Escherichia coli Isolates Harboring bla NDM Variants and 16S Methylases Belonging to Clonal Complex 131 in Southern Punjab,Pakistan

Abstract

The emergence of carbapenem-resistant Escherichia coli (CREC) especially harboring the New Delhi Metallo-β-lactamase (bla_NDM) variants are increasingly being reported from many countries, however, the data from Pakistan is limited. In the present study, 109 CREC isolates were obtained from 4,091 E. coli isolates in five tertiary care hospitals in southern Punjab, Pakistan. The antimicrobial susceptibility profiling and screening for the resistance determinants were performed followed by bla_NDM typing and multilocus sequence typing (MLST) to characterize the CREC strains. Among the carbapenemases, 57 CREC isolates were found to harbor bla_NDM. The bla_NDM-1, bla_NDM-5, bla_NDM-7, and bla_NDM-4 variants were identified in 30 (52.6%), 18 (31.6%), (12.3%), 2 (3.5%) isolates, respectively. The ESBL genes, such as bla_CTX-M and bla_TEM, were also found in different combinations, whereas the 16S methylases that is, rmtB and armA were found in 69 (63.3%) and 55 (50.5%) CREC isolates, respectively. The MLST of bla_NDM carrying E. coli revealed eight different sequence types (STs) with ST131 belonging to 21 isolates being the most prevalent. The clonal complex 131 was the predominant complex corresponding to 47 (82.5%) of bla_NDM-positive strains. Large-scale surveillance studies coupled with active infection control policies are suggested on an urgent basis to avoid an epidemic in the future.

Introduction

The emergence of carbapenem-resistant Escherichia coli (CREC) is a public health threat throughout the world.¹ An enzyme that is, New Delhi Metallo-β-lactamase-1 (bla_NDM-1), which was able to hydrolyze all the β-lactam antibiotics, except aztreonam was isolated from the Klebsiella pneumoniae and E. coli strains in Sweden during 2008.² Most of the NDM-producing strains were belonging to the family Enterobacteriaceae, however, the Pseudomonas aeruginosa and Acinetobacter spp. were also reported to harbor these carbapenemases. It is believed that the Indian subcontinent, the Middle East, and the Balkans regions are the main reservoirs of NDM-producing bacterial pathogens.³

Nevertheless, the study involving the NDM-1-producing Enterobacteriaceae isolates from the various parts of the world have shown that the spread of specific plasmids, other genetic elements, and specific clones are not responsible for the spread of the bla_NDM-1 gene.⁴ During the previous years, 29 variants of the bla_NDM-1 gene have been reported in various bacterial pathogens. Various studies have reported the increased prevalence of NDM variants for instance bla_NDM-5 or bla_NDM-7 compared with other variants.⁵ Interestingly, it is reported that the variants bla_NDM-5 and bla_NDM-7 were predominantly associated with E. coli than other bacterial species.⁶

The emergence and spread of 16S rRNA methylases among the gram-negative (GN) bacterial pathogens especially the member of Enterobacteriaceae have further worsened the condition as these enzymes confer high-level resistant phenotypes for all the clinically useful aminoglycosides, such as amikacin, gentamicin, and tobramycin.^7,8 Although various 16S rRNA methylase genes, armA, npmA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF, rmtG, and rmtH, have been reported among the GN bacteria, however, the armA and rmtB were commonly found in Enterobacteriaceae.^9,10 The transfer of these genes among the bacteria seems easier as they are usually located on the plasmids, thus can be easily transferred to other bacteria.¹¹

The E coli sequence type 131 (ST131) is considered as one of the most successful and widespread multidrug-resistant (MDR) E. coli clones that are associated with the community as well as hospital-acquired infections, such as bacteremia, urinary tract and abdominal infections with a higher rate of morbidity and mortality.¹² The resistance to the cephalosporins is mainly mediated by the acquisition of the bla_CTX-M-15 gene among the E. coli ST131 strains.^13,14 Therefore, the raised public health concerns for E. coli ST131 are due to its higher pathogenic potential and its increasing arsenal for antimicrobial resistance.

The carbapenems remained the treatment of choice for the infections caused by this clone as most E. coli ST131 strains were susceptible to carbapenems.^12,15,16 However, the bacteria are developing resistance to carbapenems mainly due to the bla_NDM, bla_OXA-48-like, or bla_KPC genes.^16–18

The detection of carbapenem-resistant strains is imperative to support the local and international efforts for effective infection control. This study has described the prevalence and molecular epidemiology of CREC isolates from the tertiary care hospitals of southern Punjab, Pakistan that were coharboring the bla_NDM variants and 16S methylases.

Materials and Methods

Ethical statement

The study got prior approval from the institutional review board of the Government College University Faisalabad, Pakistan to utilize the bacterial isolates and anonymized patient data for this study.

Clinical settings and E. coli isolates

The present study was conducted from 2019 (January) to 2020 (December) at the five tertiary care hospitals of Southern Punjab, Pakistan, including the three major cities (Multan, Bahawalpur, and Rahim Yar Khan). These tertiary care centers provide medical services to a large population residing in southern Punjab. Furthermore, southern Punjab is a gateway to Sindh and Balochistan province, therefore, the patients from these provinces also visit the tertiary care centers of Southern Punjab for treatment. A total of 4,091 E. coli isolates were collected from various clinical specimen types, including blood, urine, pus, and catheters, during the study period.

The identification of E. coli was initially carried out by colony morphology on the MacConkey, blood, and chocolate agar plates (Oxoid, United Kingdom); biochemical reactions, including motility, oxidase, methyl red, indole, citrate, Voges-Proskauer, and urease tests followed by confirmation using API 20E (BioMerieux, France). For further confirmation, the GN ID cards were used on a VITEK^® 2 system (BioMerieux).

Antimicrobial susceptibility profiling

For the antibiotic susceptibility testing of E. coli isolates, the Kirby–Bauer disc diffusion method was performed and interpreted as per Clinical and Laboratory Standards Institute (CLSI) guidelines.¹⁹ The antimicrobial agents used for this method were amikacin, gentamicin, tobramycin, ampicillin, piperacillin, amoxicillin–clavulanic acid, piperacillin–tazobactam, cefoperazone–sulbactam, cefotaxime, ceftriaxone, cefepime, imipenem, meropenem, ertapenem, ciprofloxacin, levofloxacin, trimethoprim–sulfamethoxazole, and minocycline. The antimicrobial disks used in the study were obtained from Oxoid (Oxoid).

The minimum inhibitory concentration (MIC) of the antibiotic agents, including amikacin, gentamicin, tobramycin, cefotaxime, ceftriaxone, cefepime, imipenem, meropenem, ciprofloxacin, polymyxin B, colistin, and tigecycline was tested against the ECEC strains by the broth microdilution method. The results were interpreted as per CLSI guidelines. The MIC for tigecycline was based on Food and Drug Administration (FDA) standards,²⁰ whereas the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were used to interpret the MIC results for polymyxin B and colistin.²¹ The E. coli (ATCC^® 25922) was used as a quality control strain for susceptibility testing.

Screening of antimicrobial resistance determinants

The Bacterial DNA was extracted using the bacterial genomic DNA Kit, FavorPrep™ (FAVORGEN Biotech Corp., Taiwan). For the determination of purity of DNA, the absorbance was measured using NanoDrop™ (Thermo Fisher Scientific, United Kingdom) at a wavelength of 260 and 280 nm. All the CREC isolates were screened for the ESBL-encoding genes, including bla_SHV, bla_TEM, and bla_CTX-M genes. The isolates that were found positive for the bla_CTX-M were subjected to further screening for the bla_CTX-M-1, bla_CTX-M-2, bla_CTX-M-8, bla_CTX-M-9, bla_CTX-M-10, bla_CTX-M-14, and bla_CTX-M-15. The isolates were also screened for the class B beta-lactamases, such as bla_IMP, bla_VIM, bla_SPM, bla_KPC, and bla_OXA-48, using PCR.

Furthermore, the screening for the 16S methylases that is, armA, npmA, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF, rmtG, and rmtH was also performed. All the primers used in this study were synthesized from Macrogen™ (South Korea). The primer sequences and the amplicon size of PCR products are shown in Table 1. The PCR amplicons were sequenced by the Sanger sequencing method from Macrogen (South Korea). The sequences were aligned and compared with the already available sequences in the NCBI database using the NCBI BLAST tool.

Table 1.

Primers Used for the Amplification of Antimicrobial Resistance Determinants

Target gene	Primer name	Primer sequence (5′-3′)	Amplicon (bp)	Annealing temperature (°C)
blaTEM	TEM-F	TCAACATTTCCGTGTCG	860	56
blaTEM	TEM-R	CTGACAGTTACCAATGCTTA	860	56
blaSHV	SHV-F	ATGCGTTATATTCGCCTGTG	896	56
blaSHV	SHV-R	AGATAAATCACCACAATGCGC	896	56
blaCTXM	CTXMU-F	ATGTGCAGYACCAGTAARGT	593	52
blaCTXM	CTXMU-R	TGGGTRAARTARGTSACCAGA	593	52
blaCTXM1	CTXM1-F	CCGTTTCCGCTATTACAAACCGTTG	944	56
blaCTXM1	CTXM1-R	GGCCCATGGTTAAAAAATCACTGC	944	56
blaCTXM2	CTXM2-F	ATGATGACTCACAGCATTCG	833	56
blaCTXM2	CTXM2-R	TCCCGACGGCTTTCCGCGTT	833	56
blaCTXM8	CTXM8-F	TTTGCCCGTGCGATTGG	368	50
blaCTXM8	CTXM8-R	CGACTTTCTGCCTTCTGCTCT	368	50
blaCTXM9	CTXM9-F	ATGGTGACAAAGAGAGTGCA	870	50
blaCTXM9	CTXM9-R	CCCTTCGGCGATGATTCTC	870	50
blaCTXM10	CTXM10-F	GCAGCACCAGTAAAGTGATGG	524	56
blaCTXM10	CTXM10-R	GCGATATCGTTGGTGGTACC	524	56
blaCTXM14	CTXM14-F	GAGAGTGCAACGGATGATG	941	56
blaCTXM14	CTXM14-R	TGCGGCTGGGTAAAATAG	941	56
blaCTXM15	CTXM15-F	CACACGTGGAATTTAGGGACT	996	55
blaCTXM15	CTXM15-R	GCCGTCTAAGGCGATAAACA	996	55
blaIMP	IMP-F	GGAATAGAGTGGCTTAAYTC	232	50
blaIMP	IMP-R	TCGGTTTAAYAAAACAACCACC	232	50
blaVIM	VIM-F	GATGGTGTTTGGTCGCATA	390	50
blaVIM	VIM-R	CGAATGCGCAGCACCAG	390	50
blaSPM	SPM-F	AAAATCTGGGTACGCAAACG	271	52
blaSPM	SPM-R	ACATTATCCGCTGGAACAGG	271	52
blaNDM	NDM-F	GGTTTGGCGATCTGGTTTTC	621	52
blaNDM	NDM-R	CGGAATGGCTCATCACGATC	621	52
Pre-NDM	Pre-NDM-F	CACCTCATGTTTGAATTCGCC	984	52
Pre-NDM	Pre-NDM-R	CTCTGTCACATCGAAATCGC	984	52
blaKPC	KPC-F	CGTCTAGTTCTGCTGTCTTG	798	50
blaKPC	KPC-R	CTTGTCATCCTTGTTAGGCG	798	50
blaOXA-48	OXA-48-F	TTGGTGGCATCGATTATCGG	744	52
blaOXA-48	OXA-48-F	GAGCACTTCTTTTGTGATGGC	744	52
armA	armA-F	ATTCTGCCTATCCTAATTGG	315	50
armA	armA-R	ACCTATACTTTATCGTCGTC	315	50
npmA	npmA-F	GGAGGGCTATCTAATGTGGT	371	50
npmA	npmA-R	GCCCAAAGAGAATTAAACTG	371	50
rmtA	rmtA-F	CTAGCGTCCATCCTTTCCTC	635	55
rmtA	rmtA-R	TTTGCTTCCATGCCCTTGCC	635	55
rmtB	rmtB-F	ATGAACATCAACGATGCCCT	769	50
rmtB	rmtB-R	CCTTCTGATTGGCTTATCCA	769	50
rmtC	rmtC-F	CGAAGAAGTAACAGCCAAAG	711	50
rmtC	rmtC-R	ATCCCAACATCTCTCCCACT	711	50
rmtD	rmtD-F	CGGCACGCGATTGGGAAGC	401	55
rmtD	rmtD-R	CGGAAACGATGCGACGAT	401	55
rmtE	rmtE-F	ATGAATATTGATGAAATGGTTGC	823	50
rmtE	rmtE-R	TGATTGATTTCCTCCGTTTTTG	823	50
rmtF	rmtF-F	GCGATACAGAAAACCGAAGG	589	52
rmtF	rmtF-R	ACCAGTCGGCATAGTGCTTT	589	52
rmtG	rmtG-F	AAATACCGCGATGTGTGTCC	250	55
rmtG	rmtG-R	ACACGGCATCTGTTTCTTCC	250	55
rmtH	rmtH-F	AATGACCATTGAACAGGCAGC	760	55
rmtH	rmtH-R	TCAAGCTGGGTTTGGCTGGA	760	55

Molecular characterization of bla_NDM

The screening of bla_NDM was performed by amplifying a 621-bp fragment of NDM genes using the primers listed in Table 1 by the following conditions: 95°C for 5 minutes (initial denaturation), 95°C for 30 seconds (secondary denaturation), 52°C for 45 seconds (annealing); 72°C for 45 seconds (primary extension) and 72°C for 7 minutes (final extension). Moreover, another set of primers was used to amplify the entire bla_NDM gene using the pre-NDM primers (Table 1) that were designed for the region flanking the bla_NDM gene using the following conditions: 95°C for 5 minutes (initial denaturation), 95°C for 30 seconds (secondary denaturation), 50°C for 45 seconds (annealing); 72°C for 1 minute (primary extension) and 72°C for 10 minutes (final extension).

The amplified product was sent for sequencing to Macrogen (South Korea) for sanger sequencing. The obtained DNA sequences were analyzed by MEGA software and were compared with the already available sequences in the NCBI database using the NCBI BLAST tool.

Multilocus sequence typing

The multilocus sequence typing (MLST) was performed for all the bla_NDM-positive strains. The Achtman 7 Gene MLST was performed by amplification of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) according to the conditions mentioned in the EnteroBase Database. The PCR amplicons were extracted from the agarose gel by the GeneJET Gel Extraction Kit (Thermo Fisher Scientific, MA) and sequenced by Macrogen (South Korea). The sequences were initially edited by the ChromasPro (Technelysium, Australia) and were aligned by the ClustalW algorithm (MEGA7 software). Each of the gene loci was assigned an allelic number and the sequence types (STs) were found following the respective allelic profiles for the isolates using the EnteroBase Database.

Results

Isolation and antimicrobial susceptibility of E. coli isolates

A total of 109 (2.7%) strains found resistant to carbapenems (imipenem and meropenem) were identified as CREC. The E. coli strains were isolated from the following clinical specimens: urine (n = 33), blood (n = 20), pus (n = 14), sputum (n = 13), tracheal secretion (n = 11), wound swab (n = 8), CVP tip (n = 6), fluid (n = 3) and cerebrospinal fluid (n = 1).

The antimicrobial susceptibilities for the CREC strains were performed and are listed in Fig. 1 and Table 2. The CREC strains showed 100% resistance to ampicillin, piperacillin, amoxicillin–clavulanic acid, piperacillin–tazobactam, cefoperazone, cefotaxime, ceftriaxone, cefepime, imipenem, meropenem, and ertapenem. The 98.2% resistance rates were observed for ciprofloxacin and levofloxacin as only two CREC isolates were susceptible to these drugs. The sulfamethoxazole–trimethoprim and minocycline were 85.3% and 84.4%, respectively. The resistance among the CREC isolates against the aminoglycosides was as follows: gentamicin 98.2%, tobramycin 97.2%, and amikacin 93.6%.

FIG. 1.

Antimicrobial susceptibility profiling of CREC isolates. CREC, carbapenem-resistant Escherichia coli.

Table 2.

Minimum Inhibitory Concentration Distribution of Carbapenem-Resistant Escherichia coli Isolates

Antimicrobial agents	No. of isolates at MIC (μg/mL) of
Antimicrobial agents	≤0.06	0.125	0.25	0.5	1	2	4	8	16	32	64	128	256	≥512
Amikacin	—	—	—	—	—	3	3	1	—	—	3	5	11	83
Gentamicin	—	—	—	—	1	1	—	—	2	11	—	7	39	48
Tobramycin	—	—	—	—	2	1	—	—	9	2	1	25	41	28
Cefotaxime	—	—	—	—	—	—	—	—	—	—	—	—	—	109
Ceftriaxone	—	—	—	—	—	—	—	—	—	—	—	—	—	109
Cefepime	—	—	—	—	—	—	—	—	—	—	—	—	7	102
Imipenem	—	—	—	—	—	—	33	39	26	11	—	—	—	—
Meropenem	—	—	—	—	—	—	6	46	31	21	5	—	—	—
Ciprofloxacin	—	—	—	—	—	—	—	—	—	13	34	37	15	10
Polymyxin B	—	10	—	27	69	3	—	—	—	—	—	—	—	—
Colistin	—	3	—	30	72	4	—	—	—	—	—	—	—	—
Tigecycline	—	—	2	12	37	56	2	—	—	—	—	—	—	—

MIC, minimum inhibitory concentration.

In contrast, polymyxin B, colistin, and tigecycline showed high susceptibility rates that is, 100% as all CREC strains were found susceptible suggesting these agents as the therapeutic option for infections caused by the CREC strains.

Detection of antimicrobial resistance determinants

The PCR was used to screen all the E. coli isolates for the MBLs, ESBLs, and 16S methylases among the 109 CREC isolates. Apart from the bla_NDM and bla_IMP, the other MBL encoding genes mentioned in Table 1 were not found in the E. coli strains. All the 109 strains were found positive either for bla_NDM (52.3%) or bla_IMP gene (47.7%). The bla_OXA-48 was found in 23 (21.1%) of isolates, whereas the bla_KPC was not found in any of the isolates. Additionally, the ESBL genes also existed in all the CREC strains; the bla_CTX-M was the main type (71.6%), whereas the bla_TEM accounted for 59.6% of strains as shown in Table 3. Among the 16S methylases, 69 (63.3%) were found positive for the rmtB gene, whereas the armA was found in 55 (50.5%) isolates. Other 16S methylases were not found in any of the isolates.

Table 3.

Specimen Wise Distribution of the Detected 16s Methylases, ESBLs and MBLs

Specimens	Isolates n (%)	armA n (%)	rmtB n (%)	blaIMP n (%)	blaNDM n (%)	blaOXA-48 n (%)	blaTEM n (%)	blaCTX-M n (%)
Blood	20 (18.3)	13 (65)	12 (60)	9 (45)	11 (55)	2 (10)	12 (60)	16 (80)
CSF	1 (0.9)	1 (100)	1 (100)	—	1 (100)	—	—	1 (100)
Fluid	3 (2.8)	3 (100)	1 (33.3)	2 (66.7)	1 (100)	—	2 (66.7)	3 (100)
Pus	14 (12.8)	5 (35.7)	6 (42.9)	8 (57.1)	6 (42.9)	5 (35.7)	9 (64.3)	9 (64.3)
Sputum	13 (11.9)	7 (53.8)	10 (76.9)	6 (46.2)	7 (53.8)	2 (15.4)	6 (46.2)	8 (61.5)
Tip	6 (5.5)	—	5 (83.3)	4 (66.7)	2 (33.3)	1 (16.7)	4 (66.7)	2 (33.3)
Tracheal Secretion	11 (10.1)	6 (54.5)	7 (63.6)	3 (27.3)	8 (72.7)	2 (18.2)	6 (54.5)	10 (90.9)
Urine	33 (30.3)	17 (51.5)	21 (63.6)	14 (42.4)	19 (57.6)	10 (30.3)	21 (63.6)	26 (78.8)
Wound Swab	8 (7.3)	3 (37.5)	6 (75)	6 (75)	2 (25)	1 (12.5)	5 (62.5)	3 (37.5)
Total	109	55 (50.5)	69 (63.3)	52 (47.7)	57 (52.3)	23 (21.1)	65 (59.6)	78 (71.6)

Co-occurrence of beta-lactamase and 16S methylases

The evaluation of the coexistence of genes coding the beta-lactamases and 16S methylases is presented in Table 4. The study revealed that 23 (21.1%) of E. coli strains were carrying simultaneously bla_NDM, bla_TEM, bla_CTX-M-15, armA, and rmtB genes. It was noticed that the prevalence of strains possessing bla_IMP, bla_TEM, and rmtB accounted for 14.7% of genotypes, whereas the combination of bla_IMP, bla_TEM, bla_CTX-M-1, bla_CTX-M-15, and armA was present in 13.8% of strains.

Table 4.

Co-occurrence of Genes Encoding Beta-Lactamases and 16S Methylases Among Escherichia coli Strains

Cooccurrence of gene mediating drug resistance
Beta-lactams						Aminoglycosides		n	%
—	bla _NDM	—	bla _TEM	—	bla _CTX-M-15	armA	rmtB	23	21.1
bla _IMP	—	—	bla _TEM	—	—	—	rmtB	16	14.7
bla _IMP	—	—	bla _TEM	bla _CTX-M-1	bla _CTX-M-15	armA	—	15	13.8
—	bla _NDM	—	bla _TEM	—	bla _CTX-M-15	—	rmtB	12	11.0
—	bla _NDM	bla _OXA-48	bla _TEM	—	bla _CTX-M-15	—	rmtB	8	7.3
bla _IMP	—	bla _OXA-48	—	—	—	—	—	8	7.3
—	bla _NDM	bla _OXA-48	—	—	bla _CTX-M-15	armA	rmtB	7	6.4
bla _IMP	—	—	—	—	bla _CTX-M-15	—	—	7	6.4
bla _IMP	—	—	bla _TEM	—	bla _CTX-M-15	armA	—	6	5.5
—	bla _NDM	—	—	bla _CTX-M-1	—	armA	—	2	1.8
—	bla _NDM	—	—	bla _CTX-M-1	bla _CTX-M-15	—	rmtB	2	1.8
—	bla _NDM	—	—	—	bla _CTX-M-15	armA	—	2	1.8
—	bla _NDM	—	—	bla _CTX-M-1	—	—	rmtB	1	0.9

Frequency of bla_NDM alleles

Out of 109 CREC, a high prevalence of blaNDM carbapenemases (n = 57) was observed. Of these 57 isolates, 30 (52.6%) were found to harbor bla_NDM-1 followed by bla_NDM-5 18 (31.6%), bla_NDM-7 7 (12.3%), and bla_NDM-4 was found in 2 (3.5%) isolates. The 30/57 bla_NDM-1-producing CREC strains were distributed in all the five hospitals: hospital A having the highest that is, 14 (46.7%) isolates. The blaNDM-5-harboring CREC isolates were also found highest in hospital A. The two CREC isolates harboring blaNDM-4 were obtained from hospital C only (Fig. 2). Of the 57 blaNDM-producing CREC strains, 19 (33.3%) were recovered from urine and samples followed by 11 (19.3%) from blood samples (Table 5). The antimicrobial susceptibility of various bla_NDM alleles is summarized in Table 6.

FIG. 2.

blaNDM variants distribution in different hospitals of southern Punjab.

Table 5.

Specimen Wise Distribution of blaNDM Genotypes

Sequence type	n (%)	NDM genotype	Blood	Sputum	Tracheal secretion	Urine	Pus	CSF	Fluid	CVP tip
ST131	21 (37.8)	blaNDM-1	2	4	0	1	2	1	1	—
ST131	21 (37.8)	blaNDM-5	3	0	2	4	1	—	—	—
ST3329	11 (19.3)	blaNDM-1	1	1	1	5	3	—	—	—
ST2279	8 (14)	blaNDM-5	1	—	2	4	1	—	—	—
ST8051	7 (12.3)	blaNDM-7	1	1	1	3	1	—	—	—
ST88	4 (7)	blaNDM-1	2	—	—	2	—	—	—	—
ST6293	3 (5.3)	blaNDM-1	1	—	1	—	—	—	—	1
ST209	2 (3.5)	blaNDM-4	—	1	1	—	—	—	—	—
ST3059	1 (1.8)	blaNDM-1	—	—	—	—	—	—	—	1
Total	57	—	11 (19.3)	7 (12.3)	8 (14)	19 (33.3)	8 (14)	1 (1.8)	1 (1.8)	2 (3.5)

Table 6.

Antimicrobial Susceptible Patterns of bla_NDM Producing Escherichia Coli

Antimicrobial agents	Total (n = 57)		NDM-1 (n = 30)		NDM-4 (n = 2)		NDM-5 (n = 18)		NDM-7 (n = 7)
Antimicrobial agents	S (%)	R (%)	S (%)	R (%)	S (%)	R (%)	S (%)	R (%)	S (%)	R (%)
Amikacin	0.0	100.0	0.0	100.0	0.	100.0	0.0	100.0	0.0	100.0
Gentamicin	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0
Tobramycin	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0
Ampicillin	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0
Piperacillin	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0
Amoxicillin–Clavulanate	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0
Tazobactam–Piperacillin	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0
Cefoperazone–Sulbactam	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0
Cefotaxime	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0
Ceftriaxone	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0
Cefepime	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0
Imipenem	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0
Meropenem	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0
Ertapenem	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0
Ciprofloxacin	3.5	96.5	6.7	93.3	0.0	100.0	0.0	100.0	0.0	100.0
Levofloxacin	3.5	96.5	6.7	93.3	0.0	100.0	0.0	100.0	0.0	100.0
Sulfamethoxazole–Trimethoprim	21.1	78.9	23.3	76.7	100.0	0.0	11.1	88.9	14.3	85.7
Minocycline	21.1	78.9	23.3	76.7	100.0	0.0	11.1	88.9	14.3	85.7
Polymyxin B	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0
Colistin	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0
Tigecycline	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0	100.0	0.0

All the four blaNDM variants were 100% resistant to all the tested beta-lactam agents as well as the aminoglycosides. The blaNDM-4-, blaNDM-5-, and blaNDM-7-harboring strains were 100% resistant to fluoroquinolones, whereas the resistance among blaNDM-1-harboring strains were 93.3%. The blaNDM-4-producing isolates (n = 2) were 100% susceptible to sulfamethoxazole–trimethoprim and minocycline compared with the blaNDM-1-, blaNDM-5-, and blaNDM-7-producing isolates, which were 23.3%, 11.1%, and 14.3% susceptible to both these drugs.

Sequence typing

The genetic relatedness of bla_NDM-harboring E. coli strains obtained from different specimens and hospitals were investigated by MLST. ST131 (n = 21, 37.8%) was the most prevalent genotype, which comprised 11 isolates harboring bla_NDM-1 and 10 isolates harboring the bla_NDM-5 gene (Table 7).

Table 7.

Sequence Types of Carbapenem-Resistant Escherichia coli Isolates Carrying bla_NDM Variants and 16S Methylases

Case ID	Patients details				ST	Clonal complex	MIC of antimicrobial agents												16S methylases	NDM variants	Other acquired beta-lactamases
Case ID	Specimen	Age	Gender	Hospital	ST	Clonal complex	AK	CN	TOB	CIP	CT	PB	TGC	FEP	CTX	CRO	IMP	MEM	16S methylases	NDM variants	Other acquired beta-lactamases
EC1	Blood	65	Male	C	131	ST131 Cplx	≥512	256	128	≥512	1	1	2	≥512	≥512	≥512	8	16	armA, rmtB	NDM-1	bla _CTXM15
EC2	Blood	56	Female	D	8051	ST131 Cplx	≥512	≥512	256	128	0.5	1	2	≥512	≥512	≥512	32	64	armA, rmtB	NDM-7	bla_OXA-48, bla_CTXM15
EC3	Sputum	62	Male	D	8051	ST131 Cplx	≥512	≥512	256	128	1	0.5	2	≥512	≥512	≥512	32	64	armA, rmtB	NDM-7	bla_OXA-48, bla_CTXM15
EC4	Tracheal Secretion	65	Male	E	3329	ST131 Cplx	≥512	256	128	256	1	1	2	≥512	≥512	≥512	16	32	rmtB	NDM-1	bla_TEM, bla_CTXM15
EC5	Blood	64	Female	A	131	ST131 Cplx	≥512	256	128	256	0.5	0.5	2	≥512	≥512	≥512	8	16	armA, rmtB	NDM-5	bla _CTXM15
EC6	Tracheal Secretion	75	Male	B	2279	ST131 Cplx	≥512	≥512	256	64	1	1	2	≥512	≥512	≥512	32	32	rmtB	NDM-5	bla_TEM, bla_CTXM15
EC7	Blood	55	Female	A	88	ST23 Cplx	≥512	≥512	≥512	128	1	0.5	0.5	≥512	≥512	≥512	8	16	armA, rmtB	NDM-1	bla_TEM, bla_CTXM15
EC8	Blood	70	Male	A	2279	ST131 Cplx	≥512	≥512	256	64	1	1	2	≥512	≥512	≥512	32	32	rmtB	NDM-5	bla_TEM, bla_CTXM15
EC9	Sputum	55	Male	D	131	ST131 Cplx	≥512	≥512	≥512	256	1	1	4	≥512	≥512	≥512	8	16	armA, rmtB	NDM-1	bla _CTXM15
EC10	Urine	62	Male	B	8051	ST131 Cplx	≥512	≥512	256	128	0.5	0.125	2	≥512	≥512	≥512	32	64	armA, rmtB	NDM-7	bla_OXA-48, bla_CTXM15
EC11	Tracheal Secretion	72	Female	B	2279	ST131 Cplx	≥512	≥512	256	64	1	1	1	≥512	≥512	≥512	32	32	rmtB	NDM-5	bla_TEM, bla_CTXM15
EC12	Blood	55	Male	A	3329	ST131 Cplx	≥512	256	128	≥512	1	1	1	≥512	≥512	≥512	16	32	rmtB	NDM-1	bla_TEM, bla_CTXM15
EC14	Blood	45	Female	B	6293	ST38 Cplx	≥512	128	256	64	1	1	2	≥512	≥512	≥512	8	16	rmtB	NDM-1	bla _CTXM1
EC15	Sputum	56	Female	C	3329	ST131 Cplx	≥512	256	128	256	0.5	0.125	1	≥512	≥512	≥512	16	32	rmtB	NDM-1	bla_TEM, bla_CTXM15
EC18	Tracheal Secretion	80	Male	C	209	ST10 Cplx	≥512	256	256	64	1	1	1	≥512	≥512	≥512	8	8	armA	NDM-4	bla _CTXM1
EC19	Blood	60	Female	A	131	ST131 Cplx	≥512	≥512	256	256	2	1	2	≥512	≥512	≥512	8	16	armA, rmtB	NDM-1	bla _CTXM15
EC21	Urine	50	Female	A	88	ST23 Cplx	≥512	≥512	≥512	64	1	1	1	≥512	≥512	≥512	16	32	armA, rmtB	NDM-1	bla_TEM, bla_CTXM15
EC23	Urine	2	Male	A	2279	ST131 Cplx	≥512	≥512	256	32	1	1	2	≥512	≥512	≥512	16	16	rmtB	NDM-5	bla_TEM, bla_CTXM15
EC25	Blood	51	Male	B	88	ST23 Cplx	≥512	≥512	≥512	128	0.5	0.5	2	≥512	≥512	≥512	16	16	armA, rmtB	NDM-1	bla_TEM, bla_CTXM15
EC28	Blood	12	Female	E	131	ST131 Cplx	≥512	256	128	64	0.5	1	2	≥512	≥512	≥512	8	8	armA, rmtB	NDM-5	bla_TEM, bla_CTXM15
EC29	Urine	75	Female	A	3329	ST131 Cplx	≥512	256	128	128	1	0.5	2	≥512	≥512	≥512	8	16	rmtB	NDM-1	bla_OXA-48, bla_TEM, bla_CTXM15
EC30	Urine	71	Female	C	131	ST131 Cplx	≥512	≥512	256	128	1	0.5	2	≥512	≥512	≥512	8	16	armA, rmtB	NDM-5	bla _CTXM15
EC31	Urine	71	Female	B	3329	ST131 Cplx	≥512	≥512	256	256	1	1	0.5	≥512	≥512	≥512	16	16	rmtB	NDM-1	bla_OXA-48, bla_TEM, bla_CTXM15
EC32	Tracheal Secretion	64	Male	A	8051	ST131 Cplx	≥512	≥512	256	128	0.5	0.5	2	≥512	≥512	≥512	32	64	armA, rmtB	NDM-7	bla_OXA-48, bla_CTXM15
EC34	Tracheal Secretion	63	Female	B	6293	ST38 Cplx	≥512	256	≥512	64	1	1	1	≥512	≥512	≥512	8	16	rmtB	NDM-1	bla_CTXM1, bla_CTXM15
EC37	CSF	57	Male	A	131	ST131 Cplx	≥512	256	128	≥512	0.5	1	2	≥512	≥512	≥512	8	16	armA, rmtB	NDM-1	bla _CTXM15
EC39	Urine	70	Male	B	2279	ST131 Cplx	≥512	≥512	256	64	0.5	0.5	1	≥512	≥512	≥512	32	32	rmtB	NDM-5	bla_TEM, bla_CTXM15
EC42	Pus	35	Female	D	131	ST131 Cplx	≥512	256	128	≥512	1	1	2	≥512	≥512	≥512	4	8	armA	NDM-1	bla _CTXM15
EC43	Blood	15	Male	A	131	ST131 Cplx	≥512	≥512	128	64	0.5	0.5	1	≥512	≥512	≥512	8	16	armA, rmtB	NDM-5	bla_TEM, bla_CTXM15
EC45	Sputum	55	Male	C	209	ST10 Cplx	≥512	256	256	128	0.5	1	0.5	≥512	≥512	≥512	8	16	armA	NDM-4	bla _CTXM1
EC46	Sputum	75	Female	E	131	ST131 Cplx	≥512	256	128	≥512	0.5	0.5	1	≥512	≥512	≥512	8	16	armA, rmtB	NDM-1	bla _CTXM15
EC47	Urine	70	Male	C	8051	ST131 Cplx	≥512	≥512	256	64	1	1	1	≥512	≥512	≥512	16	32	armA, rmtB	NDM-7	bla_OXA-48, bla_CTXM15
EC48	Pus	29	Female	A	3329	ST131 Cplx	≥512	≥512	256	128	2	2	2	≥512	≥512	≥512	16	32	rmtB	NDM-1	bla_OXA-48, bla_TEM, bla_CTXM15
EC50	Urine	63	Male	A	88	ST23 Cplx	≥512	≥512	≥512	64	1	1	2	≥512	≥512	≥512	16	32	armA, rmtB	NDM-1	bla_TEM, bla_CTXM15
EC51	Sputum	54	Female	A	131	ST131 Cplx	≥512	≥512	256	≥512	1	1	2	≥512	≥512	≥512	8	8	armA, rmtB	NDM-1	bla _CTXM15
EC52	Pus	27	Male	C	3329	ST131 Cplx	≥512	256	256	128	1	1	1	≥512	≥512	≥512	16	32	rmtB	NDM-1	bla_OXA-48, bla_TEM, bla_CTXM15
EC53	Urine	65	Female	D	131	ST131 Cplx	≥512	256	128	128	1	1	2	≥512	≥512	≥512	8	16	armA, rmtB	NDM-5	bla _CTXM15
EC55	Tip	41	Male	A	6293	ST38 Cplx	≥512	256	≥512	128	1	0.5	0.5	≥512	≥512	≥512	16	16	rmtB	NDM-1	bla_CTXM1, bla_CTXM15
EC56	Urine	57	Female	A	2279	ST131 Cplx	≥512	≥512	≥512	64	1	0.5	1	≥512	≥512	≥512	16	32	rmtB	NDM-5	bla_TEM, bla_CTXM15
EC57	Urine	70	Female	A	131	ST131 Cplx	≥512	256	128	128	0.5	1	1	≥512	≥512	≥512	8	8	armA, rmtB	NDM-5	bla _CTXM15
EC60	Pus	35	Male	B	131	ST131 Cplx	≥512	256	256	128	1	0.5	2	≥512	≥512	≥512	8	16	armA	NDM-5	bla _CTXM15
EC66	Sputum	52	Female	E	131	ST131 Cplx	≥512	256	128	≥512	1	1	1	≥512	≥512	≥512	8	16	armA, rmtB	NDM-1	bla _CTXM15
EC67	Urine	65	Male	A	3329	ST131 Cplx	≥512	≥512	256	256	1	1	2	≥512	≥512	≥512	16	16	rmtB	NDM-1	bla_OXA-48, bla_TEM, bla_CTXM15
EC69	Pus	14	Male	B	2279	ST131 Cplx	≥512	≥512	256	128	1	1	2	≥512	≥512	≥512	32	32	rmtB	NDM-5	bla_TEM, bla_CTXM15
EC70	Urine	65	Female	D	3329	ST131 Cplx	≥512	≥512	256	256	0.5	0.5	1	≥512	≥512	≥512	16	32	rmtB	NDM-1	bla_OXA-48, bla_TEM, bla_CTXM15
EC71	Tracheal Secretion	41	Male	A	131	ST131 Cplx	≥512	256	128	128	1	1	2	≥512	≥512	≥512	8	8	armA, rmtB	NDM-5	bla_TEM, bla_CTXM15
EC73	Tip	73	Male	A	3059	-	≥512	256	128	128	1	1	2	≥512	≥512	≥512	4	8	rmtB	NDM-1	bla_TEM, bla_CTXM15
EC75	Wound Swab	30	Female	E	131	ST131 Cplx	≥512	256	128	≥512	1	0.5	2	≥512	≥512	≥512	8	16	armA, rmtB	NDM-1	bla _CTXM15
EC80	Pus	58	Male	A	3329	ST131 Cplx	≥512	256	256	≥512	0.5	1	2	≥512	≥512	≥512	8	8	rmtB	NDM-1	bla_OXA-48, bla_TEM, bla_CTXM15
EC81	Wound Swab	30	Male	C	8051	ST131 Cplx	≥512	≥512	≥512	128	1	0.5	1	≥512	≥512	≥512	32	32	armA, rmtB	NDM-7	bla_OXA-48, bla_CTXM15
EC83	Fluid	51	Female	A	131	ST131 Cplx	≥512	≥512	≥512	≥512	0.5	1	1	≥512	≥512	≥512	16	16	armA, rmtB	NDM-1	bla _CTXM15
EC84	Tracheal Secretion	58	Male	B	131	ST131 Cplx	≥512	256	≥512	128	1	1	2	≥512	≥512	≥512	8	16	armA, rmtB	NDM-5	bla _CTXM15
EC89	Urine	53	Male	C	8051	ST131 Cplx	≥512	≥512	≥512	64	1	1	2	≥512	≥512	≥512	32	64	armA, rmtB	NDM-7	bla_OXA-48, bla_CTXM15
EC94	Urine	45	Female	E	131	ST131 Cplx	≥512	256	≥512	256	0.5	0.125	1	≥512	≥512	≥512	16	16	armA, rmtB	NDM-1	bla _CTXM15
EC95	Urine	52	Female	A	131	ST131 Cplx	≥512	256	128	128	0.5	1	2	≥512	≥512	≥512	16	32	armA, rmtB	NDM-5	bla_TEM, bla_CTXM15
EC105	Urine	50	Female	E	3329	ST131 Cplx	≥512	≥512	256	128	1	1	1	≥512	≥512	≥512	8	16	rmtB	NDM-1	bla_OXA-48, bla_TEM, bla_CTXM15
EC106	Urine	48	Female	A	2279	ST131 Cplx	≥512	≥512	256	64	1	1	0.5	≥512	≥512	≥512	16	32	rmtB	NDM-5	bla_TEM, bla_CTXM15

ST, sequence type.

ST3329 corresponded to 11 (19.3%) isolates, all of which were positive for bla_NDM-1 only. Similarly, ST2279, ST8051, ST88, ST6293, ST209, and ST3059 corresponded to 8 (14%), 7 (12.3%), 4 (7%), 3 (5.3%), 2 (3.5%), and 1 (1.8%) isolates, and harboring a single type of bla_NDM genotype (Tables 5 and 7). Of the 57 bla_NDM-harboring CREC strains, 47 (82.5%) strains belonged to clonal complex (CC) 131 containing ST131, ST8051, ST3329, and ST2279. A total of 4 (7%) isolates corresponded to CC23 (ST88), whereas the CC38 (ST6293) and CC10 (ST209) comprised 3 (5.3%) and 2 (3.5%) isolates, respectively. Among the 57 bla_NDM-positive isolates, the armA was present in 34 (59.6%) isolates, whereas the rmtB was found in 53 (93%) isolates.

Discussion

Among the Metallo-beta-lactamases, the blaNDM are the emerging β-lactamases that confer carbapenem-resistant phenotypes in GN bacteria. These are of global health concern and the situation is worsening in developing countries due to the higher medical and economic burden for the management of such infections and outbreaks.²² Therefore, more extensive, and comprehensive studies on the molecular epidemiology of the bla_NDM variants among the GN bacteria are essential to offer a clear understanding for the improvement of antibiotic policies in such endemic areas. This study describes the molecular epidemiology of E. coli strains that were nonsusceptible to carbapenems in southern Punjab by screening 4,091 E. coli strains.

The study showed that the overall prevalence of carbapenem-resistant E. coli isolates was 2.7% (109 strains). Furthermore, we have found that the blaNDM genes were the major contributor to the carbapenem resistance phenotypes as 57 (52.3%) strains were found to harbor this gene. As per available literature to date, this is the very first study that has analyzed the epidemiology of beta-lactamases producing E. coli using MLST and has described the details of resistance genes in this region.

The overall incidence of isolates harboring the bla_NDM gene is generally increasing throughout the world and these bla_NDM-carrying isolates have been reported from almost all regions of the world, such as Asia, Africa, Americas, Europe, and Australia.²³ In South India, 45.4% of carbapenem-resistant strains were carrying the bla_NDM gene from a collection of 33 isolates.²⁴ In France, 21% of CREC isolates were bla_NDM producers among a total of 140 isolates.²⁵ A nationwide study conducted in China has reported that 49% of CREC were bla_NDM producers.²⁶ In a recent study from Pakistan, 34 (27.2%) of total K. pneumoniae isolates included in the study were found to harbor the carbapenemase gene. The incidence of bla_NDM was highest as 23 strains harbored this gene, whereas the bla_IMP, bla_VIM, and bla_OXA-48 were found in nine, four, and three isolates, respectively.²⁷

A total of 117 carbapenem-resistant GN isolates were collected in a study from Pakistan, which showed that 72 (61.5%) isolates were positive for the bla_NDM belonging to three genotypes (blaNDM-1, 5, and 7). Out of these, the maximum number of bla_NDM-positive isolates, that is, 36 isolates were K. pneumoniae, whereas 11 (15.2%) isolates were identified as E. coli.²⁸ In the current study, 52.3% of CREC strains were carrying the bla_NDM gene, which is higher than the previous studies from Pakistan. Moreover, the molecular epidemiology of CREC from Southern Punjab was not reported in the previous studies.

We have demonstrated that all the CREC in the study were harboring either blaNDM (52.3%) or blaNDM (52.3%) gene and none of the isolates was carrying both MBL genes. However, the bla_OXA-48-like with an overall frequency of 21.1% was among the CREC isolates co-occurring in 15 bla_NDM-positive and 8 bla_IMP-positive isolates. In a recent study from the federal capital of Pakistan, the incidence of bla_NDM-1, bla_IMP-1, and blaOXA-48 was 35.83%, 20.83%, and 8.3%, respectively, among the CREC isolates.²⁹ However, this study also reported blaKPC-2 and bla_VIM-1 genes in 26.67% and 25% isolates, which are quite different from our findings. Such disparities of the ratios might be attributed to the disparities in the sample size, study objectives, sample source, and locations.

Furthermore, among the ESBLs, bla_CTX-M (71.6%) was the most prevalent genotype, whereas the bla_TEM was found in 59.6% isolates and none of the isolates was carrying the bla_SHV gene. The co-existence of MBLs and ESBL genes was also found in our study. The co-existence of MBL and ESBL genes had also been detected in other studies from Pakistan.^29,30

The current study also highlights the presence of 16S rRNA methylase genes among the CREC strains in the southern Punjab of Pakistan. Among the 16S rRNA methylase genes, the frequency of the rmtB gene was 69 (63.3%) CREC isolates, whereas the armA was found in 55 (50.5%) CREC isolates. Despite the data regarding the presence of 16S rRNA methylase genes in E. coli, clinical isolates from Pakistan is quite limited. In a 10-year-old study from Pakistan, 16 bla_NDM-producing CREC strains were isolated, whereas the rmtC was found in 12 isolates and armA in 7 isolates.³¹ In another study, the 16S rRNA methylases were reported in 3% of the ESBL-producing isolates that were collected in 2005 and 2009–2010.³² In a recent study from Pakistan, only a single CREC isolate was harboring bla_NDM-1 and 16S rRNA methylase, which is, rmtB.

The prevalence of 16S rRNA methylase varies in different studies from various regions of the world^33,34; however, the prevalence of these 16S methylases enzymes is quite higher in our study. In a recent study from the neighboring country (India), the prevalence of 16S rRNA methylases is comparable to our study, in which 77/117 (65.8%) isolates were positive for diverse 16S rRNA methylase genes as the rmtC, armA, rmtD, rmtF, rmtB, npmA, rmtH, rmtE, and rmtG were reported in that study.³⁵ The production of ESBLs, carbapenemases, together with 16S rRNA methylase observed in this study (Table 4) indicates an emerging and frightening combination that needs attention.

In our study, the CREC isolates were 100% susceptible to polymyxins. The reports regarding the colistin resistance especially mediated by the mcr genes among E. coli are increasing. In a study from Thailand, the blaNDM, blaOXA-48, and blaIMP were reported among the CREC strains along with the presence of mcr gene in 13 (0.3%) of carbapenem-resistant isolates.³⁶

The data about the prevalence of CREC clones from Pakistan are limited, therefore, the MLST of bla_NDM-1-harboring isolates was performed. The ST131-harboring bla_NDM-1 and bla_NDM-5 was the most prevalent (37.8%) ST in the current study. The ST131 exhibits various virulence factors and has emerged as a leading cause of extraintestinal infections caused by E. coli in different parts of the world.

The isolates corresponding to ST131 are usually MDR-expressing ESBLs, especially bla_CTX-M-15, conferring significant resistance to third-generation cephalosporins. This ST is generally considered clinically aggressive resulting in a range of nosocomial infections.³⁷ Most studies have reported that the E. coli isolates belonging to ST131 usually carry the ESBLs, especially bla_CTX-M-15,^38,39 but not the carbapenemase gene; however, the studies showing the presence of MBLs have been increasing in the past few years.

In China, the E. coli isolate carrying bla_NDM-7 gene corresponding to ST131 was reported in 2016 with two ST131 isolates carrying bla_SHV, bla_CTX-M-15, and bla_TEM. A study from Taiwan has reported that 18/25 (72%) of CREC isolates belonging to ST131 were not carrying bla_CTX-M enzymes.⁴⁰ These disparities indicate that the ST131 clone has a higher potential to acquire the resistance determinants under selective pressure. In our previous study, we have reported the ST131 E. coli isolates harboring the ESBL (bla_CTX-M, bla_TEM, and bla_SHV) and the MBLs (bla_VIM and bla_NDM) from poultry sources.¹ These findings highlight the importance of the “One Health” concept and necessitate further studies involving the isolates from the environment, poultry, livestock, and clinical sources to comprehend the transmission dynamics of such clones.

Overall, ST131 was the predominant clonal complex, including 47 isolates, containing the ST131 (21), ST3329 (11), ST2279 (8), and ST8051 (7). All of these were carrying bla_NDM-1, bla_NDM-5, or bla_NDM-7, except the ST131 that was corresponding to isolates carrying both blaNDM-1 and blaNDM-5. Despite the epidemiology of the ST131 clone that is widely studied, there are considerable gaps in understanding the mechanisms of transmission, reservoirs, and risk factors for the acquisition of ST131 isolates. The risk factors for the acquisition of ST131 include a long-term residency at a health care facility or nursing home, age, diabetes mellitus, surgical interventions, cancers, exposure to antibiotics, or lack of prior antibiotic treatment, and use of proton pump inhibitors.⁴¹

The limitation was that the clonal diversity and typing for the 57 strains carrying bla_NDM were performed and MLST and typing were not performed for the remaining isolates carrying bla_IMP genes. Therefore, further broad-scale studies involving multiple centers are essential to characterize the CREC for better understanding the molecular epidemiology of the prevalent clone in the region.

Conclusions

The present study reported the genetic diversity and emergence of bla_NDM variants and 16S rRNA methylases among the CREC in southern Punjab for the first time. The dissemination of four different NDM variants, the presence of bla_IMP-type MBLs, and 16S rRNA methylases further increases the threat owing to the limited options to treat the infections caused by such superbugs. Notably, the association of these bla_NDM variants along with other determinants with the ST131 is worrisome due to the higher virulence score of this clonal complex. The timely detection of ESBL, MBL, and 16S methylase enzyme and antimicrobial susceptibility tests are crucial in resource-limited countries for appropriate and more effective therapeutic management of such infections. Also, the surveillance and active infection control procedures should be a part of the routine procedure in clinical settings to avoid the transformation of such diverse resistant mechanisms into epidemics in the coming years.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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