Genetic Analysis and Detection of fliCH1 and fliCH12 Genes Coding for Serologically Closely Related Flagellar Antigens in Human and Animal Pathogenic Escherichia coli

The E. coli flagellar types H1 and H12 show a high serological cross-reactivity and molecular serotyping appears an advantageous method to establish a clear discrimination between these flagellar types. Analysis of fliCH1 and fliCH12 gene sequences showed that they were 97.5% identical at the nucleotide level. Because of this high degree of homology we developed a two-step real-time PCR detection procedure for reliable discrimination of H1 and H12 flagellar types in E. coli. In the first step, a real-time PCR assay for common detection of both fliCH1 and fliCH12 genes is used, followed in a second step by real-time PCR assays for specific detection of fliCH1 and fliCH12, respectively. The real-time PCR for common detection of fliCH1 and fliCH12 demonstrated 100% sensitivity and specificity as it reacted with all tested E. coli H1 and H12 strains and not with any of the reference strains encoding all the other 51 flagellar antigens. The fliCH1 and fliCH12 gene specific assays detected all E. coli H1 and all E. coli H12 strains, respectively (100% sensitivity). However, both assays showed cross-reactions with some flagellar type reference strains different from H1 and H12. The real-time PCR assays developed in this study can be used in combination for the detection and identification of E. coli H1 and H12 strains isolated from different sources.


INTRODUCTION
Strains belonging to the species of Escherichia coli are ubiquitous as commensals in the gut of humans and warm-blooded animals. Apart from their role as beneficial microbes, some E. coli strains are known to behave as human and animal pathogens, causing a wide spectrum of extraintestinal and enteric diseases, with urinary tract infection and diarrhea as most frequent (Kaper et al., 2004;Stenutz et al., 2006). Pathogenic and apathogenic E. coli cannot be discerned from each other by their morphology, cultural properties or fermentation reactions. As a consequence, serotyping is used since the 1940s as a diagnostic tool for identification of animal and human pathogenic E. coli strains (Orskov and Orskov, 1984).
E. coli serogroups are commonly defined by the antigenic properties of the lipopolysaccharide which is part of the outer membrane (O-antigen) (Stenutz et al., 2006). Motile E. coli strains can be additionally typed for their flagellar filaments (H-antigen) (Orskov and Orskov, 1984). E. coli O-and H-antisera are usually produced by immunization of rabbits with respective reference strains (Orskov and Orskov, 1984;Edwards and Ewing, 1986). At present, 182 O-antigens and 53 H-antigens have been described (Scheutz et al., 2004;Scheutz and Strockbine, 2005). The resulting O:H serotype (for example O157:H7) is commonly used for describing E. coli isolates (Bettelheim, 1978;Orskov and Orskov, 1984).
Complete serotyping of E. coli is laborious and timeconsuming and performed only in a few specialized reference laboratories worldwide. Moreover, cross-reactivity which is observed between some E. coli O-groups and H-types can complicate the interpretation of serotyping results. Last but not least, serotyping fails if autoagglutinating (O-antigen or Hantigen rough) and non-motile (NM) E. coli strains have to be examined (Orskov and Orskov, 1984;Edwards and Ewing, 1986). For these reasons, attempts were made to substitute serotyping by molecular typing of O-antigen and H-antigen encoding genes.
In the recent years, the nucleotide sequences of all known O and H-antigen genes in E. coli have been elucidated (Wang et al., 2003;Iguchi et al., 2015a). Molecular methods such as PCR and nucleotide sequencing have been successfully employed for typing of O-and H-antigen genes in E. coli (Beutin and Fach, 2014;Joensen et al., 2015;Iguchi et al., 2015b). Molecular serotyping was shown to be specific and sensitive and can substitute conventional serological detection of E. coli surface antigens (Bugarel et al., 2010;Fratamico et al., 2011;Clotilde et al., 2015;Iguchi et al., 2015b;Joensen et al., 2015). In contrast to serotyping, molecular detection of O-and H-antigen genes is easier and faster to perform and O-rough and non-motile strains can be typed on the basis of their O-and H-antigen genes (Beutin and Fach, 2014;Joensen et al., 2015).
We have previously investigated the genetic variability of flagellar types H19, H25 and H28 in E. coli (Beutin et al., 2015a,b). These flagellar types are widespread in strains belonging to numerous O-serogroups but are also associated with enterohemorrhagic E. coli O145:H25, O145:H28, and O121:H19 strains. By nucleotide sequence analysis of fliC (flagellin) genes encoding H19, H25, and H28 flagella we have observed a high genetic variability among fliC H19 , flic H25, and fliC H28 alleles, respectively. To some part, this sequence alterations were associated with some O-groups of strains which allowed the development of real-time PCR protocols for specific typing of flagellar variants encoded by enterohemorrhagic E. coli O145:H25, O145:H28, and O121:H19 strains (Beutin et al., 2015a,b). Such real-time PCR protocols were found useful for improvement of horizontal real-time PCR detection methods for EHEC from food samples (Beutin et al., 2015a,b).
In this work, we compared E. coli fliC genes that encode flagellar types H1 and H12. These flagellar types show a high serological cross-reactivity and cross-absorbed H1 and H12 antisera are used for definite H-typing (Orskov and Orskov, 1984;Edwards and Ewing, 1986). Moreover, three subtypes of H1 were detected by serological typing using factor specific antisera (Ratiner et al., 1995). Serological cross reactions may cause confounding results in diagnostic laboratories where absorbed antisera are not available. The development of molecular typing procedures for reliable detection of H1 and H12 flagellar types could overcome this specific problem.
In this work we have analyzed the nucleotide sequences of E. coli H1 and H12 strains in order to detect characteristic fliC sequence alterations corresponding with these closely related H-types. Subsequently, we have developed a real-time PCR procedure for reliable discrimination of H1 and H12 flagellar types in E. coli. The protocol should be useful for diagnostic and epidemiological investigations of human and animal pathogenic strains of E. coli.

Bacteria
E. coli strains used in this study were derived from the collections of the National Reference Laboratory for E. coli (NRL E. coli) at the Federal Institute for Risk Assessment (BfR) in Berlin, Germany and from the French Agency for Food, Environmental and Occupational Health and Safety (Anses) in Maisons-Alfort, France. E. coli strains used for specificity study included in particular the E. coli reference strains belonging to serogroups O1-O181 and H-types H1-H56 (Orskov and Orskov, 1984;Edwards and Ewing, 1986). All strains have been previously described for their serotypes and for virulence genes associated with STEC (Beutin et al., 2015a,b). All strains were grown overnight at 37 • C in Luria broth, and DNA was extracted according to manufacturers instructions using InstaGene matrix (BioRad laboratories, Marnes-La-Coquette, France).
Real-time PCR assays were performed with an ABI 7500 instrument (Applied Biosystems, Foster City, CA, USA) in 25µl reaction volumes, a LightCycler Nano (Roche Diagnostics, Meylan, France) in 10 µl reaction volumes or with a LightCycler 1536 (Roche Diagnostics, Meylan, France) in 1.5-µl reaction volumes according to the recommendations of the suppliers. Primers and TaqMan probes were used at 300 nM final concentrations. The following thermal profile was applied to all instruments: enzyme activation at 95 • C for 1-10 min as recommended followed by 40 cycles of denaturation at 95 • C and annealing at 60 • C.

PCR Detection and Mapping of E. coli O-Antigen and H-Antigen Genes
Mapping of fliC gene variants to their respective H-types was performed as previously described (Beutin et al., 2015a,b). Nucleotide sequence data obtained from thirteen fliC H1 and eight fliC H12 genes were used for designing TaqMan R real-time PCR probes and XS probes (minor groove binder replacement, Biolegio, Nijmegen, The Netherlands) and primers for specific detection of all genetic variants of thirteen fliC H1 and eight fliC H12 genes (this work). Real-time PCR probes and primers used in this work were designed with the software Primer Express V3.0 (Applied Biosystems) and are described in Table 1.

Nucleotide Sequencing
The nucleotide sequence of the PCR products were determined as described (Beutin et al., 2015b) and analyzed with the Accelrys DS Gene software package (Accelrys Inc., USA). The nucleotide sequences of the respective products for fliC homologs were determined and have been submitted to European Nucleotide Archive (ENA). The GenBank Accession numbers are listed in Table 2.

Sources and Properties of E. coli H1 and H12 Strains
The E. coli H1 and H12 strains investigated in this study were from human, animal, food, and environmental sources ( Table 3). The thirty-one flagellar type H1 strains were associated with 10 different E. coli O-serogroups, O-rough and O-untypable strains and originated from healthy and diseased humans and animals and from food. The thirty-eight H12 strains divided into thirteen different O-groups of E. coli, and in O-untypable and O-rough strains. The H12 strains were from healthy and diseased humans and animals, from food and the environment. Production of Shiga-toxins (Stx) was found in 16 (42.1%) of the H12 strains and associated with five different O-groups. Fourteen (45.2%) of the E. coli H1 strains produced Stx, however most of these were from pigs with edema disease (O139:H1, Or:H1) and harbored the stx2e gene. O:H types known to be associated with E. coli causing extraintestinal infections of humans (O2:H1, O4:H1, O6:H1, O25:H1) were detected among the investigated H1 strains. Interestingly, strains belonging to these serotypes originated not only from humans but also from animals and food. Certain strains belonged to serotypes which have not been previously associated with clinical disease and their role of pathogens for humans and animals is not yet known.   Table 2.
Nucleotide Analysis of E. coli fliC H1 and fliC H12 Genes The nucleotide sequences of the reference strains (Orskov and Orskov, 1984) for E. coli flagellar antigens H1 (strain Su1242, GenBank accession AB028471.1) and H12 (Bi 316-42, GenBank accession AY249997) (Wang et al., 2003) have been published previously. The length of coding sequence of each fliC H1 and fliC H12 gene is 1788 nucleotides and both sequences have 97.5% identity (44 nucleotide exchanges) at the nucleotide level and 98.98% identity and 99.16% similarity at the amino acid level (7 amino acids (aa) exchanges). Additional fliC nucleotide sequences from six E. coli H1 and five E. coli H12 strains were obtained in this work ( Table 2). These sequences were compared with seven fliC H1 sequences and three fliC H12 sequences already available in GenBank ( Table 2). All 21 H1 or H12 flagellin genes have a 1788 nucleotides length that codes for 595 amino acid residues.
A cluster analysis performed with thirteen fliC H1 and eight fliC H12 sequences is shown in Figure 1. Four different genotypes were detected among the thirteen fliC H1 strains. Uropathogenic E. coli O2:H1, O6:H1, O25:H1, and AIEC O83:H1 strains were identical for their fliC H1 sequences and assigned to a large cluster composed by eight strains. A smaller cluster was formed by five fliC H1 strains; four of these were Stx2e producing O139:H1 causing edema disease in pigs.
Six different genotypes were found among the eight fliC H12 strains. Identical fliC H12 sequences were only found between two O9:K9:H12 strains and each one O55:H12 and O153:H12 strain, respectively.  (Zdziarski et al., 2010). The O-serogroup of this strain was not reported but its wzx gene (position 2372093-2373353) is >99% similar to wzx of E. coli O25 strains E47a (GenBank GU014554.1) (Wang et al., 2010). Therefore, we classified ABU 83972 here as an O25:H1 strain. b The whole genome sequence of E. coli strain LF82 is available (GenBank: CU651637.1). The O-serogroup of this strain is not reported but its wzx gene (position 2127428-212804) is identical to the wzx gene of E. coli O83:H31 strain H17a GenBank: KJ778808.1 (unpublished) and of E. coli O83:H1 strain NRG857c (GenBank: CP001855.1) . Therefore, we suggest that LF82 is an O83:H1 strain. c The fliC sequence deposited under GenBank JF308285 is derived from strain EC614 reported as O157:H1 (Goulter et al., 2010). By Blast search, it is 100% identical to the fliC sequence of the H1 reference strain Su1242 (GenBank accession AY249997). Therefore, the flagellar type of EC614 was classified as H1. d Multiresistant, extended-spectrum-lactamase (ESBL)-producing E. coli from healthy human carrier. e The fliC sequence was determined in this study.

Amino acid Alterations between Flagellar Antigens H1 and H12 in E. coli Strains
An alignment of the amino acid sequences of thirteen fliC H1 and eight fliC H12 strains is shown in Table S1. All translation products had a length of 595 amino acids (aa). The eight fliC H12 strains were showing only few alterations with one or more of the strains at aa positions 249, 258, 339, and 472 (99.2% similarity) (Table S1), generating six different protein sequences (Figure 2). The thirteen fliC H1 strains split into three protein sequences (Figure 2) differing at positions 258, 431, and 481 (99.5% similarity) ( Table S1). The aa changes were all located in the variable region of fliC encoding flagellar antigen specificity (Wang et al., 2003). Differences in the aa sequence which could distinguish between all investigated fliC H1 and fliC H12 strains, respectively, were found at positions 302 (Glu/Lys), 340 (Asn/Lys), 361 (Gly/Asp), 391 (Thr/Lys), 396 (Asn/Asp), and 430 (Asn/Lys). The six flagellar type specific aa sequence differences were all located in the variable region of the fliC H1 and fliC H12 genes.

Development and Evaluation of Real-Time PCR Assays for Identification of E. coli fliC H1 and fliC H12 Strains
The close similarity between E. coli fliC H1 and fliC H12 translation products explains the serological cross-reactivity which was previously described for H1 and H12 antigens (Orskov and Orskov, 1984;Edwards and Ewing, 1986). As specific differences were found that distinguish between fliC H1 and fliC H12 sequences, molecular detection of the respective fliC genes could be more suitable than serotyping for clear identification of H1 and H12 strains of E. coli. Based on the sequence data obtained for E. coli fliC H1 and fliC H12 genes we developed a TaqMan real-time PCR assay for common detection of fliC H1 and fliC H12 genes as well as FIGURE 1 | Genetic relationships between fliC H1 and fliC H12 genes in different strains and serotypes of E. coli. Cluster analysis was performed using eight fliC H12 and thirteen fliC H1 genes listed in Table 2. GenBank accession numbers are indicated for orientation. The unweighted-pair group method using average linkages was used as a tree-building mode, and the distances were calculated according to Tajima and Nei (1984) using the Accelrys DS Gene software package.
FIGURE 2 | Genetic relationships between translation products of thirteen fliC H1 and eight fliC H12 genes in different strains and serotypes of E. coli listed in Table 2. GenBank accession numbers are indicated for orientation. The neighbor joining method with absolute differences (best tree) was used as a tree-building mode (Nei, 1996) using the Accelrys DS Gene software package.
real-time PCR assays for specific detection of fliC H1 and fliC H12 , respectively ( Table 1). Short-length XS-probes (minor groove binder replacement) had to be employed to develop real-time PCR assays specific for fliC H1 and fliC H12 sequences ( Table 1). We used two nucleotide substitutions between the sequences of fliC H1 and fliC H12 to design specific probes ( Table 1).
The assays were first tested for sensitivity and specificity on 31 E. coli H1 and 38 E. coli H12 strains ( Table 4) as well as on the E. coli H-type reference strains (H1-H56) (Orskov and Orskov, 1984;Edwards and Ewing, 1986). The real-time PCR for common detection of fliC H1 and fliC H12 reacted with all tested E. coli H1 and H12 strains ( Table 4) and not with any of the reference strains encoding all other flagellar antigens than H1 and H12.
The fliC H1 and fliC H12 gene specific assays detected all E. coli H1 and all E. coli H12 strains, respectively (Table 4). However, both assays showed cross-reactions with some flagellar type reference strains different from H1 and H12. With the fliC H1 realtime PCR, cross-reactions were observed with H6, H7, H15, H20, H34, H37, H41, H45, H46, H49, and H52 strains. The fliC H12 specific PCR reacted also with H7, H28, H31, and H41 strains ( Table 5). Although the overall sequences of the fliC genes of H-types cross-reacting with the fliC H1 and fliC H12 real-time PCR are widely different from those of fliC H1 and fliC H12 , they show high local similarities with the primers and probes sequences. In cases of cross reactivity, no or only minor differences (0-3 mismatches) were found between target-sequences and fliC H1  and fliC H12 , primers and probes ( Table 5). Three and more mismatches were found in cases of negative real-time PCR results. In respect to these findings, the assays were then tested on a second panel of 78 strains comprising strains with H-types previously found to cross-react with FlicH1 or FlicH12 PCR assays as well as strains from O-groups that can be found associated with H1 and H12, but with H-types different from H1 and H12 ( Table 6).
Overall, molecular typing of E. coli H1 and H12 strains requires first identification of H1/H12 strains with the common fliC H1 /fliC H12 real-time PCR assay, followed by specific identification of fliC H1 and fliC H12 , by their respective real-time PCR-assays. The real-time PCR for common detection of fliC H1 and fliC H12 was found 100% sensitive and 100% specific. The fliC H1 and fliC H12 gene specific assays were found 100% sensitive as they detected all E. coli H1 and all E. coli H12 strains, respectively. When used exclusively on H1 and H12 strains (as identified by the common primers/probe set in a first step), the fliC H1 and fliC H12 gene specific assays were found 100% specific. Thus, 100% of H1 and H12 strains would be accurately typed with this system.

DISCUSSION
The genetically and serologically closely related flagellar antigens H1 and H12 were found in heterogeneous types of E. coli strains belonging to 26 different O-serogroups, O-untypable and Orough strains. With one exception (O79:H1 and O79:H12), H1 and H12 strains did not share common O-serogroups which would indicate that flagellar types H1 and H12 have separated from each other not very recently in evolution. They may have evolved independently following rearrangements in the O-group loci of ancestor strains carrying the closely related H1/H12 flagellar types and do not directly derive from a common O-group ancestor.
By comparing nucleotide sequences of fliC genes from thirteen H1 and eight H12 strains we identified six H-type specific aa changes at positions 302, 340, 361, 391, 396, and 430. All these are located in the variable part of flagellin determining antigen specificity (Wang et al., 2003). As these changes are characteristic for the respective flagellar antigen, we suppose them to determine the antigen specificities of H1 and H12. The few other aa changes detected in some H1 and H12 strains might thus not be significant as specific characteristics of H1 or H12 types. However, such aa-changes could explain the finding of serological subtypes of H1 which were detected using factor specific H-antisera (Ratiner et al., 1995).
The presence of allelic subtypes within fliC genes encoding different H-types of E. coli and the finding that different flagellar types are serologically cross-reacting may complicate E. coli strain typing using H-antisera. The use of molecular typing procedures, such as real-time PCR can solve the typing problem caused by serologically closely related H-antigens, as we have shown for H1 and H12 in this work. Using primer express V3.0 software, it was not possible to design real-time PCRs specific exclusively for fliC H1 and fliC H12 , respectively. For this reason, we employed a two-step real-time detection procedure. The first step uses a real-time PCR highly specific for both H1 and H12  strains, followed by subtyping of H1/H12-positive strains with the respective fliC H1 and fliC H12 specific real-time PCRs. Short probe sequence lengths as obtained with minor groove binder (MGB) or MGB-replacements (XS-probe) are needed to ensure specificity between closely similar DNA-targets as previously shown for fliC H19 allelic discrimination (Beutin et al., 2015a). The PCRs could be used in parallel for examination of large number of isolates using high throughput PCR platforms as described previously for analysis of large numbers of Clostridia and E. coli strains Woudstra et al., 2013). Unambiguous typing of fliC H1 and fliC H12 sequences is of interest for clinical and epidemiological investigations since some H1 and H12 strains were shown to play a role as pathogens in humans and animals.
The specific molecular detection of H1 and H12 flagellins as described in this study will be useful for diagnosis and for source attribution of human and animal pathogenic ExPEC and STEC strains in outbreaks and sporadic cases of infection.