In vitro antimicrobial efficacy of Cassia alata (Linn.) leaves, stem, and root extracts against cellulitis causative agent Staphylococcus aureus

Abstract Background. Cellulitis is a common skin disease encountered in medical emergencies in hospitals. It can be treated using a combination of antibiotics therapy; however, the causative agent Staphylococcus aureus has been reported to develop resistance towards the currently used antibiotics. Therefore, the search for more alternative herbal origin antimicrobial agents is critical. Aim: In this study, maceration and Soxhlet extraction of the whole plant of Cassia alata Linn. (leaves, roots, and stem) were performed using four solvents with diferent polarities, namely n-hexane, ethyl acetate, ethanol and distilled water. The crude extracts were screened using agar well difusion, colorimetric broth microdilution, grid culture and bacterial growth curve analysis against Staphylococcus aureus. The phytochemicals in the crude extracts were identifed using Gas Chromatography-Mass Spectrometry (GC–MS). Results. Agar-well difusion analysis revealed that extraction using ethyl acetate showed the largest inhibition zone with an average diameter of 15.30 mm (root Soxhlet extract) followed by 14.70 mm (leaf Soxhlet extract) and 13.70 mm (root maceration extract). The lowest minimum inhibitory and minimum bactericidal concentration in root Soxhlet extract using ethyl acetate was 0.313 and 0.625 µg µL−1 , respectively. Our study proved that crude extract of the plant suppressed the growth of S. aureus as evidenced from a signifcant regression extension (p<0.06,p=0.00003) of lag phase for 6 h after the treatment with increased concentration. Based on the GC–MS analysis, 88 phytochemicals consist of fatty acids, esters, alkanes, phenols, fatty alcohols, sesquiterpenoids and macrocycle that possibly contributed to the antimicrobial properties were identifed, 32 of which were previously characterized for their antimicrobial, antioxidant, and anti-infammatory activities. Conclusion. Ethyl acetate crude extract was better than the other investigated solvents. The root and stem of C. alata showed signifcant antimicrobial efcacy against S. aureus in this study. The remaining 56 out of 88 phytochemicals of the plant should be intensively studied for more medicinal uses. Keywords. Cassia alata, Skin bacteria, Maceration and Soxhlet extraction, Antimicrobial, Phytochemicals, GC–MS

The characterization of Enterobacteriaceae and Pseudomonadaceae isolated from natural salt licks in Sarawak Borneo

Salt lick sites are popular hot spots for hunting among the rural community because many animals are attracted to the sites for mineral licking. The animals roaming freely within the salt lick sites may contribute to the transmission of bacterial pathogens, especially through faecal contamination of the salt lick environments. This study aimed to isolate, identify and characterise bacteria from water and soil of salt lick sites and to determine if there is evidence of the transmission of bacteria between two salt lick sites located within the vicinity of Semait river in the NCR (native customary rights) land area of Long Selaan village, Upper Baram in Malaysian Borneo. Samples of soil (n = 8) and water (n = 7) from the two salt lick sites were collected and plated on Eosine Methylene Blue Agar (EMBA) and MacConkey agar for the isolation of bacteria. Fifteen bacterial colonies were isolated and identified by Matrix-assisted laser desorption ionization time-of-flight mass spectrophotometry (MALDI-TOF-MS), 16S rRNA and API® 20E kit. The bacterial isolates were subjected to (GTG)5-PCR and antimicrobial analyses. The antibiotics used were Ampicillin (AMP), Tobramycin (TOB), Imipenem (IPM), Amikacin (AK), Trimethoprim-sulfamethoxazole (SXT), Tetracycline (TE), Chloramphenicol (C), Ceftazidime (CAZ), Nitrofurantoin (F) and Norfloxacin (NOR). The result of this study shows the presence of two families of bacteria in the salt lick samples which were Enterobacteriaceae and Pseudomonadaceae. The dendrogram plotted based on (GTG)5-PCR shows a close genetic relatedness among the bacteria from water and soil as well as in between and within the two salt lick sites. The resistance index of the bacteria from the salt licks ranged from 0.0 to 0.6. This study suggests the potential risk of antimicrobial-resistant bacteria with diverse transmission pathways associated with the salt licks within the area

Screening and characterisation of two strains of Pseudomonas aeruginosa from aquaculture and water environment

Aims: Pseudomonas has been associated with diseases occurring in people with weakened or compromised immune system after exposure to contaminated water. The diseases are commonly treated with antibiotics. However, the bacteria had developed resistances to commonly used antibiotics making treatment a difficult task. Therefore, the continuous surveillance of susceptibility of Pseudomonas especially for the human pathogen P. aeruginosa to commonly clinical and aquaculture farming used antibiotics is important to ensure that serious infections remain susceptible to those antibiotics. Methodology and results: In this study, the bacteria were screened from water, sediment and fish from rivers and aquaculture farms around Kuching, Sarawak. A total number of 38 presumptive P. aeruginosa were isolated using CHROMagarTM Pseudomonas and subjected to a series of biochemical tests. Out of all the isolates tested, only two isolates designated as AS-R10(S) and BK2-OLT2(S) fulfilled the biochemical characteristics of P. aeruginosa. 16S rRNA gene sequencing further confirmed these two isolates as P. aeruginosa based on their 100% similarity with P. aeruginosa strain GD1 and P. aeruginosa strain PA1201 in NCBI database. These two isolates were tested for their susceptibilities against nine common antibiotics used in both clinical and aquaculture farming nowadays: imipenem, piperacillin, meropenem, amikacin, gentamicin, ciprofloxacin, ceftazidime, tobramycin and norfloxacin according to CLSI standard using disk diffusion method. Conclusion, significance and impact of study: The two isolates exhibited total susceptibility to all the antibiotics analysed, suggesting the effectiveness of the antimicrobial agents towards P. aeruginosa isolated from aquaculture and water environment in the study area

Distribution and prevalence of antibiotic resistant bacteria in fish farms in East Malaysia

Aims: Aquaculture has grown tremendously in Malaysia over the past decades. However, guaranteeing aquaculture sustainability is a big challenge in terms of maintaining continuous output with a safe environment. Furthermore, the cultured species should be free from antibiotic resistance bacterial and antibiotic residue. This study aimed to monitor the existence and prevalence of antibiotic resistant bacteria associated with aquaculture farms in Sarawak. Methodology and results: Samples of water, sediment and fish were collected from five aquaculture farms within Sarawak. The samples were plated on trypticase soy agar and incubated at 28 °C for 24 h. A total of 204 bacterial isolates were isolated and analysed by (GTG)5-fingerprinting to determine genetic similarity among the bacterial isolates, so that representatives could be selected from similar clonal isolates. Based on the (GTG)5 profiles, 50 representative isolates were chosen for species identification using 16S rRNA sequencing. The identified bacteria were tested against 25 antibiotics using standard disk diffusion method. The 16S rRNA analysis revealed that the isolates constitute of 14 genera of bacteria including Bacillus (38%), Exiguobacterium (16%), Enterobacter (14%), Aeromonas (6%), Acinetobacter (4%), Citrobacter (4%), Staphylococcus (4%), Achromobacter (2%), Chitinophaga (2%), Fictibacillus (2%), Plesiomonas (2%), Pseudomonas (2%), Pseudoxanthomonas (2%) and Stenotrophomonas (2%). The antibiotic resistance analysis revealed that the highest percentage of resistance was recorded against streptomycin (75.0%), followed by ampicillin (66.0%), ceftriaxone (50.0%), rifampin (43.3%), aztreonam (36.8%) and ceftazidime (31.6%). Resistance to more than two antibiotics was observed in 40.0% of isolates with an overall multiple antibiotic resistant (MAR) index ranging from 0 to 0.79. Conclusion, significant and impact of study: The variability of antibiotic resistance patterns exhibited by different bacterial species suggests a dependence on selective pressures exhibited in different geographical locations. Our results show that the occurrence of MAR bacteria in an aquaculture environment with unknown history of antibiotics usage in the aquaculture system is possible, indicating a need to continuously monitor the presence of antibiotic resistant bacteria in the aquaculture system

Erratum to: 36th International Symposium on Intensive Care and Emergency Medicine

[This corrects the article DOI: 10.1186/s13054-016-1208-6.]

Characterization of Crude Extracts from Cassia alata Linn. against Cellulitis Causing Agent Staphylococcus aureus

Cellulitis is a type of skin disease that can be found frequently from medical emergency in hospitals. The symptoms of disease begin from a small area of tenderness, followed by swelling, and redness that spreads to neighbouring parts of the skin. It can be treated by using a combination of antibiotics; however, the causing agent Staphylococcus aureus had been reported to develop resistance towards commonly used antibiotics. Furthermore, some patients are allergic towards certain antibiotics making the treatment even more difficult. Therefore, the search for an alternative, safe and cost-effective antimicrobial agent is crucial. In this study, Soxhlet extraction and maceration were utilized to produce crude extract from leaves, root and stem of Cassia alata Linn. The crude extracts were diluted and tested against S. aureus through agar-well diffusion assay, colorimetric broth microdilution and bacterial growth curve analysis. These crude extracts were further characterized through qualitative and quantitative phytochemical screenings to determine the presence of alkaloids, flavonoids, tannins and glycosides; the phenolic and flavonoid contents were quantified in the extracts. Meanwhile, the identities of phytoconstituents in the crude extracts were then identified using Gas Chromatography-Mass Spectrometry (GC-MS). From agar-well diffusion assay, root ethyl acetate Soxhlet extract showed the largest inhibition zone with an average diameter of 15.30 mm followed by leaves ethyl acetate Soxhlet extract [14.70 mm], root ethyl acetate maceration extract [13.70 mm], stem ethyl acetate Soxhlet extract [13.30 mm], leaves ethyl acetate maceration extract [13.30 mm], leaves ethanol maceration extract [13.30 mm] and stem ethyl acetate maceration extract [13.00 mm]. These crude extracts were tested in the colorimetric broth microdilution assay where the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of root ethyl acetate Soxhlet extract were identified as low as 31.25 and 62.50 µg, respectively. The MIC/MBC ratio of the C. alata Linn. extracts tested were found to be less than 4, which indicates the extracts can be utilised as the bactericidal agents. Meanwhile, the leaves hexane Soxhlet and leaves ethyl acetate maceration extract showed MIC/MBC ratio greater than 4, suggesting the potential of the extract to be utilised as the bacteriostatic agents. Apart from that, with the exemption of leaves water Soxhlet extract, most of the water extract did not show any significant inhibition effects against the S. aureus. Besides, the bacterial growth curve analysis revealed the growth pattern of the extract treated S. aureus. The application of C. alata Linn. crude extracts in this research manage to suppress the growth of S. aureus. It recorded a significant regression extension (p<0.06, p=0.00003) of lag phase for up to 6 hours after extract treatment with the increase of extract concentration. In the phytochemical screenings conducted, alkaloids were detected in all crude extracts tested while flavonoids, tannins and glycosides were only detected in eight crude extracts. Stem ethyl acetate Soxhlet and root ethyl acetate Soxhlet extract showed the highest amount of phenolic and flavonoid contents from total phenolic content (TPC) and total flavonoid content (TFC) quantitative analyses. Based on the GC-MS analysis, about 66 phytochemicals including phenolics, steroids, fatty acid, alcohol, ester and alkane hydrocarbon that possibly contributed to the antimicrobial properties were detected in the crude extracts. Hence, from this research study, the crude extracts of C. alata Linn., especially stem ethyl acetate Soxhlet, root ethyl acetate Soxhlet, stem ethyl acetate maceration and root ethyl acetate maceration extracts should be intensively studied by purification and identification of the fractions or molecules that had potential to be used as the antimicrobial agents to treat the cellulitis infection. Keywords: Cellulitis, S. aureus, C. alata Linn., antimicrobial assay, phytochemical screening

Screening of antifungal compound producing bacteria from piper nigrum farm against phytophthora capsici

Piper nigrum, also known as the black pepper, is one of the important spice crops that is highly demanded by the world market population. It is widely used in cuisines, aromatherapy and medical. However, the infection by the Phytophthora capsici has caused the severe crop loss to the farmers and decrease in the economic values of black pepper. In order to control the growth of these pathogenic fungi, chemical fungicides such as Bordeaux mixture and copper oxychloride are widely used. However, these chemicals can cause pollutions to the environment and eliminate the non-targeted species such as the growth promoting bacteria in the soil. Therefore, the aim of this research is to isolate the endophytic bacteria that live within the roots of plants which can produce the antimicrobial compound against the growth of Phytophthora capsici. In this research project, root samples were collected from the Piper nigrum farm and the bacteria isolates were screened for their antagonistic effect via dual culture assay, mycelial growth test, spore germination test and double plate assay. Then, three bacteria showing antagonism towards Phytophthora capsici were characterized by using Polymerase Chain Reaction (PCR) and 16S rRNA sequencing. These bacteria isolates were identified as Enterobacter cancerogenus, Enterobacter cloacae P 101 and Enterobacter asburiae LF7 a by using Basic Local Alignment Search Tool (BLAST) via National Center for Biotechnology Information (NCBI)

Prevalence of chloramphenicol-resistant gene in Escherichia coli from water sources in aquaculture farms and rivers of Kuching, Northwestern Borneo

Antibiotic resistant Escherichia coli cases are increasing high especially in Southeast Asia. Illegal use of the antibiotic in the aquaculture farming may become the culprit of the outbreak and spread into environmental source. A study was conducted to: 1) detect the chloramphenicol (CAL)-resistant gene in E. coli isolated from three aquaculture farms and six rivers of northwestern Borneo and 2) investigate the correlation between cat gene with five common antibiotics used. Isolation of E. coli was done on Eosin methylene blue agar and characterized using indole, methyl red, Voges-Proskauer, citrate tests. E. coli isolates were subsequently tested for their susceptibility to five antibiotics commonly used in aqua-farming. The CAL-resistant E. coli were further analyzed for the presence of resistant genes (cat I, cat II, cat III, cat IV) using multiplex polymerase chain reaction. 42 bacterial colonies were isolated from a total of 80 individual water samples, 34 of which were identified as E. coli. Result showed 85.3% of the E. coli isolates were resistant to amoxicillin, 35.3% were resistant to tetracycline, 29.4% were resistant to CAL, 17.6% were resistant to nitrofurantoin and 8.8% were resistant to nalidixic acid. All of the 10 CAL resistant E. coli isolateswere detected with cat II genes; five isolates detected with cat IV genes; three isolates detected with cat III genes; and another two detected with cat I genes. Pearson correlation coefficient shows highly significant relationship between resistance pattern of CAL with amoxicillin; and CAL with tetracycline. Our findings provide the supplementary information of the CAL resistance gene distribution, thereby improving our understanding of the potential risk of antibiotic resistance underlying within this microbial ecosystem

Transmission pathway of Enterobacteriaceae from reared fish and surrounding environment to water body in Batang Ai reservoir, Sarawak, Borneo

This study was aimed to determine the genetic distribution and characteristics of the bacterial family in the Batang Ai reservoir and their possible sources of transmission into the lake water. Surface water samples were collected from twenty-seven sampling points scattered around the Batang Ai reservoir and four fish samples were obtained from an aquaculture farm located within the water reservoir. The samples were plated on Violet Red Bile Agar (VRBA) plates for the isolation of Enterobacteriaceae. A total of 141 bacterial colonies were isolated from the culture plates and subjected to (GTG)5 PCR analysis to determine the genetic similarities among the isolates. A dendrogram was plotted based on the (GTG)5 PCR patterns and the representative isolates were selected for identity confirmation using 16S rRNA sequencing. Based on the sequencing, five genera of Enterobacteriaceae were identified as Enterobacter, Escherichia, Shigella, Klebsiella and Pseudocitrobacter, consisting of 12 identified species, namely Enterobacter hormaechei, E. hormaechei subsp. xiangfangensis, E. cloacae, E. cloacae subsp. dissolvens, E. kobei, E. tabaci, Shigella boydii, S. flexneri, Pseudocitrobacter faecalis, Escherichia fergusonii, E. coli and Klebsiella pneumoniae. A total of 14 antibiotics from seven anti-microbial classes including penicillins, cephems, monobactams, aminoglycosides, tetracyclines, quinolones and fluoroquinolones, and phenicols were tested against the isolates. A total of seven species within the isolates were found to have multiple antibiotic resistance (MAR) index below 0.2, suggesting those isolates were derived from low risk of antibiotics contamination sources. With the exception of ampicillin and tetracycline, all antibiotics have 25% or less bacterial species displaying resistance. Most (70%) of the bacterial species showed resistance toward ampicillin. The fish isolates demonstrated multiple antibiotic resistance indexes ranging from 0.214 to 0.5. The presence of multiple antibiotic-resistant bacteria in the water reservoir may pose the risk of antibiotic-resistant bacterial infections through water-related activities and consumption of fish harbouring antimicrobial resistance bacterial species. It was proven that Enterobacteriaceae were transmitted from the activities and surrounding environments to the water reservoir, thus it is proposed that a guideline for activities to be carried out in the water reservoir is created and imposed

Screening and characterisation of two strains of Pseudomonas aeruginosa from aquaculture and water environment

Aims: Pseudomonas has been associated with diseases occurring in people with weakened or compromised immune system after exposure to contaminated water. The diseases are commonly treated with antibiotics. However, the bacteria had developed resistances to commonly used antibiotics making treatment a difficult task. Therefore, the continuous surveillance of susceptibility of Pseudomonas especially for the human pathogen P. aeruginosa to commonly clinical and aquaculture farming used antibiotics is important to ensure that serious infections remain susceptible to those antibiotics. Methodology and results: In this study, the bacteria were screened from water, sediment and fish from rivers and aquaculture farms around Kuching, Sarawak. A total number of 38 presumptive P. aeruginosa were isolated using CHROMagarTM Pseudomonas and subjected to a series of biochemical tests. Out of all the isolates tested, only two isolates designated as AS-R10(S) and BK2-OLT2(S) fulfilled the biochemical characteristics of P. aeruginosa. 16S rRNA gene sequencing further confirmed these two isolates as P. aeruginosa based on their 100% similarity with P. aeruginosa strain GD1 and P. aeruginosa strain PA1201 in NCBI database. These two isolates were tested for their susceptibilities against nine common antibiotics used in both clinical and aquaculture farming nowadays: imipenem, piperacillin, meropenem, amikacin, gentamicin, ciprofloxacin, ceftazidime, tobramycin and norfloxacin according to CLSI standard using disk diffusion method. Conclusion, significance and impact of study: The two isolates exhibited total susceptibility to all the antibiotics analysed, suggesting the effectiveness of the antimicrobial agents towards P. aeruginosa isolated from aquaculture and water environment in the study area