CRISPR-Cas Dynamics in Carbapenem-Resistant and Carbapenem-Susceptible Klebsiella pneumoniae Clinical Isolates from a Croatian Tertiary Hospital

(1) Background: CRISPR-Cas systems provide adaptive immunity against mobile genetic elements (MGEs) carrying antimicrobial resistance (AMR) genes. Carbapenem-resistant (CR) <i>Klebsiella pneumoniae</i> is a major public health concern, and the role of CRISPR-Cas in its resistance is understudied. This study explored CRISPR-Cas associations with multidrug resistance in clinical <i>K. pneumoniae</i>. (2) Methods: 400 <i>K. pneumoniae</i> isolates (200 CR and 200 carbapenem susceptible (CS)) were analyzed. Carbapenemase genes (<i>bla</i>_OXA-48, <i>bla</i>_NDM-1, <i>bla</i>_KPC-2), <i>cas1</i>, <i>rpoB</i>, and CRISPR1-3 loci were identified by PCR, while only CRISPR loci were sequenced. Genetic relatedness was assessed via PFGE, MLST, and spacer analysis. Statistical analysis utilized chi-squared and Fisher's exact tests. (3) Results: CRISPR-Cas was present in 15.8% of isolates, mainly subtypes I-E and I-E* (93.3%), with CRISPR3 loci showing the greatest spacer diversity. Clonal complexes ST14/15/101 (CR) and ST35 (CS) were identified. <i>bla</i>_OXA-48 was linked to CRISPR-Cas-negative strains, while <i>bla</i>_NDM-1 and <i>bla</i>_KPC-2 were more frequent in CRISPR-Cas-positive strains (<i>p</i> < 0.0001). Imipenem/relebactam resistance was higher in CRISPR-Cas-negative isolates. (4) Conclusions: <i>K. pneumoniae</i> CRISPR-Cas systems correlate with specific carbapenemase profiles, suggesting pressure against <i>bla</i>_OXA-48 acquisition. The coexistence of I-E and I-E* subtypes highlight synergies in targeting MGEs. CRISPR loci could be tools for subtyping organisms following MLST.