Hybrid-drug design targeting Pseudomonas aeruginosa dihydropteroate synthase and dihydrofolate reductase

In this study, we successfully present the dual-target design hypothesis to inhibit both dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) enzymes using a novel scheme that integrates our previous antibiotic-phytochemical interaction data, fragment combination and knowledge-based methods. Both the enzymes are well established antibacterial targets from folate biosynthesis pathway and their synergistic modulation by a single hybrid entity may have profound therapeutic benefits. Evaluation of the designed hybrid compounds based on their physico-chemical properties has indicated them as promising drug candidates with drug-like pharmacotherapeutic profiles. In addition, the stereo-electronic properties such as HOMO, LUMO and MEP maps calculated by quantum chemical methods gave a good correlation with the common pharmacophoric features required for dual-site interactions. Furthermore, docking and dynamics simulation studies reveal that the designed hybrid compounds have favorable binding affinity and stability in both pterin-binding site of DHPS and folate-binding site of DHFR by forming strong hydrogen bonds and hydrophobic interactions with key active-site residues. Looking forward this study could serve as a prospective lead in the process of new natural-product based hybrid-drugs development.

Bactericidal antibiotic-phytochemical combinations against methicillin resistant Staphylococcus aureus

Methicillin resistant Staphylococcus aureus (MRSA) infection is a global concern nowadays. Due to its multi-drug resistant nature, treatment with conventional antibiotics does not assure desired clinical outcomes. Therefore, there is a need to find new compounds and/or alternative methods to get arsenal against the pathogen. Combination therapies using conventional antibiotics and phytochemicals fulfill both requirements. In this study, the efficacy of different phytochemicals in combination with selected antibiotics was tested against 12 strains of S. aureus (ATCC MRSA 43300, ATCC methicillin sensitive S. aureus or MSSA 29213 and 10 MRSA clinical strains collected from National University Hospital, Singapore). Out of the six phytochemicals used, tannic acid was synergistic with fusidic acid, minocycline, cefotaxime and rifampicin against most of strains tested and additive with ofloxacin and vancomycin. Quercetin showed synergism with minocycline, fusidic acid and rifampicin against most of the strains. Gallic acid ethyl ester showed additivity against all strains in combination with all antibiotics under investigation except with vancomycin where it showed indifference effect. Eugenol, menthone and caffeic acid showed indifference results against all strains in combination with all antibiotics. Interestingly, no antagonism was observed within these interactions. Based on the fractional inhibitory concentration indices, synergistic pairs were further examined by time-kill assays to confirm the accuracy and killing rate of the combinations over time. The two methods concurred with each other with 92% accuracy and the combinatory pairs were effective throughout the 24 hours of assay. The study suggests a possible incorporation of effective phytochemicals in combination therapies for MRSA infections.

Biodegradation of Low Density Polythene (LDPE) by Pseudomonas Species.

Low density polythene (LDPE) is the most widely used packaging material primarily because of its excellent mechanical properties, barrier properties against water, light weight, low cost and high energy effectiveness. However, due to its ubiquitous nature, and resistance to biodegradability, the disposal strategies are crucial and need attention. Recently, microorganisms have become the focus of interest for environmental friendly disposal of plastic and polymer-based waste. This manuscript aims to investigate the extent of biodegradability of LDPE by four different strains of Pseudomonas bacteria-Pseudomonas aeruginosa PAO1 (ATCC 15729), Pseudomonas aeruginosa (ATCC 15692), Pseudomonas putida (KT2440 ATCC 47054) and Pseudomonas syringae (DC3000 ATCC 10862). Degradation of LDPE was determined by weight loss of the sample, morphological changes, mechanical and spectroscopic variations. The eluted compounds after degradation were analysed by gas chromatography coupled with mass spectroscopy. Our results show that Pseudomonas spp. can degrade LDPE films.

Bactericidal antibiotic-phytochemical combinations against methicillin resistant Staphylococcus aureus.

Methicillin resistant Staphylococcus aureus (MRSA) infection is a global concern nowadays. Due to its multi-drug resistant nature, treatment with conventional antibiotics does not assure desired clinical outcomes. Therefore, there is a need to find new compounds and/or alternative methods to get arsenal against the pathogen. Combination therapies using conventional antibiotics and phytochemicals fulfill both requirements. In this study, the efficacy of different phytochemicals in combination with selected antibiotics was tested against 12 strains of S. aureus (ATCC MRSA 43300, ATCC methicillin sensitive S. aureus or MSSA 29213 and 10 MRSA clinical strains collected from National University Hospital, Singapore). Out of the six phytochemicals used, tannic acid was synergistic with fusidic acid, minocycline, cefotaxime and rifampicin against most of strains tested and additive with ofloxacin and vancomycin. Quercetin showed synergism with minocycline, fusidic acid and rifampicin against most of the strains. Gallic acid ethyl ester showed additivity against all strains in combination with all antibiotics under investigation except with vancomycin where it showed indifference effect. Eugenol, menthone and caffeic acid showed indifference results against all strains in combination with all antibiotics. Interestingly, no antagonism was observed within these interactions. Based on the fractional inhibitory concentration indices, synergistic pairs were further examined by time-kill assays to confirm the accuracy and killing rate of the combinations over time. The two methods concurred with each other with 92% accuracy and the combinatory pairs were effective throughout the 24 hours of assay. The study suggests a possible incorporation of effective phytochemicals in combination therapies for MRSA infections.

Maggot metabolites and their combinatory effects with antibiotic on Staphylococcus aureus.

BACKGROUND: Maggot therapy has been in practice for effective debridement, disinfection and healing of chronic wounds. Due to their antiseptic action during wound healing, their metabolites have been investigated in the past for antibacterial activity. They have been particularly useful for treatment of wounds infected with multi-drug resistant Staphylococcus aureus. Antibiotics, on the other hand, can predispose bacteria to develop resistance. Substances that are able to modulate or delay the occurrence of resistance in bacteria are under investigation by many researchers around the world. In the present study, antibacterial activity in excretions/secretions (ES) from maggots of Lucilia cuprina blowfly was demonstrated. The extracts were also screened in combination with antibiotic, ciprofloxacin. METHODS: L. cuprina blowfly maggots were reared for extraction of its metabolites. The ES extracted was screened against S. aureus, alone and in combination with ciprofloxacin, both for short term and long term exposure analysis. A microchannel-based device and system was used for experiments instead of conventional techniques. RESULTS: The original ES had shown partial bacterial growth inhibition. However, in combination with ciprofloxacin, at sub-inhibitory concentrations, certain combinations revealed anti-staphylococcal activity, with bacterial reduction of up to 50%, after 24 hours. The six day study on S. aureus exposed to ES-ciprofloxacin combination suggested a potential delay in development of adaptive resistance as opposed to when ciprofloxacin was used as single agent. CONCLUSIONS: The combination effect of ES and ciprofloxacin at sub-MIC levels showed enhanced antibacterial activity compared to the effect of ES and ciprofloxacin as single agents. Based on the results of ES-ciprofloxacin combinations, a more effective means of treatment for S. aureus can be proposed.

Cloning and targeted disruption of two lipopolysaccharide biosynthesis genes, kdsA and waaG, of Pseudomonas aeruginosa PAO1 by site-directed mutagenesis.

The emergence of antibiotic resistance in bacterial pathogens poses a great challenge to public health and emphasizes the need for new antimicrobial targets. The recent development of microbial genomics and the availability of genome sequences allows for the identification of essential genes which could be novel and potential targets for antibacterial drugs. However, these predicted targets need experimental validation to confirm essentiality. Here, we report on experimental validation of a two potential targets in the lipopolysaccharide (LPS) biosynthesis pathway of the pathogen Pseudomonas aeruginosa PAO1 using insertion duplication. Two genes, kdsA and waaG, from LPS encoding proteins 2-dehydro-3-deoxyphosphooctonate aldolase and UDP-glucose (heptosyl) LPS α-1,3-glucosyltransferase were selected as putative target candidates for the gene disruption experiments using plasmid insertion mutagenesis to determine essentiality. The introduction of a selectable ampicillin and kanamycin resistance marker into the chromosome resulted in lack of recovery of antibiotic-resistant colonies suggesting the essentiality of these genes for the survival of P. aeruginosa. Several molecular analyses were carried out in order to confirm the essentiality of these genes. We propose that the above two validated drug targets are essential and can be screened for functional inhibitors for the discovery of novel therapeutic compounds against antibiotic-resistant opportunistic pathogen P. aeruginosa.

Activity and interactions of antibiotic and phytochemical combinations against Pseudomonas aeruginosa in vitro.

In this study the in vitro activities of seven antibiotics (ciprofloxacin, ceftazidime, tetracycline, trimethoprim, sulfamethoxazole, polymyxin B and piperacillin) and six phytochemicals (protocatechuic acid, gallic acid, ellagic acid, rutin, berberine and myricetin) against five P. aeruginosa isolates, alone and in combination are evaluated. All the phytochemicals under investigation demonstrate potential inhibitory activity against P. aeruginosa. The combinations of sulfamethoxazole plus protocatechuic acid, sulfamethoxazole plus ellagic acid, sulfamethoxazole plus gallic acid and tetracycline plus gallic acid show synergistic mode of interaction. However, the combinations of sulfamethoxazole plus myricetin shows synergism for three strains (PA01, DB5218 and DR3062). The synergistic combinations are further evaluated for their bactericidal activity against P. aeruginosa ATCC strain using time-kill method. Sub-inhibitory dose responses of antibiotics and phytochemicals individually and in combination are presented along with their interaction network to suggest on the mechanism of action and potential targets for the phytochemicals under investigation. The identified synergistic combinations can be of potent therapeutic value against P. aeruginosa infections. These findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (antibiotics and phytochemicals).

Infrared authentication of ginseng species: the use of the 2-6PC rule.

The quality of herbal products is important for ensuring efficacy and consumer safety. Traditional methods of authenticating herbs like ginseng via their morphology are hardly reliable. Different chemical constituents in herbs like ginseng tend to exhibit characteristic IR fingerprints that enable their identification. We previously introduced an IR-based protocol known as the "2-6PC rule" to categorize and identify ginseng and its products, as well as distinguishing it from morphological fakes. Here, we describe the use of this rule as a rapid and effective means of analyzing the IR spectral fingerprints of the biologically active components of ginseng, as well as distinguishing among its species. Our results show that Panax ginseng, P. quinquefolius, and P. notoginseng can be differentiated from each other. Our results also indicate the presence of starch, carbohydrates, calcium oxalate, and ginsenosides Re and Rg1 in commercial ginseng roots sold in Singapore. This work effectively demonstrates the usefulness of the 2-6PC rule as a rapid screening tool in the authentication of ginseng species.

Activity of Chitosans in combination with antibiotics in Pseudomonas aeruginosa.

Chitosan and its derivative water soluble Chitosan oligosaccharide are used in a variety of applications in pharmaceutical preparations. In this study, 2 wild (ATCC 15729 and PAO1) and 2 mutant strains (PT121 and PT149) of P. aeruginosa are investigated for drug-drug interactions in vitro. 10 antimicrobial agents (antibiotics) are combined with different degree of deacetylated Chitosans and Chitosan oligosaccharide. All the chitosans show synergistic activity with sulfamethoxazole, a sulfonamide antimicrobial agent. It is interesting to observe that the MIC value for the MexEF-OprN overexpressing mutant strain of P. aeruginosa is 5 fold higher than the other strains under investigation suggesting a possible role of this efflux pump in Sulfamethoxazole efflux. The findings suggest on the use of chitosans as enhancing agent in combination with antibiotics in pharmaceutical preparations.

A Comparative Study of Metabolic Network Topology between a Pathogenic and a Non-Pathogenic Bacterium for Potential Drug Target Identification.

Metabolic network provides a unified platform to integrate all the biological information on genes, proteins, metabolites, drugs and drug targets for a comprehensive system level study of the relationship between metabolism and disease. In recent times, drug-target identification by in silico methods has emerged causing a phenomenal achievement in the field of drug discovery. This paper focuses on describing how microbial drug target identification can be carried out using bioinformatic tools. Specifically, it highlights the use of metabolic 'choke point' and 'load point' analyses to understand the local and global properties of metabolic networks in Pseudomonas aeruginosa and allow us to identify potential drug targets. We also list out top 10 choke point enzymes based on the load point values and the number of shortest paths. A non-pathogenic bacterial strain Pseudomonas putida KT2440 and a related pathogenic bacteria P.aeruginosa PA01 was selected for the network anlaysis. A comparative study of the metabolic networks of these two microbes highlights the analogies and differences between their respective pathways. System analysis of metabolic networks will help us in identifying new drug targets which in turn will generate more in-depth understanding of the mechanism of diseases and thus provide better guidance for drug discovery.

A combined computational-experimental analyses of selected metabolic enzymes in Pseudomonas species.

Comparative genomic analysis has revolutionized our ability to predict the metabolic subsystems that occur in newly sequenced genomes, and to explore the functional roles of the set of genes within each subsystem. These computational predictions can considerably reduce the volume of experimental studies required to assess basic metabolic properties of multiple bacterial species. However, experimental validations are still required to resolve the apparent inconsistencies in the predictions by multiple resources. Here, we present combined computational-experimental analyses on eight completely sequenced Pseudomonas species. Comparative pathway analyses reveal that several pathways within the Pseudomonas species show high plasticity and versatility. Potential bypasses in 11 metabolic pathways were identified. We further confirmed the presence of the enzyme O-acetyl homoserine (thiol) lyase (EC: 2.5.1.49) in P. syringae pv. tomato that revealed inconsistent annotations in KEGG and in the recently published SYSTOMONAS database. These analyses connect and integrate systematic data generation, computational data interpretation, and experimental validation and represent a synergistic and powerful means for conducting biological research.

Biofluid mechanics of the human reproductive process: modelling of the complex interaction and pathway to the oocytes.

Recent revelations in the human reproductive process have fuelled much interest in this field of study. In particular, the once prevailing view of large numbers of ejaculated sperms racing towards the egg has been refuted recently. This is opposed to the current views derived from numerous clinical findings that state that only a very small number of sperms will ever enter the oviduct. It is believed that these few sperms must have been guided to make the long, tedious and obstructed journey to the egg. For a mature spermatozoon, its hyperactivated swimming motility upon capacitation plays an important role in the fertilization of a mature egg. Likewise, the female genital tract also provides guiding mechanisms to complement the survival of normal hydrodynamic profile sperms and thus promotes an eventual sperm-egg interaction. Understanding these mechanisms can be essential for the derivation of assisted conception techniques especially those in vitro. With the aid of computational models and simulation, suitability and effectiveness of novel assisted conception methodology can be assessed, particularly for those yet to be ready for clinical trials. This review discusses the possible bioengineering models and the mechanisms by which human spermatozoa are guided to the egg.

Study of pulse transit time oscillations during obstructive sleep apnoea by using a distributed model.

The study of arterial compliance is useful in understanding the geometrical and mechanical properties of a systemic arterial tree. Numerous mathematical models have shown their potential in relating the physical phenomena in the arterial tree to properties of the wall itself. However, limited model is available that describes the pulse transit time (PTT) oscillations of a sleeping child during tidal breathing and obstructive sleep apnoea (OSA). Data from 20 children (17 male; aged 6.4 +/- 4.1 yr) whom were recruited for overnight polysomnography (PSG) were used. A modified windkessel model with related physiological parameters was utilised to describe PTT fluctuations due to the cardiovascular system during sleep. Verification with the recorded PSG data showed similar trends with the model for both types of respiratory events. For tidal breathing, undamped PTT oscillations of 3.89 s were predicted by the model while actual data yielded a mean value of 3.72 +/- 0.79 s. Conversely, under-damping PTT responses were expected based on the model for OSA. The model estimated a Q factor of 4.23 and actual mean data were 3.86 +/- 0.64. Hence, the findings herein suggest that the proposed model has the potential to illustrate tidal breathing and OSA events in sleeping children.

Changes induced in the lower- and upper-limb pulse transit-time ratio during inspiratory resistive breathing.

The ankle brachial index (ABI) has been widely used to monitor the pathogenesis of peripheral arterial diseases such as ischemia of the extremities. Owing to the occluding nature of ABI measurement, this may not be appealing to less cooperative patients when multiple prolonged screening is required. Recently, a simple non-occluding technique termed pulse transit-time ratio (PTTR) has shown potential as a surrogate ABI marker. It is also known that abrupt changes in inspiratory efforts can lead to increased blood pressure (BP) and heart rate. Since transit-time measurements can be confounded by these parameters, it is important to understand their effects on PTTR normality. We recruited 12 healthy adults (8 males, aged 27.0+/-3.1 years) to perform three inspiratory activities. Friedman and Wilcoxon statistical results both showed that significant changes in transit-time oscillations were observed for higher inspiratory loads (p<0.05). These results were verified by a corresponding air-pressure difference measurement, for which a similar significant increase was also registered (p<0.05). However, limited changes were observed in the derived PTTR parameter (p>0.05). These findings suggest that, similar to ABI, PTTR is only confounded by abnormal local changes in either of the peripheral BPs measured.

Phonocardiographic signal analysis method using a modified hidden Markov model.

Auscultation is an important diagnostic indicator for cardiovascular analysis. Heart sound classification and analysis play an important role in the auscultative diagnosis. This study uses a combination of Mel-frequency cepstral coefficient (MFCC) and hidden Markov model (HMM) to efficiently extract the features for pre-processed heart sound cycles for the purpose of classification. A system was developed for the interpretation of heart sounds acquired by phonocardiography using pattern recognition. The task of feature extraction was performed using three methods: time-domain feature, short-time Fourier transforms (STFT) and MFCC. The performances of these feature extraction methods were then compared. The results demonstrated that the proposed method using MFCC yielded improved interpretative information. Following the feature extraction, an automatic classification process was performed using HMM. Satisfactory classification results (sensitivity > or =0.952; specificity > or =0.953) were achieved for normal subjects and those with various murmur characteristics. These results were based on 1398 datasets obtained from 41 recruited subjects and downloaded from a public domain. Constituents characteristics of heart sounds were also evaluated using the proposed system. The findings herein suggest that the described system may have the potential to be used to assist doctors for a more objective diagnosis.

Authentication of traditional Chinese medicine using infrared spectroscopy: distinguishing between ginseng and its morphological fakes.

The quality of pharmaceutical products such as ginseng is important for ensuring consumer safety and efficacy. Ginseng is an expensive herb, and adulteration with other cheaper products may occur. Quality assurance of ginseng is needed since many of its commercial products now come in various formulations such as capsules, powder, softgels and tea. Thus traditional means of authentication via smell, taste or physical appearance are hardly reliable. Herbs like ginseng tend to exhibit characteristic infrared fingerprints due to their different chemical constituents. Here we report for the first time a rapid means of distinguishing American and Asian ginsengs from two morphological fakes--sawdust and Platycodon grandiflorum, via pattern differences and principal component analysis of their infrared spectra. Our results show that ginseng can be distinguished from both sawdust and Platycodon grandiflorum, hence there is a potential of using infrared spectroscopy as a novel analytical technique in the authentication of ginseng.

Differential genome analyses of metabolic enzymes in Pseudomonas aeruginosa for drug target identification.

Complete genome sequences of several pathogenic bacteria have been determined, and many more such projects are currently under way. While these data potentially contain all the determinants of host-pathogen interactions and possible drug targets, computational tools for selecting suitable candidates for further experimental analyses are currently limited. Detection of bacterial genes that are non-homologous to human genes, and are essential for the survival of the pathogen represents a promising means of identifying novel drug targets. We used a differential pathway analyses approach (based on KEGG data) to identify essential genes from Pseudomonas aeruginosa. Our approach identified 214 unique enzymes in P. aeruginosa that may be potential drug targets and can be considered for rational drug design. About 40% of these putative targets have been reported as essential by transposon mutagenesis data elsewhere. Homology model for one of the proteins (LpxC) is presented as a case study and can be explored for in silico docking with suitable inhibitors. This approach is a step towards facilitating the search for new antibiotics.

Evaluation of blood pressure changes using vascular transit time.

Imbalance of the human haemodynamic system can provide a prognosis of syncope, dizziness or hypertension. This can be assessed by monitoring its responses to postural change. Examining variations in blood pressure (BP) is deemed an effective means to identify symptoms of this associated condition. However, conventional methods do not promote prolonged monitoring due to the discomfort caused to patients. Established correlations between BP and pulse wave transmission have shown its usefulness in clinical applications. In this study, photoplethysmography and phonocardiography were used to estimate BP changes via observed variations in delay transmission or vascular transit time (VTT) at the upper limb. Thirty-one healthy adults (21 male) were recruited to perform three test activities, namely the arm held at heart level, fully raised up and held down. Association of the three BP indices and heart rate variations with transit time changes was then computed. The results showed that observed VTT changes were related to systolic BP (R(2) = 0.820; p < 0.05), diastolic BP (R(2) = 0.517; p < 0.05), mean arterial pressure (R(2) = 0.673; p < 0.05) and heart rate (R(2) = 0.000; p > 0.05). As systolic BP had the strongest correlation, a regression equation was formulated to associate the two parameters. The non-invasive measuring nature of VTT can be more accommodating to patients, especially during continual monitoring. Moreover, it has the added advantage that the pre-ejection period is not included in its time-related derivations.

Dual-channel photoplethysmography to monitor local changes in vascular stiffness.

OBJECTIVES: Monitoring arterial distensibility changes is important to understand the cardiovascular status of patient. Pulse transit time (PTT), which is an inverse equivalent of pulse wave velocity (PWV), has shown its potential in such studies. However, its methodological approach in using an electrocardiogram and a peripheral photoplethysmography (PPG) is limited due to the inclusion of pre-ejection period (PEP) in its computation. Previous studies have suggested the using the transit time difference between two peripheral measuring sites (PTT-D) instead. However, it requires two medical instruments and may not be efficient in terms of equipment utility, especially in prolonged clinical studies. METHODS: Postural changes are known to cause complex haemodynamics adaptation and thereby affecting transit time measurements. A customised dual-channel PPG system based on discrete electronic devices was constructed to evaluate against conventional peripheral-based PTT. 10 healthy adults (7 male; mean age 27.0 yr) were recruited to assess the differences observed in PTT and PTT-D during two postural change test activities. RESULTS: PTT-D derived from the customised PPG system registered 43.3+/- 5.6 ms and - 31.1+/- 3.8 ms relative changes for the two regulated activities while conventional PTT recorded 43.6+/- 10.3 ms and -31.0+/-m 6.5 ms respectively. The former may have similar results but have significantly lower variance (< 0.05). CONCLUSIONS: Findings herein suggest that PTT-D derived from the customised PPG system shows potential. It can be used as an alternative to conventional peripheral-based PTT and possibly as a direct assessment of arterial distensibility or PWV variations as it does not include PEP in its time-related computations.

Pulse transit time based on piezoelectric technique at the radial artery.

OBJECTIVES: Pulse transit time (PTT) has shown its potential in relevant cardiovascular and cardiorespiratory studies. However, the use of photoplethysmography (PPG) in PTT measurement can be limited in events of poor peripheral perfusion. Uninterrupted PTT monitoring may also not be achievable when less cooperative patients distribute the PPG probe due to its prominent light source. Hence, there is a need for an alternative method to measure PTT in such incidents. METHODS: In this study, the piezoelectric (PIEZO) technique to detect pulsations from a human wrist above the radial artery to estimate PTT is presented. 17 healthy adults (11 male; age range of 21-33 years) were recruited to compare PTT and heart rate (HR) differences between the PPG and PIEZO methods. These time-related derivations were made with respect to an electrocardiogram (ECG). RESULTS: The timing consistency of the PIEZO transducer shows significant correlations (p < 0.01) to those derived from the ECG and a pulse oximeter. Particularly, there is a high level of agreement of < 1 beat per minute (bpm) difference in HR estimates observed when compared to the two commercial devices in the respective Bland-Altman plots. Comparison of PTT obtained from the PIEZO transducer against the PPG signal shows constantly lower values due to the shorter path length it requires to propagate. A regression equation was formulated to relate the PTT values acquired from both these signals. CONCLUSIONS: Preliminary findings herein suggest that the PIEZO technique can be useful as an alternative for PTT monitoring. This shows promise to be more accommodating for less cooperative patients or those with insufficient peripheral perfusion.