Study design and settings
Out of 590 Klebsiella pneumoniae isolated from various clinical samples as a part of the routine hospital laboratory procedure (blood culture, pus, endotracheal aspirates and pleural fluid) over a period of 8 months from February 2017 to September 2017, only 50 unrepeated clinical isolates of MDR-Klebsiella pneumoniae were OXA 48 like producers and were selected for our cross-sectional experimental study. All samples were processed in the microbiology laboratory of the Specialized Pediatric Teaching Hospital, Cairo University. The study was approved by the ethical committee of the Clinical and Chemical Pathology Department and patients’ parents provided written informed consent.
Bacterial isolates
All clinical samples were cultured on blood chocolate and MacConkey agars (Oxoid, England). Identification of all isolates was done by Vitek 2 compact system. Antimicrobial susceptibility testing was done by Kirby-Bauer method where an inoculum density equivalent to 0.5 McFarland was inoculated on Mueller-Hinton agar plates (Oxoid cop. England). Antimicrobial agents used were imipenem 10 μg, meropenem 10 μg, cefotaxime 30 μg, ciprofloxacin 5 μg, trimethoprim sulfamethoxazole 25μg, polymyxin 50μg, piperacillin-tazobactam 85μg, amikacin 30μg, levofloxacin 5μg, cefoperazone75 μg, ceftazidime 30μg, gentamicin 10 μg, ampicillin-sulbactam 20 μg, and amoxacillin-clavulunate 30 μg. Antimicrobial discs were obtained from Oxoid Limited Basingstoke, Hamsphire, England. Antimicrobials were supplied and stored according to the manufacturer’s instructions. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as reference strains for susceptibility testing. The diameters of the inhibition zones were measured for each antibiotic, and MDR Klebsiella pneumoniae was identified if the isolate showed acquisition of non-susceptibility to at least one antibiotic of ≥3 different categories [7]. Interpretation of disc diffusion zone diameters was done according to the clinical laboratory slandered institute (CLSI 2017) [8].
Detection of OXA-48-like carbapenamase-producing isolates
All Klebsiella isolates were directly inoculated on CHROMID® OXA-48 (bioMérieux, France) according to the manufacturers’ recommendations.
Determination of minimum inhibitory concentrations (MICs) of cefoperazone
The minimum inhibitory concentrations (MICs) of cefoperazone (Sigma Aldrich, UK) against indicator pathogens were determined by broth micro-dilution method according to BSAC 2012, CLSI 2017 [8, 9]. The preparation of stock solutions of antibiotic used was conducted using the following formula to determine the amount of powder needed for a standard stock solution:
$$\mathrm{Weight}\ \left(\mathrm{mg}\right)=\mathrm{volume}\ \left(\mathrm{mL}\right)\times \mathrm{concentration}\ \left(\upmu \mathrm{g}/\mathrm{mL}\right)$$
$$\mathrm{Potency}\ \left(\upmu \mathrm{g}/\mathrm{mg}\right)$$
Processing of Lactobacilli strains
Lactobacilli strains were provided by the National Research Center from different origins as follows:
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1-
The Northern Regional Research Laboratory “NRRL” Illinois, USA: Lactobacillus reuteri B-14171, Lactobacillus salivaricus NBIMCC-1589, Lactobacillus brevis NBIMCC-3448, Lactobacillus carvutus NBIMCC4562, Lactobacillus gasseri NBIMCC-1589, Lactobacillus rhamnosus B-445, and Lactobacillus helveticus CNRZ-32
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2-
Lactobacillus acidophilus was obtained from Chr. Hansen’s Lab., Denmark
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3-
Lactobacillus plantarum and Lactobacillus casei were provided by the Faculty of Agriculture Ain-Shams University.
Cell-free supernatant was prepared using Man-Rogosa-Sharpe broth (Sigma Aldrich) according to De Man et al., Liasi et al., and Eid et al. [10,11,12].
Detection of in vitro antibacterial activity of Lactobacillus spp. by agar-well diffusion method
Screening of antimicrobial activity of different Lactobacillus spps. against 10 isolates of MDR Klebsiella pneumoniae-producing OXA-48 (selection was done upon the highest MIC values to B lactams) to determine which types of CFS were the most potent to be used in combination with cefoperazone to evaluate the synergistic action.
The agar-well diffusion method was used for antimicrobial testing. The selected isolates of MDR Klebsiella pneumoniae were adjusted to 0.5 MacFarland suspension in sterile tubes. The isolates were swabbed individually on the surface of sterile Muller Hinton agar plates using a sterile cotton swab. Agar wells were prepared with sterilized cork-borer with a 10-mm diameter. One hundred microliters of CFS formulated in each of Lactobacilli spp. was added to the agar wells in the plate which were incubated at 37 °C for 24 h. The presence of inhibition zone was considered as the indicator of antimicrobial action that is reported in millimeters, and the zone diameter obtained was measured and interpreted following Shokryazdan et al., Mogna et al., Prabhurajeshwar et al., and Abed et al. [13,14,15,16].
Quality control strain Klebsiella pneumoniae (ATCC 35657) was used for comparison with CFS of different Lactobacillus strains.
Antimicrobial effect by using CFS of the most potent Lactobacillus strains (L. helviticus and L. rhamnosus alone and in combination with cefoperazone
Six wells were punched with sterile cork borer in Muller Hinton agar, after seeding the plates with fresh culture organism adjusted to 0.5 MacFarland suspension in sterile tubes. Wells were filled with 100μl CFS of probiotic Lactobacillus helviticus, 100μl CFS of probiotic Lactobacillus rhamnosus, 100μl of MIC= 512 μg/ml of cefoperazone), 50μL CFS of probiotic Lactobacillus helviticus+, 50μL of MIC= 512 μg/ml of cefoperazone, 50μL CFS of probiotic Lactobacillus rhamnosus+, 50μL of MIC= 512 μg/ml of cefoperazone, and 100μl of distilled water as a negative control. Then, the plates were covered and incubated at 37°C for 18 h. The inhibition zone around the well was measured in millimeter, and all isolates were done in duplicates.
Statistical analysis
Data management and statistical analysis were performed using Statistical Package for Social Sciences (SPSS) vs. 23. Numerical data were summarized using means and standard deviations. Categorical data were summarized as numbers and percentages. The zone of inhibition was compared between different groups using Kruskal-Wallis test. Categorical variables were compared using chi-square test. All p values are two-sided. P values < 0.05 were considered significant.
