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Journal of Clinical Microbiology, January 2004, p. 212-219, Vol. 42, No. 1
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.1.212-219.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

IS6110 Mediates Increased Transcription of the phoP Virulence Gene in a Multidrug-Resistant Clinical Isolate Responsible for Tuberculosis Outbreaks

Carlos Y. Soto,¹ M. Carmen Menéndez,^2, Esther Pérez,^1, Sofía Samper,³ Ana B. Gómez,¹ María J. García,² and Carlos Martín^1*

Grupo de Genética de Micobacterias, Departamento de Microbiología Medicina Preventiva y Salud Pública, Universidad de Zaragoza,¹ Servicio de Microbiología Hospital Miguel Servet, Zaragoza,³ Departamento de Medicina Preventiva, Universidad Autónoma de Madrid, Madrid, Spain²

Received 13 May 2003/ Returned for modification 17 July 2003/ Accepted 30 September 2003

Drug resistance in Mycobacterium tuberculosis complex strains is solely due to chromosomal mutations that could affect bacterial virulence. Molecular epidemiology studies have shown that resistant strains are less likely to be clustered than susceptible strains. However, a few multidrug-resistant (MDR) M. tuberculosis complex strains have been described as causing outbreaks, suggesting that they have restored virulence or increased transmission. One of the biggest MDR tuberculosis outbreaks documented to date was caused by the B strain of M. bovis. Restriction fragment length polymorphism fingerprinting revealed that the B strain contains two copies of IS6110. Here, we mapped and sequenced the regions flanking the two copies of IS6110 in the B strain. Ligation-mediated PCR showed that one of these IS6110 copies is located within the promoter region of phoP, a transcriptional regulator that is essential for M. tuberculosis virulence. We used PCR to screen 219 MDR M. tuberculosis complex strains (90.4% of all MDR isolates) isolated in Spain between 1998 and 2002 and found that the B strain was the only strain that contained a copy of IS6110 in the phoP promoter. To determine whether IS6110 affects phoP promoter activity in the B strain, we individually cloned the phoP gene and its promoter region (including IS6110 from the B strain and the equivalent region from M. tuberculosis without IS6110 as a control) into a mycobacterial replicative plasmid and transformed M. smegmatis with the resulting plasmid. Primer extension analysis showed that phoP transcription was strongly upregulated when the promoter region contained IS6110, as in the case of the B strain.

Present address: Division of Mycobacterial Research, NIMR, London NW7 1AA, United Kingdom.

Present address: Institut de Pharmacologie et Biologie Structurale, CNRS, 31077 Toulouse cedex, France.

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