First Report of New Delhi Metallo-Beta-Lactamase-1-Producing Klebsiella pneumoniae in Iran

Abstract

New Delhi metallo-beta-lactamase (NDM-1) is a novel metallo-beta-lactamase (MBL). Sporadic cases of NDM-1 positive strains have been reported from different countries, suggesting a widespread dissemination. The aim of this study was the detection of MBLs in Enterobacteriaceae isolated from patients in Tehran hospitals. After identification tests, the susceptibility to the antibiotics was done by Kirby–Bauer method and broth microdilution. Carbapenem-resistant isolates were tested for carbapenemase production using the modified Hodge test (MHT). Carbapenem-resistant strains screened for bla_KPC gene and genes encoding MBLs. Twenty-three isolates (6.3%) were resistant to meropenem, eleven isolates (3%) were resistant to ertapenem, and four isolates (1.1%) were resistant to imipenem. MHT was positive in 11 (47.8%) of the carbapenem-resistant isolates. In March 2011, we detected a multiple drug-resistant Klebsiella pneumoniae isolate that was resistant to all tested antibiotics except colistin. PCR confirmed that this isolate contained bla_NDM-1, bla_TEM, bla_SHV, and bla_CTX-M. This is the first report on the detection of MBL NDM-1 in Iran. The rapid spread of NDM-1-positive bacteria proved to be a major challenge for the treatment and control of infectious diseases.

Introduction

Infectious diseases caused by antibiotic-resistant Enterobacteriaceae strains remain a major public health problem in different countries.^9,32 The spread of resistant strains has compromised the utility of current treatment options for severe infections that are caused by gram-negative bacteria. Infections associated with multidrug-resistant, gram-negative bacteria present a real challenge in the treatment of patients, and the impact on hospital costs and mortality rates. In recent years, the widespread emergence of carbapenem-resistant isolates has been increasing worldwide.^19,21,28 Carbapenemases belong to class A clavulanic acid inhibitory enzymes, class B metallo-β-lactamases (MBLs), and class D oxacilinases.⁴¹ Acquired carbapenemases have been restricted to geographical areas and specific bacterial strains, and outbreaks spreading in other areas have been often associated with imported cases from countries in which the bacteria are endemic.^19,32

MBLs are important determinants of increasing resistance rate among Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii.⁸ New Delhi metallo-β-lactamase-1 (NDM-1) is a novel MBLs that is distantly related to other MBLs, sharing only 32% amino acid identity with the most closely related enzymes VIM-1 and VIM-2. In 2008, the first NDM-1 positive-gram negative isolate was detected in Sweden from a 59 year-old man previously hospitalized in India.⁴² Since 2008, NDM-1 has been detected in Klebsiella pneumoniae, Escherichia coli, A. baumannii, Enterobacter spp., Citrobacter freundii, Morganella morganii, and Providencia spp. in several parts of the world,^{7,10,16,18,24–26,31,43} which was often mediated via transfer of patients, as well as in some cases, direct transmission occurred in Europe.²⁰

NDM-1 has a higher rate of dissemination and clinical impact than other acquired MBLs.^35,40 The emergence of NDM-1 is considered a serious threat, because the risk of plasmid-mediated transfer of NDM-1 between different bacterial strains and NDM-1 producers is reported in community acquired infections.^16,24,26 The multiresistant nature of these strains severely limits treatment options. This study was designed to determine the antibiotic susceptibility patterns among Enterobacteriaceae strains. In this article, we described the first detection of bla_NDM-1 gene in Iran.

Materials and Methods

Strains and identification tests

Five hospitals in Tehran, Iran, were requested to send Enterobacteriaceae isolates to the bacteriology department of Pasteur Institute of Iran. The identification of received isolates was performed by routine biochemical tests, and some cases were tested by using API 20E strips (Biomerieux, Marcy-l'Etoile, France). The majority of collected strains were isolated from urine and feces. All strains were stored at −80°C in nutrient broth (Difco, Detroit, MI) containing 30% glycerol until processing.

Susceptibility testing

The susceptibility of isolates was performed by the disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) criteria.⁶

Minimal inhibitory concentrations (MICs) of imipenem, meropenem, ceftazidime, and cefotaxime were determined by broth microdilution as recommended by CLSI.⁵ All strains with resistance to imipenem, meropenem, and ertapenem were tested for the production of carbapenemases. The modified Hodge test (MHT) was performed according to CLSI guidelines using imipenem, meropenem, and ertapenem disks on Muller–Hinton agar plates. E. coli ATCC 25922 was used as a carbapenem susceptible strain and also for MBLs screening. MBL E-test strips (AB Biodisk, Solna, Sweden) using imipenem and imipenem-ethylenediaminetetraacetic acid (EDTA) were used according to the protocol recommended by the manufacturer. In all antimicrobial susceptibility testing, E. coli ATCC 25922, P. aeruginosa ATCC 27853, K. pneumoniae strain 7881, and P. aeruginosa, KOAS strain (Kindly provided by Patrice Nordmann, Service de Bacteriologie-Virologie, Hospital de Bicetre Paris, France) were used for quality control.

Detection of extended spectrum β-lactamase and MBLs genes by PCR

Genomic DNAs were extracted by the standard phenol-chloroform method as described by Andrysiak et al.² The detection of extended spectrum β-lactamase (ESBL) genes was carried out by PCR with specific primers to detect bla_TEM, bla_SHV_, bla_CTX-M, bla_PER, and bla_VEB genes as previously described.^1,4,13,29 K. pneumoniae strain 7881 and P. aeruginosa, KOAS strain were used as the control strain in the detection of these genes. Carbapenem-resistant strains screened for bla_KPC and bla_GES genes and genes encoding MBLs by using specific primers targeting bla_NDM-1, bla_VIM-1, bla_VIM-2, bla_IMP-1, bla_IMP-2, and bla_SPM. Primer pairs used for the amplification of the β-lactamase genes are listed in Table 1. The nucleotide sequences of positive PCR products were determined by direct sequencing at Macrogen, Inc. (Seoul, Korea). PCR products were sequenced in both directions.

Table 1.

Oligonucleotide Primers Used in This Study

Primer	Target gene/region	Sequence (5′-3′)	Product size (bp)	Annealing temperature (°C)	References
TEM-A	bla_TEM	GAGTATTCAACATTTCCGTGTC	800	52	27
TEM-B		TAATCAGTGAGGCACCTATCTC
SHV-A	bla_SHV	AAGATCCACTATCGCCAGCAG	200	60	13
SHV-B		ATTCAGTTCCGTTTCCCAGCGG
CTX_M-F	bla_CTX-M	CGCTTTGCGATGTGCAG	550	54	1
CTX_M-R		ACCGCGATATCGTTGGT
PER-F	bla_PER	AATTTGGGCTTAGGGCAGAA	925	56	4
PER-R		ATGAATGTCATTATAAAAGC
VEB-F	bla_VEB	CGACTTCCATTTCCCGATGC	643	55	29
VEB-R		GGACTCTGCAACAAATACGC
KPC-F	bla_KPC	CTTGCTGCCGCTGTGCTG	489	58	38
KPC-R		GCAGGTTCCGGTTTTGTCTC
GES-F	bla_GES	ATGCGCTTCATTCACGCAC	640	60	27
GES-R		CTATTTGTCCGTGCTCAGG
NDM-F	bla_NDM-1	ACCGCCTGGACCGATGACCA	263	58	43
NDM-R		GCCAAAGTTGGGCGCGGTTG
VIM1-F	bla_VIM-1	AGTGGTGAGTATCCGACA	261	55	36
VIM1-R		ATGAAAGTGCGTGGAGAC
VIM2-F	bla_VIM-2	ATGTTCAAACTTTTGAGTAAG	801	57	36
VIM2-R		CTACTCAACGACTGAGCG
IMP1-F	bla_IMP-1	ACCGCAGCAGAGTCTTTGCC	587	55	36
IMP1-R		ACAACCAGTTTTGCCTTACC
IMP2-F	bla_IMP-2	GTTTTATGTGTATGCTTCC	678	58	36
IMP2-R		AGCCTGTTCCCATGTAC
SPM-F	bla_SPM	GCGTTTTGTTTGTTGCTC	786	53	36
SPM-R		TTGGGGATGTGAGACTAC

Conjugation experiments

To determine whether the resistance in clinical strains was transferable, conjugation experiments were carried out by mating-out assays with E. coli K12 [F⁻ lac⁺ Nal(r)] as the recipient. Suspensions of overnight cultures were prepared with the recipient at about one-tenth of the cell density of the donor. After 18 hrs of incubation at 37°C, transconjugants were selected on MacConkey agar containing 100 μg/ml sodium azide and 5 μg/ml meropenem (MAST, Merseyside, United Kingdom).³⁰ The confirmation of putative transconjugants was performed by biochemical tests and by the detection of bla_NDM-₁ with PCR, as previously described.

Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) method was used for the molecular typing of carbapenem-resistant isolates. Genomic DNA was prepared in agarose plugs, lysed, and digested with XbaI (Fermentas, Sylvius, Lithuania). DNA fragments were separated in a CHEF-DR III PFGE apparatus (Bio-Rad Laboratories, Hercules, CA) and a gradient of 6 V/cm at a 120° angle, with the pulse time increasing from 5 to 30 sec. Electrophoresis was run at 14°C for 22 hr. DNA from Salmonella enterica serovar Braenderup strain H9812 (ATCC BAA-664) was used as a molecular size standard. The generated PFGE patterns were analyzed using the Gel Compare II (Applied Maths) software. Banding patterns were interpreted using the criteria devised by Tenover et al.³⁷

Results

A total of 360 clinical Enterobacteriaceae isolates were collected from clinical specimens in five hospitals in Tehran between 2009 and 2011. After identification tests, of the 360 collected isolates, 244 isolates (67.7%) identified as E. coli, 45 isolates (12.5%) identified as K. pneumoniae, and 34 isolates (9.4%) identified as Enterobacter spp. The other identified bacterial strains included Citrobacter spp., Shigella spp., Proteus spp., M. morganii, Serratia marcescens, Salmonella spp., and Edwardsiella tarda.

Of the isolates, 23 isolates (6.3%) were resistant to meropenem, 11 isolates (3%) were resistant to ertapenem, 4 isolates (1.1%) were resistant to imipenem, 242 isolates (67.2%) were resistant to ceftazidime, 212 isolates (58.8%) were resistant to cefepime, 257 isolates (71.3%) were resistant to cefotaxime, 221 isolates (61.3%) were resistant to ciprofloxacin, 219 isolates (60.8%) were resistant to aztreonam, 52 isolates (14.4%) were resistant to amikacin, 139 isolates (38.6%) were resistant to kanamycin, and 269 isolates (74.7%) were resistant to trimethoprim-sulfamethoxazole. The majority of the carbapenem-resistant isolates were K. pneumoniae (52.1%) and E. coli (39.1%).

MHT was positive in 11 (47.8%) of the carbapenem-resistant isolates showing carbapenemase production. Table 2 shows the antimicrobial activity and susceptibility patterns of antimicrobial agents tested against E. coli, K. pneumoniae, and Enterobacter spp. strains. Imipenem showed the highest activity (98.8% of the isolates were susceptible), and trimethoprim-sulfamethoxazole showed the lowest activity (25.2% of the isolates were susceptible) against all Enterobacteriaceae isolates in this study. Ertapenem was the most sensitive indicator antibiotic that was used for detecting carbapenemase production. Characterization of 11 MHT positive carbapenem-resistant isolates are listed in Table 3.

Table 2.

Antimicrobial Activity of Antimicrobial Agents Tested Against Escherichia coli (n=244), Klebsiella pneumoniae (n=45), and Enterobacter spp. (n=34)

	No. (%) of resistant isolates
Antimicrobial agent	Escherichia coli	Klebsiella pneumoniae	Enterobacter spp.
Cefepime	146 (59.8)	33 (73.3)	27 (79.4)
Cefotaxime	177 (72.5)	37 (82.2)	30 (88.2)
Ceftazidime	170 (69.6)	34 (75.5)	29 (85.3)
Meropenem	9 (3.7)	13 (28.8)	1 (2.9)
Imipenem	1 (0.4)	3 (6.6)	0 (0)
Ertapenem	2 (0.8)	9 (20)	0 (0)
Ciprofloxacin	159 (65.1)	32 (71.1)	24 (70.5)
Amikacin	35 (14.3)	11 (24.4)	6 (17.6)
Kanamycin	102 (41.8)	21 (46.6)	15 (44.1)
Aztreonam	153 (62.7)	32 (71.1)	28 (82.3)
Trimethoprim-sulfamethoxazole	193 (79.1)	38 (84.4)	28 (82.3)

Table 3.

Characteristics of 11 Carbapenem-Resistant Isolates

Strain no.	Identification	Antibiotic resistance pattern	MHT	MBL E-test	Detection of β-lactamase genes
NE141	Escherichia coli	CPM, CTX, CAZ, MEM, ERT, CIP, AN, K, ATM, TS	Positive	Positive	TEM, CTX-M, PER
NE142	E. coli	CPM, CTX, CAZ, MEM, ERT, CIP, TS	Positive	Positive	CTX-M
NK246	Klebsiella pneumoniae	CPM, CTX, CAZ, IMI, MEM, ERT, CIP, AN, K, ATM, TS	Positive	Positive	NDM-1, TEM, SHV, CTX-M
NK284	K. pneumoniae	CPM, CTX, CAZ, MEM, ERT, CIP, K, ATM, TS	Positive	Positive	TEM, CTX-M
NK324	K. pneumoniae	CPM, CTX, CAZ, IMI, MEM, ERT, CIP, AN, K, ATM, TS	Positive	Positive	TEM, SHV, CTX-M, PER
NK344	K. pneumoniae	CPM, CTX, CAZ, IMI, MEM, ERT, CIP, AN, K, ATM, TS	Positive	Positive	TEM, SHV, CTX-M
NK358	K. pneumoniae	CPM, CTX, CAZ, MEM, ERT, CIP, ATM, TS	Positive	Positive	CTX-M
NK360	K. pneumoniae	CPM, CTX, CAZ, MEM, ERT, AN, K, ATM, TS	Positive	Positive	SHV, CTX-M
NK361	K. pneumoniae	CPM, CTX, CAZ, MEM, ERT, CIP, AN, K, ATM, TS	Positive	Positive	TEM, SHV, CTX-M
NN371	Enterobacter spp.	CPM, CTX, CAZ, MEM, ERT, CIP, TS	Positive	Positive	TEM, CTX-M
NN375	Enterobacter spp.	CPM, CTX, CAZ, MEM, ERT, CIP, K, ATM, TS	Positive	Positive	TEM, SHV, CTX-M

CPM, cefepime; CTX, cefotaxime; CAZ, ceftazidime; IMI, imipenem; MEM, meropenem; ERT, ertapenem; CIP, ciprofloxacin; AN, amikacin; K, kanamycin; ATM, aztreonam; TS, trimethoprim-sulfamethoxazole; MHT, modified Hodge test; MBL E-test, metallo-β-lactamase E-test strips.

In March 2011, a multiple drug-resistant strain of K. pneumoniae was isolated from the urine sample of a 52-year-old male patient with high fever who was hospitalized in a general hospital in Tehran. The patient had kidney transplant rejection 2 months before hospitalization. This isolate was identified as K. pneumoniae by biochemical tests and API20E. Antibiotic susceptibility tests by the disk diffusion method showed that the isolate was resistant to meropenem, ertapenem, cefepime, cefotaxime, ceftazidime, amikacin, kanamycin, ciprofloxacin, aztreonam, and trimethoprim-sulfamethoxazole. No inhibition zone was seen around these antibiotic disks. This isolate was also resistant to imipenem, but its inhibition zone was small (12 mm). This isolate was susceptible to colistin. The patient had no history of travel to India and Pakistan. The determination of MICs confirmed resistance to meropenem (MIC; 8 μg/ml) with intermediate resistance to imipenem (MIC; 4 μg/ml).

PCR confirmed that this isolate contained bla_NDM-1, bla_TEM, bla_SHV, and bla_CTX-M. MHT for the NDM-1-producing isolate showed a weakly positive result. MBL screening by E-test strips was positive. The inhibition of MBL activity by EDTA using the combined disk test confirmed MBL production. Sequencing of bla_NDM-1 revealed that the coding region was 100% identical to the coding region of the bla_NDM-1 gene in other submitted genes in GenBank database. The nucleotide sequence of bla_NDM-1 gene determined in this study was submitted to the GenBank nucleotide sequence database under accession number JN664262. The conjugation experiment that used E. coli K12 as the recipient was unsuccessful. Resistance to carbapenems and the bla_NDM-1 gene did not see in recipient.

A PFGE analysis of 11 carbapenem-resistant isolates with the positive MBL screening test showed that these isolates were clonally unrelated, and different patterns were shown for carbapenem-resistant isolates (Fig. 1).

FIG. 1.

Dendrogram that shows the relatedness of pulsed-field gel electrophoresis patterns of 11 carbapenem-resistant isolates with positive metallo-β-lactamases screening test. New Delhi metallo-beta-lactamase-1 positive isolate (Klebsiella pneumoniae, N246) was completely unrelated to other carbapenem-resistant isolates.

Discussion

Treatment of infections associated with gram-negative bacteria is further complicated by increasing the rate of resistance to cephalosporins and, recently, carbapenems. Infectious diseases with gram-negative bacilli, particularly K. pneumoniae, are emerging as a serious challenge in hospitals.^17,23 In recent years, the increase of carbapenem-resistant Enterobacteriaceae and nonfermenting gram-negative bacilli has become a major concern worldwide.^19,23,28 During the study period, high-level antibiotic resistance has been seen among gram-negative bacilli isolated from five hospitals in Tehran. Resistance to third-generation cephalosporins in this study was 65.7% on an average: 71.3% for cefotaxime; 67.2% for ceftazidime; and 58.8% for cefepime. Klebsiella spp., E. coli, and Enterobacter spp. showed resistance rates of 79.9%, 66.6%, and 51.9% to cephalosporins, respectively. Of the cephalosporins, cefepime showed the highest activity (41.1%) against all species, and cefotaxime was the least effective against collected isolates. Imipenem showed the highest activity against all isolates (100%, 91.1%, 100% of the E.coli, K. pneumoniae, and Enterobacter spp. isolates were susceptible, respectively). In previous studies conducted in Iran, all of the Enterobacteriaceae isolates were susceptible to carbapenems³³; while previous studies reported imipenem resistance in P. aeruginosa in Iran.^14,34 Among the 11 carbapenem-resistant strains, only one strain contained a carbapenemase. The resistance in these strains could probably be due to other mechanisms such as outer membrane impermeability or efflux pumps.

In recent years, the detection of MBLs and other antibiotic resistance genes among gram-negative bacilli, particularly P. aeruginosa, has been reported in several areas in Iran, but this is the first report on the detection of MBL NDM-1 in Iran. NDM-1 is a new subclass of MBLs that possesses new amino acids near the active site, suggesting that it has a novel structure. Following the first report of NDM-1 in 2009 in Sweden,⁴² sporadic cases of NDM-1-positive strains have been reported from other countries,^{7,10,16,18,25,26,31,43} suggesting a widespread dissemination. Castanheira et al.³ detected that strains carrying bla_NDM-1 were collected in 2006 and 2007, indicating that bla_NDM-1-producing strains were present in India earlier than previously reported. NDM-1 demonstrates both β-lactamase and carbapenemase activities that are zinc dependent.^12,39,44,45 This MBL does not bind to the carbapenems as tightly as IMP-1 and VIM-2 do.⁴² NDM-1-positive bacteria have also been called Super bug, and now, they become a potentially major global health threat. Bacterial infections caused due to NDM-1 can only be treated with a few antibiotics, including colistin, tigecycline, and fluroquinolones,^15,42 but these antibiotics are often thought of as a poor choice because of its unfavorable pharmacokinetics and toxic effects. In this study, K. pneumoniae harboring NDM-1 was resistant to all tested antibiotics, including third-generation cephalosporins, carbapenems, ciprofloxacin, amikacin, kanamycin, aztreonam, and trimethoprim-sulfamethoxazole. However, Kim et al.¹⁵ showed that the orally available carbapenem, biapenem, could be considered optional treatment for infections due to NDM-1-positive bacteria. In this study, imipenem was moderately effective against the NDM-1-positive strain with low MIC (MIC 4 μg/ml), while MICs for meropenem was 8 μg/ml. Similar to other studies, the NDM-1-producing isolate had complex patterns of multidrug resistance.^16,25,42,43 It was also resistant to aztreonam. Altough aztreonam is not hydrolyzed by MBLs, but similar to our result, other studies showed that it was useless against MBLs-producing bacteria.^3,25 The aztreonam resistance observed could be due to the bla_CTX-M gene detected in this strain.

In this study, NDM-1-positive K. pneumoniae harbored bla_TEM, bla_SHV, and bla_CTX-M. In other studies, the plasmid carrying bla_NDM-1 also harbored other β-lactamase genes.^{3,16,25,26,42} These evidences would suggested that the plasmid carrying bla_NDM-1 carries several antibiotic resistance-conferring genes, and the rapid transfer of this plasmid to other pathogen bacteria can severely disseminate the antibiotic resistance among clinical bacteria. The majority of patients with NDM-1-positive bacteria have a travel background to India or Pakistan, showing an Asian source for this new MBL, but in the present study, to our knowledge, the patient had no history of travel to the Indian subcontinent, but he had traveled to Iraq and Syria before hospitalization. However, Iran is on the border of Pakistan and is near India, which this country considers the major source of NDM-1 producers.²²

In this study, negative or weakly positive MHT results were seen in NDM-1-positive isolates. This result was similar to the published results by Castanheira et al.³ This finding is very important, because CLSI recommended MHT for the detection and epidemiological investigations of carbapenemase-producing bacteria,⁶ and false-negative results for NDM-1-producing bacteria can be considered a serious alarm for clinical microbiology laboratories.

Most plasmids detected in NDM-1-positive bacteria can be easily transferred to many different gram-negative bacteria, posing a serious threat for the uncontrollable spread of multidrug-resistant bacteria.⁴⁰ K. pneumoniae is the most important cause of nosocomial infections, and it can spread rapidly in the hospital environment. The essential problem with regard to NDM-1-positive bacteria is that carbapenems are the main therapeutic option for treating infections caused by multidrug-resistant, gram-negative bacilli, particularly ESBL-producing strains, which are highly prevalent in most Iranian hospitals.^11,33 In recent years, the high prevalence of ESBL-producing bacteria made selective pressure inflicted by the use of other therapeutic options; especially carbapenems has led to the resistance to carbapenems among gram-negative bacilli. However, carbapenem resistance is still rare in Enterobacteriaceae in Iran.

Unfortunately, clinical data are still scarce, and no clonal diversity survey about the NDM-1-producing bacteria is available yet. The prevalence of NDM-1-producing bacteria as a major global health threat should prompt health authorities to design a preparedness plan for the implementation at national and global levels. The rapid spread of NDM-1-positive bacteria posed a major challenge for the treatment and control of infectious disease, and all clinicians should be aware that the NDM-1 as a potential health threat is resistant to all β-lactam and carbapenem antibiotics.

Footnotes

Acknowledgments

The authors are grateful to Dr. Christian G. Giske and Dr. Patrice Nordmann for providing standard strains. This work was supported by a grant from Pasteur Institute of Iran and the Iranian Ministry of Health and Medical Education.

Disclosure Statement

No competing financial interests exist.

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