Extracellular vesicles in mycobacteria: new findings in biogenesis, host-pathogen interactions, and diagnostics.

Since the discovery of extracellular vesicles (EVs) in mycobacterial species 15 years back, we have learned that this phenomenon is conserved in the Mycobacterium genus and has critical roles in bacterial physiology and host-pathogen interactions. Mycobacterium tuberculosis (Mtb), the tuberculosis (TB) causative agent, produces EVs both in vitro and in vivo including a diverse set of biomolecules with demonstrated immunomodulatory effects. Moreover, Mtb EVs (MEVs) have been shown to possess vaccine properties and carry biomarkers with diagnostic capacity. Although information on MEV biogenesis relative to other bacterial species is scarce, recent studies have shed light on how MEVs originate and are released to the extracellular space. In this minireview, we discuss past and new information about the vesiculogenesis phenomenon in Mtb, including biogenesis, MEV cargo, aspects in the context of host-pathogen interactions, and applications that could help to develop effective tools to tackle the disease.

The mycobacterial glycoside hydrolase LamH enables capsular arabinomannan release and stimulates growth.

Mycobacterial glycolipids are important cell envelope structures that drive host-pathogen interactions. Arguably, the most important amongst these are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are both trafficked out of the bacterium to the host via unknown mechanisms. An important class of exported LM/LAM is the capsular derivative of these molecules which is devoid of its lipid anchor. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH which specifically cleaves α-1,6-mannoside linkages within LM and LAM, driving its export to the capsule releasing its phosphatidyl-myo-inositol mannoside lipid anchor. Unexpectedly, we found that the catalytic activity of this enzyme is important for efficient exit from stationary phase cultures where arabinomannan acts as a signal for growth phase transition. Finally, we demonstrate that LamH is important for Mycobacterium tuberculosis survival in macrophages. These data provide a new framework for understanding the biological role of LAM in mycobacteria.

Immunogenicity of Mycobacterial Extracellular Vesicles Isolated From Host-Related Conditions Informs About Tuberculosis Disease Status.

Tuberculosis (TB) still represents a major global health problem affecting over 10 million people worldwide. The gold-standard procedures for TB diagnosis are culture and nucleic acid amplification techniques. In this context, both lipoarabinomannan (LAM) urine test and rapid molecular tests have been major game changers. However, the low sensitivity of the former and the cost and the prohibitive infrastructure requirements to scale-up in endemic regions of the latter, make the improvement of the TB diagnostic landscape a priority. Most forms of life produce extracellular vesicles (EVs), including bacteria despite differences in bacterial cell envelope architecture. We demonstrated that Mycobacterium tuberculosis (Mtb), the causative agent of TB, produces EVs in vitro and in vivo as part of a sophisticated mechanism to manipulate host cellular physiology and to evade the host immune system. In a previous serology study, we showed that the recognition of several mycobacterial extracellular vesicles (MEV) associated proteins could have diagnostic properties. In this study, we pursued to expand the capabilities of MEVs in the context of TB diagnostics by analyzing the composition of MEVs isolated from Mtb cultures submitted to iron starvation and, testing their immunogenicity against a new cohort of serum samples derived from TB+ patients, latent TB-infected (LTBI) patients and healthy donors. We found that despite the stringent condition imposed by iron starvation, Mtb reduces the number of MEV associated proteins relative to iron sufficient conditions. In addition, TB serology revealed three new MEV antigens with specific biomarker capacity. These results suggest the feasibility of developing a point-of-care (POC) device based on selected MEV-associated proteins.

Osmotic stress induces biofilm production by Staphylococcus epidermidis isolates from neonates.

Staphylococcus epidermidis is one of the leading causes of bloodstream infections, particularly in premature neonates, and biofilm formation is a major virulence factor. We characterized biofilm formation by 50 S. epidermidis neonatal isolates under osmotic stress and evaluated the expression of biofilm-associated genes. Phenotypical analyses of biofilm production were performed in culture medium with or without addition of NaCl or glucose. In control medium (no additions), most isolates (84%) were nonproducers or weak biofilm producers. Growth in NaCl-containing medium increased the number of moderate/strong producers, and this increase was even greater in medium containing glucose. Most of the protein-enriched biofilms (60%) could be observed only during growth in glucose, whereas 50% of the polysaccharide-enriched biofilms were observed during growth in NaCl. Studies that evaluate the conditions used to characterize biofilm production are important to help us understand the dynamics of this important virulence factor in S. epidermidis and their impact on neonatal infections.

Antimicrobial susceptibility of Neisseria meningitidis strains isolated from meningitis cases in Brazil from 2006 to 2008.

OBJECTIVE: To analyze the profile of antimicrobial susceptibility of meningococcal disease isolates collected throughout Brazil from 2006 to 2008 and forwarded to the National Reference Laboratory for Meningitis, Institute Adolfo Lutz - São Paulo. MATERIALS AND METHODS: The MIC to penicillin, ampicillin, chloramphenicol, ceftriaxone, ciprofloxacin and rifampicin was determined in a sample of 1096 (55% of the total isolates received) randomly chosen using the broth microdilution procedure. The breakpoints used were those recommended by the European Monitoring Group on Meningococci (EMGM). RESULTS: Decreased susceptibility to penicillin and ampicillin was detected in 13% and 12.9% respectively. All isolates were susceptible to chloramphenicol, ceftriaxone, and ciprofloxacin. Two strains (0.2%) showed high resistance to rifampicin and 0.5% of the isolates displayed intermediate resistance to rifampicin. CONCLUSIONS: The meningococcal strains isolated in Brazil during 2006-2008 were globally susceptible to all antibiotics currently used in treatment or chemoprophylaxis of meningococcal disease in Brazil.

Antimicrobial susceptibility of Neisseria meningitidis strains isolated from meningitis cases in Brazil from 2006 to 2008

Objective To analyze the profile of antimicrobial susceptibility of meningococcal disease isolates collected throughout Brazil from 2006 to 2008 and forwarded to the National Reference Laboratory for Meningitis, Institute Adolfo Lutz - São Paulo. Materials and methods The MIC to penicillin, ampicillin, chloramphenicol, ceftriaxone, ciprofloxacin and rifampicin was determined in a sample of 1096 (55% of the total isolates received) randomly chosen using the broth microdilution procedure. The breakpoints used were those recommended by the European Monitoring Group on Meningococci (EMGM).Results Decreased susceptibility to penicillin and ampicillin was detected in 13% and 12.9% respectively. All isolates were susceptible to chloramphenicol, ceftriaxone, and ciprofloxacin. Two strains (0.2%) showed high resistance to rifampicin and 0.5% of the isolates displayed intermediate resistance to rifampicin. Conclusions The meningococcal strains isolated in Brazil during 2006-2008 were globally susceptible to all antibiotics currently used in treatment or chemoprophylaxis of meningococcal disease in Brazil (AU)

Objetivo Analizar el perfil de susceptibilidad a los antimicrobianos de las cepas de meningococos aisladas de casos de enfermedad meningocócica en Brasil entre 2006 y 2008 y enviadas al Laboratorio Nacional de Referencia para Meningitis, Instituto Adolfo Lutz, São Paulo. Material y métodos Se determinó la CIM a penicilina, ampicilina, cloranfenicol, ceftriaxona, ciprofloxacino y rifampicina, mediante el procedimiento de microdilución seriada en caldo en una muestra de 1.096 aislados (55% de los aislados recibidos) escogida al azar. Los puntos de corte utilizados fueron los recomendados por el European Monitoring Group on Meningococci (EMGM).Resultados Se detectó disminución de la susceptibilidad a la penicilina y la ampicilina en el 13 y el 12,9% respectivamente. Todos los aislados fueron susceptibles a cloranfenicol, ceftriaxona y ciprofloxacino. Dos cepas (0,2%) mostraron alta resistencia a la rifampicina y el 0,5% de los aislados presentaron resistencia intermedia a la rifampicina. Conclusiones Las cepas de meningococos aisladas en Brasil en el periodo 2006-2008 fueron globalmente susceptibles a los antibióticos actualmente utilizados en el tratamiento o quimioprofilaxis de enfermedad meningocócica en Brasil (AU)