Acute toxicity evaluation of olive oil mill wastewaters: A comparative study of three aquatic organisms

Acute toxicity of olive mill wastewaters (traditional and continuous processes) collected from different regions of Portugal was evaluated using three test species (Vibrio fischeri formerly Photobacterium phosphoreum, Thamnocephalus platyurus, and Daphnia magna) and correlated with several physical and chemical parameters. Acute toxicity of these effluents, expressed in LC₅₀ or EC₅₀, ranged from: 0.16 to 1.24% in Microtox test, 0.73 to 12.54% in Thamnotoxkit F test, and 1.08 to 6.83% in Daphnia test. These values reflect the high toxicity of the olive mill wastewaters to all test species. Statistical analysis of the results shows a high correlation between the two microcrustacean bioassays. Microtox test did not correlate significantly with the other bioassays used. A significative correlation (p≤0.05) could also be established between L(E)C₅₀ obtained in the microcrustacean tests and some physicochemical parameters of the effluent. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 263–269, 1999     

原发性创伤弧菌感染12例

目的 :　报道 12例原发性创伤弧菌感染。方法 :　总结 1995～ 2 0 0 0年间的 12例原发性创伤弧菌感染的临床资料。结果 :　 12例原发性创伤弧菌感染 ,其中男 8例 ,女 4例。年龄 40～ 6 7岁 ,平均48.6岁。 6例有肝硬变史。好发盛夏、初秋 ,起病急。初发症状为双下肢红肿、疼痛、大疱、血疱。疱液培养发现表皮葡萄球菌 1例 ,弧菌 2例。皮损活检 :局灶性棘细胞松解 ,真皮胶原凝固性坏死 ,血管内皮坏死、脱落 ,血管壁可见纤维蛋白样坏死 ,组织内出血明显 ,可见嗜中性细胞浸润。真皮内可见众多散在分布的淡嗜伊红的近圆点状微生物。透射电镜观察 :真皮细胞内外见众多散在分布的弧形微生物可见细菌鞭毛。 1例痊愈 ,3例自动出院 ,8例死亡。 1例死于血小板减少引起的颅内出血 ,7例死于多脏器功能衰竭。死亡时间最短在发病后 5天 ,最长 10天 ,平均 6 .3天。结论 :　 (1)接触海水或生食贝壳类海鲜往往是造成感染的先决条件 ;(2 )肝硬化、糖尿病等慢性病易引起创伤弧菌感染 ;(3)常规培养致病菌发现率不高 ;(4 )双下肢出血性水肿是原发性创伤弧菌感染特征性病变之一 ;(5 )皮损病理改变 :表皮细胞灶性松解 ,表皮下水疱 ,真皮水肿、胶原纤维变性坏死 ,明显的血管炎改变、血管栓塞 ,真皮内可见细菌。 (6 )早期用抗菌素     

Ecological diversification reveals routes of pathogen emergence in endemic Vibrio vulnificus populations

Pathogen emergence is a complex phenomenon that, despite its public health relevance, remains poorly understood. Vibrio vulnificus, an emergent human pathogen, can cause a deadly septicaemia with over 50% mortality rate. To date, the ecological drivers that lead to the emergence of clinical strains and the unique genetic traits that allow these clones to colonize the human host remain mostly unknown. We recently surveyed a large estuary in eastern Florida, where outbreaks of the disease frequently occur, and found endemic populations of the bacterium. We established two sampling sites and observed strong correlations between location and pathogenic potential. One site is significantly enriched with strains that belong to one phylogenomic cluster (C1) in which the majority of clinical strains belong. Interestingly, strains isolated from this site exhibit phenotypic traits associated with clinical outcomes, whereas strains from the second site belong to a cluster that rarely causes disease in humans (C2). Analyses of C1 genomes indicate unique genetic markers in the form of clinical-associated alleles with a potential role in virulence. Finally, metagenomic and physicochemical analyses of the sampling sites indicate that this marked cluster distribution and genetic traits are strongly associated with distinct biotic and abiotic factors (e.g., salinity, nutrients, or biodiversity), revealing how ecosystems generate selective pressures that facilitate the emergence of specific strains with pathogenic potential in a population. This knowledge can be applied to assess the risk of pathogen emergence from environmental sources and integrated toward the development of novel strategies for the prevention of future outbreaks.

The emergence of human pathogens is one of the most concerning public health topics of modern times (1–4). According to the World Health Organization, over 300 emerging infectious diseases have been reported in the 1940 to 2004 period, a trend that has continued steadily with recent outbreaks of Ebola in West Africa, Cholera in Yemen, and the global pandemic caused by COVID-19 (3–5). Even though classical molecular approaches have advanced our understanding of bacterial pathogenesis, to date, the genetic adaptations and ecological drivers that facilitate selected strains within a species to emerge as pathogens and successfully colonize the human host remain poorly understood. Given the magnitude and complexity of this urgent threat, it is critical to develop tractable organismal model systems and theoretical frameworks that allow us to dissect the molecular adaptations and environmental factors that lead to the emergence of such human pathogens.Vibrio vulnificus, an emergent human pathogen, is one of the leading causes of non-Cholera, Vibrio-associated deaths globally (6). Despite being a natural inhabitant of estuarine, coastal, and brackish waters (7), this flesh-eating bacterium has gained particular notoriety as one of the fastest killing pathogens (8, 9). Humans are typically infected with V. vulnificus through ingestion of contaminated raw seafood or by direct exposure of open wounds to seawater (6). V. vulnificus infections often result in fulminant septicaemia with an alarming mortality rate exceeding 50% (6, 10–13). The bacterium is particularly lethal in some susceptible hosts, such as immunocompromised patients or those with alcohol-associated liver cirrhosis, diabetes mellitus, or hemochromatosis (14). The annual case counts of V. vulnificus infections have steadily increased over the past 20 y in the United States (15). An upsurge in its worldwide distribution over the past three decades, in correlation with climate change, has led to disease outbreaks in regions with no history of V. vulnificus infections (16–18). Furthermore, models predict this trend to continue resulting in a steady expansion of its geographical range and the subsequent increased risk of human infections (16, 19–21).Based on a series of biochemical and phenotypic traits, V. vulnificus strains have been historically classified into three Biotypes (BT): BT1, which is mostly associated with human infections (22, 23), BT2, which is primarily pathogenic to eels (24, 25), and BT3, which is geographically restricted to Israel and possesses hybrid characteristics from BT1 and BT2 (26, 27). In contrast to Vibrio cholerae, in which all strains capable of causing cholera belong to a single clade, genomic comparisons of V. vulnificus reveal a more complex pattern in the distribution of its clinical strains (28–30). Phylogenomic analyses indicate that the population of V. vulnificus is composed of four distinct groups or clusters (Cluster 1 to 4), which largely overlap with the classical BT classification system (23, 26, 28, 31, 32). Our analyses indicate that the two largest clusters, C1 and C2, exhibit high genomic divergence and appear to be speciating (28), with clinical strains from BT1 predominantly belonging to C1 (22, 23), whereas strains from C2 are primarily associated with BT2 (6, 24, 25). C3 is highly clonal and fully overlaps with BT3, and the rare C4 contains only four nonclonal strains and belongs to BT1 (28, 31). Interestingly, despite patients showing conserved clinical symptoms, C1 clinical strains arise from different clades within the cluster, suggesting independent emergence events of this deadly pathogen (28, 31, 32). To date, the unique genetic traits that allow certain C1 strains to cause severe septicemia remain mostly unknown, posing a daunting public health risk as it hinders our ability to detect potentially pathogenic V. vulnificus (33).Recently, using a combination of bioinformatic and phenotypic analyses that surveyed more than 100 strains of V. vulnificus, we determined that V. vulnificus C1 appears to be associated with a unique ecological lifestyle or ecotype (28). Nonetheless, to date, the ecological drivers that lead to the emergence of clinical V. vulnificus C1 and their pathogenic traits remain poorly understood. In order to start untangling the complex in-situ interactions between genotypes and the environment that underlie the emergence of clinical strains, in this study, we recently surveyed a large estuary in eastern Florida, the Indian River Lagoon (IRL), where outbreaks of the disease frequently occur (7, 34). We found endemic populations of V. vulnificus in the estuary and established two sampling locations to study the environmental dynamics of this bacterium in several natural reservoirs such as water, sediment, oysters, and cyanobacteria. Interestingly, the two sampling sites show major differences in the distribution of V. vulnificus clusters. One of them, Feller’s house (Site A), appears to be significantly enriched with C1 strains, whereas in the second sampling site, Shepard Park (Site B), we mostly recovered strains from C2. Genomic analyses of these strains indicate that, despite these major differences in distribution, high recombination rates as well as frequent exchange of mobile genetic elements and virulence factors between these V. vulnificus populations occur. Microdiversity analyses of these genomes revealed unique genomic markers among C1 strains in the form of clinical-associated alleles (CAAs) with a potential direct role in virulence. The isolated V. vulnificus strains are resistant to numerous commonly used antibiotics irrespective of cluster or site of isolation. However, phenotypic analyses indicate that strains from Site A exhibit traits associated with clinical outcomes, including the ability to resist serum and catabolize sialic acid, unlike those from Site B. Finally, metagenomic and physicochemical analyses of the sampling sites indicate that this marked cluster distribution is strongly associated with distinct biotic and abiotic factors (e.g., salinity, nutrients, or biodiversity) revealing how ecosystems might generate selective pressures that facilitate the emergence of specific strains in a population with pathogenic potential.     

三峡库区长江水非O1群霍乱弧菌L型的研究

为了解三峡库区长江水非Ｏ１群霍乱弧菌Ｌ型的流行病学状况,１９９４￣１９９８年作者在三峡库区选三地取长江水同时进行非Ｏ１群霍乱弧菌与非Ｏ１群霍乱弧菌Ｌ型的检测。结果发现Ｏ１群霍乱弧菌Ｌ型冬天阳性检出率较高,夏天阳性检出率较低。但各月份总阳性率无明显差异。非Ｏ１群霍乱弧菌Ｌ型返祖回复后证实为非Ｏ１群霍乱弧菌。表明非Ｏ１群霍乱弧菌Ｌ型为非Ｏ１群霍乱弧菌越冬的一种重要形式。在卫生监督中,应加强对非为群霍乱     

不同群型霍乱弧菌recA、dnaE和mdh管家基因序列分析

目的不同群型霍乱弧菌(VC)recA、dnaE和mdh管家基因序列分析。方法PCR扩增、基因测序及生物信息学分析。结果18株不同群型霍乱弧菌recA基因500bp序列中共44个位点发生变异,只有3个位点变异导致氨基酸变异;dnaE基因600bp序列中共32个位点发生变异,只有1个位点变异导致氨基酸变异;mdh基因367bp序列中共21个位点发生变异,只有1个位点变异导致氨基酸变异。O1群埃尔托生物型(EVC)产毒株和O139群VC产毒株遗传学关系高度密切;不同群型非产毒株之间遗传学关系较远;不同群型产毒株和不同群型非产毒株遗传学关系较远。结论我国不同地区流行的EVC产毒株和O139群VC产毒株可能分别来自同一克隆群,O139群VC产毒株可能起源于EVC产毒株。     

Hierarchical transitions and fractal wrinkling drive bacterial pellicle morphogenesis

Bacterial cells can self-organize into structured communities at fluid–fluid interfaces. These soft, living materials composed of cells and extracellular matrix are called pellicles. Cells residing in pellicles garner group-level survival advantages such as increased antibiotic resistance. The dynamics of pellicle formation and, more generally, how complex morphologies arise from active biomaterials confined at interfaces are not well understood. Here, using Vibrio cholerae as our model organism, a custom-built adaptive stereo microscope, fluorescence imaging, mechanical theory, and simulations, we report a fractal wrinkling morphogenesis program that differs radically from the well-known coalescence of wrinkles into folds that occurs in passive thin films at fluid–fluid interfaces. Four stages occur: growth of founding colonies, onset of primary wrinkles, development of secondary curved ridge instabilities, and finally the emergence of a cascade of finer structures with fractal-like scaling in wavelength. The time evolution of pellicle formation depends on the initial heterogeneity of the film microstructure. Changing the starting bacterial seeding density produces three variations in the sequence of morphogenic stages, which we term the bypass, crystalline, and incomplete modes. Despite these global architectural transitions, individual microcolonies remain spatially segregated, and thus, the community maintains spatial and genetic heterogeneity. Our results suggest that the memory of the original microstructure is critical in setting the morphogenic dynamics of a pellicle as an active biomaterial.

Spontaneous folding, wrinkling, and curling of soft tissues and active materials are ubiquitous in nature. For example, during the development of mammalian organs and plant leaves, collections of many cells self-organize into ordered morphological structures far larger than the size of the individual cells. Nontrivial geometric patterns and architectures often link form to function, such as the fractal branching and size scaling of leaf veins (1), the gyrification of the cerebral cortex (2–4), and the curving of villi in the gut (5, 6). Bacterial cells can also self-organize into communities known as biofilms and pellicles at interfaces. These multicellular communities are crucial in contexts such as medical infections and industrial biofouling because cells in biofilms and pellicles display enhanced resilience to antibiotics, immune clearance, and physical perturbations compared to their isogenic planktonic counterparts (7–9). Analogous to eukaryotic systems, bacterial biofilms and pellicles develop striking macroscopic morphologies including wrinkles and delaminations that are driven by combined biological, material-physics, and mechanical determinants (10–14). Studies of biofilms at the single-cell level show the emergence of internal cell ordering (15–17) and collective cellular flow (18). However, the dynamics of self-organization and the sequence of mechanical instabilities that direct morphogenesis for expanding active soft materials at fluid–fluid interfaces, such as bacterial pellicles, are undefined. Critically, overarching principles connecting microscopic cell organization to macroscopic structures, while potentially common to morphogenesis across different biological systems, are not well understood.Here, we report the sequence of architectural transitions that occur during pellicle maturation for the pathogen Vibrio cholerae. We discover a morphogenic transition characterized by a cascade of wrinkles with fractal scaling in wavelength, which is distinct from the classic wrinkle-to-fold transition widely observed in physical systems such as thin polymer films on fluid baths (19), nanoparticle thin sheets (20), and thin epitaxial layers of gold and elastomers (21, 22). In classic wrinkle-to-fold transitions, as compression increases beyond the linear regime, the initial wrinkles coalesce and localize into folds with large amplitudes. During such transitions, the shape evolution of passive films can be characterized by the minimization of the system’s total energy for different conformations (20, 23–25). In particular, elastic bending energies contained in small wavelength undulations are lost in favor of energies associated with large-scale folds. By contrast, as we demonstrate below, the opposite length-scale progression occurs for actively growing V. cholerae pellicles, in which finer wrinkles and creases continue to emerge following initial morphogenic transitions. The length scales of wrinkles in a mature V. cholerae pellicle follow fractal order, achieving a pattern with fractal dimension of ∼1.6. The additional dimensionality conferred by the fractal geometry could promote nutrient access and enhance signal transduction compared to a smooth structure (26). The fractal progression toward small length scales, as opposed to coalescence toward larger folds, stems from heterogeneous growth in the initial film. We find that the original distribution of microcolonies is preserved during pellicle architectural transitions, and, importantly, it determines the exact sequence of morphogenesis events that will occur. Indeed, changing the initial colony seeding density enabled us to identify a total of four morphogenic routes, which we termed the standard, bypass, crystalline, and incomplete modes. Our results demonstrate a direct connection between microscopic structure and macroscopic morphology for an active, growing soft biomaterial.     

Protection against Vibrio vulnificus infection by active and passive immunization with the C-terminal region of the RtxA1/MARTXVv protein

Vibrio vulnificus is a foodborne pathogen that is prevalent in coastal waters worldwide. Infection with V. vulnificus causes septicemia with fatality rates exceeding 50% even with aggressive antibiotic therapy. Several vaccine studies to prevent V. vulnificus infection have been performed but have had limited success. In this study, we identified the C-terminal region (amino acids 3491 to 4701) of the V. vulnificus multifunctional autoprocessing RTX (MARTX_Vv or RtxA1) protein, RtxA1-C, as a promising antigen that induces protective immune responses against V. vulnificus. Vaccination of mice with recombinant RtxA1-C protein with adjuvant elicited a robust antibody response and a dramatic reduction in blood bacterial load in mice infected intraperitoneally. Vaccination resulted in significant protection against lethal challenge with V. vulnificus. Furthermore, intraperitoneal passive immunization with serum raised against the recombinant RtxA1-C protein demonstrated marked efficacy in both prophylaxis and therapy. These results suggest that active and passive immunization against the C-terminal region of the RtxA1 protein may be an effective approach in the prevention and therapy of V. vulnificus infections.     

广东省茂名市副溶血弧菌涉水产品分离株病原学特征

目的 了解茂名市涉水产品中副溶血弧菌分离株的病原学特征。方法 对茂名市食品安全风险监测中分离到的副溶血弧菌进行血清分型、抗生素敏感性实验、毒力基因检测、脉冲场凝胶电泳( PFGE) 分子分型和多位点序列分型（MLST）。结果 2017—2018年共分离到涉水产品中副溶血弧菌分离株44株,O型共分7个血清型,无明显优势血清型。44株分离株除对氨苄西林和头孢唑林表现出高度的耐药性外,对其它抗生素均敏感,未发现有分离株携带tdh和trh基因。44株分离株分成44种不同的PFGE带型,MLST分型发现有30个新的ST型,呈现一定的遗传多样性。结论 广东省茂名市涉水产品中副溶血弧菌分离株血清型别多样,对大部分的抗生素敏感,均未发现携带tdh和trh 基因,且呈现一定的遗传多态性,需要继续加大监测的力度和范围。     

Potentially human‐virulent Vibrio vulnificus isolates from diseased great pompano (Trachinotus goodei)

Vibrio vulnificus is an opportunistic human pathogen responsible for the majority of seafood‐associated deaths worldwide and is also a relevant fish pathogen for the aquaculture industry. In addition to infections in aquatic livestock, V. vulnificus also represents a risk to aquarium animals. For the first time, this work describes an important mortality outbreak in Trachinotus goodei in a zoo aquarium, with the isolation of Vibrio vulnificus (Vv) from the internal organs of the diseased fish. The isolates were identified by MALDI‐TOF MS, serotyped and characterized by pulsed‐field gel electrophoresis (PFGE). Although the isolates from great pompanos did not belong to pathovar piscis (formerly biotype 2) or to any of the fish‐related serovars, they all had identical phenotypes, antimicrobial susceptibility profiles and PFGE patterns, which together with their isolation in pure culture from internal organs is strongly indicative of their clinical significance. Moreover, Vv isolates harboured important genetic markers of human virulence potential: they had the clinical variant of the vcg gene, gave the 338 bp DNA amplification product of the pilF gene and resisted the bactericidal activity of human serum. All these results strongly suggest that these Vv isolates should be considered potentially virulent for humans. These results extend the range of fish species affected by V. vulnificus, confirm the threat that this pathogen represents to aquatic animals and highlight the risk that this bacterial pathogen poses to human health.     

某酒楼1起副溶血性弧菌食物中毒事件的调查

目的 调查某酒楼食物中毒事件发生的原因和危险因素。方法 应用现场流行病学方法进行调查,对患者及厨师肛拭、剩余可疑食物和酒楼外环境样品进行实验室病原检测分析。结果 本次事件共发病11人,罹患率18.3%,主要症状为腹痛、腹泻(多为水样便)、恶心、呕吐;3名病例的肛拭及黑鸡脚(半成品)、凉菜间菜板涂抹样品中均检出副溶血性弧菌,他们的生物学特性一致。结论 此次事件为副溶血性弧菌引起的食物中毒事故,酒楼在该餐厅的食材加工、供餐及顾客的进食过程中均存在生熟交叉污染的风险。