Whole genome sequence-based molecular characterization of blood isolates of carbapenem-resistant
Enterobacter cloacae
complex from ICU patients in Kolkata, India, during 2017–2022: emergence of phylogenetically heterogeneous
Enterobacter hormaechei
subsp.
xiangfangensis
Authors: Halder Gourab , Chaudhury Bhaskar Narayan , Mandal Subhranshu , Denny Priyanka , Sarkar Deotima , Chakraborty Mandira , Khan Ujjwayini Ray , Sarkar Soma , Biswas Bedobroto , Chakraborty Arindam , Maiti Sourav , Dutta Shanta 
Published: February 2024

ABSTRACT

Blood-borne infections caused by the carbapenem-resistant
Enterobacter cloacae
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 (
n
= 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
Enterobacter hormaechei
subsp.
xiangfangensis
(47.14%) as the emerging CR-ECC subspecies in Kolkata. The predominating carbapenemase and extended-spectrum β-lactamase genes found were
bla
NDM-1
(51.42%) and
bla
CTX-M-15
(27%), respectively. Besides,
bla
NDM-4
,
bla
NDM-5
, bla
NDM-7
, bla
CMH-3
,
bla
SFO-1
,
bla
OXA-181
,
bla
OXA-232
,
bla
KPC-3
, and
bla
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
ampC
, overexpression of
acrAB,
and loss of
ompF
. 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.

IMPORTANCE

The emergence and extensive dissemination of the carbapenem-resistant
Enterobacter cloacae
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
Enterobacter hormaechei
subsp.
xiangfangensis
as the most prevalent subsp., exhibiting resistance to almost all antibiotics currently in use and harboring diverse transmissible carbapenemase genes. Besides the predominating
bla
NDM-1
and
bla
CTX-M-15
, we document diverse carbapenemase and
AmpC
genes, such as
bla
NDM-4
,
bla
NDM-7
,
bla
OXA-181
,
bla
OXA-232
,
bla
KPC-3
,
bla
CMH-3
,
bla
SFO-1
, and
bla
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.