Detection of microbial contaminants in mammalian cell cultures using a new fluorescence-based staining method

Aims: Microbial contamination of cell culture production processes is a current concern for biopharmaceutical industries. Traditional testing methods require several days to detect contamination and may advantageously be replaced by a rapid detection method. We developed a new method combining membrane filtration to microcolonies fluorescence staining method (MFSM) and compared it to epifluorescence microscopy. Methods and Results: Both methods were used to detect bacteria in CHO cells cultures. The epifluorescence microscopy showed to be limited by filterability, media interference and nonrobustness issues, whereas MFSM enabled consistent detection of Bacillus cereus, Staphylococcus epidermidis and Propionibacterium acnes after, respectively, 8, 9 and 48 h of incubation. Thanks to the nondestructive feature of the MFSM, stained membranes could be reincubated on culture media to yield visible colonies used for identification. Conclusions: The new method described in this study showed its ability to detect microbial contaminants in cell culture samples with time‐to‐results from 2–5 times shorter than the traditional testing method. Significance and Impact of the Study: The MFSM can be used as monitoring tool for cell cultures to significantly shorten detection times of microbial contamination, while preserving the ability to identify the contaminants and their viability.     

Detecting and Estimating Contamination of Human DNA Samples in Sequencing and Array-Based Genotype Data

DNA sample contamination is a serious problem in DNA sequencing studies and may result in systematic genotype misclassification and false positive associations. Although methods exist to detect and filter out cross-species contamination, few methods to detect within-species sample contamination are available. In this paper, we describe methods to identify within-species DNA sample contamination based on (1) a combination of sequencing reads and array-based genotype data, (2) sequence reads alone, and (3) array-based genotype data alone. Analysis of sequencing reads allows contamination detection after sequence data is generated but prior to variant calling; analysis of array-based genotype data allows contamination detection prior to generation of costly sequence data. Through a combination of analysis of in silico and experimentally contaminated samples, we show that our methods can reliably detect and estimate levels of contamination as low as 1%. We evaluate the impact of DNA contamination on genotype accuracy and propose effective strategies to screen for and prevent DNA contamination in sequencing studies.     

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

Mycoplasma contamination in cell culture is considered as serious problem in the manufacturing of biological products. Our goal in this research is to find the best standard and rapid method with high sensitivity, specificity, accuracy and predictive values of positive and negative results for detection of mycoplasma contamination in cell cultures of the National Cell Bank of Iran. In this study, 40 cell lines suspected to mycoplasma contamination were evaluated by three different methods: microbial culture, enzymatic mycoalert^® and molecular. Enzymatic evaluation was performed using the mycoalert^® kit while in the molecular technique, a universal primer pair was designed based on the common and fixed 16SrRNA ribosomal sequences used. Mycoplasma contaminations in cell cultures with molecular, enzymatic and microbial culture methods were determined as 57.5, 52.5 and 40 %, respectively. These results confirmed the higher rate of sensitivity, specificity and accuracy for the molecular method in comparison with enzymatic and microbial methods. Polymerase chain reaction (PCR) assay based on fixed and common sequences in the 16SrRNA, is a useful valuable and reliable technique with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. The enzymatic mycoalert^® method can be considered as a substitution for conventional microbial culture and DNA staining fluorochrome methods due to its higher sensitivity, specificity and speed of detection (<20 min).     

Immunological detection of Salmonella paratyphi A in raw prawns.

A slot blot enzyme-linked immunosorbent assay, using monoclonal antibodies specific only for Salmonella paratyphi A, to detect S. paratyphi A contamination in raw prawns has been established. When artificially contaminated prawn samples were tested. S. paratyphi A contamination could be identified correctly within 20 h. No false positives from samples artificially contaminated by other microorganisms were obtained. The sensitivity was such that as few as 1 S. paratyphi A organism per g of raw prawn could be detected. Therefore, the assay constituted a promising test for the rapid and specific detection of S. paratyphi A in prawns.     

Progress in the development of methods used for the abatement of microbial contaminants in ethanol fermentations: a review

Biofuel research and development roadmap is currently underway in several countries and is expected to pave a way for the establishment of a viable renewable energy sector that can compete with petroleum-based fuels. Ethanol fermentation has garnered increasing attention amongst various stakeholders (industries, governments, and academia) due to its economic and environmental merits. However, microbial contamination continues to be one of the major barriers in ethanologenic processes, resulting in low ethanol yields and thereby translating into economic losses. To this end, technological innovations geared towards effective elimination of microbial contamination are constantly being developed. This review explores and discusses the fermentation conditions that facilitate the growth of undesired microorganisms during ethanol fermentation processes. It highlights the methods that are currently used in biorefineries as well as innovative and advanced biotechnological methods currently being evaluated as viable alternative strategies to control or eliminate microbial contaminants in ethanol fermentations. These methods have the potential to minimize or control the contamination problem and could pave a way for the development of an efficient biofuel sector.

     

Contaminant yeast detection in industrial ethanol fermentation must by rDNA-PCR

AIMS: The present work focuses on the possibility to use conserved primers that amplify yeast ITS1-5.8S-ITS2 ribosomal DNA locus (rDNA) to detect the presence of non-Saccharomyces cerevisiae yeast in fermentation must of bioethanol fermentation process. METHODS AND RESULTS: Total DNA was extracted from pure or mixed yeast cultures containing different cell concentrations and different contaminant/fermenting yeast concentrations and submitted to PCR. Upon improvement of detection limits and DNA extraction protocol, must samples of distillery were checked for the presence of contaminant yeast. Contaminant rDNA bands were detected only in industrial samples during contamination episodes, but not in noncontaminated must. CONCLUSIONS: The method described here could detect the presence of contaminant yeast from industrial must in eight hours after sampling. SIGNIFICANCE AND IMPACT OF THE STUDY: The improved procedure may help to avoid severe contamination episodes at fermentation industries by decreasing the detection time from 5 days to 8 h and possible quantification of contaminant yeasts that can impose economical loss to the process.     

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.     

涂料微生物污染与防腐剂使用情况关系的探讨

为了解涂料的微生物污染与防腐剂使用情况之间的关系,按照ISO 9252-1989(E)检测207件市售涂料微生物指标,利用高效液相色谱检测有微生物检出涂料和40件无微生物检出涂料的防腐剂使用情况。结果发现有12件涂料微生物超标;异噻唑啉酮类防腐剂是本实验检出使用最多的防腐剂;微生物超标涂料防腐剂含量比正常低或者未检测到,种类单一。涂料的防腐剂使用情况与微生物污染程度有着密切的联系,建议建立和完善涂料限用防腐剂标准,指导涂料企业安全生产和质量监督。     

APPLICABILITY OF RAPID METHODS FOR DETECTION OF SALMONELLA SPP. IN POULTRY FEEDS: A REVIEW

The rapid detection of pathogenic microbial species in feed is of paramount importance considering its implications for animal production and food safety. More sensitive and rapid detection of contaminated feedstuffs may lead to more selective and therefore less expensive treatment of feeds, reduced rates of transmission to a poultry host and reduced carcass contamination. In order to interrupt the cycle of Salmonella spp. transmission from feed to poultry to the consumer, more rapid detection methods to monitor these sources are needed that provide conclusive results within the time frame of feed mixing or broiler processing. Within the last decade, new variations of selective media have been investigated to increase selectivity without reducing Salmonella spp. recovery. Immunological assay methods may also decrease assay time from 96 h to within 24–30 h. But all commercially available methods still require 16 to 57 h for preenrichment, enrichment, and in some cases, postenrichment to recover sublethally injured cells before the assay can be performed. Among the molecular methods that are currently available, the polymerase chain reaction (PCR) represents a tremendous potential for the detection of low levels of pathogenic bacteria. Once optimized, rapid methods may be used to quickly, reliably and inexpensively screen a variety of feedstuffs and feed components for the presence of Salmonella spp., with the goal of minimizing both the cost of feed treatment and the horizontal transmission of Salmonella spp. from feed to poultry.     

“A simple and rapid method for isolating lichen photobionts“

For decades, lichenologists have developed numerous and varied methods to isolate lichen photobionts. Most procedures are tedious, slow, and require several months after the initial isolation to obtain clones. Furthermore, the purity of the isolated photobionts obtained by more rapid methods is not sufficient to establish phycobiont axenic cultures. We have developed a new method for isolating lichen photobionts from fruticose, foliose and crustose lichens. Basically, it involves homogenization of lichen thalli (from 15 mg to 2 g), a one-step centrifugation through Percoll ®, followed by washing with Tween 20 and sonication. With this simple and rapid method (which takes less than 2 h), photobiont cells are obtained in sufficient quantity and purity to obtain dozens of axenic algal cultures.     

Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert^® assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert^® mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert^®, indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.     

Comparison of methods for the recovery and detection of low levels of injured Salmonella in ice cream and milk powder

This study compared the ability of four rapid methods and a standard cultural method to detect low levels of heat-injured cells of Salmonella typhimurium in ice cream and skimmed milk powder. The detection of Salmonella in samples contaminated with low levels (< 10 cfu 25 g-1) was significantly greater with the novel broth method than with the other methods (P 10 cfu 25 g-1, there was no significant difference between the methods except for the novel broth method and a dipstick-based immunoassay (P 相似文献   

Characterization and validation of a chemiluminescent assay based on a 1,2-dioxetane for rapid detection of enterococci in contaminated water and comparison with standard methods and qPCR

Aims: To evaluate the potential for using a novel chemiluminescence‐based enzyme assay for rapid detection of enterococci in water contaminated with faecal waste. Methods and Results: The novel assay (EntLight) was based on the enzymatic hydrolysis of the chemiluminescent 1,2‐dioxetane [(4‐methoxy‐4(3‐β‐d ‐glucoside‐4‐chlorophenyl)]spiro[1,2‐dioxetane‐3‐1,3‐tricyclo[7·3·1·0^2,7]tridec‐2,7‐ene] specific for β‐d ‐glucosidase. The specificity of the proposed EntLight assay was characterized using 26 different Enterococcus strains and 10 bacterial genera other than Enterococcus. With an analysis time of ≤8 h, the assay was found to be sensitive and specific. Validation experiments were carried out using water samples contaminated with raw municipal wastewater in comparison with qPCR and ISO standard methods. EntLight was successfully applied to detect enterococci in contaminated water within ≤8 h, and the proposed assay correlated well with both qPCR and ISO standard methods (R² > 0·776). Conclusions: EntLight can be applied to rapid and simple detection of viable enterococci in water contaminated with faecal matter. Significance and Impact of the Study: The novel EntLight assay and qPCR have the potential to be used as methods for early warning (1–7 h) of faecal pollutions in different water types.     

Key players and team play: anaerobic microbial communities in hydrocarbon-contaminated aquifers

Biodegradation of anthropogenic pollutants in shallow aquifers is an important microbial ecosystem service which is mainly brought about by indigenous anaerobic microorganisms. For the management of contaminated sites, risk assessment and control of natural attenuation, the assessment of in situ biodegradation and the underlying microbial processes is essential. The development of novel molecular methods, “omics” approaches, and high-throughput techniques has revealed new insight into complex microbial communities and their functions in anoxic environmental systems. This review summarizes recent advances in the application of molecular methods to study anaerobic microbial communities in contaminated terrestrial subsurface ecosystems. We focus on current approaches to analyze composition, dynamics, and functional diversity of subsurface communities, to link identity to activity and metabolic function, and to identify the ecophysiological role of not yet cultured microbes and syntrophic consortia. We discuss recent molecular surveys of contaminated sites from an ecological viewpoint regarding degrader ecotypes, abiotic factors shaping anaerobic communities, and biotic interactions underpinning the importance of microbial cooperation for microbial ecosystem services such as contaminant degradation.     

Advances in microbial ecosystem concepts and their consequences for ruminant agriculture

Microbial transformations in the rumen ecosystem have a major impact on our ability to meet the challenge of reducing the environmental footprint of ruminant livestock agriculture, as well as enhancing product quality. Current understanding of the rumen microbial ecosystem is limited, and affects our ability to manipulate rumen output. The view of ruminal fermentation as the sum of activities of the dominant rumen microbiota is no longer adequate, with a more holistic approach required. This paper reviews rumen functionality in the context of the microbiota of the rumen ecosystem, addressing ruminal fermentation as the product of an ecosystem while highlighting the consequences of this for ruminant agriculture. Microbial diversity in the rumen ecosystem enhances the resistance of the network of metabolic pathways present, as well as increasing the potential number of new pathways available. The resulting stability of rumen function is further promoted by the existence of rumen microbiota within biofilms. These protected, structured communities offer potential advantages, but very little is currently known about how ruminal microorganisms interact on feed-surfaces and how these communities develop. The temporal and spatial development of biofilms is strongly linked to the availability of dietary nutrients, the dynamics of which must also be given consideration, particularly in fresh-forage-based production systems. Nutrient dynamics, however, impact not only on pathway inputs but also the turnover and output of the whole ecosystem. Knowledge of the optimal balance of metabolic processes and the corresponding microbial taxa required to provide a stable, balanced ecosystem will enable a more holistic understanding of the rumen. Future studies should aim to identify key ecosystem processes and components within the rumen, including microbial taxa, metabolites and plant-based traits amenable to breeding-based modification. As well as gaining valuable insights into the biology of the rumen ecosystem, this will deliver realistic and appropriate novel targets for beneficial manipulation of rumen function.     

Comparison of enzyme-linked immunomagnetic chemiluminescence with U.S. Food and Drug Administration's Bacteriological Analytical Manual method for the detection of Escherichia coli O157:H7

Escherichia coli O157:H7, a major foodborne pathogen, has been associated with numerous cases of foodborne illnesses. Rapid methods have been developed for the screening of this pathogen in foods in order to circumvent timely plate culture techniques. Unfortunately, many rapid methods are presumptive and do not claim to confirm the presence of E. coli O157:H7. The previously developed method, enzyme-linked immunomagnetic chemiluminescence (ELIMCL), has been improved upon to allow for fewer incidences of false positives when used to detect E. coli O157:H7 in the presence of mixed cultures. The key feature of this assay is that it combines the highly selective synergism of both anti-O157 and anti-H7 antibodies in the sandwich immunoassay format. This work presents application of a newly semi-automated version of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline yielding detection limits of approximately 1 x 10(5) to 1 x 10(6) of live cells/mL. ELIMCL was further demonstrated to detect E. coli O157:H7 inoculated into artificially contaminated ground beef at ca. 400 CFU/g after a 5 h enrichment and about 1.5 h assay time for a total detection time of about 6.5 h. Finally, ELIMCL was compared with USFDA's Bacteriological Analytical Manual method for E. coli O157:H7 in a double-blind study. Using McNemar's treatment, the two methods were determined to be statistically similar for the detection of E. coli O157:H7 in ground beef inoculated with mixed cultures of select bacteria.     

A rapid method for detecting bacterial contamination in the presence of Penicillium and Streptomyces antibiotic fermentations

Contamination in antibiotic fermentation processes results in major economic and process problems. The detection and elimination of contamination is a continual objective for fermentation companies. While efforts continue to eliminate contamination by improving equipment and sterile techniques, it is still imperative to have a rapid method for detecting contamination in laboratory-stage inoculum and seed tanks. This article describes the successful studies leading to the adoption of the BACTEC, an automatic bacterial detection system, as a supplemental detection technique. The BACTEC system detects contamination by incubating samples with a selected(14)C-labeled substrate or substrates. The resulting metabolism of substrate produces (14)C-labeled CO(2) which is then quantified and expressed as a growth index, permitting detection of contamination more rapidly at a much earlier time than is possible with conventional detection techniques that involve Phenol red dextrose broth, streak plates, and microscopic examination techniques.     

Tandem use of immunofluorescent and DNA staining assays to validate nested PCR detection of mycoplasma

Mycoplasma contamination in cell culture is a serious setback to cell culturists across the world with a very high rate of reported occurrence particularly because of difficult early detection. Out of a variety of detection methods known, the double-step nested polymerase chain reaction (PCR)-based detection of mycoplasma in cell culture has been critically viewed upon because of chances of producing reliable results. A nested PCR technique, described to detect a large range of cell-culture-contaminating mycoplasma species, with greater sensitivity to detect as low a contamination as a few organisms, was compared with the results from two cytological techniques employed in tandem. These are DNA staining using Hoechst, the gold standard, and an immunofluorescent assay using a highly specific monoclonal antibody. The study undertaken on randomly collected cell cultures revealed a false-negative and several false-positive results in comparison to the cytological methods employed. The observations were particularly more unambiguous with the immunofluorescent assay employed in the study while simultaneously employed Hoechst staining serving as an indicator of bacterial contamination. There is a general apprehension that genus-specific PCR approaches could be associated with inaccurate outcome and only species-specific PCRs may be satisfactory in routine screening for mycoplasma contamination in cell cultures. At this juncture, it may be suggested that caution must be exercised while adopting the two-step nested PCR-based detection approaches, and the simultaneous employment of cytological methods used in this investigation could prove to be practicable in the proper interpretation of results.     

The microbiology of metalworking fluids

Metalworking fluids (MWFs) are complex mixtures of chemicals and are indispensable materials in industry. They are used as cooling and lubricating agents in different machining process such as grinding, milling, and cutting. The quality of MWFs is affected by physical, chemical, and microbial contaminates. In particular, MWFs are highly vulnerable to microbial contamination, which may act both as potential pathogens and deteriorgens. Microbial contamination is of major concern due to potential health hazards such as skin dermatitis and hypersensitivity pneumonitis. The contaminated MWFs can exhibit high degrees of microbial loading, ranging from 10(4) to 10(10) colony-forming units (CFU)/ml. Wide varieties of microorganisms are reported to colonize MWFs. Traditional culturing techniques are not only laborious and time consuming but also underestimate the actual distribution of the microorganisms present in the contaminated MWFs. Therefore, rapid molecular methods such as real-time PCR and fluorescent in situ hybridization are implemented to monitor the microbial load. In industry, biocides are presently used to control microbial contamination. However, it has its own disadvantages and therefore, in recent years, alternative methods such as UV irradiation were evaluated to reduce microbial contamination in MWFs. Microbes inhabiting the MWF are also capable of forming biofilm which is detrimental to the MWF system. Biofilm is resistant to common disinfectant methods, and thus further research and development is required to effectively control its formation within MWF systems. This review is intended to discuss the overall microbiological aspects of MWF.     

Metabolomics reveals stage-specific metabolic pathways of microbial communities in two-stage anaerobic fermentation of corn-stalk

Analysis of intracellular metabolites is essential to delineate metabolic pathways of microbial communities for evaluation and optimization of anaerobic fermentation processes. The metabolomics are reported for a microbial community during two stages of anaerobic fermentation of corn stalk in a biogas digester using GC–MS. Acetonitrile/methanol/water (2:2:1, by vol) was the best extraction solvent for microbial community analysis because it yielded the largest number of peaks (>200), the highest mean summed value of identified metabolites (23) and the best reproducibility with a coefficient of variation of 30 % among four different extraction methods. Inter-stage comparison of metabolite profiles showed increased levels of sugars and sugar alcohols during methanogenesis and fatty acids during acidogenesis. Identification of stage-specific metabolic pathways using metabolomics can therefore assist in monitoring and optimization of the microbial community for increased biogas production during anaerobic fermentation.