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Keywords:

  • Campylobacter jejuni;
  • RT-qPCR;
  • Vibrio cholerae;
  • Vibrio parahaemolyticus

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

A sensitive rRNA-targeted reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method was developed for detection of Vibrio cholerae/mimicus, V. parahaemolyticus/alginolyticus and Campylobacter jejuni/coli by using specific primers. Counts of the enteric pathogens spiked in human stools were quantified at the lower detection limit of 103 cells/g stool by RT-qPCR, in marked contrast with conventional quantitative polymerase chain reaction (qPCR) at the detection limit of 105 to 106 cells/g stool. The bacterial counts determined by RT-qPCR were almost equivalent to those determined by the culture method and fluorescence in situ hybridization (FISH) during the course of in vitro culture. Bacterial rRNA in the stools was stable for at least 4 weeks when the stools were kept as the suspensions in RNA-stabilizing agent, RNAlater®, even at 37oC. These data suggested that the rapid and high sensitive rRNA-targeted RT-qPCR was applicable for the accurate quantification of viable enteric pathogens, such as V. cholerae/mimicus, V. parahaemolyticus/alginolyticus and C. jejuni/coli.

List of Abbreviations: 
CFU

colony forming unit

DAPI

4′,6-diamidino-2-phenylindole

FISH

fluorescence in situ hybridization

qPCR

quantitative polymerase chain reaction

rRNA

ribosomal RNA

RT-qPCR

reverse transcription-quantitative polymerase chain reaction

VBNC

viable but non-culturable

Enteric pathogens, which exert a great threat upon human health, involve a wide range of genera. Vibrio cholerae, the etiological agent for the diarrheal disease cholera, is still one of the most important causes of morbidity and mortality worldwide. The incidence of cholera is estimated to exceed five million cases each year (1). Meanwhile, V. parahaemolyticus and Campylobacter jejuni/coli account for food-borne outbreaks worldwide. In several countries, V. parahaemolyticus accounts for over 30% of all food poisoning outbreaks of bacterial origin (2, 3). One to 7 million cases of C. jejuni/coli infections are reported per year in the USA, and they result in the development of Guillain-Barré syndrome, which is the primary cause of acute neuromuscular paralysis there (4, 5).

Identification of these enteric pathogens in patients with infectious diarrhea is therefore essential for appropriate treatment and prevention of their possible spread. Although the culture method remains the ‘gold standard’ for the isolation and identification of enteric pathogens, the time-consuming procedure makes it difficult for rapid identification. Recently, in addition to the culture method, several molecular microbiological methods, such as PCR (6–8), amplified fragment length polymorphism (9), pulsed-field gel electrophoresis (10), FISH (11) and PCR-enzyme-linked immunosorbent assay (12), have been introduced for identification and typing of bacterial strains. Among these DNA-targeted molecular methods, PCR has been the most common and provides ease and rapidity of use.

Because population levels of enteric pathogens in the intestines of patients or carriers have not yet been sufficiently investigated, it is desirable to develop sensitive methods for detection. Recently, we have reported rRNA-targeted RT-qPCR for a quantitative determination of intestinal microbiota (13–15). By this method, it was possible to determine a variety of bacterial populations existing in a concentration of more than 103 cells/g stool in the human gastrointestinal tract, as its sensitivity was approximately 100- to 1,000-times higher than that of the DNA-targeted qPCR (13, 15). This is because the copy number of rRNA per cell (approximately 104 copies per actively growing cells) is higher than that of rRNA genes (approximately 10 copies in a genome). We applied rRNA-targeted RT-qPCR for quantitative determination of V. cholerae, V. parahaemolyticus and C. jejuni/coli in stools as a more sensitive method than the DNA-targeted qPCR.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Strains and culture condition

The stock strains in our laboratory listed in Table 1 were used. All strains of Vibrio species were cultured aerobically in Nutrient Broth (Becton Dickinson Co., Sparks, MD, USA) containing 1.5% (wt/vol) NaCl. V. cholerae, V. mimicus, V. parahaemolyticus, V. alginolyticus, V. fluvialis, V. furnissii, V. vulnificus, V. metsuchinikovii and V. hollisae were cultured at 37°C for 16 hrs, whereas V. cincinnatiensis, V. harveyi, V. superstes, V. nigripulchritudo and V. neptunius were cultured at 26°C for 24 hrs. Strains of Campylobacter species except for C. rectus, C. concisus and C. curvus were subcultured on sheep blood agar (Nikken Bio Medical Laboratory Inc., Kyoto, Japan) at 37°C for 48 hrs, and a single colony of each strain was inoculated into Preston broth, which contained Bacto peptone (1.0%, wt/vol; Difco Laboratories, Detroit, MI, USA), Lab lemco powder (1.0%, wt/vol; Oxoid Co., Basingstoke, UK), PBS(−) (1.0%, wt/vol; Nissui Pharmaceutical Co., Tokyo, Japan), sodium pyruvate (0.025%, wt/vol; Kanto Chemical Co., Tokyo, Japan), sodium disulfite (0.025%, wt/vol; Kanto Chemical Co.) and iron(III)sulfate n-hydrate (0.025%, wt/vol; Kanto Chemical Co.), and cultured at 37°C for 16 hrs under the micro-aerophilic condition generated with the CampyPak plus micro-aerophilic system (Becton Dickinson Co.). Strains of C. rectus, C. concisus and C. curvus were cultured on sheep blood agar at 37°C for 48 hrs under micro-aerophilic conditions, and suspended in Preston broth. The CFU was counted using 2×YT agar containing 1.5% (wt/vol) NaCl for V. cholerae pure culture, TCBS agar (Oxoid Co.) for V. cholerae spiked in stools, TCBS agar for V. parahaemolyticus, and CCDA agar (Oxoid Co.) containing cefoperazone-amphotericin-teicoplanin (Kanto Chemical Co.) (16) for C. jejuni.

Table 1.  Specificity of the designed primers for Vibrio cholerae/mimicus, V. parahaemolyticus/alginolyticus and Campylobacter jejuni/coli
TaxonStrainReaction with the following primers†
s-Vcho-F/Rs-Vpara-F/Rs-Cjej-F/R
  1. †Specificity of the RT-qPCR assay for target bacteria with each primer was investigated using RNA extracts corresponding to 104 cells of each strain. Specificity was judged using the criteria described in Materials and Methods. In addition, negative RT-PCR results were obtained for the following bacterial strains: Salmonella enterica ss. enterica serovar typhi C6953, Salmonella enterica ss. enterica serovar typhimurium ATCC 14028, Escherichia coli ATCC 11775T, Citrobacter koseri JCM 1658T, Citrobacter freundii JCM 1657T, Citrobacter amalonaticus JCM 1661T, Enterobacter cloacae JCM 1232T, Enterobacter aerogenes JCM 1235T, Cronobacter sakazakii JCM 1233T, Enterobacter cancerogenus JCM 3943, Enterobacter amnigenus JCM 1237T, Klebsiella pneumoniae JCM 1662T, Klebsiella oxytoca JCM 1665T, Serratia marcescens JCM 1239T, Proteus mirabilis JCM 1669T, Proteus vulgaris JCM 1668, Proteus penneri JCM 3948T, Hafnia alvei JCM 1666T, Edwardsiella tarda JCM 1656T, Providencia alcalifaciens JCM 1673T, Providencia rettgeri DSM 4542T, Morganella morganii JCM 1672T, Yersinia enterocolitica DSM 4780T, Bifidobacterium adolescentis ATCC 15703T, Bifidobacterium catenulatum ATCC 27539, Bifidobacterium longum ATCC 15707T, Bacteroides fragilis DSM 2151T, Bacteroides ovatus JCM 5824T, Clostridium perfringens JCM 1290T, Ruminococcus productus ATCC 27340T, Ruminococcus obeum ATCC 29174T, Faecalibacterium prausnitzii ATCC 27768T, Collinsella aerofaciens ATCC 25986T, Prevotella melaninogenica ATCC 25845T, Eggerthella lenta ATCC 25559, Streptococcus bovis JCM 5802, Streptococcus mutans IFO 13955, Staphylococcus aureus ATCC 12600T, Enterococcus faecalis ATCC 19433T, Enterococcus faecium ATCC 19434T, Lactobacillus acidophilus ATCC 4356T, Lactobacillus casei ATCC 334T, Lactococcus lactis ATCC 19435T, Bacillus cereus JCM 2152T, Bacillus subtilis ATCC 12826, Aeromonas hydrophila ss. hydrophila JCM 1027T, Aeromonas hydrophila ss. anaerogenes JCM 1043T, Aeromonas caviae JCM 1060T, Aeromonas veronii JCM 7375T.

Vibrio cholerae569B+
Vibrio choleraeMO10+
Vibrio cholerae12/20-904+
Vibrio choleraeCVD101+
Vibrio cholerae395+
Vibrio choleraeJKB70+
Vibrio choleraeCVD103-HgR+
Vibrio choleraeO395+
Vibrio choleraeVC-20+
Vibrio choleraeMBRN-14+
Vibrio choleraeMBRN-17+
Vibrio choleraeV2+
Vibrio choleraeSO-84+
Vibrio choleraeN16961+
Vibrio choleraeIDH60+
Vibrio mimicusATCC 33653T+
Vibrio parahaemolyticusDSM 10027T+
Vibrio parahaemolyticusATCC 33844+
Vibrio parahaemolyticusATCC 33845+
Vibrio parahaemolyticusIDH2068+
Vibrio parahaemolyticusIDH2168+
Vibrio parahaemolyticusIDH2170+
Vibrio parahaemolyticusIDH2189+
Vibrio parahaemolyticusIDH2534+
Vibrio parahaemolyticusIDH2639+
Vibrio parahaemolyticusIDH2640+
Vibrio parahaemolyticusIDH2826+
Vibrio parahaemolyticusIDH3115+
Vibrio parahaemolyticusIDH3809+
Vibrio alginolyticusDSM 2171T+
Vibrio alginolyticusATCC 17750+
Vibrio alginolyticusATCC 33787+
Vibrio cincinnatiensisATCC 35912T
Vibrio fluvialisJCM 3752T
Vibrio fluvialisIDH1572
Vibrio fluvialisIDH1752
Vibrio fluvialisIDH2036
Vibrio fluvialisIDH2233
Vibrio furnissiiATCC 35016T
Vibrio furnissiiQ17
Vibrio furnissiiUM#4119
Vibrio harveyiATCC 14126T
Vibrio vulnificusATCC 33653T
Vibrio vulnificusCDC B3547
Vibrio vulnificusL-180
Vibrio vulnificusE86
Vibrio vulnificusCECT 5198
Vibrio superstesDSM 16383T
Vibrio nigripulchritudoATCC 27043T
Vibrio neptuniusDSM 17183T
Vibrio metsuchinikoviiNCTC 11171
Vibrio metsuchinikoviiIDH404
Vibrio hollisaeCDC 9041-81
Vibrio hollisaeCDC 0075-80
Campylobacter jejuniATCC 33560T+
Campylobacter jejuniATCC 43429+
Campylobacter jejuniJCM 2013+
Campylobacter jejuniCK1+
Campylobacter jejuniCK2+
Campylobacter jejuniCK3+
Campylobacter jejuniCK4+
Campylobacter jejuniCK5+
Campylobacter jejuniCK6+
Campylobacter jejuniCK7+
Campylobacter jejuniCK8+
Campylobacter coliATCC 33559T+
Campylobacter coliATCC 43478+
Campylobacter coliATCC 43482+
Campylobacter coliATCC 43473+
Campylobacter insulaenigraeDSM 17739T±
Campylobacter lariJCM 2530T±
Campylobacter upsaliensisDSM 5365T
Campylobacter fetusJCM 2527T
Campylobacter hyointestinalisATCC 35217T
Campylobacter rectusJCM 6301T
Campylobacter concisusDSM 9716T
Campylobacter curvusDSM 6644T

Salmonella enterica ss. enterica serovar typhi, S. enterica ss. enterica serovar typhimurium, Escherichia coli, Citrobacter koseri, C. freundii, C. amalonaticus, Enterobacter cloacae, Cronobacter aerogenes, E. sakazakii, E. cancerogenus, E. amnigenus, Klebsiella pneumoniae, K. oxytoca, Serratia marcescens, Proteus mirabilis, P. vulgaris, P. penneri, Hafnia alvei, Edwardsiella tarda, Providencia alcalifaciens, P. rettgeri, Morganella morganii, Yersinia enterocolitica, Staphylococcus aureus, Bacillus cereus, B. subtilis, Pseudomonas aeruginosa, Aeromonas hydrophila, A. caviae and A. veronii were cultured aerobically in Brain Heart Infusion broth (Beckton Dickinson Co.) at 37°C for 16 hrs. Bifidobacterium adolescentis, B. catenulatum, B. longum, Bacteroides fragilis, B. ovatus, Clostridium perfringens, Ruminococcus productus, R. obeum, Collinsella aerofaciens and Prevotella melaninogenica were cultured anaerobically in Modified GAM broth (Nissui Pharmaceutical Co.) containing 1.0% (wt/vol) glucose at 37°C for 24 hrs. Faecalibacterium prausnitzii and Eggerthella lenta were cultured anaerobically in Modified GAM broth containing 1.0% (wt/vol) glucose at 37°C for 72 and 48 hrs, respectively. Streptococcus bovis, S. mutans, Enterococcus faecalis, E. faecium, Lactobacillus acidophilus, L. casei and Lactococcus lactis were cultured anaerobically in Lactobacilli MRS broth (Becton Dickinson Co.) at 37°C for 24 hrs.

DAPI staining

DAPI staining was carried out as described previously (17). Briefly, fresh bacterial cultures were fixed with 3 volumes of 4% paraformaldehyde at 4°C for 16 hrs. Then, 10 μL diluted fixed-cell suspension of the appropriate dilution was smeared on a MAS-coated slide glass (Matsunami Glass Ind., Osaka, Japan), which was stained with DAPI (Vector Laboratories, Burlingame, CA, USA). The observation and acquisition were carried out with the Leica imaging system (an automatic fluorescent microscope [Leica DM6000], image acquisition software [QFluoro], and a cooled black-and-white charge-coupled display camera [Leica DFC3500FX]) (Leica Microsystems, Wetzlar, Germany). The fluorescent images obtained were analyzed using image analysis software (Image-Pro Plus v. 4.5; Media Cybernetics, Inc., Bethesda, MD, USA) to determine the fluorescent cells in each sample. Microscopic counts were determined for 10 images per sample.

Design and validation of rRNA-targeted primers

By using 16S rRNA sequences of V. cholerae (Accession no: X74695), C. jejuni (Accession no: AF372091), C. coli (Accession no: AF372092) and reference organisms including 57 other Vibrio species and 15 Campylobacter species, or 23S rRNA sequences of V. parahaemolyticus (Gene ID: 1190827) and reference organisms including 11 other Vibrio species obtained from DDBJ/GenBank/EMBL database, multiple alignments were constructed with the program Clustal X (18). After comparing the sequences, potential target sites for specific detection were identified. The 16S rRNA-targeted V. cholerae/mimicus specific primer set (s-Vcho-F, 5′-AACCTCGCAAGAGCAAAGCA-3′ and s-Vcho-R, 5′-TAGGTAACGTCAAATGATTAAGG-3′), 23S rRNA-targeted V. parahaemolyticus/alginolyticus specific primer set (s-Vpara-F, 5′-GTCCCGTAGTTGACGACGTG-3′ and s-Vpara-R, 5′-ACGCAGTCACAGGACAAAGCC-3′), and 16S rRNA-targeted C. jejuni/coli specific primer set (s-Cjej-F, 5′-AAGTCGAACGATGAAGCTCC-3′ and s-Cjej-R, 5′-CCTACTCAACTTGTGTTAAGC-3′) were newly constructed.

For further confirmation of the specificity of the primers with several species of human intestinal microbiota, total RNA fractions extracted from 104 cells of each bacterial strain (Table 1) were examined by RT-qPCR. Using the standard curve of V. cholerae 569B, V. parahaemolyticus DSM 10027T and C. jejuni ATCC 33560T, a possible cross-reaction(s) was examined. In the specificity validation of the primers for V. cholerae, V. parahaemolyticus and C. jejuni, the amplified signal was considered positive (+) when it was >100 cells, positive/negative (±) when it was in the range between 100 and 10−1 cell, and negative when it was <10−1 cell.

Preparation of stool sample

Stools from a volunteer were weighed and then suspended in 9 volumes of PBS (−) for the following CFU counting and qPCR, or suspended in 9 volumes of RNAlater® (Ambion, Austin, TX, USA) for the following RT-qPCR. Liquid bacterial cultures of V. cholerae 569B, V. parahaemolyticus DSM 10027T and C. jejuni ATCC 33560T were added to the stool suspension to obtain desired concentrations of each strain.

Extraction of RNA

Fresh culture of each bacterial strain (100 μL) was added to 200 μL RNAlater®. The stool sample (20 mg) was added to 180 μL RNAlater®. After being kept for 10 min at room temperature, the bacterial suspensions were centrifuged at 13,000 ×g for 10 min. The pellets were stored at −80oC until used for RNA extraction. The RNA was extracted from the bacterial cells and the stool samples by the method described previously (13).

Extraction of DNA

DNA was extracted by the method described previously (19) with minor modifications. Briefly, DNA was extracted from 100 μL fresh culture of each bacterial strain and 20 mg stool sample, and finally suspended in 100 μL and 1 mL Tris-EDTA buffer (pH 8.0), respectively.

RT-qPCR

RT-qPCR was carried out using a QIAGEN OneStep RT-PCR kit (QIAGEN, Hilden, Germany). Each reaction mixture (10 μL) was composed of 1 × QIAGEN OneStep RT-PCR buffer, 0.5 × Q-solution buffer, each deoxynucleoside triphosphate at a concentration of 400 μM, a 1:100,000 dilution of SYBR green I (BioWhittaker Molecular Applications, Rockland, ME, USA), 0.4 μL QIAGEN OneStep RT-PCR enzyme mixture, each of the specific primers at a concentration of 0.6 μM, and 5 μL template RNA. The reaction mixture was incubated at 50°C for 30 min for reverse transcription. The continuous amplification program consisted of one cycle at 95°C for 15 min, followed by 45 cycles at 94°C for 20 s, 55°C for 20 s, and 72°C for 50 s. The fluorescent products were detected at the last step of each cycle. A melting curve analysis was carried out after amplification to distinguish the targeted PCR products from the non-targeted ones. The melting curve was obtained by slow heating at temperatures from 60 to 95°C at the rate of 0.2°C/s with continuous fluorescence collection. Amplification and detection were carried out in 384-well optical plates with an ABI PRISM 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA).

qPCR

qPCR was carried out by using a QIAGEN OneStep RT-PCR kit (QIAGEN). Each reaction mixture (10 μL) contained the same components as those for RT-qPCR except for the replacement of 5 μL template RNA with the same amount of template DNA. The reaction mixture was incubated at 95°C for 15 min, followed by 45 cycles at 94°C for 20 s, 55°C for 20 s, and 72°C for 50 s. The following procedures were the same as those for RT-qPCR.

Determination of bacterial counts by RT-qPCR

Calibration curves to determine the bacterial counts for V. cholerae 569B, V. parahaemolyticus DSM 10027T and C. jejuni ATCC 33560T were generated as follows. Bacterial cell counts of the standard strain were determined microscopically after DAPI staining as described above. Bacterial counts in samples were determined by RT-qPCR as described previously (15).

Determination of bacterial counts by FISH

FISH analyses with the 16S rRNA-targeted bacterial universal oligonucleotide probe, Eub338 (5′-GCTGCCTCCCGTAGGAGT-3′) (20), were carried out as described previously (21). Briefly, fresh bacterial cultures were fixed with three volumes of 4% paraformaldehyde at 4°C for 16 hrs. Then, 10 or 20 μL diluted fixed-cell suspension of the appropriate dilution was smeared on a MAS-coated slide glass (Matsunami Glass Ind.), which was hybridized with the probe. The observation and acquisition were carried out in the same way as those of the DAPI staining described above. Microscopic counts were determined from 10 images.

Stability test of bacterial rRNA in the stool suspension in RNAlater®

Stool samples from three volunteers were separately weighed and then suspended in nine volumes of RNAlater®. Liquid bacterial cultures of V. cholerae 569B, V. parahaemolyticus DSM 10027T and C. jejuni ATCC 33560T were added into each stool suspension to obtain concentrations of 107–108 cells/g stool for each bacteria. The bacterial suspensions were then incubated at 37°C, 25°C or 4°C for 0, 7, 14, 21 or 28 days. RNA was extracted from an aliquot of each sample and the bacterial counts were determined by RT-qPCR.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Specificity of the designed primers

Specificity of the designed primers was validated in RT-qPCR with the total RNA fractions extracted from 104 cells of the 126 strains tested. As shown in Table 1, concerning s-Vcho-F/R (for V. cholerae and V. mimicus) and s-Vpara-F/R (for V. parahaemolyticus and V. alginolyticus), positive RT-qPCR results were obtained only for the target bacterial strains, respectively. Concerning s-Cjej-F/R (for C. jejuni and C. coli), positive results were obtained for the corresponding target bacterial strains. Weak cross-reactions with C. lari and C. insulaenigrae, which are phylogenetically close to C. jejuni and C. coli were observed. This weak cross-reaction seemed minimal because both species are an infrequent cause of human gastrointestinal disease.

Comparison of RT-qPCR and qPCR for quantitative detection of the targeted bacteria

Bacterial counts determined by direct staining (x-axis in Fig. 1) correlated highly with the Ct values determined by RT-qPCR (y-axis in Fig. 1) over the range of RNA amounts corresponding to 10-2– 104 bacterial cells/reaction for V. cholerae (Fig 1a), V. parahaemolyticus (Fig. 1b) and C. jejuni (Fig. 1c) (R2 > 0.99). The bacterial counts also correlated well with the Ct values determined by qPCR, but the detection limit was 101 cells/reaction for V. cholerae (Fig. 1a), V. parahaemolyticus (Fig. 1b) and C. jejuni (Fig. 1c). These results indicated that RT-qPCR is approximately 1,000-fold more sensitive than qPCR for quantification of these bacteria.

image

Figure 1. Comparison of bacterial counts in cultures determined by RT-qPCR and those by qPCR. (a) Vibrio cholerae 569B, (b) V. parahaemolyticus DSM 10027T and (c) Campylobacter jejuni ATCC 33560T were cultured as described in the text. RNA and DNA were extracted from cultured samples at the early stationary phase. Bacterial counts were determined microscopically by DAPI staining. Ct values were obtained by (▪) RT-qPCR and (•) qPCR using 10-fold serial dilutions of the RNA and DNA (log10 cells/reaction are from −2 to 4). Results are expressed as the mean  ±  SD of triplicate determinations.

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Comparison of RT-qPCR and qPCR for quantitative detection of the bacteria spiked in stool samples

Serial dilutions of V. cholerae, V. parahaemolyticus, and C. jejuni cell suspensions were added to the stools of a volunteer who had been confirmed in advance not to have the corresponding indigenous populations. As shown in Figure 2, bacterial counts determined by direct staining (x-axis) correlated well with the Ct values determined by RT-qPCR (y-axis) (solid lines with square marks) for V. cholerae (Fig. 2a), V. parahaemolyticus (Fig. 2b) and C. jejuni (Fig. 2c) between 103 cells (the lower detection limit) and 108 cells/g stool sample. In qPCR, bacterial counts also correlated well with the Ct values as shown by the solid lines with circular symbols. However, the detection limit was 106 cells for V. cholerae (Fig. 2a), 105 cells for V. parahaemolyticus (Fig. 2b), and 106 cells for C. jejuni (Fig. 2c) per gram stool sample, respectively. The same analyses were carried out with the pure culture of bacteria and the results are shown in Figure 2. As shown by the dotted lines (square symbols for RT-qPCR and circular symbols for qPCR) with V. cholerae (Fig. 2a), V. parahaemolyticus (Fig. 2b) and C. jejuni (Fig. 2c), similar curves were obtained to those for quantification of the bacteria spiked in the stool samples. These results indicate that RT-qPCR is 100 to 1,000-fold more sensitive than qPCR assay for detection of these bacteria in stool samples.

image

Figure 2. Comparison of bacterial counts spiked in stool samples determined by RT-qPCR and by qPCR. (a) Vibrio cholerae 569B, (b) V. parahaemolyticus DSM 10027T and (c) Campylobacter jejuni ATCC 33560T were cultured as described in the text. After bacterial counts were determined microscopically by DAPI staining, 10-fold serially diluted cultures were added to a stool sample. RNA and DNA were extracted from samples that had been inoculated with diluted culture samples (solid line) and from serially diluted cultures (dotted line), respectively. Ct values were obtained by (▪) RT-qPCR and (•) qPCR. Results are expressed as the mean  ±  SD of triplicate determinations.

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Comparison of RT-qPCR and the culture method to detect bacteria spiked in stool samples

To further confirm the accuracy of RT-qPCR to detect bacteria, pure cultures of V. cholerae, V. parahaemolyticus and C. jejuni were spiked in stool samples at final bacterial concentrations of 103, 104, 105, 106, 107 and 108 cells/g stool sample, and the bacterial counts were determined by RT-qPCR and the culture method. As shown in Figure 3, bacterial counts determined by RT-qPCR correlated well with the corresponding CFU counts.

image

Figure 3. Quantitative detection of bacteria by RT-qPCR in stool samples. (a) Vibrio cholerae 569B, (b) V. parahaemolyticus DSM 10027T, and (c) Campylobacter jejuni ATCC 33560T were cultured as described in the text. Each bacterial culture was inoculated into a stool sample to give final concentrations of 104, 105, 106, 107 and 108 cells/g stool for V. cholerae, 103, 104, 105, 106, 107 and 108 cells/g stool for V. parahaemolyticus, and 104, 105, 106, 107 and 108 cells/g stool for C. jejuni. RNA was extracted from each concentration of each bacteria, and from the Ct value of each sample by RT-qPCR. Bacterial counts were determined by using the curves generated in Figure 1. Data from three independent experiments are shown. Each CFU was determined using TCBS agar for V. cholerae and V. parahaemolyticus, and CCDA agar containing cefoperazone-amphotericin-teicoplanin for C. jejuni.

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Bacterial counts determined by RT-qPCR, qPCR, the culture method, FISH and DAPI staining during the course of in vitro culture

Bacterial counts of V. cholerae, V. parahaemolyticus and C. jejuni during the growth phases were determined periodically for 120 hrs incubation by RT-qPCR, qPCR, the culture method, FISH and DAPI staining. For all three strains tested, the counts determined by RT-qPCR correlated well with those by the culture method throughout the 120 hrs incubation, whereas the results by qPCR and DAPI staining dissociated with the results by RT-qPCR and the culture method (Fig. 4a–c). RT-qPCR counts of these strains were also similar to the corresponding counts determined by FISH, a well-established methodology targeting bacterial RNA, after 120 hrs incubation, as well as those by the culture method, although those by qPCR and DAPI staining were significantly higher than those by RT-qPCR (Table 2).

image

Figure 4. Effect of the growth phase of bacteria on bacterial counts determined by RT-qPCR, qPCR, DAPI staining and the culture method. Every 24 hrs for 120 hrs incubation in broth culture, counts of (a) Vibrio cholerae 569B, (b) V. parahaemolyticus DSM 10027T and (c) Campylobacter jejuni ATCC 33560T were determined by (○) RT-qPCR, (▴) qPCR, (□) DAPI staining and (•) the culture method as described. Each CFU was determined using 2×YT agar containing 1.5% (wt/vol) NaCl for V. cholerae, TCBS agar for V. parahaemolyticus, and CCDA agar containing cefoperazone-amphotericin-teicoplanin for C. jejuni. For RT-qPCR and qPCR, analytical curves generated in Figure 1 were used. Results are expressed as the mean  ±  SD of triplicate determinations of each bacterial count.

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Table 2.  Comparison of bacterial counts in cultures by RT-qPCR, the culture method, FISH, DAPI staining and qPCR
Methodslog10 cells/mL culture†
V. choleraeV. parahaemolyticusC. jejuni
24 hrs‡120 hrs24 hrs120 hrs24 hrs120 hrs
  1. †Counts obtained by RT-qPCR, the culture method, FISH, DAPI staining and qPCR are expressed as the number of log10 bacterial cells per milliliter of culture. Mean ± standard deviations for triplicate samples are expressed. ‡Incubation time. §NT, not tested.

RT-qPCR8.5  ±  0.17.9  ±  0.59.2  ±  0.17.8  ±  0.18.5  ±  0.26.8  ±  0.3
Culture method8.6  ±  0.37.7  ±  0.68.7  ±  0.37.9  ±  0.28.9  ±  0.46.5  ±  0.5
FISHNT§NT8.5  ±  0.18.1  ±  0.29.0  ±  0.16.3  ±  0.3
DAPI9.3  ±  0.19.4  ±  0.08.6  ±  0.18.8  ±  0.19.0  ±  0.19.1  ±  0.1
qPCR9.1  ±  0.09.5  ±  0.08.9  ±  0.08.8  ±  0.08.9  ±  0.29.2  ±  0.1

Stability of bacterial rRNA in stool sample

To examine the stability of bacterial rRNA in stool suspension, liquid cultures of V. cholerae, V. parahaemolyticus and C. jejuni were spiked in the stool suspension (10%, wt/vol) in RNAlater® to make the final bacterial concentration of 107–108 cells/g stool suspension for each bacteria, which were incubated at 37°C, 25°C or 4°C for 0, 7, 14, 21 and 28 days. RNA was extracted and bacterial counts were determined by RT-qPCR. As shown in Figure 5, bacterial counts did not change significantly through the test periods at any temperature tested. These data indicate that bacterial RNA in stool samples suspended in RNAlater® can be kept stable for no less than 4 weeks even at 37°C.

image

Figure 5. Stability of bacterial rRNAs in stools suspended in RNAlater®. (a) Vibrio cholerae 569B, (b) V. parahaemolyticus DSM 10027T and (c) Campylobacter jejuni ATCC 33560T were cultured as described in the text. Stool suspensions of each bacterium were incubated at (□) 4°C, (•) 25°C or (○) 37°C. At 0, 7, 14, 21 and 28 days after incubation, RNA was extracted from a 200 μL aliquot of each sample and bacterial counts were determined by RT-qPCR as described in the text. Results are expressed as the mean  ±  SD of triplicate determinations of each bacterial count.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

For detection of bacterial pathogens in clinical samples, PCR has currently been the most frequently used molecular technique. It has been demonstrated that the detection limit of PCR is approximately 105 to 106 cells per g stool sample in the analysis of commensal bacteria and enteropathogens (13,22). rRNA-targeted RT-qPCR was shown to be 100 to 1,000 times more sensitive than DNA targeted qPCR for the detection of V. cholerae, V. parahaemolyticus and C. jejuni both in the bacterial culture (Fig. 1a–c) and in spiked stools (Fig. 2a–c). This is because of the high copy numbers of rRNA molecules (approximately 104 molecules per cell) (23) compared to those of rRNA gene copies (approximately 10 copies) in genome DNA.

Several primers specific for enteric pathogens have been reported previously. Specific genes such as cholera toxin (ctx) and outer membrane protein (ompW) for V. cholerae (24, 25), thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh), metalloprotease (vpm) for V. parahaemolyticus (7,26), and hippuricase (hipO) for C. jejuni (27) have been used for primer targets. The detection limit of PCR using these primers is 101–102 CFU/reaction or 105 CFU/g stool (7, 22, 28). In the present study, the specific primers targeting rRNA for these species of enteropathogens were newly developed. Their high specificity and sensitivity (10−2 cells/reaction or 103 cells/g stool) were identified by RT-qPCR (Table 1, Figs 1 and 2). In spite of its advantage, there are some technical limitations in the RT-qPCR method. One is that several bacterial species closely related to each other, such as V. cholerae and V. mimicus, V. parahaemolyticus and V. alginolyticus, C. jejuni and C. coli cannot be differentiated by this method, because of the similarity of the rRNA sequences. V. mimicus is often isolated from patients with sporadic diarrheal cases (29). V. alginolyticus is mostly associated with ear infections but can occasionally cause diarrhea (30,31). The other point is that differentiation of V. cholerae O1 and O139 from non-O1, non-O139 V. cholerae, as well as of cholera toxin positive and negative V. cholerae is not possible. Therefore, combination with primers targeting specific virulence genes by RT-qPCR or nested-qPCR would be effective for further differentiation in intra- and interspecies analysis.

RNA molecules have been used as an indicator of bacterial cell viability as an alternative to DNA molecules (32,33). RT-qPCR counts correlated well with CFU counts in spiked stool samples for all the strains tested (Fig. 3a–c). In addition, RT-qPCR counts were in good agreement with the counts obtained by the culture method and FISH for all the strains tested in any condition during 120 hrs of incubation (Fig. 4a–c, Table 2). Unlike RT-qPCR and the culture counts, qPCR and DAPI staining counts remained fairly constant even at the death phase. This appears to be due to the difference of the molecular target between the methods: the target for qPCR and DAPI staining is DNA, which might not degrade rapidly in the dead cells during the late period of the culture, resulting in the quantification of DNA molecules derived from dead cells as well as those from viable cells (34). The counts of V. cholerae and C. jejuni obtained by qPCR and DAPI staining dissociated from those of the culture methods and RT-qPCR at 24 to 48 hrs, whereas this phenomenon was only demonstrated with V. parahaemolyticus at 96 hrs (Fig. 4a–c). This dissociation might be because the timing of when the bacteria start to die during in vitro culture is different from species to species. These data, taken together, suggested that RT-qPCR should be more suitable for accurate detection of viable bacteria than qPCR. It has been reported that some bacteria, including V. cholerae, V. parahaemolyticus and C. jejuni, enter the viable but non-culturable (VBNC) state as a response to some stresses (35, 36). Important pathogens might be missed by the culture method because the bacterial populations in the VBNC state fail to grow on routine media. This is very problematic because, even when in the VBNC state, bacteria have metabolic activity, maintain pathogenicity (37), and may convert to the active growth phase when optimal conditions are restored (38, 39). It is reported that the 16S rRNA level in the VBNC state was almost the same as that in the non-VBNC state, suggesting that rRNA is a valuable genetic marker for detection and quantification of viable V. cholerae (40). Therefore, RT-qPCR could be a good tool to detect V. cholerae in the VBNC state.

We used RNAlater® solution for the fixation of bacterial RNA in stool samples, which is known to be a significantly labile molecule. It has generally been recognized that RNAlater® mixed with stools or bacterial culture penetrates bacterial cells immediately after mixing and protects the RNA molecule from RNase (41). The 72 hrs storage of human tissues in RNAlater® at room temperature did not affect quantitative RNA expression (41), and RNA was kept stable for at least 7 days (42). RNA in the stool suspension was shown to be kept stable for no less than 4 weeks even at 37°C (Fig. 5a–c), suggesting that the RT-qPCR system can be applied for sampling in field trials even where research equipment such as a refrigerator is not available.

In conclusion, we have developed an RT-qPCR system for quantification of V. cholerae/mimicus, V. parahaemolyticus/alginolyticus and C. jejuni/coli as an effective tool for quantification of viable enteric pathogens in field study as well as in the laboratory.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

We thank Dr. G. B. Nair for his constructive interest during the study. We are also thankful to Ms. Debarati Ganguly for her critical reading and English correction of this paper. This work was supported in part by the Japan Initiative for Global Research Network on Infectious Diseases, Ministry of Education, Culture, Sports, Science and Technology, Japan.

DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

There is nothing to be disclosed by any of the authors.

REFERENCES

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  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
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