Rapid Microbiological Methods Research Articles

The Most Probable Limit of Detection (MPL) for rapid microbiological methods

Classical microbiological methods have nowadays unacceptably long cycle times. Rapid methods, available on the market for decades, are already applied within the clinical and food industry, but the implementation in pharmaceutical industry is hampered by for instance stringent regulations on validation and comparability with classical methods. Equivalence studies become less relevant when rapid methods are able to detect only one single microorganism. Directly testing this capability is currently impossible due to problems associated with preparing a spiked sample with low microbial counts. To be able to precisely estimate the limit of detection of rapid absence/presence tests, the method of the most probable limit is presented. It is based on three important elements; a relatively precise quantity of microorganisms, a non-serial dilution experiment and a statistical approach. For a set of microorganisms, a limit of detection of one was demonstrated using two different rapid methods.

The scope of mycoplasma contamination within the biopharmaceutical industry

Mycoplasma is well recognized as one of the most prevalent and serious microbial contaminants encountered within the manufacturing of biopharmaceuticals from the research phase to clinical development and production. The potential for mycoplasma contamination within cell culture systems was first identified by Robinson et al. in 1956 [1]. Presently, contamination rates in established cell cultures have been reported between 15 and 35% with considerably higher occurrence cited in certain selected populations [2]. In the last few years, there has been an expansion of diagnostic approaches for mycoplasma detection with the development and validation of rapid microbiological methods. The objective of this study was to determine current levels of mycoplasma infection of cell cultures, cell substrates and biologicals within a client based population. Retrospective comparison of 40,000 sample results was done to determine total contaminations rates amongst four (4) individual analytical assays. The establishment of reference data, such as existing contamination rates, becomes important in the critical appraisal of rapid microbiological methods for the detection of mycoplasma.

Reconsideration of flexibility in verifying rapid alternative food microbiological methods

Method comparison criteria for validating novel microbiology methods are discussed for aerobic plate count of fish samples using two rapid alternative methods (dissolved oxygen and automated MPN methods) and a reference method (AOAC 966.23, pour plate method). Results revealed that the present AOAC and ISO 16140 criteria such as requiring strict consistency in pairwise comparison may lead to overly strict validation disallowing advantageous rapid alternative methods in food microbiology. Pairwise comparison was not suitable for validating alternative microbiology methods, but the linear regression analysis followed by checking the fit to an ideal regression line was appropriate and easy-to-understand for validating the tested rapid alternative microbiology methods. Further discussion on an international level would benefit the development and use of rapid alternative methods.

Molecular methods for the detection and characterization of foodborne pathogens

The surveillance of foodborne pathogens in food industries has shown the urgent need for rapid and dependable methods to detect and characterize the organisms in food and environments of clinical and epidemiologic importance. Recent studies on rapid methods in microbiology have been focused on biochemical characterization, immunoassays, and molecular methods. Many molecular methods have been developed and applied to the detection and characterization of foodborne pathogens in laboratories and food industries. They can be mainly divided into DNA banding pattern-based tests and DNA sequence-based tests. The former includes nucleic acid hybridization, polymerase chain reaction (PCR), amplified restriction length polymorphism, and randomly amplified polymorphic DNA, etc. Most of these methods in commercial applications are based on PCR or hybridization techniques. The principle, characteristics, and application of molecular methods for the detection and characterization of foodborne pathogens were reviewed in this article.

The Utility of Rapid Microbiological and Molecular Techniques in Optimizing Antimicrobial Therapy

Early treatment of bloodstream infections with appropriate, definitive antimicrobial therapy has proven to reduce mortality, length of hospital stay, and healthcare costs. Culture-based testing methods require up to five days for final pathogen identification and susceptibility reporting, forcing use of broad spectrum empiric therapy. Recently, multiple rapid microbiological and molecular testing methods have been developed that reduce the time to identify the pathogen and susceptibility, allowing optimal antimicrobial therapy to be prescribed earlier. Real-time polymerase chain reaction and gene microarray have been described in literature, yet only peptide nucleic acid fluorescent in-situ hybridization has published data justifying its use based on clinical outcomes and cost savings. Target enriched multiplex polymerase chain reaction was developed to identify both the pathogen and multiple genes associated with resistance from blood within 6 hours and this methodology was studied in our hospital to assess effectiveness at optimizing antimicrobials in staphylococcal bloodstream infections.

Microbial contaminants in food: a big issue for a working group of the MoniQA NoE project

Introduction The MoniQA Network of Excellence is an EC funded project working towards the harmonization of analytical methods for monitoring food quality and safety along the food supply chain. This paper summarises both the structure and tasks of the working group on microbial contaminants within the MoniQA NoE and specifically focuses on harmonisation strategies important in the microbiological analysis of food. Objectives There is a need for rapid microbiological methods in order to quickly and efficiently identify harmful pathogens in food sources. However, one of the major problems encountered with many new methods is their market acceptance, as they have to pass extensive validation/ standardisation studies before they can be declared as official standard methods. Methods The working group on microbiological contaminants aims to contribute towards speeding up these prerequisites by collecting information on food law, quality assurance, quality control, sampling, economic impact, measurement uncertainty, validation protocols, official standard methods and alternative methods. Results The present report provides an overview of currently existing methodologies and regulations and addresses issues concerning harmonisation needs. One of the deliverables of the working group is the development of extended fact sheets and reviews based on relevant ‘hot’ topics and methods. The selection of food borne analytes for these fact sheets have been selected based on global, local and individual parameters. The working group has identified 5 groups of stakeholders (governmental bodies, standardisation/validation organisations, test kit/equipment manufacturers, food industry and consumers). Conclusion Current challenges of food microbiology are driven by new analytical methods, changes in the food market and altered consumer desires. The MoniQA NoE is contributing in overcoming these risks and challenges by providing a profound platform on microbiological rapid methods in food analysis to all stakeholders and it is expected that strong interaction within the network and beyond will foster harmonization.

Evaluation of PCR as rapid microbiological method in diagnosis of pneumococcal pneumonia

Comparison between detection of pneumococcal antigen from urine and PCR for detection of S. pneumoniae from blood is done in group of patients with pneumonia. Results are also compared according to severity of pneumonia.

Aetiological diagnosis of community acquired pneumonia: Utility of rapid microbiological methods with respect to disease severity

The present study investigated the utility of rapid microbiological methods in the aetiological diagnosis of community acquired pneumonia (CAP) according to the severity of CAP. Between 1999 and 2004, 384 adult patients with CAP were studied prospectively. In addition to standard microbiological methods, PCR and antigen detection techniques were used to identify pathogens. A total of 230 microbial agents in 209 patients were identified, with 134 (58.2%) identified by antigen detection or PCR tests. Of these 134 microbial agents, 95 (70.9%) were identified only by these rapid methods. Streptococcus pneumoniae urinary antigen detection was positive in 24.3% (81/333) of the patients with a diagnostic yield of 38.7% in those with severe pneumonia. Respiratory viral antigen detection was positive in 11.1% (35/314) of the patients with the highest diagnostic yield (20.3%) in patients with severe pneumonia. Mycoplasma pneumoniae PCR was positive in 7.5% (13/174) of the patients, all of whom were low-risk patients. Only 1 case of Chlamydia pneumoniae was identified by PCR. In conclusion, besides yielding the aetiological diagnosis rapidly, new methods add to the total diagnostic yield in CAP. The diagnostic yield of rapid methods differs according to the severity of the pneumonia.

Simple and Rapid Microbiological Method for Determination of Residual Antibacterials in Meat

A simple and rapid screening method using bioassay for the simultaneous analysis of antibacterials (penicillins, cephalosporins, macrolides, tetracyclines, quinolones, etc.) in meat has been developed. A 5 g sample was homogenized with 5 mL of methanol, and the homogenate was centrifuged for 10 min with 3,000 rpm. The pulp disk method with Bacillus subtilis BGA (Antibiotic Medium 5 (pH 8) and 8 (pH 6)), Micrococcus luteus ATCC 9341 and Geobacillus stearothermophilus as test organisms was employed for the assay of the antibacterials. Typical antibacterials (penicillin G, ampicillin, cefapirin, cefalexin, erythromycin, spiramycin, oxytetracycline, chlortetracycline, streptomycin, dihydrostreptomycin, enrofloxacin and oxolinic acid) were detected at levels of ca. 0.005-2.5 microg/g in meat. Therefore, we recommend this proposed screening method for routine analysis of residual antibacterials in livestock products.

Application of rapid methods and ultrasound imaging in the assessment of the microbiological quality of aseptically packed starch soup

Summary The application of rapid microbiological methods such as automated turbidometry and microcolony count using the direct epifluorescence technique (DEFT) was tested for the quality control of aseptically packed ultra-high temperature-treated starch-based soup. In addition, a non-destructive method using ultrasound imaging through the packages prior to the microbiological analysis was evaluated. The results showed that ultrasound could be a promising non-destructive testing method in quality control schemes for starch-based foods. Certain problems associated with calibration may arise in the application of turbidometry for heat-treated food material. Neither method studied in this work was sufficiently sensitive without a pre-incubation step.

Improved detection of Mycobacterium paratuberculosis in milk

At present there is no rapid microbiological method for the detection of viable Mycobacterium paratuberculosis in milk. By combining an extensive milk sample pretreatment with solid phase cytometry as the detection technique we were able to demonstrate viable mycobacterial cells in 50 ml of artificially contaminated pasteurized milk in less than one working day.

Implementation of Quality Control Methods (Physico‐Chemical, Microbiological and Sensory) in Conjunction with Multivariate Analysis Towards Fish Authenticity

AbstractFor Abstract see ChemInform Abstract in Full Text.

Rapid Microbiological Methods and Its Application to Pharmaceutical Water

Rapid Microbiological Methods and Its Application to Pharmaceutical Water

Rapid Microbiological Methods for Foods

Rapid Microbiological Methods for Foods

Comparison of the Traditional Three-Tube Most Probable Number Method with the Petrifilm, SimPlate, BioSys Optical, and Bactometer Conductance Methods for Enumerating Escherichia coli from Chicken Carcasses and Ground Beef

A study was conducted to compare commonly used methods, such as Petrifilm and SimPlate, and the rapid microbiological methods BioSys optical and Bactometer conductance to the standard most probable number (MPN) procedure for enumerating Escherichia coli from poultry carcasses and ground beef. Broiler carcasses and ground beef were evaluated in each of three replicate trials. Five groups of carcasses or ground beef were sampled and analyzed using Petrifilm, SimPlate, BioSys optical, and Bactometer conductance measurements after temperature abuse at 37°C for 0 (Petrifilm and SimPlate only), 2, 4, 6, or 8 h. The correlation coefficients for the regression lines comparing the standard E. coli MPN procedure to Petrifilm and SimPlate for chicken and ground beef, respectively, were as follows: 0.95, 0.94, 0.93, and 0.91. The correlation coefficients for the regression lines comparing the standard E. coli MPN procedure to BioSys optical and Bactometer conductance measurements for chicken and ground beef, respectively, were −0.91, −0.90, −0.93, and −0.96. Although Petrifilm and SimPlate performed well, E. coli could not be enumerated from 16.7 and 10% of samples, respectively, using these methods. The BioSys optical and Bactometer conductance methods performed very well when compared with Petrifilm and SimPlate. Using rapid methods (BioSys optical and Bactometer conductance), results were obtained in 1 to 11 h rather than the 48 h required to conduct Petrifilm or SimPlate or the 5 days required to conduct the MPN procedure. These methods may allow processors to test products and obtain results before shipping, avoiding the cost and loss of reputation associated with a recall or foodborne illness outbreak.

Immunomagnetic Separation with Mediated Flow Injection Analysis Amperometric Detection of Viable Escherichia coli O157

The coupling of an immunological separation (using immunomagnetic beads) with amperometric flow injection analysis detection of viable bacteria is presented. Using a solution containing Escherichia coli O157, the electrochemical response with two different mediators [potassium hexacyanoferrate(III) and 2,6-dichlorophenolindophenol] was evaluated in the FIA system. Antibody-derivatized Dynabeads were used to selectively separate E. coli O157 from a matrix. The kinetics and the capacity parameters regarding the attachment of bacteria to the immunobeads were studied. The immunomagnetic separation was then used in conjunction with electrochemical detection to measure the concentration of viable bacteria. A calibration curve of colony-forming units (cfu) against electrochemical response was obtained. The detection limit for this rapid microbiological method was 10(5) cfu mL-1, and the complete assay was performed in 2 h. Some advantages over ELISA methods are the direct detection of viable cells (and not total bacterial load) and the need for only one antibody (not enzyme-labeled), thus making the assay faster (only one washing step is necessary) and less expensive.

Microbiological quality and shelf life of cold-smoked salmon from three different processing plants

Whole fillets and slices of cold-smoked salmon from the same batch were vacuum packed and stored at 5°C for up to 50 days, sampled regularly and analysed for sensory and microbiological changes. The experiment was performed twice with salmon from three different smokehouses. The taste panel rejected sliced salmon after 21–36 days and fillets after 32–49 days of storage with no apparent relation to the microflora or salt content (4.1–6.1% in the water phase). Whole fillets developed the same spoilage characteristics, which included sweet, sour, bitter, faecal, ammonia and cabbage-like off-flavours, as sliced salmon but had a softer texture at the time of rejection. Plate count and Malthus detection times revealed that the microflora varied in numbers and composition among the three smokehouses. The microflora differed between fillets and sliced salmon, indicating the impact of the in-house flora on the size and composition of the microflora. Lactic acid bacteria,Enterobacteriaceae, psychrotrophic marine vibrio andPhotobacteriumspp. dominated the microflora of spoiled salmon. Use of conductometric measurements (Malthus) calibrated to traditional agar methods was not a suitable rapid microbiological method for determining the size and composition of the microflora, because of the variable microflora.

Rapid Microbiological Methods in Meat and Meat Products

Modern analytical techniques have to provide accurate results in time to permit forward approaches for controlling the microbiological quality of foods. Although the classical microbiological procedures of counting and identification of micro-organisms have been automated, several techniques with quite different principles are now used. The quantification of the microbial load of meat samples can be done in 4–24 hr with methods involving the growth of micro-organisms in liquid media (impedance and turbidimetric methods) and in less than 2 hr with methods counting stained microbial cells or detecting components of microbial cells. Rapid detection of undesirable (pathogens or spoilers) micro-organisms in meat samples can be done with immunological and DNA-based methods. The main techniques which can be used for the microbiological quantification and identification in the meat industries, as well as their applications are reviewed. Possibilities for rapid typing of bacterial isolates are also presented. Finally, some future trends in the microbiological analysis of foods are discussed.

Rapid microbiological methods in meat and meat products

Modern analytical techniques have to provide accurate results in time to permit forward approaches for controlling the microbiological quality of foods. Although the classical microbiological procedures of counting and identification of micro-organisms have been automated, several techniques with quite different principles are now used. The quantification of the microbial load of meat samples can be done in 4–24 hr with methods involving the growth of micro-organisms in liquid media (impedance and turbidimetric methods) and in less than 2 hr with methods counting stained microbial cells or detecting components of microbial cells. Rapid detection of undesirable (pathogens or spoilers) micro-organisms in meat samples can be done with immunological and DNA-based methods. The main techniques which can be used for the microbiological quantification and identification in the meat industries, as well as their applications are reviewed. Possibilities for rapid typing of bacterial isolates are also presented. Finally, some future trends in the microbiological analysis of foods are discussed.

Rapid Microbiological Methods with Hazard Analysis Critical Control Point

The proactive approach to ensuring food safety termed hazard analysis critical control point (HACCP) was introduced in the 1960s by the Pillsbury Company, in collaboration with the U.S. Army Natick Laboratories and National Aeronautics and Space Administration, to help guarantee that astronauts would not be incapacitated by the trauma of foodborne illness during space flights. The approach has subsequently been adopted as the standard food safety management system world-wide and is seen as forming the basis for harmonization of food inspection regulations necessitated by trade agreements such as General Agreement on Tariffs and Trade and North American Free Trade Agreement as the move toward globalization of trade in food products gains momentum. The new U.S. Department of Agriculture Mega-Reg requires mandatory introduction of HACCP, and the Food Safety Enhancement Program of Agriculture and Agri-food Canada, as well as the "due diligence" legislation of the European Union, is centered on HACCP principles.