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.     

Expanded therapeutic possibilities in hyperthyroidism in the elderly. Sclerotherapy of autonomous adenomas and thyrostatic therapy

The use of buoyant density centrifugation (BDC) to prepare samples for PCR analysis of food pathogens is described. Blue cheese and milk homogenates were inoculated with Shigella flexneri and layered on top of Percoll media. After BDC, the food homogenates remained in the upper part of the centrifuge tube, separated from the bacteria, which retained viability and were concentrated below the lighter Percoll layer. PCR inhibitors stayed in the homogenate and PCR analyses of treated samples consistently detected 10(4) cfu g-1 of blue cheese and 500 cfu ml-1 of milk, respectively. Differences in the density of live and killed Sh. flexneri and Yersinia enterocolitica were detected by BDC but were dependent on the mechanism of killing.     

Interactions of the invasive pathogens Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells and murine Peyer's patches

Invasive enteric bacteria must pass through the intestinal epithelium in order to establish infection. It is becoming clear that a common target for intestinal mucosa penetration is the specialized epithelial cell of Peyer's patches, the M cell. In order to gain a better understanding of how bacteria interact with M cells, we have compared the interactions of Salmonella typhimurium, Listeria monocytogenes, and Shigella flexneri with M cells by using a murine ligated-loop model. Our results indicate that S. typhimurium possesses a highly efficient mechanism for M cell entry that targets and destroys these cells, while L. monocytogenes and S. flexneri appear to be internalized into M cells in a less disruptive fashion. Early uptake of Listeria or Shigella into M cells appeared to lead to the death of some cells, as evidenced by the appearance of holes in the intestinal epithelium. At later time points, the follicle-associated epithelium of animals infected with these bacteria displayed extensive destruction. These data indicate that enteric pathogens use different strategies to interact with M cells and initiate infection of a host.     

The survival and growth of acid-adapted mesophilic pathogens that contaminate meat after lactic acid decontamination

Lactic acid decontamination (LAD) may adapt pathogens to lactic acid. Such organisms may have an increased resistance to acid and can contaminate meat after LAD. The survival and growth of acid adapted Campylobacter jejuni, Salmonella typhimurium. Escherichia coli O157:H7 and Staphylococcus aureus inoculated on skin surface of still warm pork belly cuts 2 h after LAD was examined during chilled (4 degrees C) storage and refrigeration abuse equivalent to 12.5 degrees C. Lactic acid decontamination included dipping in 1, 2 or 5% lactic acid solutions at 55 degrees C for 120 s. Lactic acid decontamination brought sharp reductions in meat surface pH, but these recovered with time after LAD at approximately 1-1.5 pH units below that of water-treated controls. A sharp decrease in the number of cfu of pathogens occurred on chilled 2-5% lactic acid treated pork belly cuts when the skin surface was less than pH 4.8-5.2. The reductions ranged from 0.1-0.3 log10 cfu cm-2 for E. coli O157:H7 to over 1.7-2.4 log10 cfu cm-2 for Camp. jejuni, respectively. Increase in storage temperature from 4 to 12.5 degrees C reduced delayed decrease in numbers of all pathogens except Camp. jejuni by a factor of two. Deaths in Camp. jejuni at 12.5 degrees C slightly exceeded those at 4 degrees C. After the initial sharp decline, the number of cfu of mesophilic pathogens decreased gradually at a rate similar to that on water-treated controls. Growth of all mesophilic pathogens except Camp. jejuni on 2-5% LAD meat occurred during storage at 12.5 degrees C when the meat surface pH exceeded 4.8-5.2, and was slower than on water-treated controls. Low temperature and acid-adapted E. coli O157:H7, Salm. typhimurium and Staph. aureus, and acid adapted Camp. jejuni that contaminate skin surface after hot 2-5% LAD, did not cause an increased health hazard, although microbiota and intrinsic parameters (lactic acid content, pH) were created that could advantage their survival and growth.     

Growth and survival of Shigella flexneri in common Bangladeshi foods under various conditions of time and temperature

Survival and growth of Shigella flexneri were assessed in various foods, including boiled rice, lentil soup, milk, cooked beef, cooked fish, mashed potato, mashed brinjal, and raw cucumber. Growth at 25 and 37 degrees C and survival at 5 degrees C were observed by viable counts on MacConkey agar. The organism grew well in all tested foods and growth increased from 10(5) to 10(8) to 10(10) cells per ml or g within 6 to 18 h after inoculation at 25 and 37 degrees C.     

Is undifferentiated seronegative spondyloarthropathy a forme fruste of reactive arthritis?

Undifferentiated spondyloarthropathy (USpa) may either represent a forme fruste of other spondyloarthropathies like reactive arthritis or be a different disease entity. To study the link between USpa and reactive arthritis, we studied the presence of IgA antibodies to Yersinia enterocolitica, Salmonella typhimurium, Shigella flexneri, Campylobacter jejuni and Chlamydia trachomatis in sera from 14 patients with USpa (European Spondyloarthropathy Study Group criteria) using ELISA. Escherichia coli was used as a control antigen. An OD value of more than the mean +/- 2 S.D. of 51 blood donors was considered positive. Five patients had elevated IgA antibodies to S. flexneri, while two patients each had elevated antibody levels to S. typhimurium and Chlamydia. No patient had elevated antibodies to Y. enterocolitica, C. jejuni and E. coli. Among 51 normals, 1, 4, 3, 2 and 3 had elevated antibodies to S. flexneri, S. typhimurium, Y. enterocolitica, C. jejuni and E. coli, respectively. Nine of 14 patients with USpa had antibodies to one of the bacteria implicated in reactive arthritis: of these, antibodies to Shigella were the most frequent. Thus, a proportion of patients with USpa may in fact have reactive arthritis.     

A glutamate-dependent acid resistance gene in Escherichia coli

Stationary-phase cultures of Escherichia coli can survive several hours or exposure to extreme acid (pH 2 to 3), a level well below the pH range for growth (pH 4.5 to 9). To identify the genes needed for survival in extreme acid, a microliter screening procedure was devised. Colonies from a Tn10 transposon pool in E. coli MC4100 were inoculated into buffered Luria broth, pH 7.0, in microtiter wells, grown overnight, and then diluted in Luria broth, pH 2.5, at 37 degrees C for 2 h. From 3,000 isolates screened, 3 Tet(r) strains were identified as extremely acid sensitive (<0.1% survival at pH 2.5 for 2 h). Flanking sequences of the Tn10 inserts were amplified by inverse PCR. The sequences encoded a hydrophobic partial peptide of 88 residues. A random-primer-generated probe hybridized to Kohara clones 279 and 280 at 32 min (33.7 min on the revised genomic map EcoMap7) near gadB (encoding glutamate decarboxylase). The gene was designated xasA for extreme acid sensitive. xasA::Tn10 strains grown at pH 7 to 8 showed 100-fold-less survival in acid than the parent strain. Growth in mild acid (pH 5 to 6) restored acid resistance; anaerobiosis was not required, as it is for acid resistance in rpoS strains. xasA::Tn10 eliminated enhancement of acid resistance by glutamic acid. xasA was found to be a homolog of gadC recently sequenced in Shigella flexneri, in which it appears to encode a permease for the decarboxylated product of GadB. These results suggest that GadC (XasA) participates in a glutamate decarboxylase alkalinization cycle to protect E. coli from cytoplasmic acidification. The role of the glutamate cycle is particularly important for cultures grown at neutral pH before exposure to extreme acid.     

Clearance of Shigella flexneri infection occurs through a nitric oxide-independent mechanism

Nitric oxide (NO) generated by gamma interferon (IFN-gamma) activation of macrophages mediates the killing of many intracellular pathogens. IFN-gamma is essential to innate resistance to Shigella flexneri infection. We demonstrate that NO is produced following S. flexneri infection both in mice and in activated cells in vitro and that while it is able to kill S. flexneri in a cell-free system, it is not required for clearance of S. flexneri in either infected mice or in activated cells in vitro.     

The screening detection of the antigens of pathogenic enterobacteria in the biological fluids of patients with chronic intestinal diseases

Screening detection of the antigens of some pathogenic enterobacteria in the coagglutination test with the use of Shigella (S. sonnei, S. flexneri 2a, 1b, 6), Salmonella (groups A, B, C, D, E) and Yersinia (Y. pseudotuberculosis, serovars I and III, Y. enterocolitica 03 and 09) diagnostica in patients with chronic enteric diseases, hospitalized in a noninfectious ward, has demonstrated a high incidence of the above antigens. S. flexneri antigens have shown the highest detection rate. The results thus obtained indicate that these infective agents probably take part in the development of exacerbations of chronic enteric diseases and, therefore, a comprehensive examination of this group of patients is necessary with a view to the detection of bacterial enteric infection.     

Upregulation of urokinase-type plasminogen activator by endogenous and exogenous HIV-1 Tat protein in tumour cell lines derived from BK virus/tat-transgenic mice

A universal protocol for PCR detection of 13 species of foodborne pathogens in foods was developed. The protocol used a universal culture medium and the same PCR conditions with 13 sets of specific primers. The 13 species of foodborne pathogens examined were Escherichia coli, E. coli-ETEC, E. coli-O157:H7, Shigella spp., Salmonella spp., Yersinia enterocolitica, Y. pseudotuberculosis, Vibrio cholerae, V. parahaemolyticus, V. vulnificus, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus. No interference was observed using the PCR assay when food sample was artificially inoculated with each individual bacterial species. Twelve different seafood samples and two soft cheese samples without artificial inoculation were examined by this protocol. Vibrio vulnificus, Salmonella spp., E. coli, Listeria monocytogenes and Bacillus cereus were detected in some foods. Internal probe hybridization and nested PCR procedures were used to confirm the above findings.     

Occurrence of Protozoan and Bacterial Pathogens in Water Samples from Small Water Systems in Mountainous Areas of Taiwan

Giardia, Cryptosporidium, Salmonella, and Shigella have emerged as waterborne pathogens of concern. Thirty water samples were collected from 10 small water systems in mountainous areas of Taiwan and investigated for the occurrence of these pathogens. In addition, the basic characteristics of each small water system were recorded, and each water sample was tested for several water quality parameters. The occurrence frequency was 10.0% for Giardia spp., 3.3% for Cryptosporidium spp. and Shigella flexneri, and 16.7% for Salmonella enterica. The highest correlation was found between the presence of Salmonella enterica and low water temperature, followed by the correlation between water turbidity and fecal coliform concentration. A low correlation was found between the presence of Giardia and the concentration of heterotrophic bacteria. The water samples from small water systems with filtration devices had a lower occurrence of pathogen microorganisms than those without filtration devices. The proportion of water samples with pathogenic microorganisms increased with the consumer population of the water systems, and the pathogen occurrence differed among the regions of the sampling sites. In order to prevent parasitic infection, the use of disinfection devices in small water systems would be needed.     

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.     

Bacterial entry into epithelial cells: the paradigm of Shigella

Shigella flexneri is a model for the entry of bacterial pathogens into nonphagocytic epithelial cells. On contact with the epithelial cell surface, the Ipa proteins are secreted from the bacterium. The Ipa complex then triggers a reorganization of the host-cell cytoskeleton leading to the formation of membrane ruffles, which engulf the bacterium.     

Structural determination of the O-antigenic polysaccharide from the enterotoxigenic Escherichia coli O147

The structure of the O-antigenic polysaccharide from enterotoxigenic Escherichia coli O147 has been determined by NMR spectroscopy, and component and methylation analyses. The sequence of the sugar residues could be determined by NOESY and heteronuclear-multiple-bond-connectivity NMR experiments. It is concluded that the polysaccharide is composed of tetrasaccharide repeating units with the following structure: -->4)-beta-D-GalpA-(1-->3)-beta-D-GalpNAc-(1-->2)-alpha-L-Rhap+ ++-(1-->2)-alpha-L-Rhap-(1-->, where Rha represents 6-deoxymannose. The O-antigen of E. coli O147 is identical to the repeating unit of Shigella flexneri serotype 6 lipopolysaccharide, except that the latter contains an O-acetyl group at C3 of the rhamnosyl residue substituted by the N-acetylgalactosamine residue. Immunochemical analyses using a monoclonal antibody specific for the S. flexneri serotype 6 O-antigen showed an identical reactivity with both lipopolysaccharides.     

Ampicillin resistance mediated by the R plasmid in strains of Shigella flexneri]

Forty Shigella flexneri strains isolated from children attended to at the Children's Hospital of Camagüey during an outbreak of acute diarrheal disease were studied; the minimal inhibitory concentration of ampicillin was determined. 33 strains (82.5%) were resistant to higher concentrations: 8 to 16 micrograms/mL, and 7 were susceptible to 4 micrograms/mL concentrations. Resistance plasmid (R) extraction was carried out in all the isolated strains and a common plasmid was found this plasmid was purified and transferred to Escherichia coli HE 101. Resistance transmission was tested.     

Topics in microbial risk assessment: dynamic flow tree process

Microbial risk assessment is emerging as a new discipline in risk assessment. A systematic approach to microbial risk assessment is presented that employs data analysis for developing parsimonious models and accounts formally for the variability and uncertainty of model inputs using analysis of variance and Monte Carlo simulation. The purpose of the paper is to raise and examine issues in conducting microbial risk assessments. The enteric pathogen Escherichia coli O157:H7 was selected as an example for this study due to its significance to public health. The framework for our work is consistent with the risk assessment components described by the National Research Council in 1983 (hazard identification; exposure assessment; dose-response assessment; and risk characterization). Exposure assessment focuses on hamburgers, cooked a range of temperatures from rare to well done, the latter typical for fast food restaurants. Features of the model include predictive microbiology components that account for random stochastic growth and death of organisms in hamburger. For dose-response modeling, Shigella data from human feeding studies were used as a surrogate for E. coli O157:H7. Risks were calculated using a threshold model and an alternative nonthreshold model. The 95% probability intervals for risk of illness for product cooked to a given internal temperature spanned five orders of magnitude for these models. The existence of even a small threshold has a dramatic impact on the estimated risk.     

Activation of the human immunodeficiency virus-1 long terminal repeat by respiratory burst oxidants of neutrophils

Unipolar localization of IcsA on the surface of Shigella flexneri is required for efficient formation of actin tails and protrusions in infected eucaryotic cells. Lipopolysaccharide (LPS) mutations have been demonstrated to affect either the establishment or the maintenance of IcsA in a unipolar location, although the mechanism is unknown. In order to analyze the contribution of virulence plasmid determinants on the unipolar localization of IcsA, we examined the localization of IcsA expressed from a cloned plasmid copy in two different genetic backgrounds. The localization of IcsA was first examined in a virulence plasmid-cured derivative of the wild-type S. flexneri 2a isolate 2457T. This approach examined the contribution of virulence plasmid-borne factors, including the previously identified virulence plasmid-borne protease that is responsible for cleaving IcsA in the outer membrane and releasing the 95-kDa secreted form from the cell surface. IcsA localization in a related but nonpathogenic Escherichia coli strain expressing LPS of the O8 serotype was also examined. IcsA surface presentation in both of these genetic backgrounds continued to be unipolar, demonstrating that virulence plasmid-borne determinants are not responsible for unipolar localization of IcsA. The unipolar localization of IcsA in the E. coli background suggests that a common pathway that allows IcsA to be spatially restricted to one pole on the bacterial cell surface exists in Shigella and E. coli.     

Identification of two Shigella flexneri chromosomal loci involved in intercellular spreading

The ability of Shigella flexneri to multiply within colonic epithelial cells and spread to adjacent cells is essential for production of dysentery. Two S. flexneri chromosomal loci that are required for these processes were identified by screening a pool of TnphoA insertion mutants. These mutants were able to invade cultured epithelial cells but could not form wild-type plaques. Analysis of the nucleotide sequence indicated that the sites of TnphoA insertion were within two different regions that are almost identical to Escherichia coli K-12 chromosomal sequences of unknown functions. One region is located at 70 min on the E. coli chromosome, upstream of murZ, while the other is at 28 min, downstream of tonB. The mutant with the insertion at 70 min was named vpsC because it showed an altered pattern of virulence protein secretion. The vpsC mutant formed pinpoint-sized plaques, was defective in recovery from infected tissue culture cells, and was sensitive to lysis by the detergent sodium dodecyl sulfate. Recombinant plasmids carrying the S. flexneri vpsA, -B, and -C genes complemented all of the phenotypes of the vpsC mutant. A mutation in vpsA resulted in the same phenotype as the vpsC mutation, suggesting that these two genes are part of a virulence operon in S. flexneri. The mutant with the insertion at 28 min was interrupted in the same open reading frame as S. flexneri ispA. This ispA mutant could not form plaques and was defective in bacterial septation inside tissue culture cells.