Evaluation of different molecular systems for detection and quantification of SARS-CoV-2 RNA from wastewater samples.

Wastewater-based epidemiology has proved to be a suitable approach for tracking the spread of epidemic agents including SARS-CoV-2 RNA. Different protocols have been developed for quantitative detection of SARS-CoV-2 RNA from wastewater samples, but little is known on their performance. In this study we compared three protocols based on Reverse Transcription Real Time-PCR (RT-PCR) and one based on Droplet Digital PCR (ddPCR) for SARS-CoV-2 RNA detection from 35 wastewater samples. Overall, SARS-CoV-2 RNA was detected by at least one method in 85.7% of samples, while 51.4%, 22.8% and 8.6% resulted positive with two, three or all four methods, respectively. Protocols based on commercial RT-PCR assays and on Droplet Digital PCR showed an overall higher sensitivity vs. an in-house assay. The use of more than one system, targeting different genes, could be helpful to increase detection sensitivity.

Evaluation of two commercial broth microdilution systems for dalbavancin susceptibility testing of methicillin-resistant Staphylococcus aureus and other resistant Gram-positive cocci.

OBJECTIVES: This study aims to evaluate two commercial broth microdilution (BMD) systems, E1-185-100 (Merlin) and FDANDPF (ThermoFisher), for dalbavancin susceptibility testing in comparison with reference BMD assay. METHODS: Study collection was composed of 200 non-replicate multidrug-resistant Gram-positive cocci of clinical origin, including 180 methicillin-resistant Staphylococcus aureus, 10 vancomycin-resistant enterococci, seven linezolid-resistant Staphylococcus epidermidis, and three methicillin-resistant coagulase-negative staphylococci. S. aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 reference strains were also included as controls. Testing was performed according to the ISO 20776-1 standard, starting from the same bacterial inoculum, and results were compared according to the ISO 20776-2 standard. RESULTS: Reference BMD showed that 92.6% (187/202) of the strains were susceptible to dalbavancin, whereas few staphylococci and all VanA-producing enterococci showed a resistant phenotype. In comparison with the reference method, Category Agreement and Essential Agreement were 98% (198/202; 95% CI, 95.4-99.3%) and 98% (198/202; 95% CI, 95.4-99.3%) for both Merlin and ThermoFisher panels. A few false susceptibilities were observed, for both commercial systems, with dalbavancin-resistant staphylococci. BIAS values of 11% and 3% were calculated for the Merlin and ThermoFisher systems, respectively. DISCUSSION: This study, reporting the first evaluation of the two commercially available BMD assays for dalbavancin susceptibility testing, revealed an overall good correlation with reference BMD, although with some underestimation tendency of MIC values by both commercial systems. Further studies involving a higher number of resistant isolates will be necessary to better evaluate this issue.

Novel resistance ICEs carrying the blaFIM-1 metallo-ß-lactamase gene from an ST235 Pseudomonas aeruginosa sublineage.

FIM-1 is an acquired metallo-ß-lactamase identified in a multidrug-resistant Pseudomonas aeruginosa (index strain FI-14/157) of clinical origin isolated in 2007 in Florence, Italy. Here we report on a second case of infection by FIM-1-positive P. aeruginosa (FI-17645), which occurred in 2020 in the same hospital. Both FIM-1-positive strains exhibited resistance to all anti-Pseudomonas antibiotics except colistin and cefiderocol. Comparative genomic characterization revealed that the two FIM-positive strains were closely related [core genome difference, 16 single nucleotide polymorphisms (SNPs)], suggesting a local circulation of similar strains. In the FI-14/157 index strain, the blaFIM-1 gene was associated with an ISCR19-like element that likely contributed to its capture downstream an integron platform inserted aboard a Tn21-like transposon, named Tn7703.1, which was associated with a large integrative and conjugative element (ICE) named ICE7705.1, integrated into an att site located within the 3'-end of tRNAGly CCC gene of the P. aeruginosa chromosome. In strain FI-17645, blaFIM-1 was associated with a closely related ICE, named ICE7705.2, integrated in the same chromosomal site. Similar ICE platforms, lacking the blaFIM-1-containing region, were detected in other ST235 P. aeruginosa strains from different geographic areas, suggesting a common ancestry and underscoring the role of these elements in the dissemination of resistance genes in P. aeruginosa. Sequence database mining revealed two draft P. aeruginosa genomes, one from Italy and one from the USA (both isolated in 2012), including a contig with blaFIM-1, suggesting that this resistance gene could have a broader distribution than originally anticipated.

A urokinase-associated outbreak of Ralstonia mannitolilytica bloodstream infections in haemodialysis patients in north-eastern Italy, January to April 2023.

An outbreak of Ralstonia mannitolilytica bloodstream infections occurred in four hospitals in north-eastern Italy, involving 20 haemodialysis patients with tunnelled central vascular catheter access. We identified as the outbreak source a batch of urokinase vials imported from India contaminated with R. mannitolilytica. Whole genome sequences of the clinical and urokinase strains were highly related, and only urokinase-treated patients were reported with R. mannitolilytica infections (attack rate = 34%; 95% confidence interval: 22.1-47.4). Discontinuation of the contaminated urokinase terminated the outbreak.

Evaluation of the Liquid Colony™ Produced by the FAST System for Shortening the Time of Bacterial Identification and Phenotypic Antimicrobial Susceptibility Testing and Detection of Resistance Mechanisms from Positive Blood Cultures.

BACKGROUND: the aim of this study was to evaluate the performance of the Liquid Colony™ (LC) generated directly from positive blood cultures (PBCs) by the FAST System (Qvella, Richmond Hill, ON, Canada) for rapid identification (ID) and antimicrobial susceptibility testing (AST) compared with the standard of care (SOC) workflow. METHODS: Anonymized PBCs were processed in parallel by the FAST System and FAST PBC Prep cartridge (35 min runtime) and SOC. ID was performed by MALDI-ToF mass spectrometry (Bruker, Billerica, MA, USA). AST was performed by reference broth microdilution (Merlin Diagnostika, Bornheim, Germany). Carbapenemase detection was carried out with the lateral flow immunochromatographic assay (LFIA) RESIST-5 O.O.K.N.V. (Coris, Gembloux, Belgium). Polymicrobial PBCs and samples containing yeast were excluded. RESULTS: 241 PBCs were evaluated. ID results showed 100% genus-level concordance and 97.8% species-level concordance between LC and SOC. The AST results for Gram-negative bacteria showed a categorical agreement (CA) of 99.1% (1578/1593), with minor error (mE), major error (ME), and very major error (VME) rates of 0.6% (10/1593), 0.3% (3/1122), and 0.4% (2/471), respectively. The results from Gram-positive bacteria showed a CA of 99.6% (1655/1662), with mE, ME, and VME rates of 0.3% (5/1662), 0.2% (2/1279), and 0.0% (0/378), respectively. Bias evaluation revealed acceptable results for both Gram-negatives and Gram-positives (-12.4% and -6.5%, respectively). The LC yielded the detection of 14/18 carbapenemase producers by LFIA. In terms of turnaround time, the ID, AST, and carbapenemase detection results were generally obtained one day earlier with the FAST System compared with the SOC workflow. CONCLUSIONS: The ID, AST, and carbapenemase detection results generated with the FAST System LC were highly concordant with the conventional workflow. The LC allowed species ID and carbapenemase detection within around 1 h after blood culture positivity and AST results within approximately 24 h, which is a significant reduction in the turnaround time of the PBC workflow.

Up-regulation of resident chromosomal fosB gene expression: a novel mechanism of acquired fosfomycin resistance in MRSA.

OBJECTIVES: This study investigated fosfomycin susceptibility and mechanisms of resistance in a collection of 99 Staphylococcus aureus isolated from cases of hospital-acquired pneumonia, previously collected from a multicentre survey carried out in Italy. METHODS: Fosfomycin susceptibility was tested by reference agar dilution. Bioinformatic and gene expression analysis, mutant selection experiments and WGS were executed to characterize fosfomycin resistance mechanisms. RESULTS: Fosfomycin resistance rates were 0% (0 of 35) among MSSA and 22% (14 of 64) among MRSA, with no evidence of clonal expansion. Resistance mechanisms were putatively identified in 8 of the 14 resistant strains, including: (i) chromosomal mutations causing loss of function of the UhpT transporter; (ii) overexpression of the gene encoding the Tet38 efflux pump; and (iii) overexpression of a fosB gene encoding a fosfomycin-inactivating enzyme, which was found to be resident in the chromosome of several S. aureus lineages but not always associated with fosfomycin resistance. The latter mechanism, which had not been previously described and was confirmed by results of in vitro mutant selection experiments, was associated in two cases with transposition of an IS1182 element upstream of the chromosomal fosB gene, apparently providing an additional promoter. CONCLUSIONS: This study showed that some S. aureus clonal lineages carry a resident chromosomal fosB gene and can evolve to fosfomycin resistance by overexpression of this gene.

Performance evaluation of the UMIC® Cefiderocol to determine MIC in Gram-negative bacteria.

BACKGROUND: Cefiderocol is a catechol-substituted cephalosporin with potent in vitro activity against carbapenem-resistant (CR) Gram-negative bacteria (GNB). Cefiderocol susceptibility testing is complex because iron concentrations need to be taken into consideration. Here, we assessed the clinical performance of Bruker's UMIC® Cefiderocol and corresponding iron-depleted CAMHB to determine MIC by broth microdilution (BMD) for clinically relevant GNB. METHODS: MICs of cefiderocol for 283 GN clinical isolates were determined by BMD using iron-depleted CAMHB. Frozen panels were used as a reference. The concentration range of cefiderocol was 0.03-32 mg/L. The isolates, with different degrees of susceptibility to cefiderocol, included Enterobacterales (n = 180), Pseudomonas aeruginosa (n = 49), Acinetobacter baumannii (n = 44) and Stenotrophomonas maltophilia (n = 10). RESULTS: The rates of categorical agreement (CA), essential agreement (EA) and bias were calculated to evaluate the performance of the UMIC® Cefiderocol, as compared with the reference method. Overall, the UMIC® Cefiderocol showed 90.8% EA (95% CI: 86.9%-93.7%) with a bias of -14.5% and a CA of 90.1% (95% CI: 86.1%-93.1%). For Enterobacterales, the UMIC® Cefiderocol showed 91.7% EA (95% CI: 86.7%-94.9%) with a bias of -25.0% and a CA of 87.8% (95% CI: 82.2%-91.8%). For non-fermenters, the UMIC® Cefiderocol showed 89.3% EA (95% CI: 81.9%-93.9%) (not significantly different from 90.0%, Student t-test) with a bias of -3.9% and a CA of 94.2% (95% CI: 87.7%-97.3%). CONCLUSIONS: UMIC® Cefiderocol is a valid method for the determination of cefiderocol MICs even if higher than expected discrepancies were observed with NDM-producing Enterobacterales, which presented in most cases MIC values close to the breakpoint.

Bacterial Species from Vaginal Microbiota Differently Affect the Production of the E6 and E7 Oncoproteins and of p53 and p-Rb Oncosuppressors in HPV16-Infected Cells.

Vaginal dysbiosis is characterized by a decrease in the relative abundance of Lactobacillus species in favor of other species. This condition facilitates infections by sexually transmitted pathogens including high risk (HR)-human papilloma viruses (HPVs) involved in the development of cervical cancer. Some vaginal dysbiosis bacteria contribute to the neoplastic progression by inducing chronic inflammation and directly activating molecular pathways involved in carcinogenesis. In this study, SiHa cells, an HPV-16-transformed epithelial cell line, were exposed to different representative vaginal microbial communities. The expression of the HPV oncogenes E6 and E7 and the production of relative oncoproteins was evaluated. The results showed that Lactobacillus crispatus and Lactobacillus gasseri modulated the basal expression of the E6 and E7 genes of SiHa cells and the production of the E6 and E7 oncoproteins. Vaginal dysbiosis bacteria had contrasting effects on E6/E7 gene expression and protein production. The expression of the E6 and E7 genes and the production of the relative oncoproteins was increased by strains of Gardnerella vaginalis and, to a lesser extent, by Megasphaera micronuciformis. In contrast, Prevotella bivia decreased the expression of oncogenes and the production of the E7 protein. A decreased amount of p53 and pRb was found in the cultures of SiHa cells with M. micronuciformis, and accordingly, in the same cultures, a higher percentage of cells progressed to the S-phase of the cell cycle compared to the untreated or Lactobacillus-stimulated cultures. These data confirm that L. crispatus represents the most protective component of the vaginal microbiota against neoplastic progression of HR-HPV infected cells, while M. micronuciformis and, to a lesser extent, G. vaginalis may directly interfere in the oncogenic process, inducing or maintaining the production of viral oncoproteins.

A Questionnaire Integrated with the Digital Medical Record Improved the Coverage of a Control Program for Congenital Chagas Disease in Tuscany, Italy.

The leading route of Chagas disease transmission in nonendemic countries is congenital. However, policies concerning screening, prevention, and management of congenital Chagas disease are rare in these settings. Since 2012, serological screening for Chagas disease should be provided for pregnant women at risk in Tuscany, Italy according to a Regional resolution. Due to difficulties in the implementation, in November 2019, a checklist aimed at identifying pregnant women at risk for Chagas disease was introduced in digital clinical records at Careggi University Hospital, Florence, Italy. In order to evaluate the effectiveness of the "Chagas checklist", data about the number of deliveries by women at risk and their screening coverage between 2012 and June 2022 were collected. Out of 1348 deliveries by women at risk, 626 (47%) Trypanosoma cruzi serology tests were performed during the study period. The annual screening coverage increased from an average of 40.3% between 2012 and 2019 to 75.7% between 2020 and June 2022, underlining the big impact of the checklist. Four Chagas disease serological tests out of 626 (0.6%) resulted positive, corresponding to 2 affected women. No cases of congenital transmission occurred. The study showed that a simple digital tool led to a tangible improvement in the coverage of the screening program; its application in a setting where digital charts are available will contribute to the control and elimination of congenital Chagas disease.

Multicenter study on the prevalence of colonization due to carbapenem-resistant Enterobacterales strains before and during the first year of COVID-19, Italy 2018-2020.

Background: Among multidrug-resistant (MDR) bacteria able to threaten human health, carbapenem-resistant Enterobacterales (CRE) have become a major public health threat globally. National and international guidelines point out the importance of active routine surveillance policies to prevent CRE transmission. Therefore, defining lines of intervention and strategies capable of containing and controlling the spread of CRE is considered determinant. CRE screening is one of the main actions to curb transmission and control outbreaks, outlining the presence and also the prevalence and types of carbapenemase enzymes circulating locally. Objective: The purpose of this study was to outline the epidemiology of CRE colonization in Italy, detecting CRE-colonized patients at admission and during hospitalization, before and during the first year of COVID-19. Materials and methods: A total of 11,063 patients admitted to seven different hospitals (Bologna, Catania, Florence, Genoa, Naples, Palermo, and Turin) in Intensive Care Units (ICU) and other wards (non-ICU) located in the North, Center, and South of Italy were enrolled and screened for CRE carriage at admission (T0) and during the first 3 weeks of hospitalization (T1-T3). The study spanned two periods, before (September 2018-Septemeber 2019, I observational period) and during the COVID-19 pandemic (October 2019-September 2020, II observational period). Results: Overall, the prevalence of CRE-colonized patients at admission in ICU or in other ward, ranged from 3.9 to 11.5%, while a percentage from 5.1 to 15.5% of patients acquired CRE during hospital stay. There were large differences between the I and II period of study according to the different geographical areas and enrolling centers. Overall, comparison of prevalence of CRE-positive patients showed a significant increased trend between I and II observational periods both in ICU and non-ICU wards, mostly in the Southern participating centers. KPC-producing Klebsiella pneumoniae was the most frequent CRE species-carbapenemase combination reported in this study. In particular, the presence of KPC-producing K. pneumoniae was reported in period I during hospitalization in all the CRE-positive patients enrolled in ICU in Turin (North Italy), while in period II at admission in all the CRE-positive patients enrolled in ICU in Catania and in 58.3% of non-ICU CRE-positive patients in Naples (both centers in South Italy). Conclusion: The prevalence of CRE in Italy highly increased during the COVID-19 pandemic, mostly in the Southern hospital centers. KPC-producing K. pneumoniae was the most frequent colonizing CRE species reported. The results of our study confirmed the crucial value of active surveillance as well as the importance of multicenter studies representing diverse geographical areas even in endemic countries. Differences in CRE colonization prevalence among centers suggest the need for diversified and center-specific interventions as well as for strengthening efforts in infection prevention and control practices and policies.

Epidemiology and antibiotic susceptibility profiles of obligate anaerobes in a hospital of central Italy during a one-year (2019) survey.

OBJECTIVES: In this study, we report on the epidemiology and susceptibility profiles of anaerobic pathogens consecutively isolated from various clinical samples at an Italian hospital during a one-year survey. METHODS: The collection included all non-duplicated consecutively collected anaerobic clinical isolates during 2019 in San Luca Hospital (Lucca, Italy). Antimicrobial susceptibility testing was performed using MICRONAUT-S Anaerobes MIC lyophilized plates (MERLIN Diagnostika, Germany) and interpreted using EUCAST criteria (11.0). RESULTS: A total of 169 Gram-negative and 213 Gram-positive were collected. The most frequent anaerobes were Bacteroides spp. (120, 31.4%) followed by Finegoldia magna (62, 16.2%). External ulcers were the most common source of isolates (39%), followed by blood (25.7%). In 230 patients (65%) polymicrobial aerobic/anaerobic isolates were cultured from the same external ulcer specimen. High resistance rates to clindamycin were overall detected, with the highest values among the genera Parabacteroides, Bacteroides, Prevotella, Gram-positive anaerobic cocci and Clostridium. High resistance rates were also observed to metronidazole among both Gram-positive and Gram-negative species, ranging between 10.8-50% and 13.8-46.2%, respectively. CONCLUSIONS: Our findings revealed that anaerobes susceptibility patterns have become less predictable due to an increase of resistance and suggest that periodic resistance surveillance should also be carried out for anaerobes in order to guide empirical therapy.

Antiviral Activity of Oligonucleotides Targeting the SARS-CoV-2 Genomic RNA Stem-Loop Sequences within the 3'-End of the ORF1b.

Increased evidence shows vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibited no long-term efficacy and limited worldwide availability, while existing antivirals and treatment options have only limited efficacy. In this study, the main objective was the development of antiviral strategies using nucleic acid-based molecules. To this purpose, partially overlapped 6-19-mer phosphorothioate deoxyoligonucleotides (S-ONs) designed on the SARS-CoV-2 genomic RNA stem-loop packaging sequences within the 3' end of the ORF1b were synthetized using the direct and complementary sequence. Among the S-ONs tested, several oligonucleotides exhibited a fifty percent inhibitory concentration antiviral activity ranging from 0.27 to 34 µM, in the absence of cytotoxicity. The S-ON with a scrambled sequence used in the same conditions was not active. Moreover, selected 10-mer S-ONs were tested using different infectious doses and against different SARS-CoV-2 variants, showing comparable antiviral activity that was abrogated when the central sequence was mutated. Experiments to evaluate the intracellular functional target localization of the S-ON inhibitory activity were also performed. Collectively the data indicate that the SARS-CoV-2 packaging region in the 3' end of the ORF1b may be a promising target candidate for further investigation to develop innovative nucleic-acid-based antiviral therapy.

Resistance and virulence features of hypermucoviscous Klebsiella pneumoniae from bloodstream infections: Results of a nationwide Italian surveillance study.

Among Enterobacterales, Klebsiella pneumoniae (Kp) is one of the major opportunistic pathogens causing hospital-acquired infections. The most problematic phenomenon linked to Kp is related to the dissemination of multi-drug resistant (MDR) clones producing carbapenem-hydrolyzing enzymes, representing a clinical and public health threat at a global scale. Over the past decades, high-risk MDR clones (e.g., ST512, ST307, ST101 producing bla KPC-type carbepenemases) have become endemic in several countries, including Italy. Concurrently, the spread of highly virulent Kp lineages (e.g., ST23, ST86) able to cause severe, community-acquired, pyogenic infections with metastatic dissemination in immunocompetent subjects has started to be documented. These clones, designated as hypervirulent Kp (hvKp), produce an extensive array of virulence factors and are highly virulent in previously validated animal models. While the prevalence and distribution of MDR Kp has been previously assessed at local and national level knowledge about dissemination of hvKp remains scarce. In this work, we studied the phenotypic and genotypic features of hypermucoviscous (HMV, as possible marker of increased virulence) Kp isolates from bloodstream infections (BSI), obtained in 2016-17 from 43 Italian Laboratories. Antimicrobial susceptibility testing, whole genome sequencing and the use of two animal models (G. mellonella and murine) were employed to characterize collected isolates. Over 1502 BSI recorded in the study period, a total of 19 Kp were selected for further investigation based on their HMV phenotype. Results showed that hvKp isolates (ST5, ST8, ST11, ST25) are circulating in Italy, although with a low prevalence and in absence of a clonal expansion; convergence of virulence (yersiniabactin and/or salmochelin, aerobactin, regulators of mucoid phenotype) and antimicrobial-resistance (extended-spectrum beta-lactamases) features was observed in some cases. Conventional MDR Kp clones (ST307, ST512) may exhibit an HMV phenotype, but with a low virulence potential in the animal models. To the best of our knowledge, this work represents the first systematic survey on HMV and hvKp in Italy, employing a functional characterization of collected isolates. Future surveillance programs are warranted to monitor the threatening convergence of virulence and resistance among MDR Kp and the spread of hvKp.

Nosocomial outbreak by NDM-1-producing Klebsiella pneumoniae highly resistant to cefiderocol, Florence, Italy, August 2021 to June 2022.

A nosocomial outbreak by cefiderocol (FDC)-resistant NDM-1-producing Klebsiella pneumoniae (NDM-Kp) occurred in a large tertiary care hospital from August 2021-June 2022 in Florence, Italy, an area where NDM-Kp strains have become endemic. Retrospective analysis of NDM-Kp from cases observed in January 2021-June 2022 revealed that 21/52 were FDC-resistant. The outbreak was mostly sustained by clonal expansion of a mutant with inactivated cirA siderophore receptor gene, which exhibited high-level resistance to FDC (MIC ≥ 32 mg/L) and spread independently of FDC exposure.

Molecular and Kinetic Characterization of MOX-9, a Plasmid-Mediated Enzyme Representative of a Novel Sublineage of MOX-Type Class C ß-Lactamases.

The MOX lineage of ß-lactamases includes a group of molecular class C enzymes (AmpCs) encoded by genes mobilized from the chromosomes of Aeromonas spp. to plasmids. MOX-9, previously identified as a plasmid-encoded enzyme from a Citrobacter freundii isolate, belongs to a novel sublineage of MOX enzymes, derived from the resident Aeromonas media AmpC. The blaMOX-9 gene was found to be carried on a transposon, named Tn7469, likely responsible for its mobilization to plasmidic context. MOX-9 was overexpressed in Escherichia coli, purified, and subjected to biochemical characterization. Kinetic analysis showed a relatively narrow-spectrum profile with strong preference for cephalosporin substrates, with some differences compared with MOX-1 and MOX-2. MOX-9 was not inhibited by clavulanate and sulbactam, while both tazobactam and avibactam acted as inhibitors in the micromolar range.