The incidence of Clostridium difficile infection has been elevated and becoming common in hospitals worldwide. Although antibiotics usually serve as the primary treatment for bacterial infection including C. difficile infection, limitations and failures have been evident due to drug resistance. Antibiotic resistance in C. difficile has been recognized as one of the most important factors to promote the infection and increase the level of severity and the recurrence rate. Several outbreaks in many countries have been linked to the emergence of hypervirulent drug-resistant strains. This pathogen harbours various mechanisms against the actions of antibiotics. The present study highlights three main drug-resistant strategies in C. difficile including drug inactivation, target modification and efflux pump. Other mechanisms that potentially contribute to drug-resistant traits in this organism are also discussed.
Clostridium difficile is the leading cause of nosocomial diarrhoea ranging from mild to life-threatening cases and has been attributed to approximately 20% of an antibiotic-related diarrhoea [1–3]. It has been suggested that C. difficile spreads and enters into a host body via the faecal–oral route, both vegetative cells and spores are ingested and passed to the stomach. Under low pH conditions in the stomach, only spores can survive and are able to germinate upon exposure to bile salts in the small intestinal passage. During prolonged antibiotic treatment, normal gut flora is decimated, thereby providing a niche for the drug-resistant C. difficile that prior resided in a body to overgrow and eventually outcompete commensal bacteria. The pathogenesis of C. difficile infection (CDI) is mainly developed through the production of toxins A and B. These toxins disrupt a tight junction and the cytoskeleton of colonic epithelial cells, leading to cell apoptosis [4,5]. An increase in vascular permeability allows the migration of neutrophils and monocytes to the damaged area, subsequently causing an inflammation and the formation of pseudomembrane. During the last decade, several outbreaks have been documented in North America, Europe, Oceania and South Africa, which are mostly linked to the hypervirulent drug-resistant strain, BI/NAP1/027 [6–10], whereas the prevalence of multidrug-resistant C. difficile PCR ribotypes 017 and 018 appears to be more dominant in Asia . Apart from the common prevalent ribotypes, other ribotypes including 053 and 078 have also been associated with antibiotic resistance .
This association strongly reflects the impact of antibiotic resistance towards the epidemiology and virulence of C. difficile. The present study provides an update on information regarding to the resistance mechanisms in C. difficile.
Drug resistance in
The drug resistance in C. difficile has worsen due to the inappropriate use of antibiotics as well as bacterial adaptations that apparently drive the evolution for resistance (Figure 1). Although antimicrobial agents are recommended in the practical guideline for the treatment of CDI, certain broad-spectrum antibiotics including cephalosporins, clindamycin, fluoroquinolones (FQs) and tetracycline (TET) are considered as high-risk agents that adversely induce the infection and recurrence of the disease [13,14]. Metronidazole (MTZ) and vancomycin have been recommended as the standard clinical procedure for CDI treatment over 30 years; however, resistance and reduction in antibiotic susceptibility in this organism have continuously been reported, rendering it more challenging in treatment options . Several antibiotics including fidaxomicin, fusidic acid (FDA) and rifamycins (RIFs) have alternatively been introduced to combat CDI, but the resistance to these agents during or following therapy has also been documented [16–18]. In the past few years, the emergence of hypervirulent strains, high recurrence rate and increasing level of severity of CDI have been associated with the drug resistance, therefore the knowledge on resistance mechanisms is critically required.
Timeline of the discovery of antibiotics in parallel with the emergence of resistant
Unlike other bacterial species, C. difficile harbours very low genome conservation among strains [19,20], making this pathogen owns a powerful adaptability to modulate a cellular pathway in response to certain conditions such as a host infection and an exposure to antibiotic or oxygen [21–25]. Among these, the alteration in drug susceptibility is mostly accompanied with the acquisition of mobile genetic elements (MGEs) [26,27]. More than 10% of C. difficile genome are MGEs , including IS elements, introns, bacteriophages and conjugative and mobile transposons, thereby providing genomic flexibility to C. difficile. MGEs have been associated with the virulence, pathogenicity and particularly on antibiotic resistance in C. difficile . For instance, the presence of conjugative transposon Tn5398 and Tn6194 in C. difficile confers resistance to macrolide, lincomycin and streptogramin B (MLSB). Tn4453a and Tn4453b harboured by C. difficile strain W1 have been linked to chloramphenicol (CHL) resistance . Hence, the acquisition of MGEs gives rise to the biological diversity of C. difficile and potentially associates with the development of drug resistance in C. difficile. Three main drug-resistant strategies in C. difficile include drug inactivation, target modification and efflux pump (Figure 2) and are discussed in detail as follows.
Overview of drug resistance mechanisms in
Bacteria have the common ability to inactivate drugs that enter a cell either by degrading or modifying them into a non-functional form. Similarly, C. difficile owns the typical fashion of drug inactivation by both enzymatic degradation and modification.
β-lactamases are the enzymes capable of hydrolysing β-lactam (BLT) ring present in the core structure of antibiotics belonging to the class of BLT . The genome of C. difficile strain 630 contains certain number of genes, which potentially encode for putative beta-lactamases (CD0344, CD0458, CD0464, CD0527, CD0655, CD0829, CD1802, CD2742 and CD3651), and they share significant identities among C. difficile strains [19,20,28]. Hence, drug degradation mediated by β-lactamases is proposed to play an important role to combat against BLT antibiotics in C. difficile. Although most strains are resistant to these antibiotics, the degree of resistance is varied among strains .
Antibiotic-modifying enzymes in C. difficile have been linked to CHL resistance [14,27,33]. CHL inhibits bacterial growth through the binding at the A2451 and A2452 residues of a 50S ribosomal subunit. This attachment blocks the binding of tRNA to the P-site of a large ribosomal subunit, consequently prevents the elongation of polypeptide chain . The inactivation of drug undergoes by the addition of side chain that generates a steric hindrance effect, which in turn disrupts the target-binding affinity. Two copies of catD gene encoding for CHL acetyltransferase locate at the mobile regions Tn4453a and Tn4453b of C. difficile . CHL acetyltransferase catalyses the relocation of acetyl group from acetyl-CoA to CHL, resulting in 3-O-acetyl CHL, which cannot bind to a ribosome and loses its antimicrobial action.
Prevention of antibiotic activation and detoxification
MTZ, an antibiotic in the class of nitroimidazole, is active against various anaerobic pathogens and has been recommended as the first line therapy for CDI. MTZ translocates into a bacterial cell in the form of prodrug, and requires cellular reduction to become active. MTZ radical (active) non-specifically binds to bacterial DNA, causing the strand breakage and subsequent cell death . The evidence on increase in resistance to MTZ in C. difficile has been reported in the last decade . The mechanism of resistance has been recently uncovered from genomic and proteomic analyses to associate with multiple factors [37,38].
The decrement of redox proteins including pyruvate:ferredoxin oxidoreductase (OR), pyruvate:flavodoxin OR, thioredoxin and thioredoxin reductase are thought to be responsible for MTZ resistance as they are required for drug activation . As iron is the vital component in many redox proteins, the disruption of cellular iron homoeostasis has also been mentioned to decrease the drug susceptibility. The reduction in ferric uptake, resulting either from the point mutation or alteration of expression of molecules involved in cellular iron transport has been recognized as another factor to induce MTZ resistance [36,39]. C. difficile can bypass the drug activation pathway through another catalytic reaction manipulated by putative 5-nitroimidazole reductase, NimB homologue. The homologue catalyses the conversion of prodrug into a non-toxic compound, aminoimidazole, instead of an active form. The increase up to 3-fold of NimB homologue in MTZ-resistant strain relative to a wild-type strain has been demonstrated . A reactive free radical as a consequence of active MTZ could be removed via free radical scavenging pathways. The genotypic characterization of strain conferring resistance to MTZ shows mutation in the rsbW gene encoding for serine protein kinase, the negative regulator of sigma-B. It is possible that the null mutation of rsbW gene may enhance the ability of cell to detoxify oxidative stress ascribed to the drug .
Certain drugs cannot be simply removed or inactivated, therefore the modification of drug target often represents the defensive mechanism for bacteria. Regarding to the target modification, three major mechanisms including target methylation, mutation and protection have been described in C. difficile. All of these modifications result in either complete interruption or the reduction of binding affinity between drug and target.
The cellular methylation in C. difficile has been proposed to induce resistance to macrolides (erythromycin, ERY), lincosamide (clindamycin) and streptogramin B antibiotic family [40,41]. These drugs target at a bacterial 50S ribosomal subunit, causing the inhibition of peptide chain growth by blocking the movement of ribosome . ERY ribosomal methylase B (ErmB) is responsible for ribosomal methylation at the specific site of 23S rRNA, resulting in the prevention of antibiotic binding. The genomic analysis of resistant strains revealed the location of ermB to reside on transposable regions, Tn5398, Tn6194 and Tn6215 [28,42]. Furthermore, the methylation process has been linked to the prevention of C. difficile from the action of oxazolidinones (linezolid). Linezolid binds to the peptidyl transferase catalysing region overlapped with the A-site, subsequently leads to the inhibition of protein synthesis owing to the restriction of peptide-bond formation . Linezolid resistance has been associated with the presence of cfr gene, which encodes rRNA methyltransferase . Cfr protein drives the reaction of 8-methyladenosine methylation at A2503 within the 23S rRNA of ribosomal large subunit, thereby disrupting an interaction between drug and target.
Taking into account the high adaptability of C. difficile, the genetic alterations in genes directly associated with drug target enable this pathogen to develop the resistance. FQ (moxifloxacin) inhibits bacterial growth through the disruption of enzyme responsible for negative DNA supercoiling, DNA gyrase . Mutations in the gyrA or gyrB gene within quinolone resistance-determining region lead to the reduction in fidelity or prevention of drug binding via the target conformation change. Although several amino acid substitutions have been noted in GyrA and/or GyrB, the most frequent amino acid change has been recognized at T82I in GyrA subunit [44,45].
RIFs (rifampicin and rifaximin) have recently been used as another option for CDI treatment. Nevertheless, the resistance to RIFs in C. difficile has been reported . These drugs target on a DNA-dependent RNA polymerase (RNAP), resulting in the extension of short transcript blockage . Point mutations within the rpoB gene encoding for β-subunit of RNAP cause resistance to RIFs . Among identified amino acid substitutions, the R505K substitution has been mostly evident to promote the high level of resistance . Amino acid alterations were also identified in the fidaxomicin-resistant C. difficile. Like RIF, fidaxomicin targets on RNAP and prevents the formation of RNAP–promoter complex at transcriptional initiation step . Despite both drugs share a common target, the nucleotide substitution within rpoB of fidaxomicin and RIF-resistant strains locate differently. In vitro study has revealed that amino acid substitutions in either rpoB at E1073K, Q1074K and V1143F or rpoC at D273Y confer resistance to fidaxomicin . In addition, a nucleotide deletion at T349 in CD22120 of marR homologue has also been reported to contribute to the resistance as marR encodes the transcriptional repressor for multiple antibiotics-resistant operon .
Genetic mutations have been attributed to FDA resistance in C. difficile. FDA attacks an elongation factor G (EF-G) that involves in the translocation of tRNA in protein synthesis . Both nucleotide deletion and amino acid alteration within the fusA gene, encoding for EF-G, have been found to cause the resistance. With respect to several identified mutations, amino acid changes at the interval between 432 and 459 appeared to significantly boost the resistance in this organism .
Although a resistance mechanism against vancomycin in C. difficile has not been yet fully determined, an in vitro study suggested the possibility of amino acid substitution. Vancomycin inhibits the synthesis of bacterial cell wall through the binding at C-terminal dipeptide D-alanyl-D-alanine  in which the alterations of peptidoglycan precursor probably induce vancomycin resistance . During peptidoglycan biosynthesis, N-acetylglucosaminyltransferase (MurG) converts peptidoglycan precursor from lipid I to lipid II, and this intermediate step is an important process for cell wall maturation. Therefore, the alteration in amino acid sequence within MurG may produce an unusual precursor, which subsequently affects the action of vancomycin.
TETs are commonly used for treatment of bacterial infection as the drug binds to 30S ribosomal subunit and blocks the association of aminoacyl-tRNA at the A-site, ceasing microbial protein synthesis . The emergence of TET resistance has restricted the clinical use of this antibiotic. Generally, bacteria employ three mechanisms of resistance to battle with TET: efflux, ribosome protection and enzymatic inactivation. Of the resistance mechanisms, C. difficile produces ribosomal protection protein that impedes the attachment of the drug to a ribosome. The TetM protein that functions as ribosomal protectant has been identified in TET-resistant C. difficile strains, whereas the presence of other Tet proteins such as Tet(W) and Tet(44) has also been recognized [54,55]. The TetM exhibits homology to EF-G and shares the same binding region in a ribosome. The binding of the TetM protein to a ribosome accompanying with the GTP hydrolysis allows conformational change of the ribosome, resulting in the dissociation of TET from its binding site. Cellular protein synthesis is then recovered through the binding of EF-G after the release of hydrolysed TetM.
Bacterial efflux pumps function in the diverse array of cellular processes such as nutrient uptake, cellular product export and removal of toxic substances. Efflux pumps can be classified into two major classes based on their source of required energy: ATP-binding cassette (ABC) transporters and secondary multidrug transporters . ABC transporters derive energy from ATP hydrolysis, whereas proton motive force provides energy for secondary multidrug transporters for drug extrusion. Secondary multidrug transporters can be further subdivided into four groups: resistance-nodulation-cell division, small multidrug resistance, major facilitator superfamily (MFS) and multidrug and toxic compound extrusion (MATE). In C. difficile, two secondary active transporters belonging to the MFS and MATE families have been reported to be associated with drug resistance. Heterologous expression of the clostridial Cme protein in the MFS subfamily promotes ERY resistance in Enterococcus faecalis . A sodium-dependent efflux pump of the MATE subfamily encoded by the cdeA gene of C. difficile attributes resistance to norfloxacin and ciprofloxacin when the gene was overexpressed in Escherichia coli . To date, although there is no report on ABC multidrug transporters in C. difficile, our recent findings suggested that an ABC transporter could play a role in multidrug resistance. Heterologous expression of the protein in E. coli enhanced antibiotic resistance, and the gene knockout C. difficile strain revealed higher sensitivity to certain antibiotics (Ngernsombat C, Sreesai S, Harnvoravongchai P, Chankhamhaengdecha S, and Janvilisri T, unpublished data).
Antibiotic resistance mechanisms exploited by C. difficile mostly rely on three main strategies including drug inactivation, target modification and efflux pump. C. difficile also exhibits self-protective barrier provided by biofilm formation and sporulation, enhancing the survival under the extreme environmental stresses or exposure to toxic agents.
During chronic infection, bacteria exist predominantly as biofilms, which are communities of bacteria attached to a surface and encapsulated within a polymeric matrix. Biofilm formation in C. difficile has been associated with drug resistance. A previous study in the clinical isolates revealed that C. difficile growing within biofilm exhibited 12-fold higher resistance to vancomycin than planktonic bacteria . Biofilm-cultured isolates also confer 100-fold increase in MTZ resistance compared with those cells growing planktonically . Therefore, the production of biofilm may potentially be one of the strategies manipulated by this pathogen to tackle with antibiotics. It is also noteworthy that the resistance caused by biofilm formation is likely to be strain specific.
Under harsh conditions or limited energy source, C. difficile enters a dormant state by forming resistant endospore. Common characteristics of spore are highly tolerant to both physical and chemical factors. Spore plays an important role in the virulence of C. difficile as it can survive under gastric condition and colonizes in colonic tract. During antibiotic treatment, drug enters the digestive tract and disrupts normal gut flora, whereas the C. difficile spore remains concrete. Once the concentration of antibiotic falls below lethal threshold, spore begins to germinate. Clinical data demonstrate the self-defensive system to antibiotics through spore formation that underlined the failure of vancomycin and MTZ against epidemic C. difficile strains [61,62].
Antibiotics have been extensively used throughout the world as a modern medicine in clinical remedy in order to cure infectious diseases. In parallel, antibiotic-resistant pathogens have been emerging, causing public health concern. Immunological and biological therapies are alternative ways besides antibiotics that have been utilized to prevent and treat CDI. Of these, faecal microbiota transplantation, the treatment of delivering microflora from healthy donor faeces into the intestine of the patient, replenishes host normal flora previously disrupted by antibiotics and diminishes growth of C. difficile . Generally, microorganisms develop defensive mechanisms against antimicrobial agents as well as C. difficile, a major cause of antibiotic-associated diarrhoea. Three main antibiotic resistance mechanisms including drug inactivation, target modification and active efflux pump allow C. difficile to combat against several antibiotics. The identification of individual players in each mechanism and further in-depth investigations would direct us to their biological relevance to the virulence of C. difficile, in particular the resistance to antibiotics. Ultimately, this information may shed light on the potential use of these molecules as targets for better therapeutic approach by overcoming drug resistance in this organism.
C. difficile is a major nosocomial pathogen responsible for antibiotics-associated diarrhoea.
Antibiotics disrupt normal gastrointestinal flora, thereby promoting the growth of antibiotic-resistant C. difficile in the ecological void.
The existence of mobile genetic elements provides genome flexibility, which is considered to induce the development of antibiotic resistance.
Three main mechanisms including drug inactivation, target modification and active efflux pump play a vital role in antibiotic resistance in C. difficile.
Drug inactivation either via enzymatic degradation or modification is the effective method to eliminate the antimicrobial action in this organism. In addition to a single enzymatic reaction, C. difficile is also capable of modulating metabolic pathways to respond to antibiotics.
Structural changes in drug target as a consequence of the genetic mutation, post-translational modification or target protection lead to the prevention of target accessibility and reduction in binding affinity.
Primary and secondary active efflux pumps are proposed to be responsible for reducing accumulation of antibiotics in the cell.
Biofilm and spore formation are also involved in antibiotic resistance in C. difficile.
This work was supported by government budget to Mahidol University; postdoctoral fellowship from Mahidol University (to P.H.); and the Science Achievement Scholarship of Thailand (to M.P.).
The authors declare there are no competing interests associated with the manuscript.
C. difficile infection
elongation factor G
ERY ribosomal methylase B
multidrug and toxic compound extrusion
major facilitator superfamily
mobile genetic element