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

Diserahkan: 10 Jun 2019/ Diterima: 3 Februari 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

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*Pengarang untuk surat-menyurat; email: rukman@upm.edu.my