Alicyclobacillus spp. in fruit-based products: Isolation, identification, quantitative assessment (SPME/GC-MS) of spoilage compounds and spore's resistance to thermal shocks.

Alicyclobacillus spp. is the cause of great concern for the food industry due to their spores' resistance (thermal and chemical) and the spoilage potential of some species. Despite this, not all Alicyclobacillus strains can spoil fruit juices. Thus, this study aimed to identify Alicyclobacillus spp. strains isolated from fruit-based products produced in Argentina, Brazil, and Italy by DNA sequencing. All Alicyclobacillus isolates were tested for guaiacol production by the peroxidase method. Positive strains for guaiacol production were individually inoculated at concentration of 103 CFU/mL in 10 mL of orange (pH 3.90) and apple (pH 3.50) juices adjusted to 11°Brix, following incubation at 45 °C for at least 5 days to induce the production of the following spoilage compounds: Guaiacol, 2,6-dichlorophenol (2,6-DCP) and 2,6-dibromophenol (2,6-DBP). The techniques of micro-solid phase extraction by headspace (HS-SPME) and gas-chromatography with mass spectrometry (GC-MS) were used to identify and quantify the spoilage compounds. All GC-MS data was analyzed by principal component analysis (PCA). The effects of different thermal shock conditions on the recovery of Alicyclobacillus spores inoculated in orange and apple juice (11°Brix) were also tested. A total of 484 strains were isolated from 48 brands, and the species A. acidocaldarius and A. acidoterrestris were the most found among all samples analyzed. In some samples from Argentina, the species A. vulcanalis and A. mali were also identified. The incidence of these two main species of Alicyclobacillus in this study was mainly in products from pear (n = 108; 22.3 %), peach (n = 99; 20.5 %), apple (n = 86; 17.8 %), and tomato (n = 63; 13 %). The results indicated that from the total isolates from Argentina (n = 414), Brazil (n = 54) and Italy (n = 16) were able to produce guaiacol: 107 (25.8 %), 33 (61.1 %) and 13 (81.2 %) isolates from each country, respectively. The PCA score plot indicated that the Argentina and Brazil isolates correlate with higher production of guaiacol and 2,6-DCP/2,6-DBP, respectively. Heatmaps of cell survival after heat shock demonstrated that strains with different levels of guaiacol production present different resistances according to spoilage ability. None of the Alicyclobacillus isolates survived heat shocks at 120 °C for 3 min. This work provides insights into the incidence, spoilage potential, and thermal shock resistance of Alicyclobacillus strains isolated from fruit-based products.

Evidence of defined temporal expression patterns that lead a gram-negative cell out of dormancy.

Many bacterial species are capable of forming long-lived dormant cells. The best characterized are heat and desiccation resistant spores produced by many Gram-positive species. Less characterized are dormant cysts produced by several Gram-negative species that are somewhat tolerant to increased temperature and very resistant to desiccation. While there is progress in understanding regulatory circuits that control spore germination, there is scarce information on how Gram-negative organisms emerges from dormancy. In this study, we show that R. centenum cysts germinate by emerging a pair of motile vegetative cells from a thick cyst cell wall coat ~ 6 hrs post induction of germination. Time-lapse transcriptomic analysis reveals that there is a defined temporal pattern of gene expression changes during R. centenum cyst germination. The first observable changes are increases in expression of genes for protein synthesis, an increase in expression of genes involved in the generation of a membrane potential and the use of this potential for ATP synthesis via ATPase expression. These early events are followed by expression changes that affect the cell wall and membrane composition, followed by expression changes that promote chromosome replication. Midway through germination, expression changes occur that promote the flow of carbon through the TCA cycle to generate reducing power and parallel synthesis of electron transfer components involved in oxidative phosphorylation. Finally, late expression changes promote the synthesis of a photosystem as well as flagellar and chemotaxis components for motility.

One year investigation of the prevalence and diversity of clostridial spores in raw milk from the Tokachi area of Hokkaido.

We investigated the seasonal prevalence and diversity of clostridial spores in raw milk from the Tokachi area of Hokkaido. Samples of raw milk were collected quarterly from May 2013 through February 2014. The mean clostridial spore count for the raw milk from 336 milk tankers was 27.6 CFU/100 ml. The clostridial species isolated most frequently from raw milk samples was Clostridium tyrobutyricum. The dominant species was C. tyrobutyricum regardless of the season. The percentage of samples with low spore counts (<10 CFU/100 ml) was highest (60.9%) during winter (February) and lowest (34.5%) in autumn (November). In comparison, the percentage of samples with high spore counts (>100 CFU/100 ml) was highest (5.7%) in autumn (November) and lowest (1.1%) during spring (May).

Crab-meat-isolated psychrophilic spore forming bacteria inactivation by electron beam ionizing radiation.

The present work was performed to evaluate the potential of electron beam ionizing radiation for the inactivation of three psychrophilic spore forming bacteria (Bacillus mycoides, Bacillus weihenstephanensis and Psychrobacillus psychrodurans) isolated from ready-to-eat brown crab (Cancer pagurus). Inactivation curves for the three spores were performed in both types of crab meat, brown and white. Also the effect of pH and water activity (aw) on the lethal efficacy of ionizing radiation, for the three different psychrophilic spore forming bacteria, was evaluated. The effects of pH, aw and their possible interactions were assessed in citrate-phosphate buffers of different pH, ranging between 7 and 4, and aw, ranging from <0.99 to 0.80. A reduction of aw increased the spores resistance between >0.99 and 0.90, while an aw reduction from 0.90 to 0.80 had a minor impact on their resistance. In contrast to aw, the effect of pH showed a greater variability depending on the spore species. While pH did not affect the resistance of B. weihenstephanensis at any aw, B. mycoides showed slightly higher resistance at pH 5.5 at aw of 0.90 and 0.80. pH showed a significant effect on the resistance of P. psychrodurans. For the two types of crab meat, slightly differences were observed in 6D values. B. weihenstephanensis was the most resistant, requiring 7.3-7.6 kGy to inactivate 6 Log10-cycles of this spore forming bacterium, while for B. mycoides and P. psychrodurans 6.1-6.3 and 5.4-5.3 kGy respectively were necessary to reach the same inactivation level in crab meat. An agreement between spore resistance in crab meats and lab media, with similar characteristics in pH and aw, was also observed. The results obtained in this research demonstrated the potential for ionizing radiation to achieve an appropriate inactivation level of spores naturally present in brown crab with the application of doses lower than 10 kGy.

Integration of Membrane Distillation with solar photo-Fenton for purification of water contaminated with Bacillus sp. and Clostridium sp. spores.

Although Membrane Distillation (MD) has been extensively studied for desalination, it has other applications like removing all kinds of solutes from water and concentrating non-volatile substances. MD offers the possibility of producing a clean stream while concentrating valuable compounds from waste streams towards their recovery, or emerging contaminants and pathogens present in wastewater in order to facilitate their chemical elimination. This paper analyses the elimination of bacterial spores from contaminated water with MD and the role of MD in the subsequent treatment of the concentrate with photo-Fenton process. The experiments were performed at Plataforma Solar de Almería (PSA) using a plate and frame bench module with a Permeate Gap Membrane Distillation (PGMD) configuration. Tests were done for two different kinds of spores in two different water matrixes: distilled water with 3.5wt% of sea salts contaminated with spores of Bacillus subtilis (B. subtilis) and wastewater after a secondary treatment and still contaminated with Clostridium sp. spores. An analysis of the permeate was performed in all cases to determine its purity, as well as the concentrated stream and its further treatment in order to assess the benefits of using MD. Results showed a permeate free of spores in all the cases, demonstrating the viability of MD to treat biological contaminated wastewater for further use in agriculture. Moreover, the results obtained after treating the concentrate with photo-Fenton showed a shorter treatment time for the reduction of the spore concentration in the water than that when only photo-Fenton was used.

Development of a filter to prevent infections with spore-forming bacteria in injecting drug users.

BACKGROUND: In heroin injectors, there have been a number of outbreaks caused by spore-forming bacteria, causing serious infections such as anthrax or botulism. These are, most likely, caused by injecting contaminated heroin, and our aim was to develop a filter that efficiently removes these bacteria and is also likely to be acceptable for use by people who inject drugs (i.e. quick, simple and not spoil the hit). METHODS: A prototype filter was designed and different filter membranes were tested to assess the volume of liquid retained, filtration time and efficiency of the filter at removing bacterial spores. Binding of active ingredients of heroin to different types of membrane filters was determined using a highly sensitive analytical chemistry technique. RESULTS: Heroin samples that were tested contained up to 580 bacteria per gramme, with the majority being Bacillus spp., which are spore-forming soil bacteria. To remove these bacteria, a prototype filter was designed to fit insulin-type syringes, which are commonly used by people who inject drugs (PWIDs). Efficient filtration of heroin samples was achieved by combining a prefilter to remove particles and a 0.22 µm filter to remove bacterial spores. The most suitable membrane was polyethersulfone (PES). This membrane had the shortest filtration time while efficiently removing bacterial spores. No or negligible amounts of active ingredients in heroin were retained by the PES membrane. CONCLUSIONS: This study successfully produced a prototype filter designed to filter bacterial spores from heroin samples. Scaled up production could produce an effective harm reduction tool, especially during outbreaks such as occurred in Europe in 2009/10 and 2012.

Luminescent lanthanide graphene for detection of bacterial spores and cysteine.

Here, we describe a new approach for preparation of luminescent lanthanide graphene in the presence of dipicolinic acid (DPA). Hg(2+) can competitively bind with DPA which greatly quenches the fluorescence and the resultant complex is able to selectively and sensitively detect cysteine with a detection limit of 5 nM.

Bioprocess of Kosa bioaerosols: effect of ultraviolet radiation on airborne bacteria within Kosa (Asian dust).

Kosa (Asian dust) is a well-known weather phenomenon in which aerosols are carried by the westerly winds from inland China to East Asia. Recently, the frequency of this phenomenon and the extent of damage caused have been increasing. The airborne bacteria within Kosa are called Kosa bioaerosols. Kosa bioaerosols have affected ecosystems, human health and agricultural productivity in downwind areas. In order to develop a new and useful bacterial source and to identify the source region of Kosa bioaerosols, sampling, isolation, identification, measurement of ultraviolet (UV) radiation tolerance and experimental simulation of UV radiation conditions were performed during Kosa bioaerosol transportation. We sampled these bioaerosols using a Cessna 404 airplane and a bioaerosol sampler at an altitude of approximately 2900 m over the Noto Peninsula on March 27, 2010. The bioaerosol particles were isolated and identified as Bacillus sp. BASZHR 1001. The results of the UV irradiation experiment showed that the UV radiation tolerance of Kosa bioaerosol bacteria was very high compared with that of a soil bacterium. Moreover, the UV radiation tolerance of Kosa bioaerosol spores was higher than that of soil bacterial spores. This suggested that Kosa bioaerosols are transported across the atmosphere as living spores. Similarly, by the experimental simulation of UV radiation conditions, the limited source region of this Kosa bioaerosol was found to be southern Russia and there was a possibility of transport from the Kosa source area.

Selective detection of 1000 B. anthracis spores within 15 minutes using a peptide functionalized SERS assay.

A surface-enhanced Raman spectroscopy (SERS) assay has been designed to detect Bacillus anthracis spores. The assay consists of silver nanoparticles embedded in a porous glass structure that have been functionalized with ATYPLPIR, a peptide developed to discriminately bind B. anthracis versus other species of Bacillus. Once bound, acetic acid was used to release the biomarker dipicolinic acid from the spores, which was detected by SERS through the addition of silver colloids. This SERS assay was used to selectively bind B. anthracis with a 100-fold selectivity versus B. cereus, and to detect B. anthracis Ames at concentrations of 1000 spores per mL within 15 minutes. The SERS assay measurements provide a basis for the development of systems that can detect spores collected from the air or from water supplies.

Evaluation of the relationship between the Adenosine Triphosphate (ATP) bioluminescence assay and the presence of Bacillus anthracis spores and vegetative cells.

BACKGROUND: The Adenosine triphosphate (ATP) bioluminescence assay was utilized in laboratory evaluations to determine the presence and concentration of vegetative and spore forms of Bacillus anthracis Sterne 34F2. METHODS: Seventeen surfaces from the healthcare environment were selected for evaluation. Surfaces were inoculated with 50 µL of organism suspensions at three concentrations of 104, 106, 108 colony forming units per surface (CFU/surface) of B. anthracis. Culture-based methods and ATP based methods were utilized to determine concentrations. RESULTS: When all concentrations were evaluated together, a positive correlation between log-adjusted CFU and Relative Light Units (RLU) for endospores and vegetative cells was established. When concentrations were evaluated separately, a significant correlation was not demonstrated. CONCLUSIONS: This study demonstrated a positive correlation for ATP and culture-based methods for the vegetative cells of B. anthracis. When evaluating the endospores and combining both metabolic states, the ATP measurements and CFU recovered did not correspond to the initial concentrations on the evaluated surfaces. The results of our study show that the low ATP signal which does not correlate well to the CFU results would not make the ATP measuring devises effective in confirming contamination residual from a bioterrorist event.

Uncertainty analysis of the recovery of hollow-fiber ultrafiltration for multiple microbe classes from water: a Bayesian approach.

In this study, we introduce a Bayesian approach to address uncertainty of microbial recoveries from hollow-fiber ultrafilters (HFUF) and to determine any sources of uncertainty. Microbial recoveries were measured under twenty conditions, including two types of water, two types of ultrafilters, and five types of microorganisms. The probability distributions of the recoveries were approximated using Bayesian statistics with Markov chain Monte Carlo sampling after integrating the likelihood function of the recovery data and prior information about the data. Then a variance-decomposition method was used for examining influential factors on microbial recovery by HFUF. The results revealed that HFUF efficiently recovered Escherichia coli KO11, E. coli O157:H7 and bacteriophage MS2, but recoveries for Bacillus atrophaeus spores and adenovirus 41 were markedly different between source and treated waters. The uncertainty analysis indicated that the probability distributions for recoveries had dissimilar patterns under different conditions. Among these test factors, the type of microorganisms and associated interaction effects had great impacts on the recovery. To sum up, the Bayesian approach to uncertainty analysis shows advantages in evaluating the recovery of HFUF by providing its full probability distribution.

Saliterribacillus persicus gen. nov., sp. nov., a moderately halophilic bacterium isolated from a hypersaline lake.

A novel Gram-positive, moderately halophilic bacterium, designated strain X4B(T), was isolated from soil around the hypersaline lake Aran-Bidgol in Iran and characterized taxonomically using a polyphasic approach. Cells of strain X4B(T) were motile rods and formed ellipsoidal endospores at a terminal or subterminal position in swollen sporangia. Strain X4B(T) was a strictly aerobic bacterium, catalase- and oxidase-positive. The strain was able to grow at NaCl concentrations of 0.5-22.5 % (w/v), with optimum growth occurring at 7.5 % (w/v) NaCl. The optimum temperature and pH for growth were 35 °C and pH 7.0. Analysis of 16S rRNA gene sequence revealed that strain X4B(T) is a member of the family Bacillaceae, constituting a novel phyletic lineage within this family. Highest sequence similarities were obtained with the 16S rRNA gene sequences of the type strains of Sediminibacillus albus (96.0 %), Paraliobacillus ryukyuensis (95.9 %), Paraliobacillus quinghaiensis (95.8 %) and Sediminibacillus halophilus (95.7 %), respectively. The DNA G+C content of this novel isolate was 35.2 mol%. The major cellular fatty acids of strain X4B(T) were anteiso-C(15 : 0) and anteiso-C(17 : 0) and its polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, two aminolipids, an aminophospholipid and an unknown phospholipid. The isoprenoid quinones were MK-7 (89 %) and MK-6 (11 %). The peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. On the basis of 16S rRNA gene sequence analysis in combination with chemotaxonomic and phenotypic data, strain X4B(T) represents a novel species in a new genus in the family Bacillaceae, order Bacillales for which the name Saliterribacillus persicus gen. nov., sp. nov. is proposed. The type strain of the type species (Saliterribacillus persicus) is X4B(T) ( = IBRC-M 10629(T) = KCTC 13827(T)).

Test method development to evaluate hot, humid air decontamination of materials contaminated with Bacillus anthracis ∆Sterne and B. thuringiensis Al Hakam spores.

AIMS: To develop test methods and evaluate the survival of Bacillus anthracis ∆Sterne and Bacillus thuringiensis Al Hakam spores after exposure to hot, humid air. METHODS AND RESULTS: Spores (>7 logs) of both strains were dried on six different test materials. Response surface methodology was employed to identify the limits of spore survival at optimal test combinations of temperature (60, 68, 77°C), relative humidity (60, 75, 90%) and time (1, 4, 7 days). No spores survived the harshest test run (77°C, 90% r.h., 7 days), while > 6·5 logs of spores survived the mildest test run (60°C, 60% r.h., 1 day). Spores of both strains inoculated on nylon webbing and polypropylene had greater survival rates at 68°C, 75% r.h., 4 days than spores on other materials. Electron microscopy showed no obvious physical damage to spores using hot, humid air, which contrasted with pH-adjusted bleach decontamination. CONCLUSIONS: Test methods were developed to show that hot, humid air effectively inactivates B. anthracis ∆Sterne and B. thuringiensis Al Hakam spores with similar kinetics. SIGNIFICANCE AND IMPACT OF THE STUDY: Hot, humid air is a potential alternative to conventional chemical decontamination.

Functional hybrid nickel nanostructures as recyclable SERS substrates: detection of explosives and biowarfare agents.

We present the synthesis of highly anisotropic nickel nanowires (NWs) and large area, free-standing carpets extending over cm(2) area by simple solution phase chemistry. The materials can be post-synthetically manipulated to produce hybrid tubes, wires, and carpets by galvanic exchange reactions with Au(3+), Ag(+), Pt(2+), and Pd(2+). All of these structures, especially the hybrid carpets and tubes, have been prepared in bulk and are surface enhanced Raman scattering (SERS) active substrates. Molecules of relevance such as dipicolinic acid (constituting 5-15% of the dry weight of bacterial spores of Bacillus anthracis), dinitrotoluene, hexahydro-1,3,5-triazine (RDX), and trinitrotoluene at nanomolar concentrations have been detected. An enhancement factor of ∼10(10) was observed for the Ni-Au nanocarpet. The reusability of the Ni-Au nanocarpet for SERS applications was tested 5 times without affecting the sensitivity. The reusability and sensitivity over large area have been demonstrated by Raman microscopy. Our method provides an easy and cost effective way to produce recyclable, large area, SERS active substrates with high sensitivity and reproducibility which can overcome the limitation of one-time use of traditional SERS substrates.

Hospital environment and invasive aspergillosis in patients with hematologic malignancy.

BACKGROUND: To determine whether there is a correlation between sources of Aspergillus spores in a high-efficiency particulate air (HEPA)-filtered environment and nosocomial invasive aspergillosis (IA), we performed a detailed environmental assessment and case review. METHODS: From April to October 2004, 626 bioaerosol samples, 1,257 surface samples, and 607 water samples were obtained from 74 HEPA-filtered air hospital rooms occupied by 458 patients with hematologic malignancies. Samples were collected prospectively from the room before and after cleaning within 1 hour of patient admission or discharge. Aspergillus spp was isolated from 21 surface samples and 46 bioaerosol samples. Interestingly, Aspergillus spp was not isolated from any water samples. RESULTS: Aspergillus spp was isolated from 21 surface samples and 46 bioaerosol samples. Interestingly, Aspergillus spp were not isolated from any water samples. The majority (90%) of the positive bioaerosol samples had ≤ 10 colony-forming units of Aspergillus/m3 of air. Only 2 patients developed nosocomial IA. No correlations were found between Aspergillus species isolated from the hospital rooms and those causing IA. CONCLUSION: The risk of hematologic malignancy patients acquiring nosocomial aspergillosis from water or HEPA-filtered air is very low.

Specific peptides as alternative to antibody ligands for biomagnetic separation of Clostridium tyrobutyricum spores.

Nowadays, the reference method for the detection of Clostridium tyrobutyricum in milk is the most-probable-number method, a very time-consuming and non-specific method. In this work, the suitability of the use of superparamagnetic beads coated with specific antibodies and peptides for bioseparation and concentration of spores of C. tyrobutyricum has been assessed. Peptide or antibody functionalized nanoparticles were able to specifically bind C. tyrobutyricum spores and concentrate them up to detectable levels. Moreover, several factors, such as particle size (200 nm and 1 µm), particle derivatization (aminated and carboxylated beads), coating method, and type of ligand have been studied in order to establish the most appropriate conditions for spore separation. Results show that concentration of spore is favored by a smaller bead size due to the wider surface of interaction in relation to particle volume. Antibody orientation, related to the binding method, is also critical in spore recovery. However, specific peptides seem to be a better ligand than antibodies, not only due to the higher recovery ratio of spores obtained but also due to the prolonged stability over time, allowing an optimal recovery of spores up to 3 weeks after bead coating. These results demonstrate that specific peptides bound to magnetic nanoparticles can be used instead of traditional antibodies to specifically bind C. tyrobutyricum spores being a potential basis for a rapid method to detect this bacterial target.

Use of purified Clostridium difficile spores to facilitate evaluation of health care disinfection regimens.

Clostridium difficile is a major cause of antibiotic-associated diarrheal disease in many parts of the world. In recent years, distinct genetic variants of C. difficile that cause severe disease and persist within health care settings have emerged. Highly resistant and infectious C. difficile spores are proposed to be the main vectors of environmental persistence and host transmission, so methods to accurately monitor spores and their inactivation are urgently needed. Here we describe simple quantitative methods, based on purified C. difficile spores and a murine transmission model, for evaluating health care disinfection regimens. We demonstrate that disinfectants that contain strong oxidizing active ingredients, such as hydrogen peroxide, are very effective in inactivating pure spores and blocking spore-mediated transmission. Complete inactivation of 106 pure C. difficile spores on indicator strips, a six-log reduction, and a standard measure of stringent disinfection regimens require at least 5 min of exposure to hydrogen peroxide vapor (HPV; 400 ppm). In contrast, a 1-min treatment with HPV was required to disinfect an environment that was heavily contaminated with C. difficile spores (17 to 29 spores/cm²) and block host transmission. Thus, pure C. difficile spores facilitate practical methods for evaluating the efficacy of C. difficile spore disinfection regimens and bringing scientific acumen to C. difficile infection control.

Conducting polymer nanowire-based chemiresistive biosensor for the detection of bacterial spores.

Polypyrrole nanowires (Ppy) were assembled onto microfabricated gold interdigitated microelectrodes, to construct a chemiresistive biosensor for the detection of Bacillus globigii, used as simulant of the threatening bioterrorism agent B. anthracis. The fabricated biosensor showed good linear correlation (r(2)=0.992) for low spore concentrations ranging from 1 to 100 CFU (colony forming units)/mL, a concentration that could be used in a bioterrorism attack, with a response time of 30 min, after which the sensor was saturated. The performance of the biosensor was also assessed in the absence of anti-B. globigii antibodies and in the presence of non-target bacterial cells (Escherichia coli) showing no significant non-specific interactions. We believe that Ppy nanowires are a good platform for the detection and also quantification of large molecules and biocomponents even at low concentrations.