| dc.description.abstract | The wound microbiome plays a crucial role in the development of diabetic ulcers. Understanding the pathogenic bacteria present in diabetic ulcers is essential for identifying the types of bacteria responsible for infection. Hemolysins are important virulence factors in pathogenic infections. Diabetic wounds are susceptible to bacterial infections that are resistant to multiple drugs, complicating treatment and recovery. Therefore, understanding the microbiota profiles and resistance mechanisms is essential for effective clinical management. Molecular sequencing has also improved our understanding of the complex microbial diversity found in diabetic ulcers. Pathogenic bacteria that cause infections in diabetic wounds must be isolated and controlled using bacteriophages.
The objectives of this study are as follows: (1) to reveal the diversity of bacteria in wound infections in patients with diabetes, both cultivable and uncultivable, through metagenomic analysis using Oxford Nanopore Technologies (ONT). This study examined the hemolytic capacity of pathogenic bacteria, antibiotic resistance, strong biofilm formation, resistance to the blaTEM gene, resistance to the sul1 gene, and identification of the 16S rRNA gene in selected isolates, and (2) the isolation, characterization, and efficiency of phages capable of infecting pathogenic bacteria from diabetic wounds in vitro.
Wound samples from each patient were cultured using Eosin Methylene Blue Agar (EMBA), Maltose Salt Agar (MSA), and Pseudomonas Cetrimide Agar. Single bacterial colonies that grew were tested for hemolytic activity. This study yielded 70 isolates with hemolytic activity; of these, 15 were identified as ß-hemolysin, 8 as a-hemolysin, and 47 as ?-hemolytic.
Fifteen bacterial isolates showed ß-haemolysin activity, dominated by Staphylococcus aureus and Pseudomonas aeruginosa. Six isolates showed a multiple antibiotic resistance (MAR) index >0.4 and had the ability to form biofilms. Based on their ability to form biofilms, five bacterial isolates were classified as poor (P54A and P63AC2), weak (1PACP and 14PIBC), or moderate (bacterial isolate code 13MIPP). Bacterial isolates with weak and moderate biofilm formation abilities and a MAR index = 0.4 were proceeded to the DNA isolation stage, 16s RNA gene identification, ß-lactamase gene (blaTEM) detection, sulfonamide gene (sul1) detection, and sequencing. PCR results confirmed the presence of blaTEM and sul1 in three isolates (1PACP, 13MIPP, and 14PIBC). The three isolates identified using 16s rRNA were Pseudomonas aeruginosa 1PACP, Staphylococcus aureus 13MIPP, and Pseudomonas aeruginosa 14PIBC. These findings highlight the prevalence of multidrug-resistant and biofilm-forming bacteria in diabetic wounds, emphasising the need for targeted antimicrobial strategies and resistance monitoring.
Swab samples from each patient's wound were extracted for genomic analysis using nanopore sequencing. Samples from patients receiving antibiotics were coded 4, 7AB, and 8, whereas samples from patients not receiving antibiotics were coded 11. This study showed that the species with the highest abundance in sample 11 was Acinetobacter junii, in sample 4 was Proteus mirabilis, in sample 7AB were Alcaligenes faecalis and Pseudomonas aeruginosa, and in sample 8 was Acinetobacter baumannii. Patients who did not receive antibiotic treatment and those who did receive antibiotic treatment showed the presence of significant pathogenic microorganisms.
Staphylococcus aureus 13MIPP and Pseudomonas aeruginosa 14PIBC were selected to be controlled by bacteriophages isolated from various water sources in the environment. Bacteriophage FSA_2.2DS with host Staphylococcus aureus 13MIPP was successfully isolated, while bacteriophages with host Pseudomonas aeruginosa 14PIBC that were successfully isolated were FPA_14S6A2, FPA_14S3A, FPA_14S7A, FPA_14S8A, FPA_14S8B, and FPA_14S7E. The bacteriophages FPA_14S6A2, FPA_14S3A, FPA_14S7A, FPA_14S8A, FPA_14S8B, FPA_14S7E, and FSA_2.2DS were tested for their effectiveness against their respective specific hosts and characterised for their protein molecular weight using SDS-PAGE analysis.
SDS-PAGE analysis of the six bacteriophage isolates with the host P. aeruginosa strain 14PIBC showed protein bands, except for isolate code 4 (FPA_14S8A), which had no protein bands. Isolate code 1 (FPA_14S6A2) had three protein bands with molecular weights ranging from 59.35 kDa, 29.25 kDa, to 25.99 kDa. Isolate code 2 (FPA_14S3A) had three protein bands with molecular weights ranging from 59.35 kDa, 29.25 kDa, to 25.99 kDa. Isolate code 3 (FPA_14S7A) with three protein bands with molecular weights ranging from 59.35 kDa, 29.25 kDa, 25.99 kDa. Isolate code 5 (FPA_14S8B) with 1 band with a molecular weight ranging from 29.25 kDa. Isolate code 6 (FPA_14S7E) with two bands with molecular weights ranging from 59.35 kDa to 29.25 kDa.
The effectiveness test of bacteriophages against P. aeruginosa strain 14PIBC hosts showed that FPA_14S6A2 at 3-hour, 6-hour, and 9-hour incubations showed significantly different bacterial colony growth and a decrease in the number of bacterial colonies. Meanwhile, at 12 h of incubation, the number of bacterial colonies increased (not significantly different from 6 h of incubation), but at 24 h of incubation, there was a significant decrease in the number of bacterial colonies (105 CFU/mL) compared to the control at 24 h of incubation (1011 CFU/mL).
The test of the effectiveness of bacteriophages against the S. aureus 13MIPP host showed that FSA_2.2DS at 3 and 6 h of incubation showed significantly different bacterial colony growth and an increase in the number of bacterial colonies. Meanwhile, at 9, 12, and 24 h of incubation, the number of bacterial colonies remained constant (no significant difference), whereas at 24 h of incubation, the number of bacterial colonies was 107 CFU/mL, which was significantly different from the control at 24 h of incubation (number of bacterial colonies was 1010 CFU/mL). This indicates the potential of bacteriophages to lyse Staphylococcus aureus 13MIPP and Pseudomonas aeruginosa 14PIBC bacteria originating from wound swabs of patients with diabetes. | |
