AbstractObjectives

To develop a rapid and visual loop-mediated isothermal amplification (LAMP) assay targeting the tetM gene in Clostridioides difficile (C. difficile) strains cultured from feces.

Methods

Primers were designed to recognize the tetM gene in C. difficile by LAMP, using turbidity and visual detection. The sensitivity and specificity of LAMP primers was determined. Besides, We conducted both LAMP and polymerase chain reaction (PCR) for the tcdA, tcdB, cdtA, cdtB, ermB, tetM genes in 300 toxigenic C. difficile strains cultured from feces.

Results

The target DNA was amplified and visualized within 60 minutes at a temperature of 62°C. A total of 26 bacterial strains were found negative for tetM, which manifested high specificity of the primers. The detection limit of LAMP was 36.1 pg/µl, which was 100-fold more sensitive than PCR. The positive rate of tetM in toxigenic C. difficile strains cultured from feces was 93.3% by both LAMP and PCR. The proportion of toxin types in those C. difficile strains was 95.7% for A+B+CDT−, 4% for A−B+CDT−, and 0.3% for A+B+CDT+, respectively.

Conclusions

This is the first study examining tetM gene in C. difficile strains cultured from feces by LAMP. Its high specificity and sensitivity, as well as visual detection, make the new assay a powerful diagnostic tool for rapid testing.

Key words

Introduction

Clostridium difficile, recently reclassified as Clostridioides difficile(Lawson et al., 2016), is a Gram-positive spore-forming ubiquitous bacterium (Fig 1C). Toxigenic strains can cause C. difficile infection (CDI) with various clinical manifestations ranging from mild diarrhea to life-threatening conditions, usually due to previous antibiotic treatment(Kuijper et al., 2006). The pooled incidence of toxigenic C. difficile among patients with diarrhoea was 14% in Mainland China(Tang et al., 2016). The incidence of CDI in Europe increased from 4.1 cases per 10000 patient-days in 2008 to 7.0 cases per 10000 patient-days in 2013(Bauer et al., 2011; Davies et al., 2014). In the United States, C. difficile was responsible for about half a million infections and 29000 deaths in 2011(Lessa et al., 2015). In addition, it was estimated that approximately 20% to 30% of recurrent CDI(Bakken et al., 2013). The average cost for patients with recurrent CDI ($28,218) was more than twice that for patients with primary CDI ($13,168)(Shah et al., 2016). Total annual CDI-attributable cost was estimated at $6.3 billion (Range: $1.9-$7.0 billion), with a total hospital managed stay of nearly 2.4 million days in US(Zhang et al., 2016). Generally speaking, CDI has become a perpetual problem that leads to significant morbidity and mortality, increased economic burden, and higher healthcare costs. Major risk factors for CDI include advanced age, chemotherapy, use of proton pump inhibitors, chronic renal disease, chronic liver disease, and antibiotic use, the latter including tetracycline, penicillins, cephalosporins, fluoroquinolone, clindamycin, and so on(Kelly et al., 1994; Arriola et al., 2016; Khanafer et al., 2017). What's more, tetracycline resistance was present in one-fifth of clinical C. difficile isolates, which would further increased the risk of CDI(Sholeh et al., 2020) .

Fig. 1A. Electronic colonoscopy of pseudomembranous enteritis (60×). Under the electronic colonoscopy, scattered pale yellow microscopic particles were observed, and the mucosa was hyperemic and brittle. B. Histopathologic photographs of pseudomembranous colitis (100×). Neutrophils infiltrated the lamina propria, epithelial cells were damaged, and secretions were found on the mucosal surface. C. C.difficile clinical isolated strain, Gram-positive bacillus (Gram, 1000×). D. Agarose gel electrophoresis results after PCR amplification of tcdA, tcdB, cdtA and cdtB. tcdA: 546bp, tcdB: 204bp, cdtA: 375bp, cdtB: 510bp. E. Agarose gel electrophoresis results after PCR amplification of ermB. ermB: 711bp, M: marker.

Tetracycline resistance in C. difficile is typically conferred by ribosome protection gene tetM (Roberts, 1996; Spigaglia et al., 2005). Previous studies have showed that most tetM genes in C. difficile are carried by the conjugative transposon Tn5397, which is closely related to the well-studied conjugative transposon Tn916 in the regions concerned with conjugation(Mullany et al., 1990; Roberts et al., 2001). However, Tn5397 differs from Tn916 in that it contains a Group II intron and has different integration/excision modules(Roberts et al., 2001a, 2001b). The tetM gene is inherited primarily by horizontal gene transfer (HGT) via transposons and/or plasmids, with transposon Tn916 most commonly associated with its dissemination(Dönhöfer et al., 2012; Dong et al., 2014). It has been shown that it can exchange antibiotic resistance genes with other gastrointestinal tract bacteria and that tetracycline resistance genes can be transferred between intestinal bacteria(Adams et al., 2002; Scott, 2002; Spigaglia et al., 2005). Therefore, tetracycline resistance is worthy of great attention. Here, a rapid and visual molecular assay to detect tetM gene is indispensable for efficient clinical treatment guidance and timely implementation of infection control measures.

Recently, a number of conventional polymerase chain reaction(PCR) assays have been developed for tetM detection (Adams et al., 2002; Spigaglia et al., 2007). While these assays for sensitive and specific detection appear to be promising, their equipment are quite sophisticated and consumables are expensive. Besides, the Taq DNA polymerase in PCR assays can be inactivated by inhibitors present in crude biological samples(de Franchis et al., 1988). Thus, rapid, reliable and affordable detection of tetM gene via the molecular methods still remains a challenge.

Loop-mediated isothermal amplification (LAMP) technology is a novel molecular technique for nucleic acid amplification, which depends on auto-cycling strand displacement DNA synthesis at an isothermal condition within 60 minutes by using Bst DNA polymerase(Notomi et al., 2000; Ushikubo, 2004). LAMP exhibits high specificity and selectivity because of the use of 4(or 6) primers recognizing 6(or 8) distinct regions on the target DNA, and can be completed in less than 1h due to the high amplification efficiency without the thermal cycler used in conventional PCR(Notomi et al., 2000; Parida et al., 2008). The detection limit in LAMP is higher than that in conventional PCR, and the result is determined by visual observation. LAMP assays have been widely applied to the detection of bacterium(Frisch et al., 2021; Fu et al., 2021), viruses(Li et al., 2021; Ptasinska et al., 2021), parasites(Mesquita et al., 2021; Ruang-Areerate et al., 2021) and fungi(Karakkat et al., 2018; Choopara et al., 2021), as well as detection of the hypervirulent strain, toxin types, binary toxin, ermB gene of C.difficile(Noren et al., 2011; Boyanton et al., 2012; Lin et al., 2015; Yu et al., 2017), and even in forensics(Arenas et al., 2017). In this study, four sets of primers were designed and LAMP assay for detection of tetM were optimized. Secondly, the specificity and sensitivity of the primers in the LAMP reaction were determined. Finally, the LAMP assay was comprehensively evaluated against the conventional PCR method for the identification of tetM, tcdA, tcdB, cdtA, cdtB, and ermB gene in 300 toxigenic C. difficile strains cultured from feces.

Materials and methods

Bacterial strains

In total, 27 bacterial strains were used to develop the LAMP assays (Table 1). C.difficile VPI10463 carrying tetM with the typical antimicrobial resistance was used as the positive control. Another 26 infectious bacterial strains were selected for evaluating the specificity of the LAMP assays. The sequence of tetM in the strain was validated by PCR-based sequencing and showed 100% identity with those of previously reported genes (Supplemental Materials).

Table 1Bacterial strains used in this study.

1 Lanzhou Institute of Microbiology.

2 Institute of Disease Control and Prevention, Academy of Military Medical Sciences.

Clinical stool specimens and isolation of bacterial strains

Fresh liquid or unformed stool specimens were collected from 882 hospitalized patients with suspected CDI from September 2014 to June 2020 at Nanfang hospital under IRB-approved protocols (committee ethic number:NFEC-2014-078). The above work described had been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments. C.difficile strains were cultured from feces as previously described(Lin et al., 2015). Briefly, stool specimens were grown anaerobically in cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) after ethanol shock treatment for 12-24h. Then, cycloserine cefoxitin fructose agar (CCFA) was used to further selectively isolate C. difficile species. Colonies of C. difficile were identified based on their typical morphology on agar plates, “horse barn” odor, Gram staining, and PCR for the tcdA(Kato et al., 1991), tcdB(Kato et al., 1999), cdtA(Stubbs et al., 2000), and cdtB(Stubbs et al., 2000) genes as described previously. For each isolate, toxin production and activity in vitro were assessed by cell cytotoxicity assay (CCTA). Toxigenic C. difficile strains was identified by the above methods. LAMP method was used to determine whether those toxigenic strains carried tcdA(Norén et al., 2011), tcdB(Kato et al., 2005), cdtA(Yu et al., 2017), and cdtB(Yu et al., 2017) gene. Finally, the presence of tetM (Schmidt et al., 2007) and ermB(Spigaglia et al., 2007) genes in those toxigenic C. difficile strains cultured from feces were further verified by LAMP method and PCR.

DNA extraction

Genomic DNA of all the bacterial strains and clinical isolates was extracted employing a bacterial genomic DNA extraction kit (Tiangeng, Ningbo, China) according to manufacturer's protocol and was stored immediately at -20°C until use. Pure genomic DNA was extracted from C.difficile VPI10463 by Wizard genomic DNA purification kit (Promega, Madison, Wisconsin, USA). The pure genomic DNA was serially diluted 10-fold in distilled water from 361 ng/ul to 0.000361 pg/ul. The pure genomic DNA concentration was detected before and after dilution by Spectrophotometer ND-1000 (Thermo Fisher Scientific, Inc. USA).

Primers design

Four sets of LAMP primers were designed to target six or eight distinct sequences downloaded from the NCBI GenBank database (GenBank accession no. JN846700.1). A set of four primers (Table 2) was generated employing the Primer Explorer V4 software [http:/primerexplorer.jp/lamp]. The loop primer (LB) contributed to accelerating the amplification reaction. The FIP and BIP primers were linked by a four-thymidine spacer (TTTT). Conventional PCR for tetM was conducted by the primer sets TetM1 and TetM2(Schmidt et al., 2007) (Table 2). Primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Table 2Primers used in LAMP and PCR.

LAMP reaction

The LAMP reaction was carried out in a final volume of 25 μl containing 1ul Bst DNA polymerase, 2ul DNA template and 12.5ul reaction mixtures (DNA amplification kit; Eiken Chemical Co., Ltd., Tochigi, Japan) for real-time turbidimeter and another 1ul calcein/Mn2+ complex (Fluorescent Detection Reagent; Eiken Chemical Co., Ltd.) for visual inspection directly. Primers were consisted of a concentration of 40 pmol FIP and BIP, 40 pmol LB, 5 pmol F3 and B3. The LAMP assay was performed in a reaction tube (Eiken Chemical Co. Ltd., Tochigi, Japan) for 60 minutes at an isothermal temperature of 62°C.

Detection of LAMP products

The amplification products were detected by two methods. For direct visual inspection, 1 µl of Loopamp Fluorescent Detection reagent (Eiken Chemical Co., Ltd.), containing calcein/Mn2+ complex, was added to the reaction system after the reaction mixtures were prepared. The color changed from orange to green for a positive reaction, while a negative reaction failed to turn green and remained orange. The naked eye could detect the color change under natural light or UV light at 365 nm. To monitor turbidity, a Loopamp real-time turbidimeter (LA-320c; Eiken Chemical Co., Ltd.) was chosen to monitor real-time amplification by recording the optical density (λ650 nm) at 400 nm every 6s.

PCR detection

The PCR conditions used for amplification were described previously(Kato et al., 1991; Kato et al., 1999; Stubbs et al., 2000; Schmidt et al., 2007; Spigaglia et al., 2007). A 2% agarose gel (Amresco, Solon, Ohio, USA) containing ethidium bromide was used to detect the PCR-amplified products. Images were documented by a Bio-Rad Gel Doc EQ Imaging System (Bio-Rad, Hercules, CA, USA).

Results

Optimal primers for rapid detection of tetM

We designed four sets of primers for detection of tetM. Under the same reaction conditions, we observed that all turbidity curves occurred after reaction for 10 minutes, which demonstrated that all primer sets amplified the target gene. The primers in the tetM1 set, which could amplify the target gene faster than the others, were the most optimal reaction primers (Fig. 2). Our data shown that the reaction time of the tetM1 primer with additional LB was less than one-half that of the tetM207 primer set without loop primers, which was consistent with a previous report(Norén et al., 2011).

Fig. 2Four sets of primers amplified tetM under the same reaction conditions. Turbidity was monitored to measure the optical density by a Loopamp real-time turbidimeter at 650 nm every 6s. All the primer sets were performed with Loop primer (LB) except the tetM207 primer set.

Appropriate temperature for tetM LAMP reaction

To achieve the most appropriate temperature of the LAMP reaction, we investigated different temperatures in the range from 59°C to 65°C at 1°C interval using the tetM1 primer set. Figure. 3 shown that 61°C to 64°C was the most suitable reaction temperature range. Finally, 62°C was chosen as the most appropriate temperature because of the fastest amplification.

Fig. 3Different temperatures from 59°C to 65°C at 1°C interval were detected to achieve the most appropriate tetM LAMP reaction. Turbidity was monitored by a Loopamp real-time turbidimeter at 650 nm every 6s.

Specificity of the LAMP assay

To test the specificity of LAMP for tetM, we used C. difficile VPI10463 strain carrying tetM gene as positive strain and distilled water as the negative control. Twenty-six infectious bacterial strains without tetM were selected. As depicted in Fig. 4(a), the increased turbidity curve was appeared in C.difficile VPI10463, while no changes in turbidity were recorded for the negative control (distilled water) and other bacterial species. These results manifested high specificity of the primers. For visual inspection directly, chromogenic reactions were used (Fig. 4(b)). Before the LAMP reaction, 1µl of calcein/Mn2+ complex was added to 25µl of the LAMP reaction mixture. When the reaction was finished, all positive reactions turned green, while samples with negative reactions remained orange. The results of the chromogenic reactions showed similar results to the real-time turbidity detection method.

Fig. 4Specificity of the LAMP reaction for the detection of tetM. (A) Turbidity was monitored by a Loopamp real-time turbidimeter at 650nm every 6s. (B) A visual inspection method using calcein/Mn2+ complex were carried out. Amplifification was performed at 62°C for 60 minutes. Lines: 1, Enteroadherent E.coli; 2, Enteroinvasive E.coli; 3, Enterotoxigenic E. coli; 4, Enteropathogenic E. coli 2348; 5, Neisseria meningitides; 6, Shigella sonnei 2531; 7, Shigella flexneri 4536; 8, Acinetobater baumannii; 9, Corynebacterium diphtheria CMCC38001; 10, Mycobacterium tuberculosis 4368; 11, Neisseria meningitides group B CMCC29022; 12, Burkholderia pseudomallei; 13, Vibrio parahaemolyticus 5474; 14, Vibrio cholera O139; 15, Vibrio carchariae; 16, Streptococcus pneumoniae; 17, Pseudomonas maltophilia; 18, Betahaemolytic streptococcus group A CMCC32213; 19, Bordetella pertussis ATCC 18530; 20, Bacillus anthracis; 21, Staphylococcus aureus 2740; 22, Yersinia pestis; 23, Salmonella paratyphosa 86423; 24, Pseudomonas aeruginosa; 25, Bacillus megatherium; 26, Haemophilus influenzae; 27, positive control (C. difficile VPI10463); 28, negative control (distilled water).

Sensitivity of tetM LAMP reaction

To compare the detection limit of LAMP using either real-time turbidity measurements or visual detection with traditional PCR, genomic DNA was extracted from C.difficile VPI10463, and then serially diluted 10-fold from 361 ng/µl to 0.000361 pg/µl. Distilled water was chosen as negative control. As shown in Figure 5, the detection limit of the real-time turbidity and visual detection was both 36.1 pg/µl, which was 100-fold more sensitive than traditional PCR assay.

Fig. 5Comparison of sensitivity between the LAMP and PCR for detection of the tetM. The pure genomic DNA extracted from C. difficile VPI10463 was diluted 10-fold from 361 ng/µl to 0.000361 pg/µl. Both LAMP (A, B) and PCR (C) were duplicated for each dilution point. (A) Turbidity was monitored every 6s with a Loopamp real-time turbidimeter at 650 nm. (B) For visual inspection of the color, 1 ul of calcein/Mn2+ complex was added to 25 ul of LAMP reaction mixture before the LAMP reaction; (C) PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. Amplification was performed at 62°C for 60 min. Tubes and lanes: 1, 361 ng/µl; 2, 36.1 ng/µl; 3, 3.61 ng/µl; 4, 361 pg/µl; 5, 36.1 pg/µl; 6, 3.61 pg/µl; 7, 0.361 pg/µl; 8, 0.0361 pg/µl; 9, 0.00361 pg/µl; 10, 0.000361 pg/µl; 11, negative control (distilled water); M, D2000 DNA Marker (Tiangen Biotech Co., Ltd.).

Clinical strains detection

We successfully isolated 300 toxigenic C.difficile strains from 882 fresh stool specimens (34%) suspected of CDI, from September 2014 to June 2020 at Southern Medical University Nanfang hospital. Electron colonoscopy and histopathologic photographs of pseudomembranous enteritis from our patient, a common clinical manifestation of CDI, were shown in Fig 1A and 1B. Under the electronic colonoscopy, scattered pale yellow microscopic particles were observed, and the mucosa was hyperemic and brittle. Histopathologic photographs shown that neutrophils infiltrated the lamina propria, epithelial cells were damaged, and secretions were found on the mucosal surface. Toxin types (toxin A, toxin B, and binary toxin) and resistance genes (ermB and tetM) of those toxigenic C.difficile strains cultured from feces were further evaluated by both LAMP and PCR (Fig 1D-F, Table 3). Among these strains, 287 were toxin A-positive, toxin B-positive and binary toxin-negative (A+B+CDT−) (95.7%), 12 were toxin A-negative, toxin B-positive and binary toxin-negative (A−B+CDT−) (4%), only one isolate harbored all toxin associated genes (A+B+CDT+) (0.3%). 44 A+B+CDT− isolates were negative for ermB, and the rest were positive for ermB. The ermB gene was detected in 256 (85.3%) of the 300 isolates by both LAMP and PCR assays. Both the 8 A+B+CDT− isolates and the 12 A−B+CDT− isolates were negative for tetM, while the rest of the isolates were positive for tetM (93.3%, 280/300) by both LAMP and PCR assays. In our study, all the LAMP results of toxin types and resistance genes were identical to those PCR (kappa=1), which verified the accuracy of LAMP method not only we established but also others established(Kato et al., 2005; Noren et al., 2011; Lin et al., 2015;Yu et al., 2017).

Table 3Results of toxin types and resistance genes of 300 clinical toxigenic C. difficile isolates.

Discussion

This is the first study examining tetM gene in C. difficile strains cultured from feces by LAMP method. Our data have shown that the reaction time of the tetM primer set with loop primers (LB) was less than one-half that of the original LAMP method without loop primers, which manifested that even one loop primers (LB) still could accelerated the amplification reaction. All the LAMP results of toxin types and resistance genes were identical to those PCR (kappa=1), which verified the accuracy of LAMP method. Besides, the proportion of toxin types in toxigenic C. difficile strains cultured from feces was 95.7% (287/300) for A+B+CDT−, 4% (12/300) for A−B+CDT−, and 0.3% (1/300) for A+B+CDT+ respectively, which was different from the other report from China(Wang et al., 2018). This may be due to the regional differences in C. difficile populations. It was exciting that only one isolate harbored all toxin associated genes (A+B+CDT+) was found, indicating that high-virulent C. difficile strain was not common in this area. Furthermore, the positive rate of tetM and ermB was 93.3% (280/300) and 85.3% (256/300) respectively, which were similar to the results of another 4-year study in China(Chen et al., 2018). The results indicated that C. difficile was highly resistant to tetracycline and the macrolide - lincosamide – streptogramin B (MLSB) group of antibiotics, at least within the range of medical radiation at this hospital. Therefore, rapid and accurate detection for resistance genes was an urgent need for treatment.

To meet this challenge, we evaluated and optimized a novel LAMP assay for tetM detection capable of detecting tetM in C. difficile strains cultured from feces within 60 minutes. The sensitivity of the LAMP assay was identified to be 100-fold more significant than that of the traditional PCR method. Another innovation of LAMP is its high specificity due to the use of 4-6 primers that can distinguish up to eight specific locations on the DNA template, compared to only two in typical PCR. The sensitivity and specificity are thereby greatly enhanced, and the probability of false-positive results is decreased. In addition, the PCR is carried out under temperature-cycling conditions, which is time-consuming, and the reaction also depends on the high precision of the PCR instruments. Compared to PCR, the LAMP reaction can be conducted in isothermal conditions without advanced laboratory equipment, such as a dry block heater or a water bath. LAMP assays don't involve the DNA denaturation stage, due to the strand displacement activity of the Bst DNA polymerase(Notomi et al., 2000; Nagamine et al., 2001). Moreover, purification of DNA from samples can be omitted by the LAMP method as the reaction of different components in clinical specimens is not easily affected. While the sensitivity of the PCR can be significantly reduced because of exogenous DNA and inhibitors(Kaneko et al., 2007). Finally, LAMP can visualize the reaction directly by many possibilities, such as colorimetric detection using fluorescent dyes, UV light irradiation, turbidity, real-time fluorescence, smartphone, lateral flow assay (LFA), and AC susceptometry. The fluorescent dyes calcein, the SYBR Green dyes and the EVaGreen are the most widely used for LAMP detection(Mori et al., 2004; Foo et al., 2017; Silva et al., 2019). Other dyes include malachite green dye, hydroxynaphthol blue dye, Goldview dye, GelRed dye, SYTO fluorescent dye, leuco crystal violet (LCV) dye and berberine dye (Silva et al., 2019). All fluorescent dyes bind to the double-stranded DNA and emit light of a specific length, except for calcein. Calcein can form a complex with the magnesium obtained by LAMP reaction. Its results can be observed under UV light irradiation and seen with the naked eye by color changes(Mori et al., 2004; Foo et al., 2017). In contrast, PCR need gel electrophoresis to determine the results. However, preparing the gel (agarose or polyacrylamide) and the electrophoresis itself are both time-consuming, and visualizing the products requires dyes, many of which are mutagenic or carcinogenic. Therefore, the LAMP method is more suitable than PCR for rapid detection of tetM detection, especially for routine diagnosis and infection control, due to its high specificity, sensitivity and visual detection.

Although there are many advantages of LAMP assay, it shows several drawbacks at the same time. A disadvantage of LAMP is its sensitivity to cross-contamination, i.e., material present in the aerosol. Therefore, it is recommended that the room should be ventilated and different samples should be analyzed separately. In the current study, low melting-point paraffin wax was added to the reaction tubes after adding all the reaction solutions, which can avoid the spread of amplification products. Another drawback of LAMP is that it is difficult to check the samples for reaction inhibitors, as this requires two reactions, one to detect the inhibitors and the other to amplify the material. Furthermore, LAMP products cannot always be used for further analyses, such as cloning or sequencing(Sahoo et al., 2016). Finally, any contamination of samples with exogenous genetic material may affect the results, as the target products of LAMP reaction are short. Operators need to be aware of the risk of sample contamination and follow special sterility procedures.

In conclusion, our data contributes to the present understanding of virulence and resistance genes of C.difficile isolates in China. Its high specificity and sensitivity, as well as visual detection, make the LAMP assay an exciting and powerful diagnostic tool for easy and rapid testing of tetM-bearing C. difficile strains cultured from feces. The current technique will help to guide clinical treatment effectively and implement infection control measures promptly to curb the spread of C. difficile strains in hospitals.

Acknowledgments

We are indebted to the Institute of Disease Control and Prevention, Academy of Military Medical Sciences for providing technical assistance.

Funding

This work was supported by the National Natural Science Foundation of China (grant number 82070543 and grant number 81700461).

Compliance with Ethical Standards

The authors have no conflicts of interest to declare. The Ethical Committee of Southern Medical University has approved the current study. The hospitalized patients suspected of CDI have signed informed consent.

Declaration of Competing Interest

The authors report no declarations of interest.

Conflict of interest statement

The authors have no conflicts of interest to declare.

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