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Antibiotic secretion plays a pivotal role in bacterial interference competition; yet, the impact of environmental hydration conditions on such competition is not well understood. Here, we investigate how hydration conditions affect interference competition among bacteria, studying the interactions between the antibiotic-producing Bacillus velezensis FZB42 and two bacterial strains susceptible to its antibiotics: Xanthomonas euvesicatoria 85-10 and Pseudomonas syringae DC3000. Our results show that wet-dry cycles significantly modify the response of the susceptible bacteria to both the supernatant and cells of the antibiotic-producing bacteria, compared to constantly wet conditions. Notably , X. euvesicatoria shows increased protection against both the cells and supernatants of B. velezensis under wet-dry cycles, while P. syringae cells become more susceptible under wet-dry cycles. In addition, we observed a reciprocal interaction between P. syringae and B. velezensis , where P. syringae inhibits B. velezensis under wet conditions. Our findings highlight the important role of hydration conditions in shaping bacterial interference competition, providing valuable insights into the microbial ecology of water-unsaturated surfaces, with implications for applications such as biological control of plant pathogens and mitigating antibiotic resistance.IMPORTANCEOur study reveals that hydration conditions, particularly wet-dry cycles, significantly influence antibiotic-mediated competition between bacterial species. We revealed that the effectiveness of antibiotics produced by Bacillus velezensis against two susceptible bacterial species: Xanthomonas and Pseudomonas varies based on these hydration conditions. Unlike traditional laboratory environments, many real-world habitats, such as soil, plant surfaces, and even animal skin, undergo frequent wet-dry cycles. These conditions affect bacterial competition dynamics and outcomes, with wet-dry cycles providing increased protection for some bacteria while making others more susceptible. Our findings highlight the importance of considering environmental hydration when studying microbial interactions and developing biological control strategies. This research has important implications for improving agricultural practices and understanding natural microbial ecosystems., Competing Interests: The authors declare no conflict of interest.

Bacillus megaterium ( n = 29), Bacillus velezensis ( n = 26), Bacillus amyloliquefaciens ( n = 6), Bacillus paralicheniformis ( n = 28), and Bacillus licheniformis ( n = 35) strains from different sources, origins, and time periods were tested for the MICs for nine antimicrobial agents by the CLSI-recommended method (Mueller-Hinton broth, 35°C, for 18 to 20 h), as well as with a modified CLSI method (Iso-Sensitest [IST] broth, 37°C [35°C for B. megaterium ], 24 h). This allows a proposal of species-specific epidemiological cutoff values (ECOFFs) for the interpretation of antimicrobial resistance in these species. MICs determined by the modified CLSI method were 2- to 16-fold higher than with the CLSI-recommended method for several antimicrobials. The MIC distributions differed between species for five of the nine antimicrobials. Consequently, use of the modified CLSI method and interpretation of resistance by use of species-specific ECOFFs is recommended. The genome sequences of all strains were determined and used for screening for resistance genes against the ResFinder database and for multilocus sequence typing. A putative chloramphenicol acetyltransferase ( cat ) gene was found in one B. megaterium strain with an elevated chloramphenicol MIC compared to the other B. megaterium strains. In B. velezensis and B. amyloliquefaciens , a putative tetracycline efflux gene, tet (L), was found in all strains ( n = 27) with reduced tetracycline susceptibility but was absent in susceptible strains. All B. paralicheniformis and 23% of B. licheniformis strains had elevated MICs for erythromycin and harbored ermD The presence of these resistance genes follows taxonomy suggesting they may be intrinsic rather than horizontally acquired. Reduced susceptibility to chloramphenicol, streptomycin, and clindamycin could not be explained in all species. IMPORTANCE When commercializing bacterial strains, like Bacillus spp., for feed applications or plant bioprotection, it is required that the strains are free of acquired antimicrobial resistance genes that could potentially spread to pathogenic bacteria, thereby adding to the pool of resistance genes that may cause treatment failures in humans or animals. Conversely, if antimicrobial resistance is intrinsic to a bacterial species, the risk of spreading horizontally to other bacteria is considered very low. Reliable susceptibility test methods and interpretation criteria at the species level are needed to accurately assess antimicrobial resistance levels. In the present study, tentative ECOFFs for five Bacillus species were determined, and the results showed that the variation in MICs followed the respective species. Moreover, putative resistance genes, which were detected by whole-genome sequencing and suggested to be intrinsic rather that acquired, could explain the resistance phenotypes in most cases., (Copyright © 2018 American Society for Microbiology.)

Growth of microorganisms in environments containing CO2 above its critical point is unexpected due to a combination of deleterious effects, including cytoplasmic acidification and membrane destabilization. Thus, supercritical CO2 (scCO2) is generally regarded as a sterilizing agent. We report isolation of bacteria from three sites targeted for geologic carbon dioxide sequestration (GCS) that are capable of growth in pressurized bioreactors containing scCO2. Analysis of 16S rRNA genes from scCO2 enrichment cultures revealed microbial assemblages of varied complexity, including representatives of the genus Bacillus. Propagation of enrichment cultures under scCO2 headspace led to isolation of six strains corresponding to Bacillus cereus, Bacillus subterraneus, Bacillus amyloliquefaciens, Bacillus safensis, and Bacillus megaterium. Isolates are spore-forming, facultative anaerobes and capable of germination and growth under an scCO2 headspace. In addition to these isolates, several Bacillus type strains grew under scCO2, suggesting that this may be a shared feature of spore-forming Bacillus spp. Our results provide direct evidence of microbial activity at the interface between scCO2 and an aqueous phase. Since microbial activity can influence the key mechanisms for permanent storage of sequestered CO2 (i.e., structural, residual, solubility, and mineral trapping), our work suggests that during GCS microorganisms may grow and catalyze biological reactions that influence the fate and transport of CO2 in the deep subsurface., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The apparent heat resistance of spores of Bacillus weihenstephanensis and Bacillus licheniformis was measured and expressed as the time to first decimal reduction (δ value) at a given recovery temperature and pH. Spores of B. weihenstephanensis were produced at 30°C and 12°C, and spores of B. licheniformis were produced at 45°C and 20°C. B. weihenstephanensis spores were then heat treated at 85°C, 90°C, and 95°C, and B. licheniformis spores were heat treated at 95°C, 100°C, and 105°C. Heat-treated spores were grown on nutrient agar at a range of temperatures (4°C to 40°C for B. weihenstephanensis and 15°C to 60°C for B. licheniformis) or a range of pHs (between pH 4.5 and pH 9.5 for both strains). The recovery temperature had a slight effect on the apparent heat resistance, except very near recovery boundaries. In contrast, a decrease in the recovery pH had a progressive impact on apparent heat resistance. A model describing the heat resistance and the ability to recover according to the sporulation temperature, temperature of treatment, and recovery temperature and pH was proposed. This model derived from secondary mathematical models for growth prediction. Previously published cardinal temperature and pH values were used as input parameters. The fitting of the model with apparent heat resistance data obtained for a wide range of spore treatment and recovery conditions was highly satisfactory., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Microorganisms are able to survive and grow in changing environments by activating stress adaptation mechanisms which may enhance bacterial robustness. Stress-induced enhanced robustness complicates the predictability of microbial inactivation. Using psychrotolerant Bacillus weihenstephanensis strain KBAB4 as a model, we investigated the impact of the culturing temperature on mild-oxidative-stress-induced (cross-)protection toward multiple stresses, including severe oxidative, heat, and acid stresses. Culturing at a refrigeration temperature (7°C) compared to the optimal growth temperature (30°C) affected both the robustness level of B. weihenstephanensis and the oxidative stress adaptive response. Scavengers of reactive oxygen species have a crucial role in adaptation to oxidative stresses, and this points to a possible predictive role in mild-oxidative-stress-induced robustness. Therefore, the catalase activity was determined upon mild oxidative stress treatment and was demonstrated to be significantly correlated with the robustness level of mild-stress-treated cells toward severe oxidative and heat stresses but not toward severe acid stress for cells grown at both refrigeration and optimal temperatures. The quantified correlations supported the predictive quality of catalase activity as a biomarker and also underlined that the predictive quality is stress specific. Biomarkers that are able to predict stress-induced enhanced robustness can be used to better understand stress adaptation mechanisms and might allow the design of effective combinations of hurdles to control microbial behavior.

Bacillus spp. are widely used as feed additives and probiotics. However, there is limited information on their resistance to various antibiotics, and there is a growing concern over the transfer of antibiotic resistance genes. The MIC for 8 antibiotics was determined for 85 Bacillus species strains, Bacillus subtilis subsp. subtilis (n = 29), Bacillus licheniformis (n = 38), and Bacillus sonorensis (n = 18), all of which were isolated from starters for Sudanese bread production. All the strains were sensitive to tetracycline (8.0 mg/liter), vancomycin (4.0 mg/liter), and gentamicin (4.0 mg/liter) but resistant to streptomycin. Sensitivity to clindamycin, chloramphenicol, and kanamycin was species specific. The erythromycin resistance genes ermD and ermK were detected by PCR in all of the erythromycin-resistant (MIC, ≥16.0 mg/liter) B. licheniformis strains and one erythromycin-sensitive (MIC, 4.0 mg/liter) B. licheniformis strain. Several amino acid changes were present in the translated ermD and ermK nucleotide sequences of the erythromycin-sensitive strain, which could indicate ErmD and ErmK protein functionalities different from those of the resistance strains. The ermD and ermK genes were localized on an 11.4-kbp plasmid. All of the B. sonorensis strains harbored the bacitracin synthetase gene, bacA, and the transporter gene bcrA, which correlated with their observed resistance to bacitracin. Bacitracin was produced by all the investigated species strains (28%), as determined by ultra-high-definition quadrupole time-of-flight liquid chromatography-mass spectrometry (UHD-QTOF LC/MS). The present study has revealed species-specific variations in the antimicrobial susceptibilities of Bacillus spp. and provides new information on MIC values, as well as the occurrence of resistance genes in Bacillus spp., including the newly described species B. sonorensis.

The culture supernatant of Leuconostoc mesenteroides TK41401, isolated from Japanese pickles, possessed antimicrobial activity against broad range of a bacterial genera and particularly strong activity against Bacillus coagulans, the major contaminant of pickles. An antimicrobial peptide was purified in three chromatographic steps, and its molecular mass was determined to be 6,115.59 Da by electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS). The primary structure of this peptide was determined by amino acid and DNA sequencing, and these analyses revealed that it was translated as a 63-residue precursor. This precursor showed high similarity to the precursor of lactocyclicin Q, a cyclic bacteriocin produced by Lactococcus sp. strain QU 12. The molecular weight calculated after cyclization, which was presumed to involve the same process as in lactocyclicin Q (between L3 and W63), agreed with that estimated by ESI-TOF MS. This peptide was proved to be a novel cyclic bacteriocin, and it was termed leucocyclicin Q. The antimicrobial spectrum of this bacteriocin clearly differed from that of lactocyclicin Q, even though their primary structures were quite similar. This is the first report of a cyclic bacteriocin produced by a strain of the genus Leuconostoc.

The standard test methods used to assess the efficiency of a disinfectant applied to surfaces are often based on counting the microbial survivors sampled in a liquid, but total cell removal from surfaces is seldom achieved. One might therefore wonder whether evaluations of microbial survivors in liquid-sampled cells are representative of the levels of survivors in whole populations. The present study was thus designed to determine the "damaged/undamaged" status induced by a peracetic acid disinfection for Bacillus atrophaeus spores deposited on glass coupons directly on this substrate and to compare it to the status of spores collected in liquid by a sampling procedure. The method utilized to assess the viability of both surface-associated and liquid-sampled spores included fluorescence labeling with a combination of Syto 61 and Chemchrome V6 dyes and quantifications by analyzing the images acquired by confocal laser scanning microscopy. The principal result of the study was that the viability of spores sampled in the liquid was found to be poorer than that of surface-associated spores. For example, after 2 min of peracetic acid disinfection, less than 17% ± 5% of viable cells were detected among liquid-sampled cells compared to 79% ± 5% or 47% ± 4%, respectively, when the viability was evaluated on the surface after or without the sampling procedure. Moreover, assessments of the survivors collected in the liquid phase, evaluated using the microscopic method and standard plate counts, were well correlated. Evaluations based on the determination of survivors among the liquid-sampled cells can thus overestimate the efficiency of surface disinfection procedures.

There is an increasing interest in the application of photocatalytic properties for disinfection of surfaces, air, and water. Titanium dioxide is widely used as a photocatalyst, and the addition of silver reportedly enhances its bactericidal action. However, the synergy of silver nanoparticles and TiO(2) is not well understood. The photocatalytic elimination of Bacillus atrophaeus was examined under different calcination temperatures, dip-coating speeds, and ratios of TiO(2), SiO(2), and Ag to identify optimal production conditions for the production of TiO(2)- and/or TiO(2)/Ag-coated glass for surface disinfection. Photocatalytic disinfection of pure TiO(2) or TiO(2) plus Ag nanoparticles was dependent primarily on the calcination temperature. The antibacterial activity of TiO(2) films was optimal with a high dip-coating speed and high calcination temperature (600°C). Maximal bacterial inactivation using TiO(2)/Ag-coated glass was also observed following high-speed dip coating but with a low calcination temperature (250°C). Scanning electron microscopy (SEM) showed that the Ag nanoparticles combined together at a high calcination temperature, leading to decreased antibacterial activity of TiO(2)/Ag films due to a smaller surface area of Ag nanoparticles. The presence of Ag enhanced the photocatalytic inactivation rate of TiO(2), producing a more pronounced effect with increasing levels of catalyst loading.

Release of dipicolinic acid (DPA) and its fluorescence with terbium (Tb(3+)) allow rapid measurement of the germination and viability of spores of Bacillus and Clostridium species. However, germination of coat-deficient Bacillus spores was strongly inhibited by Tb(3+) and some other multivalent cations. Tb(3+) also inhibited germination of coat-deficient Clostridium perfringens spores.

Knowledge of the nature of resistance determinants in natural habitats is fundamental to increasing our understanding of the development of antibiotic resistance in clinical settings. Here we provide the first report of a tetracycline resistance-encoding plasmid, pBHS24, from a marine sponge-associated bacterium, Bacillus sp. strain #24, isolated from Haliclona simulans.

Five parameters were evaluated with surrogates of Bacillus anthracis spores to determine effective decontamination alternatives for use in a contaminated drinking water supply. The parameters were as follows: (i) type of Bacillus spore surrogate (B. thuringiensis or B. atrophaeus), (ii) spore concentration in suspension (10(2) and 10(6) spores/ml), (iii) chemical characteristics of the decontaminant (sodium dichloro-S-triazinetrione dihydrate [Dichlor], hydrogen peroxide, potassium peroxymonosulfate [Oxone], sodium hypochlorite, and VirkonS), (iv) decontaminant concentration (0.01% to 5%), and (v) exposure time to decontaminant (10 min to 1 h). Results from 138 suspension tests with appropriate controls are reported. Hydrogen peroxide at a concentration of 5% and Dichlor or sodium hypochlorite at a concentration of 2% were highly effective at spore inactivation regardless of spore type tested, spore exposure time, or spore concentration evaluated. This is the first reported study of Dichlor as an effective decontaminant for B. anthracis spore surrogates. Dichlor's desirable characteristics of high oxidation potential, high level of free chlorine, and a more neutral pH than that of other oxidizers evaluated appear to make it an excellent alternative. All three oxidizers were effective against B. atrophaeus spores in meeting the EPA biocide standard of greater than a 6-log kill after a 10-min exposure time and at lower concentrations than typically reported for biocide use. Solutions of 5% VirkonS and Oxone were less effective as decontaminants than other options evaluated in this study and did not meet the EPA's efficacy standard for a biocide, although they were found to be as effective for concentrations of 10(2) spores/ml. Differences in methods and procedures reported by other investigators make quantitative comparisons among studies difficult.

Short- and long-term exposure to mild stress conditions can activate stress adaptation mechanisms in pathogens, resulting in a protective effect toward otherwise lethal stresses. The mesophilic strains Bacillus cereus ATCC 14579 and ATCC 10987 and the psychrotolerant strain B. weihenstephanensis KBAB4 were cultured at 12 degrees C and 30 degrees C until the exponential growth phase (i) in the absence of salt, (ii) in the presence of salt, and (iii) with salt shock after they reached the exponential growth phase and subsequently heat inactivated. Both the first-order model and the Weibull model were fitted to the inactivation kinetics, and statistical indices were calculated to select for each condition the most appropriate model to describe the inactivation data. The third-decimal reduction times (which reflected the times needed to reduce the initial number of microorganisms by three decimal powers) were determined for quantitative comparison. The heat resistance of both mesophilic strains increased when cells were salt cultured and salt shocked at 30 degrees C, whereas these salt-induced effects were not significant for the psychrotolerant strain. In contrast, only the psychrotolerant strain showed salt-induced heat resistance when cells were cultured at 12 degrees C. Therefore, culturing temperature and strain diversity are important aspects to address when adaptive stress responses are quantified. The activated adaptive stress response had an even larger impact on the number of surviving microorganisms when the stress factor (i.e., salt) was still present during inactivation. These factors should be considered when stress-integrated predictive models are developed that can be used in the food industry to balance and optimize processing conditions of minimally processed foods.

The spores of six strains of Bacillus anthracis (four virulent and two avirulent) were compared with those of four other types of spore-forming bacteria for their resistance to four liquid chemical sporicides (sodium hypochlorite at 5,000 ppm available chlorine, 70,000 ppm accelerated H2O2, 1,000 ppm chlorine dioxide, and 3,000 ppm peracetic acid). All test bacteria were grown in a 1:10 dilution of Columbia broth (with manganese) incubated at 37 degrees C for 72 h. The spore suspensions, heat treated at 80 degrees C for 10 min to rid them of any viable vegetative cells, contained 1 x 10(8) to 3 x 10(8) CFU/ml. The second tier of the quantitative carrier test (QCT-2), a standard of ASTM International, was used to assess for sporicidal activity, with disks (1 cm in diameter) of brushed and magnetized stainless steel as spore carriers. Each carrier, with 10 microl (> or = 10(6) CFU) of the test spore suspension in a soil load, was dried and then overlaid with 50 microl of the sporicide being evaluated. The contact time at room temperature ranged from 5 to 20 min, and the arbitrarily set criterion for acceptable sporicidal activity was a reduction of > or = 10(6) in viable spore count. Each test was repeated at least three times. In the final analysis, the spores of Bacillus licheniformis (ATCC 14580(T)) and Bacillus subtilis (ATCC 6051(T)) proved to be generally more resistant than the spores of the strains of B. anthracis tested. The use of one or both of the safe and easy-to-handle surrogates identified here should help in developing safer and more-effective sporicides and also in evaluating the field effectiveness of existing and newer formulations in the decontamination of objects and surfaces suspected of B. anthracis contamination.

Phenotype instability of bacterial strains can cause significant problems in biotechnological applications, since industrially useful properties may be lost. Here we report such degenerative dissociation for Aneurinibacillus migulanus (formerly known as Bacillus brevis) an established producer of the antimicrobial peptide gramicidin S (GS). Phenotypic variations within and between various strains maintained in different culture collections are demonstrated. The type strain, ATCC 9999, consists of six colony morphology variants, R, RC, RP, RT, SC, and SP, which were isolated and characterized as pure cultures. Correlations between colony morphology, growth, GS production, spore formation, and resistance to their own antimicrobial peptide were established in this study. We found the original R form to be the best producer, followed by RC, RP, and RT, while SC and SP yielded no GS at all. Currently available ATCC 9999(T) contains only 2% of the original R producer and is dominated by the newly described phenotypes RC and RP. No original R form is detected in the nominally equivalent strain DSM 2895(T) (=ATCC 9999(T)), which grows only as SC and SP phenotypes and has thus completely lost its value as a peptide producer. Two other strains from the same collection, DSM 5668 and DSM 5759, contain the unproductive SC variant and the GS-producing RC form, respectively. We describe the growth and maintenance conditions that stabilize certain colony phenotypes and reduce the degree of degenerative dissociation, thus providing a recommendation for how to revert the nonproducing smooth phenotypes to the valuable GS-producing rough ones.

Our study focused on a Mesorhizobium sp. that is phylogenetically affiliated by 16S rRNA gene sequence to other marine and saline bacteria of this genus. Liquid chromatography-mass spectrometry investigations of the extract obtained from solid-phase extraction of cultures of this bacterium indicated the presence of several N-acyl homoserine lactones (AHLs), with chain lengths of C(10) to C(16). Chromatographic separation of the active bacterial extract yielded extraordinarily large amounts of two unprecedented acylated homoserine lactones, 5-cis-3-oxo-C(12)-homoserine lactone (5-cis-3-oxo-C(12)-HSL) (compound 1) and 5-cis-C(12)-HSL (compound 2). Quorum-sensing activity of compounds 1 and 2 was shown in two different biosensor systems [Escherichia coli MT102(pSB403) and Pseudomonas putida F117(pKR-C12)]. Furthermore, it was shown that both compounds can restore protease and pyoverdin production of an AHL-deficient Pseudomonas aeruginosa PAO1 lasI rhlI double mutant, suggesting that these signal molecules maybe used for intergenus signaling. In conclusion, these data indicate that the quorum-sensing activity of compounds 1 and 2 is modulated by the chain length and functional groups of the acyl moiety. Additionally, compound 1 showed antibacterial and cytotoxic activities.

Persistence of Bacillus atrophaeus subsp. globigii spores on corroded iron coupons in drinking water was studied using a biofilm annular reactor. Spores were inoculated at 10(6) CFU/ml in the dechlorinated reactor bulk water. The dechlorination allowed for observation of the effects of hydraulic shear and biofilm sloughing on persistence. Approximately 50% of the spores initially adhered to the corroded iron surface were not detected after 1 month. Addition of a stable 10 mg/liter free chlorine residual after 1 month led to a 2-log(10) reduction of adhered B. atrophaeus subsp. globigii, but levels on the coupons quickly stabilized thereafter. Increasing the free chlorine concentration to 25 or 70 mg/liter had no additional effect on inactivation. B. atrophaeus subsp. globigii spores injected in the presence of a typical distribution system chlorine residual (approximately 0.75 mg/liter) resulted in a steady reduction of adhered B. atrophaeus subsp. globigii over 1 month, but levels on the coupons eventually stabilized. Adding elevated chlorine levels (10, 25, and 70 mg/liter) after 1 month had no effect on the rate of inactivation. Decontamination with elevated free chlorine levels immediately after spore injection resulted in a 3-log(10) reduction within 2 weeks, but the rate of inactivation leveled off afterward. This indicates that free chlorine did not reach portions of the corroded iron surface where B. atrophaeus subsp. globigii spores had adhered. B. atrophaeus subsp. globigii spores are capable of persisting for an extended time in the presence of high levels of free chlorine.

This study reports surface complexation models (SCMs) for quantifying metal ion adsorption by thermophilic microorganisms. In initial cadmium ion toxicity tests, members of the genus Geobacillus displayed the highest tolerance to CdCl2 (as high as 400 to 3,200 microM). The thermophilic, gram-positive bacteria Geobacillus stearothermophilus and G. thermocatenulatus were selected for further electrophoretic mobility, potentiometric titration, and Cd2+ adsorption experiments to characterize Cd2+ complexation by functional groups within and on the cell wall. Distinct one-site SCMs described the extent of cadmium ion adsorption by both studied Geobacillus sp. strains over a range of pH values and metal/bacteria concentration ratios. The results indicate that a functional group with a deprotonation constant pK value of approximately 3.8 accounts for 66% and 80% of all titratable sites for G. thermocatenulatus and G. stearothermophilus, respectively, and is dominant in Cd2+ adsorption reactions. The results suggest a different type of functional group may be involved in cadmium biosorption for both thermophilic strains investigated here, compared to previous reports for mesophilic bacteria.

Aerobic heterotrophs were isolated from subsurface soil samples obtained from the U.S. Department of Energy's (DOE) Field Research Center (FRC) located at Oak Ridge, Tenn. The FRC represents a unique, extreme environment consisting of highly acidic soils with co-occurring heavy metals, radionuclides, and high nitrate concentrations. Four hundred isolates obtained from contaminated soil were assayed for heavy metal resistance, and a smaller subset was assayed for tolerance to uranium. The vast majority of the isolates were gram-positive bacteria and belonged to the high-G+C- and low-G+C-content genera Arthrobacter and Bacillus, respectively. Genomic DNA from a randomly chosen subset of 50 Pb-resistant (Pb(r)) isolates was amplified with PCR primers specific for P(IB)-type ATPases (i.e., pbrA/cadA/zntA). A total of 10 pbrA/cadA/zntA loci exhibited evidence of acquisition by horizontal gene transfer. A remarkable dissemination of the horizontally acquired P(IB)-type ATPases was supported by unusual DNA base compositions and phylogenetic incongruence. Numerous Pb(r) P(IB)-type ATPase-positive FRC isolates belonging to the genus Arthrobacter tolerated toxic concentrations of soluble U(VI) (UO(2)(2+)) at pH 4. These unrelated, yet synergistic, physiological traits observed in Arthrobacter isolates residing in the contaminated FRC subsurface may contribute to the survival of the organisms in such an extreme environment. This study is, to the best of our knowledge, the first study to report broad horizontal transfer of P(IB)-type ATPases in contaminated subsurface soils and is among the first studies to report uranium tolerance of aerobic heterotrophs obtained from the acidic subsurface at the DOE FRC.

Spore formation is a sophisticated mechanism by which some bacteria survive conditions of stress and starvation by producing a multilayered protective capsule enclosing their condensed DNA. Spores are highly resistant to damage by heat, radiation, and commonly employed antibacterial agents. Previously, spores have also been shown to be resistant to photodynamic inactivation using dyes and light that easily destroy the corresponding vegetative bacteria. We have discovered that Bacillus spores are susceptible to photoinactivation by phenothiazinium dyes and low doses of red light. Dimethylmethylene blue, methylene blue, new methylene blue, and toluidine blue O are all effective, while alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin, and a benzoporphyrin derivative, which easily kill vegetative cells, are ineffective. Spores of Bacillus cereus and B. thuringiensis are most susceptible, B. subtilis and B. atrophaeus are also killed, and B. megaterium is resistant. Photoinactivation is most effective when excess dye is washed from the spores, showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores, for which conventional sporicides would have unacceptable tissue toxicity.

The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood. Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings. An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus ("Bacillus subtilis subsp. niger," also referred to as BG), a Bacillus anthracis surrogate. Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas. Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR). Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis. Culture results indicated that 10(5) to 10(6) CFU per sample were present on surfaces before decontamination. After decontamination with the foam, no culturable B. atrophaeus spores were detected. After decontamination with chlorine dioxide gas, no culturable B. atrophaeus was detected in 24 of 27 samples (89%). However, QPCR analysis showed that B. atrophaeus DNA was still present after decontamination with both methods. Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed. These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies.

The mechanism of resistance to macrolides, lincosamides, and streptogramins B was studied in four Bacillus clausii strains that are mixed in a probiotic administered to humans for prevention of gastrointestinal side effects due to oral antibiotic chemotherapy and in three reference strains of B. clausii, DSM8716, ATCC 21536, and ATCC 21537. An 846-bp gene called erm(34), which is related to the erm genes conferring resistance to these antibiotics by ribosomal methylation, was cloned from total DNA of B. clausii DSM8716 into Escherichia coli. The deduced amino acid sequence presented 61% identity with that of Erm(D) from B. licheniformis, B. halodurans, and B. anthracis. Pulsed-field gel electrophoresis of total DNA digested by I-CeuI, followed by hybridization with an erm(34)-specific probe, indicated a chromosomal location of the gene in all B. clausii strains. Repeated attempts to transfer resistance to macrolides by conjugation from B. clausii strains to Enterococcus faecalis JH2-2, E. faecium HM1070, and B. subtilis UCN19 were unsuccessful.

Stainless steel surfaces coated with paints containing a silver- and zinc-containing zeolite (AgION antimicrobial) were assayed in comparison to uncoated stainless steel for antimicrobial activity against vegetative cells and spores of three Bacillus species, namely, B. anthracis Sterne, B. cereus T, and B. subtilis 168. Under the test conditions (25 degrees C and 80% relative humidity), the zeolite coating produced approximately 3 log(10) inactivation of vegetative cells within a 5- to 24-h period, but viability of spores of the three species was not significantly affected.

An approach to decontamination of biological endospores is discussed. Specifically, the performance of an aqueous modified Fenton reagent is examined. A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions. The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions. Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu(2+)) and then subsequent conversion to hydroxyl radicals. Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii. A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature. This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O(2) is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action.

The lantibiotic mersacidin is an antimicrobial peptide of 20 amino acids which inhibits bacterial cell wall biosynthesis by binding to the precursor molecule lipid II and which is produced by Bacillus sp. strain HIL Y-85,54728. The structural gene of mersacidin as well as accessory genes is organized in a biosynthetic gene cluster which is located on the chromosome and contains three open reading frames with similarities to regulatory proteins: mrsR2 and mrsK2 encode two proteins with homology to bacterial two-component systems, and mrsR1 shows similarity to a response regulator. Both mrsR2/K2 and mrsR1 were inactivated by insertion of an antibiotic resistance marker. Disruption of mrsR1 resulted in loss of mersacidin production; however, producer self-protection was not impaired. In contrast, inactivation of mrsR2/K2 led to an increased susceptibility to mersacidin whereas biosynthesis of the lantibiotic remained unaffected. Binding of mersacidin to intact cells was significantly enhanced in the mrsR2/K2 knockout mutant. Reverse transcription-PCR analysis from total RNA preparations showed that in contrast to the wild-type strain, the structural genes of the ABC transporter MrsFGE were not transcribed in the knockout mutant. It was therefore concluded that producer self-protection against mersacidin is conferred by the ABC transporter MrsFGE and that the transcription of mrsFGE is regulated by MrsR2/K2, whereas production of the antibacterial peptide is solely activated by MrsR1.

Although metals are thought to inhibit the ability of microorganisms to degrade organic pollutants, several microbial mechanisms of resistance to metal are known to exist. This study examined the potential of cadmium-resistant microorganisms to reduce soluble cadmium levels to enhance degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) under conditions of cocontamination. Four cadmium-resistant soil microorganisms were examined in this study. Resistant up to a cadmium concentration of 275 microg ml(-1), these isolates represented the common soil genera Arthrobacter, Bacillus, and Pseudomonas. Isolates Pseudomonas sp. strain H1 and Bacillus sp. strain H9 had a plasmid-dependent intracellular mechanism of cadmium detoxification, reducing soluble cadmium levels by 36%. Isolates Arthrobacter strain D9 and Pseudomonas strain I1a both produced an extracellular polymer layer that bound and reduced soluble cadmium levels by 22 and 11%, respectively. Although none of the cadmium-resistant isolates could degrade 2,4-D, results of dual-bioaugmentation studies conducted with both pure culture and laboratory soil microcosms showed that each of four cadmium-resistant isolates supported the degradation of 500-microg ml(-1) 2,4-D by the cadmium-sensitive 2,4-D degrader Ralstonia eutropha JMP134. Degradation occurred in the presence of up to 24 microg of cadmium ml(-1) in pure culture and up to 60 microg of cadmium g(-1) in amended soil microcosms. In a pilot field study conducted with 5-gallon soil bioreactors, the dual-bioaugmentation strategy was again evaluated. Here, the cadmium-resistant isolate Pseudomonas strain H1 enhanced degradation of 2,4-D in reactors inoculated with R. eutropha JMP134 in the presence of 60 microg of cadmium g(-1). Overall, dual bioaugmentation appears to be a viable approach in the remediation of cocontaminated soils.

The synthesis of polyglutamic acid (PGA) was repressed by exogenous glutamate in strains of Bacillus licheniformis but not in strains of Bacillus subtilis, indicating a clear difference in the regulation of synthesis of capsular slime in these two species. Although extracellular gamma-glutamyltranspeptidase (GGT) activity was always present in PGA-producing cultures of B. licheniformis under various growth conditions, there was no correlation between the quantity of PGA and enzyme activity. Moreover, the synthesis of PGA in the absence of detectable GGT activity in B. subtilis S317 indicated that this enzyme was not involved in PGA biosynthesis in this bacterium. Glutamate repression of PGA biosynthesis may offer a simple means of preventing unwanted slime production in industrial fermentations using B. licheniformis.

Bacillus subtilis spores are being used for oral bacteriotherapy and bacterioprophylaxis of gastrointestinal disorders in both humans and animals. Since B. subtilis is an aerobic saprophyte, how spores may benefit the gut microbiota is an intriguing question, since other probiotics such as Lactobacillus spp. which colonize the gut are anerobes. As a first step in understanding the potential effects of ingesting spores, we have characterized five commercial products. An extensive biochemical, physiological, and phylogenetic analysis has revealed that four of these products are mislabeled. Moreover, four of these products showed high levels of antibiotic resistance.

The biosynthetic gene cluster (12.3 kb) of mersacidin, a lanthionine-containing antimicrobial peptide, is located on the chromosome of the producer, Bacillus sp. strain HIL Y-85,54728 in a region that corresponds to 348 degrees on the chromosome of Bacillus subtilis 168. It consists of 10 open reading frames and contains, in addition to the previously described mersacidin structural gene mrsA (G. Bierbaum, H. Brötz, K.-P. Koller, and H.-G. Sahl, FEMS Microbiol. Lett. 127:121-126, 1995), two genes, mrsM and mrsD, coding for enzymes involved in posttranslational modification of the prepeptide; one gene, mrsT, coding for a transporter with an associated protease domain; and three genes, mrsF, mrsG, and mrsE, encoding a group B ABC transporter that could be involved in producer self-protection. Additionally, three regulatory genes are part of the gene cluster, i.e., mrsR2 and mrsK2, which encode a two-component regulatory system which seems to be necessary for the transcription of the mrsFGE operon, and mrsR1, which encodes a protein with similarity to response regulators. Transcription of mrsA sets in at early stationary phase (between 8 and 16 h of culture).

Bacillus subtilis is currently used as an oral probiotic. We examined two commercial B. subtilis probiotic preparations, Enterogermina and Biosubtyl. Surprisingly, physiological and genetic characterization of the bacteria contained in each of these preparations has shown that neither contains B. subtilis.

Strain BAS50, isolated from a petroleum reservoir at a depth of 1,500 m and identified as Bacillus licheniformis, grew and produced a lipopeptide surfactant when cultured on a variety of substrates at salinities of up to 13% NaCl. Surfactant production occurred both aerobically and anaerobically and was optimal at 5% NaCl and temperatures between 35 and 45 degrees C. The biosurfactant, termed lichenysin A, was purified and chemically characterized. A tentative structure and composition for the surfactant are described. Lichenysin A is a mixture of lipopeptides, with the major components ranging in size from 1,006 to 1,034 Da. The lipid moiety contains a mixture of 14 linear and branched beta-hydroxy fatty acids ranging in size from C12 to C17. There are seven amino acids per molecule. The peptide moiety is composed of the following amino acids: glutamic acid as the N-terminal amino acid, asparagine, valine, leucine, and isoleucine as the C-terminal amino acid, at a ratio of 1.1:1.1:1.0:2.8:1.0, respectively. Purified lichenysin A decreases the surface tension of water from 72 mN/m to 28 mN/m and achieves the critical micelle concentration with as little as 12 mg/liter, characterizing the product as a powerful surface-active agent that compares favorably to others surfactants. The antibacterial activity of lichenysin A has been demonstrated.

Bacillus isolates from Minamata Bay sediment were selected for the ability to volatilize mercury from a range of organomercurials (including methylmercury). Chromosomal DNA from 74 of 78 such strains hybridized with the mer mercury resistance operon DNA from marine Bacillus sp. strain RC607 (Y. Wang, M. Moore, H. S. Levinson, S. Silver, C. Walsh, and I. Mahler, J. Bacteriol. 171:83-92, 1989). The most frequent classes with regard to restriction nuclease site maps of the mer operon for the new isolates were identical to or closely related to the mer determinant of strain RC607. PCR amplification analysis with primers designed from the strain RC607 mer operon gave products of precisely the predicted size with the 74 Minamata Bay isolates.

The calmodulin antagonist trifluoperazine and its analog chlorpromazine, both amphipaths, induced chemical germination of spores of various species, as do many surfactants. Cation load can greatly influence this response. Calmodulin antagonism does not seem to be involved. A new fluorometric assay for dipicolinic acid based on the fluorescence of the dipicolinic acid chelate of Tb3+ was found to be simple and sensitive.

The effects of low concentrations of nitric oxide (NO) and nitrogen dioxide (NO2) on actively dividing cultures of Staphylococcus aureus, Micrococcus luteus, Micrococcus roseus, Serratia marcescens, Bacillus subtilis, Bacillus circulans, Bacillus megaterium, and Bacillus cereus were studied. Fresh cultures of each organism were incubated for 24 h at 25 degrees C on both nutrient agar and mineral salts glucose agar plates under atmospheres containing various low concentrations of NO in air (0 to 1.9 ppm [0 to 2.0 micrograms/g of air]), NO2 in air (0 to 5.5 ppm [0 to 8.8 micrograms/g of air]), or NO and NO2 in air. Bacteria grown under air only were used as controls. After incubation, the colonies that developed on the plates were counted. None of the bacteria tested was affected by NO or NO2 at the indicated concentrations while growing on nutrient agar. Serratia marcescens, B. circulans, B. subtilis, B. megaterium, and B. cereus grown on mineral salts glucose agar were not significantly affected by NO or NO2. Low concentrations (0 to 1.9 ppm) of NO were bacteriostatic to log-phase cultures of M. roseus, M. luteus, and Staphylococcus aureus grown on mineral salts glucose agar. Bacteriostatic activity over a 24-h interval was maximal at an initial NO concentration of 1 ppm. Appreciable amounts of NO2 were produced in 24 h at initial NO concentrations greater than 1 ppm. These results suggest that NO2 may reduce the bacteriostatic activity of NO. Low concentrations (0 to 5.5 ppm) of NO2 in air did not affect any of the bacteria tested. At these low concentrations, NO affected bacterial growth, although NO2, NO2-, and NO3- did not. In addition, it was determined that the bacteriostatic activity observed in this study was not due to an increase in the acidity of the medium.

Bacteria resistant to cadmium or mercury or both were isolated from the Great Sippewissett Marsh (Cape Cod, Mass.) and from Boston Harbor. Many of these metal-resistant isolates were gram-positive aerobic sporeformers, although not necessarily isolated as spores. Although several of the isolated strains bore plasmids, cadmium and mercury resistances appeared to be, for the most part, chromosomally encoded. DNA sequence homology of the gram-positive cadmium- and mercury-resistant isolates was not demonstrable with metal resistance genes from plasmids of either gram-positive (pI258) or gram-negative (pDB7) origin. Cadmium resistance of all the marsh isolates tested resulted from reduced Cd2+ transport. On the other hand, three cadmium-resistant harbor isolates displayed considerable influx but no efflux of Cd2+. Hg-resistant strains detoxified mercury by transforming Hg2+ to volatile Hg0 via mercuric reductase.

Egg white lysozyme was demonstrated to have antibacterial activity against organisms of concern in food safety, including Listeria monocytogenes and certain strains of Clostridium botulinum. We also found that the food spoilage thermophile Clostridium thermosaccharolyticum was highly susceptible to lysozyme and confirmed that the spoilage organisms Bacillus stearothermophilus and Clostridium tyrobutyricum were also extremely sensitive. Several gram-positive and gram-negative pathogens isolated from food poisoning outbreaks, including Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Campylobacter jejuni, Escherichia coli O157:H7, Salmonella typhimurium, and Yersinia enterocolitica, were all resistant. The results of this study suggest that lysozyme may have selected applications in food preservation, especially when thermophilic sporeformers are problems, and as a safeguard against food poisoning caused by C. botulinum and L. monocytogenes.

A plasmid transformation system was developed for Bacillus polymyxa ATCC 12321 and derivatives of this strain. The method utilizes a penicillin-treated-cell technique to facilitate uptake of the plasmid DNA. Low-frequency transformation (10(-6) per recipient cell) of plasmids pC194, pBD64, and pBC16 was accomplished with this method. Selection for the transformants was accomplished on both hypertonic and nonhypertonic selective media, with the highest rates of recovery occurring on a peptone-glucose-yeast extract medium containing 0.25 M sucrose. Several additional plasmids were shown to be capable of transferring their antibiotic resistance phenotypes to B. polymyxa through the use of a protoplast transformation procedure which allowed for a more efficient transfer of the plasmid DNA. However, cell walls could not be regenerated on the transformed protoplasts, and the transformants could not be subcultured from the original selective media.

A total of 75 strains (including 5 reference strains) of Bacillus amyloliquefaciens, B. cereus, B. circulans, B. licheniformis, B. megaterium, B. pumilus, B. sphaericus, B. subtilis, and B. thuringiensis and 36 species-unidentified Bacillus strains were surveyed for plasmids by cesium chloride-ethidium bromide equilibrium centrifugation of cell lysates in a study of antibiotic resistance in host cells. Of the 111 strains, 13 (including 3 reference strains) were found to harbor plasmids, and 5 of the 13 showed antibiotic resistance. This antibiotic resistance appeared not to be due to the plasmids, however, because the trait was not cured by cultivation of cells in nutrient medium containing ethidium bromide (1 microgram/ml), sodium dodecyl sulfate (0.2 micrograms/ml), or novobiocin (1 microgram/ml), except in one strain, in which kanamycin and streptomycin resistances were cured by novobiocin. One strain of B. amyloliquefaciens, S294, was found to harbor a plasmid, pFTB14, which differed from the plasmid species of classes 1 to 6 in B. subtilis and B. amyloliquefaciens, as determined by restriction analysis and DNA contour length determination. However, in DNA-DNA hybridization on a filter after Southern blotting from an agarose gel, the pFTB14 DNA hybridized with plasmids of classes 1 to 5. Three strains of B. thuringiensis each carried at least 4 to 11 plasmid species, whereas no plasmids were detected in four strains of B. cereus, which, in relation to B. thuringiensis, is closely related taxonomically and has highly homologous DNA sequences. The plasmid DNAs prepared from species other than B. subtilis and B. amyloliquefaciens did not hybridize with that of pFTB14.

The New York Bight extends seaward some 80 to 100 miles (ca. 129 to 161 km) from the Long Island and New Jersey shorelines to the edge of the continental shelf. Over 14 x 10(6) m(3) of sewage sludge, dredge spoils, acid wastes, and cellar dirt are discharged into this area each year. Large populations of Bacillus sp. resistant to 20 mug of mercury per ml were observed in Bight sediments contaminated by these wastes. Resistant Bacillus populations were much greater in sediments containing high concentrations of Hg and other heavy metals than in sediments from areas further offshore where dumping has never been practiced and where heavy-metal concentrations were found to be low. Ampicillin resistance due mainly to beta-lactamase production was significantly (P < 0.001) more frequent in Bacillus strains from sediments near the sewage sludge dump site than in similar Bacillus populations from control sediments. Bacillus strains with combined ampicillin and Hg resistances were almost six times as frequent at the sludge dump site as in control sediments. This observation suggests that genes for Hg resistance and beta-lactamase production are simultaneously selected for in Bacillus and that heavy-metal contamination of an ecosystem can result in a selection pressure for antibiotic resistance in bacteria in that system. Also, Hg resistance was frequently linked with other heavy-metal resistances and, in a substantial proportion of Bacillus strains, involved reduction to volatile metallic Hg (Hg degrees ).

A nonmucoid colonial variant of a mucoid Bacillus subtilis strain produced less amylase activity and a transparent colonial variant of a B. licheniformis strain produced less protease activity compared with their parents. Antibiotic susceptibility patterns of the colonial variants differed, and increased resistance to beta-lactam antibiotics was correlated with increased production of extracellular beta-lactamase.