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Background: Clostridium difficile infection (CDI) is known as one of the most important causes of nosocomial infections. The main objective of this study was to evaluate the presence of Clostridium difficile in the stool of hospitalized patients with diarrhea as well as in their environments. Methods: C. difficile isolates were characterized according to the presence of toxin genes and antibiotic resistance. Multilocus Sequence Typing Analysis (MLST) was applied for finding the genetic polymorphism and relationship among strain lineages. Results: A total of 821 samples (574 stools and 247 swabs) were collected between April 2015 and May 2017. The prevalence of C. difficile isolates was 28.6% (164/574) in patients and 19% (47/247) in swabs taken from medical devices, hands of healthcare workers and skin patient sites. Finally, 11.5% (66/574) toxigenic C. difficile strains isolated from stool samples of inpatients and 4.4% (11/247) from hands of healthcare workers and skin patient sites. All the toxigenic isolates were inhibited by a low concentration of vancomycin (MIC < 0.5 μg/ml). About 43% (33/77) and 39% of isolates were resistant to Clindamycin and moxifloxacin respectively. All isolates were susceptible to metronidazole. Toxigenic C. difficile strains were analyzed by MLST and were divided into 4 different STs. The detected types were ST-54 (57.9%), followed by ST-2 (31.6. %), ST-15 (5.3%) and ST-37 (5.3%), while none of the isolates were identified as ST-1 or ST-11. Significant risk factors for CDI appear to be advanced age, undergoing chemotherapy, previous surgery, and residence in the nursing home. Conclusions: CDI is common in Iran and further studies are recommended to monitor its epidemiological variations. Moreover, greater attempts must be made to encourage antibiotic stewardship by healthcare workers and the public. Keywords: Clostridium difficile infection, Molecular characterization, Risk factor, Multilocus sequence typing analysis (MLST) Introduction symptomatic patients can spread highly transmissible Clostridium difficile is an obligate anaerobic bacillus spores, leading to healthcare challenges as high-frequency opportunistic intestinal pathogen with the capability of recurrent CDI [1, 4, 5] The risk of CDI acquisition may large glucosylate toxins production (i.e., tcdA, tcdB, cdtA, increase in a larger hospital setting due to increased envir- and cdtB)) [1, 2]. The clinical symptoms of C. difficile onmental contamination and improper disinfection . infections (CDI) ranges from mild diarrhea to pseudomem- There are few reports on C. difficile isolation from medical branous colitis . About 30% of CDIs are attributed to devices, hands of healthcare workers and its transmission health care facilities transmission, while hospitals environ- to patients in the hospital settings . The epidemiology of mental contamination with C.difficile spores may account CDI has changed during the last two decades. However, for 40% of CDI . Both asymptomatic carriers and both the incidence and severity of CDI have been increased in hospitals worldwide [3, 8]. Epidemics of CDI have hap- pened in North America and Europe recently and the epi- * Correspondence: 2018KHORVASH@gmail.com demiology of CDI in these regions is known. Circulating Nosocomial Infection Research Center, Isfahan University of Medical strains in Asia, as in other regions, have the potential to Sciences, Isfahan, Iran Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shoaei et al. Antimicrobial Resistance and Infection Control (2019) 8:12 Page 2 of 7 extend internationally . These epidemics have increased Ethical considerations the need for surveillance of C. difficile strains globally . The study was approved by the human research ethics In recent epidemiologic studies, genotyping of strains play committee of Isfahan University of Medical Sciences an important role in the identification of epidemic, hyper- (the grant No. 293347), and the study was carried out in virulent genotypes and the relations between them . In full accordance with the approved guidelines. Iran, most of the studies have been limited to PCR based ribotyping [11, 12]. The appropriate use of this method as Clostridium difficile culture a genotyping tool for small-scale analyses has been For culture analysis, Lemee L method was used . About confirmed, However, the main advantage of Multilo- 2 g of stools (or samples taken by swabs) were inoculated cus Sequence Typing Analysis (MLST) is the unam- into 10 ml of C. difficile moxalactam norfloxacin (CDMN) biguous ability to compare the results obtained in the broth culture. Then the cultures were incubated anaerobic- different regions via the internal database (accessible ally for 48 h at 37 C° using an Anoxomat system (MART at https://pubmlst.org/cdifficile)[13–15]. The infor- Microbiology B.V., The Netherlands). Alcohol shock treat- mation included in the MLST database principally ment was performed to inhibit non-sporulating organisms consists of strains from European countries, with few and enhance the isolation of C. difficile (a 1:1 suspension strains from Asia, especially from China [10, 16]. More- cultured sample with 95% ethanol was slowly vortexed and over, no molecular epidemiologic study on C. difficile held at room temperature for 30 min). The pellets were isolates from patients and their environment have been inoculated by a swab into the C. difficile moxalactam nor- conducted in Iran using the MLST method. floxacin agar (CDMN) supplemented with 7% sheep blood The main objective of the current study was to and incubated anaerobically for 48 h at 37C°. Negative determine the incidence of C. difficile in hospitalized cultures remained in the incubator for up to 7 days. patients and hospital environments. Additionally, we Irregular yellowish colonies with horse-manure odour, intended to study the genetic polymorphism and rela- ultraviolet fluorescence (365 nm), Gram stain morph- tionship among strain lineages using MLST as an ology, malachite green for spore and biochemical reac- unambiguous molecular procedure. tionssuchasL-proline aminopeptidase test (Prodisk, Remeb, Lenexa, KS, USA) were identified as C. difficile Methods strains and stored at 4 °C. These colonies were sub- Study design jected to further molecular identification [12, 19]. A cross-sectional study was conducted in medical wards of the university hospitals in Isfahan, the central of Iran from April 2015 and May 2017. Our study included the patients aged at least 18 years who were admitted to Molecular identification of C. difficile medical wards for different problems and acquired diar- All isolates were screened for the presence of the genes rhea in hospital. Diarrhea was determined as the passage encoding toxin A and B (tcd A and tcd B), binary toxin of more than two loose or watery stools per day for at (cdt A, cdt B) and triose phosphate isomerase (tpi)as least two days . A case of CDI was determined as a described by Stubbs et al. and Lemee et al. [14, 20]. diarrheal patient with positive C. difficile culture and toxin DNA extraction was performed using the procedure per- tests . Patients admitted for less than 3 days or who formed in the study by Pitcher et al. (1989). Cultures of were diagnosed with CDI within the previous 3 months, C. difficile strains grown in BHI broth were centrifuged colectomy and diarrhea on admission were excluded. and cells were treated with lysozyme and resuspended in To avoid overrepresentation, only the first stool specimen TE (Tris, 10 mM; EDTA, 50 mM; pH 8.0). Then guanidine from each patient was included. A total of 247 samples were isothiocyanate and ammonium acetate were added to the collected from the dominant hand of healthcare workers, mixture and Chloroform/isoamyl alcohol (24:1). The DNA the different skin sites of patients (hand, forearm, and was precipitated and washed with ethanol . abdomen) and medical devices using sterile swab moistened Multiplex PCR amplification performed in a thermocy- with normal saline rotated all 5× 20 cm of the surfaces. All cler (Eppendorf, Germany). An amount of 25 μl of reaction surfaces frequently touched by health care workers were mixture contained 1× PCR buffer, 250 μM of each dNTPs, documented. Fecal and environmental samples from pa- 10 pM of primers (tcdA, tcd B), 5 pM of primers (tpi), 1 U tients were collected in sterile containers and were then Taq polymerase (Cina Gene, Iran) and 100 ng of DNA. immediately transferred into the laboratory of Infectious Amplification was carried out in a touchdown protocol Diseases and Tropical Medicine Research Center, Isfahan, [12, 20]. Clostridium difficile ribotype 027 was used as Iran and preserved at − 70 °C. Variables were included the a positive control for molecular and microbiological demographic characteristics, type of comorbidities and analysis and C. perfringens 450 MTCC (Microbial Type antibiotic treatment in 8 weeks prior to CDI diagnosis. Culture Collection) was used as the negative control . Shoaei et al. Antimicrobial Resistance and Infection Control (2019) 8:12 Page 3 of 7 Multilocus sequence typing (MLST) model was used to determine clinical factors associated MLST was carried out and analyzed for C. difficile strains with C.difficle infection. First, a univariate logistic re- according to the previous studies. MLST with seven house- gression model was fitted on each clinical factors, and keeping genes (adk, atpA, dxr, glyA, recA, sodA, and tpi) then a multivariate regression model with adjustment was performed on detected isolates as described previously for the effects of other covariates was used. Variables by Griffiths et al. . Sequence types (STs) were analyzed that were significant in univariate models (P < 0.05) were by constructing a dendrogram based on the UPGMA (Un- entered into the multivariate model. Selection of vari- weighted Pair Group Method with Arithmetic mean) clus- ables in the multivariate model was based on a stepwise tering algorithm available in the BioNumerics software . procedure. Statistical analysis was performed using the statistical software (SPSS, version 16). Antibiotic susceptibility testing Antimicrobial susceptibility of toxigenic C.difficile isolates Results was determined using the Etest strips (bioMérieux, France) Among the 574 enrolled patients, 164 (28.6%) were C. for vancomycin, metronidazole, moxifloxacin, fusidic acid, ri- difficile culture positive and the tpi gene was recovered fampin, and clindamycin. Briefly, the Etest was performed by from these stool specimens. Based on PCR amplification inoculating the surface of pre-reduced Brucella Agar plates of tcdA and tcdB, 66 isolates (40.2%) carried one or both containing vitamin K1 (0.5 mg/mL), haemin (5 mg/mL) and of these genes and were considered toxigenic, while the 5% defibrinated sheep red blood cells with 1 McFarland remaining 98 isolates (59.8%) were nontoxigenic. (Fig. 1). standard matched C. difficile inoculum. The plates were in- There were no deletions found in tcdC genes from all cubated at 37 °C for 48 h under anaerobic conditions. The toxigenic isolates examined. No isolate tested positive breakpoints and interpreting of the minimum inhibitory con- for the binary toxin genes cdtA and cdtB. centrations (MICs) were carried out according to the Clinical Mean age of CDI cases was 55.4 years (standard devi- Laboratory Standards Institute (CLSI 2012) and the Euro- ation 19.4 years) and mean age of Non-CDI cases was 44 pean Committee on Antimicrobial Susceptibility Testing years(standard deviation 15.5 years). Risk of CDI increased (EUCAST) guidelines (http://www.clinical_breakpoints/clini- by nearly 4% per year (OR 1.03; 95% CI, 1.01–1.05; cal_breakpoints/clostridiumdifficle/ difficile). Clostridium P < 0.001). The use of antibiotic was identified in all perfringens MTCC 13124 and Streptococcus sp. MTCC 689 patients in the 8 weeks prior to CDI diagnosis. Patients strains were included in each run as controls. The break- were treated with one to four antibiotics, about 96% and points used were 8 μg/ ml for Clindamycin, 4 μg/ ml for 63/5% of them had been used more than one and two moxifloxacin; 2 μg/ ml, for vancomycin and 32 μg/ ml for antibiotics, respectively (Table 1). metronidazole; rifampin; fusidic acid as described previously Thestepwisemultivariatelogisticregression modelre- [23, 24]. The antimicrobial agents tested were chosen be- vealed that the following parameters were found statistically cause of the emergence of reduced susceptibility. significant between cases of CDI and cases without CDI: Advanced age, previous surgery, chemotherapy or residence Statistical analysis in a nursing home (Table 2). Among the 274 screened Data were expressed as count and percentage. All prob- samples taken from hospital environment, 47(19%) were abilities were two-tailed and a P-value of < 0.05 was positive for C. difficile colonization and 4.4% (11/247) defined as statistically significant. The logistic regression toxigenic strains. Toxigenic C.difficile strains were detected Fig. 1 Flowdiagram illustrating the number of patients and swab samples included in the study Shoaei et al. Antimicrobial Resistance and Infection Control (2019) 8:12 Page 4 of 7 Table 1 Clinical characterization of 574 hospitalized patients with nosocomial diarrhea admitted to the university hospitals, Isfahan, Iran Characteristics CDI patients n = 66 Non CDI Patients n = 508 Toxigenic C.difficile Isolates, Negative C.difficile Non-toxigenic C.difficile (A+B+, A-B+), strains n = 410 strains (A-B-), n =98 Count (%) Count (%) Count (%) Sex Male 31 (47.0) 208 (53.2) 49(52.1) Antibiotic treatment within 8 weeks Aminoglycosides 20 (30.3) 70 (17.6) 20 (21.1) Vancomycin 0 3 (0.7) 0 Cephalosporin 24 (36.4) 178 (44.8) 42 (44.2) Metronidazole 11 16.7) 6 (1.5) 2 (2.1) Clindamycin 31 (46.9) 137 (34.5) 31 (32.6) Gastroenteritis diseases 35 (53.0) 44 (11.1) 14 (14.9) Residence in nursing home 14 (21.2) 7 (1.8) 3 (3.2) Chronic renal insufficiency 26 (39.4) 47 (11.9) 16 (17.0) Previous Surgery 50 (75.8) 137 (34.7) 51 (53.7) Chemotherapy 16 (24.2) 25(6.0) 13 (13.3) Ward Internal 28 (42.4) 293 (73.4) 61(64.2) ICU 38 (57.6) 106 (26.6) 34 (35.8) Internal ward(gasteroenterology, Infectious diseasese, Diabetes,…) from different skin sites (hands and abdomens) of 7 CDI and the wards. For instance, ST-54 and ST- 2 were the patients and 4 of healthcare workers hands (Table 3). major types in the ICU and together accounted for 60% of the strains. Among other wards especially in gastroenter- Molecular epidemiology of the isolates ology ward, ST-54 and ST-2 were the most frequent STs An additional two isolates were obtained from collection and both equally represented at 35.5% of the strains. reference laboratories of C. difficile analysis (held at There was no significant difference between males and Canada, University of Guelph). They represented two females in the prevalence of ST types (P > 0.05). different PCR ribotypes (027, 078) selected to validate the MLST scheme analyses. Toxigenic C. difficile strains Antibiotic susceptibility of C. difficile from different hospitals were analyzed by MLST and We used E test strips to determine the minimum inhibitory divided into 4 different STs (Fig. 2). concentration (MIC) of each antibiotic in the toxigenic iso- The detected types were ST-54 (57.9%), followed by lates for six antibiotics. All the isolates were inhibited by a ST-2 (31.6%), ST-15 (5.3%), and ST-37 (5.3%), but none of low concentration of vancomycin (MIC < 0.5 μg/ml). Two the isolates was identified as ST-1 (BI/NAP1/027) or isolates, from 2 ICU patients aged 70 and 72 years, were re- ST-11(ribotype 078). We found a correlation between the sistant to fusidic acid, while the remaining isolates (97.4%) toxin genotypes and STs. All of the ST-54 strains were proved susceptible to it. About 42.8% (33/77) of isolates + + − + toxin type A B , and all of the ST-2 strains were A B . were resistant to Clindamycin, while it was about 38.9% for Some relations were observed between the toxin profile Moxifloxacin (30/77). Only 11.7% of isolates (9/77) were Table 2 Variables significantly associated with C.difficile infection Table 3 Summary of positive surface Clostridium difficile cultures among 574 patients with nosocomial diarrhea Category Samples Non-toxigenic C.difficile Toxigenic CdC , (n = 247) colonization n (%) n (%) Risk factors OR (95% CI) p-value HCW’ hands 73 21 (28.8) 4 (5.4) Age 0.97 (0.95–1.0) 0.004 Skin of patients 82 17 (20.7) 7 (8.5) Residence in nursing home 3.96 (1.0–15.6) 0.049 ICU devices: 52 4 (7.7) 0 Chronic renal insufficiency 2.69 (1.3–5.6) 0.008 Bed sheets 40 5 (12.5) 0 Previous surgery 4.23 (2.1–8.6) < 0.001 Clostridium difficile colonization (CdC), Chemotherapy 2.96 (1.3–6.7) 0.01 health care worker (HCW) Shoaei et al. Antimicrobial Resistance and Infection Control (2019) 8:12 Page 5 of 7 Fig. 2 Distribution of C. difficile sequence types(ST) identified by MLST resistant to rifampin. All isolates were susceptible to frequent ones found . In a similar study performed in metronidazole. There were no inner colonies suggest- Qatar, CDI prevalence was reported around 8% and RT ive of heteroresistance within the zones of inhibition 258 was shown to be the predominant ribotype .In around the metronidazole E- test strips (Table 4). European Countries, Binary toxin (CDT) has been found There was no correlation between the toxin type and in 4–12% of toxigenic C. difficile samples associated with antibiotic resistance (P > 0.05). higher mortality and recurrence rate of CDI . We found a high prevalence of non-toxigenic strains, the ab- Discussion sence of binary toxin producers and hypervirulent RTs Currently, C. difficile infection (CDI) is regarded as a 027 and 078 in the current study. Our findings were in ac- widespread issue worldwide because of its increasing mor- cordance with similar studies in Asia showing a low preva- bidity and mortality. The range of this infection is variable lence of binary toxin among isolates in various hospitals from mild diarrhea to pseudomembranous colitis espe- in China, Thailand, Korea and Japan . This may be the cially among the elderly population who have been ex- result of different circulating C. difficile strains in Asia. posed to antibiotics . CDI epidemics have indicated Our phylogenetic analysis showed that ST-54 and the need for surveillance of the international prevalence of ST-2 were the most prevalent strains. Therefore we pro- C. difficile strains. There are limited epidemiological stud- posed a low genetic diversity of toxigenic strains in ies on C. difficile in Iran. The prevalence of CDI in our Isfahan, Iran. Other similar studies in Europe, the Mid- study was lower than those reported in Western countries dle East, and North America previously identified ST2 and China [25, 26]. The prevalence of CDI at hospitals in as a common clinical strain all around the world . Kuwait was reported 9.7% in 2003, 7.8% in 2004 and 7.2% Studies performed in China and France showed various − + in 2005 with 002, 001, 126 and 140 RTs were the most STs in A B strains, yet the most prevalent STs were ST-2, ST-54, ST-37, and ST-35 in China and ST-1 in France. This might be due to different geographical loca- tions where the study was carried out [9, 10, 26]. Table 4 MIC for antimicrobial agents tested against 77 C. Most of CDI in the current study were found to be difficile isolates + + due to A B strains (ST-54). Recent studies have re- Antibiotic MIC (μg/ml) Resistant Break points ported an increasing number of infections due to A-B+ Range no. (%) (μg/ml) strains especially ST-37 in Asia though such strains do Clindamycin 0.25 > 256 33 (42.8) ≥ 8 not produce a binary toxin . Due to unavailability of Metronidazole 0.125–80 ≥ 32 C. difficile culture and toxin testing in many hospitals in Iran, awareness of circulating strains and their preva- Rifampin 0.002 > 32 9 (11.7) ≥ 32 lence is not high enough. Moxifloxacin 0.5 > 32 30 (38.9) ≥ 4 We could not establish a correlation between the hospi- Vancomycin 0.016–0.5 0 > 2 tals and detected genotypes. Our isolates were genotyped Fusidic acid 0.016 > 8 2 (2.6) ≥ 32 into 4 STs, which were not specific to Iran, a finding that Shoaei et al. Antimicrobial Resistance and Infection Control (2019) 8:12 Page 6 of 7 suggests the worldwide spread of some lineages. All Abbreviations CDI: Clostridium difficile infection; CDMN: C. difficile moxalactam norfloxacin; strains of ST54 isolates were toxin type A + B+ and all MIC: Minimum inhibitory concentration; MLST: Multilocus sequence typing strains ST2 were toxin type A-B+. In addition, there was analysis; RT: Ribotype; ST: Sequence type no correlation between the genotype and CDI risk factors Acknowledgments in our study. Based on our multivariate regression model We are thankful to the staff of Isfahan University Training Hospitals for their results, patients with CDI were significantly older and had great help in collecting our samples. Authors are thankful to Mr. Abbas daei more comorbidities than the non-CDI group. Several pre- Naser and Dr. Arash Zahed and Dr.Majid Yaran for their special cooperation in our study. vious studies have noted the association between recent hospitalization, advanced age, severe diseases and a higher Funding risk of acquiring CDI [9, 26, 30, 31]. All identified toxi- This study was funded by a grant from, Isfahan University of Medical Sciences, Isfahan, Iran (grant No. 293347). genic stains in this study showed susceptibility to vanco- mycin and metronidazole. Other similar studies showed Availability of data and materials that toxigenic C.difficile strains commonly revealed high Data generated or analyzed during this study are available and some are included in this article. (SPSS file of data; gel pictures of different PCRs are susceptibility to these most common choices for CDI available). treatment . In two studies conducted in China, the rate of resistance to moxifloxacin was reported at 46.4 and Authors‘ contributions *SP contributed in the experimental studies, and drafting the work,* KF and 13.1% . The strong correlation between CDI incidence BA contributed in the conception of design, * SHK contributed in the and resistance to these antibiotics showed that it is neces- acquisition of the data. * TH, JM, WJS, and SH contributed to the conception sary to restrict the use of fluoroquinolones to reduce CDI of design and revising the draft, * HSM contributed to the analysis of the data. All authors read and approved the final manuscript. . We have found that C. difficile frequently contami- nated hands of HCWs after caring for patients with CDI Ethics approval and consent to participate (p < 0.01). Other studies showed that skin contamination The study was approved by the human research ethics committee at Isfahan University of Medical Sciences and the study was carried out in accordance persisted in many patients after resolution of diarrhea and with the approved guidelines. was easily acquired on investigators hands  The same STs (STs 54, 2, 15) were detected from the health care Consent for publication Not applicable. workers hands, the skin of patients and their stool sam- ples. One similar study already conducted in Iran also Competing interests showed that the occurrence of C. difficile isolates with the The authors declare that they have no competing interests. same RTs in gastrointestinal imaging devices and stool specimens . While a contaminated environment is a Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in significant contributor to infection, skilled healthcare published maps and institutional affiliations. workers and effective infection control measures in the patient hospital rooms can reduce the burden of CDI in Author details Nosocomial Infection Research Center, Isfahan University of Medical health care facilities. Sciences, Isfahan, Iran. Department of Microbiology, School of Medicine, There were a number of limitations to our study includ- 3 Isfahan University of Medical Sciences, Isfahan, Iran. Nosocomial Infection ing difficulties in the sequencing of all C. difficile isolates, Research Center, Isfahan University of Medical Sciences, Isfahan, Iran. Epidemiology and Biostatics Department, Isfahan University of Medical which led to undermining the true prevalence and diver- Sciences, Isfahan, Iran. Infectious Diseases and Tropical Medicine Research sity of C. difficile STs. Due to the quick discharge of many 6 Center, Isfahan University of Medical Sciences, Isfahan, Iran. Department of of our patients; we were not able to analyze treatment and Biology, University of British Columbia, Kelowna, Canada. School of Food Science and Nutrition, Isfahan University of Medical Sciences, Isfahan, Iran. outcome characteristics of all patients with CDI. Department of Pathobiology, Ontario Veterinary College, University of Since the incidence of CDI was relatively high at Guelph, Guelph, Canada. the provincial level, a countrywide CDI surveillance Received: 16 August 2018 Accepted: 13 December 2018 (with a long period and large population size) is warranted to analyze other risk factors of CDI and contamination. References 1. Gil F, Lagos-Moraga S, Calderon-Romero P, Pizarro-Guajardo M, Paredes-Sabja D. Updates on Clostridium difficile spore biology. Anaerobe. 2017;45:3–9. Conclusion 2. 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Antimicrobial Resistance & Infection Control – Springer Journals
Published: Jan 14, 2019
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