MlrA, an Essential Enzyme for Microcystins and Nodularin on First Step Biodegradation in Microcystin-Degrading Bacteria.

Microcystin-degrading bacteria first degrade microcystins by microcystinase A (MlrA) to cleave the cyclic structure of microcystins at the Adda-Arg site of microcystin-LR, microcystin-RR, and microcystin-YR, but the cleavage of the other microcystins was not clear. In our study, the microcystin-degrading bacterium Sphingopyxis sp. C-1 as wild type and that of mlrA-disrupting mutant, Sphingopyxis sp. CMS01 were used for microcystins biodegradation. The results showed MlrA degraded microcystin-LA, microcystin-LW, microcystin-LY, microcystin-LF, and nodularin. MlrA could cleave the Adda-L-amino acid site.

Toxicity of ciprofloxacin and ofloxacin to Moina macrocopa and investigation of p-value adjustments for (eco)toxicological studies.

Ciprofloxacin (CFX) and ofloxacin (OFX) are commonly found as residual contaminants in aquatic environments, posing potential risks to various species. To ensure the safety of aquatic wildlife, it is essential to determine the toxicity of these antibiotics and establish appropriate concentration limits. Additionally, in (eco)toxicological studies, addressing the issue of multiple hypothesis testing through p-value adjustments is crucial for robust decision-making. In this study, we assessed the no observed adverse effect concentration (NOAEC) of CFX and OFX on Moina macrocopa across a concentration range of 0-400 µg L-1. Furthermore, we investigated multiple p-value adjustments to determine the NOAECs. Our analysis yielded consistent results across seven different p-value adjustments, indicating NOAECs of 100 µg CFX L-1 for age at first reproduction and 200 µg CFX L-1 for fertility. For OFX treatment, a NOAEC of 400 µg L-1 was observed for both biomarkers. However, further investigation is required to establish the NOAEC of OFX at higher concentrations with greater certainty. Our findings demonstrate that CFX exhibits higher toxicity compared to OFX, consistent with previous research. Moreover, this study highlights the differential performance of p-value adjustment methods in terms of maintaining statistical power while controlling the multiplicity problem, and their practical applicability. The study emphasizes the low NOAECs for these antibiotics in the zooplanktonic group, highlighting their significant risks to ecological and environmental safety. Additionally, our investigation of p-value adjustment approaches contributes to a deeper understanding of their performance characteristics, enabling (eco)toxicologists to select appropriate methods based on their specific needs and priorities.

Characteristics of Antibiotic Resistance and Tolerance of Environmentally Endemic Pseudomonas aeruginosa.

Antibiotic-resistant bacteria remain a serious public health threat. In order to determine the percentage of antibiotic-resistant and -tolerant Pseudomonas aeruginosa cells present and to provide a more detailed infection risk of bacteria present in the environment, an isolation method using a combination of 41 °C culture and specific primers was established to evaluate P. aeruginosa in the environment. The 50 strains were randomly selected among 110 isolated from the river. The results of antibiotic susceptibility evaluation showed that only 4% of environmental strains were classified as antibiotic-resistant, while 35.7% of clinical strains isolated in the same area were antibiotic-resistant, indicating a clear difference between environmental and clinical strains. However, the percentage of antibiotic-tolerance, an indicator of potential resistance risk for strains that have not become resistant, was 78.8% for clinical strains and 90% for environmental strains, suggesting that P. aeruginosa, a known cause of nosocomial infections, has a high rate of antibiotic-tolerance even in environmentally derived strains. It suggested that the rate of antibiotic-tolerance is not elicited by the presence or absence of antimicrobial exposure. The combination of established isolation and risk analysis methods presented in this study should provide accurate and efficient information on the risk level of P. aeruginosa in various regions and samples.

Physiological response of Simocephalus vetulus to five antibiotics and their mixture under 48-h acute exposure.

Antibiotics, widely known as major environmental xenobiotics, are increasingly being released into ecosystems due to their essential functions in human health and production. During the COVID-19 pandemic waves, antibiotic use increases remarkably in treating bacterial coinfections. Antibiotics were initially expected only to affect prokaryotes, but recent research has shown that they can disturb the biological systems of eukaryotes, especially vulnerable aquatic creatures, through both direct and indirect processes. However, their toxicity to the freshwater cladoceran Simocephalus vetulus, an essential link in the aquatic food web, has never been evaluated. The effects of four fluoroquinolones (ciprofloxacin: CFX, ofloxacin: OFX, gatifloxacin: GFX, delafloxacin: DFX), tetracycline (TET), and a mixture of these medicines (MIX) on S. vetulus thoracic limb rate (TLR) were examined in this study. After S. vetulus was exposed to 20 and 40 mg GFX L-1, 90% and 100% mortality rates were recorded. At 2.5-10 mg L-1, GFX dramatically lowered the TLR of S. vetulus, resulting in a median effective concentration of 9.69 mg L-1. TLRs increased when the organisms were exposed to 10-40 mg L-1 of CFX and 1.25-40 mg L-1 of OFX. However, DFX and TET exposures did not affect TLRs. Exposure to MIX reduced TLR only at 40 mg L-1, suggesting an antagonistic interaction among the five pharmaceuticals. This study demonstrated that S. vetulus physiological responses to antibiotics, even in the same class, are complex and elusive. Beyond a common additive concentration principle, the antagonistic interaction of antibiotic mixture indicates a high level of uncertainty in terms of ecological dangers. We initially introduce S. vetulus to ecotoxicological studies of antibiotics, presenting the species as a low-cost model for physiological investigations of environmental xenobiotics.

Chronic ecotoxicology and statistical investigation of ciprofloxacin and ofloxacin to Daphnia magna under extendedly long-term exposure.

Ciprofloxacin (CFX) and ofloxacin (OFX) are two of the most often used fluoroquinolone antibiotics, and their residues are found in large amounts in various aquatic settings. However, the toxicity tests of CFX using eukaryotic organisms such as Daphnia magna are inadequate, and the test result of OFX is currently unknown. Therefore, the chronic toxicity test for D. magna was performed during 42 days under exposure to CFX and OFX concentrations of 50, 500, and 5000 µg L-1. All exposure conditions did not cause mortality for D. magna. CFX exposure at 500 µg L-1 resulted in an earlier oogenesis date and increased brood size in the second birth. The Poisson-based generalized linear mixed-effects model revealed that the reduction of fertility was statistically significant for the CFX and OFX exposures at 5000 µg L-1. On the other hand, the production of dead eggs as offspring degradation was also found significantly as maternal D. magna exposed to antibiotics at 5000 µg L-1. In addition, following long-term exposure to antibiotics, maternal adaptation to antibiotics was established for offspring deterioration and fertility. However, the OFX exposure showed that the fertility-suppressed effects continued for a longer period than the CFX exposure. Although no rational explanation has yet been given for the more substantial effect of OFX on reducing fertility than CFX, molecular cell biology and symbiotic microbial flora derived from previous studies could explain our ecotoxicological results. This study is the first report for the OFX chronic toxicities on D. magna by comparing it to the toxicity of CFX. Our study contributes to guiding the future impact assessment of fluoroquinolone antibiotic pollution on ecosystems, including the need for new statistical methods in ecotoxicological studies.

Draft Genome Sequence of the Microcystin-Degrading Bacterium Novosphingobium sp. Strain MD-1.

This report describes the whole-genome sequence of a microcystin-degrading bacterium, Novosphingobium sp. strain MD-1, isolated from a lake in Japan. The Novosphingobium sp. strain MD-1 genome had a total length of 4,617,766 bp. Moreover, strain MD-1 showed a conserved microcystin-degrading gene cluster (mlrA to mlrF), similar to Sphingopyxis sp. strain C-1.

Effects of levofloxacin exposure on sequencing batch reactor (SBR) behavior and microbial community changes.

The adaptation mechanisms of bacterial community for nitrogen removal performance exposed to fluctuated levels of levofloxacin (LVX) during wastewater treatment in SBRs were investigated. Although LVX is completely synthetic, the results of minimum inhibitory concentration (MIC, 32 mg-LVX/L) and minimum bactericidal concentration (MBC, 512 mg-LVX/L) of the sampled sludge showed that the LVX resistance/tolerance for bacterial growth has already existed in the actual wastewater treatment plants (WWTPs). The key bacteria, i.e. Nitrosomonas sp. (ammonia-oxidizing bacteria), Nitrospira sp. (nitrite-oxidizing bacteria) and Thauera sp. (the predominant denitrifiers), decreased with LVX exposure, and the recovery of biological process in the reactor was disturbed due to LVX exposure. However, after stopping exposure their population was quickly increased and thus the performance was recovered. The results of the non-metric multidimensional scaling and microbial community by sequencing showed the LVX concentration was a crucial factor to the change of bacterial communities and controlled the quantitative evolution of the communities in our systems. This effect was more pronounced as the LVX concentration was higher. The results suggested the removal of residual antibiotics to accomplish under no effect concentration before biological treatment is important to suppress emerging and increasing of the antibiotic resistant bacteria in WWTPs.

Whole-Genome Sequence of the Microcystin-Degrading Bacterium Sphingopyxis sp. Strain C-1.

This report describes the whole-genome sequence of an alkalitolerant microcystin-degrading bacterium, Sphingopyxis sp. strain C-1, isolated from a lake in China.

Complete Genome Sequence of Pseudomonas aeruginosa Strain 8380, Isolated from the Human Gut.

Pseudomonas aeruginosa shows multidrug resistance, which is mainly attributable to its expression of xenobiotic efflux pumps. However, it is unclear how silent pumps are expressed in clinical isolates. Here, we sequenced the complete genome of P. aeruginosa strain 8380, which was isolated from a human gut.

Development of a novel antimicrobial screening system targeting the pyoverdine-mediated iron acquisition system and xenobiotic efflux pumps.

The iron acquisition systems in Pseudomonas aeruginosa are inducible in response to low-iron conditions and important for growth of this organism under iron limitation. OprM is the essential outer membrane subunit of the MexAB-OprM xenobiotic efflux pump. We designed and constructed a new model antimicrobial screening system targeting both the iron-uptake system and xenobiotic efflux pumps. The oprM gene was placed immediately downstream of the ferri-pyoverdine receptor gene, fpvA, in the host lacking chromosomal oprM and the expression of oprM was monitored by an antibiotic susceptibility test under iron depleted and replete conditions. The recombinant cells showed wild-type susceptibility to pump substrate antibiotics, e.g., aztreonam, under iron limitation and became supersusceptible to them under iron repletion, suggesting that expression of oprM is under control of the iron acquisition system. Upon screening of a chemical library comprising 2952 compounds using this strain, a compound-ethyl 2-(1-acetylpiperidine-4-carboxamido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate-was found to enhance the efficacy of aztreonam under iron limitation, suggesting that the compound inhibits either the iron acquisition system or the MexAB-OprM efflux pump. This compound was subsequently found to inhibit the growth of wild-type cells in the presence of sublethal amounts of aztreonam, regardless of the presence or absence of dipyridyl, an iron-chelator. The compound was eventually identified to block the function of the MexAB-OprM efflux pump, showing the validity of this new method.

Effect of polyphenols on reactive oxygen species production and cell growth of human dermal fibroblasts after irradiation with ultraviolet-A light.

Ultraviolet-A (UV-A) can damage microbes by generating reactive oxygen species (ROS), singlet oxygen, superoxides, hydrogen peroxide and hydroxyl radicals. These species readily react with lipids, proteins, DNA and other constituents of cells, leading to oxidative deterioration and the eventual death of the microbe. However, the oxidative ability of these reactive species also harms the viability of mammalian cells such as fibroblasts and keratinocytes, as they cause both acute and chronic damage, photo-aging, and photo-carcinogenesis. This study describes a UV-A treatment that does not affect the viability or growth of human neonate dermal fibroblasts, as determined by examining the post-irradiation cell density after the addition of polyphenols as antioxidants. The results demonstrate the possible wide applicability of UV-A sterilization. The potency of polyphenols for attenuating UV-A-induced ROS generation in cells was tested using (+)-catechin hydrate, (-)- epigallocatechin gallate hydrate, morin hydrate, quercetin hydrate and resveratrol. The lowest concentration of polyphenols required to reduce ROS by 50% in cells upon exposure to a dose of 15 J cm(-2) was determined and defined as its IC50. Pre-treatment with morin hydrate at its IC50 allowed cells irradiated with 5.0 J cm(-2) UV-A to recover to the level of the specific growth rate of cells incubated without UV-A irradiation. However, the growth rate of cells exposed to 15 J cm(-2) UV-A irradiation was scarcely influenced by co-incubation with morin hydrate; this dose of UV-A also suppressed cell growth completely in the absence of morin hydrate, although co-incubation resulted in no decrease in cell viability. This study demonstrates the potential of polyphenols for protecting both the viability of cells and their ability to proliferate from damage caused by UV-A-irradiation.

Activity of ERK regulates mucin 3 expression and is involved in undifferentiated Caco-2 cell death induced by 3-oxo-C12-homoserine lactone.

The signal molecule, 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL), is similar to a mammalian hormone in bacteria. Although most studies have examined the effects of high 3-oxo-C12-HSL concentrations (>200 µM) on mammalian cellular functions because ~600 µM 3-oxo-C12-HSL can be secreted in biofilms of Pseudomonas aeruginosa grown in vitro, we previously showed that a low 3-oxo-C12-HSL concentration (30 µM) induces the apoptosis of undifferentiated Caco-2 cells through suppressing Akt activity. Here, we found that a low concentration of 3-oxo-C12-HSL-activated ERK1/2 in undifferentiated Caco-2 cells. Incubating cells with the ERK pathway inhibitor U0126 for 30 min alleviated the mucin 3 (MUC3) expression suppressed by 3-oxo-C12-HSL, and the upregulation of MUC3 expression induced by a 48-h incubation with U0126-reduced cell death. Thus, altered MUC3 expression caused by long-term attenuated ERK1/2 activity might correlate with the death of undifferentiated Caco-2 cells induced by 3-oxo-C12-HSL.

Acyl-homoserine lactones suppresses IEC-6 cell proliferation and increase permeability of isolated rat colon.

We investigated to determine whether a variety of acyl-homoserine lactones (AHLs) influences epithelial cell proliferation and mucosal permeability. 3-Oxo-C12-homoserine lactone (HSL) and 3-oxo-C14-HSL significantly suppressed IEC-6 cell proliferation. A significant increase in mucosal permeability was observed in isolated rat colon tissue exposed to C12-HSL, 3-oxo-C12-HSL, and 3-oxo-C14-HSL. These data indicate that AHLs suppress epithelial proliferation and disrupt barrier function in intestinal mucosa.

Mucin 3 is involved in intestinal epithelial cell apoptosis via N-(3-oxododecanoyl)-L-homoserine lactone-induced suppression of Akt phosphorylation.

N-acyl-homoserine lactones (AHL) are quorum-sensing molecules in bacteria that play important roles in regulating virulence gene expression in pathogens such as Pseudomonas aeruginosa. The present study compared responses between undifferentiated and differentiated Caco-2 cells to N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL). A low concentration of 3-oxo-C12-HSL (30 µM) is sufficient to reduce viability accompanied by apoptosis via the suppression of phosphorylation by Akt in undifferentiated Caco-2 cells. The suppression of Akt phosphorylation appears specific in 3-oxo-C12-HSL, because other AHLs did not influence the phosphorylation status of Akt. The reduced viability induced by 3-oxo-C12-HSL was partially recovered by constitutively active Akt overexpression in undifferentiated Caco-2 cells. Since mucin is considered a vital component of the gut barrier, we investigated whether mucin protects cellular functions induced by 3-oxo-C12-HSL in undifferentiated Caco-2 cells. The results showed that mucin protected undifferentiated Caco-2 cells from apoptosis induced by 3-oxo-C12-HSL. 3-Oxo-C12-HSL did not induce cell death in differentiated Caco-2 cells that expressed higher levels of mucin 3 (MUC3) than undifferentiated Caco-2 cells. In addition, 3-oxo-C12-HSL promoted cell death in undifferentiated Caco-2 cells transfected with MUC3 siRNA and reduced MUC3 expression in undifferentiated Caco-2 cells. Therefore, MUC3 might be responsible for the survival of undifferentiated intestinal epithelial cells in the presence of 3-oxo-C12-HSL through regulating Akt phosphorylation. In conclusion, 3-oxo-C12-HSL might influence the survival of undifferentiated intestinal epithelial cells as well as interactions between these cells and pathogens.

Synergistic antimicrobial activity based on the combined use of a gemini-quaternary ammonium compound and ultraviolet-A light.

This study examined the utility of synergistic disinfection employing a gemini-quaternary ammonium salt (a gemini-QUAT, namely 3,3'-(2,7-dioxaoctane)bis(1-decylpyridinium bromide)), as an organic biocide in combination with irradiation by an ultraviolet-A (UV-A) light-emitting diode (LED) with a peak wavelength of 365nm. The combined system represents a novel disinfection method utilizing facilitated in situ oxidation depending on overproduction of reactive oxygen species (ROS) triggered by the initial action of the gemini-QUAT on the bacterial membrane. We demonstrate that this combination decreased the viability of pathogenic bacteria in a significant and rapid manner, and depended on doses of the gemini-QUAT and the fluence: the viability of Escherichia coli was reduced by greater than 5.0-logs by the combination procedure, but the decrease in viability was only 2.3-logs for exposure to UV at the same fluence dose in the absence of the gemini-QUAT. Adding catalase as a radical scavenger decreased bacterial inactivation by the combined disinfection procedure. Flow cytometric analysis indicated superoxide and hydrogen peroxide overproduction within cells treated with the combined disinfection procedure. The excessive superoxide, detected only in the combined system, appeared to be generated by the action of the gemini-QUAT at the bacterial membrane, leading to excessive and rapid generation of ROS in the system. Our data strongly suggested that this ROS promoted bacterial membrane peroxidation during initial treatment by the combination method, resulting in increased oxidative modification of DNA. These oxidative reactions may play an important role in the efficacy of this disinfection procedure.

Trehalose suppresses antibody aggregation during the culture of Chinese hamster ovary cells.

The aggregation of therapeutic antibodies during the manufacturing process is problematic because of the potential risks posed by the aggregates, such as an unexpected immune response. One of the hallmark effects of trehalose, a disaccharide consisting of two alpha-glucose units, is as a chemical chaperone with anti-aggregation activity. In this study, Chinese hamster ovary (CHO) cell line producing a diabody-type bispecific antibody were cultured in medium containing trehalose and the aggregation of the secreted proteins during the culture process was analyzed. An analysis of the various forms of the antibody (monomeric, dimeric, and large aggregates) showed that trehalose decreased the relative content of large aggregates by two thirds. The aggregation kinetics indicated that trehalose directly inhibited the polymerization and aggregation steps in a nucleation-dependent aggregation mechanism. Moreover, both specific and volumetric antibody production were increased in CHO cells cultured in trehalose-containing medium. Thus, the addition of trehalose to recombinant CHO cell cultures would offer a practical strategy for quality improvement in the production of therapeutic antibodies.

Synthesis of thiazole derivatives and evaluation of their antiamoebic activity and cytotoxicity.

Five ethyl (5-alkyl-2-amino-1,3-thiazol-4-yl) acetates (designated compounds 4a-e) incorporating octyl, decyl, dodecyl, tetradecyl, and hexadecyl alkyl chains, respectively, were prepared by reacting 4-alkyl-4-bromo-3-oxobutyric acid ethyl esters (3a-e) with thiourea in dried acetonitrile. Compounds 3a-e were synthesized by reacting alkylated ethyl acetoacetates with bromine. The newly synthesized compounds were characterized by mass spectrometry, NMR, and elemental analysis. Compounds 4a-c demonstrated good in vitro antiamoebic activity against Acanthamoeba polyphaga exposed to 10 mg L(-1) for 6 h at 28 °C. Compound 4b showed the highest antiamoebic activity among the tested compounds, comparable to that of chlorhexidine dihydrochloride (CHX), decreasing the number of viable cells to below the detection limit of 1 cell mL(-1). The activity of compounds 4a and 4c was similar to that of the commercial antifungal agent fluconazole (Flu). The cytotoxic and hemolytic activity of the compounds was assayed against human neonate dermal fibroblasts and sheep erythrocytes, respectively. Compounds 4a-c were less cytotoxic than Flu and CHX. Our results suggest that compound 4b, which is composed of a 2-amino-thiazole attached to a decyl group and an ethyl ester moiety, is a particularly safe and effective alternative amoebicidal agent.

Analysis of genes for succinoyl trehalose lipid production and increasing production in Rhodococcus sp. strain SD-74.

Succinoyl trehalose lipids (STLs) are promising glycolipid biosurfactants produced from n-alkanes that are secreted by Rhodococcus species bacteria. These compounds not only exhibit unique interfacial properties but also demonstrate versatile biochemical actions. In this study, three novel types of genes involved in the biosynthesis of STLs, including a putative acyl coenzyme A (acyl-CoA) transferase (tlsA), fructose-bisphosphate aldolase (fda), and alkane monooxygenase (alkB), were identified. The predicted functions of these genes indicate that alkane metabolism, sugar synthesis, and the addition of acyl groups are important for the biosynthesis of STLs. Based on these results, we propose a biosynthesis pathway for STLs from alkanes in Rhodococcus sp. strain SD-74. By overexpressing tlsA, we achieved a 2-fold increase in the production of STLs. This study advances our understanding of bacterial glycolipid production in Rhodococcus species.

Synthesis and biological activity of thiazolyl-acetic acid derivatives as possible antimicrobial agents.

5a-h, a series of (5-substituted-2-methyl-1,3-thiazole-4-yl) acetic acids as heterocyclic acetic acid derivatives, was designed and synthesized from ethyl acetoacetate. The synthesized compounds were screened for their antimicrobial activities against bacterial and fungal strains, and their characteristics were investigated by assays under various temperature and pH conditions. Cytotoxicity was evaluated with the use of sheep erythrocytes and human neonate dermal fibroblasts. Similarly, agents such as lauric acid 6 and parabens 7a-b, which are used as preservative agents for commercial cosmetics and detergents, were assayed for comparison. Although the structure of 5a is simple, comprising a thiazole attached with an octyl group and acetic acid moiety, the compound showed stronger and broader antibacterial and antifungal activities among the 5 series against the tested microbes other than gram-negative bacteria. Interestingly, 5a overcame the weak antifungal activity of parabens 7a-b. Also, the cytotoxicity of 5a was less than that of parabens 7a-b, especially to human dermal fibroblasts. These results suggest that thiazolyl-acetic acid 5a is a potentially effective biocide, and that it could be used as a preservative agent in commercially sold cosmetics and detergents, facilitated by the hydrophilic and charge properties of its carboxylic acid moiety.

Two routes of MexS-MexT-mediated regulation of MexEF-OprN and MexAB-OprM efflux pump expression in Pseudomonas aeruginosa.

The NfxC-type mutant of Pseudomonas aeruginosa produces the MexEF-OprN efflux pump and down-regulates expression of the quorum-sensing-dependent efflux pump MexAB-OprM and production of virulence factors in the presence of an active transcriptional regulator, MexT. Consequently, these cells are resistant to chloramphenicol and hypersusceptible to ß-lactam antibiotics. An upper negative regulator, MexS, has been assumed to inactivate MexT in wild-type strains, hence shutting down production of the MexEF-OprN pump. This observation was, however, reported in only one clinical strain and not confirmed in well-characterized laboratory strains. Moreover, it is not known whether MexS is involved in the quorum-sensing-dependent regulation of virulence factor production. To assess these issues, a plasmid carrying wild-type mexS was introduced into three NfxC-type mutants from laboratory strains, which carry an impaired mexS and unimpaired mexT. Unexpectedly, all the transformants produced an increased amount of MexEF-OprN proteins. Three clinical NfxC strains were similarly transformed and although MexEF-OprN was undetectable in two of these strains, one produced an increased amount of these proteins, similar to the laboratory strains. These results were interpreted to mean that P. aeruginosa takes two separate routes in MexT-mediated regulation of mexEF-oprN expression: the MexS-bypassed pathway and MexS-mediated pathway. On the other hand, the transformants of both the laboratory and clinically derived NfxC-type cells produced increased amounts of MexAB-OprM and virulence factors, suggesting that production of these proteins occurs via the MexS-mediated pathway.