Practical and economical method for using biotinylated DNA probes with bacterial colony blots to identify diarrhea-causing Escherichia coli.

A simple and economical method was developed for using biotinylated DNA probes to hybridize with bacterial colonies belonging to the various categories of diarrhea-causing Escherichia coli. Simplification and cost containment were achieved by using Whatman no. 541 filter papers instead of nitrocellulose, by minimizing the concentration of proteinase K (an expensive but necessary reagent used to pretreat the colony blots prior to hybridization with biotin-labeled DNA probes) and by reusing hybridization solution containing labeled probe DNA. After exposing the colony blots to lysing solution and steam, followed by lysozyme (1.5 mg/ml), sucrose (25%), and proteinase K (10 micrograms/ml) treatments, biotinylated probes were used to detect enterotoxigenic, enteropathogenic, enterohemorrhagic, diffuse adherence, and enteroinvasive categories of diarrhea-causing E. coli with a high level of sensitivity and specificity. Three independent observers who were experienced in reading DNA blots recorded remarkably similar results, while less satisfactory results were obtained when the blots were read by an inexperienced observer. This technique will be useful in laboratories in which radioactive isotopes are unavailable or impractical and in which budgets are restricted.     

HeLa cell-adherent enteropathogenic Escherichia coli in children under 1 year of age in Thailand.

Enteropathogenic Escherichia coli (EPEC) was isolated from 11% of 148 Hmong children under 1 year old with diarrhea at a refugee camp in northern Thailand. Of 16 children with EPEC-associated diarrhea, 11 were infected with EPEC that adhered to HeLa cells in a diffuse pattern, 3 were infected with EPEC that adhered to HeLa cells in a localized adherence (LA) pattern, and 2 were infected with EPEC that were nonadherent. In Bangkok, EPEC was isolated from 6% of 64 children under 1 year old with diarrhea and 7% of 56 children of the same age without diarrhea. Of four children with diarrhea, two were infected with EPEC with an LA pattern, and two were infected with nonadherent EPEC. Of four children without diarrhea, one was infected with EPEC with an LA pattern, one was infected with EPEC that adhered in a diffuse pattern, and two were infected with nonadherent EPEC. The 21 EPEC isolates with an LA pattern hybridized with the EPEC adherence factor DNA probe. EPEC was the only enteric pathogen identified in 16 (80%) of 20 children with EPEC-associated diarrhea. EPEC was as frequently isolated from children under 1 year old as were other bacterial enteric pathogens. The problem of identifying EPEC with pools of polyvalent antisera are described, and the need to identify additional enteropathogenic determinants of EPEC is discussed.     

Presence of specific factors in mother's milk against strains of diarrhea-causing Escherichia coli in humans

Presence of specific secretory immunoglobulin A (sIgA) against pathogenic factors of Escherichia coli related with diarrheal disease was studied in colostrum and breast-milk samples obtained longitudinally from a cohort of rural Mexican women. Levels of sIgA against heat-labile enterotoxin, Shiga-like toxin, colonization factors antigens I, II and E8775 and adherence to HEp-2 cells were detected in samples obtained from 54 rural women during the first year of lactation. Although production of specific sIgA against these pathogenic factors was almost universal it was not constant, even in the same woman. The results reflect a definite mother-infant relationship during this period. The data support the thesis of using breast-milk as a vaccination vehicle against diarrhea associated with specific organisms during the first year of life of infants born in developing areas of the world.     

Determination by DNA hybridization of Shiga-like-toxin-producing Escherichia coli in children with diarrhea in Thailand

Specific DNA probes were used to identify Shiga-like-toxin (SLT) I- and II-producing Escherichia coli from children less than 5 years of age with bloody diarrhea, in infants with diarrhea, and in controls of the same age without diarrhea in Thailand. At one hospital, SLT-producing E. coli was identified in 4 (7%) of 54 children with bloody diarrhea from whom other enteric pathogens were not identified and from 3 (6%) of 50 children without diarrhea. In the positive specimens, SLT-producing E. coli constituted only 0.3 to 4% of the 100 to 300 colonies on the replica blots. Non-toxin-encoding 933J and 933W bacteriophagelike DNA sequences were detected by colony hybridization with E. coli isolates from 18 (33%) of 54 children with bloody diarrhea and 23 (46%) of 50 controls. At another hospital, SLT-producing E. coli was not identified in 115 infants with diarrhea and 119 controls without diarrhea. One infant with diarrhea was infected with E. coli O76:H7 that hybridized with the enterohemorrhagic E. coli probe but not with the SLT probes. E. coli producing SLT I or SLT II was isolated in small numbers from a similar proportion of Thai children with bloody diarrhea in whom no other enteric pathogen was identified and from controls without diarrhea.     

Serotypes of enterotoxigenic Escherichia coli in Thailand and the Philippines.

The serotypes of 386 enterotoxigenic Escherichia coli (ETEC) isolated from 82 individuals with and without diarrhea in Thailand and the Philippines were determined. The 136 strains producing both heat-labile toxin (LT) and heat-stable toxin (ST) belonged to 12 different O serogroups; however, 83% (113/136) were of one of four serogroups (O6, O8, O25, and O78), and 76% of (104/136) belonged to one of seven O:K:H serotypes. Only 14% (28/196) of LT-only-producing ETEC belonged to serogroups most common among LT and ST strains, and these 196 strains belonged to 35 different O:K:H serotypes. Three O serogroups (O20, O27, and O78) accounted for 94% (52/54) of strains producing only ST. Although only 4% (2/54) of ST-only ETEC belonged to the seven serotypes most commonly found among strains which produced LT and ST, 85% of ETEC belonged to three other serotypes, O20:K?:H21, O27:K?:H7, and O78:H-. A total of 46% (37/80) of ETEC of serotypes O6:H16, O8:H9, O25:H42, and O78:H12 were resistant to two or more antibiotics in comparison to 68% (208/306) of ETEC of other serotypes (P less than 0.001). In Thailand and the Philippines, E. coli which produced LT and ST or ST alone, but not those which produced LT alone, were restricted in their O:K:H serotypes.     

Colonization factors associated with enterotoxigenic Escherichia coli isolated in Thailand.

Eighty-six percent (72 of 84) of heat-labile and heat-stable, none of 141 heat-labile, and 24% (27 of 111) heat-stable enterotoxigenic Escherichia coli isolates from Thailand aggregated in less than 1 M (NH4)2SO4, hemagglutinated human group A and bovine erythrocytes in 1% D-mannose, and possessed either colonization factor I or colonization factor II. No other colonization factors were identified by these two methods.     

Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coli serotype O157.

Fluorogenic procedures were used with the substrate 4-methylumbelliferyl-beta-D-glucuronide (MUG) to identify Escherichia coli. Most strains produced beta-glucuronidase and, thus, were MUG positive. A 20-min procedure was developed to detect glucuronidase activity in 1,295 bacterial cultures, representing 23 genera, of strains that were isolated from clinical specimens. Very few organisms other than E. coli were MUG positive. Of 682 E. coli strains that were isolated, 630 (92.4%) were MUG positive. When an additional 188 E. coli serotype O157 isolates were examined, 155 E. coli O157:H7, 10 E. coli O157:H-, and 1 E. coli O157:H (rough) isolate were MUG negative. All 166 cultures were verocytotoxin positive. Of the remaining 22 E. coli O157 isolates, 2 isolates were O157:H-, 1 isolate was O157:H (rough), and 19 isolates were other H types (H6, H16, H19, H25, H42, and H45); these 22 isolates were MUG positive. All 22 cultures were verocytotoxin negative. The rapid MUG procedure can be used to predict verocytotoxin-positive isolates of E. coli O157; that is, there is a very good likelihood that MUG-negative E. coli O157 isolates are verocytotoxin positive.     

Use of PCR to identify enteroaggregative Escherichia coli as an important cause of acute diarrhoea among children living in Calcutta, India.

The importance of enteroaggregative Escherichia coli (EAggEC) as a possible aetiological agent of acute diarrhoea among children in Calcutta, India, was investigated. Simultaneously the use of a previously described PCR diagnostic system was assessed for its ability to identify EAggEC infection. E. coli strains isolated during a 1-year case-control study from faecal samples of 388 children aged <5 years, with or without diarrhoea, were examined for EAggEC by HeLa cell adherence assay in parallel with a PCR assay with primers generated from an EAggEC DNA probe. A blind comparison was made between the two methods to determine their diagnostic potential. E. coli isolates that adhered to HeLa cells in an aggregative pattern were the sole isolates significantly more often in 254 cases (9%) than in 134 control (2%) children. Age stratification showed that EAggEC were isolated more frequently from children aged <36 months. The EAggEC isolates belonged to several O serogroups and showed multiple drug resistance. Both methods were positive for 26 samples, nine samples were positive by PCR alone and seven samples were positive by culture alone, thus indicating a 78% sensitivity and 97% specificity for the PCR assay. EAggEC is an important aetiological agent of acute diarrhoea among infants in and around Calcutta, and the PCR diagnostic system may be useful to identify such infection in epidemiological studies.     

Enteropathogenic Escherichia coli diarrhea in hospitalized children in Bangladesh.

The role of enteropathogenic Escherichia coli (EPEC) was evaluated in a group of children with endemic diarrhea admitted to Dhaka Shishu Hospital in Dacca, Bangladesh. EPEC was detected in fecal samples of 23% of 104 cases and 8% of 74 concurrent control children. The most commonly isolated EPEC strains were serogroups O20a, O20c:K61; O20a, O20b:K84; O26:K60; and O18a, O18c:K77. Except for O26:K60, these groups had not been reported from Bangladesh. On testing for enterotoxin production, only two strains (serogroups O26:K60, O18a, and O18c:K77) were enterotoxigenic. None was enteroinvasive as tested in the guinea pig conjunctivitis model. Our study supports the concept that EPEC may be an important cause of endemic diarrhea in Bangladesh.     

Antibiotic-resistant cell-detaching Escherichia coli strains from Nigerian children.

The properties of 23 cell-detaching Escherichia coli strains that were isolated from stool specimens in Nigeria are described. Common properties of the strains included the presence of genes encoding alpha-hemolysin (100%), pyelonephritis-associated pili (100%), and cytotoxic necrotizing factor 1 (70%) as well as lactose negativity (70%) and multiple antibiotic resistance (74%). Antibiotic resistance was shown in most cases to be transferable and associated with the presence of class 1 integrons. Phenotypic properties and pulsed-field gel electrophoresis analysis demonstrated that the majority of the strains, particularly multiply resistant, lactose-negative O4:H40 strains, were closely related. Multiply-resistant cell-detaching E. coli strains may represent an important reservoir for antibiotic resistance genes.     

Comparative study of synthetic oligonucleotide and cloned polynucleotide enterotoxin gene probes to identify enterotoxigenic Escherichia coli.

Escherichia coli isolated from 2,126 children in Thailand and the Philippines was examined for enterotoxin production and for DNA hybridization with synthetic oligonucleotide and cloned polynucleotide enterotoxin gene probes. A total of 233 infections with E. coli that were detected by one or more of these assays were identified. Of the infections, 75% (164/233) were identified by all three methods. An additional 18% (43/233) were identified by two of three methods. Isolates from 10% (19/183) of infections with E. coli that hybridized with both the oligonucleotide and cloned enterotoxin gene probes were nontoxigenic, as determined by the Y1 adrenal cell and suckling mouse assays. Although synthetic oligonucleotide probes to detect enterotoxigenic E. coli are more uniform and easier to use than cloned enterotoxin gene probes, the heat-labile toxin oligo probe used in this study did not identify 13% (11/87) of infections with E. coli that produced heat-labile toxin, as identified with the Y1 adrenal cell assay and the cloned enterotoxin gene probe. Synthetic oligonucleotide probes enable laboratories with only minimal equipment to use DNA hybridization assays to identify enterotoxigenic E. coli.     

Enterohemorrhagic Escherichia coli associated with hemolytic-uremic syndrome in Chilean children.

A clinicoepidemiological study was undertaken to determine if enterohemorrhagic Escherichia coli (EHEC) was associated with hemolytic-uremic syndrome (HUS) in children in Santiago, Valdivia, and Temuco, Chile. Prospective surveillance detected 20 hospitalized cases of HUS in children less than 4 years of age in these cities from March 1988 to March 1989. Each HUS patient was matched (by sex and age) with two control children (hospitalized elective-surgery patients). To detect EHEC, DNA from stool culture isolates of E. coli was detected by hybridization with biotin-labelled DNA probes specific for the EHEC virulence plasmid, Shiga-like toxin I (SLT-I) or SLT-II. Stool cultures from 6 of 20 cases (30%) and from 2 of 38 controls (5.3%) yielded EHEC (P = 0.0158). EHEC isolates from all HUS cases hybridized with the EHEC plasmid probe and with probes for SLT-I or -II (or both). The serogroups of the isolates included O157, O26, and O111. EHEC causes HUS in Chile, and the biotinylated gene probes are practical diagnostic tools for epidemiologic studies.     

Controlled study of Escherichia coli diarrheal infections in Bangladeshi children.

Diarrheal diseases are highly prevalent in Bangladesh. However, the relative contribution of diarrheagenic Escherichia coli organisms--those that are enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroinvasive, enterohemorrhagic, enteroaggregative, and diffuse adherent--to diarrhea in Bangladeshi populations is not known. With DNA probes specific for these diarrheagenic E. coli strains, we analyzed fecal E. coli from 451 children up to 5 years of age with acute diarrhea seeking treatment at a Dhaka hospital and from 602 matched control children without diarrhea from July 1991 to May 1992. Enteroinvasive E. coli was not isolated from any children; enterohemorrhagic E. coli was not isolated from any diarrheal children but was isolated from five control children; enteroaggregative and diffuse adherent E. coli strains were isolated with similar frequencies from children with and without diarrhea, thereby showing no association with diarrhea; ETEC was significantly associated with diarrhea in the diarrheal children as a whole and especially in the age groups of 0 to 24 months and 37 to 48 months (further analysis suggests an association with diarrhea for the heat-stable toxin only and for both heat-labile- and heat-stable-toxin-producing ETEC only); and EPEC was significantly associated with diarrhea in the diarrhea group as a whole and particularly in infants up to 1 year of age. Further analysis suggested that EPEC strains of only the traditional serogroups were significantly associated with diarrhea. ETEC and EPEC infections peaked during warm months. Our data thus suggest that EPEC and ETEC are important causes of acute diarrhea in children in this setting.     

Nonspecific resistance to Escherichia coli in mice.

Nonspecific cell-mediated immunity to a relatively virulent strain of Escherichia coli was studied in mice infected with Staphylococcus aureus and elicited with specific antigens. The infected and elicited mice were protected against as intraperitoneal challenge by E. coli for an observation period of 7 days, whereas normal mice, given the same number of bacteria, died within 18 to 24 h. However, the amount of time elapsing between elicitation and challenge greatly affected the rate of protection. Little or no protection was observed in mice injected with S. aureus but not elicited or in mice injected with staphylococcal antigens but not infected with staphylococci.     

Molecular epidemiology of Escherichia coli diarrhea in children in Hong Kong.

This pediatric hospital-based study of 388 diarrhea cases and 306 controls analyzed predominant E. coli colonies from primary culture (253 cases and 177 controls) with eight DNA probes for enteropathogenic, enterotoxigenic, enteroaggregative, and diffusely adherent E. coli. Only enteropathogenic E. coli adherence factor was identified significantly more frequently in cases (10) than in controls (0).     

Comparison between O serotyping method and multiplex real-time PCR to identify diarrheagenic Escherichia coli in Taiwan

To compare the diarrheagenic Escherichia coli (DEC) identifications obtained between traditional O serotyping and modern virulence gene detection assays, we developed a multiplex real-time PCR assay by detecting six specific virulence genes for enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC). Among 261 clinical diarrheal stool samples, a total of 137 suspected DEC (sDEC) isolates were identified by the use of commercially available antisera. The most prevalent serogroups were O1 (12/137; 8.7%), O25 (9/137; 6.5%), and O44 (9/137; 6.5%). The specific virulence genes for the 137 sDEC isolates were analyzed by the multiplex real-time PCR assay. Fifteen (10.9%) of 137 isolates were confirmed to be true DEC strains, indicating that the serotypic markers did not correlate with the specific virulence genes. ETEC (66.7%) was the most prevalent, followed by EIEC (20%) and EPEC (13.3%). No EHEC strains were identified in the specimens. Four novel serotypes were found in the study: two in EPEC strains (O111:H9 and O63:H6) and two in EIEC strains (O63:H9 and O169:H9). In conclusion, the real-time PCR assay considerably reduces the high false-positive rate from the use of serotyping alone, and thus, it is suggested that serogrouping-based methods are inadequate for the identification of DEC isolates, although they are useful for the identification of a limited number of serogroups. In addition, ETEC, EPEC, and EIEC strains were present in 5.7% (15/261) of the diarrheal patients in northern Taiwan in 2006.     

Differentiation of pathogenic Escherichia coli strains in Brazilian children by PCR.

A PCR technique to differentiate pathogenic enteric Escherichia coli strains in a field setting was evaluated. Among 76 children with acute diarrhea, this technique identified 12 children (16%) with enterotoxigenic E. coli, 6 (8%) with enteropathogenic E. coli, and 1 (1%) with enteroinvasive E. coli infection. Compared with the conventional assays, the PCR method proved to be simpler, more rapid, and inexpensive and therefore suitable for application in a developing-country field setting.     

Nationwide surveillance for Staphylococcus aureus with reduced susceptibility to vancomycin in Korea

Methicillin-resistant Staphylococcus aureus (MRSA) accounts for more than 70% of S. aureus isolates from tertiary hospitals in Korea. Clinical isolates of S. aureus were collected from eight provincial, university-affiliated hospitals during the period from June 1999 to January 2001 for nationwide surveillance. All isolates were screened for reduced susceptibility to vancomycin by using brain heart infusion agar containing 4 micro g of vancomycin per milliliter. Population analysis and the determination of the MIC of vancomycin were done for the isolates which grew on the screening agar plates. Of 682 total isolates, MRSA accounted for 64% (439 of 682). Of 27 (4%) isolates that grew on the screening agar plates, none showed the heteroresistance phenotype. No strains with reduced susceptibility to vancomycin were identified.     

Enterotoxigenic Escherichia coli strains belonging to a new serogroup, Escherichia coli O166.

Thirty-two strains of Escherichia coli belonging to a new O group, O166, were examined. Twenty-one strains had the flagella antigen H27, five had the H15 antigen, five had the H7 antigen, and one was nonmotile. All the H27 strains and the nonmotile strain produced heat-stable enterotoxin but not heat-labile enterotoxin. All the H7 strains produced heat-labile enterotoxin but not heat-stable enterotoxin. The remaining strains were nonenterotoxigenic. None of the strains possessed colonization factor antigens CFA/I, CFA/II, or PCF8775.