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1 Present address: Graduate School of Environment Department of Industrial and Environmental Engineering, Gachon University, Seongnam, 13120, Republic of Korea
Eva Bagyinszky
Footnotes
1 Present address: Graduate School of Environment Department of Industrial and Environmental Engineering, Gachon University, Seongnam, 13120, Republic of Korea
Affiliations
Veterinary Medical Research Institute, Hungária krt. 21., H-1143, Budapest, Hungary
1 Present address: Graduate School of Environment Department of Industrial and Environmental Engineering, Gachon University, Seongnam, 13120, Republic of Korea
Escherichia coli strains of the O157 serogroup include significant foodborne pathogens: enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli, which are responsible for a considerable number of hospitalizations and deaths worldwide each year. There is a constant need for rapid, reliable, and easy-to-use methods for their identification, typing, and phylogenetic classification. In this study, we proposed a new multiplex polymerase chain reaction (PCR)–based typing system for pathogenic E. coli, focusing on the O157 serogroup.
Methods
We designed primers targeting 12 lambdoid prophage regions carried by the prototypic polylysogenic strain of EHEC, the O157:H7 Sakai strain. The reactions were tested in vitro as well as in silico with the PubMLST database.
Results
The PCR assays can be grouped into four multiplex reactions, and their results can be given as a four-digit code. In vitro and in silico testing showed that these Sakai prophage regions are prevalent not only in E. coli O157 strains but also in Shiga toxigenic E. coli non-O157 strains and the method provides appropriate resolution.
Conclusions
The proposed method could be a valuable tool in epidemiologic tracing and preliminary phylogenetic grouping of this diverse group of pathogens.
Intestinal pathogenic Escherichia coli strains of the O157 serogroup include the most notorious pathogenic strains of the enterohemorrhagic (EHEC) pathotype (
). EHEC strains and especially those of the O157:H7 serotype are capable of causing hemorrhagic colitis and the life-threatening complication, hemolytic uremic syndrome (
). Shiga toxin–producing E. coli (STEC, of which EHEC is a subset producing intimin as well as Shiga toxin) are responsible for an estimated 2.8 million hospitalizations and 230 deaths worldwide annually (
In recent decades, E. coli O157 has been a subject of intense whole genome sequencing (WGS)-based studies, with valuable insights gained about their genomic structure and phylogenetic relations. Regarding the former, one of the most important notions is the significant role of prophages in the virulence and genomic variability of the strains (
). The key virulence factors of STEC and EHEC, the genes encoding Shiga toxin (Stx), are also carried by lambdoid prophages, which are inducible and transducible in several cases (
The WGS-based investigations created opportunities for the design of more precise and broad-ranged, rapid nucleotide sequence–based identification methods of pathogens, and this was also true for E. coli O157, especially because the genomic variability and abundance of its isolates warrants the need for reliable identification and classification methods.
From a practical perspective, the rapid identification of a pathogen is a key issue, both for choosing the optimal treatment as well as for epidemiological tracing. For a precise identification, polymerase chain reaction (PCR)-based methods are widely used, with multilocus variable number of tandem repeat analysis (MLVA) viewed as a precise tool for epidemiological tracing (
Effective Surveillance Using Multilocus Variable-Number Tandem-Repeat Analysis and Whole-Genome Sequencing for Enterohemorrhagic Escherichia coli O157.
New system for multilocus variable-number tandem-repeat analysis of the enterohemorrhagic Escherichia coli strains belonging to three major serogroups: O157, O26, and O111.
Multilocus Variable-Number Tandem-Repeat Analysis of Enterohemorrhagic Escherichia coli Serogroups O157, O26, and O111 Based on a De Novo Look-Up Table Constructed by Regression Analysis.
Multiple-locus variable-number tandem repeat analysis for non-O157 Shiga toxin-producing Escherichia coli: focus on serogroups O103, O121, O145, O165, and O91.
). On the other hand, prophages are widespread in E. coli, especially in the O157 serogroup, and play a role in its genomic variability and evolution (Davies et al., 2016; Fortier and Sekulovic, 2013).
In this work, we mapped and monitored the presence of prophages from one of the most well-known prototypic EHEC O157:H7 strain, the Sakai (
We proposed a PCR-based identification scheme on the basis of the sequences of the prophages. Demonstrating the prevalence of these prophages among pathogenic E. coli strains, we developed a practical tool for quick genotyping and epidemiological tracing of STEC strains and those of the O157 serogroup.
2. Materials and methods
2.1 Bacterial strains
EHEC, EPEC, and atypical (stx-, eae-) E.coli O157, non-O157 EPEC, enteroaggregative E. coli (EAEC) as well as uropathogenic (UPEC), extraintestinal pathogenic (ExPEC), nonpathogenic laboratory strain E. coli K-12 C600, one Shigella sonnei, and one S. dysenteriae strain were included among the strains on which the typing scheme was tested. All strains used in the study are listed in Table 1.
Table 1List of strains used for testing of the PCR scheme with their assigned types. The type is the result of the typing PCR reactions transformed into a four-digit numeric code.
Phage type is given according to the typing scheme of Ahmed et al., 1987. The abbreviations stand for the following: NT, nontypeable; R, phage resistant; NC, noncharacteristic PT; d, derivative; N/A, not applicable.
The Pathogenicity Island-Associated K15 Capsule Determinant Exhibits a Novel Genetic Structure and Correlates with Virulence in Uropathogenic Escherichia coli Strain 536.
a Phage type is given according to the typing scheme of Ahmed et al., 1987. The abbreviations stand for the following: NT, nontypeable; R, phage resistant; NC, noncharacteristic PT; d, derivative; N/A, not applicable.
Primers were designed manually on the basis of the nucleotide sequences of the Sakai prophages (Sp) within the genome of EHEC O157:H7 prototype strain Sakai (GenBank BA000007;
). Care was taken that the targets be present in only one copy in the Sakai genome and that their size falls between 100–400 bp but of different length each to allow the multiplexing of the reactions. For the same reason, care was taken that none of the primers of different reactions would be able hybridize with each other. The complete list and nucleotide sequences of primers are shown in Table 2.
Table 2List of primers used in the PCR typing scheme. The reaction group column indicates the reactions which can be grouped as multiplexes. The reaction number indicates the designation of the reaction within the multiplex, which is necessary for the transformation of the results into a numeric code.
The reactions were performed using deoxyribonucleic acid (DNA) extracted from overnight cultures of bacterial strains grown on Luria-Bertani agar listed in Table 1 by boiling a 10 µl loopful of cells in sterile, distilled water. From every DNA sample, 2 µl was added to a final reaction mixture of 25 µl.
All reactions were performed using Taq DNA polymerase (Fermentas/Thermofisher, Vilnius, Lithuania) according to the manufacturer's instructions. The primers were suspended in distilled water for a stock solution of 100 µM and used in a working concentration of 0.8 µM, with 0.2 µl of stock solution of each primer added to a 25 µl of total reaction volume. The heat profile of all reactions was 3 minutes of initial denaturation at 94°C, then 30 cycles consisting of a denaturation at 94°C for 30 seconds, annealing at 68°C for 30 seconds, and extension at 72°C for 30 seconds. The reactions were ended with a final extension step of 72°C for 5 minutes. Results of the reactions were visualized and evaluated by gel electrophoresis.
The reaction profile of each strain was given as a four-digit code similarly to the phage typing scheme of Farmer (1970), as shown in Table 3.
Table 3The typing scheme transforming the results of multiplex PCR reactions numbered according to Table 2 into a numeric code, analogously to the phage typing scheme outlined by Farmer (1970).
) separately for each of the primer pairs on all Escherichia genomes present in the database in December 2021 and January 2022, allowing for a 1-nucleotide mismatch and a maximum product length of 1000 nucleotides in each case. Tests were run separately for all isolates (4556 genomes), isolates labeled as EHEC (151 genomes), environmental strains of the O157 serogroup (3 genomes), and STEC (120 genomes).
3. Results
We designed and tested a PCR system targeting 12 prophage genes of the prototypic EHEC strain O157:H7 Sakai. The system could be applied as individual reactions as well as grouped into four triplex reactions, specific for groups of 3 genetic regions indicated in Table 2. Sample results of the multiplexed reactions are shown on Fig. 1.
Figure 1Sample gel electrophoresis of multiplex reactions of the typing scheme. A) reaction 1, B) reaction 2, C) reaction 3, D) reaction 4. Samples: M, marker; 1, no DNA; 2, O157:H7 Sakai (EHEC); 3, E. coli C600 (K-12, nonpathogenic); 4, O157:H7 strain 254 (EHEC); 5, O157:H7 strain 68 (EPEC); 6, O157:H43 strain T22 (atypical); 7-9, O157:NM strains (EHEC)
The schematic grouping used in interpreting the results is shown in Table 3. The results of the four multiplex reactions can lead to one of the numbered patterns listed. The patterns obtained by running all four reactions can be given as a numeric code, which is specific for a narrow group of strains.
Altogether, 40 strains were tested in vitro. The selected strains represented the enteric E. coli pathotypes as well as two ExPEC, two UPEC, and two Shigella strains. A total of 30 strains belonged to the O157 serogroup. The pathogenic strains carried at least one Sp marker gene. Most of the prophage markers genes were detected in EHEC and EPEC O157 strains, and different Sp gene patterns were observed in O157 strains, representing different patho- and serotpyes. Less genes were present in the other non-O157 enteric strains and a maximum of five genes were detected in the ExPEC isolates. One S. sonnei strain carried Stx2 phage (Sp5) marker gene, but stx2 gene was not detected. Only two (Sp9 and Sp15) of the Sp marker genes were detected in nonpathogenic laboratory strain E. coli K-12 C600.
The code for each of the test strains is given in Table 1.
The virtual PCR scanning of the Escherichia genomes contained in the PubMLST database has shown that 1775 (39%) of the 4556 isolates fell into the 8888 type, being negative for all reactions. The rest of the strains could be typed by the system in a meaningful way, carrying at least one of the target lambdoid prophage regions.
Narrowing down the scope of strains for the EHEC and STEC strains, of the 151 and 120 genomes, only four and 14 fell into the ‘untypeable’ 8888 category. The three O157 strains of environmental origin were of types 8888, 6888, and 8887. The 274 investigated strains represented 96 types altogether, the most frequent (‘2464’) being represented by 24 strains, and 52 strains representing a single type each.
Some noteworthy differences could be identified when comparing the pattern types of the EHEC and STEC strains: the ‘prototypic’ 1111 pattern were only present among the EHEC strains. Type 2462 was represented by 11 EHEC strains, whereas none of the STEC showed this type. Type 2444 and 2464 were only represented by one STEC strain each, whereas among the EHEC strains, 18 and 22 strains showed these types, respectively.
The full results of the in silico PCR, including the type codes for each isolate, are shown in Table 4.
Table 4Results of in silico PCR against EHEC, STEC, as well as atypical and commensal O157 strains in the PubMLST database. The types of the strains are presented as a four-digit code according to the scheme outlined in Table 3.
In the current study, we created a PCR-based typing system for E. coli strains of the O157 serogroup, targeting 12 prophage genes present in the prototypic EHEC strain O157:H7 Sakai, grouped analogously to the typing scheme of Farmer (1970), which was originally used in phage typing for E. coli O157:H7 (
). All the reactions target lambdoid phages, and in the overwhelming majority of cases, the targets are only present in one copy in a given genome (six of the reactions gave multiple products with the in silico PCR in a total of 57 instances; but in practice, it may not influence the results of the test, as the predicted products are of the same size in all cases).
The size of the reaction products may enable the reactions to be multiplexed, and all of them can be run with the same reaction conditions, including the annealing temperature without the loss of specificity. These features make the method rapid and easy to use.
By choosing a high number of genes, which could be grouped into multiplex reactions and its results converted into a code, we obtained a system which provides an adequate level of resolution within the target bacterial group, as the 274 in silico investigated strains were sorted into 96 types.
With the ‘democratization’ of WGS, there has been an upsurge of genomic data regarding pathogenic E. coli (
), but there is still a need for rapid classification and identification of strains, especially within the abundantly isolated and sequenced (>1200 whole genomes in GenBank as of January 2022) O157 serogroup, containing several significant pathogenic strains. Our hypothesis was that sequences of prophage origin harbored by a high number of pathogenic strains in the O157 as well as other serogroups could be reliably used for identification, as was shown earlier in the case of Salmonella serovar Typhimurium (
). The conducted in silico test showed that the multiplex PCR system is indeed reliable. The prototypic EHEC strains showed the same patterns as in vitro by testing with this method and that the scheme provides adequate resolution for simple and rapid genotyping of STEC strains as well as those of the O157 serogroup. The method could be a valuable help in epidemiologic tracing and preliminary phylogenetic grouping of this highly diverse group of pathogens.
Funding source
This work was supported by the National Research, Development, and Innovation Office (grant number K 124335). Domonkos Sváb was supported by the János Bolyai Research scholarship of the Hungarian Academy of Sciences.
Multiple-locus variable-number tandem repeat analysis for non-O157 Shiga toxin-producing Escherichia coli: focus on serogroups O103, O121, O145, O165, and O91.
New system for multilocus variable-number tandem-repeat analysis of the enterohemorrhagic Escherichia coli strains belonging to three major serogroups: O157, O26, and O111.
Effective Surveillance Using Multilocus Variable-Number Tandem-Repeat Analysis and Whole-Genome Sequencing for Enterohemorrhagic Escherichia coli O157.
The Pathogenicity Island-Associated K15 Capsule Determinant Exhibits a Novel Genetic Structure and Correlates with Virulence in Uropathogenic Escherichia coli Strain 536.
Multilocus Variable-Number Tandem-Repeat Analysis of Enterohemorrhagic Escherichia coli Serogroups O157, O26, and O111 Based on a De Novo Look-Up Table Constructed by Regression Analysis.
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