Isolation Of Mycoplasmas Research Articles

Detection of Mycoplasma spp. in free-living seabirds.

This study aimed to investigate the presence of Mycoplasma spp. and identify the species of mycoplasma isolates obtained from seabirds found on Brazilian coastal beaches. Tracheal and cloacal swab samples were collected from 50 seabirds rescued by three conservation and marine animal rehabilitation centers located in Brazil. The tracheal and cloacal samples were subjected to mycoplasma culture and the isolates were identified through PCR. A "Mollicutes-specific" 16S rRNA PCR reaction was employed for triage. Four species-specific PCR reactions were used to detect Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis, or M. gallinarum. The Mollicutes positive and species negative samples were submitted do 16S rRNA sequencing. Eighteen (36%) of 50 seabirds tested positive for mycoplasma by culture. In the PCR for the genus, 28 (56%) of 50 seabirds were positive for Mycoplasma spp., with 13 (26%) detected in the trachea, one (2%) in the cloaca, and 14 (28%) in both sites. In the species-specific PCR, M. gallisepticum was detected in 17.8%, and M. meleagridis in 17.8%. Both species were detected in 14.3%. Of the isolates not characterized at species level, we obtained ten sequences and they were divided into three clusters. The first cluster was closely related to M. meleagridis, the second to M. synoviae, and the third grouped M. tully, M. gallisepticum, and M. imitans. Four and five of nine species of seabirds studied had mycoplasma detected by culture or PCR, respectively. Mycoplasmas were found in the majority of the animals studied, with the highest prevalence proportionally found in Sula leucogaster, and the lowest in Fregata magnificens. The phylogenetic analysis identified Mycoplasma spp. adapted to aquatic birds.

Molecular determination of virulence factors of Mycoplasma cynos isolated from household dogs

Mycoplasma cynos is a member of the class Mollicutes. It is more pathogenic, more host specific than other canine Mycoplasma, and only associated with respiratory diseases in dogs due to producing many virulence factors, recently neuraminidase and hemagglutinin. The study was done to detect the neuraminidase and hemagglutinin of Mycoplasma cynos isolated from the upper respiratory tract and conjunctival infections in household dogs. Mycoplasma isolates obtained from nasal, oropharyngeal, and conjunctival swabs from household dogs suffering from respiratory diseases in different veterinary clinics in Mosul city. DNA extraction was prepared , PCR and sequencing of 16S rRNA, then Mycoplasma cynos was examined for neuraminidase and hemagglutinin using specific primers.The results showed the identification of Mycoplasma cynos strain "SM-MY-M23" under accession number“OQ446513” in percentage (67.6%), and detection of neuraminidase only. Results obtained are considered the first record of M. cynos and the important virulence factor (Neuraminidase) from household dogs in Iraq.

Pathogenic avian mycoplasmas show phenotypic differences in their biofilm forming ability compared to non-pathogenic species in vitro

Mycoplasmas are known as the minimalist microorganisms in the microbes’ world. Their minimalist nature makes them highly sensitive to the environmental conditions and limits their ability to survive for extended periods outside their animal host. Nevertheless, there are documented instances of mycoplasma transmission over significant distances and this phenomenon may be linked to relatively unexplored abilities of mycoplasmas, such as their capacity to synthesize biofilm—the predominant mode of bacterial growth in nature. The authors decided to establish a method aimed at inducing the clustering of mycoplasma planktonic cells within a biofilm in vitro and subsequently assess the capacity of certain avian mycoplasmas to synthesize a biofilm. A total of 299 avian mycoplasma isolates were included in the study, encompassing both pathogenic (Mycoplasma gallisepticum, M. synoviae, M. meleagridis, M. iowae) and non-pathogenic species (M. gallinaceum, M. gallinarum, M. iners and M. pullorum). The authors successfully demonstrated the feasibility of inducing avian mycoplasmas to synthetize in vitro a biofilm, which can be visually quantified. The only species that did not produce any biofilm was M. iowae. In general, the pathogenic mycoplasmas produced greater quantities of biofilm compared to the non-pathogenic ones. Furthermore, it was observed that the ability to produce biofilm appeared to vary, both qualitatively and quantitatively, not only among different species but also among isolates of a single species. Future studies will be necessary to determine whether biofilm production plays a pivotal epidemiological role for the pathogenic avian mycoplasmas.

Mycoplasma and Neisseria Prevalence in Asymptomatic Women and Investigation of their Susceptibility to Tamarindus indica and Syzygium aromaticum Aqueous Extracts

Neisseria and Mycoplasma are two prevalent bacteria in the female urogenital tract leading to gynecological infections and infertility. The aim of this study was to assess the antibacterial activity of Tamarindus indica and Syzygium aromaticum extracts against Neisseria and Mycoplasma isolates. It was a cross cectional study on 60 asymptomatic women at the Protestant and Regional Hospital of Ngaoundere. For this reason, a large consecutive sample of patients with several well-defined characteristics was assembled and urine and cervical-vaginal swab were collected using standard procedures. After being isolated on a specific medium, several strains of Neisseria and Mycoplasma were identified based on their morphological and biochemical characteristics. The antibacterial activity of the plant extracts was tested using the agar-well diffusion method. It was discovered that 70% of asymptomatic women had overall infection, with varying prevalence rates. The prevalence rates of Neisseria and Mycoplasma were 14.29% and 85.71% respectively. The aqueous extracts of Syzygium aromaticum against Neisseria produced inhibitory diameters of 43 mm, 40 mm and 32 mm at doses of 20 mg/mL, 10 mg/mL, and 5 mg/mL, respectively while, at the same doses, the aqueous extract of Tamarindus indica produced inhibitory diameters of 16 mm, 14 mm and 13 mm, respectively. The combined extract of Syzygium aromaticum and Tamarindus indica exhibits inhibitory diameters of 25, 23, and 30 mm at 20 mg/mL, 10 mg/mL, and 5 mg/mL, respectively. Syzygium aromaticum extract alone showed efficacy against Mycoplasma, with diameters of 16.5 mm, 13 mm and 10.5 mm at concentrations of 20 mg/mL, 10 mg/mL, and 5 mg/mL respectively. Inhibition diameters of 18 mm (for fosfomycin and ofloxacin), 22 mm (for chloramphenicol and ceftriaxone) and 26 mm (for levofloxacin) were found using Neisseria isolates. The only drugs that demonstrated efficacy against Mycoplasma were Minocycline and Josamycin. Given these findings, extracts of Syzygium aromaticum and Tamarindus indica can be investigated and used to treat infections caused by Niesseria and Mycoplasma.

Establishment of a Mycoplasma hyorhinis challenge model in 5-week-old piglets.

Mycoplasma hyorhinis is an emerging swine pathogen with high prevalence worldwide. The main lesions caused are arthritis and polyserositis, and the clinical manifestation of the disease may result in significant economic losses due to decreased weight gain and enhanced medical costs. We aimed to compare two challenge routes to induce M. hyorhinis infection using the same clinical isolate. Five-week-old, Choice hybrid pigs were inoculated on 2 consecutive days by intravenous route (Group IV-IV) or by intravenous and intraperitoneal routes (Group IV-IP). Mock-infected animals were used as control (control group). After the challenge, the clinical signs were recorded for 28 days, after which the animals were euthanized. Gross pathological and histopathological examinations, PCR detection, isolation, and genotyping of the re-isolated Mycoplasma sp. and culture of bacteria other than Mycoplasma sp. were carried out. The ELISA test was used to detect anti-M. hyorhinis immunoglobulins in the sera of all animals. Pericarditis and polyarthritis were observed in both challenge groups; however, the serositis was more severe in Group IV-IV. Statistically significant differences were detected between the challenged groups and the control group regarding the average daily weight gain, pathological scores, and ELISA titers. Additionally, histopathological scores in Group IV-IV differed significantly from the scores in the control group. All re-isolated strains were the same or a close genetic variant of the original challenge strain. Our results indicate that both challenge routes are suitable for modeling the disease. However, due to the evoked more severe pathological lesions and the application being similar to the hypothesized natural route of infection in Group IV-IV, the two-dose intravenous challenge is recommended by the authors to induce serositis and arthritis associated with M. hyorhinis infection.

Detection of Mycoplasma gallisepticum, Isolation, and Determination of Tylosin Susceptibility of Isolates from Commercial Chickens.

Mycoplasma gallisepticum (MG) is a contagious avian pathogen that causes financial losses to the poultry industry. Isolation of the pathogen is difficult and time-consuming, and therefore, far from a routine method. Serological testing methods to detect antibodies resistant to MG are widely used in routine diagnosis. Tylosin is a class of macrolide antibiotics tremendously administered in veterinary medicine for the treatment of mycoplasmosis and prophylaxis. This study aimed to detect MG by immunoassay testing, culture, and polymerase chain reaction (PCR) in commercial poultry farms and to investigate the tylosin susceptibility of the isolates. To verify the presence of antibodies resistant to MG, 750 blood samples were randomly collected from 38 broiler farms from 2019 to 2022 in Mazandaran and Golestan provinces, Iran, and rapid slide agglutination (RSA) assay was performed. Positive results were analyzed by the enzyme-linked immunosorbent assay (ELISA) for further investigation. Here, 920 swab samples were collected from 38 non-vaccinated commercial farms for culture, and PCR tests were performed for the isolated strains. The activities of tylosin were tested in vitro against these isolates using the broth microdilution method. The lowest antibiotic concentration that resulted in a color change was considered the minimum inhibitory concentration (MIC) value. Twenty-four (63.1%) farms were positive in the RSA test, and 21 (55.2%) farms were positive in the ELISA test. Nine (23.68%) of the farms grew on culture media, and 8 (21.05%) were detected as Gallisepticum species by PCR. The geometric mean of MIC for tylosin was 5.75 µg/ml, MIC50 was 4 µg/ml, and MIC90 was 8 µg/ml. The results indicated that commercial farms were infected with MG. Considering the ability of MG to spread and the probable use of the RSA test as a rapid and cheap method, it can be argued that ELISA and RSA serological tests can be used to find MG in poultry flocks, and the positive result should be confirmed by standard microbiological tests or PCR. It was also found that the isolated parts of MG changed their sensitivity to tylosin, indicating the need for routine testing to optimize treatment dose and efficiency.

Molecular Characterization and Antimicrobial Susceptibility of Mycoplasma Species Isolated from Broilers and Breeder Chickens

Mycoplasmas are considered as important avian pathogens, which cause a great economical loss in the poultry industry. As it responsible for both respiratory disease and synovitis in poultry. The aim of this study was to isolate and identify Mycoplasma gallisepticum and Mycoplasma synoviae from chickens and determine the efficacy of different antibiotics. Additionally, detection of some substantial virulence genes. Two hundred chicken samples were collected and cultured onto specific PPLO medium .The isolates were characterized by polymerase chain reaction then tested for antibiotic sensitivity by the minimum inhibitory concentration (MIC) method. The results reported that 15 samples (7.5%) were positive by culturing with prevalent of Mycoplasma synoviae and Mycoplasma gallisepticum with 12 (80%) and three (20%). Following the 16S rRNA-based detection of Mycoplasma isolates, 12 Mycoplasma synoviae and three Mycoplasma synoviae isolates were identified by different PCR-based detection methods for various virulence genes. Three Mycoplasma gallisepticum isolates contain the mgc2 gene, and nine Mycoplasma synoviae isolates were positive for the ISR (intergenic spacer region) gene. All isolates had lowest MIC values for Tylvasolin with a range of (0.062-0.125μg/mL) followed by Lincomycin, Tiamulin, Tilmicosin and Tylosin with a range of (0.062-1 μg/mL). However, the Mycoplasma synoviae isolates displayed variance in MICs for Oxytetracycline with a range of 0.5 to 8 μg/mL, and Chlorotetracycline with a range of 2 to 8 μg/mL. Meanwhile, high MIC values for Enrofloxacin were detected in all isolates with MICs ranging from 8 to 32 μg/mL. Furthermore, the MIC method identified Tylvasolin, Lincomycin, Tiamulin, Tilmicosin and Tylosin as the antibiotics of choice for the treatment of MS infections. In conclusion, these data may help in prevention and control of Mycoplasma infection in poultry.

Pathological and Molecular detection of Mycoplasma ovipneumoneae in Sheep, Basrah Province, Iraq.

Mycoplasma ovipneumonea (M. ovipneumonea) are microorganism's causes atypical pneumonia in (sheep and goat). Mycoplasma is isolated frequently from pneumonic cases (lung, trachea, and nose) of sheep but can also be found in the respiratory tract of healthy sheep. This study aimed to isolate, identify, and pathological examination of M. ovipneumonea in sheep. Samples in the current study were collected from sheep of both sex and 6-10 months of age in Basrah slaughterhouse, suffering from respiratory signs associated with ocular, nasal discharge, and coughing. Nasal swabs were collected from the nose before slaughtering; other swabs were collected from the trachea and bifurcation of bronchus for bacterial isolation on PPLOs. Tissue specimens are frozen for DNA gene-based PCR analysis and for preparing paraffin blocks for histopathological examination. The bacterial cultures revealed isolates of Mycoplasma were positive on (PPLO) broth with agar from the morphological colonies of Mycoplasmaovipeumonea "fried egg" type colony morphology. PCR results revealed the 16S rRNA gene of Mycoplasma sp. The appearance revealed different stages of pulmonary changes like respiratory congestion, edema, and hemorrhagic spots on the surface of the lungs, and their air passages contained inflammatory exudate. The microscopic lesions represent acute fibrinous-suppurative broncho-interstitial pneumonia. M. ovipneumoniae was a prevalent respiratory infectious disease in Iraqi's sheep-Basrah province with frequent bacterial isolation, pneumonic pathological changes in animals suffer from different respiratory manifestations.

Mycoplasma from the upper respiratory tract and conjunctival infections in household dogs

The study was performed to isolate and detect Mycoplasma as a causative agent of the upper respiratory tract and conjunctival infections in household dogs in Mosul city. One hundred household dogs of different ages, sex, and breed were subjected to the study, including 60 dogs suffering from moderate to severe respiratory infections and 40 healthy dogs in Mosul city from 1/2/2022 to 15/6/2022. Three hundred swabs were collected, including nasal swabs 100, oropharyngeal swabs 100, and conjunctival swabs 100. The swabs were cultured in Mycoplasma media and incubated at 37ºC with 5% CO2 in a candle jar for 4-14 days. Light and the dissecting microscope examined the growing colonies microscopically under low magnification. The colonies were also stained with modified Dienes stain. The results of the current study indicated that respiratory infections were predominant in young male household dogs compared with conjunctival infections in all examined dogs. The conjunctival infections were obvious only in Husky and Belgium dogs. The high isolation rate of Mycoplasma was from upper respiratory tract infections in diseased female dogs more than one-year age 100%. In contrast, the conjunctival infections were more dominant and had a higher isolation rate in less than one-year males. The current study revealed a high Mycoplasma isolation rate in respiratory swabs of Terrier and German dogs, while the conjunctival swabs were positive for mycoplasmal culture in Husky and Belgium dogs 100%. In conclusion, mycoplasmal infections were more dominant in upper respiratory infections in female German and Terrier dogs.

Prevalence of three Mycoplasma sp. by multiplex PCR in cattle with and without respiratory disease in central Mexico.

This study aimed to identify Mycoplasma bovis, Myc. dispar, and Myc. bovirhinis, which are involved in bovine respiratory disease through a multiplex PCR as an alternative to culture's features that hamper Mycoplasma isolation. Nasal swabs were taken from 335 cattle with and without respiratory disease background (RDB) from dairy herds in the central region of Mexico. Each sample was divided in two; the first part was processed for the direct DNA extraction of the nasal swab and the second for Mycoplasma isolation, culture, and then the multiplex PCR was performed. In the nasal swabs, Myc. bovis was identified in 21.1%; Myc. dispar, in 11.8%; and Myc. bovirhinis, in 10.8% in cattle with RDB. Isolates were identified as Myc. bovis, 20.1%; Myc. dispar, 11.8%; and Myc. bovirhinis, 6.1%. There is a strong correlation between the presence of Mycoplasma identified by PCR and the clinical history of the disease (ρ < 0.0000). In animals without RDB, Myc. bovirhinis was the only species detected in 6.1% of the samples processed directly for multiplex PCR, and in 2% of the isolates. There is an excellent correlation (kappa 0.803) between the isolation and the 16S PCR and a high correlation (kappa 0.75) between the isolation and the multiplex PCR. Therefore, we conclude that the PCR multiplex test is highly sensitive and may be used for the diagnosis and surveillance of the three species in biological samples and mycoplasma isolates.

Molecular Detection of Mycoplasma Gallisepticum and Mycoplasma Synoviae Infection in Poultry

In Pakistan, production of poultry has been evolved as a good alternate of beef and mutton. In this study multiplex PCR approaches were adopted to differentiate and detect avian mycoplasma in a single PCR reaction as a step forward in the economization of PCR detection. From the total of 25 samples, 25 (100%) had a positive Mycoplasma isolation result. The biochemical test yielded the highest number of isolations, with MG accounting for 10/25. Commercial DNA-based PCR kits have been developed as a result of recent advancements in diagnostic techniques for Mycoplasma infections. Tetracyclines, fluoroquinolones, tilmicosin, tylosin, spiramycin Infections should be reduced as much as possible.

The prevalence and species of Mycoplasma identified from dairy goats in Taiwan

Except for Mycoplasma capricolum subsp. capripneumoniae, which only causes pneumonia, Mycoplasma species can cause otitis, arthritis, septicemia, mastitis, pneumonia and encephalitis in goats. After a mycoplasma outbreak was recorded in Taiwan in 2016, an increasing number of mycoplasma infection cases have been observed. It is important to understand how many Mycoplasma species are currently prevalent in Taiwan. In this study, 57–61 bulk milk samples were collected from dairy goats in Taiwan every year from 2017 to 2020 to identify the presence of mycoplasma DNA fragments by polymerase chain reaction (PCR). The mycoplasma species were identified based on colony characteristics, PCR, and DNA sequences. In total, 39 out of 236 samples (16.52%) tested mycoplasma positive. The annual mycoplasma prevalence rates from 2017 to 2020 were 24.59% (15/61), 8.20% (5/61), 17.54% (10/57) and 15.79% (9/57), respectively. Mycoplasma mycoides subsp. capri (n = 19), Mycoplasma putrefaciens (n = 10), Mycoplasma ovipneumoniae (n = 5) and Mycoplasma bovis (n = 5) were present in Taiwan. No M. capricolum subsp. capripneumoniae was detected. Except for the large drop in 2018, the prevalence of mycoplasma infection declined to a consistent level for the final two years of the study. However, the seasonal pattern is still worth investigating. This is the first survey of mycoplasma infection in goats, and we believe that a larger survey is still needed to understand the pathogenicity and diversity of these mycoplasma isolates.

Prevalence and Antimicrobial Susceptibility of Bovine Mycoplasma Species in Egypt.

Simple SummaryBovine Mycoplasma species, particularly antimicrobial resistant Mycoplasma bovis are important causes of bovine respiratory disease (BRD) in cattle, which causes major economic losses worldwide. Thus, the current study aimed to determine the prevalence and antimicrobial resistance profiles of bovine Mycoplasma spp. isolated from cattle’s respiratory tracts, in addition to evaluating the fluoroquinolone resistance in the recovered isolates using broth microdilution and conventional PCR techniques in Egypt. Our result showed that M. bovis was the most common spp. (61%), followed by M. bovirhinis (15%). In total, mycoplasma isolates were more prevalent among all examined lung tissues (38%), followed by nasal swabs (35%), tracheal tissues (28%), and tracheal swabs (27%). All the examined mycoplasma isolates (n = 76) were 100% susceptible to spectinomycin, tulathromycin, spiramycin, and tylosin, but high doxycycline and enrofloxacin minimum inhibitory concentrations (MICs) values were observed among 43.4% and 60.5% of the tested isolates, respectively. Three and two mycoplasma isolates with high enrofloxacin MICs were confirmed to be M. bovis and M. bovirhinis, respectively, by PCR assays. All molecularly confirmed mycoplasma isolates (n = 5) were positive for the gyrA gene (100%), meanwhile, three isolates (60%) were positive for the parC gene. In conclusion, understanding antimicrobial resistance mechanisms is a significant tool for the future development of genetic-based diagnostic techniques for the rapid detection of resistant mycoplasma strains.Among many bovine Mycoplasma species (spp.), Mycoplasma bovis is recognized as a significant causative agent of respiratory diseases in cattle. In recent years, resistant M. bovis isolates, especially to fluoroquinolones, have been reported globally as a result of the extensive usage of antimicrobials in the treatment of bovine pneumonia. Therefore, the aim of this study is to investigate the prevalence and antimicrobial susceptibility patterns of bovine Mycoplasma spp. isolated from the respiratory tracts of cattle in Egypt and to assess the fluoroquinolones resistance in the recovered mycoplasma isolates via broth microdilution and conventional PCR techniques. Conventional phenotypic methods identified 128 mycoplasma isolates (32%) from 400 different samples, with M. bovis being the predominant spp. (61%), followed by M. bovirhinis (15%). Of note, mycoplasma isolates were rarely isolated from total healthy lung tissues (7/55, 12.7%), but they were frequently isolated from pneumonic lungs (31/45, 68.9%). All the examined mycoplasma isolates (n = 76) were sensitive to tilmicosin, tylosin, tulathromycin, spiramycin, and spectinomycin (100% each), while 60.5% and 43.4% of the examined isolates had high minimum inhibitory concentration (MIC) values to enrofloxacin and doxycycline, respectively. Three and two mycoplasma isolates with high enrofloxacin MICs were confirmed to be M. bovis and M. bovirhinis, respectively, by PCR assays. All molecularly confirmed mycoplasma isolates (n = 5) were positive for the gyrA gene (100%); meanwhile, three isolates (60%) were positive for the parC gene. In conclusion, our findings revealed alarming resistance to enrofloxacin and doxycycline antibiotics; thus, antimicrobial usage must be restricted and molecular techniques can help in the rapid detection of the resistant strains.

Isolation and characterization of Mycoplasma hominis from the semen of infertile men and detection of virulence factors genes

The bacterial infection of seminal fluid is one of the most important factors behind infertility in many cases. The biological diversity of the Mycoplasma have widely been reported to be associated with the infertility grade in many cases. This study was conducted to identify and characterize the identity and the phylogenetic positioning of Fourteen isolates of Mycoplasma in seminal fluids. three genetic loci were included in this study 16S rRNA, recA, and Vaa for Mycoplasma sequences. Each amplified locus was investigated in both bacterial infections to identify the identity of the possible bacterial infection of those patients and to assess the pattern of the genetic variation for each genetic locus in the investigated patients. Our results indicated a complete homology with the referring sequences of Mycoplasma hominis (GenBank acc. no. CP055150.1). Concerning the 16S rRNA fragment, no genetic variation was found in the investigated samples. It was inferred from the tree that the investigated samples were suited in the immediate vicinity to many strains of Mycoplasma hominis belonging to various European origins. Furthermore, the patterns of the neighbour phylogenetic distances in this tree indicated a specific biological diversity of the incorporated Mycoplasma hominis sequences.

Prevalence of Genomic Resistance to Macrolide in Mycoplasma Isolates among Children with Community Acquired Pneumonia

Introduction: Mycoplasma pneumonia is traditionally susceptible to macrolides, tetracycline and fluroquinolones. Since, tetracycline and fluoroquinolone are used cautiously in children, macrolides remain the antibiotic of choice for treating Mycoplasma pneumonia. But, resistance to macrolides has been reported in mycoplasma since the 2000s especially from Asia. Currently, there is no evidence on macrolide resistance of mycoplasma pneumoniae from India. Aim: To identify the prevalence of genomic resistance to macrolides in mycoplasma isolates among children hospitalised with community acquired pneumonia. Materials and Methods: This descriptive cross-sectional study was conducted in Department of Paediatrics at Institute of Child Health and Hospital for Children, Chennai, Tamil Nadu, India (tertiary care centre for children in South India), from September 2019 to August 2020. Children between 2 months to 12 years of age, who were hospitalised with community acquired pneumonia were included in the study. The sampling procedure used was induced sputum or mini bronchoalveolar lavage (in intubated children). The samples were processed for culture in Pleuropneumonia Like Organisms (PPLO) agar. The culture isolates showing the typical fried egg colonies were subjected to polymerase chain reaction to detect the presence of resistance conferring mutation in the P1 adhesin gene MPN141. Chi-square test was used to test statistical significance. Results: Among the 268 children included in the study, mycoplasma pneumonia was positive in 33 (12.3%) cases. Mycoplasma pneumoniae was most common in children aged 1-5 years old (51.5%), followed by infants (36.4%) and children aged 5-12 (12.1%). There was no significant difference in distribution among males (39.4%) and females (60.6%) (p-value=0.08). None of the mycoplasma isolates in the study showed mutation for resistance conferring genes. Conclusion: Macrolide resistance conferring genes were not identified in the study population, which may indicate that the mycoplasma strains from this part of India are still susceptible to macrolides.

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.

Evaluation of Antibacterial Activity of Selected Edible Herbs Against Genital Mycoplasmas Among University Students in Enugu State, Nigeria

<i>The prevalence rates of Mycoplasma hominis and Ureaplasma Spp</i> and the evaluation of edible herbs against genital mycoplasmas were determined among university students in Enugu state, Nigeria. Specimens from 2400 subjects comprising of 1200 male and female subjects were tested for <i>Mycoplasma hominis</i> and <i>Ureaplasma Spp</i>. Cultures were done on Mycoplasma agar, A7agar and urea-arginine LYO2 broth. Antimicrobial susceptibility pattern was determined by Mycoplasma IST2 kit. Preliminary qualitative phytochemical analysis was done using standard methods to reveal the presence of basic phytochemicals. Rizomes of <i>Curcuma longa</i>, garlic and ginger, cloves and seeds of <i>Garcina kola</i> were extracted with water, methanol and ethanol sequentially and reconstituted with dimethyl -sulfoxide to concentrations (mg/ml) of 200, 100, 50, 25, 12.5, 6.25, 3.125 and 1.56. Mycoplasma isolates were screened for sensitivity to the extracts using agar well diffusion and broth diffusion methods. <i>Mycoplasma hominis</i> occurred more in women at (81.6%) while <i>Ureaplasma Spp</i> occurred more in men at (88%). <i>Mycoplasma hominis</i> (98%) and <i>Ureaplasma Spp</i> (91.6%) showed high sensitivityto ciprofloxacin. The test plants all showed presence of alkaloids, tannis, flavonoids, terpenoids, saponins, phenols and cardiac glycosides. At 200mg/ml <i>Garcina kola</i> and <i>Curcurma longa</i> showed higher zones of inhibition at 35mm on <i>Mycoplasma hominis</i> and <i>ureaplasma Spp</i>. Syngestic activities of ethanolic extracts of Curcuma longa and garlic showed high zones of inhibition at 45mm on <i>Mycoplasma hominis</i> while at 200mg/ml <i>Garcina kola</i> and cloves showed zones of inhibition at 45mm on <i>ureaplasma spp</i>. The minimum inhibitory concentration (MIC) results of the syngestic plants were 1.56mg/ml on all the isolates. The Results from the study showed that there is high prevalence of <i>Mycoplasma hominis</i> in women while <i>Ureaplasma Spp</i> while <i>Ureaplasma Spp</i> occurred more in men in the study area. The synergistic activities of selected edible plants showed higher efficacy on resistant isolates and suggest its use in the treatment of genital Mycoplasmas.

Detection of Mycoplasma gallisepticum in broiler chickens by PCR.

Background:Mycoplasma is a significant microorganism of poultry, which can cause respiratory infections and synovial inflammation, bringing about huge financial misfortunes to poultry workmanship worldwide.Aim:The goal of existing research was to determine the infection rate of Mycoplasma gallisepticum (MG) from chronic respiratory disease cases among broilers fields in Mosul/ Iraq using the polymerase chain reaction (PCR) technique.Methods:All 92 lungs samples were collected from broilers with classical respiratory signs in different regions of the Nineveh governorate for 3 months from February to April 2021.Results:PCR tests were performed using two couple primers, one for the qualitative amplification of 16S rRNA genes (285 base pairs) in Mycoplasma spp. and the second couple for the detection of M. gallisepticum (580 base pairs). Among the samples obtained from broilers, 87 (94.7%) were positive for Mycoplasma and 79 (85.9%) were positive for M. gallisepticum.Conclusion:Our results showed that MG infection in broiler chickens leads to serious clinical symptoms and severe lesions. The rate of Mycoplasma isolation in this study is high despite the short lifespan of broiler chickens.

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.

A low-cost simple test for weekly detection of Mycoplasma hyorhinis and arginini contaminations in cell cultures and viral preparations

Mollicutes (Mycoplasma and Acholeplasma) are parasitic bacteria that adhere to cellular surfaces, naturally resistant to many antibiotics and extremely small. They are often found as contaminants in cultured cells, where they go unnoticed. They may be present in viral stocks because they are present in supernatants of cells where cultured viruses are released. The best way to keep laboratories free of Mycoplasma is to discard infected cultures, but, as judged by the very common finding of Mycoplasma-contaminated cultures in many laboratories, this is not done as often as it should be. A possible reason is that most procedures recommended take as long as performing a simple experiment and many laboratories delay testing to save money and time. Indeed, many methods exist to detect Mycoplasma infection of cell lines, but they take at least a couple of hours of hands-on work, if not more. Here we describe a procedure to screen viral stocks and tissue cultures for Mycoplasma presence. It relies on isolation of Mycoplasma on ordinary horse blood agar directly from exhausted tissue culture supernatants and does not require experienced personnel or expensive equipment. It only requires minutes of hands-on work, and, for this, it may be useful for weekly screening of cultures. It yields semiquantitative results in roughly 5 days, which is the time that usually passes between one subculture passage of cells in vitro to another. Because of its simplicity, it may be useful for detecting Mycoplasma in viral stocks and for frequent screening of cultures in research laboratories.