Methods

This was a cross-sectional analytic study conducted at Prince Mohammed Bin Nasser Hospital, Jazan, Saudi Arabia. All clinically relevant data was collected prospectively from electronic medical records between June 2023 and April 2024.

Inclusion criteria: All isolates of Enterobacterales recovered from the clinical specimens of hospitalized patients between June 2023 and April 2024 were included.

Exclusion criteria: Isolates from outpatients or emergency department patients, carbapenemase-producing organism screening isolates, repeated isolates from the same patient at the same anatomical site during the same admission, and samples yielding carbapenem-producing organisms other than Enterobacterales were excluded. To minimize over-counting, a first-isolate-per-patient–species 14-day rule was applied: only the initial isolate of a given species for each patient within any 14-day period was included in the primary analysis.

Microbiological and molecular testing was performed in the Microbiology Department of Prince Mohammed Bin Nasser Hospital, Jazan. Clinical specimens, including blood, respiratory secretions, wound swabs, and urine, were processed according to standard microbiological procedures. Specimens were inoculated onto appropriate routine culture media, including blood agar, chocolate agar, MacConkey agar, and cystine–lactose–electrolyte-deficient (CLED) agar for urine specimens, and incubated at 35–37°C for 18–24 h. After incubation, suspected Enterobacterales colonies were subcultured to obtain pure isolates and identified to the species level using the VITEK® 2 Compact automated identification system (bioMérieux, Marcy-l'Étoile, France). No additional confirmatory method was used for species identification. Antimicrobial susceptibility testing was performed using the VITEK® 2 system, and the results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) M100, 33rd edition (2023). Carbapenem susceptibility was interpreted using the CLSI breakpoints for imipenem and meropenem (susceptible ≤1 μg/mL, intermediate = 2 μg/mL, and resistant ≥4 μg/mL). Isolates demonstrating phenotypic resistance to at least one of these carbapenems were classified as carbapenem-resistant Enterobacterales (CRE) and were subsequently analyzed using the Cepheid Xpert® Carba-R assay (Cepheid, Sunnyvale, CA, USA). The assay detects the major carbapenemase gene families blaIMP, blaKPC, blaNDM, blaOXA-48, and blaVIM.

Statistical analysis: All statistical calculations were done using R version 4.2.1 and SPSS version 28.0. The demographic information, such as sex, nationality, age, and age groups, was expressed in simple terms, where categorical information was expressed as how frequently a category occurred and the percent of each category. Prevalence refers to the proportion of cases with CRE.

The relationship of clinical risk factors with CRE isolation was assessed using Pearson's chi-square test when expected cell frequencies were at least 5, or Fisher's exact test otherwise. Odds ratios with 95% confidence intervals were calculated to quantify these associations. When one or more contingency-table cells contained zero events, an uncorrected odds ratio was not estimable and was reported as not calculated (NC) rather than as 0.00. The risk factors considered included intensive care unit admission, central venous catheterization, Foley catheterization, endotracheal intubation, diabetes, hypertension, hematologic malignancies, and solid tumors. Significance was set at 0.05. The assessment of CRE genes, such as NDM and OXA-48, was done, showing their co-occurrence in different specimen types.