One-year surveillance for hypervirulent Klebsiella pneumoniae detected carbapenem-resistant superbugs.

Hypervirulent Klebsiella pneumoniae (hvKp) can cause infections in clinically healthy people, such as young and immunocompetent patients. Genes involved in the capsule synthesis or those encoding the siderophores have been adopted as predictors of hvKp. Certain sequence types, such as ST23 and ST86, have been associated with hvKp strains, too. The aim of this study was to investigate the presence of hvKp among 354 K. pneumoniae strains isolated from clinical samples of patients admitted to an Italian 900-bed hospital between 21 May 2021 and April 2022. All the isolates were screened by PCR for the amplification of virulence loci. Whole genome sequencing was performed in strains tested positive for at least one target gene. Thirteen out of 354 (3.7%) were hvKp. Five were wild type and belonged to the hypervirulent clones ST23, ST86, ST5, and ST375 and to the new clone ST6310. Six strains carried the blaKPC gene: three belonged to ST101, two to ST512, and one to ST395. Two isolates were ST147 and carried the blaNDM gene. Although hvKp isolation is not frequent, their presence should be systematically investigated to avoid the spreading of both virulent strains and strains with combined increase in virulence and resistance to antibiotics. PCR-based protocols are essential for surveillance of these strains, which do not always show a recognizable phenotype. Moreover, hvKp strains were isolated also from patients without history of recent foreign travels, indicating an increased spreading of these strains as well as an underestimated of their circulation so far.IMPORTANCEKlebsiella pneumoniae is a healthcare-associated pathogen frequently resistant to antibiotics. Hypervirulent strains of pneumoniae (hvKp) can spread from the primary site of infection to multiple sites causing life-threatening infections also in young otherwise healthy individuals. This study described the isolation of 13 isolates of K. pneumoniae with increased virulence in a large tertiary hospital over a 1-year period. Among them, eight strains were multidrug resistant and hypervirulent. Although these hypervirulent strains are still rare in Italy, their presence is particularly concerning since they can cause difficult-to-treat life-threatening infections. Moreover, not all the hypervirulent isolates were positive by the string test, so hvKp isolates were not always phenotypically detectable. Molecular biology techniques such as PCR amplification and next generation sequencing are therefore necessary for the detection of hvKp isolates, and surveillance programs exploiting molecular techniques are highly desirable.

Combining Genome Surveillance and Metadata To Characterize the Diversity of Staphylococcus aureus Circulating in an Italian Hospital over a 9-Year Period.

Staphylococcus aureus is an opportunistic pathogen and a leading cause of morbidity and mortality worldwide. Genomic-based surveillance has greatly improved our ability to track the emergence and spread of high-risk clones, but the full potential of genomic data is only reached when used in conjunction with detailed metadata. Here, we demonstrate the utility of an integrated approach by leveraging a curated collection of clinical and epidemiological metadata of S. aureus in the San Matteo Hospital (Italy) through a semisupervised clustering strategy. We sequenced 226 sepsis S. aureus samples, recovered over a period of 9 years. By using existing antibiotic profiling data, we selected strains that capture the full diversity of the population. Genome analysis revealed 49 sequence types, 16 of which are novel. Comparative genomic analyses of hospital- and community-acquired infection ruled out the existence of genomic features differentiating them, while evolutionary analyses of genes and traits of interest highlighted different dynamics of acquisition and loss between antibiotic resistance and virulence genes. Finally, highly resistant clones belonging to clonal complexes (CC) 8 and 22 were found to be responsible for abundant infections and deaths, while the highly virulent CC30 was responsible for rare but deadly episodes of infections. IMPORTANCE Genome sequencing is an important tool in clinical microbiology, as it allows in-depth characterization of isolates of interest and can propel genome-based surveillance studies. Such studies can benefit from ad hoc methods of sample selection to capture the genomic diversity present in a data set. Here, we present an approach based on clustering of antibiotic resistance profiles that allows optimal sample selection for bacterial genomic surveillance. We apply the method to a 9-year collection of Staphylococcus aureus from a large hospital in northern Italy. Our method allows us to sequence the genomes of a large variety of strains of this important pathogen, which we then leverage to characterize the epidemiology in the hospital and to perform evolutionary analyses on genes and traits of interest. These analyses highlight different dynamics of acquisition and loss between antibiotic resistance and virulence genes.

Livestock-associated methicillin-resistant Staphylococcus aureus in inpatients: a snapshot from an Italian hospital.

OBJECTIVES: This study aimed to characterize livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) strains isolated from patients admitted to Policlinico San Matteo in Pavia, located in an Italian region with high livestock density. METHODS: The starting dataset was composed by 353 MRSA strains isolated from blood cultures between 2011 and 2019 and in 954 MRSA isolated from nasal swabs, wound swabs, skin swabs, ulcer swabs, conjunctival swabs, urine and respiratory samples collected between 2018 and 2019. LA-MRSA was identified based on being MRSA resistant to tetracycline and negative for the PCR amplification of scn locus. Whole genome sequencing of the selected strains was performed, and virulence and resistance genes searched. RESULTS: Five out of 353 MRSA isolates from blood cultures (1.4%) and nine out of 904 MRSA isolates obtained from other materials (1%) were resistant to tetracycline and negative for the scn locus. The 14 strains were also negative for the lukS-pv, tsst, eta and etb loci. Nine of the 14 strains belonged to ST398, the most common ST of LA-MRSA in Europe. ST398 isolates belonged to four spa-types, of which the prevalent was t899. Eight genomes had the cassette SCCmec type V, five genomes had SCCmec type IV and one genome lacked SCCmec, mecA and mecC. CONCLUSION: The frequency of LA-MRSA in the patients of this study (1.4% in blood cultures, 1% in other samples) is low but relatively constant over time prevalence and comparable to that found in the few studies performed on patients to date.

miRNA-Signature of Irradiated Ptch1+/- Mouse Lens is Dependent on Genetic Background.

One harmful long-term effect of ionizing radiation is cataract development. Recent studies have been focused on elucidating the mechanistic pathways involved in this pathogenesis. Since accumulating evidence has established a role of microRNAs in ocular diseases, including cataract, the goal of this work was to determine the microRNA signature of the mouse lens, at short time periods postirradiation, to understand the mechanisms related to radio-induced cataractogenesis. To evaluate the differences in the microRNA profiles, 10-week-old Patched1 heterozygous (Ptch1+/-) mice, bred onto two different genetic backgrounds (CD1 and C57Bl/6J), received whole-body 2 Gy γ-ray irradiation, and 24 h later lenses were collected. Next-generation sequencing and bioinformatics analysis revealed that genetic background markedly influenced the list of the deregulated microRNAs and the mainly predicted perturbed biological functions of 2 Gy irradiated Ptch1+/- mouse lenses. We identified a subset of microRNAs with a contra-regulated expression between strains, with a key role in regulating Toll-like receptor (TLR)-signaling pathways. Furthermore, a detailed analysis of miRNome data showed a completely different DNA damage response in mouse lenses 24 h postirradiation, mainly mediated by a marked upregulation of p53 signaling in Ptch1+/-/C57Bl/6J lenses that was not detected on a CD1 background. We propose a strict interplay between p53 and TLR signaling in Ptch1+/-/C57Bl/6J lenses shortly after irradiation that could explain both the resistance of this strain to developing lens opacities and the susceptibility of CD1 background to radiation-induced cataractogenesis through activation of epithelial-mesenchymal transition.

Microdosimetric Realistic Model of a Cell with Endoplasmic Reticulum.

When investigating the biophysical effects induced by the interaction between electromagnetic fields and biological cells, it is crucial to estimate the electromagnetic field intensity at the microscopic scale (microdosimetry). This information allows to find a connection between the external applied field and the observed biological event required to establish related biomedical applications. Here, authors present a microdosimetric study based on a 2D realistic model of a cell and its endoplasmic reticulum. The microdosimetric analysis of the cell and endoplasmic reticulum was quantified in terms of electric field and transmembrane potential induced by an externally applied high amplitude 10-ns pulsed electric field. In addition, electroporated local membrane sites and pore densities were also evaluated. This study opens the way to numerically assist experimental applications of nanosecond pulsed electric fields for controlled bio-manipulation of cells and subcellular organelles.

Perspective of Family Members of Transitions to Alternative Levels of Care in Anglo-Saxon Countries.

This scoping review explores circumstances surrounding the decision about, and eventual experience of, transitioning older adults into alternative levels of housing (ALH), such as long-term care. This topic is examined from a family member perspective, given their exposure and involvement in the care of older adult relatives during this transitional period. The scoping review methodology is based on the framework of Arksey and O'Malley and subsequent recommendations from Levac, Colquhoun, and O'Brien. Approximately 470 articles were reviewed covering the period between 2000 and November 2014; 37 articles met inclusion criteria. A temporal organization of themes was used to describe the experiences of family members in the pretransition, active transition, and posttransition periods of moving older adult relatives into ALH. This paper highlights the transitional period as a time of crisis, with a lack of planning, support, and transparent discussion. This study identifies a need for future research on the potential benefits of family support groups, interim transitional housing options, different models of ALH, changing roles in the posttransition period, and the need for a comprehensive list of housing options for older adults. Results have the potential to inform policy/practice and improve the lives of older adults and their family.

Monitoring of ochratoxin A and ochratoxin-producing fungi in traditional salami manufactured in Northern Italy.

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.

Monopole patch antenna for in vivo exposure to nanosecond pulsed electric fields.

To explore the promising therapeutic applications of short nanosecond electric pulses, in vitro and in vivo experiments are highly required. In this paper, an exposure system based on monopole patch antenna is reported to perform in vivo experiments on newborn mice with both monopolar and bipolar nanosecond signals. Analytical design and numerical simulations of the antenna in air were carried out as well as experimental characterizations in term of scattering parameter (S 11) and spatial electric field distribution. Numerical dosimetry of the setup with four newborn mice properly placed in proximity of the antenna patch was carried out, exploiting a matching technique to decrease the reflections due to dielectric discontinuities (i.e., from air to mouse tissues). Such technique consists in the use of a matching dielectric box with dielectric permittivity similar to those of the mice. The average computed electric field inside single mice was homogeneous (better than 68 %) with an efficiency higher than 20 V m-1 V-1 for the four exposed mice. These results demonstrate the possibility of a multiple (four) exposure of small animals to short nanosecond pulses (both monopolar and bipolar) in a controlled and efficient way.

Microchambers for cell exposure: from the design to applications.

In the last decades, the advances in the micro and nano fabrication techniques have led to the development of microdevices that improved the possibility of analysis at cell level. These devices can be used in different applications (e.g., cell detection and identification, manipulation, cell treatments). The requisites, that are necessary to achieve, are different for various applications and represent the starting point of the project. The numerical multiphysics models can be very advantageous to analyze the performances of such devices and to predict their operation. Aim of this work is to give a look of the design rules of microchamber devices in particular for their application in electric field exposure. Two different applications for cell discrimination and characterization are reported considering time and frequency domain measurements.

A microdosimetry study for a realistic shaped nucleus.

In the last decades, the effects of ultrashort pulsed electric fields have been investigated demonstrating their capability to be involved in a great number of medical applications (e.g. cancer, gene electrotransfer, drug delivery, electrofusion). In particular, experiments in literature demonstrate that internal structures can be involved when pulse duration is reduced. Up to now, the mechanism that permits the electroporation phenomenon has not been completely understood and hence atomistic, microdosimetry and dosimetry models have been developed to help in this field. Aim of this work is to demonstrate the importance of realistically model also the internal organelles to obtain predictive results of effects at sub-cellular level with a microdosimetry model.

Effects of nanosecond pulsed electric fields on the activity of a Hodgkin and Huxley neuron model.

The cell membrane poration is one of the main assessed biological effects of nanosecond pulsed electric fields (nsPEF). This structural change of the cell membrane appears soon after the pulse delivery and lasts for a time period long enough to modify the electrical activity of excitable membranes in neurons. Inserting such a phenomenon in a Hodgkin and Huxley neuron model by means of an enhanced time varying conductance resulted in the temporary inhibition of the action potential generation. The inhibition time is a function of the level of poration, the pore resealing time and the background stimulation level of the neuron. Such results suggest that the neuronal activity may be efficiently modulated by the delivery of repeated pulses. This opens the way to the use of nsPEFs as a stimulation technique alternative to the conventional direct electric stimulation for medical applications such as chronic pain treatment.

Novel passive element circuits for microdosimetry of nanosecond pulsed electric fields.

Microdosimetric models for biological cells have assumed increasing significance in the development of nanosecond pulsed electric field technology for medical applications. In this paper, novel passive element circuits, able to take into account the dielectric dispersion of the cell, are provided. The circuital analyses are performed on a set of input pulses classified in accordance with the current literature. Accurate data in terms of transmembrane potential are obtained in both time and frequency domains for different cell models. In addition, a sensitivity study of the transfer function for the cell geometrical and dielectric parameters has been carried out. This analysis offers a new, simple, and efficient tool to characterize the nsPEFs' action at the cellular level.