Inoculum effect- a factor affecting the number of survivors

The inoculum effect is the increase in the minimum inhibitory concentration (MIC) of an antibiotic when a higher inoculum size is used. When the size of the inoculum is low, antibiotics may be able to sterilize a bacterial culture completely. However, as the initial inoculum size increases, the number of survivors also increases resulting in an increased MIC. This is especially true for many β-lactam antibiotics. The inoculum effect can be the result of the ability of the bacteria to produce enzymes that hydrolyze the antibiotics. At high inoculum sizes, the initial bacterial killing may release more β-lactamases into the medium, hydrolyzing the antibiotics, resulting in more survivors and thus a higher MIC value. Based on the inoculum effect, some researchers argue that the MIC determined by the standard dilution method may not be appropriate in vivo and that it will be better if MIC obtained from a large inoculum is used rather than using MIC obtained from the standard inoculum. However, others consider the inoculum effect to be only a laboratory phenomenon and an artifact and support the use of conventional MIC values for the pharmacokinetic/pharmacodynamic (PK/PD) assessment of antibiotics.

The phenotypic shift of persisters, as demonstrated in many experiments, is due to the effect of inoculum size only. At low inoculum size, the number of persisters is low or absent. As the inoculum size increases, the number of persisters also increases. They may remain dormant during the first few hours of incubation, but may regrow on further incubation or on transfer to fresh medium without antibiotics. Thus, the re-growth of bacteria following transfer to fresh medium without antibiotics may not be due to the phenotypic shift of persisters, but rather can be due to the inoculum effect only. Recently, the effect of inoculum size on bacterial survivors was demonstrated by Udekwu et al. (2009), using six different antibiotics. In case of linezolid, the number of survivors after 18 h of incubation with 20 times the basal MIC using a low inoculum size (5x 10⁵ cfu/ml) was less than 1% of the initial inoculum, but was almost close to 100% when the initial inoculum size was about 10⁸ cfu/ml. Similarly, with vancomycin, the number of survivors was about 1 in 10⁴ cells at low inoculum size using 20 times the basal MIC, but was much higher at high initial inoculum size. With oxacillin, the number of survivors was very high at high inoculum size and high concentration of antibiotic (i.e. a paradoxical effect, which will be discussed in the next 2 blogposts). Thus, the number of survivors was vastly different depending on the initial inoculum size, antibiotic concentration and time of incubation.

 In most of the experiments demonstrating the phenotypic shift of persisters, the initial inoculum size is kept low. The reason for the low number of persisters (less than 1% of the total population) is due to this low inoculum size. Had the researchers used a higher or lower inoculum size, they would have noticed a different result.

The bacterial survivors after antibiotic treatment with a specific inoculum size and a specified time of incubation are neither persisters nor the regrowth of bacteria after the removal of antibiotics is the phenotypic shift of persisters. The perceived phenotypic shift is only a laboratory phenomenon which may not have much clinical significance.

Next- Other factors that determine the number of survivors

Udekwu et al. (2009). Functional relationship between bacterial cell density and the efficacy of antibiotics. J Antimicrob Chemother 63(4), 745-57.