Subtilis Groups Research Articles

USE OF PLANT EXTRACT AND PROBIOTICS IN PREVENTING PSEUDOMONAS AERUGINOSA INFECTION IN CLARIAS GARIEPINUS

This study was aimed at investigating the use of plant extract and probiotics (Bacillus subtilis) in preventing bacterial infection in Clarias gariepinus. 120 post-juvenile C. gariepinus were challenged with Pseudomonas aeruginosa and neem leaves extract and B. subtilis were administered concurrently with the infected fish for fourteen days. Clinical signs and mortality were the parameters measured to observe if fresh aqueous neem leaves extract and Bacillus subtilis can prevent P. aeruginosa infection in C. gariepinus. Lesions were more in group infected with P. aeruginosa bacterial only, while lesions were absent to minimal in groups treated with neem extract and Bacillus subtilis. Mortality rate of P. aeruginosa infected group was 30%, while those treated with neem extract and Bacillus subtilis groups were 10 and 5% respectively. Both neem leave extract and B. subtilis were able to prevent P. aeruginosa infection in C. gariepinus base on the clinical sign observed, but mortality rate was higher in group administered with neem than group administered with Bacillus.

The effects of Bacillus subtilis spores and yeast cell wall supplementation on growth and health in Holstein dairy calves

The study aimed to assess the impact of Bacillus subtilis spores and yeast cell wall (YCW) on the performance and health of dairy calves during the milk-feeding period. Thirty female Holstein calves (birth body weight [BW] of 36.7 ± 4.81 kg) were randomly assigned to three treatments: Control (CTL, no additives), T1 (Bacillus subtilis spores), and T2 (Bacillus subtilis + YCW). Animals were individually housed with free access to water and commercial pellet starter. Calves received 8 L/d of pasteurized waste milk and were weaned at 63 d of age. The treatments (T1 and T2) were mixed into milk and administered daily from 3 to 63 d of age. Dry matter (DM) intake (from liquid and starter feed), fecal score, and rectal temperature were recorded daily. Additionally, heart girth was measured at 0, 15, 30, 45, and 60 d of age, and BW was measured at 0 and 60 d of age. Blood was sampled from the jugular vein at 0, 20, and 60 d of age for analysis of serum immunoglobulin G (IgG). There was treatment × time interaction (P = 0.04) for fecal consistency scores. Calves supplemented with Bacillus subtilis tended (χ2 test, P = 0.07) to have lower cases of diarrhea than other groups. T1 or T2 supplementations did not affect milk DM intake, starter DM intake, or total DM intake. Growth performance (BW at weaning and average daily gain) was similar between treatments. Dairy calves fed Bacillus subtilis plus YCW tended to have (P = 0.09) higher rectal temperature and had higher frequency (χ2 test, P < 0.01) of rectal temperature >39.1°C compared to the CTL and Bacillus subtilis groups. However, treatments had similar levels of serum IgG (CTL = 18.3, T1 = 16.7, and T2 = 18.6 mg/mL; P = 0.58). Our results suggest that Bacillus subtills and YCW may interact in the digestive tract of young calves as evidenced by the increased rectal temperature in T2. While the cause of this interaction remains uncertain, there was no negative effect on the animal health or growth performance. The use of Bacillus subtilis spores shows promise in improving fecal consistency scoring in dairy calves fed pasteurized waste milk during the pre-weaning phase.

Effect of probiotics and humate substances on blood parameters, intestinal development and immune organs of growing quail

This study aimed to evaluate impacts of dietary probiotics (Bacillus subtilis PB6) and humate substances (HS) supplementation on histomorphometry of small intestine and immune organs, blood parameters of growing quail. A total of 216 unsexed quails (seven days old) were randomly distributed to six groups. The 1st group did not receive any supplements (control), 2nd group received B. subtilis (CloSTAT: 0.5 g/kg diet), 3rd and 4th groups received HS (4 and 8 g/kg diet, respectively), 5th and 6th groups received CloSTAT + 4g HS and CloSTAT + 8g HS, respectively. Results showed that the inclusion of B. subtilis alone in quail diets significantly improved histomorphometry indices of intestine and immune organs compared to the control. Dietary supplementation of HS alone led to deteriorating histomorphometry indices of intestinal segments and immune organs compared to the control. CloSTAT, HS or both improved lipid profile and antioxidant parameters. Serum mineral levels did not differ significantly among groups except for Ca levels. In conclusion, dietary probiotics supplementation enhanced histomorphometry of intestine and immune organs and improved serum Ca, lipid profile and antioxidant indices. Moreover, the addition of HS (4 or 8 g/kg diet) improved lipid profile and antioxidant indices, but led to undesirable results in intestinal development and immune organs.

In vitro safety evaluation of Bacillus subtilis species complex isolated from Vietnam and their additional beneficial properties

Bacillus genus are Gram-positive, rod shaped, spore-forming, aerobic or facultative anaerobic bacteria, which have the ability to produce a wide variety of enzymes, antimicrobial compounds, vitamins, and carotenoids. Nowadays, Bacillus species have been increasingly proposed for use as probiotics or feed additives as B. amyloliquefaciens, B. licheniformis, B. subtilis, and B. pumilus because of their advantages such as heat-stability of the spores, storage capacity at ambient temperature, and beneficial properties for health. Since microbes are used for customers and livestock, it is critical to substantiate not only the health benefits and efficacy of unique strains but also their safety for the hosts. This study aims to evaluate the in vitro safety and some beneficial properties of Bacillus strains to select potential strains for use in humans or animals. A total of 76 Bacillus strains belonged to the B. amyloliquefaciens and B. subtilis groups were preliminary evaluated for their safety via hemolytic activity. Six non-hemolytic strains were identified and their antibiotic susceptibility, cytotoxicity on the growth of HT29 and Vero cells, extracellular enzyme production, antimicrobial activity, and identification based on 16S rRNA and rpoB gene sequences were studied. Two strains, B. subtilis VTCC 10963 and B. subtilis VTCC 11039, should be considered safe. In addition, these two strains exhibited good extracellular enzyme production (amylase, cellulase, and protease) and strong antibacterial activity against Listeria monocytogenes. Our results support the use of B. subtilis VTCC 10963 and B. subtilis VTCC 11039 for the development of probiotic products and feed additives in the future.

Bacillus subtilis Protects the Ducks from Oxidative Stress Induced by Escherichia coli: Efficacy and Molecular Mechanism.

Bacillus subtilis has been widely used in animal husbandry as a potential alternative to antibiotics due to its excellent bacteriostasis and antioxidant activity. This study aims to investigate the effects of Bacillus subtilis on the protection of ducks from Escherichia coli infection and its mechanism. The four experimental groups include the negative control group, positive control group, antibiotic group and Bacillus subtilis group. Ducks in positive, antibiotic and Bacillus subtilis groups are orally administered with Escherichia coli and equivalent saline solution for the negative group. The results show that supplements with Bacillus subtilis enhances the performance and health status of the infected ducks. Moreover, Bacillus subtilis alleviates the increase in globulin, LPS and MDA, and the decrease in albumin, T-AOC and T-SOD in the serum caused by Escherichia coli infection. Bacillus subtilis also attenuates injury in the intestine and partially reverses the increase in ROS production and the depletion of ATP in the jejunum. These effects are accompanied with the change of related genes of the ribosome (13.54%) and oxidative phosphorylation (6.68%). Collectively, Bacillus subtilis alleviates the damage caused by Escherichia coli infection in ducks by activating ribosome and oxidative phosphorylation signaling to regulate antioxidant and energy metabolism.

A Comparative Analysis of the Core Proteomes within and among the Bacillus subtilis and Bacillus cereus Evolutionary Groups Reveals the Patterns of Lineage- and Species-Specific Adaptations.

By integrating phylogenomic and comparative analyses of 1104 high-quality genome sequences, we identify the core proteins and the lineage-specific fingerprint proteins of the various evolutionary clusters (clades/groups/species) of the Bacillus genus. As fingerprints, we denote those core proteins of a certain lineage that are present only in that particular lineage and absent in any other Bacillus lineage. Thus, these lineage-specific fingerprints are expected to be involved in particular adaptations of that lineage. Intriguingly, with a few notable exceptions, the majority of the Bacillus species demonstrate a rather low number of species-specific fingerprints, with the majority of them being of unknown function. Therefore, species-specific adaptations are mostly attributed to highly unstable (in evolutionary terms) accessory proteomes and possibly to changes at the gene regulation level. A series of comparative analyses consistently demonstrated that the progenitor of the Cereus Clade underwent an extensive genomic expansion of chromosomal protein-coding genes. In addition, the majority (76–82%) of the B. subtilis proteins that are essential or play a significant role in sporulation have close homologs in most species of both the Subtilis and the Cereus Clades. Finally, the identification of lineage-specific fingerprints by this study may allow for the future development of highly specific vaccines, therapeutic molecules, or rapid and low-cost molecular tests for species identification.

Effects of Dietary Supplementation With Bacillus subtilis, as an Alternative to Antibiotics, on Growth Performance, Serum Immunity, and Intestinal Health in Broiler Chickens.

Bacillus subtilis (B. subtilis) as in-feed probiotics is a potential alternative for antibiotic growth promoters (AGP) in the poultry industry. The current study investigated the effects of B. subtilis on the performance, immunity, gut microbiota, and intestinal barrier function of broiler chickens. A 42-day feeding trial was conducted with a total of 600 1-day-old Arbor Acres broilers with similar initial body weight, which was randomly divided into one of five dietary treatments: the basal diet (Ctrl), Ctrl + virginiamycin (AGP), Ctrl + B. subtilis A (BSA), Ctrl + B. subtilis B (BSB), and Ctrl + B. subtilis A + B (1:1, BSAB). The results showed significantly increased average daily gain in a step-wise manner from the control, B. subtilis, and to the AGP groups. The mortality rate of the B. subtilis group was significantly lower than the AGP group. The concentrations of serum immunoglobulin (Ig) G (IgG), IgA, and IgM in the B. subtilis and AGP groups were higher than the control group, and the B. subtilis groups had the highest content of serum lysozyme and relative weight of thymus. Dietary B. subtilis increased the relative length of ileum and the relative weight of jejunum compared with the AGP group. The villus height (V), crypt depth (C), V/C, and intestinal wall thickness of the jejunum in the B. subtilis and AGP groups were increased relative to the control group. Dietary B. subtilis increased the messenger RNA (mRNA) expression of ZO-1, Occludin, and Claudin-1, the same as AGP. The contents of lactic acid, succinic acid, and butyric acid in the ileum and cecum were increased by dietary B. subtilis. Dietary B. subtilis significantly increased the lactobacillus and bifidobacteria in the ileum and cecum and decreased the coliforms and Clostridium perfringens in the cecum. The improved performance and decreased mortality rate observed in the feeding trial could be accrued to the positive effects of B. subtilis on the immune response capacity, gut health, and gut microflora balance, and the combination of two strains showed additional benefits on the intestinal morphology and tight junction protein expressions. Therefore, it can be concluded that dietary B. subtilis A and B could be used as alternatives to synthetic antibiotics in the promotion of gut health and productivity index in broiler production.

Comparative Genome Analysis of Bacillus sporothermodurans with Its Closest Phylogenetic Neighbor, Bacillus oleronius, and Bacillus cereus and Bacillus subtilis Groups.

Bacillus sporothermodurans currently possesses one of the most highly heat-resistant spores (HRS), which can withstand ultra-high temperature (UHT) processing. Determination of multiple whole genome sequences of B. sporothermodurans provided an opportunity to perform the first comparative genome analysis between strains and with B. oleronius, B. cereus, and B. subtilis groups. In this study, five whole genome sequences of B. sporothermodurans strains, including those belonging to the HRS clone (SAD and BR12) normally isolated from UHT milk, were compared with the aforementioned Bacillus species for gene clusters responsible for heat resistance. In the phylogenomic analysis, B. sporothermodurans, with its closest phylogenetic neighbor, B. oleronius, clustered with B. thermoamylovorans and B. thermotolerans. Heat shock proteins GrpE, GroES, GroEL, and DnaK presented identical sequences for all B. sporothermodurans strains, indicating that differences in functional efficiency are not involved in the thermal resistance variations. However, comparing all species evaluated, B. sporothermodurans exhibited a different gene configuration in the chromosomal region of the heat shock protein GrpE. Furthermore, only B. sporothermodurans strains presented the stage II sporulation protein P gene located in this region. Multisequence alignment and phylogenetic analysis of the ClpB protein showed differences for HRS and non-HRS strains. The study identified ClpC, ClpE, and ClpX as the three ATPases putatively involved in protein disaggregation in B. sporothermodurans. Bacillus sporothermodurans exhibits high homology with other Bacillus species in the DnaK, DnaJ, GroEL, and GroES cluster of genes involved in heat resistance. The data presented here pave the way to select and evaluate the phenotypic effects of genes putatively involved in heat resistance.

Combined Effects of Dietary Bacillus subtilis and Trans-cinnamic Acid on Growth Performance, Whole Body Compositions, Digestive Enzymes and Intestinal bacteria in Rainbow Trout (Oncorhynchus mykiss)

In this study, the combined effects of dietary Bacillus subtilis (BS, 10 7 g/cfu) and different levels (0.025%, 0.050%, 0.075% and 0.150%) of trans-cinnamic acid (CA) on fish growth performance, whole body compositions, digestive enzymes, intestinal bacteria and internal organ index of rainbow trout (Oncorhynchus mykiss) were investigated. Six different experimental groups including control group (C), C+BS, 0.025%CA+BS, 0.050%CA+BS, 0.075CA+BS, 0.150%CA+BS) were established. According to the results obtained, growth performance, whole body compositions and digestive pH were not statistically significant among groups. Further, no significant differences were found between experimental groups in terms of the intestinal enzymes (trypsin, alkaline phosphatase and lipase) and gastric pepsin. Significantly higher levels of intestinal amylase were found in the control+BS, 0.025%CA+BS, 0.050% CA+BS, and 0.075%CA+BS compared to the control and 0.150%CA+BS groups. Moreover, coliform and Enterobacteriaceae counts were highest in the control+B. subtilis and lowest in the 0.150% CA + B. subtilis groups.

&amp;lt;i&amp;gt;Bacillus cereus&amp;lt;/i&amp;gt; Group Exhibits More Resistant to Chlorhexidine Rather Than &amp;lt;i&amp;gt;Bacillus subtilis&amp;lt;/i&amp;gt; Group

Chlorhexidine is a widely used antiseptic agent; however, its bactericidal effect against bacterial endospores is limited. The genus Bacillus is spore-forming gram-positive bacteria that are ubiquitously found in the environment and cause opportunistic infection and food poisoning. The susceptibility of bacterial endospores to chlorhexidine was previously evaluated in Bacillus subtilis, but the primary target for disinfection with antiseptic agents, including chlorhexidine, should be harmful strains. We aimed to evaluate the susceptibility of harmful cereus group including Bacillus cereus, and to compare that with harmless Bacillus species, containing B. subtilis. We evaluated the susceptibility of the 15 strains of the cereus group to chlorhexidine in comparison with the 5 other strains, named the subtilis group in this study. Our results indicated that chlorhexidine exerted a bacteriostatic effect against Bacillus species at practical concentrations, especially during long-term exposure. The strains of B. cereus group in this study displayed relatively lower susceptibility to the antiseptic than the B. subtilis group according to the minimum inhibitory and bactericidal concentrations. We concluded that there are intrinsic differences in the susceptibility to chlorhexidine between the groups, but the molecular mechanisms are unknown. The minimum inhibitory or bactericidal concentrations of disinfectants other than chlorhexidine may also need to be clarified in the B. cereus and B. subtilis groups.

Effect of oral spray with Lactobacillus on growth performance, intestinal development and microflora population of ducklings

ObjectiveThe aim of this study is to investigate the effect of oral spray with probiotics on the intestinal development and microflora colonization of hatched ducklings.MethodsIn Exp. 1, an one-way factorial design was used to study the antibacterial activity of the probiotics and metabolites on Escherichia coli (E. coli) without antimicrobial resistance. There were four experimental groups including saline as control and Lactobacillus, Bacillus subtilis, combined Lactobacillus and Bacillus subtilis groups. In Exp. 2, 64-day-old ducklings were allotted to 2 treatments with 4 replicated pens. Birds in the control group were fed a basal diet supplemented with Lactobacillus fermentation in the feed whereas birds in the oral spray group were fed the basal diet and administrated Lactobacillus fermentation by oral spray way during the first week.ResultsIn Exp. 1, the antibacterial activities of probiotics and metabolites on E. coli were determined by the diameter of inhibition zone in order: Lactobacillus>combined Lactobacillus and Bacillus subtilis>Bacillus subtilis. Additionally, compared to E. coli without resistance, E. coli with resistance showed a smaller diameter of inhibition zones. In Exp. 2, compared to control feeding group, oral spray group increased (p<0.05) the final body weight at d 21 and average daily gain for d 1–21 and the absolute weight of the jejunum, ileum and total intestine tract as well as cecum Lactobacillus amount at d 21.ConclusionLactobacillus exhibited a lower antibacterial activity on E. coli with resistance than E. coli without resistance. Oral spray with Lactobacillus fermentation during the first week of could improve the intestinal development, morphological structure, and microbial balance to promote growth performance of ducklings from hatch to 21 d of age.

Dietary Bacillus subtilis B2A strain in laying hens challenged with Salmonella gallinarum: effects on egg production, egg quality, blood haptoglobin and targeted intestinal Salmonella shedding

ABSTRACTA total of 240 Hy-Line 40-week-old Salmonella-free layers were assigned to 5 dietary treatments composed of 12 replications, with 4 laying hens per replications in a 5-week experiment trial on laying hens challenged with Salmonella gallinarum. Dietary treatment comprised: (1) NC, basal diet; (2) PC, NC + oral S. gallinarum administration; (3) B8, PC + 0.1% Bacillus subtilis (108cfu/g); (4) B9, PC + 0.1% B. subtilis (109cfu/g) and (5) B10, PC + 0.1% B. subtilis (1010cfu/g). B. subtilis groups reduced the population of S. gallinarum in both large intestine and excreta compared with PC laying hens. The population of S. gallinarum in challenged laying hens fed B. subtilis was significantly lower in small intestine, large intestine and excreta. In the small intestine, Lactobacillus population was higher in B10 compared with PC. Addition of B. subtilis improved eggshell thickness. Egg shell strength showed an improvement in week 5 and it was higher in B9 and B10 compared with PC. Haptoglobin concentration was higher in the PC group compared with other groups. In conclusion, diets supplemented with B. subtilis as a novel anti-salmonella bacteria show potential for decreasing S. gallinarium in the intestinal tract and improving egg gravity, eggshell strength and eggshell quality during the laying period.

Effects of dietary Bacillus subtilis and inulin supplementation on performance, eggshell quality, intestinal morphology and microflora composition of laying hens in the late phase of production

Eighty Lohmann White laying hens were used to investigate the effect of dietary inclusion of Bacillus subtilis and inulin, individually or in combination, on egg production, eggshell quality, tibia traits, Ca retention, and small intestine morphology and microflora composition from 64 to 75 weeks of age. Hens were randomly distributed into 4 treatment groups, with 5 replicates per treatment and 4 hens per replicate. Treatment groups were fed basal diet (control), basal diet plus 1g/kg B. subtilis (2.3×108cfu/g), basal diet plus 1g/kg inulin, or basal diet plus a synbiotic combination of 1g/kg B. subtilis (2.3×108cfu/g) and 1g/kg inulin. Dietary supplementation of B. subtilis, inulin or synbiotic improved (P<0.05) feed conversion, egg performance, eggshell quality and calcium retention compared with the control. B. subtilis and synbiotic groups exhibited the highest (P<0.05) increase in egg production and egg weight. Inulin and synbiotic groups exhibited the highest (P<0.05) increase in eggshell thickness and eggshell calcium content, and the lowest (P<0.05) eggshell deformations. Unmarketable eggs were 8.4% (P<0.05) of the total eggs produced by the control group compared to 3.5%, 1.7%, and 1.5% for the B. subtilis, inulin and synbiotic groups, respectively. Tibia density, ash, and Ca content increased (P<0.05) by inulin and synbiotic inclusions, compared with the control. B. subtilis, inulin, and their synbiotic combination increased (P<0.05) villus height and crypt depth in all intestinal segments, compared with the control. B. subtilis and inulin modulated the ileal and caecal microflora composition by decreasing (P<0.05) numbers of Clostridium and Coliforms and increasing (P<0.05) numbers of bifidobacteria and lactobacilli, compared with the control. Colonization of the beneficial microflora along with increasing the villi–crypts absorptive area were directly associated with the improvements in performance and eggshell quality. It can be concluded that egg production and eggshell quality of laying hens can be improved (P<0.05) in the late phase of production by dietary inclusion of B. subtilis and inulin.

In vivo assessment for oral delivery of Bacillus subtilis harboring a viral protein (VP28) against white spot syndrome virus in Litopenaeus vannamei

Bacillus subtilis spores, being consumed as probiotics, have been explored as live carriers for expression and oral delivery of antigen proteins. In our initial experiment, by oral delivery of B. subtilis spores harboring VP28 (rVP28-bs) to Litopenaeus vannamei, the extremely high survival (Relative Percent Survival: 83.3%) upon challenge with white spot syndrome virus (WSSV) can be observed. After ‘vaccination’ with rVP28-bs, the hemocytic phagocytosis and immune-related gene expression levels in hemocytes were analyzed. The percentage of haemocytes phagocytosing WSSV was significantly higher ( p < 0.001) in shrimp previously fed with rVP28-bs (48.2 ± 6.3) than the controls (11.0 ± 3.5 and 8.1 ± 2.5). However, there were no significant differences ( p > 0.05) in all the experimental groups for the percentage phagocytosis of TSV (an unrelated virus of shrimp). This suggests that the heightened phagocytic activity, and thus the high-level survival of shrimp after rVP28-bs ‘vaccination’ are selective or specific towards WSSV. Moreover, immune-related genes (proPO, PE and LGBP) were significantly ( p < 0.05) upregulated in both rVP28-bs and B. subtilis feeding groups compared to the control, though no significant differences ( p > 0.05) were observed between rVP28-bs and B. subtilis groups. It was indicated that the phagocytosis enhanced by rVP28-bs was the essential one to protect shrimp from virus infection, while the rVP28-bs-stimulated humoral response only plays an assistant role in antiviral defense of shrimp. Besides, in vivo fate and dissemination assays of rVP28-bs spores showed that, as robust life forms, spores can survive transit across the gut tract, germinate to express rVP28 and exert probiotic action, and disseminate in the haemolymph to present VP28 to the shrimp defense system before being excreted. These results may raise the application prospective of this recombinant B. subtilis spores against WSSV infection in shrimp farms.

A taxonomic review of the subgenus Hyparpalus (genus Parophonus) of the Oriental and Australian regions (Coleoptera: Carabidae: Harpalini)

A taxonomic review of the subgenus Hyparpalus Alluaud, 1930 (genus Parophonus Ganglbauer, 1892) of the Oriental and Australian regions is given. Nine species are recognised in the fauna of the Oriental Region, one of which reaches the northern Australia. The Oriental species are divided in three species groups: the cyaneotinctus (two species), subtilis (three species) and javanus (four species) groups. Distinctive characters and a key for identification of these groups and all the included species are provided. Relationships between Hyparpalus of the Oriental and Afrotropical regions and status of the Afrotropical taxon Orphanixus Clarke, 1971 are discussed. The following new synonymies are established: Parophonus subtilis (Bates, 1892) = Hyparpalus iridicolor Landin, 1955, syn. nov. = H. nagpurensis Ito, 1993, syn. nov. = H. nagpurensis curvatus Ito, 1993, syn. nov. = H. pakistanensis Ito, 2008, syn. nov.; Parophonus lividus (Andrewes, 1923) = Hyparpalus hiekei Ito, 1993, syn. nov.; Parophonus vitalisi (Andrewes, 1922) = Hyparpalus maniti Ito, 1993, syn. nov. = H. maniti similis Ito, 1993, syn. nov.; Parophonus acutangulus (Bates, 1891) = Hyparpalus gracilis Andrewes, 1947, syn. nov.; Parophonus javanus (Gory, 1833) = Diaphoromerus opacus MacLeay, 1888, syn. nov. = Parophonus australicus Baehr, 1986, syn. nov. Lectotypes are designated for Hypolithus cyaneotinctus Bates, 1889, H. cyanellus Bates, 1889 and H. acutangulus Bates, 1891.

Genotypic and phenotypic diversity of Bacillus spp. isolated from steel plant waste.

BackgroundMolecular studies of Bacillus diversity in various environments have been reported. However, there have been few investigations concerning Bacillus in steel plant environments. In this study, genotypic and phenotypic diversity and phylogenetic relationships among 40 bacterial isolates recovered from steel plant waste were investigated using classical and molecular methods.Results16S rDNA partial sequencing assigned all the isolates to the Bacillus genus, with close genetic relatedness to the Bacillus subtilis and Bacillus cereus groups, and to the species Bacillus sphaericus. tDNA-intergenic spacer length polymorphisms and the 16S–23S intergenic transcribed spacer region failed to identify the isolates at the species level. Genomic diversity was investigated by molecular typing with rep (repetitive sequence) based PCR using the primer sets ERIC2 (enterobacterial repetitive intergenic consensus), (GTG)5, and BOXAIR. Genotypic fingerprinting of the isolates reflected high intraspecies and interspecies diversity. Clustering of the isolates using ERIC-PCR fingerprinting was similar to that obtained from the 16S rRNA gene phylogenetic tree, indicating the potential of the former technique as a simple and useful tool for examining relationships among unknown Bacillus spp. Physiological, biochemical and heavy metal susceptibility profiles also indicated considerable phenotypic diversity. Among the heavy metal compounds tested Zn, Pb and Cu were least toxic to the bacterial isolates, whereas Ag inhibited all isolates at 0.001 mM.ConclusionIsolates with identical 16S rRNA gene sequences had different genomic fingerprints and differed considerably in their physiological capabilities, so the high levels of phenotypic diversity found in this study are likely to have ecological relevance.

Genus-wide Bacillus species identification through proper artificial neural network experiments on fatty acid profiles

Gas chromatographic fatty acid methyl ester analysis of bacteria is an easy, cheap and fast-automated identification tool routinely used in microbiological research. This paper reports on the application of artificial neural networks for genus-wide FAME-based identification of Bacillus species. Using 1,071 FAME profiles covering a genus-wide spectrum of 477 strains and 82 species, different balanced and imbalanced data sets have been created according to different validation methods and model parameters. Following training and validation, each classifier was evaluated on its ability to identify the profiles of a test set. Comparison of the classifiers showed a good identification rate favoring the imbalanced data sets. The presence of the Bacillus cereus and Bacillus subtilis groups made clear that it is of great importance to take into account the limitations of FAME analysis resolution for the construction of identification models. Indeed, as members of such a group cannot easily be distinguished from one another based upon FAME data alone, identification models built upon this data can neither be successful at keeping them apart. Comparison of the different experimental setups ultimately led to a few general recommendations. With respect to the routinely used commercial Sherlock Microbial Identification System (MIS, Microbial ID, Inc. (MIDI), Newark, Delaware, USA), the artificial neural network test results showed a significant improvement in Bacillus species identification. These results indicate that machine learning techniques such as artificial neural networks are most promising tools for FAME-based classification and identification of bacterial species.

Species-level identification of Bacillus strains isolates from marine sediments by conventional biochemical, 16S rRNA gene sequencing and inter-tRNA gene sequence lengths analysis

The aim of this study was to compare the ability of commonly used conventional biochemical tests, sequencing analysis of 16S rRNA genes and tDNA-intergenic spacer length polymorphism (tDNA-PCR) to identify species of the genus Bacillus recovered from marine sediments. While biochemical tests were not sufficiently sensitive to distinguish between the 23 marine strains analyzed, partial 16S rRNA gene sequences allowed a correct identification, clustering them into four species belonging to Bacillus licheniformis (n = 6), Bacillus cereus (n = 9), Bacillus subtilis (n = 7) and Bacillus pumilus (n = 1). The identification results obtained with 16S rRNA sequencing were validated by tDNA-PCR analysis of 23 marine isolates that were identified by the similarities of their fingerprints to those of reference strains. tDNA-PCR fingerprinting was as discriminatory as 16S rRNA sequencing analysis. Although it was not able to distinguish among the species of the B. cereus and B. subtilis groups, it should be considered a rapid and easy approach for the reliable identification of unknown Bacillus isolates or at least for the primary differentiation of Bacillus groups.

Effect of dietary supplementation of probiotic and vitamin C on the immune response of Indian major carp, Labeo rohita (Ham.)

The immunostimulatory effect of probiotics and vitamin C has been established in many systems including fish. An investigation was carried out to study the effect of dietary supplementation of a probiotic bacterium “ Bacillus subtilis”, vitamin C in the form of ascorbyl polyphosphate and their combination on the immune response of Indian major carp, rohu, ( Labeo rohita Ham.) fingerlings fed for a period of 60 days. The total serum protein and globulin content was significantly higher ( p < 0.05) in probiotic ( B. subtilis @ 10 8 CFU/g of the feed) fed group while the respiratory burst activity of blood neutrophils was significantly high in vitamin C (ascorbyl polyphosphate @100 mg per kg diet) fed group. The antibody level was significantly high in Bacillus subtilis treated group followed by the probiotic ( B. subtilis @ 10 8 CFU/g of the feed) and ascorbyl polyphosphate (ascorbyl polyphosphate @100 mg per kg diet) combined group. The least percentage of mortality was recorded in B. subtilis treated group (25%) followed by 35 and 40% in ascorbyl polyphosphate treated and B. subtilis and ascorbyl polyphosphate combined groups, respectively.

A Molecular Comparison of Plumage and Soil Bacteria Across Biogeographic, Ecological, and Taxonomic Scales

We used molecular methods to determine the microbial community of soil and avian plumage across biogeographic, ecological, and taxonomic scales. A total of 17 soil and 116 feather samples were collected from five avian species across multiple habitat types within one Neotropical and one temperate locality. Hypotheses regarding patterns of microbial composition relative to acquisition and dispersal of plumage bacteria in the ecosystem were tested by comparing microbial communities within and between soil and plumage. Samples from the plumage of American Redstarts (Setophaga ruticilla) were collected across both habitat types and geographic scales for intraspecific comparisons. The microbial diversity in avian plumage was moderately diverse and was dominated by Pseudomonas species. Despite a highly significant individual bird effect on microbial composition of the plumage, we detected significant biogeographic and type of habitat effects. Pseudomonas species were more abundant on the temperate site when all avian species were included in the analysis, and Bacillus subtilis and Xanthomonas groups were more abundant on the Neotropical site for redstarts alone. However, 16S rDNA sequence libraries were not significantly different between Jamaican and Maryland redstarts. Biogeographic and habitat effects were significant and more pronounced for soil samples indicating lower dispersal of soil microbiota. We detected a significant difference between soil and plumage microbial communities suggesting that soil plays a small role in plumage bacterial acquisition. Our results suggest bacterial communities on the plumage of birds are dynamic and may change at different stages in a bird's annual cycle.