Genome sequencing of Mycobacterium pinnipedii strains: genetic characterization and evidence of superinfection in a South American sea lion (Otaria flavescens)

Abstract

BackgroundMycobacterium pinnipedii, a member of the Mycobacterium tuberculosis Complex (MTBC), is capable of infecting several host species, including humans. Recently, ancient DNA from this organism was recovered from pre-Columbian mummies of Peru, sparking debate over the origin and frequency of tuberculosis in the Americas prior to European colonization.ResultsWe present the first comparative genomic study of this bacterial species, starting from the genome sequencing of two M. pinnipedii isolates (MP1 and MP2) obtained from different organs of a stranded South American sea lion. Our results indicate that MP1 and MP2 differ by 113 SNPs (single nucleotide polymorphisms) and 46 indels, constituting the first report of a mixed-strain infection in a sea lion. SNP annotation analyses indicate that genes of the VapBC family, a toxin-antitoxin system, and genes related to cell wall remodeling are under evolutionary pressure for protein sequence change in these strains. OrthoMCL analysis with seven modern isolates of M. pinnipedii shows that these strains have highly similar proteomes. Gene variations were only marginally associated with hypothetical proteins and PE/PPE (proline-glutamate and proline-proline-glutamate, respectively) gene families. We also detected large deletions in ancient and modern M. pinnipedii strains, including a few occurring only in modern strains, indicating a process of genome reduction occurring over the past one thousand years. Our phylogenomic analyses suggest the existence of two modern clusters of M. pinnipedii associated with geographic location, and possibly host species, and one basal node associated with the ancient M. pinnipedii strains. Previously described MiD3 and MiD4 deletions may have occurred independently, twice, over the evolutionary course of the MTBC.ConclusionThe presence of superinfection (i.e. mixed-strain infection) in this sea lion suggests that M. pinnipedii is highly endemic in this population. Mycobacterium pinnipedii proteomes of the studied isolates showed a high degree of conservation, despite being under genomic decay when compared to M. tuberculosis. This finding indicates that further genomes need to be sequenced and analyzed to increase the chances of finding variably present genes among strains or that M. pinnipedii genome remodeling occurred prior to bacterial speciation.

Methods for Detecting Mycobacterial Mixed Strain Infections–A Systematic Review

“…Spoligotyping has been widely used in epidemiological studies to investigate the cause of recurrent TB (defined as endogenous reactivation of an initially infecting strain or exogenous reinfection with a different strain) (Small et al, 1994 ; Warren et al, 2002 ; Van Rie et al, 2005 ; Andrews et al, 2008 ), tracking epidemics (Goyal et al, 1999 ; Källenius et al, 1999 ; Caminero et al, 2001 ), and investigating laboratory cross-contamination (Nivin et al, 2000 ). Twenty (16.5%) publications reported using spoligotyping as one of the methods for detecting MSIs, with a majority employing one (Pavlic et al, 1999 ; Cox et al, 2005 ; García De Viedma et al, 2005 ; Umubyeyi et al, 2007 ; Furphy et al, 2012 ; Biffa et al, 2014 ; Chaoui et al, 2014 ; Lamine-Khemiri et al, 2014 ; Ssengooba et al, 2015 ; Wang et al, 2015 ; Egbe et al, 2017 ; Baffoe-Bonnie et al, 2019 ) or two (García De Viedma et al, 2003 ; Mokrousov et al, 2009 ; Huyen et al, 2012 ; Navarro et al, 2015 ; Silva-Pereira et al, 2019 ) additional methods ( Supplementary Table 2 ). Only three reports used spoligotyping as the sole genotyping method and reported MSIs at frequencies ranging from 11.8 to 57.1% (Andrews et al, 2008 ; Kamakoli et al, 2018 ; Guernier-Cambert et al, 2019 ).…”

“…They detected MSIs in 19% of samples from patients associated with retreatment in their study. In addition, WGS was used as second technique in three spoligotyping based studies (Witney et al, 2017 ; Baffoe-Bonnie et al, 2019 ; Silva-Pereira et al, 2019 ), one of which focused on animals. In their work, Silva-Pereira et al ( 2019 ) detected an M. pinnipedii MSI in a South American sea lion ( Otaria flavescens ) using WGS, which was not suggested by in silico spoligotyping or MIRU-VNTR initially.…”

“…In addition, WGS was used as second technique in three spoligotyping based studies (Witney et al, 2017 ; Baffoe-Bonnie et al, 2019 ; Silva-Pereira et al, 2019 ), one of which focused on animals. In their work, Silva-Pereira et al ( 2019 ) detected an M. pinnipedii MSI in a South American sea lion ( Otaria flavescens ) using WGS, which was not suggested by in silico spoligotyping or MIRU-VNTR initially. The above-mentioned studies demonstrate the importance of using more discriminatory methods along with traditional screening techniques to ensure the detection of different strains that might be present in a single sample.…”

“…In this way, the number of repeats at each VNTR locus can be determined using different DNA sizing techniques for comparing isolates. It was found that 50 studies used VNTR-typing to detect M. tuberculosis MSIs, where 28 studies used it as the sole discriminatory method for this purpose (Allix et al, 2004 ; Shamputa et al, 2004 ; Umubyeyi et al, 2007 ; Fang et al, 2008 ; Hofmann-Thiel et al, 2009 ; Mokrousov et al, 2009 ; Stavrum et al, 2009 ; Dickman et al, 2010 ; Mulenga et al, 2010 ; Cohen et al, 2011 ; Gardy et al, 2011 ; Navarro et al, 2011 , 2015 ; Cerezo et al, 2012 ; Furphy et al, 2012 ; Huyen et al, 2012 ; Hingley-Wilson et al, 2013 ; Muwonge et al, 2013 ; Peng et al, 2013 ; Biffa et al, 2014 ; Chaoui et al, 2014 ; Lamine-Khemiri et al, 2014 ; Zetola et al, 2014 ; Barletta et al, 2015 ; Mei et al, 2015 ; Pang et al, 2015 ; Shin et al, 2015 , 2018 ; Ssengooba et al, 2015 ; Streit et al, 2015 ; Wang et al, 2015 ; Zheng et al, 2015 ; Antusheva et al, 2016 ; Hajimiri et al, 2016 ; Hu et al, 2016 ; Egbe et al, 2017 ; Farmanfarmaei et al, 2017 ; Ghielmetti et al, 2017 ; Kamakoli et al, 2017 , 2019 , 2020a , b ; Khosravi et al, 2017 ; Kontsevaya et al, 2017 ; Nathavitharana et al, 2017 ; Sadegh et al, 2017 ; Silva-Pereira et al, 2019 ; Abascal et al, 2020 ; Baik et al, 2020 ; Sichewo et al, 2020 ). In addition, 24-locus MIRU-VNTR was the most commonly used method for detecting M. tuberculosis MSIs, and nearly all (47/50, 94.0%) of the reports used some form...…”

“…Of the 97 studies identified in Covidence, eight (8.2%) primarily used WGS to detect MSIs in humans (Bryant et al, 2013a ; Gan et al, 2016 ; Witney et al, 2017 ; Baffoe-Bonnie et al, 2019 ; Dippenaar et al, 2019 ; O'Donnell et al, 2019 ; Anyansi et al, 2020 ; Wollenberg et al, 2020 ), while three others (3.1%) used the method to detect MSIs in various animal species (Davidson et al, 2016 ; Pfeiffer et al, 2017 ; Silva-Pereira et al, 2019 ), only one of which focused on a MTBC bacteria (Silva-Pereira et al, 2019 ). Secondary searches found four more human studies on WGS and mycobacterial MSIs (Chan et al, 2013 ; Kay et al, 2015 ; Dheda et al, 2017 ; Nimmo et al, 2020 ), in addition to reports where the method was used to confirm such infection that were initially identified using other means ( Supplementary Table 2 ).…”