Clinical microbial laboratory investigation

Microorganisms universally attach to surfaces and produce extracellular polysaccharides, resulting in the formation of a biofilm. Biofilms pose a serious problem for public health because of the increased resistance of biofilm-associated organisms to antimicrobial agents and the potential for these organisms to cause infections in patients with indwelling medical devices. The roles of biofilms in infection, including the bloodstream and urinary tract infections, will be presented in this talk. The various collection methods for sampling and transporting biofilm-associated organisms will also be shared in the presentation. In summary, this talk provides a clear understanding on the roles and physiological aspects of biofilms that would enhance the clinical decision-making process

Critical limits for the control points for halal poultry slaughter

This study proposes critical limits (CL) for control points for halal slaughter (CPHS). Previously, 6 control points (CP) were determined, and CL for these 6 CPHS are suggested based on: 1) a literature survey for the CL for CP 1 (poultry breeding, rearing, and poultry feed) and CP 2 (welfare of poultry during transportation and lairage); 2) a field survey of slaughter plants in Kuantan (Malaysia) for CP 3 (immobilization), CP 4 (slaughter), CP 5 (time for full bleed-out), and CP 6 (washing and packaging); and 3) controlled experiments to refine the CL for CP 3, 4, and 5. The CL for CP 1 focused on stress reduction during rearing and use of substances that could compromise poultry meat wholesomeness. The CL for CP 2 emphasizes humane best-practices for handling poultry during lairage. The CL for CP 3 suggests a gap of 5 s between 2 shackles if only one shackler is employed and shackling times of <1 min for live chickens. In countries permitting water-bath electrical stunning of halal poultry, the stunning current needed to induce unconsciousness must be defined for the breed and bird size but not cause any chicken deaths. The CL for CP 4 mandates the recitation of the tasmiyah (the invocation), which if done for every chicken, will require ≥5 s between stunning and neck cutting. The CL for CP 4 also includes information about the slaughter knife. In CP 5 the recommended minimum time between neck cutting and scalding is 9.5 min. Finally, the CL for CP 6 emphasizes good supply chain hygiene and zero adulteration from haram species and substances

Critical control points and critical limits for industrial scale Halal poultry meat production

Government and non-government organizations in different countries are involved in certification of the halal (Arabic: lawful) status of products. Criteria to label a product as halal may vary depending on the predominant schools of thought of a country or nation. For instance, while water bath stunning before slaughtering is considered permissible in some countries for halal poultry meat production, others reject pre-slaughtering stunning. At the same time, to meet high demand of halal meat market, halal poultry meat production required adoption of automation and mechanical devices at different steps during slaughtering. The halal status of the food depends on the fundamental guidelines derived from the Qur’an and Hadith (the saying and living examples of Prophet Muhammad, peace be upon him). However, variations in the interpretation of these guidelines exist, making it difficult to have one universal halal standard which is acceptable for all Muslims all over the world. Despite the variations, it is possible to outline a systematic approach highlighting the critical points that need to be monitored and controlled for halal poultry meat production. We have proposed Halal Critical Control Point (HCCP) to ensure systematic production and evaluation of the halal poultry meat, from farm to table. Critical control points (CCP) are defined as the events or steps during the processing, compromise of which may render the end product as haram or shubhah. Based on the Qur’an, the Hadith, field survey and interviews with several officers of the religious councils, seven CCP were proposed. Critical limits (CL) are proposed for each CCP, either based on the field survey, experiments or existing empirical evidence. The CL for CCP1 (rearing) focus on HCCP compliant poultry feed ingredients and the genetic makeup of the chickens to be slaughtered. The CL for CCP2 (lairage) emphasises on minimum stress after transportation and before slaughtering process begins. The CL for CCP3 (immobilization) includes a gap of at least 5 sec to hang chickens onto the shackles and shackling time of less than a minute for every chicken before they are slaughtered. In countries which permit the use of water bath electrical stunning in poultry processing for halal meat production, the current to be applied for stunning must be defined according to the bodyweight and breed of the chickens and it must be shown that it does not cause the death of any of the chickens. As a part of the CL for CCP4 (tasmiyah and neck cutting), invocation of the name of Allah must be pronounced for every chicken or for each group of chickens being slaughtered. In CCP4 also, the recommended minimum time between neck cutting and scalding is 9 min. For CCP5 (scalding), counter-current water flow system must be used for scalding and the CL for the temperature is 50 to 60C. The CL for CCP6 (evisceration) is suggested as the rate of flowing water for washing eviscerated carcasses. Finally, the CL for CCP7 (deboning and packaging) emphasises zero adulteration of meat from other species, as well as maintenance of hygiene for the end products. As a conclusive remark, authors wish to declare that the proposed CCP and respective CL are not to be used as the global standard rather can be used as an example to derive required CCP and CL for a respective nation according to their Sharia law. Acknowledgement:This research was supported by International Islamic University Malaysia grant EDW B10-0392

Construction of Streptococcus pyogenes mutants by allele replacement mutagenesis

S. pyogenes secrete a large array of molecules that might contribute to resistance against antimicrobial peptides (AMPs). Some of these are anchored in the cell-wall by enzymes called sortases and others are released from the cell after secretion. One of the released proteins that have previously been reported to inhibit bactericidal effect of the AMPs are streptococcal inhibitor of complement (SIC). SIC has been recognised as a substantial virulence factor in the M1 GAS strain AP1, because Δsic mutant failed to colonise mouse throat (Lukomski et al., 2000). In fact, the colonisation of the SIC-negative strain was significantly impaired during the first four days of post-inoculation, showing that SIC is a crucial virulence factor during the early stages of infection by this strain (Lukomski et al., 2000). Hoe and colleagues then showed that GAS Δsic mutant was easily internalised and killed more effectively by human epithelial cells than the wild-type strain (Hoe et al., 2002). Previous studies used SIC-defective mutants which were constructed by insertion of transposon. The disadvantage of such mutagenesis is that the GAS mutants may have retained an intact copy of the sic gene, which could express some SIC protein. Therefore, the specific objective of the project was to construct a sic deletion mutant of GAS strain SF370 by allele-replacement mutagenesis. The constructed mutant would have a genetically clearly-defined deletion mutant, that completely removed sic gene from the S. pyogenes strain SF370 chromosome, without leaving any foreign sequences (such as an inserted plasmid) behind

The antimicrobial effect of virgin coconut oil on oral pathogen

Introduction: As time progressed, our diet changed to include the types of foods that will promote decay and so dental caries became a widespread and serious concern. Dental caries had been reported adding to the significantly high number of oral health problem worldwide, causing a burden to health care system. Despite the existing of prevention courses, the numbers did not appear to decrease. Originated from natural resources, virgin coconut oil (VCO) has been proposed as an alternative treatment as it has been reported to demonstrate antimicrobial activity on various bacteria. Hence, the present study aimed to investigate whether VCO has antimicrobial effects on certain, selected cariogenic bacteria. Materials and Methods: Crude extract of VCO was prepared by using a natural method of fermentation. The composition of VCO were screened using Gas Chromatography Mass Spectrophotometry (GCMS). The antimicrobial activities of VCO were evaluated against three selected oral pathogens; Streptococcus mutans, Lactobacillus casei and Candida albicans using disc diffusion method. The inhibitory activities of VCO were determined by measuring the diameter of inhibition zone. The antimicrobial activities were further tested using broth micro-dilution technique to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results: The inhibition zone was observed on Candida albicans plate, however no inhibitions were observed on Streptococcus mutans and Lactobacillus casei plates. The MIC and MBC results were inconsistent in 3 duplication that have been done for all the bacteria. Conclusion(s): VCO demonstrated potential antimicrobial activity against Candida albicans but not on Streptococcus mutans and Lactbacillus casei

Finding cures for the emerging yaws: analysis of Kitab Tib Muzium Terengganu and MSS1292

Yaws is a neglected tropical disease infecting underprivileged population living in tropical and subtropical regions. The disease has persisted in low-developed countries and now emerges in Europe due to increase in travel and economic factors. The threat of yaws is demonstrated by the emergence of antimicrobial resistance strain necessitating the pursuit of new cures. In this study, we used Malay medical manuscripts Kitab Tib Muzium Terengganu and MSS1292 to identify possible treatment for yaws. Malay terms synonymous with puru, are listed and categorised in terms of routes of administration and methods of preparation for the remedies. Comparative analysis using modern scientific database Pubmed was performed to find support for the use of the Malay materia medica. The study identified 17 formulations from the manuscripts for three types of yaws. The most common materials mentioned in the Malay medical manuscripts are terusi (copper sulphate), tempurung (coconut shell) and kunyit (Curcuma longa); belerang/belerang bang (arsenic sulphide), beras (rice), lada/lada sulah (pepper) and tuba tikus (arsenous acid). Comparative analysis found that there were no reports on the use of these materials as yaws treatment which open a huge potential for medical research using manuscript data for the treatment of yaws

Ancient remedies for coughs and cough-related symptoms: analysis from Malay medical manuscripts

Cough, a common symptom with various underlying causes, may lead to debilitating consequences in terms of loss of employment, healthcare utilisation and psychosocial morbidity. This study aims to document information on coughs and cough-related symptoms from six Malay medical manuscripts. The information is related to terminologies, symptoms, list of materials, method of preparation, method of administration and dosage. The materials were then compared with contemporary studies on their relevant therapeutic action. It was found that batuk, lelah and esak are the most common terms related to coughs and cough-related problems in the Malay manuscripts. Coughs can then be categorised into five types: wet cough, dry cough, cold-induced cough, asthmatic cough and physiological coughs. Asthmatic cough has the greatest number of prescriptions (n=30) than other types of coughs. In total, 62 formulations have been documented involving about 93 plant species, 6 animals and 2 minerals. Fenugreek or Trigonella foenum-graecum is the common material prescribed for all five types of coughs. Honey, gandarukam and mustaki are also commonly prescribed for all coughs except dry cough. Future study may investigate the effectiveness of these traditional formulations and their therapeutic benefits to treat coughs and cough-related problems. With proper recommendation, practice and guidelines from health practitioners, health authorities should consider the availability of local materia medica in treating coughs as an advantage to improve the quality of life for the community at large

PCR-based construction and transformation of CodY gene deletion construct in streptococcus pneumoniae

Introduction: Streptococcus pneumoniae (S. pneumoniae) is a gram-positive diplococci belonging to the genus Streptococcus and it is a well-studied pathogenic bacterium. Pneumococcal diseases such as otitis media, pneumonia, sepsis and meningitis caused by pathogenic strains of S. pneumoniae still brought significant mortality and morbidity worldwide. The pathogenicity of S. pneumoniae is exerted by various virulence factors and one of it is the enzyme hyaluronate lyase. Hyaluronate lyase plays a major role in the invasive capability of S. pneumoniae. Its mechanism of action and crystallographic structure have been determinedbut its regulatory mechanism is still poorly understood. Drawing connections between the nutritional behaviour and invasive property of S. pneumoniae, CodY regulator is hypothesized as a potential hyaluronate lyase regulator. This work was aimed to construct CodY deficient mutant of S. pneumoniae to form foundational work for the study of CodY regulatory effect on hyaluronate lyase. Materials and method: A single gene-deletion method was chosen to create CodY mutant construct containing aphA-3 gene (encoding kanamycin resistance protein) replacement cassette by employing recombinant fusion PCR method. A single band DNA product at the expected size of 2,563 bp after recombinant fusion PCR was visualized on agarose gel electrophoresis. This indicated that the linear recombinant amplicon has been successfully constructed. The recombinant amplicon was transformed into S. pneumoniae cells of Malaysian local clinical isolate by employing the competence stimulating peptide (CSP-1) to enhance and induce natural competence. Results: No viable CodY mutant was evident post-transformation. The negative transformation result was postulated to be due to the essential nature of CodY regulator. Conclusion: This work can provide basis for recombinant fusion PCR method in designing and constructing single gene deletion construct. However, further work is needed to identify the regulatory agent of hyaluronate lyas

Different slaughtering techniques and possible physiological and biomolecular effects

The aim of the study was to identify possible physiological and biomolecular changes during slaughtering. For slaughtering, before the neck cutting, chickens are immobilized manually or immobilized using shackles. Neck cutting is generally performed using automated knife, often results in decapitation. Both of these immobilization and neck cutting conditions are expected to influence muscle contraction and blood loss. We have investigated the activity and transcription of acetylcholinesterase (AChE) which terminates cholinergic synaptic transmission by hydrolysing the neurotransmitter acetylcholine that is responsible for muscle contraction and relaxation. We have also analysed the residual haemoglobin content of the skeletal muscle as indicator of blood loss. Skeletal muscle sample was collected from the chickens that were slaughtered either by decapitation (C) or by severance of the jugular veins, carotid arteries, oesophagus and trachea only (P); whilst immediately after slaughtering, chickens were either released (R) or manually constrained (T). Differences in the conditions of slaughtering: CR, PR or PT did not affect blood loss as measured by the residual Hb content and the amount of Zn and Fe either in muscle or liver, deducing no significant difference (p>0.05) in blood loss due to different type of slaughtering. No significant differences (p>0.05) were observed in AChE activity in muscles taken from all slaughter groups. However, AChE transcripts were detected in muscles from chickens from PT and CR groups which might be due to the decapitation and/or constrained muscular activity after neck cutting. Hence the results of the current study indicate that, constraining during slaughtering and decapitation might induce stress to the animals

Antibiotic susceptibility study of metal-gentamicin complexes against Pseudomonas aeruginosa biofilms

Introduction: Inherent resistance of biofilm bacteria to conventional antibiotics is alarming because biofilms induce antibiotic resistance to an order of three or more in magnitude greater than those displayed by planktonic bacteria. Pseudomonas aeruginosa is an infectious organism that causes the hallmark of chronic infections including hospital acquired infections that leads to high morbidity and mortality. One of the reasons for the occurrence of resistance is its ability to form biofilms. In this study, the resistance of P. aeruginosa biofilms against a series of metal-antibiotics, an alternative to the conventional antibiotics, was investigated. Methods: A series of metal-antibiotic complexes derived from gentamicin was synthesized to give metal-gentamicin complexes. The metal contents of all the compounds were determined using Atomic Absorption Spectroscopy (AAS). Antibiotic susceptibility testing of the gentamicin-antibiotic complexes against the biofilms was conducted using broth microdilution assay. Results: The results showed that P. aeruginosa is susceptible against all the metal-gentamicin complexes, which include, Ni(II), Fe(II), Cu(II), Zn(II) and Co(II) complexes; all were tested at 0.25 to 1 mmol concentrations. Conclusions: The results show that all metal-gentamicin complexes have higher antimicrobial activity than gentamicin (0.25 to 1 mmol) on its own. Finally, mechanisms of P. aeruginosa biofilms resistance to these metal-antibiotics are also proposed