In Vitro Antibacterial and Antibiofilm Activities of Chlorogenic Acid against Clinical Isolates of Stenotrophomonas maltophilia including the Trimethoprim/Sulfamethoxazole Resistant Strain

The in vitro antibacterial and antibiofilm activity of chlorogenic acid against clinical isolates of Stenotrophomonas maltophilia was investigated through disk diffusion, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), time-kill and biofilm assays. A total of 9 clinical S. maltophilia isolates including one isolate resistant to trimethoprim/sulfamethoxazole (TMP/SMX) were tested. The inhibition zone sizes for the isolates ranged from 17 to 29 mm, while the MIC and MBC values ranged from 8 to 16 μg mL−1 and 16 to 32 μg mL−1. Chlorogenic acid appeared to be strongly bactericidal at 4x MIC, with a 2-log reduction in viable bacteria at 10 h. In vitro antibiofilm testing showed a 4-fold reduction in biofilm viability at 4x MIC compared to 1x MIC values (0.085 < 0.397 A 490 nm) of chlorogenic acid. The data from this study support the notion that the chlorogenic acid has promising in vitro antibacterial and antibiofilm activities against S. maltophilia.


Introduction
Stenotrophomonas maltophilia is an emerging nosocomial pathogen. Although it is an organism of a limited pathogenicity, S. maltophilia infections has been associated with high morbidity and mortality especially among immunocompromised individuals. S. maltophilia, which is a high producer of bio�lm, frequently colonizes patient secretions (respiratory, urine, and wound exudates), �uids (intravenous �uids), catheters (central venous), and implants used in the hospital setting. It is associated with a wide range of infections including bacteraemia, endocarditis, meningitis, respiratory tract, and skin and so tissue infections [1]. Community acquired S. maltophilia infections have also been reported especially among patients with cystic �brosis [2,3]. S. maltophilia is intrinsically resistant to several classes of antibiotics. Nosocomial S. maltophilia strains are multiple drug resistant (MDR) and are usually less susceptible to carbapenems, cephalosporins, penicillins, chloramphenicol, and �uoroquinolones which severely complicates the treatment options. Currently, the combination drug, trimethoprim/sulfamethoxazole (TMP/SMX), remains as the main therapy of choice worldwide [1]. But, growing resistance to TMP/SMX has become a serious threat in the treatment of S. maltophilia infections. In addition, problems associated with contraindication of TMP/SMX therapy in patients due to toxicity and intolerance, spread of acquired mobile resistance determinants, and reduced susceptibilities to available antibiotics [4,5] prompt for an expanded search of new antibacterial agents.
Chlorogenic acid is a polyphenolic natural compound which is commonly present in plant materials such as apples, coffee beans, grapes, pulp, peel, and tea leaves [6]. Structurally, it is an ester of caffeic acid with the 3-hydroxyl group of a quinic acid. It has been reported to possess many health bene�ts including antibacterial, antifungal, antiviral, antiphlogistic, antioxidant, chemopreventive, and other biological activities [7][8][9][10][11][12]. Extensive in vitro antimicrobial activities and possible action mode have been reported on a variety of Gram-positive and Gram-negative bacterial pathogens [13].
Classical antibiotics target a speci�c reaction, whereas natural antimicrobial compounds like plant polyphenols inhibit several different groups of biomolecules in a pathogen. erefore, the development of resistance to such compounds is unlikely, which makes them as an attractive antibacterial candidate [14,15]. In addition, synthetic antibiotics are more focused on planktonic cells than bio�lms, while natural antibiotic compounds are proven to inhibit bacterial adhesion and bio�lm development [16]. Since there is a great demand for natural substances that inhibit bacterial planktonic cells as well as bio�lms, we investigated the in vitro antibacterial and antibio�lm e�cacy of chlorogenic acid against a collection of clinical S. maltophilia isolates.

Bacterial Isolates and Culture Conditions.
Nine clinical isolates of S. maltophilia including one TMP/SMX resistant isolate (MIC > 32 g mL −1 ) as per CLSI guidelines [17] were obtained from the Microbiology Unit at Hospital Kuala Lumpur (HKL), Malaysia. e source of the isolates included cerebrospinal �uid (CSF, 1), tracheal aspirate ( , bronchoalveolar lavage ( 1 , pus ( , and urine ( 1 . Bacterial samples were cultured onto nutrient agar slants and transported to the Medical Microbiology Laboratory at the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia (UPM). e isolates were presumptively identi�ed as S. maltophilia by Gram stain, catalase, oxidase, alpha hemolysis on blood agar, fermentation on MacConkey agar, DNase production, and mannitol fermentation on mVIA agar medium. e isolates were further genotypically con-�rmed as S. maltophilia by single stranded PCR (SS-PCR) [18,19]. Con�rmed isolates were stored in Luria-Bertani (LB) broth (Merck Pvt. Ltd., Selangor, Malaysia) supplemented with 20% glycerol at −80 ∘ C. Multiple copies of working cultures stored at −20 ∘ C were thawed in ice and used for further experiments. No culture stocks (−20 ∘ C) were used more than two times as repeated passaging may lose some of the bacterial properties.

Disk Diffusion
Assay. e antibacterial activity of chlorogenic acid against S. maltophilia was assessed by the disk diffusion method [20]. Isolates were grown in LB broth to a turbidity of 0.5 McFarland standards and, using sterile cotton swabs, the bacterial inoculum was spread onto MHA plates by lawn culture. Sterile antibiotic assay �lter paper discs (Whatman, GE Healthcare, Malaysia) of 6 mm diameter were placed on MHA plates and 20 L of chlorogenic acid from each sets ranging from 0.25 to 32 mg mL −1 (0.25, 0.5, 1, 2, 4, 8, 16, and 32) was carefully loaded onto the �lter paper discs. TMP/SMX antibiotic disc (Oxoid Limited, Hampshire, UK) was included as an antibiotic control, while paper disc impregnated with 20 L of DMSO (10%) was used as a negative control. S. maltophilia ATCC 13637 was included as a quality control. All plates were incubated at 37 ∘ C for 24 h until visible growth was observed and the inhibition zones formed around the �lter papers discs were measured and compared with the control antibiotic. e experiment was performed in triplicates.

Susceptibility
Testing. e minimum inhibitory concentration (MIC) of chlorogenic acid for S. maltophilia was determined by broth dilution method as described previously by Schwalbe et al. [21]. For MIC testing, chlorogenic acid at a concentration of 1024 g mL −1 was prepared from the previously prepared stock solution. Brie�y, 11 sterile screw capped test tubes (10 mm × 100 mm) 1 through 11 were labeled and arranged in a rack. Tubes 2-11 were loaded with 500 L of sterile MHB and 500 L of chlorogenic acid from the freshly prepared stock solution (1024 g mL −1 ) was pipetted into tubes 1 and 2. Twofold serial dilution was performed by transferring 500 L of the mixture from tube 2 to tube 3 and successive transference was continued through tube 9. To avoid carryover of antibiotic, pipette tips were changed between each tube. From tube 9, 500 L of the solution was discarded. Tube 10 was used as a growth control and received no antibiotic. Except the 11th tube (broth control), all tubes were inoculated with 500 L of bacterial inoculum (10 6 CFU mL −1 ) and incubated at 37 ∘ C for 24 h. Aer incubation, the lowest concentration of the antibiotic at which no visible growth occurred was considered as MIC. Minimum bactericidal concentration (MBC) was determined by spread plating 100 L of the broth from clear wells onto MHA plates followed by incubation at 37 ∘ C for 24 h. e lowest concentration of an antimicrobial agent at which all the cells were killed is de�ned as MBC. e experiment was performed in triplicates.

Time-Kill Assay for Detecting the Bactericidal Effect of
Chlorogenic Acid. e killing kinetics of chlorogenic acid at 0x, 1/2x, 1x, 2x, and 4x MIC values was determined by the method described previously [22] with minor modi�cations. Different concentrations of chlorogenic acid were added to each tube containing 4.5 mL of MHB. e tubes were inoculated with 5 × 10 5 colony forming unit (CFU mL −1 ) of S. maltophilia ATCC 13637 strain grown in BHI broth (Merck Pvt. Ltd., Selangor, Malaysia) and incubated at 37 ∘ C in an orbital shaker, set at 130 rpm. Tube without chlorogenic acid served as growth control (0x). An aliquot of 100 L was collected at 0 min, 2.5 h, 5 h, 10 h, and 24 h postinoculation. Tenfold serial dilution of broth culture was plated onto MHA 3 plates and the CFU mL −1 of each sample was determined by a spread plate technique incubated upon further incubation for 24 h. e experiment was performed in duplicates.

Effect of Chlorogenic Acid on S. maltophilia Performed
Bio�lms. In order to test the efficacy of chlorogenic acid on bio�lm, S. maltophilia ATCC 13637 was subjected to bio�lm formation by the method described by Peeters et al. [23] with the following modi�cations. e test strain was grown in BHI broth and when the cells reached to a turbidity of 0.5 McFarland standard, cell suspensions were added to 48 wells of a �at-bottomed polystyrene 96-well polystyrene microtiter plate (Orange Scienti�c, Belgium). To ensure proper adhesion of bacterial cells to the polystyrene surface, the plate was incubated at 37 ∘ C for 24 h at static conditions. Following 24 h of adhesion and bio�lm formation, planktonic cells were removed from the wells and the plate was rinsed with 100 L of phosphate buffered saline (PBS). Cells adhered to the polystyrene microtitre plate were treated with 100 L of chlorogenic acid at 1x, 2x, and 4x MICs. For bio�lm experiment, 24 wells of a microtiter plate inoculated with 100 L of these dilutions were considered as "treated" and 24 control wells without the test compound were considered as "nontreated. " Nontreated cells were incubated with 100 L of DMSO (10%) which served as a negative control. e plate was further incubated for 12 h at 37 ∘ C with gentle shaking and the quanti�cation of bio�lm was carried out by XTT calorimetric assay as mentioned below. e experiment was repeated two times in order to achieve reproducibility.

2.�. Bio�lm �uanti�cation �y ��� Assay.
A modi�ed XTT assay [23] was carried out to quantify the S. maltophilia performed bio�lms on polystyrene microtitre plates. Brie�y, 0.4 mg of XTT in 1 mL PBS was mixed with 10 L of menadione in 10 mL acetone (Merck Pvt. Ltd., Selangor, Malaysia). e contents were mixed thoroughly and 100 L of the XTT/menadione solution was added to all wells followed by incubation in the dark at 37 ∘ C for 5 h. e contents of the wells were microfuged at 15,000 g for 4 min and 100 L of clear supernatant from each well was transferred to a fresh 96-well �at-bottomed microtiter plate before the absorbance was read at 490 nm using (BioTek EL808, USA) a microplate reader.
Statistics. Statistical analysis was performed using SPSS version 16.0 soware 2007 (SPSS Inc., Chicago, IL). All assays were performed at least in duplicates. For bio�lm inactivation studies, mean values between treated and untreated samples were tested for signi�cance by Student�s t-test. e signi�cant difference in bio�lm reduction at different concentrations of chlorogenic acid was compared with the control (strain without the test compound was normalized as 100%). e signi�cant level was set at .

Characterization of Isolates.
All the isolates were con�rmed as S. maltophilia by phenotypic and genotypic methods. Eight isolates were susceptible to TMP/SMX by disk diffusion, whilst one isolate (isolate number 4) from CSF was found to be TMP/SMX resistant (MIC > 32 g mL −1 ) and interpreted according to CLSI guidelines.

Disk Diffusion
Assay. e results obtained from disk diffusion test are illustrated in Table 1. e zone of inhibition ranged between 17-29 mm at 32 mg mL −1 of chlorogenic acid and a consistent increase in zone sizes were observed at increasing concentrations of the chlorogenic acid. e maximum activity was observed at 32 mg mL −1 with a zone size of 29 mm. e compound was highly active against 3 isolates ( , 30, and 49) and a representative antibacterial pattern of chlorogenic acid against S. maltophilia ATCC 13637 is shown in Figure 1. Chlorogenic acid was found to be antibacterial against all the clinical isolates including the TMP/SMX resistant strain. Table 1. e MICs were in the range of 8 to 16 g mL −1 in which 7 isolates were inhibited at 8 g mL −1 and 2 isolates were inhibited at 16 g mL −1 . Isolate number 4 being TMP/SMX resistant was also susceptible to chlorogenic acid at 16 g mL −1 . Meanwhile, the MBCs of chlorogenic acid for S. maltophilia ranged between 16-32 g mL −1 .

Analysis of Bacterial Killing Kinetics.
Time-kill assay indicated that S. maltophilia ATCC 13637 cells grown in MHB maintained their viability for at least 24 h of incubation, whereas S. maltophilia treated with chlorogenic acid at increasing concentrations (8,16, and 32 g mL −1 ) killed more than 90% of the cells and no viable cells could be enumerated aer 12 h of incubation. Twofold and four fold MICs of chlorogenic acid inhibited the growth in 10 h demonstrating a dose-dependent killing property of chlorogenic acid ( Figure  2). e bactericidal endpoint for S. maltophilia at 4x and 1x MIC was achieved at 10 h and 12 h time points, respectively, with an approximate reduction of CFU by 2 log units (99.9%).

Discussion
ere exists a global threat for the existing antimicrobial agents due to the widespread emergence of microbial drug resistance. e discovery and development of new antibiotics to �ght the �superbugs� still remain a great challenge for researchers. Since the emergence of bacterial drug resistance remains at full pace, novel antimicrobial agents to counter antibiotic-resistant organisms have great demands in the market [24]. Opportunistic pathogens like S. maltophilia are frequently encountered in hospitalized patients implanted with indwelling medical devices like catheters, prosthetic heart valves, contact lenses, and so forth [25][26][27]. Both clinical and environmental isolates of S. maltophilia are capable of adhering to biotic and abiotic surfaces such as glass, plastic materials, polyvinyl chloride, and Te�on [28,29]. is study is one of the �rst reports demonstrating the antibacterial and antibio�lm activities against S. maltophilia. Our results demonstrate that chlorogenic acid is very active against S. maltophilia, including the TMP/SMX resistant strain (MIC > 32 g mL −1 ). Previously, chlorogenic acid and chlorogenic acid containing plant materials have been shown to have antiviral [30], antifungal [11,31], and strong antibacterial activities. Several Gram-positive (Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus mutans and Bacillus subtilis) [13,16,19,31] and Gram-negative (Salmonella typhimurium, Shigella dysenteriae, and Escherichia coli) [13] bacterial pathogens have been reported to be highly susceptible to chlorogenic acid. At a concentration of 32 g mL −1 , chlorogenic acid was able to produce larger zones on MHA plates ranging from 17 to 29 mm. e zone sizes are comparable with the control antibiotic TMP/SMX (30 g). Compared to the TMP/SMX susceptible isolates, chlorogenic acid was highly active against the TMP/SMX resistant strain isolated from CSF. e compound has greater solubility in DMSO which makes it more permeable as well as promoting its bactericidal property. Pure chlorogenic acid is effective in killing S. maltophilia clinical isolates with MICs ranging between 8-16 g mL −1 . e antibiotic resistant strain was inhibited at 16 g mL −1 . e compound also extended its antibacterial activity against other pathogens like Acinetobacter iwoffii, methicillin resistant S. aureus, and Pseudomonas aeruginosa determined by disk diffusion test in our earlier investigation of chlorogenic acid against an array of bacterial pathogens (data not shown). A recent study by Lou et al. [13] has shown that chlorogenic acid has broad spectrum antibacterial activity with MIC ranging from 20 to 40 g mL −1 against Gram-positive and 20 to 80 g mL −1 against Gram-negative organisms. However, this is the �rst report on a natural compound against S. maltophilia with low MIC values which is slightly less than other gram-negative organisms reported. Although the precise mechanism of action of chlorogenic acid is not completely understood, a recent study by Li et  such as FabI and FabG [22]. is supports the notion that chlorogenic acid being a polyphenolic compound has strong inhibitory effects against S. maltophilia. Meanwhile, chlorogenic acid also signi�cantly increased the permeability of outer membrane and plasma membrane in S. dysenteriae and S. pneumoniae which resulted in the leakage of cytoplasmic contents including nucleotides [13].
Previous in vitro antimicrobial studies using chlorogenic acid [13,16,19,31] have reported the antimicrobial potential of the compound, but none has investigated the bactericidal activity of chlorogenic acid against emerging pathogens like S. maltophilia. Knowledge on killing kinetics of chlorogenic acid will be highly useful in the mechanism of action, transcription analysis, and gene expression studies. In S. maltophilia, killing was observed at a lower concentration of chlorogenic acid due to its lower MIC (8-16 g mL −1 ). A concentration dependent killing was observed in case of S. maltophilia, where a 2x and 4x MICs exhibited strong bactericidal activities in 10 h. At subinhibitory concentration (0.5x MIC), a slight decrease in the colony count was observed initially. However, bactericidal activity was not detected and there was an evidence of regrowth aer 5, 10, and 24 h of incubation. A rapid decrease in colony count was observed between 5-10 h of incubation and complete killing was achieved aer 10 h at 2x and 4x MICs. At 1x MIC, the killing time of chlorogenic acid was at 12 h. ese �ndings were partly in agreement with the observations made earlier by Lou et al. [13] who reported the bactericidal activity at 12 h when tested with ethyl acetate fraction of burdock leaves containing chlorogenic acid on food related Gram-positive and Gram-negative pathogens.
Very few studies have been reported on the effect of chlorogenic acid on bio�lm formation. Against S. maltophilia bio�lms, chlorogenic acid showed bactericidal activity. A recent study by Stauder et al. [16] showed that coffee high molecular weight (cHMW) melanoidin compounds containing chlorogenic acid as one of the active components were able to abolish S. mutans (major causative agent of human dental caries) bio�lms in vitro. In this study, we observed a signi�cant 4-fold reduction in bio�lm viability at 4x MIC concentration (32 g mL −1 ) of chlorogenic acid. S. maltophilia is capable of attaching to polystyrene wells in less than 2 h of incubation; however, maximum bio�lm growth endpoint on the surface of polystyrene wells occurs at 24 h [32]. Although, bio�lm experiment remains time consuming because of the slow growing property of S. maltophilia, the microtiter well plate method and XTT assay were found to be suitable, reproducible, and quantitative methods. To the best of our knowledge, no data exist concerning natural product activity against S. maltophilia bio�lms. In the present study, for the �rst time, we tested the effects of different concentrations of chlorogenic acid on S. maltophilia performed bio�lms. �ur results showed that chlorogenic acid at 1x, 2x, and 4x MICs signi�cantly reduced the adhesion of S. maltophilia to polystyrene in a dose-dependent manner. ese �ndings could be of interest because S. maltophilia is resistant to commonly available broad spectrum antibiotics, including -lactams, aminoglycosides, and quinolones [3].

Conclusions
In conclusion, the �ndings of this study highlight the antibacterial and antibio�lm properties of chlorogenic acid against emerging nosocomial pathogen S. maltophilia. Complete eradication of microbial bio�lms still remains a crucial step and a great challenge for clinicians and researchers. Superbugs like S. maltophilia are strong bio�lm producers with intrinsic resistance to many antibiotics. Natural antimicrobial agents like chlorogenic acid which is ecologically safe and less hazardous than synthetic bactericides displayed promising antistenotrophomonas activity on planktonic as well as bio�lm forms of S. maltophilia. erefore, chlorogenic acid could be used as a safe alternative to synthetic antimicrobial drugs or in antimicrobial combinations against S. maltophilia infections.
�on��ct of �nterests e authors have no con�ict of interests.