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Antibiotics (Basel, Switzerland) Aug 2019Mycoplasma contamination detrimentally affects cellular functions and the growth of intracellular pathogens in cell cultures. Although several mycoplasmacidal agents are...
Mycoplasma contamination detrimentally affects cellular functions and the growth of intracellular pathogens in cell cultures. Although several mycoplasmacidal agents are commercially available for sterile cell cultures, they are not applicable to rickettsia-infected cells. In our attempt to find an anti-mycoplasma drug for contaminated rickettsial cultures, we determined the susceptibilities of three common species to daptomycin. and showed low-level resistance to daptomycin (minimum inhibitory concentration, MIC = 2 mg/L), whereas was high-level resistant (MIC = 32 mg/L). However, some isolates developed higher resistance to daptomycin after failed treatments with inadequate doses or durations. An aminoglycoside (gentamicin) was still active against and could be used in cultures. For complete eradication of mycoplasmas in cultures, we recommend a 3-week treatment with daptomycin at 256 mg/L. In contaminated cultures, daptomycin at 32 mg/L was effective in eradicating , whereas either gentamicin or amikacin (100 mg/L) was effective in eradicating . Unlike each drug alone, the combinations of daptomycin plus clindamycin and/or quinupristin/dalfopristin proved effective in eradicating . In summary, our study demonstrated the in vitro anti-mycoplasma activity of daptomycin and its application as a new mycoplasma decontamination method for and cultures.
PubMed: 31438510
DOI: 10.3390/antibiotics8030123 -
PDA Journal of Pharmaceutical Science... 2018This experimental study compares cell size, zeta potential, and the ability to penetrate tailor-made size exclusion membrane filters of mycoplasma cultivated in five...
UNLABELLED
This experimental study compares cell size, zeta potential, and the ability to penetrate tailor-made size exclusion membrane filters of mycoplasma cultivated in five different cultivation media. The influence of relevant filtration process parameters, in particular transmembrane pressure and filtration temperature, on their respective retention was tested. The impact of the filtration temperature was further evaluated for the Gram-negative bacteria species , the Gram-positive bacteria species , the phage PP7, and the mycoplasma species The findings were correlated to the different mechanical properties of the particles, especially also with respect to the different bacterial cell envelopes found in those species. This study suggests that mycoplasma, surrounded by a flexible lipid bilayer, are significantly susceptible to changes in temperature, altering the stiffness of the cell envelope. Mycoplasma retention could thus be increased significantly by a decreased filtration temperature. In contrast, Gram-negative and Gram-positive bacteria species, with a cell wall containing a cross-linked peptidoglycan layer, as well as bacteriophages PP7 exhibiting a rigid protein capsid, did not show a temperature-dependent retention within the applied filtration temperatures between 2 and 35 °C. The trends of the retention of with increasing temperature and transmembrane pressure were independent of cultivation media. Data obtained with mycoplasma suggest that the trend of mycoplasma retention at different filtration temperatures is also independent of the membrane pore size and thus retention level. Media in biopharmaceutical processes are sterile-filtered to prevent them from bacterial contamination. Mycoplasma represent a relevant class of bacteria. In this publication it is shown that mycoplasma cell size depends on the media they are cultivated in. Membranes used for sterile filtration retain bacteria predominantly by size exclusion. Thus, an altered cell size can result in different retention values. Another characteristic of mycoplasma is the flexible lipid bilayer and the absence of a rigid cell wall. The lipid bilayer can undergo a phase transition from a gel to a liquid-crystal phase at a certain temperature, which makes it stiffer at lower temperatures. A higher stiffness can result in higher retention values during filtration, as the deformability of the mycoplasma cell is lower and the cell does not squeeze through the membrane pores.
ABBREVIATIONS
ALCM: culture medium; ASTM: American Society for Testing and Materials; ATCC: American Type Culture Collection; CFU/mL: colony-forming units per milliliter; DLS: Dynamic light scattering; LRV: Log reduction value; PES: Polyethersulfone; PFU/mL: Plaque-forming units per milliliter; PSD: Particle size distribution; PVP: Polyvinylpyrrolidone; SDS: Sodium dodecyl sulfate; SEM: Scanning electron microscopy; SLB: Saline lactose broth; TMP: Transmembrane pressure; TSB: Tryptic soy broth.
Topics: Acholeplasma laidlawii; Culture Media; Filtration; Mycoplasma; Sterilization; Temperature
PubMed: 29343618
DOI: 10.5731/pdajpst.2017.008102 -
Applied and Environmental Microbiology Sep 2015Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the...
Nucleic acid amplification technique (NAT)-based assays (referred to here as NAT assays) are increasingly used as an alternative to culture-based approaches for the detection of mycoplasma contamination of cell cultures. Assay features, like the limit of detection or quantification, vary widely between different mycoplasma NAT assays. Biological reference materials may be useful for harmonization of mycoplasma NAT assays. An international feasibility study included lyophilized preparations of four distantly related mycoplasma species (Acholeplasma laidlawii, Mycoplasma fermentans, M. orale, M. pneumoniae) at different concentrations which were analyzed by 21 laboratories using 26 NAT assays with a qualitative, semiquantitative, or quantitative design. An M. fermentans preparation was shown to decrease the interassay variation when used as a common reference material. The preparation was remanufactured and characterized in a comparability study, and its potency (in NAT-detectable units) across different NATs was determined. The World Health Organization (WHO) Expert Committee on Biological Standardization (ECBS) established this preparation to be the "1st World Health Organization international standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection" (WHO Tech Rep Ser 987:42, 2014) with a potency of 200,000 IU/ml. This WHO international standard is now available as a reference preparation for characterization of NAT assays, e.g., for determination of analytic sensitivity, for calibration of quantitative assays in a common unitage, and for defining regulatory requirements in the field of mycoplasma testing.
Topics: DNA, Bacterial; Laboratories; Mycoplasma; Nucleic Acid Amplification Techniques; World Health Organization
PubMed: 26070671
DOI: 10.1128/AEM.01150-15 -
Journal of Oral Pathology & Medicine :... Feb 2015Mycoplasmas are the smallest free-living organisms; Mycoplasma salivarium and Mycoplasma orale are the most common species isolated from the oropharynx. Oral leukoplakia...
BACKGROUND
Mycoplasmas are the smallest free-living organisms; Mycoplasma salivarium and Mycoplasma orale are the most common species isolated from the oropharynx. Oral leukoplakia is the most prevalent potentially malignant disorder of the oral mucosa; its etiology has not been defined. Our previous study with DNA-binding fluorescent dye suggested the presence of mycoplasmas in the epithelial cells of leukoplakia tissue.
OBJECTIVE
Our aim was to detect M. salivarium in the epithelial cells of leukoplakia by immunohistochemistry.
DESIGN
We produced a polyclonal antibody (PAb) reactive to Mycoplasma by injecting a rabbit with M. salivarium cells (ATCC 23064) mixed with complete Freund's adjuvant and a monoclonal antibody specific to M. salivarium by injecting M. salivarium cells (ATCC 23557) mixed with complete Freund's adjuvant into the footpads of a rat. Then, we attempted to detect M. salivarium in the epithelium of leukoplakia tissues by immunohistochemistry.
RESULTS
We obtained an antimycoplasma rabbit PAb reactive to all seven Mycoplasma species used in this study. Three hybridoma clones producing monoclonal antibodies specific to M. salivarium were obtained, and an M. salivarium-specific monoclonal antibody, designated 7-6H, was established. Immunohistochemistry with these antibodies revealed M. salivarium in the epithelial cells of leukoplakia with hyperplasia and hyperkeratosis on histology. PCR and sequencing verified the presence of M. salivarium DNA in the epithelial cells of leukoplakia.
CONCLUSION
Intracellular M. salivarium was identified in the epithelial cells of leukoplakia.
Topics: Adult; Aged; Aged, 80 and over; Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Antibody Specificity; Bacteriological Techniques; Chlorocebus aethiops; Epithelial Cells; Female; Fluorescent Antibody Technique; Freund's Adjuvant; Humans; Immunohistochemistry; Intracellular Space; Leukoplakia, Oral; Male; Microscopy, Immunoelectron; Middle Aged; Mouth Mucosa; Mycoplasma salivarium; Polymerase Chain Reaction; Rabbits; Rats; Vero Cells; Young Adult
PubMed: 25065471
DOI: 10.1111/jop.12215 -
Transfusion Medicine and Hemotherapy :... Feb 2014Contamination of cell culture and biological material by mollicute species is an important safety issue and requires testing. We have developed a singletube real-time...
BACKGROUND
Contamination of cell culture and biological material by mollicute species is an important safety issue and requires testing. We have developed a singletube real-time polymerase chain reaction (PCR) assay for rapid detection of Mollicutes species stipulated by the European Pharmacopeia.
METHODS
Primers and TaqMan probes (FAM-labeled) were deduced from 16S rDNA sequence alignment of 18 mollicutes species. A synthetic internal control (IC) DNA and an IC-specific TaqMan probe (VIC-labeled) were included. The analytical sensitivity of the assay was determined on DNA dilutions from 12 mollicute strains. Specificity was proven by the use of DNA from other bacteria.
RESULTS
Analytical sensitivities of the PCR assay were in the range of 405-2,431 genomes/ml for 11 of the 12 tested mollicute DNA samples. The lowest sensitivity was found for Ureaplasma urealyticum (19,239 genomes/ml). Negative results for DNA samples from 3 different ubiquitous bacteria demonstrated the specificity of the PCR assay for Mollicutes. Direct testing of cell culture supernatants spiked with Mycoplasma orale revealed similar sensitivity compared to isolated DNA.
CONCLUSIONS
Our single-tube real-time PCR assay with internal reaction control enables rapid and specific detection of mollicute contaminants. The test protocol is suitable for routine quality control of cell therapeutics.
PubMed: 24659951
DOI: 10.1159/000357096 -
Brazilian Journal of Microbiology :... 2014Mycoplasma spp, belongs to the class Mollicutes and is capable to produce alterations in cellular cultures causing damages to the biotechnological industry. Bioproducts...
Mycoplasma spp, belongs to the class Mollicutes and is capable to produce alterations in cellular cultures causing damages to the biotechnological industry. Bioproducts generally require two essential inputs, bovine serum and cells. The study herein aims to evaluate the mycoplasma concentrations that affect the growing of BHK21 and Vero cells. The species used were: Mycoplasma orale, M. salivarium, M. arginini and M. hyorhinis, cultivated in a SP4 media. Two contamination tests were performed with BHK21 and Vero cells and one of them applied different concentrations of mycoplasma. In the first one, mycoplasma was applied at the day zero and, in the second one, the contamination was performed after the monolayer establishment. The both cellular cultures presented cytopathic effects with mycoplasma contamination, but the Vero cells suffered more damages than the BHK21 ones. It was also observed that the severity of the cytopathic effect depended on the mycoplasma specie, on the concentration and on the time of contact with the cellular culture, which evidences the importance of controlling the presence of mycoplasma in biotechnological industries.
Topics: Animals; Cell Line; Chlorocebus aethiops; Coculture Techniques; Cricetinae; Culture Media; Epithelial Cells; Mycoplasma
PubMed: 25763061
DOI: 10.1590/s1517-83822014000400048 -
Research in Microbiology Apr 2013The presence of foreign contamination, especially of mycoplasmas, is a major hindrance in long term in vitro cultivation of Plasmodium falciparum and may be a source of...
The presence of foreign contamination, especially of mycoplasmas, is a major hindrance in long term in vitro cultivation of Plasmodium falciparum and may be a source of false-positive results. Efforts have been made to control mycoplasma contamination by trypsinization of P. falciparum culture. Samples of accidentally contaminated cultures were used for this study. The presence of Mycoplasma orale in contaminated culture was ascertained by a species-specific PCR-based mycoplasma detection kit (Takara; Cat. No.6601). Trypsinization was carried out using trypsin-EDTA and the growth profile of P. falciparum was monitored for more than three weeks post-trypsinization. The studies were carried out with four different P. falciparum strains, various serum supplements and human erythrocytes belonging to different blood groups. It was interesting to observe that, irrespective of the different strains of P. falciparum and the variety of serum supplements and erythrocytes, mycoplasma contamination can successfully be removed from P. falciparum culture by trypsinization. No antibiotic except gentamicin, which is routinely used, was added to the medium. Results of this study indicate that the frequent appearance of mycoplasma in continuous long-term cultures of P. falciparum can be managed by trypsinization.
Topics: Anti-Bacterial Agents; Cell Culture Techniques; Culture Media; Erythrocytes; Gentamicins; Humans; Mycoplasma orale; Plasmodium falciparum; Trypsin
PubMed: 23277231
DOI: 10.1016/j.resmic.2012.12.010 -
BMC Research Notes Jan 2012During the routine laboratory cultivation of Lawsonia intracellularis, Mycoplasma contamination has been a frequent problem. When Mycoplasma contamination occurs in...
BACKGROUND
During the routine laboratory cultivation of Lawsonia intracellularis, Mycoplasma contamination has been a frequent problem. When Mycoplasma contamination occurs in laboratories that study L. intracellularis, the cultures must be discarded for 4 reasons: 1) Mycoplasma is inevitably concentrated along with L. intracellularis during the passage of L. intracellularis; 2) Mycoplasma inhibits the growth of L. intracellularis; and 3) it is impossible to selectively eliminate Mycoplasma in L. intracellularis cultures. In this study, we observed the contamination of Mycoplasma species during L. intracellularis cultivation among multiple laboratories.
RESULTS
The presence of a Mycoplasma infection in the L. intracellularis cultures was verified using polymerase chain reaction (PCR), and a sequence analysis of the partial 16S rRNA and 23S rRNA genes was performed. A PCR-based assay using genus-specific universal primers revealed that 29 (85.3%) of the 34 cultures were contaminated with Mycoplasma, including 26 with M. hyorhinis (89.2%), 2 with M. orale (6.9%), and 1 with M. fermentans (3.4%). The Mycoplasma contamination was not the result of infection with material of pig origin. McCoy cells, which are required for the cultivation of L. intracellularis, were also ruled out as the source of the Mycoplasma contamination.
CONCLUSIONS
In this study, M. hyorhinis was identified as the most common mollicute that contaminated L. intracellularis cultures. Whether L. intracellularis enhances the biological properties of Mycoplasma to promote infection in McCoy cells is not known. Because the McCoy cell line stocks that were used simultaneously were all negative for Mycoplasma, and the same worker handled both the McCoy cells to maintain the bacteria and the L. intracellularis cultures, it is possible that the L. intracellularis cultures are more vulnerable to Mycoplasma contamination. Taken together, these results suggest that continuous cultures of L. intracellularis must be tested for Mycoplasma contamination at regular intervals.The GenBank accession numbers for the sequences reported in this paper are JN689375 to JN689377.
PubMed: 22284165
DOI: 10.1186/1756-0500-5-78 -
Journal of Applied Microbiology Oct 2011To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC)...
AIMS
To optimize growth conditions for preparation of stocks of mycoplasma reference strains to obtain highly viable and disperse samples with low ratios of genomic copy (GC) number to that of colony forming units (CFU). These stocks are required for assessment of relative limits of detection (LOD) of alternative nucleic acid testing (NAT)-based methods in comparison to the conventional microbiological methods.
METHODS AND RESULTS
A kinetics study was used to assess the changes in ratios between the numbers of GC and CFU at different growth phases of six different mycoplasma cultures Acholeplasma laidlawii, Mycoplasma gallisepticum, Mycoplasma arginini, Mycoplasma fermentans, Mycoplasma orale and Mycoplasma pneumoniae. All tested mycoplasmas demonstrated low GC/CFU ratios (≤ 10) within the log and early stationary growth phases. A significant increase in GC/CFU ratios was observed at the very late stationary and death phases, when the titre of cultures has declined. Similar patterns of GC/CFU profiles were observed for A. laidlawii and Myc. gallisepticum co-cultured with suspension of Chinese hamster ovary (CHO) cells.
CONCLUSIONS
Tested mycoplasma strains harvested at the exponential-early stationary phases of growth demonstrated the lowest GC/CFU ratios and low propensity to form filamentous structures or aggregates under proposed conditions and can be used for the preparation of a mycoplasma reference panel for methods comparability study.
SIGNIFICANCE AND IMPACT OF THE STUDY
This study shows that the preparation and use of viable mycoplasma reference strains with low CG/CFU ratios is the most reliable way to adequately evaluate the LOD of alternative NAT-based mycoplasma testing methods.
Topics: Animals; Bacteriological Techniques; CHO Cells; Coculture Techniques; Colony Count, Microbial; Cricetinae; Cricetulus; DNA, Bacterial; Evaluation Studies as Topic; Gene Dosage; Limit of Detection; Mycoplasma; Polymerase Chain Reaction; Reference Standards; Validation Studies as Topic
PubMed: 21794032
DOI: 10.1111/j.1365-2672.2011.05108.x -
Journal of Applied Microbiology Jan 2011To assess the limit of detection (LOD) and the feasibility of 16S rRNA-based reverse transcription-PCR (RT-PCR) assays for advanced detection of mycoplasma contamination...
AIMS
To assess the limit of detection (LOD) and the feasibility of 16S rRNA-based reverse transcription-PCR (RT-PCR) assays for advanced detection of mycoplasma contamination in cell substrates.
MATERIALS AND METHODS
The RT-PCR approach is based on detecting the 16S rRNA molecules that, in contrast to genomic bacterial DNA, are represented by multiple copies in mycoplasma cell. The number of 16S rRNA molecules in mycoplasma cells of five species i.e. Mycoplasma arginini, Myc. fermentans, Myc. hyorhinis, Myc. orale and Acholeplasma laidlawii, all known to be frequent cell line contaminants in industrial and research laboratories, was measured using molecular methods. The results of two independently prepared mycoplasma cultures harvested at the stationary phase of their growth showed that the 16S rRNA copy number per cell varied in the range from about 400 to 2000 copies, depending on species, but stayed close between different preparations of one species. The assessment of the LOD of the in-house 16S rRNA-based RT-PCR was performed using samples of MDCK cell culture spiked with different amounts of five aforementioned mycoplasma species. To minimize the bias in methods comparison, the LOD of the RT-PCR assay was expressed in terms of genome equivalents (GEs) and compared with that determined for highly optimized 16S rDNA-based mycoplasma testing methods previously described in scientific literature.
CONCLUSIONS
The results of the study showed that the in-house 16S rRNA-based RT-PCR assay was able to reliably detect the presence of less than one mycoplasma GE that is at least 10-fold higher of the LOD previously determined for well-optimized 16S rDNA-based assays developed and described by other researchers.
SIGNIFICANCE AND IMPACT OF THE STUDY
The results of the study showed that rapid RT-PCR methods based on the detection of bacterial 16S rRNA are able to expedite mycoplasma testing of cell cultures (1-2 days vs 28 days) and to ensure the limits of detection comparable to that of currently used culture-based mycoplasma testing methods.
Topics: Animals; Cell Line; Limit of Detection; Mycoplasma; RNA, Ribosomal, 16S; Reverse Transcriptase Polymerase Chain Reaction
PubMed: 20854458
DOI: 10.1111/j.1365-2672.2010.04853.x