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© 2016 Conde et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.B

The species Mycobacterium tuberculosis variant bovis ( M. tuberculosis var. bovis ) is associated with tuberculosis, mainly in cattle and buffaloes. This pathogen has the potential to infect other mammals, including humans. Tuberculosis caused by M. tuberculosis var. bovis is a zoonosis clinically identical to tuberculosis caused by Mycobacterium tuberculosi s, and the recommended treatment in humans results in the use of antibiotics. In this study, we used the whole genome sequencing (WGS) methodology Illumina NovaSeq 6000 System platform to characterize the genome of M. tuberculosis var. bovis in cattle circulating in Mato Grosso, identify mutations related to drug resistance genes, compare with other strains of M. tuberculosis var. bovis brazilian and assess potential drug resistance. Four isolates of M. tuberculosis var. bovis of cattle origin representing the main livestock circuits, which had been more prevalent in previous studies in the state of Mato Grosso, were selected for the genomic study. The genome sizes of the sequenced strains ranged from 4,306,423 to 4,332,964 bp, and the GC content was 65.6%. The four strains from Mato Grosso presented resistance genes to pncA (pyrazinamide), characterized as drug-resistant strains. In addition to verifying several point mutations in the pncA, rpsA, rpsL, gid, rpoB, katG, gyrB, gyrA, tlyA, embA, embB, embC, fgd, fbiB , and fbiC genes, these genes were similar to antibiotic resistance in more than 92% of the Brazilian strains. Therefore, our results indicated a high genetic diversity between our isolates and other M. tuberculosis var. bovis isolated in Brazil. Thus, multiple transmission routes of this pathogen may be present in the production chain. So, to achieve a bovine tuberculosis-free health status, the use of the WGS as a control and monitoring tool will be crucial to determine these transmission routes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Anjos, Castro, Machado Filho, Suffys, Gomes, Duarte, Figueiredo and Carvalho.)

In this paper we model the spreading of the SARS-CoV-2 in Mexico by introducing a new stochastic approximation constructed from first principles, where the number of new infected individuals caused by a single infectious individual per unit time (a day), is a random variable of a time-dependent Poisson distribution. The model, structured on the basis of a Latent-Infectious-(Recovered or Deceased) (LI(RD)) compartmental approximation together with a modulation of the mean number of new infections (the Poisson parameters), provides a good tool to study theoretical and real scenarios., Competing Interests: The authors have declared that no competing interests exist.

Due to the widespread use of Maytenus ilicifolia leaves in the treatment of gastric ulcers, herbal medicines derived from such species are distributed by the national health system in Brazil. A related species, Maytenus aquifolium , is also used for the same disorders, and both are popularly known as Espinheira-santa . Due to their popular use, the quality and efficiency of the herbal medicines derived from these species is an important public health issue. The purpose of this study was to develop and test an analytical method that could quantify the content of catechin and epicatechin in dry Maytenus spp. leaves and simultaneously obtain their chemical profile to determine authenticity of the leaf samples. Ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) was used to quantify these isomers, in the selected ion monitoring (SIM) mode, while simultaneously analyzing the extract in full-scan mode. This approach was successfully applied to the analysis of commercial and authentic samples of Maytenus spp. Fewer than half the samples presented the minimum epicatechin content of 2.8 mg per g of dry leaf mass, as specified in the 6th Brazilian Pharmacopoeia (2019) for M. ilicifolia . Furthermore, by using untargeted metabolomics, it was observed that the chemical profile of most the samples was not compatible with M. ilicifolia leaves, indicating the need for stricter quality control of this material. The method described herein could be used for this control; moreover, its concept could be adapted and used for an ample variety of medicinal plant products.

Purpose: To investigate and compare the effects of therapeutic exercise, therapeutic ultrasound and photobiomodulation on pain, functionality and recruitment pattern of motor units, after a rehabilitation protocol for seamstresses with neck pain., Materials and Methods: All 36 female, randomly divided into three groups; i)exercise control group, ii)exercise and photobiomodulation group, iii)exercise and ultrasound group. The groups were composed of sewing machine operators with complaints neck pain. Clinical evaluations: Visual analog pain scale, questionnaires Nordic Musculoskeletal Questionnaire and the International Physical Activity Questionnaire, and electromyographic evaluations of the sternocleidomastoid and upper trapezius muscles. All variables were compared before and after the protocol. For statistical analysis, the values of mean, standard deviation and standard error of the mean were used. The values obtained were compared using the One-Way ANOVA with post-hoc Tukey, and Cohen's-d, with a significance coefficient of p < 0.05., Results: In the Ultrasound and laser groups there was a greater tendency to improve pain (p < 0.0001) and size of the effect on pain reduction (ultrasound = d:1.99; photobiomodulation = d:1.81). Between groups, there was a significant difference in post-treatment for the onset of right trapezius (p = 0.024) in the exercise and photobiomodulation groups (p = 0.0347). The photobiomodulation group showed pre and post-intervention differences in the left trapezius maximum onset (p = 0.010)., Conclusion: Interventions with photobiomodulation, ultrasound, and exercise assist to pain, function, and muscular activation in seamstresses with neck pain., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The chronification of ulcers or sores may result in a dramatic outcome such as amputation. Currently, the search for plant based treatments of various diseases/disorders, including complicated ones, is getting the attention of researchers worldwide. The soluble latex protein fraction (CpLP) obtained from Calotropis procera (Apocynaceae) was previously demonstrated to accelerate wound healing by topical application or when incorporated in a polyvinyl alcohol biomembrane (BioMemCpLP). Here, in vitro assays were performed to investigate and characterize the biocompatibility and bioactivity of latex proteins dressing. Macrophages (RAW 264.7), fibroblasts (L929) and keratinocytes (HaCaT) cell lines were used to evaluate the effect of CpLP. These cell lines were exposed to concentrations of CpLP comparable to those found in BioMemCpLP during 24-72 h. The cytotoxicity, proliferation, release of wound healing mediators (TGF-β, VEGF, IL-10, IL-6, IL-1β, TNF-α and NO) and migration of cells (E-cadherin and β-catenin) incubated with CpLP was assessed and the cell adhesion to BioMemCpLP as well. The results showed that CpLP has no cytotoxic effects. It induced a suitable balance between pro- and anti-inflammatory mediators, enhanced proliferation and re-epithelialization in all cell lines, but the intensity of each effect was different at various doses in all cell strains. The BioMemCpLP stimulated cell adhesion to PVA substrate. The CpLP-PVA based biomembrane can be a good option for healing of different wounds., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Bacterial biofilm on surfaces of mammary implants is a predisposing factor for several outcomes. Because Gram-positive bacteria are potential agents of biomaterial-associated infections (BAIs), their abilities to form biofilm on breast implants should be elucidated., Objectives: The aim of this study was to evaluate biofilm formation on different mammary prosthesis surfaces by major Gram-positive bacterial pathogens involved in BAIs., Methods: We initially evaluated biofilm formation on polystyrene plates with and without fibrinogen or collagen for 1 reference strain and 1 clinical isolate of Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes. We also tested the ability of clinical isolates to form biofilm on 4 different implant surfaces: polyurethane foam and smooth, microtextured, and standard textured silicone. Biofilm structure and cell viability were observed by scanning electron microscopy and confocal laser scanning microscopy., Results: All strains showed strong biofilm formation on polystyrene. After fibrinogen or collagen treatment, biofilm formation varied. With fibrinogen, reference strains of S. aureus and S. pyogenes increased biofilm formation (P < 0.05). Reference strains of all species and the clinical isolate of S. pyogenes increased biofilm formation after collagen treatment (P < 0.05). In general, S. aureus showed higher capacity to produce biofilm. Scanning electron microscopy showed that biofilm attached to all surfaces tested, with the presence of extracellular polymeric substances and voids. Viable cells were more frequent for E. faecalis and S. pyogenes., Conclusions: All species produced biofilm on all prosthesis surfaces and under different conditions. Micrographies indicated thicker bacterial biofilm formation on microtextured and/or standard textured silicone by all species, except E. faecalis., (© 2020 The Aesthetic Society. Reprints and permission: journals.permissions@oup.com.)

It was previously demonstrated that the methanol fraction of Sideroxylon obtusifolium (MFSOL) promoted anti-inflammatory and healing activity in excisional wounds. Thus, the present work investigated the healing effects of MFSOL on human keratinocyte cells (HaCaT) and experimental burn model injuries. HaCaT cells were used to study MFSOL's effect on cell migration and proliferation rates. Female Swiss mice were subjected to a second-degree superficial burn protocol and divided into four treatment groups: Vehicle, 1.0% silver sulfadiazine, and 0.5 or 1.0% MFSOL Cream (CrMFSOL). Samples were collected to quantify the inflammatory mediators, and histological analyses were performed after 3, 7, and 14 days. The results showed that MFSOL (50 μg/mL) stimulated HaCaT cells by increasing proliferation and migration rates. Moreover, 0.5% CrMFSOL attenuated myeloperoxidase (MPO) activity and also stimulated the release of interleukin (IL)-1β and IL-10 after 3 days of treatment. CrMFSOL (0.5%) also enhanced wound contraction, promoted improvement of tissue remodeling, and increased collagen production after 7 days and VEGF release after 14 days. Therefore, MFSOL stimulated human keratinocyte (HaCaT) cells and improved wound healing via modulation of inflammatory mediators of burn injuries.

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.

Background: The evaluation of procedures for drug susceptibility prediction of Mycobacterium tuberculosis based on genomic data against the conventional reference method test based on culture is realistic considering the scenario of growing number of tools proposals based on whole-genome sequences (WGS)., Objectives: This study aimed to evaluate drug susceptibility testing (DST) outcome based on WGS tools and the phenotypic methods performed on isolates of M. tuberculosis Lineage 1 from the state of Pará, Brazil, generally associated with low levels of drug resistance., Methodology: Culture based DST was performed using the Proportion Method in Löwenstein-Jensen medium on 71 isolates that had been submitted to WGS. We analysed the seven main genome sequence-based tools for resistance and lineage prediction applied to M. tuberculosis and for comparison evaluation we have used the Kappa concordance test., Findings: When comparing the WGS-based tools against the DST, we observed the highest level of agreement using TB-profiler. Among the tools, TB-profiler, KvarQ and Mykrobe were those which identified the largest number of TB-MDR cases. Comparing the four most sensitive tools regarding resistance prediction, agreement was observed for 43 genomes., Main Conclusions: Drug resistance profiling using next-generation sequencing offers rapid assessment of resistance-associated mutations, therefore facilitating rapid access to effective treatment.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: Laboratory and clinical features of childhood tuberculosis (TB) are non-specific and establishing an accurate diagnosis remains a challenge. This study evaluated a Single tube nested-PCR (STNPCR) to detect genomic DNA of Mycobacterium tuberculosis complex in blood and urine., Methods: Biological samples were obtained from children (<15 years old) with clinical suspicion of pulmonary and extrapulmonary TB at public hospitals in Recife-Pernambuco, Brazil. Cultures yielded negative results in a majority of childhood TB cases, which are generally paucibacillary. A set of clinical, epidemiological, radiological, and laboratory criteria with evident clinical improvement after anti-TB treatment were frequently used to define childhood TB cases., Results: Ninety children with clinical suspicion were enrolled in this study (44 with TB and 46 without TB). The pulmonary TB group had 20 confirmed cases and 46 negative controls, while the extrapulmonary TB group had 24 confirmed cases. The STNPCR showed sensitivities to pulmonary and extrapulmonary TB of 47.4% and 52.2% (blood) and 38.8% and 20% (urine), respectively. Considering the low performance of STNPCR on separate samples, we decided to perform a combined analysis (parallel sensitivity analysis) of the results from blood and urine samples. The parallel sensitivity increased to 65% in blood and 62.5% in urine. The specificity in both samples ranged from 93.5-97.8%., Conclusions: Although STNPCR showed moderate sensitivity, the specificity is high; therefore, the test can be used as an auxiliary tool to diagnose TB in children. It is a rapid test that demonstrated better performance than other diagnostic tests in paucibacillary samples as it does in childhood tuberculosis.

Infections related to non-tuberculous mycobacteria (NTM) have recently increased worldwide. The transmission of these microorganisms from the environment has been suggested as the main source for human infections. To elucidate the epidemiological aspects and distribution of these pathogens, many studies have evaluated several decontamination methods and protocols to properly isolate NTM from environmental samples, mainly from water. However, no satisfactory strategy has been found for isolation of most of the NTM species harboring different phenotypic characteristics. Here, we evaluated the susceptibility of 23 NTM strains presenting variable growth rate and pigmentation patterns to eight different methods: oxalic acid (2.5% and 5%), cetylpyridinium chloride (CPC) (0.0025% and 0.005%), sodium hydroxide (NaOH) (2% and 4%), and sodium dodecyl sulfate (SDS) plus NaOH (SDS 1.5%-NaOH 0.5% and SDS 3%-NaOH 1%). It was found that the viability of NTM exposed to different decontamination methods varies according to their phenotypic characteristics and two methods (SDS 1.5% plus NaOH 0.5% and CPC 0.0025%) were necessary for effective isolation of all of the species tested. These findings supply important insights for future studies on the environmental occurrence of mycobacteria and improving the sensibility of traditional strategies.

Mycobacterium bovis is the main causative agent of bovine tuberculosis (bTB) being among the animal-adapted Mycobacterium tuberculosis complex. Herds can also be infected with non-tuberculous mycobacteria (NTM) causing a negative effect on the economy and on animal and human health through zoonotic infections. Molecular tools are required for mycobacteria identification; thus, it is laborious to determine the epidemiological information of mycobacteria among herds. We aimed to describe the mycobacterial pathogens associated with cases of suspected bTB lesions in cattle/buffaloes slaughtered for consumption and to investigate bTB transmission. We evaluated 74 lesion samples from 48 animals (27 bovine/21 buffaloes) from 16 mapped farms. Positives samples from nested-PCR were cultured in Lowenstein-Jensen (LJ), 2% pyruvate (LJ + P), and 2% glycerol (LJ + G) media, followed by Ziehl-Neelsen (ZN) staining technique and partial gene sequencing (hsp65, rpoB, and 16S-rRNA). Spoligotyping and 24-MIRU-VNTR were performed. The LJ + P increased the chance of obtaining bacilli. The respiratory tract and the oral cavity were the most important infection route. In addition, the calcified part of the lesions suggested chronic bTB. Spoligotypes of M. bovis (SIT986/SB0885) differed from others found in South America, and the MIRU-VNTR 24 loci suggested that bTB was associated to highly related strains. The NTM species found are of clinical importance in humans., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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.

Lineage 1 (L1) is one of seven Mycobacterium tuberculosis complex (MTBC) lineages. The objective of this study was to improve the complex taxonomy of L1 using phylogenetic SNPs, and to look for the origin of the main L1 sublineage prevalent in Para, Brazil. We developed a high-throughput SNPs-typing assay based on 12-L1-specific SNPs. This assay allowed us to experimentally retrieve SNP patterns on nine of these twelve SNPs in 277 isolates previously tentatively assigned to L1 spoligotyping-based sublineages. Three collections were used: Pará-Brazil (71); RIVM, the Netherlands (102), Madagascar (104). One-hundred more results were generated in Silico using the PolyTB database. Based on the final SNPs combination, the samples were classified into 11 clusters (C1-C11). Most isolates within a SNP-based cluster shared a mutual spoligotyping-defined lineage. However, L1/EAI1-SOM (SIT48) and L1/EAI6-BGD1 (SIT591) showed a poor correlation with SNP data and are not monophyletic. L1/EAI8-MDG and L1/EAI3-IND belonged to C5; this result suggests that they share a common ancestor. L1.1.3/SIT129, a spoligotype pattern found in SNPs-cluster C6, was found to be shared between Pará/Brazil and Malawi. SIT129 was independently found to be highly prevalent in Mozambique, which suggests a migration history from East-Africa to Brazil during the 16th-18th slave trade period to Northern Brazil., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Tuberculosis is an infectious bacterial disease that causes millions of deaths worldwide. Current treatment recommended by WHO is effective, however it is an extensive and arduous process associated to severe adverse effects, which induces a low patient compliance and the emerging of multidrug resistant tuberculosis. Thus, as a main goal of this study, rifampicin nanoparticles were surface functionalized with a tuftsin-modifed peptide to selectively recognize receptors located on infected alveolar macrophages, enhancing nanoparticles uptake by these cells and improving antimycobacterial activity. A tuftsin-based modified peptide was synthesized and successfully attached to nanoparticles interface (NP-pRIF). In parallel, nanoparticles without peptide were also developed for comparison (NP-RIF). Physicochemical characterization demonstrated that stable and monodisperse nanodelivery systems were obtained, with a controlled drug release profile and non-cytotoxic potential. Moreover, nanoparticles containing peptide were significantly more internalized by macrophages than nanoparticles without peptide over a wide range of time. Both nanoparticles were 2-fold more effective against M. tuberculosis than free rifampicin, suggesting NP-pRIF as a promising strategy for the management of tuberculosis treatment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective:  To evaluate the accuracy of the diagnosis of fetal heart diseases obtained through ultrasound examinations performed during the prenatal period compared with the postnatal evaluation., Methods:  A retrospective cohort study with 96 pregnant women who were attended at the Echocardiography Service and whose deliveries occurred at the Complexo Hospitalar Santa Casa de Porto Alegre, in the state of Rio Grande do Sul, Brazil. Risk factor assessment plus sensitivity and specificity analysis were used, comparing the accuracy of the screening for congenital heart disease by means of obstetrical ultrasound and morphological evaluation and fetal echocardiography, considering p  < 0.05 as significant. The present study was approved by the Research Ethics Committee of the Institution., Results:  The analysis of risk factors shows that 31.3% of the fetuses with congenital heart disease could be identified by anamnesis. The antepartum echocardiography demonstrated a sensitivity of 97.7%, a specificity of 88.9%, and accuracy of 93% in the diagnosis of congenital heart disease. A sensitivity of 29.3% was found for the obstetric ultrasound, of 54.3% for the morphological ultrasound, and of 97.7% for the fetal echocardiography. The fetal echocardiography detected fetal heart disease in 67.7% of the cases, the morphological ultrasound in 16.7%, and the obstetric ultrasound in 11.5% of the cases., Conclusion:  There is a high proportion of congenital heart disease in pregnancies with no risk factors for this outcome. Faced with the disappointing results of obstetric ultrasound for the detection of congenital heart diseases and the current unfeasibility of universal screening of congenital heart diseases through fetal echocardiography, the importance of the fetal morphological ultrasound and its performance by qualified professionals is reinforced for a more appropriate management of these pregnancies., Competing Interests: The authors have no conflicts of interest to declare., (Thieme Revinter Publicações Ltda Rio de Janeiro, Brazil.)

Tuberculosis (TB) is an infectious disease with a higher risk for infection and disease among household contacts (HHC). Here, we report a molecular epidemiology-based approach to study disease transmission and the genetic characteristics of Mycobacterium tuberculosis (Mtb) strains among HHC in the city of Belem, the capital of the state of Para in north Brazil. The study included 63 TB patients belonging to 26 HHC groups (HHC1 to HHC26). Spoligotyping and 24-loci Mycobacterial Interspersed Repetitive Unit - Variable Number of Tandem Repeat (MIRU-VNTR) revealed indistinguishable bacterial genotypes among 26 patients in 14 (53.8%) HHC groups. Drug susceptibility testing (DST) revealed that 45 (71.4%) of the Mtb isolates were multidrug resistant. The major cluster composed of isolates from five HHCs and on three of these, whole genome sequencing (WGS) was performed confirming their high genetic similarity. These results pinpoint the need for improved vigilance for TB control in households in the city of Belém. When comparing WGS versus phenotypic resistance detection methods as DST and Minimum Inhibitory Concentration (MIC) our data suggest that depending on the colonies selection, results may present variation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

There is only scarce information available on genotypic diversity of the Mycobacterium tuberculosis complex (MTBC) clinical isolates circulating in the Northern part of Brazil, a relatively neglected region regarding research on tuberculosis. We therefore characterized 980 MTBC clinical isolates from the state of Pará, by spoligotyping and data was compared with patterns from around the world, besides analyzing drug susceptibility, and collecting sociodemographic data. We also performed 24 loci MIRU-VNTR typing to evaluate phylogenetic inferences among the East-African-Indian (EAI) lineage strains. The Geographic Information System analyses were performed to generate a descriptive visualization of MTBC strain distribution in the region. A total of 249 different spoligopatterns primarily belonging to evolutionary recent Euro-American lineages, as well as Central-Asian, Manu and ancestral EAI lineages, were identified, in addition to strains with reportedly unknown lineage signatures. The most frequent lineages were Latin American Mediterranean, T and Haarlem. Interestingly, EAI lineage strains were found in a significantly higher proportion in comparison with previous studies from South America. Regarding EAI lineage, the absence of spacers 4-9 and 23-24 co-related to 24 loci MIRU-VNTRs may suggest a close evolutionary relationship between such strains in Pará and those prevalent in Mozambique, which might have contributed to the genetic diversity of MTBC strains in this region., (Copyright © 2017. Published by Elsevier B.V.)

Background: Outbreaks of infections caused by rapidly growing mycobacteria have been reported worldwide generally associated with medical procedures. Mycobacterium abscessus subsp. massiliense CRM0019 was obtained during an epidemic of postsurgical infections and was characterized by increased persistence in vivo. To better understand the successful survival strategies of this microorganism, we evaluated its infectivity and proliferation in macrophages (RAW and BMDM) and alveolar epithelial cells (A549). For that, we assessed the following parameters, for both M. abscessus CRM0019 as well as the reference strain M. abscessus ATCC 19977: internalization, intracellular survival for up 3 days, competence to subvert lysosome fusion and the intracellular survival after cell reinfection., Results: CRM0019 and ATCC 19977 strains showed the same internalization rate (approximately 30% after 6 h infection), in both A549 and RAW cells. However, colony forming units data showed that CRM0019 survived better in A549 cells than the ATCC 19977 strain. Phagosomal characteristics of CRM0019 showed the bacteria inside tight phagosomes in A549 cells, contrasting to the loosely phagosomal membrane in macrophages. This observation holds for the ATCC 19977 strain in both cell types. The competence to subvert lysosome fusion was assessed by acidification and acquisition of lysosomal protein. For M. abscessus strains the phagosomes were acidified in all cell lines; nevertheless, the acquisition of lysosomal protein was reduced by CRM0019 compared to the ATCC 19977 strain, in A549 cells. Conversely, in macrophages, both M. abscessus strains were located in mature phagosomes, however without bacterial death. Once recovered from macrophages M. abscessus could establish a new intracellular infection. Nevertheless, only CRM0019 showed a higher growth rate in A549, increasing nearly 10-fold after 48 and 72 h., Conclusion: M. abscessus CRM0019 creates a protective and replicative niche in alveolar epithelial cells mainly by avoiding phagosome maturation. Once recovered from infected macrophages, CRM0019 remains infective and displays greater intracellular growth in A549 cells compared to the ATCC 19977 strain. This evasion strategy in alveolar epithelial cells may contribute to the long survival of the CRM0019 strain in the host and thus to the inefficacy of in vivo treatment.

OBJECTIVE Activated alkaline glutaraldehyde (GTA) remains one of the most widely used high-level disinfectants worldwide. However, several reports have highlighted the potential for nontuberculous mycobacteria to develop high-level resistance to this product. Because aldehyde resistance may lead to cross-resistance to other biocides, we investigated the susceptibility profile of GTA-resistant Mycobacterium chelonae and M. abscessus isolates to various disinfectant chemistries. METHODS High-level disinfectants commonly used in the reprocessing of endoscopes and other heat-sensitive, semicritical medical equipment, including different formulations of aldehyde-based products and oxidizing agents, were tested against 10 slow- and fast-growing, GTA-susceptible and GTA-resistant, Mycobacterium isolates in suspension tests and carrier tests at different temperatures. RESULTS While peracetic acid- and hydrogen peroxide-based disinfectants (S40, Resert XL, Reliance DG) efficiently killed all of the Mycobacterium isolates, GTA- and ortho-phthalaldehyde-based products (ie, Cidex, Aldahol, Cidex OPA) showed variable efficacy against GTA-resistant strains despite the ability of some formulations (Aldahol) to overcome the resistance of some of these isolates, especially when the temperature was increased from 20°C to 25°C. CONCLUSIONS Application permitting, oxidizing chemistries may provide a safe alternative to aldehyde-based products, particularly in GTA-resistant mycobacterial outbreaks. Infect Control Hosp Epidemiol 2017;38:784-791.

The phylum Actinobacteria, which comprises a great variety of Gram-positive bacteria with a high G+C content in their genomes, is known for its large production of bioactive compounds, including those with antimicrobial activity. Among the antimicrobials, bacteriocins, ribosomally synthesized peptides, represent an important arsenal of potential new drugs to face the increasing prevalence of resistance to antibiotics among microbial pathogens. The actinobacterial bacteriocins form a heterogeneous group of substances that is difficult to adapt to most proposed classification schemes. However, recent updates have accommodated efficiently the diversity of bacteriocins produced by this phylum. Among the bacteriocins, the lantibiotics represent a source of new antimicrobials to control infections caused mainly by Gram-positive bacteria and with a low propensity for resistance development. Moreover, some of these compounds have additional biological properties, exhibiting activity against viruses and tumour cells and having also potential to be used in blood pressure or inflammation control and in pain relief. Thus, lantibiotics already described in Actinobacteria exhibit potential practical applications in medical settings, food industry and agriculture, with examples at different stages of pre-clinical and clinical trials.

Background: The combination of rifapentine and moxifloxacin administered daily with other anti-tuberculosis drugs is highly active in mouse models of tuberculosis chemotherapy. The objective of this phase 2 clinical trial was to determine the bactericidal activity, safety, and tolerability of a regimen comprised of rifapentine, moxifloxacin, isoniazid, and pyrazinamide administered daily during the first 8 weeks of pulmonary tuberculosis treatment., Methods: Adults with sputum smear-positive pulmonary tuberculosis were randomized to receive either rifapentine (approximately 7.5 mg/kg) plus moxifloxacin (investigational arm), or rifampin (approximately 10 mg/kg) plus ethambutol (control) daily for 8 weeks, along with isoniazid and pyrazinamide. The primary endpoint was sputum culture status at completion of 8 weeks of treatment., Results: 121 participants (56% of accrual target) were enrolled. At completion of 8 weeks of treatment, negative cultures using Löwenstein-Jensen (LJ) medium occurred in 47/60 (78%) participants in the investigational arm vs. 43/51 (84%, p = 0.47) in the control arm; negative cultures using liquid medium occurred in 37/47 (79%) in the investigational arm vs. 27/41 (66%, p = 0.23) in the control arm. Time to stable culture conversion was shorter for the investigational arm vs. the control arm using liquid culture medium (p = 0.03), but there was no difference using LJ medium. Median rifapentine area under the concentration-time curve (AUC0-24) was 313 mcg*h/mL, similar to recent studies of rifapentine dosed at 450-600 mg daily. Median moxifloxacin AUC0-24 was 28.0 mcg*h/mL, much lower than in trials where rifapentine was given only intermittently with moxifloxacin. The proportion of participants discontinuing assigned treatment for reasons other than microbiological ineligibility was higher in the investigational arm vs. the control arm (11/62 [18%] vs. 3/59 [5%], p = 0.04) although the proportions of grade 3 or higher adverse events were similar (5/62 [8%] in the investigational arm vs. 6/59 [10%, p = 0.76] in the control arm)., Conclusion: For intensive phase daily tuberculosis treatment in combination with isoniazid and pyrazinamide, a regimen containing moxifloxacin plus low dose rifapentine was at least as bactericidal as the control regimen containing ethambutol plus standard dose rifampin., Trial Registration: www.ClinicalTrials.gov NCT00728507.

Background and Aims: Attention to patient safety has increased recently due to outbreaks of nosocomial infections associated with GI endoscopy. The aim of this study was to evaluate current cleaning and disinfection procedures of endoscope channels with high bioburden and biofilm analysis, including the use of resistant mycobacteria associated with postsurgical infections in Brazil., Methods: Twenty-seven original endoscope channels were contaminated with organic soil containing 10(8) colony-forming units/mL of Pseudomonas aeruginosa, Staphylococcus aureus, or Mycobacterium abscessus subsp bolletii. Biofilms with the same microorganisms were developed on the inner surface of channels with the initial inoculum of 10(5) colony-forming units/mL. Channels were reprocessed following current protocol, and samples from cleaning and disinfection steps were analyzed by bioluminescence for adenosine triphosphate, cultures for viable microorganisms, and confocal microscopy., Results: After contamination, adenosine triphosphate levels increased dramatically, and high bacterial growth was observed in all cultures. After cleaning, adenosine triphosphate levels decreased to values comparable to precontamination levels, and bacterial growth was demonstrated in 5 of 27 catheters, 2 with P aeruginosa and 3 with M abscessus. With regard to induced biofilm, a remarkable reduction occurred after cleaning, but significant microbial growth inhibition occurred only after disinfection. Nevertheless, viable microorganisms within the biofilm were still detected by confocal microscopy, more so with glutaraldehyde than with peracetic acid or O-phataladehyde., Conclusion: After the complete disinfection procedure, viable microorganisms could still be detected within the biofilm on endoscope channels. Prevention of biofilm development within endoscope channels should be a priority in disinfection procedures, particularly for ERCP and EUS., (Copyright © 2016 American Society for Gastrointestinal Endoscopy. Published by Elsevier Inc. All rights reserved.)

Objectives: Rapidly growing mycobacteria (RGM) have emerged as important pathogens in clinical settings, associated with esthetic procedures and postsurgical infections, pulmonary infections among cystic fibrosis patients, and other structural pulmonary diseases. Microorganisms belonging to Mycobacterium abscessus-Mycobacterium chelonae and to Mycobacterium fortuitum groups have frequently been associated with outbreaks and various epidemics. In the present study, RGM strains were characterized in order to investigate molecular markers based on proteomic analysis., Methods: Multilocus enzyme electrophoresis (MLEE) was used for species identification and clonal analysis of RGM recovered from postsurgical wound infections during an epidemic. The study included 30M. abscessus subsp. bolletii clinical isolates, most belonging to the BRA100 clone (epidemic in Rio de Janeiro city), as well as 16 RGM ATCC reference strains., Results: Molecular typing allowed the detection of diversity in the studied population and revealed species-specific isoenzymatic patterns. Additionally, the clonal relationship among M. abscessus subsp. bolletii outbreak isolates, as examined using MLEE, was markedly consistent., Conclusions: Isoenzymatic characterization was found to be a useful molecular tool to identify RGM species and to determine the relatedness among closely related M. abscessus subsp. bolletii isolates. This may be considered a powerful approach for epidemiological studies on RGM., (Copyright © 2015. Published by Elsevier Ltd.)

Interferometry was used together with the conventional microplate resazurin assay to evaluate the antimycobacterial properties of essential oil (EO) from fruits of Pterodon emarginatus and also of rifampicin against Mycobacterium bovis. The aim of this work is not only to investigate the potential antimycobacterial activity of this EO, but also to test the interferometric method in comparison with the conventional one. The Minimum Inhibitory Concentration (MIC) values of EO (625 μg/mL) and rifampicin (4 ng/mL) were firstly identified with the microplate method. These values were used as parameters in Drug Susceptibility Tests (DST) with interferometry. The interferometry confirmed the MIC value of EO identified with microplate and revealed a bacteriostatic behavior for this concentration. At 2500 μg/mL interferometry revealed bactericidal activity of the EO. Mycobacterial growth was detected with interferometry at 4 ng/mL of rifampicin and even at higher concentrations. One important difference is that the interferometric method preserves the sample, so that after weeks of quantitative observation, the sample can be used to evaluate the bactericidal activity of the tested drug., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Outbreaks associated with rapidly growing mycobacteria (RGM) have been increasingly reported worldwide, including in Brazil. Among the RGM, the Mycobacterium abscessus complex is the most pathogenic and related to multidrug resistance. The aim of this study was to evaluate the antimicrobial susceptibility and molecular profile of RGM isolates involved in new postsurgical infection outbreaks in Brazil since 2007. Of the 109 cases reported in the state of Rio Grande do Sul between 2007 and 2011, 43 (39 %) had confirmed mycobacterial growth in culture. Clinical isolates were obtained from biopsy specimens or abscess aspirates. PRA-hsp65 restriction pattern identified the isolates as M. abscessus type 2, and partial rpoB sequencing confirmed the identification as M. abscessus subsp. bolletii. All isolates were susceptible to amikacin and resistant to ciprofloxacin, doxycycline, sulfamethoxazole, moxifloxacin and tobramycin. Most isolates (72 %) were fully susceptible to cefoxitin but six isolates (14 %) were fully resistant to clarithromycin. The latter differed from the susceptibility profiles of the previously described BRA100 clone from other Brazilian regions. Nevertheless, pulsed-field gel electrophoresis analysis revealed that these isolates belonged to a single BRA100 clone. In conclusion, our study reports the persistence of an emergent single and highly resistant clone of M. abscessus subsp. bolletii for several years even after national implementation of infection control measures., (© 2014 The Authors.)

Mycobacterium leprae and Mycobacterium tuberculosis antimicrobial resistance has been followed with great concern during the last years, while the need for new drugs able to control leprosy and tuberculosis, mainly due to extensively drug-resistant tuberculosis (XDR-TB), is pressing. Our group recently showed that M. leprae is able to induce lipid body biogenesis and cholesterol accumulation in macrophages and Schwann cells, facilitating its viability and replication. Considering these previous results, we investigated the efficacies of two statins on the intracellular viability of mycobacteria within the macrophage, as well as the effect of atorvastatin on M. leprae infections in BALB/c mice. We observed that intracellular mycobacteria viability decreased markedly after incubation with both statins, but atorvastatin showed the best inhibitory effect when combined with rifampin. Using Shepard's model, we observed with atorvastatin an efficacy in controlling M. leprae and inflammatory infiltrate in the BALB/c footpad, in a serum cholesterol level-dependent way. We conclude that statins contribute to macrophage-bactericidal activity against Mycobacterium bovis, M. leprae, and M. tuberculosis. It is likely that the association of statins with the actual multidrug therapy effectively reduces mycobacterial viability and tissue lesion in leprosy and tuberculosis patients, although epidemiological studies are still needed for confirmation., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Three recently sequenced strains isolated from patients during an outbreak of Mycobacterium abscessus subsp. massiliense infections at a cystic fibrosis center in the United States were compared with 6 strains from an outbreak at a cystic fibrosis center in the United Kingdom and worldwide strains. Strains from the 2 cystic fibrosis outbreaks showed high-level relatedness with each other and major-level relatedness with strains that caused soft tissue infections during an epidemic in Brazil. We identified unique single-nucleotide polymorphisms in cystic fibrosis and soft tissue outbreak strains, separate single-nucleotide polymorphisms only in cystic fibrosis outbreak strains, and unique genomic traits for each subset of isolates. Our findings highlight the necessity of identifying M. abscessus to the subspecies level and screening all cystic fibrosis isolates for relatedness to these outbreak strains. We propose 2 diagnostic strategies that use partial sequencing of rpoB and secA1 genes and a multilocus sequence typing protocol.

Rapidly growing, non-tuberculous mycobacteria (NTM) in the Mycobacterium abscessus (MAB) species are emerging pathogens that cause various diseases including skin and respiratory infections. The species has undergone recent taxonomic nomenclature refinement, and is currently recognized as two subspecies, M. abscessus subsp. abscessus (MAB-A) and M. abscessus subsp. bolletii (MAB-B). The recently reported outbreaks of MAB-B in surgical patients in Brazil from 2004 to 2009 and in cystic fibrosis patients in the United Kingdom (UK) in 2006 to 2012 underscore the need to investigate the genetic diversity of clinical MAB strains. To this end, we sequenced the genomes of two Brazilian MAB-B epidemic isolates (CRM-0019 and CRM-0020) derived from an outbreak of skin infections in Rio de Janeiro, two unrelated MAB strains from patients with pulmonary infections in the United States (US) (NJH8 and NJH11) and one type MAB-B strain (CCUG 48898) and compared them to 25 publically available genomes of globally diverse MAB strains. Genome-wide analyses of 27,598 core genome single nucleotide polymorphisms (SNPs) revealed that the two Brazilian derived CRM strains are nearly indistinguishable from one another and are more closely related to UK outbreak isolates infecting CF patients than to strains from the US, Malaysia or France. Comparative genomic analyses of six closely related outbreak strains revealed geographic-specific large-scale insertion/deletion variation that corresponds to bacteriophage insertions and recombination hotspots. Our study integrates new genome sequence data with existing genomic information to explore the global diversity of infectious M. abscessus isolates and to compare clinically relevant outbreak strains from different continents., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

Multiple isolates of Mycobacterium abscessus subsp. bolletii, collectively called BRA100, were associated with outbreaks of postsurgical skin infections across various regions of Brazil from 2003 to 2009. We announce the draft genome sequence of a newly sequenced BRA100 strain, M. abscessus subsp. bolletii CRM-0020, isolated from a patient in Rio de Janeiro, Brazil.

The aim of this study was to evaluate the sporicidal activity of hospital disinfectants against spores of two Brazilian Clostridium difficile ribotypes and the BI/NAP1/027. Our results showed that CloroRio(®) and Cidex Opa(®) were the most efficient agents for eliminating spores of C difficile., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Introduction: The early diagnosis of mycobacterial infections is a critical step for initiating treatment and curing the patient. Molecular analytical methods have led to considerable improvements in the speed and accuracy of mycobacteria detection., Methods: The purpose of this study was to evaluate a multiplex polymerase chain reaction system using mycobacterial strains as an auxiliary tool in the differential diagnosis of tuberculosis and diseases caused by nontuberculous mycobacteria (NTM) RESULTS: Forty mycobacterial strains isolated from pulmonary and extrapulmonary origin specimens from 37 patients diagnosed with tuberculosis were processed. Using phenotypic and biochemical characteristics of the 40 mycobacteria isolated in LJ medium, 57.5% (n=23) were characterized as the Mycobacterium tuberculosis complex (MTBC) and 20% (n=8) as nontuberculous mycobacteria (NTM), with 22.5% (n=9) of the results being inconclusive. When the results of the phenotypic and biochemical tests in 30 strains of mycobacteria were compared with the results of the multiplex PCR, there was 100% concordance in the identification of the MTBC and NTM species, respectively. A total of 32.5% (n=13) of the samples in multiplex PCR exhibited a molecular pattern consistent with NTM, thus disagreeing with the final diagnosis from the attending physician., Conclusions: Multiplex PCR can be used as a differential method for determining TB infections caused by NTM a valuable tool in reducing the time necessary to make clinical diagnoses and begin treatment. It is also useful for identifying species that were previously not identifiable using conventional biochemical and phenotypic techniques.

To identify clinical and therapeutic features of pulmonary nontuberculous mycobacterial (PNTM) disease, we conducted a retrospective analysis of patients referred to the Brazilian reference center, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil, who received a diagnosis of PNTM during 1993–2011 with at least 1 respiratory culture positive for NTM. Associated conditions included bronchiectasis (21.8%), chronic obstructive pulmonary disease (20.7%), cardiovascular disease (15.5%), AIDS (9.8%), diabetes (9.8%), and hepatitis C (4.6%).Two patients had Hansen disease; 1 had Marfan syndrome. Four mycobacterial species comprised 85.6% of NTM infections: Mycobacterium kansasii, 59 cases (33.9%); M. avium complex, 53 (30.4%); M. abscessus, 23 (13.2%); and M. fortuitum, 14 (8.0%). A total of 42 (24.1%) cases were associated with rapidly growing mycobacteria. In countries with a high prevalence of tuberculosis, PNTM is likely misdiagnosed as tuberculosis, thus showing the need for improved capacity to diagnose mycobacterial disease as well as greater awareness of PNTM disease prevalence.

Croton cajucara is a shrub native to the Amazon region locally known as "sacaca". Two morphotypes are known: white and red "sacaca". The essential oils (EO) obtained by hydrodistillation from leaves of the red morphotype were, in general, rich in 7-hydroxycalamenene (28.4%-37.5%). The effectiveness of these EO regarding the antimicrobial activity against pathogenic microorganisms was initially investigated by the drop test method, showing significant inhibition zones. Among the microorganisms tested, the essential oils rich in 7-hydroxycalamenene were more effective against methicillin resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Mycobacterium tuberculosis, M. smegmatis, Mucor circinelloides and Rhizopus oryzae. The minimum inhibitory concentrations (MIC) of the oils were determined using the broth dilution assay. It was possible to observe that 7-hydroxycalamenene-rich oils presented high antimicrobial activity, with MIC of 4.76 × 10⁻³ μg/mL for MRSA, 4.88 μg/mL for M. tuberculosis, 39.06 μg/mL for M. smegmatis, and 0.152 μg/mL for R. oryzae and 3.63 × 10⁻⁸ μg/mL for M. circinelloides. The antioxidant activity of this EO suggests that 7-hydroxycalamenene provides more antioxidant activity according with EC(50) less than 63.59 μg/mL. Considering the bioactive potential of EOs and 7-hydroxycalamenene could be of great interest for development of antimicrobials for therapeutic use in treatment of bacterial and fungal infections in humans and/or veterinary practice.

A single strain of Mycobacterium abscessus subsp. bolletii, characterised by a particular rpoB sequevar and two highly related pulsed field gel electrophoresis patterns has been responsible for a nationwide outbreak of surgical infections in Brazil since 2004. In this study, we developed molecular tests based on polymerase chain reaction restriction-enzyme analysis (PRA) and sequencing for the rapid identification of this strain. Sequences of 15 DNA regions conserved in mycobacteria were retrieved from GenBank or sequenced and analysed in silico. Single nucleotide polymorphisms specific to the epidemic strain and located in enzyme recognition sites were detected in rpoB, the 3' region of the 16S rDNA and gyrB. The three tests that were developed, i.e., PRA-rpoB, PRA-16S and gyrB sequence analysis, showed 100%, 100% and 92.31% sensitivity and 93.06%, 90.28% and 100% specificity, respectively, for the discrimination of the surgical strain from other M. abscessus subsp. bolletii isolates, including 116 isolates from 95 patients, one environmental isolate and two type strains. The results of the three tests were stable, as shown by results obtained for different isolates from the same patient. In conclusion, due to the clinical and epidemiological importance of this strain, these tests could be implemented in reference laboratories for the rapid preliminary diagnosis and epidemiological surveillance of this epidemic strain.

Background: We recently described the Mycobacterium tuberculosis RD(Rio) genotype, a clonally derived sublineage within the Latin American-Mediterranean (LAM) family. Genetic diversity of M. tuberculosis likely affects the clinical aspects of tuberculosis (TB). Prospective studies that address this issue are scarce and remain controversial., Objective: To determine the association of differential clinical features of pulmonary TB with the RD(Rio) M. tuberculosis etiology., Methods: Culture-proven pulmonary TB patients (n = 272) were clinically evaluated, including history, physical examination, chest X-ray and anti-human immunodeficiency virus serology. Isolates were classified as RD(Rio) or non-RD(Rio) M. tuberculosis by multiplex polymerase chain reaction and further spoligotyped. Clinical and M. tuberculosis genotype data were analyzed., Results: RD(Rio) M. tuberculosis caused disease in 26.5% (72/270) of all TB cases. The LAM genotype, of which RD(Rio) strains are members, was responsible for 46.0% of the TB cases. Demographic data, major signs and symptoms, radiographic presentation, microbiological features and clinical outcomes were not significantly different among patients with TB caused by RD(Rio) and non-RD(Rio) strains., Conclusions: Disease caused by M. tuberculosis RD(Rio) strains was not clinically distinctive or more severe than disease caused by non-RD(Rio) strains in this series of TB patients. Larger prospective studies specifically designed to disclose differential clinical characteristics of TB caused by specific M. tuberculosis lineages are needed.

Objectives: An aqueous extract and fraction from the marine sponge Petromica citrina have antibacterial activity. We performed a chemical and biological characterization of the antibiotic substance from P. citrina and investigated its mode of action on Staphylococcus aureus cells., Methods: The inhibitory activity of the aqueous extract of P. citrina was determined against 14 bacteria belonging to type strains and clinical antibiotic-resistant strains. The aqueous extract was fractionated under bioassay guidance and the bioactive substance was identified by its (1)H-NMR, (13)C-NMR and mass spectra. The MIC and the MBC of this substance were determined. This substance was also subjected to cytotoxic bioassays. The mode of action on S. aureus cells was investigated by light and transmission electron microscopy analysis., Results: P. citrina showed a large spectrum of activity against type strains and resistant-bacteria such as S. aureus, Staphylococcus epidermidis, Enterococcus faecalis, Mycobacterium fortuitum and Neisseria gonorrhoeae. The aqueous extract was fractionated and halistanol trisulphate (24ε,25-dimethylcholestane-2β,3α,6α-triol trisodium sulphate) was isolated for the first time from P. citrina. Halistanol trisulphate had a bactericidal effect on exponentially growing S. aureus cells at the MIC (512 mg/L). Cytotoxicity biossays showed moderate toxicity against cancer cell line L929 (fibrosarcoma). This substance apparently acts by damaging the cell membrane, with subsequent cell lysis., Conclusions: Halistanol trisulphate is a broad-spectrum antibiotic isolated from P. citrina with a mode of action involving disruption of the cytoplasmic membrane. It is a new candidate for research on antibacterial substances.

Three isolates of Streptococcus agalactiae, recovered from residents of the metropolitan area of Rio de Janeiro with significant bacteriuria, were found to be resistant to levofloxacin. Determination of the minimal inhibitory concentration (MIC) confirmed one isolate as intermediate and two as resistant to levofloxacin. No reduction in levofloxacin MIC was observed with reserpine, indicating that resistance was not caused by an efflux mechanism. Typical point mutations were observed in the quinolone resistance determinant region of gyrA and parC. Other point mutations in parC generated novel altered codons: Ser80→Pro in the intermediate resistance isolate, and Gly128→Asp in a resistant isolate. Through molecular modeling, it was possible to observe that these novel substitutions might not play a role in resistance, since these amino acids were not involved in the antibiotic binding site. Pulsed field gel electrophoresis profiles revealed a non-clonal trend among these isolates. This is the first report of genetic characterization of levofloxacin-resistant S. agalactiae strains in Brazil., (Copyright © 2012 Elsevier Editora Ltda. All rights reserved.)

Aims: To compare three decontamination methods applied to paucibacillary samples for primary isolation of Mycobacterium bovis from suspect lesions. Tuberculosis caused by Myco. bovis is an important infectious disease of cattle in Brazil and also has zoonotic potential. Although a national campaign based on testing and slaughtering cattle has achieved good results, there is a strong need to develop better diagnostic methods to identify cattle with recent infections harbouring few bacilli., Methods and Results: A dairy herd (274 adult crossbred cows) located in the state of Rio de Janeiro was tested for tuberculosis with both single intradermal tuberculin test and comparative intradermal tuberculin test. Reactive cows (n=27, 9.8%) were slaughtered and suspect lesions were collected (one sample per cow). Samples considered paucibacillary (based on microscopy) were decontaminated with 0.75% hexadecylpyridinium chloride (HPC), 4% sodium hydroxide (Petroff) or 6% sulphuric acid. Using these methods, 10, five and six, respectively, of the 27 samples yielded positive cultures. Overall, Myco.bovis was isolated from 14 of 24 cows. Although the HPC method resulted in isolation of more Myco.bovis strains than either Petroff or sulphuric acid methods (P=0.015), it did not result in the recovery of Myco.bovis from all samples. However, using both HPC and 6% sulphuric acid methods for decontamination was possible to identify 13 of 14 (92·9%) of infected cows., Conclusions: At least two methods should be used concurrently for primary isolation of Myco. bovis from bovine tissues, particularly for paucibacillary samples., Significance and Impact of the Study: Detection of low numbers of Myco.bovis in tissue is an important goal in optimizing the detection of bovine tuberculosis and should assist in identification of infected cattle, in particular, those with few Myco.bovis bacilli. This was apparently the first study comparing three decontamination methods for the detection of Myco.bovis in paucibacillary samples from naturally infected cattle., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

Fluoroquinolones (FQs) have been increasingly used for effective treatment of infections caused by rapidly growing mycobacteria, and resistance to this drug has been predominantly attributed to gyrA and gyrB mutations. Accordingly, this study investigated a total of 36 Mycobacterium massiliense clinical isolates for their susceptibility to ciprofloxacin and presence of gyrA and gyrB gene mutations. The minimal inhibitory concentration (MIC) values, determined by broth microdilution method, of 35 ciprofloxacin-resistant isolates ranged between 4 and 16 μg/mL and a single susceptible isolate was obtained. A total of 31 of 35 (88.5%) ciprofloxacin-resistant isolates presented an amino acid substitution at codon 90 (Ala-90→Val) and no isolate presented mutation at position Asp-94. Moreover, 4 of 35 (11.4%) ciprofloxacin-resistant and one susceptible isolate had no mutation in Ala-90 and Asp-94. No gyrB mutation was observed in all tested M. massiliense isolates. In conclusion, our results have shown that mutations of gyrA codon 90 are frequent and may constitute an important mechanism of resistance to FQ in M. massiliense.