Lithium Promotes Osteogenesis via Rab11a-Facilitated Exosomal Wnt10a Secretion and ß-Catenin Signaling Activation.

Both bone mesenchymal stem cells (BMSCs) and their exosomes suggest promising therapeutic tools for bone regeneration. Lithium has been reported to regulate BMSC function and engineer exosomes to improve bone regeneration in patients with glucocorticoid-induced osteonecrosis of the femoral head. However, the mechanisms by which lithium promotes osteogenesis have not been elucidated. Here, we demonstrated that lithium promotes the osteogenesis of BMSCs via lithium-induced increases in the secretion of exosomal Wnt10a to activate Wnt/ß-catenin signaling, whose secretion is correlated with enhanced MARK2 activation to increase the trafficking of the Rab11a and Rab11FIP1 complexes together with exosomal Wnt10a to the plasma membrane. Then, we compared the proosteogenic effects of exosomes derived from lithium-treated or untreated BMSCs (Li-Exo or Con-Exo) both in vitro and in vivo. We found that, compared with Con-Exo, Li-Exo had superior abilities to promote the uptake and osteogenic differentiation of BMSCs. To optimize the in vivo application of these hydrogels, we fabricated Li-Exo-functionalized gelatin methacrylate (GelMA) hydrogels, which are more effective at promoting osteogenesis and bone repair than Con-Exo. Collectively, these findings demonstrate the mechanism by which lithium promotes osteogenesis and the great promise of lithium for engineering BMSCs and their exosomes for bone regeneration, warranting further exploration in clinical practice.

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.

Rapid and Visualized Detection of Virulence-Related Genes of Vibrio cholerae in Water and Aquatic Products by Loop-Mediated Isothermal Amplification.

ABSTRACT: Vibrio cholerae can cause pandemic cholera in humans. The bacterium resides in aquatic environments worldwide. Continuous testing of V. cholerae contamination in water and aquatic products is imperative for food safety control and human health. In this study, a rapid and visualized method was developed for the first time based on loop-mediated isothermal amplification (LAMP) for detection of the important virulence-related genes ace, zot, cri, and nanH for toxins and the infectious process of V. cholerae. Three pairs of molecular probes targeting each of these genes were designed and synthesized. The one-step LAMP reaction was conducted at 65°C for 40 min. Positive results were inspected by the production of a light green color under visible light or green fluorescence under UV light (302 nm). Limit of detection of the LAMP method ranged from 1.85 to 2.06 pg per reaction of genomic DNA or 2.50 × 100 to 4.00 × 102 CFU per reaction for target genes of cell culture of V. cholerae, which was more sensitive than standard PCR. Inclusivity and exclusivity of the LAMP method were 100% for all target genes. The method showed similar high efficiency to a certain extent in rapid testing of spiked or collected specimens of water and aquatic products. Target genes were detected by absence from all water samples from various sources. However, high occurrences of the nanH gene were observed in intestinal samples derived from four species of fish and one species of shellfish, indicating a risk of potentially toxic V. cholerae in commonly consumed aquatic products. The results in this study provide a potential tool for rapid and visualized detection of V. cholerae in water and aquatic products.

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.

Simple Visualized Detection Method of Virulence-Associated Genes of Vibrio cholerae by Loop-Mediated Isothermal Amplification.

Vibrio cholerae is a leading waterborne pathogenic bacterium worldwide. It can cause human cholera that is still pandemic in developing nations. Detection of V. cholerae contamination in drinking water and aquatic products is imperative for assuring food safety. In this study, a simple, sensitive, specific, and visualized method was developed based on loop-mediated isothermal amplification (LAMP) (designated sssvLAMP) to detect virulence-associated (ctxA, tcpA, hapA, mshA, pilA, and tlh) and species-specific (lolB) genes of V. cholerae. Three pairs of oligonucleotide primers (inner, outer, and loop primers) were designed and or synthesized to target each of these genes. The optimal conditions of the sssvLAMP method was determined, and one-step sssvLAMP reaction was performed at 65°C for 40 min. Positive results were simply read by the naked eye via color change (from orange to light green) under the visible light, or by the production of green fluorescence under the UV light (260 nm). The sssvLAMP method was more efficient in detecting 6.50 × 101-6.45 × 104-fold low number of V. cholerae cells, and more sensitive in V. cholerae genomic DNA (1.36 × 10-2-4.42 × 10-6 ng/reaction) than polymerase chain reaction (PCR) method. Among 52 strains of V. cholerae and 50 strains of non-target species (e.g., other Vibrios and common pathogens) examined, the sensitivity and specificity of the sssvLAMP method were 100% for all the target genes. Similar high efficiency of the method was observed when tested with spiked samples of water and aquatic products, as well as human stool specimens. Water from various sources and commonly consumed fish samples were promptly screened by this simple and efficient visualized method and diversified variation in the occurrence of the target genes was observed. V. cholerae strains could be mostly detected by the presence of hapA and tlh alone or in combination with other genes, indicating a variable risk of potentially pathogenic non-O1/O139 strains in edible food products. This novel LAMP method can be a promising tool to address the increasing need of food safety control of aquatic products.