High prevalence of Campylobacter jejuni CC21 and CC257 clonal complexes in children with gastroenteritis in Iran: A global analysis

Aims: The aim of this study was to assess the distribution of GBS-related lipo-oligosaccharide (LOS) classes and capsular genotypes among Iranian clinical C. jejuni strains and to assess its relation with dnaK gene expression. Moreover, a comprehensive study of C. jejuni MLST-genotypes and global comparison with peer sequences worldwide was intended. Methods: Distribution of sialylated-LOS classes and specic capsular genotypes were investigated in C.jejuni of clinical origin. The expression of dnaK in C. jejuni strains was measured by Real-Time-PCR. MLST-genotyping was performed to investigate the clonal relationship of clinical C. jejuni strains and comparison with global sequences worldwide. Results: C. jejuni HS23/36c was the predominant genotype (45%), followed by HS2 (20%), and HS19 and HS4 (each 10%). A total of 80% of isolates were assigned to LOS class B and C. Higher expression level of dnaK gene was detected in strains with HS23/36c, HS2 and HS4 capsular genotypes and sialylated LOS classes B or C in this study. MLST analysis showed that isolates were highly diverse and represented 6 different sequence types and 3 clonal complexes. ST-21 and ST-257 complexes were dominants (75%) in our C.jejuni strains. The CC21 was the largest CC in our collection which is consistent with global C.jejuni strains worldwide. No new ST and no common ST with our neighbor countries was detected in this study. Conclusion: Global analysis of MLST results demonstrated that ST-50 (CC21) was widely distributed in different countries, while ST-19 (CC21) and ST-257 (CC257) was less ubiquitously spread. Overall, CC21 and CC353 complexes were most frequent and most widely distributed clonal complexes around the world; although, CC353 was not detected in this study. This shows a picture of movement of dominant Campylobacter strains worldwide. The occurrence of identical clonal complexes with different capsular types and LOS classes in this study is consistent with genetic variation in circulating identical genotypes and their evolution toward different pathotypes probably through acquisition of different genetic elements including LOS and CPS gene clusters. respectively based on hippurate hydrolysis test and duplex PCR assay of cadF gene. The 20 of 23 isolates were capable to hydrolyze hippurate and identied as C. jejuni. Analysis of the duplex PCR assay of cadF gene showed that 737 and 461 bp amplicons were corresponding to C. jejuni and C. coli, respectively. HS4, HS19 and HS23/36c are usually identied in C. jejuni strains isolated from GBS patients [2, 21]. In our enteritis-related samples 85% of strains expressed one of HS23/36c, HS2, HS4 and HS19, which signies the high probability of GBS progress in this group of patients. ndings showed that the dnaK expression mean in strains with specied capsular genotype and sialylated LOS was greater than that of others (with either capsular genotype or LOS class). This nding reveals that the expression of dnaK higher in strains when sialylated LOS with particular capsular genotypes simultaneously are present in a strain. Global analysis of MLST results demonstrated that ST-50 (CC21) was widely distributed in different countries, while ST-19 (CC21) and ST-257 (CC257) was less ubiquitously spread. This study shows a picture of movement of Campylobacter strains around the world. The occurrence of identical clonal complexes with different capsular types and LOS classes in this study is consistent with genetic variation in circulating identical genotypes and their evolution toward different pathotypes probably through acquisition of different genetic elements including LOS and CPS gene clusters.


Introduction
Campylobacter jejuni (C. jejuni) is a leading cause of bacterial foodborne poisoning and acute gastroenteritis in human worldwide [1]. This bacterium often causes a moderate to severe watery regularly self-limiting diarrhea and post-infectious immune disorders such as Guillain-Barre syndrome (GBS) [2].
Campylobacter jejuni (C. jejuni) produces Capsular Polysaccharide (CPS). The CPS gene cluster is located in a hypervariable region in the C. jejuni genome. Diversity in the content and gene function together with the presence of phase variable genes in the capsule locus allows the production of broad repertoire of capsular structures [2,3]. Major serodeterminant of the CPS is in classical Penner or Heat-Stable (HS) serotyping Scheme. In the classical Penner serotyping scheme, which is based on the passive slide hemagglutination assay, C. jejuni strains are divided into 47 serotypes, of which, due to similarity in the CPS structure, 35 serotypes have been re ned, which are serotype cross-reactive pairs or complexes [4,5]. 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 [4]. Recently, CPS genotyping was used as a more effective method, since it is not sensitive to variations in capsule gene expression either in uenced by genes or gene products outside the capsule locus [2], and also it can rapidly and readily determine CPS types in C. jejuni [2,6].
The cluster of genes involved in C. jejuni Lipo-Oligosaccharide (LOS) biosynthesis, is one of the most variable regions of C. jejuni genome [7]. Among 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].
Post-infection diseases like GBS with C. jejuni have been proved to be associated with antibodies of human gangliosides. The induction of these autoantibodies is associated with molecular mimicry between human gangliosides and bacterial epitopes present at the surface of the Lipo-Oligosaccharide (LOS) [9] and the antibodies response to diarrhea is different to GBS infected by C. jejuni [10]. In addition to antiganglioside antibodies, Heat Shock Proteins (HSP) family can mediate in the autoimmune diseases. They belong to a highly protected family that is present in normal physiological conditions in prokaryotic and eukaryotic cells. These proteins are etiologic factors in many autoimmune diseases in such a way that their overexpression leads to environmental stress induction [9,11].
C. jejuni carry several of HSPs, including groELS, dnaJ, dnaK and lon [12] among which DnaK proteins (70 kDa) has a high homology in its sequence with HSP70 of human peripheral neurons [9]. A high titer of anti-HSP antibody (HSP27, HSP60 and HSP70) can be found in CSF (Cerebrospinal uid) of patients with GBS [9].
To date, no study has been reported on the distribution of CPS genotypes, LOS locus classes and Multilocus sequence typing (MLST) of C. jejuni in Iran. The aim of this study was to assess, for the rst time, the distribution of the most commonly found GBS-related LOS classes and capsular genotypes among clinical C. jejuni strains isolated from Iranian children. Moreover, the correlation of DnaK protein expression level in C. jejuni strains with selected capsular genotypes and LOS classes associated with GBS was intended. Furthermore, a comprehensive comparison of C. jejuni MLST genotypes with globally reported peer sequences worldwide was envisioned.

Materials And Methods
Phenotypic and genotypic identi cation 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 de ned 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 modi ed 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 con rm 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 con rmed by Duplex PCR [13].

Identi cation 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 identi ed in this study according to standard protocols by speci c primers (Table 1) [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, speci c set of primers ( Table 2) were used for class A, B, and C based on the DNA sequences of speci c genes related to LOS classes [7]. In order to assess the e ciency of real-time PCR ampli cation, ve 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 e ciency was measured. The main reason for calculating the e ciency 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 quanti cation 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.   [17].

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][9][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). isolates. We found no instance of class A in our collection of isolates.
Comparison of dnaK gene expression in clinical C. jejuni strains The + 3.3 ± 10% slope is a re ection of 100%±10% e ciency of real time PCR. Accordingly, the slope was − 3.38 and 3.24 and the e ciency was 97.51% and 103.54 for dnaK and 16srRNA genes, respectively. The dnaK gene expression was determined by Quantitative Real-Time PCR and according to the 2^( − ΔΔCT) method. Among isolates that showed one of the LOS classes of A-C or one of six selected capsular genotypes, 18 isolates were classi ed into three groups including group 1: with identi ed CPS genotype and sialylated LOS class (B or C) (n = 15), group 2: with identi ed CPS genotype and without sialylated LOS class (n = 2), group 3: without CPS genotype but with sialylated LOS class (n = 1), and one of the isolates that neither had the selected capsular serotypes nor the LOS locus classes was considered as a reference strain in this study.
Due to insu ciency of data, differential expression analysis could not be performed; thus, descriptive statistics was used instead. As a result, dnaK expression level in group 1 was greater than other groups ( Table 5). The dnaK expression level was much higher in clinical C. jejuni isolates with one of the CPS genotypes and the LOS classes relevant to GBS patients.
Based on the 2^ (− ΔΔCT) method, the graph of the fold change of dnaK gene expression was plotted (Fig. 2). Out of the 3 identi ed clonal complexes, ST-21 clonal complex dominated in 10 isolates (50%).
Distribution of the sequence types and the genetic link between C. jejuni strains isolated from patients with diarrhea has been shown in the dendrogram (Fig. 3). The relationship among 20 isolates based on clonal complexes is re ected in minimum spanning tree diagram (Fig. 4) [19].

Linkage between CPS genotypes and LOS class with MLST CC
Totally, 3 CCs (CC828, CC257 and CC21) were identi ed. The isolates with HS23/36c CPS and HS19 CPS genotype, were found in CC21 (Fig. 5). Majority of isolates in CC21 had LOS class B or LOS class C. In CC257, 4 isolates belonged to HS2 serotype. In this CC, LOS class C and B were observed. Two isolates were assigned to CC828 (Fig. 6).

Discussion
Epidemiological investigations have shown that a C. jejuni infection precedes GBS in 20 to 50% of cases in Europe, North and South America, Japan, and Australia [20]. Sialylated LOS loci of A, B and C classes as well as HS types of HS2, HS4, HS23/36c and HS19 are accused to be associated with GBS patients [2,20,21]. In the present study we aimed to investigate CPS types and LOS locus classes of virulent C. jejuni strains isolated from children in Iran. Among 20 isolates, the prevalent LOS class was B (11/20; 55%), followed by class C (5/20; 25%), while class A was not detected in our collection. Dominant CPS genotype was HS23/36c (9/20) followed by HS2 (4/20). The HS23/36c was not dominant in any of the reported studies and at the global picture, HS4 is the most prevalent type [22], though it was ranked no. 3 in Iran. Similar to our study, HS2 has been reported as the second most prevalent CPS type worldwide [22], while some reports rank it no. 6 in Asia and Africa and no. 7 in some developing countries [22].
A wide range of CPS types including HS1/44, HS2, HS4, HS19 and HS23/36c are usually identi ed in C. jejuni strains isolated from GBS patients [2,21]. In our enteritis-related samples 85% of strains expressed one of HS23/36c, HS2, HS4 and HS19, which signi es the high probability of GBS progress in this group of patients.
Our ndings showed that the dnaK expression mean in strains with speci ed capsular genotype and sialylated LOS was greater than that of others (with either capsular genotype or LOS class). This nding reveals that the expression of dnaK was higher in strains when sialylated LOS with particular capsular genotypes simultaneously are present in a strain.
Moreover, it was shown by HU et al., that dnaK gene expression is upregulated under in-vivo-like conditions which means it may be induced in infected human host. Considering the crucial role of dnaK in antigenic mimicry and GBS, it can be concluded that individuals infected with C. jejuni strains that have sialylated LOS classes and the selected capsular serotypes as well as a high expression pro le of dnaK may be more likely to develop GBS. Furthermore, dnaK gene can be mentioned as an antigen candidate in preventive studies or as a diagnostic marker [10].
In this study, genetic variations in 20 C. jejuni strains was also identi ed by MLST. A total of 6 STs were observed and 17/20 (85%) belonged to 3 clonal complexes (CCs), while 3 isolates belonged to STs unassigned to a CC.
The majority of C. jejuni strains were assigned to CC21; this nding supports previous observations which shows CC21 is the most prevalent CC worldwide. Within CC21, ST-50 was the dominant ST in our clinical samples, although not all of previous studies reported ST-50 as the dominant sequence type [23][24][25][26][27].
Meanwhile, consistent with our nding, ST-50 and ST-19 (CC21) and ST-257 were mostly related to human campylobacteriosis cases [26,28]. However, isolates from other sources (fresh whole retail chicken, raw milk and environmental water) also presented CC21 as dominant CCs [29].
The correlation between certain MLST clonal complexes and LOS classes and HS types were investigated in present study.
The majority of C. jejuni isolates in ST-21 (

Conclusions
For the rst time this study presents MLST typing as well as identifying CPS types and sialylated LOS classes of clinical C. jejuni strains isolated from Tehran, Iran.
In conclusion, the present study demonstrated that C. jejuni isolates had the predominant LOS class (B and C) and capsular genotypes (HS23/36, HS2, HS4 and HS19) which are supposed to be related to GBS; Isolates from this study were highly Bita Bakhshi designed and supervised the study entirely and had a major contribution in writing the manuscript and had approved the submitted version.
Shahin Najar Peeraeyeh had has a major role in analysis of the data as well as drafting the main manuscript and had approved the submitted version.
Ethics approval and consent to participate The study was reviewed and approved by the Medical Ethics Committee of Tarbiat Modares University (Code: IR.MODARES.REC) before it began and all research was performed in accordance with relevant guidelines/regulations. The clinical specimens were obtained from Microbiology Laboratory of 3 children hospitals, Tehran, Iran. The consent to participate was obtained from the parents/guardians of the minors included in this study and the data were analyzed anonymously.

Consent for publication
Not applicable.

Availability of data and materials
The datasets of the current study are available within article or can be obtained from corresponding upon request. DNA sequences of genes that have been deposited in GenBank are available in https://pubmlst.org/ campylobacter/.

Competing interests
The authors declare that they have no competing interests or personal relationships that could have in uenced the work reported in this paper.

Funding
None to declare. Figure 1 Relationship between presence of CPS type genes and LOS locus class genes.

Figure 2
Level of dnaK gene expression in clinical C. jejuni isolates of different groups.  Minimum spanning tree for categorical data (based on clonal complexes) The tree was created using GrapeTree [19]. Each clonal complex is represented by a circle, numbers in each circle related to STs. the number of isolates is shown in brackets.

Figure 5
Linkage between CPS genotypes and MLST CC.     Geographic distribution of ST-257 for 2,689Campylobacter isolates (C. jejuni (100.0%) in the world, 1990-2018. Note: The designations employed and the presentation of the material on this map do not imply the expression of any opinion whatsoever on the part of Research Square concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. This map has been provided by the authors.

Figure 11
Phylogenetic analysis of 304 C. jejuni isolates worldwide from PubMLST database. *Dendrogran plotted by Interactive Tree Of Life (iTOL) v4 [18]. White triangle: Countries with similar CC21, yellow triangle: Countries with CC828 and green triangle: Countries with similar CC257 with Iran.