ALEKSANDRA JANUSZKIEWICZ and WALDEMAR RASTAWICKI
National Institute of Public Health – National Institute of Hygiene, Department of Bacteriology, Warsaw, Poland
*Corresponding author: A. Januszkiewicz, National Institute of Public Health – National Institute of Hygiene, Warsaw, Poland; e-mail: firstname.lastname@example.org.
Submitted 3 December 2014, revised 28 July 2015, accepted 11 February 2016
Shiga toxin-producing Escherichia coli (STEC) strains also called verotoxin-producing E. coli (VTEC) represent one of the most important groups of food-borne pathogens that can cause several human diseases such as hemorrhagic colitis (HC) and hemolytic – uremic syndrome (HUS) worldwide. The ability of STEC strains to cause disease is associated with the presence of wide range of identified and putative virulence factors including those encoding Shiga toxin. In this study, we examined the distribution of various virulence determinants among STEC strains isolated in Poland from different sources. A total of 71 Shiga toxin-producing E. coli strains isolated from human, cattle and food over the years 1996 – 2010 were characterized by microarray and PCR detection of virulence genes.
As stx1a subtype was present in all of the tested Shiga toxin 1 producing E. coli strains, a greater diversity of subtypes was found in the gene stx2, which occurred in five subtypes: stx2a, stx2b, stx2c, stx2d, stx2g. Among STEC O157 strains we observed conserved core set of 14 virulence factors, stable in bacteria genome at long intervals of time. There was one cattle STEC isolate which possessed verotoxin gene as well as sta1 gene encoded heat-stable enterotoxin STIa characteristic for enterotoxigenic E. coli. To the best of our knowledge, this is the first comprehensive analysis of virulence gene profiles identified in STEC strains isolated from human, cattle and food in Poland. The results obtained using microarrays technology confirmed high effectiveness of this method in determining STEC virulotypes which provides data suitable for molecular risk assessment of the potential virulence of this bacteria.
Key words: Escherichia coli, microarray detection, Shiga toxin-producing, virulence gene
Shiga toxin-producing Escherichia coli (STEC) strains also called verotoxin-producing E. coli (VTEC) represent one of the most important groups of food-borne pathogens that can cause several human diseases such as hemorrhagic colitis (HC) and hemolytic – uremic syndrome (HUS) (EFSA, 2010; Nataro and Kaper, 1998). Over 400 serotypes of STEC have been isolated from human. However, the majority of clinical STEC infections, particularly those associated with outbreaks and serious patient outcomes, are attributable to a subset of serogroups including E. coli O157:H7 (the prototype bacterium for enterohaemorrhagic E. coli – EHEC), O26: H11, O103:H2, O111:H8, O121:H19 and O145:H28 (Nataro and Kaper, 1998). In recent years, new serotypes of EHEC have also emerged (Bielaszewska et al., 2011).
The ability of STEC strains to cause disease is associated with the presence of a wide range of identified and putative virulence factors including those encoding Shiga toxin (Nataro and Kaper, 1998). The Stx family consists of 2 major groups, Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), which are well distinguished by their amino acid sequences. Several other virulence factor are known, including genes conferring the ability to cause attaching-effacing lesions located on LEE pathogenicity island. The LEE encodes intimin (Eae), translocated intymin receptor (Tir), a type III secretion system (EspA, EspB, EspD) and effector proteins translocated by the secretion system. Beyond Stx and LEE genes, “typical STEC” associated with human disease usually carry virulence factors encoded by 60-MDa plasmid as enterohemolysin (Ehx), serine protease (EspP), catalase peroxidase (KatP) and a type II secretion system (EtpD) (Nataro and Kaper, 1998).
As more atypical STEC strains have been reported, several proteins have been proposed as potential virulence determinants as autoagglutinating adhesion (Saa), subtilase cytotoxin (SubA), non-LEE encoded effectors (Nle) (Coombes et al., 2008; Bugarel et al., 2010; Paton and Paton, 2010). Moreover, recently there have been many scientific reports describing the presence of STEC isolated from humans, animals and the environment, carrying specific virulence determinants characteristic for different E. coli pathotypes as intermediate STEC/ETEC or EAEC/STEC virulence factor profiles (Bielaszewska et al., 2011; Prager et al., 2011). Therefore determining the virulence potential of STEC relies upon the determination of somatic and flagellar antigens, together with the identification of virulence genes what can be used to make a “molecular risk assessment” of the predict potential virulence of strains (Coombes et al., 2008).
Although the number of STEC isolates reported yearly is relatively low in Poland (few cases of human STEC infections), they are often responsible for serious illnesses or HUS complications (Jakubczak et al., 2008; EFSA, 2010; Januszkiewicz et al., 2010; 2012). What is interesting one outbreak of STEC infection related to international outbreak of STEC O104:H4 in 2011 was reported in Poland (Januszkiewicz et al., 2012). There is no data containing molecular characterization of Shiga toxin – producing E. coli strains isolated in our country both from human infections as well as STEC strains isolated from animal and food. The aim of our study was to investigate the distribution of various virulence determinants among STEC strains isolated in Poland from different sources. To achieve our goal we used commercially available microtube array system designed to detect virulence genes in E. coli of various pathotypes.
Material and Methods
Bacterial strains. A total of 71 Shiga toxin-producing E. coli (STEC) strains were analyzed (Table I). Those isolates were from collection of Department of Bacteriology (n = 38), Department of Food and Consumer Articles Research (n = 4) of National Institute of Public Health–National Institute of Hygiene (NIPH–NIH) and Department of Hygiene of Food of Animal Origin in National Veterinary Research Institute (NVRI) in Puławy (n = 29). STEC strains collected during 1996 – 2010 were isolated from human (n = 45), cattle (n = 16) and food (n = 10) and were classified into following groups: serotype O157 (n = 45), serotype O26 (n = 10), serotype O111 (n = 6) and NT (not typable using available Biomex latex assay) (n = 10). More than 90% of tested human STEC O157 isolates were from reported human cases in our country. All of them were from sporadic cases. Tested STEC strains presented different band patterns when typed with pulsed-field gel electrophoresis (PFGE) using XbaI enzyme (data not showed) which indicates that they had no epidemiological link.
Identification and virulence analysis of STEC strains. Classical tube test was used for E. coli re-identification. Serotyping was performing using latex agglutination test (Biomex). Stx production was confirmed by VTEC-RPLA assay (Oxoid). The cytotoxity assay with Vero cell monolayers (VCA) in 96 – well plates was performed as described previously (Januszkiewicz et al., 2010). The presence of stx1, stx2 and eae genes were examined by PCR based methods according to protocols described previously (Januszkiewicz et al., 2010). Detection of Stx gene subtypes was performed by PCR based method according to WHO protocol (Scheutz et al., 2012). Presence of H7 antigen (fliC gene) in STEC O157 isolates was performed by PCR-RFLP (Januszkiewicz et al., 2010). A DNA microarray (Identibac Ec v. 03, Alere Technologies GmbH) was used to determine the presence of virulence genes among STEC strains. The microarrays were used according to the manufacturer’s instructions. Array images were processed in IconoClust 3.0 (Clondiag, Germany) and signals were analyzed using the gapA-positive control gene for normalization and with cut-offs as recommended by manufacturer (> 0.4 = present, 0.4 to 0.3 = ambiguous, < 0.3 = absent, relative to the gapA signal).
Phenotypic and genotypic assays of STEC identification. For a summary of results, see Table I and II. All of the STEC isolates produced a cytopathic effect on Vero cell monolayers, confirming their ability to express verotoxins. The Stx-cytotoxic activity was neutralized with antiserum. Production of appropriate verotoxins: Stx1, Stx2 or both were confirmed in tested strains by the RPLA assay. Genetic hallmarks of a appropriate toxins (stx1 and stx2) and intimin (eae) were detected by PCR (Table I). All STEC O157 strains were positive for fliC (H7) gene. PCR subtyping of the stx1 gene revealed subtype stx1a in all isolates which produced Stx1 toxin (Table II). STEC isolates produced Stx2 toxin possessed various subtypes of stx2 gene: stx2a, stx2b, stx2c, stx2d, stx2g (Table II). Stx2c subtype was produced by STEC O157 only.
Microarray analysis. All tested STEC isolates were positive for control, species- specific genes gad, encoding glutamate decarboxylase and ihfA, encoding integration host factor subunit alpha. Genes known to be present in control strain STEC O157 EDL933 and which were represented on the array were successfully detected. There were genes encoding: Shiga toxins (stx1, stx2); adhesins: an outer membrane protein important for the attachment to host cells – intimin (eaeA) and adherence-conferring protein (iha); toxins: heat stable enterotoxin (astA), enterohemolysin (ehx), cytotoxin B (toxB); type III secretion system: non-LEE-encoded effector protein A (nleA), non-LEE-encoded effector protein B (nleB), non-LEE-encoded effector protein C (nleC), EspA protein (espA), EspF effector protein (espF), non-LEE-encoded EspJ effector protein (espJ), Tir – cytoskeleton coupling protein (tccP); type II secretion pathway related protein (etpD); serin protease autotransporters – SPATE (espP); peroxidase-katalase (katP) and translocated intimin receptor (tir).
All STEC O157: H7/H- isolates (n = 45) tested positive for markers encoding adhesins (eaeA, iha), toxins (ehx, toxB), secretion systems proteins (nleA, nleB, nleC, espF, espJ, etpD, tccP), serin protease autotransporters – SPATE (espP), peroxidase-katalase (katP) and translocated intimin receptor (tir), while genes encoding heat stable enterotoxin AstA, colicin B activity protein Cba and cytolethal distending toxin subunit B CdtB, secretion III system protein EspA, Efa adhesin and assiociated with increased serum survival marker Iss were variable absent or present (Table I).
All STEC O26 (n = 11) and STEC O111 (n = 6) were positive for 13 and 11 markers presented on the array respectively (Table I). A positive spot signal in STEC O26 was detected for 13 genes encoding adhesins (eae, iha, type III secretion system EspB protein –espB and lymphocyte inhibitory factor A-efa1), toxin (ehx), secretion systems proteins (nleA, espA, espF, espJ and cell cycle inhibiting factor – cif), major fimbrial subunit (lpfA), assiociated with increased serum survival marker iss and translocated intimin receptor (tir), while few strains were additionally present for genes encoding AstA, ToxB, CelB, Cba toxins, microcin M truncated protein MchcA, microcin H47 activity protein MchcB, putative microcin L transport protein MchcF, member of the microcin operon MchcC, secretion systems proteins (NleB, NleC, TccP) and EspP and KatP.
All STEC O111 (n = 6) were positive for 11 markers presented on the array encoding: adhesins (eaeA, iha, efa1), secretion systems proteins (nleA, nleB, espA, espF, espJ, cif), major fimbrial subunit lpfA and translocated intimin receptor tir while adhesin espB, toxins (ehx, astA, celB, cba), secretion systems proteins (nleC, tccP) espP, katP and iss were variable present or absent.
Virulence gene profile. Based on presence or absence of virulence genes the tested STEC isolates were divided into 46 virulotypes (see Table I for details). Among 45 STEC O157 tested strains 21 virulotypes were distinguished (from V1 to V21) (Table I). STEC non-O157 strains isolated from cattle (n = 10), not typable using available latex assay, had different panel of virulence markers than tested isolates belonging to O157, O111 and O26 serotypes. Among STEC non-O157 strains isolated from cattle there were two strains which possessed virulence markers not present in any tested isolates. One of those strains (no. 478) possessed in subA, saa and epeA genes (Table I) which are considered to encode subtilase toxin, autoagglutinating adhesion and serine protease autotransporters of Enterobacteriaceae (SPATE) respectively. The second strain (no. 489) had st1a gene encoded heat-stable enterotoxin ST1a and the third strain (no. 449) possessed iroN marker, encoded outer membrane protein receptor for siderophore.
Besides Stx toxins, it is known that the STEC strains carry other virulence markers E. coli engagement in pathogenesis of STEC infection (Nataro and Kaper, 1998). Those virulence markers could be typical for STEC pathotype or acquire from other pathogenic E. coli. Therefore, in this study we conducted a comprehensive analysis of virulence gene profile in STEC strains from different origin isolated in Poland. To have the opportunity to detect a large number of target sequences present in genome of different E. coli pathotypes, the commercially available DNA microtube array was used. To the best of our knowledge, such a wide range of determinants of pathogenesis of STEC isolated in Poland have never been defined.
Using microarray data analysis the STEC strains isolated in Poland have been distinguished into 46 virulotypes. Interestingly, among STEC O157 strains, which constituted the most representative serological group in investigated strains collection, we observed conserved core set of virulence factors. Apart from the Stx toxin, all isolates possessed the LEE-associated genes for intimin (eaeA), the translocated intimin receptor (tir) and the effector protein (espF). Large virulence plasmid pO157 genes for enterohemolysin (ehx), toxin B (toxB), type II secretion pathway related protein (etpD) were detected in all STEC O157 isolates. Moreover all isolates tested positive for the gene for adhesin Iha, tccP and espJ markers and finally genetic markers for effector proteins NleA, NleB, NleC were present in all STEC O157 isolates assayed. Moreover, in the group of STEC O157 strains there were human, animal and food isolates which were isolated at long intervals of time. This data suggest that those panel of virulence factors might be essential for the survival of STEC O157 in a host organism and causing the infection.
These data are in agreement with the results of other researchers (Wu et al., 2008; Söderlund et al., 2010; Bugarel et al., 2011). They confirmed that the panel of genetic determinants of virulence in the STEC O157 strains genomes are highly typical for this serotype. It is worth noting that only Söderlund et al. (2010) used commercially available EC 03 array to define virulence genetic determinants in STEC O157 strains so far. Results of the Swedish researchers clearly showed the presence of stable core of virulence markers in STEC O157 genome isolated from cattle (Söderlund et al., 2010). The range of virulence determinants identified in STEC O157 strains using Array Tube Ec03 was the same as examined in the presented study.
Interestingly, we observed the stability of virulence determinants composition in STEC O157 genome for long intervals of time. It was in fact the presence of the same virulotypes in strains from different sources isolated in Poland in different years (virulotypes: V1, V2, V3, V4) (Table I). This may indicate the correlation between this virulotype and serotype and suggest that the set of genes, observed in the majority of STEC O157 isolates in the world, might be essential for the survival of these organisms in the host organism and induce them to infection.
Virulence genes in STEC isolated in Poland. Definition of virulence types (virulotypes) based on present genes/gene variants (●) as determined by Identibac Ec v. 3 microarray assay. Only genes positive for at least one STEC strain are showed. The genes present in at least 90% of STEC strains are highlighted: for STEC O157 in dark grey, for STEC O26 in grey, for STEC O111 in bright grey color. Black color marks the genetic determinants present only in single isolates. NT- isolates which were not typable using commercially available latex (Biomex Sp.z.o.o) . The stx subtypes (bold black frame) were determined by PCR according to WHO protocol (WHO protocol).
|Virulotype||Serotype||Source||No of strain||stx1 subtype||stx2 subtype||stx1AB||stx2AB||eae||tir||nleA||espJ||iha||espF||ehx||tccP||etpD||nleC||espP||katP||toxB||nleB||cif||lpfA||espA||efa1||iss||espB||astA||cba||cdtB||celB||espI||mchA||mchB||mchF||mchC||saa||subA||st1a||epeA||iroN|
Prevalence of stx1 and stx2 subtypes in the genome of 71 STEC strains.
|STEC serotype (number of strains)||Number of STEC strains with the stx gene subtype:|
|stx1||stx2||stx1 + stx2|
(n = 45)
(n = 26)
Next to Shiga toxins, intimin, encoded by LEE pathogenicity island, is the main virulence marker, whose presence is routinely determined in diagnostics of clinical STEC isolates. It is important factor in pathogenesis of EPEC and STEC isolates because it enables direct contact between the bacterial cell surface and epithelial cells of the intestine. Our results revealed that almost all STEC isolates possessed eae gene. Only four STEC non-O157 strains isolated from cattle were included in the LEE – negative group (not carry eae gene). LEE-negative STEC non-O157 strains have been isolated from human and animals from several years (Galli et al., 2010; Irino et al., 2010). Those isolates may carry other adhesion factors as LpfA, Iha and Saa which play role in pathogenesis of human infection of this bacteria (Galli et al., 2010; Irino et al., 2010). The results presented in the hearing, as well as the observations made by other authors, lead to the conclusion that the presence of LEE-negative STEC non-O157 strains is common and therefore there is a need to pay special attention in the diagnosis of infections caused by them.
Interestingly, there was one strain (no. 478) among LEE-negative STEC isolates which carried genetic determinant responsible for production of the other cytolysin – subtilase (SubAB). According to scientific reports, STEC strains producing subtilase were isolated from people with HC and HUS (Galli et al., 2010; Irino et al., 2010; Paton and Paton, 2010). Characteristic of genetic markers in subtilase – producing STEC isolates were described by other authors (Galli et al., 2010; Irino et al., 2010). The isolates no. 478 possessed saa, epeA, iha, ehx, espP, lpfA, cdtB and celB, but did not carry eae and tir markers. Study conducted in Argentina by Galli et al. (2010) have shown that among the VTEC strains isolated from humans with HC and HUS, there were isolates which had subAB, saa, lpfA, ehx, iha, cdtB markers. Similar observations made Irino et al. (2010), who showed that the subtilase – producing E. coli strains which all produced enterohemolysin, they also had the saa, lpfA, iha genes responsible for the expression of adhesins. Understanding the profile of genetic determinants in the genome of LEE – negative STEC strains may determine their pathogenicity potential, resulting from the co-occurrence of rare genetic markers present.
Recently there have been many scientific reports describing the presence of E. coli isolated from humans, animals and the environment, carrying specific virulence determinants characteristic for different E. coli pathotypes (Bielaszewska et al., 2011; Prager et al., 2011). Using microarray technology it was possible to confirm the presence of a wide range of genetic markers present in different E. coli pathotypes in a single experiment. The results of our analysis showed that among 71 STEC strains there was one isolate (no. 489) which possessed Shiga toxin gene as well as sta1 gene encoded heat-stable enterotoxin STIa characteristic for enterotoxigenic E. coli. Moreover this isolate as the only in the STEC collection in this study produced Stx2g subtypes. Our results indicate that in our country there may exist E. coli strains with specific genetic determinants for both Shiga toxin producing (STEC) and for enterotoxigenic (ETEC) E. coli. The occurrence of strains with similar properties were also found in Germany (Prager et al., 2011). Molecular characterization of 24 Shiga-toxin producing isolates producing Stx2g, obtained from clinical material, animals and the environment, have found that all tested isolates, in addition to stx2g gene carry the st1a gene and did express STIa, which typically is associated with enterotoxin-producing E. coli (Prager et al., 2011). The emergence of infections in humans strains with combination of virulence genes of different pathotypes of intestinal pathogenic E. coli may indicate the spread in the environment of a new, intermediate and emerging pathotype (Bielaszewska et al., 2011; Mellmann et al., 2011; Prager et al., 2011). This phenomenon was observed in the current year, during one of the biggest so far HUS outbreaks in Europe, which caused an epidemic strain of E. coli O104: H4. This strain exhibited features of two E. coli pathotypes: enteroaggregative (EAEC) and Shiga toxin producing (STEC) (Bielaszewska et al., 2011; Mellmann et al., 2011). Given a rising number of intermediate pathotypes becoming described among E. coli, a wider range of virulence markers should be included in the regular pathotype diagnostics.
In the routine diagnosis of infections caused by STEC tests are also used based on the amplification of gene fragments encoding Shiga toxins. The Shiga toxin genes occur in many different nucleotide sequences- subtypes (Scheutz et al., 2012). Therefore in the routine identification of the subtypes of Shiga toxins genes in the genomes of STEC strains is extremely important because it provides valuable data that are used in monitoring of the STEC infections in many countries (Leotta et al., 2008). In addition, this method is applied in the routine diagnostics of STEC infections, especially in the situation of carrying out epidemiological investigations during outbreaks caused by these microorganisms (Bielaszewska et al., 2011; Scheutz et al., 2011).
It is worth noting that among the STEC producing Stx2, isolated in Poland (n = 49), the subtype sxt2c (n = 37) was dominated. Moreover, these subtype was present only in STEC from O157 serotype and performed alone (n = 11) or with stx1a (n = 21) or stx2a (n = 5). These results are accordance with the results obtained by other authors (Leotta et al., 2008; Aspán and Eriksson, 2010; Käppeli et al., 2011). In Switzerland all STEC O157 strains (n = 44) isolated from people from 2000 to 2009 years possessed stx2a or stx2c subtypes (Käppeli et al., 2011). According to Eriksson and Aspan studies, STEC O157 strains isolated from cattle in Sweden carried stx2a or stx2c subtypes (Aspán and Eriksson, 2010). These subtypes of stx2a gene also predominated in STEC O157 strains isolated in Argentina, Australia and New Zealand (Leotta et al., 2008).
It must be underline that the determination of the subtypes of Shiga toxin genes is extremely important because it provides information about the pathogenic potential of the STEC strain. Several studies have revealed that the individual stx subtypes differ in biological activity which were observed using various animal models and in vivo cytotoxicity assay (Fuller et al., 2011). Moreover STEC strains possessed stx2a as their sole gene, or in combination with stx2c have been described as more closely associated with HUS, than STEC strains with other Stx gene combinations (Friedrich et al., 2002). Further, purified Stx2, in contrast to purified Stx1 can elicit signs of HUS in baboons (Stearns-Kurosawa et al., 2010). In addition, a recent study in mice model of disease (Fuller et al., 2011) demonstrated that Stx2b and Stx2c had potencies similar to that of Stx1, while Stx2a, Stx2d, and elastase-cleaved Stx2d were 40 to 400 times more potent than Stx1. These findings may partly explain the high virulence of STEC O104:H4 strains, which is responsible for causing the outbreak of hemolytic uremic syndrome in May 2011 in Germany (Bielaszewska et al., 2011; Prager et al., 2011; Scheutz et al., 2011). Epidemic E. coli O104: H4 strain possessed stx2a subtype, which could explain the high incidence of HUS in infected patients (one case of HUS in four cases of bloody diarrhea) (Bielaszewska et al., 2011; Prager et al., 2011; Scheutz et al., 2011). Our results showed that STEC strains isolated in the country are diverse in terms of occurrence of stx subtypes, which according to scientific publications, are more potent (stx2a, stx2d) than other subtypes of the stx genes.
In conclusion, this is the first comprehensive analysis of virulence gene profiles identified in STEC strains isolated from human, cattle and food in Poland. Simultaneous detection of virulence markers provides data suitable for molecular risk assessment of the potential virulence of STEC isolates. The results obtained using microarrays technology confirmed high effectiveness of this method in determining STEC virulotypes, which certainly supports the implementation of this method for the diagnosis of STEC isolates in our country in selected provincial sanitary – epidemiological laboratories.
This study was financially supported by the National Science Centre, grant no. N404 096838.We are deeply grateful to Professor Jacek Osek from the Department of Hygiene of Food of Animal Origin at the National Veterinary Research Institute (NVRI) in Puławy and dr Halina Ścieżyńska from the Department of Food and Consumer Articles Research in NIPH-NIH in Warsaw for providing the STEC strains. Special thanks to Professor Rafal Gierczynski for critical reading of the manuscript and many helpful suggestions.
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