Whole-genome sequencing to identify transmission of Mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study

Abstract

Background: Increasing numbers of individuals with cystic fibrosis are becoming infected with the multidrug-resistant non-tuberculous mycobacterium (NTM) Mycobacterium abscessus, which causes progressive lung damage and is extremely challenging to treat. How this organism is acquired is not currently known, but there is growing concern that person-to-person transmission could occur. We aimed to define the mechanisms of acquisition of M abscessus in individuals with cystic fibrosis.

Method: Whole genome sequencing and antimicrobial susceptibility testing were done on 168 consecutive isolates of M abscessus from 31 patients attending an adult cystic fibrosis centre in the UK between 2007 and 2011. In parallel, we undertook detailed environmental testing for NTM and defined potential opportunities for transmission between patients both in and out of hospital using epidemiological data and social network analysis.

Findings: Phylogenetic analysis revealed two clustered outbreaks of near-identical isolates of the M abscessus subspecies massiliense (from 11 patients), differing by less than ten base pairs. This variation represents less diversity than that seen within isolates from a single individual, strongly indicating between-patient transmission. All patients within these clusters had numerous opportunities for within-hospital transmission from other individuals, while comprehensive environmental sampling, initiated during the outbreak, failed to detect any potential point source of NTM infection. The clusters of M abscessus subspecies massiliense showed evidence of transmission of mutations acquired during infection of an individual to other patients. Thus, isolates with constitutive resistance to amikacin and clarithromycin were isolated from several individuals never previously exposed to long-term macrolides or aminoglycosides, further indicating cross-infection.

Interpretation: Whole genome sequencing has revealed frequent transmission of multidrug resistant NTM between patients with cystic fibrosis despite conventional cross-infection measures. Although the exact transmission route is yet to be established, our epidemiological analysis suggests that it could be indirect.

Funding: The Wellcome Trust, Papworth Hospital, NIHR Cambridge Biomedical Research Centre, UK Health Protection Agency, Medical Research Council, and the UKCRC Translational Infection Research Initiative.

Figure 1

Phylogenetic tree of M abscessus isolates indicates different modes of likely transmission (A) Maximum likelihood tree of all isolates sequenced generated by mapping reads against the M abscessus subsp abscessus reference genome and building a midpoint rooted tree using the 280 615 variable positions detected. The tree reveals the presence of the three subspecies of M abscessus (M abscessus subsp abscessus, M abscessus subsp massiliense, and M abscessus subsp bolletii) with evidence of tight clustering of isolates from each individual (denoted by a separate colour), and many examples of large genetic differences between isolates from different individuals, consistent with independent acquisition of genetically diverse organisms from the environment. We identified tight clusters consisting of multiple patient isolates within the M abscessus subsp abscessus group (M abscessus subsp abscessus cluster); (B) and the M abscessus subsp massiliense group (M abscessus subsp massiliense cluster 1 and 2); (C) which was based on mapping against an assembled M abscessus subsp massiliense reference genome. The branches between the M abscessus subsp massiliense clusters are 95 and 90 SNPs in length but have been shortened for illustration purposes. Isolates from patients from Papworth Hospital (denoted by numbers) are also compared with other published whole genome sequences (grey) for M abscessus subsp abscessus (isolates from Malaysian patients without cystic fibrosis [M93, M94, and M152]), M abscessus subsp massiliense (from Malaysian patients without cystic fibrosis [M115, M154, M139]; from a French patient without cystic fibrosis [CCUG48898]; from a patient in Birmingham UK with cystic fibrosis [47J26]; and from a surgical outbreak in Brazil [GO-06]) and M abscessus subsp bolletii (from a sputum isolate from France [CIP108541]).

Figure 2

Histograms of SNP pairwise distances between isolates of the same subspecies Distribution reveals clear distinctions between: pairwise differences of more than 10 000 SNPs, comprising comparisons of isolates from different M abscessus subspecies and from non-clustered M abscessus strains; pairwise differences of 50–200 SNPs, representing comparisons of isolates from within the M abscessus subsp abscessus cluster and between different M abscessus subsp massiliense clusters; and isolates with differences of less than 25 SNPs, constituting the diversity of samples from single individuals (red) and that of different patients' isolates within each M abscessus subsp massiliense cluster (blue).

Figure 3

Opportunities for patient-to-patient transmission within hospital The timelines of individual patients within the M abscessus subsp massiliense clusters 1 and 2 (A) and M abscessus subsp abscessus cluster (B) are shown. Short vertical lines denote hospital visits or admissions to Papworth Hospital, and circles denote sputum samples (culture negative [white]; smear negative culture positive [half-red]; smear positive [red]). Timelines become red after a positive sputum sample. Potential opportunities for transmission between patients (by virtue of being in hospital at the same time as a positive patient) are highlighted by grey vertical bars. All patients within the M abscessus subsp massiliense clusters had opportunities for transmission, whereas only two patients within the M abscessus subsp abscessus cluster had any opportunity for cross-infection.

Figure 4

Network analysis of patients with clustered and non-clustered M abscessus Social network analysis of individuals within M abscessus subsp massiliense cluster 1 (clustered cases; n=9) were compared with patients with unclustered M abscessus isolates (reference cases; n=15). We assumed that patients might acquire infection any time during a 12 month period before their first positive sample and might transmit infection at any time from this point onwards. Arrow thickness denotes duration of overlap within different areas of the hospital for individuals during periods of potential acquisition of infection with potentially infected patients. Arrow direction indicates potential route of transmission. The position of the circles represents the date of the first positive culture for M abscessus. Clustered cases were more tightly interconnected (having higher network densities) than references cases, were all exposed to individuals who had the potential to transmit infection during periods of potential acquisition, and that potential transmission events between them occurred both within the outpatients department and/or cystic fibrosis inpatient ward.

Figure 5

Date of the most recent common ancestors of putatively transmitted M abscessus subsp massiliense strains and opportunities for patient-to-patient transmission Estimates, predicted by Bayesian inference using BEAST, of when the most recent common ancestor existed for isolates from different patients within M abscessus subsp massiliense clusters 1 and 2 and from the two patients within the M abscessus subsp abscessus cluster who had transmission opportunities. In the case of both M abscessus subsp massiliense clusters, the interpatient most recent common ancestor could be dated to the period when opportunities existed for hospital-associated transmission (red circles), whereas for the grouped M abscessus subsp abscessus, it was dated to several decades before.