Isabel Chinen

Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

ABSTRACT A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

Distribution of Putative Adhesins in Different Seropathotypes of Shiga Toxin-Producing Escherichia coli

ABSTRACT The distribution of eight putative adhesins that are not encoded in the locus for enterocyte effacement (LEE) in 139 Shiga toxin-producing Escherichia coli (STEC) of different serotypes was investigated by PCR. Five of the adhesins (Iha, Efa1, LPFO157/OI-141, LPFO157/OI-154, and LPFO113) are encoded in regions corresponding to genomic O islands of E. coli EDL933, while the other three adhesins have been reported to be encoded in the STEC megaplasmid of various serotypes (ToxB [O157:H7], Saa [O113:H21], and Sfp [O157:NM]). STEC strains were isolated from humans (n = 54), animals (n = 52), and food (n = 33). They were classified into five seropathotypes (A through E) based on the reported occurrence of STEC serotypes in human disease, in outbreaks, and in the hemolytic-uremic syndrome (M. A. Karmali, M. Mascarenhas, S. Shen, K. Ziebell, S. Johnson, R. Reid-Smith, J. Isaac-Renton, C. Clark, K. Rahn, and J. B. Kaper, J. Clin. Microbiol. 41:4930-4940, 2003). The most prevalent adhesin was that encoded by the iha gene (91%; 127 of 139 strains), which was distributed in all seropathotypes. toxB and efa1 were present mainly in strains of seropathotypes A and B, which were LEE positive. saa was present only in strains of seropathotypes C, D, and E, which were LEE negative. Two fimbrial genes, lpfAO157/OI-141 and lpfAO157/OI-154, were strongly associated with seropathotype A. The fimbrial gene lpfAO113 was present in all seropathotypes except for seropathotype A, while sfpA was not present in any of the strains studied. The distribution of STEC adhesins depends mainly on serotypes and not on the source of isolation. Seropathotype A, which is associated with severe disease and frequently is involved in outbreaks, possesses a unique adhesin profile which is not present in the other seropathotypes. The wide distribution of iha in STEC strains suggested that it could be a candidate for vaccine development.

Isolation and characterization of Escherichia coli O157:H7 from retail meats in Argentina.

Between February and May 2000, 279 meat samples were collected from 136 retail stores in Gualeguaychú City, Argentina. Samples were assayed for Escherichia coli O157:H7 by selective enrichment in modified EC broth containing novobiocin, followed by immunomagnetic separation (IMS) and plating onto both sorbitol MacConkey agar supplemented with cefixime and potassium tellurite and a chromogenic medium. Eleven E. coli O157:H7 isolates were detected in 6 (3.8%) of 160 ground beef samples, in 4 (4.8%) of 83 fresh sausages, and in 1 (3.3%) of 30 dry sausages. E. coli O157:H7 was not isolated from five hamburger patties or one barbecue-type fresh sausage assayed. The isolates were tested for virulence-related genes. Ten additional Shiga toxin-producing E. coli (STEC) O157:H7 isolates of food origin, recovered from different locations in Argentina, were included for comparison purposes. All 21 isolates harbored both eae and EHEC-hlyA genes, and 12 (57.1%) encoded stx2/stx2vh-a. The isolates were of phage types 87 (seven strains), 14 (four strains), 4 (three strains), and 26 (one strain). Six strains were nontypable by phage typing. Pulsed-field gel electrophoresis (PFGE) revealed 19 XbaI-PFGE profiles. Fifteen (71%) strains were grouped in four clusters, which shared more than 80% of DNA restriction fragments. The enrichment culture method with IMS was a sensitive procedure to detect E. coli O157:H7 strains in retail meats. Some of the isolates from different stores presented a high clonal relatedness, as determined by XhaI-PFGE and phage typing, and harbored the virulence factors associated with human illness.

Risk factors for sporadic Shiga toxin-producing Escherichia coli infections in children, Argentina.

These infections can be prevented by avoiding known risk factors.

Characterisation of Shiga toxin-producing Escherichia coli O157 strains isolated from humans in Argentina, Australia and New Zealand

BackgroundShiga toxin-producing Escherichia coli (STEC) is an important cause of bloody diarrhoea (BD), non-bloody diarrhoea (NBD) and the haemolytic uraemic syndrome (HUS). In Argentina and New Zealand, the most prevalent STEC serotype is O157:H7, which is responsible for the majority of HUS cases. In Australia, on the other hand, STEC O157:H7 is associated with a minority of HUS cases. The main aims of this study were to compare the phenotypic and genotypic characteristics of STEC O157 strains isolated between 1993 and 1996 from humans in Argentina, Australia and New Zealand, and to establish their clonal relatedness.ResultsSeventy-three O157 STEC strains, isolated from HUS (n = 36), BD (n = 20), NBD (n = 10), or unspecified conditions (n = 7) in Argentina, Australia and New Zealand, were analysed. The strains were confirmed to be E. coli O157 by biochemical tests and serotyping. A multiplex polymerase chain reaction (PCR) was used to amplify the stx1, stx2 and rfbO157 genes and a genotyping method based on PCR-RFLP was used to determine stx1 and stx2 variants. This analysis revealed that the most frequent stx genotypes were stx2/stx2c (vh-a) (91%) in Argentina, stx2 (89%) in New Zealand, and stx1/stx2 (30%) in Australia. No stx1-postive strains were identified in Argentina or New Zealand. All strains harboured the eae gene and 72 strains produced enterohaemolysin (EHEC-Hly). The clonal relatedness of strains was investigated by phage typing and pulsed-field gel electrophoresis (PFGE). The most frequent phage types (PT) identified in Argentinian, Australian, and New Zealand strains were PT49 (n = 12), PT14 (n = 9), and PT2 (n = 15), respectively. Forty-six different patterns were obtained by XbaI-PFGE; 37 strains were grouped in 10 clusters and 36 strains showed unique patterns. Most clusters could be further subdivided by BlnI-PFGE.ConclusionSTEC O157 strains isolated in Argentina, Australia, and New Zealand differed from each other in terms of stx-genotype and phage type. Additionally, no common PFGE patterns were found in strains isolated in the three countries. International collaborative studies of the type reported here are needed to detect and monitor potentially hypervirulent STEC clones.

TccP2 of O157:H7 and Non-O157 Enterohemorrhagic Escherichia coli (EHEC): Challenging the Dogma of EHEC-Induced Actin Polymerization

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, TirEHEC O157 is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspFU, which mimics the function of Nck. tccP is carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-tccP gene that is carried on prophage CP-933 M/Sp4 (tccP2). Here we report that atypical, β-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact tccP and tccP2 genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically tccP negative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional tccP2 gene that encodes a secreted protein that can complement an EHEC O157:H7 ΔtccP mutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway.

Phylogenetically Related Argentinean and Australian Escherichia coli O157 Isolates Are Distinguished by Virulence Clades and Alternative Shiga Toxin 1 and 2 Prophages

ABSTRACT Shiga toxigenic Escherichia coli O157 is the leading cause of hemolytic uremic syndrome (HUS) worldwide. The frequencies of stx genotypes and the incidences of O157-related illness and HUS vary significantly between Argentina and Australia. Locus-specific polymorphism analysis revealed that lineage I/II (LI/II) E. coli O157 isolates were most prevalent in Argentina (90%) and Australia (88%). Argentinean LI/II isolates were shown to belong to clades 4 (28%) and 8 (72%), while Australian LI/II isolates were identified as clades 6 (15%), 7 (83%), and 8 (2%). Clade 8 was significantly associated with Shiga toxin bacteriophage insertion (SBI) type stx 2 (locus of insertion, argW) in Argentinean isolates (P < 0.0001). In Argentinean LI/II strains, stx 2 is carried by a prophage inserted at argW, whereas in Australian LI/II strains the argW locus is occupied by the novel stx 1 prophage. In both Argentinean and Australian LI/II strains, stx 2c is almost exclusively carried by a prophage inserted at sbcB. However, alternative q 933- or q 21-related alleles were identified in the Australian stx 2c prophage. Argentinean LI/II isolates were also distinguished from Australian isolates by the presence of the putative virulence determinant ECSP_3286 and the predominance of motile O157:H7 strains. Characteristics common to both Argentinean and Australian LI/II O157 strains included the presence of putative virulence determinants (ECSP_3620, ECSP_0242, ECSP_2687, ECSP_2870, and ECSP_2872) and the predominance of the tir255T allele. These data support further understanding of O157 phylogeny and may foster greater insight into the differential virulence of O157 lineages.

PulseNet International: Vision for the implementation of whole genome sequencing (WGS) for global food-borne disease surveillance

PulseNet International is a global network dedicated to laboratory-based surveillance for food-borne diseases. The network comprises the national and regional laboratory networks of Africa, Asia Pacific, Canada, Europe, Latin America and the Caribbean, the Middle East, and the United States. The PulseNet International vision is the standardised use of whole genome sequencing (WGS) to identify and subtype food-borne bacterial pathogens worldwide, replacing traditional methods to strengthen preparedness and response, reduce global social and economic disease burden, and save lives. To meet the needs of real-time surveillance, the PulseNet International network will standardise subtyping via WGS using whole genome multilocus sequence typing (wgMLST), which delivers sufficiently high resolution and epidemiological concordance, plus unambiguous nomenclature for the purposes of surveillance. Standardised protocols, validation studies, quality control programmes, database and nomenclature development, and training should support the implementation and decentralisation of WGS. Ideally, WGS data collected for surveillance purposes should be publicly available, in real time where possible, respecting data protection policies. WGS data are suitable for surveillance and outbreak purposes and for answering scientific questions pertaining to source attribution, antimicrobial resistance, transmission patterns, and virulence, which will further enable the protection and improvement of public health with respect to food-borne disease.

Serotypes and Shiga toxin genotypes among Escherichia coli isolated from animals and food in Argentina and Brazil

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains isolated from animals and food in Argentina (n=44) and Brazil (n=20) were examined and compared in regard to their phenotypic and genotypic characteristics to evaluate their pathogenic potential. The clonal relatedness of STEC O157 isolates (n=22) was established by phage typing (PT) and pulsed-field gel electrophoresis (PFGE). All O157 strains studied carried eae and enterohemorrhagic E. coli (EHEC)-hly sequences. In Argentina, these strains occurred both in cattle and meat, and 50% of them carried stx2/stx2vh-a genes, whereas in Brazil the O157 strains were isolated from animals, and most harbored the stx2vh-a sequence. At least 13 different O:H serotypes were identified among the non-O157 strains studied, with serotype O113:H21 being found in both countries. All but one non-O157 strains did not carry eae gene, but EHEC-hlyA gene was found in 85.7% of them, and the stx2 genotype was also more prevalent in Argentina than in Brazil (P<0.01), where stx1 alone or in association was most common (68.8%). One STEC strain isolated from a calf in Brazil harbored the new variant referred to as stx2-NV206. PFGE analysis showed that STEC O157 strains were grouped in four clusters. One Brazilian strain was considered possibly related (> or =80%) to Argentinean strains of cluster I. Differences in the pathogenic potential, especially in regard to serotypes and stx genotypes, were observed among the STEC strains recovered from animals and food in both countries.

Home-prepared Hamburger and Sporadic Hemolytic Uremic Syndrome, Argentina

To the Editor: Argentina has the highest incidence of hemolytic uremic syndrome (HUS) in the world, and 10.4 cases per 100,000 children <5 years of age were reported in 2001. HUS is the leading cause of acute renal failure in children (1); in 20% to 35% serious chronic renal failure develops, ranging from mild to serious, and HUS is the second leading cause of chronic renal failure (2,3) in Argentina. Recently, evidence of Shiga toxin–producing Escherichia coli (STEC) infection was found in 59% of Argentine HUS cases; O157:H7 was the predominant serotype isolated (4). Although outbreaks of E. coli O157:H7 have been linked to eating contaminated ground beef (5), the organism is rarely isolated from the implicated meat, and the sources of infection for sporadic cases have rarely been identified. We report a sporadic HUS case linked to the consumption of home-prepared hamburger contaminated with E. coli O157. A 2-year-old girl was brought to the emergency room of the Hospital Nacional de Pediatria “Prof. Dr. Juan Garrahan” in Buenos Aires on April 26, 2002, with a 1-day history of bloody diarrhea. Results of a physical examination were normal, and a stool culture was requested. The patient was sent home with dietary and general instructions. As watery diarrhea persisted with vomiting and fever, the girl was brought in again 3 days later. At that time, she exhibited moderate dehydration, pallor, drowsiness, and a generalized seizure of 10 to 15 min duration, tachycardia, tender and tense abdominal wall, and a history of oligoanuria for the last 48 h. Blood pressure was 128/67 mm Hg. The child was hospitalized with a presumptive diagnosis of HUS and anuric renal failure. Initial laboratory findings included the following: hematocrit, 26%; hemoglobin level, 8.8 g/dL; leukocyte count, 34,800/mm3; segmented neutrophil count, 29,928/mm3; platelet count, 91,000/mm3; serum glucose, 160 mg/dL; blood urea nitrogen (BUN), 268 mg/dL; serum creatinine, 6.3 mg/dL; albumin, 1.7 g/dL; uric acid, 14.8 mg/dL; calcium, 6.9 mg/dL; phosphorus, 6.7 mg/dL; magnesium, 2.0 mg/dL; sodium, 113 mEq/L; potassium, 7.6 mEq/L; pH 7.28; bicarbonate, 10 mmol/L; base excess, –14.9 mmol/L. Chest x-ray findings were normal with a cardiothoracic index of 0.5; results of an abdominal sonogram were normal. A sonogram of the renal system also showed that the kidneys were of normal shape and size and had increased echogenicity. Results of a brain scan showed nonspecific brain atrophy. The clinical findings and the laboratory features of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure were consistent with the diagnosis of HUS. The patient remained anuric for 17 days, required 17 peritoneal dialysis procedures, and six infusions of packed red blood cells. One month after the acute period, she had elevated BUN and serum creatinine levels and massive proteinuria. The rectal swab sample collected on April 26 was routinely cultured for E. coli, Salmonella, Shigella, Yersinia, Aeromonas, Plesiomonas, Vibrio, and Campylobacter species. Sorbitol nonfermenting colonies were recovered on sorbitol-MacConkey (SMAC) agar (Difco Laboratories, Detroit, MI) and SMAC supplemented with cefixime (50 ng/mL) and potassium tellurite (25 mg/mL) (CT-SMAC). The bacterial confluent growth zones of both SMAC and CT-SMAC were positive for stx2 and rfbO157 genes by multiplex polymerase chain reaction (PCR) using the primers described by Pollard et al. (6) and Paton et al. (7), respectively. The E. coli O157 isolates were identified by standard biochemical methods and serologic tests by using specific O157 antiserum (INPB-ANLIS “Dr. Carlos G. Malbran”) and sent to the Servicio Fisiopatogenia as National Reference Laboratory (NRL) for further characterization. As part of the case-control study conducted in the pediatric hospital to identify the risk factors associated with the STEC infection, parents of the 2-year-old girl were interviewed with a standardized questionnaire 8 days after onset of symptoms. Information was collected about her clinical illness, potential exposures in the 7 days before the onset of diarrhea, and demographic issues. Her parents reported that on April 25 the girl had eaten a home-prepared hamburger, made from ground beef purchased from a local market. No other family members reported diarrhea. Three days after the interview, on May 6, a formal complaint was presented by the mother at the Division of Public Health of Lanus, in the southern area of Buenos Aires, where the family lives. The frozen leftover ground beef from the same package used to make the hamburgers was provided by the child’s family and processed at the Laboratorio Central de Salud Publica. A 65-g portion of the ground beef was incubated overnight at 42°C in 585 mL of modified E. coli medium broth containing novobiocin (final concentration, 20 μg/mL). The sample was positive using the E. coli O157 Visual Immunoassay (Tecra Internacional Pty. Ltd., French Forest NSW, Australia) (8). Immunomagnetic separation was performed with 1 mL of the culture, according to the instructions of the manufacturer (Dynal, Inc., Oslo, Norway). The concentrate sample was plated onto CT-SMAC and O157:H7 ID medium (bioMerieux, Marcy l’Etoile, France). Up to 20 sorbitol-nonfermenting colonies were selected, confirmed as E. coli O157, and sent to NRL. At NRL, both clinical and ground beef O157 isolates were confirmed as E. coli O157:H7, susceptible to all of the antibiotics assayed, as previously described (9). Genotypic characterization showed that the isolates harbored stx2, eae, and EHEC-hlyA genes. To establish their clonal relatedness, the strains were assayed by subtyping methods (9). The identity of the strains was confirmed by the unique pulsed-field gel electrophoresis (PFGE) pattern with the restriction enzymes XbaI and AvrII, and the same phage type 4. In addition, both strains were characterized as stx2/stx2vh-a by PCR-restriction fragment length polymorphism. To our knowledge, this is the first HUS case in our country in which the source of infection was identified. No investigation was conducted to trace back the source of the ground beef. This study illustrates the importance of the surveillance of STEC infections and the usefulness of molecular subtyping techniques, such as PFGE and phage typing, to determine the relatedness of strains and assess epidemiologic associations. The public should be made aware that hamburgers, even when prepared at home, can be a source of infection. A primary strategy for preventing infection with E. coli O157:H7 is reducing risk behaviors through consumer education (10).