Sains Malaysiana 49(6)(2020): 1401-1410

http://dx.doi.org/10.17576/jsm-2020-4906-18

The Differences between the Expression Levels of axe-txe Genes in Chloramphenicol-Sensitive and Penicillin-Resistant Enterococcus faecium Isolates

(Perbezaan antara Tahap Ekspresi Gen axe-txe dalam Pencilan Enterococcus faecium yang Peka Kloramfenikol dan Tahan Penisilin)

SRI INDRA WAHYUNI MOHD IRMAL¹, SURESH KUMAR SUBBIAH¹, VASANTHA KUMARI NEELA¹, NIAZLIN MOHD TAIB¹, AZMIZA SYAWANI JASNI¹, MAHA ABDULLAH², MOHAMAD KHAIRIL RADZALI³ & RUKMAN AWANG HAMAT¹*

¹Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

²Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

³Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia

Received: 10 June 2019/Accepted: 3 February 2020

ABSTRACT

Toxin-antitoxin (TA) systems are important regulatory modules in bacterial physiological functions. In this study, Axe-Txe TA system of 20 Enterococcus faecium clinical isolates was investigated by polymerase chain reaction (PCR) using self-designed primers. The functionality of this TA system in two E. faecium isolates was evaluated by analysing the expression level of axe-txe genes using real-time quantitative PCR (RT-qPCR) in penicillin-resistant and chloramphenicol-sensitive environments at different points of time. Colony-forming units (CFU) of the bacteria were also measured at a similar point of time. The selection of these two isolates for TA functionality study was determined based on the susceptibility patterns of the two isolates to penicillin, the chloramphenicol via Kirby-Baur, and broth microdilution methods, which were then interpreted based on CLSI guidelines. Axe-Txe TA system was detected in both chromosomes and plasmids (100%, each) in all 20 isolates, while the selected two E. faecium isolates were sensitive to chloramphenicol (MIC = 4 µg/mL) and resistant to penicillin (MIC = 256 µg/mL). Although higher axe-txe genes expression was also observed in a chloramphenicol-sensitive environment at half an hour of the incubation period compared to the penicillin-resistant environment, higher expression of the axe-txe genes was found in the penicillin-resistant environment at 1 h incubation period compared to the chloramphenicol-sensitive environment. Nevertheless, E. faecium isolates in both environments exhibited higher expression of txe gene (toxin) at the 24 h incubation period. Provided that the functionality of TA systems of E. faecium isolates may vary in different antibiotic environments, various environmental conditions need to be considered in the role of TA systems as potential antimicrobial targets. Different expression of TA genes in different antibiotic environments and points of time may influence the discovery and development of drugs in the future.

Keywords: axe-txe genes; Enterococcus faecium; toxin-antitoxin system

ABSTRAK

Sistem toksin-antitoksin (TA) adalah modul kawalan penting dalam fungsi fisiologi bakteria. Dalam kajian ini, sistem Axe-Txe TA dalam 20 pencilan klinikal Enterococcus faecium dikaji menerusi kaedah tindak balas rantai polimerase (PCR) menggunakan primer-primer yang direka sendiri. Fungsi sistem TA ini dalam dua pencilan E. faecium dinilai dengan menganalisis tahap ekspresi gen axe-txe menggunakan PCR kuantitatif masa nyata (RT-qPCR) dalam persekitaran tahan penisilin dan peka terhadap kloramfenikol pada masa yang berlainan. Unit pembentuk koloni (CFU) bakteria juga diukur pada tempoh waktu yang sama. Dua pencilan yang digunakan untuk kajian fungsi TA telah dipilih berdasarkan corak kerentanannya terhadap penisilin, kloramfenikol melalui kaedah Kirby-Baur, dan kaedah pencairan-mikro bubur, yang kemudian ditafsirkan berdasarkan garis panduan CLSI. Sistem Axe-Txe TA dikesan pada kedua-dua kromosom dan plasmid (100%, masing-masing) di semua 20 pencilan, sementara dua pencilan E. faecium yang dipilih peka terhadap kloramfenikol (MIC = 4 µg/mL) dan tahan terhadap penisilin (MIC = 256 µg/mL). Walaupun ekspresi gen axe-txe yang lebih tinggi juga diperhatikan dalam persekitaran peka kloramfenikol pada setengah jam dari masa inkubasi berbanding dengan persekitaran tahan penisilin, ekspresi gen axe-txe yang lebih tinggi didapati di persekitaran tahan penisilin pada 1 jam tempoh inkubasi berbanding persekitaran peka kloramfenikol. Walaupun begitu, pencilan E. faecium di kedua-dua persekitaran menunjukkan ekspresi gen txe (toksin) yang lebih tinggi pada masa inkubasi 24 jam. Disebabkan fungsi sistem TA pencilan E. faecium mungkin berbeza dalam persekitaran antibiotik yang berbeza, pelbagai keadaan persekitaran perlu dipertimbang dalam peranan sistem TA sebagai sasaran anti-mikrob yang berpotensi. Ekspresi gen TA yang berbeza dalam persekitaran dan titik antibiotik yang berbeza dapat mempengaruhi penemuan dan perkembangan ubat pada masa hadapan.

Kata kunci: Enterococcus faecium; gen axe-txe; sistem toksin-antitoksin

REFERENCES

Aizenman, E., Engelberg-Kulka, H. & Glaser, G. 1996. An Escherichia coli chromosomal ‘addiction module’ regulated by 3’,5’-Bispyrophosphate: A model for programmed bacterial cell death. Proceedings of the National Academy of Sciences of the United States of America 93(12): 6059-6063.

Arias, C.A. & Murray, B.E. 2013. The rise of the enterococcus: Beyond vancomycin resistance. Nature Reviews Microbiology 10(4): 266-278.

Boss, L., Labudda, Ł., Węgrzyn, G., Hayes, F. & Kędzierska, B. 2013. The axe-txe complex of Enterococcus faecium presents a multilayered mode of toxin-antitoxin gene expression regulation. PLoS ONE 8(9): e73569.

Cárdenas-Mondragón, M.G., Ares, M.A., Panunzi, L.G., Pacheco, S., Camorlinga-Ponce, M., Girón, J.A., Torres, J. & De la Cruz, M.A. 2016. Transcriptional profiling of type II toxin-antitoxin genes of Helicobacter pylori under different environmental conditions: Identification of Hp0967-Hp0968 system. Frontiers in Microbiology 7: 1-14. doi: 10.3389/fmicb.2016.01872.

Chan, W.T., Espinosa, M. & Yeo, C.C. 2016. Keeping the wolves at bay: Antitoxins of prokaryotic type II toxin-antitoxin systems. Frontiers in Molecular Biosciences 3: 1-20. https://doi.org/10.3389/fmolb.2016.00009.

Christensen, S.K., Maenhaut-Michel, G., Mine, N., Gottesman, S., Gerdes, K. & Van Melderen, L. 2004. Overproduction of the Lon protease triggers inhibition of translation in Escherichia coli: Involvement of the yefM-yoeB toxin-antitoxin system. Molecular Microbiology 51(6): 1705-1717.

Chuah, E.L., Zakaria, Z.A., Suhaili, Z., Abu Bakar, S. & Desa, M.N.M. 2014. Antimicrobial activities of plant extracts against methicillin-susceptible and methicillin-resistant Staphylococcus aureus. Journal of Microbiology Research 4(1): 6-13.

CLSI. 2016. Performance Standards for Antimicrobial. 26thedition. Wayne, PA: Clinical and Laboratory Standard Institute.

Coussens, N.P. & Daines, D.A. 2016. Wake me when it’s over - bacterial toxin-antitoxin proteins and induced dormancy. Experimental Biology and Medicine 241(12): 1332-1342.

de Kraker, M.E., Jarlier, V., Monen, J.C., Heuer, O.E., van de Sande, N. & Grundmann, H. 2013. The changing epidemiology of bacteraemias in Europe: Trends from the European antimicrobial resistance surveillance system. Clinical Microbiology and Infection 19(9): 860-868.

Grady, R. & Hayes, F. 2003. Axe-Txe, a broad-spectrum proteic toxin-antitoxin system specified by a multidrug-resistant, clinical isolate of Enterococcus faecium. Molecular Microbiology 47(5): 1419-1432.

Halvorsen, E.M., Williams, J.J., Bhimani, A.J., Billings, E.A. & Hergenrother, P.J. 2011. Txe, an endoribonuclease of the enterococcal Axe-Txe toxin-antitoxin system, cleaves mRNA and inhibits protein synthesis. Microbiology 157(2): 387-397.

Hidron, A.I., Edwards, J.R., Patel, J., Horan, T.C., Sievert, D.M., Pollock, D.A. & Fridkin, S.K. 2008. Antimicrobial-resistant pathogens associated with healthcare-associated infections: Annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006-2007. Infection Control and Hospital Epidemiology 29(11): 996-1011.

Hu, M.X., Zhang, X., Li, E.L. & Feng, Y.J. 2010. Recent advancements in toxin and antitoxin systems involved in bacterial programmed cell death. International Journal of Microbiology 2010: 1-10. https://doi.org/10.1155/2010/781430.

Janssen, B.D., Garza-Sánchez, F. & Hayes, C.S. 2015. YoeB toxin is activated during thermal stress. Microbiology Open 4(4): 682-697.

Kariyama, R., Mitsuhata, R., Chow, J.W., Clewell, D.B. & Kumon, H. 2000. Simple and reliable multiplex PCR assay for surveillance isolates of vancomycin-resistant enterococci. Journal of Clinical Microbiology 38(8): 3092-3095.

Kędzierska, B. & Hayes, F. 2016. Emerging roles of toxin-antitoxin modules in bacterial pathogenesis. Molecules 21(6): 790-814.

Koch, S., Hufnagel, M., Theilacker, C. & Huebner, J. 2004. Enterococcal infections: Host response, therapeutic, and prophylactic possibilities. Vaccine 22(7): 822-830.

Lee, K.Y. & Lee, B.J. 2016. Structure, biology, and therapeutic application of toxin-antitoxin systems in pathogenic bacteria. Toxins 8(305): 1-33.

Moritz, E.M. & Hergenrother, P.J. 2007. Toxin-antitoxin systems are ubiquitous and plasmid-encoded in vancomycin-resistant enterococci. Proceedings of the National Academy of Sciences 104(1): 311-316.

Mutschler, H. & Meinhart, A. 2011. ε/ζ systems: Their role in resistance, virulence, and their potential for antibiotic development. Journal of Molecular Medicine 89(12): 1183-1194.

Palmer, K.L., Kos, V.N. & Gilmore, M.S. 2010. Horizontal gene transfer and the genomics of enterococcal antibiotic resistance. Current Opinion in Microbiology 13(5): 632-639.

Rosvoll, T.C., Pedersen, T., Sletvold, H., Johnsen, P.J., Sollid, J.E., Simonsen, G.S., Jensen, L.B., Nielsen, K.M. & Sundsfjord, A. 2010. PCR-based plasmid typing in Enterococcus faecium strains reveals widely distributed pRE25-, pRUM-, pIP501- and pHTbeta-related replicons associated with glycopeptide resistance and stabilizing toxin-antitoxin systems. FEMS Immunology and Medical Microbiology 58(2): 254-268.

Schmittgen, T.D. & Livak, K.J. 2008. Analyzing real-time PCR data by the comparative C(T) method. Nature Protocol 3(6): 1101-1108.

Sifaoui, F., Arthur, M., Rice, L. & Gutmann, L. 2001. Role of penicillin-binding protein 5 in expression of ampicillin resistance and peptidoglycan structure in Enterococcus faecium. Antimicrobial Agents and Chemotherapy 45(9): 2594-2597.

Soheili, S., Ghafourian, S., Sekawi, Z., Neela, V.K., Sadeghifard, N., Taherikalani, M., Khosravi, A., Ramli, R. & Hamat, R.A. 2015. The mazEF toxin-antitoxin system as an attractive target in clinical isolates of Enterococcus faecium and Enterococcus faecalis. Drug Design, Development and Therapy 9: 2553-2561.

Van Melderen, L. & Saavedra De Bast, M. 2009. Bacterial toxin-antitoxin systems: More than selfish entities? PLoS Genetics 5(3): e1000437-e1000442.

Wang, X. & Wood, T.K. 2011. Toxin-antitoxin systems influence biofilm and persister cell formation and the general stress response. Applied and Environmental Microbiology 77(16): 5577-5583.

Weng, P.L., Ramli, R., Shamsudin, M.N., Cheah, Y.K. & Hamat, R.A. 2013. High genetic diversity of Enterococcus faecium and Enterococcus faecalis clinical isolates by pulsed-field gel electrophoresis and multilocus sequence typing from a hospital in Malaysia. BioMed Research International 2013(938937): 1-6.

Wisplinghoff, H., Bischoff, T., Tallent, S.M., Seifert, H., Wenzel, R.P. & Edmond, M.B. 2004. Nosocomial bloodstream infections in US hospitals: Analysis of 24,179 cases from a prospective nationwide surveillance study. Clinical Infectious Diseases 39(3): 309-317.

Yang, Q.E. & Walsh, T.R. 2017. Toxin-antitoxin systems and their role in disseminating and maintaining antimicrobial resistance. FEMS Microbiology Reviews 41(3): 343-353.

*Corresponding author; email: rukman@upm.edu.my