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 are lipoarabinomannan (LAM) and its precursor, lipomannan (LM), which are trafficked from the bacterium to the host via unknown mechanisms. Arabinomannan is thought to be a capsular derivative of these molecules, lacking a lipid anchor. However, the mechanism by which this material is generated has yet to be elucidated. Here, we describe the identification of a glycoside hydrolase family 76 enzyme that we term LamH (Rv0365c in Mycobacterium tuberculosis) 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, potentially implicating arabinomannan as a signal for growth phase transition. Finally, we demonstrate that LamH is important for M. tuberculosis survival in macrophages.

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.

Dynamin-like proteins mediate extracellular vesicle secretion in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) secretes extracellular vesicles (EVs) containing a variety of proteins, lipoproteins, and lipoglycans. While emerging evidence suggests that EVs contribute to tuberculosis pathogenesis, the factors and molecular mechanisms involved in mycobacterial EV production have not been identified. In this study, we use a genetic approach to identify Mtb proteins that mediate vesicle release in response to iron limitation and antibiotic exposure. We uncover a critical role for the isoniazid-induced, dynamin-like proteins, IniA and IniC, in mycobacterial EV biogenesis. Further characterization of a Mtb iniA mutant shows that the production of EVs enables intracellular Mtb to export bacterial components into the extracellular environment to communicate with host cells and potentially modulate the immune response. The findings advance our understanding of the biogenesis and functions of mycobacterial EVs and provide an avenue for targeting vesicle production in vivo.

Maintenance of cell wall remodeling and vesicle production are connected in Mycobacterium tuberculosis.

Pathogenic and nonpathogenic mycobacteria secrete extracellular vesicles (EVs) under various conditions. EVs produced by Mycobacterium tuberculosis ( Mtb ) have raised significant interest for their potential in cell communication, nutrient acquisition, and immune evasion. However, the relevance of vesicle secretion during tuberculosis infection remains unknown due to the limited understanding of mycobacterial vesicle biogenesis. We have previously shown that a transposon mutant in the LCP-related gene virR ( virR mut ) manifested a strong attenuated phenotype during experimental macrophage and murine infections, concomitant to enhanced vesicle release. In this study, we aimed to understand the role of VirR in the vesicle production process in Mtb . We employ genetic, transcriptional, proteomics, ultrastructural and biochemical methods to investigate the underlying processes explaining the enhanced vesiculogenesis phenomenon observed in the virR mutant. Our results establish that VirR is critical to sustain proper cell permeability via regulation of cell envelope remodeling possibly through the interaction with similar cell envelope proteins, which control the link between peptidoglycan and arabinogalactan. These findings advance our understanding of mycobacterial extracellular vesicle biogenesis and suggest that these set of proteins could be attractive targets for therapeutic intervention.

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.

High rate of neonates colonized by methicillin-resistant Staphylococcus species in an Intensive Care Unit.

INTRODUCTION: Staphylococcal colonization is a risk factor for healthcare-associated infections, which are frequent in Neonatal Intensive Care Units (NICU). This study analyzed microbiology, epidemiology and clinical aspects of Staphylococcus spp. colonizing neonates. METHODOLOGY: Nasal or periumbilical swabs were evaluated from 175 newborns admitted to a NICU of a Rio de Janeiro hospital from March to September 2009. Clinical data were obtained from the medical records. SCCmec typing and the mecA and Panton-Valentine Leukocidin (PVL) genes were detected by PCR. Clonal diversity was evaluated by pulsed-field gel electrophoresis. RESULTS: Staphylococcus spp. isolates were detected in 98 (56%) neonates, 66.3% of them had birth weight ≤ 2500 g, 62.2% were preterm (˂ 37 weeks) and the mean length of hospitalization was 14.9 days. Among the 133 isolates identified, 48.1% were S. epidermidis, 23.3% S. haemolyticus and 13.5% S. aureus. Methicillin-resistant Staphylococcus isolate was detected in 77.6% of neonates. The methicillin-resistant S. aureus isolates carried the SCCmec type IV, while 94.6% of S. epidermidis and 85.7% of S. haemolyticus presented non-typeable cassettes. Among the S. aureus, 55.6% had PVL genes and the USA800 genotype was prevalent. Two genotypes of S. epidermidis and one of S. haemolyticus clustered 42.2% and 25.8% of the isolates, respectively. S haemolyticus colonization was associated with the use of parenteral nutrition and mechanical ventilation. CONCLUSION: High rate of neonates colonized by methicillin-resistant Staphylococcus species and the permanence of clones circulating in the NICU highlight the importance for continuous and preventive surveillance in this high-risk population.

Methicillin resistance and virulence genes in invasive and nasal Staphylococcus epidermidis isolates from neonates.

BACKGROUND: Staphylococcus epidermidis is an opportunistic pathogen involved in hospital-acquired infections, particularly in those related to medical devices. This study characterized 50 genetically unrelated S. epidermidis isolates from bloodstream infections (BSIs, n = 31) and nares (n = 19) of neonates in relation to staphylococcal chromosomal cassette mec (SCCmec) type, biofilm production and associated genes, and the arginine catabolic mobile elements (ACME), in order to detect virulence factors that could discriminate a potential invasiveness isolate or predict an increasing pathogenicity. RESULTS: Isolates from both groups showed no difference for biofilm production and ACME genes detection. However, BSI isolates harbored more frequently the sdrF and sesI genes (p < 0.05), whereas biofilm producer isolates were associated with presence of the aap gene. The sdrF gene was also significantly more in the biofilm producer isolates from BSI. The SCCmec type IV and the ccr2 complex were related to BSI isolates (p < 0.05), while 83% of the nasal isolates were non-typeable for the SCCmec elements, with the mec complex and ccr undetectable as the most frequent profile. CONCLUSIONS: Despite the great clonal diversity displayed by S. epidermidis isolates from neonates, BSI isolates harbored more frequently the sdrF and sesI adhesin genes, while nasal isolates were very variable in SCCmec composition. These aspects could be advantageous to improve colonization in the host increasing its pathogenicity.

Staphylococcal cassette chromosome mec elements in methicillin-resistant coagulase-negative staphylococci from a Brazilian neonatal care unit.

To characterize 46 methicillin-resistant coagulase-negative staphylococci from Brazilian neonates, we investigated their SCCmec type, susceptibility and clonality. Staphylococcus epidermidis and Staphylococcus haemolyticus were the prevalent species. SCCmec types III or IV were detected in 53.3% S. epidermidis, whereas 63.6% S. haemolyticus were nontypeable. Despite the diversity, specific clones carried specific SCCmec elements, highlighting that effective typing can help in epidemiological analysis.

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.

Frecuencia de aislamiento de Klebsiella pneumoniae productora de Beta-lacmasa de Espectro Extendido (BLEE) en muestras de hemocultivos de pacientes pediátricos internados en el Hospital Materno Infantil del Barrio San Pablo / Frequency of isolation of producing Klebsiella pneumoniae of Beta de Espectro Extendido (BLEE) in samples of hemocultures of paediatric patients committed in the Infantile Maternal Hospital of the District San Pablo

Estudio que tiene como objetivo determinar la frecuencia de aislamiento de cepas de klebsiella pneumoniae (KPN) productora de Beta-lactamosa de Espectro Extendido (BLEE) en muestras de pacientes pediátricos de un Hospital de Asunción y que fueron procesadas en el Laboratorio Central de Salud¨püblica entre enero a septiembre de 2006

Brote de intoxicación alimentaria por Salmonella Enteritidis en un Barrio de Capiatá, Paraguay / Bud of nourishing poisoning by Salmonella Enteritidis in a District of Capiatá, Paraguay

Describe un estudio de brote de intoxicación alimentaria ocurrido con los asistentes a una fiesta de cumpleaños, para el estudio se utilizaron encuestas para la obtención de datos y coprocultivos para la búsqueda del agente causal