Research Article | Open Access
Antibiofilm Activity, Compound Characterization, and Acute Toxicity of Extract from a Novel Bacterial Species of Paenibacillus
The effectiveness of many antimicrobial agents is currently decreasing; therefore, it is important to search for alternative therapeutics. Our study was carried out to assess the in vitro antibiofilm activity using microtiter plate assay, to characterize the bioactive compounds using Ultra Performance Liquid Chromatography-Diode Array Detection and Liquid Chromatography-Mass Spectrometry and to test the oral acute toxicity on Sprague Dawley rats of extract derived from a novel bacterial species of Paenibacillus strain 139SI. Our results indicate that the crude extract and its three identified compounds exhibit strong antibiofilm activity against a broad range of clinically important pathogens. Three potential compounds were identified including an amino acid antibiotic C8H20N3O4P (MW 253.237), phospholipase A2 inhibitor C21H36O5 (MW 368.512), and an antibacterial agent C14H11N3O2 (MW 253.260). The acute toxicity test indicates that the mortality rate among all rats was low and that the biochemical parameters, hematological profile, and histopathology examination of liver and kidneys showed no significant differences between experimental groups . Overall, our findings suggest that the extract and its purified compounds derived from novel Paenibacillus sp. are nontoxic exhibiting strong antibiofilm activity against Gram-positive and Gram-negative pathogens that can be useful towards new therapeutic management of biofilm-associated infections.
The effectiveness of many antimicrobial agents is currently decreasing due to the prevalence of multidrug-resistant pathogens . The emerging of these pathogens remains a serious challenge to medicine and healthcare . One of the mechanisms for such resistance is the formation of biofilms which are layers of microbial cells attached to a surface and embedded in a matrix of exopolysaccharide . Therefore, it is important to search for alternative therapeutics to control biofilm-associated infections. Although several plant-based compounds are receiving attention for their therapeutic properties , only few are reported to exhibit antibiofilm activity . Natural ecosystems are rich sources of microbes that produce a wide range of compounds that exhibit diverse and versatile biological effects [6, 7]. Many marine and soil microorganisms were recently documented for their effective antibiofilm property against pathogens [8–10]. The genus Paenibacillus represents one of the important soil bacteria that comprise strains of medical, industrial, and agricultural importance . Interest in Paenibacillus species as a source of new antimicrobials has been increasing and the probability of finding novel antibiofilm compounds from these bacterial strains is promising . It is worth mentioning that the administration of antimicrobial agents and biocide compounds in the local sites of some infection has been a useful approach to combat microbial biofilms . However, prolonged persistence of these compounds in the environment could induce toxicity towards nontarget organisms and resistance among microorganisms within biofilms . Moreover, some of these compounds may exhibit toxic effects even at therapeutic doses which makes it necessary to test their toxicity in experimental animals . This aspect has led to the development of more environment friendly compounds to combat with the issue. Acute toxicity is the toxicity produced by a compound when it is administered in one or more doses during a period of 24 hours . These studies are usually necessary for any compound intended for human use and the information obtained from them is useful in identifying the organs of toxicity and choosing the safe doses . The objective of acute studies can usually be achieved in rodents using small groups of experimental animals . Therefore, our study was carried out to assess the in vitro antibiofilm activity, characterize the bioactive compounds, and test the acute toxicity on Sprague Dawley rats of an extract derived from novel bacterial species of Paenibacillus strain 139SI.
2. Materials and Methods
2.1. Bacterial Isolates
The clinical bacterial isolates were collected from patients undergoing tonsillectomy for chronic and recurrent tonsillitis at University Malaya Medical Centre (UMMC) upon approval by the medical ethics committee (PPUM/UPP/300/02/02 reference number 744.11). Reference bacterial strains used were Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853), and Escherichia coli (ATCC 25922) . A bacterial strain 139SI originally isolated from a local agricultural soil was identified as Paenibacillus via 16S rRNA gene sequencing and deposited at the American Type Culture Collection (ATCC) with a cataloguing number (ATCC–BAA-2268) .
2.2. Experimental Animals
A total of 36 adult male and female Sprague Dawley (SD) rats were obtained from the Animal Care Unit Center (ACUC) at the Faculty of Medicine, University of Malaya. Animals were weighing 150–200 gm and were kept in wire-bottomed cages at 25°C temperature, 50% humidity, and a 12-hour light-dark cycle for at least 3 days before the experiment to allow for their acclimatization to the conditions of experiments. Animals were maintained at standard housing conditions and free access to standard diet and water ad libitum during the experiment. The experimental protocol was approved by the Animal Ethics Committee (PM/27/07/2010/MAA (R)) and all animals received humane care according to the guide for the care and use of laboratory animals  and the guide for the control of experiments on animals (CPCSEA) .
2.3. Preparation of Paenibacillus sp. Cell-Free Supernatant
A single colony from the culture of Paenibacillus species strain 139SI was transferred into sterile brain heart infusion (BHI) broth (BD Difco) followed by incubation at 37°C. We have prepared the growth curve of Paenibacillus 139SI supernatant (extract) in three different incubation periods after 24, 48, and 72 hours. However, only the 72-hour extract showed the highest activity compared to the 24 and 48 hours. This was due to the longer incubation period that allows the maximum secretion of bioactive metabolites by the Paenibacillus 139SI colonies into the culture media. Therefore, only the 72-hour incubation extract was used in our study. The Paenibacillus extract was then transferred aseptically into 50 mL conical bottom centrifuge tube (Jet Biofil) followed by centrifugation at 8000 rpm in 4°C for 20 min to separate the cell from the supernatant. The obtained supernatant was then subjected to sterile filtration to remove all unwanted particles using syringe filter with a pore size of 0.22 μm (Minisart Sartorius) . The obtained cell-free supernatant was then freeze-dried and dissolved in ultra-pure water (MilliQ, Millipore) and stored at −20°C as a stock to be used for all experiments. For each 1 mg freeze-dried supernatant powder, the amount of ultra-pure water used to resuspend the powder was 1 mL.
2.4. In Vitro Antibiofilm Activity
To assess the antibiofilm activity of Paenibacillus sp. strain 139SI extract and its purified compounds against clinically important pathogens, microtiter plate (MTP) assay was carried out using 96-well flat bottom polystyrene titer plates as described previously [21, 22]. Each well was filled with 100 μL sterile BHI broth and 50 μL overnight culture for each clinical pathogenic isolate followed by adding 150 μL Paenibacillus sp. crude extract and 150 μL of its purified compounds separately with concentrations of 1000, 1500, 2000, 2500, 3000, 3500, 4000, and 4500 μg/mL before incubation at 37°C for 24 hours. After incubation, plates were gently washed three times with phosphate-buffered saline and the planktonic cells were discarded while the weakly adherent cells were removed through two rounds of thorough washing with deionized water and allowed to air-dry before being stained. The adherent biofilm was stained by 200 μL of 0.4% crystal violet solution (w/v) for 10 min. The optical density (OD) of the biofilm was measured at 570 nm (OD570) with a microtiter absorbance reader (iMark, Bio-Rad) . To compensate for possible differences in growth rates under the different incubation conditions and/or for strains with different characteristics, the adherence index was adjusted as an estimate of the density of the biofilm which would be generated by a culture with an OD600 of 0.5 . Calculation of the adherence index was done according to the following formula: adherence index = mean density of biofilm (OD570) 0.5/mean growth (OD600).
2.5. Biofilm Inhibitory Concentration
In order to determine the lowest concentration of strain 139SI extract that can cause visible inhibition in the biofilm formation, the biofilm inhibitory concentration (BIC) test was carried out using 6-well flat bottom polystyrene titer plates as described previously with few modifications . A piece of glass cover slip was placed inside each well to allow the growth of bacterial isolates on the surface and to visualize the inhibitory effect of 139SI extract on the biofilm formation. Each well was filled with 300 μL sterile BHI broth followed by inoculation with 150 μL of overnight culture for each clinical pathogenic isolate then addition of 150 μL Paenibacillus sp. extract and 150 μL of its purified compounds separately with concentrations ranging from 1000 to 4500 μg/mL before incubation at 37°C for 24 hours. After incubation, biofilm inhibition was determined spectrophotometrically using a microtiter absorbance reader (iMark, Bio-Rad) and visualized microscopically using an upright light microscope (Eclipse LV150L, Nikon).
2.6. Characterization of Bioactive Compounds
The Paenibacillus sp. cell-free supernatant was subjected to High Performance Liquid Chromatography (HPLC). Briefly, the extract solution was filtered using an SRP-4 membrane 0.45 μm and injected into the HPLC column (Agilent Zorbax XDB-C18, 4.6 × 250 mm, 5.0 μm) at a 100 μL injection volume with a flow rate of 1.2 mL/min. The standard solvent system was a combination of acetonitrile and water at a pH of 3.55. Furthermore, the spectrum range was 200–500 nm with UV absorption of 200, 230, 254, and 320 nm. Data acquisition time was between 0 and 32 min yielding a total of 32 fractions (compounds). Further analysis to identify the chemical structure of each of the purified fractions was conducted using Ultra Performance Liquid Chromatography-Diode Array Detection (UPLC–DAD) and Liquid Chromatography-Mass Spectrometry (LC-MS). An Acquity UPLC system (Waters Corporation) equipped with a photo diode array detection detector was used for the analysis and quantification. The UPLC–ESI-MS peak identification was recorded using the UPLC system coupled with a LCQ DECA plus mass spectrometer equipped with an electrospray interface (Thermo-Finnigan Corporation). The quantification of UPLC–DAD was performed on a reversed-phase column Acquity UPLC BEH C-18 (2.1 × 50 mm) with 1.7 μm spherical porous particles. The UPLC–ESI-MS analysis was operated in positive ESI modes and compounds were identified on the basis of their UV spectra and MS fragmentation patterns by searching the dictionary of natural products on DVD, Version 20:2 (CRC Press, Taylor & Francis Group).
2.7. Acute Toxicity Procedure
The virulence of our novel bacterial species of Paenibacillus strain 139SI was tested in experimental mice using the LD50 test as described previously . For acute toxicity test, the selected experimental rats were randomly divided into six groups of six rats each as the following: group 1: normal saline (5 mL/Kg, oral) daily for 14 days (male control group), group 2: normal saline (5 mL/Kg, oral) daily for 14 days (female control group), group 3: 139SI extract (5 mL/Kg, oral) with a concentration of (2 gm/Kg) daily for 14 days (male low dose group), group 4: 139SI extract (5 mL/Kg, oral) with a concentration of (2 gm/Kg) daily for 14 days (female low dose group), group 5: 139SI extract (5 mL/Kg, oral) with a concentration of (4 gm/Kg) daily for 14 days (male high dose group), group 6: 139SI extract (5 mL/Kg, oral) with a concentration of (4 gm/Kg) daily for 14 days (male high dose group).
The body weight of all animals was measured daily. Mortalities, clinical signs, and time of onset were recorded. In addition, gross general observations were observed on the basis of behavioral signs such as food intake, salivation, muscular weakness, reflexes, piloerection, respiration (dyspnea), convulsion, and changes in locomotion . All rats were sacrificed 24 hours after the last oral administration and overnight fasting prior to anesthesia with an intramuscular combination of Ketamine and Xylazine (1 mL of 100 mg/mL Xylazine and 9 mL of 100 mg/mL Ketamine) given at a dose of 0.1 mL/100 gm of body weight followed by necropsy. Blood samples were collected and the liver and kidneys were harvested, washed in normal saline, blotted with filter paper, and weighed. Gross examination was conducted in a blind fashion to examine the macroscopic abnormalities on the organs compared to the control. Moreover, liver and kidneys were subsequently subjected to a histopathological evaluation to examine the microscopic abnormalities on the organs compared to the control.
2.8. Biochemical Parameters and Hematological Profile
Upon sacrifice, blood was drawn from the jugular vein under anesthesia and samples were immediately collected and then referred to the clinical diagnostic laboratories (CDL) at University Malay Medical Centre (UMMC) for assessment of the biochemical parameters and hematological profile. For the biochemical parameters, blood was collected into yellow caped VACUETTE clot activator. Liver function tests were assessed including total protein, albumin, globulin, total bilirubin, conjugate bilirubin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and g-glutamyltransferase. In addition, renal function tests were assessed including sodium, potassium, chloride, carbon dioxide, anion gap, urea, and creatinine. For the hematological profile, blood was collected into violet caped VACUETTE EDTA tubes and the complete blood count (CDC) test was assessed including hemoglobin (HGB), hematocrit (HCT), red blood cells (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width (RDW), white blood cell (WBC), and platelet. In addition, differential blood count test was assessed including neutrophil, lymphocyte, monocyte, eosinophil, and basophil.
2.9. Histopathology Examination
Upon sacrifice, the thoracic cavity was opened by an excision through the peritoneum that was extended through the sternum and the rib cage was fully opened followed by the collection of liver and kidneys. The collected organs were fixed with 10% neutral buffered formalin (NBF) for 24 hours and then sliced into smaller pieces to be fixed again with NBF for another 24 hours. Histopathology examination was performed as described previously . Briefly, fixed tissues were embedded in paraffin wax using an embedding center (Leica EG1160, Leica Biosystems), sectioned using a microtome (Leica RM2135, Leica Biosystems), and fixed onto glass slides using a water bath (Leica HI1210, Leica Biosystems). The paraffin sections were then stained with hematoxylin and eosin (H&E) stain mounted with diphenyl xylene (DPX) and visualized using an upright light microscope (Eclipse LV150L, Nikon).
2.10. Statistical Analysis
Statistical analysis was carried out using the Statistical Product and Service Solutions software (IBM SPSS statistics 21). Categorical data were compared by the χ2 test, while unpaired differences in continuous data were compared by both the Mann-Whitney test and the analysis of variance (ANOVA) test. All values were reported as standard error mean (S.E.M ±) and a probability value of was considered to be statistically significant.
3.1. In Vitro Antibiofilm Activity
The results of MTP assay for the crude extract of Paenibacillus sp. strain 139SI showed significant inhibition of the biofilm formation when assessed spectrophotometrically. The lowest and most effective concentration that caused the reduction in the biofilm’s adherence index was 4500 μg/mL. Among all the 32 purified fractions (compounds) of the crude extract, only 3 compounds showed the highest antibiofilm activity selected against Gram-negative and Gram-positive clinical isolates (Tables 1 and 2), respectively. Moreover, compound number 5 (FR5) was the most active with significant decrease in the adherence index when compared to other compounds and controls. The results of MTP assays were compatible with the BIC test in which there was an 80% inhibition in the biofilm when visualized under light microscope showing scattered bacterial cells with no extracellular matrix.
|OD >0.24 is positive biofilm former isolate.|
OD >0.12–<0.24 is weak biofilm former isolate.
OD <0.12 is negative biofilm former isolate.
represents the most active compound.
|OD >0.24 is positive biofilm former isolate.|
OD >0.12–<0.24 is weak biofilm former isolate.
OD <0.12 is negative biofilm former isolate.
*The most active compound.
3.2. Characterization of Potential Compounds
The results of characterizing the compounds from Paenibacillus sp. extract using HPLC showed a total of 32 purified fractions in which only 3 fractions exhibited antibiofilm activity in vitro when assessed spectrophotometrically . From these 3 fractions, a total of 3 potential compounds were identified in which the first compound was Leucine 2-(hydroxymethoxyphosphinyl)-2-methylhydrazide with a molecular weight of 253.237 and a molecular formula of C8H20N3O4P described as an amino acid antibiotic with an activity against Gram-positive and Gram-negative bacteria. The second compound was 4-Hydroxy-5-(hydroxymethyl)-3-(14-methylpentadecanoyl) tetronicacid-2(5H)-furanone with a molecular weight of 368.512 and a molecular formula of C21H36O5 described as a phospholipase A2 inhibitor. The third compound was 6-(hydroxymethyl)-1-phenazinecarboxyamide with a molecular weight of 253.260 and a molecular formula of C14H11N3O2 described as an antibacterial agent.
3.3. Gross General Observation of Experimental Rats
Gross general observations showed that experimental rats grew at relatively constant rates. Following the 14 days oral ingestion of Paenibacillus sp. extract, there was no significant difference () in the overall growth among the groups except for high dose group where a decrease in growth was observed in the last two days of experiment. These results suggested that the 14 days acute oral ingestion of extract did not affect the weight of rats. Moreover, there was an irregular dose-dependent mortality in both sexes for which only one rat from each sex died after 72 hours ingestion of the high dose (1 out of 6 males and 1 out of 6 females). Moreover, the observed symptoms of toxicity included minor hypoactivity, loss of appetite, hyperventilation, convulsion, dizziness, and salivation; however, they were statistically insignificant when compared to the controls.
3.4. Biochemical Parameters and Hematological Profile of Blood
Despite minor discrepancies between sexes, the results of biochemical parameters showed no significant differences () in the liver and kidney function tests among males and females (Tables 3 and 4), respectively. However, there were elevated levels in globulin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, g-glutamyltransferase, potassium, urea, and creatinine. Moreover, there were increased levels of anion gap among female rats only. Overall, our results indicate that the 139SI extract has no detectable differences on both liver and kidney functions. Moreover, the results of hematological profile (Tables 5 and 6) showed no significant differences () in both the complete and differential blood count except for elevated levels in red blood cells (RBC), white blood cell (WBC), platelet, neutrophil, lymphocyte, and monocyte particularly among the high dose groups (4 gm/Kg).
|Values are expressed as the standard error mean ± S.E.M. and the significant value was at .|
*Values that are above or below the control reference range.
g/L: gram per liter, μmol/L: micromole per liter, and IU/L: international unit per liter.