An octopus gift: Mycobacterium marinum multiple skin lesions.

Mycobacterium marinum is a nontuberculous mycobacterium responsible of infections in humans, ranging from skin infection to disseminated infection in immunocompromised hosts. Clinical suspicion and prompt diagnosis are crucial to prescribe appropriate antimycobacterial treatment and avoid sequelae.

Symbiotic organs shaped by distinct modes of genome evolution in cephalopods.

Microbes have been critical drivers of evolutionary innovation in animals. To understand the processes that influence the origin of specialized symbiotic organs, we report the sequencing and analysis of the genome of Euprymna scolopes, a model cephalopod with richly characterized host-microbe interactions. We identified large-scale genomic reorganization shared between E. scolopes and Octopus bimaculoides and posit that this reorganization has contributed to the evolution of cephalopod complexity. To reveal genomic signatures of host-symbiont interactions, we focused on two specialized organs of E. scolopes: the light organ, which harbors a monoculture of Vibrio fischeri, and the accessory nidamental gland (ANG), a reproductive organ containing a bacterial consortium. Our findings suggest that the two symbiotic organs within E. scolopes originated by different evolutionary mechanisms. Transcripts expressed in these microbe-associated tissues displayed their own unique signatures in both coding sequences and the surrounding regulatory regions. Compared with other tissues, the light organ showed an abundance of genes associated with immunity and mediating light, whereas the ANG was enriched in orphan genes known only from E. scolopes Together, these analyses provide evidence for different patterns of genomic evolution of symbiotic organs within a single host.

A Pseudoalteromonas Clade with Remarkable Biosynthetic Potential.

Pseudoalteromonas species produce a diverse range of biologically active compounds, including those biosynthesized by nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). Here, we report the biochemical and genomic analysis of Pseudoalteromonas sp. strain HM-SA03, isolated from the blue-ringed octopus, Hapalochlaena sp. Genome mining for secondary metabolite pathways revealed seven putative NRPS/PKS biosynthesis gene clusters, including those for the biosynthesis of alterochromides, pseudoalterobactins, alteramides, and four novel compounds. Among these was a novel siderophore biosynthesis gene cluster with unprecedented architecture (NRPS-PKS-NRPS-PKS-NRPS-PKS-NRPS). Alterochromide production in HM-SA03 was also confirmed by liquid chromatography-mass spectrometry. An investigation of the biosynthetic potential of 42 publicly available Pseudoalteromonas genomes indicated that some of these gene clusters are distributed throughout the genus. Through the phylogenetic analysis, a particular subset of strains formed a clade with extraordinary biosynthetic potential, with an average density of 10 biosynthesis gene clusters per genome. In contrast, the majority of Pseudoalteromonas strains outside this clade contained an average of three clusters encoding complex biosynthesis. These results highlight the underexplored potential of Pseudoalteromonas as a source of new natural products.IMPORTANCE This study demonstrates that the Pseudoalteromonas strain HM-SA03, isolated from the venomous blue-ringed octopus, Hapalochalaena sp., is a biosynthetically talented organism, capable of producing alterochromides and potentially six other specialized metabolites. We identified a pseudoalterobactin biosynthesis gene cluster and proposed a pathway for the production of the associated siderophore. A novel siderophore biosynthesis gene cluster with unprecedented architecture was also identified in the HM-SA03 genome. Finally, we demonstrated that HM-SA03 belongs to a phylogenetic clade of strains with extraordinary biosynthetic potential. While our results do not support a role of HM-SA03 in Hapalochalaena sp. venom (tetrodotoxin) production, they emphasize the untapped potential of Pseudoalteromonas as a source of novel natural products.

Recipient sepsis caused by Lactococcus garvieae contamination of platelets from a donor with colon cancer.

Lactococcus garvieae is a well-known fish pathogen that has low virulence in humans and is rarely isolated from the blood cultures of endocarditis patients. We describe herein the first reported case of transfusion-transmitted L. garvieae sepsis caused by a contaminated platelet concentrate from a donor who consumed raw octopus before blood donation. Retrospective examination of the laboratory results of the index donor revealed that his haemoglobin levels had been steadily decreasing, which led to the detection of a latent colon cancer. The donors with colon lesions involving a latent cancer may relate an asymptomatic bacteremia.

The bacteriolytic mechanism of an invertebrate-type lysozyme from mollusk Octopus ocellatus.

As an important economic mollusk in coastal areas, Octopus ocellatus dependents on innate immune system to resist the invasion of microorganisms. Lysozyme is a crucial effector owing to its significant lytic activity against bacterial pathogens during the immune responses. In this study, characteristic and immune function of an I-type lysozyme from O. ocellatus (OoLyz) was investigated. OoLyz shared a close relationship with the lysozymes from other bivalve mollusks. The mRNA of OoLyz exhibited a broad transcript in different tissues/organs, and with the greatest expression in hepatopancreas. The expression of OoLyz was significantly raised when O. ocellatus was infected by Vibrio anguillarum or Micrococcus luteus, suggesting OoLyz participated in innate immune response of host. Prokaryotic recombinant OoLyz (rOoLyz) exhibited obvious bacteriolysis ability towards both gram-negative bacteria V. anguillarum and Escherichia coli, and gram-positive bacteria M. luteus and Staphylococcus aureus. The bacteriolysis activities of rOoLyz towards gram-negative but not gram-positive bacteria was heat stable, indicating that OoLyz might clear gram-positive bacterium by enzyme-dependent mechanisms, but eliminate gram-negative microbe via enzymatic activity independent way. Scanning electron microscopy analysis showed that rOoLyz destroyed microbes by damaging cell wall. More importantly, the fact that rOoLyz could directly degrade the peptidoglycan, further revealed its bactericidal mechanism as a muramidase. Our results revealed the essential role of I-type lysozyme in the innate immunity of O. ocellatus, and shed new light to understand the mechanism of immune defense of mollusks.

Experimental infection of octopus vulgaris (Cuvier, 1797) with Photobacterium damsela subsp. piscicida. Immunohistochemical tracking of antigen and tissue responses.

Adult common octopus individuals were intramuscularly infected with Photobacterium damsela subsp. piscicida in order to investigate if this species is sensitive to this common and important fish pathogen. The fate of the bacterial antigens and the tissue responses of Octopus vulgaris were studied employing immunohistochemical techniques. Strong reaction at the site of injection was evident from day 2 post-infection that continued until day 14. Great numbers of hemocytes that were attracted at the site of infection were involved in phagocytosis of bacteria. Very early in the infection, a transition of cells to fibroblasts and an effort to isolate the infection was observed. During the course of the study, very large necrotic cells were seen at the site of infection, whereas during the later stages hemocytes with phagocytosed bacteria were observed in well-defined pockets inside the muscle tissue. None of the internal organs tested for the presence of the bacterium were positive with the exception of the digestive gland where antigen staining was observed which was not associated with hemocyte infiltration. The high doses of bacterial cells used in this experimental infection and the lack of disease signs from Octopus vulgaris suggest that, under normal conditions, octopus is resistant to Photobacterium damsela subsp. piscicida.

[Microbiological analysis of red octopus in fishing ports of Campeche, Mexico]. / Análisis microbiológicodel pulpo rojo en puertos pesqueros de Campeche, México.

OBJECTIVE: In this work we studied the microbiological quality of the red octopus given its important economic and social impact on the region South-Southeast of Mexico. MATERIALS AND METHODS: Samples were taken in different areas of capture of the species and analyzed with biochemical tests described in the Mexican official standards, identifying strains belonging to the genus Vibrio, Salmonella and faecal coliforms, and E. coli O157: H7. We used the BAx System for the identification of microorganisms through their bacterial DNA. The results obtained in biochemical and molecular methods were confirmed. RESULTS: Bland-Altman statistical method pointed out that both techniques can be used interchangeably. McNemar test showed that both methods have the same efficacy for the identification of pathogens (value X2=0.5 ρ=0.4795). CONCLUSION: The microbiological quality of the octopus in the South-Southeast region of Mexico is deficient due to the presence of pathogenic intestinal flora that might represent an epidemiological risk. The indexes established by the regulations suggest the need to apply effective and rapid identification technologies, such as the BAx System.This alternative method of analysis can contribute to the implementation of effective strategies that allow compliance with the minimal sanitary specifications during the processing of fishing products, thus strengthening the control systems to decrease the risks of epidemiological outbreaks in the region.

Involvement of a Serpin serine protease inhibitor (OoSerpin) from mollusc Octopus ocellatus in antibacterial response.

Serpin is an important member of serine protease inhibitors (SPIs), which is capable of regulating proteolytic events and involving in a variety of physiological processes. In present study, a Serpin homolog was identified from Octopus ocellatus (designated as OoSerpin). Full-length cDNA of OoSerpin was of 1735 bp, containing a 5' untranslated region of 214 bp, a 3' UTR of 282 bp, and an open reading frame of 1239 bp. The open reading frame encoded a polypeptide of 412 amino acids which has a predicted molecular weight of 46.5 kDa and an isoelectric point of 8.52. The OoSerpin protein shares 37% sequence identity with other Serpins from Mus musculus (NP_941373) and Ixodes scapularis (XP_002407493). The existence of a conserved SERPIN domain strongly suggested that OoSerpin was a member of the Serpin subfamily. Expression patterns of OoSerpin, both in tissues and towards bacterial stimulation, were then characterized. The mRNA of OoSerpin was constitutively expressed at different levels in all tested tissues of untreated O. ocellatus, including mantle (lowest), muscle, renal sac, gill, hemocyte, gonad, systemic heart, and hepatopancreas (highest). The transcriptional level of OoSerpin was significantly up-regulated (P<0.01) in O. ocellatus upon bacterial challenges with Vibrio anguillarum and Micrococcus luteus, indicating its involvement in the antibacterial immune response. Furthermore, rOoSerpin, the recombinant protein of OoSerpin, exhibited strong abilities to inhibit proteinase activities of trypsin and chymotrypsin as well as the growth of Escherichia coli. Our results demonstrate that OoSerpin is a potential antibacterial factor involved in the immune response of O. ocellatus against bacterial infection.

Skin lesion-associated pathogens from Octopus vulgaris: first detection of Photobacterium swingsii, Lactococcus garvieae and betanodavirus.

The common octopus Octopus vulgaris Cuvier, 1798 is extremely important in fisheries and is a useful protein source in most Mediterranean countries. Here we investigated pathogens associated with skin lesions in 9 naturally deceased specimens that included both cultured and wild common octopus. Within 30 min after death, each octopus was stored at 4°C and microbiologically examined within 24 h. Bacterial colonies, cultured from swabs taken from the lesions, were examined using taxonomical and biochemical analyses. Vibrio alginolyticus and V. parahaemolyticus were only isolated from cultured animals. A conventional PCR targeting the 16S ribosomal RNA (rRNA) gene and sequencing were performed on 2 bacterial isolates that remained unidentified after taxonomical and biochemical analysis. The sequence results indicated that the bacteria had a 99% identity with Lactococcus garvieae and Photobacterium swingsii. L. garvieae was confirmed using a specific PCR based on the 16S-23S rRNA internal transcribed spacer region, while P. swingsii was confirmed by phylogenetic analyses. Although all animals examined were found to be infected by the protozoan species Aggregata octopiana localised in the intestines, it was also present in skin lesions of 2 of the animals. Betanodavirus was detected in both cultured and wild individuals by cell culture, PCR and electron microscopy. These findings are the first report of L. garvieae and betanodavirus from skin lesions of common octopus and the first identification of P. swingsii both in octopus skin lesions and in marine invertebrates in Italy.

Diversity and biosynthetic potential of culturable microbes associated with toxic marine animals.

Tetrodotoxin (TTX) is a neurotoxin that has been reported from taxonomically diverse organisms across 14 different phyla. The biogenic origin of tetrodotoxin is still disputed, however, TTX biosynthesis by host-associated bacteria has been reported. An investigation into the culturable microbial populations from the TTX-associated blue-ringed octopus Hapalochlaena sp. and sea slug Pleurobranchaea maculata revealed a surprisingly high microbial diversity. Although TTX was not detected among the cultured isolates, PCR screening identifiedsome natural product biosynthesis genes putatively involved in its assembly. This study is the first to report on the microbial diversity of culturable communities from H. maculosa and P. maculata and common natural product biosynthesis genes from their microbiota. We also reassess the production of TTX reported from three bacterial strains isolated from the TTX-containing gastropod Nassarius semiplicatus.

Ontogeny of tetrodotoxin levels in blue-ringed octopuses: maternal investment and apparent independent production in offspring of Hapalochlaena lunulata.

Many organisms provision offspring with antipredator chemicals. Adult blue-ringed octopuses (Hapalochlaena spp.) harbor tetrodotoxin (TTX), which may be produced by symbiotic bacteria. Regardless of the ultimate source, we find that females invest TTX into offspring and offspring TTX levels are significantly correlated with female TTX levels. Because diversion of TTX to offspring begins during the earliest stages of egg formation, when females are still actively foraging and looking for mates, females may face an evolutionary tradeoff between provisioning larger stores of TTX in eggs and retaining that TTX for their own defense and offense (venom). Given that total TTX levels appear to increase during development and that female TTX levels correlate with those of offspring, investment may be an active adaptive process. Even after eggs have been laid, TTX levels continue to increase, suggesting that offspring or their symbionts begin producing TTX independently. The maternal investment of TTX in offspring of Hapalochlaena spp. represents a rare examination of chemical defenses, excepting ink, in cephalopods.

A method for isolating RNA from metabolically active bacterial flora associated with octopus.

AIM: Development of a method for the isolation and purification of metagenomic RNA (mgRNA) from the ectopic bacterial flora of octopus. METHODS AND RESULTS: Modifications were made to the methods of Valenzuela-Avendaño et al. (Plant Mol Biol Rep, 2005, 23, 199a) and Chomczynski and Sacchi (Anal Biochem, 1987, 162, 156) to develop a protocol based on chemical lysis with Trizol. This proposed protocol effectively isolated mgRNA. The resulting bacterial RNA transcripts were amplified with universal primers directed to the hypervariable regions of the 16S rRNA gene by complementary DNA synthesis. Protocol efficacy in the study of metabolically active bacterial flora was proven using DGGE, which produced a banding pattern that recovered sequences mainly related to the Vibrionaceae family. CONCLUSION: The analysed samples were clearly complex, and the proposed protocol was proven to effectively isolate mgRNA from the metabolically active bacterial flora associated with octopus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first protocol proposed for the isolation of bacterial mgRNA that allows identification and study of metabolically active bacterial flora associated with octopus. This is an important step forward in understanding and controlling the microbial community of this economically important fishery resource, aimed at detecting its potentially pathogenic bacteria.

Combined effect of vacuum-packaging and oregano essential oil on the shelf-life of Mediterranean octopus (Octopus vulgaris) from the Aegean Sea stored at 4 degrees C.

The present study evaluated the use of vacuum packaging (alone) or with addition of oregano essential oil (EO), as an antimicrobial treatment for shelf-life extension of fresh Mediterranean octopus stored under refrigeration for a period of 23 days. Four different treatments were tested: A, control sample; under aerobic storage in the absence of oregano essential oil; VP, under vacuum packaging in the absence of oregano essential oil; and VO1, VO2, treated samples with oregano essential oil 0.2 and 0.4% (v/w), respectively, under VP. Of all the microorganisms enumerated, Pseudomonas spp., H2S-producing bacteria and lactic acid bacteria (LAB) were the groups that prevailed in octopus samples, irrespective of antimicrobial treatment. With regard to the chemical freshness indices determined, thiobarbituric acid (TBA) values were low in all octopus samples, as could have been expected from the low fat content of the product. Both trimethylamine nitrogen (TMA-N) and total volatile basic nitrogen (TVB-N) values of oregano treated under VP octopus samples were significantly lower compared to control samples during the entire refrigerated storage period. Based primarily on sensory evaluation (odor), the use of VP, VO1 and VO2 extended the shelf-life of fresh Mediterranean octopus by ca. 3, 11 and 20 days, respectively.

Analysis of the bacterial microbiome in the small octopus, Octopus variabilis, from South Korea to detect the potential risk of foodborne illness and to improve product management.

The small octopus (Octopus variabilis) is a popular seafood in many countries including South Korea. Because it is often consumed uncooked, the microorganisms in it often cause food poisoning. Therefore, analyzing the microbiome of the small octopus can help to understand the risk of food poisoning and manage octopus products better. A total of 40 small octopuses were collected from four sites in November and August. The microbiota was analyzed using Illumina Miseq sequencing, and the amount of bacteria was quantified by real-time PCR. In addition, we analyzed the influence of Vibrio vulnificus infection on the microbiome of the small octopus through artificial infection experiments. Bacteroidetes was the predominant phylum in August, and Proteobacteria was predominant in November. The composition of the microbiota in octopus depended on sampling region and season. The potential risk of foodborne illness from small octopus consumption might be higher in August than in November due to the abundance of potential pathogens. In the infection experiment, the proportion of V. vulnificus increased only at 27°C. The composition and functional gene profiles of the microbiota varied in a similar manner between non-infected and infected samples over time at the same temperature. These results indicated that the indigenous microbiota in small octopus could inhibit colonization by V. vulnificus during storage. Although further studies are necessary to clarify these results, our results could help us better understand food poisoning through octopus ingestion and manage products.

Preservation of shelf life of pota and octopus in chilled storage under controlled atmospheres.

Whole ungutted pota and octopus were stored in bulk in stainless steel refrigerated containers under controlled atmospheres. In order to evaluate the effectiveness of this treatment, physicochemical tests (pH, trimethylamine nitrogen, total volatile basic nitrogen, 2-thiobarbituric acid) and sensory assessments (inspection) were done. For both pota and octopus, the control lot had the highest physicochemical values and the lowest sensory values, and of the two assayed atmospheres, the 60/15/25% atmosphere (CO2/O2/N2) had the best physicochemical results. pH, trimethylamine nitrogen, and total volatile basic nitrogen levels were lower (P < or = 0.05) in octopus than in pota, and the inspection results were significantly (P < or = 0.05) higher. However, the octopus had a higher 2-thiobarbituric acid index than the pota. Therefore, the application of controlled atmospheres on these cephalopods during chilled bulk storage considerably increases their shelf life.

Análisis microbiológicodel pulpo rojo en puertos pesqueros de Campeche, México / Microbiological analysis of red octopus in fishing ports of Campeche, Mexico

Resumen: Objetivo: Estudiar la calidad microbiológica del pulpo rojo dado su importante impacto económico y social en la región sur-sureste de México. Material y métodos: Se tomaron muestras en diversas zonas de captura de la especie y se analizaron con pruebas bioquímicas descritas en las normas oficiales mexicanas. Se identificaron cepas pertenecientes al género Vibrio, Salmonella, coliformes fecales y E. coli O157:H7. Con el empleo del Sistema BAx, se logró la identificación de microorganismos a través de su ADN bacteriano. Los resultados obtenidos en los métodos bioquímicos y moleculares fueron contrastados. Resultados: El método estadístico de Bland-Altman indicó que ambas técnicas pueden usarse indistintamente. La prueba de McNemar demostró que ambos métodos cuentan con la misma eficacia para la identificación de patógenos (valor X2=0.5 ρ=0.4795). Conclusión: La calidad microbiológica del pulpo en la región sur-sureste de México es deficiente debido a la presencia de flora bacteriana patógena que podría representar un riesgo epidemiológico. Los índices establecidos por las normas sugieren la necesidad de aplicar técnicas de identificación eficaces y rápidas como el Sistema BAx. Este método alternativo de análisis puede coadyuvar a la implementación de estrategias efectivas que permitan cumplir con especificaciones mínimas sanitarias durante el procesamiento de los productos pesqueros, y así elevar los sistemas de control para disminuir los riesgos de brotes epidemiológicos en la región.

Abstract: Objective: In this work we studied the microbiological quality of the red octopus given its important economic and social impact on the region South-Southeast of Mexico. Materials and methods: Samples were taken in different areas of capture of the species and analyzed with biochemical tests described in the Mexican official standards, identifying strains belonging to the genus Vibrio, Salmonella and faecal coliforms, and E. coli O157: H7. We used the BAx System for the identification of microorganisms through their bacterial DNA. The results obtained in biochemical and molecular methods were confirmed. Results: Bland-Altman statistical method pointed out that both techniques can be used interchangeably. McNemar test showed that both methods have the same efficacy for the identification of pathogens (value X2=0.5 ρ=0.4795). Conclusion: The microbiological quality of the octopus in the South-Southeast region of Mexico is deficient due to the presence of pathogenic intestinal flora that might represent an epidemiological risk. The indexes established by the regulations suggest the need to apply effective and rapid identification technologies, such as the BAx System.This alternative method of analysis can contribute to the implementation of effective strategies that allow compliance with the minimal sanitary specifications during the processing of fishing products, thus strengthening the control systems to decrease the risks of epidemiological outbreaks in the region.