Laura Pérez-Lago

Whole Genome Sequencing Analysis of Intrapatient Microevolution in Mycobacterium tuberculosis: Potential Impact on the Inference of Tuberculosis Transmission

BACKGROUND It has been accepted that the infection by Mycobacterium tuberculosis (M. tuberculosis) can be more heterogeneous than considered. The emergence of clonal variants caused by microevolution events leading to population heterogeneity is a phenomenon largely unexplored. Until now, we could only superficially analyze this phenomenon by standard fingerprinting (RFLP and VNTR). METHODS In this study we applied whole genome sequencing for a more in-depth analysis of the scale of microevolution both at the intrapatient and interpatient scenarios. RESULTS We found that the amount of variation accumulated within a patient can be as high as that observed between patients along a chain of transmission. Intrapatient diversity was found both at the extrapulmonary and respiratory sites, meaning that this variability can be transmitted and impact on the inference of transmission events. One of the events studied allowed us to track for a single strain the complete process of (i) interpatient microevolution, (ii) intrapatient respiratory variation, and (iii) isolation of different variants at different infected sites of this patient. CONCLUSIONS Our study adds new data to the understanding of variability in M. tuberculosis in a wide clinical scenario and alerts about the difficulties of establishing thresholds to differentiate relatedness in M. tuberculosis with epidemiological purposes.

Systematic Survey of Clonal Complexity in Tuberculosis at a Populational Level and Detailed Characterization of the Isolates Involved

ABSTRACT Clonally complex infections by Mycobacterium tuberculosis are progressively more accepted. Studies of their dimension in epidemiological scenarios where the infective pressure is not high are scarce. Our study systematically searched for clonally complex infections (mixed infections by more than one strain and simultaneous presence of clonal variants) by applying mycobacterial interspersed repetitive-unit (MIRU)–variable-number tandem-repeat (VNTR) analysis to M. tuberculosis isolates from two population-based samples of respiratory (703 cases) and respiratory-extrapulmonary (R+E) tuberculosis (TB) cases (71 cases) in a context of moderate TB incidence. Clonally complex infections were found in 11 (1.6%) of the respiratory TB cases and in 10 (14.1%) of those with R+E TB. Among the 21 cases with clonally complex TB, 9 were infected by 2 independent strains and the remaining 12 showed the simultaneous presence of 2 to 3 clonal variants. For the 10 R+E TB cases with clonally complex infections, compartmentalization (different compositions of strains/clonal variants in independent infected sites) was found in 9 of them. All the strains/clonal variants were also genotyped by IS6110-based restriction fragment length polymorphism analysis, which split two MIRU-defined clonal variants, although in general, it showed a lower discriminatory power to identify the clonal heterogeneity revealed by MIRU-VNTR analysis. The comparative analysis of IS6110 insertion sites between coinfecting clonal variants showed differences in the genes coding for a cutinase, a PPE family protein, and two conserved hypothetical proteins. Diagnostic delay, existence of previous TB, risk for overexposure, and clustered/orphan status of the involved strains were analyzed to propose possible explanations for the cases with clonally complex infections. Our study characterizes in detail all the clonally complex infections by M. tuberculosis found in a systematic survey and contributes to the characterization that these phenomena can be found to an extent higher than expected, even in an unselected population-based sample lacking high infective pressure.

Current knowledge and pending challenges in zoonosis caused by Mycobacterium bovis: a review.

Mycobacterium bovis is both the causative agent of bovine tuberculosis (TB) and a zoonotic pathogen. In humans, considerably fewer cases of TB are caused by M. bovis than M. tuberculosis; nevertheless, diagnostic limitations mean that currently available data on prevalence grossly underestimate the true dimension of the problem. The routes of transmission from animals to humans are well known and include direct exposure to infected animals or consumption of contaminated animal products. Application of fingerprinting tools facilitates analysis of the molecular epidemiology of M. bovis in animal-to-human and human-to-human transmission. Apart from cattle and M. bovis, other animal species and members within the M. tuberculosis complex can contribute to the zoonosis. Improvements in diagnostic techniques, application of more advanced discriminatory genotyping tools, and collaboration between veterinary and human health care researchers are key to our understanding of this zoonosis.

Characterization of Microevolution Events in Mycobacterium tuberculosis Strains Involved in Recent Transmission Clusters

ABSTRACT Under certain circumstances, it is possible to identify clonal variants of Mycobacterium tuberculosis infecting a single patient, probably as a result of subtle genetic rearrangements in part of the bacillary population. We systematically searched for these microevolution events in a different context, namely, recent transmission chains. We studied the clustered cases identified using a population-based universal molecular epidemiology strategy over a 5-year period. Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units–variable-number tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1- to 5-year period. The IS6110 insertion sites from 16 representative isolates of reference and microevolved variants were mapped by ligation-mediated PCR in order to characterize the genetic background involved in microevolution. Both intragenic and intergenic IS6110 locations resulted from these microevolution events. Among those cases of IS6110 locations in intergenic regions which could have an effect on the regulation of adjacent genes, we identified the overexpression of cytochrome P450 in one microevolved variant using quantitative real-time reverse transcription-PCR. Our results help to define the frequency with which microevolution can be expected in M. tuberculosis transmission chains. They provide a snapshot of the genetic background of these subtle rearrangements and identify an event in which IS6110-mediated microevolution in an isogenic background has functional consequences.

A non-canonical mismatch repair pathway in prokaryotes

Mismatch repair (MMR) is a near ubiquitous pathway, essential for the maintenance of genome stability. Members of the MutS and MutL protein families perform key steps in mismatch correction. Despite the major importance of this repair pathway, MutS–MutL are absent in almost all Actinobacteria and many Archaea. However, these organisms exhibit rates and spectra of spontaneous mutations similar to MMR-bearing species, suggesting the existence of an alternative to the canonical MutS–MutL-based MMR. Here we report that Mycobacterium smegmatis NucS/EndoMS, a putative endonuclease with no structural homology to known MMR factors, is required for mutation avoidance and anti-recombination, hallmarks of the canonical MMR. Furthermore, phenotypic analysis of naturally occurring polymorphic NucS in a M. smegmatis surrogate model, suggests the existence of M. tuberculosis mutator strains. The phylogenetic analysis of NucS indicates a complex evolutionary process leading to a disperse distribution pattern in prokaryotes. Together, these findings indicate that distinct pathways for MMR have evolved at least twice in nature.

Differences in gene expression between clonal variants of Mycobacterium tuberculosis emerging as a result of microevolution

Clonal variants of Mycobacterium tuberculosis can emerge as a result of microevolution in a single host or after sequential infection of different hosts. The significance of subtle genotypic variations is still unknown. In three of the four loci analyzed from clonal variants differing in only one MIRU-VNTR locus, we found that the expression of the adjacent genes was modulated differently. These data highlight the potential advantages that acquisition of subtle variability may have in M. tuberculosis.

Fast and low-cost decentralized surveillance of transmission of tuberculosis based on strain-specific PCRs tailored from whole genome sequencing data: a pilot study

Molecular epidemiology has transformed our knowledge of how tuberculosis (TB) is transmitted. Whole genome sequencing (WGS) has reached unprecedented levels of accuracy. However, it has increased technical requirements and costs, and analysis of data delays results. Our objective was to find a way to reconcile speed and ease of implementation with the high resolution of WGS. The targeted regional allele-specific oligonucleotide PCR (TRAP) assay presented here is based on allele-specific PCR targeting strain-specific single nucleotide polymorphisms, identified from WGS, and makes it possible to track actively transmitted Mycobacterium tuberculosis strains. A TRAP assay was optimized to track the most actively transmitted strains in a population in Almería, Southeast Spain, with high rates of TB. TRAP was transferred to the local laboratory where transmission was occurring. It performed well from cultured isolates and directly from sputa, enabling new secondary cases of infection from the actively transmitted strains to be detected. TRAP constitutes a fast, simple and low-cost tool that could modify surveillance of TB transmission. This pilot study could help to define a new model to survey TB transmission based on a decentralized multinodal network of local laboratories applying fast and low-cost TRAPs, which are developed by central reference centres, tailored to the specific demands of transmission at each local node.

Co-infection with Drug-Susceptible and Reactivated Latent Multidrug-Resistant Mycobacterium tuberculosis.

To the Editor: Genotyping of Mycobacterium tuberculosis (MTB) has identified mixed infections involving >1 MTB strain (1–4), which are clinically relevant when different susceptibility patterns are involved (4–7). We describe a tuberculosis (TB) case-patient with mixed infection in an area of moderate incidence. In a low-resistance setting (monoresistance 4.6%; multiresistance 1.7%), 1 of the strains was drug susceptible and the other was multidrug-resistant (MDR). Molecular fingerprinting and epidemiologic research revealed that the infection corresponded to a recent infection by a susceptible strain and reactivation of an MDR TB strain. The patient was an HIV-negative woman, 47 years of age, who had immigrated to Spain from Romania and had been living in Almeria for >3 years. TB was diagnosed in May 2014; she had experienced symptoms for 2 months. Her diagnosis was confirmed 3 years after being studied in Almeria as a close contact of her husband, also from Romania, who had tested positive for MDR TB (resistant to rifampin and isoniazid). When she was observed in the contact trace, she tested positive for purified protein derivative, had been vaccinated against the M. bovis bacillus Calmette-Guerin strain, and had no radiologic findings or clinical symptoms. Based on the susceptibility profile of her husband, prophylaxis was not prescribed. Her husband adhered to anti-TB treatment for 20 months; all microbiological control test results had been negative since 2 months after starting therapy. Because her infection was thought to originate from previous contact with an MDR TB case-patient, we assessed her sputum samples for resistance using GenoType-MTBDR-plus (Hain-Lifescience, Nehren, Rhineland-Paltinate, Germany). The test showed hybridization with the same mutant probes (rpoB-MUT3; katG-MUT1) as those of her husband, the assumed index MDR TB case-patient (Figurepanel A). The pattern was indeterminate because the hybridization for the mutant probes was faint, and intense hybridization was observed for all the wt probes (Figure panel A). Results suggested the simultaneous presence of an MDR strain and a susceptible strain in a respiratory specimen. The presence of the MDR TB strain was confirmed by the phenotypic antibiogram in a BBL MGIT mycobacteria growth indicator tube (Becton Dickinson, Franklin Lakes, NJ, USA) after the isolate had been cultured. GeneXpert (Cepheid, Sunnyvale, CA, USA) was used to analyze 2 respiratory specimens. Results indicated susceptibility to rifampin, revealing the limitations of this test: the use of probes targeting the wt sequences failed to detect resistant strains that coexist with a susceptible strain (7). Figure Identification of co-infection with drug-susceptible and reactivated latent multidrug-resistant Mycobacterium tuberculosis (MDR TB). A) Genotype of multidrug-resistant tuberculosis (MDR TB) and results for the MDR TB index patient and for 2 respiratory ... To ascertain the likelihood of 2 co-infecting strains, we analyzed the specimen and the cultured isolate by mycobacterial interspersed repetitive unit–variable number tandem repeat (MIRU-VNTR), which is highly sensitive for detecting complex infections (8,9). Double alleles were found at 12 loci (Figure, panel B), confirming co-infection. Peaks in the electropherograms suggested that 1 of the 2 strains was under-represented and its proportion was lower in the cultured isolates (Figure, panel C), indicating that culturing diminished its representation of the minority strain. This finding was consistent with the inability of the GenoType test to detect the MDR strain when applied to the cultured isolate (Figure, panel A). We also detected lower fitness for the MDR strain compared to the susceptible strain (p<0.01) (Technical Appendix Table). Proportions of the resistant and susceptible strains were determined by plating on Middlebrook 7H11 +/− isoniazid (0.4 μL/mL) and counting single colonies. Because 2% of the colonies were of the resistant strain, we separated the strains and concluded: 1) the strain cultured in the presence of an antimicrobial drug coincided in the population exclusively with the MDR strain from the husband (as shown by MIRU-VNTR); and 2) the co-infecting MDR strain was a minority strain that was under-represented in the cultured isolate. To analyze the origin of the susceptible strain, we investigated its MIRU-VNTR type in the population-based molecular epidemiology survey (10) and found another 4 cases (from 2008, 2011 [2 cases], and 2014). Three case-patients had emigrated from Romania, and all 5 case-patients lived in the same area of Almeria. These data indicated that the susceptible strain was circulating in the geographic/epidemiological context of the current case-patient before and when she tested positive for that strain; therefore, she likely acquired the susceptible strain through recent transmission. The presence of susceptible and resistant strains in a patient should be considered even in moderate incidence settings and where resistance rates are not high. Underdetection of these cases could lead to misinterpretation when MDR became apparent after treatment of susceptible strains. Diagnostic laboratories could easily screen for mixed infections by applying MIRU-VNTR. However, only by integrating clonal analysis, refined molecular typing, and epidemiologic data from universal genotyping programs can we clarify the reasons underlying complex MTB infections. For this case-patient, a recent infection with a susceptible strain coincided with or could have triggered reactivation of a latent infection involving an MDR strain acquired through close contact years previously. We emphasize the alteration of the true clonal complexity of an infection induced by culturing specimens and that some commercial tests do not identify complex MTB infections. These findings are particularly relevant when the infection involves resistant strains such as those found in this case-patient. Technical Appendix: In vitro fitness of the multidrug-resistant and susceptible Mycobacterium tuberculosis strains involved in the mixed infection. Click here to view.(106K, pdf)

Unmasking subtle differences in the infectivity of microevolved Mycobacterium tuberculosis variants coinfecting the same patient.

Clonal variants of Mycobacterium tuberculosis can emerge as a result of microevolution phenomena. The functional significance of these subtle genetic rearrangements is normally disregarded. We show that clonal variants from two patients had different infective behaviours in some in vitro cellular infection models but not in others. Microevolution may have a subtle impact on infectivity, but specific experimental conditions are needed to unmask it.

Persistent Infection by a Mycobacterium tuberculosis Strain That Was Theorized To Have Advantageous Properties, as It Was Responsible for a Massive Outbreak

ABSTRACT The strains involved in tuberculosis outbreaks are considered highly virulent and transmissible. We analyzed the case of a patient in Madrid, Spain, who was persistently infected over an 8-year period by the same Beijing Mycobacterium tuberculosis strain. The strain was responsible for a severe outbreak on Gran Canaria Island. The case provides us with a unique opportunity to challenge our assumptions about M. tuberculosis Beijing strains. No clinical/radiological findings consistent with a virulent strain were documented, and the in vitro growth rate of the strain in macrophages was only moderate. No secondary cases stemming from this prolonged active case were detected in the host population. The strain did not acquire resistance mutations, despite constant treatment interruptions, and it remained extremely stable, as demonstrated by the lack of single-nucleotide-polymorphism (SNP)-based differences between the sequential isolates. Our data suggest that the general assumption about M. tuberculosis Beijing strains having advantageous properties (in terms of virulence, transmissibility, and the tendency to acquire mutations and resistance) is not always accurate.