Usefulness of Microscan System panels with EUCAST clinical breakpoints to evaluate the antimicrobial susceptibility of ß-lactamase producing- Gram negative isolates

The study aimed to evaluate the ability of NBC45, NBC46 and NB40 Microscan (MS) panels, updated to 2010 EUCAST breakpoints, to identify at species level and to correctly define the susceptibility to ß-lactams of 61 ß-lactamases (BLs) producing Gram-negative isolates. A collection of 73 fully identified strains was analyzed: 21 Klebsiella spp., 17 E. coli, 15 P. mirabilis, 9 A. baumannii (Ab), 7 P. aeruginosa and 4 Enterobacter spp.. 61/73 were BLs and/or carbapenemases producers: 15 were CTX-M-1/-2/-14/-15 positive, and among them two were also VIM-1 positive. Four were TEM-52/-92, 3 PER-1, 2 SHV-12/-18 and 6 CMY-16 producers, while 11 were KPC-2/-3, 9 OXA-51/-58/-23, 8 VIM-1 and 2 IMP-13 positive. One K. oxytoca K-1 iper-producer, 11 non-BL producers/ATCC control strains and a OprD2 porin lacking P. aeruginosa were also included. All isolates were identified by Api-20E and VITEK-2 System and antibiotic susceptibilities were obtained by broth microdilution method. Resistance genes were identified by PCR and sequencing. All 73 isolates were correctly identified and a complete agreement for susceptibility patterns was observed for both ATCC control strains and non-BL clinical isolates. MS failed to detect a BL/Extended-Spectrum-ß-Lactamase (ESâL) production in 5/61 cases: any ESßL alert was detected using NBC46 panel for 3/15 CTX-M positive strains and 2 VIM-1/CTX-M-15 producing K. pneumoniae isolates. Intermediate resistance to cefoxitin (MIC 16 mg/L), susceptibility to cefepime (MIC 8 mg/L for ertapenem (ETP), according to previously results. All VIM-1 producers resulted intermediate/resistant to imipenem (IP) and meropenem (MP); decreased MIC values were observed in 2/8 cases. Carbapenem MICs >8 mg/L were detected for IP-13 P. aeruginosa producers; 6/9 OXA carbapenemases- producing Ab showed IP MIC >8 mg/L and 3/6 MP MIC >8 mg/L. 3/9 Ab OXA-58/-51 producers, tested using NB40 panel, were intermediate or resistant to doripenem and meropenem. Regarding the detection of BLs overall agreement between MS and reference methods was 91.9%. Carbapenems MIC values resulted a fold lower than previously determined. Nevertheless using 2010 EUCAST breakpoints for ETP, MP and IP was possible to detect all carbapenemases- producers. MS System represents a useful tool to perform identification of BL- producing Gram negative bacteria

Safe use of human anatomical preparations in frontal and interactive teaching

In the institute of Human Anatomy of Pavia, the use of cadaver dissection is not economically feasible. In order to improve students’ preparation related to topography of the central nervous system, we decided to use formalin-fixed brains and cranial sections belonging to the collection of cadaveric specimens. These specimens, preserved in formalin, however cannot be manipulated as such by the students because formalin can cause headaches, burning sensation in the throat, difficult breathing and can trigger or aggravate asthma symptoms [1, 2]. Furthermore, formalin is known to be a human carcinogen [3]. In order to minimize toxic effects, whole brains were extensively washed in running water and then sliced according to different reference planes using a “home-made” device enabling cuts according to parallel planes. Finally, the resulting sections were inserted into transparent plastic envelopes, immerged in a solution composed by 0.5% agar and 1% sodium azide as preservative. Medical students can now use these human brain sections to test their own ability to recognize nervous system structures. This strategy optimize specimen’s choice and focalize student’s attention on peculiar, selected human samples in full compliance with current security laws

Teaching and learning human Anatomy in the University of Pavia: from models and clinical specimens to prosection on 3D models from our museum collection

Due to decline of resources and support for teaching human anatomy, in our Institute the use of cadaver dissection is not economically feasible. After a few years in which I was able to perform prosection on fixed organs belonging to the Institute collection, over the last years students learned topographical anatomy on commercial plastic models (bones, muscles, joints, trunks and brains). New perspectives recently occurred thanks to a collaboration with Prof. Auricchio’s group, which is involved in the strategic plan “3DPRINTING” (http://www. unipv.eu/site/home/area-stampa/articolo12952.html). First, we have segmented DICOM images of Computed Tomography (CT) to reconstruct 3D models of all the feet’s bones from a patient. Then, these 3D models have been post-processed to obtain suitable file for 3D printing. A 3DSYSTEMS ProJet 460 Plus, professional, full-color binder jetting printer (property of General Surgery2), has been used to create 3D models of feet’s bone by chalk powder binding. Medical students will use these models to test their own ability to recognize feet’s bones shape and to recompose them. Second, a plastic 3D anatomical model has been scanned by Artec Eva 3D Object Scanner to obtain a 3D virtual model of the physical one; this model has been modified to create a new modular model, printed with a process similar to one described above. Our Anatomy Institute is enriched by a Museum, established in the late eighteenth century by universally known anatomists (Rezia, Scarpa, Panizza, Zoja). This historical collection contains several sections (osteology, angiology, splanchnology, neurology and topographic anatomy). It is impossible to use these anatomical specimens of historical interest for prosection, but their life-size copies will constitute a cheap and effective method of learning. This strategy could not replace cadaver dissection experience but we hope that it could assist students in the comprehension of anatomical systems in a cost effective way within a systemic anatomy course. Besides, this method should optimize specimen’s choice and focalize student’s attention on peculiar, selected samples, preparing more appropriately medical students to their clinical practice

Evaluation of the effects of a dynamic culture on osteogenic differentiation of oral-periosteal cells grown on PLGA sponges

Oral-periosteum derived stem cells represent an innovative cell source for bone tissue engineering applications in terms of accessibility and self-commitment towards osteogenic lineage [1]. In this scenario, biomaterials play a pivotal role in tissue engineering in supporting stem cells growth and regeneration of tissue defects [2]. Among these biomaterials, Fisiograft®, a synthetic co-polymer composed of polylactic and polyglycholic acids produced by Ghimas (Bologna, Italy), is highly biocom- patible and completely absorbed within 4-6 months. In particular, Fisiograft® sponges are normally used in dental applications to fix completely periodontal defects without damage Schneider’s membrane. We evaluated the osteogenic potential of Fisiograft® sponges on oral-periosteal cells derived from patients undergoing dental extractions. For this purpose, we created a dynamic culture based on a rotating apparatus in which we seeded periosteal cells with Fisiograft® sponges for 7, 14 and 21 days without adding osteogenic supplement in the medium. Osteoblast differen- tiation of cells was evaluated by Alizarin Red S staining and by qRT-PCR on genes involved in bone development. Results show that Fisograft® sponges promote greater osteogenic differentiation of cells in the dynamic culture with respect to standard condition already at 14 days, as demonstrated by Alizarin Red staining. BMP-2 and Osteoprotegerin genes are highly expressed by cells grown on Fisiograft® sponges in dynamic culture at 14 days with respect to plastic culture. Taken together, these results confirm the osteogenic potential of Fisiograft® sponges in accelerating the dif- ferentiation of cells to an osteoblast phenotype (already to 14 days of culture) without any osteogenic induction. The combination of this PLGA biomaterial and oral-peri- osteal cells could represent a promising bio-complex in maxillo-facial tissue repair

Essential amino acids activate mTOR/p70 pathway in soleus muscle of chronically supplemented rats

Aim: Using an in vitro assay we assessed whether the acute exposure to essential amino acids (EAA) enriched mixture (EAAem) is able to activate mTOR signaling pathway (mTOR and p70S6K) after prolonged supplementation with the same mixture in soleus muscle of adult and elderly rats. Method: Adult (9 months of age at the end of treatment, n=10) and elderly (19 months of age at the end of treatment, n=10) male Wistar rats were chronically treated with EAA enriched mixture for 6 months (1.5 gr/kg/day in tap water). For each group 5 untreated rats served as matched control. At the end of treatment rats were grouped as follows (n=5 each): AD adults untreated controls ; EL, elderly untreated controls; AD+EAAem, AD supplemented with EAAem; EL+EAAem, EL supplemented with EAAem; AD+EAAem incubated with EAAem (1% or in Krebs solution for 30 min); EL+EAAem incubated with EAAem. Following treatment the activation level of mTOR and p70S6K was measured by Western blot. Results: The basal level of mTOR and p70S6K activation appeared to be higher in untreated AD compared with EL. Following incubation with EAAem a significant change in the level of p70S6K activation unlike mTOR was observed in EL whereas no change was observed in AD. In chronically treated AD muscles the basal level of p70S6K unlike mTOR activation appeared to decrease and the acute exposure to EAAem produced a significant reduction of mTOR activation. Contrarily to untreated rats, in chronically treated EL muscles the acute exposure to EAAem produced a significant activation of mTOR unlike p70S6K. Conclusion: Results in the young indicate a higher basal level of activation and a reduced responsiveness of the pathway to acute and chronic exposure to EAA enriched mixture. On the contrary, in the elderly, a lower basal level of activation was associated with a higher responsiveness to EAA enriched mixture. In particular although with a different timing, acute exposure activates mTOR signaling even following prolonged supplementation

Molecular characterisation of the nucleic acids recovered from aged forensic samples

The molecular composition of the genetic substrate recovered from seven aged forensic samples has been extensively investigated. A simple enzymatic test based on DNAseI incubation of the extracts showed that the UV-fluorescent material from the forensic specimens is composed of nucleic acids, with the DNA fraction representing at least 90% of the total amount. Since spectrophotometric determinations of the extracts showed unreliable results due to anomalous OD260/OD280 ratios, quantification of the nuclease-sensitive genetic material was performed by a slightly modified agarose plate method. The first quantitative data on exogenous contamination in aged forensic samples are provided by slot-blot hybridisation of the extracts to human, bacterial and fungal probes. Only limited amounts of human and contaminant DNA were detected in the samples. The molecular integrity of the primary structure of these aged DNA samples was analysed by reversed-phase HPLC/MS. The data show a good correlation between the degree of chemical damage and the ability to hybridise to molecular probes. The ability to achieve specific genetic profiles was assessed by multiplex PCR amplification of STR loci. Our data show that accurate determination of the molecular composition of the DNA recovered from forensic samples can be extremely useful for a reliable evaluation of the PCR typing results

Safe use of human anatomical preparations in frontal and interactive teaching

In the institute of Human Anatomy of Pavia, the use of cadaver dissection is not economically feasible. In order to improve students’ preparation related to topogra-phy of the central nervous system, we decided to use formalin-fixed brains and cra-nial sections belonging to the collection of cadaveric specimens. These specimens, preserved in formalin, however cannot be manipulated as such by the students be-cause formalin can cause headaches, burning sensation in the throat, difficult breathing and can trigger or aggravate asthma symptoms [1, 2]. Furthermore, for-malin is known to be a human carcinogen [3]. In order to minimize toxic effects, whole brains were extensively washed in running water and then sliced according to different reference planes using a "home-made" device enabling cuts according to parallel planes. Finally, the resulting sections were inserted into transparent plastic envelopes, immerged in a solution composed by 0.5% agar and 1% sodium azide as preservative. Medical students can now use these human brain sections to test their own ability to recognize nervous system structures. This strategy optimize speci-men’s choice and focalize student’s attention on peculiar, selected human samples in full compliance with current security laws