Genetic Diversity of Antibacterial Resistant Bacterial strains from Industrial Effluents in Nairobi County, Kenya.

Description

Antimicrobial resistant genes (ARGs) in addition to antimicrobial resistant (AMR) exist in a variety of environmental sources, including industrial waste waters. Antimicrobial resistance genes and profiles of bacteria isolated from industrial effluents were investigated in this study. Identification was performed using API® 20E identification kit. Disc diffusion technique was used to ascertain antibiotic susceptibility profiles of isolated bacteria, where classes of antibiotics used included: penicillin, tetracyclines, aminoglycosides, sulfonamides, cephalosporins, and fluoroquinolones. Multiplex PCR was used to detection of genes that encoded antibiotic resistance. Five bacterial species; Aeromonas, Pseudomonas, Escherichia coli, Klebsiella and Bacilli were isolated. Escherichia coli Was resistant to penicillin (100%), moderate resistance to sulfonamides and tetracycline (33.3%). Klebsiella isolates showed strong resistance to sulfonamides (80%) and penicillin (100%). Bacilli exhibited high level of resistance to sulfonamides and penicillin (100%) with lower of resistance toward aminoglycosides (28.57%). Pseudomonas strains demonstrated total resistance to tetracycline, sulfonamides, and penicillin. Aeromonas isolates did not show any resistance to fluoroquinolones, aminoglycosides, or carbapenems, they showed resistance to penicillin, tetracycline, sulfonamides and cephalosporins. Molecular screening revealed presence of blaTEM, blaOXA, blaKPN, blaParC, blaNDM genes. Genes sequences aligned portrayed 80% and above relatedness indicating significant relatedness. The results highlighted the critical need for ongoing AMR surveillance, both in the environment and clinical settings where antibiotic abuse hastens development of resistance. The study also emphasizes how critical it is to create new therapeutic approaches to fight MDR pathogens, particularly given rising incidence of strains that are resistant to carbapenem.

Steps to reproduce

Sample Collection Sterile 10 mL screw cap bottles were used to collect industrial effluent samples aseptically. Samples were put in cool boxes and transported to the microbiology laboratory for examination. To ensure precise microbiological analysis and to reduce alterations in bacterial populations, all samples were processed in 24 hours after collection. Bacterial Isolation and Identification A serial dilution technique was used to isolate bacteria from the corrected effluents samples. A set of dilutions, from 10⁻¹ to 10⁻⁵, was created by mixing a portion of each sample with sterile distilled water. Nutrient agar plates (Himedia Lab, India) were used where 0.1 mL aliquot from every dilution was plated, and the plates incubated at 37°C for 18-24 hours. Distinct bacterial colonies were chosen after incubation and sub-cultured onto MacConkey agar and Mannitol salt agar in order to isolate and distinguish bacterial species. For further identification of the bacteria species, gram negative bacteria were identified using API® 20E identification kit (BioMérieux, France). After being preserved in tryptic soy broth with 10% glycerol, isolated bacteria were kept at -80°C for additional examination. Antimicrobial Susceptibility Testing The disc diffusion method was used where Mueller-Hinton agar (Himedia Lab, India) was utilized in testing antibiotic susceptibility. Imipenem (10µg), levofloxacin (30µg), ciprofloxacin (25µg), ampicillin (10µg), tetracycline (30µg), amoxicillin (30µg), gentamicin (10µg), sulfamethoxazole/trimethoprim (25 µg) cefepime (30µg), and ceftriaxone (30µg), were antibiotics that were tested (Oxoid Limited, United Kingdom). Pure bacterial colonies were streaked on Mueller-Hinton agar culture plates, then on the agar surface antibiotic discs were placed. The inhibition zones diameter was measured in millimeters after incubating the agar plates at 37°C for 18 to 24 hours. Clinical and Laboratory Standards Institute (CLSI) guidelines were followed in interpretating the results. DNA Extraction and resistance genes detection DNA extraction of antibiotic-resistant isolates was done using ZyppyTM Plasmid Miniprep Kit (Zymo Research USA), in accordance with manufacture instructions. Extracted DNA was observed using a UV transilluminator at 1.5% agarose gel to assess the quality of the. The reaction mixture comprised of 25μL and included a master mix containing 0.5μL of 10μM forward primer, 0.5μL of 10μM reverse primer, 12.5μL Master mix (one Taq quick load 2X master mix with standard buffer), Nuclease-free water 9.5μL together with 2μL of DNA template. Amplification was performed using following conditions: 30 cycles at 94°C for 30sec, 30 seconds at 55°C, and concluding with 30seconds at 72°C. Final extension was conducted at 72°C for 10 minutes. PCR products Gel electrophoresis was executed using 80V of voltage on a 1.5% of agarose gel for duration of 30 minutes, with DNA staining SYBR green dye.

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