Rifampicin- and Rifabutin-Resistant Listeria monocytogenes Strains Isolated from Food Products Carry Mutations in rpoB Gene

Abstract

The aim of this study was to investigate the mechanism of rifampicin resistance in Listeria monocytogenes strains isolated from different types of food and the impact of specific mutations in the rpoB gene on susceptibility to different antimicrobial agents and on fitness cost. Fifteen spontaneous rifampicin-resistant strains were selected. The DNA regions corresponding to clusters I–II, III, and N-terminal end of the rpoB gene of Escherichia coli were amplified and sequenced, leading to the identification of 10 different substitutions, nine of which (Ser466Pro, Gln470Lys Asp473Asn, Gly479Asp, His483Tyr/Arg/Asp, Arg486His, and Leu490Pro) were located in cluster I and one (Pro521Leu) in cluster II. From among these mutations, substitutions at positions 466, 470, 486, 490, and 521 have not been described for L. monocytogenes. Only substitutions at positions 470, 479, 483, and 486 lead to resistance to very high concentrations of rifampicin (minimum inhibitory concentration [MIC] ≥256 μg/mL) and rifabutin (MIC 128 μg/mL). Furthermore, mutations at positions 473, 490, and 521 had different effects on susceptibility to rifampicin compared to other bacterial species. A correlation between rifampicin resistance and susceptibility to a wide range of antimicrobials was determined. Substitutions in RpoB did not change the susceptibility of the mutants to different antimicrobials. The fitness of the mutants was assessed by paired competition experiments. Mutations at positions 470 and 479 were not associated with a reduction in fitness level. There was no correlation between the MIC of rifampicin and fitness cost. The risk of transmission of resistant strains through the food chain highlights the need for monitoring resistance, identifying mutant organisms, their genotypes, and their altered phenotypes to understand their dissemination.

Introduction

L isteria monocytogenes is a Gram-positive, nonspore-forming foodborne pathogen that can cause serious human infections, such as bacteremia and central nervous system infections, primarily in neonates and immunocompromised adults. It can also cause perinatal infections that may result, for example, in abortions (Hof, 2004). The treatment of listeriosis is currently based on the synergistic association of high doses of ampicillin and gentamicin. Nonetheless, the efficacy of dual ampicillin–gentamicin therapy is not supported by recent epidemiological studies (Fernández-Guerrero et al., 2012; Muñoz et al., 2012). Trimethoprim/sulfamethoxazole is generally used in cases of intolerance to beta-lactams, but rifampicin, erythromycin, vancomycin, linezolid, and meropenem have been proposed as possible alternatives for treatment (Hof, 2004; Janakiraman, 2008). Rifampicin has been successfully used in combination with ampicillin (Barocci et al., 2015). Despite antibiotic therapy, listeriosis represents a grave public health problem since it was fatal in around 20% of cases reported in the last two decades (Muñoz et al., 2012). These organisms are among the most important causes of death from foodborne infections in industrialized countries (EFSA, 2015).

Bacterial DNA-dependent RNA polymerase (RNAP) is responsible for the transcription of DNA. This enzyme is the target of several low-molecular-weight inhibitors. The best-known RNAP inhibitor, rifampicin (Rif), acts selectively on bacterial RNA polymerase by binding to its β-subunit and blocking the path of RNA elongation when the transcript becomes 2 or 3 nucleotides long (Campbell et al., 2001). Rifampicin displays a broad spectrum of antibiotic activity against Gram-positive and, to a lesser extent, Gram-negative bacteria (Floss and Tin-Wein, 2005). Depending on its concentration, this antibiotic can have a bacteriostatic or bactericidal effect. Resistance to rifampicin is commonly due to a single point mutation in the β-subunit of RNAP encoded by the rpoB gene and is less frequently associated with deletions or insertions. Such mutations have been described in a wide variety of organisms (Floss and Tin-Wein, 2005; Goldstein, 2014). Ninety-five percent of these mutations map to four regions in the N-terminal half of the β-subunit polypeptide involved in the binding of rifampicin. In Escherichia coli, Rif resistance mutations are usually located in the central region of the polypeptide, within cluster I (amino acids 505–537), cluster II (amino acids 562–575), and cluster III (amino acids 684–690) and they can also occur near the N-terminus (Campbell et al., 2001). Other mechanisms of rifampicin resistance have also been described, but these are much rarer (Goldstein, 2014).

Little is known about the resistance of L. monocytogenes to rifampicin and the molecular basis of such resistance.

In this study, the relationship between resistance to rifampicin and rifabutin and genetic alterations in the rpoB gene of 15 spontaneous rifampicin-resistant mutants of L. monocytogenes isolated from different types of food was evaluated. The impact of RpoB alterations on susceptibilities to a diverse collection of other antibiotics and fitness cost was also investigated.

Materials and Methods

Bacterial strains

Fifteen rifampicin-resistant isolates (five from smoked fish, four from frozen vegetables, three from cold cuts, and three from dairy products) were obtained from a previous study of L. monocytogenes strains collected from different types of food and food-related sources in Poland during 2004–2010 (Korsak et al., 2012).

Determination of rifampicin and rifabutin susceptibility

The minimum inhibitory concentrations (MICs) of rifampicin and rifabutin were determined using a broth microdilution method according to guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2010).

To ensure reproducibility, MIC determinations were repeated at least twice.

Very limited guidelines for interpreting the MIC results for L. monocytogenes are available; CLSI (2010) provides criteria only for penicillin G and trimethoprim/sulfamethoxazole.

However, the monomodal distribution of the MICs allowed delineation of the wild-type populations. MIC ranges for rifampicin are <0.25–0.5 μg/mL.

Serotyping of L. monocytogenes strains

Resistant and isogenic isolates were serotyped by multiplex polymerase chain reaction (PCR), according to the method described by Doumith et al. (2004).

Detection of mutations in the rpoB gene

To identify mutations in the rpoB gene of rifampicin-resistant L. monocytogenes isolates, the DNA region corresponding to the Rif clusters I–II, III, and N-terminal of E. coli was amplified by PCR. Primers were designed on the basis of the genome sequence of L. monocytogenes EGD-e. The primers rpoB-F (5′-GGTTCGTGAACGTATGTCTA-3′) (nucleotides 1293–1312) and rpoB-R (5′-GCGCTACTACGTAATTATCC-3′) (nucleotides 1710–1729) were designed to flank clusters I and II. The primers: rpoB-III-F (5′-TGACTACATGGACGTATCCGCC-3′ (nucleotides 1827–1847), rpoB-III-R (5′-CATGTTTGGCAGTTACAGCA-3′) (nucleotides 2028–2038), rpoB-N-F (5′-CTAAGAACCGTGATGCAAAC-3′) (nucleotides 251–270), and rpoB-N-R (5′-GTTTCATACTCAAGCCAAGC-3′) (nucleotides 510–530) were designed to amplify the regions that correspond to the E. coli rpoB III and N-terminal clusters, respectively. Amplification was carried out in a 25 μL reaction mixture containing 0.2 mM of each primer, 0.2 mM dNTPs, 1 U Pfu polymerase (Fermentas), and 50 ng chromosomal DNA. The conditions were heating for 1 cycle at 94°C for 5 min; 30 cycles at 94°C for 30 s of primer-specific annealing for 30 s and at 72°C for 1 min followed by 1 cycle at 72°C for 10 min. Primer pair rpoB-F-rpoB-R and rpoB-III-F-rpoB-III-R were annealed at 50°C, while primer pair rpoB-N-F and rpoB-N-R were annealed at 53°C.

All PCR products were purified using QIAquick PCR Purification Kit (Qiagen). Amplicons were sequenced for rifampicin-resistant isolates and parental strains.

Drug susceptibility testing

The sensitivities of the resistant mutants and respective parental strains to a wide range of antibiotics were assayed by agar diffusion tests on Mueller-Hinton agar plates containing 5% sheep blood (Oxoid). The disks contained the following antibiotics: ceftibuten, ceftizoxime, ceftazidime, cefepime, cefoperazone, cefotetan, cefotaxime, cefadroxil, ceftriaxone, aztreonam, tetracycline, chloramphenicol, vancomycin (30 μg), ciprofloxacin (5 μg), trimethoprim/sulfamethoxazole (1.25 and 23.75 μg), gentamicin, ampicillin (10 μg), and erythromycin (15 μg) (Oxoid). For each antibiotic, at least three independent assays were performed per strain.

Determination of competitive fitness in vitro

Fitness costs associated with rifampicin resistance mutation were determined by pairwise competition of resistant mutant with the drug-susceptible progenitor strain, essentially as described previously (Cohan et al., 1994). The competition assays were performed for 10 mutant isolates with different rpoB genotypes. Briefly, brain heart infusion broth (BHI) was inoculated with a mixture of overnight cultures of rifampicin-susceptible parental strain and a rifampicin-resistant mutant in ratio 10:1. Dilutions of the mixed cultures were plated onto both non-selective BHI and selective BHI containing rifampicin at a concentration of 0.32 μg/mL for the parental strains at time 0 and after 24 h incubation at 37°C.

Relative competitive fitness (W) was calculated according to the equation W = ln [N_R(24)/N_R(0)]/ln[N_S(24)/N_S(0)], where N_R(t) and N_S(t) represent viable counts of the resistant strain and susceptible counterpart, respectively, at time t (either 0 or 24 h) (Lenski, 1988). Fitness determinations were conducted at least in triplicate.

Statistical analysis

Statistical analysis was carried out using STATISTICA v.6.0. Data were analyzed using the one-sample t-test.

Results

Rifampicin and rifabutin resistance

We previously investigated the MICs of rifampicin against 471 L. monocytogenes isolates recovered from different types of food and food-related sources (Korsak et al., 2012). Although all the isolates showed susceptibility to rifampicin, in the case of 15, single colonies growing in the inhibition growth zone surrounding the E-strip were observed. All these colonies were isolated and their susceptibilities to rifampicin and rifabutin were determined. The MICs of rifampicin for these isolates range from 1 to >256 μg/mL, while for rifabutin 0.5–128 μg/mL (Table 1). Nine mutants were associated with high-level resistance to rifampicin (MIC ≥256 μg/mL) and six with low resistance (MIC range from 1 to 2 μg/mL). In the case of the parental strains, MICs of rifampicin and rifabutin ranged from 0.008 to 0.03 μg/mL and 0.02 to 0.06 μg/mL, respectively (data not shown).

Table 1.

Characteristic of Rifampicin/Rifabutin-Resistant L. monocytogenes Isolates

		MIC, μg/mL
Strain	Genoserogroup	Rifampicin	Rifabutin	Amino acid position	Alteration in rpoB gene	Amino acid substitution	Relative fitness (mean ± SEM) ^a,b
142/05	IIc	2	2	466	TCG→CCG	Ser→Pro	0.839 ± 0.052^c
163/03	IIc	2	2	466	TCG→CCG	Ser→Pro	—
13/03	IIa	2	2	466	TCG→CCG	Ser→Pro	—
23/04	IIa	>256	128	470	CAG→AAG	Gln→Lys	0.991 ± 0.026
24/07	IIa	>256	128	470	CAG→AAG	Gln→Lys	—
122/04	IIc	2	2	473	GAT→AAT	Asp→Asn	0.893 ± 0.028^c
39/07	IIb	>256	128	479	GGC → GAC	Gly→Asp	0.812 ± 0.094
2268/03	IIc	>256	128	479	GGC → GAC	Gly→Asp	—
966/09	IIa	256	256	483	CAT→TAT	His→Tyr	0.703 ± 0.064^c
148/11	IIc	256	256	483	CAT→TAT	His→Tyr	—
2783/08	IIa	256	>256	483	CAT → CGT	His→Arg	0.885 ± 0.015^d
4670/07	IIc	256	>256	483	CAT→GAT	His→Asp	0.877 ± 0.035^c
59/04	IIa	256	128	486	CGT → CAT	Arg→His	0.76 ± 0.028^d
131/03	IIa	1	1	490	CTT → CCT	Leu→Pro	0.846 ± 0.053^c
233/03	IIa	1	0.5	521	CCA → CTA	Pro→Leu	0.055 ± 0.032^e

Underlined letters indicate nucleotide substitutions.

Fitness costs associated with rifampicin resistance mutation were determined for Rif mutants representing different rpoB genotypes.

Mean values and SEMs were derived from a total of three different fitness assays.

The fitness reduction is significant, as determined by one-sample t-test (p < 0.05).

The fitness reduction is significant, as determined by one-sample t-test (p < 0.01).

The fitness reduction is significant, as determined by one-sample t-test (p < 0.005).

MIC, minimum inhibitory concentration; SEMs, standard errors of the mean.

Molecular mechanism of rifampicin and rifabutin resistance

The 15 isolates phenotypically resistant to rifampicin and rifabutin and their susceptible counterparts were further characterized by sequencing three regions of gene rpoB homologous to the Rif clusters I–II, III, and N-terminal of E. coli, respectively.

Sequence analysis revealed that all resistant isolates had a single base pair alteration in region I and region II of the rpoB gene resulting in an amino acid substitution. Ten different changes at eight positions were identified: nine clustered from nucleotide positions 1396–1469 and one was at position 1562 (Table 1). The mutations in the rpoB gene were located mainly in cluster I. Only one was in cluster II.

Four rifampicin-resistant isolates showed a mutation at codon 483. Two mutants revealed a C1447T transition (His/Tyr), one A1448G transition (His/Arg), and one C1447G transversion (His/Asp). Three isolates exhibited T1396C transitions (Ser466Pro). Two mutants exhibited a A1436G transition (Gly479Asp) and two others a C1408A transversion (Gly470Lys). The other four isolates showed the transitions: G/A at position 1417 (Asp473Asn) and 1454 (Arg486His), T/C at position 1469 (Leu490Pro), and C/T at position 1562 (Pro521Leu).

Substitutions at positions: 470, 479, 483, and 486 were associated with high-level resistance to rifampicin and rifabutin.

Antimicrobial susceptibility

A specific mutation in rpoB can alter susceptibility to different antimicrobials. A correlation between rifampicin resistance and susceptibility to a wide range of antimicrobial agents was determined. None of the Rif^r mutants exhibited differences in susceptibility toward antibiotics that target penicillin binding proteins (extended- and broad-spectrum cephalosporins and ampicillin), antibiotics that target other aspects of cell wall biosynthesis (vancomycin), or antibiotics with non-cell wall targets (tetracycline, chloramphenicol, ciprofloxacin, trimethoprim/sulfamethoxazole, gentamicin, and erythromycin).

Fitness costs of the rpoB mutation

The Rif mutants representing ten different rpoB genotypes with their Rif^s isogenic counterpart were subjected to competition assays, which revealed that 8 of 10 mutations within the rpoB gene were associated with a reduction in their level of fitness. Only two mutational changes: Gln470Lys and Gly479Asp, which were capable of conferring high-level resistance, were not associated with a reduction in fitness level (Table 1).

No relationship was detected between the cost of rpoB mutation and the level of resistance to rifampicin.

Discussion

Rifampicin penetrates well into host cells and is active against intracellular L. monocytogenes (Hof, 2004). However, the value of rifampicin for therapy of listeriosis remains a matter of debate. One could argue that for the initial therapy of listeriosis, which most often manifests itself as meningitis, rifampicin is not essential, but for a complete cure, which would include the eradication of hidden intracellular bacteria, the use of rifampicin can be considered important (Hof et al., 1997; Allerberger and Wagner, 2010). At least in the immunocompromised host, ampicillin in combination with rifampicin could be a good alternative regimen to minimize the risk of treatment failures (Barocci et al., 2015). Rifampicin has been used in case reports of listerial infection (e.g., Mylonakis et al., 2002; Barocci et al., 2015).

The molecular characterization of mutations leading to the Rif^r phenotype has revealed that gene rpoB encoding for β subunit of RNA polymerase is the target of these mutations in E. coli. The same is demonstrated also in many other Gram-positive and Gram-negative bacteria (Goldstein, 2014).

In this study, we report the results of the analysis of rifampicin-resistant mutants of L. monocytogenes from food origin. From among 15 spontaneous mutants, nine revealed high resistance to rifampicin and six low resistance. The isolates were also rifabutin resistant. These results show that all the isolates were resistant to both rifamycins.

Sequence analysis revealed that all resistant mutants of L. monocytogenes had a single base pair change in the region of rpoB gene that resulted in an amino acid substitution. In nine cases, a point mutation was found in the so-called rifampicin resistance cluster I, covering in L. monocytogenes amino acid residues 466–490 corresponding to residues 509–533 in E. coli. Only one resistant isolate had acquired a mutation at position 521, which corresponds to residue 564 in E. coli cluster II (Campbell et al., 2001). Certain mutations in cluster I were frequently found: the substitution serine to proline at position 466 in three isolates, glutamine to lysine at position 470, and the substitution glycine to aspartic acid (479) in two mutants, respectively. Four isolates had three different substitutions at position 483. The genetic background, in which a mutation in rpoB occurred, did not appear to influence the MIC.

To our knowledge, Ser466Pro, Gln470Lys, Arg486His, Leu490Pro, and Pro521Leu substitutions have not yet been described for L. monocytogenes (Morse et al., 1999; Chenal-Francisque et al., 2014).

As shown previously, specific mutations in rpoB alleles lead to the development of cross-resistance to all rifamycins (Wichelhaus et al., 1999; Xu et al., 2005; Glocker et al., 2007), while a subset of mutations was associated with resistance to rifampicin, but not to rifabutin (Williams et al., 1998; Yang et al., 1998; Casavoglu et al., 2004; Sirgel et al., 2013; Jamieson et al., 2014). All identified mutations in the rpoB gene of L. monocytogenes led to cross-resistance to rifampicin and rifabutin.

Structural studies of the Rif-RNA polymerase complex with Thermus aquaticus RNAP have permitted the identification of the binding pocket and the description of the mechanism of Rif action (Campbell et al., 2001). The disposition of the Rif binding site of T. aquaticus RNA polymerase with respect to the active site is identical to E. coli RNAP and presumably other prokaryotic RNA polymerases. The residues: Gln510, Leu511, Gln513, Phe514, Asp516, His526, Arg529, Ser531, Leu533, Gly534, Glu565, and Ile572 in E. coli RNAP are close enough to Rif to participate in direct interaction. All but one, Glu565, are known to mutate to strong Rif^r (Campbell et al., 2001).

Comparative analysis of the level of resistance to rifampicin and rifabutin in L. monocytogenes and the mutation sites indicated that high-level resistance correlated with mutations at codons 470, 479, 483, and 486 that correspond to E. coli 513, 522, 526, and 529, respectively. Only three of these residues belong to the putative Rif-binding pocket. Four other changes at position 466, 473, 490, and 521 (corresponding to E. coli 509, 516, 533, and 564, respectively) caused low resistance to these antibiotics. From these, the residues at position 473 and 490 belong to the putative Rif-binding pocket.

Identical substitutions at the same position were also found in other bacteria. For example, the amino acid substitution Gln470Lys, which was not previously described in L. monocytogenes, was identified in Staphylococcus aureus (corresponding position 468) (Aubry-Damon et al., 1998), Helicobacter pylori (position 527) (Heep et al., 1999), Streptococcus pneumoniae (position 486) (Ferrándiz et al., 2005), Pseudomonas aeruginosa, and Pseudomonas putida (position 518) (Jatsenko et al., 2010) and also conferred high-level rifampicin resistance. In turn, the alteration Gly479Asp was found in high-resistant Enterococcus faecalis strains (Enne et al., 2004). Alterations at position 483 were found in E. faecalis (position 489) (Enne et al., 2004), H. pylori (position 540) (Heep et al., 1999), and S. pneumoniae (position 499) (Ferrándiz et al., 2005). The changed Arg486His was found in S. aureus (position 484) (Aubry-Damon et al., 1998; Wichelhaus et al., 2002), E. faecalis (position 492) (Enne et al., 2004), E. coli (position 529), and T. aquaticus (position 409) (Campbell et al., 2005) and led in all species to high-level resistance. However, the same substitution had a lesser effect in P. aeruginosa (position 534) (Jatsenko et al., 2010).

The substitution Ser to Pro at position 466, which conferred low-level rifampicin resistance, was identified in L. monocytogenes for the first time in this study. It was also found in S. aureus (Aubry-Damon et al., 1998; Wichelhaus et al., 2002) and S. pneumoniae (position 481) (Ferrándiz et al., 2005). The mutation mapped at position 473 (Asp to Asn) and caused weak resistance against rifampicin, but the same substitution had strong effects in H. pylori (corresponding 530) (Heep et al., 1999; Glocker et al., 2007), E. coli (position 516), and T. aquaticus (position 396) (Campbell et al., 2005). Another previously undescribed mutation in L. monocytogenes, Leu490Pro, results in low-level resistance. It is interesting that the same substitution had a strong phenotypic effect in E. coli (position 533) and T. aquaticus (position 413) (Campbell et al., 2005). Another change was identified for the first time in L. monocytogenes mapped in cluster II at position 521 and resulted in low-level resistance. This mutation had been also found in E. coli (position 564). Contrary to L. monocytogenes, in E. coli, this substitution causes a very high level of resistance (Reynolds, 2000).

As shown by others, a specific mutation in the rpoB gene could alter susceptibility to different antimicrobials in other bacteria. For example, reduced vancomycin susceptibility in S. aureus (Watanabe et al., 2011), trimethoprim/sulfamethoxazole susceptibility in Brucella abortus (Sandalakis et al., 2012), susceptibility to ofloxacin in Mycobacterium tuberculosis (Louw et al., 2011), or enhanced intrinsic cephalosporin resistance in enterococci (Kristich and Little, 2012). Listeria strains show a high natural resistance to cephalosporins, especially to those that are broad spectrum, such as cefotaxime, and to monobactams. In this aspect, Listeria spp. resemble enterococci (Hof et al., 1997). In this study, we also examined the effects of mutations in the β subunit of RNA polymerase on the susceptibility of rifampicin-resistant isolates to various antimicrobial agents that target a range of alternative cellular processes. We found no significant difference between the resistant and parental strains. Therefore, we conclude that these substitutions in the rpoB of L. monocytogenes-resistant isolates did not affect susceptibility with regard to diverse antimicrobials.

Resistance determinants that interfere with normal physiological processes in the bacterial cell usually cause a reduction in biological fitness, for example, reduction of growth rate, invasiveness, and loss of virulence. We investigated the intrinsic detriments in biological fitness associated with mutations in the β subunit of RNA polymerase that confer rifampicin resistance in L. monocytogenes. The competition assay of selected resistant mutants with their sensitive isogenic counterpart revealed that only two rpoB genotypes displayed no fitness burden, whereas the other mutations were associated in some cases with a considerable loss of fitness. The magnitude of this cost was variable depending on the specific mutation causing rifampicin resistance. As found in Bacillus subtilis (Cohan et al., 1994), E. coli (Reynolds, 2000), or S. aureus (Wichelhaus et al., 2002), the cost of resistance did not correlate with the level of rifampicin resistance. In contrast, in Enterococcus faecium, higher fitness costs were associated with higher MICs (Enne et al., 2004).

In conclusion, while spontaneous rifampicin-resistant L. monocytogenes strains have been previously obtained in laboratory conditions through multiple stepwise passages of reference strain on medium containing rifampicin (Morse et al., 1999) or isolated from patients (Chenal-Francisque et al., 2014), this is the first report on the molecular basis of spontaneous rifampicin-resistant L. monocytogenes mutants isolated from food.

We have established that cross-resistance to rifampicin and rifabutin in L. monocytogenes is due to mutations in cluster I and II of the rpoB gene and that the resistance levels are dependent on the location of the amino acid substitution. Five of these substitutions are newly identified in L. monocytogenes, and three of these have different effects on resistance level compared to mutations corresponding to these residues in other bacterial species. The identified changes had no influence on susceptibility to a wide range of antimicrobial agents, but influenced the intrinsic fitness burden of most of the studied isolates.

Since the food origin of human infections is now recognized, it is thus important to test for antibiotic resistance and molecular basis of resistance in foodborne and clinical isolates of L. monocytogenes.

Footnotes

Acknowledgment

This work was supported financially by the Ministry of Science and Higher Education (Grant No. N312 255335).

Disclosure Statement

No competing financial interests exist.

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