A Two-Step Approach for Diagnosing Glutamate Dehydrogenase Genes by Conventional Polymerase Chain Reaction from Clostridium difficile Isolates.

BACKGROUND Clostridium difficile is the major causative agent of nosocomial antibiotic-associated colitis. The gold standard for C. difficile detection is stool culture followed by cytotoxic assay, although it is laborious and time-consuming. We developed a screening test based on a two-step conventional polymerase chain reaction (PCR) approach to detect gluD, the glutamate dehydrogenase (GDH) enzyme gene, which is a marker for screening of C. difficile. Targeting gluD comparing to the conserved stable genetic element of pathogenicity locus (PaLoc), with an accessory gene of Cdd3, was an effective method for the detection of this pathogen from patients with enterocolitis. METHODS Fresh fecal samples of the patients who were clinically suspicious for antibiotic-associated colitis were collected. Stool specimens were cultured on the cycloserine-cefoxitin fructose agar (CCFA) in an anaerobic condition, following alcohol shock treatment and enrichment in Clostridium difficile Brucella broth (CDBB). On confirmed colonies, PCR was carried out for detection of PaLoc subsidiary gene, Cdd3, and toxicogenic genes, tcdA and tcdB. The gluD that is GDH gene detection was performed by conventional PCR on the extracted DNA from 578 fresh stool samples. RESULTS 57 (9.8%) strains of C. difficile were approved by conventional PCR for gluD and Cdd3 genes, in which 37 (6.4%) colonies had tcdA+/tcdB+ genotype, 2 (0.3%) tcdA+/tcdB-, 4 (0.7%) tcdA-/ tcdB+ and the remaining 14 (2.4%) colonies were tcdA and tcdB negative. CONCLUSION These results demonstrate that targeting gluD by PCR is quite promising for rapid detection of C. difficile from fresh fecal samples. Furthermore, the multiple-gene analysis for tcdA and tcdB assay proved a reliable approach for diagnosing of toxigenic strains among clinical samples.

INTRODUCTION

Clostridium difficile (C. difficile) is an anaerobic spore-forming gram-positive bacillus, which is the major cause of widespread diseases such as self-limiting diarrhea or fatal colitis upon antibiotic treatments. The main toxicogenic causes of C. difficile infections are two large Clostridia toxins; an enterotoxin (TcdA) and a cytotoxin (TcdB). As well as inflammation and fluid secretion, Clostridial exotoxins bind to the human intestinal cells and are responsible for the damage to the intestinal mucosa. Various types of virulence factors contribute to the pathogenicity of C. difficile within the gastrointestinal tract. The most significant change in cells treated with TcdA or TcdB is the loss of cytoskeletal structure. TcdB binds and enters the colonic epithelium, which causes a series of inflammatory reactions as a cytotoxin; otherwise, TcdA is an enterotoxin.[1,2] The symptoms of the related infection depend on toxin-encoding pathogenicity locus (PaLoc) in the bacterial genome. The PaLoc is a conserved and stable genetic unit, which is 19.6 kb and contains the tcdA and tcdB genes. Other PaLoc genes are tcdR and tcdC, which encode positive and negative regulators for tcdA and tcdB toxins. The PaLoc is located at the same site on the chromosome and includes three additional tcd open reading frames (ORFs), tcdD, tcdE, and tcdC, and ORFs for the insertion sequences cdd-2, cdd-3 that located upstream and downstream of the PaLoc, present in both non-toxigenic and toxigenic strains.[3-5]

Studies on C. difficile indicated that it has the ability to ferment low molecular weight substrates of amino acids. Glutamate has the main dependence of the other amino acid metabolism. Glutamate dehydrogenase (GDH) is a constructor enzyme produced in large amounts by all strains of C. difficile independent of their toxigenic or non-toxigenic forms.[6,7] Detection of this enzyme by immunoassay methods has been considered a valid technique with proper sensitivity for the screening of C. difficile in stool samples.[8] More comprehensive diagnostic assays for C. difficile infection are based on clinical symptoms in combination with laboratory tests for screening C. difficile toxins and GDH.[9] GDH enzyme detection methods do not distinguish between toxigenic and non-toxigenic strains, thus a toxin assay is required while a definitive diagnosis is necessary. In addition, approaching toxin is essential for epidemiological research, optimal management, and prevention programs.[10] Molecular methods for the diagnosis of C. difficile infection have been studied far less than those used to diagnose other infectious diseases. Various nucleic acid amplification tests are commonly used for detection of toxigenic C. difficile.[11] The known high sensitivity (93% to 100%) of nucleic acid amplification assays is useful in the confirmation of GDH gene or other toxin assays, but it should be noted that it is only useful in the acute disease and to verify false positive cases.[12,13] The European Society of Clinical Microbiology and Infectious Diseases recommended a two-step algorithm detecting GDH as a screening method combined with toxigenic culture or cytotoxic assays.[14]

We developed a two-step approach by toxigenic culture and conventional PCR assay for comparing the outbreak of GDH gene (gluD) to the PaLoc accessory gene of Cdd3. For an additional confirmation survey, we also detected toxin-positive samples by conventional PCR test for tcdA & tcdB genes.

MATERIALS AND METHODS

Patients’ samples:

A total of 578 fresh diarrheal stool specimens of patients with enterocolitis were collected from hospital inpatients with a history of prolonged antibiotic therapy (1 to 8 weeks) in the period from 2015 to 2018.

Toxigenic culture:

The stool samples were transported at room temperature and cultured within 8 hours after collection, or stored at 4° C, for no more than 48 hours before being cultured. Toxigenic culture based on the anaerobic culture of stool samples from 2-5 days was used as a reference procedure for C. difficile diagnosis. Since the bacteria are spore-forming, all the stool samples were treated with the alcoholic shock. The samples were divided to two equal parts, one exposed to 96% ethanol for 1 hour and the other was enriched in Clostridium difficile Brucella broth (CDBB) for 1 minute supporting the vegetative forms of C. difficile.[15] The treated samples were subsequently plated on selective agar, cycloserine-cefoxitin fructose agar (CCFA), and incubated in an anaerobic chamber (Gas Pack Anaerocult® A Merk) at 37°C for at least 2-5 days. After isolation in CCFA medium, the presence of C. difficile was confirmed by gram stain, colony morphology, and detection of “horse-barn” odor.[16]

DNA extraction:

Each isolated strain from cultures was transferred with an inoculating loop into a 1.5 mL microcentrifuge tube containing 200 mL of sterile PBS buffer, and bacterial DNA was extracted using the QIAamp kit (Qiagen, Germany), according to the manufacturer’s instructions.

Nucleic acid amplification:

Detection of Cdd3, glutamate dehydrogenase (gluD), toxin A (tcdA), and toxin B (tcdB) genes was performed by multiplex conventional PCR test, in which melting temperature for both primer sets was over 600C so that the annealing and the extension steps could be combined into a single step. The specific primer sequences are presented in table 1. The PCR reactions were carried out using 2× Mix RED Master Mix (Amplicon, Denmark), 40 pmol of the respective primer pair for each reaction, and 5 µL of the extracted DNA. The PCR procedure was included a denaturing step for 1 min at 95°C and annealing at 52°C for 1 min, followed by a 1 min extension at 72°C for 40 cycles. A final extension step was done at 72°C for 10 min.

Table 1

Primers sequence used for amplification of Cdd3, gluD, tcdB, and tcdA genes

Gene	Primer	Sequence: 5 ----> 3'	Product size (bp)	Ref.
tcdA	TA1TA2	5´-ATG ATA AGG CAA CTT CAG TGG-3´5´-TAA GTT CCT CCT GCT CCA TCA A-3´	624 bp	[17]
tcdB	TB1TB2	5´-GAG CTG CTT CAA TTG GAG AGA-3´5´-GTA ACC TAC TTT CAT AAC ACC AG-3´	412 bp	[17]
Cdd3	Tim6Struppi6	5′-TCC AAT ATA ATA AAT TAG CAT TCC A -3′5′-GGC TAT TAC ACG TAA TCC AGA TA -3′	622 bp	[17]
GDH	gluD1gluD2	5`-TGTCAGGAAAAGATGTAAATGTCTTCGAG-3′5`-TTAGTACCATCCTCTTAATTTCATAGCTTC-3′	1278 bp	[18]

Detection of amplified products:

Amplified products were visualized by running 5µL of the reaction mixture on a 1% agarose gel immersed in Tris-acetate-EDTA(TAE) at 100 V for 60 min. Gels were stained by adding DNA Safe Stain (SinaClon BioScience Co.) and visualized under UV light.

RESULTS

Study population

This study was performed on 578 samples obtained from Shariati and Milad General Hospitals, Tehran, Iran. The appearances of stool samples were unformed, loose, bloody, or watery. All of the patients had a history of antibiotic therapy from 1-8 weeks, most of them treated with beta-lactam, quinolone, and some with vancomycin and metronidazole.

Routine toxigenic culture:

A total of 62 (10.7%) stool specimens grew in toxigenic culture. DNA of C. difficile colonies was extracted using the Qiagen DNA extraction kit. The PCR results for the gluD gene in 57 (9.8%) strains of the cultured samples were positive. Moreover, all of the colonies were also positive by PCR on the Cdd3 gene. Detection of tcdA & tcdB genes were subsequently carried out by conventional PCR assay from the extracted DNA of isolated C. difficile from toxigenic culture.

Toxigenic strains were retrieved in 57 colonies (9.8% frequency), tcdA 39 (6.7%), and tcdB 41 (7.1%), in which 37 (6.4%) colonies had the tcdA+/tcdB+ genotype, 2 (0.3%) had tcdA+/tcdB-, 4 (0.7%) had tcdA-/tcdB+, and the remaining 14 (2.4%) colonies had tcdA and tcdB negative genotype (table 2, figure 1).

Table 2

Comparison of the conventional PCR results with the toxigenic culture of Clostridium difficile

C. difficile strains	tcdA	tcdB	gluD	Cdd3
37 (6.4%)	+	+	+	+
2 (0.3%)	+	-	+	+
4 (0.7%)	-	+	+	+
14 (2.4%)	-	-	+	+
0	-	-	-	-
Total57 (9.8%)	39(6.7%)	41(7.1%)	57(9.8%)	57(9.8%)

Fig. 1

Column chart of the frequency of the gluD, Cdd3, tcdA, and tcdB

DISCUSSION

We used a two-step method to detect C. difficile from stool specimens including toxigenic cultures followed by conventional PCR method. The toxigenic culture was performed after fecal samples treatment with the alcoholic shock, which was twice and also selective enrichment medium (CDBB) that was three times more effective than direct plating for detection of C. difficile disable in forming spores. The main finding of our study is the high number of the clinical samples tested for detection of Clostridium difficile by toxigenic culture. Also comparing the GDH based PCR assay with the specific gene of Cdd3 does not differentiate between toxigenic and non-toxigenic C. difficile species.[19-21]

C. difficile has been identified as the causative organism of antibiotic-associated colitis in humans.[22] Prevention and management of Clostridium difficile infection are challenging issues. The optimized laboratory diagnosis of C. difficile infection remains controversial.[23] Many laboratories designed algorithms for screening the presence of a C. difficile common antigen, GDH, as a marker for the presence of the organism in the stool.[24] The increase in the outbreak of Clostridium difficile infection in different populations has been proportional to the rate of hypervirulent species. The presence of GDH is not specific to toxin-producing C. difficile types.[25] However, molecular assays detecting GDH or C. difficile toxin genes (tcdA and tcdB) represent high sensitivity for differentiating the toxin-producing strains.[26,27]

Our study showed 9.8% frequency of antibiotic-associated colitis and demonstrated that PCR of tcdA and tcdB toxin genes could be used as a rapid method for confirmation of the toxigenic potential of C. difficile isolates. The toxin gene profile results have shown that among 57 strains of C. difficile isolated from patients’ stools, 14 (2.4%) strains were non-toxigenic A-B-, 37 (6.4%) strains were toxigenic A+B+, 2 (0.3%) strains were toxin A+B-, and the remaining 4 (0.7%) strains were A-B+. The reports of our survey for the frequency of 9.8% highlight the consistent outbreak of C. difficile infection rated from 6% to 21% in several countries such as Europeans and American ones.[28-31] There are several reports about the prevalence of C. difficile in Iran. Rates differ from 6% in Crohn’s disease to 15.3-20% in hospitalized C. difficile infection detected by alternative methods such as PCR or Loop mediated isothermal amplification (LAMP) and also molecular typing.[32-35] There are fewer reports of antibiotic-associated Clostridium difficile infection (CDI) outbreaks in our country. That is why we suggest a local epidemic occurrence should be confirmed by further epidemiological methods such as pulse-field electrophoresis or ribotyping.

Removal of PCR inhibitors from fecal samples through the PCR reaction is critical which is why the direct use of molecular methods of the samples are limited. C. difficile infections are not commonly diagnosed and reported in Iran. Moreover, in the studied intensive care units where documented cases have been admitted, infection rates are not clear. It may be due to inadequate knowledge on the part of the clinicians concerning the clinical detection of the infection. Another reason may be related to the use of a low sensitivity toxin assays employed in the diagnostic laboratories instead of the toxigenic culture and cytotoxic assays, which has caused further problems and is time-consuming.

CONCLUSION

Availability of proper laboratory methods has a significant influence on documentation of CDI frequency. The use of a procedure for microbiological analysis, which can implement a unique method improves the monitoring of CDI incidence and helps in understanding its epidemiological prevalence. Furthermore, it is important that clinicians be instructed to adopt and apply the most reliable diagnostic strategies, which targeting nucleic acids, including endpoint or real-time PCR methods for detection of the genes like those encoding GDH and TcdA and/or TcdB toxins.