New Delhi metallo-β-lactamase-1-producing Klebsiella pneumoniae isolates in hospitalized patients in Kashan, Iran.

BACKGROUND AND OBJECTIVES: New Delhi metallo-ß-lactamase (NDM) is a newly emerging metallo-ß-lactamases, which can destroy all β-lactams including carbapenems. Therefore, this study aimed at evaluating New Delhi metallo-ß-lactamase-1-production in clinical isolates of Klebsiella pneumoniae in Kashan, Iran.
MATERIALS AND METHODS: In a cross-sectional study, 181 K. pneumoniae isolates were collected from clinical samples of patients, who referred to Shahid Beheshi hospital in Kashan during November 2013 and October 2014. Antimicrobial susceptibility patterns were determined using disk diffusion method, according to CLSI guidelines. Metallo-ß-lactamase (MBL) production was identified among imipenem-resistant K. pneumoniae isolates using imipenem-EDTA double disk synergy test (EDTA-IMP DDST). PCR method and sequencing were used to detect integron Class 1 and blaNDM-1 gene. Statistical analyses were performed using SPSS software Version 16.
RESULTS: Of the 181 K. pneumoniae isolates, 36 (19.9 %) were imipenem-resistant strains. A total of 28 out of 36 (77.7%) imipenem-resistant K. pneumoniae isolates were identified as MBL producer strains. Also, 150 (82.9%) K. pneumoniae isolates carried intI1 gene, and 20 (11.1%) K. pneumoniae isolates harbored blaNDM-1 gene.
CONCLUSION: Our study revealed a high frequency of MBL production and the presence of blaNDM-1 among K. pneumoniae strains, especially among hospitalized patients, which is alarming. Moreover, the presence of Class 1 integrons in all multi-drug resistant K. pneumoniae isolates highlights the risk of rapid spread of the resistance genes, especially in clinical settings.

INTRODUCTION

Carbapenems includes a Class of ß-lactams that can kill most bacteria and are recommended for treatment of infections caused by extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, mainly K. pneumoniae (1, 2). Carbapenem resistance due to the carbapenemase enzymes is one of the complicated health issues worldwide because carbapenemase producing clinical isolates simultaneously show resistance to carbapenems and to all other ß-lactam antibiotics (3). Metallo-ß-lactamases (MBLs) belong to Class B ß-lactamase and need zinc for their activity (4). MBLs have a wide spectrum of β-lactamase activity and can affect a wide range of β-lactam antibiotics including carbapenems (4). Bulk of the resistance genes including carbapenemases in K. pneumoniae are carried on Class 1 integrons. It has been documented that metallo-β-lactamases are associated with gene cassettes carried on integrons (5). Integrons as transferable genetic elements facilitate the transfer of resistance genes among different bacteria (6).

Among the newly identified metallo-β-lactamases, New Delhi metallo-ß-lactamase (NDM) is a recently described enzyme conferring resistance to all ß-lactams, especially carbapenems except monobactams (7). Since its first identification in New Delhi, India, in 2008, NDM has been reported by different countries around the world as an important health concern (8).

The blaNDM-1 gene, responsible for producing NDM in clinical isolates, is carried on transferable genetic elements, leading to rapid dissemination of these genes (7). There is limited data on NDM production and carriage of Class 1 integrons in clinical isolates of K. pneumoniae in our regain. Thus, the present study was conducted to identify the blaNDM metallo-β-lactamase gene and the presence of Class 1 integrons in clinical isolates of K. pneumoniae in Kashan, Iran.

MATERIALS AND METHODS

In this cross-sectional study, 181 clinical isolates of K. pneumoniae including urine (n = 124), respiratory tract samples (n = 43), blood (n = 5), wound (n = 6), cerebrospinal fluid (CSF) (n = 1), and catheter (n = 2) were isolated from patients, who referred to Shahid Beheshi hospital in Kashan during November 2013 to October 2014. The isolates were identified using standard microbiological methods (9). Antibiotic resistance profiles to the following antibiotics were determined using disk diffusion method: gentamicin (10 μg), ampicillin (30 μg), amoxicillin/clavulanic acid (20/10 μg), aztreonam (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefoxitin (30 μg), cefteriaxone (30 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), trimethoprim-sulfamethoxazole (25 μg), ertapenem (10 μg) and imipenem (10 μg), (Mast, UK). Antibiotic susceptibility protocol was in accordance with the guidelines of Clinical and Laboratory Standards Institute (CLSI). The Escherichia coli (ATCC 25922) standard strains were used to perform quality control in susceptibility testing. To identify MBL-producing K. pneumoniae isolates, imipenem resistant isolates were studied using Imipenem-EDTA double disk synergy Test (EDTA-IMP DDST). Boiling method was used for DNA extraction of K. pneumoniae isolates. PCR amplification was used to detect blaNDM-1 gene using specific primers including NDM-F (5′-GGTTTGGCGATCTGGTTTTC-3′) and NDM-R (5′-CGGAATGGCTCATCACGATC-3′) (10). Strains that were confirmed to carry blaNDM-1 gene by sequencing were used as positive controls. The carriage of the Class 1 integrons was investigated by amplification of the intI1 gene among MDR K. pneumoniae isolates using PCR assays. The primers used were as follow: IntI1-F (5′-TCTCGGGTAACATCAAGG-3′) and IntI-R (5′-AGGAGATCCGAAGACCTC-3′) (6). The PCR products were separated by electrophoresis on 1.2% agarose gel and seen in gel document system (Bio-Rad, UK). Some purified PCR products were sequenced (Bioneer, South Korea) and compared by online BLAST software (http://www.ncbi.nlm.nih.gov/BLAST/). SPSS software Version 16 (SPSS, Inc.) was used for statistical analyses. Differences were considered by the χ2 test, and p-values of less than 0.05 were determined as statistically significant.

RESULTS

Of the 181 K. pneumoniae isolates of hospitalized patients, 78 (43.1%) were collected from male and 103 (56.9%) from female patients. The patients’ age ranged from 1 to 97, and their mean age was 50.36 years.

The antibiotic resistance profile is demonstrated in Table 1. Disk diffusion method revealed that 36 (19.9 %) of K. pneumoniae isolates were imipenem-resistant strains. MBL production was observed in 28 (77.7%) isolates of imipenem–resistant K. pneumoniae strains. A total of 150 (82.9%) K. pneumoniae isolates carried intI1 gene and were Class 1 integron-positive isolates. Moreover, 20 (11.1%) K. pneumoniae isolates harbored blaNDM-1 gene and were recognized as NDM-producing isolates. The blaNDM-1 gene carriage was found in all hospital wards except Critial Care Unit (CCU), while the most frequent ward, which harbored the blaNDM-1 gene, was intensive care unit (ICU) (Table 2). The nucleotide sequencing of the PCR products of blaNDM-1 (the GenBank accession number: KP340793.1) and intI1 genes were equal to those deposited in the GenBank.

Table 1.

Antimicrobial resistance profile of Klebsiella pneumoniae isolates busing disk diffusion method

Antibiotic	Isolates (n)%
	Sensitive	Intermediate	Resistant
Gentamicin	117 (64.6)	5 (2.8)	59 (32.6)
Ampicillin	5 (2.8)	3 (1.7)	173 (95.5)
Amoxicillin-clavulanic acid	63 (34.8)	26 (14.4)	92 (50.8)
Aztreonam	82 (45.3)	16 (8.8)	83 (45.9)
Cefotaxime	68 (37.6)	11 (6.1)	102 (56.3)
Ceftazidime	80 (44.2)	10 (5.5)	91 (50.3)
Cefoxitin	117 (64.6)	14 (7.8)	50 (27.6)
Ceftriaxone	78 (43.1)	6 (3.3)	97 (53.6)
Nalidixic acid	56 (30.9)	34 (18.8)	91 (50.3)
Ciprofloxacin	89 (49.2)	12 (6.6)	80 (44.2)
Trimethoprim-sulfamethoxazole	104 (57.5)	25 (13.8)	52 (28.7)
Imipenem	137 (75.7)	8 (4.4)	36 (19.9)
Ertapenem	130 (71.8)	12 (6.6)	39 (21.6)

Table 2.

Association among age, sex, sample type, patient admission and blaNDM-1 gene in Klebsiella pneumoniae isolates

Patient Characteristics	blaNDM-1 Positive n= 20 (%)	blaNDM-1 Negative n= 161 (%)	p-value
Age
<50 years	13 (65)	128 (79.5)	0.4
≥50 years	7 (35)	33 (20.5)
Sex
Male	9 (45)	69 (42.9)	0.06
Female	11 (55)	92 (57.1)
Sample Type
Urine	10 (50)	114 (70.8.)	0.001
CSF	1 (5)	0 (0)
Blood	0 (0)	5 (3.1)
Catheters	2 (10)	0 (0)
Wound	2 (10)	4 (2.5)
Respiratory	5 (25)	38 (23.6)
Patient admission
Hospitalized	20 (100)	107 (66.5)	0.001≥
Out patients	0 (0)	54 (33.5)
Hospitalized ward
ICU	10 (50)	28 (17.4)	0.001≥
Infectious diseases	3 (15)	16 (9.9)
Surgery	1 (5)	17 (10.6)
maternity	2 (10)	9 (5.6)
Pediatric	1 (5)	11 (6.9)
Internal medicine	2 (10)	15 (9.3)
Emergency	1 (5)	6 (3.7)
CCU	0 (0)	5 (3.1)
Outpatients	0 (0)	54 (33.5)

The statistical analysis revealed a correlation (P < 0.05) among the hospitalized patients’ ward, sample type, and patient admission with NDM production (Table 2).

DISCUSSION

Carbapenem resistant K. pneumoniae strains are increasing, and infections due to these strains are accompanied with higher mortality, length of hospitalization, and cost of treatment (11).

Our results revealed that 77.7% of imipenem–resistant K. pneumoniae strains produced MBL. Different frequencies of MBL production have been reported among K. pneumoniae strains (8). In contrast with our results, in a study conducted by Fazeli et al. in Isfahan, 10.2% of carbapenem-resistant K. pneumoniae isolates have been reported to be MBL producers (12). In another study in Greece, the prevalence of metallo-beta-lactamases in K. pneumoniae isolates from blood was 50% (13). The reason for the diverse prevalence of MBL production among K. pneumonia strains in different studies may be due to the use of different methods or different clinical samples. In addition, the discrepancy of phenotypic and genotypic features of bacterial isolates and factors such as cultural-economic status in diverse geographical areas could also be the reason. The results of our PCR assays demonstrated that 11.1% of K. pneumoniae isolates carried blaNDM-1 gene and produced NDM. In accordance with our findings, in a study conducted in Isfahan, 12% of carbapenem-resistant K. pneumoniae isolates were expressed New Delhi metallo-beta-lactamase (12), whereas, the prevalence of blaNDM-1 among Enterobacteriaceae in countries such as India and Kuwait has been reported to be higher (7, 14). Although the blaNDM-1 carrying K. pneumoniae strains are not very common in Iran, the results of this study is alarming. In most studies, NDM producer K. pneumoniae strains are resistant to most generally used antibiotics including β-lactams, β-lactamase inhibitors, fluoroquinolones, aminoglycosides and carbapenems (9, 15). The analysis of antibiotic resistance profiles of NDM producer K. pneumoniae strains in this study revealed that all NDM-producing K. pneumoniae isolates were multi-drug resistant strains, with resistance to almost all tested antibiotics; and this is in agreement with the results of other studies (12, 15). According to the literature, New Delhi metallo-beta-lactamase is a kind of beta-lactamase, which confers resistance to carbapenems and all β-lactam antibiotics except monobactams, such as aztreonam. In this study, in agreement with other reports, NDM-producing K. pneumoniae strains showed resistance to aztreonam along with other tested antibiotics (7, 12). The resistance to aztreonam in these blaNDM-1 positive K. pneumoniae strains may probably be due to other mechanism of resistance. The association between metallo-β-lactamase related genes including blaNDM-1 and mobile genetic elements, such as plasmid and integrons, has been documented (12, 13). We found that all multi-drug resistant K. pneumoniae isolates carried Class 1 integrons. The concomitance of NDM genes and mobile genetic elements, especially Class 1 integrons, facilitates their widespread propagation, which is a serious threat to the management of hospital-acquired infections. Furthermore, in this study, all NDM positive K. pneumoniae strains were isolated from hospitalized patients, and this is similar to reports by Jamal et al. indicating a nosocomial acquirement (7). Also, half of our NDM-producing K. pneumoniae strains were identified among hospitalized patients in ICU, where patients commonly have underlying diseases and experience long-term hospitalization and prolonged treatment with antibiotics, which facilitate the selection and spread of these resistant strains.

CONCLUSION

This study revealed that the high frequency of MBL production and presence of blaNDM-1 among K. pneumoniae strains, especially among hospitalized patients, are highly alarming. Also, the presence of Class 1 integrons in all multi-drug resistant K. pneumoniae isolates highlights the risk of rapid spread of the resistance genes, especially in clinical settings.