Rapid diagnostics and ceftazidime/avibactam for KPC-producing Klebsiella pneumoniae bloodstream infections: impact on mortality and role of combination therapy.

This study was aimed at investigating risk factors for mortality in patients suffering from KPC-producing Klebsiella pneumoniae (KPC-Kp) bloodstream infections (BSIs), evaluating the impact of rapid diagnostics and ceftazidime/avibactam use. This observational retrospective study (January 2017-May 2021) included all patients with a KPC-Kp BSI. Uni-multivariable analyses were carried out to evaluate the effect of clinical variables on both in-hospital death (IHD) and 30-day all-cause mortality, and the role of the combination of ceftazidime/avibactam plus polymyxin. One hundred and ninety-six patients met the study's inclusion criteria. Older age, having undergone renal replacement therapy during the 30 days preceding the KPC-Kp BSI onset, having an INCREMENT-CPE score ≥ 8, and having suffered from a superimposed and/or following KPC-Kp BSI treatment candidemia were found to be the main factors associated with both mortality rates. Among protective factors, the centrality of ceftazidime/avibactam in monotherapy (IHD: OR: 0.34; CI 95%: 0.11-1.00-30-day all-cause mortality: OR: 0.18; CI 95%: 0.04-0.77) or combination (IHD: OR: 0.51; CI 95%: 0.22-1.19-30-day all-cause mortality: OR: 0.62; CI 95%: 0.21-1.84) emerged and became even more evident once the effect of ceftazidime/avibactam plus polymyxin was removed. Rapid diagnostics may be useful to adopt more effective strategies for the treatment of KPC-Kp BSI patients and implement infection control measures, even if not associated with higher patient survival. Ceftazidime/avibactam, even when used alone, represents an important option against KPC-Kp, while combined use with polymyxin might not have altered its efficacy. Patient comorbidities, severity of BSI, and complications such as candidemia were confirmed to have a significant burden on survival.

Burden of primary influenza and respiratory syncytial virus pneumonia in hospitalised adults: insights from a 2-year multi-centre cohort study (2017-2018).

BACKGROUND: Viral community-acquired pneumonia (CAP) is a potentially serious illness, particularly in adult patients with underlying chronic conditions. In addition to the most recent SARS-CoV-2, influenza, and respiratory syncytial virus (RSV) are considered the most relevant causes of viral CAP. AIMS: To describe the clinical features of hospitalised adults admitted for influenza-A/B and RSV pneumonia and analyse, according to aetiology, factors associated with non-invasive ventilation (NIV) failure and in-hospital death (IHD). METHODS: This was a retrospective and multi-centre study of all adults who were admitted for laboratory-confirmed influenza-A/B or RSV pneumonia, during two consecutive winter seasons (October-April 2017-2018 and 2018-2019) in three tertiary hospitals in Portugal, Italy and Cyprus. RESULTS: A total of 356 adults were included in the study. Influenza-A, influenza-B and RSV were deemed to cause pneumonia in 197 (55.3%), 85 (23.9%) and 74 (20.8%) patients, respectively. Patients with both obstructive sleep apnoea or obesity hypoventilation syndrome and influenza-A virus pneumonia showed a higher risk for NIV failure (odds ratio (OR) 4.66; 95% confidence interval (CI) 1.42-15.30). Patients submitted to NIV showed a higher risk for IHD, regardless of comorbidities (influenza-A OR 3.00; 95% CI 1.35-6.65, influenza-B OR 4.52; 95% CI 1.13-18.01, RSV OR 5.61; 95% CI 1.26-24.93). CONCLUSION: The increased knowledge of influenza-A/B and RSV pneumonia burden may contribute to a better management of patients with viral CAP.

Rapid determination of ceftazidime/avibactam susceptibility of carbapenemase-producing Enterobacterales directly from blood cultures: a comparative evaluation of EUCAST disc diffusion RAST and direct Etest® RAST.

OBJECTIVES: To evaluate the performance of two rapid antimicrobial susceptibility testing (RAST) methods to determine ceftazidime/avibactam susceptibility directly from blood cultures (BCs). METHODS: A total of 246 Escherichia coli or Klebsiella pneumoniae isolates were tested for ceftazidime/avibactam susceptibility directly from BC bottles using EUCAST RAST and Etest® RAST. Results obtained after 4, 6 and 8 h of incubation were compared with those obtained by reference broth microdilution on pure overnight subcultures. RESULTS: In total, the proportion of readable zones after 4 h of incubation was 96.7% and reached 100% after 6 and 8 h of incubation. EUCAST RAST yielded >98% of categorical agreement (CA) with all reading times. Major error (ME) and very major error (VME) rates were inferior to 3%, for each of the reading times. The proportion of results in the area of technical uncertainty (ATU) was almost similar (3.8%-4.1%) at the different reading times. DET-RAST yielded 97.5%, 98% and 99.6% of CA with readings at 4, 6 and 8 h, respectively. One (0.6%) ME was observed at each reading time, whereas five (5.9%) and four (4.5%) VMEs were observed analysing readings at 4 and 6 h, respectively. No VME was observed with readings at 8 h. CONCLUSIONS: EUCAST RAST was accurate to determine ceftazidime/avibactam susceptibility of carbapenemase-producing K. pneumoniae and E. coli directly from BC bottles. DET-RAST has the advantage of determining MIC values and avoiding ATU results but showed to be an accurate method only with reading at 8 h.

Occurrence of multi-carbapenemases producers among carbapenemase-producing Enterobacterales and in vitro activity of combinations including cefiderocol, ceftazidime-avibactam, meropenem-vaborbactam, and aztreonam in the COVID-19 era.

PURPOSE: To evaluate the prevalence of multi-carbapenemase-producing Enterobacterales (EB) and the activity of cefiderocol (CFDC), meropenem-vaborbactam (MEV), ceftazidime-avibactam (CZA), and combinations of CZA plus aztreonam (ATM), MEV plus ATM and CFDC plus CZA against them. METHODS: A collection of carbapenemase-producing EB clinical isolates (n = 1242) was investigated by lateral flow immunoassay NG-Test CARBA-5 and molecular testing. Cefiderocol MICs were determined using broth microdilution SensititreTM panel. MICs of CZA and MEV were determined by the gradient diffusion method. Antimicrobial synergy testing was performed using gradient diffusion strip crossing. RESULTS: KPC were the most frequent carbapenemases (83.2%), followed by VIM (9.2 %), OXA-48-like (4.3 %) and NDM enzymes (4.1%). Multi-carbapenemase producers were found in 10 (0.8%) isolates. Three combinations of two different carbapenemases were observed: KPC+VIM (n = 4), NDM+OXA-48-like (n = 4), and VIM+OXA-48-like (n = 2). CFDC showed potent activity against eight out of ten dual-carbapenemases producers, while resistance or reduced susceptibility was shown towards CZA and MEV. CFDC in combination with CZA showed no synergistic effects and only two additive effects on seven (87.5%) of the CFDC-susceptible strains. Conversely, CZA plus ATM and MEV plus ATM combinations were synergistic against all ATM-resistant strains regardless of dual-carbapenemases phenotype. CONCLUSIONS: The occurrence of multi-carbapenemase producers is not uncommon in Northern Italy area. MEV in combination with ATM might be considered as a potential therapeutic option, alternative to CZA plus ATM. CFDC susceptibility testing and synergy evaluation of ATM-based combinations should be performed in the lab routine to evaluate the most in vitro active antimicrobial regimen.

Direct detection of extended-spectrum-ß-lactamase-producers in Enterobacterales from blood cultures: a comparative analysis.

Accurate detection of extended-spectrum-ß-lactamase (ESBL)-producing Enterobacterales from bloodstream infection (BSI) is of paramount importance for both epidemiological and clinical purposes, especially for optimization of antibiotic stewardship interventions. Three phenotypic methods for the detection of ESBL phenotype in Klebsiella pneumoniae and Escherichia coli BSI were compared over a 4-month period (May-August 2021) in a main University Hospital from Northern Italy. The methods were the biochemical Rapid ESBL NP®, the immunological NG-Test CTX-M MULTI®, and the E-test technique based on ESBL E-test®. One hundred forty-two blood cultures (BCs) positive for K. pneumoniae or E. coli were included. ESBL and carbapenemase phenotype were detected in 26.1% (n = 37) and 16.9% (n = 24), respectively. The Rapid ESBL NP®, NG-Test CTX-M MULTI®, and direct ESBL E-test® positive and negative predictive values with 95% confidence intervals were 1 (0.87-1) and 0.97 (0.92-0.99), 1 (0.87-1) and 0.97 (0.92-0.99), and 1 (0.88-1) and 1 (0.96-1), respectively. The three phenotypic methods evaluated showed good performance in the detection of ESBL phenotype from K. pneumoniae- or E. coli-positive BCs. Rapid ESBL NP® and NG-test CTX-M® offer the important advantage of a turnaround time of 15 to 45 min, and the Rapid ESBL NP test in addition detects any type of ESBL producers.

In vitro activity of cefiderocol against ceftazidime-avibactam susceptible and resistant KPC-producing Enterobacterales: cross-resistance and synergistic effects.

PURPOSE: To assess the in vitro activity of cefiderocol (CFDC) against a collection of both ceftazidime-avibactam (CZA) susceptible and resistant KPC-producing Enterobacterales (KPC-EB) isolates. Secondly, to assess its synergistic activity in combination with different antibiotics. METHODS: One hundred KPC-EB isolates were tested: 60 CZA susceptible and 40 CZA resistant. Among them, 17 pairs of CZA susceptible and resistant KPC-producing Klebsiella pneumoniae (KPC-Kp) isolates were collected from 17 distinct patients before and after CZA treatment, respectively. CFDC susceptibility was evaluated by both broth microdilution (lyophilized panels; Sensititre; Thermo Fisher) and disk diffusion testing. Results were interpreted using EUCAST breakpoints. Synergistic activity of CFDC in combination with CZA, meropenem-vaborbactam, imipenem, and amikacin against six characterized KPC-Kp strains, before and after acquisition of CZA resistance, was evaluated using gradient diffusion strip crossing method. RESULTS: CFDC resistance rate was significantly higher in CZA resistant EB subset than in the susceptible one (p < 0.001): 82.5% vs 6.7%. MIC50 and MIC90 values were 0.25 and 2 mg/L, 8 and 64 mg/L in CZA-susceptible and CZA-resistant subset, respectively. KPC-Kp isolates harboring KPC-D179Y or KPC-Δ242-GT-243 variants showed CFDC MICs ranging from 4 to 64 mg/L. CFDC showed in vitro synergistic effect mostly with CZA, against both CZA susceptible and resistant isolates, resulting in a synergy rate of 66.7%. CONCLUSIONS: CZA resistance mechanisms in KPC-EB impair the in vitro activity of CFDC, often leading to co-resistance. CFDC in combination with the new ß-lactamases inhibitors might represent a strategy to enhance its activity.

Severity of RSV infection in Southern European elderly patients during two consecutive winter seasons (2017-2018).

In Europe, the respiratory syncytial virus (RSV) surveillance system is very heterogeneous and there is growing evidence of the importance of RSV infections resulting in hospitalization of elderly patients. The aim of this study was to assess the severity of RSV infection in the elderly living in the aged Southern European countries. We conducted a retrospective study of elderly patients ( ≥65-year old) admitted for laboratory-confirmed RSV infection in three tertiary hospitals in Portugal, Italy, and Cyprus over two consecutive winter seasons (2017-2018). Uni-multivariable analyses were carried out to evaluate the effect of clinical variables on radiologically confirmed pneumonia, use of noninvasive ventilation (NIV), and in-hospital death (IHD). A total of 166 elderly patients were included. Pneumonia was evident in 29.5%. NIV was implemented in 16.3%, length of stay was 11.8 ± 12.2 days, and IHD occurred in 12.1%. Multivariable analyses revealed that the risk of pneumonia was higher in patients with chronic kidney disease (CKD) (odds ratio [OR]: 2.57; 95% confidence interval [CI]: 1.12-5.91); the use of NIV was higher in patients with obstructive sleep apnea or obesity hypoventilation syndrome (OSA or OHS) (OR: 5.38; 95% CI: 1.67-17.35) and CKD (OR: 2.52; 95% CI: 1.01-6.23); the risk of IHD was higher in males (OR: 3.30; 95% CI: 1.07-10.10) and in patients with solid neoplasm (OR: 9.06; 95% CI: 2.44-33.54) and OSA or OHS (OR: 8.39; 95% CI: 2.14-32.89). Knowledge of factors associated with RSV infection severity may aid clinicians to set priorities and reduce disease burden. Development of effective antiviral treatment and vaccine against RSV is highly desirable.

Fast-track identification of CTX-M-extended-spectrum-ß-lactamase- and carbapenemase-producing Enterobacterales in bloodstream infections: implications on the likelihood of deduction of antibiotic susceptibility in emergency and internal medicine departments.

This study aims at presenting a reliable fast-track diagnostics for the detection of CTX-M ESBL- (CTX-M-p) and carbapenemase-producers (CA-p) directly from blood cultures (BCs) of patients with Enterobacterales (EB) bloodstream infections (BSIs) admitted in emergency and internal medicine departments and its contribution in estimation of in vitro antibiotic susceptibility. A fast-track workflow including MALDI-TOF species identification and two lateral flow immunochromatographic assays for the detection of CTX-M-p and CA-p directly from BCs was performed in parallel with conventional routine, and results were compared. A total of 236 BCs of patients suffering from EB BSI were included. Accuracy of the fast-track workflow ranged from 99.6 to 100%. Among E. coli isolates, CTX-M-p (20.5%) were susceptible to ceftolozane-tazobactam (C/T, 97%), ceftazidime-avibactam (CZA, 100%), and piperacillin-tazobactam (TZP, 84.8%), whereas CTX-M-and-main-carbapenemases-non-producer (CTX-M-CA-np, 79.5%) isolates were susceptible to all the antibiotics tested. Among K. pneumoniae isolates, CTX-M-p (23.3%) were poorly susceptible to TZP (40%) but widely susceptible to C/T (90%), CZA (100%), and amikacin (90%), whereas CTX-M-CA-np (55.8%) were also susceptible to cefepime. CA-p K. pneumoniae (20.9%) were susceptible to CZA (88.9%). All the species other than E. coli and K. pneumoniae were CTX-M-CA-np and were widely susceptible to the antibiotics tested except for isolates of the inducible and derepressed AmpC- or AmpC/ESBL-p species. Rapid identification of species and phenotype together with knowledge of local epidemiology may be crucial to determine the likelihood of deduction of in vitro antibiotic susceptibility on the same day of positive BC processing.

Comparative virulence of seasonal viruses responsible for lower respiratory tract infections: a southern European multi-centre cohort study of hospital admissions.

OBJECTIVES: Our study aimed to compare the clinical severity of lower respiratory tract infections (LRTI's) caused by Influenza and Respiratory Syncytial Virus (RSV). METHODS: We conducted a retrospective cohort study of LRTI admissions with positive PCR results for Influenza or RSV from 2017 to 2019 in three teaching hospitals in southern Europe. Data on clinical characteristics, viral agents and disease outcome were collected. Nosocomial infection was excluded. Main outcomes were invasive mechanical ventilation and in-hospital death. RESULTS: A total of 984 patients were included. Median age was 75 years. Influenza A was the most frequently identified virus (56.5%), of which 27.1% were subtype H1N1 and 53.0% H3N2. Influenza B was isolated in 22.3% and RSV in 21.0%. There were 10.5% of patients who died during admission and 8.3% submitted to IMV. Influenza A H1N1 was associated with lower age and less co-morbidity, while the opposite was observed for RSV. Influenza A H1N1 was independently associated with both higher risk of death (adjusted odds ratio 2.0 [1.2-3.4] p = 0.008) and IMV (adjusted odds ratio 5.1 [3.0-8.5] p < 0.001). CONCLUSION: Influenza A H1N1 was an independent predictor of mortality and IMV. These findings may have implications on hospital resource planning and vaccination policies.

Influenza and respiratory syncytial virus infections in the oldest-old continent.

SARS-CoV-2 dramatically revealed the sudden impact of respiratory viruses in our lives. Influenza and respiratory syncytial virus (RSV) infections are associated with high rates of morbidity, mortality, and an important burden on healthcare systems worldwide, especially in elderly patients. The aim of this study was to identify severity predictors in the oldest-old admitted with influenza and/or RSV infections. This is a multicenter, retrospective study of all oldest-old patients (≥ 85 years old) admitted for laboratory-confirmed influenza and/or RSV infection in three tertiary hospitals in Portugal, Italy, and Cyprus over two consecutive winter seasons. The outcomes included the following: pneumonia on infection presentation, use of non-invasive ventilation (NIV), and in-hospital death (IHD). The association with possible predictors, including clinical features and type of virus infection, was assessed using uni- and multivariable analyses. A total of 251 oldest-old patients were included in the study. Pneumonia was evident in 32.3% (n = 81). NIV was implemented in 8.8% (n = 22), and IHD occurred in 13.9% (n = 35). Multivariable analyses revealed that chronic obstructive pulmonary disease (COPD) or asthma was associated with pneumonia (OR 1.86; 95% CI 1.02-3.43; p = 0.045). COPD or asthma (OR 4.4; 95% CI 1.67-11.6; p = 0.003), RSV (OR 3.12; 95% CI 1.09-8.92; p = 0.023), and influenza B infections (OR 3.77; 95% CI 1.06-13.5; p = 0.041) were associated with NIV use, respectively, while chronic kidney disease was associated with IHD (OR 2.50; 95% CI 1.14-5.51; p = 0.023). Among the oldest-old, chronic organ failure, such as COPD or asthma, and CKD predicted pneumonia and IHD, respectively, beyond the importance of viral virulence itself. These findings could impact on public health policies, such as fostering influenza immunization campaigns, home-based care programs, and end-of-life care. Filling knowledge gaps is crucial to set priorities and advise on transition model of care that best fits the oldest-old.

Direct ß-Lactam Inactivation Method: a New Low-Cost Assay for Rapid Detection of Carbapenemase- or Extended-Spectrum-ß-Lactamase-Producing Enterobacterales Directly from Positive Blood Culture Bottles.

We validate and evaluate a new phenotypic assay, named the direct ß-lactam inactivation method (dBLIM), for the rapid and simultaneous detection of carbapenemase or extended-spectrum-cephalosporinase activity directly from Enterobacterales (EB)-positive blood cultures (BCs). It originates from the carbapenem inactivation method (CIM), an inexpensive and highly sensitive assay for carbapenemase activity detection. dBLIM cutoff values to detect extended-spectrum ß-lactamase (ESBL) and carbapenemase activities resulted in diameters of ≤12 mm for a 5-µg-cefotaxime disk and for a 10-µg-meropenem disk. dBLIM assessment was determined with both aerobic and anaerobic BC bottles spiked with 422 characterized EB strains, classifiable into the following 4 phenotypic groups: (i) ESBL/AmpC-type ß-lactamase (ACBL)/carbapenemase (CARB)-nonproducing (np-ESBL/ACBL/CARB) EB (n = 116), (ii) ESBL-producing EB (n = 111), (iii) AmpC-ß-lactamase-producing EB (n = 33), and (iv) carbapenemase-producing EB (n = 162). No false-positive results were obtained in any of the np-ESBL/ACBL/CARB EB, ESBL, and AmpC groups, demonstrating an overall assay specificity of 100%. There were no significant discrepancies in dBLIM performance between aerobic and anaerobic BCs across all groups, except with VIM-type carbapenemase-expressing EB. Interestingly, among BCs spiked with blaVIM-harboring EB, the sensitivity rates of the assay in anaerobic and aerobic bottles were 53.6% and 100%, respectively. In contrast, dBLIM performance was deemed excellent for the KPC, OXA-48, and NDM carbapenemase producers regardless of the type of bottle being tested, with a sensitivity rate ranging between 99% and 100%. Concerning the detection of the extended-spectrum cephalosporinases of the ESBL-producing and AmpC types, dBLIM sensitivities was 100% and 84 to 87%, respectively. dBLIM may be a cost-effective and highly robust phenotypic screening method for the reliable detection of carbapenemases or extended-spectrum cephalosporinases directly from BCs on the same day of bottle positivity detection.

Host-microbiota interactions shed light on mortality events in the striped venus clam Chamelea gallina.

Mass mortalities due to disease outbreaks have recently affected a number of major taxa in marine ecosystems. Climate- and pollution-induced stress may compromise host immune defenses, increasing the risk of opportunistic diseases. Despite growing evidence that mass mortality events affecting marine species worldwide are strongly influenced by the interplay of numerous environmental factors, the reductionist approaches most frequently used to investigate these factors hindered the interpretation of these multifactorial pathologies. In this study, we propose a broader approach based on the combination of RNA-sequencing and 16S microbiota analyses to decipher the factors underlying mass mortality in the striped venus clam, Chamelea gallina, along the Adriatic coast. On one hand, gene expression profiling and functional analyses of microbial communities showed the over-expression of several genes and molecular pathways involved in xenobiotic metabolism, suggesting potential chemical contamination in mortality sites. On the other hand, the down-regulation of several genes involved in immune and stress response, and the over-representation of opportunistic pathogens such as Vibrio and Photobacterium spp. indicates that these microbial species may take advantage of compromised host immune pathways and defense mechanisms that are potentially affected by chemical exposure, resulting in periodic mortality events. We propose the application of our approach to interpret and anticipate the risks inherent in the combined effects of pollutants and microbes on marine animals in today's rapidly changing environment.

Casein composition and differential translational efficiency of casein transcripts in donkey's milk.

The amount of the four caseins (αs1, αs2, ß and κ-CN) in donkey milk was evaluated by Urea-PAGE analysis at pH 8.6, followed by immuno-detection with polyclonal antibodies, coupled to densitometric analysis. The results showed the percentage of each casein in decreasing order: ß (54.28) > αs1 (35.59) > αs2 (7.19) > κ-CN (2.79). The mRNA quantification of donkey casein transcripts, carried out by RT-qPCR, showed that the average percentage of corresponding gene transcripts (CSN2, CSN1S1, CSN1S2 I and CSN3) was 70.85, 6.28, 14.23 and 8.65, respectively. The observed translation efficiency, assessed as percentage of single milk casein fraction out of single percentage of transcript, was 0.76, 5.66, 0.50 and 0.32, respectively. The analysis of the sequences flanking the start codon, the codon usage frequencies and the coding sequence length might explain, at least in part, the differential transcriptional and translational rate observed among the casein transcripts.

A fast and reliable polymerase chain reaction method based on short interspersed nuclear elements detection for the discrimination of buffalo, cattle, goat, and sheep species in dairy products.

OBJECTIVE: Aim of present study was the set up of a fast and reliable protocol using species-specific markers for the quali-quantitative analysis of DNA and the detection of ruminant biological components in dairy products. For this purpose, the promoter of the gene coding for the α-lactoalbumin (LALBA) was chosen as possible candidate for the presence of short interspersed nuclear elements (SINEs). METHODS: DNA was isolated from somatic cells of 120 individual milk samples of cattle (30), Mediterranean river buffalo (30), goat (30), and sheep (30) and the gene promoter region (about 600/700 bp) of LALBA (from about 600 bp upstream of exon 1) has been sequenced. For the development of a single polymerase chain reaction (PCR) protocol that allows the simultaneous identification of DNA from the four species of ruminants, the following internal primers pair were used: 5'-CACTGATCTTAAAGCTCAGGTT-3' (forward) and 5'-TCAGA GTAGGCCACAGAAG-3' (reverse). RESULTS: Sequencing results of LALBA gene promoter region confirmed the presence of SINEs as monomorphic "within" and variable in size "among" the selected species. Amplicon lengths were 582 bp in cattle, 592 bp in buffalo, 655 in goat and 729 bp in sheep. PCR specificity was demonstrated by the detection of trace amounts of species-specific DNA from mixed sources (0.25 ng/µL). CONCLUSION: We developed a rapid PCR protocol for the quali-quantitative analysis of DNA and the traceability of dairy products using a species-specific marker with only one pair of primers. Our results validate the proposed technique as a suitable tool for a simple and inexpensive (economic) detection of animal origin components in foodstuffs.

Milk microRNA-146a as a potential biomarker in bovine tuberculosis.

In this research communication we exploited the potential use of milk microRNAs (miRs) as biomarkers for bovine tuberculosis (bTB). bTB is a zoonotic disease caused by Mycobacterium bovis which affects animal health, influencing herd economic sustainability. Diagnosis is based on skin delayed-type hypersensitivity reaction and quantification of interferon gamma but both techniques are influenced by several confounding factors. Thus, new methods for early diagnosis are required. In this context, microRNAs have been used as promising biomarkers for both infectious and non-infectious diseases. To determine the possible involvement of microRNAs in bTB, we analysed the expression of four immune-related miRs in 200 cows grouped in cases and controls with respect to positivity to tuberculosis. The analysis showed a different magnitude of expression in the groups indicating that active tuberculosis could influence miRs expression. We used expression values of miR-146a, the highest differentially expressed miR, for Receiver operating characteristic (ROC) curve analysis. In order to determine a test cut-off value for miR-146a expression that would differentiate cases and controls, a value for the miR-146a expression higher than 8 was selected as this gave a test specificity and sensitivity of 80·0% and 86·0% respectively. These values confirm the possibility of using miR-146a as a milk prognostic biomarker for bovine tuberculosis.

Molecular characterisation, genetic variability and detection of a functional polymorphism influencing the promoter activity of OXT gene in goat and sheep.

The purpose of the study described in this Research Communication was to report the full characterisation of the goat and sheep oxytocin-neurophysin I gene (OXT), their promoters and amino acid sequences. Using the genomic DNA as template, we sequenced and compared the whole OXT gene (3 exons), plus 958/960 nucleotides at the 5' flanking region and 478/477 nucleotides at the 3' flanking region, in 46 sheep and 24 goats belonging to different breeds/genetic types reared in Italy, Greece and Germany. The comparison of the obtained sequences showed a high degree of genetic variability at these loci. In particular, we focused on the SNP g.438T > C as possible example of trans-specific polymorphism. This SNP alters a putative binding site of the transcription factor Oct-1. The set-up of a luciferase assay confirmed that the C variant of this SNP negatively affects the promoter activity of the sheep OXT gene. The results of this study suggest that the SNP g.438T > C might be useful to promote association studies with traits/physiological processes controlled by this hormone.

Extracellular Self-DNA Effects on Yeast Cell Cycle and Transcriptome during Batch Growth.

The cell cycle and the transcriptome dynamics of yeast exposed to extracellular self-DNA during an aerobic batch culture on glucose have been investigated using cytofluorimetric and RNA-seq analyses. In parallel, the same study was conducted on yeast cells growing in the presence of (heterologous) nonself-DNA. The self-DNA treatment determined a reduction in the growth rate and a major elongation of the diauxic lag phase, as well as a significant delay in the achievement of the stationary phase. This was associated with significant changes in the cell cycle dynamics, with slower exit from the G0 phase, followed by an increased level of cell percentage in the S phase, during the cultivation. Comparatively, the exposure to heterologous DNA did not affect the growth curve and the cell cycle dynamics. The transcriptomic analysis showed that self-DNA exposure produced a generalized downregulation of transmembrane transport and an upregulation of genes associated with sulfur compounds and the pentose phosphate pathway. Instead, in the case of the nonself treatment, a clear response to nutrient deprivation was detected. Overall, the presented findings represent further insights into the complex functional mechanisms of self-DNA inhibition.