Identification of bacterial isolate
The bacterial isolate which was resistant to ampicillin and gentamicin was identified using 16s rRNA sequencing. One of the best and most efficient techniques for identifying species and determining evolutionary relationships is 16s rRNA sequencing. There are conserved and variable regions in 16s rRNA. The variable sections are of greater interest when it comes to identification, even though the conserved portions are used to determine the evolutionary history of the species (Rahman et al., 2007). It is possible to identify an organism by comparing its small subunit ribosomal ribonucleic acid (ribosomal SSU), which is present in all bacteria and has species-specific variability that is specific to each bacterium. From the sequencing, the organism was identified as Aeromonas caviae according to Fig. 3. This phylogenetic tree indicates 16s rRNA sequence is more related to Aeromonas caviae CMFRI/ACa-01. These results were examined in the BLAST tool and the final alignment was checked and aligned using MEGA11 software. These Aeromonads are known for their role in causing lower respiratory tract and urinary infections in humans (Janda and Abbott 2010).
Antibacterial Potential of NaHS with antibiotics on A. caviae
In this study, we examined the antimicrobial resistance of NaHS with antibiotics (AB + NaHS) against antibiotic-resistant bacteria A. caviae. We evaluated their effectiveness both qualitatively and quantitatively by observing the presence or absence of a zone of inhibition and determining the minimum inhibitory concentration values. The results from the well diffusion assay are presented in Table 2, the NaHS combined with ampicillin and gentamicin at concentrations ranging from 10, 50 and 100 µg/mL against A. caviae. Figure 4 shows the inhibitory effect of NaHS combined with ampicillin (8, 8.75 and 10.25 mm) and gentamicin (9, 9.5 and 11.25mm) at concentrations ranging from 10, 50 and 100 µg/mL against A. caviae. Our findings revealed that the resistance of A. caviae was re-sensitized when using AB + NaHS. Notably, the combination of NaHS with the antibiotics exhibited the most effective inhibitory result, with the most pronounced inhibition zones observed at 100µg concentration (measuring 10.25 mm and 11.25 mm, respectively).
Table 2
Antibacterial activity of Aeromonas caviae: NaHS with Ampicillin and NaHS with Gentamicin (According to EUCAST)
S.No | Concentration (µg/ml) | Zone of inhibition (mm) |
NaHS + Ampicillin | NaHS + Gentamicin |
1 | 10 µg | 8 ± 0.0 | 9 ± 0.0 |
2 | 50 µg | 8.75 ± 0.5 | 9.5 ± 0.57 |
3 | 100 µg | 10.25 ± 0.5 | 11.25 ± 0.5 |
AB + NaHS generate significant intracellular ROS in A. caviae.
The DCFH-DA (2’,7’-dichlorofluorescein diacetate is a fluorescent probe) assay was used to evaluate the intracellular formation of ROS to examine the mechanism involved in the inhibitory impact of ampicillin, gentamicin, and antibiotics with NaHS on A. caviae. ROS levels in antibiotics with NaHS-treated cells were high compared with the control (A. caviae without antibiotic or NaHS), as indicated in Fig. 5. This implies that cells exposed to H2S stress experienced a burst of ROS generation. Furthermore, ROS production in negative control and in antibiotics alone was negligible thus indicating significant ROS formation in the presence of AB + NaHS.
AB + NaHS induced lipid peroxidation in A. caviae.
In this study, malondialdehyde (MDA) formation was utilised as an indicator to quantify lipid peroxidation. MDA levels in A. caviae cells treated with AB + NaHS were significantly higher than in cells treated with ampicillin and gentamicin alone, as shown in Fig. 6. This suggests that releasing AB + NaHS causes oxidative stress in bacterial cells. According to the data presented in Fig. 6, the concentration of MDA in A. caviae cells treated with AB + NaHS is notably higher in comparison to the MDA levels observed in the control group of A. caviae cells.
AB + NaHS induced enzyme activities of SOD, CAT, GST, and GSH in A. caviae.
To evaluate the impact of ROS generated by A. caviae cells without antibiotics or NaHS (control) and those treated with ampicillin, gentamicin, and antibiotics along with H2S, we assessed the activity of ROS-related enzymatic and non-enzymatic parameters including SOD, CAT, GST, and GSH. In A. caviae cells treated with AB + NaHS, the activity of SOD was notably diminished compared to cells treated with gentamicin and ampicillin alone, with SOD levels decreasing to 45 and 55 U/mg, respectively, in contrast to the control (Fig. 7). AB + NaHS-treated cells also exhibited decreased CAT activity (Fig. 8). Therefore, the decrease in the levels of antioxidants in AB + NaHS-treated A. caviae might possibly be linked to the disturbance of electron transport structures in the cell's membrane.
We examined GST activity to further validate these results. GST, an enzyme that utilizes GSH and is primarily involved in detoxification processes, displayed a remarkable increase in specific activity when A. caviae cells were exposed to antibiotics combined with H2S, as depicted in Fig. 9. The total specific GST activity surpassed that of the control. The results indicated a significant disparity between A. caviae cells treated with AB + NaHS and the control group. Notably, A. caviae cells subjected to AB + NaHS exhibited higher GST activity. In bacterial cells exposed to antibiotics in conjunction with H2S, the concentration of the cellular antioxidant metabolite GSH decreased significantly. We observed a 45% and 30% reduction in GSH levels in A. caviae cells treated with ampicillin + NaHS and gentamicin + NaHS, respectively, in comparison to the control (85%) (Fig. 10). The results suggest that the reduction of GSH in AB + NaHS-treated cells led to increased ROS production, surpassing the antioxidant defense system, causing oxidative stress and ultimately, reduced cell viability.
Oxidative stress induced by AB + NaHS burst respiratory chain in A. caviae.
LDH is a mitochondrial enzyme susceptible to denaturation in conditions of oxidative stress. It serves as a dependable indicator to evaluate the adverse impact of ROS on the respiratory chain of bacterial cells. As demonstrated in Fig. 11, LDH activity is notably decreased in bacterial cells treated with AB + NaHS compared to cells treated with only gentamicin and ampicillin or with negative control (A. caviae without AB + NaHS). This demonstrates that the combination of NaHS and antibiotics causes oxidative stress, which ruptures the respiratory chain and causes the LDH enzyme to become denatured, finally killing the bacterial cells.
Examination of viable and nonviable bacteria through SEM.
The effect of AB + NaHS in A. caviae cells using scanning electron microscopic images shows severe detrimental effects on morphology where Fig. 12 has a clear disruption in membrane integrity and altered cellular shape (Fig. 12C and E). The cellular morphology of negative control (A. caviae without AB + NaHS) (Fig. 12A) and cells treated with Ampicillin alone (Fig. 12B) and Gentamicin alone (Fig. 12D) were observed with normal growth and smooth surface. Thus, it was established that AB + NaHS effects observed include bacterial abnormal growth, lysis, and disruption.