Is the effectiveness of mannitol (Allison et al. 2011) due to elimination of persisters?

Allison et al. (2011) claims that aminoglycosides given in combination with specific metabolites like glucose, fructose, mannitol or pyruvate can be used to treat biofilm-related infections. They demonstrated that catheters colonized with E. coli biofilms, implanted in the urinary tract of mice, had reduced biofilm viability and lower kidney bacterial load when gentamicin was given in combination with mannitol than with gentamicin alone. Based on their findings, it is also proposed that inhaled tobramycin along with mannitol can be effective in treating Pseudomonas aeruginosa infection in cystic fibrosis (CF) (Cain 2011).

However, it is already reported that inhaled mannitol can improve the lung function in CF (Robinson et al. 1999; Jaques et al. 2008). The background information in the abstract of Jaques et al. (2008) is reproduced below.

“The airways in patients with cystic fibrosis (CF) are characterized by the accumulation of tenacious, dehydrated mucus that is a precursor for chronic infection, inflammation, and tissue destruction. The clearance of mucus is an integral component of daily therapy. Inhaled mannitol is an osmotic agent that increases the water content of the airway surface liquid, and improves the clearance of mucus with the potential to improve lung function and respiratory health”.

If mannitol along with aminoglycosides is effective in reducing the bacterial load in CF patients, is it due to the osmotic activity of mannitol or is it due to the ability of mannitol to eliminate persisters? It is already reported that antibiotics may not be effective without clearing mucus and other secretions in the airway.

Similarly, the choice of using mannitol in mouse urinary tract infection model (Allison et al. 2011) raises some questions. Mannitol at 1.5g/kg is an osmotic diuretic, which increases the urinary output. Can the reduction in the number of persisters be due to the increased urine flow that wash away some of the bacteria in the biofilm attached to the urinary catheter? Washing away some of the bacteria not only reduce the bacterial number but also exposes some of the deep seated bacteria, otherwise protected by the biofilm, to the antibiotic more effectively. If I am guessing it correctly, use of other diuretics with aminoglycosides may also give same results (although some combinations are contraindicatory).

In conclusion, it is predictable that

1. administration of glucose with aminoglycosides may not have any effect in reducing bacterial load in CF (on the other hand, it is possible that it may actually worsen the condition)

2. administration of mannitol along with aminoglycosides may reduce bacterial load (but it may not be due to metabolite-enabled eradication of persisters, but due to the osmotic activity of mannitol that improves the clearance of mucus, thus improving the  lung function).

3. even the above combination may not be able to kill the persisters completely.

Allison et al. (2011). Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature 473: 216-220.

Cain, C. (2011). Sweetening antibiotic treatments. SciBX 4(23): doi:10.1038/scibx.2011.647.

Baker et al. (2008). Inhaled mannitol improves lung function in cystic fibrosis. Chest 133(6): 1388-1396.

Robinson et al. (1999). The effect of inhaled mannitol on bronchial mucus clearance in cystic fibrosis patients: a pilot study. European Respiratory Journal 14(3): 678-685.

Can a spoonful of sugar help in the treatment of persistent infections?

A recent article published in the journal Nature suggests that taking antibiotics with certain sugars can improve their effectiveness against persistent infections. As per Allison et al. (2011), bacterial persisters in biofilms can be eradicated by facilitating aminoglycoside uptake when used in combination with specific metabolites. They found that 10 mM glucose, fructose, mannitol and 20 mM pyruvate given along with gentamicin could reduce the number of persisters in vitro by more than 99.9% than with gentamicin alone. This activity was found to be antibiotic specific (i.e. only with aminoglycosides, but not with other groups of antibiotics). The authors found that the metabolites induced an elevated proton motive force in persisters which facilitated the uptake of aminoglycosides resulting in their rapid killing. Since persisters are dormant bacteria with reduced PMF, they usually overcome the bactericidal activity of aminoglycosides by not taking up the antibiotic.

However, if 10mM glucose along with gentamicin can significantly reduce the number of persisters, why should infections persist in our body? The average blood glucose levels in humans fluctuate around 5mM and can go as high as 8-12 mM after a rich meal. Even at 5mM glucose, a significant number of persisters are killed (suppl. Fig. 6a, Allison et al. (2011)). It means that the normal glucose level is high enough to eradicate persisters in biofilms when aminoglycosides are used for treatment.

However, blood glucose levels can vary in different tissues or fluids. For example, glucose concentration is very low in airway secretions of healthy, normal individuals (Philips et al. 2003, Baker et al.2007). Hence it can be argued that addition of sugars with aminoglycosides can work well in chronic infections like cystic fibrosis (CF) or tuberculosis. However, glucose concentrations in nasal or bronchial secretions are usually elevated in hyperglycemia and in inflammatory conditions of lungs including CF (Baker et al. 2007). Equally important is that elevated bronchial glucose is associated with increased respiratory infections (Baker et al. 2007). Thus, in CF and tuberculosis, even though the glucose level in respiratory fluids may be high enough for ‘waking up’ persisters from dormancy, the infection persists.

Similarly, suppl. Fig. 6 (Allison et al. 2011) shows that the eradication of persisters is dependent on metabolite concentration. Does it mean that a patient with diabetes mellitus have greater chance for successful treatment of biofilm related infections with aminoglycosides, as their blood glucose level is higher? As indicated above, respiratory fluid glucose concentration is elevated in hyperglycemic conditions.

However, mannitol given in combination with aminoglycosides can be effective in reducing bacterial load in infections such as CF. But this may not be due to metabolite-enabled elimination of persisters. This aspect will be discussed next.

Allison et al. (2011). Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature 473: 216-220.

Baker et al. (2007). Hyperglycemia and cystic fibrosis alter respiratory fluid glucose concentrations estimated by breath condensate analysis. J Appl Physiol 102: 1969-1975.

Philips et al. (2003). Factors determining the appearance of glucose in upper and lower respiratory tract secretions. Intensive Care Med 29:2204-2210

What difference it will make if the number of persisters is 10^5 or 10^6?

Researchers claim that antibiotics may not be able to sterilize a bacterial culture completely and that a small subpopulation of less than 1% of bacteria (persisters) can survive antibiotic treatment which, on removal of antibiotic, may undergo phenotypic shift and cause infections. Some strategies have been proposed to eliminate persisters that have the potential in the treatment of chronic infections and biofilm-related infections. One of the strategies is to wake the persisters from dormancy and make them succeptible to antibiotics.

Allison et al. (2011) proposed that addition of specific metabolites can kill persisters when given along with aminoglycosides. They reported that metabolites such as glucose, fructose or mannitol can induce proton motive force in persisters which will help in the uptake of aminoglycosides resulting in their killing. They noticed that this strategy might work in vivo also. In their experiment, catheters colonized with E.coli biofilms were implanted in the urinary tracts of mice and treated with antibiotic (gentamicin) alone and also with a combination of antibiotic and mannitol. After three days of treatment, catheter tubing was extracted to determine biofilm viability and kidneys were removed to determine bacterial load. They found that gentamicin alone was not effective, whereas gentamicin in combination with mannitol reduced the viability of the catheter biofilms by nearly 1.5 orders of magnitude. They also found that the kidney bacterial load was approximately 10⁷ cfu/g of kidney for untreated animals, slightly higher than 10⁶ cfu/g for gentamicin alone treatment group but only 10⁵ cfu/g of kidney for the combination treatment group. Based on these findings, authors claimed that in vivo results demonstrate the feasibility of their approach for clinical use.

However, were the researchers able to eliminate persisters? Persisters are a small subpopulation of less than 1% bacteria that can survive antibiotic therapy and which undergo phenotypic shift once the antibiotics are removed. The number of bacteria that survived the combination treatment was still more than 1% of the total population of untreated control. How can the researchers claim that the above approach was successful against persisters?

What will happen to the 10⁶ cfu/g of kidney that persisted following gentamicin alone treatment? Once the antibiotic is removed from the body, those persisters will undergo ‘phenotypic shift’, re-grow and cause infection.

And what will happen to the 10⁵ cfu/g of kidney that persisted following the combination therapy? The same…..

Hence, is there any difference between antibiotic alone treatment and combination treatment as far as re-growth of persisters and treatment of biofilms or chronic infections are concerned? No...

In order to claim that their strategy is effective, the researchers should either

a. be able to sterilize the biofilms without leaving any persisters or

b. assume that 100% bacterial killing is not necessary (i.e. a reduction in bacterial load is sufficient for successful treatment). However, if this is the case, what is the significance of persisters?

Next- Can a spoonful of sugar helps in the treatment of persistent infections?

Allison et al. (2011). Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature 473: 216-220

Can we use MIC99 or MBC 99 in determining the extent of persister killing?

MIC and MBC are the major pharmacodynamics parameters used to quantify antibiotic activity against bacteria. MIC is defined as the minimum concentration of antibiotic that inhibit the growth of at least 99% of the population whereas MBC is the minimum concentrations of antibiotic required to kill at least 99% of the population. However, Keren et al. (2004) argues that MBC do not take into account of persisters which constitute less than 1% of the population since persisters will not be reported in conventional MBC tests as it do not fall within the above accepted range.

Byrne et al. (2007) reported that diethyldithiocarbamate (DETC) and pyrrolidine dithiocarbamate (PDTC) are active against growing and non-growing persisters of Mycobacterium tuberculosis. The activities of above agents were determined using the conventional methods of determining MIC99 and MBC99. They also found that DETC and PDTC enhanced the activities of antituberculosis drugs against both young and old cultures. They noticed a reduction in colony forming units when antituberculosis drugs were used in combination with DETC and PDTC.

However, what is the basis of using MBC99 in determining the activities of those agents against persisters? By using MBC99, aren’t they missing the same persisters they are supposed to kill? Similarly, the combination of the above agents with antituberculosis drugs has only reduced the number of CFU, but has not completely killed all the bacteria. How can it be concluded that the above agents are active against persisters?

To me, that article only indicates that DETC and PDTC enhance the killing activities of antituberculoss drugs, but does not give any indication of their activities against persisters.

Next- What difference it will make if the number of persisters is 10⁵ or 10⁶?

Keren et al. (2004). Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 230(1), 13-8.

Byrne et al. (2007). Pyrrolidine dithiocarbamate and diethyldithiocarbamate are active against growing and nongrowing persister Mycobacterium tuberculosis. Antimicrobial Agents and Chemotherapy 51(12):4495-4497.

Is 100% in vitro bacterial killing the most important parameter determining the in vivo efficacy of antibiotics?

Persisters are implicated in chronic recurrent infections and in biofilms because the antibiotic treatment may leave a small subset of persisters which may later repopulate and cause infections (Lewis 2007). This small subpopulation is considered to be the nucleus for chronic infections. However, the significance of these in vitro survivors is questionable as they may not affect the outcome of treatment.

If 100% killing is necessary, how can one justify the determination of dosage regimen of an antibiotic using the kinetic parameters based on MIC? After all, MIC only indicates that bacteria are inhibited from growth. The important PK/PD parameters such as the percentage of time above the MIC (t > MIC), ratio of peak concentration to MIC (Cmax/MIC) and the ratio of the area under the curve to MIC (AUC/MIC) are based on MIC. If 100% in vitro killing is a necessity, will it be more appropriate to use MBC values? Even MBC may not be ideal as, by definition, it kills only 99.9% of bacteria. Should we consider MBC₁₀₀ as the single most important parameter that determine the in vivo antibiotic efficacy?

Next- Can we use MIC99 or MBC 99 in determining the extent of persister killing?

Lewis, K. (2007). Persister cells, dormancy and infectious diseases. Nat Rev Microbiol. 5(1): 48-56.

Genes implicated in persistence

Fauvart et al. (2011) provides an exhaustive overview of genes implicated in persistence. They divide the genes into true persister genes (mutants which exhibit the same MIC as wild type), probable persister genes (MIC values were not determined) and probable persister or resistant genes (MIC different from that of wild type).

True persister genes include hipBA, glpD, glpABC, plsB, dnaK, dksA, apaH, surA, ygfA, ygfB, yigB, hupB, yafQ, tisAB, istR-1, istR-2 and ihfB in E.coli., relA, relA spoT, rpoS, rpoN, lasR, lasI, ding, spuC, edpA, fosA, glpT, algR, ycgM, pheA, plH and yfiR in P.aerogenosa and vcnS in Streptococcus pneumoniae.

Probable persister genes include lexA3, recA13, hhA, hokA, cspD, mqsR, ybfM, hfq, recA, recB, xerC, xerD, relE, dnaJ, pmrC, oppA and mazF/chpAI in E.coli, relE2 and relE3 in M. tuberculosis. 

Probable persister/resistant genes include phoU, phoY2, sucB and ubiF.

Even though hipA or tisB are best studies persister genes, neither hipA not tisB is present in the genomes of M. tuberculosis, P. aeruginosa or S. aureus (Fauvert et al. 2011).

So, how specific is the role of hipA or other genes in persistence?

Fauvart et al. (2011). Role of persister cells in chronic infections: clinical relevance and perspectives on anti-persister therapies. J Med Microbiol 60(6): 699-709.

Is there a clear relationship between hipA and the phenotypic shift of persisters?

The number of persisters increases during late exponential and stationary phases. Also, the overexpression of HipA produces a high frequency of persisters (Falla and Chopra 1998). Similarly, mutation in hipA also produces a high frequency of persisters (Moyed and Bertrand 1983). Based on these findings, it was proposed that persister formation is governed by both deterministic mechanisms and by stochastic fluctuations in the expression of HipA. However, researchers have not provided any evidence to demonstrate such stochastic fluctuations in the levels of HipA following antibiotic treatment. Moreover, overexpression of a number of proteins can generate a high frequency of persisters, as indicated  previously.

hipA mutants can not be easily isolated even though persisters are present in any bacterial cultures. After plating approximately 10¹⁰ bacteria on agar plates containing different concentrations of ciprofloxacin for 96 h, Marcusson et al. (2005) found that some bacteria could survive the lethal action of the antibiotic. However, the survival of this subpopulation was not associated with any mutation in the hipA or hipB genes, indicating that the persistence of bacteria in the presence of antibiotics may not be due to hipA or hipB mutations.

Moreover, hip locus is restricted to relatively few bacterial species, (not even all strains of E. coli), and hence the antibiotic tolerance due to hip mutations may not be clinically significant (Falla and Chopra 1998). Similarly, persistence in many species of bacteria may not be related to hip (Falla and Chopra 1998).

Thus the role of hipA in the phenotypic shift of persisters is vague and obscure.

Falla, T. J., and Chopra, I. (1998). Joint tolerance to beta-lactam and fluoroquinolone antibiotics in Escherichia coli results from overexpression of hipA. Antimicrob Agents Chemother 42(12), 3282-4.

Moyed, H. S., and Bertrand, K. P. (1983). hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. J Bacteriol 155(2), 768-75.

Marcusson et al. (2005). Mutant prevention concentration of ciprofloxacin for urinary tract infection isolates of Escherichia coli. J Antimicrob Chemother 55(6): 938-43.

How specific is the role of hipA in persister generation?

The toxin-antitoxin (TA) system, found in the plasmids and chromosomes of many bacteria, is implicated in persister formation. This system consists of a pair of genes, one of which encodes a stable toxin and the other of which encodes an unstable antitoxin. Examples for such systems include hipBA, relBE, mazEF etc. In the presence of the antitoxin, the expression of the toxin, which inhibits translation, is down regulated. However, when the antitoxin level is reduced, the expression of the toxin becomes predominant, resulting in the inhibition of translation and thus the protein synthesis that finally results in programmed cell death (PCD).

The major TA system involved in persister cell formation is hipBA. Mutations in hipA produce a high frequency of persisters (Moyed and Bertrand 1983). Similarly, overexpression of HipA results in 10-to-1000 fold increases in persister formation (Falla and Chopra 1998; Keren et al. 2004; Korch and Hill 2006). In addition, overexpression of RelE, the toxin protein of another TA system, also results in a high frequency of persisters (Keren et al. 2004). Thus, specific roles for these toxins in persister formation were proposed.

However, the importance of TA modules in persister formation is questioned by some researchers. Vazquez-Laslop et al. (2006) found that the cells overexpressing proteins unrelated to TA modules, also resulted in a high frequency of persisters. They found that the proteins that are toxic to the cell, when overexpressed, would result in a high frequency of persisters, thus questioning the specific roles of hipA or other TA modules in persister generation. This raises the question whether the in vitro over-expression of proteins is a reliable method to demonstrate the phenotypic shift of persisters.

Falla, T. J., and Chopra, I. (1998). Joint tolerance to beta-lactam and fluoroquinolone antibiotics in Escherichia coli results from overexpression of hipA. Antimicrob Agents Chemother 42(12), 3282-4.

Keren et al. (2004). Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 230(1), 13-8.

Korch, S. B., and Hill, T. M. (2006). Ectopic overexpression of wild-type and mutant hipA genes in Escherichia coli: effects on macromolecular synthesis and persister formation. J Bacteriol 188(11), 3826-36.

Moyed, H. S., and Bertrand, K. P. (1983). hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. J Bacteriol 155(2), 768-75.

Vazquez-Laslop et al. (2006). Increased persistence in Escherichia coli caused by controlled expression of toxins or other unrelated proteins. J Bacteriol 188(10), 3494-7.

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.

Don't miss those 1.5 hours to treat persistent infections

One of the strategies proposed for the treatment of persistent infections is to kill bacterial cells with a high dose of an antibiotic and allowing the antibiotic concentration to decrease to enable the growth of persisters followed by the administration of a second dose of antibiotic shortly after persisters start to grow (Lewis 2007). Later Gefen et al. (2008) found that even the persisters are vulnerable to the action of antibiotics over a narrow time window after the exit from stationary phase. They found that, during the first 1.5 h after the transfer to fresh medium, the non-growing cells are vulnerable to the action of ampicillin. However, after this period, those cells get differentiated into the dormant state that protects them from the bactericidal action of ampicillin. Authors suggest that, by subjecting the bacterial populations to antibiotics within this time window, before the onset of full dormancy, it might be possible to prevent persistence.

However, translating this knowledge to in vivo situations will be difficult. In the above in vitro experiment, an overnight bacterial culture was diluted in fresh medium and at each time point after the dilution, an aliquot of the culture was directly exposed to ampicillin for 5 h, and the number of persisters was calculated to determine their vulnerability to the antibiotic. In vivo, the antibiotics can not be removed completely at once since the concentration of the antibiotic decreases only gradually depending on the half life of the antibiotic. The concentration of the antibiotic at which persisters start to grow is not known. The knowledge of this concentration is important because the time window during which the persisters are vulnerable to the action of antibiotics is very narrow (i.e. 1.5 h for ampicillin). How can we subject the bacterial populations to antibiotics within this time window without knowing the antibiotic concentration at which the persisters start to grow?

The fundamental problem with these type of conclusions is that the researchers try to extrapolate in vitro results as such to in vivo conditions. The effects of metabolism and hence the half-life of the antibiotic, post-antibiotic effect, plasma protein binding, tissue distribution etc. are never taken into consideration in in vitro experiments. Hence the chances of errors are high when extrapolating results from the static in vitro experiments as such to in vivo conditions.

The above article has shown that, even though persisters are intransigent to the action of antibiotics, they have a generous side also. They offer us a window of opportunity to eliminate them. Clinicians can now utilize this 1.5 h window of opportunity to treat persistent infections!!

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

Lewis, K. (2007). Persister cells, dormancy and infectious diseases. Nat Rev Microbiol. 5(1): 48-56.

Gefen et al. (2008). Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria. Proc Natl Acad Sci USA 105(16): 6145–6149.

   

Is it possible to treat persister infections serendipitously?

Even though persister infections are difficult to treat with antibiotics, it may be possible to eliminate persister bacteria serendipitiously as per Lewis (2007).   

“The goal of established therapies is to maintain the plasma level of an antibiotic at a maximum concentration, in order to discourage the development of resistance. Most importantly, an optimal pulse-dosing regimen would probably vary from patient to patient. However, it seems that some patients might have inadvertently taken solving the problem of intractable persistent infections into their own hands. Individuals who suffer from persistent infections that require a lengthy therapy are often cured, but why a year-long regimen is better than a month-long one is unclear. An efficacious fluctuating dose of antibiotics administered serendipitously by the patient might be responsible for persister eradication in these cases. The patients might adjust drug dosing simply through being absent-minded, which sooner or later could produce the perfect drug-administration regimen. Curing persistent infections might therefore result from patient non-compliance. Analysing how persistent infections are cured might shed light on the likelihood of developing a rational regimen for the pulse-dosing sterilization of infection.”

Is maintaining the plasma level of antibiotics at maximum concentration always the goal of antibiotic therapy? This goal may be true for those antibiotics exhibiting concentration-dependent killing. However, for time-dependent killing antibiotics, percentage of time the concentration of the antibiotic is maintained above MIC (t>MIC) is the most important parameter determining the efficacy of the antibiotic.

Can the suggestion that patient non-compliance or serendipitous administration of antibiotics by the patient may cure persistent infections be supported by any pharmacokinetic/pharmacodynamic models? Isn’t true that, one of the reasons for the development of drug resistance leading to antibiotic treatment failure is the non-adherence to antibiotic regimen by patients?

Next- Don't miss those 1.5 hours to treat persistent infections

Lewis, K. (2007). Persister cells, dormancy and infectious diseases. Nat Rev Microbiol. 5(1): 48-56.

If persisters can be killed by a second dose of antibiotic, what is their clinical significance?

The experiments demonstrating phenotypic shift have used only a static approach; i.e., a culture of bacteria incubated with a single dose and a single concentration of antibiotic for a relatively small period of time. However, this static approach has a number of disadvantages and is not a true reflection of in vivo conditions. In vivo, antibiotics are administered as multiple doses for several days. Antimicrobial efficacy results from the exposure of bacteria to variable antibiotic concentrations. Thus, in vitro conditions with a single dose and concentration of antibiotics may not reflect the true dynamic situation in the target organ and hence the result can not be extrapolated as such to in vivo conditions.

As reviewed by Lewis (2007), it may be possible to sterilize an infection by using a simple approach:

“A disarmingly simple approach to sterilize an infection was first proposed by Bigger in 1944. The proposal is to kill bacterial cells with a high dose of an antibiotic, then allow the antibiotic concentration to decrease, which will enable persisters to resuscitate and start to grow. If a second dose of antibiotic is administered shortly after persisters start to grow, a complete sterilization might be achieved. This approach is successful in vitro, and a P. aeruginosa biofilm can essentially be sterilized with 2 consecutive applications of a fluoroquinolone (K. L., unpublished observations). Perhaps understandably, this approach has not been received with enthusiasm by specialists in clinical microbiology.”

Is this not the same ‘approach’ we follow during the treatment of bacterial infections with antibiotics? After the first dose of the antibiotic, the concentration of the antibiotic decreases depending on its half-life and is followed by the next dose. In clinical infections, most antibiotics are administered as multiple doses for several days. If a P. aeruginosa biofilm can essentially be sterilized with 2 consecutive applications of a fluoroquinolone or if a second dose of antibiotic can kill all persisters and achieve complete sterilization, what is the clinical significance of persisters?

Next- Is it possible to treat persister infections serendipitously?

Lewis, K. (2007). Persister cells, dormancy and infectious diseases. Nat Rev Microbiol. 5(1): 48-56.

Can treatments with bacteriostatic drugs produce persisters?

Antibiotics, in general, can be divided into bactericidal and bacteriostatic drugs, depending on their mechanism of action. Bactericidal drugs like penicillin, aminoglycosides etc., act by killing the bacteria whereas bacteriostatic drugs like tetracycline, chloramphenicol, macrolides etc., act by inhibiting bacterial growth. However, it is difficult to classify antibiotics into true bactericidal or bacteriostatic antibiotics (Pankey and Sabath 2004). Under certain conditions, bactericidal drugs may only inhibit the growth of bacteria. Similarly, bacteriostatic drugs also kill bacteria depending on the concentration of antibiotic, total time of incubation and the sensitivity of the organism. Many bacteriostatic drugs kill 90-99% of the bacteria after 18-24 h, but do not kill 99.9% of them to be termed as bactericidal drugs (for a detailed review of the clinical significance of bactericidal and bacteriostatic antibiotics, see Pankey and Sabath 2004). Here again, the survivors remain in a dormant stage in the presence of the bacteriostatic antibiotic but may regrow once the antibiotic is removed. Are those 1-10% or more of the survivors persisters? If they are, will it be easy to isolate persisters after treatment with bacteriostatic drugs? If they are not, how can we distinguish the survivors following treatment with bactericidal and bacteriostatic antibiotics? Or in other words, why are the survivors following bactericidal drugs persisters and those following bacteriostatic drugs not?

Even though the static drugs, in general, do not kill 99.9% of bacteria after 18-24 h of incubation, they are powerful antibiotics widely used in treatment of many bacterial infections. If persisters are responsible for recurrent infections, one has to assume that the use of bacteriostatic drugs almost always carries the risk of chronic or recurrent infections. Is it safe to use bacteriostatic antibiotics since they carry the risk of chronic infections?

Next- If persisters can be killed by a second dose of antibiotic, what is their clinical significance?

Pankey, G. A., and Sabath, L. D. (2004). Clinical relevance of bacteriostatic versus bactericidal mechanisms of action in the treatment of Gram-positive bacterial infections. Clin Infect Dis 38(6), 864-70.

Persisters induced by antibiotics- Old wine in new bottle?

Persisters are considered to be a preformed subpopulation in a bacterial culture which are not induced by antibiotics. However, Dorr et al. (2009) reported an inducible mechanism of persister formation mediated by SOS response. They found that ciprofloxacin treatment killed a majority of the population except for a small fraction of persisters. Those persisters were not a pre-formed subpopulation, but were induced by antibiotic treatment, a finding contrary to previous views. They noticed that an increase in ciprofloxacin concentration from 0.02 µg/ml to 0.5 µg/ml increased the average SOS induction and reduced the number of persister cells (Fig.2., Dorr et al. 2009). It was suggested that the bacterial persistence in the presence of ciprofloxacin was dependent on a functional SOS response. However, since all cells exposed to ciprofloxacin could induce SOS, but only a fraction became persisters, it was suggested that a specific high or low level of SOS induction was required for persister formation (Dorr et al. 2009).

However, it can be noticed that, when the ciprofloxacin concentration was increased from 0.5 µg/ml to 1 or 2 µg/ml, the persister fraction also increased with increase in SOS induction (Fig.2., Dorr et al. 2009). Increase in the number of survivors following exposure to high concentrations of quinolones has already been reported. It can be observed with a variety of quinolones at different incubation temperatures (Malik et al. 2009). Paradoxical effect of ciprofloxacin (quinolone) and oxacillin (β-lactam antibiotics) has been demonstrated by Udekwu et al. (2009) also.

Even though quinolones induce an SOS response, the SOS-DNA repair system may not play any role in protecting the bacteria from damage or cause the death of bacteria (Lewin et al. 1989). SOS induction upon treatment with quinolones may not have any effects on bacterial survival (Lewin et al. 1989). Similarly, SOS response may not be required for the paradoxical survival of bacterial cells at high quinolone concentration (Malik et al. 2009).

Dorr et al. (2009) also noticed that bacterial cultures pretreated with a low concentration of ciprofloxacin produced more persisters when incubated with higher ciprofloxacin concentrations. Based on these findings, the authors concluded that persisters are formed upon ciprofloxacin treatment rather than performing. However, the finding is not at all surprising or new. It is already reported that sub-inhibitory ciprofloxacin can induce adaptive resistance (Gould et al. 1990; Brazas et al. 2007) and that subinhibitory concentrations alter the expression of many chaperones and heat shock proteins (Brazas et al. 2007). Adaptive resistance has been reported with other antibiotics and biocides also.

Thus, Dorr et al. (2009) failed to correlate the paradoxical effect and inducible persister formation by ciprofloxacin. Even though the authors were noticing the paradoxical effect of ciprofloxacin, they failed to report it. Paradoxical effect could have been more evident, had the authors used still higher concentrations of ciprofloxacin. There is an ideal concentration of quinolone where survivors are minimal. Concentrations above or below this critical level may generate more survivors in vitro and induce an SOS response. However, the induction of SOS may not be responsible for the survival of these persisters. Similarly, adaptive resistance induced by subinhibitory concentrations of ciprofloxacin (an already known phenomenon) is made into a new topic by using the term persisters.

Next- Can treatment with bacteriostatic drugs result in persister formation?

Dorr et al. (2009). SOS response induces persistence to fluoroquinolones in Escherichia coli. PLoS Genet 5(12), e1000760.

Malik et al. (2009). Lon protease is essential for paradoxical survival of Escherichia coli exposed to high concentrations of quinolone. Antimicrob Agents Chemother 53(7), 3103-5.

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

Lewin et al. (1989). 4-quinolones and the SOS response. J Med Microbiol 29(2), 139-44.

Gould et al. 1990. Concentration-dependent bacterial killing, adaptive resistance and post-antibiotic effect of ciprofloxacin alone and in combination with gentamicin. Drugs Exp. Clin. Res. 16:621-628.

Brazas et al. 2007. Role of Lon, an ATP-dependent protease homolog, in resistance of Pseudomonas aeruginosa to ciprofloxacin. Antimicrob Agents Chemother, 51(12): 4276-4283.

Other factors that determine the number of survivors

Apart from the initial inoculum size, the concentration of the antibiotic, the time of incubation and the type of antibiotics influence the number of bacterial survivors following exposure to antibiotics.

Antibacterial activity of antibiotics, in general, is proportional to its concentration. However, at very high concentrations of some antibiotics, this may not be true. Antibiotics are most effective in a narrow range of concentration above the MIC. When the concentration of antibiotics is well above the MIC, a paradoxical effect can be noticed, wherein some bacteria are not killed but are only inhibited from growing. This is well documented with nalidixic acid and also reported with other antibiotics such as β-lactam antibiotics. Paradoxical effect of oxacillin and ciprofloxacin was recently demonstrated by Udekwu et al. (2009). With high inoculum size, the number of survivors after 18 h of antibiotic treatment was more with 100 times the basal MIC (bMIC) than with 5 times the bMIC. This was true for ciprofloxacin also.

Total time of incubation also influence the number of survivors. The importance of this factor was also demonstrated by Udekwu et al. (2009). In certain cases, the number of survivors is high after a short incubation period of 3 h but progressively decreases following 18 h of incubation (ex: vancomycin at 20 times the bMIC against low cell density), whereas in other cases the number of survivors increases following 18 h of incubation (ex: daptomycin at 5 times the bMIC against low cell density), which was not due to the re-growth of mutant population.

The type of antibiotics is another factor in determining the number of survivors. Concentration dependent antibiotics such as aminoglycosides (ex: gentamicin or tobramycin) produce low number of persisters. This group of antibiotics exhibit minimal inoculum effect and paradoxical effect. The lack of persisters following exposure to aminoglycosides, reported in many experiments, is due to the property of the antibiotic only.

The demonstration of persisters following incubation of a single inoculum size of bacterial culture with a single antibiotic concentration for a specific period of time (3-6 h) is questionable. The persisters and their phenotypic shift demonstrated in many experiments may not be a survival strategy of the bacterial population, but rather the property of antibiotics itself which have been known and reported decades back. The dosage regimen of the antibiotics have been optimized only after considering all these factors.

Next- Persisters induced by antibiotics- Old wine in new bottle?

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

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.

Where 94% is a small subpopulation…

Persisters are defined as a small subpopulation of bacteria, constituting less than 1% of the total population, that are either dormant or slow growing and capable of evading death by antibiotics by exhibiting transient multi-drug tolerance.

Recently it was shown that, following exposure to levofloxacin and vancomycin, Staphylococcus epidermidis RP62a biofilms contained 28% and 94% persisters respectively (Shapiro et al. 2011). Are they persisters, by definition? Similarly, following exposure to the above antibiotics, the number of planktonic persister cells was less than 1% of the total population during the log phase, but produced almost the same number of cells as the antibiotic-untreated control group during stationary phase. Are these cells persisters? As per Lewis (2007), the maximum number of persisters is ~1% of cells which are produced during the stationary phase. “examination of the rate of E. coli persister-cell formation over time showed that few of these cells are formed in early exponential phase, followed by a sharp increase in persister-cell formation in mid-exponential phase, reaching a maximum of ~1% of cells forming persisters in the non-growing stationary phase”.

Low number of persisters is considered as a barrier to the discovery of persister genes (Lewis 2007). However, if persisters constitute more than 90% of the population (Shapiro et al. 2011), why it is difficult to isolate persisters and study persister genes? Similarly, why researchers have to depend on expensive and complicated microfluidic devices to isolate persisters (Balaban et al. 2004)?

The reason for the discrepancy is that the definition for persisters is based on particular experimental condition only and that the researchers have ‘misused’ the term persisters for any type of survivors following antibiotic treatment. As for another example, Harrison et al. (2005) found that 24 h of exposure of E. coli JM109 to antibiotics did not produce any survivors whereas a short incubation for 2 h resulted in many survivors. Similarly, they found that the highly tolerant E. coli JM109 survivors could be eradicated by metal oxyanions by 24 h exposure. Are the survivors after 2 h exposure to antibiotics or metal oxyanions persisters?

Next- Inoculum effect- a factor affecting the number of survivors

Shapiro et al. (2011). Evidence for persisters in Staphylococcus epidermidis RP62a planktonic cultures and biofilms. J. Med. Micro. DOI: 10.1099/jmm.0.026013-0

Harrison et al. (2005). Persister cells mediate tolerance to metal oxyanions in Escherichia coli. Microbiology 151: 3181-3195.

Lewis, K. (2007). Persister cells, dormancy and infectious diseases. Nat Rev Microbiol. 5(1): 48-56.

Balaban et al. (2004). Bacterial persistence as a phenotypic switch. Science 305 (5690): 1622-1625.

Experimental demonstration of persisters

In most of the experiments demonstrating persisters, bacterial cultures were incubated in the presence of antibiotics for 3-6 h. At the end of the incubation period, the majority of the bacteria were found to be dead, except for a small subpopulation of persisters. During the incubation period, the persisters were either growing slowly or not at all, but once the antibiotic was removed, they were able to grow normally.

Researchers had incubated bacterial cultures using a single concentration and dose of the antibiotic against a specific inoculum size for a very short incubation time and then assumed that the bacteria that survived the antibiotic treatment are persisters that have the capability to undergo a phenotypic shift. However, a different result would have been obtained had the researchers used  

1. different inoculum sizes (inoculum effect)

2. different concentrations of antibiotic

3. different total time of incubaton and

4. multiple doses of antibiotics

The definition of persisters as a small subpopulation of bacteria (less than 1% of the total populaton) that are not killed in the presence of bacteria is based on a particular experimental condition only. This definition can change once the conditions are also changed. For example, had the researchers incubated the bacterial culture with antibiotic for 24-48 h, rather than 3-6 h (at the same antibiotic concentration and inoculum size used in their experiments), they would have noticed one of three conditions.

1. Complete sterilization of the culture without any persisters;

2. Re-growth of the remaining survivors even in the presence of antibiotics;

3. Or, less frequently, the surviving bacteria remaining in the dormant stage itself without growth.

Similarly, had the researchers tested different antibiotic concentrations and inoculum sizes followed by incubation for 24-48 h, they would have noticed the optimal conditions for complete sterilization. The perceived phenotypic shift demonstrated in many experiments can be the result of the failure of antibiotics to kill all bacteria owing to the suboptimal conditions of killing provided in the experiments.

Next- Where 94% is a small subpopulation…

Persister bacteria and their role in chronic infections- a brief review

Complete sterilization of a bacterial culture is expected when an antibiotic-sensitive bacterial population is treated with optimal concentrations of antibiotics. However, Joseph Bigger in 1944 noticed that penicillin could not sterilize a Staphylococcal culture completely even in the absence of a penicillin-resistant population. While most of the bacteria were killed by penicillin, Bigger noticed that a small subpopulation somehow survived which were neither killed nor grown in the presence of antibiotics. Upon removal of penicillin, the survivors grew abundantly just like the parent population. They were as sensitive as the parent population to the bactericidal action of the antibiotic but again left a small percentage of survivors. It is argued that the survival of this small subpopulation of persisters in the presence of antibiotics is not due to antibiotic resistance, but rather to their ability to undergo a phenotypic shift by remaining in a dormant, non-dividing state that enable them to survive the lethal actions of antibiotics.

Some of the properties of persisters are

-they are not induced by antibiotics nor are they mutants.

-their formation depends on the growth stage of bacteria but they are not cells at a particular stage in the cell cycle. During the stationary phase, the number of persisters increases.

-they represent a very small subpopulation of less than 1% of the total population.

-persisters are tolerant to antibiotics and are neither killed nor grown in the presence of antibiotics. They exhibit increased MBC (minimum bactericidal concentration) but show almost the same MIC (minimum inhibitory concentration) as the wild type

-they are pre-formed rather than being generated during the antibiotic treatment. However, an inducible mechanism of persister formation mediated by SOS response has been recently reported

-the phenotypic shift of persisters is reported to be under genetic control since overexpression of genes such as hipA, relE, mazF results in high number of persisters.

Isolating persisters is difficult because of their low number in the presence of antibiotics and because of their ability to undergo phenotypic shift upon the removal of the antibiotic. However, high persister (hiP) mutants of E. coli, first isolated by Moyed and Bertrand (1983), can be used to obtain large number of persisters.

Persisters are implicated in chronic and recurrent infections as they can survive antibiotic treatment and re-grow later on the removal of antibiotics. Similarly, they are implicated in biofilm-associated infections. Persisters in the biofilm may be more important than the planktonic cells in recurrent infections (Lewis 2007). Antibiotic treatment may kill majority of the biofilm and planktonic cells but may leave the persisters intact. Whereas the planktonic persister cells may be removed by the immune system, the biofilm persisters may be protected by the matrix from the immune cells. Once the antibiotic is removed, those persisters in the biofilm may start to grow and repopulate the biofilm, which may later release some planktonic cells causing recurrent infections.

Lewis, K. (2007). Persister cells, dormancy and infectious diseases. Nat Rev Microbiol. 5(1): 48-56.