Accepted Article
Article Type: Original Article
Novel sequence types of non-O157 Shiga toxin-producing E. coli isolated from cattle
Joshua Isiko1,2, Margaret Khaitsa1,3, and Teresa M. Bergholz1,* 1
Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND
58102
2
College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, P.O Box 7062,
Kampala, Uganda
3
Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi
State University, Mississippi State, MS 39762
*
corresponding author
Teresa M. Bergholz 130A Van Es North Dakota State University Fargo, ND 58102 701-231-7692
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12404 This article is protected by copyright. All rights reserved.
Accepted Article
[email protected]
Running title: Novel STs of non-O157 STEC
SIGNIFICANCE and IMPACT OF THE STUDY The foodborne pathogen Shiga toxin-producing Escherichia coli (STEC) is a significant public health concern. One of the main reservoirs for STEC are cattle, which can directly or indirectly contribute to STEC in the food supply. The genetic subtype data presented here highlight the diversity of STEC that can be isolated from cattle. These results further our understanding of the ecology of STEC in the primary production environment, which is important for developing effective control measures to reduce this pathogen in the food supply.
ABSTRACT The objective of this study was to assess the genetic diversity of non-O157 STEC isolates from cattle. Multi-locus sequence typing (MLST) was used to identify and compare the sequence types (STs) of 43 non-O157 STEC cattle isolates using the EcMLST database curated by the STEC Center at Michigan State University. For the 43 isolates, 19 STs were identified and 10 of those STs were novel compared to those in EcMLST. For the 43 isolates, 19 different serotypes were identified. STEC O22:H8, O174:H28, and O8:H19 were most common, and STEC O8 isolates were the most diverse, with 7 different STs for isolates with that O-group. STEC strains with O-types identified in this study have been isolated from cattle by other researchers, as well as from cases of human gastroenteritis. Of the 10 novel STs
This article is protected by copyright. All rights reserved.
Accepted Article
identified, 6 were found to be closely related to previously identified STs, indicating that populations of non-O157 STEC in cattle are similar to those from other sources, including human clinical cases.
Keywords: non-O157 STEC, cattle, MLST, sequence typing, E. coli, non-O157 VTEC
INTRODUCTION Shiga toxin-producing Escherichia coli (STEC), also known as Vero toxin-producing E. coli (VTEC), are
human pathogens of zoonotic origin which have been linked to serious disease such as hemorrhagic colitis and hemolytic uremic syndrome (Smith, et al., 2014). Over 400 serotypes of STEC/VTEC are identified to be pathogenic due to the presence of one or more genes encoding Shiga toxin (Blanco, et al., 2004, Brooks, et al., 2005, Hussein, 2007, Tozzoli and Scheutz, 2014). The most studied serotype of STEC to date has been O157:H7, due to large outbreaks in the 1980’s and 1990’s associated with strains of this serotype (Goldwater and Bettelheim, 1998). The importance of non-O157 STEC serotypes has been increasingly recognized, and the most recent estimates from the U.S. Centers for Disease Control and Prevention indicate that non-O157 STEC are responsible for the majority of human illness caused by STEC (Scallan, et al., 2011). Surveillance data of foodborne outbreaks in the U.S. from 1998-2008 report that 308 outbreaks during this period were linked to STEC, and of those outbreaks that could be attributed to a specific food source, 78 were linked to beef (Gould, et al., 2013). Cattle are considered to be the main reservoir of STEC, and either directly or indirectly contribute to the presence of STEC in the food supply. The prevalence of STEC O157 in cattle can range from 0.3% to 27.3%, and depends on a number of factors such as feed type and season (Hussein, 2007). The prevalence of non-O157 STEC in cattle is also variable, and has been found to range from 4.6% to 55.9%
This article is protected by copyright. All rights reserved.
Accepted Article
(Hussein, 2007). Overall, the current data indicate that non-O157 STEC are just as prevalent in cattle as STEC O157. A considerable amount of research has been conducted to understand the dynamics of STEC colonization in bovine herds and to determine how STEC may be transmitted between cattle or from the environment to cattle, though the majority of this research to date has been focused on STEC O157. In many cases, molecular subtyping data are used to assess transfer of STEC strains among cattle (Dunn, et al., 2004), between cattle and their environment (LeJeune, et al., 2004), among cattle farms within a geographic region (Renter, et al., 2004, Wetzel and LeJeune, 2006), or between cattle and beef during processing (Dodd, et al., 2010).
While molecular subtyping data have provided insights into the diversity of STEC O157 and how it can spread between cattle and their environment, comparatively less is known about the diversity of nonO157 STEC from cattle and on cattle farms. A greater understanding of the ecology of STEC in food production animals, in foods, and in the environment is needed to develop effective control strategies for this pathogen. To assess the genetic diversity of non-O157 STEC from cattle, we used multi-locus sequencing typing (MLST) to determine genetic differences among a set of isolates from cattle. We compared the sequence type data to those in the EcMLST database, and identified 10 novel sequence types from the set of 43 non-O157 isolates from cattle.
RESULTS AND DISCUSSION Serotypes of the non-O157 STEC isolates from cattle All of the non-O157 STEC isolates had previously been identified by PCR as possessing stx1 and/or stx2
(Ekiri, et al., 2014) (Table 1). The serotypes of these isolates were determined by O-typing at the E. coli
This article is protected by copyright. All rights reserved.
Accepted Article
Article Type: Original Article
Novel sequence types of non-O157 Shiga toxin-producing E. coli isolated from cattle
Joshua Isiko1,2, Margaret Khaitsa1,3, and Teresa M. Bergholz1,* 1
Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND
58102
2
College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, P.O Box 7062,
Kampala, Uganda
3
Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi
State University, Mississippi State, MS 39762
*
corresponding author
Teresa M. Bergholz 130A Van Es North Dakota State University Fargo, ND 58102 701-231-7692
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12404 This article is protected by copyright. All rights reserved.
Accepted Article
STEC 022:H8 and O22:H53 isolates were identified as either ST 1059, a novel ST, or ST 145. ST 1059 differs from ST 145 by a single nucleotide in icdA, and was assigned to CG 31. Of the data available in EcMLST, CG 31 contains 38 different STs, and includes STEC strains of serotype O22. STEC O22 strains have been frequently isolated from cattle (Pradel, et al., 2000, Urdahl, et al., 2003, Blanco, et al., 2004), and more rarely from human cases of illness (Gould, et al., 2013). Two of the four ST 145 strains in EcMLST were from humans, and no source data are available for the other two strains. All seven O174:H28 isolates were ST 160, which is part of CG 21. STEC O174 isolates have been found to belong to 4 distinct phylogenetic groups, including CG 21, with an isolate from cattle identified as ST 160 (Tarr, et al., 2008). STEC O91 isolates were identified as ST 89 (2 isolates), which belongs to CG 34, or to novel ST 1062, which is related to, but outside of CG 34 (Fig. 1). ST 1062 differs from ST 89 by 10 nucleotides. STEC O91 strains have commonly been isolated from cattle and sheep (Urdahl, et al., 2003, Blanco, et al., 2004), while accounting for 1% of non-O157 STEC isolated from human cases (Gould, et al., 2013).
Cattle isolates of serotype O8 were found to be the most diverse, with 10 STEC O8 isolates belonging to
7 different STs. Of these STs, 5 were assigned to CG 43, which is characterized as STEC O8:H19 in the EcMLST database. Two novel STs were found to be part of this CG, ST 1063 differs from ST 221 by 4 nucleotides, and from ST 254 by 2 nucleotides. ST 1060 differs from ST 1063 by 6 nucleotides. Two recent studies using multi-locus variable number of tandem repeats analysis (MLVA) to subtype nonO157 STEC found varying degrees of diversity among STEC O8 isolates. STEC O8 isolates from raw beef products in Argentina were found to be quite diverse, with three different MLVA profiles (Franci, et al., 2011), while STEC O8 isolates from cattle in Mexico all had the same MLVA profile (Amezquita-Lopez, et al., 2012).
This article is protected by copyright. All rights reserved.
Accepted Article
A number of cattle STEC isolates were found to belong to CGs that are not commonly associated with STEC strains. Two isolates of O103:H2 had ST 119, which belongs to CG 17. The majority of isolates in this CG are classified as enteropathogenic E. coli with the H2 antigen. An O2:H34 STEC cattle isolate belongs to CG 23, which contains mostly isolates from healthy humans. Two novel STs were also found to belong to CGs not typically associated with STEC strains. STEC O8:H11 with ST 1064 belongs to CG 13, which mainly contains enterotoxigenic E. coli isolates, and STEC O2:H33 with ST 1065 belongs to CG 35, which mainly contains E. coli isolated from extra-intestinal infections. It is important to note that the majority of isolates in the EcMLST database are from humans – of the 3812 isolates with 7 gene MLST data, for those with source information 582 isolates are from humans, while only 57 are from cattle.
MATERIALS AND METHODS Non-O157 STEC isolates Non-O157 STEC isolates from cattle fecal samples were collected from the North Dakota State University Research Extension Center at Dickinson, North Dakota, as described previously (Ekiri, et al., 2014). Ekiri et al. sampled 48 cows (4 times) and 48 calves (5 times) over an approximately 1 year period. Cows were housed the entire time at the Dickinson Research Extension Center, while calves were transferred to the University of Nebraska Panhandle Research Feedlot for finishing. A total of 208 isolates were identified as STEC due to the presence of stx1 and/or stx2 (detected by PCR; Gannon et al., (1992) as
described previously (Ekiri, et al. 2014). Here we randomly selected a subset of 43 of the 208 isolates which had stx1 and/or stx2 for further characterization using multi-locus sequence typing (MLST). Of
these 43 isolates, 29 were from fecal samples from adult cows and 14 were from fecal samples from calves (
Article Type: Original Article
Novel sequence types of non-O157 Shiga toxin-producing E. coli isolated from cattle
Joshua Isiko1,2, Margaret Khaitsa1,3, and Teresa M. Bergholz1,* 1
Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND
58102
2
College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, P.O Box 7062,
Kampala, Uganda
3
Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi
State University, Mississippi State, MS 39762
*
corresponding author
Teresa M. Bergholz 130A Van Es North Dakota State University Fargo, ND 58102 701-231-7692
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12404 This article is protected by copyright. All rights reserved.
Accepted Article
[email protected]
Running title: Novel STs of non-O157 STEC
SIGNIFICANCE and IMPACT OF THE STUDY The foodborne pathogen Shiga toxin-producing Escherichia coli (STEC) is a significant public health concern. One of the main reservoirs for STEC are cattle, which can directly or indirectly contribute to STEC in the food supply. The genetic subtype data presented here highlight the diversity of STEC that can be isolated from cattle. These results further our understanding of the ecology of STEC in the primary production environment, which is important for developing effective control measures to reduce this pathogen in the food supply.
ABSTRACT The objective of this study was to assess the genetic diversity of non-O157 STEC isolates from cattle. Multi-locus sequence typing (MLST) was used to identify and compare the sequence types (STs) of 43 non-O157 STEC cattle isolates using the EcMLST database curated by the STEC Center at Michigan State University. For the 43 isolates, 19 STs were identified and 10 of those STs were novel compared to those in EcMLST. For the 43 isolates, 19 different serotypes were identified. STEC O22:H8, O174:H28, and O8:H19 were most common, and STEC O8 isolates were the most diverse, with 7 different STs for isolates with that O-group. STEC strains with O-types identified in this study have been isolated from cattle by other researchers, as well as from cases of human gastroenteritis. Of the 10 novel STs
This article is protected by copyright. All rights reserved.
Accepted Article
identified, 6 were found to be closely related to previously identified STs, indicating that populations of non-O157 STEC in cattle are similar to those from other sources, including human clinical cases.
Keywords: non-O157 STEC, cattle, MLST, sequence typing, E. coli, non-O157 VTEC
INTRODUCTION Shiga toxin-producing Escherichia coli (STEC), also known as Vero toxin-producing E. coli (VTEC), are
human pathogens of zoonotic origin which have been linked to serious disease such as hemorrhagic colitis and hemolytic uremic syndrome (Smith, et al., 2014). Over 400 serotypes of STEC/VTEC are identified to be pathogenic due to the presence of one or more genes encoding Shiga toxin (Blanco, et al., 2004, Brooks, et al., 2005, Hussein, 2007, Tozzoli and Scheutz, 2014). The most studied serotype of STEC to date has been O157:H7, due to large outbreaks in the 1980’s and 1990’s associated with strains of this serotype (Goldwater and Bettelheim, 1998). The importance of non-O157 STEC serotypes has been increasingly recognized, and the most recent estimates from the U.S. Centers for Disease Control and Prevention indicate that non-O157 STEC are responsible for the majority of human illness caused by STEC (Scallan, et al., 2011). Surveillance data of foodborne outbreaks in the U.S. from 1998-2008 report that 308 outbreaks during this period were linked to STEC, and of those outbreaks that could be attributed to a specific food source, 78 were linked to beef (Gould, et al., 2013). Cattle are considered to be the main reservoir of STEC, and either directly or indirectly contribute to the presence of STEC in the food supply. The prevalence of STEC O157 in cattle can range from 0.3% to 27.3%, and depends on a number of factors such as feed type and season (Hussein, 2007). The prevalence of non-O157 STEC in cattle is also variable, and has been found to range from 4.6% to 55.9%
This article is protected by copyright. All rights reserved.
Accepted Article
(Hussein, 2007). Overall, the current data indicate that non-O157 STEC are just as prevalent in cattle as STEC O157. A considerable amount of research has been conducted to understand the dynamics of STEC colonization in bovine herds and to determine how STEC may be transmitted between cattle or from the environment to cattle, though the majority of this research to date has been focused on STEC O157. In many cases, molecular subtyping data are used to assess transfer of STEC strains among cattle (Dunn, et al., 2004), between cattle and their environment (LeJeune, et al., 2004), among cattle farms within a geographic region (Renter, et al., 2004, Wetzel and LeJeune, 2006), or between cattle and beef during processing (Dodd, et al., 2010).
While molecular subtyping data have provided insights into the diversity of STEC O157 and how it can spread between cattle and their environment, comparatively less is known about the diversity of nonO157 STEC from cattle and on cattle farms. A greater understanding of the ecology of STEC in food production animals, in foods, and in the environment is needed to develop effective control strategies for this pathogen. To assess the genetic diversity of non-O157 STEC from cattle, we used multi-locus sequencing typing (MLST) to determine genetic differences among a set of isolates from cattle. We compared the sequence type data to those in the EcMLST database, and identified 10 novel sequence types from the set of 43 non-O157 isolates from cattle.
RESULTS AND DISCUSSION Serotypes of the non-O157 STEC isolates from cattle All of the non-O157 STEC isolates had previously been identified by PCR as possessing stx1 and/or stx2
(Ekiri, et al., 2014) (Table 1). The serotypes of these isolates were determined by O-typing at the E. coli
This article is protected by copyright. All rights reserved.
Accepted Article
Article Type: Original Article
Novel sequence types of non-O157 Shiga toxin-producing E. coli isolated from cattle
Joshua Isiko1,2, Margaret Khaitsa1,3, and Teresa M. Bergholz1,* 1
Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND
58102
2
College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, P.O Box 7062,
Kampala, Uganda
3
Department of Pathobiology and Population Medicine, College of Veterinary Medicine, Mississippi
State University, Mississippi State, MS 39762
*
corresponding author
Teresa M. Bergholz 130A Van Es North Dakota State University Fargo, ND 58102 701-231-7692
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/lam.12404 This article is protected by copyright. All rights reserved.
Accepted Article
STEC 022:H8 and O22:H53 isolates were identified as either ST 1059, a novel ST, or ST 145. ST 1059 differs from ST 145 by a single nucleotide in icdA, and was assigned to CG 31. Of the data available in EcMLST, CG 31 contains 38 different STs, and includes STEC strains of serotype O22. STEC O22 strains have been frequently isolated from cattle (Pradel, et al., 2000, Urdahl, et al., 2003, Blanco, et al., 2004), and more rarely from human cases of illness (Gould, et al., 2013). Two of the four ST 145 strains in EcMLST were from humans, and no source data are available for the other two strains. All seven O174:H28 isolates were ST 160, which is part of CG 21. STEC O174 isolates have been found to belong to 4 distinct phylogenetic groups, including CG 21, with an isolate from cattle identified as ST 160 (Tarr, et al., 2008). STEC O91 isolates were identified as ST 89 (2 isolates), which belongs to CG 34, or to novel ST 1062, which is related to, but outside of CG 34 (Fig. 1). ST 1062 differs from ST 89 by 10 nucleotides. STEC O91 strains have commonly been isolated from cattle and sheep (Urdahl, et al., 2003, Blanco, et al., 2004), while accounting for 1% of non-O157 STEC isolated from human cases (Gould, et al., 2013).
Cattle isolates of serotype O8 were found to be the most diverse, with 10 STEC O8 isolates belonging to
7 different STs. Of these STs, 5 were assigned to CG 43, which is characterized as STEC O8:H19 in the EcMLST database. Two novel STs were found to be part of this CG, ST 1063 differs from ST 221 by 4 nucleotides, and from ST 254 by 2 nucleotides. ST 1060 differs from ST 1063 by 6 nucleotides. Two recent studies using multi-locus variable number of tandem repeats analysis (MLVA) to subtype nonO157 STEC found varying degrees of diversity among STEC O8 isolates. STEC O8 isolates from raw beef products in Argentina were found to be quite diverse, with three different MLVA profiles (Franci, et al., 2011), while STEC O8 isolates from cattle in Mexico all had the same MLVA profile (Amezquita-Lopez, et al., 2012).
This article is protected by copyright. All rights reserved.
Accepted Article
A number of cattle STEC isolates were found to belong to CGs that are not commonly associated with STEC strains. Two isolates of O103:H2 had ST 119, which belongs to CG 17. The majority of isolates in this CG are classified as enteropathogenic E. coli with the H2 antigen. An O2:H34 STEC cattle isolate belongs to CG 23, which contains mostly isolates from healthy humans. Two novel STs were also found to belong to CGs not typically associated with STEC strains. STEC O8:H11 with ST 1064 belongs to CG 13, which mainly contains enterotoxigenic E. coli isolates, and STEC O2:H33 with ST 1065 belongs to CG 35, which mainly contains E. coli isolated from extra-intestinal infections. It is important to note that the majority of isolates in the EcMLST database are from humans – of the 3812 isolates with 7 gene MLST data, for those with source information 582 isolates are from humans, while only 57 are from cattle.
MATERIALS AND METHODS Non-O157 STEC isolates Non-O157 STEC isolates from cattle fecal samples were collected from the North Dakota State University Research Extension Center at Dickinson, North Dakota, as described previously (Ekiri, et al., 2014). Ekiri et al. sampled 48 cows (4 times) and 48 calves (5 times) over an approximately 1 year period. Cows were housed the entire time at the Dickinson Research Extension Center, while calves were transferred to the University of Nebraska Panhandle Research Feedlot for finishing. A total of 208 isolates were identified as STEC due to the presence of stx1 and/or stx2 (detected by PCR; Gannon et al., (1992) as
described previously (Ekiri, et al. 2014). Here we randomly selected a subset of 43 of the 208 isolates which had stx1 and/or stx2 for further characterization using multi-locus sequence typing (MLST). Of
these 43 isolates, 29 were from fecal samples from adult cows and 14 were from fecal samples from calves (
