Abstract

Mycoplasma amphoriforme has been associated with infection in patients with primary antibody deficiency (PAD). Little is known about the natural history of infection with this organism and its ability to be transmitted in the community. The bacterial load was estimated in sequential sputum samples from 9 patients by quantitative polymerase chain reaction. The genomes of all available isolates, originating from patients in the United Kingdom, France, and Tunisia, were sequenced along with the type strain. Genomic data were assembled and annotated, and a high-resolution phylogenetic tree was constructed. By using high-resolution whole-genome sequencing (WGS) data, we show that patients can be chronically infected with M. amphoriforme manifesting as a relapsing-remitting bacterial load, interspersed by periods when the organism is undetectable. Importantly, we demonstrate transmission of strains within a clinical environment. Antibiotic resistance mutations accumulate in isolates taken from patients who received multiple courses of antibiotics. Mycoplasma amphoriforme isolates form a closely related species responsible for a chronic relapsing and remitting infection in PAD patients in the United Kingdom and from immunocompetent patients in other countries. We provide strong evidence of transmission between patients attending the same clinic, suggesting that screening and isolation may be necessary for susceptible patients. This work demonstrates the critical role that WGS can play in rapidly unraveling the biology of a novel pathogen.

Highlights

  • Mycoplasma amphoriforme has been associated with infection in patients with primary antibody deficiency (PAD)

  • By using high-resolution whole-genome sequencing (WGS) data, we show that patients can be chronically infected with M. amphoriforme manifesting as a relapsing-remitting bacterial load, interspersed by periods when the organism is undetectable

  • This work demonstrates the critical role that WGS can play in rapidly unraveling the biology of a novel pathogen

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Summary

Methods

The bacterial load was estimated in sequential sputum samples from 9 patients by quantitative polymerase chain reaction. 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 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

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