Synergistic antimicrobial combination of carvacrol and thymol impairs single and mixed-species biofilms of Candida albicans and Staphylococcus epidermidis.

Candida albicans and Staphylococcus epidermidis are important opportunistic human pathogens, which form mixed-species biofilms and cause recalcitrant device associated infections in clinical settings. Further to many reports suggesting the therapeutic potential of plant-derived monoterpenoids, this study investigated the interaction of the monoterpenoids carvacrol (C) and thymol (T) against mono- and mixed-species growth of C. albicans and S. epidermidis. C and T exhibited synergistic antimicrobial activity. The time-kill study and post-antimicrobial effect results revealed the effective microbicidal action of the C + T combination. Filamentation, surface coating assays and live-dead staining of biofilms determined the anti-hyphal, antiadhesion, and anti-biofilm activities of the C + T combination, respectively. Notably, this combination killed highly tolerant persister cells of mono-species and mixed-species biofilms and demonstrated less risk of resistance development. The collective data suggest that the C + T combination could act as an effective therapeutic agent against biofilm associated mono-species and mixed-species infections of C. albicans and S. epidermidis.

In vitro and in silico attenuation of quorum sensing mediated pathogenicity in Pseudomonas aeruginosa using Spirulina platensis.

Biofilm forming pathogenic bacteria showing resistance towards antimicrobial agents has increased the urge to find an alternative treatment strategy. Among the biofilm forming pathogenic bacteria, Pseudomonas aeruginosa is a well-recognised Gram negative biofilm former causing nosocomial infection and other disease among immunocompromised patients. The aim of the current study is to evaluate the antipathogenic potentials of S. platensis against P. aeruginosa. S. platensis methanolic extract (SME) inhibited the biofilm (89%), extracellular polymeric substances (EPS), cell surface hydrophobicity (CSH) (44%), pyocyanin, pyoveridin and swarming motility of P. aeruginosa. Partial purification of SME using thin layer chromatography (TLC), column chromatography and gas chromatography mass spectroscopy (GCMS) revealed the major component as hexadecanoic acid (HxD). Further analysis through in silico approach showed the efficient binding of HxD with the biofilm regulatory proteins (Las R and salidase enzyme) of P. aeruginosa. The coating of HxD over different material surfaces efficiently prevented the adhesion of P. aeruginosa biofilm. The results of the toxicity assay revealed that the SME is non-toxic towards Artemia salina at the given concentration. Hence the overall work proves the potential non-toxic effects of SME against P. aeruginosa pathogenicity.

Microfouling inhibition of human nosocomial pathogen Pseudomonas aeruginosa using marine cyanobacteria.

Pseudomonas aeruginosa is a Gram negative, opportunistic biofilm forming pathogenic bacterium which is developing as a serious problem worldwide. The pathogenicity of P. aeruginosa mainly depends upon biofilm and quorum sensing (QS) mechanism. Targeting biofilm and QS regulated factor will automatically reduce the pathogenicity of P. aeruginosa. Therefore it is compulsory to identify naturally derived biofilm and QS inhibitors against P. aeruginosa. In the present study Oscillatoria subuliformis, a marine cyanobacterium was used against the biofilm and QS of P. aeruginosa. O. subuliformis intracellular methanolic extract (OME) at different concentration (1.5 µg mL-1, 3 µg mL-1 and 5 µg mL-1) was tested against several virulence factors like Extracellular Polymeric Substance (EPS), Cell Surface Hydrophobicity (CSH), elastase, pyocyanin and swarming motility. OME inhibited biofilm (56%), EPS (40%), CSH (56%), pyocyanin (27%), elastase activity and swarming motility in P. aeruginosa without interfering in their survival. Characterization of the OME using FTIR and GCMS confirmed palmitic acid and oleic acid as active compound. CLSM analysis of catheter coated with OME, palmitic and oleic acid proved biofilm inhibition over urinary catheters. The results postulates that oleic and palmitic acid could be an effective attenuator of P. aeruginosa pathogenesis.

2-Furaldehyde diethyl acetal from tender coconut water (Cocos nucifera) attenuates biofilm formation and quorum sensing-mediated virulence of Chromobacterium violaceum and Pseudomonas aeruginosa.

The aim of this study was to evaluate the anti-biofilm and quorum sensing inhibitory (QSI) potential of tender coconut water (TCW) against Chromobacterium violaceum and Pseudomonas aeruginosa. TCW significantly inhibited the QS regulated violacein, virulence factors and biofilm production without affecting their growth. qRT-PCR analysis revealed the down-regulation of autoinducer synthase, transcriptional regulator and virulence genes. Mass-spectrometric analysis of a petroleum ether extract of the TCW hydrolyte revealed that 2-furaldehyde diethyl acetal (2FDA) and palmitic acid (PA) are the major compounds. In vitro bioassays confirmed the ability of 2FDA to inhibit the biofilm formation and virulence factors. In addition, the combination of PA with 2FDA resulted in potent inhibition of biofilm formation and virulence factors. The results obtained strongly suggest that TCW can be exploited as a base for designing a novel antipathogenic drug formulation to treat biofilm mediated infections caused by P. aeruginosa.

One pot synthesis and anti-biofilm potential of copper nanoparticles (CuNPs) against clinical strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic pathogen frequently associated with nosocomial infections, is emerging as a serious threat due to its resistance to broad spectrum antimicrobials. The biofilm mode of growth confers resistance to antibiotics and novel anti-biofilm agents are urgently needed. Nanoparticle based treatments and therapies have been of recent interest because of their versatile applications. This study investigates the anti-biofilm activity of copper nanoparticles (CuNPs) synthesized by the one pot method against P. aeruginosa. Standard physical techniques including UV-visible and Fourier transform infrared spectroscopy, X-ray diffraction and transmission electron microscopy were used to characterize the synthesized CuNPs. CuNP treatments at 100 ng ml(-1) resulted in a 94, 89 and 92% reduction in biofilm, cell surface hydrophobicity and exopolysaccharides respectively, without bactericidal activity. Evidence of biofilm inhibition was also seen with light and confocal microscope analysis. This study highlights the anti-biofilm potential of CuNPs, which could be utilized as coating agents on surgical devices and medical implants to manage biofilm associated infections.

Evaluation of bacterial diversity in Palk Bay sediments using terminal-restriction fragment length polymorphisms (T-RFLP).

Although it is known that Palk Bay sediments harbor diverse and novel bacteria with important ecological and environmental functions, a comprehensive view of their molecular diversity is still lacking. In the present study, bacterial diversity in Palk Bay sediments was characterized using the molecular method terminal-restriction fragment length polymorphisms (T-RFLP). The bacterial assemblages detected by T-RFLP analysis revealed that the nearshore sediment harbored high number of bacterial count, whereas the 2.5-m sediment harbored diverse and distinct bacterial composition with fine heterogeneity. The major bacterial groups detected in all the three sediment samples were Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria (including alpha (α), gamma (γ), delta (δ), and epsilon (ε)-Proteobacteria), and uncultured bacteria. This is the first study that reveals the presence of Bacteroidetes, delta (δ)- and epsilon (ε)-Proteobacteria, and uncultured bacteria in Palk Bay sediments. The hitherto unexplored wide microbial diversity of Palk Bay coastal area was unraveled in the current study through culture-independent approach. These data suggest that the continued use of cultivation-independent techniques will undoubtedly lead to the discovery of additional bacterial diversity and provide a direct means to learn more about the ecophysiology and biotechnological potential of Palk Bay coastal area.

A novel compound from the marine bacterium Bacillus pumilus S6-15 inhibits biofilm formation in gram-positive and gram-negative species.

Biofilm formation is a critical problem in nosocomial infections and in the aquaculture industries and biofilms show high resistance to antibiotics. The aim of the present study was to reveal a novel anti-biofilm compound from marine bacteria against antibiotic resistant gram-positive and gram-negative biofilms. The bacterial extract (50 µg ml(-1)) of S6-01 (Bacillus indicus = MTCC 5559) showed 80-90% biofilm inhibition against Escherichia coli, Shigella flexneri, Proteus mirabilis and S6-15 (Bacillus pumilus = MTCC 5560) showed 80-95% biofilm inhibition against all the 10 tested organisms. Furthermore, they also reduced the hydrophobicity index and extracellular polymeric substances (EPS) production. Structural elucidation of the active principle in S6-15 using GC-MS, (1)H NMR, and (13)C NMR spectral data revealed it to be 4-phenylbutanoic acid. This is the first report of 4-phenylbutanoic acid as a natural product. The purified compound (10-15 µg ml(-1)) showed potential activity against a wide range of biofilms. This study for the first time, reports a novel anti-biofilm compound from a marine bacterium with wide application in medicine and the aquaculture industry.

Assessment and characterization of heavy metal resistance in Palk Bay sediment bacteria.

The present study aimed at characterizing the heavy metal resistance and assessing the resistance pattern to multiple heavy metals (300 mmol L⁻¹) by Palk Bay sediment bacteria. From 46 isolates, 24 isolates showed resistance to more than eight heavy metals. Among the 24 isolates S8-06 (Bacillus arsenicus), S8-10 (Bacillus pumilus), S8-14 (B. arsenicus), S6-01 (Bacillus indicus), S6-04 (Bacillus clausii), SS-06 (Planococcus maritimus) and SS-08 (Staphylococcus pasteuri) exhibited high resistance against arsenic, mercury, cobalt, cadmium, lead and selenium. Plasmid curing confirmed that the heavy metal resistance in S8-10 is chromosomal borne. Upon treatment with the heavy metals, the strain S8-10 showed many morphological and physiological changes as shown by SEM, FTIR and AAS analysis. S8-10 removed 47% of cadmium and 96% of lead from the growth medium. The study suggests that sediment bacteria can be biological indicators of heavy metal contamination.

The in vitro antibiofilm activity of selected marine bacterial culture supernatants against Vibrio spp.

The aim of the work is to investigate the effect of marine bacterial culture supernatants on biofilm formation of Vibrio spp., a major menace in aquaculture industries. Vibrio spp. biofilm cause life-threatening infections in humans and animals. Forty-three marine bacterial culture supernatants were screened against the hydrophobicity index, initial attachment and biofilm formation in Vibrio spp. Twelve culture supernatants showed antibiofilm activity. The bacterial culture supernatants S8-07 (Bacillus pumilus) and S6-01 (B. indicus) inhibited the initial attachment, biofilm formation and dispersed the mature biofilm at 5% v/v concentration without inhibiting the growth. Analysis by light microscopy and confocal laser scanning microscopy showed that the architecture of the biofilm was destroyed by bacterial supernatants when compared to the control. The bacterial supernatants also reduce the surface hydrophobicity of Vibrio spp. which is one of the important requirements for biofilm formation. Further characterization of antibiofilm activity in S8-07 culture supernatant confirmed that it is an enzymatic activity and the size is more than 10 kDa and in S6-01, it is a heat-stable, non-protein compound. Furthermore, both the supernatants failed to show any biosurfactant activity. The culture supernatants of S8-07 and S6-01 with promising antibiofilm property have potential for application in medicine and marine aquaculture.

Marine bacterial isolates inhibit biofilm formation and disrupt mature biofilms of Pseudomonas aeruginosa PAO1.

According to the Centers for Disease Control and Prevention, biofilms cause 65% of infections in developed countries. Pseudomonas aeruginosa biofilm cause life threatening infections in cystic fibrosis infection and they are 1,000 times more tolerant to antibiotic than the planktonic cells. As quorum sensing, hydrophobicity index and extracellular polysaccharide play a crucial role in biofilm formation, extracts from 46 marine bacterial isolates were screened against these factors in P. aeruginosa. Eleven extracts showed antibiofilm activity. Extracts of S6-01 (Bacillus indicus = MTCC 5559) and S6-15 (Bacillus pumilus = MTCC 5560) inhibited the formation of PAO1 biofilm up to 95% in their Biofilm Inhibitory Concentration(BIC) of 50 and 60 microg/ml and 85% and 64% in the subinhibitory concentrations (1/4 and 1/8 of the BIC, respectively). Furthermore, the mature biofilm was disrupted to 70-74% in their BIC. The antibiofilm compound from S6-15 was partially purified using solvent extraction followed by TLC and silica column and further characterized by IR analysis. Current study for the first time reveals the antibiofilm and antiquorum-sensing activity of B. pumilus, B. indicus, Bacillus arsenicus, Halobacillus trueperi, Ferrimonas balearica, and Marinobacter hydrocarbonoclasticus from marine habitat.

Bacillus pumilus of Palk Bay origin inhibits quorum-sensing-mediated virulence factors in Gram-negative bacteria.

The aim of the current study was to inhibit quoring-sensing(QS)-mediated virulence factors of representative Gram-negative bacteria by marine bacterial isolates. Bacteria isolated from Palk Bay sediments were screened for anti-QS activity. Eleven strains inhibited QS signals in Chromobacterium violaceum (ATCC 12472) and C. violaceum CV026. The marine bacterial strain S8-07 reduced the accumulation of N-acyl homoserine lactone (AHLs) and showed significant inhibition of LasA protease(76%), LasB elastase(84%), caseinase(70%), pyocyanin (84%), pyoverdin and biofilm formation(87%) in Pseudomonas aeruginosa PAO1. Strain S8-07 also showed highly significant reduction (90%) in prodigiosin, secreted casienase (92%), hemolytic activity (73%) and biofilm formation (61%) in Serratia marcescens. Strain S8-07, identified as Bacillus pumilus (accession number FJ584416), showed distinct profiles of inhibition against the virulence factors of both P. aeruginosa PAO1 (las, rhl) and S. marcescens (shl). Polar extraction and proteinase K treatment of the culture supernatant confirmed that the anti-QS activity of S8-07 was indeed due to a protein molecule. Acidification assay and HPLC analysis revealed that the degradation of AHL was not due to lactonase activity, but rather, was due to acylase activity of S8-07. Thus, novel anti-QS acylase activity is reported for the first time from a B. pumilus strain of marine origin.

Isolation of heterotrophic bacteria from Palk Bay sediments showing heavy metal tolerance and antibiotic production.

Analysis of culturable and unculturable bacteria and their potential bioactive compounds from Palk Bay is yet to be explored. The present study for the first time characterizes the culturable bacteria from Palk Bay sediment using 16S rRNA gene sequencing. The characterized bacteria were also screened for antibacterial activity against human and aquaculture pathogens. In the 16S rRNA gene sequence analysis characterized that most of the bacteria were affiliated to members of Firmicutes and less with Gammaproteobacteria, Actinobacteria and Alphaproteobacteria. A high portion of (39%) of the bacteria showed antibacterial activity against both Gram positive and Gram negative test strains. The antibiotics from the strain S6-05 were partially purified using solvent extraction followed by TLC and silica column and further characterized by IR analysis. Two active principles A and B showed difference in the activity against Gram positive and Gram negative pathogens. But in the synergistic application they showed excellent activity against all the test pathogens. This study provides the first evidence on the existence of certain Bacillus species in the marine environment, namely Bacillus arsenicus, Bacillus indicus, Bacillus boroniphilus, Bacillus cibi and Bacillus niabensis which also had antibacterial activity. Several of the isolates exhibited tolerance to arsenic and boron to a new level of 25 and 100 mM, respectively. The current study reveals the fact that a great deal remains in the bacterial diversity of Palk Bay region.