Isolation and antibiotic resistance screening of bacterial strains
A total of 12 strains were isolated, of which five were obtained from pharmaceutical and seven from domestic effluents. All strains were grown on general-purpose, selective, and differential media. Isolated strains were tested for their resistance against five antibiotics, including ERY, STRP, CIP, SXT, and MET. Of 12 strains, 9 showed complete resistance to SXT as no inhibition zone was observed around the antibiotic disc. For ERY, 4 isolates showed absolutely no inhibition zone, whereas 3 isolates showed zones within the selective range for resistance. Only one strain showed resistance to CIP, and the same was the case with STRP. None of the isolates showed resistance against MET. Six isolates PC, PD, PE, D1A, D2D, and D2F, showed the highest resistance against SXT and ERY and were selected for further experiments. These results indicated the presence of multiple drug resistance patterns among these isolates except D2D, which was resistant to SXT only. The antibiotic resistance profile and percentages of all 12 isolates are given in Figure 1A and 1B.
MIC of antibiotics for resistant strains
The MIC of ERY and SXT for six selected isolates is summarized in Table 2. For ERY, three strains, PC, D2F, and D1A, showed the highest resistance with the MIC of 512 µg/ml. Same strains, D1A and D2F, were observed to be highly resistant to SXT as well, the highest MIC observed for both strains was 1024 µg/ml.
Identification of isolated strains:
Molecular characterization of antibiotic resistant strains:
Amplicon size for all three bacterial strains was ~200 bps (Figure 2). According to BLAST analysis, isolate PC, D1A, and D2F displayed the highest sequence similarity to Comamonas jiangduensis, Aeromonas hydrophila, and Aeromonas caviae, respectively. The accession no. for strain PC is MN587995, D1A is MN587996 and D2F is MT560322. A phylogenetic tree was constructed for all three isolates to determine their evolutionary relationship with closely related species (Figure 3A and 3B).
Optimization of culture conditions:
Optimization of culture conditions, including temperature, pH, and inoculum size, was done for the three highly resistant strains PC, D1A, and D2F, to achieve maximum biotransformation of antibiotics. Optimization of incubation temperature was carried out by incubating NB tubes containing antibiotics (MIC) and culture from selected isolates at different incubation temperatures, including 30, 35, 37, 40, and 45°C. A similar temperature requirement was observed for the maximum growth (OD600) of all three isolates irrespective of the presence of either ERY or SXT. For ERY, the temperature at which all three strains, PC, D1A, and D2F, showed maximum growth was found to be 35°C (Figure 4A). Maximum growth in the presence of SXT was also observed at 35°C for both D1A and D2F (Figure 4B). However, the growth of all three isolates decreased dramatically with further increase in temperature.
Maximum growth of strains PC and D1A was obtained at pH 7.5 whereas, strain D2F gave highest OD at pH 7 in the presence of ERY. A significant decrease in growth was observed with the increase in pH from 7.5 to 9.5 (Figure 5A). The optimum pH for the growth of strains D1A and D2F in the presence of SXT was found to be 9, where OD600 drastically increased from pH 5.5 to 9 and decreased afterward (Figure 5B).
For all three strains, varied inoculum size requirements were observed, suggesting its significant role in growth kinetics of bacteria. In the presence of ERY, strains PC, D1A and D2F showed maximum growth at 0.66, 1.66, and 0.066%, respectively (Figure 6A). Surprisingly, the two strains D1A and D2F, which were resistant to ERY as well, also showed different inoculum size requirement of 1% for maximum growth in the presence of SXT in contrast to ERY (Figure 6B).
The growth curve of antibiotic-resistant strains was studied to determine their viability in culture medium over the course of time and the events (lag, log, stationary, and decline phase) taking place in the bacterial population. For all three isolates, the lag phase was observed for an hour, which shifted towards the log phase with an exponential increase in cell densities till 72 h after which stationary phase was achieved as no significant increase in the OD was observed afterward. These results indicated that the time required to reach the stationary phase is not affected by the presence of two different antibiotics, even for the common strains (Figure 7A and 7B).
Determination of biotransformation of antibiotics
Microbiological assay for the evaluation of antibiotic potency
Reduction in the potency of antibiotics treated with bacterial strains was observed by reduction in inhibition zone over time. These zones were compared to the standards of known antibiotic concentrations (Table 3) to assess the extent of reduction in test antibiotic’s antimicrobial activity. For ERY, Isolate PC showed a significant reduction in the initial concentration (512 µl) within 3h of incubation. This concentration reduces to another half after 24 h, and a much smaller inhibition zone was observed at 96 h (Figure 8). Almost similar results were obtained with the strain D2F for ERY (Figure 8). More than a 2-fold reduction in ERY concentration was observed after 3h of incubation with the strain D1A, which drastically reduced in further 24 h of incubation. A very slight zone was observed around the test sample after 96 h of incubation (Figure 8). For SXT, strain D2F showed an almost 4-fold reduction in the initial concentration (1024 µl) within 3h of incubation, which decreased further by the factor of 2 in 24 h and ultimately a very slight to a negligible zone of inhibition was observed at 96 h (Figure 9). Strain D1A showed similar pattern of reduction of SXT as for ERY in the three studied time intervals with almost complete reduction in zone of inhibition within 96 h (Figure 9).
In summary, obvious biotransformation of ERY and SXT was achieved by PC, D1A, and D2F.Out of three strains, D1A was found to be the most potential candidate for this purpose as no considerable inhibition of susceptible strain was observed around the test sample of maximum growth time (96 h).