Surveillance of healthcare acquired infections in hospital and community: a retrospective study in Local Healthcare Organization of Rovigo

Background: antimicrobial resistance is recognized as one of the greatest threats to human health worldwide. Infections caused by multidrug-resistant bacteria are associated with higher incidences of mortality, morbidity, prolonged hospital stay and increase of costs. Surveillance of alert organism/conditions and bacterial resistance to antimicrobials is a systematic and dynamic system of data collection that analyses and monitors trends of bacterial resistance. This study was conducted to detect antimicrobial susceptibility patterns in order to inform treatment choices and generate hospital-wide baseline data. Methods: the Local Healtcare Oganization of Rovigo has started a program of surveillance on antimicrobial resistance in hospital and community. In this work some results of the surveillance of microorganism isolated and related antimicrobial resistance are reported, collected in the period 2009-2010 in Rovigo and Trecenta Hospitals, Territorial Nursing Homes (TNH) and community from patients’ blood, urine and respiratory samples. Results: data show a significant difference in the level of antibiotic resistance between the two Hospitals. High rates of extended-spectrum β-lactamase (ESBL)-producing organisms are detected and carbapenems are the only reliable agents for the treatment of many infections in the Hospital of Trecenta and TNH. Conclusions: because ESBL producing bacteria are emerging pathogens in the community, the rational use of available antibiotics or the appropriate antimicrobial prescribing are imperative. Local surveillance is a powerful tool to detect and monitor hospital and community infections and provides information useful as a guide to medical practice, including therapeutics and disease-control activities

Development of a repressible mycobacterial promoter system based on two transcriptional repressors

Tightly regulated gene expression systems represent invaluable tools for studying gene function and for the validation of drug targets in bacteria. While several regulated bacterial promoters have been characterized, few of them have been successfully used in mycobacteria. In this article we describe the development of a novel repressible promoter system effective in both fast- and slow-growing mycobacteria based on two chromosomally encoded repressors, dependent on tetracycline (TetR) and pristinamycin (Pip), respectively. This uniqueness results in high versatility and stringency. Using this method we were able to obtain an ftsZ conditional mutant in Mycobacterium smegmatis and a fadD32 conditional mutant in Mycobacterium tuberculosis, confirming their essentiality for bacterial growth in vitro. This repressible promoter system could also be exploited to regulate gene expression during M. tuberculosis intracellular growt

Essentiality of mmpL3 and impact of its silencing on Mycobacterium tuberculosis gene expression

MmpL3 is an inner membrane transporter of Mycobacterium tuberculosis responsible for the export of trehalose momomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. MmpL3 represents an emerging target for tuberculosis therapy. In this paper, we describe the construction and characterization of an mmpL3 knockdown strain of M. tuberculosis. Downregulation of mmpL3 led to a stop in bacterial division and rapid cell death, preceded by the accumulation of TDM precursors. MmpL3 was also shown to be essential for growth in monocyte-derived human macrophages. Using RNA-seq we also found that MmpL3 depletion caused up-regulation of 47 genes and down-regulation of 23 genes (at least 3-fold change and false discovery rate <= 1%). Several genes related to osmoprotection and metal homeostasis were induced, while several genes related to energy production and mycolic acids biosynthesis were repressed suggesting that inability to synthesize a correct outer membrane leads to changes in cellular permeability and a metabolic downshift

Development of a repressible mycobacterial promoter system based on two transcriptional repressors

Tightly regulated gene expression systems represent invaluable tools for studying gene function and for the validation of drug targets in bacteria. While several regulated bacterial promoters have been characterized, few of them have been successfully used in mycobacteria. In this article we describe the development of a novel repressible promoter system effective in both fast- and slow-growing mycobacteria based on two chromosomally encoded repressors, dependent on tetracycline (TetR) and pristinamycin (Pip), respectively. This uniqueness results in high versatility and stringency. Using this method we were able to obtain an ftsZ conditional mutant in Mycobacterium smegmatis and a fadD32 conditional mutant in Mycobacterium tuberculosis, confirming their essentiality for bacterial growth in vitro. This repressible promoter system could also be exploited to regulate gene expression during M. tuberculosis intracellular growth

Supercritical CO2 for the drying and microbial inactivation of apple's slices

Supercritical CO2 (Sc-CO2) drying has been recognized as a promising low temperature drying technique for food products. In this regard, this work focuses on the feasibility of Sc-CO2 drying of apple’s slices: both the microbiological stability and mechanical behavior of the test product after the process have been investigated in dependence from different process parameters, namely drying time, pressurization time, and depressurization time. The microbiological stability was determined for both inoculated pathogenic bacteria (Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes) and naturally present microorganisms (yeasts and molds, mesophilic bacteria and spores and Enterobacteriaceae). Results demonstrated a complete inactivation of pathogenic bacteria under the detection limit (&lt;1 CFU/g) just after the pressurization (10 min) and depressurization (20 min) phases. After the same steps, a strong reduction of vegetative bacteria and yeasts and molds was also observed in comparison with air drying and freeze drying samples. As regards the mechanical behavior, the Young Modulus, measured before and after the CO2 processes to provide a measurement of samples’ stiffness, resulted dependent from the final water activity, but independent from the length of pressurization and depressurization phases at longer drying time. Overall, these results are promising to foster the development of the technology at industrial level

Sviluppo di un sistema dell' espressione inducibile per l' espressione condizionale in micobatterio basato sull' utilizzo dei repressori Pip e TetR

Inducible gene expression systems are powerful tools for studying gene function and for the validation of drug targets in bacteria. We report the development of a novel gene regulation system that allows negative-controlled gene expression. The system is based on two repressors: Pip and TetR. The gene of interest is placed under the transcriptional control of a Pip-dependant promoter (ptr); the Pip encoding gene, however, is placed under TetR transcriptional regulation. Both pip and tetR were integrated into the chromosome using an integrative plasmid. In the absence of ATc, TetR represses pip and the gene of interest can be transcribed. However, upon addiction of anhydrotetracycline (ATc), TetR allows pip expression, resulting in the stringent repression of the gene of interest. Using lacZ as a reporter gene, we showed that supplementation with subinhibitory concentrations of ATc allows stringent repression of its transcription in M. smegmatis and M. tuberculosis both in liquid and in solid media. Time-course and dose-response experiments showed that repression could be modulated in response to ATc concentration. One of the most useful applications of a reverse-inducible system is the possibility to construct conditional mutants of essential genes, allowing their characterization and validation as drug targets. At this purpose we constructed a M. smegmatis mutant where the ftsZ promoter was replaced with a Pip regulated promoter. Upon integration of the integrative plasmid carrying pip and tetR, the recombinant strain was unable to growth in sub- inhibitory ATc concentration, confirming ftsZ repression. The same technique was used in M. tuberculosis where fadD32 operon (essential in M. tuberculosis) and rv3213c (not essential in M. tuberculosis) were placed under Pptr transcriptional control. In the presence of ATc (in the concentration of 200ng/ml) only the conditional mutant of rv3213c was able to grow demonstrating that fadD32 operon is essential in M. tuberculosis. A different strategy to construct conditional mutant of Rv3790 was elaborated; this gene belongs to a large operon in M. tuberculosis so it was elaborated a technique that allows to have an dependant ATc- regulation only in Rv3790 while the other genes continue to be transcribed by their wild- type promoter. The development of a novel reverse-inducible system based on ATc will represent an invaluable tool to study essential genes and virulence-associated genes in mycobacteria.I sistemi d'espressione inducibili rappresentano ad oggi un potente mezzo con il quale studiare la funzione dei geni essenziali e nell'individuazione di bersagli farmacologici. Essi infatti si dimostrano fondamentali nello studio della funzione di geni quando questa è sconosciuta in quanto permettono di dosarne l'attività in risposta ad un semplice stimolo come l'aggiunta al terreno di un metabolita. Questo sarà un importante strumento per facilitare sia lo studio di nuovi regolatori che lo studio di geni essenziali o importanti per la virulenza. Durante questo triennio di dottorato la mia attività di ricerca si è occupata dello sviluppo e della caratterizzazione di un sistema di regolazione genica per micobatterio che permettesse un controllo negativo basato sull'attività di due repressori: Pip e TetR. In questo sistema il gene d'interesse è posto sotto il controllo trascrizionale di un promotore dipendente da Pip (ptr); Pip a sua volta è posto sotto la regolazione di TetR attraverso l'inserimento nel suo promotore, di due operatori (tetO) riconosciuti specificamente da TetR. L'induttore utilizzato per questo sistema è l'antibiotico anidrotetraciclina (ATc), prodotto di degradazione della tetraciclina, meno tossico ma ancora in grado di interagire con il regolatore TetR. In assenza di ATc, TetR reprime pip e il gene d'interesse puಠessere trascritto; con l'aggiunta di ATc invece si ottiene come effetto l'espressione di pip e di conseguenza lo spegnimento trascrizionale del gene di interesse. Un'applicazione molto utile per un sistema d'espressione inducibile è la costruzione di mutanti condizionali per lo studio di geni essenziali con lo scopo di valutare la possibilità di validare il loro impiego come bersagli farmacologici. A tal fine è stato costruito un mutante condizionale in M. smegmatis, dove il promotore del gene ftsZ, è stato sostituito dal promotore regolato dal repressore pip. Tale mutante in seguito, è stato trasformato con il plasmide integrativo avente entrambi i repressori ed il ricombinante ottenuto è risultato incapace di crescere già a concentrazioni sub-inibitorie di ATc, confermando cosଠla repressione trascrizionale di ftsZ. Lo stesso metodo è stato impiegato per ottenere due mutanti condizionali in M. tuberculosis H37Rv dove l'operone di fadD32 (essenziale in M. smegmatis), o il gene rv3213c (non essenziale; utilizzato come controllo) sono stati posti sotto il controllo trascrizionale di Pptr. In presenza di ATc (in concentrazione pari a 200ng/ml) solo il mutante condizionale di rv3213c è stato in grado di crescere dimostrando come l'operone di fadD32 sia essenziale anche in M. tuberculosis. Una tecnica alternativa per l'ottenimento di un mutante condizionale è stata applicata con il gene Rv3790 di M. tuberculosis; poiché esso appartiene ad un operone costituito da 12 geni, non è stato possibile utilizzare la strategia prevista per ftsZ e fadD32 in quanto un silenziamento di Rv3790 si sarebbe trasmesso anche all'intero operone. Per poter analizzare gli effetti dovuti unicamente alla presenza/ assenza di Rv3790 nella cellula batterica, è stato sviluppato un sistema dove la regolazione ATc- dipendente è stata vincolata solamente al gene suddetto mentre i geni appartenenti al medesimo operone continuavano ad essere regolati a livello trascrizionale dal loro promotore wild- type. Lo sviluppo di un innovativo sistema d'espressione inducibile che utilizza l'anidro- tetraciclina come induttore, rappresenta uno strumento prezioso per lo studio di geni che sono essenziali ed associati alla virulenza nel micobatterio

Characterization of a Mycobacterium tuberculosis ESX-3 Conditional Mutant: Essentiality and Rescue by Iron and Zinc ▿

Recently, a novel type of secretory pathway, type VII secretion systems (T7SSs), has been characterized in mycobacteria. The chromosomes of Mycobacterium tuberculosis and Mycobacterium bovis encode five T7SSs (ESX-1 to ESX-5). The best characterized of them, ESX-1, is involved in host-pathogen interactions, and its deletion is one of the main causes of M. bovis BCG attenuation. Another T7SS, ESX-3, has been previously shown to be transcriptionally controlled by the zinc uptake repressor (Zur) and by the iron-dependent transcriptional repressor (IdeR), suggesting that it might be involved in zinc and iron homeostasis. In this study, we characterized an M. tuberculosis conditional mutant in which transcription of the ESX-3 gene cluster can be downregulated by anhydrotetracycline. We showed that this T7SS is essential for growth and that this phenotype can be complemented by zinc, iron, or supernatant from a wild-type parental strain culture, demonstrating that the ESX-3 secretion system is responsible for the secretion of some soluble factor(s) required for growth that is probably involved in optimal iron and zinc uptake

Global transcriptional responce to Vancomycin in Mycobacterium tuberculosis

In order to gain additional understanding of the physiological mechanisms used by bacteria to maintain surface homeostasis and to identify potential targets for new antibacterial drugs, we analysed the variation of the Mycobacterium tuberculosis transcriptional profile in response to inhibitory and subinhibitory concentrations of vancomycin. Our analysis identified 153 genes differentially regulated after exposing bacteria to a concentration of the drug ten times higher than the MIC, and 141 genes differentially expressed when bacteria were growing in a concentration of the drug eightfold lower than the MIC. Hierarchical clustering analysis indicated that the response to these different conditions is different, although with some overlap. This approach allowed us to identify several genes whose products could be involved in the protection from antibiotic stress targeting the envelope and help to confer the basal level of M. tuberculosis resistance to antibacterial drugs, such as Rv2623 (UspA-like), Rv0116c, PE20-PPE31, PspA and proteins related to toxin-antitoxin systems. Moreover, we also demonstrated that the alternative sigma factor sigma(E) confers basal resistance to vancomycin, once again underlining its importance in the physiology of the mycobacterial surface stress response

Stimulating agency in preschool children using feedback (in formative assessment): An exploratory research

The research aims to analyse what feedback can promote agency in preschool children, whether given by educators or peers themselves, also assuming that giving (good) feedback to peers improves agentivity. Some authors focused on university, primary, and secondary school contexts about the effectiveness of feedback in teaching processes (Hattie, 2012), and the role of peer feedback (Nicol, 2018; Grion & Restiglian, 2019). This study aims to observe which feedback (received/given) improves the sense of agency in preschool children. Activating peer feedback practices has positive effects on learning and represents a valid alternative to the feedback given by the teacher (Tseng & Tsai, 2007). Also, for the agency, there is a considerable amount of research about teacher agency (Cong-Lem, 2021) but very little research about children's agency in the curriculum, even if the students' role as active agents in learning is strategic (Ebrahim, 2011). Starting from an educational theory of progressivism focused on improving 41 communication (Tan, 2006), exploratory qualitative research was conducted through a three-month video observation in a preschool (5-year-old children). The videos were analysed using an observation protocol supported by a research team blind review. Data were collected according to GDPR, and research intents were explained to children's families and preschool management. The research led to the definition of a framework including the main characteristics of effective feedback. Some insights into the child's self-regulation also emerged. According to the idea of educational continuity formative assessment in school, the results will support other research about children's feedback for assessment