Molecular basis of metronidazole resistance

Molecular basis of
metronidazole resistance
in pathogenic bacteria and
protozoa
Kirkwood M. Land, Patricia J. Johnson

Department of Microbiology and Immunology, UCLA School of Medicine, Los

Angeles, CA 90095-1489, USA

Abstract The molecular basis of metronidazole resistance has

been examined in anaerobic bacteria, such as Bacteroides,
Clostridium, and Helicobacter, and anaerobic parasitic protists
such as Giardia, Entamoeba, and trichomonads.A variety of
enzymatic and cellular alterations have been shown to
correlate with metronidazole susceptibility in these pathogens;
however, a common theme has been revealed. Resistant cells
are typically deficient in drug activation.The frequent
correlation between metronidazole resistance and ineffective
drug activation suggests that drug resistance is the result of
modification of proteins involved in drug activation.
Fig. 1 Metronidazole is administered as an inactive prodrug. It
is activated to its cytotoxic form via the transfer of an
electron to the nitro group of the drug, which converts it to a
INTRODUCTION nitroso free radical form.The donor of electrons varies
depending on the organism.
etronidazole (commonly known as Flagyl) is a

M 5-nitroimidazole compound used to treat a wide

variety of infections caused by pathogenic bacteria
and protists, such as Helicobacter, Clostridium, Bacteroides
leads to further uptake of the inactive form.Thus, the rate of
intracellular activation is important as it affects drug uptake
(bacteria), Trichomonas, Entamoeba, and Giardia via a concentration gradient.
(human-infective parasitic protists) and Tritrichomonas In this review article, we present an overview of our
(a cattle-infective parasitic protist). Metronidazole is adminis- current understanding of the mechanisms underlying
tered in an inactive form and becomes activated in either the metronidazole resistance in several bacterial and protozoal
cytoplasm (in the case of infections caused by bacteria and pathogens (Table 1).
Entamoeba and Giardia) or in a specialized organelle called
the hydrogenosome (in Trichomonas and Tritrichomonas).
METRONIDAZOLE RESISTANCE IN BACTERIA
Clinical resistant strains of each of these pathogens have
been described, but little is known about the molecular basis It has long been recognized that metronidazole is an effec-
for metronidazole resistance. tive antimicrobial against many types of anaerobic bacterial
pathogens, including Clostridium and Bacteroides. In recent
years, it has been observed that this drug is also effective
SELECTIVE ACTIVATION OF METRONIDAZOLE TO
against a variety of microaerophilic bacteria such as
FREE-RADICAL, CYTOTOXIC FORMS
Helicobacter pylori and Campylobacter spp.. With its
The cytotoxic selectivity of metronidazole results from the increased use against microaerophiles, resistant isolates of
necessity of the drug to be reduced to an active cytotoxic these different bacteria have emerged.
form. The nitro group of metronidazole is reduced via
metabolic pathways of low redox potential. The absence of Bacteroides and Clostridium
electron-transport proteins with sufficiently negative redox Genes implicated in 5-nitroimidazole resistance in
potential to allow drug activation renders the drug ineffec- Bacteroides have been isolated from several species, includ-
tive in aerobic cells. This selective cytotoxicity of metro- ing B. vulgatus and B. fragilis.3–5 Most of these genes, called
nidazole relies on biochemical properties of these pathogens nim, are found on low-copy number plasmids, although
that are lacking in the aerobic cells of the eukaryotic hosts one gene, nimB from B. fragilis, has been mapped to the
that harbor them. chromosome. The transfer of nim genes to susceptible
Metronidazole is administered in an inactive form and recipients has been shown to increase metronidazole resis-
enters the cell by passive diffusion (Fig. 1).1,2 In anaerobic tance3–5 and these loci are currently described as undefined
cells, conversion of the drug from inactive to active form 5-nitroimidazole resistance determinants of unknown origin.

 1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 289–294 289
 Land and Johnson
Table 1 Summary of mutations/alterations observed in metronidazole resistant cells

Organism Proposed mechanism of resistance

Anaerobic and microaerophilic bacteria

Bacteroides spp. Inactivation of metronidazole by products of nim gene locus?
(Trinh et al. 1995;Trinh and Reysset 1996)
Clostridium spp. Unknown: alterations in flavodoxins or hydrogenase?
(Church et al. 1988, 1990; Knight et al. 1967; Santangelo et al. 1991)
Helicobacter pylori Inactivation of rdxA: reduced or no drug activation in resistant cells
Reduced activities of pyruvate:flavodoxin oxidoreductase and α-ketoglutarate reductase?
(Hoffman et al. 1996; Goodwin et al. 1998)
Anaerobic parasitic protists
Giardia lamblia Reduced or altered pyruvate:ferredoxin oxidoreductase (PFO) and/or ferredoxin (Fd)?
(Townson et al. 1994, 1996; Upcroft and Upcroft 1999)
Entamoeba histolytica No change or increase in PFO levels and activity?
Increased superoxide dismutase activity:specific or generalized stress response?
Decreased ferredoxin I and flavin reductase?
(Samarawickrema et al. 1997;Wassmann et al. 1999)
Trichomonas vaginalis Deficiency of hydrogenosomal ferredoxin, a protein needed for drug activation?
Ineffective oxygen scavening: competition with oxygen for electrons needed for activation?
(Quon et al. 1992;Yarlett et al. 1986; Kulda et al. 1993; Brown et al. 1999)
Tritrichomonas foetus Deficiencies in hydrogenosomal proteins involved in drug activation and organellar function?
(Cerkasovova et al. 1984)

The function of nim genes is unknown. Haggoud and of strains from the US and Western Europe.13
colleagues6 examined the hydrophobicity profiles of the Proposed mechanisms of resistance in H. pylori include:
putative nim gene products; no predictable membrane- (1) decreased drug uptake or active efflux; (2) deficiency in
spanning region was identified, as one might expect if the metronidazole activation or modification; (3) target modifica-
gene product served as a membrane transporter. Recently, it tion or loss; and (4) and increased DNA repair or oxygen
has been suggested that nim genes may encode a 5-nitroimi- scavenging capabilities.14 The inactivation of recA, a gene
dazole reductase that could convert 5-nitroimidazoles to a needed for generalized DNA repair and recombination,
non-toxic amino derivative, eliminating its cytotoxic effects.7 enhanced the susceptibility of H. pylori to metronidazole.15
The relevance of nim genes in metronidazole susceptibility Expression of a cloned recA gene from a metronidazole
in Bacteroides requires further studies. resistant strain seemed to increase the level of resistance in
Studies on metronidazole resistance in Clostridium have E. coli.16
provided biochemical evidence that the bidirectional hydro- Early biochemical studies on resistant isolates focused
genase (hydrogenase 1) of C. pasteuranium plays a critical on the metabolic enzymes of H. pylori; specifically on
enzymatic role in the reduction of metronidazole via a ferre- pyruvate:ferredoxin/flavodoxin oxidoreductase (POR) and
doxin-linked mechanism.8,9 Genes controlling the activation α-ketoglutarate oxidoreductase (KOR).14 The premise for this
of metronidazole in C. acetobutylicum have also been work was based on studies in anaerobic protists which
identified. Among the genes shown to confer high levels of showed that POR activity was decreased in resistant strains
sensitivity to metronidazole were those encoding a flavo- (see below). Studies by Hoffman and colleagues14 demon-
doxin and a hydrogenase.10 These proteins could serve as strated that in H. pylori POR and KOR activities in resistant
electron donors to metronidazole for drug activation. Along strains were repressed in cells grown in the presence of the
these lines, flavodoxins from Clostridium have been shown drug, but not in the absence of the drug. These findings
to be capable of replacing ferredoxins as electron carriers.10 suggested a model where metronidazole could regulate the
activity of the metabolic enzymes POR and KOR in H. pylori;
Helicobacter pylori the presence of the drug would down-regulate the activity of
Helicobacter pylori is a spiral gram-negative bacterium that the enzymes, and vice versa. Whether this regulatory effect
was first isolated in 1982 from the gastric mucosa of a on enzyme activity by metronidazole was the primary cause
patient inflicted with gastritis and peptic ulceration.11 Since of resistance or was a secondary manifestation of another
then, there has been increasing evidence that H. pylori plays mutation was not demonstrated.
an important role in duodenal ulceration.12 Metronidazole Recently, work by Hoffman and colleagues17 showed that
has been used in multiple antimicrobial therapeutic strate- inactivation of an H. pylori gene encoding an oxygen-
gies to eradicate H. pylori. Interest in metronidazole insensitive NADPH nitroreductase, called rdxA (designated
resistance in H. pylori has increased with the emergence of HP0954 in the entire genome sequence),18 is the cause of
resistant isolates in developing countries and some 10–30% naturally acquired metronidazole resistance in H. pylori.The

290 Drug Resistance Updates (1999) 2, 289–294  1999 Harcourt Publishers Ltd
 Molecular basis of metronidazole resistance
strategy used to identify the metronidazole-resistance deter- The purified Giardia ferredoxin has likewise been shown to
minant in H. pylori extended from an earlier observation that be capable of activating metronidazole in vitro.22 Given the
H. pylori were naturally competent for uptake of chromo- role of ferredoxin and PFO in electron transport pathways in
somal DNA, and that DNA from resistant clinical strains could Giardia, an analysis of their expression or activities in drug
transform sensitive strains to resistant ones.19 resistant strains should lend insight into the molecular basis
It was not clear whether the resistance determinant was for resistance.Those readers interested in an update on pro-
due to mutations in the genome or the acquisition of an addi- posed mechanisms of drug resistance in Giardia are encour-
tional gene.To overcome this problem, a cosmid library was aged to consult a review article by Upcroft in this journal.24
first constructed with genomic DNA from a resistant strain; Metronidazole resistance in E. histolytica has been
cosmid DNAs were then tested for their ability to transform described, but the molecular basis of resistance is not clear
metronidazole-sensitive H.pylori to a metronidazole-resistant and has not been studied extensively. Research on drug resis-
phenotype. Open reading frames identified included a gene tance in E. histolytica has been primarily centered on study-
with protein-level homology to classical oxygen-insensitive ing emetine resistance, a drug that is unrelated to the
NADPH nitroreductases of several other Gram-negative bac- nitroimidazoles. Emetine resistance appears to be modulated
teria.17 PCR amplification and sequencing of the correspond- by expression of P-glycoproteins, transmembrane proteins
ing segment from the metronidazole sensitive strain revealed that are postulated to pump the drug out of the cell.
an ORF that was 14 codons longer at the 3′ end. This indi- Overexpression of the E. histolytica P-glycoprotein (Eh
cates that metronidazole resistance in H. pylori is caused by pgp1) gene confers emetine resistance.25 Other P-glycopro-
inactivation of rdxA, which in the resistant strain examined tein genes have been cloned from E. histolytica (Eh pgp5
occurred by a nonsense mutation that resulted in a truncated and Eh pgp6) and all three Pgp genes have been shown to be
rdxA protein. overexpressed in the resistant strain C2.26 Readers interested
To further explore the possibility that alterations in rdxA in mechanisms of emetine resistance in E. histolytica are
could confer resistance to metronidazole, rdxA was encouraged to consult a review article by Orozco and
expressed in E. coli, which is known to be metronidazole colleagues.27
resistant.The expression of rdxA in E. coli rendered the cells With regard to metronidazole resistance, both Fd28 and
susceptible to metronidazole. Moreover, allelic exchange PFO29 genes have been cloned from E. histolytica. Like
mutagenesis and complementation demonstrated that rdxA Giardia, E. histolytica does not possess hydrogenosomes
inactivation is sufficient for metronidazole resistance.17 and the activation of metronidazole occurs in the cytoplasm,
Interestingly, it was observed that growth of rdxA mutants in where the electron-transfer components PFO and Fd are
metronidazole containing medium also resulted in the dis- located.The possible role of these proteins, as well as others
appearance of POR activity. Furthermore, during growth in of unrelated function, in development of metronidazole
metronidazole-free medium, this mutant strain exhibited resistant strains of E. histolytica is unclear. In light of this,
only half as much POR activity as its parental strain. This Upcroft and colleagues30 generated a metronidazole-resistant
suggests that mutations in rdxA may indirectly affect the strain of E. histolytica by prolonged exposure of a suscepti-
level of POR activity. This study establishes that null muta- ble strain to metronidazole in order to examine changes
tions in just a single gene, rdxA, are responsible for metron- which occur in the development of resistance. Using these
idazole resistance in H. pylori. isogenic strains, PFO levels and activity were assayed in the
resistant and susceptible strains. Using immunofluorescence
and immunoblot analysis, it appeared that the levels of PFO
METRONIDAZOLE RESISTANCE IN PARASITIC
were either the same between the two strains, or slightly
PROTISTS
increased in the resistant cells. Spectrophotometric analysis
In addition to treating bacterial pathogens, metronidazole is of PFO activities in resistant and susceptible cells revealed an
used to treat infections with anaerobic protozoan parasites, increased activity in resistant cells. Parallel to these experi-
including Trichomonas, Tritrichomonas, Giardia and ments, the activity of iron superoxide dismutase (FeSOD)
Entamoeba. was analyzed in both strains; and interestingly, there was an
increase in FeSOD activity in the resistant cells. Furthermore,
Giardia lamblia and Entamoeba histolytica another recent study strengthens the potential role of SOD
Treatment-refractory cases of patients with persistent in metronidazole resistant E. histolytica.31 In this study,
giardiasis have been documented20 and these resistant metronidazole resistant cells were shown to overexpress
isolates, when examined at the biochemical level, appear to FeSOD and have decreased expression of ferredoxin I and
have defective pyruvate:ferredoxin oxidoreductase (PFO) flavin reductase. Using transfection techniques, overexpres-
activity. The components of the electron transport pathway sion of FeSOD and peroxiredoxin in sensitive cells gave rise
involved in metronidazole activation have not yet been to cells with increased resistance to metronidazole, demon-
completely defined in this parasite, but two of the crucial strating a direct role for these proteins in metronidazole
components, PFO and ferredoxin (Fd), have been puri- susceptibility.31
fied.21–23 Giardia PFO, one of two major cytosolic 2-oxoacid
oxidoreductases, transfers electrons to Giardia ferredoxin Trichomonas vaginalis and Tritrichomonas foetus
(Fd 1) with simultaneous reduction of metronidazole.23 Trichomonas vaginalis is a flagellated human parasitic
Ferredoxins from C. pasteurianum and T. foetus are also able protist which infects the urogenital tract. Resistant strains
to act as acceptors of electrons from purified Giardia PFO. isolated from patients who were refractory to treatment

 1999 Harcourt Publishers Ltd Drug Resistance Updates (1999) 2, 289–294 291
 Land and Johnson
with metronidazole have been described and catalogued (Tvpgp) was found to be present in two copies in all exam-
with the ATCC.32 ined drug sensitive T. vaginalis strains. Upon examination of
Drug activation in trichomonad parasites is thought to seven clinical resistant strains, overexpression of Tvpgp
occur via similar metabolic pathways as previously (2–20 fold) was observed in three strains. Interestingly, in
described for E. histolytica and G. lamblia, with the excep- four of the seven drug resistant strains studied, only one
tion that these biochemical pathways are compartmental- copy of Tvpgp was present. No clear correlation between
ized into a specialized organelle called the hydrogenosome. levels of Tvpgp expression and gene copy number was
This unusual organelle is the site of carbohydrate metabo- observed. For example, two of three strains that overexpress
lism, as well as drug activation.33 Once the drug has entered Tvpgp contained two genes; however, the third strain con-
the organelle by passive diffusion, the process of activation tained only one gene. Likewise, there is no correlation
involves a transfer of a single electron from the hydrogeno- between levels of Tvpgp expression and levels of drug resis-
somal protein pyruvate:ferredoxin oxidoreductase (PFO) tance. It is possible that these genes have point mutations
and ferredoxin (Fd) to the nitro group of metronidazole, which alter the interaction of the encoded protein with the
converting the drug to its cytotoxic form (Fig. 1). PFO and drug. Alternatively, Tvpgp may not interact with metronida-
ferredoxin are part of an electron transfer chain that medi- zole. The lack of a consistent relationship between metron-
ates the oxidation of pyruvate to acetyl-CoA within the idazole resistance and levels of expression of Tvpgp in
hydrogenosome.34,35 For the details of metronidazole activa- T. vaginalis is reminiscent of that observed in other drug-
tion by PFO and ferredoxin, the reader is referred to studies resistant protists.44–49 These observations have led to the
described by Moreno and colleagues36,37 and Yarlett and suggestion that the P-glycoprotein in parasitic protists may
colleagues.38–40 be involved in a general stress response that allows the
Early work on metronidazole resistance in Trichomonas organism to survive until a specific response is mounted.47
vaginalis using resistant clinical isolate CDC085 and suscep- However, it is impossible to test whether this is the case,
tible laboratory strain ATCC30001 suggested two possible as the parental strains which gave rise to these clinically-
hypotheses for resistance: (1) that a defective ferredoxin pro- resistant isolates are not available. For this reason, we and
tein existed in the resistant isolate; or (2) reduced levels of others have turned to the use of isogenic strains, where
the ferredoxin protein were present in CDC085.39 With the resistance was generated in the laboratory to allow one to
cloning of the T. vaginalis ferredoxin gene,41 a detailed examine changes that occur during development of resis-
molecular analysis of the ferredoxin gene and its expression tance and to make comparisons with the drug sensitive
in sensitive and resistant cells was carried out. Quon et al.42 parental strain.
were able to demonstrate a strict correlation between Isogenic metronidazole-resistant strains of T. foetus and
altered transcription of the ferredoxin gene and drug resis- T. vaginalis have been generated in the laboratory of Kulda
tance in four clinically-resistant strains (including CDC085) and colleagues by continuous challenge of a drug sensitive
when compared to two drug sensitive isolates. Detailed mol- strain (KV1) with metronidazole over a long period of time
ecular analysis revealed that clinically resistant strains of T. until stable resistance lines were present (MR-100) which
vaginalis have decreased levels of the ferredoxin protein could grow continuously on high levels of metronida-
and its mRNA.The reduced levels of protein and mRNA were zole.50,51 The slow development of cells with increasing
attributed to reduced transcription of the ferredoxin gene as levels of stable resistance over time suggests that there is a
determined by nuclear run-on assays.42 These findings multi-step process involved in the acquisition of the stable
strongly correlate metronidazole resistance with altered resistant phenotype (cells with intermediate levels of resis-
regulation of ferredoxin gene transcription in clinically tance are unstable). The eventual loss of hydrogenosomal
resistant strains of T. vaginalis. This reduction in gene activity in highly resistant cells would intuitively involve
transcription results in decreased intracellular levels of multiple mutations.
ferredoxin, and this, in turn, may limit the ability of the cell to Detailed biochemical analyses of these resistant cells
activate metronidazole. However, whether ferredoxin plays a show ablated enzymatic activities of PFO and another
direct role in drug susceptibility is unknown. Mutations in hydrogenosomal protein, hydrogenase (HYD), the enzyme
other cellular components could possibly have a ‘down- which catalyzes formation of molecular hydrogen under
stream effect’ on the expression of ferredoxin and subse- anaerobic conditions.52,53 More recently, using metronidazole
quently affect the susceptibility of the cell to the drug. resistant strains generated in a similar fashion to that of
The molecular mechanisms underlying metronidazole Kulda and coworkers, Brown and colleagues have demon-
resistance in trichomonads appear to involve either deficien- strated a loss of PFO and ferredoxin mRNA in resistant cells.
cies or defects in hydrogenosomal components necessary for Further analysis of these resistant cells demonstrated alterna-
drug activation. However, studies on other parasitic protists tive pathways for utilizing substrates for energy while
(such as Entamoeba) suggest a role for the overexpression circumventing the activation of metronidazole.54
of a P-glycoprotein in conferring resistance to other drugs,
such as emetine, by possibly transporting the drug out of the
A COMMON THEME
cell. Whether such transporter proteins play a role in drug
resistance in T. vaginalis is not clear. In light of this possi- Based on studies of metronidadazole resistance in H. pylori
bility, a P-glycoprotein-like gene related to members of the and trichomonad parasites, it appears that lack of drug
ABC (ATP-binding cassette) family of transporter proteins activation is the primary mechanism of drug resistance in
was cloned and characterized from T. vaginalis.43 This gene these organisms. Alteration of rdxA in H. pylori would

292 Drug Resistance Updates (1999) 2, 289–294  1999 Harcourt Publishers Ltd
 Molecular basis of metronidazole resistance
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