Pathogenomics analysis of high-risk clone ST147 multidrug-resistant Klebsiella pneumoniae isolated from a patient in Egypt.

BACKGROUND: The emergence of multi-drug-resistant Klebsiella pneumoniae (MDR-KP) represents a serious clinical health concern. Antibiotic resistance and virulence interactions play a significant role in the pathogenesis of K. pneumoniae infections. Therefore, tracking the clinical resistome and virulome through monitoring antibiotic resistance genes (ARG) and virulence factors in the bacterial genome using computational analysis tools is critical for predicting the next epidemic. METHODS: In the current study, one hundred extended spectrum ß-lactamase (ESBL)-producing clinical isolates were collected from Mansoura University Hospital, Egypt, in a six-month period from January to June 2022. One isolate was selected due to the high resistance phenotype, and the genetic features of MDR-KP recovered from hospitalized patient were investigated. Otherwise, the susceptibility to 25 antimicrobials was determined using the DL Antimicrobial Susceptibility Testing (AST) system. Whole genome sequencing (WGS) using Illumina NovaSeq 6000 was employed to provide genomic insights into K. pneumoniae WSF99 clinical isolate. RESULTS: The isolate K. pneumoniae WSF99 was phenotypically resistant to the antibiotics under investigation via antibiotic susceptibility testing. WGS analysis revealed that WSF99 total genome length was 5.7 Mb with an estimated 5,718 protein-coding genes and a G + C content of 56.98 mol%. Additionally, the allelic profile of the WSF99 isolate was allocated to the high-risk clone ST147. Furthermore, diverse antibiotic resistance genes were determined in the genome that explain the high-level resistance phenotypes. Several ß-lactamase genes, including blaCTX-M-15, blaTEM-1, blaTEM-12, blaSHV-11, blaSHV-67, and blaOXA-9, were detected in the WSF99 isolate. Moreover, a single carbapenemase gene, blaNDM-5, was predicted in the genome, positioned within a mobile cassette. In addition, other resistance genes were predicted in the genome including, aac(6')-Ib, aph(3')-VI, sul1, sul2, fosA, aadA, arr-2, qnrS1, tetA and tetC. Four plasmid replicons CoIRNAI, IncFIB(K), IncFIB(pQil), and IncR were predicted in the genome. The draft genome analysis revealed the occurrence of genetic mobile elements positioned around the ARGs, suggesting the ease of dissemination via horizontal gene transfer. CONCLUSIONS: This study reports a comprehensive pathogenomic analysis of MDR-KP isolated from a hospitalized patient. These findings could be relevant for future studies investigating the diversity of antimicrobial resistance and virulence in Egypt.

Phenotypic and molecular characterization of multi-drug resistant Klebsiella spp. isolates recovered from clinical settings.

Klebsiella pneumoniae is a Gram-negative bacterium that colonizes the gastrointestinal tract and nasopharynx with many being linked to nosocomial infections. Extended-spectrum ß-lactamases (ESBL)-producing and carbapenem-resistant K. pneumoniae is recognized by the World Health Organization (WHO) as a critical public health concern. In this study, whole-genome sequencing (WGS) - based analysis was performed to understand the molecular epidemiology of multi-drug resistant Klebsiella spp. clinical isolates. Genome comparison, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and whole-genome-SNP-based phylogenetic analysis (wg-SNP) were used for in-depth molecular characterization. in silico typing was used to determine the resistance genes, virulence factors, Inc. groups, and capsular types. All except one isolate were non-susceptible to meropenem and 89% were non-susceptible to ertapenem and imipenem. blaNDM, blaOXA-48, and blaKPC were the detected carbapenemases with blaNDM-1 found in half of the sequenced genomes. Resistance to colistin was detected in one isolate and was linked to several genetic alterations in crrB, pmrB, and pmrC genes. The most common plasmid type was IncFIB followed by IncR, and the Type 3 fimbriae, encoded by the mrkABCDF operon, was conserved among all isolates. The most common sequence- (ST) and K-type detected were ST147 and K64. The prevelance and the genomic relatedness of ST147 isolates, as shown by the data from SNP-based phylogenetic analysis, PFGE, and genomic clustering, may be an outbreak marker. However, this can only be validated through a more comprehensive study encompassing a wider sampling scheme and over an extended timeframe.

One-year surveillance for hypervirulent Klebsiella pneumoniae detected carbapenem-resistant superbugs.

Hypervirulent Klebsiella pneumoniae (hvKp) can cause infections in clinically healthy people, such as young and immunocompetent patients. Genes involved in the capsule synthesis or those encoding the siderophores have been adopted as predictors of hvKp. Certain sequence types, such as ST23 and ST86, have been associated with hvKp strains, too. The aim of this study was to investigate the presence of hvKp among 354 K. pneumoniae strains isolated from clinical samples of patients admitted to an Italian 900-bed hospital between 21 May 2021 and April 2022. All the isolates were screened by PCR for the amplification of virulence loci. Whole genome sequencing was performed in strains tested positive for at least one target gene. Thirteen out of 354 (3.7%) were hvKp. Five were wild type and belonged to the hypervirulent clones ST23, ST86, ST5, and ST375 and to the new clone ST6310. Six strains carried the blaKPC gene: three belonged to ST101, two to ST512, and one to ST395. Two isolates were ST147 and carried the blaNDM gene. Although hvKp isolation is not frequent, their presence should be systematically investigated to avoid the spreading of both virulent strains and strains with combined increase in virulence and resistance to antibiotics. PCR-based protocols are essential for surveillance of these strains, which do not always show a recognizable phenotype. Moreover, hvKp strains were isolated also from patients without history of recent foreign travels, indicating an increased spreading of these strains as well as an underestimated of their circulation so far.IMPORTANCEKlebsiella pneumoniae is a healthcare-associated pathogen frequently resistant to antibiotics. Hypervirulent strains of pneumoniae (hvKp) can spread from the primary site of infection to multiple sites causing life-threatening infections also in young otherwise healthy individuals. This study described the isolation of 13 isolates of K. pneumoniae with increased virulence in a large tertiary hospital over a 1-year period. Among them, eight strains were multidrug resistant and hypervirulent. Although these hypervirulent strains are still rare in Italy, their presence is particularly concerning since they can cause difficult-to-treat life-threatening infections. Moreover, not all the hypervirulent isolates were positive by the string test, so hvKp isolates were not always phenotypically detectable. Molecular biology techniques such as PCR amplification and next generation sequencing are therefore necessary for the detection of hvKp isolates, and surveillance programs exploiting molecular techniques are highly desirable.

Genomic surveillance of carbapenem-resistant Klebsiella pneumoniae reveals a prolonged outbreak of extensively drug-resistant ST147 NDM-1 during the COVID-19 pandemic in the Apulia region (Southern Italy).

OBJECTIVES: The recent worldwide spread of New Delhi metallo-beta-lactamase-producing Klebsiella pneumoniae (NDM-KP) in health-care settings remains a concern. The aim of the study was to describe an outbreak of extensively drug-resistant ST147 NDM-1-KP in the Apulia region of Southern Italy that occurred between 2020 and 2022 through genomic surveillance of carbapenem-resistant Enterobacterales. METHODS: A total of 459 carbapenem-resistant KP isolates collected from patients hospitalised with bloodstream infections were tested using a commercial multiplex real-time polymerase chain reaction to identify carbapenemase genes. A subset of 27 isolates was subjected to whole-genome sequencing. Core-genome multilocus sequence typing was performed by analysing a panel of 4884 genes. RESULTS: Molecular testing revealed that 104 (22.6%) isolates carried the carbapenemase NDM gene. Phylogenetic analysis of the 27 isolates subjected to whole-genome sequencing revealed high genetic relatedness among strains. All isolates were resistant to all first-line antibiotics. Virulome analysis identified the ybt locus, the two well-recognised virulence factors iucABCDiutA and rmpA, and the genes encoding the type 3 pilus virulence factor. Plasmids IncFIB(pkPHS1), IncFIB(pNDM-Mar), IncFIB(pQil), IncHI1B(pNDM-MAR), IncR, and Col(pHAD28) were identified in all isolates. Moreover, further analysis identified the IncFIB-type plasmid carrying the NDM-1 genes. CONCLUSION: The increasing circulation of extensively drug-resistant NDM-1 ST147 KP strains in Southern Italy in recent years is worrisome, because these clones pose a real risk, particularly in hospital settings. Genomic surveillance is a crucial tool for early identification of emerging threats such as the spread of high-risk pathogens. Rapid infection control measures and antimicrobial stewardship are key to preventing further spread of hypervirulent KP strains.

Various arrangements of mobile genetic elements among CC147 subpopulations of Klebsiella pneumoniae harboring blaNDM-1: a comparative genomic analysis of carbapenem resistant strains.

BACKGROUND: Certain clonal complexes (CCs) of Klebsiella pneumoniae such as CC147 (ST147 and ST392) are major drivers of blaNDM dissemination across the world. ST147 has repeatedly reported from our geographical region, but its population dynamics and evolutionary trajectories need to be further studied. METHODS: Comparative genomic analysis of 51 carbapenem-nonsusceptible strains as well as three hypervirulent K. pneumoniae (hvKp) recovered during 16-months of surveillance was performed using various bioinformatics tools. We investigated the genetic proximity of our ST147 strains with publicly available corresponding genomes deposited globally and from neighbor countries in our geographic region. RESULTS: While IncL/M plasmid harboring blaOXA-48 was distributed among divergent clones, blaNDM-1 was circulated by twenty of the 25 CC147 dominant clone and were mostly recovered from the ICU. The NDM-1 core structure was bracketed by a single isoform of mobile genetic elements (MGEs) [ΔISKpn26-NDM-TnAs3-ΔIS3000-Tn5403] and was located on Col440I plasmid in 68.7% of ST392. However, various arrangements of MGEs including MITESen1/MITESen1 composite transposon or combination of MITESen1/ISSen4/IS903B/IS5/ISEhe3 on IncFIb (pB171) were identified in ST147. It seems that ST392 circulated blaNDM-1 in 2018 before being gradually replaced by ST147 from the middle to the end of sample collection in 2019. ST147 strains possessed the highest number of resistance markers and showed high genetic similarity with four public genomes that harbored blaNDM-1 on the same replicon type. Mainly, there was a convergence between clusters and isolated neighboring countries in the minimum-spanning tree. A conserved arrangement of resistance markers/MGEs was linked to methyltransferase armA which was embedded in class 1 integron in 8 isolates of ST147/ST48 high-risk clones. CONCLUSION: Our findings highlight the dynamic nature of blaNDM-1 transmission among K. pneumoniae in Iran that occurs both clonally and horizontally via various combinations of MGEs. This is the first analysis of Iranian ST147/NDM + clone in the global context.

Dissemination of the high-risk cloneST147 carbapenem-resistant klebsiella pneumoniae from a local tertiary care hospital in the Republic of Korea.

BACKGROUND: The emergence of carbapenem-resistant Enterobacterales (CRE) infections is rapidly increasing and represents a serious public threat. In 2020, a total of 16,883 carbapenemase-producing Enterobacterales strains were collected; among these isolates, 21 strains were repeatedly isolated in a local tertiary care hospital. METHODS: Antimicrobial susceptibility testing was performed using the broth microdilution method. All 21 strains of CRKP were analyzed by PFGE after XbaI digestion. The 21 CRKP strains were sequenced on the Illumina Miseq and Oxford Nanopore GridION platforms. RESULTS: These 21 CRKP isolates showed an identical antimicrobial resistance profile, including resistance to ampicillin, carbapenems, cephems, chloramphenicol, fluoroquinolone, macrolides and trimethoprim/sulfamethoxazole. Based on whole-genome analysis, these 21 CRKP isolates shared a common genetic structure (ISAba125-IS630-blaNDM-1-bleMBL) and harbored additional resistance determinants (blaOXA-1, blaCTX-M-15, blaSHV-11, blaSHV-67, aac(6')-Ib-cr, qnrS1, OqxA, OqxB, catB3, mph(A), sul1, and dfrA12) and mutations in the quinolone resistance-determining regions of gyrA (S83I) and parC (S80I). These isolates belonged to the ST147 and KL64 capsular types, which were carried on IncFIB replicon plasmids. The 21 CRKP strains collected from one hospital were divided into five PFGE patterns, and they were closely related with a minimum similarity value of 95.2%. These isolates were found to be highly related based on the presence of between 2 and 27 SNPs. CONCLUSIONS: These findings indicate that NDM-1-producing K. pneumoniae ST147 may have been introduced via a common source, implying nosocomial transmission; furthermore, continuous monitoring is necessary to prevent endemic transmission.

Continuous infusion of meropenem-vaborbactam for a KPC-3-producing Klebsiella pneumoniae bloodstream infection in a critically ill patient with augmented renal clearance.

PURPOSE: To demonstrate the feasibility of continuous infusion of meropenem-vaborbactam to optimize the treatment of carbapenem-resistant Enterobacterales. METHODS: Report of a case of a Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae bloodstream infection comfirmed by whole genome sequencing and therapeutic drug monitoring (TDM) of meropenem. RESULTS: A patient with augmented renal clearance (ARC) went into septic shock caused by an ST11 KPC-3-producing K. pneumoniae bloodstream infection that was successfully treated with a continuous infusion of meropenem-vaborbactam at a dosage of 1 g/1 g q4h as a 4-h infusion. TDM confirmed sustained concentrations of meropenem ranging from 8 to 16 mg/L throughout the dosing interval. CONCLUSION: Continuous infusion of meropenem-vaborbactam was feasible. It could be appropriate for optimizing the management of critically ill patients with ARC, as it resulted in antibiotic concentrations above the minimum inhibitory concentration for susceptible carbapenem-resistant Enterobacterales (up to 8 mg/L) throughout the dosing interval.

Unravelling the Evolutionary Dynamics of High-Risk Klebsiella pneumoniae ST147 Clones: Insights from Comparative Pangenome Analysis.

BACKGROUND: The high prevalence and rapid emergence of antibiotic resistance in high-risk Klebsiella pneumoniae (KP) ST147 clones is a global health concern and warrants molecular surveillance. METHODS: A pangenome analysis was performed using publicly available ST147 complete genomes. The characteristics and evolutionary relationships among ST147 members were investigated through a Bayesian phylogenetic analysis. RESULTS: The large number of accessory genes in the pangenome indicates genome plasticity and openness. Seventy-two antibiotic resistance genes were found to be linked with antibiotic inactivation, efflux, and target alteration. The exclusive detection of the blaOXA-232 gene within the ColKp3 plasmid of KP_SDL79 suggests its acquisition through horizontal gene transfer. The association of seventy-six virulence genes with the acrAB efflux pump, T6SS system and type I secretion system describes its pathogenicity. The presence of Tn6170, a putative Tn7-like transposon in KP_SDL79 with an insertion at the flanking region of the tnsB gene, establishes its transmission ability. The Bayesian phylogenetic analysis estimates ST147's initial divergence in 1951 and the most recent common ancestor for the entire KP population in 1621. CONCLUSIONS: Present study highlights the genetic diversity and evolutionary dynamics of high-risk clones of K. pneumoniae. Further inter-clonal diversity studies will help us understand its outbreak more precisely and pave the way for therapeutic interventions.

Hospital sanitary facilities on wards with high antibiotic exposure play an important role in maintaining a reservoir of resistant pathogens, even over many years.

BACKGROUND: Hospitals with their high antimicrobial selection pressure represent the presumably most important reservoir of multidrug-resistant human pathogens. Antibiotics administered in the course of treatment are excreted and discharged into the wastewater system. Not only in patients, but also in the sewers, antimicrobial substances exert selection pressure on existing bacteria and promote the emergence and dissemination of multidrug-resistant clones. In previous studies, two main clusters were identified in all sections of the hospital wastewater network that was investigated, one K. pneumoniae ST147 cluster encoding NDM- and OXA-48 carbapenemases and one VIM-encoding P. aeruginosa ST823 cluster. In the current study, we investigated if NDM- and OXA-48-encoding K. pneumoniae and VIM-encoding P. aeruginosa isolates recovered between 2014 and 2021 from oncological patients belonged to those same clusters. METHODS: The 32 isolates were re-cultured, whole-genome sequenced, phenotypically tested for their antimicrobial susceptibility, and analyzed for clonality and resistance genes in silico. RESULTS: Among these strains, 25 belonged to the two clusters that had been predominant in the wastewater, while two others belonged to a sequence-type less prominently detected in the drains of the patient rooms. CONCLUSION: Patients constantly exposed to antibiotics can, in interaction with their persistently antibiotic-exposed sanitary facilities, form a niche that might be supportive for the emergence, the development, the dissemination, and the maintenance of certain nosocomial pathogen populations in the hospital, due to antibiotic-induced selection pressure. Technical and infection control solutions might help preventing transmission of microorganisms from the wastewater system to the patient and vice versa, particularly concerning the shower and toilet drainage. However, a major driving force might also be antibiotic induced selection pressure and parallel antimicrobial stewardship efforts could be essential.

Emergence of multidrug-resistant ST11 blaKPC-2 producing Klebsiella pneumoniae coharboring blaCTX-M and blaSHV in Pakistan.

INTRODUCTION: Carbapenemases are primarily responsible for the intensified spread of multidrug-resistant (MDR) K. pneumoniae by virtue of antibiotics overuse. Therefore, frequent investigation of high-risk clones especially from developing world is crucial to curtail global spread. METHODOLOGY: In this observational study, 107 K. pneumoniae were retrieved and confirmed genotypically from April 2018 to March 2020 from tertiary care hospitals in Lahore, Pakistan. Carbapenemases and extended-spectrum ß-lactamases were verified by Polymerase Chain Reaction and Sanger sequencing. Multilocus sequence typing and plasmid replicon typing were used to assign clonal lineages and plasmid replicons. RESULTS: Among the K. pneumoniae, 72.9% (78/107) strains were carbapenem resistant (CR) with 65.4% (51/78) exhibiting carbapenemase producing phenotype. Among CR K. pneumoniae 38.5% (30/78) strains exhibited the following carbapenemase genotypes: blaNDM-1 (26.7%, 8/30), blaOXA-48 (26.7%, 8/30), blaKPC-2 (20.0%, 6/30), blaVIM (10.0%, 3/30), blaNDM-1/blaOXA-48 (10.0%, 3/30), blaOXA-48/blaVIM (3.3%, 1/30) and blaOXA-48/blaIMP (3.3%, 1/30). Tigecycline and polymyxin-B retained susceptible profile. ß-lactam drugs showed intermediate to high resistance. The occurrence of CR K. pneumoniae infections was significantly associated with wound (39.7%, p = 0.0007), pus (38.5%, p = 0.009), general surgery (34.6%, p = 0.002) and intensive-care unit (26.9%, p = 0.04). blaKPC-2 producing K. pneumoniae coharboring blaCTX-M/blaSHV (66.7%) and blaCTX-M (33.3%) exhibited sequence type (ST) 258 (n = 4) and ST11 (n = 2) sequence types with IncFII, IncN, IncFIIA, IncL/M and IncFIIK plasmids. CONCLUSIONS: This is the first report describing the emergence of MDR blaKPC-2 producing K. pneumoniae ST11 coharboring blaCTX-M and blaSHV in Pakistan.

Fusion plasmid enhanced the endemic extensively drug resistant Klebsiella pneumoniae clone ST147 harbored blaOXA-48 to acquire the hypervirulence and cause fatal infection.

BACKGROUND: Klebsiella Pneumoniae (Kp) sequence type (ST) 147 has emerged globally and spread rapidly, particularly the extensively drug resistant (XDR) isolates. However, the infections caused by this subtype is rare reported in China for now. The clinical, microbiological and genomic characteristics are unclear. METHODS: A systemic retrospective study was conducted in a Chinese tertiary hospital. Clinical information of the infection cases was collected, and whole-genome sequencing and phenotypic experiments were performed on the ST147 isolates. The resistance and virulence genes were identified, and the plasmids harboring these genes were further studied. RESULTS: Six ST147 isolates from six patients among 720 available clincial Kp isolates were detected. Notably, two isolates, PEKP4035 and PEKP4265, represented both XDR and hypervirulence by acquiring blaOXA-48, blaCTX-M-15 and key virulence genes, iucA + rmpA2, representing no fitness cost and resulting fatal infection. Four of the six ST147 isolates presented with more nucleotide differences, whereas the PEKP4035 and PEKP4265 both isolated from the intensive care unit possessed 20 single nucleotide polymorphisms among one year, indicating the prolonged survive and transmission. Interestingly, the two isolates harbored the same fused plasmid composed of sul2 and iucA + rmpA2, which might be generated by recombination of a plasmid like KpvST101_OXA-48 with the pLVPK plasmid via IS26. Besides, two ~ 70 kb plasmids conferring multiple-drug resistance were also identified among the two isolates, which presented resistance genes including blaOXA-48, blaCTX-M-16, strA and strB. Interestingly, we reported that blaCTX-M-15, a common resistance gene within ST147, has successfully transferred into the chromosome by ISEcp1. CONCLUSIONS: XDR hypervirulent ST147 Kp is emerging, suggesting enhanced surveillance is essential.

Clinical and Microbiological Risk Factors for 30-Day Mortality of Bloodstream Infections Caused by OXA-48-Producing Klebsiella pneumoniae.

Bloodstream infections (BSI) caused by carbapenem-resistant Klebsiella pneumoniae are associated with high morbidity and mortality, and the therapy options available for their treatment are frequently scarce. The aim of this study was to analyze risk factors for 30-day mortality in patients with BSI caused by OXA-48-producing K. pneumoniae. The clinical and treatment features of the patients, who attended a single hospital over a five-year period, were retrospectively reviewed. The microbiological features, including the sequence types (ST) and the somatic (O) and capsular (K) antigens, as well as their resistance properties, comprising phenotypes and genetic background, were also considered. To identify the risk factors for 30-day mortality, uni- and multivariate statistical analyses were performed. The univariate analysis revealed statistically significant correlations for age, male gender, lower respiratory system infection, infection by ST147 isolates, and infection by isolates expressing the K64 antigen. The multivariate analysis, applied to variables yielding p-values close to or lower than 0.05 in the univariate analysis, confirmed gender, lower respiratory system infection, and infection with ST147 isolates, but not age or infection with K64 isolates, as risk factors for 30-day mortality. Moreover, the multivariate analysis showed that patients suffering from hematological malignancies or having been treated with inappropriate therapy, both having p-values slightly higher than 0.05 in the univariate analysis, exhibited significantly poorer outcomes in the multivariant analysis. The association of the ST147 clone with an increased risk of mortality is a novel finding that deserves further attention. Studies like the one presented here can certainly benefit the management of patients with nosocomial BSI caused by carbapenemase-producing K. pneumoniae.

Klebsiella pneumoniae ST147: and then there were three carbapenemases.

Gram-negative bacteria containing three different carbapenemases are extremely rare. Klebsiella pneumoniae (N22-925) with KPC-2, NDM-1, and OXA-48 was obtained from a Canadian patient with recent hospitalization in Romania. Short and long read whole genome sequencing showed that the blaKPC-2 was situated on a 214 kb IncFIB(K)/IncFII(K) plasmid, the blaNDM-1 on a 104 kb IncFIB (pQil)/IncFII(K) plasmid, and the blaOXA-48 on a 64 kb IncL plasmid. These plasmids were conjugated to Escherichia coli J53. N22-925 belonged to a unique ST147 cluster that is likely endemic in Romania. This case emphasizes the need for rapid carbapenemase screening in patients from endemic regions. We described the first complete genome sequence of a K. pneumoniae isolate with three different carbapenemases, providing a reference for future studies on this rarely reported occurrence.

Whole-Genome Sequencing Enables Molecular Characterization of Non-Clonal Group 258 High-Risk Clones (ST13, ST17, ST147 and ST307) among Carbapenem-Resistant Klebsiella pneumoniae from a Tertiary University Hospital Centre in Portugal.

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.

Characteristics in the whole-genome sequence of Klebsiella pneumoniae ST147 from Turkey.

The study aimed to analyze antibiotic resistance determinants in a carbapenem-resistant Klebsiella pneumoniae by whole-genome sequencing (WGS). K. pneumoniae was isolated from a urine sample and it was characterized by 16S rDNA sequencing in Turkey. This strain was named as Kpn Rize-53-TR. Antimicrobial susceptibility testing was performed for seventeen antibiotics by VITEK-2 and the result was confirmed by MIC. The whole genome of isolate was sequenced by Illumina and was analysed by bioinformatic tools for MLST, replicon types, and antimicrobial resistance genes. The whole genome data was submitted to NCBI. The isolate was found to be resistant to all tested ß-lactam antibiotics and the highest MIC values were found for piperacillin, piperacillin/tazobactam (≥128). No resistance to colistin and moderate susceptibility to amikacin and tetracycline was observed. The isolate carried 12 resistance genes belonging to 10 resistance classes; ere(A), fosA, oqxB, cmlA1, aac(a)-IIa, bla KPC-2, bla TEM-1A, bla SHV-67, bla CTX-M-15, bla OXA-1-2-9. Mutations were detected in gyrA (83Y) and parC (80I) genes. Clonal subtype of the isolate was ST147, and it had wzi420 and wzc38 alleles. Its serotype was O3/O3a. The bla KPC-2 was firstly found in both ST147 clonal group in Turkey and in serotype O3/O3a in the world. By plasmid replicon typing, five plasmids IncFII(K), Col(BS512), IncR, IncFIA(HI1) and IncFIB(pQil) were determined in Kpn Rize-53-TR and bla KPC-2 was located on IncFII(K) plasmid. The presence of bla KPC-2 on the plasmid with other resistance genes accelerates its own spread together with other resistance genes.

Epidemiological features of nosocomial Klebsiella pneumoniae: virulence and resistance determinants.

Aim: The authors aimed to examine antibiotic resistance genes and representative virulence determinants among 100 Klebsiella pneumoniae isolates with an emphasis on capsular serotypes and clonality of some of the isolates. Methods: PCR amplification of (rmpA, rmpA2, iutA, iroN and IncHI1B plasmid) and (NDM, OXA-48, KPC, CTX-M-15, VIM, IMP, SPM) was conducted. Wzi sequencing and multilocus sequence typing (MLST) were performed. Results: K2 was the only detected serotype in the authors' collection. RMPA2 was the most common capsule-associated virulence gene detected. All studied isolates harbored OXA-48-like (100%) and NDM (43%) (n = 43). ST147 was the most common sequence type. Conclusion: This work provides insight into the evolution of the coexistence of virulence and resistance genes in a tertiary healthcare setting in Cairo, Egypt.

Dissemination of carbapenem resistant bacteria from hospital wastewater into the environment.

Infections with antibiotic resistant pathogens threaten lives and cause substantial costs. For effective interventions, knowledge of the transmission paths of resistant bacteria to humans is essential. In this study, carbapenem resistant bacteria were isolated from the wastewater of a maximum care hospital during a period of two years, starting in the patient rooms and following the sewer system to the effluent of the wastewater treatment plant (WWTP). The bacteria belonged to six different species and 44 different sequence types (STs). The most frequent STs, ST147 K. pneumoniae (blaNDM/blaOXA-48) and ST235 P. aeruginosa (blaVIM) strains, were present at nearly all sampling sites from the hospital to the WWTP effluent. After core genome multi-locus sequence typing (cgMLST), all ST147 K. pneumoniae strains presented a single epidemiological cluster. In contrast, ST235 P. aeruginosa formed five cgMLST clusters and the largest cluster contained the strain from the WWTP effluent, indicating without doubt, a direct dissemination of both high-risk clones into the environment. Thus, there are - at least two - possible transmission pathways to humans, (i) within the hospital by contact with the drains of the sanitary installations and (ii) by recreational or irrigation use of surface waters that have received WWTP effluent. In conclusion, remediation measures must be installed at both ends of the wastewater system, targeting the drains of the hospital as well as at the effluent of the WWTP.

Whole-Genome Analysis of a High-Risk Clone of Klebsiella pneumoniae ST147 Carrying Both mcr-1 and blaNDM-1 Genes in Peru.

The increasing prevalence and dissemination of carbapenemase-producing Enterobacterales represent a serious concern for public health. We studied the genetic features of a multidrug-resistant isolate of high-risk clone ST147 Klebsiella pneumoniae coharboring mcr-1 and blaNDM-1 recovered from a human clinical urine sample in 2017 in Peru. Whole-genome sequencing and conjugation assays identified mcr-1 and blaNDM-1 genes on two different conjugative plasmids, which belong to IncI2 and IncFIB/HI1B incompatibility groups, respectively. The presence of blaCTX-M-15 (in the studied isolate, located on the chromosome) and mutations in GyrA S83I and ParC S80I were detected, as expected for ST147. In addition, other ß-lactamases (blaTEM-26 and blaOXA-1) and PMQR (qnrE2 and aac(6')-Ib-cr) among several resistance determinants were identified. The coexistence not previously described of these genes in the same high-risk clone is a cause for serious concern that supports the need for implementation of genomic surveillance studies.

Multidrug-Resistant Klebsiella pneumoniae Clones from Wild Chimpanzees and Termites in Senegal.

Antibiotic resistance genes exist naturally in various environments far from human usage. Here, we investigated multidrug-resistant Klebsiella pneumoniae, a common pathogen of chimpanzees and humans. We screened antibiotic-resistant K. pneumoniae from 48 chimpanzee stools and 38 termite mounds (n = 415 samples) collected in protected areas in Senegal. The microsatellite method was used to identify chimpanzee individuals (n = 13). Whole-genome sequencing was performed on K. pneumoniae complex isolates to identify antibiotic-resistant genes and characterize clones. We found a high prevalence of carbapenem-resistant K. pneumoniae among chimpanzee isolates (18/48 samples from 7/13 individuals) and ceftriaxone resistance among both chimpanzee individuals (19/48) and termite mounds (7/415 termites and 3/38 termite mounds). The blaOXA-48 and the blaKPC-2 genes were carried by international pOXA-48 and pKPC-2 plasmids, respectively. The ESBL plasmid carried blaCTX-M-15, blaTEM-1B, and blaOXA-1 genes. Genome sequencing of 56 isolates identified two major clones associated with hospital-acquired infections of K. pneumoniae (ST307 and ST147) in chimpanzees and termites, suggesting circulation of strains between the two species, as chimpanzees feed on termites. The source and selection pressure of these clones in this environment need to be explored.

Regional Spread of blaNDM-1-Containing Klebsiella pneumoniae ST147 in Post-Acute Care Facilities.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) harboring blaKPC have been endemic in Chicago-area healthcare networks for more than a decade. During 2016-2019, a series of regional point-prevalence surveys identified increasing prevalence of blaNDM-containing CRE in multiple long-term acute care hospitals (LTACHs) and ventilator-capable skilled nursing facilities (vSNFs). We performed a genomic epidemiology investigation of blaNDM-producing CRE to understand their regional emergence and spread. METHODS: We performed whole-genome sequencing on New Delhi metallo-beta-lactamase (NDM)+ CRE isolates from 4 point-prevalence surveys across 35 facilities (LTACHs, vSNFs, and acute care hospital medical intensive care units) in the Chicago area and investigated the genomic relatedness and transmission dynamics of these isolates over time. RESULTS: Genomic analyses revealed that the rise of NDM+ CRE was due to the clonal dissemination of an sequence type (ST) 147 Klebsiella pneumoniae strain harboring blaNDM-1 on an IncF plasmid. Dated phylogenetic reconstructions indicated that ST147 was introduced into the region around 2013 and likely acquired NDM around 2015. Analyzing the relatedness of strains within and between facilities supported initial increases in prevalence due to intrafacility transmission in certain vSNFs, with evidence of subsequent interfacility spread among LTACHs and vSNFs connected by patient transfer. CONCLUSIONS: We identified a regional outbreak of blaNDM-1 ST147 that began in and disseminated across Chicago area post-acute care facilities. Our findings highlight the importance of performing genomic surveillance at post-acute care facilities to identify emerging threats.