The Use of CRISPR-Cas9 as an Alternative Therapy to Combat Antibiotic Resistance

ii. The use of CRISPR-Cas9 as an alternative therapy to combat antibiotic resistance

Antibiotic overuse coupled with the rapid evolution of bacteria in response has resulted in the spread and increase in antibiotic resistant infections worldwide.  This has led to a growing need to research viable alternative treatments providing a more effective strategy for dealing with such infections.  Using a combination of primary studies and secondary, summary papers the use of alternative therapies, with a focus on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated protein 9 was reviewed and its viability discussed.  The most concerning bacteria to healthcare workers are the six bacteria which cause antibiotic resistant infections - Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter species, or ESKAPE bacteria.  Alternative therapies to infections caused by these bacteria include, but are not limited to, the use of lysins, probiotics, faecal transplants, bacterioricins, and bacteriophages.  Studies show some success in the use of lysins and faecal transplants although recent problems with faecal transplants indicate further research is required. Additionally, the use of CRISPR and its associated proteins to alter genomes offers an exciting opportunity to explore.  Horizontal gene transfer (HGT) between bacteria is a key way in which antibiotic resistant genes spread.  An important area of research is the prevention of HGT.  CRISPR-Cas9 has been used effectively to prevent conjugation in Staphylococcus spp using the insertion of mutations, the deletion of sequences, and shown the importance of the Cas protein to achieve this.  CRISPR-Cas9 is an important tool in science and research concerning its use in the challenge of antibiotic resistance is valuable.  Although success has been shown in the alteration of bacterial genomes and the prevention of HGT, further work must be done to support the initial studies and to combat the current disadvantages of the method; the cost and the large scale applications.

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  i.The use of CRISPR-Cas9 as an alternative therapy to combat antibiotic resistance

Antimicrobial resistance (AMR) and the infections caused by antibiotic resistant bacteria is a growing problem which is predicted will lead to the deaths of 10 million people annually by 2050 (de la Fuente-Nunez et al., 2017).  As well as the impact this will have on the structure and provision of healthcare, it will also impact the environment and on society socially and economically.  In response to AMR, scientists have been studying alternative ways to combat antibiotic resistance.  One such method is the use of Clustered Regularly Interspaced Short Palindromic Repeats and associated protein 9 (CRISPR – Cas9) system of defence which exists naturally within bacteria (Bikard et al., 2012)  In order to research the history of antibiotic resistance and the steps taken using CRISPR to combat it Web of Science was utilised to search for antibiotic resistance in order to gain a background to the subject.  This led to an article titled 'Alternatives to conventional antibiotics in the era of antimicrobial resistance'.  This was good overview of the methods to combat antibiotic resistance which are currently being studied and focused my search on research involving CRISPR.  I followed up on the most relevant articles referenced within this paper and started to use search terms which were relevant to these, including ‘CRISPR-Cas’, ‘sequence specific antimicrobials’, and ‘RNA guided’.  Once papers had been organised into the objectives they were suited to using Endnote, notes were made under the heading of each objective.  This aimed to organise the key points for the study and could be used as a starting point for writing the review.  My study has been focused on the following objectives;

1. Discuss the history, biology of and global impact of antibiotic resistance and the emergence of antibiotic resistance genes in microbes
2.  Briefly compare and contrast the alternative therapies available to treat infections caused by resistant bacteria
3. Discuss the biology and use of bacteriophages as a delivery method of DNA into bacteria and the effectiveness of this approach
4. Describe the use of the CRISPR-Cas system as an alternative therapy to bacterial infections and the modes of action employed in this approach
5. Critically analyse the research evidence for the benefits and pitfalls of CRISP-Cas9 as a therapy
6. Discuss the future of CRISPR-Cas9 and the opportunities for further research 

The scope of this literature review covers alternative therapies to antibiotics and will not cover the development of, or lack of research into novel antibiotics and possible sources for them.  Nor will it delve into prevention of the ongoing misuse of antibiotics.  These topics have been well covered in other literature and it is more productive to now look at alternatives.  There are several areas of research concerning alternative therapies to antibiotic use.  CRISPR is a fascinating approach as the applications for CRISPR are far reaching and can be applied to other disorders and illnesses. This review focuses on the use of CRISPR-Cas9 as a therapy linked to antibiotic resistance but will not cover the other uses of CRISPR-Cas9. 

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References

• Bikard, D., Hatoum-Aslan, A., Mucida, D. and Marraffini, L. A. (2012) 'CRISPR Interference Can Prevent Natural Transformation and Virulence Acquisition during In Vivo Bacterial Infection', Cell Host & Microbe, Vol. 12, No. 2, pp. 177-186. [Online] Available at https://doi.org/10.1016/j.chom.2012.06.003 (Accessed 29 Jun 19)
• de la Fuente-Nunez, C., Torres, M. D. T., Mojica, F. J. M. and Lu, T. K. (2017) 'Next-generation precision antimicrobials: towards personalized treatment of infectious diseases', Current Opinion in Microbiology, Vol. 37, pp. 95-102. [Online] Available at https://doi.org/10.1016/j.mib.2017.05.014 (Accessed 27 Apr 19)

4. Chapter 1

1.1   Introduction - Background to topic

Since the introduction of penicillin to common use in the 1940s, when Alexander Fleming noted that the overuse of penicillin would lead to problems, there have been concerns about resistance to antibiotics.  By the late 1940’s resistance to Staphylococcus aureus had been noted in hospitals worldwide and was a concern to clinicians (Barber, 1947 in Podolsky, 2018).  In 1955 Lindsay Batten noted that “We may run clean out of effective ammunition and then how the bacteria and moulds will lord it” (Batten, 1955 in Podolsky, 2018).  Despite these warnings antibiotics were still over prescribed in general, and attempts to tackle the issue were seen at a local level but not addressed on a global platform (Podolsky, 2018).   

Bacterial resistance to drugs causes 700,000 deaths worldwide annually and costs the NHS in the UK £180 million per year (House of Commons, 2018).  In 2011 the Chief Medical Officer’s report in the UK focused on the rising threat of antimicrobial resistance (AMR) to antibiotics and infections in which the author referred to AMR as ‘a ticking time bomb’ (Davies, S. 2011).  The report discussed the issue in detail and made specific recommendations to named organisations to aid in the battle against AMR.  Following this report, in 2013 the UK government set up a 5 year committee to tackle AMR which covered antibiotic use in humans, animals, and the environment. 

1.2   Methodology

Following initial searches for papers on antibiotic resistance a more focused search on alternative therapies and their uses was utilised.  A mixture of primary research papers and reviews was used to inform the literature review on the use of CRISPR-Cas9 as an alternative therapy and its comparison to and discussion regarding other, alternative therapies.

1.3   Justification for topic and scope

Hospital workers are increasingly finding the need to look to alternatives to treat antibiotic resistant infections, particularly in immune comprised patients. 

Chapter 2

2.1   Discuss the history, biology of and global impact of antibiotic resistance and the emergence of antibiotic resistance genes in microbes

There is evidence that humans have been using substances with antibiotic properties since 350-550 CE.  Studies of skeletal fragments from Sudanese Nubia showed tetracycline was present, indicating it was consumed by the individuals the bones belonged to (Nelson et al, 2010 in Aminov, 2010).  Further studies have shown that tetracycline was present in remains found in Egypt which dated to the late Roman period (Cook et al, 1989 in Aminov, 2010).  In addition to this, traditional Chinese medicine uses a variety of remedies which contain antimicrobial properties (Aminov, 2010).  Their presence in different locations and cultures indicate a conscious use to relieve symptoms of illness, even if the cause of the illness was unknown.  The value of these two studies involving tetracycline shows the same result achieved, from two time periods, using two methods.  The advantage of this is that each supports the other without necessarily being the aim of either.  The combination of these instances of substances with antimicrobial properties being used throughout history, and for long before the current overuse of antibiotics in recent history, may have been contributing to the rise of genes which confer antibiotic resistance in bacteria.  One example of this is Klebsiella oxytoca which has been developing a resistance to β-lactamase, although concomitantly to the bacteria expressing antibiotic resistance (ABR) (Fevre et al., 2005).This suggests that perhaps ABR has been evolving for thousands of years and it is only the current overuse of antibiotics after the development in penicillin in 1928 that has sped up the evolution of the antibiotic phenotype.

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There are six species of bacteria which exist and cause infections in both healthcare and non-healthcare settings worldwide and which concern researchers in terms of their development of antibiotic resistant genes.  These are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter species, shortened to the acronym ESKAPE (Rice, L. 2008).  The knowledge that the majority of ABR infections come from just six bacteria is an advantage to researchers and allows them to focus their attempts at a solution to the problem and pinpoint areas of weakness.  These bacteria can represent model organisms that can be used in the future should other organisms start to present the same problems, hopefully providing researchers a head start on how to proceed. 

2.2   Briefly compare and contrast the alternative therapies available to treat infections caused by resistant bacteria

In recent years researchers have started to develop a number of alternatives to antibiotics to combat the growing problem of resistance including; bacterioricins, bacteriophages, faecal transplant, phage lysins, and probiotics (Brussow, 2017; de la Fuente-Nunez et al., 2017).  Phage lysins attack bacterial cell walls and lyse Gram positive bacteria.  No resistance has yet been described but it has been shown to be effective against a number of infections including meningitis, pneumonia, sepsis, and pharyngitis in animal models as shown in a study by Pastagia et al (2013) in Brussow(2017).  However they do not diffuse away once they have been used and their action is not renewed so a way to remove the lysins is required.  A separate treatment which can be used which does not require a method of removal is faecal microbiota therapy (FMT), in which healthy microbiota is introduced into the gut of an infected individual, allowing the healthy microbiota to overtake and exclude the pathogenic bacteria (Ghosh et al., 2018).  This method has been shown to be effective in the treatment ofinfection caused by Clostridium difficile, Salmonella, andEscherichia coli (Lahtinen et al., 2017).  Although some side effects were noted in this study they could not be attributed to the faecal transplant and it was ultimately concluded that the treatment was safe for those who are immune compromised.  However, more recently the US Food and Drug Administration (FDA) have announced their intention to halt FMT trials due to the death of an immune comprised patient who contracted an antibiotic resistant infection from FMT (Hou, 2019). 

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Chapter 3

3.1 Discuss the biology and use of bacteriophages as a delivery method of DNA into bacteria and the effectiveness of this approach

Bacteriophages, viruses which infect bacteria, are the most ubiquitous biological organism.  CRISPR-Cas9 can be used to introduce genes, mutations, and deletions in the genome into bacteriophages which could then go on to infect bacteria (Martel and Moineau, 2014).

3.2 Describe the use of the CRISPR-Cas system as an alternative therapy to bacterial infections and the modes of action employed in this approach

Small CRISPR RNA’s (crRNAs) are formed of sequences of spacers and repeats which have been processed within the cell.  These crRNAs rely on the relationship between the spacers in the CRISPR sequence and the bacteriophages.  These sequences can then produce an acquired immunity against any infection which would have been caused by the bacteriophage (Marraffini and Sontheimer, 2008). 

Horizontal gene transfer between bacteria is a source of the spread of ABR.    This can be through transduction, transformation, or conjugation and can occur both within and between species.  Two bacteria which spread antibiotic resistant genes using conjugation for HGT are methicillin resistant Staphylococcus aureus and vancomycin resistant Staphylococcus aureus (MRSA and VRSA) (Marraffini and Sontheimer, 2008).  The resistant genes in the plasmid of one species of bacteria can spread to another species in this manner.  The Marraffini and Sontheimer study aimed to disrupt the delicate balance of sequence matching between CRISPR and cell RNA to prevent gene transfer.  This was achieved by creating a mutated conjugative plasmid by introducing nine silent mutations which changed the gene sequence, using the CRISPR sequence spc1 and the nes gene conferring resistance.  There are several strengths to this study.  The clinically isolated Staphylococcus epidermis, RP62a,used in the study is a valid choice to study conjugative plasmid spread of genes due to its CRISPR sequence which is homologous to a spacer (spc1) found in the nickase (nes) gene.  This gene occurs in the conjugative plasmids of all sequenced Staphylococci allowing for the potential replication of the study with further Staphylococci spp to support the results in this study.  An attempt to support the results was also conducted by the authors.  Whilst initially they added mutations to the CRISPR sequence, in a follow up experiment the authors used a deletion to affect the sequence, and found results concurrent with the first experiment, and further were able to show that the cas sequence was required to alter the plasmid.  Obtaining multiple results which all support the aim of the paper allows it to act as a base point for other researchers who are looking at similar methods. Additionally, the research was focused on one specific method of transference of ABR genes.  An advantage of this is that the results are clear and can be more easily compared both within and across studies.   

Chapter 4

4.1 Critically analyse the research evidence for the benefits and pitfalls of CRISP-Cas9 as a therapy

Although there are benefits to using CRISPR-Cas as an alternative therapy to bacterial infections there are also pitfalls and some further considerations before it can be used as a therapy.  The major disadvantage of this therapy is the lack of a clear way to administer this as a therapy and once it can be consistently shown to be effective the development of this should become a priority.  One possibility of this could be the introduction of bacteriophages to a person with an antibiotic resistant infection, or as a local application added to surfaces to disrupt colonies of bacteria and infect them with bacteria which aren’t resistant to antibacterials so that they can be cleaned more effectively.

Another disadvantage to the therapy is the scalability of it.  Currently, CRISPR-Cas therapies are tailored to an individual, making it costly and rendering it unsuitable for widespread applications on a large scale.  Currently, this means that only patients with severe infections would be treated and other treatments would still be required for the large majority of patients.

Ongoing mutations and evolution of bacteria mean that finding an alternative therapy is still a race.  Whilst the evidence from studies suggests that the removal, or introduction, of sections of the genome is successful care must be taken when altering the genome of bacteria in case unforeseen repercussions of this make the bacteria more virulent or result in mutations which benefit the bacteria.  

4.2 Discuss the future of CRISPR-Cas9 and the opportunities for further research. 

Chapter 5

 Conclusion

The increase in the number and variety of studies concentrated on the problem of antibiotic resistance is an improvement on the lack of movement in research and healthcare communities in the past.   The spread of antibiotic resistant infections and the predicted deaths these will cause require immediate attention.  The uses of bacteriophages and CRISPR-Cas9 as alternative methods of treatment provide valuable areas of research which should be further explored.  Whilst CRISPR-Cas9 has advantages it also comes with disadvantages which must be overcome.        

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References

• Aminov, R. I. (2010) 'A brief history of the antibiotic era: lessons learned and challenges for the future', Frontiers in Microbiology, Vol. 1. [Online] Available at https://doi.org/10.3389/fmicb.2010.00134 (Accessed 25 Mar 19)
• Bikard, D., Hatoum-Aslan, A., Mucida, D. and Marraffini, L. A. (2012) 'CRISPR Interference Can Prevent Natural Transformation and Virulence Acquisition during In Vivo Bacterial Infection', Cell Host & Microbe, Vol. 12 No. 2, pp. 177-186. [Online] Available at https://doi.org/10.1016/j.chom.2012.06.003 (Accessed 29 Jun 19)
• Brussow, H. (2017) 'Infection therapy: the problem of drug resistance - and possible solutions', Microbial Biotechnology, Vol. 10, No. 5, pp. 1041-1046. [Online] Available at https://doi.org/10.1111/1751-7915.12777 (Accessed 25 Mar 19)
• Davies, S. (2011) ' Infections and the rise of antimicrobial
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• de la Fuente-Nunez, C., Torres, M. D. T., Mojica, F. J. M. and Lu, T. K. (2017) 'Next-generation precision antimicrobials: towards personalized treatment of infectious diseases', Current Opinion in Microbiology, 37, pp. 95-102. [Online] Available at https://doi.org/10.1016/j.mib.2017.05.014 (Accessed 27 Apr 19)
• Fevre, C., Jbel, M., Passet, V., Weill, F. X., Grimont, P. A. D. and Brisse, S. (2005) 'Six groups of the OXY beta-lactamase evolved over millions of years in Klebsiella oxytoca', Antimicrobial Agents and Chemotherapy, Vol. 49, No. 8, pp. 3453-3462. [Online] Available at https://doi.org/10.1128/aac.49.8.3453-3462.2005 (Accessed 25 Jun 19)
• Hou, C. (2019) ‘FDA Suspends Clinical Trials Involving Fecal Transplants’, The Scientist, [Online] Available at https://www.the-scientist.com/news-opinion/fda-suspends-clinical-trials-involving-fecal-transplants-66009?utm_content=94952047&utm_medium=social&utm_source=facebook&hss_channel=fbp-242730579188418&fbclid=IwAR3F88E1uRna-B7ge8d7G6vwdnw5sXHPAwu96tEYKFfaA_9S-1tuV_D1I7M (Accessed 26 Jun 2019)
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• Lahtinen, P., Mattila, E., Anttila, V. J., Tillonen, J., Teittinen, M., Nevalainen, P., Salminen, S., Satokari, R. and Arkkila, P. (2017) 'Faecal microbiota transplantation in patients with Clostridium difficile and significant comorbidities as well as in patients with new indications: A case series', World Journal of Gastroenterology, Vol. 23, No. 39, pp. 7174-7184. [Online] Available at https://doi.org/10.3748/wjg.v23.i39.7174 (Accessed 26 Jun 19)
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• Martel, B. and Moineau, S. (2014) 'CRISPR-Cas: an efficient tool for genome engineering of virulent bacteriophages', Nucleic Acids Research, Vol. 42, No. 14, pp. 9504-9513. [Online] Available at https://doi.org/10.1093/nar/gku628 (Accessed 06 Apr 19)
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