2.1 Study area
This in-vitro experimental study was carried out during the period from October 2019 to October 2020. The study protocol was approved by the Ethics Committee at the High Institute of Public Health (HIPH), and an informed consent was obtained from each patient.
2.2 Sampling
An interview questionnaire sheet was filled for each patient including all relevant information (age, sex, type of surgical wounds, duration of skin wound or burn, current or previous use of urinary catheters, recurrent symptomatic UTIs, antibiotics treatment and history of hospitalization). All inpatients and outpatients with UTIs, skin and wound infections were included (as these are the most common infections caused by ESBL bacteria) with no exclusion criteria. Most of the patients were females. Patients age range were from 16 > 51 years.
The minimal sample size was calculated based on a previous study that aimed to detect AgNPs enhancement of the bactericidal activity of inactive antibiotics against multiresistant Enterobacteriaceae, which reported a mean FIC of antibiotic in combination with different AgNPs concentrations of 0.4 with standard deviation of 0.1. By using precision of 0.03 and confidence interval of 95%, the minimum required sample size is 43 ESBL isolates.
The study enrolled ESBL producing E. coli and K. pneumoniae isolates from 43 clinical samples obtained from patients attending or admitted to main hospital with different types of infections (urinary tract, wound and burn skin infections).
2.3 Laboratory investigation
2.3.1 Processing of samples
2.3.1.1 Swabs from infected wounds and burns
Swabs were inoculated on blood agar (BA) (Himedia M144) and MacConkey’s agar plates (Himedia M081).
2.3.1.2 Urine samples
A standard calibrated loop 10 µl (0.01 ml) was inserted vertically in the well-mixed uncentrifuged urine and was then streaked over the surface of well dried CLED agar plate (Himedia M792).
2.3.2 Identification procedures
All isolated colonies were identified according to the standard microbiological procedures following their morphology in Gram stain, cultural characteristics and biochemical properties [8].
2.3.2.1 Wound and burn isolates
Isolates that appeared as medium sized, circular, grayish colonies, smooth, either hemolytic or non-hemolytic on BA plates, pink lactose fermenter (LF) on MacConkey's agar plates and appeared microscopically as gram negative bacilli were considered as members of Enterobacteriaceae.
2.3.2.2 Urine isolates
Oxidase negative isolates that appeared microscopically as gram negative bacilli and appeared as opaque yellow colonies with slightly deeper yellow center on CLED agar were considered as E. coli. Isolates that appeared as large yellow to whitish-blue and extremely mucoid colonies on CLED agar were considered as K. pneumoniae.
2.3.3 Detection of ESBLs production among E. coli and K. pneumoniae isolates by double-disk synergy test (DDST)
A lawn culture of each isolate was made on MHA plate, as recommended by CLSI. A disc of amoxicillin-clavulanic acid (20/10 μg) was placed in the center of the plate at 20 mm distance to ceftazidime 30 μg (CAZ) and cefotaxime 30 μg (CTX) (Bioanalyse, Turkey). ESBLs production was detected by the appearance of keyhole effect toward clavulanic acid disc due to the enhanced activities of ceftazidime and cefotaxime [8].
2.3.4 Determination of MICs of ineffective antibiotics by broth microdilution method
MICs of ineffective antibiotics and AgNPs were determined by broth microdilution assay [9, 10]. Antibiotic dilutions were prepared using Muller Hinton broth to get a concentration range between 128 and 0.25 µg/ml for ampicillin, cefotaxime and ceftazidime, and between 32 and 0.06 µg/ml for ciprofloxacin. A stock AgNPs solution (108 µg/ml) was used, and then two-fold serial dilutions were done using MHB to obtain the following dilutions: 54, 27, 13.5, 6.8, 3.4, 1.7, 0.8, 0.4, 0.2 and 0.1 µg/ml [11]. Quality control was performed using E. coli (ATCC 25,922) strain as a positive control.
The effect of the combination of ineffective antibiotics with AgNPs was evaluated by checkerboard method [12]. The mean MIC values obtained for the combinations were used for the calculation of the fractional inhibitory concentration (FIC) index.
FIC index was calculated using the following formula:
$$\mathrm{FIC}=\frac{\mathrm{MICAg\, in\, combination }}{\mathrm{MIC\, Ag\, alone}}+\frac{\mathrm{MIC\, Ab\, in\, combination}}{\mathrm{MIC\, Ab\, alone}}$$
An average FIC was calculated from four FIC from each raw to classify the combined antibacterial effect of antibiotics and AgNPs as synergistic (FIC ≤ 0.5), additive (0.5 < FIC ≤ 1), indifferent (1 < FIC < 2) and antagonistic (FIC ≥ 2) [13].
Effect of AgNPs on beta-lactamases production was evaluated by DDST. AgNPs solution was added to liquid MHA to achieve a final silver concentration equal to MICAg/2.
MHA plate that contained AgNPs was inoculated with two loopful of bacterial suspension of ESBL producing E. coli and K. pneumoniae isolates (108 CFU/ml). Cefotaxime disc (30 µg) and ceftazidime (30 μg) were placed 15 mm apart (edge to edge) from an amoxicillin-clavulanic acid disc (20/10 μg). Then, plates were incubated for 16–20 h at 35 °C. A positive result was indicated when the inhibition zones around cefotaxime and ceftazidime discs were not augmented in the direction of the disc containing clavulanic acid (inhibition of enhancement) [11].
2.4 Statistical analysis of data
Data were collected and entered to the computer using Statistical Package for Social Science (SPSS) program for statistical analysis (version 21). Chi-square test (χ2) was used to test association between qualitative variables. Monte Carlo corrections were carried out when indicated (n × m table or expected cells less than 5). Fisher exact (FE) correction and correction for Chi-square were done when more than 20% of the cells have expected count less than 5. An alpha level was set to 5% with a significance level of 95%, and a beta error accepted up to 20% with a power of study of 80%.
