The Problem

Antibiotic Resistance

The European Centre for Disease Prevention and Control (ECDC) estimated about 670.000 infections per year in the EU/EEA due to AMR bacteria, connected directly to 33.000 deaths and a related cost to the health care systems of around 1.1 billion Euros. In fact, along with an increase in mortality, it must be considered an additional cost in using alternative drugs and procedures and in extending hospitalization, together with possible disabilities. Between 2020 and 2021, the number of reported cases increased for all pathogens under surveillance. The World Bank points out the consequences of a wide spread of AMR, such as a reduction in the economic output because of lower effective labor supply, making some of the 2030 Goals, like ending poverty, hardest to reach.

The Global Antimicrobial Resistance and Use Surveillance System (GLASS) collected the data of 2019 in 70 countries: the total number of laboratory-confirmed infections was more than 3,100 millions, and a number of AST (Antimicrobial susceptibility testing) of more than 3,000 millions. The most recent estimates predict that 4,95 million deaths are associated with bacterial AMR in 2019 [1], of which 1,27 million deaths attributable to bacterial AMR, and between the major responsible pathogens there were Escherichia coli, followed by Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa [1].

Italy, our country, is the most severely affected nation in the European region, as reported by the latest data in 2019, which documented approximately 11,000 deaths for infections associated to AMR bacteria [2]. AMR spreads not only in health-care centers and among human beings, but also in animals and crops. Some data indicate that the use of antibiotics in livestock is significant, same for agriculture, and many studies show a correlation between these key factors and AMR among humans, while animals and crops affected by diseases lead to a food availability and economic problem. A serious case in Italy, but also China, Japan and New Zealand, regards the spread of Pseudomonas syringae pv. actinidiae responsible for canker disease in Actinidia chinensis Planch var. chinensis and Actinidia chinensis var. deliciosa, damaging the production of kiwifruit with an important economic impact. Copper pesticide combined with antibiotics are useless and increase the risk of MDR organisms [3].

Additionally, AMR has a severe impact also on the environment, with a particular interest in the agri-food sector [4]. As reported by the Food and Agriculture Organization of the United Nations (FAO), the impact on these fields will cause production losses and damages to livelihoods [4], also due to the fact that AMR can spread among different hosts, thus contaminating the entire food chain. As a result, it is estimated that, if not faced, AMR may cumulatively result in a 3.4 trillion USD loss in the world annual gross domestic product in ten years [4]. Measures must be implemented immediately, starting from the population. In fact, WHO and ECDC raised the need for an information and sensibilization campaign among people about AMR.

Acinetobacter baumannii

Acinetobacter baumannii is a gram-negative bacteria, an obligate aerobe, and one of the most prevalent causes of nosocomial infections [5]. The estimated annual incidence of infections caused by this bacteria is 1 million cases, with a high mortality rate. The most common infections it causes include skin, soft tissue and urinary tract infections, meningitis, and pneumonia.

The main problem lies in its high multidrug-resistant rate against antibiotics, its ability to acquire antibiotic resistance, and its biofilm production. Indeed, the biofilm facilitates horizontal gene transfer while protecting the bacteria from antibiotics. Moreover, multidrug resistant (MDR) A. baumannii forms strong biofilms on both abiotic and biotic surfaces. This bacteria also has the ability to survive for long periods to either dry or moist conditions remaining viable for months, this capability linked to the biofilm production on most of the plastics that are used in hospitals, leads to a high transmissibility of this bacteria[5, 6]. We can see in the image the main mechanism of antibiotic resistance of A. baumannii (Figure1).

AMR mechanism of A. baumannii

Figure1. The main antibiotic resistance mechanisms of Acinetobacter baumannii are here divided into six categories: (A) Permeability defects result from modifications in porins, such as the carbapenem-associated outer membrane protein (CarO) and the OMP family. (B) One-step or two-step drug extrusion from the cytosol to the outer membrane occurs via the efflux pump family. Among these, the resistance-nodulation-division superfamily (RND-superfamily) facilitates drug transport from the cytoplasm or the periplasm through its AdeABC, AdeIJK, or AdeFGH efflux pump systems. The major facilitator superfamily (MFS; e.g., TetA, TetB, CmlA, CraA, AmvA, AbaF), the multidrug and toxic compound extrusion (MATE) transporter family (e.g., AbeM), and the small multidrug resistance (SMR) transporter (e.g., AbeS) are H+ and Na+-coupled multidrug efflux pumps located at the inner membrane. (C) Hydrolysis of β-lactam antibiotics is catalyzed by β-lactamases. Acinetobacter baumannii β-lactamases are classified into four molecular classes: class A (e.g., TEM, GES, PER, CTX-M, SCO, VEB, KPC, CRAB enzyme family), class B (e.g., IMP, VIM, NDM, SIM enzyme family), class C (e.g., Amp family), and class D (e.g., OXA subgroups enzyme family).(D) Complete loss of lipopolysaccharides (LPS) due to inactivation of lipid A biosynthesis genes (lpxA, lpxC, and lpxD) results in colistin resistance. (E) Aminoglycoside-modifying enzymes are classified into three classes: acetyltransferases (e.g., AAC3, AAC(6′)), adenyltransferases (e.g., ANT(2′′), ANT(3′′)), and phosphotransferases (e.g., APH(3′′), APH(3′)). (F) Alterations in targeted sites of TetM confer ribosomal protection against tetracycline, and modification of the GyrA subunit of DNA gyrase confers resistance to quinolones.[6]

The choice of antibiotics for treating MDR A. baumannii infection is limited; polymyxin B and colistin can be effective against urinary tract infections, infected wounds, and bloodstream infections; combining imipenem with aminoglycosides, glycylcyclines, ampicillin, rifampicin, aztreonam, sulbactam, and lipopeptides can produce a synergistic effect. However, these combinations are not always effective [7].

Klebsiella pneumoniae

Klebsiella pneumoniae is a member of the Enterobacterales family, a gram-negative bacillus commonly endowed with a capsule that can cause various types of nosocomial and community-acquired infections, including urinary tract infections, pneumonia, surgical site infections, and bloodstream infections.

K. pneumoniae is inherently resistant to ampicillin due to the presence of a gene called SHV-1 penicillinase on its main chromosome. Resistance to other drugs can occasionally develop due to the acquisition of genes through horizontal transfer, mainly via large conjugative plasmids. The accumulation of resistance determinants in a single strain can lead to the formation of pan-resistant strains that cannot be treated with currently available antibiotics. Recently, there has been a rapid increase in the number of multi-resistant K. pneumoniae strains, with particular attention focused on those producing extended-spectrum beta-lactamases (ESBL). These strains show resistance to penicillins, cephalosporins (including third-generation cephalosporins), and aztreonam. [1]. Furthermore, of notable clinical relevance is the presence of K. pneumoniae strains belonging to the Enterobacteriaceae family (CRE) that are resistant to carbapenems, as carbapenems often constitute the last therapeutic option for persistent infections caused by gram-negative bacteria [8, 9].

In addition to being a significant clinical issue in itself, K. pneumoniae has a broader ecological distribution, a more diverse DNA composition, greater antimicrobial resistance (AMR) genetic diversity, and a higher number of plasmids compared to other opportunistic gram-negative bacteria. Therefore, it is likely that K. pneumoniae plays a key role in the spread of antimicrobial resistance genes from environmental microorganisms to clinically relevant pathogens. Supporting this notion, K. pneumoniae genomes contain over 400 acquired resistance genes, twice as many as E. coli and 50% more than other species, as well as an average accessory gene G+C content ranging from 20% to over 70%, indicating they come from taxonomically diverse donors.

Furthermore, it has been observed that K. pneumoniae typically harbors three plasmids per organism, a significantly higher value compared to other species, demonstrating considerable permissiveness towards plasmid acquisition and transmission. Therefore, this bacteria is an important vehicle for the dissemination of antibiotic resistance genes among different bacterial populations associated with humans.

It is also important to emphasize that distinct subpopulations of K. pneumoniae do not appear to exist among human, animal, and environmental sources, suggesting that isolates from different niches can interact and exchange resistance genes. This phenomenon contributes to genetic diversification and the spread of resistance genes[10, 11].

Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous Gram-negative aerobic bacterium that can cause disease in both plants and animals, including humans. It is of considerable medical relevance because of the large distribution of multidrug-resistant strain. P. aeruginosa with carbapenem-resistance is listed among the “critical” group of pathogens by WHO, which urgently need novel antibiotics in the clinics. Nosocomial infection caused by P. aeruginosa persists in developing resistance to commonly efficient antibiotics, emerging as a significant issue in healthcare.

In 2021 in Italy, P. aeruginosa was responsible for 7.6% of invasive infections caused by antibiotic-resistant pathogens (source: Istituto Superiore di Sanità). It can be found in soil, decomposing organic matter, water, and wet surfaces, making it particularly suitable for spreading in a hospital environment. Its wide environmental distribution is made possible by its simple nutritional requirements, resistance to a wide range of temperatures, and the ability to utilize various organic compounds as sources of carbon and nitrogen. Additionally, it possesses certain structural factors, enzymes, and toxins that naturally make it resistant to many antibiotics. Most P. aeruginosa infections are opportunistic, with patients at greatest risk being those undergoing broad-spectrum antibiotic therapy, cystic fibrosis patients, patients with urinary catheters, burn patients, and generally immunocompromised individuals. P. aeruginosa infections primarily affect the respiratory tract, skin, and urinary tract, but can escalate to bloodstream infections and endocarditis in severe cases.

P. aeruginosa exhibits numerous virulence factors, some of which are related to antibiotic resistance. It produces alginate capsules, a mucoid polysaccharide that enhances adhesion to surfaces and protects against phagocytosis. It is capable of producing biofilms in response to specific environmental stimuli (quorum sensing), which act as a barrier to drug diffusion. It possesses efflux pumps on its cell wall that rapidly expel drugs and can acquire resistance through horizontal transfer. Treatment of P. aeruginosa infections is further hindered by the bacterium's high mutation rate, which allows for rapid adaptation and development of resistance. The most common resistances in P. aeruginosa strains are to aminoglycosides, fluoroquinolones, carbapenems, ceftazidime, and beta-lactams [12].


For these reasons we felt it necessary to develop functional, fast and easy adaptable therapy for MDR bacteria as ESKAPE.

References

  1. C. J. L. Murray et al., ‘Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis’, The Lancet, vol. 399, no. 10325, pp. 629–655, Feb. 2022, doi: 10.1016/S0140-6736(21)02724-0.
  2. https://www.epicentro.iss.it/antibiotico-resistenza/epidemiologia-europa
  3. Yu JG, Lim JA, Song YR, Heu S, Kim GH, Koh YJ, Oh CS. Isolation and Characterization of Bacteriophages Against Pseudomonas syringae pv. actinidiae Causing Bacterial Canker Disease in Kiwifruit. J Microbiol Biotechnol. 2016 Feb;26(2):385-93. doi: 10.4014/jmb.1509.09012. PMID: 26628254.
  4. FAO. What is it? | Antimicrobial Resistance. Food and Agriculture Organization (FAO) website, visited on 2 July 2023.
  5. Morris FC, Dexter C, Kostoulias X, Uddin MI and Peleg AY (2019) The Mechanisms of Disease Caused by Acinetobacter baumannii. Front. Microbiol. 10:1601. doi: 10.3389/fmicb.2019.01601
  6. Cavallo I, Oliva A, Pages R, Sivori F, Truglio M, Fabrizio G, Pasqua M, Pimpinelli F and Di Domenico EG (2023) Acinetobacter baumannii in the critically ill: complex infections get complicated. Front. Microbiol. 14:1196774. doi: 10.3389/fmicb.2023.1196774
  7. Rahimzadeh G, Rezai MS, Farshidi F. Genotypic patterns of multidrug‑resistant Acinetobacter baumannii: A systematic review. Adv Biomed Res 2023;12:56.
  8. S. Gualtero et al., “Factors associated with mortality in Infections caused by Carbapenem-resistant Enterobacteriaceae,” Journal Infect Dev Ctries, vol. 14, no. 6, pp. 654–659, Jun. 2020, doi: 10.3855/jidc.12267.
  9. K. L. Wyres and K. E. Holt, “Klebsiella pneumoniae as a key trafficker of drug resistance genes from environmental to clinically important bacteria,” Current Opinion in Microbiology, vol. 45. Elsevier Ltd, pp. 131–139, Oct. 01, 2018. doi: 10.1016/j.mib.2018.04.004.
  10. V. Takhaveev and M. Heinemann, “Metabolic heterogeneity in clonal microbial populations,” Current Opinion in Microbiology, vol. 45. Elsevier Ltd, pp. 30–38, Oct. 01, 2018. doi: 10.1016/j.mib.2018.02.004.
  11. Y. Wang et al., “CRISPR-Cas9 and CRISPR-Assisted Cytidine Deaminase Enable Precise and Efficient Genome Editing in Klebsiella pneumoniae,” aem.asm.org 1 Applied and Environmental Microbiology Downloaded, vol. 84, pp. 1834–1852, 2018, doi: 10.1128/AEM.
  12. Qin S, Xiao W, Zhou C, Pu Q, Deng X, Lan L, Liang H, Song X, Wu M. Pseudomonas aeruginosa: pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics. Signal Transduct Target Ther. 2022 Jun 25;7(1):199. doi: 10.1038/s41392-022-01056-1. PMID: 35752612; PMCID: PMC9233671.