Cytotoxicity of some pesticides is a disadvantage for the Salmonella/microsome assay with regard to the equivalence assessment of pesticide technical grade active ingredients to the original products ...and detection of low-level impurities. The technical grade active ingredients (TGAIs) of pesticides from certain chemical classes were found to be toxic for Salmonella typhimurium strains. Among the highly cytotoxic compounds were sulfonylureas, which include 20 active ingredients. In addition, this class includes active pharmaceutical ingredients used for the manufacture of antidiabetics drugs.
A traditional selection methodology was applied using the cultivation of S. typhimurium TA100 in the presence of high concentrations of thifensulfuronmethyl (TFSM) to obtain a resistant test strain insusceptible to sulfonylurea toxic effect. Two strains resistant not only to sulfonylureas (SFU) but also triazolepyrimidines were received. The first mutant strain (deposited as S. typhimurium VKPM B-14099 in the Russian National Collection of Industrial Microorganisms) demonstrated the TA100 phenotypic characteristics: hisG46, rfa, ΔuvrB-bio, pKM101. The second strain (deposited as S. typhimurium VKPM B-14359) showed the TA1535 phenotypic characteristics and probably lost the R-factor due to the selection using the poor Gm-media with TFSM. Positive controls caused pronounced mutagenic effects (±S9) in both strains, consequently the mutants did not lose the ability to respond to induction of the reverse gene mutations. The maximum non-cytotoxic concentrations of SFUs and triazole-pyrimidines for the Ames test strains did not exceed 0.05–0.125 mg/plate, while no evidence of cytotoxicity was observed for the mutants up to 5.0 mg/plate. Electron microscopy of the ultrathin sections of Salmonella cells grown with and without TFSM showed an obvious difference in the structure of the cell wall and cytoplasm in mutant and parental cultures. The concurrent resistance both to SFU and triazolepyrimidines was assumed to be mediated by the same mechanism of action of the pesticides from these classes – inhibition of acetohydroxyacid synthase. To confirm this hypothesis, the tests in the presence of branched-chain amino acids were carried out. The enrichment of agar with isoleucine prevented the toxic effects of SFU and triazolepyrimidines for all Ames test strains used in the study, while strong cytotoxicity was observed in the presence of valine and leucine.
Considering the tolerance of strains both to SFU and triazolpyrimidines and the results with branched-chain amino acids, the modification of target acetohydroxyacid synthase was supposed the key to the acquired resistance. The new strains resistant to sulfonylureas and triazole-pyrimidines expands the possibilities to reveal mutagenic impurities that may occur in TGAIs in small amounts.
•Improvement of the Ames tester strains for the detection of cytotoxic chemicals.•Mutants resistant to acetohydroxyacid synthase inhibitors were derived from TA100.•Enrichment of agar with isoleucine prevented the toxicity for the Ames test strains.•Cultivation with thifensulfuron-methyl leads to alteration of cell envelope.
Planctomycetes of the family
Pirellulaceae
are commonly addressed as budding aquatic bacteria with a complex lifestyle. Although this family is well represented by cultured and taxonomically ...characterized isolates, nearly all of them were obtained from brackish or marine habitats. The examples of described freshwater
Pirellulaceae
planctomycetes are limited to two species only,
Pirellula staley
and ‘
Anatilimnocola aggregata
’. In this study, we characterized a novel freshwater planctomycete of the genus ‘
Anatilimnocola
’, strain PX40
T
, which was isolated from a boreal eutrophic lake. Strain PX40
T
was represented by budding, unpigmented, ellipsoidal to pear-shaped cells, which often occurred in characteristic flower-like rosettes. Cells were covered by bundles of fimbriae; crateriform-like structures were localized on a reproductive cell pole only. These planctomycetes were obligately aerobic, heterotrophic bacteria that utilized various sugars and some polysaccharides, and were highly sensitive to NaCl. Growth occurred in the pH range 5.0–7.5 (with an optimum at pH 6.5–7.0), and at temperatures between 15 and 30 °C (with an optimum at 22–25 °C). The major fatty acids of strain PX40
T
were C18:1
ω
9c, C16:0, and 16:1
ω
7c; cells also contained a wide variety of hydroxy- and dihydroxy-fatty acids and a C31:9 alkene. The major intact polar lipids were diacylglyceryl-(N,N,N)-trimethylhomoserines. The 16S rRNA gene sequence of strain PX40
T
displayed 96.6% similarity to that of ‘
Anatilimnocola aggregata
’ ETA_A8
T
. The genome of strain PX40
T
was 8.93 Mb in size and contained one copy of rRNA operon, 76 tRNA genes and 7092 potential protein-coding genes. The DNA G+C content was 57.8%. The ANI value between strain PX40
T
and ‘
Anatilimnocola aggregata
’ ETA_A8
T
was 78.3%, suggesting that these planctomycetes represent distinct species. We, therefore, propose a novel species of the genus ‘
Anatilimnocola
’, ‘
A. floriformis’
sp. nov., with strain PX40
T
(= KCTC 92369
T
= VKM B-3621
T
= UQM 41463
T
) as the type strain.
This study of lichens in the subarctic zone of the northern hemisphere has resulted in the detection of new representatives of the order
Rhizobiales
. The16S rRNA gene sequence phylogeny placed the ...strains as a separate branch inside the
Rhizobiales
clade. Strain RmlP001
T
exhibits 91.85% similarity to
Roseiarcus fermentans
strain Pf56
T
and 91.76% to
Beijerinckia doebereinerae
strain LMG 2819
T
, whilst strain RmlP026
T
is closely related to
B. doebereinerae
strain LMG 2819
T
(91.85%) and
Microvirga pakistanensis
strain NCCP-1258
T
(91.39%). A whole-genome phylogeny of the strains confirmed their taxonomic positions. The cells of both strains were observed to be Gram-negative, motile rods that multiplied by binary fission. The cells were found to contain poly-
β
-hydroxybutyrate and polyphosphate, to grow at pH 3.5–8.0 and 10–30 °C, and could not fix atmospheric nitrogen. Their major cellular fatty acid identified was C
18:1
ω
7c (68–71%) and their DNA G + C contents determined to be 70.5–70.8%. Beta-carotene was identified as their major carotenoid pigment; Q-10 was the only ubiquinone detected. Strains RmlP001
T
and RmlP026
T
are distinguishable from related species by the presence of β-carotene, the absence of C1 metabolism and the ability to grow in the presence of 3.5% NaCl. Based on their phylogenetic, phenotypic and chemotaxonomic features, we propose a novel genus
Lichenibacterium
and two novel species
, Lichenibacterium ramalinae
(the type species of the genus) and
Lichenibacterium minor,
to accommodate these bacteria within the family
Lichenibacteriaceae
fam. nov. of the order
Rhizobiales
. The
L. ramalinae
type strain is RmlP001
T
(= KCTC 72076
T
= VKM B-3263
T
) and the
L. minor
type strain is RmlP026
T
(= KCTC 72077
T
= VKM B-3277
T
).
Biodegradation of phenol is an effective method for removing this toxicant from contaminated sites. Phenol is a toxic compound for living cells, so many bacteria degrade phenol in relatively low ...concentrations, up to 0.75 g L
. The
strain 1CP is an effective destructor of a wide range of pollutants. In the absence of a carbon source in the medium, cells of the
1CP strain easily form cyst-like resting cells (CLC). The purpose of this work was to evaluate the viability of cells during long-term storage and the efficiency of the process of phenol destruction by
1CP cells germinating after dormancy. Resting cells were obtained by simple cultivation in a rich medium followed by storage under static conditions. This is a simple approach to obtain a large amount of biomass. Decomposition of phenol proceeded via catechol followed by
-cleavage of aromatic ring. The induction of three phenol hydroxylases was detected by RT-PCR in cells germinated in a mineral medium with phenol as the carbon source. The stability of the genome of cells germinating after dormancy is shown by box-PCR. Dormant
1CP cells, both suspended and immobilized, can be directly used for the decomposition of phenol after 4-12 months storage. In addition to phenol, after 9 months of storage, immobilized germinating cells easily metabolized 4-chlorophenol and 2,4,6-trichlorophenol. The results demonstrate a potential and simple approach toward achieving long-term storage of cells for further use in bioremediation.
The intensive development of agriculture leads to the depletion of land and a decrease in crop yields and in plant resistances to diseases. A large number of fertilizers and pesticides are currently ...used to solve these problems. Chemicals can enter the soil and penetrate into the groundwater and agricultural plants. Therefore, the primary task is to intensify agricultural production without causing additional damage to the environment. This problem can be partially solved using microorganisms with target properties. Microorganisms that combine several useful traits are especially valuable. The aim of this work was to search for new microbial strains, which are characterized by the ability to increase the bioavailability of nutrients, phytostimulation, the antifungal effect and the decomposition of some xenobiotics. A few isolated strains of the genera
and
were characterized by high activity against fungal phytopathogens. One of the bacterial strains identified as
on the basis of the 16S rRNA gene sequence was characterized by an unusual cellular morphology and development cycle, significantly different from all previously described bacteria of this genus. All isolated bacteria are capable of benzoate degradation as a sign of the ability to degrade aromatic compounds. Isolated strains were shown to be prospective agents in biotechnologies.
Purpose
Crude oil and oil products are the most widespread environmental pollutants. The most efficient bioremediation is performed by using specific oil-degrading strains. Our objectives were to ...assess the role of soil bacteria, belonging to the following genera
Arthrobacter
,
Microbacterium
,
Rhodococcus
,
Gordonia
, and
Acinetobacter
in reduction of toxicity of environmental pollutants. Bacteria with different versatility were chosen: isolates from aromatic compounds or crude oil-contaminated soils and common representatives of the soil microflora.
Materials and methods
In this work, crude oil from the field Aschisay (Kazakhstan) of the following composition: alkanes 78%, naphthenes 6.7%, arenes 3.7%, and other compounds 11.6% was used as carbon source. To investigate the metabolic activity of microorganisms, they were cultured in flasks for 10 days under different conditions (variations in pH range, temperature, salinity, carbon source). Infrared spectrophotometry method was employed to determine the residual oil content after cultivation of bacteria. The ability of bacteria to produce biosurfactants was assessed by measuring surface tension and emulsifying activity (the Francey et al. method); localization of biosurfactants was detected.
Results and discussion
Forty-six strains from oil-spilled soils were isolated, with seven of these isolates showing the high degradation ability. Analysis of 16S-RNA gene sequences assigns these cultures to the genus
Rhodococcus
. Their degradation activity was then compared with the one of two rhodococci isolated from soil contaminated with chloroaromatics. The strains under study degraded crude oil, diesel fuel, and phenol; some of them destroyed benzene and naphthalene. The most active strains utilized up to 55–59% of crude oil hydrocarbons. The behavior of strains in the presence of petroleum components (benzene, toluene, nonane, decane, hexadecane) revealed bacterial persistence under severe conditions. Bacteria proved to be more sensitive to aromatic solvents than to aliphatic hydrocarbons. Most of the strains produced biosurfactants when grown on hydrophobic substrates.
Conclusions
The obtained results show that bacteria highly adapted to oil contaminations play an important role in the biodegradation of recalcitrant pollutants. Such strains may serve as the basis of bioaugmentation approach for soil remediation in sites with high contamination degree. Furthermore, this study highlights a significant role of common representatives of soil microflora in reducing pollution level in the soil owing to various, however, not necessary high destructive activities of soil strains.
In this work, a new Ch2 strain was isolated from soils polluted by agrochemical production wastes. This strain has a unique ability to utilize toxic synthetic compounds such as
-caprolactam (CAP) as ...a sole carbon and energy source and the herbicide glyphosate (GP) as a sole source of phosphorus. Analysis of the nucleotide sequence of the 16S rRNA gene of Ch2 revealed that the strain belongs to the species
. This strain grew in the mineral medium containing CAP in a concentration range of 0.5 to 5.0 g/L and utilized 6-aminohexanoic acid and adipic acid, which are the intermediate products of CAP catabolism. The ability of strain Ch2 to degrade CAP is determined by a conjugative megaplasmid that is 550 kb in size. When strain Ch2 is cultured in a mineral medium containing GP (500 mg/L), more intensive utilization of the herbicide occurs in the phase of active growth. In the phase of declining growth, there is an accumulation of aminomethylphosphonic acid, which indicates that the C-N bond is the first site cleaved during GP degradation (glyphosate oxidoreductase pathway). Culture growth in the presence of GP during the early step of its degradation is accompanied by unique substrate-dependent changes in the cytoplasm, including the formation of vesicles of cytoplasmic membrane consisting of specific electron-dense content. There is a debate about whether these membrane formations are analogous to metabolosomes, where the primary degradation of the herbicide can take place. The studied strain is notable for its ability to produce polyhydroxyalkanoates (PHAs) when grown in mineral medium containing GP. At the beginning of the stationary growth phase, it was shown that, the amount and size of PHA inclusions in the cells drastically increased; they filled almost the entire volume of cell cytoplasm. The obtained results show that the strain
Ch2 can be successfully used for the PHAs' production. Moreover, the ability of
Ch2 to degrade CAP and GP determines the prospects of its application for the biological cleanup of CAP production wastes and in situ bioremediation of soil polluted with GP.
In the process of evolution, living organisms develop mechanisms for population preservation to survive in unfavorable conditions. Spores and cysts are the most obvious examples of dormant forms in ...microorganisms. Non-spore-forming bacteria are also capable of surviving in unfavorable conditions, but the patterns of their behavior and adaptive reactions have been studied in less detail compared to spore-forming organisms. The purpose of this work was to study the features of transition from dormancy to active vegetative growth in one of the non-spore-forming bacteria,
135, which is known as a destructor of such aromatic compounds as benzoate, 3-chlorobenzoate, and phenol. It was shown that
135 under unfavorable conditions forms cyst-like cells with increased thermal resistance. Storage for two years does not lead to complete cell death. When the cells were transferred to fresh nutrient medium, visible growth was observed after 3 h. Immobilized cells stored at 4 °C for at least 10 months regenerated their metabolic activity after only 30 min of aeration. A study of the ultrathin organization of resting cells by transmission electron microscopy combined with X-ray microanalysis revealed intracytoplasmic electron-dense spherical membrane ultrastructures with significant similarity to previously described acidocalcisomas. The ability of some resting
135 cells in the population to secrete acidocalcisome-like ultrastructures into the extracellular space was also detected. These structures contain predominantly calcium (Ca) and, to a lesser extent, phosphorus (P), and are likely to serve as depots of vital macronutrients to maintain cell viability during resting and provide a quick transition to a metabolically active state under favorable conditions. The study revealed the features of transitions from active growth to dormant state and vice versa of non-spore-forming bacteria
135 and the possibility to use them as the basis of biopreparations with a long shelf life.
Previously, the protective role of the S-layer protein 2 (Slp2) of the vaginal
2029 (LC2029) strain against foodborne pathogens
,
serovar Enteritidis, and
O157:H was demonstrated. We demonstrate the ...new roles of the Slp2-positive LC2029 strain and soluble Slp2 against
infections. We show that LC2029 bacteria can adhere to the surface of the cervical epithelial HeLa cells, prevent their contact with
, and block yeast transition to a pathogenic hyphal form. Surface-bound Slp2 provides the ability for LC2029 to co-aggregate with various
strains, including clinical isolates.
-induced necrotizing epithelial damage is reduced by colonization with the Slp2-positive LC2029 strain. Slp2 inhibits the adhesion of various strains of
to different human epithelial cells, blocks yeast transition to a pathogenic hyphal form, and prevents the colonization and pathogenic infiltration of mucosal barriers. Only Slp2 and LC2029 bacteria stimulate the production of protective human β-defensin 3 in various epithelial cells. These findings support the anti-
potential of the probiotic LC2029 strain and Slp2 and form the basis for further research on their ability to prevent and manage invasive
infections.
The Ligilactobacillus salivarius 7247 (LS7247) strain, originally isolated from a healthy woman’s intestines and reproductive system, has been studied for its probiotic potential, particularly ...against Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) as well as its potential use in synbiotics. LS7247 showed high tolerance to gastric and intestinal stress and effectively adhered to human and animal enterocyte monolayers, essential for realizing its probiotic properties. LS7247 showed high anti-Salmonella activity. Additionally, the cell-free culture supernatant (CFS) of LS7247 exhibited anti-Salmonella activity, with a partial reduction upon neutralization with NaOH (p < 0.05), suggesting the presence of anti-Salmonella factors such as lactic acid (LA) and bacteriocins. LS7247 produced a high concentration of LA, reaching 124.0 ± 2.5 mM after 48 h of cultivation. Unique gene clusters in the genome of LS7247 contribute to the production of Enterolysin A and metalloendopeptidase. Notably, LS7247 carries a plasmid with a gene cluster identical to human intestinal strain L. salivarius UCC118, responsible for class IIb bacteriocin synthesis, and a gene cluster identical to porcine strain L. salivarius P1ACE3, responsible for nisin S synthesis. Co-cultivation of LS7247 with SE and ST pathogens reduced their viability by 1.0–1.5 log, attributed to cell wall damage and ATP leakage caused by the CFS. For the first time, the CFS of LS7247 has been shown to inhibit adhesion of SE and ST to human and animal enterocytes (p < 0.01). The combination of Actigen prebiotic and the CFS of LS7247 demonstrated a significant combined effect in inhibiting the adhesion of SE and ST to human and animal enterocytes (p < 0.001). These findings highlight the potential of using the LS7247 as a preventive strategy and employing probiotics and synbiotics to combat the prevalence of salmonellosis in animals and humans caused by multidrug resistant (MDR) strains of SE and ST pathogens.
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