Enabling mRNA medicine for brain tumors

mRNA is a new class of drugs that has the potential to revolutionize the treatment of brain tumors. Thanks to the COVID-19 mRNA vaccines and numerous therapy-based clinical trials, it is now clear that lipid nanoparticles (LNPs) are a clinically viable means to deliver RNA therapeutics. However, LNP-mediated mRNA delivery to brain tumors remains elusive. Over the past decade, numerous studies have shown that tumor cells communicate with each other via small extracellular vesicles, which are around 100 nm in diameter and consist of lipid bilayer membrane similar to synthetic lipidbased nanocarriers. We hypothesized that rationally designed LNPs based on extracellular vesicle mimicry would enable efficient delivery of RNA therapeutics to brain tumors without undue toxicity. We synthesized LNPs using four components similar to the formulation used in the mRNA COVID19 vaccines (Moderna and Pfizer): ionizable lipid, cholesterol, helper lipid and polyethylene glycol (PEG)-lipid. For the in vitro screen, we tested ten classes of helper lipids based on their abundance in extracellular vesicle membranes, commercial availability, and large-scale production feasibility while keeping rest of the LNP components unchanged. The transfection kinetics of GFP mRNA encapsulated in LNPs and doped with 16 mol% of helper lipids was tested using GL261, U87 and SIM-A9 cell lines. Several LNP formations resulted in stable transfection (upto 5 days) of GFP mRNA in all the cell lines tested in vitro. The successful LNP candidates (enabling >80% transfection efficacy) were then tested in vivo to deliver luciferase mRNA to brain tumors via intrathecal administration in a syngeneic glioblastoma (GBM) mouse model, which confirmed luciferase expression in brain tumors in the cortex. LNPs were then tested to deliver Cre recombinase mRNA in syngeneic GBM mouse model genetically modified to express tdTomato under LoxP marker cassette that enabled identification of LNP targeted cells. mRNA was successfully delivered to tumor cells (70-80% transfected) and a range of different cells in the tumor microenvironment, including tumor-associated macrophages (80-90% transfected), neurons (31- 40% transfected), neural stem cells (39-62% transfected), oligodendrocytes (70-80% transfected) and astrocytes (44-76% transfected). Then, LNP formulations were assessed for delivering Cas9 mRNA and CD81 sgRNA (model protein) in murine syngeneic GBM model to enable gene editing in brain tumor cells. Sanger sequencing showed that CRISPR-Cas9 editing was successful in ~94% of brain tumor cells in vivo. In conclusion, we have developed a library of safe LNPs that can transfect GBM cells in vivo with high efficacy. This technology can potentially be used to develop novel mRNA therapies for GBM by delivering single or multiple mRNAs and holds great potential as a tool to study brain tumor biology.

The Hidden Cost of COVID-19: Focus on Antimicrobial Resistance in Bloodstream Infections.

Antibiotic resistance is one of the greatest growing public health threats and a worldwide priority. According to the WHO, drug-resistant diseases may cause 10 million deaths a year by 2050 and have a substantial impact on the global economy, driving up to 24 million people into poverty. The ongoing COVID-19 pandemic has exposed the fallacies and vulnerability of healthcare systems worldwide, displacing resources from existing programs and reducing funding for antimicrobial resistance (AMR) fighting efforts. Moreover, as already seen for other respiratory viruses, such as flu, COVID-19 is often associated with superinfections, prolonged hospital stays, and increased ICU admissions, further aggravating healthcare disruption. These events are accompanied by widespread antibiotic use, misuse, and inappropriate compliance with standard procedures with a potential long-term impact on AMR. Still, COVID-19-related measures such as increasing personal and environmental hygiene, social distancing, and decreasing hospital admissions could theoretically help the AMR cause. However, several reports have shown increased antimicrobial resistance during the COVID-19 pandemic. This narrative review focuses on this "twindemic", assessing the current knowledge of antimicrobial resistance in the COVID-19 era with a focus on bloodstream infections and provides insights into the lessons learned in the COVID-19 field that could be applied to antimicrobial stewardship initiatives.

Ventilation indices for evaluation of airborne infection risk control performance of air distribution.

Air distribution is an effective engineering measure to fight against respiratory infectious diseases like COVID-19. Ventilation indices are widely used to indicate the airborne infection risk of respiratory infectious diseases due to the practical convenience. This study investigates the relationships between the ventilation indices and airborne infection risk to suggest the proper ventilation indices for the evaluation of airborne infection risk control performance of air distribution. Besides the commonly used ventilation indices of the age of air (AoA), air change effectiveness (ACE), and contaminant removal effectiveness (CRE), this study introduces two ventilation indices, i.e., the air utilization effectiveness (AUE) and contaminant dispersion index (CDI). CFD simulations of a hospital ward and a classroom served by different air distributions, including mixing ventilation, displacement ventilation, stratum ventilation and downward ventilation, are validated to calculate the ventilation indices and airborne infection risk. A three-step correlation analysis based on Spearman's rank correlation coefficient, Pearson correlation coefficient, and goodness of fit and a min-max normalization-based error analysis are developed to qualitatively and quantitatively test the validity of ventilation indices respectively. The results recommend the integrated index of AUE and CDI to indicate the overall airborne infection risk, and CDI to indicate the local airborne infection risk respectively regardless of the effects of air distribution, supply airflow rate, infectivity intensity, room configuration and occupant distribution. This study contributes to airborne transmission control of infectious respiratory diseases with air distribution.

A Data-Driven Framework for Identifying Intensive Care Unit Admissions Colonized With Multidrug-Resistant Organisms.

Background: The rising prevalence of multi-drug resistant organisms (MDROs), such as Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-resistant Enterococci (VRE), and Carbapenem-resistant Enterobacteriaceae (CRE), is an increasing concern in healthcare settings. Materials and Methods: Leveraging data from electronic healthcare records and a unique MDRO universal screening program, we developed a data-driven modeling framework to predict MRSA, VRE, and CRE colonization upon intensive care unit (ICU) admission, and identified the associated socio-demographic and clinical factors using logistic regression (LR), random forest (RF), and XGBoost algorithms. We performed threshold optimization for converting predicted probabilities into binary predictions and identified the cut-off maximizing the sum of sensitivity and specificity. Results: Four thousand six hundred seventy ICU admissions (3,958 patients) were examined. MDRO colonization rate was 17.59% (13.03% VRE, 1.45% CRE, and 7.47% MRSA). Our study achieved the following sensitivity and specificity values with the best performing models, respectively: 80% and 66% for VRE with LR, 73% and 77% for CRE with XGBoost, 76% and 59% for MRSA with RF, and 82% and 83% for MDRO (i.e., VRE or CRE or MRSA) with RF. Further, we identified several predictors of MDRO colonization, including long-term care facility stay, current diagnosis of skin/subcutaneous tissue or infectious/parasitic disease, and recent isolation precaution procedures before ICU admission. Conclusion: Our data-driven modeling framework can be used as a clinical decision support tool for timely predictions, characterization and identification of high-risk patients, and selective and timely use of infection control measures in ICUs.

Targeted intracellular delivery of Cas13 and Cas9 nucleases using bacterial toxin-based platforms.

Targeted delivery of therapeutic proteins toward specific cells and across cell membranes remains major challenges. Here, we develop protein-based delivery systems utilizing detoxified single-chain bacterial toxins such as diphtheria toxin (DT) and botulinum neurotoxin (BoNT)-like toxin, BoNT/X, as carriers. The system can deliver large protein cargoes including Cas13a, CasRx, Cas9, and Cre recombinase into cells in a receptor-dependent manner, although delivery of ribonucleoproteins containing guide RNAs is not successful. Delivery of Cas13a and CasRx, together with guide RNA expression, reduces mRNAs encoding GFP, SARS-CoV-2 fragments, and endogenous proteins PPIB, KRAS, and CXCR4 in multiple cell lines. Delivery of Cre recombinase modifies the reporter loci in cells. Delivery of Cas9, together with guide RNA expression, generates mutations at the targeted genomic sites in cell lines and induced pluripotent stem cell (iPSC)-derived human neurons. These findings establish modular delivery systems based on single-chain bacterial toxins for delivery of membrane-impermeable therapeutics into targeted cells.

The Tissue Distribution of SARS-CoV-2 in Transgenic Mice With Inducible Ubiquitous Expression of hACE2.

The novel coronavirus disease COVID-19 has become one of the most socially significant infections. One of the main models for COVID-19 pathogenesis study and anti-COVID-19 drug development is laboratory animals sensitive to the virus. Herein, we report SARS-CoV-2 infection in novel transgenic mice conditionally expressing human ACE2 (hACE2), with a focus on viral distribution after intranasal inoculation. Transgenic mice carrying hACE2 under the floxed STOP cassette [(hACE2-LoxP(STOP)] were mated with two types of Cre-ERT2 strains (UBC-Cre and Rosa-Cre). The resulting offspring with temporal control of transgene expression were treated with tamoxifen to induce the removal of the floxed STOP cassette, which prevented hACE2 expression. Before and after intranasal inoculation, the mice were weighed and clinically examined. On Days 5 and 10, the mice were sacrificed for isolation of internal organs and the further assessment of SARS-CoV-2 distribution. Intranasal SARS-CoV-2 inoculation in hACE2-LoxP(STOP)×UBC-Cre offspring resulted in weight loss and death in 6 out of 8 mice. Immunostaining and focus formation assays revealed the most significant viral load in the lung, brain, heart and intestine samples. In contrast, hACE2-LoxP(STOP) × Rosa-Cre offspring easily tolerated the infection, and SARS-CoV-2 was detected only in the brain and lungs, whereas other studied tissues had null or negligible levels of the virus. Histological examination revealed severe alterations in the lungs, and mild changes were observed in the brain tissues. Notably, no changes were observed in mice without tamoxifen treatment. Thus, this novel murine model with the Cre-dependent activation of hACE2 provides a useful and safe tool for COVID-19 studies.

The Role of the Perioperative Nurse in Implementing Contact Precautions to Prevent Transmission of Multidrug-Resistant Organisms.

The World Health Organization and Centers for Disease Control and Prevention consider the global increase in multidrug-resistant organisms (MDROs) to be one of the greatest modern threats to public health. Limited treatment options exist for microorganisms such as carbapenem-resistant Enterobacterales and Candida auris; as a result, infected patients may experience poor outcomes. Perioperative nurses should use infection prevention measures (eg, contact precautions) to prevent the spread of emerging MDROs when transporting patients to and from procedures, caring for patients during procedures, and completing between-procedure cleaning. Because nurses are involved with all phases of perioperative care, they are well-positioned to serve as infection prevention champions and provide education to personnel, patients, and caregivers. This article describes actions and steps the perioperative nurse should take during implementation of contact precautions to prevent the transmission of MDROs-specifically, emerging pathogens carbapenem-resistant Enterobacterales and C auris.

Synergistic Combination of AS101 and Azidothymidine against Clinical Isolates of Carbapenem-Resistant Klebsiella pneumoniae.

Owing to the over usage of carbapenems, carbapenem resistance has become a vital threat worldwide, and, thus, the World Health Organization announced the carbapenem-resistant Enterobacteriaceae (CRE) as the critical priority for antibiotic development in 2017. In the current situation, combination therapy would be one solution against CRE. Azidothymidine (AZT), a thymidine analog, has demonstrated its synergistically antibacterial activities with other antibiotics. The unexpected antimicrobial activity of the immunomodulator ammonium trichloro(dioxoethylene-o,o')tellurate (AS101) has been reported against carbapenem-resistant Klebsiella pneumoniae (CRKP). Here, we sought to investigate the synergistic activity between AS101 and AZT against 12 CRKP clinical isolates. According to the gene detection results, the blaOXA-1 (7/12, 58.3%), blaDHA (7/12, 58.3%), and blaKPC (7/12, 58.3%) genes were the most prevalent ESBL, AmpC, and carbapenemase genes, respectively. The checkerboard analysis demonstrated the remarkable synergism between AS101 and AZT, with the observable decrease in the MIC value for two agents and the fractional inhibitory concentration (FIC) index ≤0.5 in all strains. Hence, the combination of AS101 and azidothymidine could be a potential treatment option against CRKP for drug development.

Hydrophobic Optimization of Functional Poly(TPAE-co-suberoyl chloride) for Extrahepatic mRNA Delivery following Intravenous Administration.

Messenger RNA (mRNA) has generated great attention due to its broad potential therapeutic applications, including vaccines, protein replacement therapy, and immunotherapy. Compared to other nucleic acids (e.g., siRNA and pDNA), there are more opportunities to improve the delivery efficacy of mRNA through systematic optimization. In this report, we studied a high-throughput library of 1200 functional polyesters for systemic mRNA delivery. We focused on the chemical investigation of hydrophobic optimization as a method to adjust mRNA polyplex stability, diameter, pKa, and efficacy. Focusing on a region of the library heatmap (PE4K-A17), we further explored the delivery of luciferase mRNA to IGROV1 ovarian cancer cells in vitro and to C57BL/6 mice in vivo following intravenous administration. PE4K-A17-0.2C8 was identified as an efficacious carrier for delivering mRNA to mouse lungs. The delivery selectivity between organs (lungs versus spleen) was found to be tunable through chemical modification of polyesters (both alkyl chain length and molar ratio in the formulation). Cre recombinase mRNA was delivered to the Lox-stop-lox tdTomato mouse model to study potential application in gene editing. Overall, we identified a series of polymer-mRNA polyplexes stabilized with Pluronic F-127 for safe and effective delivery to mouse lungs and spleens. Structure-activity relationships between alkyl side chains and in vivo delivery were elucidated, which may be informative for the continued development of polymer-based mRNA delivery.

Association between tocilizumab and emerging multidrug-resistant organisms in critically ill patients with COVID-19: A multicenter, retrospective cohort study.

BACKGROUND: Tocilizumab is an IgG1 class recombinant humanized monoclonal antibody that directly inhibits the IL-6 receptor. Several randomized clinical trials have evaluated its safety and efficacy in patients with coronavirus disease 2019 (COVID-19), and these studies demonstrate conflicting results. Our study aimed to determine the association between tocilizumab treatment and microbial isolation and emergence of multidrug-resistant bacteria in critically ill patients with COVID-19. METHODS: A multicenter retrospective cohort study was conducted at two tertiary government hospitals in Saudi Arabia. All critically ill patients admitted to intensive care units with a positive COVID-19 PCR test between March 1 and December 31, 2020, who met study criteria were included. Patients who received tocilizumab were compared to those who did not receive it. RESULTS: A total of 738 patients who met our inclusion criteria were included in the analysis. Of these, 262 (35.5%) received tocilizumab, and 476 (64.5%) were included in the control group. Patients who received tocilizumab had higher odds for microbial isolation (OR 1.34; 95% CI 0.91-1.94, p = 0.13); however, the difference was not statistically significant. Development of resistant organisms (OR 1.00; 95% CI 0.51-1.98, p = 0.99) or detection of carbapenem-resistant Enterobacteriaceae (CRE) (OR 0.67; 95% CI 0.29-1.54, p = 0.34) was not statistically significant between the two groups. CONCLUSIONS: Tocilizumab use in critically ill patients with COVID-19 is not associated with higher microbial isolation, the emergence of resistant organisms, or the detection of CRE organisms.

A View on 20 Years of Antimicrobial Resistance in Japan by Two National Surveillance Systems: The National Epidemiological Surveillance of Infectious Diseases and Japan Nosocomial Infections Surveillance.

The Ministry of Health, Labour and Welfare (MHLW) of Japan has conducted two national surveillance systems for approximately 20 years to monitor antimicrobial resistance (AMR) in bacteria: the National Epidemiological Surveillance of Infectious Diseases (NESID) and the Japan Nosocomial Infections Surveillance (JANIS). Data accumulated for 20 years by these two surveillance systems have helped depict the epidemiology of the representative AMR bacteria in Japan chronologically. The epidemiology of methicillin-resistant Staphylococcus aureus teaches us that once AMR bacteria have established their high endemicity, controlling such AMR bacteria requires time and is challenging. On the other hand, the epidemiology that multidrug-resistant Acinetobacter sp. exhibits when a strict containment policy for AMR bacteria was introduced in the early phase of its emergence and spread reveals that it is possible to control it. Detailed epidemiology provided by these two different national surveillance systems in Japan enabled us to set up the goal for controlling each AMR bacteria at the hospital level to the prefecture/national level. It is the public health authorities' responsibility to maintain a good surveillance system for AMR bacteria and share the data and findings with healthcare professionals and academicians.

Trends of Multidrug-Resistant Pathogens, Difficult to Treat Bloodstream Infections, and Antimicrobial Consumption at a Tertiary Care Center in Lebanon from 2015-2020: COVID-19 Aftermath.

INTRODUCTION: We studied the trend of antimicrobial resistance and consumption at Saint George Hospital University Medical Center (SGHUMC), a tertiary care center in Beirut, Lebanon, with a focus on the SARS-CoV-2 pandemic. MATERIALS AND METHODS: We calculated the isolation density/1000 patient-days (PD) of the most isolated organisms from 1 January 2015-31 December 2020 that included: E. coli (Eco), K. pneumoniae (Kp), P. aeruginosa (Pae), A. baumannii (Ab), S. aureus (Sau), and E. faecium (Efm). We considered March-December 2020 a surrogate of COVID-19. We considered one culture/patient for each antimicrobial susceptibility and excluded Staphylococcus epidermidis, Staphylococcus coagulase-negative, and Corynebacterium species. We analyzed the trends of the overall isolates, the antimicrobial susceptibilities of blood isolates (BSI), difficult-to-treat (DTR) BSI, carbapenem-resistant Enterobacteriaceae (CRE) BSI, and restricted antimicrobial consumption as daily-defined-dose/1000 PD. DTR implies resistance to carbapenems, beta-lactams, fluoroquinolones, and additional antimicrobials where applicable. RESULTS AND DISCUSSION: After applying exclusion criteria, we analyzed 1614 blood cultures out of 8314 cultures. We isolated 85 species, most commonly Eco, at 52%. The isolation density of total BSI in 2020 decreased by 16%: 82 patients were spared from bacteremia, with 13 being DTR. The isolation density of CRE BSI/1000 PD decreased by 64% from 2019 to 2020, while VREfm BSI decreased by 34%. There was a significant decrease of 80% in Ab isolates (p-value < 0.0001). During COVID-19, restricted antimicrobial consumption decreased to 175 DDD/1000 PD (p-value < 0.0001). Total carbapenem consumption persistently decreased by 71.2% from 108DDD/1000 PD in 2015-2019 to 31 DDD/1000 PD in 2020. At SGHUMC, existing epidemics were not worsened by the pandemic. We attribute this to our unique and dynamic collaboration of antimicrobial stewardship, infection prevention and control, and infectious disease consultation.

Secondary infection in severe and critical COVID-19 patients in China: a multicenter retrospective study.

BACKGROUND: Since 2020 COVID-19 pandemic became an emergent public sanitary incident. The epidemiology data and the impact on prognosis of secondary infection in severe and critical COVID-19 patients in China remained largely unclear. METHODS: We retrospectively reviewed medical records of all adult patients with laboratory-confirmed COVID-19 who were admitted to ICUs from January 18th 2020 to April 26th 2020 at two hospitals in Wuhan, China and one hospital in Guangzhou, China. We measured the frequency of bacteria and fungi cultured from respiratory tract, blood and other body fluid specimens. The risk factors for and impact of secondary infection on clinical outcomes were also assessed. RESULTS: Secondary infections were very common (86.6%) when patients were admitted to ICU for >72 hours. The majority of infections were respiratory, with the most common organisms being Klebsiella pneumoniae (24.5%), Acinetobacter baumannii (21.8%), Stenotrophomonas maltophilia (9.9%), Candida albicans (6.8%), and Pseudomonas spp. (4.8%). Furthermore, the proportions of multidrug resistant (MDR) bacteria and carbapenem resistant Enterobacteriaceae (CRE) were high. We also found that age ≥60 years and mechanical ventilation ≥13 days independently increased the likelihood of secondary infection. Finally, patients with positive cultures had reduced ventilator free days in 28 days and patients with CRE and/or MDR bacteria positivity showed lower 28-day survival rate. CONCLUSIONS: In a retrospective cohort of severe and critical COVID-19 patients admitted to ICUs in China, the prevalence of secondary infection was high, especially with CRE and MDR bacteria, resulting in poor clinical outcomes.

Present and Future Perspectives on Therapeutic Options for Carbapenemase-Producing Enterobacterales Infections.

Carbapenem-resistant Enterobacterales (CRE) are included in the list of the most threatening antibiotic resistance microorganisms, being responsible for often insurmountable therapeutic issues, especially in hospitalized patients and immunocompromised individuals and patients in intensive care units. The enzymatic resistance to carbapenems is encoded by different ß-lactamases belonging to A, B or D Ambler class. Besides compromising the activity of last-resort antibiotics, CRE have spread from the clinical to the environmental sectors, in all geographic regions. The purpose of this review is to present present and future perspectives on CRE-associated infections treatment.

Periphery-aware COVID-19 diagnosis with contrastive representation enhancement.

Computer-aided diagnosis has been extensively investigated for more rapid and accurate screening during the outbreak of COVID-19 epidemic. However, the challenge remains to distinguish COVID-19 in the complex scenario of multi-type pneumonia classification and improve the overall diagnostic performance. In this paper, we propose a novel periphery-aware COVID-19 diagnosis approach with contrastive representation enhancement to identify COVID-19 from influenza-A (H1N1) viral pneumonia, community acquired pneumonia (CAP), and healthy subjects using chest CT images. Our key contributions include: 1) an unsupervised Periphery-aware Spatial Prediction (PSP) task which is designed to introduce important spatial patterns into deep networks; 2) an adaptive Contrastive Representation Enhancement (CRE) mechanism which can effectively capture the intra-class similarity and inter-class difference of various types of pneumonia. We integrate PSP and CRE to obtain the representations which are highly discriminative in COVID-19 screening. We evaluate our approach comprehensively on our constructed large-scale dataset and two public datasets. Extensive experiments on both volume-level and slice-level CT images demonstrate the effectiveness of our proposed approach with PSP and CRE for COVID-19 diagnosis.

Novel transgenic mice with Cre-dependent co-expression of GFP and human ACE2: a safe tool for study of COVID-19 pathogenesis.

The current coronavirus disease (COVID-19) pandemic remains one of the most serious public health problems. Increasing evidence shows that infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes a very complex and multifaceted disease that requires detailed study. Nevertheless, experimental research on COVID-19 remains challenging due to the lack of appropriate animal models. Herein, we report novel humanized mice with Cre-dependent expression of hACE2, the main entry receptor of SARS-CoV-2. These mice carry hACE2 and GFP transgenes floxed by the STOP cassette, allowing them to be used as breeders for the creation of animals with tissue-specific coexpression of hACE2 and GFP. Moreover, inducible expression of hACE2 makes this line biosafe, whereas coexpression with GFP simplifies the detection of transgene-expressing cells. In our study, we tested our line by crossing with Ubi-Cre mice, characterized by tamoxifen-dependent ubiquitous activation of Cre recombinase. After tamoxifen administration, the copy number of the STOP cassette was decreased, and the offspring expressed hACE2 and GFP, confirming the efficiency of our system. We believe that our model can be a useful tool for studying COVID-19 pathogenesis because the selective expression of hACE2 can shed light on the roles of different tissues in SARS-CoV-2-associated complications. Obviously, it can also be used for preclinical trials of antiviral drugs and new vaccines.

Antimicrobial Stewardship Program, COVID-19, and Infection Control: Spread of Carbapenem-Resistant Klebsiella Pneumoniae Colonization in ICU COVID-19 Patients. What Did Not Work?

The Italian burden of disease associated with infections due to antibiotic-resistant bacteria has been very high, largely attributed to Carbapenem-Resistant Klebsiella pneumoniae (CR-Kp). The implementation of infection control measures and antimicrobial stewardship programs (ASP) has been shown to reduce healthcare-related infections caused by multidrug resistance (MDR) germs. Since 2016, in our teaching hospital of Terni, an ASP has been implemented in an intensive care unit (ICU) setting, with the "daily-ICU round strategy" and particular attention to infection control measures. We performed active surveillance for search patients colonized by Carbapenem-Resistant Enterobacteriaceae (CRE). In March 2020, coronavirus disease 2019 (COVID-19) arrived and the same ICU was reserved only for COVID-19 patients. In our retrospective observational study, we analyzed the bimonthly incidence of CRE colonization patients and the incidence of CRE acquisition in our ICU during the period of January 2019 to June 2020. In consideration of the great attention and training of all staff on infection control measures in the COVID-19 era, we would have expected a clear reduction in CRE acquisition, but this did not happen. In fact, the incidence of CRE acquisition went from 6.7% in 2019 to 50% in March-April 2020. We noted that 67% of patients that had been changed in posture with prone position were colonized by CRE, while only 37% of patients that had not been changed in posture were colonized by CRE. In our opinion, the high intensity of care, the prone position requiring 4-5 healthcare workers (HCWs), equipped with personal protective equipment (PPE) in a high risk area, with extended and prolonged contact with the patient, and the presence of 32 new HCWs from other departments and without work experience in the ICU setting, contributed to the spread of CR-Kp in our ICU, determining an increase in CRE acquisition colonization.