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Brazilian Journal of Microbiology logoLink to Brazilian Journal of Microbiology
. 2021 Jan 22;52(2):597–606. doi: 10.1007/s42770-021-00433-2

Assessment of the in vitro activity of azithromycin niosomes alone and in combination with levofloxacin on extensively drug-resistant Klebsiella pneumoniae clinical isolates

Hoda Mohamed Owais 1, Manal Mohammad Baddour 1, Hala Abd El-Raouf El-Metwally 1, Heba Soliman Barakat 2, Nour Sherif Ammar 3, Marwa Ahmed Meheissen 1,✉
PMCID: PMC8105464  PMID: 33483896

Abstract

Background and aim

Extensively drug-resistant (XDR) Klebsiella pneumoniae represent a major threat in intensive care units. The aim of the current study was to formulate a niosomal form of azithromycin (AZM) and to evaluate its in vitro effect on XDR K. pneumoniae as a single agent or in combination with levofloxacin.

Material and methods

Forty XDR K. pneumoniae isolates (23 colistin-sensitive and 17 colistin-resistant) were included in the study. Formulation and characterization of AZM niosomes were performed. The in vitro effect of AZM solution/niosomes alone and in combination (with levofloxacin) was investigated using the checkerboard assay, confirmed with time-kill assay and post-antibiotic effect (PAE).

Results

The AZM niosome mean minimal inhibitory concentration (MIC) (187.4 ± 209.1 μg/mL) was significantly lower than that of the AZM solution (342.5 ± 343.4 μg/mL). AZM niosomes/levofloxacin revealed a 40% synergistic effect compared to 20% with AZM solution/levofloxacin. No antagonistic effect was detected. The mean MIC values of both AZM niosomes and AZM solution were lower in the colistin-resistant group than in the colistin-sensitive group. The mean PAE time of AZM niosomes (2.3 ± 1.09 h) was statistically significantly longer than that of the AZM solution (1.37 ± 0.5 h) (p = 0.023).

Conclusion

AZM niosomes were proved to be more effective than AZM solution against XDR K. pneumoniae, even colistin-resistant isolates.

Keywords: Niosomes, Azithromycin, Klebsiella pneumoniae, Colistin

Introduction

The problem of antimicrobial resistance continues to be a global threat. Antimicrobial resistance is now caused by superbugs, which are resistant to almost all clinically available antimicrobial agents [1]. Klebsiella pneumoniae (K. pneumoniae) is one of these superbugs and recognized as a major cause of hospital-acquired infections. The dissemination of extensively drug-resistant (XDR) K. pneumoniae is causing difficult-to-treat infections worldwide. These infections have been related to increased morbidity, mortality, long hospital stay, and increased healthcare costs [1].

A major advance in nanomedicine holds promise for the treatment of XDR bacterial infections. The nanoparticles act as antibacterial or carriers of antimicrobial agents [2]. Antibiotics in nanoform drug delivery systems provide several functions such as prolonged antibiotic systemic circulation half-life with sustained antibiotic release, and improved antibiotic solubility, which will in turn improve drug effectiveness, reduce systemic side effects, and allow administration of lower drug doses [3]. On the other hand, clinical trials of nanodelivery systems are still limited, due to their high cost and possible unknown side effects [2].

Niosomes are nonionic surfactant–based vesicles. They are attractive and promising nanotools for enhancing antibacterial activity and reducing antimicrobial resistance. This can be due to their role in the alteration of bacterial cell membrane permeability and adhesion with possible fusion with the bacterial cell wall, together with inhibition of biofilm formation. Furthermore, niosomes can be designed to be orally, parenterally, or topically prescribed. Niosomes can remain for a considerable time in blood, and they are also more stable and more cost-effective than liposomes [4].

In view of the increasing problem of XDR K. pneumoniae in our university hospitals and in an attempt to make use of these new drug delivery systems, the present study was designed. This study aims to formulate an azithromycin (AZM) nanodelivery system (niosomes), to investigate its in vitro activity and post-antibiotic effect on XDR K. pneumoniae clinical isolates alone and in combination with levofloxacin (LEV).

Material and methods

Clinical isolates

Forty XDR K. pneumoniae collected from intensive care units (ICUs) at Alexandria University Hospitals constituted the material of this study. The 40 isolates were divided into two groups according to their susceptibility to colistin sulfate (by the reference broth microdilution method) [5]: colistin-sensitive ≤ 2 μg/mL (23 isolates; 57.5%) and colistin-resistant > 4 μg/mL (17 isolates; 42.5%) groups.

Klebsiella sp. initial identification was done via conventional biochemical methods (triple sugar iron agar positive, citrate positive, urease positive, nonmotile, and Voges-Proskauer test positive) [6]. Confirmation of identification to the species level was performed using the Vitek 2 compact system (BioMérieux, Marcy, L’Étoile, France). XDR was defined according to the European Centre for Disease Prevention and Control (ECDC) and the Centers for Disease Control and Prevention (CDC) as nonsusceptibility to at least one agent in all but two or fewer antimicrobial categories [7].

The study was approved by the Research Ethics Committee of Alexandria Faculty of Medicine. Laboratory workup was performed on isolates collected through routine clinical work with anonymous patient’s identifiable information. No informed consent was necessary.

Formulation and characterization of AZM nanodelivery system

AZM niosomes were prepared by the thin-film hydration method using a 1:1 M ratio of Span 60 and Tween 40 surfactants together with cholesterol (total surfactant-cholesterol molar ratio 1:1). Surfactants and cholesterol were dissolved in 3 mL chloroform which was then evaporated under reduced pressure at 60 °C in a rotary evaporator (Buchi Rotavapor, Switzerland) until complete evaporation of all chloroform. The film was hydrated with 5 mL AZM solution (2% w/v) in sterile distilled water at 60 °C for 1 h. AZM niosomes were allowed to cool to room temperature (25 °C) and left overnight at 4–5 °C for complete swelling of the vesicles [8].

The mean diameter of niosomes and zeta potential were determined using the Malvern dynamic light scattering particle size and zeta potential analyzer (Zetasizer Nano ZS90, Malvern Instruments, Worcestershire, UK) in triplicate. A drop of niosome dispersion was examined by transmission electron microscopy (TEM) (Jeol-100 CX, Japan) equipped with a digital camera at 80 kV accelerating voltage. AZM release was tested by a dialysis method as previously described [9]. A linear regression analysis of the release data (2–12 h) was performed to determine release kinetics. Niosomes were stored in their original solution at 4 °C for 12 months. The change in size and zeta potential of AZM niosomes as well as their release pattern was monitored during the storage period.

All K. pneumoniae isolates were subjected to:

  1. Broth microdilution reference method

The minimal inhibitory concentration (MIC) of AZM solution, AZM niosomes, and LEV (Sigma-Aldrich, USA) for K. pneumoniae isolates was tested using the broth microdilution (BMD) method according to the Clinical Laboratory Standards Institute (CLSI) guidelines [10]. Twofold serial dilutions ranging from 512 to 0.5 μg/mL for AZM and AZM niosomes and from 16 to 0.25 μg/mL for LEV were prepared using cation-adjusted Mueller-Hinton broth (CAMHB). The MIC of LEV was interpreted according to the CLSI 2020 recommended breakpoints [11]. Escherichia coli ATCC 25922 was included as control. Since AZM is considered originally an anti-gram-positive antibiotic, and no CLSI or EUCAST (The European Committee on Antimicrobial Susceptibility Testing) breakpoints are provided, its MIC and that of AZM niosomes were just recorded.

  • 2.

    Checkerboard synergy assay

Testing of in vitro synergy between AZM solution/AZM niosomes and LEV was performed using the standard checkerboard synergy assay (CBA) as previously described [12]. Antibiotic powders used were the formulated AZM niosomes, AZM, and LEV (Sigma-Aldrich, USA). AZM niosomes or AZM solution was tested in the range of 0.5–512 μg/mL in the horizontal wells of the microtiter plate, while LEV was tested in the vertical wells following the range of 0.25–16 μg/mL. The inoculum was prepared as a 1:100 dilution of a half McFarland bacterial suspension. The plates were inoculated and incubated at 37 °C for 24 h. The presence or absence of microbial growth was determined visually. The fractional inhibitory concentration index (FICI) for each antibiotic was calculated according to the following formula: FICI = FIC of agent A + FIC of agent B, where FIC of agent A or B = MIC of agent A or B in combination/MIC of agent A or B alone. The combination value was derived from the highest dilution of antibiotic combination giving no visible growth. For interpretation of the results, FIC index values of ≤ 0.5 indicate synergism, FIC index values of between 0.5 and 4 indicate indifference, and FIC index values of > 4 indicate antagonism [13].

  • 3.

    Multiple combination bactericidal test

The CBA test was followed by the multiple combination bactericidal test (MCBT), where the plate was placed on a shaker mixer for 1 min. Then, the content of the clear wells was mixed by a pipette for 5–6 times, and 10 μL from clear wells was inoculated on blood agar and incubated at 35 °C for 24 h and examined for bactericidal activity (99.9% kill on the next day denotes that this combination was bactericidal) [14].

  • 4.

    Time-kill assay

The activity of single antibiotics as well as of combinations was investigated by time-kill studies performed in the logarithmic growth phase using an initial inoculum of ~ 5 × 105 CFU/mL. The following antibiotic concentrations were used: 1/4X MIC and 1/8X MIC. A growth control with no antibiotic was also included. Samples were taken at defined time points (0, 2, 4, 6, and 24 h post-inoculation). After incubation on blood agar plates, colonies were counted, and the results were recorded as the number of CFU/mL. Synergy was defined as a ≥ 2 log10 decrease in CFU/mL between the combination and the most active single agent tested at the same concentration after 24 h. Indifference was defined as a < 2 log10 increase or decrease in colony count at 24 h by the combination compared with that by the most active drug alone. Antagonism was defined as a ≥2 log10 increase in colony count at 24 h by the combination compared with that by the most active drug alone. Bactericidal activity was defined as ≥ 3 log10 CFU/mL reduction of the initial bacterial count at each time point. All tests were performed in duplicate [13].

  • 5.

    Post-antibiotic effect

Post-antibiotic effect (PAE) was determined using a standard viable counting method by testing the antibiotic combinations that showed synergy according to the CBA. Test isolates were exposed to antibiotics at 1X MIC (alone and in combinations) for 1 h at 37 °C, followed by washout, centrifugation (5000 rpm for 10 min), and resuspension in 10 mL of CAMHB. One hundred microliters from each suspension was 10-fold serially diluted in CAMHB and then inoculated onto blood agar plates at defined time points (0, 1, 2, 4, 6 h post-inoculation). Control curves were done using the same method without antibiotics. PAE = T − C, where T is the time (in hours) required for the viability count in the test culture to increase 1 log10 above the count observed immediately after centrifugation, and C is the corresponding time for the controls [15].

Statistical analysis

Data were coded and processed using the statistical package for the Social Sciences (SPSS) version 25 (IBM Corp., Armonk, NY, USA). Data were summarized and described using the mean, median, range, and frequencies. Categorical and continuous variables were analyzed using the appropriate tests. A chi-square test was used to study the significant association between two categorical variables. Independent sample t test and Wilcoxon’s test were used to detect the significant difference in the mean and median of the quantitative variables. One-way independent ANOVA was performed for normally distributed data in more than two samples. A p value of less than 0.05 was considered statistically significant [16]. A diagnostic analysis was used to detect the diagnostic ability of the checkerboard test compared to the time-kill assay test.

Results

The characterization of prepared niosomes revealed that the mean size of AZM-loaded niosomes after several passages was 98 ± 50, PDI = 0.4. Zeta potential values indicated negatively charged AZT-loaded noisome (− 20.5 mV). Figure 1 shows the TEM micrographs of AZM-loaded niosomes after extrusion. Release results at 37 °C for AZM-loaded niosomes compared to standard drug solution under sink conditions are shown in Fig. 2. The release of free AZM was completed in about 4 h, confirming drug dialyzability. On the other hand, a slower sustained release of AZM from freshly prepared niosomes for 12 h confirmed vesicle integrity. Release data produced a biphasic profile, characteristic of vesicular systems. A faster initial phase of 2 h during which free drug is released followed by the slower phase (2 to 12 h) of diffusion of entrapped AZM out of the niosomes. AZM release reached approximately 90% after 12 h. Storage of AZM niosomes in their original solution at 4 °C for 12 months produced almost no change in their original size. This indicates the lack of neither vesicle fusion nor aggregation attributed to the negative zeta potential of the vesicles. Also, assessing the stability of the release pattern by repeating the release experiment at the end of the storage period revealed reproducibility of the release profile, which reflects the maintenance of niosome structural integrity during the storage period. Furthermore, examining the microbiological stability revealed no change in AZM niosome MICs against XDR K. pneumoniae when stored in their original solution at 4 °C for 12 months.

Fig. 1.

Fig. 1

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Transmission electron micrograph (TEM) of azithromycin niosomes

Fig. 2.

Fig. 2

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In vitro release of azithromycin niosomes (2–12 h)

Out of the 40 XDR K. pneumoniae, 17 (42.5%) isolates were from blood culture samples, 15 (37.5%) from bronchoalveolar lavage, and eight (20%) from pus specimens.

According to the results of BMD, the 40 isolates were resistant to LEV (MIC ≥ 2 μg/mL) with a mean MIC of 38.2 ± 22.4 μg/mL ranging from 8 to 64 μg/mL, with no statistically significant difference in mean MIC between the colistin-sensitive (42.4 μg/mL ± 22.3) and colistin-resistant groups (32.5 μg/mL ± 22.5) (p = 0.17). Although the range of MIC values of AZM solution was similar to that of the AZM niosomes (4–1024 μg/mL), the AZM niosome mean MIC (187.4 ± 209.1 μg/mL) was statistically significantly lower than that of the AZM solution (342.5 ± 343.4 μg/mL) (t = 3.9, p = 0.0001) in all isolates. Additionally, the mean MIC of both AZM solution and AZM niosomes was statistically significantly lower in the colistin-resistant (139.5 μg/mL ± 240.8, 101.8 μg/mL ± 204.6, respectively) than in the colistin-sensitive group (492.5 μg/mL ± 333.9, 250.6 μg/mL ± 159.9, respectively) (p = 0.0007, p = 0.01, respectively).

Concerning the CBA results, the AZM niosome/LEV combination revealed a statistically higher number of isolates showing synergy than the AZM solution/levofloxacin (16/40 versus 8/40 isolates) (X2 = 14.9, p = 0.0001). Out of the 16 isolates, which showed synergy, a higher percentage was observed in the colistin-resistant (8/17; 47%) than in the colistin-sensitive group (8/23; 34.8%). The remaining isolates in the two combinations showed indifference with no antagonism detected with any combination. All isolates that showed synergy with AZM solution/LEV combination were also synergistic with AZM niosome/LEV combination. There was a significant reduction in the mean FICI (0.7 ± 0.32, range: 0.125–1.03) of AZM niosome/LEV combination compared to the mean FICI (0.932 ± 0.51; range: 0.125–2.06) of AZM solution/LEV combination for the 40 isolates (p = 0.004) indicating that the AZM niosomes augment the antimicrobial efficacy of the combination. However, in isolates that showed synergy, the mean FICI of AZM niosomes/LEV (0.372 ± 0.138, range = 0.125–0.5) was comparable to that of the AZM solution/LEV combination (0.353 ± 0.13, range = 0.125–0.5) (p = 0.971).

When combining AZM and LEV, there was a 2.6-fold reduction in AZM solution MIC, while a 3.8-fold reduction in AZM niosome MIC. In addition, the mean MIC values of both AZM niosomes and AZM solution were lower in the colistin-resistant group (7.5 μg/mL ± 7 and 54 μg/mL ± 82.7) than in the colistin-sensitive group (79.5 μg/mL ± 128 and 261 μg/mL ± 345.2, respectively). Moreover, LEV MIC decreased below the susceptible breakpoints (≤ 0.5 μg/mL) in 13 isolates (13/40; 32.5%) when combined with AZM solution and in 11 isolates (11/40; 27.5%) when combined with AZM niosomes.

Out of the eight isolates that showed synergy with AZM solution/LEV combination, the MCBT identified only one isolate (12.5%) with a bactericidal effect, while out of the 16 isolates showing synergy with AZM niosome/LEV combination, the MCBT results in five isolates (31.3%) showing bactericidal effect.

The results of the CBA of AZM solution/LEV combination were evaluated using the time-kill assay (TKA). Synergy with a 2 log10 reduction was confirmed in seven out of the eight isolates showing synergy with the CBA, at 2 and 4 h (one isolate), at 2, 4, and 6 h (one isolate), at 4 h (two isolates), at 4 and 6 h (two isolates), and at 6 h (one isolate). Regrowth occurred at 24 h with all isolates. Regarding AZM niosome/LEV combination, 15 out of 16 isolates showed synergy with a 2 log10 reduction. Synergy was observed at 2 and 4 h (one isolate), at 2, 4, and 6 h (five isolates), at 4 h (one isolate), at 4 and 6 h (three isolates), and at 6 h (five isolates). Regrowth also occurred at 24 h in all isolates. The remaining isolates showed indifference with either formula and none showed antagonism (Fig. 3). Thus, in comparison to the golden standard TKA, the CBA displayed a very good sensitivity (100%) and specificity (96%) with both combinations.

Fig. 3.

Fig. 3

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Time-kill assay of K. pneumoniae isolates. a Time-kill assay of the seven K. pneumoniae isolates (showing synergy) with a mean azithromycin solution concentration of 128 μg/mL and a mean levofloxacin concentration of 8 μg/mL. Synergy was observed at mean time of 4 h. b Time-kill assay of the 15 K. pneumoniae isolates with a mean azithromycin niosome concentration of 60 μg/mL and a mean levofloxacin concentration of 10 μg/mL. Synergy was detected at a mean time of 2 h

A bactericidal effect with 3 log10 reductions in the viable count of K. pneumoniae was detected only in three isolates with AZM niosome/LEV combination at 6 h, while none of the isolates exhibited bactericidal activity with AZM solution/LEV combination.

The results of PAE testing revealed that the mean PAE time of AZM niosomes (2.3 ± 1.09; ranging from 1 to 5 h) was statistically significantly longer than that of the AZM solution (1.37 ± 0.5, ranging from 1 to 2 h) (p = 0.023). The duration of PAE of LEV ranged from 0 to 2 h. Although the mean time of PAE of AZM niosome/LEV combination (1.8 ± 0.75 h) was slightly longer than the mean time of AZM solution/LEV combination (1.5 ± 0.5 h), this difference was not statistically significant (p = 0.31). (Figs. 4 and 5).

Fig. 4.

Fig. 4

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The mean post-antibiotic effect (PAE) values for K. pneumoniae isolates that showed synergy with azithromycin solution/levofloxacin (seven isolates) and azithromycin niosome/levofloxacin (15 isolates) combinations at 1X MIC for 1 h

Fig. 5.

Fig. 5

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The post-antibiotic effect of azithromycin solution/niosomes and in combination with levofloxacin. a Post-antibiotic effect (PAE) of azithromycin solution/levofloxacin alone and in combination. a Post-antibiotic effect (PAE) of azithromycin niosomes/levofloxacin alone and in combination

Discussion

Infections caused by XDR K. pneumoniae continue to increase and currently represent one of the main challenges for the treatment of infections in the intensive care units. Colistin has been considered one of the last resort antibiotics to treat such infections. However, the increased reporting of colistin resistance calls for the urgent need of unordinary solutions (novel compounds or combinations) to control these XDR pathogens [17].

The study of colistin resistance prevalence among XDR K. pneumoniae isolates showed a high percentage (42.5%). A lower prevalence was proved in previous studies; Santimaleeworagun et al. [18] investigated the prevalence of colistin resistance among the gram-negative bacteria, and the highest percentage was found among K. pneumoniae isolates (17.3%). Giacobbe et al. [19] also reported that 19.5% of carbapenem-resistant K. pneumoniae were resistant to colistin. The higher prevalence of colistin resistance in the present study could be explained by the difference in the resistance pattern of the studied isolates, as all our isolates were XDR. Moreover, colistin is being used as an empiric antibiotic treatment in ICUs of our university hospitals.

Niosomes are nonionic surfactant vesicles with attractive drug carriers’ characteristics since they have the ability to encapsulate hydrophobic and hydrophilic drugs staying stable and less toxic. Niosomes’ vesicle size is crucial to achieve the desired therapeutic effect. Small-sized vesicles offer several advantages as low toxicity, high stability, and selective delivery to the target site with good phagocytic uptake [4]. In the current study, the niosomes’ size was similar to the findings of Ullah et al. [20]. Moreover, the hydroxyl group in cholesterol molecule with uneven distribution of polarity could lead to the negatively charged azithromycin niosomes. The ionic dissociation of nonionic surfactants with the formation of ionic impurities could also confer a highly negative charge niosomes. These will eventually prolong the antibiotic effect in blood rather than those with a positive or neutral charge, leading to increased effectiveness of the azithromycin-loaded vesicles in the treatment of infections. Furthermore, sufficiently high negative zeta potential values produce electrostatic stabilization of the vesicles [4].

The hydrophobic nature of the AZM makes it impermeable through the outer membrane of K. pneumoniae [21]; it was possibly the cause of the negligible activity of AZM solution against the studied K. pneumoniae isolates. However, the MIC of AZM in the niosomal form resulted in significant lowering of MIC by nearly 2-folds (from 342.5 ± 343.4 to 187.4 ± 209.1 μg/mL). The present results indicated that the AZM niosomes could be considered an attractive and successful form of the antimicrobial delivery system. Improvement of antimicrobial activity by niosomal formulation has been attributed to a decrease in the hydrophobic nature of the AZM and increase in the antibiotic permeability by an interaction between niosomes and bacterial cell membranes by fusion and contact release to unload the entrapped drug directly inside the bacterial cell [22]. Previous studies also reported that niosomes enhance the antibiotic activity against their studying microorganisms [23, 24].

Aiming to increase the antibiotic effect of AZM solution/AZM niosomes as a single agent, it was combined with LEV. The CBA revealed that 20% of the isolates showed synergy with AZM solution/LEV combination, while a more pronounced synergistic effect (40%) was noted with AZM niosome/LEV combination. No antagonistic effect appeared in both combinations.

These results corroborate with those of previous studies showing an in vitro synergistic effect between AZM and quinolones against pseudomonas and other gram-negative bacteria [25, 26]. However, our results showed lower synergistic rates than those of the studies testing the clinical effect of macrolide/quinolone combinations, as in the study of Kolumbic et al. [27], which showed a 90% microbiological cure of patients using ciprofloxacin/AZM combination. Magri et al. [28] also assessed the treatment effect of ciprofloxacin and azithromycin combination in patients with chronic bacterial prostatitis and reported an 83.9% eradication rate with a lower relapse rate as compared with ciprofloxacin or azithromycin single-agent therapy. The results of the previous in vivo studies may explain the difference between the effect of antibiotic combinations in vivo and in vitro and would refer to the need of further testing our combinations in vivo to prove their effect.

Although the AZM is not the primary treatment option for K. pneumoniae infection, the synergy detected in our study was most probably due to its ability to inhibit the expression of various virulence factors with alteration of bacterial outer membrane as observed in several other bacterial species (Pseudomonas, Stenotrophomonas), along with its antibacterial effect and the bactericidal activity of LEV [25, 29, 30].

In the current study, there was a 2.6-fold reduction in AZM solution MIC, while a 3.8-fold reduction in AZM niosome MIC, when combined with LEV. Additionally, 32.5% and 27.5% of the isolates were sensitive to LEV when combined with AZM solution and AZM niosomes, respectively. This was in agreement with Gordon et al. [31] who hypothesized that antibiotic combination could help in decreasing the antibiotic dose required for bacterial inhibition. The results of the present study further point to the better in vitro synergistic effect of AZM niosomes/LEV compared to the AZM solution/LEV combination for the treatment of XDR K. pneumoniae infections.

One interesting finding in the current study is that the mean MIC values of both AZM solution and AZM niosomes were significantly lower in the colistin-resistant K. pneumoniae group compared to those in the colistin-sensitive group, when used singly as well as in combination with LEV. This may suggest that the colistin resistance is beneficial to the in vitro activity of unconventional antibiotic. This agreed with the observation of Vidaillac et al. [32] who noticed that colistin resistance is favorable to the in vitro activity of unusual antibiotic combinations.

The MCBT test confirmed that the AZM solution/LEV combination exerted a bactericidal effect in only one isolate (this isolate showed synergy with high azithromycin MIC) while, in the AZM niosome/LEV combination, five isolates exhibited a bactericidal effect. The niosomal formulation of AZM would have enhanced the bactericidal effect of the combination owing to the ability of the niosomes to interact with bacterial cells, fuse, and contact release of the antibiotic inside the cell, overcoming several resistance mechanisms and eventually enhancing the accumulation of the drug inside the cell [23]. The bactericidal activity of AZM against several gram-negative organisms including K. pneumoniae was proved in previous studies [29, 30, 33, 34].

In TKA, synergy with a 2 log10 reduction in the viable bacterial count was confirmed in seven isolates showing synergy with a mean time of 4 h and in 15 isolates with a mean of 2 h, when AZM solution/LEV and AZM niosome/LEV combinations were tested, respectively. The CBA displayed a high sensitivity and specificity in comparison to TKA. This was approved in other studies, which tested the agreement between the CBA and TKA of various combinations against their corresponding organisms and reported a high sensitivity and specificity [35, 36].

It is noteworthy to mention that in TKA, the activity of AZM solution/LEV combination was not bactericidal, while it showed a bactericidal effect with three isolates only after 6 h when using AZM niosome/LEV combination. The discrepancy between the bactericidal results of TKA and MCBT was possibly due to different experimental conditions and different antibiotic concentrations. Similarly, Mezzatesta et al. [37] reported a disagreement between the MCBT and TK assay results in some tested combinations.

The ability of the tested combinations to produce PAE had an important significant role in timing the regimen doses for XDR isolates. The PAE of AZM solution in the current study was on the average of 1.37 ± 0.5 h. The same mean PAE time (2 h) was reported by Debbia et al. [38] for K. pneumoniae isolates. The mean PAE induced by LEV alone in our work was (1 ± 0.5 h) despite that all strains were LEV resistant. This finding may explain the role of LEV in both combinations. When searching in literature, very scarce data are available on the PAE of AZM/AZM niosomes to compare our findings to others.

Nevertheless, there are some limitations in this study. The in vitro activity and results of tested antibiotics and their combinations could not predict the in vivo effects, as AZM antibiotic was proved to have other nonbactericidal auxiliary effects, in addition to its ability to concentrate intracellularly. Thus, AZM solution and AZM niosomes should be tested in vivo to confirm their efficacy against K. pneumoniae and to validate their possible synergistic efficacy with LEV.

Conclusions

AZM niosomal form was proved to be more potent and effective than AZM solution against XDR K. pneumoniae. Although the clinical efficacy of AZM against K. pneumoniae is still controversial, our results suggest that the niosomal formulation might provide a way to overcome the resistance of K. pneumoniae to AZM and give a promising treatment option especially for XDR and even colistin-resistant isolates. Moreover, a combination of AZM niosomes and LEV increased its effect by synergism. It was obvious that AZM niosomal form induced a significantly extended and potent PAE activity, which would affect the dosing and regimen protocol for K. pneumoniae infections.

Authors’ contributions

MMB and HAE were responsible for the idea, study design, and revision of the manuscript. HSB was responsible for the formulation and characterization of niosomes. NSA was responsible for the data collection. HAO was responsible for the laboratory work, data analysis, and drafting of the manuscript. MAM was responsible for the idea, study design, laboratory work supervision, data analysis and interpretation, and writing of the manuscript. All authors contributed to the interpretation of data and approved the final manuscript.

Data availability (data transparency)

Available

Compliance with ethical standards

Ethics approval and consent to participate

Not required

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The author accepts the responsibility for releasing this material on behalf of any and all co-authors. This transfer of publication rights covers the nonexclusive right to reproduce and distribute the article, including reprints, translations, photographic reproductions, microform, electronic form (offline, online), or any other reproductions of similar nature.

Conflict of interest

The authors declare that they have no conflict of interest.

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Footnotes

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References

  • 1.Effah CY, Sun T, Liu S, Wu Y. Klebsiella pneumoniae: an increasing threat to public health. Ann Clin Microbiol Antimicrob. 2020;19(1):1. doi: 10.1186/s12941-019-0343-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Yeh YC, Huang TH, Yang SC, Chen CC, Fang JY. Nano-based drug delivery or targeting to eradicate bacteria for infection mitigation: a review of recent advances. Front Chem. 2020;8:286. doi: 10.3389/fchem.2020.00286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Patra JK, Das G, Fraceto LF, Campos E, Rodriguez-Torres M, Acosta-Torres LS, Diaz-Torres LA, Grillo R, Swamy MK, Sharma S, Habtemariam S, Shin HS. Nano based drug delivery systems: recent developments and future prospects. J Nanobiotechnol. 2018;16(1):71. doi: 10.1186/s12951-018-0392-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Ge X, Wei M, He S, Yuan WE. Advances of non-ionic surfactant vesicles (niosomes) and their application in drug delivery. Pharmaceutics. 2019;11(2):55. doi: 10.3390/pharmaceutics11020055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.The European Committee on Antimicrobial Susceptibility Testing. Breakpoint tables for interpretation of MICs and zone diameters. Version 10.0, 2020. http://www.eucast.org."
  • 6.Tille P (2017) Bailey and Scott’s diagnostic microbiology. 14th. St Louis, Missouri, Mosby Elsevier
  • 7.Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, Paterson DL, Rice LB, Stelling J, Struelens MJ, Vatopoulos A, Weber JT, Monnet DL. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect. 2012;18(3):268–281. doi: 10.1111/j.1469-0691.2011.03570.x. [DOI] [PubMed] [Google Scholar]
  • 8.Barakat HS, Kassem MA, El-Khordagui LK, Khalafallah NM. Vancomycin-eluting niosomes: a new approach to the inhibition of staphylococcal biofilm on abiotic surfaces. AAPS PharmSciTech. 2014;15(5):1263–1274. doi: 10.1208/s12249-014-0141-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Manconi M, Sinico C, Valenti D, Lai F, Fadda AM. Niosomes as carriers for tretinoin. III A study into the in vitro cutaneous delivery of vesicle-incorporated tretinoin. Int J Pharm. 2006;311(1–2):11–19. doi: 10.1016/j.ijpharm.2005.11.045. [DOI] [PubMed] [Google Scholar]
  • 10.Clinical and Laboratory Standards Institute (CLSI) (2012) Methods for dilution antimicrobial susceptibility tests f or bacteria that grow aerobically; Approved Standard—Ninth Edition. CLSI document M07-A9. Wayne: Clinical and Laboratory Standards Institute
  • 11.Clinical and Laboratory Standards Institute (CLSI) (2020) Performance standards for antimicrobial susceptibility testing. 30th ed. CLSI supplement M100. Wayne, Pennsylvania, USA: Clinical and Laboratory Standards Institute
  • 12.Schwalbe R, Steele-Moore L, Goodwin AC. Antimicrobial susceptibility testing protocols. New York: CRC Press; 2007. [Google Scholar]
  • 13.Moody J. Synergism testing: broth microdilution checkerboard and broth macrodilution methods. In: Garcia LS, Isenberg HD, editors. Clinical microbiology procedures handbook, 2nd edition update. Washington, DC: ASM Press; 2007. pp. 5.12.1–5.12.23. [Google Scholar]
  • 14.Aaron SD, Ferris W, Henry DA, Speert DP, Macdonald NE. Multiple combination bactericidal antibiotic testing for patients with cystic fibrosis infected with Burkholderia cepacia. Am J Respir Crit Care Med. 2000;161(4Pt1):1206–1212. doi: 10.1164/ajrccm.161.4.9907147. [DOI] [PubMed] [Google Scholar]
  • 15.Lavigne JP, Bonnet R, Michaux-Charachon S, Jourdan J, Caillon J, Sotto A. Post-antibiotic and post-beta-lactamase inhibitor effects of ceftazidime plus sulbactam on extended-spectrum beta-lactamase-producing Gram-negative bacteria. J Antimicrob Chemother. 2004;53(4):616–619. doi: 10.1093/jac/dkh140. [DOI] [PubMed] [Google Scholar]
  • 16.Chan Y. Biostatistics 102: quantitative data–parametric & non-parametric tests. Blood Press. 2003;140(24.08):79. [PubMed] [Google Scholar]
  • 17.Ma YX, Wang CY, Li YY, Li J, Wan QQ, Chen JH, Tay FR, Niu LN. Considerations and caveats in combating ESKAPE pathogens against nosocomial infections. Adv Sci (Weinh) 2019;7(1):1901872. doi: 10.1002/advs.201901872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Santimaleeworagun W, Thunyaharn S, Juntanawiwat P, Thongnoy N, Harindhanavudhi S, Nakeesathit S, Teschumroon S. The prevalence of colistin-resistant Gram-negative bacteria isolated from hospitalized patients with bacteremia. J Appl Pharm Sci. 2020;10(02):056–059. doi: 10.7324/JAPS.2020.102009. [DOI] [Google Scholar]
  • 19.Giacobbe DR, Del Bono V, Trecarichi EM, De Rosa FG, Giannella M, Bassetti M, Bartoloni A, Losito AR, Corcione S, Bartoletti M, Mantengoli E, Saffioti C, Pagani N, Tedeschi S, Spanu T, Rossolini GM, Marchese A, Ambretti S, Cauda R, Viale P, Viscoli C, Tumbarello M, ISGRI-SITA (Italian Study Group on Resistant Infections of the Società Italiana Terapia Antinfettiva) Risk factors for bloodstream infections due to colistin-resistant KPC-producing klebsiella pneumoniae: results from a multicenter case-control-control study. Clin Microbiol Infect. 2015;21(12):1106.e1–1106.e8. doi: 10.1016/j.cmi.2015.08.001. [DOI] [PubMed] [Google Scholar]
  • 20.Ullah S, Shah MR, Shoaib M, Imran M, Shah SW, Ali I, Ahmed F. Creatinine-based non-phospholipid vesicular carrier for improved oral bioavailability of azithromycin. Drug Dev Ind Pharm. 2017;43(6):1011–1022. doi: 10.1080/03639045.2017.1291667. [DOI] [PubMed] [Google Scholar]
  • 21.Gomes C, Martínez-Puchol S, Palma N, Horna G, Ruiz-Roldán L, Pons MJ, Ruiz J. Macrolide resistance mechanisms in Enterobacteriaceae: focus on azithromycin. Crit Rev Microbiol. 2017;43(1):1–30. doi: 10.3109/1040841X.2015.1136261. [DOI] [PubMed] [Google Scholar]
  • 22.Eleraky NE, Allam A, Hassan SB, Omar MM. Nanomedicine fight against antibacterial resistance: an overview of the recent pharmaceutical innovations. Pharmaceutics. 2020;12(2):142. doi: 10.3390/pharmaceutics12020142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Khan S, Akhtar MU, Khan S, Javed F, Khan AA. Nanoniosome-encapsulated levoflaxicin as an antibacterial agent against Brucella. J Basic Microbiol. 2020;60(3):281–290. doi: 10.1002/jobm.201900454. [DOI] [PubMed] [Google Scholar]
  • 24.Ghafelehbashi R, Akbarzadeh I, Tavakkoli Yaraki M, Lajevardi A, Fatemizadeh M, Heidarpoor Saremi L. Preparation, physicochemical properties, in vitro evaluation and release behavior of cephalexin-loaded niosomes. Int J Pharm. 2019;569:118580. doi: 10.1016/j.ijpharm.2019.118580. [DOI] [PubMed] [Google Scholar]
  • 25.Saini H, Chhibber S, Harjai K. Azithromycin and ciprofloxacin: a possible synergistic combination against Pseudomonas aeruginosa biofilm-associated urinary tract infections. Int J Antimicrob Agents. 2015;45(4):359–367. doi: 10.1016/j.ijantimicag.2014.11.008. [DOI] [PubMed] [Google Scholar]
  • 26.Saiman L, Chen Y, Gabriel PS, Knirsch C. Synergistic activities of macrolide antibiotics against Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans isolated from patients with cystic fibrosis. Antimicrob Agents Chemother. 2002;46(4):1105–1107. doi: 10.1128/aac.46.4.1105-1107.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Kolumbić Lakos A, Skerk V, Maleković G, Dujnić Spoljarević T, Kovacic D, Pasini M, Markotić A, Magri V, Perletti G. A switch therapy protocol with intravenous azithromycin and ciprofloxacin combination for severe, relapsing chronic bacterial prostatitis: a prospective non-comparative pilot study. J Chemother. 2011;23(6):350–353. doi: 10.1179/joc.2011.23.6.350. [DOI] [PubMed] [Google Scholar]
  • 28.Magri V, Montanari E, Škerk V, Markotić A, Marras E, Restelli A, Naber KG, Perletti G. Fluoroquinolone-macrolide combination therapy for chronic bacterial prostatitis: retrospective analysis of pathogen eradication rates, inflammatory findings and sexual dysfunction. Asian J Androl. 2011;13(6):819–827. doi: 10.1038/aja.2011.36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Kumaraswamy M, Lin L, Olson J, Sun CF, Nonejuie P, Corriden R, Döhrmann S, Ali SR, Amaro D, Rohde M, Pogliano J, Sakoulas G, Nizet V. Standard susceptibility testing overlooks potent azithromycin activity and cationic peptide synergy against MDR Stenotrophomonas maltophilia. J Antimicrob Chemother. 2016;71(5):1264–1269. doi: 10.1093/jac/dkv487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Meerwein M, Tarnutzer A, Böni M, Van Bambeke F, Hombach M, Zinkernagel AS. Increased azithromycin susceptibility of multidrug-resistant gram-negative bacteria on RPMI-1640 agar assessed by disk diffusion testing. Antibiotics (Basel) 2020;9(5):E218. doi: 10.3390/antibiotics9050218. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Gordon NC, Png K, Wareham DW. Potent synergy and sustained bactericidal activity of a vancomycin-colistin combination versus multidrug-resistant strains of Acinetobacter baumannii. Antimicrob Agents Chemother. 2010;54(12):5316–5322. doi: 10.1128/AAC.00922-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Vidaillac C, Benichou L, Duval RE. In vitro synergy of colistin combinations against colistin-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae isolates. Antimicrob Agents Chemother. 2012;56(9):4856–4861. doi: 10.1128/AAC.05996-11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ulloa ER, Kousha A, Tsunemoto H, Pogliano J, Licitra C, LiPuma J, Sakoulas G, Nizet V, Kumaraswamy M. Azithromycin exerts bactericidal activity and enhances innate immune mediated killing of MDR Achromobacter xylosoxidans. Infect Microb Dis. 2020;2(1):10–17. doi: 10.1097/IM9.0000000000000014. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Lin L, Nonejuie P, Munguia J, Hollands A, Olson J, Dam Q, Kumaraswamy M, Rivera H, Jr, Corriden R, Rohde M, Hensler ME, Burkart MD, Pogliano J, Sakoulas G, Nizet V. Azithromycin synergizes with cationic antimicrobial peptides to exert bactericidal and therapeutic activity against highly multidrug-resistant gram-negative bacterial pathogens. EBioMedicine. 2015;2(7):690–698. doi: 10.1016/j.ebiom.2015.05.021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Bremmer DN, Bauer KA, Pouch SM, Thomas K, Smith D, Goff DA, Pancholi P, Balada-Llasat JM. Correlation of checkerboard synergy testing with time-kill analysis and clinical outcomes of extensively drug-resistant Acinetobacter baumannii respiratory infections. Antimicrob Agents Chemother. 2016;60(11):6892–6895. doi: 10.1128/AAC.00981-16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Flamm RK, Rhomberg PR, Lindley JM, Sweeney K, Ellis-Grosse EJ, Shortridge D. Evaluation of the bactericidal activity of fosfomycin in combination with selected antimicrobial comparison agents tested against gram-negative bacterial strains by using time-kill curves. Antimicrob Agents Chemother. 2019;63(5):e02549–e02518. doi: 10.1128/AAC.02549-18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Mezzatesta ML, Caio C, Gona F, Zingali T, Salerno I, Stefani S. Colistin increases the cidal activity of antibiotic combinations against multidrug-resistant Klebsiella pneumoniae: an in vitro model comparing multiple combination bactericidal testing at one peak serum concentration and time-kill method. Microb Drug Resist. 2016;22(5):360–363. doi: 10.1089/mdr.2015.0160. [DOI] [PubMed] [Google Scholar]
  • 38.Debbia EA, Molinari G, Paglia P, Schito GC. Post-antibiotic effect of azithromycin on respiratory tract pathogens. Drugs Exp Clin Res. 1990;16(12):615–619. [PubMed] [Google Scholar]

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