Phenotypic characterisation of Klebsiella-specific phage and the study of antibiofilm activity and synergistic effect with meropenem on multidrug-resistant Klebsiella pneumoniae causing respiratory infections
A
A
Dr
EthelSuman1✉Emailethel.suman@manipal.eduAnsaRahman1
AnjanaAP1
MonishaMBhandary1
SHarshaPaul2
HimaniKotian3
SuchitraShenoy1
M1
1Department of Microbiology, Kasturba Medical College MangaloreManipal Academy of Higher EducationManipalManipalIndia
2Department of Microbiology, Science, Commerce and ManagementThe Yenepoya Institute of Arts, Yenepoya (Deemed to be University)MangaloreKarnatakaIndia
3Department of Community Medicine, Kasturba Medical College MangaloreManipal Academy of Higher EducationManipalIndia
Authors: Ethel Suman a*, Ansa Rahman a, Anjana AP a, Monisha M Bhandary a, S Harsha Paul b, Himani Kotian c, Suchitra Shenoy Ma
Affiliation: a Department of Microbiology, Kasturba Medical College Mangalore, Manipal Academy of Higher Education, Manipal, India
b Department of Microbiology, The Yenepoya Institute of Arts, Science, Commerce and Management, Yenepoya (Deemed to be University), Mangalore, Karnataka, India
c Department of Community Medicine, Kasturba Medical College Mangalore, Manipal Academy of Higher Education, Manipal, India
Corresponding author: Dr Ethel Suman, Department of Microbiology, Kasturba Medical College Mangalore, Manipal Academy of Higher Education, Manipal, India. Email: ethel.suman@manipal.edu
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Abstract
Background
Klebsiella pneumoniae is a major pathogen that causes ventilator-associated pneumonia and can produce biofilms on endotracheal tubes. This can act as a diffusive barrier, hindering the treatment of infections caused by multidrug-resistant strains of the pathogen. Treatment of such multidrug-resistant strains has become a major challenge and a global problem. Among the various alternative methods used to combat this problem, the use of specific bacteriophages appears to be quite encouraging.
This study aimed to isolate and perform phenotypic characterisation of Klebsiella-specific phage from sewage water and compare the effects of Klebsiella-specific phage and subinhibitory concentrations of meropenem on in vitro biofilm production by the respiratory clinical isolates of Klebsiella pneumoniae.
Methods
Isolation and phenotypic characterisation of Klebsiella-specific phage from sewage water was done using K.pneumoniae subspp.pneumoniae ATCC 33495 as the host bacterium. The antibiofilm activity of the isolated phage was studied on 40 respiratory clinical isolates of Klebsiella pneumoniae. Their biofilm production was performed on endotracheal tubing pieces in microtiter plates as well as in the presence of Klebsiella-specific phage, subinhibitory concentrations of meropenem, and a combination of Klebsiella-specific phage and meropenem.
Results
Transmission Electron Microscopy (TEM) confirmed a classic Myoviridae structure—an icosahedral head (50–100 nm) with a contractile tail sheath and a complex baseplate. Other characteristics included a latent period of 20 minutes and a burst size of 69 phage particles per cell, with a multiplicity of infection being 10, with strong host-killing ability and was stable and infective at 25°C and a neutral pH range (6–7)
Subinhibitory concentration of meropenem brought about a significant decrease in biofilm production (p < 0.001) in 91.3% of meropenem-sensitive but significantly increased in 100% of meropenem-resistant strains. The combination of bacteriophage and meropenem had a synergistic effect, resulting in significant reduction in both the meropenem-sensitive and resistant strains
Conclusion
This study suggests the potential application of specific phage-coated and antibiotic-coated endotracheal tubing in preventing biofilms, thereby reducing the risk of ventilator-associated pneumonia.
Keywords:
Klebsiella pneumoniae
Bacteriophage
VAP
Meropenem
Endotracheal tube
Biofilm
1. Introduction
Klebsiella pneumoniae is a facultative anaerobic, nonsporing, capsulated bacillus that is a gram-negative member of the family Enterobacteriaceae. This organism can cause several infections in the community as well as in hospitals [1, 2]. The evolutionary feature of Klebsiella pneumoniae is its occurrence in two pathotypes, namely, hypervirulent K. pneumoniae (hKp) and classical K. pneumoniae (cKp), which make it an important nosocomial pathogen [3].
The important property of pathogenesis in these organisms is the formation of biofilms and fimbriae, which are recognised as the major appendages that mediate adhesion to biotic and abiotic surfaces, especially in K. pneumoniae type 3[1].
Healthcare-associated infections are caused mainly by the presence of indwelling medical devices. Endotracheal tubes (ETTs), which are introduced into the trachea of a patient to maintain open airways, impair mucociliary clearance and thereby disrupt the cough reflex. This in turn promotes the accumulation of secretions in the lungs and contributes to the development of pneumonia in patients on ventilators (VAPs) [4, 5, 6]. The abiotic surface of the endotracheal tube facilitates the adherence of the microorganisms, and the organisms grow in this niche and produce biofilms [7]. Endotracheal intubation facilitates the movement of various microorganisms to the sterile bronchoalveolar area and thereby increases the risk of pneumonia [8].
A
Although only moderate levels of chromosomally encoded penicillinases are present in Klebsiella pneumoniae, a substantial collection of drug-resistant plasmids encoding resistance to aminoglycosides and extended-spectrum beta-lactamases (ESBLs), predominantly SHVs (sulphydryl variables) and TEMs (Temoniera), which are active against cephalosporins, are available. The presence of these plasmids, chromosomal mutations, and biofilm production contribute to the organism's resistance, making antibiotic treatment difficult by hindering diffusion, especially in healthcare-associated infections [1]. The use of antiseptic agents such as gardine-coated ETT inhibited biofilm formation by Klebsiella pneumoniae, as evidenced by electron microscopy [8].Multidrug resistance in Klebsiella pneumoniae has made treatment strategies difficult. An alternative therapy using bacteriophages specific to multiple drug-resistant Klebsiella pneumoniae has a beneficial effect through the inhibition of biofilms [9]. Experimental studies using phages on biofilms produced by individual species of bacteria as well as multiple species of bacteria, have revealed that phages have the capacity to lyse biofilms produced by individual or multiple host cells [10, 11]. The use of bacteriophages has been applied in a large variety of settings, including clinical and industrial settings. In industry, they have been used in filtration membranes, whereas in the clinic, they have been used for therapeutic purposes, including phage therapy for infectious diseases as well as antibiofilm agents in biofilm-infected indwelling devices [12, 13]. Phages are known to produce polysaccharide depolymerases, which degrade extracellular polymeric substances that are present in bacterial biofilms, thereby allowing phages to enter bacteria and cause lysis of bacterial cells [14].
This study focused on the isolation and phenotypic characterisation of a bacteriophage specifically targeting K. pneumoniae from sewage water. The phage was phenotypically characterised using techniques such as transmission electron microscopy, determination of optimal multiplicity of infection(MOI), pH stability assay, temperature stability assay, one-step growth curve, and time-kill assay. The antibacterial effect of Klebsiella pneumoniae-specific phage on the biofilm production by Klebsiella pneumoniae isolates from respiratory tract infections was studied. To do this, the biofilm-forming ability of the Klebsiella pneumoniae isolates from respiratory tract infections was first studied on endotracheal tubing pieces. It is to be noted that of the various antibiotics, meropenem is the key antibiotic used for treating moderate to severe pneumonia. It is a second-generation carbapenem and is commonly prescribed for Gram-negative bacterial infections, particularly those in the respiratory tract caused by even extended-spectrum beta-lactamase-producing bacteria. Hence, this study also used meropenem to compare the effect of Klebsiella-specific phage and to determine the synergistic effects of meropenem and Klebsiella-specific phages on biofilms [15].
2. Materials and methods
A
This was a cross-sectional, time-bound, prospective, institution-based study, and sampling was performed via a non-probability convenience sampling method.The study was conducted in accordance with the guidelines of the Declaration of Helsinki. It was approved by the Institutional Ethics Committee, which waived patient consent to participate as the research was based solely on the isolates from specimens received in the laboratory and associated laboratory data.
Biological samples consisted of the cultures of K. pneumoniae isolated from respiratory tract samples, such as sputum, bronchoalveolar lavage, endotracheal secretion tip, endotracheal aspirate and tracheal secretion.
Klebsiella pneumoniae isolated from other samples, such as urine, blood, and other exudates, as well as other organisms isolated, were excluded from the study. The total number of isolates used in the study was 40 in number. All the isolates were multidrug-resistant (resistant to more than three classes of antibiotics) and 15/40 (37.5%) were resistant to all the antibiotics (Extensively drug-resistant - XDR). Of the 40 isolates, 23 were sensitive to meropenem while 17 were resistant to meropenem. The isolates were preserved at − 80°C in 20% glycerol broth.
K.pneumoniae subspp.pneumoniae ATCC 33495 was used as the host bacterium for the isolation of Klebsiella-specific phage from sewage [15]. (This bacterium was commercially purchased from HiMedia, Pvt Ltd, Mumbai, India). This strain was also used as the organism to check the growth control in the various assays.
2.1. Bacterial isolates
Respiratory specimens received in the Microbiology laboratory were cultured on Blood agar, Chocolate agar, MacConkey medium, and Thioglycolate broth. All media were procured from HiMedia, Pvt Ltd, Mumbai, India. The plates were incubated at 37°C for 18h. Identification and antibiotic sensitivity testing of the isolates were performed via the VITEK-2 system (Biomerieux, USA). Interpretation of antibiotic susceptibility was performed according to CLSI guidelines [16].
2.1.1 Source of the Biological Samples
The source of the phage was sewage water, and the host bacterium was K. pneumoniae subspp. pneumoniae ATCC 33495 (purchased commercially from HiMedia, Pvt Ltd, Mumbai, India). The 40 Klebsiella pneumoniae isolates were obtained from respiratory specimens received in the Microbiology laboratory, as described earlier.
2.2. Preparation and purification of the Klebsiella-specific phage suspension
A specific Klebsiella pneumoniae bacteriophage was isolated via the standard method. An overnight broth culture of K.pneumoniae subspp.pneumoniae ATCC 33495 (HiMedia, Pvt Ltd, Mumbai, India) was inoculated with sewage water, to which nutrient broth was added, and the mixture was incubated at 37°C for 24 h. This was followed by centrifugation and filtration of the supernatant through a 0.45 µm filter. The filtrate was stored at 4°C until titer determination.
Serial dilutions of the lysate-containing phages were performed via SM buffer (100 mM sodium chloride, 8 mM magnesium sulfate, 50 mM Tris. HCl (pH 7.5)) All the chemicals were procured from HiMedia, Pvt Ltd, Mumbai, India. The dilutions (100–300 µl) were mixed with 100 µl of K. pneumoniae subsp. pneumoniae ATCC 33495 (HiMedia, Pvt Ltd, Mumbai, India). Following incubation at 37°C (20 min), the suspensions were added to molten tryptone agar (5 ml) and overloaded onto nutrient agar plates (HiMedia, Pvt Ltd, Mumbai, India), followed by incubation for 24 h at 37°C, after which the plaque-forming units/ml (PFU/ml) were counted [17, 18].
2.2.1 Phenotypic characterisation of the phage
Phenotypic characterisation of the isolated K. pneumoniae specific phage was performed by transmission electron microscopy (TEM), determination of optimal multiplicity of infection (MOI), pH stability assay, temperature stability assay, one-step growth curve, and time-kill assay. The results were tabulated.
2.2.1.1 One-step growth curve [19]:
Using one-step growth curve, the burst size and latent period of the phage were determined.
A freshly made bacterial culture (OD 0.4) was centrifuged for 15 mins at 10,000 g, 4℃. The pellet was resuspended in LB (5ml) broth and incubated at 37℃ for 10 mins. Bacteriophage was mixed with bacterial culture at an MOI of 10 (108 CFU/mL bacteria and 10⁶ PFU/mL phage). For the adsorption of phage, this mixture was incubated for 10 mins at 37℃. Centrifugation was done at 10,000 rpm for 10 minutes. The bacterial-phage pellet was resuspended in 5ml of LB broth after removing the supernatant, and then incubated at 37℃. Using a plaque assay, the phage titer was determined at 10-minute intervals up to 2 h followed by hourly intervals up to 9h. Burst size and latent period were determined.
Burst size = (Final Plaque forming units-Initial Plaque forming units)/Number of infected bacterial cells
2.2.1.2 Bacterial growth inhibition assay [20]:
According to the study by Menon et al. (2021), a bacterial growth inhibition assay was conducted using the microtiter plate method. It was carried out in 96-well plates over 9 hours at different MOIs of phage to determine the phage’s infectivity on bacteria.
Phage buffer was used to treat the phage controls, and at 37℃, the microtiter plates were incubated for nine hours; the OD600 values were taken every hour and tabulated.
2.2.1.3 Determination of Optimal Multiplicity of Infection (MOI) of the isolated phage [21]:
Multiplicity of infection was determined by growing the host bacteria, Klebsiella pneumoniae subspp. pneumoniae ATCC 3349 to have a cell density of 1.0 at OD600.
Mixtures to obtain ratios of phage to host cells were made from 0.001, 0.01, 0.1, 1, 10 PFU/CFU, and after 1h of incubation at 37oC each of the mixtures was assayed for plaque formation using the double-layer agar overlay method. The highest titer of the phage showing plaque formation
was considered as the optimal multiplicity of infection (MOI) of the isolated phage.
2.2.1.4 Determination of pH stability of phage [22]:
500µl of phage was combined with SM buffer with varying pH values (2,3,4,5,6,7,8,9 and 10) at a ratio of 1:10 (v/v), and the mixture was incubated for 1h at 37℃. Double-layer agar plate method was used to determine the phage titer. The experiment was conducted in duplicate.
2.2.1.5 Determination of temperature stability of phage [23]:
The phage diluted at the MOI was incubated at various temperatures (-20 ºC, 4ºC. 25º C, 37º C, 50 ºC, 80º C and 100º C) after which it was plated on Luria Bertani medium by the double-layer agar plate method to determine the plaque-forming units/ml. The experiment was conducted in duplicate.
2.2.1.6 Time Kill Assay [15]:
Time kill assay was done using the following procedure:
K.pneumoniae subspp.pneumoniae ATCC 33495 was inoculated into LB broth to achieve 108 CFU/ml cell density. K. pneumoniae specific phage at a concentration of 106 PFU/ml (MOI of 10) was mixed with the bacterial culture. At the time points of 0, 2, 4, 6, and 24h, plating was done on LB agar and incubated for 48 h to determine bacterial plate counts. Depletion in bacterial counts compared to the control indicates the lytic activity of the phage.
2.2.1.7 Transmission electron microscopy (TEM) [24]:
The phage was attached to carbon-containing copper grids, and this was negatively stained using 2% uranyl acetate solution. The copper grids were analysed using TEM (120 kV) after drying.
2.3. Measurement of the subinhibitory concentration of meropenem for each isolate
2.3.1. Sub-inhibitory concentration of Meropenem:
The tube dilution method was used to determine the subinhibitory concentration of meropenem for the clinical isolates by preparing a stock solution in sterile distilled water and making two-fold dilutions. [25]. The sub-inhibitory concentration of Meropenem was found to be 0.25µg/ml for 57.5% (23 out of 40 sensitive to meropenem) isolates and 8µg/ml for 42.5% (17 out of 40 resistant to meropenem) isolates. The same concentration was used to determine the biofilm formation in the presence of meropenem, and Klebsiella-specific phage as well as to evaluate the synergistic effect of meropenem and Klebsiella-specific phage in biofilm formation.
2.3.2. Bacterial inoculum preparation
The clinical isolates were subcultured on plates of nutrient agar (HiMedia, Pvt Ltd, Mumbai, India) and incubated for 24 h at 37°C. Four to five isolated colonies of similar morphology were inoculated into sterile Mueller–Hinton broth (MHB- HiMedia, Pvt Ltd, Mumbai, India). This mixture was incubated for 4–6 h at 37°C. The growth was matched to that of 0.5 MacFarland (1.5 × 108 cfu/ml) using the DensiCHEK plus instrument (Biomerieux, USA)
2.3.3. Preparation of the stock solution and dilution range of the antibiotic:
1000µg/ml was prepared using sterile distilled water as the solvent. The antibiotic dilution range was prepared from 128µg/ml to 0.062µg/ml. 10µl of the bacterial inoculum was inoculated into each tube, followed by incubation at 37°C for 24 h. K. pneumoniae ATCC 33495 was used as growth control in each test. The dilution preceding the inhibitory concentration was considered as the sub-inhibitory concentration of meropenem.
Studies using sub-inhibitory concentrations of antimicrobial agents have been documented to be the most effective method to investigate and assess the antibacterial effects of other substances without causing excessive killing. This concentration is thought to reflect the exact condition that the bacteria may encounter in the tissues or in the environment [26]
2.4. Biofilm formation by the microtiter plate method [27]
In vitro biofilm production of the clinical isolates of Klebsiella pneumoniae was done by the microtiter plate method. Biofilms were generated in the presence or absence of the antibiotic meropenem (subinhibitory concentration) and the presence of Klebsiella-specific phages. In addition, a combination of a Klebsiella-specific phage and meropenem (subinhibitory concentration) was also used to determine whether there was any synergistic effect. The phage‒antibiotic combination was applied to 5 samples that were resistant to meropenem and 5 samples that were sensitive to meropenem. The brain heart infusion broth (BHI from HiMedia Pvt Ltd, Mumbai, India) culture of the isolate (after 18 h of growth) was matched to a 0.5 MacFarland culture (1.5 × 108 cfu/ml) using the DensiCHEK plus instrument (Biomerieux, USA). Endotracheal tube (Siliconised PVC from Smiths Medical (REF 100/199/085 made in Mexico) was cut into 0.5 cm2 pieces using the Slitting and Milling machine (Bharat Fritz Werner Limited, Bangalore, India) at the Hebich Technical Training Institute, Balmatta, Mangalore, India and sterilized in an autoclave (M.C.Dalal Agencies, Chennai, India).
Biofilm formation was studied in different sets.
1.
The first set consisted of sterile 0.5cm2 ETT pieces to which the culture of the isolates (200 µl) was added to 96 well-round bottomed microtiter plates (HiMedia Pvt Ltd, Mumbai, India)
2.
The second set consisted of biofilms formed on sterile 0.5 cm2 ETT pieces in the presence of the antibiotic meropenem (subinhibitory concentration). This was done by dispensing 100µl of a sub-inhibitory concentration of meropenem and 100µl of bacterial inoculum, making a total volume of 200µl into microwells containing 0.5 cm2 ETT pieces.
3.
The third set consisted of the formation of biofilm on sterile 0.5 cm2 ETT pieces in the presence of Klebsiella-specific bacteriophage. This was done by dispensing 100µl of suspension of bacteriophage and 100µl of isolate culture, making a total volume of 200µl into microwells containing 0.5 cm2 ETT pieces.
4.
The fourth set consisted of the formation of biofilm on sterile 0.5 cm2 ETT pieces in the presence of both Klebsiella-specific bacteriophage and the antibiotic meropenem (sub-inhibitory concentration). This was done by dispensing 50µl of bacteriophage suspension, 50µl of meropenem (sub-inhibitory concentration) and 50µl of bacterial inoculum into the microwells containing 0.5 cm2 ETT pieces.
Following incubation for 24 h at 37°C, the ETT pieces were washed with PBS at pH 7.4, followed by vortexing (2 mins). The spread plate method was performed for the quantification of bacteria [28].
Spread plate method: 900µl of BHI broth was taken in test tubes and 100µl of bacterial suspension in phosphate-buffered saline was added to the first tube constituting 1:1000 dilution.100µl of bacterial suspension was mixed and transferred from the first tube to the second tube constituting the 1:100 dilution.100µl of bacterial suspension was discarded from the 1:100 dilution to compensate the corresponding volume. 20µl of bacterial suspension from a 1:100 dilution was plated onto sterile nutrient agar plates and swabbed using sterile cotton swabs. Following incubation for 24h at 37°C colony count was done and the colony forming unit of each isolate was calculated accordingly.
2.5. Scanning electron microscopy
Scanning electron microscopy was used to confirm the biofilms on the ETT pieces for the representative samples from each group. This was done by using air-dried ETT pieces after the biofilm assay. The air-dried pieces were placed on brass stubs, and gold sputtering was performed using an Ion sputtering 10mA unit (JFC-1600, Autofine Coater from JEOL, Tokyo, Japan). After gold sputtering, scanning electron microscopy was performed by placing the stubs in the vacuum chamber (JSM-6380 LA, JEOL, Akshima, Japan)
2.6. Detection of biofilms produced in the microtiter plates
Spectrophotometric detection of biofilms produced in the microtiter plates was performed following the steps of washing with PBS (7.4), fixation with Bouin fixative for 10 min at 25°C, discarding the contents and staining with crystal violet (1%). After the excess stain was rinsed away with water, each well was dispensed with glacial acetic acid (33%), and the absorbance at 570 nm was detected spectrophotometrically via an ELISA reader to calculate optical density (OD) values. The test was repeated to calculate the mean value. [29–32]. K.pneumoniae subspp.pneumoniae ATCC 33495 served as the organism to check the growth control.
2.7. Statistical analysis
The OD values of the biofilms were reported in terms of the minimum, maximum mean and standard deviation, and the OD values of the biofilms produced by the isolates in the presence/absence of an antibiotic (meropenem), presence/absence of a specific phage, and presence/absence of a combination of meropenem and phage were compared via one-way Kruskal‒Wallis nonparametric ANOVA, and pairwise comparisons of the OD values were performed via Dwass‒Steel‒Critchlow‒Fligner pairwise comparisons. P < 0.05 was considered to indicate statistical significance.
3. Results
3.1. Klebsiella-specific bacteriophage- Isolation from sewage water
K. pneumoniae subspp. pneumoniae ATCC 33495 was used as the host organism for isolating Klebsiella-specific phage from sewage water. Visible clear plaques ranging from 1.0 to 2.0 mm with well-defined boundaries indicated that the isolated phages were virulent (Fig. 1).
3.2 Phenotypic characterisation of the isolated Klebsiella-specific phage:
Transmission electron microscopy revealed an icosahedral head ranging from 50–100 nm in diameter, with a contractile tail sheath and a complex baseplate containing kinked fibres (Fig. 2-a)
Morphology and the size of plaques produced suggest that the bacteriophage belongs to the Order Caudovirales and the family Myoviridae [33].
One-step growth curve of the isolated phage showed a burst size of 69 which means that 69 phage particles are released from one bacterium during the lytic phase, and a latent period of 20 minutes as depicted in the graph (Fig. 2-b).
Thermal stability assay showed that at 25°C the infectivity of the phage was maximum as revealed by the Plaque forming units/ml (Fig. 2-c)
pH stability assay showed that the highest activity of the phage was between pH 6 to pH 7 (Fig. 2-d)
Time kill assay showed that after 2 h there was a significant reduction in the number of bacterial colonies (Fig. 2-e).
Optimal multiplicity of infection was found to be 10 by using the formula as follows:
Multiplicity of infection = Plaque-forming units (PFU/ml)/colony-forming units of bacteria(CFU/ml). This suggests that 10 phage particles are required to lyse one bacterial cell. Figure 2-f shows the TEM image of the bacterial call infected with the phages.
Bacterial growth inhibition test results performed with various MOI are depicted in the graph (Fig. 3).
3.3 Antibiogram of the isolates
Out of the total 40 strains of K. pneumoniae, 22 isolates were sensitive to cefoperazone-sulbactam, 16 isolates to piperacillin-tazobactam and 26 isolates to amikacin. Highest resistance was seen against cefuroxime (n = 25), piperacillin-tazobactam (n = 23) and ceftriaxone (n = 23), respectively (Fig. 3). 26 and 23 isolates were sensitive to the aminoglycosides, namely, gentamicin and amikacin, respectively. Two isolates showed resistance to netilmicin. Regarding the susceptibility to carbapenems it was found that 17 isolates of Klebsiella pneumoniae showed resistance to meropenem and 15 isolates resistance to imipenem. The isolates were moderately sensitive to cefoperazone-sulbactam (n = 22) and cefuroxime (n = 25). Twenty three isolates showed sensitivity to 3rd generation, cephalosporin-ceftriaxone, while 21 clinical isolates exhibited resistance to ciprofloxacin.
3.4. Subinhibitory concentration of meropenem
The subinhibitory concentration of meropenem was 0.25 µg/ml for 57.5% (23 out of 40 sensitive) of the samples and 8 µg/ml for 42.5% (17 out of 40 resistant) of the samples. The same concentration was used to determine biofilm formation in the presence of meropenem, a Klebsiella-specific phage, and to evaluate the synergistic effect of meropenem and the Klebsiella-specific phage on biofilm formation (Fig. 3).
3.5. Biofilm formation in microtiter plates
In the presence of meropenem (subinhibitory concentration), 91.3% of meropenem-sensitive strains (21 out of 23) presented a significant decrease in biofilm production (p < 0.001), whereas all meropenem-resistant strains (17 out of 17) presented an increase in biofilm production (Fig. 4).
There was a marked reduction in biofilm production by K. pneumoniae isolated from clinical samples when treated with Klebsiella-specific phages (82.5%; p = 0.029). The strains that were resistant to meropenem when treated with a specific phage presented 100% sensitivity to the phage, and biofilm production was significantly reduced (p = 0.029), as shown in Fig. 5.
The mean OD 570 of biofilms produced by K. pneumoniae isolates in the presence of the Klebsiella-specific phage and meropenem is shown in Fig. 6.
The combination of bacteriophage and meropenem had a synergistic effect on 100% of meropenem-sensitive strains and 100% of meropenem-resistant strains, resulting in a significant reduction in the production of biofilms, as evidenced by their OD values (Fig. 7) and colony counts (Table 1, Fig. 8 and Fig. 9).
In the absence of meropenem/phage | In subinhibitory concentration of Meropenem | In Klebsiella specific phage | Combination of both subinhibitory concentration of meropenem and Klebsiella specific phage | |
|---|---|---|---|---|
Mean CFU/ml of sensitive samples | 5.3x107 | 2.7x106 | 5.2x105 | 2.2x104 |
Mean CFU/ml of resistant samples | 5.9x107 | 2.4x107 | 2.6x105 | 1.1x104 |
Furthermore, the SEM images confirmed biofilm reduction in ETTs caused by the combination of meropenem with phages in representative samples of meropenem-resistant and meropenem-sensitive isolates (Fig. 10 and Fig. 11).
The present study revealed statistically significant p values for all three methods, with p values of 0.012, < 0.001, and 0.029 for plain, meropenem, and phage, respectively. The interquartile range and median of phage treatment for endotracheal pieces were 0.0940 and 0.184, respectively (Tables 2 and 3).
Method | N | Min mean | Max mean | Median | SD | IQR | Shapiro‒Wilk W | Shapiro-wilk (p value) |
|---|---|---|---|---|---|---|---|---|
Plain | 40 | 0.127 | 0.366 | 0.207 | 0.0711 | 0.115 | 0.0928 | 0.012 |
Meropenem | 40 | 0.125 | 2.21 | 0.204 | 0.685 | 0.926 | 0.746 | < 0.001 |
Phage | 40 | 0.105 | 0.350 | 0.184 | 0.0601 | 0.0940 | 0.939 | 0.029 |
| N: Number of isolates | ||||||||
| SD: Standard deviation | ||||||||
| IQR: Interquartile range | ||||||||
| Min: Minimum | ||||||||
| Max: Maximum | ||||||||
Methods | W | P value |
|---|---|---|
Plain + meropenem | 0.400 | 0.957 |
Meropenem + phage | -3.187 | 0.063 |
Plain + phage | -2.709 | 0.134 |
| W: Wilcoxon rank sum test statistic | ||
4. Discussion
Resistance to multiple antibiotics, especially by Klebsiella pneumoniae has become a global concern. This spread of MDR Klebsiella pneumoniae has resulted in the increased acquisition of resistant Klebsiella pneumoniae strains in the community as well as at the hospital level (1). The inner side of endotracheal devices inserted into the mouth of patients can act as an excellent reservoir for the attachment of biofilm-producing bacteria and thereby contribute to ventilator-associated pneumonia (VAP) (7).
The prevalence of MDR infections of K. pneumoniae has been exponentially increasing for all available antimicrobials (34). Antimicrobial resistance of Klebsiella pneumoniae may be due to the production of broad-spectrum and extended-spectrum β-lactamases (35). Due to the side effects related to antibiotic therapy, treatment with specific phage therapy gains importance in the field of treatment of nosocomial bacterial infections. Clinical usage of phage has aided in the successful treatment of antibiotic-resistant infections (36). The use of a novel bacteriophage on a mouse model has shown a significant decrease in the load of bacteria in the lungs and a reduction in the lesion severity in vivo against the virulent strains of K. pneumoniae which reveals the exponential use of phage in therapy as an alternative treatment (26).
In the present study, the sub-inhibitory concentration of meropenem and the effectiveness of specific Klebsiella phage isolated from sewage water were used to determine the production of biofilm in endotracheal tubing. The colony count of adherent bacteria on endotracheal tubing pieces treated with a sub-inhibitory concentration of meropenem and Klebsiella-specific phage was greatly reduced when compared to that of plain inoculum. Sensitive isolates showed a reduction in the formation of biofilm and colony count was reduced from a count of 107 to 106 (Table 2). On the other hand, there was an increase in biofilm production even when there was the presence of the antibiotic meropenem by the antibiotic-resistant isolates. This carbapenem resistance offered by the isolates can be due to increased expression of beta-lactamases, and loss of outer membrane proteins, which, among carbapenemase production, is an important mechanism. Studies have suggested that since the effect of β-lactams might be hampered by the presence of β-lactamase in the biofilm matrix, carbapenems like meropenem are generally ineffective against carbapenem-resistant organisms that are embedded in the biofilm. β-lactamase contained in the extracellular matrix of biofilms is also thought to attenuate the activities of β-lactam antibiotics, as described in previous studies (37–39). However, the treatment of the resistant isolates with specific Klebsiella phage showed a drastic reduction in the colony count and biofilm production on the endotracheal tubing pieces which implies the use of bacteriophage in treatment strategies to treat infections caused by multidrug-resistant Klebsiella pneumoniae. The synergistic action of Klebsiella-specific phage and meropenem was again found to be more efficient than the antibiotic and phage itself in terms of colony count.
Several studies have documented the lytic effect of bacteriophages on biofilm production. Phage degrades the extracellular matrix of biofilm and tends to replicate in large numbers at the site of infection. This suggests the usage of specific bacteriophages for effective reduction in biofilm production. (40). Endotracheal tubing coated with phage as well as antibiotic-coated endotracheal tubing is effective in the prevention of biofilm production on endotracheal devices to date (41). The evolution of phage-resistant mutants and phage-resistant subspecies is an important challenge in the use of phage therapy. The defence mechanisms exhibited by bacteria to various phages can contribute to the evolution of phage-resistant mutants both genotypically and phenotypically. This implies the need to understand more about the mechanisms of bacterial resistance to phage to make them useful in the treatment modality of various infections (42). The biofilm can provide protection and act as a chemical barrier for the penetration and subsequent interaction between the phage receptors and phage-binding proteins, making it more difficult to administrate phage therapy to control clinical pathogens (43) effectively. A systematic study of the interaction between phage and biofilm can evade this challenge effectively.
The use of a sub-inhibitory concentration of meropenem along with phage helped to evade the phage resistance mechanism that can arise in the phage population. The combination showed a pronounced effect on biofilm prevention and thereby in the reduction of colony count of clinical isolates rather than the phage and antibiotic itself. Treatment of resistant isolates with Klebsiella-specific phage favoured the inhibition of adhesion of bacteria onto the surfaces of endotracheal tubing and thereby contributed to the significant reduction of colony count. The scanning electron microscopic pictures of the endotracheal tubing also show similar results (Fig. 10, 11).
A recent study done to isolate and characterize the phage of Klebsiella for therapeutic use showed that when the Klebsiella-specific phage, was applied singly on Klebsiella, they failed to suppress Klebsiella growth. The study also pointed to the fact that the failure to inhibit the bacteria could be due to the development of mutants that were resistant to the phage. The study further suggested the use of a mixture (cocktail) of phages for the treatment of infections by Klebsiella [44].
Inter quartile range and median of phage treatment on endotracheal pieces were found to be 0.0940 and 0.184 which was less than that of plain (0.115,0.207) and meropenem (0.926,0.204) respectively (Tables 2 and 3). Statically similar results were found in a study by Edison J. Cano et.al in their study in 2021 [45] and Verma et.al in 2010 [46]. This implies that the treatment of phage produces an effective reduction in the production of biofilm, followed by plain broth. The increased biofilm formation in the presence of meropenem can be due to the production of carbapenemase by different mechanisms by the isolates [47]. Therefore, we found that the use of Klebsiella-specific phage has a pronounced effect in the reduction of biofilm in endotracheal tubing pieces and can be used in the treatment modalities individually or as a cocktail.
5. Limitations of the study:
A bacteriophage specific to Klebsiella pneumoniae was isolated and propagated on its host strain, K. pneumoniae subsp. pneumoniae ATCC 33495. Isolation was confirmed via a soft agar overlay assay, which revealed clear plaques indicative of lytic activity. Preliminary phenotypic characterisation, including transmission electron microscopy (TEM), was conducted. Although the genome of this phage remains unsequenced, future genomic analysis would offer a better perspective on key traits such as phage-host interactions and mechanisms for evading bacterial defences. Such data is essential for the rational selection of phages for human therapeutic applications.
6. Future perspectives:
Although phages are being tried as potential therapeutic agents, there are many challenges regarding their use. The most important challenge is the reliability of their efficiency, quality and performance. This is a potential area for further research.
7. Conclusion:
Within limitations, this study suggests the use of a combination of specific phage-coated and antibiotic-coated endotracheal tubing in the prevention of biofilm, thereby reducing the risk in the incidence of ventilator-associated pneumonia. Phage therapy has a significant role in the treatment modality of multidrug-resistant infections. The study notes that while the phage significantly reduces bacterial count and biofilm, it is not completely bactericidal on its own for the clinical pathogen tested, hence the proposed combination approach.
The result of this study leads to the innovative use of a novel preventive strategy, the use of phage and antibiotic combinations for the treatment of MDR and XDR isolates. The study also sheds light on the possible use of a specific phage cocktail for treating K. pneumoniae infections. Hence, phage therapy can serve as a primary alternative method to treat emerging resistance to antimicrobials. Moreover, this approach can be beneficial in managing nosocomial and other community-acquired MDR K.pneumoniae infections, especially in immunocompromised hosts.
Acknowledgments:
The authors are grateful to Kasturba Medical College, Mangalore, Manipal Academy of Higher Education, Manipal, India, for providing the infrastructure and invaluable support throughout the conduct of the research project.
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Funding:
None
Declaration of competing interest
s: None declared
Patient consent to participate
Waiver of consent obtained from the Institutional Ethics Committee
Ethical approval and consent to participate:
The study was conducted in accordance with the guidelines of the Declaration of Helsinki. It was approved by the Institutional Ethics Committee of Kasturba Medical College, Mangalore (IEC KMC MLR06/2022/269, IECKMCMIR05/2025/229, and IECKMCMLR05/2025/230), which waived patient consent to participate as the research was based solely on the isolates from specimens received in the laboratory and associated laboratory data. There is no risk to the patient and no direct contact between the researcher and the patient. The identity of the patient and images related to the patient are not displayed in the manuscript.
Consent for publication:
Not applicable
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Data Availability
The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files, and are available from the corresponding author upon reasonable request.
Clinical trial number: Not applicable
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Author Contribution
ES: Conceptualisation, investigation, methodology, data curation, supervision, writing – original draft, revision, and editing.AR: Investigation, methodology, data curation, writing – original draft.AAP: Investigation, methodology, data curationMMB: Investigation, methodology, data curationSHP: Investigation, methodology, data curation, writing – original draft.HK: Data curation, statistical work, writing – original draft, and editing.SSM: Investigation, SupervisionAll the authors have read and approved the submitted version of the manuscript.
AR: Investigation, methodology, data curation, writing – original draft.
AAP: Investigation, methodology, data curation
MMB: Investigation, methodology, data curation
SHP: Investigation, methodology, data curation, writing – original draft.
HK: Data curation, statistical work, writing – original draft, and editing.
SSM: Investigation, Supervision
All the authors have read and approved the submitted version of the manuscript.
References
1.
Vuotto, C., Longo, F., Balice, M., Donelli, G. & Varaldo, P. Antibiotic resistance related to biofilm formation in Klebsiella pneumoniae. Pathogens 3 (3), 743–758 (2014).
2.
Tan, D. et al. Characterization of Klebsiella pneumoniae ST11 isolates and their interactions with lytic phages. Viruses 11 (11), 1080 (2019).
3.
Liu, C. et al. Hypervirulent Klebsiella pneumoniae is emerging as an increasingly prevalent K. pneumoniae pathotype responsible for nosocomial and healthcare-associated infections in Beijing, China. Virulence 11 (1), 1215–1224 (2020).
4.
Chung, P. Y. The emerging problems of Klebsiella pneumoniae infections: Carbapenem resistance and Biofilm Formation. FEMS Microbiol. Lett. 363 (20), fnw219 (2016).
5.
McCartney, C. & Wilkinson, J. D. A history of tracheal intubation. Current Anaesth. Critical Care. 6 (1), 54–58 (1995).
6.
Chastre, J. & Fagon, J. Y. Ventilator-associated pneumonia. Am. J. Respir Crit. Care Med. 165 (7), 867–903 (2002).
7.
Costerton, J. W., Stewart, P. S. & Greenberg, E. P. Bacterial biofilms: a common cause of persistent infections. Science 284 (5418), 1318–1322 (1999).
8.
Raad, I. I. et al. The prevention of biofilm colonization by multidrug-resistant pathogens that cause ventilator-associated pneumonia with antimicrobial-coated endotracheal tubes. Biomaterials 32 (11), 2689–2694 (2011).
9.
Tabassum, R. et al. Complete genome analysis of a Siphoviridae phage TSK1 showing biofilm removal potential against. Klebsiella pneumoniae Sci. Rep. 8 (1), 17904 (2018).
10.
Sutherland, I. W., Hughes, K. A., Skillman, L. C. & Tait, K. The interaction of phages and biofilms. FEMS Microbiol. Lett. 232 (1), 1–6 (2004).
11.
Doolittle, M., Cooney, J. & Caldwell, D. Lytic infection of Escherichia coli biofilms by bacteriophage T4. Can. J. Microbiol. 41 (1), 12–18 (1995).
12.
Sillankorva, S., Neubauer, P. & Azeredo, J. Phage control of dual-species biofilms of Pseudomonas fluorescens and Staphylococcus lentus. Biofouling 26 (5), 567–575 (2010).
13.
Ruoting, P., Gisella, R. & Lamas, S. Inhibition of biofilm formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes. Appl. Environ. Microbiol. 80 (17), 5340–5348 (2014).
14.
Rydman, P. S. & Bamford, D. H. The lytic enzymes of bacteriophage PRDI associated with the viral membrane. J. Bacteriol. 184 (1), 104–110 (2002).
15.
Sundaramoorthy, N. S., Thothathri, S., Bhaskaran, M., Ganesh Prasad, A. & Nagarajan, S. Phages from Ganges River curtail in vitro biofilms and planktonic growth of drug-resistant Klebsiella pneumoniae in a zebrafish infection model. AMB Express. 11 (1), 27 (2021).
16.
CLSI. Performance standards for antimicrobial susceptibility testing; Twenty-sixth edition, informational supplement CLSI document, M 100-S26; Clinical and Laboratory Standards Institute.
17.
Bonilla, N. et al. Phage on tap-a quick and efficient protocol for the preparation of bacteriophage laboratory stocks. Peer J. 4, e2261 (2016).
18.
Townsend, E. M. et al. Isolation and characterization of Klebsiella phages for phage therapy. Phage (New Rochelle). 2 (1), 26–42 (2021).
19.
Mirza, K. A. et al. Characterization and genome analysis of novel Klebsiella pneumoniae phage vbKpUKJ_2 isolated from hospital sewage water. BMC Microbiol. 25 (1), 96 (2025).
20.
Menon, N. D. et al. A Novel N4-Like Bacteriophage Isolated from a Wastewater Source in South India with Activity against Several Multidrug-Resistant Clinical Pseudomonas aeruginosa Isolates. mSphere 6 (1), e01215–e01220 (2021).
21.
Yang, H., Liang, L., Lin, S. & Jia, S. Isolation and characterization of a virulent bacteriophage AB1 of Acinetobacter baumannii [Internet]. BMC Microbiol. 10, 131 (2010).
22.
Wang, R. et al. Characterization of phage HZY2308 against Acinetobacter baumannii and identification of phage-resistant bacteria. Virol. J. 21 (1), 283. 10.1186/s12985-024-02556-y (2024). Published 2024 Nov 7.
23.
Liu, J., Gao, S., Dong, Y., Lu, C. & Liu, Y. Isolation and characterization of bacteriophages against virulent Aeromonas hydrophila. BMC Microbiol. ;20(1):141. (2020). 10.1186/s12866-020-01811-w. PMID: 32487015.
24.
Melo, L. D. R. et al. Isolation and characterization of a new Staphylococcus epidermidis broad-spectrum bacteriophage. J. Gen. Virol. 95, 506–515 (2014).
25.
Kowalska-Krochmal, B. & Dudek-Wicher, R. The Minimum Inhibitory Concentration of Antibiotics: Methods, Interpretation, Clinical Relevance. Pathogens 10 (2), 165 (2021).
26.
Narimisa, N., Amraei, F., Kalani, B. S., Mohammadzadeh, R. & Jazi, F. M. Effects of sub-inhibitory concentrations of antibiotics and oxidative stress on the expression of type II toxin-antitoxin system genes in Klebsiella pneumoniae. J Glob Antimicrob. Resist ; (21):53–56. (2020).
27.
O’Toole and Kolter. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 occurs via multiple convergent signaling pathways. A genetic analysis. Mol. Microbiol. 28 (3), 449–461 (1998).
28.
Sanders, E. R. Aseptic laboratory techniques: plating methods. J. Vis. Exp. (63), e3064. 10.3791/3064 (2012).
29.
Suman, E., Varghese, B., Joseph, N., Nisha, K. & Kotian, M. S. The Bacterial biofilms in dialysis water systems and the effect of subinhibitory concentration of chlorine on them. J. Clin. Diagn. Res. 7 (5), 849–852 (2013).
30.
Suman, E., Jose, J., Varghese, S. & Kotian, M. S. Study of biofilms production in Escherichia coli causing urinary tract infection. Indian J. Med. Microbiol. 25 (3), 305–306 (2007).
31.
Amankwah, S., Abdusemed, K. & Kassa, T. Bacterial biofilm destruction: A focused review on the recent use of phage-based strategies with other antibiofilm agents. Nanotechnology Science Applications. 14, 161–177 (2021).
32.
Raval, Y. S. et al. Hydrogen-peroxide generating electrochemical bandage is active in vitro against mono- and dual-species biofilms. Biofilm 3, 100055 (2021).
33.
Kęsik-Szeloch, A. et al. Characterising the biology of novel lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae. Virol. J. 10, 100. https://doi.org/10.1186/1743-422X-10-100 (2013).
34.
Tian, X. et al. Meropenem for children with severe pneumonia: Protocol for a randomized controlled trial. Front. Pharmacol. 13, 1021661 (2022).
35.
Navon-Venezia, S., Kondratyeva, K. & Carattoli, A. Klebsiella pneumoniae: A major worldwide source and shuttle for antibiotic resistance. FEMS Microbiol. Rev. 41 (3), 252–275 (2017).
36.
Oliveira, V. C. et al. Identification and characterization of new bacteriophages to control multidrug-resistant Pseudomonas aeruginosa biofilm on endotracheal tubes. Front. Microbiol. 11, 580779 (2020).
A
37.Pinto, A. M., Faustino, A., Pastrana, L. M., Bañobre-López, M. & Sillankorva, S. Pseudomonas aeruginosa PAO 1 In Vitro Time-Kill Kinetics Using Single Phages and Phage Formulations-Modulating Death, Adaptation, and Resistance. Antibiot. (Basel). 10 (7), 877 (2021).
A
38.Anand, T. et al. Phage therapy for treatment of virulent Klebsiella pneumoniae infection in a mouse model. J. Global Antimicrob. Resist. 21, 34–41 (2020).
A
39.Hengzhuang, W. et al. High -lactamase levels change the pharmacodynamics of beta-lactam antibiotics in Pseudomonas aeruginosa biofilms. Antimicrob. Agents Chemother. 57, 196–204 (2013).
40.
Dibdin, G. H., Assinder, S. J., Nichols, W. W. & Lambert, P. A. Mathematical model of beta-lactam penetration into a biofilm of Pseudomonas aeruginosa while undergoing simultaneous inactivation by released beta-lactamases. J. Antimicrob. Chemother. 38, 757–769 (1996).
41.
Rahdar, H. A. et al. Correlation between biofilm formation and carbapenem resistance among clinical isolates of Klebsiella pneumoniae. Ethiop. J. Health Sci. 29 (6), 745–750 (2019).
42.
Hennequin, C., Robin, F., Cabrolier, N., Bonnet, R. & Forestier, C. Characterization of a dha-1-producing Klebsiella pneumoniae strain involved in an outbreak and role of the AMPR regulator in virulence. Antimicrob. Agents Chemother. 56 (1), 288–294 (2012).
43.
Lan, P., Jiang, Y., Zhou, J. & Yu, Y. A global perspective on the convergence of hypervirulence and carbapenem resistance in Klebsiella pneumoniae. J. Glob Antimicrob. Resist. 25, 26–34 (2021).
44.
Fu, W. et al. Bacteriophage cocktail for the prevention of biofilm formation by Pseudomonas aeruginosa on catheters in an in vitro model system. Antimicrob. Agents Chemother. 54 (1), 397–404 (2009).
45.
Cano, E. J. et al. Phage therapy for limb-threatening prosthetic knee Klebsiella pneumoniae infection: Case report and in vitro characterization of anti-biofilm activity. Clin. Infect. Dis. 73 (1), e144–e51 (2020).
46.
Verma, V., Harjai, K. & Chhibber, S. Structural changes induced by a lytic bacteriophage make ciprofloxacin effective against older biofilm of Klebsiella pneumoniae. Biofouling 26 (6), 729–737 (2010).
47.
Soleimani Sasani, M., Eftekhar, F. & Hosseini, M. Isolation and Characterization of a Klebsiella pneumoniae Specific Lytic Bacteriophage from a Hospital Waste-water Treatment Plant. J. Med. Microbiol. Infect. Dis. 7 (1), 6–11 (2019).
Tables
A
Fig. 1
Soft agar overlay plate showing clear plaques of bacteriophages against
Klebsiella pneumoniae ATCC 33495
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Fig. 2
Phenotypic characterisation of Klebsiella-specific phage- a) Transmission Electron Microscopic (TEM)image of the Klebsiella -specific phage b) Graph showing one-step growth curve at MOI 10 − 6 c) Effect of temperature on the stability of the phage d) Effect of pH on the stability of the phage e)Time kill curve at MOI of 10 f) Klebsiella pneumoniae infected with Klebsiella specific phage
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Fig. 3
Bacterial inhibition assay in the presence of various concentrations of the phage
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Fig. 4
Antibiogram pattern of K. pneumoniae isolates
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Fig. 5
Subinhibitory concentrations (sub-MICS) of meropenem for the various
K. pneumoniae isolates
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Fig. 6
Mean OD 570 of biofilms produced by K. pneumoniae upon exposure to subinhibitory concentrations of meropenem
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Fig. 7
Mean OD 570 of biofilms produced by K. pneumoniae isolates upon exposure to Klebsiella-specific phages
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Fig. 8
Mean OD 570 of biofilms produced by K. pneumoniae isolates in the presence of the Klebsiella-specific phage and meropenem
A
Fig. 9
Comparison of the absorbance (mean OD570) of biofilms produced by meropenem-sensitive and meropenem-resistant K. pneumoniae strains
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Fig. 10
Colony counts by spread plate method-plain inoculum (A), in the presence of subinhibitory concentrations of meropenem (B), in the presence of phage (C), and in the presence of phage + meropenem (D) in a representative resistant sample.
A
Fig. 11
Colony counts via the surface plating method in the presence of plain inoculum (A) and subinhibitory concentrations of meropenem (B), phage (C), and phage + meropenem (D) in a representative sensitive sample.
A
A
Fig. 12
Scanning electron microscopy images of biofilms produced by representative resistant isolates of Klebsiella pneumoniae - plain inoculum (A), in the presence of subinhibitory concentrations of meropenem (B), phage (C), and phage + meropenem combination (D)
A
A
Fig. 13
Scanning electron microscopy images of biofilms produced by representative sensitive isolates of Klebsiella pneumoniae - plain inoculum (A), in the presence of subinhibitory concentrations of meropenem (B), phage (C), and phage + meropenem combination (D)
Figure legends
Figure 1. Soft agar overlay plate showing clear plaques of bacteriophages against
Klebsiella pneumoniae ATCC 33495
Figure 2. Phenotypic characterisation of Klebsiella-specific phage- a) Transmission Electron Microscopic (TEM)image of the Klebsiella -specific phage b) Graph showing one-step growth curve at MOI 10 − 6 c) Effect of temperature on the stability of the phage d) Effect of pH on the stability of the phage e)Time kill curve at MOI of 10 f) Klebsiella pneumoniae infected with Klebsiella specific phage
Figure 3. Bacterial inhibition assay in the presence of various concentrations of the phage
Figure 4. Antibiogram pattern of K. pneumoniae isolates
A
Fig. 5
Subinhibitory concentrations of meropenem for various K. pneumoniae isolates
Figure 6. Mean OD 570 of biofilms produced by K. pneumoniae upon exposure to subinhibitory concentrations of meropenem
Figure 7. Mean OD 570 of biofilms produced by K. pneumoniae isolates upon exposure to Klebsiella-specific phages
Figure 8. Mean OD 570 of biofilms produced by K. pneumoniae isolates in the presence of the Klebsiella-specific phage and meropenem
A
Fig. 9
Comparison of the absorbance (mean OD570) of biofilms produced by sensitive and resistant K. pneumoniae strains
A
Fig. 10
Colony counts via the surface plating method in the presence of plain inoculum (A), subinhibitory concentrations of meropenem (B), phage (C), and phage + meropenem (D) in a representative resistant sample
Figure 11. Colony counts via the surface plating method in the presence of plain inoculum (A) and subinhibitory concentrations of meropenem (B), phage (C), and phage + meropenem (D) in a representative sensitive sample
A
Fig. 12
Scanning electron microscopy images of biofilms produced by representative resistant isolates of Klebsiella pneumoniae in the presence of plain inoculum (A), antibiotics (B), phages (C), or phage‒antibiotic combinations (D)
A
Fig. 13
Scanning electron microscopy images of biofilms produced by representative sensitive isolates of Klebsiella pneumoniae in the presence of plain inoculum (A), antibiotics (B), phages (C), and phage‒antibiotic combinations (D)