In Vitro Activity of Cefiderocol on Multiresistant Bacterial Strains and Genomic Analysis of Two Cefiderocol Resistant Strains.

Cefiderocol is a new siderophore cephalosporin that is effective against multidrug-resistant Gram-negative bacteria, including carbapenem-resistant strains. The aim of this study was to evaluate the activity of this new antimicrobial agent against a collection of pathogens using broth microdilution assays and to analyze the possible mechanism of cefiderocol resistance in two resistant Klebsiella pneumoniae isolates. One hundred and ten isolates were tested, comprising 67 Enterobacterales, two Acinetobacter baumannii, one Achromobacter xylosoxidans, 33 Pseudomonas aeruginosa and seven Stenotrophomonas maltophilia. Cefiderocol showed good in vitro activity, with an MIC < 2 µg/mL, and was able to inhibit 94% of the tested isolates. We observed a resistance rate of 6%. The resistant isolates consisted of six Klebsiella pneumoniae and one Escherichia coli, leading to a resistance rate of 10.4% among the Enterobacterales. Whole-genome sequencing analysis was performed on two cefiderocol-resistant Klebsiella pneumoniae isolates to investigate the possible mutations responsible for the observed resistance. Both strains belonged to ST383 and harbored different resistant and virulence genes. The analysis of genes involved in iron uptake and transport showed the presence of different mutations located in fhuA, fepA, iutA, cirA, sitC, apbC, fepG, fepC, fetB, yicI, yicJ, and yicL. Furthermore, for the first time, to the best of our knowledge, we described two Klebsiella pneumoniae isolates that synthesize a truncated fecA protein due to the transition from G to A, leading to a premature stop codon in the amino acid position 569, and a TonB protein carrying a 4-amino acid insertion (PKPK) after Lysine 103. In conclusion, our data show that cefiderocol is an effective drug against multidrug-resistant Gram-negative bacteria. However, the higher resistance rate observed in Enterobacterales underlines the need for active surveillance to limit the spread of these pathogens and to avoid the risks associated with the emergence of resistance to new drugs.

Varicella-Zoster Virus Prevalence among Pregnant Women: A European Epidemiological Review.

Europe has faced a massive spread of the varicella-zoster virus through the years. Since the introduction of an effective vaccine, complications and severe forms of chickenpox have been restricted. Nevertheless, among the population, some categories need specific care, such as pregnant women, who present one of the most fragile conditions facing this infection, both for the mother and the fetus. In this review, we highlight how the varicella-zoster virus can be dangerous during pregnancy, underlining the problem of treatment and vaccination, and collect information about the European epidemiology among this particular category of women.

Emergence of Pandemic Clonal Lineage Sequence Types 131 and 69 of Extraintestinal Escherichia coli as a Cause of Meningitis: Is It Time To Revise Molecular Assays?

Two Escherichia coli strains, respectively responsible for neonatal and adult meningitis, were isolated and their phenotypic antibiotic susceptibility and genomic features characterized by whole-genome sequencing (WGS). Multiplex real-time PCR targeting the principal microorganisms involved in meningitis etiology failed to identify either isolate. Afterwards, matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify the isolates as E. coli strains. Genomic analysis showed that they belonged to sequence types 131 and 69 (ST131 and ST69). Neither of the isolates harbored the K1 capsular antigen or belonged to other capsular serotypes, but they shared different virulence factors, including ibe genes, responsible for invasion of brain endothelial cells. IMPORTANCE The extraintestinal pathogenic Escherichia coli group is characterized by the presence of uropathogenic E. coli (UPEC), sepsis-associated E. coli (SEPEC), and neonatal meningitis E. coli (NMEC). All of these members exhibit many virulence factors, such as lipopolysaccharides, toxins, iron acquisition factors, invasins, fimbriae, and capsules. Urinary infections are the most common infections caused by this group, followed by globally increasing numbers of both community- and nosocomially acquired bloodstream infections, associated with considerable patient morbidity and mortality. Some lineages tend to become dominant; in addition to enhanced fitness, this epidemiological success stems from increased virulence, antibiotic resistance, gut colonization, and greater host-to-host transmission. Our results underline the importance of continuous surveillance of these new emerging lineages and the need to develop new meningitis molecular assay panels able to identify them.

Competition for dominance within replicating quasispecies during prolonged SARS-CoV-2 infection in an immunocompromised host.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) emerge for their capability to better adapt to the human host aimed and enhance human-to-human transmission. Mutations in spike largely contributed to adaptation. Viral persistence is a prerequisite for intra-host virus evolution, and this likely occurred in immunocompromised patients who allow intra-host long-term viral replication. The underlying mechanism leading to the emergence of variants during viral persistence in the immunocompromised host is still unknown. Here, we show the existence of an ensemble of minor mutants in the early biological samples obtained from an immunocompromised patient and their dynamic interplay with the master mutant during a persistent and productive long-term infection. In particular, after 222 days of active viral replication, the original master mutant, named MB610, was replaced by a minor quasispecies (MB61222) expressing two critical mutations in spike, namely Q493K and N501T. Isolation of the two viruses allowed us to show that MB61222 entry into target cells occurred mainly by the fusion at the plasma membrane (PM), whereas endocytosis characterized the entry mechanism used by MB610. Interestingly, coinfection of two human cell lines of different origin with the SARS-CoV-2 isolates highlighted the early and dramatic predominance of MB61222 over MB610 replication. This finding may be explained by a faster replicative activity of MB61222 as compared to MB610 as well as by the capability of MB61222 to induce peculiar viral RNA-sensing mechanisms leading to an increased production of interferons (IFNs) and, in particular, of IFN-induced transmembrane protein 1 (IFITM1) and IFITM2. Indeed, it has been recently shown that IFITM2 is able to restrict SARS-CoV-2 entry occurring by endocytosis. In this regard, MB61222 may escape the antiviral activity of IFITMs by using the PM fusion pathway for entry into the target cell, whereas MB610 cannot escape this host antiviral response during MB61222 coinfection, since it has endocytosis as the main pathway of entry. Altogether, our data support the evidence of quasispecies fighting for host dominance by taking benefit from the cell machinery to restrict the productive infection of competitors in the viral ensemble. This finding may explain, at least in part, the extraordinary rapid worldwide turnover of VOCs that use the PM fusion pathway to enter into target cells over the original pandemic strain.

Role of Q675H Mutation in Improving SARS-CoV-2 Spike Interaction with the Furin Binding Pocket.

Genotype screening was implemented in Italy and showed a significant prevalence of new SARS-CoV-2 mutants carrying Q675H mutation, near the furin cleavage site of spike protein. Currently, this mutation, which is expressed on different SARS-CoV-2 lineages circulating worldwide, has not been thoughtfully investigated. Therefore, we performed phylogenetic and biocomputational analysis to better understand SARS-CoV-2 Q675H mutants' evolutionary relationships with other circulating lineages and Q675H function in its molecular context. Our studies reveal that Q675H spike mutation is the result of parallel evolution because it arose independently in separate evolutionary clades. In silico data show that the Q675H mutation gives rise to a hydrogen-bonds network in the spike polar region. This results in an optimized directionality of arginine residues involved in interaction of spike with the furin binding pocket, thus improving proteolytic exposure of the viral protein. Furin was predicted to have a greater affinity for Q675H than Q675 substrate conformations. As a consequence, Q675H mutation could confer a fitness advantage to SARS-CoV-2 by promoting a more efficient viral entry. Interestingly, here we have shown that Q675H spike mutation is documented in all the VOCs. This finding highlights that VOCs are still evolving to enhance viral fitness and to adapt to the human host. At the same time, it may suggest Q675H spike mutation involvement in SARS-CoV-2 evolution.