Rabbit Ileal Loop Assay Research Articles

ASSESSMENT OF THE MICROBIOLOGICAL AND PHYSIOCHEMICAL QUALITIES OF WATER SOURCED FROM DIFFERENT TREATMENT TANKS AT A MUNICIPAL DAM LOCATED IN BENIN CITY, EDO STATE, NIGERIA

The culturable microbial profile of water and biofilms associated with the internal walled surface of different treatment tanks at the Benin City water dam facility were investigated using techniques such as serial dilution and pour plate methods. The physiochemical attributes of the stored water were determined using methods which included the use of appropriate meters and titrimetery.The pathogenicity of the biofilm bacterial isolates was determined using the rabbit ileal loop assay. The mean heterotrophic bacterial, fungal and coliform counts ranged from 0.2 × 102cfu/ml to 9.3 × 105cfu/ml, 3.7 ×105 cfu/ml to 6.7 ×105cfu/ml and 18 MPN/100 ml to 180 MPN/100 ml respectively. Several microbial isolates which included Bacillus sp.,Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, Staphylococcus epidermidis, Klebsiella mobilis, Micrococcus sp., Flavobacterium sp, Aeromonas sp, Klebsiella sp., Rhizopus stolonifer, Aspergillus flavus, Aspergillus niger, Penicilliumsp., Fusariumoxysporum, Neurospora sp., Mucorsp. and Rhizopus stolonifer were present in both the water and swabbed tank walls. There was fluid accumulation in ileal loops inoculated with Escherichia coli and Staphylococcus epidermidis whilst fluid accumulation was absent in ileal loops seeded with P. aeruginosa, P. mirabilis, K. pneumoniae and B. subtilis respectively.The pH and turbidity readings ranged from 6.6 to 7.2 and 2 NTU to 10 NTU. The authorities managing the water dam facility should improve on their current treatment and disinfection methods

Comparison of toxicities between Vibrio fluvialis and Vibrio furnissii strains isolated from environmental samples

Gastrointestinal episodes associated with Vibrio species have been rising worldwide in the last few years. In this study, toxicities of two very well characterized V. fluvialis and V. furnissii strains, isolated from environmental samples, were used to investigate the comparative pathogenicity of these strains. The results showed that there is a positive relationship between toxin production as well as a high interspecies correlation. In the analysis of toxicity, both species showed positive results with close similarity in cell death on BHK-21 cell line and destruction of RBC, which were indicative of cytotoxic and hemolytic activity, respectively. Besides, molecular analysis showed the absence of ctx, tdh, trh, stx1 and stx2 genes. On the other hand, reduced fluid accumulation ratio in rabbit ileal loop assay and paralysis of hind legs of Swiss Albino mice were observed for V. furnissi strain, which indicates the differences in pathogenicity between the two species.
 Bangladesh J Microbiol, Volume 38, Number 2, December 2021, pp 73-77

Characterization of Pathogenic Vibrio parahaemolyticus Isolated From Fish Aquaculture of the Southwest Coastal Area of Bangladesh.

Vibrio parahaemolyticus is a major foodborne pathogen responsible for significant economic losses in aquaculture and a threat to human health. Here, we explored the incidence, virulence potential, and diversity of V. parahaemolyticus isolates from aquaculture farms in Bangladesh. We examined a total of 216 water, sediment, Oreochromis niloticus (tilapia), Labeo rohita (rui), and Penaeus monodon (shrimp) samples from the aquaculture system where 60.2% (130/216) samples were positive for V. parahaemolyticus. Furthermore, we identified 323 V. parahaemolyticus strains from contaminated samples, 17 of which were found positive for trh, a virulence gene. Four isolates out of the 17 obtained were able to accumulate fluid in the rabbit ileal loop assay. The correlation between the contamination of V. parahaemolyticus and environmental factors was determined by Pearson correlation. The temperature and salinity were significantly correlated (positive) with the incidence of V. parahaemolyticus. Most of the pathogenic isolates (94.1%) were found resistant to ampicillin and amoxicillin. O8: KUT was the predominant serotype of the potentially pathogenic isolates. ERIC-PCR reveals genetic variation and relatedness among the pathogenic isolates. Therefore, this region-specific study establishes the incidence of potential infection with V. parahaemolyticus from the consumption of tilapia, rui, and shrimp raised in farms in Satkhira, Bangladesh, and the basis for developing strategies to reduce the risk for diseases and economic burden.

Characterization of vibrio parahaemolyticus organisms isolated from shrimps and shrimp fields located in the coastal areas of Bangladesh

Background: In recent years, this group isolated trh positive Vibrio parahaemolyticus serotype 01:K56 from fresh water sediments around Dhaka city, Bangladesh. V. parahaemolyticus is a well known marine bacterium with pandemic effects of causing enteric diseases and the presence of trh positive V. parahaemolyticus serotype 01;K56 in the rivers of Bangladesh imply great public health concern. On the other hand, shrimp export sector is the second largest export earner for Bangladesh and contamination of shrimp with pathogenic V. parahaemolyticus could be a threat to the shrimp export sector. Therefore, this study was carried out to characterize V. parahaemolyticus isolates from shrimps and shrimp fields located in the coastal areas of Bangladesh. Methods and materials: Thirty four V. parahaemolyticus organisms were isolated and confirmed by standard biochemical, serological and molecular techniques. All these isolates belonged to 10 serogroups (O10:KUT, O1:K25, OUT:K61, O11:K33, O2:K28, O1:K33, O1:KUT, O5:KUT, O11:KUT and O8:KUT) confirmed by duplicate testing. Along with the occurrence of thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH), the incidence of two sets of gene clusters that encode the type 3 secretion system apparatus (T3SS1 & T3SS2α) which contribute in pathogenicity of V. parahaemolyticus were also investigated. Results: All V. parahaemolyticus isolates were found to be negative for both tdh and trh genes, which were confirmed by PCR. T3SS1 was found to be present in all V. parahaemolyticus strains as confirmed by the PCR. All these environmental isolates were found to be negative for T3SS2α, which confirmed that T3SS2α is not associated with non pathogenic environmental strains. We also phenotypically characterized the environmental isolates for the enterotoxicity test, which showed no fluid accumulation in rabbit ileal loop assay. All the above mentioned tests were done twice and the variances were less than 5 percent. Conclusion: All these findings clearly indicate that the V.parahaemolyticus organisms, which are associated with the shrimp and shrimp fields of the coastal areas are non pathogenic in nature and thus, would not be a threat for shrimp export sector, as long as the concerned bacterium is V. parahaemolyticus.

Characterization of ctx gene Negative Vibrio fluvialis Organisms Isolated from the Environment

A total of five Vibrio fluvialis organisms were isolated from the different environmental samples collected from Dhaka, Satkhira and Khulna. All these isolates were confirmed following API 20NE tests. Molecular analysis showed the absence of ctx, toxR, tdh, trh, stx1, and stx2 genes in these organisms. However, culture filtrates and crude proteins prepared from these organisms showed fluid accumulation in rabbit ileal loop assay, haemolysis of sheep red blood cess, rounding of BHK-21, HeLa and MDCK cells in cell culture assay, hind limb paralysis and death of mice in mice lethality assay and morphological changes in mouse neuronal cell assay. All these results indicated that the environmental V. fluvialis organisms, may not contain different virulence genes, including the ctx gene. However, the other in vivo and in vitro assays indicate that the toxins produced by the V. fluvialis organisms may contain enterotoxin, haemolysin, cytotoxin and neurotoxin.
 Bangladesh J Microbiol, Volume 36 Number 2 December 2019, pp 91-97

Investigation of the antibiotics susceptibility patterns and pathogenic potential of bacteria isolated from poultry wastes

The antibiotic resistant patterns and pathogenic potential of bacteria isolated from poultry wastes were investigated in this study. A total of 20 samples of poultry wastes were collected from different sites of poultry houses in Uli, Ihiala Local Government Areas, Anambra State, Nigeria using sterile polythene bags. The pathogens were isolated and identified using streaking plating technique, biochemical characterization and API Identification System. The disc diffusion method and plasmid curing were used to determine the antibiotic resistant patterns and resistant gene location. The pathogenicity study involves orally or intraperitonially inoculation of 0.5 mL of the different bacterial pathogens into three months’ immuno - competent albino mice for two weeks; and rabbit ileal loop assay for enterotoxin production in five healthy adult rabbit for 18 to 24 hrs. The isolated pathogens were Staphylococcus aureus, E. coli, Pseudomonas aeruginosa, Clostridium, Salmonella, Shigella, V. cholerae and V. parahaemolyticus. The antibiotics sensitivity test revealed that the bacterial pathogens were most susceptible to ciprofloxacin, rifampicin, levofloxacin and most resistant to ceporex, amoxil, ampiclox and norfloxacin with significance in the Gram negative antibiotics sensitivity testing (P < 0.05), but no significance in the Gram antibiotics sensitivity positive testing (P > 0.05). 12.5% of the strains lost their antibiotic resistance plasmids marker after sodium dodecyl sulfate (SDS) mediated curing. The pathogenicity testing revealed that the intestine had the highest significant microbial count (9 log CFU/g x 105) and the liver recorded the least significant count (7 log CFU/g x105) compare to the control (inoculated with water) that had no count. The enterotoxin testing revealed that Staphylococcus aureus recorded the highest enterotoxin production (0.45 mL/cm) while Pseudomonas aeruginosa recorded the least (0.16 mL/cm). Thus, the test isolates have shown reasonable pathogenic potentials and hence proper enlightenment and sensitization of the public health problems associated with these wastes should be encouraged.

Novel Hybrid of Typical Enteropathogenic Escherichia coli and Shiga-Toxin-Producing E. coli (tEPEC/STEC) Emerging From Pet Birds.

Exotic psittacine birds have been implicated as reservoir of diarrheagenic Escherichia coli (E. coli), including enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC). Here, we present a genotypic and phenotypic characterization of typical EPEC/STEC hybrid strains isolated from exotic psittacine birds. The strains were positive for eae, bfpA, and stx2f genes, belong to serotype O137:H6 and ST2678. Two strains were subject to whole genome sequencing, confirming the presence of the virulence factors of both E. coli pathotypes. Phenotypical in vitro tests confirmed their ability to adhere to HeLa cells and cause cytotoxicity to Vero cells. The rabbit ileal loop assays showed the attaching and effacing lesion, in addition to inflammatory process and overproduction of intestinal mucus. This is the first report of hybrid typical EPEC/STEC (O137:H6/ST2678) strains isolated from companion psittacine birds and the results suggest zoonotic risks.

Highly conserved type 1 pili promote enterotoxigenic E. coli pathogen-host interactions.

Enterotoxigenic Escherichia coli (ETEC), defined by their elaboration of heat-labile (LT) and/or heat-stable (ST) enterotoxins, are a common cause of diarrheal illness in developing countries. Efficient delivery of these toxins requires ETEC to engage target host enterocytes. This engagement is accomplished using a variety of pathovar-specific and conserved E. coli adhesin molecules as well as plasmid encoded colonization factors. Some of these adhesins undergo significant transcriptional modulation as ETEC encounter intestinal epithelia, perhaps suggesting that they cooperatively facilitate interaction with the host. Among genes significantly upregulated on cell contact are those encoding type 1 pili. We therefore investigated the role played by these pili in facilitating ETEC adhesion, and toxin delivery to model intestinal epithelia. We demonstrate that type 1 pili, encoded in the E. coli core genome, play an essential role in ETEC virulence, acting in concert with plasmid-encoded pathovar specific colonization factor (CF) fimbriae to promote optimal bacterial adhesion to cultured intestinal epithelium (CIE) and to epithelial monolayers differentiated from human small intestinal stem cells. Type 1 pili are tipped with the FimH adhesin which recognizes mannose with stereochemical specificity. Thus, enhanced production of highly mannosylated proteins on intestinal epithelia promoted FimH-mediated ETEC adhesion, while conversely, interruption of FimH lectin-epithelial interactions with soluble mannose, anti-FimH antibodies or mutagenesis of fimH effectively blocked ETEC adhesion. Moreover, fimH mutants were significantly impaired in delivery of both heat-stable and heat-labile toxins to the target epithelial cells in vitro, and these mutants were substantially less virulent in rabbit ileal loop assays, a classical model of ETEC pathogenesis. Collectively, our data suggest that these highly conserved pili play an essential role in virulence of these diverse pathogens.

Potential probiotic attributes of a new strain of Bacillus coagulans CGMCC 9951 isolated from healthy piglet feces.

A new strain of Bacillus coagulans CGMCC 9551, which has a broad range of antibacterial activities against six main pathogenic bacteria including Escherichia coli O8, Staphylococcus aureus, Salmonella enterica subsp. enterica serovar enteritidis, Streptococcus suis, Listeria monocytogenes and Pasteurella multocida, was isolated from healthy piglet feces. In adhesion assay, the isolate exhibited a stronger adhesion to pig intestinal mucus than that of B. subtilis JT143 and L. acidophilus LY24 respectively isolated from BioPlus(®)2B and FloraFIT(®) Probiotics (P < 0.05). The adhesion activity reached 44.5 ± 3.2, 48.9 ± 2.6, 42.6 ± 3.3 and 37.6 ± 2.4% to jejunum, ileum, transverse colon and sigmoid colon, separately. The survival rate of B. coagulans CGMCC 9551 was reduced by only 20% at 4 h exposure under 0.9% w/v bile salt. The strain was fully resistant to pH 2 for 2 h with 90.1 ± 3.5% survival and susceptible to 15 antibiotics commonly used in veterinary medicine. Additionally, the bacteria showed amylase, protease and cellulase activities. The safety assessment demonstrated the lack of toxicity potential in B. coagulans CGMCC 9551 by ligated rabbit ileal loop assay, acute and subchronic toxicity test. These results implied that that the new strain of B. coagulans CGMCC 9951 isolated from healthy piglet feces has promising probiotic characteristics and offers desirable opportunities for its successful commercialization as one excellent candidate probiotic.

Role of integrons, plasmids and SXT elements in multidrug resistance of Vibrio cholerae and Providencia vermicola obtained from a clinical isolate of diarrhea.

The isolates of Vibrio cholerae and Providencia vermicola obtained from a diarrheal patient were investigated for genetic elements governing their drug resistance phenotypes. Out of 14 antibiotics tested, V. cholerae Vc IDH02365 isolate showed resistance to nine antibiotics, while P. vermicola Pv NBA2365 was found to be resistant to all the antibiotics except polymyxin B. Though SXT integrase was depicted in both the bacteria, class 1 integron was found to be associated only with Pv NBA2365. Integrons in Pv NBA2365 conferred resistance to β-lactams, aminoglycosides, and trimethoprim. Pv NBA2365 carried two transformable plasmids imparting distinct antibiotic resistance traits to their Escherichia coli transformants. In rabbit ileal loop assays, Pv NBA2365 did not show any fluid accumulation (FA) in contrast with Vc IDH02365 that showed high FA. To the best of our knowledge, this is the first report of a highly drug resistant P. vermicola and additionally co-existence of multidrug resistant V. cholerae and P. vermicola. Both the microbes appeared to possess a wide array of mobile genetic elements for a large spectrum of antimicrobial agents, some of which are being used in the treatment of acute diarrhea.

BEC, a Novel Enterotoxin of Clostridium perfringens Found in Human Clinical Isolates from Acute Gastroenteritis Outbreaks

Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.

CHARACTERIZATION OF Escherichia coli STRAINS ISOLATED FROM SOME LOCALLY BREWED BEERS IN JOS METROPOLIS

This study investigated the microbiological quality of some traditionally brewed and sold beers in Jos, Nigeria. A total of 72 samples, 24 each of mash, wort and beer were collected from six localities namely: Angwan-Rukuba, Gada-Biyu, Tudun-Wada, Ojukwu Street, Kugiya-Bukuru and Jenta Adamu. The different kinds of local beer brands collected from the areas where Moskas, Redcorn, Pito and 9inches. The physico-chemical properties of tap and well waters from the six areas were analysed. The pH and the total titratable acidity of the samples of mash, wort and beer were determined. Samples of raw materials and final products were cultured on various selected media to determine the total bacterial, fungal and faecal coliform. Isolated faecal coliforms were plated on Eosin-Methylene Blue Agar for E.coli identification. Isolates of Escherichia coli were further subjected to rabbit ileal loop assay for enterotoxin production. The result of the physico-chemical parameters of the tap and well waters from these six localities showed only turbidity to exceed acceptable limits, with the highest average value from Jenta Adamu with 10.76 ± 0.21. The pH value of the samples was lowest in the beer samples from Moskas with 4.18 ± 0.15, while the total acidity was highest in Moskas with 0.078 ± 0.0098 mg/L. The average values of bacterial plate counts in all the locations and brands recorded the highest in the mash of the Redcorn with 9.5 x 107. Cfu/ml of Fungi was highest in all beers brands when compared to their mash and wort samples with the highest fungi plate count in the final product of 9inches with 23.3 x 107 cfu/ml. Out of the 36 Escherichia coli assayed for enterotoxin producing attributes only 13 (36.11 %) exhibited fluid inducible capability in animal model. Effective education of food production safety and hygienic handling methods should be encouraged among the local brewers.

Synergistic Effect of Various Virulence Factors Leading to High Toxicity of Environmental V. cholerae Non-O1/ Non-O139 Isolates Lacking ctx Gene : Comparative Study with Clinical Strains

Background Vibrio cholerae non-O1/ non-O139 serogroups have been reported to cause sporadic diarrhoea in humans. Cholera toxins have been mostly implicated for hypersecretion of ions and water into the small intestine. Though most of the V. cholerae non-O1/ non-O139 strains lack these cholera toxins, several other innate virulence factors contribute towards their pathogenicity. The environmental isolates may thus act as reservoirs for potential spreading of these virulence genes in the natural environment which may cause the emergence of epidemic-causing organisms.ResultsThe environmental isolates of vibrios were obtained from water samples, zooplanktons and phytoplanktons, from a village pond in Gandhinagar, Gujarat, India. They were confirmed as Vibrio cholerae non-O1/ non-O139 using standard biochemical and serotyping tests. PCR experiments revealed that the isolates lacked ctxA, ctxB, tcpA, zot and ace genes whereas other pathogenicity genes like toxR, rtxC, hlyA, hapA and prtV were detected in these isolates. Compared with epidemic strain V. cholerae O1 El Tor N16961, culture supernatants from most of these isolates caused higher cytotoxicity to HT29 cells and higher hemolytic, hemagglutinin and protease activities. In rabbit ileal loop assays, the environmental isolates showed only 2-4 folds lesser fluid accumulation in comparison to N16961 and a V. cholerae clinical isolate IDH02365 of 2009. Pulsed Field Gel electrophoresis and Random amplification of Polymorphic DNA indicated that these isolates showed considerable diversity and did not share the same clonal lineage even though they were derived from the same water source. All the isolates showed resistance to one or more antibiotics.ConclusionThough these environmental isolates lacked the cholera toxins, they seem to have adopted other survival strategies by optimally utilising a diverse array of several other toxins. The current findings indicate the possibility that these isolates could cause some gastroenteric inflammation when ingested and may serve as progenitors for overt disease-causing organisms.

Role of 6-gingerol in reduction of cholera toxin activity in vitro and in vivo.

Vibrio cholerae is one of the major bacterial pathogens responsible for the devastating diarrheal disease called cholera. Chemotherapy is often used against V. cholerae infections; however, the emergence of V. cholerae with multidrug resistance (MDR) toward the chemotherapeutic agents is a serious clinical problem. This scenario has provided us with the impetus to look into herbal remediation, especially toward blocking the action of cholera toxin (CT). Our studies were undertaken to determine the antidiarrheal potential of 6-gingerol (6G) on the basis of its effect on CT, the virulence factor secreted by V. cholerae. We report here that 6G binds to CT, hindering its interaction with the GM1 receptor present on the intestinal epithelial cells. The 50% inhibitory concentration (IC50) was determined to be 10 μg/ml. The detailed mechanistic study was conducted by enzyme-linked immunosorbent assay (ELISA), fluorescence spectroscopy, and isoelectric focusing. These results were validated with in vitro studies performed with the CHO, HeLa, and HT-29 cell lines, whereas a rabbit ileal loop assay was done to estimate the in vivo action, which confirms the efficacy of 6G in remediation of the choleragenic effects of CT. Thus, 6G can be an effective adjunctive therapy with oral rehydration solution for severe CT-mediated diarrhea.

Application of protein purification methods for the enrichment of a cytotoxin from Campylobacter jejuni

BackgroundCampylobater jejuni, a major foodborne diarrhoeal pathogen is reported to produce a number of cytotoxins of which only a cytolethal distending toxin (CDT) has been characterised so far. One or more additional cytotoxins other than CDT, including a Chinese hamster ovary (CHO) cell active, Vero cell inactive cytotoxin, may mediate inflammatory diarrhoea. Our objective was to develop a method to enrich and thus partially characterise this cytotoxin, as a pathway to the eventual identification and characterisation of the toxin.ResultsA number of biochemical methods including cation- and anion-exchange chromatography were evaluated to enrich the cytotoxin from a cell lysate of a known cytotoxin-producing C. jejuni, C31. The cytotoxin in crude lysate was initially prepared by size-exclusion desalting and then subjected to high pressure liquid chromatography (HPLC) ion-exchange fractionation. One pooled fraction (pool B) was cytotoxic for CHO cells equivalent to crude toxin (tissue culture infectivity dose 50 [TCID50] of 1–2 μg/ml). The proteins of pool B were identified by mass spectrometry (MS) after separation by SDS-PAGE and trypsin digestion. Also, pool B was directly digested with trypsin and then subjected to liquid chromatography tandem mass spectrometry (LCMS) analysis for identification of lesser abundant proteins in the fraction. A total of 41 proteins were found in the fraction, which included enzymes involved in metabolic and transport functions. Eighteen non-cytoplasmic proteins including 2 major antigenic peptide proteins (PEB2 and PEB3) and 3 proteins of unknown function were also identified in the screen. Cytotoxicity in pool B was trypsin-sensitive indicating its protein nature. The cytotoxic activity was heat-stable to 50°C, and partially inactivated at 60-70°C. The pool B fraction also induced fluid accumulation in the adult rabbit ileal loop assay with cytotoxicity for mucosa confirming the presence of the cytotoxin.ConclusionsWe report the enrichment and partial purification of C. jejuni cytotoxin by HPLC ion-exchange chromatography. Further purification may be achieved using additional complementary chromatographic techniques. A short-list of six candidate cytotoxin proteins was identified using an LCMS screen of pool B. Successful isolation of the cytotoxin will initiate steps for the determination of the role of this cytotoxin in the pathogenesis of C. jejuni diarrhoea.

Purification and Characterization of Cytotoxin Produced by a Clinical Isolate of Vibrio cholerae O54 TV113

Vibrio cholerae O54 TV113 isolated from a diarrheal patient produces an extracellular cytotoxin that caused alteration in the morphology of Chinese hamster ovary cells manifested as cell shrinkage with intact cell boundaries and finally causing cell death. Syncase medium supplemented with lincomycin (50 μg/ml), pH 7.2, and 18 h incubation with shaking at 37 °C supported optimal cytotoxin production. We isolated and purified this cytotoxin to homogeneity by ultrafiltration, 40-80 % ammonium sulfate precipitation, gradient-anion exchange chromatography, stepwise-anion exchange chromatography, and size exclusion chromatography increasing the specific activity by 866-fold. The cytotoxin is heat-labile, sensitive to protease and papain, and has a molecular weight of 64 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and enterotoxic activity in rabbit ileal loop assay. Both cytotoxic and enterotoxic activity could be inhibited or neutralized by antiserum raised against purified cytotoxin but not by preimmune serum. Immunodiffusion test between purified cytotoxin and its antiserum gave a single well-defined precipitin band showing reaction of complete identity and a well-defined single band in an immunoblot assay. This study thus indicate that the cytotoxin expressed by strain TV113 has both cytotoxic and enterotoxic activity and appears to contribute in pathogenesis of non-O1, non-O139 strains.

Construction and evaluation of chimeric heat-labile toxin B subunit and N-terminal(1–75) fragment of colonization factor antigen I gene of enterotoxigenic Escherichia coli

A significant percentage of enterotoxigenic Escherichia coli (ETEC) diarrheal disease worldwide is caused by colonization factor antigen I-producing isolates of ETEC either as infant diarrhea with high mortality or traveler’s diarrhea. Bacterial adhesion to intestinal epithelial cells and elaboration of enterotoxins are the main steps in pathogenesis of ETEC, and antibodies to these antigens have been associated with protection. Colonization factor antigen I (CFA/I) is the most frequently encountered adhesin in epidemiological studies. In this study, a DNA fragment encoding the first 25 amino acids of the N-terminal end of CFA/I B subunit was genetically fused to the C-terminal end of the B subunit of heat-labile enterotoxin gene of Escherichia coli with five glycine residues as a linker and expressed in E. coli. The N-terminal fragment was selected because it is relatively conserved among several ETEC fimbriae and strongly reacts with monoclonal antibodies against CFA/I, and several B-cell epitopes have been mapped to this region. The B subunit of heat labile enterotoxin (LTB) of E. coli is a potent immunogen and also elicits antitoxin response. The recombinant protein was recognized by anti-LTB MAb and anti-rCfaB polyclonal antibody and bound GM1 gangliosides receptors in enzyme-linked immunosorbant assay (ELISA). Polyclonal antibody raised in rabbit reacted with the dissociated as well as intact fimbriae expressed on the surface of bacteria in western blot and ELISA, respectively, and blocked fluid accumulation in the rabbit ileal loop assay. This demonstrates that the rLTB-CfaB(1–25) protein could be used for production of anti-LTB and anti-CfaB and that combining the two fragments has not impaired the function of either part. Moreover, complete lack of fluid accumulation in rabbit ileal loops might have resulted from both anti-adhesive and anti-toxin activity of the antibodies raised against the fusion protein.

Filamentous vibriophage fs2 encoding therstC gene integrates into the same chromosomal region as cholera toxin X

The genome of the filamentous phage of Vibrio cholerae fs2 was found to contain rst C and rst B1 (truncated) genes downstream of ORF500. att -fs2-dir and att- fs2-rev sequences homologous to that of att -CTXφ were found between orf 500 and rst C of the fs2 genome. This prompted us to search for the integration site of fs2 in the genomes of V. cholerae O1 and O139. The genome of fs2 was found to integrate downstream of att RS of the CTXφ phage, which integrated into chromosome I of V. cholerae O1 and O139. When infected with fs2, a fimbriate strain of V. cholerae O1 appeared to reduce fimbrial production in an adult rabbit ileal loop assay.

Filamentous vibriophage fs2 encoding therstC gene integrates into the same chromosomal region as cholera toxin X

The genome of the filamentous phage of Vibrio cholerae fs2 was found to contain rstC and rstB1 (truncated) genes downstream of ORF500. att-fs2-dir and att-fs2-rev sequences homologous to that of att-CTXphi were found between orf500 and rstC of the fs2 genome. This prompted us to search for the integration site of fs2 in the genomes of V. cholerae O1 and O139. The genome of fs2 was found to integrate downstream of attRS of the CTXphi phage, which integrated into chromosome I of V. cholerae O1 and O139. When infected with fs2, a fimbriate strain of V. cholerae O1 appeared to reduce fimbrial production in an adult rabbit ileal loop assay.

Ocorrência de Bacillus cereus em leite integral e capacidade enterotoxigênica das cepas isoladas

Pesquisaram-se a presença de Bacillus cereus e a produção de enterotoxinas produzidas por esses microrganismos em 120 amostras de diversos tipos de leite. Bacillus cereus foi isolado e identificado em 22 (73,3%), 15 (50,0%), 29 (96,7%) e quatro (13,3%) amostras de leite em pó, cru, pasteurizado e UAT (longa vida), respectivamente. Para a detecção de enterotoxinas pela técnica da alça ligada de coelho, foram positivos, respectivamente, três (13,6%), um (7,1%) e 10 (35,7%) microrganismos isolados das amostras de leite em pó, leite cru e leite pasteurizado. Pelo teste de aumento de permeabilidade vascular, dois (9,1%), um (7,1%), um (3,6%) e um (4,0%) microrganismos isolados de leite em pó, cru, pasteurizado e UAT apresentaram-se enterotoxigênicos, respectivamente. O uso da técnica de aglutinação passiva em látex demonstrou a produção da toxina diarréica por três (33,3%), sete (63,6%), quatro (30,8%) e oito (80,0%) microrganismos isolados, respectivamente, de leite em pó, cru, pasteurizado e UAT. Os resultados indicam um risco potencial, podendo colocar em risco a saúde dos consumidores desses produtos.