Abstract
Escherichia coli O104 isolates collected from different sources in the United States were examined for virulence genes typical of enterohemorrhagic E. coli and those identified in the O104:H4 isolate associated with the 2011 German outbreak. The unexpected presence of virulence markers in these isolates highlights the importance of screening unusual and potentially pathogenic Shiga toxin-producing E. coli serotypes.
TEXT
Shiga toxin-producing Escherichia coli (STEC) can cause various degrees of human illnesses, including diarrhea, hemorrhagic colitis (HC), and life-threatening hemolytic uremic syndrome (HUS) (11). A number of STEC serotypes have been implicated in food-borne illnesses worldwide; although STEC serotype O157:H7 is the most common serotype associated with severe consequences such as HUS, many “non-O157 STEC” serotypes are gaining importance as food-borne pathogens (10). Among them, serogroups O26, O45, O103, O111, O121, and O145 accounted for 83% of cases of non-O157 STEC infections in the United States from 1983 to 2002 (4). In May 2011, a rare serotype, O104:H4, caused a major outbreak in Europe (mostly in Germany) with 4,321 illnesses and an unusually high number of HUS cases (∼852), including 50 deaths (15). In the United States, six cases of O104:H4 infections, with one death linked to travel to Germany, were reported (8). This O104:H4 strain was found to be a clone that combines the virulence characteristics of enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC). It harbored Shiga toxin gene 2 (stx2) and several EAEC marker genes such as aatA, aggA, aggR, set1, pic, and aap but was negative for other EHEC markers such as Stx1, intimin (eae), and enterohemolysin (hly) (2). Knowledge about this serotype is limited, largely because E. coli O104:H4 has only recently been recognized as a food-borne pathogen capable of causing severe disease and overshadowing past outbreaks by other serotypes. The severity of the recent O104:H4 outbreak, the uncommon vehicle of transmission (seeds of fenugreek), and the unique characteristics of the outbreak strain signify the need for a thorough investigation of this serotype in the United States.
We obtained 58 E. coli O104 isolates with diverse flagellar (H) antigens from different animal and environmental sources in the collections of the E. coli Reference Center at Pennsylvania State University and examined their genetic diversity along with the presence and distribution of virulence markers identified in the 2011 E. coli O104:H4 outbreak strain. Isolates were characterized using several molecular and phenotypic assays. We also looked for virulence loci specific for the E. coli O104:H4 outbreak strain and other EHEC markers and established genetic relationships among them. Because of the increased public health significance of E. coli O104, this study aimed to determine the presence of unique characteristics of O104:H4 that may also have promoted the emergence of this pathogen in the United States and to evaluate the pathogenic potential of those strains found harboring these factors.
The E. coli isolates listed in Table 1 were confirmed using a Vitek 2 Compact system (bioMérieux, Durham, NC) following the manufacturer's instructions. The O104 serotype (rfbO104) and the presence of stx1, stx2, terD, and fliCH4 were confirmed using PCR as previously described (1, 2). Individual PCR analyses were performed to determine the prevalence of other E. coli virulence genes (eae, iha, lpfAO26, irp2, fyuA, aatA, aggR, set1, and pic) as described previously (3, 5–7, 9, 12, 16–18). None of the isolates (Table 1) carried fliCH4. The prevalence of the stx2 gene (2%) was lower than that of stx1 gene (20%) in the isolates analyzed, with only one isolate (9.0124) carrying stx2 and 11 isolates harboring stx1. Although none of the isolates were found to carry intimin (eae), other adhesion marker genes (iha, lpfAO26) were present in more than half of the isolates (62%).
Table 1.
Virulence loci, antimicrobial susceptibility, and hemolysis activity of E. coli O104 isolates
| Isolate | No. of isolates with indicated serotypea |
Sourceb |
Virulence gene or locus |
Heme | R-typef | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| EHECc |
EAECd |
||||||||||||||||
| O | H | Carrier | Location, date of collection | stx | terD | ipAfO26 | irp2 | fyuA | iha | eae | aatA | aggR | set1 | pic | |||
| EDL933g | 157 | 7 | Human | 1, 2 | + | − | − | − | + | + | − | − | − | − | + | NA | |
| GER | 104 | 4 | Human, HUS | Germany, 2011 | 2 | + | + | + | + | + | − | + | + | + | + | − | Amp, Tio, Axo, Nal, Str, Tet, Cot |
| MN | 104 | 21 | Human | MN, 2001 | 2 | − | + | + | − | + | − | − | − | − | − | + | NA |
| GEO | 104 | 4 | Human | GA, 2004 | 2 | + | + | + | + | + | − | + | + | + | + | − | Amp, Nal, Str, Tet, Cot |
| 0.0122 | 104 | ? | ? | ? | − | − | − | − | − | − | − | − | − | − | − | − | Amp |
| 0.3194 | 104 | ? | Water | NY | − | − | − | + | − | − | − | − | − | − | − | + | − |
| 0.3359 | 104 | ? | Water | NY | − | − | − | + | − | − | − | − | − | − | − | + | − |
| 0.3362 | 104 | ? | Water | NY | − | − | − | + | − | − | − | − | − | − | − | + | − |
| 1.1952 | 104 | ? | Human | MN | − | − | + | + | − | + | − | − | − | − | + | − | − |
| 1.2185 | 104 | ? | Water | NY | − | + | + | + | − | − | − | − | − | − | − | + | Kan, Tet |
| 1.2386 | 104 | ? | Goat | CA | − | + | + | + | − | − | − | − | − | − | − | − | Kan, Tet, Cot |
| 1.2633 | 104 | ? | Cow | NE | 1 | − | + | + | − | − | − | − | − | − | − | + | − |
| 1.2673 | 104 | 12 | Cow | NE | 1 | − | + | + | − | − | − | − | − | − | − | + | − |
| 1.2806 | 104 | ? | Cow | NE | 1 | − | + | + | − | − | − | − | − | − | − | + | − |
| 1.2807 | 104 | ? | Cow | NE | 1 | − | + | + | − | − | − | − | − | − | − | + | − |
| 1.2824 | 104 | ? | Cow | NE | 1 | − | + | + | − | − | − | − | − | − | − | + | − |
| 1.2825 | 104 | ? | Cow | NE | 1 | − | + | + | − | − | − | − | − | − | − | + | − |
| 1.3285 | 104 | ? | Water | NY | − | − | − | + | − | − | − | − | − | − | − | − | − |
| 2.0936 | 104 | ? | Water | NY | − | − | − | + | − | − | − | − | − | − | − | − | − |
| 3.3790 | 104 | ? | Goose | CA | − | − | − | + | − | − | − | − | − | − | − | + | − |
| 4.0898 | 104 | 11 | ? | OH | − | − | + | + | − | − | − | − | − | − | − | − | Amp, Chl, Nal, Str, Tet |
| 4.2039 | 104 | 49 | Avian | CA | − | − | − | + | − | − | − | − | − | − | − | − | − |
| 4.2366 | 104 | ? | Water | NY | − | − | − | + | − | − | − | − | − | − | − | + | − |
| 4.2458 | 104 | ? | Water | NY | − | − | − | + | − | − | − | − | − | − | − | − | − |
| 5.1515 | 104 | ? | ? | NY | − | − | + | + | − | − | − | − | − | − | − | − | − |
| 5.3526 | 104 | 7 | Sheep | NE | 1 | − | + | + | − | − | − | − | − | − | − | + | − |
| 6.0778 | 104 | 7 | Carcass | NE | 1 | − | + | + | − | + | − | − | − | − | − | − | − |
| 6.0779 | 104 | 7 | Carcass | NE | 1 | − | + | + | − | − | − | − | − | − | − | − | − |
| 6.0800 | 104 | 7 | Carcass | NE | 1 | − | + | + | − | − | − | − | − | − | − | − | − |
| 6.0829 | 104 | 7 | Carcass | NE | 1 | − | + | + | − | − | − | − | − | − | − | + | − |
| 6.0830 | 104 | 7 | Carcass | NE | 1 | − | + | + | − | − | − | − | − | − | − | + | − |
| 6.1342 | 104 | ? | Goose | NY | − | − | − | − | − | − | − | − | − | − | − | − | − |
| 6.1343 | 104 | ? | Goose | NY | − | − | − | − | − | − | − | − | − | − | − | − | − |
| 6.1344 | 104 | ? | Goose | NY | − | − | − | − | − | − | − | − | − | − | − | − | − |
| 6.1345 | 104 | ? | Goose | NY | − | − | − | − | − | − | − | − | − | − | + | − | − |
| 6.1349 | 104 | ? | Goose | NY | − | − | − | − | − | − | − | − | − | − | − | − | − |
| 6.1353 | 104 | ? | Goose | NY | − | − | − | − | − | − | − | − | − | − | − | − | − |
| 6.1354 | 104 | ? | Goose | NY | − | − | − | − | − | − | − | − | − | − | − | − | − |
| 7.0441 | 104 | ? | Water | CA | − | − | + | − | − | − | − | − | − | − | − | − | − |
| 7.1708 | 104 | 27 | Cow | NE | − | − | + | − | − | − | − | − | − | + | − | + | − |
| 9.0124 | 104 | ? | Cow | CA | 2 | − | + | − | − | + | − | − | − | − | − | + | − |
| 10.0293 | 104 | ? | Human | IA | − | − | + | + | − | − | − | − | − | − | − | − | Kan |
| 82.0531 | 104 | 21 | Cow | PA | − | − | − | + | − | − | − | − | − | − | − | − | − |
| 82.0833 | 104 | 6 | Human | NY | − | − | + | + | − | − | − | − | − | − | − | − | − |
| 83.0711 | 104 | 21 | ? | NY | − | − | + | − | − | − | − | − | − | − | − | − | − |
| 83.0712 | 104 | ? | ? | NY | − | − | + | − | + | − | − | − | − | + | − | + | − |
| 83.0713 | 104 | ? | ? | NY | − | − | + | + | − | − | − | − | − | − | − | − | − |
| 84.0801 | 104 | 7 | ? | IA | − | − | + | + | − | − | − | − | − | + | − | − | − |
| 85.0858 | 104 | ? | Dog | CA | − | − | − | − | + | − | − | − | − | − | − | + | − |
| 86.1602 | 104 | ? | Turkey | CA | − | + | − | + | − | − | − | − | − | − | − | + | − |
| 92.1238 | 104 | ? | Goose | NY | − | − | − | − | − | − | − | − | − | − | − | + | − |
| 95.3781 | 104 | ? | Human | VA | − | − | + | + | − | − | − | − | − | − | − | − | − |
| 95.3782 | 104 | ? | Human | VA | − | − | + | − | − | − | − | − | − | − | − | − | − |
| 95.3783 | 104 | ? | Human | VA | − | − | + | + | − | − | − | − | − | − | − | − | − |
| 95.3784 | 104 | ? | Human | VA | − | − | + | + | − | − | − | − | − | − | − | − | − |
| 97.0061 | 104 | ? | Water | VA | − | − | + | − | − | − | − | − | − | − | − | − | − |
| 97.0562 | 104 | ? | Human | VA | − | − | + | + | − | − | − | − | − | − | − | − | − |
| 97.0563 | 104 | ? | Human | VA | − | − | + | + | − | − | − | − | − | − | − | − | − |
| 99.0230 | 104 | ? | Cow | CO | 1 | − | + | + | − | − | − | − | − | − | − | − | − |
| 99.1282 | 104 | ? | Deer | PA | − | − | − | − | − | − | − | − | − | − | − | + | − |
| 99.1324 | 104 | ? | ? | PA | − | − | + | + | − | − | − | − | − | − | − | − | − |
| 99.1936 | 104 | ? | Food | MD | − | − | − | − | − | − | − | − | − | − | − | + | Str, Tet |
O and H serotyping results from the E. coli Reference Center, Penn State University.
Source as documented by the E. coli Reference Center, Penn State University.
EHEC, PCRs were used to test for the presence of STEC virulence loci.
EAEC, PCRs were used to test for the presence of EAEC virulence loci.
Hem, hemolysis; washed blood agar.
R-type, Antimicrobial susceptibility resistance phenotype; Amp, ampicillin; Axo, ceftriaxone; Chl, chloramphenicol; Cot, trimethoprim/sulfamethoxazole; Kan, kanamycin; Nal, nalidixic acid; Str, streptomycin; Tet, tetracycline; Tio, ceftiofur. All strains were sensitive to amikacin, amoxicillin/clavulanic acid, cefoxitin, ciprofloxacin, gentamicin, and sulfisoxazole. NA, not applicable.
Control strain.
Pulsed-field gel electrophoresis (PFGE) analysis with XbaI-digested genomic DNA was conducted using a rapid standardized protocol for E. coli O157:H7 (13) and generated 40 PFGE patterns that divided the 58 isolates into two main distinct clusters with a similarity index of about 64% (Fig. 1). The German outbreak strain was part of the mostly non-hemolysin-producing cluster 1. The majority of isolates belonging to cluster 2 showed typical EHEC enterohemolysin activity on 4% washed blood agar with a heart infusion broth base (Table 1). Strains showing hemolysis activity, presence of irp2, an iron acquisition system marker gene encoded on a pathogenicity island (17), and lpfAO26 grouped into subclusters 1A and 1B and subclusters 2A and 1B. Most members of cluster 2 were hemolytic, with most subcluster 2A members harboring stx1. The single isolate carrying stx2 belonged to subcluster 1A, however, showing a hemolytic phenotype differing from that of other cluster 1 members. Although the majority of the isolates carried the irp2 gene, other markers for EHEC (fyuA, iha) and EAEC (set1 [Shigella enterotoxin 1] and pic [Pic protein involved in intestinal colonization]) were detected in only two or three isolates. None of the isolates appeared to carry aatA, specific for the EAEC virulence plasmid pAA, or aggR, encoding the transcriptional regulator AggR.
Fig 1.
Pulsed-field gel electrophoresis of XbaI-digested DNA. Data for antibiograms, EHEC markers, and EAEC markers and strain identification (ID) number, location, source, and H-type are shown. The dendrogram created by the unweighted-pair group method using average linkages was generated with BioNumerics software (Applied Maths, St-Martens-Latem, Belgium) by using a Dice coefficient with a 1.0% lane optimization and 1.0% band position tolerance. The scale above the dendrogram indicates percent similarity. AMI, amikacin; AMC, amoxicillin/clavulanic acid; AMP, ampicillin; FOX, cefoxitin; AXO, ceftriaxone; CHL, chloramphenicol; CIP, ciprofloxacin; GEN, gentamicin; KAN, kanamycin; NAL, nalidixic acid; STR, streptomycin; SUL, sulfisoxazole; TET, tetracycline; COT, trimethoprim/sulfamethoxazole; TIO, ceftiofur. EHEC and EAEC virulence gene markers are as follows: terD (tellurite resistance gene), stx1 (Shiga toxin 1), stx2 (Shiga toxin 2), ipAfO26 (adhesin of STEC O26), irp2 and fyuA (iron acquisition system markers encoded on high pathogenicity island), iha (ashesin), hly (enterohemolysin gene hlyA), eae (γ-intimin), aatA (pAA virulence plasmid marker gene), aggA (aggregative adherence fimbriae I), set1 (Shigella enterotoxin 1), and pic (intestinal colonization protein gene).
Antimicrobial susceptibility testing was performed following CLSI (Clinical and Laboratory Standards Institute) guidelines and using a Gram-negative (CMV1AGNF) panel provided by Trek Diagnostics, Cleveland, OH. Few isolates exhibited antibiotic resistance and were not restricted or typical for a specific cluster. One O104:H11 isolate (4.0898; OH) shared resistance to four antimicrobials with the German O104:H4 outbreak strain and also grouped closely with it in the PFGE dendrogram (cluster 1B). However, this isolate lacked most other virulence traits associated with the German strain.
Several isolates analyzed in this study carried some of the typical O104:H4 virulence-associated loci and showed distinct PFGE profiles scattered among the two clusters. Some markers (irp2, lpfAO26) were shared by most isolates, although hemolysis activity was limited to cluster 2 isolates. This finding showed that virulence traits could be acquired by O104 strains from diverse locations and different sources and with various H-types. E. coli isolates harboring genes linked to EHEC are of particular importance due to their association with serious human infections (14); therefore, all STEC isolates found in this analysis have to be considered potentially pathogenic. However, non-H4 STEC O104 isolates appeared to be only distantly related to the pathogenic O104:H4 serotype (Fig. 1) and carried only a few virulence marker genes (Table 1). The elevated pathogenicity of O104:H4 is speculated to be linked to the unusual acquisition of stx2 in an EAEC strain combining both EAEC and EHEC virulence genes, which were indeed present in some of the isolates screened here (EAEC toxins pic and set1).
It is now evident that, through rapid acquisition of different virulence factors, an E. coli serotype, previously naïve or rarely associated with food-borne disease, can evolve to cause major outbreaks of severe illness and death. The combination of EAEC and EHEC virulence factors in the German outbreak strain of the summer of 2011 emphasizes the importance of being able to detect diarrheagenic E. coli by the use of appropriate methods that identify not only the most prevalent serotypes but also less-recognized ones. It is difficult to predict which less-common serotype could be implicated in the next outbreak. Therefore, efforts are urgently needed to better understand the evolution and emergence of bacterial pathogens and to develop rapid measures for identification of other serotypes even though they have been associated only rarely with HUS in the past.
ACKNOWLEDGMENT
The study was supported in part by the Joint Institute for Food Safety & Applied Nutrition (JIFSAN), University of Maryland, College Park, MD.
Footnotes
Published ahead of print 30 December 2011
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