Multidrug-Resistant Nontuberculous Mycobacteria Isolated from Cystic Fibrosis Patients

Worldwide, nontuberculous mycobacteria (NTM) have become emergent pathogens of pulmonary infections in cystic fibrosis (CF) patients, with an estimated prevalence ranging from 5 to 20%. This work investigated the presence of NTM in sputum samples of 129 CF patients (2 to 18 years old) submitted to longitudinal clinical supervision at a regional reference center in Rio de Janeiro, Brazil. From June 2009 to March 2012, 36 NTM isolates recovered from 10 (7.75%) out of 129 children were obtained. Molecular identification of NTM was performed by using PCR restriction analysis targeting the hsp65 gene (PRA-hsp65) and sequencing of the rpoB gene, and susceptibility tests were performed that followed Clinical and Laboratory Standards Institute recommendations. for evaluating the genotypic diversity, pulsed-field gel electrophoresis (PFGE) and/or enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) was performed. the species identified were Mycobacterium abscessus subsp. bolletii (n = 24), M. abscessus subsp. abscessus (n = 6), Mycobacterium fortuitum (n = 3), Mycobacterium marseillense (n = 2), and Mycobacterium timonense (n = 1). Most of the isolates presented resistance to five or more of the antimicrobials tested. Typing profiles were mainly patient specific. the PFGE profiles indicated the presence of two clonal groups for M. abscessus subsp. abscessus and five clonal groups for M. abscesssus subsp. bolletii, with just one clone detected in two patients. Given the observed multidrug resistance patterns and the possibility of transmission between patients, we suggest the implementation of continuous and routine investigation of NTM infection or colonization in CF patients, including countries with a high burden of tuberculosis disease.Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)PDTIS-FIOCRUZUniv Fed Rio de Janeiro, Programa Posgrad Clin Med, Hosp Univ Clementino Fraga Filho, Rio de Janeiro, BrazilUniv Fed Rio Grande do Sul, Programa Posgrad Ciencias Med, Porto Alegre, RS, BrazilUniv Fed Rio de Janeiro, Fac Ciencias Med, Dept Microbiol Imunol & Parasitol, Rio de Janeiro, BrazilInst Fernandes Figueira Fiocruz, Rio de Janeiro, BrazilUniv Estado Rio de Janeiro, Hosp Univ Pedro Ernesto, Rio de Janeiro, BrazilUniv Fed Rio de Janeiro, Inst Microbiol, BR-21941 Rio de Janeiro, BrazilFundacao Oswaldo Cruz, Inst Pesquisa Evandro Chagas, Rio de Janeiro, BrazilInst Doencas Torax, Rio de Janeiro, BrazilJohns Hopkins Univ, Baltimore, MD USAUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv Fed Fluminense, Inst Biomed, Niteroi, RJ, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPERJ: 103.225/2011FAPERJ: 103.287/2011FAPERJ: 110.272/2010FAPERJ: 110.761/2010FAPERJ: 111.497/2008CNPq: 476536/2012-0CNPq: 473444/2010-0CNPq: 567037/2008-8Web of Scienc

Cell Surface Remodeling of Mycobacterium abscessus under Cystic Fibrosis Airway Growth Conditions.

Understanding the physiological processes underlying the ability of Mycobacterium abscessus to become a chronic pathogen of the cystic fibrosis (CF) lung is important to the development of prophylactic and therapeutic strategies to better control and treat pulmonary infections caused by these bacteria. Gene expression profiling of a diversity of M. abscessus complex isolates points to amino acids being significant sources of carbon and energy for M. abscessus in both CF sputum and synthetic CF medium and to the bacterium undergoing an important metabolic reprogramming in order to adapt to this particular nutritional environment. Cell envelope analyses conducted on the same representative isolates further revealed unexpected structural alterations in major cell surface glycolipids known as the glycopeptidolipids (GPLs). Besides showing an increase in triglycosylated forms of these lipids, CF sputum- and synthetic CF medium-grown isolates presented as yet unknown forms of GPLs representing as much as 10% to 20% of the total GPL content of the cells, in which the classical amino alcohol located at the carboxy terminal of the peptide, alaninol, is replaced with the branched-chain amino alcohol leucinol. Importantly, both these lipid changes were exacerbated by the presence of mucin in the culture medium. Collectively, our results reveal potential new drug targets against M. abscessus in the CF airway and point to mucin as an important host signal modulating the cell surface composition of this pathogen

Caracterização da resistência a quinolonas em micobactérias de crescimento rápido de importância médica

As micobactérias de crescimento rápido (MCR) estão amplamente distribuídas no ambiente, tendo emergido como agentes de infecção pós-cirúrgica. Recentemente, foram relatados no Brasil surtos de infecções por Mycobacterium massiliense em pacientes submetidos a procedimentos invasivos, nos quais os instrumentais médicos não sofreram esterilização e desinfecção adequadas. As quinolonas representam uma opção para o tratamento de infecções por MCR, sendo sugeridas para a terapêutica de infecções por algumas espécies. Seu mecanismo de ação envolve a interrupção da replicação do DNA pela formação de um complexo com a DNA girase. Essa enzima é constituída de 2 subunidades A e 2 subunidades B codificadas, respectivamente, pelos genes gyrA e gyrB. Alterações em aminoácidos nas regiões determinantes da resistência a quinolonas (RDRQ) de gyrA e gyrB implicam em resistência a quinolonas pela redução da interação com a DNA girase. Com o objetivo de caracterizar a resistência a quinolonas em MCR, as atividades in vitro de quinolonas de diferentes gerações foram determinadas por microdiluição em caldo em 54 cepas de M. massiliense, incluindo 43 cepas pertencentes ao clone BRA100 e duas cepas não clonais, isoladas no estado do Rio de Janeiro, e 9 cepas isoladas em diferentes estados do Brasil. Cepas de referência de M. abscessus, M. chelonae, M. fortuitum, M. massiliense e M. smegmatis também foram incluídas no estudo. Além disso, as sequências peptídicas das RDRQ de gyrA e gyrB foram obtidas por sequenciamento de DNA em 40 cepas dessa mesma espécie, incluindo 38 pertencentes ao clone BRA100, isoladas no estado do Rio de Janeiro. Todas as 54 cepas apresentaram resistência a todas as gerações de quinolonas, mas entre espécies diferentes de MCR houve variação na susceptibilidade a essa classe de antimicrobianos. As 40 cepas sequenciadas tiveram os mesmos aminoácidos nas RDRQ, incluindo a Ala-83 em gyrA e Arg-447 e Asp-464 em gyrB, já descritos como responsáveis por uma resistência intrínseca a quinolonas em micobactérias. Considerando esses aspectos, as quinolonas podem ser ativas in vitro contra MCR como M. fortuitum, M. chelonae e M. smegmatis, mas não devem utilizadas para o tratamento de infecções por M. massiliense. A resistência a quinolonas em M. massiliense apresentou, nas RDRQ gyrA e gyrB, os mesmos mecanismos de resistência em cepas de clones diferentes, mas outros mecanismos podem estar envolvidos com a resistência a altos níveis desses antimicrobianos.Rapidly growing mycobacteria (RGM) are widely distributed in the environment and have emerged as agents of postsurgical infections. Recently, outbreaks of infections caused by Mycobacterium massiliense have been reported in patients who had undergone invasive procedures, in which medical instruments have not been properly sterilized or disinfected, in many states of Brazil. The quinolones represent an option for the treatment RGM infections. Their mechanism of action involves the interruption the DNA replication by forming a complex with DNA gyrase. This enzyme is composed by two A and B subunits codified by the gyrA and gyrB genes. The amino acid changes in the quinolone resistance determining regions (QRDR) of gyrA and gyrB may decrease the interaction between the quinolones and the DNA gyrase. In order to characterize the resistance to quinolones among RGM, the in vitro activities of different generations of quinolones were determined by broth microdilution for 54 strains of M. massiliense, including 43 strains belonging to BRA100 clone and 2 non clonal strains, isolated from Rio de Janeiro state, and 9 strains isolated from different states of Brazil. Reference strains of M. abscessus, M. chelonae, M. fortuium, M. massiliense and M. smegmatis were also studied. Furthermore, the peptide sequences of the QRDR of gyrA and gyrB were obtained by DNA sequencing for 40 strains of M. massiliense, including 38 strains that belong to BRA100 clone, isolated from Rio de Janeiro state and compared with other RGM sequences previously described. All 54 M. massiliense strains were resistant to all generations of quinolones, but different species presented distinct susceptibilities to this class of antimicrobials. The 40 strains sequenced presented the same amino acids in the QRDR, including the Ala-83 in gyrA, Arg-447 and Asp-464 in gyrB, which have already been described as responsible for an intrinsic resistance to quinolones in mycobacteria. Considering these aspects, the quinolones might be active against RGM, such as M. fortuitum, M. chelonae and M. smegmatis, but they should not be chosen for treating M. massiliense infections. The quinolone resistance among M. massiliense showed, in the QRDR of gyrA and gyrB, the same mechanisms of resistance in strains belonging to different clones, however, other mechanisms may also be involved with the resistance to high levels of these antimicrobials

Characterization of quinolone resistance in Mycobacterium abscessus subsp. bolletii and other related rapidly growing mycobacteria

Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorEm diversos estados do Brasil, foram relatadas epidemias de infecções causadas por micobactérias de crescimento rápido (MCR) desde o ano 2000. A maioria dos casos foi principalmente associada ao clone BRA100 de Mycobacterium massiliense, recentemente renomeada para Mycobacterium abscessus subsp. bolletii, isolado de pacientes submetidos a procedimentos invasivos nos quais os instrumentos médicos não foram adequadamente esterilizados e/ou desinfetados. Sendo as quinolonas uma opção no tratamento de infecções por MCR e sugerida para esquemas terapêuticos para esses surtos, foram avaliadas nesse trabalho as atividades in vitro de quatro gerações de quinolonas para cepas clinicas e de referência de MCR através da microdiluição em caldo. Também foram analisadas as sequências peptídicas das regiões determinantes da resistência a quinolonas (RDRQ) das subunidades A e B da DNA gyrase (GyrA e GyrB) após o seqüenciamento de DNA seguido pela tradução da sequência de aminoácidos. Cinquenta e quatro cepas de M. abscessus subsp bolletii, incluindo o clone BRA100, isoladas em diferentes estados do Brasil, e 19 cepas de referência de MCR foram caracterizadas. Todas as 54 cepas clínicas de M. abscessus subsp. bolletii foram resistentes a todas as gerações de quinolonas e mostraram o mesmo resíduo nas RDRQ, incluindo Ala-83 em GyrA, Arg-447 e Asp-464 em GyrB, descritos como sendo responsáveis por gerar um baixo nível de resistência a quinolonas em micobactérias. Porém, outras espécies de MCR apresentaram diferentes susceptibilidade e padrões de mutações contrários aos classicamente já definidos, sugerindo que outros mecanismos de resistência, diferentes de mutações em gyrA e gyrB também possam estar envolvidos na alta resistência a quinolonas.Several outbreaks of infections caused by rapidly growing mycobacteria (RGM) have been reported in many Brazilian states since 2000. Most of the cases were mainly associated to Mycobacterium massiliense, recently renamed as Mycobacterium abscessus subsp. bolletii, BRA100 clone recovered from patients who had undergone invasive procedures, in which medical instruments have not been properly sterilized and / or disinfected. Since quinolones have represented an option for the treatment of general RGM infections and suggested for therapeutic schemes for these outbreaks, we evaluated the in vitro activities of four generations of quinolones for clinical and reference RGM by broth microdilution, and analysis of peptide sequences of the quinolone resistance determining regions (QRDR) of GyrA and GyrB after DNA sequencing followed by amino acid translation. Fifty four isolates of M. abscessus subsp bolletii, including clone BRA100, recovered in different states of Brazil, and 19 reference strains of RGM species were characterized. All 54 M. abscessus subsp. bolletii isolates were resistant to all generations of quinolones and showed the same amino acids in the QRDR including the Ala-83 in GyrA, Arg-447 and Asp-464 in GyrB, described as responsible for an intrinsic low level of resistance to quinolones in mycobacteria. But other RGM species presented distinct susceptibilities to this class of antimicrobials and patterns of mutations contrary to what has been traditionally defined, suggesting that other mechanisms of resistance, different from gyrA or gyrB mutations, may also be involved in resistance to high levels of quinolones

Gene Replacement in <i>Mycobacterium chelonae</i>: Application to the Construction of Porin Knock-Out Mutants

<div><p><i>Mycobacterium chelonae</i> is a rapidly growing mycobacterial opportunistic pathogen closely related to <i>Mycobacterium abscessus</i> that causes cornea, skin and soft tissue infections in humans. Although <i>M. chelonae</i> and the emerging mycobacterial pathogen <i>M. abscessus</i> have long been considered to belong to the same species, these two microorganisms considerably differ in terms of optimum growth temperature, drug susceptibility, pathogenicity and the types of infection they cause. The whole genome sequencing of clinical isolates of <i>M. chelonae</i> and <i>M. abscessus</i> is opening the way to comparative studies aimed at understanding the biology of these pathogens and elucidating the molecular bases of their pathogenicity and biocide resistance. Key to the validation of the numerous hypotheses that this approach will raise, however, is the availability of genetic tools allowing for the expression and targeted mutagenesis of genes in these species. While homologous recombination systems have recently been described for <i>M. abscessus</i>, genetic tools are lacking for <i>M. chelonae</i>. We here show that two different allelic replacement methods, one based on mycobacteriophage-encoded recombinases and the other on a temperature-sensitive plasmid harboring the counterselectable marker <i>sacB</i>, can be used to efficiently disrupt genes in this species. Knock-out mutants for each of the three porin genes of <i>M. chelonae</i> ATCC 35752 were constructed using both methodologies, one of which displays a significantly reduced glucose uptake rate consistent with decreased porin expression.</p></div

Glucose uptake by <i>M. chelonae</i> ATCC 35752 and its isogenic porin knock-out mutants.

<p>The accumulation of [U-¹⁴C]glucose by the strains over time was measured as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094951#s2" target="_blank">Materials and Methods</a>. Glucose uptake rates were calculated on the first 10 min of the reactions. Uptake experiments were performed in triplicates and are shown with their standard deviations.</p

Comparative efficiency of the <i>Ts-sacB</i> system using <i>zeo</i> and <i>kan</i> disrupted allelic exchange substrates in <i>M. chelonae</i> ATCC 35752.

<p>One to three transformants (T1, T2 and T3) were selected on plates upon transformation with the pPR27-derived plasmids, grown in 7H9-OADC broth at 30°C for 5 to 7 days, and finally plated onto 7H11-OADC containing Kan or Zeo and 10% sucrose at 37°C. The percentage of CFUs presenting the expected phenotype for allelic exchange mutants at the last selection step of the Ts-SacB procedure (sucrose resistant; Kan^R or Zeo^R and XylE⁻) is indicated for each construct. Four to ten candidate mutants were analyzed by PCR in each case and the number of double crossover mutants identified is indicated in the last column.</p

Gene replacement at the <i>MCH_4689c</i>, <i>MCH_4690c</i> and <i>MCH_4691c</i> porin loci of <i>M. chelonae</i> ATCC 35752 using the <i>Ts-sacB</i> and recombineering systems.

<p>(A) Porin gene cluster of <i>M. chelonae</i> ATCC 35752. The positions of the primers used to generate the allelic exchange substrates and analyze the candidate mutants are indicated. IGR1 and IGR2 represent the intergenic regions. (B) Candidate mutants obtained for each of the porin genes using the <i>Ts-sacB</i> or the recombineering systems were analyzed by PCR as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094951#s2" target="_blank">Materials and Methods</a> and confirmed by sequencing the regions flanking the resistance cassette. The expected size of the PCR fragments is 3.3 kb for the wild-type parent strain and 3.8 kb for the knock-out mutants. MWM, molecular weight marker. WT, wild-type. (C) Immunoblot analysis of porin production in the wild-type, mutant and complemented mutant strains. Strains were grown in 7H9-OADC-Tween 80 broth at 30°C to mid-log phase (OD600 = 1) and porins were selectively extracted from whole cells at 100°C using 0.5% <i>n</i>-octylpolyoxyethylene as a detergent as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094951#pone.0094951-Heinz1" target="_blank">[44]</a>. Protein samples prepared from the same amount of cells for each strain were denatured by boiling in 80% DMSO followed by acetone precipitation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094951#pone.0094951-Stahl1" target="_blank">[23]</a>. Denatured proteins were loaded volume to volume, separated by SDS-PAGE, blotted onto a nitrocellulose membrane, and porins were detected using rabbit antiserum to purified MspA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094951#pone.0094951-Stahl1" target="_blank">[23]</a>. Immune complexes were detected by chemiluminescence (Pierce, ELC) and semi-quantified using the Image Lab software (Biorad).</p