Typing and clustering of Yersinia pseudotuberculosis isolates by restriction fragment length polymorphism analysis using insertion sequences.

Yersinia pseudotuberculosis is an enteropathogen that has an animal reservoir and causes human infections, mostly in temperate and cold countries. Most of the methods previously used to subdivide Y. pseudotuberculosis were performed on small numbers of isolates from a specific geographical area. One aim of this study was to evaluate the typing efficiency of restriction fragment length polymorphism of insertion sequence hybridization patterns (IS-RFLP) compared to other typing methods, such as serotyping, ribotyping, and multilocus sequence typing (MLST), on the same set of 80 strains of Y. pseudotuberculosis of global origin. We found that IS100 was not adequate for IS-RFLP but that both IS285 and IS1541 efficiently subtyped Y. pseudotuberculosis. The discriminatory index (DI) of IS1541-RFLP (0.980) was superior to those of IS285-RFLP (0.939), ribotyping (0.944), MLST (0.861), and serotyping (0.857). The combination of the two IS (2IS-RFLP) further increased the DI to 0.998. Thus, IS-RFLP is a powerful tool for the molecular typing of Y. pseudotuberculosis and has the advantage of exhibiting well-resolved banding patterns that allow for a reliable comparison of strains of worldwide origin. The other aim of this study was to assess the clustering power of IS-RFLP. We found that 2IS-RFLP had a remarkable capacity to group strains with similar genotypic and phenotypic markers, thus identifying robust populations within Y. pseudotuberculosis. Our study thus demonstrates that 2IS- and even IS1541-RFLP alone might be valuable tools for the molecular typing of global isolates of Y. pseudotuberculosis and for the analysis of the population structure of this species.

Characterization of the anisotropic mechanical behaviour of colonic tissues: experimental activity and constitutive formulation.

The aim was to investigate the biomechanical behaviour of colonic tissues by a coupled experimental and numerical approach. The wall of the colon is composed of different tissue layers. Within each layer, different fibre families are distributed according to specific spatial orientations, which lead to a strongly anisotropic configuration. Accounting for the complex histology of the tissues, mechanical tests must be planned and designed to evaluate the behaviour of the colonic wall in different directions. Uni-axial tensile tests were performed on tissue specimens from 15 fresh pig colons, accounting for six different loading directions (five specimens for each loading direction). The next step of the investigation was to define an appropriate constitutive framework and develop a procedure for identification of the constitutive parameters. A specific hyperelastic formulation was developed that accounted for the multilayered conformation of the colonic wall and the fibre-reinforced configuration of the tissues. The parameters were identified by inverse analyses of the mechanical tests. The comparison of model results with experimental data, together with the evaluation of satisfaction of material thermomechanics principles, confirmed the reliability of the analysis developed. This work forms the basis for more comprehensive activities that aim to provide computational tools for the interpretation of surgical procedures that involve the gastrointestinal tract, considering the specific biomedical devices adopted.

Investigation of the mechanical behaviour of the foot skin.

BACKGROUND/PURPOSE: The aim of this work was to provide computational tools for the characterization of the actual mechanical behaviour of foot skin, accounting for results from experimental testing and histological investigation. Such results show the typical features of skin mechanics, such as anisotropic configuration, almost incompressible behaviour, material and geometrical non linearity. The anisotropic behaviour is mainly determined by the distribution of collagen fibres along specific directions, usually identified as cleavage lines. METHODS: To evaluate the biomechanical response of foot skin, a refined numerical model of the foot is developed. The overall mechanical behaviour of the skin is interpreted by a fibre-reinforced hyperelastic constitutive model and the orientation of the cleavage lines is implemented by a specific procedure. Numerical analyses that interpret typical loading conditions of the foot are performed. The influence of fibres orientation and distribution on skin mechanics is outlined also by a comparison with results using an isotropic scheme. RESULTS: A specific constitutive formulation is provided to characterize the mechanical behaviour of foot skin. The formulation is applied within a numerical model of the foot to investigate the skin functionality during typical foot movements. Numerical analyses developed accounting for the actual anisotropic configuration of the skin show lower maximum principal stress fields than results from isotropic analyses. CONCLUSION: The developed computational models provide reliable tools for the investigation of foot tissues functionality. Furthermore, the comparison between numerical results from anisotropic and isotropic models shows the optimal configuration of foot skin.

Resistance to plague of Mus spretus SEG/Pas mice requires the combined action of at least four genetic factors.

We have previously described SEG/Pas as the first mouse inbred strain able to survive subcutaneous injection of virulent Yersinia pestis, the agent of plague, and we identified Yprl1, Yprl2 and Yprl3 as three quantitative trait loci (QTLs) controlling this exceptional phenotype in females from a backcross between SEG/Pas and C57BL/6 strains. We have now developed congenic strains to further characterize the extent and effect of these genomic regions. In this study, we confirm the importance of two of these regions, both in males and females, while the third one may well be a spurious association. We show that no genomic region alone is able to increase the survival of C57BL/6 mice, but that C57BL/6 mice carrying both Yprl2 and Yprl3 exhibit intermediate resistance. Each of these two QTLs contains at least two subregions, which are required to increase survival. Finally, through the analysis of congenic strains in an F1 background, we establish the mode of inheritance of the SEG-derived resistance alleles. Altogether, this study has clarified and enhanced our understanding of the genetic architecture of resistance to plague in SEG/Pas mice.

Mus spretus SEG/Pas mice resist virulent Yersinia pestis, under multigenic control.

Laboratory mice are well known to be highly susceptible to virulent strains of Yersinia pestis in experimental models of bubonic plague. We have found that Mus spretus-derived SEG/Pas (SEG) mice are exceptionally resistant to virulent CO92 and 6/69 wild type strains. Upon subcutaneous injection of 10(2) colony-forming units (CFU), 90% of females and 68% of males survived, compared with only an 8% survival rate for both male and female C57BL/6 mice. Furthermore, half of the SEG mice survived a challenge of up to 10(7) CFU. The time required for mortality was similar between B6 and SEG, suggesting that survival is dependent on early rather than late processes. The analysis of 322 backcross mice identified three significant quantitative trait loci (QTLs) on chromosomes 3, 4 and 6, with dominant SEG protective alleles. Each QTL increased the survival rate by approximately 20%. The three QTLs function additively, thereby accounting for 67% of the difference between the parental phenotypes. Mice heterozygous for the three QTLs were just as resistant as SEG mice to Y. pestis challenge. The SEG strain therefore offers an invaluable opportunity to unravel mechanisms and underlying genetic factors of resistance against Y. pestis infection.

Biomechanical behaviour of heel pad tissue: experimental testing, constitutive formulation, and numerical modelling.

This paper deals with the constitutive formulation of heel pad tissue and presents a procedure for identifying constitutive parameters using experimental data, with the aim of developing a computational approach for investigating the actual biomechanical response. The preliminary definition of constitutive parameters was developed using a visco-hyperelastic formulation, considering experimental data from in vitro compression tests on specimens of fat pad tissue and data from in vivo tests to identify the actual trend of tissue stiffness. The discrepancy between model results and experimental data was evaluated on the basis of a specific cost function, adopting a stochastic/deterministic procedure. The parameter evaluation was upgraded by considering experimental tests performed on the fat pad tissues of a cadaveric foot using in situ indentation tests at 0.01 and 350 mm/s strain rates. The constitutive formulation was implemented in a numerical model. The comparison of data from in situ tests and numerical results led to an optimal domain of parameters based on an admissible discrepancy criterion. Numerical results evaluated for different sets of parameters inside the domain are reported and compared with experimental data for a reliability evaluation of the proposed procedure.

Computational Tools for the Reliability Assessment and the Engineering Design of Procedures and Devices in Bariatric Surgery.

Obesity is one of the main health concerns worldwide. Bariatric Surgery (BS) is the gold standard treatment for severe obesity. Nevertheless, unsatisfactory weight loss and complications can occur. The efficacy of BS is mainly defined on experiential bases; therefore, a more rational approach is required. The here reported activities aim to show the strength of experimental and computational biomechanics in evaluating stomach functionality depending on bariatric procedure. The experimental activities consisted in insufflation tests on samples of swine stomach to assess the pressure-volume behaviour both in pre- and post-surgical configurations. The investigation pertained to two main bariatric procedures: adjustable gastric banding (AGB) and laparoscopic sleeve gastrectomy (LSG). Subsequently, a computational model of the stomach was exploited to validate and to integrate results from experimental activities, as well as to broad the investigation to a wider scenario of surgical procedures and techniques. Furthermore, the computational approach allowed analysing stress and strain fields within stomach tissues because of food ingestion. Such fields elicit mechanical stimulation of gastric receptors, contributing to release satiety signals. Pressure-volume curves assessed stomach capacity and stiffness according to the surgical procedure. Both AGB and LSG proved to reduce stomach capacity and to increase stiffness, with markedly greater effect for LSG. At an internal pressure of 5 kPa, outcomes showed that in pre-surgical configuration the inflated volume was about 1000 mL, after AGB the inflated volume was slightly lower, while after LSG it fell significantly, reaching 100 mL. Computational modelling techniques showed the influence of bariatric intervention on mechanical stimulation of gastric receptors due to food ingestion. AGB markedly enhanced the mechanical stimulation within the fundus region, while LSG significantly reduced stress and strain intensities. Further computational investigations revealed the potentialities of hybrid endoscopic procedures to induce both reduction of stomach capacity and enhancement of gastric receptors mechanical stimulation. In conclusion, biomechanics proved to be useful for the investigation of BS effects. Future exploitations of the biomechanical methods may largely improve BS reliability, efficacy and penetration rate.

First isolation of Yersinia entomophaga in human urinary tract.

Yersinia entomophaga is an insect pathogen first isolated from larvae of Coleoptera in New Zealand in 2011. We report here the first isolation of Y. entomophaga from human urine. Using whole-genome sequencing, we confirmed the presence of specific chromosomal virulence genes and identified a plasmid harbouring a quinolone resistance gene.

Experimental-numerical analysis of minipig's multi-rooted teeth.

The paper pertains to the analysis of the biomechanical behaviour of the periodontal ligament (PDL) by using a combined experimental and numerical approach. Experimental analysis provides information about a two-rooted pig premolar tooth in its socket with regard to morphological configuration and deformational response. The numerical analysis developed for the present investigation adopts a specific anisotropic hyperelastic formulation, accounting for tissue structural arrangement. The parameters to be adopted for the PDL constitutive model are evaluated with reference to data deducted from experimental in vitro tests on different specimens taken from literature. According to morphometric data relieved, solid models are provided as basis for the development of numerical models that adopt the constitutive formulation proposed. A reciprocal validation of experimental and numerical data allows for the evaluation of reliability of results obtained. The work is intended as preliminary investigation to study the correlation between mechanical status of PDL and induction to cellular activity in orthodontic treatments.

Interaction phenomena between oral implants and bone tissue in single and multiple implant frames under occlusal loads and misfit conditions: A numerical approach.

An investigation is carried out on the effects induced in bone tissue surrounding oral implants placed in the premolar region of a mandible by using a numerical approach. In particular, a single implant and a multiple implant frame under loading are considered. The effects of accuracy in the coupling of the connecting bar and implants are evaluated. The mechanical response of the bone-oral implant system, depending on the different mechanical properties assumed for the peri-implant bone tissue during the evolutionary trend of osseointegration, is studied. A further task regard to the comparison of the mechanical state induced in the bone depending on the loading conditions considered. Effects of physiological occlusal loads are compared with ones given by framework defects arising from the specific manufacturing process, such as misfit between the implants and the connecting bar. The investigation offers the basis for an integrated clinical and biomechanical evaluation of the effects induced on peri-implant bone, depending on bone properties, implant system configuration, and the actions induced. Analyses performed show that stress states induced by the investigated type of misfit are comparable to those arising from the application of physiological loading conditions.

3 IS-RFLP: a powerful tool for geographical clustering of global isolates of Yersinia pestis.

Multiple copies of several classes of insertion sequences (IS) are found in the genome of Yersinia pestis, the causative agent of bubonic and pneumonic plague. We used the genetic instability generated by these IS to develop a method (designated 3IS-RFLP) based on the restriction fragment length polymorphism of the IS100, IS285 and IS1541 elements for studying Y. pestis strains of worldwide origin. We show that 31S-RFLP is a powerful tool to group Y. pestis isolates according to their geographical origin, and therefore that this method may be valuable for investigating the origin of new or re-emerging plague foci or for addressing forensic issues.

Detection, seroprevalence and antimicrobial resistance of Yersinia enterocolitica and Yersinia pseudotuberculosis in pig tonsils in Northern Italy.

Yersiniosis is the third most common reported zoonoses in Europe, with Y. enterocolitica and Y. pseudotuberculosis responsible for 98.66% and 0.94% of the confirmed human cases in 2013. From June 2013 to October 2014, 201 pigs at slaughter belonging to 67 batches were tested for Y. enterocolitica and Y. pseudotuberculosis in tonsils. Diaphragm muscle samples were tested for antibodies against Yersinia by a commercially available ELISA test. Y. enterocolitica 4/O:3 was detected in 55/201 pig tonsils (27.4%; 95% CI 23.1-37.1). The positive pigs came from 38/67 batches (56.7%) and were reared in 36/61 farms (59.0%). There was no statistical difference between farrow-to-finish and finishing farms. The mean count of Y. enterocolitica was 3.56±0.85log10CFU/g with a minimum of 2.0log10CFU/g and a maximum of 4.78log10CFU/g. Y. pseudotuberculosis was isolated from 4/201 pig tonsils (2.0%; 95% CI 0.0-4.5). Three isolates belonged to serotype O:3 and one to serotype O:1. The positive pigs belonged to 4/67 batches (6.0%) and came from finishing farms only. Y. pseudotuberculosis could be enumerated in one sample only (4.27log10CFU/g). The ELISA test demonstrated that 56.1% of the meat juice samples were positive for Yersinia antibodies. Serological positivity was found in 67.9% (36/53) of the Y. enterocolitica- and 75.0% (3/4) of the Y. pseudotuberculosis positive pigs. A significant association was found between serological results and the presence of Y. enterocolitica in tonsils (OR=1.97, p=0.044). All the Y. enterocolitica 4/O:3 isolates were susceptible to amoxicillin-clavulanic acid, gentamicin, ceftazidime, ertapenem and meropenem, 94.5% to cefotaxime, 89.1% to kanamycin and 78.2% to tetracycline. The highest resistance rates were observed for ampicillin (100%), sulphonamides (98.2%) and streptomycin (78.2%). Y. pseudotuberculosis strains were sensitive to all the antimicrobials tested, i.e. amoxicillin, amoxicillin/clavulanic acid, azithromycin, cephalothin, cefoxitin, ceftriaxone, ciprofloxacin, nalidixic acid, sulphonamide, tetracycline and ticarcillin. The study shows that Italian fattening pigs are frequently infected with human pathogenic Y. enterocolitica 4/O:3. Although the isolation rate is slightly lower than in other European countries, the serological test demonstrates that the infection is widespread among pig population. In fact, seroprevalence is similar to other EU countries. The detection of Y. pseudotuberculosis serotypes O:1 and O:3 in pig tonsils is of concern. Since tonsils may represent a contamination source for pig meat at slaughter, further studies regarding human infections by both microbial species are strongly recommended.

Anisotropic elasto-damage constitutive model for the biomechanical analysis of tendons.

The aim of this work is to investigate the instantaneous mechanical response of tendons by the use of an anisotropic elasto-damage constitutive model. This study addresses the analysis of the mechanical behaviour of healthy tendons during physiological loading and to degeneration phenomena. These are correlated with aging or traumatic events such as chronic or acute overloading during sporting activities. Histo-morphometric considerations suggest the adoption of a transversally isotropic constitutive model that describes the anisotropy of the material. The non-linearity of its overall mechanical response is taken into account by using a hyperelastic approach and also evaluates softening behaviour related to damage phenomena. The values of the parameters adopted within the analytical model are estimated both for human tendons previously subjected to cyclical loading and for specimens not subjected to cyclical loading. The results obtained by adopting this analytical model are compared with the experimental data in order to evaluate the capability of the model to describe the mechanical response of the tissue.

Bladder tissue biomechanical behavior: Experimental tests and constitutive formulation.

A procedure for the constitutive analysis of bladder tissues mechanical behavior is provided, by using a coupled experimental and computational approach. The first step pertains to the design and development of mechanical tests on specimens from porcine bladders. The bladders have been harvested, and the specimens have been subjected to uniaxial cyclic tests at different strain rates along preferential directions, considering the distribution of tissue fibrous components. Experimental results showed the anisotropic, non-linear and time-dependent stress-strain behavior, due to tissue conformation with fibers distributed along preferential directions and their interaction phenomena with ground substance. In detail, experimental data showed a greater tissue stiffness along transversal direction. Viscous behavior was assessed by strain rate dependence of stress-strain curves and hysteretic phenomena. The second step pertains the development of a specific fiber-reinforced visco-hyperelastic constitutive model, in the light of bladder tissues structural conformation and experimental results. Constitutive parameters have been identified by minimizing the discrepancy between model and experimental data. The agreement between experimental and model results represent a term for evaluating the reliability of the constitutive models by means of the proposed operational procedure.

A highly efficient electroporation system for transformation of Yersinia.

The various pathogenic Yersinia species are not readily and efficiently transformed by classical methods. For this reason, the electroporation technique was applied for genetic transformation of these species. Using optimal conditions, we were able to transform the six Yersinia strains studied with the two most widely used groups of plasmids: pSU2718 (a pACYC184 derivative) and pK19 (a pUC19 derivative). Only Yersinia enterocolitica (Y. e.) serotype 0:8 gave poor results (less than 5 x 10(2) transformants/microgram) DNA). Electrical transformation of the other species resulted in high efficiencies, up to 10(5) transformants/microgram DNA for Y. e. serotypes 0:3 and 0:9, 10(6) for Y. pseudotuberculosis and 10(7) for Y. pestis. The results varied for each strain with the type of plasmid used. Neither the introduced foreign plasmid nor the resident 72-kb virulence plasmid underwent detectable deletions. Transformation was most efficient with supercoiled DNA, decreasing by one and four orders of magnitude for relaxed circular and linearized plasmids, respectively. The ability to easily and efficiently transfer plasmid DNA via electroporation will greatly facilitate the application of recombinant DNA technology for direct cloning and analysis of significant genes into Yersinia.

The Yersinia high-pathogenicity island: an iron-uptake island.

Highly pathogenic Yersinia carry a pathogenicity island termed high-pathogenicity island (HPI). The Yersinia HPI comprises genes involved in the synthesis of the siderophore yersiniabactin and can thus be regarded as an iron-uptake island. A unique characteristic of the HPI is its wide distribution among different enterobacteria such as Escherichia coli, Klebsiella, Citrobacter and Salmonella. Other types of iron-uptake systems are also carried by different pathogenicity islands in enterobacteria.

Infrequent detection of Yersinia enterocolitica in childhood diarrhea in Bangladesh.

In a search for Yersinia infection in Bangladesh, one isolate of Y. enterocolitica serotype 0:3 was obtained from stools of 1,450 children with fever and diarrhea and one of Y. enterocolitica serotype 0:8 was recovered from intestinal contents of 80 fatal diarrheal cases during postmortem examination. These results suggest that Yersinia infection is an infrequent cause of tropical diarrhea.

Use of pulsed field gel electrophoresis to size the chromosome of the bacterial fish pathogen Yersinia ruckeri.

Field inversion gel electrophoresis (FIGE) and contour-clamped homogeneous field (CHEF) electrophoresis were used to analyse the chromosome of Yersinia ruckeri. The 8 base-pair recognition endonucleases, NotI and SfiI, generated less than 47 DNA fragments whose size and distribution were appropriate for pulsed field separation. Each isolate displayed a characteristic restriction pattern, with about 20% of bands in common. Depending on the strain used, the estimated genome size for this bacterial fish pathogen ranged from 4460 to 4770 kilobase pairs.

The Yersinia high-pathogenicity island is present in different members of the family Enterobacteriaceae.

A pathogenicity island termed high-pathogenicity island (HPI) is present in pathogenic Yersinia. This 35 to 45 kb island carries genes involved in synthesis, regulation and transport of the siderophore yersiniabactin. Recently, the HPI was also detected in various strains of Escherichia coli. In this study, the distribution of the HPI in the family Enterobacteriaceae was investigated. Among the 67 isolates pertaining to 18 genera and 52 species tested, nine (13.4%) harbored the island. These isolates were three E. coli, one Citrobacter diversus and five Klebsiella of various species (Klebsiella pneumoniae, Klebsiella rhinoscleromatis, Klebsiella ozaenae, Klebsiella planticola, and Klebsiella oxytoca). As in Yersinia sp., all nine isolates synthesized the HPI-encoded iron-repressible proteins HMWP1 and HMWP2. In the K. oxytoca strain, the right-end portion of the HPI was deleted, whereas the entire core region of the island was present in the eight other enterobacteria strains analyzed. In most of these isolates, the HPI was bordered by an asn tRNA locus, as in Yersinia sp. This report thus demonstrates the spread of the HPI among various members of the family Enterobacteriaceae.

Local hopping of IS3 elements into the A+T-rich part of the high-pathogenicity island in Yersinia enterocolitica 1B, O:8.

The high-pathogenicity island (Yen HPI) of Yersinia enterocolitica biogroup (BG) 1B strains is associated with mouse virulence. Three repeated sequences are clustered on the A+T-rich part of the Yen HPI downstream of the fyuA yersiniabactin receptor gene in Y. enterocolitica O:8 strains WA-314 and 8081. In addition to IS1328 and IS1400, the RS3 repeated sequence consists of a novel insertion sequence, IS1329, inserted into the remnants of IS1222. This partial IS retains both 44-bp inverted terminal repeats (ITRs) of IS1222 but has suffered deletions of different sizes in strains WA-314 and 8081. IS1329 is 1243-bp long, carries 25-bp imperfect ITRs and two consecutive orfs capable to encode 110-amino acid (aa) and 249-aa proteins, respectively. IS1329 is present only in BG 1B Y. enterocolitica strains. Similarly to IS1400, IS1329 and IS1222 belong to the IS3 group of mobile elements and seem to have preference for the 'local hopping' into the A+T-rich part of the Yen HPI. These insertion sequences may be responsible for the imprecise deletions of the Yen HPI in strain WA-314.