Thermo-resistant enzyme-producing microorganisms isolated from composting

Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Efektivitas Sanitizer Komersial Berbasiskan Asam Perasetat terhadap Biofilm Bacillus cereus

Bacillus cereus is known to have the ability to adhere and form biofilms on the surface of stainless steel that causes problems in the food industries. Bacterial biofilms generally can increase resistance to sanitizer treatment. This study aimed to evaluate the ability of peracetic acid-based commercial sanitizer to inactivate B. cereus biofilm on stainless steel (SS) surfaces. Biofilm of B. cereus ATCC 10876 was developed on SS surfaces and treated with 7 commercial peracetic acid-based sanitizers at their recommended dosages. Two sanitizers, i.e. B (peracetic acid and QAC) and F (peracetic acid and acidified water) showing the ability to inactivate B. cereus on solid media at concentration of 200, 400, and 800 ppm were further tested on biofilms with contact times of 1, 3, and 5 minutes. The 48 hours biofilms B. cereus contained 2.78-3.78 CFU/cm2. Both sanitizers B and F had significant effects in inactivating B. cereus biofilm. In general, sanitizer B could reduce more biofilm bacteria at any contact time than sanitizer F. Use of 200 ppm of sanitizer B or F 5 minutes could inactivate 3.04 log CFU/cm2 and 2.68 log CFU/cm2 biofilm, respectively. Exposure of B. cereus biofilm to peracetic acid-based sanitizer resulted in the damage of the extracellular matrix of the biofilms. This study showed that commercial sanitizers containing peracetic acid and quaternary ammonium compounds were effective in inactivating B. cereus biofilms.

Antimicrobial Susceptibility of Persister Biofilm Cells of Bacillus cereus and Pseudomonas fluorescens

The present study evaluates the antimicrobial susceptibility of persister cells of Bacillus cereus and Pseudomonas fluorescens after their regrowth in suspension and as biofilms. Two conventional (benzalkonium chloride—BAC and peracetic acid—PAA) and two emerging biocides (glycolic acid—GA and glyoxal—GO) were selected for this study. Persister cells resulted from biofilms subjected to a critical treatment using the selected biocides. All biocide treatments developed B. cereus persister cells, except PAA that effectively reduced the levels of vegetative cells and endospores. P. fluorescens persister cells comprise viable and viable but non-culturable cells. Afterwards, persister cells were regrown in suspension and in biofilms and were subjected to a second biocide treatment. In general, planktonic cultures of regrown persister cells in suspension lost their antimicrobial tolerance, for both bacteria. Regrown biofilms of persister cells had antimicrobial susceptibility close to those regrown biofilms of biocide-untreated cells, except for regrown biofilms of persister P. fluorescens after BAC treatment, which demonstrated increased antimicrobial tolerance. The most active biocide against persister cells was PAA, which did not promote changes in susceptibility after their regrowth. In conclusion, persister cells are ubiquitous within biofilms and survive after critical biocide treatment. The descendant planktonic and biofilms populations showed similar properties as the original ones.

Phylogenetic and protein prediction analysis reveals the taxonomically diverse distribution of virulence factors in the Bacillus cereus group

Bacillus cereus is a food contaminant with widely varying enterotoxic potential of its virulence proteins. In this article, phylogenetic analysis of the whole-genome amino acid sequences of 41 strains, evolutionary distance calculation of the amino acid sequences of the virulence genes, and functional and structural prediction of the virulence proteins were performed to reveal the taxonomically diverse distribution of virulence factors. The genome evolution of the strains showed a clustering trend based on the coding virulence genes. The strains of B. cereus have evolved into non-toxic risk and toxic risk clusters with medium-high- and medium-low-risk clusters. The distances of evolutionary transfer relative to housekeeping genes of incomplete virulence genes were greater than those of complete virulence genes, and the distance values of HblACD were higher than those of nheABC and CytK among the complete virulence genes. Cytoplasmic localization was impossible for all the virulence proteins, and NheB, NheC, Hbl-B, and Hbl-L 1 were extracellular according to predictive analysis. Nhe and Hbl proteins except CytK had similar spatial structures. The predicted structures of Nhe and Hbl mainly showed ‘head’ and ‘tail’ domains. The ‘head’ of NheA and Hbl-B, including two α-helices separated by β-tongue strands, might play a special role in Nhe trimers and Hbl trimers, respectively. The ‘cap’ of CytK, which includes two ‘latches’ with many β-sheets, formed a β-barrel structure with pores, and a ‘rim’ balanced the structure. The evolution of B. cereus strains showed a clustering tendency based on the coding virulence genes, and the complete virulence-gene operon combination had higher relative genetic stability. The beta-tongue or latch associated with β-sheet folding might play an important role in the binding of virulence structures and pore-forming toxins in B. cereus .

Lactobacillus rhamnosus Ameliorates Multi-Drug-Resistant Bacillus cereus-Induced Cell Damage through Inhibition of NLRP3 Inflammasomes and Apoptosis in Bovine Endometritis

Bacillus cereus, considered a worldwide human food-borne pathogen, has brought serious health risks to humans and animals and huge losses to animal husbandry. The plethora of diverse toxins and drug resistance are the focus for B. cereus. As an alternative treatment to antibiotics, probiotics can effectively alleviate the hazards of super bacteria, food safety, and antibiotic resistance. This study aimed to investigate the frequency and distribution of B. cereus in dairy cows and to evaluate the effects of Lactobacillus rhamnosus in a model of endometritis induced by multi-drug-resistant B. cereus. A strong poisonous strain with a variety of drug resistances was used to establish an endometrial epithelial cell infection model. B. cereus was shown to cause damage to the internal structure, impair the integrity of cells, and activate the inflammatory response, while L. rhamnosus could inhibit cell apoptosis and alleviate this damage. This study indicates that the B. cereus-induced activation of the NLRP3 signal pathway involves K+ efflux. We conclude that LGR-1 may relieve cell destruction by reducing K+ efflux to the extracellular caused by the perforation of the toxins secreted by B. cereus on the cell membrane surface.