Prevalence and Molecular Characterization of New Delhi Metallo-Beta-Lactamases in Multidrug-Resistant Pseudomonas aeruginosa and Acinetobacter baumannii from India

Abstract

New Delhi metallo-beta-lactamase (NDM)-mediated carbapenem resistance in Pseudomonas aeruginosa and Acinetobacter baumannii is a major concern. We investigated the presence of NDM and its variants in P. aeruginosa and A. baumannii at a tertiary hospital in North India. A total of 236 isolates (130 P. aeruginosa and 106 A. baumannii) were included; 38 (29.23%) P. aeruginosa and 20 A. baumannii isolates (18.8%) were resistant to carbapenems and all of them were bla_NDM positive. All 38 carbapenem-resistant P. aeruginosa harbored bla_NDM-1, while 12 (60%) of 20 A. baumannii harbored bla_NDM-2. Pulsed-field gel electrophoresis showed that all 58 isolates were clonally unrelated. By Southern blot analysis, bla_NDM-2 was located on chromosome. The bla_NDM-2-positive isolates were more frequently recovered from tracheal aspirate (67% vs.16%; p = 0.02) and intensive care unit (67% vs. 20%; p = 0.001) than bla_NDM-1. Among other carbapenemases, VIM was significantly associated with bla_NDM-1 than bla_NDM-2 (61% vs. 17%; p = 0.006). Mortality between bla_NDM-1- and bla_NDM-2-infected patients was comparable. When expressed in Escherichia coli, bla_NDM-2 transformant conferred one doubling dilution higher MIC value for cefotaxime, piperacillin/tazobactam than bla_NDM-1. The study shows the emergence of bla_NDM-mediated resistance among P. aeruginosa and A. baumannii and rapid evolution of bla_NDM-2 in A. baumannii with its chromosomal localization.

Introduction

P seudomonas aeruginosa and Acinetobacter baumannii are important pathogens, which cause a wide range of serious infections in hospitalized and in intensive care unit (ICU) patients.¹ These nonfermenters often are multidrug resistant (MDR) with increasing trend of resistance to oxyimino cephalosporins and carbapenems over the last two decades.² Carbapenem resistance is considered a significant healthcare problem because of the limited treatment options.³ New Delhi metallo-β-lactamase (NDM) is one of the recently described major carbapenemases, and this enzyme compromises the efficacy of almost all β-lactams (except aztreonam), including the last resort carbapenems.⁴ Currently, 18 NDM variants (NDM-1 to −18) have been identified (www.lahey.org/Studies). Although NDM-1 is mostly detected in Enterobacteriaceae, this carbapenemase has also been reported in P. aeruginosa and Acinetobacter spp.³ Till date, NDM variants had not been reported in P. aeruginosa; however, NDM-2 had been reported in A. baumannii from Israel and Middle East.^5–7 These bacteria are considered environmental pathogens with intense colonization capacity and ability to persist for indefinite periods in the hospital environment.^3,8 Hence, it is important to know the prevalence of NDM and its variants in these pathogens in hospital settings for patients' management. The present study was designed to investigate the prevalence of bla_NDM-1 and its variants in P. aeruginosa and A. baumannii and their molecular characterization.

Materials and Methods

Bacterial isolates

A total of 236 consecutive P. aeruginosa and A. baumannii recovered from various clinical specimens between July and December 2012 at Sanjay Gandhi Postgraduate Institute of Medical Sciences, (Lucknow, India), a 900-bed tertiary care referral hospital in North India, were included in the study. The isolates were initially identified by standard biochemical tests⁹ and further confirmed by the Vitek MS identification system (BioMérieux Marcy-l'Étoile, France).

Antimicrobial susceptibility testing

Susceptibility testing was done by the disk diffusion method and minimum inhibitory concentration (MIC) was determined by the broth microdilution method to different beta-lactam antibiotics and aminoglycosides, as per breakpoints and procedure recommended in Clinical and Laboratory Standards Institute (CLSI) guidelines.¹⁰ Antibiotic disks and media were procured from Difco (BD Biosciences, San Jose, CA) and pure antibiotic powders from Sigma-Aldrich (St. Louis, MO). Escherichia coli strain ATCC 25922 was used as quality control.

DNA amplification and analysis

The cell lysate was prepared from all carbapenem-resistant isolates by boiling. Briefly, a fresh bacterial colony was suspended in 150 μl of sterile distilled water and boiled at 100°C for 10 min. After centrifugation of cell lysate at 15,000 g for 15 min at 4°C, the supernatant was collected and stored at −20°C until further analysis. PCR was performed for the detection of genes encoding acquired carbapenemases (bla_NDM, bla_IMP, bla_VIM, bla_SIM, bla_GIM, bla_SPM, bla_OXA-48/-181, bla_BIC, bla_DIM, and bla_KPC) following previously described methods.^11,12 Isolates were also examined for the presence of additional plasmid-encoded beta-lactamases such as bla_TEM, bla_SHV, bla_CTX-M, and bla_CMY as well as genes for 16S rRNA methyltransferase (armA and rmtA-rmtF) for aminoglycoside resistance by PCR as described elsewhere.^13–16

Sequencing

For the identification of bla_NDM-1 and its variants, DNA fragments amplified for the bla_NDM region (984 bp encompassing the entire bla_NDM) were purified using a QIA quick PCR purification kit (QIAGEN, Hilden, Germany). Purified PCR amplicons (984 bp) were sequenced using bla_NDM specific primers.^17,18 Sequencing was performed by Amnion Biosciences Pvt. Ltd. (Bengaluru, India) and sequences were analyzed using the BLAST program at the National Center for Biotechnology Information. Amino acid sequences of NDMs were predicted by the standard code and aligned using the ExPASy online tool for translation.

Comparative analysis of bla_NDM variants

To evaluate and compare the susceptibility between NDM-1 and NDM variants (NDM-2) to different antibiotics, cloning of the bla_NDM-1 and bla_NDM-2 was performed using a Zero Blunt TOPO PCR cloning kit followed by expression in E. coli TOPO10 (Invitrogen, Carlsbad, CA). PCR amplicons of 984 bp encompassing the entire bla_NDM with the same promoter were amplified using bla_NDM full-length specific primers as described previously.^17,18

Nucleotide sequence accession numbers

The nucleotide sequence data reported in the present study had been assigned EMBL/GenBank nucleotide accession nos. KF284076- KF284132.

Pulsed-field gel electrophoresis and Southern hybridization

Genomic DNA of the isolates positive for bla_NDM was prepared in agarose blocks, digested with restriction enzymes SpeI or ApaI, and separated using a CHEF II D-Mapper XA PFGE system (Bio-Rad, Hercules, CA) with running conditions as described previously.¹⁹ Digested products were analyzed by pulsed-field gel electrophoresis (PFGE) to study clonality of the isolates. For location of bla_NDM-2, the probe of 984 bp was developed by PCR amplification of bla_NDM-2 using DIG high prime DNA labeling kit II (Roche Diagnostics, Mannheim, Germany). Genomic DNA prepared in agarose plug from all bla_NDM-2-positive isolates was digested with I-Ceu-1 and S1 nuclease separately and subjected to PFGE. PFGE separated digested products were hybridized using bla_NDM-2 probe.²⁰

Statistical analysis

Statistical analysis was performed using SPSS statistics for Windows v.17.0 (SPSS, Inc., Chicago, IL). Categorical variables expressed as numbers and percentages were compared using Pearson's chi-square test and Fisher's test as appropriate. A p-value of <0.05 was considered statistically significant, and all the tests were two tailed.

Results

Prevalence of bla_NDM-₁ and its variant, their susceptibility testing, and MIC determination

A total of 236 isolates (130 P. aeruginosa and 106 A. baumannii) were included in this study; 58 (24.6%) isolates were nonsusceptible to meropenem and imipenem by disk diffusion and broth microdilution (MIC range, 4 μg/ml to >512 μg/ml) and their distributions were as follows: P. aeruginosa 38 (29.2%) of 130 and A. baumannii 20 (18.8%) of 106 isolates. All carbapenem-nonsusceptible isolates were bla_NDM positive by PCR. Sequencing results of bla_NDM-positive amplicons revealed that 8/20 (40%) A. baumannii and all 38 P. aeruginosa harbored bla_NDM-1, while 12/20 (60%) of A. baumannii had a C to G substitution at position 82 resulting in an amino acid change from proline to alanine (Pro→Ala) at position 28 and were identified as bla_NDM-2. The nucleotide sequence data of 46 bla_NDM-1 and 12 bla_NDM-2 reported in the present study had been assigned EMBL/GenBank nucleotide accession nos. KU510336- KU510393.

Presence of additional antibiotic resistance genes in isolates positive for bla_NDM-1 and bla_NDM-2

All 58 bla_NDM-positive isolates were also resistant to aminoglycosides such as amikacin and gentamicin (MIC ≥256 μg/ml) and different beta-lactams and beta-lactam/beta-lactamase inhibitor combinations, but susceptible to colistin and tigecycline. The frequency of additional beta-lactamase and 16S rRNA methyltransferase genes (either alone or in combination) was compared between bla_NDM-1-positive (n = 46) and bla_NDM-2-positive (n = 12) isolates (Table 1). All genes were found comparable except bla_VIM, which had a significant association with bla_NDM-1 (bla_NDM-1 vs. bla_NDM-2; 61% vs. 17%, p = 0.006). Among other carbapenemases, SPM was detected in 12 (26%) of bla_NDM-1-positive and 05 (42%) of bla_NDM-2-positive isolates for the first time in India. IMP was detected in 17 (37%) and 02 (17%) of bla_NDM-1- and bla_NDM-2-positive isolates, respectively. All the strains were negative for bla_OXA-48/-181, bla_BIC, bla_DIM, bla_KPC, bla_SIM, and bla_GIM. Among the ESBLs, bla_TEM, bla_CTX-M, and bla_SHV, and among the 16S rRNA methyltransferases, armA, rmtB, rmtC, rmtD, and rmtF were detected in almost equal proportions in bla_NDM-1- and bla_NDM-2-positive isolates. Their frequencies of distributions are shown in Table 1.

Table 1.

Presence of Additional Antibiotic-Resistant Genes in bla_NDM-1- and bla_NDM-2-Positive Clinical Isolates of Pseudomonas aeruginosa and Acinetobacter baumannii

Overall gene frequency	NDM-1 (n = 46)	NDM-2 (n = 12)	p-Value
IMP	17 (37%)	2 (17%)	0.46
VIM	28 (61%)	2 (17%)	0.006
SPM	12 (26%)	5 (42%)	0.29
CTX-M	8 (17%)	5 (42%)	0.07
TEM	8 (17%)	5 (42%)	0.07
SHV	5 (11%)	2 (17%)	0.58
bla _CMY	14 (30%)	2 (17%)	0.34
armA	23 (50%)	7 (58%)	0.60
rmtB	7 (15%)	1 (8%)	0.53
rmtC	5 (11%)	0 (0%)	0.57
rmtD	1 (2%)	1 (8%)	0.37
rmtF	6 (13%)	1 (8%)	0.65
Genes either alone or in combination
Carbapenemases
Only IMP	2 (4%)	0 (0%)	0.50
Only VIM	14 (30%)	1 (8%)	0.11
Only SPM	4 (9%)	2 (17%)	0.78
IMP+VIM	8 (17%)	0 (0%)	0.11
IMP+SPM	2 (4%)	2 (17%)	0.18
VIM+SPM	1 (2%)	1 (8%)	0.37
IMP+VIM+SPM	5 (11%)	0 (0%)	0.57
Extended-spectrum beta-lactamases (ESBLs)
Only CTX-M	2 (4%)	1 (8%)	0.50
Only TEM	2 (4%)	2 (17%)	0.18
Only SHV	1 (2%)	0 (0%)	1.00
CTX-M+TEM	2 (4%)	2 (17%)	0.18
CTX-M+SHV	0 (0%)	1 (8%)	0.20
CTX-M+TEM+SHV	4 (9%)	1 (8%)	1.00
Methyltransferases
Only armA	14 (30%)	4 (33%)	0.84
Only rmtB	3 (7%)	0 (0%)	1.00
Only rmtC	1 (2%)	0 (0%)	1.00
Only rmtF	3 (7%)	0 (0%)	1.00
armA + rmtB	4 (9%)	1 (8%)	1.00
armA + rmtC	2 (4%)	0 (0%)	1.00
armA + rmtD	1 (2%)	1 (8%)	0.37
armA + rmtF	1 (2%)	1 (8%)	0.37
rmtC + rmtF	1 (2%)	0 (0%)	1.00
armA + rmtC + rmtF	1 (2%)	0 (0%)	1.00
bla_CMY + carbapenemase + ESBL + 16S rRNA methyltransferase	3 (7%)	0 (0%)	1.00
ESBL +16S rRNA methyltransferase	1 (2%)	1 (8%)	0.37

Demographic and clinical data of patients infected with bla_NDM-1- and bla_NDM-2-positive isolates

Clinical and demographic data were compared between patients infected with bla_NDM-1-positive (n = 46) and bla_NDM-2-positive (n = 12) isolates (Table 2). The highest number of bla_NDM-positive (both bla_NDM-1 and bla_NDM-2) isolates was recovered from ICU (17/58; 29.3%) followed by surgical gastroenterology (13/58; 22.4%), and the most common clinical specimen was endotracheal aspirate (19/58; (32.8%) followed by pus (16/58; 27.6%). When compared between bla_NDM-1 and bla_NDM-2, the frequency of bla_NDM-2 isolation was significantly higher in ICU (67% vs. 20%; p = 0.001) and from clinical specimen, tracheal aspirate (67% vs. 16%; p = 0.02) (Table 2). All 58 patients infected with bla_NDM-positive isolates received broad-spectrum antibiotics before and after hospital admission. Mortality rate was found comparable between patients infected with bla_NDM-1-positive and bla_NDM-2-positive (26% vs. 50%; p = 0.11) isolates. All 12 bla_NDM-2-positive A. baumannii patients had hospital-acquired infections; 9 of them were on mechanical ventilation and 3 had undergone major surgery (Table 3).

Table 2.

Demographic and Clinical Data of Patients Infected with bla_NDM-1- and bla_NDM-2-Positive Isolates

Factors	NDM-1 (n = 46)	NDM-2 (n = 12)	p Value
Age (Years)
Mean (±SD)	48.12 ± 12.23	45.83 ± 18.83
Sex
Male	29 (63%)	8 (64%)	0.81
Female	17 (37%)	4 (33%)	0.18
Specimens
Blood	8 (17%)	3 (33%)	0.54
Urine	12 (7%)	0 (0%)	0.18
Pus	15 (39%)	1 (8%)	0.09
Tracheal aspirate	11 (16%)	8 (67%)	0.02
Hospital wards
Intensive care unit	9 (20%)	8 (67%)	0.001
Nephrology	2 (4.0%)	2 (17%)	0.13
Urology	7 (15%)	0 (0%)	0.14
Transplant unit	2 (4%)	0 (0%)	1.00
Neurosurgery	4 (9.0%)	0 (0%)	0.57
Endocrine surgery	1 (2%)	0 (0%)	1.00
Pulmonary medicine	3 (7%)	0 (0%)	0.36
Surgical gastroenterology	12 (26%)	1 (8%)	0.18
Gastroenterology	3 (7%)	0 (0%)	0.36
Neurology	2 (4%)	1 (8%)	0.46
Cardiology	1 (2%)	0 (0%)	0.37
Patients
Hospitalized	41 (93%)	12 (100%)	0.23
Nonhospitalized	5 (10.86%)	0 (0%)	0.57
Outcome
Recovered	34 (74%)	6 (50%)	0.11
Death	12 (26%)	6 (50%)	0.11

Table 3.

Demographic and Clinical Features of Patients Infected with bla_NDM-2-Positive Acinetobacter baumannii (n = 12)

	KnAb21	KnAb22	KnAb23	KnAb24	KnAb25	KnAb26	KnAb27	KnAb28	KnAb29	KnAb210	KnAb211	KnAb212
Patient's age (year)	70	16	44	28	45	18	71	55	20	30	70	60
Sex	Female	Male	Female	Male	Female	Male	Male	Male	Male	Female	Male	Male
Department	Neurology	Intensive care unit (ICU)	ICU	Surgical gastroenterology	ICU	ICU	ICU	Nephrology	Nephrology	ICU	ICU	ICU
Specimen	ET aspirate	Maxillary sinus aspirate from left middle meatus nasal cavity	Blood	Blood	ET aspirate	ET aspirate	ET aspirate	ET aspirate	ET aspirate	ET aspirate	ET aspirate	Blood
Diagnosis	Cerebrovascular accident, septic shock	Complicated malaria with dengue coinfection	Subarachnoid hemorrhage	Acute chronic pancreatitis, sepsis with colocutaneous fistula	Cardiomyopathy, lower respiratory tract infection, multiorgan dysfunction	Tubercular meningitis, ventriculo-peritoneal (VP) shunt malfunction	Right frontoparietal hemorrhage	Chronic kidney disease with septic shock	Acute kidney injury, lower respiratory tract infection with septic shock	Chronic kidney disease, pneumonia, Koch's abdomen	Right parietal lobe hemorrhage, aspiration pneumonia	Cerebrovascular accident, septic shock
Procedure, if any	Mechanical ventilation	Mechanical ventilation	Right pterional craniotomy and clipping of aneurysm	Laparotomy, lavage and loop ileostomy	Mechanical ventilation	VP Shunt and Mechanical ventilation	Mechanical ventilation	Mechanical ventilation, hemodialysis	Hemodialysis	Mechanical ventilation	Mechanical ventilation	Mechanical ventilation
Infection	Hospital acquired (HA)	HA	HA	HA	HA	HA	HA	HA	HA	HA	HA	HA
Comorbidity	Hypertension	—	Hypertension	—	Diabetes mellitus (DM), hypertension	—	Coronary artery disease, hypertension	DM, hypertension	-	-	-	DM, Hypertension
Outcome	Death	Recovered	Recovered	Death	Death	Recovered	Recovered	Recovered	Death	Death	Recovered	Death

ET, endotracheal.

PFGE and Southern blot analysis for isolates positive for bla_NDM

PFGE was performed on 38 NDM-positive P. aeruginosa and 20 A. baumannii isolates separately; PFGE profile of 38 P. aeruginosa and 20 A. baumannii was clonally unrelated because of different PFGE band patterns. Attempts to transfer bla_NDM-2 from all 12 isolates (KnAb21- KnAb212) to E. coli J53 (azide resistant) were unsuccessful. Southern hybridization using bla_NDM-2 probes revealed that bla_NDM-2 was located on chromosomal DNA.

Comparative analysis of susceptibility between bla_NDM-1- and bla_NDM-2-positive isolates

One for each A. baumannii clinical isolate positive for bla_NDM-1 (KnAb11) and bla_NDM-2 (KnAb21) was selected for full-length gene amplification for bla_NDM and cloning in E. coli TOPO10. It gave rise to E. coli TOPO10 carrying recombinant plasmids, pbla_NDM-1 and pbla_NDM-2. The MICs for carbapenems (imipenem and meropenem) and different beta-lactams and beta-lactam/beta-lactamase inhibitor combinations were compared (Table 4). E. coli TOPO10 carrying pbla_NDM-2 showed one doubling dilution higher MIC value for cefotaxime and piperacillin/tazobactam than pbla_NDM-1 and the results were consistent when repeated; however, there was no difference in MIC range for carbapenems (imipenem and meropenem).

Table 4.

Minimum Inhibitory Concentration (MIC μg/ml) of Escherichia coli TOPO10 and Its Transformants with pbla_NDM-1 and pbla_NDM-2 of Different Antibiotics

MIC of bla_NDM-positive Acinetobacter baumanii (MIC μg/ml)			MIC of E. coli TOPO10 and its transformants (MIC μg/ml)
Antibiotics	A. baumannii with bla_NDM-1 (KNAb11)	A. baumannii with bla_NDM-2 (KnAb21)	E. coli TOPO10 with pbla_NDM-1	E. coli TOPO10 with pbla_NDM-2	E. coli TOPO10
Ceftazidime	>512	>512	512	512	2
Cefotaxime	>512	>512	256	512	4
Cefepime	512	>512	256	256	4
Aztreonam	>512	>512	0.12	0.12	0.12
Cefoperazone/sulbactam	>512	256	>512	>512	0.12
Piperacillin/tazobactam	>512	>512	512	>512	0.06
Cefoxitin	512	128	64	64	2
Imipenem	64	512	32	32	0.01
Meropenem	256	256	128	128	0.06

Discussion

NDM producers are now alarmingly on the increase worldwide and they pose a potential risk for therapeutic failure with the empirical treatments currently in place.³ Till date, 18 NDM variants (NDM-1 to −18) have been reported from different groups of bacteria (www.lahey.org/Studies). Interestingly, all 18 NDM variants carrying isolates had nosocomial origin substantiating that the NDM harboring bacteria are rapidly evolving in hospitals. In India, we previously reported the prevalence of NDM-1, NDM-5, NDM-6, and NDM-7 in multidrug-resistant Enterobacteriaceae.²¹ The prevalence of bla_NDM-positive Acinetobacter and Pseudomonas spp. has been reported from India previously.^22,23 In this study, besides NDM-1 in P. aeruginosa and A. baumannii, we report for first time the prevalence of NDM-2 and its characterization in carbapenem-resistant A. baumannii from North India. The bla_NDM was detected in 29.3% P. aeruginosa and 18.8% A. baumannii in the present study, in contrast to a previous report of 8% in P. aeruginosa from South India²³ and 4% in A. baumannii from North India.²² This high prevalence of bla_NDM in the present study might be due the selection pressure caused by excessive use of carbapenems in our hospital. Overall, the frequency of recovery of bla_NDM-positive isolates was higher in the ICU. The observation justifies the previous study, suggesting ICUs as the epicenter and major source of emergence of antimicrobial resistance.^1,24 Surgical gastroenterology was the second most common site for bla_NDM-positive isolates. This is in concordance with our earlier finding, which might be due to higher exposure of patients to carbapenems after surgery.²¹ The bla_NDM-2 was detected only in A. baumannii; 12 (60%) of 20 carbapenem-resistant isolates of A. baumannii carried bla_NDM-2 and the remaining 8 (40%) harbored bla_NDM-1. This is the first study from India showing the emergence of NDM-2 harboring A. baumannii. The presence of a higher proportion of NDM-2 compared to NDM-1 in A. baumannii indicates that bla_NDM-2 is rapidly evolving in A. baumannii in our hospital. So far, NDM-2 had been reported in A. baumannii only from Israel and Middle East.^5–7 None of our carbapenem-resistant P. aeruginosa isolates was positive for bla_NDM-2 or for any other NDM variant(s). Till date, NDM variants had not been reported in P. aeruginosa. Overall, mortality was comparable between patients infected with bla_NDM-1- and bla_NDM-2-positive isolates (26% vs. 50%; p = 0.11). Among other carbapenemase genes, only bla_VIM was significantly associated with bla_NDM-1 compared to bla_NDM-2 (p = 0.006). An earlier study from India reported the co-occurrence of VIM-2 with NDM-1 in P. aeruginosa on two different plasmids.²⁵ In this study, we detected multiple metallo-beta-lactamases such as IMP+VIM+SPM along with NDM-1 in 5 (13.15%) of 38 P. aeruginosa. The genetic background and transmission dynamics of multiple metallo-beta-lactamases in P. aeruginosa call for further investigation. Till date, SPM was not reported from India. This is the first study from India where SPM was detected in 26% and 42% of bla_NDM-1- and bla_NDM-2-positive isolates, respectively.

Clonal dissemination of NDM-2 producing A. baumannii isolate in Israel and United Arab Emirates had been reported.^5–7 However, in the present study, NDM-2-positive isolates were clonally unrelated, suggesting the existence and spread of multiple clones. The localization study for bla_NDM-2 by Southern hybridization revealed that the gene was located on the chromosome, substantiating that the NDM genes are getting stabilized in the genome of A. baumannii. Similar to the present observation, the earlier studies also reported the localization of bla_NDM-2 on chromosome.^5–7

Comparative analysis between bla_NDM-1 and bla_NDM-2 showed that bla_NDM-2 conferred one doubling dilution higher MIC value for cefotaxime and piperacillin/tazobactam, when expressed in E. coli TOP10. However, in a previous study, no significant difference in MICs to different beta-lactams was observed when bla_NDM-1 and bla_NDM-2 were cloned.¹⁷ Difference by ±1 doubling dilution may be due to normal technical variability of antimicrobial susceptibility testing.²⁶ Since the MICs of the cloned bla_NDM-2 were repeated with consistent results, the higher MIC value to cefotaxime and piperacillin/tazobactam for NDM-2 is unlikely because of technical variability; hence, it needs to be elucidated by kinetic studies using pure enzyme.

Conclusion

Both P. aeruginosa and A. baumannii are ubiquitously present in the hospital environment and are considered important nosocomial pathogens, especially in intensive care settings. Hence, the emergence of bla_NDM in these bacterial pathogens is a matter of grave concern. Prevalence of bla_NDM-2 in A. baumannii in higher proportions suggests that bla_NDM-2 is rapidly evolving in this organism and getting stabilized on the chromosome. Further studies are required to understand the molecular epidemiology on the emergence of bla_NDM and its variants in these organisms to develop strategies to combat their emergence and spread.

Footnotes

Ethical Approval

This study was approved by the Institutional Ethics Committee of Sanjay Gandhi Postgraduate Institute of Medical Sciences (Lucknow, India) [PGI/BE/619/2014].

Acknowledgments

The authors are grateful to the Department of Animal Health, Universidad Complutense de Madrid (Madrid, Spain) for providing positive controls for different 16S rRNA methyltransferases (ArmA and RmtA-F) and sodium-azide-resistant E. coli J53AzR.

This study was supported by an Indo-Spanish research grant from the Department of Science and Technology (New Delhi, India) [DST/INT/SPAIN/P-28/2011-C] and by the Spanish Ministry of Science and Innovation [PRI-PIBIN-2011-0915]. Indian Council for Medical Research, New Delhi, India (Ref no 80/895/2014-ECD I), is gratefully acknowledged for providing Senior Research Fellowship to M.R.

Disclosure Statement

No competing financial interests exist.

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Prevalence and Molecular Characterization of New Delhi Metallo-Beta-Lactamases in Multidrug-Resistant Pseudomonas aeruginosa and Acinetobacter baumannii from India

Abstract

Introduction

Materials and Methods

Bacterial isolates

Antimicrobial susceptibility testing

DNA amplification and analysis

Sequencing

Comparative analysis of blaNDM variants

Nucleotide sequence accession numbers

Pulsed-field gel electrophoresis and Southern hybridization

Statistical analysis

Results

Prevalence of blaNDM-1 and its variant, their susceptibility testing, and MIC determination

Presence of additional antibiotic resistance genes in isolates positive for blaNDM-1 and blaNDM-2

Demographic and clinical data of patients infected with blaNDM-1- and blaNDM-2-positive isolates

PFGE and Southern blot analysis for isolates positive for blaNDM

Comparative analysis of susceptibility between blaNDM-1- and blaNDM-2-positive isolates

Discussion

Conclusion

Footnotes

Ethical Approval

Acknowledgments

Disclosure Statement

References

Comparative analysis of bla_NDM variants

Prevalence of bla_NDM-₁ and its variant, their susceptibility testing, and MIC determination

Presence of additional antibiotic resistance genes in isolates positive for bla_NDM-1 and bla_NDM-2

Demographic and clinical data of patients infected with bla_NDM-1- and bla_NDM-2-positive isolates

PFGE and Southern blot analysis for isolates positive for bla_NDM

Comparative analysis of susceptibility between bla_NDM-1- and bla_NDM-2-positive isolates