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.

Oral microbiota signatures associated with viremia and CD4 recovery in treatment-naïve HIV-1-infected patients.

PURPOSE: Few reports focused on the role of oral microbiome diversity in HIV infection. We characterized the microbiota-immunity axis in a cohort of treatment-naïve HIV-1-infected patients undergoing antiretroviral therapy (ART) focusing on the oral microbiome (OM) and immunological responsivity. METHODS: The sequencing of 16S rRNA V3-V4 hypervariable region was performed on salivary samples of 15 healthy control (HC) and 12 HIV + patients before starting ART and after reaching virological suppression. Then, we correlated the OM composition with serum cytokines and the Short Chain Fatty acids (SCFAs). RESULTS: The comparison between HIV patients and HC oral microbiota showed differences in the bacterial α-diversity and richness. We documented a negative correlation between oral Prevotella and intestinal valeric acid at before starting ART and a positive correlation between oral Veillonella and gut acetic acid after reaching virological suppression. Finally, an increase in the phylum Proteobacteria was observed comparing saliva samples of immunological responders (IRs) patients against immunological non-responders (INRs). CONCLUSIONS: For the first time, we described an increase in the oral pro-inflammatory Proteobacteria phylum in INRs compared to IRs. We provided more evidence that saliva could be a non-invasive and less expensive approach for research involving the oral cavity microbiome in HIV patients.

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.

Citrobacter freundii resistant to novel ß-lactamase inhibitor combinations and cefiderocol, co-producing class A, B and D carbapenemases encoded by transferable plasmids.

OBJECTIVES: To characterize a carbapenem-resistant Citrobacter freundii (Cf-Emp) co-producing class A, B and D carbapenemases, resistant to novel ß-lactamase inhibitor combinations (BLICs) and cefiderocol. METHODS: Carbapenemase production was tested by an immunochromatography assay. Antibiotic susceptibility testing (AST) was performed by broth microdilution. WGS was performed using short- and long-read sequencing. Transfer of carbapenemase-encoding plasmids was assessed by conjugation experiments. RESULTS: Cf-Emp was isolated on selective medium for carbapenem-resistant Enterobacterales from the surveillance rectal swab taken at hospital admission from a patient of Moroccan origin. Cf-Emp produced three different carbapenemases, including KPC-2, OXA-181 and VIM-1, and was resistant to all ß-lactams including carbapenems, novel BLICs (ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam) and cefiderocol. MIC of aztreonam/avibactam was 0.25 mg/L. The strain belonged to ST22, one of the C. freundii lineages of global diffusion, known to be associated with carbapenemase production. Each carbapenemase gene was located aboard a different plasmid (named pCf-KPC, pCf-OXA and pCf-VIM, respectively), which also carried other clinically relevant resistance genes, such as armA (pCf-KPC), blaSHV-12 (pCf-VIM) and qnrS1 (pCf-OXA). Transferability to Escherichia coli J53 by conjugation was observed for all plasmids. CONCLUSIONS: The finding of enterobacterial strains carrying multiple carbapenemase genes on transferable plasmids is alarming, because similar strains could provide an important reservoir for disseminating these clinically relevant resistance determinants.

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.

Combination regimen of nirmatrelvir/ritonavir and molnupiravir for the treatment of persistent SARS-CoV-2 infection: A case report and a scoping review of the literature.

Immunocompromised patients still experience unpredictable courses of COVID-19, despite that effective vaccines and drugs against SARS-CoV-2 are now available. Antiviral combination regimens may have a role in SARS-CoV-2 infection in immunocompromised hosts, but current knowledge is still limited. We describe the case of a 73-year-old Italian man affected by follicular lymphoma with persistent SARS-CoV-2 infection who was successfully treated with co-administration of oral antivirals (10-day molnupiravir and nirmatrelvir/ritonavir). The therapy was well tolerated both from a clinical and biochemical standpoint, with no signs of toxicity. We also performed a scoping review, to sum up available knowledge on combined antiviral regimens including remdesivir, molnupiravir, or nirmatrelvir/ritonavir. Pending further studies on larger cohorts of patients, our report is consistent with available pre-clinical and clinical data, supporting the possible use of combination therapy in selected difficult-to-treat COVID-19 cases.

Deciphering variable resistance to novel carbapenem-based ß-lactamase inhibitor combinations in a multi-clonal outbreak caused by Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae resistant to ceftazidime/avibactam.

OBJECTIVES: Carbapenemase-producing Enterobacterales represent a major cause of difficult-to-treat infections world-wide. Novel ß-lactam/ß-lactamase inhibitor combinations, including ceftazidime/avibactam (CZA), meropenem/vaborbactam (MVB), and imipenem/relebactam (IMR), represented a break-through in the treatment of some carbapenemase-producing Enterobacterales infections. However, acquired resistance to these agents has been reported in Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales. Herein, we reported an outbreak caused by CZA-resistant, KPC-producing Klebsiella pneumoniae (KPC-Kp), which was also variably resistant to carbapenem-based ß-lactam/ß-lactamase inhibitor combinations. METHODS: Bacterial isolates were subjected to antimicrobial susceptibility testing, whole-genome sequencing, determination of blaKPC gene dosage, and analysis of carbapenemase activity. RESULTS: Overall, 15 KPC-Kp, nine CZA-resistant (CZAR), and six CZA-susceptible isolates were collected from an outbreak involving six patients in a neurorehabilitation facility. Of the nine CZAR isolates, seven were also resistant to MVB and one was also resistant to IMR. Whole-genome sequencing revealed that the outbreak was multi-clonal, with CZAR KPC-Kp belonging to the ST101, ST1519, and two ST512 sub-lineages, which were involved in two independent transmission clusters. Resistance to CZA was primarily mediated by overproduction of KPC-3 associated with increased gene dosage, a mechanism accounting for cross-resistance to MVB in most cases, and to IMR in a single KPC-Kp isolate; multiple OmpK36 aletarions were also detected. Mutated KPC (KPC-53) was detected in a single case. Positivity for CZAR KPC-Kp was inconstantly associated with previous CZA exposure. CONCLUSIONS: In this multi-clonal outbreak of KPC-Kp, the overproduction of KPC-3 was the leading mechanism of cross-resistance to CZA and MVB, whereas resistance to IMR appeared less affected. The emergence and dissemination of similar resistance mechanisms may have relevant clinical and diagnostic implications, and their surveillance is warranted.

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.

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.

Isothermal microcalorimetry vs checkerboard assay to evaluate in-vitro synergism of meropenem-amikacin and meropenem-colistin combinations against multi-drug-resistant Gram-negative pathogens.

OBJECTIVES: To evaluate the activity of meropenem-amikacin and meropenem-colistin combinations with checkerboard broth microdilution (CKBM) compared with isothermal microcalorimetry (ITMC) assays against a multi-centric collection of multi-drug-resistant Gram-negative clinical isolates; and to compare the fractional inhibitory concentration (FIC) index and time to results of CKBM and ITMC. METHODS: A collection of 333 multi-drug-resistant Gram-negative clinical isolates showing reduced susceptibility to meropenem (121 Klebsiella pneumoniae, 14 Escherichia coli, 130 Pseudomonas aeruginosa and 68 Acinetobacter baumannii) isolated from different centres (Florence, Madrid, Rotterdam and Stockholm) was included in the study. The antimicrobial activity of meropenem-amikacin and meropenem-colistin combinations was evaluated with CKBM and ITMC. FIC index results were interpreted as synergistic/additive and indifferent for values ≤0.5/0.51, respectively. Whole-genome sequencing data of a subset of strains were used to evaluate their clonality. RESULTS: In total, 254 and 286 strains were tested with meropenem-colistin and meropenem-amikacin combinations with ITMC and CKBM, respectively. Synergistic/additive effects were observed for 46 strains (20 K. pneumoniae, four E. coli, 22 P. aeruginosa) and 20 strains (three K. pneumoniae, 11 P. aeruginosa and six A. baumannii) with meropenem-amikacin and meropenem-colistin combinations, respectively, with CKBM. ITMC showed good concordance with CKBM, with 89.5% and 92.2% of cases interpreted within the same FIC index category for meropenem-amikacin and meropenem-colistin combinations, respectively. Most of the synergistic/additive effects were detected within 6 h by ITMC. CONCLUSIONS: ITMC showed very good concordance with CKBM against a large collection of multi-drug-resistant Gram-negative clinical isolates, and could be implemented for the rapid evaluation of in-vitro activity of antimicrobial combinations.

Long-term SARS-CoV-2 Asymptomatic Carriage in an Immunocompromised Host: Clinical, Immunological, and Virological Implications.

PURPOSE: SARS-CoV-2 infection in immunocompromised hosts is challenging, and prolonged viral shedding can be a common complication in these patients. We describe the clinical, immunological, and virological course of a patient with eosinophilic granulomatosis with polyangiitis, who developed the status of long-term asymptomatic SARS-CoV-2 carrier for more than 7 months. METHODS: Over the study period, the patient underwent 20 RT-PCR tests for SARS-CoV-2 detection on nasopharyngeal swabs. In addition, viral cultures and genetic investigation of SARS-CoV-2 were performed. As for immunological assessment, serological and specific T-cell testing was provided at different time points. RESULTS: Despite the patient showing a deep drug-induced B and T adaptive immunity impairment, he did not experience COVID-19 progression to severe complications, and the infection remained asymptomatic during the follow-up period, but he was not able to achieve viral clearance for more than 7 months. The infection was finally cleared by SARS-CoV-2-specific monoclonal antibody treatment, after that remdesivir and convalescent plasma failed in this scope. The genetic investigations evidenced that the infection was sustained by multiple viral subpopulations that had apparently evolved intra-host during the infection. CONCLUSION: Our case suggests that people with highly impaired B- and T-cell adaptive immunity can prevent COVID-19 progression to severe complications, but they may not be able to clear SARS-CoV-2 infection. Immunocompromised hosts with a long-term infection may play a role in the emergence of viral variants.

Resistance to Ceftazidime/Avibactam, Meropenem/Vaborbactam and Imipenem/Relebactam in Gram-Negative MDR Bacilli: Molecular Mechanisms and Susceptibility Testing.

Multidrug resistance (MDR) represents a serious global threat due to the rapid global spread and limited antimicrobial options for treatment of difficult-to-treat (DTR) infections sustained by MDR pathogens. Recently, novel ß-lactams/ß-lactamase inhibitor combinations (ßL-ßLICs) have been developed for the treatment of DTR infections due to MDR Gram-negative pathogens. Although novel ßL-ßLICs exhibited promising in vitro and in vivo activities against MDR pathogens, emerging resistances to these novel molecules have recently been reported. Resistance to novel ßL-ßLICs is due to several mechanisms including porin deficiencies, increasing carbapenemase expression and/or enzyme mutations. In this review, we summarized the main mechanisms related to the resistance to ceftazidime/avibactam, meropenem/vaborbactam and imipenem/relebactam in MDR Gram-negative micro-organisms. We focused on antimicrobial activities and resistance traits with particular regard to molecular mechanisms related to resistance to novel ßL-ßLICs. Lastly, we described and discussed the main detection methods for antimicrobial susceptibility testing of such molecules. With increasing reports of resistance to novel ßL-ßLICs, continuous attention should be maintained on the monitoring of the phenotypic traits of MDR pathogens, into the characterization of related mechanisms, and on the emergence of cross-resistance to these novel antimicrobials.

Assessment of the feasibility of pool testing for SARS-CoV-2 infection screening.

BACKGROUND: SARS-CoV-2 pandemic represented a huge challenge for national health systems worldwide. Pooling nasopharyngeal (NP) swabs seems to be a promising strategy, saving time and resources, but it could reduce the sensitivity of the RT-PCR and exacerbate samples management in terms of automation and tracing. In this study, taking advantage of the routine implementation of a screening plan on health workers, we evaluated the feasibility of pool testing for SARS-CoV-2 infection diagnosis in the presence of low viral load samples. METHOD: Pools were prepared with an automated instrument, mixing 4, 6 or 20 NP specimens, including one, two or none positive samples. Ct values of positive samples were on average about 35 for the four genes analyzed. RESULTS: The overall sensitivity of 4-samples and 6-samples pools was 93.1 and 90.0%, respectively. Focussing on pools including one sample with Ct value ≥35 for all analyzed genes, sensitivity decreased to 77.8 and 75.0% for 4- and 6-samples, respectively; pools including two positive samples, resulted positive in any size as well as pools including positive samples with Ct values <35. CONCLUSION: Pool testing strategy should account the balance between cost-effectiveness, dilution effect and prevalence of the infection. Our study demonstrated the good performances in terms of sensitivity and saving resources of pool testing mixing 4 or 6 samples, even including low viral load specimens, in a real screening context possibly affected by prevalence fluctuation. In conclusion, pool testing strategy represents an efficient and resources saving surveillance and tracing tool, especially in specific context like schools, even for monitoring changes in prevalence associated to vaccination campaign.

Clinical consequences of very major errors with semi-automated testing systems for antimicrobial susceptibility of carbapenem-resistant Enterobacterales.

OBJECTIVES: In this study we investigated the rate of susceptibility testing discrepancies between semi-automated and reference systems with carbapenem-resistant Enterobacterales (CRE) and the impact of alleged errors by semi-automated systems on guiding targeted therapy for CRE bloodstream infection (BSI). METHODS: This was a multicentre, retrospective study enrolling patients with monomicrobial BSI caused by CRE from January 2013 to December 2016. Nonduplicate isolates from index blood cultures tested locally with semi-automated systems were centralized at a referral laboratory and retested with a reference broth microdilution or agar dilution method. RESULTS: We enrolled 366 patients with CRE-BSI; 220 (60%) were male, and the median age was 67 years (interquartile range, 54-76 years). When compared with the results of the reference methods, those of the semi-automated systems exhibited variable rates of very major errors (VMEs; i.e. false susceptibilities) and major errors (MEs; i.e. false resistances). The highest rates of VMEs were observed with fosfomycin (14%) and colistin (13.9%), and the highest rates of MEs were observed with gentamicin (21%), fosfomycin (7.7%), and tigecycline (34%). Overall, VMEs and MEs led clinicians to prescribe or confirm ineffective therapy in 25 of 341 patients (7%). Receipt of ineffective therapy supported by a misleading susceptibility test was associated with higher 30-day mortality rates by Kaplan-Meier survival curves rates compared with receipt of active therapy (56% vs. 26%; p = 0.002), and the difference was confirmed after adjustment for confounders in a Cox regression model (adjusted hazard ratio: 2.91; 95% CI, 1.62-5.22; p < 0.001). DISCUSSION: MEs and VMEs were relatively common with semi-automated susceptibility testing systems. VMEs were associated with inappropriate use of antibiotics and poorer outcomes.