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Cystic fibrosis (CF) is a life-threatening genetic disease characterised by chronic lung infections sustained by opportunistic pathogens such as Pseudomonas aeruginosa. During the chronic long-lasting lung infections, P. aeruginosa adapts to the host environment. Hypermutability, mainly due to defects in the DNA repair system, resulting in an increased spontaneous mutation rate, represents a way to boost the rapid adaptation frequently encountered in CF P. aeruginosa isolates. We selected 609 isolates from 51 patients with CF chronically colonised by P. aeruginosa to study, by full-length genome sequencing, the longitudinal evolution of the bacterium. We recovered at least one hypermutable (mutator) isolate in 57% of patients. By combining genomic information and phenotypic analyses, we followed the evolutionary pathways of the P. aeruginosa mutator strains, identifying their contribution to multi-drug resistance and the emergence of new sub-lineages. By implementing patient clinical data, we observed that mutators preferentially follow a specific evolutionary trajectory in patients with a negative clinical outcome and that maintenance antibiotic polytherapy, based on alternating molecules, apparently reduces the occurrence of hypermutability. Finally, we draw attention to the possibility that modulator-induced changes in the pulmonary environment may be associated with the onset of hypermutability. [ABSTRACT FROM AUTHOR]

Background: Group 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. Methods: GAS 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. Results: Twenty-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). Conclusions: This 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 FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bacterial SOS response is an inducible system of DNA repair and mutagenesis. Streptococci lack a canonical SOS response, but an SOS-like response was reported in some species. The mef(A)-msr(D)-carrying prophage Ф1207.3 of Streptococcus pyogenes contains a region, spanning orf6 to orf11, showing homology to characterized streptococcal SOS-like cassettes. Genome-wide homology search showed the presence of the whole Φ1207.3 SOS-like cassette in three S. pyogenes prophages, while parts of it were found in other bacterial species. To investigate whether this cassette confers an SOS-mutagenesis phenotype, we constructed Streptococcus pneumoniae R6 isogenic derivative strains: (i) FR172, streptomycin resistant, (ii) FR173, carrying Φ1207.3, and (iii) FR174, carrying a recombinant Φ1207.3, where the SOS-like cassette was deleted. These strains were used in survival and mutation rate assays using a UV-C LED instrument, for which we designed and 3D-printed a customized equipment, constituted of an instrument support and swappable-autoclavable mini-plates and lids. Upon exposure to UV fluences ranging from 0 to 6,400 J/m² at four different wavelengths, 255, 265, 275, and 285 nm, we found that the presence of Φ1207.3 SOS-like cassette increases bacterial survival up to 34-fold. Mutation rate was determined by measuring rifampicin resistance acquisition upon exposure to UV fluence of 50 J/m² at the four wavelengths by fluctuation test. The presence of Φ1207.3 SOS-like cassette resulted in a significant increase in the mutation rate (up to 18-fold) at every wavelength. In conclusion, we demonstrated that Φ1207.3 carries a functional SOS-like cassette responsible for an increased survival and increased mutation rate in S. pneumoniae. [ABSTRACT FROM AUTHOR]