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Klebsiella pneumoniae multidrug-resistant (MDR) high-risk clones drive the spread of antimicrobial resistance (AMR) associated infections, resulting in limited therapeutic options. This study described the genomic characteristics of K. pneumoniae MDR high-risk clones in Gauteng, South Africa. Representative carbapenem-resistant [K. pneumoniae carbapenemase (KPC)-2, New-Delhi metallo-beta (β)-lactamase (NDM)-1, oxacillinase (OXA)-181, OXA-232, OXA-48, Verona integron-encoded metallo-β-lactamase (VIM)-1] K. pneumoniae isolates (n = 22) obtained from inpatient and outpatient's urine (n = 9) and inpatients rectal carriage (n = 13) were selected for short-read whole genome sequencing. Klebsiella pneumoniae population include sequence type (ST)-307 (n = 3), ST2497 (n = 5) and ST17 (n = 4). The ST17 strains were exclusively obtained from rectal screening. Ten isolates co-harboured carbapenemase genes including β-lactamase gene encoding KPC-2 + OXA-181, NDM-1 + OXA-48 and NDM-1 + OXA-181. One ST307 isolate (UP-KT-73CKP) co-harboured three carbapenemase genes (blaNDM-1 + blaOXA-48 + blaOXA-181), while all the ST2497 strains co-harboured (blaNDM-1 + blaOXA-232). Phenotypically, hypermucoviscosity was observed in a single ST307 isolate. The ST307 isolate UP-KT-151UKP harboured colibactin genotoxins. The following mobile genetic elements were detected: plasmids [incompatibility group (Inc)-FIB(K), IncX3], and bacteriophages [e.g. Klebsi_ST16_OXA48phi5.4_NC_049450, Klebsi_3LV2017_NC_047817(36)]. The study highlights the importance of local genomic surveillance systems to characterise K. pneumoniae MDR high-risk clones. This data will aid in designing infection and prevention measures for limiting the spread of carbapenemase-producing K. pneumoniae in Gauteng, South Africa. [ABSTRACT FROM AUTHOR]

Abstract Klebsiella pneumoniae multidrug-resistant (MDR) high-risk clones drive the spread of antimicrobial resistance (AMR) associated infections, resulting in limited therapeutic options. This study described the genomic characteristics of K. pneumoniae MDR high-risk clones in Gauteng, South Africa. Representative carbapenem-resistant [K. pneumoniae carbapenemase (KPC)-2, New-Delhi metallo-beta (β)-lactamase (NDM)-1, oxacillinase (OXA)-181, OXA-232, OXA-48, Verona integron-encoded metallo-β-lactamase (VIM)-1] K. pneumoniae isolates (n = 22) obtained from inpatient and outpatient’s urine (n = 9) and inpatients rectal carriage (n = 13) were selected for short-read whole genome sequencing. Klebsiella pneumoniae population include sequence type (ST)-307 (n = 3), ST2497 (n = 5) and ST17 (n = 4). The ST17 strains were exclusively obtained from rectal screening. Ten isolates co-harboured carbapenemase genes including β-lactamase gene encoding KPC-2 + OXA-181, NDM-1 + OXA-48 and NDM-1 + OXA-181. One ST307 isolate (UP-KT-73CKP) co-harboured three carbapenemase genes (bla NDM-1 + bla OXA-48 + bla OXA-181), while all the ST2497 strains co-harboured (bla NDM-1 + bla OXA-232). Phenotypically, hypermucoviscosity was observed in a single ST307 isolate. The ST307 isolate UP-KT-151UKP harboured colibactin genotoxins. The following mobile genetic elements were detected: plasmids [incompatibility group (Inc)-FIB(K), IncX3], and bacteriophages [e.g. Klebsi_ST16_OXA48phi5.4_NC_049450, Klebsi_3LV2017_NC_047817(36)]. The study highlights the importance of local genomic surveillance systems to characterise K. pneumoniae MDR high-risk clones. This data will aid in designing infection and prevention measures for limiting the spread of carbapenemase-producing K. pneumoniae in Gauteng, South Africa.

Introduction: Aim of the study was the molecular characterization of 21 ceftazidime/avibactam resistant (CZA-R) Klebsiella pneumoniae strains, collected in the period October 2021–March 2022 from an Intensive Care COVID Unit in a Northern Italian Hospital. Methods: After growth on selective/chromogenic culture media and susceptibility tests assessment, resistance genes content was ascertained for all the isolates by the HybriSpot 12 multiplexing, PCR and Whole-Genome Sequencing (WGS). Clonality was assessed by PFGE and MLST according to the Pasteur scheme. A SNPs-based phylogenetic tree was obtained comparing representative isolates and global genomes. The blaKPC gene horizontal transmission was evaluated by conjugation experiments. blaKPC-166 was cloned in a pCR2.1 vector and transformed in chemically competent TOP10 cells. Results: Sixteen inpatients resulted positive for colonization and/or infection by KPC-producing K. pneumoniae (KPC-Kp) strains. The 21 CZA-R KPC-Kp isolates obtained showed MDR phenotype; susceptibility to meropenem was always retained. All the CZA-R KPC-Kp presented a novel blaKPC variant, named blaKPC-166, showing a single nucleotide substitution (T811C) compared to the blaKPC-94; but related to blaKPC-2. Two different pulsotypes were detected: A in 18/21 and B in 1/21 cases, two strains from the same patient being untypable by PFGE. Interestingly, the outbreak was sustained by the high-risk clone ST307, although the ST22, ST6342, ST6418 and ST6811 have also been identified and associated to KPC-166. Worryingly, blaKPC-166 could be transferred horizontally and, after cloning, it conferred resistance to CZA. Discussion: This novel variant confers CZA–resistance and carbapenems susceptibility restoration. As KPC-166 was found expressed by multiple Kp clones, greater efforts should be made to prevent the further dissemination of such strains in Italian clinical settings.

The Klebsiella pneumoniae ST307 clone, identified in the mid-1990s, has emerged as a global antimicrobial-resistant (AMR) high-risk clone, significantly contributing to the global health challenge also posed by other AMR K. pneumoniae lineages. The acquisition of a blaCTX-M-15-carrying plasmid has facilitated its widespread dissemination. At Europe's major transport hub for the movement of live animals, Frankfurt Airport, a shipment of 20 live leopard tortoises was sampled during German border control in 2014. Phylogenetic analysis (MLST) identified a K. pneumoniae ST307 strain, prompting further investigation. Our analysis revealed the presence of a ~193 kb plasmid carrying a broad range of AMR genes, including blaCTX-M-15, blaTEM-1B, blaOXA-1, aac(3)-IIa, aac(6′)-Ib-cr, aph(3″)-Ib, aph(6)-Id, and qnrB1. Additionally, mutations in the quinolone resistance-determining region in gyrA (S83I) and parC (S80I) were detected. Phenotypic testing demonstrated resistance of the isolate to the most common antimicrobials used in both human and veterinary medicine; exceptions included carbapenems and newer β-lactamase inhibitor combinations. Because the role of imported exotic animals in the dissemination of AMR genes is largely deficient, the present study fills yet missing mosaic pieces in the complete picture of extended-spectrum β-lactamase (ESBL)-producing Enterobacterales. [ABSTRACT FROM AUTHOR]

ABSTRACT: 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.

The Klebsiella pneumoniae ST307 clone, identified in the mid-1990s, has emerged as a global antimicrobial-resistant (AMR) high-risk clone, significantly contributing to the global health challenge also posed by other AMR K. pneumoniae lineages. The acquisition of a blaCTX-M-15-carrying plasmid has facilitated its widespread dissemination. At Europe’s major transport hub for the movement of live animals, Frankfurt Airport, a shipment of 20 live leopard tortoises was sampled during German border control in 2014. Phylogenetic analysis (MLST) identified a K. pneumoniae ST307 strain, prompting further investigation. Our analysis revealed the presence of a ~193 kb plasmid carrying a broad range of AMR genes, including blaCTX-M-15, blaTEM-1B, blaOXA-1, aac(3)-IIa, aac(6′)-Ib-cr, aph(3″)-Ib, aph(6)-Id, and qnrB1. Additionally, mutations in the quinolone resistance-determining region in gyrA (S83I) and parC (S80I) were detected. Phenotypic testing demonstrated resistance of the isolate to the most common antimicrobials used in both human and veterinary medicine; exceptions included carbapenems and newer β-lactamase inhibitor combinations. Because the role of imported exotic animals in the dissemination of AMR genes is largely deficient, the present study fills yet missing mosaic pieces in the complete picture of extended-spectrum β-lactamase (ESBL)-producing Enterobacterales.

ABSTRACT: Objectives: Extended spectrum β-lactamase (ESBL)- and ampicillinase C (AmpC)-carrying Enterobacteriaceae have been widely reported among companion animals. According to previous studies, dogs with a shelter or stray background might be at risk of carrying such bacteria. The aim of this study was to explore, with whole-genome sequencing (WGS), the genomic characteristics of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolated from imported dogs with a stray or shelter background. Methods: E. coli (n = 58) and K. pneumoniae (n = 2) isolates from imported dogs originating from seven countries were included. Phenotypic resistance was investigated by selective isolation and antibiotic susceptibility testing. Whole-genome sequencing was used to study the genomic characteristics and the presence of antimicrobial resistance genes (ARGs) and virulence determinants of the ESBL/AmpC-producing E. coli and K. pneumoniae isolates. Results: A high diversity of different ARGs (n = 56) and sequence types (STs) (n = 32), including high-risk clonal lineages ST410 (n = 3) and ST307 (n = 1), was identified in E. coli and K. pneumoniae isolates, respectively. Genes encoding resistance to β-lactams accounted for the majority, with the most frequent being blaCTX-M-15. Moreover, 17 (29%) E. coli isolates qualified as presumptive extraintestinal pathogenic and/or uropathogenic E. coli. Conclusions: Our results highlight the multiplicity of genetic backgrounds disseminating ESBL/AmpC-genes in the studied dogs, calling for further investigation of possible drivers responsible for the dissemination of ARGs in animal shelters and amongst stray dogs. From a public health perspective, enhanced genomic surveillance of ESBL/AmpC-producing Enterobacteriaceae in dogs is needed in Finland.

A clinical strain of Klebsiella pneumoniae typed as sequence type 307 carrying three different alleles of the flu gene encoding the Escherichia coli virulence factor antigen 43 associated with biofilm formation was detected and characterized. The flu alleles are located in the chromosome inside putative integrative conjugative elements. The strain displays the phenotypes associated with Ag43, i.e. bi-phasic colony morphology and enhanced biofilm production. Furthermore, the strain produces low amount of capsule known to affect Ag43 function. Analysis of 1431 worldwide deposited genomes revealed that 3.7% Klebsiella pneumoniae carry one or two flu alleles. [ABSTRACT FROM AUTHOR]

Background. The global spread of carbapenemase-producing Enterobacterales has become an epidemiological risk for healthcare systems by limiting available antimicrobial treatments. The COVID-19 pandemic worsened this scenario, prompting the emergence of extremely resistant microorganisms. Methods. Between March 2020 and September 2021, the NRL confirmed 82 clinical Enterobacterales isolates harboring a combination of blaKPC and MBL genes. Molecular typing was analyzed by PFGE and MLST. Modified double-disk synergy (MDDS) tests were used for phenotypic studies. Results. Isolates were submitted from 28 hospitals located in seven provinces and Buenos Aires City, including 77 K. pneumoniae, 2 K. oxytoca, 2 C. freundii, and 1 E. coli. Almost half of K. pneumoniae isolates (n = 38; 49.4%), detected in 15 hospitals, belong to the CC307 clone. CC11 was the second clone, including 29 (37.7%) isolates (22, ST11 and 7, ST258) from five cities and 12 hospitals. Three isolates belonging to CC45 were also detected. The carbapenemase combinations observed were as follows: 55% blaKPC-2 plus blaNDM-5; 32.5% blaKPC-2 plus blaNDM-1; 5% blaKPC-3 plus blaNDM-1; 5% blaKPC-2 plus blaIMP-8; and 2.5% strain with blaKPC-2 plus blaNDM-5 plus blaOXA-163. Aztreonam/avibactam and aztreonam/relebactam were the most active combinations (100% and 91% susceptible, respectively), followed by fosfomycin (89%) and tigecycline (84%). Conclusions. The MDDS tests using ceftazidime-avibactam/EDTA and aztreonam/boronic acid disks improved phenotypic classification as dual producers. The successful high-risk clones of K. pneumoniae, such as hyper-epidemic CC307 and CC11 clones, drove the dissemination of double carbapenemase-producing isolates during the COVID-19 pandemic. [ABSTRACT FROM AUTHOR]

ABSTRACT: Background: The spread of carbapenemase-producing Klebsiella pneumoniae (CP-Kp) strains belonging to high-risk sequence types (STs) is a concern. For Switzerland, national data about the molecular features (especially the STs) of CP-Kp of human origin is not available. In veterinary clinics, ST11 and ST307 blaOXA-48-possessing K. pneumoniae strains have been recently reported. Methods: We analysed a collection of 285 K. pneumoniae genomes (170 were CP-Kp) isolated in Switzerland from human and non-human sources during 2006–2020. Whole-genome sequencing, core genome phylogenies and public databases were used to present a detailed overview regarding carbapenemases, STs and plasmids. Results: The top five STs were (main carbapenemase gene) ST512 (blaKPC-3), ST258 (blaKPC-2) and ST101 (blaOXA-48), consisting of strains of human origin only, and ST11 (blaOXA-48) and ST307 (blaOXA-48) strains isolated from human, animal and environmental sources. However, during 2016–2020, the main STs for CP-Kp were ST11 (17.6%), ST307 and ST101 (both 14.7%), whereas ST258 (5.9%) and ST512 (4.4%) significantly declined. Most carbapenemase genes were carried on plasmids already described. Core genome analysis revealed that ST11 K. pneumoniae of animal and human origin were closely related, whereas those of ST307 were distant. Conclusions: We described, for the first time, the features of the CP-Kp circulating in Switzerland in human and non-human settings. Our genomic analysis revealed that the emerging high-risk ST11 and ST307 lineages were often isolated from non-human settings. This study provided a baseline for further whole-genome sequencing-based One-Health surveillance of CP-Kp and emphasized the need for metadata to track dissemination routes between the different settings.

ABSTRACT The global epidemiology of multidrug resistant Klebsiella pneumoniae, a serious threat to both animal and human health, is dominated by the spread of pathogenic clones, each separately evolving via acquisition of transferable antibiotic resistance or niche-specific virulence determinants. In horses, K. pneumoniae infection can lead to severe respiratory illness. Here, we characterized multiple isolates recovered from bronchial aspirates of a mare with pneumonia refractory to antibiotics. First, we used a combination of standard microbiology, bacteriophage cross-susceptibility and antibiotic resistance testing to profile the infecting K. pneumoniae population. The genomes of isolates with distinct fingerprints (pulsed-field gel electrophoresis) and unique combined bacteriophage/antibiotic profiles were then further analyzed using whole-genome sequencing. Adhesion to human epithelial cells and biofilm production were also measured as virulence indicators. Although it is commonly expected for one clone to dominate an infection episode, we identified five coexisting multidrug resistant K. pneumoniae sharing the same niche. One was a novel sequence type (ST4656), while the other four were all members of emerging human pathogenic clonal groups (ST307, ST628, ST893 and ST392). These isolates did not display significant differences from one another in terms of virulence or resistance and differed only in plasmid content from isolates implicated in severe human infections, with equal potential to prolong duration and severity of infection when sharing the same niche. This study highlights the importance of more precise surveillance and detection measures to uncover bacterial heterogeneity, reminding us that the “single clone” concept is not an absolute in invasive bacterial infections. IMPORTANCE Multidrug resistant Klebsiella pneumoniae are agents of life-threatening infections in animals and humans, with several multidrug resistant clones causing outbreaks of disease worldwide. It is generally accepted that only one clone will be dominant in an infection episode. In this study, we investigated K. pneumoniae isolates from a horse with severe pneumonia and demonstrated co-occurrence of multiple sequence types previously identified as emerging human pathogens. The equine isolates are not significantly different from one another in terms of virulence or resistance, with equal potential to prolong duration and severity of infection, and are indistinguishable from isolates recovered from humans, except for plasmid content. Our study highlights how the “one dominant clone” concept is not an absolute in severe infection, illustrating the need for improved diagnostics to track heterogeneity of infection, and reinforces the importance of cross-monitoring of environmental and human reservoirs of multidrug resistant pathogens.

ABSTRACT The ability of extensively drug-resistant (XDR) Klebsiella pneumoniae to rapidly acquire resistance to novel antibiotics is a global concern. Moreover, Klebsiella clonal lineages that successfully combine resistance and hypervirulence have increasingly occurred during the last years. However, the underlying mechanisms of counteracting fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we investigated whether and how an XDR sequence type (ST)307 K. pneumoniae strain developed resistance against the novel drug combination ceftazidime-avibactam (CAZ-AVI) using experimental evolution. In addition, we performed in vitro and in vivo assays, molecular modeling, and bioinformatics to identify resistance-conferring processes and explore the resulting decrease in fitness and virulence. The subsequent amelioration of the initial costs was also addressed. We demonstrate that distinct mutations of the major nonselective porin OmpK36 caused CAZ-AVI resistance that persists even upon following a second experimental evolution without antibiotic selection pressure and that the Klebsiella strain compensates the resulting fitness and virulence costs. Furthermore, the genomic and transcriptomic analyses suggest the envelope stress response regulator rpoE and associated RpoE-regulated genes as drivers of this compensation. This study verifies the crucial role of OmpK36 in CAZ-AVI resistance and shows the rapid adaptation of a bacterial pathogen to compensate fitness- and virulence-associated resistance costs, which possibly contributes to the emergence of successful clonal lineages. IMPORTANCE Extensively drug-resistant Klebsiella pneumoniae causing major outbreaks and severe infections has become a significant challenge for health care systems worldwide. Rapid resistance development against last-resort therapeutics like ceftazidime-avibactam is a significant driver for the accelerated emergence of such pathogens. Therefore, it is crucial to understand what exactly mediates rapid resistance acquisition and how bacterial pathogens counteract accompanying fitness and virulence costs. By combining bioinformatics with in vitro and in vivo phenotypic approaches, this study revealed the critical role of mutations in a particular porin channel in ceftazidime-avibactam resistance development and a major metabolic regulator for ameliorating fitness and virulence costs. These results highlight underlying mechanisms and contribute to the understanding of factors important for the emergence of successful bacterial pathogens.

Klebsiella pneumoniae is an opportunistic pathogen that can colonize the gastrointestinal tract (GIT) of humans. The mechanisms underlying the successful translocation of this pathogen to cause extra-intestinal infections remain unknown, although virulence and antimicrobial resistance traits likely play significant roles in the establishment of infections. We investigated K. pneumoniae strains isolated from GIT colonization (strains Kp_FZcol-1, Kp_FZcol-2 and Kp_FZcro-1) and from a fatal bloodstream infection (strain Kp_HM-1) in a leukemia patient. All strains belonged to ST307, carried a transferable IncF plasmid containing the bla CTX-M-15 gene (pKPN3–307 TypeA-like plasmid) and showed a multidrug-resistance phenotype. Phylogenetic analysis demonstrated that Kp_HM-1 was more closely related to Kp_FZcro-1 than to the other colonizing strains. The Kp_FZcol-2 genome showed 81 % coverage with the Kp_HM-1 246,730 bp plasmid (pKp_HM-1), lacking most of its putative virulence genes. Searching public genomes with similar coverage, we observed the occurrence of this deletion in K. pneumoniae ST307 strains recovered from human colonization and infection in different countries. Our findings suggest that strains lacking the putative virulence genes found in the pKPN3–307 TypeA plasmid are still able to colonize and infect humans, highlighting the need to further investigate the role of these genes for the adaptation of K. pneumoniae ST307 in distinct human body sites. • Distinct but related genotypes of K. pneumoniae ST307 colonized the patient's gut. • K. pneumoniae ST307 likely translocated from the human gut into the bloodstream. • Colonizing and infecting K. pneumoniae ST307 carried distinct pKPN3–307 plasmids. • Deletion of putative virulence genes was observed in a colonizing strain. [ABSTRACT FROM AUTHOR]

Background. The global spread of carbapenemase-producing Enterobacterales has become an epidemiological risk for healthcare systems by limiting available antimicrobial treatments. The COVID-19 pandemic worsened this scenario, prompting the emergence of extremely resistant microorganisms. Methods. Between March 2020 and September 2021, the NRL confirmed 82 clinical Enterobacterales isolates harboring a combination of blaKPC and MBL genes. Molecular typing was analyzed by PFGE and MLST. Modified double-disk synergy (MDDS) tests were used for phenotypic studies. Results. Isolates were submitted from 28 hospitals located in seven provinces and Buenos Aires City, including 77 K. pneumoniae, 2 K. oxytoca, 2 C. freundii, and 1 E. coli. Almost half of K. pneumoniae isolates (n = 38; 49.4%), detected in 15 hospitals, belong to the CC307 clone. CC11 was the second clone, including 29 (37.7%) isolates (22, ST11 and 7, ST258) from five cities and 12 hospitals. Three isolates belonging to CC45 were also detected. The carbapenemase combinations observed were as follows: 55% blaKPC-2 plus blaNDM-5; 32.5% blaKPC-2 plus blaNDM-1; 5% blaKPC-3 plus blaNDM-1; 5% blaKPC-2 plus blaIMP-8; and 2.5% strain with blaKPC-2 plus blaNDM-5 plus blaOXA-163. Aztreonam/avibactam and aztreonam/relebactam were the most active combinations (100% and 91% susceptible, respectively), followed by fosfomycin (89%) and tigecycline (84%). Conclusions. The MDDS tests using ceftazidime-avibactam/EDTA and aztreonam/boronic acid disks improved phenotypic classification as dual producers. The successful high-risk clones of K. pneumoniae, such as hyper-epidemic CC307 and CC11 clones, drove the dissemination of double carbapenemase-producing isolates during the COVID-19 pandemic.

Abstract Background Carbapenem-resistant Klebsiella pneumoniae (CR-KP) is an urgent public health issue in Italy. This pattern of resistance is due mainly to dissemination of carbapenemase genes. Molecular characterization of carbapenem-resistant Klebsiella pneumoniae (CR-KP) strains was performed over a three-year period. In-depth analysis was performed on a subset of emerging CR-KP ST101 and ST307 clones. Methods A prospective study was performed on 691 patients with CR-KP bloodstream infections hospitalized in 19 hospitals located in three large provinces in Southern Italy. Carbapenemase genes were identified via genotyping methods. Multi-locus sequence typing (MLST) and Whole Genome Sequencing (WGS) were carried out on ST101 and ST307 isolates. Results Among the CR-KP isolates, bla KPC was found in 95.6%, bla VIM was found in 3.5%, bla NDM was found in 0.1% and bla OXA-48 was found in 0.1%. The bla KPC-3 variant was identified in all 104 characterized KPC-KP isolates. MLST of 231 representative isolates revealed ST512 in 45.5%, ST101 in 20.3% and ST307 in 18.2% of the isolates. cgMLST of ST307 and ST101 isolates revealed presence of more than one beta-lactam resistance gene. Amino acid substitution in the chromosomal colistin-resistance gene pmrB was found in two ST101 isolates. Conclusions ST512 is widespread in Southern Italy, but ST101 and ST307 are emerging since they were found in a significant proportion of cases. Aggressive infection control measures and a continuous monitoring of these high-risk clones are necessary to avoid rapid spread of CR-KP, especially in hospital settings.

The worldwide spread of Klebsiella pneumoniae carbapenemase–producing Klebsiella pneumoniae (KPC-Kp) isolates was reported to be caused by dissemination of 1 clonal complex (i.e., clonal group [CG] 258, which includes sequence types [STs] 258 and 512). We conducted whole-genome sequencing and epidemiologic analysis of all KPC-Kp isolates in France in 2018 and found that new successful high-risk clones of ST147, ST307, ST231, and ST383 are now the main drivers of blaKPC genes. The blaKPC genes were mostly carried by Tn4401a and Tn4401d structures and a new non–Tn4401 element. Our epidemiologic investigations showed that the emergence of these non-CG258 KPC-Kp isolates in France was linked to dissemination of these clones from Portugal. Thus, KPC-Kp epidemiology has changed in Europe, at least in several non–KPC-endemic countries of western Europe, such as France and Portugal, where CG258 is not the most prevalent clone.

Abstract Background In this study, we evaluated the genetic relatedness of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KPN) isolates from an outbreak in a neonatal intensive care unit (NICU) in August 2017, We implemented an active countermeasure to control this outbreak successfully. Methods The incidence of healthcare-associated ESBL-KPN bacteremia was evaluated before and after initiating enhanced infection control (IC) practices in January 2018. Surveillance cultures were set up and monitored for neonates, medical personnel, and NICU environments. Molecular analyses, including pulse-field gel electrophoresis (PFGE), sequence typing, and ESBL genotyping, were performed for the isolated KPN strains. Results After implementing the enhanced IC procedures, the healthcare-associated bacteremia rate decreased from 6.0 to 0.0 per 1000 patient-days. Samples from neonates (n = 11/15, 73.3%), medical personnel (n = 1/41, 2.4%), and medical devices and the environments (6/181, 3.3%) tested positive for ESBL-KPN in the surveillance cultures in December 2017. Active surveillance cultures revealed that 23 of 72 neonates who were screened (31.9%) were colonized with ESBL-KPN between January and March 2018. All the isolates demonstrated closely related PFGE patterns and were identified as ST307 strain carrying the CTX-M-15 gene. Conclusions Contaminated NICU environments and medical devices, as well as transmission by medical personnel, appeared to be the source of the outbreak of ESBL-KPN infection. We employed an enhanced IC strategy during January–March 2018 and successfully controlled the clonal outbreak of CTX-M-15-positive KPN. ST307 has emerged as an important bacteremia-causing pathogen in the NICU and should be carefully monitored.

β-lactams and fluoroquinolones are extensively used worldwide in the treatment of infections caused by Enterobacterales. In this study, we investigated the prevalence of extended-spectrum β-lactamases (ESBL), their correlation with plasmid-mediated quinolone resistance determinants (PMQR) and clonal distribution among the cefotaxime-resistant K. pneumoniae isolates. In Korea, a total of 429 K. pneumoniae collected in 2015 were studied. Antimicrobial susceptibility test for cefotaxime, ciprofloxacin and levofloxacin was performed by broth microdilution method. By PCR and/or sequencing, mutations in gyrA and parC genes, PMQR genes and ESBL were identified. Multilocus-sequence-type (MLST) was determined for isolates harboring CTX-M-15. Among the 149 K. pneumoniae showing cefotaxime MICs of >1 μg/ml, 142 (95.3%) isolates were ESBL-producers and CTX-M-15 was predominant (99 isolates). Among the 142 ESBL-producers, mutations in gyrA and parC were found in 112 (78.9%) and 93 isolates (65.5%), respectively. PMQR genes were detected in 141 isolates and the non-susceptibility rate to ciprofloxacin and levofloxacin was 95.1% (135/142) and 82.4% (117/142), respectively. The most frequently found PMQR combination was qnrB-aac(6′)-Ib-cr-oqxAB , (58/142, 40.8%). By MLST, four major STs/CC: ST48, ST392, ST307 and CC15 accounted for 67% of the CTX-M-15 producers and the prevalence of qnrB was significantly higher in these four major STs/CC than other groups (P = 0.004). Of note, we found the additive effect of PMQR genes; the more PMQR genes, the higher ciprofloxacin MICs. CTX-M-15 was predominant among the cefotaxime-resistant K. pneumoniae and co-harboring CTX-M-15 and PMQR genes, especially qnrB , seems to contribute the spread of high risk clones. [ABSTRACT FROM AUTHOR]

A novel Klebsiella pneumoniae carbapenemase (KPC) variant, KPC-55, produced by a K. pneumoniae ST307 strain was characterized. K. pneumoniae strain BS407 was recovered from an active surveillance rectal swab of a patient newly admitted to a general hospital in Busan, South Korea. Carbapenemase production was confirmed by the modified Hodge test, and the MICs of β-lactams were determined by the broth microdilution method. The whole genome was sequenced. Cloning and expression of the blaKPC–55 gene in Escherichia coli and MIC determination were performed. The enzyme KPC-55 was used for kinetic assays against β-lactams and compared with the KPC-2 enzyme. The new allele of the blaKPC gene had a T794A alteration compared to the blaKPC–2 gene, resulting in the amino acid substitution Y264N in the middle of the β9-sheet. Compared to the KPC-2-producing strain, the KPC-55-producing strain exhibited a lower level of resistance to most β-lactam drugs tested, however, the KPC-55 enzyme catalyzed aztreonam and meropenem at an increased efficiency compared to the catalytic activity of KPC-2. KPC subtypes could have varied phenotypes due to alterations in amino acid sequences, and such an unexpected resistance phenotype emphasizes the importance of detailed characterizations for the carbapenemase-producing Enterobacterales.

ABSTRACT An emerging multidrug-resistant Klebsiella pneumoniae high-risk clone of sequence type 307 (ST307) has been increasingly reported worldwide. Here, we described the genomic characteristics of an IMP-38-producing ST307 K. pneumoniae strain and investigated the prevalence of blaIMP-38 among carbapenem-resistant Klebsiella pneumoniae isolates from a tertiary care hospital in central China. A total of 14 IMP-38-producing ST307 K. pneumoniae strains were identified from 2013 to 2016, with 13 strains isolated from patients with neonatal sepsis in the neonatal ward. PacBio and Illumina whole-genome sequencing analysis performed on a representative IMP-38-producing K. pneumoniae strain, WCGKP294, showed that it contained a circular chromosome and two plasmids. Carbapenemase gene blaIMP-38 is colocated with blaCTX-M-3 in transposon Tn6382 on an IncHI5 plasmid (pWCGKP294-2). WCGKP294 harbors another IncFIB plasmid, pWCGKP294-1, carrying three copies of tandem-repeated IS26-blaSHV-2A-deoR-ygbJ-ygbK-fucA-IS26 composite transposon elements. Phylogenetic analysis placed WCGKP294 in the global ST307 cluster, distant from the U.S. (Texas) and South Africa clusters. Nevertheless, WCGKP294 does not contain the chromosomal fluoroquinolone resistance-associated mutations and IncFIIK/IncFIBK plasmid-associated blaCTX-M-15 gene that are frequently found in other global ST307 strains. IMPORTANCE We described the genome and resistome characterization of a carbapenem-resistant Klebsiella pneumoniae ST307 strain carrying blaIMP-38 in China. This report highlights that the high-risk ST307 clone continues to acquire different antimicrobial resistance genes, posing significant challenges to clinical practice, and should be closely monitored.

A novel Klebsiella pneumoniae carbapenemase (KPC) variant, KPC-55, produced by a K. pneumoniae ST307 strain was characterized. K. pneumoniae strain BS407 was recovered from an active surveillance rectal swab of a patient newly admitted to a general hospital in Busan, South Korea. Carbapenemase production was confirmed by the modified Hodge test, and the MICs of β-lactams were determined by the broth microdilution method. The whole genome was sequenced. Cloning and expression of the bla KPC–55 gene in Escherichia coli and MIC determination were performed. The enzyme KPC-55 was used for kinetic assays against β-lactams and compared with the KPC-2 enzyme. The new allele of the bla KPC gene had a T794A alteration compared to the bla KPC–2 gene, resulting in the amino acid substitution Y264N in the middle of the β9-sheet. Compared to the KPC-2-producing strain, the KPC-55-producing strain exhibited a lower level of resistance to most β-lactam drugs tested, however, the KPC-55 enzyme catalyzed aztreonam and meropenem at an increased efficiency compared to the catalytic activity of KPC-2. KPC subtypes could have varied phenotypes due to alterations in amino acid sequences, and such an unexpected resistance phenotype emphasizes the importance of detailed characterizations for the carbapenemase-producing Enterobacterales. [ABSTRACT FROM AUTHOR]

Extended-spectrum beta-lactamase (ESBL)- and carbapenemase (CP)-producing Klebsiella pneumoniae isolates are a public health concern at clinical level, mainly in Southern European countries. However, there are scarce data on the role of companion animals in the emergence of resistance to clinically relevant antibiotics. Therefore, our study aimed to determine the presence of K. pneumoniae with relevant beta-lactamases in fecal samples from healthy dogs (kennel and house dogs) and sick dogs in seven different hospitals in Portugal. Fecal samples from 125 healthy dogs and 231 sick dogs (one per animal) were collected during April–August 2017. Samples were screened on MacConkey agar supplemented with meropenem, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) was used for K. pneumoniae identification. Genotypic detection of ESBLs or CPs was carried out by PCR/sequencing. Moreover, the presence of other antimicrobial resistance genes and multilocus sequence typing was tested by PCR/sequencing. K. pneumoniae isolates were obtained from 16 tested samples (4.4%), and 3 of them were ertapenem and/or meropenem intermediate/resistant (all of them imipenem susceptible and negative for CP genes). Fifteen K. pneumoniae isolates were ESBL producers, and they carried the following beta-lactamase genes: blaCTX-M-15+blaSHV-28 (four isolates, in three cases associated with blaTEM-1), blaCTX-M-15+blaSHV-1 (five isolates, associated with TEM-1 in three cases), and blaSHV-28+blaTEM-1 (six isolates). Three ESBL-producing K. pneumoniae isolates of different origins and beta-lactamase genotypes (CTX-M-15+SHV-28, CTX-M-15+SHV-28+TEM-1, or SHV-28+TEM-1) belonged to the lineage ST307, and one isolate was identified as ST15 (CTX-M-15+SHV-1). These findings highlight that dogs are frequent carriers of ESBL-producing K. pneumonia isolates, harboring mostly genes encoding CTX-M-15 or SHV-28, associated in some cases with the high-risk clones ST307 and ST15. [ABSTRACT FROM AUTHOR]

The worldwide spread of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) isolates was reported to be caused by dissemination of 1 clonal complex (i.e., clonal group [CG] 258, which includes sequence types [STs] 258 and 512). We conducted whole-genome sequencing and epidemiologic analysis of all KPC-Kp isolates in France in 2018 and found that new successful high-risk clones of ST147, ST307, ST231, and ST383 are now the main drivers of blaKPC genes. The blaKPC genes were mostly carried by Tn4401a and Tn4401d structures and a new non-Tn4401 element. Our epidemiologic investigations showed that the emergence of these non-CG258 KPC-Kp isolates in France was linked to dissemination of these clones from Portugal. Thus, KPC-Kp epidemiology has changed in Europe, at least in several non-KPC-endemic countries of western Europe, such as France and Portugal, where CG258 is not the most prevalent clone. [ABSTRACT FROM AUTHOR]

Background: Carbapenem-resistant Klebsiella pneumoniae (CR-KP) is an urgent public health issue in Italy. This pattern of resistance is due mainly to dissemination of carbapenemase genes. Molecular characterization of carbapenem-resistant Klebsiella pneumoniae (CR-KP) strains was performed over a three-year period. In-depth analysis was performed on a subset of emerging CR-KP ST101 and ST307 clones. Methods: A prospective study was performed on 691 patients with CR-KP bloodstream infections hospitalized in 19 hospitals located in three large provinces in Southern Italy. Carbapenemase genes were identified via genotyping methods. Multi-locus sequence typing (MLST) and Whole Genome Sequencing (WGS) were carried out on ST101 and ST307 isolates. Results: Among the CR-KP isolates, blaKPC was found in 95.6%, blaVIM was found in 3.5%, blaNDM was found in 0.1% and blaOXA-48 was found in 0.1%. The blaKPC-3 variant was identified in all 104 characterized KPC-KP isolates. MLST of 231 representative isolates revealed ST512 in 45.5%, ST101 in 20.3% and ST307 in 18.2% of the isolates. cgMLST of ST307 and ST101 isolates revealed presence of more than one beta-lactam resistance gene. Amino acid substitution in the chromosomal colistin-resistance gene pmrB was found in two ST101 isolates. Conclusions: ST512 is widespread in Southern Italy, but ST101 and ST307 are emerging since they were found in a significant proportion of cases. Aggressive infection control measures and a continuous monitoring of these high-risk clones are necessary to avoid rapid spread of CR-KP, especially in hospital settings. [ABSTRACT FROM AUTHOR]

Background: In this study, we evaluated the genetic relatedness of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KPN) isolates from an outbreak in a neonatal intensive care unit (NICU) in August 2017, We implemented an active countermeasure to control this outbreak successfully.Methods: The incidence of healthcare-associated ESBL-KPN bacteremia was evaluated before and after initiating enhanced infection control (IC) practices in January 2018. Surveillance cultures were set up and monitored for neonates, medical personnel, and NICU environments. Molecular analyses, including pulse-field gel electrophoresis (PFGE), sequence typing, and ESBL genotyping, were performed for the isolated KPN strains.Results: After implementing the enhanced IC procedures, the healthcare-associated bacteremia rate decreased from 6.0 to 0.0 per 1000 patient-days. Samples from neonates (n = 11/15, 73.3%), medical personnel (n = 1/41, 2.4%), and medical devices and the environments (6/181, 3.3%) tested positive for ESBL-KPN in the surveillance cultures in December 2017. Active surveillance cultures revealed that 23 of 72 neonates who were screened (31.9%) were colonized with ESBL-KPN between January and March 2018. All the isolates demonstrated closely related PFGE patterns and were identified as ST307 strain carrying the CTX-M-15 gene.Conclusions: Contaminated NICU environments and medical devices, as well as transmission by medical personnel, appeared to be the source of the outbreak of ESBL-KPN infection. We employed an enhanced IC strategy during January-March 2018 and successfully controlled the clonal outbreak of CTX-M-15-positive KPN. ST307 has emerged as an important bacteremia-causing pathogen in the NICU and should be carefully monitored. [ABSTRACT FROM AUTHOR]

In the past years, the Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae sequence type 258 (ST258) became an important worldwide spread nosocomial pathogen. Recent evidence shows that the global epidemiology is changing, with the rise of new lineages. In this study we report the microbiological and genomic features of two VIM-1-producing K. pneumoniae isolates belonging to the emerging ST307. Two extensively drug-resistant K. pneumoniae strains, collected between May and June 2017, were confirmed as blaVIM positive by GeneXpert system. The whole-genome sequencing revealed that both KpV_S_1 and KpV_S_2 isolates harbored blaVIM-1 and blaCTX-M-15 genes, besides qnrS1 and qnrB1, strB, mphA, tetR, and tetA determinants. KpV_S_1 and KpV_S_2 isolates belonged to ST661 and ST307, respectively. Both STs have been recently reported as responsible of outbreaks in several European countries. The detection of blaVIM-1 gene in nonpredominant K. pneumoniae clones in a hospital setting should alert on the changing of the epidemiological situation in Italy, usually endemic reservoir of KPC enzyme. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae sequence type (ST) 307 is an emerging global antimicrobial drug-resistant clone. We used whole-genome sequencing and PCR to characterize K. pneumoniae ST307 with oxacillinase (OXA) 181 carbapenemase across several private hospitals in South Africa during 2014-2016. The South Africa ST307 belonged to a different clade (clade VI) with unique genomic characteristics when compared with global ST307 (clades I-V). Bayesian evolution analysis showed that clade VI emerged around March 2013 in Gauteng Province, South Africa, and then evolved during 2014 into 2 distinct lineages. K. pneumoniae ST307 clade VI with OXA-181 disseminated over a 15-month period within 42 hospitals in 23 cities across 6 northeastern provinces, affecting 350 patients. The rapid expansion of ST307 was most likely due to intrahospital, interhospital, intercity, and interprovince movements of patients. This study highlights the importance of molecular surveillance for tracking emerging antimicrobial clones. [ABSTRACT FROM AUTHOR]

Over a 5-month period between the end of June and the beginning of November in 2015, a KPC-producing Enterobacteriaceae outbreak occurred in a general hospital in Busan, South Korea, being associated with a total of 50 clinical isolates from 47 patients. Multilocus sequence typing and pulsed-field gel electrophoresis were carried out for strain typing and whole-genome sequencing was performed to characterize the plasmids. A clonal spread of K. pneumoniae sequence type 307 (ST307) carrying a self-transferable IncX3-type plasmid harboring bla KPC-2 was responsible for the outbreak. Sporadic emergence of K. pneumoniae ST697 carrying an IncFII-type plasmid and a ST11 isolate harboring a small plasmid devoid of any known origin of replication were observed to be associated with bla KPC-3 , but no further dissemination of these strains was identified. The results indicated a healthcare-associated infection associated with a bla KPC -harboring plasmid dissemination and a clonal spread of KPC-producing Enterobacteriaceae . [ABSTRACT FROM AUTHOR]

배경 In healthcare infections, Carbapenem-resistant Klebsiella pneumoniae (CRKP), as multi-drug resistant bacteria, constitutes a serious threat to public health. The occurrence of CRKP infection cases is highly concentrated in Gyeongsang-do. In this study, we investigated the molecular characteristics of CRKP collected from Gyeongsang-do. 방법 Based on identified Klebsiella pneumoniae carbapenemase (KPC) type and the antimicrobial resistance profile (MICs > 16 mg/L), 44 CRKP isolates from 19 hospitals were subjected to XbaI-pulsed field gel electrophoresis (PFGE), and whole-genome sequencing (WGS) by Miseq platform. The molecular genetic analysis was determined by CLC genome workbench and Center for genomic epidemiology services. 결과 All CRKP isolates exhibited multidrug resistance to carbapenems, cephems and fluoroquinolones, harboring a blaKPC-2 gene. Twenty-four unique XbaI-PFGE patterns were identified and tightly clustered on similarity value of ≥ 90 %. In whole genome analysis, isolates were identified as multilocus sequence typing (MLST) sequence type 307, resistance gene profiles (blaKPC-2, blaCTX-M-15, blaOXA-1, blaTEM-1B, aac (6′)-Ib-cr, oqxAB, catB3, qnrB1, dfrA14, and sul2) and plasmid replicons (IncFIB(K), IncFII(K), IncX3) were detected. The genetic relationships showed that the KPC-2-producing CRKP ST307 highly related by whole genome multilocus sequence typing (wgMLST). 결론 The finding suggested that laboratory surveillance is necessary to control the dissemination of CRKP. Therefore, more comprehensive surveillance is required to strengthen monitoring for prevent the spread of high-risk clone. [ABSTRACT FROM AUTHOR]

The carbapenem-resistant Enterobacterales (CRE) strains have been identified by the World Health Organization as critical priority pathogens in research and development of diagnostics, treatments, and vaccines. However, recent molecular information about carbapenem-resistant K. pneumoniae (CRK) epidemiology in Portugal is still scarce. Thus, this study aimed to provide the molecular epidemiology, resistome, and virulome of CRK clinical strains recovered from a tertiary care hospital centre (2019–2021) using polymerase chain reaction (PCR) and the advanced molecular technique whole-genome sequencing (WGS). PCR amplification of carbapenemase genes was performed in 437 carbapenem-resistant K. pneumoniae strains. The most frequent carbapenemases were: KPC-3 (42%), followed by OXA-181 (20%), GES-5 (0.2%), and NDM-1 (0.2%). Additionally, 10 strains (2%) coproduced KPC-3 and OXA-181, and 1 strain coproduced KPC-3 and OXA-48 (0.2%). The genomic population structure of 68 strains characterized by WGS demonstrated the ongoing dissemination of four main high-risk clones: ST13, ST17, ST147, and ST307, while no clones belonging to the European predominant clonal groups (CG15 and CG258) were found. Moreover, we describe one K. pneumoniae ST39-KL62 that coproduced the NDM-1 carbapenemase and the extended-spectrum beta-lactamase CTX-M-15, and one K. pneumoniae ST29-KL54 GES-5 and BEL-1 coproducer. Furthermore, a high prevalence of iron siderophores were present in all CRK strains, with several strains presenting both colibactin and the hypermucoviscosity phenotype. Thus, the data presented here highlight an uncommon molecular epidemiology pattern in Portugal when compared with most European countries, further supporting the emergence and dissemination of nonclonal group 258 hypervirulent multidrug high-risk clones and the need to promote in-depth hospital molecular surveillance studies. [ABSTRACT FROM AUTHOR]

During active surveillance at the Mediterranean Institute for Transplantation and Advanced Specialized Therapies (ISMETT, Palermo, Italy) with the CARBA screening medium, five pairs of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae and Escherichia coli strains were isolated in each of five colonized patients. In each patient, lateral gene transfer was demonstrated by comparing K. pneumoniae and E. coli strains, both possessing KPC-3, Tn 4401a and pKpQIL-IT elements. The isolates were found to be multiclonal by multilocus sequence typing (sequence type (ST) 512 related to ST258, and ST307 belonging to a clonal complex different from the habitual sequence clone ST258 isolated in Italy) and pulsed-field gel electrophoresis. The results of our study highlight the easy transfer of KPC among Enterobacteriaceae colonizing the human intestine, and the active and careful surveillance required to identify and prevent the spread of these multidrug-resistant microorganisms. [ABSTRACT FROM AUTHOR]