The Open Microbiology Journal




ISSN: 1874-2858 ― Volume 14, 2020
RESEARCH ARTICLE

Molecular Detection and PCR-RFLP Analysis of Mucoviscosity-Associated Gene A (magA) in Clinical Isolates of Multidrug-Resistant Klebsiella pneumoniae in Bangladesh



Md. Hazrat Ali1, *, Saeed Anwar1, Nusrat Jahan Toma1, , Ikram Rafid1, , Md. Kamrul Hasan2, Md. Javed Foysal1, 3
1 Department of Genetic Engineering and Biotechnology, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh
2 Department of Biochemistry and Molecular Biology, Tejgaon College, National University, Gazipur 1704, Bangladesh
3 School of Molecular and Life Sciences, Curtin University, Kent Street, Bentley, Perth, WA6102, Australia

Abstract

Background and Objective:

The mucoviscosity associated gene A (magA) in the hypermucoviscous variants of K. pneumoniae is reported to be associated with invasive infections and considered a virulence factor. We sought to analyze the magA genes in K. pneumoniae isolates in the clinical specimen collected from Bangladesh.

Methods:

We established a multicenter cohort of patients with Klebsiella infection hospitalized at 05 different hospitals between September 2016 and April 2017. We collected 313 K. pneumoniae isolates from patients who consented to participate in the study. The isolates were evaluated for harboring the magA genes using a single-tube multiplexed polymerase chain reaction. The magA genes were analyzed by PCR-RFLP technique using two enzymes, namely PciI and SmaI. Antibiogram assay using 12 commercially available antibiotic discs was performed on all the isolates.

Results:

The presence of K. pneumoniae specific gene (ureD) was confirmed in all the isolates. The percentage of isolates harboring the magA gene was 7.34%(23 isolates), the majority of which was collected from the patients admitted in intensive care units (16 isolates, 69.6%), and infectious diseases wards (5 isolates, 21.7%). PCR-RFLP analysis revealed that for 7 out of 23 isolates, where Sma1 could not cleave the magA gene. All the isolates showed resistance to ampicillin, carbenicillin cefradine, chloramphenicol, erythromycin, kanamycin, and sulphamethoxazole, though the extent was varying. However, imipenem showed 100% sensitivity to all the tested isolates.

Conclusion:

This study demonstrates the presence of the magA gene in multidrug-resistant clinical isolates of K. pneumoniae collected from Bangladesh.

Keywords: Klebsiella pneumoniae, Polysaccharide capsule, ureD, magA, Multidrug-resistance, PCR-RFLP, Imipenem, Antibiogram.


Article Information


Identifiers and Pagination:

Year: 2020
Volume: 14
First Page: 196
Last Page: 204
Publisher Id: TOMICROJ-14-196
DOI: 10.2174/1874285802014010196

Article History:

Received Date: 1/4/2020
Revision Received Date: 14/7/2020
Acceptance Date: 8/8/2020
Electronic publication date: 05/09/2020
Collection year: 2020

© 2020 Ali et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: https://creativecommons.org/licenses/by/4.0/legalcode. This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


These authors contributed equally to this work.
* Address correspondence to this author at Department of Genetic Engineering and Biotechnology, 211 - Academic Building E, Shahjalal University Avenue, Shahjalal University of Science and Technology, Sylhet 3114, Bangladesh; Tel: +88 01716 299 245; E-mail:hazratsust05@gmail.com






1. INTRODUCTION

Klebsiella pneumoniae is an encapsulated gram-negative bacterium of the Enterobacteriaceae family [1Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998; 11(4): 589-603.
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transmitted into other parts of the body. Nevertheless, infections are infrequent as healthy immune systems of men can resist the bacterium effectively. However, in immunocompromised patients, it causes severe diseases, e.g., septicemia, pneumonia, urinary tract infections (UTI), liver abscess, chronic lung disorders, and nosocomial infections [1Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev 1998; 11(4): 589-603.
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K. pneumoniae are intrinsically resistant to ampicillin. Besides, they ubiquitously possess an inducible chromosomally encoded ß-lactamase (cephalosporinase), making them resistant to first and second-generation cephalosporins [3Wand ME, Baker KS, Benthall G, et al. Characterization of pre-antibiotic era Klebsiella pneumoniae isolates with respect to antibiotic/disinfectant susceptibility and virulence in Galleria mellonella. Antimicrob Agents Chemother 2015; 59(7): 3966-72.
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Among the Gram-negative bacteria, K. pneumoniae are the second-most frequent hospital-acquired and nosocomial pathogens [11Lin WH, Wang MC, Tseng CC, et al. Clinical and microbiological characteristics of Klebsiella pneumoniae isolates causing community-acquired urinary tract infections. Infection 2010; 38(6): 459-64.
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]. In clinical settings, K. pneumoniae infections are usually diagnosed by examining samples of the infected tissue, e.g., sputum, urine, or blood by culture-based methods [15Viau R, Frank KM, Jacobs MR, et al. Intestinal Carriage of Carbapenemase-Producing Organisms: Current Status of Surveillance Methods. Clin Microbiol Rev 2016; 29(1): 1-27.https://cmr.asm.org/content/29/1/1 [Internet].
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]. Cutting-edge technologies, e.g., Sanger and next-generation sequencing, have gained a foothold in health care for diagnosis of infection at the individual level, outbreak detection, transmission mapping, and source tracing [21Wendt JM, Kaul D, Limbago BM, et al. Transmission of methicillin-resistant Staphylococcus aureus infection through solid organ transplantation: confirmation via whole genome sequencing. Am J Transplant 2014; 14(11): 2633-9.
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[http://dx.doi.org/10.1126/scitranslmed.3004129]
]. However, high-throughput screening through targeted sequencing is expensive, labor extensive, and requires expertise. Simple molecular techniques, e.g., PCR, PCR-RFLP assays are, in contrast, rapid and easy to perform, but have considerable sensitivity, specificity, and wide-range of application in infection detection [15Viau R, Frank KM, Jacobs MR, et al. Intestinal Carriage of Carbapenemase-Producing Organisms: Current Status of Surveillance Methods. Clin Microbiol Rev 2016; 29(1): 1-27.https://cmr.asm.org/content/29/1/1 [Internet].
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Several virulence factors, e.g., adhesion, siderophore, O antigen, and capsule attribute to its pathogenesis [24Kil KS, Darouiche RO, Hull RA, Mansouri MD, Musher DM. Identification of a Klebsiella pneumoniae strain associated with nosocomial urinary tract infection. J Clin Microbiol 1997; 35(9): 2370-4.
[http://dx.doi.org/10.1128/JCM.35.9.2370-2374.1997] [PMID: 9276418]
]. Among the 77 forms of capsular antigens, K1 and K2 are mostly studied. Between these two, K2 is the most common type isolated from patients with pneumonia, bacteremia, and UTI. Previous genetic studies identified that the genomic map of K. pneumoniae capsule contains a gene cluster subsuming the cps (capsular polysaccharide synthesis), rmpA, rmpA1 and rmpA2 (regulator of the mucoid phenotype A, A1, and A2, respectively), wb (the wb gene cluster codes for O-specific polysaccharide), and magA (mucoviscosity associated gene A) [25Regué M, Izquierdo L, Fresno S, et al. A second outer-core region in Klebsiella pneumoniae lipopolysaccharide. J Bacteriol 2005; 187(12): 4198-206.
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[http://dx.doi.org/10.1128/AEM.29.6.819-825.1975] [PMID: 1098574]
]. Functional aspects of these genes include the synthesis of capsular polysaccharide, the regulation of the extracellular polysaccharide capsule's synthesis, and the production of lipopolysaccharide, respectively. In most of the isolates of K. pneumoniae, these four genes remain conserved. Among the genes mentioned above, magA is a novel virulent gene that causes acute infection of Klebsiella, e.g., septicemia, bacteremia, pneumonia, and lung and liver abscesses [27Chan K-S, Chen C-M, Cheng K-C, Hou C-C, Lin H-J, Yu W-L. Pyogenic liver abscess: a retrospective analysis of 107 patients during a 3-year period. Jpn J Infect Dis 2005; 58(6): 366-8.
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, 28Chung DR, Lee SS, Lee HR, et al. Emerging invasive liver abscess caused by K1 serotype Klebsiella pneumoniae in Korea. J Infect 2007; 54(6): 578-83.
[http://dx.doi.org/10.1016/j.jinf.2006.11.008] [PMID: 17175028]
]. It spans about 1 kb region and involves in biosynthesis, transfer, and glycosylation of lipopolysaccharide [27Chan K-S, Chen C-M, Cheng K-C, Hou C-C, Lin H-J, Yu W-L. Pyogenic liver abscess: a retrospective analysis of 107 patients during a 3-year period. Jpn J Infect Dis 2005; 58(6): 366-8.
[PMID: 16377869]
]. The presence of chromosomal magA gene confers a hyper-viscous phenotype, which can be characterized by a positive string test (formation of a mucoviscous string of 5 mm diameter during passing loop through a colony) and resistance to phagocytosis. Among 77 characterized capsular serotypes (K), a major portion belongs to K1 and K2 serotypes. Based on these observations, the genetic locus containing magA can be targeted as a new pathogenicity island responsible for increasing the virulence of K. pneumoniae strains [29Struve C, Bojer M, Nielsen EM, Hansen DS, Krogfelt KA. Investigation of the putative virulence gene magA in a worldwide collection of 495 Klebsiella isolates: magA is restricted to the gene cluster of Klebsiella pneumoniae capsule serotype K1. J Med Microbiol 2005; 54(Pt 11): 1111-3. [1].
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].

In the present study, we sought to investigate the presence of magA gene in isolates of K. pneumoniae obtained from patients from different wards of Bangladeshi hospitals.

2. MATERIALS AND METHODS

2.1. Samples

From September 2016 to April 2017, we collected 313 clinical specimens from patients, including urine, blood, trachea, and wounds. Authorized personnel collected the specimen at the bedside from the patients (one isolate per patient) admitted in 05 hospitals of 05 different regions, namely Sylhet, Dhaka, Mymensingh, Rajshahi, and Khulna districts of Bangladesh. All isolates were subjected to standard confirmatory tests, which included microscopic evaluation, Gram staining, oxidase test, catalase test, and growth on SIM (sulfide, indole, motility), lysine iron agar, phenylalanine agar, urea agar, malonate, blood agar, and MacConkey agar. Bacterial isolates were stored in tryptic soy broth (TSB) supplemented with 20% glycerol at -20°C.

2.2. Design of Primers

Primer3Plus tool was utilized to design the specific primers for PCR-based identification of magA and ureD genes in K. pneumoniae isolates [30Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JAM. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 2007; 35(SUPPL.2)
[http://dx.doi.org/10.1093/nar/gkm306]
]. The ureD (urease D, an accessory gene in the urease gene cluster) was coamplified as an internal control, as it is a specific gene for K. pneumoniae [31Alimohammadi L, Peymani A, Habibollah-Pourzereshki N. Detection of MagA gene among clinical isolates of klebsiella pneumoniae collected from hospitals of Qazvin, Iran. Malaysian J Sci 2017; 36(1): 22-31.
[http://dx.doi.org/10.22452/mjs.vol36no1.3]
]. Since we took advantage of a single tube multiplexed PCR for this study, careful optimization of Ta of the primers was critically important. We used Primer3Plus, OligoAnalyzer 3.1, and Multiple Primer Analyzer tools to optimize the annealing temperature (Ta) of the primers (Table 1) [30Untergasser A, Nijveen H, Rao X, Bisseling T, Geurts R, Leunissen JAM. Primer3Plus, an enhanced web interface to Primer3. Nucleic Acids Res 2007; 35(SUPPL.2)
[http://dx.doi.org/10.1093/nar/gkm306]
, 32Hung JH, Weng Z. Designing polymerase chain reaction primers using Primer3Plus. Cold Spring Harb Protoc 2016; 2016(9): 821-6.
[http://dx.doi.org/10.1101/pdb.prot093096] [PMID: 27574202]
-34Integrated DNA Technologies OligoAnalyzer 31 2014.]. The designed primers were commercially purchased from Integrated DNA Technologies.

Table 1
List of primers used for the detection of ureD and magA genes in K. pneumoniae isolates.


2.3. Genomic DNA Extraction

We extracted genomic DNA from the obtained isolates using a commercially available bacterial DNA isolation kit pursuing the manufacturer's instructions (GenElute™ Bacterial Genomic DNA Kit, Sigma-Aldrich, Cat.: NA2120). The DNA quality was examined by electrophoresis on 0.7% agarose gel (Ultra-pure Agarose, Invitrogen, Cat. No.: 6500-25).

2.4. Single Tube Multiplex PCR Amplification of ureD and magA Genes

The single-tube multiplexed PCR was carried out with 3.0 µL of template DNA, 1 µL of each primer (10 μM), 12.5 µL of master mix (Promega Hot Start, Cat No.: M7122) and 5.5 µL of nuclease-free Water (Promega, USA) in a total volume of 25 µL. The magA and ureD genes were amplified simultaneously using the ProFlex™ 2 x 96-well PCR System (Applied Biosystems, Cat No.: 4484076). The PCRs were initially heated to 96°C for 7 minutes, followed by 35 cycles of denaturation (96°C, 1 minute), annealing (49°C, 1 minute), and extension (72°C, 1 minute). A final extension was carried at 72°C for 7 minutes. PCR products were evaluated by electrophoresis on 1.2% agarose gels ((Ultra-pure Agarose, Invitrogen, Cat. No.: 6500-25). 1-kb DNA ladder (Promega, Cat. No.: G5711) was used to compare DNA band sizes.

Using commercially available DNA purification kits, we purified 15 randomly selected PCR products of both genes from the TBE buffered agarose gels (GeneJET Gel Extraction Kit, Cat.: K0692). Purified PCR products were sent to Macrogen Corp. (Geumcheon-gu, Seoul, Rep. of Korea) for Sanger sequencing. The sequence outcomes were aligned and compared with the reference sequences of the genes using the BLAST program at the web interface of the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi) [35Mount DW. Using the Basic Local Alignment Search Tool (BLAST). Cold Spring Harb Protoc 2007; 2007(7): pdb.top17.]. The confirmed samples were used as the control for the rest of the analysis.

2.5. Restriction Digestion

10 µL of the PCR products, which produced positive outcomes for both the genes, were transferred to a separate 0.5 µL-Eppendorf vial and added with 17 µL of nuclease-free water. Then 1.5 µL of each of PciI (Thermo Fisher Scientific, Cat: ER1871) and SmaI (Thermo Fisher Scientific, Cat: ER0661) restriction enzymes were added and mixed carefully to the solution and incubated at 32°C for an overnight period (Table 2). The incubation temperature was set at the mean temperature of the optimum incubation temperatures of the enzymes mentioned above. Using UV illumination, the restriction fragments were compared against a 1-kb plus DNA ladder (Thermo Scientific, Cat. No.: FERSM1331) on 1.5% agarose gels. This step was repeated multiple times to confirm that the restriction digestion was not incidental.

2.6. Antibiogram Assay of the Isolates

A disc diffusion method was adopted to investigate antibiotic profiling of K. pneuomoniae isolates using 11 commercially available discs [36El Bouamri MC, Arsalane L, El Kamouni Y, Zouhair S. Antimicrobial susceptibility of urinary Klebsiella pneumoniae and the emergence of carbapenem-resistant strains: A retrospective study from a university hospital in Morocco, North Africa. Afr J Urol 2015; 21(1): 36-40.
[http://dx.doi.org/10.1016/j.afju.2014.10.004]
, 37Kang CI, Song JH. Antimicrobial resistance in Asia: current epidemiology and clinical implications. Infect Chemother 2013; 45(1): 22-31.
[http://dx.doi.org/10.3947/ic.2013.45.1.22] [PMID: 24265947]
]. High-quality absorbent paper discs impregnated with the following antibiotics were used: ampicillin (10 µg/disc), carbenicillin (10 µg/disc), cefradine (25 µg/disc), chloramphenicol (30 µg/disc), ciprofloxacin (30 µg/disc), erythromycin (15 µg/disc), gentamycin (10 µg/disc), imipenem (10 µg/disc), kanamycin (30 µg/disc), levofloxacin (5 µg/disc), streptomycin (10 µg/disc) and sulphamethoxazole (25 µg/disc). A fresh bacterial culture inoculated on Tryptocasein Soy Agar plates (Sigma-Aldrich, Cat.: 22091) by spreading. An antibiotic disc and a blank disc (as negative control) were placed carefully onto each culture plates and incubated overnight at 37°. After the incubation period, the zone of inhibition due to the activity of antibiotic-containing discs was measured following the protocol described by Sharmeen et al. [38Sharmeen R, Hossain MN, Rahman MM, Foysal MJ, Miah MF. In-vitro antibacterial activity of herbal aqueous extract against multi-drug resistant Klebsiella sp. isolated from human clinical samples Int Curr Pharm J 2012; 1(6): 133-7.
[http://dx.doi.org/10.3329/icpj.v1i6.10534]
].

2.7. Statistical Analysis

All data were analyzed using IBM-SPSS version 17.0. We evaluated statistical significance using both one-tailed and two-tailed experiments by Student t-test and χ2 test at 99% (p ≤ 0.01) and 95% (p ≤ 0.05) confidence intervals.

3. RESULTS

3.1. Patient Characteristics and the Isolates Obtained

In the current study, 313 isolates of K. pneumoniae were obtained from the intensive care units (ICU), internal medicine and infectious diseases wards of the five hospitals (Table 3). Out of these 313 samples, 192 isolates (61.3%) were collected from females and the rest (n = 121, 38.7%) were from males. The age of the patients ranged from 13 to 84 years, with a mean of 49.50 ± 18.24 years.

Table 2
Restriction enzymes used for restriction digestion.


Table 3
General characteristics of the patients and the specimen collected.


3.2. Multiplex PCR Outcomes

All the isolates carried the ureD gene (served as an internal control), and 23 isolates harbored the magA gene (Fig. 1). Out of these 23 isolates, 07 were collected from Dhaka (10.14%, n = 69). Among the rest 16 isolates, 05 were from Mymensingh (8.77%, n = 57), 08 were from Sylhet (8.51%, n = 94) and 03 were from Rajshahi (5.77%, n = 52). None of the isolates collected from the Khulna region harbored the magA gene. Hence, the overall prevalence of the magA gene in our studied subjects was 7.34% (n = 313). The isolates harboring magA were mostly isolated from the patients hospitalized in the intensive care unit (ICU) (Table 4). Twenty-three out of the 313 isolates harbored the magA gene, and most of these isolates were collected from the patients admitted in intensive care units (16 isolates, 69.6%) and infectious diseases wards (05 isolates, 21.7%).

Fig. (1)
Identification of ureD and magA genes by single-tube multiplex PCR amplification. K1 - K13 and K14 – K23 denotes different isolates that contained magA gene. Neg denotes negative control (water), which served as a loading control.


Table 4
The frequency of magA genes in K. pneumoniae isolates based on the clinical samples and hospital wards.


Fig. (2)
PCR-RFLP analysis of ureD and magA genes. K1 - K13 and K14 – K23 denotes different isolates that were double digested with SmaI and PciI restriction enzymes. Neg denotes negative control (water), which served as a loading control. Udg refers to DNA solution that contained undigested ureD and magA genes.


3.3. PCR-RFLP Analysis

After overnight incubation, PciI could digest the magA gene into two fragments (958 and 269 bp) in 16 out of the 23 isolates. On the contrary, SmaI digested the ureD gene into the desired fragments (453 and 372 bp) in all 23 isolates (Fig. 2).

3.4. Antibiotic Sensitivity of K. pneumoniae Isolates

All the isolates exhibited resistance to ampicillin, carbenicillin, cefradine, chloramphenicol, erythromycin, kanamycin, and sulphamethoxazole. Ciprofloxacin and levofloxacin showed ~75% sensitivity, whereas gentamycin and streptomycin exhibited ~12% sensitivity. Two isolates, both collected from Mymensingh district, showed a 10% sensitivity for streptomycin. Imipenem was the only antibiotic used, which showed 100% sensitivity to all the tested isolates (Fig. 3). Among the 23 isolates that contained the magA gene, imipenem, ciprofloxacin, levofloxacin showed 100%, 69.6%, and 65.28% sensitivity, respectively. The sensitivity of both gentamycin and streptomycin was 13.04% (Fig. 3).

Fig. (3)
Antibiotic susceptibility analysis of the K. pneumoniae isolates. A. Overall antibiotic susceptibility analysis of the K. pneumoniae isolates (n = 313). This includes both magA containing and magA not-containing isolates. B. Antibiotic susceptibility of the magA containing isolates (n = 23).


4. DISCUSSION

K. pneumoniae are responsible for several nosocomial infections among the debilitated elderly and children [39Bagheri P, Sepand M. A meta-analytical study of intentional and accidental non-food poisoning incidences in Iran (1991-2013). Koomesh [Internet] 2015; 16(3): 443-53. http://koomeshjournal.semums.ac.ir/article-1-2494-en.html]. They cause significant morbidity following urinary tract infections, septicemia, and pneumonia due to high resistance to extensively administered antibiotics [9Navon-Venezia S, Kondratyeva K, Carattoli A. Klebsiella pneumoniae: a major worldwide source and shuttle for antibiotic resistance. FEMS Microbiol Rev 2017; 41(3): 252-75.
[http://dx.doi.org/10.1093/femsre/fux013] [PMID: 28521338]
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[http://dx.doi.org/10.1093/cid/cir482] [PMID: 21865189]
].

The magA gene, which plays a critical role in polysaccharide capsule production of the bacterium, is one of the critical factors in K. pneumoniae associated virulence [41Lin JC, Chang FY, Fung CP, et al. High prevalence of phagocytic-resistant capsular serotypes of Klebsiella pneumoniae in liver abscess. Microbes Infect 2004; 6(13): 1191-8.
[http://dx.doi.org/10.1016/j.micinf.2004.06.003] [PMID: 15488738]
]. Previous studies reported that the secondary acquired infections, e.g., eye infection, bacteremia, sepsis, and meningitis, are magA related diseases [42Lederman ER, Crum NF. Pyogenic liver abscess with a focus on Klebsiella pneumoniae as a primary pathogen: an emerging disease with unique clinical characteristics. Am J Gastroenterol 2005; 100(2): 322-31.
[http://dx.doi.org/10.1111/j.1572-0241.2005.40310.x] [PMID: 15667489]
, 43Yeh KM, Chang FY, Fung CP, Lin JC, Siu LK. Serotype K1 capsule, rather than magA per se, is really the virulence factor in Klebsiella pneumoniae strains that cause primary pyogenic liver abscess. J Infect Dis 2006; 194(3): 403-4.
[http://dx.doi.org/10.1086/505153] [PMID: 16826492]
]. In the present study, over 7% isolates were positive for the presence of magA gene (Table 3). Amrai et al., studied 173 K. pneumoniae isolates collected from different clinical samples taken from Iran and reported 2.3% prevalence of the magA gene [44Amraie H, Shakib P, Rouhi S, Bakhshandeh N, Zamanzad B. Prevalence assessment of magA gene and antimicrobial susceptibility of Klebsiella pneumoniae isolated from clinical specimens in Shahrekord, Iran. Iran J Microbiol 2014; 6(5): 311-6.
[PMID: 25848520]
]. The prevalence of the gene in Bangladeshi subjects was significantly higher (p = 0.0203; χ2= 5.386). Previous studies on clinical samples of K. pneumoniae, invasive and non-invasive metastatic infections reported that the prevalence of the magA gene was 08 – 100% [45Liu C, Shi J, Guo J. High prevalence of hypervirulent Klebsiella pneumoniae infection in the genetic background of elderly patients in two teaching hospitals in China. Infect Drug Resist 2018; 11: 1031-41.
[http://dx.doi.org/10.2147/IDR.S161075] [PMID: 30104891]
-50Fang CT, Chuang YP, Shun CT, Chang SC, Wang JT. A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J Exp Med 2004; 199(5): 697-705.
[http://dx.doi.org/10.1084/jem.20030857] [PMID: 14993253]
]. Another remarkable observation was found studying isolates collected from the liver abscess that none of the samples were positive for the presence of the magA gene.

Previously, the magA gene was considered one of the primary reasons for the prevalence of these bacteria in East and South Asian countries [51Rahimian J, Wilson T, Oram V, Holzman RS. Pyogenic liver abscess: recent trends in etiology and mortality. Clin Infect Dis 2004; 39(11): 1654-9.
[http://dx.doi.org/10.1086/425616] [PMID: 15578367]
]. Several reports have suggested the magA gene as a root among geographical location, genetic predisposition, and susceptibility to the infections [51Rahimian J, Wilson T, Oram V, Holzman RS. Pyogenic liver abscess: recent trends in etiology and mortality. Clin Infect Dis 2004; 39(11): 1654-9.
[http://dx.doi.org/10.1086/425616] [PMID: 15578367]
-54Shon AS, Bajwa RPS, Russo TA. Hypervirulent (hypermucoviscous) Klebsiella pneumoniae: a new and dangerous breed. Virulence 2013; 4(2): 107-18.
[http://dx.doi.org/10.4161/viru.22718] [PMID: 23302790]
]. Struve et al., sorted out that only K1 serotype of Klebsiella spp. isolated from patients with liver abscesses carries the magA gene [29Struve C, Bojer M, Nielsen EM, Hansen DS, Krogfelt KA. Investigation of the putative virulence gene magA in a worldwide collection of 495 Klebsiella isolates: magA is restricted to the gene cluster of Klebsiella pneumoniae capsule serotype K1. J Med Microbiol 2005; 54(Pt 11): 1111-3. [1].
[http://dx.doi.org/10.1099/jmm.0.46165-0] [PMID: 16192445]
]. In the current study, most of the species containing magA gene were isolated from patients in ICUs, and trachea and urine samples. It was noticeable that long-time hospitalization and the use of invasive medical tools enhanced the susceptibility of patients towards a Klebsiella infection. Our study suggests that the evaluation of magA could be considered a suitable index for rapid molecular identification and diagnosis of these bacteria. Besides, it can be used for the timely treatment of infections caused by K. pneumoniae.

This study describes a PCR-RFLP based technique for precise detection and analysis of magA gene in K. pneumoniae isolates collected from clinical specimens. Our analysis revealed that all the isolates were uniformly cleaved by Pci1. Nearly 1 in every 3 isolates (30.43%) produced negative results for Sma1. This variation by restriction endonuclease digestion of PCR products exerted significant diversity in the magA gene. What it translates into is, the magA gene in K. pneumoniae is significantly evolving in clinical isolates of Bangladesh.

Previous studies affirmed that K. pneumoniae are resistant to most of the antibiotics and thus, they are termed as multidrug-resistant. Our study revealed that K. pneumoniae isolates collected from clinical samples are 100% resistant to several antibiotics, including ampicillin, cefradine, chloramphenicol, erythromycin, kanamycin, and sulphamethoxazole. Only imipenem was found fully effective against all the isolates. These outcomes support previous reports from Dhaka and Sylhet regions of Bangladesh [23Mahmudunnabi G, Majlish ANK, Momtaz F, Foysal MJ, Rahman MM, Islam K. Molecular detection and PCR-RFLP analysis using Pst1 and Alu1 of multidrug resistant Klebsiella pneumoniae causing urinary tract infection in women in the eastern part of Bangladesh. J Genet Eng Biotechnol 2018; 16(1): 77-82.
[http://dx.doi.org/10.1016/j.jgeb.2017.12.004] [PMID: 30647708]
, 55Rahim MA, Samad T, Mitra P, Zaman S, Habib SH, Afroze SR, et al. Frequency, Risk Factors and Antibiotic Sensitivity Pattern of Extended-Spectrum Beta-Lactamase Producing Escherichia coli and Klebsiella pneumoniae Causing Urinary Tract Infection: Experience from a Tertiary Care Hospital of Bangladesh. BIRDEM Med J 2017; 7(2): 155-9.
[http://dx.doi.org/10.3329/birdem.v7i2.32455]
, 56Jobayer M, Afroz Z, Nahar SS, Begum A, Begum SA, Shamsuzzaman SM. Antimicrobial susceptibility pattern of extended-spectrum beta-lactamases producing organisms isolated in a Tertiary Care Hospital, Bangladesh. Int J Appl Basic Med Res 2017; 7(3): 189-92.
[http://dx.doi.org/10.4103/ijabmr.IJABMR_28_16] [PMID: 28904920]
]. Our study suggests that multi-drug resistant strains of K. pneumoniae are prevalent across the country.

The antibiotic-resistant pattern has continuously been increasing at an alarming rate, primarily due to the overdose and unregulated use of antibiotics. Nowadays, powerful antibiotics are prescribed in the treatment of most of the diseases, contributing to the spread of multidrug-resistant bacteria [36El Bouamri MC, Arsalane L, El Kamouni Y, Zouhair S. Antimicrobial susceptibility of urinary Klebsiella pneumoniae and the emergence of carbapenem-resistant strains: A retrospective study from a university hospital in Morocco, North Africa. Afr J Urol 2015; 21(1): 36-40.
[http://dx.doi.org/10.1016/j.afju.2014.10.004]
]. For effective treatment, it is critically important to evaluate the antibiotic-sensitivity status of the causative agents [36El Bouamri MC, Arsalane L, El Kamouni Y, Zouhair S. Antimicrobial susceptibility of urinary Klebsiella pneumoniae and the emergence of carbapenem-resistant strains: A retrospective study from a university hospital in Morocco, North Africa. Afr J Urol 2015; 21(1): 36-40.
[http://dx.doi.org/10.1016/j.afju.2014.10.004]
, 37Kang CI, Song JH. Antimicrobial resistance in Asia: current epidemiology and clinical implications. Infect Chemother 2013; 45(1): 22-31.
[http://dx.doi.org/10.3947/ic.2013.45.1.22] [PMID: 24265947]
].

CONCLUSION

Infection with multi-drug resistant K. pneumoniae is now a global public health concern. Our study suggests that magA producing K. pneumoniae strains are prevalent in the clinical samples of Bangladesh. It also points out that this gene continues to evolve in K. pneumoniae strains in this country. We suggest that the magA gene, coupled with the ureD gene (as an internal control) in the single-tube multiplexed PCR, can be used as a reliable genetic marker for the rapid diagnosis of multi-drug K. pneumoniae infections. Considering the presence of the magA gene among clinical isolates of K. pneumoniae, especially in the isolates collected from ICUs, it is necessary to adopt suitable infection controlling tools and appropriate therapeutic approaches to prevent further spread of this virulent organism in our hospital settings.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

This study was approved by the Ethical and Animal Care Committee of the Shahjalal University of Science and Technology.

HUMAN AND ANIMAL RIGHTS

Not applicable.

CONSENT FOR PUBLICATION

Written informed consent was obtained from all the participants.

AVAILABILITY OF DATA AND MATERIALS

The authors confirm that the data supporting the findings of this research are available within the article.

FUNDING

The authors received no formal funding for this study.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

The authors wholeheartedly praise the cooperation by the hospital authorities who helped in sample collection for this investigation. We also appreciate the wholehearted cooperation of Prof. Md. Faruque Miah and his lab at Shahjalal University of Science and Technology.

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"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


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