associated infections is highly attractive [3]. Peptidoglycan (PG) is the major component of the bacterial cell wall and the most commonly cited source of D-amino acids in bacteria. PG is comprised of long glycan chains linked by short peptide stems. It is a plastic structure that provides a protective barrier for the cell, enabling cells to survive under variable physicochemical conditions [12]. Stems from adjacent glycan strands can be linked either directly or by an interpeptide bridge, which is itself comprised of amino acids. D-amino acids can be incorporated into both the peptide stem and the interpeptide bridge [13]. Incorporation of a D-amino acid into PG was also first reported to induce biofilm disassembly in Bacillus subtilis (B. subtilis) [14,15]. In addition to their incorporation into PG, free Damino acids may directly modulate the activity of periplasmic transpeptidase enzymes [16], and certain D-amino acids may serve as signaling compounds to mediate biofilm dispersal. On the basis of these observations, we hypothesized that combining dispersal agents with antimicrobials may be an effective therapeutic strategy for biofilms, functionally restoring the susceptibility of biofilms to antimicrobials through the release of bacteria from the biofilm. The dispersal activity of D-AAs on the biofilms of standard strain (PAO1) and clinical isolates was evaluated, and it was also investigated whether the combination of D-Tyr with antibiotics enhanced the activity against biofilm-producing bacteria in vitro. 2. Materials and methods 2.1. Strains and growth conditions P. aeruginosa PAO1 Strain (ATCC 15692) was used in this study. Six biofilm-forming clinical isolates, (designated stains JYK-0001, JYK-0002, JYK-0016, JYK-0004, JYK-0047 and JYK-0071) were chosen from a collection of 72 clinical isolates of P. aeruginosa during Jan 2014 to Dec 2014, from the Third Xiangya Hospital of Central South University, Changsha, China. No special ethical permit was required for this study according to the Chinese law. Cultures were stored at 80 C, and all isolates were routinely grown in LuriaeBertani broth (LB) (10 g tryptone per liter, 5 g yeast extract per liter, 5 g NaCl per liter) at 37 C with constant shaking (160 rpm). 2.2. Antibiotics and D-AAs Amikacin (AMK) was purchased from SigmaeAldrich (St. Louis, MO). D-AAs were purchased from SigmaeAldrich and prepared as a concentrated stock solution in distilled water or 1.0 N HCl, followed by filter sterilization. From the prepared stock solutions, D-AAs was diluted into Mueller-Hinton (MH) broth to a final concentration of 50 mM and neutralized when necessary with NaOH (1 M) (pH 7 to 7.4). All subsequent working concentrations of D-AAs were prepared by diluting the neutralized 50 mM stock into MH broth to yield a final working concentration. Because D-Tyr is the most
39
effective among those D-AAs (D-leucine, D-methionine, D-Tyrptophan, and D-Tryptophan) that disassemble the biofilms (Fig. 2), it was selected for the following investigation. 2.3. Biofilm development of PAO1 To determine the kinetics of biofilm formation, an overnight culture was diluted at a ratio of 1:200 with fresh media, and 200 ml was deposited in a 6-well plate with 4 ml LB broth and a 9 9 mm glass slide inside. The plate was incubated at 37 C without agitation, and the biofilm formation was determined by crystal violet (CV) staining after removing the culture and washing the slides vigorously with 1 PBS every 6 h for a total of 24 h. The biofilm was observed using an optical microscope at 10 100 magnification. 2.4. Minimal inhibitory concentration (MIC) and minimal biofilminhibiting concentration (MBIC) MICs were determined by a microtitre broth dilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI). The MIC was defined as the lowest antibiotic concentration that yielded no visible growth. The test medium was MH broth, and the density of bacteria was 5 105 colony-forming units (CFU)/ mL. Cell suspensions (100 mL) were inoculated into the wells of 96well microtitre plates in the presence of AMK with different final concentrations (0, 1, 2, 4, 6, 8, 16, 32, 64, 128, 256, 512, and 1024 mg/ ml). The inoculated microplates were incubated at 37 C for 24 h before being read. Antimicrobial susceptibility assays were performed in triplicate. The MBICs was calculated as the concentration of the antibiotic in which biofilms were eradicated (i.e. killed and/or dispersed) to a level 50% for MBIC50 or 90% for MBIC90 in comparison to control wells only treated with LB broth for 24 h [17]. 2.5. Assessment of the effects of D-Try alone or D-Tyr combined with AMK on biofilms in 96-well plates The semi-quantitative determination of biofilm formation was performed in 96-well sterile tissue culture plates (Corning, NY, America) based on the modified method reported by Christensen et al. [18]. Briefly, 1:200 diluted overnight bacterial culture was added to a 96-well plate, and bacteria were grown at 37 C in LB medium. After 24 h of growth, the plates were washed with 1 phosphate-buffered saline (PBS), and 200 ml of media without or supplemented with D-Tyr or D-Tyr combined with AMK at the designated concentrations in MH broth was added to each well for an additional 8, 16, or 24 h, respectively. Following exposure, the cells were washed as described above, and the biomass was determined by measuring the A490nm of 0.5% (w/v) crystal violet solubilized in ethanol. Experimental assays were performed in triplicate. 2.6. Timeekill assay The 24 h biofilms formed in 96-well plates were washed with 1 PBS to remove the planktonic cells. After 8, 16, and 24 h of incubation in AMK alone or combined with D-Tyr at the designated concentrations, the cells that adhered to the wells were mixed with the planktonic cells by thoroughly rubbing the surfaces with two moistened swabs, then mixing vigorously with a 1 ml pipette. The mixture was serially diluted (101e105) in saline, and aliquots of 100 mL were plated onto sterile NA plates. The plates were incubated for 24 h at 37 C. After the incubation period, the number of viable cells were counted, and the results were expressed in Log10 cfu/ml.
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Fig. 1. The biofilm development of PAO1 on glass covers is shown. Representative images (magnification, 10 100) of PAO1 biofilm grown on glass covers for 6 h (A), 12 h (B), 18 h (C), and 24 h (D), respectively, are shown and were stained with crystal violet to assess biofilm formation.
temperature. Cells were washed twice in 1 PBS. Samples were then removed from the PBS rinse solution and inverted onto a slide glass. Biofilms or adhered cells were imaged through the cover glass using the following excitation and emission wavelengths: 488 and 500 nm for SYTO9 and 490 and 635 nm for PI, respectively. Only SYTO9 staining was used to detect the dispersal ability of DTyr on JYK-0047 at the designated concentration of 5 mM.
2.8. Statistical analysis The normal distribution of the data was checked using the ShapiroeWilk test. Differences between groups were examined for statistical significance using analysis of variance (ANOVA). Statistics were determined using SPSS software, version 19.0. P < 0.05 denoted a statistically significant difference. All experiments were performed in triplicate and repeated three times.
Fig. 2. The effects of D-amino acids against PAO1 biofilms are shown. 24 h mature biofilms were cultured overnight in 96-well plates with individual D-amino acids at concentrations ranging from 0.01 to 50 mM and the quantification of biofilm formation determined by crystal violet staining. Biofilm was significantly impaired by D-Tyrosine at concentrations of 5 mM *, P < 0.05 versus untreated control. Error bars represent SD.
2.7. Confocal laser scanning microscopy (CLSM) Confocal microscopy images were obtained by a Leica TCS SP8 spectral confocal inverted microscope. The 24 h biofilms of PAO1 formed in 6-well plates with 18 18 mm glass cover slides inside were washed with 1 PBS to remove the planktonic cells, transferred into new wells, and submerged in AMK alone or AMK combined with D-Tyr at a designated final concentration. After 8, 16, and 24 h of incubation, the planktonic cells were removed with 1 PBS. Pre-mixed dyes SYTO9 (green) and PI (red) [LIVE/DEAD BacLight Bacterial Viability Kit (L7012), USA] were added to the slides and incubated without shaking for 10 min at room
3. Results 3.1. Biofilm development of PAO1 PAO1 formed biofilms on glass covers. These biofilms were detected by staining the adhered cells with crystal violet. The development of a bacterial biofilm could be divided into three phases, which involved specific molecular factors: attachment, maturation, and detachment [19]. Fig. 1 shows the biofilm development of PAO1 from the stage of attachment to the surface to the growth of the cells into a sessile biofilm (Fig. 1AeD). Individual planktonic cells formed cell-to-surface and cell-to-cell contacts, resulting in the formation of microcolonies. The development involved the synthesis of an extracellular polymeric matrix, which held the bacterial cells together in a mass and firmly attached the bacterial mass to the underlying surface (Fig. 1AeB). Continued growth of bacterial cells on surfaces led to the development of mature biofilm colonies containing millions of tightly packed cells gathered into a three-dimensional structure (Fig. 1CeD).
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3.2. D-AAs causes disassembly of existing PAO1 biofilm To evaluate the potential clinical applications of D-AAs, it was tested whether D-AAs effectively dispersed the preformed biofilm of PAO1 at various concentration (0.01e50 mM). The results showed that the dispersal activity of D-AAs were significant (p < 0.05) at the concentration of 5, 1, 25 and 25 mM, respectively (Fig. 2). But D-Tyr is the most effective among those D-AAs that disassemble the biofilms at the same effective concentration. The 24-h treatment with 5 mM D-Tyr resulted in a reduction of the biomass by nearly 50%. Based on the initial screening of D-AAs dispersal activity, D-Tyr was chosen at the concentration of 5 mM for use in all subsequent in vitro assays. 3.3. The dispersive effects of D-Tyr on the 24 h mature biofilms of clinical airway isolates When tested against biofilms of PAO1, D-Tyr was observed to have the strongest significant dispersal activity compared with other D-AAs at the same concentration of 5 mM, as indicated by the reductions in biofilm biomass determined by crystal violet assay (Fig. 2). Then, when we tested against biofilms of six clinical isolates of P. aeruginosa, D-Tyr at 5 mM was also observed to have significant dispersal activity (Fig. 3A). Consistent with the result, the confocal microscopy analysis of biofilms of JYK-0047 treated with D-Tyr (5 mM) demonstrated a significant reduction of the biomass compared to that of the untreated controls (Fig. 3B). 3.4. D-AAs had no significant effects on the growth of P. aeruginosa The reduction in the biomass led to further investigation on whether this was due to cell death or detachment. Overnight culture of PAO1 was inoculated into LB broth in the presence or
Fig. 4. D-amino acids do not inhibit cell growth. Overnight culture of PAO1 was inoculated into 25 mL of LB broth and grown at 37 C with agitation in the presence or absence of individual D-amino acids (D-Tyr, D-Trp, D-Met and D-Leu) at 5 or 10 mM respectively. Absorbance at 600 nm was measured every 2 h up to 14 h. The average of two replicates is shown.
absence of individual D-AAs at 5 or 10 mM, respectively. Absorbance at 600 nm was measured every 2 h up to 14 h. The results showed that D-Tyr had no significant effects on the growth of PAO1, indicating that biofilm dispersive activity was not associated with growth inhibition (Fig. 4).
3.5. D-Tyr enhanced the activity of antimicrobials against P. aeruginosa Antimicrobial susceptibility assays with and without D-Tyr were performed on both the planktonic cells and biofilms of PAO1 and clinical isolates. The MICs of AMK for planktonic cells were determined for PAO1 (0.5 mg/ml), JYK-0001 (>256 mg/ml), JYK-0002 (1 mg/ml), JYK-0016 (1 mg/ml), JYK-0045 (0.5 mg/ml), JYK-0047
Fig. 3. The biofilm dispersive activity of D-Tyrosine (5 mM) on clinical isolates are shown. (A) Biofilm dispersal was assessed by measuring the absorbance of solubilized crystal violet from stained biofilms following treatment with D-Tyrosine at 490 nm *, P < 0.05 versus untreated control. Error bars represent SD. (B) Representative CLSM images of biofilms of JYK-0047 treated with D-Tyrosine for 24 h are shown. Biofilms were stained with SYTO9 (green).
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Table 1 MIC, MBIC50 and MBIC90 of AMK alone or combined with D-Tyr. Antimicrobial agent
PAO1 JYK-0001 JYK-0002 JYK-0004 JYK-0016 JYK-0047 JYK-0071
MIC (mg/ml)
MBIC50 (mg/ml)
MBIC90 (mg/ml)
AMK
AMK þ D-Tyr
AMK
AMK þ D-Tyr
AMK
AMK þ D-Tyr
0.5 >256 1 1 0.5 0.5 0.5
0.5 >256 1 1 0.5 0.5 0.5
32 256 512 128 32 32 >1024
8 64 64 32 16 16 512
128 >1024 >1024 512 256 512 >1024
32 1024 512 128 128 256 >1024
MIC, minimal inhibitory concentration, for planktonic cells; MBIC, minimal biofilm-inhibiting concentration, for biofilm cells; AMK, amikacin; D-Tyr, D-Tyrosine.
(0.5 mg/ml) and JYK-0071 (0.5 mg/ml) (Table 1). Moreover, the results showed that the combination of D-Tyr (5 mM) with AMK did not alter the susceptibilities of planktonic cells (MICs). Conversely, the MBIC50 and MBIC90 for the P. aeruginosa stains, respectively, were determined for PAO1 (32 and 128 mg/ml), JYK-0001 (256 and > 1024 mg/ml), JYK-0002 (512 and > 1024 mg/ml), JYK-0004 (128 and 512 mg/ml), JYK-0016 (32 and 256 mg/ml), JYK-0047 (32 and 512 mg/ml) and JYK-0071 (>1024 and > 1024 mg/ml) when used AMK alone, which exceeded the range of the MICs for planktonic cells. As anticipated, exposure to AMK combined with D-Tyr (5 mM) enhanced the activity of AMK by reducing the concentration of the MBIC50 and MBIC90 to 8 and 32 mg/ml for PAO1, 64 and 1024 mg/ml for JYK-0001, 64 and 512 mg/ml for JYK-0002, 32 and 128 mg/ml for JYK-0004, 16 and 128 mg/ml for JYK-0016, 16 and 256 mg/ml for JYK0047, 512 and > 1024 mg/ml for JYK-0071, respectively (Table 1). The results indicated that the combined exposure resulted in a reduction of the observed MBIC50 and MBIC90 from 16 to 512 mg/ml lower than with AMK used alone. The related fluorescent images showed that the exposure of PAO1 to the combined treatment at the experimentally determined MBIC50 described above for AMK (8 mg/ ml) and D-Tyr (5 mM) resulted in a greater reduction of the biomass (Fig. 5 A, C) as well as viable bacteria (Fig. 5 B,C) from the biofilm than treatment with AMK alone. 4. Discussion The differentiation process that transformed small groups of adherent bacteria into a thick matrix-enclosed biofilm community on a colonized surface was observed as a series of morphological changes (Fig. 1AeD). Stage 1 showed the initial attachment of cells to the surface (Fig. 1A). The individual adherent cells that initiated biofilm formation on a surface were surrounded only by small amounts of exopolymeric material, and many were capable of independent movement [20]. However, these adherent cells sometimes actually left the surface to resume the planktonic lifestyle. Before they exuded enough expolysaccharide and adhered irreversibly (Fig. 1B), the bacteria exhibited several behaviors including rolling, creeping, aggregate formation, and “windrow” formation [21]. As biofilms matured, they developed the basic threedimensional structure containing water channels, marking the early development of biofilm architecture (Fig. 1C). Also, many cells altered their physiological processes in response to conditions in their particular niches. During the period of maturation and dispersion for PAO1 (Fig. 1D), biofilm architecture improved, and individual microcolonies sometimes detached from the surface or increased in size as planktonic cells, leaving empty spaces that became part of the water channels [21]. P. aeruginosa is the most relevant pathogen colonizing the respiratory tract of CF patients and causes an important decline in pulmonary function [22]. In some of the stages of the process (planktonic dispersal), the microorganisms are frequently
susceptible to antibiotics, but compared to their planktonic counterparts, biofilm-derived bacteria have distinctive phenotypes in regards to growth, gene expression, and protein production that confer resistance to antimicrobial agents as well as host mechanisms of clearance [23,24]. Above all, the resistance is mainly because bacteria are encased in an exopolysaccharide (EPS) matrix that can act as a barrier, preventing antibiotics from penetrating the biofilm [25]. Finally, a small population (i.e., 0.1e10%) of “persister cells” survives antimicrobial therapy and rapidly grows after the cessation of antibiotic therapy, potentially resulting in recurrent infections [26]. Recent studies have shown that the D-amino acids can disperse biofilms formed by a diverse range of bacterial species, including S. aureus and P. aeruginosa. In contrast to other biofilm dispersal agents, D-AAs have minimal cellular toxicity [14,27,28]. Given the importance of the biofilms in disease and the limitations of conventional antimicrobials against this phenotype, it was herein assessed whether the use of D-AAs, the potential biofilm dispersal signaling molecules, could enhance the activity of antimicrobials against biofilms. Consistent with these studies, it was observed that D-AAs had significant dispersive activity on 24 h biofilms of P. aeruginosa at different concentrations (Fig. 1). Importantly, and in contrast to previous studies with B. subtilis, significant effects on either cell viability or bacterial growth were not observed at the effective concentrations (Fig. 4). Therefore, the effects of D-AAs seem to be specific for biofilm dispersal. The inhibition of biofilm formation by D-AAs is largely, if not entirely, mediated by misincorporation into protein, presumably resulting in proteotoxicity. It has been proposed that D-AAs affect the union of the TasA protein to the cell wall in B. subtilis NCBI3610 biofilms [29]. The protein TasA is polymerized to form amyloid-like fibers, which are considered to be the fundamental structural units in Bacillus biofilms that bind to the bacterial cell wall. Recently, scientists found that TapA, linking the TasA fibers to the cell wall, was separated from the cell wall in a similar fashion as the amyloid TasA fibers when treated with D-AAs [30]. Conversely, D-AAs inhibited the expressions of biofilm matrix genes epsA and tapA at concentrations that inhibited biofilm formation [29], but Kolodkin-Gal I found that D-AAs did not inhibit the expressions of the matrix operons epsA-O and yqxM-sipW-tasA [14]. We will rectify these contradictory results in our future study. AMK, a semisynthetic analog of kanamycin, is very active against most gram-negative bacteria, including gentamicin- and tobramycin-resistant strains. The effectiveness of AMK in the treatment of serious gram-negative bacillary infections is welldocumented [31]. As anticipated, the antimicrobial agent tested against biofilms of P. aeruginosa was ineffective at the tested concentration against the planktonic phenotype. However, the combination of D-Tyr (5 mM) significantly enhanced the antimicrobial activity of AMK and reduced the MBIC50 and MBIC90 (Table 1). AMK (10 mg/ml) combined with D-Tyr (5 mM) demonstrated the enhancement of bactericidal activity with 1.25 Log10 CFU/ml, 0.75
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Fig. 5. The effects of the combination of D-Tyrosine (5 mM) and AMK (8 mg/ml) against the biofilms of PAO1. (A) The kinetic dispersing effects of AMK alone or combined with DTyrosine on PAO1 biofilms are shown. Biofilm dispersal was assessed by measuring the absorbance of solubilized crystal violet at 490 nm. (B) The kinetic bactericidal effects of DTyrosine and AMK against biofilms of PAO1 are shown. The biofilm of PAO1 was developed in a 96-well plate for 24 h, followed by exposure to AMK in the absence or presence of DTyrosine for 8, 16, and 24 h, respectively. Viable bacteria formed biofilms were determined by plating serial dilutions, followed by the removal of adherent bacteria by swabbing from wells. (C) Representative CLSM images about the effects of D-Tyrosine and AMK on PAO1 biofilm morphology are shown. The biofilms were developed on glass covers for 24 h, followed by exposure to AMK in the absence or presence of D-Tyrosine for 8, 16, and 24 h, respectively. The cells were fixed and stained for fluorescence microscopy imaging.
Log10 CFU/ml, and 1.68 Log10 CFU/ml decreases compared to AMK (10 mg/ml) alone at 8, 16, and 24 h, respectively. The timeekill curve and timeedisperse curve were consistent with the results (Fig. 5AeB). Confocal microscopy gave a more detailed picture of biofilm dispersal during exposure to AMK (10 mg) alone or combined with D-Tyr (5 mM) for 8, 16, and 24 h, respectively [LIVE/ DEAD BacLight Bacterial Viability Kit (L7012), Invitrogen, USA] (Fig. 5C). Since the antimicrobial concentrations required for biofilm eradication may be reduced by co-delivery with D-Tyr, the toxicity of the liver and kidney due to AMK can potentially be mitigated. In summary, this work demonstrated the ability of D-Tyr to disperse biofilms in P. aeruginosa. The data suggest that combining AMK with D-Tyr may increase the success of a treatment by increasing the efficacy of AMK against biofilm-related infections. Because D-AAs are not bactericidal, they are anticipated to be most
effective as an adjuvant therapy to conventional treatment with systemic antibiotics. Most studies of biofilms have taken a reductionist approach, where single-species biofilms have been extensively investigated. However, biofilms in human beings can sometimes comprise multiple species. For example, about 100e200 bacterial species were found to colonize and form biofilms on hard and soft tissues of the oral cavity, where the interspecies interactions play important roles in determining the development, structure, and function of these biofilms [32]. Thus, we will continue studying the effects of D-amino acids combined with antibiotics on multi-species biofilms. References [1] R.D. Monds, G.A. O'Toole, The developmental model of microbial biofilms: ten years of a paradigm up for review, Trends Microbiol. 17 (2009) 73e87.
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