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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 7  |  Issue : 1  |  Page : 17-22

Phenotypic and molecular study of carbapenem-resistant Enterobacteriaceae in a referral hospital in the East coast Malaysia


Department of Basic Medical Sciences, Faculty of Medicine, International Islamic University Malaysia, Kuantan, Pahang, Malaysia

Date of Web Publication1-Mar-2018

Correspondence Address:
Dr. Saleh Mahdi Yahya Mohsen
Department of Basic Medical Sciences, Faculty of Medicine, International Islamic University Malaysia, Kuantan, Pahang
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijhas.IJHAS_96_17

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  Abstract 


INTRODUCTION: Infections due to carbapenemase-producing Enterobacteriaceae have been increasingly reported worldwide and are causing significant health concerns. In this study, we investigated the occurrence of New Delhi metallo-beta-lactamase (NDM) carbapenemase-producing Enterobacteriaceae (CPE) in Enterobacteriaceae isolates from Hospital Tengku Ampuan Afzan (HTAA) and verified the presence of associated carbapenemases genes.
MATERIALS AND METHODS: Standard antimicrobial susceptibility testing was performed to analyze the isolates' susceptibility pattern. Modified Hodge test was used to detect carbapenemase production. Genes associated with CPE were detected by standard polymerase chain reaction amplification with a series of established primers, followed by DNA sequencing.
RESULTS: Out of 259 Enterobacteriaceae isolates, four Klebsiella pneumoniae isolates (1.5%) were found to be phenotypically carbapenem-resistant. NDM-1 carbapenemase was detected in three of the four isolates. Two of them were recovered from blood and the third strain was isolated from a urine sample. The three isolates were found to be resistant to all β-lactams antibiotics and the majority of other nonβ-lactam antibiotics. Meanwhile, all the isolates were susceptible to polymyxin B. They also carried other β-lactamase genes along with the blaNDMgene. Nucleotide sequence of the blaNDM-1gene showed no base variation with other NDM-1 sequences from the NCBI nucleotide database.
CONCLUSION: The presence of NDM-producing K. pneumoniae in the HTAA is alarming and of serious concern due to the high-level resistance of this gene and the risk of an outbreak occurrence. Further studies to detect the genetic relatedness and clonality of these resistance organisms are highly recommended.

Keywords: Carbapenemases, genes, hospital, New Delhi metallo-beta-lactamase


How to cite this article:
Mohsen SM, Hamzah HA, Al-Deen Mustafa MI. Phenotypic and molecular study of carbapenem-resistant Enterobacteriaceae in a referral hospital in the East coast Malaysia. Int J Health Allied Sci 2018;7:17-22

How to cite this URL:
Mohsen SM, Hamzah HA, Al-Deen Mustafa MI. Phenotypic and molecular study of carbapenem-resistant Enterobacteriaceae in a referral hospital in the East coast Malaysia. Int J Health Allied Sci [serial online] 2018 [cited 2018 Jun 22];7:17-22. Available from: http://www.ijhas.in/text.asp?2018/7/1/17/226263




  Introduction Top


Since the past 3 decades, carbapenems have been used as the last resort therapy for serious infections caused by extended-spectrum β-lactamase (ESBL)-producing bacteria. However, carbapenem-resistant strains with the ability of hydrolyzing almost all β-lactam antibiotics and several nonbeta-lactam antibiotics have emerged and have dramatically increased worldwide causing a grave concern.[1],[2] Species belonging to the family of Enterobacteriaceae, in particular  Escherichia More Details coli and Klebsiella pneumoniae, are the most frequently associated with nosocomial and acquired infection. According to the Centers for Disease Control and Prevention of the United States of America,[3] the percentage of carbapenem-hydrolyzing Enterobacteriaceae has increased over the past decade from 1.2% in 2001 to 4.2% in 2011.

Several chromosomal and plasmid-encoded carbapenemases such as K. pneumoniae carbapenamase (KPC), metallo-β-lactamase (VIM, IMP, and NDM) and oxa-48, have been identified worldwide. These enzymes are responsible for resistance to carbapenem. Production of KPC and New Delhi metallo-beta-lactamase-1 (NDM-1) carbapenemases enzyme is currently the most common resistance mechanisms.[4],[5],[6] Resistance to carbapenem can also occur due to the combination of some structural mutations with the production of other β-lactamase such as the combination of ESBL/AmpCs production with the loss of porins and/or active efflux pumps.[1],[4],[7]

The KPCs belong to class A serine carbapenemases based on Ambler classification system.[5],[8] KPC-producing isolates have been reported in many countries after the first identification in K. pneumoniae isolate in North Carolina 1996.[9] The endemicity and nosocomial outbreaks of KPC-producing bacteria have also been reported in several regions such as in the eastern USA, Colombia, Argentina, and Greece.[10],[11]

Another important carbapenemases is NDM-1, which belongs to class B metallo-B-lactamase according to Ambler classification and is currently the most distributed carbapenemase worldwide.[12] The activity of NDM-1 is strongly linked to the crystal structure and flexible zinc active site of this enzyme that make it capable of hydrolyzing several antibiotic substrates.[13] This carbapenemase was initially identified from K. pneumoniae strain in 2009 from a Swedish resident who had received medical care in New Delhi, India.[14] Although NDM-1-positive strains are found commonly in the Indian subcontinent and Pakistan, they have been detected in more than fifteen countries throughout the world, nearly from all continents.[12],[15] To date, seven different NDM-carbapenemases variants have been reported from many countries worldwide.[12] This study sought to investigate the presence of NDM-1 and KPC carbapenemases among K. pneumoniae and E. coli isolates in Hospital Tengku Ampuan Afzan (HTAA), Kuantan, Malaysia.


  Materials and Methods Top


Bacterial strains

A total of 259 nonduplicate E. coli and K. pneumoniae isolates were obtained from the bacteriology laboratory in HTAA over a period from May to September, 2014. These isolates were recovered from a range of clinical samples, including blood, urine, and swabs, (n = 101), (n = 135), and (n = 23), respectively, from patients who were admitted to the hospital. The isolates were identified to the species level using API20E (bioMerieux, France) according to the manufacturer's instructions.

Antimicrobial susceptibility testing

Kirby-Bauer diffusion method using Muller-Hinton Agar was used for susceptibility testing for all identified clinical isolates according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Thirteen β-lactam antibiotics along with six non-β-lactam antibiotics were used, including ampicillin (10 μg), amoxicillin/clavulanic acid (20/10), gentamicin (10 μg), amikacin (30 μg), ciprofloxacin (5 μg), piperacillin (100 μg), piperacillin/tazobactam (100/10 μg), sulbactam/ampicillin (10/10 μg), ceftazidime (30 μg), cefotaxime (30 μg), (30/10 μg), cefuroxime (30 μg), cefepime (30 μg), chloramphenicol (30 μg), trimethoroprim/sulfamethozaxole (25 μg), imipenem (10 μg), meropenem (10 μg), ertapenem (10 μg), cefoperazone/sulbactam (105 μg), and polymixine B (300 μg). Escherichia coli ATCC 25922 was used as a negative control.

Phenotypic detection of carbapenemase-producing strains

Isolates with decreased susceptibility or resistance to carbapenems were phenotypically screened for carbapenemase production using Modified Hodge test (MHT), as recommended by CLSI. Enhance growth of the ATCC E. coli 25922 at the junction with the tested organism and formation of a clover leaf-like indentation within the diffusion zone was considered a positive result. The lack of clover leaf-like indentation was considered as a negative result. K. pneumoniae ATCC BAA-1706 and ATCC BAA-1705 were used as negative and positive controls, respectively.

Molecular identification of carbapenemase genes

Plasmid and genomic DNA preparation

Plasmid DNAs were obtained using innuPREP Plasmid Rapid Kit (Analytik Jena, Germany) following the manufacturer's instruction. Genomic DNAs were isolated using the boiling method similar to that described by Mathers [16] with some modifications. One milliliter of the fresh overnight bacterial broth was centrifuged for 3 min at 8000 × g. The cell pellet was then resuspended in 300 μl sterile distilled water and placed at 95°C for 20 min and re-centrifuged at 10,000 × g for 3 min. The supernatants were used as DNA template for polymerase chain reaction (PCR) amplification. Biophotometer with UV absorption was used to determine the purity and quality of the extracted DNA before proceeding for the PCR amplification.

Polymerase chain reaction amplification, detection and sequencing

Carbapenem-resistant and MHT positive isolates were analyzed to check for the presence of carbapenemases resistance genes using conventional PCR followed by gel electrophoresis. PCR was performed using published primers specific to certain genes [Table 1].[32] In addition, carbapenem-resistant isolates were also screened for other β-lactamase-mediated genes, including CTX-M, TEM, and SHV using primers described by Wiegand,[17] [Table 1].
Table 1: Primer sequences used in polymerase chain reaction amplification and sequencing

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PCR amplification protocol was carried out using 50 μL reaction mixture, each reaction contained; 3 μL of 25 mM MgCl 2, 10 μL of 5X of PCR buffer, 1 μL 10 mM dNTP, 0.5 μL of Taq polymerase, 1 μL 10 μmol/L of each primers (forward and reverse), and 10 μL of DNA template. The reactions were placed onto a PCR thermal cycler for amplification using a standard PCR protocol. Annealing temperature was adjusted for each gene according to the melting temperature of the primer of which, 52°C was used for KPC and 54°C for NDM-1. The other cycling parameters used were 5 min, initial denaturation at 95°C; followed by 35 cycles at 94°C for 60s, annealing temperature as described above for 1 min, and 72°C for 1 min; and extension for 7 min at 72°C. K. pneumoniae ATCC BAA-1706, K. pneumoniae ATCC BAA-1705 were included in each run as a negative and positive blaKPC control, respectively. In addition to that, one reaction with water instead of the DNA template was included as template control. From each PCR product, 10 μL was separated by electrophoresis in 1.5% agarose using TAE buffer.

Sequencing was done with primers used in the PCR to deduce the nucleotide sequence from the real-time PCR amplification products. Each amplicon was sequenced from both directions. The sequencing was carried out employing ABI PRISM ® automated dye-terminator system. Sequences were scanned and multiple sequence alignment was conducted with CLUSTAL W in BioEdit version 7.0.9.0 software package. Sequence similarity among query strains from this study was established using BLAST program (NCBI Homepage).


  Results Top


Over the study period, 3.6%, 3.6%, and 4.5% of K. pneumoniae and 0.0%, 0.0%, and 0.7% of E. coli isolates were found resistant to meropenem, imipenem, and ertapenem, respectively. Three of the K. pneumoniae isolates were MHT positive, which indicated that these strains produced carbapenemase, [Figure 1] while all the other isolates were MHT negative. Susceptibility testing showed that isolates number 1 and 3 were resistant to all tested β-lactams and nonβ-lactam antibiotics except polymyxin B. On the other hand, isolate number 2 was susceptible to polymyxin B and chloramphenicol and resistant to the other tested antibiotics. Isolate number 4 showed a different susceptibility pattern, where it was found to be susceptible to amikacin, gentamicin, ciprofloxacin, and polymyxin B and resistant to other antibiotics, including carbapenems. However, isolate number 1 (U897) was phenotypically resistant to all carbapenems, but no carbapenemase (KPC nor NDM) genes were detected in this isolate.
Figure 1: Positive Modified Hodge(MHT)Test using ertapenem disc showing clear clover leaf like-indentation in both tested organism and positive control, and no indentation with negative control streak.

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PCR amplification revealed that NDM-1 carbapenemase was detected among three of the carbapenem-resistant K. pneumoniae isolates [Figure 2], and no carbapenemases genes were detected among all E. coli isolates. No blaKPC gene was detected from all bacterial isolates during the study. In addition, NDM-1 positive strains were also positive to at least one of other β-lactamase genes (CTX-M, SHV, and TEM) [Table 2]. The three known most common ESBLs genes, (CTX-M, SHV, and TEM) were detected in this strain [Table 2].
Figure 2: Gel electrophoresis of polymerase chain reaction products of the carbapenem-resistant isolates using New Delhi metallo-beta-lactamase -1 primer. M =100 bp DNA ladder; 1 = New Delhi metallo-beta-lactamase -1 negative isolate; 2, 3 and 4 = New Delhi metallo-beta-lactamase - 1 positive strains; C = Negative control. All the isolates in the gel were resistant to carbapenems and each lane number corresponds to the isolate number in Table 2

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Table 2: Characteristics of carbapenem-resistant isolates

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All positive PCR products were successfully sequenced from both forward and reverse directions. Partial nucleotide sequences (528 bp) of NDM-1 gene sequences were aligned with the reference sequence coded for NDM. The sequences were submitted to GenBank (www.ncbi.nlm.nih.gov) and were given accession numbers KT380916, KT380917, and KT380918. BLAST analysis revealed 100% similarity with other sequences from different strains, isolated in other places.


  Discussion and Conclusion Top


Since the past 2 decades, carbapenems have been used as the ultimate option for antibiotics against serious infections caused by both Gram-negative and Gram-positive bacteria. However, the extensive use and misuse of these agents accelerated the emergence of carbapenem-hydrolyzing bacteria and limited the potency of such agents.[7],[10],[18] In Malaysia, the resistance rate to carbapenems is still relatively low compared to other Asian countries. According to the National Surveillance of Antimicrobial Resistance, Ministry of Health Malaysia, only <0.6% of K. pneumoniae were carbapenem-resistant in 2011 and this increased to 1.5% in 2013.[19],[20]

The findings in this study are relatively high compared to national surveillance results as they revealed that only 1.7% and 1.5% of K. pneumoniae and 0.3% and 0.3% of E. coli were resistant to meropenem and imipenem, respectively. However, this result is low compared to a study conducted in a tertiary hospital in Mumbai, India which revealed that 12.3% of Enterobacteriaceae isolates were found to be carbapenems resistant.[21] The prevalence and extent of carbapenemases in Southeast Asia are not clearly determined yet however, carbapenemases are frequently reported in many Asian countries.

In Malaysia, only a few reports of NDM-1 carbapenemase identification and characterization have been published. Ahmad et al.,[22] described the phenotypic and genotypic characteristics of NDM-1-positive K. pneumoniae strain isolated from the urine of patients in a Malaysian hospital. They revealed that the strain was resistant to ertapenem and meropenem but was sensitive to imipenem. In addition, they reported that other β-lactamase gene, including CTX-M 15, TEM-1, and SHV-11 were also detected in the same strain.

In the present study, NDM-1 carbapenemase was detected among three K. pneumoniae isolates, and no carbapenemases genes were detected among all E. coli isolates. These three strains were also positive to at least one of other β-lactamase genes (CTX-M, SHV and TEM), thus making them as multidrug-resistant bacteria. The three NDM-1 positive isolates were resistant to almost all tested antibiotics besides carbapenems but were susceptible to polymyxin B [Table 2]. This co-resistance to other antibacterial drugs among these isolates calls for the need to identify other resistant genes and mechanisms. This finding is in agreement with several studies worldwide that showed NDM-1-bearing strains are commonly resistant to all available antibiotics except polymyxins.[23] NDM-1 carbapenemase has been identified in many regions worldwide mostly in India, Pakistan, Canada, and Britain.[11] Several sporadic cases have been reported from Asia-Pacific region, including Singapore, Taiwan, Japan,[24] Hong Kong,[25] Australia,[26] and Malaysia,[22] in addition to continental and Eastern Europe and Africa,[27] both in hospital settings and community. This rapid dissemination could be due to globalization and escalating international travel, that is in addition, to the location of the genes on a readily transferable plasmids and transposons.[11],[28] NDM-1 was found to be the most dominant carbapenemase in a study conducted in Asia-Pacific region for monitoring the trends of resistance among Enterobacteriaceae isolates.[29] In 2011, a study from Singapore revealed that 23% (n = 12) of all carbapenem-resistant Enterobacteriaceae were identified as NDM-1-producing isolates.[23]

Partial nucleotide sequence analysis of the coding region of the blaNDM-1 gene revealed no new mutation occurred in the studied region and confirmed the identity of the gene. This suggests that the gene is in the conserved nature among the K. pneumoniae isolates. However, the blaNDM-1 gene is transferable in other Enterobacteriaceae as well as in Pseudomonas aeruginosa and Acinetobacter baumanni.[30] Dissemination and failure to eradicate the bacteria may lead to an outbreak at the hospital. Therefore, stringent control measure should be taken to curb the infections.

One of the carbapenem-resistant isolates was phenotypically resistant to all carbapenems. However, no carbapenemase genes were detected in this isolate. The three most common ESBLs genes known (CTX-M, SHV and TEM), were also detected in this strain [Table 2]. The actual resistance mechanism is unknown. This resistance mechanism could be due to the combination of ESBLs production and porin alteration, which is worth investigating further. Carbapenem resistance can also occur through a combination of ESBL/AmpCs production with the loss of porins or alteration of efflux pump.[4],[5],[31]

No blaKPC gene was detected from all the bacterial isolates during the study, and this finding corroborated several other studies proving that KPC carbapenemase is rarely detected in Asia. Sheng et al.,[29] reported the absence of KPCs from the isolates in a longitudinal study conducted in the Asia-Pacific region for monitoring the trends of resistance among Enterobacteriaceae.


  Conclusion Top


This study demonstrated the presence of NDM-1 carbapenemase-producing K. pneumoniae i solates in HTAA. The presence of NDM-1 in Malaysia could be related to the vast Indian community and the significant movement between Malaysia and the Indian subcontinent suggesting the possibility of the spread of this gene from India. Thus, there is a need for national surveillance, identification and characterization of carbapenemase-producing isolates and implementation of an appropriate effective strategy for controlling or limiting their further spread to prevent the occurrence of outbreaks.

Financial support and sponsorship

This study was financed by endowment grant type B (EDW B14-145-1030) provided by IIUM.

Conflicts of interest

There are no conflicts of interest.



 
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