Characterization and Insilico Analysis of Cassia auriculata against Multi drug Resistant Klebsiella pneumoniae Isolated from Inanimate Origin

J. Mary Sheela¹*, Ravuri. Durga Bhavani², A. Pugazhendhi³

¹Associate Professor, Department of Microbiology, Ethiraj College for Women, Chennai.

²PG Student, Department of Microbiology, Ethiraj College for Women, Chennai.

³Product Manager, J Mitra & Co. Pvt Ltd., Chennai.

*Corresponding Author E-mail: marysheela_j@ethirajcollege.edu.in, Pugazhendhi.a@gmail.com

ABSTRACT:

Infection with carbapenem-resistant Enterobacteriaceae (CRE) or carbapenemase-producing Enterobacteriaceae is emerging as an important challenge in health-care.50 swabs were collected from inanimate origins located in Chennai and the organisms isolated were identified by performing various preliminary tests like gram staining, capsule staining, motility, catalase, oxidase and biochemicals. antibiotic sensitivity testing was done for all the isolates using Ampicillin (AMP), Tetracycline (TE), Nitrofurantoin (NIT), Colistin (CL), Piperacillin (PIT), Noroxin (NX), Cefepime (CPM), Cefeparazone (CPZ), Ceftazidime (CFZ), Ciprofloxacin (CIP), Cotrimazole (COT), Ceftriaxone (CFX), Tigecycline (TGC), Tobramycin (TOB), Gentamicin (GEN), Amikacin (AK) and Imipenem (IMP) drugs and highest resistant against all the gram negative drugs was shown by Klebsiella which was further confirmed by MIC. The isolate was analysed for species by 16S rRNA sequencing which was reported as Klebsiella pneumoniae. The main objective of the present study is to identify the genes responsible for showing MDR and given for PCR reported as NDM, OXA-48 and K2A are the genes. Molecular docking was done by retrieving the gene structures of OXA-48 NDM and K2A of K.pneumoniae from protein data bank with proteins present in the leaf part of Cassia auriculata in patchdock and the binding interactions were interpreted and visualized using pyMOL to give a breakpoint by designing a drug for MDR bacterial strains.

KEYWORDS: Klebsiella pneumonia; Drug resistance; Cassia auriculata; Antibiotics.

1. INTRODUCTION:

Klebsiella organisms are often resistant to multiple antibiotics [20]. Current evidence implicates plasmids as the primary source of the resistance genes [6]. Klebsiella species with the ability to produce extended-spectrum beta-lactamases (ESBL) are resistant to all beta-lactam antibiotics, except carbapenems [9].

Carbapenems are a class of highly effective antibiotic agents commonly used for the treatment of severe or high-risk bacterial infections. [9]. This class of antibiotics are usually reserved for known or suspected multidrug-resistant (MDR) bacterial infections [5]. The concern is that carbapenem is often used as a drug of last resort when battling resistant bacterial strains [15].

A number of mechanisms cause carbapenem resistance in the Enterobacteriaceae. These include hyper production of ampCbeta-lactamase with an outer membrane porin mutation [2], CTX-M extended-spectrum beta-lactamase with a porin mutation or drug efflux, and carbapenemase production. The most important mechanism of resistance by carbapenem-resistant Klebsiella pneumoniae is the production of a carbapenemase enzyme, blakpc [2]. Patients with unrecognized carbapenem-resistant K. pneumoniae colonization have been reservoirs for transmission during nosocomial outbreaks [8].

In 2009, strains of K. pneumoniae with gene called New Delhi metallo-beta-lactamase (NDM-1) that even gives resistance against intravenous antibiotic carbapenem, were discovered in India and Pakistan [1]. Loss of OmpK35 play a major role in antibiotic resistance as depicted in the K. pneumoniae CSUB10R deletion mutant (ΔOmpK35 and ΔOmpK36 under complementation following transformation with a plasmid-encoded OmpK35 gene [2]. Since K. pneumoniae normally lacks a chromosomally encoded, class C β-lactamase, acquisition of a plasmid-encoded, bla AMPC or another broad spectrum β-lactamase can confer high level carbapenem resistance in porin-deficient strains [15].

2. MATERIALS AND METHODS:

2.1 Sample collection:

A total of 50 samples were collected from mobile toilets and washrooms located in Chennai by using sterile swabs and transported into laboratory and were inoculated into saline after one hour of incubation at 37°C, the samples were processed for further identification.

2.2 Preliminary tests:

All the samples were inoculated into saline and cultured on Nutrient agar, MacConkey, Hicrome agar and on blood agar. The colonies were identified by performing preliminary tests such as gram staining, motility, catalase and oxidase further performed biochemical and sugars utilization test for species level identification.

2.3 Antimicrobial sensitivity testing:

Kirby Bauer method was performed to determine the susceptibility profiles of the isolates using Muller Hinton agar and tested for their resistance against the following antibiotic discs Ampicillin (AMP), Tetracycline (TE), Nitrofurantoin (NIT), Colistin (CL), Piperacillin (PIT), Noroxin (NX), Cefepime (CPM), Cefeparazone (CPZ), Ceftazidime (CFZ), Ciprofloxacin (CIP), Cotrimazole (COT), Ceftriaxone (CFX), Tigecycline (TGC), Tobramycin (TOB), Gentamicin (GEN), Amikacin (AK), Imipenem (IMP),which was further confirmed by automated MIC [11].

2.4 String test:

Hypervirulent K. pneumoniae is unique and emerging strain of Klebsiella species enhances the multi drug resistant genes and thereby increases the capsule thickness and making the strain resistant to phagocytosis and helps in action of spreading infection to distant organs [10]. From the single mucoid colony cultured on blood agar plates were obtained and studied for their ability to form viscous strings.

2.5 Modified Hodge Test:

Carbapenemase production is detected by the Modified Hodge Test when the test isolate produces the enzyme and allows growth of a carbapenem susceptible strain towards a carbapenem disc. The result is a characteristic cloverleaf-like indication [13].

2.6 DNA extraction:

DNA was isolated from the given culture and Fragment of16SrRNA gene was amplified by using Polymerase Chain Reaction (PCR). Forward and reverse DNA sequence of an amplicon was carried out with 16SF and 16SR primers by using Cycle sequencing kit on ABI3730xl Genetic Analyzer. The 16SrRNA gene sequence was used to carry out BLAST with NCBI gen bank database. Based on maximum identity score for first ten sequences were selected to identify the organism genus and species [3].

2.7 Resistant genes determination:

The obtained isolate of K. pneumoniae was sub-cultured and genomic DNA was isolated by using the DNA Mini kit (Qiagen, USA). The obtained genomic DNA was estimated using the NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, USA). Polymerase chain reaction was performed by using the Taq DNA Polymerase with isolated genomic DNA as the template. Primers specific to K2A, MagA NDM and Oxa-48 genes were targeted [7].

Table1: Primers used in PCR.

Target gene	Primer
K2A	F-CAACCATGGTGGTCGATTAG R-TGGTAGCCATATCCCTTTGG
MagA	F-GGTGCTCTTACATCATTGC R-GCAATGGCCATTTGCGTTAG
NDM	F-GCAGCTTGTCGGCCATGCGGC R-GGTCGCGAAGCTGAGCACCGCAT
OXA-48	F-GCGTGGTTAAGGATGAACAC R-CATCAAGTTCAACCCAACCG

The reaction was set up on ice and PCR was performed in Bio-Rad T100 Thermal cycler (Biorad Laboratories, USA). The PCR amplified samples were loaded on to a 1% agarose gel, and electrophoresis was performed. The bands were visualized using ethidium bromide and documented.

2.8 Protein data bank:

PDB is a database for the three-dimensional structural data of large biological molecules such as proteins and nucleic acids. The necessary gene structures for binding with the targeted genes were retrieved from the search box of the data bank and saved as a file format for docking by using patchdock [11].

2.9 Protein-protein docking using patch dock:

It is used to perform protein-protein docking. PATCH Dock is an algorithm to perform molecular docking. The input is two molecules of any type, i.e., protein, DNA, peptide, drugs. The output is a list of potential complexes sorted by shape complementarity criteria. The algorithm has 3 major stages: (1) molecular shape representation; (2) surface patch matching; (3) filtering and sorting.

2.10 Molecular graphics with PyMOL:

PyMOLis an open source molecular visualization system created by Warren Lyford Delano used to visualise the protein structure. It is currently commercialized by Schrodinger. PyMOL can produce high-quality 3D images of small molecules and biological macromolecules, such as proteins [14]. Using PyMOL, structural data can be visualized in nearly 3D different ways.

3. RESULTS:

Out of 50 samples, 3 isolates were suspected and confirmed as Klebsiella with preliminary testing and further screened for multi-drug resistance using Kirby Bauer disc diffusion method of the three isolates only 1 out of 3 showed resistance for all the gram-negative antibiotic drugs and determined by MIC. The resistant showed isolate was given for 16SrRNA sequencing for identification of genus and species and confirmed as K. pneumoniae. To identify the genes responsible for showing multidrug resistance the isolate was given for polymerase chain reaction and reported as K2A, NDM and OXA-48 are the genes present in the isolate.

3.1 Antibiotic Sensitivity Test:

Samples showing sensitivity and resistant pattern.

Fig: 1 ATCC Klebsiella

Fig: 2 Test sample

3.2 STRING TEST:

From the single mucoid colony cultured on blood agar plates were used and studied for their ability to form viscous strings. The hyper muco viscosity was determined by the formation of viscous strings about 5 mm length with the test sample.

3.3 MODIFIED HODGE TEST:

The Multi drug resistant Klebsiella pneumoniae showing clover leaf like indentation with E coli growing along the test organism, zone formation can be seen around the carbapenem disc showing sensitive to E coli but resistant to test organism. Thus the isolated Klebsiella pneumoniae showing positive MHT by producing carbapenemase.

Fig:4 Test Sample

Fig:5 ATCC Klebsiella

3.4 Identification of target gene responsible for showing MDR:

The obtained isolate of K. pneumoniae was sub-cultured and genomic DNA was isolated by using the DNA Mini kit (Qiagen, USA). The obtained genomic DNA was estimated using the NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, USA). Primers specific to K2A, MagA, NDM and Oxa-48 genes were targeted.

4. DISCUSSION:

Now-a-days Carbapenem resistant K. pneumoniae strains becoming epidemic and pandemic because of showing resistance to penicillins, aminoglycosidases and even cephalosporins particularly with problematic combinations of carbapenemases (OXA-48) and metallo beta lactamases (NDM) with loss of outer membrane porin leads difficulty in detection and treating infectious diseases. Hence, became limited treatment options thereby causing increase in mortality [12]. The present study states that multi drug resistant K. pneumoniae strain can show sensitive with C. auriculata comparing all other plants. This leads to the new choice in treatment for resistant strains of Klebsiella. For molecular docking the reported gene structures of K. pneumoniae and C. auriculata were retrieved from protein data bank and protein protein docking was done by patchdock and visualized in pyMOL with the genes NDM and OXA-48 of K. pneumoniae with the genes present in leaf part of C. auriculata.

5. CONCLUSION:

There is a high prevalence the rate of CRKP (carbapenem resistant K. pneumoniae) isolation remains on the rise and has become a major threat to public health, especially to those in critical conditions. An accurate and convenient prediction model for recognizing the risk of CRKP may improve empiric antibiotic prescription and decrease the rate of treatment failure and adverse effects, helping to identify the high-risk and provide precise antibiotics treatment [13]. Bacterial strains becoming more virulent and showing resistance to almost all the antibiotics and becoming limited treatment options thereby causing increase in mortality. Screening by MHT for these carbapenemases is a good epidemiological tool to assess the severity of the infection. The knowledge of the resistant pattern of bacterial strains will help to guide the appropriate antibiotic use and molecular docking will help to formulate an empirical antibiotic to treat gram negative infections.

6. REFERENCES:

1. Ahmed Abduljabbar Jaloob Ajnaby and Janeen Mohammed Reda Jaber Alhasnawi. 2017. Phenotypic and Molecular Characterization of Multidrug Resistance Klebsiella pneumoniae Isolated from Different Clinical Sources in AI-Najaf Province-Iraq. Book of microbiology.20:217-232.

2. Ahmed Abduljabbar Jaloob Aljanaby, Israa Abduljabbar Jaloob Aljanaby. Profile of Antimicrobial Resistance of Aerobic Pathogenic Bacteria isolated from Different Clinical Infections in Al-Kufa Central Hospital –Iraq During period from 2015 to 2017. Research J. Pharm. and Tech 2017; 10(10):3264-3270. doi: 10.5958/0974-360X.2017.00579.0

3. Antonio Domenech-Sanchez, Santiago Hernandez-Alles, Luis Martinez-Martinez, Vicente J. Beneda, and Sebastian Alberta. 1999. Identification and Characterization of a New porin Gene of Klebsiella pneumoniae: Its Role in β-Lactam Antibiotic Resistance. Journal of Bacteriology, 2726+2732.

4. Abu Hena Md Saiful Karim Chowdhury, Sukumar Nandi, Mahbubur Rahman, A S M Ashanul Karim, Syeda Shanoor Hasina Mamtaz, Nura Nasrin Rowshan Ara, et al. 2016. Comparision Between Phenotypic Confirmatory Test & Double Disc Synergy Test in Detection of Extended Spectrum β-Lactamase Produers Among Gram-Negative Bacilli. Journal of microbiology:P. 3-8.

5. B. Reshmi, P. Gopinath. Detection of blaNDM-1gene for the production of MBL in Clinical Strains of Klebsiella pneumoniae. Research J. Pharm. and Tech 2016; 9(10):1618-1620.

6. Cornelius J. Clancy, Liang Chen, Jae H. Hong, Shaoji Cheng, Binghua Hao, Ryan K. Shields, et al. 2013. Mutations of the OmpK36 Porin Gene and Promoter Impact Responses of Sequence Type 258, KPC-2-Producing Klebsiella pneumoniae Strains to Doripenem and Doripenem-Colistin. Antimicrobial Agents and Chemotherapy. p5258-5265.

7. Frank M. Kaczmarek, Faida Dib-Hajj, Wenchi Shang, and Thomas D. Gootz. 2006. High-Level Carbapenem Resistance in a Klebsiella pneumoniae Clinical Isolate Is Due to the combination of bla_ACT-1β-Lactamase Production, Porin OmpK35/36 Inservational Inactivation, and Down-Regulation of the Phosphate Transport. Journal of Antimicrobial Agents and Chemotherapy. p, 3396-3406.

8. Hisham A. Abbas, Ashraf A. Kadry, Ghada H. Shaker, Reham M. Goda. Resistance of Escherichia coli and Klebsiella pneumoniae isolated from different Sources to β-lactam Antibiotics. Research J. Pharm. and Tech. 2017; 10(2): 589-591. doi: 10.5958/0974-360X.2017.00116.0

9. K.R Jeya, M. VeeraPagu. Screening for the Antimicrobial Activity of Lippia nodiflora Leaf Extract Against Selective Bacterial Population. Research J. Pharm. and Tech. 4(11): Nov. 2011; Page 1669-1672.

10. Lena Ahmed Saleh Al-Faqeeh, Rafiuddin Naser, Kagne SR. Determination of antibiotic resistant profiles for bacteria isolated from clinical samples in Aurangabad, India. Research J. Pharm. and Tech. 2020; 13(8):3813-3816.

11. Muhammed Ahmad, Carl Urban, Noriel Mariano, Patricia A. Bradford, Ellen Calcagni, Steven J. Projan, Karen Bush, and James J. Rahal. 1999. Clinical Characteristics and Molecular Epidemiology Associated with Imipenem Resistant Klebsiella pneumoniae. Clinical Infectious Diseases 29:352-5.

12. Mandour S. A. 2016. Epidemiological studies using biotyping, serotyping and bacteriocin typing as marker systems to identify clinical multidrug-resistant klebsiella spp. Journal of Antimicrobial Agents and Chemotherapy. p: (378-386).

13. Mohammed Ahad, Gopinath P. Detection of Carbapenemase Resistance among Clinical Isolates of Klebsiella pneumoniae. Research J. Pharm. and Tech. 2016; 9(10):1585-1587.

14. Neil Woodford, Philip M. Tierno Jr., Katherine Young, Luke Tysall, Marie-France I. Palepou, Elaine Ward, et al. 2004. Outbreak of klebsiella pneumoniae producing a New Carbapenem-Hydrolyzig class A β-lactamase, KPC-3, in a New York Medical center. Society of Biotechnology and Microbiology. p:48(12); 4793-4799.

15. Patricia A. Bradford, Carl Urban, Noriel Mariano, Steven J. Projan, James J. Rahal and Karen Bush. 1997. Imipenem Resistance in Klebsiella pneumoniae Is Associated with the Combination of ACT-1, a Plasmid-Medicated AmpC β-Lactamase, and the Loss of na Outer Membrane Protien. Pakistan Journal of Microbiology. p. 563±569.

16. Patricia A. Bradford, Simona Bratu, Carl Urban, Melissa Visalli, Noriel Mariano, David Landman, et al. 2004. Emergence of Carbapenem-Resistant Klebsiella Species Possessing the Class a Carbapenem-Hydrolyzig KPC-2 and Inhibitor -Resistant TEM-30 β-Lactamases in New York City. Society of Clinical Infectious Diseases and Microbiology. p:39:55-60.

17. Poothakuzhiyil Remya, Mariappan Shanthi, Uma Sekhar. 2018. Occurrence and Characterization of Hyperviscous K1 and K2 Serotype in Klebsiella pneumoniae. J. Lab Physicians. P. 10:283-8.

18. R Adrizian, F Suryaningrat, A Alam and D Setiabudi. 2018. Incidence of multidrug-resistant, extensively drug-resistant and pan-drug-resistant bacteria in children hospitalized at Dr. Hasan Sadikin general hospital. Journal of Chicago. p:10.1088/1755.

19. Roumayne L. Ferreira, of Klebsiella pneumoniae strains producing carbapenemases and increase of resistance to colistin in an Italian teaching hospital from January 2012 to December. Journal of clinical Microbiology. p:2014. 15:244.

20. Sara Banu, Gopinath P. Detection of blactx-m Gene for Esbl Resistance among Clinical Isolates of Klebsiella pneumoniae. Research J. Pharm. and Tech 2016; 9(10):1615-1617.

21. Shireen Rana, Siddesh Basawaraj Sirwar, Vijayaraghavan. Prevalence and Antibiogram of Extended Spectrum beta- Lactamase Producing Klebsiella pneumoniae and Proteus mirabilis in UTI. Research J. Pharm. and Tech. 8(11): Nov., 2015; Page 1465-1468.

22. Sukanth kumar Enmozhi, Vijayalakshmi Ganesan, Manigundan Kaari, Bharathi Selvaraj, Gopikrishnan Venugopal, Jerrine Joseph, Radhakrishnan Manikkam. Anti-infective potential of marine actinobacteria against carbapenem resistant Klebsiella pneumoniae ATCC13882. Research J. Pharm. and Tech. 2020; 13(8):3653-3660.

23. Campanini, Marcia C. A. Brito, Eulalia M. L. da Silva, Calo Cesar de Melo Freire Anderson F. da Cunha and Maria-Cristina da Silva Pranchevicius. 2019. High Prevalence of Multidrug-Resistant Klebsiella pneumoniae Harboring Several Virulence and β-Lactmase Encoding Genes in a Brazilian Intensive Care Unit. Frontiers in Microbiology P: 2018.03198.

24. Rashid Iqbal, Nadeem Ikram, Muhammad Shoaib, Muhammad Javaid Asad, Raja Tahir Mehmood, Abida Niazi, et al. 2017. Phenotypic Confirmatory Disc Diffusion Test (PCDDT) Double Disc Syergy Test (DDST), E-Test OS Diagnostic tool for Detection of Extended Spectrum Beta Lactamase (ESBL) Producing Uropthogens. Jornal of Applied Biotechnology & Bioengineering. P: 344-349.

25. Yee-Huang Ku, Chi- Chung Chen, Mei-Feng Lee, Yin- Ching Chuang, Hung-Jen Tang, Wen- Liang Yu. 2016. Comparison of Synergism between Colistin, Fosfomycin and tigecycline against extended-spectrum β-Lactamase-producing Klebsiella pneumoniae isolates or with Carbapenem resistance. Journal of Microbiology, P:50. 931-939.

Received on 26.11.2020 Modified on 03.02.2021

Asian J. Res. Pharm. Sci. 2021; 11(3):199-204.

DOI: 10.52711/2231-5659.2021.00032