Whole genome sequence-based molecular characterization of blood isolates of carbapenem-resistant <i>Enterobacter cloacae</i> complex from ICU patients in Kolkata, India, during 2017–2022: emergence of phylogenetically heterogeneous <i>Enterobacter hormaechei</i> subsp. <i>xiangfangensis</i>

Blood-borne infections caused by the carbapenem-resistant <i>Enterobacter cloacae</i> complex (CR-ECC) are major public threats with respect to the challenges encountered during treatment. This study describes the whole genome sequencing-based molecular characteristics of blood isolates (<i>n</i> = 70) of CR-ECC from patients admitted to the intensive care unit of tertiary care hospitals in Kolkata, India, during 2017-2022 with respect to species identification, antimicrobial resistance (AMR) profiling, mechanism of drug resistance, and molecular subtypes. Vitek2 MALDI and species-specific PCR identified <i>Enterobacter hormaechei</i> subsp. <i>xiangfangensis</i> (47.14%) as the emerging CR-ECC subspecies in Kolkata. The predominating carbapenemase and extended-spectrum β-lactamase genes found were <i>bla</i>_NDM-1 (51.42%) and <i>bla</i>_CTX-M-15 (27%), respectively. Besides, <i>bla</i>_NDM-4, <i>bla</i>_NDM-5<i>, bla</i>_NDM-7<i>, bla</i>_CMH-3, <i>bla</i>_SFO-1, <i>bla</i>_OXA-181, <i>bla</i>_OXA-232, <i>bla</i>_KPC-3, and <i>bla</i>_DHA-7 genes were also detected, which were not previously reported from India. A multitude of Class 1 integrons (including In180, In4874, In4887, and In4888, which were novel) and plasmid replicon types (IncFIB, IncFII, IncX3, IncHI1-HI2, IncC, and IncR) involved in AMR dissemination were identified. Reverse transcription-PCR and western blot revealed that carbapenem resistance in non-carbapenemase-producing CR-ECC isolates was contributed by elevated levels of <i>ampC</i>, overexpression of <i>acrAB,</i> and loss of <i>ompF</i>. A total of 30 distinct sequence types (STs) were ascertained by multi-locus sequence typing; of which, ST2011, ST2018, ST2055, ST2721, and ST2722 were novel STs. Pulsed-field gel electrophoresis analysis showed heterogeneity (69 pulsotypes with a similarity coefficient of 48.40%) among the circulating isolates, suggesting multiple reservoirs of infections in humans. Phylogenetically and genetically diverse CR-ECC with multiple AMR mechanisms mandates close monitoring of nosocomial infections caused by these isolates to forestall the transmission and dissemination of AMR.IMPORTANCEThe emergence and extensive dissemination of the carbapenem-resistant <i>Enterobacter cloacae</i> complex (CR-ECC) have positioned it as a critical nosocomial global pathogen. The dearth of a comprehensive molecular study pertaining to CR-ECC necessitated this study, which is the first of its kind from India. Characterization of blood isolates of CR-ECC over the last 6 years revealed <i>Enterobacter hormaechei</i> subsp. <i>xiangfangensis</i> as the most prevalent subsp., exhibiting resistance to almost all antibiotics currently in use and harboring diverse transmissible carbapenemase genes. Besides the predominating <i>bla</i>_NDM-1 and <i>bla</i>_CTX-M-15, we document diverse carbapenemase and <i>AmpC</i> genes, such as <i>bla</i>_NDM-4, <i>bla</i>_NDM-7, <i>bla</i>_OXA-181, <i>bla</i>_OXA-232, <i>bla</i>_KPC-3, <i>bla</i>_CMH-3, <i>bla</i>_SFO-1, and <i>bla</i>_DHA-7, in CR-ECC, which were not previously reported from India. Furthermore, novel integrons and sequence types were identified. Our findings emphasize the need for strengthened vigilance for molecular epidemiological surveillance of CR-ECC due to the presence of epidemic clones with a phylogenetically diverse and wide array of antimicrobial resistance genes in vulnerable populations.