Antibiotic tolerance among clinical isolates: mechanisms, detection, prevalence, and significance

Abstract

SUMMARYAntibiotic treatment failures in the absence of resistance are not uncommon. Recently, attention has grown around the phenomenon of antibiotic tolerance, an underappreciated contributor to recalcitrant infections first detected in the 1970s. Tolerance describes the ability of a bacterial population to survive transient exposure to an otherwise lethal concentration of antibiotic without exhibiting resistance. With advances in genomics, we are gaining a better understanding of the molecular mechanisms behind tolerance, and several studies have sought to examine the clinical prevalence of tolerance. Attempts have also been made to assess the clinical significance of tolerance through in vivo infection models and prospective/retrospective clinical studies. Here, we review the data available on the molecular mechanisms, detection, prevalence, and clinical significance of genotypic tolerance that span ~50 years. We discuss the need for standardized methodology and interpretation criteria for tolerance detection and the impact that methodological inconsistencies have on our ability to accurately assess the scale of the problem. In terms of the clinical significance of tolerance, studies suggest that tolerance contributes to worse outcomes for patients (e.g., higher mortality, prolonged hospitalization), but historical data from animal models are varied. Furthermore, we lack the necessary information to effectively treat tolerant infections. Overall, while the tolerance field is gaining much-needed traction, the underlying clinical significance of tolerance that underpins all tolerance research is still far from clear and requires attention.

Keywords: animal models; antibiotic tolerance; clinical microbiology; diagnostics; treatment failure.

Fig 1

Schematic depiction of (A) minimum inhibitory concentration (MIC) and (B) time-kill assay results with tolerant bacteria. (A) In an MIC assay, a resistant strain exhibits an MIC above the susceptibility breakpoint, while a tolerant strain exhibits a susceptible MIC. (B) In a time-kill assay, the entire population of a tolerant strain dies at a slower rate than a susceptible strain with the same MIC; therefore, it will have a greater minimum duration of killing (MDK), for example, 99% of the population. In contrast, a persistent strain exhibits a biphasic killing curve, whereby the majority of the population dies at the same rate as a susceptible strain and it has a similar MDK₉₉, but a small subpopulation dies as a slower rate.

Fig 2

Molecular mechanisms of genotypic antibiotic tolerance. Schematic depiction of the six key metabolic pathways/cellular processes in which mutations have been reported to confer tolerance. Purple ovals indicate enzymes/proteins for which tolerance mutations have been identified. Glycolysis (not boxed) produces glucose-6-phosphate, which is converted into ribose-5-phosphate (R5P) via the pentose phosphate pathway (not shown). During purine synthesis (1), R5P is converted into the key metabolite phosphoribosyl pyrophosphate (PRPP) by the enzyme Prs. PRPP is then transformed via a 10-enzyme pathway into inosine monophosphate, which feeds into the two branches that ultimately produce the ATP and GTP. PRPP also acts as a key precursor in the purine salvage pathway, in which free bases are converted into ATP and GTP. The pur operon, encoding many of the enzymes involved in the de novo purine synthesis pathway, is repressed by PurR. Glycolysis also produces pyruvate, which is transformed into acetyl CoA that feeds into the TCA cycle. Within the TCA cycle (2), CitZ (also known as GltA) acts as a citrate synthase, while OdhA forms part of the α-ketoglutarate dehydrogenase complex that converts α-ketoglutarate into succinyl CoA. The TCA cycle generates NADH, which is used by the electron transport chain (3) to ultimately generate ATP. NuoN forms part of NADH dehydrogenase complex I, while HemB and MenD contribute to the synthesis of heme and menadione, respectively. These complexes and compounds are key components of the electron transport pathway. GTP/GDP and ATP produced by the purine synthesis pathway are substrates for the enzyme Rel, which synthesizes the stringent response “alarmone” (p)ppGpp (4). Classically, Rel synthesizes (p)ppGpp in response to uncharged tRNAs entering the ribosome, which can occur when the activity of a tRNA synthetase (such as MetG, LeuS, ProS, and IleS) is compromised. Small alarmone synthetases, like RelQ, can also synthesize (p)ppGpp. (p)ppGpp binds to, and inhibits, the activity of many enzymes within the purine biosynthesis pathway. During translation, ribosome biogenesis (5) is dependent on the action of GTPases, including RsgA. Similar to the purine biosynthesis pathway, (p)ppGpp binds to, and regulates, the activity of many of these GTPases. (6) RpoB and RpoC constitute the β and β′ subunits of RNA polymerase, which synthesizes the mRNA needed for translation. In γ- and β-proteobacteria, such as Escherichia coli, (p)ppGpp binds to RNA polymerase in conjunction with DksA to modulate its activity.

Fig 3

Experimental outline of different methods for antibiotic tolerance detection. (A) Time-kill assay. At different time points post-antibiotic addition, culture samples are serially diluted and plated on agar for viable counting. (B) MBC/MIC ratio. A standard broth microdilution assay is set up to determine the minimum inhibitory concentration (MIC). Wells at and above the MIC are then plated out on agar to determine the minimum bactericidal concentration (MBC). (C) TDtest. An antibiotic disk is applied to a lawn of bacteria and a zone of inhibition is observed. Additional nutrients are then added to the disk to allow tolerant bacteria present within the zone to form colonies. (D) REPTIS. Bacteria are plated on agar containing an inhibitory concentration of antibiotic; any colonies that grow at this point are resistant. The plate is then replica plated onto an antibiotic-free plate to allow the growth of tolerant colonies. (E) SPOCK. Bacteria are grown to stationary phase on a filter and then transferred to an antibiotic plate. The filter is then transferred to a plate containing a metabolic dye, which stains live cells red while dead cells exhibit green autofluorescence.

Fig 4

Prevalence of antibiotic tolerance reported in the literature. Box and whisker plots showing values for the reported prevalence of antibiotic tolerance (data taken from Table 2). Many studies have more than one reported value depending on the method or antibiotic used. Boxes extend from the 25th to 75th percentiles, whiskers indicate the range, and the lines in the middle represent the medians.