Detection of High-Level Rifaximin Resistance in Enteric Bacteria by Agar Screen

Abstract

Objectives:

This study aimed at determining the prevalence of rifaximin resistance in a large collection of Enterobacterales resistant to third-generation cephalosporins. A simple agar screen was developed to detect high-level resistance.

Methods:

A total of 401 isolates nonsusceptible to third-generation cephalosporins (including 342 Escherichia coli and 39 Klebsiella spp. and 20 Enterobacter spp.) were tested by microdilution for their MICs of rifaximin and rifampicin. Isolates with a confirmed rifaximin minimal inhibitory concentration (MIC) of >64 mg/L and a number of high-level resistant, and susceptible control isolates were tested for growth on Mueller-Hinton agar supplemented with rifaximin or rifampicin at a concentration of 256 mg/L. Amino acid mutations in rpoB and the presence of rifaximin resistance-associated genes arabidopsis response regulator (arr) 2/3 were investigated.

Results:

Microdilution assays identified rifaximin resistance in nine E. coli and three Klebsiella spp. isolates with complete cross-resistance to rifampicin (MICs of both >64 mg/L). The rifaximin agar screen correctly identified 9/9 clinical E. coli isolates, 2/2 E. coli controls, and 3/3 Klebsiella spp. with high-level rifaximin resistance, and was negative in 45 control clinical isolates with rifaximin MICs ranging between 2 and 32 mg/L according to broth microdilution. All nine high-level rifaximin agar screen-positive E. coli clinical isolates (vs. none of the tested controls) had rpoB mutations or carried arr2/3.

Conclusions:

Our agar screen test has the potential to detect high-level rifaximin-resistant Enterobacterales. Such strains remain rare among extended spectrum beta-lactamase (ESBL)-positive enteric bacteria, but may emerge among patients receiving rifaximin for prevention of hepatic encephalopathy and spontaneous bacterial peritonitis or among patients receiving rifaximin for other indications.

Introduction

Resistance to beta-lactam antibiotics among gram-positive and -negative organisms remains a significant threat¹ and is of great concern, in particular regarding gram-negative multidrug-resistant (MDR) organisms. Such MDR organisms now include Klebsiella pneumoniae, Escherichia coli (E. coli), and Enterobacter species,² many of which are resistant to third-generation cephalosporins due to extended spectrum beta-lactamases (ESBLs). Apart from ESBLs, resistance to third-generation cephalosporins can also be caused by the production of the AmpC beta-lactamase found particularly in Enterobacter, Citrobacter, Morganella, and Serratia spp.

The rifamycins are a group of bactericidal antibiotics that are produced either by the bacterium Amycolatopsis rifamycinica or are semisynthetic rifamycin derivatives. The group comprises rifampicin, rifaximin, and two other derivatives, rifabutin and rifapentine. The antibacterial activity of rifamycins relies on the inhibition of bacterial DNA-dependent RNA synthesis.³ Rifampicin (Rmp), also known as rifampin, is an antibiotic used to treat several types of bacterial infections, including tuberculosis, leprosy, and Legionnaire's disease. Rifaximin (Rfx) is a nonabsorbable rifamycin developed and marketed for the treatment of traveler's diarrhea with good susceptibility of diarrhea-causing E. coli.^4,5 The agent has also been approved for treatment of irritable bowel syndrome with diarrhea, and for the prevention of overt hepatic encephalopathy recurrence in adults with liver disease. In hepatic encephalopathy, the agent has improved effectiveness compared with nonabsorbable disaccharides and similar activity as neomycin, paromomycin, vancomycin, or metronidazole.^6,7 Rifaximin is also being prescribed in selected cases with inflammatory bowel disease and has some role as second-line therapy for Clostridioides difficile infection.⁸

Rifamycin resistance can occur due to mutations within four highly conserved regions of rpoB, the chromosomal gene that encodes the beta subunit of the messenger RNA (mRNA) polymerase.^9–11 It has been reported that rpoB mutations in E. coli convey complete cross-resistance between the rifamycins¹² with minimal inhibitory concentrations (MICs) >500 mg/L. Another mechanism is acquisition of plasmid-mediated arabidopsis response regulator (arr) genes, which encode ADP-ribosyltransferases that inactivate rifamycins.^13,14 Finally, reported mechanisms of resistance include reduced intracellular accumulation presumably due to enhanced efflux although direct evidence for the relevance of this mechanism is missing.¹⁰ Alterations in virulence and plasmid curing at subinhibitory concentration of the drug have also been observed.¹⁵

The aim of the present study was to investigate the prevalence of rifamycin resistance among a large collection of Enterobacterales obtained in a German multicenter study Antibiotika-Therapie-Optimierungsstudie (ATHOS) investigating the admission prevalence of third-generation cephalosporin resistance.¹⁶ We further developed a method to detect high-level rifamycin resistance with the help of an agar screen test. We believe that such a test could be further developed as a helpful screening test among patients receiving rifaximin for prevention of hepatic encephalopathy and spontaneous bacterial peritonitis or among patients receiving rifaximin for other indications.

Materials and Methods

Bacterial strains

Details of the multicenter study performed in 2014 and 2015 are reported elsewhere.¹⁶ In brief, from a total of 4,376 patients enrolled at 6 centers, a total of 401 isolates nonsusceptible to third-generation cephalosporins were available for further testing: 342 E. coli, 39 Klebsiella spp. (3 Klebsiella oxytoca and 36 Klebsiella pneumoniae [K. pneumoniae]), and 20 Enterobacter spp. (2 Enterobacter aerogenes and 18 Enterobacter cloacae). Species identification of isolates growing on ESBL agar was performed using mass spectrometry Matrix-assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS) or the Vitek2 GN ID card (bioMérieux). Susceptibility testing was carried out using Vitek2 (bioMérieux). All isolates which were confirmed to be nonsusceptible to cefotaxime, ceftriaxone, or ceftazidime according to EUCAST breakpoints were included in the study and further characterized. Phenotypic detection of ESBL production was performed with the combination disc test as recommended by EUCAST, using cefotaxime, ceftazidime, and cefepime±clavulanate and tested for AmpC production by cefoxitin-cloxacillin disc test.

As control isolates we used E. coli strains 19678M6 (rpoB mutation D516G) and 19678M3 (rpoB mutation H526N), kindly obtained from Dr. Joaquim Ruiz at the Microbiology Department, Hospital Clínic, School of Medicine, University of Barcelona in Spain. For a first validation of the agar screen test, we used an international collection of 34 MDR E. coli clinical isolates in addition to that were previously investigated by whole genome sequencing (WGS) in our laboratory. The strains had been kindly provided by Murat Akova (Ankara, Turkey), Mutasim E. Ibrahim (Abha, Saudi Arabia), Can Imirzalioglu (Gießen, Germany), Gunnar Kahlmeter (Växjö, Sweden), Michael Kresken (Bonn-Rheinbach, Germany), Rumyana Markovska (Sofia, Bulgaria), Yasufumi Matsumura (Kyoto, Japan), Marie-Hélène Nicholas-Chanoine (Clichy, France), and Stefania Stefani (Catania, Italy). They were resistant to third-generation cephalosporins, ciprofloxacin, trimethoprim-sulfamethoxazole, tetracycline, gentamicin, and/or tobramycin.

Broth microdilution susceptibility testing

Study isolates underwent initial susceptibility testing for rifampicin and rifaximin by broth microdilution with the MICRONAUT system (Merlin Diagnostika, Bornheim-Hersel, Germany) according to standard procedures ISO 20776-1:2006.¹⁷ The following concentration ranges were included: Rmp 0.063–128 mg/L and Rfx 0.063–64 mg/L.

MICs were also determined by an in-house microdilution assay. Rmp or Rfx (dissolved in dimethyl sulfoxide [DMSO]) was pipetted into Mueller Hinton (MH) broth, diluted twofold (in 50 μL volumes), and 50 μL of a bacterial suspension (2 μL of the McFarland standard per mL of MH broth) was added to give final concentrations of 1–128 mg/L for Rmp and 2–256 mg/L for Rfx, respectively.

Agar screen for detection of high-level Rfx and Rmp resistance

All isolates with elevated Rfx and/or Rmp MICs were inoculated on a fresh MH agar supplemented with Rmp and Rfx, each at a concentration of 256 mg/L. The isolates were incubated overnight at 37°C. MH agar plates without supplement served as control. Growth was assessed visually. The assay was repeated three times. We selected as negative control strains 14 E. coli and 1 Klebsiella spp. (from the ATHOS study isolate collection) and 30 (out of 32) MDR E. coli strains (from the international collection) with an MIC of ≤32 mg of rifaximin and rifampicin per liter each (determined by broth microdilution). We also tested 39 isolates that had discrepancies in microdilution test results for rifaximin versus rifampicin (high MICs of rifaximin but low MICs of rifampicin).

Polymerase chain reaction and nucleotide sequencing

The rpoB gene and the arr2/3 gene were detected by real-time polymerase chain reaction as described previously.^18,19 Selected isolates underwent further Sanger sequencing of the rpoB gene according to the manufacturer's standards (Sequence Laboratories, Göttingen, Germany). We furthermore included the 34 MDR strains from our international collection for Sanger sequencing. Sequence analysis was performed with CLC Genomics Work Bench 9 (Qiagen, Hilden, Germany). The sequences were aligned and compared with a sequence consensus of E. coli E24377/A, K. pneumoniae HS11281, and E. cloacae ATCC13047.

Results

Seven E. coli and 3 Klebsiella spp. strains had elevated MICs in the MICRONAUT system for Rfx and Rmp of >64 mg/L each. Thirteen E. coli, 13 Klebsiella spp., and 13 Enterobacter spp. had discrepant results for Rfx and Rmp MICs. Results are shown in Table 1. All isolates with a Rfx MIC of >64 mg/L were retested with the in-house MIC dilution assay. This assay was considered the “gold standard” of MIC determination by us. Retesting resulted in a rate of confirmed (>64 mg/L) Rfx resistance of (only) 7 E. coli, 3 Klebsiella spp., and none out of 20 Enterobacter spp., without a single discrepancy between Rfx and Rmp MICs (at >64 mg/L) (Table 1). The prevalence of Rfx resistance among the German admission prevalence study isolates of third-generation cephalosporin-resistant E. coli, thus, was 7/324 (2%), whereas it was somewhat higher in the international collection of MDR E. coli (2/34, 6%).

Table 1.

Results of Broth Microdilution MIC and High-Level Rifamycin Resistance Agar Screen Tests

Isolates according to MIC	MIC of rifaximin (mg/L)		Growth on HL-Rfx agar screen (256 mg/L)	MIC of rifampicin (mg/L)		Growth on HL-Rmp agar screen (256 mg/L)
Isolates according to MIC	MICRONAUT plates	In-house microdilution	Growth on HL-Rfx agar screen (256 mg/L)	MICRONAUT plates	In-house microdilution	Growth on HL-Rmp agar screen (256 mg/L)
High-level resistant to both Rfx and Rmp
Escherichia coli 19678M3	>64	>256	Yes	>128	>128	Yes
E. coli 19678M6	>64	>256	Yes	>128	>128	Yes
E. coli (n = 7; ATHOS)	>64	>256	7/7	≥128	≥128	6/7
E. coli (n = 2; international MDR collection)	ND	>256	2/2	ND	>128	2/2
Klebsiella spp. (n = 3)	>64	>256	3/3	>128	>128	3/3
Discrepant results (microbroth dilution)
E. coli (n = 13)	>64	2 to >256	0/13	8–32	2–16	0/13
Klebsiella spp. (n = 13)	>64	32 to >256	0/13	16–32	16–32	0/13
Enterobacter spp. (n = 13)	>64	16 to 64	0/13	16–32	8–16	0/13
Negative controls
E. coli (n = 14; ATHOS)	≤32	2 to 16	0/14	4–16	2–8	0/14
E. coli (n = 30; international MDR collection)	ND	8 to 16	0/30	ND	4–16	0/30
Klebsiella spp. (n = 1)	≤32	32	0/1	16	16	0/1

ATHOS, Antibiotika-Therapie-Optimierungsstudie; HL, high-level; MDR, multidrug-resistant; MIC, minimal inhibitory concentration; ND, not determined; Rfx, rifaximin; Rmp, rifampicin.

Rfx agar screen correctly identified 7/7 E. coli with confirmed Rfx-R (>256 mg/L), whereas it was negative in 13 E. coli control clinical isolates with Rfx MICs ranging between 2 and 16 mg/L in broth microdilution (Merlin Diagnostika). The Rfx agar screen also correctly identified 3/3 Klebsiella spp. with Rfx MICs >256 mg/L and Rmp MICs >128. The single agar-screen-negative Klebsiella strain had discrepant Rfx (>256 mg/L) and Rmp (32/L) MICs. Sequencing results of rpoB mutations and presence of the arr2/3 gene are shown in Table 2. Among the 34 MDR strains from the international collection, only 2 were determined to be high-level rifamycin-resistant. rpoB sequencing in these 34 strains revealed only 2 SNPs, leading to an amino acid exchange: Q513L in strain SA 128 (high-level resistant) and E1272A in strain 2012-0633 (not high-level resistant).

Table 2.

High-Level Rifamycin-Resistant Escherichia coli Isolates with rpoB Mutations and/or Presence of arr2/3

Strain		MICs		rpoB mutations	arr2/3 Gene
E. coli		Rifaximin	Rifampicin
	A763	>256	>128	Asp516Tyr	—
	A866	>256	>128	Leu511Pro	—
	B22	>256	>128	—	Present
	B75	>256	>128	—	Present
	B159	>256	128	Ser574Phe	—
	L17	>256	>128	His526Leu	Present
	M4	>256	>128	Ser531Phe	—
	SA128	>256	>128	Gln513Leu	—
	PEG 10-68-51	>256	>128	—	Present
Klebsiella spp.
	A921	>256	>128	Insertion of Leu and Ser between Gln510 and Leu511; Glu626Asn; Val723Thr	—
	B148	>256	>128	Glu625Asp; Val723Leu	Present
	AS7	>256	>128	Glu626Asn; Val723Thr; Asp483Gly	Present
	B83	>256	>128	Glu626Asn; Val723Thr	Present

Nucleotide sequences were compared with those of E. coli E24377/A.

arr, arabidopsis response regulator.

Discussion

Until now, the frequency of Rfx and Rmp resistance has not been investigated systematically. Studies to date indicate that rifamycins have good in vitro activity against bacterial pathogens mainly focusing on traveler's diarrhea.^20–22 Rifamycin combination with polymyxins are under discussion for fecal decontamination of multiresistant Enterobacterales.²³ There are no clinical breakpoints or published epidemiologic cutoff values (ECOFFs) for rifamycins against Enterobacterales. Hopkins et al. used a provisional value of ≤32 mg/L to distinguish wild-type isolates from those likely to have an acquired resistance mechanism.¹⁸ Others applied ≥32 mg/L to define resistance among bacterial enteropathogens isolated from traveler's diarrhea.²¹

An important aim of this study was to estimate rifamycin, that is, Rfx and Rmp resistance among Enterobacterales. The prevalence among the species tested of <10% is reassuring and likely to be the result of the presumably very low frequency of rifaximin prescribing. Higher percentages (25%) have been reported for E. coli isolated from patients with prior rifaximin treatment.²⁴ In a study of E. coli clinical isolates isolated from travelers returning to the U.K., Rfx MICs were ≥128 mg/L for five E. coli out of 90 (6%), three of which were carbapenemase producers.¹⁸ Using selective media (as in our agar screen) for clinical samples might enhance the detection of such strains. Rmp and Rfx susceptibility of our isolates correlated well with the agar screening test. Further clinical studies are needed to reassess the performance and diagnostic accuracy of such an agar screen among patients during or after treatment with rifaximin. Our agar screening test might offer some advantages: simultaneous investigation of several strains, possible feasibility directly with stool samples, being cost-effective and less time-consuming the conventional culture and MIC determination.

Among E. coli, we detected rpoB mutations at positions 511, 513, 516, 526, 531, and 574 (Table 2) that have been described earlier,^24,25 whereas the mutations identified by Gomes et al. (positions 626 and 723) were not found in this study. Our results underline the importance of the region around the amino acids 513–526 in the rpoB gene in E. coli for a high-level Rfx resistance. The analysis of the rpoB gene in the 34 MDR E. coli shows that rpoB is highly conserved even in MDR strains subjected to high selection pressure. Furthermore, it lends additional credibility to the assumption that Q513L is a significant factor in rifamycin resistance. The low genetic variability of the rpoB gene might suggest that mutations in this area entail considerable fitness-costs in strains not under rifamycin-induced selection pressure. Interestingly, Enterobacter spp. did not grow on Rfx or Rmp agar.

Table 2 shows the result of the mutation analysis of rpoB and arr2/3 in the highly Rfx-resistant E. coli (n = 9) and Klebsiella (n = 4) isolates. In 5/9, E. coli rpoB was modified, whereas 3/9 harbored arr2/3. Only one E. coli harbored arr2/3 and showed rpoB mutation. We assume that especially arr2/3 could possibly explain elevated Rfx MICs. The arr2/3 gene is known to be associated with MDR in Salmonella enterica.^26,27 For Klebsiella, 3/4 isolates with an MIC of Rfx >256 mg/L harbored arr2/3 with rpoB mutations at the same time. Only 1/4 Klebsiella had rpoB mutations only. The relevance of this finding is completely unclear, as there are no comparable data for other species such as Klebsiella. We suggest that here the mutations at positions 483, 625, 626, and 723 may be of importance.

A limitation of the present study is that we cannot guarantee that the collection of isolates is representative. We tested only cephalosporin-resistant E. coli and a relatively small collection of international MDR isolates. A limitation was also that a rifamycin concentration >256 mg/L could not be exceeded due to solubility problems. Therefore, MICs >256 mg/L could not be further differentiated. Studies from Mexico used 400 and 800 μg/mL for in vitro testing of bacteria isolated from patients with intestinal infections and found only few strains resistant to Rfx at these concentrations.^4,5

In conclusion, we show that high-level Rfx resistance seems to be rare in E. coli and other Enterobacterales is frequently associated with rpoB mutations and can be easily detected through an agar screen with 256 μg Rfx per mL. This test should be evaluated to enhance detection of Rfx-resistant Enterobacterales in patients on long-term treatment with Rfx.

Footnotes

Acknowledgments

Members of the ATHOS study group are as follows: Sabina Armean, Tübingen; Anne C. Boldt, Berlin; Minh Trang Bui, Berlin; Dirk Busch, Munich; Gesche Först, Freiburg; Federico Foschi, Tübingen; Georg Häcker, Freiburg; Markus Heim, Munich; Vera Ihle, Freiburg; Klaus Kaier, Freiburg; Marina Kipnis, Berlin; Hans-Peter Lipp, Tübingen; Nayana Märtin, Berlin; Mathias Nordmann, Berlin; Luis-Alberto Penadiaz, Berlin; Gabriele Peyerl-Hoffmann, Freiburg; Jan Rupp, Lübeck; Christian Schneider, Freiburg; Christine Schröder, Berlin; Katrin Spohn, Tübingen; Michaela Steib-Bauert, Freiburg; Maria Vehreschild, Cologne; Ulrich vor dem Esche, Freiburg; and Thorsten Wille, Cologne.

Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported, in part, by the German Center for Infection Research (DZIF).

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