Genomic Investigations unmask Mycoplasma amphoriforme, a new respiratory pathogen

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Mycoplasma amphoriforme; infection control; primary antibody deficiency; respiratory infection; whole genome sequencing.

Figure 1.

Natural history of a patient with Mycoplasma amphoriforme infection (patient 1). Colony-forming units are estimated using the udg quantitative polymerase chain reaction (PCR). The symbols represent the bacteria isolated and the antibiotic treatment used. *udg PCR was not performed, but the patient was positive by culture or 16S ribosomal RNA PCR. Abbreviations: AMX, amoxicillin; AZM, azithromycin; CM, clarithromycin; CPR, ciprofloxacin; DOX, doxycycline; H Inf, Haemophilus influenzae; M Catt, M. cattarhalis.

Figure 2.

Maximum likelihood phylogenetic tree for 20 isolates of Mycoplasma amphoriforme from 9 patients distinguished by color and the strain designated by its code (8 from Royal Free London National Health Service Foundation Trust and 1 of 3 French/Tunisian isolates). The table was constructed with randomized axelerated maximum likelihood using a Generalised time reversible evolutionary model and a γ-correction for among-site rate variation. Single-nucleotide polymorphisms (SNPs) are noted for the 23S ribosomal RNA, and nonsynonymous SNPs for gyrA, gyrB, and parC genes using M. amphoriforme numbering. For each, a red or salmon-pink bar indicates evidence of association of the SNP with phenotypic antibiotic resistance, pink a possible association, and blue represents the ancestral, sensitive allele. Bootstrap support values for the relationships shown in the phylogeny can be found in Supplementary Figure 3.

Figure 3.

A, Accumulation of single-nucleotide polymorphisms (SNPs) detected in available Mycoplasma amphoriforme isolates within patient 1 (orange) and 8 (green or blue) from 1999 to 2006. Numbers indicate the number of SNPs the isolates have compared to the A39 type strain sequence, which was also isolated from patient 1. B, Measure of clinical isolate diversity detected at different sampling times for patient 1. The numbers represent the numbers of minority variants detected, corrected for depth of coverage. Minority variants were counted if they were supported by at least 4 reads, with 2 reads on each strand and a base and mapping quality of 50 and 30, respectively. There is a positive nonsignificant trend between time and number of minority variants, indicating an increase of diversity over the course of the infection (linear regression model: r² = 0.532, P = .099).