JOURNAL OF CLINICAL MICROBIOLOGY, May 2006, p. 1863–1866 0095-1137/06/$08.00⫹0 doi:10.1128/JCM.44.5.1863–1866.2006 Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 44, No. 5
Isolation, Characterization, and Epidemiological Assessment of Shiga Toxin-Producing Escherichia coli O84 Isolates from New Zealand Adrian L. Cookson,1* Dawn Croucher,2 Chris Pope,2 Jenny Bennett,2 Fiona Thomson-Carter,2 and Graeme T. Attwood1 Metabolism and Microbial Genomics Section, Food and Health Group, AgResearch, Grasslands Research Centre, Tennent Drive, Palmerston North,1 and Enteric Reference Laboratory, ESR Keneperu Science Centre, Porirua,2 New Zealand Received 9 February 2006/Accepted 17 February 2006
Shiga toxin-producing Escherichia coli O84 isolates (n ⴝ 22) were examined using culture- and molecularly based methods in order to compare their phenotypic and genotypic characteristics. These analyses directly linked Shiga toxin-producing Escherichia coli O84 isolates from cattle and sheep with human isolates indicating that New Zealand livestock may be a reservoir of infection. strains detected from human clinical cases are serogroup O157, but whether this is a true reflection of most STEC infections being associated with O157 or whether this is a consequence of screening methods is unknown. Recent investigations detected only non-O157 STEC strains, such as O5, O26, O84, O91, and O128) from New Zealand cattle and sheep
The first recorded case in New Zealand of Shiga toxinproducing Escherichia coli (STEC) O157:H7 was in 1993 (3), and since then, STEC strains, including O157, have been isolated from sporadic cases of infection in increasing numbers, reaching a maximum of 91 cases (88 O157 and 3 non-O157) isolates in 2003 (1). Within New Zealand, over 90% of STEC
TABLE 1. STEC O84 isolates used in this study and their phenotypic and genotypic characteristicsa Plasmid characteristics Serotype
O84:H⫺ O84:H2 O84:H2 O84:H⫺ O84:H⫺ O84:H⫺ O84:H2 O84:H⫺ O84:H⫺ O84:H2 O84:H⫺ O84:H⫺ O84:H⫺ O84:H⫺ O84:H2 O84:H⫺ O84:H⫺ O84:H⫺ O84:H⫺ O84:H⫺ O84:H⫺ O84:H⫺ a b
Strain
AGR066 AGR069 AGR081 AGR091 AGR133 AGR349 AGR353 AGR563 AGR748 AGR769 AGR867 03-1204 02-2853 03-3976 04-3069 05-0494 4177 4178 4179 4180 537/89 IHIT3669
Sourceb
Bovine (New Zealand) Bovine (New Zealand) Bovine (New Zealand) Bovine (New Zealand) Bovine (New Zealand) Bovine (New Zealand) Bovine (New Zealand) Ovine (New Zealand) Bovine (New Zealand) Bovine (New Zealand) Ovine (New Zealand) Human, sinus (New Zealand) Human ND (New Zealand) Human D (New Zealand) Human D (New Zealand) Human D (New Zealand) Bovine (Scotland) Bovine (Scotland) Bovine (Scotland) Bovine (Scotland) Bovine (Germany) Bovine (Germany)
SF
-Glucuronidase
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹
⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺
⫹, positive for process or enzyme; ⫺, negative for process or enzyme. ND, no details; D, diarrhea.
* Corresponding author. Mailing address: Metabolism and Microbial Genomics Section, Food and Health Group, AgResearch, Grasslands Research Centre, Tennent Drive, Private Bag 11008, Palmerston North, New Zealand. Phone: 64 6 351 8229. Fax: 64 6 351 8003. E-mail:
[email protected]. 1863
PFGE pattern
K I J H E G G H M L J F E D C C A B A A O N
EcoRI fragment size (kb) by hybridization with STEC ehxA probe
BamHI
EcoRI
6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 6.7 10.0 6.7 6.7 6.7 6.7 6.7 6.7 6.7 3.2 4.5
A B A A B A A B B A A B C D E F G G G G H I
A B A A B A A B B A A B C A D C E E E E F G
Digest pattern
1864
NOTES
J. CLIN. MICROBIOL.
FIG. 1. Restriction enzyme digests and Southern hybridization of O84:H⫺/H2 plasmids. Plasmids were cut with BamHI (a) or EcoRI (b) and run on 0.8% (wt/vol) agarose gel or hybridization of an EcoRI-cut plasmid digest with STEC ehxA purified PCR product (c). Lane 1, AGR066; lane 2, AGR069; lane 3, AGR081; lane 4, AGR091; lane 5, AGR133; lane 6, AGR349; lane 7, AGR353; lane 8, AGR563; lane 9, AGR748; lane 10, AGR769; lane 11, AGR867; lane 12, 03-1204; lane 13, 02-2853; lane 14, 03-3976; lane 15, 04-3069; lane 16, 05-0494; lane 17, 4177; lane 18, 4178; lane 19, 4179; lane 20, 4180; lane 21, 537/90; lane 22, IHIT3669; lane 23, O157:H7 NCTC12900. M, ⬎1-kb molecular mass ladder.
(7, 8). In this report, we outline the emergence of serogroup O84 as a causative agent of diarrheal disease in New Zealand, its source, its phenotypic and genotypic analysis, and comparison with several O84 isolates obtained overseas. Twenty-two STEC O84 isolates were examined in this study. Of these, nine O84:H⫺/H2 strains were isolated from cattle and two O84:H⫺ strains were isolated from sheep (7, 8). Four bovine isolates from Scotland (10), two bovine isolates from Germany (11), and five human clinical O84 isolates from New Zealand were also included in the study (Table 1). In contrast to the 16 New Zealand O84 isolates that were non-sorbitol fermenting (NSF) and -glucuronidase negative, the four Scottish isolates and IHIT3669 were sorbitol fermenting (SF) and -glucuronidase negative (Table 1). The remaining German isolate, 537/89, was positive for SF and -glucuronidase positive (Table 1). stx and/or eae detection was performed using a multiplex PCR method outlined previously (6). Primers intF and (zeta)
intR, based on GenBank sequence AJ298279 from STEC O84 537/89 (11), were used to detect the eae subtype. The STEC enterohemolysin (ehxA), serine protease (espP), katalase peroxidase (katP), and type II secretion system (etpD) genes were detected using primers outlined previously (5, 6, 12). PCR analysis indicated that all 22 of the isolates were stx1, eae ( subtype), ehxA, and espP positive (Table 1). Plasmids were isolated and digested with BamHI or EcoRI to compare plasmid profiles and Southern blot analysis was performed using a 534-bp fragment from the ehxA locus from E. coli O157:H7 (NCTC12900) as a probe against EcoRI-cut plasmid DNA that was transferred to a nylon membrane. Nine distinctive BamHI plasmid digest profiles and seven distinctive EcoRI plasmid digest profiles were observed (Fig. 1). The New Zealand O84 isolates could be grouped into at least four EcoRI plasmid profile types although profiles A and B could be readily distinguished only through extended electrophoresis (data not shown). The STEC ehxA probe hybridized to a single
VOL. 44, 2006
NOTES
1865
FIG. 2. XbaI PFGE profiles of STEC O84H⫺/H2 from cattle, sheep and New Zealand clinical isolates. The scale at the top shows similarity by the Dice coefficient. Sco, Scotland; NZ, New Zealand; Ger, Germany.
EcoRI fragment of approximately 6.8 kb in 14 of 15 New Zealand isolates (Fig. 1c) but hybridized to a fragment of approximately 10-kb associated with 02-2853. Pulse-field gel electrophoresis (PFGE) of the 22 O84 isolates was performed using the PulseNet standardized protocol (2). Sixteen different PFGE profiles were identified among the 22 O84 isolates. Although the PFGE pattern E derived from a bovine O84 isolate, AGR133, was indistinguishable from that of the human isolate 02-2853 (Fig. 2) and their BamHI plasmid digest profiles were relatively similar (Fig. 1a), the 02-2853 EcoRI plasmid digest profile differed from that of AGR133 by an insertion of approximately 3 kb in the corresponding 7.2-kb band (Fig. 1b). The apparent significance in the isolation of five STEC O84 isolates from cases of human illness in New Zealand over a period of approximately three years led us to examine whether there were any phenotypic or genotypic links between the STEC O84 samples recently isolated from cattle and sheep (8). While O84:H⫺ has been associated with diarrhea and/or hemolytic-uremic syndrome in Germany (9, 13) and Spain (4), disease associated with the O84:H2 serotype has not been recorded. STEC O84 has been isolated worldwide, mainly from healthy cattle in Canada, Belgium, Germany, Japan,
France, Hong Kong, United Kingdom, the United States, and Australia but also from cattle with diarrhea and from healthy sheep (www.microbionet.com.au/vtecfullref1u.htm), indicating that healthy cattle and sheep are a reservoir for STEC O84 worldwide. The worldwide distribution of the NSF, -glucuronidase-negative clone resistant to cefixime and tellurite is unknown, but none of the overseas isolates had this phenotype. The cefixime- and tellurite-resistant, NSF, -glucuronidasenegative phenotype, typical of the New Zealand O84 isolates may be a factor in their comparative frequency of isolation from recent human diarrheal cases in New Zealand. In contrast, the apparent rarity of the O84 serogroup as a potential causative agent of diarrheal disease in Europe and the rest of the world may be due to its ability to ferment sorbitol, a trait that may be overlooked when the focus is upon NSF STEC strains such as O157:H7. In this study, however, we have been able to examine only sorbitol fermentation of six non-New Zealand O84 isolates. PFGE has been proven as a powerful tool for the epidemiological analysis of bacterial isolates isolated from human disease and comparison with isolates from animals or from the environment through visualization of the whole bacterial genome and provides a high degree of discrimination within STEC serogroups. This study, besides linking an
1866
NOTES
O84:H⫺ isolate isolated from cattle with an O84:H⫺ isolate associated with human disease through PFGE analysis, also indicates that STEC O84 from cattle, sheep, and clinical cases have sufficiently similar virulence levels and PFGE profiles to imply that they may be a significant risk to human health in New Zealand through ingestion of contaminated food or direct contact with animals or fecally contaminated material. This link has been made completely independently of any epidemiological investigations associated with outbreaks or sporadic cases of STEC infection and is an example of how routine bacterial surveillance from cattle and sheep may be used to identify possible reservoirs of pathogens having similar PFGE profiles. Ideally, all human clinical fecal samples should be screened by a stx toxin-based test, and even if diarrheal episodes are confirmed as not being caused by serogroup O157, it is important to be aware that non-sorbitol fermenting, -glucuronidase-negative isolates that are also resistant to cefixime and tellurite, and that do not react with anti-E. coli O157 antisera or latex reagent, should be assessed for the presence of stx and other associated virulence factors in order to preclude non-O157 STEC infection. In summary, this is the first study to note the apparent link between STEC O84 from cattle and sheep with human disease through PFGE analysis; however, the actual routes of transmission and the minimal infectious dose for disease to occur are unknown. Therefore, further focus is required to evaluate the significance of STEC O84 as a cause of human disease both in New Zealand and worldwide and to establish its pathogenic mechanisms.
J. CLIN. MICROBIOL.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
This study was funded through AgResearch Repositioning funds. REFERENCES 1. Anonymous. 2004. New Zealand public health surveillance report. Institute of Environmental Science and Research Limited, Wellington, New Zealand. [Online.] http://www.surv.esr.cri.nz/surveillance/NZPHSR.php?we_objectID⫽303. 2. Anonymous. 2004. PulseNet USA standardized molecular subtyping of food-
13.
borne bacterial pathogens by pulsed-field gel electrophoresis (manual). National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Ga. [Online.] http://www.cdc.gov/pulsenet/protocols/ecoli _salmonella_shigella_protocols.pdf. Baker, M., R. Eyles, J. Bennett, C. Nicol, W. Wong, and N. Garrett. 1999. Emergence of verotoxigenic Escherichia coli (VTEC) in New Zealand. N. Z. Public Health Rep. 6:9–12. Blanco, J., M. Blanco, J. E. Blanco, A. Mora, M. P. Alonso, E. A. Gonza ´lez, and M. I. Berna ´rdez. 2001. Epidemiology of verocytotoxigenic Escherichia coli (VTEC) in ruminants. In G. Duffy, P. Garvey, and D. McDowell (ed.), Verocytotoxigenic Escherichia coli. Food and Nutrition Press Inc., Trumbull, Conn. Brunder, W., H. Schmidt, and H. Karch. 1996. KatP, a novel catalaseperoxidase encoded by the large plasmid of enterohaemorrhagic Escherichia coli O157:H7. Microbiology 142:3305–3315. Cookson, A. L., C. M. Hayes, G. R. Pearson, J. M. Roe, A. D. Wales, and M. J. Woodward. 2002. Isolation from sheep of an attaching and effacing Escherichia coli O115:H⫺ with a novel combination of virulence factors. J. Med. Microbiol. 51:1041–1049. Cookson, A. L., S. C. S. Taylor, and G. T. Attwood. 2006. The prevalence of Shiga toxin-producing Escherichia coli in cattle and sheep in the lower North Island, New Zealand. N. Z. Vet. J. 54:28–33. Cookson, A. L., S. C. S. Taylor, J. Bennett, F. Thomson-Carter, and G. T. Attwood. 2006. Serotypes and analysis of distribution of Shiga toxin-producing Escherichia coli in cattle and sheep in the lower North Island, New Zealand. N. Z. Vet. J. 54:78–84. Friedrich, A. W., M. Bielaszewska, W. L. Zhang, M. Pulz, T. Kuczius, A. Ammon, and H. Karch. 2002. Escherichia coli harboring Shiga toxin 2 gene variants: frequency and association with clinical symptoms. J. Infect. Dis. 185:74–84. Jenkins, C., M. C. Pearce, H. Chart, T. Cheasty, G. A. Willshaw, G. J. Gunn, G. Dougan, H. R. Smith, B. A. Synge, and G. Frankel. 2002. An eight-month study of a population of verocytotoxigenic Escherichia coli (VTEC) in a Scottish cattle herd. J. Appl. Microbiol. 93:944–953. Jores, J., K. Zehmke, J. Eichberg, L. Rumer, and L. H. Wieler. 2003. Description of a novel intimin variant (type zeta) in the bovine O84:NM verotoxin-producing Escherichia coli strain 537/89 and the diagnostic value of intimin typing. Exp. Biol. Med. 228:370–376. Paton, A. W., and J. C. Paton. 1998. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157. J. Clin. Microbiol. 36:598–602. Schmidt, H., J. von Maldeghem, M. Frosch, and H. Karch. 1998. Antibiotic susceptibilities of verocytotoxin-producing Escherichia coli O157 and nonO157 isolates isolated from patients and healthy subjects in Germany during 1996. J. Antimicrob. Chemother. 42:548–550.
