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Background: Mycobacterium avium subsp. hominissuis (MAH) is an environmental opportunistic pathogen for humans and swine worldwide; in humans, the vast majority of MAH infections is due to strains belonging to specific genotypes, such as the internal transcribed spacer (ITS)-sequevars Mav-A and Mav-B that mostly cause pulmonary infections in elderly patients and severe disseminated infections in acquired immunodeficiency syndrome patients, respectively. To test whether the different types of infections in distinct patients' populations might reflect a different virulence of the infecting genotypes, MAH human isolates, genotyped by ITS sequencing and MIRU-VNTR minisatellite analysis, were studied for the capacity to infect and replicate in human macrophages in vitro. Methods: Cultures of human peripheral blood mononuclear cells and phagocytic human leukemic cell line THP-1 cells were infected with each MAH isolate and intracellular colony-forming units (CFU) were determined. Results: At 2 h after infection, i.e., immediately after cell entry, the numbers of intracellular bacteria did not differ between Mav-A and Mav-B organisms in both phagocytic cell types. At 5 days, Mav-A organisms, sharing highly related VNTR-MIRU genotypes, yielded numbers of intracellular CFUs significantly higher than Mav-B organisms in both phagocytic cell types. MIRU-VNTR-based minimum spanning tree analysis of the MAH isolates showed a divergent phylogenetic pathway of Mav-A and Mav-B organisms. Conclusion: Mav-A and Mav-B sequevars might have evolved different pathogenetic properties that might account for their association with different human infections. [ABSTRACT FROM AUTHOR]

Background: In Italy, the prevalence of non-tuberculous mycobacteria (NTM) in human infections is largely unknown. Herein, we report the epidemiology of NTM infections in a region of central Italy, Tuscany, over the last 11 years, and provide a review of the recent literature on NTM isolation rates in different geographic regions.Methods: The complete collection of NTM strains isolated from a total of 42,055 clinical specimens at the Laboratory of Clinical Mycobacteriology of Pisa University Hospital, Italy, from 1 January 2004 to 31 December 2014 was included.Results: In our setting, in the period 2004-2014 a total of 147 patients had cultures positive for NTM. The number of NTM isolates increased considerably from five isolates in 2004 to 29 in 2014; a sharp increase occurred in the last 3 years. Overall, 16 NTM species were isolated; the most common were M. avium, M. intracellulare and M. gordonae detected in respectively in 41.5, 14.3 and 11.6% of NTM patients. In general, NTM isolates were largely prevalent in people older than 60 (57.8%); patients aged 1-10 year-old almost exclusively yielded M. avium and M. intracellulare. Of the 147 NTM clinical isolates, 76.2% were from respiratory specimens, 10.9% from lymph nodes, 2.7% from blood (yielding exclusively M. avium), and the remaining 10.2% from other clinical specimens.Conclusions: The observed increase in NTM isolation rate in our setting is in keeping with the general increase in NTM infections reported worldwide in the past two decades, although the distribution of the NTM prevalent species differs by geographic region. [ABSTRACT FROM AUTHOR]

A sample of 260 Mycobacterium tuberculosis strains assigned to the Euro-American family was studied to identify phylogenetically informative genomic regions of difference (RD). Mutually exclusive deletions of regions RD115, RD122, RD174, RD182, RD183, RD193, RD219, RD726 and RD761 were found in 202 strains; the RDRio deletion was detected exclusively among the RD174-deleted strains. Although certain deletions were found more frequently in certain spoligotype families (i.e., deletion RD115 in T and LAM, RD174 in LAM, RD182 in Haarlem, RD219 in T and RD726 in the “Cameroon” family), the RD-defined sublineages did not specifically match with spoligotype-defined families, thus arguing against the use of spoligotyping for establishing exact phylogenetic relationships between strains. Notably, when tested for katG463/gyrA95 polymorphism, all the RD-defined sublineages belonged to Principal Genotypic Group (PGG) 2, except sublineage RD219 exclusively belonging to PGG3; the 58 Euro-American strains with no deletion were of either PGG2 or 3. A representative sample of 197 isolates was then analyzed by standard 15-locus MIRU-VNTR typing, a suitable approach to independently assess genetic relationships among the strains. Analysis of the MIRU-VNTR typing results by using a minimum spanning tree (MST) and a classical dendrogram showed groupings that were largely concordant with those obtained by RD-based analysis. Isolates of a given RD profile show, in addition to closely related MIRU-VNTR profiles, related spoligotype profiles that can serve as a basis for better spoligotype-based classification. [ABSTRACT FROM AUTHOR]

Background: The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th, 2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution. Methods: The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results. Results: Seven out of the 11 laboratories (63 %), perfectly typed more than 90% of isolates, 3 scored between 80- 90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus (Mostly spacers 15, 14, 18, 37, 39, 40). Conclusions: We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors occur randomly either in the process of re-using membranes, or during the reading of the results and transferring of data from the film to a digital file. Last, the performance of the microbead-based method was excellent as previously shown by Cowan et al. (J. Clin. Microbiol. 2004) and Zhang et al. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping. [ABSTRACT FROM AUTHOR]

PPE44 is a member of the Mycobacterium tuberculosis PPE proteins, a polymorphic family of 69 glycine-rich proteins that predictively represent a source of antigenic variation. The genetic diversity of gene ppe44 among clinical isolates has been studied. No genomic polymorphism of ppe44 was found by a PCR-restriction fragment length polymorphism assay using three restriction enzymes. Nucleotide sequencing of gene ppe44 of a number of isolates, selected to represent the major phylogenetic lineages of M. tuberculosis, showed no nucleotide substitution, with the exception of isolates of the Beijing genotype. These findings indicate that gene ppe44 is basically conserved among M. tuberculosis strains. The expression of gene ppe44 was then determined at the transcriptional level by a real-time reverse transcriptase PCR assay. Extremely high quantitative variations in ppe44 expression were found among the isolates; ppe44 expression of the Beijing strains was significantly higher than the non-Beijing strains. To test whether differential expression of gene ppe44 has the potential to provide a dynamic antigen display, antibodies to PPE44 were titered in the sera of M. tuberculosis-infected subjects. Variation of antibody response to PPE44 was found with regard to both antibody titers and the proportion of responding subjects. These results indicate that the differential expression of genes ppe could influence the host's immune responsiveness, thus having implications in the immunopathogenesis of tuberculosis. [ABSTRACT FROM AUTHOR]

An 8.3 kDa protein (SA5K), secreted by Mycobacterium tuberculosis/Mycobacterium bovis bacillus Calmette–Gue´rin (BCG) in culture filtrate, has been previously described in our laboratory. In the present study, analysis of the distribution of SA5K gene (Rv1174c) among M. tuberculosis strains, isolated from a wide variety of clinical specimens, revealed that the gene is present in all clinical isolates analyzed (29/29). SA5K expression inside human macrophages infected with BCG was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) on RNA extracted from bacterial cells following 24 and 48 h of infection. In addition, in order to evaluate whether SA5K gene was also expressed at the site of infection in the lung, a nested RT-PCR assay was developed to detect specific mRNA in sputum samples collected from smear positive tuberculosis patients. SA5K mRNA was detected in all the samples containing high numbers of tubercle bacilli demonstrating that the corresponding gene is expressed during the course of clinical infection. [Copyright &y& Elsevier]

DNA synthesis of human hepatoblastoma HepG2 cells is reversibly inhibited by butyrate. When butyrate is removed from the culture medium, cells re-enter the cell cycle, synthesizing DNA with a time lag of about 12 h. HepG2 cells, growth-inhibited for 30 h with butyrate, synthesize and accumulate a nuclear protein, called D. Protein D synthesis is inhibited in cells which, released from the butyrate block, have resumed DNA synthesis as well as in growing cells never exposed to butyrate. Protein D has been purified from growth-arrested cells and partially sequenced. The amino acid sequences of five internal trypsin peptides indicate that protein D is a novel nuclear protein. [ABSTRACT FROM AUTHOR]

The depression of contact sensitivity to oxazolone in mice infected with Pseudomonas aeruginosa was studied. In oxazolone-sensitized mice, P. aeruginosa infection affects cell proliferation in the lymph nodes draining the site of sensitization. This impaired cell proliferation does not seem to be due to an altered lymphocyte reactivity, since lymph node and spleen cells from infected animals show a normal mitotic responsiveness to both T and B cell mitogens. In addition, the draining lymph nodes and spleens of mice exhibiting a depressed response to oxazolone contain a cell population able actively to suppress the response to the same antigen of syngeneic recipients sensitized immediately before the cell transfer. These suppressor cells require antigenic stimulation and appear to act on the induction phase of contact sensitivity. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa infection depresses contact sensitivity to oxazolone in mice. To test whether an altered lymphocyte circulation plays a role in this depressionCr-labeled lymphocytes from P. aeruginosa-infected and oxazolone-sensitized donors were injected intravenously into infected and sensitized recipients, and the radioactivity uptake of several organs was determine. The controls consisted of normal mice receiving labeled lymphocytes from normal donors. While the radioactivity recovered from the liver, spleen, and mesenteric lymph nodes was similar in the test and the control group, significantly more radioactivity was recovered from the draining lymph nodes of infected and sensitized recipients. The concentration of labeled lymphocytes from sensitized donors in the draining lymph nodes of sensitized recipients was 18% greater than that of the controls but 31% lower than that of infected and sensitized animals receiving cells from infected and sensitized donors. P. aeruginosa infection enhances lymphocyte entrapment within the draining lymph nodes of oxazolone-sensitized mice. [ABSTRACT FROM AUTHOR]

A total of 60 Mycobacterium tuberculosis strains isolated in the area of Pisa, Italy, over a period from April 1993 to December 1995, were analyzed for the IS6110-based restriction fragments length polymorphism (RFLP). Isolates were found to show a great heterogeneity and only few isolates shared identical DNA banding patterns. In particular, 55 distinct IS6110 patterns were found (average number of isolates per pattern: 1.09) and only 9 strains (15%) occurred in 4 clusters of 2–3 identical clones. Computer analysis of genetic similarities among the strains revealed a family of 17 isolates including the clustered clones implicated in recently acquired infections. No correlation was found between the RFLP DNA patterns of the isolates and drug susceptibility. Of the 5 isolates from immigrants only one showed abnormal DNA fingerprinting. Our data indicate that the patterns of M. tuberculosis isolates in Pisa area are comparable to those of countries with low-prevalence TB and that a low level of TB transmission occurs in this area. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa depresses contact sensitivity to oxazolone in mice by activating B suppressor cells within the lymph nodes that drain the site of sensitization. These suppressor cells affect the induction phase of the immune response by blocking the cooperation between 'stimulator' macrophages and 'initiator' T cells on one hand and 'recruited' T cells on the other. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor about the molecular typing of Mycobacterium tuberculosis strains.