For an infecting bacterium the human body provides several potential ecological niches with both internally (e.g. host immunity) and externally (e.g. antibiotic use) imposed growth restrictions that are expected to drive adaptive evolution in the bacterium, including the development of antibiotic resistance. To determine the extent and pattern of heterogeneity generated in a bacterial population during long-term antibiotic treatment, we examined in a monoclonal Mycobacterium tuberculosis infection antibiotic resistant mutants isolated from one patient during a 9-years period. There was a progressive accumulation of resistance mutations in the infecting clone. Furthermore, apparent clonal sweeps as well as co-existence of different resistant mutants were observed during this time, demonstrating that during treatment there is a high degree of dynamics in the bacterial population. These findings have important implications for diagnostics and treatment of drug resistant tuberculosis infections.

We present a novel denaturing gradient gel electrophoresis (DGGE) method which characterizes multiclonal communities of Staphylococcus aureus. The spa PCR-based DGGE method simultaneously separates strains that differ in only one base, thereby revealing multiclonal colonization and infections.Staphylococcus aureus causes a wide range of infections and is responsible for a considerable portion of hospital-acquired infections.In 30% to 70% of healthy individuals, S. aureus is a transient or persisting part of the residential flora (5, 10).Cespedes et al. investigated the frequency of simultaneous nasal carriage of multiple S. aureus strains by picking three bacterial colonies from plates derived from each colonized individual. Fewer than 7% of them were predicted to carry Ͼ1 strain (1). The simultaneous presence of an invasive and a carrier strain of methicillin-resistant S. aureus (MRSA) in one individual was reported by Soderquist and Berglund (12). The issue of multiclonal colonization is important, but conventional laboratory methods for detection of S. aureus are based on culture of a single colony. This might result in the identification of an antibiotic-susceptible commensal strain rather than a second, more resistant strain, which may impair the antibiotic treatment and bias epidemiological conclusions.We have developed a species-specific denaturing gradient gel electrophoresis (DGGE) method for S. aureus, utilizing spa, to characterize multiclonal colonization and infection. The novel assay was used to investigate a MRSA outbreak, revealing colonization with two different strains in some of the individuals.spa typing has been documented to be a useful tool in investigations of MRSA epidemiology (7) and for studies of S. aureus transmission (6). Thus, we based our DGGE method on spa and primer pairs, described by Kahl et al. (4), which were modified for DGGE analyses by the attachment of a GC clamp (11) at either the forward or the reverse primer. Annealing temperature and MgCl 2 concentrations for two primer combinations were optimized for PCR specificity and efficiency (data not shown).Eight S. aureus isolates of known spa types were acquired from the