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    Distinguishing Between Resistance, Tolerance and Persistence To Antibiotic Treatment

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    Kenny BT

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    © All Rights Reserved
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    PERSPECTIVES OPINION Distinguishing between resistance, tolerance and persistence to antibiotic treatment Asher Brauner, Ofer Fridman, Orit Gefen and Nathalie Q. Balaban Abstract | Antibiotic tolerance is associated with the failure of antibiotic treatment and the relapse of many bacterial infections. However, unlike resistance, whichis commonly measured using the minimum inhibitory concentration (MIC) metric, tolerance is poorly characterized, owing to the lack of a similar quantitative indicator. Thismaylead to the misclassification of tolerant strainsasresistant,or vice ‘versa, and resuitin ineffective treatments. In this Opinion article, we describe recent studies of tolerance, resistance and persistence, outlining how aclearand distinct, definition for each phenotype can be developed from these findings. We propose a framework for classifying the drug response of bacterial strains according to these definitions that is based on the measurement of the MIC together witha recently defined quantitative indicator of tolerance, the minimum duration for killing (MOK) Finally, we discuss genes that are associated with increased tolerance — the ‘tolerome’ —as targets for treating tolerant bacterial strains .iglation and genetic characterization of antibioic-resistant bacterial strains hae uncovered many molecular mechanisms ofesistance, including mutations in the drug target, enzymatic activity that direcly inactivates the antibiotic and the activation of efflux pumps thal pump fut the antibiotic The genes that ace involved in these mechanisms ate termed 5. However, as long, ago as 1946 twas observed that bacteria were able to survive extensive antibiotic ‘eatments without acquiring resistance mutations. The terms tolerance RF 5) and persistence’ (4) were coined to distinguish these modes of survival from ‘resistance, but the definitions ofthese diferent terms, and their distinction from one another, have remained somewhat ambiguous" Resistance’ ie used to describe ‘he inherited ability of microorganisms to grow athigh concentrations ofan antibiotic, irrespective of the duration of treatment, and ie quantified by the ‘minimum inhibitory concentration (MIC) the ‘esistome biotic 6-18, of the particular a ‘whereas tolerance’ is more generally used to describe the ability, whether inherited oF not, of microorganisms to survive transient ‘exposure to high concentrations ofan antibiotic without a change in the MI ‘which i often achieved by slowing dawn, an essential bacterial proces F'6. 1) In this Opinion article, we follow the tolerance terminology defined by Kester and Fortune’, namely tha tolerance enables bacterial celle tosurvive a transient exposure to antibiotics a concentrations that would otherwise be lethal’. For example, tolerance to Peactams say occur when bacteria grow slowly”, which ie associated with slower cell wall, assembly. As B-lactams requie active cell, wall assembly to kill bacteria, slower growth wil result in a longer minimum treatment duration to achieve the same level of illing, regardless ofthe concenteation of the antibiotic. Dormancy may be viewed as an ‘extreme case of slow growth, and dormancy that leade to tolerance may also be termed “drug indifference’ (| ‘Tolerance may be acquired through genetic mutation or conferred by environmental conditions" for example, poor growth conditions have been shown to increase tolerance to several classes of antibiti. This tolerance was exploited by Lederberg and Zinder to isolate auxotrophic ‘mutants as only non-growing auxotrophs are able to survive when a mutagenized bacterial population is exposed to penicillin in the absence of an amino aid! ‘A non-groving state that leads to tolerance ‘an also be induced by the antibiotic ite ‘This drug-induced (lerance subsequently protects the bacteria from the lethal activity ofthe antibiotic In contrat o resistance and tolerance, Which ate atrbutes of whole bacterial populations, persistence’ isthe ability ‘fa subpopulation of clonal bacterial population to survive exposure to high concentrations of an antibiotic”. Persistence is typically observed when the majority ‘ofthe bacterial population is rapily Julled while a subpoptlation persists for ‘much longer period of time, despite the population being clonal. The resulting {inse-kill curve willbe biphasic", owing to the heterogeneous response of persistent and nnon-persstent subpopulations. The dower rate of illing ofthe persistent subpopulation isnon-hertable: when persistent bacterial cells are isolated, regrown and re-exposed to the same antibiotic treatment, the same heterogeneous response to the drug will be ‘observed asin the original population, with the division ofthe population into persistent and non-persistent subpopulations G19, The first direct observations of persistence a the single cell level showed, that slow geowth, as well as dormancy, of small subpopulation ofbacteral cells can underlie the high ate of survival ‘ofa whole population" Additional, generally dose-dependent, mechanisms of persistence that also display biphasic killing have been observed subsequent to these ‘initial observations" Experimental discrimination between the different strategies used by bacterial cells for survival during exposure to antibiotics is important for several reasons. Fist hese survival strategies, despite supesficial similarities, dif in their 1.20% Maclin blisters ied Mets eer a b eee 2 4 8 16 7 18 aos 16 32 62 128 or4as 00000~~ (© ° ras eo Ps © tess Fraction of survivor Susceptible versus tolerant bacterial strains 16 eo, ®OOQOO®»~~ (eo, ®QOQOO® 16 jQQQO@~ PERSPECTIVES Susceptible versus persistent bacterial stains @ 8 48 6 3 8 178 [egeese ®OQ»- e178 02 4 8 6 2 6 28 ®OOQOO®»~ 2 @) suscep © Tolerant Wi suscepubdie © ersstene Fraction of survivors Time hous) Figure 1] Characteristic drug responses of resistance, tolerance and persistence. Ihe survival strategies of resistance, tolerance and pers's- fence to antibiotic treatment each manifest s a characteristic drug response. |The minimum inhibitory concentration (MIC) fra strain of bocteria tht isresistan to an antibiotics substantially highor than the IMIC fr a susceptibie strain. Coloured wells represent bacterial growth ‘whereas wellsinwhich the anibiotie concentration i high enough toil ‘the bacteria areinlight brown. b| The MIC for atolerantstrainof bacteria issimilarto that of a susceptible strain; however, the minimum ferkiling (MDK: for example for 9% of bacteria cells in the population (MOK, ora tolerant strain is substantially higher than the MOK, for & susceptible strain. [A persistent stain of bacteria hasa similar MIC and asimlor MOK,, toa susceptible stain: however, the MDK for 99.99% of bacteria cellein the population (MDK,,,) substantially higher fora persistent stan than the MD, fora susceptible strain, Concentrations nd timescales are chosen for lcstration purposes only Time hous) basic made of action, which means that a treatment will offen be ineffective ite applied respective ofthe survival strategy. Second, the underlying mechanisms and the experiments that are required to investigate them, may be very different for each strategy ‘Thied, the range of antibiotics that is affected by the drug response can differ according to the survival strategy. For example, tolerance by low growth will often confer an advantage to several asses of antibiotic, ‘whereas most resistance mechanisms are specific to one class of antibiotics. Finally, the quantitative measurement of resistance, tolerance or persistence require different ‘metrics and experimental procedures for cach survival strategy. Th this Opinion atiele, we discuss the current asi for and the strategies used to distinguish between resistance, tolerance and persistence to antibiotic in bacterial strain, without any a priori knowledge ‘ofthe molecular mechanisms tha are involved, These terms have often been ‘used interchangeably in the literature, but ‘we propose a clear and distinct definition foreach term, and an experimental rmework for distinguishing between these phenotypes that user standardized and ‘measureable metric to detect tolerance to drug exposure — the minimum duration for killing (MK). We hope thatthe ‘combination ofthe MIC and the MDK may ‘be used as standards forthe in vitro charac terization of sensitivity to antibiotics, which ‘ultimately may lead to beter treatments for recaleitrant infections Resistance or tolerance? Resistance. Resistance to antibiotics, which is typically caused by inherited mutations, is azociated with numerous molecular ‘mechanisms that have been comprehensively reviewed lsewhere™. tis important to note that mechanisms of bacterial resistance decrease the effectiveness ofthe antibiotic tha ea higher concentration of the antibiotic is required to produce the same cflect in a resistant strain a is produced ina susceptible strain! (IC. 9, Resistance fs quantified by the MIC, which can be defined a the minimum concentration ‘ofan antibiotic that is required to prevent net growth ofthe culture. In practice the [MIC is measured by exposing a bacterial population to incressing concentrations ofthe antibiotic in a standardized growth ‘medium, Ths enables the measurement of the minimum concentration at which growth, fs not detected, typically after 16-20 hours ‘of exposure tothe antibiotic, The range of concentrations thats tested in a lineal ‘microbiology laboratory i usally Limited to the concentrations ofthe antibiotic used fn the clinic, The MIC thatis determined by these test is viewed ta convenient metsic 1.2016 Maclin blisters ied Mets eer PERSPECTIVES for resistance, and a bacterial stain with ‘higher MIC than another strain ill be regarded as more resistant” Measurements ofthe MIC that indicate total insuseeptibilty to an antibiatc may be viewed as an extreme case of resistance, ‘The MIC has two major limitations as a general metric for measuring the response of bacterial strain to an antibioti, First, itis ‘ot informative for bacterial strain that are tolerant, rather than resistant. Second the MIC measured in vitro can vary according to the experimental conditions that are ‘sed, which may affect the usefulnes of this matric as apredicor ofthe effectiveness ofthe abiotic in vivo®, However, the case of measuring the MIC means that tis currently the only metric that is routinely ‘sed to inform treatment decisions for ‘bacterial strain eolated in the clinic Tolerance. Tolerance i generally understood. tobe the ability ofa bacterial population to survive a transient exposure to antibiotic, coven at concentrations that far exceed the ‘MIC. Unlike resistance, tolerance applies only to bactericidal antibiotics and not to bacteriostatic antibiotics, sll bacteria, are expected to survive transient exposure lobacteriostaic antibioice™, which axe not lethal and instead merely arrest growth, Therefore, all discussion of drug exposure in this Opinion article should ‘be assumed to rele to concentrations of ‘bactericidal antibiotic. Importantly. a longer exposure to an antibiotic, rather than a higher concentration of an antibiotic, required to produce the same lve of iling n tolerant is produced ina susceptible strain (16,10 Ac tolerant bacteria can have the same MIC as non-tolerant bacteria, the MIC isnot informative aa metric to evaluate tolerance’, One suggested approach for the evaluation of tolerance is the ‘measurement oftime-kil curves at diferent concentrations ofan antibiotic’. However, without standard method for interpreting these curves the results that ae obtained in different aborstories are dificult to compare". Another measure of tolerance {hat has been proposed isthe MBC/MIC. ratio®, where MBC represents the minimum bactericidal concentration, namely the concentration ofan antibiotic that is required to kill 299.9% of cells in a bacterial culture, ‘ypiealy after 24 hours of incubation. For cases in which concentrations of antibiotic that are near the MIC cause only growth arrest but the MBC results in death, the [MBC/MIC ratio wil produce a large val “Therefore this metric may accurately evaluate the level of drug-induced tolerance ‘bat wae shown to correlate poorly with other forms of tolerance’ Recently, the MDK was described as ‘quantitative messure of tolerance that cam be ‘extracted from time-kill curves, based on the notion that a tolerant bacterial strain requires more exposure time to be effectively killed than a susceptible strain The MDK is defined asthe typical duration of antibiotic treatment ‘that ie required toll given proportion of the bacterial population” at concentrations that far exceed the MIC (thai, when. the rate of cell death is independent ofthe ‘concentration of antibiotic). For example, ‘the minimum duration of treatment that i requited to il 9% ofa bacterial population (MD, ), which can be extracted from a time-Kill curve 6.10, The assumption ‘that undezkies the MDK ara measure of tolerance ie that the killing effect reaches saturation at high concentrations of an antibiotic o that ite almost insensitive to ‘concentration and dependent only on the duration of exposure”, Similarly tothe MIC, which ean be used to compate the level of resistance ‘between bacterial strains, the MDK ean ‘be used to compare the level of tolerance between strains In contrast to the killing rate (thats, the rate at which survival ‘decreases exponentially), which can only ‘be extracted from exponential killing ‘curves, the MDK simply integrates all of the different factors tha together undesie «faster or slower overall king eficacy, such az delays in ling or killing curves that are not exponential, Therelor, thie ‘quantification of tolerance i not dependent ‘on any particular molecular mechanism, We argue thatthe MDK should be the preferred metric for the measurement of tolerance, as itenables a quantitative comparison between, different bacterial strains and conditions Furthermore, an evaluation framework that ‘measures both the MDK and the MIC would ‘enable a clear distinction to be made between, resistance (an increase i the MIC) and tolerance (an increase in the MDX) Reports of tolerance in the hterature are generally associated with slow growth and reduced metabolism!™*”. As inthe lactam example, the slowing or complete ‘cestaton of growth revultein a reduced or diminished susceptibiliy to many antibiotics "Thisisa direct result ofthe evolution ofthese antibiotics in microorgenisms competing {or resources, in which the production of antibiotics that target fast growing bacterial ‘ells which are the most competitive for resources is selected for" Different classes of antibiotic have evolved to target different processes that are required for growth andit 4s sometimes posible to artificially decouple the efficacy ofthe antibiotic from the growth ate (that is, decouple target production and growth), once the process thats targeted bythe antibiotics known, For example in Escherichia col ell that are growth-arrested by the stringent response, treatment with chloramphenicol enables cell wall assembly to resume without the fll resumption of call growth, Asa result of the resumption, ‘of cll wall assembly, the bacterial cells are sensitive to lactams, eventhough they remain essentially grovth-arested”. However, reports that Ecol cells ean be killed by B-retams during growth artes ate often based on experiments that measure the grovth arrest ofthe batch culture, which means that a dynamic balance of growth and death —in which 8 lactams only target growing calls but the overall growth ofthe culture ie stationary — cannot be ruled out. ‘Although some studies have assayed growth arrestin single cells, these studies assayed the absence of growth a the beginning of the ‘reatment™ and cannot rule out thet grove, ‘occurred daring treatment. Which form of tolerance? ‘We identify two main forms of tolerance, which we term tolerance by slow growth and ‘tolerance by lag! Although these two forms of tolerance share an increased MDK compared with susceptible bacterial cells, the ‘mathematical description and measutement protocol difer between them. The distinction rises because tolerance by slow growih, ‘occurs at steady state, whereas tolerance bylag isa transient state that is induced by sarvation or ates, Tolerance by slow growth. Coniltons that decrease the rate of growth have long been ‘know to increat tolerance to numerous antibiotics!” asthe mechanisms of action ofthese drugs share a requirement for growth. For example, the mechanism of ction of f-lactams relies onthe eruption ‘of bonds in the peptidoglycan layer that ‘occurs during bacterial growth, -actams exploit this proces by preventing the reassembly ofthe peptidoglycan bonds, which eventually leads to cll sis. Therefore, the numberof defect inthe peptidoglycan layer ‘increases in proportion with the growth rate, Indeed, che killing rte of bacteria that ate exposed to f-actams ha even been shown tobe proportional othe growth rate, which demonstrates the strength of corelation 1.2016 Mac Hin Pslstes ied Migs eared ‘between slow growth and tolerance for these antibiotics Similarly the exposure of ‘bacterial cll to DNA gyrase inhibitor, such as fluoroquinolones, results in DNA damage and killing ata rate thai proportional to the growth rat! Indeed, plotting the MDK,, versus the grow rate, using values extracted from time-Kill curves published in the literature, confims the positive corzelation ofiling activity and growth rate for several species of bacteria and different classes of antibiotic BC. 2, Tolerance by slow growth can either be inherited, when a bacterial species or strain hasan inherently slow growth rate, or non-inherited, when slow growth occurs ‘because the conditions for growth ae poor Species with inherently slow growth rates include Mycobacteriune tuberculosis, which Ihae« doubling time in nutrient-rich medium of approximately 24 hours", This doubling Lume is approximately 40 times longer than that of Ecol and, not surprisingly, the MDK,, of M, tuberculosis strains is also approximately 40 umes longer than the MDK, of E col Anxotrophs and other bacterial strains with ‘mutations that reduce thei intrinsic growth rate also show inherited tolerance”, ‘Non-inherited tolerance by sow growth occurs when bacterial growth is impaired, suchas by poorer growth conditions ‘the location of ell within bioflm ‘or exposure to inhibitors When the antibiotic is added in the presence of these _grovth-reducing conditions, ling wall ‘be reduced. Note that dormancy can be viewed a the extreme case of dow growth, {in which the growth rate ie zero, Important, a decrease in growth rate ha been observed for intracellular bacteria when within a hos ell For example, Salmonella enterica subsp. enterica serovar Typhimurium cells ‘with arrested growth have been detected iningected macrophages" Accordingly. infections by intracellular pathogens are otoriouly dificult to eradicate, even when treated with antibiotics that readily penetrate Iho cells, The notion that tolerance rather than resistance underlies the resilience of these infections ie sppported by in vito assays ‘that showed that intracellular Staphylococcus ‘aureus treated with diloraclin hada fivefold inerease in the MDK, but no change in the MIC, compared with extracellular S. aureus, which suggest that tolerance ‘enables this intracellular pathogen to survive treatment with dicloxacilin ‘A special case of tolerance by slow growth is drug-induced tolerance, which occurs ‘when bacterial cells respond to antibiotic ‘exposure by reducing or arresting ther ‘growth, This effect has been observed in chum fo Taerance by log riserascey slow growth PERSPECTIVES various Gram-negative and Gram-positive bacteria when exposed to antibiotics, including an antbiotc that nbibits cell, wall synthesis’, whereby the growth azest ‘was mediated by the defective induction ‘of autolysins* and the fluoroquinolone Ciprofloxacin, whereby the grovrh arrest war mediated by the induction of stress response” In summary, non inherited tolerance can be triggered by external stress factors that include starvation® hos factors* and even the antibiotic tel, As might be expected, tolerance by slow growth also ‘occurs when antibiotics are added athe stationary phase of growth, in which the net {growth rte ofthe bacterial population is zero (but conaltions are permissive for abalance between the growth and desth of individual cell) In addition, in what may be viewed as an extreme cae of tolerance by slow growth, tolerance atthe stationary phase can aecur ‘when the growth rat of individual bacterial cells is er0" which can produce an extemely long MDK®. The protective eflet of growth arrest asa passive survival strategy ca be enhanced by the aetvation of stress response ‘mechanisms that provide futher protection from antibiotic strese™ Some of these additional protective mechanisms, such a8 the production of efflux pumps, may also reduce [= Growing [= Nongrowing Doubling time fours) Time hows) Figure 2 Tolerance arses from slow growth or lag phase. a [The min- imum duration for ling (MOK) Tor 99% of bacterial ells in population (MOK, j'sploted agains doubling time for several combinations afbac- terial strain orspecies and antiboltc, sextracted from timo-kll curves in ‘the literature", The dashed ine shows the bes fit for the relationship between the MDK,, and the doubling time for strins a bae~ teriathataretolerantby slow growth, which demonstrates the correlation between these twovarables, The shaded area highlights the distribution of bacterial strains hat ae tolerant by lg; hese strains were detected by exposure to the drug directly an dilution from the stationary phase. b|Aschematic growth curve that shove the importance of subculturing toreach strietiy exponential growth, An inital 1m 100,000 dilution af & bacterial population from a culture inthe stationary ahase ofthe ravth cycles followed by seria n100 dilutions: ineach instance, the colony's grown unilthe population density aches 10'colony erming uits(CFU} mi before dilution, Fach dilution reduces the numberof residual rnon-growing bacterial cells— thats, cellin thelag phase— inthe po- Uulation and several dilution steps may be required until the populations composed only of calls in the exponential growth phase, with no cals lag phase 1.20% Mac Hin blisters ied Migs eer PERSPECTIVES the effective concentation ofthe antibiotic, ‘which increases the MIC and results in a mixed phenotype of resistance and tolerance. ‘Tolerance by lag. n alton tothe stationary phase, another phase ofthe bacterial growth, cycle during which bacteria do not grow, and _may therefore be transiently protected from Jalling by antbiotcs, isthe lag phase. The lag phase is defined as the time stakes for growth-arrested bacterial cells (for example, Under starvation conditions) to resume exponential growth when adjusting to an environment that i permissive for growth (forexample, when starved bacterial cells axe diluted into fresh nutrient conditions). ‘The typical mean lag time of E. coliK-12 populations when diluted from an overnight culture is 30 minutes, but this time can be substantially longer when a culture hasbeen in the stationary phate for seve before dilution into fresh nutrients. Although, these two growth phases are often thought tobe simula to each other, the lag phase hes now been chown tobe a distinct metabolic stat om the stationary phase, in which bacterial cells must fist adapt to the increase in nutrient concentration before resuming exponential growth. Therefore, tolerance by lag dflere from the extreme cate of tolerance by sow growth that occurs a the stationary phase. imilasly tothe lag phase that occurs Mer stationary growth, transient growth arrest can also occur during lag phase {hat follows transitions between growth conditions — for example, when bacterial cells enter the host envionment or sitch between diferent niches. Importantly, tolerance bylag i transient phenotype that isnot sustained when the culture has suficient me to fully adjust to the new conditions. Therefore, tolerance bylag ‘modelled a decay process whereas tolerance by slow growth isa steady-state ‘phenotype thatis characterized by a reduction in killing rte Thos, the mathematical descriptions of these two forme of wlerance are inherently diferent Tolerance by lag occurs when the antibiotic treatment is shorter than the duration of the growth ares", The protective effect ofthe lg phase on the survival of the bactesial population is very broad a tan, enable tolerance to different antbiotics™, in addition to other streses, inclu exposure tothe host immune system" and the induction of prophages, Despite the ‘wansience of growth arrest at the lag phase, tolerance by lag canbe very efletive, reaching an MDK of many hours or days For example, ithas been shown thatthe intermittent ‘exposure ofE colito a lactam antibiotic ‘an select fora lag phase that 10 times longer than the lg phase of the ancestral population reaching an MDK,, of more than a day, Remarkably, the duration of the lag phase evolved to match the duration ‘of antibiotic treatment in as few ae eight ‘exposures tothe drug. Owing tothe tolerance that was conferred by the extended lg time, antibiotic treatment eventually became ineffective, even when changing the cass ‘of anubiotic, at long asthe duration ofthe treatment was the same, Several genes were repeatedly mutated in these populations (204, which led to an inherited tlerance by lag. By contrast, no change in the MIC wae detected, which cuggests that the phenotype ‘oftolerance by lag may evolve more rapidly than the emergence of resistance. The rapid evolution to tolerance by lag that wa ‘observed in thi in vitro aay in which ‘bacterial populations adapted tothe duration ‘ofthe treatment rather than tits chemical ‘composition, calls for an evaluation of the ‘importance of the evolution of tolerance in the host environment, Measuring tolerance. Iisimportant to realize thal the experimental protocal for the in vtro measurement of tolerance differs according to whether the measurement is for tolerance by slow growth or tolerance by lag 1202. To measure tolerance at the lag phase, ‘exposure tothe antibiotic must oceur dieetly ‘on transition tothe lag phase (generally, when alti from a stationary-phase culture), Ifthe culture is instead frst diluted into feesh ‘medium for an undetermined period of time before exposure to antibiotics, a mixed population of exponentially growing and non-dividing bacterial celis arises (16 2b, "The proportion ofthe bacterial poplation that survives exposure tothe antibiotic will then depend on the time between dilution into fresh medium and exposure othe drug owing tothe complex population dynamic of the exit from the lag phase, ‘which i heterogencous atthe single-cell level”, The dependence on experimental parameters that can be challenging to control say result in non-reproducible evults and ambiguous measurements of tolerance ot persistence (see below). By contrat, tolerance by slove growth should be measured ina steady-state clture during exponential growth, ensuring that no lagging bacterial ells are carted over from the stationary phase. Ths steady-state cclkure can be achieved in chemostats orn ‘clues that are sub-cultured several times ‘during the exponential phase Note that true exponential grow ends long before the transition to the stationary phase that occurs thigh cell density, typically already at an (OD (optical density a 600m) of 0.1 in rich mediums" Persistence and heterogeneity For thoze abiotic treatments that effectively al the majority ofthe bacterial population, subpopulations that are not killed by the antibiotic can nevertheless emerge even in dona cultures, When these surviving subpopulations are grovn in the presence ofthe same antbiai, the heterogeneous response is repested”®. This phenomenon fs termed bacterial persistence’ and the surviving bacterial els are referred to 35 perssters, We note tat persistence’ is aso ‘used more generally to describe infections that are not cured effectively and persiet inthe host including those infections that maybe “unrelated tothe definition of persistence used inthis Opinion article to denote the presence ‘ofa subpopulation of persitersin a onal population of bacteria, ‘As opposed to tolerance and resistance, persistence only ceurs in a subpopulation of bacterial cells, Persistence canbe detected by the presence ofa bimodal (or multimodal) ‘ime-Kil curve that deviates fom the simple decay expected from a uniform bacterial population. Inthe simple ese of two ‘coesisting subpopulations, persistence ie characterized bya switching betsscen two phenotypes — suscepribleand persistent Perssters constitute the less numerous subpopulation (typically less than 196) and sre lle ata slower rate than the susceptible cells", We propose that the firs step towards characterizing the heterogeneity of bacterial populations under antibiotic treatment to determine whether persisters survive the exposure tothe antibiotic because they are transiently more resistant or because they are transiently more tolerant than the majority ofthe population 2082) Time-dependent persistence. “Time-dependent persistence is characterized bythe presence of «subpopulation of tolerant bacteria, which typically has ether aonger lag time (tolerance by lg) or slower growth rte (tolerance by slow grow:h) than the majority ofthe population, These ‘oro types of persistence have very diferent dynamic and were previously defined as ‘Type I persistence and Type TI persistence, respectively", All ofthe characteristics of tolerance that are descrved above fora whole bacterial population canals be applied toa subpopulation with time-dependent 1.2016 Mac Hin Pslstes ied Migs eared Box |The tolerome: genetic factors that increase the MK. PERSPECTIVES ‘Aninereasein the minimum duration for kiln (MD) occurs when the Ling ate of bacterial cells that are exposedto antibiotics slowed ‘down by one or more of numerous mechanisms Norinhertd tolerance canbe wiggered by external sress factors. such as starvation low ‘temperature’, host factors” and even te antibiotic itsl!"™, However, ‘weUse the term Yolerome’ to describe the geneti Tactrsthat have been repeately shown to inereaee tolerance ortime-dependent persistence Forthe lactam and Muoraquinolone classes of antibiotic the stringent response which inhibits bacterial growth, hasbeen shown tohavea centaltoein tolerance. Duting nuteitions stress the decrease inthe ‘valailty of amine acids leads ta an accumulation of uncharged &RNAS thot triggers the production of guanosine tetaphosphate(ppGpp | ‘alarmanestresssgnal that mediates the stinger response”. The fst high-prsistence mutants tobe iolated in the laborato echerichacalimutants found to have mutation inthe hip toxin-anttonin module, which encodes Hip. toxin, and its cognate _atitoxin, Hip Hip was later shown to inactivate an essential minoraeyl RNA synthetase, glutamate-2RNA ligase (GLK thus producing high levels of paGpp owing tothe accumulation of uncharged, {RNAs When hip isexpressed above threshold set by the abundance of HipB astringent response isinduced. importantly the stringent response involves the induction of ala phase thats a transient growth ares). hasbeen shown inboth Gram-negative” and Gram-positive bacteria”, ‘sth expression of hp increases, the lag phase becomes longer, which results ina longer MDK end a phenotype of tolerance by lag Asie from hip, the overexpression of other toxin enesin toxin-atitonin modules ‘canals produce similar tolerance phenotypes”. The toleromehas been studied using mutational screens for tolerance, ‘which have dentfied numerous mutations that are related othe ‘activation ofthe stringent response, including mutations in hip, hipBt and methionine-

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