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Genomic analyses of Klebsiella isolates sampled from multiple human, animal and environmental sources in Northern Italy explore Klebsiella population diversity and show that transmission of multidrug-resistant clones between clinical and environmental settings is scarce.The Klebsiella group, found in humans, livestock, plants, soil, water and wild animals, is genetically and ecologically diverse. Many species are opportunistic pathogens and can harbour diverse classes of antimicrobial resistance genes. Healthcare-associated Klebsiella pneumoniae clones that are non-susceptible to carbapenems can spread rapidly, representing a high public health burden. Here we report an analysis of 3,482 genome sequences representing 15 Klebsiella species sampled over a 17-month period from a wide range of clinical, community, animal and environmental settings in and around the Italian city of Pavia. Northern Italy is a hotspot for hospital-acquired carbapenem non-susceptible Klebsiella and thus a pertinent setting to examine the overlap between isolates in clinical and non-clinical settings. We found no genotypic or phenotypic evidence for non-susceptibility to carbapenems outside the clinical environment. Although we noted occasional transmission between clinical and non-clinical settings, our data point to a limited role of animal and environmental reservoirs in the human acquisition of Klebsiella spp. We also provide a detailed genus-wide view of genomic diversity and population structure, including the identification of new groups.

The COVID-19 pandemic, especially during the first wave, posed a great challenge to the hospital management and generally promoted nosocomial pathogen dissemination. MDR A. baumannii can easily spread and persist for a long time on surfaces, causing outbreaks in health care settings.

Consumption of raw food, especially smoked fish, meat, soft cheeses, and vegetables, contaminated with Listeria monocytogenes can cause listeriosis, which can be invasive in pregnant women, elderly...

Acinetobacter baumanniiis a nosocomial pathogen that poses a serious threat due to the rise of incidence of multidrug resistant (MDR) strains. During the COVID-19 pandemic, MDRA.baumanniiclones have caused several outbreaks worldwide. Here we describe a detailed investigation of an MDRA. baumanniioutbreak that occurred at Fondazione IRCCS Policlinico San Matteo (Pavia, Italy). A total of 96A. baumanniistrains, isolated between January and July 2020 from 41 inpatients (both SARS-CoV-2 positive and negative) in different wards, were characterized by phenotypic and genomic analyses combining Illumina and Nanopore sequencing. Antibiotic susceptibility testing revealed that all isolates were resistant to carbapenems and the sequence analysis attributed this to the carbapenemase geneblaOXA-23. Screening of virulence factors unveiled that all strains carried determinants for biofilm formation, while plasmid analysis revealed the presence of two plasmids, one of which was a ⍰100kbp long and encoded a phage sequence.A core genome-based phylogeny was inferred to integrate outbreak strain genomes with background genomes from public databases and from the local surveillance program. All strains belonged to the globally disseminated ST2 clone and were mainly divided into two clades. Isolates from the outbreak clustered with surveillance isolates from 2019, suggesting that the outbreak was caused by two strains that were already circulating in the hospital before the start of the pandemic. The intensive spread ofA. baumanniiin the hospital was enhanced by the extreme emergency situation of the first COVID-19 pandemic wave that resulted in minor attention to infection prevention and control practices.ImportanceThe COVID-19 pandemic, especially during the first wave, posed a great challenge to the hospital management and generally promoted nosocomial pathogen dissemination. Multidrug resistant (MDR)A. baumanniican easily spread and persist for a long time on surfaces, causing outbreaks in healthcare settings. Infection prevention and control practices, epidemiological surveillance and microbiological screening are fundamental in order to control such outbreaks.Here, we sequenced the genomes of 96 isolates from an outbreak of MDRA. baumanniistrains using both short- and long-read technology in order to reconstruct the outbreak events in fine detail. The sequence data demonstrated that two endemic clones of MDRA. baumanniiwere the source of this large hospital outbreak during the first COVID-19 pandemic wave, confirming the effect of COVID-19 emergency disrupting the protection provided by the use of the standard prevention procedures.

TheKlebsiellagroup is highly diverse both genetically and ecologically, being commonly recovered from humans, livestock, plants, soil, water, and wild animals. Many species are opportunistic pathogens, and can harbour diverse classes of antimicrobial resistance (AMR) genes.K. pneumoniaeis responsible for a high public-health burden, due in part to the rapid spread of health-care associated clones that are non-susceptible to carbapenems.Klebsiellathus represents a highly pertinent taxon for assessing the risk to public health posed by animal and environmental reservoirs. Here we report an analysis of 6548 samples and 3,482 genome sequences representing 15Klebsiellaspecies sampled over a 15-month period from a wide range of clinical, community, animal and environmental settings in and around the city of Pavia, in the northern Italian region of Lombardy. Despite carbapenem-resistant clones circulating at a high frequency in the hospitals, we find no genotypic or phenotypic evidence for non-susceptibility to carbapenems outside of the clinical environment. The non-random distribution of species and strains across sources point to ecological barriers that are likely to limit AMR transmission. Although we find evidence for occasional transmission between settings, hierarchical modelling and intervention analysis suggests that direct transmission from the multiple non-human (animal and environmental) sources included in our sample accounts for less than 1% of hospital disease, with the vast majority of clinical cases originating from other humans.

During a citywide microbiological screening project in Pavia (Italy) a bacterial strain isolated from the surface of an Automated Teller Machine was classified as a Klebsiella sp. by MALDI-TOF spectrometry, and shown to be susceptible to the most antimicrobial classes by phenotypic testing. After Illumina genome sequencing and subsequent assembly, a high-quality draft genome was obtained (size = 5,051,593 bp, N50 = 615,571 bp, largest contig = 1,328,029 bp, N_contig = 17, GC content = 51.58%, coverage = 141.42), absence of antimicrobial resistance genes was confirmed, but the strain resulted to be highly divergent from all Klebsiella, and more related to other Enterobacteriaceae. The higher values of 16S rRNA identity were with members of the genera Citrobacter, Salmonella, and “Superficieibacter.” An ortholog-based phylogenomic analysis indicated a sister group relationship with “Superficieibacter electus,” in a distinct clade from other members of the Enterobacteriaceae family. In order to evaluate whether the novel genome represents a new species of “Superficiebacter,” average nucleotide identity (ANI) and Hadamard analysis were performed on a dataset of 78 Enterobacteriaceae. The novel genome showed an ANI of 87.51% with S. electus, which compared on identity values between other members of the family, clearly indicates that the genome represents a new species within the genus “Superficieibacter.” We propose for the new species the name “Superficieibacter maynardsmithii.”

Consumption of raw food, especially smoked fish, meat, soft cheeses, and vegetables, contaminated with

Clostridioides difficile infection (CDI) is the leading cause of antibiotic-associated diarrhea, especially in hospitalized elderly patients, representing a global public health concern. Clinical presentations vary from mild diarrhea to severe pseudomembranous colitis that may progress to toxic megacolon or intestinal perforation. Antibiotic therapy is recognized as a risk factor and exacerbates dysbiosis of the intestinal microbiota, whose role in CDI is increasingly acknowledged. A clinically challenging complication is the development of recurrent disease (rCDI). In this study, using amplicon metagenomics, we compared the fecal microbiota of CDI and rCDI patients (sampled at initial and recurrent episode) and of non-infected controls. We also investigated whether CDI severity relates to specific microbiota compositions. rCDI patients showed a significantly decreased bacterial diversity as compared to controls (p &lt, 0.01). The taxonomic composition presented significant shifts: both CDI and rCDI patients displayed significantly increased frequencies of Firmicutes, Peptostreptococcaceae, Clostridium XI, Clostridium XVIII, and Enterococcaceae. Porphyromonadaceae and, within it, Parabacteroides displayed opposite behaviors in CDI and rCDI, appearing discriminant between the two. Finally, the second episode of rCDI was characterized by significant shifts of unclassified Clostridiales, Escherichia/Shigella and Veillonella. No peculiar taxa composition correlated with the severity of infection, likely reflecting the role of host-related factors in determining severity.

Fungi have a crucial role in the correct maturation of salami, but special attention should be addressed to the production of the nephrotoxic, immunotoxic, and carcinogenic mycotoxin ochratoxin A (OTA). In a monitoring study conducted in Northern Italy, OTA was detected by liquid chromatography coupled with mass spectrometry in 13 out 133 samples of traditional salami (9.8% of the total count). Mycological analysis of these samples yielded 247 fungal isolates which were identified to species level. The most frequent species were Penicillium nalgiovense, P. solitum, and P. chrysogenum. P. nordicum, an OTA-producing species commonly found in proteinaceous food, was not found in these samples. Three isolates were found to be Aspergillus westerdijkiae, an OTA-producing species. In order to check the results of the microbiological identification, 19 different strains of Aspergillus and 94 of Penicillium were tested for the presence of a sequence common to OTA-producing fungi by real-time PCR. None of the studied isolates, including the three A. westerdijkiae, possessed the otanpsPN target which is common to OTA-producing strains. Two out of three isolates of the A. westerdijkiae were also PCR-negative for the otanpsPN gene and did not produce OTA in culture. Conversely, this target sequence was amplified from the DNA purified from 14 salami casings including three casings harboring A. westerdijkiae. The amplification of sequences specific for OTA-producing strains performed on total genomic DNA extracted directly from salami casings provided a more suitable approach than PCR analysis of isolates from salami for the OTA-related otanpsPN gene to evaluate the risk of OTA contamination.

The recurrence of multi-drug resistant (MDR) pathogens to the latest antibiotics and the limited development of new antibacterial agents have reduced the options for the treatment of severe infections. The reintroduction of old antibiotics, such as colistin, represents an effective strategy, since the latest antibiotics are over-consumed and ineffective against MDR pathogens. In 2015, Liu (Lancet Infect Dis 16:161-168, 2016) reported Escherichia coli (E. coli) isolates carrying plasmid-mediated colistin resistance gene mcr-1. The first of mcr-1 positive colistin-resistant (col-R) E. coli from a human blood culture was observed in 2012 in Latin America, while in Italy was reported for the first time by our center in 2016. The present study aimed to describe the prevalence of mcr-1 positive col-R strains in E. coli-related bloodstream infection among patients hospitalized in Fondazione IRCCS Policlinico San Matteo in Pavia, Italy, from 2012 to 2018, including the three cases already published.All col-R E. coli strains isolated from blood cultures collected during the study period were analyzed. The minimal inhibitory concentration of colistin was determined using broth microdilution and detection of mcr-1 and mcr-2 genes was performed by PCR. The sequence type of E. coli mcr-1 positive was determined according to Multilocus sequence typing.Out of 1557 samples, 14 strains (0.90%) were col-R. and positive for the presence of the mcr-1 gene, with no mcr-2 detected. The most common ST was ST10 (n = 3), followed by ST410 (n = 2). The remaining strains exhibited different MLST profiles, indicating that they were genetically unrelated.Proper reporting of the presence of mcr-1 genes is an essential component to anticipate the spread of colistin resistance. This public health issue is particularly alarming in Italy due to the consistent circulation of MDR bacteria.

This paper presents a new approach for the recording of the total quantity of energy exchanged during the micro electrical discharge machining (EDM) process. In particular, this approach allows for the estimation of the percentage of energy absorbed by the two electrodes (tool and workpiece) using a combination of theoretical models and experimental results, thanks to an advanced discharges measuring approach. An experimental campaign was executed on a Sarix SX200 micro-EDM machine with a tungsten carbide tool electrode. In particular, the process was analyzed for two different electrode shapes, a triangular and a rectangular based parallelepipeds, and two sets of machining parameters. After the execution of the experimental campaign it estimate the total amount of energy that occurred during the entire die sinking process. The energy effectively lost into the tool, into the workpiece and into the dielectric was estimated from the measurement of the volume of material actually removed both from the tool and from the workpiece. The preliminary results appears to fit those presented in the literature and the validity of several theoretical crater models was then assessed. The validation of the presented acquisition method could enable the use of information related to energy repartition in the simulation of the micro-EDM process. The reported research was partly funded by the Engineering and Physical Sciences Research Council (EPSRC) under the grant EP/J004901/1.

RÉSUMÉCette étude a exploré les expériences des aidants devant s’occuper de la transition d’une personne âgée vers une institution de soins de longue durée (SLD). Une approche de description qualitative a été utilisée pour l’analyse d’entretiens semi-structurés effectués auprès de 13 aidants de personnes âgées hébergées en centres de soins de longue durée dans le sud de l’Ontario. Nos résultats illustrent que les aidants ressentent des soucis et un lourd fardeau de manière chronique lorsqu’il s’agit de prendre une décision sur l’hébergement ou de faire une demande pour un centre de soins de longue durée. Le sentiment de ne pas avoir le contrôle était un thème récurrent dans les entretiens, particulièrement dans les cas où les aidants requéraient une place en SLD. Les participants se sont sentis bousculés, sous pression et punis à travers le système de santé, mais soulagés et privilégiés après l’acceptation de l’offre de la place en CHSLD. Cette expérience tumultueuse a encouragé les aidants à anticiper les prochaines transitions. Il est recommandé que les aidants aient accès à une préparation et à un soutien ciblé pour mieux gérer les transitions. Une amélioration de la communication entre les professionnels de la santé est essentielle. Les amendes imposées lors du refus d’une place en SLD devraient être réexaminées, considérant l’impact négatif de celles-ci sur les aidants.

Salmonella enterica serovar Typhimurium (STm) and its monophasic variant 4,[5],12:i:- (VMSTm) have been responsible for an increased number of foodborne infections in humans in Europe in recent years. The aim of this study was to investigate the origin of three foodborne salmonellosis outbreaks that occurred in Pavia Province (Lombardy region, northern Italy) in 2010. Phenotypic and genetic characteristics of the STm and VMSTm isolates from patients and from food that were recovered in the framework of the three outbreaks were evaluated through serotyping, phage typing, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multiple-locus variable-number tandem repeat analysis (MLVA). Salami from three artisan producers, which had all purchased meat from the same slaughterhouse, was the food source of infection in outbreak I. STm isolates were recovered from salami and patients with symptoms of gastroenteritis. These isolates had the same PFGE type and the same rare MLVA profile (3-18-9-NA-211). The same molecular profiles were found in an STm isolate from a salami, which likely was the source of another family outbreak (II). A VMSTm strain with common phenotypic and molecular profiles was isolated from three hospitalized patients and identified as the cause of another putative outbreak (III). During the following 3 years (2011 through 2013), 360 salami produced in Pavia Province were monitored for the presence of S. enterica . In 2011, no STm and VMSTm isolates were recovered from 159 salami tested. During 2012 and 2013, 13.9% of 201 tested salami harbored S. enterica , and half of the isolates were VMSTm, mainly in salami from those artisan producers involved in the previous outbreaks. These isolates were genetically variable, especially in terms of MLVA profiles. The data collected suggest that from 2012, VMSTm has replaced STm in the environments of the salami producers monitored in this study, and these data confirm the dominance of this emergent serovar along the pork supply chain.

The recent miniaturization trend in manufacturing, has enhanced the production of new and highly sophisticated systems in various industrial fields. In recent years, machining of the so called difficult to cut materials has become an important issue in several sectors. Micro Electrical Discharge Machining (micro-EDM) thanks to its contactless nature, is one of the most important technologies for the machining of this type of materials and it can be considered as one of the most promising manufacturing technologies for the fabrication of micro components. One of the most relevant applications of micro-EDM is micro-drilling. Micro holes in fact, are widely used for example in micro-electromechanical systems (MEMS), serving as channels or nozzles to connect two micro-features, and in micro-mechanical components. The present study is about micro drilling of metal plates by means of micro-EDM technology. In particular, the aim of this work is to investigate the effects of the downsizing of the micro holes diameter on the drilling performances. The influence of the reduction of the diameters in terms of both process performances (e.g., tool wear, taper rate, diametrical overcut) and general quality of the holes was investigated. Steel plates having thickness equal to 0.8 mm were taken into account. The drilling process was carried out using a micro-EDM machine Sarix SX 200 with carbide electrodes having diameter equal to 300, 200, 100 and 50 μm. Since the standard electrodes adopted in this study had a diameter equal to 300 μm, a wire EDM unit was used to obtain the other electrodes. The relationship between the process parameters considered the most significant and the final output, was studied. Furthermore, the geometrical and dimensional properties of the micro-holes were analyzed using both optical and scanning electron microscopes. In particular, it is demonstrated that the diameter size has a significant influence on the final value of the diametrical overcut while peak current and frequency parameters have a negligible effect.

In micro-EDM machining the most commonly used dielectrics are mineral oil and deionized water, that are characterised by different dielectric permittivity. As a matter of fact, the use of these dielectrics, especially mineral oil, requires proper air filtering and a correct dismissal of the used dielectric. The spread of an environment friendly approach for a more conscious manufacturing enhanced the study of micro-EDM technology in different dielectric conditions. In particular, it was underlined the possibility to conduct EDM and micro-EDM tests with different dielectrics, in other words the so called "dry’’conditions. Aim of this paper is to investigate the influence of the dielectric fluid on the process performance and on the geometrical characteristics of the micro holes. A comparison between the conventional mineral oil, compressed air and oxygen was carried out. The experimental campaign was executed with a Sarix SX 200 machine for the machining of magnesium sheets having thickness of 1 mm.

Micro injection moulding is one of the most used thermoplastic technology for the manufacturing of miniaturized products, due to the high productivity of the process. Thermoplastic micro manufacturing has reached nowadays an incredible spread: polymeric components are used in several fields, such as biomedical, IT, telecommunication, automotive and aerospace. In recent years, an innovation in polymer area was the addition of fillers on the pure polymer, in order to improve the physical properties and/or to reduce the cost of the composite. The aim of the present work is to carry out a comparison between the mechanical properties of POM base material and POM filled with carbon nanotubes micro injected using different process parameters - melting temperature, injection speed and holding pressure. Uniaxial mechanical tests were performed to obtain the main mechanical properties: yield stress, deformation at break and stress at break. The results, analyzed with Analysis of Variance statistical approach show that the mechanical response of the micro¬ specimens is mainly affected by the CNT presence, whereas the other factors seems to play only a secondary role. The presence of CNT induces a remarkable increase in the mechanical resistance, but drastically reduces the material ductility.

International audience; The present paper introduces the use of voxels embedded in an octree structure in order to numerically simulate manufacturing processes. In particular, micro electrical discharge machining (μEDM) is used here as a case study. The involved elements (tool and workpiece) are modelized in a volumetric manner using voxels and the process is simulated on a step-by step basis. Comparisons using the Hausdorff metric with experimental results are included and discussed.

In recent years, the need for products containing micro-features has shown a pronounced and steady growth in several fields of application. For the development of micro-holed devices, one of the most important technologies is micro-EDM (Electro Discharge Machining). Micro-EDM can be considered as an ideal process to obtain burr-free micron-size features with high aspect ratios. In particular, micro-EDM is a non-contact material removal process in which rapid electric spark discharges remove the material composing the workpiece by means of melting and vaporizing phenomena. The present work deals with the fabrication of micro holes using micro-EDM technology. The investigation focuses on the influence of different electrodes and workpiece materials on the process performance, expressed in terms of tool wear ratio. In particular, the influence of four different workpiece materials (stainless steel, titanium, magnesium and brass), three electrode materials (copper, brass and tungsten carbide) and two different electrode shapes (cylindrical and tubular) was investigated. Moreover, an analysis of the geometrical characteristics of the micro holes in terms of conicity and diametrical overcut was carried out. An influence of electrode geometries, electrode material and workpiece material on the final output was found.

Klebsiella oxytocacauses opportunistic human infections and post-antibiotic haemorrhagic diarrhoea. ThisEnterobacteriaceaespecies is genetically heterogeneous and is currently subdivided into seven phylogroups (Ko1 to Ko4, Ko6 to Ko8). Here we investigated the taxonomic status of phylogroups Ko3 and Ko4. Genomic sequence-based phylogenetic analyses demonstrate that Ko3 and Ko4 formed well-defined sequence clusters related to, but distinct from,Klebsiella michiganensis(Ko1),Klebsiella oxytoca(Ko2),K. huaxiensis(Ko8) andK. grimontii(Ko6). The average nucleotide identity of Ko3 and Ko4 were 90.7% withK. huaxiensisand 95.5% withK. grimontii, respectively. In addition, three strains ofK. huaxiensis, a species so far described based on a single strain from a urinary tract infection patient in China, were isolated from cattle and human faeces. Biochemical and MALDI-ToF mass spectrometry analysis allowed differentiating Ko3, Ko4 and Ko8 from the otherK. oxytocaspecies. Based on these results, we propose the namesKlebsiella spallanzaniifor the Ko3 phylogroup, with SPARK_775_C1T(CIP 111695T, DSM 109531T) as type strain, andKlebsiella pasteuriifor Ko4, with SPARK_836_C1T(CIP 111696T, DSM 109530T) as type strain. Strains ofK. spallanzaniiwere isolated from human urine, cow faeces and farm surfaces, while strains ofK. pasteuriiwere found in faecal carriage from humans, cows and turtles.Accession numbersThe nucleotide sequences generated in this study were deposited in ENA and are available through the INSDC databases under accession numbersMN091365(SB6411T= SPARK775C1T), MN091366 (SB6412T= SPARK836C1T) and MN104661 to MN104677 (16S rRNA), MN076606 to MN076643 (gyrAandrpoB), and MN030558 to MN030567 (blaOXY). Complete genomic sequences were submitted to European Nucleotide Archive under the BioProject number PRJEB15325.AbbreviationsANI, average nucleotide identity; HCCA, a-cyano-4-hydroxycinnamic acid; isDDH,in silicoDNA-DNA hybridization; SCAI, Simmons citrate agar with inositol; MALDI57 ToF MS: Matrix-assisted laser desorption/ionization time of flight mass spectrometry