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  Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 36  |  Issue : 1  |  Page : 104-107
 

Association of glycerol kinase gene with class 3 integrons: A novel cassette array within Escherichia coli


1 Department of Microbiology, Assam University, Silchar, Assam, India
2 Department of Microbiology, Silchar Medical College and Hospital, Silchar, Assam, India

Date of Web Publication2-May-2018

Correspondence Address:
Dr. Amitabha Bhattacharjee
Department of Microbiology, Assam University, Silchar, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_17_188

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 ~ Abstract 

Background: Integrons are genetic elements which are known for their role in capturing and spreading of antibiotic resistance determinants among Gram-negative bacilli. So far, there is no study regarding Class 3 integron and their genetic organisation in India. Objective: This study investigates the occurrence of Class 3 integron and their gene cassette array among Escherichia coli. Materials and Methods: In this study, a total of 200 E. coli isolates were collected from indoor and outdoor patients from Silchar Medical College and Hospital during September 2015 to February 2016. Detection of the integrase genes and gene cassettes within the Class 3 integron was performed by polymerase chain reaction which was further analysed by sequencing. Results: Twenty-seven isolates were found to harbour Class 3 integron. Sequencing of the gene cassettes and whole Class 3 integron revealed the presence of nine different types of cassettes array, out of which the arrangement with glycerol kinase gene cassette was found to be the most prevalent. Arrangement with blaCTX-Mgene cassette was also detected in few isolates. Conclusion: This study provides epidemiological profiling of Class 3 integrons in this geographical area. The data generated in this study are helpful in infection control programme, anti-infective research and search for epidemiological markers.


Keywords: Class 3 integron, Enterobacteriaceae, gene cassette, integrase, multidrug resistance


How to cite this article:
Elizabeth R, Chanda DD, Chakravarty A, Paul D, Chetri S, Bhowmik D, Wangkheimayum J, Bhattacharjee A. Association of glycerol kinase gene with class 3 integrons: A novel cassette array within Escherichia coli. Indian J Med Microbiol 2018;36:104-7

How to cite this URL:
Elizabeth R, Chanda DD, Chakravarty A, Paul D, Chetri S, Bhowmik D, Wangkheimayum J, Bhattacharjee A. Association of glycerol kinase gene with class 3 integrons: A novel cassette array within Escherichia coli. Indian J Med Microbiol [serial online] 2018 [cited 2018 Nov 15];36:104-7. Available from: http://www.ijmm.org/text.asp?2018/36/1/104/231661



 ~ Introduction Top


Integrons are the natural gene capture system which can incorporate circularised open reading frames, called gene cassettes by site-specific recombination, and convert them to functional genes. Integrons are coupled with mobile DNA elements, such as insertion sequences (ISs), transposons and conjugative plasmids.[1] Integron platform comprises the integrase gene (int I), a recombination site att I and a promoter gene (Pc), which facilitates expression of cassette-associated genes when cassettes are inserted at att I.[2]

Integrons have been classified into several classes based on the differences in the int I sequences [3] and integron Classes 1–3 are called resistant integrons, which appear to be able to acquire same gene cassettes. Class 4 and 5 integrons are termed super integron. Class 1 integron are the most prevalent among clinical isolates as well as commensals out of the five classes.[4] Class 3 integron has been observed in Acinetobacter spp., Alcaligenes, Citrobacter freundii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas putida, Salmonella spp. and Serratia marcescens.[5],[6],[7] However, in this geographical area, no study regarding Class 3 integron and their genetic organisation and association with cassette arrays have been on record. Thus, this study was performed to detect the emergence and proliferation of Class 3 integrons from this part of the world.


 ~ Materials and Methods Top


Bacterial isolates

A total of consecutive non-duplicate 200 isolates of E. coli collected from patients admitted to various wards and clinics of Silchar Medical College and Hospital, Silchar, Assam, India, between September 2015 and February 2016 were used for the study. The clinical isolates were identified by standard biochemical tests and 16s rDNA sequencing.

DNA extraction

DNA extraction from single colonies of E. coli was obtained by boiling centrifugation method. For this, overnight culture was suspended in 200 μl of sterile distilled water and heated at 90°C for 10 min. Cellular debris was removed by centrifugation and the supernatant was used as a template for polymerase chain reaction (PCR).

Screening of Class 3 integron

Screening of the isolates for the presence of Class 3 integrase gene was done by PCR using bacterial DNA as the template. The PCR conditions were 94°C for 2 min followed by 34 cycles at 94°C for 15 s, 55°C for 40 s, 72°C for 1 min and final extension at 72°C for 5 min using primers intI3 F and intI3 R [Table 1].
Table 1: List of primers

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Amplification of gene cassettes

Isolates which were found to be positive for Class 3 integron were further processed to amplify the gene cassettes present within them using primers attI3 L and Class 3R [Table 1]. The PCR conditions were 95°C for 3 min followed by 34 cycles at 95°C for 20 s, 50°C for 45 s, 72°C for 2 min and final extension at 72°C for 7 min.

Amplification of whole Class 3 integron

Class 3 integrase-positive isolates were further subjected to PCR for amplification of whole Class 3 integron using primers intI3 P and HS287 [Table 1]. The PCR conditions were 95°C for 2 min followed by 30 cycles at 95°C for 15 s, 50°C for 1 min, 72°C for 2 min and final extension at 72°C for 7 min. The amplified products were purified and sequenced using Sanger's method. Sequences were compared using BLAST search (http://www.ncbi.nlm.nih.gov/BLAST).

Cloning of Class 3 integron

The genetic array of gene cassettes within the variable region of Class 3 integron was determined by cloning the amplified products on to pGEM-T vector (Promega, Madison, USA) and transformed into E. coli DH5α. Transformants were selected by blue-white screening method. The whole procedure was done as per the manufacturer's instruction (https://www.promega.com/-/./pgem-t-and-pgem-t-easy-vector-systems-protocol.pdf).

Screening of Class 1 and 2 integron

Isolates showing Class 3 integrase-positive were subjected to multiplex PCR for the presence of Class 1 and 2 integron. The PCR conditions followed were 94°C for 3 min followed by 32 cycles at 94°C for 20 s, 54°C for 20 s, 72°C for 1 min and final extension at 72°C for 5 min using primers int1 F and int1 R for Class 1 integron and int2 F and int2 R for Class 2 integron [Table 1].

Antibiogram profiling

Antimicrobial susceptibility testing was carried out by Kirby–Bauer disk diffusion method. The applied antimicrobials in disk diffusion method were as follows: amikacin (30 μg), ampicillin (10 μg), ciprofloxacin (5 μg), co-trimoxazole (25 μg), gentamicin (10 μg), imipenem (10 μg), piperacillin/tazobactam (100/10 μg), polymyxin B (300 units) and tigecycline (15 μg) (Hi-Media, Mumbai, Maharashtra, India). The results were interpreted as per the Clinical and Laboratory Standards Institute criteria.[8]

Typing of the isolates

The isolated organisms were typed by enterobacterial repetitive intergenic consensus (ERIC) PCR.[9] The PCR conditions were 95°C for 3 min followed by 30 cycles at 95°C for 20 s, 46°C for 40 s, 72°C for 3 min and final extension at 72°C for 10 min


 ~ Results Top


All the confirmed isolates of E. coli were subjected to screening for the presence of Class 3 integron and were observed that 27 isolates were harbouring this class [Table 2] and [Figure 1]. Gene cassettes with size ranging from 300 bp to 1.5 kb were found in 21 isolates. The variable size of whole Class 3 integron investigated has size ranging from 500 bp to 1.5 kb and was found in 16 isolates. Sequencing results revealed nine different types of cassettes array. Arrangement I with insB, orfA, orfB and orfC was found in two isolates. In arrangement II, single-cassette iroB was found which was observed in only one isolate. This is followed by arrangement III with single-gene cassette glycerol kinase (glpK) which is the most prevalent and found in six isolates. Arrangement IV consists of single gene cassette glpX which is observed in only one isolate [Figure 2]. One isolate with arrangement V containing gene cassette array traO and traM was observed. Arrangement VI with gene cassettes traM, traN and blaCTX-M was found in two isolates. This is the only arrangement containing antibiotic resistance gene. Single-gene cassette lolA consists of arrangement VII which is observed in one isolate. Arrangement VIII and arrangement IX showed the presence of single gene cassette traM and traO, respectively, in one isolate each [Figure 2]. While testing the susceptibility pattern of the clones of arrangement VI, it was observed that the clones were resistant towards broad-spectrum penicillins and third-generation cephalosporin such as cefotaxime, ceftazidime and ceftriaxone.
Table 2: Characterization and screening of class 3 integron in clinical isolates

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Figure 1: Characterisation of Class 3 integron

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Figure 2: Schematic representation of Class 3 integron

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Co-existence of more than one type of integron was also observed as 14 isolates were found to be positive of both Class 1 and 3 integron, three isolates harbour both Class 2 and 3 and only one isolate harbour all the three types, i.e. Class 1, 2 and 3 integron [Table 3] and [Figure 3]. Susceptibility profiling was done for all the Class 3 integrin-positive isolates against nine different types of antibiotics, and the results had shown that high levels of resistance were found against piperacillin/tazobactam (88.88%) followed by ciprofloxacin (85.18%) and ampicillin (70.37%) [Table 4]. While testing the clonal diversity of the 27 Class 3 integrin-positive isolates by ERIC PCR, 13 different haplotypes of E. coli were observed [Figure 4]a and [Figure 4]b.
Table 3: Screening of class 1 and 2 integron in clinical isolates

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Figure 3: Screening of Class 1 and 2 integron

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Table 4: Antibiotic susceptibility profiling

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Figure 4: (a and b) Clonal diversity of Escherichia coli isolates shown by enterobacterial repetitive intergenic consensus polymerase chain reaction

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 ~ Discussion Top


In this study, the existence of Class 3 integrons was confirmed in 13.5% of clinical E. coli isolates which was found to be lower compared to the previous studies.[10],[11] Although our finding showed less frequency than other studies, this can be considered as a unique finding as Class 3 integron is rarely found, and also, there are no reports regarding the detection of Class 3 integron from this part of the world. Sequencing of gene cassettes within Class 3 integron indicates the presence of nine types of gene cassette arrangements; ins B-orf A-orf B-orf C, tra O-tra M, tra M-tra N-blaCTX-M, iro B, glp K, glp X, lol A, tra M and tra O in which glp K, coding for glpK responsible for metabolic functions in E. coli, was found to be the most prevalent gene even though an extended spectrum β-lactamase gene blaCTX-M gene showing resistance to third-generation cephalosporins such as cefotaxime, ceftazidime and ceftriaxone was also recorded which was also evidenced elsewhere.[12] Our finding revealed that more than one type of gene capture mechanism is present which helps in the transmission of antibiotic resistance. Presence of Class 1 integron is indicative of lateral transfer of resistance determinants among clinical isolates in this setting. The co-existence of more than one type of integron indicates the high mobility of the resistance gene within intra- and inter-species level that is also in agreement with previous studies.[13],[14],[15],[16],[17] As the Class 3 integrin-positive E. coli showed the least resistance pattern towards the cationic antimicrobial peptide (CAP), polymyxin B, it may be stated that this group of antibiotics is still effective and could be a treatment option of bacterial infections from this region.


 ~ Conclusion Top


Although Class 3 integrons are previously reported for carriage of antibiotic resistance, we observed in our study that this gene capture mechanism is predominantly associated for carriage of different genes responsible for bacterial metabolic activities. This finding is unique and not reported elsewhere. Therefore, it could be established that in this region, bacterial survival by essential metabolic activities is also encoded through Class 3 integron. Besides carrying genes for metabolism, two isolates were found to harbour blaCTX-M with Class 3 integron which emphasises its role in carriage of cephalosporin resistance in these hospital settings. Our study advocates the need for continued surveillance as well as further investigation of those Class 3 integrons and their gene cassette to determine the impact and conditions for molecular acquisition of these genes within E. coli and how they help in survivability and adaptability of bacteria. This study is of epidemiological interest and provides an effective device for surveying genetic environment of hospital isolates in this geographical area.

Acknowledgement

We would like to acknowledge Head, Department of Microbiology, Assam University and Assam University Biotech Hub, for providing the infrastructural facility.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

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Rowe-Magnus DA, Mazel D. The role of integrons in antibiotic resistance gene capture. Int J Med Microbiol 2002;292:115-25.  Back to cited text no. 1
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2.
Hall RM, Brookes DE, Stokes HW. Site-specific insertion of genes into integrons: Role of the 59-base element and determination of the recombination cross-over point. Mol Microbiol 1991;5:1941-59.  Back to cited text no. 2
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Ceccarelli D, Colombo MM. Role, circulation and molecular characterization of integrons and ices in clinical and environmental Vibrio. Commun Curr Res Educ Top Trends Appl Microbiol 2007;1:221-9.  Back to cited text no. 3
    
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Lin Z, Lai YM, Zaw MT. Prevalence of class 1, class 2, class 3 integron in antibiotic resistant uropathogenic Eschericheria coli isolates. Indian J Med Res Pharm Sci 2015;2:1-9.  Back to cited text no. 4
    
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Arakawa Y, Murakami M, Suzuki K, Ito H, Wacharotayankun R, Ohsuka S, et al. A novel integron-like element carrying the metallo-beta-lactamase gene blaIMP. Antimicrob Agents Chemother 1995;39:1612-5.  Back to cited text no. 5
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Correia M, Boavida F, Grosso F, Salgado MJ, Lito LM, Cristino JM, et al. Molecular characterization of a new class 3 integron in Klebsiella pneumoniae. Antimicrob Agents Chemother 2003;47:2838-43.  Back to cited text no. 6
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Ploy MC, Chainier D, Tran Thi NH, Poilane I, Cruaud P, Denis F, et al. Integron-associated antibiotic resistance in Salmonella enterica serovar typhi from Asia. Antimicrob Agents Chemother 2003;47:1427-9.  Back to cited text no. 7
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Clinical and Laboratory Standard Institute. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-First Informational Supplement. M100-S21. Wayne PA, USA: Clinical and Laboratory Standard Institute; 2011.  Back to cited text no. 8
    
9.
Versalovic J, Koeuth T, Lupski JR. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res 1991;19:6823-31.  Back to cited text no. 9
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Kargar M, Mohammadalipour Z, Doosti A, Lorzadeh S, Japoni-Nejad A. High prevalence of class 1 to 3 integrons among multidrug-resistant diarrheagenic Escherichia coli in southwest of Iran. Osong Public Health Res Perspect 2014;5:193-8.  Back to cited text no. 10
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Pour PH, Momtaz H, Serajyan AA, Tajbakhsh E. Investigating class I, II and III integrons in multidrug resistance in Pseudomonas aeruginosa isolated from hospital infections in Ahvaz. Int J Med Lab 2015;2:168-76.  Back to cited text no. 11
    
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Barraud O, Casellas M, Dagot C, Ploy MC. An antibiotic-resistant class 3 integron in an Enterobacter cloacae isolate from hospital effluent. Clin Microbiol Infect 2013;19:E306-8.  Back to cited text no. 12
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Shibata N, Doi Y, Yamane K, Yagi T, Kurokawa H, Shibayama K, et al. PCR typing of genetic determinants for metallo-beta-lactamases and integrases carried by gram-negative bacteria isolated in Japan, with focus on the class 3 integron. J Clin Microbiol 2003;41:5407-13.  Back to cited text no. 13
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Goldstein C, Lee MD, Sanchez S, Hudson C, Phillips B, Register B, et al. Incidence of class 1 and 2 integrases in clinical and commensal bacteria from livestock, companion animals, and exotics. Antimicrob Agents Chemother 2001;45:723-6.  Back to cited text no. 14
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Hall RM, Collis CM, Kim MJ, Partridge SR, Recchia GD, Stokes HW, et al. Mobile gene cassettes and integrons in evolution. Ann N Y Acad Sci 1999;870:68-80.  Back to cited text no. 15
    
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Poirel L, Carattoli A, Bernabeu S, Bruderer T, Frei R, Nordmann P, et al. A novel incQ plasmid type harbouring a class 3 integron from Escherichia coli. J Antimicrob Chemother 2010;65:1594-8.  Back to cited text no. 16
    
17.
Taherikalani M, Maleki A, Sadeghifard N, Mohammadzadeh D, Soroush S, Asadollahi P, et al. Dissemination of class 1, 2 and 3 integrons among different multidrug resistant isolates of Acinetobacter baumannii in Tehran hospitals, Iran. Pol J Microbiol 2011;60:169-74.  Back to cited text no. 17
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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