Publications by authors named "Gian Maria Rossolini"

357 Publications

Summary of the Available Molecular Methods for Detection of SARS-CoV-2 during the Ongoing Pandemic.

Int J Mol Sci 2021 Jan 28;22(3). Epub 2021 Jan 28.

Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy.

Since early 2020, the COVID-19 pandemic has caused an excess in morbidity and mortality rates worldwide. Containment strategies rely firstly on rapid and sensitive laboratory diagnosis, with molecular detection of the viral genome in respiratory samples being the gold standard. The reliability of diagnostic protocols could be affected by SARS-CoV-2 genetic variability. In fact, mutations occurring during SARS-CoV-2 genomic evolution can involve the regions targeted by the diagnostic probes. Following a review of the literature and an in silico analysis of the most recently described virus variants (including the UK B 1.1.7 and the South Africa 501Y.V2 variants), we conclude that the described genetic variability should have minimal or no effect on the sensitivity of existing diagnostic protocols for SARS-CoV-2 genome detection. However, given the continuous emergence of new variants, the situation should be monitored in the future, and protocols including multiple targets should be preferred.
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http://dx.doi.org/10.3390/ijms22031298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865767PMC
January 2021

Effective antimicrobial combination in vivo treatment predicted with microcalorimetry screening.

J Antimicrob Chemother 2021 Jan 14. Epub 2021 Jan 14.

Department of Clinical Microbiology, Rigshospitalet, 2200 Copenhagen N, Denmark.

Objectives: The worldwide emergence of antibiotic resistance calls for effective exploitation of existing antibiotics. Antibiotic combinations with different modes of action can synergize for successful treatment. In the present study, we used microcalorimetry screening to identify synergistic combination treatments against clinical MDR isolates. The synergistic effects were validated in a murine infection model.

Methods: The synergy of meropenem combined with colistin, rifampicin or amikacin was tested on 12 isolates (1 Escherichia coli, 5 Klebsiella pneumoniae, 3 Pseudomonas aeruginosa and 3 Acinetobacter baumannii) in an isothermal microcalorimeter measuring metabolic activity. One A. baumannii strain was tested with two individual pairings of antibiotic combinations. The microcalorimetric data were used to predict in vivo efficacy in a murine peritonitis/sepsis model. NMRI mice were inoculated intraperitoneally and after 1 h treated with saline, drug X, drug Y or X+Y. Bacterial load was determined by cfu in peritoneal fluid and blood after 4 h.

Results: In vitro, of the 13 combinations tested on the 12 strains, 3 of them exhibited a synergistic reduction in MIC (23% n = 3/13), 5 showed an additive effect (38.5% n = 5/13) and 5 had indifferent or antagonistic effects (38.5% n = 5/13). There was a significant correlation (P = 0.024) between microcalorimetry-screening FIC index values and the log reduction in peritoneal fluid from mice that underwent combination treatment compared with the most effective mono treatment. No such correlation could be found between chequerboard and in vivo results (P = 0.16).

Conclusions: These data support microcalorimetic metabolic readout to predict additive or synergistic effects of combination treatment of MDR infections within hours.
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http://dx.doi.org/10.1093/jac/dkaa543DOI Listing
January 2021

Evaluating Cefiderocol in the Treatment of Multidrug-Resistant Gram-Negative Bacilli: A Review of the Emerging Data.

Infect Drug Resist 2020 29;13:4697-4711. Epub 2020 Dec 29.

Clinica Malattie Infettive, Ospedale Policlinico San Martino - IRCCS, Genoa, Italy.

Infections due to multidrug-resistant Gram-negative bacteria (MDR-GNB), especially when carbapenem resistant, have been very difficult to manage in the last fifteen years, owing to the paucity of dependable therapeutic options. Cefiderocol is a siderophore cephalosporin recently approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) that may have the potential to fill some of the remaining gaps in the treatment of MDR-GNB infections. Among others, cefiderocol demonstrated in vitro activity against carbapenem-resistant and metallo-β-lactamases producers. Clinical data from both registrative studies and post-marketing experiences are essential to confirm whether these promises from in vitro studies could readily translate into clinical practice, as well as to delineate the precise place in therapy for cefiderocol for the treatment of MDR-GNB in the near future. Because of its unique potential, it is essential to provide both randomized controlled trials (RCT) and real-life data to improve the ability of clinicians to exploit its benefit in both empirical and targeted treatment of MDR-GNB infections. In this narrative review, we discuss the emerging data from pivotal RCT and initial real-life experiences on the use of cefiderocol for the treatment of MDR-GNB infections.
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http://dx.doi.org/10.2147/IDR.S205309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7778378PMC
December 2020

Detection of Oxazolidinone Resistance Genes and Characterization of Genetic Environments in Enterococci of Swine Origin, Italy.

Microorganisms 2020 Dec 17;8(12). Epub 2020 Dec 17.

Department of Life and Environmental Sciences, Polytechnic University of Marche, 60121 Ancona, Italy.

One hundred forty-five florfenicol-resistant enterococci, isolated from swine fecal samples collected from 76 pig farms, were investigated for the presence of , , and genes by PCR. Thirty florfenicol-resistant isolates had at least one linezolid resistance gene. was found to be the most widespread linezolid resistance gene (23/30), while and were detected in 6/30 and 7/30 enterococcal isolates, respectively. WGS analysis also showed the presence of the (D) gene in ( = 2 isolates) and in ( = 1 isolate). The linezolid resistance genes hybridized both on chromosome and plasmids ranging from ~25 to ~240 kb. Twelve isolates were able to transfer linezolid resistance genes to enterococci recipient. WGS analysis displayed a great variability of genetic contexts identical or related to transposons (Tn and Tn), plasmids (pE035 and pWo27-9), and chromosomal regions. environments showed identities with Tn-like transposon and a region from p12-2300 plasmid; (D) genetic contexts were related to the corresponding region of the plasmid 4 of E8014; was always found on Tn. Circular forms were obtained only for - and -carrying genetic contexts. Clonality analysis revealed the presence of (ST16, ST27, ST476, and ST585) and (ST21) clones previously isolated from humans. These results demonstrate a dissemination of linezolid resistance genes in enterococci of swine origin in Central Italy and confirm the spread of linezolid resistance in animal settings.
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http://dx.doi.org/10.3390/microorganisms8122021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766396PMC
December 2020

Evaluation of the commercial AD fosfomycin test for susceptibility testing of multidrug-resistant Enterobacterales and Pseudomonas aeruginosa.

Clin Microbiol Infect 2020 Dec 5. Epub 2020 Dec 5.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy; Microbiology and Virology Unit, Florence Careggi University Hospital, Florence, Italy.

Objectives: To compare fosfomycin susceptibility testing with the commercial agar dilution (AD) test, AD Fosfomycin (Liofilchem, Roseto degli Abruzzi, Italy) and the reference AD method, using a collection of multidrug-resistant (MDR) Enterobacterales and Pseudomonas aeruginosa clinical isolates.

Methods: The collection included 119 carbapenemase-producing Enterobacterales, 53 Enterobacterales producing acquired AmpC-type and/or extended-spectrum β-lactamases and 38 carbapenemase-producing P. aeruginosa, including representatives of different high-risk clones. AD Fosfomycin and AD reference method (ISO 20776-1:2019) were performed starting from the same microbial suspension. Results were interpreted according to EUCAST clinical breakpoints (10.0). Essential agreement (EA), category agreement (CA) and error rates were calculated as described by the International Organization for Standardization.

Results: Of 172 Enterobacterales, 143 (83.1%, including 92.9% (52 of 56) of the NDM-producers and 84.2% (48 of 57) of the KPC-producers) were susceptible to fosfomycin using reference AD. A CA of 91.9% (158 of 172; 95% CI 87.1%-95.3%) and an EA of 92.5% (136 of 147; 95% CI 87.4%-96.0%), respectively, were calculated for the commercial AD Fosfomycin test, with 9.8% (14 of 128) of major errors and no very major errors (0 of 29). Overall, 86.8% (33 of 38) of P. aeruginosa showed a fosfomycin MIC ≤128 mg/L using reference AD. An EA of 84.8% (95% CI 66.3%-92.0%) was calculated for the commercial AD Fosfomycin test, with a CA of 100% (95% CI 93.6%-100%) when considering a tentative breakpoint at 128 mg/L.

Conclusions: AD Fosfomycin showed an overall good concordance compared with reference AD.
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http://dx.doi.org/10.1016/j.cmi.2020.11.029DOI Listing
December 2020

Antibacterial and Anti-Inflammatory Activity of an Antimicrobial Peptide Synthesized with D Amino Acids.

Antibiotics (Basel) 2020 Nov 24;9(12). Epub 2020 Nov 24.

Department of Medical Biotechnologies, University of Siena, 53100 Siena, Italy.

The peptide SET-M33 is a molecule synthesized in tetra-branched form which is being developed as a new antibiotic against Gram-negative bacteria. Its isomeric form with D amino acids instead of the L version (SET-M33D) is also able to kill Gram-positive bacteria because of its higher resistance to bacterial proteases (Falciani et al., , 2012, 7, e46259). Here we report the strong in vitro activity of SET-M33D (MIC range 0.7-6.0 µM) against multiresistant pathogens of clinical interest, including Gram-positives , , and , and various Gram-negative enterobacteriaceae. SET-M33D antibacterial activity is also confirmed in vivo against a MRSA strain of with doses perfectly compatible with clinical use (5 and 2.5 mg/Kg). Moreover, SET-M33D strongly neutralized lipopolysaccharide (LPS) and lipoteichoic acid (LTA), thus exerting a strong anti-inflammatory effect, reducing expression of cytokines, enzymes, and transcription factors (TNF-α, IL6, COX-2, KC, MIP-1, IP10, iNOS, NF-κB) involved in the onset and evolution of the inflammatory process. These results, along with in vitro and in vivo toxicity data and the low frequency of resistance selection reported here, make SET-M33D a strong candidate for the development of a new broad spectrum antibiotic.
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http://dx.doi.org/10.3390/antibiotics9120840DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760307PMC
November 2020

The changing epidemiology of carbapenemase-producing Klebsiella pneumoniae in Italy: toward polyclonal evolution with emergence of high-risk lineages.

J Antimicrob Chemother 2021 Jan;76(2):355-361

Department of Infectious Diseases, Istituto Superiore di Sanità, Rome, Italy.

Background: Previous studies showed that the epidemic of carbapenem-resistant Klebsiella pneumoniae (CR-KP) observed in Italy since 2010 was sustained mostly by strains of clonal group (CG) 258 producing KPC-type carbapenemases. In the framework of the National Antibiotic-Resistance Surveillance (AR-ISS), a countrywide survey was conducted in 2016 to explore the evolution of the phenotypic and genotypic characteristics of CR-KP isolates.

Methods: From March to July 2016, hospital laboratories participating in AR-ISS were requested to provide consecutive, non-duplicated CR-KP (meropenem and/or imipenem MIC >1 mg/L) from invasive infections. Antibiotic susceptibility was determined according to EUCAST recommendations. A WGS approach was adopted to characterize the isolates by investigating phylogeny, resistome and virulome.

Results: Twenty-four laboratories provided 157 CR-KP isolates, of which 156 were confirmed as K. pneumoniae sensu stricto by WGS and found to carry at least one carbapenemase-encoding gene, corresponding in most cases (96.1%) to blaKPC. MLST- and SNP-based phylogeny revealed that 87.8% of the isolates clustered in four major lineages: CG258 (47.4%), with ST512 as the most common clone, CG307 (19.9%), ST101 (15.4%) and ST395 (5.1%). A close association was identified between lineages and antibiotic resistance phenotypes and genotypes, virulence traits and capsular types. Colistin resistance, mainly associated with mgrB mutations, was common in all major lineages except ST395.

Conclusions: This WGS-based survey showed that, although CG258 remained the most common CR-KP lineage in Italy, a polyclonal population has emerged with the spread of the new high-risk lineages CG307, ST101 and ST395, while KPC remained the most common carbapenemase.
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http://dx.doi.org/10.1093/jac/dkaa431DOI Listing
January 2021

In Vitro Alteration by Dentine and Protein of the Antimicrobial Activity of Two Endodontic Irrigants: HybenX and Sodium Hypochlorite.

Antibiotics (Basel) 2020 Nov 10;9(11). Epub 2020 Nov 10.

Department of Experimental and Clinical Medicine, University of Florence, 50134 Florence, Italy.

Irrigant solutions commonly used for the treatment of endodontic infections can be inhibited by both organic and inorganic substances. The aim of this study was to evaluate the in vitro antimicrobial activity of the novel irrigant HybenX and 2.5% and 5% sodium hypochlorite against , in presence of dentine powder (DP) or bovine serum albumin 20% (BSA) as inhibitory agents. An American Type Culture Collection (ATCC) 29212 suspension was added to the irrigants (Hybenx or NaOCl) and one or two different inhibitors (BSA and DP) either after one-hour pre-incubation at 35 ± 1 °C or not. The antimicrobial activity of HybenX against was already proved at 15 min and was neither affected by BSA nor by DP or combinations thereof. NaOCl 2.5% showed an effective antimicrobial activity starting from 15 min and this activity was partially inhibited by BSA and BSA plus DP combination within one hour when pre-incubation occurred. NaOCl 5% showed antimicrobial activity within 15 min, which was inhibited within one hour only in the presence of both BSA and DP regardless of the pre-incubation period. HybenX could represent a good alternative to common irrigants for the treatment of endodontic infections, showing a rapid antimicrobial activity not inhibited by organic and inorganic inhibitors.
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http://dx.doi.org/10.3390/antibiotics9110792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696392PMC
November 2020

Arbo-Score: A Rapid Score for Early Identification of Patients with Imported Arbovirosis Caused by Dengue, Chikungunya and Zika Virus.

Microorganisms 2020 Nov 4;8(11). Epub 2020 Nov 4.

Department of Experimental and Clinical Medicine, University of Florence, 50134 Florence, Italy.

Background: Chikungunya (CHIKV), Dengue (DENV), and Zika (ZIKV) viruses present significant clinical and epidemiological overlap, making an accurate and rapid diagnosis challenging. Timely activation of preventive vector control measures is crucial to avoid outbreaks in non-endemic settings. Diagnosis is based on combination of serological and molecular assays which could be time consuming and sometimes disappointing.

Methods: We report the results of a retrospective case-control study carried out at a tertiary teaching hospital in Italy, including all febrile subjects returning from tropical countries during the period 2014-2019. Controls were travelers with other febrile illnesses who tested negative in laboratory analysis for CHIKV, DENV, ZIKV arbovirosis. A score weighted on the regression coefficients for the independent predictors was generated.

Results: Ninety patients were identified: 34 cases (22 DENV, 4 CHIKV, and 8 ZIKV) and 56 controls. According to our results, myalgia, cutaneous rash, absence of respiratory symptoms, leukopenia, and hypertransaminasemia showed the strongest association with arbovirosis. Combining these variables, we generated a scoring model that showed an excellent performance (AUC 0.93). The best cut-off (>=2) presented a sensitivity of 82.35% and specificity of 96.43%.

Conclusion: A handy and simple score, based on three clinical data (myalgia, cutaneous rash and absence of respiratory symptoms) and two laboratory results (leukopenia and hypertransaminasemia), provides a useful tool to help diagnose arboviral infections and appropriately activate vector control measures in order to avoid local transmission.
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http://dx.doi.org/10.3390/microorganisms8111731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716211PMC
November 2020

Performance Evaluation of a Commercial Real-Time PCR Assay and of an In-House Real-Time PCR for DNA Detection in a Tropical Medicine Reference Center, Northern Italy.

Microorganisms 2020 Oct 30;8(11). Epub 2020 Oct 30.

Department of Infectious-Tropical Diseases and Microbiology, IRCCS SacroCuore Don Calabria Hospital, Negrar di Valpolicella, 37024 Verona, Italy.

Chagas disease, a neglected protozoal disease endemic in Latin America, is also currently considered an emerging threat in nonendemic areas because of population movements. The detection of DNA is increasingly being considered as important evidence to support Chagas disease diagnoses. However, further performance evaluation of molecular assays is useful for a standardization of strategy considering the whole process in routine diagnosis, especially for the different settings such as endemic and nonendemic countries. Seventy-five samples were collected from subjects screened for Chagas disease in Italy. The DNA was isolated from blood using automated extraction. We evaluated the performance of the commercial RealCycler CHAG kit (pmPCR) based on satellite DNA (SatDNA) and of an in-house real-time PCR (ihPCR) targeting Sat and kinetoplast (k) DNAs, using the concordance of two serology assays as a reference standard. The sensitivity of kDNA and SatDNA tests by ihPCR and SatDNA by pmPCR were 14.29% (95% confidence interval (CI) 6.38 to 26.22), 7.14% (95% CI 1.98 to 17.29), and 7.14% (95% CI 1.98 to 17.29), respectively. Specificity was 100% for all PCR assays and targets. Overall, our results suggest that the preferred approach for clinical laboratories is to combine the kDNA and SatDNA as targets in order to minimize false-negative results increasing sensitivity.
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http://dx.doi.org/10.3390/microorganisms8111692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692395PMC
October 2020

Treatment of severe infections due to metallo-β-lactamases-producing Gram-negative bacteria.

Future Microbiol 2020 11 3:1489-1505. Epub 2020 Nov 3.

Infectious Diseases Unit, Ospedale Policlinico San Martino - IRCCS, Genoa, Italy.

In the last decades, there was an important paucity of agents for adequately treating infections due to metallo-β-lactamases-producing Gram-negative bacteria (MBL-GNB). Cefiderocol, a novel siderophore cephalosporin showing activity against MBL-GNB, has been recently marketed, and a combination of aztreonam and ceftazidime/avibactam has shown a possible favorable effect on survival of patients with severe MBL-GNB infections in observational studies. Other agents showing activity against MBL-GNB are currently in clinical development (e.g., cefepime/taniborbactam, LYS228, cefepime/zidebactam) that could be an important addition to our future armamentarium for severe MBL-GNB infections. Nonetheless, we should not discontinue our efforts to optimize the use of non-β-lactams agents, since they could remain an essential last-resort or alternative option in selected cases.
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http://dx.doi.org/10.2217/fmb-2020-0210DOI Listing
November 2020

KPC-53, a KPC-3 Variant of Clinical Origin Associated with Reduced Susceptibility to Ceftazidime-Avibactam.

Antimicrob Agents Chemother 2020 12 16;65(1). Epub 2020 Dec 16.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy

This study reports on the characterization of a clinical isolate showing high-level resistance to ceftazidime-avibactam associated with the production of KPC-53, a KPC-3 variant exhibiting a Leu167Glu168 duplication in the Ω-loop and a loss of carbapenemase activity. Whole-genome sequencing (WGS) revealed the presence of two copies of , located on a pKpQIL-like plasmid and on a plasmid prophage of the family, respectively. The present findings provide new insights into the mechanisms of resistance to ceftazidime-avibactam.
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http://dx.doi.org/10.1128/AAC.01429-20DOI Listing
December 2020

Cell-mediated and humoral adaptive immune responses to SARS-CoV-2 are lower in asymptomatic than symptomatic COVID-19 patients.

Eur J Immunol 2020 Dec 9;50(12):2013-2024. Epub 2020 Nov 9.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

The characterization of cell-mediated and humoral adaptive immune responses to SARS-CoV-2 is fundamental to understand COVID-19 progression and the development of immunological memory to the virus. In this study, we detected T-cells reactive to SARS-CoV-2 proteins M, S, and N, as well as serum virus-specific IgM, IgA, IgG, in nearly all SARS-CoV-2 infected individuals, but not in healthy donors. Virus-reactive T cells exhibited signs of in vivo activation, as suggested by the surface expression of immune-checkpoint molecules PD1 and TIGIT. Of note, we detected antigen-specific adaptive immune response both in asymptomatic and symptomatic SARS-CoV-2 infected subjects. More importantly, symptomatic patients displayed a significantly higher magnitude of both cell-mediated and humoral adaptive immune response to the virus, as compared to asymptomatic individuals. These findings suggest that an uncontrolled adaptive immune response contribute to the development of the life-threatening inflammatory phase of the disease. Finally, this study might open the way to develop effective vaccination strategies.
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http://dx.doi.org/10.1002/eji.202048915DOI Listing
December 2020

Integrated chromosomal and plasmid sequence analyses reveal diverse modes of carbapenemase gene spread among .

Proc Natl Acad Sci U S A 2020 10 23;117(40):25043-25054. Epub 2020 Sep 23.

Centre for Genomic Pathogen Surveillance, Wellcome Genome Campus, Hinxton, CB10 1SA Cambridge, United Kingdom;

Molecular and genomic surveillance systems for bacterial pathogens currently rely on tracking clonally evolving lineages. By contrast, plasmids are usually excluded or analyzed with low-resolution techniques, despite being the primary vectors of antibiotic resistance genes across many key pathogens. Here, we used a combination of long- and short-read sequence data of isolates ( = 1,717) from a European survey to perform an integrated, continent-wide study of chromosomal and plasmid diversity. This revealed three contrasting modes of dissemination used by carbapenemase genes, which confer resistance to last-line carbapenems. First, genes have spread primarily via the single epidemic pOXA-48-like plasmid, which emerged recently in clinical settings and spread rapidly to numerous lineages. Second, and genes have spread via transient associations of many diverse plasmids with numerous lineages. Third, genes have transmitted predominantly by stable association with one successful clonal lineage (ST258/512) yet have been mobilized among diverse plasmids within this lineage. We show that these plasmids, which include pKpQIL-like and IncX3 plasmids, have a long association (and are coevolving) with the lineage, although frequent recombination and rearrangement events between them have led to a complex array of mosaic plasmids carrying Taken altogether, these results reveal the diverse trajectories of antibiotic resistance genes in clinical settings, summarized as using one plasmid/multiple lineages, multiple plasmids/multiple lineages, and multiple plasmids/one lineage. Our study provides a framework for the much needed incorporation of plasmid data into genomic surveillance systems, an essential step toward a more comprehensive understanding of resistance spread.
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http://dx.doi.org/10.1073/pnas.2003407117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7587227PMC
October 2020

Low risk of serological cross-reactivity between dengue and COVID-19.

Mem Inst Oswaldo Cruz 2020 14;115:e200225. Epub 2020 Aug 14.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

In the near future, the overlap of Coronavirus disease 2019 (COVID-19) and dengue epidemics is a concrete threat in tropical regions. Co-epidemics of COVID-19 and dengue could be an overwhelming challenge for health systems in low- and middle-income countries. In this work, we investigated potential serological cross-reactions between COVID-19 and dengue patients. Among 32 COVID-19 positive sera, no positive Dengue virus (DENV) IgG/IgM results were observed. On the other hand, one false-positive result was observed among 44 DENV-positive sera tested for COVID-19 antibodies with each of the two rapid tests used. Further data on accuracy of COVID-19 diagnostic test are urgently warranted.
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http://dx.doi.org/10.1590/0074-02760200225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7427313PMC
August 2020

Assessment of the in vitro activity of ceftazidime/avibactam against a global collection of multidrug-resistant Klebsiella spp. from the INFORM surveillance programme (2015-2017).

Int J Antimicrob Agents 2020 Sep 25;56(3):106111. Epub 2020 Jul 25.

Pfizer Inc., 558 Eastern Point Road, Groton, CT 06340, USA.

Infections caused by Klebsiella spp. are difficult to treat when these pathogens exhibit multidrug resistance (resistance to ≥3 drug classes). This study determined rates of multidrug-resistant (MDR) Klebsiella spp. among clinical isolates collected globally (Africa/Middle East, Asia, Oceania, Europe and Latin America, but not the USA) as part of the INFORM surveillance programme (2015-2017). In vitro antimicrobial activities of ceftazidime/avibactam (CAZ/AVI) and comparator antimicrobials against these MDR isolates were determined using CLSI broth microdilution methodology. MICs were interpreted using EUCAST 2019 breakpoints. By species, 38.4% (4555/11 864) of Klebsiella pneumoniae isolates were MDR, followed by 28.3% (452/1598) of Klebsiella aerogenes. The majority (69.5%) of MDR K. pneumoniae isolates were extended-spectrum β-lactamase (ESBL)-positive and carbapenemase-negative compared with 1.3% of MDR K. aerogenes. Globally, >92% of MDR K. pneumoniae isolates were resistant to amoxicillin/clavulanic acid, aztreonam, cefepime, ceftazidime or ceftriaxone. CAZ/AVI, colistin and tigecycline MIC values were 1-2 mg/L against the global collection of MDR K. pneumoniae and MDR K. aerogenes. Approximately 5% (216/4555) of all MDR K. pneumoniae isolates and 1.1% (5/452) of all MDR K. aerogenes isolates were resistant to CAZ/AVI. Rates of resistance to CAZ/AVI and colistin were low for MDR, ESBL-positive, carbapenemase-negative K. pneumoniae (0.1% and 3.0%, respectively). Resistance to CAZ/AVI was highest among MDR, carbapenemase-positive, metallo-β-lactamase (MBL)-positive K. pneumoniae (98.0%), whereas colistin resistance was highest among MDR, carbapenemase-positive, MBL-negative isolates (30.2%). The results of this study suggest that CAZ/AVI, colistin and tigecycline are potential treatment options for nosocomial infections caused by MDR Klebsiella spp. isolates.
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http://dx.doi.org/10.1016/j.ijantimicag.2020.106111DOI Listing
September 2020

Phenotypical and molecular assessment of the virulence potential of KPC-3-producing Klebsiella pneumoniae ST392 clinical isolates.

Microbiol Res 2020 Nov 6;240:126551. Epub 2020 Jul 6.

Fondazione Ri.MED, Palermo, Italy; IRCCS-ISMETT, Palermo, Italy. Electronic address:

Klebsiella pneumoniae is a Gram-negative bacterium of clinical importance, due to its resistance to several antibiotic classes. We have identified 4 clinical isolates of K. pneumoniae sequence type (ST) 392 KPC-3-producing strains from patients at the Istituto Mediterraneo per i Trapianti e Terapie ad Alta Specializzazione (IRCCS-ISMETT), a Southern Italian transplantation health facility, during a routine surveillance for carbapenemase-producing Enterobacterales from in-house clinical samples. Since those were among, to the best of our knowledge, the first KPC-producing K. pneumoniae ST392 isolated in Europe, we assessed their virulence potential, to understand if this particular ST can become an endemic clinical threat. ST392 isolates were investigated to assess their virulence potential, namely resistance to human sera, formation of abiotic biofilms, adhesion to biotic surfaces, exopolysaccharide production and in vivo pathogenesis in the wax moth Galleria mellonella animal model. ST392-belonging strains were highly resistant to human sera. These strains also have a high capacity to form abiotic biofilms and high levels of adhesion to the human epithelial colorectal adenocarcinoma HT-29 cell line. An increase of transcriptional levels of genes involved in serum resistance (aroE and traT) and adhesion (pgaA) was observed when compared with the Klebsiella quasipneumoniae subsp. similipneumoniae strain ATCC 700603 reference strain. Infection of G. mellonella larvae with ST392 clinical isolates showed that the latter were not highly pathogenic in this model. Together, our results indicate that ST392 isolates have the potential to become a strain of clinical relevance, especially in health settings where patients are immunosuppressed, e.g., transplant recipients.
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http://dx.doi.org/10.1016/j.micres.2020.126551DOI Listing
November 2020

A novel colistin adjuvant identified by virtual screening for ArnT inhibitors.

J Antimicrob Chemother 2020 09;75(9):2564-2572

Department of Sciences, 'Department of Excellence 2018 - 2022', Roma Tre University, Rome, Italy.

Background: Colistin is a last-resort treatment option for many MDR Gram-negative bacteria. The covalent addition of l-aminoarabinose to the lipid A moiety of LPS is the main colistin resistance mechanism in the human pathogen Pseudomonas aeruginosa.

Objectives: Identification (by in silico screening of a chemical library) of potential inhibitors of ArnT, which catalyses the last committed step of lipid A aminoarabinosylation, and their validation in vitro as colistin adjuvants.

Methods: The available ArnT crystal structure was used for a docking-based virtual screening of an in-house library of natural products. The resulting putative ArnT inhibitors were tested in growth inhibition assays using a reference colistin-resistant P. aeruginosa strain. The most promising compound was further characterized for its range of activity, specificity and cytotoxicity. Additionally, the effect of the compound on lipid A aminoarabinosylation was verified by MS analyses of lipid A.

Results: A putative ArnT inhibitor (BBN149) was discovered by molecular docking and demonstrated to specifically potentiate colistin activity in colistin-resistant P. aeruginosa isolates, without relevant effect on colistin-susceptible strains. BBN149 also showed adjuvant activity against colistin-resistant Klebsiella pneumoniae and low toxicity to bronchial epithelial cells. Lipid A aminoarabinosylation was reduced in BBN149-treated cells, although only partially.

Conclusions: This study demonstrates that in silico screening targeting ArnT can successfully identify inhibitors of colistin resistance and provides a promising lead compound for the development of colistin adjuvants for the treatment of MDR bacterial infections.
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http://dx.doi.org/10.1093/jac/dkaa200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443731PMC
September 2020

Gene Expression Modulates the Inflammatory Response of Human Macrophages to Escherichia coli.

Infect Immun 2020 07 21;88(8). Epub 2020 Jul 21.

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

MCR-1 is a plasmid-encoded phosphoethanolamine transferase able to modify the lipid A structure. It confers resistance to colistin and was isolated from human, animal, and environmental strains of , raising serious global health concerns. In this paper, we used recombinant -expressing to study the impact of MCR-1 products on -induced activation of inflammatory pathways in activated THP-1 cells, which was used as a model of human macrophages. We found that infection with recombinant -expressing significantly modulated p38-MAPK and Jun N-terminal protein kinase (JNK) activation and pNF-κB nuclear translocation as well as the expression of genes for the relevant proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and IL-1β compared with -negative strains. Caspase-1 activity and IL-1β secretion were significantly less activated by -positive strains than the -negative parental strain. Similar results were obtained with clinical isolates of -positive , suggesting that, in addition to colistin resistance, the expression of allows the escape of early host innate defenses and may promote bacterial survival.
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http://dx.doi.org/10.1128/IAI.00018-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375756PMC
July 2020

Autochthonous Human and Canine Infection in Europe: Report of a Human Case in An Italian Teen and Systematic Review of the Literature.

Pathogens 2020 Jun 3;9(6). Epub 2020 Jun 3.

Department of Experimental and Clinical Medicine, University of Florence, 50134 Florence, Italy.

Autochthonous human and canine strongyloidiasis is reported in Europe but is unclear whether the transmission of infection still occurs. We report a previously unpublished human case in an Italian teen and perform a systematic review of literature on autochthonous human and canine strongyloidiasis in Europe to investigate the current dynamic of transmission. Overall, 109 papers published after 1987 were included and one previously unpublished Italian case was added. Eighty case reports were retrieved and 42 of them (52.5%) had severe strongyloidiasis. Most cases were diagnosed in Spain, Italy and France. The median age was 58, the most represented age group was 61-70 years, 11 patients were under 30, and 7 of them were diagnosed after 2000. Epidemiological studies on human strongyloidiasis showed prevalence ranging from 0.56% to 28%. Overall, agriculture work, mine work and walking barefoot were the most commonly reported risk factors for infection. Canine strongyloidiasis was reported mainly in Italy (68 cases), but a few cases occurred also in Iceland, Finland, England, Germany, France, Switzerland, Russia, Slovakia, Romania and Greece. Autochthonous strongyloidiasis is still reported in Europe and sporadic transmission still occurs. Health care professionals should be aware of this issue to identify infected subjects and avoid adverse outcomes, especially in immunosuppressed patients. Further investigations are needed to clarify the zoonotic transmission of this nematode.
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http://dx.doi.org/10.3390/pathogens9060439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350350PMC
June 2020

Impaired immune cell cytotoxicity in severe COVID-19 is IL-6 dependent.

J Clin Invest 2020 09;130(9):4694-4703

Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy.

BACKGROUNDCoronavirus disease 19 (COVID-19) is an emerging infectious disease caused by SARS-CoV-2. Antiviral immune response is crucial to achieve pathogen clearance; however, in some patients an excessive and aberrant host immune response can lead to an acute respiratory distress syndrome. The comprehension of the mechanisms that regulate pathogen elimination, immunity, and pathology is essential to better characterize disease progression and widen the spectrum of therapeutic options.METHODSWe performed a flow cytometric characterization of immune cell subsets from 30 patients with COVID-19 and correlated these data with clinical outcomes.RESULTSPatients with COVID-19 showed decreased numbers of circulating T, B, and NK cells and exhibited a skewing of CD8+ T cells toward a terminally differentiated/senescent phenotype. In agreement, CD4+ T and CD8+ T, but also NK cells, displayed reduced antiviral cytokine production capability. Moreover, a reduced cytotoxic potential was identified in patients with COVID-19, particularly in those who required intensive care. The latter group of patients also showed increased serum IL-6 levels that inversely correlated to the frequency of granzyme A-expressing NK cells. Off-label treatment with tocilizumab restored the cytotoxic potential of NK cells.CONCLUSIONThe association between IL-6 serum levels and the impairment of cytotoxic activity suggests the possibility that targeting this cytokine may restore antiviral mechanisms.FUNDINGThis study was supported by funds from the Department of Experimental and Clinical Medicine of University of Florence (the ex-60% fund and the "Excellence Departments 2018-2022 Project") derived from Ministero dell'Istruzione, dell'Università e della Ricerca (Italy).
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http://dx.doi.org/10.1172/JCI138554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456250PMC
September 2020

Abundance of Colistin-Resistant, OXA-23- and ArmA-Producing Belonging to International Clone 2 in Greece.

Front Microbiol 2020 15;11:668. Epub 2020 Apr 15.

bioMérieux, Data Analytics Unit, La Balme-les-Grottes, France.

Carbapenem resistant (CRAB) represents one of the most challenging pathogens in clinical settings. Colistin is routinely used for treatment of infections by this pathogen, but increasing colistin resistance has been reported. We obtained 122 CRAB isolates from nine Greek hospitals between 2015 and 2017, and those colistin resistant (ColR; = 40, 32.8%) were whole genome sequenced, also by including two colistin susceptible (ColS) isolates for comparison. All ColR isolates were characterized by a previously described mutation, PmrB, which was associated with low-level colistin resistance. Some isolates were characterized by additional mutations in PmrB (E140V or L178F) or PmrA (K172I or D10N), first described here, and higher colistin minimum inhibitory concentrations (MICs), up to 64 mg/L. Mass spectrometry analysis of lipid A showed the presence of a phosphoethanolamine (pEtN) moiety on lipid A, likely resulting from the PmrA/B-induced overexpression. Interestingly, also the two ColS isolates had the same lipid A modification, suggesting that not all lipid A modifications lead to colistin resistance or that other factors could contribute to the resistance phenotype. Most of the isolates ( = 37, 92.5%) belonged to the globally distributed international clone (IC) 2 and comprised four different sequence types (STs) as defined by using the Oxford scheme (ST 425, 208, 451, and 436). Three isolates belonged to IC1 and ST1567. All the genomes harbored an intrinsic group carbapenemase gene, where and were associated with IC2 and IC1, respectively. Carbapenem resistance was due to the most commonly reported acquired carbapenemase gene , with IS located upstream of the gene and likely increasing its expression. The gene, associated with high-level resistance to aminoglycosides, was detected in 87.5% of isolates. Collectively, these results revealed a convergent evolution of different clonal lineages toward the same colistin resistance mechanism, thus limiting the effective therapeutic options for the treatment of CRAB infections.
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http://dx.doi.org/10.3389/fmicb.2020.00668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212473PMC
April 2020

Meropenem/vaborbactam: a next generation β-lactam β-lactamase inhibitor combination.

Expert Rev Anti Infect Ther 2020 07 3;18(7):643-655. Epub 2020 May 3.

Dipartimento di Scienze di Laboratorio e Infettivologiche, Fondazione Policlinico Universitario A. Gemelli IRCCS , Rome, Italy.

Introduction: infections due to carbapenem-resistant (CRE) constitute a worldwide threat and are associated with significant mortality, especially in fragile patients, and costs. Meropenem-vaborbactam (M/V) is a combination of a group 2 carbapenem with a novel cyclic boronic acid-based β-lactamase inhibitor which has shown good efficacy against KPC carbapenemase-producing , which are amongst the most prevalent types of CRE.

Areas Covered: This article reviews the microbiological and pharmacological profile and current clinical experience and safety of M/V in the treatment of infections caused by CRE.

Expert Opinion: M/V is a promising drug for the treatment of infections due to KPC-producing CRE (KPC-CRE). It exhibited an almost complete coverage of KPC-CRE isolates from large surveillance studies and a low propensity for resistance selection, retaining activity also against strains producing KPC mutants resistant to ceftazidime-avibactam. Both meropenem and vaborbactam have a favorable pharmacokinetic profile, with similar kinetic properties, a good intrapulmonary penetration, and are efficiently cleared during continuous venovenous hemofiltration (CVVH). According to available data, M/V monotherapy is associated with higher clinical cure rates and lower rates of adverse events, especially in terms of nephrotoxicity, if compared to 'older' combination therapies.
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http://dx.doi.org/10.1080/14787210.2020.1756775DOI Listing
July 2020

Determination of the capsular polysaccharide structure of the Klebsiella pneumoniae ST512 representative strain KPB-1 and assignments of the glycosyltransferases functions.

Int J Biol Macromol 2020 Jul 26;155:315-323. Epub 2020 Mar 26.

Department of Life Sciences, University of Trieste, 34127 Trieste, Italy. Electronic address:

Klebsiella pneumoniae strain KPB-1 was isolated in early 2011 from the pleural fluid of an inpatient admitted at an Italian hospital. It was characterized to produce the KPC-3 carbapenemase and to belong to sequence type 512, a derivative of sequence type 258 clade II characterized by the cps-2 gene cluster. The K-antigen of K. pneumoniae KPB-1 was purified and its structure determined by using GLC-MS of appropriate carbohydrate derivatives and 1D and 2D NMR spectroscopy of the native polysaccharide. All the collected data demonstrated the following repeating unit for the K. pneumoniae KPB-1 capsular polysaccharide: The reactions catalysed by each glycosyltransferase in the cps-2 gene cluster were assigned on the basis of structural homology with other Klebsiella K antigens.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.03.196DOI Listing
July 2020

The role of dalbavancin in the treatment of acute bacterial skin and skin structure infections (ABSSSIs).

Expert Rev Anti Infect Ther 2020 05 29;18(5):415-422. Epub 2020 Mar 29.

Department of Medicine, University of Udine, Udine, Italy.

: Acute bacterial skin and skin-structure infections (ABSSSI) are a subgroup of skin and soft tissue infections and are a common source of morbidity in both the community and the hospital setting. The most common cause of ABSSSI is , which also includes methicillin-resistant (MRSA), together with beta-hemolytic streptococci, enterococci, and Gram-negative bacteria. Since the emergence of MRSA, the management of ABSSSI has become more challenging. Novel therapies alternative to teicoplanin and vancomycin, intravenous agents commonly used against MRSA and employed in hospitalized patients, and to other antibiotics which are used as standard of care for MRSA infection, with a higher efficacy and safer profile are worth evaluating.: This review presents and discusses current evidence on the use of dalbavancin in the treatment of ABSSSI.: Dalbavancin represents a promising therapeutic choice in patients with ABSSSI, thanks to its favorable pharmacokinetic profile, valuable antimicrobial spectrum, and good safety profile.
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http://dx.doi.org/10.1080/14787210.2020.1746643DOI Listing
May 2020

Genomic Epidemiology of Carbapenem- and Colistin-Resistant Isolates From Serbia: Predominance of ST101 Strains Carrying a Novel OXA-48 Plasmid.

Front Microbiol 2020 21;11:294. Epub 2020 Feb 21.

bioMérieux, Data Analytics Unit, La Balme-les-Grottes, France.

is a major cause of severe healthcare-associated infections and often shows MDR phenotypes. Carbapenem resistance is frequent, and colistin represents a key molecule to treat infections caused by such isolates. Here we evaluated the antimicrobial resistance (AMR) mechanisms and the genomic epidemiology of clinical isolates from Serbia. Consecutive non-replicate clinical isolates ( = 2,298) were collected from seven hospitals located in five Serbian cities and tested for carbapenem resistance by disk diffusion. Isolates resistant to at least one carbapenem ( = 426) were further tested for colistin resistance with Etest or Vitek2. Broth microdilution (BMD) was performed to confirm the colistin resistance phenotype, and colistin-resistant isolates ( = 45, 10.6%) were characterized by Vitek2 and whole genome sequencing. Three different clonal groups (CGs) were observed: CG101 (ST101, = 38), CG258 (ST437, = 4; ST340, = 1; ST258, = 1) and CG17 (ST336, = 1). genes, encoding for acquired colistin resistance, were not observed, while all the genomes presented mutations previously associated with colistin resistance. In particular, all strains had a mutated MgrB, with MgrB being the prevalent mutation and associated with ST101. Isolates belonging to ST101 harbored the carbapenemase OXA-48, which is generally encoded by an IncL/M plasmid that was no detected in our isolates. MinION sequencing was performed on a representative ST101 strain, and the obtained long reads were assembled together with the Illumina high quality reads to decipher the genetic background. The gene was located in a novel IncFIA-IncR hybrid plasmid, also containing the extended spectrum β-lactamase-encoding gene and several other AMR genes. Non-ST101 isolates presented different MgrB alterations (C28S, C28Y, K2, K3, Q30, adenine deletion leading to frameshift and premature termination, IS-mediated inactivation) and expressed different carbapenemases: OXA-48 (ST437 and ST336), NDM-1 (ST437 and ST340) and KPC-2 (ST258). Our study reports the clonal expansion of the newly emerging ST101 clone in Serbia. This high-risk clone appears adept at acquiring resistance, and efforts should be made to contain the spread of such clone.
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http://dx.doi.org/10.3389/fmicb.2020.00294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7047997PMC
February 2020

Prolonged outbreak of New Delhi metallo-beta-lactamase-producing carbapenem-resistant Enterobacterales (NDM-CRE), Tuscany, Italy, 2018 to 2019.

Euro Surveill 2020 02;25(6)

The members of the network are acknowledged at the end of the article.

In Tuscany, Italy, New Delhi metallo-beta-lactamase-producing carbapenem-resistant Enterobacterales (NDM-CRE) have increased since November 2018. Between November 2018 and October 2019, 1,645 samples were NDM-CRE-positive: 1,270 (77.2%) cases of intestinal carriage, 129 (7.8%) bloodstream infections and 246 (14.9%) infections/colonisations at other sites. were prevalent (1,495; 90.9%), with ST147/NDM-1 the dominant clone. Delayed outbreak identification and response resulted in sustained NDM-CRE transmission in the North-West area of Tuscany, but successfully contained spread within the region.
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http://dx.doi.org/10.2807/1560-7917.ES.2020.25.6.2000085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7029447PMC
February 2020