Ethical considerations

Ethical approval for this study was obtained from the Adeleke University Ethical Review Committee (AUERC) with reference number AUERC/FOS/MCB/10. The study adhered strictly to ethical guidelines for research involving human participants. Informed consent was obtained from all participants prior to specimen collection, and participants were assured of the confidentiality and anonymity of their personal and medical information. All data and specimens collected were securely stored and used solely for this research.

Bacterial collection

UNIOSUN Teaching Hospital, formerly known as LAUTECH Teaching Hospital, is a prominent healthcare institution in Osogbo, Osun State, Southwest Nigeria, serving as a pivotal healthcare hub for the region. Between January and April 2022, a total of 200 clinical specimens were randomly collected from the hospital’s Microbiology department. These specimens included blood, sputum, urine, wound, stool, and oral cavity samples. All isolates were patient-unique, ensuring no duplicate samples from the same individual. The selection process was conducted randomly, with no attempts to balance isolates based on the various body sites of origin.

The samples were collected from patients at the study location using sterile swabs. The swab sticks were then placed in sterile coolers with icepacks maintained at 4–8°C upon collection and transported to the laboratory for analysis. Upon arrival at the laboratory, all samples were stored in freezers for long-term preservation until cultivation.

Patient information, including age, gender, admission class, diagnosis, and patient status, was obtained from medical records.

Cultivation of samples and screening for enterobacteriaceae

Each sample was enriched in 225 mL of nutrient broth (Huankai Ltd., Guangzhou, China) and incubated at 37°C for 24 hours. After enrichment, the broth was streaked onto MacConkey agar (Huankai Ltd., Guangzhou, China) and incubated under the same conditions for an additional 24 hours. From the MacConkey agar plates, three pink mucoid colonies were selected and subcultured onto nutrient agar for 24 hours at 37°C.

The bacterial isolates were initially identified using Gram staining, capsule tests, and motility tests based on their morphological characteristics. Biochemical identification was conducted through a series of tests, including catalase, oxidase, citrate, urease, and indole tests. Sugar fermentation tests were performed using glucose, lactose, and sucrose, and Methyl Red/Voges-Proskauer (MR/VP) tests were also included. The identification of likely bacterial species was guided by Bergey’s Manual of Systematic Bacteriology. Confirmed cultures were preserved in Luria-Bertani broth supplemented with 20% glycerol and stored at −80°C for subsequent analyses.

Antimicrobial sensitivity testing

AST was performed using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar. This method was chosen for its simplicity, practicality, and standardization. A bacterial inoculum of approximately 1–2 × 10⁸ CFU/mL was evenly applied to the surface of Mueller-Hinton agar plates.

Six commercially prepared antibiotic disks with fixed concentrations were placed on the inoculated agar surface. The antibiotics tested were Ceftriaxone (5 μg), Cefotaxime (30 μg), Colistin (25 μg), Meropenem (10 μg), Imipenem (10 μg), and Ertapenem (10 μg). The plates were incubated at 35°C for 16–24 hours. After incubation, the growth inhibition zones around each antibiotic disk were measured in millimeters. The diameter of the inhibition zone was used to determine the isolates’ susceptibility, considering the antibiotics’ diffusion rate through the agar medium. The zone diameters of each drug were interpreted according to CLSI criteria.¹² The classification of isolates as susceptible, intermediate (moderately resistant), or resistant was performed according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints. E. coli ATCC 25922 was used as the quality control strain throughout the analysis.

DNA extraction and multiplex PCR

DNA extraction was performed using a commercial kit from Stratec Biomedical Systems (Birkenfeld, Germany). A 1mL volume of bacterial suspension, standardized to .5 McFarland from an overnight culture, was centrifuged, and DNA was extracted from the resulting pellet according to the manufacturer’s instructions. The extracted DNA was resuspended in 100μL of TE buffer.

Serotyping primers (K1, K2, K3, and K5) and virulence-associated genes (fimH, ramA, traT, mrkD) were used for the identification and confirmation of K. pneumoniae strains. Primers for K. pneumoniae 16S–23S ITS were also used for strain confirmation, as described previously.^{Reference Turton, Perry, Elgohari and Hampton13} The primers used in this study are shown in Table 1.

Table 1.

Primers used for serotype and virulence gene identification

From the eventually used isolates, 20 isolates were randomly selected for serotyping and virulence gene testing. These isolates were chosen to represent diverse resistance patterns and clinical backgrounds, ensuring a representative sample for analysis. This selection allowed the study to focus on specific genetic traits linked to virulence and capsular types within a manageable sample size.

Two independent Multiplex PCR assays were performed to identify capsular serotypes and capsule-associated genes based on the protocol described previously.^{Reference Candan and Aksöz14} Each PCR was conducted in a 25 μL reaction mixture containing 5 pmol of each primer, 2 μL of extracted DNA, and the Master PCR mixture (Yekta Tajhiz Azma®, Iran). Amplified products were subjected to electrophoresis on a 1% agarose gel and stained with CyberSafe stain for visualization. The thermal cycling conditions for both Multiplex PCR assays were identical and followed established protocols.

Statistical analysis

Statistical analyses were conducted using PyCharm (version 2024.1.2) with the scipy.stats library. The chi-square test was employed to examine associations between categorical variables, such as age group, sex, admission class, patient status, and sample type. Observed frequencies were compared to expected frequencies using the chi-square statistic, with a p-value of less than .05 considered statistically significant.

ANOVA was used to investigate differences in mean ages across categorical groups. Age was treated as a continuous variable, while sex, admission class, patient status, and sample type were considered categorical factors. Post hoc tests, including Tukey’s HSD, were conducted to determine specific group differences if ANOVA yielded significant results.