A preliminary study of in vitro and in vivo synergistic effects of ciprofloxacin and D-tyrosine against Pseudomonas aeruginosa isolates

Purpose: To investigate the synergistic antimicrobial effects of ciprofloxacin and D-tyrosine against drug-resistant bacteria. Method: The antimicrobial effects of ciprofloxacin and D-tyrosine on clinical isolates of multidrugresistant (MDR) Pseudomonas aeruginosa (P. aeruginosa) no. 3556 were determined in vitro based on time-kill curve, and in vivo in P. aeruginosa-zebrafish infection model. Furthermore, 30 clinical isolates of multidrug-resistant P. aeruginosa were used in vitro to ascertain the synergistic effect of the two agents. Results: Combined use of ciprofloxacin and D-tyrosine produced synergistic effects against the clinical isolate of P. aeruginosa no. 3556 in vitro and in vivo. Synergism occurred in 96.67 % (95 % CI, range 83.33 99.41 %) of the clinical isolates, and ciprofloxacin dose was reduced in 90 % (95 % CI, range 74.38 96.54 %) of the clinical isolates in vitro. Conclusion: These preliminary results suggest that the combination of ciprofloxacin and D-tyrosine is a promising therapeutic strategy against MDR P. aeruginosa infections.


INTRODUCTION
Pseudomonas aeruginosa has become an increasingly notorious opportunistic pathogen in common bacterial infections, including community-acquired, healthcare-associated, and nosocomial infections [1]. The emergence of multidrug-resistant P. aeruginosa, including those resistant to broad-spectrum cephalosporins and carbapenems, has increased dramatically over the last two decades, and it is now a global health problem [1]. It is estimated that P. aeruginosa accounts for 5 to 10 % of infections in developing countries, 25 % of which are caused by multidrug-resistant P. aeruginosa [2]. Studies have shown that the mortality due to in-hospital infection caused by multidrugresistant P. aeruginosa may be up to 31.1 % [2].
A major problem associated with antibiotic resistance is that pre-existing antibiotics are rapidly becoming ineffective against new and emerging antibiotic-resistant bacteria [3]. For these reasons, there is need to evolve novel and improved treatments against drug-resistant P. aeruginosa infections.
One promising solution is based on the use of combination of two or more conventional antimicrobial agents. This produces increased antimicrobial effects when compared with a single agent. Synergistic effect could offer a potential benefit for treatment of bacterial infections through drug combinations [4]. Many researchers have demonstrated that drug combinations produce synergistic effects.
Examples are the use of vancomycin and βlactams against Staphylococcus aureus highly resistant to vancomycin, and the use of ciprofloxacin-tobramycin combination against P. aeruginosa [5,6]. Moreover, the combination of a chemical compound and a conventional antimicrobial agent results in increased antibacterial effects, for example, silver nanoparticles in combination with antibiotics against gram-positive and gram-negative bacterial pathogens [7,8]. D-Amino acids have been found to affect biofilm formation. Studies have demonstrated that combination of D-amino acids and antimicrobial agents produces synergistic antimicrobial effects against pathogens [9][10][11][12]. However, studies on the effect of D-tyrosine as an antimicrobial enhancer against bacteria have resulted in controversial outcomes [12][13][14].
This study was aimed at determination of the synergistic antimicrobial effects of ciprofloxacin -D-tyrosine combination on clinically isolated P. aeruginosa No. 3556 in vitro based on time-kill curve, and in vivo in P. aeruginosa-zebrafish infection model.

EXPERIMENTAL Bacterial strains, culture and preparation
Thirty-one clinical isolates of P. aeruginosa strains used in this study were kept in our laboratory. Multidrug-resistant P. aeruginosa strain No. 3556 selected from the above strains was used as reference strain in vitro based on time-kill curve, and in vivo in P. aeruginosazebrafish infection model. The strain was inoculated in Luria-Bertani (LB) agar plates, followed by overnight incubation at 37 °C. Then, a single colony on the plate was inoculated into 5-mL fresh LB broth and incubated, with shaking at 180 rpm, for 12 h at 37 °C. Bacterial suspension (500 µL) was inoculated into 5 mL fresh LB broth, followed by incubation at 37 °C, with shaking at 180 rpm, for 6 h. The bacterial cultures were subjected to centrifugation at 6,000 × g for 5 min. The harvested cell pellets were washed twice with phosphate buffered saline (PBS, pH 7.2) and serially diluted for use in this study.

Antibiotic and D-tyrosine preparation
Ciprofloxacin was purchased from Sigma-Aldrich (St. Louis, MO, United States). It was dissolved in sterile water to a concentration of 256 μg/mL. The drug susceptibility of P. aeruginosa strain No. 3556 was chosen according to the Clinical and Laboratory Standards Institute (Clinical and Laboratory Standards Institute (CLSI), 2019) [15]. D-Tyrosine was purchased from Sigma-Aldrich (St. Louis, MO, United States), and was dissolved in PBS, followed by filtration through 0.22 µm membrane filters before use.

Optimization of D-tyrosine concentration
Pseudomonas aeruginosa No. 3556 was prepared in the same manner as described above. The concentration of D-tyrosine was optimized by co-culturing of 100 µL of bacterial suspension and 100 µL of serially diluted D-tyrosine (0 to 16 µM) in a 96-well plate. Eight hours later, the OD600 was read in a microplate reader. The experiment was done in 3 replicates. Bacteria culture without D-tyrosine was set as control.

In vitro time-kill curves for single and combination drugs
The effectiveness of ciprofloxacin and D-tyrosine, alone and in combination, against clinical isolate of multidrug-resistant P. aeruginosa No. 3556 was determined using the time-kill curve method. Time-kill assays were carried out according to the CLSI guidelines. In these assays, 5 × 10 5 CFU/mL inoculum was incubated in 96-well plate containing adjusted Mueller Hinton broth supplemented with ciprofloxacin and D-tyrosine, alone or in combination, in separate wells. In the entire experiment, the concentration of ciprofloxacin varied from 16 to 0.5 μg/mL, while the concentration of D-tyrosine was kept at 8 µM, based on the optimization result. Aliquots were removed from each tube and diluted serially (1:10) with PBS for use in determination of cell viability after 8 h of culture in Luria-Bertani (LB) agar plates. Time-kill experiment was performed in triplicate. According to a previous description [16], a combination of two antimicrobial agents was considered synergistic if it caused a ≥ 2 log unit reduction in CFU/mL, relative to the sum of the reductions observed with the individual compounds at the end of the experiment.

Evaluation
of the effectiveness of ciprofloxacin and D-tyrosine, in combination and singly, against clinical isolate of P. aeruginosa The degrees of effectiveness of ciprofloxacin and D-tyrosine, alone and in combination, against 30 clinical isolates of P. aeruginosa in vitro were determined based on the procedure of time-kill curve for multiple-drug-resistant P. aeruginosa No. 3556. The concentration of ciprofloxacin varied from 32 μg/mL to 0.06 μg/mL.

In vivo assessment of the effectiveness of ciprofloxacin and D-tyrosine in combination and singly, on P. aeruginosa-zebrafish infection model
Each zebrafish was infected with 10 μL of a 5 × 10 5 CFU/ml bacterial suspension in the treatment group (n= 10 per group). The treatment groups were exposed to ciprofloxacin (8 μg/mL) and Dtyrosine (8 µM), singly and in combination. Zebrafish without bacteria served as the control group. The percentage survival in each experimental group was monitored from 0 h to 144 h post-infection.

Statistical analysis
Results from in vitro and in vivo experiments are presented as mean ± standard deviation (SD). GraphPad InStat version 6.01 (GraphPad Software, CA, USA) was used for statistical analysis of the differences amongst group means, based on one-way analysis of variance (ANOVA). Differences were assumed statistically significant at p < 0.05.

Optimized D-tyrosine concentration
The results showed that D-tyrosine interfered with the formation of biofilm, as shown in Figure  1 A. The formation of P. aeruginosa No. 3556 biofilm decreased with increase in the concentration of D-tyrosine from 0 to 8 µM, and subsequent microplate reader results showed that the bacterial concentrations were significantly decreased (p = 0.01 < 0.05). When D-tyrosine concentration was increased to 16 µM, there was no change in the formation of P. aeruginosa biofilm. Subsequent microplate reader results showed that there was no significant change in the bacterial concentration

Single and combined effects of ciprofloxacin and D-tyrosine in vitro based on time-kill curve test
The antimicrobial effects of ciprofloxacin and Dtyrosine, singly and in combination, were determined based on the growth properties of P. aeruginosa No. 3556. As shown in Figure 2, the bacteria growth when treated with ciprofloxacin or D-tyrosine alone was set as control group, and the results indicated that P. aeruginosa No. 3556 was resistant to both agents. In contrast, the combination of ciprofloxacin and D-tyrosine reduced the bacteria from 10 log CFU/mL to 6 log CFU/mL when the concentration of ciprofloxacin used ranged from 8 to 16 µM. The reduction margin in bacteria (4 log CFU/mL) indicated that the ciprofloxacin -D-tyrosine combination resulted in synergistic/additive effect in vitro. Obviously, the dose of ciprofloxacin could be decreased during combination of ciprofloxacin and D-tyrosine, indicating that the combination was better at killing drug-resistant bacteria than any of the agents when used alone.

Effectiveness of ciprofloxacin and D-tyrosine, alone and in combination, on clinical isolates of P. aeruginosa in vitro
As shown in Table 1

Effect of ciprofloxacin and D-tyrosine on P. aeruginosa-zebra fish infection model
As shown in Figure 3, the survival of untreated zebrafish was 100 % (from 0 to 144 h). This served as control. In the group injected with P.

DISCUSSION
Bacterial resistance has always been a serious problem. Combined treatment with multiple drugs or chemical compounds may be a solution to the problem of drug resistance. In this preliminarily study, the effects of ciprofloxacin and D-tyrosine, singly and in combination, on multidrug-resistant P. aeruginosa were investigated in vitro and in vivo. D-Tyrosine is a bioactive substance. However, the report that Damino acids may inhibit biofilms has remained controversial [13,14,17]. The results of the present study showed that D-tyrosine did not completely prevent the formation of biofilm, but inhibited the growth of bacteria by influencing the formation of biofilm to some extent. This might explain the synergistic effect of ciprofloxacin and D-tyrosine against the clinical isolates of P. aeruginosa in vitro and in vivo.
Time-kill curve results showed that combination of ciprofloxacin and D-tyrosine produced synergistic effect against P. aeruginosa in vitro. As explained above, it might be that D-tyrosine reduced the bacteria biofilm formation and accelerated the entry of ciprofloxacin into the cell, thereby increasing the efficacy of the drug. Furthermore, the use of D-tyrosine reduced the dose of ciprofloxacin in the time-kill curve. The decrease in drug dose during treatment could reduce the risk of drug-resistance. The combination of ciprofloxacin and D-tyrosine improved the survival time of zebrafish in P.
aeruginosa-zebrafish infection model experiment. This result indicates that the synergistic effect seen in vitro was also produced in vivo.

Limitations of the study
This study has some limitations. Although it has demonstrated that combination of ciprofloxacin and D-tyrosine produced positive synergistic effect in vitro and in vivo in time-kill curve and P. aeruginosa-zebrafish infection model, respectively, single MDR P. aeruginosa is not sufficient as an index strain for a viable study. More MDR bacterial strains should be investigated in future in order to determine the precise mechanism involved in the observed synergism.

CONCLUSION
The findings of this study suggest that the ciprofloxacin/D-tyrosine combination produces synergistic effect in vitro and in vivo. This phenomenon offers a new strategy for tackling the current problem of drug-resistant bacteria.

Conflict of interest
No conflict of interest is associated with this work.