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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 2  |  Page : 176-182
 

Prevalence, toxin gene profile, genotypes and antibiotic susceptibility of Clostridium difficile in a tertiary care hospital in Taif, Saudi Arabia


1 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, Taif, Saudi Arabia; Department of Medical Microbiology and Immunology, Faculty of Medicine, Zagazig University, Zagazig, Egypt
2 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, Taif, Saudi Arabia; Department of Molecular and Clinical Parasitology, National Liver Institute, Menoufia University, Menoufia, Egypt
3 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, Taif, Saudi Arabia; Department of Parasitology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
4 Department of Medical Microbiology and Immunology, Faculty of Pharmacy, Taif University, Taif, Saudi Arabia; Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
5 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, Taif, Saudi Arabia
6 Department of Internal Medicine, Faculty of Medicine, Zagazig University, Zagazig, Egypt
7 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, Taif, Saudi Arabia; Department of Medical Microbiology and Immunology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission01-Jul-2020
Date of Decision10-Jul-2020
Date of Acceptance17-Jul-2020
Date of Web Publication29-Aug-2020

Correspondence Address:
Dr. Khadiga A Ismail
Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Taif University, Taif

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_20_300

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 ~ Abstract 


Purpose: Clostridium difficile (C. difficile) is an important causative agent of nosocomial diarrhoea and has become a major worldwide public health concern. The current study was conducted to determine the prevalence of C. difficile infection (CDI) amongst patients with nosocomial diarrhoea in a large tertiary care hospital in Taif, Saudi Arabia, and to define molecular characteristics and antimicrobial sensitivity profiles of C. difficile strains isolated from those patients. Materials and Methods: Stool specimens were collected from 456 patients and were cultured for C. difficile isolation. The isolates were subjected to multiplex polymerase chain reaction (PCR) for detecting genes encoding the toxins (toxin A, toxin B and binary toxin [CDT]), genotyping by PCR ribotyping method and antimicrobial sensitivity testing using E test strips. Results: Seventy-four C. difficile strains were recovered, of which 44 (59.5%) were A+B+CDT, 14 (18.9%) were AB+CDT, 4 (5.4%) were A+B+CDT+ and 12 (16.2%) were ABCDT. Toxigenic strains, and hence CDI, were detected in 13.6% of the patients (62/456). Fourteen different ribotypes were distinguished amongst bacterial isolates, of which ribotypes 002, 001, 017, 014 and 020 were the most prevalent (20.3%, 18.9%, 18.9%, 9.5% and 8.1%, respectively). Four isolates (5.4%) belonged to ribotype 027. All bacterial isolates showed sensitivity to metronidazole, vancomycin and piperacillin-tazobactam. The isolates exhibited resistance to linezolid (2.7%), chloramphenicol (5.4%), rifampicin (13.5%), tetracycline (21.6%), moxifloxacin (48.6%), clindamycin (54%) and imipenem (83.8%). Multiple drug resistance was observed in 56.8% of the isolates. Conclusion: Further larger studies are required for an accurate understanding of CDI epidemiology in Saudi Arabia.


Keywords: Antibiotic susceptibility, Clostridium difficile, polymerase chain reaction ribotyping, prevalence, toxin genes


How to cite this article:
Saber T, Hawash YA, Ismail KA, Khalifa AS, Alsharif KF, Alghamdi SA, Saber T, Eed EM. Prevalence, toxin gene profile, genotypes and antibiotic susceptibility of Clostridium difficile in a tertiary care hospital in Taif, Saudi Arabia. Indian J Med Microbiol 2020;38:176-82

How to cite this URL:
Saber T, Hawash YA, Ismail KA, Khalifa AS, Alsharif KF, Alghamdi SA, Saber T, Eed EM. Prevalence, toxin gene profile, genotypes and antibiotic susceptibility of Clostridium difficile in a tertiary care hospital in Taif, Saudi Arabia. Indian J Med Microbiol [serial online] 2020 [cited 2020 Sep 26];38:176-82. Available from: http://www.ijmm.org/text.asp?2020/38/2/176/293910





 ~ Introduction Top


Clostridium difficile (C. difficile) is a Gram-positive, obligate anaerobe, spore-forming rod.[1] C. difficile infection (CDI) is recognised amongst the most important nosocomial infections, and its prevalence has increased significantly worldwide.[2] In addition, the morbidity and severity of CDI have resulted in an increased economic burden on health-care systems due to high treatment costs and prolonged stays in hospitals.[3] CDI symptoms vary from mild diarrhoea to severe manifestations, including toxic megacolon and pseudomembranous colitis.[4] The principal risk factor for CDI is disruption of normal intestinal flora by antimicrobial therapy.[5] Other important risk factors include advanced age, prolonged hospital stays and underlying diseases such as chronic renal disease, inflammatory diseases of the bowel and immune deficiency.[6] However, CDIs have been reported increasingly in the community amongst young people who do not exhibit the traditional risk factors.[7] The main virulence factor of C. difficile is the secretion of toxin A (enterotoxin), encoded by the tcdA gene, and toxin B (cytotoxin), encoded by the tcdB gene. These virulence genes are found together with regulatory genes in a 19.6 kb region named Paloc on the chromosome of C. difficile.[8] In addition, some strains of C. difficile secrete binary toxin (CDT) that is encoded by two genes named cdtA and cdtB. CDT is a member of the family ADP-ribosyltransferase and causes actin depolymerisation in the cytoskeleton, leading to increased adhesiveness of such bacteria.[9] Toxigenic strains of C. difficile, which are those that induce disease, always bear toxin B, frequently bear toxin A and less frequently secrete CDT.[10] The C. difficile strains that are positive for toxin B (tcdB+) and negative for toxin A (tcdA) have received great attention.[11] Severe infections and outbreaks induced by tcdA tcdB+ C. difficile strains have been increasingly reported, with a higher frequency in East Asian countries.[12] Molecular typing provides information on the emergence, origin, distribution and identification of the highly virulent strains and thereby supports understanding of C. difficile epidemiology. Various molecular tools have been utilised for typing C. difficile strains, such as polymerase chain reaction (PCR) ribotyping, pulsed-field gel electrophoresis, restriction endonuclease analysis, multilocus sequence typing and amplified fragment length polymorphism.[13] The PCR ribotyping method is the most frequently used due to its high discriminatory power and low cost;[14] this method categorises C. difficile into various ribotypes based on the banding patterns created by PCR of the intergenic spacer region between the 16S and 23S ribosomal RNA genes.[15] Molecular epidemiology studies worldwide have recovered the hypervirulent C. difficile strain known as BI/NAP1/027 (BI: restriction endonuclease analysis group BI; NAP1: North American pulsed-field type 1; PCR ribotype 027), which is particularly predominant in North America and Europe.[7] In addition to molecular typing, studying the patterns of antimicrobial sensitivity of C. difficile is necessary to monitor the spread of this organism.[16] Due to the role of antimicrobial therapy in CDI development, antibiotic stewardship and surveillance are necessary to determine the resistance patterns of C. difficile and to identify the antimicrobial agents that might support the selection of particular C. difficile isolates.[17] The antimicrobial resistance rates differ across geographic regions and are related to the local antibiotic prescribing policies.[18] The first-line drugs for CDI treatment are metronidazole and vancomycin; however, many studies have reported decreased sensitivity or resistance to both drugs.[19] Recently, C. difficile was reported as a microorganism with an urgent threat level due to its antimicrobial resistance.[20] Information concerning the prevalence, molecular characterisation and antimicrobial resistance of C. difficile in Saudi Arabia is limited. Therefore, the present study was performed to estimate the prevalence of CDI amongst patients suffering from nosocomial diarrhoea in a large tertiary care hospital in Taif, located in the western region of Saudi Arabia. In addition, the study aimed at assessing the molecular epidemiology and antimicrobial resistance patterns of C. difficile isolates from these patients through the detection of toxin genes, PCR ribotyping and antimicrobial susceptibility testing.


 ~ Materials and Methods Top


Study design and population

In this cross-sectional study, faecal specimens were collected from 456 patients (314 males and 142 females) hospitalised at a large tertiary care hospital in Taif, Saudi Arabia, from December 2018 to December 2019. Study approval was obtained from the Research Ethics Committee at Taif University, and each patient provided written consent before participation in the study. The patients' ages ranged between 23 and 79 years (mean ± standard deviation = 49 ± 5.9). Patients experienced healthcare-associated diarrhoea, as defined by more than three loose stools during a period ≤24 h, that developed at least 72 h after hospital admission.[21],[22] A single diarrheal stool specimen was collected from each patient.

Bacterial isolation and identification

Stool specimens were treated with absolute ethanol at a ratio of 1:1 for 1 h at room temperature followed by culture on cefoxitin-cycloserine-fructose agar plates (Oxoid, Basingstoke, UK) and incubation under anaerobic conditions for 48–72 h at 37°C. Suspected colonies were identified as C. difficile by Gram staining, colony morphology characteristic odour and yellowish-green fluorescence under long-wave ultraviolet light (365 nm). The identification of the isolates was confirmed by biochemical testing using API 20A (bioMerieux, Marcy I'Etoile, France).[23],[24] All C. difficile strains were preserved at −70°C in brain–heart infusion broth with 10% glycerol until further analysis.

Molecular investigations

DNA was extracted from the isolated C. difficile strains using the QIAamp DNA mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The presence of toxin genes (tcdA, tcdB, cdtA, and cdtB) was determined by multiplex PCR, according to the European Centre for Disease Prevention and Control.[25] [Table 1] shows the sequence of the primers used and the size of amplicons. The PCR reactions were achieved in a final volume of 25 μl containing 12.5 μl of Taq Hot Start PCR Master Mix (Qiagen, Hilden, Germany), 2.5 μl of DNA extract, the primers by volumes [Table 1], and 7.07 μl of PCR-grade distilled water. The thermocycler conditions were 15 min at 94°C, followed by 35 cycles of 45 s at 94°C, 45 s at 50°C, and 1 min at 72°C and a final extension at 72°C for a further 30 min. The amplified products were subjected to separation by electrophoresis in agarose gel (1.5%) containing ethidium bromide and visualised under ultraviolet illumination. A molecular size standard 100 bp DNA ladder was used to determine the size of bands. Primers 16S (5́-CTGGGGTGAAGTCGTAACAAGG-3̀) and 23S (5́-GCGCCCTTTGTAGCTTGACC-3̀) were used for PCR ribotyping, as previously reported by Stubbs et al.[15]
Table 1: Primers used for the detection of Clostridium difficile toxin genes[25]

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Antimicrobial sensitivity testing

The C. difficile strains were tested for sensitivity to the following antimicrobial agents: vancomycin, rifampicin, linezolid, clindamycin, metronidazole, piperacillin-tazobactam, imipenem, tetracycline, chloramphenicol and moxifloxacin. The sensitivity testing was accomplished by determining the minimal inhibitory concentrations (MICs) of the above-mentioned antibiotics using the E test strips (bioMerieux, Marcy I'Etoile, France), according to the manufacturer's instructions, on BD Brucella blood agar with hemin and Vitamin K1 (Becton Dickinson GmbH, Germany). For vancomycin, the MIC was interpreted as defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST,[26]), while the MIC breakpoints for rifampicin and linezolid were based on a previous publication.[27] For the remaining antibiotics, the MIC interpretation was conducted according to the Clinical and Laboratory Standards Institute breakpoint criteria.[28]

Statistical analysis

Data analysis was conducted using SPSS software, version 22.0 for Windows (SPSS Inc., Chicago, IL, USA). Fisher's exact test was used to compare the antibiotic resistance rates according to the toxin gene profile. P ≤ 0.05 was considered statistically significant.


 ~ Results Top


Clostridium difficile isolation and toxin gene detection

A total of 456 faecal specimens from hospitalised patients with nosocomial diarrhoea were cultured for C. difficile, and 16.2% (74/456) were culture positive. Of the 74 C. difficile isolates recovered, 59.5% (44/74) were positive for tcdA and tcdB and were negative for cdtA/cdtB (A + B + CDT ); 18.9% (14/74) were negative for tcdA, positive for tcdB, and negative for cdtA/cdtB (A B + CDT ); 5.4% (4/74) were positive for the genes of the three toxins (A + B + CDT +); and 16.2% (12/74) were non-toxigenic (A B CDT ). Thus, the overall prevalence of toxigenic C. difficile amongst the studied patients was 13.6% (62/456).

PCR ribotyping

All isolated C. difficile strains were analysed by PCR ribotyping, and 14 different ribotypes were identified. The most prevalent ribotypes were 002 (20.3%, 15/74), 001 (18.9%, 14/74), 017 (18.9%, 14/74), 014 (9.5%, 7/74) and 020 (8.1%, 6/74). Four of the isolates (5.4%) belonged to the ribotype 027. The 14 ribotypes identified, along with their respective toxin profiles, are presented in [Table 2].
Table 2: Toxin gene profiles and ribotypes of the 74 Clostridium difficile isolates

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Antimicrobial susceptibility

[Table 3] shows the results of antibiotic susceptibility testing in terms of MIC range, MIC50 and MIC90 (MICs required to inhibit 50% and 90% of the isolates, respectively), and the percentage of the resistant strains. All C. difficile strains showed sensitivity to metronidazole, vancomycin, and piperacillin-tazobactam. A low rate of resistance to linezolid (2.7%, 2/74) and chloramphenicol (5.4%, 4/74) was found. The most prevalent resistance detected was for imipenem (83.8%, 62/74). The rates of resistance to the remaining antimicrobial agents were as follows: clindamycin, 54% (40/74); moxifloxacin, 48.6% (36/74); tetracycline, 21.6% (16/74) and rifampicin, 13.5% (10/74). Multiple-drug resistance (MDR), as defined by resistance to at least three various classes of antibiotics, was observed in 56.8% (42/74) of the isolates. [Table 4] shows the rates of resistance to different antibiotics and the rates of MDR amongst the C. difficile strains according to toxin production, where the rate of resistance to most antibiotics as well as MDR was significantly higher amongst the toxigenic strains compared to the non-toxigenic strains. Similarly, the A B + C. difficile strains were more significantly resistant to most antibiotics than were the A + B + strains.
Table 3: Interpretive minimal inhibitory concentration breakpoints and resistance of the 74 Clostridium difficile isolates to the various antimicrobial agents

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Table 4: Antibiotic resistance rates of Clostridium difficile according to toxin production

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 ~ Discussion Top


In recent years, the incidence of CDIs showed a significant increase, which represented an important health problem, particularly in hospitals.[29] To the best of our knowledge, this is the first study on the prevalence, antibiotic susceptibility and molecular characterisation of CDIs in the western region of Saudi Arabia. The prevalence of CDIs in the current study was determined to be 13.6% because CDI is caused by toxigenic strains,[30] and the overall prevalence of toxigenic C. difficile was 62/456. This result is in accordance with that of a previous Saudi study conducted in Riyadh, where toxigenic C. difficile strains were detected in 14.8% of the studied patients.[31] However, the prevalence of CDIs in the current study is lower than that reported by two other Saudi studies. One study conducted in the major hospitals of Al-Madinah found the prevalence of CDIs to be 21.7%,[32] while the other study involved four major hospitals in the Eastern Province of Saudi Arabia and showed an 18.9% overall prevalence for toxigenic C. difficile.[33] The prevalence of CDIs in the neighbouring countries is also variable, where toxigenic C. difficile was responsible for 6.6%, 12.7% and 21% of nosocomial diarrhoea in Egypt,[34] Jordan [35] and Iraq,[36] respectively. In comparison to Asian countries, the CDI prevalence in this study is greater than that recorded in 2017 in Thailand, 9.2%[37] and Indonesia, 10.9%.[38] In general, the prevalence of CDIs differs amongst regions and hospitals,[24] and this discrepancy may stem from the variations in geographical distribution, infection control policies, and/or regulations of antibiotic use.[29],[34] Moreover, the method of laboratory detection and the characteristics of the investigated population affect the results of prevalence studies.[37] The present study revealed that most of the recovered C. difficile isolates (64.9%, 48/74) carry the genes of A and B toxins. This finding is similar to a previous report in Saudi Arabia [33] and previous reports elsewhere around the world.[39] C. difficile isolates positive for these two toxins share the greatest role in CDI pathogenesis. However, many studies recorded that the A B + strains are increasing significantly in cases of CDI.[40] The prevalence of A B + strains varies in relation to the studied population.[41] In this study, A B + strains accounted for 18.9% (14/74) of the total isolates, which is higher than the percentage (3.4%) reported in the Eastern Province of Saudi Arabia.[33] Several studies in/China reported isolation rates of 6.5%–37% for A B + strains.[16],[27],[39],[42] The prevalence of these strains was 28.4% in Indonesia,[38] 25.7% in South Korea,[41] 32% in Thailand,[43] and 13.8% in Japan.[44] In addition to the large clostridial A and B toxins, the binary toxin produced by C. difficile strains has been recorded in recent years. The presence of CDT genes in C. difficile strains is closely correlated with higher rates of recurrence and mortality. The prevalence of these genes differs amongst geographic regions and may differ amongst hospitals due to the variations in patient or strain selection.[40] In this study, 5.4% (4/74) of the isolated C. difficile strains carried the CDT genes (CDT +). The CDT + strains were reported recently in Saudi Arabia with a similar prevalence (6.8%)[33] but were reported at a much higher rate (38.7%) in another Saudi study.[31] The PCR ribotyping method has been utilised globally in epidemiological studies to detect relatedness among strains due to the simplicity of the procedure.[43] Data regarding the prevalent ribotypes of C. difficile in Saudi Arabia are scarce. The five most prevalent ribotypes in this study were 002 (20.3%), 001 (18.9%), 017 (18.9%), 014, (9.5%) and 020 (8.1%). These five ribotypes constituted 75.7% of the total strains. In a recent Saudi study,[33] ribotype 001 was the most common ribotype identified. Similar to the present study, the ribotypes 002 and 001 were the two most frequent ribotypes observed in Kuwait.[45] C. difficile ribotypes 002, 001, 014 and 020 have been reported to be common in hospitals worldwide.[46] The PCR ribotype 017, A B + C. difficile isolates are widely detected throughout Asian countries,[28] including China,[39],[47] Thailand [37],[43] and Indonesia.[38] The majority of CDI cases recorded in recent years are increasingly linked with the emergence of the epidemic ribotype 027 strains that show high resistance to fluoroquinolones as well as higher rates of severe disease and mortality.[14],[48] These hypervirulent strains were reported most frequently in North America and Europe, The increasing use of fluoroquinolones in health-care facilities in the United States may have provide a selective advantage for these strains.[41],[44] In most Asian countries, in contrast, the 027 strains were detected only sporadically,[14],[39],[41],[47] and there were no reports of 027 strains in Saudi Arabia until 2013, when Alzahrani and Al Johani [49] reported four cases of infection by C. difficile 027 strains over 1 year amongst patients hospitalised in King Abdul-Aziz Medical City, Riyadh. These strains represented 3.4% of the C. difficile isolates in the recent Saudi study conducted in the Eastern Province.[33] The current study detected four (5.4%) strains belonging to the ribotype 027. Like most of the 027 strains detected worldwide, these four isolates were binary toxin gene positive [47] and as expected, showed resistance to moxifloxacin.[42] The antimicrobial resistance patterns of C. difficile differ greatly across various populations and countries.[50] Periodical analysis of the antimicrobial sensitivity of C. difficile recovered from clinical specimens is essential for assessing the currently used empiric therapies and clarifying the effect of other antibiotics on the development of CDIs.[51] The isolates were tested for their sensitivity to 10 antimicrobials to investigate current trends in the antibiotic susceptibility of C. difficile. Metronidazole is the first-line antibiotic to treat mild-to-moderate CDIs, while vancomycin is used for severe infections.[52] In the present study, no resistance to vancomycin and metronidazole was detected, and many previous studies cited that C. difficile isolates retained full susceptibility to such drugs.[17],[42],[43],[53] In recent years, vancomycin and/or metronidazole resistance has been observed.[33], 34, [54],[55],[56],[57] This resistance demonstrates the need for continuous surveillance [42] and controlled prescription of these antimicrobials to inhibit the emergence of resistant strains.[58] The current study also revealed 100% sensitivity of the isolated C. difficile to piperacillin-tazobactam, and a similar result was reported by previous studies in Egypt,[34] Korea,[41] China [27],[42] and Kuwait.[45] Linezolid has been found to reduce the level of C. difficile toxins, but resistance to linezolid has been occasionally identified amongst the clinical C. difficile isolates. Selection and transmission of linezolid-resistant strains could increase the occurrence of CDIs, principally in the health care facilities where linezolid is frequently used.[59] In the current study, only two isolates (2.7%) were found to be linezolid resistant. A low rate of chloramphenicol resistance (5.4%) was observed in this study, a finding that is in accordance with the results of Gao et al. in China,[27] El-Sokkary et al. in Egypt,[34] and Peng et al. in Florida, the United States.[57] However, greater rates of chloramphenicol resistance among the C. difficile isolates were reported in Thailand, 12%,[43] and Iran, 18%.[54] A high rate of imipenem resistance was found among the isolated C. difficile strains in this study (83.8%). C. difficile clinical isolates from various countries exhibited a great variation in resistance to imipenem, as follows: Kuwait, 89%,[45] Egypt, 79.4%,[34] China, 4.9%[27] and North America, 2.3%.[55] Some antibiotics, including fluoroquinolones and clindamycin, have been found to be strongly associated with the development of CDIs.[60] In addition, the incidence of CDIs in England was reduced by about 80% by limiting the national prescription of fluoroquinolones and eliminating isolates resistant to these drugs. This underscores the significance of fluoroquinolone restriction and antimicrobial stewardship in the prevention and control of CDIs.[47] Amongst the 74 C. difficile isolates in the present study, 48.6% were resistant to moxifloxacin. High proportions of moxifloxacin resistance were observed in Japan, 42%,[44] Iran, 42.2%,[58] China, 45%[27] and Korea, 55.7%.[56] In contrast, limited moxifloxacin resistance has been observed Lebanon, 5.6%.[17] In addition, in Hudhaiah and Elhadi's study of Saudi Arabia,[33] moxifloxacin resistance was limited (2.3%). The rate of clindamycin resistance in the present study was 54%, which is consistent with previous studies conducted in Kuwait, 47.4%,[45] China, 53.4%,[42] Egypt, 55.2%[34] and Iran, 60%.[47] In contrast, lower rates of clindamycin resistance (8.3%–36%) were reported in other studies.[55],[60] The prevalence of MDR in this study was 56.8% (42/74), which is similar to the rate of 53.4% reported by Zhou et al. in China [42] but lower than the rate (69.3%) that Mohammadbeigi et al. reported in Iran.[54] The toxigenic strains showed higher rates of resistance to most antibiotics, as well as a higher prevalence of MDR, than the non-toxigenic strains. The association between toxin production and resistance phenotype in C. difficile was also reported by previous studies.[16],[43],[54] Furthermore, the A B + isolates were found to be associated with more antibiotic resistance than were the A + B + isolates, which is in accordance with several previous reports.[27],[41],[43]


 ~ Conclusion Top


This study contributes to the current knowledge on the prevalence of CDIs as well as molecular epidemiology and antibiotic resistance profiles of C. difficile clinical isolates in Saudi Arabia. Vancomycin and metronidazole remain options conducive to the initial treatment of CDI. Antibiotic stewardship is necessary to prevent emergence of additional drug-resistant strains and to reduce the incidence of CDI. In addition, further studies examining samples from more patients from different regions of Saudi Arabia are recommended for a better understanding of the phenotypic and genotypic characters of C. difficile and to monitor the rapidly changing epidemiology of CDI.

Financial support and sponsorship

This study was funded by the Deanship of Scientific Research, Taif University, KSA (Project number 1-439-6081).

Conflicts of interest

There are no conflicts of interest.



 
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  [Table 1], [Table 2], [Table 3], [Table 4]



 

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2004 - Indian Journal of Medical Microbiology
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