Comparative Study of Campylobacter spp . Isolated from Children With Gastroenteritis in Bahonar Hospital , Karaj , Using PCR and RFLP

Background: Campylobacter species are responsible for the majority of cases of food-borne gastroenteritis. The sources of the disease outbreaks are often contaminated water or milk, and consumption of undercooked poultry product is the main cause of sporadic campylobacteriosis cases. Objectives: The aims of this study were to determine the prevalence of Campylobacter gastroenteritis in children and to differentiate the interfering species using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) methods at the Bahonar hospital in Karaj, Iran. Patients and Methods: A total of 150 stool samples were collected from children under 10 years old during the summer of 2014. PCR was performed using genusand species-specific primers and RFLP was done using AluI and TasI enzymes. Results: The results showed the amplification of 400 and 491 bp segments and Campylobacter contamination in 30 (20%) samples; 5 out of 30Campylobacter positive samples (16.66%) were identified asC. jejuni, 20 (66.66%) asC. coli, 3 (10%) asC. jejuni andC. coli (mixed infection), and 2 (6.66%) were identified as non-jejuni, non-coli Campylobacter using the PCR method. Following the evaluation of RFLP results, 7 positive samples (23.33%) showed the electrophoretic pattern of C. jejuni, 21 (70%) showed the electrophoretic pattern of C. coli, and 2 (6.6%) showed both of the patterns and mixed contamination with jejuni and coli species. The results of digestion with TasI did not show any C. lari or C. upsaliensis patterns. Conclusions: The results of this study showed high percentage of Campylobacter contamination in the tested stool samples. The other surprising finding was the high rate of Campylobacter coli positive samples; the difference between the results of PCR using species-specific primers (hipo and asp) and the RFLP method (electrophoretic patterns) in some of the positive samples confirms the hypothesis of variations in nucleotide sequences of the hipo primer binding site in some Iranian isolates of Campylobacters.


Background
Campylobacter species are responsible for the majority of food-borne gastroenteritis in many developed and developing countries.Infection by Campylobacter can result in severe problems outside the intestine such as Guillain-Barre syndrome (GBS).Campylobacter species are estimated to be responsible for 400 to 500 million cases of diarrhea each year worldwide.The incidence of diarrhea in children under five years of age is estimated at 40,000 to 100,000 cases yearly in developed countries.The sources of disease outbreaks are often contaminated water or milk, and consumption of undercooked poultry product is the main cause of sporadic campylobacteriosis cases (1)(2)(3)(4)(5)(6)(7)(8).Campylobacter detection is based on culturing stool samples, which requires specific selective media and microaerophilic conditions at 37°C to 42°C for 48 hours.The sodium hippurate hydrolysis reaction is a culture-based biochemical test used to differentiate Campylobacter jejuni from C. coli.However, detection of Campylobacter using polymerase chain reaction (PCR) based methods is currently more common because of their high sensitivity and specificity and the above-mentioned difficulties and limitations of culture and biochemical methods (9,10).

Objectives
According to the results of recent molecular surveys on Campylobacter gastroenteritis, variation in the nucleotide sequence of hipo and asp primer binding sites in Iranian isolates of Campylobacter is probable, and the aim of this study was to test this hypothesis by comparing the results of PCR (using the species-specific primers) and restriction fragment length polymorphism (RFLP) methods.

Bacterial Strains and Stool Samples
C. jejuni RTCC 1097 and C. coli RTCC 1113 standard strains were prepared from Razi type culture collection (Razi vaccine and serum research institute).Children's stool specimens (n = 150) were collected from Bahonar hospital in Karaj from July to September 2014.These samples were collected from children under 10 years of age suffering from symptoms of gastroenteritis.Collected samples were stored at -20°C until DNA extraction.

DNA Extraction
DNA extraction from stool samples was performed by using CinnaPure DNA kits (CinnaGen, Iran) according to the manufacturer's recommended procedure.First, distilled water was added to the collection tube to provide a homogenous suspension.Then, 100 µL of this suspension was transferred to a sterile micro tube.In the next step, 100 µL pre-lysis buffer and 10 µL ributinase were added and shaken for a few minutes.Finally, the tube was placed for 45 minutes in a heater at 55°C, and every 10 minutes the sample was shaken again and transferred back to the heater.The obtained DNA was stored at -20°C.

Polymerase Chain Reaction
PCR was performed using primers targeting genes, namely cadf (genus-specific virulence gene), 23S rRNA (genus-specific gene), hipo (hippuricase gene for C. jejuni) and asp (aspartokinase gene for C. coli).The primer sets are listed in Table 1 (Bioneer, South Korea).All extracted DNA from specimens was checked by genus-specific cadF primer, then positive samples were analyzed with hipo and asp primers to differentiate jejuni and coli species (Figure 1).All cadf positive samples were tested by PCR using 23S rRNA (another Campylobacter genus-specific gene) primer, and the products were digested with AluI and TasI enzymes using the RFLP method (9)(10)(11)(12)(13)(14).The PCR mixture in a total volume of 25 µL for each primer contained: 12.5 µL Taq Mix (2×) with 1× final concentration (DELTA life sciences), 1 µL reverse primer (10 µM), 1 µL forward primer (10 µM), 5.5 µL sterile water, and 5 µL template DNA.The PCR amplification was performed for 35 cycles for each set of primers, which have different thermal conditions as shown in Table 2.The amplified PCR products were stained by DNA safe stain and were visualized via a UV transilluminator after electrophoresis on 1.3% agarose gel.

PCR-RFLP Conditions
In this step, positive PCR products of 23S rRNA were digested using two restriction enzymes, AluI and TasI (Thermo Scientific) in order to differentiate Campylobacter species (11,13,14).The RFLP mixture for a final AluI volume of 32 µL contained: 10 µL PCR product of 23S rRNA, 1 µL restriction enzyme AluI, 2 µL (10×) tango buffer, and 18 µL nuclease-free water.Finally, the mixture was incubated at 37°C for 150 minutes.For TasI: 10 µL of PCR product of 23S rRNA, 1 µL restriction enzyme TasI, 2 µL (10×) buffer B, and 18 µL nuclease-free water was incubated at 65°C for 4 hours after adding paraffin oil.Electrophoretic patterns obtained after digestion of the PCR-amplified DNA of the positive samples were analyzed on 1.7% agarose gel.

Sensitivity of PCR
Digestion of PCR products by AluI and TasI endonucleases yielded known electrophoretic patterns (the RFLP method confirms our PCR results) (15,16).

Results
Evaluation of the PCR products showed amplification of 400 and 491 bp genus-specific segments in 30 out of 150 tested samples.The results of PCR using species-specific primers and RFLP methods for speciation of Campylobacter in positive samples are shown in Table 3, Figures 2 and 3. Analysis using the t-test showed a significantly higher rate of infection in children under 1 year of age (P = 0.37), but there was no statistically significant difference between the rates of infection in male and female groups (Tables 4  and 5).

Discussion
Campylobacter spp., especially C. jejuni and C. coli, are recognized as one of the major bacterial agents of foodborne gastroenteritis worldwide over recent decades (1).Culture-based methods have a number of limitations for detection of Campylobacter spp. in human fecal samples, such as the requirement for 48 hours incubation to isolate the organisms (10).Therefore, PCR-based methods have been developed for detection of Campylobacter spp. in different specimens, including fecal samples (11,13,14).The main source of Campylobacter disease outbreaks is associated with consumption of contaminated water or milk and consumption of undercooked food; in particular, poultry products are the main cause of sporadic Campylobacteriosis cases (1)(2)(3)(4)(5)(6)(7)(8).Contamination of milk with Campylobacter spp. is often seen after pasteurization or due to poor pasteurization; for example, milk bottles that were pecked by jackdaws and magpies, contaminating free school milk, were a probable source of human Campylobacter infection in Great Britain in an outbreak affecting around 2500 children (6).Particular groups of people, such as infants, children, pregnant women, and individuals with immunodeficiency, are more at risk of developing campylobacteriosis (9,11,(17)(18)(19)(20)(21).
Many PCR-based molecular surveys have been performed on Campylobacter contamination in different clinical samples during the last two decades.Al Amri et al. ( 9) evaluated 114 culture-positive stool specimens (54 human and 60 chicken) using multiplex PCR.According to the results, 70 (61.4%)were identified as C. jejuni, 35 (30.7%) as C. coli, and 9 (7.9%) as a mixed infection with C. jejuni and C. coli.In addition, two culture-negative samples were identified as C. jejuni.This study showed that multiplex PCR is a highly sensitive and specific tool for direct detection and identification of Campylobacters spp. in stool samples.

4
Int J Enteric Pathog.2016; 4(2):e33867.In this study PCR using genus-specific primers (cadf and 23S rRNA) was performed to determine the frequency of Campylobacter contamination in the cases of children with gastroenteritis in Bahonar hospital, Karaj, Iran, and PCR using species-specific primers (hipo for C. jejuni and asp for C. coli) and RFLP were carried out to differentiate the interfering species.The results of PCR using both genusspecific primers (cadf and 23S rRNA) revealed Campylobacter contamination in 20% of tested stool samples, which is relatively high rate compared to the results of most other published surveys, i.  (15).The higher frequency of Campylobacter gastroenteritis could be due to increased consumption of undercooked poultry products in recent years as well as the high sensitivity of molecular techniques in comparison with culture-based methods.Cadf, hipo, and asp genes have all been the targets of primers used in much independent research.Although these genes are reported to be highly conserved among isolates from food and clinical samples (9), according to the results of recent molecular surveys on Campylobacters (15,16) variation in the sequence of hipo and asp primer binding sites in Iranian isolates is probable, and the aim of this study was to test this hypothesis.Comparing the results of PCR and RFLP using AluI RE in the speciation of positive samples (Table 3) confirmed the above-mentioned hypothesis.It can be concluded that the lack of amplification of species-specific segments in two of seven C. jejuni isolates (detected based on jejuni species-specific electrophoretic pattern, yielding 202, 166, and 124 bp segments) is due to differences in the nucleotide sequence of the hipo primer Int J Enteric Pathog.2016; 4(2):e33867.binding site in these isolates compared with the standard strain.Another finding supporting the hypothesis is that increasing the annealing temperature using a gradient thermal cycler weakened the corresponding speciesspecific bands in some of the positive samples.
Both PCR (with hipo and asp primers) and RFLP results showed remarkable and high percentages of C. coli contamination in tested samples, which is in contrast to the results of most other studies.Of course, Wardak et al. (17) in Poland and Ronveaux et al. (18) in Belgium reported similar findings.High frequency of C. coli could be due to transmission of the bacterium from person to person as well as a common source of infection at the same time and in the same area.None of the Campylobacterpositive samples were identified as C. lari or C. upsaliensis using the RFLP method, which shows low contamination with these species in the cases of gastroenteritis in children.The results of this study showed a high percentage of Campylobacter contamination in the tested stool samples.The other surprising finding was the high rate of Campylobactercoli-positive samples.In addition, the difference between the results of PCR using species-specific primers (hipo and asp) and the RFLP method (electrophoretic patterns) in some of the positive samples confirms the hypothesis of variations in the nucleotide sequences of the hipo primer binding site in some Iranian isolates of Campylobacters.

Figure 1 .
Figure 1.PCR Amplification Products of Samples for Identification and Speciation of C. jejuni and C. coli

Table 3 .a
PCR and RFLP Results a , b Did not identify any C. lari and C. upsaliensis.b Values are expressed as No. (%).

Figure 2 .
Figure 2. Electrophoretic Patterns of Positive Samples Following Digestion With AluI

Figure 3 .
Figure 3. Electrophoretic Patterns of Positive Samples Following Digestion With TasI

Fermer
et al. (11) reported on the PCR-RFLP method using THERM1 and THERM4 primers and AluI and Tsp5091 restriction enzymes as a high-potential technique for detection and speciation of thermophilic Campylobacters (C.jejuni, C. coli, C. lari, and C. upsaliensis).Following molecular analysis of 118 Campylobacter isolates from human and veterinary clinical samples, they identified 95.5% of human isolates as C. jejuni, 3% as C. coli, and 1.5% as C. upsaliensis.Steinhauserova et al. (14) reported on the PCR-RFLP method using THERM1 and THERM4 primers and AluI and Tsp5091 restriction enzymes as a high-potential technique for detection and speciation of thermophilic Campylobacters (C.jejuni, C. coli, C. lari, and C. upsaliensis).Following molecular analysis of 204 Campylobacter isolates from human, dog, and pig clinical samples, they identified 64.4% of human isolates as C. jejuni and 35.6% as C. coli.

Table 1 .
List of Oligonucleotide Primers used for PCR

Table 2 .
Reagents and Thermal Conditions of PCR

Table 4 .
Campylobacter spp.Contamination of Stool Specimens in Different Age Groups

Table 5 .
Campylobacter spp.Contamination of Stool Specimens by Gender Groups a a Values are expressed as No. (%).