Eriochrome Black T-Eu3+ Complex as a Ratiometric Colorimetric and Fluorescent Probe for the Detection of Dipicolinic Acid, a Biomarker of Bacterial Spores.

A novel ratiometric colorimetric and fluorescent dual probe based on Eriochrome Black T (EBT)-Eu3+ complex was designed to detect dipicolinic acid (DPA), a major constituent of bacterial spores, with high sensitivity and selectivity. UV-vis titration experiments demonstrated that EBT and Eu3+ ions formed a 1:1 coordination pair in water. In the presence of Eu3+ ions, the blue solution of EBT changed to magenta, however, upon the addition of DPA, the magenta color changed to blue immediately and characteristic fluorescence emission from DPA-Eu3+ complex was observed. In addition, the sensitivity of the system was further evaluated on Geobacillus stearothermophilus spores and as low as 2.5 × 105 spores were detected.

Insights into the Geobacillus stearothermophilus species based on phylogenomic principles.

BACKGROUND: The genus Geobacillus comprises bacteria that are Gram positive, thermophilic spore-formers, which are found in a variety of environments from hot-springs, cool soils, to food manufacturing plants, including dairy manufacturing plants. Despite considerable interest in the use of Geobacillus spp. for biotechnological applications, the taxonomy of this genus is unclear, in part because of differences in DNA-DNA hybridization (DDH) similarity values between studies. In addition, it is also difficult to use phenotypic characteristics to define a bacterial species. For example, G. stearothermophilus was traditionally defined as a species that does not utilise lactose, but the ability of dairy strains of G. stearothermophilus to use lactose has now been well established. RESULTS: This study compared the genome sequences of 63 Geobacillus isolates and showed that based on two different genomic approaches (core genome comparisons and average nucleotide identity) the Geobacillus genus could be divided into sixteen taxa for those Geobacillus strains that have genome sequences available thus far. In addition, using Geobacillus stearothermophilus as an example, we show that inclusion of the accessory genome, as well as phenotypic characteristics, is not suitable for defining this species. For example, this is the first study to provide evidence of dairy adaptation in G. stearothermophilus - a phenotypic feature not typically considered standard in this species - by identifying the presence of a putative lac operon in four dairy strains. CONCLUSIONS: The traditional polyphasic approach of combining both genotypic and phenotypic characteristics to define a bacterial species could not be used for G. stearothermophilus where many phenotypic characteristics vary within this taxon. Further evidence of this discordant use of phenotypic traits was provided by analysis of the accessory genome, where the dairy strains contained a putative lac operon. Based on the findings from this study, we recommend that novel bacterial species should be defined using a core genome approach.

Genotypic and phenotypic characterization of foodborne Geobacillus stearothermophilus.

Geobacillus stearothermophilus is the main thermophilic spore former involved in flat sour spoilage of canned foods. Three typing methods were tested and applied to differentiate strains at intra-species level: panC sequence analysis, REP-PCR and M13-PCR. panC gene was highly conserved within the studied strains, suggesting a low intra-specific diversity. This was supported by REP-PCR primary assays and M13-PCR results. M13-PCR profile analysis succeeded in differentiating six closely related groups (at 79% threshold similarity) among 127 strains from a range of spoiled canned food products and from different canneries. Phenotypic traits were investigated among 20 selected strains representing groups and origins. Ranges of growth under different temperatures (from 40 °C to 70 °C), pH (from 5.0 to 6.5), NaCl concentrations (from 1 to 5%) and sporulation conditions poorly differed between strains, but wet heat resistance of spores showed a 20-fold variation between strains. Furthermore, in this study, strains that belonged to the same M13-PCR genetic group did not share phenotypic characteristics or common origin. The work emphasizes a low diversity within the G. stearothermophilus species but data from this study may contribute to a better control of G. stearothermophilus spoilage in canned food.

Characterization of thermophilic bacilli from a milk powder processing plant.

AIMS: To determine whether strains of Geobacillus stearothermophilus isolated from a milk powder manufacturing plant were different in their ability to form biofilms and produce spores. In addition, this study evaluated whether there were other physiological characteristics that could differentiate these strains. METHODS AND RESULTS: Ten G. stearothermophilus strains and one Anoxybacillus species were isolated from a milk powder manufacturing plant. A microtitre plate assay was used to show that these strains differed in their abilities to form biofilms and produce spores. Scanning electron microscopy showed differences in the biofilm morphologies of three of the G. stearothermophilus strains. Biochemical profiling, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and fatty acid profiling further showed that they had distinct characteristics. CONCLUSIONS: These G. stearothermophilus strains, isolated from the same environment, showed differences in their ability to form biofilms and produce endospores. Based on the multiple characterization methods used in this study, these strains of G. stearothermophilus isolated from one manufacturing plant are diverse. SIGNIFICANCE AND IMPACT OF THE STUDY: Differences in the ability of G. stearothermophilus to form biofilms and produce spores may influence the cleaning method used to control the growth of thermophilic bacilli in a dairy processing environment.

Identification of a microscopically selected microorganism in milk samples.

Identification of unwanted microbial contaminants microscopically observed in food products is challenging due to their low abundance in a complex matrix, quite often containing other microorganisms. Therefore, a selective identification method was developed using laser capture microdissection in combination with direct-captured cell PCR. This procedure was validated with Geobacillus stearothermophilus and further used to identify microbial contaminants present in some industrial milk samples. The microscopically observed contaminants were identified as mainly Methylobacterium species.

Transformation of chenodeoxycholic acid by thermophilic Geobacillus stearothermophilus.

We performed a series of experiments with Geobacillus stearothermophilus, a thermophile isolated from oil-contaminated soil in the Kuwaiti desert. The organism has a good potential for the transformation of a broad spectrum of organic molecules such as steroids, amino acids, and aromatic hydrocarbons. In the present study, we tested its potential for the transformation of a bile component, chenodeoxycholic acid (CDCA). Five transformed products, namely, cholic acid, methylcholate, methylchenodeoxycholate, 3α-hydroxy-7-oxo-5ß-cholanic acid, and 7α-hydroxy-3-oxo-5ß-cholanic acid, were the major transformation products catalyzed by G. stearothermophilus. Under aerobic conditions, no evidence of side chain degradation, ring cleavage, or dehydrogenation was found among the metabolites of CDCA. CDCA transformation by a thermophile is reported for the first time.

Optimization, purification, and biochemical characterization of thermoalkaliphilic lipase from a novel Geobacillus stearothermophilus FMR12 for detergent formulations.

This study was aimed to produce a high compatible thermoalkaliphilic lipase (TA) with detergents from new thermophilic bacterial strains utilizing fish wastes for industrial application. Among bacterial isolates, a new Geobacillus stearothermophilus FMR12 efficiently utilized fish wastes at a concentration of 20% (w/v), exhibiting highly lipolytic activity at extreme thermal and alkaline pH conditions. Optimized fermentation parameters of TA lipase production were ascertained, promoting the productivity of the TA lipase from 424 to 1038 U/ml. Purification results of TA lipase exposed prominent specific activity of 4788 U/mg, purification fold of 12.44, and 7.8% yield. The purified TA lipase demonstrated outstanding activity and stability in a temperature range of 40-95 °C and pH (4-11), revealing optimal activity at 70 °C and pH 9. The molecular weight of the enzyme was estimated to be 63 kDa. Compared to control, the TA lipase activity was promoted in the presence of calcium chloride. Likewise, Triton X-100 enhanced the activity of the TA lipase, recording 128% of the control enzyme. Interestingly, the TA lipase conserved higher than 90% of its activity after blending with commercial detergents, emphasizing its competence for detergent formulations.

Identification of a New Heavy-Metal-Resistant Strain of Geobacillus stearothermophilus Isolated from a Hydrothermally Active Volcanic Area in Southern Italy.

Microorganisms thriving in hot springs and hydrothermally active volcanic areas are dynamically involved in heavy-metal biogeochemical cycles; they have developed peculiar resistance systems to cope with such metals which nowadays can be considered among the most permanent and toxic pollutants for humans and the environment. For this reason, their exploitation is functional to unravel mechanisms of toxic-metal detoxification and to address bioremediation of heavy-metal pollution with eco-sustainable approaches. In this work, we isolated a novel strain of the thermophilic bacterium Geobacillus stearothermophilus from the solfataric mud pool in Pisciarelli, a well-known hydrothermally active zone of the Campi Flegrei volcano located near Naples in Italy, and characterized it by ribotyping, 16S rRNA sequencing and mass spectrometry analyses. The minimal inhibitory concentration (MIC) toward several heavy-metal ions indicated that the novel G. stearothermophilus isolate is particularly resistant to some of them. Functional and morphological analyses suggest that it is endowed with metal resistance systems for arsenic and cadmium detoxification.

Heat resistance of spores of 18 strains of Geobacillus stearothermophilus and impact of culturing conditions.

In this study, different methods were evaluated for enumeration of spores of G. stearothermophilus, different sporulation methods were assessed for yields and wet heat resistances of obtained spores, and subsequently, the variation in heat resistances of spores was determined. Overall, tryptone soya agar (TSA) was the most suitable medium for enumeration of spores of this thermophilic bacterium. Sporulation on different media both at 55 and at 61 °C led to considerable variation in spore heat resistance. The heat resistance of spores was highest upon sporulation on medium supplemented with free ions of calcium, potassium, magnesium and manganese (CaKMgMn). For 18 different G. stearothermophilus strains that were isolated from various sources, spores were subsequently produced on nutrient agar supplemented with CaKMgMn at 55 °C. Strain ATCC 12980T, also known as 9A20, which is commonly used in steam sterilization tests was included. The survival of spores of all strains was assessed at 125 °C and 130 °C using two independent spore batches per strain. The mean D125°C for spores of the 18 strains was 1.1 min (95% PI 0.48-2.3 min) and the mean D130°C was 0.37 min (95% PI 0.17-0.82 min). For spore inactivation of these 18 strains, a z-value of 11.1 °C was estimated, resulting in an estimated D-value of 2.4 min (95% PI 1.1-5.2) at the reference temperature 121.1 °C. Based on the data sets obtained in this study, it was found that the variability in spore heat resistance could largely be attributed to strain variability and conditions used during sporulation (especially the sporulation medium); reproduction and experimental variabilities were much smaller. The established variabilities were compared with the overall variability in spore heat resistance of G. stearothermophilus based on a meta-analysis of reported D-values. The data presented indicate that strain variability and history of sporulation each account for approximately half of the overall variability observed with respect to the heat resistance of spores of G. stearothermophilus. The findings presented in this study allow for optimal recovery of G. stearothermophilus spores from foods and a better understanding of factors that determine the heat resistance properties of spores of G. stearothermophilus. Moreover, this study once more underlines the limited effects of heat treatments used in the food industry on inactivation of spores of this bacterium.

Convergence of Bigelow and Arrhenius models over a wide range of heating temperatures.

The heat resistance of the bacterial spores of Moorella thermoacetica, Clostridium sporogenes, Geobacillus stearothermophilus and Bacillus coagulans was determined over a wide range of temperatures using the capillary method and thermoresistometer Mastia. The results showed that the two experimental methods gave similar heat resistance values excepted for Geobacillus stearothermophilus. The effect of temperature on thermal resistance was evaluated using the Arrhenius and Bigelow models. The fit of the heat sensitivity parameters of the Arrhenius and Bigelow models on the heat resistance parameter values obtained over a wide temperature range was equally good. Despite the apparent mathematical incompatibility of the two equations, it is recognized that they yield the same goodness of fit. This paper finds a mathematical reason for this convergence and explains why inside a temperature range of at least 100 °C, no significant difference in the quality of fit between these two models can be found.

Elevating the standard of endoscope processing: Terminal sterilization of duodenoscopes using a hydrogen peroxide-ozone sterilizer.

BACKGROUND: The health care community is increasingly aware of the processing challenges and infection risks associated with duodenoscopes owing to published reports of outbreaks and regulatory recalls. Studies have demonstrated that the current practices are inadequate for consistently producing patient-ready endoscopes. Alternatively, terminal sterilization would offer a greater margin of safety and potentially reduce the risk of patient infection. The purpose of this study was to evaluate the efficacy of a hydrogen peroxide-ozone sterilizer with regulatory clearance for terminal sterilization of duodenoscopes. METHODS AND RESULTS: Validation studies were performed under laboratory simulated-use and clinical in-use conditions. The overkill method study demonstrated a reduction of at least 6-log of Geobacillus stearothermophilus spores at half-cycle, providing a sterility assurance level of 10-6. In addition, the sterilizer achieved a 6-log reduction of G stearothermophilus in the presence of inorganic and organic soils in a simulated-use study. The clinical in-use study confirmed that the sterilizer achieved sterilization of patient-soiled duodenoscopes under actual use conditions. CONCLUSIONS: Simulated-use and clinical in-use studies demonstrated the efficacy of a hydrogen peroxide-ozone sterilizer for terminal sterilization of duodenoscopes. This offers health care facilities a viable alternative for duodenoscope processing to enhance patient safety as part of a comprehensive infection control strategy.

Scrubbing technique for needleless connectors to minimize contamination risk.

This study aimed to investigate the appropriate scrubbing technique for needleless connectors to minimize contamination risk. To demonstrate a highly effective scrubbing technique to physically eliminate bacteria, needleless connectors were contaminated with Geobacillus stearothermophilus spores and then scrubbed. The study showed that the highest bacterial elimination rate was achieved by scrubbing an access port in a straight line with an alcohol cotton swab, applying a force that was almost equal to an arterial compression haemostasis to the access port, and repeating this procedure once using a new alcohol cotton swab.

Purification, crystallization and preliminary X-ray analysis of the BseCI DNA methyltransferase from Bacillus stearothermophilus in complex with its cognate DNA.

The DNA methyltransferase M.BseCI from Bacillus stearothermophilus (EC 2.1.1.72), a 579-amino-acid enzyme, methylates the N6 atom of the 3' adenine in the sequence 5'-ATCGAT-3'. M.BseCI was crystallized in complex with its cognate DNA. The crystals were found to belong to the hexagonal space group P6, with unit-cell parameters a = b = 87.0, c = 156.1 A, beta = 120.0 degrees and one molecule in the asymmetric unit. Two complete data sets were collected at wavelengths of 1.1 and 2.0 A to 2.5 and 2.8 A resolution, respectively, using synchrotron radiation at 100 K.

Propensity for biofilm formation by aerobic mesophilic and thermophilic spore forming bacteria isolated from Chinese milk powders.

Biofilms on the surface of dairy manufacturing plants are potential reservoirs of microbial contamination. These microbial aggregates may harbour pathogenic and spoilage organisms which contaminate dairy products. The biofilm forming capacity of many spore forming isolates of dairy origin has not been given much attention. The present study explored the biofilm forming potential of 148 isolates, comprising mesophilic and thermophilic bacteria, with particular emphasis on Bacillus licheniformis on polystyrene and stainless steel (SS) surfaces. We concluded that only four species are of significance for biofilm development on the surface of SS in the presence of skimmed milk, namely, B. licheniformis, Geobacillus stearothermophilus, Geobacillus thermoleovorans group and Anoxybacillus flavithermus. The maximum number of cells recovered from the biofilms developed on SS coupons in the presence of skimmed milk for these four species was as follows: 4.8, 5.2, 4.5 and 5.3logCFU/cm2, respectively. Number of cells recovered from biofilms on 1cm2 SS coupons increased in the presence of tryptic soy broth (TSB) for all mesophiles including B. licheniformis, while decreased for G. stearothermophilus, G. thermoleovorans group and A. flavithermus. The crystal violet staining assay on polystyrene proved to be inadequate to predict cell counts on SS for the bacteria tested in our trial in the presence of either TSB or skimmed milk. The results support the idea that biofilm formation is an important part of bacterial survival strategy as only the most prevalent isolates from milk powders formed good biofilms on SS in the presence of skimmed milk. Biofilm formation also proved to be a strain-dependent characteristic and interestingly significant variation in biofilm formation was observed within the same RAPD groups of B. licheniformis which supports the previously reported genetic and phenotypic heterogeneity within the same RAPD based groups. The work reported in this manuscript will broaden our knowledge on biofilm formation of a large number of dairy isolates and emphasize strain and substrate dependence.

Toward a Philosophy and Theory of Volumetric Nonthermal Processing.

Nonthermal processes for food preservation have been under intensive investigation for about the past quarter century, with varying degrees of success. We focus this discussion on two volumetrically acting nonthermal processes, high pressure processing (HPP) and pulsed electric fields (PEF), with emphasis on scientific understanding of each, and the research questions that need to be addressed for each to be more successful in the future. We discuss the character or "philosophy" of food preservation, with a question about the nature of the kill step(s), and the sensing challenges that need to be addressed. For HPP, key questions and needs center around whether its nonthermal effectiveness can be increased by increased pressures or pulsing, the theoretical treatment of rates of reaction as influenced by pressure, the assumption of uniform pressure distribution, and the need for (and difficulties involved in) in-situ measurement. For PEF, the questions include the rationale for pulsing, difficulties involved in continuous flow treatment chambers, the difference between electroporation theory and experimental observations, and the difficulties involved in in-situ measurement and monitoring of electric field distribution.

Effectiveness of the SYSTEM 1E Liquid Chemical Sterilant Processing System for reprocessing duodenoscopes.

BACKGROUND: A troubling number of health care-acquired infection outbreaks and transmission events, some involving highly resistant microbial pathogens and resulting in serious patient outcomes, have been traced to reusable, high-level disinfected duodenoscopes in the United States. The Food and Drug Administration (FDA) requested a study be conducted to verify liquid chemical sterilization efficacy of SYSTEM 1E(®) Liquid Chemical Sterilant Processing System (STERIS Corporation, Mentor, OH) with varied duodenoscope designs under especially arduous conditions. Here, we describe the system's performance under worst case SYSTEM 1E(®) processing conditions. METHODS: The test protocol challenged the system's performance by running a fractional cycle to evaluate reduction of recoverable test spores from heavily contaminated endoscopes, including all channels and each distal tip, under worst case SYSTEM 1E(®) processing conditions. RESULTS: All devices were successfully liquid chemically sterilized, showing greater than a 6 log10 reduction of Geobacillus stearothermophilus spores at every inoculation site of each duodenoscope tested, in less than half the exposure time of the standard cycle. CONCLUSIONS: The successful outcome of the additional efficacy testing reported here indicates that the SYSTEM 1E(®) is an effective low-temperature liquid chemical sterilization method for duodenoscopes and other critical and semicritical devices. It offers a fast, safe, convenient processing alternative while providing the assurance of a system expressly tested and cleared to achieve liquid chemical sterilization of specific validated duodenoscope models.

A RAPD based study revealing a previously unreported wide range of mesophilic and thermophilic spore formers associated with milk powders in China.

Aerobic spore forming bacteria are potential milk powder contaminants and are viewed as indicators of poor quality. A total of 738 bacteria, including both mesophilic and thermophilic, isolated from twenty-five powdered milk samples representative of three types of milk powders in China were analyzed based on the random amplified polymorphic DNA (RAPD) protocol to provide insight into species diversity. Bacillus licheniformis was found to be the most prevalent bacterium with greatest diversity (~43% of the total isolates) followed by Geobacillus stearothermophilus (~21% of the total isolates). Anoxybacillus flavithermus represented only 8.5% of the total profiles. Interestingly, actinomycetes represented a major group of the isolates with the predominance of Laceyella sacchari followed by Thermoactinomyces vulgaris, altogether comprising of 7.3% of the total isolates. Out of the nineteen separate bacterial species (except five unidentified groups) recovered and identified from milk powders, twelve proved to belong to novel or previously unreported species in milk powders. Assessment and characterization of the harmful effects caused by this particular micro-flora on the quality and safety of milk powders will be worth doing in the future.

Development of a Quantitative PCR Assay for Thermophilic Spore-Forming Geobacillus stearothermophilus in Canned Food.

The thermophilic spore forming bacteria Geobacillus stearothermophilus is recognized as a major cause of spoilage in canned food. A quantitative real-time PCR assay was developed to specifically detect and quantify the species G. stearothermophilus in samples from canned food. The selected primer pairs amplified a 163-bp fragment of the 16S rRNA gene in a specific PCR assay with a detection limit of 12.5 fg of pure culture DNA, corresponding to DNA extracted from approximately 0.7 CFU/mL of G. stearothermophilus. Analysis showed that the bacterial species G. stearothermophilus was not detected in any canned food sample. Our approach presented here will be useful for tracking or quantifying species G. stearotethermophilus in canned food and ingredients.

Contamination pathways of spore-forming bacteria in a vegetable cannery.

Spoilage of low-acid canned food during prolonged storage at high temperatures is caused by heat resistant thermophilic spores of strict or facultative bacteria. Here, we performed a bacterial survey over two consecutive years on the processing line of a French company manufacturing canned mixed green peas and carrots. In total, 341 samples were collected, including raw vegetables, green peas and carrots at different steps of processing, cover brine, and process environment samples. Thermophilic and highly-heat-resistant thermophilic spores growing anaerobically were counted. During vegetable preparation, anaerobic spore counts were significantly decreased, and tended to remain unchanged further downstream in the process. Large variation of spore levels in products immediately before the sterilization process could be explained by occasionally high spore levels on surfaces and in debris of vegetable combined with long residence times in conditions suitable for growth and sporulation. Vegetable processing was also associated with an increase in the prevalence of highly-heat-resistant species, probably due to cross-contamination of peas via blanching water. Geobacillus stearothermophilus M13-PCR genotypic profiling on 112 isolates determined 23 profile-types and confirmed process-driven cross-contamination. Taken together, these findings clarify the scheme of contamination pathway by thermophilic spore-forming bacteria in a vegetable cannery.

Mitochondrial DNA Fragmentation as a Molecular Tool to Monitor Thermal Processing of Plant-Derived, Low-Acid Foods, and Biomaterials.

Cycle threshold (Ct) increase, quantifying plant-derived DNA fragmentation, was evaluated for its utility as a time-temperature integrator. This novel approach to monitoring thermal processing of fresh, plant-based foods represents a paradigm shift. Instead of using quantitative polymerase chain reaction (qPCR) to detect pathogens, identify adulterants, or authenticate ingredients, this rapid technique was used to quantify the fragmentation of an intrinsic plant mitochondrial DNA (mtDNA) gene over time-temperature treatments. Universal primers were developed which amplified a mitochondrial gene common to plants (atp1). These consensus primers produced a robust qPCR signal in 10 vegetables, 6 fruits, 3 types of nuts, and a biofuel precursor. Using sweet potato (Ipomoea batatas) puree as a model low-acid product and simple linear regression, Ct value was highly correlated to time-temperature treatment (R(2) = 0.87); the logarithmic reduction (log CFU/mL) of the spore-forming Clostridium botulinum surrogate, Geobacillus stearothermophilus (R(2) = 0.87); and cumulative F-value (min) in a canned retort process (R(2) = 0.88), all comparisons conducted at 121 °C. D121 and z-values were determined for G. stearothermophilus ATCC 7953 and were 2.71 min and 11.0 °C, respectively. D121 and z-values for a 174-bp universal plant amplicon were 11.3 min and 9.17 °C, respectively, for mtDNA from sweet potato puree. We present these data as proof-of-concept for a molecular tool that can be used as a rapid, presumptive method for monitoring thermal processing in low-acid plant products.