Comparative genome analysis and characterization of the Salmonella Typhimurium strain CCRJ_26 isolated from swine carcasses using whole-genome sequencing approach.

Salmonella pathogenicity relies on virulence factors many of which are clustered within the Salmonella pathogenicity islands. Salmonella also harbours mobile genetic elements such as virulence plasmids, prophage-like elements and antimicrobial resistance genes which can contribute to increase its pathogenicity. Here, we have genetically characterized a selected S. Typhimurium strain (CCRJ_26) from our previous study with Multiple Drugs Resistant profile and high-frequency PFGE clonal profile which apparently persists in the pork production centre of Rio de Janeiro State, Brazil. By whole-genome sequencing, we described the strain's genome virulent content and characterized the repertoire of bacterial plasmids, antibiotic resistance genes and prophage-like elements. Here, we have shown evidence that strain CCRJ_26 genome possible represent a virulence-associated phenotype which may be potentially virulent in human infection. SIGNIFICANCE AND IMPACT OF THE STUDY: Whole-genome sequencing technologies are still costly and remain underexplored for applied microbiology in Brazil. Hence, this genomic description of S. Typhimurium strain CCRJ_26 will provide help in future molecular epidemiological studies. The analysis described here reveals a quick and useful pipeline for bacterial virulence characterization using whole-genome sequencing approach.

Usefulness of IS6110-restriction fragment length polymorphism typing of Brazilian strains of Mycobacterium tuberculosis and comparison with an international fingerprint database.

Strains of Mycobacterium tuberculosis isolated from 219 different tuberculosis patients, 115 from patients residing in Rio de Janeiro, 79 from Rio Grande do Sul and the remaining from other regions of the country, were analyzed by IS6110-restriction fragment length polymorphism fingerprinting. The IS6110-DNA patterns from these strains were highly polymorphic: 174 different patterns were observed and 25 patterns were shared by 70 isolates (32%). Most strains (93.4%) had multicopy patterns and only 17% of clustered strains had less than six IS6110 copies. Strain clustering was significantly higher for isolates from Rio Grande do Sul (36.7%) in comparison with strains from Rio de Janeiro (22.6%), but only when using high stringency during cluster analysis. Upon screening of an international database containing 3,970 fingerprints of M. tuberculosis strains, 15% of the patterns of Brazilian strains (21% of the strains) were identical to a fingerprint of an isolate from another country and one particular eight-band pattern forming the largest Brazilian cluster was detected in seven additional countries, suggesting that international transmission of tuberculosis from and to Brazil could be occurring frequently. Alternatively,preferential use of certain IS6110 integration sites could also be important in high-copy number strains, having important consequences for the use of databases for epidemiological studies on a large scale.

Use of PCR-mediated amplification of Mycobacterium leprae DNA in different types of clinical samples for the diagnosis of leprosy.

DNA of Mycobacterium leprae, obtained by a highly efficient nucleic acid extraction procedure, was used for standardisation of the amplification of an M. leprae-specific repetitive sequence by use of the polymerase chain reaction (PCR). With pure DNA, M. leprae-specific amplification was obtained with as low as 100 ag (1 ag = 10(-18) g) of target DNA, a quantity equal to about one-tenth of the bacterial genome. Optimal processing of different types of clinical samples such as biopsy material, blood and lymph fluid, from multibacillary leprosy patients, was studied. Simple freezing-boiling cycles in the presence of Triton X100, with some additional sample-specific modifications such as pre-treatment with NaOH to eliminate PCR inhibitors, was found to be sufficient to yield amplification of bacterial DNA in samples from paucibacillary patients. Clinical samples from 27 untreated leprosy patients, covering the various clinical forms of the disease, and with a bacterial index ranging from 5+ to 0, were collected and processed for PCR analysis. After hybridisation of the amplified material with a specific sequence, 25 of 27 patients analysed gave positive results for M. leprae in at least one of the samples. The potential of PCR for the diagnosis of leprosy is discussed.

Use of the polymerase chain reaction in the diagnosis of leprosy.

One of the main limitations for successful epidemiological control of leprosy is the lack of a method for its diagnosis in subclinical cases. Because of the long incubation period of the disease, liberation and spread of Mycobacterium leprae during subclinical stages-principally in cases of untreated multibacillary forms of leprosy-constitute the main source of infection. This report describes the use of the polymerase chain reaction (PCR) for the detection of M. leprae in different types of tissue samples (blood, lymph, nasal secretion and hair) from an individual who was suspected of having leprosy. Although no conclusive diagnosis could be made by traditional diagnostic methods, the individual was found to be infected with M. leprae after amplification of the bacterial DNA.

Evaluation of PCR mediated DNA amplification in non-invasive biological specimens for subclinical detection of Mycobacterium leprae.

DNA from Mycobacterium leprae, present in non-invasive clinical samples from leprosy patients, such as nasal secretion and hair bulbs, was submitted to amplification by the polymerase chain reaction using a M. leprae-specific repetitive sequence as a target. After optimization of sample processing and of the PCR conditions, we were able to detect DNA from M. leprae in both types of clinical samples, even from paucibacillary leprosy patients. The use of hair bulbs and nasal secretion as clinical samples for screening of household contacts and for the evaluation of a risk population, or for the follow-up of patients under chemotherapy, and monitoring of bacterial load is discussed.

Use of PCR-restriction fragment length polymorphism analysis of the hsp65 gene for rapid identification of mycobacteria in Brazil.

Polymerase chain reaction amplification of part of the gene coding for the heat shock protein hsp65 followed by restriction enzyme analysis (PRA) is a recently described tool for rapid identification of mycobacteria. In this study, the speed and simplicity of PRA for identification of isolates of mycobacteria from patients with clinical symptoms of tuberculosis was evaluated and compared with identification results obtained by commercially available methods. Established PRA patterns were observed for nineteen isolates of Mycobacterium tuberculosis, eleven belonging to the complex M. avium-intracellulare, four of M. kansasii, one of M. fortuitum, one of M. abscessus, three of M. gordonae and one of the recently described species M. lentiflavum, as identified by commercially available methods. Two isolates of M. fortuitum and one of M. gordonae had unique and so far undescribed PRA patterns, suggesting geographically-related intra-species variation within the hsp65 sequence. We propose the inclusion of these new patterns in the PRA identification algorithm and have defined more accurately the molecular weight values of the restriction fragments. This is the first report on the isolation of M. lentiflavum in Brazil suggesting that identification by means of PRA could be useful for detection of mycobacterial species that are usually unnoticed. Where the use of several commercial techniques in combination was necessary for correct identification, PRA demonstrated to be a simple technique with good cost-benefit for characterization of all mycobacterial isolates in this study.

Pathogenesis of nerve damage in leprosy: genetic polymorphism regulates the production of TNF alpha.

Studies carried out over the last decade have strongly suggested that TNF alpha both overtly participates in the cell-mediated immune response against Mycobacterium leprae, and is overproduced during reaction. In addition, reactions are intimately related to the onset of nerve damage. Finally, TNF alpha has been implicated in the pathogenesis of many human and experimental autoimmune peripheral neuropathies that, as in leprosy, result in demyelination and axonal lesions. Because of recent findings associating human TNF alpha mutant alleles at the -308 position with increased production of TNF alpha in many immunological and infectious diseases, an investigation of the role of TNF2 in predisposing leprosy patients to reaction has been undertaken. Analysis of 300 patients with leprosy--210 multibacillary and 90 paucibacillary--has shown that the percentage of reactional patients was similar among both carriers and non-carriers of the TNF2 allele. However, a separate analysis of 57 carriers of TNF2 found that reactions occurred much more frequently among heterozygous than among homozygous patients. Moreover, the frequency of neuritis was somewhat greater among the heterozygous patients than among the non-carriers. Enhanced serum levels of TNF alpha have been noted in both TNF-1 and TNF-2 mutant patients in the course of leprosy reaction. Our observations to date suggest that other factors not related to the presence of the mutant gene may lead to the TNF alpha hyper-responsiveness observed during reaction.

Evaluation of chemiluminescence, procoagulant activity and antigen presentation by monocytes from lepromatous leprosy patients with or without reactional episodes.

In this study, we evaluated the activity of peripheral blood mononuclear cells (PBMC), isolated from treated and untreated lepromatous leprosy patients, from lepromatous leprosy patients during and after reactional episodes (erythema nodosum leprosum (ENL) and reversal reaction (RR)), and from normal healthy individuals. We determined reactive oxygen intermediate (ROI) production, procoagulant activity (PCA) and HLA-DR antigen expression of monocytes, besides lymphoproliferation, both in the presence and absence of various stimulatory agents. Phorbol myristate acetate (PMA) stimulated ROI production by monocytes from all the groups studied, with patients during reactional episodes (ENL and RR) showing a significantly higher response (p < 0.009 and p < 0.00001). Irradiated Mycobacterium leprae, although having little effect when added alone, strongly suppressed PMA-stimulated ROI production. Muramyl dipeptide (MDP) had no influence on either basal or on PMA-induced ROI production. Basal monocyte PCA, as well as M. leprae or concanavalin A (ConA)-induced monocyte PCA was comparable in monocytes from all the groups studied. ConA was able to induce mitogenic activity in mononuclear cells isolated from all the groups studied. M. leprae, although stimulatory for normal individuals, did not induce lymphoproliferation in lepromatous leprosy patients, except for cells from patients during RR, which responded equally to M. leprae and to ConA. The absence of M. leprae-induced lymphoproliferation in lepromatous leprosy patients is not caused by the lack of basal HLA-DR expression, as PBMC from all individuals studied showed the same level of this antigen. Our results suggest an increase of spontaneous or PMA-induced monocyte activity, as detected by ROI production, during the reactional episode; addition of M. leprae suppressed this response. The increase in monocyte activity could be correlated with the increase of lymphoproliferation response to M. leprae during RR, but not during ENL. The importance of a possible immune suppressive action of M. leprae is discussed.

DNA fingerprinting of Mycobacterium tuberculosis from patients with and without AIDS in Rio de Janeiro.

Isolates of Mycobacterium tuberculosis derived from patients with AIDS from a single hospital in Rio de Janeiro were typed using a standardized RFLP technique detecting IS6110 polymorphism. Nineteen isolates were obtained from 15 different patients. Eleven distinct IS6110 patterns were found, with 4 banding patterns shared by 2 patients. The clustering value of 53% was much higher in comparison with clustering of M. tuberculosis strains from TB patients without clinical signs for HIV infection from randomly selected health centers. We present these results as preliminary data on M. tuberculosis strain polymorphism in Brazil and on the higher risk for recent transmission amongst patients with AIDS.

PCR-based identification of Mycobacterium murphy causing Canine Leproid Granuloma Syndrome in Niterói, southeast Brazil - case report / Identificação baseada em PCR de Mycobacterium murphy causando Síndrome do Granuloma Lepróide Canino em Niterói, sudeste do Brasil ˗ Relato de Caso

Canine Leproid Granuloma Syndrome (CLGS), also known as canine leprosy, is a cutaneous nodular infectious disease caused by Mycobacterium sp.. Despite being reported worldwide, it is still quite unknown and underdiagnosed. Diagnosis may be achieved by cytopathology or histopathology of skin lesions, but identification of the infectious agent is complex, since bacterial in vitro growth is not possible, relying upon molecular techniques such as PCR to confirm Mycobacterium DNA in the sample. We report a CLGS case in Niteroi, Rio de Janeiro state, Brazil, diagnosed by cytopathology and submitted to molecular identification of the agent. PCR amplification of hsp65 gene was performed and revealed 100% genetic homology to M. murphy strain. This is the first CLGS report with molecular identification in Rio de Janeiro state, and this finding should raise awareness about CLGS as a differential diagnosis among granulomatous skin diseases in this region.(AU)

A síndrome de granuloma leproide canino (SGLC), também conhecida como lepra canina, é uma doença infecciosa cutânea nodular causada por Mycobacterium sp. Apesar de ser relatada mundialmente, ainda é bastante desconhecida e subdiagnosticada. O diagnóstico pode ser conseguido por citopatologia ou histopatologia de lesões cutâneas, mas a identificação do agente infeccioso é complexa, uma vez que o crescimento in vitro bacteriano não é possível, dependendo de técnicas moleculares como a PCR para confirmar o DNA de Mycobacterium na amostra. Relatou-se um caso da SGLC em Niterói, estado do Rio de Janeiro, Brasil, diagnosticado por citopatologia e submetido à identificação molecular do agente. Foi realizada amplificação por PCR do gene hsp65, que revelou 100% de homologia genética com a cepa M. murphy. Este é o primeiro relato da SGLC com identificação molecular no estado do Rio de Janeiro, o que mostra a importância de se acrescentar a SGLC ao diagnóstico diferencial das doenças granulomatosas de pele nessa região.(AU)

Understanding the relationship between Mycobacterium bovis spoligotypes from cattle in Latin American countries.

Spoligotyping is the most frequently used method for genotyping isolates of Mycobacterium bovis worldwide. In the current work, we compared spoligotypes from 1684 M. bovis isolates from Argentina (816), Brazil (412), Chile (66), Mexico (274) and Venezuela (116), obtained from cattle, humans, pigs, wild boars, farmed deer, goats, buffaloes, cats, and wild animals. A total of 269 different spoligotypes were found: 142 (8.4%) isolates presented orphan spoligotypes, whereas 1542 (91.6%) formed 113 different clusters. In cattle, SB0140 was the most representative spoligotype with 355 (24.6%) isolates, followed by SB0121 with 149 (10.3%) isolates. Clustering of spoligotypes ranged from 95.2% in Argentina to 85.3% in Mexico. Orphan spoligotypes were also variable, ranging from 23.7% in Mexico to 4.1% in Brazil. A large proportion of spoligotypes were common to the neighboring countries Argentina, Brazil and Chile. In conclusion, despite the diversity of spoligotypes found in the five countries studied, there are major patterns that predominate in these neighboring countries. These clusters may reflect a long-lasting active transmission of bovine tuberculosis or common historical origins of infection.

The changing face of the epidemiology of tuberculosis due to molecular strain typing--a review.

About one third of the world population is infected with tubercle bacilli, causing eight million new cases of tuberculosis (TB) and three million deaths each year. After years of lack of interest in the disease, World Health Organization recently declared TB a global emergency and it is clear that there is need for more efficient national TB programs and newly defined research priorities. A more complete epidemiology of tuberculosis will lead to a better identification of index cases and to a more efficient treatment of the disease. Recently, new molecular tools became available for the identification of strains of Mycobacterium tuberculosis (M. tuberculosis), allowing a better recognition of transmission routes of defined strains. Both a standardized restriction-fragment-length-polymorphism-based methodology for epidemiological studies on a large scale and deoxyribonucleic acids (DNA) amplification-based methods that allow rapid detection of outbreaks with multidrug-resistant (MDR) strains, often characterized by high mortality rates, have been developed. This review comments on the existing methods of DNA-based recognition of M. tuberculosis strains and their peculiarities. It also summarizes literature data on the application of molecular fingerprinting for detection of outbreaks of M. tuberculosis, for identification of index cases, for study of interaction between TB and infection with the human immuno-deficiency virus, for analysis of the behavior of MDR strains, for a better understanding of risk factors for transmission of TB within communities and for population-based studies of TB transmission within and between countries.

In vitro tumor necrosis factor production by mononuclear cells from lepromatous leprosy patients and from patients with erythema nodosum leprosum.

The production of tumor necrosis factor (TNF) by Mycobacterium leprae-stimulated phagocyte cells, isolated from lepromatous leprosy patients (LL) and normal individuals, was evaluated, using the highly TNF-sensitive mouse fibrosarcoma cell line WEHI164cl13. Mononuclear cells, isolated from all individuals studied, showed a low level of spontaneous TNF production, except for patients undergoing erythema nodosum leprosum (ENL), in which we found significantly higher levels of TNF. Addition of M. leprae to the phagocyte cell culture enhanced TNF production in all groups studied, except in the group with untreated leprosy patients. Strongest M. leprae-induced TNF release was found in mononuclear cell cultures derived from ENL patients. Patients in the postreactional state showed significantly higher TNF levels than healthy controls. These findings support the idea that TNF plays a key role in the complex symptomatology of ENL.

Molecular epidemiology of methicillin resistant Staphylococcus aureus isolated from newborns in a hospital in Rio de Janeiro, Brazil.

Methicillin resistant Staphylococcus aureus (MRSA) is an organism that is frequently transmitted in hospitals and perinatal units. The MRSA is considered a public health problem in neonatology because of its strong potential for dissemination in the wards associated with high rates of morbidity and mortality. In this study we describe the bacteriological, epidemiological and molecular characteristics of MRSA isolated from anterior nares and blood cultures of newborns hospitalized in a public maternity hospital in the city of Rio de Janeiro, Brazil. The frequency of MRSA isolated from nasal swabs of newborns was 47.8% (43/90). The genetic analysis of MRSA strains from anterior nares, showed 8 different pulsed field gel electrophoresis patterns (PFGE). Upon analysis of PFGE patterns of the 12 MRSA strains isolated from blood cultures, 8 different patterns were observed, 9 (75%) strains were genetic related to nasal secretion isolates patterns. In conclusion, our data demonstrate the importance of screening of newborns for the presence of MRSA in Brazilian hospitals and the usefulness of genetic typing of these pathogen during epidemiologic studies. This should lead to a better knowledge on the significancy and spreading of MRSA in the hospitals.

Detection of Mycobacterium leprae DNA by polymerase chain reaction in the blood of individuals, eight years after completion of anti-leprosy therapy.

Thirty eight patients with indeterminate leprosy (HI), at least 4 to 6 years after discharge from multibacillary (MB) or paucibacillary (PB) schemes of anti leprosy multidrug therapy (MDT), were submitted to traditional diagnostic procedures for leprosy and to polymerase chain reaction (PCR) analysis of different clinical samples for detection of Mycobacterium leprae DNA. No significant difference was observed for any of the parameters analyzed between PB or MB schemes of treatment and no indications were found for more efficient outcome of HI using the MB scheme. Remarkably, 18 (54.5%) of the individuals were PCR positive in at least one of the samples: positivity of PCR was highest in blood samples and four individuals were PCR positive in blood and some other sample. Upon comparison of PCR results with clinical and histopathological parameters, no correlation was found between PCR-positivity and eventual relapse. This is the first report on detection of M. leprae DNA in PB patients, more than half a decade after completion of MDT, suggesting that live bacilli are present and circulating much longer than expected, although reinfection of the individuals can not be excluded. Overall, we feel that because of the high sensitivity of the assay, extreme care should be taken about association of PCR results, efficacy of treatment and disease status.

Leprosy and AIDS: two cases of increasing inflammatory reactions at the start of highly active antiretroviral therapy.

Reported here are the cases of two HIV-positive patients with skin lesions suggestive of leprosy, based on clinical and pathological analysis, which worsened during the few weeks following initiation of highly active antiretroviral therapy. The lesions improved after a few weeks of multidrug therapy for leprosy. Mycobacterium leprae was confirmed by polymerase chain reaction analysis of blood in case 1 and of a biopsy sample in case 2. Neither Mycobacterium avium complex nucleic acid, which is usually associated with immune restoration syndrome, nor mycobacterial cutaneous manifestations were detected in either case.

Use of PCR for the determination of the frequency of the delta F508 mutation in Brazilian cystic fibrosis patients.

The delta F508 mutation in the cystic fibrosis (CF) gene was studied in a population of 18 Brazilian CF patients and their 17 families by use of PCR and differential hybridization with oligonucleotides. In a total of 34 chromosomes considered, 12 (35%) carried the F508 deletion, a frequency much lower than that reported in most other populations. As a consequence, CF in Brazil would be predominantly caused by mutations different from the F508 deletion.

Novel allelic variants of Mycobacteria isolated in Brazil as determined by PCR-restriction enzyme analysis of hsp65.

Human isolates of Mycobacterium collected in 16 different states of Brazil were submitted to PCR-restriction analysis (PRA) of a 439-bp fragment of the hsp65 gene with HaeIII and BstEII. Fourteen allelic variants not described in clinical isolates so far were observed among 36 (10%) of 356 Brazilian strains, including a new pattern for Mycobacterium scrofulaceum, M. intracellulare, and M. flavescens, two new patterns for M. fortuitum, three new patterns each for M. gordonae and M. terrae, and one new pattern for M. avium complex-like strains. Two unidentified strains each also presented a new pattern, strongly suggesting that Mycobacterium genotypes are distributed biogeographically. The PRA procedure was also performed with 43 reference isolates belonging to 34 species, adding a further six new patterns to the identification algorithm. A database containing the normalized restriction patterns of both enzymes was constructed. Patterns available on the Internet can be introduced into this database, which will make possible the comparison of genotypes from isolates from different parts of the world.