Anatomy of an extensively drug-resistant Klebsiella pneumoniae outbreak in Tuscany, Italy.

A protracted outbreak of New Delhi metallo-ß-lactamase (NDM)-producing carbapenem-resistant Klebsiella pneumoniae started in Tuscany, Italy, in November 2018 and continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug-resistant, NDM-producing K. pneumoniae isolates cultured over a 20-mo period from 76 patients at several healthcare facilities in southeast Tuscany. All isolates belonged to high-risk clone ST-147 and were typically nonsusceptible to all first-line antibiotics. Albeit sporadic, resistances to colistin, tigecycline, and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST-147 isolates circulating in Tuscany were monophyletic and highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates), and shared a recent ancestor with clinical isolates from the Middle East. While the blaNDM-1 gene was carried by an IncFIB-type plasmid, our investigations revealed that the ST-147 lineage from Italy also acquired a hybrid IncFIB/IncHIB-type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST-11 and ST-307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of an archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats.

Characterization of pFOX-7a, a conjugative IncL/M plasmid encoding the FOX-7 AmpC-type ß-lactamase, involved in a large outbreak in a neonatal intensive care unit.

OBJECTIVES: FOX-type enzymes are a lineage of AmpC-type ß-lactamases from Aeromonas spp. whose genes have been mobilized to plasmids spreading among Enterobacteriaceae, where they can be responsible for resistance to extended-spectrum cephalosporins and ß-lactamase inhibitor combinations. Little is known about the genetic context and plasmid vehicles of bla(FOX) determinants. Here, we have characterized a plasmid encoding the FOX-7 ß-lactamase, which was involved in a large outbreak caused by two Klebsiella pneumoniae clones in a neonatal intensive care unit. METHODS: Plasmid transferability was tested in conjugation experiments using Escherichia coli recipients. Plasmids from different strains were compared by restriction profiling and PCR mapping. The complete sequence of pFOX-7a plasmid was determined by a next-generation sequencing approach followed by gap filling using PCR and sequencing. RESULTS: An apparently identical conjugative plasmid encoding FOX-7 was detected in representatives of the K. pneumoniae clones that caused the outbreak and in sporadic FOX-7-producing strains of other species from the same ward. The plasmid, named pFOX-7a, has an IncL/M-type backbone and two separate resistance modules including a Tn3-like transposon and a novel Tn1696 derivative, named Tn6234, which carries an integron platform, a hybrid (but still functional) mercury resistance module and a novel putative transposon of original structure, named Tn6240, associated with the bla(FOX-7) gene. CONCLUSIONS: pFOX-7a is the first completely characterized plasmid encoding a FOX-type ß-lactamase. The bla(FOX-7) gene was associated with a putative transposable element of original structure, which was likely involved in its mobilization from the Aeromonas metagenome.

Clonal relatedness and conserved integron structures in epidemiologically unrelated Pseudomonas aeruginosa strains producing the VIM-1 metallo-{beta}-lactamase from different Italian hospitals.

Three epidemiologically independent Pseudomonas aeruginosa isolates, representative of the first VIM-1 metallo-beta-lactamase producers detected at three different hospitals in northern Italy, were investigated to determine their genomic relatedness and to compare the structures of the genetic supports for the VIM-1 determinants. The three isolates, all of serotype O11, appeared to be clonally related according to the results of genotyping by macrorestriction analysis of genomic DNA by pulsed-field gel electrophoresis and random amplification of polymorphic DNA. Investigation of the genetic support for the bla(VIM-1) determinant revealed that it was carried on identical or almost identical integrons (named In70.2 and In70.3) located within a conserved genomic context. The integrons were structurally related to In70 and In110, two plasmid-borne bla(VIM-1)-containing integrons from Achromobacter xylosoxidans and Pseudomonas putida isolates, respectively, from the same geographic area (northern Italy) and were found to be inserted close to the res site of a Tn5051-like transposon, different from any of those described previously, that was apparently carried on the bacterial chromosome. The present findings suggest that the three VIM-1-producing isolates are members of the same clonal complex which have been spreading in hospitals in northern Italy since the late 1990s and point to a common ancestry of their bla(VIM-1)-containing integrons.

Resistance determinants and clonal diversity in group A streptococci collected during a period of increasing macrolide resistance.

Susceptibility to macrolides and lincosamides was investigated with 299 consecutive nonduplicate Streptococcus pyogenes clinical isolates collected over a 6-year period (1992 to 1997) from an area of central Italy. During this period, macrolide resistance rates steadily increased (from 9% in 1992 to 53% in 1997; P < 0.001). The increase was caused by isolates with a macrolide-lincosamide-streptogramin B resistance phenotype, carrying mostly erm(B) but also erm(TR) genes, that were not detected in the first 2 years and were detected with increasing prevalence (8, 5, 26, and 37%, respectively) during the following 4 years. During the same period, the prevalence of isolates with a macrolide resistance phenotype, carrying mef(A) determinants, did not vary significantly; on average it was 13%, with modest rate fluctuations in different years and no definite trend. Molecular typing revealed a remarkable clonal diversity among susceptible and resistant isolates and a notable heterogeneity of the genetic environment of the resistance genes. The analysis of clonal diversity in relation with resistance phenotypes and genotypes revealed that increased macrolide resistance rates were due to a complex interplay of different mechanisms, with a relevant contribution played by an "epidemic" spread of genetic elements carrying the erm(B) gene among the circulating streptococcal population.