Efficacy of ceftiofur N-acyl homoserine lactonase niosome in the treatment of multi-resistant Klebsiella pneumoniae in broilers

In this study, the efficiency of the ceftiofur N-acyl homoserine lactonase niosome against multi-resistant Klebsiella pneumoniae in broilers was evaluated. Fifty-six K. pneumoniae isolates previously recovered from different poultry and environmental samples were screened for the ahlK gene. The lactonase enzyme was extracted from eight quorum-quenching isolates. The niosome was formulated, characterized, and tested for minimal inhibitory concentration (MIC) and cytotoxicity. Fourteen-day-old chicks were assigned to six groups: groups Ӏ and П served as negative and positive controls, receiving saline and K. pneumoniae solutions, respectively. In groups Ш and IV, ceftiofur and niosome were administrated intramuscularly at a dose of 10 mg/kg body weight for five consecutive days, while groups V and VI received the injections following the K. pneumoniae challenge. Signs, mortality, and gross lesions were recorded. Tracheal swabs were collected from groups П, V, and VI for counting K. pneumoniae. Pharmacokinetic parameters were evaluated in four treated groups at nine-time points. The niosome was spherical and 56.5 ± 4.41 nm in size. The viability of Vero cells was unaffected up to 5 × MIC (2.4 gml−1). The niosome-treated challenged group showed mild signs and lesions with lower mortality and colony count than the positive control group. The maximum ceftiofur serum concentrations in treated groups were observed 2 h following administration. The elimination half-life in niosome-treated groups was longer than that reported in ceftiofur-treated groups. This is the first report of the administration of N-acyl homoserine lactonase for the control of multi-resistant K. pneumoniae infections in poultry. Supplementary Information The online version contains supplementary material available at 10.1007/s11259-023-10161-7.


Introduction
Klebsiella pneumoniae is an opportunistic Gram-negative bacterial pathogen that normally inhabits the mucosal surfaces of humans and animals, and is also commonly found in different environmental sources such as water and soil (Struve and Krogfelt 2004;Hosny and Fadel 2021).It causes a wide variety of community-acquired infections in human, including urinary tract infections, pneumonia, septicemia, and liver abscesses (Hayati et al. 2019).K. pneumoniae is also found in livestock and poultry; causing respiratory symptoms, production loss, and even deaths if untreated or immunosuppressed (Hamza et al. 2016;Hayati et al. 2019).K. pneumoniae is one of the most common causes of yolk sac infections in chicks (Khan et al. 2004;Hosny and Fadel 2021).K. pneumoniae have been isolated from chickens suffered from omphalitis, dermatitis, cellulitis, and inflamed respiratory mucosa (Kowalczyk et al 2022).The general isolation rates of the bacteria in poultry are 10% to 35% (Aly et al 2014;Hamza et al. 2016;Hosny and Fadel 2021;Khelfa and Morsy 2015;Zhai et al. 2020).
The expression of virulence factors such as capsular polysaccharides, lipopolysaccharides, adherence factors, siderophore production, and biofilm formation have been reported to be under the regulation of quorum sensing in Klebsiella species (Clegg and Murphy 2017;Santajit et al. 2022).K. pneumoniae has the typical LuxR/LuxI quorum sensing system and the most commonly produced signal molecules is N-acyl-homoserine lactones (AHLs) (Chan 2013).It was reported that the most quantifiable AHLs produced by K. pneumoniae isolated from poultry samples were C4-HSL, C6-HSL, and C12-HSL (Hosny and Fadel 2021).
Beta-lactams are the most commonly used antibiotics for treating infections caused by Enterobacteriaceae in the veterinary sector (Rubin and Pitout 2014).Ceftiofur is a thirdgeneration cephalosporin antibiotic with a wide spectrum of activity against Gram-negative and Gram-positive pathogens, that exerts its bactericidal action by inhibition of the bacterial cell wall synthesis (El-Sayed et al. 2015).It has worldwide approval for respiratory diseases in chicks and turkey poults (Hornish and Katarski 2002).K. pneumoniae resistance to broad-spectrum β-lactams has become an alarming and growing public health challenge based on the recent reports on antimicrobial resistance from the US Centers for Disease Control and Prevention (CDC) and the European Centre for Disease Prevention Control (CDC 2019;ECDC 2020).The increasing incidence of multidrug-resistant bacteria poses a significant challenge for physicians and veterinarians, limiting treatment choices and raising concerns about transmission to humans through the food chain and the emergence of super-resistant bacteria (Li et al. 2022).
Since quorum sensing regulates different biological functions associated with virulence, and as the emergence of multi-drug resistant bacterial strains is on the rise, there is increasing pressure to discover new antimicrobial agents targeting virulence, including bacterial adhesion, toxin function, regulation of virulence expression (D' Angelo et al. 2018).
The AiiA enzyme was the first enzyme identified in a Bacillus species strain and expressed in the agricultural pathogen Erwinia carotovora, causing a reduction in virulence (Reina et al. 2022).Subsequently, many AHL-degrading enzymes, such as AhlD, AiiM, AidC, and MomL, were identified in Arthrobacter, Microbacterium, Chryseobacterium, and Muricauda, respectively (Cai et al. 2018).Many AHL-degrading QQ bacteria, such as Klebsiella, Pseudomonas, Ralstonia, Variovorax, Comamonas, and Agrobacterium genera were isolated from a wide range of aquatic animals and environments (Chan 2013;Chen et al. 2013).In K. pneumoniae, AhlK encodes an AHL-degrading enzyme, and the main two AHLs degraded by AhlK were C6-HSL and 3-oxo-C6-HSL (Park et al. 2003;Chan 2013).Previous studies have displayed that QQ strains can be applied as biocontrol agents in aquaculture and agriculture (Dong et al. 2000;Torres et al. 2016;Zhang et al. 2019;Chen et al. 2020).However, little is known about their potential use as biocontrol agents in the poultry sector.
Although the approach against virulence factors has become a promising strategy for combating different bacterial pathogens, one of its limitations is its inability to completely eradicate the infection (Dehbanipour and Ghalavand 2022).This limitation is overcome by combining antivirulence drugs with antibiotics (Bortolotti et al. 2019;Zhao et al. 2020;Dehbanipour and Ghalavand 2022).Furthermore, traditional drug delivery systems for these therapeutic compounds have significant disadvantages, including a lack of compatibility at the required level, poor biodistribution, disturbed release, and limited accuracy to reach the target sites (Gao et al. 2018).Recently, the development of nanoparticles for drug delivery had a significant impact on the pharmacokinetic profile and therapeutic index of drugs (Gao et al. 2018).Niosomes, defined as non-ionic surfactant-based liposomes, are one of several nanoparticle delivery systems that offer several advantages over other delivery systems through enhancing drug solubility, immune evasion, modulating drug release, and delivering drug molecules to target sites (Kumar and Rajeshwarrao 2011).They are distinguished from other nanoparticles due to their ability to incorporate both hydrophilic and hydrophobic drugs, proteins, enzymes, and genes directly into infection sites (Kumar and Rajeshwarrao 2011;Ag Seleci et al. 2016;Ge et al. 2019).
On this basis, QQ K. pneumoniae isolates previously recovered from different poultry and environmental samples were identified and characterized with the aim of formulating and characterizing the extracted AhlK lactonase enzyme in a ceftiofur N-acyl homoserine lactonase niosome form to be assessed in the control of multi-resistant K. pneumoniae infection in broilers.

Identification of K. pneumoniae isolates
A total of 56 K. pneumoniae isolates were previously recovered from eggs, organs, cloacal swabs, and different environmental niches collected from different markets, farms and backyards with history of enteritis in Giza governorate and summarized as follows: (eight from chicken eggs, four from duck eggs, one from geese egg, four from pigeon eggs, ten from chicken organs, four from duck organs, eight from duck cloacal swabs, two from pigeon cloacal swabs, one from water, two from chicken litter, twelve from duck litter) (Hosny and Fadel 2021).The identification was done as described by (Hamza et al. 2016;Hosny and Fadel 2021) using buffered peptone water (Oxoid Limited, Thermo Fisher Scientific Inc., UK) and Mac-Conkey agar plates (Oxoid Limited, Thermo Fisher Scientific Inc., UK).The plates were examined for mucoid and pink colonies growth.Confirmation of the K. pneumoniae isolates was done as described by (Hansen et al. 2004) using the API20E system (BioMerieux, Marcy l'Etoile, France) present in the Reference Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Dokki, Egypt (RLQP, Egypt).

Screening of K. pneumoniae quorum quenching gene ahlK by conventional PCR
The DNAs of 56 K. pneumoniae isolates were extracted using a QIAGEN kit (Qiagen, Germany, GmbH).The ahlK gene was amplified using forward and reverse primers: KgInF, 5-GCA CTC TGA TCA TAC GGG AGC AAT -3 and KgInR, 5-TGG CCC GGT GAA TGC CCT GGG GTG -3.The amplification was conducted according to Chan (2013) in a Thermal Cycler (Applied biosystem 2720).Each PCR reaction was performed in 25 µL total reaction volume containing 12.5 µL of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µL of each primer with a concentration of 20 pmol, and 5 µL of genomic DNA extract 5.5 µL of DNAse and RNAse free water.The amplification conditions were as follows: initial denaturation at 95 °C for 5 min, followed by 40 cycles for denaturation at 95 °C for 45 s, annealing at 65 °C for 45 s, extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min.The products of PCR were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1 × TBE buffer at room temperature.The expected molecular weight for the electrophorized product is 250 bp.

Sequencing of ahlK gene from K. pneumoniae isolates
The PCR products for the selected six QQ isolates containing ahlK gene recovered from duck were subsequently purified by a QIAquick Gel Extraction Kit (Qiagen, Germany, GmbH).The purified PCR products (30 ng/μl) were sequenced in RLQP, Egypt using a BigDye Terminator V3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) and DNA Analyzer 3500 XL (Applied Biosystems, Tokyo, Japan).The alignment of the sequences was performed using BIOEDIT software.The Basic Local Alignment Search Tool (BLAST®) was used to compare the results of the ahlK gene sequencing to the gene bank database (NCBI) to establish identification at the isolate level.

Extraction of N-acyl homoserine lactonase
The enzyme extract was prepared as described by (See-Too et al. 2017;Hosny and Fadel 2021).Briefly, eight QQ K. pneumoniae isolates containing ahlK gene were grown overnight in Luria broth agar plates (LB) (Oxoid Limited, Thermo Fisher Scientific Inc., UK) buffered with 50 mM 3-(N-morpholino) propane sulfonic acid (MOPS) (pH 6.5) (Santa Cruz Biotechnology, Inc, Canada).The isolates were ultracentrifuged and the supernatant was extracted using acidified ethyl acetate (0.1%, v/v glacial acetic acid) (Fischer Scientific, Leicestershire, UK).The residues were dissolved in deionized water and used for biosensor and chromatographic assays.

Reverse phase high-performance liquid chromatography
Chemicals and reagents Acetonitrile (ACN) and methanol (MeOH) were obtained from Fischer Scientific (Leicestershire, UK).Ethylenediaminetetraacetic acid (EDTA) was purchased from Merck (Darmstadt, Germany).De-ionized water of HPLC grade was obtained using a Milli-Q system (Waters Corp., Milford, MA, USA).

Preparation of AHL standards
The AHL standards stock solutions were prepared as described by (Hosny and Fadel 2021) by dissolving AHL standards in acetonitrile to obtain a concentration of 1mgml −1 .The calibration standard concentrations were diluted from stock solution at a range from 5 to 50 µgml −1 .
Chromatographic assay Analysis was determined as described by Hosny and Fadel (2021) using an Agilent 1200 series (Agilent Co., Santa Clara, CA, USA) equipped with a quaternary pump, vacuum degasser autosampler injector, and ultraviolet detector.To analyze AHL-degradation activity, 10µgml −1 of C6-HSL and 3-oxo-C6-HSL were incubated with the extract at 20 °C in buffered LB and aqueous media (0.07%NaCl) with MOPS (pH 6.5) for 0 and 24 h.The C6-HSL and 3-oxo-C6-HSL in buffered LB and aqueous media were used as negative controls.HPLC analysis was performed using optimized chromatographic parameters outlined in Table 1 to obtain high separation efficiency.

Validation of the HPLC method
The validation was performed according to (ICHQ2(R1) 2005; USP 2021) guidelines for different parameters such as linearity, range, recovery, accuracy, detection limit (DL), and quantification limit (QL).

Thermal and pH stability of N-acyl homoserine lactonase extract
The stability of the N-acyl homoserine lactonase against different thermal and pH conditions was assessed according to Sakr et al. (2013) with some modifications in the thermal temperatures and pH.Briefly, 1 mL of the enzyme extract was incubated in the water bath at 40, 60, and 90 ∘ C for 60 min.The pH of the enzyme extract (1 mL) was adjusted at 4, 6, 8, and10 using a pH meter (Jenway, UK); the buffers used were 1 mol HCl (AppliChem, Darmstadt, Germany) and 1 mol NaOH (AppliChem).After heat and pH treatments, the quenching activity of the enzyme extracts was assayed against A. tumefaciens NTL4(pZLR4) biosensor strain using a well diffusion assay as previously described.Following 48 h of incubation, the plates were observed for the inhibition zones of pigment production.

Chemicals and reagents
Ceftiofur sodium (Naxcel ® ) was obtained from Zoetis, USA in a form of a water-soluble powder, where each 1 mL contains 50 mL of ceftiofur base.Liposome-base (phospholipid) was purchased from Chemajet Chemical Company, Alexandria, Egypt.Tween 20 and phosphate-buffered saline (PBS) were obtained from (Sigma-Aldrich, St. Louis, USA) and de-ionized water.
Ceftiofur N-acyl homoserine lactonase niosome was prepared according to (Kazi et al. 2010;Rasti et al. 2012) in the Nanomaterial Research and Synthesis unit, AHRI, Giza, Egypt.Briefly, 0.0129 g of ceftiofur N-acyl homoserine lactonase (75% v/25%v) and (50% v/50%v) were solubilized in 10 ml of PBS solution as an aqueous phase.The solution was mixed with 10 ml of liposome suspension, 20 ml of tween 20, and 60 ml of deionized water.The mixture was subjected to sonication using a homogenous blender of 1000 watts at 25 °C for 10 min.The deionized water was slowly added to the oilphase mixture, followed by sonication for 30 min.

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
for ceftiofur, N-acyl homoserine lactonase, and ceftiofur N-acyl homoserine lactonase niosome using broth macro-dilution method The broth macro-dilution technique was used to determine the MIC and MBC for ceftiofur, N-acyl homoserine lactonase, and ceftiofur N-acyl homoserine lactonase niosome (75% v/25%v) and (50% v/50%v) following Clinical and Laboratory Standards Institute guidelines (CLSIM07-A9 2012; CLSI 2017).Twofold serial dilutions of the tested solutions (1mgml −1 ) in distilled water were prepared.The selected range to define the K. pneumoniae organism as resistant (resistance breakpoint ≥ 8) according to (CLSIVET01S 2015).The medium used in the macro-dilution testing is Mueller-Hinton broth (MHB) (Oxoid Limited, Thermo Fisher Scientific Inc., UK).The K. pneumoniae isolate (KP19) used was previously isolated from a chicken organ that was an AHL producer (C4, C6, C8, and C12), resistant to cefotaxime, cefaclor, cefoxitin, tetracycline, gentamycin, and amoxicillin-clavulanic acid (Hosny and Fadel 2021).Furthermore, the KP19 isolate was ESBL and AmpC producer as it produced positive amplification for blaTEM, blaSHV, and AmpC genes (data not shown).For bacterial inoculum preparation, pure mucoid colonies cultured overnight on MacConkey medium were suspended in MHB medium and adjusted to 5 × 10 5 CFUml −1 .The bacterial inoculum (0.1 ml) was added to 2 ml of a liquid MHB medium containing ceftiofur, N-acyl homoserine lactonase, and ceftiofur N-acyl homoserine lactonase niosome in the dilution series.The tubes were then incubated for 16 to 20 h at 35 ± 2 °C.The last two tubes contained positive control of K. pneumoniae bacteria and negative control of ceftiofur.The MIC was considered the lowest concentration of the tested solutions that produced no visible growth (no turbidity recorded).The MBC value was determined after subculturing of each tested dilution which showed no turbidity on to plate count agar media (Oxoid Limited, Thermo Fisher Scientific Inc., UK) for 16 to 20 h at 35 ± 2 °C.MBC was defined as the lowest concentration of the tested solutions that produced no bacterial growth on the agar plates.

Determination of synergy between the ceftiofur and N-acyl homoserine lactonase using checkerboard broth micro-dilution method
The fractional inhibitory concentration (FIC) of combined ceftiofur and N-acyl homoserine lactonase was determined by a checkerboard broth micro-dilution method as described by (Fadel et al. 2022).The fractional inhibitory concentration (FIC) was calculated as follows: FIC of N-acyl homoserine lactonase = MIC of N-acyl homoserine lactonase in combination with ceftiofur / MIC of N-acyl homoserine lactonase alone, FIC of ceftiofur = MIC of ceftiofur in combination with N-acyl homoserine lactonase / MIC of ceftiofur alone and FIC index (FICI) = FIC of N-acyl homoserine lactonase + FIC of ceftiofur.Synergism is defined as FICI < 0.5, additive effect as FICI 0.5-1, indifference as FICI = 1-4, and antagonism as FICI > 4.

Cytotoxicity of the ceftiofur N-acyl homoserine lactonase niosome
The cytotoxicity of the niosome (50% v/50%v) was evaluated according to (Vichai and Kirtikara 2006;Borin et al. 2018) in African green monkey kidney cells (Vero cell) using Sulforhodamine B assay (SRB).Briefly, 100 μL of the cell suspension (5 × 10 4 cells ml −1 ) was added in 96-well plates (Corning Incorporated -Life Sciences, NY, United States) and incubated for 24 h.Cells were then treated with 100 μL of the niosome solution at various concentrations (0.01, 0.1, 1,10, 100 µgml −1 ) in triplicates.After 72 h, plates were washed twice with 1X PBS and the cells were fixed by 150 μL of 10% Trichloroacetic acid (TCA) (Fischer Scientific, Leicestershire, UK) and incubated at 4 °C for 1 h.The TCA solution was removed, and the cells were washed 5 times with distilled water.After that, 70 μL of SRB solution (0.4% w/v) was added and incubated in a dark place at room temperature for 10 min.Plates were then washed 3 times with 1% acetic acid and allowed to air-dry overnight.Finally, 150 μL of Tris-EDTA buffer (10 mM) was added to dissolve the protein-bound SRB stain and the absorbance was measured at 540 nm using a BMG FLUOstar Omega microplate reader (BMG LABTECH, Ortenberg, Germany).Unexposed cells were used as negative controls.The absorbance values were represented as mean ± standard deviation (SD).The cell viability was calculated according to the following equation (Vatan 2022): Cell viability % = 1-(A experimental /A control ) × 100, where A experimental is the absorbance of the cells exposed to the niosome, and A control is the absorbance of the negative control.The half-maximal inhibitory concentration (IC 50 ) was obtained from the fitting of the dose-response curve as described by Ihling et al. (2022).

Efficacy of ceftiofur N-acyl homoserine lactonase niosome in the treatment of multi-resistant K. pneumoniae in broilers
Materials The present study was executed using ceftiofur sodium Naxcel® (Zoetis, USA The calibration standard concentrations were diluted from stock solution at a range from 0.05 to 10 µgml −1 .The quality control samples were prepared at three levels in blank chicken serum to be 0.1, 1, and 10 µgml −1 .Isooctane was obtained from Fischer Scientific (Leicestershire, UK). 5% Trichloroacetic acid (TCA) was obtained from Fischer Scientific (Leicestershire, UK).Spectrophotometric grade trifluoroacetic acid (TFA) ≥ 99.9% and ammonium acetate buffer 0.05 M; pH5 were purchased from Sigma-Aldrich (St. Louis, MO, USA).Acetonitrile (ACN), methanol (MeOH), trichloroacetic acid (TCA), and de-ionized water of HPLC grade.

Bacterial inoculum
The K. pneumoniae strain (KP19) used in the experimental study was the same one used in the determination of MIC and MBC (Hosny and Fadel 2021).
Experimental birds and facilities A total of 180 commercial 14-day-old Ross broilers were purchased from a commercial hatchery in Behera.Birds were housed in battery cages in a biosafety level Π experimental room in the RLQP, Egypt, which has temperature, lighting, and ventilation controls according to Ross Broiler management specifications (Aviagen 2018).Birds were provided with ad-libitum water and a grower diet according to Ross 308 broiler Nutrition specifications guidelines (Aviagen 2014).Tracheal and environmental swabs were taken from chicks (extra than 180), experimental room, and cages before the housing of chicks and examined for K. pneumoniae infection.

Experimental design
The experimental design of this study was conducted according to (El-Sayed et al. 2015;Tantawy et al. 2018) with a modification in the age of birds.Chickens were divided into six groups of three replicates (10 birds /group): group Ӏ was received 1 ml of saline solution and assigned as a negative control, group П served as a positive control group that was challenged orally with 1 ml of K. pneumoniae isolate (KP19) containing 10 9 CFU/ml on day 15.Groups Ш and IV were administrated with ceftiofur and niosome (10 mg/kg BW, I.M), respectively on days 16 th to 20 th .Groups V and VI were challenged orally with K. pneumoniae culture containing 10 9 CFU/ml on day 15 followed by administration of ceftiofur and niosome, respectively (10 mg/kg BW, I.M) on days 16 th to 20 th .
Samples Clinical signs and mortality were recorded twice daily (09:00 a.m. and 17:00 p.m.).Tracheal swabs were collected from groups П, V, and VI on days 16-21 for the counting of K. pneumoniae (Table S1).Blood samples were collected from the wing veins of five birds from groups Ш and IV, V, and VI at nine-time points (0.16, 0.5, 1, 2, 4, 8, 10, 12 and 24 h) following administration of ceftiofur and noisome on day 16 for studying of pharmacokinetic (PK) parameters (Table S1).Blood samples were subsequently centrifuged for 10 min at 2000 rpm and stored at − 20 °C until assayed.Birds were euthanized on day 21 and samples were taken from lung, liver, spleen, and intestine to examine gross lesions (Table S1).

Quantification of ceftiofur in serum samples
The concentration of ceftiofur in serum samples was determined by the HPLC method using an Agilent 1200 series (Agilent Co., Santa Clara, CA, USA) at AHRI, Giza, Egypt as described by Abd Elhafeez and Fadel (2021).Ceftiofur was extracted as described by Abd Elhafeez and Fadel (2021).Briefly, 200 µL of quality control and serum samples were mixed with 100 µL of 5% TCA.The mixtures were centrifuged for 10 min at 4000 × g and the clear aqueous phase was transferred into HPLC vials.The chromatographic conditions were outlined in Table 1, and validation was performed according to (ICHQ2(R1) 2005; USP 2021) guidelines.

Assessment of PK parameters
Mean serum levels of ceftiofur versus the time course were investigated using a compartmental approach (El-Sayed et al. 2015).The pharmacokinetic parameters, including maximum serum drug concentration (Cmax), absorption half-life t 1/2 ka , elimination half-life (t1/2), apparent volume of distribution (V/F), apparent total clearance of the drug from serum CL/F, time to reach maximum serum concentration (T max ), area under the concentration-time curve from 0 to 24 h (AUC 0−24 ), mean residence time (MRT) were calculated according to Zhang et al. (2010) using pk solver an add-in program for Microsoft Excel, version 2.

Statistical analysis
Statistical analysis was carried out using IBM SPSS Statistics (version.21.0.Armonk, NY: IBM Corp.).Kolmogorov-Smirnov and Shapiro-Wilk tests were used to check the normality of the data of bacterial count and pharmacokinetic parameters.A Kruskal-Wallis's test was used to determine the differences in the bacterial count between ceftiofur and niosome treated challenged groups and the positive control group.A Mann-Whitney test was used to determine the difference between the ceftiofur and niosome treated challenged groups.An independent sample t-test was used to examine the significance of differences in the pharmacokinetic parameters of ceftiofur in four treated groups.The P-values were considered significant at values ≤ 0.05.

DNA sequencing and alignment of ahlK gene
The comparison of the ahlK sequences in the present study with other known AHL lactonase nucleotide data available in the GenBank database for ahlK revealed high similarity (up to 100%).

Pairwise identity matrix of nucleotide and amino acid sequences
The pairwise identity matrix revealed a high degree of similarity among the six K. pneumoniae isolates (95% to 100%) to the amino acid data available in the GenBank database for ahlK group.These isolates were very similar to K. pneumoniae strain USA (accession number CP067563.1),K. pneumoniae strain China (accession number CP045193.1),and K. pneumoniae strain United Kingdom (accession number CP057459.1)(Fig. S1).

Screening of AHL-quenching activity of N-acyl homoserine lactonase extract using A. tumefaciens NTL4 biosensor
All eight K. pneumoniae isolates (2 from duck cloacal swabs, 5 from duck litters, and 1 from water) were able to degrade C6-HSL and 3-oxo-C6-HSl producing inhibition zones with mean diameter sizes of 31 and 25, respectively; suppressing the activation of the indicator strain.

Screening of AHL-quenching activity of N-acyl homoserine lactonase extract using reverse phase high-performance liquid chromatography
The method was accurate with high recovery for C6-HSL and 3-oxo-C6-HSL standards in a percentage of 93% and 111%, respectively.The standards displayed good linearity (˃ 0.99) with a low detection limit (LOD) and quantification limit (LOQ) as 1.9 and 1.4 μg/ml, respectively for LOD and 5.5 and 4.4 μg/L, respectively for LOQ.Specificity and selectivity of standards revealed retention times of 5.9 and 10.17 min, respectively (Fig. 1).It revealed the complete disappearance of two AHL chromatograms (C6-HSL and 3-oxo-C6-HSL) following incubation of standards with lactonase extract in aqueous medium at 0 and 24 h and in LB medium at 24 h (Fig. 2a,  b).Partial degradation of two AHL chromatograms (C6-HSL and 3-oxo-C6-HSL) was obtained following incubation of standards with lactonase extract in LB medium at zero hours (Fig. 2c).These findings suggested that ahlK lactonase has quenching activity towards C6-HSLs and 3-oxo-C6-HSLs.

Thermal and pH stability of N-acyl homoserine lactonase
The enzyme retained its activity when incubated at 40 ∘ C and 60 ∘ C for 60 min evidenced by the presence of inhibition zones against C6-HSL, and 3-oxo-C6-HSl with mean diameter sizes of 30 and 24, respectively.Furthermore, it maintained its activity at pH 6, 8, and 10 evidenced by the presence of inhibition zones against C6-HSL and 3-oxo-C6-HSl with mean diameter sizes of 31 and 25, respectively at pH 6 and 28, and 23, respectively at pH 8, 25, and 21, respectively at pH 10.However, the enzyme activity was lost completely when incubated at 90 ∘ C for 60 min and at pH 4 evidenced by the absence of inhibition zones.

Determination of synergy between the ceftiofur and N-acyl homoserine lactonase using checkerboard broth micro-dilution method
The combination of ceftiofur and N-acyl homoserine lactonase has a synergistic effect on the tested K. pneumoniae strain, as the FIC index of ceftiofur / N-acyl homoserine lactonase was 0.042.

Efficacy of ceftiofur N-acyl homoserine lactonase niosome in the treatment of multi-resistant K. pneumoniae in broilers
Clinical signs and mortality Groups Ӏ, Ш, and IV showed no clinical signs, while moderate to severe signs such as diarrhea, respiratory signs, ruffling feathers, and progressive Fig. 2 a, b, and c.HPLC chromatograms of C6-HSL and 3-oxo-C6-HSL degraded by ahlK lactonase activity.A, B) Complete degradation of C6-HSL and 3-oxo-C6-HSL chromatograms in A) an aqueous medium after incubation for 0 and 24 h and B) in LB medium after incubation for 24 h.C) Partial degradation of C6-HSL and 3-oxo-C6-HSL chromatograms in LB medium after incubation for zero hours weakness were recorded in groups V and П.On the other hand, group VI showed mild respiratory signs and diarrhea (Table 3).
Group П showed 10% mortality on the fourth day postinfection compared to the other groups (Ӏ, Ш, IV, V, and VI) that revealed no evidence of mortalities (Table 3).

Gross lesions
No gross lesions were recorded in groups Ӏ, Ш, and IV.Groups (П and V) showed noticeable severe lesions, such as polyserositis in their internal organs, enteritis, and congestion of the lung, liver, and spleen.In contrast, mild lesions of polyserositis, enteritis, and congestion in the lung, liver, and spleen were recorded in 20-30% of chickens in group VI (Table 4).

Enumeration of K. pneumoniae
There were significant differences in the K. pneumoniae counts in the tracheal swab samples between the positive control group (П), niosome and ceftiofur treated challenged groups (VI and V), by a Kruskal-Walli's test (P = 0 000).The niosome and ceftiofur treated challenged groups (VI and V) showed a decrease in the count of K. pneumoniae in the tracheal swabs by 3.39 and 0.15 log 10 CFU, respectively compared to the positive control groups (II) that showed an increase in the count by 1.92 log 10 CFU (Table 5).There were significant differences in the K. pneumoniae counts between ceftiofur and niosome treated challenged groups (V and VI) by the Mann-Whitney test (P = 0 000).

Quantification of ceftiofur in serum samples
The method was accurate with a high recovery of (93-111%) and good linearity (correlation coefficient ˃ 0.99).The detection limit and quantification limit of the method for serum were 0.01 and 0.03 μgml −1 , respectively.Ceftiofur was detected as a Fig. 3 Characterization of ceftiofur N-acyl homoserine lactonase niosome using Transmission Electron Microscope.TEM analysis revealed that the niosome had spherical shape with an average size of 56.5 ± 4.41 nm single sharp peak after 7.004 min and showed no interference in the matrix (Fig. 4).

Pharmacokinetics results
The pharmacokinetic parameters of ceftiofur administrated IN groups Ш, IV, V, and VI (10 mg/kg BW I.M) in were outlined in Table 6.There was a significant increase in the absorption half-life (t 1/2 ka ), elimination half-life (t 1/2Beta ), maximum serum concentration (C max ), area under curve (AUC), and mean residence time (MRT) in the niosome-treated groups (IV and VI) compared with ceftiofur treated groups (Ш and V) (p < 0.05).In contrast; the total clearance of ceftiofur (Cl/F) was significantly lower in the niosome-treated groups (IV and VI) than ceftiofur-treated groups (Ш and V) (p < 0.05).Ceftiofur was quantifiable in the serum in groups Ш, IV, V, and VI at different time points after single intramuscular administration of 10 mg/kg BW (Fig. 5).

Discussion
Antimicrobial-resistant infections pose a major threat to animal and human health as a result of antibiotic abuse or overuse, causing high mortalities (Serwecińska 2020).The emergence of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae over the past few years in poultry raises critical concerns for therapies against multidrug-resistant infections (Li et al. 2022).The risk of zoonotic transmission of MDR K. pneumoniae from poultry to humans through direct contact or food chain is still unknown.Some investigations have reported the dissemination of ESBL-producing Escherichia coli from poultry to farm workers (Huijbers et al. 2014;Leversteinvan Hall et al. 2011;Tansawai et al. 2019).Antivirulence drugs have been identified as a promising option for controlling antibiotic-resistant bacteria through targeting  -1.92 ± 0.01 b 0.15 ± 0.00 a 3.39 ± 0.01 c 0.000 Fig. 4 HPLC chromatogram of ceftiofur standard at a concentration of 5 µg/ml.Ceftiofur standard expressed a single sharp peak with a retention time of 7.004 min Table 6 Kinetics of ceftiofur after single intramuscular injection of 10mgKg −1 in treated non-challenged groups (Ш and IV) and treated challenged groups (V, and VI) All values are expressed as mean ± SD, *Significant change at p < 0.05 with respect to Group Ш using t-test, ** Significant change at p < 0.05 with respect to Group V using t-test, t 1/2 ka : absorption half-life , t 1/2Beta : elimination half-life, V/F: apparent volume of distribution, CL/F: apparent total clearance of the drug from serum, T max : time to reach maximum serum concentration , C max : maximum serum drug concentration, AUC 0-24 : area under the serum concentration-time curve from time zero to time 24 hs, MRT: mean residence time Kinetic Parameters Group Ш (Non-challenged treated with ceftiofur) Group IV (Non-challenged treated with niosome) Group V (Ceftiofur-treated group after challenge with K. pneumoniae) Group VI (Niosome-treated group after challenge with K. pneumoniae) t 1/2 ka (h) 0.7 ± 0.9 0.98 ± 0.8* 0.59 ± 0.6 1.1 ± 0.9** t 1/2Beta (h) 5.5 ± 0.15 6.8 ± 0.7* 4.9 ± 0.5 5.7 ± 0.7** V/F (mg) (µg/ml) 0.22 ± 0.12 0.3 ± 1.2* 0.28 ± 0.7 0.22 ± 0.3 CL/F(mg)(µg/ml)/hr 0.04 ± 0.54 0.03 ± 0.9* 0.09 ± 0.8 0.04 ± 0.8** T max (h) 2.3 ± 0.7 2.4 ± 0.6 2.2 ± 0.6 2.5 ± 0.7 C max (μg/ml) 24.3 ± 0.5 26.3 ± 0.8* 23.7 ± 0.8 25.4 ± 0.4** AUC 0-24 (μg h/ml) 247.9 ± 0.7 254.7 ± 0.9* 228.02 ± 0.7 236.8 ± 0.9** MRT (h) 8.9 ± 0.6 10.2 ± 0.8* 8.2 ± 0.8 9.8 ± 0.7** virulence functions and behaviors rather than pathogen viability (Tang and Zhang 2014).Many macromolecular QQ agents have been discovered, but none of them have been commercialized.The greatest challenge for the commercialization of antivirulence drugs is technological and economic, since the purification of bioactive compounds is necessary for their use on a commercial scale, which is expensive to produce in enough quantities for the drug design and delivery industry (Clatworthy et al. 2007;Tang and Zhang 2014;D'Angelo et al. 2018).Furthermore, current limitations on the administration of macromolecular QQ agents, such as their narrow spectrum and low bioavailability have prompted the search for new formulation options (Park et al. 2011;Tang and Zhang 2014).In this study, the QQ lactonase enzyme AhlK, produced by eight K. pneumoniae isolates, was formulated with ceftiofur in a niosomal form to determine its efficacy in the treatment of multi-beta lactam-resistant K. pneumoniae infection in broilers.Ceftiofur sodium was selected to be used as it is the most commercially available formulation of ceftiofur tested in avian species which permits once-daily treatment in mammals.Eight QQ K. pneumoniae isolates containing ahlK gene showing resistance to beta-lactam antibiotics have been recovered.According to Kusada et al. (2019), this phenotypic bi-functionality was reported in bacteria inhabiting natural ecosystems, including P. aeruginosa PAO1, Acidovorax species MR-S7, Sphingomonas species S1, Acidovorax species M2, Acidovorax species M6, Diaphorobacter species S2, Stenotrophomonas species S17, and Mycobacterium species M1.To date, there are no published reports on the isolation of AHL-degrading K. pneumoniae from poultry and environmental samples.The percentage of QQ isolates recovered from environmental samples in this study was 75% (6 of 8) higher than reported in previous studies of other species in soil, where percentages ranged from 2% to 4.8% (Dong et al. 2000(Dong et al. , 2002;;D'Angelo-Picard et al. 2005).Two QQ K. pneumoniae isolates were recovered from cloacal swabs collected from ducks; these bacteria may have been driven from the gastrointestinal tract, which could reflect the ability of bacteria to enter and colonize this ecosystem (Ghanei-Motlagh et al. 2019).
A blast search for the ahlK gene for six K. pneumoniae isolates showed high nucleotide sequence similarity (up to 99%) with other ahlK genes existing in the NCBI database.The selection of the six isolates was based on the high intensity of bands obtained in the PCR reaction.Similarly, a high genetic similarity, greater than 90%, was observed in AiiA lactonase recovered from the Bacillus genus (Sakr et al. 2013).
The combined AHLs synthesis and degradation were reported in six K. pneumoniae isolates in this study, which suggests that bacteria can control their own AHL production and repression depending on the growth phase (Chan et al. 2011).These results support previous studies that revealed the co-existence of AHL synthesis and degradation in Agrobacterium tumefaciens, marine Shewanella, and ginger rhizosphere Burkholderia GG4 strains (Zhang et al. 2002(Zhang et al. , 2004;;Tait et al. 2009;Chan et al. 2011).
The QQ activity of the extracted lactonase enzyme was assessed against different synthetic AHLs using biosensor and HPLC assays.The results revealed that the selected strains can degrade C6-HSL and 3-oxo-C6-HSl, which supports earlier studies that displayed C6-HSL and 3-oxo-C6-HSL degradation by the AhlK lactonase enzyme produced by K. pneumoniae (Park et al. 2003;Chan 2013).The use of MOPS solution (pH 6.5) in the extraction of lactonase enzyme and during HPLC analysis was to ensure that there was no change in the pH values with time to avoid recyclization of the opening lactone ring (See-Too et al. 2017).It was observed complete degradation of C6-HSL and 3-oxo-C6-HSL in buffered aqueous media containing 0.07% NaCl compared to buffered LB medium containing 10 gl −1 of NaCl.This might be attributed to the higher solubility of sodium chloride in the aqueous medium than in the LB medium and the interference of yeast extract present in the LB medium with AhlK lactonase quenching activity (Dor et al. 2021).These findings suggested that AhlK lactonase might be halophilic and reflected the increased salt content of the collected litter samples, which might be related to the repeated reuse and composting of litter on the farms (Wang et al. 2016).According to Liu et al. (2019), AhlX was the first halophilic AHL lactonase isolated from the marine Salinicola salaria MCCC1A01339 that could tolerate 25% NaCl.
The stability of the AhlK lactonase enzyme was further assessed against different thermal and pH conditions.The lactonase enzyme showed high thermal stability; it retained 96.7% of its activity when incubated at 40 ∘ C and 60 ∘ C for 60 min but lost its activity when incubated at 90 ∘ C for 60 min.This is consistent with previous findings of Sakr et al. (2013), who reported that the lactonase enzyme from B. weihenstephanensis retained > 90% of its activity when incubated at 50 °C for 60 min and lost its activity when the enzyme was incubated at 90 °C for 60 min.On the other hand, Wang et al. (2004) reported the inactivation of recombinant lactonase enzyme AiiA 240 isolated from Bacillus species at 45 ∘ C. Cao et al. (2012) reported that lactonase enzyme Bacillus sp.Strain AiiA AI96 retained about 60% of its activity after incubation for 3 min at 90 ∘ C. Furthermore, the activity of the AhlK lactonase enzyme remained unchanged at pH 6, retained > 90% and 80%, of its activity at pH 8 and 10, respectively, and completely lost its activity at pH 4. Similarly, previous studies have displayed high stability of AHL lactonase enzymes over a pH range of 6-9 and completely lost their activity at acidic pH (Wang et al. 2004;Cao et al. 2012;Sakr et al. 2013).
Two nano-liposomal formulations were evaluated to select the most suitable one, enabling efficient quenching activity and delivery of ceftiofur N-acyl homoserine lactonase.The results revealed that niosome (50% v/50%v) had antibacterial properties against the tested K. pneumoniae KP19 isolate with a MIC value 13-fold lower than obtained using ceftiofur alone.Furthermore, there were synergistic interactions between ceftiofur and the AhlK lactonase enzyme, as indicated by the FIC index.It was reported that beta-lactam antibiotics were one of the most antimicrobial groups associated with a positive interaction potential (Haroun and Al-Kayali 2016).Bortolotti et al. (2019) reported that the combination of LasR quorum sensing inhibitors with ciprofloxacin could reduce the formation of biofilm and the antibiotic tolerance of P. aeruginosa.According to (Vinoj et al. 2015), the AiiA lactonase loaded on gold nanoparticles has a promising antibiofilm activity against multi-drug resistant Proteus species.Gupta and Chhibber (2019) reported that the AiiA lactonase loaded on silver nanoparticles has a significant effect on the reduction in exopolysaccharide production, cell surface hydrophobicity, and metabolic activity, and has promising anti-biofilm activity against multidrug-resistant K. pneumoniae.Tween 20 was selected to be used as a non-ionic surfactant in the niosome formulation due to its higher stability, compatibility, solubility, and lower toxicity compared to anionic and cationic counterparts (Kumar and Rajeshwarrao 2011).Sonication was used during niosome preparation to obtain vesicles with homogenous size distribution and no aggregation (Hallaj-Nezhadi and Hassan 2015); this allowed the mean diameters of niosome to be reduced from 528 nm to 20.54 ± 1.28 nm.
The niosome (50% v/50%v) was further characterized using TEM and zetasizer.According to Ag Seleci et al. (2016), niosomes were divided into three types based on their sizes and bilayer composition: small unilamellar vesicles, which range in size from 10 to 100 nm, large unilamellar vesicles which range in size from (100-3000 nm), and multilamellar vesicles, which include several bilayers.In this study, TEM analysis revealed that the prepared niosome was a spherical small unilamellar vesicle with no aggregates.Previous studies displayed that AiiA Lactonases loaded on gold and silver nanoparticles were spherical and their sizes ranged from 10-30 nm (Vinoj et al. 2015;Gupta and Chhibber 2019).Zeta potential is an important parameter that can influence the stability, pharmacokinetics, bio-distribution, and toxicity of niosomes (Ge et al. 2019).In this study, the niosome had a high negative charge, which leads to an increase in its stability for longer periods and makes it less likely to aggregate (Ge et al. 2019).
The cytotoxicity of niosome (50% v/50%v) was investigated in Vero cell lines.The results revealed the absence of cytotoxic effects at concentrations of 0.01, 0.1, 1, and 10 μgmL −1 but the cell viability was affected at 100 μgmL −1 .This is consistent with a previous study by Vinoj et al. (2015) who reported that the treatment of a mouse macrophage cell line with AHL lactonase coated on gold nanoparticles isolated from Bacillus licheniformis (AiiA AuNPs) at concentrations of 2 to 8 M did not have a cytotoxic effect but still had an impact on cell viability at concentrations higher than 8 M. According to Gupta and Chhibber (2019), AHL Lactonase coated on silver nanoparticles (AiiA AgNPs) isolated from Bacillus sp.ZA12 did not have a cytotoxic effect on mouse macrophage cells at concentrations of 1.5, 3, and 6 µg/mL but still had a great effect on cell viability at concentrations of 50 gmL −1 , 75 gmL −1 , and 100 gmL −1 .
The potential use of ceftiofur N-acyl homoserine lactonase niosome in treating multi-resistant K. pneumoniae in broilers was evaluated.The selection of the intramuscular route for the delivery of the formulated niosome was based on the results of previous studies, which reported that ceftiofur is poorly absorbed after oral administration and the delivery of proteins after oral administration is hindered by proteolytic enzymes, pH gradients, and the epithelial barrier (Deghmane et al. 2016;Haddadzadegan et al. 2022).Cao et al. (2012) reported that N-acyl homoserine lactonase AiiAB546 was unable to protect zebrafish against Aeromonas hydrophila infection by oral administration.The present results reported the effectiveness of ceftiofur N-acyl homoserine lactonase niosome in controlling mortalities in the niosome-treated birds in groups (IV and VI).This result is consistent with previous studies that revealed the ability of recombinant N-acyl homoserine lactonase Ahl-1and the combination of lactonase with ciprofloxacin to reduce mortality in murine models (Gupta et al. 2015;Sakr et al. 2021).
Furthermore, it was found that the largest reductions in the K. pneumoniae count in tracheal swab samples were in group VI on day 21 of the study period following the administration of ceftiofur N-acyl homoserine lactonase niosome for five consecutive days.According to Sakr et al. (2021) employing a mouse model, topically applied recombinant N-acyl homoserine lactonase Ahl-1 reduced P. aeruginosa count in blood, lung, and liver by 4 logs after three days of treatment.Gupta et al. (2015) reported that topical application of the combination of lactonase and ciprofloxacin in a murine model revealed the reduction of the P. aeruginosa load in blood, lung, and liver by a range of 1.5 to 2.4 logs after three days of treatment.
Certain pharmacokinetic parameters were studied for establishing a rational dosage schedule that achieves the desired therapeutic outcome.The serum concentration of ceftiofur can be quantified by measuring desfuroylceftiofur, the active metabolite of ceftiofur, which is produced by cleaving the thioester bond (CVMP 1999).Ceftiofur's antimicrobial properties are time-dependent, meaning the bactericidal activity is determined by the duration of exposure to the drug over MIC (Hooper et al. 2016).Depending on this fact, the detected serum ceftiofur concentration in the non-challenged group Ш was less than MIC (16 µg/ml) at 8 h following administration of ceftiofur at a dose of 10 mg/ kg BW I.M in broilers.These findings suggest the mandatory repetition of ceftiofur every 8 h which would be inconvenient and impractical in the treatment of K. pneumoniae.On the contrary, the ceftiofur serum concentration fell below the detected MIC (2.4 µgml −1 ) in the non-challenged group IV at 24 h following administration of niosome at a dose of 10 mg/kg BW in broilers.As a result, the niosome is the most convenient treatment regimen for K. pneumoniae.The obtained serum levels of ceftiofur in the niosome-treated groups (IV and VI) were lower than those obtained in the ceftiofur-treated groups (Ш and V).This might be attributed to the higher penetrating power of niosome in the tissues.These results support a previous study that showed the great efficacy of nano-antimicrobials against infectious bacteria (Agnihotri and Dhiman 2017).
The t 1/2 ka , t 1/2Beta , C max , AUC, and MRT findings of the present study were higher in the niosome treated groups (IV and VI) compared to ceftiofur treated groups (Ш and V).This might be attributed to the slow release of drugs from liposome molecules (Akbarzadeh et al. 2013).The long elimination half-life (t 1/2 ßeta) reported in the niosometreated groups (IV and VI) (6.8 ± 0.7 and 5.7 ± 0.7 h, respectively) reflected its high absorption and low clearance time (0.03 ± 0.9 and 0.04 ± 0.8), respectively (Jackson et al. 2012).The maximum serum concentration (Cmax) of ceftiofur in tested groups (Ш, IV, V, and VI) was observed after 2 h following administration of ceftiofur and niosome.This is in line with the fact that maximum blood concentrations of ceftiofur and metabolites were achieved within 0.5 and 2 h of dosing (CVMP 1999).In addition, the Cmax, Tmax, and t 1/2ß values of ceftiofur in the non-challenged ceftiofur treated group were 24.3 ± 0.15, 2.3 ± 0.7, and 5.5 ± 0.15, respectively.This is in line with the findings of (El-Sayed et al. 2015), who reported that a C max of 25.94 μgml −1 achieved at a maximum time of t max = 2.51 h with 5.47 h as t1/2 ß value.

Conclusions
Finally, we conclude that the encapsulation of ceftiofur N-acyl homoserine lactonase in a niosomal formulation provided higher efficacy in the treatment of multi-resistant K. pneumoniae infection in broilers.Furthermore, this study provided new insight into the diversity of organisms that display both multiple beta-lactam antibiotic resistance and QQ activities, as well as quorum sensing and quorum quenching activity.Further studies on the correlation between betalactam antibiotic resistance and QQ activities and the salt tolerance of QQ molecules are needed.
sequencing for the ahlK gene isolated in six isolates.Dalia M.A. Elmasry formulated and characterized the ceftiofur N-acyl homoserine lactonase niosome using Transamination Electron Microscope and Zetasizer and evaluated the cytotoxicity potential of the niosome using the Sulforhodamine B assay.Mai A. Fadel screened the AHL-degrading ability of K. pneumoniae strains using HPLC, statistically analyzed the HPLC results, determined the MIC of ceftiofur N-acyl homoserine lactonase niosome, evaluated the synergism between ceftiofur and Nacyl homoserine lactonase, and assessed the pharmacokinetic parameters of the experiment.Reham A. Hosny, Zeinab Abdelbadiea, Dalia M.A. Elmasry, and Mai A. Fadel designed and carried out the experiment, interpreted the results, and wrote and revised the manuscript.All authors read and approved the manuscript.
Funding Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB).

Data availability
The authors confirm that the data supporting the findings of this study are available within the article and its supplementary information.

Fig. 5
Fig.5The concentration-time semilogarithmic plot for ceftiofur in serum after intramuscular administrations of ceftiofur and ceftiofur N-acyl homoserine lactonase niosome in groups III, IV, V, VI.The

Table 1
Chromatographic conditions used in the detection of N-acyl homoserine lactonase and ceftiofur using HPLC method methanol: water was used for 15 min.Finally, methanol (35:65, v/v) was used for 8 min

Table 3
Clinical signs in the different groups of the experimental study

Table 4
Gross lesions in the positive control group and experimental challenged groups treated with ceftiofur and ceftiofur N-acyl homoserine lactonase niosome

Table 5
Effect of ceftiofur and ceftiofur N-acyl homoserine lactonase niosome treatments on the count quantification of K. pneumoniae in the experimental groups V and VI at the age of 16 to 21 days of the experimental study Counts were expressed by mean ± standard deviation of the three replicated groups.Values of mean reduction count with different superscript letters (a, b, and c) were significantly different (p < 0.05)