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

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 blaNDM-1 (51.42%) and blaCTX-M-15 (27%), respectively. Besides, blaNDM-4, blaNDM-5, blaNDM-7, blaCMH-3, blaSFO-1, blaOXA-181, blaOXA-232, blaKPC-3, and blaDHA-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 blaNDM-1 and blaCTX-M-15, we document diverse carbapenemase and AmpC genes, such as blaNDM-4, blaNDM-7, blaOXA-181, blaOXA-232, blaKPC-3, blaCMH-3, blaSFO-1, and blaDHA-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.

Multidrug resistance is a common trait in ECC globally, partly due to "intrinsic resistance" to β-lactam drugs and the ability to rapidly acquire mobile genetic ele ments through horizontal transfer (6).The ECC members are the second most common β-lactam-resistant genus in Enterobacteriaceae (8).ECC are resistant to penicillins and first-and second-generation cephalosporins due to efflux pumps and an inducible chromosomal AmpC-type (class C) cephalosporinase (9).Resistance to third-generation cephalosporins and aztreonam is often caused by mutations in ampD, resulting in constitutive hyperproduction (derepression) of AmpC and plasmid-mediated extendedspectrum β-lactamases (ESBLs) such as bla TEM , bla SHV , and bla CTX-M (9).Due to rising resistance to aminoglycosides, fluoroquinolones, and third-generation cephalosporins, the carbapenem group (meropenem, imipenem, and ertapenem) "last-resort antibiotics, " have replaced them in multi drug resistant (MDR) ECC treatment (9).However, carba penem resistance in ECC has risen expeditiously in the last decade, drawing clinical attention.ECC acquire carbapenem resistance by acquiring plasmid-encoded carbapene mase genes (such as bla OXA-48 -like variants, bla VIM , bla IMP , bla KPC , and bla NDM-1 ) and the constitutively overexpressing AmpC (especially the bla ACT-type ) and/or ESBL in conjunc tion with a disruption in membrane permeability [the loss or reduction of the outer membrane proteins (OMPs) OmpF and/or OmpC] and efflux pumps (5,10,11).Recently, a novel plasmid-mediated bla CMH-3 gene has also been discovered, which is closely related to chromosomal bla ACT AmpC-type β-lactamases (12).
In clinical settings, ESBL genes like bla TEM , bla SHV , and bla CTX-M types are frequently observed (13).Over the last decade, reports of a rare ESBL gene bla SFO-1 (a plasmidmediated non-TEM, non-SHV, and non-CTX-M class A ESBL) existing alongside carbape nemase genes have gained importance. bla SFO-1 is an inducible β-lactamase, capable of hydrolyzing cefotaxime very efficiently and ceftazidime poorly and is apparently inactive toward cephamycins and carbapenems (14).
The pathogenicity of Enterobacteriaceae can be enhanced by introducing additional virulence factors in addition to carbapenem resistance (15).Like other Enterobacterales, ECC strains have multiple secretion systems (OMPs), siderophores, biofilm mediators, heavy metal resistance, and stress response mechanisms (6,15).Unlike other Enterobac terales, however, only a few of ECC members have undergone a complete genome-wide analysis, characterizing specific virulence-associated genetic determinants.
Currently, whole genome sequencing (WGS) has become an indispensable tool for generating in-depth epidemiological data on genomic diversity, bacterial distributions, accurate species identification (3), antimicrobial resistance (AMR) gene profiles, and molecular subtypes.Diversity in AMR genes, sequence types (STs), and wide taxo nomic varieties among ECC were observed through worldwide surveillance.Addition ally, carbapenem-resistant Enterobacter cloacae complex (CR-ECC) exhibited regional distribution, with bla NDM predominating in China, bla VIM and bla OXA-48 in Europe, and bla KPC in North America (9).However, there is a dearth of data from the Indian subcon tinent.This study, therefore, focuses on the molecular mechanisms of AMR, plasmid profiles, and molecular subtypes of ECC isolated from hospitalized patients in Kolkata with special emphasis on resistance to carbapenems.This study will help researchers comprehend the burden of AMR caused by this newly discovered carbapenem-resist ant pathogen, which is crucial for developing effective treatment and stricter infection prevention and control measures.

CR-ECC subspecies
A total of 70 CR-ECC blood isolates obtained from eight hospitals in Kolkata during January 2017 and December 2022 were included in this study.The rate of isolation of CR-ECC from blood samples in Kolkata cannot be ascertained because the total number of samples screened in each hospital was inaccessible.The 16S rRNA PCR and speciesspecific multiplex PCR identified E. hormaechei (n = 42) as the predominating CR-ECC among blood isolates, followed by E. cloacae (n = 22).The most common subspecies of E. hormaechei was found to be E. hormaechei subsp.xiangfangensis (Enterobacter xiangfangensis) (n = 33).In addition, E. hormaechei subsp.steigerwaltii (Enterobacter steigerwaltii) (n = 8) and E. hormaechei subsp.hoffmannii (n = 1) were also found in circulation.Furthermore, sporadic cases of carbapenem-resistant E. kobei (n = 2), Enterobacter sichuanensis (n = 1), Enterobacter roggenkampii (n = 1), and Pseudentero bacter timonenensis (n = 1) were also recorded in this study.

Carbapenem resistance
Fifty-three of the 70 study strains were found to be carbapenemase producers with bla NDM (n = 46) being the predominant, followed by bla Oxa-48 -like variant (n = 9).Additionally, one isolate was found to be a bla KPC (KPC-3) producer.The most common bla NDM found in the study was bla NDM-1 (n = 36), followed by bla NDM-5 (n = 5), bla NDM-7 (n = 4), and bla NDM-4 (n = 1).The different bla OXA-48 -like variants found in the study strains were bla OXA-181 (n = 7) and bla OXA-232 (n = 2).A total of six study strains were found to be coproducers of bla NDM and bla oxa-48 -like variants.No additional carbapene mase genes were found.At least two different carbapenemase genes were present in eight strains.The genetic environment of the different carbapenemase genes is depicted in Fig. 1.

ESBL and ampC genes
The bla CTX-M-15 (n = 19) was the principal ESBL gene found in the study isolates.Besides, bla OXA-10 (n = 3) and bla OXA-21 (n = 1) were also found.Notably, all the ESBL genes co-existed with carbapenemase genes.The bla SFO-1 gene was detected in five CR-ECC isolates, of which three were found in E. hormaechei subsp.xiangfangensis.All five bla SFO-1 positive isolates co-harbored bla NDM-1 and/or bla OXA-232 genes.

Other resistance genes and integrons
Among the 16S methyltransferase genes conferring resistance to aminoglycosides, nine isolates were positive for armA, while three isolates were co-producer of armA and rmtB.

Virulence determinants
The study isolates possessed the biofilm-forming wcaABCDEFKLM, wza, and csgABCEFG genes, while other related genes like wzb, wzc, or manC genes were absent.The characteristic aerobactin (siderophore) genes like fhuAB and iucAB were present in all study isolates but the complete operon (fhuABCD and iucABCD) was missing.The heavy metal resistance genes like arsH (arsenic), pcoABCDEF (copper), terABCDWYZ (tellurite), and zitB (zinc) were found in all strains.Environmental stress response genes (clpB, hsp, cspCE, and ompR) and antibiotic stress response genes (marAB, araC, and ramA) were detected in all CR-ECC strains.Notably, the carbapenemase-producing ECC strains were found to be lacking the efflux pump regulator soxSR gene.All except two isolates carried the genes mdtABCDGHIJKL, macA, and macB which mediate inherent resistance to a wide variety of drugs.In addition, genes like fimH and papCD, which help in adhesion to extracellular matrix were also present.All carbapenem-producing isolates carried the T6SS (impABCDEFGHJKM, vasDJ, and evpB) in addition to the T1SS (hlyD and tolC), T2SS (ftsY, tatABC, yajC, and yidC), T4SS (traABCDEFGHIJKLMUW, trbBCJL, and rhs), and T5SS (bamABCD).The presence or absence of the virulence determinant genes is depicted in Fig. 3B.

Phenotypic detection of the role of efflux pump in AMR
The MIC of ertapenem showed a fourfold decrease or more in the presence of all three efflux pump inhibitors (NMP and PAβN) tested (Table 2), suggesting the existence of an association between efflux pump and carbapenem resistance.

Western blotting
The protein levels of AcrA and AcrB were found to be higher than the control in CR-ECC isolates.Among the OMPs, increased levels of OMP C and lower levels of OMP D were  observed in some isolates.Furthermore, loss of OMP F and unchanged levels of OMP A were found in all isolates (Fig. 4; Fig. S1).

Real-time PCR of detection of the role of efflux pump genes, porins, and ampC in AMR
Reverse transcription (RT-PCR) of the efflux pump gene (acrAB, tolC) and regulatory genes (soxR, ramA) showed significantly elevated levels of gene expression (Table 2), validating the phenotypic association between AMR and efflux pump in CR-ECC.Furthermore, all strains showed increased ampC gene expression.Additionally, ompC was more highly expressed in 29 ECC isolates than in the control ATCC13047, while ompF was significantly less expressed in these ECCs.No change in the expression of ompA was observed in the study isolates.

Pulsed-field gel electrophoresis
Restriction digestion of 70 isolates with Xba-I revealed the presence of 69 pulsotypes in pulsed-field gel electrophoresis (PFGE) with a similarity coefficient of 48.40% (Fig. 7).No correlation was found among the strain pulsotype and hospital.There was no predominance of any pulsotype in any hospital.However, only one pair of isolates (P955: NRCR 5 and 22: NRCR 6) was found to be clonal (100% similarity), which was isolated in 2019 from the same hospital (Hospital N) but in different months and from different patients.The phylogenetic diverse ECC strains in circulation suggest diverse reservoirs of infection and enhanced risk of dissemination of CR-ECC strains.

DISCUSSION
In India, no comprehensive study solely focused on carbapenem-resistant ECC strains has been done in 15 years.A handful of studies on the Enterobacteriaceae family from southern India have mentioned the isolation of E. cloacae harboring bla NDM-1 (sample type was not specified) (16,17) and E. cloacae possessing bla NDM-1 and bla OXA-48 -like variants (bla OXA-181 ) from blood samples (17).One carbapenem-resistant E. hormaechei isolate has also been reported from western India (18).However, unlike this study, the previous studies from India have neither explored the detailed mechanisms of carbape nem resistance in ECC nor engaged in any whole genome-based study.Additionally, the speciation of ECC strains causing BSI and their molecular types including multi-locus sequence types were not mentioned in any of the previous studies carried out in India.In this study, E. hormaechei was found to be the major blood isolate among all the Enterobacter sp.surpassing E. cloacae which was in agreement with earlier studies globally (19).To the best of our knowledge.This is the first study from India reporting the emergence of carbapenem, colistin, and ceftazidime-avibactam resistance in E. xiangfangensis among the ECC complex.
A diverse array of carbapenemases were found in our study (Fig. 1).Both intraspecies and interspecies difference was observed in the genetic environment (upstream and downstream) of all the carbapenem genes identified in this study.This was mainly due to differences in the insertion sequence (IS) elements, transposons, and recombinase genes.The Indian subcontinent is a major hub (20) for bla NDM carbapenemase, and consequently, the bla NDM-1 gene was found to be the dominant carbapenemase in this study.Earlier studies from China, Brazil, and Lebanon have also reported bla NDM-1 in E. hormaechei species (19).It is noteworthy that besides E. hormaechei and E. cloacae, bla NDM-1 was also discovered in E. kobei, E. sichuanensis, E. roggenkampii, and P. timonen sis in this study, which is the first of its kind from India (Fig. 3).In addition, bla NDM-4 and the rare allele bla NDM-7 were also identified in E. hormaechei, which was previously reported from Spain and Canada only (19).The presence of bla NDM-5 in E. xiangfangensis and E. steigerwaltii strains was also not previously reported in India.
The ECC strains with bla KPC gene alleles have been geographically restricted to north, south, and central America (19).In this study, we identified bla KPC-3 -producing E. xiangfangensis, which has not been previously reported from India, although it was reported in E. hormaechei in the USA (19).Interestingly, this isolate also harbored bla NDM-1 and both these genes were transmissible but via two different plasmids of IncC and IncF2 types, respectively.A novel 78 kb untypeable pBT202 plasmid (Fig. 8A) carrying bla NDM-1 in E. kobei was a surprise finding.
The bla OXA-48 -like variants carbapenemases are not very frequently reported in Enterobacter sp., although few variants have been reported in E. steigerwaltii and Enterobacter oharae from middle-eastern countries, Vietnam, Belgium, and Brazil in the past (19).In this study, we report emergence of bla OXA-181 and bla OXA-232 in E. xiangfan gensis and E. kobei, which has not been previously documented in Enterobacter sp.This is also the first report from India demonstrating the co-transmission of bla NDM-5 and bla OXA181 via the same plasmid (IncC) in E. xiangfangensis (Fig. 8B).The conjugative IncX3 plasmid of 48 kb size carrying bla OXA-181 gene in E. kobei was also unique (Fig. 8C), confirmed by Plasmid Finder curator.
In non-CP-ECC strains (E.cloacae subsp.cloacae), resistance to carbapenem was due to increased expression of efflux pump genes (acrA-acrB and its regulator soxS/ramA), along with elevated expression of ampC cephalosporinase, upregulation of ompC, and downregulation of ompF (Fig. 4).These findings substantiated earlier reports, which suggested that a decreased expression of ompF could result in a compensatory increase in ompC expression (21,22).
This study identified a plethora of STs (n = 30) not previously reported from India (Fig. 5), which was supported by STs from China and the USA (9,21,23).The findings of our study demonstrate an extensive variety of CR-ECC strains and variations in genetic relatedness, in line with previous research (24).The most epidemic STs in ECC worldwide were ST114, ST171, and ST78, but the predominating ST varied by country and region (9,21).None of the STs were predominated in this study.The only subsp.-specificST in this study was ST520, which was previously reported for E. kobei (8).STs are usually linked to an AMR phenotype, but this study found no such association.Four common STs associated with ESBLs in Enterobacter spp.worldwide are ST66, ST78, ST108, and ST114, all of which carry bla CTX-M-15 (9).Out of these four common ESBL STs, only ST114 was found in three E. xiangfangensis study isolates.Two of these isolates had bla NDM-7 and bla CTX-M-15 on a transmissible IncX3 plasmid, while another ST114 isolate did not.ST171 has become the most common CR-ECC clone in the USA due to the widespread use of carbapenems and fluoroquinolones.Most ST171 isolates carry bla KPC-3 , but some may carry bla KPC-2 or bla KPC-4 (9,24).Interestingly, two ST171 isolates in this study carried the bla NDM-1 gene on IncX3 or IncFII (Yp) plasmids instead of bla KPC genes.PFGE was found to be more discriminatory than MLST.Similar to earlier reports, the study isolates with identical STs had different pulsotypes, suggesting the circulation of diverse clones of Enterobacter spp. in this region.Unlike PFGE dendrogram, wg-MLST clustered ECC study strains from the same subspecies into the same clades more efficiently (Fig. 6).
There are a number of limitations in this study.The sample collection was limited to a few hospitals in Kolkata, so it may not accurately represent CR-ECC across India.
The hybrid assembly could not be performed for all isolates, limiting plasmid mapping analysis for all CR-ECC isolates.WGS identified a number of virulence factors in Entero bacter spp., but expression levels of virulence genes were not evaluated as it was beyond the scope of this study.

Conclusion
The CR Enterobacterales family causes most nosocomial infections worldwide.WGS analysis identified E. xiangfangensis as the major CR-ECC pathogen.However, clinical microbiology laboratories cannot accurately identify ECC at the species level.Variations in study methods may also skew results.Sequencing-based identification studies are crucial because pan-resistant nosocomial pathogens may usher in a "post-antibiotic era." In ECC study isolates, carbapenemase (bla NDM ), overexpression of ESBL (bla CTX- M-15 ), and/or AmpC (bla DHA and bla ACT ), and porin loss were the main mechanisms of carbapenem resistance.WGS could detect emerging and/or low-incidence genes like bla SFO-1 and bla CMH-3 that were not routinely monitored for AMR.CR-ECC's diversity and high plasmid uptake suggest a specialized antibiotic resistance prevention strategy.

Strain collection and identification
CR-ECC strains were isolated from the blood of patients admitted to the intensive care unit of eight tertiary care hospitals in Kolkata from January 2017 to December 2022.Patients had not received any carbapenem antibiotic prior to hospital admission.Pure cultures of the isolates were sent to the bacteriology division at ICMR-NICED after they had been identified at the respective hospitals using the VITEK MS and VITEK 2 Compact system (bioMérieux, France).The speciation of ECC was confirmed at ICMR-NICED by both 16S rRNA PCR (6) and species-specific multiplex PCR (25).

Phenotypic detection of carbapenemases
Phenotypic detection of carbapenemases was carried out by Rapidec Carba NP-kit (bioMérieux, Marcy-l'Étoile, France) following the user's manual.The production of carbapenemase by bacteria is indicated by a color change from red to yellow within 30 min.

Determination of transferability of AMR genes by conjugation and characteri zation of transconjugants
The conjugal transfer of AMR genes from test strains (donor) to plasmid-free sodium azide-resistant E. coli J53 (recipient) was tested by liquid mating assay at 37°C.An isolated fresh colony of donor and recipient cells from Chrome Agar plates (Difco) was inoculated into separate tubes with 3 mL LB broth and incubated at 37°C for 18 h with shaking.Donor and recipient were subsequently mixed in fresh LB broth in a 1:5 ratio and incubated without shaking.After 18 h, cells were serially diluted and spread on chrome agar plates with 100 mg/L sodium azide and meropenem (2 mg/L) (Sigma-Aldrich, USA).After overnight culture, biochemical profiling confirmed transconjugants (TC).The MICs of antimicrobials were determined by BMD, and AMR determinants in the TC were confirmed by PCRs using TC plasmid DNA as a template.

Determination of plasmid profiles and their replicon-typing
Plasmid DNA was isolated from the wild-type strains and the TCs by both the Kado-Liu method (35) and Qiagen Plasmid Midikit (Qiagen, Germany).The extracted plasmid DNA was electrophoresed in 0.8% agarose gel at 70 V using 1× TBE running buffer and stained with ethidium bromide (0.5 g/mL).Plasmid sizing was done by FP-Quest (Bio-Rad, USA) software using E. coli V517 and Shigella flexneri YSH6000 as molecular weight markers.Replicon-typing PCR elucidated the plasmid incompatibility types (36).

Determination of the role of efflux pump in AMR
The role of the efflux pump in carbapenem resistance was determined phenotypically by measuring the MICs of ertapenem both in the absence and in the presence of efflux pump inhibitors (EPI) 1-(1-naphthylmethyl)-piperazine (NMP; 25 mg/L) and PAβN (1 mg/L) and (Sigma-Aldrich).MIC was determined by the BMD method and carried out in triplicates in separate experiments.A fourfold reduction or more in MIC in the presence of EPI was considered significant.
To eliminate any chances of an incorrect reading of protein levels in western blot, real-time PCR was subsequently performed to determine the expression levels of efflux pump and outer-membrane porins.

RT-PCR for determining efflux pump (acrA, acrB, and tolC) and their regulators (ramA, marA, and soxS) and outer membrane porin (OmpC, OmpD, and OmpF) gene expression
The bacterial isolates were cultured overnight at 37℃ in LB broth under antibiotic (meropenem) stress.Total RNA was extracted from the log-phase of two replicate cultures, using Trizol Reagent.Quantification and quality check of the extracted RNA was done using NanoDrop Biophotometer Plus (Eppendorf, Germany).Total RNA was digested with RNase-free DNaseI (New England Biolabs, USA) to remove any contami nating genomic DNA.Thereafter, cDNA was synthesized employing QuantiNova cDNA Synthesis Kit (Qiagen) and DNaseI-treated RNA as a template.To monitor gene expres sion levels, real-time RT-PCR using specific gene primers (as listed in Table S1) was carried out in Step One Plus Real-Time PCR System (Applied Biosystems).The 2 -ΔΔCT method (38) was used to calculate relative RNA expression levels, after normalizing with ribosomal housekeeping gene rpoB.All real-time RT-PCR experiments were repeated thrice, with rpoB gene as an internal control (39).Each target gene's relative expression was then calibrated against the expression of an E. cloacae ATCC 13047 pan susceptible isolate (expression = 1).A twofold increase in gene expression in comparison to the reference strain was considered significant overexpression.

PFGE
Clonality of the CR-ECC isolates was determined by PFGE following standard proto col (CDC, 2013), using XbaI (50 U/plug) restriction digestion enzyme and CHEF-DRIII pulsed-field electrophoresis systems (Bio-Rad), with switch times of 5 and 35 s, and run duration of 21 h.The XbaI-digested Salmonella Braenderup H9812 was used as a molecular weight ladder, while XbaI-digested E. cloacae ATCC 13047 and E. hormaechei ATCC 700323 were used as positive controls.FP-Quest v4.5 (Bio-Rad) generated the dendrogram based on the Dice coefficient with 1.5% position tolerance.The PFGE band patterns were interpreted as previously described (40).

Whole genome shotgun sequencing analysis
The Qiagen DNeasy blood & tissue kit (Qiagen) was used for genomic DNA isolation.The DNA was quantified using Qubit 3.0.(Thermo Fisher Scientific, USA).Nextera XT was employed for DNA library preparation for paired-end sequencing (2 × 501 cycles) and sequenced on Illumina Novaseq 6000 platform using the protocol for v1.5 chemistry and a 2 × 250 bp read length (Illumina Inc., San Diego, CA).

FIG 1 FIG 2
FIG 1 Schematic representation of the genetic environment of the various carbapenemases (bla oxa-48 -like variant, bla KPC , and bla NDM ) identified in our study.

FIG 3 (
FIG 3 (A) Heatmap of CR-ECC study isolates depicting respective AMR phenotype, AMR gene, plasmid replicon typing, and sequence types.Cluster analysis was performed on the heatmap using Euclidean distance.(B) Heatmap depicting the presence or absence of different virulence determinants found in CR-ECC study isolates.

FIG 4
FIG 4 Western blot of AcrA, AcrB, and outer membrane porin (Omp) protein expression in CR-ECC isolates that do not produce carbapenemases.Enterobacter.cloacae ATCC 13047 was used as a control strain.(A) The AcrA protein in the U3306 isolate displayed increased protein expression in comparison to the control strain, while the SD25 isolate displayed decreased band intensity.The AcrA levels in isolates U3639 and P2253 were unchanged.(B) In the case of AcrB, all four test samples showed increased protein intensity when compared to the control.(C) No differences were observed between test and control strains for OmpA expression.(D) The expression of porin OmpF was completely lost in test strains.(E) The test strains showed overproduction of OmpC.(F) Complete loss of OmpD was found in U3306, along with decreased expression in U3262 and U3639, and increased expression in P2253 as compared to control.

FIG 5
FIG 5 Minimum spanning tree based on MLST allelic profile: pictorial depiction of genetic relationship of different STs of ECC-CR clinical isolates found in this study by goeBURST and PHYLOViZ 2.0.Each kurtosis or node (circle) corresponds to one ST designated by a particular number, and the size of the circle is proportional to the number of isolates (genomes).Each circle is divided into cross-sections of different colors.Each color represents the different countries in which the STs are found (genomes downloaded from the pubMLST database, last accessed on 25 September 2023).The numbers on the branches represent the allelic differences.Branches with a length greater than three locus variants are outlined in dashes.All the STs obtained in this study (India) are represented in red color; all have been validated and curated by the pubMLST curator, and this is now available on the pubMLST website.The red-colored circles with yellow halo (ST2011, ST2018, ST2055, ST2721, and ST2722) depict the novel STs discovered in this study.Although ST96, ST124, ST346, and ST265 are represented only in red color, these are not exclusive to India but rather our study.This representation is because the pubMLST has only the Sanger sequence but no whole genome records for these STs.

FIG 7
FIG 7 Dendrogram of PFGE cluster analysis of XbaI-digested patterns of 70 carbapenem non-susceptible ECC isolates along with their year of isolation and hospital names (abbreviation).

TABLE 2
Effect of efflux pump inhibitor PAβN on different antibiotic susceptibilities and fold change in efflux pump, regulators, and OMP by RT-PCR