Characterization of Clostridioides difficile Strains from an Outbreak Using MALDI-TOF Mass Spectrometry

The epidemiology of Clostridioides difficile infection (CDI) has changed over the last two decades, due to the emergence of C. difficile strains with clinical relevance and responsible for nosocomial outbreaks with severe outcomes. This study reports an outbreak occurred in a Long-term Care Unit from February to March 2022 and tracked by using a Matrix-Assisted Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) typing approach (T-MALDI); subsequently, a characterization of the toxigenic and antimicrobial susceptibility profiles of the C. difficile isolates was performed. A total of 143 faecal samples belonging to 112 patients was evaluated and C. difficile DNA was detected in 51 samples (46 patients). Twenty-nine C. difficile isolates were obtained, and three different clusters were revealed by T-MALDI. The most representative cluster accounted 22 strains and was considered to be epidemic, in agreement with PCR-Ribotyping. Such epidemic strains were susceptible to vancomycin (MIC <= 0.5 mg/mL) and metronidazole (MIC <= 1 mg/mL), but not to moxifloxacin (MIC > 32 mg/mL). Moreover, they produced only the Toxin A and, additionally, the binary toxin. To our knowledge, this is the first reported outbreak referable to a tcdA+/tcdB-/cdt+ genotypic profile. In light of these results, T-MALDI is a valid and rapid approach for discovering and tracking outbreaks

Distribution of human Cytomegalovirus gB genotypes in samples from pediatric patients in Parma during the period 1995-2003

Background. Human Cytomegalovirus (HCMV) infection is a leading cause of developmental disability and late neurological sequelae in children. Several literature data indicate that HCMV pericapsidic glycoprotein B (gB) is highly immunogenic and is involved in virus-cell interaction.The gB gene has hypervariable regions producing four genotypes (gB1, gB2, gB3, gB4); however, the correlation between gB genotypes and HCMV infection outcome remains unclear. Objectives. The main goal of this study was that of evaluating the distribution of HCMV gB genotypes in samples from pediatric population in Parma with congenital, perinatal or post-natal infections, in order to find a correlation between viral gB genotypes and the clinical outcome of the infection. Study design. Forty eight urine samples, selected between 1999 and 2003 and stored at -80°C, underwent DNA extraction, nested PCR amplification of a gB gene region and restriction polymorphism analysis (RFLP). Results. The gB genotypes distribution in the considered pediatric population was as follows: gB1 was the most diffused (45.83%) followed by gB2 (22.92%), gB3 (16.67%) and gB4 (14.58%). Conclusions. In the considered population, gB1 was the most represented genotype and was often found in congenital and perinatal symptomatic infections, as well as in post-natal, asymptomatic infections

Active surveillance for carbapenemaseproducing Klebsiella pneumoniae and correlation with infection in subjects attending an Italian tertiary-care hospital: a 7-year retrospective study

Objectives The distribution of carbapenemase-producing Klebsiella pneumoniae (CPKP) phenotypes and genotypes in samples collected during 2011–2018 was evaluated. The association between patients with CPKP-positive rectal swab and those with CPKP infection, as well as the overall analysis of CPKP-infected patients, was performed. Setting The study was performed in a tertiary-care hospital located in Northern Italy. Participants Two groups were considered: 22 939 ‘atrisk’ patients submitted to active surveillance for CPKP detection in rectal swabs/stools and 1094 CPKP-infected patients in which CPKP was detected in samples other than rectal swabs. Results CPKP-positive rectal swabs were detected in 5% (1150/22 939). A CPKP infection was revealed in 3.1% (719/22 939) of patients: 582 with CPKP-positive rectal swab (50.6% of the 1150 CPKP-positive rectal swabs) and 137 with CPKP-negative rectal swab. The 49.4% (568/1150) of the patients with CPKP-positive rectal swab were carriers. The overall frequency of CPKP-positive patients (carriers and infected) was almost constant from 2012 to 2016 (excluding the 2015 peak) and then increased in 2017–2018. blaKPC was predominant followed by blaVIM. No difference was observed in the frequency of CPKP-positive rectal swab patients among the different material groups. Among the targeted carbapenemase genes, blaVIM was more significantly detected from urine than from other samples. Conclusions The high prevalence of carriers without evidence of infection, representing a potential reservoir of CPKP, suggests to maintain the guard about this problem, emphasising the importance of active surveillance for timely detection and separation of carriers, activation of contact precautions and antibiotic treatment guidance on suspicion of infection

Leptospira species and serovars identified by MALDI-TOF mass spectrometry after database implementation

Background: Leptospirosis, a spirochaetal zoonotic disease of worldwide distribution, endemic in Europe, has been recognized as an important emerging infectious disease, though yet it is mostly a neglected disease which imparts its greatest burden on impoverished populations from developing countries. Leptospirosis is caused by the infection with any of the more than 230 serovars of pathogenic Leptospira sp. In this study we aimed to implement the MALDI-TOF mass spectrometry (MS) database currently available in our laboratory with Leptospira reference pathogenic (L. interrogans, L. borgpetersenii, L. kirschneri, L. noguchii), intermediate (L. fainei) and saprophytic (L. biflexa) strains of our collection in order to evaluate its possible application to the diagnosis of leptospirosis and to the typing of strains. This was done with the goal of understanding whether this methodology could be used as a tool for the identification of Leptospira strains, not only at species level for diagnostic purposes, but also at serovar level for epidemiological purposes, overcoming the limits of serological and molecular conventional methods. Twenty Leptospira reference strains were analysed by MALDI-TOF MS. Statistical analysis of the protein spectra was performed by ClinProTools software. Results: The spectra obtained by the analysis of the reference strains tested were grouped into 6 main classes corresponding to the species analysed, highlighting species-specific protein profiles. Moreover, the statistical analysis of the spectra identified discriminatory peaks to recognize Leptospira strains also at serovar level extending previously published data. Conclusions: In conclusion, we confirmed that MALDI-TOF MS could be a powerful tool for research and diagnostic in the field of leptospirosis with broad applications ranging from the detection and identification of pathogenic leptospires for diagnostic purposes to the typing of pathogenic and non-pathogenic leptospires for epidemiological purposes in order to enrich our knowledge about the epidemiology of the infection in different areas and generate control strategies

MALDI-TOF MS: A Reliable Tool in the Real Life of the Clinical Microbiology Laboratory

Matrix-Assisted Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) in the last decade has revealed itself as a valid support in the workflow in the clinical microbiology laboratory for the identification of bacteria and fungi, demonstrating high reliability and effectiveness in this application. Its use has reduced, by 24 h, the time to obtain a microbiological diagnosis compared to conventional biochemical automatic systems. MALDI-TOF MS application to the detection of pathogens directly in clinical samples was proposed but requires a deeper investigation, whereas its application to positive blood cultures for the identification of microorganisms and the detection of antimicrobial resistance are now the most useful applications. Thanks to its rapidity, accuracy, and low price in reagents and consumables, MALDI-TOF MS has also been applied to different fields of clinical microbiology, such as the detection of antibiotic susceptibility/resistance biomarkers, the identification of aminoacidic sequences and the chemical structure of protein terminal groups, and as an emerging method in microbial typing. Some of these applications are waiting for an extensive evaluation before confirming a transfer to the routine. MALDI-TOF MS has not yet been used for the routine identification of parasites; nevertheless, studies have been reported in the last few years on its use in the identification of intestinal protozoa, Plasmodium falciparum, or ectoparasites. Innovative applications of MALDI-TOF MS to viruses’ identification were also reported, seeking further studies before adapting this tool to the virus’s diagnostic. This mini-review is focused on the MALDI-TOF MS application in the real life of the diagnostic microbiology laboratory

Aspetti morfologici e funzionali dei proteasomi durante il processo di differenziamento di cellule muscolari ed in corso di infezione da citomegalovirus umano in vitro

Deux volets thématiques se rapportant à la biologie des protéasomes ont été abordés au cours de ce travail de thèse. Le premier a consisté à préciser la localisation des protéasomes au sein de la structure sarcomérique de muscles striés de rat et d'élaborer des outils moléculaires appropriés, permettant de suivre la distribution des protéasomes au cours de la différenciation myogénique. Ces mêmes outils ont également été élaborés au cours de la deuxième partie du travail, qui a consisté à évaluer la participation de ces complexes multicatalytiques lors de l'infection de fibroblastes par le cytomegalovirus humain. L'expression des protéines virales très précoces a été étudiée dans les fibroblastes synchronisés ou non au cours de l'infection, à la suite de l'utilisation ou non d'un inhibiteur de la fonction protéolytique des protéasomes. On a aussi étudié la distribution des protéasomes dans les cellules infectées, afin d'évaluer si celle-ci se modifiait au cours de l'infection virale.Two different topics about proteasomes have been evaluated during this doctoral thesis. In the first part we have showed the proteasome localization at the level of the sarcomeric structure of rat skeletal muscle and we have developed useful molecular tools to follow proteasome distribution during myogenic differentiation. The same tools have been developed during the second part of this project. The goal of this work was to evaluate the involvement of this multicatalytic complex in fibroblasts infected by human cytomegalovirus. Viral immediate-early protein expression was studied using fibroblasts synchronized or not during viral infection, using an inhibitor of the proteolytic activity of proteasomes to examine the effects of the lack of this proteasomal function on viral protein expression. In addition, we investigated the intracellular distribution of proteasomes in infected cells with the aim to evaluate if proteasome distribution changed during viral infection.PARIS12-CRETEIL BU Multidisc. (940282102) / SudocSudocItalyFranceITF

Inhibition of late influenza virus genome expression by diamidinophenylindole.

Abstract The growth cycle of influenza virus strain FPV, Ulster 73, was altered by treatment of LLC-MK2 cells with diamidinophenylindole. Viral protein synthesis was restricted to the early pattern of virus multiplication, and post-treatment experiments showed the ability of the drug to block virus replication until the 4th hour p.i. Drug addition (followed by removal) revealed the inhibition of synthesis of late viral products, and especially of membrane protein. Kinetic studies on the production of viral RNA indicated a decrease in the synthesis of late virus-induced RNA species, suggesting that the target of DAPI is probably the late transcription of the virus genome. The nonpermissive condition mediated by the drug could represent a suitable model to study cellular intervention during viral growth. PMID: 2974708 [PubMed - indexed for MEDLINE