Publications by authors named "Cecilia Ambrosi"

29 Publications

  • Page 1 of 1

Urinary tract infections: Can we prevent uropathogenic infection with dietary intervention?

Int J Vitam Nutr Res 2021 Apr 21:1-5. Epub 2021 Apr 21.

San Raffaele Roma Open University, Rome, Italy.

Urinary tract infections (UTIs) are among the most common causes of infections in women. Via the fecal-perineal-urethral route, uropathogenic (UPEC) can cause ascending urinary tract infections, including cystitis and pyelonephritis. These infections re-occur within six months or they account for, at least, three episodes within a year of recurrent UTIs (rUTIs). Long term and continuous antibiotic treatment or prophylaxis should be considered as the last options in rUTIs. Conversely, updated European Association of Urology guidelines recommend non-antimicrobial approaches to prevent rUTIs. Accordingly, several studies reported the efficacy of number of natural molecules in inhibiting UPEC adhesion to bladder cells, restraining bacterial growth, as well as stimulating the host innate immune defenses, and protecting the bladder and the kidney mucosa. Therefore, we propose an "anti-UPEC" diet enriched of foods containing natural compounds that were proven effective against UPEC, such as D-mannose, cranberry extracts and medicinal plants. Being a valuable and safe clinical approach to reduce UTI recurrence and limiting the detrimental effects of long and continuous antibiotic prophylaxis, dietary interventions should be evaluated in future clinical trials.
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http://dx.doi.org/10.1024/0300-9831/a000704DOI Listing
April 2021

: An Ancient Commensal with Weapons of a Pathogen.

Pathogens 2021 Mar 24;10(4). Epub 2021 Mar 24.

IRCCS San Raffaele Pisana, 00166 Rome, Italy.

is regarded as a life-threatening pathogen associated with community-acquired and nosocomial infections, mainly pneumonia. The rise in the number of antibiotic-resistant strains reduces effective therapies and increases mortality. Bacterial comparative genomic studies have unraveled the innate and acquired virulence factors of These virulence factors are involved in antibiotic resistance, environmental persistence, host-pathogen interactions, and immune evasion. Studies on host-pathogen interactions revealed that evolved different mechanisms to adhere to in order to invade host respiratory cells as well as evade the host immune system. In this review, we discuss current data on genetic features and virulence factors. An emphasis is given to the players in host-pathogen interaction in the respiratory tract. In addition, we report recent investigations into host defense systems using in vitro and in vivo models, providing new insights into the innate immune response to infections. Increasing our knowledge of pathogenesis may help the development of novel therapeutic strategies based on anti-adhesive, anti-virulence, and anti-cell to cell signaling pathways drugs.
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http://dx.doi.org/10.3390/pathogens10040387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8063835PMC
March 2021

Acinetobacter baumannii Targets Human Carcinoembryonic Antigen-Related Cell Adhesion Molecules (CEACAMs) for Invasion of Pneumocytes.

mSystems 2020 Dec 22;5(6). Epub 2020 Dec 22.

IRCCS San Raffaele Pisana, Department of Human Sciences and Promotion of the Quality of Life, San Raffaele Roma Open University, Rome, Italy.

Multidrug-resistant is regarded as a life-threatening pathogen mainly associated with nosocomial and community-acquired pneumonia. Here, we show that can bind the human carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors CEACAM1, CEACAM5, and CEACAM6. This specific interaction enhances internalization in membrane-bound vacuoles, promptly decorated with Rab5, Rab7, and lipidated microtubule-associated protein light chain 3 (LC3). Dissecting intracellular signaling pathways revealed that infected pneumocytes trigger interleukin-8 (IL-8) secretion via the extracellular signal-regulated kinase (ERK)1/2 and nuclear factor-kappa B (NF-κB) signaling pathways for clearance. However, in CEACAM1-L-expressing cells, IL-8 secretion lasts only 24 h, possibly due to an -dependent effect on the CEACAM1-L intracellular domain. Conversely, the glycosylphosphatidylinositol-anchored CEACAM5 and CEACAM6 activate the c-Jun NH-terminal kinase (JNK)1/2-Rubicon-NOX2 pathway, suggestive of LC3-associated phagocytosis. Overall, our data show for the first time novel mechanisms of adhesion to and invasion of pneumocytes by via CEACAM-dependent signaling pathways that eventually lead to bacterial killing. These findings suggest that CEACAM upregulation could put patients at increased risk of lower respiratory tract infection by This work shows for the first time that binds to carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), CEACAM5, and CEACAM6. This binding significantly enhances internalization within alveolar host cell epithelia. Intracellular trafficking involves typical Rab5 and Rab7 vacuolar proteins as well as light chain 3 (LC3) and slowly progresses to bacterial killing by endosome acidification. CEACAM engagement by leads to distinct and specific downstream signaling pathways. The CEACAM1 pathway finely tunes interleukin-8 (IL-8) secretion, whereas CEACAM5 and CEACAM6 mediate LC3-associated phagocytosis. The present study provides new insights into -host interactions and could represent a promising therapeutic strategy to reduce pulmonary infections caused by this pathogen.
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http://dx.doi.org/10.1128/mSystems.00604-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7762790PMC
December 2020

SARS-CoV-2: Comparative analysis of different RNA extraction methods.

J Virol Methods 2021 01 4;287:114008. Epub 2020 Nov 4.

San Raffaele Roma Open University, 00166 Rome, Italy; IRCCS San Raffaele Pisana, 00166 Rome, Italy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of the COVID-19 pandemic. Although other diagnostic methods have been introduced, detection of viral genes on oro- and nasopharyngeal swabs by reverse-transcription real time-PCR (rRT-PCR) assays is still the gold standard. Efficient viral RNA extraction is a prerequisite for downstream performance of rRT-PCR assays. Currently, several automatic methods that include RNA extraction are available. However, due to the growing demand, a shortage in kit supplies could be experienced in several labs. For these reasons, the use of different commercial or in-house protocols for RNA extraction may increase the possibility to analyze high number of samples. Herein, we compared the efficiency of RNA extraction of three different commercial kits and an in-house extraction protocol using synthetic ssRNA standards of SARS-CoV-2 as well as in oro-nasopharyngeal swabs from six COVID-19-positive patients. It was concluded that tested commercial kits can be used with some modifications for the detection of the SARS-CoV-2 genome by rRT-PCR approaches, although with some differences in RNA yields. Conversely, EXTRAzol reagent was the less efficient due to the phase separation principle at the basis of RNA extraction. Overall, this study offers alternative suitable methods to manually extract RNA that can be taken into account for SARS-CoV-2 detection.
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http://dx.doi.org/10.1016/j.jviromet.2020.114008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7640895PMC
January 2021

The Global Emergency of Novel Coronavirus (SARS-CoV-2): An Update of the Current Status and Forecasting.

Int J Environ Res Public Health 2020 08 5;17(16). Epub 2020 Aug 5.

Department of Public Health and Infectious Diseases, Sapienza University of Rome, Laboratory affiliated to Institute Pasteur Italia- Cenci Bolognetti Foundation, 00185 Rome, Italy.

Over the past two decades, there have been two major outbreaks where the crossover of animal to humans has resulted in severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). In December 2019, a global public health concern started with the emergence of a new strain of coronavirus (SARS-CoV-2 or 2019 novel coronavirus, 2019-nCoV) which has rapidly spread all over the world from its origin in Wuhan, China. SARS-CoV-2 belongs to the genus, which includes human SARS-CoV, MERS and two other human coronaviruses (HCoVs), HCoV-OC43 and HCoV-HKU1. The fatality rate of SARS-CoV-2 is lower than the two previous coronavirus epidemics, but it is faster spreading and the large number of infected people with severe viral pneumonia and respiratory illness, showed SARS-CoV-2 to be highly contagious. Based on the current published evidence, herein we summarize the origin, genetics, epidemiology, clinical manifestations, preventions, diagnosis and up to date treatments of SARS-CoV-2 infections in comparison with those caused by SARS-CoV and MERS-CoV. Moreover, the possible impact of weather conditions on the transmission of SARS-CoV-2 is also discussed. Therefore, the aim of the present review is to reconsider the two previous pandemics and provide a reference for future studies as well as therapeutic approaches.
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http://dx.doi.org/10.3390/ijerph17165648DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7459861PMC
August 2020

Fecal microRNAs as Innovative Biomarkers of Intestinal Diseases and Effective Players in Host-Microbiome Interactions.

Cancers (Basel) 2020 Aug 5;12(8). Epub 2020 Aug 5.

Research Laboratories, Bambino Gesù Children's Hospital, IRCCS, 00146 Rome, Italy.

Over the past decade, short non-coding microRNAs (miRNAs), including circulating and fecal miRNAs have emerged as important modulators of various cellular processes by regulating the expression of target genes. Recent studies revealed the role of miRNAs as powerful biomarkers in disease diagnosis and for the development of innovative therapeutic applications in several human conditions, including intestinal diseases. In this review, we explored the literature and summarized the role of identified dysregulated fecal miRNAs in intestinal diseases, with particular focus on colorectal cancer (CRC) and celiac disease (CD). The aim of this review is to highlight one fascinating aspect of fecal miRNA function related to gut microbiota shaping and bacterial metabolism influencing. The role of miRNAs as "messenger" molecules for inter kingdom communications will be analyzed to highlight their role in the complex host-bacteria interactions. Moreover, whether fecal miRNAs could open up new perspectives to develop novel suitable biomarkers for disease detection and innovative therapeutic approaches to restore microbiota balance will be discussed.
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http://dx.doi.org/10.3390/cancers12082174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7463924PMC
August 2020

FimH and Anti-Adhesive Therapeutics: A Disarming Strategy Against Uropathogens.

Antibiotics (Basel) 2020 Jul 10;9(7). Epub 2020 Jul 10.

Department of Public Health and Infectious Diseases, Sapienza University of Rome, 00185 Rome, Italy.

Chaperone-usher fimbrial adhesins are powerful weapons against the uropathogens that allow the establishment of urinary tract infections (UTIs). As the antibiotic therapeutic strategy has become less effective in the treatment of uropathogen-related UTIs, the anti-adhesive molecules active against fimbrial adhesins, key determinants of urovirulence, are attractive alternatives. The best-characterized bacterial adhesin is FimH, produced by uropathogenic (UPEC). Hence, a number of high-affinity mono- and polyvalent mannose-based FimH antagonists, characterized by different bioavailabilities, have been reported. Given that antagonist affinities are firmly associated with the functional heterogeneities of different FimH variants, several FimH inhibitors have been developed using ligand-drug discovery strategies to generate high-affinity molecules for successful anti-adhesion therapy. As clinical trials have shown d-mannose's efficacy in UTIs prevention, it is supposed that mannosides could be a first-in-class strategy not only for UTIs, but also to combat other Gram-negative bacterial infections. Therefore, the current review discusses valuable and effective FimH anti-adhesive molecules active against UTIs, from design and synthesis to in vitro and in vivo evaluations.
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http://dx.doi.org/10.3390/antibiotics9070397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7400442PMC
July 2020

d-Mannose Treatment neither Affects Uropathogenic Properties nor Induces Stable FimH Modifications.

Molecules 2020 Jan 13;25(2). Epub 2020 Jan 13.

IRCCS San Raffaele Pisana, Department of Human Sciences and Promotion of the Quality of Life, San Raffaele Roma Open University, 00166 Rome, Italy.

Urinary tract infections (UTIs) are mainly caused by uropathogenic (UPEC). Acute and recurrent UTIs are commonly treated with antibiotics, the efficacy of which is limited by the emergence of antibiotic resistant strains. The natural sugar d-mannose is considered as an alternative to antibiotics due to its ability to mask the bacterial adhesin FimH, thereby preventing its binding to urothelial cells. Despite its extensive use, the possibility that d-mannose exerts "antibiotic-like" activity by altering bacterial growth and metabolism or selecting FimH variants has not been investigated yet. To this aim, main bacterial features of the prototype UPEC strain CFT073 treated with d-mannose were analyzed by standard microbiological methods. FimH functionality was analyzed by yeast agglutination and human bladder cell adhesion assays. Our results indicate that high d-mannose concentrations have no effect on bacterial growth and do not interfere with the activity of different antibiotics. d-mannose ranked as the least preferred carbon source to support bacterial metabolism and growth, in comparison with d-glucose, d-fructose, and l-arabinose. Since small glucose amounts are physiologically detectable in urine, we can conclude that the presence of d-mannose is irrelevant for bacterial metabolism. Moreover, d-mannose removal after long-term exposure did not alter FimH's capacity to bind to mannosylated proteins. Overall, our data indicate that d-mannose is a good alternative in the prevention and treatment of UPEC-related UTIs.
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http://dx.doi.org/10.3390/molecules25020316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7024335PMC
January 2020

A simple, fast and reliable scan-based technique as a novel approach to quantify intracellular bacteria.

BMC Microbiol 2019 11 12;19(1):252. Epub 2019 Nov 12.

Department of Public Health and Infectious Diseases, Sapienza University of Rome, 00185, Rome, Italy.

Background: Quantification of intracellular bacteria is fundamental in many areas of cellular and clinical microbiology to study acute and chronic infections. Therefore, rapid, accurate and low-cost methods represent valuable tools in determining bacterial ability to persist and proliferate within eukaryotic cells.

Results: Herein, we present the first application of the immunofluorescence In-Cell Western (ICW) assay aimed at quantifying intracellular bacteria in in vitro infection models. The performance of this new approach was evaluated in cell culture infection models using three microorganisms with different lifestyles. Two facultative intracellular bacteria, the fast-growing Shigella flexneri and a persistent strain of Escherichia coli, as well as the obligate intracellular bacterium Chlamydia trachomatis were chosen as bacterial models. The ICW assay was performed in parallel with conventional quantification methods, i.e. colony forming units (CFUs) and inclusion forming units (IFUs). The fluorescence signal intensity values from the ICW assay were highly correlated to CFU/IFUs counting and showed coefficients of determination (R), ranging from 0,92 to 0,99.

Conclusions: The ICW assay offers several advantages including sensitivity, reproducibility, high speed, operator-independent data acquisition and overtime stability of fluorescence signals. All these features, together with the simplicity in performance, make this assay particularly suitable for high-throughput screening and diagnostic approaches.
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http://dx.doi.org/10.1186/s12866-019-1625-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849193PMC
November 2019

Insights into the Periplasmic Proteins of AB5075 and the Impact of Imipenem Exposure: A Proteomic Approach.

Int J Mol Sci 2019 Jul 13;20(14). Epub 2019 Jul 13.

Department of Public Health and Infectious Diseases, Sapienza University of Rome, 00185 Rome, Italy.

Carbapenem-resistant strains cause life-threatening infections due to the lack of therapeutic options. Although the main mechanisms underlying antibiotic-resistance have been extensively studied, the general response to maintain bacterial viability under antibiotic exposure deserves to be fully investigated. Since the periplasmic space contains several proteins with crucial cellular functions, besides carbapenemases, we decided to study the periplasmic proteome of the multidrug-resistant (MDR) AB5075 strain, grown in the absence and presence of imipenem (IMP). Through the proteomic approach, 65 unique periplasmic proteins common in both growth conditions were identified: eight proteins involved in protein fate, response to oxidative stress, energy metabolism, antibiotic-resistance, were differentially expressed. Among them, ABUW_1746 and ABUW_2363 gene products presented the tetratricopeptide repeat motif, mediating protein-protein interactions. The expression switch of these proteins might determine specific protein interactions to better adapt to changing environmental conditions. ABUW_2868, encoding a heat shock protein likely involved in protection against oxidative stress, was upregulated in IMP-exposed bacteria. Accordingly, the addition of periplasmic proteins from cultured with IMP increased bacterial viability in an antioxidant activity assay. Overall, this study provides the first insights about the composition of the periplasmic proteins of a MDR strain, its biological response to IMP and suggests possible new targets to develop alternative antibiotic drugs.
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http://dx.doi.org/10.3390/ijms20143451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679007PMC
July 2019

Colonic adenoma-associated Escherichia coli express specific phenotypes.

Microbes Infect 2019 Aug - Sep;21(7):305-312. Epub 2019 Feb 11.

Department of Public Health and Infectious Diseases, Sapienza University of Rome, P.le A. Moro 5, 00185, Rome, Italy; Dani Di Giò Foundation-Onlus, Rome, Italy. Electronic address:

Specific Escherichia coli strains have been associated to colorectal cancer, while no data are available on genotypic and phenotypic features of E. coli colonizing premalignant adenomatous polyps and their pathogenic potential. This study was aimed at characterizing isolates collected from polyps and adjacent tissue in comparison with those from normal mucosa. From colonoscopy biopsies, 1500 E. coli isolates were retrieved and genotyped; 272 were characterized for phylogroup and major phenotypic traits (i.e., biofilm formation, motility, hemolysins, and proteases). Selected isolates were analyzed for extraintestinal pathogenic E. coli (ExPEC)-associated virulence genes and in vivo pathogenicity using Galleria mellonella. The majority of isolates collected from polyps were strong biofilm and poor protease producers, whereas those isolates from normal mucosa were highly motile, proteolytic and weak biofilm formers. Isolates from adjacent tissues shared features with those from both polyps and normal mucosa. Among selected E. coli isolates, ExPEC gene content/profile was variable and uncorrelated with the tissue of collection and larval mortality. Despite the heterogeneous virulence-gene carriage of the E. coli intestinal population, E. coli colonizing colonic adenomatous polyps express specific phenotypic traits that could represent an initial pathoadaptation to local environmental changes characterizing these lesions.
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http://dx.doi.org/10.1016/j.micinf.2019.02.001DOI Listing
February 2020

Cutaneous candidiasis caused by Candida albicans in a young non-immunosuppressed patient: an unusual presentation.

Int J Immunopathol Pharmacol 2018 Jan-Dec;32:2058738418781368

3 Plastic and Reconstructive Surgery Unit, Campus Bio-Medico University, Rome, Italy.

Candidiasis is a fungal infection caused by yeasts that belong to the genus Candida. There are over 20 species of Candida yeasts that can cause infection in humans, the most common of which is Candida albicans. Candida yeasts normally reside in the intestinal tract and can be found on mucous membranes and skin without causing infection. However, under immunocompromised conditions, Candida can cause significant infections in susceptible patients. Herein, we report a peculiar presentation of a C. albicans cutaneous infection in an immunocompetent young subject. This case widens our knowledge on the C. albicans infections both in terms of host susceptibility and cutaneous manifestations.
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http://dx.doi.org/10.1177/2058738418781368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398342PMC
October 2018

in Italy: A Case of Septicemia and Abdominal Aortic Aneurysm Infection.

Front Med (Lausanne) 2018 24;5:156. Epub 2018 May 24.

Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy.

We report a case of septicemia in a 63-year-old patient admitted to the Vascular Surgery Department of Umberto I Hospital (Rome, Italy) for an abdominal aortic aneurysm. The microorganism, recovered from both peripheral blood cultures and aneurysmatic aortic wall specimens, was identified as using matrix-assisted laser desorption ionization-time of flight analysis (MALDI-TOF MS) and 16S rDNA gene sequencing. The isolate responsible for septicemia belonged to the O:9 serotype (biogroup 2). A genetic screening of the isolate made it possible to detect the presence of both the and genes, encoding a heat-stable enterotoxin and a protein involved in invasion/adherence and serum resistance, respectively. Our case contributes in enriching epidemiological data concerning infections, which might represent severe complications in patients suffering from cardiovascular diseases. Moreover, this study, together with the others, should be regarded as valuable and useful tools for monitoring the rate of infections worldwide.
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http://dx.doi.org/10.3389/fmed.2018.00156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5978273PMC
May 2018

Virulence Traits: A Comparative Study of a Novel Sequence Type with Other Italian Endemic International Clones.

Front Microbiol 2017 12;8:1977. Epub 2017 Oct 12.

Department of Public Health and Infectious Diseases, Sapienza University of Rome, Laboratory Affiliated to Institute Pasteur Italia, Cenci-Bolognetti Foundation, Rome, Italy.

Carbapenem-resistant (CRAb) have emerged in recent decades as major causes of nosocomial infections. Resistance is mainly due to overexpression of intrinsic and/or acquired carbapenemases, especially oxacillinases (OXA). In Italy, although the sequence type (ST) 2 and the ST78 are the most frequently detected, we recently reported ST632, a single locus variant of ST2. Therefore, this study was aimed at unraveling common bacterial surface virulence factors involved in pathogenesis and antibiotic resistance in representative CRAb of these ST genotypes. Outer membrane protein (OMP) composition together with motility, biofilm formation, adherence to, invasion of, and survival within pneumocytes were analyzed. Differently from the carbapenem-susceptible reference strain ATCC 17978, either overexpressed OXA-51 or both OXA-23 and OXA-51 co-purified with OMPs in CRAb. This tight association ensures their maximal concentration on the inner surface of the outer membrane to provide the best protection against carbapenems. These findings led us to propose for the first time a common behavior of OXA enzymes in CRAb. Despite the presence of both OmpA and phosphorylcholine-porinD and the ability of all the strains to adhere to cells, invasion, and survival within pneumocytes was shown only by ST2 and ST78 isolates, sharing the highest number of identified OMPs. Conversely, notwithstanding genetic and OMPs similarities with ST2, ST632 was unable to invade and survive within epithelial cells. Overall, our study shows that different STs share a specific OMP composition, also shaped by overexpressed OXA, that is needed for invasiveness and survival of CRAb.
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http://dx.doi.org/10.3389/fmicb.2017.01977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5643476PMC
October 2017

Genetic diversity, phylogroup distribution and virulence gene profile of pks positive Escherichia coli colonizing human intestinal polyps.

Microb Pathog 2017 Nov 5;112:274-278. Epub 2017 Oct 5.

Department of Public Health and Infectious Diseases, Sapienza University of Rome, Rome, Italy.

Some Escherichia coli strains of phylogroup B2 harbor a (pks) pathogenicity island that encodes a polyketide-peptide genotoxin called colibactin. It causes DNA double-strand breaks and megalocytosis in eukaryotic cells and it may contribute to cancer development. Study of bacterial community that colonizes the adenomatous polyp lesion, defined as precancerous lesions, could be helpful to assess if such pathogenic bacteria possess a role in the polyp progression to cancer. In this cross-sectional study, a total of 1500 E. coli isolates were obtained from biopsies of patients presenting adenomatous colon polyps, the normal tissues adjacent to the polyp lesion and patients presenting normal mucosa. pks island frequency, phylogenetic grouping, fingerprint genotyping, and virulence gene features of pks positive (pks) E. coli isolates were performed. We found pksE. coli strongly colonize two patients presenting polypoid lesions and none were identified in patients presenting normal mucosa. Predominant phylogroups among pksE. coli isolates were B2, followed by D. Clustering based on fragment profiles of composite analysis, typed the pks isolates into 5 major clusters (I-V) and 17 sub-clusters, demonstrating a high level of genetic diversity among them. The most prevalent virulence genes were fimH and fyuA (100%), followed by vat (92%), hra and papA (69%), ibeA (28%), and hlyA (25%). Our results revealed that pksE. coli can colonize the precancerous lesions, with a high distribution in both the polyp lesions and in normal tissues adjacent to the lesion. The high differences in fingerprinting patterns obtained indicate that pksE. coli strains were genetically diverse, possibly allowing them to more easily adapt to environmental variations.
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http://dx.doi.org/10.1016/j.micpath.2017.10.009DOI Listing
November 2017

The OmpA amino acid residues EVQ are essential for the interaction with the virulence factor PhoN2.

Biochem Biophys Rep 2016 Dec 12;8:168-173. Epub 2016 Aug 12.

Dip. Scienze Mediche, Orali e Biotecnologiche, Università "G. D'Annunzio" di Chieti, Chieti, Italy.

is an intracellular pathogen that deploys an arsenal of virulence factors promoting host cell invasion, intracellular multiplication and intra- and inter-cellular dissemination. We have previously reported that the interaction between apyrase (PhoN2), a periplasmic ATP-diphosphohydrolase, and the C-terminal domain of the outer membrane (OM) protein OmpA is likely required for proper IcsA exposition at the old bacterial pole and thus for full virulence expression of (Scribano et al., 2014). OmpA, that is the major OM protein of Gram-negative bacteria, is a multifaceted protein that plays many different roles both in the OM structural integrity and in the virulence of several pathogens. Here, by using yeast two-hybrid technology and by constructing an 3D model of OmpA from 5a strain M90T, we observed that the OmpA residues EVQ are likely essential for PhoN2-OmpA interaction. The EVQ amino acids are located within a flexible region of the OmpA protein that could represent a scaffold for protein-protein interaction.
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http://dx.doi.org/10.1016/j.bbrep.2016.08.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5613738PMC
December 2016

Molecular characterisation of extensively drug-resistant Acinetobacter baumannii: First report of a new sequence type in Italy.

J Glob Antimicrob Resist 2016 12 1;7:154-156. Epub 2016 Nov 1.

Institute Pasteur Italia, Fondazione Cenci Bolognetti, Department of Public Health and Infectious Diseases, Sapienza University, Rome, Italy; San Raffaele Pisana IRCCS, Telematic University, Rome, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.jgar.2016.10.002DOI Listing
December 2016

The Adherent/Invasive Escherichia coli Strain LF82 Invades and Persists in Human Prostate Cell Line RWPE-1, Activating a Strong Inflammatory Response.

Infect Immun 2016 Nov 17;84(11):3105-3113. Epub 2016 Oct 17.

Department of Public Health and Infectious Diseases, Sapienza University, Rome, Italy.

Adherent/invasive Escherichia coli (AIEC) strains have recently been receiving increased attention because they are more prevalent and persistent in the intestine of Crohn's disease (CD) patients than in healthy subjects. Since AIEC strains show a high percentage of similarity to extraintestinal pathogenic E. coli (ExPEC), neonatal meningitis-associated E. coli (NMEC), and uropathogenic E. coli (UPEC) strains, here we compared AIEC strain LF82 with a UPEC isolate (strain EC73) to assess whether LF82 would be able to infect prostate cells as an extraintestinal target. The virulence phenotypes of both strains were determined by using the RWPE-1 prostate cell line. The results obtained indicated that LF82 and EC73 are able to adhere to, invade, and survive within prostate epithelial cells. Invasion was confirmed by immunofluorescence and electron microscopy. Moreover, cytochalasin D and colchicine strongly inhibited bacterial uptake of both strains, indicating the involvement of actin microfilaments and microtubules in host cell invasion. Moreover, both strains belong to phylogenetic group B2 and are strong biofilm producers. In silico analysis reveals that LF82 shares with UPEC strains several virulence factors: namely, type 1 pili, the group II capsule, the vacuolating autotransporter toxin, four iron uptake systems, and the pathogenic island (PAI). Furthermore, compared to EC73, LF82 induces in RWPE-1 cells a marked increase of phosphorylation of mitogen-activated protein kinases (MAPKs) and of NF-κB already by 5 min postinfection, thus inducing a strong inflammatory response. Our in vitro data support the hypothesis that AIEC strains might play a role in prostatitis, and, by exploiting host-cell signaling pathways controlling the innate immune response, likely facilitate bacterial multiplication and dissemination within the male genitourinary tract.
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http://dx.doi.org/10.1128/IAI.00438-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5067744PMC
November 2016

The Shigella flexneri OspB effector: an early immunomodulator.

Int J Med Microbiol 2015 Jan 11;305(1):75-84. Epub 2014 Nov 11.

Dipartimento di Scienze Sperimentali e Cliniche, Università "G. D'Annunzio", 66100 Chieti, Italy. Electronic address:

Through the action of the type three secretion system (T3SS) Shigella flexneri delivers several effectors into host cells to promote cellular invasion, multiplication and to exploit host-cell signaling pathways to modulate the host innate immune response. Although much progress has been made in the understanding of many type III effectors, the molecular and cellular mechanism of the OspB effector is still poorly characterized. In this study we present new evidence that better elucidates the role of OspB as pro-inflammatory factor at very early stages of infection. Indeed, we demonstrate that, during the first hour of infection, OspB is required for full activation of ERK1/2 and p38 MAPKs and the cytosolic phospholipase A(2) (cPLA(2)). Activation of cPLA(2) ultimately leads to the production and secretion of PMN chemoattractant metabolite(s) uncoupled with release of IL-8. Moreover, we also present evidence that OspB is required for the development of the full and promptly inflammatory reaction characteristic of S. flexneri wild-type infection in vivo. Based on OspB and OspF similarity (both effectors share similar transcription regulation, temporal secretion into host cells and nuclear localization) we hypothesized that OspB and OspF effectors may form a pair aimed at modulating the host cell response throughout the infection process, with opposite effects. A model is presented to illustrate how OspB activity would promote S. flexneri invasion and bacterial dissemination at early critical phases of infection.
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http://dx.doi.org/10.1016/j.ijmm.2014.11.004DOI Listing
January 2015

Bdellovibrio bacteriovorus directly attacks Pseudomonas aeruginosa and Staphylococcus aureus Cystic fibrosis isolates.

Front Microbiol 2014 5;5:280. Epub 2014 Jun 5.

Microbiology Section, Department of Public Health and Infectious Diseases, "Sapienza" University Rome, Italy.

Bdellovibrio bacteriovorus is a predator bacterial species found in the environment and within the human gut, able to attack Gram-negative prey. Cystic fibrosis (CF) is a genetic disease which usually presents lung colonization by Pseudomonas aeruginosa or Staphylococcus aureus biofilms. Here, we investigated the predatory behavior of B. bacteriovorus against these two pathogenic species with: (1) broth culture; (2) "static" biofilms; (3) field emission scanning electron microscope (FESEM); (4) "flow" biofilms; (5) zymographic technique. We had the first evidence of B. bacteriovorus survival with a Gram-positive prey, revealing a direct cell-to-cell contact with S. aureus and a new "epibiotic" foraging strategy imaged with FESEM. Mean attaching time of HD100 to S. aureus cells was 185 s, while "static" and "flow" S. aureus biofilms were reduced by 74 (at 24 h) and 46% (at 20 h), respectively. Furthermore, zymograms showed a differential bacteriolytic activity exerted by the B. bacteriovorus lysates on P. aeruginosa and S. aureus. The dual foraging system against Gram-negative (periplasmic) and Gram-positive (epibiotic) prey could suggest the use of B. bacteriovorus as a "living antibiotic" in CF, even if further studies are required to simulate its in vivo predatory behavior.
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http://dx.doi.org/10.3389/fmicb.2014.00280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046265PMC
June 2014

Polar localization of PhoN2, a periplasmic virulence-associated factor of Shigella flexneri, is required for proper IcsA exposition at the old bacterial pole.

PLoS One 2014 27;9(2):e90230. Epub 2014 Feb 27.

Dipartimento di Scienze Sperimentali e Cliniche, Università "G. D'Annunzio", Chieti, Italy.

Proper protein localization is critical for bacterial virulence. PhoN2 is a virulence-associated ATP-diphosphohydrolase (apyrase) involved in IcsA-mediated actin-based motility of S. flexneri. Herein, by analyzing a ΔphoN2 mutant of the S. flexneri strain M90T and by generating phoN2::HA fusions, we show that PhoN2, is a periplasmic protein that strictly localizes at the bacterial poles, with a strong preference for the old pole, the pole where IcsA is exposed, and that it is required for proper IcsA exposition. PhoN2-HA was found to be polarly localized both when phoN2::HA was ectopically expressed in a Escherichia coli K-12 strain and in a S. flexneri virulence plasmid-cured mutant, indicating a conserved mechanism of PhoN2 polar delivery across species and that neither IcsA nor the expression of other virulence-plasmid encoded genes are involved in this process. To assess whether PhoN2 and IcsA may interact, two-hybrid and cross-linking experiments were performed. While no evidence was found of a PhoN2-IcsA interaction, unexpectedly the outer membrane protein A (OmpA) was shown to bind PhoN2-HA through its periplasmic-exposed C-terminal domain. Therefore, to identify PhoN2 domains involved in its periplasmic polar delivery as well as in the interaction with OmpA, a deletion and a set of specific amino acid substitutions were generated. Analysis of these mutants indicated that neither the (183)PAPAP(187) motif of OmpA, nor the N-terminal polyproline (43)PPPP(46) motif and the Y155 residue of PhoN2 are involved in this interaction while P45, P46 and Y155 residues were found to be critical for the correct folding and stability of the protein. The relative rapid degradation of these amino acid-substituted recombinant proteins was found to be due to unknown S. flexneri-specific protease(s). A model depicting how the PhoN2-OmpA interaction may contribute to proper polar IcsA exposition in S. flexneri is presented.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0090230PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3937361PMC
October 2014

Outer membrane protein A (OmpA): a new player in shigella flexneri protrusion formation and inter-cellular spreading.

PLoS One 2012 14;7(11):e49625. Epub 2012 Nov 14.

Dip. di Scienze Sperimentali e Cliniche, Università "G. D'Annunzio' di Chieti, Chieti, Italy.

Outer membrane protein A (OmpA) is a multifaceted predominant outer membrane protein of Escherichia coli and other Enterobacteriaceae whose role in the pathogenesis of various bacterial infections has recently been recognized. Here, the role of OmpA on the virulence of Shigella flexneri has been investigated. An ompA mutant of wild-type S. flexneri 5a strain M90T was constructed (strain HND92) and it was shown to be severely impaired in cell-to-cell spreading since it failed to plaque on HeLa cell monolayers. The lack of OmpA significantly reduced the levels of IcsA while the levels of cell associated and released IcsP-cleaved 95 kDa amino-terminal portion of the mature protein were similar. Nevertheless, the ompA mutant displayed IcsA exposed across the entire bacterial surface. Surprisingly, the ompA mutant produced proper F-actin comet tails, indicating that the aberrant IcsA exposition at bacterial lateral surface did not affect proper activation of actin-nucleating proteins, suggesting that the absence of OmpA likely unmasks mature or cell associated IcsA at bacterial lateral surface. Moreover, the ompA mutant was able to invade and to multiply within HeLa cell monolayers, although internalized bacteria were found to be entrapped within the host cell cytoplasm. We found that the ompA mutant produced significantly less protrusions than the wild-type strain, indicating that this defect could be responsible of its inability to plaque. Although we could not definitely rule out that the ompA mutation might exert pleiotropic effects on other S. flexneri genes, complementation of the ompA mutation with a recombinant plasmid carrying the S. flexneri ompA gene clearly indicated that a functional OmpA protein is required and sufficient for proper IcsA exposition, plaque and protrusion formation. Moreover, an independent ompA mutant was generated. Since we found that both mutants displayed identical virulence profile, these results further supported the findings presented in this study.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0049625PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3498225PMC
June 2013

The NUTRA-SNACKS project: basic research and biotechnological programs on nutraceutics.

Adv Exp Med Biol 2010 ;698:1-16

Institute of Crystallography, Department of Agrofood, National Council of Research, Monterotondo Scalo, Rome, Italy.

The Nutra-Snacks project aims at creating novel high quality ready-to-eat foods with functional activity, useful for promoting public health. The team is composed of seven research institutes and three SMEs from different countries whose activities span from basic to applied research providing the right technological transfer to small and medium industries involved in the novel food production chain. Strategic objectives include the application of plant cell and in vitro culture systems to create very large amounts of high-value plant secondary metabolites with recognized anticancer, antilipidemic, anticholesterol, antimicrobial, antiviral, antihypertensive and anti-inflammatory properties and to include them in specific food products. To this end, the screening of a vast number of working organisms capable of accumulating the desired compounds and the characterization of their expression profiles represent fundamental steps in the research program. The information allows the identification of plant species hyper-producing metabolites and selection of those metabolites capable of specifically counteracting the oxidative stress that underlies the development of important pathologies and diseases. In addition, devising safe metabolite extraction procedures is also crucial in order to provide nutraceutical-enriched extracts compatible with human health. New biotechnological approaches are also undertaken including the exploitation of photosynthetic algal strains in bio-farms to enhance the synthesis ofantioxidant compounds and the design of novel bioreactors for small and large scale biomass production. Further outstanding objectives include the development of (i) safety and quality control protocols (ii) biosensor techniques for the analysis of the emerging ready-to-eat food and (iii) a contribution to define a standard for new regulations on nutraceutics.
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http://dx.doi.org/10.1007/978-1-4419-7347-4_1DOI Listing
May 2011

Membrane-association determinants of the omega-amino acid monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aeruginosa.

Microbiology (Reading) 2008 Sep;154(Pt 9):2804-2813

National Institute for Infectious Diseases IRCCS, 'Lazzaro Spallanzani', Via Portuense 292, I-00149 Rome, Italy.

The L-ornithine N(delta)-oxygenase PvdA catalyses the N(delta)-hydroxylation of L-ornithine in many Pseudomonas spp., and thus provides an essential enzymic function in the biogenesis of the pyoverdine siderophore. Here, we report a detailed analysis of the membrane topology of the PvdA enzyme from the bacterial pathogen Pseudomonas aeruginosa. Membrane topogenic determinants of PvdA were identified by computational analysis, and verified in Escherichia coli by constructing a series of translational fusions between PvdA and the PhoA (alkaline phosphatase) reporter enzyme. The inferred topological model resembled a eukaryotic reverse signal-anchor (type III) protein, with a single N-terminal domain anchored to the inner membrane, and the bulk of the protein spanning the cytosol. According to this model, the predicted transmembrane region should overlap the putative FAD-binding site. Cell fractionation and proteinase K accessibility experiments in P. aeruginosa confirmed the membrane-bound nature of PvdA, but excluded the transmembrane topology of its N-terminal hydrophobic region. Mutational analysis of PvdA, and complementation assays in a P. aeruginosa DeltapvdA mutant, demonstrated the dual (structural and functional) role of the PvdA N-terminal domain.
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http://dx.doi.org/10.1099/mic.0.2008/018804-0DOI Listing
September 2008

Involvement of AlgQ in transcriptional regulation of pyoverdine genes in Pseudomonas aeruginosa PAO1.

J Bacteriol 2005 Aug;187(15):5097-107

Dipartimento di Biologia, Università di Roma Tre, Viale G. Marconi 446, 00146 Roma, Italy.

In response to iron limitation, Pseudomonas aeruginosa produces the fluorescent siderophore pyoverdine. Transcription of pyoverdine biosynthetic (pvd) genes is driven by the iron starvation sigma factor PvdS, which is negatively regulated by the Fur-Fe(II) holorepressor. We studied the effect of AlgQ, the Escherichia coli Rsd orthologue, on pyoverdine production by P. aeruginosa PAO1. AlgQ is a global regulatory protein which activates alginate, ppGpp, and inorganic polyphosphate synthesis through a cascade involving nucleoside diphosphate kinase (Ndk). AlgQ is also capable of interacting with region 4 of RpoD. In a reconstituted E. coli system, PvdS-dependent transcription from the pvdA promoter was doubled by the multicopy algQ gene. The P. aeruginosa DeltaalgQ mutant exhibited a moderate but reproducible reduction in pyoverdine production compared with wild-type PAO1, as a result of a decline in transcription of pvd genes. PvdS expression was not affected by the algQ mutation. Single-copy algQ fully restored pyoverdine production and expression of pvd genes in the DeltaalgQ mutant, while ndk did not. An increased intracellular concentration of RpoD mimicked the DeltaalgQ phenotype, whereas PvdS overexpression suppressed the algQ mutation. E. coli rsd could partially substitute for algQ in transcriptional modulation of pvd genes. We propose that AlgQ acts as an anti-sigma factor for RpoD, eliciting core RNA polymerase recruitment by PvdS and transcription initiation at pvd promoters. AlgQ provides a link between the pyoverdine and alginate regulatory networks. These systems have similarities in responsiveness and physiological function: both depend on alternative sigma factors, respond to nutrient starvation, and act as virulence determinants for P. aeruginosa.
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http://dx.doi.org/10.1128/JB.187.15.5097-5107.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1196021PMC
August 2005

Is there an answer? Is it better for a bacterium to be gram-positive or gram-negative?

IUBMB Life 2004 Jun;56(6):361-3

Department of Biology, University Roma Tre, Rome, Italy.

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http://dx.doi.org/10.1080/15216540400000884DOI Listing
June 2004

Expression of L-ornithine Ndelta-oxygenase (PvdA) in fluorescent Pseudomonas species: an immunochemical and in silico study.

Biochem Biophys Res Commun 2004 Jan;313(2):245-57

Unità di Microbiologia Molecolare, Istituto Nazionale per le Malattie Infettive I.R.C.C.S. Lazzaro Spallanzani, Via Portuense 292, 00149 Roma, Italy.

Omega-amino acid monooxygenases (EC 1.14.13.-), catalysing the formation of hydroxamate precursors of microbial siderophores (e.g., pyoverdine), have so far eluded structural and biochemical characterisation. Here, the expression of recombinant L-ornithine-Ndelta-oxygenase (PvdA) from Pseudomonas aeruginosa PAO1 is reported. A library of eight monoclonal antibodies (MAbs) directed against PvdA has been generated. Two MAb families recognising the N- and C-terminal regions of PvdA were identified. The MAbs made it possible to demonstrate that 45-48 kDa PvdA homologues are expressed in response to iron limitation by different species and strains of fluorescent pseudomonads. Despite the different degrees in sequence similarity between P. aeruginosa PvdA and putative homologues from Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, Burkholderia cepacia, and Ralstonia solanacearum, in silico domain scanning predicts an impressive conservation of putative cofactor and substrate binding domains. The MAb library was also used to monitor PvdA expression during the transition of P. aeruginosa from iron-sufficient to iron-deficient growth.
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http://dx.doi.org/10.1016/j.bbrc.2003.11.116DOI Listing
January 2004

Different responses of pyoverdine genes to autoinduction in Pseudomonas aeruginosa and the group Pseudomonas fluorescens-Pseudomonas putida.

Appl Environ Microbiol 2002 Aug;68(8):4122-6

Unità di Microbiologia Molecolare, I.R.C.C.S. Lazzaro Spallanzani, 00149 Rome, Italy.

We investigated the regulation of the psbA and pvdA pyoverdine biosynthesis genes, which encode the L-ornithine N(5)-oxygenase homologues in Pseudomonas strain B10 and Pseudomonas aeruginosa PAO1, respectively. We demonstrate that pyoverdine(B10), as the end product of its biosynthetic pathway, is a key participant of the control circuit regulating its own production in Pseudomonas strain B10. In P. aeruginosa PAO1, however, pyoverdine(PAO1) has no apparent role in the positive regulation of the pvdA gene.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC124028PMC
http://dx.doi.org/10.1128/aem.68.8.4122-4126.2002DOI Listing
August 2002

Transcriptional regulation of pseudobactin synthesis in the plant growth-promoting Pseudomonas B10.

FEMS Microbiol Lett 2002 Mar;208(2):219-25

Dipartimento di Biologia, Università di Roma Tre, Viale G. Marconi 446, 00146 Rome, Italy.

We have investigated the iron-dependent regulation of the psbA gene, encoding the enzyme L-ornithine N(5)-oxygenase in the rhizobacterium Pseudomonas B10. We have cloned and characterized a Pseudomonas B10 gene, designated psbS, required for psbA expression. PsbS is endowed with structural and functional features of extracytoplasmatic function (ECF) sigma factors, and is closely related to the iron starvation sigmas PvdS, PbrA, and PfrI, which mediate the iron-repressible expression of pseudobactin biosynthesis genes in different Pseudomonas species. Expression of psbA was found to be indirectly controlled by Fur, which abrogates psbS transcription in the presence of sufficient iron.
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http://dx.doi.org/10.1111/j.1574-6968.2002.tb11085.xDOI Listing
March 2002