Primer sequences, target genes, amplicon sizes, and cycling conditions.
A total of 100 broiler chickens were examined for the presence of Pseudomonas aeruginosa by standard microbiological techniques. Susceptibility pattern for amikacin and cefotaxime was performed by Kirby-Bauer and microdilution assays. Then, checkerboard titration in trays was applied and FIC was measured to identify the type of interaction between the two antibiotics. The ability of isolates to form in vitro biofilm was detected by two methods, one qualitative (CRA) and the other quantitative (MTP), followed by investigating the effect of each antibiotic alone and in combination on the expression of biofilm genes. The overall isolation percentage of P. aeruginosa was 21%. Resistance to each antibiotic was more than 50%; the range of cefotaxime MIC was 8–512 μg/ml, while amikacin MIC range was 1–64 μg/ml. The FIC index established a synergistic association between tested two drugs in 17 (81%) of isolates and the remaining represent partially synergism. The qualitative technique showed that only 66.6% of the isolates were considered biofilm producers, while the quantitative technique showed that 90.4% of the isolates were biofilm producers. Further to RT-PCR investigation, significant repression against biofilm-forming genes (filC, pelA, and pslA) was observed for the combination of antibiotics when compared with monotherapy.
- P. aeruginosa
- combination therapy
- gene expression
The infection with
Due to this intrinsic resistance to antibiotics, its ability to easily develop new resistance, its ability to create biofilms, and the recent decline in drug discovery programs,
Concomitant use of antibiotics (combination therapy) is recommended for severe infections when
Concerning bacterial biofilms, Batoni et al.  and Grassi et al.  proved a strong interaction between the effectiveness of combination therapy and biofilms formed by
2. Material and methods
2.1 Sampling and isolate characterization
A total of 500 samples of the liver, heart, kidney, spleen, and lung (100 each) was aseptically collected from 100 freshly dead and diseased with respiratory manifestations broiler chickens from different ages and localities in Sharkia province, Egypt, from November 2018 to February 2019. All samples were subjected to conventional methods for isolation and identification of pseudomonas recommended by the Health Protection Agency .
2.2 Antibiotic susceptibility testing
2.2.1 Disk diffusion method
The antimicrobial susceptibility test of the isolates was performed by Kirby-Bauer disk diffusion test . In brief, each test isolate was swabbed uniformly onto the surface of Mueller-Hinton agar plates. Antibiotic sterile disks including cefotaxime (CTX: 30 μg) and amikacin (AK: 30 μg) were then placed on to the agar surface of the plate. Following incubation, the inhibition zones, in millimeters, were measured in duplicate and scored as sensitive, intermediate, and resistant categories by the critical breakpoints recommended by the Clinical and Laboratory Standards Institute (CLSI) .
2.2.2 Preparation of antibiotic stock solution
Standard powder forms of cefotaxime and amikacin were stored at 4°C till usage. The stock solution of each antibiotic was prepared by weighing and consequently dissolving suitable amounts of the antibiotics reaching a concentration of 1000 μg/mL in Mueller-Hinton broth.
2.2.3 Determination of the minimum inhibitory concentration (MIC)
MIC values of antibiotics were determined by the microdilution method following the recommendations of Papich . Stock solutions of antibiotics were prepared and added to the bottom of a 96-well microtiter plate (Nunc Inc., Roskilde, Denmark). 100 mL of this solution was added to the first well of the 96-well plate and serially diluted. 100 mL of an overnight culture of
2.2.4 Test for synergism
The synergistic effect of the antibiotic combinations was detected using a checkerboard dilution assay . The initial concentration of each drug should be fourfold greater than the desired concentration (MIC concentration) and then diluted twofold. In a screw cap test tube, 0.25 mL of broth of each two drugs to be tested was added to 0.5 mL of broth containing a suspension of the organism to be tested to reach the final volume of 1 mL. The inoculum of the bacterial suspension (in 0.5 mL of broth) should be approximately 2 × 105 colony-forming unit (CFU) to produce a final inoculum of 1 × 105 CFU per mL after the addition of an equal volume of the antimicrobial solutions. Each test composed of 36 tubes set horizontally and vertically, 6 rows in one direction contained twofold serial dilutions of antibiotic 1, and 6 rows in the other direction contained twofold serial dilutions of antibiotic 2; two additional rows contained twofold serial dilution of antibiotic 1 or antibiotic 2 alone. The tubes were incubated at 37°C for 24 and 48 h, the tubes were read as those showing turbidity (+) and those showing no turbidity (−). A fractional inhibitory concentration index was used to interpret the results.
2.2.5 Estimation of FIC index
FIC of each agent was calculated by dividing the MIC of the drug in combination by the MIC of the drug alone. The sum of both FICs (ƩFIC = FIC of antibiotic A + FIC of antibiotic B) in each well was used to categorize the combined activity of antimicrobial agents at the given concentrations as synergistic (ƩFIC <= 0.5), partially synergistic (ƩFIC >0.5 and < 1), additive (ƩFIC = 1), indifferent (ƩFIC >1 and < 4), and antagonistic (ƩFIC > = 4) .
2.3 Phenotypic characterization of biofilm production
2.3.1 Congo red agar test
Freeman et al.  have described a simple qualitative method to detect biofilm production by using a Congo red agar (CRA) medium. CRA medium was prepared with brain heart infusion agar (Oxoid, UK) 37 g/L, sucrose 50g/L, and Congo red indicator (Oxoid, UK) 8 g/L. The first Congo red dye was prepared as a concentrated aqueous solution and autoclaved (121°C for 15 min) separately from the other medium constituents. Then, it was added to the autoclaved brain heart infusion agar with sucrose at 55°C. In this test, the Congo red dye was used as a pH indicator, showing black coloration at pH ranges between 3.0 and 5.2. Plates with the Congo red agar medium were seeded and incubated in an aerobic environment for 24–48 h at 37°C. Isolates were interpreted according to their colony phenotypes. Black colonies with dry constancy and rough surface and edges were suspected as a positive sign of slime formation, while both black colonies with a smooth, round, and shiny surface and red colonies of dry texture and rough edges and surface were suspected as intermediate slime producers. Red colonies with smooth, round, and shiny surfaces were indicators for negative slime formation.
2.3.2 Quantitative detection of biofilm by microtiter plate method
The biofilm assay is performed by using flat-bottom microtiter plates (Techno Plastic Products, Switzerland) as described by O’Toole .
For biofilm quantification, 200 μL of 0.1% aqueous crystal violet solution was added to each well, and the plates were allowed to stand for 15 min. The wells were subsequently washed three times with sterile PBS to wash off the excess crystal violet. Crystal violet bound to the biofilm was extracted with 200 μL of an 80:20 (v/v) mixture of ethyl alcohol and acetone, and the absorbance of the extracted crystal violet was measured at 545 nm on ELISA reader (stat fax 2100, USA). A negative control, crystal violet binding to wells was measured for wells exposed only to the medium with no bacteria. All biofilm assays were performed in triplicate. The interpretation of biofilm production was according to the criteria described by Stepanović et al. . Based on these criteria, optical density cutoff value (ODc) is defined as an average OD of negative control +3 × SD (standard deviation) of the negative control. The ability to produce biofilm of each
2.4 Molecular evaluation
2.4.1 DNA extraction
DNA extraction from isolates was performed using the QIAamp DNA Mini Kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Concisely, 10 μL of proteinase K and 200 μL of lysis buffer were added to 200 μL of the sample suspension and incubated at 56°C for 10 min. Then, 200 μL of 100% ethanol was added to the lysate followed by washing and centrifugation according to the manufacturer’s recommendations. Nucleic acid was eluted with 100 μL of elution buffer.
2.4.2 PCR amplification of biofilm virulence genes
The obtained DNA was examined for the presence of biofilm in a 25 μL reaction comprising 12.5 μL of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 μL of each primer of 20 pmol concentration, 4.5 μL of water, and 6 μL of DNA template. The reaction was implemented in an Applied Biosystems 2720 Thermal Cycler for the investigation of the presence of biofilm genes. The properties of all used primers, as well as amplicon length and cycling conditions, were synopsized by Ghadaksaz et al.  and listed in Table 1 .
|Target gene||Primer sequences||Amplified segment (bp)||Primary denaturation||Amplification (35 cycles)||Final extension|
|TCCCTACCTCAGCAGCAAGC||656||94°C, 5 min||94°C, 30 s||60°C, 40 s||72°C, 45 s||72°C, 10 min|
|CATACCTTCAGCCATCCGTTCTTC||786||94°C, 5 min||94°C, 30 s||60°C, 40 s||72°C, 45 s||72°C, 10 min|
|TGAACGTGGCTACCAAGAACG||180||94°C, 5 min||94°C, 30 s||56.2 °C, 30 s||72°C, 30 s||72°C, 7 min|
2.4.3 Analysis of the PCR products
The products of PCR were separated by electrophoresis on 1.5% agarose gel (AppliChem, Germany, GmbH) in 1× TBE buffer at room temperature using gradients of 5 V/cm. For gel analysis, 20 μL of the products were loaded in each gel slot. A GelPilot 100 bp DNA ladder (Qiagen, Germany, GmbH) and GeneRuler 100 bp ladder (Fermentas, Germany) were used to verify the size of fragments. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra), and the data were assessed through computer software.
2.4.4 Quantitative analysis of biofilm gene expression
Biofilm gene expression was analyzed by quantitative real-time PCR (qRT-PCR), and the 16S rRNA housekeeping gene of
|Target gene||Reverse transcription||Primary denaturation||Amplification (40 cycles)||Dissociation curve (1 cycle)||Reference|
|Secondary denaturation||Annealing||Extension||Secondary denaturation||Annealing||Final denaturation|
|50°C, 30 min||94°C, 15 min||94°C, 15 s||52°C, 30 s||72°C, 30 s||94°C, 1 min||52°C, 1 min||94°C, 1 min||Spilker et al. |
|60°C, 30 s||60°C, 1 min|
|60°C, 30 s||60°C, 1 min||Ghadaksaz et al. |
|56.2 °C, 30 s||56.2 °C, 1 min|
2.5 Statistical analysis
Data analysis was performed by SPSS version 22 for windows. A t-test was used to detect statistical differences of the experiments including antibiotic combination treatment versus single antibiotic therapy. Moreover, one-way ANOVA was used for contrasting the influence of these remedies on the fold change of biofilm gene expression. A P ≤ 0.05 value was suspected as statistically significant.
3.1 The recovery rate of isolation and identification
|Sample||No. of examined samples|
Young (1–10 days)
|Old broilers (11–35 days)||39||3||7.6%||1||2.5%|
3.2 Antimicrobial activity
According to the disk diffusion method, 76.2% of isolates were resistant to cefotaxime, 14.3% were intermediate, and 9.5% were sensitive. Regarding amikacin, 57.2% of isolates were resistant, 9.5% were intermediate, and 33.3% were sensitive. Of interest, 57.2% of isolates were resistant to both tested antibiotics.
According to the microdilution assay, the range of cefotaxime MIC was 8–512 μg/mL, while the amikacin MIC range was 1–64 μg/mL as depicted in Table 4 .
|Isolates no.||MIC of CTX||MIC of AK||MIC of CTX in combination||MIC of AK in combination||FIC of CTX||FIC of AK||Ʃ FIC||Interpretation|
|17||32||8||8||4||0.25||0. 5||0.75||Partially synergistic|
In the checkerboard technique, the interaction between the combination of cefotaxime and amikacin against
3.3 Congo red test
About 66.6% of the isolates were positive for biofilm production with varying degrees. Out of 21
3.4 Microtiter plate test (MTP)
Biofilm quantification analyses showed that 90.4% of the isolates were biofilm producers, indicating that this technique was more efficient than Congo red agar for the detection of biofilm production. The obtained isolates of this study had the following results for the categories of biofilm production: 9.6% were non-adherent, 33.4% weakly adherent, 42.8% moderately adherent, and 14.2% strongly adherent as shown in Figure 2 .
A comparison of results obtained by the CRA method versus that of MTP assay is declared in Table 5 . Out of 21 biofilm
|Sample code no.||CRA||No.||MTP|
|1, 16, 20, 21||Dry black||4||2||2||0||0|
|4, 7, 9, 10, 14, 15||Smooth black||6||1||3||2||0|
|3, 5, 6, 17||Dry red||4||0||2||2||0|
|2, 8, 11, 12, 13, 18, 19||Smooth red||7||0||0||5||2|
3.5 Detection of biofilm genes in strong biofilm
P. aeruginosaisolates by conventional multiplex PCR
All strong biofilm producers
3.6 Quantitative assessment effect of each antibiotic alone and in combination on biofilm gene expression
By RT-PCR, comparing the amount of examining biofilm gene products before and after each treatment with a sub-inhibitory concentration (SIC) of each antibiotic alone and combination, results revealed that the amount of examining gene products was relatively increased in untreated samples with drugs than those treated, which leads to high threshold cycle (Ct) value in treated than untreated. Interestingly, we found that drug combination was more effective in significantly reducing the expression of biofilm genes than each antibiotic alone.
Statistical data assessed that fold changes in
|Genes||Isolate no.||Fold change|
Here we isolated 21
In the current investigation, all the isolates were tested against cefotaxime and amikacin to determine the antibiotic susceptibility patterns. A high-resistance rate was detected for both antibiotics at which 76.2% were resistant to cefotaxime and 57.2% to amikacin. This might be due to the indiscriminate use of antibiotics in the feed of broiler breeders or other environmental possibilities .
The increased observance of multiple resistances (mainly to beta-lactam antibiotics) in pseudomonas isolates is making it increasingly difficult to treat infections caused by this pathogen. Resistance to antimicrobials in pseudomonas strains develops via several mechanisms, including the production of specific enzymes (b-lactamases, enzymes that modify aminoglycosides, for example), changes in cell-membrane permeability, and active efflux systems .
Interpretative reading was used to detect the bactericidal activity of each antibiotic against isolates with cefotaxime MICs of 8–512 and amikacin MICs of 1–64. These data are reinforced by findings from other countries, including Kuwait , Canada , China , and the USA .
Synergy testing has shown evidence of an interaction of two antibiotics in combination against pseudomonas bacterial isolates where statistical analysis provides important insights into drug synergism where the FIC index calculations exemplified a significant synergism of both drugs achieving an enhanced overall effect which is substantially greater than the sum of their ones. These results were consistent with the previous studies of Saiman , Dundar and Otkun , and Hawkey et al. . The possible explanation for this synergism is the ability of beta-lactam cefotaxime to penetrate the outer membrane of pseudomonas bacteria which thereby increases the permeability of the bacterium to the aminoglycoside amikacin binding to 30S ribosome inhibiting the protein synthesis, thus leading to a synergistic effect in the in vitro studies .
To investigate the effect of a synergistic combination of the repositionable drugs against
In this study, biofilm production was examined qualitatively, depending on colony morphology of 21
The qualitative technique revealed that only 66.6% of the isolates were considered biofilm producers, while the biofilm quantitative technique (MTP method) revealed that 90.4% of the isolates were biofilm producers, indicating that the quantitative technique was more efficient than the qualitative technique for the detection of biofilm production. There was also high biofilm production by the evaluated tested isolates of
Biofilms are surface-associated communities embedded within an extracellular matrix . The extracellular matrix consists of polysaccharides, proteins, nucleic acids, and lipids and is a distinguishing feature of biofilms, capable of functioning as both a structural scaffold and a protective barrier . Extracellular polysaccharides are a crucial component of the matrix and carry out a range of functions including promoting attachment to surfaces and other cells, building and maintaining biofilm structure, as well as protecting the cells from antimicrobials and host defenses [50, 51].
Conventional PCR was carried out for detection of
Moreover, Suriyanarayanan et al.  mentioned that the effects of
Regarding the qRT-PCR results, the suppressing effects in fold change of previously mentioned biofilm gene expression were detected for drug combination in comparison with each antibiotic alone. Exposure to each antibiotic caused a decreased level of biofilm expression ranging between 0.1- and 0.7-fold changes, while the repression was strong and most significant with amikacin-cefotaxime combination treatment with fold change reaching 0.08, i.e., the consequence of treatment on the average expression profile among all biofilm involving genes constituting the bacterial communities studied. As described in this paper and by others [59, 60, 61], sub-MICs of combinations have potent effects on attenuating biofilm formation which are totally different from each antibiotic alone.
The treatment of biofilm-related
Conflict of interest
The authors manifested that they have no conflicts of interest.
|FIC||fractional inhibitory concentration|
|MIC||minimum inhibitory concentration|
|CRA||Congo red agar|
|RT-PCR||reverse transcriptase-polymerase chain reaction|