Implications of ammonia stress for the pathogenicity of Shewanella spp. in Oreochromis niloticus: effects on hematological, biochemical, immunological, and histopathological parameters.

Environmental stressors (such as ammonia) in aquaculture could increase the risk of pathogenicity, posing a more severe threat to farmed fish. The aim of this study was to investigate the effects of ammonia stress on the pathogenicity of Shewanella spp. in Oreochromis niloticus. First, a 96-hour static test was used to determine the median lethal concentration (LC50) of unionized ammonia to Nile tilapia. After 96 h of exposure, the Un-ionized ammonia (UIA) LC50 was estimated to be 4.26 mg/L. Second, an experiment was conducted to test the effect of unionized ammonia stress on the pathogenicity of Shewanella spp. in O. niloticus for 30 days. A study involved 180 fish divided into six groups, with the first group serving as a control. The second group (AMN1/10) and the third group (AMN1/20) were not challenged and were exposed to 1/10 (0.42 mg/L) and 1/20 (0.21 mg/L) of the 96-hour LC50 of UIA, respectively. Then 0.2 mL (0.14 × 105) of Shewanella spp. was intraperitoneally injected into the fourth (SH), fifth (SH + AMN1/10), and sixth (SH + AMN1/20) groups, which were subjected to 0, 1/10 (0.42 mg/L), and 1/20 (0.21 mg/L) of the 96-hour LC50 of UIA, respectively. The survival rate, hematological indices, immunological parameters, and antioxidant activity of the fish significantly decreased when they were exposed to ammonia and Shewanella infection separately or together. Histopathological changes were also observed in the kidney and liver. Furthermore, both individual and combined exposures significantly altered renal and hepatic function, with notable increases in glucose and cortisol levels, as well as in the expression of proinflammatory cytokine genes (TNF-α and IL-1ß). However, the detrimental effects of co-exposure to ammonia stress and Shewanella infection were greater than those of separate exposures. As a result, we may say that increased ammonia concentrations enhance the infection of Shewanella spp. These findings could contribute to a better understanding of Shewanella infection in Nile tilapia.

Analysis of clinical characteristics of infections caused by Shewanella species.

PURPOSE: The Shewanella genus is a rare pathogen of marine origin. In recent years, there has been a continuous increase in infection cases caused by this bacterium, and we have observed the uniqueness of infections caused by this microorganism. MATERIALS AND METHODS: This study conducted a retrospective analysis of the medical history and laboratory examination data of patients infected with the Shewanella genus over the past decade. Additionally, it employed bioinformatics methods to analyze the relevant virulence factors and antibiotic resistance genes associated with the Shewanella genus. RESULTS: Over the past 10 years, we have isolated 51 cases of Shewanella, with 68.82% being Shewanella putrefaciens (35/51 cases) and 31.37% being Shewanella algae (16/51 cases). Infected individuals often had underlying diseases, with 39.22% (20/51) having malignant tumors and 25.49% (13/51) having liver and biliary system diseases primarily characterized by stones. The majority of patients, 62.74% (32/51), exhibited mixed infections, including one case with a combination of infections from three other types of bacteria and five cases with a combination of infections from two other types of bacteria. The identified microorganisms were commonly resistant to ticarcillin-clavulanic acid (23.5%), followed by cefoperazone-sulbactam (19.6%), ciprofloxacin (17.6%), and cefotaxime (17.6%). Bioinformatics analysis indicates that Shewanella can express bile hydrolysis regulators and fatty acid metabolism regulators that aid in adapting to the unique environment of the biliary tract. Additionally, it expresses abundant catalase, superoxide dismutase, and two-component signal transduction system proteins, which may be related to environmental adaptation. Shewanella also expresses various antibiotic resistance genes, including beta-lactamases and aminoglycoside modification enzymes. Iron carriers may be one of its important virulence factors. CONCLUSIONS: We speculate that the Shewanella genus may exist as a specific colonizer in the human body, and under certain conditions, it may act as a pathogen, leading to biliary infections in the host.

Detection of S83V GyrA mutation in quinolone-resistant Shewanella algae using comparative genomics.

BACKGROUND: Shewanella algae is a zoonotic pathogen that poses a serious health threat to immunocompromised hosts. Treatment of S. algae infections is challenging due to the pathogen's intrinsic resistance to a variety of ß-lactam antibiotics. Therapeutic options have become further limited by the emergence of quinolone-resistant strains. Currently, there are few studies concerning the genetic and molecular mechanisms underlying acquired quinolones resistance in S. algae. qnrA was once proposed as the candidate gene related to quinolones resistance in S. algae. However, recent studies demonstrated qnrA are highly conservative and does not confer resistance to quinolones in S. algae. METHODS: A total of 27 non-duplicated isolates of S. algae strains were examined. MICs of ciprofloxacin were determined using Vitek 2. Whole genome sequencing was performed using MiSeq platform. Comprehensive Antibiotic Resistance Database and ResFinder were used for annotation of quinolones resistance genes. Multiple sequence alignment by EMBOSS Clustal Omega were used to identified mutation in quinolone resistance-determining regions. To investigation of the alteration of protein structure induced by mutation, in silico molecular docking studies was conducted using Accryl Discovery studio visualizer. RESULTS: All S. algae harbored the quinolone-resistance associated genes (qnrA, gyrA, gyrB, parC, and parE) regardless its resistance to ciprofloxacin. Comparison of these genomes identified a nonsynonymous mutation (S83V) in chromosome-encoded gyrase subunits (GyrA) in quinolone-resistant strain. We found this mutation disrupts the water-metal ion bridge, reduces the affinity of the quinolone-enzyme complex for the metal ions and therefore decrease the capability of quinolones to stabilize cleavage complexes. CONCLUSIONS: The study provides insight into the quinolone resistance mechanisms in S. algae, which would be helpful for the evolution of antibiotic resistance in this bacterium.

Searching for putative virulence factors in the genomes of Shewanella indica and Shewanella algae.

Bacterial pathogens are a major threat to both humans and animals worldwide. It is crucial to understand the mechanisms of various disease processes at the molecular level. Shewanella species are widespread in the environment and some are considered as emerging opportunistic human and marine mammal pathogens. In this study, putative virulence factors on the genome of Shewanella indica BW, a bacterium isolated from the Bryde's whale (Balaenoptera edeni), were determined. Additionally, for comparative purposes, putative virulence factors from two other S. indica and ten S. algae strains were also determined using the Pathosystems Resource Integration Center (PATRIC) pipeline. We confirmed the presence of previously reported virulence factors and we are proposing several new candidate virulence factors. Interestingly, the putative virulence factors were very similar between the two species with the exception of microbial collagenase which was present in all S. algae genomes, but absent in all S. indica genomes.

Effect of mannan oligosaccharides on the microbiota and productivity parameters of Litopenaeus vannamei shrimp under intensive cultivation in Ecuador.

The white leg Litopenaeus vannamei shrimp is of importance to the eastern Pacific fisheries and aquaculture industry but suffer from diseases such as the recently emerged early mortality syndrome. Many bacterial pathogens have been identified but the L. vannamei microbiota is still poorly known. Using a next-generation sequencing (NGS) approach, this work evaluated the impact of the inclusion in the diet of mannan oligosaccharide, (MOS, 0.5% w/w), over the L. vannamei microbiota and production behavior of L. vannamei under intensive cultivation in Ecuador. The MOS supplementation lasted for 60 days, after which the shrimp in the ponds were harvested, and the production data were collected. MOS improved productivity outcomes by increasing shrimp survival by 30%. NGS revealed quantitative differences in the shrimp microbiota between MOS and control conditions. In the treatment with inclusion of dietary MOS, the predominant phylum was Actinobacteria (28%); while the control group was dominated by the phylum Proteobacteria (30%). MOS has also been linked to an increased prevalence of Lactococcus- and Verrucomicrobiaceae-like bacteria. Furthermore, under the treatment of MOS, the prevalence of potential opportunistic pathogens, like Vibrio, Aeromonas, Bergeyella and Shewanella, was negligible. This may be attributable to MOS blocking the adhesion of pathogens to the surfaces of the host tissues. Together, these findings point to the fact that the performance (survival) improvements of the dietary MOS may be linked to the impact on the microbiota, since bacterial lines with pathogenic potential towards shrimps were excluded in the gut.

Shewanella khirikhana sp. nov. - a shrimp pathogen isolated from a cultivation pond exhibiting early mortality syndrome.

Early mortality syndrome (EMS) in cultivated shrimp is of complex aetiology. One of the causes is acute hepatopancreatic necrosis disease (AHPND) caused by unique Vibrio isolates that carry two Pirvp toxin genes, but other causes of EMS remain mostly unexplained. Here, we describe the discovery of a Shewanella isolate TH2012T from an EMS/AHPND outbreak pond and demonstrate its virulence for shrimp (the mean lethal concentration of 105 colony-forming units per millilitre by immersion challenge) accompanied by distinctive histopathology, particularly of the ventral nerve cord and lymphoid organ but also including the digestive tract. On the basis of its complete genome sequence, multilocus phylogenetic trees, digital DNA-DNA hybridization analysis and differential phenotypic characteristics, we propose that Shewanella isolate TH2012T represents a novel species, separated sufficiently from the type strains S. litorisediminis and S. amazonensis to justify naming it Shewanella khirikhana sp. nov. Analysis of the TH2012T genome revealed no homologues of the Pirvp toxin genes but revealed a number of other potential virulence factors. It constitutes the first Shewanella isolate reported to be pathogenic to shrimp.

Systematic analysis of lysine acetylome reveals potential functions of lysine acetylation in Shewanella baltica, the specific spoilage organism of aquatic products.

Protein lysine acetylation is a major post-translational modification and plays a critical regulatory role in almost every aspect in both eukaryotes and prokaryotes, yet there have been no data on Shewanella baltica, which is one of the specific spoilage organism (SSO) of aquatic products. Here, we performed the first global acetylproteome analysis of S. baltica. 2929 lysine acetylation sites were identified in 1103 proteins, accounting for 26.1% of the total proteins which participate in a wide variety of biological processes, especially in the constituent of ribosome, the biosynthesis of aminoacyl-tRNA, the amino acids and fatty acid metabolism. Besides, 14 conserved acetylation motifs were detected in S. baltica. Notably, various directly or indirectly spoilage-related proteins were prevalently acetylated, including enzymes involved in the unsaturated fatty acids biosynthesis closely related to the cold adaptability, cold shock proteins, pivotal enzymes involved in the putrescine biosynthesis, and a LuxR-type protein in quorum sensing system. The acetylome analysis in Shewanella can supplement the database and provide new insight into uncovering the spoilage mechanisms of S. baltica. The provided dataset illuminates the potential role of reversible acetylation in S. baltica, and serves as an important resource for exploring the physiological role of lysine acetylation in prokaryotes. SIGNIFICANCE: The psychrotrophic nature and the ability of S. baltica to make good use of "habitat" nutrients explain its importance in spoilage of seafood stored at low temperatures. However, the underlying mechanism of spoilage potential from the perspective of protein post-translational modification was rarely studied. This work identifies the first comprehensive survey of a lysine acetylome in S. baltica and uncovers the involvement of lysine acetylation in the diverse biological processes, especially in the closely spoilage-related pathways. This study provides a resource for functional analysis of acetylated proteins and creates opportunities for in-depth elucidation of the physiological role of protein acetylation in Shewanella spp.

Kinetics of trifurcated electron flow in the decaheme bacterial proteins MtrC and MtrF.

The bacterium Shewanella oneidensis has evolved a sophisticated electron transfer (ET) machinery to export electrons from the cytosol to extracellular space during extracellular respiration. At the heart of this process are decaheme proteins of the Mtr pathway, MtrC and MtrF, located at the external face of the outer bacterial membrane. Crystal structures have revealed that these proteins bind 10 c-type hemes arranged in the peculiar shape of a staggered cross that trifurcates the electron flow, presumably to reduce extracellular substrates while directing electrons to neighboring multiheme cytochromes at either side along the membrane. Especially intriguing is the design of the heme junctions trifurcating the electron flow: they are made of coplanar and T-shaped heme pair motifs with relatively large and seemingly unfavorable tunneling distances. Here, we use electronic structure calculations and molecular simulations to show that the side chains of the heme rings, in particular the cysteine linkages inserting in the space between coplanar and T-shaped heme pairs, strongly enhance electronic coupling in these two motifs. This results in an [Formula: see text]-fold speedup of ET steps at heme junctions that would otherwise be rate limiting. The predicted maximum electron flux through the solvated proteins is remarkably similar for all possible flow directions, suggesting that MtrC and MtrF shuttle electrons with similar efficiency and reversibly in directions parallel and orthogonal to the outer membrane. No major differences in the ET properties of MtrC and MtrF are found, implying that the different expression levels of the two proteins during extracellular respiration are not related to redox function.

Combined intra- and extracellular reduction involved in the anaerobic biodecolorization of cationic azo dye by Shewanella oneidensis MR-1.

Microbial reduction decolorization is a promising strategy for cationic azo dye pollution remediation, but the reduction mechanism is unclear yet. In this work, the anaerobic reduction decolorization mechanism of cationic red X-GRL (X-GRL) by Shewanella oneidensis MR-1 (MR-1) was investigated from both intracellular and extracellular aspects. The exogenous additional riboflavin treatment test was used to analyze the extracellular reduction mechanism of X-GRL, and the actual role of riboflavin during the reduction of X-GRL was identified by three-dimensional fluorescence analysis for the first time. The proteinase K and the electron competitor treatment tests were used to analyze the intracellular reduction mechanism of X-GRL. Moreover, the effect of external environment on the reduction mechanism of X-GRL was elucidated by the decolorization performance of MR-1 wild type and its mutants, ΔomcA/mtrC, ΔmtrA, ΔmtrB and ΔcymA, under different external pH conditions. The results indicated that X-GRL could be decolorized by MR-1 in both extracellular and intracellular spaces. The extracellular decolorization of X-GRL could be caused by Mtr respiratory pathway or the indirect reduction of riboflavin, while the intracellular decolorization might occur due to the intracellular reduction depending on CymA pathway and a NADH-dependent reduction catalyzed by intracellular azoreductases. Furthermore, the proportion of extracellular decolorization decreased, whereas that of intracellular decolorization increased as the environmental pH rose.

Proteomics Goes to Court: A Statistical Foundation for Forensic Toxin/Organism Identification Using Bottom-Up Proteomics.

Bottom-up proteomics is increasingly being used to characterize unknown environmental, clinical, and forensic samples. Proteomics-based bacterial identification typically proceeds by tabulating peptide "hits" (i.e., confidently identified peptides) associated with the organisms in a database; those organisms with enough hits are declared present in the sample. This approach has proven to be successful in laboratory studies; however, important research gaps remain. First, the common-practice reliance on unique peptides for identification is susceptible to a phenomenon known as signal erosion. Second, no general guidelines are available for determining how many hits are needed to make a confident identification. These gaps inhibit the transition of this approach to real-world forensic samples where conditions vary and large databases may be needed. In this work, we propose statistical criteria that overcome the problem of signal erosion and can be applied regardless of the sample quality or data analysis pipeline. These criteria are straightforward, producing a p-value on the result of an organism or toxin identification. We test the proposed criteria on 919 LC-MS/MS data sets originating from 2 toxins and 32 bacterial strains acquired using multiple data collection platforms. Results reveal a > 95% correct species-level identification rate, demonstrating the effectiveness and robustness of proteomics-based organism/toxin identification.

Microfluidic dielectrophoresis device for trapping, counting and detecting Shewanella oneidensis at the cell level.

Shewanella oneidensis, a model organism for electrochemical activity bacteria, has been widely studied at the biofilm level. However, to obtain more information regarding this species, it is essential to develop an approach to trap and detect S. oneidensis at the cell level. In this study, we report a rapid and label-free microfluidic platform for trapping, counting and detecting S. oneidensis cells. A microfluidic chip was integrated with a modified dielectrophoresis (DEP) trapping technique and hole arrays of different hole sizes. By numerical simulation and an elaborate electric field distribution design, S. oneidensis cells were successfully trapped and positioned in the hole arrays. Real time fluorescence imaging was also used to observe the trapping process. With the aid of a homemade image program, the trapped bacteria were accurately counted, and the results demonstrated that the amount of bacteria correlated with the hole sizes. As one of the significant applications of the device, Raman identification and detection of countable S. oneidensis cells was accomplished in two kinds of holes. The microfluidic platform provides a quantitative sample preparation and analysis method at the cell level that could be widely applied in the environmental and energy fields.

Whole genome sequence to decipher the resistome of Shewanella algae, a multidrug-resistant bacterium responsible for pneumonia, Marseille, France.

UNLABELLED: We characterize and decipher the resistome and the virulence factors of Shewanella algae MARS 14, a multidrug-resistant clinical strain using the whole genome sequencing (WGS) strategy. The bacteria were isolated from the bronchoalveolar lavage of a hospitalized patient in the Timone Hospital in Marseille, France who developed pneumonia after plunging into the Mediterranean Sea. RESULTS: The genome size of S. algae MARS 14 was 5,005,710 bp with 52.8% guanine cytosine content. The resistome includes members of class C and D beta-lactamases and numerous multidrug-efflux pumps. We also found the presence of several hemolysins genes, a complete flagellum system gene cluster and genes responsible for biofilm formation. Moreover, we reported for the first time in a clinical strain of Shewanella spp. the presence of a bacteriocin (marinocin). CONCLUSION: The WGS analysis of this pathogen provides insight into its virulence factors and resistance to antibiotics.

From red to green: the propidium iodide-permeable membrane of Shewanella decolorationis S12 is repairable.

Viability is a common issue of concern in almost all microbial processes. Fluorescence-based assays are extensively used in microbial viability assessment, especially for mixed-species samples or biofilms. Propidium iodide (PI) is the most frequently used fluorescence indicator for cell viability based on the membrane permeability. Our results showed that the accumulation of succinate from fumarate respiration could induce PI-permeability in Shewanella decolorationis biofilm cells. Confocal laser scanning microscope further showed that the PI-permeable membrane could be repaired in situ when the extracellular succinate was eliminated by switching fumarate respiration to electrode respiration. Simultaneously with the membrane repair, the electrode respiring capacity of the originally PI-permeable cells was recovered. Agar-colony counts suggested that a major portion of the repaired cells were viable but nonculturable (VBNC). The results evidenced that S. decolorationis S12 has the capacity to repair PI-permeable membranes which suggests a reevaluation of the fate and function of the PI-permeable bacteria and expanded our knowledge on the flexibility of bacterial survival status in harsh environments.

The genus Shewanella: from the briny depths below to human pathogen.

The genus Shewanella is currently composed of more than 50 species that inhabit a range of marine environs and ecosystems. Several members of this genus, including S. oneidensis, have been identified that could potentially play key roles in environmental processes such as bioremediation of toxic elements and heavy metals and serving as microbial fuel cells. In contrast to this beneficial role, shewanellae are increasingly being implicated as human pathogens in persons exposed through occupational or recreational activities to marine niches containing shewanellae. Documented illnesses linked to Shewanella include skin and soft tissue infections, bacteremia, and otitis media. At present, it is unclear exactly how many Shewanella species are truly bona fide human pathogens. Recent advances in the taxonomy and phylogenetic relatedness of members of this genus, however, support the concept that most human infections are caused by a single species, S. algae. Some phylogenetic data further suggest that some current members of the genus are not true Shewanella species sensu stricto. The current review summarizes our present knowledge of the distribution, epidemiology, disease spectrum, and identification of microbial species focusing on a clinical perspective.

Biological characteristics and pathogenicity of a highly pathogenic Shewanella marisflavi infecting sea cucumber, Apostichopus japonicus.

Shewanella marisflavi isolate AP629 is described as a novel pathogen of sea cucumber. The LD(50) values (14 days) in sea cucumber, mice and swordtail fish were 3.89 × 10(6) , 6.80 × 10(4) and 4.85 × 10(4) CFU g(-1) body weight, respectively. Studies on S. marisflavi were conducted, including morphology, physiological and biochemical characteristics, haemolysis, whole-cell protein and 16S rDNA gene sequence. Colonies of S. marisflavi appeared faint red on marine agar and green on thiosulphate-citrate-bile salt-sucrose media. Shewanella marisflavi had polar flagella. The cells were Gram-negative, oxidase- and catalase-positive and not sensitive to O/129. The bacterium exhibited ß-haemolysis on sheep blood agar and produced H(2) S. Shewanella marisflavi survived and grew at 4-35°C, pH 6.0-9.2 and in the presence of 0-8% NaCl. The whole-cell proteins included 13 discrete bands, and proteins of molecular weight 87, 44 and 39 kDa were found in all five strains of Shewanella spp. The difference in 16S rDNA gene sequences in S. marisflavi was at the 446 bp site: S. marisflavi (KCCM 41822) - G, isolate AP629 - A. This is the first report that Shewanella is pathogenic to sea cucumber.

Epidemiology and clinical features of Shewanella infection over an eight-year period.

Shewanella is a rare human pathogen that can lead to fatal infections. However, clinical information about this bacterium remains scarce. In this study, we retrospectively reviewed all patients with laboratory isolates of Shewanella over an 8-y period to assess risk factors, clinical manifestations and outcome. Twenty-nine patients were identified. Shewanella was most commonly isolated from intra-abdominal specimens (48.2%), followed by skin and soft tissue specimens (27.6%), blood (13.8%) and sputum (10.3%). Malignancy, hepatobiliary disease and diabetes mellitus were common underlying diseases. The overall 30-day mortality rate was 20.6%. Shewanella was considered a definite causative pathogen in 7 patients, and a recurrent infection occurred in 2 patients. Colonization of the biliary tract was common. Among co-isolated pathogens, the enteric flora was most represented. All isolates were susceptible to ceftazidime and aminoglycosides, but 1 isolate was resistant to imipenem. In conclusion, Shewanella may become a colonizing bacterium, subsequently causing invasive diseases in patients with an underlying disease.

First case of human spondylodiscitis due to Shewanella algae.

We present the first case of human spondylodiscitis due to Shewanella algae. Our patient did not have any predisposing factors. The portal of entry was probably a cutaneous lesion on the leg, exposed to seawater. Bacteria were isolated in pure culture from a needle biopsy specimen of the vertebral disk. Automated identification systems identified the organism as Shewanella putrefaciens. However, molecular biology identified it as S. algae. Treatment with ceftriaxone and amikacin, then ciprofloxacin successfully addressed the infection. We also review four published cases of human osteoarticular infections caused by Shewanella spp: two cases of arthritis and two cases of osteomyelitis. Two patients had predisposing factors, and contact with water was found in two cases. The clinical, radiological and biological characteristics of S. algae spondylodiscitis are indistinguishable from those of spondylodiscitis of other causes. A cutaneous lesion with exposure to water is a potential portal of entry. Molecular typing is necessary to obtain a precise bacteriological identification.

A large-scale epidemiological study to identify bacteria pathogenic to Pacific oyster Crassostrea gigas and correlation between virulence and metalloprotease-like activity.

A 4-year bacteriological survey (2003-2007) of four molluscs cultivated in France and faced with mortality episodes was performed by the French shellfish pathology network. The more abundant bacteria isolated during 92 mortality episodes, occurring mainly in Pacific oyster Crassostrea gigas, were identified by genotyping methods. It allowed us both to confirm the representativeness of Vibrio splendidus and Vibrio aestuarianus bacterial strains and to identify both a large number of Vibrio harveyi-related strains mainly detected during 2007 oyster mortality outbreaks and to a lesser extent bacterial strains identified as Shewanella colwelliana. Because metalloprotease has been reported to constitute a virulence factor in a few Vibrio strains pathogenic for C. gigas, several bacterial strains isolated in this study were screened to evaluate their pathogenicity in C. gigas spat by experimental infection and their ability to produce metalloprotease-like activity in the culture supernatant fluids. A high level (84%) of concordant results between azocaseinase activities and virulence of strains was obtained in this study. Because bacterial metalloprotease activities appeared as a common feature of pathogenic bacteria strains associated with mortality events of C. gigas reared in France, this phenotypic test could be useful for the evaluation of virulence in bacterial strains associated with such mortality episodes.

Primary Shewanella algae bacteremia mimicking Vibrio septicemia.

Shewanella algae infections are rare in humans. Previously reported cases of S. algae have mainly been associated with direct contact with seawater. We report a case of primary S. algae bacteremia occurring after the ingestion of raw seafood in a patient with liver cirrhosis that presented a fulminent course of necrotizing fasciitis.

Skin and soft tissue infections (patera foot) in immigrants, Spain.

An unusual skin and soft tissue infection of the lower limbs has been observed in immigrants from sub-Saharan Africa who cross the Atlantic Ocean crowded on small fishing boats (pateras). Response to conventional treatment is usually poor. Extreme extrinsic factors (including new pathogens) may contribute to the etiology of the infection and its pathogenesis.