Large-Scale Biogeographical Shifts of Abundance of Antibiotic Resistance Genes and Marine Bacterial Communities as Their Carriers along a Trophic Gradient.

The role of marine environments in the global spread of antibiotic resistance still remains poorly understood, leaving gaps in the One Health-based research framework. Antibiotic resistance genes (ARGs) encoding resistance to five major antibiotic classes, including sulfonamides (sul1, sul2), tetracyclines (tetA, tetB), ß-lactams (blaCTX-M, blaTEMblaVIM), macrolides (ermB, mphA), aminoglycosides (aac3-2), and integrase gene (intl1) were quantified by RT-qPCR, and their distribution was investigated in relation to environmental parameters and the total bacterial community in bottom layer and surface waters of the central Adriatic (Mediterranean), over a 68 km line from the wastewater-impacted estuary to coastal and pristine open sea. Seasonal changes (higher in winter) were observed for antibiotic resistance frequency and the relative abundances of ARGs, which were generally higher in eutrophic coastal areas. In particular, intl1, followed by blaTEM and blaVIM, were strongly associated with anthropogenic influence and Gammaproteobacteria as their predominant carriers. Water column stratification and geographic location had a significant influence on ARGs distribution in the oligotrophic zone, where the bacterial community exhibited a seasonal shift from Gammaproteobacteria in winter to Marine group II in summer.

Significant Benefits of Environmentally Friendly Hydrosols from Tropaeolum majus L. Seeds with Multiple Biological Activities.

Tropaeolum majus L. is a traditional medicinal plant with a wide range of biological activities due to the degradation products of the glucosinolate glucotropaeolin. Therefore, the goals of this study were to identify volatiles using gas chromatography-mass spectrometry analysis (GC-MS) of the hydrosols (HYs) isolated using microwave-assisted extraction (MAE) and microwave hydrodiffusion and gravity (MHG). Cytotoxic activity was tested against a cervical cancer cell line (HeLa), human colon cancer cell line (HCT116), human osteosarcoma cell line (U2OS), and healthy cell line (RPE1). The effect on wound healing was investigated using human keratinocyte cells (HaCaT), while the antibacterial activity of the HYs was tested against growth and adhesion to a polystyrene surface of Staphylococcus aureus and Escherichia coli. Antiphytoviral activity against tobacco mosaic virus (TMV) was determined. The GC-MS analysis showed that the two main compounds in the HYs of T. majus are benzyl isothiocyanate (BITC) and benzyl cyanide (BCN) using the MAE (62.29% BITC and 15.02% BCN) and MHG (17.89% BITC and 65.33% BCN) extraction techniques. The HYs obtained using MAE showed better cytotoxic activity against the tested cancer cell lines (IC50 value of 472.61-637.07 µg/mL) compared to the HYs obtained using MHG (IC50 value of 719.01-1307.03 µg/mL). Both concentrations (5 and 20 µg/mL) of T. majus HYs using MAE showed a mild but statistically non-significant effect in promoting gap closure compared with untreated cells, whereas the T. majus HY isolated using MHG at a concentration of 15 µg/mL showed a statistically significant negative effect on wound healing. The test showed that the MIC concentration was above 0.5 mg/mL for the HY isolated using MAE, and 2 mg/mL for the HY isolated using MHG. The HY isolated using MHG reduced the adhesion of E. coli at a concentration of 2 mg/mL, while it also reduced the adhesion of S. aureus at a concentration of 1 mg/mL. Both hydrosols showed excellent antiphytoviral activity against TMV, achieving100% inhibition of local lesions on the leaves of infected plants, which is the first time such a result was obtained with a hydrosol treatment. Due to the antiphytoviral activity results, hydrosols of T. majus have a promising future for use in agricultural production.

Picoplankton diversity in an oligotrophic and high salinity environment in the central Adriatic Sea.

By combining qualitative 16S metabarcoding and quantitative CARD-FISH methods with neural gas analysis, different patterns of the picoplankton community were revealed at finer taxonomic levels in response to changing environmental conditions in the Adriatic Sea. We present the results of a one-year study carried out in an oligotrophic environment where increased salinity was recently observed. We have shown that the initial state of community structure changes according to environmental conditions and is expressed as qualitative and quantitative changes. A general pattern of increasing diversity under harsh environmental conditions, particularly under the influence of increasing salinity at the expense of community abundance was observed. Considering the trend of changing seawater characteristics due to climate change, this study helps in understanding a possible structural change in the microbial community of the Adriatic Sea that could affect higher levels of the marine food web.

Microbiome profiling and characterization of virulent and vancomycin-resistant Enterococcus faecium from treated and untreated wastewater, beach water and clinical sources.

Vancomycin-resistant Enterococcus faecium (VREfm) is an opportunistic pathogen among the highest global priorities regarding public and environmental health. Following One Health approach, we determined for the first time the antibiotic resistance and virulence genes, and sequence types (STs) affiliation of VREfm recovered simultaneously from marine beach waters, submarine outfall of a wastewater treatment plant and an offshore discharge of untreated sewage, and compared them with the surveillance VREfm from regional university hospital in Croatia to assess the hazard of their transmission and routes of introduction into the natural environment. Importantly, VREfm recovered from wastewater, coastal bathing waters and hospital shared similar virulence, multidrug resistance, and ST profiles, posing a major public health threat. All isolates carried the vanA gene, while one clinical isolate also possessed the vanC2/C3 gene. The hospital strains largely carried the aminoglycoside-resistance genes aac(6')-Ie-aph(2″)-Ia, and aph(2″)-Ib and aph(2″)-Id, which were also predominant in the environmental isolates. The hyl gene was the most prevalent virulence gene. The isolates belonged to 10 STs of the clonal complex CC17, a major epidemic lineage associated with hospital infections and outbreaks, with ST117 and ST889 common to waterborne and hospital isolates, pointing to their sewage-driven dissemination. To gain better insight into the diversity of accompanying taxons in the surveyed water matrices, microbiome taxonomic profiling was carried out using Illumina-based 16S rDNA sequencing and their resistome features predicted using the PICRUSt2 bioinformatics tool. An additional 60 pathogenic bacterial genera were identified, among which Arcobacter, Acinetobacter, Escherichia-Shigella, Bacteroides and Pseudomonas were the most abundant and associated with a plethora of antibiotic resistance genes and modules, providing further evidence of the hazardous effects of wastewater discharges, including the treated ones, on the natural aquatic environment that should be adequately addressed from a sanitary and technological perspective.

A Plant Worthy of Further Study-Volatile and Non-Volatile Compounds of Portenschlagiella ramosissima (Port.) Tutin and Its Biological Activity.

New and detailed data are presented on the phytochemical composition of the volatile and non-volatile organic compounds of the Mediterranean endemic species Portenschlagiella ramosissima (Port.) Tutin. Both the essential oil and hydrosol were obtained from the air-dried plant by hydrodistillation and analyzed by gas chromatography-mass spectrometry. The volatile compounds from the fresh and air-dried plants and from the hydrosol were isolated for the first time by headspace solid-phase microextraction using two fibres of different polarity. The benzene derivative group was the predominant group in all samples, with myristicin being the most abundant component of all. The non-volatile compounds of the methanol extract were analyzed by ultra-high-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionisation, and three flavonoid glycosides, one anthocyanidin glycoside, and lipid derivatives were detected. Both the chemical composition and biological activities of this plant have been described in a very limited number of publications, making it an interesting source for further study. The antiphytoviral activity of the essential oil and hydrosol showed that both extracts significantly reduced the number of lesions on the leaves of local host plants infected with tobacco mosaic virus. Moderate antiproliferative activity of the methanol extract was detected in three cancer cell lines, cervical cancer cell line, human colon cancer cell line and human osteosarcoma cell line, using the MTS-based cell proliferation assay. Based on the results, we highlight this plant as a new source of bioactive compounds and natural phytotherapeutic agent that deserves further investigation.

Submarine Outfalls of Treated Wastewater Effluents are Sources of Extensively- and Multidrug-Resistant KPC- and OXA-48-Producing Enterobacteriaceae in Coastal Marine Environment.

The rapid and ongoing spread of carbapenemase-producing Enterobacteriaceae has led to a global health threat. However, a limited number of studies have addressed this problem in the marine environment. We investigated their emergence in the coastal waters of the central Adriatic Sea (Croatia), which are recipients of submarine effluents from two wastewater treatment plants. Fifteen KPC-producing Enterobacteriaceae (nine Escherichia coli, four Klebsiella pneumoniae and two Citrobacter freundii) were recovered, and susceptibility testing to 14 antimicrobials from 10 classes showed that four isolates were extensively drug resistant (XDR) and two were resistant to colistin. After ERIC and BOX-PCR typing, eight isolates were selected for whole genome sequencing. The E. coli isolates belonged to serotype O21:H27 and sequence type (ST) 2795, while K. pneumoniae isolates were assigned to STs 37 and 534. Large-scale genome analysis revealed an arsenal of 137 genes conferring resistance to 19 antimicrobial drug classes, 35 genes associated with virulence, and 20 plasmid replicons. The isolates simultaneously carried 43-90 genes encoding for antibiotic resistance, while four isolates co-harbored carbapenemase genes bla KPC-2 and bla OXA-48. The bla OXA-48 was associated with IncL-type plasmids in E. coli and K. pneumoniae. Importantly, the bla KPC-2 in four E. coli isolates was located on ~40 kb IncP6 broad-host-range plasmids which recently emerged as bla KPC-2 vesicles, providing first report of these bla KPC-2-bearing resistance plasmids circulating in E. coli in Europe. This study also represents the first evidence of XDR and potentially virulent strains of KPC-producing E. coli in coastal waters and the co-occurrence of bla KPC-2 and bla OXA-48 carbapenemase genes in this species. The leakage of these strains through submarine effluents into coastal waters is of concern, indicating a reservoir of this infectious threat in the marine environment.

Chemical Composition and Biological Activity of Essential Oil and Extract from the Seeds of Tropaeolum majus L. var. altum.

Research background: Plant Tropaeolum majus L. (garden nasturtium) belongs to the family Tropaeolaceae and contains benzyl glucosinolate. The breakdown product of benzyl glucosinolate, benzyl isothiocyanate (BITC), exhibits various biological activities such as antiproliferative, antibacterial and antiinflammatory. In order to optimize the content of biologically active volatile compounds in plant extract and essential oil, the use of appropriate extraction technique has a crucial role. Experimental approach: The current study investigates the effect of two modern extraction methods, microwave-assisted distillation (MAD) and microwave hydrodiffusion and gravity (MHG), on the chemical composition of volatile components present in the essential oil and extract of garden nasturtium (T. majus L. var. altum) seeds. Investigation of the biological activity of samples (essential oil, extract and pure compounds) was focused on the antiproliferative effect against different cancer cell lines: cervical cancer cell line (HeLa), human colon cancer cell line (HCT116) and human osteosarcoma cell line (U2OS), and the antibacterial activity which was evaluated against the growth and adhesion of Staphylococcus aureus and Escherichia coli to polystyrene surface. Results and conclusions: Essential oil and extract of garden nasturtium (T. majus) seeds were isolated by two extraction techniques: MAD and MHG. BITC and benzyl cyanide (BCN) present in the extract were identified by gas chromatography-mass spectrometry. Essential oil of T. majus showed higher antiproliferative activity (IC50<5 µg/mL) than T. majus extract (IC50<27 µg/mL) against three cancer cell lines: HeLa, HCT116 and U2OS. BITC showed much higher inhibitory effect on all tested cells than BCN. The essential oil and extract of T. majus showed strong antimicrobial activity against S. aureus and E. coli. Novelty and scientific contribution: This work represents the first comparative report on the antiproliferative activity of the essential oil and extract of T. majus seeds, BITC and BCN against HeLa, HCT116 and U2OS cells as well as their antimicrobial activity against S. aureus and E. coli. This study demonstrates that the essential oil of T. majus seeds exhibits stronger antiproliferative and antimicrobial activity than the plant extract.

Free Volatile Compounds of Veronica austriaca ssp. jacquinii (Baumg.) Eb. Fisch. and Their Biological Activity.

The composition of free volatile compounds of essential oils (EO) and hydrosols (Hy) from four different localities of the species Veronica austriaca ssp. jacquinii (Baumg.) Eb. Fisch. were analyzed by gas chromatography coupled with mass spectrometry. In the EOs, the most abundant compounds identified were hexahydrofarnesyl acetone (23.34-52.56%), hexadecanoic acid (palmitic acid, 26.71-58.91%) and octadecanol acetate (0-6.24%). The hydrosols were characterized by high abundance of methyl eugenol (23.35-57.93%), trans-p-mentha-1(7),8-dien-2-ol (5.24-7.69%) and thymol (3.48-9.45%). Glandular trichomes were analyzed using SEM (Scanning Electron Microscopy), as they are the sites of synthesis of free volatile compounds. We have detected glandular trichomes, consisting of a one stalk cell and two elliptically shaped head cells, and non-glandular (unbranched, bi-cellular to multicellular) trichomes on stems, leaves and the sepals. Data for volatile compounds from EOs and hydrosols were analyzed using Principal Component Analyses (PCA) to demonstrate variations in the composition of the volatile compounds identified. Isolated samples of EO and hydrosols were analyzed for their antioxidant activity using two methods, DPPH (2,2-diphenyl-1-picrylhydrazyl) and ORAC (Oxygen Radical Absorbance Capacity). The essential oils showed higher antioxidant activity than the hydrosols in ORAC method, but lower activity by the DPPH method. The isolates were also tested for their antiproliferative activity on different types of cancer cells and also on two lines of healthy cells, and the results showed that the extracts were not toxic to the cell lines tested. Total polyphenols, total tannins, total flavonoids and total phenolic acids were also analyzed and determined spectrophotometrically. The free volatile compounds of Veronica austriaca ssp. jacquinii can be considered as a safe natural product.

Not Only a Weed Plant-Biological Activities of Essential Oil and Hydrosol of Dittrichia viscosa (L.) Greuter.

With the increasing interest in obtaining biologically active compounds from natural sources, Dittrichia viscosa (L.) Greuter (Asteraceae) came into our focus as a readily available and aromatic wild shrub widely distributed in the Mediterranean region. This work provides a phytochemical profile of D. viscosa in terms of parallel chemical composition in the lipophilic fraction (essential oil) and the water fraction (hydrosol). GC-MS analysis identified 1,8-cineole, caryophyllene oxide, α-terpenyl acetate, and α-muurolol as the major components of the essential oil, while in the hydrosol p-menth-1-en-9-ol, 1,8-cineole, linalool, cis-sabinene hydrate, and α-muurolol were the major volatile components. 3,4-Dihydroxybenzoic acid was found to be the predominant compound in the hydrosol composition by HPLC analysis. The antimicrobial potential of both extracts was evaluated against thirteen opportunistic pathogens associated with common skin and wound infections and emerging food spoilage microorganisms. The antimicrobial activity of the essential oil suggests that the volatiles of D. viscosa could be used as novel antimicrobial agents. The antiproliferative results of D. viscosa volatiles are also new findings, which showed promising activity against three cancer cell lines: HeLa (cervical cancer cell line), HCT116 (human colon cancer cell line), and U2OS (human osteosarcoma cell line). The decrease in GSH level observed in hydrosol-treated HeLa cells suggests oxidative stress as a possible mechanism of the antiproliferative effect of hydrosol on tumor cells. The presented results are also the first report of significant antiphytoviral activity of hydrosol against tobacco mosaic virus (TMV) infection. Based on the results, D. viscosa might have the potential to be used in crop protection, as a natural disinfectant and natural anticancer agent.

Bacteria tolerant to colistin in coastal marine environment: Detection, microbiome diversity and antibiotic resistance genes' repertoire.

The global spread of mobilized colistin resistance (mcr) genes in clinical and natural environments dangerously diminishes the effectiveness of this last-resort antibiotic, becoming an urgent health threat. We used a multidisciplinary approach to detect mcr-1 gene and colistin (CL)-resistant bacteria in seawater from two Croatian public beaches. Illumina-based sequencing of metagenomic 16S rRNA was used to assess the taxonomic, functional, and antibiotic resistance genes (ARGs) profiling of the bacterial community tolerant to CL regarding different culture-based isolation methodologies. Data revealed that the choice of methodology alters the diversity and abundance of taxa accounting for the CL-resistance phenotype. The mcr-1 gene was identified by cloning and sequencing in one sample, representing the first report of mcr-1 gene in Croatia. Culturing of CL-resistant strains revealed their resistance phenotypes and concurrent production of clinically significant ß-lactamases, such as CTX-M-15, CTX-M-3 and SHV-12. We also report the first identification of blaCTX-M-15 gene in Klebsiella huaxiensis and K. michiganensis, as well as the blaTEM-1+CTX-M-3 in Serratia fonticola. ARGs profiles derived from metagenomic data and predicted by PICRUSt2, showed the highest abundance of genes encoding for multidrug efflux pumps, followed by the transporter genes accounting for the tetracycline, macrolide and phenicol resistance. Our study evidenced the multidrug resistance features of CL-tolerant bacterial communities thriving in surface beach waters. We also showed that combined application of the metagenomic approaches and culture-based techniques enabled successful detection of mcr-1 gene, which could be underreported in natural environment.

Chemical Composition and New Biological Activities of Essential Oil and Hydrosol of Hypericum perforatum L. ssp. veronense (Schrank) H. Lindb.

The chemical profile, antiproliferative, antioxidant and antiphytoviral activities of the species Hypericum perforatum ssp. veronense (Schrank) H. Lindb. (Clusiaceae) were investigated. Free volatiles were isolated and the chemical composition was determined in the lipophilic fraction (essential oil) and for the first time in the water fraction (hydrosol). The aim is to provide phytochemical data for H. perforatum ssp. veronense useful for distinguishing ssp. veronense from ssp. angustifolium, as there are taxonomic disagreements between them and the composition of the secretory products may be helpful in this respect. In the essential oil, the most abundant compounds identified were α-pinene and n-nonane, while in the hydrosol, myrtenol, carvacrol and α-pinene were the most abundant. Overall, the class of monoterpenes and oxygenated monoterpenes dominated in the EO and hydrosol samples. The essential oil showed high antioxidant activity, in contrast to the antiproliferative activity, where the hydrosol showed exceptional activity against three cancer cell lines: Hela (cervical cancer cell line), HCT116 (human colon cancer cell line) and U2OS (human osteosarcoma cell line). Both the essential oil and hydrosol showed antiphytoviral activity against tobacco mosaic virus infection on the local host plants. This is the first report dealing with biological activities of hydrosol of H. perforatum ssp. veronense, and the obtained results suggest that this traditional medicinal plant is a valuable source of volatiles with promising antiproliferative, antioxidant and antiphytoviral activities.

Phytochemical Characterization and Screening of Antioxidant, Antimicrobial and Antiproliferative Properties of Allium × cornutum Clementi and Two Varieties of Allium cepa L. Peel Extracts.

Onions are one of the most widely grown vegetable crops. As production increases, so does the generation of waste from various parts of the onion, raising the need for efficient ecological disposal and use of such waste products. However, onion waste products are a rich source of antioxidants with a range of biological properties, therefore, they could potentially be used in food and pharmaceutical industries. In the present study, we identified the main flavonols and anthocyanins in peel extracts of Allium × cornutum Clement ex Visiani, 1842, and two varieties of Allium cepa L. and tested their antioxidant, antimicrobial and antiproliferative properties. Quercetin 3,4'-diglucolside, quercetin 4'-monoglucoside and quercetin are the most abundant flavonols in all onion extracts detected by high-performance liquid chromatography (HPLC) method. The composition of anthocyanins varied in all extracts. 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) assays showed that the triploid onion A. × cornutum had the highest antioxidant power. Evaluation of antimicrobial activity by broth microdilution assay also showed that A. × cornutum had higher antimicrobial activity compared to the red and yellow onion varieties. Comparable antiproliferative activity was confirmed for all onion extracts tested on three cancer cell lines: Hela (cervical cancer cell line), HCT116 (human colon cancer cell line) and U2OS (human osteosarcoma cell line). The most abundant onion flavonols (quercetin 3,4'-diglucoside and quercetin 4'-monoglucoside) showed weaker antimicrobial as well as antiproliferative properties compared to the extracts, leading to the conclusion that other phytochemicals besides flavonols contribute to the biological activity of onion peel extracts. The results demonstrate the antioxidant and antimicrobial properties of onion peels, which have promising potential as cancer cell proliferation inhibitors.

Comparison of Organosulfur and Amino Acid Composition between Triploid Onion Allium cornutum Clementi ex Visiani, 1842, and Common Onion Allium cepa L., and Evidences for Antiproliferative Activity of Their Extracts.

Species that belong to the genus Allium have been widely used for human food and traditional medicine. Their beneficial health effects, as well as the specific aroma, are associated with their bioactive chemical compounds, such as sulfur compounds and flavonoids. Gas chromatography and mass spectrometry (GC-MS) and reverse-phase high-performance liquid chromatography (reverse-phase HPLC) were used to identify organosulfur and amino acid content of triploid hybrid onion, Allium cornutum Clement ex Visiani, 1842, and common onion, Allium cepa L. Allium extracts were tested for their antiproliferative activity in three human cancer cell lines (HeLa, HCT116, and U2OS). DNA fragmentation and DAPI staining analysis were performed on HeLa cells to evaluate the effect of extracts on DNA damage and cell morphology. The mRNA expression of p53, Bax, and Caspase-3 genes involved in apoptosis were analyzed by real-time PCR. Using GC-MS, 27 compounds were found in two Allium species headspaces. Differences were noted among the main compound abundance in the headspace (although the major thiols and disulfides were qualitatively identic in both Allium species) and dipropyl disulfide, diisopropyl trisulfide, and (Z)-prop-1-enyl propyl trisulfide were predominant sulfides. Identification of amino acids and their quantities were determined by reverse-phase HPLC. Most abundant amino acids in both onions were arginine (Arg) and glutamic acid (Glu). The results of cytotoxicity testing confirmed antiproliferative effects of both species. The DNA fragmentation assay, DAPI staining and real time PCR analysis confirmed that A. cornutum and A. cepa extracts induced apoptosis in HeLa cells. This study presents the evidence for possible therapeutic use of A. cornutum and A. cepa extracts against human cervical carcinoma cell line.

Broad-spectrum resistance of Pseudomonas aeruginosa from shellfish: infrequent acquisition of novel resistance mechanisms.

Pseudomonas aeruginosa is one the most common multidrug-resistant pathogens worldwide. It has been previously detected in marine shellfish, but its antibiotic resistance in such environment has not been explored. By combining PCR detection of acquired genes, and resistance-nodulation-cell division (RND) efflux studying, we investigated the multifactorial resistance traits of 108 P. aeruginosa isolates recovered from wild-growing Mediterranean mussels (Mytilus galloprovincialis) in Croatia. Eleven different resistance profiles were found, with the main mechanism being the overexpression of intrinsic efflux pump(s), particularly MexAB-OprM. Several acquired resistance determinants were detected, including the ß-lactamase gene blaTEM-116, sulfamethoxazole resistance gene sul1, and the class 1 integron gene cassette carrying the streptomycin resistance gene aadA7. This study evidenced the multiple resistance in P. aeruginosa in shellfish from human-impacted marine environment, pointing to the underestimated role of the marine habitat for maintenance of multiresistant P. aeruginosa and, consequently, the potential risk for human and environmental health.

Chemical Composition and Biological Activity of Allium cepa L. and Allium × cornutum (Clementi ex Visiani 1842) Methanolic Extracts.

Here, we report a comparative study of the phytochemical profile and the biological activity of two onion extracts, namely Allium cepa L. and Allium × cornutum (Clementi ex Visiani 1842), members of the family Amaryllidaceae. The identification of flavonoids and anthocyanins, and their individual quantities, was determined by high-performance liquid chromatography (HPLC). The potency of both extracts to scavenge free radicals was determined by the DPPH (2,2'-diphenyl-1-picrylhydrazyl) radical-scavenging activity and oxygen radical absorbance capacity (ORAC) methods. The DNA protective role was further tested by the single-cell gel electrophoresis (COMET) assay and by Fenton's reagent causing double-strand breaks on the closed circular high copy pUC19 plasmid isolated from Escherichia coli. In the presence of both extracts, a significant decrease in DNA damage was observed, which indicates a protective role of Allium cepa and Allium × cornutum on DNA strand breaks. Additionally, cytotoxicity was tested on glioblastoma and breast cancer cell lines. The results showed that both extracts had antiproliferative effects, but the most prominent decrease in cellular growth was observed in glioblastoma cells.

Urban riverine environment is a source of multidrug-resistant and ESBL-producing clinically important Acinetobacter spp.

Some Acinetobacter species have emerged as very important opportunistic pathogens in humans. We investigated Acinetobacter spp. from the polluted urban riverine environment in Croatia in regard to species affiliation, antibiotic resistance pattern, and resistance mechanisms. Considerable number of isolates produced acquired extended-spectrum ß-lactamase(s) (ESBLs), CTX-M-15 solely or with TEM-116. By Southern blot hybridization, bla TEM-116 was identified on plasmids ca. 10, 3, and 1.2 kb in Acinetobacter junii, A. gandensis, and A. johnsonii. The bla TEM-116-carrying plasmid in A. gandensis was successfully transferred by conjugation to azide-resistant Escherichia coli J53. A. radioresistens isolate also carried an intrinsic carbapenemase gene bla OXA-133 with ISAba1 insertion sequence present upstream to promote its expression. Majority of ESBL-producing isolates harbored integrases intI1 and/or intI2 and the sulfamethoxazole resistance gene sul1. Almost all isolates had overexpressed resistance-nodulation-cell division (RND) efflux system, indicating that this mechanism may have contributed to multidrug resistance phenotypes. This is the first report of environmental CTX-M-15-producing Acinetobacter spp. and the first identification of CTX-M-15 in A. johnsonii, A. junii, A. calcoaceticus, A. gandensis, A. haemolyticus, and A. radioresistens worldwide. We identified, also for the first time, the environmental Acinetobacter-producing TEM ESBLs, highlighting the potential risk for human health, and the role of these bacteria in maintenance and dissemination of clinically important antibiotic resistance genes in community through riverine environments.

Prevalence and diversity of extended-spectrum-ß-lactamase-producing Enterobacteriaceae from marine beach waters.

A total of 1,351 Enterobacteriaceae isolates from 144 seawater samples were collected over a four-year period from three public beaches in the eastern Adriatic Sea in Croatia. Approximately 35% of the strains were multidrug-resistant. BlaESBL genes were detected in 4.2% of the isolated Enterobacteriaceae, the main species of which were Escherichia coli, Enterobacter cloacae and Klebsiella pneumoniae. BlaTEM-1+SHV-12 was the most dominant genotype, followed by blaCTX-M-15.Raoultella terrigena and E. intermedius simultaneously harboured blaTEM-1,blaSHV-11/12 and blaCTX-M-15. Isolate fingerprinting revealed that marine E. coli isolates were clonally related to CTX-M-producing strains from a regional university hospital. These results indicate that marine beach waters are reservoirs of ESBL-producing Enterobacteriaceae and thus constitute a public health problem with further potential to act as mediators in gene flow between marine coastal areas and clinical settings.

Triparental origin of triploid onion, Allium × cornutum (Clementi ex Visiani, 1842), as evidenced by molecular, phylogenetic and cytogenetic analyses.

BACKGROUND: Reconstruction of the parental origins of cultivated plants from wild relatives, especially after long periods of domestication, is not a trivial task. However, recent advances in molecular phylogenetics, among other approaches, have proved to be very informative in analyses of the origin and evolution of polyploid genomes. An established minor garden crop, triploid onion Allium × cornutum (Clementi ex Visiani, 1842) (2n = 3x = 24), is widespread in southeastern Asia and Europe. Our previous cytogenetic analyses confirmed its highly heterozygous karyotype and indicated its possible complex triparental genome origin. Allium cepa L. and Allium roylei Stearn were suggested as two putative parental species of A. × cornutum, whereas the third parental species remained hitherto unknown. RESULTS: Here we report the phylogenetic analyses of the internal transcribed spacers ITS1-5.8S-ITS2 of 35S rDNA and the non-transcribed spacer (NTS) region of 5S rDNA of A. × cornutum and its relatives of the section Cepa. Both ITS and NTS sequence data revealed intra-individual variation in triploid onion, and these data clustered into the three main clades, each with high sequence homology to one of three other species of section Cepa: A. cepa, A. roylei, and unexpectedly, the wild Asian species Allium pskemense B. Fedtsh. Allium pskemense is therefore inferred to be the third, so far unknown, putative parental species of triploid onion Allium × cornutum. The 35S and 5S rRNA genes were found to be localised on somatic chromosomes of A. × cornutum and its putative parental species by double fluorescent in situ hybridisation (FISH). The localisation of 35S and 5S rDNA in A. × cornutum chromosomes corresponded to their respective positions in the three putative parental species, A. cepa, A. pskemense, and A. roylei. GISH (genomic in situ hybridisation) using DNA of the three putative parental diploids corroborated the results of the phylogenetic study. CONCLUSIONS: The combined molecular, phylogenetic and cytogenetic data obtained in this study provided evidence for a unique triparental origin of triploid onion A. × cornutum with three putative parental species, A. cepa, A. pskemense, and A. roylei.

Aeromonas spp. simultaneously harbouring bla(CTX-M-15), bla(SHV-12), bla(PER-1) and bla(FOX-2), in wild-growing Mediterranean mussel (Mytilus galloprovincialis) from Adriatic Sea, Croatia.

Aeromonas species are becoming renowned as emerging pathogens by increasingly giving rise to a wide spectrum of food and waterborne infections in humans. Another worrisome feature of aeromonads is the growing frequency of antibiotic resistance as a consequence of their prominent diversity in terms of resistance determinants. This study aimed at determining the antimicrobial resistance pattern, prevalence and characterization of acquired ß-lactamases, including extended-spectrum-ß-lactamases (ESBLs) and AmpC cephalosporinases, as well as the presence of class 1 and 2 integrons, in Aeromonas isolates from wild-growing Mediterranean mussel (Mytilus galloprovincialis) of the eastern coast of Adriatic Sea, Croatia. Isolates were tested for susceptibility to 16 antibiotics and ß-lactam/ß-lactamase inhibitor combinations. Cephalosporin-resistant isolates were further screened by PCR for genes encoding AmpC (bla(FOX), bla(CMY), bla(MOX), bla(LAT), bla(BIL), bla(DHA), bla(ACC), bla(MIR), bla(ACT)), ESBLs (bla(TEM), bla(SHV), bla(CTX-M), bla(PER), bla(VEB), bla(GES/IBC), bla(OXA)) and integrases (intI1, intI2, intI3). Location of bla genes was characterized by plasmid DNA fingerprinting and Southern blot hybridization. Plasmids carrying ESBL genes were investigated for transferability by conjugation and PCR-based replicon typed. Out of 147 Aeromonas isolates recovered, 30 (20%) demonstrated multiple resistance profile, with co-resistance most frequently detected against penicillins, piperacillin/sulbactam and tetracycline. ESBL-encoding genes were detected in 21 (13 Aeromonas caviae and 8 Aeromonas hydrophila) isolates, with bla(CTX-M-15) gene identified in 19 and bla(SHV-12) in 12 isolates. Among them, 10 isolates simultaneously harboured bla(CTX-M-15) and bla(SHV-12), while 3 isolates additionally carried an AmpC ß-lactamase bla(FOX-2) gene. bla(PER-1) gene was identified in a single isolate also harbouring the bla(CTX-M-15) gene. While bla(SHV-12) was chromosomally encoded, bla(CTX-M-15) was located on conjugative IncFIB-type plasmids of ~40 kb in A. caviae isolates. IntI1 and intI2 genes were detected in 57.1% and 33.3% of ESBL-producing isolates. To the best our knowledge, this is the first report of environmental A. caviae isolates producing CTX-M-15, and isolation of SHV-12-producing A. hydrophila and A. caviae strains worldwide. This is also believed to be the first report of the FOX-2, CTX-M-15 and SHV-12 simultaneous production in aeromonads, highlighting both the potential risk for human health, and a role of these foodborne pathogens as reservoirs of resistance determinants in coastal marine environment.