There are several rapid microbiological methods becoming available that have useful applications in pharmaceutical and medical devices. They are ATP bioluminescence, fluorescent labeling, electrical resistance, and nucleic acid probes. In choosing to employ rapid methods, the microbiologist should examine their prospective performances against the specific requirements for that sector. Some methods may require expensive equipment and offer full automation, and others represent only a small investment. The regulatory view of these methods is changing and they still officially have not been approved in medical and pharmaceutical area, but it will still be up to the microbiologist to demonstrate that the method chosen is fit for the purpose intended.
Water for injection (WFI) and purified water are the most widely used and stringently regulated raw material in pharmaceutical manufacturing. WFI is utilized for a parenteral drug product. Water system is monitored at frequent and routine frequency for demonstrating the overall system control and stability of performance. The critical ports demonstrating systemic control should be monitored more frequently. For reducing the overall risk of microbial contamination or microbial build-up, it is important to develop appropriate alert and action levels. The assignment of alert and action levels should be performance-based, derived from the historic data and well below water specifications. These levels and overall excursion rates should be assessed annually. An action level should not be established at a level equivalent to the specification. Consecutive or multiple alert level excursions and each action level excursion should be comprehensively investigated with appropriate corrective and preventive action. It is important to analyze the efficacy of the corrective and preventive action to reduce the overall excursion rates.
Prediction of competitive microbial growth is becoming important for microbial food safety. There would be two approaches to predict competitive microbial growth with mathematical models. The first approach is the development of a growth model for competitive microbes. Among several candidates for the competition model considered, the combination of the primary growth model of the new logistic (NL) model and the competition model of the Lotka-Vorttera (LV) model showed the best performance in predicting microbial competitive growth in the mixed culture of two species. This system further successfully predicted the growth of three competitive species in mixed culture. The second approach is the application of the secondary model especially for the parameter of the maximum cell population in the primary growth model. The combination of the NL model and a polynomial model for the maximum population successfully predicted Salmonella growth in raw ground beef. This system further successfully predicted Salmonella growth in beef at various initial concentrations and temperatures. The first approach requires microbial growth data in monoculture for analysis. The second approach to the prediction of competitive growth from the viewpoint of microbial food safety would be more suitable for practical application.
Bacterial contamination in ready-to-eat (RTE) foods prepared and sold by small businesses in middle Thailand was surveyed. The 135 samples were randomly purchased from open markets and supermarkets in Bangkok and Pathum Thani provinces during May 2013 to February 2015. The results indicated that the rate of contamination in cooked pork, chicken meat and cooked aquatic items were 13/39 (33%), 18/45 (40%) and 14/57 (25%), respectively and the rate of bacterial contamination of collected samples from open market and supermarket were 26/67 (39%) and 19/68 (27%), respectively. Therefore, no statistically significant difference of contamination rate between two kinds of market or among three categories of food. The most contaminated pathogenic bacteria was Staphylococcus aureus 27/135 (20%) while that of Salmonella spp. was the lowest 5/135 (4%) in each categories of collected food samples. Implementation of suitable hygienic practices in the small food businesses are thought to be required to reduce the risk of foodborne illnesses caused by the consumption of RTE foods sold in middle Thailand.
With the object of developing new biocides milder for human use than the current antiseptics, we synthesized a series of bis-quaternary ammonium compounds (bis-QACs). The antimicrobial activity of the newly synthesized bis-QACs and common biocides used as antiseptics was compared by examining minimum inhibitory concentrations and minimum bactericidal concentrations (MBCs). Moreover, the cytotoxicity of these compounds against human cells was determined to calculate the biocompatibility index (BI) of these compounds. BI was the ratio of the concentration of a biocide giving a 50% lethal effect on normal human epidermal keratinocytes to its MBC against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The commonly used antiseptics tested were benzalkonium chloride (BAC), octenidine dihydrochloride (OCT), chlorhexidine digluconate (CHG) and polyhexamethylene biguanide (PHMB). In comparison with these antiseptics, it was shown that some of new bis-QACs exhibited a wider and more potent antimicrobial spectrum than OCT. The cytotoxicity of these bis-QACs was equal or lower compared to that of the quaternary ammonium compounds (BAC and OCT), although these bis-QACs showed higher toxicity than the biguanide-based compounds (CHG and PHMB). Finally, the comparison of BIs revealed that new bis-QACs such as N-dodecyl ｛4,4ʼ-(2,4,8,10-tetraoxaspiro［5.5］undecan-3,9-diyl) ｝dipyridinium dibromide (4TOSU-12), 3,3'-［1,4-Phenylenebis (oxy)］bis (1-dodecylpyridinium) dibromide (3PHBO-12) and 3-(3-Hydroxy-2-(hydroxymethyl)-2-｛［(1-dodecylpyridinium-3-yl) oxy］methyl｝propoxy)-1-dodecylpyridinium dibromide (3HHDMP-12) had equal or greater biocompatibility than the commonly used biocides tested. Thus, these results strongly suggested that 4TOSU-12, 3PHBO-12 and 3HHDMP-12 could be useful as antiseptics for topical application to the skin.
Food is a basic necessity for human survival, but it is still the vehicle for the transmission of food borne disease. Various studies have examined the roles of spices, herbs, nuts, and semi-dried fruits, making the need for safe and convenient methods of decontamination a necessity. The current study determined the bacterial and fungal loads of 26 spices and herbs, 5 nuts, 10 semi-dried fruits and 5 other foods. Spices, herbs and semi-dried foods demonstrated the highest bacterial and fungal loads with the majority showing over 104 CFU/mL. Nuts and other foods showed growths ranging from 102 to 106 CFU/mL. The current study also attempted to determine the effects of heat and plasma treatment. The log reduction of bacterial growth after heat treatment (maximum: 120 min for 60℃) was between 0.08 to 4.47, and the log reduction after plasma treatment (maximum: 40 min) ranged from 2.37 to 5.75. Spices showed the lowest rates of reduction, whereas the semi-dried and other foods showed moderate to high levels of decrease after heat treatment. The log reduction of fungal growth after heat treatment ranged from 0.27 to 4.40, and log reduction after plasma treatment ranged from 2.15 to 5.91．Furthermore, we validated the sterilization effect of plasma treatment against Bacillus spp. and Staphylococcus spp. by using scanning electron microscopy. Both treatment methods could prove to be advantageous in the agriculture related fields, enhancing the quality of the foods.
Manganese contamination in water is one of the most serious problems in Southeast Asian countries, including Vietnam. Bioremediation using microorganisms, especially from the brewing yeast Saccharomyces cerevisiae, is expected to be a useful technique to remove manganese from contaminated water. Yeast strain S. cerevisiae BY4741 as the wild-type strain and some manganese-accumulating mutants bred from BY4741 were examined for cell growth and manganese accumulation in YPD liquid medium containing various concentrations of Mn2+.
Variants accumulating larger concentrations of manganese were isolated by the repeated screening of survivors in YPD media containing10mM Mn2+. Manganese was accumulated by the yeast cells during growth, but the growth of BY4741 was retarded with increasing Mn2+ concentrations and almost inhibited at 15mM Mn2+. One variant isolate, named IM3, showed no retardation of growth up to 15 mM Mn2+ and could absorb over 4-fold more manganese than the BY4741 strain. Effects of culture temperature and pH on the growth and manganese accumulation were analyzed for IM3. Maximum accumulation was shown at 30℃, pH 6.0 while the optimal growth was shown at 37℃, pH 5.0 – 7.0. Interestingly, IM3 could grow a little at pH 9.0 when manganese was added to the culture media, while it could not grow without the addition of manganese.
Presence of Escherichia coli O157:H7 on fresh goat meat samples (n= 40) of Dhaka city was analyzed using conventional and molecular methods. A total of 86 presumptive E. coli O157:H7 colonies were isolated from 60% of the samples using selective agar plating method. After conventional biochemical assay followed by API 20E assay, only 11 isolates were found to be E. coli O157:H7. Further serological test identified only four isolates that has strong agglutination reaction against anti-H7 sensitized latex. The biochemically and serologically confirmed isolates were then screened for major virulence factors include eaeA, rfbE, fliC, stx1 and stx2 genes by PCR. PCR analysis of positive isolates showed, 10 isolates were eaeA and rfbE genes positive but fliC gene was only in six, indicating that these isolates were H7 positive with flagellum antigens which might not expressed or detected in serotyping tests. Multiplex PCR against eaeA, stx1 and stx2 genes of the isolates showed similar results as when done individually. These results revealed that only 7% of the primary presumptive E. coli O157:H7 was found to be stx producing E. coli O157:H7 and thus greatly influenced the detection of the pathogen in meat samples.
The inactivation of Vibrio parahaemolyticus cells that were unattached or attached to a polyethylene terephthalate (PET) disc in pH-controlled sodium hypochlorite (NaOCl) solutions was studied under turbulent conditions. No significant desorption of attached cells occurred at the free available chlorine (FAC) concentrations from 0.1 to 1.0 mg/l. The number of viable cells was estimated by microbial calorimetry. The logarithmic relative reduction of viable cells was proportional to the product of the FAC concentration and time. In the pH range of 5.6 to 9.3, the first-order inactivation rate constants for unattached and attached cells increased with decreasing solution pH. It was found that the rate constants for unattached cells were approximately 6 to 7 times higher than those for attached cells at all pH values examined. It was confirmed that attached cells were more resistant to NaOCl solutions than unattached cells even when accessibility of attached cells to HOCl/OCl- was enhanced under turbulent conditions.