Resistance of Bacillus Endospores to Extreme Terrestrial and Extraterrestrial Environments

Endospores of Bacillus spp., especially Bacillus subtilis, have served as experimental models for exploring the molecular mechanisms underlying the incredible longevity of spores and their resistance to environmental insults. In this review we summarize the molecular laboratory model of spore resistance mechanisms and attempt to use the model as a basis for exploration of the resistance of spores to environmental extremes both on Earth and during postulated interplanetary transfer through space as a result of natural impact processes.     

Multiple-Antibiotic Resistance of Enterococcus spp. Isolated from Commercial Poultry Production Environments

The potential impact of food animals in the production environment on the bacterial population as a result of antimicrobial drug use for growth enhancement continues to be a cause for concern. Enterococci from 82 farms within a poultry production region on the eastern seaboard were isolated to establish a baseline of susceptibility profiles for a number of antimicrobials used in production as well as clinical environments. Of the 541 isolates recovered, Enterococcus faecalis (53%) and E. faecium (31%) were the predominant species, while multiresistant antimicrobial phenotypes were observed among all species. The prevalence of resistance among isolates of E. faecalis was comparatively higher among lincosamide, macrolide, and tetracycline antimicrobials, while isolates of E. faecium were observed to be more frequently resistant to fluoroquinolones and penicillins. Notably, 63% of the E. faecium isolates were resistant to the streptogramin quinupristin-dalfopristin, while high-level gentamicin resistance was observed only among the E. faecalis population, of which 7% of the isolates were resistant. The primary observations are that enterococci can be frequently isolated from the poultry production environment and can be multiresistant to antimicrobials used in human medicine. The high frequency with which resistant enterococci are isolated from this environment suggests that these organisms might be useful as sentinels to monitor the development of resistance resulting from the usage of antimicrobial agents in animal production.     

Extremophilic Bacillus cereus MVK04 Isolated from Thorium Ore Sample Possesses Self-Assembled Surface Layer Protein on Cell Wall to Resist Extreme Environments

The present study investigates the extremophilic nature of bacteria present in thorium rich mine ore samples collected from Manavalakuruchi, Tamilnadu, India. Six different bacteria strains were isolated from these ore samples and screened for its resistance against varying pH, uranium and gamma radiation. Deinococcus radiodurans ATCC 13939 and Escherichia coli MTCC 1687 was used as positive and negative control respectively. Among the six different bacterial strains, MVK04 strain was found to resist higher pH, uranium concentration and gamma radiation. The organism was identified as Bacillus cereus based on biochemical, 16S rRNA and MALDI TOF fingerprinting studies. The interaction of uranium ions with MVK04 bacterial cell wall was investigated using high resolution transmission electron microscopy (HRTEM) and energy dispersive spectroscopy (EDS). The bacterial MVK04 was further investigated for the presence of surface layer protein (S-layer protein) on the bacterial cell wall. The S-layer protein was isolated, partially purified and self-assembled onto TEM copper grids and the self-assembled protein nanostructures were characterized using HRTEM. The presence of surface layer protein on bacterial cell wall may be the possible reason for its extremophilic characteristics and its escape from lethal effect of higher concentration of uranium, lower pH and increased gamma radiation.     

Molecular Characterization of Fluoroquinolone-Resistant Aeromonas spp. Isolated from Imported Shrimp

Sixty-three nalidixic acid-resistant Aeromonas sp. isolates were obtained from imported shrimp. Phylogenetic analysis of gyrB sequences indicated that 18 were A. enteropelogenes, 26 were A. caviae, and 19 were A. sobria. Double missense mutations in the quinolone resistance-determining region (QRDR) of gyrA at codon 83 (Ser→Val/Ile) and codon 92 (Leu→Met) coupled with a point mutation of parC at codon 80 (Ser→Ile/Phe) conferred high levels of quinolone resistance in the isolates. A majority of A. enteropelogenes and A. caviae strains harbored toxin genes, whereas only a few A. sobria strains harbored these genes. The fluoroquinolone-resistant Aeromonas spp. exhibited higher cytotoxicity than fluoroquinolone-sensitive, virulent Aeromonas spp. to rat epithelial cells.     

Antagonism of Bacillus spp. Isolated from Marine Biofilms Against Terrestrial Phytopathogenic Fungi

We aimed at determining the antagonistic behavior of bacteria derived from marine biofilms against terrestrial phytopathogenic fungi. Some bacteria closely related to Bacillus mojavensis (three isolates) and Bacillus firmus (one isolate) displayed antagonistic activity against Colletotrichum gloeosporioides ATCC 42374, selected as first screen organism. The four isolates were further quantitatively tested against C. gloeosporioides, Colletotrichum fragariae, and Fusarium oxysporum on two culture media, potato dextrose agar (PDA) and a marine medium-based agar [yeast extract agar (YEA)] at different times of growth of the antagonists (early, co-inoculation with the pathogen and late). Overall antagonistic assays showed differential susceptibility among the pathogens as a function of the type of culture media and time of colonization (P < 0.05). In general, higher suppressive activities were recorded for assays performed on YEA than on PDA; and also when the antagonists were allowed to grow 24 h earlier than the pathogen. F. oxysporum was the most resistant fungus while the most sensitive was C. gloeosporioides ATCC 42374. Significant differences in antagonistic activity (P < 0.05) were found between the different isolates. In general, Bacillus sp. MC3B-22 displayed a greater antagonistic effect than the commercial biocontrol strain Bacillus subtilis G03 (Kodiak). Further incubation studies and scanning electronic microscopy revealed that Bacillus sp. MC3B-22 was able to colonize, multiply, and inhibit C. gloeosporioides ATCC 42374 when tested in a mango leaf assay, showing its potential for fungal biocontrol. Additional studies are required to definitively identify the active isolates and to determine their mode of antifungal action, safety, and biocompatibility.     

Reaerosolization of Bacillus spp. in Outdoor Environments: A Review of the Experimental Literature

Reaerosolization or resuspension-that is, the reintroduction of previously airborne particles into the atmosphere-is a complex phenomenon. Microbial reaerosolization is particularly poorly understood because few studies have been done in this area, and many of the studies that have been performed are not in the peer-reviewed literature. The reaerosolization of Bacillus anthracis in outdoor environments is of particular concern because of its stability and potential for use as a biological weapon. This review pulls together data from more than 30 publications, spanning field and laboratory experiments, to summarize the current state of our understanding of Bacillus spp. reaerosolization in outdoor environments.     

Molecular Characterization of Coccidioides spp. Strains Isolated from Patients in the Argentine Republic

Current Fungal Infection Reports - This review summarizes the analysis of the internal transcribed spacers (ITS) sequencing of Coccidioides spp. strains isolated in the endemic region of...     

Isolation and Characterization of Thermophilic Bacillus spp. from Geothermal Environments on Deception Island,South Shetland Archipelago

Abstract Ten bacterial strains isolated from water and sediment samples taken from geothermal areas of Deception Island, in the South Shetland archipelago, were found to represent six distinct types of thermophilic, Gram-positive, aerobic, catalase positive, endospore-forming rods, identified as Bacillus sp. Six representative strains were subjected to routine phenotypic characterization, numerical taxonomic, and chemotaxonomic analyses. Two isolates were identified as thermophilic strains of B. licheniformis and B. megaterium, but the four other strains could not be identified as known species of Bacillus and, hence, may represent new ones. Received: 28 March 1996; Accepted: 29 August 1996     

Molecular Epidemiology and Characterization of Campylobacter spp. Isolated from Wild Bird Populations in Northern England

Campylobacter infections have been reported at prevalences ranging from 2 to 50% in a range of wild bird species, although there have been few studies that have investigated the molecular epidemiology of Campylobacter spp. Consequently, whether wild birds are a source of infection in humans or domestic livestock or are mainly recipients of domestic animal strains and whether separate cycles of infection occur remain unknown. To address these questions, serial cross-sectional surveys of wild bird populations in northern England were carried out over a 2-year period. Fecal samples were collected from 2,084 wild bird individuals and screened for the presence of Campylobacter spp. A total of 56 isolates were recovered from 29 birds sampled at 15 of 167 diverse locales. Campylobacter jejuni, Campylobacter lari, and Campylobacter coli were detected by PCR, and the prevalences of different Campylobacter spp. in different avian families ranged from 0% to 33%. Characterization of 36 C. jejuni isolates by multilocus sequence typing revealed that wild birds carry both livestock-associated and unique strains of C. jejuni. However, the apparent absence of unique wild bird strains of C. jejuni in livestock suggests that the direction of infection is predominantly from livestock to wild birds. C. lari was detected mainly in wild birds sampled in an estuarine or coastal habitat. Fifteen C. lari isolates were analyzed by macrorestriction pulsed-field gel electrophoresis, which revealed genetically diverse populations of C. lari in Eurasian oystercatchers (Haematopus ostralegus) and clonal populations in magpies (Pica pica).Infection with Campylobacter spp. continues to be the leading cause of human infectious intestinal disease in the United Kingdom and has a significant economic impact (39). Consequently, there is a continuing effort to identify effective control methods. The majority of human infections (∼90%) are caused by Campylobacter jejuni subsp. jejuni (46). Other Campylobacter species, including Campylobacter coli and Campylobacter lari, can also cause enteritis in humans, but their prevalence is lower. Most C. jejuni infections are believed to result from consumption of contaminated food, including poultry meat (27, 40), red meat (52), and milk (13), which is thought to be contaminated primarily by feces. It is well established that most livestock species, including poultry, ruminants, and pigs, carry C. jejuni asymptomatically (27), making control at the farm level difficult. However, the epidemiology of C. jejuni cannot be explained solely by food-borne exposure; C. jejuni has also been isolated from a range of environmental samples, including samples of soil, water, sand, and the feces of a number of wildlife species, including wild birds (1-3). However, the role that non-food-borne exposure plays in the epidemiology of C. jejuni is currently not well defined.High prevalences of Campylobacter species infections have been found in a wide range of wild bird species, although there is great variation between taxa (2, 4, 7, 16, 35, 47, 48). Given their ability to fly long distances and their ubiquity, wild birds have the potential to play an important role in the epidemiology and evolution of Campylobacter spp. However, whether wild birds are a source of infection for humans or domestic livestock or are mainly recipients of domestic animal strains or, indeed, whether separate cycles of infection occur remain unknown. These questions remain unanswered in part because investigations of the epidemiology of Campylobacter spp. have been complicated by their high inter- and intraspecies genetic diversity (6).The methods that have been routinely used to characterize Campylobacter isolates are restricted due to genomic instability in Campylobacter populations (10, 38, 45). Multilocus sequence typing (MLST) is a method that has the advantage of being objective since it is sequence based, which allows comparison of isolates from different laboratories and accurate determination of relationships between isolates from diverse sources (11). MLST studies of C. jejuni in farm animals and the environment, including wildlife, suggest that some strains may be associated with particular host groups (6, 10, 15, 30). However, in the same studies other strains were found to occur in several host species or habitats. Few studies have investigated the molecular epidemiology of Campylobacter infection in wild bird populations using MLST, and because only a relatively small number of isolates from wild birds have been characterized by MLST, conclusions have not been drawn yet about how wild bird isolates fit into the overall phylogenetic scheme or whether wild birds act as reservoirs, amplifiers, or merely indicators of infection of domestic animals with zoonotic genotypes.In the current study a large cross-sectional survey of wild bird populations in northern England was undertaken to investigate the epidemiology of Campylobacter infection. Previous studies that have focused on the epidemiology of Campylobacter spp. solely in wild birds have investigated either a narrow range of taxonomic groups (2, 5, 17, 23, 29, 33, 43, 50) or wild birds from a limited range of habitats (18, 25, 48). Studies that have investigated a broad range of wild bird species have used Campylobacter characterization techniques that do not allow conclusions about possible host associations to be drawn or comparison of the genetic diversity of isolates between studies (21, 25, 34, 47, 53). Therefore, the aims of this study were (i) to determine the host range and prevalence of Campylobacter spp. in a wild bird population and (ii) through molecular characterization of isolates to determine whether wild birds were a likely source of infection in humans or domestic livestock and whether separate cycles of infection with host-adapted strains of Campylobacter spp. were maintained in the wild bird population.     

Bacillus endospores isolated from granite: close molecular relationships to globally distributed Bacillus spp. from endolithic and extreme environments

As part of an ongoing effort to catalog spore-forming bacterial populations in environments conducive to interplanetary transfer by natural impacts or by human spaceflight activities, spores of Bacillus spp. were isolated and characterized from the interior of near-subsurface granite rock collected from the Santa Catalina Mountains, AZ. Granite was found to contain approximately 500 cultivable Bacillus spores and approximately 10(4) total cultivable bacteria per gram. Many of the Bacillus isolates produced a previously unreported diffusible blue fluorescent compound. Two strains of eight tested exhibited increased spore UV resistance relative to a standard Bacillus subtilis UV biodosimetry strain. Fifty-six isolates were identified by repetitive extragenic palindromic PCR (rep-PCR) and 16S rRNA gene analysis as most closely related to B. megaterium (15 isolates), B. simplex (23 isolates), B. drentensis (6 isolates), B. niacini (7 isolates), and, likely, a new species related to B. barbaricus (5 isolates). Granite isolates were very closely related to a limited number of Bacillus spp. previously found to inhabit (i) globally distributed endolithic sites such as biodeteriorated murals, stone tombs, underground caverns, and rock concretions and (ii) extreme environments such as Antarctic soils, deep sea floor sediments, and spacecraft assembly facilities. Thus, it appears that the occurrence of Bacillus spp. in endolithic or extreme environments is not accidental but that these environments create unique niches excluding most Bacillus spp. but to which a limited number of Bacillus spp. are specifically adapted.     

芽胞杆菌菌体蛋白提取方法的比较

目的：比较不同破碎方法下3种芽胞杆菌菌体蛋白的提取效果。方法：以菊酯类农药降解菌巨大芽胞杆菌、球形芽胞杆菌、弯曲芽胞杆菌为研究对象,以裂解液为提取介质,分别采用超声波破碎法、水煮法和机械破碎法提取芽胞杆菌蛋白,并通过革兰染色电镜观察菌株破碎程度,用SDS-PAGE和Bradford法测定蛋白浓度。结果：革兰染色电镜观察发现玻璃珠机械破碎法对3种芽孢杆菌的细胞壁破碎程度最明显,其次为超声波破碎法,水煮法破碎效果不明显。SDS-PAGE和Bradford法测定结果表明,巨大芽胞杆菌、球形芽胞杆菌、弯曲芽胞杆菌经玻璃珠机械破碎后,提取的蛋白图谱条带清晰,丰度高,重复性好,含量分别为20.247、19.902和18.893 mg/mL;经超声波破碎提取的蛋白图谱条带较清晰,丰度一般,重复性不好,含量分别为10.572、9.438和10.424 mg/mL;经沸水浴破碎提取的蛋白图谱条带模糊,丰度低,重复性差,含量分别为1.366、1.119和1.136 mg/mL。结论：玻璃珠机械破碎法是破碎3种芽胞杆菌的最优方法,破碎后提取的蛋白含量高,条带清晰,重复性好。     

Archaeabacterial Seryl-tRNA Synthetases: Adaptation to Extreme Environments and Evolutionary Analysis

The aminoacyl-tRNA synthetases are ubiquitous enzymes which catalyze a crucial step of the cell life, the specific attachment of amino acids to their cognate tRNA. The amino acid sequences of three archaeal seryl-tRNA synthetases (SerRS) from Haloarcula marismortui and Methanococcus jannaschii, both belonging to the group of Euryarchaeota, and from Sulfolobus solfataricus, of the group of Crenarchaeota, were aligned with other eubacterial and eukaryal available SerRS sequences. In an attempt to identify some features of adaptation to extreme environments of these organisms, amino acid composition and amino acid substitutions between mesophilic and thermophilic SerRS were analyzed. In addition, universal phylogenetic trees of SerRS including the three known archaeal sequences, rooted by the threonyl-tRNA synthetases were inferred. Amino acid analyses of the SerRS revealed two ways of adaptation to thermophilic environments between the Eubacteria and the Archaea; most of the usually described amino acid substitutions were nonsignificant in the case of archaeal thermophilic SerRS and most amino acid composition biases seemed to be linked to the genome G+C content pressure. The phylogenetic analysis of the SerRS showed the Archaea to be paraphyletic, H. marismortui emerging with the Gram-positive Bacteria, M. jannaschii being near the root of the tree, and S. solfataricus branching with Eucarya. Received: 30 March 1998 / Accepted: 14 July 1998     

Hemolytic and Nonhemolytic Enterotoxin Genes are Broadly Distributed among Bacillus thuringiensis Isolated from Wild Mammals

The presence of cytotoxin K (cytK), nonhemolytic (NHE), and hemolytic (HBL) enterotoxin genes was investigated in 74 Bacillus thuringiensis strains recovered from the intestines of wild mammals from northeast Poland, using polymerase chain reaction amplification and Southern hybridization. All the isolates harbored genes coding for toxin(s) that could cause diarrhea. The B. thuringiensis strains containing the nhe genes were found more frequently (nheA 100%, nheB 77%, nheC 96%) than those with the hblACD (74%) and cytK (73%) genes. The presence/absence of the nheA, hblA, and cytK genes was confirmed in all of the B. thuringiensis strains by Southern hybridization. Interestingly, these experiments also indicated that the nheA locus is located on a more variable chromosome region compared with hblA and, to a lesser degree, cytK. Detection of the 41-kDa component of NHE enterotoxin by the TECRA assay revealed various protein levels by B. thuringiensis strains. These results indicate the existence of environmental B. thuringiensis strains bearing the potential virulence arsenal for the production of diarrheal toxins, and emphasize the importance of small animals in the spread of B. cereus–like enterotoxin genes in nature. However, further investigation is needed to clarify any possible involvement of environmental B. thuringiensis strains in human health issues.     

Molecular Relationships in Biofilm Formation and the Biosynthesis of Exoproducts in Pseudoalteromonas spp.

Marine Biotechnology - Most members of the Pseudoalteromonas genus have been isolated from living surfaces as members of epiphytic and epizooic microbiomes on marine macroorganisms. Commonly...     

Purification and Characterization of Xylanases from Alkalophilic Thermophilic Bacillus spp.

Xylanases from alkalophilic thermophilic Bacillus spp. Wl and W2 were purified and characterized. The xylanases from the two strains were fractionated into two active components (I and II) by DEAE-Toyopearl 650M chromatography. Components I from the two strains had similar properties: optimum pH, 6.0; optimum temperature, 65°C; isoelectric point, pH 8.5 and 8.3; molecular weight, 21,500 and 22,500; and Michaelis constant, 4.5 and 4.0mg-xylan/ml. Components II from the two strains also had similar properties: optimum pH, 7.0~9.0 and 7.0~9.5; optimum temperature, 70°C; isoelectric point, pH 3.6 and 3.7; molecular weight, 49,500 and 50,000; and Michaelis constant, 0.95 and 0.57mg-xylan/ml. The activities of components I and II were inhibited by Hg⁺⁺ and Cu⁺⁺. Components I hydrolyzed xylan to yield xylobiose and higher oligomers, but components II produced xylose other than xylobiose and xylooligomers.     

Novel alkaline serine proteases from alkalophilic Bacillus spp.: purification and some properties

Two extracellular alkaline proteases produced by an alkalophilic Bacillus isolate were purified and characterized using acetone precipitation, DEAE- and CM-Sepharose CL-6B ion exchange and Sephacryl S-200 gel filtration chromatographic techniques. Analysis of the purified proteases by SDS–PAGE revealed that both proteases, AP-1 and AP-2 were homogenous with molecular weight estimates of 28 and 29 kDa, respectively. The optimum activity of AP-1 and AP-2 were at temperatures of 50 and 55°C and pHs of 11 and 12, respectively. The enzymes were also stable in the pH range of 6.0–12.0 for a period of 4 h with and without Ca²⁺ (5 mM) and temperatures of up to 50°C. The half-lives of the enzymes recorded at 50°C were 50 and 40 min for proteases AP-1 and AP-2, respectively. The inhibition profile of the enzymes by phenylmethanesulphonyl fluoride, confirmed these enzymes to be alkaline serine proteases. The purified proteases hydrolysed native protein substrates such as casein, elastin, keratin, albumin and the synthetic chromogenic peptide substrates Glu-Gly-Ala-Phe-pNA and Glu-Ala-Ala-Ala-pNA. The K_m values for the purified proteases were calculated as 1.05 mM and 1.29 mM, respectively, for Glu-Gly-Ala-Phe-pNA, and 3.81 mM and 4.79 mM, respectively, for Glu-Ala-Ala-Ala-pNA as substrates. The kinetic data also indicated that small aliphatic and aromatic amino acids were the preferred residues at the P₁ position.     

Identification and characterization of clinical Bacillus spp. isolates phenotypically similar to Bacillus anthracis

Bacillus anthracis, the etiological agent of anthrax, is a gram-positive, spore-forming rod, with colonies exhibiting a unique ground-glass appearance, and lacking hemolysis and motility. In addition to these phenotypes, several others traits are characteristic of B. anthracis such as susceptibility to gamma phage, the presence of two virulence plasmids (pX01 and pX02), and specific cell wall and capsular antigens that are commonly detected by direct fluorescent-antibody assays. We report on the identification and characterization of 14 Bacillus megaterium and four Bacillus sp. clinical isolates that are nonhemolytic, nonmotile, and produce a capsule antigenically similar to B. anthracis. This work furthers our understanding of Bacillus diversity and the limitations of the assays and phenotypes that are used to differentiate species in this genus. Further work is necessary to understand whether these strains are opportunistic pathogens or just contaminates.