RESUMEN
A novel strictly anaerobic chemoorganotrophic bacterium, designated Mahy22T, was isolated from sulfidic bottom water of a shallow brackish meromictic lake in Japan. Cells of the strain were Gram-stain-negative, non-motile and coccoid in shape with diameters of about 600-800 nm. The temperature range for growth was 15-37 °C, with optimum growth at 30-32 °C. The pH range for growth was pH 6.2-8.9, with optimum growth at pH 7.2-7.4. The strain grew with NaCl concentrations of 5% or below (optimum, 2-3%). Growth of the strain was enhanced by the addition of thiosulfate. The major cellular fatty acids were C16:0 and anteiso-C15:0. Respiratory quinones were not detected. The complete genome sequence of strain Mahy22T possessed a 1â885â846 bp circular chromosome and a 12â782 bp circular genetic element. The G+C content of the genome sequence was 30.1 mol%. Phylogenetic analysis based on the 16S rRNA gene revealed that the novel strain belonged to the family Acholeplasmataceae, class Mollicutes. The closest relative of strain Mahy22T with a validly published name was Acholeplasma palmae J233T with a 16S rRNA gene sequence similarity of 90.5%. Based on the results of polyphasic analysis, the name Mariniplasma anaerobium gen. nov., sp. nov. is proposed to accommodate strain Mahy22T, along with reclassification of some Acholeplasma species into Alteracholeplasma gen. nov., Haploplasma gen. nov. and Paracholeplasma gen. nov.
Asunto(s)
Acholeplasmataceae/clasificación , Filogenia , Aguas Salinas , Microbiología del Agua , Acholeplasma , Acholeplasmataceae/aislamiento & purificación , Anaerobiosis , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Ácidos Grasos/química , Japón , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
For the first time it was shown that the development of resistance to ciprofloxacin in vitro in Acholeplasma laidlawii, a mycoplasma which is widely spread in nature and which is the main contaminant of cell cultures and vaccines, is associated with diverse pathways of virulence evolution: virulome and virulence differ significantly between ciprofloxacin-resistant strains, including those with the same level of antimicrobial resistance.
Asunto(s)
Antiinfecciosos , Mycoplasma , Acholeplasma , Acholeplasma laidlawii , Ciprofloxacina/farmacología , VirulenciaRESUMEN
Members of the family Plectroviridae produce particles that are non-enveloped rigid rods (70-280×10-16 nm). The supercoiled, circular, single-stranded DNA genome of about 4.5-8.3 kb, encodes 4-13 proteins. Viruses of this family infect cell wall-less bacteria, adsorbing to the bacterial surface, replicating their DNA by a rolling-circle mechanism or transposition, and releasing progeny from cells by extrusion, without killing the host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Plectroviridae which is available at ictv.global/report/plectroviridae.
Asunto(s)
Bacteriófagos/clasificación , Virus ADN/clasificación , Acholeplasma/virología , Bacteriófagos/fisiología , Bacteriófagos/ultraestructura , Virus ADN/fisiología , Virus ADN/ultraestructura , ADN de Cadena Simple , Genoma Viral , Especificidad del Huésped , Virión/ultraestructura , Replicación ViralRESUMEN
In March 2017, a wild-caught female common mudpuppy Necturus maculosus from Iowa, USA, with an enlarged posterior abdomen was submitted for diagnostic assessment. The cause of the abdominal distension was a large fluid-filled abdominal mass, diagnosed as a nephroblastoma. Parasites and numerous bacteria were isolated and identified from the mudpuppy but were determined to be incidental. Samples of the neoplasm inoculated onto an American toad Anaxyrus americanus cell line (BufoTad) yielded cytopathic effect during several passages. However, standard molecular testing of the cell culture supernatant failed to identify any viruses. Next-generation sequencing identified the replicating agent as a bacterium of the genus Acholeplasma. Immunohistochemistry confirmed the presence of Acholeplasma within the nephroblastoma, including within tumor cells. This is the first report of nephroblastoma and the second report of neoplasia in this species. The results also suggest that certain bacteria of the genus Acholeplasma might be oncogenic.
Asunto(s)
Acholeplasma/aislamiento & purificación , Infecciones por Bacterias Gramnegativas/veterinaria , Necturus maculosus , Tumor de Wilms/veterinaria , Animales , Femenino , Infecciones por Bacterias Gramnegativas/microbiología , Iowa , Tumor de Wilms/microbiologíaRESUMEN
During previous routine inspections of bluegill fry (BF-2) and rainbow trout gonad (RTG-2) cells incubated with organ samples from asymptomatic Arctic char Salvelinus alpinus, brook trout Salvelinus fontinalis, and rainbow trout Oncorhynchus mykiss, a distinctive, reproducible cytopathic effect (CPE) appeared. The striking CPE, involving progressive vacuolation turning into slowly proceeding pyknotic degeneration, was originally attributed exclusively to enhanced growth of Acholeplasma sp. However, at a recent re-examination of re-infected BF-2 cells using electron microscopy (EM), conventional PCR, and quantitative PCR (qPCR), a virus was also detected. Two days post inoculation (dpi), EM revealed characteristic virions inside cytoplasmic vacuoles and next to bacteria outside the cells. The nucleotide sequences of the viral nsP3 gene fragment obtained from supernatants of infected cells were 100% identical and representative for salmonid alphavirus type 2 (SAV 2). The 16S RNA gene (16S rDNA) fragment sequences of the Mollicutes-specific PCR product obtained from SAV-infected as well as virus-free BF-2 control cells were identical with Acholeplasma laidlawii. In addition, qPCR results indicated enhanced propagation of virus and bacteria increasing with vacuolation between 5 and 8 dpi. Advanced vacuolation can be regarded as a CPE of both SAV and A. laidlawii, suggesting a viral impact on the bacterial infection that turns a latent intracellular stage into an apparent degenerative condition.
Asunto(s)
Alphavirus , Enfermedades de los Peces , Oncorhynchus mykiss , Acholeplasma , Infecciones por Alphavirus , Animales , Línea CelularRESUMEN
We describe two novel species of Acholeplasma sp. strain N93 and Mycoplasma sp. strain LR5794 which were isolated from the nasopharynx of a horse from the United Kingdom and from the oral cavity of a North American raccoon from Canada, respectively. These strains were phenotypically and genetically characterized and compared to other established Mycoplasma and Acholeplasma species. Both strains are facultative anaerobes, resistant to penicillin, and produce acid from glucose but do not hydrolyze arginine and urea. Both strains grew well in microaerophilic and anaerobic atmospheric conditions at 35-37 °C using PPLO (pleuropneumonia-like organisms) medium. Acholeplasma sp. N93 does not require serum for growth. Colonies of both strains showed a typical fried-egg appearance and transmission electron microscopy of bacterial cells revealed a typical mycoplasma cellular morphology. Molecular characterization included assessment of several genetic loci. The genetic analysis indicated that Acholeplasma sp. N93 and Mycoplasma sp. LR5794 were most closely related to A. hippikon and A. equifetale, and M. molare and M. lagogenitalium, respectively. However, both novel strains were genetically unique in comparison to other well-known Mycoplasma and Acholeplasma species. Based on the isolation source history, phenotypic, genotypic, and phylogenetic characteristics of these novel strains, we propose the name Acholeplasma equirhinis sp. nov. for Acholeplasma sp. isolated from the nasopharynx of a horse [the type strain is N93T (= DSM 106692T = ATCC TSD-139T = NCTC 14351T)], and the name Mycoplasma procyoni sp. nov. for the Mycoplasma sp. isolated from the oral cavity of a North American raccoon [the type strain is LR5794T (= DSM 106703T = ATCC TSD-141T = NCTC 14309T)].
Asunto(s)
Acholeplasma/aislamiento & purificación , Caballos/microbiología , Boca/microbiología , Mycoplasma/aislamiento & purificación , Nasofaringe/microbiología , Mapaches/microbiología , Acholeplasma/clasificación , Acholeplasma/genética , Animales , Canadá , ADN Bacteriano/genética , Mycoplasma/clasificación , Mycoplasma/genética , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Reino UnidoRESUMEN
BACKGROUND: Mycoplasma mastitis is increasingly posing significant impact on dairy industry. Although the effects of major conventional mastitis pathogens on milk components has been widely addressed in the literature, limited data on the effects of different Mycoplasma and Acholeplasma spp. on milk quality and quantity is available. The aim of this study was to determine the casual relationship of Mycoplasma spp. and A. laidlawii to mastitis and compare them to subclinical mastitis caused by conventional mastitis pathogens from a single dairy herd in South Australia; Mycoplasma spp. and A. laidlawii were detected using PCR applied directly to milk samples. The herd had mastitis problem with high somatic cell count and low response rate to conventional antimicrobial therapy. A total of 288 cow-level milk samples were collected aseptically and used in this study. RESULTS: Conventional culture showed a predominance of coagulase-negative staphylococci, followed by coagulase-positive staphylococci, Streptococcus spp., Enterococcus spp., E. coli, and Klebsiella spp. PCR results showed a high prevalence of mycoplasmas (76.7%), including A. laidlawii (10.8%), M. bovis (6.2%), M. bovirhinis (5.6%), M. arginini (2%), and (52.1%) of cows were co-infected with two or more Mycoplasma and Acholeplasma species. Mycoplasma co-infection significantly increased somatic cell counts (SCC) similar to conventional mastitis pathogens and compared to non-infected cows with 389.3, 550.3 and 67.3 respectively; and decreased the milk yield with 29.0, 29.9 and 34.4 l, respectively. Mycoplasma co-infection caused significant increase in protein percentage, and significant decrease in fat percentage and total milk solids, similar to other conventional mastitis pathogens. In contrast, changes in milk composition and yield caused by various individual Mycoplasma species were non-significant. CONCLUSIONS: Mycoplasma mastitis had on-farm economic consequences similar to common conventional mastitis pathogens. Results of our study indicate that co-infection Mycoplasma mastitis caused similar effect on milk composition to other mastitis pathogens and we hope these findings raise the awareness of the importance of their detection on routine diagnostic panels.
Asunto(s)
Glándulas Mamarias Animales/microbiología , Mastitis Bovina/microbiología , Leche/química , Infecciones por Mycoplasma/veterinaria , Acholeplasma/aislamiento & purificación , Animales , Bovinos , Femenino , Leche/citología , Leche/microbiología , Mycoplasma/aislamiento & purificación , Infecciones por Mycoplasma/microbiología , Australia del SurRESUMEN
Mycoplasma bacteria are able to pass through sterilizing grade filters due to their small size and lack of a cell wall, making them a common contaminant of biopharmaceutical productions. The classical method for detecting Mycoplasma is described in the European Pharmacopeia (Ph.Eur) 2.6.7. The method takes 28 days to perform, due to the slow growing nature of some Mycoplasma species. The Ph.Eur has described Nucleic Acid Testing (NAT) as a rapid alternative to the classical method. Here we present the development of a quantitative polymerase chain reaction (qPCR) assay capable of unambiguous detection of Mycoplasma with high sensitivity and specificity. The broadness of detection and the specificity towards Mycoplasma has been investigated by in silico analysis of the primer sequences followed by testing on purified Mycoplasma DNA as well as DNA from closely related genera. The assay will in all probability detect at least 356 species and strains of Mycoplasma, Spiroplasma and Acholeplasma with high sensitivity. To our knowledge this assay has the most uniform amplification efficiency over the broadest range of species and it is extremely specific towards Mycoplasma. With appropriate validation, the assay can be applied as a powerful tool for rapid Mycoplasma detection in the biopharmaceutical industry.
Asunto(s)
Acholeplasma/genética , ADN Bacteriano/genética , Mycoplasma/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Spiroplasma/genética , Cartilla de ADN/genética , Contaminación de Medicamentos/prevención & control , Humanos , Mycoplasma/clasificación , ARN Ribosómico 16S/genética , Reproducibilidad de los Resultados , Tecnología Farmacéutica/métodos , Tenericutes/clasificación , Tenericutes/genéticaRESUMEN
Mycoplasma mastitis is a contagious and costly disease of dairy cattle that significantly affects animal health and milk productivity. Mycoplasma bovis is the most prevalent and invasive agent of mycoplasma mastitis in dairy cattle, and early detection is critical. Other mycoplasma have been isolated from milk; however, the role and prevalence of these species as mastitis pathogens are poorly understood. Routine screening of milk for mycoplasma by bacteriological culture is an important component of a farm control strategy to minimize a herd mycoplasma outbreak, but phenotypic methods have limited ability to speciate mycoplasma, affecting how farms and practitioners can understand the role and effect of species other than M. bovis in herd health. Fastidious mycoplasma culture can be lengthy and inconclusive, resulting in delayed or false negative reports. We developed and validated a multitarget PCR assay that can in the same day confirm or reject a presumptive positive mycoplasma culture found upon bacteriological testing of clinical specimens, further discriminate between Acholeplasma and Mycoplasma, and identify M. bovis. Coupled with sequence analysis isolates can be further identified as bovine mycoplasma Mycoplasma arginini, Mycoplasma alkalescens, Mycoplasma canadense, Mycoplasma bovirhinis, Mycoplasma bovigenitalium, Mycoplasma californicum, Acholeplasma laidlawii, and Acholeplasma oculi. Assay validation included analysis of 845 mycoplasma representing these species and 30 additional bacterial species obtained from routine milk submissions to the Quality Milk Production Services from New York State farms and veterinary clinics between January 2012 and December 2015. Among 95 herds, we found 8 different Mycoplasma species and 3 different Acholeplasma species, with an overall prevalence of M. bovirhinis of 1%, A. oculi of 2%, M. arginini of 2%, M. californicum of 3%, M. canadense of 10%, M. bovigenitalium of 10%, A. laidlawii of 11%, M. alkalescens of 17%, and M. bovis of 78%. More than one mycoplasma was found in 14% of the herds tested, and both M. bovis and Acholeplasma were found in 6% of the farms. Incorporation of the validated molecular diagnostic assay into routine bacteriological screening as a supportive confirmation and identification tool will lead to an improved assessment of Mycoplasma and Acholeplasma prevalence data, which will facilitate increased knowledge about the role of these mycoplasma in mastitis.
Asunto(s)
Enfermedades de los Bovinos/epidemiología , Infecciones por Bacterias Gramnegativas/veterinaria , Leche/microbiología , Patología Molecular/métodos , Reacción en Cadena de la Polimerasa/veterinaria , Análisis de Secuencia de ADN/veterinaria , Acholeplasma/aislamiento & purificación , Animales , Bovinos , Enfermedades de los Bovinos/diagnóstico , Enfermedades de los Bovinos/microbiología , Femenino , Infecciones por Bacterias Gramnegativas/diagnóstico , Infecciones por Bacterias Gramnegativas/epidemiología , Infecciones por Bacterias Gramnegativas/microbiología , Mycoplasma/aislamiento & purificación , Infecciones por Mycoplasma/diagnóstico , Infecciones por Mycoplasma/epidemiología , Infecciones por Mycoplasma/microbiología , Infecciones por Mycoplasma/veterinaria , Mycoplasma bovis/aislamiento & purificación , New York/epidemiología , Patología Molecular/instrumentación , Reacción en Cadena de la Polimerasa/métodos , Prevalencia , Análisis de Secuencia de ADN/métodosRESUMEN
As a result of comparative analysis of complete genomes as well as cell and vesicular proteomes of A. laidlawii strains differing in sensitivity to ciprofloxacin, it was first shown that the mycoplasma resistance to the antibiotic is associated with the reorganization of genomic and proteomic profiles, which concerns many genes and proteins involved in fundamental cellular processes and realization of bacterial virulence.
Asunto(s)
Acholeplasma/genética , Antibacterianos/farmacología , Ciprofloxacina/farmacología , Farmacorresistencia Bacteriana/genética , Genoma Bacteriano , Proteoma , Acholeplasma/clasificación , Acholeplasma/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismoRESUMEN
AIMS: To examine whether co-administration of intestinal alkaline phosphatase (IAP) with antibiotics early in life may have a preventive role against metabolic syndrome (MetS) in mice. METHODS: A total of 50 mice were allocated to four treatment groups after weaning. Mice were treated with azithromycin (AZT) ± IAP, or with no AZT ± IAP, for three intermittent 7-day cycles. After the last treatment course, the mice were administered a regular chow diet for 5 weeks and subsequently a high-fat diet for 5 weeks. Body weight, food intake, water intake, serum lipids, glucose levels and liver lipids were compared. 16S rRNA gene pyrosequencing was used to determine the differences in microbiome composition. RESULTS: Exposure to AZT early in life rendered mice susceptible to MetS in adulthood. Co-administration of IAP with AZT completely prevented this susceptibility by decreasing total body weight, serum lipids, glucose levels and liver lipids to the levels of control mice. These effects of IAP probably occur as a result of changes in the composition of specific bacterial taxa at the genus and species levels (e.g. members of Anaeroplasma and Parabacteroides). CONCLUSIONS: Co-administration of IAP with AZT early in life prevents mice from susceptibility to the later development of MetS. This effect is associated with alterations in the composition of the gut microbiota. IAP may represent a novel treatment against MetS in humans.
Asunto(s)
Fosfatasa Alcalina/uso terapéutico , Antibacterianos/efectos adversos , Azitromicina/efectos adversos , Suplementos Dietéticos , Disbiosis/prevención & control , Mucosa Intestinal/enzimología , Síndrome Metabólico/prevención & control , Acholeplasma/clasificación , Acholeplasma/efectos de los fármacos , Acholeplasma/crecimiento & desarrollo , Acholeplasma/aislamiento & purificación , Fosfatasa Alcalina/efectos adversos , Animales , Bacteroides/clasificación , Bacteroides/efectos de los fármacos , Bacteroides/crecimiento & desarrollo , Bacteroides/aislamiento & purificación , Bovinos , Dieta Alta en Grasa/efectos adversos , Suplementos Dietéticos/efectos adversos , Disbiosis/inducido químicamente , Disbiosis/microbiología , Disbiosis/fisiopatología , Heces/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Masculino , Síndrome Metabólico/complicaciones , Síndrome Metabólico/etiología , Síndrome Metabólico/microbiología , Ratones Endogámicos C57BL , Tipificación Molecular , Obesidad/complicaciones , Obesidad/etiología , Obesidad/microbiología , Obesidad/prevención & control , Destete , Aumento de Peso/efectos de los fármacosRESUMEN
BACKGROUND: Acholeplasma oculi belongs to the Acholeplasmataceae family, comprising the genera Acholeplasma and 'Candidatus Phytoplasma'. Acholeplasmas are ubiquitous saprophytic bacteria. Several isolates are derived from plants or animals, whereas phytoplasmas are characterised as intracellular parasitic pathogens of plant phloem and depend on insect vectors for their spread. The complete genome sequences for eight strains of this family have been resolved so far, all of which were determined depending on clone-based sequencing. RESULTS: The A. oculi strain 19L chromosome was sequenced using two independent approaches. The first approach comprised sequencing by synthesis (Illumina) in combination with Sanger sequencing, while single molecule real time sequencing (PacBio) was used in the second. The genome was determined to be 1,587,120 bp in size. Sequencing by synthesis resulted in six large genome fragments, while the single molecule real time sequencing approach yielded one circular chromosome sequence. High-quality sequences were obtained by both strategies differing in six positions, which are interpreted as reliable variations present in the culture population. Our genome analysis revealed 1,471 protein-coding genes and highlighted the absence of the F1FO-type Na+ ATPase system and GroEL/ES chaperone. Comparison of the four available Acholeplasma sequences revealed a core-genome encoding 703 proteins and a pan-genome of 2,867 proteins. CONCLUSIONS: The application of two state-of-the-art sequencing technologies highlights the potential of single molecule real time sequencing for complete genome determination. Comparative genome analyses revealed that the process of losing particular basic genetic features during genome reduction occurs in both genera, as indicated for several phytoplasma strains and at least A. oculi. The loss of the F1FO-type Na+ ATPase system may separate Acholeplasmataceae from other Mollicutes, while the loss of those genes encoding the chaperone GroEL/ES is not a rare exception in this bacterial class.
Asunto(s)
Acholeplasma/genética , Genómica , Evolución Molecular , Genoma Bacteriano/genética , Análisis de Secuencia , Especificidad de la EspecieRESUMEN
Microbiological culture of milk samples has been used as a standard diagnosis for Mycoplasma mastitis. This technique is effective in isolating mollicutes that are Mycoplasma-like; however, isolates may be misinterpreted as Acholeplasma species, which are indistinguishable from Mycoplasma species by culture. A study to contrast the abilities of 2 culture-based tests, digitonin and nisin disc diffusion assays and a conventional polymerase chain reaction (PCR) technique, to discriminate between Mycoplasma and Acholeplasma was performed using 16S ribosomal RNA gene partial sequencing as the gold standard of comparison. A total of 288 bovine mollicute field isolates (248 from milk and 40 from other organ sites) and 13 reference strains were tested. Results obtained from the digitonin disc diffusion assay when it was performed with all field isolates were 92.7% and 99.0% in agreement with the gold standard using 5 mm and 3 mm of zone of growth inhibition as thresholds, respectively. Considering only milk isolates, agreements between the digitonin disc diffusion assay with the gold standard were 97.2% and 100% using 5 mm and 3 mm of zone of growth inhibition as thresholds, respectively. Culture identification using the nisin disc diffusion assay and the PCR was in a 100% agreement with the gold standard. Comparable results using culture-based nisin and digitonin disc diffusion assays, and PCR, to distinguish Mycoplasma and Acholeplasma species was found, especially for isolates from bovine milk.
Asunto(s)
Acholeplasma , Enfermedades de los Bovinos/microbiología , Digitonina , Pruebas Antimicrobianas de Difusión por Disco/veterinaria , Infecciones por Bacterias Gramnegativas/veterinaria , Infecciones por Mycoplasma/veterinaria , Mycoplasma , Nisina , Reacción en Cadena de la Polimerasa/veterinaria , Acholeplasma/genética , Acholeplasma laidlawii/genética , Animales , Bovinos , Enfermedades de los Bovinos/diagnóstico , Pruebas Antimicrobianas de Difusión por Disco/métodos , Femenino , Infecciones por Bacterias Gramnegativas/diagnóstico , Infecciones por Bacterias Gramnegativas/microbiología , Mycoplasma/genética , Infecciones por Mycoplasma/diagnóstico , Infecciones por Mycoplasma/microbiología , Reacción en Cadena de la Polimerasa/métodos , ARN Ribosómico 16S/genéticaRESUMEN
Mycoplasma gallisepticum is a bacterial pathogen of poultry that is estimated to cause annual losses exceeding $780 million. The National Poultry Improvement Plan guidelines recommend regular surveillance and intervention strategies to contain M. gallisepticum infections and ensure mycoplasma-free avian stocks, but several factors make detection of M. gallisepticum and diagnosis of M. gallisepticum infection a major challenge. Current techniques are laborious, require special expertise, and are typically plagued by false results. In this study, we describe a novel detection strategy which uses silver nanorod array-surface-enhanced Raman spectroscopy (NA-SERS) for direct detection of avian mycoplasmas. As a proof of concept for use in avian diagnostics, we used NA-SERS to detect and differentiate multiple strains of avian mycoplasma species, including Acholeplasma laidlawii, Mycoplasma gallinarum, Mycoplasma gallinaceum, Mycoplasma synoviae, and M. gallisepticum, including vaccine strains 6/85, F, and ts-11. Chemometric multivariate analysis of spectral data was used to classify these species rapidly and accurately, with >93% sensitivity and specificity. Furthermore, NA-SERS had a lower limit of detection that was 100-fold greater than that of standard PCR and comparable to that of real-time quantitative PCR. Detection of M. gallisepticum in choanal cleft swabs from experimentally infected birds yielded good sensitivity and specificity, suggesting that NA-SERS is applicable for clinical detection.
Asunto(s)
Acholeplasma/aislamiento & purificación , Técnicas Bacteriológicas/métodos , Aves/microbiología , Mycoplasma/aislamiento & purificación , Nanotubos , Plata/metabolismo , Espectrometría Raman/métodos , Acholeplasma/química , Acholeplasma/clasificación , Animales , Análisis por Micromatrices/métodos , Mycoplasma/química , Mycoplasma/clasificación , Sensibilidad y EspecificidadRESUMEN
Animal-derived materials such as animal sera represent a low, but finite, risk for introduction of an adventitious agent (virus or mollicute) into a biological bulk harvest during upstream manufacturing processes involving mammalian cell substrates. Viral and mollicute (Mycoplasma sp. and Acholeplasma sp.) contamination events have been relatively rare, but many of those that have been reported have been attributed to use of infected animal sera in growth media during cell expansion. The risk of introduction of viruses and mollicutes may be mitigated by elimination of the use of animal sera and implementation instead of chemically defined or serum- and animal-derived material-free cell culture media. When use of animal sera is unavoidable, however, mitigation of the risk of introducing an adventitious contaminant may involve treatment of the sera to inactivate potential contaminants. Gamma irradiation is one of the most widely employed methods for viral and mollicute inactivation in animal sera. In this article, we review the inactivation results reported for viral and mollicute inactivation in frozen serum. Studies performed to assess the impact of gamma irradiation on serum quality and performance are also discussed. The available data indicate that inactivation of mollicutes in serum is essentially complete at the gamma radiation doses normally employed (25-40 kGy), while the efficacy and kinetics for viral inactivation in serum by gamma irradiation appear to be dependent in part upon the size of the target virus.
Asunto(s)
Acholeplasma/efectos de la radiación , Rayos gamma , Mycoplasma/efectos de la radiación , Suero/efectos de la radiación , Virus/efectos de la radiación , Animales , Medios de Cultivo/química , Medios de Cultivo/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Contaminación de Medicamentos/prevención & control , Suero/microbiología , Suero/virología , Inactivación de Virus/efectos de la radiaciónRESUMEN
Infections with Mollicutes species (such as Mycoplasma, Acholeplasma, and Ureaplasma) can induce a variety of problems in living organisms and laboratory cell cultures. Therefore, it is necessary to establish a routine diagnostic protocol for Mycoplasma infection in order to ensure reliable research results, as well as the safety of commercial biological products. For that purpose a novel PCR-based procedure using specific designed primers complementary to 16S rRNA genome region of mollicute species was evaluated. PCR was optimized and sensitivity and specificity was evaluated by defined cell count concentrations (2-31250 CFU/ml) of different strains of Mycoplasma, Acholeplasma and Ureaplasma. Amplicon (272 bp) was cloned by PCR-cloning and sequenced by dideoxy chain termination. PCR, was found to be able to detect 10 copies of mollicute target DNA. No cross-reactivity with genomic DNA of non-mollicute bacteria or human cell lines was observed. Forty seven human and animal cell lines were evaluated for mollicute contamination. Twenty five cell lines (53%) were correctly identified as contaminated by this molecular approach. The results of this study demonstrated that this PCR-based method is not only fast and reproducible, but also highly sensitive and specific for detecting contaminant mycoplasmas in cell cultures.
Asunto(s)
Acholeplasma/aislamiento & purificación , Técnicas Bacteriológicas/métodos , Mycoplasma/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Ureaplasma/aislamiento & purificación , Acholeplasma/genética , Animales , Técnicas de Cultivo de Célula , Línea Celular , Cartilla de ADN/genética , Genes de ARNr , Humanos , Mycoplasma/genética , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Sensibilidad y Especificidad , Ureaplasma/genéticaRESUMEN
An oligonucleotide array was developed to detect and genotype mollicutes based on the internal transcribed spacer (ITS) sequence. The results of the assay were compared with those of a PCR-RFLP assay. The proposed oligonucleotide array containing 5 genus- and 23 species-specific probes was able to detect Mycoplasma species, including M. penetrans and M. spermatophilum, that were not detected by the PCRRFLP assay. Therefore, the results demonstrated that the proposed oligonucleotide array was effective for the detection and discrimination of 23 species, including an acholeplasma, 21 mycoplasmas, and a ureaplasma, and showed promise as a countermeasure to ensure that biological products are safe and of good quality.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos , Ribotipificación/métodos , Tenericutes/genética , Acholeplasma/genética , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , ADN Bacteriano/aislamiento & purificación , ADN Espaciador Ribosómico/aislamiento & purificación , Genes de ARNr , Mycoplasma/genética , Filogenia , Control de Calidad , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Especificidad de la Especie , Ureaplasma/genéticaAsunto(s)
Técnicas de Cultivo de Célula , Células Cultivadas/microbiología , Contaminación de Equipos , Peróxido de Hidrógeno , Acholeplasma/efectos de los fármacos , Técnicas de Laboratorio Clínico , Gases , Peróxido de Hidrógeno/química , Peróxido de Hidrógeno/farmacología , Mycoplasma/efectos de los fármacosRESUMEN
The partial nucleotide sequences of the rpoB and gyrB genes as well as the complete sequence of the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known Acholeplasma species. The same genes of Mesoplasma and Entomoplasma species were also sequenced and used to infer phylogenetic relationships among the species within the orders Entomoplasmatales and Acholeplasmatales. The comparison of the ITS, rpoB, and gyrB phylogenetic trees with the 16S rRNA phylogenetic tree revealed a similar branch topology suggesting that the ITS, rpoB, and gyrB could be useful complementary phylogenetic markers for investigation of evolutionary relationships among Acholeplasma species. Thus, the multilocus phylogenetic analysis of Acholeplasma multilocale sequence data (ATCC 49900 (T) = PN525 (NCTC 11723)) strongly indicated that this organism is most closely related to the genera Mesoplasma and Entomoplasma (family Entomoplasmataceae) and form the branch with Mesoplasma seiffertii, Mesoplasma syrphidae, and Mesoplasma photuris. The closest genetic relatedness of this species to the order Entomoplasmatales was additionally supported by the finding that A. multilocale uses UGA as the tryptophan codon in its gyrB and gyrA sequences. Use of the UGA codon for encoding tryptophan was previously reported as a unique genetic feature of Entomoplasmatales and Mycoplasmatales but not of Acholeplasmatales. These data, as well as previously published data on metabolic features of A. multilocale, leads to the proposal to reclassify A. multilocale as a member of the family Entomoplasmataceae.
Asunto(s)
Acholeplasma/genética , Genes Bacterianos/genética , Filogenia , ARN Ribosómico 16S/genética , Secuencia de Bases , Marcadores Genéticos , Proteínas de Plantas/genética , ARN Ribosómico 23S/genética , Transcripción Genética/genéticaRESUMEN
We evaluated the applicability of tRNA gene PCR in combination with fluorescent capillary electrophoresis with an ABI310 genetic analyzer (Applied Biosystems, Calif.) for the identification of different mollicute species. A total of 103 strains and DNA extracts of 30 different species belonging to the genera Acholeplasma, Mycoplasma, and Ureaplasma were studied. Reproducible peak profiles were generated for all samples, except for one M. genitalium isolate, the three M. gallisepticum isolates, and 8 of the 24 Ureaplasma cultures, where no amplification could be obtained. Clustering revealed numerous discrepancies compared to the identifications that had been previously obtained by means of biochemical and serological tests. Final identification was obtained by 16S rRNA gene amplification followed by sequence analysis and/or restriction digestion. This confirmed the identification obtained by tRNA gene PCR in all cases. Seven samples yielded an unexpected tRNA gene PCR profile. Sequence analysis of the 16S rRNA genes showed that six of these samples were mixed and that one had a unique sequence that did not match any of the published sequences, pointing to the existence of a not-yet-described species. In conclusion, we found tRNA gene PCR to be a rapid and discriminatory method to correctly identify a large collection of different species of the class of Mollicutes and to recognize not-yet-described groups.