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Abstract Objectives Genomic surveillance of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) is crucial for virulence, drug-resistance monitoring, and outbreak containment. Methods Genomic analysis on 87 KPC-Kp strains isolated from 3 Northern Italy hospitals in 2019-2021 was performed by whole genome sequencing (WGS), to characterize resistome, virulome, and mobilome, and to assess potential associations with phenotype resistance and clinical presentation. Maximum Likelihood and Minimum Spanning Trees were used to determine strain correlations and identify potential transmission clusters. Results Overall, 15 different STs were found; the predominant ones included ST307 (35, 40.2%), ST512/1519 (15, 17.2%), ST20 (12, 13.8%), and ST101 (7, 8.1%). 33 (37.9%) KPC-Kp strains were noticed to be in five transmission clusters (median number of isolates in each cluster: 5 [3-10]), four of them characterized by intra-hospital transmission. All 87 strains harbored Tn4401a transposon, carrying bla KPC-3 (48, 55.2%), bla KPC-2 (38, 43.7%), and in one case (1.2%) bla KPC-33, the latter gene conferred resistance to ceftazidime/avibactam (CZA). Thirty strains (34.5%) harbored porin mutations; of them, 7 (8.1%) carried multiple Tn4401a copies. These strains were characterized by significantly higher CZA minimum inhibitory concentration compared with strains with no porin mutations or single Tn4401a copy, respectively, even if they did not overcome the resistance breakpoint of 8 ug/mL. Median 2 (IQR:1-2) virulence factors per strain were detected. The lowest number was observed in ST20 compared to the other STs (p

Abstract Since its emergence, SARS-CoV-2 Omicron clade has shown a marked degree of variability and different clinical presentation compared with previous clades. Here we demonstrate that at least four Omicron lineages circulated in children since December 2021, and studied until November 2022: BA.1 (33.6%), BA.2 (40.6%), BA.5 (23.7%) and BQ.1 (2.1%). At least 70% of infections concerned children under 1 year, most of them being infected with BA.2 lineages (n = 201, 75.6%). Looking at SARS-CoV-2 genetic variability, 69 SNPs were found to be significantly associated in pairs, (phi 0.3 and p-value 0.75). One of these clusters (A23040G, A27259C, T23617G, T23620G) was also positively associated with moderate/severe COVID-19 presentation (AOR [95% CI] 2.49 [1.26–4.89] p-value: 0.008) together with comorbidities (AOR [95% CI] 2.67 [1.36–5.24] p-value: 0.004). Overall, these results highlight the extensive SARS-CoV-2 Omicron circulation in children, mostly aged

BackgroundGroup A Streptococcus (GAS) causes multiple clinical manifestations, including invasive (iGAS) or even life-threatening (severe-iGAS) infections. After the drop in cases during COVID-19 pandemic, in 2022 a sharp increase of GAS was reported globally.MethodsGAS strains collected in 09/2022–03/2023 in two university hospitals in Milan, Italy were retrospectively analyzed. Clinical/epidemiological data were combined with whole-genome sequencing to: (i) define resistome/virulome, (ii) identify putative transmission chains, (iii) explore associations between emm-types and clinical severity.ResultsTwenty-eight isolates were available, 19/28 (67.9%) from adults and 9/28 (32.1%) from pediatric population. The criteria for iGAS were met by 19/28 cases (67.9%), of which 11/19 (39.3%) met the further criteria for severe-iGAS. Pediatric cases were mainly non-invasive infections (8/9, 88.9%), adult cases were iGAS and severe-iGAS in 18/19 (94.7%) and 10/19 (52.6%), respectively. Thirteen emm-types were detected, the most prevalent being emm1 and emm12 (6/28 strains each, 21.4%). Single nucleotide polymorphism (SNP) analysis of emm1.0 and emm12.0 strains revealed pairwise SNP distance always >10, inconsistent with unique transmission chains. Emm12.0-type, found to almost exclusively carry virulence factors speH and speI, was mainly detected in children and in no-iGAS infections (55.6 vs. 5.3%, p = 0.007 and 66.7 vs. 0.0%, p < 0.001, respectively), while emm1.0-type was mainly detected in severe-iGAS (0.0 vs. 45.5%, p = 0.045).ConclusionsThis study showed that multiple emm-types contributed to a 2022/2023 GAS infection increase in two hospitals in Milan, with no evidence of direct transmission chains. Specific emm-types could be associated with disease severity or invasiveness. Overall, these results support the integration of classical epidemiological studies with genomic investigation to appropriately manage severe infections and improve surveillance.

Abstract Background Since its emergence in November 2021, SARS-CoV-2 Omicron clade has quickly become dominant, due to its increased transmissibility and immune evasion. Different sublineages are currently circulating, which differ in mutations and deletions in regions of the SARS-CoV-2 genome implicated in the immune response. In May 2022, BA.1 and BA.2 were the most prevalent sublineages in Europe, both characterized by ability of evading natural acquired and vaccine-induced immunity and of escaping monoclonal antibodies neutralization. Case presentation A 5-years old male affected by B-cell acute lymphoblastic leukemia in reinduction was tested positive for SARS-CoV-2 by RT-PCR at the Bambino Gesù Children Hospital in Rome in December 2021. He experienced a mild COVID-19 manifestation, and a peak of nasopharyngeal viral load corresponding to 15.5 Ct. Whole genome sequencing identified the clade 21 K (Omicron), sublineage BA.1.1. The patient was monitored over time and tested negative for SARS-CoV-2 after 30 days. Anti-S antibodies were detected positive with modest titre (3.86 BAU/mL), while anti-N antibodies were negative. 74 days after the onset of the first infection and 23 days after the last negative test, the patient was readmitted to hospital with fever, and tested positive for SARS-CoV-2 by RT-PCR (peak of viral load corresponding to 23.3 Ct). Again, he experienced a mild COVID-19. Whole genome sequencing revealed an infection with the Omicron lineage BA.2 (21L clade). Sotrovimab administration was started at the fifth day of positivity, and RT-PCR negativity occurred 10 days later. Surveillance SARS-CoV-2 RT-PCR were persistently negative, and in May 2022, anti-N antibodies were found positive and anti-S antibodies reached titres > 5000 BAU/mL. Conclusions By this clinical case, we showed that SARS-CoV-2 reinfection within the Omicron clade can occur and can be correlated to inadequate immune responses to primary infection. We also showed that the infection’s length was shorter in the second respect to first episode, suggesting that pre-existing T cell-mediated immunity, though not preventing re-infection, might have limited the SARS-CoV-2 replication capacity. Lastly, Sotrovimab treatment retained activity against BA.2, probably accelerating the viral clearance in the second infectious episode, after which seroconversion and increase of anti-S antibodies titres were observed.

A proof-of-concept study on the genomic evolution of SARS-CoV-2 during antiviral treatment reveals that Molnupiravir induces a higher in vivo intra-host variability compared to Paxlovid and drug-naive.

ABSTRACT Streptococcus pyogenes prophage Φ1207.3 (formerly Tn1207.3) carries the mef(A)-msr(D) resistance genes, responsible for type M macrolide resistance. To investigate if Φ1207.3 is a functional bacteriophage, we transferred the element from the original S. pyogenes host in a prophage-free and competence-deficient S. pneumoniae strain. Pneumococcal cultures of the Φ1207.3-carrying lysogen were treated with mitomycin C to assess if Φ1207.3 enters the lytic cycle. Mitomycin C induced a limited phage burst and a growth impairment, resulting in early entrance into the stationary phase. To determine if Φ1207.3 is able to produce mature phage particles, we prepared concentrated supernatants recovered from a mitomycin C-induced pneumococcal culture by sequential centrifugation and ultracentrifugation steps. Negative-staining transmission electron microscopy (TEM) of supernatants revealed the presence of phage particles with an icosahedral, electron-dense capsid and a long, noncontractile tail, typical of a siphovirus. Quantification of Φ1207.3 was performed by quantitative PCR (qPCR) and semiquantitatively by TEM. PCR quantified 3.34 × 104 and 6.06 × 104 excised forms of phage genome per milliliter of supernatant obtained from the untreated and mitomycin C-treated cultures, respectively. By TEM, we estimated 3.02 × 103 and 7.68 × 103 phage particles per milliliter of supernatant. The phage preparations of Φ1207.3 infected and lysogenized pneumococcal recipient strains at a frequency of 7.5 × 10−6 lysogens/recipient but did not show sufficient lytic activity to form plaques. Phage lysogenization efficiently occurred after 30 min of contact of the phages with the recipient cells and required a minimum of 103 phage particles. IMPORTANCE Bacteriophages play an important role in bacterial physiology and genome evolution. The widespread use of genome sequencing revealed that bacterial genomes can contain several different integrated temperate bacteriophages, which can constitute up to 20% of the genome. Most of these bacteriophages are only predicted in silico and are never shown to be functional. In fact, it is often difficult to induce the lytic cycle of temperate bacteriophages. In this work, we show that Φ1207.3, a peculiar bacteriophage originally from Streptococcus pyogenes, which can lysogenize different streptococci and carries the macrolide resistance mef(A)-msr(D) gene pair, is capable of producing mature virions, but only at a low level, while not being able to produce plaques. This temperate phage is probably a partially functional phage, which seems to have lost lytic characteristics to specialize in lysogenization. While we are not used to conceiving phages separately from lysis, this behavior could actually be more frequent than expected.

Background: Whole-genome sequencing (WGS) provides important information for the characterization, surveillance, and monitoring of antimicrobial resistance (AMR) determinants, particularly in cases of multi- and extensively drug-resistant microorganisms. We reported the results of a WGS analysis carried out on carbapenemases-producing Klebsiella pneumoniae, which causes hospital-acquired infections (HAIs) and is characterized by a marked resistance profile. Methods: Clinical, phenotypic, and genotypic data were collected for the AMR surveillance screening program of the University Hospital of Sassari (Italy) during 2020–2021. Genomic DNA was sequenced using the Illumina Nova Seq 6000 platform. Final assemblies were manually curated and carefully verified for the detection of antimicrobial resistance genes, porin mutations, and virulence factors. A phylogenetic analysis was performed using the maximum likelihood method. Results: All 17 strains analyzed belonged to ST512, and most of them carried the blaKPC-31 variant blaOXA-48-like, an OmpK35 truncation, and an OmpK36 mutation. Phenotypic analysis showed a marked resistance profile to all antibiotic classes, including β-lactams, carbapenems, aminoglycosides, fluoroquinolone, sulphonamides, and novel β-lactam/β-lactamase inhibitors (BL/BLI). Conclusion: WGS characterization revealed the presence of several antibiotic resistance determinants and porin mutations in highly resistant K. pneumoniae strains responsible for HAIs. The detection of blaKPC-31 in our hospital wards highlights the importance of genomic surveillance in hospital settings to monitor the emergence of new clones and the need to improve control and preventive strategies to efficiently contrast AMR.

Abstract Background Tn5253, a composite Integrative Conjugative Element (ICE) of Streptococcus pneumoniae carrying tet(M) and cat resistance determinants, was found to (i) integrate at specific 83-bp integration site (attB), (ii) produce circular forms joined by a 84-bp sequence (attTn), and (iii) restore the chromosomal integration site. The purpose of this study is to functionally characterize the attB in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, and to investigate the presence of Tn5253 attB site into bacterial genomes. Results Analysis of representative Tn5253-carryng transconjugants obtained in S. pneumoniae strains with different genetic backgrounds and in other bacterial species, namely Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes, and Enterococcus faecalis showed that: (i) Tn5253 integrates in rbgA of S. pneumoniae and in orthologous rbgA genes of other bacterial species, (ii) integration occurs always downstream of a 11-bp sequence conserved among streptococcal and enterococcal hosts, (iii) length of the attB site corresponds to length of the duplication after Tn5253 integration, (iv) attB duplication restores rbgA CDS, (v) Tn5253 produced circular forms containing the attTn site at a concentration ranging between 2.0 × 10−5 to 1.2 × 10−2 copies per chromosome depending on bacterial species and strain, (vi) reconstitution of attB sites occurred at 3.7 × 10−5 to 1.7 × 10−2 copies per chromosome. A database search of complete microbial genomes using Tn5253 attB as a probe showed that (i) thirteen attB variants were present in the 85 complete pneumococcal genomes, (ii) in 75 pneumococcal genomes (88.3 %), the attB site was 83 or 84 nucleotides in length, while in 10 (11.7 %) it was 41 nucleotides, (iii) in other 19 bacterial species attB was located in orthologous rbgA genes and its size ranged between 17 and 84 nucleotides, (iv) the 11-bp sequence, which correspond to the last 11 nucleotides of attB sites, is conserved among the different bacterial species and can be considered the core of the Tn5253 integration site. Conclusions A functional characterization of the Tn5253 attB integration site combined with genome analysis contributed to elucidating the potential of Tn5253 horizontal gene transfer among different bacterial species.

Abstract Objectives In streptococci, the type M resistance to macrolides is due to the mef(A)–msr(D) efflux transport system of the ATP-Binding cassette (ABC) superfamily, where it is proposed that mef(A) codes for the transmembrane channel and msr(D) for the two ATP-binding domains. Phage ϕ1207.3 of Streptococcus pyogenes, carrying the mef(A)–msr(D) gene pair, is able to transfer the macrolide efflux phenotype to Streptococcus pneumoniae. Deletion of mef(A) in pneumococcal ϕ1207.3-carrying strains did not affect erythromycin efflux. In order to identify candidate genes likely involved in complementation of mef(A) deletion, the Mef(A) amino acid sequence was used as probe for database searching. Results In silico analysis identified 3 putative candidates in the S. pneumoniae R6 genome, namely spr0971, spr1023 and spr1932. Isogenic deletion mutants of each candidate gene were constructed and used in erythromycin sensitivity assays to investigate their contribution to mef(A) complementation. Since no change in erythromycin sensitivity was observed compared to the parental strain, we produced double and triple mutants to assess the potential synergic activity of the selected genes. Also these mutants did not complement the mef(A) function.

Background: Carbapenem resistant Klebsiella pneumoniae (cr-Kp) causes serious infections associated with a high mortality rate. The clinical efficacy of ceftazidime/avibactam (CZA), meropenem/vaborbactam (M/V), and imipenem/relebactam (I/R) against cr-Kp is challenged by the emergence of resistant strains, making the investigation and monitoring of the main resistance mechanisms crucial. In this study, we reported the genome characterization of a Klebsiella pneumoniae strain isolated from a critically ill patient and characterized by a multidrug resistant (MDR) profile, including resistance to CZA, M/V, and I/R. Methods: An antimicrobial susceptibility test (AST) was performed by an automated system and E-test and results were interpreted following the EUCAST guidelines. Genomic DNA was extracted using a genomic DNA extraction kit and it was sequenced using the Illumina Nova Seq 6000 platform. Final assembly was manually curated and carefully verified for detection of antimicrobial resistance genes, porins modifications, and virulence factors. Results: The K. pneumoniae isolate belonged to sequence type ST512 and harbored 23 resistance genes, conferring resistance to all antibiotic classes, including blaKPC-31 and blaOXA-181, leading to carbapenems resistance. The truncation of OmpK35 and mutation OmpK36GD were also observed. Conclusions: The genomic characterization demonstrated the high resistant profile of new cr-Kp coharboring class A and D carbapenemases. The presence of KPC-31, as well as the detection of OXA-181 and porin modifications, further limit the therapeutic options, including the novel combinations of β-lactam/β-lactamase inhibitor antibiotics in patients with severe pneumonia caused by cr-Kp.

Disbalancing envelope stress responses was investigated as a strategy for sensitization of Escherichia coli to antimicrobial agents. Seventeen isogenic strains were selected from the KEIO collection with deletions in genes corresponding to the σE, Cpx, Rcs, Bae, and Psp responses. Antimicrobial activity against 20 drugs with different targets was evaluated by disk diffusion and gradient strip tests. Growth curves and time-kill curves were also determined for selected mutant-antimicrobial combinations. An increase in susceptibility to ampicillin, ceftazidime, cefepime, aztreonam, ertapenem, and fosfomycin was detected. Growth curves for Psp response mutants showed a decrease in optical density (OD) using sub-MIC concentrations of ceftazidime and aztreonam (ΔpspA and ΔpspB mutants), cefepime (ΔpspB and ΔpspC mutants) and ertapenem (ΔpspB mutant). Time-kill curves were also performed using 1xMIC concentrations of these antimicrobials. For ceftazidime, 2.9 log10 (ΔpspA mutant) and 0.9 log10 (ΔpspB mutant) decreases were observed at 24 and 8 h, respectively. For aztreonam, a decrease of 3.1 log10 (ΔpspA mutant) and 4 log1010 (ΔpspB mutant) was shown after 4–6 h. For cefepime, 4.2 log10 (ΔpspB mutant) and 2.6 log10 (ΔpspC mutant) decreases were observed at 8 and 4 h, respectively. For ertapenem, a decrease of up to 6 log10 (ΔpspB mutant) was observed at 24 h. A deficient Psp envelope stress response increased E. coli susceptibility to beta-lactam agents such as cefepime, ceftazidime, aztreonam and ertapenem. Its role in repairing extensive inner membrane disruptions makes this pathway essential to bacterial survival, so that disbalancing the Psp response could be an appropriate target for sensitization strategies.

DNA sequencing of whole bacterial genomes has revealed that the entire set of mobile genes (mobilome) represents as much as 25% of the bacterial genome. Despite the huge availability of sequence data, the functional analysis of the mobile genetic elements (MGEs) is rarely reported. Therefore, established laboratory protocols are needed to investigate the biology of this important part of the bacterial genome. Conjugation is a mechanism of horizontal gene transfer which allows the exchange of MGEs among strains of the same or different bacterial species. In streptococci and enterococci, integrative and conjugative elements (ICEs) represent a large part of the mobilome. Here, we describe an efficient and easy-to-perform plate mating protocol for in vitro conjugative transfer of ICEs in streptococci (Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus gordonii, Streptococcus pyogenes), Enterococcus faecalis, and Bacillus subtilis. Conjugative transfer is carried out on solid media and selection of transconjugants is performed with a multilayer plating. This protocol allows the transfer of large genetic elements with a size up to 81 kb, and a transfer frequency up to 6.7 × 10−3 transconjugants/donor cells.