Is it an indication of the current peer-review process?

The following news articles published last week are worth reading

1. Fraud case we might have seen coming (http://www.nature.com/news/2011/110728/full/news.2011.437.html)

2. Paper on genetics of longevity retracted (http://www.nature.com/news/2011/110721/full/news.2011.429.html)

3. Scientist in hot water over polar bears, Al Gore (http://www.msnbc.msn.com/id/43933715/ns/us_news-environment/?gt1=43001)

4. Peer review in science can be improved, MPs report (http://www.bbc.co.uk/news/science-environment-14314501)

Does this say anything about the current policies in science and research?

Is it not important to incorporate the knowledge on PK/PD when studying bacterial killing kinetics?

Many researchers studying bacterial killing by antibiotics use single culture conditions as indicated in the earlier blogs. However, such conditions do not take into account of

1. variable drug levels that occur under in vivo conditions

2. protein binding. In in vitro conditions, drug is available as free drug whereas in in vivo conditions, drugs can be bound to proteins. Only the unbound drug is pharmacologically effective and relevant in predicting therapeutic efficacy

3. concentration of drug at target sites- the drug penetration into the target sites can be influenced by a number of factors which are absent in in vitro conditions

4. tissue distribution- depending on the extent of binding of drug molecules to tissue proteins, the concentration of unbound (free) drug can vary

5. importance of immune cells

6. the role of inoculum effect, paradoxical effect etc. (discussed earlier)

Because of these disadvantages, MIC or MBC or MBC₁₀₀ can not be considered as ideal pharmacodynamic parameters. They can only give approximate information about the effect of antibiotics.

Is it a good practice to extrapolate the results of single culture conditions as such to in vivo situations?

Persisters and phenotypic shift- some concluding remarks

Researchers have considered any bacteria that survive antibiotic therapy as persisters. Persisters, thus include

1. a small subpopulation of bacteria that survive antibiotic treatment in vitro

2. bacteria in biofilms, not killed by antibiotics

3. stationary phase bacteria, tolerant to antibiotics

4. mutant bacteria including hipA mutants

5. bacteria surviving  in chronic and recurrent infections in spite of antibiotic therapy

Even though they are all termed persisters, the reason why they survive may be different for each of the above cases and some of those persisters (as demonstrated in many experiments) may not have any clinical significance.

Similarly, some of the statements on persisters and their phenotypic shift are contradictory

1. Even though they are termed non-mutants, majority of persisters have a genetic background (refer July 18 blogpost)

2. isolation of persisters is considered difficult as they constitute less than 1% of the population. However, persisters may form upto 94% in biofilms and hence isolation should not be difficult (June 3)

3. Even though hipA is the best studied persister gene, it is not present in the genomes of M. tuberculosis, P. aeruginosa or S. aureus, the organisms responsible for many chronic infections (June 23 and 27)

4. Moreover, hipA mutants have not been isolated either in vitro or in vivo for decades (July 16)

5. Even though researchers argue that MBC do not take into account of persisters, they still use MBC values in determining the activities of antimicrobial agents against persisters (June 26)

6. Many of the treatment strategies against persisters are questionable (June 10, 13, 15 and 28)

It is known that chronic infections and biofilm-associated infections are difficult to treat with conventional antibiotic therapy. However, using this knowledge, researchers have created an in vitro illusion through retrospective thinking to demonstrate that such infections are caused by a phenotypic shift of persisters.

Why some antibiotic combination treatments are effective against persisters?

Researchers claim that persisters are tolerant to multiple antibiotics. In such experiments, they isolate persisters in vitro after treating with one antibiotic and then test the killing rate of persisters with other antibiotics. Based on these results, they report that persisters are multi-drug tolerant cells.

However, in many instances, persisters can be killed by combinations of antibiotics. Minardi et al. (2011) found that a combination of intraperitoneal tygecycline and stent-bonded rifampin was effective in sterilizing both urine culture and ureteral stent culture (biofilms) against Enterococcus faecalis whereas treatment with tygecycline or rifampin alone were not effective in sterilizing the culture (tygecycline alone treatment left a small subpopulation of bacteria which constituted less than 1% of the control group). Similar results were also reported with combinations of clarithromycin and amikacin against P.aeruginosa biofilms in an animal model of ureteral stent infection (Cirioni et al. 2011).

Why combination treatment was better against the persisters in biofilms in the above case? Or in other words, how did 3.8x10³ cfu/ml of bacteria in the stent culture that survived tygecycline alone treatment were killed completely when rifampin was given along with tygecycline? If the same experiment were done with tygecycline alone, one would have considered those bacteria as persisters (in the above articles, authors have not used the term 'persisters'). This raises the question whether the persisters reported in many experiments are ‘true persisters’ or simply bacteria that survived antibiotic therapy due to suboptimal conditions.

Earlier, I had argued that many of the experiments demonstrating ‘persisters’ are the result of suboptimal conditions provided in their experiments.  

Minardi et al. (2011). Efficacy of tygecycline and rifampin alone and in combination against Enterococcus faecalis biofilm infection in a rat model of ureteral stent. Journal of surgical research doi:10.1016/j.jss.2011.05.002.

Cirioni et al. (2011). Effect of the combination of clarithromycin and amikacin on Pseudomonas aeruginosa biofilm in an animal model of ureteral stent infection. J Antimicrob Chemother 66(6): 1318-1323. 

If persisters are non-mutants, are hip mutants persisters?

Persisters are defined as non-mutants that form a small subpopulation of bacteria exhibiting transient antibiotic tolerance. i.e. in the presence of antibiotics, they are tolerant to the bactericidal activity of antibiotics, but once the antibiotics are removed, they again become sensitive to antibiotics. Researchers have studied a number of hip (high persister) mutants and showed that the hip mutants produce a high frequency of persisters. Example includes the most commonly studied hipA mutants. In addition, other hip mutants whose genetic background is not known, is also reported. For example, Lafleur et al. (2010) suggested that the fifteen hip isolates they had identified had an underlying genetic change. Similarly, Mulcahy et al. (2010) found that the late isolates of P.aeroginosa from cystic fibrosis patients that showed a 100-fold increase in persister levels carried a large number of mutations including mutS and mexZ.

If persisters are non-mutants, can those mutants be considered as persisters?

Some explanations are available..

“The formation of hip mutants may be a general feature of recalcitrant fungal infections, although it is likely that persisters from both wild-type strains and hip mutants contribute to drug tolerance and the survival of the pathogen.” (Lafleur et al. 2010)

“While the persister state is a temporary one for a bacterial cell, the frequency with which cells enter this temporary state can be increased by mutations. A mutant that has an increased frequency of persister formation then is a hip mutant. Once these mutants are selected, the population stably forms more persisters than the wild-type strain from which it was derived.” (Mulcahy et al. 2010)

Can we consider the persisters from both wild type bacteria and hip mutants the same even though the latter has an underlying genetic change? If they are the same, why persisters are defined as non-mutants?

Lafleur et al. (2010). Patients with long-term oral carriage harbor high-persister mutants of Candida albicans. Antimicrob Agents Chemother 54(1), 39-44.

Mulcahy et al. (2010). Emergence of Pseudomonas aeroginosa strains producing high levels of persister cells in patients with cystic fibrosis. Journal of Bacteriology 192(23): 6191-6199.

When was the last time researchers isolated hipA mutants either in vitro or in vivo?

It was Moyed and Bertrand (1983) who isolated hipA7 mutants for the first time. They were screening for mutants showing high persistence and isolated ‘high persistence’ mutants that included hipA7 mutant. The hipA7 mutant generates high frequency of persisters than wild type strains when exposed to antibiotics. Similarly, it was later found that the overexpression of hipA results in high frequency of persisters. Based on these findings, it was concluded that hipA gene is very important in persister generation.

However, isolation of this mutant in vitro or in vivo has not been reported after Moyed and Bertrand (1983). In fact, some of the researchers failed to isolate hipA mutants among the persisters generated in vitro. 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 in the above case was not due to hipA or hipB mutations.

In vivo, isolation of high persister (hip) strains has been reported among fungal and bacterial species (Lafleur et al. 2010; Mulcahy et al. 2010). However, in both cases, ‘hip’ is only a general term for high persisters and  the genetic background of hip phenotype is unknown (researchers assumed that the hip phenotype was caused by an underlying genetic change).  

Given the fact that persisters are isolated following antibiotic administration with any bacterial species tested, one would expect isolation of hipA mutants with high frequency. It is interesting to note that a mutant that has not been isolated either in vitro or in vivo for decades is considered highly important for a very common phenomenon.

Moyed and Bertrand (1983). hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murien synthesis. J. Bacteriol. 155:768-775

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

Lafleur et al. (2010). Patients with long-term oral carriage harbor high-persister mutants of Candida albicans. Antimicrob Agents Chemother 54(1), 39-44.

Mulcahy et al. (2010). Emergence of Pseudomonas aeroginosa strains producing high levels of persister cells in patients with cystic fibrosis. Journal of Bacteriology 192(23): 6191-6199.

An example for in vivo persisters?

Except for a few, most of the experiments demonstrating phenotypic shift of persisters are in vitro experiments only. However, a recent article reported the isolation of in vivo persisters of Candida albicans and C. glabrata from cancer patients (LaFleur et al. 2010). Those patients were on long-term treatment with topical chlorhexidine as they were at high risk of oral candidiasis. The number of persisters isolated from patients treated daily with topical chlorhexidine was determined by growing biofilms in vitro followed by  challenging the biofilms with high concentrations of amphotericin B, and plating for survivors. Persister levels were found to be higher in patients with long-term carriage than in patients with transient carriage. Fifteen hip isolates that were isolated from different patients were considered as mutants, indicating that hip mutants are involved in recalcitrant fungal infections.

However, some of the conclusions made by authors are questionable

1. authors identified isolates that showed increased survival in the presence of amphotericin B and suggested that the high persister phenotype was caused by an underlying genetic change. It is unclear how the authors concluded that the survivors had an underlying genetic change without doing any relevant studies.

2. If they had an underlying genetic change, can they be considered as persisters? (persisters are non-mutants which exhibit a transient phenotypic tolerance)

3. Moreover, the strains isolated from those patients did not show any signs of infections, as admitted by the authors. Hence, it is not known whether the presence of these persisters can cause chronic infections.

Lafleur et al. (2010). Patients with long-term oral carriage harbor high-persister mutants of Candida albicans. Antimicrob Agents Chemother 54(1), 39-44.

Robustness of colony biofilm antibiotic tolerance

The conclusions of Zuroff et al. (2010) is given below.

“Biofilm antibiotic tolerance can vary in unpredictable manners based on modest changes in culturing conditions. Common antimicrobial testing methods, which only consider a single culturing condition, are not desirable since slight culturing variations can lead to very different outcomes. The presented data suggest it is essential to test antimicrobial strategies over a range of culturing perturbations relevant to the targeted application. In addition, the highly dynamic antibiotic tolerance responses observed here may explain why some current antimicrobial strategies occasionally fail.”

The authors found that in vitro colony biofilm antibiotic tolerance can vary considerably depending on perturbations in nutrient availability, temperature, age of biofilm etc. Earlier, other researchers have used only a single culturing condition (i.e. incubated bacterial cultures with a single dose and concentration of antibiotic, for a specified time of incubation using a specific inoculum size. The effect of nutrients and temperature has not been considered) in their experiments and extrapolated those results to derive at certain conclusions. However, the conclusions derived from single culturing conditions may not be reliable. In fact, this is one of the points stressed in my book and the previous blogposts as far as demonstration of persisters and their phenotypic shift is concerned. This highly dynamic antibiotic tolerance response may also explain why it is taking long time for antibiofilm agents to be available in the market (Romero and Kolter 2011).

Zuroff et al. (2010). Robustness analysis of culturing perturbations on Escherichia coli biofilm beta-lactam and aminoglycoside antibiotic tolerance. BMC Microbiology 10:185  

Romero, D. and Kolter, R. (2011). Will biofilm disassembly agents make it to the market? Trends in Microbiology 19(7): 304-306.

Safety of sugar coated antibiotics- comparing Allison et al. (2011) and Zuroff et al. (2010)

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 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 like β-lactam antibiotics or quinolones).

However, this finding is in stark contrast with Zuroff et al. (2010) who reported that addition of glucose to E.coli biofilms resulted in an increase in cfu/biofilm by an order of more than 6log₁₀ than without glucose when aminoglycoside was used but a decrease in cfu/biofilm of nearly the same magnitude when ampicillin was used. They also noticed the same effect with other metabolites like fructose, sorbitol and gluconate. This effect was found to be concentration dependent also.

This contrasting finding raises the question whether addition of glucose or other metabolites (probably with the exception of mannitol, which is discussed in the earlier blogposts) along with aminoglycosides (Allison et al. 2010) will be safe in the treatment of bacterial persisters and biofilm-related persistent infections.

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

Zuroff et al. (2010). Robustness analysis of culturing perturbations on Escherichia coli biofilm beta-lactam and aminoglycoside antibiotic tolerance. BMC Microbiology 10:185  

Why antibiofilm agents are unavailable in the market?

The abstract of the review article “Will biofilm disassembly agents make it to the market?” (Romero and Kolter 2011) is given below.

“Nearly 12 years after promising results suggested that antibiofilm agents might be developed into novel therapeutics, there are no such products on the market. In our opinion, the reasons for this have been predominantly economic. Recent developments, however, suggest that there could still be emerging opportunities for the developments of such products.”

The authors attribute the failure to develop antibiofilm products mainly on economic factors.

“We do not think that the failure to develop a product has been as a result of the fact that the antibiofilm approach is intrinsically flawed. Rather, we feel that the path to product development is long and expensive and the potential market is not as lucrative as corporations would like it to be.”

Is the economical factor mainly responsible for the failure? Isn’t true that at least some of the aspects of biofilm research are intrinsically flawed? Take persisters and their phenotypic shift as an example. Persisters are implicated in chronic infections and biofilm-associated infections. Researchers claim that by targeting this small subpopulation of bacteria, it may be possible to treat chronic infections successfully. However, is it possible to achieve their claim?

Earlier, I had argued in my book that the current knowledge on persisters is fundamentally flawed. The aim of the last few blogposts was also to question the significance of many of the research findings on persisters.

Authors suggest that the continuing research on biofilms might yield some promising avenues that might someday be translated to products. We can hope that biofilm disassembly products using enzymes, phages or small molecules will be available in the market in future.  However, at least with persisters, future outlook is pessimistic. The reason is simple; when the hypothesis is fundamentally flawed, the chances of product development based on the hypothesis are also very low.

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

Romero, D. and Kolter, R. (2011). Will biofilm disassembly agents make it to the market? Trends in Microbiology 19(7): 304-306.