Arch Pediatr Infect Dis. 2017 January; 5(1):e38888. doi: 10.5812/pedinfect.38888.

Published online 2016 September 4. Research Article

Comparison of the Detection Limits of the Culture and PCR Methods

for the Detection of Clostridium difficile, Clostridium perfringens,
Campylobacter jejuni, and Yersinia enterocolitica in Human Stool
Leila Ganji,1 Masoumeh Azimirad,2 Nastaran Farzi,2,3 Masoud Alebouyeh,2,3,* Mohammad Hassan

Shirazi,1,* Seyed Saeed Eshraghi,1 Abbas Mirshafiey,1 Naser Ebrahimi Daryani,1 and Mohammad Reza
2,3
Zali
1
Department of Pathobiology, School of Public Health, University of Medical Sciences, Tehran, Iran
2
Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran,
Iran
3
Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran,
Iran
*
Corresponding authors: Masoud Alebouyeh, Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid
Beheshti University of Medical Sciences, Tehran, Iran, E-mail: [email protected].; Mohammad Hassan Shirazi, Department of Pathobiology, School of Public
Health, University of Medical Sciences, Tehran, Iran, E-mail: [email protected]

Received 2016 May 01; Revised 2016 July 20; Accepted 2016 August 02.

Abstract

Background: Detection of fastidious enteropathogenic bacteria in fecal samples of patients with gastroenteritis is a challenge in
clinical microbiological laboratories.
Objectives: The aim of this study was to compare the detection limits of the PCR and culture methods for the diagnosis of Campy-
lobacter spp., Yersinia spp., Clostridium perfringens, and Clostridium difficile in human stool samples.
Methods: Healthy human stool and sterile phosphate-buffered saline (PBS) samples were separately spiked with 10-fold dilutions
of C. jejuni, C. difficile, Y. enterocolitica, and C. perfringens reference strains to obtain final concentrations of 101 - 108 colony forming
units (CFU) per gram. Dilutions of each suspension were inoculated onto specific culture media and colony counts were determined.
Polymerase chain reaction (PCR) was carried out on DNA extracts of each dilution using specific primers. All of the assays were
performed in two separate replicas.
Results: In the cases of the culture and PCR assays, detection limits of 101 and 102 CFU/g for C. difficile, 2 × 104 and 2 × 104 CFU/g for
C. perfringens, 104 and 102 CFU/g for C. jejuni, and 102 and 104 CFU/g for Y. enterocolitica, respectively, were obtained. In the cases of the
spiked PBS samples, a detection limit of 101 for C. jejuni and Y. enterocolitica was obtained using the culture method. While 102 -fold
higher sensitivity was observed for C. jejuni via PCR compared with the culture assay, equal (C. perfringens) or lower sensitivity limits
(C. difficile and Y. enterocolitica) were detected for the spiked stool samples with other bacteria.
Conclusions: These results showed differences in the bacterial culture and PCR methods for quantitative detection of fastidious
bacteria in human stool samples. However, a bacterial load of 104 CFU per gram of stool was measured as a sufficient amount for
detection of the fastidious bacteria by either culture or PCR assays. More suitable PCR methods could be used for rapid diagnosis of
the slow-growing bacteria in the patients’ stool samples.

Keywords: Culture, PCR, Campylobacter jejuni, Clostridium difficile, Clostridium perfringens, Yersinia enterocolitica

1. Background tion of metabolites that dysregulate the normal signaling

pathways of the intestinal cells is involved in the disease
There are increasing data regarding the roles of the in- progression in infected patients. More than 200 transmit-
testinal microbiota in the pathogenesis of several human ted microbial agents from foodstuffs are associated with
diseases (1). Prolonged interaction of these bacteria with gastroenteritis in the human population (5). Among these
the intestine or their overgrowth seems to be responsible agents, the most important enteric bacterial pathogens
for chronic diseases in this organ (2). Even in low num- that are responsible for gastrointestinal diseases are Es-
bers, pathogenic bacteria which are not common mem- cherichia coli, Campylobacter jejuni, Shigella spp., Salmonella
bers of the gut microbiota can cause different human ill- spp., Vibrio cholera, and Yersinia spp. (6). Clostridium difficile
nesses under some conditions (3, 4). Invasion of these is also considered the most frequently identified enteric
pathogens into the intestinal barrier layer or the produc-

Copyright © 2016, Pediartric Infections Research Center. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0
International License (http://creativecommons.org/licenses/by-nc/4.0/) which permits copy and redistribute the material just in noncommercial usages, provided the
original work is properly cited.
 Ganji L et al.

pathogen in hospitalized patients with a recent history of 3. Methods

medication (7). The infectious dose of these pathogens
varies depending on their virulence potency and level of 3.1. Bacterial Strains and Growth Conditions
resistance to the harsh conditions of the gut environment The strains used in this study were the reference
(8). In some cases, gastrointestinal infectious diseases are strains of Campylobacter jejuni (ATCC strain 33560), C. diffi-
acquired by the consumption of contaminated foods or cile (research center of gastroenterology and liver disease
water that are infected with fewer than 10 microorganisms [RIGLD]-141), Yersinia enterocolitica (ATCC strain 101776), and
(9). Detection of responsible bacterial agents in environ- C. perfringens (; RIGLD-2). Selective culture media, includ-
mental or fecal samples mainly depends on the validity of ing Brucella agar (Merck, Germany; supplemented with
the laboratory tests used and the sampling procedure, in- 5% sheep blood and Campylobacter selective supplement),
cluding time of sampling and transport conditions. It is Clostridium difficile agar (Mast, UK; supplemented with de-
hard to resolve challenges that exist for the detection and fibrinated horse blood and Clostridium difficile selectavial),
enumeration of these bacteria, since the causative bacte- Yersinia selective agar (Merck; supplemented with Yersinia
ria are often present in low numbers within these samples selective supplement CIN), and Egg Yolk agar (Merck; sup-
or their presence is influenced by food materials or high plemented with neomycin), were used for the subculture
counts of indigenous bacteria (10). Therefore, a rapid yet of these strains. In the cases of C. perfringens and C. diffi-
sensitive and specific diagnostic assay would be advanta- cile, the cultures were incubated under anaerobic condi-
geous to clinicians for the early recognition of disease and tions (Anoxomat, MART Microbiology B.V.; 0% O2 , 10% H2 ,
to infection control practitioners for the swift implemen- 10% CO2 , and 80% N2 ) at 37°C for 48 hours. Campylobacter je-
tation of control measures (11). juni was grown under microaerobic conditions (6% O2 , 6%
Bacterial culture is considered the “gold standard” for CO2 , 3% H2 , and 85% N2 ) for 24 hours at 42°C; Y. enterocolitica
identification of diarrheagenic bacteria from stool spec- was grown under ambient air conditions at 25°C.
imens. However, this method is time consuming and
laborious, requiring prolonged incubation, selective en- 3.2. Spiked Stool Experiments
richment, and reduction of the background flora, which Serial tenfold dilutions of each bacterial species
should be followed by biochemical identification tests (12, were freshly prepared at defined concentration (101 -
13). Most laboratories are unable to diagnose the anaero- 108 CFU/mL) in a control stool specimen and phosphate-
bic, microaerophilic, and fastidious bacteria responsible buffered saline (PBS). A total of 100 µL of each dilution was
for human gastrointestinal disorders using conventional spread onto the selective media for enumeration of the
microbiological methods. Recently, more rapid DNA-based colony counts. In the cases of C. difficile, the inoculated
methods for direct identification and even subtyping of samples were initially treated with alcohol and yeast
these pathogens in stool specimens have been developed extract broth to remove the common intestinal microbial
(14-21). Polymerase chain reaction (PCR) is a powerful tech- flora. For alcohol treatment, about 1 g of stool was mixed
nique for the detection of the target DNA in various clinical with an equal volume of 95% methanol and then slowly
specimens, including fecal samples. However, fecal speci- vortexed and held at room temperature for 2 minutes.
mens often contain substances that may interfere with the The treated suspensions were cultured on the selective
PCR assay, leading to false-positive or false-negative results media supplemented with 5% horse blood (CDSA). For the
(2, 4, 11, 13, 15, 22). Accordingly, its application in clinical lab- enrichment of Clostridium, nearly 1 g of the stool samples
oratories needs validation. Comparison of results for con- were mixed with an equal volume of yeast extract broth
ventional culture- and molecular-based methods, which (Yeast extract; Merck). The treated suspensions were then
are designed for each bacterial species, is essential. cultured on the selective medium. To detect C. perfrin-
gens spores, the methanol (95%) and heat-treated spiked
samples (90°C, 20 minutes) were cultured on Neomycin
Egg Yolk agar. Cold enrichment in PBS and direct culture
2. Objectives
of the inoculated stool samples on CIN (agar 25°C) and
MacConkey agar (37°C) were used for isolation of Y. en-
In this study, we aimed to compare the detection limit terocolitica. Two replicas of the tests were performed for
and performance of PCR and culture methods for diagno- analysis of the variations in our results (23).
sis of Campylobacter spp., Yersinia spp., C. perfringens, and C.
difficile. As enteric pathogens, these are among most sensi- 3.3. DNA Extraction and PCR
tive to the ambient culture conditions commonly used for DNA was extracted from the prepared stools accord-
diagnosis in clinical laboratories. ing to the manufacturer’s instructions using the DNA Stool

2 Arch Pediatr Infect Dis. 2017; 5(1):e38888.

 Ganji L et al.

Kit (Bioneer, South Korea). The concentration of DNA sam- 5. Discussion

ples was 30 µg. All of the DNA extracts were stored at -
20°C until use. Amplification of target genes for the detec- The prevalence levels of Campylobacter, Y. enterocolitica,
tion of Campylobacter spp. (16S rRNA), Yersinia enterocolit- and C. difficile are about 10.8%, 1.2%, and 21%, respectively, in
ica (ompF), C. difficile (Cdd-3), and C. perfringens (16S rRNA) stool samples of patients with gastroenteritis in develop-
was performed using the specific primers depicted in Ta- ing countries (28-30). These rates are lower than those re-
ble 1. All PCR amplifications were performed in 25-µL vol- ported for developed countries (4, 31). Despite differences
umes containing 3 µL of DNA template, 0.5-mM concen- in geographic area and culture in these countries, inaccu-
trations of deoxynucleoside triphosphates, 2.5 µL of 10 × rate results could be obtained due to the need for individ-
PCR buffer (gene fanavaran), 0.75 mM MgCl2 , 0.3 µM con- ual equipment and defined culture media for the detection
centrations of each forward and reverse primer, 0.2 U of of these pathogens in patients’ stool samples. Because of
Taq DNA polymerase (Gene Fanavaran, Iran) under the fol- their advantages, molecular tests are now widely used to
lowing conditions: initial denaturation step at 95°C for 5 detect these bacteria in clinical specimens. These tests are
minutes, followed by 35 cycles of denaturation at 94°C for extremely useful diagnostic tools and are particularly valu-
1 minute, annealing for 1 minute, and extension at 72°C for able for the detection of infectious agents that are difficult
1 minute. After the last cycle, the mixture was incubated at to grow in conventional culture media. However, the pres-
72°C for 5 minutes. The amplification products were ana- ence of PCR inhibitors and variation of procedures that are
lyzed by electrophoresis on a 1.5% agarose gel. Images were used for sample preparation or extraction of nucleic acids
obtained after staining of the gels with ethidium bromide affects their accuracy, precision, specificity, and sensitivity
and under an ultraviolet (UV) light imaging system. (23). The interpretation of results for these assays should
be based on their limit of detection.
In this study, analysis of culture and PCR methods for
the detection of C. jejuni showed a sensitivity limit of 104
4. Results
and 102 CFU/g, respectively, for the spiked stool samples. In
a study by Singh et al. (32), higher sensitivity of PCR com-
The results of the PCR assays showed specificity of pared with culture method was indicated in the spiked
primer pairs that were used for detection of the target bac- fecal samples (Table 3). Our results showed lower detec-
teria. Accordingly, the species-specific primers provide a tion limits for both the culture and PCR assays compared
single PCR amplicon in the spiked stool samples (Figure 1). with those reported by Persson et al. (33) (pure culture,
Analysis of culture method results for the detection of C. 101-2 CFU; spiked stool, 105 CFU/g). The obtained detec-
jejuni showed a sensitivity limit of 104 CFU/g for the spiked tion level for conventional PCR in our study was similar
stool samples. This detection level was as low as 101 CFU to those reported for C. jejuni and C. coli using the real-
when the bacterium was directly inoculated on the culture time PCR method (2.5 × 102 CFU/g of feces) (34). The in-
medium from the PBS suspensions. PCR results showed congruent results could be explained as relating to differ-
a lower detection limit for the spiked stool samples (102 ences in the primer sequences and DNA extraction meth-
CFU/g). The validity of these results was confirmed by ob- ods used in this study. Recovery of C. jejuni from stool
taining the same results for both of the studied strains in samples may be affected by the types of culture media
two separate tests. Analysis of the culture results for de- used. Potturi-Venkata et al. (35) showed a higher isola-
tection of C. difficile on selective medium showed its sensi- tion rate for modified charcoal cefoperazone deoxycholate
tivity for the detection of 10 organisms in 1 gram of stool (mCCDA) compared with Brucella agar–based media for
sample. The PCR detection limit for this bacterium was isolation of Campylobacter spp. from fecal samples (Table
100 CFU/g; however, an increased detection limit of up to 3). C. jejuni is a microaerophilic bacterium that requires
10 genome copies per PCR reaction was obtained on pre- specific incubation conditions to grow in synthetic culture
pared dilutions of genomic DNA. In the case of Y. enterocol- media. Although usage of an effective culture medium
itica, there was also discordance between results obtained will improve the isolation rate of the bacterium, the need
by culture and those using the PCR method. In the cul- to provide microaerophilic conditions for its growth and
ture method, Y. enterocolitica was detected in all dilutions supplements to prevent the growth of fecal microbiota are
of bacteria in PBS. In contrast, a lower limit of detection considered the main limitations of this method.
was observed via PCR (104 CFU/g). The results of anaerobic Alcohol pretreatment of stool specimens together
culture for C. perfringens showed a detection limit of 2 × with appropriate incubation time (up to 1 week) seems to
104 CFU/g that was similar to those were obtained by PCR be an effective method for the detection of C. difficile spores
assay results. in the stool samples (7, 8); however, usage of other sensitive

Arch Pediatr Infect Dis. 2017; 5(1):e38888. 3

 Ganji L et al.

Table 1. Primers Used in This Study

Bacterium Target Gene Primer Sequence (5´ to Amplicon Size Tm°C Reference
3´) (bp)
F: GGATGA-
Campylobacter CACTTTTCGGAGC
16S rRNA C412F; C1228R 816 48 (24)
spp. R: CATTGTAGCACGT-
GTGTC
F:
GTCTGGGCTTTGCTG-
GTC
Yersinia spp. 16S rRNA 227Fmod; 669R 428 - 465 43 (25)
R: GCGTCGTATTTAG-
CACCAACG
F: TCC
AATAATAAATTAG-
CATTCC
C. difficile Cdd-3 Tim6; Struppi6 622 54 (26)
R: GGCTATACACG-
TAATCCAGATA
F: AAAGATGGCAT-
CATCATTCAAC
C. perfringens 16S rRNA 184-205; 441-462 279 50 (27)
R: TACCGTCAT-
TATCTTCCCCAAA

Table 2. Detection Limits of Campylobacter, Clostridium and Yersinia spp. (CFU/gram stool) in the Culture and PCR Assays Using Spiked Stool Samples

Limit of Detection, CFU/Gram Stool

C. jejuni C. difficile C. perfringens Y. enterocolitica

Culture 104 10 2 × 104 10

PCR 102 102 2 × 104 104

Table 3. Comparison of the Culture and Molecular Assay Methods for Quantitative C. difficile, and their detection limit was estimated to be as
Detection of Fastidious Enteropathogens in the Spiked Human Stool Samples high as 5 × 104 CFU per gram of feces. Using the prepared
dilutions of genomic DNA, an increased detection limit of
Bacterium Method Results, CFU References
g-1 /DNA Copya up to 10 genome copies per PCR reaction was obtained in
Culture and PCR 104 /102 This study
our experiment. According to these results, the incongru-
5 2
ence of the PCR results compared with the culture results
Campylobacter Culture and PCR 10 /10 (33)
spp.
could be explained by the low yield of DNA that was ex-
Real-time PCR 2.5 × 102 (36)
tracted from the C. difficile spores in the spiked stool sam-
qPCR 102 (37)
ples or the existence of mutations in the cdd3 locus in the
Culture and PCR 10/104 This study
regions where our primers adhered. The existence of mu-
Multiplex PCR 105 (38) tations was not supported by our data, since cdd3 was de-
Y. enterocolitica
Real-time PCR 102 (39) tected in the DNA extracts of diluted DNA samples at the
Culture and PCR 4 × 103 /4 × 102 (40) lower concentration. In a study by Luna et al. (43), where
Culture and PCR 10/102 This study tcdA and tcdB were targeted in the spiked stool samples
C. difficile
Real-time PCR 5 × 104 (41) by real-time PCR, the lower limit of detection of C. difficile
Culture and PCR 2 × 104 /2 × 104 This study was 250 CFU/mL for tcdA and 500 CFU/ml for tcdB. These
C. perfringens
Multiplex PCR 102-4 (42)
researchers similarly concluded that the sensitivity of the
a tests can only be increased in the more concentrated sam-
CFU/g, colony forming unit/gram stool; DNA copy of target bacterium per
gram stool was represented for all the molecular assays. ples.

Discordance between results of culture and PCR meth-

and rapid tests is preferable. In our experiment, the PCR ods was also determined in the case of Y. enterocolitica. This
detection limit for this bacterium was 100 CFU/g. It seems finding was supported by Weimer et al. (38), who used mul-
that anaerobic culture has a much lower detection limit (10 tiplex real-time PCR for the simultaneous detection of Y.
CFU/g) than PCR assay for the detection of C. difficile. Be- enterocolitica and other bacteria in stool samples; they re-
langer et al. (41) used real-time PCR assay for detection of ported a sensitivity limit of 105 CFU/g in their research. In

4 Arch Pediatr Infect Dis. 2017; 5(1):e38888.

 Ganji L et al.

Figure 1. PCR Results

A, C. difficile; B, C. jejuni; C, C. perfringens; D, Y. enterocolitica.

another study, detection limits of 102 CFU/mL and 103 CFU/g both the culture and PCR methods. This amount was sim-
for the pure culture and stool sample, respectively, were ilar to those reported by Wise et al. (42) using a multiplex
obtained using real-time PCR (39). However, Boyapalle et PCR assay. In their study, C. perfringens alpha and entero-
al. (40) reported a lower detection limit for PCR (4 × 102 toxin genes were targeted for detection of the bacterium
CFU/g) compared with culture method (4 × 103 CFU/g). The in spiked fecal samples of domestic animals, and an aver-
lower sensitivity of the PCR method compared with the cul- age sensitivity of 102-4 CFU/g was reported. Our results il-
ture method was also confirmed by the results of our as- lustrated a correlation between the PCR assay and tradi-
say using DNA extracts of the provided dilutions of Y. en- tional culture method for the detection of C. perfringens in
terocolitica in PBS (71.4%, 103 CFU/mL). In a study by Wan- fecal spiked samples. Since the studied samples were sub-
net et al. (44), those primers that targeted ail and 16s rRNA jected to heat treatment for the elimination of non-spore-
genes showed a sensitivity of 100% (one genomic copy) in forming bacteria (which may affect the germinating cells
pure culture of Y. enterocolitica. Differences in the primers of C. perfringens), it seems that the detection limits of these
that target the ompF gene for PCR and the type of DNA tests are lower than 103 CFU/g in human fecal samples.
extraction kit used in our experiment may explain these
contradictory results. Re-analysis of the tests using differ- The PCR results for all bacteria mentioned in this study
ent primers and extraction kits will provide more accurate were available on the same day as the assays were per-
data about their limits of detections. formed, whereas the culture results took 24 hours for Y.
enterocolitica and 48 - 72 hours for C.jejuni, C. difficile, and
C. perfringens is not only a member of human micro- C. perfringens. These results collectively showed that di-
biota in the gastrointestinal tract, but it is also considered rect plating can be used successfully for the detection of
as a common cause of food poisoning in foodborne out- anaerobic enteric bacterial pathogens (C. difficile and C. per-
breaks (45). In general, detection of > 106-8 CFU per gram fringens) and fastidious bacteria (Yersinia spp. and Campy-
of this bacterium in stool samples of patients with gas- lobacter spp.) in human fecal samples when a bacterial
troenteritis is considered clinically important (42). Our re- load of greater than 104 CFU/gram is present. The specified
sults showed a similar detection limit (2 × 104 CFU/g) for PCR assays showed acceptable results with respect to detec-

Arch Pediatr Infect Dis. 2017; 5(1):e38888. 5

 Ganji L et al.

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