Evaluation of the Anti-Shigellosis Activity of Dietary Isothiocyanates in Galleria mellonella Larvae.

Cruciferous vegetables, widely present in daily diets, are a rich source of organosulfur compounds with proven health benefits, especially chemopreventive or antioxidative effects. Isothiocyanate derivatives (ITCs) exhibit a broad spectrum of biological and pharmacological activity and recently, their antibacterial properties have been of particular importance. Here, we have focused on the anti-shigellosis activity of sulforaphane (SFN) and phenethyl ITC (PEITC). The genus Shigella causes gastroenteritis in humans, which constitutes a threat to public health. Production of a potent Stx toxin by S. dysenteriae type 1 results not only in more severe symptoms but also in serious sequela, including the hemolytic uremic syndrome. Here, we present evidence that two aliphatic and aromatic ITCs derivatives, SFN and PEITC, have an effective antibacterial potency against S. dysenteriae, also negatively regulating the stx gene expression. The molecular mechanism of this effect involves induction of the global stress-induced stringent response. ITCs also inhibit bacterial virulence against the Vero and HeLa cells. We present evidence for the therapeutic effect of sulforaphane and phenethyl ITC against a S. dysenteriae infection in the Galleria mellonella larvae model. Thus, our results indicate that isothiocyanates can be effectively used to combat dangerous bacterial infections.

Adaptations in the physiological heterogeneity and viability of Shigella dysenteriae, Shigella flexneri and Salmonella typhimurium, after exposure to simulated gastric acid fluid.

Stomach acidity is an important barrier of the human body to protect itself from microbial pathogens entering the small intestine and causing infection. This study examined the survival adaptations of non-acid adapted diarrheal Shigella and Salmonella strains in an environment mimicking the human stomach. The bacterial responses to the challenge of acidic simulated gastric fluid were studied using flow cytometry physiological heterogeneity, membrane integrity and survival (culturability) respectively. Flow cytometry showed that bacterial cells, when exposed to gastric fluid, transformed distinctly, into physiologically heterogeneous sub-populations: intact, stressed and damaged cells, when stained with propidium iodide and thiazole orange. Shigella and Salmonella cells became membrane compromised during initial acid shock (0-30 min), and 80% of these cells shifted to the stressed state throughout gastric fluid exposure. Approximately 10-30% of bacterial strains remained culturable after 60 min of gastric fluid exposure at pH 2.5-4.5, with the percentage increasing with an inoculum size of 102 CFU/ml. This ability of non-acid adapted Shigella and Salmonella sp. to adapt and survive low pH gastric fluid, even though the bacterial numbers decreased or changed to a stressed state, further supports the possible risk of infection when consumed.

A two-step method for extraction of lipopolysaccharide from Shigella dysenteriae serotype 1 and Salmonella typhimurium: An improved method for enhanced yield and purity.

Bacterial lipopolysaccharide (LPS) has been widely used as an antigen and adjuvant in immunological applications. Amongst the methods developed for extraction of LPS, hot phenol extraction (HPE) method is the gold standard. However, the HPE method provides poor yield of LPS (~4.5% by weight), is associated with relatively higher impurities of proteins and nucleic acids, and the acidic hot phenol can cause a degradative effect on LPS. In this work a two-step extraction (TSE) method was developed using a non-capsulated, [Shigella dysenteriae serotype-1] (Sd1) and capsulated [Salmonella typhimurium type B (StB)] species as model pathogens. The TSE method takes advantage of growth kinetics of bacteria wherein a two-step sequential approach for LPS extraction was employed. In step-1, culture supplemented with CaCl2 during early log phase of growth was induced to release LPS by the effect of EDTA at their late exponential phase of growth. In step-II, cells with left over LPS were subjected to modified HPE method that reduced both the degradative effect of acidic hot phenol and associated impurities. The LPS produced using TSE method enabled not only enhanced yield (~2.78 and ~2.91 fold higher for Sd1 and StB respectively) requiring nearly similar duration of extraction, but also was structurally and functionally comparable with LPS produced using HPE method and commercially procured LPS. Overall, the developed TSE method is relatively more efficient (enhanced yield), clean (healthy extraction with reduced impurities), safe (reduced handling of larger pathogenic culture) and cost-effective for LPS extraction with potential for scale up.

Characterization of new Myoviridae bacteriophage WZ1 against multi-drug resistant (MDR) Shigella dysenteriae.

Shigella dysenteriae is a normal inhabitant of the human gastrointestinal tract, but sometimes it causes severe infection known as shigellosis (bacillary dysentery). Bacteriophages are considered very safe and effective agents for controlling bacterial infections and contaminations. In this study, we describe the isolation and characterization of bacteriophage WZ1, isolated from waste water which inhibits the growth of S. dysenteriae. Phage WZ1 showed maximum stability at 37 °C and was stable up to 65 °C but was totally inactive at 70 °C. The pH stability increased from low to high and was totally inactive at pH 3 while maximum stability was observed at optimal pH 7. Phage WZ1 adsorption rate to the host bacterium was significantly enhanced by the addition of CaCl2 . It has a latent time and burst time of 24 min and about 430 virions/cell, respectively. Transmission electron microscopy of phage WZ1 revealed a head width of 10 ± 0.5 nm and length of 10 ± 0.2 nm with a contractile tail of 128 ± 25 nm long and 21 ± 0.5 nm wide and belongs to family Myoviridae of order Caudovirales. Twelve structural proteins ranging from 22 to 150 kDa were detected by Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). The genome was found to be double stranded DNA with an approximate size of 38 kb. It has a very good reduction potential for S. dysenteriae by lowering abruptly the optical density of the planktonic S. dysenteriae culture. Phage WZ1 is a very promising candidate for phage therapy and other applications such as phage typing.

Oxalis corniculata (Oxalidaceae) leaf extract exerts in vitro antimicrobial and in vivo anticolonizing activities against Shigella dysenteriae 1 (NT4907) and Shigella flexneri 2a (2457T) in induced diarrhea in suckling mice.

In this study, the extract of a green leafy vegetable Oxalis corniculata (Oxalidaceae) was evaluated for its in vitro antibacterial and in vivo anti colonizing effect against common intestinal pathogenic bacteria. Methanolic extract (80%) of Oxalis corniculata (Oxalidaceae) leaf contained a polyphenol content of 910 mg gallic acid equivalent per gram of dry weight and the yield was 8%. The flavonoid content was 2.353 g quercetin equivalent per 100 g of the extract. In vitro studies indicated that the extract inhibited numerous pathogenic bacteria like Staphylococcus aureus (ATCC 25922), Escherichia coli (ATCC 25923), Shigella dysenteriae 1 (NT4907), Shigella flexneri 2a (2457T), Shigella boydii 4 (BCH612), and Shigella sonnie phase I (IDH00968). The minimum inhibitory concentration (MIC) against E. coli (ATCC 25923) was minimal (0.08 mg/mL), whereas MIC against S. flexneri 2a (2457T) was higher (0.13 mg/mL). A suckling mouse model was developed which involved challenging the mice intragastrically with S. flexneri 2a (2457T) and S. dysenteriae 1 (NT4907) to study the anticolonization activity. It was revealed that the extract was more potent against S. dysenteriae 1 (NT4907) as compared to S. flexneri 2a (2457T). It was also found that simultaneous administration of extract along with bacterial inoculums promoted good anticolonization activity. Significant activity was observed even when treated after 3 h of bacterial inoculation.

Monitoring of growth and physiological activities of biofilm during succession on polystyrene from activated sludge under aerobic and anaerobic conditions.

The present research work monitored the successive biofilm development and its catabolic role in the degradation of polystyrene (PS). PS material was artificially colonized with biofilm by incubating it with activated sludge under aerobic and anaerobic conditions. Biofilm formation was monitored by gravimetric weight analysis, spectrophotometric absorbance technique, heterotrophic plate count, and scanning electron microscopy under aerobic and anaerobic conditions. The wet weight (1.59 and 1.17 g) and dry weight (0.41 and 0.08 g) of a biofilm showed a significant constant increase under aerobic and anaerobic conditions, respectively, from first till 9 weeks of incubation. Plate count of the selected bacteria (Escherichia coli, Salmonella typhimurium, Shigella dysenteriae, Pseudomonas aeruginosa) considerably declined (90-99 %) in the biofilm after seventh and fifth weeks of incubation under aerobic and anaerobic conditions, respectively, indicating a positive shift from pathogenic to beneficial microbial community. While most probable number index of fecal coliforms and E. coli in the sludge showed more reduction (98 and 99 %) under aerobic as compare to anaerobic conditions (86 and 91 %) after 9 weeks of biofilm formation on PS cubes. Correspondingly, the decreasing levels of chemical oxygen demand and biochemical oxygen demand (up to 73 %) showed signs of sludge digestion. Scanning electron microscope coupled with energy dispersive X-ray spectroscope revealed nature of PS media containing high carbon content. However, biofilm development proved to be involved in the biochemical transformation of the PS medium as indicated by Fourier transform infrared spectroscopy.

[Microcalorimetric investigation on the interaction of six alkaloids from rhizoma coptidis].

How to identify active constituents of traditional Chinese medicines (TCMs) and study their interactions are key problems in the development of TCMs. The inhibitory effect of six alkaloids from Rhizoma Coptidis (RC) on Shigella dysenteriae (S. dysenteria) growth had been investigated by microcalorimetry in this study. Main active constituents of RC were confirmed by comparing their contributions to the bacteriostatic effect, and the interactions among active constituents were further researched. According to the result, in 0.8 mg-mL-1 extract of RC, the contributions of six active alkaloids including berberine, coptisine, epiberberine, palmatine and the combination of jatrorrhizine and columbamine were 52.83%, 36.31%, 2.49%, 4.27% and 3.21%, respectively. Therefore, berberine and coptisine were the main active constituents of RC that inhibited the growth of S. dysenteria. The study of interactions among the six alkaloids indicated that, 1 there were some contstituents antagonizing the inhibitory effect of RC, 2 there was a synergy effect between berberine and coptisine, 3 there were additive effects between other four alkaloids and the main active constituents. These results may provide some useful references for the establishment of the quality standard for RC and the development of multi-component TCMs.

[Effects of lamivudine on growth of intestinal characteristic bacteria by microcalorimetry in vitro].

The study is aimed to investigate the effect of lamivudine on growth and metabolism of three intestinal characteristic bacteria (namely, Bifidobacterium adolescentis, Escherichia coli and Shigella dysenteriae). The growth condition of the three bacteria was quantitatively evaluated by microcalorimetry with four characteristic parameters of the thermal power-time curves, including the growth rate constant (k), thermal power (p), time to peak (t) and calorific value (Q). The results showed that the IC50 value of lamivudine on B. adolescentis was 200 microg x mL(-1), and the IC50 values of lamivudine on S. dysenteriae and E. coli were higher than 3 000 microg x mL(-1) and 6 000 microg x mL(1), respectively. Therefore, lamivudine made different inhibitory effects on the three bacteria, in which the B. adolescentis was most susceptible to lamivudine. This work showed that taking lamivudine chronically is likely to affect the balance of good flora in the intestinal tract, and might increase endotoxin release, leading to inflammation and disease progression in hepatopathy.

In vivo versus in vitro protein abundance analysis of Shigella dysenteriae type 1 reveals changes in the expression of proteins involved in virulence, stress and energy metabolism.

BACKGROUND: Shigella dysenteriae serotype 1 (SD1) causes the most severe form of epidemic bacillary dysentery. Quantitative proteome profiling of Shigella dysenteriae serotype 1 (SD1) in vitro (derived from LB cell cultures) and in vivo (derived from gnotobiotic piglets) was performed by 2D-LC-MS/MS and APEX, a label-free computationally modified spectral counting methodology. RESULTS: Overall, 1761 proteins were quantitated at a 5% FDR (false discovery rate), including 1480 and 1505 from in vitro and in vivo samples, respectively. Identification of 350 cytoplasmic membrane and outer membrane (OM) proteins (38% of in silico predicted SD1 membrane proteome) contributed to the most extensive survey of the Shigella membrane proteome reported so far. Differential protein abundance analysis using statistical tests revealed that SD1 cells switched to an anaerobic energy metabolism under in vivo conditions, resulting in an increase in fermentative, propanoate, butanoate and nitrate metabolism. Abundance increases of transcription activators FNR and Nar supported the notion of a switch from aerobic to anaerobic respiration in the host gut environment. High in vivo abundances of proteins involved in acid resistance (GadB, AdiA) and mixed acid fermentation (PflA/PflB) indicated bacterial survival responses to acid stress, while increased abundance of oxidative stress proteins (YfiD/YfiF/SodB) implied that defense mechanisms against oxygen radicals were mobilized. Proteins involved in peptidoglycan turnover (MurB) were increased, while ß-barrel OM proteins (OmpA), OM lipoproteins (NlpD), chaperones involved in OM protein folding pathways (YraP, NlpB) and lipopolysaccharide biosynthesis (Imp) were decreased, suggesting unexpected modulations of the outer membrane/peptidoglycan layers in vivo. Several virulence proteins of the Mxi-Spa type III secretion system and invasion plasmid antigens (Ipa proteins) required for invasion of colonic epithelial cells, and release of bacteria into the host cell cytosol were increased in vivo. CONCLUSIONS: Global proteomic profiling of SD1 comparing in vivo vs. in vitro proteomes revealed differential expression of proteins geared towards survival of the pathogen in the host gut environment, including increased abundance of proteins involved in anaerobic energy respiration, acid resistance and virulence. The immunogenic OspC2, OspC3 and IpgA virulence proteins were detected solely under in vivo conditions, lending credence to their candidacy as potential vaccine targets.

Survival of Salmonella Typhi and Shigella dysenteriae in dehydrated infant formula.

UNLABELLED: Powdered infant formula has previously been linked to the transmission of various bacterial pathogens in infants resulting in life-threatening disease and death. Survival studies of 2 common foodborne pathogens, Salmonella enterica serovar Typhi and Shigella dysenteriae, in powdered infant formula have not been previously studied despite the potentially devastating consequences from ingestion of these organisms, particularly by newborns, in case of a natural or deliberate contamination event. Therefore, to better predict the risk of S. Typhi and S. dysenteriae infection from consumption of infant formula, the present study was undertaken to determine survival of these microorganisms in dry infant formula under varying atmospheric conditions. A 2-strain cocktail of S. Typhi and a 3-strain cocktail of S. dysenteriae were stored for up to 12 wk in dehydrated infant formula in an ambient air or nitrogen atmosphere. Viable counts of S. Typhi at 12 wk in infant formula revealed a 2.9- and 1.69-log decrease in ambient air and nitrogen atmosphere, respectively. Viable counts of S. dysenteriae at 12 wk in infant formula revealed a 0.81- and 0.42-log decrease in ambient air and nitrogen atmosphere, respectively. These results show that S. Typhi and S. dysenteriae can remain viable for prolonged periods of time in powdered infant formula, and the presence of nitrogen enhances survival. PRACTICAL APPLICATION: Our goal in this work was to study the survival of S. Typhi and S. dysenteriae in dehydrated storage conditions in infant formula. This interest is partially generated by the possibility of using these 2 microorganisms to deliberately contaminate the food supply. The outcome of this study will help us to have a better idea how to respond and react to the risk of deliberate food contamination.

Acanthamoeba castellanii an environmental host for Shigella dysenteriae and Shigella sonnei.

The interaction between Shigella dysenteriae or Shigella sonnei and Acanthamoeba castellanii was studied by viable counts, gentamicin assay and electron microscopy. The result showed that Shigella dysenteriae or Shigella sonnei grew and survived in the presence of amoebae for more than 3 weeks. Gentamicin assay showed that the Shigella were viable inside the Acanthamoeba castellanii which was confirmed by electron microscopy that showed the Shigella localized in the cytoplasm of the Acanthamoeba castellanii. In conclusion, the relationship between Shigella dysenteriae and Shigella sonnei with Acanthamoeba castellanii is symbiotic, and accordingly free-living amoebae may serve as a transmission reservoir for Shigella in water.

Lactobacilli facilitate maintenance of intestinal membrane integrity during Shigella dysenteriae 1 infection in rats.

OBJECTIVE: Lactobacilli are used in various dairy products and fermented foods for their potential health beneficial effects. Recently we reported the protective role of Lactobacillus rhamnosus and Lactobacillus acidophilus during Shigella dysenteriae 1 infection. Nevertheless, investigations on the membrane-stabilizing effect of L. rhamnosus and L. acidophilus have not been done. Hence, the present study evaluated the effect of L. rhamnosus and L. acidophilus on the maintenance of intestinal membrane integrity during S. dysenteriae 1-induced diarrhea in rats. METHODS: Rats were divided into eight groups (n = 6 in each group). Induced rats received single oral dose of S. dysenteriae (12 x 10(8) colony-forming units [cfu]/mL). Treated rats received L. rhamnosus (1 x 10(7)cfu/mL) or L. acidophilus (1 x 10(7)cfu/mL) orally for 4 d, alone or in combination, followed by Shigella administration. At the end of the experimental period, animals were sacrificed and the assay of membrane-bound adenosine triphosphatases (Na(+)/K(+)-ATPase, Ca(2+)-ATPase, and total ATPase), immunoblot analysis of tight junctional proteins (claudin-1 and occludin), and transmission electron microscopic studies were performed. RESULTS: Induced rats showed a significant (P < 0.05) reduction in the membrane-bound ATPases and reduced expression of tight junction proteins in the membrane, coupled with their increased expression in the cytosol, indicating membrane damage. Transmission electron microscopic studies correlated with biochemical parameters. Pretreatment with combination of L. rhamnosus and L. acidophilus significantly prevented these changes. CONCLUSION: Lactobacillus rhamnosus and L. acidophilus synergistically offered better protection to the intestinal membrane when compared with individual treatments with these strains during S. dysenteriae 1 infection.

Protective role of lactobacilli in Shigella dysenteriae 1-induced diarrhea in rats.

OBJECTIVE: Studies on lactic acid bacteria exemplify their use against various enteropathogens in vitro. Nevertheless, in vivo effects of Lactobacillus during Shigella infection have not been evaluated. The present study evaluated the effect of Lactobacillus rhamnosus and Lactobacillus acidophilus on neutrophil infiltration and lipid peroxidation during Shigella dysenteriae 1-induced diarrhea in rats. METHODS: The rats were divided into eight groups (n = 6 in each group). Induced rats received single oral dose of S. dysenteriae (12 x 10(8) colony-forming units [cfu]/mL). Treated rats received L. rhamnosus (1 x 10(7) cfu/mL) or L. acidophilus (1 x 10(7) cfu/mL) orally for 4 d, alone or in combination, followed by Shigella administration. At the end of the experimental period, animals were sacrificed and the assay of the activity of alkaline phosphatase, myeloperoxidase, and antioxidants and the estimation of lipid peroxides were performed. Activity staining of superoxide dismutase and catalase was done in addition to gelatin zymography for matrix metalloproteinase (MMP; MMP-2 and MMP-9) activity. A portion of the intestinal tissue was fixed in 10% formalin for histologic studies. RESULTS: Administration of S. dysenteriae 1 alone resulted in increased levels of myeloperoxidase, lipid peroxidation, alkaline phosphatase, and the expression of MMP-2 and MMP-9 with concomitant decrease in the antioxidant levels. Pretreatment with the combination of L. rhamnosus (1 x 10(7) cfu/mL) and L. acidophilus (1 x 10(7) cfu/mL) significantly attenuated these changes when compared with the diseased group. Histologic observations were in correlation with biochemical parameters. CONCLUSION: Lactobacillus rhamnosus plus L. acidophilus offered better protection when compared with individual treatment with these strains during Shigella infection.

Construction, detection and microarray analysis on Shigella dysenteriae a1 IroN, ShuA single, double mutants.

In this study, we constructed single mutants MTS-1, MTS-2 of IroN and ShuA gene and double mutant MTS of them in Shigella dysenteriae A1 strain 51197 by insert and absence. The functional detection of every mutant was performed at the level of culture medium and cell experiment. The gene expression profiles of the mutants and the wild-type strains under iron-enriched and iron-limited conditions were analyzed by the SD51197 whole genomic microarray. The results showed that all the mutants grew obviously less well than the wild-type strains in L broth appending iron chelator DIP. The addition of iron to the cultures can stimulate the growth of mutants back to wild-type levels. In either the experiments on the ability of intracellular multiplication or the cell-to-cell spread in HeLa and U937 cell lines, mutants showed no obvious change in virulence compared with the parental strain SD51197. However when DIP was added to the cultured HeLa cells, the ability of intracellular multiplication of MTS-1, MTS-2, MTS has reduced about 23.4%, 25.2%, 43.6% respectively. The analysis of expression profiles under the iron-limited condition showed that the mutants were more sensitive for the changes of iron deficiency than the wild-type strains, many genes have been altered. Up-regulated genes mainly involved genes of transcription, coenzyme metabolism, amino acid transport and metabolism, and unknown functional genes, while down-regulated genes mainly involved genes of energy and carbohydrate metabolism and unknown function genes; the expression levels of known iron-transport associated genes generally showed up-regulated. The results demonstrated that iron-transport associated genes IroN, ShuA were likely to have some effects on the virulence and growth of S. dysenteriae.

Activities of aqueous extracts of Mallotus oppositifolium on Shigella dysenteriae A1-induced diarrhoea in rats.

1. Mallotus oppositifolium is reported to possess medicinal properties and is traditionally used in Cameroon for the treatment of diarrhoea. In the present study, we have evaluated the acute toxicity, in vitro antibacterial and in vivo antidiarrhoeal effects of an aqueous extract of these plant leaves. 2. Shigella dysenteriae A(1) (Sd1)-induced diarrhoeal rats were obtained by oral administration of increasing densities of the Sd1 strain isolated from bloody diarrhoea occurring in East Cameroon. When diarrhoea appeared, rats were treated for 5 consecutive days with 120, 240 or 360 mg/kg extract or norfloxacin (5.7 mg/kg). The weight and frequencies of faeces, as well as the number of Sd1, were assessed during the treatment period and the death rate was recorded. 3. The M. oppositifolium extract was not toxic. In vitro, the minimal inhibitory and minimal bactericidal concentrations of the extracts were 1,172 and 9,375 microg/mL, respectively. In vivo, 12 x 10(8) Sd1 provoked diarrhoea within 24 h, which was characterized by soft or liquid stools, that were moulded, smooth and mucus or blood coated. Diarrhoea went along with an increase in faeces weight and frequency (P < 0.001 by the 3rd day), as well as an increase in the bacterial population to a maximum on the 2nd day after infection (P < 0.05). The death rate was 67% by day 6. 4. Whereas norfloxacin significantly (P < 0.01) reduced Sd1 growth, M. oppositifolium extracts (240 and 360 mg/kg) restored bacterial growth to its initial density and no deaths were recorded. There was a significant (P < 0.05) reduction in stools weight and frequency with 240 mg/kg extract. 5. The results suggest that M. oppositifolium leaves could be a therapeutic alternative for bacterial aetiological diarrhoea in Central Africa, where multidrug supply and access to modern health centres are public health problems.

Batch process solar disinfection is an efficient means of disinfecting drinking water contaminated with Shigella dysenteriae type I.

AIMS: The mortality and morbidity rate caused by Shigella dysenteriae type I infection is increasing in the developing world each year. In this paper, the possibility of using batch process solar disinfection (SODIS) as an effective means of disinfecting drinking water contaminated with Sh. dysenteriae type I is investigated. METHODS: Phosphate-buffered saline contaminated with Sh. dysenteriae type I was exposed to simulated solar conditions and the inactivation kinetics of this organism was compared with that of Sh. flexneri, Vibrio cholerae and Salmonella typhimurium. SIGNIFICANCE: Recovery of injured Sh. dysenteriae type I may be improved by plating on medium supplemented with catalase or pyruvate. Sh. dysenteriae type I is very sensitive to batch process SODIS and is easily inactivated even during overcast conditions. Batch process SODIS is an appropriate intervention for use in developing countries during Sh. dysenteriae type I epidemics.

GASP phenotype: presence in enterobacteria and independence of sigmaS in its acquisition.

The appearance of growth advantage in stationary phase or GASP was originally detected in Escherichia coli. The presence of this phenotype in other enterobacteria such as Enterobacter cloacae, Salmonella typhimurium, Providencia stuartii and Shigella dysenteriae is described in this work. E. cloacae GASP strains presented lower levels of RpoS than the parental strain, although no mutation in the gene or its promoter was detected. This work offers evidence of GASP rpoS-independent pathways as GASP was also acquired in knock-out rpoS E. cloacae and E. coli strains.

Survival of Shigella dysenteriae type 1 on fomites.

Studies have shown that various objects, such as utensils, toys, and clothes, can serve as vehicles for transmission of Shigella spp. Shigellae can become viable but non-culturable (VBNC) when exposed to various environmental conditions as shown in earlier studies. The present study was carried out to detect VBNC Shigella dysenteriae type 1 on various fomites by direct viable counting, polymerase chain reaction (PCR), and fluorescent antibody methods. S. dysenteriae type 1 was inoculated onto cloth, wood, plastic, aluminum, and glass objects. Results showed that 1.5-4.0 hours after inoculation, S. dysenteriae type 1 became non-culturable, and after five days, non-culturable but viable S. dysenteriae type 1 could be detected by both PCR and fluorescent antibody techniques. Fomites can be considered an important potential route of transmission of VBNC S. dysenteriae type 1 and a significant factor in the epidemiology of shigellosis.

TonB is required for intracellular growth and virulence of Shigella dysenteriae.

To assess the importance of TonB-dependent iron transport systems to growth of Shigella in vivo, a tonB mutant of Shigella dysenteriae was isolated and tested in cultured cells. The tonB mutant invaded epithelial cells, but did not form plaques in confluent monolayers of Henle cells, indicating an inability of this mutant to spread from cell to cell. The rate of intracellular multiplication of the tonB mutant was reduced significantly compared to that of the wild type. The loss of virulence in the tonB mutant was not due to loss of either Shu or Ent, the TonB-dependent systems which allow for transport of heme and ferrienterobactin, respectively. A shuA mutant lacking the outer membrane receptor for heme, an entB mutant defective in enterobactin synthesis, and a shuA entB double mutant each were able to invade cultured cells, multiply intracellularly, and form wild-type plaques. The ability of S. dysenteriae to access iron during intracellular growth was assessed by flow cytometric analysis of an iron- and Fur-regulated shuA-gfp reporter construct. Low levels of green fluorescent protein expression in the intracellular environment were observed in all strains, indicating that iron is available to intracellular bacteria, even in the absence of TonB-dependent iron transport. The failure of the tonB mutant to grow well in an iron-replete intracellular environment suggests that TonB plays a role in addition to heme- and siderophore-mediated iron acquisition in vivo, and this function is required for the intracellular growth and intercellular spread of S. dysenteriae.

Spontaneous tandem amplification and deletion of the shiga toxin operon in Shigella dysenteriae 1.

Only one species of Shigella, Shigella dysenteriae 1, has been demonstrated to produce Shiga toxin (Stx). Stx is closely related to the toxins produced by Shiga toxin-producing Escherichia coli (STEC). In STEC, these toxins are often encoded on lambdoid bacteriophages and are major virulence factors for these organisms. Although the bacteriophage-encoded stx genes of STEC are highly mobile, the stx genes in S. dysenteriae 1 have been believed to be chromosomally encoded and not transmissible. We have located the toxin genes of S. dysenteriae 1 to a region homologous to minute 30 of the E. coli chromosome, within a 22.4 kbp putative composite transposon bracketed by IS600 insertion sequences. This region is present in all the S. dysenteriae 1 strains examined. Tandem amplification occurs via the flanking insertion sequences, leading to increased toxin production. The global regulatory gene, fnr, is located within the stx region, allowing deletions of the toxin genes to be created by anaerobic growth on chlorate-containing medium. Deletions occur by recombination between the flanking IS600 elements. Lambdoid bacteriophage genes are found both upstream and within the region, and we demonstrate the lysogeny of Shigella species with STEC bacteriophages. These observations suggest that S. dysenteriae 1 originally carried a Stx-encoding lambdoid prophage, which became defective due to loss of bacteriophage sequences after IS element insertions and rearrangements. These insertion sequences have subsequently allowed the amplification and deletion of the stx region.