Detection of Mycoplasma Contamination in Transplanted Retinal Cells by Rapid and Sensitive Polymerase Chain Reaction Test

Contamination of cells/tissues by infectious pathogens (e.g., fungi, viruses, or bacteria, including mycoplasma) is a major problem in cell-based transplantation. In this study, we tested a polymerase chain reaction (PCR) method to provide rapid, simple, and sensitive detection of mycoplasma contamination in laboratory cultures for clinical use. This mycoplasma PCR system covers the Mycoplasma species (spp.) listed for testing in the 17th revision of the Japanese Pharmacopoeia, and we designed it for use in transplantable retinal cells. Here, we analyzed mycoplasma contamination in induced pluripotent stem cell (iPS cell)-derived transplantable retinal pigment epithelium (RPE) cells. In the spike tests to RPE cells with nine species of class Mollicutes bacteria, including seven Mycoplasma spp. and one of each Acholeplasma spp. and Ureaplasma spp., contamination at the concentration of 100 and 10 CFU/mL were detected with 100% probability in all cases, while 1 CFU/mL had a detection rate of 0–75%. DNA prepared from bacteria species other than class Mollicutes species was not detectable, indicating the specificity of this PCR. While iPS cells and iPS-RPE cells established in our laboratory were all negative by this PCR, some of the commercially available cell lines were positive. Cells for transplantation should never have infection, as once pathogens are implanted into the eyes, they can cause severe intraocular inflammation. Thus, it is imperative to monitor for infections in the transplants, although generally, mycoplasma infection is difficult to detect.

273. Neurological Involvement Caused by Intracellular Bacteria

Background Infection of the central nervous system is a severe and fatal disease. Causative agents include bacteria, viruses or fungi. Intracellular bacteria are not only overlooked, but also underdiagnosed. We aimed to study the clinical, laboratory and evolutionary features of neurological involvement caused by intracellular bacteria. Methods We conducted a retrospective study including all patients hospitalized in the infectious disease department for neurological involvement caused by intracellular bacteria between 1995 and 2020. The diagnosis was confirmed by serology. Results We encountered 76 cases among which 43 were males (56.6%). The mean age was 32±18 years. The revealing symptoms included fever (97.4%), cephalalgia (73.7%), vomiting (64.5%) and arthralgia (51.3%). Lumbar puncture revealed a median white blood cell count of 120[56-340]/mm3. Lymphocytic pleocytosis was noted in 62% of the cases. Elevated cerebrospinal fluid (CSF) protein level was noted in 37 cases (48.7%) with a median of 0.84[0.6-1.37] g/L. Low CSF fluid glucose level was noted in 14 cases (18.4%). There were 70 cases (92.1%) of meningitis and 6 cases of meningoencephalitis (7.9%). The causative agent included Rickettsia species in 47 cases (61.8%), Brucella species in 17 cases (22.4%) and Mycoplasma species in 12 cases (15.8%). Laboratory investigations included elevated C-reactive protein levels (40.7%), thrombocytopenia (32.8%) and increase in hepatic enzyme levels (21%). Anemia was noted in 27 cases (35.5%), leukocytosis in 24 cases (31.5%) and leucopoenia in 6 cases (7.8%). Blood and CSF cultures were positive for Brucella in 2 cases (2.6%) and 5 cases (6.5%), respectively. The mean duration of treatment was 156±94 days for brucellosis cases, 9±4 days for rickettsiosis cases and 10±6 days for Mycoplasma cases. The disease evolution was favorable in 72 cases (94.7%). Four patients were dead (5.3%). Complications were noted in 5 cases (6.5%) and sequelae in 2 cases (2.6%). Conclusion Intracellular bacteria including Brucella, Rickettsia and Mycoplasma species should be considered in front of neurological symptoms. Meningitis with lymphocytic pleocytosis was the most common clinical presentation. An early diagnosis followed by the adequate treatment might avoid complications and death. Disclosures All Authors: No reported disclosures

Minimalistic mycoplasmas harbor different functional toxin-antitoxin systems

Mycoplasmas are minute bacteria controlled by very small genomes ranging from 0.6 to 1.4 Mbp. They encompass several important medical and veterinary pathogens that are often associated with a wide range of chronic diseases. The long persistence of mycoplasma cells in their hosts can exacerbate the spread of antimicrobial resistance observed for many species. However, the nature of the virulence factors driving this phenomenon in mycoplasmas is still unclear. Toxin-antitoxin systems (TA systems) are genetic elements widespread in many bacteria that were historically associated with bacterial persistence. Their presence on mycoplasma genomes has never been carefully assessed, especially for pathogenic species. Here we investigated three candidate TA systems in M. mycoides subsp. capri encoding a (i) novel AAA-ATPase/subtilisin-like serine protease module, (ii) a putative AbiEii/AbiEi pair and (iii) a putative Fic/RelB pair. We sequence analyzed fourteen genomes of M. mycoides subsp. capri and confirmed the presence of at least one TA module in each of them. Interestingly, horizontal gene transfer signatures were also found in several genomic loci containing TA systems for several mycoplasma species. Transcriptomic and proteomic data confirmed differential expression profiles of these TA systems during mycoplasma growth in vitro. While the use of heterologous expression systems based on E. coli and B. subtilis showed clear limitations, the functionality and neutralization capacities of all three candidate TA systems were successfully confirmed using M. capricolum subsp. capricolum as a host. Additionally, M. capricolum subsp. capricolum was used to confirm the presence of functional TA system homologs in mycoplasmas of the Hominis and Pneumoniae phylogenetic groups. Finally, we showed that several of these M. mycoides subsp. capri toxins tested in this study, and particularly the subtilisin-like serine protease, could be used to establish a kill switch in mycoplasmas for industrial applications.

Identification of Mycoplasma species and related organisms from ruminants in England and Wales during 2005–2019

Background Mycoplasma species have been associated with economically important diseases affecting ruminants worldwide and include contagious bovine pleuropneumonia (CBPP), contagious caprine pleuropneumonia (CCPP) and contagious agalactia, listed by the World Organisation for Animal Health (OIE). The Mycoplasma Team at the Animal and Plant Health Agency provides an identification service for Mycoplasma and Ureaplasma species of veterinary importance to the United Kingdom (UK), supporting the detection of new and emerging pathogens, as well as contributing to the surveillance of endemic, and the OIE listed diseases exotic to the UK. Mycoplasma and other Mollicutes species were identified from diagnostic samples from farmed ruminants in England and Wales using a combination of culture and 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis, submitted between 2005 and 2019. Results A total of 5578 mollicutes identifications, which include mycoplasmas and the related acholeoplasmas and ureaplasmas, were made from farmed ruminant animals during the study period. Throughout the study period, the pathogen Mycoplasma bovis was consistently the most frequently identified species, accounting for 1411 (32%) of 4447 molecular identifications in cattle, primarily detected in the lungs of pneumonic calves, followed by joints and milk of cattle showing signs of arthritis and mastitis, respectively. M. bovirhinis, M. alkalescens, M. dispar, M. arginini and Ureaplasma diversum, were also common. Mixed species, principally M. bovis with M. alkalescens, M. arginini or M. bovirhinis were also prevalent, particularly from respiratory samples. The non-cultivable blood-borne haemoplasmas Candidatus ‘Mycoplasma haemobos’ and Mycoplasma wenyonii were identified from cattle, with the latter species most often associated with milk-drop. M. ovipneumoniae was the predominant species identified from sheep and goats experiencing respiratory disease, while M. conjunctivae preponderated in ocular samples. The UK remains free of the ruminant mycoplasmas listed by OIE. Conclusions The continued high prevalence of M. bovis identifications confirms its ongoing dominance and importance as a significant pathogen of cattle in England and Wales, particularly in association with respiratory disease. M. ovipneumoniae has seen a general increase in prevalence in recent years, notably in coughing lambs and should therefore be considered as a primary differential diagnosis of respiratory disease in small ruminants.

Septic arthritis due to Mycoplasma orale in a young patient with hypogammaglobulinemia

Mycoplasma orale 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 M. orale 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 past medical history was significant for Burkitt’s lymphoma, the treatment of which had included rituximab 6 years earlier. M. orale 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. Mycoplasma species should be suspected in patients with humoral immunodeficiency and compatible findings.

Integrating the Human and Animal Sides of Mycoplasmas Resistance to Antimicrobials

Mycoplasma infections are frequent in humans, as well as in a broad range of animals. However, antimicrobial treatment options are limited, partly due to the lack of a cell wall in these peculiar bacteria. Both veterinary and human medicines are facing increasing resistance prevalence for the most commonly used drugs, despite different usage practices. To date, very few reviews have integrated knowledge on resistance to antimicrobials in humans and animals, the latest dating back to 2014. To fill this gap, we examined, in parallel, antimicrobial usage, resistance mechanisms and either phenotype or genotype-based methods for antimicrobial susceptibility testing, as well as epidemiology of resistance of the most clinically relevant human and animal mycoplasma species. This review unveiled common features and differences that need to be taken into consideration in a “One Health” perspective. Lastly, two examples of critical cases of multiple drug resistance are highlighted, namely, the human M. genitalium and the animal M. bovis species, both of which can lead to the threat of untreatable infections.

Clinical, histopathological, and molecular characterization of Mycoplasma species in sheep and goats in Egypt

Background and Aim: Mycoplasma infection in small ruminants is a serious problem in sheep and goat herds around the world. It is responsible for high economic losses and decreased animal productivity. This study aimed to highlight the clinical, histopathological, minimum inhibitory concentration (MIC), and molecular characterization of Mycoplasma species in sheep and goats in Menoufiya Governorate, Egypt. Materials and Methods: A total of 234 samples were collected; 104 samples were collected from pneumonic lung tissues from the abattoir, in addition, 10 and 20 samples collected from apparently and diseased sheep, respectively, and 40 and 60 samples were collected from apparently and diseased goats, respectively, which were subjected to isolation onto pleuropneumonia-like organism medium. Polymerase chain reaction (PCR), histopathological examination, and determination of the MIC were also performed. Results: Of 104 samples of lung tissues showing pneumonic lesions, 56 (53.84%) were positive for Mycoplasma isolation. The positive isolation of Mycoplasma from 10 and 20 samples from apparently and diseased sheep was 30% and 40%, respectively as well as the positive isolation of Mycoplasma was 17% and 56.66% out of 40 and 60 apparently healthy and diseased field goat's cases, respectively. All the diseased sheep and goats showed respiratory manifestations, including cough, bilateral nasal discharge, conjunctivitis, and systemic reaction. Evaluation of the MIC for Mycoplasma ovipneumoniae revealed that lincospectin and tylosin were the most effective antibiotics at 2.5 μg/mL. Histopathological examination of affected lung tissue showed extensive hemorrhagic pneumonia with extensive alveolar hemorrhage. The PCR technique proved to be a rapid, specific, and sensitive method for the detection of M. ovipneumoniae and Mycoplasma arginini at 390 and 326 bp, respectively. Conclusion: M. ovipneumoniae and M. arginini were the most prevalent species associated with respiratory infections in sheep and goats in the study area. Further studies are needed to investigate the role of these species in dissemination of the disease within herds of small ruminants.