Detection of Shigella and enteroinvasive Escherichia coli using polymerase chain reaction

Two oligonucleotide primers were used in a polymerase chain reaction (PCR) procedure to amplify a region of the invasive-associated locus (ial) of Shigella and enteroinvasive Escherichia coli (EIEC). Detection of the amplified product can be done by agarose gel electrophoresis, which is specific and sensitive enough for routine diagnosis of these two pathogens. PCR is done using DNA extracted directly from faeces. The procedure can be completed in 7 h. These findings demonstrate a novel method for rapid, sensitive, specific, and simple diagnosis of diarrhoea caused by Shigella and EIEC.     

Using equipment in unfamiliar clinical settings: audiology screening

To develop a better and selective medium for the isolation of Shigella spp., MacConkey's Agar (MAC) was modified by adding potassium tellurite (K2TeO3) at a concentration of 1 microgram/ml. The formulation designated Teknaf Enteric Agar (TEA) was studied for the inhibitory effect of potassium tellurite on the growth of different enteric bacteria, and as a medium for isolating Shigella spp. from clinical stool samples (n = 3125). We observed that the growth of E. coli was effectively inhibited on TEA with no effect on the growth of S. dysenteriae type 1 and S. flexneri. A total of 2019 Shigellae were isolated through the combined use of TEA, MAC, and Salmonella-Shigella Agar (SS). On TEA, 1921 S. dysenteriae type 1 and S. flexneri were isolated as compared to 1765 from the combined use of MAC and SS. A total of 194 of S. dysenteriae type 1 and S. flexneri were exclusively isolated from TEA as compared to 38 which were only made from MAC and SS. We conclude that TEA significantly increased the overall isolation rate of Shigella spp. as compared to the combined use of MAC and SS (P < 0.0001), although it is not suitable for the isolation of S. sonnei.     

"Black holes" and bacterial pathogenicity: a large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli

Plasmids, bacteriophages, and pathogenicity islands are genomic additions that contribute to the evolution of bacterial pathogens. For example, Shigella spp., the causative agents of bacillary dysentery, differ from the closely related commensal Escherichia coli in the presence of a plasmid in Shigella that encodes virulence functions. However, pathogenic bacteria also may lack properties that are characteristic of nonpathogens. Lysine decarboxylase (LDC) activity is present in approximately 90% of E. coli strains but is uniformly absent in Shigella strains. When the gene for LDC, cadA, was introduced into Shigella flexneri 2a, virulence became attenuated, and enterotoxin activity was inhibited greatly. The enterotoxin inhibitor was identified as cadaverine, a product of the reaction catalyzed by LDC. Comparison of the S. flexneri 2a and laboratory E. coli K-12 genomes in the region of cadA revealed a large deletion in Shigella. Representative strains of Shigella spp. and enteroinvasive E. coli displayed similar deletions of cadA. Our results suggest that, as Shigella spp. evolved from E. coli to become pathogens, they not only acquired virulence genes on a plasmid but also shed genes via deletions. The formation of these "black holes," deletions of genes that are detrimental to a pathogenic lifestyle, provides an evolutionary pathway that enables a pathogen to enhance virulence. Furthermore, the demonstration that cadaverine can inhibit enterotoxin activity may lead to more general models about toxin activity or entry into cells and suggests an avenue for antitoxin therapy. Thus, understanding the role of black holes in pathogen evolution may yield clues to new treatments of infectious diseases.     

Multiple sample PCR amplification and electrophoretic analysis on a microchip

Polymerase chain reactions (PCRs) were carried out on as many as four DNA samples at a time on a microchip device. The PCR products were then analyzed, either individually or together on the same device, by microchip gel electrophoresis. A standard PCR protocol was used to amplify 199- and 500-base pair (bp) regions of bacteriophage lambda DNA and 346- and 410-bp regions of E. coli genomic and plasmid DNAs, respectively. Thermal lysis of the bacteria was integrated into the PCR cycle. A product sizing medium, poly(dimethylacrylamide), and an intercalating dye for fluorescence detection were used in the electrophoretic analysis of the products. PCR product sizes were determined by coelectrophoresis with marker DNA.     

Evaluation of a technique for identification of Shiga-like toxin-producing Escherichia coli by using polymerase chain reaction and digoxigenin-labeled probes

A polymerase chain reaction (PCR) technique for the identification of Shiga-like toxin (SLT)-producing Escherichia coli was assessed by using 95 strains of SLT-producing E. coli and 5 Shigella dysenteriae type 1 strains. PCR was used for the amplification of slt gene sequences from whole bacterial colonies. A digoxigenin-labeled DNA probe was used for identification of the PCR products in a spot blot hybridization assay. Modifications were made to adapt this technique for the proper identification of 10 SLT-producing isolates which were refractory to the heat lysis step that was used to liberate whole-cell DNA for PCR and 6 isolates which gave nonspecific amplification products. The sensitivity and specificity of this assay were each 99% when compared with toxin neutralization results by using SLT-specific monoclonal antibodies. These values indicate that this detection technique could be suitable for use in a clinical laboratory.     

H-NS regulates DNA repair in Shigella

We report a new role for H-NS in Shigella spp.: suppression of repair of DNA damage after UV irradiation. H-NS-mediated suppression of virulence gene expression is thermoregulated in Shigella, being functional at 30 degrees C and nonfunctional at 37 to 40 degrees C. We find that H-NS-mediated suppression of DNA repair after UV irradiation is also thermoregulated. Thus, Shigella flexneri M90T, incubated at 37 or 40 degrees C postirradiation, shows up to 30-fold higher survival than when incubated at 30 degrees C postirradiation. The hns mutants BS189 and BS208, both of which lack functional H-NS, show a high rate of survival (no repression) whether incubated at 30 or 40 degrees C postirradiation. Suppression of DNA repair by H-NS is not mediated through genes on the invasion plasmid of S. flexneri M90T, since BS176, cured of plasmid, behaves identically to the parental M90T. Thus, in Shigella the nonfunctionality of H-NS permits enhanced DNA repair at temperatures encountered in the human host. However, pathogenic Escherichia coli strains (enteroinvasive and enterohemorrhagic E. coli) show low survival whether incubated at 30 or 40 degrees C postirradiation. E. coli K-12 shows markedly different behavior; high survival postirradiation at both 30 and 40 degrees C. These K-12 strains were originally selected from E. coli organisms subjected to both UV and X irradiation. Therefore, our data suggest that repair processes, extensively described for laboratory strains of E. coli, require experimental verification in pathogenic strains which were not adapted to irradiation.     

Identification of enteroinvasive Escherichia coli and Shigella strains in pediatric patients by an IpaC-specific enzyme-linked immunosorbent assay

A new method, a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) recognizing a secreted, invasion plasmid-coded protein antigen (IpaC), was used to identify enteroinvasive Escherichia coli and Shigella strains among colonies from 859 cultures of fecal samples from children in Kuwait. A total of 33.8% of the samples were diarrheal. By the immunoassay, enteroinvasive E. coli strains were identified from two diarrheal samples but from none of the samples from children without diarrhea. These strains were fully virulent and belonged to serogroup O28ac. In addition, 26 Shigella strains were also recognized by the ELISA, while only 23 were isolated by routine biotyping and serotyping. For two diarrheal patients, Shigella was identified by culture only. The study showed that the IpaC-specific immunoassay is a simple and useful tool for identifying enteroinvasive strains. Furthermore, by reporting the first enteroinvasive E. coli isolates from Kuwait, the study indicates the presence of this group of pathogens as a potential source of diarrhea in the region.     

Sexually selected vigilance behaviour of the grey partridge is affected by plasma androgen levels

Polymerase chain reaction (PCR) diagnostic methods have rarely been used in epidemiologic studies of Shigella and enteroinvasive Escherichia coli (EIEC) infections. In this study, amplification of the invasion plasmid antigen H (ipaH) gene by PCR and standard culture methods was used to identify Shigella species or EIEC among 154 patients with dysentery, 154 age-matched controls, and family contacts in Thailand. The ipaH PCR system increased the detection of Shigella species and EIEC from 58% to 79% among patients with dysentery and from 6% to 22% among 527 family contacts; 75% of infections in family members were asymptomatic. Detection of the ipaH gene was statistically associated with dysentery. Household contacts of patients with shigellosis diagnosed only by PCR had significantly higher rates of shigellosis than household contacts of patients who did not have Shigella or EIEC infections. Detection of the ipaH gene by PCR is far more sensitive than detection by standard culture and is highly correlated with evidence of Shigella transmission among family contacts.     

Investigation of film curing stages by dielectric analysis and physical characterization

A previously published sequence of the 23S rRNA gene of Coxiella burnetii has been reported to contain an intervening sequence of 444 base pairs (bp). The sequence information on the intervening sequence and the 23S rRNA gene was exploited to develop a specific PCR-based assay for C. burnetii. A primer set was designed that amplified a 477-bp fragment encompassing part of the intervening sequence and part of the 23S rDNA. From all of nine C. burnetii strains tested, a fragment of the expected size was amplified. As predicted from the published sequence, restriction endonuclease digestion of the PCR product from the Coxiella strains with RsaI produced two distinct fragments approximately 210- and 270-bp in size. The PCR-based method showed a detection limit of 10(2) bacteria as determined by visualization of the amplicon on an agarose gel. When experimentally infected blood was analyzed, the detection limit was 10(3) bacteria. No visible amplicons were observed when 41 bacterial strains, representing 29 species other than C. burnetii, were tested. The presence of the DNA in all bacterial samples was confirmed by amplification of a 350-bp fragment of the 16S rDNA using two universal primers. The described method proved to be specific for C. burnetii and may become a rapid and sensitive diagnostic assay for C. burnetii. The results also demonstrate that the intervening sequence within the 23S rRNA gene is generally found among isolates of C. burnetii.     

Short-term clinical study on a new aqueous humor drainage implant for refractory glaucoma

Alkaline phosphatase-conjugated oligonucleotide probes were developed to detect the gene coding for Vero toxin 1 (VT1) and Vero toxin 2 (VT2). Using these probes, 3 hr was enough to detect VT genes when suspicious colonies of enterohaemorrhagic Escherichia coli (EHEC) were obtained on an agar plate. The results of a hybridization test with 144 isolates of EHEC O157 and one isolate of Shigella dysenteriae Type 1 agreed exactly with the immunological detection, reversed passive latex agglutination (RPLA) test, of VTs in their culture supernatants. The sensitivity levels of these probes for the detection of VT genes were 100%. The specificity of these probes were also tested with a total of 1,002 strains of Escherichia coli other than EHEC and 8 strains of Shigella sp. other than Shigella dysenteriae Type 1; the results showed 100% specificity.     

Isolation of Shigella dysenteriae serotypes 11, 12, and 13 from patients with diarrhea in Bangladesh

Nine isolates of bacteria biochemically resembling Shigella dysenteriae but not belonging to the 10 recognized serotypes were isolated from patients with diarrhea in Bangladesh. Further studies suggested that two, one, and six isolates belonged to the recently recognized S. dysenteriae serotypes 11, 12, and 13, respectively.     

Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria

The ability to transport and use haemin as an iron source is frequently observed in clinical isolates of Shigella spp. and pathogenic Escherichia coli. We found that many of these haem-utilizing E. coli strains contain a gene that hybridizes at high stringency to the S. dysenteriae type 1 haem receptor gene, shuA. These shuA-positive strains belong to multiple phylogenetic groups and include clinical isolates from enteric, urinary tract and systemic infections. The distribution of shuA in these strains suggests horizontal transfer of the haem transport locus. Some haem-utilizing pathogenic E. coli strains did not hybridize with shuA, so at least one other haem transport system is present in this group. We also characterized the chromosomal region containing shuA in S. dysenteriae. The shuA gene is present in a discrete locus, designated the haem transport locus, containing eight open reading frames. Several of the proteins encoded in this locus participate with ShuA in haem transport, as a Salmonella typhimurium strain containing the entire haem transport locus used haem much more efficiently than the same strain containing only shuA. The haem transport locus is not present in E. coli K-12 strains, but the sequences flanking the haem transport locus in S. dysenteriae matched those at the 78.7 minute region of E. coli K-12. The junctions and flanking sequences in the shuA-positive pathogenic E. coli strains tested were nearly identical to those in S. dysenteriae, indicating that, in these strains, the haem transport locus has an organization similar to that in S. dysenteriae, and it is located in the same relative position on the chromosome.     

Molecular biology detection and antibiotic sensitivities of Shigella spp. and entero-invasive Escherichia coli (EIEC) in patients returning from the tropics

1579 stool samples from patients with travel-associated diarrhea were examined by conventional culture methods to detect Shigella spp. (48 positive samples or 3.2%) and by PCR to detect shigellae and enteroinvasive Escherichia coli (EIEC) (87 positive samplers or 5.8%). The numerical relation of shigellae to EIEC in PCR-positive samples was about 60 to 40%. However, the lack of discrimination between shigellae and EIEC is not important for the physician because the choice of antibiotics remains the same.     

Studies on bacillary dysentery cases of overseas travellers--during 1979 to 1995

A total of 36,780,440 overseas travellers during 1979-1995 (17 years) were quarantined at Osaka and Kansai Airport Quarantine Station, 84,777 travellers reported themselves suffer from diarrhoea. Stools from 29,587 persons were bacteriologically examined. Various enteropathogenic bacteria were isolated from 9,766 (33.0%) patients of the stools examined. Isolated species were as follows: Plesiomonas shigelloides (3,234 cases); Salmonella spp. (2,236 cases); enterotoxgenic Escherichia coli (1,621 cases); Vibrio parahaemolyticus (1,959 cases); and Shigella spp. (1,242 cases). 1,278 different Shigella strains were isolated from 1,242 cases who were thus diagnosed as bacillary dysentery patients. The suspected regions or countries for infection of these cases were analysed. The serovars and antibiotic-sensitivities of the isolated strains were examined. Colicine typing of S. sonnei strains were also done. The results can be summarized as follows. 1) The most cases (53.4%) were infected in India. 2) The percentage distribution of sub-species of the strains was as follows; S. sonnei (57.8%), S flexneri (29.8%), S. boydii (8.4%), and S. dysenteriae (4.0%), respectively. 3) The major colicine type of S. sonnei strains were type 6 and 0. 4) The percentage of Antibiotic-resistant strains of each sub-species was S. dysenteriae (92.2%), S. sonnei (89.4%), S. flexneri (87.1%), and S. boydii (84.9%), respectively. The percentage of Antibiotic-resistant strains of S. flexneri were increased annually.     

Antimicrobial susceptibility of Shigella from a rural community in Bangladesh

Of the 63 Shigella strains isolated from stool cultures from 200 patients who attended a district hospital in Bangladesh with bloody diarrhoea, 37 (59%) were S. dysenteriae type 1, 25 (39%) were S. flexneri and only one (2%) was S. sonnei. Over half (54%) of the Shigella isolates came from children aged < 10 years. Most (89%) of the isolates of S. dysenteriae type 1 were resistant to ampicillin, cotrimoxazole, nalidixic acid, tetracycline and chloramphenicol. Although many (60%) of the isolates of S. flexneri were resistant to ampicillin and cotrimoxazole, only 4% of them were resistant to nalidixic acid. However, all of the S. dysenteriae and S. flexneri were sensitive to ciprofloxacin. The need for periodic monitoring to determine the resistance pattern in remote areas is emphasised.     

Cloning and sequencing of the gene encoding a 31-kilodalton antigen of Haemophilus somnus

Immunoblots using bovine antibody against Haemophilus somnus as the primary antibody consistently identified 31-, 40- and 78-kDa proteins in Sarkosyl-insoluble extracts of H. somnus. A genomic library of H. somnus 8025 DNA was constructed in plasmid pUC19, and 45 recombinants expressed proteins which were recognized by bovine antiserum in Western blots (immunoblots). Ten of the recombinants expressing a 31-kDa protein caused the lysis of bovine erythrocytes. Restriction endonuclease mapping indicated that the hemolytic recombinants shared an approximately 1.7-kb BglII fragment. Southern blot analysis using the BglII fragment as a probe revealed homology among the recombinants and the presence of an identically sized BglII fragment in the chromosome of all H. somnus isolates tested. Sequence analysis indicated the presence of an 822-bp open reading frame within the 1.7-kb BglII fragment. Deletion of this open reading frame resulted in the loss of hemolytic activity and protein expression in recombinant Escherichia coli, suggesting the possible role of the 31-kDa protein as a hemolysin. An amino acid sequence deduced from the DNA sequence shared homology with outer membrane protein A of E. coli, Salmonella typhimurium, and Shigella dysenteriae, with P6 of Haemophilus influenzae, and with PIII of Neisseria gonorrhoeae. An amino acid analysis of the recombinant 31-kDa protein agreed with the amino acid composition deduced from the DNA sequence.     

First melanoma metastases after 10 years and more of remission

The aim of this study was to combine immunomagnetic capture and a polymerase chain reaction (PCR) followed by hybridization with a DNA probe for the detection of Bacteroides forsythus. Magnetic beads were coated with the immunoglobulin G fraction of an antiserum specific for B. forsynthus. Aliquots were incubated with various concentrations of a suspension of B. forsythus or with a suspension containing 16 bacterial species, at a concentration of 10(10) cells/ml, spiked with dilutions of B. forsythus. Beads with bound bacteria were boiled, and the target DNA in the supernatant was amplified to generate a 392-bp PCR fragment specific for B. forsythus. The amplified product was detected by dot-blot hybridization with a digoxigenin-labeled 392-bp probe. The detection limit was determined to be 10 cells/ml using immunocapture on a suspension of B. forsythus and 100 on spiked bacterial suspensions. Subgingival plaque samples were obtained from 39 Bolivian individuals with poor oral hygiene. Each sample was analyzed by the above procedure and by immunofluorescence. The overall prevalence of individuals harboring B. forsythus was 62% by immunofluorescence and 82% by PCR-DNA probe assay. The immunocapture, PCR. DNA-probe procedure should be useful for the detection of B. forsythus, particularly in false-negative samples obtained by less sensitive techniques.     

Nucleic acid amplification for rapid detection of Rhodococcus equi in equine blood and tracheal wash fluids

OBJECTIVE: To evaluate the ability of nucleic acid amplification techniques to detect Rhodococcus equi in equine buffy coat, blood, and tracheal wash fluid and to differentiate between virulent and avirulent strains of the bacteria. SAMPLE POPULATION: Blood anticoagulated with EDTA and tracheal wash fluid from healthy horses. PROCEDURE: Logarithmic dilutions of virulent and avirulent strains of R equi were added to equine buffy coat and tracheal wash fluid samples. The DNA was extracted and amplified by polymerase chain reaction (PCR), using primers specific for the 16S ribosomal subunit gene and the virulence plasmid of R equi. RESULTS: PCR with 16S ribosomal subunit primers amplified a 441-bp segment of DNA from virulent and avirulent strains of R equi, but not from samples containing other species of bacteria. The virulence plasmid primers amplified an 875-bp segment of DNA from virulent strains of R equi, but not from avirulent R equi, or from other species of bacteria. Virulent strains of R equi could be identified by PCR and differentiated from avirulent strains within 12 to 24 hours after sample collection, with as few as 10 to 100 organisms present. CONCLUSIONS: PCR can be used to rapidly and accurately identify R equi in equine blood and tracheal wash fluid samples and can differentiate between virulent and avirulent strains of the organism. CLINICAL RELEVANCE: Because PCR can confirm a diagnosis of R equi infection in horses more rapidly and specifically than use of standard culture techniques, extrapolation of this assay to soil and fecal samples could be useful in epidemiologic studies and studies of environmental disinfection or decontamination.     

A randomized study of the efficacy of the PROPATH Program for patients with Parkinson disease

By polymerase chain reaction (PCR) using a pair of primers specific for Salmonella phoE gene a 365-bp specific gene fragment could be amplified from yolk of infertile eggs and dead-in-shell chicken embryos, and from environmental samples. Out of 45 dead-in-shell embryo samples, 20 (44.4%) were found positive for Salmonella DNA by PCR compared to 11 (24.4%) by bacteria isolation. Salmonella DNA could also be detected from infertile eggs, chicken faeces, floor litter and chick fluff, which incidence was higher than that by bacteria isolation.     

Strategy for the detection of Helicobacter species by amplification of 16S rRNA genes and identification of H. felis in a human gastric biopsy

The aim of the present work was to develop polymerase chain reactions (PCRs) based on the conserved nucleotide sequence of the 16S rRNA gene for detection of bacteria of the Helicobacter genus in human antral biopsy samples. The assay for Helicobacter spp was developed by amplifying a 399-bp 16S rRNA gene sequence specific to the genus Helicobacter. The identity of the amplicon was confirmed by hybridization with an internal probe and by restriction by endonuclease VspI showing two expected fragments of 295 and 104 base pairs. A total of 65 dyspeptic patients from France and New Caledonia were screened for Helicobacter spp infection through the use of the following diagnostic assays on biopsy specimens collected through endoscopy: direct detection of bacteria in histological sections by Giemsa and Warthin Starry staining, urease test and bacterial isolation, PCR for Helicobacter pylori ureC/glmM gene, and PCR targeted to 16S rRNA genes. The 16S rRNA gene PCR assay was able to detect down to 680 bacterial cells, as assessed by agarose gel electrophoresis, and down to 4 bacterial cells by hybridization of amplicon with the internal probe. The 16S rRNA PCR test was 100% specific and sensitive; results obtained with this test were in agreement with the visualization of bacteria by histology. Urease test and culture were 86.4% and 22.7% sensitive, and 96.5 and 100% specific, respectively. The H. pylori ureC/glmM gene-based PCR was 100% specific and only 95.4% sensitive, since one biopsy from a Melanesian patient contained a Helicobacter strain other than H. pylori. For this Melanesian patient, a branch-specific PCR targeting the epsilon branch of Proteobacteria was used to amplify a 967-bp amplicon. This amplicon was sequenced and matched with the H. felis sequence. This was confirmed using an H. felis-specific urease PCR test.