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Prevalence, genetic relatedness and antibiotic resistance of hospital-acquired clostridium difficile PCR ribotype 018 strains
Accepted Manuscript Title: Prevalence, genetic relatedness and antibiotic resistance of hospitalacquired clostridium difficile PCR ribotype 018 strains Author: Mi-Ran Seo, Jieun Kim, Yangsoon Lee, Dong-Gyun Lim, Hyunjoo Pai PII: DOI: Reference:
S0924-8579(18)30036-0 https://doi.org/10.1016/j.ijantimicag.2018.01.025 ANTAGE 5367
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
26-10-2017 27-1-2018
Please cite this article as: Mi-Ran Seo, Jieun Kim, Yangsoon Lee, Dong-Gyun Lim, Hyunjoo Pai, Prevalence, genetic relatedness and antibiotic resistance of hospital-acquired clostridium difficile PCR ribotype 018 strains, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.01.025. 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.
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TITLE PAGE
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Prevalence, genetic relatedness and antibiotic resistance of hospital-acquired
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Clostridium difficile PCR ribotype 018 strains
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Mi-Ran Seo1a, Jieun Kim1a, Yangsoon Lee2, Dong-Gyun Lim 3b, Hyunjoo Pai1b
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Department of Internal Medicine1 and Laboratory Medicine2, College of Medicine, Hanyang
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University, Center for Chronic Diseases, Research Institute, National Medical Center3, Seoul, Korea
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Corresponding author: Hyunjoo Pai, M.D., Ph.D. Division of Infectious Disease, Department
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of Internal Medicine, College of Medicine, Hanyang University. 232 Wangsimni-ro,
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Seongdong-gu, Seoul, 133-792, Korea
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Tel: 82-2-2290-8356, Fax: 82-2-2298-9183
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E-mail address: [email protected]
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Dong-Gyun Lim, M.D., Ph.D. Center for Chronic Diseases, Research Institute, National Medical
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Center 245 Euljiro, Jung-gu, Seoul 04564, Korea
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Tel: +82-2-2276-2300
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E-mail: [email protected]
Fax: +82-2-2276-2319
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a Mi-Ran Seo and Jieun Kim contributed equally to this work.
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b. Hyunjoo Pai and Dong-Gyun Lim contributed equally to this work
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Highlights
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Clonal change of endemic Clostridium difficile strains was investigated during five-year period
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During 5 years, resistance of antibiotics promoting C. difficile infections increased.
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Resistance to antibiotics for the treatment of CDI did not increase over the same time period.
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Abstract
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Clostridium difficile infection (CDI) is a major healthcare-associated infection. Aim of this
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study was to investigate the genetic relatedness of the endemic C. difficile PCR ribotype 018
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strains in an institution and changes to their characteristics during five-year period.
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A total of 207 isolates obtained from inpatients at Hanyang University Hospital from 2009
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to 2013 were analyzed using multilocus variable-number tandem-repeat analysis (MLVA).
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Minimum inhibitory concentrations (MICs) of several antibiotics were determined.
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In total, 204 (98.6%) were genetically related, with a summed tandem-repeat distance
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(STRD) ≤10. Minimum-spanning-tree analysis identified 78 MLVA types, categorized into
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six clonal complexes (CCs). The largest cluster, CC-I, included 51 MLVA types from 148
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isolates (71.5%) and the second largest cluster, CC-II, included 10 MLVA types from 36
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isolates (17.4%). Resistance rates for antibiotics are as follows: clindamycin (CLI), 97.6%;
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moxifloxacin (MXF), 98.6%; vancomycin (VAN), 1.4%; rifaximin (RFX), 8.2%. All isolates
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were susceptible to TZP and MTZ. Comparing the MICs of antibiotics for the isolates each
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year from 2009 to 2013, MICs of antibiotics which promote CDI such as CLI, MXF, TZP and
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RFX increased over the 5-year period (P value by Kruskal-Wallis test: <0.0001, <0.0001,
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<0.0001, and <0.0001 respectively); however, MICs of VAN or MTZ, antibiotics for
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treatment of CDI, did not increase or decreased over the same time period (P value by
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Kruskal-Wallis test = 0.166, <0.0001).
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C. difficile RT018 isolates in a tertiary hospital over a five-year period presented a close
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clonal relationship. MICs of antibiotics promoting CDI increased with this clonal expansion.
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Key Words: Clostridium difficile, PCR ribotype 018, MLVA, Antimicrobials, Minimum
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inhibitory concentration
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1. Introduction
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Clostridium difficile causes symptoms ranging from mild diarrhea to pseudomembranous
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colitis (PMC), and affects mainly elderly people who have been exposed to antibiotics. The
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increased incidence and severity of C. diffficile infections (CDI) due to an epidemic of PCR
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ribotype 027 (RT027; also called NAP1) strain in North America are well documented [1]. In
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Asian countries, the most prevalent C. diffficile RTs are RT017, RT018, RT014, RT002 and
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RT001 [2-7]. The PCR RT018 strains are positive for C. difficile toxins A and B, but negative
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for the binary toxin, CDT (A+B+CDT–).
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PCR RT018 strains are the most prevalent RT in Korea and Japan [2, 5, 6, 8, 9], and also
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the predominant genotype in Italy [10]. RT018 is highly transmissible and accounted for 95.7%
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of the secondary cases (caused by nosocomial transmission) in a hospital in Italy [10]. RT018
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was reported to cause more severe infections with more toxin-positive stools in a study in
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Korea [11]. The RT018 strains have a higher resistance rate to many antimicrobials, including
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to fluoroquinolones, than other RTs [2, 10, 10], which facilitates successful settlement and
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spread in hospitals. In our previous studies, we reported that RT018 strains cause 30.8% of
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hospital-acquired CDIs in our hospital [11], including multiple outbreaks [2], but the clonal
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distribution of the RT018 strains has not been clearly documented. The aim of this study was
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to investigate the genetic relatedness of the RT018 strains, and to describe the change in
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RT018 strains over a period of 5 years in a single institution, using multilocus variable-
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number tandem-repeat analysis (MLVA) and antibiotic susceptibility tests (ASTs).
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2. Materials and methods
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2.1. Study design and definition
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The study was conducted at the Hanyang University Hospital, a 900-bed tertiary care
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facility in Seoul, Korea. All patients who had a CDI (as defined in the following paragraph)
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from January 2009 to December 2013 were identified through a review of medical charts, and
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were included in this study. The study was approved by the institutional review board of
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Hanyang University Hospital (HYUH IRB 2016-01-031). Informed consent was waived by
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the board.
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Diarrhea was defined as unformed stools more than three times per day on consecutive
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days or six times within 36 hours [13]. We diagnosed CDI using several techniques: (1) when
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stool cultures were positive for C. difficile toxin (tcdA and tcdB) or the binary toxin (cdtA or
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cdtB) genes as identified by multiplex PCR [14]; (2) when positive results were obtained
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from stool samples using a commercial toxin A&B assay kit (VIDASⓇC. difficile toxin A & B;
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BioMerieux SA, Marcy l’Etoile, France), (3) and/or pseudomembrane was identified using
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endoscopy or histology [2]. The patients with CDI who developed diarrhea at least 72 hours
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after hospitalization or within two months of discharge (during which the patient did not
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reside in healthcare facilities) were considered to have healthcare-associated CDI (HA-CDI)
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[15].
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2.2. Isolation of C. difficile and detection of toxin genes by multiplex PCR
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After alcohol shock treatment, stool specimens were cultivated on C. difficile
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moxalactam–norfloxacin–taurocholate agar (CDMN-TA agar; Oxoid Ltd., Cambridge, UK)
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supplemented with 7% horse blood [16]. Colonies of C. difficile were identified by Rapid ID
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32A (BioMerieux SA, Marcy I’Etoile, France). To identify toxin genes, multiplex PCR was
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performed using template DNA, as described previously [17]. The positive controls were
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ATCC 43598 (PCR RT017), ATCC 9689 (PCR RT027), VPI 10643 (ATCC 43255, PCR
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RT087) and ATCC 700057, representing A–B+CDT–, A+B+CDT+, A+B+CDT–, and A–B–CDT–
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RTs, respectively.
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2.3. PCR ribotyping of C. difficile strains
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The PCR ribotyping was performed using genomic DNA, as described elsewhere [18].
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After electrophoresis of the amplified products, the clustering of banding patterns was
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checked visually. Each unique pattern was assigned its own RT code and was compared with
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the PCR RTs of the reference strains RT027 [ATCC 9689], RT017 [ATCC 43598] and
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standard strains from the European Centre for Disease Prevention and Control–Brazier
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collection [19].
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2.4. Multilocus variable-number tandem-repeat analysis
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Molecular genotyping using MLVA was performed using the six variable-number tandem-
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repeat loci: CDR4, CDR5, CDR9, CDR48, CDR49 and CDR60, as described elsewhere [20,
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21]. The forward primer of each loci was labeled at the 5′ end with either 6-
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carboxyfluorescein or hexachlorofluorescein. The PCR products were sent to Solgent Inc
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(Seoul, Korea; http://www.solgent.com/) for GENSCAN analysis. The genetic relationships
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between the genotypes were determined by clustering them according to MLVA type using
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the number of differing loci and the summed absolute distance as coefficients for calculating
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the minimum-spanning tree, using BioNUMERICS software (Version 5.0; Applied Maths
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NV, Sint-Martens-Latem, Belgium). Clusters containing two or more isolates whose MLVA
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types generated a summed tandem-repeat difference (STRD) of ≤2 defined a clonal complex
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(CC), and genetically related clusters were defined by an STRD of ≤10 [20].
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2.5. ASTs
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The minimum inhibitory concentrations (MICs) of six antibiotics— metronidazole (MTZ),
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vancomycin (VAN), piperacillin/tazobactam (TZP), clindamycin (CLI), moxifloxacin (MXF)
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and rifaximin (RFX) — were determined. Brucella agar containing hemin (5ug/mL), vitamin
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K1 (10ug/mL), and 5% horse blood was used for the ASTs, as recommended by the Clinical
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and Laboratory Standards Institute (CLSI) [22]. The MICs of CLI, MXF, VAN were
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determined by Etest (AB-BIODISK, Solna, Sweden) and those of MTZ, RFX and TZP were
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determined by the agar dilution test (Sigma-Aldrich, St. Louis, MO, USA). C. difficile ATCC
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700057 was used as a control strain for the susceptibility tests. Resistance breakpoints (which
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define resistance to an antibiotic) were as defined by the CLSI and the European Committee
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on Antimicrobial Susceptibility Testing (EUCAST) [22, 23].
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2.6. Statistical analysis
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SPSS 21.0 version (SPSS, Chicago, IL, USA) was used for statistical analysis. To analyze
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difference of MIC values by year, we used the Kruskal–Wallis test followed by the
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Bonferroni multiple comparison test. A P-value of <0.05, as determined by a two-tailed test,
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was considered statistically significant.
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3. Results
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3.1. Prevalence of C. difficile strains
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Among a total of 1341 C. difficile isolates obtained from inpatients in Hanyang University
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Hospital from January 2009 to December 2013, 789 (58.8%) were HA-CDI, and 44 (3.3%)
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were community-onset CDI (CO-CDI). A further 250 (18.6%) isolates were from patients
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with toxigenic colonization and 258 (19.2%) were toxin negative. The 789 isolates defined as
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HA-CDI were included in the study.
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3.2. PCR ribotype of C. difficile strains
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PCR ribotyping distinguished 789 C. diffficile strains isolated from patients with HA-CDI.
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Table 1 shows the distribution of PCR ribotypes over the five-year study period; 207 (26.2%)
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isolates were RT018, 201 (25.5%) RT017, and 381 (48.3%) were other RTs. The most
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common RTs after RT017 and RT018 were RT001 (4.7%), RT002 (4.2%), RT015 (2.5%) and
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RT014 (2.3%), but it was rare for RTs other than RT018 and RT017 to account for more than
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10% of total isolates in any given year. The prevalence of RT018 was 28.9% (37/128) in 2009,
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33% (60/182) in 2010, 28.5% (45/158) in 2011, 30.1% (52/173) in 2012 and 8.8% (13/148)
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in 2013, indicating a variable distribution.
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Of the 789 C. difficile isolates, 24 (3%) produced CDT: four RT130 isolates, three RT027
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isolates, three RT078 isolates, two RT267 isolates, one RT122 isolate and 11 isolates of
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unknown ribotype.
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3.3. Clonal distribution of C. difficile RT018 isolates
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To describe the clonal distribution of C. difficile in our hospital during a five-year period,
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MLVA was used to determine the relationship between the 207 RT018 isolates, the most
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prevalent hospital strains in our institution. The 207 isolates included 78 different MLVA
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types, which belonged to one big genetically related cluster. The number of MLVA types by
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year was as follows: 21 types/37 isolates in 2009, 27 types/60 isolates in 2010, 22 types/45
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isolates in 2011, 23 types/52 isolates in 2012 and 10 types/13 isolates in 2013. RT018 isolates
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from 2010 and 2012 showed the least diversity, and the isolates from 2013 showed the most
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diversity. The CDR4 locus was the most diverse variable-number tandem-repeat (VNTR)
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with 22 alleles, and the CDR5 and CDR48 loci showed the least diversity with three alleles
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each. As a clonal complex (CC) is defined as a STRD ≤2 and two or more isolates of the
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same MLVA type, the 78 MLVA types were categorized into six CCs, including two major
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CCs, denoted CC-I and CC-II (Figure 1). CC-I and CC-II are single-locus variants with a
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STRD of 9, and CC-I and CC-III are single-locus variants with a STRD of 4. CC-I is the
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largest CC, with 51 MLVA types from 148 isolates (71.5%), and was identified mostly in
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2009, 2010 and 2013. CC-II includes 10 MLVA types from 36 isolates, and was mostly
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distributed in 2011 and 2012 (Figure 2). Of the 207 RT018 isolates, 204 (98.6%) were
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genetically related (STRD ≤10).
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3.4. Change in antimicrobial susceptibility of C. difficile RT018 isolates over time
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The resistance rates of the 207 RT018 isolates for antibiotics are as follows: CLI, 97.6%
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(202/207); MXF, 98.6% (204/207); VAN, 1.4% (3/207); RFX, 8.2% (17/207). All isolates
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were susceptible to TZP and MTZ. To investigate any changes in antibiotic resistance of the
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clonally related RT018 isolates over five years, we compared the MICs of antibiotics for the
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isolates each year from 2009 to 2013 (Figure 3). Although most isolates were resistant to CLI
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and MXF, the geometric means of the MIC values for CLI and MXF in 2009 were lower than
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in other years (P value by Kruskal-Wallis test : <0.0001, <0.0001). All the isolates were
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susceptible to TZP, but MIC values increased significantly during 5 years (P value by
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Kruskal-Wallis test <0.0001). RFX resistance rate were diverse through 5 years, and the
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increasing trend in geometric mean of MIC values were noticed as well (P value by Kruskal-
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Wallis test <0.0001). However, the MIC value of VAN did not increase and MTZ MIC
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decreased over the same time period (P value by Kruskal-Wallis test = 0.166, <0.0001).
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Three isolates from 2009, 2010 and 2013 were resistant to VAN, and their MIC values were 3,
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3 and 4 mg/L, respectively. As for MTZ, most isolates distributed in MICs from 0.125 to
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1mg/L: 25 isolates with the MIC value of 1mg/L, but no strain with MIC ≥ 2mg/L.
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4. Discussion The aim of the current study was to investigate the prevalence and genetic relatedness of C.
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difficile RT018 strains, and to describe changes in their antibiotic resistance over five years.
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Using MLVA, we described the clonal distribution of RT018 in a single institution, including
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the increased genetic diversity among the RT018 strains over the five-year period (Fig. 1).
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The CDR4 locus was the most diverse VNTR (22 alleles) in RT018, which indicates that
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CDR4 was evolving most rapidly and could achieve high copy numbers. Given those changes,
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the predominant MLVA types appear to be changing.
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We then investigated whether antibiotic resistance is a driving force for clonal change.
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Indeed, the MIC values of several antibiotics for RT018 strains showed an increasing trend
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year-on-year, whether or not the strains were susceptible to those antibiotics. However,
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because we could not compare the MICs of antibiotics for different MLVA types, we could
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not demonstrate that genetic divergence occurs because of increasing MICs. Whole genome
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sequencing of the isolates to further characterize genetic relatedness will provide more
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information.
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Antibiotic resistance has become a worldwide problem for the treatment of C. difficile,
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although most antibiotics are not effective against CDIs, and this provides important selective
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pressure for resistant organisms to survive in hospital setting. In a study conducted in 14
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European countries during 2005, the resistance rates of C. difficile to CLI and MXF were
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46.1% and 37.5%, respectively [24], and in a study from China the resistance rates to MXF,
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CLI, TET and rifampicin were 46.4%, 71.4%, 35.7% and 25.0%, respectively [25]. A study
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from Korea reported that resistance rates of imipenem, cefotetan, MXF, ampicillin, and CLI
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were 25%, 34%, 42%, 51% and 60%, respectively among 120 C. difficile isolates collected
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between 2006 and 2008 from 12 hospitals [19].
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In the present study, C. difficile RT018 was highly resistant to CLI and MXF (97.6% and
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98.6%, respectively), and our results show a similar trend with those reported from Italy [10].
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In a European study comparing the geometric mean MICs of several antibiotics for different
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strains of C. difficile, RT018 showed the highest MICs for MXF, along with RT027 and
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RT017 [12]. Antimicrobials from the rifamycin group, including rifampin (RFP) and RFX,
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have been used for CDI treatment failures and recurring CDI. Although EUCAST determines
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epidemiological MIC breakpoint of RFP as 0.004mg/L and RFP resistance is known to
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correlate with RFX [23], MIC breakpoint of RFX is not determined. In the absence of CLSI-
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or EUCAST-defined MIC breakpoints for RFX against C. difficile, we used a MIC ≥32mg/L
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as the criterion for reduced in vitro susceptibility [12]. In the current study, 8.2% (17/207) of
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the RT018 C. difficile isolates were resistant to RFX, with a MIC50 of 0.004mg/L and a
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MIC90 of 0.25mg/L. The previously reported MICs of RFP and RFX for C. difficile isolates
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were either very low (≤0.002mg/L) or very high (≥32mg/L) [26, 27]. In the present study, 17
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RFX-resistant isolates exhibited MIC values ≥64mg/L, but MICs of 16 susceptible isolates
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ranged from 0.008 to 2mg/L.
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MTZ and VAN are the current drugs of choice for treatment of CDI, and are generally
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used as first-line therapy; however, recent studies of clinical isolates from the UK have
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reported decreased rates of susceptibility to MTZ and VAN [28]. The geometric mean of
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MICs of MTZ for the historical C. difficile RT001 were 1.03 mg/L (range 0.25–2mg/L)
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compared with 5.94mg/L (range 4–8mg/L) (P <0.001) for recent isolates with reduced MTZ
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susceptibility (24.4% of isolates) [28]. In the present study, MTZ, TZP and VAN were
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generally effective against RT018. It is interesting that no MTZ-resistant isolate was found
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among multidrug-resistant RT018 strains and the geometric mean MICs of MTZ and VAN
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did not increase over 5 years, while the MICs of several antibiotics promoting CDI
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development showed a MIC creep among these strains. These findings indicate that
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antibiotics which promote CDI is a major selective force for endemic multidrug-resistant
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strains to survive in an institution. Nevertheless, in our CD collection during the same period,
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2% of the isolates with a diverse susceptibility to other antibiotics and diverse PCR RTs
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exhibited a MIC value of MTZ ≥ 4 mg/L (data not shown).
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5. Conclusions
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In this study, we describe the clonal change in C. difficile RT018 strains, the most
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prevalent nosocomially transmitted strains of C. difficile (26.2%) in a tertiary hospital in
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Korea, during a five-year period using MLVA. MICs of CLI, MXF, RFX and TZP for the
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clonally related RT018 strains increased gradually over five years.
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Declarations
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Funding: This research was supported by Research Program funded by National Medical
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Center, Research Institute (grant number: NMC2016-MS-02).
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Competing Interests: None to declare.
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Ethical Approval: Yes. HYUH IRB 2016-01-031
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Kim H, Jeong SH, Roh KH, Hong SG, Kim JW, Shin MG, et al. Investigation of toxin gene diversity,
Marsh JW, O'Leary MM, Shutt KA, Pasculle AW, Johnson S, Gerding DN, et al. Multilocus variable-
Curry SR, Muto CA, Schlackman JL, Pasculle AW, Shutt KA, Marsh JW, et al. Use of multilocus
Clinical and Laboratory Standards Institute. Methods for antimicrobial susceptibility testing of
European Committee on Antimicrobial Susceptibility Testing (EUCAST). 2017-03-10 (version
Page 14 of 23
329
[24]
330
difficile infections in Europe with phenotypic and genotypic characterisation of the isolates. Clin Microbiol
331
Infect 2007;13:1048-57.
332
[25]
333
Shanghai hospital: antimicrobial resistance, toxin profiles and ribotypes. Int J Antimicrob Agents 2009;33:339-
334
342.
335
[26]
336
test for in vitro susceptibility testing of Clostridium difficile. J Med Microbiol 2011;60:1206-12.
337
[27]
338
rifaximin resistance in clinical isolates of Clostridium difficile. Antimicrob Agents Chemother 2008;52:2813-7.
339
[28]
340
reduced susceptibility to metronidazole in Clostridium difficile. J Antimicrob Chemother 2008;2:1046-52.
Barbut F, Mastrantonio P, Delmee M, Brazier J, Kuijper E, Poxton I. Prospective study of Clostridium
Huang H, Wu S, Wang M, Zhang Y, Fang H, Palmgren AC, et al. Clostridium difficile infections in a
Huhulescu S, Sagel U, Fiedler A, Pecavar V, Blaschitz M, Wewalka G, et al. Rifaximin disc diffusion
O'Connor JR, Galang MA, Sambol SP, Hecht DW, Vedantam G, Gerding DN, et al. Rifampin and
Baines SD, O'Connor R, Freeman J, Fawley WN, Harmanus C, Mastrantonio P, et al. Emergence of
341 342 343 344
Fig. 1. Minimum spanning tree analysis of Clostridium difficile RT018 isolates classified by
345
multilocus variable-number tandem-repeat analysis (MLVA). Each circle represents a unique MLVA
346
type. Numbers between the circles represent the summed tandem–repeat differences (STRD) between
347
MLVA types. Clonal complexes (CC-I to CC-VI) are marked clusters containing two or more isolates
348
whose MLVA types generated a STRD of ≤2.
349 350
Fig. 2. Annual distribution of clonal complexes of Clostridium difficile PCR ribotype 018.
351 352
Fig. 3. Antimicrobial susceptibilities and geometric mean MIC of Clostridium difficile PCR ribotype
353
018 from 2009 to 2012. Light gray areas indicate resistance, and dark gray areas indicate
354
susceptibility to various antimicrobial agents. (A) clindamycin (P value by Kruskal-Wallis test :
355
<0.0001), (B) moxifloxacin (P value by Kruskal-Wallis test : <0.0001), (C)
356
piperacillin/tazobactam (P value by Kruskal-Wallis test : <0.0001), (D) rifaximin (P value by
Page 15 of 23
357
Kruskal-Wallis test : <0.0001), (E) vancomycin (P value by Kruskal-Wallis test : 0.166), (F)
358
metronidazole (P value by Kruskal-Wallis test : <0.0001).
359 360 361
Page 16 of 23
Table 1. Distribution of Clostridium difficile PCR ribotypes in a single hospital between 2009 and 2013
Year
PCR ribotype 001
002
012
2009 (n=128)
14 (10.9)
5 (3.9)
0.0
2010 (n=182)
7 (3.8)
1 (0.5)
0.0
2011 (n=158)
6 (3.8)
0.0
0.0
2012 (n=173)
7 (4.0)
5 (2.9)
6 (3.5)
2013 (n=148)
3 (2.0)
22 (14.9)
5 (3.4)
37 (4.7)
33 (4.2)
11 (1.4)
Total
014
015
017*
018
027*
078*
*
*
085
106
112
122*
130*
*
*
163
267*
*
293
Unknown* **
6 (4.7 ) 5 (2.7 ) 3 (1.9 )
0.0
20 (15.6)
37 (28.9)
3 (2.3)
0.0
2 (1.6 )
0.0
4 (3.1)
1 (0.8)
4 (3.1)
2 (1.6)
1 (0.8)
3 (2.3)
26 (20.3)
2 (1.1)
59 (32.4)
60 (33.0)
0.0
1 (0.5)
0.0
0.0
3 (1.6)
0.0
0.0
3 (1.6)
0.0
0.0
41 (22.5)
1 (0.6)
50 (31.6)
45 (28.5)
0.0
2 (1.3)
0.0
0.0
6 (3.8)
0.0
0.0
1 (0.6)
0.0
2 (1.3)
42 (26.6)
0.0
15 (8.7)
29 (16.8)
52 (30.1)
0.0
0.0
0.0
1 (0.6)
4 (2.3)
0.0
0.0
2 (1.2)
1 (0.6)
5 (2.9)
46 (26.6)
2 (1.4)
43 (29.1)
13 (8.8)
0.0
0.0
0.0
1 (0.7)
9 (6.1)
0.0
0.0
2 (1.4)
0.0
1 (0.7)
43 (29.1)
20 (2.5)
201 (25.5)
207 (26.2)
3 (0.4)
3 (0.4)
2 (0.3 )
2 (0.3)
26 (3.3)
1 (0.1)
4 (0.5)
10 (1.3)
2 (0.3)
11 (1.4)
198 (25.1)
4 (2.7 ) 18 (2.3 )
All data are number (%), unless otherwise stated. *All A–B+ strains were RT017. **PCR ribotypes 027, 078, 122, 130, and 267 were A+B+CDT+. *** PCR ribotypes unknown.
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S0924-8579(18)30036-0 https://doi.org/10.1016/j.ijantimicag.2018.01.025 ANTAGE 5367
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
26-10-2017 27-1-2018
Please cite this article as: Mi-Ran Seo, Jieun Kim, Yangsoon Lee, Dong-Gyun Lim, Hyunjoo Pai, Prevalence, genetic relatedness and antibiotic resistance of hospital-acquired clostridium difficile PCR ribotype 018 strains, International Journal of Antimicrobial Agents (2018), https://doi.org/10.1016/j.ijantimicag.2018.01.025. 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.
1
TITLE PAGE
2 3
Prevalence, genetic relatedness and antibiotic resistance of hospital-acquired
4
Clostridium difficile PCR ribotype 018 strains
5 6
Mi-Ran Seo1a, Jieun Kim1a, Yangsoon Lee2, Dong-Gyun Lim 3b, Hyunjoo Pai1b
7 8
Department of Internal Medicine1 and Laboratory Medicine2, College of Medicine, Hanyang
9
University, Center for Chronic Diseases, Research Institute, National Medical Center3, Seoul, Korea
10 11 12 13
Corresponding author: Hyunjoo Pai, M.D., Ph.D. Division of Infectious Disease, Department
14
of Internal Medicine, College of Medicine, Hanyang University. 232 Wangsimni-ro,
15
Seongdong-gu, Seoul, 133-792, Korea
16
Tel: 82-2-2290-8356, Fax: 82-2-2298-9183
17
E-mail address: [email protected]
18 19
Dong-Gyun Lim, M.D., Ph.D. Center for Chronic Diseases, Research Institute, National Medical
20
Center 245 Euljiro, Jung-gu, Seoul 04564, Korea
21
Tel: +82-2-2276-2300
22
E-mail: [email protected]
Fax: +82-2-2276-2319
23 24
a Mi-Ran Seo and Jieun Kim contributed equally to this work.
25
b. Hyunjoo Pai and Dong-Gyun Lim contributed equally to this work
Page 1 of 23
26
Highlights
27 28
Clonal change of endemic Clostridium difficile strains was investigated during five-year period
29 30
During 5 years, resistance of antibiotics promoting C. difficile infections increased.
31 32
Resistance to antibiotics for the treatment of CDI did not increase over the same time period.
33
Abstract
34
Clostridium difficile infection (CDI) is a major healthcare-associated infection. Aim of this
35
study was to investigate the genetic relatedness of the endemic C. difficile PCR ribotype 018
36
strains in an institution and changes to their characteristics during five-year period.
37
A total of 207 isolates obtained from inpatients at Hanyang University Hospital from 2009
38
to 2013 were analyzed using multilocus variable-number tandem-repeat analysis (MLVA).
39
Minimum inhibitory concentrations (MICs) of several antibiotics were determined.
40
In total, 204 (98.6%) were genetically related, with a summed tandem-repeat distance
41
(STRD) ≤10. Minimum-spanning-tree analysis identified 78 MLVA types, categorized into
42
six clonal complexes (CCs). The largest cluster, CC-I, included 51 MLVA types from 148
43
isolates (71.5%) and the second largest cluster, CC-II, included 10 MLVA types from 36
44
isolates (17.4%). Resistance rates for antibiotics are as follows: clindamycin (CLI), 97.6%;
45
moxifloxacin (MXF), 98.6%; vancomycin (VAN), 1.4%; rifaximin (RFX), 8.2%. All isolates
46
were susceptible to TZP and MTZ. Comparing the MICs of antibiotics for the isolates each
47
year from 2009 to 2013, MICs of antibiotics which promote CDI such as CLI, MXF, TZP and
48
RFX increased over the 5-year period (P value by Kruskal-Wallis test: <0.0001, <0.0001,
49
<0.0001, and <0.0001 respectively); however, MICs of VAN or MTZ, antibiotics for
50
treatment of CDI, did not increase or decreased over the same time period (P value by
51
Kruskal-Wallis test = 0.166, <0.0001).
52
C. difficile RT018 isolates in a tertiary hospital over a five-year period presented a close
Page 2 of 23
53
clonal relationship. MICs of antibiotics promoting CDI increased with this clonal expansion.
54 55
Key Words: Clostridium difficile, PCR ribotype 018, MLVA, Antimicrobials, Minimum
56
inhibitory concentration
57
Page 3 of 23
58
1. Introduction
59
Clostridium difficile causes symptoms ranging from mild diarrhea to pseudomembranous
60
colitis (PMC), and affects mainly elderly people who have been exposed to antibiotics. The
61
increased incidence and severity of C. diffficile infections (CDI) due to an epidemic of PCR
62
ribotype 027 (RT027; also called NAP1) strain in North America are well documented [1]. In
63
Asian countries, the most prevalent C. diffficile RTs are RT017, RT018, RT014, RT002 and
64
RT001 [2-7]. The PCR RT018 strains are positive for C. difficile toxins A and B, but negative
65
for the binary toxin, CDT (A+B+CDT–).
66
PCR RT018 strains are the most prevalent RT in Korea and Japan [2, 5, 6, 8, 9], and also
67
the predominant genotype in Italy [10]. RT018 is highly transmissible and accounted for 95.7%
68
of the secondary cases (caused by nosocomial transmission) in a hospital in Italy [10]. RT018
69
was reported to cause more severe infections with more toxin-positive stools in a study in
70
Korea [11]. The RT018 strains have a higher resistance rate to many antimicrobials, including
71
to fluoroquinolones, than other RTs [2, 10, 10], which facilitates successful settlement and
72
spread in hospitals. In our previous studies, we reported that RT018 strains cause 30.8% of
73
hospital-acquired CDIs in our hospital [11], including multiple outbreaks [2], but the clonal
74
distribution of the RT018 strains has not been clearly documented. The aim of this study was
75
to investigate the genetic relatedness of the RT018 strains, and to describe the change in
76
RT018 strains over a period of 5 years in a single institution, using multilocus variable-
77
number tandem-repeat analysis (MLVA) and antibiotic susceptibility tests (ASTs).
78 79
2. Materials and methods
80
2.1. Study design and definition
81
The study was conducted at the Hanyang University Hospital, a 900-bed tertiary care
82
facility in Seoul, Korea. All patients who had a CDI (as defined in the following paragraph)
Page 4 of 23
83
from January 2009 to December 2013 were identified through a review of medical charts, and
84
were included in this study. The study was approved by the institutional review board of
85
Hanyang University Hospital (HYUH IRB 2016-01-031). Informed consent was waived by
86
the board.
87
Diarrhea was defined as unformed stools more than three times per day on consecutive
88
days or six times within 36 hours [13]. We diagnosed CDI using several techniques: (1) when
89
stool cultures were positive for C. difficile toxin (tcdA and tcdB) or the binary toxin (cdtA or
90
cdtB) genes as identified by multiplex PCR [14]; (2) when positive results were obtained
91
from stool samples using a commercial toxin A&B assay kit (VIDASⓇC. difficile toxin A & B;
92
BioMerieux SA, Marcy l’Etoile, France), (3) and/or pseudomembrane was identified using
93
endoscopy or histology [2]. The patients with CDI who developed diarrhea at least 72 hours
94
after hospitalization or within two months of discharge (during which the patient did not
95
reside in healthcare facilities) were considered to have healthcare-associated CDI (HA-CDI)
96
[15].
97 98
2.2. Isolation of C. difficile and detection of toxin genes by multiplex PCR
99
After alcohol shock treatment, stool specimens were cultivated on C. difficile
100
moxalactam–norfloxacin–taurocholate agar (CDMN-TA agar; Oxoid Ltd., Cambridge, UK)
101
supplemented with 7% horse blood [16]. Colonies of C. difficile were identified by Rapid ID
102
32A (BioMerieux SA, Marcy I’Etoile, France). To identify toxin genes, multiplex PCR was
103
performed using template DNA, as described previously [17]. The positive controls were
104
ATCC 43598 (PCR RT017), ATCC 9689 (PCR RT027), VPI 10643 (ATCC 43255, PCR
105
RT087) and ATCC 700057, representing A–B+CDT–, A+B+CDT+, A+B+CDT–, and A–B–CDT–
106
RTs, respectively.
107
Page 5 of 23
108
2.3. PCR ribotyping of C. difficile strains
109
The PCR ribotyping was performed using genomic DNA, as described elsewhere [18].
110
After electrophoresis of the amplified products, the clustering of banding patterns was
111
checked visually. Each unique pattern was assigned its own RT code and was compared with
112
the PCR RTs of the reference strains RT027 [ATCC 9689], RT017 [ATCC 43598] and
113
standard strains from the European Centre for Disease Prevention and Control–Brazier
114
collection [19].
115 116
2.4. Multilocus variable-number tandem-repeat analysis
117
Molecular genotyping using MLVA was performed using the six variable-number tandem-
118
repeat loci: CDR4, CDR5, CDR9, CDR48, CDR49 and CDR60, as described elsewhere [20,
119
21]. The forward primer of each loci was labeled at the 5′ end with either 6-
120
carboxyfluorescein or hexachlorofluorescein. The PCR products were sent to Solgent Inc
121
(Seoul, Korea; http://www.solgent.com/) for GENSCAN analysis. The genetic relationships
122
between the genotypes were determined by clustering them according to MLVA type using
123
the number of differing loci and the summed absolute distance as coefficients for calculating
124
the minimum-spanning tree, using BioNUMERICS software (Version 5.0; Applied Maths
125
NV, Sint-Martens-Latem, Belgium). Clusters containing two or more isolates whose MLVA
126
types generated a summed tandem-repeat difference (STRD) of ≤2 defined a clonal complex
127
(CC), and genetically related clusters were defined by an STRD of ≤10 [20].
128 129
2.5. ASTs
130
The minimum inhibitory concentrations (MICs) of six antibiotics— metronidazole (MTZ),
131
vancomycin (VAN), piperacillin/tazobactam (TZP), clindamycin (CLI), moxifloxacin (MXF)
132
and rifaximin (RFX) — were determined. Brucella agar containing hemin (5ug/mL), vitamin
Page 6 of 23
133
K1 (10ug/mL), and 5% horse blood was used for the ASTs, as recommended by the Clinical
134
and Laboratory Standards Institute (CLSI) [22]. The MICs of CLI, MXF, VAN were
135
determined by Etest (AB-BIODISK, Solna, Sweden) and those of MTZ, RFX and TZP were
136
determined by the agar dilution test (Sigma-Aldrich, St. Louis, MO, USA). C. difficile ATCC
137
700057 was used as a control strain for the susceptibility tests. Resistance breakpoints (which
138
define resistance to an antibiotic) were as defined by the CLSI and the European Committee
139
on Antimicrobial Susceptibility Testing (EUCAST) [22, 23].
140 141
2.6. Statistical analysis
142
SPSS 21.0 version (SPSS, Chicago, IL, USA) was used for statistical analysis. To analyze
143
difference of MIC values by year, we used the Kruskal–Wallis test followed by the
144
Bonferroni multiple comparison test. A P-value of <0.05, as determined by a two-tailed test,
145
was considered statistically significant.
146 147
3. Results
148
3.1. Prevalence of C. difficile strains
149
Among a total of 1341 C. difficile isolates obtained from inpatients in Hanyang University
150
Hospital from January 2009 to December 2013, 789 (58.8%) were HA-CDI, and 44 (3.3%)
151
were community-onset CDI (CO-CDI). A further 250 (18.6%) isolates were from patients
152
with toxigenic colonization and 258 (19.2%) were toxin negative. The 789 isolates defined as
153
HA-CDI were included in the study.
154 155
3.2. PCR ribotype of C. difficile strains
156
PCR ribotyping distinguished 789 C. diffficile strains isolated from patients with HA-CDI.
157
Table 1 shows the distribution of PCR ribotypes over the five-year study period; 207 (26.2%)
Page 7 of 23
158
isolates were RT018, 201 (25.5%) RT017, and 381 (48.3%) were other RTs. The most
159
common RTs after RT017 and RT018 were RT001 (4.7%), RT002 (4.2%), RT015 (2.5%) and
160
RT014 (2.3%), but it was rare for RTs other than RT018 and RT017 to account for more than
161
10% of total isolates in any given year. The prevalence of RT018 was 28.9% (37/128) in 2009,
162
33% (60/182) in 2010, 28.5% (45/158) in 2011, 30.1% (52/173) in 2012 and 8.8% (13/148)
163
in 2013, indicating a variable distribution.
164
Of the 789 C. difficile isolates, 24 (3%) produced CDT: four RT130 isolates, three RT027
165
isolates, three RT078 isolates, two RT267 isolates, one RT122 isolate and 11 isolates of
166
unknown ribotype.
167 168
3.3. Clonal distribution of C. difficile RT018 isolates
169
To describe the clonal distribution of C. difficile in our hospital during a five-year period,
170
MLVA was used to determine the relationship between the 207 RT018 isolates, the most
171
prevalent hospital strains in our institution. The 207 isolates included 78 different MLVA
172
types, which belonged to one big genetically related cluster. The number of MLVA types by
173
year was as follows: 21 types/37 isolates in 2009, 27 types/60 isolates in 2010, 22 types/45
174
isolates in 2011, 23 types/52 isolates in 2012 and 10 types/13 isolates in 2013. RT018 isolates
175
from 2010 and 2012 showed the least diversity, and the isolates from 2013 showed the most
176
diversity. The CDR4 locus was the most diverse variable-number tandem-repeat (VNTR)
177
with 22 alleles, and the CDR5 and CDR48 loci showed the least diversity with three alleles
178
each. As a clonal complex (CC) is defined as a STRD ≤2 and two or more isolates of the
179
same MLVA type, the 78 MLVA types were categorized into six CCs, including two major
180
CCs, denoted CC-I and CC-II (Figure 1). CC-I and CC-II are single-locus variants with a
181
STRD of 9, and CC-I and CC-III are single-locus variants with a STRD of 4. CC-I is the
182
largest CC, with 51 MLVA types from 148 isolates (71.5%), and was identified mostly in
Page 8 of 23
183
2009, 2010 and 2013. CC-II includes 10 MLVA types from 36 isolates, and was mostly
184
distributed in 2011 and 2012 (Figure 2). Of the 207 RT018 isolates, 204 (98.6%) were
185
genetically related (STRD ≤10).
186 187
3.4. Change in antimicrobial susceptibility of C. difficile RT018 isolates over time
188
The resistance rates of the 207 RT018 isolates for antibiotics are as follows: CLI, 97.6%
189
(202/207); MXF, 98.6% (204/207); VAN, 1.4% (3/207); RFX, 8.2% (17/207). All isolates
190
were susceptible to TZP and MTZ. To investigate any changes in antibiotic resistance of the
191
clonally related RT018 isolates over five years, we compared the MICs of antibiotics for the
192
isolates each year from 2009 to 2013 (Figure 3). Although most isolates were resistant to CLI
193
and MXF, the geometric means of the MIC values for CLI and MXF in 2009 were lower than
194
in other years (P value by Kruskal-Wallis test : <0.0001, <0.0001). All the isolates were
195
susceptible to TZP, but MIC values increased significantly during 5 years (P value by
196
Kruskal-Wallis test <0.0001). RFX resistance rate were diverse through 5 years, and the
197
increasing trend in geometric mean of MIC values were noticed as well (P value by Kruskal-
198
Wallis test <0.0001). However, the MIC value of VAN did not increase and MTZ MIC
199
decreased over the same time period (P value by Kruskal-Wallis test = 0.166, <0.0001).
200
Three isolates from 2009, 2010 and 2013 were resistant to VAN, and their MIC values were 3,
201
3 and 4 mg/L, respectively. As for MTZ, most isolates distributed in MICs from 0.125 to
202
1mg/L: 25 isolates with the MIC value of 1mg/L, but no strain with MIC ≥ 2mg/L.
203 204 205
4. Discussion The aim of the current study was to investigate the prevalence and genetic relatedness of C.
206
difficile RT018 strains, and to describe changes in their antibiotic resistance over five years.
207
Using MLVA, we described the clonal distribution of RT018 in a single institution, including
Page 9 of 23
208
the increased genetic diversity among the RT018 strains over the five-year period (Fig. 1).
209
The CDR4 locus was the most diverse VNTR (22 alleles) in RT018, which indicates that
210
CDR4 was evolving most rapidly and could achieve high copy numbers. Given those changes,
211
the predominant MLVA types appear to be changing.
212
We then investigated whether antibiotic resistance is a driving force for clonal change.
213
Indeed, the MIC values of several antibiotics for RT018 strains showed an increasing trend
214
year-on-year, whether or not the strains were susceptible to those antibiotics. However,
215
because we could not compare the MICs of antibiotics for different MLVA types, we could
216
not demonstrate that genetic divergence occurs because of increasing MICs. Whole genome
217
sequencing of the isolates to further characterize genetic relatedness will provide more
218
information.
219
Antibiotic resistance has become a worldwide problem for the treatment of C. difficile,
220
although most antibiotics are not effective against CDIs, and this provides important selective
221
pressure for resistant organisms to survive in hospital setting. In a study conducted in 14
222
European countries during 2005, the resistance rates of C. difficile to CLI and MXF were
223
46.1% and 37.5%, respectively [24], and in a study from China the resistance rates to MXF,
224
CLI, TET and rifampicin were 46.4%, 71.4%, 35.7% and 25.0%, respectively [25]. A study
225
from Korea reported that resistance rates of imipenem, cefotetan, MXF, ampicillin, and CLI
226
were 25%, 34%, 42%, 51% and 60%, respectively among 120 C. difficile isolates collected
227
between 2006 and 2008 from 12 hospitals [19].
228
In the present study, C. difficile RT018 was highly resistant to CLI and MXF (97.6% and
229
98.6%, respectively), and our results show a similar trend with those reported from Italy [10].
230
In a European study comparing the geometric mean MICs of several antibiotics for different
231
strains of C. difficile, RT018 showed the highest MICs for MXF, along with RT027 and
232
RT017 [12]. Antimicrobials from the rifamycin group, including rifampin (RFP) and RFX,
Page 10 of 23
233
have been used for CDI treatment failures and recurring CDI. Although EUCAST determines
234
epidemiological MIC breakpoint of RFP as 0.004mg/L and RFP resistance is known to
235
correlate with RFX [23], MIC breakpoint of RFX is not determined. In the absence of CLSI-
236
or EUCAST-defined MIC breakpoints for RFX against C. difficile, we used a MIC ≥32mg/L
237
as the criterion for reduced in vitro susceptibility [12]. In the current study, 8.2% (17/207) of
238
the RT018 C. difficile isolates were resistant to RFX, with a MIC50 of 0.004mg/L and a
239
MIC90 of 0.25mg/L. The previously reported MICs of RFP and RFX for C. difficile isolates
240
were either very low (≤0.002mg/L) or very high (≥32mg/L) [26, 27]. In the present study, 17
241
RFX-resistant isolates exhibited MIC values ≥64mg/L, but MICs of 16 susceptible isolates
242
ranged from 0.008 to 2mg/L.
243
MTZ and VAN are the current drugs of choice for treatment of CDI, and are generally
244
used as first-line therapy; however, recent studies of clinical isolates from the UK have
245
reported decreased rates of susceptibility to MTZ and VAN [28]. The geometric mean of
246
MICs of MTZ for the historical C. difficile RT001 were 1.03 mg/L (range 0.25–2mg/L)
247
compared with 5.94mg/L (range 4–8mg/L) (P <0.001) for recent isolates with reduced MTZ
248
susceptibility (24.4% of isolates) [28]. In the present study, MTZ, TZP and VAN were
249
generally effective against RT018. It is interesting that no MTZ-resistant isolate was found
250
among multidrug-resistant RT018 strains and the geometric mean MICs of MTZ and VAN
251
did not increase over 5 years, while the MICs of several antibiotics promoting CDI
252
development showed a MIC creep among these strains. These findings indicate that
253
antibiotics which promote CDI is a major selective force for endemic multidrug-resistant
254
strains to survive in an institution. Nevertheless, in our CD collection during the same period,
255
2% of the isolates with a diverse susceptibility to other antibiotics and diverse PCR RTs
Page 11 of 23
256
exhibited a MIC value of MTZ ≥ 4 mg/L (data not shown).
257 258
5. Conclusions
259
In this study, we describe the clonal change in C. difficile RT018 strains, the most
260
prevalent nosocomially transmitted strains of C. difficile (26.2%) in a tertiary hospital in
261
Korea, during a five-year period using MLVA. MICs of CLI, MXF, RFX and TZP for the
262
clonally related RT018 strains increased gradually over five years.
263 264
Declarations
265
Funding: This research was supported by Research Program funded by National Medical
266
Center, Research Institute (grant number: NMC2016-MS-02).
267
Competing Interests: None to declare.
268
Ethical Approval: Yes. HYUH IRB 2016-01-031
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341 342 343 344
Fig. 1. Minimum spanning tree analysis of Clostridium difficile RT018 isolates classified by
345
multilocus variable-number tandem-repeat analysis (MLVA). Each circle represents a unique MLVA
346
type. Numbers between the circles represent the summed tandem–repeat differences (STRD) between
347
MLVA types. Clonal complexes (CC-I to CC-VI) are marked clusters containing two or more isolates
348
whose MLVA types generated a STRD of ≤2.
349 350
Fig. 2. Annual distribution of clonal complexes of Clostridium difficile PCR ribotype 018.
351 352
Fig. 3. Antimicrobial susceptibilities and geometric mean MIC of Clostridium difficile PCR ribotype
353
018 from 2009 to 2012. Light gray areas indicate resistance, and dark gray areas indicate
354
susceptibility to various antimicrobial agents. (A) clindamycin (P value by Kruskal-Wallis test :
355
<0.0001), (B) moxifloxacin (P value by Kruskal-Wallis test : <0.0001), (C)
356
piperacillin/tazobactam (P value by Kruskal-Wallis test : <0.0001), (D) rifaximin (P value by
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357
Kruskal-Wallis test : <0.0001), (E) vancomycin (P value by Kruskal-Wallis test : 0.166), (F)
358
metronidazole (P value by Kruskal-Wallis test : <0.0001).
359 360 361
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Table 1. Distribution of Clostridium difficile PCR ribotypes in a single hospital between 2009 and 2013
Year
PCR ribotype 001
002
012
2009 (n=128)
14 (10.9)
5 (3.9)
0.0
2010 (n=182)
7 (3.8)
1 (0.5)
0.0
2011 (n=158)
6 (3.8)
0.0
0.0
2012 (n=173)
7 (4.0)
5 (2.9)
6 (3.5)
2013 (n=148)
3 (2.0)
22 (14.9)
5 (3.4)
37 (4.7)
33 (4.2)
11 (1.4)
Total
014
015
017*
018
027*
078*
*
*
085
106
112
122*
130*
*
*
163
267*
*
293
Unknown* **
6 (4.7 ) 5 (2.7 ) 3 (1.9 )
0.0
20 (15.6)
37 (28.9)
3 (2.3)
0.0
2 (1.6 )
0.0
4 (3.1)
1 (0.8)
4 (3.1)
2 (1.6)
1 (0.8)
3 (2.3)
26 (20.3)
2 (1.1)
59 (32.4)
60 (33.0)
0.0
1 (0.5)
0.0
0.0
3 (1.6)
0.0
0.0
3 (1.6)
0.0
0.0
41 (22.5)
1 (0.6)
50 (31.6)
45 (28.5)
0.0
2 (1.3)
0.0
0.0
6 (3.8)
0.0
0.0
1 (0.6)
0.0
2 (1.3)
42 (26.6)
0.0
15 (8.7)
29 (16.8)
52 (30.1)
0.0
0.0
0.0
1 (0.6)
4 (2.3)
0.0
0.0
2 (1.2)
1 (0.6)
5 (2.9)
46 (26.6)
2 (1.4)
43 (29.1)
13 (8.8)
0.0
0.0
0.0
1 (0.7)
9 (6.1)
0.0
0.0
2 (1.4)
0.0
1 (0.7)
43 (29.1)
20 (2.5)
201 (25.5)
207 (26.2)
3 (0.4)
3 (0.4)
2 (0.3 )
2 (0.3)
26 (3.3)
1 (0.1)
4 (0.5)
10 (1.3)
2 (0.3)
11 (1.4)
198 (25.1)
4 (2.7 ) 18 (2.3 )
All data are number (%), unless otherwise stated. *All A–B+ strains were RT017. **PCR ribotypes 027, 078, 122, 130, and 267 were A+B+CDT+. *** PCR ribotypes unknown.
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