Motile aeromonads from farmed and wild freshwater fish in northern Italy: an evaluation of antimicrobial activity and multidrug resistance during 2013 and 2016

Background Antimicrobial resistant bacteria are emerging biological contaminants of the environment. In aquatic ecosystems, they originate mainly from hospitals, livestock manure and private households sewage water, which could contain antimicrobial agents and resistant microorganisms. Aeromonas spp. occur ubiquitously in aquatic environments and they cause disease in fish. Motile aeromonads are also associated with human gastrointestinal and wound infections and fish can act as a transmission route of antimicrobial resistance (AMR) aeromonads to humans. The environmental ubiquity, the natural susceptibility to antimicrobials and the zoonotic potential of Aeromonas spp. make them optimal candidates for studying the AMR in aquatic ecosystems. Results The AMR patterns of 95 motile aeromonads isolated from freshwater fish during 2013 and 2016 were analyzed. All samples from fish came from farms and natural water bodies located in northern Italy, which is an area characterized by high anthropic impact on the environment. The isolates were biochemically identified as Aeromonas hydrophila, Aeromonas sobria or Aeromonas caviae and AMR was determined by the standard disk diffusion method. All isolates were resistant to cloxacillin, spiramycin and tilmicosin. High AMR frequencies (>\u200995%) were detected for tylosin, penicillin and sulfadiazine. AMR to danofloxacin, enrofloxacin, flumequine, ceftiofur, aminosidine, colistin, doxycycline, gentamicin, marbocyl and florfenicol was observed at low levels (<\u200910%). No AMR to cefquinome was found. Logistic regression showed several differences in antimicrobial activity between complexes. According to the source of aeromonads, only few differences in AMR between isolates from farmed and wild fish were observed. Conclusions Our data revealed an increasing trend of AMR to neomycin and apramycin among Aeromonas isolates during the study period, while resistance to erythromycin, tetracycline and thiamphenicol decreased. All isolates were multidrug resistance (MDR), but A. caviae showed the highest number of MDR per isolate. In most isolates, various degrees of MDR were detected to macrolides, quinolones, fluoroquinolones, polymyxins and cephalosporins (third and fourth generations), which are listed, by the World Health Organisation, to be among the highest priority and critically important antimicrobials in human medicine. Our findings underlined that freshwater fish can act as potential source of MDR motile aeromonads. Due to their zoonotic potential, this can pose serious threat to human health

Esbl/ampc-producing escherichia coli in wild boar: Epidemiology and risk factors

The complex health problem of antimicrobial resistance (AMR) involves many host species, numerous bacteria and several routes of transmission. Extended-spectrum β-lactamase and AmpC (ESBL/AmpC)-producing Escherichia coli are among the most important strains. Moreover, wildlife hosts are of interest as they are likely antibiotics free and are assumed as environmental indicators of AMR contamination. Particularly, wild boar (Sus scrofa) deserves attention because of its increased population densities, with consequent health risks at the wildlife–domestic–human interface, and the limited data available on AMR. Here, 1504 wild boar fecal samples were microbiologically and molecularly analyzed to investigate ESBL/AmpC-producing E. coli and, through generalized linear models, the effects of host-related factors and of human population density on their spread. A prevalence of 15.96% of ESBL/AmpC-producing E. coli, supported by blaCTX-M (12.3%), blaTEM (6.98%), blaCMY (0.86%) and blaSHV (0.47%) gene detection, emerged. Young animals were more colonized by ESBL/AmpC strains than older subjects, as observed in domestic animals. Increased human population density leads to increased blaTEM prevalence in wild boar, suggesting that spatial overlap may favor this transmission. Our results show a high level of AMR contamination in the study area that should be further investigated. However, a role of wild boar as a maintenance host of AMR strains emerged

Nonredundant role of CCRL2 in lung dendritic cell trafficking.

Chemokine CC motif receptor-like 2 (CCRL2) is a heptahelic transmembrane receptor that shows the highest degree of homology with CCR1, an inflammatory chemokine receptor. CCRL2 mRNA was rapidly (30 minutes) and transiently (2-4 hours) regulated during dendritic cell (DC) maturation. Protein expression paralleled RNA regulation. In vivo, CCRL2 was expressed by activated DC and macrophages, but not by eosinophils and T cells. CCRL2(-/-) mice showed normal recruitment of circulating DC into the lung, but a defective trafficking of antigen-loaded lung DC to mediastinal lymph nodes. This defect was associated to a reduction in lymph node cellularity and reduced priming of T helper cell 2 response. CCRL2(-/-) mice were protected in a model of ovalbumin-induced airway inflammation, with reduced leukocyte recruitment in the BAL (eosinophils and mononuclear cells) and reduced production of the T helper cell 2 cytokines, interleukin-4 and -5, and chemokines CCL11 and CCL17. The central role of CCRL2 deficiency in DC was supported by the fact that adoptive transfer of CCRL2(-/-) antigen-loaded DC in wild-type animals recapitulated the phenotype observed in knockout mice. These data show a nonredundant role of CCRL2 in lung DC trafficking and propose a role for this receptor in the control of excessive airway inflammatory responses. (Blood. 2010;116(16):2942-2949

Selective accumulation of langerhans-type dendritic cells in small airways of patients with COPD

<p>Abstract</p> <p>Background</p> <p>Dendritic cells (DC) linking innate and adaptive immune responses are present in human lungs, but the characterization of different subsets and their role in COPD pathogenesis remain to be elucidated. The aim of this study is to characterize and quantify pulmonary myeloid DC subsets in small airways of current and ex-smokers with or without COPD.</p> <p>Methods</p> <p>Myeloid DC were characterized using flowcytometry on single cell suspensions of digested human lung tissue. Immunohistochemical staining for langerin, BDCA-1, CD1a and DC-SIGN was performed on surgical resection specimens from 85 patients. Expression of factors inducing Langerhans-type DC (LDC) differentiation was evaluated by RT-PCR on total lung RNA.</p> <p>Results</p> <p>Two segregated subsets of tissue resident pulmonary myeloid DC were identified in single cell suspensions by flowcytometry: the langerin+ LDC and the DC-SIGN+ interstitial-type DC (intDC). LDC partially expressed the markers CD1a and BDCA-1, which are also present on their known blood precursors. In contrast, intDC did not express langerin, CD1a or BDCA-1, but were more closely related to monocytes.</p> <p>Quantification of DC in the small airways by immunohistochemistry revealed a higher number of LDC in current smokers without COPD and in COPD patients compared to never smokers and ex-smokers without COPD. Importantly, there was no difference in the number of LDC between current and ex-smoking COPD patients.</p> <p>In contrast, the number of intDC did not differ between study groups. Interestingly, the number of BDCA-1+ DC was significantly lower in COPD patients compared to never smokers and further decreased with the severity of the disease. In addition, the accumulation of LDC in the small airways significantly correlated with the expression of the LDC inducing differentiation factor activin-A.</p> <p>Conclusions</p> <p>Myeloid DC differentiation is altered in small airways of current smokers and COPD patients resulting in a selective accumulation of the LDC subset which correlates with the pulmonary expression of the LDC-inducing differentiation factor activin-A. This study identified the LDC subset as an interesting focus for future research in COPD pathogenesis.</p

Activin A Induces Langerhans Cell Differentiation In Vitro and in Human Skin Explants

Langerhans cells (LC) represent a well characterized subset of dendritic cells located in the epidermis of skin and mucosae. In vivo, they originate from resident and blood-borne precursors in the presence of keratinocyte-derived TGFβ. Ιn vitro, LC can be generated from monocytes in the presence of GM-CSF, IL-4 and TGFβ. However, the signals that induce LC during an inflammatory reaction are not fully investigated. Here we report that Activin A, a TGFβ family member induced by pro-inflammatory cytokines and involved in skin morphogenesis and wound healing, induces the differentiation of human monocytes into LC in the absence of TGFβ. Activin A-induced LC are Langerin+, Birbeck granules+, E-cadherin+, CLA+ and CCR6+ and possess typical APC functions. In human skin explants, intradermal injection of Activin A increased the number of CD1a+ and Langerin+ cells in both the epidermis and dermis by promoting the differentiation of resident precursor cells. High levels of Activin A were present in the upper epidermal layers and in the dermis of Lichen Planus biopsies in association with a marked infiltration of CD1a+ and Langerin+ cells. This study reports that Activin A induces the differentiation of circulating CD14+ cells into LC. Since Activin A is abundantly produced during inflammatory conditions which are also characterized by increased numbers of LC, we propose that this cytokine represents a new pathway, alternative to TGFβ, responsible for LC differentiation during inflammatory/autoimmune conditions

Indagine sulla patogenicit&#224; di ceppi mobili di aeromonas spp. isolati da specie ittiche

Lo studio della patogenicit\ue0 di Aeromonas spp. \ue8 caratterizzato da notevole complessit\ue0, sviluppandosi in genere su pi\uf9 livelli e comprendendo saggi biologici in vitro (linee cellulari) ed in vivo (modelli animali), esami molecolari ed analisi delle propriet\ue0 fenotipiche degli isolati. Nell\u2019ambito di una tesi di specialit\ue0 (Allevamento, Igiene, Patologia delle Specie Acquatiche e Controllo dei Prodotti Derivati) sulla caratterizzazione in vitro della virulenza di questi microrganismi, sono stati esaminati 101 ceppi di Aeromonas mobili, provenienti da specie ittiche selvatiche (69), allevate (26), ed ornamentali (6). L\u2019isolamento \ue8 avvenuto da pesci setticemici (27) e apparentemente sani (74). Per ciascuno degli isolati, identificati biochimicamente a livello di complex, sono state analizzate le propriet\ue0 citotossiche su tre diverse linee cellulari (EPC, BF2, Vero) ed una serie di caratteristiche colturali (crescita a 37\ub0C, emolisi), biochimiche (test VP, decarbossilazione della lisina, fenomeno del suicidio) ed enzimatiche (lipasi, gelatinasi) indicate in letteratura come potenziali fattori di virulenza di Aeromonas spp. In particolare, si \ue8 indagato sulla correlazione fra questi fattori, la fenospecie dei ceppi (A. hydrophila, A. sobria, A. caviae), la provenienza (selvatica, allevata) ed il quadro clinico delle specie ittiche d\u2019isolamento. In generale, lo studio ha registrato alte prevalenze (54.5-99.0%) dei potenziali indicatori di patogenicit\ue0 all\u2019interno del campione testato. La produzione di lipasi (98.0%) e gelatinasi (99.0%) \ue8 stata il fattore pi\uf9 frequentemente riscontrato. Anche le caratteristiche colturali/biochimiche hanno mostrato un\u2019ampia diffusione fra i ceppi esaminati, con prevalenze variabili dal 70.3% (crescita a 37\ub0C) al 94.1% (fenomeno del suicidio: attivit\ue0 non suicida). L\u2019attivit\ue0 citotossica ha rappresentato il fattore di virulenza meno prevalente (54.5%). Dai risultati \ue8 emersa una correlazione della capacit\ue0 di crescita a 37\ub0C, attivit\ue0 emolitica e citotossica con la fenospecie dei ceppi, in particolare con i complex A. caviae (crescita a 37\ub0C) ed A. hydrophila (attivit\ue0 emolitica e citotossica). Ad esclusione dell\u2019azione decarbossilasica sulla lisina, prevalente negli isolati selvatici, nessuno dei markers di virulenza \ue8 risultato associato all\u2019origine dei ceppi. Solamente la crescita a 37\ub0C \ue8 stata correlata con presenza di patologia nei pesci, indicando una scarsa significativit\ue0 dei fattori fenotipici analizzati nella caratterizzazione in vitro in tal senso della patogenicit\ue0 di Aeromonas spp. Dei sistemi cellulari testati, le cellule Vero si sono dimostrate le pi\uf9 sensibili alle citotossine prodotte da Aeromonas spp. Si ritiene comunque che l\u2019importanza di utilizzare le cellule Vero sia legata, oltre ad una migliore visualizzazione dell\u2019effetto citopatico, anche alla possibilit\ue0 di individuare ceppi in grado di esprimere il loro potenziale patogeno nei mammiferi e nell\u2019uomo, il quale andrebbe per\uf2 confermato mediante l\u2019impiego dei tradizionali modelli animali. Ulteriori studi si rendono necessari per meglio definire il significato clinico dei fattori di virulenza di Aeromonas spp. nei pesci, da eseguirsi su un maggior numero di isolati ed associando l\u2019esame dei caratteri fenotipici dei ceppi alle analisi molecolari e, soprattutto, alle prove biologiche in vivo

5 KWE + 5 KWT PEM-FC generator from bioethanol: fuel processor and development of new reforming

A power unit constituted by a reformer, a H2 purification section and a fuel cell is being tested c/o the Dept. of Physical Chemistry and Electrochemistry of Universit\ue0 degli Studi di Milano, on the basis of a collaboration with Helbio S.A. Hydrogen and Energy Production Systems (supplier of the unit) and some sponsors (Linea Energia S.p.A., Parco Tecnologico Padano and Provincia di Lodi). The system size allows to cogenerate 5 kWe (a.c.) + 5 kWt (hot water at 65\ub0C) as peak output. Bioethanol, obtainable by different non-food competitive biomass is transformed into syngas by a prereforming and a reforming stage and the reformate is purified from CO to a concentration below 20 ppmv, suitable to feed the proton exchange membrane fuel cells (PEMFC) stack integrated in the fuel processor. This result is achieved by feeding the reformate to two water gas shift reactors, connected in series and operating at high and low temperature, respectively. CO concentration in the outcoming gas is ca. 0.7 vol% and the final CO removal to meet the specifications is accomplished by two methanation stages in series. The second methanation step acts as a guard since ca. 15 ppmv of CO are obtained even after the first reactor. The purified H2 is suitable for feeding a 5 kWe PEMFC stack, which should have an expected overall efficiency higher than 80% (including thermal output). The main goal of the present project is to check system performance under different operating conditions, to verify the effectiveness of the proposed technology and to suggest adequate improvements. In particular, the system will be tested under different load, to check for the readyness of response. Another point will be the effect of bioethanol origin, purity and concentration, so to open the way to separation processes different from distillation. Due to the demonstrative character of the project the main part of the experimentation focuses on the accumulation of a suitable amount of hours-on-stream to validate the system feasibility. A parallel investigation is active on the development of alternative nanostructured catalysts for the present application. In particular, Ni, Co and Cu-based catalysts, supported over La2O3, TiO2 and SiO2 were tested at 500, 625 and 750\ub0C. At the moment no perfect candidate has been found to operate the steam reforming at the lowest temperature, due to unsatisfactory material balances and by-products formation at 500\ub0C with most catalysts. Good H2 productivity, with 100% C balance has been achieved at higher temperature ( 65625\ub0C)

Activin A induces dendritic cell migration through the polarized release of CXC chemokine ligands 12 and 14

Activin A is a dimeric protein, member of the transforming growth factor (TGF)– family that plays a crucial role in wound repair and in fetal tolerance. Emerging evidence also proposes activin Aas a key mediator in inflammation. This study reports that activin A induces the directional migration of immature myeloid dendritic cells (iDCs) through the activation of ALK4 and ActRIIA receptor chains. Conversely, activin A was not active on plasmacytoid dendritic cells (DCs) or mature myeloid DCs. iDC migration to activin A was phosphatidylinositol 3-kinase –dependent, Bordetella pertussis toxin– and cycloheximide-sensitive, and was inhibited by M3, a viral-encoded chemokinebinding protein. In a real-time video microscopy-based migration assay, activin A induced polarization of iDCs, but not migration. These characteristics clearly differentiated the chemotactic activities of activin A from TGF- and classic chemokines. By the use of combined pharmacologic and low-density microarray analysis, it was possible to define that activin-A–induced migration depends on the selective and polarized release of 2 chemokines, namely CXC chemokine ligands 12 and 14. This study extends the proinflammatory role of activin A to DC recruitment and provides a cautionary message about the reliability of the in vitro chemotaxis assays in discriminating direct versus indirect chemotactic agonists

Activin A induces dendritic cell migration through the polarized release of CXC chemokine ligands 12 and 14

Activin A is a dimeric protein, member of the transforming growth factor (TGF)-β family that plays a crucial role in wound repair and in fetal tolerance. Emerging evidence also proposes activin A as a key mediator in inflammation. This study reports that activin A induces the directional migration of immature myeloid dendritic cells (iDCs) through the activation of ALK4 and ActRIIA receptor chains. Conversely, activin A was not active on plasmacytoid dendritic cells (DCs) or mature myeloid DCs. iDC migration to activin A was phosphatidylinositol 3-kinase γ-dependent, Bordetella pertussis toxin- and cycloheximide-sensitive, and was inhibited by M3, a viral-encoded chemokine-binding protein. In a real-time video microscopy-based migration assay, activin A induced polarization of iDCs, but not migration. These characteristics clearly differentiated the chemotactic activities of activin A from TGF-β and classic chemokines. By the use of combined pharmacologic and low-density microarray analysis, it was possible to define that activin-A-induced migration depends on the selective and polarized release of 2 chemokines, namely CXC chemokine ligands 12 and 14. This study extends the proinflammatory role of activin A to DC recruitment and provides a cautionary message about the reliability of the in vitro chemotaxis assays in discriminating direct versus indirect chemotactic agonists