pKBuS13, a KPC-2-Encoding Plasmid from Klebsiella pneumoniae Sequence Type 833, Carrying Tn4401b Inserted into an Xer Site-Specific Recombination Locus

Here, we report the first detection of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae strain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. Its bla KPC determinant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn4401b::bla KPC-2 transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn4401b detected a region that did not have significant matches in databases other than the genome of a carbapenem-resistant Escherichia coli strain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence of tra genes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported

Antimicrobial and Cytotoxic Properties of Extracts from Plants Traditionally Used in North-East Brazil

The north-eastern region of Brazil is home to communities of diverse ethnic origins that still depend significantly on folk medicine, with emphasis on plant extracts. The present work aims at investigating medicinal plants used in the traditional medicine of north-eastern Brazil with an assessment of the antimicrobial profile of their extracts. The antimicrobial activity of 12 extracts from 11 plant species from eight higher plant families traditionally used was evaluated against three human pathogens (Staphylococcus aureus, Escherichia coli, Streptococcus pneumoniae) using the Agar Disk Diffusion and Broth Microdilution assays. The cytotoxic effects were evaluated on human keratinocytes. A hydroalcoholic extract from Anadenanthera colubrina presented the most promising in vitro antimicrobial activity against S.aureus associated with low cytotoxicity towards human keratinocytes. The information gathered in the present study represents a starting point for further research aiming at providing scientific evidence to the empirical usage of medicinal plants in traditional practices

Interplay of OpdP Porin and Chromosomal Carbapenemases in the Determination of Carbapenem Resistance/Susceptibility in Pseudomonas aeruginosa

8noCarbapenem resistance in Pseudomonas aeruginosa strains responsible for chronic lung infections in cystic fibrosis (CF) patients is mainly due to loss of the OprD protein and, limited to meropenem and doripenem, to overexpression of efflux pumps. However, recent reports of isolates showing inconsistent genotype-phenotype combinations (e.g., susceptibility in the presence of resistance determinants and vice versa) suggest the involvement of additional factors whose role is not yet fully elucidated. Among them, the OpdP porin as an alternative route of entry for carbapenems other than OprD and the overexpression of two chromosomal carbapenemases, the Pseudomonas-derived cephalosporinase (PDC) and the PoxB oxacillinase, have recently been reconsidered and studied in specific model strains. Here, the contribution of these factors was investigated by comparing different phenotypic variants of three strains collected from the sputum of colonized CF patients. Carbapenem uptake through OpdP was investigated both at the functional level, by assessing the competition exerted by glycine-glutamate, the OpdP's natural substrate, against imipenem uptake, and at the molecular level, by comparing the expression levels of opdP genes by quantitative real-time PCR (qRT-PCR). Moreover, overexpression of the chromosomal carbapenemases in some of the isolates was also investigated by qRT-PCR. The results showed that, even if OprD inactivation remains the most important determinant of carbapenem resistance in strains infecting the CF lung, the interplay of other determinants might have a nonnegligible impact on bacterial susceptibility, being able to modify the phenotype of part of the population and consequently complicating the choice of an appropriate therapy. IMPORTANCE This study examines the interplay of multiple factors in determining a pattern of resistance or susceptibility to carbapenems in clinical isolates of Pseudomonas aeruginosa, focusing on the role of previously poorly understood determinants. In particular, the impact of carbapenem permeability through OprD and OpdP porins was analyzed, as well as the activity of the chromosomal carbapenemases AmpC and PoxB, going beyond the simple identification of resistance determinants encoded by each isolate. Indeed, analysis of the expression levels of these determinants provides a new approach to determine the contribution of each factor, both individually and in coexistence with the other factors. The study contributes to understanding some phenotype-genotype discordances closely related to the heteroresistance frequently detected in P. aeruginosa isolates responsible for pulmonary infections in cystic fibrosis patients, which complicates the choice of an appropriate patient-specific therapy.openopenAtrissi, Jad; Milan, Annalisa; Bressan, Raffaela; Lucafò, Marianna; Petix, Vincenzo; Busetti, Marina; Dolzani, Lucilla; Lagatolla, CristinaAtrissi, Jad; Milan, Annalisa; Bressan, Raffaela; Lucafò, Marianna; Petix, Vincenzo; Busetti, Marina; Dolzani, Lucilla; Lagatolla, Cristin

Evaluation of a biomimetic 3D substrate based on the Human Elastin-like Polypeptides (HELPs) model system for elastolytic activity detection

Elastin is a fibrous protein that confers elasticity to tissues such as skin, arteries and lung. It is extensively cross-linked, highly hydrophobic and insoluble. Nevertheless, elastin can be hydrolysed by bacterial proteases in infectious diseases, resulting in more or less severe tissue damage. Thus, development of substrates able to reliably and specifically detect pathogen-secreted elastolytic activity is needed to improve the in vitro evaluation of the injury that bacterial proteases may provoke. In this work, two human biomimetic elastin polypeptides, HELP and HELP1, as well as the matrices derived from HELP, have been probed as substrates for elastolytic activity detection. Thirty strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients were analyzed in parallel with standard substrates, to detect proteolytic and elastolytic activity. Results point to the HELP-based 3D matrix as an interesting biomimetic model of elastin to assess bacterial elastolytic activity in vitro. Moreover, this model substrate enables to further elucidate the mechanism underlying elastin degradation at molecular level, as well as to develop biomimetic material-based devices responsive to external stimuli

Aseptic central nervous system infections in adults: what predictor for unknown etiological diagnosis?

Despite the availability of nucleic acid amplification tests (NAATs), most of aseptic acute meningitides, encephalitides, and meningoencephalitides (AAMEMs) in adults remain of unknown etiology so far. To shed light on such topic, we aimed to evaluate potential predictors for AAMEMs of unknown origin. We collected retrospectively data from all consecutive cases of AAMEMs in adults discharged from an Italian referral hospital, from January 2004 to December 2016. Laboratory analysis included common immunometric methods and NAATs. Potential predictors for unknown etiology (age, seasonality, serum C-reactive protein value, antibiotic use before lumbar puncture, immunodeficiency conditions, clinical symptoms and signs) were evaluated by a logistic regression analysis model. A p value 64\u20090.05 was considered to indicate statistical significance. The study included 92 patients (median age 39 years; 54.3% males) affected by meningitis (n\u2009=\u200957), encephalitis (n\u2009=\u200925), and meningoencephalitis (n\u2009=\u200910). The identified agents that cause AAMEMs were herpesviruses (20.7%), enteroviruses (5.4%), tick-borne encephalitis virus (3.3%), influenza virus A (2.2%), West Nile virus (1.1%), and parvovirus B19 (1.1%), while 66.3% of cases were of unknown etiology. No predictor was found to be significantly associated with AAMEMs of unknown etiology. We suggest that potential infectious agents causing undiagnosed cases should be investigated among non-bacterial, non-opportunistic, and non-seasonal organisms

A Real-Time Polymerase Chain Reaction-Based Protocol for Low/Medium-Throughput Y-Chromosome Microdeletions Analysis

We describe a real-time polymerase chain reaction (PCR) protocol based on the fluorescent molecule SYBR Green chemistry, for a low- to medium-throughput analysis of Y-chromosome microdeletions, optimized according to the European guidelines and aimed at making the protocol faster, avoiding post-PCR processing, and simplifying the results interpretation. Methods: We screened 156 men from the Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Institute for Maternal and Child Health IRCCS Burlo Garofolo (Trieste, Italy), 150 not presenting Y-chromosome microdeletion, and 6 with microdeletions in different azoospermic factor (AZF) regions. For each sample, the Zinc finger Y-chromosomal protein (ZFY), sex-determining region Y (SRY), sY84, sY86, sY127, sY134, sY254, and sY255 loci were analyzed by performing one reaction for each locus. Results: AZF microdeletions were successfully detected in six individuals, confirming the results obtained with commercial kits. Conclusion: Our real-time PCR protocol proved to be a rapid, safe, and relatively cheap method that was suitable for a low- to medium-throughput diagnosis of Y-chromosome microdeletion, which allows an analysis of approximately 10 samples (with the addition of positive and negative controls) in a 96-well plate format, or approximately 46 samples in a 384-well plate for all markers simultaneously, in less than 2\u2009h without the need of post-PCR manipulation

Intestinal-mucosa anti-transglutaminase antibody assays to test for genetic gluten intolerance

Celiac disease (CD) is an autoimmune enteropathy characterized by gluten-triggered intestinal mucosa lesions in genetically susceptible individuals carrying the HLA DQ2 or DQ8. CD diagnosis is based on the concentration of IgA serum antitransglutaminase (anti-tTG) antibodies together with mucosal damage at intestinal biopsy.1 However, it is now known that in subjects with genetic gluten intolerance, gastrointestinal and extra-intestinal symptoms may be present even when both mucosal morphology and serum anti-tTGs are normal.2,3 In this context, the anti-tTG in the intestinal mucosal seem to be the specific CD immunological marker that is detectable before the development of intestinal atrophy and the appearance of serum anti-tTG.4 This prospective study investigates the presence of intestinal anti-tTG antibodies in patients with differing clinical spectrums of genetic gluten intolerance by using two immunoassays: double immunofluorescence test for anti-tTG on the intestinal mucosa and flow cytometry assay to measure acid-eluted intestinal anti-tTG

Circulation of SARS-CoV-2 Variants among Children from November 2020 to January 2022 in Trieste (Italy)

Introduction: The ongoing coronavirus disease 19 (COVID-19) outbreak involves the pediatric population, but to date, few reports have investigated the circulation of variants among children. Material and Methods: In this retrospective study, non-hospitalized pediatric patients with SARS-CoV-2-positive nasopharyngeal swabs (NPS) were enrolled at the Institute for Maternal and Child Health-IRCCS Burlo Garofolo, Trieste (Italy), from November 2020 to January 2022. SARS-CoV-2 variants were identified by in vitro viral isolation, amplification, automatic sequencing of the receptor binding domain (RBD) of the SARS-CoV-2 spike coding gene, and subsequent next-generation sequencing. The growth curves of the isolated strains were defined in vitro by infecting Vero-E6 cells and quantifying the viral load in the supernatants up to 72 h post-infection by qRT–PCR. The neutralization activity of sera obtained from a COVID-19 vaccinated subject, recovered (2020) patient, vaccinated and recovered (2021) patient, and seronegative subject was assessed by microneutralization assay against the different variants. Results: In total, 32 SARS-CoV-2-positive children, 16 (50%) females, with a median age of 1.4 years (range: 1 day–13 years), were enrolled. The D614G amino acid substitution was detected in all isolated and amplified viral strains. Of the 32 isolates, 4 (12.5%) carried a nonsynonymous nucleotide mutation leading to the N439K (3/4), lineage B.1.258 (∆H69/∆V70), and S477N (1/4) substitution. In 7/32 (21.8%) isolates, amino acid substitutions allowed the identification of a delta variant, lineage B.1.617.2-AY.43, and in 1/32 (3.1%), the Omicron strain (B.1.1.529.BA1) was identified. The growth curves of the B.1, B.1.258 (∆H69/∆V70), B.1.617.2-AY.43, and B.1.1.529.BA1 variants did not show any significant differences. A reduction in the serum neutralizing activity against B.1.258 (∆H69/∆V70) only in a vaccinated subject (1.7-fold difference), against B.1.617.2-AY.43 in a vaccinated subject and in recovered patients (12.7 and ≥2.5-fold differences, respectively), and against B.1.1.529.BA1 variant (57.6- and 1.4-fold differences in vaccinated and in vaccinated and recovered patients) were observed compared to the B.1 variant. Conclusions: SARS-CoV-2 variants carrying the B.1.258 (∆H69/∆V70) and S477N substitutions were reported here in a pediatric population for the first time. Although the growth rates of the isolated strains (B.1.258, B.1.617.2-AY.43, B.1.1.529.BA1) did not differ from the B.1 variant, neutralizing activity of the sera from vaccinated subjects significantly decreased against these variants. Attention should be devoted to the pediatric population to prevent the spread of new SARS-CoV-2 variants in an unvaccinated and predominantly naive population