Microbiological Quality Control Research Articles

A culture-independent nucleic acid diagnostics method for use in the detection and quantification of Burkholderia cepacia complex contamination in aqueous finished pharmaceutical products.

The Burkholderia cepacia complex (Bcc) is the number one bacterial complex associated with contaminated Finished Pharmaceutical Products (FPPs). This has resulted in multiple healthcare related infection morbidity and mortality events in conjunction with significant FPP recalls globally. Current microbiological quality control of FPPs before release for distribution depends on lengthy, laborious, non-specific, traditional culture-dependent methods which lack sensitivity. Here, we present the development of a culture-independent Bcc Nucleic Acid Diagnostic (NAD) method for detecting Bcc contaminants associated with Over-The-Counter aqueous FPPs. The culture-independent Bcc NAD method was validated to be specific for detecting Bcc at different contamination levels from spiked aqueous FPPs. The accuracy in Bcc quantitative measurements was achieved by the high degree of Bcc recovery from aqueous FPPs. The low variation observed between several repeated Bcc quantitative measurements further demonstrated the precision of Bcc quantification in FPPs. The robustness of the culture-independent Bcc NAD method was determined when its accuracy and precision were not significantly affected during testing of numerous aqueous FPP types with different ingredient matrices, antimicrobial preservative components and routes of administration. The culture-independent Bcc NAD method showed an ability to detect Bcc in spiked aqueous FPPs at a concentration of 20 Bcc CFU/mL. The rapid (≤ 4 hours from sample in to result out), robust, culture-independent Bcc NAD method presented provides rigorous test specificity, accuracy, precision, and sensitivity. This method, validated with equivalence to ISO standard ISO/TS 12869:2019, can be a valuable diagnostic tool in supporting microbiological quality control procedures to aid the pharmaceutical industry in preventing Bcc contamination of aqueous FPPs for consumer safety.

The Credibility of Microbiological Diagnostics in MedicalDiagnostic Laboratories Based on the Results of POLMICROExternal Laboratory Quality Control POLMICRO 2023

<br>Introduction: The article describes results of the 30th edition of the Polish National ExternalQuality Assessment Scheme in Microbiology POLMICRO organised by the Centre of QualityControl in Microbiology (CQCM).</br><br>Aim: The evaluation of the reliability of the microbiological diagnostics performed bymedical diagnostic laboratories in Poland.</br><br>Materials and methods: The organiser analysed the results achieved by participants ofprogrammes conducted by CQCM in 2023 concerning important stages of microbiologicaldiagnostics such the as microscopic analysis of Gram-stained slides, identificationof etiological factors of infections, assessment of antimicrobial susceptibility ofmicroorganisms and the detection of antimicrobial resistance mechanisms.</br><br>Results: A positive annual assessment in the POLMICRO 2023 programme was achieved by98.33%, so 413 of 420 participants of the POLMICRO 2023 programme, and all laboratories participatingin the POLMICRO/SSE (n = 132) and POLMICRO/MIKOLOGIA (n = 139) programmes.</br><br>Conclusions: The results of external quality control programmes confirm the reliability oftesting and the high level of microbiological diagnostics performed by medical diagnosticlaboratories in Poland.</br>

The Use of Microbiological Quality Control Techniques Adopted in a Human Tissue Bank

The Brazilian Ministry of Health’s Consolidation Ordinance No. 4 of September 28, 2017, provides the requisites for the evaluation of services for human tissue banks (bancos de tecidos humanos [BTH]) in terms of the microbiological control of environments (ISO 5 classification processing room) and products (culture for aerobic, anaerobic, and fungal pathogens), with a view to guaranteeing harmlessness in tissue transplants. Objectives: To highlight the techniques for microbiological control of distributed tissues adopted by a BTH over 5 years, as well as the results obtained from the application and use of these techniques. Methods: Using standard operating procedures (SOPs), the methodology, and frequency of collections, tests and room cleaning were established in advance. Microbiological cultures were taken from the tissues during all the collections and processing. During tissue processing, fingerprint samples on the processor’s gloves and in the processing room were seeded on blood agar plates. The processing room was cleaned weekly with biguanide and quaternary ammonium. The processing bench was sanitized with sterile 70% alcohol and the microbiological environmental control was carried out every 6 months by a qualified outsourced company. Results: In the period analyzed, the techniques proved effective in 46 (96%) cases, with contamination identified in only two (4%) processed and collected samples. The effectiveness and results were documented. Conclusion: Effective biological quality control methods are legitimately required, but they can be improved, raising the need to develop safe protocols for the quality of services and the safety of transplanted tissues.

Microbiological Skip Test: Fundamentals and Evaluation of Water Activity in Pharmaceutical Ingredients

Quality control is essential for Good Manufacturing Practices in the pharmaceutical industry. The time and costs associated with microbiological quality control are well known. It is proposed the reduction of traditional microbiological methods of counting the total number of microorganisms and research of specific pathogens using the water activity (Aw) method, a practice known as the skip test. The raw material selected as a prototype for this study met the criteria for the feasibility of reductions in analyses having synthetic origins, solid physical state, low or high pH in aqueous solution, and Aw results below 0.75 at a temperature of 25°C, and absence of nonconformity records. The risk management process was implemented using risk analysis with the mode, effect, criticality, failure analysis tool, and opening change control with the replacement of microbiological standard by Aw in 20 sequential batches after complete analysis of the first batch received in the year. It was possible to certify the microbiological quality of the selected pharmaceutical ingredients using the AW results, thereby confirming the feasibility of the skip test.

Phase change materials for portable isothermal incubators as a solution to shorten effective analysis times in microbiological quality control of drinking water.

The microbiological quality control of water for human consumption of parameters relevant as E.coli and total coliforms does not start on the field despite the existence of test methods that could make it possible. One of the things that makes this difficult is the possibility of initiating an effective and reliable incubation at the sampling site. The appearance of isothermal media with phase change materials solves this limitation. When phase change materials combine a relatively high melting heat with a suitable melting temperature adapted to the application temperature, they become excellent materials for thermal protection and for thermal energy storage. Starting the test at the same sampling point means that the effective times to obtain a result are shorter, improving water quality control. On the other hand, operationally, it also allows longer sampling routes. Both aspects are essential for managers responsible for controlling water quality for human consumption. In this work, the evidence that demonstrates the feasibility of this approach is presented.

Next-generation Sequencing Results Require Higher Inoculum for Cutibacterium acnes Detection Than Conventional Anaerobic Culture.

Cutibacterium acnes has been described as the most common causative microorganism in prosthetic shoulder infections. Conventional anaerobic culture or molecular-based technologies are usually used for this purpose, but little to no concordance between these methodologies (k = 0.333 or less) has been observed. (1) Is the minimum C. acnes load for detection higher for next-generation sequencing (NGS) than for anaerobic conventional culture? (2) What duration of incubation is necessary for anaerobic culture to detect all C. acnes loads? Five C. acnes strains were tested for this study: Four strains were causing infection and were isolated from surgical samples. Meanwhile, the other was a reference strain commonly used as a positive and quality control in microbiology and bioinformatics. To create inoculums with varying degrees of bacterial load, we began with a standard bacterial suspension at 1.5 x 10 8 colony-forming units (CFU)/mL and created six more diluted suspensions (from 1.5 x 10 6 CFU/mL to 1.5 x 10 1 CFU/mL). Briefly, to do so, we transferred 200 µL from the tube with the highest inoculum (for example, 1.5 x 10 6 CFU/mL) to the following dilution tube (1.5 x 10 5 CFU/mL; 1800 µL of diluent + 200 µL of 1.5 x 10 6 CFU/mL). We serially continued the transfers to create all diluted suspensions. Six tubes were prepared per strain. Thirty bacterial suspensions were tested per assay. Then, 100 µL of each diluted suspension was inoculated into brain heart infusion agar with horse blood and taurocholate agar plates. Two plates were used per bacterial suspension in each assay. All plates were incubated at 37°C in an anaerobic chamber and assessed for growth after 3 days of incubation and daily thereafter until positive or Day 14. The remaining volume of each bacterial suspension was sent for NGS analysis to identify bacterial DNA copies. We performed the experimental assays in duplicate. We calculated mean DNA copies and CFUs for each strain, bacterial load, and incubation timepoint assessed. We reported detection by NGS and culture as a qualitative variable based on the identification or absence of DNA copies and CFUs, respectively. In this way, we identified the minimum bacterial load detected by NGS and culture, regardless of incubation time. We performed a qualitative comparison of detection rates between methodologies. Simultaneously, we tracked C. acnes growth on agar plates and determined the minimum incubation time in days required for CFU detection in all strains and loads examined in this study. Growth detection and bacterial CFU counting were performed by three laboratory personnel, with a high intraobserver and interobserver agreement (κ > 0.80). A two-tailed p value below 0.05 was considered statistically significant. Conventional cultures can detect C. acnes at a load of 1.5 x 10 1 CFU/mL, whereas NGS can detect bacteria when the concentration was higher, at 1.5 x 10 2 CFU/mL. This is represented by a lower positive detection proportion (73% [22 of 30]) for NGS than for cultures (100% [30 of 30]); p = 0.004). By 7 days, anaerobic cultures were able to detect all C. acnes loads, even at the lowest concentrations. When NGS is negative and culture is positive for C. acnes , there is likely a low bacterial load. Holding cultures beyond 7 days is likely unnecessary. This is important for treating physicians to decide whether low bacterial loads necessitate aggressive antibiotic treatment or whether they are more likely contaminants. Cultures that are positive beyond 7 days likely represent contamination or bacterial loads even below the dilution used in this study. Physicians may benefit from studies designed to clarify the clinical importance of the low bacteria loads used in this study at which both methodologies' detection differed. Moreover, researchers might explore whether even lower C. acnes loads have a role in true periprosthetic joint infection.

Revisiting transformative WASH: measuring impact

Revisiting transformative WASH: measuring impact

Improvement of the Pour Plate Method by Separate Sterilization of Agar and Other Medium Components and Reduction of the Agar Concentration.

Although the pour plate method is widely employed in microbiological quality control, it has certain drawbacks, including having to melt the culture medium before seeding. In this study, the preparation of the culture medium was modified by using a lower concentration of agar (10 g/L), which was separated from the nutrients during sterilization. The new protocol was assessed in media frequently used in microbiological quality control of food, cosmetics, and pharmaceutical products, with tryptic soy agar (TSA), Sabouraud 4% dextrose agar (SDA), and violet red bile glucose agar (VRBG). In comparison with the conventionally produced media, the modifications significantly improved the growth of Saccharomyces cerevisiae in SDA, Staphylococcus aureus, Salmonella enterica subsp. enterica serovar Typhimurium, and Candida albicans in TSA and Escherichia coli ATCC 8739 and ATCC 25922 and S. Typhimurium in VRBG. The modified VRBG was also more selective for Pseudomonas aeruginosa. Regarding physicochemical properties, a significantly lower pH was observed in TSA and VRBG and lower strength values in TSA. Sterilizing agar separately from the other components of the medium and reducing the agar concentration to 10 g/L can improve microorganism growth and enhance the selectivity of differential media in the pour plate method. These modifications could facilitate the automation of this culture technique. IMPORTANCE In the era of rapid microbiological methods, there is a need to improve long-established culture techniques. Drawbacks of the pour plate method include having to melt each medium separately before seeding. For this technique, we demonstrate that separating the agar from the other components of commonly used media during sterilization and reducing the agar concentration to 10 g/L can enhance microbial growth. The new protocol could have advantages in routine laboratory practice because less agar is required and the same molten agar suspension can be used to prepare different media. Moreover, these modifications could facilitate the automation of the pour plate method.

Ultra-Violet (UV): A Good Bacterial Sterilizer?

Introduction: The aim of this study was to improve the DISTER-UV and to perform microbiological quality control at the biomedical laboratory of the West African Polytechnic University from January 2022 to November 2022. Methodology: During this eleven-month prospective study, we set up a quality control device (QCD). For microbiological quality control, we performed different cultures of bacteria with different bacteriological and morphological characteristics at T0 (no sterilization) and at T30 (after 30 minutes of sterilization under DISTER-UV). Results: After the realization, the DCQ attached to the DISTER-UV1 allows to display of the UV wavelength present in the light box. This device also displays and alerts when the UV intensity emitted by the lamps is below 250 nm. During microbiological quality control, the cultures carried out at T30 and incubated for 24 hours did not reveal any bacterial colonies. This shows the bactericidal character of DISTER-UV-2. Conclusion: The improvement and the microbiological quality control allowed us to switch from DISTER-UV1 (without sensor) to DISTER-UV-2 (with sensor or DCQ). The biological control allowed us to affirm that the DISTER-UV-2 is bactericidal.

Analysis of anthocyanins content and microbiological quality control in fresh and frozen raspberry fruit

Raspberry (Rubus idaeus L.) is a plant from the rose family and the most important berry-type fruit in our country. The most significant sorts of raspberries grown in Serbia are the red sorts, namely Willamette, Meeker, Gradina, Malling exploit and Skina. Raspberry fruit may be used either fresh or as a raw material in food industry, for the production of fruit juice, nectar, syrup, concentrate, dried and frozen fruit. The goal of this paper is to determine pH, titratable acidity (as citric acid), soluble solids, concentration of anthocyanins, vitamin C and total polyphenols, and to perform sensory analysis and microbiological quality control in both fresh and frozen fruit. pH value was measured with pH-meter. The concentration of acids was determined by the volumetric method, by titration with NaOH. The dry matter was measured by a refractometer. The concentration of anthocyanins was determined by the spectrophotometry method. The concentration of vitamin C was determined using the volumetric method. The concentration of polyphenol compounds was determined according to the Singleton method. The microbiological methods of detection and counting microorganisms were performed according to ISO standards. The results of this study have shown that both fresh and frozen raspberries are a good source of anthocyanins, indicating that their consumption is favorable for human health and that they may be useful in the production of functional food. The microbiological analyses have shown the absence of L. monocytogenes and Salmonella spp., while the concentration of Enterobacteriaceae and E. coli was below 10 cfu/g in both fresh and frozen raspberry fruit.

Development and validation of analytical method for microbiological quality control of Cannabis oil solution

Development and validation of analytical method for microbiological quality control of Cannabis oil solution

Development and validation of analytical method for microbiological quality control of Cannabis oil solution: THC 40 mg + CBD 40 mg/1 ml

Development and validation of analytical method for microbiological quality control of Cannabis oil solution: THC 40 mg + CBD 40 mg/1 ml

A regulatory perspective: Examples from microbiological quality control of non-sterile medicinal products

A regulatory perspective: Examples from microbiological quality control of non-sterile medicinal products

Nanotechnology-Enabled Biosensors: A Review of Fundamentals, Design Principles, Materials, and Applications.

Biosensors are modern engineering tools that can be widely used for various technological applications. In the recent past, biosensors have been widely used in a broad application spectrum including industrial process control, the military, environmental monitoring, health care, microbiology, and food quality control. Biosensors are also used specifically for monitoring environmental pollution, detecting toxic elements' presence, the presence of bio-hazardous viruses or bacteria in organic matter, and biomolecule detection in clinical diagnostics. Moreover, deep medical applications such as well-being monitoring, chronic disease treatment, and in vitro medical examination studies such as the screening of infectious diseases for early detection. The scope for expanding the use of biosensors is very high owing to their inherent advantages such as ease of use, scalability, and simple manufacturing process. Biosensor technology is more prevalent as a large-scale, low cost, and enhanced technology in the modern medical field. Integration of nanotechnology with biosensors has shown the development path for the novel sensing mechanisms and biosensors as they enhance the performance and sensing ability of the currently used biosensors. Nanoscale dimensional integration promotes the formulation of biosensors with simple and rapid detection of molecules along with the detection of single biomolecules where they can also be evaluated and analyzed critically. Nanomaterials are used for the manufacturing of nano-biosensors and the nanomaterials commonly used include nanoparticles, nanowires, carbon nanotubes (CNTs), nanorods, and quantum dots (QDs). Nanomaterials possess various advantages such as color tunability, high detection sensitivity, a large surface area, high carrier capacity, high stability, and high thermal and electrical conductivity. The current review focuses on nanotechnology-enabled biosensors, their fundamentals, and architectural design. The review also expands the view on the materials used for fabricating biosensors and the probable applications of nanotechnology-enabled biosensors.

The credibility of Polish diagnostic microbiological testing in 2021 based on the results of the POLMICRO external laboratory control

<br>Introduction: The paper presents the results of the 28th edition of the Polish National External Quality Assessment Scheme in Microbiology POLMICRO organised by the Centre of Quality Control in Microbiology (CQCM).</br> <br>Aim: The aim of the scheme is the assessment of the credibility of microbiological testing carried out by medical laboratories in Poland.</br> <br>Material and methods: The assessment is based on the analysis of the results obtained by the participants in each round of the POLMICRO, POLMICRO/MIKOLOGIA (MICOLOGY), and POLMICRO/SSE programmes in 2021.</br> <br>Results: Positive results in the full annual cycle were obtained by 95.73% of laboratories participating in the POLMICRO programme and all participants of POLMICRO/MIKOLOGIA (MICOLOGY) and POLMICRO/SSE.</br> <br>Conclusions: Medical diagnostic laboratories participating in the CQCM programmes of external laboratory control demonstrated high competence in such areas of microbiological diagnostics as the identification of pathogenic microorganisms, antimicrobial susceptibility testing, and detection of resistance mechanisms.</br>

Research on Thermal Imaging Usage as a Method for Detecting Bacillus subtilis Bacteria in Mortadella

Meat and meat products are important sources of valuable proteins and other nutrients, but they are also a conducive environment for microorganisms’ growth and can easily corrupt. In order to ensure the safety and quality of these products throughout its entire shelf-life, reliable microbial contamination assessment techniques must be used. Despite their effectiveness, traditional diagnostics methods are expensive and time consuming. Therefore, new timesaving, reliable techniques are searched for. Infrared thermography could be a good alternative method in this area. As a quick technique to detect microorganisms, it can overcome the limitations of traditional microbiological food-quality assessment methods. It has numerous advantages, such as the possibility of taking immediate temperature measurements and short processing times for obtaining a thermal image, non-contact and non-destructive measurements, and, unlike other methods, real-time measurement monitoring. Real-time monitoring is particularly important for modern production systems. The purpose of this research study is to develop a methodology for microbiological quality control of mortadella inoculated with Bacillus subtilis ATCC 6633 bacteria using a thermal imaging camera with an uncooled microbolometric detector. It was found that the thermal imaging measurements used in this research study enabled the distinction of contaminated samples (min. 106 CFU/g) from sterile samples. The tests should only record the temperature of the samples during the first 25 s after previously performing activations at −18 °C ± 1 °C for 60 s. This is where differences between the samples are most pronounced. Estimating the trend line of the sample’s cooling process is advisable.

Evaluation of exhaust air dust polymerase chain reaction as a supplement method for soiled bedding sentinel monitoring in specific pathogen free mouse facility using two different individually ventilated cage racks.

Health monitoring is essential for ensuring animal health and reliable research results. Each animal facility should establish adequate health monitoring methods, and microbiological quality control should be implemented through regular health surveillance. Recently, specific pathogen free (SPF) mice have been housed in individually ventilated cage (IVC) racks in the majority of mouse facilities globally, and health monitoring is implemented using a soiled bedding sentinel (SBS). Even though SBS monitoring is a standard method, it has a limitation in that some pathogens are not sufficiently transmitted to the sentinel housed in the IVC. The exhaust air dust polymerase chain reaction (EAD PCR) method has been reported to be a reliable complementary method to SBS monitoring based on research findings. In Korea, health monitoring programs using EAD PCR have not yet been applied to laboratory animal facilities. The microbiological status of mouse colonies housed in the two IVC racks was compared using SBS and EAD PCR monitoring in our SPF mouse facility. Except for Helicobacter spp. and Staphylococcus aureus, the detection of 16 pathogens did not differ between the two methods. In the detection of Helicobacter spp., EAD PCR was found to be more sensitive than SBS. Helicobacter spp. were not found by SBS, whereas four S. aureus positive samples were detected by either SBS or EAD PCR test. According to our findings, EAD PCR can be used as a supplement to SBS monitoring. Moreover, EAD PCR can reduce the number of animals used, making it a 3R (Replacement, Reduction, Refinement)-consistent method.

Microbiological quality control of wastewater by species identification of microorganisms using MALDI-TOF MS

Introduction. One of the main tasks of medical care for infectious diseases is the rapid identification of an infectious agent. The purpose of the study is to evaluate the effectiveness of the MALDI mass spectrometry for identification bacteria for microbiological control of wastewater quality. Materials and methods. Samples of wastewater samples at the treatment stage from the Kuryanovskaya aeration station were analyzed in accordance with MU 2.1.5.800-99 “Management of state sanitary and epidemiological supervision of wastewater disinfection” by the identification method in accordance with MUK 4.2.1884-04 “Sanitary-microbiological and sanitary - parasitological water analysis of surface water bodies” with application MALDI-TOF MS and 16S rRNA gene sequencing. Results. 5 strains of museum reference cultures and 22 bacterial isolates from wastewater samples grown on selective media of Endo, Enterococcus and Chromococcus coliform agar were studied, identified by MALDI-TOF MS, and confirmed by sequencing of specific regions of the 16S rRNA gene in bacteria of the genus Salmonella by MALDI-TOF MS identified only gender. Conclusion. In the routine practice of microbiological research, the identification of microorganisms is based on the determination of their cultural, tinctorial properties, and biochemical activity, the determination of which requires large financial and time costs. The use of the MALDI-TOF MS method makes it possible to reduce the time of identification of a microorganism when visible growth of microorganisms appears.

Recommendations on Describing the Requirements for Microbiological Quality and Sterility of Medicinal Products

The paper presents recommendations on describing analytical procedures for evaluation of medicinal products in terms of sterility and microbiological quality in accordance with the State Pharmacopoeia of the Russian Federation and the Pharmacopoeia of the EAEU. These quality attributes should be covered in Product specification files for any medicinal product. The paper describes specific considerations for preparation of the corresponding sections of the Russian or EAEU Product specification files. It is necessary to provide and justify quality requirements, refer to or describe the recommended analytical procedure and indicate differences, if any, from the standard one. Standardisation of the approach to microbiological quality control of medicinal products will allow analysts to accurately perform the tests, get reliable results and ensure the safety of medicinal products.

External quality control of microbiological testing in Polish medical diagnostic laboratories in 2020

<br><b>Introduction:</b>The paper presents the course of external quality control programmes organised in 2020 by the Centre of Quality Control in Microbiology (CQCM), including the 27<sup>th</sup> edition of the Polish National External Quality Assessment Scheme in Microbiology POLMICRO.</br> <br><b>Aim:</b>The assessment of the microbiological diagnostics’ quality carried out by Polish laboratories based on results of the POLMICRO, POLMICRO/MIKOLOGIA (MYCOLOGY), and POLMICRO/SSE programmes.</br> <br><b>Material and methods:</b>The participants’ responses from all rounds conducted in 2020 were analysed, i.e. five rounds of the POLMICRO programme, two rounds of the POLMICRO /MIKOLOGIA programme, and one round of POLMICRO/SSE.</br> <br><b>Results:</b>Positive evaluation in all rounds in the annual cycle were obtained by 90,1% of POLMICRO, 92,5% POLMICRO/MIKOLOGIA, and 100% POLMICRO/SSE participants.</br> <br><b>Conclusions:</b> Laboratories participating in the CQCM programmes demonstrated skills in the identification, antimicrobial susceptibility testing, and detection of resistance mechanisms of pathogenic microorganisms. The area of internal quality control of antimicrobial susceptibility testing requires improvement, especially the selection of reference strains.</br>