Sodium dehydroacetate exposure decreases locomotor persistence and hypoxia tolerance in zebrafish.

Environmental exposure to sodium dehydroacetate (DHA-S) is inevitable as DHA-S is a high-volume preservative widely used in cosmetics, processed foods and personal care products. DHA-S is absorbed rapidly when administered orally or on the skin and generally considered to be safe and well tolerated. However, DHA-S has recently been reported to induce weight loss and allergic contact dermatitis, yet little is known about how DHA-S affect the related biological processes. Here, we characterize the biological effects of DHA-S on zebrafish model by directly waterborne exposure. Zebrafish is susceptible to DHA-S exposure at early developmental stage. DHA-S decreased the hatch rate and locomotor persistence of zebrafish, and eventually induced lethality during the continuous exposure at relatively low concentrations of commonly addition. Acute DHA-S exposure decreased respiration capacity in larval zebrafish, promoted the expression of HIF-1α (hypoxia-inducible factor-1α) and caused rapid adult zebrafish death in 30 h. We further demonstrated that DHA-S inhibited the activity of succinate dehydrogenase (SDH) inducing respiratory chain interruption, energy deficiency and organic acids accumulation. These results suggest that the approved DHA-S may pose serious environmental/ecological pressures on the aquatic animal's migration.

Sodium dehydroacetate induces cardiovascular toxicity associated with Ca2+ imbalance in zebrafish.

The environmental effects of additives have attracted increasing attention. Sodium dehydroacetate (DHA-S), as an approved preservative, is widely added in processed foods, cosmetics and personal care products. However, DHA-S has been recently reported to induce hemorrhage and coagulation aberration in rats. Yet little is known about the ecotoxicological effect and underlying mechanisms of DHA-S. Here, we utilized the advantage of zebrafish model to evaluate such effects. DHA-S induced cerebral hemorrhage, mandibular dysplasia and pericardial edema in zebrafish after 24 h exposure (48-72 hpf) at 50 mg/L. We also observed the defective heart looping and apoptosis in DHA-S-treated zebrafish through o-dianisidine and acridine orange staining. Meanwhile, DHA-S induced the deficiency of Ca2+ and vitamin D3 in zebrafish. We further demonstrated that DHA-S stimulated Ca2+ influx resulting in Ca2+-dependent mitochondrial damage in cardiomyocytes. Additionally, DHA-S inhibited glucose uptake and repressed the biosynthesis of amino acids. Finally, we identified that sodium bicarbonate could rescue zebrafish from DHA-S induced cardiovascular toxicity. Altogether, our results suggest that DHA-S is a potential risk for cardiovascular system.

A TaqMan-based multiplex real-time PCR assay for the rapid detection of tigecycline resistance genes from bacteria, faeces and environmental samples.

BACKGROUND: Tigecycline is a last-resort antibiotic used to treat severe infections caused by extensively drug-resistant bacteria. Recently, novel tigecycline resistance genes tet(X3) and tet(X4) have been reported, which pose a great challenge to human health and food security. The current study aimed to establish a TaqMan-based real-time PCR assay for the rapid detection of the tigecycline-resistant genes tet(X3) and tet(X4). RESULTS: No false-positive result was found, and the results of the TaqMan-based real-time PCR assay showed 100% concordance with the results of the sequencing analyses. This proposed method can detect the two genes at the level of 1 × 102 copies/µL, and the whole process is completed within an hour, allowing rapid screening of tet(X3) and tet(X4) genes in cultured bacteria, faeces, and soil samples. CONCLUSION: Taken together, the TaqMan-based real-time PCR method established in this study is rapid, sensitive, specific, and is capable of detecting the two genes not only in bacteria, but also in environmental samples.

Nonribosomal antibacterial peptides that target multidrug-resistant bacteria.

Covering: 2000 to 2018, particularly from 2010 to early 2018 The increase in the incidence of antibiotic resistant infections is threatening to overwhelm healthcare practices worldwide. Most antibiotics in clinical use are becoming ineffective, so therefore it is imperative to develop new antibiotics and novel therapeutic strategies. Traditionally, the chemical and mechanistic diversity of nonribosomal antibacterial peptides (NRAPs) as lead compounds have meant that their structures are ideal for antibiotic discovery. Here, we summarize the state of our current knowledge about the mechanisms of antibiotic resistance, which can be used to guide the development of new antibiotics. Furthermore, we provide an overview of NRAPs for treating multi-drug resistant bacteria, including innovative approaches for screening NRAPs from new sources and the underlying mechanisms of antibacterial activity. Finally, we discuss the design of NRAP scaffolds for precise medicine and combinatorial NRAP therapies with existing antibiotics to overcome resistance, which will help to control infections in the post-antibiotic era.

Multifaceted toxin profile, an approach toward a better understanding of probiotic Bacillus cereus.

Strains of the Bacillus cereus group have been widely used as probiotics for human beings, food animals, plants, and environmental remediation. Paradoxically, B. cereus is responsible for both gastrointestinal and nongastrointestinal syndromes and represents an important opportunistic food-borne pathogen. Toxicity assessment is a fundamental issue to evaluate safety of probiotics. Here, we summarize the state of our current knowledge about the toxins of B. cereus sensu lato to be considered for safety assessment of probiotic candidates. Surfactin-like emetic toxin (cereulide) and various enterotoxins including nonhemolytic enterotoxin, hemolysin BL, and cytotoxin K are responsible for food poisoning outbreaks characterized by emesis and diarrhea. In addition, other factors, such as hemolysin II, Certhrax, immune inhibitor A1, and sphingomyelinase, contribute to toxicity and overall virulence of B. cereus.

Ca2+ protect zebrafish embryos from water acidification.

Ionizable strategies are routinely used to enhance the solubility and dissolution rates of various pharmaceuticals. These chemicals may directly affect aquatic environment once discharged from factories, hospitals or livestock farms. Here, we assessed the potential side effect of tetracyclines (TCs) on the development of zebrafish embryos. Tetracycline hydrochloride decreased water pH from 6.4 to 4.4 at 30 mg/L. Acidified water exceeded the tolerance of zebrafish embryos in pure water during the early ten hours post fertilization (hpf). Interestingly, we found that Ca2+ in the embryo medium could increase the tolerance of embryos to acidified water. Furthermore, we found that the protection of Ca2+ was not due to the formation of TCs-Ca2+ complexes under acidic condition, based on spectral analysis. Meanwhile we showed that exogenous addition of Ca2+ could inhibit the accumulation of Ca2+ from the cytoplasm to the surface of embryos. These results may shed light on the strategies for protecting aquatic animals from acidic environments.

Non-hemolytic enterotoxin of Bacillus cereus induces apoptosis in Vero cells.

Bacillus cereus is an opportunistic pathogen that often causes foodborne infectious diseases and food poisoning. Non-hemolytic enterotoxin (Nhe) is the major toxin found in almost all enteropathogenic B. cereus and B. thuringiensis isolates. However, little is known about the cellular response after Nhe triggered pore formation on cell membrane. Here, we demonstrate that Nhe induced cell cycle arrest at G0 /G1 phase and provoked apoptosis in Vero cells, most likely associated with mitogen-activated protein kinase (MAPK) and death receptor pathways. The influx of extracellular calcium ions and increased level of reactive oxygen species in cytoplasm were sensed by apoptosis signal-regulating kinase 1 (ASK1) and p38 MAPK. Extrinsic death receptor Fas could also promote the activation of p38 MAPK. Subsequently, ASK1 and p38 MAPK triggered downstream caspase-8 and 3 to initiate apoptosis. Our results clearly demonstrate that ASK1, and Fas-p38 MAPK-mediated caspase-8 dependent pathways are involved in apoptotic cell death provoked by the pore-forming enterotoxin Nhe.

Magnetic-assisted biotinylated single-chain variable fragment antibody-based immunoassay for amantadine detection in chicken.

A sensitive competitive immunoassay with simple operation was developed for the detection of the anti-virus drug amantadine (AMD). The single-chain variable fragment (scFv) antibody against AMD was site-specific biotinylated and overexpressed as a secreted body in Escherichia coli AVB101. Horseradish peroxidase-labeled streptavidin-biotinylated scFv antibody (HRP-SA-BIO-scFv) could specifically bind to AMD-functionalized magnetic beads (MBs) and then the immune complexes were separated from the matrix solution by magnet. The concentration of the AMD could be known by the measurement of the signal produced by the horseradish peroxidase. The newly established assay provides a significant improvement in comparison to the conventional ELISA without SA-BIO signal amplification and MBs separation. The limit of detection and assay time was 0.64 vs. 8.4 ng/mL and 50 vs. 150 min, respectively. The recoveries ranged from 77.8 to 112% with the coefficient of variation less than 13%. The immunoassay exhibited an obvious cross-reactivity to rimantadine (84%), 1-(1-adamantyl)ethylamine (72%), and somantadine (63%). These results demonstrated that the developed immunoassay provided a sensitive, rapid, and accurate approach for the detection of AMD in chicken by employing MBs as solid phase and SA-BIO as signal amplification. When applied in natural chicken samples, the newly established method provided results consistent with those from UPLC-MS/MS, suggesting that the proposed method could be used for rapid screening of the target of interest; the new immunoassay could also be extended to other small molecular contaminants and thus represents a universal strategy for food safety analysis. Graphical abstract ᅟ.

A Biosurfactant-Inspired Heptapeptide with Improved Specificity to Kill MRSA.

The emergence and rapid spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a serious threat to public health. New antibiotics and strategies are urgently needed to combat S. aureus associated infections. Bacaucin, a novel cyclic lipopeptide from Bacillus subtilis CAU21, is reported. Bacaucin shows broad antibacterial activity against Gram-positive bacteria, but is also hemolytic and cytotoxic. However, bacaucin-1, a bacaucin-inspired ring-opened heptapeptide, shows specific antibacterial activity against MRSA by a membrane-disruptive mechanism without detectable toxicity to mammalian cells or induction of bacterial resistance. Bacaucin-1 was efficient in preventing infections in both in vitro and in vivo models and is a valuable prototype antibiotic with high potential against S. aureus infections.

Untargeted metabolomic profiling of amphenicol-resistant Campylobacter jejuni by ultra-high-performance liquid chromatography-mass spectrometry.

Campylobacter jejuni, an important foodborne microorganism, poses severe and emergent threats to human health as antibiotic resistance becomes increasingly prevalent. The mechanisms of drug resistance are hard to decipher, and little is known at the metabolic level. Here we apply metabolomic profiling to discover metabolic changes associated with amphenicol (chloramphenicol and florfenicol) resistance mutations of Campylobacter jejuni. An optimized sample preparation method was combined with ultra-high-performance liquid chromatography-time-of-flight mass spectrometry (UHPLC-TOF/MS) and pattern recognition for the analysis of small-molecule biomarkers of drug resistance. UHPLC-triple quadrupole MS operated in multiple reaction monitoring mode was used for quantitative analysis of metabolic features from UHPLC-TOF/MS profiling. Up to 41 differential metabolites involved in glycerophospholipid metabolism, sphingolipid metabolism, and fatty acid metabolism were observed in a chloramphenicol-resistant mutant strain of Campylobacter jejuni. A panel of 40 features was identified in florfenicol-resistant mutants, demonstrating changes in glycerophospholipid metabolism, sphingolipid metabolism, and tryptophan metabolism. This study shows that the UHPLC-MS-based metabolomics platform is a promising and valuable tool to generate new insights into the drug-resistant mechanism of Campylobacter jejuni.

Cloning, expression, purification and characterization of a bispecific single-chain diabody against fluoroquinolones and sulfonamides in Escherichia coli.

A recombinant bispecific single-chain diabody (scDb), recognizing fluoroquinolones (FQs) and sulfonamides (SAs), was successfully constructed with two single-chain variable fragment antibodies (scFvs). The scDb gene was cloned into the expression vector pJB33, and 6×His-tagged scDb was expressed as soluble bodies in Escherichia coli RV308 host, then purified by one step affinity chromatography of immobilized metal ion affinity chromatography (IMAC). SDS-PAGE and Western blotting analysis of the purified scDb indicated that the prepared scDb was successfully expressed as a ∼60 kDa and the final purity of the scDb protein was up to 95% with yields of approximately 6 mg/L of bacterial culture. The scDb was further characterized by indirect competitive enzyme linked immunosorbent assay (icELISA), showing that the affinity and specificity of scDb were fully retained from the two parental scFvs, capable of simultaneously binding FQs and SAs. The 50% inhibition concentration (IC50) values of the optimized immunoassay were 0.45 ng mL(-1) for FQs and 0.75 ng mL(-1) for SAs, respectively. The scDb exhibited high affinity to 20 FQs and 14 SAs. Taken together, these findings suggested that the prepared scDb could be used to develop future novel immunoassay for simultaneous determination of 20 FQs and 14 SAs.

High resolution melting curve analysis as a new tool for rapid identification of canine parvovirus type 2 strains.

A high resolution melting (HRM) curve method was developed to identify canine parvovirus type 2 (CPV-2) strains by nested PCR. Two sets of primers, CPV-426F/426R and CPV-87R/87F, were designed that amplified a 52 bp and 53 bp product from the viral VP2 capsid gene. The region amplified by CPV-426F/426R included the A4062G and T4064A mutations in CPV-2a, CPV-2b and CPV-2c. The region amplified by CPV-87F/87R included the A3045T mutation in the vaccine strains of CPV-2 and CPV-2a, CPV-2b and CPV-2c. Faecal samples were obtained from 30 dogs that were CPV antigen-positive. The DNA was isolated from the faecal samples and PCR-amplified using the two sets of primers, and genotyped by HRM curve analysis. The PCR-HRM assay was able to distinguish single nucleotide polymorphisms between CPV-2a, CPV-2b and CPV-2c using CPV-426F/426R. CPV-2a was distinguished from CPV-2b and CPV-2c by differences in the melting temperature. CPV-2b and CPV-2c could be distinguished based on the shape of the melting curve after generating heteroduplexes using a CPV-2b reference sample. The vaccine strains of CPV-2 were identified using CPV-87F/87R. Conventional methods for genotyping CPV strains are labor intensive, expensive or time consuming; the present PCR-based HRM assay might be an attractive alternative.

A novel phenicol exporter gene, fexB, found in enterococci of animal origin.

OBJECTIVES: To investigate two porcine Enterococcus isolates for the genetic basis of phenicol resistance and to determine the location and the genetic environment of the novel resistance gene. METHODS: A total of 391 isolates with reduced florfenicol susceptibility (MIC ≥ 16 mg/L), obtained from 557 nasal swabs of individual pigs, were screened by PCR for the known florfenicol resistance genes. Isolates that were negative in these PCRs were analysed for their species assignment and antimicrobial susceptibility. Plasmids were extracted and subjected to transformation and conjugation assays. Restriction fragments of the phenicol resistance plasmids were cloned and sequenced. The sequences obtained were analysed and compared with sequences deposited in the databases. RESULTS: The two isolates, Enterococcus faecium EFM-1 and Enterococcus hirae EH-1, exhibited MICs of chloramphenicol and florfenicol of 64 mg/L and carried a new phenicol resistance gene, designated fexB. This gene codes for a phenicol exporter of 469 amino acids organized in 14 transmembrane domains. The fexB gene was located on the 35 kb pEFM-1 from E. faecium and on the 25.3 kb pEH-1 from E. hirae, respectively. Both plasmids were non-conjugative. The fexB gene was found to be embedded in virtually the same genetic environment of 14.8 kb in both plasmids. CONCLUSION: To the best of our knowledge, this is the first report of the new florfenicol exporter gene fexB. Based on its plasmid location, horizontal transfer from the enterococci to other bacteria is possible.

Mucoid Acinetobacter baumannii enhances anti-phagocytosis through reducing C3b deposition.

Background: Multidrug resistant (MDR) Acinetobacter baumannii causes serious infections in intensive care units and is hard to be eradicated by antibiotics. Many A. baumannii isolates are identified as the mucoid type recently, but the biological characteristics of mucoid A. baumannii and their interactions with host cells remains unclear. Methods: The mucoid phenotype, antimicrobial susceptibility, biofilm-forming ability, acid resistance ability, peroxide tolerance, and in vivo toxicity of clinical ICUs derived A. baumannii isolates were first investigated. Secondly, the phagocytic resistance and invasive capacity of A. baumannii isolates to macrophages (MH-S, RAW264.7) and epithelial cells (A549) were analyzed. Furthermore, the abundance of C3b (complement factor C3 degradation product) deposition on the surface of A. baumannii was investigated. Last, the relationship between C3b deposition and the abundance of capsule in A. baumannii isolates were analyzed. Results: These A. baumannii strains showed different mucoid phenotypes including hyper mucoid (HM), medium mucoid (MM), and low mucoid (LM). All tested strains were MDR with high tolerance to either acid or hydrogen peroxide exposure. Notably, these mucoid strains showed the increase of mortality in the Galleria mellonella infection models. Besides, the HM strain exhibited less biofilm abundance, higher molecular weight (MW) of capsule, and greater anti-phagocytic activity to macrophages than the LM strain. Together with the increased abundance of capsule, high expression of tuf gene (associated with the hydrolysis of C3b), the HM strain effectively inhibits C3b deposition on bacterial surface, resulting in the low-opsonization phenotype. Conclusion: Capsular characteristics facilitate the anti-phagocytic activity in hyper mucoid A. baumannii through the reduction of C3b deposition. Mucoid A. baumannii exhibits high phagocytosis resistance to both macrophages and epithelial cells.

Genomic Insights into the Increased Occurrence of Campylobacteriosis Caused by Antimicrobial-Resistant Campylobacter coli.

Campylobacter is the leading bacterial cause of diarrheal illnesses worldwide. Campylobacter jejuni and C. coli are the most common species accounting for campylobacteriosis. Although the proportion of campylobacteriosis caused by C. coli is increasing rapidly in China, the underlying mechanisms of this emergence remain unclear. In this study, we analyzed the whole-genome sequences and associated environments of 1,195 C. coli isolates with human, poultry, or porcine origins from 1980 to 2021. C. coli isolates of human origin were closely related to those from poultry, suggesting that poultry was the main source of C. coli infection in humans. Analysis of antimicrobial resistance determinants indicated that the prevalence of multidrug-resistant C. coli has increased dramatically since the 2010s, coinciding with the shift in abundance from C. jejuni to C. coli in Chinese poultry. Compared with C. jejuni, drug-resistant C. coli strains were better adapted and showed increased proliferation in the poultry production environment, where multiple antimicrobial agents were frequently used. This study provides an empirical basis for the molecular mechanisms that have enabled C. coli to become the dominant Campylobacter species in poultry; we also emphasize the importance of poultry products as sources of campylobacteriosis caused by C. coli in human patients. IMPORTANCE The proportion of campylobacteriosis caused by C. coli is increasing rapidly in China. Coincidentally, the dominant species of Campylobacter occurring in poultry products has shifted from C. jejuni to C. coli. Here, we analyzed the whole-genome sequences of 1,195 C. coli isolates from different origins. The phylogenetic relationship among C. coli isolates suggests that poultry was the main source of C. coli infection in humans. Further analysis indicated that antimicrobial resistance in C. coli strains has increased dramatically since the 2010s, which could facilitate their adaptation in the poultry production environment, where multiple antimicrobial agents are frequently used. Thus, our findings suggest that the judicious use of antimicrobial agents could mitigate the emergence of multidrug-resistant C. coli strains and enhance clinical outcomes by restoring drug sensitivity in Campylobacter.

Collateral sensitivity to pleuromutilins in vancomycin-resistant Enterococcus faecium.

The acquisition of resistance to one antibiotic sometimes leads to collateral sensitivity to a second antibiotic. Here, we show that vancomycin resistance in Enterococcus faecium is associated with a remarkable increase in susceptibility to pleuromutilin antibiotics (such as lefamulin), which target the bacterial ribosome. The trade-off between vancomycin and pleuromutilins is mediated by epistasis between the van gene cluster and msrC, encoding an ABC-F protein that protects bacterial ribosomes from antibiotic targeting. In mouse models of vancomycin-resistant E. faecium colonization and septicemia, pleuromutilin treatment reduces colonization and improves survival more effectively than standard therapy (linezolid). Our findings suggest that pleuromutilins may be useful for the treatment of vancomycin-resistant E. faecium infections.

Metabolism of olaquindox in rat and identification of metabolites in urine and feces using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry.

Olaquindox (OLA), N-(2-hydroxyethyl)-3-methyl-2-quinoxalincarboxamide-1,4-dioxide, is an antimicrobial and growth-promoting agent for animals, which has been banned or allowed only limited use for its potential toxicity. To thoroughly understand the metabolic pathways, metabolism of OLA in rat was studied using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry with MS(E) and mass defect filtering techniques. Twenty metabolites (M1-M20) were detected in rat feces and urine, of which nine phase I metabolites (M6, M7, M11-M16) and four phase II metabolites (M17-M20) were found in vivo for the first time. The structures of metabolites were reliably characterized on the basis of accurate mass and fragment ions in MS(E) spectra. The major metabolic pathways reported previously in pigs, including reduction of N→O groups, oxidation of the alcohol and hydrolysis, were also confirmed in this study. In addition, hydroxylation of the methyl group, N-dehydroxyethylation and glucuronidation were also proved to be the important metabolic pathways, which contribute to improving our knowledge about in vivo metabolism of OLA.

Genomic and Phenotypic Analysis of Persistent Carbapenem-Resistant Klebsiella pneumoniae Isolates from a 5-Year Hospitalized Patient.

The emerging epidemic of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a global public health crisis. However, the phylogenetic affiliation and pathotypic status of CRKP strains in the host colonization period under consistent antibiotic treatments are not well characterized. In this study, a 5-year tracking study was performed, in which a patient admitted to an intensive care unit was recruited and then screened for the carriage of CRKP based on microbiological culture. Nine isolates from the sputum or stool samples were acquired and subjected to real-time whole-genome sequencing, antimicrobial susceptibility testing, Galleria mellonella larval infection, and epithelial cell invasion assay. All nine isolates showed phenotypic resistance to carbapenems, quinolones, and aminoglycosides. Altogether, blaKPC-2 and 10 other antibiotic resistance genes were identified and all nine CRKP isolates exhibited low virulence with more than 38 virulence factors. All but one variant belonged to ST11 with a novel sequence type, differing at the phoE locus. The isolates shared the same plasmid replicon type, prophages, and capsular serotype (K47) with few single-nucleotide polymorphism variations, consistent with epidemiological clones. Furthermore, these CRKP isolates displayed the ability of moderate invasion of lung epithelial cells. Meanwhile, a deficiency of chromosomal type IV secretion system-related gene cluster was detected after 2 years of carriage. Our findings demonstrated that low-virulence CRKP clones could colonize in the gut and respiratory tract under multiple antibiotic stresses, suggesting the strong colonization adaptability of CRKP to the host. Ethical approval was given by The Second Affiliated Hospital of Zhejiang University, School of Medicine, (2018-039).

A Marine Antibiotic Kills Multidrug-Resistant Bacteria without Detectable High-Level Resistance.

Antibiotic resistance nowadays is spreading much faster than the introduction of new antibiotics into clinical practice. There is an urgent need for potential compounds to combat multidrug-resistant (MDR) bacteria. Marine fungi provide a promising source for chemical diversity with antibiotic-like molecules. To identify structurally distinct compounds that effectively eradicate MDR pathogens and to control the development of antibiotic resistance, we have reinvestigated equisetin, a previously reported meroterpenoid isolated from a marine sponge-derived fungus. Equisetin exerted efficient antibacterial activities against either MRSA or VRE without detectable high-level resistance. Meanwhile, equisetin, as an antibiotic adjuvant, restores colistin susceptibility to colistin-resistant bacteria toward diverse Gram-negative pathogens. Intriguingly, the low-level equisetin-resistant Staphylococcus aureus displayed collateral sensitivity to multiple classes of existing antibiotics with decreased capacity to produce biofilm. Lastly, equisetin showed efficacy with MRSA in three infected animal models. This work suggests that equisetin derived from marine natural products is a promising lead to overcome antibiotic resistance, providing new insight in future antibiotic discovery and development.