A joint study led by the University of Copenhagen has revealed previously unknown properties and molecular functions of a lesser-studied CRISPR-Cas type. The study demonstrates its effectiveness in combating antimicrobial resistance in a high-risk bacterial strain affecting hospitalised and physically vulnerable patients.

The World Health Organization has identified antibiotic resistance as «one of the biggest global threats to public health and development», with an estimated 4.95 million deaths annually worldwide. The OECD (Organisation for Economic Co-operation and Development) predicts a «twofold increase in resistance by 2035 compared to 2005 levels».

The most concerning resistance rates, highlighted in the 2022 report of the Global Antimicrobial Resistance and Use Surveillance System, involve the so-called ESKAPE pathogens – an acronym for Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species – which have become increasingly resistant to most available antimicrobials.

While the continuous rise in antimicrobial resistance is largely attributed to the massive – and often unnecessary – use of antibiotics among humans, animals, and plants, the role of hospitals (which use large quantities of antibiotics daily over prolonged periods) in shaping resistance profiles cannot be ignored.

A systematic investigation detailed in “Antimicrobial resistance bacteria and genes detected in hospital sewage provide valuable information in predicting clinical antimicrobial resistance” (Science of The Total Environment, November 2021 issue), conducted by a team of scientists from Shantou University in China, highlighted the extensive use of antimicrobial drugs in hospitals. According to the authors, this has led to the development of new bacteria and antibiotic-resistant genes over the past two decades. But let’s take a step back.

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TAKEAWAYS

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The presence of antibiotic-resistant bacteria in wastewater treatment processes

In 2019, a study by researchers from the University of Southern California (“Antibiotic resistance is spreading from wastewater treatment plants” – Science Daily) was among the first to highlight the correlation between antibiotic resistance and wastewater treatment. “Treatment” encompasses all procedures aimed at removing organic pollutants and waste products from wastewater. But what are the specifics of this correlation?

In essence, our bodies, after metabolising antibiotics, excrete the residues through faeces and urine, which are then conveyed to wastewater treatment plants.

Here, the research team explains, one of the most common methods is treatment using a membrane bioreactor.

This method «involves a filtration system and a biological process in which microscopic bacteria attack and consume organic waste, also encountering antibiotic residues. In response, they develop “resistance genes” that can be passed from parent to offspring cells and among neighbouring cells through a process known as “horizontal gene transfer“». Over time, as these antibiotic-resistant bacteria reproduce and grow, a type of “biomass” forms:

«A typical wastewater treatment plant produces tons of antibiotic-resistant DNA biomass every day. After treatment, this biomass is either disposed of in landfills or used as fertiliser for agricultural and livestock production»

the authors note. In a more alarming scenario, small amounts of these antibiotic-resistant bacteria escape the plant, reaching the final watercourse. This water is partially discharged into rivers and seas and partially recycled for irrigation or to replenish groundwater, a common source of drinking water.

The issue of hospital wastewater

Wastewater from hospitals and other inpatient facilities contains the highest quantities of organic waste contaminated with antimicrobial drug residues, often taken by patients over extended periods. This, according to scientists from Shantou University, who curated the referenced research, contributes to the proliferation of significant quantities of bacteria and antibiotic-resistant genes in the wastewater.

The most recent and illustrative data on this issue dates back to 15 January 2024, thanks to a study led by Walailak University in Thailand. This research, titled “Multidrug antibiotic resistance in hospital wastewater as a reflection of antibiotic prescription and infection cases,” was published in the journal “Science of The Total Environment.” The study examined water samples from wastewater treatment plants servicing 144 residential facilities in Nakhon Si Thammarat province, including 24 samples from four hospital wastewater treatment sites in the same area. Two of these samples included water from a receiving canal, where treated water was discharged, aiming to assess the presence of antibiotic-resistant bacteria.

Analysis of the pathogenic microorganisms in the samples revealed that 87.5% of the bacteria belonged to the Enterobacteriaceae family, with Klebsiella pneumoniae being the predominant species (47.9%).

«The antimicrobial sensitivity test showed that 57.6% of the isolated bacteria were resistant to amoxicillin/clavulanic acid, the most commonly used antibiotic in the hospital under study. The overall resistance rate before and after water treatment was 27.7% and 28.0%, respectively, with an overall multidrug resistance rate of 33.3%», the research team concluded.

These alarming findings confirm the initial hypotheses of the researchers.

Antibiotic resistance genes and their transmission

Resistance genes that develop in bacteria after encountering antibiotic-laden organic waste during wastewater treatment processes are «mobile genetic elements capable of traveling between microorganisms via horizontal gene transfer, even from dead cells to living cells. » These genes are found in so-called “plasmids,” extra-chromosomal DNA elements that carry antibiotic resistance genes, spreading them rapidly within a bacterial population and among different bacterial species. This process, known as “horizontal gene transfer,” is responsible for the evolution of ever-new resistance strains [source: “Antibiotic Resistance Genes” – Science Direct].

Given this dynamic, it is not only urgent to find pharmacological alternatives to replace antibiotics that have become ineffective, but also essential to understand the mechanism of gene transfer and disrupt it by developing inhibitors against it. This could be a new approach, according to a study by Jawaharlal Nehru University in New Delhi, described in “Antibiotic resistance: a global crisis, problems and solutions” (Critical Reviews in Microbiology, 21 February 2024).

Another perspective in the fight against antimicrobial resistance, suggested by Indian researchers, is to consider specific factors within host cells on which resistance genes rely for their survival and to combat them with targeted therapies.

Antibiotic resistance: focus on type IV CRISPR-Cas systems

Among the research efforts exploring potential solutions to the global challenge of antibiotic resistance is a recent study titled “Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans.” Published in the journal “Cell Host and Microbe” and available online from 15 May 2024, this international collaboration led by the University of Copenhagen specifically investigates the novel functions of one of the CRISPR-Cas systems and their efficacy in combating antimicrobial resistance in clinically relevant bacterial strains.

CRISPR, an acronym for Clustered Regularly Interspaced Short Palindromic Repeats, refers to a class of DNA segments found in bacteria, serving as a natural defence mechanism against external attacks. Since the initial studies on this bacterial defence mechanism, increasingly sophisticated genetic engineering techniques have been developed for manipulating DNA in plants, animals, and humans. This has led to the discovery within the bacterium “Streptococcus pyogenes” of a CRISPR system that utilises the Cas9 protein, functioning as a molecular scissor to defend against pathogens.

In 2012, scientists Emmanuelle Charpentier and Jennifer A. Doudna transformed the CRISPR-Cas9 system into a new genome-editing tool, which, compared to its predecessors, could identify and cut target DNA sequences within plant, animal, and human genomes more simply, precisely, and rapidly, removing and replacing them with others.

CRISPR-Cas systems are currently classified into two classes, six types, and 33 subtypes. The class 1 CRISPR-Cas system includes types I, III, and IV, involving multiple Cas proteins, while class 2, which includes types II (such as CRISPR-Cas9), V, and VI, uses a single protein. Types I, II, and V systems recognise and cleave DNA, type VI targets RNA, and type III can cut both DNA and RNA. Only type IV does not include any of these functions.

Characteristics of type IV-A3 CRISPR-Cas systems

All CRISPR-Cas systems (excluding type IV) protect bacteria from the invasion of external genetic elements and are defined by the Danish study authors as «bacterial adaptive immune systems that target and cleave the nucleic acids (DNA and RNA) of invasive genetic parasites such as bacteriophages and viruses, which can infect and ultimately kill bacterial cells». Regarding type IV, they specify:

«Unlike the others, type IV CRISPR-Cas systems have been little studied until now. They are considered the ‘odd cousins’ of the other Cas systems, as they are the only ones lacking the immune memory acquisition module and the DNA-cutting component, properties that have made the other CRISPR systems famous. Like other class 4 systems, they form multi-protein complexes and are divided into distinct subtypes (from IV-A to IV-E) and variants (from IV-A1 to IV-A3), based on their molecular architecture».

The study led by the University of Copenhagen is not the first to focus on the applications of CRISPR systems in the fight against antimicrobial resistance. For at least six years, the international research community has been examining the potential of CRISPR-Cas as a new technology in this field. However, previous studies have always and only considered CRISPR-Cas types capable of cutting DNA, a crucial ability for physically eliminating individual antibiotic-resistant bacterial strains. As highlighted in the 2022 publication “The Application of the CRISPR-Cas System in Antibiotic Resistance” (National Library of Medicine):

«The specific cutting capabilities of CRISPR-Cas, targeting genetic elements carrying resistance genes, open the prospect of preventing and controlling horizontal gene transfer and limiting the spread of antibiotic resistance».

It is the unique characteristics and particular association with plasmids (as we shall see later) that have made type IV-A3 CRISPR-Cas systems the focus of the Danish authors’ attention, who have finally delved into their role and underlying molecular functions.

The role of type IV-A3 CRISPR-Cas in restoring antibiotic sensitivity to a high-risk pathogen

The cited study reveals that although type IV-A3 CRISPR-Cas systems lack the molecular scissors to cut the DNA of invading parasites, they contain a protein capable of forcing the double helix of DNA to unwind like a skein of yarn. «This unique function, known as ‘helicase,’ could have significant biotechnological applications»,the authors note.

Additionally, another important finding observed in the laboratory is that type IV-A3 systems are carried by plasmids. As previously discussed, these plasmids harbour antibiotic resistance genes, which are developed by bacteria exposed to organic waste containing antibiotics during wastewater treatment processes.

This pairing is highly beneficial, as the team discovered that type IV-A3 CRISPR-Cas systems tend to act on plasmids using the helicase, effectively silencing their main functions. This implies two major outcomes:

• impacting horizontal gene transfer, through which plasmids spread antibiotic resistance genes, creating new strains
• affecting the stability of target plasmids, thereby breaking the chain of dissemination

Inspired by this natural function, the research team sought to recreate in the laboratory a scenario where a type IV-A3 CRISPR-Cas system selectively silences antimicrobial resistance genes carried by the high-risk pathogen Klebsiella pneumoniae, a significant threat to hospitalised patients.

The findings from this initial experiment showed that silencing plasmid functions resulted in Klebsiella pneumoniae regaining sensitivity to antibiotics. The choice of this particular bacterium was intentional, as type IV-A3 CRISPR-Cas systems are notably prevalent among Klebsiella bacteria. This opens up significant implications for future research, which can further explore the mechanisms of horizontal gene transfer by plasmids to fully understand and combat the dynamics of antibiotic resistance.

Glimpses of Futures

Combating antibiotic resistance with the most innovative genetic engineering tools allows us to directly address the core of the issue – the DNA of bacteria resistant to antimicrobials – by silencing their key functions without cutting their segments.

To anticipate possible future scenarios, let’s analyse – using the STEPS matrix – the impacts that the evolution of type IV-A3 CRISPR-Cas systems in countering antibiotic resistance in prevalent pathogenic microorganisms (not just Klebsiella pneumoniae) could have in social, technological, economic, political, and sustainability contexts.

S – SOCIAL: for several years now, antimicrobial resistance has been flagged as a crucial global public health issue, impacting not only human health but also animal and plant health, as well as food security. In Europe, specifically, where antimicrobial resistance directly causes an estimated 35,000 deaths annually, the European Commission in July 2022 classified antibiotic resistance as «one of the top three priority health threats in the Union». Looking ahead, once laboratory studies and tests on the most resistant bacterial strains (which pose a high risk, especially for hospitalised patients and physically vulnerable individuals) are successfully completed, CRISPR-Cas type IV-A3 systems could potentially, alongside the development of new classes of antibiotics, become a tool to disrupt the links of horizontal gene transfer – responsible for spreading antibiotic resistance genes -without the need for molecular scissors, which are still at risk of off-target effects. This would prevent the continuous emergence of new strains and subsequent social alarms.

T – TECHNOLOGICAL: although research on the topic is still in its early stages and much remains to be discovered about type IV-A3 CRISPR-Cas systems, their subtypes, and variants, the emerging prospects are numerous. The methodologies and techniques employed, as well as the results already obtained from this initial work, could prove fundamental in the search for alternative treatments for other types of infections (such as fungal infections), which are often multi-resistant to existing drugs. Additionally, in the future, the association of type IV-A3 CRISPR-Cas with plasmids could be strategically significant for research monitoring plasmids that carry other clinically important genes with the potential for rapid spread.

E – ECONOMIC: in recent years, global antibiotic resistance has led to the rapid depletion of effective antimicrobial stocks and inadequate replenishment of new supplies and antimicrobial products. Considering the high costs of pharmaceutical research – estimated by analysts at the Center for Global Development to «require incentives of around $3.1 billion for the development of new classes of antibiotics» -the economic burden is significant. In the EU, the cost of antimicrobial resistance to healthcare systems is about €1.1 billion annually, including second and third-line drugs necessary when an infection does not respond to initial antibiotic treatment, and hospital stays due to treatment complications. Against this bleak backdrop, the future interruption of horizontal gene transfer, through which antimicrobial resistance genes are spread, thanks to the action of type IV-A3 CRISPR-Cas systems, would have a direct positive impact on global GDP. This would also affect trade in agricultural and animal products, whose productivity is also compromised by antibiotic resistance.

P – POLITICAL: The European Council’s Recommendation on enhancing EU actions to combat antimicrobial resistance, published in the Official Journal of the European Union on 22 June 2023, does not mention exploring solutions leveraging genetic engineering techniques, particularly CRISPR systems. However, this topic is discussed in an Italian document from the National Committee for Biosafety, Biotechnology and Life Sciences, distributed by the Prime Minister’s Office in April 2022. The document suggests future actions «to create a bridge with the group on advanced biotechnologies, examining the possibility of using CRISPR-Cas9 for editing to correct bacterial resistance enzymes». In anticipation of a potential future where type IV-A3 CRISPR-Cas systems play a recognised role in combating antibiotic resistance, it will be necessary, for greater transparency, to officially integrate CRISPR-Cas tools into clinical and laboratory practices to end the transmission of antimicrobial resistance genes. As of now, all DNA manipulation interventions directly or indirectly involving humans are overseen by legislators, with a strong focus on short- and long-term risk analysis. The key question in this context is what precautions are necessary when eliminating specific pathogens from a microbial community by forcibly opening the double helix of plasmid DNA. Currently, all this is at the experimental stage in the laboratory. However, if these practices were to be concretely implemented within the next decade, the legitimate question is whether the approval of Governments and Authorities will be required.

S – SUSTAINABILITY: the socio-economically fragile countries suffer the most from antibiotic resistance. According to the Global Research on Antimicrobial Resistance, Africa is the continent most exposed to this phenomenon, partly due to its historically high mortality rate from airborne infections. Here, the number of deaths related to antimicrobial resistance (over one million annually) «even exceeds those caused by HIV and malaria, marking a fundamental shift in the health challenges the region faces». In this particular context, the future application of type IV-A3 CRISPR-Cas systems to interrupt the gene transmission chain of antibiotic resistance, thereby halting the continual emergence of new bacterial strains, must be ensured to the same extent as in other countries, in line with the principles of health equity promoted by the World Health Organization.