SUMMER DIARRHEA IN THE SAN JOAQUIN VALLEY

In laboratory, epidemiologic and clinical studies of 85 patients with diarrhea admitted to the General Hospital of Fresno County and the San Joaquin County Hospital during part of the summer of 1949 the following features were noted:1. Cultures were positive for Shigella in about 45 per cent of the cases in the San Joaquin County Hospital and in about 15 per cent of those in the Fresno County Hospital.2. In 80 per cent of cases in which there was gross blood in the stools before the third day of hospitalization, Shigella grew on cultures. No gross blood was noted in 27 per cent of cases in which Shigella was demonstrated.3. The cases in which Shigella was demonstrated did not differ greatly from the others with regard to fever, leukocytosis, or response to therapy.4. Salmonella organisms were not grown on cultures in any case.5. Most of the patients were infants younger than one year of age.6. Poor socio-economic and hygienic conditions characterized the households from which these county hospital patients came.7. Household follow-up studies indicated that there had been one or more instances of diarrhea among household associates of approximately half the hospitalized patients at some time during the month prior to admission.     

Delivery of biologically active anti-inflammatory cytokines IL-10 and IL-1ra in vivo by the Shigella type III secretion apparatus

Pathogenicity of many Gram-negative bacteria relies on a type III secretion (T3S) apparatus, which is used for delivery of bacterial effectors into the host cell cytoplasm allowing the bacteria to manipulate host cell cytoskeleton network as well as to interfere with intracellular signaling pathways. In this study, we investigated the potential of the Shigella flexneri T3SA as an in vivo delivery system for biologically active molecules such as cytokines. The anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist (IL-1ra) were genetically fused to the first 30 or 60 residues of the Shigella T3S effector IpaH9.8 or to the first 50 residues of the Yersinia enterocolitica effector YopE and the recombinant fusion proteins were expressed in S. flexneri. YopE(50)-IL-10, IpaH(60)-IL-10, and IpaH(60)-IL-1ra were efficiently secreted via the T3S apparatus of Shigella. Moreover, these recombinant proteins did not impair the invasive ability of the bacteria in vitro. In a murine model, Shigella strains expressing YopE(50)-IL-10, IpaH(60)-IL-10, and IpaH(60)-IL-1ra induced a lower mortality in mice that was associated with reduced inflammation and a restricted localization of bacteria within the lung tissues as compared with wild-type Shigella. Moreover, the level of TNF-alpha and IL-1beta mRNA were reduced in the lungs following infection by IL-10- and IL-1ra-secreting Shigella, respectively. These findings demonstrate that the Shigella T3S apparatus can deliver biologically active cytokines in vivo, thus opening new avenues for the use of attenuated bacteria to deliver proteins for immunomodulation or gene therapy purposes.     

The determination in saliva of IgA antibodies to Shigella ribosomes for the diagnosis of dysentery

The levels of antiribosomal antibodies to Shigella ribosomes in serum and saliva samples from 38 dysentery patients (15 S. sonnei cases and 23 S. flexneri cases), 14 patients with salmonellosis and 136 healthy adults were determined in ELISA with ribosomes from S. sonnei R-mutant used as solid-phase antigen. High levels of "normal" antiribosomal IgA, IgG and IgM antibodies were revealed in the sera of healthy persons while the level of salivary IgA antibodies was very low. In dysentery infection no increase in the levels of serum IgG and IgM antibodies and only a slight increase in the level of IgA antibodies were revealed. Local immune response was manifested by the early (on days 2-4 from the onset of infection) and significant augmentation (12- to 16-fold) of salivary antiribosomal IgA antibodies. An increase in the level of these antibodies was registered in 95-100% of dysentery patients but not in patients with salmonellosis, which made it possible to recommend the method for diagnosing shigellosis. Immune response to Shigella ribosomal antigens, in contrast to the response induced by Shigella O-antigen, is almost exclusively local.     

Frequency of serovars and antimicrobial resistance in Shigella spp. from Brazil

A total of 296 Shigella spp. were received from State Public Health Laboratories, during the period from 1999 to 2004, by National Reference Laboratory for Cholera and Enteric Diseases (NRLCED)--IOC/Fiocruz, Rio de Janeiro, Brazil. The frequency of Shigella spp. was: S. flexneri (52.7%), S. sonnei (44.2%), S. boydii (2.3%), and S. dysenteriae (0.6%). The most frequent S. flexneri serovars were 2a and 1b. The highest incidence rates of Shigella isolation were observed in the Southeast (39%) and Northeast (34%) regions and the lowest rate in the South (3%) of Brazil. Strains were further analyzed for antimicrobial susceptibility by disk diffusion method as part of a surveillance program on antimicrobial resistance. The highest rates of antimicrobial resistance were to trimethoprim-sulfamethozaxole (90%), tetracycline (88%), ampicillin (56%), and chloramphenicol (35%). The patterns of antimicrobial resistance among Shigella isolates pose a major difficulty in the determination of an appropriate drug for shigellosis treatment. Continuous monitoring of antimicrobial susceptibilities of Shigella spp. through a surveillance system is thus essential for effective therapy and control measures against shigellosis.     

Up-regulation of MUC2 and IL-1β expression in human colonic epithelial cells by Shigella and its interaction with mucins

Background

The entire gastrointestinal tract is protected by a mucous layer, which contains complex glycoproteins called mucins. MUC2 is one such mucin that protects the colonic mucosa from invading microbes. The initial interaction between microbes and mucins is an important step for microbial pathogenesis. Hence, it was of interest to investigate the relationship between host (mucin) and pathogen interaction, including Shigella induced expression of MUC2 and IL-1β during shigellosis.

Methods

The mucin-Shigella interaction was revealed by an in vitro mucin-binding assay. Invasion of Shigella dysenteriae into HT-29 cells was analyzed by Transmission electron microscopy. Shigella induced mucin and IL-1β expression were analyzed by RT-PCR and Immunofluorescence.

Results

The clinical isolates of Shigella were found to be virulent by a congo-red binding assay. The in vitro mucin-binding assay revealed both Shigella dysenteriae and Shigella flexneri have binding affinity in the increasing order of: guinea pig small intestinal mucinShigella dysenteriae into HT-29 cells occurs within 2 hours. Interestingly, in Shigella dysenteriae infected conditions, significant increases in mRNA expression of MUC2 and IL-1β were observed in a time dependent manner. Further, immunofluorescence analysis of MUC2 shows more positive cells in Shigella dysenteriae treated cells than untreated cells.

Conclusions

Our study concludes that the Shigella species specifically binds to guinea pig colonic mucin, but not to guinea pig small intestinal mucin. The guinea pig colonic mucin showed a greater binding parameter (R), and more saturable binding, suggesting the presence of a finite number of receptor binding sites in the colonic mucin of the host. In addition, modification of mucins with TFMS and sodium metaperiodate significantly reduced mucin-bacterial binding; suggesting that the mucin-Shigella interaction occurs through carbohydrate epitopes on the mucin backbones. Overproduction of MUC2 may alter adherence and invasion of Shigella dysenteriae into human colonic epithelial cells.     

Inhibition of Shigella flexneri by the Normal Intestinal Flora I. Mechanisms of Inhibition by Klebsiella

Growth curves were plotted for Shigella flexneri and Klebsiella (Aerobacter aerogenes) multiplying in pure and mixed culture. In mixed culture, Klebsiella inhibited Shigella. Exponential growth was interrupted and Shigella entered into a logarithmic death phase. An analysis of cultures at the time inhibition occurred revealed that formic and acetic acids produced by Klebsiella were responsible for the inhibition of Shigella. Klebsiella strongly reduced the culture medium. The volatile fatty acids, operating under reduced conditions, exerted a bactericidal effect on Shigella. Results are discussed with reference to the possible role of volatile fatty acids as factors responsible for Shigella inhibition in vivo.     

Inhibition of Shigella flexneri by the Normal Intestinal Flora II. Mechanisms of Inhibition by Coliform Organisms

Of 15 strains of coliform bacteria, all isolated from human feces, 14 inhibited the growth of Shigella flexneri in mixed culture. In every case, when inhibition occurred, exponential growth of Shigella was interrupted in the mixed culture and the organisms entered into either a stationary or a death phase. None of the test coliform strains produced colicines active against Shigella. An analysis of mixed-culture environments at the time Shigella inhibition occurred revealed that the inhibition was not due to nutrient depletion nor to the development of adverse pH or oxidation-reduction potentials in themselves. In mixed cultures, the coliform strains produced formic and acetic acids in concentrations that inhibited Shigella growth. With one exception, the coliform strains also greatly reduced the culture medium. In average concentrations produced, the formic and acetic acids exerted a bactericidal effect on Shigella under the reduced conditions found in mixed cultures. The acids were only moderately toxic for the coliform strains under the same conditions. Results indicate that volatile acid production and concomitant reduction of the medium are the mechanisms by which coliform bacteria inhibit Shigella growth in mixed cultures.     

Resistance of Shigella to antibiotics

Antibiotic sensitivity of 104 Shigella clinical strains and 104 Escherichia coli strains isolated from patients with acute dysentery not treated with antibiotics in 1986-1987 was studied. It was shown that 100 per cent of the dysentery pathogens and colon bacilli were antibiotic resistant. Strains resistant simultaneously to chloramphenicol, ampicillin, streptomycin, tetracycline, monomycin and kanamycin were the most frequent among the dysentery pathogens. Colon bacilli and dysentery pathogens isolated from the same patient had specific sets of antibiotic resistance markers. Pathogenetic therapy of dysentery and exclusion of antibiotic use for several years did not result in lower numbers of Shigella antibiotic resistant strains.     

Significance of determination of Shigella antibiotic resistance in bacteriological diagnosis of dysentery]

The results of the Shigella antibiotic susceptibility assay within 1995-2002 are presented. 1472 cultures from 1158 patients with intestinal infections and bacteria carriers were isolated. The isolates were tested for their susceptibility to tetracycline, chloramphenicol, gentamicin, kanamycin, ampicillin and ofloxacin. It was shown that S. flexneri and S. sonnei were resistant to tetracycline. The S. flexneri isolates were highly resistant to chloramphenicol (73.3 to 96.0%) while resistance to it in the isolates of S. sonnei varied from 7.7 to 88.5%. In this connection the Levin medium with tetracycline was used to increase the Shigella isolation. In the study of the culture media efficiency with respect to isolation of Shigella it was observed that the Levin medium with tetracycline provided higher rates of S. flexneri and S. sonnei isolation (2.3- and 1.7-fold increase respectively) vs. the Shigella isolation on the Ploskirev medium without the antibiotic.     

Mobilization of phase I plasmid in Shigella sonnei and stability of its inheritance in rough strains of Shigella and Escherichia

The plasmid pSS120, determining the synthesis of species specific I phase antigen of Shigella sonnei is mobilized for genetic transfer into E. coli K12 recipient cells with the frequency 12-41%. The frequency depends on the type of mobilized plasmid and recipient strain. The I phase antigen is normally expressed in II phase recipient cells and in E. coli cells. During mobilization pSS120 forms cointegrates representing a recombinant of mobilizing and mobilized plasmids DNA. The study of pSS120 inheritance stability has shown the plasmid to be unstable during culturing of bacteria and to be partially lost from the parent Shigella sonnei strains as well as from the "hybrid" transconjugants obtained. The 60 Md plasmid present in the donor strains of Shigella sonnei is prone to structural fragmentation particularly expressed in Shigella sonnei/E. coli hybrids.     

Antibiotic sensitivity of Shigella isolated from patients from 1974-1985

Antibiotic sensitivity of 3524 Shigella cultures isolated from patients in 1974-1982 and 414 cultures isolated in 1983-1985 was assayed with standard paper disks. The isolates of 1974-1982 were mostly responsive to ampicillin, carbenicillin, kanamycin, gentamicin, monomycin, neomycin and chloramphenicol. Certain differences in the level of the antibiotic resistance were observed in the Shigella isolates belonging to diverse species. Polyresistant cultures of Shigella amounted to 96.5% and ranged from 88.5 to 99.4% in different years. The number of the cultures with multiple resistance among Shigella sonnei was somewhat higher than that among the Flexneri and Newcastle bacilli. The Shigella isolates of 1983-1985 were mostly responsive to gentamicin, carbenicillin, neomycin, kanamycin and monomycin. 55.5% of the Shigella isolates were responsive to chloramphenicol and only 3.1% to tetracycline. Almost all the causative agents of dysentery isolated within that period were polyresistant. Phenotypic characteristics of multiple resistance in the Shigella cultures were studied.     

Depressed Cellular Formation of Antibody to Shigella Antigens In Vitro by Spleen Cell Cultures from Unresponsive Mice

Specific antibody plaque-forming cells (PFC) to Shigella-soluble antigen did not appear in spleen cell cultures from Shigella-tolerant mice, as occurred with similar cultures prepared from normal mice immunized with Shigella antigen prior to sacrifice. Cultures from tolerant mice also failed to form serologically detectable amounts of agglutinins in vitro. Exposure of cell cultures from tolerant mice in vitro to additional antigen had little or no effect on appearance of plaque-forming cells to Shigella. Spleen cells from normal control mice formed readily detectable levels of antibody, as well as specific antibody plaque-forming cells, after similar stimulation with antigen either in vivo or in vitro. The absence of antibody-forming cells in cultures prepared from spleens of tolerant mice was specific since such cultures, as well as those from normal control mice, formed numerous antibody plaques to unsensitized sheep erythrocytes in vitro after in vivo challenge of the mice with sheep erythrocytes. Tolerance to Shigella antigen, as assessed by absence of antibody-forming cells in vitro, persisted for several months. Spleen cell cultures from tolerant mice less than 3 to 4 months of age did not form significant numbers of antibody plaques, even after in vitro exposure to specific antigen. However, spleen cultures prepared from neonatally treated mice, approximately 6 to 8 months old, formed essentially normal numbers of specific PFC in vitro, indicating that the animals had "recovered" from tolerance and that their lymphoid cells were capable of responding to Shigella antigen in vitro. Absence of specific PFC in cell cultures from tolerant animals supports the concept that tolerance is due to a central failure of specific immunocompetent cells and not due to an inhibitory effect caused by either "excess" antigen or humoral antibody.     

Risk factors in the epidemiology of dysentery

The general tendency of a decrease in the morbidity rates of dysentery induced by Shigella sonnei and Shigella flexneri (separately) from Monday to the end of the week (Saturday-Sunday) has been revealed, and at the same time the "infection risk" for both kinds of salmonellosis has been found to fall on the last days of the week (Friday-Sunday).     

Gene cloning and characterization of alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei.

Alanine racemase genes (alr) from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei were cloned and expressed in Escherichia coli JM109. All genes encoded a polypeptide of 359 amino acids, and showed more than 99% sequence identities with each other. In particular, the S. dysenteriae alr was identical with the S. flexneri alr. Differences in the amino acid sequences between the four Shigella enzymes were only two residues: Gly138 in S. dysenteriae and S. flexneri (Glu138 in the other) and Ile225 in S. sonnei (Thr225 in the other). The S. boydii enzyme was identical with the E. coli K12 alr enzyme. Each Shigella alr enzyme purified to homogeneity has an apparent molecular mass about 43,000 by SDS-gel electrophoresis, and about 46,000 by gel filtration. However, all enzymes showed an apparent molecular mass about 60,000 by gel filtration in the presence of a substrate, 0.1 M l-alanine. These results suggest that the Shigella alr enzymes having an ordinary monomeric structure interact with other monomer in the presence of the substrate. The enzymes were almost identical in the enzymological properties, and showed lower catalytic activities (about 210 units/mg) than those of homodimeric alanine racemases reported.     

Further Evaluation of Immunofluorescence Techniques for Detection of Shigellae in Fecal Specimens

The fluorescent antibody (FA) tests for group B and group D Shigella were reevaluated. Duplicate swab specimens from patients suspected of having shigellosis were cultured shortly after collection and after transport in a soft-agar holding medium. Smears for FA examination were made at the same time. Results obtained for the group D Shigella agree closely with those obtained in previous studies. The percentage of isolations of group B Shigella from transported specimens which were positive by FA was 59.6% as compared to 39.3 and 53.3% in previous studies. S. flexneri was isolated from 76.7% of the FA-positive specimens when they were examined shortly after collection. More isolates of Shigella were obtained when specimens were examined by both methods than when either method was used alone. The results indicated that FA tests for both group B and group D Shigella are practical and may be useful to laboratories engaged in Shigella isolation.     

The needle component of the type III secreton of Shigella regulates the activity of the secretion apparatus

Gram-negative bacteria commonly interact with eukaryotic host cells by using type III secretion systems (TTSSs or secretons). TTSSs serve to transfer bacterial proteins into host cells. Two translocators, IpaB and IpaC, are first inserted with the aid of IpaD by Shigella into the host cell membrane. Then at least two supplementary effectors of cell invasion, IpaA and IpgD, are transferred into the host cytoplasm. How TTSSs are induced to secrete is unknown, but their activation appears to require direct contact of the external distal tip of the apparatus with the host cell. The extracellular domain of the TTSS is a hollow needle protruding 60 nm beyond the bacterial surface. The monomeric unit of the Shigella flexneri needle, MxiH, forms a superhelical assembly. To probe the role of the needle in the activation of the TTSS for secretion, we examined the structure-function relationship of MxiH by mutagenesis. Most point mutations led to normal needle assembly, but some led to polymerization or possible length control defects. In other mutants, secretion was constitutively turned "on." In a further set, it was "constitutively on" but experimentally "uninducible." Finally, upon induction of secretion, some mutants released only the translocators and not the effectors. Most types of mutants were defective in interactions with host cells. Together, these data indicate that the needle directly controls the activity of the TTSS and suggest that it may be used to "sense" host cells.     

Effect of bovine lactoferrin on the minimum inhibitory concentrations of ampicillin and trimethoprim-sulfamethoxazole for clinical Shigella spp. strains

Here, we determined the effect of bovine lactoferrin (bLF) on the minimum inhibitory concentration (MIC) of ampicillin and trimethoprim-sulfamethoxazole in Shigella . Using a microdilution method, the MIC was determined in the presence or absence of bovine lactoferrin (10 mg/mL) on 88 Shigella strains (56 Shigella flexneri , 15 Shigella boydii , 13 Shigella sonnei , and 4 Shigella dysenteriae ) previously isolated from peruvian children <2 years old. A fold change of 2 or more in MIC values was considered significant. For ampicillin, 67 (76%) strains were highly resistant; one-third of the strains (32%) showed a decrease in ampicillin MIC in the presence of LF. This was more typical of MIC values in less resistant strains. For 7 (8%) ampicillin-resistant strains, the decrease in the MIC resulted in the strains reaching the cutoff for susceptible in the presence of bLF. For trimethoprim-sulfamethoxazole, 93% of the isolates (n = 82) were highly resistant and only 4 isolates (5%) decreased their MIC in the presence of bLF. None of the trimethoprim-sulfamethoxazole resistant strains became susceptible in the presence of LF. The decrease in the MIC in the presence of bLF seems to depend on the mechanisms of action of each antibiotic. In vivo studies are needed to further evaluate bLF as a coadjuvant to antibiotic treatment of resistant Shigella.     

Red重组系统在痢疾杆菌基因敲除中的应用研究

利用λ噬菌体的Red系统在细菌中进行基因敲除的技术,最近发展较快,但多在大肠杆菌中进行。以alkA、wcaJ、yphF和dam 4个基因为例,分别在大肠杆菌和痢疾杆菌中利用Red系统进行基因敲除。对于大肠杆菌,除dam基因外,其余3个基因均能被有效敲除,而在痢疾杆菌中只能敲除一个alkA基因。结果表明,为使该系统能有效地应用于痢疾杆菌和其它细菌,还需对该系统进行改进。     

Lipopolysaccharide core structures and their correlation with genetic groupings of Shigella strains. A novel core variant in Shigella boydii type 16

Bacteria Shigella, the cause of shigellosis, evolved from the intestinal bacteria Escherichia coli. Based on structurally diverse O-specific polysaccharide chains of the lipopolysaccharides (LPSs; O-antigens), three from four Shigella species are subdivided into multiple serotypes. The central oligosaccharide of the LPS called core is usually conserved within genus but five core types called R1-R4 and K-12 have been recognized in E. coli. Structural data on the Shigella core are limited to S. sonnei, S. flexneri and one S. dysenteriae strain, which all share E. coli core types. In this work, we elucidated the core structure in 14 reference strains of S. dysenteriae and S. boydii. Core oligosaccharides were obtained by mild acid hydrolysis of the LPSs and studied using sugar analysis, high-resolution mass spectrometry and two-dimensional NMR spectroscopy. The R1, R3 and R4 E. coli core types were identified in 8, 3 and 2 Shigella strains, respectively. A novel core variant found in S. boydii type 16 differs from the R3 core in the lack of GlcNAc and the presence of a D-glycero-D-manno-heptose disaccharide extension. In addition, the structure of an oligosaccharide consisting of the core and one O-antigen repeat was determined in S. dysenteriae type 8. A clear correlation of the core type was observed with genetic grouping of Shigella strains but not with their traditional division to four species. This finding supports a notion on the existing Shigella species as invalid taxa and a suggestion of multiple independent origins of Shigella from E. coli clones.     

Dynamics of the change in sensitivity of shigellae to gentamycin and cephaloridine in vitro]

The dynamics of the changes in the Shigella sensitivity to gentamicin and cephaloridin was studied in vitro using liquid nutrient media with gradually increasing concentrations of the drugs. 50 passages were performed. It was found that Shigella flexner and sonnei decreased their sensitivity to gentamicin to a little extent and remained middle sensitive. Sensitivity of Shigella flexner to cephaloridin also changed to a little extent, while Shigella sonnei became moderately resistant.