Insights into the genomic and phenotypic characteristics of Bacillus spp. strains isolated from biofilms in broiler farms.

The characterization of surface microbiota living in biofilms within livestock buildings has been relatively unexplored, despite its potential impact on animal health. To enhance our understanding of these microbial communities, we characterized 11 spore-forming strains isolated from two commercial broiler chicken farms. Sequencing of the strains revealed them to belong to three species Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis. Genomic analysis revealed the presence of antimicrobial resistance genes and genes associated with antimicrobial secretion specific to each species. We conducted a comprehensive characterization of the biofilm formed by these strains under various conditions, and we revealed significant structural heterogeneity across the different strains. A macro-colony interaction model was employed to assess the compatibility of these strains to coexist in mixed biofilms. We identified highly competitive B. velezensis strains, which cannot coexist with other Bacillus spp. Using confocal laser scanning microscopy along with a specific dye for extracellular DNA, we uncovered the importance of extracellular DNA for the formation of B. licheniformis biofilms. Altogether, the results highlight the heterogeneity in both genome and biofilm structure among Bacillus spp. isolated from biofilms present within livestock buildings.IMPORTANCELittle is known about the microbial communities that develop on farms in direct contact with animals. Nonpathogenic strains of Bacillus velezensis, Bacillus subtilis, and Bacillus licheniformis were found in biofilm samples collected from surfaces in contact with animals. Significant genetic and phenotypic diversity was described among these Bacillus strains. The strains do not possess mobile antibiotic resistance genes in their genomes and have a strong capacity to form structured biofilms. Among these species, B. velezensis was noted for its high competitiveness compared with the other Bacillus spp. Additionally, the importance of extracellular DNA in the formation of B. licheniformis biofilms was observed. These findings provide insights for the management of these surface microbiota that can influence animal health, such as the use of competitive strains to minimize the establishment of undesirable bacteria or enzymes capable of specifically deconstructing biofilms.

In vitro assessment of the immunomodulatory effects of probiotic Bacillus strains on chicken PBMCs.

The beneficial effects of feeding probiotic Bacillus subtilis DSM 32315 (BS) and Bacillus velezensis CECT 5940 (BV) to chickens in vivo are well-documented, with potential immune modulation as a key mechanism. In this study, we investigated the direct interactions of chicken peripheral blood mononuclear cells (PBMCs) with BS or BV in vitro through whole transcriptome profiling and cytokine array analysis. Transcriptome profiling revealed 20 significantly differentially expressed genes (DEGs) in response to both Bacillus treatments, with twelve DEGs identified in BS-treated PBMCs and eight in BV-treated PBMCs. Pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) indicated significant regulation of immune-related pathways by both BS and BV. Notably, BS treatment upregulated genes associated with immune cell surface markers (CD4, CD25, CD28), anti-inflammatory cytokine interleukin-10 (IL-10), and C-C motif chemokine ligand 5 (CCL5), while downregulating the gene encoding pro-inflammatory IL-16. BV treatment similarly affected genes associated with immune cell surface markers, IL-16, and CCL5, with no impact on the gene encoding IL-10. Both treatments induced higher expression of the gene encoding the avian ß-defensin 1 (AvBD1). The results of this in vitro study indicate an immunomodulatory effect of BS and BV in chicken PBMCs by regulating genes involved in anti-inflammatory, bacteriostatic, protective, and pro-inflammatory responses. Consequently, BS and BV may serve to augment the immune system's capacity to defend against infection by modulating immune responses and cytokine expression. Thus, the administration of these probiotics holds promise for reducing reliance on antimicrobials in farming practices.

Supplementing a Bacillus-based direct-fed microbial improves feed efficiency in lactating dairy cows.

This experiment was conducted to evaluate the effects of feeding a Bacillus-based direct-fed microbial (DFM) on performance and nutrient digestibility of lactating dairy cows. Seventy-six lactating (42 ±â€…6 days in milk [DIM]) Holstein-Friesian primiparous and multiparous cows were enrolled to a 16-wk experiment. Cows were blocked by lactation number and DIM and within blocks, assigned to 1 of the 2 treatments: 1) basal partial-mixed ration (PMR) without DFM addition (n = 38; CON) or 2) basal PMR with the addition of 3 g/head/d of a DFM containing B. licheniformis 809 and B. subtilis 810 (n = 38; BOVACILLUS, Chr. Hansen A/S, Hørsholm, Denmark; DFM). The DFM was mixed in a protein-based pellet, whereas the CON group was fed the same pellet without DFM (0.6 kg/cow/d). The PMR contained (dry matter [DM] basis) 50% of forage and 48% of a concentrate feed based on corn meal, soybean meal, wheat meal, wheat middlings, and a mineral-vitamin premix, with the remaining part of the diet being represented by the pellet used as a carrier for the treatments (CON and DFM). Dry matter intake (DMI), milk yield, and production efficiency were recorded daily, whereas milk protein and fat concentrations were recorded using electronic milk meters. An additional milk sample was collected every second week of the study for milk composition. On week 15 of the study, fecal samples were collected from each cow for apparent nutrient digestibility calculation. All data were analyzed using the MIXED procedure of SAS (version 9.4; SAS Inst. Inc., Cary, NC). No treatment effects were observed on cow final body weight, daily DMI, milk yield, energy-corrected milk (ECM), ECM efficiency, milk composition (yield or content), and somatic cell count (SCC) (P ≥ 0.12). However, cows fed DFM had a greater feed and N efficiency (P ≤ 0.03) compared to cows fed CON. Moreover, DM digestibility tended to be greater for DFM-fed cows when compared to CON (P = 0.10), whereas no further nutrient digestibility differences were observed (P ≥ 0.24). In summary, supplementing a DFM containing Bacillus licheniformis and B. subtilis benefited feed efficiency of lactating dairy cows fed a PMR, while also tending to improve the digestibility of DM.

Bacillus spp. as potential probiotics: promoting piglet growth by improving intestinal health.

The application of Bacillus spp. as probiotics in the swine industry, particularly for piglet production, has garnered significant attention in recent years. This review aimed to summarized the role and mechanisms of Bacillus spp. in promoting growth and maintaining gut health in piglets. Bacillus spp. can enhance intestinal barrier function by promoting the proliferation and repair of intestinal epithelial cells and increasing mucosal barrier integrity, thereby reducing the risk of pathogenic microbial invasion. Additionally, Bacillus spp. can activate the intestinal immune system of piglets, thereby enhancing the body's resistance to diseases. Moreover, Bacillus spp. can optimize the gut microbial community structure, enhance the activity of beneficial bacteria such as Lactobacillus, and inhibit the growth of harmful bacteria such as Escherichia coli, ultimately promoting piglet growth performance and improving feed efficiency. Bacillus spp. has advantages as well as challenges as an animal probiotic, and safety evaluation should be conducted when using the newly isolated Bacillus spp. This review provides a scientific basis for the application of Bacillus spp. in modern piglet production, highlighting their potential in improving the efficiency of livestock production and animal welfare.

Auxin-Producing Bacteria Used as Microbial Biostimulants Improve the Growth of Tomato (Solanum lycopersicum L.) Seedlings in Hydroponic Systems.

Seven auxin-producing endophytic bacterial strains (Azospirillum spp., Methylobacterium symbioticum, Bacillus spp.), and two different combinations of these strains were used to verify their influence on tomato during germination and development in hydroponic conditions where, as a novelty for Canestrino di Lucca cultivar, endophytic bacteria were inoculated. To emphasize the presence of bacterial auxins in roots and stems of seedlings, both in situ staining qualitative assessment and quantitative analysis were carried out. Moreover, hypogeal and epigeal growth of the plantlets were measured, and correlation analyses were conducted to examine the relationship between the amount of indolacetic acid (IAA) produced by the bacterial strains and root and stem parameters. Plantlets treated with microbial inoculants showed a significant increase in the survival rate compared to the control treatment. The best results as IAA producers were from Azospirillum baldaniorum Sp245 and A. brasilense Cd, which also induced significant root growth. On the other hand, Bacillus amyloliquefaciens and B. licheniformis induced the best rates in stem growth. These findings highlight the potential for using endophytic bacterial strains in a hydroponic co-cultivation system that enables inoculating plantlets, at an early stage of growth (5 days old).

Bio-perfume guns: Antifungal volatile activity of Bacillus sp. LNXM12 against postharvest pathogen Botrytis cinerea in tomato and strawberry.

Gray mold disease, caused by Botrytis cinerea is a major postharvest disease impacting fruits such as strawberries and tomatoes. This study explores the use of volatile organic compounds (VOCs) produced by Bacillus spp. as eco-friendly biocontrol agents against B. cinerea. In vitro experiments demonstrated that VOCs from Bacillus sp. LNXM12, B. thuringiensis GBAC46, and B. zhanghouensis LLTC93-VOCs inhibited fungal growth by 61.2%, 40.5%, and 21.6%, respectively, compared to the control. LNXM12 was selected for further experiments due to its highest control efficacy of 58.3% and 76.6% on tomato and strawberry fruits, respectively. The LNXM12 VOCs were identified through gas chromatography-mass spectrometry (GC-MS) analysis, and 22 VOCs were identified. Synthetic VOCs with the highest probability percentage, namely ethyloctynol, 3-methyl-2-pentanone (3M2P), 1,3-butadiene-N, N-dimethylformamide (DMF), and squalene were used in experiments. The results showed that the synthetic VOCs ethyloctynol and 3M2P were highly effective, with an inhibition rate of 56.8 and 57.1% against fungal mycelium radial growth at 120 µg/mL on agar plates. Trypan blue staining revealed strongly disrupted, deeper blue, and lysed mycelium in VOC-treated B. cinerea. The scanning and transmission electron microscope (SEM and TEM) results showed that fungal mycelium was smaller, irregular, and shrunken after synthetic VOC treatments. Furthermore, the synthetic VOCs Ethyloctynol and 3M2P revealed high control efficacy on tomatoes and strawberries infected by B. cinerea. The control efficacy on leaves was 67.2%, 66.1% and 64.5%, 78.4% respectively. Similarly, the control efficiency on fruits was 45.5%, 67.3% and 46.3% 65.1%. The expression of virulence genes in B. cinerea was analyzed, and the results revealed that selected genes BcSpl1, BcXyn11A, BcPG2, BcNoxB, BcNoxR, and BcPG1 were downregulated after VOCs treatment. The overall result revealed novel mechanisms by which Bacillus sp. volatiles control postharvest gray mold disease.

Search for biocontrol agents among endophytic lipopeptide-synthesizing bacteria Bacillus spp. to protect wheat plants against Greenbug aphid (Schizaphis graminum).

Beneficial endophytic bacteria can suppress the development of insect pests through direct antagonism, with the help of metabolites, or indirectly by the induction of systemic resistance through the regulation of hormonal signaling pathways. Lipopeptides are bacterial metabolites that exhibit direct antagonistic activity against many organisms, including insects. Also, lipopeptides are able to trigger induced systemic resistance (ISR) in plants against harmful organisms, but the physiological mechanisms of their action are just beginning to be studied. In this work, we studied ten strains of bacteria isolated from the tissues of wheat and potatoes. Sequencing of the 16S rRNA gene showed that all isolates belong to the genus Bacillus and to two species, B. subtilis and B. velezensis. The genes for lipopeptide synthetase - surfactin synthetase (Bs_srf ), iturin synthetase (Bs_ituA, Bs_ituB) and fengycin synthetase (Bs_fenD) - were identified in all bacterial isolates using PCR. All strains had high aphicidal activity against the Greenbug aphid (Schizaphis graminum Rond.) due to the synthesis of lipopeptides, which was proven using lipopeptide-rich fractions (LRFs) isolated from the strains. Endophytic lipopeptide-synthesizing strains of Bacillus spp. indirectly affected the viability of aphids, the endurance of plants against aphids and triggered ISR in plants, which manifested itself in the regulation of oxidative metabolism and the accumulation of transcripts of the Pr1, Pr2, Pr3, Pr6 and Pr9 genes due to the synthesis of lipopeptides, which was proven using LRF isolated from three strains: B. subtilis 26D, B. subtilis 11VM, and B. thuringiensis B-6066. We have for the first time demonstrated the aphicidal effect of fengycin and the ability of the fengycin-synthesizing strains and isolates, B. subtilis Ttl2, Bacillus sp. Stl7 and B. thuringiensis B-6066, to regulate components of the pro-/antioxidant system of aphid-infested plants. In addition, this work is the first to demonstrate an elicitor role of fengycin in triggering a systemic resistance to S. graminum in wheat plants. We have discovered new promising strains and isolates of endophytes of the genus Bacillus, which may be included in the composition of new biocontrol agents against aphids. One of the criteria for searching for new bacteria active against phloem-feeding insects can be the presence of lipopeptide synthetase genes in the bacterial genome.

Stimulating the biofilm formation of Bacillus populations to mitigate soil antibiotic resistome during insect fertilizer application.

Antibiotic resistance in soil introduced by organic fertilizer application pose a globally recognized threat to human health. Insect organic fertilizer may be a promising alternative due to its low antibiotic resistance. However, it is not yet clear how to regulate soil microbes to reduce antibiotic resistance in organic fertilizer agricultural application. In this study, we investigated soil microbes and antibiotic resistome under black soldier fly organic fertilizer (BOF) application in pot and field systems. Our study shows that BOF could stimulate ARB (antibiotic resistant - bacteria) - suppressive Bacillaceae in the soil microbiome and reduce antibiotic resistome. The carbohydrate transport and metabolism pathway of soil Bacillaceae was strengthened, which accelerated the synthesis and transport of polysaccharides to form biofilm to antagonistic soil ARB, and thus reduced the antibiotic resistance. We further tested the ARB - suppressive Bacillus spp. in a microcosm assay, which resulted in a significant decrease in the presence of ARGs and ARB together with higher abundance in key biofilm formation gene (epsA). This knowledge might help to the development of more efficient bio-fertilizers aimed at mitigating soil antibiotic resistance and enhancing soil health, in particular, under the requirements of global "One Health".

Restoration of the soil fertility under Cr(VI) and artificial drought condition by the utilization of plant growth-promoting Bacillus spp. SSAU2.

The study explores the potential of an indigenous halo-tolerant microbe identified as Bacillus spp. SSAU-2 in enhancing soil fertility and promoting plant growth for sustainable agricultural practices under the influence of multiple abiotic stresses such as Cr(VI), high salinity, and artificial drought condition. The study investigated various factors influencing IAA synthesis by SSAU-2, such as pH (5 to 11), salinity (10 to 50 g/L), tryptophan concentration (0.5 to 1%), carbon (mannitol mand lactose), and nitrogen sources (peptone and tryptone). The highest IAA concentration was observed at pH 10 (1.695 mg/ml) and pH 11 (0.782 mg/ml). IAA synthesis was optimized at a salinity level of 30 g/l, with lower and higher salinity levels resulting in decreased IAA concentrations. Notably, the presence of mannitol and lactose significantly augmented IAA synthesis, while glucose and sucrose had inhibitory effects. Furthermore, peptone and tryptone played a pivotal role in enhancing IAA synthesis, while ammonium chloride exerted an inhibitory influence. SSAU-2 showed a diverse array of capabilities, including the synthesis of gibberellins, extracellular polymeric substances, siderophores, and hydrogen cyanide along with nitrogen fixation and ammonia production. The microbe could efficiently tolerate 45% PEG-6000 concentration and effectively produce IAA in 15% PEG concentration. It could also tolerate high concentration of Cr(VI) and synthesize IAA even in 50 ppm Cr(VI). The findings of this study provide valuable insights into harnessing the potential of indigenous microorganisms to promote plant growth, enhance soil fertility, and establish sustainable agricultural practices essential for restoring the health of ecosystems.

Efficacy of DAP coated with bacterial strains and their metabolites for soil phosphorus availability and maize growth.

Phosphorus (P) use efficiency in alkaline/calcareous soils is only 20% due to precipitation of P2O5 with calcium and magnesium. However, coating Diammonium Phosphate (DAP) with phosphorus solubilizing bacteria (PSB) is more appropriate to increase fertilizer use efficiency. Therefore, with the aim to use inorganic fertilizers more effectively present study was conducted to investigate comparative effect of coated DAP with PSB strains Bacillus subtilis ZE15 (MN003400), Bacillus subtilis ZR3 (MN007185), Bacillus megaterium ZE32 (MN003401) and Bacillus megaterium ZR19 (MN007186) and their extracted metabolites with uncoated DAP under axenic conditions. Gene sequencing was done against various sources of phosphorus to analyze genes responsible for phosphatase activity. Alkaline phosphatase (ALP) gene amplicon of 380bp from all tested strains was showed in 1% w/v gel. Release pattern of P was also improved with coated fertilizer. The results showed that coated phosphatic fertilizer enhanced shoot dry weight by 43 and 46% under bacterial and metabolites coating respectively. Shoot and root length up to 44 and 42% with metabolites coated DAP and 41% with bacterial coated DAP. Physiological attributes also showed significant improvement with coated DAP over conventional. The results supported the application of coated DAP as a useful medium to raise crop yield even at lower application rates i.e., 50 and 75% DAP than non-coated 100% DAP application which advocated this coating technique a promising approach for advancing circular economy and sustainable development in modern agriculture.

Aerobic spore-forming bacteria associated with ropy bread: Identification, characterization and spoilage potential assessment.

Aerobic spore-forming (ASF) bacteria have been reported to cause ropiness in bread. Sticky and stringy degradation, discoloration, and an odor reminiscent of rotting fruit are typical characteristics of ropy bread spoilage. In addition to economic losses, ropy bread spoilage may lead to health risks, as virulent strains of ASF bacteria are not uncommon. However, the lack of systematic approaches to quantify physicochemical spoilage characteristics makes it extremely difficult to assess rope formation in bread. To address this problem, the aim of this study was to identify, characterize and objectively assess the spoilage potential of ASF bacteria associated with ropy bread. Hence, a set of 82 ASF bacteria, including isolates from raw materials and bakery environments as well as strains from international culture collections, were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and their species identity confirmed by 16S rRNA and gyrA or panC gene sequencing. A standardized approach supported by objective colorimetric measurements was developed to assess the rope-inducing potential (RIP) of a strain by inoculating autoclaved bread slices with bacterial spores. In addition, the presence of potential virulence factors such as swarming motility or hemolysis was investigated. This study adds B. velezensis, B. inaquosorum and B. spizizenii to the species potentially implicated of causing ropy bread spoilage. Most importantly, this study introduces a standardized classification protocol for assessing the RIP of a bacterial strain. Colorimetric measurements are used to objectively quantify the degree of breadcrumb discoloration. Furthermore, our results indicate that strains capable of inducing rope spoilage in bread often exhibit swarming motility and virulence factors such as hemolysis, raising important food quality considerations.

Identification of differentially expressed genes and metabolism signaling pathway in the spleen of broilers supplemented with probiotic Bacillus spp.

Probiotics are essential in the body's nutrients, improving the ratio of meat to meat, immune response, and preventing diseases. In this study, RNA-sequencing (RNA-seq) was used to identify the differentially expressed genes (DEGs), enriched related pathways, and Gene Ontology (GO) terms among blank negative control (NC), supplemented with Bacillus spp. (BS) and commercial probiotic (PC) groups after a 42-day fed supplementation. The results showed that 2005, 1356, and 2189 DEGs were significantly altered in BS vs. NC, PC vs NC, and BS vs PC groups, respectively. On the other hand, 9 DEGs were further validated by qRT-PCR, indicating that the qRT-PCR and RNA-Seq results were more consistent. Therefore, the GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of DEGs showed that the DEGs were mainly enriched to metabolism signalling pathways (alpha-linolenic acid metabolism, linoleic acid metabolism, tryptophan metabolism, tyrosine metabolism, ether lipid metabolism, and metabolic pathway, etc) and immune response pathways (cytokine-cytokine receptor interaction, MAPK signalling pathway, and intestinal immune network for IgA production, neuroactive ligand-receptor interaction etc). These results will provide a better understanding of the role of probiotics in chicken development and provide basic information on the genetic development of chickens.

Biocontrol manufacturing and agricultural applications of Bacillus velezensis.

Many microorganisms have been reported as bioagents for producing ecofriendly, cost-effective, and safe products. Some Bacillus species of bacteria can be used in agricultural applications. Bacillus velezensis in particular has shown promising results for controlling destructive phytopathogens and in biofungicide manufacturing. Some B. velezensis strains can promote plant growth and display antibiotic activities against plant pathogen agents. In this review, we focus on the often-overlooked potential properties of B. velezensis as a bioagent for applications that will extend beyond the traditional agricultural uses. We delve into its versatility and future prospects, the challenges such uses may encounter, and some drawbacks associated with B. velezensis-based products.

Unleashing Bacillus species as versatile antagonists: Harnessing the biocontrol potentials of the plant growth-promoting rhizobacteria to combat Macrophomina phaseolina infection in Gloriosa superba.

Charcoal rot caused by Macrophomina phaseolina is one of the most devastating diseases that cause severe yield loss in Gloriosa superba cultivation. Plant growth-promoting rhizobacteria (PGPR) are extensively harnessed as biocontrol agents due to their effectiveness in combating a wide array of plant pathogens through a multifaceted approach. The present study delved into the mechanisms underlying its ability to inhibit root rot pathogen and its capacity to promote plant growth in G. superba, commonly known as glory lily. PGPR isolated from the rhizosphere of glory lily were subjected to in vitro assessments using the dual plate technique. The isolated Bacillus subtilis BGS-10 and B. velezensis BGS-21 showed higher mycelial inhibition (61%) against M. phaseolina. These strains also promote plant growth by producing indole-3-acetic acid, siderophore, ammonia, amylase, cellulase, pectinase, xylanase, and lipase chemicals. Genome screening of BGS-10 and BGS-21 revealed the presence of antimicrobial peptide genes such as Iturin (ituD gene), surfactin (srfA and sfp genes) along with the mycolytic enzyme ß-1,3-glucanase. Further, the presence of secondary metabolites in the bacterial secretome was identified through gas chromatography-mass spectrometry (GC/MS) analysis. Notably, pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl), 9 H-pyrido[3,4-b] indole and L-leucyl-D-leucine exhibited the highest docking score against enzymes responsible for pathogen growth and plant cell wall degradation. Under glasshouse conditions, tuber treatment and soil application of talc-based formulation of B. subtilis BGS-10 and B. velezensis BGS-21 suppress the root rot incidence with a minimal disease incidence of 27.78% over untreated control. Concurrently, there was a notable induction of defense-related enzymes, including peroxidase (PO), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL), in glory lily. Therefore, it can be concluded that plant growth-promoting Bacillus strains play a significant role in fortifying the plant's defense mechanisms against the root rot pathogen.

Characterization of heavy metal-associated bacteria from petroleum-contaminated soil and their resistogram and antibiogram analysis.

The aim of the current study was to screen and identify heavy metal (chromium, cadmium, and lead) associated bacteria from petroleum-contaminated soil of district Muzaffarabad, Azad Jammu and Kashmir, Pakistan to develop ecofriendly technology for contaminated soil remediation. The petroleum-contaminated soil was collected from 99 different localities of district Muzaffarabad and the detection of heavy metals via an atomic absorption spectrometer. The isolation and identification of heavy metals-associated bacteria were done via traditional and molecular methods. Resistogram and antibiogram analysis were also performed using agar well diffusion and agar disc diffusion methods. The isolated bacteria were classified into species, i.e., B. paramycoides, B. albus, B. thuringiensis, B. velezensis, B. anthracis, B. pacificus Burkholderia arboris, Burkholderia reimsis, Burkholderia aenigmatica, and Streptococcus agalactiae. All heavy metals-associated bacteria showed resistance against both high and low concentrations of chromium while sensitive towards high and low concentrations of lead in the range of 3.0 ± 0.0 mm to 13.0 ± 0.0 mm and maximum inhibition was recorded when cadmium was used. Results revealed that some bacteria showed sensitivity towards Sulphonamides, Norfloxacin, Erythromycin, and Tobramycin. It was concluded that chromium-resistant bacteria could be used as a favorable source for chromium remediation from contaminated areas and could be used as a potential microbial filter.

Emergence of multidrug-resistant Bacillus spp. derived from animal feed, food and human diarrhea in South-Eastern Bangladesh.

BACKGROUND: Antimicrobial resistance poses a huge risk to human health worldwide, while Bangladesh is confronting the most severe challenge between the food supply and the huge consumption of antibiotics annually. More importantly, probiotics containing Bacillus spp. are claimed to be an alternative to antimicrobial stewardship programs. However, their antibiotic resistance remains elusive. Thus, we employed the antimicrobial susceptibility test and PCR to assess the prevalence of resistance, including multidrug resistance (MDR) and resito-genotyping of isolated Bacillus spp. RESULTS: The phenotypic profile showed that Bacillus spp. were 100% sensitive to gentamicin (2 µg/mL), whereas lowered sensitivity to levofloxacin (67.8%, 0.5-1 µg/mL), ciprofloxacin (62.3%, 0.5-1 µg/mL), clindamycin (52.2%, 0.25-0.5 µg/mL), amoxicillin-clavulanic acid (37.6%, 0.06 µg/mL), azithromycin (33.4%, 1-2 µg/mL), tetracycline (25.6%, 2-4 µg/mL), nitrofurantoin (21.1%, 16-32 µg/mL), co-trimoxazole (19.2%, 2 µg/mL), and erythromycin (18.8%, 0.25-0.5 µg/mL). The strains were completely resistant to penicillin, amoxicillin-clavulanic acid, cefixime, ceftriaxone, vancomycin, and co-trimoxazole, and a species-specific trend was seen in both phenotypic and genotypic resistance patterns. Genotypic resistance indicated prevalence of the bla1 (71.5%), tetA (33%), erm1 (27%), blaTEM (13.1%), blaCTX-M-1/blaCTX-M-2 /sul1 (10.1%), blaSHV (9.6%), and qnrS (4.1%) genes. The ß-lactamase resistance gene bla1 was found in all penicillin-resistant (MIC ≥ 32 µg/mL) Bacillus spp. One hundred ninety-one isolates (89.6%) were MDR, with 100% from diarrhea, 90.3% from food, and 88.7% from animal feed. CONCLUSION: Based on the MIC value and profile analysis of antibiotic resistance genes, this is the first study that Bacillus spp. antimicrobial susceptibilities have been identified in Bangladesh, and our study will shed light on the adverse effects of feed-borne Bacillus spp. emerging from animal feed to the food chain. A comprehensive investigation is urgently needed by policymakers on tolerance limits and harmful effects in the animal industry.

Bacillus-based biocontrol beyond chemical control in central Africa: the challenge of turning myth into reality.

Agricultural productivity in the Great Lakes Countries of Central Africa, including Burundi, Rwanda, and the Democratic Republic of Congo, is affected by a wide range of diseases and pests which are mainly controlled by chemical pesticides. However, more than 30% of the pesticides used in the region are banned in European Union due to their high toxicity. Globally available safe and eco-friendly biological alternatives to chemicals are virtually non-existent in the region. Bacillus PGPR-based biocontrol products are the most dominant in the market and have proven their efficacy in controlling major plant diseases reported in the region. With this review, we present the current situation of disease and pest management and urge the need to utilize Bacillus-based control as a possible sustainable alternative to chemical pesticides. A repertoire of strains from the Bacillus subtilis group that have shown great potential to antagonize local pathogens is provided, and efforts to promote their use, as well as the search for indigenous and more adapted Bacillus strains to local agro-ecological conditions, should be undertaken to make sustainable agriculture a reality in the region.

Microbiological evaluation of the indigenous fermented condiment okpeye available at various retail markets in the south-eastern region of Nigeria.

In Africa, indigenous fermented condiments contribute to food security as a low-cost source of protein. Okpeye is an indigenous fermented condiment produced from Prosopis africana seeds. The reliance on spontaneous fermentation processes and unhygienic practices during production often results in the contamination of the final product with microbial hazards. A microbiological evaluation of 18 commercial samples of okpeye purchased from six markets in two cities in southeastern Nigeria was conducted. Fifty-nine (59) bacteria were isolated and identified at the species level by phenotyping and sequencing the 16S rRNA, gyrB and rpoB genes. Bacillus (47.4 %) and Staphylococcus (42.3 %) were the predominant bacterial genera in okpeye. Overall, B. amyloliquefaciens and S. simulans were the most frequently occurring bacteria and were present in all samples. In addition, B. cereus was isolated in samples obtained from all markets. Other bacterial species included B. velezensis, Oceanobacillus caeni, S. cohnii, Escherichia fergusonni and Vagacoccus lutrae. The B. cereus isolates (10) were screened for the presence of 8 enterotoxin genes (hblA, hblC, hblD, nheA, nheB, nheC, cytK, entFM) and one emetic gene (cesB). The non-haemolytic enterotoxin (nheABC) and haemolytic enterotoxin (hblABD) complexes were present in 70 % and 50 % of B. cereus respectively. The positive rate of cytK and entFM genes was 70 %, while the cesB gene was 30 %. Antibiotic susceptibility assessment showed that most of the isolates were susceptible to gentamicin, tetracycline, streptomycin, and erythromycin but resistant to ciprofloxacin and vancomycin. These findings highlight the need for further controls to reduce contamination with potential pathogenic bacteria in indigenous fermented condiments such as okpeye. There is also a need to educate producers regarding hygienic practices to safeguard public health and food security.

Effect of deficit irrigation combined with Bacillus simplex on water use efficiency and growth parameters of maize during vegetative stage.

The production of crops depending on many factors including water, nutrient, soil types, climate and crops types, water stress and drought is in one of the important factors affecting crop productivity. The experiment was conducted in pots to evaluate the effect of biofertilizers (Bacillus simplex) with deficit irrigations on the early development and growth of maize crop under greenhouse condition. Pre sowing seed was inoculated with strain of bacteria (B+/B-) and different irrigation levels (no stress: 100% (I1) and deficit irrigation: 75 (I2), 50 (I3), 25 (I4) % of required water amount to reach pot capacity) was performed. Data was collected on different morphological characteristics and root characteristic of maize crop. Highest plant height (125 cm), stem diameter (18.02 mm), leaf area (350 cm- 2), plant weight (180.42 g in fresh, 73.58 g in dry), root length (92.83 cm) root ((91.70 g in fresh, (28.66 g in dry) weight were recorded in pots applied with 100% irrigation followed by 75%. Bacillus treated plants showed significant increase in leaf area (214.20 cm- 2), plant fresh weight (91.65 g) and dry weight (42.05 g), root length (79.20 cm), root fresh (53.52 g) and dry weight (16.70 g) compared with control (without bacteria). Likewise highest relative water content of leaf was observed with I3 followed by I2 and I1 respectively. Highest water use efficiency was recorded as 0.67 g pot- 1 mm- 1 in I1 with B + treatment. Likewise, Bacillus inoculated pots resulted in increased water use efficiency (0.44 g pot- 1 mm- 1) compared with no application (0.36 g pot- 1 mm- 1). It can be endorsed from the outcome that Bacillus inoculation increased plant biomass, root biomass of maize and water use efficiency during early growth stage of maize despite of water stress and can be used under limited water condition for crop combating during moderate to lower stress conditions.

Association of Bacillus subtilis and Bacillus amyloliquefaciens: minimizes the adverse effects of necrotic enteritis in the gastrointestinal tract and improves zootechnical performance in broiler chickens.

This study aimed to evaluate the efficiency and capacity of the probiotic composed of Bacillus subtilis and Bacillus amyloliquefaciens, in improving the zootechnical performance of broiler chickens challenged with Eimeria spp. and Clostridium perfringens. The broilers were distributed in a completely randomized design in poultry isolators (12 birds each), resulting in 3 treatments: T1 (control, no challenge and no Bacillus in diet), T2 (challenged with Eimeria spp., followed by Clostridium perfringens infection and no Bacillus in the diet), and T3 (challenged with Eimeria spp., Clostridium perfringens and treated with Bacillus subtilis and Bacillus amyloliquefaciens). They were evaluated for a period of 29 d, divided into preinitial (1-7 d of age), initial (8-21 d), and growth (22-29 d) phases. Assessments of body weight, weight gain, feed consumption, and feed conversion were conducted, along with the classification of the scores and optical microscopy of the tract gastrointestinal. The animals challenged and treated with the probiotic containing Bacillus spp. showed improved indicators of zootechnical performance. Additionally, the animals challenged and treated (T3) had a better score for intestinal lesions compared to the other treatment groups. Therefore, the probiotic consisting of Bacillus subtilis and Bacillus amyloliquefaciens could be considered an effective option for disease prevention and improving the zootechnical performance of broiler chickens.