Phenotypic and genotypic identification of C. jejuni strains from fecal samples
Based on Cochran formula for calculating of sample size, a total of 3000 gastroenteritis cases were examined for suspected cases of sporadic campylobacteriosis. Gastroenteritis was characterized as abdominal pain with ≥ 3 episodes per day. Children with underlying gastrointestinal disease, physiologic diarrhea or history of antibiotic intake were excluded, which were defined by physicians in hospitals. Suspected cases of bacterial gastroenteritis were subjected to specimen collection, among which 280 cases accompanied WBC (white blood cell) and RBC (red blood cell) shedding in the majority of cases and were considered as suspected cases of campylobacteriosis.
Fecal specimens were collected from children with gastroenteritis, aged 0-5 years, referred to 3 Children’s Medical Center and Hospitals at Tehran, Iran, from May to October 2018. Information on age, clinical symptoms, history of non-pasteurized dairy products consumption, animal contact as well as laboratory results was recorded. Then specimens were transferred from laboratory of hospital to the laboratory of Tarbiat Modares University using Carry-Blair Transport Media (Micro Media-Hungary) and immediately streaked on Brucella agar and modified charcoal-cefoperazone-deoxycholate agar (mCCDA) (Merck-Germany). Plates were incubated at 42° C for 48 hours under microaerophilic condition using Gas Pack C (Merck-Germany). Gram staining, spiral morphology, catalase and oxidase production, nitrate reduction and indoxyl acetate hydrolysis test were used to confirm C. jejuni colonies. Also, hippurate hydrolysis test was used for phenotypic distinguishing of C. jejuni from C. coli from enteritis patients. Eventually, twenty C. jejuni and three C. coli strains were confirmed by Duplex PCR [13] and in the following, twenty C. jejuni was studied.
Twenty-three Campylobacter isolates were identified from 280 stool samples among which 20 and 3 isolates were C. jejuni and C. coli, respectively. All confirmed C. jejuni strains (n=20) were designated and used for further analyses.
Identification of capsular genotypes and LOS locus classes
The capsular genotypes of C. jejuni strains were identified using specific primers for most commonly found genotypes HS1, HS2, HS4c, HS19, HS23/36c and HS41 (Table 1) [4, 5].
Table 1
The primer sequences included in the C. jejuni capsule typing scheme
Primer | Forward sequence | Reverse sequence | Product size (bp) | Reference |
HS1 | GCAAGAGAAACATCTCGCCTA | TTGGCGGTAAGTTTTTGAAGA | 610 | [5] |
HS2 | CATCCTAGCACAACTCACTTCA | CAGCATTGGAGGATTTACAATATAT | 62 | [5] |
HS4A | CCTAACATATCATACACTACGGT | TATATTTGGTTAGGGATCCA | 370 | [5] |
HS19 | GGCAACAAACAAACATATTCAGA | CGAGGATGAAAATGCCTCAA | 450 | [5] |
HS23/36 | GCTTTATATCTATCCAGTCCATTATCA | GCTTGGGAGATGAATTTACCTTTA | 161 | [5] |
HS41 | TGCAATCTCTAAAGCCCAAG | CTTACATATGCTGGTAGAGATGATATG | 279 | [5] |
From 19 different LOS locus classes from A to S, 3 classes (A, B and C) play a key role in the biosynthesis of the sialic acid and are often found in isolates from the stools of patients with GBS [2, 6, 8]. Therefore, sets of primers specific for class A, B, and C classes were used for characterization of LOS identities (Table 2) [6].
Table 2
Primer used for identification of classes A, B and C
ORF | Forward sequence | Reverse sequence | Product size (bp) | Reference |
Orf7ab | ACTACACTTTAAAACATTTAATCC AAAATCA | CCATAAGCCTCACTAGAAGGTATGAGTATA | 580 | [7] |
Orf 6ab1 | CAAGGGCAATAGAAAGCTGTATCA | ACAAGCACTTCATTCTTAGTATTACAAAT | 631 | [7] |
Orf6ab2 | TCATCTTGCCAACTTATAATGTGGA | TCTAGCGATATTAAACCAACAGCCT | 517 | [7] |
Orf5bII | CTGTGATGATGGGAGTGAAGAGC | GGTAATCGTTTCGGCGGTATT | 539 | [7] |
Orf6c | GTAGTAGATGATTGTGGTAATGATAAA | ATAGAATTGCTATTTACATGCTGG | 554 | [7] |
Orf7c | TTGAAGATAGATATTTTGTGGGTAAA | CTTTAAGTAGTGTTTTATGTCACTTGG | 746 | [7] |
Genomic DNA was extracted using a genomic DNA extraction kit (GeNetBio, Korea), according to the manufacturer instructions. PCRs was carried out within a thermal cycler (Eppendorf, Germany) in a final volume of 25 μL containing 1-10 ng DNA template, 2.5 μL 10X PCR buffer, 1 unit of Taq DNA polymerase, 2.0 mM MgCl2, 0.2 μM of each primer, 0.3 mM each dNTP and sterile deionized water. Amplification conditions was as follow: 95°C for 5 min, followed by 30 amplification cycles including denaturation at 94°C for 1 min, annealing at 52°C for 1 min and extension at 72°C for 1 min. The reaction was ended with a final extension at 72°C for 5 min and followed by electrophoresis of amplicons on 1% agarose gel.
Real-Time PCR for dnaK gene expression in clinical C. jejuni isolates
Microbial DnaK is a bacterial conserved chaperone protein which has a sequence homology with human peripheral nerve HSP70 and its high level expression is supposed to be related to GBS promotion.
In order to assess the efficiency of real-time PCR amplification, five serial 1:10 dilutions of cDNA was used as template for qRT-PCR reaction of the dnaK and 16srRNA genes. The CTs values and the concentrations of the template were used to plot the standard curve and calculate the primer efficiency.
RNA extraction was performed on 20 C. jejuni strains, which were previously checked for the presence of capsular types and LOS locus classes using a Favoren Biotec Corp kit (Taiwan). Subsequently, the RNA molecules were treated using the DNase I kit (TaKaRa). A cDNA synthesis kit (Yekta Tajhiz Azma-Iran) was used to generate a single-strand cDNA. The cDNAs were kept at -20°C. Quantitative Real Time-PCR was performed using SYBR Green (RealQ Plus Master Mix Green-Denmark) in Qiagen-Rotor-Gene Q with HRM. 16S rRNA gene was used as the internal control. One of the isolates that neither had the selected capsular serotypes nor the LOS locus classes was considered as a reference gene. The PCR reaction mixture consisted of 100 ng to1 mg of cDNA (for dnaK and 16S rRNA genes), 1 mM of each primer (Table 3) [16] and 12.5 mL of SYBR Green I Master Mix. Cycling conditions included an initial denaturing step of 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C. 2-∆∆Ct is a relative quantification method for analyze the relative changes in gene expression from real-time quantitative PCR [16]. SPSS software version 20 was used for the analysis of data.
Table 3
Specific primers used for Real-Time PCR
Primer | Forward sequence | Reverse sequence | Product size (bp) | Reference |
dnaK | AAACGCCAAGCGGTAACTAA | TTCTTTAGCCGCGTCTTCAT | 90 | [15] |
16SrRNA | AAGGGCCATGATGACTTGACG | AGCGCAACCCACGTATTTAG | 107 | [15] |
Multilocus Sequence Typing (MLST)
The MLST method was performed according to Dingle, et al. [17]. Each 25 μL amplification reaction mixture comprised 1-10 ng DNA template, 1X PCR buffer, 1.25 unit of Taq DNA polymerase, 1.5 mM MgCl2, 1 μM of each primer (Table 4), 0.8 mM each dNTP and sterile deionized water.
Table 4
Primers used for Campylobacter jejuni MLST
Primer | Forward | Reverse | Product size (bp) | Reference |
asp | CCAACTGCAAGATGCTGTACC | TTAATTTGCGGTAATACCATC | 625 | [16] |
gln | CATGCAATCAATGAAGAAAC | TTCCATAAGCTCATATGAAC | 722 | [16] |
glt | GTGGCTATCCTATAGAGTGGC | CCAAAGCGCACCAATACCTG | 575 | [16] |
gly | AGCTAATCAAGGTGTTTATGCGG | AGGTGATTATCCGTTCCATCGC | 648 | [16] |
pgm | GGTTTTAGATGTGGCTCATG | TCCAGAATAGCGAAATAAGG | 700 | [16] |
tkt | GCTTAGCAGATATTTTAAGTG | ACTTCTTCACCCAAAGGTGCG | 691 | [16] |
unc | TGTTGCAATTGGTCAAAAGC | TGCCTCATCTAAATCACTAGC | 631 | [16] |
The PCR conditions were as follows: denaturation at 94 °C for 2 min; annealing at 50 °C for 1 min, extension at 72 °C for 1 min for 35 cycles. DNA Sequences of each housekeeping gene were submitted to C. jejuni MLST database (http://pubmlst.org/campylobacter) and the related allelic numbers, Sequence Types (ST) and Clonal Complexes (CC) were identified [18]. The Accession Numbers of DNA Sequences of 20 C. jejuni isolates were deposited in GenBank (https://pubmlst.org/campylobacter). Dendrogram was plotted using Interactive Tree Of Life (iTOL) v4 [19].
Geographic distribution of STs and CCs
A circular dendrogram was plotted for comparison of the peer sequences reported worldwide. A total of 72,392 isolates were downloaded from PubMLST website and analyzed [18]. Based on the inclusion and exclusion criteria, a total of 304 isolates were included in dendrogram. The inclusion criteria were: C. jejuni strains from the five recent years (2014-2018), C. jejuni strains from human stool, C. jejuni strains from the sporadic cases and gastroenteritis. The inclusion criteria were based on parameters that well warrant comparisons with our strains. Excluding criteria were unspecified ST or CC as well as repeated ST samples of each country.
The recorded MLST data in PubMLST database were also used to compare STs and CCs between our isolates with those from our neighbor countries. Among Iran neighboring countries only Turkey and Pakistan had recorded data about C. jejuni in PubMLST. Eleven isolates from human stool samples has been reported form these countries which were included for the final analysis. Dendrograms was plotted using Interactive Tree Of Life (iTOL) v4 [19]. Various sequence types were obtained from and plotted using PubMLST database tools [18].
Statistical analysis
To assess the presence of sialylated LOS classes A, B and C and CPS types in 20 C. jejuni strains isolated from children with gastroenteritis, we used the Cochran's Q test. Data were analyzed with the Statistical Package for Social Sciences (Version 25.0, SPSS Inc., Chicago, IL, USA); p<0.05 was considered statistically significant.
Table 6
Top 10 Clonal complexes based on frequency and diversity extracted from circle dendrogram
Rank | Clonal Complex | Diversity | Rank | Clonal Complex | Frequency |
1 | ST-353 Complex | 12 | 1 | ST-21 Complex | 51 |
2 | ST-21 Complex | 10 | 2 | ST-353 Complex | 42 |
3 | ST-206 Complex | 7 | 3 | ST-206 Complex | 17 |
4 | ST-48 Complex | 6 | 4 | ST-45 Complex | 17 |
5 | ST-464 Complex | 5 | 5 | ST-48 Complex | 14 |
6 | ST-52 Complex | 5 | 6 | ST-354 Complex | 13 |
7 | ST-574 Complex | 5 | 7 | ST-464 Complex | 13 |
8 | ST-607 Complex | 5 | 8 | ST-403 Complex | 10 |
9 | ST-354 Complex | 4 | 9 | ST-443 Complex | 10 |
10 | ST-1034 Complex | 3 | 10 | ST-52 Complex | 9 |