2.1 Bacterial isolates
A total number of 200 urine isolates of E. coli (n=100) and K. pneumoniae (n=100) were used in this study. These clinical pathogens were collected (2018-2019) from the Pathkind Labs, Gurugram, India, isolated from urine samples of urinary tract infections (UTIs) patients. We collected these isolates of E. coli and K. pneumoniae on Mueller Hinton Agar (MHA, HiMedia) plates and characterised by using various differential media like MacConkey agar (HiMedia), eosin methylene blue (EMB, HiMedia) agar and by performing the biochemical tests like Indole, Methyl Red, Voges Proskauer and Citrate (IMViC) tests [20]. For the combination study five MDR clinical isolates of each of E. coli and K. pneumoniae were selected that were meropenem and colistin resistant. All of the clinical isolates were stored as aliquots in Muller Hinton Broth (MHB, HiMedia) supplemented with 25% glycerol at -80 °C.
2.2 Quality control strains
The E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as the quality control strains for antimicrobial susceptibility testing and phenotypic characterization of ESBLs producing clinical isolates. E. coli ATCC 25922 and K. pneumoniae ATCC 2470 strains were used as the negative and positive controls, respectively, in blue carba NP test [21]. The positive and negative controls for Modified Hodge test (MHT) were K. pneumoniae ATCC BAA-1705 and K. pneumoniae ATCC BAA-1706, respectively [22].
2.3 Antimicrobial susceptibility testing (AST)
Minimum inhibitory concentrations (MIC) of the clinical isolates were analysed by using the broth microdilution method as per the Clinical and Laboratory Standards Institute (CLSI) guidelines [23]. The 1 mg/mL stock solutions of the meropenem (MEM, Zydus Healthcare LTD), ciprofloxacin (CIP), aztreonam (AZT, Fluka), cefotaxime (CFT), colistin (COL), nitrofurantoin (NIT), levofloxacin (LEV), amikacin (AMK), ceftazidime (CAZ), ampicillin (AMP), tigecycline (TIG), trimethoprim (TMP), tetracycline (TET, Sigma Aldrich) and were prepared and stored as aliquots at -80 °C for further use. Working solutions of these antimicrobial agents were prepared just before the use in the cation-adjusted Muller-Hinton Broth (CAMHB, HiMedia).
The overnight grown fresh cultures were used to prepare the 0.5 McFarland bacterial suspension to get the 1.5x108 CFU/mL from which the culture inoculums were prepared in the ratio of 1:100 suspensions (106 CFU/mL) and inoculated into the 96 well culture plates containing the different dilutions of the drugs chosen for AST. Plates were incubated in the ambient air for 16-18 h at 37 °C. Interpretation of the MIC (expressed as the lowest concentration of the antimicrobials that inhibits visible growth after incubation) was done according to the CLSI guidelines and for colistin according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST).
2.4 Phenotypic characterization of the ESBLs (extended-spectrum beta-lactamases) producing bacterial isolates by disc diffusion method
For this study, the pure cultures of clinical isolates grown overnight on MacConkey agar (HiMedia) were used to prepare the culture inoculums matching the 0.5 McFarland standard by using the instrument densitometer (DEN1-B). The bacterial isolates were spread on the MHA plates with the help of sterile cotton swabs to obtain a fine lawn culture. The plates were then allowed to dry. The discs of CAZ (30 µg) and CAZ + clavulanic acid (30 µg + 10 µg) respectively, were placed on the surface of the plates and incubated overnight at 37 °C in an ambient air incubator. The diameters of the zones of inhibition for the CAZ and CAZ + clavulanic acid were measured and if the diameters of combinations increased by >5 mm than alone CAZ were confirmed as the ESBL-producing isolates [24].
2.5 Phenotypic characterization of the carbapenemase-producing bacterial isolates by performing Blue Carba NP test
Overnight grown cultures on MHA were used to perform this test. The test solution was freshly prepared to consist of the aqueous solution of bromothymol blue (0.04%, pH 6, Sigma), 0.1 mmol/L ZnSO4 (Sigma) and imipenem drug (3 mg/ml, Sigma) with a final pH 7. The negative solution was prepared to contain only bromothymol blue (pH 6). In a U-bottom 96 well micro-dilution culture plate 100 µL of the test, the solution was added in duplicates (row A and row B) and in the next row, 100 µL of the negative solution was added. The test clinical strains from the MHA plates were inoculated in the test solutions and negative solutions with the help of sterile inoculation loops. The plates were incubated at 37 °C in the shaker incubator for about 2-3 h. The plates were monitored for colour change from red to orange or red in the antibiotic containing wells at intervals of time like after 30 mins, 90 mins and finally after 2-3 h [25].
2.6 Modified Hodge Test (MHT)
The clinical isolates were screened for the production of carbapenemases by using the MHT also. The E. coli ATCC 25922 strain grown overnight on TSA plates was used to prepare the culture inoculums of 0.5 McFarland standard by using the instrument densitometer. The bacterial cultures of 1:10 dilutions were streaked on the MHA plates to obtain a fine lawn culture. A disc containing 10 µg of meropenem was placed in the centre of the test area. The single isolated colony of the test bacterial strain grown overnight on the TSA pates was taken and streaked in a straight line from the edge of the disk to the edge of the plate. The plates were then incubated at 37 °C in an ambient air incubator overnight. Interpretation of the result was done on the basis of the presence or absence of the clover-leaf-like indentation of the ATCC 25922 growing along the test organism growth streaked within the disk diffusion zone. MHT negative test showed no growth of the E. coli 25922 along with the test organism growth streak within the disk diffusion [22].
2.7 Genotypic characterization of the genes responsible for the resistance in clinical isolates of E. coli and K. pneumoniae
Genotypic characterization of the AMR in these clinical isolates were performed by using the polymerase chain reaction (PCR) targeted the different resistance gene markers. Template DNA was prepared by boiling method and the purity of the DNA was confirmed by using the gel electrophoresis technique [26]. The clinical isolates were analysed for the presence of blaNDM-1 (New Delhi Metallo-beta-lactamase-1), blaKPC (beta-lactamase K. pneumoniae Carbapenemase), OXA-48, AmpC and MCR-1 (mobile colistin resistance gene-1) (Table S1). We used 1X Buffer (HiMedia) with 0.5 µL (2.5 mM) of dNTPs (HiMedia), 1 µL (3 pmol/µL) of each of reverse and forward primers (Eurofins genomics), 1 U/µL Taq DNA polymerase (HiMedia), 2 µL of DNA sample from bacterial lysate and deionised water to make the final volume of 25 µL as the PCR master mix. The PCR conditions in the thermal cycler (CFX96 Real-Time System) were as follows: 5 min at 94 °C and 30 cycles of 30 sec at 94 °C, 90 sec at an annealing temperature standardized by gradient PCR for each primer set and 1 min at 72 °C, followed by 10 min at 72 °C and stored at -4 °C. The amplified PCR products were analysed in 1.5% agarose gel stained with EtBr (Ethidium Bromide) with 50-1500 bp DNA ladder and visualized under UV light by Gel Doc System (XR+ BIO-RAD) [27].
2.8 In vitro combination study by checker-board method
The stock solution (1 mg/mL) of the MMV675968 compound was prepared in dimethyl sulfoxide (DMSO) and stored at -20 °C and in the same way auranofin was also prepared and stored. For the colistin based combination study, seven different dilutions of drug A (colistin) and 10 different dilutions of drug B (auranofin, MMV675968 compound, levofloxacin and nitrofurantoin) were prepared. For meropenem based combinations the drug A were auranofin, MMV675968 compound, levofloxacin and nitrofurantoin while meropenem was used as drug B. The overnight grown fresh cultures were used to prepare the 0.5 McFarland culture suspensions to get the 1.5X108 CFU/mL from which the culture inoculums were prepared in the ratio of 1:100 suspensions (106 CFU/mL). In 96 well culture plates, the seven dilutions of drug A and 10 dilutions of drug B were dispensed in combination. An equal volume of the culture inoculums was added and the plates were incubated overnight in the ambient air at 37 °C. Fractional inhibitory concentrations (FIC) of agent A or B = MIC of agent A or B in combination/MIC of agent A or B alone. The interpretation of the FICI (Fractional Inhibitory Concentration Index) was done by adding the FIC of drug A and FIC of drug B. Synergistic values were considered as a FICI ≤ 0.5 and additive/indifference as between > 0.5 - < 4 and antagonistic as FICI ≥ 4. The combinations of colistin and meropenem (separately) were performed with MMV675968 compound, auranofin, nitrofurantoin and levofloxacin against the meropenem and colistin resistant clinical isolates of E. coli and K. pneumoniae.
2.9 Statistical methods
Isolates and antimicrobial resistance data were analysed in Microsoft Excel using descriptive statistics as applicable and Pearson’s Chi-squared test and prop test of equal proportion in R programming (version 4.2.3). The p-value <0.05 is considered statistically significant.