Tools for Detection of Mycoplasma amphoriforme: a Primary Respiratory Pathogen?

Abstract

h Mycoplasma amphoriforme is a recently described organism isolated from the respiratory tracts of patients with immunodeficiency and evidence of chronic infection. Novel assays for the molecular detection of the organism by real-time quantitative PCRs (qPCRs) targeting the uracil DNA glycosylase gene (udg) or the 23S rRNA gene are described here. The analytical sensitivities are similar to the existing conventional M. amphoriforme 16S rRNA gene PCR, with the advantage of being species specific, rapid, and quantitative. By using these techniques, we demonstrate the presence of this organism in 17 (19.3%) primary antibody-deficient (PAD) patients, 4 (5%) adults with lower respiratory tract infection, 1 (2.6%) sputum sample from a patient attending a chest clinic, and 23 (0.21%) samples submitted for viral diagnosis of respiratory infection, but not in normal adult control subjects. These data show the presence of this microorganism in respiratory patients and suggest that M. amphoriforme may infect both immunocompetent and immunocompromised people. Further studies to characterize this organism are required, and this report provides the tools that may be used by other research groups to investigate its pathogenic potential. Mycoplasma amphoriforme was first isolated in 1999 from a patient with primary antibody deficiency (PAD) with chronic bronchitis. It has also been isolated subsequently from both immunocompromised and immunocompetent patients with respiratory tract infections (RTI) in London, Denmark, France, and Tunisia (1-3). Based on 16S rRNA sequencing, M. amphoriforme belongs to the same phylogenetic group as other human pathogenic Mycoplasma species, the pneumoniae group (1, 2). The closest species phylogenetically for which there is a whole-genome sequence is Mycoplasma gallisepticum, a bird pathogen. Phenotypic studies have demonstrated that M. amphoriforme has features in common with this group, including gliding motility, a protruding polar tip resembling that of M. gallisepticum, and a cytoskeletal structure at its polar tip with homology to that of M. pneumoniae's attachment organelle (1, 4).To understand the role that this novel agent plays in human health, better laboratory tools are required. M. amphoriforme is fastidious, requiring specialized media for cultivation, and it takes approximately 2 weeks for colonies to appear on agar. The colonial morphology resembles granular droplets, making detection difficult, as the droplets can blend into the sample matrix and be overlooked. This paper reports the development and evaluation of two real-time quantitative PCR (qPCR) assays: one assay targeting M. amphoriforme's uracil DNA glycosylase gene (udg) and one assay targeting the variable region of the 23S rRNA gene that is unique to M. amphoriforme. The new qPCR assays were compared with a previously reported 16S rRNA gene assay (2) and used to test a range of human samples from the United Kingdom. MATERIALS AND METHODSPatients, samples, and ethical approval. Clinical samples from two hospitals were u...

Genomic Investigations Unmask Mycoplasma amphoriforme, a New Respiratory Pathogen

“…The Ethics Committee of the Royal Free London National Health Service Foundation Trust (RFL) approved these studies. Sputum samples were collected from a total of 88 adult patients with PAD attending the PAD Clinic at the RFL and tested for MAM using Mycoplasma culture, a 16S rRNA gene MAM-specific polymerase chain reaction (PCR) (16S PCR), and a uracil DNA glycosylase MAM-specific quantitative PCR ( udg quantitative polymerase chain reaction [qPCR]) [ 4 ]. A total of 19 sequential isolates from 9 of the 17 MAM positive patients were available for WGS.…”

“…Extraction of DNA from sputum samples was performed using a Chelex-based method as previously described [ 4 ]. DNA for WGS was extracted using the Wizard Genomic DNA extraction kit (Promega, Southampton, UK) following the manufacturer's instructions using the protocol for gram-negative bacteria, and amplified using the illustra Genomiphi V2kit (GE Healthcare), according to the manufacturer's instructions.…”

“…The MAM-specific conventional 16S PCR was performed prospectively, and the real-time qPCR targeting udg was performed retrospectively on DNA extracts from PAD patient samples as described previously [ 4 ].…”

“…Considering the burden of disease from LRTIs on human health and the recent association of MAM with LRTI in immunocompetent patients [ 1 , 4 ], it is important to understand the dynamics of chronic infection with this organism and to determine whether it is being transmitted among vulnerable patients and how these strains relate to other isolated strains. Using the available clinical case notes and whole-genome sequencing (WGS), we provide evidence for multiple strains circulating internationally, chronic relapsing respiratory infection, and, crucially, patient-to-patient transmission within a hospital environment.…”

Molecular detection of Mycoplasma amphoriforme and Ureaplasma spp. from patient samples previously investigated for Mycoplasma pneumoniae infection

“…Uracil DNA glycosylase gene (UDG) targeting primers were used as a confirmatory step to ensure reliable positives. Both these primer sets were designed by Ling et al 4 .…”