Genomic Characterization of Two NDM-5-Producing Isolates of Klebsiella pneumoniae ST11 from a Single Patient.

Two metallo-ß-lactamase-producing Klebsiella pneumoniae (HA30 and HA31) were isolated in a hospital in Argentina during 2018. K. pneumoniae HA30 was isolated from a rectal swab during the epidemiological surveillance for carbapenemase-producing strains, while K. pneumoniae HA31 was collected from the same patient 4 days after hospitalization. The aim of the present study was to identify the clonal relationships and resistome of these two NDM-producing K. pneumoniae strains isolated from a patient with a fatal outcome. Whole-genome sequencing (WGS) was performed using Illumina MiSeq-I, and subsequent analysis involved genome assembly, annotation, antibiotic resistance gene identification, multilocus sequence typing (MLST), and plasmid characterization using bioinformatics tools. Conjugation assays to E. coli J53 was conducted as previously described. K. pneumoniae HA30 exhibited extensively drug-resistant phenotype, while HA31 was multidrug-resistant as defined by Magiorakos et al., including both resistance to carbapenems, aminoglycosides and ciprofloxacin with blaNDM-5, blaCTX-M-15 and rmtB genes found in both strains. MLST analysis showed that both strains belonged to ST11, differing by only 4 cgSNPs, indicating that K. pneumoniae HA30 and HA31 were the same strain. Conjugation assays revealed that K. pneumoniae HA31 strain possessed a transferable plasmid to E. coli J53. Bioinformatics studies identified that the same strain colonizing an inpatient during hospital admission subsequently caused the infection leading to a fatal outcome, being the first report of blaNDM-5, rmtB and blaCTX-M-15 genes in a K. pneumoniae ST11 strain from Latin America. Our results also highlighted the importance of focusing on epidemiological surveillance programs.

Replacement of KPC-producing pandemic lineages and dissemination of plasmids associated with antimicrobial resistance determinants during inpatient's hospitalization.

OBJECTIVES: The emergence of blaKPC-2 within nosocomial settings has become a major public health crisis worldwide. Our aim was to perform whole-genome sequencing (WGS) of three KPC-producing Gram-negative bacilli (KPC-GNB) strains isolated from a hospitalized patient to identify acquired antimicrobial resistance genes (ARGs). METHODS: WGS was performed using Illumina MiSeq-I, and de novo assembly was achieved using SPAdes. Bioinformatics analysis was done using Resfinder, AMRFinder, ISFinder, plasmidSPAdes, PlasmidFinder, MOB-suite, PLSDB database, and IntegronFinder. Conjugation assays were performed to assess the ability of blaKPC-2 to transfer via a plasmid-related mobilization mechanism. RESULTS: High-risk clone KPC-producing Klebsiella pneumoniae sequence type (ST) 258 (HA3) was colonizing an inpatient who later was infected by KPC-producing Escherichia coli ST730 (HA4) and subsequently by KPC-producing K. pneumoniae ST11 (HA15) during hospitalization. Although belonging to different species, both strains causing infections harbored the same gene configuration for dissemination of blaKPC-2 in related IncM1 plasmids recently found in other KPC-GNB isolated from Hospital Alemán at Ciudad Autónoma de Buenos Aires. Conjugation assays revealed that only pDCVEA4-KPC from E. coli HA4 was successfully transferred with a conjugation frequency of 3.66 × 101. CONCLUSIONS: Interchange of multidrug-resistant K. pneumoniae lineages ST258 replaced by ST11 in the framework of colonization and infection by KPC-GNB of an inpatient from our institution was found. In addition, the transfer of the gene configuration of blaKPC-2 between infecting strains may have occurred in the nosocomial environment, but we cannot rule out that the event took place in vivo, within the patient, during hospitalization.

Genomic data reveals the emergence of the co-occurrence of blaKPC-2 and blaCTX-M-15 in an Escherichia coli ST648 strain isolated from rectal swab within the framework of hospital surveillance.

OBJECTIVES: The worldwide dissemination of carbapenemase-producing Escherichia coli lineages belonging to high-risk clones poses a challenging public health menace. The aim of this work was to investigate genomic features of a colonizing multidrug-resistant strain of Klebsiella pneumoniae carbapenemase (KPC)-producing E. coli from our institution. METHODS: Whole-genome sequencing was done by Illumina MiSeq-I, and de novo assembly was achieved using SPAdes. Resistome, mobilome, plasmids, virulome, and integrons were analysed using ResFinder, AMRFinder, ISFinder, PlasmidFinder, MOB-suite, VirulenceFinder, and IntegronFinder. Sequence types (STs) were identified with pubMLST and BIGSdb databases. Conjugation assays were also performed. RESULTS: Escherichia coli HA25pEc was isolated from a rectal swab sample taken within the framework of the hospital epidemiological surveillance protocol for detection of carbapenemase-producing Enterobacterales. Escherichia coli HA25pEc corresponded to the first report of ST648 co-harbouring blaKPC-2 and blaCTX-M-15 in Latin America from a colonized patient. It had 19 antibiotic resistance genes (ARGs), including blaKPC-2, located on a Tn4401a isoform. Conjugation assays revealed that blaKPC-2 was not transferred by conjugation to E. coli J53 under our experimental conditions. CONCLUSION: Escherichia coli ST648 has been detected previously in companion and farm animals as well as in hospital- and community-acquired infections worldwide. Although scarcely reported as KPC-producers, our finding in a culture surveillance with several acquired ARGs, including blaCTX-M-15, alerts the potential of this clone for worldwide unnoticed spreading of extreme drug resistance to ß-lactams. These data reinforce the importance of carrying out molecular surveillance to identify reservoirs and warn about the dissemination of new international clones in carbapenemase-bearing patients.

Emergence of colistin resistance in Klebsiella pneumoniae ST15 disseminating blaKPC-2 in a novel genetic platform.

OBJECTIVES: Isolation of colistin- and carbapenem-resistant Klebsiella pneumoniae (CCR-Kp) is increasing in hospital settings worldwide, which is related to increased morbidity, mortality and healthcare costs. The aim of this work was to perform whole-genome sequencing (WGS), genomic and phylogenetic analysis, and conjugation assays of an extensively drug-resistant (XDR) CCR-Kp isolate from Argentina. METHODS: WGS of strain KpS26 isolated from a bloodstream infection was performed using Illumina MiSeq-I, and de novo assembly was achieved using SPAdes v.3.11. A maximum likelihood tree was created using MEGA7 based on core genome single nucleotide polymorphisms from whole-genome alignment of K. pneumoniae isolates identified in silico as sequence type 15 (ST15). The resistome, plasmids and integrons were analysed using ResFinder, AMRFinderPlus, ISfinder, plasmidSPAdes, PlasmidFinder and IntegronFinder. Standard conjugation was performed. RESULTS: KpS26 belonged to ST15, which is less common than ST258, ST25 and ST11 that are globally reported as responsible for CCR-Kp outbreaks. Fourteen transferable antimicrobial resistance genes (ARGs), including blaKPC-2 in a novel genetic platform transferable by conjugation, were detected contributing to the XDR phenotype. The amino acid substitution T157P in the protein encoded by the pmrB gene of KpS26, previously reported as being responsible for resistance to colistin in K. pneumoniae lineages globally disseminated, was also identified in this strain. CONCLUSION: The XDR CCR-Kp isolate analysed here shows that ST15 is also disseminating blaKPC-2 in Argentina alongside other ARGs, evidencing that KPC epidemiology continues to be shaped by intricate and assorted ways of lateral gene transfer.

Novel insights related to the rise of KPC-producing Enterobacter cloacae complex strains within the nosocomial niche.

According to the World Health Organization, carbapenem-resistant Enterobacteriaceae (CRE) belong to the highest priority group for the development of new antibiotics. Argentina-WHONET data showed that Gram-negative resistance frequencies to imipenem have been increasing since 2010 mostly in two CRE bacteria: Klebsiella pneumoniae and Enterobacter cloacae Complex (ECC). This scenario is mirrored in our hospital. It is known that K. pneumoniae and the ECC coexist in the human body, but little is known about the outcome of these species producing KPC, and colonizing or infecting a patient. We aimed to contribute to the understanding of the rise of the ECC in Argentina, taking as a biological model both a patient colonized with two KPC-producing strains (one Enterobacter hormaechei and one K. pneumoniae) and in vitro competition assays with prevalent KPC-producing ECC (KPC-ECC) versus KPC-producing K. pneumoniae (KPC-Kp) high-risk clones from our institution. A KPC-producing E. hormaechei and later a KPC-Kp strain that colonized a patient shared an identical novel conjugative IncM1 plasmid harboring bla KPC-2. In addition, a total of 19 KPC-ECC and 58 KPC-Kp strains isolated from nosocomial infections revealed that high-risk clones KPC-ECC ST66 and ST78 as well as KPC-Kp ST11 and ST258 were prevalent and selected for competition assays. The competition assays with KCP-ECC ST45, ST66, and ST78 versus KPC-Kp ST11, ST18, and ST258 strains analyzed here showed no statistically significant difference. These assays evidenced that high-risk clones of KPC-ECC and KPC-Kp can coexist in the same hospital environment including the same patient, which explains from an ecological point of view that both species can exchange and share plasmids. These findings offer hints to explain the worldwide rise of KPC-ECC strains based on the ability of some pandemic clones to compete and occupy a certain niche. Taken together, the presence of the same new plasmid and the fitness results that showed that both strains can coexist within the same patient suggest that horizontal genetic transfer of bla KPC-2 within the patient cannot be ruled out. These findings highlight the constant interaction that these two species can keep in the hospital environment, which, in turn, can be related to the spread of KPC.