The functional repertoire of AmpR in the AmpC β-lactamase high expression and decreasing β-lactam and aminoglycosides resistance in ESBL Citrobacter freundii

Falak Naz Tariq; Mehreen Shafiq; Nadeem Khawar; Gul Habib; Haji Gul; Azam Hayat; Mujaddad Ur Rehman; Ihab Mohamed Moussa; Eman A Mahmoud; Hosam O Elansary

doi:10.1016/j.heliyon.2023.e19486

The functional repertoire of AmpR in the AmpC β-lactamase high expression and decreasing β-lactam and aminoglycosides resistance in ESBL Citrobacter freundii

Heliyon. 2023 Aug 25;9(9):e19486. doi: 10.1016/j.heliyon.2023.e19486. eCollection 2023 Sep.

Authors

Affiliations

¹ Department of Microbiology, Abbottabad University of Science and Technology, Havelian, Abbottabad, 22500, Pakistan.
² Institute of Pathology and Diagnostic Medicine, Khyber Medical University, Peshawar, 25000, Pakistan.
³ College of Animal Science and Technology, Anhui Agricultural University, No. 130 Changjiang West Road, Hefei, 230036, China.
⁴ Department of Botany and Microbiology, College of Science, King Saud University, P.O. Box 2455, Riyadh, 11451, Saudi Arabia.
⁵ Department of Food Industries, Faculty of Agriculture, Damietta University, Damietta, Egypt.
⁶ Department of Plant Production, College of Food & Agriculture Sciences, King Saud University, P.O. Box 2460, Riyadh, 11451, Saudi Arabia.

Abstract

Citrobacter freundii is characterized by AmpC β-lactamases that develop resistance to β-lactam antibiotics. The production of extended-spectrum β-lactamase (ESBL) is substantially high in Escherichia coli, C. freundii, Enterobacter cloacae, and Serratia marcescens, but infrequently explored in C. freundii. The present investigation characterized the ESBL C. freundii and delineated the genes involved in decrease in antibiotics resistance. We used the VITEK-2 system and Analytical Profile Index (API) kit to characterize and identify the Citrobacter isolates. The mRNA level of AmpC and AmpR was determined by RT-qPCR, and gel-shift assay was performed to evaluate protein-DNA binding. Here, a total of 26 Citrobacter strains were isolated from COVID-19 patients that showed varying degrees of antibiotic resistance. We examined and characterized the multidrug resistant C. freundii that showed ESBL production. The RT-qPCR analysis revealed that the AmpC mRNA expression is significantly high followed by a high level of AmpR. We sequenced the AmpC and AmpR genes that revealed the AmpR has four novel mutations in comparison to the reference genome namely; Thr64Ile, Arg86Ser, Asp135Val, and Ile183Leu while AmpC remained intact. The ΔAmpR mutant analysis revealed that the AmpR positively regulates oxidative stress response and decreases β-lactam and aminoglycosides resistance. The AmpC and AmpR high expression was associated with resistance to tazobactam, ampicillin, gentamicin, nitrofurantoin, and cephalosporins whereas AmpR deletion reduced β-lactam and aminoglycosides resistance. We conclude that AmpR is a positive regulator of AmpC that stimulates β-lactamases which inactivate multiple antibiotics.

Keywords: Aminoglycosides resistance; AmpC; AmpR mutation; C. freundii; Oxidative stress; β-lactamases.