The emergence of clonally diverse carbapenem-resistant Enterobacter cloacae complex in West Bengal, India: A dockyard of β-lactamases periling nosocomial infections

doi:10.21203/rs.3.rs-3148026/v1

Download PDF

Research Article

The emergence of clonally diverse carbapenem-resistant Enterobacter cloacae complex in West Bengal, India: A dockyard of β-lactamases periling nosocomial infections

https://doi.org/10.21203/rs.3.rs-3148026/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 21 Nov, 2023

Read the published version in International Microbiology →

You are reading this latest preprint version

Carbapenem-resistant Enterobacter cloacae complex (CRECC) constitutes a global public health threat challenging clinical treatment and infection control, especially in low- and middle-income countries such as India. We analyzed the antimicrobial susceptibility, major β-lactamase genes, plasmid profiles, and genetic relatedness to understand the molecular epidemiology of CRECC clinical isolates (n=44) in West Bengal, India, during 2021-2022. The majority (>55%) of the isolates were resistant to fluoroquinolones, aminoglycosides, and co-trimoxazole, even >20% for tigecycline and >35% were extensively drug-resistant. Co-β-lactamase production was categorized into twenty-seven types, importantly NDM (84%), OXA-48 (40%), TEM (61%), CTX-M (46%), OXA-1 (55%), and MIR (27%). The NDM-1 and OXA-181 were major variants with the observations of two novel NDM-24 and -29 variants. Wide-range of plasmids (2 to >212kb) were harbored by the β-lactamase-producing isolates: small (91%), medium (27%), large (9%), and mega (71%). IncX3, ColE1, and HI2 were noted in about 30% of isolates, while IncF and R were carried by <20% of isolates. The clonally diverse CRECC isolates were noted to cause cross-infections, especially at superficial site, bloodstream, and urinary-tract. This is the first molecular surveillance on CRECC in India. The study isolates serve as the dockyard of NDM, TEM, and CTX-M harboring a wide range of plasmids. The outcomes of the study may strengthen local and national policies for infection prevention and control practices, clarifying the genetic diversity among CRECC. Extensive genomic study may further intersect the relationships between these different plasmids, especially with their sizes, types, and antibiotic resistance markers.

Enterobacter cloacae complex

carbapenem resistance

β-lactamases

epidemiology

plasmid

clone

India

Enterobacter cloacae complex (ECC), which belongs to the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), is now emerging as a potential drug-resistant pathogen, attributing morbidity and mortality and aggravating antimicrobial resistance (AMR) [1]. ECC, which comprises 22 species of Enterobacter, are saprophytes colonizing diverse niches, including soil, sewage, and the human gut. ECC is the third-major Enterobacterales species after Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae), causing several hospital-acquired infections (HAIs) and occasionally community-acquired infections, importantly soft tissue infections, urinary tract infections, pneumonia, and septicemia, resulting in severe case fatalities [1, 2]. These organisms are intrinsically-resistant to penicillins and first- and second-generation cephalosporins due to chromosomally-encoded AmpC cephalosporinases, which also confer resistance to third-generation cephalosporins and monobactams by induction and de-repression [2, 3]. Fourth-generation cephalosporins and/or carbapenems are the drugs of choice for ECC infections [2, 4]. Hit-or-miss use of broad-spectrum antibiotics, especially β-lactams, spirals the issue of AMR, promoting horizontal transmission of plasmid-borne antibiotic resistance markers among Gram-negative pathogens primarily in low- and middle-income countries (LMICs) [4, 5]. ECC is the second-largest β-lactam-resistant nosocomial pathogen in the family of Enterobacterales, catering to diverse extended-spectrum β-lactamase (ESBL) genes (TEM, SHV, and CTX-M) and carbapenemase genes (KPC, NDM, IMP, and VIM) [1, 2]. The lower isolation (2–10%) of ECC off-tracks its importance as a human pathogen, but the sustained increase of metallo-β-lactamase (MBL) carbapenemase-driven carbapenem resistance among them is confronting patient management and infection prevention practices in several nations, including India [5, 6]. Hence, the World Health Organization (WHO) recognized carbapenem-resistant (CR) ECC as a high-priority pathogen fostering new antibiotics and diagnostics [7]. The presence of large plasmids among the isolates of ECC potentiates the co-carrying and transfer of multiple antibiotic-resistant traits, attributing multidrug-resistant (MDR) or extensively drug-resistant (XDR) phenotypes [8, 9]. Over the past few decades, genomic heterogeneity within ECC has dispersed numerous high-risk MDR clones across the globe, twisting their containment strategy [2, 9]. To curb AMR, precise perception of the molecular epidemiology of CRECC is pivotal to breaking the chain of infection as well as the transmission of clinically important β-lactamases, especially in hospital settings. However, molecular studies have been hardly conducted in CRECC, particularly in AMR affected countries such as India. Owing to the public health concern of CRECC, the present study was designed to unveil the burden of clinically relevant β-lactamases, plasmid profiling, and clonality among CRECC isolates in West Bengal, India.

Bacterial isolates

The consecutive non-duplicate CRECC isolates were collected from inpatients at two Government district-level tertiary-care hospitals located in West Bengal, India: Nil Ratan Sircar Medical College and Hospital (NRSMCH), Kolkata, and Malda Medical College and Hospital (MMCH), Malda, between February 2021 and December 2022, and sent to Vidyasagar University, Midnapore for molecular characterization. The isolates were identified to species level using the VITEK2 instrument (bioMérieux SA, Marcy l’Etoile, France) and maintained as frozen stock at − 70°C in brain/heart infusion broth with 15% glycerol for further testing. HAI was defined as an infection occurring > 48 h after admission to the hospital. This was a retrospective cross-sectional study that was approved by the Institutional Ethical Committee (Ref. VU/IEC/122/2020) of Vidyasagar University, India. Patients were classified as paediatric (< 15 years), adult (15–64 years), and elderly (≥ 65 years).

Phenotypic resistotyping

The isolate was considered carbapenem nonsusceptible at meropenem and/or imipenem minimum inhibitory concentrations (MICs) of ≥ 2µg/ml using VITEK2 (bioMérieux). Additionally, susceptibility to ciprofloxacin, levofloxacin, amikacin, gentamicin, co-trimoxazole, and tigecycline was recorded. Fosfomycin and colistin susceptibilities were determined by Kirby-Bauer disc diffusion and colistin broth disc elution methods, respectively [10]. Quality control was assessed using E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853. Susceptibility results were interpreted by the Clinical Laboratory and Standard Institute guideline, except for tigecycline, for which the European Committee on Antimicrobial Susceptibility Testing guideline was followed [10, 11]. Resistant and intermediately susceptible categories were designated as non-susceptible. The XDR was described as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e., bacterial isolates remain susceptible to only one or two categories) [12].

Screening of β-lactamase genes

The DNA templates were prepared with a boiling process by dissolving a single bacterial colony in 200µl of distilled water, incubating at 95°C for 10min, freezing on ice for 5min, and collecting the supernatant following centrifugation. Sixteen major β-lactamase gene families were screened by end-point simplex and multiplex-PCRs using published primers with modifications (Supp. Table 1): penicillinase (OXA-1); AmpC cephalosporinases (ACC; DHA; MIR: ACT, CHE, and GC1; CMY-1: MOX and FOX; and CMY-2: CFE, LAT and BIL); ESBLs (TEM, SHV, CTX-M-1, CTX-M-2, and CTX-M-9) and carbapenemases (KPC, NDM, VIM, IMP, and OXA-48) [13, 14]. The 16S rRNA gene was amplified with a carbapenemase multiplex-PCR as an internal extraction control. Simplex- and multiplex-PCRs were adjusted at a total reaction volume of 15µl, including 3µl template, and 25µl, including 5µl template by Takara Taq DNA polymerase (DSS Takara Bio Inc., India) and QIAGEN multiplex-PCR master mix kit (QIAGEN, GmbH, Germany), respectively. The amplification was carried out on a Mastercycler nexus gradient (Eppendorf, Hamburg, Germany). PCR amplicons were electrophoresed on a 2% agarose gel pre-stained with EtBr and visualized by the Gel-Doc system (Bio-Rad, Hercules, US).

Table 1

Diversity of β-lactamases and plasmids among the study CREC isolates (n = 44) in West Bengal, India
β-lactamase				Plasmid
Category (n)	Class	Pattern (n)	Type	Inc type (n)	Number; size range (kb)
SBLP (4)	ESBL	CTX-M-1 (1)^Δ	B01	ColE1	1; 21.9
	Carbapenemase	NDM (1)	B02	Untypable	1; 21.9
		OXA-48 (2)	B03	X3 (1), R (1)	2; 2.5->212
DBLP (10)	AmpC/Carbapenemase	MIR/NDM (2)	B04	ColE1 (1)	2; 6.4 and 21.9
		CMY-2/NDM (1)	B05	FIB, FII	1; 2.1
	Penicillinase/Carbapenemase	OXA-1/NDM (1)	B06	HI2	2; 2.1 and 2.7
	ESBL/Carbapenemase	TEM/NDM (5)β	B07	X3 (2), A/C (1), HI1 (1), R (1)^ε	2–7; 2.2->212^*
		TEM/OXA-48 (1)	B08	R	1; 46.3
TBLP (11)	Penicillinase/Carbapenemase	OXA-1/OXA-48/NDM (3)	B09	ColE1 (2)^±	3–4; 2.1–212
	AmpC/Penicillinase/Carbapenemase	MIR/OXA-1/NDM (3)	B10	X3 (1), HI2 (1), R (1), ColE1 (1)	1–4; 2.1->212
	Penicillinase/ESBL/Carbapenemase	OXA-1/TEM/NDM (2)	B11	HI2 (2), X3 (1)	3–8; 2.1–212
		OXA-1/CTX-M-1/NDM (2)	B12	FIA (1), FIB (2), FII (2)	1–5; 2.1–212
	ESBL/Carbapenemase	TEM/CTX-M-1/NDM (1)	B13	FII	3; 6.9–212
QBLP (8)	AmpC/Penicillinase/ESBL	DHA/OXA-1/TEM/CTX-M-1 (1)^Δ	B14	HI2	2; 2.4–4.9
	AmpC/Penicillinase/ESBL/Carbapenemase	MIR/OXA-1/CTX-M-1/NDM (1)	B15	X3	5; 2.5–170
	Penicillinase/ESBL/Carbapenemase	OXA-1/TEM/CTX-M-1/NDM (2)	B16	Untypable	2–3; 3.3–150
		OXA-1/TEM/NDM/OXA-48 (1)	B17	Untypable	1; 212
	ESBL/Carbapenemase	TEM/CTX-M-1/NDM/OXA-48 (2)	B18	FII (1); HI2 (1), R (1)	3–6; 2.1–212
		TEM/SHV/CTX-M-1/NDM (1)	B19	FIA, FIB, X3, HI1	8; 2.1–212
PBLP (10)	AmpC/ESBL/Carbapenemase	MIR/CMY-2/TEM/NDM/OXA-48 (1)	B20	X3, HI2, ColE1	8; <2.1–212
		MIR/TEM/CTX-M-2/NDM/OXA-48 (1)	B21	X3, HI1	8; 2.5->212^@
		CMY-2/TEM/CTX-M-1/NDM/OXA-48 (1)	B22	FIA, FIB, FII	4; 2.2–212
	AmpC/Penicillinase/ESBL/Carbapenemase	MIR/ OXA-1/TEM/NDM/OXA-48 (1) ^$	B23	X3, ColE1	3; 5.5–51.7
		MIR/OXA-1/TEM/CTX-M-1/NDM (1)	B24	X3, HI2, ColE1	7; 2.4–212^#
		MIR/OXA-1/TEM/CTX-M-1/OXA-48 (1)	B25	Untypable	4; 2.6–162
	Penicillinase/ESBL/Carbapenemase	OXA-1/TEM/CTX-M-1/NDM/OXA-48 (4)	B26	X3 (1), ColE1 (1)^†	1–6; 2.1->212^&
HBLP (1)	AmpC/Penicillinase/ESBL/Carbapenemase	MIR/OXA-1/TEM/SHV/CTX-M-1/VIM (1)	B27	FIA, FIC, ColE1	8; <2.1–212
Abbreviations: SBLP, single β-lactamase producer; DBLP, double β-lactamase producer; TBLP, triple β-lactamase producer; QBLP, quadruple β-lactamase producer; PBLP, penta β-lactamase producer; HBLP, hexa β-lactamase producer.
Δ The isolates did not contain carbapenemase.
* Each of two isolates carried two mega-plasmids of 140 and 175kb, and 106 and 212kb, respectively.
@ The isolate carried three mega-plasmids of ≥ 200kb
# The isolate carried two mega-plasmids of 140 and 212kb
& One of the isolates carried two mega-plasmids of ≥ 212kb
β One of them carried NDM-29
$ The isolate carried NDM-7
† Two isolates did not harbor any Inc
± One isolates did not harbor any Inc
ε Two isolates did not harbor any Inc

Sequencing of NDM and OXA-48

The amplicons were purified by NucleoSpin Gel and PCR Clean-Up Kit (DSS Takara Bio Inc., India) following direct sequencing with published primers (Supp. Table 1) [14]. The sequencing results were analyzed by the Basic Local Alignment Search Tool (BLAST) database search program of the National Center for Biotechnology Information (NCBI).

Plasmid profiling

Small-to-large plasmids from the study isolates were extracted by the Kado and Liu method using E. coli V517 and Shigella flexneri YSH6000 as the reference strains [15]. Extracted plasmid DNA was electrophoresed on 0.8% SeaKem LE agarose (Lonza, Basel, Switzerland) in 1X TAE buffer at 80V for 2h, followed by EtBr staining and visualization with the Gel-Doc system (Bio-Rad). Plasmid incompatibility (Inc) types FIA, FIB, FIC, FII, A/C, HI1, HI2, L/M, R, X1-X4, and ColE1 were detected by the PCR-based replicon typing (PBRT) as described earlier (Supp. Table 1) [16, 17, 18, 19, 20].

Clonality assessment

Bacterial genomic DNA was extracted from overnight grown broth cultures with the Wizard Genomic DNA Purification Kit (Promega, Madison, US) and stored at -20⁰C for further use. The purity and quantity of extracted DNA were checked by a BioPhotometer Plus instrument (Eppendorf). Enterobacterial repetitive intergenic consensus (ERIC)-PCR was performed to assess clonal relatedness among the isolates [21]. Briefly, the amplification mixture (25µl) contained 1X ExPrime Taq buffer supplied with 2mM MgCl₂, 200µM of each dNTPs, 0.8µM of ERIC2 primer, 2U of ExPrime Taq DNA polymerase (Genet Bio, Chungnam, Korea), and 150ng of template DNA. PCR was performed in a Bio-Rad T100 thermocycler (Bio-Rad) as follows: an initial denaturation (95°C, 5min) followed by 35 cycles of denaturation (94°C, 45s), annealing (52°C, 1min), and extension (68°C, 3min) with a final extension (68°C, 10min). PCR amplicons were stored at 4⁰C until they were electrophoresed on 1.5% (w/v) SeaKem LE agarose gels at 80V for 2h containing 0.5X TBE buffer and 0.5µg/ml EtBr with 100bp DNA ladder and visualized by the Gel-Doc system (Bio-Rad).

Gel image analysis

Plasmid profiles of the isolates were analyzed with E. coli V517 (plasmid sizes: 54kb, 5.6kb, 5.1kb, 3.0kb, 2.7kb, 2.1kb) and Shigella flexneri YSH6000 (212kb) as the reference strains using Image Lab software ver. 5.2.1 for Windows (Bio-Rad). Extracted plasmids were categorized as small (≤ 25kb), medium (> 25 to 50kb), large (> 50 to 100kb), and mega (> 100kb) size. A dendrogram was constructed from ERIC-PCR fingerprint bands normalized against a 100bp DNA ladder using GelJ software ver. 2.0 for Windows [22]. Similarity analysis and clustering of matched bands were calculated using the Dice coefficient with 1.5% tolerance and the unweighted pair group method with arithmetic mean (UPGMA), respectively. The isolates showing ≥ 80% similarity were considered clonally related [21]. Different ERIC-PCR patterns were assigned for a new ERIC type. Each cluster of the dendogram was alphabetically marked.

Clinical information

Study isolates (n = 44) were cultured from superficial infection sites (n = 19, 43.2%), blood (n = 12, 27.3%), urine (n = 8, 18.2%), lower respiratory samples (n = 2, 4.5%), and sterile space fluid (n = 3, 6.8%) at NRSMCH (n = 34) and MMCH (n = 10) during the study period. The median age of patients was 31 years (range, 3 months to 74 years); the majority of them were adults (32, 72.7%) followed by paediatrics (9, 20.5%); and 26 (59.1%) patients were male.

Resistotypes

The CRECC isolates (n = 44) were largely resistant to fluoroquinoles (ciprofloxacin: 33, 75%; levofloxacin: 26, 59.1%), followed by aminoglycosides (gentamicin: 25, 56.8%; amikacin: 16, 36.4%) and sulfonamides (co-trimoxazole: 27, 61.4%) (Fig. 1). Resistance to colistin and tigecycline were noted in two (4.5%; MICs, 4 and ≥ 16µg/ml) and ten (22.7%; MIC range, 2-≥8µg/ml) isolates, respectively. All isolates remained susceptible to fosfomycin. Strikingly, sixteen (36.4%) isolates were XDR.

Versatility of β-lactamases

Of the 44 CRECC isolates, NDM was observed in 37 (84.1%) isolates, followed by OXA-48 (18, 40.1%) and VIM (1, 2.3%). Fourteen (31.8%) were double-carbapenemase producing (DCP) isolates of NDM and OXA-48. KPC and IMP were not detected. The isolates presented a total of twenty-seven β-lactamase types (B01 to B27), categorized as single β-lactamase producing (n = 4, B01-B03), double β-lactamase producing (n = 10, B04-08), triple β-lactamase producing (n = 11, B09-B13), quadruple β-lactamase producing (n = 8, B14-B19), penta β-lactamase producing (n = 10, B20-B26), and hexa β-lactamase producing (n = 1, B27) (Table 1). Thirty isolates (68.2%) contained ≥ 3 β-lactamases. TEM was detected in 27 isolates (61.4%), followed by OXA-1 (24, 54.5%) and CTX-M (20, 45.5%). CTX-M-1 (n = 19) predominated over CTX-M-2 (n = 1). Sixteen isolates (36.4%) were TEM-CTX-M-producers, and even one (B27) added SHV. Fifteen isolates (34.1%) carried AmpC cephalosporinase, including MIR (12, 27.3%), CMY-2 (3, 6.8%), and DHA (1, 2.3%) (Table 1, Fig. 2). Notably, one pus isolate (B27) co-carried six β-lactamases, including one AmpC (MIR); one penicillinase (OXA-1); three ESBLs (TEM, SHV, and CTX-M-1); and one MBL (VIM). Two isolates (meropenem MIC, 4µg/ml), did not accommodate any of the tested carbapenemases belonged to types B01 (CTX-M-1) and B14 (DHA/ OXA-1/TEM /CTX-M-1), respectively (Table 1).

NDM and OXA-48 variants

Twenty-three and eight representative isolates were analyzed for allelic discrimination of NDM and OXA-48, respectively. The NDM-1, -4, and − 5 were noted in eleven, seven, and five isolates, respectively. One showed NDM-7, while two novel variants (NDM-24 and − 29) were detected in each isolate. All were positive for the OXA-181 variant. DCP isolates carried either NDM-1/OXA-181 or NDM-4/OXA-181.

Prevalence of plasmids

The isolates (n = 44) contained up to eight plasmids ranging in size from 2 to > 212kb (Table 1, Fig. 3). Twenty-five isolates (56.8%) carried ≥ 3 plasmids. Notably, mega-plasmids of > 150kb were found in thirty-one isolates (70.5%), among which five (16.1%) co-carried two such plasmids. Large (60kb) and medium sized (> 25 to 50kb) plasmids were isolated from four (9.1%) and twelve (27.3%) isolates, respectively. Forty isolates (90.9%) carried small plasmids of ≤ 25kb, and the majority (32, 80%) were under < 10kb. Multiple small plasmids, even up to seven, were noted among the isolates. Seven isolates carried small plasmids only, were positive for up to five β-lactamases of the family AmpC, penicillinase, ESBL and carbapenemase. Plasmid carrying capability among single- to triple-β-lactamase producing isolates (n = 25) was found with a median of two, while it was four for tetra- to hexa-β-lactamase producers (n = 19).

Plasmid replicons

A total of 33 (75%) isolates belonged to one or more of the tested Inc types. IncX3 was present in twelve isolates (36.4%), followed by ColE1 (10, 30.3%) and IncHI2 (9, 27.3%) (Table 1). IncFI and II plasmids were carried by each of six isolates (18.2%), among which four contained both of them. The IncFIA combined with FIB and FIC in three and one isolate(s), respectively. IncA/C, HI1, and R were observed in one, three, and five isolate(s), respectively. IncF and HI were noted in isolates carrying plasmids of size ranges from 50 to > 200kb, except in three isolates with small plasmids of < 5kb. IncX3 was found in isolates harboring medium-size plasmids (> 25 to 50kb). Four isolates co-harbored three plasmid types, combining IncX3 with IncFI and HI1 or HI2 and ColE1 or A/C and R. IncL/M was not observed. The isolates with novel NDM-24 and − 29 variants co-carried plasmids in combination with IncFIA-FIB-X3-HI1 and IncX3-A/C-R, respectively (Fig. 4). The hexa-β-lactamase producing isolate (B27) was typed for IncFIA, FIC, and ColE1, carrying a total of eight plasmids of sizes ranging from < 2.1 to 212kb. Strikingly, nine of the Inc-untypable isolates contained medium- to mega-size plasmids along with different β-lactamases, including MIR, OXA-1, TEM, CTX-M, NDM, and OXA-48.

Genetic relatedness

The CRECC isolates (n = 30) represented 29 ERIC-types (E1 to E29) with > 60% similarity, mainly distributed under cluster A (Fig. 4). Of the thirty isolates, 23 and 7 were from NRSMCH and MMCH, respectively. Cluster A contained 22 isolates (E08 to E29) with 72% similarity, segregating into two sub-clusters: A1 (5 isolates at 78% similarity), and A2 (17 isolates at 76% similarity). Under cluster A, the related clones with ≥ 80% genetic similarity were as follows: E08 and E09, E10 to E12, E13 to E15, E16 and E17, E18 to E20, and E21 to E29. Two isolates, collected from different study sites, were found to be 100% genetically similar (E02). Twelve isolates under sub-cluster A2.2 carried two to four β-lactamases with > 80% of NDM as the sole carbapenemase producer, while sub-clusters A1 and A2.1 isolates (n = 10) predominantly harbored five β-lactamases (60%) and dual-carbapenemase (NDM/OXA-48; 70%). The isolates of cluster A carried up to three plasmids, among which IncX3 was common (6/22), while the rest were mostly IncF (4/8). Ten ERIC-types did not harbor any of the tested plasmids, and nine of them clustered under A. The NDM-5 variant isolates were limited to subcluster A2.2, while the NDM-1 and − 4 were genetically diverse. One urine isolate (E03) harboring a novel NDM-24 variant was distantly related to the rest of the isolates. One biopsy fluid-derived isolate (E08) positive for the novel NDM-29 allele belonged to subcluster A1 and showed > 90% genetic similarity with a urine isolate (E09). One NDM-7 positive blood isolate (E17), which belonged to subcluster A2.1, was in clonal relation (84%) with a urine isolate (E16) unique for CTX-M-2. Clonally diverse isolates indicated their capability to cause cross-infections at different anatomical sites, most importantly the superficial, bloodstream and urinary tract.

Since the first passage of NDM-1-producing Enterobacterales, including CRECC, from India to the US in 2010, the global situation of AMR has been remarkably worsened due to carbapenem resistance hard-hitting the good health and well-being goal under the 2030 Agenda for Sustainable Development [1, 5]. Recently, a few AMR surveillances recorded the highest resistance to carbapenems among ECC, posing a high-risk for healthcare fraternities in India [6, 23]. These observations prompted the present study, which focuses on clinically-relevant β-lactamases and plasmid profiling as well as genetic-relatedness among the nosocomial isolates of CRECC in West Bengal, India.

Polymyxins, tigecycline, aminoglycosides, and fosfomycin are recommended in treating MBL and OXA-48-producing CRE infections [1, 4, 21]. The present study showed high resistance rates (> 60%) to fluoroquinolones, aminoglycosides, and co-trimoxazole (Fig. 1). Recently, the ICMR reported 25–35% resistance to fluoroquinolones and > 24% to aminoglycosides among ECC in India [6]. Variable resistance rates were documented to fluoroquinolones (30–80%), aminoglycosides (10–80%), and co-trimoxazole (23–90%) from China, Nepal, Myanmar, Lebanon, Spain, and Italy [12, 24, 25, 26, 27, 28]. The in vitro efficacy of levofloxacin and amikacin was better than that of ciprofloxacin (27.2% vs. 20.5%) and gentamicin (52.3% vs. 36.4%) among the study isolates, respectively. These comparative findings among fluoroquinolones and aminoglycosides were in tandem with other studies [3, 6, 25, 29]. We had noted tigecycline resistance in > 22% of isolates, which opposed the findings of other researchers, even in CRECC [12, 25, 28, 29]. Two study isolates (4.5%) were colistin-resistant (MIC, ≥ 4µg/ml). Recently, dual resistance to colistin-carbapenem in ECC emerged from Myanmar (2/31) and China (20/113) [3, 12]. Combinations of polymyxins and aminiglycosides were preferred as empirical therapy in NDM and OXA-48-endemic regions like India [4, 6, 21]. But the emergence of aminoglycoside- and/or polymyxin-resistant CRECC complicated the management of CRECC-infected patients [7, 12, 24]. Fosfomycin resistance was not observed in this study. Strikingly, a twenty-year Italian surveillance study 2000–2019 documented a high resistance rate to fosfomycin (37%, 455/1230) in ECC [7]. Overall, we described > 36% of isolates as XDR, which was in parallel with few global studies [1, 3, 12, 26].

A diverse array of β-lactamases was detected among the study CRECC isolates. NDM was the common carbapenemase (> 80%), followed by OXA-48 (> 40%) among the study isolates (Fig. 2). The Indian subcontinent is an endemic region for NDM-producing Enterobacterales, causing several outbreaks [4, 5]. Since last decade, dominance of NDM-producing CRECC has been noted in India (36%, 13/36), Myanmar (100%, 31/31), Lebanon (59%, 24/41), and China (76%, 14/18) [12, 24, 27, 30]. The NDM-1 variant was prevalent (47.8%) among the representative NDM-positive study isolates (n = 23) followed by NDM-4 (30.4%) and 5 (21.7%). We had noted one NDM-7 and two novel variants, NDM-24 and NDM-29 (Fig. 4). Among NDM-producing ECC isolates, the NDM-1 was commonly noted than the NDM-5 in Lebanon (n = 41, 41% vs. 2%), China (n = 36, 67% vs. 8%), and Myanmar (n = 31, 81% vs. 4%) [12, 27, 31]. Oshiro et al. rarely observed NDM-4 and − 7 in four and one Myanmarese isolate(s), respectively [12]. This study was the first to docket NDM-24 and NDM-29 among CRECC in India. Recently, both of these variants of NDM were reported in E. coli and K. pneumoniae from China and Russia [31, 32]. OXA-48-type, the second-most common carbapenemase among CP-CRE, circulates in North Africa, the Middle East, and Europe and has silently spread in India in the absence of molecular surveillance [1, 4, 5]. Strikingly, OXA-48 was rarely (< 10%) reported in CRECC from India, Nepal, and Lebanon [5, 26, 27], while several nations did not trace its existence [2, 3, 12]. The OXA-181 variant was noted among the study isolates and was also detected in Indian and Lebanese isolates [5, 27]. On the other hand, the scarcity of VIM in this study support the earlier observations, except for one Indian study with a high occurrence of this MBL (63%, 23/36) [3, 5, 28, 30]. We did not find KPC- and IMP-producing isolates mimicking the previous reports from India, except for a few sporadic cases of IMP [5, 30]. The circulation of KPC-producing ECC was tracked in Brazil and the US, while IMP isolates were restricted in Japan [8, 21, 33]. Interestingly, Liu et al. detected both KPC and IMP in Chinese CRECC isolates [3].

In comparison to E. coli and K. pneumoniae, plasmidic AmpC production in ECC has been rarely investigated due to its inducible and constitutive nature [1, 2]. In this study, we observed the moderate presence of plasmidic AmpC-producing isolates (34%), among which MIR (> 27%) predominated, with a rare occurrence (< 7%) of CMY-2 and DHA (Fig. 2). Of note, our study did not discriminate between the variants of MIR. The MIR, especially ACT variants, was also highlighted in Nepal (98%, 125/127), Lebanon (54%, 32/59), and Myanmar (25.8%, 8/31), while it was rarely detected in China (< 3%, 6/212) [9, 12, 26, 27]. Rare or no findings of DHA-producing ECC were registered in different locations, except in China [9, 12, 27].

The global crisis of ESBL-producing ECC is an immediate threat for the healthcare fraternity. A high frequency of the study CREC isolates (> 70%) were ESBL producers, especially TEM (> 60%) and CTX-M-1 (> 45%) (Fig. 2). But, Mogasale et al. reported a low prevalence of ESBL-ECC (< 8%, 11/162) compared to CRECC (58%, 94/162) in India during 2016–2019 [23]. TEM and CTX-M genes were noted as major ESBLs in Brazil (94% and 100%), China (27% and 55%), and Nepal (82% and 94%) in ECC [3, 21, 26]. We detected one CTX-M-2-producing ECC, but none was CTX-M-9 (Fig. 4). Interestingly, Chinese researchers' monitored the co-prevalence of CTX-M-1, -2, and − 9 groups among ECC isolates [3, 24]. Rare detection of SHV was corroborated with other studies from India, Brazil, and Korea, except China (up to > 50%) [3, 9, 21, 25, 27, 30]. OXA-1 was detected in > 50% of the study isolates. This penicillinase was prevalent in Nepal (76%, 60/79) and Lebanon (54%, 32/59), but it was limited in China [9, 25, 26, 27].

Large plasmids get priority in AMR investigation, while small ones are involved in bacterial evolution and infection [8, 34]. The study isolates harbored several plasmids with a wide-range of sizes and replicon types. The prevalence of small (> 90%) and mega (> 70%) plasmids was noted among the isolates, while medium plasmids were carried by 27% of the isolates (Table 1). Based on the study plasmid profile, we selected the following Inc-types known to belong in these size ranges: FI and II (45-200kb), H1 and 2 (75-400kb), A/C (18-230kb), R (40-160kb), L/M (50-80kb), X (30-50kb), and ColE1 (≤ 25kb) [8, 16, 34]. The IncX3 (37%) was the most common among the CREC study isolates, with a moderate prevalence of IncFI and II (< 20%). IncX3 was rarely reported in ECC from China, Australia, and Gabon [8], while IncFI and II was frequently captured in India, Nepal, China, the US, Mexico, Spain, Australia, and Mozambique [8, 26, 28, 29, 30]. Recently, Mehri et al. noted the prevalence of IncFII (56%), followed by IncFIB (34%), and IncX3 (22%), among CREC in Lebanon [27].

ColE1 (31%), and IncHI2 (28%), were the second- and third-most common plasmids among the study isolates, respectively (Table 1). Low-to-moderate isolation of IncHI2 was reported, especially in non-NDM-producing CREC from Australia, China, the USA, India, and Nepal [8, 26, 30]. Small plasmids such as ColE1 were hardly considered for AMR investigation. ColE1 was the sole plasmid present in five study isolates carrying up to five β-lactamases (Table 1). Interestingly, three untypable study isolates carried small plasmids of < 10kb and were positive for up to four β-lactamases. In 2016, one study reported Col plasmids carrying TEM-1, SHV-12, KPC-2, and qnrB in CRECC from the USA [8]. Ramirez et al. explained the potential of small plasmids (≤ 25kb) that mostly belong to ColE1 among K. pneumoniae for carrying diverse β-lactamases, including CMY, TEM, CTX-M, and OXA-48 [34]. The rare detection of IncR, HI1, and A/C among the study isolates was corroborated with the findings from India, Nepal, Lebanon, and Canada [8, 26, 27, 30]. Eleven (25%) study isolates carrying medium- to mega-plasmids remained untypable by the tested Inc-types. The IncF and HI2 were detected in three study isolates carrying plasmids of < 5kb. But other researchers noted these Inc-types in large-to-mega plasmids [8, 16]. Yet, we have to investigate the individual plasmids in relation with Inc-types and β-lactamases.

ERIC-PCR, a band-based molecular typing method, is widely used for epidemiological investigations, especially in HAIs, due to its discriminatory power, reliability, rapidity, and low-cost. Polyclonal to monoclonal emergence and transmission of the ECC isolates was assessed in different geographic regions, verifying their pulsotypes and sequence-types, but rarely by ERIC-types [1, 2]. The study CRECC isolates (n = 30) were found to be clonally diverse, segregating 29 genotypes (Fig. 4). In cluster A, twenty-two isolates eventually formed six clonally-related ERIC-types. ERIC-PCR-based clonal and/or clonally-related transmission of ECC in association with or without carbapenem resistance was evidenced in Brazil, Spain, China, and India [21, 25, 28, 30]. The study isolates derived from superficial infection sites were noted to be genetically more diverse than blood and urine isolates. Trevino et al. depicted the clonal relatedness among the Spanish isolates, mainly from blood, urine, and superficial infection sites [28]. We did not observe any correlation between the ERIC-types and the β-lactamases and plasmid-types among the isolates; even the clonally related isolates carried versatile AMR traits. Cabrel et al. described the variable profiles of β-lactamase and plasmid among clonally-related Brazilian ECC hospital isolates [21]. Long-duration hospital- and community-based study may further categorically interpret the sustainability of individual CRECC clones in disseminating the clinically-relevant β-lactamases through plasmids.

To the best of our knowledge, this is the first molecular study merely on CRECC recovered from clinical settings in a border-state of eastern India focusing on the convenient and timely detection of β-lactamases, plasmids, and clonality. The clonally diverse fluoroquinolones-, aminoglycosides-, colistin-, and/or tigecycline-resistant isolates challenge treatment regimens and cross-border transmission of outbreak-prone β-lactamases. The prevalence of both large and small plasmids may exacerbate the crisis of AMR, along with bacterial evolution and infection. Our study was constrained by the limited number of CRECC isolates, the short duration, the lack of in-depth whole genome analysis, and the characterization of plasmids. Further, in-depth genomic surveillance studies are required to interpret the individual clones of CRECC for a better understanding of their transmission in hospital as well as community settings in India.

Author contributions

SD: Conceptualization, funding acquisition, supervision, formal analysis, methodology, and writing original draft and editing; AM: Formal analysis, validation and evaluation; MB: Formal analysis and data curation; SS: Formal analysis, resources of study isolates, and writing review and editing; PS: Resources of study isolates

All authors have read and approved the final version.

Funding

The study was financially assisted by the antimicrobial resistance project (grant no. AMR/Adhoc/ 241/2020-ECD-II) under the Indian Council of Medical Research (ICMR), New Delhi, India, including the fellowship of both AM and MB.

Availability of data and material

All data generated or analyzed during this study are included in this published article. Data are available from corresponding author on reasonable request.

Ethics approval

The study was approved by the Institutional Ethical Committee of Vidyasagar University, India (Ref. VU/IEC/122/2020).

Consent to participate

This was a retrospective study.

Consent for publication

Not applicable.

Conflicts of interest

No potential conflict of interest was reported by the author(s).

Annavajhala MK, Gomez-Simmonds A, Uhlemann A-C (2019) Multidrug-resistant Enterobacter cloacae complex emerging as a global, diversifying threat. Front. Microbiol 10:44. http://doi.org/10.3389/fmicb.2019.00044.
Bolourchi N, Giske CG, Nematzadeh S, Mirzaie A, Abhari SS, Solgi H et al (2022) Comparative resistome and virulome analysis of clinical NDM-1-producing carbapenem-resistant Enterobacter cloacae complex. J Glob Antimicrob Resist 28:254-263. https://doi.org/10.1016/j.jgar.2022.01.021.
Liu S, Huang N, Zhou C, Lin Y, Zhang Y, Wang L et al (2021) Molecular mechanisms and epidemiology of carbapenem-resistant Enterobacter cloacae complex Isolated from Chinese patients during 2004–2018, Infect Drug Resist 14:3647-3658. http://doi.org/10.2147/IDR.S327595.
Tilahun M, Kassa Y, Gedefie A, Ashagire M (2021) Emerging carbapenem-resistant Enterobacteriaceae infection, its epidemiology and novel treatment options: a review. Infect Drug Resist 14:4363-4374. https://doi.org/10.2147/IDR.S337611.
Das S (2023) The crisis of carbapenemase-mediated carbapenem resistance across the human-animal-environmental interface in India. Infect Dis Now 53:104628. https://doi.org/10.1016/j.idnow.2022.09.023.
The Indian Council of Medical Research (2022) Antimicrobial resistance research and surveillance network. Annual report, January 2021 to December 2021. https://main.icmr.nic.in/sites/default/files/upload_documents/AMR_Annual_Report_2021.pdf
Intra J, Carcione D, Sala RM, Siracusa C, Brambilla P, Leoni V (2023) Antimicrobial resistance patterns of Enterobacter cloacae and Klebsiella aerogenes strains isolated from clinical specimens: a twenty-year surveillance study. Antibiotics 12:775. https://doi.org/10.3390/antibiotics12040775.
Kopotsa K, Sekyere JO, Mbelle NM (2019) Plasmid evolution in carbapenemase-producing Enterobacteriaceae: a review. Ann N Y Acad Sci 1457:61-91. https://doi.org/10.1111/nyas.14223.
Wu C, Lin C, Zhu X, Liu H, Zhou W, Lu J et al (2018) The β-lactamase gene profile and a plasmid-carrying multiple heavy metal resistance genes of Enterobacter cloacae. Int J Genomics 2018:4989602. https://doi.org/10.1155/2018/4989602.
The Clinical and Laboratory Standards Institute (2021) Performance standards for antimicrobial susceptibility testing. 31st ed., CLSI M100. Wayne, PA. https://clsi.org/standards/products/microbiology/documents/m100/
European Committee on Antimicrobial Susceptibility Testing (2021) Breakpoint tables for interpretation of MICs and zone diameters. version 11.0. EUCAST. Vaxjo, Sweden. https://www.eucast.org/clinical_breakpoints.
Oshiro S, Tada T, Watanabe S, Tohya M, Hishinuma T, Uchida H et al (2020) Emergence and spread of carbapenemresistant and aminoglycoside-panresistant Enterobacter cloacae complex isolates coproducing NDM-type metallo-β-lactamase and 16S rRNA methylase in Myanmar. mSphere 5:e00054-20. https://doi.org/10.1128/mSphere.00054-20.
Mallick A, Roy A, Sarkar S, Mondal KC, Das S (2023) Customized molecular diagnostics of bacterial bloodstream infections for carbapenem resistance: a convenient and affordable approach. Pathog Glob Health 17:1-8. https://doi.org/10.1080/20477724.2023.2201982.
Lee JJ, Lee JH, Kwon DB, Jeon JH, Park KS, Lee C-R et al (2015) Fast and accurate large-scale detection of ß-lactamase genes conferring antibiotic resistance. Antimicrob Agents Chemother 59:5967-5975. https://doi.org/10.1128/AAC.04634-14.
Kado CI, Liu ST (1981) Rapid procedure for detection and isolation of large and small plasmids. J Bact 145:1365-1373. https://doi.org/10.1128/jb.145.3.1365-1373.1981.
Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ (2005) Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 63:219-228. https://doi.org/10.1016/j.mimet.2005.03.018.
Garcia-Fernandez A, Fortini D, Veldman K, Mevius D, Carattoli A (2009) Characterization of plasmids harbouring qnrS1, qnrB2 and qnrB19 genes in Salmonella. J Antimicrobial Chemother 63:274-281. https://doi.org/10.1093/jac/dkn470.
Villa L, Garcia-Fernandez A, Fortini D, Carattoli A (2010) Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. J Antimicrob Chemother 65:2518-2529. https://doi.org/10.1093/jac/dkq347.
Johnson TJ, Bielak EM, Fortini D, Hansen LH, Hasman H, Debroy C et al (2012) Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae. Plasmid 68:43-50. https://doi.org/10.1016/j.plasmid.2012.03.001.
Ares-Arroyo M, Bernabe-Balas C, Santos-Lopez A, Baquero MR, Prasad KN, Cid D et al (2018) PCR-based analysis of ColE1 plasmids in clinical isolates and metagenomic samples reveals their importance as gene capture platforms. Front. Microbiol 9:469. https://doi.org/10.3389/fmicb.2018.00469.
Cabral AB, Maciel MAV, Barros JF, Antunes MM, Barbose de Castro CMM, Lopes ACS (2017) Clonal spread and accumulation of β-lactam resistance determinants in Enterobacter aerogenes and Enterobacter cloacae complex isolates from infection and colonization in patients at a public hospital in Recife, Pernambuco, Brazil. J Med Microbiol 66:70-77. https://doi.org/10.1099/jmm.0.000398.
Heras J, Dominguez C, Mata E, Pascual V, Lozano C, Torres C et al (2015) GelJ- a tool for analyzing DNA fingerprint gel images. BMC Bioinformatics 16:270. https://doi.org/10.1186/s12859-015-0703-0.
Mogasale VV, Saldanha P, Pai V, Rekha PD, Mogasale V (2021) A descriptive analysis of antimicrobial resistance patterns of WHO priority pathogens isolated in children from a tertiary care hospital in India. Scientific Reports 11:5116. https://doi.org/10.1038/s41598-021-84293-8.
Chen J, Tian S, Nian H, Wang R, Li F, Jiang N et al (2021) Carbapenem-resistant Enterobacter cloacae complex in a tertiary Hospital in Northeast China, 2010–2019. BMC Infect Dis 21:611. https://doi.org/10.1186/s12879-021-06250-0.
Liu J, Li GM, Lin LY, Wu XL, Huang SL, Zhou Y et al (2016) Association of antibiotic resistance with SHV-12 extended-spectrum β-lactamase in Enterobacter cloacae. Exp Ther Med 11:269-276. https://doi.org/10.3892/etm.2015.2851.
Manandhar S, Nguyen Q, Nguyen T, Nguyen T, Pham DT, Rabaa MA et al (2022) Genomic epidemiology, antimicrobial resistance and virulence factors of Enterobacter cloacae complex causing potential community-onset bloodstream infections in a tertiary care hospital of Nepal. JAC Antimicrob Resist 4:dlac050. https://doi.org/10.1093/jacamr/dlac050.
Merhi G, Amayri S, Bitar I, Araj GF, Tokajian S (2022) Whole genome-based characterization of multi-drug resistant Enterobacter and Klebsiella aerogenes isolates from Lebanon. Microbiol Spectrum 11:e02917-22. https://doi.org/10.1128/spectrum.02917-22.
Trevino M, Moldes L, Martinez-Lamas L, Varon C, Regueiro BJ (2009) Carbapenem-resistant Enterobacter cloacae and the emergence of metallo-beta-lactamase-producing strains in a third-level hospital (Santiago de Compostela, NW Spain). Eur J Clin Microbiol Infect Dis 28:1253–1258. https://doi.org/10.1007/s10096-009-0765-x.
Sumbana J, Santona A, Fiamma M, Taviani E, Deligios M, Chongo V et al (2022) Polyclonal emergence of MDR Enterobacter cloacae complex isolates producing multiple extended spectrum beta-lactamases at Maputo Central Hospital, Mozambique. Rend Fis Acc Lincei 33:39–45. https://doi.org/10.1007/s12210-021-01039-4.
Khajuria A, Praharaj AK, Kumar M, Grover N (2014) Carbapenem resistance among Enterobacter species in a tertiary care hospital in central India. Chemother Res Pract 2014:972646. http://dx.doi.org/10.1155/2014/972646.
Han R, Shi Q, Wu S, Yin D, Peng M, Dong D et al (2020) Dissemination of carbapenemases (KPC, NDM,OXA-48, IMP, and VIM) among carbapenem-resistant Enterobacteriaceae isolated from adult and children patients in China. Front Cell Infect Microbiol 10:314. http://dx.doi.org/10.3389/fcimb.2020.00314.
Zhu Y, Jia X, Jia P, Li X, Yang Q (2021) Genetic and phenotypic characterization of the novel metallo-β-lactamase NDM-29 from Escherichia coli. Front Microbiol 12:743981. http://dx.doi.org/10.3389/fmicb.2021.743981.
Kananizadeh P, Oshiro S, Watanabe S, Iwata S, Kuwahara-Arai K, Shimojima M et al (2020) Emergence of carbapenem-resistant and colistin-susceptible Enterobacter cloacae complex co-harboring blaIMP-1 and mcr-9 in Japan. BMC Infect Dis 20:282. https://doi.org/10.1186/s12879-020-05021-7.
Ramirez MS, Iriarte A, Reyes-Lamothe R, Sherrattn DJ, Tolmasky ME (2019) Small Klebsiella pneumoniae plasmids: neglected contributors to antibiotic resistance. Front Microbiol 10:2182. https://doi.org/10.3389/fmicb.2019.02182.

No competing interests reported.

SuppTable1.docx

Download PDF

Journal Publication

published 21 Nov, 2023

Read the published version in International Microbiology →

Editorial decision: Major revision
15 Jul, 2023
Editor assigned by journal
15 Jul, 2023
Submission checks completed at journal
14 Jul, 2023
First submitted to journal
07 Jul, 2023

You are reading this latest preprint version

The emergence of clonally diverse carbapenem-resistant Enterobacter cloacae complex in West Bengal, India: A dockyard of β-lactamases periling nosocomial infections

Status:

Journal Publication

Version 1

Abstract

Figures

Introduction

Materials and methods

Bacterial isolates

Phenotypic resistotyping

Screening of β-lactamase genes

Sequencing of NDM and OXA-48

Plasmid profiling

Clonality assessment

Gel image analysis

Results

Clinical information

Resistotypes

Versatility of β-lactamases

NDM and OXA-48 variants

Prevalence of plasmids

Plasmid replicons

Genetic relatedness

Discussion

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1