Evaluation of an immunoassay for direct detection of Escherichia coli O157 in stool specimens.

An enzyme-linked immunosorbent assay (ELISA) produced by LMD Laboratories, Inc., Carlsbad, Calif., was compared with culture for the detection of Escherichia coli O157. Nine of 185 stool specimens evaluated had positive results by the LMD E. coli O157 ELISA and grew E. coli O157 on culture; 174 had negative by LMD E. coli O157 ELISA results and did not grow E. coli O157 on culture. Of 174 specimens negative by LMD E. coli O157 ELISA, 117 specimens grew other enteric pathogens: Campylobacter spp. (46 isolates), Salmonella spp. (43 isolates), Yersinia spp. (20 isolates), and Shigella spp. (8 isolates). There were two indeterminant results by the LMD E. coli O157 ELISA. One stool specimen did not grow other enteric pathogens on culture, and one grew a Campylobacter sp. on culture. Both had negative LMD E. coli O157 ELISA results upon repeat testing. The LMD E. coli O157 ELISA is an accurate, easy-to-read screening method for the detection of E. coli O157 in fecal specimens.     

Duplex real-time SYBR green PCR assays for detection of 17 species of food- or waterborne pathogens in stools

A duplex real-time SYBR Green LightCycler PCR (LC-PCR) assay with DNA extraction using the QIAamp DNA Stool Mini kit was evaluated with regard to detection of 8 of 17 species of food- or waterborne pathogens in five stool specimens in 2 h or less. The protocol used the same LC-PCR with 20 pairs of specific primers. The products formed were identified based on a melting point temperature (T(m)) curve analysis. The 17 species of food- or waterborne pathogens examined were enteroinvasive Escherichia coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enterotoxigenic E. coli, enteroaggregative E. coli, Salmonella spp., Shigella spp., Yersinia enterocolitica, Yersinia pseudotuberculosis, Campylobacter jejuni, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Aeromonas spp., Staphylococcus aureus, Clostridium perfringens, and Bacillus cereus. No interference with the LC-PCR assay was observed when stool specimens were artificially inoculated with each bacterial species. The detection levels were approximately 10(5) food- or waterborne pathogenic bacteria per g of stool. The protocol for processing stool specimens for less than 10(4) food- or waterborne pathogenic bacteria per g of stool requires an overnight enrichment step to achieve adequate sensitivity. However, the rapid amplification and reliable detection of specific genes of greater than 10(5) food- or waterborne pathogenic bacteria per g in samples should facilitate the diagnosis and management of food- or waterborne outbreaks.     

实时荧光PCR方法快速检测空肠弯曲菌方法的建立

目的建立实时荧光PCR快速检测空肠弯曲菌的方法。方法以空肠弯曲杆菌HipO基因的保守序列为模板设计特异性引物探针,建立一种能快速检测样本中空肠弯曲杆菌的实时荧光PCR方法;对方法的特异性和敏感性进行评价,并以正常人粪便为空白样本,添加一定量空肠弯曲菌标准株菌液进行检测,以对方法的检测效果进行初步评价。结果该实时荧光PCR方法只对空肠弯曲杆菌进行特异扩增,同种属的结肠弯曲菌及其他常见食源性病原菌均不能扩增;整个检测过程只需要80min,对空肠弯曲菌菌悬液可检测至5个细菌,对加标粪便样本可检测至10-100个细菌。结论本研究建立的实时荧光PCR检测空肠弯曲菌方法不仅能实现对空弯菌的快速检测,而且还为空弯菌的快速诊断及其引起的食源性疾病的监控溯源提供有意义的参考。     

Epidemiology of bacterial pathogens associated with infectious diarrhea in Djibouti.

During a survey examining the causes of diarrhea in the East African country of Djibouti, 140 bacterial pathogens were recovered from 209 diarrheal and 100 control stools. The following pathogens were isolated at comparable frequencies from both diarrheal and control stools: enteroadherent Escherichia coli (EAEC) (10.6 versus 13%), enterotoxigenic E. coli (ETEC) (11 versus 10%), enteropathogenic E. coli (EPEC) (7.7 versus 12%), Salmonella spp. (2.9 versus 3%), and Campylobacter jejuni-C. coli (3.3 versus 5%). Surprisingly, the EAEC strains isolated did not correspond to well-recognized EPEC serogroups. No Yersinia spp., enteroinvasive E. coli, or enterohemorrhagic E. coli were isolated during the course of this study. Only the following two genera were recovered from diarrheal stools exclusively: Shigella spp. (7.7%) and Aeromonas hydrophila group organisms (3.3%). Shigella flexneri was the most common Shigella species isolated. Patients with Shigella species were of a higher average age than were controls (27 versus 13 years), while subjects with Campylobacter or Salmonella species belonged to younger age groups (2.6 and 1.6 years, respectively). Salmonella cases were more often in females. Shigella diarrhea was associated with fecal blood or mucus and leukocytes. ETEC was not associated with nausea or vomiting. Anorexia, weight loss, and fever were associated with the isolation of Salmonella and Aeromonas species. EAEC, ETEC, EPEC, and Shigella species were resistant to most drugs used for treating diarrhea in Africa, while the antibiotic most active against all bacteria tested was norfloxacin. We conclude that in Djibouti in 1989, Shigella and Aeromonas species must be considered as potential pathogens whenever they are isolated from diarrheal stools and that norfloxacin should be considered the drug of choice in adults for treating severe shigellosis and for diarrhea prophylaxis in travelers.     

Bacterial aetiology of diarrhoea in young children: high prevalence of enteropathogenic Escherichia coli (EPEC) not belonging to the classical EPEC serogroups

Diarrhoeal disease continues to be one of the most common causes of admittance in Children hospital emergency. The aim of the present study was to investigate the relative contribution of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) as a cause of infectious bacterial diarrhoea in children from the region of Toulouse. We analysed 280 samples of stools from 280 children (<2 years) with diarrhoea admitted in the "Hopital des Enfants" from January to August 2005. Classic pathogens (Salmonella, Campylobacter, Yersinia, Shigella, Aeromonas and Vibrio) were detected by standard culture methods. Enterotoxigenic Clostridium difficile were identified after culture by immuno-enzyme assay (IEA). Virulence genes of EPEC and EHEC were detected by using PCR. Shiga-toxin production of EHEC strains was confirmed with an IEA test. Potential enteric pathogens were identified in 55 patients. EPEC was the most frequently identified agent (30 patients), followed by Campylobacter (9 cases: 7 C. jejuni and 2 C. coli) and C. difficile (8 patients), then EHEC (5 patients) and Salmonella (3 patients). No Shigella, Yersinia, Aeromonas or other pathogenic bacteria were detected during this period in that class of children. EPEC not belonging to the classical EPEC serogroups were highly prevalent (24 versus 6). EHEC possessed different genotypes and serogroups: O26 (2 strains), O157 (2 strains) and one un-typable strain. This study demonstrates the importance of EPEC (55 % of positive cases) and of EHEC (more frequent than Salmonella) in the aetiology of diarrhoeal diseases of young children. We confirm the usefulness of the PCR methodology: it allows the detection of virulent E. coli and thus increases by two fold the diagnosis of bacterial diarrhoea.     

In vitro activity of norfloxacin and other antibacterial agents against gastro-intestinal pathogens isolated in Sweden

The in vitro activity of norfloxacin was compared to that of ampicillin, doxycycline, chloramphenicol, trimethoprim in combination with sulfamethoxazole (1/20), and erythromycin, against 272 clinical isolates of gastro-intestinal pathogens. Norfloxacin was the most active compound of those tested with MICs in the range 0.004-2 mg/l. Concentrations inhibiting 90% of the strains (MIC 90) were 0.008 mg/l for Vibrio cholerae, 0.016 mg/l for Aeromonas hydrophila, 0.032 mg/l for Vibrio cholerae non 01, 0.064 mg/l for Vibrio parahaemolyticus, Yersinia enterocolitica 03, enterotoxigenic (ETEC) and enteropathogenic (EPEC) Escherichia coli and Shigella species, 0.125 mg/l for Salmonella species, and 0.5 mg/l for Campylobacter species. Resistance to one or several of the other drugs was seen with higher or lower frequency in all the bacterial species tested. No cross-resistance between any of the other agents and norfloxacin was recorded.     

Specific detection of Campylobacter jejuni and Campylobacter coli by using polymerase chain reaction.

Development of a routine detection assay for Campylobacter jejuni and Campylobacter coli in clinical specimens was undertaken by using the polymerase chain reaction (PCR). An oligonucleotide primer pair from a conserved 5' region of the flaA gene of C. coli VC167 was used to amplify a 450-bp region by PCR. The primer pair specifically detected 4 strains of C. coli and 47 strains of C. jejuni; but it did not detect strains of Campylobacter fetus, Campylobacter lari, Campylobacter upsaliensis, Campylobacter cryaerophila, Campylobacter butzleri, Campylobacter hyointestinalis, Wolinella recta, Helicobacter pylori, Escherichia coli, Shigella spp., Salmonella spp., Vibrio cholerae, Citrobacter freundii, or Aeromonas spp. By using a nonradioactively labeled probe internal to the PCR product, the assay could detect as little as 0.0062 pg of purified C. coli DNA, or the equivalent of four bacteria. In stools seeded with C. coli cells, the probe could detect between 30 and 60 bacteria per PCR assay. The assay was also successfully used to detect C. coli in rectal swab specimens from experimentally infected rabbits and C. jejuni in human stool samples.     

Cadmium chloride susceptibility, a characteristic of Campylobacter spp.

We report a simple diagnostic characteristic useful in the presumptive identification of Campylobacter jejuni and Campylobacter coli. Filter paper disks impregnated with cadmium chloride were placed on streaked agar medium. Zones of growth inhibition for Campylobacter spp. occurred at 1.25 micrograms per disk. Other enteropathogens (Salmonella spp., Shigella spp., Vibrio cholerae, Vibrio parahaemolyticus, Escherichia coli, and Yersinia enterocolitica) were resistant to at least 40 micrograms per disk, with the exception of a strain of Shigella flexneri, which showed first susceptibility at 10 micrograms per disk. Most of the 52 Campylobacter strains, which were isolated from human clinical and animal sources, showed zones of inhibition greater than 10 mm with 2.5 micrograms of cadmium chloride per disk. At 20 micrograms per disk, Campylobacter isolates from clinical sources were significantly (P less than 0.01) more susceptible to cadmium chloride inhibition than were those from meat samples.     

A method for detecting Shiga toxin and Shiga-like toxin-I in pure and mixed culture

Shiga toxin and Shiga-like toxins (SLTs, syn. Verotoxins) are currently detected by tissue culture assays that are expensive, time-consuming and require specialised facilities and experienced personnel. We have developed a rapid method to detect Shiga toxin and SLT-I (Verotoxin 1) based on their binding to globotriosyl ceramide (Gb3). Bound toxin was then detected by an enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. The direct detection of cytotoxins from pure culture plates and from a mixed bacterial culture was studied. Using polymyxin extraction (0.1 g/L, 30 min, 37 degrees C) and Gb3-based ELISA we detected toxin from reference strains Shigella dysenteriae 1 strain 60R (Shiga toxin) and Escherichia coli O26:H11 strain H30 (SLT-I), and from clinical isolates of E. coli O157:H7 and O26:H11 (both SLT-I) from 11 patients with diarrhoea, haemorrhagic colitis or haemolytic uraemic syndrome. Toxin production by these strains was confirmed by a radiolabelled HeLa cell assay and the structural genes were detected by DNA hybridisation. The Gb3-based ELISA could detect SLT-I in extracts of a mixed culture even when the toxin-positive strains represented only 1% of the mixture. No cross-reactivity was found with bacteria that produce other cytotoxins, such as other E. coli and Shigella, Salmonella, Aeromonas and Campylobacter spp.     

Two-year survey of etiologic agents of diarrheal disease at San Lazaro Hospital, Manila, Republic of the Philippines.

The prevalence of bacterial pathogens and rotavirus in 2,908 patients with diarrhea who were admitted to San Lazaro Hospital in Manila in 1983 and 1984 was determined. One or more enteric pathogens were isolated or detected in samples from 1,698 (58.4%) patients. Isolation rates for the various enteropathogens were as follows: rotavirus, 30.6%; Shigella spp., 11.6%; Salmonella spp., 9.2%; enterotoxigenic Escherichia coli (1983 only), 7.8%; Vibrio cholerae biotype eltor, 3.8%; non-O1 V. cholerae, 2.8%; Vibrio parahaemolyticus, 1.7%; other Vibrio spp., 1.1%; Campylobacter jejuni, 3.0%; Aeromonas hydrophila, 1.3%; and Plesiomonas shigelloides 1.1%. Giardia lamblia and Entamoeba histolytica were detected in 0.6 and 0.1%, respectively, of stool samples examined. Determination of the etiologic role of isolates was complicated by one or more of the following factors: isolation of multiple enteric pathogens (302 cases); isolation of Salmonella spp., enterotoxigenic E. coli, and C. jejuni from a similar proportion of asymptomatic control patients and patients with diarrhea; and isolation of a high proportion of certain pathogens (especially Salmonella spp.) only from enrichment broth, suggesting infection with a small number of organisms. Isolation of V. cholerae eltor was seasonal, with the majority of cases occurring in the rainy months. In addition, the number of patients with diarrhea increased with the onset of the monsoon rains and peaked during the months of maximum rainfall. Rotavirus infection occurred in both children and adults throughout the year and was the most frequently identified cause of diarrhea in children under 5 years of age. Shigella spp. were the most common agents of diarrhea in adults.     

Development and testing of invasion-associated DNA probes for detection of Shigella spp. and enteroinvasive Escherichia coli.

Genetic determinants of the invasive phenotype of Shigella spp. and enteroinvasive Escherichia coli (EIEC), two common agents of bacillary dysentery, are encoded on large (180- to 210 kilobase), nonconjugative plasmids. Several plasmid-encoded antigens have been implicated as important bacterial ligands that mediate the attachment and invasion of colonic epithelial cells by the bacteria. Selected invasion plasmid antigen (ipa) genes have recently been cloned from Shigella flexneri serotype 5 into the lambda gt11 expression vector. Portions of three ipa genes (ipaB, ipaC, and ipaD) were tested as DNA probes for diagnostic detection of bacillary dysentery. Under stringent DNA hybridization conditions, all three DNA sequences hybridized to a single 4.6-kilobase HindIII fragment of the invasion plasmids of representative virulent Shigella spp. and EIEC strains. No hybridization was detected in isogenic, noninvasive Shigella mutants which had lost the invasion plasmid or had deleted the ipa gene region. Furthermore, these probes did not react with over 300 other enteric and nonenteric gram-negative bacteria tested, including Salmonella, Yersinia, Edwardsiella, Campylobacter, Vibrio, Klebsiella, Aeromonas, Enterobacter, Rickettsia, and Citrobacter spp. and various pathogenic E. coli strains. The use of unique invasion-essential gene segments as probes for the specific detection of invasive dysentery organisms should benefit both epidemiologic and diagnostic analyses of Shigella spp. and EIEC.     

Etiology of children's diarrhea in Montevideo, Uruguay: associated pathogens and unusual isolates

We studied microorganisms associated with infant diarrhea in a group of 256 children admitted to a public pediatric hospital in Montevideo, Uruguay. Diagnostic procedures were updated to optimize detection of potential pathogens, which were found in 63.8% of cases, and to be able to define their characteristics down to molecular or antigenic type. Coinfection with two or more agents was detected in more than one-third of positive studies. Escherichia coli enteric virotypes, especially enteropathogenic E. coli (EPEC), were shown to be prevalent. Rotavirus, Cryptosporidium, Campylobacter (mainly Campylobacter jejuni), and Shigella flexneri were also often identified. Enterotoxigenic E. coli, Salmonella, and Giardia lamblia were sporadically recognized. Unusual findings included two enteroinvasive E. coli strains, one Shigella dysenteriae 2 isolate, and a non-O:1 Vibrio cholerae culture. EPEC bacteria and S. flexneri (but not Salmonella) showed unusually frequent antimicrobial resistance, especially towards beta-lactam antibiotics, which is the subject of ongoing work.     

An evaluation of three commercial fecal transport systems for the recovery of enteric pathogens

Twenty-five isolates, including six strains of Shigella species, six strains of Salmonella species, five strains of Yersinia enterocolitica, six strains of Campylobacter jejuni, and two strains of Vibrio parahaemolyticus, were inoculated at a concentration of 1.5 x 10(4) colony-forming units/mL into the following transport systems: Fekal Enteric Plus (Trend Scientific, Inc., St. Paul, MN), Cary Blair Transport Medium (Remel, Inc., Lenexa, KS), and Para-Pak C & S (Meridian Diagnostics, Inc., Cincinnati, OH). The Fekal Enteric Plus system showed a greater than or equal to 50% recovery of the original bacterial inoculum after 96 hours for all Salmonella, Shigella, and Yersinia strains tested and after as long as 72 hours for Vibrio strains. The Cary Blair Transport System showed greater than or equal to 50% recovery of the initial inoculum at 96 hours for five of six Salmonella strains, four of six Shigella strains, all Yersinia strains, and one of two Vibrio strains. With the use of the Para-Pak C & S, greater than or equal to 50% recovery of the original inoculum after 96 h was demonstrated for five of six Salmonella strains, four of six Shigella strains, all Yersinia strains, and one of two Vibrio strains. All three systems did not demonstrate even 10% recovery of the initial C. jejuni inoculum at 24 hours when held at room temperature.     

Production of Shiga-like toxin among Escherichia coli strains and other bacteria isolated from diarrhea in S?o Paulo, Brazil

An elevated level of Shiga-like toxin I (SLT-I) production was found in 1 of 466 Escherichia coli strains studied. Among the 34 sonic lysates obtained from classical enteropathogenic E. coli, 5 produced SLT-I. The Aeromonas, Citrobacter, Edwardsiella, Enterobacter, Klebsiella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Yersinia, and Vibrio strains also studied were not SLT producers, except for a Shigella dysenteriae type 1 strain. Although SLT-I-producing E. coli strains were isolated from diarrhea, they seem to be an uncommon cause of disease in children less than 1 year old in our community.     

The role of Proteae in diarrhea

A survey was undertaken on the occurrence of Protease in the human fecal flora and its coincidence with other well-documented enteropathogens such as Campylobacter, Salmonella, Shigella, Staphylococcus aureus, Yersinia, protozoa and rotavirus. A total of 2000 fecal specimens was investigated, 1000 from patients suffering from diarrhea and 1000 from healthy persons which served as controls. Proteus mirabilis was isolated more frequently from diarrhea cases than from healthy people. The difference was statistically significant (P less than 0.001). There was no correlation between its occurrence and isolation of Campylobacter, Salmonella, Shigella, Staphylococcus aureus, and protozoa. However, a questionable coincidence was found with rotavirus (P less than 0.2) and a more certain correlation with Yersinia enterocolitica (P less than 0.025). Proteus mirabilis in patients suffering from infection by other known enteropathogens was largely absent, suggesting that the organisms were independent causative agents of intestinal disorders. Notwithstanding this, they may additionally play a role as opportunists in enteric diseases due to some other pathogens.     

Evaluation of Statens Serum Institut Enteric Medium for Detection of Enteric Pathogens

The efficacy of the Statens Serum Institut (SSI) enteric medium for isolation and direct identification of enteric pathogens was evaluated. Six different biochemical reactions can be read by using the SSI enteric medium, allowing direct identification of a range of enteric pathogens. All 248 gram-negative bacterial species that were tested grew on the SSI enteric medium. Only 10 of 248 bacteria (4%) showed discrepant results in the biochemical reactions, and none of these were enteric pathogens. Forty-three of 47 enteric pathogens (92%) produced identical rates of semiquantitative growth on the SSI enteric medium and 5% blood agar, whereas three Vibrio spp. and one Aeromonas spp. showed reduced growth. Gram-positive bacteria did not grow on the SSI enteric medium. Most enteric pathogens had a detection limit of 50 bacteria per ml of feces, but higher numbers of Vibrio spp. and some Shigella spp. were required for detection. The growth rates of 125 enteric pathogens and 12 Yersinia spp. on the SSI enteric medium, xylose lysine deoxycholate (XLD), Hektoen enteric (HE), Salmonella-Shigella (SS), and cefsulodin-irgasan-novobiocin (CIN) agar were compared. Detection rates after application of 200 CFU were 99% for SSI enteric medium, 92% for XLD, 88% for HE, and 82% for SS agar. The 12 Yersinia spp. grew excellently on both the SSI enteric medium and CIN agar. We conclude that the performance of the SSI enteric medium compares favorably to those of other media tested. Its ability to detect Yersinia spp. may limit the number of media needed in the typical laboratory. The direct identification of enteric pathogens on the medium may also provide a more rapid diagnosis.     

Use of the duplex TaqMan PCR system for detection of Shiga-like toxin-producing Escherichia coli O157

Real-time PCR assays have been applied for the detection and quantification of pathogens in recent years. In this study two combinations of primers and fluorescent probes were designed according to the sequences of the rfb(Escherichia coli O157) and stx2 genes. Analysis of 217 bacterial strains demonstrated that the duplex real-time PCR assay successfully distinguished the Escherichia coli O157 serotype from non-E. coli O157 serotypes and that it provided an accurate means of profiling the genes encoding O antigen and Shiga-like toxin 2. On the other hand, bacterial strains that lacked these genes were not detected by this assay. The quantitative ranges of the real-time PCR assay for these two genes were linear for DNA concentrations ranging from 10(3) to 10(9) CFU/ml of E. coli O157:H7 in pure culture and milk samples. The real-time PCR allowed the construction of standard curves that facilitated the quantification of E. coli O157:H7 in feces and apple juice samples. The detection sensitivity of the real-time PCR assay ranged from 10(4) to 10(9) CFU/g (or 10(4) to 10(9) CFU/ml) for feces and apple juice and 10(5) to 10(9) CFU/g for the beef sample without enrichment. After enrichment of the food samples in a modified tryptic soy broth, the detection range was from 10(0) to 10(3) CFU/ml. The real-time PCR assays for rfb(E. coli) (O157) and stx2 proved to be rapid tests for the detection of E. coli O157 in food matrices and could also be used for the quantification of E. coli O157 in foods or fecal samples.     

Etiology of diarrhea in young children in Denmark: a case-control study

Infectious gastroenteritis is one of the most common diseases in young children. To clarify the infectious etiology of diarrhea in Danish children less than 5 years of age, we conducted a 2-year prospective case-control study. Stools from 424 children with diarrhea and 870 asymptomatic age-matched controls were examined, and their parents were interviewed concerning symptoms. Rotavirus, adenovirus, and astrovirus were detected by enzyme-linked immunosorbent assay, and norovirus and sapovirus were detected by PCR. Salmonella, thermotolerant Campylobacter, Yersinia, Shigella, and Vibrio spp. were detected by standard methods. Shiga toxin-producing (STEC), attaching-and-effacing (A/EEC), enteropathogenic (EPEC), enterotoxigenic, enteroinvasive, and enteroaggregative Escherichia coli were detected by using colony hybridization with virulence gene probes and serotyping. Parasites were detected by microscopy. Overall, a potential pathogen was found in 54% of cases. More cases than controls were infected with rotavirus, Salmonella, norovirus, adenovirus, Campylobacter, sapovirus, STEC, classical EPEC, Yersinia, and Cryptosporidium strains, whereas A/EEC, although common, was not associated with illness. The single most important cause of diarrhea was rotavirus, which points toward the need for a childhood vaccine for this pathogen, but norovirus, adenovirus, and sapovirus were also major etiologies. Salmonella sp. was the most common bacterial pathogen, followed by Campylobacter, STEC, Yersinia, and classical EPEC strains. A/EEC not belonging to the classical EPEC serotypes was not associated with diarrhea, underscoring the importance of serotyping for the definition of EPEC.     

Laboratory investigation of diarrhea in travelers to Mexico: evaluation of methods for detecting enterotoxigenic Echerichia coli.

A laboratory investigation was conducted on cultures collected from travelers before, during, and after a trip to Mexico to characterize the etiology of traveler's diarrhea. Four laboratory methods for detecting enterotoxigenicity of Escherichia coli were evaluated: the infant mouse assay, the Chinese hamster ovary (CHO) cell assay, the Y1 adrenal cell assay, and the rabbit ileal loop. Although a number of common enteric pathogens were identified as a cause of traveler's diarrhea, including six serotypes of Salmonella, two serotypes of Shigella, Vibrio parahaemolyticus, Giardia lamblia, and Entamoeba histolytica, enterotoxigenic Escherichia coli was most commonly isolated. Strains were identified that produced only heat-labile enterotoxin (LT), only heat-stable enterotoxin (ST), or both LT and ST. The infant mouse assay yielded results falling into two distinct groups, providing a clear separation of positive and negative cultures. The CHO assay also formed two groups, with positive cultures producing 11% or more of the elongated cells. There was good agreement between the CHO and the Y1 adrenal cell assays for detection of LT. The adrenal cell system for detection of LT was more suitable than the CHO assay for processing large numbers of specimens because of the miniculture modification of this method utilized in this study. The infant mouse method was a simple and reliable method for detecting ST.     

Shigella flexneri invasion plasmid antigens B and C: epitope location and characterization with monoclonal antibodies.

Invasion plasmid antigens B (IpaB) and C (IpaC) are associated with the ability of shigellae to invade cultured mammalian cells. Monoclonal antibodies against IpaB and IpaC polypeptides were produced and used in a whole-cell enzyme-linked immunosorbent assay to show that both IpaB and IpaC polypeptides were exposed on the surface of virulent shigellae. Moreover, these surface epitopes were shown to be highly conserved among different serotypes of Shigella spp. and enteroinvasive Escherichia coli. Cross-reactive epitopes were not found on noninvasive Shigella strains or on other enteric bacteria including Salmonella, Yersinia, Campylobacter, Vibrio, and Aeromonas spp. and various pathogenic strains of E. coli. The monoclonal antibodies were used in competitive binding assays to define three unique epitopes of the IpaB polypeptide and four unique epitopes of the IpaC polypeptide. Epitope locations and their corresponding DNA-encoding regions were defined by examining the IpaB and IpaC products expressed by lambda gt11 recombinants and by constructing a genetic map of the insert DNAs of these recombinants. Three IpaB epitopes (2F1, 1H4, 4C8) were found to be encoded on three contiguous DNA regions comprising a 700-base-pair (bp) segment that corresponded to the amino-terminal end of the IpaB polypeptide. Similarly, a 640-bp DNA segment that corresponded to the amino-terminal end of the IpaC polypeptide was found to encode three clustered IpaC epitopes (5H1, 9B6, 5B1). Approximately 50 bp downstream from this region a fourth IpaC epitope-encoding region (2G2) was found. The effect of the monoclonal antibodies on plaque formation by virulent Shigella flexneri on a monolayer of cultured mammalian cells (a sensitive measure of invasiveness) was determined. Only the IpaB-specific monoclonal antibody 2F1 was able to reduce the plaque-forming capacity by greater than 50%, suggesting that this epitope of the IpaB polypeptide is involved in the invasion process.