Short Communication: Enterotoxin Genes and Antibiotic Susceptibility of Bacillus cereus Isolated from Garlic Chives and Agricultural Environment.

This study aims to investigate the enterotoxin profiles and antibiotic susceptibility of Bacillus cereus isolated from garlic chives and environmental samples. A total of 103 B. cereus isolates were used to identify enterotoxin genes, including hblA, hblC, hblD, nheA, nheB, and nheC. The hemolysin BL enterotoxin complex (hblACD) was detected in 38 isolates (36.9%), and the non-hemolytic enterotoxin complex (nheABC) was detected in 8 (7.8%) isolates. Forty-five isolates (43.7%) had hblACD and nheABC genes. B. cereus was resistant to ß-lactam antibiotics and susceptible to non-ß-lactam antibiotics. However, some B. cereus strains showed intermediate resistance to ß-lactam and non-ß-lactam antibiotics. B. cereus isolated from garlic chives showed intermediate resistance to cefotaxime (7.7%), rifampin (15.4%), clindamycin (30.8%), erythromycin (7.7%), and tetracycline (7.7%). B. cereus isolates from the agricultural environment were moderately resistant to cefotaxime (18.9%), rifampin (15.6%), clindamycin (12.2%), erythromycin (4.4%), and tetracycline (5.6%). Moreover, B. cereus isolates from garlic chives and cultivation environments could change their antibiotic resistance profile from susceptible to intermediate-resistant to rifampin, clindamycin, erythromycin, and tetracycline and exhibit multidrug resistance. These results indicate that continuous monitoring of B. cereus contamination in the produce and agricultural environment might be needed to ensure the safety of consuming fresh vegetables.

[The first identification of epidemic clone of enterotoxic Escherichia coli O∶6 serogroup highly associated with azithromycin resistance in Shanghai].

Objective: To investigate the molecular characterization of adult diarrhea cases caused by enterotoxic Escherichia coli (ETEC) and explore the practical model of epidemiology for laboratory technique and data needs based on the surveillance network. Methods: Epidemiological design and sampling targeted adult cases ETEC caused diarrhea in epidemic season. The enterotoxin type, serogroup, resistance, colonization factor and molecular type of ETEC were identified. Multiple dynamic phenotypic characteristics of ETEC were indicated by multidimensional and multivariable data. Results: From 2016 to 2018, 84 eligible ETEC strains were detected. The dominant serums/toxins were O∶6 (STh), O∶25 (LT), O∶159 (STh), O∶153 (STh). O∶6 (STh+CS21), which replaced O∶25 and O∶159 as the popular clones in 2018. Six cases of O∶153 (STh+CFA/I+CS8+PT34) in outbreak in 2017 were imported ones. The resistance rates of ETEC strains detected in adults to sulfasoxazole, naproxinic acid, ampicillin and azithromycin were more than 30%, multidrug resistance (MDR) reached 58.3%. Serum/toxin types suggested that attenuated strains were more likely to become MDR. Molecular typing confirmed that the genetic similarity of the dominant clone of O∶6 serogroup (PT20-24) was higher than O∶25 and O∶159. There was a high correlation between the minimal inhibitory concentration (MIC) of azithromycin and the resistant gene mphA (87.5%, 28/32). O∶6 (STh+CS21+mphA) resistant clone was first detected in 2016. Conclusion: A new epidemic clone in adult ETEC diarrhea cases in Shanghai was O∶6 (STh+CS21+mphA). For the first time the association between azithromycin resistance gene mphA and a serum group of ETEC was observed. Multidimensional and multivariate analysis techniques based on epidemiology can help reveal the potential transmission pattern of ETEC for the accurate surveillance and early warning of outbreaks.

Berberine blocks the relapse of Clostridium difficile infection in C57BL/6 mice after standard vancomycin treatment.

Vancomycin is a preferred antibiotic for treating Clostridium difficile infection (CDI) and has been associated with a rate of recurrence of CDI of as high as 20% in treated patients. Recent studies have suggested that berberine, an alternative medical therapy for gastroenteritis and diarrhea, exhibits several beneficial effects, including induction of anti-inflammatory responses and restoration of the intestinal barrier function. This study investigated the therapeutic effects of berberine on preventing CDI relapse and restoring the gut microbiota in a mouse model. Berberine was administered through gavage to C57BL/6 mice with established CDI-induced intestinal injury and colitis. The disease activity index (DAI), mean relative weight, histopathology scores, and levels of toxins A and B in fecal samples were measured. An Illumina sequencing-based analysis of 16S rRNA genes was used to determine the overall structural change in the microbiota in the mouse ileocecum. Berberine administration significantly promoted the restoration of the intestinal microbiota by inhibiting the expansion of members of the family Enterobacteriaceae and counteracting the side effects of vancomycin treatment. Therapy consisting of vancomycin and berberine combined prevented weight loss, improved the DAI and the histopathology scores, and effectively decreased the mortality rate. Berberine prevented CDIs from relapsing and significantly improved survival in the mouse model of CDI. Our data indicate that a combination of berberine and vancomycin is more effective than vancomycin alone for treating CDI. One of the possible mechanisms by which berberine prevents a CDI relapse is through modulation of the gut microbiota. Although this conclusion was generated in the case of the mouse model, use of the combination of vancomycin and berberine and represent a novel therapeutic approach targeting CDI.

Plant compounds enhance the assay sensitivity for detection of active Bacillus cereus toxin.

Bacillus cereus is an important food pathogen, producing emetic and diarrheal syndromes, the latter mediated by enterotoxins. The ability to sensitively trace and identify this active toxin is important for food safety. This study evaluated a nonradioactive, sensitive, in vitro cell-based assay, based on B. cereus toxin inhibition of green fluorescent protein (GFP) synthesis in transduced monkey kidney Vero cells, combined with plant extracts or plant compounds that reduce viable count of B. cereus in food. The assay exhibited a dose dependent GFP inhibition response with ~25% inhibition at 50 ng/mL toxin evaluated in culture media or soy milk, rice milk or infant formula, products associated with food poisonings outbreak. The plant extracts of green tea or bitter almond and the plant compounds epicatechin or carvacrol were found to amplify the assay response to ~90% inhibition at the 50 ng/mL toxin concentration greatly increasing the sensitivity of this assay. Additional studies showed that the test formulations also inhibited the growth of the B. cereus bacteria, likely through cell membrane disruption. The results suggest that the improved highly sensitive assay for the toxin and the rapid inactivation of the pathogen producing the toxin have the potential to enhance food safety.

Expression of the cholera toxin B subunit (CT-B) in maize seeds and a combined mucosal treatment against cholera and traveler's diarrhea.

The non-toxic B subunit (CT-B) of cholera toxin from Vibrio cholerae is a strong immunogen and amplifies the immune reaction to conjugated antigens. In this work, a synthetic gene encoding for CT-B was expressed under control of a γ-zein promoter in maize seeds. Levels of CT-B in maize plants were determined via ganglioside dependent ELISA. The highest expression level recorded in T(1) generation seeds was 0.0014% of total aqueous soluble protein (TASP). Expression level of the same event in the T(2) generation was significantly increased to 0.0197% of TASP. Immunogenicity of maize derived CT-B was evaluated in mice with an oral immunization trial. Anti-CTB IgG and anti-CTB IgA were detected in the sera and fecal samples of the orally immunized mice, respectively. The mice were protected against holotoxin challenge with CT. An additional group of mice was administrated with an equal amount (5 µg per dose each) of mixed maize-derived CT-B and LT-B (B subunit of E. coli heat labile toxin). In the sera and fecal samples obtained from this group, the specific antibody levels were enhanced compared to either the same or a higher amount of CT-B alone. These results suggest that a synergistic action may be achieved using a CT-B and LT-B mixture that can lead to a more efficacious combined vaccine to target diarrhea induced by both cholera and enterotoxigenic strains of Escherichia coli.

Antibacterial activities of semipurified fractions of Quercus infectoria against enterohemorrhagic Escherichia coli O157:H7 and its verocytotoxin production.

Escherichia coli O157:H7 is one of the most important foodborne pathogens, causing nonbloody and bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. Use of antibiotics has been demonstrated to result in increased levels of verocytotoxin (VT) production as well as antibiotic resistance. Quercus infectoria was investigated for its antibacterial activity against E. coli O157:H7 and other VT-producing enterohemorrhagic E. coli (VTEC). The MIC was determined by a broth microdilution method, and the MBC was assessed by subculturing the bacteria from the wells that showed no apparent growth onto Mueller-Hinton agar. The fractions Qi2, Qi3, and Qi4 of Q. infectoria were demonstrated to possess good antibacterial activity, with MICs and MBCs ranging from 250 to 500 microg/ml. The effect of the effective fraction, Qi4, on the production of VT was determined using a reversed passive latex agglutination. The results indicate that at 20 h, fraction Qi4 markedly inhibits the release of VT1 and VT2 from VTEC cells at both inhibitory and subinhibitory concentrations. Furthermore, verotoxicity assay demonstrated that bacterial cultures treated with fraction Qi4 exerted less toxic effect on Vero cells. These in vitro results clearly indicate that the fraction Qi4 might constitute a promising natural food additive for the control of food poisoning by E. coli O157:H7 as well as other VTEC strains.

An outbreak of staphylococcal food poisoning caused by enterotoxin H in mashed potato made with raw milk.

Mashed potato made with raw bovine milk was suspected to have been the source of a food poisoning outbreak. Almost 8 x 10(8)Staphylococcus aureus CFU g(-1) were detected in the mashed potato. S. aureus was also found in bulk milk from the farm that had supplied milk for the mashed potato. Isolates from mashed potato and bulk milk were positive for the gene encoding staphylococcal enterotoxin H (seh), and the corresponding protein toxin, SEH, was detected by ELISA in the mashed potato. Production of SEH by S. aureus isolates from mashed potato (n = 4) and bulk milk (n = 4) was also demonstrated by ELISA. Sequencing of seh from one mashed potato isolate and one bulk milk isolate confirmed that the gene was a variant seh, and that the genes in both isolates were identical. Macrorestriction of chromosomal DNA with Sma1 followed by pulsed-field gel electrophoresis of seh-positive S. aureus from mashed potato and bulk milk revealed indistinguishable banding patterns between isolates from both sources. It seems likely that raw bovine milk used in the preparation of mashed potato contained S. aureus that subsequently produced sufficient SEH in the mashed potato to cause food poisoning.

Eradication of pathogenic bacteria and restoration of normal pouch flora: comparison of metronidazole and ciprofloxacin in the treatment of pouchitis.

PURPOSE: Pouchitis is the major long-term complication after ileal pouch-anal anastomosis for ulcerative colitis. Metronidazole and ciprofloxacin are commonly used for treatment; however, nothing is known about the effects on the pouch flora during and after pouchitis episodes. This study was designed to evaluate the effect of both antibiotics on eradication of pathogens and the restoration of normal pouch flora. METHODS: The fecal flora obtained from 13 patients with ulcerative colitis was examined at the beginning of a pouchitis episode before treatment, during treatment with metronidazole or ciprofloxacin, and during pouchitis-free periods. Some patients experienced more than one pouchitis episode. Therefore, a total of 104 samples was obtained. Each sample was cultured under aerobic and anaerobic conditions and the isolated bacteria were identified. Furthermore, the clinical response to both antibiotics was compared using the Pouchitis Disease Activity Index score. RESULTS: During pouchitis-free periods, the patients had a flora characterized by high numbers of anaerobes and no or low numbers of pathogens. This flora resembles normal colon flora. During pouchitis episodes, we found a significant decrease of anaerobes ( P = 0.01), a significant increase of aerobic bacteria ( P = 0.01), and significantly more numbers of pathogens, such as Clostridium perfringens (in 95 percent of the samples; P < 0.01) and hemolytic strains of Escherichia coli (in 57 percent of the samples; P = 0.05). Treatment with metronidazole resulted in a complete eradication of the anaerobic flora, including C. perfringens. However, no changes in the numbers of E. coli were found. In contrast, when the patient was treated with ciprofloxacin, not only C. perfringens, but also all coliforms including hemolytic strains of E. coli disappeared. The larger part of the anaerobic flora was left undisturbed during the administration of ciprofloxacin. Patients treated with ciprofloxacin experienced significant larger reductions in Pouchitis Disease Activity Index score compared with patients treated with metronidazole ( P = 0.04). CONCLUSIONS: This study strongly suggests a role of pathogenic bacteria ( C. perfringens and/or hemolytic strains of E. coli) in pouchitis. From a microbiologic and a clinical point of view, ciprofloxacin is preferable to metronidazole, because treatment with ciprofloxacin eradicates both pathogens and results in an optimal restoration of normal pouch flora.

Real time biosensor analysis of staphylococcal enterotoxin A in food.

Currently there is no 'real-time' detection system to identify food borne toxins. In order to develop such a system, we have used a evanescent wave biosensor for real time detection of staphylococcal enterotoxin A (SEA) in foods. The approach used here is sandwich biosensor, a method utilizing two antibodies. The toxin binds initially to a capturing antibody which is bound covalently on the surface of the biosensor detector. The second antibody binds to the captured toxin. We were able to measure SEA in foods with little or no background interference, demonstrating that biosensor-based measurement of SEA was possible not only with purified SEA but also in complex food matrices such as hot dogs, potato salad, milk and mushrooms. Autoclaved samples of SEA did not evoke a positive response. With both purified SEA and SEA-spiked foods, the assay sensitivity is 10-100 ng/g depending on the material tested and the assay is rapid ( <4 min) when a single antibody is used.

Cecal carriage of Clostridium perfringens in broiler chickens given Mucosal Starter Culture.

Day-of-hatch broiler chicks housed in isolation units were each given, by oral gavage, 0.1 ml of Mucosal Starter Culture (MSC) or saline control. Each of the treated and control chicks was subsequently given a composite culture of three strains of bacitracin-resistant Clostridium perfringens (Cp) previously isolated from chickens with symptoms of necrotic enteritis. Some chicks were maintained on a corn-based diet provided ad libitum. Others were given the feed supplemented with 50% rye (a predisposing factor for necrotic enteritis). At 7, 14, and 21 days after receiving Cp, chicks were euthanatized, and cecal contents were diluted and plated on selective agar containing bacitracin. For chicks on corn feed, Cp numbers were similar in control birds and birds given MSC in three of four trials. In two of the trials that demonstrated no effect of MSC on Cp numbers, enterotoxin presence was determined. The number of birds with detectable Cp enterotoxin in their small intestine and the mean toxin levels were lower in the MSC-treated birds. In a fourth trial with birds on corn-based feed, mean Cp numbers and the number of Cp-positive birds were lower in the MSC-treated birds. For the two trials involving chickens on rye-supplemented feed, Cp numbers and the percentage of Cp-positive birds were significantly reduced in MSC-treated birds compared with control birds. Enterotoxin in birds receiving the 50% rye diet was at low levels or not detected in control and MSC-treated birds. Results suggest that MSC may reduce intestinal proliferation of Cp, a causative agent of necrotic enteritis in poultry and of foodborne disease in humans.

Influence of temperature shifts on survival, growth, and toxin production by psychrotrophic and mesophilic strains of Bacillus cereus in potatoes and chicken gravy.

A study was done to determine the influence of temperature on growth and toxin production characteristics of psychrotrophic and mesophilic strains of Bacillus cereus when inoculated into mashed potatoes and chicken gravy containing various concentrations of sodium chloride and held at temperatures different from those at which cells had been cultured. Logarithmic growth phase cells (10 h, 30 degrees C) of psychrotrophic (F3802A/84) and mesophilic (B4ac-1) strains of Bacillus cereus were inoculated into rehydrated commercially processed instant mashed potatoes and chicken gravy supplemented with 0, 2, or 4% sodium chloride. Growth, survival, and diarrheal toxin production in potatoes and gravy held at 30, 37, and 10 degrees C (strain F3802A/84) or 30, 40, and 10 degrees C (strain B4ac-1) were monitored. Both strains grew in both foods containing no added sodium chloride or 2% sodium chloride when held at 30, 37, or 40 degrees C for 2 days. Strain B4ac-1 grew better than strain F3802A/84 in foods containing 4% sodium chloride. Maximum amounts of enterotoxin (1024 ng/g) were produced by strain B4ac-1 in chicken gravy held at 30 and 40 degrees C. Strain F3802A/84 grew to populations of 7 log10 CFU/g in foods containing no added sodium chloride or 2% sodium chloride at 10 degrees C. Strain F3802A/84 produced the highest amount of enterotoxin (1024 ng/g) at 30 degrees C in chicken gravy containing 0.7 or 2% sodium chloride; however, little or low amounts of toxin (4-16 ng/g) were produced in chicken gravy at 10 degrees C. Compared to strain B4ac-1, cells of strain F3802A/84 subjected to a downward shift in incubation temperature (10 degrees C) grew more rapidly in chicken gravy. Strain B4ac-1 produced the highest amount of toxin (1024 ng/g) at 30 degrees C in gravy containing 4% sodium chloride and at 40 degrees C in gravy containing 0.7% sodium chloride. Toxin was not detected in inoculated mashed potatoes. Results of this study indicate that shifts in incubation temperature influence growth and toxin production by psychrotrophic and mesophilic strains of B. cereux differently. It is important to store pasteurized, ready-to-eat foods at a temperature low enough to prevent the growth of B. cereus.

Prevalência de Staphylococcus aureus resistentes à meticilina (MRSA) produtores de leite humano ordenhado e seu crescimento em colostro humano / Prevalence of enterotoxigenic Staphylococcus aureus resistant to meticilin (MRSA) in milked samples of human milk and their growth in human colostrum

Foram coletados e analisadas 500 amostras de leite humano ordenhado, oriundas de cinco cidades brasileiras (100 de cada), visando detectar cepas de staphylococcus aureus resistentes à meticilina (MRSA) produtoras de enterotoxinas. Nestas amostras foram encontradas 57 cepas de mMRSA, sendo confirmada a presença do gene mecA, responsável pela resitência, por meio de sonda molecular específica. Posteriormente, 40 das 57 cepas isoladas, foram selecionadas de modo a conter todas as cepas representantes de cada regiäo, com exceçäo da que apresentava a maioria, da qual foram selecionadas aleatoriamente 10 cepas para completar as 40 a serem analisadas. As cepas foram submetidas a pesquisa de quatro enterotoxinas por meio do Kit SET-RPLA (oxoid). Dentre estas, foram encontradas duas produtoras de enteroxina B, com as quais foram feitas curvas de crescimento em colostro humano e em " Trypticase Soy Broth", Tendo por fim avaliar o impacto dos fatores de defesa do colostro sobre o desenvolvimento e produçäo de toxinas pelos microorganismos em questäo. Para tal, foram inoculadas 5,0 x 10 elevado ao quadrado UFC/ml, que representava aproximadamente a média das contagens das cepas S. aureus nas amostras que continham as cepas resitentes à meticilina. Após cinco horas de incubaçäo a 37 graus celcius as populaçöes eram superiores a 4,9 x 10 elevado a quatro UFC/ml e apresentavam liberaçäo de enterotoxinas no meio de cultura e no colostro. Nossos resultados ressaltam a importância dos cuidados higiênicos-sanitários e das técnicas apropriadas de preservaçäo do leite humano ordenhado, para evitar a proliferaçäo do S. aureus e a conseqüente produçäo de enterotoxinas no produto, que por näo poderem ser removidas nem destruídas pelos procedimentos normais, levariam risco de vida a seus receptores

A prospective randomized controlled trial of oral ciprofloxacin in acute ulcerative colitis.

OBJECTIVES: The aim of this prospective, randomized, controlled trial was to evaluate the role of ciprofloxacin as an adjunct to corticosteroids in acute ulcerative colitis. METHODS: Seventy consecutive patients with mild (n = 37) or moderately active (n = 33) ulcerative colitis were randomized to receive oral ciprofloxacin (250 mg b.i.d., n = 34) or placebo (n = 36) for 14 days. In addition, they were given oral prednisolone (initial dose 20 or 40 mg for mild and moderately active ulcerative colitis, respectively) and rectal betamethasone enemas (2 g at night) for 7-9 weeks. All patients were receiving olsalazine (0.5 g twice daily). At study entry, the groups were similar with respect to age, sex, extent, duration, and severity of disease, and previous treatments. Patients were assessed clinically, endoscopically, and histologically before, at the end of the trial (day 14), and on completion of steroid treatment, or at any time worsening of symptoms or a complication of ulcerative colitis occurred. RESULTS: At the end of the study, 24 patients (70.5%) in the ciprofloxacin group and 26 patients (72%) in the placebo group achieved remission (p > 0.1, Yates chi 2). Ten patients in each group necessitated higher doses of oral (n = 12) or intravenous (n = 8) steroids. Of the latter patients, two underwent emergency colectomy without perioperative deaths. Clostridium difficile toxin A was not detected in nonresponders to ciprofloxacin treatment. CONCLUSIONS: A short course of oral ciprofloxacin treatment does not seem to increase the proportion of patients with active ulcerative colitis going into remission.

Escherichia coli heat-stable enterotoxin in feces and intestines of calves with diarrhea.

Two experiments were conducted to evaluate detection of Escherichia coli heat-stable enterotoxin (ST) in the feces of calves as a method for implicating E coli in neonatal calf diarrhea. The first experiment evaluated the use of the infant mouse test for detection of ST in the feces of calves with naturally occurring diarrhea. Simultaneous identification of bovine enteropathogenic strains of E coli (EEC) and of other infective agents implicated in neonatal calf diarrhea was attempted in these samples. The ST was detected with certainty in only 7 of 41 samples from calves less than or equal to 3 weeks old. Enteropathogenic E coli, however, was detected in 27 samples. In 23 of these 27 samples, EEC was the only recognizable diarrheagenic agent. In a small percentage of the samples, Salmonella, rotavirus, coronavirus, and cryptosporidium were recognized alone, in combination with each other, or with EEC. In the second experiment, 6 calves were fed colostrum from cows inoculated with the bovine EEC strain B44; 6 were given colostrum from cows vaccinated with non-EEC strain 28F, and 4 were given milk from nonvaccinated heifers. Two of the calves that were given colostrum from cows inoculated with strain B44 were challenge exposed with the non-EEC strain 28F. The remaining calves were challenge exposed with the EEc strain B44. Fecal samples were taken from these calves at intervals and were examined for the presence of ST and of the challenge-exposure organism. The ST was detected in approximately one half of the fecal samples obtained, and it was most often detected in the early stages of the induced diarrhea. Calves were observed to shed the challenge-exposure EEC strain for long periods in the absence of diarrhea or detectable amounts of ST in the feces. The ST was detectable in fecal samples when the diarrhea was severe and when the dry matter content of the fecal samples was low.