Microbiological Quality Control Research Articles

Exserohilum rostratum: Anatomy of a national outbreak of fungal meningitis

On 18 September 2012, an alert physician caring for a patient with fungal meningitis who had received an epidural methylprednisolone acetate injection approximately 6 weeks earlier, recognized its possible significance and reported this case to the Tennessee Department of Health, who in turn notified the Centers for Disease Control and Prevention. Shortly thereafter, additional cases of fungal meningitis were reported, and so began a large, unprecedented, multistate fungal meningitis outbreak. The predominant etiologic agent was Exserohilum rostratum, a black mould that was not known to have previously caused an outbreak of such devastating disease of this magnitude. The contaminated medication was prepared and distributed by a compounding pharmacy located in Massachusetts. This article reviews (i) the development and handling of the outbreak; (ii) the clinical manifestations and management of the infections; (iii) the detection, identification, and characteristics of E. rostratum, including the factors that might enhance its virulence; (iv) the regulatory concerns for microbiological quality control; and (v) what we have learned from this experience.

Detection of Resistance to Beta-Lactamase Inhibitors in Strains with CTX-M Beta-Lactamases: a Multicenter External Proficiency Study Using a Well-Defined Collection of Escherichia coli Strains

Under the auspices of the Spanish Society for Infectious Diseases and Clinical Microbiology Quality Control program, 14 Escherichia coli strains masked as blood culture isolates were sent to 68 clinical microbiology laboratories for antimicrobial susceptibility testing to β-lactam antibiotics. This collection included three control strains (E. coli ATCC 25922, an IRT-2 producer, and a CMY-2 producer), six isogenic strains with or without the OmpF porin and expressing CTX-M β-lactamases (CTX-M-1, CTX-M-15, and CTX-M-14), one strain carrying a double mechanism for β-lactam resistance (i.e., carrying CTX-M-15 and OXA-1 enzymes), and four strains carrying CTX-M variants with different levels of resistance to β-lactams and β-lactam-β-lactamase inhibitor (BLBLI) combinations. The main objective of the study was to ascertain how these variants with reduced susceptibilities to BLBLIs are identified in clinical microbiology laboratories. CTX-M variants with high resistance to BLBLIs were mainly identified as inhibitor-resistant TEM (IRT) enzymes (68.0%); however, isogenic CTX-M mutant strains with reduced susceptibilities to BLBLIs and cephalosporins were mainly associated with extended-spectrum β-lactamase production alone (51 to 80%) or in combination with other mechanisms (14 to 31%). Concerning all β-lactams tested, the overall interpretative discrepancy rate was 11.5%, of which 38.1% were the consequence of postreading changes in the clinical categories when a resistance mechanism was inferred. Therefore, failure to recognize these complex phenotypes might contribute to an explanation of their apparent absence in the clinical setting and might lead to inadequate drug treatment selection. A proposal for improving recognition is to adhere strictly to the current CLSI or EUCAST guidelines for detecting reduced susceptibility to BLBLI combinations, without any interpretative modification.

Molecular Methods for Microbiological Quality Control of Meat and Meat Products: A Review

Achieving food safety is a global health goal and the food-borne diseases take a major check on global health. Therefore, detection of microbial pathogens in food is the solution to the prevention and recognition of problems related to health and safety. Conventional and standard bacterial detection methods such as culture and colony counting methods and immunology-based methods may take up to several hours or even a few days to yield a result. Obviously, this is inadequate, and recently many researchers are focusing towards the progress of rapid diagnostic methods. The advent of molecular techniques has led to the development of a diverse array of assay for quality control of meat and meat products. Rapid analysis using DNA hybridization and amplification techniques offer more sensitivity and specificity to get results than culture based methods as well as dramatic reduction in the time to get results. Many methods have also achieved the high level automation, facilitating their application as routine sample screening assays. This review is intended to provide an overview of the molecular methods for microbiological quality control of meat and meat products.

Zn2+ and Pyrophosphate Sensing: Selective Detection in Physiological Conditions and Application in DNA-Based Estimation of Bacterial Cell Numbers

A diformyl-quinoline based receptor (L1) exhibits selective colorimetric and fluorometric sensing of Zn(2+) in aqueous medium at pH 7.4 based on the intraligand charge transfer (ICT) process. The in situ formed phenoxo-bridged complex, L1·2Zn can selectively and specifically sense PPi among all the other biologically important anions including ATP through reversible binding. The detection limit for Zn(2+) and PPi were found to be approximately 56 and 2 ppb, respectively. The unique selectivity of the PPi by the L1-Zn ensemble could be used as an analytical tool to probe PPi generation in a prototype polymerase chain reaction (PCR) setup and track DNA amplification with higher sensitivity as compared to conventional agarose gel electrophoresis. Interestingly, the principle of PPi estimation in PCR rendered rapid estimation of bacterial cell numbers with a limit of detection of 10 CFU of Escherichia coli MTCC 433 in as early as 10 PCR cycles. The proposed method of PPi sensing offers interesting application potential in PCR-based rapid diagnostics for pathogenic agents and microbiological quality control.

Assessment of Microbiological Quality of Yogurt Sold By Street Vendors in Onitsha Metropolis, Anambra State, Nigeria

Four brands of yogurt sold by street vendors in Onitsha Metropolis, Anambra State, Eastern Nigeriia were sampled, the pH was determined and microbiological assessments were conducted in order to ascertain the total heterotrophic bacteria, coliforms and yeast in the samples (A – D) during a seven day period. The results revealed that values of pH monitored varied from 3.69 – 4.50 while a total of five bacteria species belonging to Escherichia coli, Staphylococcus aureus, Streptococcus, Lactobacillus and Bacillus species, and three fungi species belonging to Aspergillus, Rhizopus and Saccharomyces were isolated from the samples. Sample B had the highest mean heterotrophic bacteria count with a value of 6.1 x 10 cfu/ml. Statistical analysis of heterotrophic bacteria count among the 4 sample groups had p-value =0.0000374. There is a significant difference in the heterotrophic bacteria count among the groups. Low titre values of starter cultures were recorded in the control samples. Escherichia coli, an indicator of coliform was detected in all the samples and the value of 4.4 x 10 cfu/ml was observed in sample B. Coliforms, S. aureus, Bacillus species and fungi were not detected from control samples purchased directly from the producing companies. Statistical analysis of coliform count in the four groups had p-value=0.529296. There was no significant difference in the coliform count among the 4 sample groups at α =0.05 and p-value =0.529296. The findings suggest that Research Paper British Microbiology Research Journal, 3(2): 198-205, 2013 199 the yogurt traded by street vendors in Onitsha Metropolis has poor microbiological quality control. This poses danger to public health. Therefore, attention of the appropriate government agencies and manufacturers is needed to ensure that sale of yogurt by vendors is done in most appropriate condition and in a mobile refrigerator to maintain adequate temperature, thereby, reduce contamination.

Quality in diagnostic microbiology: Experiential note to emphasize value of internal control programs

Background:Quality control (QC) in diagnostic microbiology is a matter of effective, efficient, accurate reporting in the expected turnaround time. Major stages of the analytical “standard operational procedures” where QC could be easily affected include organism identification and antibacterial susceptibility testing.Aim:The objective of this experiential technical note is to provide an evidence base to highlight the value of internal QC program in evaluating the effectiveness and efficiency of a laboratory's standard operational procedures; and the competences of individual scientific/technical staff.Materials and Methods:This report is based on four different scenarios requiring internal QC, including cases that are not reported within the turnaround time of standard operational procedures. Small-scale evaluations of (i) internal QC program, (ii) ciprofloxacin vs. moxifloxacin susceptibilities, and (iii) calibrated dichotomous susceptibility vs. directed susceptibility testing were performed.Results:The internal QC program identified sources of discrepancies in laboratory results. Evidence base for decision on new methodology and antibiotic testing were developed. For instance, it is observed that calibrated dichotomous susceptibility gives greater annular radius than directed susceptibility (P < 0.01).Conclusions:Internal QC program continues to be valuable means of identifying discrepancies, and vetting new ideas. This report presents evidence base to reaffirm that the need for internal QC is ever present.

Cytofluorometric detection of wine lactic acid bacteria: application of malolactic fermentation to the monitoring

In this study we report for the first time a rapid, efficient and cost-effective method for the enumeration of lactic acid bacteria (LAB) in wine. Indeed, up to now, detection of LAB in wine, especially red wine, was not possible. Wines contain debris that cannot be separated from bacteria using flow cytometry (FCM). Furthermore, the dyes tested in previous reports did not allow an efficient staining of bacteria. Using FCM and a combination of BOX/PI dyes, we were able to count bacteria in wines. The study was performed in wine inoculated with Oenococcus oeni (10(6)CFUml(-1)) stained with either FDA or BOX/PI and analyzed by FCM during the malolactic fermentation (MLF). The analysis show a strong correlation between the numbers of BOX/PI-stained cells determined by FCM and the cell numbers determined by plate counts (red wine: R (2)≥0.97, white wine R (2)≥0.965). On the other hand, we found that the enumeration of O. oeni labeled with FDA was only possible in white wine (R (2)≥0.97). Viable yeast and LAB populations can be rapidly discriminated and quantified in simultaneous malolactic-alcoholic wine fermentations using BOX/PI and scatter parameters in a one single measurement. This rapid procedure is therefore a suitable method for monitoring O. oeni populations during winemaking, offers a detection limit of <10(4)CFUml(-1) and can be considered a useful method for investigating the dynamics of microbial growth in wine and applied for microbiological quality control in wineries.

Single‐cell analysis of cell viability after a biocide treatment unveils an absence of positive correlation between two commonly used viability markers

Discrimination among viable/active or dead/inactive cells in a microbial community is a vital question to address issues on ecological microbiology or microbiological quality control. It is commonly assumed that metabolically active cells (ChemchromeV6 [CV6] procedure) correspond to viable cells (direct viable count procedure [DVC]), although this assumption has never been demonstrated and is therefore a matter of debate. Indeed, simultaneous determination of cell viability and metabolic activity has never been performed on the same cells. Here, we developed a microfluidic device to investigate the viability and the metabolic activity of Escherichia coli cells at single-cell level. Cells were immobilized in a flow chamber in which different solutions were sequentially injected according to different scenarios. By using time-lapse microscopy combined with automated tracking procedures, we first successfully assessed the ability of cells to divide and their metabolic activity at single-cell level. Applying these two procedures on the same cells after a hypochlorous acid (HOCl) treatment, we showed that the ability of cells to divide and their metabolic activity were anticorrelated. These results indicate that the relation between CV6 uptake and cell viability may be partially incorrect. Care must be taken in using the terms “CV6-positive” and “viable” synonymously.

Electronic Nose for Microbiological Quality Control of Food Products

Electronic noses (ENs) have recently emerged as valuable candidates in various areas of food quality control and traceability, including microbial contamination diagnosis. In this paper, the EN technology for microbiological screening of food products is reviewed. Four paradigmatic and diverse case studies are presented: (a)Alicyclobacillusspp. spoilage of fruit juices, (b) early detection of microbial contamination in processed tomatoes, (c) screening of fungal and fumonisin contamination of maize grains, and (d) fungal contamination on green coffee beans. Despite many successful results, the high intrinsic variability of food samples together with persisting limits of the sensor technology still impairs ENs trustful applications at the industrial scale. Both advantages and drawbacks of sensor technology in food quality control are discussed. Finally, recent trends and future directions are illustrated.

Pseudomonas aeruginosa in a hydropathic facility: diversity, susceptibility and imipenem resistance mutation

To detect Pseudomonas aeruginosa in water and treatment equipment biofilms of a thermae hydropathic facility and to study antibiotic susceptibility and genetic diversity. One hundred and fifty-four planktonic isolates were obtained from 2220 water samples during 4 years. Seventy-two biofilm isolates were obtained from 23 samples of inner parts of three inhalation equipments. Antibiotic susceptibility was determined by disc diffusion. All isolates were susceptible to tested antimicrobials, except two biofilm isolates and one planktonic isolate. Twenty-one resistant mutants were observed (nine from biofilms), mostly with imipenem (IP) resistance (81%), by diminished expression of OprD porin, as it was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Random amplification polymorphic DNA showed a genetically heterogeneous population that is spread through the entire system and persistent in time. IP resistance mutation ability was spread through the population. The permanent assessment of Ps. aeruginosa is necessary not only in water, as expressed in official programmes, but also in equipments where biofilms are evident. Ps. aeruginosa was more prevalent in biofilm populations and presented higher ability to adapt to antibiotic pressure. Twenty-one million people use thermae in Europe. Official microbiological quality control programmes only consider water surveillance. Present study proves the need of a review on current official programmes.

Aspects of investigating microflora contamination of drugs (A Review)

Various aspects of investigating microorganisms present in drugs are considered. Sources of contamination in nonsterile drugs are indicated. Problems related to changes in the organoleptic properties and therapeutic quality of contaminated drugs and the hazard of microorganisms for human health are analyzed. Data on the qualitative and quantitative composition of contaminant microflora isolated from various drugs are presented. Effective methods of contaminant isolation are described. Modern culture media and methods for the identification of bacteria and fungi during the microbiological quality control of drugs are presented.

Rapid enumeration of low numbers of moulds in tea based drinks using an automated system

Aseptically prepared cold drinks based on tea have become popular worldwide. Contamination of these drinks with harmful microbes is a potential health problem because such drinks are kept free from preservatives to maximize aroma and flavour. Heat-tolerant conidia and ascospores of fungi can survive pasteurization, and need to be detected as quickly as possible. We were able to rapidly and accurately detect low numbers of conidia and ascospores in tea-based drinks using fluorescent staining followed by an automated counting system. Conidia or ascospores were inoculated into green tea and oolong tea, and samples were immediately filtered through nitrocellulose membranes (pore size: 0.8 μm) to concentrate fungal propagules. These were transferred onto potato dextrose agar and incubated for 23 h at 28 °C. Fungi germinating on the membranes were fluorescently stained for 30 min. The stained mycelia were counted selectively within 90 s using an automated counting system (MGS-10LD; Chuo Electric Works, Osaka, Japan). Very low numbers (1 CFU/100 ml) of conidia or ascospores could be rapidly counted, in contrast to traditional labour intensive techniques. All tested mould strains were detected within 24 h while conventional plate counting required 72 h for colony enumeration. Counts of slow-growing fungi ( Cladosporium cladosporioides) obtained by automated counting and by conventional plate counting were close (r 2 = 0.986). Our combination of methods enables counting of both fast- and slow-growing fungi, and should be useful for microbiological quality control of tea-based and also other drinks.

Inactivation of Bacteria in Freshwater by Momentary Decompression Following High Pressurization

Rapid and continuous pressure treatment was realized using a hydraulic pump and the momentary decompression following high pressurization was used to inactivate bacteria. The number of colony-forming E. coli decreased to 1/1000 in response to 10 cycles of pressure treatment. In groundwater samples, repeated pressure treatment led to a two-log decrease in the number of colony-forming bacteria. These findings suggest that repeated cycles of momentary decompression following high pressurization enabled a marked decrease in bacterial growth activity. The results presented herein may contribute to microbiological quality control and the safety of freshwater.

Controle de qualidade da raiz de Operculina macrocarpa (Linn) Urb., Convolvulaceae

Considering the quality of the phytotherapic agents, it is important to point out that it includes rigorous attendance of the different steps of the development and production of these products, from the collection of the vegetable to the availability of the final product. In this work the quality control of the Operculina macrocarpa (Linn) Urb. roots, popularly known as 'batata-de-purga', was carried out. Pharmacopoeic and no pharmacopoeic methodologies were employed to physico-chemical and microbiological quality control. The obtained results showed that the roots presents a content of resin of 9,85%, The microbiological analysis did not present pathogenic growth among the other accomplished tests. The work stands out the importance of the establishment of norms for the quality control for the plants, so that they are found able to be used for phytotherapic reasons.

Microbiological quality and preservative capacity of commonly available cosmetics in Dar es Salaam, Tanzania

Ten brands of cosmetics were randomly purchased from shops in Dar es Salaam, and were subjected to microbiological assessment for microbial quality, preservative capacity and identification of microbial contaminants. Aliquots of each cosmetic were uniformly spread-plated on agar plates to quantify, isolate and identify microbial contaminants using conventional microbiological methods. The cup-plate technique complemented by the dilution test was used for evaluation of cosmetic preservative capacity. Microbial contaminants were present in 70% of the cosmetics. The most frequently isolated and identified microbial contaminants were attributable to Proteus mirabilis, Staphylococcus aureus, Bacillus and Trichophyton species. The cosmetics displayed inadequate preservative capacity evidenced by inability to lower the inherent bio-burdens to acceptable levels and to inhibit growth of the tested microorganisms. Such products can have detrimental effects on health status of consumers as consequence of their altered stability profiles and secondary microbial infections. Therefore, microbiological quality control of cosmetics available in the Tanzanian market should be re-enforced. Keywords: Preservative capacity, cosmetics, microbial quality, microorganisms East and Central African Journal of Pharmaceutical Sciences Vol. 13 (2010) 3- 11

Microbiological Quality Assessment of Laboratory Mice in Korea and Recommendations for Quality Improvement

Regular monitoring of commercial laboratory rodents and institutional research animal residents is essential for microbiological quality control programs. The objective of our study was to investigate the recent prevalence of infectious pathogens in laboratory mice from eight experimental animal vendors and 56 institutional animal facilities in Korea. Our investigation was conducted in 2006-2007. Specific Pathogen Free (SPF) mice from four commercial breeders were clean according to serological, bacteriological, parasitological, and histopathological examination results. However, mice from one intermediate vendor that distributed SPF animals from main commercial vendors to local districts had Syphacia obvelata and Mycoptes musculinus infections. Additionally, mice from conventional animal breeders were highly contaminated. Among the 56 institutional animal facilities, mouse hepatitis virus (MHV), Sendai virus and Mycoplasma pulmonis positive results were obtained in 23.2, 8.9, and 1.8% of animals tested, respectively. These results indicate that quarantine and eradication efforts of infectious pathogens in these facilities are sub-optimal and need to be improved. The use of commercial conventional mice for research should be eliminated and appropriate vendor selection as well as thorough quarantine before releasing animals into a facility are needed. Finally we suggest qualified veterinary experts are needed at each animal facility to ensure an adequate health surveillance program.

Survival of Clostridium spores in river water and in sand from a slow sand filter

Spores of sulphite-reducing clostridia (SSRC; incl. C. perfringens) have been introduced in water quality monitoring for microbial safety of drinking water because of their higher resistance to water disinfection processes than E. coli. Interpretation of this parameter, however, is hampered by lack of quantitative data on the spore decay rates. We determined decay rates of environmental Clostridium spores in river water and in filter sand in a twelve year microcosm study at different temperatures. Decay was not significant at 3°C and limited at 15°C. First order decay rates in water and sand were in the same order of magnitude and ranged from of 0.001 up to 0.0023 d−1. Molecular typing of isolates of surviving SSRC showed diversity in the species at different temperatures. From the results in combination with literature data it was concluded that there is heterogeneity in the longevity of environmental SSRC. There is a need for more information on the survival of C. perfringens spores which is essential for interpretation of this parameter in water microbiological quality control.

Guidelines to Implement Quality Management Systems in Microbiology Laboratories for Tissue Banking

Human tissues for implants are a biomedical product that is being used more frequently by many medical disciplines. There are infections in the patients related to the implanted tissues. The early detection of infections transmitted by blood and the microbiological study of tissues before their clinical use are strategies in tissue banks to prevent these situations. This work sought to contribute to establish the bases for the operation of a laboratory applied to the microbiological quality control of tissues. Based on classical microbiological principles, we defined the operation of microbiological control and tissues sterilization since 2003. We determine lists of acceptable microorganisms for every tissue, criteria for the interpretation of results, and a diagnostic algorithm of microbiological quality. We observed that the circumstances of donor death can be a determinant of the quality. The environment and the operator should be investigated as probable sources of contamination in outbreaks. The criteria of work based on a solid methodology must help to avoid the transmission of infections between donor and recipient. This is a critical point in the quality management of a tissue bank.

Microbiological quality and quality control of purified water and ultrapure dialysis fluids for online hemodiafiltration in routine clinical practice

During online hemodiafiltration, patients are directly infused with sterile substitution solutions to maintain fluid balance. Adequate water treatment and a well-organized quality control process are essential to provide non-pyrogenic fluids with consistent optimal quality. We sought to assess water quality, the water treatment system, and the methods for surveillance of microbiological water quality in 10 Dutch dialysis centers that routinely treat patients with hemodiafiltration. Microbiological monitoring results (micro-organisms and endotoxins) were collected over a 1-year period representing 11,258 hemodiafiltration sessions covering 97 patients. In all centers, water purification was based on a reverse osmosis module in combination with a second reverse osmosis and/or an electrodeionizer. All centers regularly and routinely monitored the microbiological purity of the dialysis water with adequate analytical methods but with variable monitoring frequency. Microbiological assessments were compliant with reference quality levels in 3923 of 3961 samples. Our study suggests that non-pyrogenic substitution fluids can be produced online for a prolonged period of time. It is likely that the current Dutch Quality of Care Guideline has contributed to high-quality water treatment and a well-organized control process.

Controles físico, físico-químico, químico e microbiológico dos frutos de Dimorphandra mollis Benth., Fabaceae

Considering the quality of phytotherapic agents, it is important to point out that the concern with this subject includes rigorous attendance the different steps of development and production of these products, from the collection of the vegetable to the availability of the final product. The quality control for the plants and their extracts is essential when they are used in the development of phytotherapics. In this work, methodologies (pharmacopoeic and no pharmacopoeic) were employed to establish norms for the quality control of Dimorphandra mollis Benth. (Leguminosae) fruit, popularly known as faveira. Pharmacopoeic and no pharmacopoeic methodologies were employed to physic, physic-chemical, chemical and microbiological quality control. The results obtained established the physic-chemical characters of the drug and showed that the fruits and the ethanol 70% extract present a content of flavonoids of 10.25% and 17.21%, respectively. The preliminary phytochemical analysis of the D. mollis fruits evidenced tannins, saponnins and flavonoids. The microbiological analysis did not present pathogenic growth among the accomplished tests.