Vegetative Bacterial Cells Research Articles

Sporicidal Effects of High‐Intensity 405 nm Visible Light on Endospore‐Forming Bacteria

Resistance of bacterial endospores to treatments, including biocides, heat and radiation is a persistent problem. This study investigates the susceptibility of Bacillus and Clostridium endospores to 405 nm visible light, wavelengths which have been shown to induce inactivation of vegetative bacterial cells. Suspensions of B. cereus endospores were exposed to high-intensity 405 nm light generated from a light-emitting diode array and results demonstrate the induction of a sporicidal effect. Up to a 4-log(10) CFU mL(-1) reduction in spore population was achieved after exposure to a dose of 1.73 kJ cm(-2). Similar inactivation kinetics were demonstrated with B. subtilis, B. megaterium and C. difficile endospores. The doses required for inactivation of endospores were significantly higher than those required for inactivation of B. cereus and C. difficile vegetative cells, where ca 4-log(10) CFU mL(-1) reductions were achieved after exposure to doses of 108 and 48 J cm(-2), respectively. The significant increase in dose required for inactivation of endospores compared with vegetative cells is unsurprising due to the notorious resilience of these microbial structures. However, the demonstration that visible light of 405 nm can induce a bactericidal effect against endospores is significant, and could have potential for incorporation into decontamination methods for the removal of bacterial contamination including endospores.

Evaluation of fluorescence in situ hybridization to detect encapsulated Bacillus pumilus SAFR‐032 spores released from poly(methylmethacrylate)

Bacillus pumilus SAFR-032 spores originally isolated from the Jet Propulsion Laboratory spacecraft assembly facility clean room are extremely resistant to UV radiation, H(2)O(2), desiccation, chemical disinfection and starvation compared to spores of other Bacillus species. The resistance of B. pumilus SAFR-032 spores to standard industrial clean room sterilization practices is not only a major concern for medical, pharmaceutical and food industries, but also a threat to the extraterrestrial environment during search for life via spacecraft. The objective of the present study was to investigate the potential of Alexa-FISH (fluorescence in situ hybridization with Alexa Fluor® 488 labeled oligonucleotide) method as a molecular diagnostic tool for enumeration of multiple sterilant-resistant B. pumilus SAFR-032 spores artificially encapsulated in, and released via organic solvent from, a model polymeric material: poly(methylmethacrylate) (Lucite, Plexiglas). Plexiglas is used extensively in various aerospace applications and in medical, pharmaceutical and food industries. Alexa-FISH signals were not detected from spores via standard methods for vegetative bacterial cells. Optimization of a spore permeabilization protocol capitalizing on the synergistic action of proteinase-K, lysozyme, mutanolysin and Triton X-100 facilitated efficient spore detection by Alexa-FISH microscopy. Neither of the Alexa-probes tested gave rise to considerable levels of Lucite- or solvent-associated background autofluorescence, demonstrating the immense potential of Alexa-FISH for rapid quantification of encapsulated B. pumilus SAFR-032 spores released from poly(methylmethacrylate).

Feasibility of selected prophylactic barriers in arrestance of airborne bacterial vegetative cells and endospores

Transmission of infection by airborne agents is a risk for health care personnel, patients, and visitors. This risk is heightened in regions without access to environmental controls and personal protective equipment. The ability of 2 prophylactic barriers (ie, semitransparent netting for insect control) to arrest bioaerosols was assessed for potential use within the malarial zones. Barriers (pore sizes of 0.8 mm and 0.25 mm) were challenged with bioaerosols of vegetative cells and endospores of Bacillus anthracis strain Sterne 34F2 using a bioaerosol chamber. Barriers were also challenged with airborne inert polystyrene latex particles of known diameters (0.1, 0.43, 0.6, 1.3, 3.2, and 8.0 μm), and the arrestance provided by barrier with the 0.25 mm pore size was expressed as a function of aerodynamic diameter of challenge aerosols. Barrier with the 0.8 mm pore size provided no significant arrestance of aerosols, whereas the barrier with the 0.25 mm pore size provided an 8% arrestance of vegetative cells and a 13% arrestance of endospores. No arrestance at or below the 0.6 μm particle size was observed. The level of arrestance provided by these prophylactic barriers does not justify their use as a sole method of preventing transmission.

On the Difference Between Surface-Enhanced Raman Scattering (SERS) Spectra of Cell Growth Media and Whole Bacterial Cells

It has been recently suggested [N. E. Marotta and L. A. Bottomley, Appl. Spectrosc. 64, 601-606 (2010)] that previously reported surface-enhanced Raman scattering (SERS) spectra of vegetative bacterial cells are due to residual cell growth media that were not properly removed from samples of the lab-cultured microorganism suspensions. SERS spectra of several commonly used cell growth media are similar to those of bacterial cells, as shown here and reported elsewhere. However, a multivariate data analysis approach shows that SERS spectra of different bacterial species grown in the same growth media exhibit different characteristic vibrational spectra, SERS spectra of the same organism grown in different media display the same SERS spectrum, and SERS spectra of growth media do not cluster near the SERS spectra of washed bacteria. Furthermore, a bacterial SERS spectrum grown in a minimal medium, which uses inorganics for a nitrogen source and displays virtually no SERS features, exhibits a characteristic bacterial SERS spectrum. We use multivariate analysis to show how successive water washing and centrifugation cycles remove cell growth media and result in a robust bacterial SERS spectrum in contrast to the previous study attributing bacterial SERS signals to growth media.

Germicidal UV Sensitivity of Bacteria in Aerosols and on Contaminated Surfaces

We compared the UV sensitivity of Bacillus atrophaeus (a surrogate for B. anthracis), Pantoea agglomerans (a bacterial simulant frequently used in biodefense studies), and Yersinia ruckeri (a surrogate for Yersinia pestis) either airborne or deposited on a semisolid (wet) agar surface. Bacterial vegetative cells were aerosolized into an exposure chamber and exposed for various lengths of time to an ultraviolet (UV) light source emitting at 254 nanometer (nm) (in the UVC region also known as UVGI). Aerosols were collected onto gelatin filters, which were dissolved, diluted, plated, and incubated to enumerate colony formation. In darkness (with the UV light switched off), it took between 170 and 330 s airborne (depending on the bacterial species) to decrease by 90% of the original load (i.e., by 1 Log10) the number of viable organisms originally present. The fluence of UVC required to inactivate 90% (F–1 Log10) of bacteria aerosolized into an atmosphere with 80–90% relative humidity corresponded to 70.3 J.m...

Application of Mid-infrared and Raman Spectroscopy to the Study of Bacteria

Infrared spectroscopy and Raman spectroscopy provide complementary technologies for rapid and precise detection of microorganisms and are emerging methods in food analysis. It is possible to use either of these techniques to differentiate and quantify microorganisms in relatively simple matrices such as liquid media and simple solutions with determinations taking less than an hour. Vibrational spectroscopy, unlike other techniques used in microbiology, is a relatively simple method for studying structural changes occurring within a microbial cell following environmental stress and applications of food processing treatments. Vibrational spectroscopy provides a wide range of biochemical properties about bacteria in a single spectrum, most importantly characteristics of the cell membrane. These techniques are especially useful for studying properties of bacterial biofilms on contact surfaces, the presence and viability of bacterial vegetative cells and spores, the type and degree of bacterial injury, and assessment of antibiotic susceptibility. Future trends in food analysis will involve combining vibrational spectroscopy with microscopy, mass spectroscopy, or DNA-based methods to comprehensively study bacterial stress. Further advances in selectivity, sensitivity, and improved chemometric methods, along with reduction in the cost of instrumentation, may lead to the development of field-ready and real-time analytical systems.

Acoustic lysis of vegetative bacterial cells: Method and device development

A critical problem of many pathogen detection assays is the availability of intracellular protein and deoxyribonucleic acid (DNA). Acoustic lysis of suspended vegetative bacterial cells in a microfluidic system offers several advantages over conventional lysis techniques. The intracellular proteins and DNA are released and available for detection. A novel acoustic lysing alternative technique to the existing lysing methods for sample preparation and lysis step is proposed. We report here an efficient lysis device that uses acoustic excitation for performing lysis of Gram-positive and Gram-negative vegetative cells and has a high yield in a short amount of time. We also verified the condition of released protein since one of the major uses of vegetative cells lysis is for protein expression studies. Fluorimetry and flow cytometry were used to assess the degree of damage induced on the cells by the actual lysis method. The acoustic device allows the delivery of proteins in a non-denatured form, without adding chemicals, particles or other substances (e.g. enzymes) that could complicate the process or the detection procedure. The lysis device operates at low power (50–400 mW) and short time (3 min) and has high efficiency in comparison to current lysis standards (>85% vs. 12–50%).

Accuracy, precision, and method detection limits of quantitative PCR for airborne bacteria and fungi.

Real-time quantitative PCR (qPCR) for rapid and specific enumeration of microbial agents is finding increased use in aerosol science. The goal of this study was to determine qPCR accuracy, precision, and method detection limits (MDLs) within the context of indoor and ambient aerosol samples. Escherichia coli and Bacillus atrophaeus vegetative bacterial cells and Aspergillus fumigatus fungal spores loaded onto aerosol filters were considered. Efficiencies associated with recovery of DNA from aerosol filters were low, and excluding these efficiencies in quantitative analysis led to underestimating the true aerosol concentration by 10 to 24 times. Precision near detection limits ranged from a 28% to 79% coefficient of variation (COV) for the three test organisms, and the majority of this variation was due to instrument repeatability. Depending on the organism and sampling filter material, precision results suggest that qPCR is useful for determining dissimilarity between two samples only if the true differences are greater than 1.3 to 3.2 times (95% confidence level at n = 7 replicates). For MDLs, qPCR was able to produce a positive response with 99% confidence from the DNA of five B. atrophaeus cells and less than one A. fumigatus spore. Overall MDL values that included sample processing efficiencies ranged from 2,000 to 3,000 B. atrophaeus cells per filter and 10 to 25 A. fumigatus spores per filter. Applying the concepts of accuracy, precision, and MDL to qPCR aerosol measurements demonstrates that sample processing efficiencies must be accounted for in order to accurately estimate bioaerosol exposure, provides guidance on the necessary statistical rigor required to understand significant differences among separate aerosol samples, and prevents undetected (i.e., nonquantifiable) values for true aerosol concentrations that may be significant.

Inactivation of Escherichia coli and Lactobacillus plantarum in relation to membrane permeabilization due to rapid chilling followed by cold storage

The relationship between membrane permeabilization and loss of viability by chilling depending on the chilling rate was investigated in two bacterial models: one Gram-positive bacterium, Lactobacillus plantarum, and one Gram-negative bacterium, Escherichia coli. Cells were cold shocked slowly (2 degrees C/min) or rapidly (2,000 degrees C/min) from physiological temperature to 0 degrees C and maintained at this temperature for up to 1 week. Loss of membrane integrity was assessed by the uptake of the fluorescent dye propidium iodide (PI). Cell death was found to be strongly dependent on the rate of temperature downshift to 0 degrees C. Prolonged incubation of cells after the chilling emphasized the effect of treatment on the cells, as the amount of cell death increased with the length of exposure to low temperature, particularly when cells were rapidly chilled. More than 5 and 3-log reductions in cell population were obtained with L. plantarum and E. coli after the rapid cold shock followed by 7-day storage, respectively. A correlation between cell inactivation and membrane permeabilization was demonstrated with both bacterial strains. Thus, loss of membrane integrity due to the chilling treatments was directly involved in the inactivation of vegetative bacterial cells.

Effect of Low-Pressure Carbonation on Heat Inactivation of Yeast and Bacterial Vegetative Cells

Effect of low-pressure carbonation (LPC) on heat inactivation of yeast and bacterial vegetative cells was investigated. Microbial cell suspensions were carbonated at 0.6 MPa and 12°C for 15 min and subsequently heated for 1 min at temperatures ranging from 50°C to 70°C at 1 MPa. As a control experiment, suspensions were heat treated for 1 min under atmospheric pressure without LPC. The heat inactivation effect on yeast was not significantly changed by LPC; however, the heat inactivation efficiency on several bacteria was enhanced. The promoted inactivation was also observed for heat treatment under atmospheric pressure after LPC. The enhanced inactivation was not notable by heat treatment followed by LPC. The results suggest enhanced heat inactivation against bacteria under LPC, and the increased heat inactivation of bacteria may be due to LPC-mediated sensitization to heat.

Bacterial Inactivation by Solar Ultraviolet Radiation Compared with Sensitivity to 254 nm Radiation

Our goal was to derive a quantitative factor that would allow us to predict the solar sensitivity of vegetative bacterial cells to natural solar radiation from the wealth of data collected for cells exposed to UVC (254 nm) radiation. We constructed a solar effectiveness spectrum for inactivation of vegetative bacterial cells by combining the available action spectra for vegetative cell killing in the solar range with the natural sunlight spectrum that reaches the ground. We then analyzed previous studies reporting the effects of solar radiation on vegetative bacterial cells and on bacterial spores. Although UVC-sensitive cells were also more sensitive to solar radiation, we found no absolute numerical correlation between the relative solar sensitivity of vegetative cells and their sensitivity to 254 nm radiation. The sensitivity of bacterial spores to solar exposure during both summer and winter correlated closely to their UVC sensitivity. The estimates presented here should make it possible to reasonably predict the time it would take for natural solar UV to kill bacterial spores or with a lesser degree of accuracy, vegetative bacterial cells after dispersion from an infected host or after an accidental or intentional release.

Influence of microwave radiation on soil bacteria

Cultures of different soil bacteria were used in the experiment. Both the suppression and the stimulation of the growth were observed for different bacterial species under the impact of microwaves. Spore suspensions responded to microwave radiation upon a shorter time of exposure than suspensions of vegetative bacterial cells. The influence of microwave radiation on the biomass accumulation and the intensity of other physiological processes in different species of streptomycetes led to changes in the number and activity of these microorganisms in the soil microbial complex. Changes in the capacity of streptomycetes for assimilation of substrates under the impact of microwaves were identified by the method of multisubstrate testing.

Classification of Organic and Biological Materials with Deep Ultraviolet Excitation

We show that native fluorescence can be used to differentiate classes or groups of organic molecules and biological materials when excitation occurs at specific excitation wavelengths in the deep ultraviolet (UV) region. Native fluorescence excitation-emission maps (EEMs) of pure organic materials, microbiological samples, and environmental background materials were compared using excitation wavelengths between 200-400 nm with emission wavelengths from 270 to 500 nm. These samples included polycyclic aromatic hydrocarbons (PAHs), nitrogen- and sulfur-bearing organic heterocycles, bacterial spores, and bacterial vegetative whole cells (both Gram positive and Gram negative). Each sample was categorized into ten distinct groups based on fluorescence properties. Emission spectra at each of 40 excitation wavelengths were analyzed using principal component analysis (PCA). Optimum excitation wavelengths for differentiating groups were determined using two metrics. We show that deep UV excitation at 235 (+/-2) nm optimally separates all organic and biological groups within our dataset with >90% confidence. For the specific case of separation of bacterial spores from all other samples in the database, excitation at wavelengths less than 250 nm provides maximum separation with >6sigma confidence.

Development and Validation of a Pilot Scale Enhanced Biosafety Level Two Containment for Performance Evaluation of Produce Disinfection Technologies

The development and validation of the Biosafety Level 2 (BSL-2) enhanced containment system located at the produce pilot plant facility of the U.S. Department of Agriculture—Eastern Regional Research Center is presented here. This multi-purpose containment is used to enclose pilot and pilot scale washing and sanitizing equipment for fruits and vegetables, or other decontamination equipment where aerosol generation is likely, and complete protection is required for researchers. This containment is operated under a negative pressure with all exhausted air (approximately eight containment air exchanges per hour) being passed in parallel through two hydrophobic HEPA filters. During operation, personnel are excluded from the containment, materials are introduced into and removed from the containment via pass-throughs, and equipment is operated via computer control and glove ports.At the completion of any series of processing trials, vegetative bacterial cells remaining within the containment, and in the proces...

Microbial Growth and the Effects of Mild Acidification and Preservatives in Refrigerated Sweet Potato Puree

Refrigerated sweet potato puree is a convenient form of sweet potato that can be used as an ingredient in formulated foods. The microbiology of refrigerated sweet potato puree during storage for up to 5 weeks was evaluated. Because the puree was made by comminuting steam-cooked sweet potatoes before refrigeration, no naturally occurring vegetative bacterial cells were detected during a 4-week period of refrigerated storage at 4°C. However, if postprocessing microbial contamination of the puree were to occur, contaminating microorganisms such as Listeria monocytogenes could grow during refrigerated storage. The effects of acidification or the addition of potassium sorbate and sodium benzoate on a population of L. monocytogenes inoculated into refrigerated (4°C) sweet potato puree were determined. Inoculation of the refrigerated puree with L. monocytogenes at 106 CFU/ml resulted in a 3-log increase after 3 weeks storage of nonsupplemented puree. Supplementation of the sweet potato puree with 0.06% (wt/vol) sorbic acid or benzoic acid plus mild acidification of the sweet potato puree with citric acid to pH 4.2 prevented growth of L. monocytogenes during storage at 4°C.

Highly Efficient Classification and Identification of Human Pathogenic Bacteria by MALDI-TOF MS

Accurate and rapid identification of pathogenic microorganisms is of critical importance in disease treatment and public health. Conventional work flows are time-consuming, and procedures are multifaceted. MS can be an alternative but is limited by low efficiency for amino acid sequencing as well as low reproducibility for spectrum fingerprinting. We systematically analyzed the feasibility of applying MS for rapid and accurate bacterial identification. Directly applying bacterial colonies without further protein extraction to MALDI-TOF MS analysis revealed rich peak contents and high reproducibility. The MS spectra derived from 57 isolates comprising six human pathogenic bacterial species were analyzed using both unsupervised hierarchical clustering and supervised model construction via the Genetic Algorithm. Hierarchical clustering analysis categorized the spectra into six groups precisely corresponding to the six bacterial species. Precise classification was also maintained in an independently prepared set of bacteria even when the numbers of m/z values were reduced to six. In parallel, classification models were constructed via Genetic Algorithm analysis. A model containing 18 m/z values accurately classified independently prepared bacteria and identified those species originally not used for model construction. Moreover bacteria fewer than 10(4) cells and different species in bacterial mixtures were identified using the classification model approach. In conclusion, the application of MALDI-TOF MS in combination with a suitable model construction provides a highly accurate method for bacterial classification and identification. The approach can identify bacteria with low abundance even in mixed flora, suggesting that a rapid and accurate bacterial identification using MS techniques even before culture can be attained in the near future.

High pressure in combination with elevated temperature as a method for the sterilisation of food

Application of high-pressure processing to foods can effect a decrease in the number of vegetative bacterial cells, and hence can result in pasteurisation. Inactivation of bacterial spores, however, is required for the sterilisation of foods. This article reviews the current status of the application of high-pressure treatments for the inactivation of bacterial spores, and particularly examines the requirement for a combination of high pressure and high temperature processing to achieve the sterilisation of foods.

Evaluation of Physical Capture Efficiency and Disinfection Capability of an Iodinated Biocidal Filter Medium

Poly(styrene-divinylbenzene)-4-(methyltrimethylammonium) triiodide (PMT) has recently been applied onto nonwoven air filter media to purportedly combine filtration and iodine disinfection to achieve enhanced attenuation of viable airborne pathogens without aggravating the pressure drop of the medium. This paper reports and compares the physical capture efficiency and biological removal efficiency of the novel biocidal filter medium. During challenges with inert fluorescent particles, bothiodine-treated and untreated media displayed statistically equivalent physical capture efficiencies ＞ 97%, and typically ＞ 99%. The pressure drag (3.2 kPa•s/m) was less than 10% that of a glass fiber HEPA (38 kPa•s/m) medium. Biological disinfection by the media was evaluated using aerosols containing M. luteus and E. coli vegetative bacterial cells. Biological removal efficiency (99.997%) was observed to be two logs greater than inert particle capture. Viable penetration through the biocidal filters was observed in only two of 10 experiments. The results suggest that an antimicrobial-coated filter medium can provide effective protection against airborne pathogens with a significantly lower pressure drop than that imposed by conventional high-efficiency filtration systems. A near-contact mechanism is proposed in which the distances of nearest approach to treated fiber surfaces as a microbe penetrates through the filter define the probability that charge sites on the microbe’s surface will capture enough iodine molecules from triiodide complexes at the surfaces to terminate viability.

The BclB Glycoprotein of Bacillus anthracis Is Involved in Exosporium Integrity

Anthrax is a highly fatal disease caused by the gram-positive, endospore-forming, rod-shaped bacterium Bacillus anthracis. Spores, rather than vegetative bacterial cells, are the source of anthrax infections. Spores of B. anthracis are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. Filaments of the hair-like nap are made up largely of a single collagen-like glycoprotein called BclA. A second glycoprotein, BclB, has been identified in the exosporium layer. The specific location of this glycoprotein within the exosporium layer and its role in the biology of the spore are unknown. We created a mutant strain of B. anthracis DeltaSterne that carries a deletion of the bclB gene. The mutant was found to possess structural defects in the exosporium layer of the spore (visualized by electron microscopy, immunofluorescence, and flow cytometry) resulting in an exosporium that is more fragile than that of a wild-type spore and is easily lost. Immunofluorescence studies also indicated that the mutant strain produced spores with increased levels of the BclA glycoprotein accessible to the antibodies on the surface. The resistance properties of the mutant spores were unchanged from those of the wild-type spores. A bclB mutation did not affect spore germination or kinetics of spore survival within macrophages. BclB plays a key role in the formation and maintenance of the exosporium structure in B. anthracis.

Virulence of Bacillus cereus: A multivariate analysis

Biological activity and presence of DNA sequences related to virulence genes were studied in 21 strains of the Bacillus cereus group. The activity of spent culture supernatants and the effect of infection by vegetative bacterial cells were assessed on cultured human enterocytes (Caco-2 cells). The effect of extracellular factors on the detachment, necrosis and mitochondrial dehydrogenase activity of cultured human enterocytes was studied. Hemolytic activity on rabbit red blood cells was also evaluated and the effect of direct procaryotic–eucaryotic interactions was assessed in infection assays with vegetative bacterial cells. Concerning virulence genes, presence of the DNA sequences corresponding to the genes entS, entFM, nhe ( A, B and C), sph, hbl ( A, B, C and D), piplC and bceT was assessed by PCR. Ribopatterns were determined by an automated riboprinting analysis after digestion of the DNA with EcoRI. Principal component analysis and biplots were used to address the relationship between variables. Results showed a wide range of biological activities: decrease in mitochondrial dehydrogenase activity, necrosis, cell detachment and hemolytic activity. These effects were strain-dependent. Concerning the occurrence of the DNA sequences tested, different patterns were found. In addition, ribotyping showed that strains under study grouped into two main clusters. One of these clusters includes all the strains that were positive for all the DNA sequences tested. Positive and negative correlations between variables under study were evidenced. Interestingly, high detaching strains were positively correlated with the presence of the sequences entS, nheC and sph. Within gene complexes, high correlation was found between sequences of the hbl complex. In contrast, sequences of the nhe complex were not correlated. Some strains clustered together in the biplots. These strains were positive for all the DNA sequences tested and they were able to detach enterocytes upon infection. Our results highlight the multifactorial character of the virulence of the B. cereus group and show the correlation between ribopatterns, occurrence of toxin genes and biological activity of the strains under study.