Genomic characterization of carbapenemase-producing Klebsiella pneumoniae ST307 revealed multiple introductions in Buenos Aires, Argentina.

OBJECTIVES: To describe at genomic level nine carbapenemase-producing Klebsiella pneumoniae ST307 (Kp-ST307) clinical isolates recovered in Buenos Aires during 2017 to 2021, investigating their resistome, virulome, and phylogeny. METHODS: Antimicrobial susceptibility was determined according to Clinical and Laboratory Standards Intitute (CLSI). Genomic DNA was sequenced by Illumina MiSeq and analysed using SPAdes, PROKKA, and Kleborate. Phylogeny of 355 randomly selected Kp-ST307 genomes and those from nine local isolates was inferred by a maximum-likelihood approach. The tree was visualized using Microreact. RESULTS: Besides resistance to ß-lactams and fluoroquinolones, six out of nine Kp-ST307 were also resistant to ceftazidime/avibactam (CZA). This difficult-to-treat resvistance phenotype was mediated by blaSHV-28 and GyrA-83I/ParC-80I mutations in addition to carbapenemase coding genes. Among CZA susceptible isolates, two of them harboured blaKPC-3 while the other harboured blaKPC-2+blaCTX-M-15. Regarding CZA-resistant isolates, three harboured blaKPC-3+blaNDM-1+blaCMY-6, two carried blaKPC-2+blaNDM-5+blaCTX-M-15, and blaNDM-5+blaCTX-M-15 were detected in the remaining isolate. Furthermore, five colistin-resistant isolates presented a nonsense mutation in mgrB. Global Kp-ST307 isolates were distributed in two deep-branching lineages while local isolates were set in the main clade of the phylogenetic tree. The five isolates from the same hospital, harbouring blaKPC-3 or blaKPC-3+blaNDM-1+blaCMY-6, clustered in a monophyletic subclade with Italian isolates. Also, an isolate harbouring blaKPC-2+blaNDM-5+blaCTX-M-15 recovered in another hospital was closed to this group. The remaining local Kp-ST307 were grouped in other subclades containing isolates of diverse geographical origin. CONCLUSION: The inferred resistome was consistent with the resistant phenotype. Phylogeny suggested multiple introduction events in our region and a single major introduction in one hospital followed by local spread.

Genetic and Biochemical Characterization of AXC-2 from Achromobacter ruhlandii.

Achromobacter spp. are intrinsically resistant to multiple antibiotics and can also acquire resistance to those commonly used for the treatment of respiratory infections, especially in patients with cystic fibrosis. The aim of this study was to perform the genetic and biochemical characterization of AXC-2 from A. ruhlandii and to analyze all available AXC variants. Steady-state kinetic parameters were determined on a purified AXC-2 enzyme. It exhibited higher catalytic efficiencies towards amino-penicillins and older cephalosporins, while carbapenems behaved as poor substrates. Phylogenetic analysis of all blaAXC variants available in the NCBI was conducted. AXC was encoded in almost all A. ruhlandii genomes, whereas it was only found in 30% of A. xylosoxidans. AXC-1 was prevalent among A. xylosoxidans. AXC variants were clustered in two main groups, correlating with the Achromobacter species. No association could be established between the presence of blaAXC variants and a specific lineage of A. xylosoxidans; however, a proportion of AXC-1-producing isolates corresponded to ST 182 and ST 447. In conclusion, this study provides valuable insights into the genetic context and kinetic properties of AXC-2, identified in A. ruhlandii. It also provides a thorough description of all AXC variants and their association with Achromobacter species and various lineages.

Outbreak of Klebsiella pneumoniae ST11 Resistant To Ceftazidime-Avibactam Producing KPC-31 and the Novel Variant KPC-115 during COVID-19 Pandemic in Argentina.

We describe an outbreak of Klebsiella pneumoniae sequence type 11 (ST11) producing KPC variants resistant to ceftazidime-avibactam. Six patients hospitalized in the intensive care unit (mostly due to critical COVID pneumonia) presented infection or colonization by this bacterium. They had several comorbidities and required mechanical ventilation, central venous catheters, and urinary catheters. All 6 patients had a history of fecal colonization with KPC-producing Enterobacterales (KPC-E). Three of them had previous episodes of infection with ceftazidime-avibactam-susceptible KPC-producing K. pneumoniae, which were treated with ceftazidime-avibactam. Several phenotypic methods failed to detect carbapenemase production in these 6 ceftazidime-avibactam-resistant isolates, and they showed in vitro susceptibility to imipenem and meropenem. All of them rendered positive results for blaKPC by PCR, and amplicon sequencing identified blaKPC-31 variant in 5 isolates and a novel variant, named blaKPC-115, in the other. Moreover, matrix-assisted laser desorption ionization-time of flight mass spectrometry was able to detect KPC in all isolates. Ceftazidime-avibactam-resistant isolates, as well as those recovered from previous infection episodes (KPC-3-producing K. pneumoniae, ceftazidime-avibactam susceptible), displayed a unique pulse type and belonged to ST11. Based on whole-genome sequencing results of selected isolates, less than 7 single-nucleotide polymorphisms were identified among them, which was indicative of the presence of a unique clone. Both in vivo selection and horizontal transmission seemed to have occurred in our hospital. Detection of these strains is challenging for the laboratory. History of previous KPC-E infections or colonization and systematic testing for resistance to ceftazidime-avibactam might help raise awareness of this possibility. IMPORTANCE Klebsiella pneumoniae is one of the main bacteria that cause infections in health care settings. This pathogen has developed a high level of resistance to many antibiotics. Some K. pneumoniae isolates can produce an enzyme known as carbapenemase KPC, making carbapenems (considered the last line for therapy) not effective to treat their infections. The combination ceftazidime-avibactam, approved by FDA in 2015, is useful to treat infections caused by KPC-producing K. pneumoniae. This study describes the emergence, in one hospital in Argentina, of K. pneumoniae isolates that produce KPC variants (KPC-31 and KPC-115) resistant to ceftazidime-avibactam. The ceftazidime-avibactam-resistant bacteria were isolated in inpatients, including some that previously received this combination as treatment. Transmission of this strain to other patients also occurred in the studied period. Detection of these bacteria is challenging for the laboratory. The knowledge and awareness of the emergence of this pathogen in our region are highly valuable.

Characterisation of blaKPC-2-harbouring plasmids recovered from Pseudomonas aeruginosa ST654 and ST235 high-risk clones.

OBJECTIVE: The main objectives were to describe two blaKPC-2 plasmids recovered from Pseudomonas aeruginosa isolates belonging to the ST654 and ST235 high-risk clones, and to compare with complete sequences of blaKPC-2 harbouring plasmids available in public databases. METHODS: Antimicrobial susceptibility was determined according to CLSI (Clinical and Laboratory Standards Institute) guidelines. Genomes were sequenced using an Illumina MiSeq platform, and blaKPC-2 plasmid sequences were achieved using MinION platform. Sequences were analysed using Unicycler and RAST. In silico predictions of the isolates sequence type (ST), antimicrobial resistance genes, plasmid replicon typing and MOB relaxases were fulfilled using bioinformatics tools. RESULTS: PA_2047 and PA_HdC isolates corresponded to the high-risk clones ST654 and ST235, respectively. The carbapenem resistance was mediated by KPC-2. Both blaKPC-2 harbouring plasmids, pPA_2047 and pPA_HdC, were different among them, non-conjugative and untypable by PlasmidFinder. pPA_2047 presented high identity with a Pae-13 plasmid, and these both located blaKPC-2 in Tn4401b isoform. pPA_HdC displayed a novel architecture, and the genetic context of blaKPC-2 was original. Besides the blaKPC-2 gene, resistance genes to aminoglycosides and quinolones were detected, including the novel phosphotransferase CrpP in PA_HdC. CONCLUSION: This study expands the limited knowledge about the molecular epidemiology of blaKPC-2 in P. aeruginosa from Latin America. Two novel plasmids harbouring blaKPC-2 were described that were untypable by their incompatibility group. The plasmid recovered from P. aeruginosa PA_HdC (ST235) displayed a novel architecture and an original context for blaKPC-2. On the other hand, the genetic platform carrying blaKPC-2 in P. aeruginosa PA_2047 (ST654) seems to a be a classical one.