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Journal Concepts in Structural Biology & Bioinformatics (JSBB)

ANTIBIOTICS RESISTANCE ARTICLES

Review

Could bacteriophage, the ancient enemy of bacteria, be the

solution to antibiotic resistance?

BENABBOU Taha Ahmed

1

., BELLIL Yahia

1,2

., BENREGUIEG Mokhtar

1

., CHAHROUR Wassila

1,2

1. Laboratory of Biotoxicology, Pharmacognosy and biological valorisation of plants Compus Ain El Hadjar, Faculty of Sciences,

Department of Biology, University Dr Tahar Moulay Saida, 20100 Saida, Algeria.

2. Laboratory of Applied Microbiology, Department of Biology, Faculty of Nature Science and Life, University of Oran 1 Ahmed

BENBELLA, B.P. 16, Es-Sénia,31100 Oran, Algeria.

Correspondence: BENABBOU Taha Ahmed Department of Biology, University Dr Tahar Moulay Saida, 20100 Saida, Algeria

E. mail : t.a.benabbou@gmail.com

Abstract

The threat of antibiotic-resistant bacterial pathogens has led to a resurgence of

bacteriophages in the medical field. Although knowledge of these bacterial viruses has

progressed considerably after almost a century of study, their therapeutic use is far from being

mastered. The objective of this literature review was to provide an overview of bacteriophages

while emphasizing the need not to underestimate phage therapy in the era of epidemic

antibiotic resistance.

Key words

Bacteriophages, bacterial infections, antibiotic resistance, phage therapy, phage-antibiotic

synergy.

Introduction

Humans today are victims of tiny predators, the most threatening of which are

pathogenic bacteria. This enemy has long been fought by antibiotics, but their excessive use in

recent decades has led to the inexorable development of bacterial resistance, with more and

more therapeutic failures reported worldwide (WHO, 2022). Fortunately, bacteria are also

victims of natural predators since, like all other living organisms, they can be infected by viruses

that outnumber their bacterial hosts by 10 to 15 times (Suttle, 2007). These viruses of bacteria,

the bacteriophages or phages, are the most abundant in the biosphere and are present in all

areas where bacteria grow. Virtually all bacterial species can be infected by many different

phages, the majority of which are unknown, and what has been discovered represents only a

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tiny fraction of their true diversity. Phages and since their discovery in the early 20th century by

Felix d'Herelle (Golkar, Bagasra and Pace, 2014), they have been used to combat certain

bacterial infections under the name of phage therapy, which seems to be within reach, with well

described efficacy in the literature (Hatfull, Dedrick and Schooley, 2022), this concept spread in

the medical community until it was quickly supplanted by the discovery and use of antibiotics,

yet phage therapy has continued to exist to this day through various laboratories and institutes,

most notably in the Republic of Georgia, thanks to the George Eliava Institute co-founded by

d'Herelle and Eliava in the 1920s (Bradbury, 2004), resulting in some of the biotechnology,

pharmaceutical and agri-food companies attempting to commercially exploit the antibacterial

activity of phages (Comeau et al., 2008; Sigg et al., 2022).

Discovery of bacteriophages

In 1896, Ernest Hankin was the first to notice that the waters of the Jumna and Ganges

rivers in India were bactericidal. After filtration, they had the capacity to cure patients suffering

from cholera, a disease caused by the Vibrio cholerae bacterium. He assumed that this

substance was responsible for the end of a cholera epidemic by ingesting water from these

rivers (Hankin, 1896). A few years later, lysis phenomena in bacterial cultures were observed

without being explained. It was not until 1915 and 1917 that two scientists, Frederick Twort and

Felix d'Herelle, studied this phenomenon in more detail and formulated hypotheses as to its

origin. Twort imagined an "ultra-microscopic virus" and D'Herelle, an "invisible microbe".

D'Herelle quickly isolated the mysterious infectious agent and named it bacteriophage, literally

"the eater of bacteria" (Golkar, Bagasra and Pace, 2014). It was not until 1940 that the first

bacteriophage was observed under an electron microscope by Ernst Ruska. The development of

molecular biology then allowed to deepen the knowledge on their structure, their composition

and their functioning (Akhverdyan et al., 2011; Jassim and Limoges, 2013).

Structure and composition of bacteriophages

Bacteriophages are between 20 and 200 nm in size (the size of a virus is generally

between 10 and 400 nm) (Walker, 2006). Their descriptions have allowed to highlight an

important diversity of viruses classified according to their morphology (morphotype), their

composition (in particular the nature of the nucleic acid which can be DNA or RNA, single or

double stranded) and their host spectrum (Hanlon, 2007). Most of the bacteriophages currently

described from electron microscopy observations belong to the order Caudovirales (Fig 1).

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Fig 1: Transmission electron microscopy (TEM) images of corresponding types of phages.

Scale bar: 100 nm. (Image adopted from Ackermann and Francis Jr, (2012))

One of the most studied is the T4 bacteriophage which infects the bacterium Escherichia

coli. It will therefore be taken here as a reference (Fig 2).

Fig 2: Electron micrographs of bacterial viruses. Bacteriophage T4 of Escherichia coli. The

tail components function in attachment of the virion to the host and injection of the nucleic

acid. The head is about 85 nm in diameter. The structure has been adopted from Madigan et

al., (2008).

Life cycle

During the process of infection, a phage attaches to a bacterium and injects its genetic

material into the bacterial cytoplasm (Fig 3). The phage's genetic information then takes control

of the bacterial cell's machinery by disabling the synthesis of bacterial components and

redirecting the bacterial synthetic machinery to the manufacture of other phage components

(Guttman and Raya, 2005). The newly made phage heads are individually filled with replicas of

the phage chromosome. Eventually, many phages progeny are made and are released upon

rupture of the bacterial cell wall (Hanlon, 2007). This breaking process is called lysis.

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Fig 3: Two types of phage replication cycles. The model has been adopted from (Reece et al., 2012)

Within 15 hours after one single phage particle infects a single bacterial cell, the effects

are visible to the naked eye as a clear area, or plaque, in the opaque lawn of bacteria covering

the surface of a plate of solid medium (Anderson et al., 2011) (Fig 4).

Fig 4: Phage plaques. Through repeated infection and production of progeny phage, a

single phage produces a clear area, or plaque, on the opaque lawn of bacterial cells (Griffiths et

al., 2005).

Temperate phages can remain within the host cell for a period without killing it (Verheust

et al., 2010). Their DNA either integrates into the host chromosome to replicate with it or

replicates like a plasmid, separately in the cytoplasm. A phage integrated into the bacterial

genome is called a prophage. A bacterium harboring a quiescent phage is called lysogenic (Fig

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3). Occasionally a lysogenic bacterium lyses spontaneously (Wagner and Waldor, 2002;

Verheust et al., 2010).

Phage therapy: bacteriophages as therapeutic agents

Bacteriophages have been widely used to combat various bacterial infections after their

discovery in the early 20th century. For example, d’Herelle had the idea of using them as

therapeutic agents to treat children with dysentery. The result was a success and, little by little,

curative treatment with bacteriophages spread throughout the world (Rao and Lalitha, 2015).

But very soon, due to the lack of knowledge about the nature and functioning of

bacteriophages, this practice became controversial and subject to strong criticism. In 1928, the

discovery of penicillin by Alexander Fleming (Fleming, Sheehan and JM, 1928), followed by the

discovery of other antibiotics, heralded the decline of phagotherapy. The latter, much simpler

to manufacture, were quickly mass-produced by the pharmaceutical industry and then

marketed. However, today, in view of the increase in cases of antibiotic resistance, the

therapeutic potential of bacteriophages is once again being studied (Jaiswal et al., 2013; Hatfull,

Dedrick and Schooley, 2022; Uyttebroek et al., 2022). Unlike antibiotics, bacteriophages have a

very narrow spectrum of action, allowing them to target pathogenic bacteria more precisely.

Their use, generally in the form of a cocktail of bacteriophages (a combination of several

bacteriophages), would make it possible to fight infectious bacteria in a more targeted manner

and to overcome the appearance of resistance. Finally, as bacteriophages do not attack

eukaryotic cells, they are eliminated once their target has been eradicated (Wittebole, De Roock

and Opal, 2014; Sompalli et al., 2022). With this in mind, bacteriophage therapy can be very

effective in treating a variety of infections, promising results have been observed in curing

anthrax, caused by Bacillus anthracis (Jamal et al., 2019) or to reduce nasopharyngeal (nose and

pharynx) carriage of pathogens (Loeffler, Nelson and Fischetti, 2001). Phages have also been

used in neonates for the treatment of brain and spinal cord meningitis. This deadly infection

can be successfully treated with phages (Macneal, Frisbee and Blevins, 1943). Additionally, for

the most deadly infections such as methicillin-resistant Staphylococcus aureus (MRSA), oral

therapy could be used to treat the disease or infections (Álvarez et al., 2019).

Synergy between antibiotic therapy and phage production

Phage propagation is essential for phage therapy; it also has an enormous impact on

microbial ecology in all ecosystems. Nevertheless, the effects of environmental factors on

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phage propagation remain virtually unexplored. In this area, the use of phages in combination

with antibiotics that block cell division seems to be a better idea to facilitate bacteriophage

overproduction. The synergistic effect of this combination was first demonstrated in 1941,

when Zaytzeff-Jern et Meleney showed that the combination of phages with sulfanilamide and

sulfapyridine inhibited the in vivo growth of Staphylococcus aureus and Escherichia coli

(Zaytzeff-Jern and Meleney, 1941). Years later, in 1945, and this time using a β-lactam,

Himmelweit was able to prove that the phage-penicillin combination reduced the time to

complete lysis of Staphylococcus aureus from 6 hr and 3 min for penicillin alone, to 1 hr and 55

min for phages alone, and to 1 hr and 25 min for the phage-penicillin combination

(Himmelweit, 1945). Furthermore, in (2007) Comeau et al. found that the addition of low doses

of cefotaxime to bacterial cultures of uro-pathogenic Escherichia coli at a non-lethal dose

increased phage production by more than 7-fold, thereby accelerating bacterial lysis (Fig 5).

Fig 4. The phage ΦMFP antibiotic effect on E. coli MFP. Only disks containing the β-

lactams aztreonam and cefixime (indicated by "+" symbols) produced large phage plaques in

their vicinity (Comeau et al., 2007).

Conclusion

The practice of using bacteriophages has recently been seen as a way to end the cycle of

bacterial infections and as the safest treatment to eradicate multidrug-resistant bacteria

without any side effects. However, this practice still has to overcome many obstacles, especially

regarding the manufacturing process. The latter must be mastered because currently, even if

bacteriophages seem to meet the definition of a drug and more precisely of a bio-drug, there

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is no official manufacturing guide. Another difficulty is the lack of knowledge on the effects of

bacteriophages in the long term on bacterial ecosystems, both in the body and in our

environment.

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