Screening for Escherichia coli O157:H7 in Illinois

Objective: To determine the incidence of infection with Escherichia coli O157:H7 in a tertiary referral center in Chicago, where a similar study had been performed in 1984, to evaluate cases of disease reported to the Illinois Department of Public Health (IDPH) in 1993, and to determine laboratory practices used to detect this infection throughout the state.
Methods: During a 6-month period in 1993, all stool specimens at Rush-Presbyterian-St Luke's Medical Center (RPSLMC) were tested for E. coli O157:H7. Reports of diagnosed E. coli O157:H7 cases investigated by IDPH were also reviewed. A survey of 73 hospitals in the Chicago area was performed to determine routine culturing practices, specifically, the selection of stool specimens for evaluation for this pathogen.
Results: In the RPSLMC survey, two cases were identified among 1985 samples (incidence 0.1%), similar to the 0.08% incidence detected in a similar study conducted at the same institution in 1984. Through passive surveillance, the IDPH received 44 reports of E. coli O157:H7 in 1993. The hospital survey revealed that, in the seven labs testing all stool specimens for E. coli O157:H7, an incidence of 16/8137 specimens (0.2%) was determined.
Conclusions: These data suggest that sporadic E. coli O157:H7 remains uncommon in Illinois and that the incidence may not have changed over a 9-year period. The low yield and substantial cost of culturing all stools suggest that only specimens from patients with bloody diarrhea should be evaluated routinely in areas of low endemicity.     

Verocytotoxin-producing Escherichia coli (VTEC) O157 and other VTEC from human infections in England and Wales: 1995-1998

A total of 3429 isolations of verocytotoxin-producing Escherichia coli O157 (VTEC O157) was confirmed from human sources in England and Wales during the period 1995-1998. The largest annual total was 1087 in 1997. Most infections occurred in the third quarter of each year. The overall rate of infection ranged from 1.28 to 2.10/100,000 population and showed regional variation. The highest incidence was in children aged 1-4 years. Annually, between 5% and 11% of strains were from patients who had travelled abroad. There were 67 general outbreaks of infection represented by 407 (11.9%) VTEC O157 isolates. Outbreaks involved transmission by contaminated food or water, person-to-person spread and direct or indirect animal contact, and five were associated with foreign travel. The majority (76%) of strains carried verocytotoxin (VT) 2 genes and 23.3% were VT1+VT2. Most strains had the flagellar antigen H7, but c. 14% were non-motile. Approximately 20% of isolates were resistant to antimicrobial agents, predominantly streptomycin, sulphonamides and tetracycline. In addition to VTEC O157, strains of serogroup O157 that did not possess VT genes were identified. These were either derivatives of VTEC O157 that had lost VT genes or were strains with H antigens other than H7 that have never been associated with VT production. Strains of VTEC other than O157 were characterised. Most were associated with diarrhoea, bloody diarrhoea or haemolytic uraemic syndrome and had virulence markers in addition to VT.     

Characterization of Escherichia coli serotype O157:H7.

A total of 174 strains of Escherichia coli serotype O157:H7 representing human isolates obtained from outbreaks and sporadic cases of hemorrhagic colitis, hemolytic-uremic syndrome, and nonbloody diarrheal illnesses as well as from asymptomatic carriers across Canada and the United States were examined. E. coli serotype O157:H7 possessed distinct biochemical markers, a 100% negative reaction for beta-glucuronidase and sorbitol, and a 100% positive reaction for raffinose and dulcitol; all strains otherwise were biochemically typical of E. coli. The vast majority (97%) of the strains were susceptible to commonly used antimicrobial agents. All strains produced readily detectable levels of Verotoxin; however, with polymyxin extraction, nearly 50% of the strains showed up to a 10-fold increase in the toxin level. None were found to mediate hemagglutination of human group A erythrocytes with or without D-mannose. The majority (approximately 70%) of the strains showed localized and diffuse adherence to HEp-2 cells and Henle 407 cells, and the adherence patterns were not very different from those observed among other E. coli strains. Twenty phage types were recognized, with phage types 1 and 2 accounting for 65% of the test strains. Plasmid analysis indicated three basic plasmid profiles: profile I was characterized by 68.7- and 4.2-megadalton (MDa) plasmids (62% of strains), profile II was characterized by 66.2- and 1.8-MDa plasmids (20% of strains), and profile III was characterized by a 62.5-MDa plasmid (18% of strains). A small number (19%) of the strains carried at least one additional plasmid over the basic complements, and these could be considered to constitute a miscellaneous category. None of the above-described characteristics of E. coli serotype O157:H7 could be directly correlated with one another, with the nature of infection, or with the geographical distribution of strains.     

Antimicrobial resistance patterns of Shiga toxin-producing Escherichia coli O157:H7 and O157:H7- from different origins

Shiga toxin-producing Escherichia coli (STEC) serotypes including O157:H7 (n = 129) from dairy cows, cull dairy cow feces, cider, salami, human feces, ground beef, bulk tank milk, bovine feces, and lettuce; and O157:H7- (n = 24) isolated from bovine dairy and bovine feedlot cows were evaluated for antimicrobial resistance against 26 antimicrobials and the presence of antimicrobial resistance genes (tetA, tetB, tetC, tetD, tetE, tetG, floR, cmlA, strA, strB, sulI, sulII, and ampC). All E. coli exhibited resistance to five or more antimicrobial agents, and the majority of isolates carried one or more target antimicrobial resistance gene(s) in different combinations. The majority of E. coli showed resistance to ampicillin, aztreonam, cefaclor, cephalothin, cinoxacin, and nalidixic acid, and all isolates were susceptible to chloramphenicol and florfenicol. Many STEC O157:H7 and O157:H7-isolates were susceptible to amikacin, carbenicillin, ceftriaxone, cefuroxime, ciprofloxacin, fosfomycin, moxalactam, norfloxacin, streptomycin, tobramycin, trimethoprim, and tetracycline. The majority of STEC O157:H7 (79.8%) and O157:H7- (91.7%) carried one or more antimicrobial resistance gene(s) regardless of whether phenotypically resistant or susceptible. Four tetracycline resistant STEC O157:H7 isolates carried both tetA and tetC. Other tetracycline resistance genes (tetB, tetD, tetE, and tetG) were not detected in any of the isolates. Among nine streptomycin resistant STEC O157:H7 isolates, eight carried strA-strB along with aadA, whereas the other isolate carried aadA alone. However, the majority of tetracycline and streptomycin susceptible STEC isolates also carried tetA and aadA genes, respectively. Most ampicillin resistant E. coli of both serotypes carried ampC genes. Among sulfonamide resistance genes, sulII was detected only in STEC O157:H7 (4 of 80 sulfonamide-resistant isolates) and sulI was detected in O157:H7- (1 of 16 sulfonamide resistant isolates). The emergence and dissemination of multidrug resistance in STEC can serve as a reservoir for different antimicrobial resistance genes. Dissemination of antimicrobial resistance genes to commensal and pathogenic bacteria could occur through any one of the horizontal gene transfer mechanisms adopted by the bacteria.     

Bovine immune response to shiga-toxigenic Escherichia coli O157:H7

Although cattle develop humoral immune responses to Shiga-toxigenic (Stx+) Escherichia coli O157:H7, infections often result in long-term shedding of these human pathogenic bacteria. The objective of this study was to compare humoral and cellular immune responses to Stx+ and Stx- E. coli O157:H7. Three groups of calves were inoculated intrarumenally, twice in a 3-week interval, with different strains of E. coli: a Stx2-producing E. coli O157:H7 strain (Stx2+ O157), a Shiga toxin-negative E. coli O157:H7 strain (Stx- O157), or a nonpathogenic E. coli strain (control). Fecal shedding of Stx2+ O157 was significantly higher than that of Stx- O157 or the control. Three weeks after the second inoculation, all calves were challenged with Stx2+ O157. Following the challenge, levels of fecal shedding of Stx2+ O157 were similar in all three groups. Both groups inoculated with an O157 strain developed antibodies to O157 LPS. Calves initially inoculated with Stx- O157, but not those inoculated with Stx2+ O157, developed statistically significant lymphoproliferative responses to heat-killed Stx2+ O157. These results provide evidence that infections with STEC can suppress the development of specific cellular immune responses in cattle, a finding that will need to be addressed in designing vaccines against E. coli O157:H7 infections in cattle.     

多重PCR-DHPLC技术检测肠出血性大肠杆菌O157:H7基因型的方法学研究

目的 开发肠出血性大肠杆菌O157:H7的多重PCR-变性高效液相色谱(DHPLC)检测方法 .方法 以编码大肠杆菌0157抗原的rfbE 基因、编码毒力因子的类志贺毒素(SLT)基因为目的 基因,选择2对引物,建立并优化了大肠杆菌O157:H7的多重PCR-DHPLC检测体系.扩增产物分别为224 bp和499 bp.结果 采用37株细菌验汪了该多重PCR具有良好的特异件.PCR榆测的灵敏度可达到4 CFU/ml.结论 实验证明,研究建立的多重PCR-DHPLC方法 可特异、灵敏地实现对大肠杆菌O157:H7的检测.     

Enzyme-linked immunomagnetic chemiluminescent detection of Escherichia coli O157:H7

E. coli O157:H7 is a pathogenic microorganism that has been implicated in numerous cases of foodborne illnesses. A variety of rapid methods exist that show promise for the presumptive detection of this pathogen without the immediate need for incubating test samples for hours to days in microbial enrichment and culture media. In recent years, highly sensitive chemiluminescence has become a more affordable and portable detection method. Chemiluminescent detection has been coupled with the selectivity of antibodies, magnetic microparticle separation/isolation, and enzymatic signal amplification in order to develop a rapid method, termed enzyme-linked immunomagnetic chemiluminescence (ELIMCL). This work presents the application of ELIMCL to the detection of E. coli O157:H7 in pristine buffered saline with a detection limit of 7.6 x 10(3) for live cells in approx. 75 min assay time. The blocking agent casein and the surfactant Tween 20 were used to lower background luminescence and thus maximize signal-to-noise ratios. After 5.5 h of enrichment culture, ELIMCL was demonstrated to detect E. coli O157:H7 inoculated in ground beef at 10 CFU/g in a total assay time of about 7 h.     

大肠埃希菌O157∶H7的pO157-cured突变株构建

目的建立从肠出血性大肠埃希菌O157∶H7中去除pO157的方法,构建pO157-cured突变株(O157Cu).方法分别将mini-R100系质粒pKP2368和mini-F系质粒pIndigoBAC-5通过电穿孔转化入O157∶H7 Sakai、EDL933、85-170、China2364、Arami505和Mida2684菌株中,筛选转化子,进行质粒图谱分析,PCR检测转化子中pKP2368和pIndigoBAC-5基因;并扩增pO157中的复制不相容区incB、incC和incD序列,DNA测序验证PCR结果.结果采用mini-F系质粒pIndigoBAC-5转化,菌株Sakai、EDL933、China2364、Arami505、Mida2684的pO157丢失率(分别为94.44%、52.08%、91.67%、96.67%和63.33%)明显高于采用mini-R100系质粒pKP2368转化时其pO157丢失率(分别为15.22%、9.26%、0、0、0),差异有显著性(P值分别为0.001);对于菌株85-170,无论采用pKP2368还是采用pIndigoBAC-5转化,其pO157丢失率均为0.菌株Sakai、China2364、Arami505、Mida2684的pO157中均含有incB、incC和incD序列,EDL933的pO157中含有incC和incD序列,而85-170的pO157中只含有incC序列.结论用携带与pO157相同活性复制子的R100系质粒pKP2368构建pO157-cured突变株,结果并不理想;但携带RepFIA区、含有incB、incC和incD序列的mini-F系质粒pIndigoBAC-5却适用于构建多种菌株的pO157-cured突变株,成功率高、重复性好.pO157中所含mini-F序列incB、incC和incD的存在情况可能与pO157丢失率有关.     

Vaccination of pregnant dams with intimin(O157) protects suckling piglets from Escherichia coli O157:H7 infection

Cattle are important reservoirs of enterohemorrhagic Escherichia coli (EHEC) O157:H7 that cause disease in humans. Both dairy and beef cattle are asymptomatically and sporadically infected with EHEC. Our long-term goal is to develop an effective vaccine to prevent cattle from becoming infected and transmitting EHEC O157:H7 to humans. We used passive immunization of neonatal piglets (as a surrogate model) to determine if antibodies against EHEC O157 adhesin (intimin(O157)) inhibit EHEC colonization. Pregnant swine (dams) with serum anti-intimin titers of < or =100 were vaccinated twice with purified intimin(O157) or sham-vaccinated with sterile buffer. Intimin(O157)-specific antibody titers in colostrum and serum of dams were increased after parenteral vaccination with intimin(O157). Neonatal piglets were allowed to suckle vaccinated or sham-vaccinated dams for up to 8 h before they were inoculated with 10(6) CFU of a Shiga toxin-negative (for humane reasons) strain of EHEC O157:H7. Piglets were necropsied at 2 to 10 days after inoculation, and intestinal samples were collected for determination of bacteriological counts and histopathological analysis. Piglets that ingested colostrum containing intimin(O157)-specific antibodies from vaccinated dams, but not those nursing sham-vaccinated dams, were protected from EHEC O157:H7 colonization and intestinal damage. These results establish intimin(O157) as a viable candidate for an EHEC O157:H7 antitransmission vaccine.     

Escherichia coli O157:H7 Requires Intimin for Enteropathogenicity in Calves

Enterohemorrhagic Escherichia coli (EHEC) strains require intimin to induce attaching and effacing (A/E) lesions in newborn piglets. Infection of newborn calves with intimin-positive or intimin-negative EHEC O157:H7 demonstrated that intimin is needed for colonization, A/E lesions, and disease in cattle. These results suggest that experiments to determine if intimin-based vaccines reduce O157:H7 levels in cattle are warranted.     

Implication of Virulence Factors in Escherichia coli O157:H7 Pathogenesis

Since the first documented outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7 in 1982, numerous publications have demonstrated or proposed putative components implicated in the pathogenesis of this gastrointestinal infection. Indeed, Escherichia coli O157:H7 pathogenesis is linked to several potential virulent factors such as verotoxins (or Shiga-like toxins), components implicated in attaching/effacing of microvilli, and the enterohemolysin phenotypes. Defining the precise molecular mechanisms involved in the pathogenesis of E. coli O157:H7 implies detailed comprehension of the virulent factors that provoke a wide range of pathophysiological symptoms. The public health significance of this emerging worldwide menace has been demonstrated by clinical complications during treatment, its low infectious dose, and the severity of clinical manifestations. In this review I describe current knowledge of Escherichia coli O157:H7 pathogenesis with emphasis on known and potential virulent factors.     

Escherichia coli O157:H7 in microbial flora of sheep.

We found naturally occurring, potentially virulent Escherichia coli O157:H7 strains in sheep. The incidence of E. coli O157:H7 was transient and ranged from 31% of sheep in June to none in November. The use of a sensitive culture technique and the choice of the proper sampling season were both essential for detecting this bacterium in sheep. DNA hybridizations showed that 80% of the E. coli O157:H7 isolates had at least two of the Shiga-like toxin types I or II or the attaching-effacing lesion genes.     

Performance of the ImmunoCard STAT! E. coli O157:H7 test for detection of Escherichia coli O157:H7 in stools

ImmunoCard STAT! E. coli O157:H7 (Meridian Diagnostics, Inc., Cincinnati, Ohio) is a novel rapid (10-min) test for the presence of Escherichia coli O157:H7 in stools. The test may be performed either directly on stool specimens or on an overnight broth culture of stool. In a multicenter prospective study, 14 of 14 specimens positive by culture for E. coli O157:H7 were positive by the ImmunoCard STAT! O157:H7 test, and there were no false positives from 263 culture-negative specimens. In a retrospective study, the test was positive in 339 (81%) of 417 stored culture-positive specimens and the specificity was 95% (98 of 103 specimens). No false positives were associated with alternate stool pathogens. The ImmunoCard STAT! O157:H7 test has high sensitivity and specificity.     

Sterilization of Escherichia coli O157:H7 using micro corona ionizer

We demonstrated in vitro sterilization of Escherichia coli O157:H7 bacteria on agar by a pin-between-planes micro corona ionizer. The gap between the pin and the grid was ~1.1 mm, the length of the grid was ~2.1 mm and the height was ~1.0 mm. The effective pin radius and discharge length were both approximated to be 200 μm. Ozone generation rates of ~2.3?×?10^?3 mg/s, ~2.7?×?10^?3 mg/s and ~3.5?×?10^?3 mg/s at 1,500 V were calculated for relative humidity (RH) of 35 %, 25 % and 10 % respectively. Analytical ozone generation rate increases as RH decreases and it is consistent with experimental observations. Using target and control petri dishes with E. coli plated agar, the sterilization capability of the micro corona ionizer at 37 °C for 24 h was evaluated. A ~60 % reduction in bacterial colony was shown with plate counting and its kill radius could be tuned from?~?20 mm to ~5 mm by reducing the duty cycle from 100 % to 50 % with 30 min pulse width. The results suggested that the micro corona ionizer might be suitable as a tunable ozone source in wound dressing for chronic wound management.     

Detection of Escherichia coli O157:H7 by multiplex PCR.

In order to develop a PCR assay for Escherichia coli O157:H7, a portion of the 60-MDa plasmid harbored by enterohemorrhagic E. coli (EHEC) was sequenced and PCR primers were designed. A multiplex PCR method was then designed by employing primers specific for the EHEC eaeA gene, conserved sequences of Shiga-like toxins I (SLT-I) and II (SLT-II), and the 60-MDa plasmid. PCR products of 1,087 bp (eaeA), 227 and/or 224 bp (SLT-I and/or SLT-II), and 166 bp (plasmid) were successfully amplified simultaneously in a single reaction. The multiplex PCR method can be used to specifically identify EHEC of serogroup O157.     

Shiga toxin-producing Escherichia coli in children: diagnosis and clinical manifestations of O157:H7 and non-O157:H7 infection

Shiga toxin-producing Escherichia coli (STEC), a cause of food-borne colitis and hemolytic-uremic syndrome in children, can be serotype O157:H7 (O157) or other serotypes (non-O157). E. coli O157 can be detected by culture with sorbitol-MacConkey agar (SMAC), but non-O157 STEC cannot be detected with this medium. Both O157 and non-O157 STEC can be detected by immunoassay for Shiga toxins 1 and 2. The objectives of this study were first to compare the diagnostic utility of SMAC to that of the Premier EHEC enzyme immunoassay (Meridian Diagnostics) for detection of STEC in children and second to compare the clinical and laboratory characteristics of children with serotype O157:H7 STEC and non-O157:H7 STEC infections. Stool samples submitted for testing for STEC between April 2004 and September 2009 were tested by both SMAC culture and the Premier EHEC assay at Children's Hospital Boston. Samples positive by either test were sent for confirmatory testing and serotyping at the Hinton State Laboratory Institute (HSLI). Chart review was performed on children with confirmed STEC infection. Of 5,110 children tested for STEC, 50 (0.9%) had STEC infection confirmed by culture; 33 were O157:H7 and 17 were non-O157:H7. The Premier EHEC assay and SMAC culture detected 96.0% and 58.0% of culture-confirmed STEC isolates (any serotype), respectively, and 93.9% and 87.9% of STEC O157:H7 isolates, respectively. There were no significant differences in disease severity or laboratory manifestations of STEC infection between children with O157:H7 and those with non-O157 STEC. The Premier EHEC assay was significantly more sensitive than SMAC culture for diagnosis of STEC, and O157:H7 and non-O157:H7 STEC caused infections of similar severity in children.     

Four laboratory-associated cases of infection with Escherichia coli O157:H7

An investigation of four cases of infection with Escherichia coli O157:H7 among laboratorians from different clinical laboratories revealed that the DNA fingerprint pattern of each case isolate was indistinguishable from that of an isolate handled in the laboratory prior to illness. These data suggest that the infections were laboratory acquired, and they demonstrate the importance of laboratorians strictly adhering to biosafety practices recommended for the handling of infectious materials.