Isolation, characterization and anti-biofilm efficacy of a novel Klebsiella pneumoniae phage

doi:10.21203/rs.3.rs-3311342/v1

Download PDF

Research Article

Isolation, characterization and anti-biofilm efficacy of a novel Klebsiella pneumoniae phage

https://doi.org/10.21203/rs.3.rs-3311342/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

The Multi-Drug-Resistant (MDR) Klebsiella pneumoniae (K. pneumoniae) is an important pathogen that threatens public health directly with life threatening infections. The need for the development of new effective and safe alternative treatments for these infections is crucial. Therefore, the interest in phage therapy as a promising alternative is increasing. Here, a novel phage named ΦAYH was isolated from the Tigris River water, Baghdad, IRAQ near sewage of Baghdad Medical City with its specific host from the same site. Phage ΦAYH belongs to Podoviridae family in the order Caudovirales. The ΦAYH maintained stability at different temperatures (-10- 60°C) and pH values (5-11). For one-step growth, latent period was 10 min with burst size ~64 virions/ cell at MOI 10. The phage was able to lyse 8 from 32 clinical K. pneumoniae isolates in vitro. The SDS-PAGE test revealed one major structural protein and different structural proteins ranging from 28 to 89 kDa in size. The phage host and 32 clinical K. pneumoniaeisolates were tested for phenotypic identification and antibiotics profile by VITEK-2 system and genotypically using rpob gene. All clinical K. pneumoniae isolates showed resistance to the most antibiotics tested while phage host was resistant only to amoxicillin. Biofilm production by all clinical isolates including the host isolate was tested. These isolates showed different ability as following: 72.72 % as weak, 6.06% as moderate, and 21.21% as strong biofilm producer. Together these results demonstrate that ΦAYH is a promising alternative against MDR K. pneumoniae.

Klebsiella pneumoniae

bacteriophages

Multidrug-resistant

anti-biofilm

A new phage named ΦAYH was isolated from Tigris River water, Baghdad, IRAQ and characterized.
The Klebsiella pneumoniae host was isolated from the same phage site.
Phage ΦAYH belonged to the family Podoviridae in the order Caudovirales.
ΦAYH exhibited a wide temperature tolerance range (-10-60°C) and pH tolerance in a range (5-11).
Phage ΦAYH can infect and lyse Klebsiella pneumonia clinical isolates.
The ΦAYH phage showed a promising anti-biofilm activity.

Klebsiella pneumoniae (K. pneumoniae) is considered one of the important pathogens in hospital-acquired infections responsible for a broad range of infections including septicemia, pneumoniae and urinary tract infections (Tabassum et al., 2018). K. pneumoniae possess a various mechanisms for antibiotic resistance (Qin et al., 2017). In addition, this bacterium can quickly develop strains with multidrug resistance, which leads to failure to treat these strains with antibiotics. It has been noticed that K. pneumoniae can easily develop resistance to antibiotics throughout the production of different enzymes such as carbapenemase and extended spectrum β-lactamase (ESBLs) (Munita and Arias, 2016). Furthermore, the increase in occurrence of carbapenem-resistant K. pneumoniae has become a fatal factor in the failure of antibiotics effectiveness against infections treated by these antibiotics (Tesfa et al., 2022). Different local studies suggest the rapid and uncontrolled spread of antibiotic resistant bacteria between Iraqi patients (Alzaidi and Mohammed, 2022; Kanaan and Khashan, 2022; Salman et al., 2022). In addition, several studies on Iraqi rivers indicated their contamination with antibiotic-resistant bacteria (Abbas, 2021; Abdulsattar et al., 2020; Alwash and Al-Rafyai, 2019). K. pneumoniae are able to form biofilms on various surfaces: biotic and abiotic surfaces. In a case of biotic surfaces include host various tissues like the urinary, respiratory, and gastrointestinal tract mucosa and abiotic surfaces involve catheters and medical devices (Guerra et al., 2022). The biofilm is defined as communities of bacteria surrounded by an extracellular matrix and this matrix consists of exopolysaccharides, proteins, lipopeptides and DNA (Ashurst and Dawson, 2018). These structured microbial communities play a role in increased antimicrobial resistance by reducing penetration of these agents and resistance to defense mechanisms of the host (Vuotto et al., 2014). Several factors contribute in ability to form biofilm for K. pneumoniae include fimbriae and pili, iron metabolism, polysaccharide capsule, and the presence of different bacterial species. The bacteria that can form biofilms able to resist antibiotics than planktonic bacteria by 10–1000 times (Patel, 2005). Attention for alternative antimicrobial agents like phages have been increased for the treatment of human bacterial infections in the recent years (Chen et al., 2022; Koberg et al., 2017; Nazir et al., 2022; Obradović et al., 2023; Pallavali et al., 2017). The most abundant organisms on earth are bacteriophages that are capable of infecting bacteria specifically (Doss et al., 2017). Bacteriophages are isolated easily from the environment and not expensive to use. In addition, they are safe and their immunological complications are few compared to drug side effects (Ji et al., 2019; Soleimani Sasani and Eftekhar, 2020). A recent study indicated the efficiency of bacteriophages in treating device-associated infections caused by K. pneumoniae (Cano et al., 2021). Due to the rapid emergence of MDR K. pneumoniae, the isolation and characterization of more different lytic phages are needed. The present study aimed to isolate a novel bacteriophage against MDR K. pneumoniae, characterize the isolated phage based on morphology, titre, physiological and chemical parameters, host range, optimal multiplicity of infection, one-step growth curve and proteomic analysis. Furthermore, the anti biofilm ability of bacteriophage against host and other MDR clinical isolates was assessed.

2.1 Collection of clinical K. pneumoniae isolates

This study included no human subjects; therefore, ethical approval was not required. The K. pneumoniae clinical isolates were collected from some laboratories in Baghdad city hospitals including: Children’s Hospital, Teaching Laboratories of Medical City, Al Karkh General Hospital and Baghdad Teaching Hospital from October 2022 to February 2023. The isolates were cultured on both blood and MacConkey agar plates for primary isolation and phenotypic identification.

2.2 Clinical isolates identification

All clinical isolates of K. pneumoniae were identified by VIETK 2 system (Bio- Merieux/France) as K. pneumoniae ssp. pneumoniae. Final identification was done by molecular method using rpoB gene (He et al., 2016). Genomic DNA was isolated by boiling method as following: the isolates were cultured on fresh agar plate and 10 single colonies of each isolate were added to 400 µl ddH2O in eppendorf tubes. Then, the samples were left in water bath at 100ºC for 10 min to lyse the cells. The tubes were kept cooled on ice immediately followed by frozen the tubes at -20 for 20 min. The following step, these tubes left to thaw at room temperature and homogenized by vortex for 10s. Then, the samples were centrifuged at 13,362 xg at 4°C for 15 min and the upper aqueous layer was taken and transferred into new sterile eppendorf tubes. All DNA samples were kept frozen until used (Dashti et al., 2009). All K. pneumoniae isolates identified in this study were transferred to 1.5 ml eppendorf tubes contain nutrient broth with 20% (v/v) glycerol and kept at -80ºC for long-term storage.

2.3 Molecular identification of k. pneumoniae isolates

PCR was used to detect rpob gene and the PCR reaction was prepared in a total volume of 25 µl as the following: Promega Master mix 12.5 µl, forward primer (F 5-GTTGGCGAAATGGCGGAAAAC-3) 1 µl; reverse primer (R 5-ACGTCCATGTAGTCAACCTGG-3) 1 µl; nuclease-free distilled water 5.5 µl; DNA template 5 µl. PCR conditions were used for 30 cycles as the following: 95°C for 5 min as the initial denaturation; 95°C for 30s denaturation step of DNA; 57°C for 30s as the annealing step of the primers, 72°C for 45 s as the elongation step, 72°C for 5 min as final extension step. The PCR products were run along DNA ladder (100bp, Cleaver scientific/UK) using electrophoresis at 100 V for 45 min in a 1% agarose gel with SYBR safe DNA gel stain (Invitrogen). The bands were visualized using a UV transilluminator.

2.4 Collection of samples for phage and host isolation

Samples were collected from the Tigris River near Baghdad Medical City in September 2022 (33.3474351, 44.3722910). Briefly, the water samples (50 ml) were taken from 50 cm in depth and 1 meter away from Tigris River edge, collected in sterile dark bottles and stored in 4 ° C (ice box). In lab, water samples (10 ml) were centrifuged at 5000 rpm for 10 min to remove the solid impurities. A 100 µl from the supernatant were cultured on MacConkey and Chromagar and incubated at 37°C for overnight to the host. A large mucoid colonies and blue colonies on MacConkey and Chromagar, respectively were picked and subjected for further biochemical tests for identification. A glycerol stock was prepared and stored at -80° C. While the supernatant from centrifugation step were passed through 0.45µm and 0.22µm pore size membrane filter (Millipore, USA) to remove residual cells for phage isolation.

2.5 Phage isolation and purification

Each identified K. pneumoniae isolate from water sample at exponential phase was mixed with the filtered sample (1:1) and left for 10 min at room temperature (RT) for phage adsorption. Later, nutrient semisolid medium (4 ml) was mixed with the host-phage mixture, poured onto fresh nutrient agar plate quickly and allowed to dry. After incubation for 18 hours, a single plaque was collected using sterile pipette tips and re-suspended in phosphate buffered saline (PBS). The phage purification was repeated for five rounds using agar overlay method by picking new plaque to obtain uniform pure plaques (Gorodnichev et al., 2021).

2.6 Phage titration

The double layer plate technique was used to determine phage titre. Briefly, a serial of dilutions (ten-fold) from phage was prepared with PBS. Each dilution was mixed with the host K. pneumoniae at log-phase (1:1), left at room temperature for 10 min, fresh soft agar added (4ml), and mixture poured onto a fresh nutrient agar plate for each dilution. Following overnight incubation, the plaque number on each plate was counted and the titre calculated as a plaque-forming unit per ml (pfu/ml). The high titre was stored at -80°C (Balcão et al., 2022).

2.7 Transmission Electron Microscopy (TEM) and Plaque morphology

To observe bacteriophage morphology, the plate containing phage plaques was emerged with 10 ml PBS and left at room temperature for 2 hours and then centrifuged for 30 min at 1500 rpm. Further, the obtained supernatant was passed through 0.22µm pore size membrane. A drop from the phage lysate on a copper grid (200mesh coated with Formvar film thickness 50nano) was stained negatively with (2% w/v) uranyl acetate and lead citrate drops. The grid was allowed to dry on a filter paper. The phage morphology was examined with ZEISS FESEM Supra 55vp (Germany) at Basra University- Pharmacy College, Basra, Iraq. For plaque morphology, a plate containing uniform plaques was incubated (24,48 and 72) hours to observe plaque morphology (Abdulhussein and O. Abdulsattar, 2022).

2.8 Thermal Tolerance of ΦAYH phage

The effect of different temperatures (-10, 4 ◦C, 25 ◦C (representing phage storage room temperature), 37 ◦C, 50 ◦C, 60 ◦C, 70 ◦C, and 80 ◦C on phage ΦAYH stability was determined. The fresh phage lysate was diluted in PBS buffer to 10⁸ pfu/ml and incubated at each temperature for one hour. The lysates at high temperatures were allowed to cool and then the double agar overlay method was used to determine the phage titre at each temperature. This experiment was carried out in triplicates (Wintachai et al., 2020).

2.9 pH stability of ΦAYH phage

The phage ΦAYH viability at different pH (2,3,4,5,6,7,8,9,10,11,12, and 13) was tested. With minor modification, the phage lysate (10⁸ pfu/ml) was incubated at each pH at 37 ◦C. After one-hour incubation, the titre of phage samples was determined using the double layer plate method. The experiment was conducted three times and pH = 7 used as a control (Martins et al., 2022).

2.10 Host range determination

The spot assay was performed to determine the host range of ΦAYH phage. A total of 32 clinical Klebsiella pneumoniae, 3 of Escherichia coli, 3 of Pseudomonas aeruginosa, 4 of Acinetobacter baumannii, 2 of Staphylococcus aureus, and 2 isolates of Serratia marcescens were used. Briefly, each clinical isolate at exponential phase was mixed with 4 ml molten agar (0.7%). The mixture was poured on fresh nutrient agar and after solidification a 3 µl of purified fresh phage lysate (10⁸ pfu/ml) was pipetted on each of the selected isolate. On the next day, the presence of phage plaques was observed for each isolate and the host was used as a positive control. The experiment was performed in triplicate (Peng et al., 2020).

2.11 Optimal MOI

The host bacteria at mid-log phase (~ 108 cfu/ml) was mixed with phage ΦAYH at a different MOI (0.0001, 0.001, 0.1, 1, and 10). The mixture was left at 37°C for 10 hours and the titre was determined using an agar overlay method for each MOI. This experiment was conducted in triplicate and the optimal MOI was determined.

2.12 One step growth curve

The host K. pneumoniae (log-phase) was infected with phage ΦAYH at optimal MOI and left for 5 minutes to allow phage adsorption. The mixture was centrifuged (5000xg, 10 min) at 4°C. The pellet washed twice with fresh nutrient broth and suspended in fresh nutrient broth (10 ml). Then, the samples were incubated in a shaker (180 rpm) for 2 hours at 37°C. A sample at each 10 min interval for 120 min was collected and titre at each time was measured using double-layer method. The burst size and latent period was determined. This experiment was conducted three times (Li et al., 2020).

2.13 Bacterial biofilm susceptibility

The ability of all clinical K. pneumoniae isolates including the phage host to form biofilm was detected by Microtiter plates method as following: The isolates were cultured on brain heart infusion agar for overnight at 37^оC and few single colonies (10) were selected, suspended in 5 ml of PBS and mixed by vortex. A 20µl of each bacterial suspension was added to brain heart infusion (180 µl) in 96-well flat-bottomed microtiter plate. The controls wells contained 200 µl un-inoculated broth. Following step, the plates were incubated at 37ºC for 48h followed by removing the well contents. Then, the wells were washed three times with PBS (pH 7.2) and left at room temperature for 15 minutes to dry. The wells were stained with crystal violet (1%) for 15 minutes and followed by removing the crystal violet, and washing the wells three times with PBS (pH 7.2) to remove the unbounded dye. The plate was allowed to dry at ambient temperature and 200 µl ethanol was added to the wells before measuring the OD. The absorbance of each well was measured at 630 nm using ELISA reader. Each assay was repeated three times and the adherence capabilities of the bacterial were calculated as follow: non-biofilm producers (OD ≤ ODc), weak biofilm producers (ODc < OD ≤ 2ODc), moderate biofilm producers (2 ODc < OD ≤ 4 ODc) and strong biofilm producers (4 ODc < OD). (ODc) represents OD cut-off (three standard deviations above the mean OD of the negative control) (Babapour et al., 2016).

2.14 Bacterial biofilm susceptibility to phage ΦAYH

Bacterial biofilm susceptibility was done as previously described but instead of adding 20µl of each bacterial suspension to 180 µl of brain heart infusion, 20µl of each bacterial suspension and 2µl of the phage was added to 178 µl of medium in each well. The incubation, washing and staining steps were followed as previously described (Taylor, 2020).

2.15 Proteomics analysis

Purified phage particles were subjected to Amicon Ultra centrifugal filter units (50 kDa), Millipore (Merck, France) to a final volume of 100 µl by centrifugation at 4 ºC for 30 min at 5000 rpm. The concentrated sample was mixed with LDS sample buffer (4:1) and denatured for 10 min at 99 ºC, followed by centrifugation for 2 min (13000 rpm) and loaded into SDS-PAGE gel (10%). After electrophoresis (25 mA, 70v for 2.5 h) in a 1× MES running buffer, the gel was then stained with Coomassie blue R-250.

3.1 Host bacterium isolation and identification

As shown in (Fig. 1A and B) different bacterial isolates including K. pneumoniae were obtained from the Tigris River water samples. The colonies that showed mucoid colonies on MacConkey and blue colonies on CHROM agar were picked and further purified (Fig. 1C and D) for further species identification. The biochemical test including oxidase and catalase tests were performed. The K. pneumoniae showed negative and positive results for oxidase and catalase test respectively. The VIETK 2 system and genotypic identification by ropB confirmed the identification of K. pneumoniae and indicated the K. pneumoniae isolate chosen, as a host for phage isolation and characterization was resistance to Amoxicillin.

3.2 Isolation and identification of clinical bacterial isolates

The clinical K. pneumoniae isolates were isolated from different hospitals in Baghdad city and sources of these isolates were distributed as the following: 51.5% from urine, 12.5% from sputum, 12.5% from blood, 6.25% from wound swab, 9.37% from endotracheal tube, 3.12% from kidney pus, and 3.12% from foley tip. These clinical isolates including host were identified by the automated VITEK 2 system and amplification of rpob gene. This gene, which encodes β-subunit of RNA polymerase was suggested as a powerful tool for K. pneumoniae isolates identification (He et al., 2016). Thus, PCR products for this gene were confirmed the identification of the isolates in this study (Fig. 2).

3.3 Phage isolation, purification

A phage named ΦAYH was isolated from the Tigris River near Baghdad Medical City, Baghdad, IRAQ in September 2022 using host from the same site. The initial screening included using different K. pneumoniae isolates obtained from the same site as a host. For plaque morphology, the phage formed clear plaques (3 m) in diameter surrounded by large translucent halo as shown in (Fig. 3A). The TEM analysis showed the phage exhibited a Podoviridae family features belonging to Caudovirales order with an icosahedral head (50 nm) and a short tail (5mm) according to the latest classification by the International Committee on the Taxonomy of Viruses (ICTV) (Fig. 3B). All phage characterization tests were performed using host K. pneumoniae isolated from the same site for phage isolation

After prolong incubation, the plaque morphology changed, translucent halos surrounded phage plaques diameter was 3 mm after 24 hours incubation, reached to 5 mm after 48 hours and 7 mm in 72 hours while clear center remained constant.

3.4 Thermal stability

To determine phage stability at different temperatures, suspensions of the phage (10⁸ pfu/ml) with the host at exponential phase were mixed and incubated at different temperatures from − 10 to 80 ◦C for one hour. After incubation, phage titre was measured for each temperature in triplicates and phage maintained active between − 10–50 ◦C. When temperature reached 60 ◦C, the phage activity declined and no phage was detected at temperature 70◦C and 80◦C (Fig. 5).

3.5 pH stability

The stability of phage ΦAYH at various pH values was tested using agar overlay method. The phage was stable at pH from 5 to 11. However, no phage detected at a pH 2 and 3 (acidic) or pH above 12 (alkaline) as shown in (Fig. 6).

3.6 Host range

The agar overlay method was applied to determine host range of the phage ΦAYH. A total number of 32 different clinical isolates were tested. Results showed that phage ΦAYH could infect the K. pneumoniae host and 8/32 of K. pneumoniae clinical isolates suggesting that phage ΦAYH is highly specific and showed did not lyse other species tested (Fig. 7) (Table 1).

Table 1

Bacterial strains, source, antimicrobial sensitivity, serotypes and host range analysis.
Bacterial strain	Source	Antibiotic resistance	Sub class	Spot assay
K. pneumoniae Host	Sewage	Amoxicillin	Resistant to one class	+
K. pneumoniae12	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin Trimethoprim/Sulfamethoxazole ,minocycline(I)	MDR	-
K. pneumoniae15	Wound swab	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Minocycline(I), Trimethoprim/Sulfamethoxazole	MDR	+
K. pneumoniae20	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Doxycycline ,Minocycline, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumoniae22	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam,Imipenem, Meropenem ,Amikacin, Gentamicin Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin ,Doxycycline ,Minocycline, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumoniae23	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Trimethoprim/Sulfamethoxazole	MDR	+
K. pneumoniae32	Sputum	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem(I), Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Doxycycline ,Minocycline, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR	+
K. pneumoniae 35	Sputum	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Doxycycline ,Minocycline, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumoniae 36	Foley tip	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumoniae39	Endo Tracheal Tube	Ampicillin ,Ceftriaxone, Aztreonam, Piperacillin/Tazobactam, Cefepime, Amikacin, Gentamicin, Ciprofloxacin ,Ceftazidime, levofloxacin, Tetracycline	MDR	+
K. pneumoniae42	Urine	Ampicillin ,Ceftriaxone, Aztreonam, Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumoniae49	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam,Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin ,Pefloxacin, Doxycycline ,Minocycline, Tetracycline, Colistin ,Rifampicin, Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumoniae55	Sputum	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Doxycycline ,Minocycline, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumoniae56	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin ,Minocycline, Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumoniae57	Endo Tracheal Tube	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin Doxycycline ,Minocycline, Tetracycline	MDR	-
K. pneumoniae74	Blood	Ampicillin ,Ceftriaxone Piperacillin/Tazobactam, Cefepime, Gentamicin, ,Ceftazidime, Tetracycline	MDR	-
K. pneumonia 89	Urine	Ampicillin ,Ceftriaxone, Aztreonam (I),, Cefepime, Gentamicin ,Ceftazidime	MDR	+
K. pneumoniae 95	Blood	Ampicillin ,Ceftriaxone, Cefepime, Gentamicin, Trimethoprim/Sulfamethoxazole ,Ceftazidime	MDR	-
K. pneumoniae 99	Blood	Ampicillin ,Ceftriaxone, Aztreonam, Piperacillin/Tazobactam (i), Cefepime, Gentamicin, Trimethoprim/Sulfamethoxazole ,Ceftazidime, ,Tetracycline (I)	MDR	-
K. pneumoniae108	Urine	Ampicillin ,Ceftriaxone, Aztreonam, Trimethoprim/Sulfamethoxazole, ,Ceftazidime, levofloxacin, Tetracycline	MDR	-
K. pneumoniae117	Blood	Ampicillin ,Ceftriaxone, Aztreonam, Cefepime ,Ceftazidime, Tetracycline	MDR	-
K. pneumoniae289	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam (I) ,Ceftazidime, Cefepime, Aztreonam, Amikacin(I), Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Minocycline (I), Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumoniae315	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem, Meropenem ,Amikacin(I), Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin Doxycycline ,Minocycline, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumoniae326	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Ceftazidime, Cefepime, Aztreonam, Tobramycin, Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Doxycycline ,Minocycline, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR	+
K. pneumoniae342	Wound swab	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin, Aztreonam, Minocycline, Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumoniae366	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam, Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin ,Minocycline(I), Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumoniae368	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,PiperacillinPiperacillin/Tazobactam(I),Cefpodoxime, ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Doxycycline ,Minocycline, Trimethoprim/Sulfamethoxazole	MDR	+
K. pneumoniae 400	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin , Doxycycline ,Minocycline, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumoniae 418	Endo Tracheal tube	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem, Meropenem ,Amikacin, Gentamicin(I), Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin Doxycycline ,Minocycline, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR
K. pneumoniae 422	Pus from kidney	Ticarcillin ,Piperacillin ,Ceftazidime, Aztreonam, ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Trimethoprim/Sulfamethoxazole	MDR	-
K. pneumonia 429	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam, Doxycycline ,Minocycline, Tetracycline	MDR	+
K. pneumonia432	Sputum	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam, Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin , Trimethoprim/Sulfamethoxazole	MDR	-
Pseudomonas aeruginosa 23	c.v.I tip	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,cefotaxime ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime,Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin	MDR
Pseudomonas aeruginosa 55	Sputum	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,cefotaxime ,Ceftazidime ,Ceftriaxone, Cefepime, Imipenem, Ertapenem ,Meropenem, Gentamicin Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR
Pseudomonas aeruginosa 64	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime, cefotaxim ,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam ,Imipenem, Meropenem ,Amikacin, Gentamicin, Netilmicin, Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin	MDR
Acinetobacter Baumannii 3	Pleural fluid	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,cefotaxime ,Ceftazidime ,Ceftriaxone, Cefepime, Imipenem, Ertapenem ,Meropenem ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Tetracycline	MDR
Acinetobacter Baumannii 26	Sputum	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,cefotaxime ,Ceftazidime ,Ceftriaxone, Cefepime, Imipenem, Ertapenem ,Meropenem, Gentamicin , Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR
Acinetobacter Baumannii 55	Sputum	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam ,Cefixime ,Cefpodoxime ,cefotaxime ,Ceftazidime ,Ceftriaxone, Cefepime, Imipenem, Ertapenem ,Meropenem, Gentamicin Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR
Acinetobacter Baumannii 59	Wound swab	Piperacillin\Tazobactam, Cefazolin, Ceftazidime, Ceftriaxone, Cefepime, Imipenem, Gentamycin, Ciprofloxacin, Levofloxacin, Trimethoprim / Sulfamethoxazole.	MDR
Serratia marcescens 7	Ear swab	Cefixime ,Cefpodoxime, Doxycycline ,Minocycline, Tetracycline	MDR
Serratia marcescens 22	Ear swab	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Cefixime ,Cefpodoxime ,cefotaxime ,Ceftazidime, Aztreonam, Gentamicin, Tobramycin, Norfloxacin, Doxycycline ,Minocycline,Tetracycline	MDR
Escherichia coli 53	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam,Cefpodoxime ,Ceftazidime ,Ceftriaxone, Cefepime, Aztreonam, Gentamicin, Tobramycin ,Ciprofloxacin, levofloxacin,Moxifloxacin, Norfloxacin ,Ofloxacin Doxycycline ,Minocycline, Tetracycline Trimethoprim/Sulfamethoxazole	MDR
Escherichia coli 359	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam Cefpodoxime ,Ceftazidime, Cefepime, Aztreonam, Gentamicin Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin Doxycycline ,Minocycline, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR
Escherichia coli 4	Urine	Ticarcillin ,Ticarcillin /Clavulanic acid ,Piperacillin ,Piperacillin/Tazobactam Cefpodoxime ,Ceftazidime, Cefepime, Aztreonam, Gentamicin Tobramycin ,Ciprofloxacin, levofloxacin, Moxifloxacin, Norfloxacin ,Ofloxacin Doxycycline ,Minocycline, Tetracycline, Trimethoprim/Sulfamethoxazole	MDR
Staphylococcus aureus 403	Urine	Cefoxitin ,Benzylpenicillin ,Oxacillin, Gentamicin ,Inducible Clindamycin resistance,Teicoplanin, Vancomycin ,Tetracycline , Fusidic Acid	MDR
Staphylococcus aureus 405	Ear swab	Cefoxitin Screen(+) ,Benzylpenicillin ,Oxacillin Inducible Clindamycin Resistance (-) ,Teicoplanin, Vancomycin ,Tetracycline, Fusidic Acid	MDR

3.7 MOI

All the tested MOI could reduce viability of the host K. pneumoniaeand phage ΦAYH produced highest titre at MOI 10 and that MOI considered the optimal MOI as shown in (Fig. 8) and was used later for one-step growth curve experiment.

3.8 One-step growth curve (OSGC)

As mentined above, MOI 10 was used for OSGC test. Based on the result of a single growth experiment, the latent period of phage ΦAYH was 10 min, and the burst size was ~ 64 particles/cell (Fig. 9).

3.9 Proteomic analysis

SDS- PAGE was used to analyze phage ΦAYH structural proteins. One major protein band was observed with molecular weight ~ 62 and several protein bands, with molecular weights ranging from 28 to around 98kDa on the gel as shown in (Fig. 10).

3.10 Biofilm formation

Table 2: The ability of K. pneumoniae isolates to form biofilm and antibiofilm activity of bacteriophage ΦAYH

K. pneumoniae isolates	The ability of the isolates to form biofilm %				Total number of the isolates
Before phage treatment	Weak	Moderate	Strong	Non
Number of the isolates	24 (72.72)	2 (6.06%)	0	7 (21..21%)	33 (100%)
After phage treatment
Number of the isolates	8(30.77%)	0	0	18 (69.23%) (7 isolates were excluded, which were non biofilm producer	26 (100%)

The ability of 33 isolates of K. pneumoniae to form biofilm including the host was tested by Microtiter Plate method. The isolates showed a variable ability for biofilm production as the following: 72.72% as weak, 6.06% as moderate and 21.21% as non-biofilm producer .The Antibiofilm Activity of bacteriophage ΦAYH to prevent biofilm was tested against the host and clinical isolates of K. pneumoniae. The results showed that bacteriophage ΦAYH had antibiofilm activity against the most clinical isolates tested expect the host, which remained weak biofilm producer, Table 2.

As a result of antibiotic misuse, MDR K. pneumoniae is considered as one of the major threats to global public health (Li et al., 2020). It requires immediate solutions to find alternatives that can substitute available antibiotic treatment. Moreover, the ability of K. pneumoniae to form biofilms on medical devices causing devices associated with nosocomial infections can increase bacterial survival rate (Wu et al., 2019). In this study, phage ΦAYH was isolated from the Tigris River, near Baghdad Medical City sewage and characterized using a host from the same site. Sewage wastewater is discharged from hospitals to rivers offers a source for a high diversity of phages to obtain against different pathogens (Abdelrahman et al., 2022; Ali et al., 2023; Rai et al., 2022; Samir et al., 2022). The plaques formed by phage ΦAYH were transparent with a approximately of 3 mm in diameter surrounded by a halo (Fig. 3A), indicating that phage ΦAYH is a lytic phage (Bai et al., 2022; Peng and Yuan, 2018). The presence of halos suggests depolymerase enzyme activity (Domingo-Calap et al., 2020). Morphological analysis observation using TEM indicated that phage ΦAYH belongs to Podoviridae family from order Caudovirales according to the latest classification by the International Committee on the Taxonomy of Viruses (ICTV) (Fig. 3B).

Phage therapy requires investigating the stability of candidate phages at different temperatures and pH tolerance. The temperature plays an important role in phage viability, existence, and storage (Jończyk et al., 2011). The phage ΦAYH remains stable at temperature between − 10°C and 50°C. Thermal stability of different phage strains varies according to phage structure and isolation sites (Martins et al., 2022). However, high temperatures affect phage stability (Ateba and Akindolire, 2019; Crothers-Stomps et al., 2010; Domingo-Calap et al., 2020). Another important factor in phage survival is the acidic and alkaline of the environment (Krasowska et al., 2015). For pH tolerance, phage ΦAYH showed stability at pH ranging from 5 to 11. Acidic conditions of the gastric environment cause loss of phage viability and thus prevent oral administration. To overcome phage ΦAYH gastric acid inactivation, phage protection by alginate and chitosan is one of solutions (Silva Batalha et al., 2021). The multiplicity of infection 10 was considered the optimal and applied in one-step growth curve. The latent period of phage ΦAYH was 10 min, and the average burst size was 64 pfu/cell in one-step growth curve. A previous study on a phage belonging to the Podoviridae family exhibited a burst size of approximately 50 to 60 vrion/ infected cell with a latent period of 15 minutes (Chaturongakul and Ounjai, 2014) .While another study showed a burst size of 120 virion/infected cell and 40 min latent period (Manohar et al., 2019). Our result on latent period agrees with another result for the KPP-5 phage from Podoviridae that showed same latency period (10 min) (D’Andrea et al., 2017). The host rang assay showed narrow range, which is preferred in phage therapy as it lower probability for affecting other normal flora members but at the same time it may limit the phage therapy to very specific bacterial infections. To overcome this limitation, phage cocktail is a promising option to prevent emergence of phage/ bacterial resistant (Manohar et al., 2019). A study showed that Podoviridae family members resist high pH, thermal inactivation and with narrow host range (Karumidze et al., 2013). Through SDS-PAGE analysis of purified concentrated phage ΦAYH proteins, one major protein band was observed with molecular size approximately 62 kDa and several protein bands ranging from 28 to 98 kDa were observed on the gel. The major protein is an indicative for capsid protein which agree with a study for phage KP34 that where the capsid protein (Drulis-Kawa et al., 2011). Another study showed that the SDS and the sequence homology of phages vB_KpnP_SU503 (SU503) and vB_KpnP_SU552A (SU552A) are within the Autographivirinae subfamily of Podoviridae family that show one major capsid protein (Eriksson et al., 2015).

K. pneumoniae can form biofilm, which is a bacterial communities embedded an extracellular matrix. These communities assist in an increased resistance to antimicrobial agents and host defense mechanisms (e.g., antimicrobial peptides, the complement system, and phagocytosis) (Guerra et al., 2022; Marks et al., 2014; Rabin et al., 2015). Increased antibiotics resistance threat and their inability in breaking the biofilm structure has urged the need for finding novel strategies for preventing or delaying the biofilm (Górski and Weber-Dabrowska, 2005). As bacterial predators, phages can be used to eradicate biofilms by several mechanisms, and these mechanisms affect the target bacterial cells. Depolymerases and lysins are the most crucial mechanisms to break down the defense barrier during infections of the host bacteria (Topka-Bielecka et al., 2021). Depolymerases can be involved in depolymerize the polysaccharides of capsular polysaccharide (CPS) and formation of biofilm, which assist in access of bacteriophage to the bacterial surfaces (Drulis-Kawa et al., 2015). Furthermore, depolymerization of CPS or Extra Poly Saccharides (EPS) from encapsulated bacteria remove an important shield of these bacteria, which render them more susceptible to antimicrobial agents and the host immune defense system (Bansal et al., 2014; Mushtaq et al., 2004). Thus, in our study, we investigated the ability of K. pneumoniae host and clinical isolates to form biofilm and all of them showed various biofilm abilities. Antibiofilm activity of ΦAYH bacteriophage to prevent biofilm formation was tested against these isolates and our study showed that the phage had ability to prevent the biofilm formation of the clinical isolates. These isolates with moderate and weak ability to form biofilm became a non-biofilm producer after phage treatment. This could present unconventional therapies to control biofilm formation of MDR isolates. However, there are still limitations in phage therapy against biofilm such as slow penetration through biofilm could be a problem for phages (Hu et al., 2010). In addition, host range as some phages with broad spectrum and some phages narrow spectrum, the narrow spectrum phage could be obstacles especially in polymicrobial biofilms on medical devices (Donlan and Costerton, 2002; Donlan, 2009). Furthermore, this specificity can be considered as one of phage therapy advantages, which target specific bacteria without affecting other helpful bacterial species. For phage therapy success, different factors maybe considered including phage injection, phage/bacteria ratio, immune response of the host and phage clearance, phage burst size, phage half-life, and in vivo bacterial resistance (Ly-Chatain, 2014).

To summarize, phage ΦAYH and its host were isolated from the same site indicating that hospital sewage is a good source for the isolation of phages against MDR K. pneumoniae isolates. Morphological characteristics of phage ΦAYH demonstrated that it belongs to the Podoviridae family. Isolated phage has a promising heat and pH stability. It possessed a burst size of 64virions/ cell and a latent period of 10 min. The phage ΦAYH eradicated K. pneumoniae biofilm. Thus, isolated phage maybe a valuable candidate for therapeutic alternative to overcome antibiotic resistant and biofilm forming infections caused by K. pneumoniae. Investigation is required to improve efficacy and clinical impact of phage therapy. Furthermore, genomic studies are required to determine present or absence of toxic or virulent genes in the phage.

Acknowledgment

Authors would like to express thankful to Mustansiriyah University (www.uomustansiriyah.edu.iq) Baghdad/ Iraq for its supporting in the present work.

Author Contributions: All authors contributed equally in writing—original draft preparation, all authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Conflicts of Interest: The authors declare no conflict of interest.

Ethical approval: the Ethics Committee of the Mustansiriyah University approved and oversaw this study.

Abbas, F.M., 2021. Genetic characterization of OXA-1 β-lactamase producing carbapenem-resistant Klebsiella pneumoniae from Al-Hillah river water, Babylon Province, Iraq. J. Genet. Environ. Resour. Conserv. 9, 197–201.
Abdelrahman, F., Rezk, N., Fayez, M.S., Abdelmoteleb, M., Atteya, R., Elhadidy, M., El-Shibiny, A., 2022. Isolation, characterization, and genomic analysis of three novel E. coli bacteriophages that effectively infect E. coli O18. Microorganisms 10, 589.
Abdulhussein, A.A., O. Abdulsattar, B., 2022. Isolation and characterization of two novel phages as a possible therapeutic alternative against multi-drug resistant E. coli. Gene Rep. 28, 101644. https://doi.org/10.1016/j.genrep.2022.101644
Abdulsattar, B.O., Abdulsattar, J.O., Rasool, K.H., Abdulhussein, A.-R.A., Abbas, M.H., 2020. Study of Antimicrobial Resistance Pattern of Escherichia coli and Klebsiella Strains and Multivariate Analysis for Water Quality Assessment of Tigris River, Baghdad, Iraq. Nat. Environ. Pollut. Technol. 19, 1327–1334.
Ali, S., Karaynir, A., Salih, H., Öncü, S., Bozdoğan, B., 2023. Characterization, genome analysis and antibiofilm efficacy of lytic Proteus phages RP6 and RP7 isolated from university hospital sewage. Virus Res. 326, 199049.
Alwash, M.S., Al-Rafyai, H.M., 2019. Antibiotic resistance patterns of diverse Escherichia coli phylogenetic groups isolated from the Al-Hillah River in Babylon Province, Iraq. Sci. World J. 2019.
Alzaidi, J.R., Mohammed, A.S., 2022. First Record of Dissemination of BLBLI-Resistant from Public Hospitals in Baghdad, Iraq. Open Microbiol. J. 16.
Ashurst, J.V., Dawson, A., 2018. Klebsiella pneumoniae.
Ateba, C.N., Akindolire, M.A., 2019. Isolation and characterisation of bacteriophages with lytic activity against virulent Escherichia coli O157: H7: Potential bio-control agents.
Babapour, E., Haddadi, A., Mirnejad, R., Angaji, S.-A., Amirmozafari, N., 2016. Biofilm formation in clinical isolates of nosocomial Acinetobacter baumannii and its relationship with multidrug resistance. Asian Pac. J. Trop. Biomed. 6, 528–533.
Bai, J., Zhang, F., Liang, S., Chen, Q., Wang, W., Wang, Y., Martín-Rodríguez, A.J., Sjöling, \AAsa, Hu, R., Zhou, Y., 2022. Isolation and Characterization of vB_kpnM_17 – 11, a Novel Phage Efficient Against Carbapenem-Resistant Klebsiella pneumoniae. Front. Cell. Infect. Microbiol. 12.
Balcão, V.M., Moreli, F.C., Silva, E.C., Belline, B.G., Martins, L.F., Rossi, F.P., Pereira, C., Vila, M.M., da Silva, A.M., 2022. Isolation and molecular characterization of a novel lytic bacteriophage that inactivates MDR Klebsiella pneumoniae strains. Pharmaceutics 14, 1421.
Bansal, S., Harjai, K., Chhibber, S., 2014. Depolymerase improves gentamicin efficacy during Klebsiella pneumoniae induced murine infection. BMC Infect. Dis. 14, 1–11.
Cano, E.J., Caflisch, K.M., Bollyky, P.L., Van Belleghem, J.D., Patel, R., Fackler, J., Brownstein, M.J., Horne, B., Biswas, B., Henry, M., Malagon, F., Lewallen, D.G., Suh, G.A., 2021. Phage Therapy for Limb-threatening Prosthetic Knee Klebsiella pneumoniae Infection: Case Report and In Vitro Characterization of Anti-biofilm Activity. Clin. Infect. Dis. 73, e144–e151. https://doi.org/10.1093/cid/ciaa705
Chaturongakul, S., Ounjai, P., 2014. Phage–host interplay: examples from tailed phages and Gram-negative bacterial pathogens. Front. Microbiol. 5, 442.
Chen, X., Tang, Q., Li, X., Zheng, X., Li, P., Li, M., Wu, F., Xu, Z., Lu, R., Zhang, W., 2022. Isolation, characterization, and genome analysis of bacteriophage P929 that could specifically lyase the KL19 capsular type of Klebsiella pneumoniae. Virus Res. 314, 198750.
Crothers-Stomps, C., Høj, L., Bourne, D.G., Hall, M.R., Owens, L., 2010. Isolation of lytic bacteriophage against Vibrio harveyi. J. Appl. Microbiol. 108, 1744–1750.
D’Andrea, M.M., Marmo, P., Henrici De Angelis, L., Palmieri, M., Ciacci, N., Di Lallo, G., Demattè, E., Vannuccini, E., Lupetti, P., Rossolini, G.M., 2017. φBO1E, a newly discovered lytic bacteriophage targeting carbapenemase-producing Klebsiella pneumoniae of the pandemic Clonal Group 258 clade II lineage. Sci. Rep. 7, 2614.
Dashti, A.A., Jadaon, M.M., Abdulsamad, A.M., Dashti, H.M., 2009. Heat treatment of bacteria: a simple method of DNA extraction for molecular techniques. Kuwait Med J 41, 117–122.
Domingo-Calap, P., Beamud, B., Mora-Quilis, L., González-Candelas, F., Sanjuán, R., 2020. Isolation and characterization of two Klebsiella pneumoniae phages encoding divergent depolymerases. Int. J. Mol. Sci. 21, 3160.
Donlan, R.M., 2009. Preventing biofilms of clinically relevant organisms using bacteriophage. Trends Microbiol. 17, 66–72.
Donlan, R.M., Costerton, J.W., 2002. Biofilms: survival mechanisms of clinically relevant microorganisms. Clin. Microbiol. Rev. 15, 167–193.
Doss, J., Culbertson, K., Hahn, D., Camacho, J., Barekzi, N., 2017. A review of phage therapy against bacterial pathogens of aquatic and terrestrial organisms. Viruses 9, 50.
Drulis-Kawa, Z., Mackiewicz, P., Kęsik-Szeloch, A., Maciaszczyk-Dziubinska, E., Weber-Dąbrowska, B., Dorotkiewicz-Jach, A., Augustyniak, D., Majkowska-Skrobek, G., Bocer, T., Empel, J., 2011. Isolation and characterisation of KP34—a novel φKMV-like bacteriophage for Klebsiella pneumoniae. Appl. Microbiol. Biotechnol. 90, 1333–1345.
Drulis-Kawa, Z., Majkowska-Skrobek, G., Maciejewska, B., 2015. Bacteriophages and phage-derived proteins–application approaches. Curr. Med. Chem. 22, 1757–1773.
Eriksson, H., Maciejewska, B., Latka, A., Majkowska-Skrobek, G., Hellstrand, M., Melefors, Ö., Wang, J.-T., Kropinski, A.M., Drulis-Kawa, Z., Nilsson, A.S., 2015. A suggested new bacteriophage genus,“Kp34likevirus”, within the Autographivirinae subfamily of Podoviridae. Viruses 7, 1804–1822.
Gorodnichev, R.B., Volozhantsev, N.V., Krasilnikova, V.M., Bodoev, I.N., Kornienko, M.A., Kuptsov, N.S., Popova, A.V., Makarenko, G.I., Manolov, A.I., Slukin, P.V., 2021. Novel Klebsiella pneumoniae K23-specific bacteriophages from different families: similarity of depolymerases and their therapeutic potential. Front. Microbiol. 12, 669618.
Górski, A., Weber-Dabrowska, B., 2005. The potential role of endogenous bacteriophages in controlling invading pathogens. Cell. Mol. Life Sci. 62, 511–519.
Guerra, M.E.S., Destro, G., Vieira, B., Lima, A.S., Ferraz, L.F.C., Hakansson, A.P., Darrieux, M., Converso, T.R., 2022. Klebsiella pneumoniae biofilms and their role in disease pathogenesis. Front. Cell. Infect. Microbiol. 555.
He, Y., Guo, X., Xiang, S., Li, J., Li, X., Xiang, H., He, J., Chen, D., Chen, J., 2016. Comparative analyses of phenotypic methods and 16S rRNA, khe, rpoB genes sequencing for identification of clinical isolates of Klebsiella pneumoniae. Antonie Van Leeuwenhoek 109, 1029–1040.
Hu, J., Miyanaga, K., Tanji, Y., 2010. Diffusion properties of bacteriophages through agarose gel membrane. Biotechnol. Prog. 26, 1213–1221.
Ji, Y., Cheng, M., Zhai, S., Xi, H., Cai, R., Wang, Z., Zhang, H., Wang, X., Xue, Y., Li, X., 2019. Preventive effect of the phage VB-SavM-JYL01 on rabbit necrotizing pneumonia caused by Staphylococcus aureus. Vet. Microbiol. 229, 72–80.
Jończyk, E., K\lak, M., Międzybrodzki, R., Górski, A., 2011. The influence of external factors on bacteriophages. Folia Microbiol. (Praha) 56, 191–200.
Kanaan, M.H.G., Khashan, H.T., 2022. Molecular typing, virulence traits and risk factors of pandrug-resistant Acinetobacter baumannii spread in intensive care unit centers of Baghdad city, Iraq. Rev. Med. Microbiol. 33, 51–55.
Karumidze, N., Kusradze, I., Rigvava, S., Goderdzishvili, M., Rajakumar, K., Alavidze, Z., 2013. Isolation and characterisation of lytic bacteriophages of Klebsiella pneumoniae and Klebsiella oxytoca. Curr. Microbiol. 66, 251–258.
Koberg, S., Brinks, E., Fiedler, G., Hüsing, C., Cho, G.-S., Hoeppner, M.P., Heller, K.J., Neve, H., Franz, C.M., 2017. Genome sequence of Klebsiella pneumoniae bacteriophage PMBT1 isolated from raw sewage. Genome Announc. 5, e00914–16.
Krasowska, A., Biegalska, A., Augustyniak, D., \Loś, M., Richert, M., \Lukaszewicz, M., 2015. Isolation and characterization of phages infecting Bacillus subtilis. BioMed Res. Int. 2015.
Li, M., Guo, M., Chen, Long, Zhu, C., Xiao, Y., Li, P., Guo, H., Chen, Liang, Zhang, W., Du, H., 2020. Isolation and characterization of novel lytic bacteriophages infecting epidemic carbapenem-resistant Klebsiella pneumoniae strains. Front. Microbiol. 11, 1554.
Ly-Chatain, M.H., 2014. The factors affecting effectiveness of treatment in phages therapy. Front. Microbiol. 5, 51.
Manohar, P., Tamhankar, A.J., Lundborg, C.S., Nachimuthu, R., 2019. Therapeutic characterization and efficacy of bacteriophage cocktails infecting Escherichia coli, Klebsiella pneumoniae, and Enterobacter species. Front. Microbiol. 10, 574.
Marks, L.R., Mashburn-Warren, L., Federle, M.J., Hakansson, A.P., 2014. Streptococcus pyogenes biofilm growth in vitro and in vivo and its role in colonization, virulence, and genetic exchange. J. Infect. Dis. 210, 25–34.
Martins, W.M., Cino, J., Lenzi, M.H., Sands, K., Portal, E., Hassan, B., Dantas, P.P., Migliavacca, R., Medeiros, E.A., Gales, A.C., 2022. Diversity of lytic bacteriophages against XDR Klebsiella pneumoniae sequence type 16 recovered from sewage samples in different parts of the world. Sci. Total Environ. 839, 156074.
Munita, J.M., Arias, C.A., 2016. Mechanisms of antibiotic resistance. Virulence Mech. Bact. Pathog. 481–511.
Mushtaq, N., Redpath, M.B., Luzio, J.P., Taylor, P.W., 2004. Prevention and cure of systemic Escherichia coli K1 infection by modification of the bacterial phenotype. Antimicrob. Agents Chemother. 48, 1503–1508.
Nazir, A., Qi, C., Shi, N., Gao, X., Feng, Q., Qing, H., Li, F., Tong, Y., 2022. Characterization and Genomic Analysis of a Novel Drexlervirial Bacteriophage IME268 with Lytic Activity Against Klebsiella pneumoniae. Infect. Drug Resist. 1533–1546.
Obradović, M., Malešević, M., Di Luca, M., Kekić, D., Gajić, I., McAuliffe, O., Neve, H., Stanisavljević, N., Vukotić, G., Kojić, M., 2023. Isolation, Characterization, Genome Analysis and Host Resistance Development of Two Novel Lastavirus Phages Active against Pandrug-Resistant Klebsiella pneumoniae. Viruses 15, 628.
Pallavali, R.R., Degati, V.L., Lomada, D., Reddy, M.C., Durbaka, V.R.P., 2017. Isolation and in vitro evaluation of bacteriophages against MDR-bacterial isolates from septic wound infections. PLOS ONE 12, e0179245. https://doi.org/10.1371/journal.pone.0179245
Patel, R., 2005. Biofilms and antimicrobial resistance. Clin. Orthop. Relat. Res. 1976–2007 437, 41–47.
Peng, Q., Fang, M., Liu, X., Zhang, C., Liu, Y., Yuan, Y., 2020. Isolation and characterization of a novel phage for controlling multidrug-resistant Klebsiella pneumoniae. Microorganisms 8, 542.
Peng, Q., Yuan, Y., 2018. Characterization of a novel phage infecting the pathogenic multidrug-resistant Bacillus cereus and functional analysis of its endolysin. Appl. Microbiol. Biotechnol. 102, 7901–7912.
Qin, J., Qiu, Y., Guo, S., LI, M., Lin, F., Wan, R., Wen, Y., 2017. Distribution and antimicrobial resistance profile of Klebsiellapneumoniae. Chin. J. Infect. Chemother. 269–272.
Rabin, N., Zheng, Y., Opoku-Temeng, C., Du, Y., Bonsu, E., Sintim, H.O., 2015. Biofilm formation mechanisms and targets for developing antibiofilm agents. Future Med. Chem. 7, 493–512.
Rai, P., Shetty, S.S., Prabell, S., Kuntar, A., Pinto, D., Kumar, B.K., Divyashree, M., Raj, J.R.M., Premanath, R., Deekshit, V.K., 2022. Characterisation of broad-spectrum phiKZ like jumbo phage and its utilisation in controlling multidrug-resistant Pseudomonas aeruginosa isolates. Microb. Pathog. 172, 105767.
Salman, H.A., Alsallameh, S.M.S., Muhamad, G., Taha, Z., 2022. Prevalence of Multi-Antibiotic Resistant Bacteria Isolated from Children with Urinary Tract Infection from Baghdad, Iraq. Microbiol. Biotechnol. Lett. 50, 147–156.
Samir, S., El-Far, A., Okasha, H., Mahdy, R., Samir, F., Nasr, S., 2022. Isolation and characterization of lytic bacteriophages from sewage at an egyptian tertiary care hospital against methicillin-resistant Staphylococcus aureus clinical isolates. Saudi J. Biol. Sci. 29, 3097–3106.
Silva Batalha, L., Pardini Gontijo, M.T., Vianna Novaes de Carvalho Teixeira, A., Meireles Gouvêa Boggione, D., Soto Lopez, M.E., Renon Eller, M., Santos Mendonça, R.C., 2021. Encapsulation in alginate-polymers improves stability and allows controlled release of the UFV-AREG1 bacteriophage. Food Res. Int. 139, 109947. https://doi.org/10.1016/j.foodres.2020.109947
Soleimani Sasani, M., Eftekhar, F., 2020. Potential of a bacteriophage isolated from wastewater in treatment of lobar pneumonia infection induced by Klebsiella pneumoniae in mice. Curr. Microbiol. 77, 2650–2655.
Tabassum, R., Shafique, M., Khawaja, K.A., Alvi, I.A., Rehman, Y., Sheik, C.S., Abbas, Z., Rehman, S.U., 2018. Complete genome analysis of a Siphoviridae phage TSK1 showing biofilm removal potential against Klebsiella pneumoniae. Sci. Rep. 8, 17904.
Taylor, C.F., 2020. Potential of Klebsiella pneumoniae Phage Depolymerases as Antimicrobial Agents. (PhD Thesis). Manchester Metropolitan University for the degree of Master of Science (by …
Tesfa, T., Mitiku, H., Edae, M., Assefa, N., 2022. Prevalence and incidence of carbapenem-resistant K. pneumoniae colonization: systematic review and meta-analysis. Syst. Rev. 11, 1–15.
Topka-Bielecka, G., Dydecka, A., Necel, A., Bloch, S., Nejman-Faleńczyk, B., Węgrzyn, G., Węgrzyn, A., 2021. Bacteriophage-derived depolymerases against bacterial biofilm. Antibiotics 10, 175.
Vuotto, C., Longo, F., Balice, M.P., Donelli, G., Varaldo, P.E., 2014. Antibiotic resistance related to biofilm formation in Klebsiella pneumoniae. Pathogens 3, 743–758.
Wintachai, P., Naknaen, A., Thammaphet, J., Pomwised, R., Phaonakrop, N., Roytrakul, S., Smith, D.R., 2020. Characterization of extended-spectrum-β-lactamase producing Klebsiella pneumoniae phage KP1801 and evaluation of therapeutic efficacy in vitro and in vivo. Sci. Rep. 10, 1–18.
Wu, Y., Wang, R., Xu, M., Liu, Y., Zhu, X., Qiu, J., Liu, Q., He, P., Li, Q., 2019. A novel polysaccharide depolymerase encoded by the phage SH-KP152226 confers specific activity against multidrug-resistant Klebsiella pneumoniae via biofilm degradation. Front. Microbiol. 10, 2768.

No competing interests reported.

TableS1.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Isolation, characterization and anti-biofilm efficacy of a novel Klebsiella pneumoniae phage

Status:

Version 1

Abstract

Figures

Highlights

1. Introduction

2. Materials and Methods

2.1 Collection of clinical K. pneumoniae isolates

2.2 Clinical isolates identification

2.3 Molecular identification of k. pneumoniae isolates

2.4 Collection of samples for phage and host isolation

2.5 Phage isolation and purification

2.6 Phage titration

2.7 Transmission Electron Microscopy (TEM) and Plaque morphology

2.8 Thermal Tolerance of ΦAYH phage

2.9 pH stability of ΦAYH phage

2.10 Host range determination

2.11 Optimal MOI

2.12 One step growth curve

2.13 Bacterial biofilm susceptibility

2.14 Bacterial biofilm susceptibility to phage ΦAYH

2.15 Proteomics analysis

3. Results

3.1 Host bacterium isolation and identification

3.2 Isolation and identification of clinical bacterial isolates

3.3 Phage isolation, purification

3.4 Thermal stability

3.5 pH stability

3.6 Host range

3.7 MOI

3.8 One-step growth curve (OSGC)

3.9 Proteomic analysis

3.10 Biofilm formation

4. Discussion

5. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1