Mycoplasma Species Research Articles

Prevalence and Molecular Characterization of Mycoplasma Species, Pasteurella multocida, and Staphylococcus aureus Isolated from Calves with Respiratory Manifestations.

Simple SummaryRespiratory infection is one of the most common problems facing the livestock industry in Egypt, and results in substantial economic losses. Several bacterial pathogens are implicated in respiratory infections in young calves. In our study, Mycoplasma bovis, Mycoplasma bovigenitalium, Pasteurella multocida, and Staphylococcus aureus were the most common bacterial pathogens isolated from calves suffering from respiratory manifestations in Menoufiya Governorate, Egypt. However, the results revealed a low prevalence of these pathogens compared to other studies carried out in Egypt. This may be due to the low number of samples or application of vaccination programs in the study area. Mixed infection is the main perceived criteria in this study. The genetic characterization of these pathogens revealed the high identity and similarities within several global and local linages, proposing the probability of disseminating these bacterial pathogens through several routes during animal contact and movements and animal trading between different geographic locations around the world.Bovine respiratory disease (BRD) is a complex syndrome associated with high mortality in young calves and causes severe economic losses in the cattle industry worldwide. The current study investigated the prevalence and molecular characterization of common bacterial pathogens associated with respiratory symptoms in young calves from Sadat City, one of the largest industrial cities in Menoufiya Governorate, Egypt. In between December 2020 and March 2021, 200 mixed-breed young calves of 6–12 months were examined clinically. Of them, sixty (30%) calves showed signs of respiratory manifestations, such as coughing, serous to mucopurulent nasal discharges, fever, and abnormal lung sound. Deep nasal (Nasopharyngeal) swabs were collected from the affected calves for bacteriological investigation. Phenotypic characterization and identification revealed Mycoplasma bovis, Mycoplasma bovigenitalium, Pasteurella multocida, and Staphylococcus aureus in 8.33%, 5%, 5%, and 5% of the tested samples, respectively. The PCR technique using species-specific primer sets successfully amplified the target bacterial DNA in all culture-positive samples, confirming the identity of the isolated bacterial species. Partial gene sequencing of 16S rRNA gene of M. bovigenitalium, P. multocida, and S. aureus, and mb-mp 81 gene of M. bovis revealed high nucleotide similarity and genetic relationship with respective bacterial species reported from Egypt and around the world, suggesting transmission of these bacterial species between animal host species and localities. Our study highlights the four important bacterial strains associated with respiratory disorders in calves and suggests the possible spread of these bacterial pathogens across animal species and different geographic locations. Further studies using WGS and a large number of isolates are required to investigate the realistic lineage of Egyptian isolates and globally.

Prevalence of Genital Mycoplasma in Pregnancies with Shortened Cervix

Objective: To determine whether colonization with genital Mycoplasma species (spp.) in patients presenting with a shortened cervix before 34th week of pregnancy is associated with preterm birth. Methods: The collective of this retrospective study consisted of 100 pregnant women who presented to a German Tertiary Perinatal Center between 2017 and 2020 due to a shortened cervix defined as a cervical length of 25mm or shorter measured by transvaginal ultrasound before 34 weeks of gestation. At the time of admission gestational age ranged from 18+4 and 33+3 weeks of pregnancy. All patients underwent urine Polymerase Chain Reaction (PCR) for genital Mycoplasma (Ureaplasma (U.) urealyticum, U. parvum, M. hominis or M. genitalium). Patients who were tested positive underwent a therapy with macrolides (azithromycin or clarithromycin). Results: 37% of the patients were positive for Ureaplasma spp. whereas 5% (5 patients) were Mycoplasma spp positive. All the latter were simultaneously colonized with Ureaplasma spp. Ureaplasma positive patients were significantly younger than those who were tested negative. Median maternal age at examination was 30 years (a) versus 31a (p=0.04). There was no difference between Ureaplasma positive and negative patients regarding median maternal body-mass-index (BMI) (kg/m²) (23.4 versus 22.3, p=0.41), cervical length at admission (mm) (15 versus 17, p=0.17), gestational age at examination (days, d) (198 versus 197, p=0.97) or gestational age at birth (d) (250 versus 257, p=0.33), respectively. Comparing U. parvum positive and U. urealyticum positive patients, there was some weak indication that U. parvum positive patients may get a shortening of the cervix earlier in pregnancy, as the median gestational age at examination was 196d versus 215d (p=0.06). Regarding Mycoplasma positive and negative patients, there was no difference in all examined parameters. Conclusions: Overall, one third of all women in our study with a shortened cervix before 34th week of pregnancy were colonized with genital Mycoplasma spp. We were able to show that pregnant women, who were treated with antibiotics when tested-positive for genital Mycoplasma, gave birth at the same gestational age as patients with a shortened cervix without detected Mycoplasma. This raises the question of whether routine testing and early antibiotic treatment should be established in prenatal care.

Evidence for the Rapid and Divergent Evolution of Mycoplasmas: Structural and Phylogenetic Analysis of Enolases.

Mycoplasmas are a group of prokaryotes without cell walls that have evolved through several rounds of degenerative evolution. With a low cell DNA G + C content and definitively long genetic lineages, mycoplasmas are thought to be in a state of rapid evolution. However, little associated evidence has been provided. Enolase is a key enzyme in glycolysis that is widely found in all species from the three domains, and it is evolutionarily conserved. In our previous studies, enolase acted as a virulence factor and participated in cell-surface adhesion in Mycoplasma hyopneumoniae. Furthermore, unique loop regions were first found in the crystal structure of Mhp Eno. Here, enolase structures from Mycoplasma pneumoniae and Mycoplasma bovis were determined. An extra helix 7 is specific and conservatively found in almost all mycoplasma enolases, as confirmed by crystal structures and sequence alignment. Particular motifs for helix 7, which is composed of F-K/G-K-L/F-K-X-A-I, have been proposed and could be regarded as molecular markers. To our surprise, the genetic distances between any two mycoplasma enolases were obviously longer than those between the two corresponding species themselves, indicating divergent evolution of mycoplasma enolases, whereas no horizontal gene transfer was detected in mycoplasma enolase genens. Furthermore, different evolutionary patterns were adopted by different loop regions of mycoplasma enolase. Enolases from different Mycoplasma species also showed different affinities for PLG and fibronectin. Our results indicate the rapid and divergent evolution of mycoplasma enolase and mycoplasmas. This study will also aid understanding the independent evolution of Mycoplasma species after separation from their common ancestor.

Occurrence of \u2018Candidatus Mycoplasma haemosuis\u2019 in fattening pigs, sows and piglets in Germany using a novel gap-based quantitative real-time PCR assay

BackgroundThe appearance of the novel porcine haemotrophic mycoplasma (HM) species ‘Candidatus Mycoplasma haemosuis’ was reported in apparently healthy but also in clinically sick animals in China, Korea and in a case report from Germany. Outside of Asia, however, nothing further is known about the frequency of ‘Ca. M. haemosuis’ in pigs to date. To investigate the distribution of this novel HM species in Germany, fattening pigs, sows and pre-suckling piglets were examined using a herein developed quantitative real-time PCR assay (qPCR). Because the piglets were sampled before the first colostrum uptake, additional information on a possible vertical transmission from dams to their offspring was obtained.ResultsOur novel qPCR assay successfully detected ‘Ca. M. haemosuis’ in all blood samples from the ‘Ca. M. haemosuis’-infected pigs. No cross-reactivity was detected when DNA from non-target Mycoplasma spp. and other bacterial species representing 105 bacteria/reaction were used as a template. The lower limit of detection of the qPCR was thus 10 gap gene copies per reaction and 2.5 x 103 genome equivalents (GE) per mL blood.‘Candidatus M. haemosuis’ was detected by this qPCR in blood samples from a total out of 6.25% sows (13/208), 4.50% pre-suckling piglets (28/622) and 17.50% fattening pigs (35/200). On farm level, 3 out of 21 piglet producing farms (14.28%) and 9 out of 20 fattening farms (45.00%) were positive for ‘Ca. M. haemosuis’. Co-infections with M. suis were evident in all age groups.Conclusion‘Candidatus M. haemosuis’ infection is present in German pig farms and the detection of the novel porcine HM species in piglets immediately after birth before colostrum intake indicates vertical transmission. The novel qPCR assay specific for ‘Ca. M. haemosuis’ described herein will be a prerequisite for future studies on the prevalence, epidemiology as well as the clinical and economic impact of ‘Ca. M. haemosuis’ infections.

Isolation of Mycoplasma spp. from Geese with Pneumonia and Identification of Microbial Isolates via Molecular Methods

This study aimed to investigate Mycoplasma species in the lungs of 500 geese with pneumonia from the Kars region (Turkey) via cultural and molecular methods. The samples were cultured on Frey’s Broth and Agar media. To identify Mycoplasma species a Growth Inhibition Test was used. The identification was continued with species-specific PCR and sequence analysis which provide amplification of the genes dnaX, pcrA, rpoB, and the sequence of the 16S rRNA gene, respectively. In addition, Mycoplasma gallisepticum and Mycoplasma synoviae from pneumonic lung samples were directly analyzed via Multiplex Real-time PCR. As a result, 51 Mycoplasma strains were isolated and 32 were identified as Mycoplasma anatis, 9 as Mycoplasma anseris, 5 as Mycoplasma cloacale and 3 as Mycoplasma anserisalpingitis. Two Mycoplasma isolates that could not be identified were grouped in the same branch as a result of 16S RNA sequencing and their nearest neighbour was found to be Mycoplasma sp. 2045 (GenBankNo.MK615061.1). M. gallisepticum DNA was detected in 3 pneumonic lung samples and M. gallisepticum/M. synoviae DNAs were found simultaneously in 1 sample. While some Mycoplasma species identified in this study consolidated their place as pneumonic agents, some increased their potential to become a pneumonic agent when compared with cases caused by well-recognized Mycoplasma strains. Two isolates were identified as -Mycoplasma spp. as their 16S rRNA gene sequence identity levels scored below the threshold of 98.7% for species demarcation and still need to be defined whether they are possible representatives of a novel Mycoplasma species.

Mycoplasmosis of farm animals

The article presents an overview characterizing the spread of Mycoplasma infections among various kinds of farm animals. It also demonstrates the urgency of the pathogen of the disease - Mycoplasma spp., its characteristics and structural features, which provide significant differences from other types of microorganisms. Most species of Mycoplasma spp. are strictly specific to the host, but some of them pose a danger to humans. Mycoplasmas in animals cause diseases ranging from acute forms of the disease to an asymptomatic course. With the development of pathology, damage to various organs and tissues is observed, which indicates a high tropism of the pathogen, which contributes to the development of a generalized form of pathology. The most susceptible is the respiratory and reproductive tract, as a result of which farms are inflicted with significant economic damage. At the same time, some of the mycoplasmas build a symbiotic relationship with the host organism. Attempts to use antimicrobial therapy, including in various combinations, do not always lead to a positive result, which is due to the development of antibiotic resistance of the pathogen. Thanks to the change in the genome, mycoplasmas have become the smallest bacteria capable of self-replication. Mycoplasmas are classified as parasites or symbionts of animals, insects and plants, while the disease itself is opportunistic. Diagnosis of the disease consists mainly of three methods: serological, molecular biological and bacteriological, which are often used simultaneously. The cultivation of mycoplasmas has its own characteristic difficulties and features due to the structure of bacteria.

The Prevalence and Species of Mycoplasma Identified from Dairy Goats in Taiwan

The Prevalence and Species of Mycoplasma Identified from Dairy Goats in Taiwan

The expression of GapA and CrmA correlates with the Mycoplasma gallisepticum in vitro infection process in chicken TOCs

Mycoplasma (M.) gallisepticum is the most pathogenic mycoplasma species in poultry. Infections cause mild to severe clinical symptoms associated with respiratory epithelial lesion development. Adherence, biofilm formation, and cell invasion of M. gallisepticum contribute to successful infection, immune evasion, and survival within the host. The important M. gallisepticum membrane-bound proteins, GapA and CrmA, are key factors for host cell interaction and the bacterial life-cycle, including its gliding motility, although their precise role in the individual infection step is not yet fully understood. In this study, we investigated the correlation between the host–pathogen interaction and the GapA/CrmA expression in an environment that represents the natural host’s multicellular compartment. We used an in vitro tracheal organ culture (TOC) model, allowing the investigation of the M. gallisepticum variants, Rlow, RCL1, RCL2, and Rhigh, under standardised conditions. In this regard, we examined the bacterial adherence, motility and colonisation pattern, host lesion development and alterations of mucociliary clearance. Compared to low virulent RCL2 and Rhigh, the high virulent Rlow and RCL1 were more efficient in adhering to TOCs and epithelium colonisation, including faster movement from the cilia tips to the apical membrane and subsequent cell invasion. RCL2 and Rhigh showed a more localised invasion pattern, accompanied by significantly fewer lesions than Rlow and RCL1. Unrelated to virulence, comparable mucus production was observed in all M. gallisepticum infected TOCs. Overall, the present study demonstrates the role of GapA/CrmA in virulence factors from adherence to colonisation, as well as the onset and severity of lesion development in the tracheal epithelium.

High-speed Atomic Force Microscopy Observation of Internal Structure Movements in Living Mycoplasma.

Dozens of Mycoplasma species belonging to the class Mollicutes bind to solid surfaces through the organelle formed at a cell pole and glide in its direction by a unique mechanism. In Mycoplasma mobile, the fastest gliding species in Mycoplasma, the force for gliding is generated by ATP hydrolysis on an internal structure. However, the spatial and temporal behaviors of the internal structures in living cells were unclear. High-speed atomic force microscopy (HS-AFM) is a powerful method to monitor the dynamic behaviors of biomolecules and cells that can be captured while maintaining their active state in aqueous solution. In this protocol, we describe a method to detect their movements using HS-AFM. This protocol should be useful for the studies of many kinds of microorganisms. Graphic abstract: Scanning Mycoplasma cell.

The Evaluation of Humoral Immune Response to Contagious Bovine Pleuropneumonia in Cattle with Respiratory Disorders by Western Blot Technique, Competitive Enzyme Linked Immunosor-bent Assay and Complement Fixation Test

In this study 253 serum samples from cattle showing respiratory disorders were tested by confirmatory Western Immunoblotting test (IBT), complement fixation test (CFT) and competitive enzyme linked immunosorbent assay (c-ELISA). Two (0.8%) out of 253 serum samples were found to be positive and two were (0.8%) doubtful by CFT while 7 (2.8%) serum samples were found as positive and 25 (9.9%) of them were found to be doubtful by c-ELISA. On the other hand, a core profile of antigenic bands needed to be identified at 110, 98, 95, 62/60 and 48 kDa was not detected in any of the test serum sample. Immunoblot analysis some of serum samples displayed the number of bands between one or three and some of them were highly faint. In order to detect similarities between MmmSc strains related to their electrophoretic profiles, whole cell proteins of Mycoplasma species isolated from cattle and MmmSC reference strains (Mycoplasma mycoides subsp. mycoides SC Botswana (African strain), Mycoplasma mycoides subsp. mycoides SC PG1 (reference strain), Mycoplasma mycoides subsp. mycoides SC V5 (Australian strain), M. bovigenitalium, M. bovirhinis, M. capricolum subsp. capricolum, M.canadense, M.alkalescens and M. bovis) were analysed by sodium dodecyl sulphate polyacrilamide gel elektrophoresis (SDS-PAGE). The result of this analysis indicated that antigens of 220, 78, 82, 68, 24 kDa were common among all tested strains. According to these results, it was thought that positive results by both CFT and c-ELISA, also non specific band patterns observed by IBT might be caused by a cross reaction between tested mycoplasma strains. As conclusion, it is recommended that all the positive results by CFT and c-ELISA be confirmed by IBT.

Mycoplasma tauri sp. nov. isolated from the bovine genital tract

Since 2006, a Mycoplasma species unidentifiable to the species level has been regularly isolated from the semen and prepuce of apparently healthy bulls, and occasionally from cattle displaying inflammatory disease of the genital tract. Seven of these Mycoplasma isolates were subjected to a comprehensive taxonomic study. The strains investigated grew well in modified Hayflick’s medium and colonies on agar exhibited typical fried egg morphology and produced ‘film and spots’. Transmission electron microscopy revealed a cell morphology characteristic of mycoplasmas with spherically shaped cells bounded by a bi-layered cell membrane. The strains studied neither produced acid from sugar carbon sources nor did hydrolyse arginine or urea, and genome annotation indicated that organic acids (pyruvate, lactate) are used as energy sources. Phylogenetic analyses of 16S rRNA gene sequences, the 16S-23S intergenic spacer region, and partial rpoB gene and protein sequences placed the strains within the Mycoplasma (M.) bovis cluster of the Hominis group with M. primatum, M. agalactiae, and M. bovis being their closest relatives. Genomic information including whole-genome similarity metrics (ANIb, ANIm, TETRA, dDDH, AAI) and phylogenomics, proteomic features revealed by matrix-assisted laser desorption ionization time of flight (MALDI-ToF) mass spectrometry as well as serological reactions and polar lipid profiling strongly indicated that the strains examined were representatives of a hitherto unclassified species of genus Mycoplasma, for which the name Mycoplasma tauri sp. nov. with type strain Zaradi2T (=ATCC BAA-1891T = DSM 22451T) is proposed.

Evaluating the Genetic Capacity of Mycoplasmas for Coenzyme A Biosynthesis in a Search for New Anti-mycoplasma Targets.

Mycoplasmas are responsible for a wide range of disease states in both humans and animals, in which their parasitic lifestyle has allowed them to reduce their genome sizes and curtail their biosynthetic capabilities. The subsequent dependence on their host offers a unique opportunity to explore pathways for obtaining and producing cofactors – such as coenzyme A (CoA) – as possible targets for the development of new anti-mycoplasma agents. CoA plays an essential role in energy and fatty acid metabolism and is required for membrane synthesis. However, our current lack of knowledge of the relevance and importance of the CoA biosynthesis pathway in mycoplasmas, and whether it could be bypassed within their pathogenic context, prevents further exploration of the potential of this pathway. In the universal, canonical CoA biosynthesis pathway, five enzymes are responsible for the production of CoA. Given the inconsistent presence of the genes that code for these enzymes across Mycoplasma genomes, this study set out to establish the genetic capacity of mycoplasmas to synthesize their own CoA de novo. Existing functional annotations and sequence, family, motif, and domain analysis of protein products were used to determine the existence of relevant genes in Mycoplasma genomes. We found that most Mycoplasma species do have the genetic capacity to synthesize CoA, but there was a differentiated prevalence of these genes across species. Phylogenetic analysis indicated that the phylogenetic position of a species could not be used to predict its enzyme-encoding gene combinations. Despite this, the final enzyme in the biosynthesis pathway – dephospho-coenzyme A kinase (DPCK) – was found to be the most common among the studied species, suggesting that it has the most potential as a target in the search for new broad-spectrum anti-mycoplasma agents.

273. Neurological Involvement Caused by Intracellular Bacteria

BackgroundInfection 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.MethodsWe 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.ResultsWe 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%).ConclusionIntracellular 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

Expression of miRNA-601 and PD-L1 among Iranian Patients with Lung Cancer and Their Relationship with Smoking and Mycoplasma Infection.

Objective microRNAs (miRNAs) are highly conserved noncoding RNA molecules that mainly function to regulate gene expressions, and have a significant role in tumourigenesis. Programmed cell death-ligand 1 (PD-L1) is a major co-inhibitory checkpoint signal that controls T cell activities, maintains peripheral tolerance and is contribute to the development of cancer. The aim of this study is to examine miRNA-601 and PD-L1 gene expression in patients with non-small-cell lung cancer (NSCLC) and its relation with Mycoplasma infection. Materials and Methods In this case-control study, respiratory secretions and blood samples were collected from 80 healthy people and 80 NSCLC patients. The expression levels of miRNA-601 and PD-L1 were evaluated using real-time polymerase chain reaction (qRT-PCR). The presence of Mycoplasma species in respiratory secretions was detected by biochemical assays and PCR. Results There was no significant difference in the expression level of miRNA-601 between control and patients with tumour stage I, but miRNA-601 expression was significantly downregulated in patients with tumour stages II, III, and IV (P<0.05). A significant, negative relationship was found between miRNA-601 expression and tumour stage (P<0.001). Overexpression of PD-L1 was found in all of the disease stages. PCR results showed the presence of Mycoplasma pneumoniae (M. pneumoniae) in respiratory secretions from patients with stages III and IV NSCLC. We observed that 72% of patients with stages III and IV NSCLC had a positive smoking history and 65.3% were positive for Mycoplasma. ConclusionSerum miRNA-601 may act as a potential noninvasive biomarker for lung cancer and Mycoplasma infection prognosis.

Laboratory animal mycoplasmosis: A mini review

Mycoplasmas are the smallest, free-living, cell wall less prokaryotes from the class mollicutes, and these are relatively slow-growing microorganisms. Most of the laboratory animals may get the mycoplasma infection naturally. A large number of mycoplasma species (Mycoplasma pulmonis, M. muris, M. neurolyticum, M. collis, M. arthritidis and M. caviae etc.) have been reported from the laboratory animals. These are responsible for the development of a large numbers of clinical conditions in laboratory animals hence they may cause interference in the results of on-going research/experimental works on those laboratory animals. This review article gives insights on laboratory animal mycoplasma infections including pathogenesis, clinical signs, lesions, diagnosis, and interference in the experimental work, prevention, and control.

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.

Development of an immunochromatographic test for the detection of Mycoplasma pneumoniae GroES antigen

Mycoplasma pneumoniae frequently causes community-acquired pneumonia in children; β-lactam antibiotics are ineffective against this bacterium because of its lack of a cell wall. Hence, a rapid and simple detection method is required to ensure appropriate treatment. In this study, we developed a rapid and simple immunochromatography-based detection method using monoclonal antibodies that react with the co-chaperone GroES of M. pneumoniae. Mice were immunized with recombinant GroES, and hybridoma cells producing anti-GroES monoclonal antibodies were established. For the development of the immunochromatographic test, antibody pairs with superior reactivity and specificity were selected. The developed immunochromatographic test could detect 0.1 ng/mL of recombinant GroES within 20 min. Moreover, no cross-reaction was observed with other microorganisms, including six Mycoplasma species, 20 other bacterial species, and one yeast species. Macrolide-resistant and -susceptible M. pneumoniae clinical isolates were detected at approximately 104 to 105 colony-forming units/mL. The study indicates that immunochromatographic tests targeting GroES are useful for rapid and simple detection of M. pneumoniae.

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.

Genome Mosaicism in Field Strains of Mycoplasma bovis as Footprints of In-Host Horizontal Chromosomal Transfer.

Horizontal gene transfer was long thought to be marginal in Mollicutes, but the capacity of some of these wall-less bacteria to exchange large chromosomal regions has been recently documented. Mycoplasma chromosomal transfer (MCT) is an unconventional mechanism that relies on the presence of a functional integrative conjugative element (ICE) in at least one partner and involves the horizontal acquisition of small and large chromosomal fragments from any part of the donor genome, which results in progenies composed of an infinite variety of mosaic genomes. The present study focuses on Mycoplasma bovis, an important pathogen of cattle responsible for major economic losses worldwide. By combining phylogenetic tree reconstructions and detailed comparative genome analyses of 36 isolates collected in Spain (2016 to 2018), we confirmed the mosaic nature of 16 field isolates and mapped chromosomal transfers exchanged between their hypothetical ancestors. This study provides evidence that MCT can take place in the field, most likely during coinfections by multiple strains. Because mobile genetic elements (MGEs) are classical contributors of genome plasticity, the presence of phages, insertion sequences (ISs), and ICEs was also investigated. Data revealed that these elements are widespread within the M. bovis species and evidenced classical horizontal transfer of phages and ICEs in addition to MCT. These events contribute to wide-genome diversity and reorganization within this species and may have a tremendous impact on diagnostic and disease control. IMPORTANCE Mycoplasma bovis is a major pathogen of cattle that has significant detrimental effects on economics and animal welfare in cattle rearing worldwide. Understanding the evolution and the adaptative potential of pathogenic mycoplasma species in the natural host is essential to combating them. In this study, we documented the occurrence of mycoplasma chromosomal transfer, an atypical mechanism of horizontal gene transfer, in field isolates of M. bovis that provide new insights into the evolution of this pathogenic species in their natural host. Although these events are expected to occur at low frequency, their impact is accountable for genome-wide variety and reorganization within M. bovis species, which may compromise both diagnostic and disease control.

Evidence of Mycoplasma spp. transmission by migratory wild geese

Mycoplasma infections have been found in different species of waterfowl worldwide. However, the question of how the pathogens have been transmitted and dispersed is still poorly understood. Samples collected from clinically healthy greater white-fronted geese (Anser albifrons) (N = 12), graylag geese (Anser anser) (N = 6), taiga bean geese (Anser fabalis) (N = 10), and barnacle geese (Branta leucopsis) (N = 1) were tested for Mycoplasma spp. All Mycoplasma-positive samples were specified by species-specific PCR for Mycoplasma anserisalpingitidis (formerly known as Mycoplasma sp. 1220), M. anseris, M. anatis, and M. cloacale. The presence of Mycoplasma spp. was confirmed in 22 of 29 sampled birds (75.9%). Mycoplasma anserisalpingitidis was the most frequently detected species (15 of 22, 68.2%). However, we did not detect any of the other Mycoplasma spp. typical for geese, among which are M. anatis, M. anseris, and M. cloacale. Phylogenetic analysis revealed that Polish sequences of M. anserisalpingitidis formed a distinct branch, along with 2 Hungarian isolates obtained from domestic geese. Eight of the samples identified as Mycoplasma spp.-positive were negative for the aforementioned Mycoplasma species. A phylogenetic tree constructed based on partial 16S rRNA gene analysis showed that Mycoplasma spp. sequences collected from Polish wild geese represent a distinct phylogenetic group with Mycoplasma sp. strain 2445 isolated from a domestic goose from Austria. The results of our study showed that wild geese could be a reservoir and vector of different species of the Mycoplasma genus that can cause significant economic losses in the domestic goose industry.