Kirnpal Kaur Banga Singh

Antibacterial properties of tualang honey and its effect in burn wound management: a comparative study

BackgroundThe use of honey as a natural product of Apis spp. for burn treatment has been widely applied for centuries. Tualang honey has been reported to have antibacterial properties against various microorganisms, including those from burn-related diagnoses, and is cheaper and easier to be absorbed by Aquacel dressing. The aim of this study is to evaluate the potential antibacterial properties of tualang honey dressing and to determine its effectiveness as a partial thickness burn wound dressing.MethodsIn order to quantitate the bioburden of the swabs, pour plates were performed to obtain the colony count (CFU/ml). Swabs obtained from burn wounds were streaked on blood agar and MacConkey agar for bacterial isolation and identification. Later, antibacterial activity of Aquacel-tualang honey, Aquacel-Manuka honey, Aquacel-Ag and Aquacel- plain dressings against bacteria isolated from patients were tested (in-vitro) to see the effectiveness of those dressings by zone of inhibition assays.ResultsSeven organisms were isolated. Four types of Gram-negative bacteria, namely Enterobacter cloacae, Klebsiella pneumoniae, Pseudomonas spp. and Acinetobacter spp., and three Gram-positive bacteria, namely Staphylococcus aureus, coagulase-negative Staphylococcus aureus (CONS) and Streptococcus spp., were isolated. Total bacterial count decreased on day 6 and onwards. In the in-vitro antibacterial study, Aquacel-Ag and Aquacel-Manuka honey dressings gave better zone of inhibition for Gram positive bacteria compared to Aquacel-Tualang honey dressing. However, comparable results were obtained against Gram negative bacteria tested with Aquacel-Manuka honey and Aquacel-Tualang honey dressing.ConclusionsTualang honey has a bactericidal as well as bacteriostatic effect. It is useful as a dressing, as it is easier to apply and is less sticky compared to Manuka honey. However, for Gram positive bacteria, tualang honey is not as effective as usual care products such as silver-based dressing or medical grade honey dressing.

A pentaplex PCR assay for the rapid detection of methicillin-resistant Staphylococcus aureus and Panton-Valentine Leucocidin

BackgroundStaphylococcus aureus is a major human pathogen, especially methicillin-resistant S. aureus (MRSA), which causes a wide range of hospital and community-acquired infections worldwide. Conventional testing for detection of MRSA takes 2–5 days to yield complete information of the organism and its antibiotic sensitivity pattern.ResultsThe present study focused on the development of a pentaplex PCR assay for the rapid detection of MRSA. The assay simultaneously detected five genes, namely 16S rRNA of the Staphylococcus genus, fem A of S. aureus, mec A that encodes methicillin resistance, luk S that encodes production of Panton-Valentine leukocidin (PVL), a necrotizing cytotoxin, and one internal control. Specific primer pairs were successfully designed and simultaneously amplified the targeted genes. The analytical sensitivity and specificity of the pentaplex PCR assay was evaluated by comparing it with the conventional method. The analytical sensitivity of the pentaplex PCR at the DNA level was found to be 10 ng DNA. The analytical specificity was evaluated with 34 reference staphylococci and non-staphylococcal strains and was found to be 100%. The diagnostic evaluation of MRSA carried out using 230 clinical isolates, showed 97.6% of sensitivity, 99.3% of specificity, 98.8% of positive predictive value and 98.6% of negative predictive value compared to the conventional method. The presence of an internal control in the pentaplex PCR assay is important to exclude false-negative cases.ConclusionThe pentaplex PCR assay developed was rapid and gave results within 4 h, which is essential for the identification of Staphylococcus spp., virulence and their resistance to methicillin. Our PCR assay may be used as an effective surveillance tool to survey the prevalence of MRSA and PVL-producing strains in hospitals and the community.

Wound contraction effects and antibacterial properties of Tualang honey on full-thickness burn wounds in rats in comparison to hydrofibre

BackgroundFull-thickness burn wounds require excision and skin grafting. Multiple surgical procedures are inevitable in managing moderate to severe full-thickness burns. Wound bed preparations prior to surgery are necessary in order to prevent wound infection and promote wound healing. Honey can be used to treat burn wounds. However, not all the honey is the same. This study aims to evaluate the wound contraction and antibacterial properties of locally-produced Tualang honey on managing full-thickness burn wounds in vivo.MethodsThirty-six female Sprague Dawley rats were randomly divided into three groups. Under anaesthesia, three full-thickness burn wounds were created on the dorsum of the rats. The full-thickness burn wounds were inoculated with a specific organism (104), namely Pseudomonas aeruginosa (n = 12), Klebsiella pneumoniae (n = 12), or Acinetobacter baumannii (n = 12). The three burn wounds were dressed with Tualang honey, hydrofibre and hydrofibre silver respectively. Swab samples were obtained every 3 days (day 3, 6, 9, 12, 15, 18 and 21) for quantitative and semi-quantitative microbiological analyses. Clinical assessments, including observations concerning the appearance and wound size, were measured at the same time.ResultsThere was a rapid 32.26% reduction in wound size by day 6 (p = 0.008) in the Tualang honey-treated wounds, and 49.27% by day 15 (p = 0.005). The wounds remained smaller by day 18 (p < 0.032). Tualang honey-treated rats demonstrated a reduction in bacterial growth in Pseudomonas aeruginosa inoculated wounds (p = 0.005). However, hydrofibre silver and hydrofibre-treated wounds are superior to honey-treated wounds with Acinetobacter baumannii (p = 0.035). There was no statistical significant of antibacterial property in Klebsiella pneumonia inoculated wounds.ConclusionsTualang honey has better results with regards to its control of Pseudomonas aeruginosa and its wound contraction effects on full-thickness burn wound in vivo.

A Pentaplex PCR Assay for the Detection and Differentiation of Shigella Species

The magnitude of shigellosis in developing countries is largely unknown because an affordable detection method is not available. Current laboratory diagnosis of Shigella spp. is laborious and time consuming and has low sensitivity. Hence, in the present study, a molecular-based diagnostic assay which amplifies simultaneously four specific genes to identify invC for Shigella genus, rfc for S. flexneri, wbgZ for S. sonnei, and rfpB for S. dysenteriae, as well as one internal control (ompA) gene, was developed in a single reaction to detect and differentiate Shigella spp. Validation with 120 Shigella strains and 37 non-Shigella strains yielded 100% specificity. The sensitivity of the PCR was 100 pg of genomic DNA, 5.4 × 104 CFU/ml, or approximately 120 CFU per reaction mixture of bacteria. The sensitivity of the pentaplex PCR assay was further improved following preincubation of the stool samples in Gram-negative broth. A preliminary study with 30 diarrhoeal specimens resulted in no cross-reaction with other non-Shigella strains tested. We conclude that the developed pentaplex PCR assay is robust and can provide information about the four target genes that are essential for the identification of the Shigella genus and the three Shigella species responsible for the majority of shigellosis cases.

A 9-year study of shigellosis in Northeast Malaysia: Antimicrobial susceptibility and shifting species dominance

AimsIn Malaysia, Shigella spp. is the third most common bacterial agent responsible for childhood diarrhoea. This study was conducted to determine the prevalence and antimicrobial susceptibility patterns of Shigella spp. isolated from patients admitted to the Hospital Universiti Sains Malaysia from January 2001 to December 2009.Subjects and methodsA hospital-based retrospective study was used. Stool samples from patients were cultured using a standard culture method. Shigella spp. isolates were identified by biochemical and serological methods, and the antimicrobial susceptibility pattern was evaluated using the Kirby-Bauer disc-diffusion method.ResultsA total of 138 Shigella spp. were isolated from a total of 14,830 routine stool specimens, yielding an isolation rate of 0.93% that corresponded to 9.99% of the 1,381 bacterial pathogens isolated. Of these isolates, S. sonnei was the predominant species, followed by S. flexneri and S. boydii. Seasonal variation was noticed, and no significant differences were detected in the demographic data for S. flexneri and S. sonnei. The susceptibility of all isolated Shigella strains was tested against seven antibiotics. Ceftriaxone (99.1%), ciprofloxacin (98.4%), and nalidixic acid (93.8%) were effective against the Shigella strains, whereas tetracycline and trimethoprim-sulfamethoxazole exhibited high frequencies of resistance (58.4% and 53.8%, respectively).ConclusionThis study is important for public health education aimed at reducing the morbidity and mortality associated with Shigella spp. infection. Our results also will be helpful for paediatricians and microbiologists in the selection of appropriate antibiotics for the management of diarrhoea.

Evaluations of bacterial contaminated full thickness burn wound healing in Sprague Dawley rats Treated with Tualang honey

Aim: The effect of Tualang honey on wound healing in bacterial contaminated full-thickness burn wounds was evaluated in 36 male Sprague Dawley rats. Materials and Methods: The rats were randomly divided into three groups (n = 12/group). Three full-thickness burn wounds were created on each rat. Each group of rats was inoculated with a different organism in the burn wounds: Group A was inoculated with Pseudomonas aeruginosa, Group B was inoculated with Klebsiella pneumoniae and Group C was inoculated with Acinetobacter baumannii. One wound on each rat was dressed with either Tualang honey, Chitosan gel or Hydrofibre silver. Each wound size was measured on day 3, 6, 9, 12, 15, 18 and 21 of the study. Results: The mean wound size of the Tualang honey-treated wounds was not statistically different than that of the Chitosan gel or Hydrofibre silver-treated wounds when the wounds were compared throughout the entire experiment (P > 0.05). However, comparing the mean wound size on day 21 alone revealed that the Tualang honey-treated wounds were smaller in comparison to that of the Chitosan gel and Hydrofibre silver-treated groups. Conclusions: This study shows that topical application of Tualang honey on burn wounds contaminated with P. aeruginosa and A. baumannii gave the fastest rate of healing compared with other treatments.

Enzymatic electrochemical detection of epidemic-causing Vibrio cholerae with a disposable oligonucleotide-modified screen-printed bisensor coupled to a dry-reagent-based nucleic acid amplification assay.

In this study, we developed a nucleic acid-sensing platform in which a simple, dry-reagent-based nucleic acid amplification assay is combined with a portable multiplex electrochemical genosensor. Preparation of an amplification reaction mix targeting multiple DNA regions of interest is greatly simplified because the lyophilized reagents need only be reconstituted with ultrapure water before the DNA sample is added. The presence of single or multiple target DNAs causes the corresponding single-stranded DNA (ssDNA) amplicons to be generated and tagged with a fluorescein label. The fluorescein-labeled ssDNA amplicons are then analyzed using capture probe-modified screen-printed gold electrode bisensors. Enzymatic amplification of the hybridization event is achieved through the catalytic production of electroactive α-naphthol by anti-fluorescein-conjugated alkaline phosphatase. The applicability of this platform as a diagnostic tool is demonstrated with the detection of toxigenic Vibrio cholerae serogroups O1 and O139, which are associated with cholera epidemics and pandemics. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 168 spiked stool samples. The limit of detection was low (10 colony-forming units/ml) for both toxigenic V. cholerae serogroups. A heat stability assay revealed that the dry-reagent amplification reaction mix was stable at temperatures of 4-56 °C, with an estimated shelf life of seven months. The findings of this study highlight the potential of combining a dry-reagent-based nucleic acid amplification assay with an electrochemical genosensor in a more convenient, sensitive, and sequence-specific detection strategy for multiple target nucleic acids.

Development of a dry-reagent-based nucleic acid-sensing platform by coupling thermostabilised LATE-PCR assay to an oligonucleotide-modified lateral flow biosensor

In this study, we report for the first time the development of a dry-reagent-based nucleic acid-sensing platform by combining a thermostabilised linear-after-the-exponential (LATE)-PCR assay with a one-step, hybridisation-based nucleic acid lateral flow biosensor. The nucleic acid-sensing platform was designed to overcome the need for stringent temperature control during transportation or storage of reagents and reduces the dependency on skilled personnel by decreasing the overall assay complexity and hands-on time. The platform was developed using toxigenic Vibrio cholerae as the model organism due to the bacteriums propensity to cause epidemic and pandemic cholera. The biosensor generates result which can be visualised with the naked eyes and the limit of detection was found to be 1pg of pure genomic DNA and 10CFU/ml of toxigenic V. cholerae. The dry-reagent-based nucleic acid-sensing platform was challenged with 95 toxigenic V. cholerae, 7 non-toxigenic V. cholerae and 66 other bacterial strains in spiked stool sample and complete agreement was observed when the results were compared to that of monosialoganglioside (GM1)-ELISA. Heat-stability of the thermostabilised LATE-PCR reaction mixes at different storage temperatures (4-56°C) was investigated for up to 90days. The dry-reagent-based genosensing platform with ready-to-use assay components provides an alternative method for sequence-specific detection of nucleic acid without any cold chain restriction that is associated with conventional molecular amplification techniques.

Serum neopterin is elevated in patients infected with Shigella

BackgroundNeopterin is produced by human macrophages/monocytes when stimulated with interferon-gamma. Production of neopterin is found in serum, cerebrospinal fluid (CSF) and urine of patients with infections by viruses, intracellular bacteria and parasites, autoimmune diseases, malignant tumors and patients in allograft rejection episodes.MethodsIn this study, the level of neopterin was determined in serum samples obtained from patients infected with Shigella (all four species) and healthy individuals. The study population comprised of 14 patients infected with Shigella and 14 normal controls. Serum neopterin was measured using an enzyme-linked immunosorbent assay (ELISA).ResultsThe mean of serum neopterin concentration was 36.32 ± 9.71 nmol/L among patients infected with Shigella and 2.88 ± 0.77 nmol/L in the control group. The mean serum neopterin levels were significantly higher in the test group as compared to the normal group (p = 0.002).ConclusionThis study revealed that neopterin was elevated in patients infected with Shigella.

An impedimetric micro-immunosensing assay to detect Alzheimer's disease biomarker: Aβ40

A miniaturized biosensing platform, based on monoclonal amyloid-beta antibodies (mAβab) that were immobilized on a disc-shaped platinum/iridium (Pt/Ir) microelectrode surface coupled with an impedimetric signal transducer, was developed for the label-free and sensitive detection of amyloid-beta peptide fragment 1-40 (Aβ40); a reliable biomarker for early diagnosis of Alzheimers disease (AD). A Pt/Ir microelectrode was electropolymerized with poly (ortho-phenylenediamine), a conducting free amine-containing aromatic polymer; followed by crosslinking with glutaraldehyde for subsequent coupling of mAβab on the microelectrode surface. This modification strategy efficiently improved the impedimetric detection performance of Aβ40 in terms of charge transfer resistance (∼400-fold difference) and normalized impedance magnitude percentage change (∼40% increase) compared with a passive adsorption-based immobilization method. The sensitivity of the micro-immunosensing assay was found to be 1056 kΩ/(pg/mL)/cm2 and the limit of detection was found to be 4.81 pg/mL with a dynamic range of 1-104 pg/mL (R2 = 0.9932). The overall precision of the assay, as measured by relative standard deviation, ranged from 0.84 to 5.15%, demonstrating its reliability and accuracy; while in respect to assay durability and stability, the immobilized mAβab were able to maintain 80% of their binding activity to Aβ40 after incubation for 48 h at ambient temperature (25 °C). To validate the practical applicability, the assay was tested using brain tissue lysates prepared from AD-induced rats. Results indicate that the proposed impedimetric micro-immunosensing platform is highly versatile and adaptable for the quantitative detection of other disease-related biomarkers.