Phylogenomic and genomic analysis reveals unique and shared genetic signatures of Mycobacterium kansasii complex species.

Species belonging to the Mycobacterium kansasii complex (MKC) are frequently isolated from humans and the environment and can cause serious diseases. The most common MKC infections are caused by the species M. kansasii (sensu stricto), leading to tuberculosis-like disease. However, a broad spectrum of virulence, antimicrobial resistance and pathogenicity of these non-tuberculous mycobacteria (NTM) are observed across the MKC. Many genomic aspects of the MKC that relate to these broad phenotypes are not well elucidated. Here, we performed genomic analyses from a collection of 665 MKC strains, isolated from environmental, animal and human sources. We inferred the MKC pangenome, mobilome, resistome, virulome and defence systems and show that the MKC species harbours unique and shared genomic signatures. High frequency of presence of prophages and different types of defence systems were observed. We found that the M. kansasii species splits into four lineages, of which three are lowly represented and mainly in Brazil, while one lineage is dominant and globally spread. Moreover, we show that four sub-lineages of this most distributed M. kansasii lineage emerged during the twentieth century. Further analysis of the M. kansasii genomes revealed almost 300 regions of difference contributing to genomic diversity, as well as fixed mutations that may explain the M. kansasii's increased virulence and drug resistance.

Unraveling the metabolism of Mycobacterium caprae using comparative genomics.

In our laboratory, Mycobacterium caprae has poor growth in standard medium (SM) 7H9-OADC supplemented with pyruvate and Tween-80. Our objectives were to identify mutations affecting M. caprae metabolism and use this information to design a culture medium to improve its growth. We selected 77 M. caprae genomes and sequenced M. caprae NLA000201913 used in our experiments. Mutations present in >95% of the strains compared to Mycobacterium tuberculosis H37Rv were analyzed in silico for their deleterious effects on proteins of metabolic pathways. Apart from the known defect in the pyruvate kinase, M. caprae has important lesions in enzymes of the TCA cycle, methylmalonyl cycle, B12 metabolism, and electron-transport chain. We provide evidence of enzymatic redundancy elimination and epistatic mutations, and possible production of toxic metabolites hindering M. caprae growth in vitro. A newly designed SM supplemented with l-glutamate allowed faster growth and increased final microbial mass of M. caprae. However, possible accumulation of metabolic waste-products and/or nutritional limitations halted M. caprae growth prior to a M. tuberculosis-like stationary phase. Our findings suggest that M. caprae relies on GABA and/or glyoxylate shunts for in vitro growth in routine media. The newly developed medium will improve experiments with this bacterium by allowing faster growth in vitro.

Whole-Genome Sequencing of Alcaligenes faecalis HZ01, with Potential to Inhibit Nontuberculous Mycobacterial Growth.

Alcaligenes faecalis is a Gram-negative rod that is ubiquitous in the environment and is an opportunistic human pathogen. Here, we report the whole-genome sequencing analysis of A. faecalis HZ01, which presents mycobacterial growth inhibitory activity and was isolated from a contaminated culture of Mycobacterium chubuense ATCC 27278.

Molecular epidemiology of Mycobacterium tuberculosis in Brazil before the whole genome sequencing era: a literature review.

Molecular-typing can help in unraveling epidemiological scenarios and improvement for disease control strategies. A literature review of Mycobacterium tuberculosis transmission in Brazil through genotyping on 56 studies published from 1996-2019 was performed. The clustering rate for mycobacterial interspersed repetitive units - variable tandem repeats (MIRU-VNTR) of 1,613 isolates were: 73%, 33% and 28% based on 12, 15 and 24-loci, respectively; while for RFLP-IS6110 were: 84% among prison population in Rio de Janeiro, 69% among multidrug-resistant isolates in Rio Grande do Sul, and 56.2% in general population in São Paulo. These findings could improve tuberculosis (TB) surveillance and set up a solid basis to build a database of Mycobacterium genomes.

Phenotypic and Genotypic Drug Susceptibility Assessment of Mycobacterium bovis Bacillus Calmette-Guérin Clinical Strains.

PURPOSE: Mycobacterium bovis Bacillus Calmette-Guérin (BCG) is the only vaccine licensed against tuberculosis. Despite the protection offered by the vaccine, in some circumstances children and immunocompromised individuals can develop associated infections, known as BCGitis. Drug susceptibility patterns of BCG clinical strains have rarely been described. We aimed to describe the susceptibility pattern of BCG clinical strains isolated in two different countries. METHODS: We performed culture-based drug susceptibility testing of thirty one BCG strains isolated from patients in Brazil (n=5, 16%) and Argentina (n=26, 84%) using the broth micro-dilution method (phenotypic method). Final antibiotic concentrations for susceptibility testing ranged from 0.5 to 16 mg/L for amikacin, 0.3125 to 10 mg/L for ethambutol, 0.05 to 1.6 mg/L for isoniazid and 0.25 to 8 mg/L for rifampicin, streptomycin and ofloxacin. Additionally, we compared the results with genetic data obtained by whole genome sequencing. RESULTS: By using the phenotypic method we detected one strain resistant to ethambutol, three strains resistant to rifampicin and one resistant to isoniazid. Additionally, two strains that were phenotypically resistant to both isoniazid and rifampicin carried mutations in the katG and rpoB genes simultaneously. CONCLUSION: There is evidence of the emergence of BCG-resistant strains isolated from vaccine-related complications. We recommend drug susceptibility testing of the BCG strain causing the infection in order to prevent treatment failure.

Characterization of Mycobacterium tuberculosis var. africanum isolated from a patient with pulmonary tuberculosis in Brazil.

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC), including Mycobacterium tuberculosis var. tuberculosis (MTB) and Mycobacterium tuberculosis var. africanum (MAF). While MTB is isolated worldwide, MAF is almost completely restricted to the African continent, and despite the historical proximity between Brazil and Africa during the slave trade, no case of TB being caused by MAF has been reported in Brazil to date. We hereby describe the first case of TB caused by MAF in Brazil comparing its genome against the published ones. A female patient who had never visited Africa presented with clinical symptoms typical of pulmonary TB. Based on 16S rRNA gene sequencing, the cultured isolate was identified as belonging to MTBC and partial sequence of the hsp65 gene was identical to that of MAF. This was confirmed by genotyping based on detection of Single Nucleotide Polymorphism (SNP), Region of Difference (RD) and spoligotyping. The isolate presented the Shared International Typing (SIT) 181. In the whole-genome comparison against MAF genomes available on published EMBL-EBI European Nucleotide Archive (ENA), the Brazilian genome (MAFBRA00707) was identified as belonging to Lineage 6 and clustered with isolates from The Gambia. This is the first report of the isolation of MAF from a patient from Brazil, without evidence of having any contact with an African index case.

Newly sequenced genomes of four Bacillus Calmette Guerin vaccines.

Bacillus Calmette Guerin (BCG) vaccines comprise a family of related strains. Whole genome sequencing has allowed the better characterisation of the differences between many of the BCG vaccines. As sequencing technologies improve, updating of publicly available sequence data becomes common practice. We hereby announce the draft genome of four commonly used BCG vaccines in Brazil, Argentina and Venezuela.

Nasopharyngeal carriage of respiratory pathogens in Warao Amerindians: significant relationship with stunting.

OBJECTIVE: To assess risk factors for nasopharyngeal carriage of potential pathogens in geographically isolated Warao Amerindians in Venezuela. METHODS: In this point prevalence survey, nasopharyngeal swabs were obtained from 1064 Warao Amerindians: 504 children aged 0-4 years, 227 children aged 5-10 years and 333 caregivers. Written questionnaires were completed to obtain information on demographics and environmental risk factors. Anthropometric measurements were performed in children aged 0-4 years. RESULTS: Carriage rates of Streptococcus pneumoniae, Staphylococcus aureus, Haemophilus influenzae and Moraxella catarrhalis were 51%, 7%, 1% and 13%, respectively. Crowding index, method of cooking and tobacco exposure were not associated with increased carriage. In multivariable analysis, an increase in height-for-age Z score (i.e. improved chronic nutritional status) was associated with decreased odds of S. pneumoniae colonisation (OR 0.76, 95% CI 0.70-0.83) in children aged 0-4 years. CONCLUSIONS: Better knowledge of demographic and environmental risk factors facilitates better understanding of the dynamics of colonisation with respiratory bacteria in an Amerindian population. Poor chronic nutritional status was associated with increased pathogen carriage in children <5 years of age. The high rates of stunting generally observed in indigenous children may fuel the acquisition of respiratory bacteria that can lead to respiratory and invasive disease.

Stunting correlates with high salivary and serum antibody levels after 13-valent pneumococcal conjugate vaccination of Venezuelan Amerindian children.

OBJECTIVE: To determine the impact of pre-vaccination nutritional status on vaccine responses in Venezuelan Warao Amerindian children vaccinated with the 13-valent pneumococcal conjugate vaccine (PCV13) and to investigate whether saliva can be used as read-out for these vaccine responses. METHODS: A cross-sectional cohort of 504 Venezuelan Warao children aged 6 weeks - 59 months residing in nine geographically isolated Warao communities were vaccinated with a primary series of PCV13 according to Centers for Disease Control and Prevention (CDC)-recommended age-related schedules. Post-vaccination antibody concentrations in serum and saliva of 411 children were measured by multiplex immunoassay. The influence of malnutrition present upon vaccination on post-vaccination antibody levels was assessed by univariate and multivariable generalized estimating equations linear regression analysis. RESULTS: In both stunted (38%) and non-stunted (62%) children, salivary antibody concentrations correlated well with serum levels for all serotypes with coefficients varying from 0.61 for serotype 3-0.80 for serotypes 5, 6A and 23F (all p < 0.01). Surprisingly, higher serum and salivary antibody levels were observed with increasing levels of stunting in children for all serotypes. This was statistically significant for 5/13 and 11/13 serotype-specific serum and saliva IgG concentrations respectively. CONCLUSION: Stunted Amerindian children showed generally higher antibody concentrations than well-nourished children following PCV13 vaccination, indicating that chronic malnutrition influences vaccine response. Saliva samples might be useful to monitor serotype-specific antibody levels induced by PCV vaccination. This would greatly facilitate studies of vaccine efficacy in rural settings, since participant resistance generally hampers blood drawing.

PCR deduction of invasive and colonizing pneumococcal serotypes from Venezuela: a critical appraisal.

INTRODUCTION: Serotype surveillance of Streptococcus pneumoniae is indispensable for evaluating the potential impact of pneumococcal conjugate vaccines. Serotyping by the standard Quellung reaction is technically demanding, time consuming, and expensive. A simple and economical strategy is multiplex PCR-based serotyping. We evaluated the cost effectiveness of a modified serial multiplex PCR (mPCR), resolving 24 serotypes in four PCR reactions and optimally targeting the most prevalent invasive and colonizing pneumococcal serotypes found in Venezuela. METHODOLOGY: A total of 223 pneumococcal isolates, 140 invasive and 83 carriage isolates, previously serotyped by the Quellung reaction and representing the 18 most common serotypes/groups identified in Venezuela, were serotyped with the adapted mPCR. RESULTS: The mPCR serotyped 76% of all the strains in the first two PCR reactions and 91% after four reactions, correctly identifying 17 serotypes/groups. An isolate could be serotyped with mPCR in less than 2 minutes versus 15 minutes for the Quellung reaction, considerably lowering labor costs. A restrictive weakness of mPCR was found for the detection of 19F strains. Most Venezuelan 19F strains were not typeable using the mPCR, and two 19F cps serotype variants were identified. CONCLUSIONS: The mPCR assay is an accurate, rapid, and economical method for the identification of the vast majority of the serotypes from Venezuela and can be used in place of the standard Quellung reaction. An exception is the identification of serotype 19F. In this setting, most 19F strains were not detectable with mPCR, demonstrating a need of serology-based quality control for PCR-based serotyping.