AP-1γ2 is an adaptor protein 1 variant required for endosome-to-Golgi trafficking of the mannose-6-P receptor (CI-MPR) and ATP7B copper transporter.

Selective retrograde transport from endosomes back to the trans-Golgi network (TGN) is important for maintaining protein homeostasis, recycling receptors, and returning molecules that were transported to the wrong compartments. Two important transmembrane proteins directed to this pathway are the Cation-Independent Mannose-6-phosphate receptor (CI-MPR) and the ATP7B copper transporter. Among CI-MPR functions is the delivery of acid hydrolases to lysosomes, while ATP7B facilitates the transport of cytosolic copper ions into organelles or the extracellular space. Precise subcellular localization of CI-MPR and ATP7B is essential for the proper functioning of these proteins. This study shows that both CI-MPR and ATP7B interact with a variant of the clathrin adaptor 1 (AP-1) complex that contains a specific isoform of the γ-adaptin subunit called γ2. Through synchronized anterograde trafficking and cell-surface uptake assays, we demonstrated that AP-1γ2 is dispensable for ATP7B and CI-MPR exit from the TGN while being critically required for ATP7B and CI-MPR retrieval from endosomes to the TGN. Moreover, AP-1γ2 depletion leads to the retention of endocytosed CI-MPR in endosomes enriched in retromer complex subunits. These data underscore the importance of AP-1γ2 as a key component in the sorting and trafficking machinery of CI-MPR and ATP7B, highlighting its essential role in the transport of proteins from endosomes.

HIV-1 Nef Changes the Proteome of T Cells Extracellular Vesicles Depleting IFITMs and Other Antiviral Factors.

Extracellular vesicles (EVs) are biomolecule carriers for intercellular communication in health and disease. Nef is a HIV virulence factor that is released from cells within EVs and is present in plasma EVs of HIV-1 infected individuals. We performed a quantitative proteomic analysis to fully characterize the Nef-induced changes in protein composition of T cell-derived EVs and identify novel host targets of HIV. Several proteins with well-described roles in infection or not previously associated with HIV pathogenesis were specifically modulated by Nef in EVs. Among the downregulated proteins are the interferon-induced transmembrane 1, 2, and 3 (IFITM1-3) proteins, broad-spectrum antiviral factors known to be cell-to-cell transferable by EVs. We demonstrate that Nef depletes IFITM1-3 from EVs by excluding these proteins from the plasma membrane and lipid rafts, which are sites of EVs biogenesis in T cells. Our data establish Nef as a modulator of EVs' global protein content and as an HIV factor that antagonizes IFITMs.

Molecular diagnosis of Cytomegalovirus infection: clinical performance of the Aptima transcription-mediated amplification assay toward conventional qPCR chemistry on whole blood samples.

Human Cytomegalovirus (HCMV) infection is life-threatening for immunocompromised patients. Quantitative molecular assays on whole blood or plasma are the gold standard for the diagnosis of invasive HCMV infection and for monitoring antiviral treatment in individuals at risk of HCMV disease. For these reasons, an accurate standardization toward the WHO 1st International Standard among different centers and diagnostic kits represents an effort for better clinical management of HCMV-positive patients. Herein, we evaluate, for the first time, the performance of a new transcription-mediated amplification (TMA) assay versus quantitative polymerase chain reaction (qPCR) chemistry, used as a routine method, on whole blood samples. A total of 755 clinical whole blood specimens were collected and tested simultaneously with TMA and qPCR assays. The data showed a qualitative agreement of 99.27% for positive quantified samples and 89.39% for those undetected between the two tested methods. Evaluation of viremia in positive samples highlighted a good correlation between TMA and qPCR chemistries in terms of International Units (ΔLog10 IU/mL: -0.29 ± 0.40). The TMA assay showed a significant correlation with qPCR in patients monitored for up to 3 months, thus allowing an accurate assessment of viremia in transplant patients. Therefore, TMA chemistry showed good agreement with qPCR testing, used as a current diagnostic routine. It also offers important advantages, such as FDA approval on plasma and In Vitro Diagnostic (IVD) on both plasma and whole blood, automated workflow with minimal hands-on time, and random access loading, thus enabling a rapid and reliable diagnostic in HCMV-infected patients. IMPORTANCE: In this paper, we describe the clinical performance of a novel transcription-mediated amplification (TMA) assay for the detection and quantification of human Cytomegalovirus (HCMV) DNA from whole blood samples. This is a pivotal analysis in immunocompromised patients [transplanted, HIV-positive, and Hematopoietic Stem Cell (HSC) recipients], and molecular tests with high sensitivity and specificity are necessary to evaluate the HCMV viral load in these patients. To our knowledge, this is the first in-depth evaluation of TMA chemistry for HCMV diagnosis on whole blood samples. Moreover, also technical aspects of this assay make it suitable for clinical diagnostics.

Detection of volatile organic compounds as new paradigm to accelerate antimicrobial susceptibility testing: performance evaluation of VITEK® REVEAL™.

OBJECTIVES: The measurement of VOCs release in the headspace of a bacterial culture represents a new approach to rapidly assess antimicrobial susceptibility. Herein, we evaluated the diagnostic performance of the VITEK® REVEAL™ system directly from a collection of Gram-negative positive blood cultures. MATERIALS AND METHODS: One hundred and twenty-eight positive blood cultures were included in the analysis (Enterobacterales, n = 95; Pseudomonas aeruginosa, n = 21; Acinetobacter baumannii complex, n = 12). Samples were processed using VITEK® REVEAL™ according to the manufacturer's recommendations, and MICs of 22 antimicrobials were compared with those obtained using reference methods. Categorical agreement (CA), essential agreement (EA) and categorical errors were calculated. RESULTS: Overall, 2220 strain/antibiotic pair combinations were analysed. Of these, most were classified as resistant by reference antimicrobial susceptibility testing (1091/2220; 48.7%). The overall CA and EA were 97.6% and 97.7%, respectively. CA ranged from 97.5% in Enterobacterales to 97.9% in both P. aeruginosa and A. baumannii complex. The overall number of categorical discrepancies were: 18 very major errors (1.6%), 13 major errors (1.2%) and 22 minor errors (2.4%). EA ranged from 95.2% in P. aeruginosa to 98.1% in Enterobacterales. Screening test for ESBL phenotype was positive, indeterminate and negative in 13.7%, 32.6% and 27.4% of Enterobacterales isolates tested by both VITEK® REVEAL™ and the reference method, showing 100% CA. CONCLUSIONS: VITEK® REVEAL™ represents a reliable tool to obtain antimicrobial susceptibility results of the main Gram-negative species directly from positive blood cultures with time to results of less than 8 h.

Multidrug-resistant pathogens and ventilator-associated pneumonia in critically ill COVID-19 and non-COVID-19 patients: a prospective observational monocentric comparative study.

BACKGROUND: The COVID-19 pandemic has increased the incidence of ventilator-associated pneumonia (VAP) among critically ill patients. However, a comparison of VAP incidence in COVID-19 and non-COVID-19 cohorts, particularly in a context with a high prevalence of multidrug-resistant (MDR) organisms, is lacking. MATERIAL AND METHODS: We conducted a single-center, mixed prospective and retrospective cohort study comparing COVID-19 patients admitted to the intensive care unit (ICU) of the "Città della Salute e della Scienza" University Hospital in Turin, Italy, between March 2020 and December 2021 (COVID-19 group), with a historical cohort of ICU patients admitted between June 2016 and March 2018 (NON-COVID-19 group). The primary objective was to define the incidence of VAP in both cohorts. Secondary objectives were to evaluate the microbial cause, resistance patters, risk factors and impact on 28 days, ICU and in-hospital mortality, duration of ICU stay, and duration of hospitalization). RESULTS: We found a significantly higher incidence of VAP (51.9% - n = 125) among the 241 COVID-19 patients compared to that observed (31.2% - n = 78) among the 252 NON-COVID-19 patients. The median SOFA score was significantly lower in the COVID-19 group (9, Interquartile range, IQR: 7-11 vs. 10, IQR: 8-13, p < 0.001). The COVID-19 group had a higher prevalence of Gram-positive bacteria-related VAP (30% vs. 9%, p < 0.001), but no significant difference was observed in the prevalence of difficult-to-treat (DTR) or MDR bacteria. ICU and in-hospital mortality in the COVID-19 and NON-COVID-19 groups were 71% and 74%, vs. 33% and 43%, respectively. The presence of COVID-19 was significantly associated with an increased risk of 28-day all-cause hospital mortality (Hazard ratio, HR: 7.95, 95% Confidence Intervals, 95% CI: 3.10-20.36, p < 0.001). Tracheostomy and a shorter duration of mechanical ventilation were protective against 28-day mortality, while dialysis and a high SOFA score were associated with a higher risk of 28-day mortality. CONCLUSION: COVID-19 patients with VAP appear to have a significantly higher ICU and in-hospital mortality risk regardless of the presence of MDR and DTR pathogens. Tracheostomy and a shorter duration of mechanical ventilation appear to be associated with better outcomes.

Performance evaluation of Bruker UMIC® microdilution panel and disc diffusion to determine cefiderocol susceptibility in Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Achromobacter xylosoxidans and Burkolderia species.

PURPOSE: Cefiderocol susceptibility testing (AST) represents an open challenge for clinical microbiology. Herein, we evaluated the performance of the UMIC® Cefiderocol broth microdilution (BMD) test and disc diffusion on Gram-negative species. METHODS: UMIC® Cefiderocol BMD test, disc diffusion and reference BMD were in parallel performed on a collection of 256 clinical isolates. Categorical agreement (CA), essential agreement (EA), bias, major errors (MEs) and very major errors (VMEs) were calculated for both AST methods. RESULTS: The UMIC® Cefiderocol BMD strip exhibited an EA < 90% (85.5%), a CA higher than 90% (93.7%) and a low number of VMEs (n = 4, 4.2%) and MEs (n = 12, 7.4%). UMIC® Cefiderocol identified 96.2% of the resistant isolates [Enterobacterales, (39/40); P. aeruginosa (19/19); A. xylosoxidans (5/6); S. maltophilia (5/6); Burkholderia spp. (8/8)]. Disc diffusion showed a high CA (from 94.9 to 100%) regardless of disc manufacturer in Enterobacterales, P. aeuroginosa, A. baumannii and S. maltophilia. However, high rates of results falling in the area of technical uncertainty (ATU) were observed in Enterobacterales (34/90, 37.8%) and P. aeruginosa (16/40, 40%). Disc diffusion showed a poor performance in A. xylosoxidans and Burkholderia spp. if PK/PD breakpoint was used (overall, 5/9 VMEs; in contrast, the use of P. aeruginosa-specific breakpoints resulted in 100% of CA with 24.6% of results in the ATU). CONCLUSION: In conclusion, disc diffusion and UMIC® Cefiderocol are valid methods for the determination of cefiderocol susceptibility. Given the high number of results in the ATU by disc diffusion, a combined use of both AST methods may represent a solution to overcome the challenge of cefiderocol susceptibility testing in routine microbiology laboratories.

Prevalence and mortality of ceftazidime/avibactam-resistant KPC-producing Klebsiella pneumoniae bloodstream infections (2018-2022).

INTRODUCTION: Ceftazidime/avibactam-resistance in Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) is a topic of great interest for epidemiological, diagnostic, and therapeutical reasons. However, data on its prevalence and burden on mortality in patients with bloodstream infection (BSI) are lacking. This study was aimed at identifying risk factors for mortality in patients suffering from ceftazidime/avibactam-resistant KPC-Kp BSI. METHODS: An observational retrospective study (January 2018-December 2022) was conducted at a tertiary hospital including all consecutive hospitalized adult patients with a ceftazidime/avibactam-resistant KPC-Kp BSI. Data on baseline clinical features, management, and admission outcomes were analyzed. RESULTS: Over the study period, among all the KPC-Kp BSI events recorded, 38 (10.5%) were caused by ceftazidime/avibactam-resistant KPC-Kp strains, 37 events being finally included. The ceftazidime/avibactam-resistant KPC-Kp strains revealed susceptibility restoration to at least one carbapenem in more than 60% of cases. In-hospital and 30-day all-cause mortality rates were 22% and 16.2%, respectively. Non-survivors suffered from more baseline comorbidities and experienced a more severe ceftazidime/avibactam-resistant KPC-Kp BSI presentation (i.e., both the Pitt Bacteremia and INCREMENT-CPE scores were significantly higher). Presenting with a higher Charlson Comorbidity Index, chronic kidney disease-KDIGO stage 3A or worse-having recently gone through renal replacement therapy, having suffered from an acute kidney injury following the ceftazidime/avibactam-resistant KPC-Kp BSI, and being admitted for cardiac surgery were the strongest predictors of mortality. CONCLUSION: Ceftazidime/avibactam resistance in KPC-Kp BSI easily emerged in our highly KPC-Kp endemic area with remarkable mortality rates. Our findings might provide physicians possibly actionable information when managing patients with a ceftazidime/avibactam-resistant KPC-Kp BSI.

Effects of temperature up-shift and UV-A radiation on fatty acids content and expression of desaturase genes in cyanobacteria Microcystis aeruginosa: stress tolerance and acclimation responses.

Temperature up-shift and UV-A radiation effects on growth, lipid damage, fatty acid (FA) composition and expression of desaturase genes desA and desB were investigated in the cyanobacteria Microcystis aeruginosa. Although UV-A damaging effect has been well documented, reports on the interactive effects of UV radiation exposure and warming on cyanobacteria are scarce. Temperature and UV-A doses were selected based on the physiological responses previously obtained by studies with the same M. aeruginosa strain used in this study. Cells pre-grown at 26 °C were incubated at the same temperature or 29 °C and exposed to UV-A + PAR and only PAR for 9 days. Growth rate was significantly affected by UV-A radiation independently of the temperature throughout the experiment. High temperature produced lipid damage significantly higher throughout the experiment, decreasing at day 9 as compared to 26 °C. In addition, the cells grown at 29 °C under UV-A displayed a decrease in polyunsaturated FA (PUFA) levels, with ω3 PUFA being mostly affected at the end of exposure. Previously, we reported that UV-A-induced lipid damage affects differentially ω3 and ω6 PUFAs. We report that UV-A radiation leads to an upregulation of desA, possibly due to lipid damage. In addition, the temperature up-shift upregulates desA and desB regardless of the radiation. The lack of lipid damage for UV-A on ω3 could explain the lack of transcription induction of desB. The significant ω6 decrease at 26 °C in cells exposed to UV-A could be due to the lack of upregulation of desA.

First case of Chryseobacterium gallinarum bloodstream infection: a diagnostic and therapeutic challenge for an emerging pathogen.

Chryseobacterium spp. belongs to the Flavobacteriaceae family and is a rod-shaped gram-negative, glucose non-fermenting, non-motile bacterium ubiquitous in the environment. In humans, Chryseobacterium may be responsible for infections such as urinary tract infections (UTI) and ventriculitis with a pathogenic burden increasing in recent years. Chryseobacterium gallinarum was isolated for the first time in 2014 in a pharyngeal scrape sample of chicken and, until now, only one case of human UTI has been described in a pregnant 20-year-old Indian patient. Herein, we report the first case of bloodstream infection caused by C. gallinarum in a 67-year-old female burn patient, correctly identified by 16S-rRNA sequencing and successfully treated with cefepime and fosfomycin.

Is Silver Addition to Scaffolds Based on Polycaprolactone Blended with Calcium Phosphates Able to Inhibit Candida albicans and Candida auris Adhesion and Biofilm Formation?

Candida spp. periprosthetic joint infections are rare but difficult-to-treat events, with a slow onset, unspecific symptoms or signs, and a significant relapse risk. Treatment with antifungals meets with little success, whereas prosthesis removal improves the outcome. In fact, Candida spp. adhere to orthopedic devices and grow forming biofilms that contribute to the persistence of this infection and relapse, and there is insufficient evidence that the use of antifungals has additional benefits for anti-biofilm activity. To date, studies on the direct antifungal activity of silver against Candida spp. are still scanty. Additionally, polycaprolactone (PCL), either pure or blended with calcium phosphate, could be a good candidate for the design of 3D scaffolds as engineered bone graft substitutes. Thus, the present research aimed to assess the antifungal and anti-biofilm activity of PCL-based constructs by the addition of antimicrobials, for instance, silver, against C. albicans and C. auris. The appearance of an inhibition halo around silver-functionalized PCL scaffolds for both C. albicans and C. auris was revealed, and a significant decrease in both adherent and planktonic yeasts further demonstrated the release of Ag+ from the 3D constructs. Due to the combined antifungal, osteoproliferative, and biodegradable properties, PCL-based 3D scaffolds enriched with silver showed good potential for bone tissue engineering and offer a promising strategy as an ideal anti-adhesive and anti-biofilm tool for the reduction in prosthetic joints of infections caused by Candida spp. by using antimicrobial molecule-targeted delivery.

Role of Pel and Psl polysaccharides in the response of Pseudomonas aeruginosa to environmental challenges: oxidative stress agents (UVA, H2O2, sodium hypochlorite) and its competitor Staphylococcus aureus.

Pseudomonas aeruginosa is a versatile bacterium capable of adapting to a wide range of stress factors, including solar UVA radiation (400-315 nm). High UVA doses produce lethal effects due to the action of reactive oxygen species. Sublethal UVA doses also induces oxidative damage, but, in addition, it triggers a variety of adaptive responses, including the overexpression of pelA and pslA genes in P. aeruginosa. These genes encode the synthesis of Pel and Psl, which are essential polysaccharides in biofilm formation. The present study analysed the role of Pel and Psl in the adaptive responses generated by exposure to low UVA doses, and their importance in the response to lethal doses of UVA, hydrogen peroxide (H2O2), and sodium hypochlorite, in both planktonic cells and submerged and air-liquid interface (ALI) biofilms. It also studied the roles of Pel and Psl in P. aeruginosa-Staphylococcus aureus interaction. The results demonstrate that the capacity of sublethal UVA exposure to increase cell hydrophobicity and cell attachment and generate cross-protection phenomena in P. aeruginosa depends on the presence of Pel and Psl. The study also shows that Pel and Psl have a key role in the tolerance to lethal doses of UVA radiation, sodium hypochlorite and H2O2, in both biofilms and planktonic cells. Finally, co-culture assays showed total inhibition of S. aureus growth in presence of P. aeruginosa. This phenomenon depends, at least in part, on the simultaneous presence of Pel and Psl in planktonic cells and biofilms, suggesting a relevant role of these polysaccharides in the interaction between these species.

Long-term locoregional recurrence in patients treated for breast cancer.

BACKGROUND/PURPOSE: Locoregional control in breast cancer is a fundamental part of treatment and determinant for survival outcomes. It has been reported that most locoregional recurrence (LRR) events occur in the first 5 years after treatment. However, LRR continue to occur after this timeline, with unclear risk factors and unknown survival impact. METHODS: Retrospective singe-centered cohort of patients treated for primary breast cancer, between January 2002 and December 2004. Primary outcome was LRR; secondary outcomes were overall survival (OS), disease-free survival (DFS), and predictive factors for LRR. RESULTS: This analysis included 1001 patients, of which 959 (95%) had invasive carcinoma. A mastectomy was performed in 501 (50%) and 500 (50%) had breast conservative surgery (BCS). Median follow-up time was 197 [Inter-quartile range (IQR) 96-211] months. Global LRR rate was 7.6%, with median time to recurrence of 45 [IQR 21-91] months. There was no difference in LRR rate after mastectomy vs BCS, adjusted to tumor stage (p > 0.05). The 10-year OS and DFS rates were 68.4 and 77.8%, respectively. Factors associated with LRR were metastatic axillary lymph nodes and high histologic grade (p < 0.05). Estrogen-negative (ER) tumors had higher LRR rates than ER-positive tumors in the first 5 years (p < 0.05); but no difference was observed with longer follow-up (p > 0.05). LRR was associated with OS (p < 0.05). DISCUSSION AND CONCLUSIONS: Global LRR in this cohort was 7.6% (with over 16 years of follow-up). LRR associates with decreased OS. Time to LRR varies significantly with tumor biology, supporting differentiation of follow-up regimens.

Antibodies against hyaluronan oligosaccharides in xenotransplantation.

Carbohydrate-specific antibodies are significant mediators of xenograft rejection. This study analyzed the carbohydrate specificity of antibodies in baboons before and after xenotransplantation of organs or injection of porcine red blood cells from hDAF transgenic pigs, using a glycan array with structurally defined glycans. Antibodies against hyaluronic acid disaccharide (HA2) showed the highest reactivity at baseline and rose after xenogeneic exposure. We also investigated in the serum of baboons that underwent xenotransplantation with either hDAF or hDAF/hMCP transgenic pig organs and Lewis rats after hamster-skin xenotransplantation the specificity of anti-HA antibodies on a glycan microarray representing HA oligosaccharides containing from two to 40 saccharides. Notably, the HA oligosaccharides ranging from 32 to 40 saccharides exhibited the highest antibody binding intensities at baseline in baboon and rat sera. After xenotransplantation, antibodies against HA38 and HA40 in baboons, and HA32, HA34, and HA36 in rats showed the highest titer increases. The changes of anti-HA IgM and IgG antibodies in rats after skin xenotransplantation was also confirmed by an ELISA specific for HA2, HA24, and HA85 antibodies. Thus, xenotransplantation is associated with increased antibodies against HA-oligosaccharides, which may represent a new target for intervention.

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.

Increased blaKPC Copy Number and OmpK35 and OmpK36 Porins Disruption Mediated Resistance to Imipenem/Relebactam and Meropenem/Vaborbactam in a KPC-Producing Klebsiella pneumoniae Clinical Isolate.

Herein, we report the in vivo evolution of imipenem/relebactam-resistance in KPC-producing K. pneumoniae (KPC-Kp) isolated from a critically-ill patient treated with multiple combination therapies based on ceftazidime-avibactam or meropenem-vaborbactam. Imipenem/relebactam-resistance was associated to meropenem-vaborbactam cross-resistance and was due to truncated OmpK35 and OmpK36 porins and increased copy of blaKPC copy number. Genome analysis demonstrated that imipenem/relebactam-resistant KPC-Kp harbored a second copy of blaKPC-carrying Tn4401 in a ColRNAI plasmid as a consequence of a transposition event.

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.

Evaluation of a diagnostic algorithm for rapid identification of Gram-negative species and detection of extended-spectrum ß-lactamase and carbapenemase directly from blood cultures.

OBJECTIVES: To evaluate a rapid diagnostic algorithm based on MALDI-TOF MS, lateral flow immunoassays (LFIAs) and molecular testing performed directly from positive blood cultures (BCs) for Gram-negative species identification and detection of CTX-M extended-spectrum ß-lactamases and main carbapenemases. METHODS: Non-duplicate BCs positive to Gram-negative bacteria at microscope examination were subjected to species identification by direct MALDI-TOF MS following recovery of bacterial pellet by Rapid MBT Sepsityper® kit. Subsequently, NG-Test® CARBA 5 and NG-Test® CTX-M MULTI LFIAs were performed according to identified microbial species. Eazyplex® SuperBug CRE molecular assay was performed in cases of NG-Test® CARBA 5 negative results in patients with documented carbapenemase-producers carriage. Results of rapid diagnostic workflow were compared with those obtained by conventional diagnostic routine. RESULTS: Overall, the direct MALDI-TOF MS protocol allowed reliable identification to the species level of 92.1% of the 2133 monomicrobial BCs. Rate of matched identification was significantly higher for Enterobacterales (97.3%) in comparison to non-fermenting Gram-negative species (80.2%), obligate anaerobic bacteria (42.1%) and fastidious Gram-negative species (41.5%). The overall sensitivity of NG-Test® CARBA 5 and NG-Test® CTX-M MULTI was 92.2% and 91.6%, respectively. Integration of Easyplex® SuperBug CRE allowed the detection of blaKPC mutants associated with ceftazidime/avibactam resistance, reaching 100% sensitivity in carbapenemase detection. Both LFIAs and molecular testing showed no false-positive results. CONCLUSIONS: Algorithms based on MALDI-TOF MS, LFIAs and molecular testing may represent a cost-effective tool to timely identify Gram-negative species and detect resistance markers directly from BCs. According to local epidemiology, these results may allow antimicrobial stewardship interventions including prompt use of new approved drugs.

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.

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.