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Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54) obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70% of the samples. PCR confirmed the identification of 23 samples (100%) that grew in culture, 9 samples (60%) that failed to grow in culture, plus 6 (37.5%) samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes.

Five mycobacterial isolates from sewage were classified as members of the genus Mycobacterium but presented inconclusive species assignments. Thus, the isolates (MYC017, MYC098, MYC101, MYC123 and MYC340) were analyzed by phenotypical, biochemical, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and genomic features to clarify their taxonomic position. Phenotypic analysis and biochemical tests did not distinguish these isolates from other non-pigmented mycobacteria. In contrast, MALDI-TOF MS analysis showed that isolates were not related to any previously described Mycobacterium species. Comparative genomic analysis showed values of ANI and dDDH between 81.59-85.56% and 24.4-28.8%, respectively, when compared to the genomes of species of this genus. In addition, two (MYC101 and MYC123) presented indistinguishable protein spectra from each other and values of ANI = 98.57% and dDDH = 97.3%, therefore being considered as belonging to the same species. Phylogenetic analysis grouped the five isolates within the Mycobacterium terrae complex (MTC) but in a specific subclade and separated from the species already described and supported by 100% bootstrap value, confirming that they are part of this complex but different from earlier described species. According to these data, we propose the description of four new species belonging to the Mycobacterium genus: (i) Mycobacterium defluvii sp. nov. strain MYC017 T (= ATCC TSD-296 T = JCM 35364 T ), (ii) Mycobacterium crassicus sp. nov. strain MYC098 T (= ATCC TSD-297 T = JCM 35365T), (iii) Mycobacterium zoologicum sp. nov. strain MYC101 T (= ATCC TSD-298 T = JCM 35366 T ) and MYC123 (= ATCC BAA-3216 = JCM 35367); and (iv) Mycobacterium nativiensis sp. nov. strain MYC340 T (= ATCC TSD-299 T = JCM 35368 T )., 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 © 2024 Romagnoli, Conceição, Machado, Barreto, Sharma, Silva, Marques, Juliano, da Silva Lourenço, Digiampietri, Suffys, Leão and Viana-Niero.)

Polycyclic aromatic hydrocarbons (PAHs) are chemical compounds that are widespread in the environment, arising from the incomplete combustion of organic material, as well as from human activities involving petrol exploitation, petrochemical industrial waste, gas stations, and environmental disasters. PAHs of high molecular weight, such as pyrene, have carcinogenic and mutagenic effects and are considered pollutants. The microbial degradation of PAHs occurs through the action of multiple dioxygenase genes ( nid ), which are localized in genomic island denominate region A, and cytochrome P450 monooxygenases genes ( cyp ) dispersed in the bacterial genome. This study evaluated pyrene degradation by five isolates of Mycolicibacterium austroafricanum using 2,6-dichlorophenol indophenol (DCPIP assay), gas chromatography/mass spectrometry (CG/MS), and genomic analyses. Two isolates (MYC038 and MYC040) exhibited pyrene degradation indexes of 96% and 88%, respectively, over a seven-day incubation period. Interestingly, the genomic analyses showed that the isolates do not have nid genes, which are involved in PAH biodegradation, despite their ability to degrade pyrene, suggesting that degradation may occur due to the presence of cyp 150 genes, or even genes that have not yet been described. To the best of our knowledge, this is the first report of isolates without nid genes demonstrating the ability to degrade pyrene.

Introduction: SARS-CoV-2 is the pathogen of COVID-19. The virus is composed of the spike, membrane and envelope. On physiological smell, odoriferous substances bind to proteins secreted by sustentacular cells in order to be processed by olfactory receptor neurons. Olfactory disorder is one of the main manifestations of COVID-19, however, research is still required to clarify the mechanism involved in SARS-CoV-2 induced anosmia., Objective: This article aims to analyze current scientific evidence intended to elucidate the pathophysiological relationship between COVID-19 and the cause of olfactory disorders., Methods: Pubmed, Embase, Scopus and ScienceDirect were used to compose this article. The research was conducted on November 24th, 2020. Original articles with experimental studies in human, animal and in vitro, short communications, viewpoint, published in the English language and between 2019 and 2020 were included, all related to the pathophysiological relationship between olfactory disorders and COVID-19 infection., Results: Both human cell receptors ACE2 and TMPRSS2 are essential for the SARS-CoV-2 entrance. These receptors are mostly present in the olfactory epithelium cells, therefore, the main hypothesis is that anosmia is caused due to damage to non-neuronal cells which, thereafter, affects the normal olfactory metabolism. Furthermore, magnetic resonance imaging studies exhibit a relationship between a reduction on the neuronal epithelium and the olfactory bulb atrophy. Damage to non-neuronal cells explains the average recovery lasting a few weeks. This injury can be exacerbated by an aggressive immune response, which leads to damage to neuronal cells and stem cells inducing a persistent anosmia. Conductive anosmia is not sufficient to explain most cases of COVID-19 induced anosmia., Conclusion: Olfactory disorders such as anosmia and hyposmia can be caused by COVID-19, the main mechanism is associated with olfactory epithelium damage, targeting predominantly non-neuronal cells. However, neuronal cells can also be affected, worsening the condition of olfactory loss., (Copyright © 2021 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.)

Severe Acute Respiratory Syndrome Coronavirus 2 is part of the Cononaviridae family and is the causative agent of the 2019 (Covid-19) Coronavirus pandemic declared by the World Health Organization in March, 2020. This virus has a high rate of transmission, affecting several individuals, and has caused thousands of deaths. The clinical manifestations of Severe Acute Respiratory Syndrome Coronavirus 2 infection are not restricted only to the respiratory tract, and there is an express involvement of the cardiovascular system with a higher risk of death in this group. In such patients there is an overactivation of renin-angiotensin-aldosterone system, which promotes an increase in the expression of angiotensin-converting enzyme - 2 that acts as a receptor for the SPIKE protein expressed by the virus and enables the interaction between the host cell and Severe Acute Respiratory Syndrome Coronavirus 2. This process of infection causes a hyperinflammatory state that increases the inflammatory markers of cardiac injury. Hence, an adequate understanding and clinical guidance regarding the monitoring, and controlling the damage in these patients is essential to avoid worsening of their clinical condition and to prevent death.

Purpose: In laparoscopic incisional hernia repair, meshes with a tissue-separating barrier are positioned intraperitoneally. Despite this property, the close contact between mesh and viscera involves a risk of adhesion formation. Some natural products, such as red propolis (RP), could reduce these adhesions owing to their anti-inflammatory properties. This study aimed to compare two different intraperitoneal meshes with respect to their characteristics of adhesion formation, histological findings and evaluate the role of RP in the development of these adhesions., Methods: 40 Wistar rats received placement of two different meshes (Symbotex and Dynamesh IPOM) on peritoneum. The animals were divided into two groups: control group (mesh) and treatment group (mesh and RP). After 7 and 14 days, 20 animals of each group underwent midline laparotomy to determine the adhesions and histological characteristics., Results: Out of the 40 animals, there were two deaths in the test group and two in the control group. All animals in both groups developed adherence to the mesh. At postoperative day (POD) 7, two Symbotex meshes presented firm adhesions and at POD 14, two Dynamesh meshes had firm adhesions as well. The comparison between the meshes under the effect of RP in relation to the control group showed no statistical difference., Conclusions: Both meshes showed intraperitoneal adhesions in all evaluated samples with similar results on the characteristics of adhesions. RP showed no effect on the incidence or gradation of intraperitoneal adhesions with the mesh.

We investigated the species diversity of Mycobacteriaceae in surface water samples from six environments at the zoological park in São Paulo, Brazil. Three hundred and eighty isolates were cultivated and identified by phenotypic characteristics (growth rate and pigmentation) and sequencing of hsp65, rpoB and 16S rRNA genes. The results revealed that almost 48% of the isolates could be identified at the species level; about 50% were classified at the genus level, and only less than 2% of the isolates showed an inconclusive identification. The isolates classified at the genus level and not identified were then evaluated by phylogenetic analyses using the same three concatenated target genes. The results allowed us to identify at the genus level some isolates that previously had inconclusive identification, and they also suggested the presence of putative candidate species within the sample, demonstrating that this zoological park is an important source of diversity., Competing Interests: The authors have declared that no competing interests exist.

BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB) has increased considerably worldwide. OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution. METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB). FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression. MAIN CONCLUSIONS The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.

Purpose: Nontuberculous mycobacteria keratitis is a rare but challenging complication of laser in situ keratomileusis (LASIK). This study was conducted to determine the source(s) of infection in a cluster of cases of keratitis after LASIK and to describe this outbreak and patients' outcomes., Methods: In this retrospective, case series, single-center study, 86 patients were included who underwent LASIK or photorefractive keratectomy between December 2011 and February 2012. Corneal scrapes from the affected eyes, samples of tap and distilled water, water from the reservoir of the distilling equipment, steamer, and autoclave cassette; antiseptic and anesthetic solutions and surgical instrument imprints were cultivated in liquid and on solid media. Gram-negative bacteria and yeasts were identified using automated systems and mycobacteria by polymerase chain reaction-restriction enzyme analysis of the hsp65 gene (PRA-hsp65) and DNA sequencing. Mycobacterial isolates were typed by pulsed-field gel electrophoresis. The cases and outcomes are described. The main outcome measure was identification of the source(s) of the mycobacterial infections., Results: Eight (15 eyes) of 86 patients (172 eyes) who underwent LASIK developed infections postoperatively; no patients who underwent photorefractive keratectomy developed infections. Mycobacterium chelonae was isolated from 4 eyes. The distilled water collected in the surgical facility contained the same M. chelonae strain isolated from the patients' eyes. Different gram-negative bacteria and yeasts were isolated from samples collected at the clinic but not from the patients' eyes., Conclusions: Tap water distilled locally in surgical facilities may be a source of infection after ocular surgery and its use should be avoided.

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.

Isolates of the Mycobacterium chelonae - M. abscessus complex are subdivided into four clusters (CHI to CHIV) in the INNO-LiPA® Mycobacterium spp DNA strip assay. A considerable phenotypic variability was observed among isolates of the CHII cluster. In this study, we examined the diversity of 26 CHII cluster isolates by phenotypic analysis, drug susceptibility testing, whole genome sequencing and single-gene analysis. Pairwise genome comparisons were performed using several approaches, including average nucleotide identity (ANI) and genome-to-genome distance (GGD) among others. Based on ANI and GGD the isolates were identified as M. chelonae (14 isolates), M. franklinii (2 isolates) and M. salmoniphium (1 isolate). The remaining 9 isolates were subdivided into three novel putative genomospecies. Phenotypic analyses including drug susceptibility testing, as well as whole genome comparison by TETRA and delta differences, were not helpful in separating the groups revealed by ANI and GGD. The analysis of standard four conserved genomic regions showed that rpoB alone and the concatenated sequences clearly distinguished the taxonomic groups delimited by whole genome analyses. In conclusion, the CHII INNO-LiPa is not a homogeneous cluster; on the contrary, it is composed of closely related different species belonging to the M. chelonae-M. abscessus complex and also several unidentified isolates. The detection of these isolates, putatively novel species, indicates a wider inner variability than the presently known in this complex.

An epidemic of post-surgical wound infections, caused by a non-tuberculous mycobacterium, has been on-going in Brazil. It has been unclear whether one or multiple lineages are responsible and whether their wide geographical distribution across Brazil is due to spread from a single point source or is the result of human-mediated transmission. 188 isolates, collected from nine Brazilian states, were whole genome sequenced and analysed using phylogenetic and comparative genomic approaches. The isolates from Brazil formed a single clade, which was estimated to have emerged in 2003. We observed temporal and geographic structure within the lineage that enabled us to infer the movement of sub-lineages across Brazil. The genome size of the Brazilian lineage was reduced relative to most strains in the three subspecies of Mycobacterium abscessus and contained a novel plasmid, pMAB02, in addition to the previously described pMAB01 plasmid. One lineage, which emerged just prior to the initial outbreak, is responsible for the epidemic of post-surgical wound infections in Brazil. Phylogenetic analysis indicates that multiple transmission events led to its spread. The presence of a novel plasmid and the reduced genome size suggest that the lineage has undergone adaptation to the surgical niche.

The objective of this study was to demonstrate the presence of mycobacterial nucleic acid sequences in peripheral blood and arteries from patients with Takayasu arteritis (TA). Polymerase chain reaction was performed to detect mycobacterial DNA from three different nucleic acid sequences including the insertion sequence (IS) 6110, the 65-kDa heat shock protein gene (HSP65), and the 16S ribosomal RNA (rRNA) gene in peripheral blood from 32 TA patients and in arterial specimens from 10 TA patients. Twenty-eight HIV-negative patients with pulmonary tuberculosis prior to therapy were tested for IS6110 in peripheral blood as positive controls, and 24 blood donors were evaluated as healthy controls (HC). All TA patients were negative for the insertion sequence IS6110 and for HSP65 and 16S rRNA genes in blood samples and in arterial specimens. IS6110 sequence was found in peripheral blood from 22 (78.5 %) patients with pulmonary tuberculosis but not in HC. In conclusion, the strategy of mycobacterial-specific nucleic acid amplification in the peripheral blood and arterial specimens of TA patients was unable to lend support to the association between TA and tuberculosis long suggested in the literature.

Food-producing animals can harbor Escherichia coli strains with potential to cause diseases in humans. In this study, the presence of enteropathogenic E. coli (EPEC) was investigated in fecal samples from 130 healthy sheep (92 lambs and 38 adults) raised for meat in southern Brazil. EPEC was detected in 19.2% of the sheep examined, but only lambs were found to be positive. A total of 25 isolates was characterized and designated atypical EPEC (aEPEC) as tested negative for bfpA gene and BFP production. The presence of virulence markers linked to human disease as ehxA, paa, and lpfA O113 was observed in 60%, 24%, and 88% of the isolates, respectively. Of the 11 serotypes identified, eight were described among human pathogenic strains, while three (O1:H8, O11:H21 and O125:H19) were not previously detected in aEPEC. Associations between intimin subtypes and phylogroups were observed, including eae-θ2/A, eae-β1/B1, eae-α2/B2 and eae-γ1/D. Although PFGE typing of 16 aEPEC isolates resulted in 14 unique pulsetypes suggesting a genetic diversity, specific clones were found to be distributed in some flocks. In conclusion, potentially pathogenic aEPEC strains are present in sheep raised for meat, particularly in lambs, which can better contribute to dissemination of these bacteria than adult animals., (Copyright © 2016. Published by Elsevier B.V.)

The taxonomic position of members of the Mycobacterium abscessus complex has been the subject of intensive investigation and, in some aspects confusion, in recent years as a result of varying approaches to genetic data interpretation. Currently, the former species Mycobacterium massiliense and Mycobacterium bolletii are grouped together as Mycobacterium abscessus subsp. bolletii. They differ greatly, however, as the former M. bolletii has a functional erm(41) gene that confers inducible resistance to macrolides, the primary therapeutic antimicrobials for M. abscessus, while in the former M. massiliense the erm(41) gene is non-functional. Furthermore, previous whole genome studies of the M. abscessus group support the separation of M. bolletii and M. massiliense. To shed further light on the population structure of Mycobacterium abscessus, 43 strains and three genomes retrieved from GenBank were subjected to pairwise comparisons using three computational approaches: verage ucleotide dentity, enome to enome istance and single nucleotide polymorphism analysis. The three methods produced overlapping results, each demonstrating three clusters of strains corresponding to the same number of taxonomic entities. The distances were insufficient to warrant distinction at the species level, but met the criteria for differentiation at the subspecies level. Based on prior erm(41)-related phenotypic data and current genomic data, we conclude that the species M. abscessus encompasses, in adjunct to the presently recognized subspecies M. abscessus subsp. abscessus and M. abscessus subsp. bolletii, a third subspecies for which we suggest the name M. abscessus subsp. massiliense comb. nov. (type strain CCUG 48898T=CIP 108297T=DSM 45103T=KCTC 19086T).

Background: A large collection of sequenced mycobacteriophages capable of infecting a single host strain of Mycobacterium smegmatis shows considerable genomic diversity with dozens of distinctive types (clusters) and extensive variation within those sharing evident nucleotide sequence similarity. Here we profiled the mycobacterial components of a large composting system at the São Paulo zoo., Results: We isolated and sequenced eight mycobacteriophages using Mycobacterium smegmatis mc(2)155 as a host. None of these eight phages infected any of mycobacterial strains isolated from the same materials. The phage isolates span considerable genomic diversity, including two phages (Barriga, Nhonho) related to Subcluster A1 phages, two Cluster B phages (Pops, Subcluster B1; Godines, Subcluster B2), three Subcluster F1 phages (Florinda, Girafales, and Quico), and Madruga, a relative of phage Patience with which it constitutes the new Cluster U. Interestingly, the two Subcluster A1 phages and the three Subcluster F1 phages have genomic relationships indicating relatively recent evolution within a geographically isolated niche in the composting system., Conclusions: We predict that composting systems such as those used to obtain these mycobacteriophages will be a rich source for the isolation of additional phages that will expand our view of bacteriophage diversity and evolution.

Five isolates of non-pigmented, rapidly growing mycobacteria were isolated from three patients and,in an earlier study, from zebrafish. Phenotypic and molecular tests confirmed that these isolates belong to the Mycobacterium chelonae-Mycobacterium abscessus group, but they could not be confidently assigned to any known species of this group. Phenotypic analysis and biochemical tests were not helpful for distinguishing these isolates from other members of the M. chelonae–M.abscessus group. The isolates presented higher drug resistance in comparison with other members of the group, showing susceptibility only to clarithromycin. The five isolates showed a unique PCR restriction analysis pattern of the hsp65 gene, 100 % similarity in 16S rRNA gene and hsp65 sequences and 1-2 nt differences in rpoB and internal transcribed spacer (ITS) sequences.Phylogenetic analysis of a concatenated dataset including 16S rRNA gene, hsp65, and rpoB sequences from type strains of more closely related species placed the five isolates together, as a distinct lineage from previously described species, suggesting a sister relationship to a group consisting of M. chelonae, Mycobacterium salmoniphilum, Mycobacterium franklinii and Mycobacterium immunogenum. DNA–DNA hybridization values .70 % confirmed that the five isolates belong to the same species, while values ,70 % between one of the isolates and the type strains of M. chelonae and M. abscessus confirmed that the isolates belong to a distinct species. The polyphasic characterization of these isolates, supported by DNA–DNA hybridization results,demonstrated that they share characteristics with M. chelonae–M. abscessus members, butconstitute a different species, for which the name Mycobacterium saopaulense sp. nov. is proposed. The type strain is EPM10906T (5CCUG 66554T5LMG 28586T5INCQS 0733T).

Objectives: to evaluate and indicate the procedure to be followed in the health unit, both for diagnosis and the treatment of acute exogenous intoxications by carbamates or organophosphates., Methods: a descriptive study based on retrospective analysis of the clinical history of patients diagnosed with intoxication by carbamates or organophosphates admitted at the emergency unit of the Hospital de Urgências de Sergipe Governador João Alves (HUSE) between January and December of 2012. Some criteria were evaluated, such as: intoxicating agent; patient's age and gender; place of event, cause, circumstances and severity of the intoxication; as well as signs and symptoms of the muscarinic, nicotinic and neurological effects., Results: seventy patients (average age: 25 ± 19.97) formed the study's population. It was observed that 77.14% of them suffered carbamate intoxication. However, organophosphate intoxications were more severe, with 68.75% of patients presenting moderate to severe forms. Suicide attempt was the leading cause of poisoning, with 62 cases (88.57% of total). Atropine administration was an effective therapeutic approach for treating signs and symptoms, which included sialorrhea (p = 0.0006), nausea (p = 0. 0029) and emesis (p < 0.0001). The use of activated charcoal was shown effective, both in combating the signs and symptoms presented by both patient groups (p < 0.0001)., Conclusion: it is concluded that the use of atropine and activated charcoal is highly effective to treat the signs and symptoms developed by patients presenting acute exogenous intoxication by carbamates or organophosphates.

Objective: to produce improvements in transfusion practices through the implementation of an educational program for health professionals in a university hospital., Methods: this is an interventional and prospective study, with pre- and postanalysis of an educational intervention. The research was developed at the University Hospital of the Universidade Federal de Sergipe, involving participation of health professionals in the stage of training, during the month of February 2011, in addition to the monitoring of blood transfusions performed in the pre- and post-intervention periods. Transfusion practices were investigated upon request for transfusion or devolution of unused blood components. Knowledge of health professionals was assessed based on the responses to a questionnaire about transfusion practices., Results: during the educative campaign, 63 professionals were trained, including 33 nurses or nursing technicians and 30 physicians. Among the doctors, there was a statistically significant gain of 20.1% in theoretical knowledge (p=0.037). Gain in the nursing group was even higher: 30.4% (p=0.016). The comparative analysis of transfusion request forms showed a non-significant decrease from 26.7 to 19.5% (p=0.31) in all forms with incomplete information. We also observed a statistically significant improvement in relation to the filling of four items of transfusion request., Conclusion: there was a significant improvement of the entire process related to blood transfusions after interventional project conducted in February 2011.

Drug susceptibility testing (DST) of rapidly growing mycobacteria (RGM) are recommended for guiding the antimicrobial therapy. We have evaluated the use of resazurin in Mueller-Hinton medium (MHR) for MIC determination of RGM and compared the results with those obtained with the reference standard broth microdilution in Mueller-Hinton (MH) and with the resazurin microtiter assay (REMA) in 7H9 broth. The MIC of eight drugs: amikacin (AMI), cefoxitin (FOX), ciprofloxacin (CIP), clarithromycin (CLA), doxycycline (DOX), linezolid (LZD), moxifloxacin (MXF) and trimethoprim-sulfamethoxazole (TMP-SMX) were evaluated against 76 RGM (18 species) using three methods (MH, MHR, and REMA) in a 96-well plate format incubated at 37 °C over 3-5 days. Results obtained in the MH plates were interpreted by the appearance of turbidity at the bottom of the well before adding the resazurin. MHR and 7H9-REMA plates were read by visual observation for a change in color from blue to pink. The majority of results were obtained at day 5 for MH and 1 day after for MHR and 7H9-REMA. However, the preliminary experiment on time to positivity results using the reference strain showed that the resazurin can be added to the MH at day 2 to produce the results at day 3, but future studies with large sets of strains are required to confirm this suggestion. A high level of agreement (kappa 1.000-0.884) was obtained between the MH and the MHR. Comparison of results obtained with 7H9-REMA, on the other hand, revealed several discrepancies and a lower level of agreement (kappa 1.000-0.111). The majority of the strains were resistant to DOX and TMP-SMX, and the most active antimicrobials for RGM were AMI and FOX. In the present study, MHR represented an excellent alternative for MIC determination of RGM. The results could be read reliably, more easily, and more quickly than with the classical MH method.

Introduction: The most common pathogen in bacteria lpharyngotonsillitis is group A β-hemolytic streptococcus, although groups B, C, F,and G have also been associated with pharyngotonsillitis., Objective: To assess the levels of the cytokines TNF-α, IL-6,IL-4, and IL-10 in bacterial pharyngotonsillitis caused by group A and non-A (groups B, C, F and G) β-hemolytic streptococcus., Methods: The study was conducted at a pediatric emergency care unit. The sample comprised children (5-9 years old) with acute bacterial pharyngotonsillitis diagnosed between December of 2011 and May of 2012. The research involved collection of blood samples from the patients, enzyme-linked immunosorbent assay detection of TNF-α, IL-6,IL-4, and IL-10, and collection of two oropharyngeal swabs for bacterial isolation. Additionally, the medical history of the study participants was also collected., Results: In the studied group (mean age: 5.93 years), higher pharyngotonsillitis incidence was observed in the female gender (64.76%). Higher incidence of tonsillar exudates was observed with groups A and C. No statistically significant differences in cytokine levels were observed among groups. However, the group A and the control group showed a difference in the IL-6 level (p=0.0016)., Conclusions: The Groups A and C showed higher cytokine levels than the Groups B and control, suggesting similar immunological patterns., (Copyright © 2015 Associação Brasileira de Otorrinolaringologia e Cirurgia Cérvico-Facial. Published by Elsevier Editora Ltda. All rights reserved.)

Introduction: Rheumatic Fever represents a serious public health problem in developing countries, with thousands of new cases each year. It is an autoimmune disease, which occurs in response to infection by streptococcus A., Objective: The aim of this study was to evaluate the immunolabeling and protein expression for endothelin-1 and 3 (ET-1, ET-3) and its receptors (ETA, ETB) in rheumatic mitral valves., Methods: Immunohistochemistry was used to identify ET-1/ET-3 and ETA/ETB receptors in rheumatic and control mitral valves. Quantitative analysis of immunostaining for ET-1/ET-3 and ETA/ETB receptors was performed. In addition, western blot analysis was carried out to assess protein levels in tissue samples., Results: ET-1 and ETA receptor immunostaining predominated in stenotic valves, mainly associated with fibrotic regions, inflammatory areas and neovascularization. Quantitative analysis showed that the average area with positive expression of ET-1 was 18.21 ± 14.96%. For ETA and ETB, the mean expressed areas were respectively 15.06 ± 13.13% and 9.20 ± 11.09%. ET-3 did not have a significant expression. The correlation between the expression of both endothelin receptors were strongly positive (R = 0.74, P = 0.02), but the correlation between ET-1 and its receptor were negative for both ETA (R = -0.37, P = 0.25), and ETB (R = -0.14, P = 0.39). This data was supported by western blot analysis., Conclusion: The strong correlation between ET-1 and its receptors suggests that both play a role in the pathophysiology of rheumatic mitral valve stenosis and may potentially act as biomarkers of this disease.

Sheep constitute an important source of zoonotic pathogens as Shiga toxin-producing Escherichia coli (STEC). In this study, the prevalence, serotypes and virulence profiles of STEC were investigated among 130 healthy sheep from small and medium farms in southern Brazil. STEC was isolated from 65 (50%) of the tested animals and detected in all flocks. A total of 70 STEC isolates were characterized, and belonged to 23 different O:H serotypes, many of which associated with human disease, including hemolytic-uremic syndrome (HUS). Among the serotypes identified, O76:H19 and O65:H- were the most common, and O75:H14 and O169:H7 have not been previously reported in STEC strains. Most of the STEC isolates harbored only stx1, whereas the Stx2b subtype was the most common among those carrying stx2. Enterohemolysin (ehxA) and intimin (eae) genes were detected in 61 (87.1%) and four (5.7%) isolates, respectively. Genes encoding putative adhesins (saa, iha, lpfO113) and toxins (subAB and cdtV) were also observed. The majority of the isolates displayed virulence features related to pathogenesis of STEC, such as adherence to epithelial cells, high cytotoxicity and enterohemolytic activity. Ovine STEC isolates belonged mostly to phylogenetic group B1. PFGE revealed particular clones distributed in some farms, as well as variations in the degree of genetic similarity within serotypes examined. In conclusion, STEC are widely distributed in southern Brazilian sheep, and belonged mainly to serotypes that are not commonly reported in other regions, such as O76:H19 and O65:H-. A geographical variation in the distribution of STEC serotypes seems to occur in sheep., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Outbreaks of infections by rapidly growing mycobacteria following invasive procedures, such as ophthalmological, laparoscopic, arthroscopic, plastic, and cardiac surgeries, mesotherapy, and vaccination, have been detected in Brazil since 1998. Members of the Mycobacterium chelonae-Mycobacterium abscessus group have caused most of these outbreaks. As part of an epidemiological investigation, the isolates were typed by pulsed-field gel electrophoresis (PFGE). In this project, we performed a large-scale comparison of PFGE profiles with the results of a recently developed multilocus sequence typing (MLST) scheme for M. abscessus. Ninety-three isolates were analyzed, with 40 M. abscessus subsp. abscessus isolates, 47 M. abscessus subsp. bolletii isolates, and six isolates with no assigned subspecies. Forty-five isolates were obtained during five outbreaks, and 48 were sporadic isolates that were not associated with outbreaks. For MLST, seven housekeeping genes (argH, cya, glpK, gnd, murC, pta, and purH) were sequenced, and each isolate was assigned a sequence type (ST) from the combination of obtained alleles. The PFGE patterns of DraI-digested DNA were compared with the MLST results. All isolates were analyzable by both methods. Isolates from monoclonal outbreaks showed unique STs and indistinguishable or very similar PFGE patterns. Thirty-three STs and 49 unique PFGE patterns were identified among the 93 isolates. The Simpson's index of diversity values for MLST and PFGE were 0.69 and 0.93, respectively, for M. abscessus subsp. abscessus and 0.96 and 0.97, respectively, for M. abscessus subsp. bolletii. In conclusion, the MLST scheme showed 100% typeability and grouped monoclonal outbreak isolates in agreement with PFGE, but it was less discriminative than PFGE for M. abscessus., (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.)

Background: Phospholipases C (PLCs) are virulence factors found in several bacteria. In Mycobacterium tuberculosis (Mtb) they exhibit cytotoxic effects on macrophages, but the mechanisms involved in PLC-induced cell death are not fully understood. It has been reported that induction of cell necrosis by virulent Mtb is coordinated by subversion of PGE2, an essential factor in cell membrane protection., Results: Using two Mtb clinical isolates carrying genetic variations in PLC genes, we show that the isolate 97-1505, which bears plcA and plcB genes, is more resistant to alveolar macrophage microbicidal activity than the isolate 97-1200, which has all PLC genes deleted. The isolate 97-1505 also induced higher rates of alveolar macrophage necrosis, and likewise inhibited COX-2 expression and PGE2 production. To address the direct effect of mycobacterial PLC on cell necrosis and PGE2 inhibition, both isolates were treated with PLC inhibitors prior to macrophage infection. Interestingly, inhibition of PLCs affected the ability of the isolate 97-1505 to induce necrosis, leading to cell death rates similar to those induced by the isolate 97-1200. Finally, PGE2 production by Mtb 97-1505-infected macrophages was restored to levels similar to those produced by 97-1200-infected cells., Conclusions: Mycobacterium tuberculosis bearing PLCs genes induces alveolar macrophage necrosis, which is associated to subversion of PGE2 production.

Plasmid-mediated kanamycin resistance was detected in a strain of Mycobacterium abscessus subsp. bolletii responsible for a nationwide epidemic of surgical infections in Brazil. The plasmid did not influence susceptibility to tobramycin, streptomycin, trimethoprim-sulfamethoxazole, clarithromycin, or ciprofloxacin. Plasmid-mediated drug resistance has not been described so far in mycobacteria.

The Retrovirus Epidemiology Donor Study (REDS) program was established in the United States in 1989 with the purpose of increasing blood transfusion safety in the context of the HIV/AIDS and human T-lymphotropic virus epidemics. REDS and its successor, REDS-II were at first conducted in the US, then expanded in 2006 to include international partnerships with Brazil and China. In 2011, a third wave of REDS renamed the Recipient Epidemiology and Donor Evaluation Study-III (REDS-III) was launched. This seven-year research program focuses on both blood banking and transfusion medicine research in the United States of America, Brazil, China, and South Africa. The main goal of the international programs is to reduce and prevent the transmission of HIV/AIDS and other known and emerging infectious agents through transfusion, and to address research questions aimed at understanding global issues related to the availability of safe blood. This article describes the contribution of REDS-II to transfusion safety in Brazil. Articles published from 2010 to 2013 are summarized, including database analyses to characterize blood donors, deferral rates, and prevalence, incidence and residual risk of the main blood-borne infections. Specific studies were developed to understand donor motivation, the impact of the deferral questions, risk factors and molecular surveillance among HIV-positive donors, and the natural history of Chagas disease. The purpose of this review is to disseminate the acquired knowledge and briefly summarize the findings of the REDS-II studies conducted in Brazil as well as to introduce the scope of the REDS-III program that is now in progress and will continue through 2018.

We developed a multilocus sequence typing (MLST) scheme for Mycobacterium abscessus sensu lato, based on the partial sequencing of seven housekeeping genes: argH, cya, glpK, gnd, murC, pta and purH. This scheme was used to characterize a collection of 227 isolates recovered between 1994 and 2010 in France, Germany, Switzerland and Brazil. We identified 100 different sequence types (STs), which were distributed into three groups on the tree obtained by concatenating the sequences of the seven housekeeping gene fragments (3576bp): the M. abscessus sensu stricto group (44 STs), the "M. massiliense" group (31 STs) and the "M. bolletii" group (25 STs). SplitTree analysis showed a degree of intergroup lateral transfers. There was also evidence of lateral transfer events involving rpoB. The most prevalent STs in our collection were ST1 (CC5; 20 isolates) and ST23 (CC3; 31 isolates). Both STs were found in Europe and Brazil, and the latter was implicated in a large post-surgical procedure outbreak in Brazil. Respiratory isolates from patients with cystic fibrosis belonged to a large variety of STs; however, ST2 was predominant in this group of patients. Our MLST scheme, publicly available at www.pasteur.fr/mlst, offers investigators a valuable typing tool for M. abscessus sensu lato in future epidemiological studies throughout the world., (Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Objective: To evaluate the immunological profile and gene expression of endothelin-1 (ET-1) in mitral valves of patients with rheumatic fever originated from a reference service in cardiovascular surgery., Methods: This was a quantitative, observational and cross-sectional study. Thirty-five subjects (divided into four groups) participated in the study, 25 patients with chronic rheumatic heart disease and ten control subjects. The mean age of the sample studied was 34.5 years. Seventeen of them (48.58%) were male and 18 (51.42%) were female. Inflammatory cytokines (TNF-α, IL-4 and IL-10) were measured and ten mitral valves of patients who underwent first valve replacement were collected for determination of gene expression of endothelin-1 by real time PCR., Results: Among the groups studied (patients vs. controls), there was a statistically significant difference in IL-10 levels (P=0.002), and no differences in other cytokines. Expression of endothelin-1 was observed in 70% of samples. Quantitatively, average of ET-1 expression was 62.85±25.63%., Conclusion: Inflammatory cytokine IL-10 participates in the maintenance of chronicity of rheumatic fever in patients who underwent valve replacement and those who are undergoing medical treatment. The expression of endothelin-1 in heart valve lesions in patients undergoing mitral valve replacement confirms its association with inflammatory activity in rheumatic fever.

An epidemic of surgical-site infections by a single strain of Mycobacterium abscessus subsp. bolletii affected >1,700 patients in Brazil from 2004 to 2008. The genome of the epidemic prototype strain M. abscessus subsp. bolletii INCQS 00594, deposited in the collection of the National Institute for Health Quality Control (INCQS), was sequenced.

Background: Although risk factors for HIV infection are known, it is important for blood centres to understand local epidemiology and disease transmission patterns. Current risk factors for HIV infection in blood donors in Brazil were assessed., Methods: A case-control study was conducted at large public blood centres located in four major cities between April 2009 and March 2011. Cases were persons whose donations were confirmed positive by enzyme immunoassays followed by Western blot confirmation. Audio computer-assisted structured interviews (ACASI) were completed by all cases and controls. Multivariable logistic regression was used to estimate adjusted odds ratios (AORs) and associated 95% confidence intervals (CIs)., Results: There were 341 cases, including 47 with recently acquired infection, and 791 controls. Disclosed risk factors for both females and males were sex with an HIV-positive person AOR 11.3, 95% CI (4.1, 31.7) and being an IVDU or sexual partner of an IVDU [AOR 4.65 (1.8, 11.7)]. For female blood donors, additional risk factors were having male sex partners who also are MSM [AOR 13.5 (3.1, 59.8)] and having unprotected sex with multiple sexual partners [AOR 5.19 (2.1, 12.9)]. The primary risk factor for male blood donors was MSM activity [AOR 21.6 (8.8, 52.9)]. Behaviours associated with recently acquired HIV were being a MSM or sex partner of MSM [13.82, (4.7, 40.3)] and IVDU [11.47, (3.0, 43.2)]., Conclusion: Risk factors in blood donors parallel those in the general population in Brazil. Identified risk factors suggest that donor compliance with selection procedures at the participating blood centres is inadequate., (© 2013 International Society of Blood Transfusion.)

Background: We evaluate the current prevalence of serologic markers for hepatitis B virus (HBV) and hepatitis C virus (HCV) in blood donors and estimated HCV incidence and residual transfusion-transmitted risk at three large Brazilian blood centers., Study Design and Methods: Data on whole blood and platelet donations were collected from January through December 2007, analyzed by center; donor type; age; sex; donation status; and serologic results for hepatitis B surface antigen (HBsAg), antibody to hepatitis B core antigen (anti-HBc), and anti-HCV. HBV and HCV prevalence rates were calculated for all first-time donations. HCV incidence was derived including interdonation intervals that preceded first repeat donations given during the study, and HCV residual risk was estimated for transfusions derived from repeat donors., Results: There were 307,354 donations in 2007. Overall prevalence of concordant HBsAg and anti-HBc reactivity was 289 per 100,000 donations and of anti-HCV confirmed reactivity 191 per 100,000 donations. There were significant associations between older age and hepatitis markers, especially for HCV. HCV incidence was 3.11 (95% confidence interval, 0.77-7.03) per 100,000 person-years, and residual risk of HCV window-phase infections was estimated at 5.0 per million units transfused., Conclusion: Improvement in donor selection, socioeconomic conditions, and preventive measures, implemented over time, may have helped to decrease prevalence of HBV and HCV, relative to previous reports. Incidence and residual risk of HCV are also diminishing. Ongoing monitoring of HBV and HCV markers among Brazilian blood donors should help guide improved recruitment procedures, donor selection, laboratory screening, and counseling strategies., (© 2012 American Association of Blood Banks.)

Background and Objectives: Although the incidence of TRALI is unknown in Brazil, some blood centers have adopted strategies to prevent TRALI. We evaluated the impact of three policies to mitigate TRALI on the supply of blood products: to divert the production of whole blood-derived plasma from female donors; to defer all female donors from apheresis platelet collections, and to defer only multiparous female donors from apheresis platelet collections., Materials and Methods: Data from allogeneic whole blood and apheresis platelet donations from April 2008 to December 2009 were collected in three Brazilian blood centers and the impact of the aforementioned strategies was evaluated., Results: Of 544,814 allogeneic blood donations, 30.8% of whole blood plasma and 24.1% of apheresis platelet donations would be reduced if only male donor plasma was issued for transfusion and all female donors were deferred from apheresis donation, respectively. If only multiparous donors were deferred from apheresis donation, there would be a 5% decrease of all apheresis platelet collections., Conclusion: Restricting the use of whole blood derived plasma to male-only donors and deferring all female apheresis platelet donors would impact two out of three Brazilian blood centers. A deferral policy on multiparous apheresis platelet donors may be acceptable as a temporary measure, but may cause more stress on a system that is already working at its limit., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Background: An extended outbreak of mycobacterial surgical infections occurred in Brazil during 2004-2008. Most infections were caused by a single strain of Mycobacterium abscessus subsp. bolletii, which was characterized by a specific rpoB sequevar and two highly similar pulsed-field gel electrophoresis (PFGE) patterns differentiated by the presence of a ∼50 kb band. The nature of this band was investigated., Methodology/principal Findings: Genomic sequencing of the prototype outbreak isolate INCQS 00594 using the SOLiD platform demonstrated the presence of a 56,267-bp [corrected] circular plasmid, designated pMAB01. Identity matrices, genetic distances and phylogeny analyses indicated that pMAB01 belongs to the broad-host-range plasmid subgroup IncP-1β and is highly related to BRA100, pJP4, pAKD33 and pB10. The presence of pMAB01-derived sequences in 41 M. abscessus subsp. bolletii isolates was evaluated using PCR, PFGE and Southern blot hybridization. Sixteen of the 41 isolates showed the presence of the plasmid. The plasmid was visualized as a ∼50-kb band using PFGE and Southern blot hybridization in 12 isolates. The remaining 25 isolates did not exhibit any evidence of this plasmid. The plasmid was successfully transferred to Escherichia coli by conjugation and transformation. Lateral transfer of pMAB01 to the high efficient plasmid transformation strain Mycobacterium smegmatis mc(2)155 could not be demonstrated., Conclusions/significance: The occurrence of a broad-host-range IncP-1β plasmid in mycobacteria is reported for the first time. Thus, genetic exchange could result in the emergence of specific strains that might be better adapted to cause human disease.

Background: Seasonal distribution of blood donation hinders efforts to provide a safe and adequate blood supply leading to chronic and persistent shortages. This study examined whether holidays, geographical area and donation type (community versus replacement) has any impact on the fluctuation of donations., Methods: The numbers of blood donations from 2007 through 2010 in three Brazilian Retrovirus Epidemiological Donor Study II (REDS-II) participating centers were analyzed according to the week of donation. The weeks were classified as holiday or non-holiday. To compare donations performed during holiday versus non-holiday weeks, tabulations and descriptive statistics for weekly donations by blood center were examined and time series analysis was conducted., Results: The average weekly number of donations varied according to the blood center and type of week. The average number of donations decreased significantly during Carnival and Christmas and increased during the Brazilian National Donor Week. The fluctuation was more pronounced in Recife and Belo Horizonte when compared to São Paulo and higher among community donors., Conclusion: National bank holidays affect the blood supply by reducing available blood donations. Blood banks should take into account these oscillations in order to plan local campaigns, aiming at maintaining the blood supply at acceptable levels.

Objectives: Rheumatic fever is a highly prevalent disease in Brazil, and it poses a major public health problem. It is the leading cause of acquired heart disease in childhood and adolescence. The aim of this study was to evaluate the gene expression of ET-3 and its receptors, in replaced rheumatic mitral valves., Methods: We studied the gene expression of endothelin-3 (ET-3) and its receptors, endothelin receptor A and endothelin receptor B (ETr-A and ETr-B), in the rheumatic mitral valves of 17 patients who underwent valve replacement surgery. The samples also underwent a histological analysis., Results: Our data showed that almost all patients, regardless of individual characteristics such as gender or age, expressed the endothelin receptor genes, but did not express the genes for ET-3. In quantitative analysis, the ETr-A/GAPDH mean ratio was 33.04 ± 18.09%; while the ETr-B/GAPDH mean ratio was 114.58 ± 42.30%. Regarding histopathological individual features, the frequency of fibrosis is 100%, 88.23% of mononuclear infiltrate, 52.94% of neovascularization, 58.82% of calcification and absence of ossification., Conclusion: The presence of receptors ETr-A and ETr-B in rheumatic mitral valves suggests its interaction with the system of circulating endothelins, particularly ETr-B (known for acting in the removal of excess endothelin) detected in a greater proportion, which could explain the lack of expression of endothelin in rheumatic mitral valve, process to be elucidated.

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: In a previous study, we detected the presence of a Mycobacterium avium species-specific insertion sequence, IS1245, in Mycobacterium kansasii. Both species were isolated from a mixed M. avium-M. kansasii bone marrow culture from an HIV-positive patient. The transfer mechanism of this insertion sequence to M. kansasii was investigated here., Methodology/principal Findings: A linear plasmid (pMA100) was identified in all colonies isolated from the M. avium-M. kansasii mixed culture carrying the IS1245 element. The linearity of pMA100 was confirmed. Other analyses suggested that pMA100 contained a covalently bound protein in the terminal regions, a characteristic of invertron linear replicons. Partial sequencing of pMA100 showed that it bears one intact copy of IS1245 inserted in a region rich in transposase-related sequences. These types of sequences have been described in other linear mycobacterial plasmids. Mating experiments were performed to confirm that pMA100 could be transferred in vitro from M. avium to M. kansasii. pMA100 was transferred by in vitro conjugation not only to the M. kansasii strain from the mixed culture, but also to two other unrelated M. kansasii clinical isolates, as well as to Mycobacterium bovis BCG Moreau., Conclusions/significance: Horizontal gene transfer (HGT) is one of most important mechanisms leading to the evolution and diversity of bacteria. This work provides evidence for the first time on the natural occurrence of HGT between different species of mycobacteria. Gene transfer, mediated by a novel conjugative plasmid, was detected and experimentally reproduced.

Mycobacterium abscessus, Mycobacterium bolletii, and Mycobacterium massiliense (Mycobacterium abscessus sensu lato) are closely related species that currently are identified by the sequencing of the rpoB gene. However, recent studies show that rpoB sequencing alone is insufficient to discriminate between these species, and some authors have questioned their current taxonomic classification. We studied here a large collection of M. abscessus (sensu lato) strains by partial rpoB sequencing (752 bp) and multilocus sequence analysis (MLSA). The final MLSA scheme developed was based on the partial sequences of eight housekeeping genes: argH, cya, glpK, gnd, murC, pgm, pta, and purH. The strains studied included the three type strains (M. abscessus CIP 104536(T), M. massiliense CIP 108297(T), and M. bolletii CIP 108541(T)) and 120 isolates recovered between 1997 and 2007 in France, Germany, Switzerland, and Brazil. The rpoB phylogenetic tree confirmed the existence of three main clusters, each comprising the type strain of one species. However, divergence values between the M. massiliense and M. bolletii clusters all were below 3% and between the M. abscessus and M. massiliense clusters were from 2.66 to 3.59%. The tree produced using the concatenated MLSA gene sequences (4,071 bp) also showed three main clusters, each comprising the type strain of one species. The M. abscessus cluster had a bootstrap value of 100% and was mostly compact. Bootstrap values for the M. massiliense and M. bolletii branches were much lower (71 and 61%, respectively), with the M. massiliense cluster having a fuzzy aspect. Mean (range) divergence values were 2.17% (1.13 to 2.58%) between the M. abscessus and M. massiliense clusters, 2.37% (1.5 to 2.85%) between the M. abscessus and M. bolletii clusters, and 2.28% (0.86 to 2.68%) between the M. massiliense and M. bolletii clusters. Adding the rpoB sequence to the MLSA-concatenated sequence (total sequence, 4,823 bp) had little effect on the clustering of strains. We found 10/120 (8.3%) isolates for which the concatenated MLSA gene sequence and rpoB sequence were discordant (e.g., M. massiliense MLSA sequence and M. abscessus rpoB sequence), suggesting the intergroup lateral transfers of rpoB. In conclusion, our study strongly supports the recent proposal that M. abscessus, M. massiliense, and M. bolletii should constitute a single species. Our findings also indicate that there has been a horizontal transfer of rpoB sequences between these subgroups, precluding the use of rpoB sequencing alone for the accurate identification of the two proposed M. abscessus subspecies.

An epidemic of infections by rapidly growing mycobacteria related to surgical procedures between 2004 and 2008 in Brazil was caused by a unique strain showing the Mycobacterium abscessus type 2 pattern when it was analyzed by the molecular method of PCR-restriction enzyme analysis of the hsp65 gene (PRA-hsp65). In order to investigate the diversity of M. abscessus type 2 clinical isolates and to assess whether this epidemic strain was present in specimens from nonsurgical patients, we studied 52 isolates from 38 patients showing this characteristic PRA-hsp65 pattern obtained between 2005 and 2009. All isolates were identified by sequencing of region V of the rpoB gene and typed by pulsed-field gel electrophoresis (PFGE) using two restriction enzymes, DraI and AseI. Seven isolates obtained from sputum, bronchoalveolar lavage fluid, and urine in three different Brazilian states showed rpoB sequences 100% similar to the rpoB sequence of epidemic strain INCQS 594 and PFGE patterns highly related to the patterns of isolates, evidencing the presence of the epidemic strain in isolates from patients not associated with the surgical epidemic. The remaining isolates showed diverse rpoB sequences, with the highest similarities being to the corresponding sequences of M. massiliense(T) CIP 108297 (21 isolates), M. bolletii(T) CIP 108541 (19 isolates), or M. abscessus(T) ATCC 19977 (5 isolates). Two additional clusters could be detected by PFGE. PFGE showed 100% typeability and reproducibility and discriminatory powers, calculated by Simpson's index of diversity, of 0.978 (DraI) and 0.986 (AseI), confirming its suitability for the discrimination of M. abscessus type 2 isolates.

Background: Opportunistic Mycobacterium avium typically causes disease in immunocompromised patients and in some groups of apparently healthy individuals. The high virulence of some bacterial lineages increases the disease risk. High-resolution molecular genotyping studies of M. avium clinical isolates demonstrated that some genotype patterns were more prevalent than others, suggesting that close genetic relatedness of these successful isolates sharing a similar genotype could determine similar biological properties associated with high virulence., Methods and Findings: In this study, we aimed to compare the virulence and pathogenic properties of two epidemiologically unrelated M. avium isolates sharing an indistinguishable DNA fingerprint in a well-characterized model of pulmonary infection in mice, resistant or susceptible to mycobacteria. The mice, C57BL/6 wild- type or IFN-gamma gene disrupted (GKO), respectively, were intratracheally infected with two isolates, H27 (human blood isolate) and P104 (pig lymph node isolate), and the lungs were examined for bacterial loads, histopathology and cytokine gene expression. The obtained data demonstrated significant differences in the virulence properties of these strains. Although the H27 strain grew significantly faster than P104 in the early stage of infection, this bacterium induced protective immunity that started to reduce bacterial numbers in the wild-type mice, whereas the P104 strain established a chronic infection. In the GKO mice, both strains were capable of causing a chronic infection, associated with higher bacterial burdens and severe lung pathology, in a similar manner., Conclusions/significance: The results demonstrated that the studied isolates differed in the pathogenic properties although were indistinguishable by actually widely used genotyping techniques demonstrating that the genotype similarity does not predict similarity in virulence of M. avium isolates.

Transforming growth factor beta (TGF-β) has been implicated in the pathogenesis of several diseases including infection with intracellular pathogens such as the Mycobacterium avium complex. Infection of macrophages with M. avium induces TGF-β production and neutralization of this cytokine has been associated with decreased intracellular bacterial growth. We have previously demonstrated that epithelioid cell surrogates (ECs) derived from primary murine peritoneal macrophages through a process of differentiation induced by IL-4 overlap several features of epithelioid cells found in granulomas. In contrast to undifferentiated macrophages, ECs produce larger amounts of TGF-β and inhibit the intracellular growth of M. avium. Here we asked whether the levels of TGF-β produced by ECs are sufficient to induce a self-sustaining autocrine TGF-β signaling controlling mycobacterial replication in infected-cells. We showed that while exogenous addition of increased concentration of TGF-β to infected-macrophages counteracted M. avium replication, pharmacological blockage of TGF-β receptor kinase activity with SB-431542 augmented bacterial load in infected-ECs. Moreover, the levels of TGF-β produced by ECs correlated with high and sustained levels of ERK1/2 activity. Inhibition of ERK1/2 activity with U0126 increased M. avium replication in infected-cells, suggesting that modulation of intracellular bacterial growth is dependent on the activation of ERK1/2. Interestingly, blockage of TGF-β receptor kinase activity with SB-431542 in infected-ECs inhibited ERK1/2 activity, enhanced intracellular M. avium burden and these effects were followed by a severe decrease in TGF-β production. In summary, our findings indicate that the amplitude of TGF-β signaling coordinates the strength and duration of ERK1/2 activity that is determinant for the control of intracellular mycobacterial growth.