Phenotypic and genotypic identification of C. jejuni strains from fecal samples
Two-hundred and eighty fecal specimens collected from children with diarrhea 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 were recorded. A total of 280 suspected cases of sporadic Campylobacteriosis without history of antibiotic intake were selected from approximately 3000 diarrheal cases. Diarrhea was defined as ≥ 3 episodes per day, accompanied with WBC (white blood cell) and RBC (red blood cell) shedding in a majority of cases. Specimens were transferred to the laboratory 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. Finally, twenty C. jejuni and three C. coli strains were confirmed by Duplex PCR [13].
Identification Of Capsular Genotypes And Los Locus Classes
Since the C. jejuni Penner serotypes associated with Guillain-Barre syndrome (GBS) often belong to HS1, HS4c, HS19, HS23/36c and HS41, furthermore, the most common serotypes among sporadic cases are reported in the HS4c, HS2 and HS1 cases [4], the capsular genotypes including HS1, HS2, HS4, HS19, HS23/36c and HS41, PCR were identified in this study according to standard protocols by specific primers (Table 1) [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 isolated from the stools of patients with GBS [2, 7, 8]. Therefore, specific set of primers (Table 2) were used for class A, B, and C based on the DNA sequences of specific genes related to LOS classes [7].
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] |
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; denaturation at 94 °C for 1 min, annealing at 52 °C for 1 min, and extension at 72 °C for 1 min. Finally, an additional extension step (5 min, 72 °C) was performed. Finally, amplicons were electrophoresed on 1% agarose gel.
Real-Time PCR for dnaK gene expression in clinical C. jejuni isolates
Determination Of Real-time Pcr Primer Efficiency
In order to assess the efficiency of real-time PCR amplification, five serial template dilution of 1:10 served as DNA 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. In the next step, based on the slope of the standard curve, the primer efficiency was measured. The main reason for calculating the efficiency of primers is to accurately calculate the fold change, which is the output of qRT-PCR reaction [14].
Real-Time PCR for dnaK gene expression
RNA extraction was performed on pure cultures of 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 primers (Table 3) [15] 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 methods for analyze the relative changes in gene expression from real-time quantitative PCR [15]. 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 relies on the sequence analysis of seven housekeeping genes. The MLST method was performed according to Dingle, et al. [16]. 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 primers (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. Allelic numbers were identified for each of the housekeeping genes, Sequence Types (ST), and Clonal Complexes (CC) with submitted DNA Sequences in the C. jejuni MLST database (http://pubmlst.org/campylobacter) [17]. Dendrogram was plotted using Interactive Tree Of Life (iTOL) v4 [18].
Geographic Distribution Of Sts And Ccs
A phylogenetic analysis revealed that there was no common STs or CCs between Iran and its neighbor countries, Turkey and Pakistan (Fig. 7). This may be due to scarcity of published data from these countries. No data is available from other neighboring countries of Iran. World map related to C. jejuni strains from 1980 to 2018 shows the distribution of ST19 (CC21), ST50 (CC21), and ST257 (CC257) among different continents (Figs. 8–10). The frequency of CC21 strains was associated to sporadic cases of human gastroenteritis which was recorded from America (USA and Canada), Europe (UK, The Netherlands and Germany), Asia (Iran, this study) and Australia. Global analysis of CC257 (including ST-257) strains in the same time period showed recorded strains from Asia (Iran, this study), Europe (UK and Spain), America (Chile) and Africa (South Africa).
In a dendrogram constructed by global analysis of MLST results during 2014–2018 a similar picture was depicted for distribution of CC21 and CC257 clonal complexes. Moreover, neighbor-joining results indicated that CC21 and CC353 complexes are the most divers, most frequent and most widely distributed clonal complexes around the world, respectively; although, CC353 was not detected in this study (Fig. 11) (Table 6).
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 |