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Genomic Investigations Unmask Mycoplasma Amphoriforme, a New

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Genomic Investigations Unmask Mycoplasma Amphoriforme, a New MAJOR ARTICLE Genomic Investigations Unmask Mycoplasma amphoriforme, a New Respiratory Pathogen Downloaded from https://academic.oup.com/cid/article-abstract/60/3/381/311894 by University of Glasgow user on 27 March 2019 Stephen H. Gillespie,1 Clare L. Ling,2,3 Katarina Oravcova,1 Miguel Pinheiro,1 Louise Wells,4 Josephine M. Bryant,4 Timothy D. McHugh,3 Cecile Bébéar,5 David Webster,6 Simon R. Harris,4 Helena M. B. Seth-Smith,4,a and Nicholas R. Thomson4 1School of Medicine, University of St Andrews, United Kingdom; 2Shoklo Malaria Research Unit, Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Mae Sot, Thailand; 3University College London Centre for Clinical Microbiology, Royal Free Campus, and 4Wellcome Trust Sanger Institute, Hinxton, United Kingdom; 5University of Bordeaux, France; and 6Department of Immunology, Royal Free Hospital NHS Trust, London, United Kingdom 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 trans- mitted in the community. Methods. The bacterial load was estimated in sequential sputum samples from 9 patients by quantitative poly- merase 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. Antibi- otic 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 relaps- ing 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; whole genome sequencing; respiratory infection; infection control; primary antibody deficiency. Lower respiratory tract infection (LRTI) is an important amphoriforme was first isolated from a patient with X- cause of morbidity and mortality in all age groups, linked agammaglobulinemia with chronic bronchitis in especially in immunocompromised patients. An etiolog- 1999. The patient was expectorating a large volume of ical agent is only found in approximately 70% of sputum that tested negative for all other recognized cases despite intensive investigation [1]. Mycoplasma lower respiratory tract pathogens [2]. The organism was identified as a novel bacterial species and given the Received 23 June 2014; accepted 7 September 2014; electronically published 23 name M. amphoriforme (MAM) [2]. Based on 16S ribo- October 2014. somal RNA (rRNA) gene sequencing, the closest species a fi Present af liation: Functional Genomics Center Zürich, University of Zürich, is Mycoplasma testudinis [2]. MAM grows poorly on Switzerland. Correspondence: Stephen H. Gillespie, MD, DSc, FRCP(Edin), FRCPath, School of standard mycoplasma media and lacks the appearance Medicine, University of St Andrews, North Haugh, St Andrews KY16 9TF, UK of other Mycoplasma species. Only a limited number ([email protected]). of strains have been isolated worldwide, and the majority Clinical Infectious Diseases® 2015;60(3):381–8 © The Author 2014. Published by Oxford University Press on behalf of the Infectious of isolates are from patients with primary antibody DiseasesSocietyofAmerica.ThisisanOpenAccessarticledistributedundertheterms deficiency (PAD) attending a single specialist clinic in of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any London, with additional isolates from immunocompe- medium, provided the original work is properly cited. tent patients in Denmark, France, and Tunisia [3]. DOI: 10.1093/cid/ciu820 M. amphoriforme Clinical Genomics • CID 2015:60 (1 February) • 381 Considering the burden of disease from LRTIs on human was finished using long-range PCR and pair read information. health and the recent association of MAM with LRTI in immu- The genome was annotated as described previously, using Arte- nocompetent patients [1, 4], it is important to understand the mis [5–7]. The genome was sequenced to a level classified as dynamics of chronic infection with this organism and to deter- “finished.” The MAM genomic DNA sequence was compared mine whether it is being transmitted among vulnerable patients against the European Molecular Biology Laboratory (EMBL) and how these strains relate to other isolated strains. Using the prokaryote database using BLASTN and BLASTX [8]. Transfer available clinical case notes and whole-genome sequencing RNAs were predicted by tRNAscan-SE [9]. Potential coding se- (WGS), we provide evidence for multiple strains circulating in- quences were predicted using GLIMMER [10] and the results ternationally, chronic relapsing respiratory infection, and, cru- were combined and checked manually. The predicted protein cially, patient-to-patient transmission within a hospital sequences were searched against a nonredundant protein data- Downloaded from https://academic.oup.com/cid/article-abstract/60/3/381/311894 by University of Glasgow user on 27 March 2019 environment. base using WUBLASTP and FASTA. The complete 6-frame translation was used to search PROSITE [11], and the predicted METHODS proteins were compared against the Pfam database of protein domain [12] using hidden Markov models and the Conserved Patients, Samples, and Ethical Approval Domain Search tool against the Conserved Domain Database The Ethics Committee of the Royal Free London National Health from the National Center for Biotechnology Information [13]. Service Foundation Trust (RFL) approved these studies. Sputum The results of these analyses were compiled using Artemis and samples were collected from a total of 88 adult patients with PAD used for a manual gene-by-gene annotation of the sequence and attending the PAD Clinic at the RFL and tested for MAM using predicted proteins. Annotation was based, wherever possible, Mycoplasma culture, a 16S rRNA gene MAM-specific polymerase on characterized proteins or genes. Repeat sequences were iden- fi chain reaction (PCR) (16S PCR), and a uracil DNA glycosylase ti ed using the Dotter program [14] and manually. MAM-specific quantitative PCR (udg quantitative polymerase WGS: Clinical Isolates chainreaction[qPCR])[4]. A total of 19 sequential isolates WGS was performed on single isolates on a MiSeq Instrument from 9 of the 17 MAM positive patients were available for (Illumina, San Diego, California). A 300-cycle MiSeq Reagent WGS. Additionally three patients from three patients reported Kit version 2 was used to generate 75-bp paired-end reads. The previously provided by the University of Bordeaux (Supplemen- reads obtained for each isolate were mapped against the MAM tary Table 1)[3]. The clinical information was reviewed for reference strain A39 with SMALT (Available at: http://www. evidence of symptoms associated with LRTI. sanger.ac.uk/resources/software/smalt/). Single-nucleotide poly- morphisms (SNPs) were identified using SamTools Mpileup Extraction of DNA [15] and bcftools, and filtered as previously described [16]. Extraction of DNA from sputum samples was performed using a Chelex-based method as previously described [4]. DNA for Accession Numbers WGS was extracted using the Wizard Genomic DNA extraction The raw sequence data are available under the accession number ’ kit (Promega, Southampton, UK) following the manufacturer s ERP000340. The sequence and annotation for MAM A39 have instructions using the protocol for gram-negative bacteria, and been deposited with EMBL (accession number HG937516). amplified using the illustra Genomiphi V2kit (GE Healthcare), according to the manufacturer’s instructions. Phylogenetic Analysis Regions with high-SNP density or SNPs within repetitive re- MAM-Specific PCR gions were excluded from the phylogenetic analysis as described The MAM-specific conventional 16S PCR was performed pro- previously [17]. Maximum likelihood phylogenetic trees were spectively, and the real-time qPCR targeting udg was performed constructed with randomized axelerated maximum likelihood retrospectively on DNA extracts from PAD patient samples as using a generalised time reversible evolutionary model and a described previously [4]. γ-correction for among-site rate variation. Support for relation- ships in the maximum-likelihood phylogenetic tree was as- WGS: Reference Genome sessed by running 100 bootstrap replicates. The genome of the reference MAM strain A39 was Sanger se- quenced to a depth of 8 times coverage using cloning vector Detecting Minority Variants p0TWI2 with a selection of insert sizes (2–3kb,3–4kb,and Minority variants were extracted from the mapping data using 4–5
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