Biological Function of Prophage-Related Gene Cluster ΔVpaChn25_RS25055~ΔVpaChn25_0714 of Vibrio parahaemolyticus CHN25.

Vibrio parahaemolyticus is the primary foodborne pathogen known to cause gastrointestinal infections in humans. Nevertheless, the molecular mechanisms of V. parahaemolyticus pathogenicity are not fully understood. Prophages carry virulence and antibiotic resistance genes commonly found in Vibrio populations, and they facilitate the spread of virulence and the emergence of pathogenic Vibrio strains. In this study, we characterized three such genes, VpaChn25_0713, VpaChn25_0714, and VpaChn25_RS25055, within the largest prophage gene cluster in V. parahaemolyticus CHN25. The deletion mutants ΔVpaChn25_RS25055, ΔVpaChn25_0713, ΔVpaChn25_0714, and ΔVpaChn25_RS25055-0713-0714 were derived with homologous recombination, and the complementary mutants ΔVpaChn25_0713-com, ΔVpaChn25_0714-com, ΔVpaChn25_RS25055-com, ΔVpaChn25_RS25055-0713-0714-com were also constructed. In the absence of the VpaChn25_RS25055, VpaChn25_0713, VpaChn25_0714, and VpaChn25_RS25055-0713-0714 genes, the mutants showed significant reductions in low-temperature survivability and biofilm formation (p < 0.001). The ΔVpaChn25_0713, ΔVpaChn25_RS25055, and ΔVpaChn25_RS25055-0713-0714 mutants were also significantly defective in swimming motility (p < 0.001). In the Caco-2 model, the above four mutants attenuated the cytotoxic effects of V. parahaemolyticus CHN25 on human intestinal epithelial cells (p < 0.01), especially the ΔVpaChn25_RS25055 and ΔVpaChn25_RS25055-0713-0714 mutants. Transcriptomic analysis showed that 15, 14, 8, and 11 metabolic pathways were changed in the ΔVpaChn25_RS25055, ΔVpaChn25_0713, ΔVpaChn25_0714, and ΔVpaChn25_RS25055-0713-0714 mutants, respectively. We labeled the VpaChn25_RS25055 gene with superfolder green fluorescent protein (sfGFP) and found it localized at both poles of the bacteria cell. In addition, we analyzed the evolutionary origins of the above genes. In summary, the prophage genes VpaChn25_0713, VpaChn25_0714, and VpaChn25_RS25055 enhance V. parahaemolyticus CHN25's survival in the environment and host. Our work improves the comprehension of the synergy between prophage-associated genes and the evolutionary process of V. parahaemolyticus.

Adult-born neurons in critical period maintain hippocampal seizures via local aberrant excitatory circuits.

Temporal lobe epilepsy (TLE), one common type of medically refractory epilepsy, is accompanied with altered adult-born dentate granule cells (abDGCs). However, the causal role of abDGCs in recurrent seizures of TLE is not fully understood. Here, taking advantage of optogenetic and chemogenetic tools to selectively manipulate abDGCs in a reversible manner, combined with Ca2+ fiber photometry, trans-synaptic viral tracing, in vivo/vitro electrophysiology approaches, we aimed to test the role of abDGCs born at different period of epileptogenic insult in later recurrent seizures in mouse TLE models. We found that abDGCs were functionally inhibited during recurrent seizures. Optogenetic activation of abDGCs significantly extended, while inhibition curtailed, the seizure duration. This seizure-modulating effect was attributed to specific abDGCs born at a critical early phase after kindled status, which experienced specific type of circuit re-organization. Further, abDGCs extended seizure duration via local excitatory circuit with early-born granule cells (ebDGCs). Repeated modulation of "abDGC-ebDGC" circuit may easily induce a change of synaptic plasticity, and achieve long-term anti-seizure effects in both kindling and kainic acid-induced TLE models. Together, we demonstrate that abDGCs born at a critical period of epileptogenic insult maintain seizure duration via local aberrant excitatory circuits, and inactivation of these aberrant circuits can long-termly alleviate severity of seizures. This provides a deeper and more comprehensive understanding of the potential pathological changes of abDGCs circuit and may be helpful for the precise treatment in TLE.

First Report of Potentially Pathogenic Klebsiella pneumoniae from Serotype K2 in Mollusk Tegillarca granosa and Genetic Diversity of Klebsiella pneumoniae in 14 Species of Edible Aquatic Animals.

Klebsiella pneumoniae can cause serious pneumonitis in humans. The bacterium is also the common causative agent of hospital-acquired multidrug-resistant (MDR) infections. Here we for the first time reported the genetic diversity of K. pneumoniae strains in 14 species of edible aquatic animals sampled in the summer of 2018 and 2019 in Shanghai, China. Virulence-related genes were present in the K. pneumoniae strains (n = 94), including the entB (98.9%), mrkD (85.1%), fimH (50.0%), and ybtA (14.9%) strains. Resistance to sulfamethoxazole-trimethoprim was the most prevalent (52.1%), followed by chloramphenicol (31.9%), and tetracycline (27.7%), among the strains, wherein 34.0% had MDR phenotypes. Meanwhile, most strains were tolerant to heavy metals Cu2+ (96.8%), Cr3+ (96.8%), Zn2+ (91.5%), Pb2+ (89.4%), and Hg2+ (81.9%). Remarkably, a higher abundance of the bacterium was found in bottom-dwelling aquatic animals, among which mollusk Tegillarca granosa contained K. pneumoniae 8-2-5-4 isolate from serotype K2 (ST-2026). Genome features of the potentially pathogenic isolate were characterized. The enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR)−based genome fingerprinting classified the 94 K. pneumoniae strains into 76 ERIC genotypes with 63 singletons, demonstrating considerable genetic diversity in the strains. The findings of this study fill the gap in the risk assessment of K. pneumoniae in edible aquatic animals.

Antibacterial Activity and Components of the Methanol-Phase Extract from Rhizomes of Pharmacophagous Plant Alpinia officinarum Hance.

The rhizomes of Alpinia officinarum Hance (known as the smaller galangal) have been used as a traditional medicine for over 1000 years. Nevertheless, little research is available on the bacteriostatic activity of the herb rhizomes. In this study, we employed, for the first time, a chloroform and methanol extraction method to investigate the antibacterial activity and components of the rhizomes of A. officinarum Hance. The results showed that the growth of five species of pathogenic bacteria was significantly inhibited by the galangal methanol-phase extract (GMPE) (p < 0.05). The GMPE treatment changed the bacterial cell surface hydrophobicity, membrane fluidity and/or permeability. Comparative transcriptomic analyses revealed approximately eleven and ten significantly altered metabolic pathways in representative Gram-positive Staphylococcus aureus and Gram-negative Enterobacter sakazakii pathogens, respectively (p < 0.05), demonstrating different antibacterial action modes. The GMPE was separated further using a preparative high-performance liquid chromatography (Prep-HPLC) technique, and approximately 46 and 45 different compounds in two major component fractions (Fractions 1 and 4, respectively) were identified using ultra-HPLC combined with mass spectrometry (UHPLC-MS) techniques. o-Methoxy cinnamaldehyde (40.12%) and p-octopamine (62.64%) were the most abundant compounds in Fractions 1 and 4, respectively. The results of this study provide data for developing natural products from galangal rhizomes against common pathogenic bacteria.

Diverse Aquatic Animal Matrices Play a Key Role in Survival and Potential Virulence of Non-O1/O139 Vibrio cholerae Isolates.

Vibrio cholerae can cause pandemic cholera in humans. The waterborne bacterium is frequently isolated from aquatic products worldwide. However, current literature on the impact of aquatic product matrices on the survival and pathogenicity of cholerae is rare. In this study, the growth of eleven non-O1/0O139 V. cholerae isolates recovered from eight species of commonly consumed fish and shellfish was for the first time determined in the eight aquatic animal matrices, most of which highly increased the bacterial biomass when compared with routine trypsin soybean broth (TSB) medium. Secretomes of the V. cholerae isolates (draft genome size: 3,852,021-4,144,013 bp) were determined using two-dimensional gel electrophoresis (2DE-GE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques. Comparative secretomic analyses revealed 74 differential extracellular proteins, including several virulence- and resistance-associated proteins secreted by the V. cholerae isolates when grown in the eight matrices. Meanwhile, a total of 8,119 intracellular proteins were identified, including 83 virulence- and 8 resistance-associated proteins, of which 61 virulence-associated proteins were absent from proteomes of these isolates when grown in the TSB medium. Additionally, comparative genomic and proteomic analyses also revealed several strain-specific proteins with unknown functions in the V. cholerae isolates. Taken, the results in this study demonstrate that distinct secretomes and proteomes induced by the aquatic animal matrices facilitate V. cholerae resistance in the edible aquatic animals and enhance the pathogenicity of the leading waterborne pathogen worldwide.

Prophage-encoded gene VpaChn25_0734 amplifies ecological persistence of Vibrio parahaemolyticus CHN25.

Vibrio parahaemolyticus is a waterborne pathogen that can cause acute gastroenteritis, wound infection, and septicemia in humans. The molecular basis of its pathogenicity is not yet fully understood. Phages are found most abundantly in aquatic environments and play a critical role in horizontal gene transfer. Nevertheless, current literature on biological roles of prophage-encoded genes remaining in V. parahaemolyticus is rare. In this study, we characterized one such gene VpaChn25_0734 (543-bp) in V. parahaemolyticus CHN25 genome. A deletion mutant ΔVpaChn25_0734 (543-bp) was obtained by homologous recombination, and a revertant ΔVpaChn25_0734-com (543-bp) was also constructed. The ΔVpaChn25_0734 (543-bp) mutant was defective in growth and swimming mobility particularly at lower temperatures and/or pH 7.0-8.5. Cell surface hydrophobicity and biofilm formation were significantly decreased in the ΔVpaChn25_0734 (543-bp) mutant (p < 0.05). Based on the in vitro Caco-2 cell model, the deletion of VpaChn25_0734 (543-bp) gene significantly reduced the cytotoxicity of V. parahaemolyticus CHN25 to human intestinal epithelial cells (p < 0.05). Comparative secretomic and transcriptomic analyses revealed a slightly increased extracellular proteins, and thirteen significantly changed metabolic pathways in the ΔVpaChn25_0734 (543-bp) mutant, showing down-regulated carbon source transport and utilization, biofilm formation, and type II secretion system (p < 0.05), consistent with the observed defective phenotypes. Taken, the prophage-encoded gene VpaChn25_0734 (543-bp) enhanced V. parahaemolyticus CHN25 fitness for survival in the environment and the host. The results in this study facilitate better understanding of pathogenesis and genome evolution of V. parahaemolyticus, the leading sea foodborne pathogen worldwide.

First Experimental Evidence for the Presence of Potentially Virulent Klebsiella oxytoca in 14 Species of Commonly Consumed Aquatic Animals, and Phenotyping and Genotyping of K. oxytoca Isolates.

Klebsiella oxytoca is a recently emerging pathogen that can cause necrotizing enterocolitis, hemorrhagic colitis, sepsis-associated purpura fulminans, and infective endocarditis in humans. The bacterium is ubiquitous in water and soil environments. Nevertheless, current literature on K. oxytoca in aquatic products is rare. In this study, we surveyed K. oxytoca contamination in 41 species of consumable aquatic animals sold in July, August, and September of 2018 and 2019 in Shanghai, China, 40 of which had no history of carrying this bacterium. K. oxytoca was for the first time isolated from 14 species with high abundance in benthic animals. None of the K. oxytoca isolates (n = 125) harbored toxin genes mviM, tisB, and yqgB. However, a high occurrence of virulence-associated genes was observed, including brkB (73.6%), cdcB (66.4%), pduV (64.8%), and virk (63.2%). Resistance to sulphamethoxazole-trimethoprim (56.0%) was the most predominant among the isolates, followed by chloramphenicol (6.4%), tetracycline (5.6%), and kanamycin (3.2%). Approximately 8.0% of the isolates displayed multidrug resistant phenotypes. Meanwhile, high percentages of the isolates tolerated the heavy metals Cu2+ (84.8%), Pb2+ (80.8%), Cr3+ (66.4%), Zn2+ (66.4%), and Hg2+ (49.6%). Different virulence and resistance profiles were observed among K. oxytoca isolates in 3 types and 14 species of aquatic animals. The ERIC-PCR-based genome fingerprinting of the 125 K. oxytoca isolates revealed 108 ERIC genotypes with 79 singletons, which demonstrated the genetic diversity of the isolates. The results of this study fill gaps for policy and research in the risk assessment of K. oxytoca in consumable aquatic animals.

First Experimental Evidence for the Presence of Potentially Toxic Vibrio cholerae in Snails, and Virulence, Cross-Resistance and Genetic Diversity of the Bacterium in 36 Species of Aquatic Food Animals.

Vibrio cholerae is the most common waterborne pathogen that can cause pandemic cholera in humans. Continuous monitoring of V. cholerae contamination in aquatic products is crucial for assuring food safety. In this study, we determined the virulence, cross-resistance between antibiotics and heavy metals, and genetic diversity of V. cholerae isolates from 36 species of aquatic food animals, nearly two-thirds of which have not been previously detected. None of the V. cholerae isolates (n = 203) harbored the cholera toxin genes ctxAB (0.0%). However, isolates carrying virulence genes tcpA (0.98%), ace (0.5%), and zot (0.5%) were discovered, which originated from the snail Cipangopaludina chinensis. High occurrences were observed for virulence-associated genes, including hapA (73.4%), rtxCABD (68.0-41.9%), tlh (54.2%), and hlyA (37.9%). Resistance to moxfloxacin (74.9%) was most predominant resistance among the isolates, followed by ampicillin (59.1%) and rifampicin (32.5%). Approximately 58.6% of the isolates displayed multidrug resistant phenotypes. Meanwhile, high percentages of the isolates tolerated the heavy metals Hg2+ (67.0%), Pb2+ (57.6%), and Zn2+ (57.6%). Distinct virulence and cross-resistance profiles were discovered among the V. cholerae isolates in 13 species of aquatic food animals. The ERIC-PCR-based genome fingerprinting of the 203 V. cholerae isolates revealed 170 ERIC-genotypes, which demonstrated considerable genomic variation among the isolates. Overall, the results of this study provide useful data to fill gaps for policy and research related to the risk assessment of V. cholerae contamination in aquatic products.

A sensitive and specific nanosensor for monitoring extracellular potassium levels in the brain.

Extracellular potassium concentration affects the membrane potential of neurons, and, thus, neuronal activity. Indeed, alterations of potassium levels can be related to neurological disorders, such as epilepsy and Alzheimer's disease, and, therefore, selectively detecting extracellular potassium would allow the monitoring of disease. However, currently available optical reporters are not capable of detecting small changes in potassium, in particular, in freely moving animals. Furthermore, they are susceptible to interference from sodium ions. Here, we report a highly sensitive and specific potassium nanosensor that can monitor potassium changes in the brain of freely moving mice undergoing epileptic seizures. An optical potassium indicator is embedded in mesoporous silica nanoparticles, which are shielded by an ultrathin layer of a potassium-permeable membrane, which prevents diffusion of other cations and allows the specific capturing of potassium ions. The shielded nanosensor enables the spatial mapping of potassium ion release in the hippocampus of freely moving mice.

Longitudinal Changes in Crude Protein and Amino Acids in Human Milk in Chinese Population: A Systematic Review.

OBJECTIVE: More than 20% of the world population live in China, which has made significant achievement in human milk research. Part of the data that were published in Chinese were, however, unavailable to non-Chinese speakers. There was also no comprehensive overview of crude protein and amino acid levels in human milk in Chinese population. This systematic review aimed to compile the data on human milk crude protein and amino acid levels in Chinese population. METHODS: After searching for and screening original research articles in both English and Chinese, 23 published from 1987 to 2019 were identified (18 in Chinese and 5 in English). The data were pooled into 9 defined lactation stages. RESULTS: Crude protein and amino acids (protein bound plus nonprotein bound) concentrations gradually decreased during the first 60 postpartum days and remained relatively static thereafter. The concentrations and dynamic change of crude protein and amino acids were similar to those in other populations. By contrast, the longitudinal changes in free amino acids (nonprotein bound) were less clear due to the limited data available. Several common weaknesses were identified in these studies. CONCLUSIONS: Our study represented the most comprehensive overview on crude protein and amino acid concentrations in human milk in Chinese population, and enhanced the knowledge of protein and amino acid intakes and requirements by Chinese infants.