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. 2024;17(3):304–312. doi: 10.22037/ghfbb.v17i3.2982

Comparison of toxin gene expression levels and molecular typing of Clostridioides difficile strains isolated from patients with diarrhea

Leili Shokoohizadeh 1,2, Mahnaz Moomivand 2, Abbas Yadegar 3, Masoumeh Azimirad 3, Seyyed Hamid Hashemi 1,4, Mohammad Yousef Alikhani 1,2
PMCID: PMC11413378  PMID: 39308537

Abstract

Aim:

This study aimed to evaluate the expression of tcdA, tcdB, and binary toxin genes (cdtA and cdtB) by Real-Time PCR and molecular typing of Clostridioides difficile isolated from patient diarrhea samples from Hamadan Hospitals, west of Iran.

Background:

The concentration of C. difficile toxins (CDTs) is associated with the severity of the disease and the mortality rate. Measuring CDT levels could provide a reliable and objective means of determining the severity of C. difficile infection (CDI).

Methods:

From November 2018 to September 2019, 130 diarrhea samples were collected from hospitalized patients in three hospitals in Hamadan. C. difficle isolates were detected by culture and PCR. The presence of the genes encoding the toxin was identified by PCR, whereas the measurement of toxin expression was conducted using a relative Real-Time PCR technique. Genetic linkage of the isolates was also assessed by Ribotyping and Repetitive Extragenic Palindromic (rep-PCR) methods.

Results:

Among 130 diarrhea samples, 16 (12.3%) were positive for C. difficile. Genes encoding cdtA and tcdB were detected in all isolates, and 8 (50%) and 6 (37.5%) isolates were positive for the cdtA and cdtB genes. Real-time PCR results showed different expression levels of the toxin genes. A significant increase in the expression of the tcdA gene was observed compared with the control strain (P<0.05). Besides, more expression of cdtA gene was observed in the strains compared with cdtB gene. Ribotyping and rep-PCR results showed high genetic diversity of C. difficile among hospitals investigated.

Conclusion:

We encountered toxigenic C. difficile strains with various toxin expression levels, ribotypes, and rep types based on the findings of this study. This indicated that various clones from various sources circulate in the hospitals and among patients.

Key Words: Clostridioides difficile, Gene expression, Real-Time PCR, Molecular typing

Introduction

Clostridioides difficile is a Gram-positive, obligate anaerobic spore-forming bacterium that was identified as an important human pathogen, especially in hospitalized patients. C. difficle infection (CDI) is primarily associated with severe diarrhea followed by antibiotic use (1, 2). Toxigenic C. difficile strains produce two major exotoxins called toxin A (enterotoxin) and toxin B (cytotoxin). They are principally involved in the pathophysiology of CDI (2). The main symptoms of CDI are diarrhea, inflammation, and tissue necrosis, which are caused by the toxins' complicated cascade of cellular reactions in the host. It is difficult to comprehend the factors that contributed to the epidemic of some C. difficile strains. Debilitating recurrent infections are common. For accurate epidemiological research, as well as the most effective strategies for management and prevention, toxins should be detected for diagnosis (3, 4). The level of C. difficile toxins (CDTs) in feces is linked to disease severity and mortality rate. It may be possible to accurately and objectively define the severity of CDI by measuring CDT levels (5). Consequently, it is possible to predict the pathogenicity of C. difficile toxins by measuring their gene expression levels using a molecular technique like Real-Time PCR. Although Real-time PCR was used in the majority of studies to directly detect C. difficile from stool samples (6-8). Molecular typing methods are highly beneficial for epidemiological studies and bacterial genetic associations due to the paramount significance of regulating bacterial infections, particularly nosocomial infections (9). Various molecular typing methods were used in epidemiological studies to study genetic diversity and genetic relatedness and find the origin of CDI (10-12). Among the molecular methods of bacterial typing, PCR ribotyping is considered the standard method for molecular typing of C. difficile (10, 11, and 13). Most studies in Iran have focused on PCR-based ribotyping (13-15). Rep-PCR is a PCR-based typing technology that generates fingerprints directly, without the need for the endonuclease enzyme. Since rep-PCR is an affordable and rapid method; it is cost-effective and can be used to molecular typing of C. difficile strains (16-17). As one of the main goals of this study was the molecular typing of C. difficile strains, it was necessary to compare different genotypes based on antibiotic resistance patterns and toxin profiles; therefore, we used the results of our previous study, which determined antibiotic resistance patterns and toxin profiles of C. difficile strains (18). This study aimed to assess the gene expression levels of tcdA and tcdB genes, as well as the binary toxin genes of C. difficile, using Real-time PCR assay. Furthermore, the study aimed to analyze the genetic diversity of C. difficile strains via rep-PCR and Ribotyping.

Methods

Identification of C. difficile isolates

In a previous cross-sectional study, we isolated 16 strains of C. difficile from 130 diarrheal samples collected from patients hospitalized in Hamadan hospitals in western Iran between November 2018 and September 2019 (18). C. difficile strains were identified and confirmed by microbiological tests and PCR technique. C. difficile colonies were maintained in cooked meat broth at 4 ⁰C (18). The antimicrobial susceptibility to vancomoycin, metronidazole, and clindamycin was assessed using the agar dilution method in accordance with the CLSI and EUCAST guidelines in the preceding study. The frequency of tcdA, tcdB, cdtA and cdtB genes was detected by PCR technique in our previous study (18). All data for identification, antimicrobial susceptibility and toxin genes frequencies of were presented in ref. 18 in this study (18).

DNA extraction and PCR

Genomic DNAs were extracted from C. difficile colonies on CCFA plates using a commercial DNA extraction kit (Qiagen, Hilden, Germany). All C. difficile isolates were subjected to the rep-PCR typing and Ribotyping.

RNA Extraction and cDNA synthesis

All C. difficile strains were subjected to Real-time PCR (RT-PCR) to determine toxin genes expression levels. Total RNA was extracted using an RNA extraction kit (SinaClon, Iran), and cDNA was subsequently created using a cDNA synthesis kit (Yekta Tajhiz Azma, Iran), following the manufacturer's instructions.

Real-Time PCR reaction

Real-time quantification of cDNA was performed with the detection system (Roche, Germany) using the SYBR Green PCR Master Mix. The optimized reaction consisted of a Master Mix (10X), 0.4 µl of each primer of toxin genes (10 pmol) as previously described (19, 20), 2 µl of cDNA (100 μg/ml), and 7.2 µl of DEPC-water in a total volume of 20 µl. Toxin genes expression levels (fold changes) of all genes were calculated using the 2−ΔΔCt method (21). The expression levels of toxin-encoding genes were compared to the expression levels of these genes in standard positive control strains of C. difficile. C. difficile strain VPI 10463 was used as a positive control for the tcdA and tcdB genes and C. difficile strain RIGLD 141 was used as a positive control for the cdtA and cdtB genes. The real-time PCR procedure was programmed as follows. Initial denaturation at (95°C for 15 min), followed by 40 cycles at (95°C for 5 sec), (60°C for 34 sec), (72°C for 30 sec), and Melting curve at (60°C for 1 min) and (95°C for 15 sec).

rep-PCR typing

Diversity and genetic linkage among C. difficile isolates were assessed by rep-PCR using the primer REP-F: 5’-ICGICTTATCIGGCCTAC-3’ and REP-R: 5’-IIIICGICGICATCIGGC -3’ (16), according to the following protocol: Initial denaturation (95°C for 2 min) followed by 45 cycles of denaturation (95°C for 30 sec), annealing (38°C for 1 min), extension (72°C for 2 min), and a final cycle of extension at 72°C for 16 min. The PCR products were electrophoresed on a 1% agarose gel at a voltage of 70 V for duration of 1 hour. The resulting band patterns were captured using a gel documentation system (16). The observed band patterns were analyzed and grouped together using BioNumeric software version 7.1 (Applied Maths, Belgium).

Ribotyping

To determine the ribotypes of C. difficile isolates, PCR was performed using specific primers (P3: 5ʹ-CTGGGGTGAAGT CGTAACAAG-3ʹ and P5: 5ʹ-GCGCCCTTTGTAGCTTGACC-3ʹ) according to the following program: Initial denaturation (95°C for 2 min) followed by 35 cycles of denaturation (94°C for 60 s), annealing (55°C for 30 sec), extension (72 °C for 90 s), and a final cycle of extension at 72°C for 10 min (22). Different profiles or ribotypes of C. difficile strains were analyzed using BioNumeric software version 7.1 (Applied Maths).

Statistical analysis

Statistical analysis was conducted using SPSS software, version 21 for Windows (SPSS Inc., Chicago, IL, USA). A p-value of <0.05 was deemed statistically significant.

Results

Our study results showed that out of 130 stool samples, 16 samples (12.3.6%) were positive for C. difficile. All C. difficile isolates (100%) carried both tcdA and tcdB genes (tcdA+/tcdB+) and were considered toxin producing strains. Binary toxin genes (cdtA and cdtB) were detected in 6 (37.5%) and 8 (50%) isolates, respectively. Co-presence of the cdtA and cdtB genes was not observed in any isolates.

The results of Real-Time PCR showed that various gene expression levels were observed. Based on real-time PCR, increased expression was mainly observed in the tcdA gene. Out of the eight isolates, 43.7% exhibited elevated levels of tcdA gene expression compared to the control strain. Five isolates exhibited reduced tcdB gene expression compared to the reference strain, but there was no indication of an upregulation in tcdB gene expression (Figure 1). Therefore, based on Real-time PCR results of the binary toxin genes (Figure 2), an increased cdtA gene expression was observed in 6 (37.5 %) strains. Only one strain had higher expression of the cdtB gene compared to the control strain.

Figure 1.

Figure 1

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Comparison of tcdA and tcdB gene expression levels in C. difficile isolated from diarrhea samples of patients in Hamadan hospitals. (Calibrator: strain VPI 10463 as a positive control strain for the tcdA and tcdB genes)

Figure 2.

Figure 2

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Comparison of cdtA and cdtB gene expression levels in C. difficile isolated from diarrhea samples of patients in Hamadan hospitals (Calibrator: strain RIGLD 141 as a positive control strain for the cdtA and cdtB genes)

In every strain of C. difficilis, ribotyping analysis showed distinct banding patterns. Figure 3 depicts the sixteen distinct profiles that were observed. However, some of these strains were isolated from the same hospital and wards and shared the same resistance pattern and toxins. Toxin profiles and patterns of resistance in ribotypes did not significantly correlate. This study's molecular typing analysis reveals that ribotyping is more effective at distinguishing C. difficile strains. The ribotyping method had a higher discriminatory power than rep-PCR method when online formula for calculating discriminatory power was used (http://insilico.ehu.es/mini_tools/discriminatory_power/index.php). The discriminatory power of rep-PCR was 0.97, while that of ribotyping was 1.0.

Figure 3.

Characterization of 16 C. difficile strains isolated from diarrhea samples of patients in Hamadan hospitals by ribotype, binary toxin pattern, antibiotic resistance pattern, hospital and ward (all strains are tcdA+/tcdB+)

Figure 3

Neg: negative, Int: internal, ICU: Intensive care unit, Van: vancomycin, Met: Metronidazole, Clin: clindamycin, S: sensitive, R: resistant

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Based on band samples obtained by the electrophoresis of PCR products of REP regions in different C. difficile isolates, the size of the fragments varied from 500 bp to 2000 bp. It was found that there was high genetic diversity in C. difficile strains (Figure 4), so more than 80% of the isolates were classified into different types or categories. Based on rep-PCR analysis, only three isolates were categorized into a shared class, whereas 14 distinct kinds were identified using REP sample profiles. In fact, three isolates could not be differentiated by rep-PCR and to better identify them, more powerful molecular or sequencing-based methods are needed. All 16 isolates were similar in their tcdA and tcdB toxin gene profiles but differed in their binary toxin gene profiles and antibiotic resistance patterns (Figure 3). There was no significant correlation between REP types with resistance profile and toxin profile (P>0.05).

Figure 4.

Figure 4

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Dendrogram of rep-PCR patterns of 16 C. difficile strains isolated from diarrhea samples of patients at Hamadan hospitals. The numbers of isolates, binary toxin patterns, antibiotic resistance patterns, hospitals, and wards are the same in Figures 3 and 4. No rep-PCR products were detected in isolates number 4, 6, and 8, thus clustering them into a common type.

Discussion

This study found that 12% of hospitalized patients in Hamadan hospitals were at risk for CDI. The fact that all C. difficile strains were toxin-producing or toxinogenic is a key finding. All C. difficile isolates tested were positive for the tcdA and tcdB. Some C. difficile strains contained genes encoding the binary toxins cdtA and cdtB. Real-Time PCR findings indicated varying expression levels of the toxin genes. A notable rise in tcdA gene expression was noted compared to the control strain (P<0.05). Furthermore, the strains exhibited a greater level of expression of the cdtA gene compared to the cdtB gene. The ribotyping and rep-PCR data demonstrated a significant genetic variability of C. difficile across the hospitals examined. Multiple prevalence reports of C. difficile infection (CDI) have been recorded in different geographic areas (23-27). Our study's CDI prevalence was lower than that of other Iranian studies (28-30). Variation in CDI prevalence may be affected by the subject population, C. difficile detection methods, and hospital infection control strategies (18).

C. difficile strains were more resistant to vancomycin and metronidazole in our study, but less to clindamycin. In Iran and other regions, numerous reports of C. difficile antibiotic resistance were published (15, 24- 32). More than 50% and 30% of C. difficile strains isolated from Tehran hospitals were found to be resistant to metronidazole in a recently published Iranian study (31).

The results of our study demonstrate the distribution of different ribotypes of C. difficile strains in hospitals in Hamadan. C. difficile ribotype diversity was high, but no predominant ribotype was found (Figure 2). This study's findings are consistent with those of previous studies carried out in Iran and other countries (13-15, 33- 35). In this study, we were compelled to conduct ribotyping for C. difficile strains using P3 and P3 primers and standard gel agarose electrophoresis due to restrictions and a lack of facilities to transport ribotyping-PCR products abroad for capillary electrophoresis. Unfortunately, it was not possible to compare the ribotype patterns with known reference strains. However, based on the results of ribotyping by capillary electrophoresis, distinct ribotypes were identified in hospitals in Tehran and Isfahan (13, 36). From 2004 to 2018, ribotypes 001, 0126, and 084 were found to be the most prevalent in Tehran in a study by Azimirad et al (13). Kuhsari et al.'s study from Tehran found that in three tertiary care hospitals in Tehran, ribotypes 039, AI-12, and AI-21 were found to be the predominant ribotypes among clinical and non-clinical C. diff isolates (36). According to a research conducted at one of Isfahan's teaching hospitals, the most prevalent ribotype among C. difficile strains recovered from stool samples of patients who experienced diarrhea was ribptype 078. (37). One case of the highly virulent ribotype (027) in children younger than five years old was reported from Iran (38). The tcdA/tcdB toxin profiles of all ribotypes in our study were comparable, but their patterns of antibiotic resistance and cdtA/cdtB profiles were distinct. Moreover, it is possible that the environment provided distinct conditions for the relevant C. difficile strains such as endogenous infection and spread of strains from outside and inside the hospital. Relatively effective infection control strategies appear to be responsible for the lack of spread of the dominant clones of C. difficile among patients in different hospital wards. C. difficile strains isolated from hospitalized patients in the wards did not come from the same source because they had distinct ribotypes in the studied hospitals.

The research used the rep-PCR approach in addition to ribotyping to identify the strains of C. difficile. Both of these methods rely on PCR. There are currently no reports from Iran about the molecular typing of C. difficile using the rep-PCR approach. More than 80% of the isolates in this study were classified as belonging to one of several types or categories, and only three were classified as belonging to a single type, indicating a high genetic diversity among C. difficile isolates.

Our findings showed that rep-PCR methods were less effective at discrimination than ribotyping. Ribotyping identified 16 different ribotypes and rep-PCR identified 14 different types. Some epidemiological studies have used rep-PCR for molecular typing of C. difficile strains. Russello et al. studied the molecular characterization of C. difficile-associated diarrhea using rep-PCR in Italy. Their results indicated that rep-PCR could be used as a reliable method for molecular typing of C. difficile and as a useful method for better infection control in hospitals (39). Rahmati et al. conducted a research in which they used three REP-PCR-based approaches, including rep-PCR, ERIC-PCR, and the BOX method, to discriminate fifty C. difficile strains with ribotype 001 from the UK. Analysis of the results showed that all methods exhibited satisfactory levels of performance and reproducibility. However, rep-PCR is more specific and provides more information about the epidemiology of C. difficile disease in UK hospitals (40).

For the purpose of typing 205 C. difficile isolates, the researchers in Finland compared an automated rep-PCR method, PCR ribotyping, and pulsed-field gel electrophoresis (PFGE). In the local clinical microbiology labs, the automated rep-PCR-based typing method is an option for first-line molecular typing. Because this method was faster and easier to use than PCR ribotyping or PFGE typing, it required less hands-on time. However, there are differences between their rep-PCR method and ours. In their investigation, the researchers used microfluidics lab-on-a-chip technology to detect the rep-PCR products and then separate the amplicons. Hence, it is essential to enhance and adapt fundamental molecular typing techniques to enhance results (41).There were differences in the expression levels of toxin genes according to the Real-Time PCR results in this study. Some toxin genes were either not expressed at all in some strains or were expressed differently in others. The highest expression levels were observed in the expression of the tcdA gene. In 43.7% of isolates, tcdA gene expression was higher than control strains or calibrators. It is suggested that the mechanisms affecting the expression of these genes to be further investigated in terms of the difference in the expression of various toxin genes in C. difficile strains.

In Iran, there haven't been any Real-Time PCR studies on the levels of tcdA, tcdB, cdtA, and cdtB gene expression. Real-time PCR method, which is mostly used for diagnostic purposes, was used in numerous studies to directly detect C. difficile from stool samples (7, 8, 42). Following the confirmation of the samples by PCR and culture, Real-Time PCR was used in our investigation to examine the expression levels of toxin genes in different strains of C. difficile. Real-Time PCR confirmed the presence of C. difficile colonies in our investigation, and various strains displayed varying levels of expression. The studies that have utilized the Real-Time PCR method in Iran and other nations are the subject of the discussion that follows.

Luna et al. used Real-Time PCR at Texas Children's Hospital to diagnose CDI. Real-Time PCR, conducted directly on feces samples, demonstrated an ideal performance with a sensitivity of 95%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99% (7). Kim et al. conducted a study in which a colony of toxigenic C. difficile strains (two distinct strains with tcdA+/tcdB-, tcdA+/tcdB- profiles) was used as a standard for the diagnosis of CDI in stools in South Korea using Real-Time PCR method. The device-defined cut-offs were used to automatically interpret the results (43). Real-time PCR was used to test how quickly C. difficile could be found in feces, based on Belanger et al. They came to the conclusion that because measuring the cytotoxin in tissue cultures is the gold standard for identifying C. difficile and detecting toxicogenic C. difficile, it takes a long time and requires 24 hours of incubation. Consequently, they chose Real-Time PCR because it is fast, specific, and sensitive. Real-Time PCR can also directly identify C. difficile in feces (19).

Song et al. used Real-Time PCR as a quick diagnostic method for C. difficile infection. Out of 207 samples, 117 cases were found to have CDI. Real-Time PCR, toxin assay, and tissue culture were all 87.2%, 48.7%, and 65% sensitive, respectively. Their findings indicate that Real-Time PCR is a very effective method for properly and rapidly diagnosing CDI. Furthermore, their study demonstrates that Real-Time PCR is the optimal approach for accurately and swiftly diagnosing CDI and identifying toxin-producing genes (44). Because this was the first study in the west of Iran to compare the level of toxin gene expression and molecular typing of C. difficile isolates, we encountered a number of challenges and limitations, including low hospital cooperation in providing samples and financial constraints brought on by economic sanctions. Antimicrobial susceptibility selective culture media, supplements, and antibiotic powders were required for the isolation and identification of C. difficile strains. In most cases, we were unable to obtain these materials, which presented numerous challenges and prevented us from collecting additional samples, and examining additional strains. Even sending ribotyping results to capillary electrophoresis was impossible.

Conclusion

Based on the results of molecular typing of C. difficile strains, different C. difficile clones circulating in Hamadan hospital and CDI may be in terms of the acquisition of different endogenous or environmental pathogenic strains. Besides, strains differed from each other based on the expression of toxin genes. Further studies are recommended for factors or mechanisms that affect the expression level of C. difficile toxins. In order to obtain more precise and cohesive results, it is advised to employ a variety of molecular typing methodologies and to examine additional strains of C. difficile that have been collected from a variety of regions.

Acknowledgements

We extend our gratitude to all the individuals at the Foodborne and Waterborne Disease Research Center, Research Institute for Gastroenterology and Liver Disease, Shahid Beheshti University of Medical Sciences, Tehran, IRAN, for facilitating the subculture of our samples and confirming the C. difficile isolates. We also acknowledge the staff of the microbiology laboratory at Sina and Beheshti Hospitals in Hamadan.

Ethical approval

The present study was ethically approved by the Institutional Review Board of Hamadan University of Medical Sciences (IR.UMSHA.REC.1397.510).

Funding

This research has been supported by Vice Chancellor for Research & Technology of Hamadan University of Medical Sciences, Hamadan, IRAN (Grant no: 9708295113).

Conflict of interests

The authors declare that they have no competing interests.

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