Traceability of potential enterotoxigenic Bacillus cereus in bee-pollen samples from Argentina throughout the production process.

Bee-pollen is a functional food sold for human and animal consumption but also is a favorable microhabitat for many spore-forming bacteria. Among them, Bacillus cereus can produce several toxins and other virulence factors, causing an emetic or diarrheal syndrome after ingestion. The study involved 36 bee-pollen samples obtained from different sampling points throughout the production process (collecting, freezing, drying, and cleaning) in Argentina. Fifty isolates of B. cereus yielded 24 different fingerprint patterns with BOX and ERIC primers. Only three fingerprint patterns were maintained throughout the production process. In contrast, others were lost or incorporated during the different steps, suggesting that cross-contamination occurred as shown by differences in fingerprint patterns after freezing, drying, and cleaning steps compared to the initial collection step. Genes encoding for cereulide (ces), cytotoxin K (cytK), sphingomyelinase (sph), the components of hemolysin BL (hblA, hblB, hblC, hblD) and non-hemolytic complex (nheAB) were studied. All the isolates displayed one or more enterotoxin genes. The most frequent virulence genes detected belong to the HBL complex, being the most abundant hblA (98%), followed by hblD (64%), hblB (54%), and hblC (32%), respectively. Ten strains (20%), present at all sampling points, carried all the subunits of the HBL complex. The non-hemolytic enterotoxic complex (nheAB) was found in 48 strains (96%), while seven strains (14%) present at all sampling points showed the amplification product for sphingomyelinase (sph). One cereulide-producer was isolated at the cleaning step; this strain contained all the components for the hemolytic enterotoxin complex HBL, the NHE complex, and cytotoxin K related to the foodborne diarrhoeal syndrome. In total, 11 different virulence patterns were observed, and also a correlation between rep-fingerprint and virulence patterns. The results suggest that bee-pollen can be contaminated at any point in the production process with potential enterotoxic B. cereus strains, emphasizing the importance of hygienic processing.

Toxicological evaluation of lotus, ginkgo, and garlic tailored fermented Korean soybean paste (Doenjang) for biogenic amines, aflatoxins, and microbial hazards.

The present study aimed to develop a consortium of nutritive fermented food products, supplemented with phytochemicals, with reduced toxicological contents. We developed new flavored Doenjang products (protein rich) fermented with lotus, ginkgo, and garlic plant extract-based Meju (termed as EMD) as the starter culture and by using traditional Meju (termed as TMD), where these plant extracts were added later during the fermentation process. Fermented Doenjang samples were analyzed for reduced levels of biogenic amines (BAs), aflatoxins, and microbial hazards, (including Bacillus cereus) as well as for their nutritive contents and antioxidant potential, after varying periods of fermentation (0, 3, 6, 9 and 12 months). All Doenjang samples prepared using plant extracts and their mixtures (1% and 10%) showed desired reduction in B. cereus counts, BAs, aflatoxins, and other foodborne pathogens as well as showed potent antioxidant abilities, including phenolic/flavonoid contents. Based on the higher efficiency in reducing various toxicants, Ginkgo biloba leaf extract added TMD samples were selected for the development of Doenjang products as an innovative approach, with great potential to improve the quality and safety of soybean fermented products in the Korean market, offering enhanced health benefits and reduced risks of toxicity.

Optimization of physicochemical parameters for maximum amylase production by indigenously isolated Bacillus cereus AS2 strain.

Amylases are enzymes that catalyze the hydrolysis of starch into highly valuable products of economic significance. Amylases are used extensively in various industrial sectors. Microbial sources particularly Bacillus species are well known for the cost effective commercial production of amylase enzyme. Present study focuses on the enhancement of amylase enzyme production from an indigenously isolated Bacillus cereus AS2 strain via one variable at a time (OVAT) optimization of different physical and chemical factors. Purposely, eight parameters possibly affecting the amylase production including temperature, pH, incubation time, inoculum size, substrate concentration, metal ions, carbon and nitrogen sources were investigated. According to the results, amylase production was significantly boosted at maximum when the Bacillus cereus AS2 was grown at 45°C on pH 7.0 for 72 hours in the medium supplemented with 4% starch and 0.5% glycine. Among the different metal ions tested, CaCl2 (0.05%) was found significant to accelerate extracellular amylase production.

Characterization of Bacillus cereus group isolates from powdered food products.

Mashed potato powder as well as powdered infant formula (PIF) are frequently contaminated with Bacillus cereus sensu lato (B. cereus s.l.), mainly with its spores. These products have also been implicated in foodborne illnesses. Here, we characterized B. cereus s.l. isolates originating from powdered products based on sporulation assays, toxin gene profiling, and panC typing combined with a SplitsTree analysis. Furthermore, cytotoxicity assays with B. cytotoxicus isolates were performed. 78% of PIF tested positive for B. cereus s.l., whereas 92% of all mashed potato powders were positive. In total, 43 isolates were further characterized. The nhe and cytK2 genes were most frequently detected. Moreover, a cereulide-producer was detected from PIF. Most isolates were assigned to panC group III, but members of group II, IV, V, and VII could also be found. Nine B. cytotoxicus were isolated out of nine mashed potato powders. All panC group VII isolates were positive for cytK1. Cytotoxicity assays of these nine isolates revealed one highly cytotoxic strain, while all other isolates exhibited no detectable cytotoxicity, underpinning that cytotoxicity of a certain B. cereus group strain cannot be deduced from the sole presence or absence of toxin genes.

Novel Bacillus cereus strain from electrokinetically remediated saline soil towards the remediation of crude oil.

A new strain SWH-15 was successfully isolated after initial electrokinetic remediation experiment using the same saline soil sampled from Shengli Oilfield, China. Four methods (morphological and biochemical characteristics, whole-cell fatty acid methyl esters (FAMEs) analysis, 16S rRNA sequence analysis and DNA G + C content and DNA-DNA hybridization analysis) were used to identify the taxonomic status of SWH-15 and confirmed that SWH-15 was a novel species of the Bacillus (B.) cereus group. Then, we assessed the degrading ability of the novel strain SWH-15 to crude oil through a microcosm experiment with four treatments, including control (CK), bioremediation using SWH-15 (Bio), electrokinetic remediation (EK), and combined bioremediation and electrokinetic remediation (Bio + EK). The results showed that the Bio + EK combined remediation treatment was more effective than the CK, Bio, and EK treatments in degrading crude oil contaminants. Bioaugmentation, by addition of the strain SWH-15 had synergistic effect with EK in Bio + EK treatment. Bacterial community analysis showed that electrokinetic remediation alone significantly altered the bacterial community of the saline soil. The addition of the strain SWH-15 alone had a weak effect on the bacterial community. However, the strain SWH-15 boosted the growth of other bacterial species in the metabolic network and weakened the impact of electrical field on the whole bacterial community structure in the Bio + EK treatment.

Atividade antibacteriana de óleos essenciais de café verde e torrado (Coffea arabica), Cacau (Theobroma cacao), casca e folha de canela do ceilão (Cinnamomum zeylanicun) / Antibacterial activity of essential oils from green and roasted coffee (Coffea arabica), Cacau (Theobroma cacao), bark and cinnamon leaf from Ceylon (Cinnamomum zeylanicun)

Diversos conservantes naturais têm sido utilizados na inativação de micro-organismos, sem efeitos adversos com relação aos valores nutricionais dos alimentos e da saúde humana. Entretanto, estudos apontam reações adversas aos aditivos sintéticos, tais como reações tóxicas e o possível desenvolvimento de cânceres específicos. A busca por agentes antimicrobianos naturais em alternativa aos conservantes sintéticos tem sido constante; a fim de proporcionar o controle microbiológico e a extensão da vida de prateleira, excluindo, portanto, as desvantagens trazidas pelo uso de aditivos artificiais, através de ação antimicrobiana de óleos essenciais. O objetivo do trabalho foi verificar a ação antibacteriana dos óleos essenciais de café verde e torrado (Coffea arabica), cacau (Theobroma cacao), casca e folha de canela-do- -Ceilão (Cinnamomum zeylanicun) sobre as bactérias Bacillus cereus, Bacillus subtilis, Escherichia coli, Staphylococcus aureus, Salmonella Enteritidis e Salmonella Typhimurium. Os óleos essenciais foram impregnados em discos de papel filtro de 6 mm de diâmetro, próprios para antibiograma; sendo posteriormente colocados em placas de Petri contendo Ágar Nutriente previamente semeado com os micro-organismos. As mesmas foram incubadas a 35°C por 24 e 48 horas. Após este período foi possível observar e medir o diâmetro dos halos e, halos iguais ou superiores a 10 mm foram considerados significativos de atividade antimicrobiana. Os óleos essenciais de cacau, canela casca e canela folha inibiram significativamente todos os micro-organismos testados. E. coli foi inibida significativamente por todos os óleos essenciais. O óleo essencial de canela casca demonstrou os melhores efeitos inibitórios, sendo o mais significativo sobre a E. coli (halo de 36 mm).(AU)

Elucidation of enterotoxigenic Bacillus cereus outbreaks in Austria by complementary epidemiological and microbiological investigations, 2013.

Identifying Bacillus cereus as the causative agent of a foodborne outbreak still poses a challenge. We report on the epidemiological and microbiological investigation of three outbreaks of food poisoning (A, B, and C) in Austria in 2013. A total of 44% among 32 hotel guests (A), 22% among 63 employees (B) and 29% among 362 residents of a rehab clinic (C) fell sick immediately after meal consumption. B. cereus isolated from left overs or retained samples from related foods were characterized by toxin gene profiling, and molecular typing using panC sequencing and M13-PCR typing (in outbreak A and C). We identified two B. cereus strains in outbreak A, and six B. cereus strains, each in outbreak B and C; we also found Staphylococcus aureus and staphylococcal enterotoxins in outbreak A. The panC sequence based phylogenetic affiliation of the B. cereus strains, together with findings of the retrospective cohort analyses, helped determining their etiological role. Consumption of a mashed potatoes dish in outbreak A (RR: ∞), a pancake strips soup in outbreak B (RR 13.0; 95% CI 1.8-93.0) and for outbreak C of a fruit salad (RR 1.50; 95% CI 1.09-2.00), deer ragout (RR: 1.99; 95% CI 1.23-3.22) and a cranberry/pear (RR 2.46; 95% CI 1.50-4.03)were associated with increased risk of falling sick. An enterotoxigenic strain affiliated to the phylogenetic group with the highest risk of food poisoning was isolated from the crème spinach and the strawberry buttermilk, and also from the stool samples of the one B. cereus positive outbreak case-patient, who ate both. Our investigation of three food poisoning outbreaks illustrates the added value of a combined approach by using epidemiological, microbiological and genotyping methods in identifying the likely outbreak sources and the etiological B. cereus strains.

Assessment of the microbial load of some medicinal plants commonly used in Romania.

AIM: This study aims to assess the microbial load of some medicinal plants (Matricaria chamomilla, Achillea millefolium, Ocimum basilicum, Calendula officinalis, Tilia cordata, Hypericum perforatum) commonly used as medicinal teas. MATERIAL AND METHODS: A total of 24 samples collected from the local market, as well as from the spontaneous flora of Galati County, were analyzed in terms of the total number of germs, the content of coliform bacteria, yeasts and molds, and the presence of specific pathogenic bacteria (Escherichia coli, Bacillus cereus, Coagulase-positive Staphylococcus, Salmonella spp.). RESULTS: The values obtained for total number of germs, yeasts and molds were in accordance with the limits set by the European Pharmacopoeia for plant products to which hot water is added before use. In the case of specific pathogenic bacteria, the presence of E. coli was confirmed in 41.66% of the samples, the presence of Coagulase-positive Staphylococcus in 16.66% of the samples, and the presence of Bacillus cereus in 33.33% of the samples. Salmonella spp. was absent in all samples. CONCLUSIONS: The study highlights a certain microbial load of medicinal plants analyzed, emphasizing the importance of quality control in all stages of production.

Dechlorination of chloroorganics, decolorization, and simultaneous bioremediation of Cr6+ from real tannery effluent employing indigenous Bacillus cereus isolate.

A native Bacillus cereus isolate has been employed, for the first time, for simultaneous decolorization, dechlorination of chloroorganics, and Cr(6+) remediation from the real tannery effluent. Most of the physicochemical variables in 3:1 diluted effluent were well above the standard prescribed limits, which decreased substantially upon microbial treatment. The extent of bioremediation was better in diluted (3:1) as compared to undiluted effluent supplemented with nutrients and augmented with B. cereus isolate. Maximum growth, effluent decolorization (42.5 %), dechlorination (74.1 %), and Cr(6+) remediation (34.2 %) were attained with 4.0 % (v/v) inoculum, 0.8 % glucose, and 0.2 % ammonium chloride in 3:1 diluted effluent at natural pH (8.1) within 72 h of incubation. The efficiency of bioremediation in a bioreactor was higher as compared to a flask trial during 72 h of incubation: decolorization (47.9 %) was enhanced by 5.4 %, dechlorination (77.4 %) by 3.3 %, and Cr(6+) removal (41.7 %) by 7.5 % at an initial color of 286 Pt-Co units and initial concentration of 62 mg chloride ions and 108 mg l(-1) Cr(6+). Immobilized biomass of Pseudomonas putida and B. cereus coculture enhanced the extent of Cr(6+) remediation (51.9 %) by 10.2 % compared to the bioreactor trial. Chromate reductase activity and reduced Cr directly correlated and were mainly associated with soluble fraction of B. cereus plus effluent natural microflora. The GC-MS analyses revealed the formation of metabolites such as acetic acid and 2-butenoic acid in bacterially treated effluent. The supplementation of nutrients along with B. cereus augmentation is required for efficient effluent bioremediation.

[Diarrhoea and oedema in two show horses after feeding a pelleted supplemental feed for horses according to VDLUFA's perspective of microbial quality classified as safe for use in horses]. / Auftreten von Durchfall und Odemen bei zwei Turnierpferden nach Fütterung eines laut VDLUFA-verbandsmethode mikrobiologisch unbedenklichen Ergänzungsfuttermittels für Pferde.

A new batch of a supplemental feed was fed as pellets (diameter 8 mm) to two Warmblood-type horses. One horse developed watery diarrhoea within two days. Pronounced oedema due to hypalbuminemia was seen about ten days later. The feed was replaced by pellets of identical composition and mixing process, but lower diameter (5 mm). After one week of feeding, oedema regressed and faeces were normally formed. At refeeding the larger sized pellets, the symptoms recurred, but now both horses were affected. After a change to the smaller pellets, the horses recovered soon. Water activity (aw-value) of the larger and the smaller sized pellets was 0.68 and 0.56, respectively. In the larger sized pellets crude fat increased, whereas crude protein and nitrogen-free extracts decreased, giving a hint to microbial activity. Samples of both pellets were examined by VDLUFA methods and the microbial quality was classified in quality step 1. Though the quality parameters complied with recommendations for the product, it contained large numbers of spoilage indicating bacteria. The content of sulfite-reducing clostridia was higher in the 8 mm pellets than in the 5 mm pellets, with 3.3 x 10(2) and 1.1 x 10(2) colony forming units, respectively. The larger sized pellets produced remarkable quantities of gas. Bacillus cereus of non probiotic origin was identified.

A nanoporous membrane-based impedimetric immunosensor for label-free detection of pathogenic bacteria in whole milk.

We introduce a nanoporous membrane based impedimetric immunosensor for the label-free detection of bacterial pathogens in whole milk. A simple and rapid method to modify a commercially available alumina nanoporous membrane with hyaluronic acid (HA) effectively reduced the non-specific binding of biomolecules and other cells, and permitted successful immobilization of antibodies. Escherichia coli O157:H7, one of the most harmful food-borne pathogenic bacteria, was tested as a model pathogen in this study. The ionic impedance of electrolytes through nanopores, due to antibody-pathogen interactions, was monitored by impedance spectra and analyzed by normalized impedance change (NIC). The regression equation for the NIC at 1 kHz versus concentration of E. coli O157:H7 (10-10(5)cfu/ml) was obtained, and the detection limit found to be as low as 10 cfu/ml. In addition, the proposed immunosensor was successfully used for the detection of E. coli O157:H7 in whole milk samples with the detection limit as low as 83.7 cfu/ml with 95% probability. The specificity of the immunosensor was also demonstrated using non-target bacteria, including Staphylococcus aureus, Bacillus cereus, and non pathogenic E. coli DH5α. This study shows that a HA-functionalized nanoporous membrane-based impedimetric sensor is capable of detecting pathogenic bacteria in whole milk without any pretreatment. This is a significant step for evaluating the safety of food and environmental samples and other medical diagnostics.

Molecular and toxigenic characterization of Bacillus cereus and Bacillus thuringiensis strains isolated from commercial ground roasted coffee.

Thirty samples of roasted ground coffee beans from 10 different commercial brands were analyzed to investigate the occurrence and levels of Bacillus cereus and Bacillus thuringiensis strains. Strains were evaluated for their genetic diversity by repetitive element sequence polymorphism PCR (Rep-PCR) and for their toxigenic profiles, i.e., the presence of hblA, hblC, hblD, nheA, nheB, nheC, cytK, ces, and entFM. Survival and multiplication of B. cereus sensu lato in the ready-to-drink coffee was determined to evaluate this beverage as a possible vehicle for B. cereus infection. B. cereus was detected in 17 (56.7%) of the 30 samples, and B. thuringiensis was detected in 8 (26.7%) of the 30 samples. Five samples did not produce any characteristic growth. The most common gene, entFM, was detected in 23 strains (92%). The NHE complex (nheA, nheB, and nheC genes) was found in 19 strains (76%). The HBL complex (hblA, hblC, and hblD) was found in 16 strains (64%). All strains were negative for ces. The cytK gene was found in 16 strains (64%). The computer-assisted cluster analysis of Rep-PCR profiles using a clustering criterion of 80% similarity revealed four main clusters. Cluster 1 was the predominant and comprised three B. thuringiensis strains with 100% similarity, cluster 2 comprised two B. cereus strains (100% similarity), cluster 3 comprised two B. thuringiensis strains (90% similarity), and cluster 4 comprised one B. thuringiensis strain and one B. cereus strain (85% similarity). The cluster analysis of fingerprints generated by Rep-PCR revealed a high genetic diversity among the B. cereus strains, suggesting that the contamination could have originated from different sources. In our experiments, when sugar was added and the beverage was kept in thermic bottles there was a significant increase in B. cereus sensu lato levels, which may increase the risk of food poisoning. These results highlight the need for additional studies on this subject to better evaluate coffee as a food poisoning vehicle.

Biogenesis of antibacterial silver nanoparticles using the endophytic bacterium Bacillus cereus isolated from Garcinia xanthochymus.

OBJECTIVE: To synthesize the ecofriendly nanoparticles, which is viewed as an alternative to the chemical method which initiated the use of microbes like bacteria and fungi in their synthesis. METHODS: The current study uses the endophytic bacterium Bacillus cereus isolated from the Garcinia xanthochymus to synthesize the silver nanoparticles (AgNPs). The AgNPs were synthesized by reduction of silver nitrate solution by the endophytic bacterium after incubation for 3-5 d at room temperature. The synthesis was initially observed by colour change from pale white to brown which was confirmed by UV-Vis spectroscopy. The AgNPs were further characterized using FTIR, SEM-EDX and TEM analyses. RESULTS: The synthesized nanoparticles were found to be spherical with the size in the range of 20-40 nm which showed a slight aggregation. The energy-dispersive spectra of the nanoparticle dispersion confirmed the presence of elemental silver. The AgNPs were found to have antibacterial activity against a few pathogenic bacteria like Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Klebsiella pneumoniae. CONCLUSIONS: The endophytic bacteria identified as Bacillus cereus was able to synthesize silver nanoparticles with potential antibacterial activity.

Gram-positive bacteria on grass pollen exhibit adjuvant activity inducing inflammatory T cell responses.

BACKGROUND: Recently, it has been established that pollen grains contain Th2-enhancing activities besides allergens. OBJECTIVE: The aim of this study was to analyse whether pollen carry additional adjuvant factors like microbes and what immunological effects they may exert. METHODS: Timothy pollen grains were collected and disseminated on agar plates, and the growing microorganisms were cultivated and defined. Furthermore, the immunologic effects of microbial products on DC and T cell responses were analysed. RESULTS: A complex mixture of bacteria and moulds was detected on grass pollen. Besides Gram-negative bacteria that are known to favour Th1-directed immune responses, moulds were identified as being sources of allergens themselves. Herein, we focused on Gram-positive bacteria that were found in high numbers, e.g. Bacillus cereus and Bacillus subtilis. Contact of immature dendritic cells (DC) from grass pollen allergic donors with supernatants of homogenized Gram-positive bacteria induced maturation of DC as measured by up-regulation of CD80, CD83 and CD86 and by enhanced production of IL-6, IL-12p40 and TNF-α, which was less pronounced compared with effects induced by lipopolysaccharide (LPS). Consequently, stimulation of autologous CD4(+) T cells with supernatants of homogenized Gram-positive bacteria plus grass pollen allergen-pulsed DC led to an enhanced proliferation and production of IL-4, IL-13, IL-10, IL-17, IL-22 and IFN-γ production compared with T cells that were stimulated with allergen-pulsed immature DC alone, whereas production of the transcription factor for regulatory T cells FoxP3 was not significantly affected. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that grass pollen is colonized by several microorganisms that influence the immune response differently. Similar to LPS, supernatants of homogenized Gram-positive bacteria may serve as adjuvants by augmenting DC maturation and inflammatory Th1, Th2 and Th17 responses helping to initiate allergic immune responses.

Antibacterial compounds from mushrooms II: lanostane triterpenoids and an ergostane steroid with activity against Bacillus cereus isolated from Fomitopsis pinicola.

Anti- Bacillus cereus bioassay-guided fractionation of a crude extract of the American mushroom, Fomitopsis pinicola, was performed using thin-layer chromatography, Sephadex LH-20 column chromatography, and preparative-scale HPLC. Five lanostane triterpenoids (1-5) and one ergostane steroid (6) were isolated and identified. Compound 1 is a new lanostane triterpenoid, and its structure was determined using 1D and 2D NMR experiments, HR-MS, and physical data. Each of the purified compounds (1-6) was tested for antibacterial activity against B. cereus using standard MIC assays. Compounds 1-6 had MIC values of 32, 16, 32, 32, 128, and 64 microg/mL, respectively.

Identification of Bacillus cereus in a chungkukjang that showed high anticancer effects against AGS human gastric adenocarcinoma cells.

Anticancer effects of chungkukjang (a Korean short-term fermented soy paste) were studied in human gastric adenocarcinoma cells, and Bacillus strains from chungkukjang were isolated and identified. Before the experiments, six different chungkukjang products (K-, M-, Mn-, O-, Os-, and H-chungkukjangs) were purchased from a folk village in the Sunchang region, Jeonbuk, Republic of Korea. Based on sensory evaluation tests and general chemical and quality studies, K-, H-, and M-chungkukjangs were selected for the experiments. All chungkukjang samples exhibited in vitro anticancer activities; however, K-chungkukjang revealed the highest anticancer activity in the previous studies. In this experiment, K-chungkukjang again showed the highest anticancer effect in the AGS cells. At the concentration of 1 mg/mL, K-chungkukjang (87%) showed the highest growth inhibitory effect, followed by H-chungkukjang (85%) and MC-chungkukjang (69%) (P < .05). K-chungkukjang induced apoptosis as determined by 4,6-diamidino-2-phenylindole staining and exhibited increased bax and decreased bcl-2 mRNA expression. Three representative Bacillus strains from K-chungkukjang were isolated and identified by recA gene sequencing as Bacillus cereus, Bacillus amyloliquefaciens, and Bacillus subtilis. Identifying B. cereus in the chungkukjang means that when chungkukjang is prepared by the traditional method, B. cereus, which is a common cause of foodborne disease, can grow during the natural fermentation process. All B. cereus strains, of course, are not pathogens, but its presence causes food safety concerns. Therefore, using a starter culture is safer than the traditional natural fermentation for the industrialization of chungkukjang in Korea.

[Effect of phytosubstances with antioxidant activity on persistence properties of microorganisms].

The in vitro effect of phytosubstances containing flavonoids and phenylpropanoids on the antilysozyme activity of varioous groups of microorganisms, such as staphylococci, bacilli, enterobacteria and nonfermenting bacteria was studied. The second fraction of the pink carbonic acid extract showed high antioxidant activity and most efficiently inhibited the ability of various microorganisms to inactivate the host lysozyme. The selected phytosubstance could be useful in clinical practice as an additional antimicrobial agent in etiotropic therapy or prophylaxis of some infections.

Fulminant septicemia of Bacillus cereus resistant to carbapenem in a patient with biphenotypic acute leukemia.

We report a case of fulminant septicemia with Bacillus cereus resistant to carbapenem. A 33-year-old man was suffering from febrile neutropenia (FN) on day 15 after the start of remission-induction therapy for biphenotypic acute leukemia under gut decontamination with polymyxin B and nystatin. Meropenem, a carbapenem, was administered according to the guideline for FN. Two days later (on day 17), he complained of severe abdominal pain, lost consciousness, went into sudden cardiopulmonary arrest, and died. Autopsy showed multiple spots of hemorrhage and necrosis caused by bacterial plaque in the brain, lungs, and liver. B. cereus was isolated from a blood sample obtained in the morning on day 17 and it was after his death that the isolated B. cereus was revealed to be resistant to carbapenem. B. cereus obtained from blood samples has been reported to be usually sensitive to carbapenem and also to vancomycin, new quinolones, and clindamycin. If B. cereus resistant to carbapem increases, our method of gut decontamination with polymyxin B and nystatin may have to be changed to one containing a new quinolone for the prevention of septicemia. Careful watching to determine whether B. cereus resistant to carbapem increases may be also important for empiric therapy, because carbapenem is often selected as the initial therapy for FN in patients with severe neutropenia.

Isolation and characterization of ZH14 with antiviral activity against Tobacco mosaic virus.

A large number of bacteria were isolated from plant samples and screened for antiviral activity against the Tobacco mosaic virus (TMV). The bacterium ZH14, which was isolated from Chinese Anxi oolong tea, secreted the antiviral substances, having 94.2% virus inhibition when the bacterial culture filtrate and TMV extract were mixed at a ratio of 1:1. The ZH14 strain is a gram-positive, spore-forming rod and has the ability to degrade ribonucleic acid. Based on its effectiveness on virus inhibition, ZH14 was selected for characterization and was identified as a strain of the Bacillus cereus group based on phenotypic tests and comparative analysis of its 16S rDNA sequence. At the same time, we determined the antiviral product of ZH14 as an extracellular protein with high molecular mass, having an optimum temperature of 15-60 degrees C and an optimum pH of 6-10. Hence, the ZH14 strain and its culture filtrate have potential application in controlling plant diseases caused by TMV.

Identification of a non-pathogenic surrogate organism for chlorine dioxide (ClO2) gas treatment.

The identification of non-pathogenic surrogate microorganisms is beneficial for determining and validating the efficacy of antimicrobial treatments in food manufacturing environments. A surrogate organism was identified to aid in the decontamination process of fresh produce when treated with chlorine dioxide (ClO(2)) gas. Thirty-two known strains of pathogenic and non-pathogenic microorganisms and seven unknown microbial isolates from mushroom, tomatoes, and strawberries were evaluated. The primary goal was to find alternative non-pathogenic organisms that had an equal or higher resistance compared to Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes. Among the strains tested, MR1 (mushroom isolate), E. coli O157:H7 C7927, E. coli O157:H7 204P, STB2 (strawberry isolate), and vegetative cells of Bacillus cereus 232 in wet inoculum were found to be the most resistant to gaseous ClO(2) treatment at 0.3 mg/l for 1 min and D-values at 0.3 mg/l ClO(2) were 3.53, 1.95, 1.72, 1.68, and 1.57 min, respectively. For identification, the MR1 and STB2 strains were identified using a Ribotyper with the EcoRI restriction enzyme of 16S rDNA sequence. MR1 was identified as Hafnia alvei with a similarity value of 94% using the ribotype pattern and with a 93.6% similarity using an API 20E strip, and with a 99% similarity using 16S rDNA analysis. The Ped-2E9-based cytotoxicity assay was conducted for the MRI strain extracellular toxin and whole cell toxicity and did not show cytotoxicity. Analysis, using multiplex PCR, was performed to verify absence of the eaeA gene. H. alvei is a suitable non-pathogenic surrogate, with higher resistance to ClO(2) gas compared to pathogens studied, that may be useful to establish optimum conditions of ClO(2) gas decontamination systems.