Klebsiella pneumoniae is an opportunistic pathogen that is very difficult to treat mainly due to its high propensity to acquire complex resistance traits. Notably, multidrug resistance (MDR)-Klebsiella pneumoniae (KP) infections are responsible for 22%–72% of mortality among hospitalized and immunocompromised patients. Although treatments with new drugs or with combined antibiotic therapies have some degree of success, there is still the urgency to investigate and develop an efficient approach against MDR-KP infections. In this study, we have evaluated, in an in vitro model of human macrophages, the efficacy of a combined treatment consisting of apoptotic body-like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) and φBO1E, a lytic phage specific for the major high-risk clone of KPC-positive MDR-KP. Results show that ABL/PI5P did not affect in a direct manner KKBO-1 viability, being able to reduce only the intracellular KKBO-1 bacterial load. As expected, φBO1E was effective mainly on reducing extracellular bacilli. Importantly, the combination of both treatments resulted in a simultaneous reduction of both intracellular and extracellular bacilli. Moreover, the combined treatment of KKBO-1-infected cells reduced proinflammatory TNF-α and IL-1β cytokines and increased anti-inflammatory TGF-β cytokine production. Overall, our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with MDR pathogens such as MDR-KP.

The improper use of antibiotics by humans may promote the dissemination of resistance in wildlife. The persistence and spread of acquired antibiotic resistance and human-associated bacteria in the environment, while representing a threat to wildlife, can also be exploited as a tool to monitor the extent of human impact, particularly on endangered animal species. Hence, we investigated both the associated enterobacterial species and the presence of acquired resistance traits in the cloacal microbiota of the critically endangered lesser Antillean iguana (Iguana delicatissima), by comparing two separate populations living in similar climatic conditions but exposed to different anthropic pressures. A combination of techniques, including direct plating, DNA sequencing and antimicrobial susceptibility testing allowed us to characterize the dominant enterobacterial populations, the antibiotic resistant strains and their profiles. A higher frequency of Escherichia coli was found in the samples from the more anthropized site, where multi-drug resistant strains were also isolated. These results confirm how human-associated bacteria as well as their antibiotic-resistance determinants may be transferred to wildlife, which, in turn, may act as a reservoir of antibiotic resistance.

Phage therapy is now reconsidered with interest in the treatment of bacterial infections. A major piece of information for this application is the definition of the molecular targets exploited by phages to infect bacteria. Here, the genetic basis of resistance to the lytic phage φBO1E by its susceptible host Klebsiella pneumoniae KKBO-1 has been investigated. KKBO-1 phage-resistant mutants were obtained by infection at high multiplicity. One mutant, designated BO-FR-1, was selected for subsequent experiments, including virulence assessment in a Galleria mellonella infection model and characterization by whole-genome sequencing. Infection with BO-FR-1 was associated with a significantly lower mortality when compared to that of the parental strain. The BO-FR-1 genome differed from KKBO-1 by a single nonsense mutation into the wbaP gene, which encodes a glycosyltransferase involved in the first step of the biosynthesis of the capsular polysaccharide (CPS). Phage susceptibility was restored when BO-FR-1 was complemented with the constitutive wbaP gene. Our results demonstrated that φBO1E infects KKBO-1 targeting the bacterial CPS. Interestingly, BO-FR-1 was less virulent than the parental strain, suggesting that in the context of the interplay among phage, bacterial pathogen and host, the emergence of phage resistance may be beneficial for the host.

vB_StuS_MMDA13 is a lytic bacteriophage, belonging to the Siphoviridae family. It was isolated from a surface freshwater sample, in a pond near Viterbo (Italy), using the Sphingomonas turrisvirgatae MCT13 as host. The host range was determined on a total of 21 strains, including 8 Sphingomonas spp type strains (S. koreensis NBRC_16723, S. cynarae DSM 25525, S. hankookensis DSM 23329, S. insulae DSM 21792, S. pseudosanguinis DSM 19512, S. panni DSM 15761, S. soli DSM 18313 and S. naasensis DSM 100060), 6 type strains belonging to other genera of the Sphingomonadaceae family (Sphingobium yanoikuyae DSM 7462, Sphingobium chlorophenolicum DSM 7098, Novosphingobium aromaticivorans DSM 12444, Novosphingobium capsulatum DSM 30196, Sphingopyxis alaskensis DSM 13593 and Sphingopyxis macrogoltabida DSM 8826), Agrobacterium tumefaciens DSM 5172, Burkholderia cenocepacia J2315, and five Pseudomonas aeruginosa isolates. The results demonstrated that the lytic spectrum of this phage is restricted to S. turrisvirgatae MCT13 .

Ancient parchments record an immense part of our cultural heritage, having been used as the main written support material for centuries. Parchment easily undergoes biodeterioration, whose main signs are the so-called purple spots, which often lead to detachment of the superficial written layer. Up to recent years, several studies have been analyzing damaged parchments from different world’s archives, trying to trace back the culprit of the purple spots. However, standard cultivation and early molecular techniques have been demonstrated to be unsuccessful, leading the parchment damage issue remaining unsolved for many years. Nowadays, some studies have explored the parchment biodeterioration dynamics by adopting a multidisciplinary approach combining standard microbiological methods with high-throughput molecular, chemical and physical techniques. This approach allowed an unprecedented level of knowledge on the complex dynamics of parchment biodeterioration. This mini review discusses the application of the combination of basic and high-throughput techniques to study historical parchments, highlighting the strengths and weaknesses of this approach. In particular, it focuses on how metagenomics has been paramount for the unequivocal identification of the microbial main actors of parchment biodeterioration and their dynamics, but also on how metagenomics may suffer the distortion inflict by the historical perspective on the analysis of ancient specimens. As a whole, this mini review aims to describe the scenario of information on parchment biodeterioration obtained so far by using the integration of metagenomic with recent chemical (Raman spectroscopy) and physical (Light Transmission Analysis) approaches, which might have key implications in the preservation of many ancient documents.

A yellow pigmented and agar-pitting settlement was confined from a water test acquired from a waste dump inside a neglected arrangement of built wetlands. The strain was cleaned and named MCT13T. This pole molded, Gram-negative, oxidase-and catalase-positive, oxygen consuming, non-spore-shaping, and non-motile strain framed round provinces and developed ideally at pH 7.5±0.2, at 28–30 °C on LB agar, with 0–0.5 % NaCl. The 16S rRNA quality succession investigation put the MCT13T seclude inside the Sphingomonas (sensu stricto) bunch. The DNA G+C content was 65.3 %. The main watched ubiquinone was Q10. The significant unsaturated fats included C17 : 1ω6c and C18 : 1ω7c/C18 : 1ω6c. The major polar lipids were sphingoglycolipid, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major polyamine was spermidine. The 16S rRNA quality phylogenetic examination performed all in all succession, demonstrated the nearest relative of MCT13T to be Sphingomonas koreensis (98.52 %); notwithstanding, there are a few genotypic and phenotypic contrasts between the novel disengage and the sort strain JSS26T of S. koreensis. Based on these outcomes, strain MCT13T speaks to a novel animal types in the family Sphingomonas, for which the name Sphingomonas turrisvirgatae sp. nov. is proposed. The sort strain is MCT13T (=DSM 105457T=BAC RE RSCIC 7T). Abbreviations: DDH, DNA–DNA hybridization; GGDC, Genome-to-Genome Distance Calculator; LB, lysogeny stock; LBA, lysogeny stock, agarized; L-E.S.A., Laboratorio di Ecologia Sperimentale e Acquacoltura (Laboratory of Experimental Ecology and Aquaculture); MCA, MacConkey agar; NA, supplement agar; SEM, examining electron microscopy; TSA, tryptone soy agar. Introduction: At the hour of its first definition, the Sphingomonas (sensu stricto) variety included just nine species; at this point this sort incorporates in excess of 90 legitimately distributed or approved species. Its immense biodiversity is seen by the few recently portrayed species, the high number of draft genomes of still uncharacterized disengages, and the assortment of situations where these small scale living beings, or their 16S rDNA quality, have been found. Strain MCT13T was fortunately recuperated during the portrayal of the high-impact bacterial network in a waste jettison inside a neglected arrangement of developed wetlands in the L-E.S.A. region of the University of Rome Tor Vergata, Rome, Italy (41° 51′ 4.1″ N 12° 37′ 48.7″ E 115 m). Water tests were spread on a few culture media containing distinctive salt fixations. Following seven days of brooding at room temperature a yellow-pigmented agar-pitting settlement, encompassed by an obvious clearing zone was seen on ZoBell w/o NaCl. In the wake of being over and over cleansed by sub-refined on agarized lysogeny medium (LB/LBA; Liofilchem s.r.l.), the novel strain, assigned MCT13T, was put away at −70 °C in 40 % glycerol, and refined routinely on LB, agar or stock, at 30 °C. The ordered situation of MCT13T was researched with a polyphasic approach. To decide the phylogenetic situation of the seclude, the genomic DNA was removed with a business pack (Qiagen) and a nearly complete 16S rRNA quality of strain MCT13T (1451 bp – MG077083) was intensified utilizing the bacterial general preliminaries 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) and sequenced on the two strands at an outside office (Macrogen, Seoul, Republic of Korea). The Classifier program at the rdp site (http://rdp.cme.msu.edu/classifier/classifier.jsp) distinguished the MCT13T confine as having a place with the family Sphingomonas, with 100 % certainty. The entirety of the 16S rRNA quality nucleotide marks explicit of the Sphingomonas bunch I, The chose successions were lined up with clustal_w (rendition 2.1) and the unshared terminal nucleotides were physically cut. The separation grid, remade with Kimura's two-parameter model, uncovered that the nearest MCT13T relative, among the sort strains of species with truly distributed names, was Sphingomonas koreensis JSS26T (98.43 %) followed (97.33 %) by both Sphingomonas mali NBRC 15500T and Sphingomonas pruni NBRC 15498T. The personality to the sort types of the family, Sphingomonas paucimobilis, was 94.31 %. The draft genome of MCT13T (MDDS01000000) has been gotten and the entire length 16S rRNA quality was utilized to rehash the arrangements. The likeness to S. koreensis JSS26T was equivalent to 98.52 %, still underneath the 98.7 % edge for another species. This outcome was affirmed by the correlation of the draft genome with both the accessible genome arrangements of S. koreensis (JSS26T/NBRC 16723T, PGEN01000001 and ABOJV, CP018820), performed with the Genome-to-Genome Distance Calculator 2.1 instrument (GGDC: http://ggdc.dsmz.de/ggdc.php#), that brought about a DNA–DNA hybridization (DDH) estimation of 23.5 % (20.5 %–25.2 % certainty span), far underneath the limit estimation of 70 %, which depicts bacterial species. The DDH esteem with S. paucimobilis was 21.20 % (19%–23.7 %). The normal G+C content of the novel strain was 65.3 mol%, predictable with the range portrayed for Sphingomonas . Phylogenetic and sub-atomic developmental examinations dependent on 16S rRNA qualities were led with the product mega7.0, by utilizing the most extreme probability strategy and 1000 bootstraps. In the recreated phylogenetic tree, the MCT13T fell inside Sphingomonas bunch I, framing a vigorous clade with S. koreensis, upheld by a high bootstrapping esteem. Further bunching with different clades yielded low bootstrap esteems. These outcomes were affirmed by both the base advancement and greatest miserliness calculations. In this tree MCT13T and the described S.koreensis strains JSS26T/NBRC 16723Tand ABOJV, structure adjacent however particular clades with certain successions acquired from ecological examples or potentially uncharacterized separates. The phylogenetic separations between MCT13T 16S rRNA quality and those of the set up Sphingomonas species, the determined DDH values, the interesting phenotypic attributes and the chemotaxonomic information, show that MCT13T speaks to a novel animal groups in the class Sphingomonas, for which the name Sphingomonas turrisvirgatae sp. nov. is proposed.

Sea turtles have been proposed as health indicators of marine habitats and carriers of antibiotic-resistant bacterial strains, for their longevity and migratory lifestyle. Up to now, a few studies evaluated the antibacterial resistant flora of Mediterranean loggerhead sea turtles (Caretta caretta) and most of them were carried out on stranded or recovered animals. In this study, the isolation and the antibiotic resistance profile of 90 Gram negative bacteria from cloacal swabs of 33 Mediterranean wild captured loggerhead sea turtles are described. Among sea turtles found in their foraging sites, 23 were in good health and 10 needed recovery for different health problems (hereafter named weak). Isolated cloacal bacteria belonged mainly to Enterobacteriaceae (59%), Shewanellaceae (31%) and Vibrionaceae families (5%). Although slight differences in the bacterial composition, healthy and weak sea turtles shared antibiotic-resistant strains. In total, 74 strains were endowed with one or multi resistance (up to five different drugs) phenotypes, mainly towards ampicillin (70%) or sulfamethoxazole/trimethoprim (more than 30%). Hence, our results confirmed the presence of antibiotic-resistant strains also in healthy marine animals and the role of the loggerhead sea turtles in spreading antibiotic-resistant bacteria.

Members of Sphingomonas genus have gained a notable interest for their use in a wide range of biotechnological applications, ranging from bioremediation to the production of valuable compounds of industrial interest. To date, knowledge on phages targeting Sphingomonas spp. are still scarce. Here, we describe and characterize a lytic bacteriophage, named vB_StuS_MMDA13, able to infect the Sphingomonas turrisvirgatae MCT13 type strain. Physiological characterization demonstrated that vB_StuS_MMDA13 has a narrow host range, a long latency period, a low burst size, and it is overall stable to both temperature and pH variations. The phage has a double-stranded DNA genome of 63,743 bp, with 89 open reading frames arranged in two opposite arms separated by a 1186 bp non-coding region and shows a very low global similarity to any other known phages. Interestingly, vB_StuS_MMDA13 is endowed with an original nucleotide modification biosynthetic gene cluster, which greatly differs from those of its most closely related phages of the Nipunavirus genus. vB_StuS_MMDA13 is the first characterized lytic bacteriophage of the Siphoviridae family infecting members of the Sphingomonas genus.

In nature, many plants or their extracted compounds have been found to possess anti-inflammatory features and therapeutic properties against infectious as well as non-infectious diseases, including cancer. In this study, we analysed the immunomodulatory effects on innate immune cells of hydroalcoholic extract from Origanum vulgare L. ssp. hirtum (HyE-Ov), a plant traditionally known for its anti-oxidative properties. The effects of HyE-Ov were tested on human monocyte derived dendritic cells (DC), type-1 (M1) and type-2 macrophages (M2) infected with M. bovis Bacille Calmette-Guerin (BCG), used as a model of persistent intracellular bacterium. DC, M1 and M2 treated with HyE-Ov significantly enhanced their mycobactericidal activity, which was associated with phagosomal acidification in M1 and M2 and increase of phagosomal, but not mitochondrial ROS production in M1, M2, and DC. Treatment of BCG-infected DC with HyE-Ov significantly reduced TNF-alpha and IL-12 production and increased TGF-beta synthesis. Finally, experiments were repeated using eight different HPLC fractions of HyE-Ov. Results showed that the capability to activate antimicrobial and anti-inflammatory response is shared by different fractions, suggesting that diverse bioactive molecules are present within the hydroalcoholic extract. Altogether, these results show that HyE-Ov promotes anti-mycobacterial innate immunity and limits inflammatory response in vitro and suggest that this plant extract may be exploitable as phytocomplex or nutraceutical for novel host-directed therapeutic approaches.

Animal hides are one of man's earliest and mostly used materials; many rawhide products, primarily leather, have for centuries been used for several purposes. The peculiar mechanical properties of leather depend on the hide composition, a dense collagen feltwork. Unfortunately, due to their proteic composition, rawhides may undergo microbial attack and biodeterioration. Over centuries, different processes and treatments (brining, vegetal or chrome tanning, tawing, etc.) were set up to face the biological attack and modify/stabilise the hide's mechanical properties. Nevertheless, even present-day rawhides are subjected to biological colonisation, and traces of this colonisation are clearly shown in Chrome(III) tanned leathers (in the wet blue stage), with obvious economic damages. The colonisation traces on tanned leathers consist of isolated or coalescent red patches, known as red heat deterioration. Parchments are rawhide products, too; they derive from another manufacturing procedure. Even parchments undergo microbial attack; the parchment biodeterioration seems comparable to leather red heat deterioration and is known as purple spots. Recently, an ecological succession model explained the process of historical parchment purple spot deterioration; the haloarchaea Halobacterium salinarum is the pioneer organism triggering this attack. The marine salt used to prevent rawhide rotting is the carrier of haloarchaea colonisers (Migliore et al., 2019). The aim of this study was to investigate the dynamics of biodeterioration on Chrome(III) tanned leathers and its effects on the stability/integrity of collagen structure. To this end, standard cultivation methods were integrated with three updated technologies, Next-Generation Sequencing (NGS), Raman spectroscopy, and Light Transmitted Analysis (LTA). A bioinformatic comparison between chrome tanned leather vs. historical parchment colonisers was performed to evaluate if leather and parchment share common culprits; furthermore, the effect of the biodeterioration on the physical properties of the hide product was evaluated.

Objectives: The aim of this study is to characterize a new bacteriophage able to infect Enterococcus faecalis, and to evaluate its ability to disrupt biofilm. Methods: The vB_EfaH_EF1TV (EF1TV) host-range was determined by spot test and efficiency of plating using a collection of 15E. faecalis clinical strains. The phage genome was sequenced with a next generation sequencing approach. Anti-biofilm activity was tested by crystal violet method and confocal laser scanning microscopy. Phage-resistant mutants were selected and sequenced to investigate receptors exploited by phage for infection. Results: EF1TV is a newly discoveredE. faecalis phage which belongs to the Herelleviridae family. EF1TV, whose genome is 98% identical to φEF24C, is characterized by a linear dsDNA genome of 143,507 bp with direct terminal repeats of 1,911 bp. The phage is able to infect E. faecalis and shows also the ability to degrade biofilm produced by strains of this species. The results were confirmed by confocal laser scanning microscopy analyzing the biofilm reduction in the same optical field before and after phage infection. Conclusions: The EF1TV phage shows promising features such as an obligatory lytic nature, an anti-biofilm activity and the absence of integration-related proteins, antibiotic resistance determinants and virulence factors, and therefore could be a promising tool for therapeutic applications.

The preservation of cultural heritage is one of the major challenges of today's society. Parchments, a semi-solid matrix of collagen produced from animal skin, are a significant part of the cultural heritage, being used as writing material since ancient times. Due to their animal origin, parchments easily undergo biodeterioration: the most common biological damage is characterized by isolated or coalescent purple spots, that often lead to the detachment of the superficial layer and the consequent loss of written content. Although many parchments with purple spot biodegradative features were studied, no common causative agent had been identified so far. In a previous study a successional model has been proposed, basing on the multidisciplinary analysis of damaged versus undamaged samples from a moderately damaged document. Although no specific sequences were observed, the results pointed to Halobacterium salinarum as the starting actor of the succession. In this study, to further investigate this topic, three dramatically damaged parchments were analysed; belonging to a collection archived as Faldone Patrizi A 19, and dated back XVI-XVII century A.D. With the same multidisciplinary approach, the Next Generation Sequencing (NGS, Illumina platform) revealed DNA sequences belonging to Halobacterium salinarum; the RAMAN spectroscopy identified the pigment within the purple spots as haloarchaeal bacterioruberin and bacteriorhodopsine, and the LTA technique quantified the extremely damaged collagen structures through the entire parchments, due to the biological attack to the parchment frame structures. These results allowed to propose a model of the progressive degradation pattern of the parchment collagen. Overall, these data validate a multi-phase microbial succession model. This demonstration is pivotal to possible new restoration strategies, important for a huge number of ancient documents.

Antibiotic resistance in bacterial pathogens has currently reached very high and alarming levels [...].

Resistance to carbapenems in Enterobacteriaceae, including Klebsiella pneumoniae, represents a major clinical problem given the lack of effective alternative antibiotics. Bacteriophages could provide a valuable tool to control the dissemination of antibiotic resistant isolates, for the decolonization of colonized individuals and for treatment purposes. In this work, we have characterized a lytic bacteriophage, named vB_Kpn_F48, specific for K. pneumoniae isolates belonging to clonal group 101. Phage vB_Kpn_F48 was classified as a member of Myoviridae, order Caudovirales, on the basis of transmission electron microscopy analysis. Physiological characterization demonstrated that vB_Kpn_F48 showed a narrow host range, a short latent period, a low burst size and it is highly stable to both temperature and pH variations. High throughput sequencing and bioinformatics analysis revealed that the phage is characterized by a 171 Kb dsDNA genome that lacks genes undesirable for a therapeutic perspective such integrases, antibiotic resistance genes and toxin encoding genes. Phylogenetic analysis suggests that vB_Kpn_F48 is a T4-like bacteriophage which belongs to a novel genus within the Tevenvirinae subfamily, which we tentatively named "F48virus". Considering the narrow host range, the genomic features and overall physiological parameters phage vB_Kpn_F48 could be a promising candidate to be used alone or in cocktails for phage therapy applications.

A yellow pigmented and agar-pitting colony was isolated from a water sample obtained from a drainage ditch within a disused system of constructed wetlands. The strain was purified and named MCT13T. This rod-shaped, Gram-negative, oxidase- and catalase-positive, aerobic, non-spore-forming, and non-motile strain formed round colonies and grew optimally at pH 7.5±0.2, at 28-30 °C on LB agar, with 0-0.5 % NaCl. The 16S rRNA gene sequence analysis placed the MCT13T isolate within the Sphingomonas (sensu stricto) cluster. The DNA G+C content was 65.3 %. The only observed ubiquinone was Q10. The major fatty acids included C17 : 1ω6c and C18 : 1ω7c/C18 : 1ω6c. The major polar lipids were sphingoglycolipid, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major polyamine was spermidine. The 16S rRNA gene phylogenetic analysis performed on the whole sequence, showed the closest relative of MCT13T to be Sphingomonas koreensis (98.52 %); however, there are several genotypic and phenotypic differences between the novel isolate and the type strain JSS26T of S. koreensis. On the basis of these results, strain MCT13T represents a novel species in the genus Sphingomonas, for which the name Sphingomonas turrisvirgatae sp. nov. is proposed. The type strain is MCT13T (=DSM 105457T=BAC RE RSCIC 7T).

The pandemic dissemination of KPC carbapenemase-producing Klebsiella pneumoniae (KPC-KP) represents a major public health problem, given their extensive multidrug resistance profiles and primary role in causing healthcare-associated infections. This phenomenon has largely been contributed by strains of Clonal Group (CG) 258, mostly of clade II, which in some areas represent the majority of KPC-KP isolates. Here we have characterized a newly discovered lytic Podoviridae, named φBO1E, targeting KPC-KP strains of clade II lineage of CG258. Genomic sequencing revealed that φBO1E belongs to the Kp34virus genus (87% nucleotide identity to vB_KpnP_SU552A). ΦBO1E was stable over a broad pH and temperature range, exhibited strict specificity for K. pneumoniae strains of clade II of CG258, and was unable to establish lysogeny. In a Galleria mellonella infection model, φBO1E was able to protect larvae from death following infection with KPC-KP strains of clade II of CG258, including one colistin resistant strain characterized by a hypermucoviscous phenotype. To our best knowledge φBO1E is the first characterized lytic phage targeting K. pneumoniae strains of this pandemic clonal lineage. As such, it could be of potential interest to develop new agents for treatment of KPC-KP infections and for decolonization of subjects chronically colonized by these resistant superbugs.

Bacteria are used in ecotoxicology for their important role in marine ecosystems and their quick, reproducible responses. Here we applied a recently proposed method to assess the ecotoxicity of nanomaterials on the ubiquitous marine bacterium Vibrio anguillarum, as representative of brackish and marine ecosystems. The test allows the determination of 6-h EC50 in a wide range of salinity, by assessing the reduction of bacteria actively replicating and forming colonies. The toxicity of copper oxide nanoparticles (CuO NPs) at different salinities (5-20-35 ‰) was evaluated. CuSO4 5H2O and CuO bulk were used as reference toxicants (solubility and size control, respectively). Aggregation and stability of CuO NP in final testing dispersions were characterized; Cu2+ dissolution and the physical interactions between Vibrio and CuO NPs were also investigated. All the chemical forms of copper showed a clear dose-response relationship, although their toxicity was different. The order of decreasing toxicity was: CuSO4 5H2O > CuO NP > CuO bulk. As expected, the size of CuO NP aggregates increased with salinity and, concurrently, their toxicity decreased. Results confirmed the intrinsic toxicity of CuO NPs, showing modest Cu2+ dissolution and no evidence of CuO NP internalization or induction of bacterial morphological alterations. This study showed the V. anguillarum bioassay as an effective tool for the risk assessment of nanomaterials in marine and brackish environments.