- European Supplement 6.6. – 2.6.14: Bacterial endotoxins

- European Supplement: n.5.1.10. – Guidelines for using the test for bacterial endotoxins

One of the greatest contributions to the field of microbiology came from the kitchen. In 1881, scientist Walter Hesse was searching for a solid medium that could be used to cultivate bacteria. Unlike gelatin, the growth medium of choice at that time, the material had to be stable at high temperatures, allow a variety of microorganisms to be separated easily, and resist digestion or liquefaction by certain microbial species. Fanny Angelina, Hesse’s wife and laboratory assistant, had the answer: Agar Agar, a gelling agent that she used in her jellies and puddings. This simple kitchen ingredient revolutionized the science of microbiology, allowing the separation and culturing of microbes to become a routine procedure. Now, almost 125 years later, microbiology agar is the most important and widely used microbial growth medium available today. Fanny would be proud… but should we be proud as well?

Although the growth of microbial cells on agar surfaces provides the laboratory with critical information about the amount and the type of organisms that may be present in a sample under evaluation, the time to result is usually longer than what is desired. Days and even weeks may elapse before microbial colonies are visually detected, and in most cases, confluent growth prevents individual organisms from being isolated, necessitating sub-culture onto additional agar media, delaying the time to result even further. Additionally, many laboratories are discovering that microorganisms, when stressed due to nutrient deprivation, or following exposure to sub-lethal concentrations of antimicrobial agents, such as preservatives, disinfectants, heat or decontaminating gases, may not replicate when cultured on artificial media, because the environment is not truly optimal for the resuscitation and subsequent proliferation of organisms that may be present. For these and many other technical, quality and business reasons, the modern microbiological laboratory has been motivated toward developing innovative approaches for the detection, quantification and identification of microorganisms, and this includes the implementation of rapid microbiological methods.

The recently published Volume 4 of the Encyclopedia of Rapid Microbiological Methods is playing an important role in providing guidance for companies wishing to implement rapid methods as alternatives to conventional microbiological assays. Since the first three volumes were published in 2005, many new technologies that have been developed, validation and statistical strategies have improved, and most importantly, regulatory guidance and new policies have paved the way for an easier path to acceptance and implementation.  The following is a brief overview of what is discussed:

- The application of modern microbial methods to the Quality Control testing of probiotics, including master and working cell banks, release and stability testing, viable cell counts, identification and strain typing, and absence of bacterial pathogens.

- Considerations when aligning a RMM with an end-user’s particular needs, such as the drivers for rapid methods, time savings, same day results, sample compatibility, automation, using a qualitative method as a screening tool, validation, identification, integration with LIMS and other data management platforms.

- Overview of rapid and automated microbial identification systems, including MALDI-TOF and SELDI-TOF mass spectrometry, FT-IR, elastic and inelastic light scattering, ribotyping, PCR, gene sequencing, microfluidics and microarrays.

- A case study for using MALDI-TOF mass spectrometry for the identification of microorganisms. Sample preparation, OQ, PQ, accuracy, precision, robustness and computer validation are discussed.

- A second case study on microbial identification focusing on genotypic methods, amplification of DNA, automation and validation (accuracy, precision, robustness and specificity).

- A novel case study of a new growth-based rapid microbiological method that detects the presence of specific organisms
 and provides an estimation of viable cell count. Data from the validation studies, inclusivity and exclusivity testing, and a comparison to USP <61> are provided.

- An end-user case study that describes an evaluation of a relatively new growth-based rapid method that utilizes a membrane filtration workflow coupled with a viability staining technique. A review of the technology and evaluation results are offered followed by a discussion of the system’s use for monitoring mammalian cell cultures.

- A comprehensive end-user evaluation using an optical spectroscopy technology for the real-time and continuous monitoring of airborne microorganisms in cleanroom and isolator environments.

- Validation and workflow of an ATP bioluminescence RMM for the release testing of both sterile (sterility testing) and non-sterile products (bioburden assessments).

- An end-user’s validation approach for a rapid, growth-based detection system as an alternate sterility test for cellular immunotherapy products.

- A case study by an end-user of a rapid, solid-phase cytometry technology. The validation strategy, use of statistical models, and considerations for stressed cells and sample matrix effects are offered.

- Understanding how to use statistics when validating an alternative sterility test, including probabilities and multiplicity, limit of detection and what is statistically “different” versus what is statistically “equivalent.” This is a must read for anyone wanting to validate a rapid sterility test and how to design the studies and use statistics to justify the results.

- The use of a novel qPCR-based system for the rapid detection of specific microorganisms.

- An end-user case study using a nucleic acid amplification platform for the detection of Mycoplasma. A review of regulatory requirements for nucleic acid amplification systems is also presented.

- An analysis of the science and workflow of a new nucleic acid amplification and microarray-based rapid method for the detection of Mycoplasma.

- An end-user’s overview of rapid viral detection methods. Experiences with Vesivirus, MVM and other public viral incidents are explored, as well as other topics associated with the future directions in using molecular methods for viral detection.

- A review of biopharmaceutical manufacturing, regulations, testing requirements and contamination events, and how the application of rapid methods fits in with the future of bioprocessing.

In addition to the chapters listed above, the new volume starts with an excellent introduction by FDA’s Dr. Bryan Riley, New Drug Microbiology Staff at the Center for Drug Evaluation and Research.  Dr. Riley explains that modern approaches to process control (including Process Analytical Technology) require the availability of results in real-time (or at least close to real-time) to enable the operator to use the test results to make process decisions and adjustments. Furthermore, current rapid microbiological test methods are now able to start providing some of the advantages (from a process control and economic return standpoint) long enjoyed by our colleagues in the clinical and food microbiology labs. He concludes that pharmaceutical microbiologists would be well served by considering which of their samples would provide a benefit with a more rapid result and then assessing the current alternate microbiological methods to see if any of them are a good fit for their needs.

Volume 4 of the Encyclopedia of Rapid Microbiological Methods provides new insights, validation and implementation guidance, and most importantly, encouragement for the pharmaceutical industry to embrace the next generation of microbiology testing platforms and strategies.

- Volume 4 may be obtained through the PDA at https://store.pda.org/ProductCatalog/Product.aspx?ID=1899. 
- Volumes 1-3 may be accessed at https://store.pda.org/ProductCatalog/Product.aspx?ID=513.

By Michael J. Miller, Ph.D.
President, Microbiology Consultants, LLC and Owner of rapidmicromethods.com

http://visit.microbiologics.com/l/8752/2013-07-24/dbjn3

The purpose of the Growth Promotion Test is to determine the suitability of culture media that is used in pharmaceutical tests. The test is performed by inoculating the media with a small number of microorganisms (less than 100 colony forming units) to ensure the nutritive properties of the media are adequate to support even a small number of microorganisms. Pseudomonas aeruginosa is a fastidious microorganism that is notoriously difficult to grow. For this reason, it is an ideal strain to use for the Growth Promotion Test because it will meaningfully challenge the media.

Fonte: Microbiologics

The research project¹ utilized Seward’s Stomacher 400 Circulator to prepare the preserved samples and bacterial inocula, as well as to evaluate the recovery of the microorganisms after exposure of catfish to fish and chip vinegar in a simple challenge test.

The results showed that vinegar diluted to 0.5% acetic acid on catfish fillets would be suitable for prolonging shelf life and did not make the product unappealing to consumers. This presents a simple opportunity at an affordable price to reduce economic loss due to spoilage in food products, even where the flavour of vinegar is not desirable.

The Stomacher has also been used recently in the evaluation of red wine as a food preservative². The exact mechanisms responsible for the antimicrobial activity of wine are not fully understood, but different components of wine have been proposed to contribute to its antimicrobial activity. Some authors give emphasis to the role of wine phenolics and others suggest the role of non-phenolic constituents of wine, such us organic acids, ethanol.

The aim of the study² was to investigate the antibacterial efficiency of three phenolic compound combinations and total phenolic compounds at concentrations of 100-200mg/l of three Argentinean red wine varieties on E. coli and L. monocytogenes viability in a fish-meat model system, at 4 °C and 20 °C.

The results of this investigation suggest phenolic compounds found in wine could be used to extend the shelf life of fish. The results also indicate the use of rutin–quercetin in combination as a preservative during the transport and conservation of fish meat to the fish market. This is an effective antibacterial agent even when there is an interruption in the refrigeration of the product.

References:

1. Antimicrobial Activity of Vinegar on Bacterial Species Isolated from Retail and Local Channel Catfish. Talaysha Lingham, Samuel Besong, Gulnihal Ozbay and Jung-Lim Lee (September 2012). J Food Process Technol S11-001

2. Phenolic Compounds from Wine as Natural Preservatives of Fish Meat. María José Rodríguez Vaquero, Pedro Aredes Aredes Fernández and María Cristina Manca de Nadra (April 2012). 

SOURCE: Seward Limited

Basic bactericidal - BS EN 1040
Basic fungicidal - BS EN 1275
Bactericidal - BS EN 1276
Fungicidal - BS EN 1650
Surface Bactericidal - BS EN 13697
Surface fungicidal - BS EN 13697
Viricidal - BS EN 13610
Sporicidal - BS EN 13704
Veterinary bactericidal - BS EN 1656
Veterinary fungicidal - BS EN 1657
Hygienic handwashing - BS EN 1499
Hygienic handrub - BS EN 1500

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Key to the effort was input from the AOAC Methods Committee on Antimicrobial Efficacy Testing and from stakeholders, who were critical to the editorial review process and consensus-building activities.

The methods were revised editorially to improve consistency (for example, cautions and notes, media preparation, and operating technique). In addition, minimum and maximum log density values were established for test microbes on inoculated carriers and retesting guidance was added. Each method was revised at least twice in the last 5 years. Revised methods include 955.14, 955.15, and 964.02 (Use-Dilution Methods); 960.09 (Germicidal and Detergent Sanitizing Action of Disinfectants); 961.02 (Germicidal Spray Products as Disinfectants); 965.12(Tuberculocidal Activity of Disinfectants); 966.04 (Sporicidal Activity of Disinfectants Test); and 2008.05 (Three-Step Method). The revised methods are available on the OMA Online at www.eoma.aoac.org.

Clostridium difficile

The ERP on Clostridium difficile was formed to establish performance requirements and overall direction for validation of an AOAC method for production of spores and testing the efficacy of disinfectants against C. difficile. The ERP established a fitness-for-purpose statement, identified recovery and enumeration media, and validated two sporulation protocols for producing high-quality and high-titer C. difficile spores. Although the EPA contract ended January 11, 2013, panel members agreed to collaborate on two papers: a white paper detailing the ERP process, objectives, and outcome, and a collaborative study manuscript comparing the two sporulation protocols.

Formulation Chemistry of Disinfectants

The contract work also included an LC method for the determination of mixed phenolic compounds. The successful single-laboratory validation (SLV) study was used to seek a full AOAC collaborative study validation of the method. The method was approved as First ActionOfficial Method^SM 2011.26 and is available on OMA Online.

Link: www.aoac.org

Are you mostly interested in a challenge test for milk? I saw in Chapter 6, Microbiological Challenge Testing, that the pathogens that may be considered for use in challenge studies for milk are Salmonellae, S. aureus, enterohemorrhagic E. coli (E. coli 0157:H7), and L. monocytogenes. We carry all these organisms. We do not sell one of the organisms recommended. It is Clostridium botulinum. Chapter 6 says that Clostridium sporogenes can be used as a surrogate for C. botulinum for certain applications. We also sell some of the other organisms listed in Chapter 6. They are Bacillus cereus, Clostridium perfringens, Listeria innocua, Shigella species, and Vibrio species. In addition we sell Campylobacter and Yersinia enterocolitica but Chapter 6 recommends using surrogates because these organisms are difficult to grow. Customers may also be interested in Chronobacter muytjensii because it can contaminate infant formula. We also sell organisms that may be considered normal flora in some foods. These organisms can indicate spoilage if found in high numbers. Lactobacillus fermentum and rhamnosus are examples. I made a few microorganism suggestions at the end of this letter. The suggestions are based on the list of pathogens at the end of Chapter 6.

Products

Several different types of products which can be used for challenge testing.

1. KWIK-STIK (P and S), LYFO DISK (V), KWIK-STIK Plus, Lab Elite: For a challenge test, the customer would grow the organism on an agar plate, make a suspension of the organism, calibrate it using a spectrophotometer of turbidometer, make serial dilutions as necessary, and then inoculate his test with the number of organisms which his test calls for. For example, it the customer wanted to inoculate his test with 100-990 microorganisms, he would use a turbidometer to make a suspension of organisms equivalent to a 0.5 McFarland (which is approximately 108 microorganisms). Then he would serially dilute the suspension until he had the concentration he needed in order to inoculate his test with 100-990 microorganisms. It would be much easier to perform the test with an enumerated product but some organisms may not be available in an enumerated form. Also, some customers may prefer to use fresh instead of lyophilized microorganisms.

The article from the Journal of Food Protection says, “When reviving strains from the frozen or lyophilized state, there should be one to two successive transfers in a non-selective growth medium.” Directions for maintaining organisms can be found on the Microbiologics website. If the customer wants a non-enumerated product, it might be best to use KWIK-STIK Plus (X) or Lab Elite (CRM) microorganisms because the journal says, “ The number of times a culture is transferred to product new working stock cultures should be minimized to avoid genetic changes…APAC International guidelines for laboratories indicate there should be not more than five passages from primary reference material. In order to keep passage numbers down, the customer can use KWIK-STIK Plus (2 passages) or Lab Elite (PFGE shows that it is equivalent to the original ATCC strain.) The article you sent says , “Biochemical characteristics, serology, genetic profile, virulence, or toxicity should be periodically reconfirmed as appropriate.” Genetic information is included with the Lab Elite product. The Lab Elite product has a unique genetic profile so it can be distinguished from other strains of the same species. http://www.microbiologics.com/Products/Lab-Elite If a laboratory is ISO:17025 accredited, they should use certified reference material (Lab Elite) when available. Sometimes you may prefer lyophilized organisms. The Journal article says, “A dry inoculum may be required for studies in low-moisture foods or when added moisture needs to be avoided. Inoculum can be prepared by freeze drying.”

2. EZ-FPC: EZ-FPC products are available at concentrations of 100-990 cfu per pellet (qualitative FPC) and at 1000-9900 (quantitative FPC) cfu per pellet. They are sold 10 pellets to a vial. They do not come with hydration fluid. They come with a Certificate of Assay. The following is a link to the product list. http://www.microbiologics.com/Products/EZ-FPC Just click on the orange box at the bottom of the page to see the list of microorganisms.

Qualitative EZ-FPC – 100-990 cfu per pellet
In the article you sent me from the Journal of Food Protection (Parameters for Determining Inoculated Pack?Challenge Study Protocols) it says under 5.1, Growth Studies, “When conducting studies to determine whether a pathogen grows in a product, ideally the number of organisms used should reflect the numbers normally expected in the product. Typically, an inoulum level of between 2 and 3 log CFU/g is use, even when this exceeds expected numbers because this level allows enumeration by direct plating. ” Our qualitative FPC product would meet this requirement because it ranges from 100 to 990 cfu per pellet.

If a Very Low Number of Organisms is needed: <100 cfu per inoculums.
If the customer wants to use a very low population of cells (<100 cfu per sampling unit), he can either dilute the EZ-FPC product further or use a product such as EZ-Accu Shot (A), EZ-CFU One Step (Z), or EZ-CFU (C). These 3 products deliver less than 100 cfu per inoculums when used as directed. Customers should always test the organisms on nonselective agar as well as selective agar because the organisms grow best on nonselective agar. Non selective agar is the control and is used to determine the number of cfu in the inoculum.

Quantitative EZ-FPC – 1000-9900 cfu per pellet
These products are used when testing the accuracy of one’s counting methods. A laboratory may perform counts when testing the shelf life of a food product.

When a laboratory performs a challenge test by inoculating a food product with a microorganism suspension, they should concurrently test the suspension on non-selective agar in order to determine the number of cfu per ml of inoculums.

The article that you sent me from the Journal of Food Protection, says in 4.3 that challenge studies should generally be conducted using three to five bacterial strains either individually or in combination (a cocktail). Your customers will probably want to purchase more than one strain for a study.

3. Epower: Epower products are sold as a vial of 10 pellets at a specific concentration for example, 104, 106, 108). They come with a Certificate of Assay. The following is a link to the product list. http://www.microbiologics.com/Products/Epower Just click on the orange box at the bottom of the page to see the list of microorganisms.

Epower products can be used in inactivation studies because (according to the journal article) “high numbers of organisms are typically used, e.g. 6 to 7 log CFU/g. the target level of reduction, which influences the inoculums level used, may depend on the regulations for specific food types, e.g., a 5-log reduction of the appropriate pathogen in juice (21CFR 120.24)”

We sell some strains of organisms at high concentrations. If we do not have the microorganism which the customer needs at a high Epower concentration, you can fill out a Lyophilized Microorganism Request Form. When the demand for a certain product is high we try to add it to our catalog. An alternative is to ask the technical support department what the approximate concentration of an organism is. Another solution is to purchase the KWIK-STIK or LYFO DISK and make an inoculom from freshly grown organisms.

4. Lab Elite: The article you sent says , “Biochemical characteristics, serology, genetic profile, virulence, or toxicity should be periodically reconfirmed as appropriate.” Genetic information is included with the Lab Elite product. The Lab Elite product has a unique profile so it can be distinguished from other strains of the same species. http://www.microbiologics.com/Products/Lab-Elite If a laboratory is ISO:17025 accredited, they should use certified reference material (Lab Elite) when available.

Font: Microbiologics