Review Article

Mechanisms of intrinsic resistance in enterococci Alexander Kiruthiga1,2, Kesavaram Padmavathy1*

ABSTRACT

Enterococci are considered as serious nosocomial pathogens as they are likely to exhibit resistance effectively to all meant for clinical use. The most predominant species encountered frequently among human infections includes faecalis and Enterococcus faecium. Antibiotic resistance in enterococci may be either intrinsic or acquired through mutation of the intrinsic genes or horizontal gene transfer of resistance determinants. This paper reviews the mechanisms of intrinsic resistance in enterococci.

KEY WORDS: Enterococcus faecalis, Enterococcus faecium, Enterococcus, Intrinsic resistance

INTRODUCTION species and is not attributed to horizontal gene transfer.[4] The genes encoding intrinsic resistance Among Enterococci, Enterococcus faecalis and may either be expressed constitutively (always Enterococcus faecium are the most often encountered expressed) or induced (expressed only upon antibiotic species in various human infections ranging from exposure).[5] Due to the limited choice of antibiotics uncomplicated urinary tract infection to serious against enterococci, monotherapy with a single class bacteremia. Enterococci are considered as serious of antimicrobial agents often results in poor treatment nosocomial pathogens due to their intrinsic resistance outcomes and is significantly associated with and their potential to acquire resistance to various intrinsic resistance exhibited by them. Enterococci antimicrobial agents.[1] Besides exhibiting natural are proven to be intrinsically resistant to β-lactams, intrinsic resistance to multiple antimicrobial classes aminoglycosides, and sulfonamides.[6] (beta-lactams, aminoglycosides, and glycopeptides), they possess a remarkable ability to acquire resistance Intrinsic resistance in enterococci is found to be mediated to last resort of antibiotics (quinupristin-dalfopristin, by different mechanisms of resistance (Table 1). (1) Low , , and tigecycline) that are or altered permeability, (2) class of modifying used to treat drug resistant enterococci.[2] Hence, enzymes such as ribosome modifying methyl transferase the therapeutic management of serious enterococcal and aminoglycoside modifying enzymes, (3) production infections is a cause of concern.[3] Recent reviews of penicillin binding proteins (PBPs), and (4) ATP- related to enterococcal resistance have mainly focused binding cassette (ABC) efflux pumps.[7] on acquired resistance in enterococci. Hence, this review narrates the different mechanisms of intrinsic INTRINSIC RESISTANCE resistance exhibited by E. faecalis, E. faecium, and TOWARDS VARIOUS CLASSES other clinically relevant Enterococcal species. OF ANTIBIOTICS Intrinsic resistance is an inherent property present β-lactams universally in the genome of a particular bacterial Ampicillin/penicillin

Access this article online Due to low affinity PBPs synthesis and bacterial viability Website: jprsolutions.info ISSN: 0975-7619 have been a major target in antimicrobial therapy

1Department of Microbiology, Research Laboratory for Oral and Systemic Health, Sree Balaji Dental College and Hospital, Bharath Institute of Higher Education and Research, Chennai, Tamil Nadu, India, 2Department of Microbiology, Priyadarshini Dental College and Hospital, Pandur, Tamil Nadu, India

*Corresponding author: Kesavaram Padmavathy, Department of Microbiology, Sree Balaji Dental College and Hospital, Bharath Institute of Higher Education and Research, Velachery Main Road, Chennai - 600 100, Tamil Nadu, India. Phone: 91-9884164212. E-mail: [email protected]

Received on: 09-01-2020; Revised on: 19-02-2020; Accepted on: 15-03-2020

896 Drug Invention Today | Vol 13 • Issue 6 • 2020 Alexander Kiruthiga and Kesavaram Padmavathy ..) Contd. ( [8] [1] [8] [3] [12] [13] [18] [19] [22] [23] [28] [34] [8,9] [10,11] [14,16] [29-32] References Species involved E. faecalis E. faecium E. faecalis E. faecium E. faecium E. faecium E. faecalis E. faecalis E. faecalis E. faecalis E. faecium E. faecium E. gallinarum E. casseliflavus E. flavescens E. faecium E. faecium E. faecalis Confers intrinsic to resistance β-lactam antibiotics Low-level resistance to penicillin β- lactam agents β- lactam agents β-lactam agents Cephalosporins Enhances intrinsic resistance to cephalosporins Low-level aminoglycoside resistance Amikacin, kanamycin, netilmicin, and tobramycin and kanamycin Tobramycin Low-level resistance to but not to teicoplanin Low to high for both vancomycin and teicoplanin Low-level resistance to vancomycin Ofloxacin and ciprofloxacin C4 codes AA AA gene mur gene homolog qnr ) E.faecalis Mechanism of intrinsic resistance (1) Production of β-lactamases, which destroy the β-lactam ring (2) Presence of altered PBPs, which for β-lactams have lower affinity PBP4 PBP5 Altered cell wall (3) (1) PBP-5 cross-linkage of peptidoglycan (2) Regulation of signal transduction pathway (3) Kinase activity of IreK (4) Presence of (1) Low cell wall permeability Aminoglycoside modifying (2) enzymes (3) Ribosome modifying enzymes phenotypes reported till date Van Presence of 9 vanC; vanC1, C2, C3, for intrinsic resistance vanD; vanD1, D2, D3, D4, D5 vanN Presence of (qnr Intrinsic resistance mechanisms of clinically effective antibiotics against enterococci Intrinsic resistance Mode of action Interferes with the synthesis of peptidoglycan by inhibiting penicillin- binding proteins Inhibition of cell wall synthesis of Binds to the 16S rRNA the 30S ribosomal subunit and interferes with protein synthesis Inhibits bacterial growth by interfering with the synthesis of peptidoglycan Inhibition of bacterial topoisomerase enzymes, gyrase and namely DNA topoisomerase IV Name of the antibiotic β – lactams Ampicillin Penicillin Cephalosporins Aminoglycosides Glycopeptides Fluoroquinolones S. No. 1. 2. 3. 4. Table 1: Intrinsic resistance mechanisms in enterococci 1: Intrinsic resistance Table

Drug Invention Today | Vol 13 • Issue 6 • 2020 897 Alexander Kiruthiga and Kesavaram Padmavathy [7] [7] [43] [8, 44] [46,50] [8, 45, 46] References , TMP-SMX: Trimethoprim/sulfamethoxazole TMP-SMX: , species Species involved E. faecalis E. faecalis E. faecium E. faecalis E. avium E. gallinarum E. casseliflavus Enterococcal E. faecalis E. faecium

Confers intrinsic to resistance Quinupristin (streptogramin B) dalfopristin (streptogramin class) Clindamycin A Clindamycin, streptogramin and B A Intrinsic resistance to lincosamides and streptogramins TMP- Intrinsic resistance to SMX in the presence of folic acid Moderate resistance folate synthesis gene gene de novo msrC lsa Mechanism of intrinsic resistance pump ABC efflux (1) (2) Absorption of folic acid without the Reduced uptake and enzymatic inactivation Intrinsic resistance mechanisms of clinically effective antibiotics against enterococci Intrinsic resistance Intrinsic resistance mechanisms of least clinically important antibiotics against enterococci Intrinsic resistance Mode of action Inhibition of protein synthesis by binding to the 50S subunit of ribosome Inhibition of tetrahydrofolate synthesis and dihydropteroate synthetase Inhibition of protein synthesis by hindering both peptide translocation and ribosome disassembly ) Continued Name of the antibiotic Macrolides/lincosamides/ streptogramins Trimethoprim- sulfamethoxazole Fusidic acid Table 1: ( Table S. No. 5. 6. 7. gallinarum casseliflavus, E. gallinarum: Enterococcus avium, E. casseliflavus: Enterococcus faecium, E. avium: Enterococcus faecalis, E. faecium Enterococcus E. faecalis: Enterococcus

898 Drug Invention Today | Vol 13 • Issue 6 • 2020 Alexander Kiruthiga and Kesavaram Padmavathy for decades. Cell wall active β-lactam antibiotics the presence of mur AA and mur AB (homolog such as ampicillin and penicillin inhibit of mur A in E. coli) of which mur AA enhances its peptidoglycan synthesis. PBPs considered essential intrinsic resistance to cephalosporin.[3] for the peptidoglycan synthesis can be divided as Class A (bifunctional enzymes responsible for D, Aminoglycosides D-transpeptidase, and transglycosylase activity) Enterococci exhibit a low-level intrinsic resistance and Class B (contains only transpeptidase domain to aminoglycosides. Nevertheless, the degree of and depends on the transglycosylase activity of resistance varies among various aminoglycosides with other enzymes). Six putative PBP genes have been minimum inhibitory concentrations (MICs) ranging identified among E. faecalis and E. faecium, of which from as low as 4 µg/mL to as high as 256 µg/mL.[14,15] three genes (ponA, pbpF, pbpZ) belong to Class A Decreased uptake of aminoglycosides leads to low- and other three genes (pbp5, pbpA, pbpB) belong level aminoglycoside resistance among Enterococcal to Class B.[8] Intrinsic resistance of enterococci to species. Enterococci being a facultative anaerobe, β-lactams is associated with the expression of species- its metabolic pathway interferes with the proteins specific low-affinity PBPs generally, PBP5 (in E. involved in electron transport and hence limits the faecium) and PBP4 (in E. faecalis) that bind weakly to uptake of aminoglycosides and thereby contributes beta-lactam antibiotics.[1,8] to intrinsic low level aminoglycoside resistance.[14,16] Nevertheless, a combination of aminoglycosides with β-lactamase-mediated resistance a cell wall active agent significantly enhances the Another mechanism of ampicillin resistance uptake of aminoglycosides in enterococci.[17] E. faecium described in E. faecalis and E. faecium is mediated predominantly produces an enzyme 6’acetyl transferase- by β-lactamase enzyme that cleaves the β-lactam ring, acetylase [AAC’6’-Ii] which renders intrinsic thereby inactivates the antibiotic.[8] resistance to amikacin, kanamycin, netilmicin, and tobramycin.[18] Other additional intrinsic mechanisms of L, D-transpeptidase-mediated resistance in E. faecium aminoglycoside resistance among enterococci include Resistance to β-lactam antibiotics is also found to be ribosomal target modification conferring resistance to mediated by an enzyme called L,D-transpeptidase kanamycin and tobramycin.[19] enzyme (Ldtfm), especially in E. faecium. However, L,D-transpeptidase is distinct from the penicillin- Glycopeptides sensitive DD-transpeptidase.[8,9] Glycopeptides, vancomycin, and teicoplanin are often used for the treatment of infections caused by Gram- Bacteriostatic effect is exhibited by Enterococcal positive that exhibit resistance to β-lactams.[20] species as they exhibit low intrinsic resistance to cell Thirty years after the introduction of vancomycin in wall active agents such as β-lactam antibiotics and clinical practice, vancomycin-resistant enterococci glycopeptides. The species of genus Enterococcus was first reported in 1986.[21] Since then, dissemination usually show a low intrinsic resistance to β-lactam of glycopeptide resistance in enterococci has become antibiotics such as penicillin, ampicillin, piperacillin, a serious cause of concern in the nosocomial and imipenem, which exert on them a bacteriostatic settings.[20] Glycopeptide resistance can either be effect. Bacteriostatic effect is insufficient to treat intrinsic or acquired and enterococcal strains may serious enterococcal infections, and in turn, they exhibit resistance to both vancomycin and teicoplanin require a synergistic bactericidal activity. These or to vancomycin alone. Glycopeptide resistance can antibiotics, like the glycopeptides, have bacteriostatic be intrinsic or acquired and strains may be resistant to activity against these microorganisms, a reason why a vancomycin and teicoplanin or to vancomycin only.[20] synergistic bactericidal association is required in case Nine types of vancomycin resistance, namely, vanA, of serious infections as endocarditis or .[9] vanB, van C, vanD, vanE, van G, van L, van M, and van N have been reported in enterococci till date.[22] Cephalosporins The most common being, VanA and VanB phenotypes Mechanism of intrinsic resistance to cephalosporins frequently reported among the clinical isolates of is found to be determined by triad of factors.[3] (1) E. faecalis and E. faecium.[22] VanA phenotype is PBP5 – that catalyzes the cross-linkage of peptidoglycan characterized by inducible high-level resistance in the presence of cephalosporins,[10,11] (2) regulation to both vancomycin and teicoplanin; nevertheless, of CroRS, a two component signal transduction VanB phenotypes exhibit variable levels of inducible pathway that regulates the expression of the gene(s) resistance to vancomycin but are susceptible to which augments resistance to cephalosporins,[12] and teicoplanin. Other acquired glycopeptide resistance (3) IreK (formerly PrkC), a Ser/Thr Kinase whose types include, VanD,[23] VanE,[20,24] van G,[25] van L,[26] activity is critical for cephalosporin resistance.[13] In van M,[27] and van N,[28] exhibit variable levels of addition, genomic analysis of E. faecalis had proven resistance to vancomycin and teicoplanin, and VanE

Drug Invention Today | Vol 13 • Issue 6 • 2020 899 Alexander Kiruthiga and Kesavaram Padmavathy phenotype is characterized by low level resistance to possess poor/moderate antibacterial activity against vancomycin and susceptibility to teicoplanin. enterococci with higher MIC range (E. faecalis ATCC 29212, MIC: 2–4 μg/ml) compared to Gram- Of the van types known, the intrinsic resistance negative (Escherichia coli ATCC 25922, MIC: to low-level vancomycin is exhibited by vanC 0.03–0.06 μg/ml).[34,41] phenotype characteristic of Enterococcus gallinarum (vanC1 genotype), Enterococcus casseliflavus (vanC Macrolides/Lincosamides/Streptogramins [29,30] 2-4 genotype), and Enterococcus flavescens. Increasing reports of resistance to macrolides and VanC enterococci possess two distinct pathways lincosamides among clinical isolates of Gram-positive [30] for the PG precursor synthesis, one that produces bacteria are being documented in the recent years.[42] PG precursors with D-Ala–D-Ala termini, and the This may be attributed to (a) ribosomal modification, other produces PG precursors with D-alanyl–D- (b) efflux of the antibiotic, and (c) drug inactivation.[42] serine termini. vanC gene clusters mediate the latter E. faecalis exhibits intrinsic resistance to quinupristin pathway that confers poor binding of vancomycin (streptogramin B class),[43] dalfopristin (streptogramin and hence confer intrinsic vancomycin resistance of A class), and clindamycin (a lincosamide).[7] Compared E. gallinarum and E. casseliflavus.[31] Two enzymes to other Enterococcal species, higher macrolide VanX and VanY are involved in the hydrolysis of PG resistance in E. faecium is due to a species – specific precursors with D-Ala termini that are synthesized intrinsic gene msrC associated with ABC transport by the host. VanX (VanX ), a D,D- dipeptidase B efflux.[44] Broad-spectrum resistance to macrolides hydrolyzes D- Ala-D- Ala, while VanY (VanY ) a B and lincosamides is conferred by the ribosomal D,D-carboxypeptidase hydrolyzes the terminal D- Ala modification while antibiotic efflux as well inactivation residue of late PG precursors. In VanC resistant of the drug can have reduced effect. Modification phenotypes, both the above activities (performed by of the ribosomal target confers broad-spectrum VanX and VanY) are encoded by a single gene VanXY . C resistance to macrolides and lincosamides, whereas VanXY binds to zinc, thereby stabilizes the binding C efflux and inactivation affect only some of these of substrate and catalyzes the hydrolysis of the D-Ala molecules.[42] Resistance to these drugs is conferred termini of the PG precursor, D-Ala-D-Ala dipeptide, and late PG precursor, UDP MurNAc-pentapeptide. by the expression of an intrinsic gene, lsa which is structurally related to ABC-efflux pumps, thereby VanXYC exhibits lower dipeptidase activity against [7] D-alanyl–D-serine (unlike VanX) and no activity suggesting drug efflux mechanism. lsa encoding a against UDP MurNAc-pentapeptide (D-ser) (unlike drug efflux pump is reported among clinically relevant VanY).[31,32] Enterococcal species, E. faecalis, Enterococcus avium, E. gallinarum, and E. casseliflavus conferring Fluoroquinolones intrinsically resistance to lincosamides and [45,46] Quinolones exhibit a unique mode of action by streptogramins. inhibiting DNA gyrase and DNA IV. Quinolone Trimethoprim-sulfamethoxazole resistance in enterococci may be either low level – intrinsic or high level – acquired.[33] The Trimethoprim-sulfamethoxazole inhibits two primary target for fluoroquinolones varies for Gram- successive steps in the tetrahydrofolate synthesis positive and Gram-negative bacteria, Topoisomerase pathway; the former inhibits dihydrofolate IV Gram-positive bacteria and DNA gyrase for Gram- reductase, while the latter inhibits dihydropteroate negative bacteria.[34,35] Fluoroquinolone resistance synthetase. Hence, trimethoprim-sulfamethoxazole in Gram-positive bacteria is largely attributed to the act synergistically inhibiting folate synthesis following mechanisms: (1) Mutations in the quinolone and exhibit a broad spectrum of activity against resistance determining region (QRDR) present with susceptible bacteria. In vitro testing of enterococcal the target genes gyrA and parC (frequently described susceptibility to trimethoprim-sulfamethoxazole in in E. faecalis and E. faecium while not indicated in a media devoid of folate may yield a susceptible E. gallinarum and E. casseliflavus) that alter the result.[7] In vitro susceptibility results of trimethoprim- binding affinity of the antibiotic,[33,36,37] (2) efflux pump sulfamethoxazole do not correlate well with its mediated externalization of the fluoroquinolones, efficacy in vivo.[47] Although enterococci appear to be exemplified by NorA in E. faecium[38] and emeA susceptible to trimethoprim-sulfamethoxazole in vitro, in E. faecalis,[39] (3) qnr mediated resistance in they are reported to be ineffective in the management E. faecalis is due to reduced binding of the antibiotic of serious enterococcal infections.[48,49] Synthesis of to DNA, thereby protecting DNA gyrase and hence folic acid through de novo pathway is a prerequisite the subsequent formation of Quinolone-DNA for nucleic acid production in bacteria, as many gyrase complex described.[34,40] qnr like homolog is bacteria could not absorb folate from the surrounding documented to play a role in the intrinsic resistance environment.[7] Nevertheless, enterococci can absorb of E. faecalis to fluoroquinolones. Fluoroquinolones folic acid, thereby escape the action of trimethoprim-

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