In-depth characterization of multidrug-resistant NDM-1 and KPC-3 co-producing Klebsiella pneumoniae bloodstream isolates from Italian hospital patients.

Bloodstream infection (BSI) caused by carbapenem-resistant Klebsiella pneumoniae (KP) poses significant challenges, particularly when the infecting isolate carries multiple antimicrobial resistance (AMR) genes/determinants. This study, employing short- and long-read whole-genome sequencing, characterizes six New Delhi metallo-ß-lactamase (NDM) 1 and KP carbapenemase (KPC) 3 co-producing KP isolates, the largest cohort investigated in Europe to date. Five [sequence type (ST) 512] and one (ST11) isolates were recovered from patients who developed BSI from February to August 2022 or February 2023 at two different hospitals in Rome, Italy. Phylogenetic analysis revealed two distinct clusters among ST512 isolates and a separate cluster for the ST11 isolate. Beyond blaNDM-1 and blaKPC-3, various AMR genes, indicative of a multidrug resistance phenotype, including colistin resistance, were found. Each cluster-representative ST512 isolate harbored a blaNDM-1 plasmid (IncC) and a blaKPC-3 plasmid [IncFIB(pQil)/IncFII(K)], while the ST11 isolate harbored a blaNDM-1 plasmid [IncFII(pKPX1)] and a blaKPC-3 plasmid [IncFIB(K)/IncFII(K)]. The blaNDM-1 plasmids carried genes conferring resistance to clinically relevant antimicrobial agents, and the aminoglycoside resistance gene aac(6')-Ib was found on different plasmids. Colistin resistance-associated mgrB/pmrB gene mutations were present in all isolates, and the yersiniabactin-encoding ybt gene was unique to the ST11 isolate. In conclusion, our findings provide insights into the genomic context of blaNDM-1/blaKPC-3 carbapenemase-producing KP isolates.IMPORTANCEThis study underscores the critical role of genomic surveillance as a proactive measure to restrict the spread of carbapenemase-producing KP isolates, especially when key antimicrobial resistance genes, such as blaNDM-1/blaKPC-3, are plasmid borne. In-depth characterization of these isolates may help identify plasmid similarities contributing to their intra-hospital/inter-hospital adaptation and transmission. Despite the lack of data on patient movements, it is possible that carbapenem-resistant isolates were selected to co-produce KP carbapenemase and New Delhi metallo-ß-lactamase via plasmid acquisition. Studies employing long-read whole-genome sequencing should be encouraged to address the emergence of KP clones with converging phenotypes of virulence and resistance to last-resort antimicrobial agents.

Genomic distribution of the insertion sequence IS711 reveal a potential role in Brucella genome plasticity and host preference.

The Insertion Sequence 711 (IS711) is linked to the Brucella genus. Mapping the genomic distribution of IS711 can help understand this insertion element's biological and evolutionary role. This work aimed to delineate the genomic distribution of the IS711 element and to study its association with Brucella evolution. A total of 124 genomes representing 9 Brucella species were searched using BLASTn sequence alignment tool to identify complete and truncated copies of IS711. Based on the genomic context, each IS711 locus was assigned a code using the initial letters of its neighboring genes. Various tools were used to annotate the neighboring genes and determine the shared synteny around orthologous IS711 loci. The tool Islandviewer 4 was used to scan for genomic islands. The Codon Tree method was used to build phylogenetic trees of B. melitensis, B. abortus, and B. suis genomes. The phylogenetic trees of the three species were analyzed, taking into account the genomic distribution patterns of IS711. The result of IS711 frequency analysis showed a relatively conserved number of copies/genome for the different species and for some biovars. The analysis showed that Brucella species with a relatively low IS711 copy number (4-8 copies/genome) are linked to domestic animals as primary hosts and have potential for zoonotic transmission. However, species with a relatively higher copy number (12-30 copies/genome) are less zoonotic and tend to be linked with wild animals as primary hosts. Analyzing the genomic distribution map of IS711 loci showed several unique patterns of IS711 distribution that are correlated with the evolution of Brucella species and biovars. The results also showed that 46.2% of the conserved IS711 elements are located within genomic islands. Based on our results and previous data, we postulate a model explaining the IS711 role in Brucella evolution. We assume that during the transition from a free-living to an intracellular lifestyle, a descendant of the Brucella genus had acquired a progenitor sequence of the IS711. Subsequently, a burst in IS711 transposition occurred. This parasitic expansion can be deleterious and has to be counteracted by evolutionary forces to prevent lineage extension and to promote adaptation to host. Similar to other plasmid-free pathogenic α-Proteobacteria bacteria, the balance of expansion and reduction of insertion elements could be one of the mechanisms to control genome reduction and streamlining. We hypothesize that the IS711-mediated genomic changes and other small sequence nucleotide changes in specific orthologous genes could significantly contribute to Brucella's evolution and adaptation to different animal hosts.

Brucellosis re-emergence after a decade of quiescence in Palestine, 2015-2017: A seroprevalence and molecular characterization study.

Brucellosis is an endemic disease in many developing countries and ranked by the World Health Organization among the top seven "neglected zoonoses". Although a Palestinian brucellosis control program was launched in 1998, the disease re-emerged after 2012. Interestingly, a similar re-emerging pattern was reported in the neighbouring Israeli regions. The aim of this work was to characterize the re-emerging strains and delineate their genetic relatedness. During 2015-2017, blood samples from 1324 suspected human cases were analyzed using two serological tests. Seropositive samples were cultured, and their DNAs were analyzed by different genetic markers to determine the involved Brucella species and rule out any possible involvement of the Rev.1 vaccine strain. The rpoB gene was sequenced from nine isolates to screen for rifampicin resistance mutations. Multi locus VNTR analysis (MLVA-16) was used for genotyping the isolates. The molecular analysis showed that all isolates were Brucella melitensis strains unrelated to the Rev.1 vaccine. The rpoB gene sequences showed four single nucleotide variations (SNVs) not associated with rifampicin resistance. MLVA-16 analysis clustered the isolates into 22 unique genotypes that belonged to the East Mediterranean lineage. Altogether, our findings show that the re-emergence of brucellosis was due to B. melitensis strains of local origin, the Palestinian and Israeli control programs' weaknesses could be a major factor behind the re-emergence of the disease. However, other socioeconomic and environmental factors must be investigated. Moreover, strengthening brucellosis control programs and enhancing cooperation between all stakeholders is essential to ensure long-term program outcomes to fight brucellosis.