Why did Balaban et al. (2004) miss small colony variants?

Small colony variants (SCVs) constitute a naturally occurring, slow-growing subpopulation of bacteria that form small colonies (less than one-tenth of the size of parent colonies) on solid media (Proctor et al. 2006). SCVs have been reported in a wide range of bacterial genera and species. The major characteristics of SCVs include slow growth rate, formation of small colonies on agar and increased antibiotic resistance especially to aminoglycosides. Since the growth rate of SCVs is approximately nine times lower than the parent strains, they require a longer incubation time (48-72 h) to form pinpoint colonies on agar. The slow growth rate and formation of small colonies is often due to the inability of the bacteria to synthesize certain substances required for their growth; thus, supplementation of these substances in the growth medium can result in a normal growth rate. They are implicated in a number of chronic infections, especially cystic fibrosis and chronic osteomyelitis.

The frequency of SCVs in the population is about 10^-5. SCVs can be selected by aminoglycosides both in vitro and in vivo. Treatment of a bacterial culture with aminoglycoside kills the normal, fast dividing population, whereas SCVs are spared due to reduced uptake of the antibiotic by SCVs. Thus, aminoglycosides can be used to select SCVs, which are normal subpopulation present in a bacterial culture.

Using a microfluidic device, Balaban et al. (2004) showed that persisters can be of two types: Type-I and Type-II persisters. Type-I persisters are a pre-existing population of non-growing cells produced during the stationary phase and take a longer time to exit the stationary phase. The switching rate of normal cells to Type-I persisters during the exponential phase is negligible. Type-II persisters, on the other hand, are not in a growth-arrested state but constitute slow-growing cells and are generated continuously. They are formed by a phenotype-switching mechanism wherein a normal cell spontaneously becomes a Type-II persister and vice versa. A wild type population thus consists of three subpopulations: normal cells that grow fast and are quickly killed by antibiotics; Type-I persisters that are generated during the stationary phase of the previous cycle; and Type-II persisters that are generated continuously. Both Type-I and Type-II persisters can avoid being killed by antibiotics and undergo phenotypic shift in the absence of antibiotics.

Why Balaban et al. (2004) missed SCVs in their experiments using the microfluidic device for detecting persisters? Even though ampicillin (the antibiotic used in their experiments) can not select SCVs, they should have detected SCVs, which are normal subpopulations of bacteria in a culture that grow slowly but do not revert to normal growth (unless auxotrophic agents are added).

Next- How specific is the role of hipA in persister generation?

Balaban et al. (2004). Bacterial persistence as a phenotypic switch. Science 305(5690), 1622-5.

Proctor et al. (2006). Small colony variants: a pathogenic form of bacteria that facilitates persistent and recurrent infections. Nat Rev Microbiol 4(4), 295-305.