Piperine, a phytochemical prevents the biofilm city of methicillin-resistant Staphylococcus aureus: A biochemical approach to understand the underlying mechanism.

Methicillin-resistant Staphylococcus aureus (MRSA), a drug-resistant human pathogen causes several nosocomial as well as community-acquired infections involving biofilm machinery. Hence, it has gained a wide interest within the scientific community to impede biofilm-induced MRSA-associated health complications. The current study focuses on the utilization of a natural bioactive compound called piperine to control the biofilm development of MRSA. Quantitative assessments like crystal violet, total protein recovery, and fluorescein-di-acetate (FDA) hydrolysis assays, demonstrated that piperine (8 and 16 µg/mL) could effectively compromise the biofilm formation of MRSA. Light and scanning electron microscopic image analysis confirmed the same. Further investigation revealed that piperine could reduce extracellular polysaccharide production by down-regulating the expression of icaA gene. Besides, piperine could reduce the cell-surface hydrophobicity of MRSA, a crucial factor of biofilm formation. Moreover, the introduction of piperine could interfere with microbial motility indicating the interaction of piperine with the quorum-sensing components. A molecular dynamics study showed a stable binding between piperine and AgrA protein (regulator of quorum sensing) suggesting the possible meddling of piperine in quorum-sensing of MRSA. Additionally, the exposure to piperine led to the accumulation of intracellular reactive oxygen species (ROS) and potentially heightened cell membrane permeability in inhibiting microbial biofilm formation. Besides, piperine could reduce the secretion of diverse virulence factors from MRSA. Further exploration revealed that piperine interacted with extracellular DNA (e-DNA), causing disintegration by weakening the biofilm architecture. Conclusively, this study suggests that piperine could be a potential antibiofilm molecule against MRSA-associated biofilm infections.

Application of cuminaldehyde and ciprofloxacin for the effective control of biofilm assembly of Pseudomonas aeruginosa: A combinatorial study.

Pseudomonas aeruginosa is widely associated with biofilm-mediated antibiotic resistant chronic and acute infections which constitute a persistent healthcare challenges. Addressing this threat requires exploration of novel therapeutic strategies involving the combination of natural compounds and conventional antibiotics. Hence, our study has focused on two compounds; cuminaldehyde and ciprofloxacin, which were strategically combined to target the biofilm challenge of P. aeruginosa. The minimum inhibitory concentration (MIC) of cuminaldehyde and ciprofloxacin was found to be 400 µg/mL and 0.4 µg/mL, respectively. Moreover, the fractional inhibitory concentration index (FICI = 0.62) indicated an additive interaction prevailed between cuminaldehyde and ciprofloxacin. Subsequently, sub-MIC doses of cuminaldehyde (25 µg/mL) and ciprofloxacin (0.05 µg/mL) were selected for an array of antibiofilm assays which confirmed their biofilm inhibitory potential without exhibiting any antimicrobial activity. Furthermore, selected doses of the mentioned compounds could manage biofilm on catheter surface by inhibiting and disintegrating existing biofilm. Additionally, the test combination of the mentioned compounds reduced virulence factors secretion, accumulated reactive oxygen species and increased cell-membrane permeability. Thus, the combination of cuminaldehyde and ciprofloxacin demonstrates potential in combating biofilm-associated Pseudomonal threats.

Identification of a novel quinoline-based UV-protective pigment from a psychrotrophic Arctic bacterium.

AIMS: Psychrotrophs are extremophilic microorganisms that grow optimally in low temperature having many unique bioactive molecules of biotechnological applications. In this study, we characterized a pigment from an arctic bacterium with protective activity towards UV exposure. METHODS AND RESULTS: The present research reports isolation and characterization of a psychrotrophic bacteria, RSAP2, from the soil sample of NyAlesund (78°56"N, 11°54"E), Svalbard, Norway. The strain showed closest 16S rRNA gene sequence similarity (99.9%) with Kocuria indica NIO-1021. RSAP2 is a Gram-positive, coccoid aerobe which produces a yellow pigment. The optimal parameters for pigment production while grown in LB medium were 3% (w/v) NaCl and 4 days of incubation of the culture at 20°C and pH 9 with shaking (180 rpm). The pigment was extracted in methanol and acetone (2:1) and further purified through column chromatography. It was characterized by mass spectrometry, UV-visible, fluorescence, IR, 1 H NMR, 13 C NMR spectroscopy and CHNS/O analysis. The pigment has a molecular weight of about 258 daltons and the molecular formula was determined as C15 H18 N2 O2 and is a quinoline derivative. We show that the pigment can protect Escherichia coli against UV-mediated mutagenesis. We further demonstrate that the pigment displays a significant antimicrobial effect and in sublethal concentrations it impairs biofilm formation ability of the model organism Staphylococcus aureus. CONCLUSIONS: The pigment of a psychrotrophic Arctic bacterium, most likely a strain of K. indica, was purified and its chemical structure was determined. The quinoline-based pigment has the ability to protect live cells from UV induced damage. SIGNIFICANCE AND IMPACT OF STUDY: Analysis and characterization of this newly isolated quinoline-based pigment is a potential candidate for future application in skin care products.

Serratia sp. scl1: isolation of a novel thermostable lipase producing microorganism which holds industrial importance.

Lipase being a hydrolysable enzyme plays a major role in serving various purposes of the industries. Thus, it is very important to have a sustainable and efficient source of this enzyme. In this present study, several microorganisms were isolated from medicinal effluent of a pharmaceutical industry that could produce efficient lipase activity. Among these isolates, a designated strain scl1 was isolated and based on the molecular and biochemical characterisation was tentatively assigned to the genus Serratia. Preliminary studies confirmed the strain scl1 was found to exhibit the highest production of lipase at a temperature and pH of 35 °C and 7, respectively under the incubation for 48 h. Further, the lipase activity was measured by following spectrophotometric method using pNPP as the substrate in which the Km and Vmax of the crude enzyme was found to be 3.349 × 10-3 M and 5.68 × 10-1 unit/mL, respectively. The extracellular crude lipase was found to show a temperature and pH optima of 75 °C and 8, respectively which gave a strong indication that the enzyme appeared to be highly thermostable. This study revealed the strain scl1 is able to produce a thermostable lipase which can meet the needs of the modern-day industrialization techniques. However, more work is required to purify the enzyme and get it ready for commercial applications.

Bioaugmentation of Enterobacter cloacae AKS7 causes an enhanced degradation of low-density polyethylene (LDPE) in soil: a promising approach for the sustainable management of LDPE waste.

Enterobacter cloacae AKS7 was previously reported to degrade UV-treated low-density polyethylene (LDPE) more efficiently than UV-untreated LDPE. However, the degradation of LDPE by Enterobacter cloacae AKS7 at the LDPE-contaminated soil remained unaddressed. To address this issue, soil microcosms were prepared in which an equal amount of either UV-treated or UV-untreated LDPE was added. Then, the microcosms were either augmented with AKS7 or left non-augmented. We observed that the bioaugmented microcosms exhibited approximately twofold greater polymer degradation than non-bioaugmented microcosms. To investigate the underlying cause, we found that the abundance of LDPE-degrading organisms got increased by approximately fivefold in bioaugmented microcosms than non-bioaugmented microcosms. The microbial biomass carbon and nitrogen content got enhanced by approximately twofold in bioaugmented microcosms as contrasted to non-bioaugmented microcosms. Furthermore, the bioaugmented microcosms showed almost twofold increase in the level of dehydrogenase and fluorescein diacetate (FDA) hydrolyzing activity than the non-bioaugmented microcosms. To add on, Shannon-diversity index and Gini coefficient were determined in each microcosm to measure the microbial richness and evenness, respectively, using the results of carbon source utilization pattern of BiOLOG ECO plate. The bioaugmented microcosms exhibited ~ 30% higher functional richness and ~ 30% enhanced functional evenness than the non-bioaugmented microcosms indicating the formation of an enriched ecosystem that could offer various functions including polymer degradation. Taken together, the results suggested that Enterobacter cloacae AKS7 could be used as a promising bioaugmenting agent for the sustainable degradation of LDPE waste at a contaminated site.

1,4-Naphthoquinone accumulates reactive oxygen species in Staphylococcus aureus: a promising approach towards effective management of biofilm threat.

Staphylococcus aureus, a Gram-positive opportunistic microorganism, promotes pathogenicity in the human host through biofilm formation. Microorganisms associated with biofilm often exhibit drug-resistance property that poses a major threat to public healthcare. Thus, the exploration of new therapeutic approaches is the need of the hour to manage biofilm-borne infections. In the present study, efforts are put together to test the antimicrobial as well as antibiofilm activity of 1,4-naphthoquinone against Staphylococcus aureus. The result showed that the minimum bactericidal concentration (MBC) of this compound was found to be 100 µg/mL against Staphylococcus aureus. In this regard, an array of experiments (crystal violet, biofilm protein measurement, and microscopic analysis) related to biofilm assay were conducted with the sub-MBC concentrations (1/20 and 1/10 MBC) of 1,4-naphthoquinone. All the results of biofilm assay demonstrated that these tested concentrations (1/20 and 1/10 MBC) of the compound (1,4-naphthoquinone) showed a significant reduction in biofilm development by Staphylococcus aureus. Moreover, the tested concentrations (1/20 and 1/10 MBC) of the compound (1,4-naphthoquinone) were able to reduce the microbial motility of Staphylococcus aureus that might affect the development of biofilm. Further studies revealed that the treatment of 1,4-naphthoquinone to the organism was found to increase the cellular accumulation of reactive oxygen species (ROS) that resulted in the inhibition of biofilm formation by Staphylococcus aureus. Hence, it can be concluded that 1,4-naphthoquinone might be considered as a promising compound towards biofilm inhibition caused by Staphylococcus aureus.

1,4-Naphthoquinone disintegrates the pre-existing biofilm of Staphylococcus aureus by accumulating reactive oxygen species.

Staphylococcus aureus causes several nosocomial and community-acquired infections in human host involving biofilm. Thus, strategies need to be explored to curb biofilm threats by either inhibiting the formation of biofilm or disintegrating the pre-existing biofilm. Towards this direction, we had already revealed the biofilm inhibiting properties of 1,4-naphthoquinone against S. aureus. In this study, we have investigated whether this compound can act on pre-existing biofilm. Hence, biofilm of S. aureus was developed first and challenged further with 1,4-naphthoquinone. Experiments such as crystal violet assay, fluorescence microscopy, and estimation of total biofilm protein were performed to confirm the biofilm disintegration properties of 1,4-naphthoquinone. The disintegration of pre-existing biofilm could be attributed to the generation of reactive oxygen species (ROS). To investigate further, we observed that extracellular DNA (eDNA) was found to play an important role in holding the biofilm network as DNaseI treatment could cause an efficient disintegration of the same. To examine the effect of ROS on the eDNA, we exposed pre-existing biofilm to either 1,4-naphthoquinone or a combination of both 1,4-naphthoquinone and ascorbic acid for different length of time. Post-incubation, ROS generation and the amount of eDNA associated with the biofilm were determined wherein an inversely proportional relationship was observed between them. The result indicated that with the increase of ROS generation, the amount of eDNA associated with biofilm got decreased substantially. Thus, the results indicated that the generation of ROS could degrade the eDNA thereby compromising the integrity of biofilm which lead to the disintegration of pre-existing biofilm.

Piperine exhibits promising antibiofilm activity against Staphylococcus aureus by accumulating reactive oxygen species (ROS).

Staphylococcus aureus causes numerous community-acquired and nosocomial infections in humans by exploiting biofilm. In this context, this study aims to impede the formation of Staphylococcus aureus biofilm by exposing the cells to a plant-based alkaloid, piperine. Our study revealed that piperine exhibited considerable antimicrobial activity against the test organism. However, we had tested the lower concentrations (up to 32 µg/mL) of piperine to observe whether they could show any antibiofilm activity against the same organism. Several experiments, like crystal violet (CV) assay, estimation of total biofilm protein, and fluorescence microscopic observations, established that lower concentrations (up to 16 µg/mL) of piperine showed efficient antibiofilm activity against Staphylococcus aureus. In this connection, we also noticed that the lower concentrations (8 and 16 µg/mL) of piperine showed a considerable reduction in microbial metabolic activity. Besides, it was also observed that the mentioned concentrations of piperine did not compromise the microbial growth of the target organism while exhibiting antibiofilm activity. To understand the underlying mechanism of microbial biofilm inhibition under the influence of piperine, we observed that the compound was found to accumulate reactive oxygen species in the bacterial cells that could play an important role in the inhibition of biofilm formation. Furthermore, the tested concentrations (8 and 16 µg/mL) of piperine were able to inhibit the motility of the test organism that might compromise the development of biofilm. Thus, piperine could be considered as a potential agent for the effective management of biofilm threat caused by Staphylococcus aureus.

Degradation of low-density poly ethylene (LDPE) by Enterobacter cloacae AKS7: a potential step towards sustainable environmental remediation.

Plastics composed of polyethylene are non-biodegradable and are mostly harmful to the environment. Literature studies documented that the extent of microbial degradation of low-density polyethylene (LDPE) seems to be insufficient and the underlying mechanisms of such degradation remain unexplored. In the present study, efforts were given to degrade LDPE by a recently isolated bacteria Enterobacter cloacae AKS7. Scanning electron microscopic (SEM) image, tensile strength, and weight loss analysis confirmed the efficient degradation of LDPE by AKS7. To investigate the mechanism, it was observed that with the progression of time, the extent of microbial colonization got increased considerably over the LDPE surface. It was also observed that the organism (AKS7) gradually increased the secretion of extracellular polymeric substances (EPS) suggesting the formation of efficient biofilm over the LDPE surface. Furthermore, to comprehend the role of cell-surface hydrophobicity towards biofilm formation, two mutants of AKS7 were screened that showed a considerable reduction in cell-surface hydrophobicity in contrast to its wild type. The result showed that the mutants revealed compromised LDPE degradation than wild-type cells of AKS7. Further investigation revealed that the mutant cells of AKS7 were incapable of adhering to LDPE in contrast to wild-type cells. Thus, the results demonstrated that the cell-surface hydrophobicity of AKS7 favors the development of microbial biofilm over LDPE that leads to the enhanced degradation of LDPE by AKS7. Therefore, the organism holds the assurance to be considered as a promising bio-remediating agent for the sustainable degradation of polythene-based hazardous waste.

Inhibition of biofilm formation of Pseudomonas aeruginosa by caffeine: a potential approach for sustainable management of biofilm.

Pseudomonas aeruginosa is a potent biofilm forming organism causing several diseases on host involving biofilm. Several natural and synthetic molecules have been explored towards inhibiting the biofilm formation of Pseudomonas aeruginosa. In the current report, the role of a natural molecule namely caffeine was examined against the biofilm forming ability of P. aeruginosa. We have observed that caffeine shows substantial antimicrobial activity against P. aeruginosa wherein the minimum inhibitory concentration (MIC) of caffeine was found to be 200 µg/mL. The antibiofilm activity of caffeine was determined by performing a series of experiments using its sub-MIC concentrations (40 and 80 µg/mL). The results revealed that caffeine can significantly inhibit the biofilm development of P. aeruginosa. Caffeine has been found to interfere with the quorum sensing of P. aeruginosa by targeting the swarming motility. Molecular docking analysis further indicated that caffeine can interact with the quorum sensing proteins namely LasR and LasI. Thus, the result indicated that caffeine could inhibit the formation of biofilm by interfering with the quorum sensing of the organism. Apart from biofilm inhibition, caffeine has also been found to reduce the secretion of virulence factors from Pseudomonas aeruginosa. Taken together, the results revealed that in addition to biofilm inhibition, caffeine can also decrease the spreading of virulence factors from Pseudomonas aeruginosa.

Exploration of strategies to increase the nitrogen and phosphate content of solid waste landfill soil.

Several strategies were undertaken to increase the fertility of landfill soil as rapid urbanization remarkably decreases the agricultural land, posing challenges to the fast-growing human population. Towards this direction, soil microcosms were prepared wherein the addition of nutrient or biofertilizer or the combination of both increased the soil nitrogen and phosphate content considerably. The maximum amount of nitrogen fixation and phosphate solubilization occurred in microcosm treated with biofertilizer and nutrient. To investigate the underlying cause, we observed that separate application of nutrient or biofertilizer or combined application of both increased the abundance of nitrogen-fixing and phosphate-solubilizing bacteria in the microcosms. However, the highest abundance of nitrogen-fixing and phosphate-solubilizing bacteria was spotted in a microcosm challenged with nutrient and biofertilizer together. It was detected that with increasing population of nitrogen-fixing and phosphate-solubilizing bacteria, the soil nitrogen and phosphate level also got enhanced, respectively, thus establishing a strong positive correlation between them. The microcosm treated with biofertilizer and nutrient manifested the highest degree of heterotrophic microbial growth and microbial activity than the microcosms either treated with nutrient or biofertilizer. The microcosm treated with nutrient and biofertilizer was found to exhibit the highest functional diversity compared to others. A surface plot was constructed to demonstrate the association among microbial activity, functional diversity, and the availability of soil nitrogen and phosphate content of soil. The result indicates that the combined application of nutrient and biofertilizer increases the microbial activity leading to the formation of a heterogeneous ecosystem that enhances the nitrogen and phosphate content of landfill soil considerably.

Free tryptophan residues inhibit quorum sensing of Pseudomonas aeruginosa: a potential approach to inhibit the development of microbial biofilm.

Microbial biofilm reveals a cluster of microbial population aggregated on a surface. Pseudomonas aeruginosa, a strong biofilm forming organism, often causes several human diseases. Microorganism-based diseases become more difficult to manage when the causative organism develops biofilm during the course of disease progression as the organism attains alarming drug resistance in biofilm form. Agents inhibiting microbial biofilm formation could be considered as a potential tool to weaken the extent of microbial pathogenesis. Tryptophan has already been reported as a promising agent against the biofilm development by P. aeruginosa. In the current study, we had focused on the underlying mechanism of microbial biofilm inhibition of P. aeruginosa under the influence of tryptophan. The expression level of the mRNA of the genes (lasR, lasB and lasI) associated with quorum sensing was compared between tryptophan treated and untreated cells under similar conditions using real time polymerase chain reaction (RT-PCR). The results showed that the tested concentrations of tryptophan considerably reduced the expression of those genes (lasR, lasB and lasI) that are required during the occurrence of quorum sensing in P. aeruginosa. Molecular docking also revealed that tryptophan can interact with the proteins responsible for the occurrence of quorum sensing in P. aeruginosa. The cytotoxicity assay was carried out wherein we observed that the tested concentration of tryptophan did not show any considerable cytotoxicity against the RAW 264.7 macrophage cell line. From this study, it may be concluded that the tryptophan-mediated inhibition of biofilm formation is associated with interference of quorum sensing in P. aeruginosa. Hence, tryptophan could be used as a potential agent against the microbial biofilm mediated pathogenesis.

Zinc oxide nanoparticle inhibits the biofilm formation of Streptococcus pneumoniae.

Biofilms are structured consortia of microbial cells that grow on living and non living surfaces and surround themselves with secreted polymers. Infections with bacterial biofilms have emerged as a foremost public health concern because biofilm growing cells can be highly resistant to both antibiotics and host immune defenses. Zinc oxide nanoparticles have been reported as a potential antimicrobial agent, thus, in the current study, we have evaluated the antimicrobial as well as antibiofilm activity of zinc oxide nanoparticles against the bacterium Streptococcus pneumoniae which is a significant cause of disease. Zinc oxide nanoparticles showed strong antimicrobial activity against S. pneumoniae, with an MIC value of 40 µg/ml. Biofilm inhibition of S. pneumoniae was also evaluated by performing a series of experiments such as crystal violet assay, microscopic observation, protein count, EPS secretion etc. using sub-MIC concentrations (3, 6 and 12 µg/ml) of zinc oxide nanoparticles. The results showed that the sub-MIC doses of zinc oxide nanoparticles exhibited significant anti-biofilm activity against S. pneumoniae, with maximum biofilm attenuation found at 12 µg/ml. Taken together, the results indicate that zinc oxide nanoparticles can be considered as a potential agent for the inhibition of microbial biofilms.

Allee effect: the story behind the stabilization or extinction of microbial ecosystem.

A population exhibiting Allee effect shows a positive correlation between population fitness and population size or density. Allee effect decides the extinction or conservation of a microbial population and thus appears to be an important criterion in population ecology. The underlying factor of Allee effect that decides the stabilization and extinction of a particular population density is the threshold or the critical density of their abundance. According to Allee, microbial populations exhibit a definite, critical or threshold density, beyond which the population fitness of a particular population increases with the rise in population density and below it, the population fitness goes down with the decrease in population density. In particular, microbial population displays advantageous traits such as biofilm formation, expression of virulence genes, spore formation and many more only at a high population density. It has also been observed that microorganisms exhibiting a lower population density undergo complete extinction from the residual microbial ecosystem. In reference to Allee effect, decrease in population density or size introduces deleterious mutations among the population density through genetic drift. Mutations are carried forward to successive generations resulting in its accumulation among the population density thus reducing its microbial fitness and thereby increasing the risk of extinction of a particular microbial population. However, when the microbial load is high, the chance of genetic drift is less, and through the process of biofilm formation, the cooperation existing among the microbial population increases that increases the microbial fitness. Thus, the high microbial population through the formation of microbial biofilm stabilizes the ecosystem by increasing fitness. Taken together, microbial fitness shows positive correlation with the ecosystem conservation and negative correlation with ecosystem extinction.

Pre-oxidation of low-density polyethylene (LDPE) by ultraviolet light (UV) promotes enhanced degradation of LDPE in soil.

Polyethylene represents nearly 64% of all the synthetic plastics produced and are mainly used for domestic and industrial applications. Their extensive use poses a serious environmental threat because of their non-biodegradable nature. Among all the polyethylene remediation strategies, in situ bioremediation happens to be the safest and efficient one. In the current study, efforts had been given to compare the extent of LDPE degradation under UV-treated and UV-untreated conditions by soil microcosm. Landfill soil was collected and UV-treated and UV-untreated LDPE were added separately to the soil following incubation under similar conditions. Electron microscopic images as well as the weight loss and the tensile strength results clearly revealed that UV-treated LDPE showed better degradation than the non-treated ones in soil. To elucidate the mechanism of this enhanced biodegradation, the bond spectra of differentially treated LDPE were analyzed by FTIR. The results obtained from bond spectra studies revealed that UV treatment increases both carbonyl and terminal double-bond index of the LDPE, thereby making it highly susceptible for microbial degradation. Moreover, incubation of UV-treated LDPE with soil favors better adherence of metabolically active and significantly higher number of microorganisms on it. Taken together, all these results demonstrate the higher microbial association and their better metabolic potential to the UV-treated LDPE that lead to enhanced degradation of the LDPE by the soil microorganisms.

Biofilm, pathogenesis and prevention--a journey to break the wall: a review.

Biofilms contain group(s) of microorganisms that are found to be associated with the biotic and abiotic surfaces. Biofilms contain either homogenous or heterogeneous populations of bacteria which remain in the matrix made up of extracellular polymeric substances secreted by constituent population of the biofilm. Biofilms can be either single or multilayered. Biofilms are an increasing issue of concern that is gaining importance with each passing day. Due to the ubiquitous nature of biofilms, it is difficult to eradicate them. It has been seen that many infectious diseases harbour biofilms of bacterial pathogens as the reservoir of persisting infections which can prove fatal at times. The presence of biofilms can be seen in diseases like endocarditis, cystic fibrosis, periodontitis, rhinosinusitis and osteomyelitis. The presence of biofilms has been mostly seen in medical implants and urinary catheters. Various signalling events including two-component signalling, extra cytoplasmic function and quorum sensing are involved in the formation of biofilms. The presence of an extracellular polymeric matrix in biofilms makes it difficult for the antimicrobials to act on them and make the bacteria tolerant to antibiotics and other drugs. The aim of this review was to discuss about the basic formation of a biofilm, various signalling cascades involved in biofilm formation, possible mechanisms of drug resistance in biofilms and recent therapeutic approaches involved in successful eradication of biofilms.

Piperine, a Plant Alkaloid, Exhibits Efficient Disintegration of the Pre-existing Biofilm of Staphylococcus aureus: a Step Towards Effective Management of Biofilm Threats.

Staphylococcus aureus causes a range of chronic infections in humans by exploiting its biofilm machinery and drug-tolerance property. Although several strategies have been proposed to eradicate biofilm-linked issues, here, we have explored whether piperine, a bioactive plant alkaloid, can disintegrate an already existing Staphylococcal biofilm. Towards this direction, the cells of S. aureus were allowed to develop biofilm first followed by treatment with the test concentrations (8 and 16 µg/mL) of piperine. In this connection, several assays such as total protein recovery assay, crystal violet assay, extracellular polymeric substances (EPS) measurement assay, fluorescein diacetate hydrolysis assay, and fluorescence microscopic image analysis confirmed the biofilm-disintegrating property of piperine against S. aureus. Piperine reduced the cellular auto-aggregation by decreasing the cell surface hydrophobicity. On further investigation, we observed that piperine could down regulate the dltA gene expression that might reduce the cell surface hydrophobicity of S. aureus. It was also observed that the piperine-induced accumulation of reactive oxygen species (ROS) could enhance biofilm disintegration by decreasing the cell surface hydrophobicity of the test organism. Together, all the observations suggested that piperine could be used as a potential molecule for the effective management of the pre-existing biofilm of S. aureus.

Cuminaldehyde and Tobramycin Forestall the Biofilm Threats of Staphylococcus aureus: A Combinatorial Strategy to Evade the Biofilm Challenges.

Staphylococcus aureus, an opportunistic Gram-positive pathogen, is known for causing various infections in humans, primarily by forming biofilms. The biofilm-induced antibiotic resistance has been considered a significant medical threat. Combinatorial therapy has been considered a reliable approach to combat antibiotic resistance by using multiple antimicrobial agents simultaneously, targeting bacteria through different mechanisms of action. To this end, we examined the effects of two molecules, cuminaldehyde (a natural compound) and tobramycin (an antibiotic), individually and in combination, against staphylococcal biofilm. Our experimental observations demonstrated that cuminaldehyde (20 µg/mL) in combination with tobramycin (0.05 µg/mL) exhibited efficient reduction in biofilm formation compared to their individual treatments (p < 0.01). Additionally, the combination showed an additive interaction (fractional inhibitory concentration value 0.66) against S. aureus. Further analysis revealed that the effective combination accelerated the buildup of reactive oxygen species (ROS) and increased the membrane permeability of the bacteria. Our findings also specified that the cuminaldehyde in combination with tobramycin efficiently reduced biofilm-associated pathogenicity factors of S. aureus, including fibrinogen clumping ability, hemolysis property, and staphyloxanthin production. The selected concentrations of tobramycin and cuminaldehyde demonstrated promising activity against the biofilm development of S. aureus on catheter models without exerting antimicrobial effects. In conclusion, the combination of tobramycin and cuminaldehyde presented a successful strategy for combating staphylococcal biofilm-related healthcare threats. This combinatorial approach holds the potential for controlling biofilm-associated infections caused by S. aureus.

Synergistic interaction of cuminaldehyde and tobramycin: a potential strategy for the efficient management of biofilm caused by Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic pathogen, has been found to cause several chronic and acute infections in human. Moreover, it often shows drug-tolerance and poses a severe threat to public healthcare through biofilm formation. In this scenario, two molecules, namely, cuminaldehyde and tobramycin, were used separately and in combination for the efficient management of biofilm challenge. The minimum inhibitory concentration (MIC) of cuminaldehyde and tobramycin was found to be 150 µg/mL and 1 µg/mL, respectively, against Pseudomonas aeruginosa. The checkerboard assay revealed that the fractional inhibitory concentration (FIC) index of cuminaldehyde and tobramycin was 0.36 suggesting a synergistic association between them. The sub-MIC dose of cuminaldehyde (60 µg/mL) or tobramycin (0.06 µg/mL) individually did not show any effect on the microbial growth curve. However, the same combinations could affect microbial growth curve of Pseudomonas aeruginosa efficiently. In connection to biofilm management, it was observed that the synergistic interaction between cuminaldehyde and tobramycin could inhibit biofilm formation more efficiently than their single use (p < 0.01). Further investigation revealed that the combinations of cuminaldehyde and tobramycin could generate reactive oxygen species (ROS) that resulted in the increase of membrane permeability of bacterial cells leading to the efficient inhibition of microbial biofilm formation. Besides, the synergistic interaction between cuminaldehyde (20 µg/mL) and tobramycin (0.03 µg/mL) also showed significant biofilm dispersal of the test microorganism (p < 0.01). Hence, the results suggested that synergistic action of cuminaldehyde and tobramycin could be applied for the efficient management of microbial biofilm.

Piperine Exhibits Potential Antibiofilm Activity Against Pseudomonas aeruginosa by Accumulating Reactive Oxygen Species, Affecting Cell Surface Hydrophobicity and Quorum Sensing.

Gram-positive and Gram-negative bacteria often develop biofilm through different mechanisms in promoting pathogenicity. Hence, the antibiofilm molecule needs to be examined separately on both organisms to manage the biofilm threat. Since the antibiofilm activity of piperine against Staphylococcus aureus was already reported; here, we aimed to examine the antibiofilm activity of it against Pseudomonas aeruginosa. P. aeruginosa is an opportunistic Gram-negative pathogen that can cause several healthcare-associated infections by exploiting biofilm. Several experiments like crystal violet assay, estimation of total protein, measurement of extracellular polymeric substance, and microscopic analysis confirmed that lower concentrations (8 and 16 µg/mL) of piperine could inhibit the microbial biofilm formation considerably. Besides, it could also reduce the secretion of virulence factors from P. aeruginosa. Further investigation showed that the cell surface hydrophobicity and microbial motility of the test organism got reduced under the influence of piperine. Piperine exposure was found to increase the accumulation of reactive oxygen species (ROS) that resulted in the inhibition of biofilm formation. Furthermore, the molecular simulation studies suggested that piperine could affect the quorum sensing network of P. aeruginosa. Towards this direction, we noticed that piperine treatment could decrease the expression of the quorum sensing gene (lasI) that resulted in the inhibition of biofilm formation. Besides biofilm inhibition, piperine was also found to disintegrate the pre-existing biofilm of P. aeruginosa without showing any antimicrobial property to the test organism. Thus, piperine could be used for the sustainable protection of public-healthcare by compromising the biofilm assembly of P. aeruginosa.