Daniela Bonanni

Immunogenicity and protective efficacy of tuberculosis subunit vaccines expressing PPE44 (Rv2770c)

In this study we have evaluated the vaccine potential of a Mycobacterium tuberculosis antigen of the PPE protein family, namely PPE44 (Rv2770c). PPE44-specific immune responses could be detected in mice acutely, chronically and latently infected with M. tuberculosis. Vaccination of mice with a plasmid DNA vaccine coding for PPE44 or recombinant PPE44 protein formulated in adjuvant generated strong cellular and humoral immune responses; immunodominant T cell epitopes were identified. Most importantly, vaccination of mice with both subunit vaccines followed by an intratracheal challenge with M. tuberculosis resulted in a protective efficacy comparable to the one afforded by BCG. Taken together these results indicate that PPE44 of M. tuberculosis is a protective antigen that could be included in novel subunit TB vaccines and that warrants further analysis.

Genetic Diversity, Determined on the Basis of katG463 and gyrA95 Polymorphisms, Spoligotyping, and IS6110 Typing, of Mycobacterium tuberculosis Complex Isolates from Italy

ABSTRACT Mycobacterium tuberculosis complex isolates (n = 248) collected during a 1-year period in Tuscany, Italy, were genotyped for the katG463 and gyrA95 polymorphisms and by standard spacer oligonucleotide typing (spoligotyping) and IS6110 restriction fragment length polymorphism (RFLP) assays. Most of the isolates (n = 212; 85.5%) belonged to genotypic groups 2 and 3, which included most isolates from Italian-born patients. The remaining isolates were genotypic group 1 organisms, which were prevalent among foreign-born patients (29 of 36; 80.6%). Spoligotype analysis detected 116 unique patterns and 34 clusters including 166 isolates. The combination of spoligotyping and IS6110 RFLP analyses yielded 28 distinct clusters including 65 identical isolates (26.2%)—22 clusters with 2 isolates, 4 clusters with 3 isolates, 1 cluster with 4 isolates, and 1 cluster with 5 isolates—thus proving a low transmission rate in the community. Predominant spoligotypes representing 50% of clustered isolates were found in six clusters that included widespread type ST53 (clade T1) with 29 isolates (11.7% of total isolates); types ST50 and ST47 (Haarlem family) with 18 isolates (7.3%) and 8 isolates (3.2%), respectively; type ST42 (Latino-American and Mediterranean clade) with 13 isolates (5.2%); new type ST1737 (named “Tuscany”) with 8 isolates (3.2%); and type ST1 (W-Beijing family) with 7 isolates (2.8%). Other spoligotype families, such as the Mycobacterium africanum, East African-Indian (EAI2/Manila), and central Asia 1 (CAS1/Delhi) families (all including organisms of genotypic group 1) and the Cameroun family (genotypic group 2), were detected especially among immigrant patients. The occurrence of genotypes originally found in distant geographic areas with a high prevalence of tuberculosis may represent a hallmark for changes in the dynamics of transmission of tuberculosis in the region in the near future.

Involvement of the fadD33 gene in the growth of Mycobacterium tuberculosis in the liver of BALB/c mice

The potential pathogenic role of Mycobacterium tuberculosis H37Rv fadD33, a gene encoding an acyl-CoA synthase that is underexpressed in the attenuated strain H37Ra, was investigated. In a first approach, fadD33 was cloned and expressed in strain H37Ra to restore gene expression and fadD33-complemented bacteria were used to investigate whether fadD33 might confer any growth advantage to M. tuberculosis H37Ra in an infection model of BALB/c mice. No differences were found in the growth rates of M. tuberculosis H37Rv, H37Ra and fadD33-complemented H37Ra in the lungs and spleen. In contrast, in the liver, where the attenuated strain H37Ra showed impaired growth compared to the virulent strain H37Rv, complementation of the attenuated strain H37Ra with fadD33 restored bacterial replication. In a further approach, the fadD33 gene of strain H37Rv was disrupted by allelic exchange mutagenesis and the virulence of the mutant strain was tested by mouse infection. It was found that disruption of fadD33 decreased M. tuberculosis H37Rv growth in the liver, but not in the lungs or spleen, and complementation of the fadD33-disrupted mutant with fadD33 restored bacterial replication in the liver, but did not affect replication in the lungs and spleen. These findings suggest that fadD33 plays a role in M. tuberculosis virulence by supporting bacterial growth in the liver.

Molecular analysis of clinical isolates of Mycobacterium bovis recovered from humans in Italy

ABSTRACT In order to achieve a better knowledge of Mycobacterium bovis epidemiology in Italy, 42 clinical isolates from humans were genotyped. Predominant molecular patterns were found in one cluster of 15 isolates sharing spoligotype (ST482), variable-number tandem repeat (VNTR), and IS6110-based restriction fragment length polymorphism (one 1.9-kb band) profiles and in two clusters of 6 and 3 Mycobacterium bovis BCG isolates differing by one VNTR character. The remaining 18 isolates yielded unique profiles. Our results confirm the potential utility of spoligotyping and VNTR typing as a major typing system of M. bovis isolates.

A real-time PCR assay for detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates.

A real-time PCR genotypic assay was developed for the detection of isoniazid (INH) resistance in Mycobacterium tuberculosis. The assay detects mutations C(-15)T and, possibly, G(-24)T in the regulatory region of the inhA gene and proved as sensitive and specific as nucleotide sequencing in all the clinical isolates tested. Our assays mapped the mutations efficiently in 10 out of 35 resistant isolates, thereby covering 29% of all resistant strains.

Beijing/W Mycobacterium tuberculosis in Italy.

To the Editor: Molecular typing of Mycobacterium tuberculosis strains isolated in several countries in recent years has shown that a group of strains known as “Beijing” is widespread around the world (1). The Beijing group of M. tuberculosis has been associated with drug resistance; one multidrug-resistant strain, designated “W,” was found in New York City in the early 1990s and caused large institutional outbreaks of tuberculosis (TB) in the United States (2). M. tuberculosis strains of Beijing/W genotype are mostly prevalent in Asia (1), but recent data suggest that they have been spreading in Indochina and are prevalent among younger persons in Vietnam (3). Beijing/W strains are also widespread in Eastern Europe (1); during the last decade, the Beijing/W genotype of M. tuberculosis, with more prevalent drug-resistant mutations than non-Beijing strains, has been identified in 40% to 50% of clinical isolates studied in Russia (4). We studied a total of 245 M. tuberculosis strains collected during a 1-year period, from January to December 2002, from the same number of TB patients hospitalized in Tuscany, Italy. All the isolates were typed by the standardized IS6110 restriction fragment length polymorphism (RFLP) and the spoligotyping (spacer oligonucleotide typing) techniques. A total of 216 distinct IS6110 RFLP patterns were found among the 245 isolates; 51 isolates (20.8%) occurred in 23 clusters, each constituting strains with an identical IS6110 RFLP and spoligotype pattern; 19 clusters contained two isolates each, 3 contained three isolates, and 1 contained four isolates. Spoligotype analysis showed seven isolates with the typical Beijing/W pattern of probe hybridization only to spacer sequences 35–43. The Beijing/W isolates yielded distinct IS6110 RFLP profiles with similarity coefficient >57.8%. Characteristics of the Beijing/W strains and respective patients, obtained from clinical records, are reported in the Table. Although the overall prevalence of Beijing/W strains was low (7/245, 2.9%), five of the seven strains were from recent immigrants to Italy from China who live in the same area; the other two strains were from Italian citizens also living in that area. Recent immigration from high-prevalence areas is therefore likely to be associated with the occurrence of the Beijing/W genotype in Italy. None of the Beijing/W strains was associated with TB outbreaks; nonetheless, infection of Italian residents with Beijing strains suggests that spread of this genotype is ongoing. Table Characteristics of Mycobacterium tuberculosis strains of Beijing/W genotype isolated in 2002 in Tuscany, Italya Beijing/W strains have been strongly associated with drug resistance in a number of countries (2,4–6), but elsewhere the association was weak or absent. In our survey, no substantial drug resistance was observed; all Beijing/W strains isolated in Tuscany were susceptible to rifampin, ethambutol, pirazinamide, and streptomycin (tested only in two strains), and all but one were susceptible to isoniazid. Although we detected only a few cases, our data do not show a trend of Beijing/W strains’ being associated with infection in young people, as has been observed in other settings (3). The age of immigrants with Beijing/W TB (mean 33.2 years, standard deviation [SD] 8.2 years) did not significantly differ from that of immigrants infected with non-Beijing/W strains (30.7 years, SD 7.4 years), a find that indicates that, at least in our setting, immigrant status, rather than M. tuberculosis genotype, is associated with infection in young people. The few cases of Beijing/W infections in Italian-born patients do not allow us to draw conclusions regarding nonimmigrant patients. In conclusion, M. tuberculosis strains of Beijing/W genotype are becoming widespread worldwide, including in countries with a low prevalence of TB. Their association with drug resistance and infection in young people, clearly shown in certain settings, remains to be defined. Further molecular epidemiologic surveillance is needed to monitor trends in prevalence and spread of these strains.

Most Human Isolates of Mycobacterium avium Mav-A and Mav-B Are Strong Producers of Hemolysin, a Putative Virulence Factor

ABSTRACT Hemolysin was quantified in 58 isolates of Mycobacterium avium from human, animal, and environmental sources. Human Mav-A and Mav-B isolates were the strongest producers; in contrast, animal and environmental Mav-A isolates and human, animal, and environmental Mav-C organisms were low-level producers. Hemolysin production was not restricted to isolates causing invasive infections.