To achieve reliable results in antibiotic susceptibility assays, labs need to hit target standards for organism or inoculum load, researchers concluded in a study published in Antimicrobial Agents and Chemotherapy. “Although we have no idea about how often clinical labs deviate even beyond the allowable range, we expect this happens with some frequency and would further skew results,” said study co-author James Kirby, MD, medical director of clinical microbiology at Beth Israel Deaconess Medical Center in Boston, in a statement.

The Clinical and Laboratory Standards Institute (CLSI) has set the target inoculum at 5 x 105 CFU mL-1, allowing some leeway with a second acceptable target range of 2-8 x 105 CFU mL-1. The “inoculum effect” (IE) refers to the number of organisms initially inoculated into an antibiotic lab test.

Labs typically perform IE testing at 100fold greater than CLSI’s recommended inoculum. There are several reasons for this, according to Kirby. For one thing, it’s easy to do, as it’s as close to an undiluted bacterial suspension as you can get. In comparison, the target CLSI inoculum is usually prepared by diluting the original bacterial suspension 150- to 300fold. Tradition is another reason. IE testing at 100fold greater than CLSI’s recommendation is typically reported in scientific literature, Kirby told CLN Stat.

Researchers wanted to find out if IE could undermine the accuracy of minimal inhibitory concentrations (MIC) measurements. “One of the most important functions of the clinical microbiology laboratory is to determine the susceptibility of microorganisms to antibiotics,” Kirby said. 

“The importance of the MIC is it predicts whether antibiotic therapy in a patient will be successful or not. We need to give an antibiotic to patients at specific doses to avoid toxicity,” Kirby indicated. “Therefore, accurate determination of the MIC is paramount. If the clinical lab determines an MIC that is spuriously too high or too low for a given pathogen, the wrong therapeutic decisions may be made.”

To see if deviations around the target inoculation impacted results, the researchers employed inkjet printing technology to test the IE of several pathogens cited as urgent drug resistance threats by the World Health Organization and Centers for Disease Control and Prevention: extended spectrum beta-lactamase producers (ESBL) and carbapenem-resistant Enterobacteriaceae (CRE). “Currently, clinical labs don’t typically test for the resistance elements themselves but just determine the MICs for these organisms as for all others and report out MIC results without further qualification,” Kirby said.

Researchers determined IE by testing orthogonal titrations of antibiotic and organism inoculum. According to Kirby, the inkjet printing technology “allowed us to do this in a very reproducible and accurate manner and with extremely fine granularity.” Through this method, they discovered that the susceptibility determination against meropenem and cefepime—two drugs commonly used to treat drug-resistant Gram negative pathogens—were greatly affected by inoculum differences within the allowable range of inoculum, said Kirby.

Researchers reported that a twofold rise in inoculum led to a 1.6 Log2-fold increase in MIC for cefepime resistant and susceptible dose-dependent strains. “For carbapenemase-producing strains, each twofold reduction in inoculum resulted in a 1.26 Log2-fold reduction in meropenem MIC. At the lower end of the CLSI allowable inoculum range, minor error rates of 34.8% were observed for meropenem when testing a resistant strain set,” the researchers summarized.

In an interesting finding, ceftazidime-avibactam, a new drug developed to treat CRE infections, was not similarly affected. This suggests that issues associated with laboratory-determined MIC values are less likely with this drug, Kirby said.

In future research, Kirby plans to explore IE for additional drugs and pathogens. “One of our goals is to increase the accuracy and reproducibility of MIC testing so we can make wiser therapeutic choices,” he said. “The inoculum effect is only one potential source of variation in the determination of MIC values.”