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Mutations of rpoB associated with rifampin resistance were studied in 37 multidrug-resistant (MDR) clinical strains of Mycobacterium tuberculosis isolated in Italy. At least one mutated codon was found in each MDR strain. It was always a single-base substitution leading to an amino acid change. Nine different rpoB alleles, three of which had not been reported before, were found. The relative frequencies of specific mutations in this sample were different from those previously reported from different geographical areas, since 22 strains (59.5%) carried the mutated codon TTG in position 531 (Ser→Leu) and 11 (29.7%) had GAC in position 526 (His→Asp).

The in vitro activity of 16 antimicrobial agents against 46 drug-resistant strains of Mycobacterium tuberculosis recently isolated from Italian patients was determined. As for first-line antituberculosis drugs, while isoniazid was ineffective against all the strains tested, resistance to streptomycin, rifampicin, pyrazinamide, and ethambutol was 80.4%, 71.7%, 39.1%, and 8.7%, respectively. Among second-line antituberculous drugs, resistance to ciprofloxacin, ofloxacin, and sparfloxacin and to amikacin and kanamycin was around 20%. About 10% of the strains were resistant to capreomycin and cycloserine and 4.3% were resistant to ethionamide; no strain was found to be resistant to thiacetazone, para-aminosalicylic acid, and viomycin. Although all strains displayed a rather continuous distribution of minimal inhibitory concentrations (MICs), a bimodal distribution was observed for rifampicin, amikacin, and kanamicin, with very high MIC values for resistant strains; relatively low MICs were found for fluoroquinolone-resistant strains. Among the small number of strains resistant to second-line agents, low resistant levels were observed. Restriction fragment length polymorphism analysis showed few strain clusters with resistance to first-line antituberculous drugs and aminoglycosides, fluoroquinolones, or both. Altogether, these results showed that second-line agents were still active against the isoniazid-resistant and multiply first-line resistant strains tested, with none or low resistance levels; these observations can be of importance for the treatment of multidrug-resistant tuberculosis in Italy.

The bacterial growth and the production of tumor necrosis factor alpha (TNF-α) and TNF receptors (TNF-Rs) in the spleen and blood of BALB/c mice challenged with Mycobacterium avium complex (MAC) were monitored. Infection developed in two phases: the first, up to day 21, was associated with rapid MAC multiplication in the spleen and a drop in the mycobacteremia, and the second was associated with control of the infection in both compartments. In the spleen, TNF-α and TNF-RII mRNA levels peaked on day 21 and then slowly decreased; however, no increase in the level of TNF-RI mRNA was observed throughout these experiments. The level of circulating soluble TNF-RII (sTNF-RII) was transiently increased after day 21. In a model in which overproduction of bioactive TNF-α was triggered in response to a second infection with MAC, an increased production of sTNF-RII by cultured splenocytes was also observed. Administration of an antagonist anti-TNF-RII monoclonal antibody (MAb 6G1) to infected mice inhibited the bacterial growth in the spleen, suggesting that the TNF-RII and/or sTNF-RII was functionally involved in the mechanisms that control the infection. Overall, these observations suggest that upregulation of TNF-RII or sTNF-RII contributes to modulation of the TNF-α antibacterial activity in MAC infections.