Accepted Manuscript Rifaximin-resistant Clostridium difficile strains isolated from symptomatic patients E. Reigadas, P. Muñoz-Pacheco, L. Alcalá, M. Marín, A. Martin, E. Bouza PII:
S1075-9964(17)30197-X
DOI:
10.1016/j.anaerobe.2017.10.002
Reference:
YANAE 1802
To appear in:
Anaerobe
Received Date: 28 July 2017 Revised Date:
14 September 2017
Accepted Date: 5 October 2017
Please cite this article as: Reigadas E, Muñoz-Pacheco P, Alcalá L, Marín M, Martin A, Bouza E, Rifaximin-resistant Clostridium difficile strains isolated from symptomatic patients, Anaerobe (2017), doi: 10.1016/j.anaerobe.2017.10.002. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Rifaximin-Resistant Clostridium difficile Strains Isolated From
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Symptomatic Patients
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Reigadas E., PharmD, PhD1, 2, 3
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Muñoz-Pacheco P., PhD1
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Alcalá L., PharmD 1, 3, 4
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Marín M., PharmD, PhD 1, 2, 3, 4
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Martin A., PhD1, 3
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Bouza E., MD, PhD1, 2, 3, 4
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Department of Clinical Microbiology and Infectious Diseases, Hospital General Universitario
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Gregorio Marañón, Madrid, Spain
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Madrid, Spain
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Instituto de Investigación Sanitaria Gregorio Marañón, Madrid, Spain
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CIBER de Enfermedades Respiratorias (CIBERES CB06/06/0058), Madrid, Spain
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Running title: Rifaximin-resistant C. difficile clinical isolates
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Keywords: C. difficile, antimicrobial susceptibility, rifaximin, rifampicin, ribotype.
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Corresponding author:
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Reigadas Ramírez Elena, PharmD, PhD
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Servicio de Microbiología Clínica y Enfermedades Infecciosas
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Hospital General Universitario “Gregorio Marañón”
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C/ Dr. Esquerdo, 46
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28007 Madrid, Spain
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Phone: +34- 91- 586 84 53/Fax: +34- 91- 504 49 06
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E-mail:
[email protected]
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Alternative corresponding author:
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Bouza Santiago Emilio, MD, PhD
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Servicio de Microbiología Clínica y Enfermedades Infecciosas
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Hospital General Universitario “Gregorio Marañón”
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C/ Dr. Esquerdo, 46
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Medicine Department, School of Medicine, Universidad Complutense de Madrid (UCM),
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28007 Madrid, Spain
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Phone: +34- 91- 586 84 53/Fax: +34- 91- 504 49 06
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E-mail:
[email protected]
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ACCEPTED MANUSCRIPT 34 ABSTRACT
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Background: Rifaximin has been proposed as an alternative treatment for
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specific cases of Clostridium difficile infection (CDI) and intestinal
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decontamination. Rifaximin-resistant C. difficile has occasionally been reported.
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Antibiotic susceptibility testing relies on anaerobic agar dilution (reference
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method), which is cumbersome and not routinely used. There is no commercial
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test for detection of resistance to rifaximin.
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Objectives: To assess resistance to rifaximin by C. difficile and to evaluate the
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correlation between the results of the rifampicin E-test and susceptibility to
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rifaximin.
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Methods: We compared the in vitro susceptibility of clinical CDI isolates to
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rifaximin over a 6-month period using the agar dilution method with
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susceptibility to rifampicin using the E-test. All isolates were characterized using
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PCR-ribotyping. Clinical data were recorded prospectively.
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Results: We recovered 276 consecutive C. difficile isolates and found that
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32.2% of episodes were caused by rifaximin-resistant strains. The MICs for
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rifaximin ranged from 256 mg/L. Rifaximin and rifampicin MICs were
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comparable, and all strains classed as resistant by agar dilution were correctly
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classified as resistant by E-test. The most common ribotypes were 001 (37.2%),
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078/126 (14.3%), and 014 (12.0%). Ribotype 001 exhibited the highest MICs for
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rifaximin.
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Conclusions: Resistance to rifaximin was common; resistance rates were
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higher in ribotype 001 strains. Susceptibility to rifaximin determined by agar
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dilution correlated with susceptibility to rifampicin determined using the E-test,
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including rifaximin-resistant strains. Our results suggest that the rifampicin E-
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test is a valid method for the prediction of rifaximin-resistant C. difficile.
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INTRODUCTION Clostridium difficile infection (CDI) is the leading cause of hospital-
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acquired diarrhea in developed countries, yet the current standard treatment for
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CDI is not always effective. The antibiotics of choice for the treatment of CDI
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are metronidazole and vancomycin. However, despite the fact that both
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antibiotics have a very high cure rate, both have high recurrence rates
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(estimated at 15%-25%)[1, 2]. Recently developed antibiotics such as
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fidaxomicin appear to be more efficacious than traditional drugs in preventing
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recurrences [3].
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The rifamycin derivative rifaximin has been proposed as an adjunctive
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treatment to decrease recurrent CDI [4, 5], although resistance to this
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antimicrobial has been reported. Antibiotic susceptibility testing is based on
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anaerobic agar dilution, which is considered the reference method for C.
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difficile; however, this approach is cumbersome and time-consuming and is not
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widely used in routine testing of clinical isolates. There is no commercially
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available test for resistance to rifaximin.
Our objectives were to assess resistance to rifaximin and to evaluate the
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correlation between results with the rifampicin E-test and susceptibility to
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rifaximin.
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MATERIAL AND METHODS
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Setting
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Our institution is a large teaching hospital with 1,550 beds. The clinical
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microbiology laboratory receives samples from patients hospitalized at our
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center and from all the outpatient institutions in our catchment area.
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Bacterial Isolates We analyzed the in vitro susceptibility of consecutive clinical C. difficile
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isolates collected from patients with clinically confirmed CDI over a 6-month
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period.
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Antimicrobial Susceptibility Testing
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Susceptibility to rifaximin was tested using the agar dilution method
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according to the procedures of the Clinical and Laboratory Standards Institute
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(CLSI)[6]. Rifaximin was obtained from Sigma Chemical Company (St Louis,
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Missouri, USA). Quality control strains included C. difficile ATCC 700057, B.
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fragilis ATCC 25285, and B. thetaiotamicron ATCC 29741[7]. The range of
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rifaximin concentrations tested was 0.0009 to 256 g/L. Antimicrobial
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susceptibility testing for rifampicin was performed by E-test according to the
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manufacturer’s instructions (AB Biodisk, Solna, Sweden) on sheep blood agar
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with 1 µg of vitamin K1 and 5 µg of hemin (Oxoid, Basingstoke, United
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Kingdom). The MICs were recorded for each isolate after 48 hours of incubation
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following CLSI recommendations [6]. Isolates that exhibited MICs >32 mg/L for
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rifaximin and rifampicin were classed as resistant.
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Ribotyping
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All isolates were characterized using PCR-ribotyping[8]. Phylogenetic analysis
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of ribotyping profiles was conducted using the unweighted pair group method
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with arithmetic mean (UPGMA) and Dice coefficients (Bionumerics 5.0).
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Ribotypes were named using an international designation[8].
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Definitions 6
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A CDI episode was defined as the presence of a positive result for
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toxigenic C. difficile testing and the presence of diarrhea (≥3 unformed stools in
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24 hours) or colonoscopic findings demonstrating pseudomembranous colitis. Severity of CDI was defined according to the guidelines of the Society of
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Healthcare Epidemiology of America (SHEA) and the Infectious Diseases
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Society of America (IDSA)[9].
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An episode was considered a recurrence if after recovery from a previous
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episode (at least 3 days without diarrhea and clinical improvement), symptoms
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returned and a stool sample separated from the former by between 15 and 60
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days was positive. Episodes occurring more than 60 days after the previous one
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were not considered recurrences but new episodes.
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Death was considered CDI-related when it was not attributable to other
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unrelated causes occurring within 10 days of the CDI diagnosis and/or due to
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well-known complications of CDI.
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Clinical data
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For clinical assessment only first episodes were assessed. The patients’
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data collected included age, sex, and hospital department or outpatient clinic at
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diagnosis of CDI. Data regarding the underlying condition were recorded using
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the McCabe and Jackson score for prognosis of underlying diseases, and
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comorbidity factors were assessed according to the Charlson index[10, 11].
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Clinical data recorded for the CDI episode included antibiotic treatment,
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severity, recurrence, mortality, and CDI-related mortality.
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Ethical Issues This study was approved by the Ethics Committee of Hospital General Universitario Gregorio Marañón.
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We recovered 276 consecutive C. difficile isolates from clinical
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samples that fulfilled our institutional criteria for CDI. MIC ranges, geometric
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means (GM), and the minimum concentrations inhibiting 50% (MIC50) and 90%
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(MIC90) of the 276 isolates are shown in Table 1. The MICs for rifaximin against
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the 276 clinical isolates ranged from 256 mg/L, with a GM of
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0.256 mg/L, an MIC50 of 0.015 mg/L, and an MIC90 of >256 mg/L. Overall, the
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MICs for rifaximin were either very low or very high (≤0.125 mg/L, n=185
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isolates; 64 mg/L, n=1 isolate; and ≥256 mg/L, n=88 isolates).
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observed also in the case of rifampicin, for which all strains were either ≤0.32
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mg/L (n=186 mg/L) or >32 mg/L (n=90).
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Overall, we found that 32.2% of the C. difficile strains were
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rifaximin-resistant, this corresponded to 31.8 % of our initial CDI episodes being
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caused by C. difficile rifaximin-resistant strains. Median age was 69.6 years,
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and 52.5% of patients were male. Overall, 78.9% of the patients were
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hospitalized at the time of the diagnosis of the CDI episode. Most patients were
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hospitalized in medical wards (70.8%), followed by surgical wards (15.5%),
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intensive care units (6.1%), oncological wards (4.3%) and other (3.1%). Eighty-
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five percent of the patients had received antibiotic treatment in the month prior
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to the CDI episode.
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rifaximin-resistant strains and rifaximin-susceptible strains regarding severity of
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CDI (mild to moderate 81.3% vs 85.4, p=0.535; severe 17.2% vs 13.1%,
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p=0.519; and severe complicated 1.6% vs 1.5%, p=1.000). Similarly, no
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differences were found for recurrence rate (17.2% vs 14.6% p=0.677), overall
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mortality (9.4% vs 8.0%, p=0.788), or related mortality (3.1% vs 2.2% p=0.654).
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Rifaximin and rifampicin MICs for C. difficile isolates were
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comparable. With respect to the breakpoint classification for resistance, there
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was agreement in 99.6% of the isolates: only 1 isolate was classified as
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rifampicin-resistant by the E-test and rifaximin-susceptible by agar dilution. All
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strains shown to be resistant by agar dilution were correctly classified as
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resistant by E-test.
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A total of 258 strains were available for PCR-ribotyping. The 3
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most common ribotypes were 001 (37.2%), 078/126 (14.3%), and 014 (12.0%).
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The distribution of the ribotypes is shown in Figure 1. The analysis of
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antimicrobial susceptibility according to the most frequently encountered
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ribotypes is shown in Table 2. Ribotype 001 exhibited the highest MICs for
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rifaximin (MIC50/90, >256/>256; GM, 59.521 mg/L) and the highest resistance
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rate for rifaximin (86.3%; n=95), followed by 046 (20%; n=4) and 078/126
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(5.4%; n= 37).
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DISCUSSION
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A high proportion of the CDI episodes at our institution were caused by
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rifaximin-resistant strains. The results of the rifampicin E-test correlated closely
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with those of the rifaximin anaerobic agar dilution method, and the E-test
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correctly classified all rifaximin-resistant strains. For the past 30 years, metronidazole and vancomycin have been the
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mainstay of treatment of CDI. Newer antibiotics such as fidaxomicin and
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recently developed approaches such as monoclonal antibodies aim to
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overcome the main disadvantage of conventional treatments, namely, their high
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recurrence rate [9, 12], although these will probably prove more expensive.
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Rifaximin has been proposed as an adjunctive treatment to decrease
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recurrences [4, 5].Most in vitro studies on rifaximin showed good antibacterial
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activity against C. difficile and rare development of resistant clones[13-15].
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Consistent with previous findings, we observed that the MICs for the isolates
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were either very high or very low [13, 14, 16]. In addition, we observed a very
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high rate of resistance to rifaximin (32.2%, 89 isolates). Similarly high rates
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have only been reported in a study from China (29%) [17], although the authors
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analyzed fewer strains. Other study had previously documented high resistance
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rates to other rifamycin, rifampicin in an epidemic clone at University of
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Pittsburgh Medical Center–Presbyterian Hospital[18].
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Whether the rifampicin E-test can reliably predict susceptibility to
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rifaximin remains a controversial issue [15, 16] [19] [20]. The total number of
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resistant isolates in the studies assessing this issue was quite low. In our study,
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we found that a high number of strains were resistant and that all of the
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rifaximin-resistant isolates were correctly classified using the rifampicin E-test.
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We found no differences between episodes caused by rifaximin-resistant strains and rifaximin-susceptible strains regarding severity of CDI, recurrence,
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overall mortality, or CDI-related mortality. These observations suggest that
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resistance to rifaximin is not a marker for hypervirulent strains. Since none of
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the episodes were treated with rifaximin, we were not able to determine the
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clinical implications of receiving rifaximin in patients infected with resistant
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strains.
The most common ribotype in the present study was 001, which has
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been reported to have one of the highest percentages of resistance to 3 or more
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classes of antibiotics[21]. We observed that 86.3% of ribotype 001 isolates were
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rifaximin-resistant. In the few studies that have further assessed this
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observation, resistant strains corresponded mostly to ribotypes 017 and 027
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[16, 17, 22].
Our findings are limited by the fact that we were not able to establish a
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correlation between the antimicrobial susceptibility testing methods in ribotype
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027 strains owing to the limited number of 027 strains in our center at the time
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of the study. However, to the best of our knowledge, this is the only study to
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assess susceptibility to rifaximin and its correlation with the E-test and to report
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such a high number of rifaximin-resistant strains from patients with clinically
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confirmed CDI. Moreover, this is the only study to have established a correlation
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between these results and the clinical characteristics of the patients.
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In conclusion, resistance to rifaximin was common: resistance rates were
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higher in ribotype 001 strains at our hospital. Susceptibility to rifaximin
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determined using the agar dilution method correlated significantly with
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susceptibility to rifampicin detected using the E-test, including rifaximin resistant
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for the prediction of rifaximin-resistant C. difficile. Clinicians should take local
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epidemiology into account before considering treating a CDI case with rifaximin.
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In this sense, the rifampicin E-test is a simple tool that would enable any
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laboratory to carry out this type of surveillance study.
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Acknowledgments
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We thank Thomas O’Boyle for his help in the preparation of the
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manuscript.
Some of the results of this study were previously presented in poster
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form at the “27th European Congress of Clinical Microbiology and Infectious
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Diseases” (22nd-25th April, 2017; Vienna, Austria).
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Transparency declarations/Potential conflicts of interest. The authors
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declare no conflicts of interest.
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Role of funding source
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This study was financed by Fondo de Investigaciones Sanitarias (FIS),
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Research Project number PI13/00687 and PI16/00490, and by the European
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Regional Development Fund (FEDER) “A way of making Europe”.
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[1] J.G. Bartlett. Clostridium difficile: progress and challenges. Ann N Y Acad Sci 1213 (2010) 62-9. [2] A.N. Ananthakrishnan. Clostridium difficile infection: epidemiology, risk factors and management. Nat Rev Gastroenterol Hepatol 8 (2011) 17-26. [3] S.M. McGlone, R.R. Bailey, S.M. Zimmer, M.J. Popovich, Y. Tian, P. Ufberg, et al. The economic burden of Clostridium difficile. Clin Microbiol Infect 18 (2012) 282-9. [4] K.W. Garey, S.S. Ghantoji, D.N. Shah, M. Habib, V. Arora, Z.D. Jiang, et al. A randomized, double-blind, placebo-controlled pilot study to assess the ability of rifaximin to prevent recurrent diarrhoea in patients with Clostridium difficile infection. J Antimicrob Chemother 66 (2011) 2850-5. [5] S. Johnson, C. Schriever, M. Galang, C.P. Kelly, D.N. Gerding. Interruption of recurrent Clostridium difficile-associated diarrhea episodes by serial therapy with vancomycin and rifaximin. Clin Infect Dis 44 (2007) 846-8. [6] Clinical and Laboratory Standards Institute. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard M11-A8 7th ed. Wayne, PA, USA, 2012
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[7] C.a.L.S. Institute. Performance standards for antimicrobial susceptibility testing: 24th informational supplement M100-S24. . Clinical and Laboratory Standards Institute, Wayne, PA; 2014. [8] S.L. Stubbs, J.S. Brazier, G.L. O'Neill, B.I. Duerden. PCR targeted to the 16S-23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes. J Clin Microbiol 37 (1999) 461-3. [9] S.H. Cohen, D.N. Gerding, S. Johnson, C.P. Kelly, V.G. Loo, L.C. McDonald, et al. Clinical practice guidelines for Clostridium difficile infection in adults: 2010 update by the society for healthcare epidemiology of America (SHEA) and the infectious diseases society of America (IDSA). Infect Control Hosp Epidemiol 31 (2010) 431-55. [10] M.E. Charlson, P. Pompei, K.L. Ales, C.R. MacKenzie. A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J Chronic Dis 40 (1987) 373-83. [11] M. WR, J. GG. Gram-negative bacteremia: I. Etiology and ecology. . Arch Intern Med 110 (1962) 847–55. [12] S.B. Debast, M.P. Bauer, E.J. Kuijper. European Society of Clinical Microbiology and Infectious Diseases: update of the treatment guidance document for Clostridium difficile infection. Clin Microbiol Infect 20 Suppl 2 (2014) 1-26. [13] A. Marchese, A. Salerno, A. Pesce, E.A. Debbia, G.C. Schito. In vitro activity of rifaximin, metronidazole and vancomycin against Clostridium difficile and the rate of selection of spontaneously resistant mutants against representative anaerobic and aerobic bacteria, including ammonia-producing species. Chemotherapy 46 (2000) 253-66. [14] D.W. Hecht, M.A. Galang, S.P. Sambol, J.R. Osmolski, S. Johnson, D.N. Gerding. In vitro activities of 15 antimicrobial agents against 110 toxigenic clostridium difficile clinical isolates collected from 1983 to 2004. Antimicrob Agents Chemother 51 (2007) 2716-9.
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[15] Z.D. Jiang, H.L. DuPont, M. La Rocco, K.W. Garey. In vitro susceptibility of Clostridium difficile to rifaximin and rifampin in 359 consecutive isolates at a university hospital in Houston, Texas. J Clin Pathol 63 (2010) 355-8. [16] J.R. O'Connor, M.A. Galang, S.P. Sambol, D.W. Hecht, G. Vedantam, D.N. Gerding, et al. Rifampin and rifaximin resistance in clinical isolates of Clostridium difficile. Antimicrob Agents Chemother 52 (2008) 2813-7. [17] H. Huang, A. Weintraub, H. Fang, S. Wu, Y. Zhang, C.E. Nord. Antimicrobial susceptibility and heteroresistance in Chinese Clostridium difficile strains. Anaerobe 16 (2010) 633-5. [18] S.R. Curry, J.W. Marsh, K.A. Shutt, C.A. Muto, M.M. O'Leary, M.I. Saul, et al. High frequency of rifampin resistance identified in an epidemic Clostridium difficile clone from a large teaching hospital. Clin Infect Dis 48 (2009) 425-9. [19] M.A. Miller, R. Blanchette, P. Spigaglia, F. Barbanti, P. Mastrantonio. Divergent rifamycin susceptibilities of Clostridium difficile strains in Canada and Italy and predictive accuracy of rifampin Etest for rifamycin resistance. J Clin Microbiol 49 (2011) 4319-21. [20] S. Huhulescu, U. Sagel, A. Fiedler, V. Pecavar, M. Blaschitz, G. Wewalka, et al. Rifaximin disc diffusion test for in vitro susceptibility testing of Clostridium difficile. J Med Microbiol 60 (2011) 1206-12. [21] P. Spigaglia, F. Barbanti, P. Mastrantonio. Multidrug resistance in European Clostridium difficile clinical isolates. J Antimicrob Chemother 66 (2011) 2227-34. [22] J.W. Cheng, M. Xiao, T. Kudinha, F. Kong, Z.P. Xu, L.Y. Sun, et al. Molecular Epidemiology and Antimicrobial Susceptibility of Clostridium difficile Isolates from a University Teaching Hospital in China. Front Microbiol 7 (2016) 1621.
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Table 1. In vitro activity of the antimicrobials tested against 276 clinical
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isolates of Clostridium difficile. MIC Range
MIC50/90
Geometric Mean
Agent
(mg/L)
(mg/L)
(mg/L)
Rifaximin
256
0.015/>256
0.256
Rifampicin
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0.020/>32
0.230
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Antimicrobial
MIC, minimum inhibitory concentration
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isolates of Clostridium difficile according to the most frequent ribotypes
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encountered in our study.
MIC Range (mg/L) Rifampicin 32 Rifaximin 0.009->256 MIC50/90 (mg/L) Rifampicin >32/>32 Rifaximin >256/>256 Geometric Mean (mg/L) Rifampicin 12.601 Rifaximin 59.521 356
MIC, minimal inhibitory concentration
Ribotype 014/020 (n=31)
32 0.004->256
