A Whole-Genome Sequencing-Based Approach for the Characterization of Klebsiella pneumoniae Co-Producing KPC and OXA-48-like Carbapenemases Circulating in Sardinia, Italy.

BACKGROUND: Whole-genome sequencing (WGS) provides important information for the characterization, surveillance, and monitoring of antimicrobial resistance (AMR) determinants, particularly in cases of multi- and extensively drug-resistant microorganisms. We reported the results of a WGS analysis carried out on carbapenemases-producing Klebsiella pneumoniae, which causes hospital-acquired infections (HAIs) and is characterized by a marked resistance profile. METHODS: Clinical, phenotypic, and genotypic data were collected for the AMR surveillance screening program of the University Hospital of Sassari (Italy) during 2020-2021. Genomic DNA was sequenced using the Illumina Nova Seq 6000 platform. Final assemblies were manually curated and carefully verified for the detection of antimicrobial resistance genes, porin mutations, and virulence factors. A phylogenetic analysis was performed using the maximum likelihood method. RESULTS: All 17 strains analyzed belonged to ST512, and most of them carried the blaKPC-31 variant blaOXA-48-like, an OmpK35 truncation, and an OmpK36 mutation. Phenotypic analysis showed a marked resistance profile to all antibiotic classes, including ß-lactams, carbapenems, aminoglycosides, fluoroquinolone, sulphonamides, and novel ß-lactam/ß-lactamase inhibitors (BL/BLI). CONCLUSION: WGS characterization revealed the presence of several antibiotic resistance determinants and porin mutations in highly resistant K. pneumoniae strains responsible for HAIs. The detection of blaKPC-31 in our hospital wards highlights the importance of genomic surveillance in hospital settings to monitor the emergence of new clones and the need to improve control and preventive strategies to efficiently contrast AMR.

Infection Prevention Control Strategies of New Delhi Metallo-ß-lactamase Producing Klebsiella pneumoniae.

The spread of multi-drug resistant organisms (MDROs) is increasing at an alarming rate worldwide. Among these, Carbapenemase-producing New Delhi Metallo-ß-lactamase (NDM) poses a significant clinical threat, and appropriate measures must be taken to prevent or limit its penetration into still-free territories. The present report describes two independent cases of patients from Ukraine colonized by NDM-producing Klebsiella pneumoniae and admitted to two separate wards of an acute university hospital in a territory not yet affected by Carbapenemase producers of this class. Moreover, this report illustrates the infection prevention control (IPC) strategies promptly implemented by the IPC operational team to verify the possible spread of the microorganism in the ward and avoid any possible further contamination. The identification of genes coding for Carbapenemases, performed using real-time PCR, revealed no other cases within the wards involved. These cases emphasize the importance of early case recognition of multidrug-resistant bacteria, the necessity of effective inter-hospital communication, the need for effective antimicrobial stewardship protocol, and the importance of adequate IPC policies. Additionally, we highlight the need to improve screening procedures in the case of patients from countries with a high prevalence of MDRO, as essential measures to prevent potential nosocomial outbreaks and/or endemization.

Impact of the COVID-19 Pandemic on the Prevalence of HAIs and the Use of Antibiotics in an Italian University Hospital.

The COVID-19 pandemic has massively affected healthcare systems globally, causing a possible reduction in attention to traditional infection prevention programs. The objective of this study was to estimate the prevalence of healthcare-associated infections (HAIs) and the use of antimicrobials in an Italian University Hospital and to investigate whether the intensification of hospital infection control measures during the COVID-19 pandemic has affected the prevalence of bacterial HAIs. A point prevalence survey was conducted according to the simplified ECDC protocol. The survey identified a local HAI prevalence of 9.0%, revealing an increase compared to pre-pandemic values (7.3%). The survey also identified an antimicrobial exposure of 40.8%, revealing a decrease in their use compared to the study carried out in the pre-pandemic era (44.6%). Among the organizational challenges experienced during the COVID-19 pandemic, despite the greater attention paid to infection prevention measures aimed at reducing SARS-CoV-2, many healthcare facilities had to contend with the controlled availability of personnel, physical space limitations and a large number of patients. Active surveillance in hospital wards and the consequent reporting by personnel specialized in infection control is fundamental for hospitals to recognize gaps in prevention and report any observed increases in HAIs.

Controlling Infectious Risk in Transfusion: Assessing the Effectiveness of Skin Disinfection in Blood Donors.

Bacterial infectious risk is a major problem in transfusion medicine. The type of micro-organisms isolated during bacterial contamination of blood products indicates that the donor's skin is its main source. In this context, the primary measures to reduce this risk are: (a) optimal disinfection of the donor's arm and (b) satellite bag diversion of the initial volume of blood collected. This work aimed to verify the effectiveness of skin disinfection of the blood donor's venipuncture site. Two methodological approaches were used: (a) qualitative and quantitative microbiological testing of the skin at the collection site, before and post-disinfection; (b) qualitative microbiological testing of the first deviated blood. Pre-disinfection testing showed skin microbial load values between 3 and >200 CFU/plate. More than two-thirds of the isolates were Gram-positive bacteria (77.8%) of which 57.7% were staphylococci. Among Gram-negative bacteria, Pseudomonadaceae, Enterobacteriaceae, and Acinetobacter spp. were isolated from the blood donors (BDs). Post-disinfection, a 100% reduction in microbial load was observed in 84.4% of BDs. Microbiological testing of the first blood diverted sample revealed the presence of microbial flora in 1.9% samples; of the isolates, 83.3% were non-aureus staphylococci. This study highlights the importance of the correct application of skin disinfection procedures in order to ensure blood safety.

High-Risk Clone of Klebsiella pneumoniae Co-Harbouring Class A and D Carbapenemases in Italy.

Background: Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is endemic globally, causing severe infections in hospitalized patients. Surveillance programs help monitor and promptly identify the emergence of new clones. We reported the rapid spread of a novel clone of K. pneumoniae co-harbouring class A and D carbapenemases in colonized patients, and the potential risk factors involved in the development of infections. Methods: Rectal swabs were used for microbiological analyses and detection of the most common carbapenemase encoding genes by real-time PCR (i.e., blaKPC, blaOXA-48, blaNDM, blaVIM, and blaIMP). All strains co-harbouring KPC and OXA-48 genes were evaluated. For each patient, the following variables were collected: age, sex, length and ward of stay, device use, and outcome. Clonality of CR-Kp was assessed by preliminary pulsed field gel electrophoresis (PFGE), followed by multi-locus sequence typing (MLST) analyses. Results: A total of 127 isolates of K. pneumoniae co-harbouring KPC and OXA-48 were collected between September 2019 and December 2020. The median age (IQR) of patients was 70 (61-77). More than 40% of patients were admitted to intensive care unit (ICU). Around 25% of patients developed an invasive infection, the majority of which were respiratory tract infections (17/31; 54.8%). ICU stay and invasive infection increased the risk of mortality (OR: 5.39, 95% CI: 2.42-12.00; OR 6.12, 95% CI: 2.55-14.69, respectively; p-value ≤ 0.001). The antibiotic susceptibility test showed a resistance profile for almost all antibiotics considered. Monoclonal origin was confirmed by PFGE and MLST showing a similar restriction pattern and belonging to ST-512. Conclusions: We report the spread and the marked antibiotic resistance profiles of K. pneumoniae strains co-producing KPC and OXA-48. Further study could clarify the roles of clinical and microbiological variables in the development of invasive infection and increasing risk of mortality, in colonized patients.

Ten-Year Evaluation of Thermal Comfort in Operating Rooms.

The microclimate is a particularly important environmental aspect in operating rooms (ORs), where more than in other hospital environments, it is extremely important, and at the same time extremely difficult, to reconcile the needs of different types of occupants (patients and operators). Moreover, unsuitable microclimatic conditions may affect the onset of infection. The present study aimed to analyze the periodic monitoring of the microclimatic conditions carried out in ORs over 10 years, to verify the adequacy of the thermal comfort conditions for all occupants. The evaluation of thermal comfort was carried out using the Fanger indices and the standards required by current legislation and specific guidelines. Non-compliant values for at least one parameter were found in 98.8% of the examinations performed in the ORs. A condition of thermal discomfort was calculated for 3.6% of healthcare professionals and 98.3% of patients. The monitoring of microclimatic conditions is particularly important in the OR as an indicator of inadequate functioning of the air conditioning system, which might affect the thermal comfort of all occupants and lead to microbial contamination of the room.

Influenza Vaccination Strategies in Healthcare Workers: A Cohort Study (2018-2021) in an Italian University Hospital.

Vaccinating healthcare workers (HCWs) is the most effective intervention for preventing nosocomial influenza infection. However, influenza vaccination coverage (VC%) among HCWs remains low. The aim of the study was to analyse the trend of VC% among healthcare workers in an Italian hospital through a three-year vaccination project covering three influenza seasons (2018-2019, 2019-2020, and 2020-2021). A gap analysis was performed at the case base (2018-2019), on-site vaccination was trialled in the 2019-2020 season, and an integrated vaccination offer (on-site vaccination and the classic offer at a vaccination clinic) was implemented for the 2020-2021 season. For each unit of vaccinated HCWs, the following variables were recorded: main demographic details, area of affiliation (medical/surgical/services), and professional category. Logistic regression analyses were performed to assess the relationship between outcome (undergoing vaccination in 2020-2021) and other variables related to the healthcare workers' characteristics. In the three seasons, VC% values of 13.2%. 27.7%, and 58.9% were recorded, respectively (p < 0.005). The highest VC% was recorded among physicians (94.93%), in the medical area (63.27%), and males (62.59%) and in general among the youngest HCWs. Comparison of the coverage values recorded in the three seasons showed that in the last season considered (2020-2021) about 80% of health workers preferred to be vaccinated in the workplace instead of using the standard vaccination delivery method (invitation to attend the vaccination clinic). Our study suggests that the integrated vaccination offer may lead to an increase in VC% among HCWs compared to the classical offer modalities.

ATP Bioluminescence for Assessing the Efficacy of the Manual Cleaning Procedure during the Reprocessing of Reusable Surgical Instruments.

Achieving sterilization by adopting proper practices is essential to ensure that surgical instruments do not transmit microorganisms to patients. As the effectiveness of sterilization mandates effective cleaning, it is necessary to verify the success of cleaning procedures. In this study, we used the adenosine triphosphate (ATP) bioluminescence method for assessing the efficacy of the manual cleaning procedure during the reprocessing of reusable surgical instruments. The ATP bioluminescence assay was performed on 140 surgical instruments of 12 different types, both before being cleaned (baseline) and after each of the cleaning procedures (i.e., decontamination, manual washing, drying, and visual inspection). For each instrument, two swabs were used as follows: one to sample the entire surface (test point 1) and the other to sample the most difficult part of the surface to clean (test point 2). Overall, for each type of instrument, there was a decrease in contamination ranging from 99.6 to >99.9% (log reduction from 2.40 to 3.76). Thus, in order to standardize the assessment of cleanliness, it may be useful to introduce the bioluminescence method into the daily routine or, at least, at regular time intervals as a complementary check combined with visual inspection. This would allow real-time verification of the achievement of an adequate level of cleanliness.

Harmonised LUCAS in-situ land cover and use database for field surveys from 2006 to 2018 in the European Union.

Accurately characterizing land surface changes with Earth Observation requires geo-located ground truth. In the European Union (EU), a tri-annual surveyed sample of land cover and land use has been collected since 2006 under the Land Use/Cover Area frame Survey (LUCAS). A total of 1351293 observations at 651780 unique locations for 106 variables along with 5.4 million photos were collected during five LUCAS surveys. Until now, these data have never been harmonised into one database, limiting full exploitation of the information. This paper describes the LUCAS point sampling/surveying methodology, including collection of standard variables such as land cover, environmental parameters, and full resolution landscape and point photos, and then describes the harmonisation process. The resulting harmonised database is the most comprehensive in-situ dataset on land cover and use in the EU. The database is valuable for geo-spatial and statistical analysis of land use and land cover change. Furthermore, its potential to provide multi-temporal in-situ data will be enhanced by recent computational advances such as deep learning.

Antimicrobial Effect of Thymus capitatus and Citrus limon var. pompia as Raw Extracts and Nanovesicles.

In view of the increasing interest in natural antimicrobial molecules, this study screened the ability of Thymus capitatus (TC) essential oil and Citrus limon var. pompia (CLP) extract as raw extracts or incorporated in vesicular nanocarriers against Streptococcus mutans and Candida albicans. After fingerprint, TC or CLP were mixed with lecithin and water to produce liposomes, or different ratios of water/glycerol or water/propylene glycol (PG) to produce glycerosomes and penetration enhancer vesicles (PEVs), respectively. Neither the raw extracts nor the nanovesicles showed cytotoxicity against human gingival fibroblasts at all the concentrations tested (1, 10, 100 µg/mL). The disc diffusion method, MIC-MBC/MFC, time-kill assay, and transmission electron microscopy (TEM) demonstrated the highest antimicrobial potential of TC against S. mutans and C. albicans. The very high presence of the phenol, carvacrol, in TC (90.1%) could explain the lethal effect against the yeast, killing up to 70% of Candida and not just arresting its growth. CLP, rich in polyphenols, acted in a similar way to TC in reducing S. mutans, while the data showed a fungistatic rather than a fungicidal activity. The phospholipid vesicles behaved similarly, suggesting that the transported extract was not the only factor to be considered in the outcomes, but also their components had an important role. Even if other investigations are necessary, TC and CLP incorporated in nanocarriers could be a promising and safe antimicrobial in caries prevention.

Population Distrust of Drinking Water Safety. Community Outrage Analysis, Prediction and Management.

The aim of the work is to evaluate the effectiveness of the risk communication strategies in cases of unsafe drinking water supply in Sardinia, the Italian context with the highest population distrust in drinking water safety. During the period 2010⁻2015, the ordinances published on the institutional websites were analyzed, and the population risk perception was evaluated by applying, for the first time in public health threats, the "OUTRAGE Prediction & Management" software released by Sandman. Overall, 417 ordinances issued by the Sardinian Municipalities were found. Only 1.5% of the ordinances reported information about parameters, concentrations, and risks to health, whereas 4.8% indicated only the parameters and non-standard levels. By contrast, 53.2% specified only the non-standard parameter, and 40.5% indicated a generic non-drinking motivation. The outrage assessment showed values exceeding the threshold of risk acceptance, attributable to the lack and low clarity of the information reported by the ordinances. The present study allowed us to highlight critical issues in risk communication of the quality of drinking water.

Citrus limon Extract Loaded in Vesicular Systems for the Protection of Oral Cavity.

Background: The nanoincorporation of the extract of Citrus limon (L.) Osbeck var. pompia into liposomes was aimed at improving its antioxidant and antibacterial effects. Methods: The extract of the rind of Citrus limon (L.) Osbeck var. pompia was obtained by maceration in ethanol, evaporation, and freeze-drying. The extract phytochemical fingerprint was obtained by HPLC and mass spectrometry, and it was determined that gallic acid, neohesperidin, eriocitrin, and neoeriocitrin were the most abundant components. The freeze-dried extract was loaded in liposomes, glycerosomes, and penetration-enhancer-containing vesicles prepared with propylene glycol (PG-PEVs). Results: Capability of the vesicles of improving efficacy of the extract in counteracting oxidative stress was studied in vitro in keratinocytes, along with antimicrobial activity against planktonic cultures of Streptococcus mutans, Lactobacillus acidophilus, and Streptococcus sanguinis. Conclusion: Results showed that the vesicles, especially glycerosomes and PG-PEVs, prevented oxidative damage and cell death, and inhibited bacterial proliferation.

Thymus essential oil extraction, characterization and incorporation in phospholipid vesicles for the antioxidant/antibacterial treatment of oral cavity diseases.

The aim of the work was to extract, characterize, and formulate Thymus capitatus (Tymbra capitata) essential oil in phospholipid vesicles: liposomes, glycerosomes and Penetration Enhancer-containing Vesicles (PEVs). The steam-distilled essential oil was mainly composed of carvacrol. The oil was mixed with lecithin and water to produce liposomes, or different ratios of water/glycerol or water/propylene glycol (PG) to produce glycerosomes and PG-PEVs, respectively. Cryo-TEM showed the formation of unilamellar, spherical vesicles, and light scattering disclosed that their size increased in the presence of glycerol or PG, which improved long-term stability. The formulations were highly biocompatible, and capable of counteracting oxidative stress and favouring wound repair in keratinocytes, thanks to enhanced uptake. The antibacterial activity of the oil was demonstrated against cariogenic Streptococcus mutans, Lactobacillus acidophilus, and commensal Streptococcus sanguinis. The combination of antioxidant and antibacterial activities of Thymus essential oil formulations may be useful for the treatment of oral cavity diseases.

[Microbial contamination of surfaces in the departments of Medicine and Surgery: single center prevalence study in Sassari (Italy)]. / Contaminazioni microbiche di superfici di frequente contatto in reparti di medicina e chirurgia: studio monocentrico di prevalenza a Sassari.

INTRODUCTION: Healthcare-associated infections (HAIs) represent a clinical and public health problem worldwide. Microbial transmission can frequently occurs between patients or between patients and health-care workers; however, several devices and surfaces could act as reservoir and source of microorganisms. Aim of this cross-sectional study was to show the microbial contamination of devices or surfaces located in the departments of Medicine and Surgery of an Italian University Hospital. METHODS: Swabs were used to sample devices (keyboards, phones) and surfaces (door handles, water closed, light switches), at two different time-points. Samples were then evaluated in the UOC Hygiene and Preventive Medicine laboratory of the same University Hospital. RESULTS: 189 swabs were collected, 95 (53.3%) from the Medicine and 94 (49.7%) from the Surgery Department. The bacterial contamination prevalence was 42.9%, significantly higher in the Medicine than in the Surgery Department (51.6% vs 34%; p=0.015). A greater contamination was observed in water closed (22/36, 61.1%), phones (22/40, 55%), and keyboards of personal computers (18/36, 51.4%; p<0.001). No statistical differences were detected in the contamination rates when the different time-points were compared, as well as in the isolation rate of pathogenic bacterial strains. DISCUSSION: This survey highlights the potential role of devices and surfaces in the HAI pathogenesis. Further longitudinal and analytical studies might better assess the HAI risk associated with bacterial contamination in nosocomial settings.

Molecular typing of XDR Acinetobacter baumannii strains in an Italian ICU.

OBJECTIVE: To investigate the antimicrobial susceptibility and clonal relationship of Acinetobacter baumannii strains isolated in an Italian ICU. DESIGN: Epidemiological, observational, retrospective, longitudinal study. SETTING AND PARTICIPANTS: The ICU of the University Hospital of Sassari, Italy. MAIN OUTCOME MEASURES: Pulsed Field Gel Electrophoresis (PFGE) and Multi Locus Sequence Typing (MLST) were used to evaluate the genomic features of the isolated strains. RESULTS: Drug susceptibility testing for all isolated strains showed the same resistance pattern, characterized by resistance to the most important antibiotics, with the only exception of colistin. PFGE showed a very poor between-strain variability; three distinct clusters, 11, 4, and 1 isolates in size, were identified (Dice's coefficient: 92.11%). MLST showed that all isolated strains belonged to sequence type 2 (ST2). All isolates collected from the environment and the human samples were positive for the following genes: blaOXA-23, blaOXA-51-like, blaVIM-like, blaIMP-like, andISAba1; however, blaOXA-24-like, blaOXA-58-like, and blaNDM-like were not detected. CONCLUSIONS: The survey identified XDR strains belonging to the same cell clone, confirming the wide circulation and environmental persistence of this microorganism.

Prevalence of canine coronavirus antibodies by an enzyme-linked immunosorbent assay in dogs in the south of Italy.

An enzyme-linked immunosorbent assay (Elisa), using as antigen canine coronavirus-infected CrFK cell supernatant, was developed to detect antibodies against canine coronavirus (CCoV). Out of a total of 109 dog serum samples, 80 which were positive by routine virus neutralisation test were also Elisa positive. Seventeen samples which were negative by the virus neutralisation test, were positive by Elisa and by the confirmatory Western blotting test. The Elisa was substantially more sensitive than the virus neutralisation test in detecting antibodies to CCoV and may be used as an alternative technique to virus neutralisation.