Abdullah Safar Althubiani

Leaf Extracts of Mangifera indica L. Inhibit Quorum Sensing – Regulated Production of Virulence Factors and Biofilm in Test Bacteria

Quorum sensing (QS) is a global gene regulatory mechanism in bacteria for various traits including virulence factors. Disabling QS system with anti-infective agent is considered as a potential strategy to prevent bacterial infection. Mangifera indica L. (mango) has been shown to possess various biological activities including anti-QS. This study investigates the efficacy of leaf extracts on QS-regulated virulence factors and biofilm formation in Gram negative pathogens. Mango leaf (ML) extract was tested for QS inhibition and QS-regulated virulence factors using various indicator strains. It was further correlated with the biofilm inhibition and confirmed by electron microscopy. Phytochemical analysis was carried out using ultra performance liquid chromatography (UPLC) and gas chromatography–mass spectrometry (GC-MS) analysis. In vitro evaluation of anti-QS activity of ML extracts against Chromobacterium violaceum revealed promising dose-dependent interference in violacein production, by methanol extract. QS inhibitory activity is also demonstrated by reduction in elastase (76%), total protease (56%), pyocyanin (89%), chitinase (55%), exopolysaccharide production (58%) and swarming motility (74%) in Pseudomonas aeruginosa PAO1 at 800 μg/ml concentration. Biofilm formation by P. aeruginosa PAO1 and Aeromonas hydrophila WAF38 was reduced considerably (36–82%) over control. The inhibition of biofilm was also observed by scanning electron microscopy. Moreover, ML extracts significantly reduced mortality of Caenorhabditis elegans pre-infected with PAO1 at the tested concentration. Phytochemical analysis of active extracts revealed very high content of phenolics in methanol extract and a total of 14 compounds were detected by GC-MS and UPLC. These findings suggest that phytochemicals from the ML could provide bioactive anti-infective and needs further investigation to isolate and uncover their therapeutic efficacy.

Multidrug resistance and transferability of blaCTX-M among extended-spectrum β-lactamase-producing enteric bacteria in biofilm

This study aimed to investigate the occurrence of biofilm-forming extended-spectrum β-lactamase (ESBL)-producing enteric bacteria in hospital wastewater and to evaluate their antibiotic resistance behaviour and transferability of the plasmid-encoded blaCTX-M gene in biofilm. ESBL production was confirmed using the combined disc test and Etest. Amplification of blaCTX-M was performed by PCR. Antibiotic susceptibility was evaluated using the disc diffusion assay and broth dilution method. Transfer of blaCTX-M in planktonic and biofilm state was performed by broth mating and filter mating experiments, respectively. Among 110 enteric bacteria, 24 (21.8%) isolates belonging to Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae were found to produce ESBL and formed varying levels of biofilm in vitro. Presence of blaCTX-M was detected in 18 (75%) ESBL-producing isolates. A many fold increase in resistance to antibiotics was observed in biofilm. Among ESBL-producers, seven isolates could transfer the blaCTX-M gene by conjugation, with transfer frequencies ranging from 2.22×10(-4) to 7.14×10(-2) transconjugants/recipient cell in the planktonic state and from 3.04×10(-3) to 9.15×10(-1) in biofilm. The transfer frequency of blaCTX-M was significantly higher in biofilm compared with the planktonic state, and co-transfer of ciprofloxacin resistance was also detected in five isolates. This study demonstrates that biofilm-forming ESBL-producing enteric bacteria with a greater transfer frequency of resistance genes will lead to frequent dissemination of β-lactam and fluoroquinolone resistance genes in environmental settings. The emergence and spread of such multidrug resistance is a serious threat to animal and public health.

Identification and characterization of a novel antimicrobial peptide compound produced by Bacillus megaterium strain isolated from oral microflora

In recent years, the decreased efficacy of existing antibiotics toward management of emergent drug-resistant strains has necessitated the search for novel antibiotics from natural products. In this regard, Bacillus sp is well known for producing variety of secondary metabolites of potential use. Therefore, we performed an investigation to isolate and identify Bacillus sp from oral cavity for production of novel antimicrobial compounds. We extracted, purified, and identified a novel bioactive compound by B. megaterium (KC246043.1). The optimal production of compound was observed on de Man Rogosa and Sharpe broth by incubating at 37 °C, and pH 7.0 for 4 days. The bioactive compound was extracted by using n-butanol (2:1 v/v), purified on TLC plates with detection at Rf 7.8 cm; further characterized and identified as a cyclic ploypeptide sharing structural similarity with bacitracin. Minimum inhibitory concentration of bioactive compound was found to be 0.25, 0.5, 1.0, 3.125 and 6.25 μg/ml against Micrococcus luteus ATCC10240, Salmonella typhi ATCC19430, Escherichia coli ATCC35218. Pseudomonas aeruginosa ATCC27853 and Staphylococcus aureus ATCC25923 respectively, with no activity against Candida albicans ATCC10231. Our findings have revealed a novel cyclic peptide compound from B. megaterium with broad spectrum antimicrobial activity against both Gram positive and Gram negative bacteria.

Bioactive extracts of Carum copticum L. enhances efficacy of ciprofloxacin against MDR enteric bacteria

The widespread occurrence of extended spectrum β-lactamases (ESβLs) producing enteric bacteria and their co-resistance with flouroquinolones has impaired the current antimicrobial therapy. This has prompted the search for new alternatives through synergistic approaches with herbal extracts. In this study Carum copticum (seeds) was extracted first in methanol and then subsequently extracted in different organic solvents. MIC of plant extracts, ciprofloxacin and thymol was determined by broth micro-dilution method using TTC. Synergism between plant extracts and ciprofloxacin was assayed by the checkerboard method. Chemical constituents of active extracts were analyzed by GC-MS. Methanolic, hexane and ether extract of Carum copticum exhibited significant antibacterial activity with MIC values ranged from 0.25 mg/ml to 2.0 mg/ml. Synergy analysis between Carum copticum extracts and ciprofloxacin combinations revealed FIC index in the range of 0.093–0.25. About 81% ciprofloxacin resistant ESβL producing enteric bacteria were re-sensitized in the presence of 15.6–250 μg/ml of methanolic extract of Carum copticum. Moreover, ciprofloxacin showed 8 to 64 folds reduction in MIC in presence of 250 and 500 μg/ml of hexane extract. Whereas, 4–32 folds reduction in MIC of ciprofloxacin was achieved in the presence of 31.25 and 62.5 μg/ml of ether extract, indicating synergistic enhancement of drug activity. The chemical analysis of hexane and ether extracts by GC-MS revealed the common occurrence of one or more phenolic hydroxyl at different locations on benzene ring. This study demonstrated the potential use of herbal extract of Carum copticum in combination therapy against ESβL producing bacteria.

Rhizobacterial Biofilms: Diversity and Role in Plant Health

The diverse nature of rhizobacteria and their interaction with plant roots involves complex processes and provides a unique microbial niche in the rhizosphere both beneficial and harmful to plant health depending on nature of bacteria. Biofilms are defined as the bacterial populations which stick to living and nonliving surfaces and encased in a self-produced extracellular polymeric substances (EPS). Both disease-causing and beneficial plant growth-encouraging bacteria may form biofilm on abiotic and biotic surfaces including plant surface and in soil. It is now well known that a microbe under natural condition forms mixed/polymicrobial biofilm. The process of biofilm development and their regulation are well studied among human pathogenic bacteria such as Pseudomonas aeruginosa. However, recent investigations indicated an increased interest in the research on biofilm on plant-associated rhizobacteria such as Azotobacter, Acinetobacter, Bacillus, Burkholderia, Klebsiella, Pantoea, Pseudomonas and Rhizobium. In this chapter we have made an attempt to review recent studies on rhizobacterial biofilms and their possible impact on plant health under natural and stress conditions.

The Prebiotic Properties of Date Palm (Phoenix dactylifera L.) Seeds in Stimulating Probiotic Lactobacillus

Date seeds/ kernels are currently used in the feeding of animals such as cattle, sheep, and camel, and in the poultry and fish industries as well. Due to the presence of a large quantity of total dietary fibre they are considered to have potential health benefits for human as prebiotics. The present study was aimed to investigate the effects of the date palm (Phoenix dactylifera L.) seed products in the form of (i) the finely ground date seeds powder (FGDSP) and (ii) the aqueous extract of date seed powder (AEDSP) on the growth of the lactic acid bacteria (LAB) Lactobacillus paracasei ssp paracasei as probiotics. Subsequently, to investigate the prebiotic properties of the date palm (Phoenix dactylifera L.) seed product i.e dietary fibre concentrate (DFC) in stimulating the growth of the probiotic. The total numbers of bacterial populations at 0, 24, 48, 72 and 96 h of fermentation were checked for each sample during the fermentation period. The data obtained clearly demonstrated that both the form of test date palm (Phoenix dactylifera L.) viz. FGDSP and AEDSP could be used as a carbon source for bacterial fermentations. The DFC presented the potential to be applied as a novel source of prebiotics, by increasing the population of Lactobacillus paracasei ssp paracasei as probiotics in addition to decreasing the pH values. This research proposed that date seeds could potentially be utilized as a starting material to manufacture functional food ingredients especially as they are a common waste product, especially in Middle Eastern countries. Hence, further investigations should be carried out to identify active biological molecules in date seeds and their potential application in encouraging the growth of probiotics and other health benefits.

Diversity, Quorum Sensing, and Plant Growth Promotion by Endophytic Diazotrophs Associated with Sugarcane with Special Reference to Gluconacetobacter diazotrophicus

Endophytic bacteria are widely distributed among plants and colonize both intracellular and intercellular spaces and do not harm the host plant. However, the distributions of endophytic diazotrophs are limited. Endophytic diazotrophs like Gluconacetobacter diazotrophicus are mainly associated with sugarcane and some other plants and responsible for significant contribution of biological nitrogen fixation with sugarcane. In this article, we described the diversity and role of quorum sensing. We also discussed the contributions of different bacterial traits that are necessary for successful colonization of the plant interior part. Further mechanisms of plant growth promotion are elaborated. Molecular characterization and identification of endophytic diazotrophs will further help in better understanding of plant colonization and plant growth promotion.