Characterization of six Shiga toxin-producing Escherichia coli (STEC) strains carrying Stx2-phages from Colombia

Shiga toxin producing Escherichia coli (STEC) is a bacterial pathogen that causes diarrhea and severe human diseases like HUS; its main virulence factor are the Shiga toxins (Stx1 and Stx2). Some Stx2 subtypes have been identified and associated with the risk of developing severe illness. Stx toxins are encoded in temperate bacteriophages which control their expression through the lytic cycle that is regulated by the late genes and Q anti-terminator protein. The aim of this work was to characterize six STEC strains carrying Stx2-phages to provide preliminary information and understanding about Stx2 strains from Colombia regarding the Stx production, lytic cycle induction and subtyping of Stx2. Two highlighted strains with higher levels of Stx production and lytic cycle’s induction were observed. All the evaluated strains carried either the Stx2a, Stx2c, or Stx2d subtypes. In addition, most of the strains carried the qO111 allele, and only one strain showed differences in the ninG region. Differences in the evaluated characteristics of the strains were observed, which could indicate the variability of these six STEC strains carrying Stx2-phages.


Introduction
Shiga toxin-producing Escherichia coli (STEC), is an emerging pathogen involved in food-borne infections which causes diarrheal disease outbreaks, like hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). The HUS is one of the most severe human diseases caused by STEC, characterized by producing thrombocytopenia and severe kidney failure, mainly affecting children under 5 years old and older adults [1]. In this sense, cattle is considered the main reservoir of pathogenic STEC for humans and its transmission may occur through the consumption of food contamination by the cattle feces, such as meat, cheese, and unpasteurized milk. Likewise, other ways of transmission are possible by the intake of contaminated water, fruits, and vegetables, causing the STEC infection outbreaks [2].
The main STEC virulence factor associated with HUS is Shiga toxins (Stx). The Shiga toxins belong to the AB 5 family of protein toxins, with an enzymatically active A moiety and a nontoxic B moiety responsible for binding to cellular receptors on the target cells, such as kidney cells, the gastrointestinal tract and central nervous system cells. There are two types of Stx: Stx1 and Stx2, which share approximately 56 % of the homology in their amino acid sequence. However, Stx2 has been epidemiologically linked to the development of severe diseases, like HUS [3]. Studies on the human brain microvascular endothelial cells (HBMC) have shown to be up to 1000 times more susceptible to Stx2 than to Stx1 [4], in this sense Stx2 has received greater research attention.
Seven subtypes of Stx2 have been identified: Stx2a, Stx2b, Stx2c, Stx2d, Stx2e, Stx2f, Stx2g. The most frequent subtypes identified so far in strains of human and bovine origin are Stx2a, Stx2c and Stx2d; these three subtypes have shown high toxicity in vitro and are often associated with development of hemorrhagic colitis (HC) and HUS. Unlike the Stx2b, Stx2e, Stx2f and Stx2g subtypes that are rarely associated with severe diseases in humans, and are frequently isolated from animal reservoirs different from cattle. [5,6]. The amino acid sequences of the Stx2a, Stx2c, and Stx2d subtypes showed to be closely related, and shared between 97 % and 99 % identity; however, due to their biological differences and toxicity, they are classified as different subtypes [7].
Shiga toxin genes (stx) are encoded by temperate bacteriophages inserted in the bacterial chromosome, phages encoding stx (Stx-phages) can be induced by DNA damaging agents such as mitomycin C. The Stx expression is linked to the lytic cycle of the bacteriophage and its release depends on the lysis of the bacteria. As a result of the induction process, Stx-phages play an important role in STEC pathogenesis due to the regulation of the Stx expression and their contribution to the propagation of the stx genes in other bacteria [8]. The stx genes are located in the late region of the Stx-phages where genes implicated in the lytic cycle are found. Likewise, late genes are regulated by a transcription anti-terminator protein Q. In absence of the Q protein, the phages cannot carry out their lytic cycle, which implies they will not produce Stx [9]. Three allelic variants: q933, q21, and qO111, have been identified for the Q protein gene. which are related with differences on the levels of expression of the Stx and the virulence of the strains that carry them [10,11].
In Colombia, the knowledge about STEC is still limited. The few studies have focused on the detection of the O157:H7 serotype from different sources without the use of molecular techniques [12,13]; in addition, a characterization for the isolated native strains is not currently existing. The aim of this study is to characterize six STEC strains carrying Stx2-phages to provide preliminary information and understanding about Stx2 production, lytic cycle induction and subtyping of Stx2 in STEC strains from Colombia.

Bacterial strains
This study was descriptive cross-sectional. Six cattle STEC strains (102, 10610, 600 5052, 615, and N108) carrying Stx2-phages were selected, belonging to the Center of Biomedical Research strains collection, Universidad del Quindío. Quiguanas et al. [14] characterized previously these strains according to the presence-absence of STEC virulence genes (stx2, eae, saa, and hlyA). Briefly, fecal samples were collected by rectal swabbing from cattle in the farm El Cofre, located in Ulloa-Valle del Cauca, Colombia. Each sample was transported in test tubes with peptone water at room temperature to the Biomedical Research Center (CIBM) of the Universidad del Quindío for processing. By multiplex PCR, stx1, stx2 or both genes were detected. Positive samples for one or both toxin genes were considered STEC positive. Strains were isolated in MacConkey agar. Individual colonies were characterized by multiplex PCR for detection of genes, encoding different virulence factors (eae, saa, and hlyA). However, the serotype of these strains is still unknown ( Table 1). For this study, we selected only STEC strains carrying Stx2-phages due to

102
Stx2d Stx2d C c a Positive for primers QATG and Q3 and negative for primers Q-stx-f and 595 b Positive for primers SF1-F and SF1-R, and SF1-R and 595 c Size of amplified 1200 pb its epidemiological importance worldwide, since it is the predominant type in STEC infections in humans, and it is associated with a greater severity of disease. The STEC reference strain EDL933 was used as positive control and the E. coli DH5˛strain was used as negative control for its great capacity to host Stx-phages.

Induction of the lytic cycle
The six strains were grown individually in LB Broth Base (Lennox, powder, L Broth Base) overnight at 37 ı C, 100 rpm. These cultures were refreshed in LB medium at 37 ı C, 180 rpm, when the culture reached the exponential growth phase (optical density OD 600 D 0:2 nm to 0:3 nm); it was divided into two subcultures and Mitomycin C (MMC) was added to a fraction at a final concentration of 0:5 µg ml 1 (inducer of the lytic cycle) [15]. The OD 600 of the cultures both with and without MMC were monitored at different times (0 h, 1 h, 2 h, 3 h, 4 h, and 5 h) to construct growth curves/lysis; the growth was measured with a microplate spectrophotometer Epoch™ (BioTek). All assays were done at least three times and independently for each strain.

Stx2 production
To compare the titers of Stx2, after overnight incubation (37 ı C, 180 rpm) the cultures with and without MMC were centrifuged at 11:500 rpm, 4 ı C during 10 min and the supernatants were evaluated for the presence of Stx2 by using an enzyme immunoassay (Ridascreen ® Verotoxin, R-Biopharm) according to the manufacturer's instructions. The ELISA plates were read in the microplate spectrophotometer Epoch™ (BioTek) at OD 650 . However, when the OD 650 values exceeded the limit permitted by the Epoch™ (BioTek), serial dilutions were conducted to obtain a value within the equipment's reading range. The toxin titers were expressed as the result of the multiplication between the OD 650 and the reciprocal of the dilution factor; all the assays were carried out at least three times.

Analysis of the late region of Stx2-phages
The upstream region of the stx 2 gene was analyzed by PCR in Veriti 96-Well Thermal Cycler (Applied Biosystems™). The proximity of the ninG gene with stx 2 was evaluated using NinG/526 and 595 primers [16], the variability in this gene was evaluated by comparing the sizes of the amplified fragments. The presence of the different alleles of q gene (q933, qO111, and q21) was evaluated in all of the strains as well as its closeness with the stx 2 gene. To detect the q933 allele, the QATG5' and Q3' primers were used [17], and the closeness with the stx 2 gene was evaluated using the Q-stx-f and 595 primers [16]. The presence of the qO111 allele was evaluated with the SF1-F and SF1-R primers [11] and its closeness with the stx 2 gene was evaluated with the SF1-F and 595 primers. The q21 allele and its closeness with the stx 2 gene was evaluated with the Q21 and 595 primers [18] (Figure 1). All the primers used here are shown in Table 2. The conditions for the PCR were the following: 94 ı C for 5 min, followed by 30 cycles of 94 ı C for 1 min, 60 ı C for 1 min, 72 ı C for 3 min, ending with 72 ı C for 10 min. For the QATG5' and Q3 primers annealing temperature was 53 ı C.

Identification of Stx2 subtypes
The identification of Stx2 subtypes was performed through analysis of sequences with the method proposed by Persson et al. [19] and Scheutz et al. [7]. A partial sequence of stx 2 gene was obtained by using the F4 and R1 sequencing primers (Table 2), these primers amplifying a 491 bp fragment which was translated into 159 amino acids, covering 95 residues from the C-terminal region of the A subunit and 64 residues from the N-terminal region of the B subunit, where the sequences of the toxins have greater variability. The PCRs were performed with the Platinum™ Taq DNA Polymerase High Fidelity kit (Invitrogen™) with the following conditions: 95 ı C for 15 min, followed by 35 cycles at 94 ı C for 50 s, 56 ı C for 40 s, and 72 ı C for 1 min, and an ending temperature of 72 ı C for 3 min. The amplicons obtained with forward and reverse PCR primers were sequenced by the Sanger method in an ABI3500 (Applied Biosystems™). The DNA used for the different PCRs were purified by using the Wizard ® Genomics Kit (Promega). AB048227; X65949. Although the sequences of the reference strains were complete sequences, in order to perform the analysis, these sequences were cut in the region shown in the alignment ( Figure 4A). The sequences obtained in this study were deposited in the GenBank with the following access numbers: MT680394; MT680395; MT680396; MT680397; MT680398; MT680399.

Induction of the lytic cycle
Analysis of the growth curves showed that all the cultures without MMC had an exponential growth with OD 600 values of approximately 1.4; the same behavior was observed with the positive and negative control cultures: EDL933 and DH5˛. However, upon inducing the phages with MMC, we observed different kinetics in the induction of the stx2 phages (Figure 2). Two strains (10610 and 600) showed a high induction with OD 600 values lower than 0.2 (5 h after MMC induction) and the others (102, 5052, 615 and N108) showed a more reduced induction with OD 600 values of approximately 0.7 (5 h after MMC induction).

Shiga toxin production
All the strains evaluated showed Stx2 production; nevertheless, the titers obtained were different among each other. The 10610-strain showed higher Stx production, presenting similar titers to the positive control strain. The 5052, 600, and N108 strains showed lower titers than the control strain, but with moderate Stx production. The 102 and 615 strains produced the lowest titers compared with the other strains (Figure 3). In general, all the strains revealed an increase in the toxin titers when the cultures were induced with MMC. We observed that Stx was also detected under non-induction conditions, but always with low titers, demonstrating a basal toxin production in all the strains. The results of absorbance and dilutions carried out are shown in supplementary material: Table S1.

Analysis of the late region of Stx2-phages
The proximity of the ninG gene with the stx 2 gene was confirmed in all the strains, where an amplified of 1700 bp fragment was observed, except for the N108 strain that obtained a fragment of 1200 bp, suggesting that there are differences in the regulating region of these phages. Regarding the q gene alleles, the q933 allele was detected in all the strains; however, upon evaluating its closeness with the stx 2 gene, the amplicon was not obtained. As for the qO111 allele, its presence and closeness with the stx 2 gene were confirmed in five strains. None of the strains studied carried the q21 allele, the results are shown in Table 1.

Identification of Stx2 subtypes
In some strains, the chromatograms showed double peaks in specific positions that corresponded to different nucleotides, in these cases, the IUPAC nucleotide nomenclature was used to solve these ambiguities. Once the consensus sequence was obtained and translating it into amino acids, we observed that in most cases the ambiguities did not alter the amino acid sequences (synonymous mutations); only in two strains (10610 and 600) we observed a codon change in the position 137 and it could be translated into two different amino acids: Alanine or Aspartic acid ( Figure 4A).

Analysis of the intergenic region between the A and B subunit genes of Stx2
, showed that all the strains had the same sequence (AGGAGTTAAGT); this sequence has been reported for the Stx2a, Stx2c, and Stx2d subtypes. Analyzing the alignment, we observed that all subtype sequences were highly conserved and only differ in some positions ( Figure 4A). The combination of these positions was used to classify the subtypes, obtaining the following results: Stx2a subtype; one strain, Stx2c subtype; three strains and Stx2d subtype; two strains. Specific changes in amino acid residues were observed in some sequences; however, these are not strictly restricted to a subtype. In the dendrogram both the studied and the reference sequences formed separate groups, confirming what was observed in the alignment ( Figure 4B).

Discussion
Presence of inducible phages in the studied strains was evaluated through the analysis of the growth curves/lysis; two strains (10610 and 600) showed bacteriolysis by phage induction with MMC. Likewise, their cultures had the highest Stx titers together with the 5052 strain under induced conditions. Demonstrating that these strains display inducible phages capable of producing Stx2. Various studies showed that STEC strains with inducible Stx-phages increase substantially the Stx production, when the cultures were treated with MMC, evidencing a direct relationship between induction and Stx production [20,21]. Additionally, Ogura et al. [22] have proposed that variability in Stx production among STEC strains may be related with the genetic characteristics of the phages that carry them.
Analyzing the bacterial growth/lysis curves constructed, two strains (102 and 615) showed a different behavior; both had low response to induction and low Stx titers, even under induced conditions. Indeed, there are studies demonstrating that bacteria can have a high frequency of defective prophages [20,21,22,23,24], which are not capable of carrying out its lytic cycle, limiting its capacity to kill the host bacterial cell; therefore, this would avoid (in the case of Stxphages) Stx production. Even though Johansen et al. [25] suggest that the level of Stx production in bacteria carrying apparently defective phages is lower than bacteria carrying inducible phages. In this sense, the low Stx production in the 102 and 615 strains may be explained by the lack of Stx2-phage induction observed through the OD 600 kinetics, which could be due to the presence of a defective Stx2-phage.
In this study we detected Stx production in cultures without MMC, suggesting that stx2 genes are carried by prophages with spontaneous induction, leading to high release of Stx independently of the bacterial SOS system, involved in the activation of the lytic cycle [21]. In agreement with our results, Gamage et al. [26] by analyzing pure supernatants, also reported that all stx 2 strains produced high levels of Stx both in presence and absence of inductors of the lytic cycle. Shimizu et al. [27] also reported that some Stx phages have a relatively high spontaneous induction, detecting the presence of Stx2 in the extracellular fraction in absence of any applied induction.
Three alleles encoding Q protein have been described (q933, qO111 and q21), all of them differ in their activity as anti-terminators [10, 11]. The qO111 allele was detected in five strains (102, 10610, 5052, 615, and N108), which was described recently in sorbitol-fermenting STEC strains isolated from patients with diarrheic diseases and HUS in several European countries [28,29,11] and related with high progression to HUS [30]. In this study it was not possible to demonstrate the closeness of the q933 allele with the stx 2 genes. Perhaps, Stx2 is encoded in a defective Stx-phage or some phages encoded in these strains do not carry the stx 2 gene. Regarding the q21 allele, none strains were found carrying this allele, principally identified in STEC strains with low or null Stx production and frequently isolated from beef, animal origin or the environment in Asian countries, where these are widely distributed [31,32,33,10].
Subtyping the STEC strains permitted identifying three Stx2 subtypes: one Stx2a-positive strain, three Stx2c-positive strains and two Stx2d-positive strains. According to the literature, the STEC strains carrying these three subtypes are frequently isolated from patients with HUS [5, 34], which has led to be strongly related with this disease. The most relevant subtypes identified in this study were: the Stx2a; according to the literature, it has demonstrated to be the most potent and active on the endothelial cells [4, 6] and the Stx2d, due to an "activatable tail" that causes an increase of up to 25 times its toxicity on Vero cells when it is previously incubated with elastases [35].
Although the three subtypes share a high percentage of identity regarding their amino acid sequence, differences were found in positions 89, 95 at the end of C-terminal of the A subunit, and in the END or EDD motif in the B subunit. The END motif of the B subunit plays an important role in the interaction of the toxin with its receptor; therefore, mutations in these positions would cause changes in the affinity of the toxin with its receptor [6], which could explain the differences in toxicity of these Stx2 subtypes. Finally, in the evaluated strains, the Stx2b, Stx2e, Stx2g, and Stx2f subtypes were not identified; these subtypes are rarely associated with human diseases and contrary to the other subtypes, they have been isolated from pigs and pigeons. In this study, these subtypes were not detected, since all the strains were isolated from cattle [36,37].

Conclusion
The results obtained in this study show that the six native STEC strains evaluated had differences in the Stx2 titers, induction of the phage's lytic cycle, Stx2 subtypes, and late region of the Stx2-phages. The characteristics of the STEC strains found in this study will help to improve the understanding of STEC from Colombia, since these strains are similar to STEC strains that cause diseases in humans reported in the literature. However, more studies are necessary with broader samples that permit determining their virulence.