Antibacterial Activity of River Water Bacteriophage against Multidrug-resistant Gram-negative Bacteria, An In vitro Study

Microbes show a high antimicrobial resistance due to a high rate of mutations predisposed by many factors, especially the abuse of antibiotics. Therefore, there is a great need for an alternative therapeutic agent for infectious diseases caused by microbes resistant to antibiotics. Bacteriophages are viruses parasitizing microbes, that got a big scientist’s attention due to their ability as an alternative therapy for severe bacterial infections. This study is devoted to identifying bacteriophage from river water on tested pathogenic isolates isolated from clinical cases of UTI in vitro and finding out the effect of phage on these bacterial isolates as an initial step of further in vivo phage therapeutic study on the same tested isolates. The results showed a significant bactericidal effect of the isolated bacteriophages against the pathogenic bacterial isolates.


INTRODUCTION
Many bacterial species over several years are able to avoid the effect of antimicrobial agents, because of bacterial genomic DNA transfer or spontaneous bacterial genome mutations due to the abuse and overuse of antibiotics.These microbial mechanisms defect the activity of certain antibacterial antibiotics turning them ineffective. 1nfectious bacterial pathogens that are resistant to antibiotics are categorised as Mutable Drug-Resistant pathogens (MDR) and they become a significant health threat worldwide widely. 2 This turned out to be even more problematic, especially with the slower production of new antibiotic agents over recent years. 3Therefore, it became urgent to use an alternative agent for treating diseases caused by infectious bacteria to decrease antibiotic resistance emerging and disseminating. 4,5acteriophages are viruses that infect and replicate inside bacterial cells. 6They got big scientists' attention due to their ability as an alternative therapy for severe bacterial infections.Bacteriophage or simply phage infection can lead to the production of progeny phage via the lytic replication cycle inside bacterial cells.Lytic replication describes the process by which the phage uses the bacterial host cell as a factory to produce new phage capsids, tails and other structural proteins to form new phage particles within the bacterium. 7Recent theoretical developments have revealed that bacteriophages could take the role of solving the pathogenic bacterial resistance problem as alternative antibacterial therapy. 8,9The mechanisms used by phage are promiscuous compared to therapeutic regimes of antibiotics.Phage infection is so specific, each bacterial species has its specific phage that could be infected with, even if those bacteria are Mutable Drug-Resistant pathogens. 10,11In addition to the highly precise specificity of its bacterial host, the phage is abundant, effective in small doses and they are safe for humans and animals.Furthermore, phage particles decreased in their number with their host declined. 12 large number of therapeutic, in vitro and in vivo, approaches using phage particles have been developed over the last few decades testing different bacterial species and have achieved some very good results. 13For example, bacteriophage was evaluated for the treatment of infection caused by Klebsiella pneumoniae in mice. 14Moreover, several in vitro assays reported the efficiency of host-specific bacteriophage in the control of antibiotic-resistant bacteria for vancomycin-resistant Enterococcus faecium in experimental animals, as well as the efficiency of bacteriophage species isolated from some river water or swage water in the treatment against some enteric pathogens. 15,16These are examples of using a host-specific phage as an alternative treatment against Mutable Drug-Resistant bacterial pathogens.
In Iraq, antimicrobial resistance among pathogenic bacteria becomes predominant, and little effort has been forced to control this problem. 17At the same time, this country possesses very good water recourses with two big rivers; therefore, looking for lytic phage in river water or sewage water could be an alternative solution for antimicrobial resistance.
In this study, we examined the effect of phage from Euphrates River water, near Ramadi city, Iraq, against multidrug-resistant bacteria isolated from patients with urinary tract infections (UTI).

Water samples
Samples of river water were taken from virial places of the Euphrates River near Ramadi city, Iraq.The static surface of the side water was used to collect samples in 100 ml sterile containers.Samples were centrifuged to remove big particulates from the water for 30 minutes at 3000 rpm.The supernatant was poured into a sterile wight tube after it was filtered using a 0.22µm syringe filter.

Bacterial Isolates
Eighty-six Bacterial isolates were isolated from 123 patients suffering from urinary tract infections at the Ramadi Teaching Hospital for Maternity and Children.Bacterial isolates were then investigated for their bacterial species and antibiotic sensitivity using VITEK 2 system (bioMeriex).Clinical bacterial isolates were the only strains used in this study as the availability of commercial strains could not be achieved.MacFarland standards of turbidity on 0.5 O.D. for each isolate were obtained.This was by inoculating two or three bacterial colonies in 20 ml of Nutrient broth after 4 hours of incubation at 37°C.

Detection of bacteriophage effect against host bacteria
The host range of possible water bacteriophage was identified with 86 bacterial isolates using the spot test.Bacterial lawn culture was made using a sterile cotton swab, after being moistened with bacterial broth culture with 0.5 O.D. on tryptic soy agar plates from each bacterial isolate.A drop of five microliters of each filtered water sample was spotted on the agar plate.After they were allowed to dry, the plates were incubated at 37°C for 24 hours.The plates were then examined for the effectiveness zone on the spotted places. 16

Plaque assay for bacteriophage isolation
After it was filtrated, one ml of phage fluid was added to a sterile, 100 ml conical flask already containing 50 ml of a bacterial suspension of the respective host.The mixture was mixed very well before being left for 10 minutes at an appropriate temperature.The mixture was then mixed well with 3 ml of 07% molten agar at 50°C, before it was poured into the top of a nutrient agar plate.
After incubation at 37°C, the plates were absorbed for plaque formation over the surface of the top agar for each plate. 18

RESULTS
To find out whether the study hypothesis is verified or not, eighty-six (86) clinical bacterial isolates were isolated from 123 patients suffering from urinary tract infections.These clinical isolates are Escherichia coli(60 isolates), Enterobacter cloacae (23 isolates), Klebsiella pneumoniae (24 isolates), Pseudomonas aeruginosa (12 isolates), and Proteus mirabilis (27 isolates).On another hand, eight samples of river water were taken from the static surface of the riverside to find out whether these water samples contained phage species or not.
The antibiotic sensitivity of bacterial isolates was tested using VITEK 2 system (BioMeriex), this enabled the detection of the bacterial phenomenon of multidrug resistance (Figure 1).
The results revealed 88.33 % of Escherichia coli isolates were resistant to a wide range of antibiotics.Similarly, a high range of resistance was found when we tested Pseudomonas aeruginosa against used antibiotics.The antibiotic-sensitivity test of Enterobacter cloacae and Proteus mirabilis isolates showed medium resistance, with 34.68 Figure 1.The sensitivity pattern of bacterial isolate used in this study.Escherichia coli and Pseudomonas aeruginosa showed high resistance to the used antibiotics, while medium antibiotics resistance was reported with Enterobacter cloacae and Proteus mirabilis.And Klebsiella pneumoniae isolates were the most sensitive isolates to antibiotics % and 37.03%, respectively.The most antibioticsensitive isolates were Klebsiella pneumoniae isolates, with 16.6% bacterial resistance (Figure 1).
Regarding the bacterial isolates' sensitivity to the bacteriophage, the host range of possible water bacteriophage was detected using the plate spot method on bacterial lawns.
The results showed a wide sensitivity range of most bacterial isolates to water samples phage, which is considered significant as the P value is 0.024 using T-test.This result was so obvious, as there was a clear zoon usually created in the place of the water spot on each bacterial lawn (Figure 2 and 3).Although we got a significant detection of water bacteriophage against most bacterial isolates, however, we did not get any positive results of phage plaque assays, this unfortunate result was obtained after ten times repeating plaque assays with several parameter changes throughout about ten months of lab work.

DISCUSSION
Antibiotics resistant bacteria or what is called multidrug-resistant bacteria (MDR) become a serious problem for the human population throughout the world.Many bacterial pathogens have emerged as new serotypes with high resistance phenomenon to a high range of reliable antibiotics.Moreover, in recent years, the emergence of newly resistant bacterial serotypes was combined with less development of new antibiotics generations, which leads to minimised drug options for some severe bacterial infections. 19he use of bacteriophage as an antibacterial infection treatment started even before the discovery of antibiotics.However, the antibiotic emergence in the 1940s especially those with broad-spectrum besides the high bacterial sensitivity to them, at that time, replaced the development of bacteriophage as a therapeutic agent against bacterial infections. 20n this study, we examined the effect of suspected phage isolates that might be present in Euphrates River water, near Ramadi city, Iraq, against some Gram-negative bacterial isolates taken from patients with UTI.All bacterial isolates antibiotics sensitivity patrons were tested using VITEK 2 system (bioMeriex).The bacterial isolates' sensitivity to antibiotics was tested against a wide range of antibiotics belonging to groups of Penicillins, several generations of Cephalosporins, Fluoroquinolones, Aminoglycosides, Monobactams, Carbapenems, Folic acid Synthesis Inhibitors and Nitrofurans.
According to the results of this study, most E. coli isolates were resistant to antibiotics used.In this study, E.coli represents a considerable part of the commensal microbiota of both humans and animals; however, they are also an illness pathogen that could cause serious public health problems. 21E. coli has a variety of antibiotic resistance genes which might be the reason behind their treatment failure in human and animal infections.Significant resistance genes have been detected in E. coli genomes recently.Most of these genes were disseminated within the same bacterial generation by horizontal DNA transfer.Furthermore, E. coli acts as a resistance genes reservoir for other so related enterobacterial species which can receive these genes and can also act as a donor, so they pass these resistance genes to E. coli as well as other enterobacteria. 22ome of these genes are β-Lactams genes, which are present in numerous numbers in the E. coli genome.Many E. coli strains have a wide range of extended-spectrum β-lactamases (ESBLs) as well as cephalosporinases genes that could switch off the activity of penicillins, aminopenicillins, and the third and fourthgeneration cephalosporins including cefovecin, ceftiofur and cephalosporin cefquinome. 23Some other E. coli strains have the resistance ability to quinolones and fluoroquinolones.The target of these antibiotics is to invalidate the activity of both DNA gyrase, which has two subunits GyrA and GyrB, and topoisomerase, which consists of ParC and ParE subunits, in E. coli and other Gram-negative bacteria.Antibiotic resistance to quinolones and fluoroquinolones takes place due to a mutation in the genes for these enzymes in the E. coli genome. 24egarding the result of antibiotic sensitivity of Pseudomonas aeruginosa isolates, a similar result was seen as the result of E. coli isolates, as most P. aeruginosa isolates have high resistance to antibiotics used in this study.Pseudomonas aeruginosa is considered an opportunistic pathogen that is causing a significant health problem, particularly in compromised immunity patients and individuals with cystic fibrosis illness.It was reported that P. aeruginosa has a resistance phenomenon to many antibiotics including-lactamase, aminoglycosides, and quinolones. 25,26These bacteria are remarkably known for their various mechanisms of antibiotic resistance including intrinsic, adaptive, and acquired resistance mechanisms.As intrinsic, the bacteria could make their cells' outer membrane less permeable, they could also drive the antibiotic out of the cell by efflux pump expression, and the creation of enzymes that inactivate antibiotics.As an adaptive ability of resistance, the bacteria have the ability of biofilm formation particularly in lower respiratory tract infections which limits the access of antibiotics to the bacterial cells.The acquired ability of resistance can be accrued by either mutational change or by antibiotic genes' horizontal transfer. 27,28Therefore, the use of alternative treatment strategies for the therapy of P. aeruginosa infections becomes urgently needed particularly for patients with antibiotic-resistant infections.
The results of this study revealed that both Enterobacter cloacae and Proteus mirabilis isolates showed medium antibiotic resistance.Enterobacter spp., are within Enterobacteriaceae that resistant to carbapenem antibiotic (CRE) in the United States and this is the reason behind the distribution of carbapenem-resistant infectious diseases. 29Enterobacter cloacae particularly resistant strains area causative agent of a wide range of hospital-acquired infections, including urinary tract infections, pneumonia, and septicaemia. 30,31It was reported that Enterobacter cloacae can acquire antibiotic resistance genes, including wide deferent carbapenems genes.
Proteus mirabilis is a member of Enterobacteriaceae that is considered as normal bacterial flora of the human genitive tract.However, these bacteria are also opportunistic pathogens that cause serious urinary tract infections.Proteus mirabilis has a natural resistance phenomenon to some antibiotics and has low susceptibility to antibiotics like imipenem.Proteus mirabilis has the ability to lose porins and reduce the expression of bacterial genes that encode penicillin-binding proteins (PBPs). 32lebsiella pneumoniae isolates showed high sensitivity to the most used antibiotics in this study, this is not a bizarre outcome, similar results were obtained by Ameshe et al. who stated in their study, in Northwest Ethiopia, in 2022; that Klebsiella pneumoniae isolates had highly sensitive patterns to chloramphenicol, gentamicin, ciprofloxacin, cefoxitin, meropenem, ceftazidime, cefotaxime and nitrofurantoin.33 Nevertheless, many studies reported that multidrug resistance Klebsiella pneumoniae become a public health problem in many parts of the world.34,35 Pathogenetic bacteria with multiantibiotic resistance is a significant globule problem that is estimated to be as serious as the reason behind about ten million mortalities annually by 2050.36 Therefore, new alternative therapeutic approaches are strongly needed.Using bacteriophages therapeutic strategies is one of these approaches.Phage in their nature, like other viruses, are abundant agents that cannot grow or replicate without the use of bacterial cells with all their mechanisms to make new phases and spread after destroying the bacterial cell.Furthermore, phage are innocuous to plants, animals and even humans.For these reasons, they could be used as an antibacterial agent instead of using antibiotics or they could be used in combination with antibiotics to treat bacterial infections in plants, animals and humans.37 According to our results using the plate spot method on bacterial lawns, most of the used bacterial isolates showed a high range of sensitivity to water samples' phage.This result confirms many previous studies' outcomes. Siilar results were stated by Bhetwal et al. as they tested the effect of river-isolated bacteriophage on bacterial isolates that had MDR or ESBL bacteria.16 Many previous studies confirmed our presented results, as they confirmed the therapeutic effect of bacteriophage on pathogenic bacteria.[38][39][40][41]

Figure 2 .Figure 3 .
Figure 2.The effect of water's phage samples on bacterial isolates used in this study.Bacteria that highly be inhabited by bacteriophage was Enterobacter cloacae followed byE.coli, while the less be affected bacteria was Proteus mirabilis