-
Cold Spring Harbor Protocols Jul 2021Because mycoplasmas are a diverse group of organisms and are difficult to culture, several different strategies for detecting mycoplasma contamination have been...
Because mycoplasmas are a diverse group of organisms and are difficult to culture, several different strategies for detecting mycoplasma contamination have been developed. To date, no one test is suitable for detecting all of the possible mycoplasmas that may contaminate hybridoma or myeloma cultures. Therefore, it is sensible to consider using several methods. The most commonly used techniques are described here. screening by growth on microbial medium can be performed on agar plates or in broth culture. Cultures are grown under both aerobic and anaerobic conditions because some common strains of prefer the lack of oxygen (, , , and ). colonies form a characteristic "fried egg" appearance on agar plates, and this is the diagnostic feature used to confirm mycoplasma contamination. The colonies are small and are most easily seen with an inverted microscope. A quicker method for testing for mycoplasma takes advantage of the DNA-intercalating dye Hoechst 33258. Fixed cells are stained with the dye, and contaminated cultures are detected by the bright, punctate cytoplasmic staining of the DNA. Finally, commercial kits for the detection of mycoplasmas using colorimetric assays or reporter cells are also described.
Topics: Culture Media; Hybridomas; Mycoplasma
PubMed: 34210772
DOI: 10.1101/pdb.prot103283 -
Open Forum Infectious Diseases Sep 2022is a rare cause of invasive infection in immunodeficient hosts. Phosphatidylinositol 3-kinase, regulatory subunit 1 () mutations predispose patients to sinopulmonary...
is a rare cause of invasive infection in immunodeficient hosts. Phosphatidylinositol 3-kinase, regulatory subunit 1 () mutations predispose patients to sinopulmonary infections, alongside bronchiectasis autoimmunity and lymphoproliferation. We report 2 cases of deficiency with invasive and effective treatment options.
PubMed: 36196300
DOI: 10.1093/ofid/ofac472 -
PDA Journal of Pharmaceutical Science... 2020Capture bioprocessing unit operations were previously shown to clear or kill several log of a model mycoplasma in lab-scale spike/removal studies. Here, we confirm this...
Capture bioprocessing unit operations were previously shown to clear or kill several log of a model mycoplasma in lab-scale spike/removal studies. Here, we confirm this observation with two additional mollicute species relevant to biotechnology products for human use: and Clearance of and from protein A column purification was similar to that seen with , though some between cycle carryover was evident, especially for However, on-resin growth studies for all three species revealed that residual mycoplasma in a column slowly die off over time rather than expanding further. Solvent/detergent exposure completely inactivated though detectable levels of remained. A small-scale model of a commercial low-pH hold step did inactivate live , but this inactivation required a lower pH set point and occurred with slower kinetics than previously seen with Additionally, ultraviolet-C irradiation was shown to be effective for and inactivation whereas virus-retentive filters for upstream and downstream processes, as expected, cleared These data argue that and overall would be largely cleared by early bioprocessing steps as shown previously for and that barrier technologies can effectively reduce the risk from media components. For some unit operations, and may be hardier, and require more stringent processing or equipment cleaning conditions to assure effective mycoplasma reduction. By exploring how some of the failure modes in commercial antibody manufacturing processes can still eliminate mycoplasma burden, we demonstrate that required best practices assure biotechnology products will be safe for patients.
Topics: Animals; CHO Cells; Chemistry, Pharmaceutical; Coculture Techniques; Cricetinae; Cricetulus; Drug Contamination; Mycoplasma; Mycoplasma orale
PubMed: 31519782
DOI: 10.5731/pdajpst.2018.009613 -
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 -
Case Reports in Infectious Diseases 2020A young woman with mixed connective tissue disease complicated by erosive arthritis, secondary hypogammaglobulinemia due to rituximab, and a history of many infectious...
A young woman with mixed connective tissue disease complicated by erosive arthritis, secondary hypogammaglobulinemia due to rituximab, and a history of many infectious complications developed multiple nonhealing wounds, polyarticular joint pain, and leukocytosis. Radiographic studies demonstrated multiple scattered areas of osteomyelitis and complex abscesses. Purulent fluid drained from multiple sites did not yield a microbiologic diagnosis by standard culture technique, but was ultimately identified using 16 S ribosomal RNA gene amplification and sequencing. We describe this unique case and review the literature.
PubMed: 32850161
DOI: 10.1155/2020/8852115 -
Current Microbiology Jan 2021Mycoplasma is the smallest self-replicating bacteria, figuring as common contaminant of eukaryotic cell cultures. Production inputs and operator's manipulation seem to...
Mycoplasma is the smallest self-replicating bacteria, figuring as common contaminant of eukaryotic cell cultures. Production inputs and operator's manipulation seem to be the main sources of such contamination. Many analytical approaches have been applied for mycoplasma detection in cell cultures and also in biological products. However, unless they were validated, only indicator cell culture and bacteriological culture are considered as compendial methods for quality control of biological products. Nano-flow cytometry has been pointed out as an alternative technique for addressing prokaryotic and eukaryotic cell viability being a substantial tool for reference material production. In this study, a viability-flow-cytometry assay was standardized for M. gallisepticum and then applied to other cell-culture-contaminant mycoplasmas. For this, M. galliseticum's growth rate was observed and different treatments were evaluated to establish low viability cultures (cell death-induced control). Distinct viability markers and their ideal concentrations (titration) were appraised. Ethanol treatment showed to be the best death-inducing control. CFDA and TOPRO markers revealed to be the best choice for detecting live and dead mycoplasma frequencies, respectively. The standardized methodology was applied to Mycoplasma arginini, M. hyorhinis, M. orale, Spiroplasma citri and Acholeplasma laidlawii. Significant statistical difference was observed in the percentage of viable cells in comparison to ethanol treatment for A. laidlawii in CFDA and in both markers for M. gallisepticum, M. hyorhinis and S. citri. In summary, we standardized a flow cytometry assay for assessing M. gallisepticum - and potentially other species - viability and ultimately applied for reference material production improving the quality control of biological products.
Topics: Cell Culture Techniques; Flow Cytometry; Mycoplasma; Mycoplasma gallisepticum; Tenericutes
PubMed: 33159562
DOI: 10.1007/s00284-020-02255-1 -
Journal of the Association of Medical... Dec 2021is an obligate intracellular bacterium usually found as a commensal in the human oral cavity. Symptomatic infections with this organism are rare, but severe disease has...
is an obligate intracellular bacterium usually found as a commensal in the human oral cavity. Symptomatic infections with this organism are rare, but severe disease has been described in the setting of impaired humoral immunity. Here, we describe a case in which was identified from the joint fluid of a patient with septic arthritis, splenic lesions, and agammaglobulinemia. A 15-year-old boy was admitted to the hospital with fever, progressive left knee swelling, and pain. His medical history was significant for Burkitt's lymphoma, the treatment of which had included rituximab 6 years earlier. was identified in the synovial fluid using 16S ribosomal RNA gene sequencing. He was also found to be hypogammaglobulinemic, and imaging revealed multiple splenic lesions. He was treated with doxycycline and intravenous immunoglobulin, which resulted in complete resolution of his arthritis and other symptoms. species should be suspected in patients with humoral immunodeficiency and compatible findings.
PubMed: 36338458
DOI: 10.3138/jammi-2021-0002 -
Cell Discovery Apr 2021Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of Coronavirus disease 2019 (COVID-19). However, the microbial composition of...
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a global pandemic of Coronavirus disease 2019 (COVID-19). However, the microbial composition of the respiratory tract and other infected tissues as well as their possible pathogenic contributions to varying degrees of disease severity in COVID-19 patients remain unclear. Between 27 January and 26 February 2020, serial clinical specimens (sputum, nasal and throat swab, anal swab and feces) were collected from a cohort of hospitalized COVID-19 patients, including 8 mildly and 15 severely ill patients in Guangdong province, China. Total RNA was extracted and ultra-deep metatranscriptomic sequencing was performed in combination with laboratory diagnostic assays. We identified distinct signatures of microbial dysbiosis among severely ill COVID-19 patients on broad spectrum antimicrobial therapy. Co-detection of other human respiratory viruses (including human alphaherpesvirus 1, rhinovirus B, and human orthopneumovirus) was demonstrated in 30.8% (4/13) of the severely ill patients, but not in any of the mildly affected patients. Notably, the predominant respiratory microbial taxa of severely ill patients were Burkholderia cepacia complex (BCC), Staphylococcus epidermidis, or Mycoplasma spp. (including M. hominis and M. orale). The presence of the former two bacterial taxa was also confirmed by clinical cultures of respiratory specimens (expectorated sputum or nasal secretions) in 23.1% (3/13) of the severe cases. Finally, a time-dependent, secondary infection of B. cenocepacia with expressions of multiple virulence genes was demonstrated in one severely ill patient, which might accelerate his disease deterioration and death occurring one month after ICU admission. Our findings point to SARS-CoV-2-related microbial dysbiosis and various antibiotic-resistant respiratory microbes/pathogens in hospitalized COVID-19 patients in relation to disease severity. Detection and tracking strategies are needed to prevent the spread of antimicrobial resistance, improve the treatment regimen and clinical outcomes of hospitalized, severely ill COVID-19 patients.
PubMed: 33850111
DOI: 10.1038/s41421-021-00257-2 -
Journal of Thoracic Disease Feb 2022The current COVID-19 pandemic is posing a major challenge to public health on a global scale. While it is generally believed that severe COVID-19 results from...
BACKGROUND
The current COVID-19 pandemic is posing a major challenge to public health on a global scale. While it is generally believed that severe COVID-19 results from over-expression of inflammatory mediators (i.e., a "cytokine storm"), it is still unclear whether and how co-infecting pathogens contribute to disease pathogenesis. To address this, we followed the entire course of the disease in cases with severe or critical COVID-19 to determine the presence and abundance of all potential pathogens present-the total "infectome"-and how they interact with the host immune system in the context of severe COVID-19.
METHODS
We examined one severe and three critical cases of COVID-19, as well as a set of healthy controls, with longitudinal samples (throat swab, whole blood, and serum) collected from each case. Total RNA sequencing (meta-transcriptomics) was performed to simultaneously investigate pathogen diversity and abundance, as well as host immune responses, in each sample. A Bio-Plex method was used to measure serum cytokine and chemokine levels.
RESULTS
Eight pathogens, SARS-CoV-2, (), (), (), (), , herpes simplex virus (HSV) and human cytomegalovirus (CMV), identified in patients with COVID-19 appeared at different stages of the disease. The dynamics of inflammatory mediators in serum and the respiratory tract were more strongly associated with the dynamics of the infectome compared with SARS-CoV-2 alone. Correlation analysis revealed that pulmonary injury was directly associated with cytokine levels, which in turn were associated with the proliferation of SARS-CoV-2 and co-infecting pathogens.
CONCLUSIONS
For each patient, the cytokine storm that resulted in acute lung injury and death involved a dynamic and highly complex infectome, of which SARS-CoV-2 was a component. These results indicate the need for a precision medicine approach to investigate both the infection and host response as a standard means of infectious disease characterization.
PubMed: 35280492
DOI: 10.21037/jtd-21-1284 -
Diagnostics (Basel, Switzerland) May 2021, , and sp. are atypical bacteria responsible for in vitro cell culture contaminations that can warp the results. These bacteria also cause human and animal infections...
, , and sp. are atypical bacteria responsible for in vitro cell culture contaminations that can warp the results. These bacteria also cause human and animal infections and may lead to chronic diseases. In developed polymerase chain reaction (PCR) in this study a quantitative PCR with SYBR Green I fluorochrome was applied to facilitate the , , and sp. DNA detection and identification. Screening Test-1 v.1 (triplex qPCR) allowed for the detection of 11 species. Test-1 v.2 (three single qPCRs) pre-identified three subgroups, allowing for the reduction of using single qPCRs in Test-2 for species identification. The range of both tests was consistent with pharmacopeial requirements for microbial quality control of mammal cells and included detection of , , , , , , , , , , and . Limit of detection values varied between 125-300 and 50-100 number of copies per milliliter in Test-1 and Test-2, respectively. Test-1 and Test-2 showed fully concordant results, allowed for time-saving detection and/or identification of selected species from , , and in tested cell cultures.
PubMed: 34068904
DOI: 10.3390/diagnostics11050876