The emergence and global dissemination of carbapenemaseproducing Gram-negative bacteria (CPGNB) pose a significant public health threat due to the limited therapeutic options for CPGNB infections, which are associated with high mortality rates and a considerable economic and societal burden. 1 Early identification of CPGNB is useful for timely infection prevention and control measures and therapeutic stewardship, and identification of the resistance mechanisms has become essential following the availability of several new antimicrobial agents with variable coverage depending on resistance mechanism. 2 Lateral flow immunoassays (LFIAs) are rapid (15-30 min) systems for detection of clinically relevant resistance mechanisms, including carbapenemases.LFIAs require limited technical expertise in the absence of expensive equipment, and are generally less expensive than genotypic methods. 3,4 Moreover, unlike the latter, LFIA systems detect proteins and are not affected by false positivity in case of silenced genes. 4 However, false negativity may also occur with LFIA systems in case of low level of production of the target protein (e.g. with weakly expressed MBLs), 5,6 or of amino acid changes in the epitopes of the target protein recognized by the antibody probe used by the system (e.g.changes at position 179 in the Ω-loop of KPC carbapenemase variants, which confer resistance to avibactam and exhibit impaired carbapenemase activity). [7][[9][10][11] In this work we evaluated three commercial LFIA systems for detection of the five most common families of carbapenemases encountered among CPGNB, namely the KPC/IMP/NDM/VIM/ OXA-48 Combo Test Kit (KINVO, Medomics Medical Technology, Jiangsu, China), the O.K.

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.

Pseudomonas aeruginosa is an uncommon cause of infective endocarditis (IE), accounting for less than 1% episodes worldwide (1). However, P. aeruginosa IE is challenging due to the multidrug resistance (MDR) or even difficult-to-treat (DTR) resistance profiles possibly exhibited by P. aeruginosa strains, along with their ability to grow as biofilms (2).

Objectives: Carbapenemase-producing Enterobacterales represent a major cause of difficult-to-treat infections world-wide. Novel beta-lactam/Enterobacterales-lactamase inhibitor combinations, including ceftazidime/avibactam (CZA), meropenem/vaborbactam (MVB), and imipenem/relebactam (IMR), represented a breakthrough 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 beta-lactam/beta-lactamase inhibitor combinations. Methods: Bacterial isolates were subjected to antimicrobial susceptibility testing, whole-genome sequencing, determination of bla(KPC) gene dosage, and analysis of carbapenemase activity. Results: Overall, 15 KPC-Kp, nine CZA-resistant (CZA(R)), and six CZA-susceptible isolates were collected from an outbreak involving six patients in a neurorehabilitation facility. Of the nine CZA(R) 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 CZA(R) KPC-Kp belonging to the ST101, ST1519, and two ST512 sublineages, 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 CZA(R) 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. (c) 2022 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Objectives To investigate the in vitro activity of fosfomycin, colistin and combinations thereof against planktonic and biofilm cultures of Gram-negative pathogens, mostly showing MDR phenotypes, at concentrations achievable via inhalation of aerosolized drugs. Methods Activity against planktonic cultures was tested by the chequerboard assay with 130 strains, including 52 Pseudomonas aeruginosa, 47 Klebsiella pneumoniae, 19 Escherichia coli, 7 Stenotrophomonas maltophilia and 5 Acinetobacter baumannii. Activity against biofilm cultures was tested by biofilm chequerboard and quantitative antibiofilm assays with a subset of 20 strains. In addition, 10 of these strains were tested in mutant prevention concentration (MPC) assays. Results Against planktonic cultures, synergism between fosfomycin and colistin was detected with a minority (10%) of strains (eight K. pneumoniae and five P. aeruginosa), while antagonism was never observed. Synergism between fosfomycin and colistin against biofilms was observed with the majority of tested strains (16/20 in biofilm chequerboard assays, and 18/20 in the quantitative antibiofilm assays), including representatives of each species and regardless of their resistance genotype or phenotype. Furthermore, combination of fosfomycin and colistin was found to significantly reduce the MPC of individual drugs. Conclusions Fosfomycin and colistin in combination, at concentrations achievable via inhalation of nebulized drugs, showed notable synergy against MDR Gram-negative pathogens grown in biofilm, and were able to reduce the emergence of fosfomycin- and colistin-resistant subpopulations.

Background Carbapenemase-producing Enterobacterales (CPE), particularly those producing metallo-beta-lactamases, are among the most challenging antibiotic-resistant pathogens, causing outbreaks of difficult-to-treat nosocomial infections worldwide. Since November 2018, an outbreak of New Delhi metallo-beta-lactamases-positive CPE (NDM-CPE) has emerged in Tuscany, Italy. In this study, we aimed to investigate the NDM-CPE associated with the outbreak and characterise the responsible Klebsiella pneumoniae clone. Methods We used whole-genome sequencing and bioinformatic analysis to characterise NDM-CPE isolates that caused bloodstream infections in 53 patients at 11 hospitals in Tuscany and that were collected between Jan 1, 2018, and July 5, 2019 (ie, the early phase of the outbreak and preceding months). The CPE isolates characterised in this study were isolated and identified at the species level and as NDM producers by six diagnostic microbiology laboratories that serve the 11 hospitals. We used comparative genomic analysis, antimicrobial susceptibility testing, plasmid conjugal transfer assays, evaluation of virulence potential in the Galleria mellonella infection model, and serum bactericidal assays to further characterise the clone causing the outbreak. Findings The outbreak was sustained by an ST147 K pneumoniae producing NDM-1, which had a complex resistome that mediated resistance to most antimicrobials (except cefiderocol, the aztreonam-avibactam combination, colistin, and fosfomycin). The clone belonged to a sublineage of probably recent evolution, occurred by the sequential acquisition of an integrative and conjugative element encoding the yersiniabactin siderophore, an FIB(pQil)-type multiresistance plasmid carrying bla(NDM-1), and a transferable chimeric plasmid, derived from virulence elements of hypervirulent K pneumoniae, carrying several resistance and virulence determinants. Infection of G mellonella larvae revealed a variable virulence potential. The behaviour in serum bactericidal assays was different from typical hypervirulent K pneumoniae strains, with variable grades of serum resistance apparently associated with mutations in specific chromosomal loci (csrD, pal, and ramR). Interpretation This description of a sublineage of ST147 K pneumoniae with a complex resistome and virulome that is capable of sustaining a large regional outbreak adds to existing research on the evolutionary trajectories within high-risk clones of K pneumoniae. Global surveillance programmes are warranted to track the dissemination of these lineages, and to prevent and control their spread. Copyright (C) 2022 The Author(s). Published by Elsevier Ltd.

Although accumulating data have investigated the effect of SARS-CoV-2 mutations on antibody neutralizing activity, less is known about T cell immunity. In this work, we found that the ancestral (Wuhan strain) Spike protein can efficaciously reactivate CD4+ T cell memory in subjects with previous Alpha variant infection. This finding has practical implications, as in many countries only one vaccine dose is currently administered to individuals with previous COVID-19, independently of which SARS-CoV-2 variant was responsible of the infection. We also found that only a minority of Spike-specific CD4+ T cells targets regions mutated in Alpha, Beta and Delta variants, both after natural infection and vaccination. Finally, we found that the vast majority of Spike-specific CD4+ T cell memory response induced by natural infection or mRNA vaccination is conserved also against Omicron variant. This is of importance, as this newly emerged strain is responsible for a sudden rise in COVID-19 cases worldwide due to its increased transmissibility and ability to evade antibody neutralization. Collectively, these observations suggest that most of the memory CD4+ T cell response is conserved against SARS-CoV-2 variants of concern, providing an efficacious line of defense that can protect from the development of severe forms of COVID-19.

Objective: Evaluate the real-world accuracy of Myxovirus resistance protein A (MxA) detected by the rapid, point-of-care FebriDx test during the second-wave pandemic in Italy in patients with acute respiratory infection (ARI) and a clinical suspicion of COVID-19. Design and methods: Prospective, observational, diagnostic accuracy study whereby hospitalized patients with ARI were consecutively enrolled in a single tertiary care center in Italy from August 1, 2020 to January 31, 2021. Results: COVID-19 was diagnosed in 136/200 (68.0%) patients and Non-COVID-19 was diagnosed in 64/200 (32.0%) patients. COVID-19 patients were younger and had a lower Charlson comorbidity index compared to Non-COVID-19 patients (p < 0.001). Concordance between FebriDx, MxA and rt-PCR for SARS-CoV-2 (gold standard) was good (k 0.93, 95% CI 0.87–0.99). Overall sensitivity and specificity were 97.8% [95% CI 93.7–99.5] and 95.3% [95% CI 86.9%–99.0%], respectively. FebriDx demonstrated a negative predictive value of 95.3% (95% CI 86.9–99.0) for an observed disease prevalence of 68%. Conclusions: FebriDx MxA showed high diagnostic accuracy to identify COVID-19 and could be considered as a real-time triage tool to streamline the management of suspected COVID-19 patients. FebriDx also detected bacterial etiology in Non-COVID-19 patients suggesting good performance to distinguish bacterial from viral respiratory infection.

BACKGROUND:The emergence of SARS-CoV-2 variants of concern (VOCs) for increased transmissibility and being potentially capable of immune-escape mandates for epidemiological surveillance. Genomic alterations present in VOCs can affect the results of RT-qPCR assays for routine diagnostic purposes, leading to peculiar profiles that can be used for rapid screening of variants. This study reports a peculiar profile observed with the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay and VOC-Alpha (202012/01, lineage B.1.1.7, also named VOC-UK), which was the first identified SARS-CoV-2 VOC. METHODS:Samples were analyzed by two RT-qPCR assays: the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay (ASFR, Seegene Technologies Inc; Seoul, South Korea) and the TaqPath COVID-19 RT-PCR (Thermo Fisher Scientific, USA). Definition of the SARS-CoV-2 variant was carried out by Sanger sequencing of the relevant S-gene regions and, in some cases, by whole genome sequencing (WGS) using the ARTIC-nCoV workflow on a MiniION (Oxford Nanopore Technologies, Oxford, UK) or a Illumina MiSeq platform (San Diego, California, USA). RESULTS:Of the 173 SARS-CoV-2-positive specimens, all those of lineage B.1.1.7 (N=71) showed an average Cq difference between the N and S genes of +11±2 (range, +8/+15). None of the other specimens, including several different lineages (Wild-type for the analyzed regions, N=22; Gamma, N=63; Delta, N=9; B.1.258Δ, N=3; B.1.160, N=3; B.1.177.7, N=1; B.1.1.420, N=1), exhibited a similar difference in Cq values. CONCLUSIONS:The peculiar pattern of delayed N gene positivity could constitute a convenient method for VOC-Alpha screening, simultaneous to viral detection, when using the Allplex™ SARS-CoV-2/FluA/FluB/RSV assay.

Limited data about New Delhi metallo-β-lactamase (NDM) bacteremia are available. Blood isolates from 40 patients with NDM bacteremia were studied for antibiotic susceptibility and whole-genomic sequencing. NDM bacteremia has high 30-day mortality. In most cases, aztreonam-avibactam is active in vitro. Ceftazidime-avibactam plus aztreonam may represent a feasible therapeutic option.

This study reports on the characterization of a Klebsiella pneumoniae clinical isolate showing high-level resistance to ceftazidime-avibactam associated with the production of KPC-53, a KPC-3 variant exhibiting a Leu167Glu168 duplication in the Ω-loop and a loss of carbapenemase activity. Whole-genome sequencing (WGS) revealed the presence of two copies of bla KPC-53 , located on a pKpQIL-like plasmid and on a plasmid prophage of the Siphoviridae family, respectively.

We investigated the colistin resistance mechanism in an Escherichia coli strain (LC711/14) isolated in Italy in 2014, from an urinary tract infection, which was previously shown to express a colistin resistance mechanism different from mcr-1. LC711/14 was found to carry a novel mutation in the pmrB gene, resulting in a leucine to proline amino acid substitution at position 10 of the PmrB sensor kinase component of the PmrAB signal transduction system. The role of this substitution in colistin resistance was documented by expression of the wild-type and mutated alleles in a pmrB deletion derivative of the E. coli reference strain MG1655, in which expression of the mutated allele conferred colistin resistance and upregulation of the endogenous pmrHFIJKLM lipid A modification system. Complementation of LC711/14 with the wild- type pmrB allele restored colistin susceptibility and decreased expression of pmrHFIJKLM, confirming the role of this PmrB mutation. Substitution of leucine at position 10 of PmrB with other amino acids (glycine and glutamine) resulted in loss of function, underscoring a key role of this residue which is located in the cytoplasmic secretion domain of the protein. This work demonstrated that mutation in this domain of the PmrB sensor kinase can be responsible for acquired colistin resistance in E. coli strains of clinical origin.