Undetected multidrug-resistant tuberculosis amplified by first-line therapy in mixed infection

Abstract

Infections with >1 Mycobacterium tuberculosis strain(s) are underrecognized. We show, in vitro and in vivo, how first-line treatment conferred a competitive growth advantage to amplify a multidrug-resistant M. tuberculosis strain in a patient with mixed infection. Diagnostic techniques that identify mixed tubercle bacilli populations are needed to curb the spread of multidrug resistance.

Keywords: Mycobacterium tuberculosis; TB; antimicrobial resistance; bacteria; co-infection; drug sensitive; multidrug resistant; treatment resistance; tuberculosis and other mycobacteria.

Figure 1

Mycobacterium tuberculosis co-culture competition experiment in a study of the amplification of multidrug resistance induced by first-line treatment of a mixed M. tuberculosis infection. The results suggest competitive advantages in vitro, which may account for patient strain phenotype in vivo. S, drug sensitive; R, drug resistant; OADC, oleic acid, albumin, dextrose, catalase growth supplement; OD₆₀₀, optical density read at 600 nm; −INH, without isoniazid; +INH, with INH; MIRU-VNTR, mycobacterial interspersed repetitive unit–variable number tandem repeat; CFU, colony-forming units.

Figure 2

Analyses of the amplification of multidrug-resistant Mycobacterium tuberculosis during treatment of a drug-sensitive (S) strain in a mixed infection (i.e., infection with drug-resistant [R] and S strains). In the presence of isoniazid (INH), the faster growing S strain lost its competitive advantage, and the R strain became more prevalent. A–C) Data are means of 3 independent replicates with SE bars. A) Single strain growth analyses of S (black circles) and R (gray triangles) M. tuberculosis strains. Growth was measured by optical density at 600 nm (OD₆₀₀). *p<0.05. B) Competitive growth analyses of mixed strains alone (black squares) and with 0.2 μg/mL INH (gray triangles). Growth, in triplicate, in 7H9 broth plus OADC (oleic acid, albumin, dextrose, and catalase growth supplement), glycerol, and Tween 80 was measured by optical density at OD₆₀₀. Statistical analyses were performed on triplicate samples by using 2-way analysis of variance. *p<0.05. C) Identification of the predominant strain in mixed cultures with and without 0.2 μg/mL INH (INH/plate+ and INH/plate−, respectively). Strains were identified on day 7 by plating a 10-fold dilution series of co-cultures onto 7H10 agar, plus OADC and glycerol, with or without 0.2 μg/mL INH (0/plate+ and 0/plate−, respectively) and incubating for 2 weeks at 37°C. Statistical analyses were conducted on triplicate samples by using a 2-tailed t-test. *p<0.02. CFU, colony-forming units.