Prevalence and Distribution of bla-CTX-M Genogroups Among Cephalosporin-Resistant Clinical Isolates of Escherichia coli and Klebsiella pneumoniae in Misrata, Libya: A Cross-Sectional Study

Abstract

CTX-M enzymes are the most prevalent extended-spectrum β-lactamase (ESBL) family worldwide, yet genogroup-level data from Libya in general, and from Misrata in particular, remain limited. This laboratory-based cross-sectional study aimed to investigate the prevalence and distribution of bla-_CTX-M genogroups among cephalosporin-resistant clinical isolates of Escherichia coli and Klebsiella pneumoniae in Misrata, Libya, and to explore the differences between the two bacterial species. From December 2024 to May 2025, 120 non-duplicated cephalosporin-resistant isolates (43 E. coli and 77 K. pneumoniae) were collected from public and private diagnostic laboratories across Misrata. Species identification and disc-diffusion susceptibility testing were performed at the source laboratories. DNA was extracted using a standard heat-lysis method. All isolates were initially screened for bla-_CTX-M genes by singleplex PCR with universal primers. Positive isolates were subsequently assigned to bla-_CTX-M genogroups 1, 2, 8, and 25 by multiplex PCR. Proportions were compared using the chi-squared or Fisher's exact test. Overall, bla-_CTX-M was detected in (90. %; 95% CI 83.3–94.2), with no significant difference between E. coli (90.7%) and K. pneumoniae (89.6%; p = 1.00). Among the five targeted bla-_CTX-M genogroups, only bla-_CTX-M genogroups1 and 9 were detected. Genogroup 1 was the predominant genotype, accounting for 78.7% (85/108), and genogroup 9 accounted for 57.4% (62/108).  Co-carriage of both genogroups 1 and 9 was detected in 36.1 (39/108) of the positive isolates and was significantly more frequent in K. pneumoniae than in E. coli (43.5% versus 23.1%, p = 0.034). This difference was sensitive to the statistical test used (p = 0.056 with continuity correction) and may be partly confounded by specimen source, as E. coli isolates were more often obtained from urine. Overall, bla-_CTX-M genes were highly prevalent among cephalosporin-resistant E. coli and K. pneumoniae investigated in this study, with bla-_CTX-M genogroup 1 and 9 accounting for all detected bla-_CTX-M genogroups. The considerable frequency of co-carriage, particularly among K. pneumoniae isolates, highlights the importance of continued molecular surveillance. Further investigation through variant-level identification and plasmid characterisation would help clarify the genetic basis and potential dissemination of these resistance determinants, providing valuable information to support local infection prevention and control measures and antimicrobial stewardship strategies.