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Background: In this study, the mechanisms implicated in delafloxacin resistance in Klebsiella pneumoniae strains were investigated. Delafloxacin is a novel, broad-spectrum fluoroquinolone that has been approved for clinical application. Methods: In our study, 43 K. pneumoniae strains were assessed, antimicrobial susceptibility testing was performed via the broth microdilution method, and the minimum inhibitory concentration (MIC) values for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, and imipenem were determined. Four delafloxacin-resistant K. pneumoniae strains were selected for whole-genome sequencing (WGS). Results: The MIC50 values for the 43 K. pneumoniae strains were as follows: ciprofloxacin 0.5 mg/L, levofloxacin 0.25 mg/L, moxifloxacin 0.5 mg/L, and delafloxacin 0.25 mg/L. All four selected delafloxacin-resistant K. pneumoniae strains showed extended-spectrum beta-lactamase production, and one strain exhibited carbapenem resistance. WGS enabled us to determine the sequence types (STs) of these strains, namely, ST307 (two strains), ST377, and ST147. Multiple mutations in quinolone-resistance-determining regions (QRDRs) were detected in all the delafloxacin-resistant K. pneumoniae strains; specifically, gyrA Ser83Ile and parC Ser80Ile were uniformly present in the strains of ST307 and ST147. However, in the ST377 strain, gyrA Ser83Tyr, Asp87Ala, and parC Ser80Ile, amino acid substitutions were detected. We also identified OqxAB and AcrAB efflux pumps in all delafloxacin-resistant K. pneumoniae strains. The association between beta-lactamase production and delafloxacin resistance was determined; specifically, CTX-M-15 production was detected in the ST147, ST307, and ST377 strains. Moreover, NDM-1 was detected in ST147. Conclusions: We conclude that multiple mutations in QRDRs, in combination with OqxAB and AcrAB efflux pumps, achieved delafloxacin resistance in K. pneumoniae. In our study, we report on NDM-1-producing K. pneumoniae ST147 in Hungary. [ABSTRACT FROM AUTHOR]

Multidrug-resistant Acinetobacter baumannii is a major concern in healthcare institutions worldwide. Several reports described the dissemination of A. baumannii high-risk clones that are responsible for a high number of difficult-to-treat infections. In our study, 19 multidrug-resistant A. baumannii strains from Budapest, Hungary, were investigated based on whole-genome sequencing (WGS). The obtained results were analysed together with data from 433 strains of A. baumannii from the Pathogenwatch database. WGS analysis of 19 A. baumannii strains detected that 12 belonged to ST2 and seven belonged to ST636. Among ST2 strains, 11 out of 12 carried either blaOXA-23 or blaOXA-58 genes; however, all strains of ST636 uniformly carried blaOXA-72 gene. All strains of ST2 and ST636 carried blaOXA-66 and blaADC-25 genes. Based on core genome multilocus sequence typing (cgMLST), 10 strains of ST2 belonged to cgMLST906, one strain to cgMLST458, and one strain to cgMLST1320; by contrast, all strains of ST636 belonged to cgMLST1178. Certain virulence determinants were present in all strains of both ST2 and ST636, namely, Ata, Bap, BfmRS, T2SS and PNAG. Interestingly, OmpA was present in all strains of ST2, but it was absent in all strains of ST636. Comparative analysis of 19 strains of this study and the collection of 433 isolates from Pathogenwatch database, proved a diverse clonal distribution of high-risk A. baumannii clones in Europe. The major clone in Europe is ST2, which is present all over the continent. However, ST636 has been mainly reported in Eastern Europe. Interestingly, cgMLSTs of ST2 correspond to the production of different beta-lactamases, namely, OXA-82 in cgMLST116, OXA-72 in cgMLST506, and cgMLST556, PER-1 in cgMLST456 and cgMLST1041. Our study demonstrates that the ST2 high-risk clone of A. baumannii is the most widespread in Europe; however, based on cgMLST analysis, a detailed detection of beta-lactamase production can be determined. [ABSTRACT FROM AUTHOR]

Abstract In this study delafloxacin resistance mechanisms in Escherichia coli strains were analyzed. Delafloxacin is a new fluoroquinolone, that is approved for clinical application however, resistance against this agent is scarcely reported. In our study 37 E. coli strains were included and antimicrobial susceptibility testing was performed for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, imipenem. Six delafloxacin resistant E. coli strains were selected for whole-genome sequencing and all of them exhibited resistance to other fluoroquinonlones and showed an extended-spectrum beta-lactamase phenotype. The six delafloxacin resistant E. coli strains belonged to different sequence types (STs) namely, ST131 (2 strains), ST57 (2 strains), ST162 and ST15840. Each delafloxacin resistant strain possessed multiple mutations in quinolone resistance-determining regions (QRDRs). Notably, three mutations in gyrA Ser83Leu, Asp87Asn and parC Ser80Ile were in strains of ST162, ST57 and ST15840. However, the two strains of ST131 carried five combined mutations namely, gyrA Ser83Leu, Asp87Asn, parC Ser80Ile, Glu84Val, parE Ile549Leu. Association of delafloxacin resistance and production of CTX-M-15 in ST131, CMY-2 in ST162 and ST15840 was detected. In this study a new ST, ST15840 of clonal complex 69 was identified. Our results demonstrate, that at least three mutations in QRDRs are required for delafloxacin resistance in E. coli.

In this study delafloxacin resistance mechanisms in Escherichia coli strains were analyzed. Delafloxacin is a new fluoroquinolone, that is approved for clinical application however, resistance against this agent is scarcely reported. In our study 37 E. coli strains were included and antimicrobial susceptibility testing was performed for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, imipenem. Six delafloxacin resistant E. coli strains were selected for whole-genome sequencing and all of them exhibited resistance to other fluoroquinonlones and showed an extended-spectrum beta-lactamase phenotype. The six delafloxacin resistant E. coli strains belonged to different sequence types (STs) namely, ST131 (2 strains), ST57 (2 strains), ST162 and ST15840. Each delafloxacin resistant strain possessed multiple mutations in quinolone resistance-determining regions (QRDRs). Notably, three mutations in gyrA Ser83Leu, Asp87Asn and parC Ser80Ile were in strains of ST162, ST57 and ST15840. However, the two strains of ST131 carried five combined mutations namely, gyrA Ser83Leu, Asp87Asn, parC Ser80Ile, Glu84Val, parE Ile549Leu. Association of delafloxacin resistance and production of CTX-M-15 in ST131, CMY-2 in ST162 and ST15840 was detected. In this study a new ST, ST15840 of clonal complex 69 was identified. Our results demonstrate, that at least three mutations in QRDRs are required for delafloxacin resistance in E. coli. [ABSTRACT FROM AUTHOR]

BackgroundEpidemiological risk factors such as the demography of a place, environment, food, livestock, and companion animals are known sources of Klebsiella pneumoniae infection. Whole-genome sequencing (WGS) has become a powerful tool to complement traditional microbiological characterization of foodborne pathogens. Moreover, K. pneumoniae has several species complexes (KpSC) and is very difficult to differentiate using routine microbiological methods. The present study aims to investigate the prevalence of K. pneumoniae in fish available in the retail market using WGS.MethodsIsolation of K. pneumoniae, identification of K. pneumoniae isolates, and determination of the minimum inhibitory concentration (MIC) were performed. Whole-genome sequencing of K. pneumoniae genomes and phylogenomic analysis were conducted for visual comparison of the genomes. Furthermore, genomes of non-human origin that were submitted from India to the NCBI database were downloaded and included in the comparative analysis.ResultsThe findings showed that many antibiotic-resistant genes (ARGs) are prominent, including acrD, BaeR, cpxA, mdtB, mdtC, CRP, H-NS, KpnE, KpnF, KpnG, KpnH, acrA, acrB, marA, ramB, oqxA, oqxB, LptD, and emrR. Four fish-sourced isolates had different blaSHV resistance gene variants. The presence of ARGs for aminoglycosides [aac(3)-IId], fluoroquinolones (oqxA, oqxB), and fosfomycin (fosA5, fosA6) in these K. pneumoniae isolates from fish sources was found. One of the CIFT-K6 isolates had the uncommon serotype of K. pneumoniae O3b with the high-risk clone “ST37.” The ST515 sequence type was present in two K. pneumoniae isolates (CIFT-K7 and CIFT-K8), but the O3b serotype and ST192 allele type were present in the CIFT-K10 isolate.ConclusionTo the best of our knowledge, this research study represents the first Indian report of K. pneumoniae linked to fish, specifically the high-risk clone ‘ST37’ and two other STs, 515 and 192. The most common plasmid type detected in all four isolates was IncFIB, and 75% of the isolates were IncFII and IncHI1B. The prevalence of ARGs linked to efflux pump resistance mechanisms is highlighted by the analysis of genome sequence data.

The present study aimed to explore the genomic characteristics of eight New Delhi metallo-β-lactamase-1 (NDM-1)-producing carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates from a Bulgarian tertiary hospital (2021–2023) in comparison to bla NDM-1-positive strains originating from the Balkans. Antimicrobial susceptibility testing, phenotypic assays for carbapenemase activity, PCR screening, whole-genome sequencing (WGS), and phylogenomic analysis were performed. Seven of the CRPA isolates investigated (Minimum inhibitory concentration values of imipenem and meropenem >32 mg L−1) were also resistant to piperacillin-tazobactam, ceftazidime, ceftazidime-avibactam, cefepime, ceftolozane-tazobactam, amikacin, tobramycin, ciprofloxacin, and levofloxacin, but were susceptible to colistin (0.5–2 mg L−1) and cefiderocol (0.25–1 mg L−1). The P. aeruginosa Pae57 isolate (designated Pae57) remained susceptible to aminoglycosides as well. WGS uncovered the co-existence of bla NDM-1 and bla GES-1. The isolates belonged to the ST654 high-risk clone, except for Pae57 (ST611). Alignment against reference sequences revealed the presence of a Tn 21 transposon harboring ble MBL– bla NDM-1–IS Aba125. It was similar to that found in the P. aeruginosa ST654 NDM1_1 strain (GCA_020404785.1) from Serbia. Phylogenomic analysis of our isolates indicated that seven of them (ST654) differed from each other in no more than 44 single-nucleotide polymorphisms (SNPs). Pae57 (ST611) was strikingly different (>21,700 SNPs) compared to all Balkan strains. In conclusion, to our knowledge this is the first report of bla NDM-1-positive P. aeruginosa ST611 isolation, which indicates the transmission dynamics of this determinant between high-risk and potentially high-risk P. aeruginosa clones. Obtained results unveil the dissemination of clonally related NDM-1-producing P. aeruginosa strains in the monitored hospital for approximately a 2-year period. [ABSTRACT FROM AUTHOR]

Background: In this study, the mechanisms implicated in delafloxacin resistance in Klebsiella pneumoniae strains were investigated. Delafloxacin is a novel, broad-spectrum fluoroquinolone that has been approved for clinical application. Methods: In our study, 43 K. pneumoniae strains were assessed, antimicrobial susceptibility testing was performed via the broth microdilution method, and the minimum inhibitory concentration (MIC) values for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, and imipenem were determined. Four delafloxacin-resistant K. pneumoniae strains were selected for whole-genome sequencing (WGS). Results: The MIC50 values for the 43 K. pneumoniae strains were as follows: ciprofloxacin 0.5 mg/L, levofloxacin 0.25 mg/L, moxifloxacin 0.5 mg/L, and delafloxacin 0.25 mg/L. All four selected delafloxacin-resistant K. pneumoniae strains showed extended-spectrum beta-lactamase production, and one strain exhibited carbapenem resistance. WGS enabled us to determine the sequence types (STs) of these strains, namely, ST307 (two strains), ST377, and ST147. Multiple mutations in quinolone-resistance-determining regions (QRDRs) were detected in all the delafloxacin-resistant K. pneumoniae strains; specifically, gyrA Ser83Ile and parC Ser80Ile were uniformly present in the strains of ST307 and ST147. However, in the ST377 strain, gyrA Ser83Tyr, Asp87Ala, and parC Ser80Ile, amino acid substitutions were detected. We also identified OqxAB and AcrAB efflux pumps in all delafloxacin-resistant K. pneumoniae strains. The association between beta-lactamase production and delafloxacin resistance was determined; specifically, CTX-M-15 production was detected in the ST147, ST307, and ST377 strains. Moreover, NDM-1 was detected in ST147. Conclusions: We conclude that multiple mutations in QRDRs, in combination with OqxAB and AcrAB efflux pumps, achieved delafloxacin resistance in K. pneumoniae. In our study, we report on NDM-1-producing K. pneumoniae ST147 in Hungary.

Multidrug-resistant Acinetobacter baumannii is a major concern in healthcare institutions worldwide. Several reports described the dissemination of A. baumannii high-risk clones that are responsible for a high number of difficult-to-treat infections. In our study, 19 multidrug-resistant A. baumannii strains from Budapest, Hungary, were investigated based on whole-genome sequencing (WGS). The obtained results were analysed together with data from 433 strains of A. baumannii from the Pathogenwatch database. WGS analysis of 19 A. baumannii strains detected that 12 belonged to ST2 and seven belonged to ST636. Among ST2 strains, 11 out of 12 carried either blaOXA-23 or blaOXA-58 genes; however, all strains of ST636 uniformly carried blaOXA-72 gene. All strains of ST2 and ST636 carried blaOXA-66 and blaADC-25 genes. Based on core genome multilocus sequence typing (cgMLST), 10 strains of ST2 belonged to cgMLST906, one strain to cgMLST458, and one strain to cgMLST1320; by contrast, all strains of ST636 belonged to cgMLST1178. Certain virulence determinants were present in all strains of both ST2 and ST636, namely, Ata, Bap, BfmRS, T2SS and PNAG. Interestingly, OmpA was present in all strains of ST2, but it was absent in all strains of ST636. Comparative analysis of 19 strains of this study and the collection of 433 isolates from Pathogenwatch database, proved a diverse clonal distribution of high-risk A. baumannii clones in Europe. The major clone in Europe is ST2, which is present all over the continent. However, ST636 has been mainly reported in Eastern Europe. Interestingly, cgMLSTs of ST2 correspond to the production of different beta-lactamases, namely, OXA-82 in cgMLST116, OXA-72 in cgMLST506, and cgMLST556, PER-1 in cgMLST456 and cgMLST1041. Our study demonstrates that the ST2 high-risk clone of A. baumannii is the most widespread in Europe; however, based on cgMLST analysis, a detailed detection of beta-lactamase production can be determined.

ABSTRACT: Objectives: The various sequence types (STs) of Pseudomonas aeruginosa (P. aeruginosa) high-risk clones (HiRiCs) have been sporadically reported in China, but the systematic analysis of genomes for these STs remains limited. This study aimed to address the evolutionary pathways underlying the emergence of HiRiCs and their routes of dissemination from Chinese and global perspectives. Methods: The phylogenetic analysis was performed based on 416 newly sequenced clinical P. aeruginosa strains from Guangdong (GD), published genome sequences of 282 Chinese isolates, and 868 HiRiCs isolates from other countries. The genomic comparison study of global HiRiC ST244 was conducted to detect the model of global dissemination and local separation driven by association regional-specific antibiotic resistance genes. Furthermore, the evolutionary route of the emerging, China-specific HiRiC ST1971 was explored using Most Recent Common Ancestor (MRCA) analysis. Results: Based on comparative genomics analysis, we found a clear geographical separation of ST244 isolates, yet with an association between ST244 isolates from GD and America. We identified a set of 38 AMR genes that contribute to the geographical separation in ST244, and we identified genetic determinants either positively (MexB) and negatively (opmD) associated with GD ST244. For the China-unique HiRiC ST1971, its evolutionary history across different continents before emerging as ST1971 in China was also deduced. Conclusion: This study provides insight into the specific genetics underlying regional differences among globally disseminated P. aeruginosa HiRiCs (ST244) as well as new understanding of the dissemination and evolution of a regional HiRiC (ST1971). Understanding the genetics of these and other HiRiCs may assist in controlling their emergence and further spread.

ABSTRACT Pseudomonas aeruginosa is a major opportunistic pathogen that can cause severe systemic infections. Here, we found that the type III secretion system effector ExoU was the main determinant of pathogenicity of a highly virulent clinical isolate strain, BSI_S5, of P. aeruginosa, which caused severe bloodstream infections and belongs to ST463/O4. Deletion of exoU showed significantly attenuated cytotoxicity and virulence in vivo, while deletion of two other unique genes from strain BSI_S5, chr_1696 and chr_4238, which encodes type I secretion system permease/ATPase or polysaccharide biosynthesis protein, respectively, caused no apparent loss of toxicity compared to the parent strain, and complementation of the ΔexoU mutant restored its virulence. The presence of ExoU-induced lung lesions and the mucosal damage of gallbladder in mice, and the replication of the ΔexoU mutant was hindered in vivo compared with the parent strain. We show here for the first time that a clinical isolate with intact exoU harbored a mutation of T→G substitution in the adjacent specific Pseudomonas chaperone for ExoU (spcU), which caused phenylalanine to valine change at amino acid 94 of SpcU, resulting in significant loss of cytotoxicity. This finding suggests that intact exoU is not sufficient for cytotoxicity, but a functional downstream SpcU is required for ExoU secretion and cytotoxicity. Finally, we found that BSI_S5 harbored two types of insertion sequences adjacent to exoU, suggesting the potential of exoU to be transferred to other strains. Our study opens an avenue for further mechanistic study of P. aeruginosa systemic infections regarding its high pathogenicity and clinical spread. IMPORTANCE The identification of decisive virulence-associated genes in highly pathogenic P. aeruginosa isolates in the clinic is essential for diagnosis and the start of appropriate treatment. Over the past decades, P. aeruginosa ST463 has spread rapidly in East China and is highly resistant to β-lactams. Given the poor clinical outcome caused by this phenotype, detailed information regarding its decisive virulence genes and factors affecting virulence expression needs to be deciphered. Here, we demonstrate that the T3SS effector ExoU has toxic effects on mammalian cells and is required for virulence in the murine bloodstream infection model. Moreover, a functional downstream SpcU is required for ExoU secretion and cytotoxicity. This work highlights the potential role of ExoU in the pathogenesis of disease and provides a new perspective for further research on the development of new antimicrobials with antivirulence ability.

Wild birds have emerged as novel reservoirs and potential spreaders of antibiotic-resistant priority pathogens, being proposed as sentinels of anthropogenic activities related to the use of antimicrobial compounds. The aim of this study was to investigate the occurrence and genomic features of extended-spectrum β-lactamase (ESBL)-producing bacteria in wild birds in South America. In this regard, we have identified two ESBL (CTX-M-55 and CTX-M-65)-positive Escherichia coli (UNB7 and GP188 strains) colonizing Creamy-bellied Thrush (Turdus amaurochalinus) and Variable Hawk (Geranoaetus polyosoma) inhabiting synanthropic and wildlife environments from Brazil and Chile, respectively. Whole-genome sequence (WGS) analysis revealed that E. coli UNB7 and GP188 belonged to the globally disseminated clone ST602, carrying a wide resistome against antibiotics (β-lactams), heavy metals (arsenic, copper, mercury), disinfectants (quaternary ammonium compounds), and pesticides (glyphosate). Additionally, E. coli UNB7 and GP188 strains harbored virulence genes encoding hemolysin E, type II and III secretion systems, increased serum survival, adhesins and siderophores. SNP-based phylogenomic analysis, using an international genome database, revealed genomic relatedness (19–363 SNP differences) of GP188 with livestock and poultry strains, and genomic relatedness (61–318 differences) of UNB7 with environmental, human and livestock strains (Table S1), whereas phylogeographical analysis confirmed successful expansion of ST602 as a global clone of One Health concern. In summary, our results support that ESBL-producing E. coli ST602 harboring a wide resistome and virulome have begun colonizing wild birds in South America, highlighting a potential new reservoir of critical priority pathogens.

The environmental contamination plays a significant role in the emergence of antimicrobial resistance. In this study, we report a genomic analysis of an extensively drug-resistant and blaNDM-1-producing Klebsiella pneumoniae (EW807) strain recovered from a surface water sample. Strain EW807 belonged to sequence type (ST) 340 and serotype O4:KL15, a high-risk clone of the clonal group 258. This strain carried a broad resistome, including blaNDM-1 and blaCTX-M-15. The core genome multilocus sequence typing phylogenetic analysis revealed that the EW807 strain was most related to strains from Brazil and the USA. An IncX3 plasmid was identified harboring the blaNDM-1 gene, while an IncFIB(K) plasmid was detected carrying the blaCTX-M-15 in addition to multidrug resistance and multimetal tolerance regions. IncX3 and IncFIB(K) plasmids shared high similarity with plasmids from a human in China and a dog in Brazil, respectively. The regions harboring the blaNDM-1 and blaCTX-M-15 genes contained sequences from the Tn3 family. These findings suggest that IncX3 plasmid could play a role in the spread of NDM-1 in a post-pandemic scenario. To the best of our knowledge, this is the first report of blaNDM-1-producing K. pneumoniae ST340 O4:KL15 strain in the environment. Therefore, the presence of high-risk clones of K. pneumoniae carrying carbapenemases in the environment requires strict surveillance. [ABSTRACT FROM AUTHOR]

Mutations leading to upregulation of efflux pumps can produce multiple drug resistance in the pathogen Pseudomonas aeruginosa. Changes in their DNA binding regions, i.e., palindromic operators, can compromise pump depression and subsequently enhance resistance against several antibacterials and biocides. Here, we have identified (pseudo)palindromic repeats close to promoters of genes encoding 13 core drug-efflux pumps of P. aeruginosa. This framework was applied to detect mutations in these repeats in 17,292 genomes. Eighty-nine percent of isolates carried at least one mutation. Eight binary genetic properties potentially related to expression were calculated for mutations. These included palindromicity reduction, mutation type, positioning within the repeat and DNA-bending shift. High-risk ST298, ST308 and ST357 clones commonly carried four conserved mutations while ST175 and the cystic fibrosis-linked ST649 clones showed none. Remarkably, a T-to-C transition in the fourth position of the upstream repeat for mexEF-oprN was nearly exclusive of the high-risk ST111 clone. Other mutations were associated with high-risk sublineages using sample geotemporal metadata. Moreover, 1.5% of isolates carried five or more mutations suggesting they undergo an alternative program for regulation of their effluxome. Overall, P. aeruginosa shows a wide range of operator mutations with a potential effect on efflux pump expression and antibiotic resistance. [ABSTRACT FROM AUTHOR]

Objective: Escherichia coli ST131 is a pandemic clone associated with multidrug resistance, starting with beta-lactamase production and fluoroquinolone resistance in the first place, leading to significant systemic infections. Clones that develop due to the frequency of antimicrobial resistance and the rate of spread in our country are important issues that need to be investigated. This study aims to investigate the incidence of ST131which is a "high-risk pandemic clone E. coli" in ESBL-producing and non-ESBL-producing strains, as well as their biofilm-forming abilities and antibiotic resistance rates. Materials and methods: A total of 86 E. coli isolates were used in the study. Bacterial identifications were performed by conventional and automated methods. The double disc synergy method was used to demonstrate the presence of ESBL. Molecular studies in all E. coli strains were performed by real-time PCR method. Findings: 86 strains were studied, of which 83.72% were urine, 6.98% were wound, 4.65% were blood, and 2.33% were tracheal aspirate and sputum. 79.07% of these strains were ESBL-positive. 58.1% of the strains were female, whereas 41.9% were male patients, and the average age was 46.2. Out of 86 strains, 38.72% were ST131 positive, the H30 subclone was detected in 27.27% of them, and the H30-Rx subclone was detected in all of the H30 subclone positive strains. The presence of the ESBL resistance gene was detected at the rate of TEM 41.86%, SHV 37.21%, CTX-M 36.04%, and OXA 4.65%. Most commonly SHV gene (54.54%) was seen in ST131 clone-positive samples. Finally, while it was found that 48.83% of the strains formed biofilm by any method, biofilm formation was detected in 69.7% of the samples that were positive for the ST131 clone. Result: Our study can reveal the dramatic prevalence of the ESBL-producing E. coli strains along with the high-risk ST131 clone, the dominance of the H30Rx subclone of this risky clone, as well as the importance of the influence of resistance mechanisms along with resistance and biofilm. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The ever-increasing prevalence of infections produced by multidrug-resistant or extensively drug-resistant Pseudomonas aeruginosa is commonly linked to a limited number of aptly-named epidemical 'high-risk clones' that are widespread among and within hospitals worldwide. The emergence of new potential high-risk clone strains in hospitals highlights the need to better and further understand the underlying genetic mechanisms for their emergence and success. P. aeruginosa related high-risk clones have been sporadically found in China, their genome sequences have rarely been described. Therefore, the large-scale sequencing of multidrug-resistance high-risk clone strains will help us to understand the emergence and transmission of antibiotic resistances in P. aeruginosa high-risk clones. In this study, 212 P. aeruginosa strains were isolated from 2 tertiary hospitals within 3years (2018-2020) in Guangdong Province, China. Whole-genome sequencing, multi-locus sequence typing (MLST) and antimicrobial susceptibility testing were applied to analyze the genomic epidemiology of P. aeruginosa in this region. We found that up to 130 (61.32%) of the isolates were shown to be multidrug resistant, and 196 (92.45%) isolates were Carbapenem-Resistant Pseudomonas aeruginosa. MLST analysis demonstrated high diversity of sequence types, and 18 reported international high-risk clones were identified. Furthermore, we discovered the co-presence of exoU and exoS genes in 5 collected strains. This study enhances insight into the regional research of molecular epidemiology and antimicrobial resistance of P. aeruginosa in China. The high diversity of clone types and regional genome characteristics can serve as a theoretical reference for public health policies and help guide measures for the prevention and control of P. aeruginosa resistance. [ABSTRACT FROM AUTHOR]

Antimicrobial-resistant Escherichia coli isolates have emerged in various ecologic compartments and evolved to spread globally. We sought to (1.) investigate the occurrence of ESBL-producing E. coli (ESBL-Ec) in feces from free-range chickens in a rural region and (2.) characterize the genetic background of antimicrobial resistance and the genetic relatedness of collected isolates. Ninety-five feces swabs from free-range chickens associated with two households (House 1/House 2) in a rural region in northern Tunisia were collected. Samples were screened to recover ESBL-Ec, and collected isolates were characterized for phenotype/genotype of antimicrobial resistance, integrons, and molecular typing (pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)). Overall, 47 ESBL-Ec were identified, with the following genes detected: 35 blaCTX-M-1, 5 blaCTX-M-55, 5 blaCTX-M-15, 1 blaSHV-2, and 1 blaSHV-12. Resistance to fluoroquinolones, tetracycline, sulfonamides, and colistin was encoded by aac(6′)-Ib-cr (n = 21), qnrB (n = 1), and qnrS (n = 2); tetA (n = 17)/tetB (n = 26); sul1 (n = 29)/sul2 (n = 18); and mcr-2 (n = 2) genes, respectively. PFGE and MLST identified genetic homogeneity of isolates in House 1; however, isolates from House 2 were heterogeneous. Notably, among nine identified sequence types, ST58, ST69, ST224, and ST410 belong to pandemic high-risk clonal lineages associated with extrapathogenic E. coli. Minor clones belonging to ST410 and ST471 were shared by chickens from both households. The virulence genes fyuA, fimH, papGIII, and iutA were detected in 35, 47, 17, and 23 isolates, respectively. Findings indicate a high occurrence of ESBL-Ec in free-range chickens and highlight the occurrence of pandemic zoonotic clones. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the frequency of carbapenem resistant Klebsiella pneumoniae (CRKP) sequence types (STs) in Iran. Samples were collected from three university hospitals in Sanandaj, Iran, from December 2016 to March 2018. Antibiotic susceptibility testing, phenotypic and genotypic detection of carbapenemases were performed. Common K. pneumoniae capsular types were sought for all isolates. The genetic relatedness of isolates was investigated by multilocus sequence typing (MLST). Plasmids were detected by PCR-based Replicon Typing (PBRT). During the study, 67 K. pneumoniae isolates were identified. Of which, 18 (26.9%) isolates were detected as carbapenem-resistant. The most effective antibacterial agent was tigecycline (97%, 65 isolates) followed by imipenem and ertapenem (73.13%, 49 isolates). PCR showed that 13 isolates (19.4%) had bla NDM-1 gene and 5 (7.5%) harbored bla OXA-48. Examination of common capsular types showed that 2 isolates had K2 and 2 others had K54. REP-PCR revealed 10 clones and 11 singleton strains. MLST analysis of CRKP found ST15 as the most common type (13 isolates, 72.2%), but other STs were also detected namely, ST19, ST117, ST1390, and ST1594. ColE1 and IncL/M plasmids were the carriers of bla NDM-1 and bla OXA-48, respectively. The results showed that CRKP spread in our health centers. Our results, therefore, indicate a worrying trend of resistance to carbapenems in K. pneumoniae. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae is considered an opportunistic pathogen frequently involved with healthcare-associated infections. The genome of K. pneumoniae is versatile, harbors diverse virulence factors and easily acquires and exchanges resistance plasmids, facilitating the emergence of new threatening clones. In the last years, ST16 has been described as an emergent, clinically relevant strain, increasingly associated with outbreaks, and carrying virulence factors (such as ICEKp, iuc, rmpADC/2) and a diversity of resistance genes. However, a far-reaching phylogenetic study of ST16, including geographically, clinically and temporally distributed isolates is not available. In this work, we analyzed all publicly available ST16 K. pneumoniae genomes in terms of virulence factors, including capsular lipopolysaccharide and polysaccharide diversity, plasmids and antimicrobial resistance genes. A core genome SNP analysis shows that less than 1% of studied sites were variant sites, with a median pairwise single nucleotide polymorphism difference of 87 SNPs. The number and diversity of antimicrobial resistance genes, but not of virulence-related genes, increased consistently in ST16 strains during the studied period. A genomic comparison between ST16 and the high-risk clone ST11 K. pneumoniae, showed great similarities in their capacity to acquire resistance and virulence markers, differing mostly in the great diversity of capsular lipopolysaccharide and polysaccharide types in ST11, in comparison with ST16. While virulence and antimicrobial resistance scores indicated that ST11 might still constitute a more difficult-to-manage strain, results presented here demonstrate the great potential of the ST16 clone becoming critical in public health. [ABSTRACT FROM AUTHOR]

Mutations leading to upregulation of efflux pumps can produce multiple drug resistance in the pathogen Pseudomonas aeruginosa. Changes in their DNA binding regions, i.e., palindromic operators, can compromise pump depression and subsequently enhance resistance against several antibacterials and biocides. Here, we have identified (pseudo)palindromic repeats close to promoters of genes encoding 13 core drug-efflux pumps of P. aeruginosa. This framework was applied to detect mutations in these repeats in 17,292 genomes. Eighty-nine percent of isolates carried at least one mutation. Eight binary genetic properties potentially related to expression were calculated for mutations. These included palindromicity reduction, mutation type, positioning within the repeat and DNA-bending shift. High-risk ST298, ST308 and ST357 clones commonly carried four conserved mutations while ST175 and the cystic fibrosis-linked ST649 clones showed none. Remarkably, a T-to-C transition in the fourth position of the upstream repeat for mexEF-oprN was nearly exclusive of the high-risk ST111 clone. Other mutations were associated with high-risk sublineages using sample geotemporal metadata. Moreover, 1.5% of isolates carried five or more mutations suggesting they undergo an alternative program for regulation of their effluxome. Overall, P. aeruginosa shows a wide range of operator mutations with a potential effect on efflux pump expression and antibiotic resistance.

The increasing number of infections from multidrug-resistant P. aeruginosa (MDRPA) has compromised the selection of appropriate treatment in critically ill patients. Recent investigations have shown the existence of MDRPA global clones that have been disseminated in hospitals worldwide. We aimed to describe the molecular epidemiology and genetic diversity of the MDRPA acquired by Intensive Care Units (ICU) patients in our hospital. We used phenotypic methods to define the MDRPA and molecular methods were used to illustrate the presence of carbapenemase encoding genes. To characterize the MDRPA isolates, we used MALDI-TOF biomarker peaks, O-antigen serotyping, and multi-locus sequence typing analyses. Our data show that the most widely distributed MDRPA clone in our ICU unit was the ST175 strain. These isolates were further investigated by the whole-genome sequencing technique to determine the resistome profile and phylogenetic relationships, which showed, as previously described, that the MDR profile was due to the intrinsic resistance mechanisms and not the carbapenemase encoding genes. In addition, this study suggests that the combination of environmental focus and cross-transmission are responsible for the spread of MDRPA clones within our ICU unit. Serotyping and MALDI-TOF analyses are useful tools for the early detection of the most prevalent MDRPA clones in our hospital. Using these methods, semi-directed treatments can be introduced at earlier stages and healthcare professionals can actively search for environmental foci as possible sources of outbreaks. [ABSTRACT FROM AUTHOR]

ABSTRACT The aim of this study was to investigate the genomic features of an extensively drug-resistant (XDR) Pseudomonas aeruginosa isolate (P-469) emerging in Chile. Antibiotic susceptibility was determined by disk diffusion and “colistin agar” test. Whole-genome sequencing (WGS) was performed by the Illumina NextSeq 2000 platform, and epidemiologically and clinically relevant data (i.e., sequence-type, serotype, mobile genetic elements, virulome, resistome, plasmidome, prophages, and CRISPR-Cas systems) were retrieved using multiple bioinformatic tools. The P-469 strain displayed an XDR profile, remaining susceptible to colistin. Genomic analysis revealed that this isolate belonged to the “high-risk” clone ST654 (CC654), serotype O4, and genotype exoS+. Strikingly, two CRISPR-Cas systems, five intact prophages sequences, and a broad resistome that included blaNDM-1 and the novel blaVIM-80 carbapenemase genes were predicted. Our results revealed the genomic characteristics of P. aeruginosa belonging to the high-risk clone ST654/O4 coproducing NDM-1 and VIM-80 in Chile, supporting that genomic surveillance is necessary to track the emergence and spread of epidemiologically successful WHO’s critical priority pathogens in order to prevent their rapid dissemination.

ABSTRACT: Objectives: Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen causing a wide range of community- and hospital-acquired infections. Here we report the complete genome sequence of an extensively drug-resistant (XDR) P. aeruginosa strain (PA790) in order to understand the antibiotic resistance genes (ARGs) harboured by such a strain. Methods: Whole-genome sequencing (WGS) was performed using Illumina HiSeq and Nanopore MinION platforms. Genome assembly was performed using Unicycler v.0.4.8. The genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). In silico predictions were fulfilled using curated bioinformatics tools. Results: Pseudomonas aeruginosa PA790 was classified as XDR and belongs to sequence type 773 (ST773). The complete genome size is 6 932 250 bp with a G+C content of 66.02% and a BUSCO (Benchmarking Universal Single-Copy Orthologs) score of 100. Strain PA790 harboured 12 different ARGs conferring resistance to eight different classes of antibiotics. It was identified as the nineteenth ST773 strain among 5785 whole-genome sequences of P. aeruginosa available in the NCBI database. Conclusion: Pseudomonas aeruginosa PA790 belongs to ST773 and was identified as the nineteenth such isolate to be submitted to NCBI and the first complete ST773 genome from India. The WGS data with multiple ARGs of P. aeruginosa PA790 (ST773) will aid in understanding the evolution and phylogeny of such high-risk clones and provide a solid basis for further research on XDR strains.

Aamir Jamal Gondal,1,2 Sidrah Saleem,1 Shah Jahan,3 Nakhshab Choudhry,4 Nighat Yasmin2 1Department of Microbiology, University of Health Sciences, Lahore, Pakistan; 2Department of Biomedical Sciences, King Edward Medical University, Lahore, Pakistan; 3Department of Immunology, University of Health Sciences, Lahore, Pakistan; 4Department of Biochemistry, King Edward Medical University, Lahore, PakistanCorrespondence: Aamir Jamal GondalDepartment of Biomedical Sciences, King Edward Medical University, Nelagumbad Chowk, Anarkali Bazar, Lahore 54000, PakistanTel +92-300-8822055Email ajgondal119@gmail.comPurpose: The emergence of multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) is associated with the acquisition of multiple carbapenemases. Their clonal spread is a worldwide concern due to their critical role in nosocomial infections. Therefore, the identification of high-risk clones with antibiotic resistance genes is very crucial for controlling its global spread.Materials and Methods: A total of 227 K. pneumoniae strains collected during April 2018 to November 2019 were confirmed by PCR. Carbapenemases and extended-spectrum β-lactamases (ESBL) were detected phenotypically. Confirmation of carbapenemases was carried out by PCR and Sanger sequencing. The clonal lineages were assigned to selected isolates by multilocus sequence typing (MLST), and the plasmid analysis was done by PCR-based detection of the plasmid replicon typing.Results: Of the total K. pneumoniae, 117 (51.5%) were carbapenem resistant (CRKP) and 140 (61.7%) were identified as ESBL producers. Intermediate to high resistance was detected in the tested β-lactam drugs while polymyxin-B and tigecycline were found to be susceptible. Among CRKP, 91 (77.8%) isolates were detected as carbapenemase producing, while 55 (47%) were positive for blaNDM-1 23.9% (n=28), blaOXA-48 22.2% (n=26) and blaVIM 0.85% (n=1) while 12.7% (n=7) carried both blaNDM-1 and blaOXA-48 genes. The CRKP coharboring blaNDM-1 and blaOXA-48 genes (n=7) were positive for blaCTX-M blaSHV (n=3), blaSHV (n=1) and blaCTX-M (n=3). The novel CRKP with the coexistence of blaNDM-1, blaOXA-48, blaCTX-M and blaSHV genes were associated with the high-risk clone ST147 (n=5) and ST11 (n=2). The assigned replicon types were IncL/M, IncFII, IncA/C and IncH1.Conclusion: This is the first report of the coexistence of blaNDM-1, blaOXA-48, blaCTX-M and blaSHV genes on a high-risk lineage ST147 from Pakistan. This study highlights the successful dissemination of carbapenemase resistance genes in the high-risk clones that emphasizes the importance of monitoring and controlling the spread of these diverse clones globally.Keywords: high-risk clone, New Delhi metallo-β-lactamase, MLST, K. pneumoniae, carbapenem resistance

We set out to study the prevalence of the mcr-1 gene in carbapenemase-producing Klebsiella pneumoniae (CPKP) strains, and to determine whether its presence is associated with a fitness cost. A total of 234 clinical CPKP isolates were collected from a tertiary medical center in Taiwan from January 2018 to January 2019. The mcr-1 and carbapenemase genes were detected by polymerase chain reaction (PCR) followed by Sanger sequencing. The mcr-1-positive carbapenemase-producing strain was characterized by whole genome sequencing, a plasmid stability test and a conjugation assay. In vitro growth rate and an in vivo virulence test were compared between the parental mcr-1-positive strain and its mcr-1 plasmid-cured strain. We identified only one mcr-1 positive strain (KP2509), co-harboring blaKPC–2 and blaOXA–48, among 234 (1/234, 0.43%) CPKP strains. KP2509 and its Escherichia coli mcr-1 transconjugant showed moderate colistin resistance (MIC = 8 mg/L). The mcr-1 is located on a large conjugative plasmid (317 kb), pKP2509-MCR, with three replicons, IncHI, IncFIB, and IncN. Interestingly, a complete Type IV-A3 CRISPR-Cas system was identified in pKP2509-MCR. Plasmid pKP2509-MCR was highly stable in KP2509 after 270 generation of passage, and the pKP2509-MCR cured strain PC-KP2509 showed similar growth rate and in vivo virulence in comparison to KP2509. The prevalence of mcr-1 in CPKP strains remains low in our center. Notably, we identified a large plasmid with multiple replicons containing both the mcr-1 and the Type IV-3A CRISPR-Cas genes. The further spread of this highly stable plasmid raises concern that it may promote the increase of mcr-1 prevalence in CPKP.

Objectives: The study aimed to characterize the clonal spread of resistant bacteria and dissemination of resistance plasmids among carbapenem-resistant Enterobacterales at a tertiary hospital in Catalonia, Spain. Methods: Isolates were recovered from surveillance rectal swabs and diagnostic samples. Species identification was by matrix-assisted laser desorption ionization-time time of flight mass spectrometry (MALDI-TOF MS). Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Antimicrobial susceptibility was assessed by gradient-diffusion and carriage of bla genes was detected by PCR. Plasmid typing, conjugation assays, S1-PFGE studies and long-read sequencing were used to characterize resistance plasmids. Results: From July 2018 to February 2019, 125 Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales were recovered from 101 inpatients from surveillance (74.4%) or clinical samples (25.6%), in a tertiary hospital in Barcelona. Clonality studies identified a major clone of Klebsiella pneumoniae belonging to sequence type ST15 and additional isolates of K. pneumoniae , Escherichia coli and Enterobacter sp. from different STs. All isolates but one carried the bla KPC–2 allelic variant. The bla KPC–2 gene was located in an IncFIIk plasmid of circa 106 Kb in a non-classical Tn 4401 element designated NTEKPC-pMC-2-1. Whole-genome sequencing revealed different rearrangements of the 106 Kb plasmid while the NTEKPC-pMC-2-1 module was highly conserved. Conclusion: We report a hospital outbreak caused by the clonal dissemination of KPC-producing ST15 K. pneumoniae but also the intra- and inter-species transmission of the bla KPC–2 gene associated with plasmid conjugation and/or transposon dissemination. To our knowledge, this is the first report of an outbreak caused by KPC-producing Enterobacterales isolated from human patients in Catalonia and highlights the relevance of surveillance studies in the early detection and control of antibiotic resistant high-risk clones. [ABSTRACT FROM AUTHOR]

We set out to study the prevalence of the mcr-1 gene in carbapenemase-producing Klebsiella pneumoniae (CPKP) strains, and to determine whether its presence is associated with a fitness cost. A total of 234 clinical CPKP isolates were collected from a tertiary medical center in Taiwan from January 2018 to January 2019. The mcr-1 and carbapenemase genes were detected by polymerase chain reaction (PCR) followed by Sanger sequencing. The mcr-1 -positive carbapenemase-producing strain was characterized by whole genome sequencing, a plasmid stability test and a conjugation assay. In vitro growth rate and an in vivo virulence test were compared between the parental mcr-1 -positive strain and its mcr-1 plasmid-cured strain. We identified only one mcr-1 positive strain (KP2509), co-harboring bla KPC– 2 and bla OXA– 48, among 234 (1/234, 0.43%) CPKP strains. KP2509 and its Escherichia coli mcr-1 transconjugant showed moderate colistin resistance (MIC = 8 mg/L). The mcr-1 is located on a large conjugative plasmid (317 kb), pKP2509-MCR, with three replicons, IncHI, IncFIB, and IncN. Interestingly, a complete Type IV-A3 CRISPR-Cas system was identified in pKP2509-MCR. Plasmid pKP2509-MCR was highly stable in KP2509 after 270 generation of passage, and the pKP2509-MCR cured strain PC-KP2509 showed similar growth rate and in vivo virulence in comparison to KP2509. The prevalence of mcr-1 in CPKP strains remains low in our center. Notably, we identified a large plasmid with multiple replicons containing both the mcr-1 and the Type IV-3A CRISPR-Cas genes. The further spread of this highly stable plasmid raises concern that it may promote the increase of mcr-1 prevalence in CPKP. [ABSTRACT FROM AUTHOR]

Objectives: Escherichia coli sequence type 131 (ST131) is an international multiresistant high-risk clone associated with a large number of clinical infections. Here we report the draft genome sequence of a ST131 clinical isolate (EC538) obtained from a patient with bloodstream infection (BSI) in China co-harbouring the blaKPC-2, blaCTX-M-3, blaCTX-M-14, qnrS1, aac(3)-IIa and aac(6ʹ)-Ib-cr genes. Methods: Antimicrobial susceptibility testing of E. coli EC538 was performed. DNA of E. coli EC538 was extracted and was sequenced using an Illumina HiSeqTM X Ten platform. Generated sequence reads were assembled using CLC Genomics Workbench. Contigs were annotated using Rapid Annotation using Subsystem Technology (RAST), and further bioinformatics analyses were performed. Results: The total number of assembled bases was 5 420 040 bp, with 5611 protein-coding sequences. Escherichia coli EC538 belongs to ST131 by multilocus sequence typing (MLST). The presence of blaKPC-2, blaCTX-M-3 and blaCTX-M-14 genes was detected in addition to other antimicrobial resistance genes conferring resistance to fluoroquinolones, aminoglycosides, trimethoprim, sulfonamides, tetracyclines, macrolides and rifampicin. Conclusion: To our knowledge, this is the first report of an E. coli ST131 strain from a BSI co-harbouring blaKPC-2, blaCTX-M-3, blaCTX-M-14, qnrS1, aac(3)-IIa and aac(6ʹ)-Ib-cr genes in China. The presented genome sequence of a carbapenemase-producing E. coli ST131 strain could provide further insight into the genomic diversity of this highly virulent, multiresistant and successfully pandemic bacterial pathogen.

Objective: Carbapenemase-producing Acinetobacter baumannii has been recognized as a critical priority pathogen by the World Health Organization. We hereby report the identification and the draft genome sequence of a carbapenem-resistant A. baumannii isolated from a patient with community-onset urinary tract infection, in a Chilean Patagonian city. Methods: The whole genome was sequenced on an Illumina NextSeq platform and de novo assembled using Unicycler v.0.4. Resistome analysis and epidemiological investigation (based on MLST data and Pasteur scheme) were performed using bioinformatics tools available from the Center for Genomic Epidemiology. Results: The genome size was calculated at 3 890 824 bp, with a GC content of 39.1%, comprising 3864 total genes, 30 tRNAs, 3 rRNAs, 4 ncRNAs, and 109 pseudogenes. Carbapenem-resistant A. baumannii Ab3_Ch strain belonged to the international sequence type ST15 (clonal complex, CC15), and harboured the ISAba-1-blaOXA-219 gene array, along to blaTEM-1B and blaADC-6 β-lactamase genes, and aac(3)-IIa and aph(3′)-VIa aminoglycoside resistance genes. Additionally, efflux pump encoding genes (abaF, abaQ, abeS, adeI, adeK, adeL, adeN, adeR, adeS, and amvA) were identified, and mutations in the quinolone resistance-determining region of gyrA (Ser81Leu) and parC (Ser84Leu) were considered responsible for fluoroquinolone resistance. Conclusion: This genome sequence data could be used for comparative genomic studies of critical priority A. baumannii strains, as well as to understand the specific features of hospital-associated A. baumannii lineages of international clonal complexes emerging in community settings.

Klebsiella pneumoniae is considered an opportunistic pathogen frequently involved with healthcare-associated infections. The genome of K. pneumoniae is versatile, harbors diverse virulence factors and easily acquires and exchanges resistance plasmids, facilitating the emergence of new threatening clones. In the last years, ST16 has been described as an emergent, clinically relevant strain, increasingly associated with outbreaks, and carrying virulence factors (such as ICEKp, iuc, rmpADC/2) and a diversity of resistance genes. However, a far-reaching phylogenetic study of ST16, including geographically, clinically and temporally distributed isolates is not available. In this work, we analyzed all publicly available ST16 K. pneumoniae genomes in terms of virulence factors, including capsular lipopolysaccharide and polysaccharide diversity, plasmids and antimicrobial resistance genes. A core genome SNP analysis shows that less than 1% of studied sites were variant sites, with a median pairwise single nucleotide polymorphism difference of 87 SNPs. The number and diversity of antimicrobial resistance genes, but not of virulence-related genes, increased consistently in ST16 strains during the studied period. A genomic comparison between ST16 and the high-risk clone ST11 K. pneumoniae, showed great similarities in their capacity to acquire resistance and virulence markers, differing mostly in the great diversity of capsular lipopolysaccharide and polysaccharide types in ST11, in comparison with ST16. While virulence and antimicrobial resistance scores indicated that ST11 might still constitute a more difficult-to-manage strain, results presented here demonstrate the great potential of the ST16 clone becoming critical in public health.

The increasing number of infections from multidrug-resistant P. aeruginosa (MDRPA) has compromised the selection of appropriate treatment in critically ill patients. Recent investigations have shown the existence of MDRPA global clones that have been disseminated in hospitals worldwide. We aimed to describe the molecular epidemiology and genetic diversity of the MDRPA acquired by Intensive Care Units (ICU) patients in our hospital. We used phenotypic methods to define the MDRPA and molecular methods were used to illustrate the presence of carbapenemase encoding genes. To characterize the MDRPA isolates, we used MALDI-TOF biomarker peaks, O-antigen serotyping, and multi-locus sequence typing analyses. Our data show that the most widely distributed MDRPA clone in our ICU unit was the ST175 strain. These isolates were further investigated by the whole-genome sequencing technique to determine the resistome profile and phylogenetic relationships, which showed, as previously described, that the MDR profile was due to the intrinsic resistance mechanisms and not the carbapenemase encoding genes. In addition, this study suggests that the combination of environmental focus and cross-transmission are responsible for the spread of MDRPA clones within our ICU unit. Serotyping and MALDI-TOF analyses are useful tools for the early detection of the most prevalent MDRPA clones in our hospital. Using these methods, semi-directed treatments can be introduced at earlier stages and healthcare professionals can actively search for environmental foci as possible sources of outbreaks.

Objectives: The aim of this research was to investigate the clinical and microbiological characteristics of a case of community-acquired carbapenem-resistant Escherichia coli isolated from a patient with a bloodstream infection in China.Methods:Escherichia coli Huamei202001 was recovered from the first blood culture from a patient hospitalised in China. An antimicrobial susceptibility test was performed, and the genome was sequenced on an Illumina HiSeq X 10 platform with a 150-bp paired-end approach. The generated sequence reads were assembled using Unicycler, and the whole genome sequence data were analysed using bioinformatics tools. Moreover, the patient and her main family members obtained a faecal sample screening test for CRE, the positive strain was further isolated and the identification and antimicrobial susceptibility testing was performed.Results:Escherichia coli Huamei202001 belonged to sequence type 410. In addition, a blaNDM-5-encoding IncX3-type plasmid was responsible for the spreading of carbapenem resistance. Only the patient was detected as having a positive faecal sample screening test for CRE. Strain Fec01 was identified as E. coli, and the antibiotic susceptibility profile was the same as that of E. coli Huamei202001.Conclusions:Escherichia coli Huamei202001 is defined as community-acquired carbapenem-resistant Enterobacteriaceae. The clone ST410 that harbours the blaNDM-5-encoding IncX3-type plasmid is causing new high-risk clones globally. Thus, infection control measures should be strengthened to curb the dissemination of IncX3.

Objective  Antimicrobial resistance poses a serious threat to children's health. In recent years, high-risk Escherichia coli ST131 has become an important target for global surveillance studies. The E.coli ST131 clone is associated with extended spectrum β-lactamase (ESBL) production, as well as multidrug resistance and treatment failure. Studies on this clone in the pediatric age group are limited. We aim to investigate the rate of high-risk E. coli ST131 clone in ESBL-positive E. coli isolates obtained from pediatric patients. Methods  A total of 292 ESBL-positive E. coli isolates from clinical samples of pediatric patients was included in the study. MALDI-TOF MS system was used for bacterial identification. Susceptibility tests were performed using BD Phoenix automated system. ST131 detection was done by MALDI-TOF-MS. Fisher's exact test was used to compare the groups (significance <0.05). Results  A total of 292 isolates was analyzed. The high-risk ST131 clone was detected in 117 (40%) of the 292 ESBL-positive isolates. ST131 rates were found to be significantly higher in children under the age of 5 years compared with children over the age of 5 years (49.3 vs. 31.1%, p  = 0.0019). Ciprofloxacin resistance was higher in ST131 isolates (45.6 vs. 31.7%; p  < 0.05). Conclusion  The rate of the ST131 clone was found to be high in the pediatric population. The significantly high rate of resistance to ciprofloxacin, which is not commonly used in the pediatric population, in ST131 isolates reveals the importance of the spread of high-risk clones for the development of resistance. [ABSTRACT FROM AUTHOR]

ABSTRACT Genotyping of 2,882 Pseudomonas aeruginosa isolates that had been collected during the last 40 years identified the ExoU-positive lineages PA14 (ST253) and ST235 as the second and third most frequent clones in the P. aeruginosa population. Both clones were approximately 2-fold more frequently detected in animate habitats than in soil or aquatic habitats. While ST253 clone isolates were causing mainly acute and chronic infections in humans, ST235 isolates had been preferentially collected from hospitalized patients with severe acute infections, particularly, keratitis, urinary tract infections, burn wounds, and ventilator-associated pneumonia. The two major exoU clones differed substantially in the composition and flexibility of the accessory genome and by more than 8,000 amino acid sequences. Pronounced sequence variation between orthologs was noted in genes encoding elements of secretion systems and secreted effector molecules, including the type III secretion system, indicating the modes of action of the different clones. When comparing representatives of the two clones in batch culture, the PA14 strain orchestrated the quorum sensing circuitry for the expression of pathogenic traits and stopped growing in batch culture when it entered the stationary phase, but the quorum sensing-deficient ST235 strain expressed high type III secretion activity and continued to grow and to divide. In summary, unrestricted growth, high constitutive type III secretion activity, and facilitated uptake of foreign DNA could be major features that have made ST235 a global high-risk clone associated with poor outcomes of acute nosocomial infections. IMPORTANCE The ubiquitous and metabolically versatile environmental bacterium Pseudomonas aeruginosa can cause infections in a wide variety of hosts, including insects, plants, animals, and humans. P. aeruginosa is one of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens that are the major cause of nosocomial infections in the United States and are a threat all over the world because of their capacity to become increasingly resistant to all available antibiotics. Most experimental work on P. aeruginosa has been performed with reference strains PAO1 and PA14, providing deep insight into key metabolic and regulatory pathways thought to be applicable to all P. aeruginosa strains. However, this comparative study on the two most common exoU-positive clones taught us that there are major lineages in the population such as the global high-risk clone ST235 that exhibit uncommon traits of lifestyle, genome mobility, and pathogenicity distinct from those in our knowledge gained from the studies with the reference strains.

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.

Acinetobacter baumannii is one of the most important nosocomial pathogens able to rapidly develop extensive drug resistance. Here, we study the role of accessory genome in the success of the globally disseminated clone 1 (GC1) with functional and genomic approaches. Comparative genomics was performed with available GC1 genomes (n = 106) against other A. baumannii high-risk and sporadic clones. Genetic traits related to accessory genome were found common and conserved along time as two novel regions of genome plasticity, and a CRISPR-Cas system acquired before clonal diversification located at the same loci as “sedentary” modules. Although identified within hotspot for recombination, other block of accessory genome was also “sedentary” in lineage 1 of GC1 with signs of microevolution as the AbaR0-type genomic island (GI) identified in A144 and in A155 strains which were maintained one month in independent experiments without antimicrobial pressure. The prophage YMC/09/02/B1251_ABA_BP was found to be “mobile” since, although it was shared by all GC1 genomes, it showed high intrinsic microevolution as well as mobility to different insertion sites. Interestingly, a wide variety of Insertion Sequences (IS), probably acquired by the flow of plasmids related to Rep_3 superfamily was found. These IS showed dissimilar genomic location amongst GC1 genomes presumably associated with promptly niche adaptation. On the other hand, a type VI secretion system and three efflux pumps were subjected to deep processes of genomic loss in A. baumannii but not in GC1. As a whole, these findings suggest that preservation of some genetic modules of accessory genome harbored by strains from different continents in combination with great plasticity of IS and varied flow of plasmids, may be central features of the genomic structure of GC1. Competition of A144 and A155 versus A118 (ST 404/ND) without antimicrobial pressure suggested a higher ability of GC1 to grow over a clone with sporadic behavior which explains, from an ecological perspective, the global achievement of this successful pandemic clone in the hospital habitat. Together, these data suggest an essential role of still unknown properties of “mobile” and “sedentary” accessory genome that is preserved over time under different antibiotic or stress conditions.

ABSTRACTHere, we presented 11 cases with colistin-resistant Pseudomonas aeruginosa infection and co-existence of OXA-48 and NDM-1 in the ST235 high-risk clone. The molecular analyses were performed by Sanger sequencing and RT–PCR. The eight patients (72.7%) had an invasive infection and three (27.3%) had colonization. The 30-day mortality rate was 87.5% (7/8). Three patients (37.5%, 3/8) received colistin therapy before isolation of P. aeruginosa. In the Multilocus sequence typing (MLST) analysis of 11 isolates, eight (72.7%) isolates belonged to P. aeruginosa ST235 clone. All isolates were NDM-1 positive, and nine isolates (81.8%) were found to be positive for both OXA-48 and NDM-1. Sequences of pmrAB and phoPQ revealed numerous insertions and deletions in all isolates. In 10 isolates pmrAB and phoPQ were found to be upregulated. In conclusion, the co-existence of OXA-48 and NDM-1 genes in colistin-resistant P. aeruginosa ST235 high-risk clone indicates the spread of carbapenemases in clinical isolates and highlights need of continuous surveillance for high-risk clones of P. aeruginosa.

Background Antimicrobial resistance (AMR) is a major challenge in the treatment of infections caused by Pseudomonas aeruginosa. Highly drug-resistant infections are disproportionally caused by a small subset of globally distributed P. aeruginosa sequence types (STs), termed "high-risk clones." We noted that clonal complex (CC) 446 (which includes STs 298 and 446) isolates were repeatedly cultured at 1 medical center and asked whether this lineage might constitute an emerging high-risk clone. Methods We searched P. aeruginosa genomes from collections available from several institutions and from a public database for the presence of CC446 isolates. We determined antibacterial susceptibility using microbroth dilution and examined genome sequences to characterize the population structure of CC446 and investigate the genetic basis of AMR. Results CC446 was globally distributed over 5 continents. CC446 isolates demonstrated high rates of AMR, with 51.9% (28/54) being multidrug-resistant (MDR) and 53.6% of these (15/28) being extensively drug-resistant (XDR). Phylogenetic analysis revealed that most MDR/XDR isolates belonged to a subclade of ST298 (designated ST298*) of which 100% (21/21) were MDR and 61.9% (13/21) were XDR. XDR ST298* was identified repeatedly and consistently at a single academic medical center from 2001 through 2017. These isolates harbored a large plasmid that carries a novel antibiotic resistance integron. Conclusions CC446 isolates are globally distributed with multiple occurrences of high AMR. The subclade ST298* is responsible for a prolonged epidemic (≥16 years) of XDR infections at an academic medical center. These findings indicate that CC446 is an emerging high-risk clone deserving further surveillance. [ABSTRACT FROM AUTHOR]

Purpose: The emergence of multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) is associated with the acquisition of multiple carbapenemases. Their clonal spread is a worldwide concern due to their critical role in nosocomial infections. Therefore, the identification of high-risk clones with antibiotic resistance genes is very crucial for controlling its global spread. Materials and Methods: A total of 227 K. pneumoniae strains collected during April 2018 to November 2019 were confirmed by PCR. Carbapenemases and extended-spectrum β-lactamases (ESBL) were detected phenotypically. Confirmation of carbapenemases was carried out by PCR and Sanger sequencing. The clonal lineages were assigned to selected isolates by multilocus sequence typing (MLST), and the plasmid analysis was done by PCR-based detection of the plasmid replicon typing. Results: Of the total K. pneumoniae, 117 (51.5%) were carbapenem resistant (CRKP) and 140 (61.7%) were identified as ESBL producers. Intermediate to high resistance was detected in the tested β-lactam drugs while polymyxin-B and tigecycline were found to be susceptible. Among CRKP, 91 (77.8%) isolates were detected as carbapenemase producing, while 55 (47%) were positive for blaNDM-1 23.9% (n=28), blaOXA-48 22.2% (n=26) and blaVIM 0.85% (n=1) while 12.7% (n=7) carried both blaNDM-1 and blaOXA-48 genes. The CRKP coharboring blaNDM-1 and blaOXA-48 genes (n=7) were positive for blaCTX-MblaSHV (n=3), blaSHV (n=1) and blaCTX-M (n=3). The novel CRKP with the coexistence of blaNDM-1, blaOXA-48, blaCTX-M and blaSHV genes were associated with the high-risk clone ST147 (n=5) and ST11 (n=2). The assigned replicon types were IncL/M, IncFII, IncA/C and IncH1. Conclusion: This is the first report of the coexistence of blaNDM-1, blaOXA-48, blaCTX-M and blaSHV genes on a high-risk lineage ST147 from Pakistan. This study highlights the successful dissemination of carbapenemase resistance genes in the high-risk clones that emphasizes the importance of monitoring and controlling the spread of these diverse clones globally. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is one of the most important nosocomial pathogens able to rapidly develop extensive drug resistance. Here, we study the role of accessory genome in the success of the globally disseminated clone 1 (GC1) with functional and genomic approaches. Comparative genomics was performed with available GC1 genomes (n = 106) against other A. baumannii high-risk and sporadic clones. Genetic traits related to accessory genome were found common and conserved along time as two novel regions of genome plasticity, and a CRISPR-Cas system acquired before clonal diversification located at the same loci as "sedentary" modules. Although identified within hotspot for recombination, other block of accessory genome was also "sedentary" in lineage 1 of GC1 with signs of microevolution as the AbaR0-type genomic island (GI) identified in A144 and in A155 strains which were maintained one month in independent experiments without antimicrobial pressure. The prophage YMC/09/02/B1251_ABA_BP was found to be "mobile" since, although it was shared by all GC1 genomes, it showed high intrinsic microevolution as well as mobility to different insertion sites. Interestingly, a wide variety of Insertion Sequences (IS), probably acquired by the flow of plasmids related to Rep_3 superfamily was found. These IS showed dissimilar genomic location amongst GC1 genomes presumably associated with promptly niche adaptation. On the other hand, a type VI secretion system and three efflux pumps were subjected to deep processes of genomic loss in A. baumannii but not in GC1. As a whole, these findings suggest that preservation of some genetic modules of accessory genome harbored by strains from different continents in combination with great plasticity of IS and varied flow of plasmids, may be central features of the genomic structure of GC1. Competition of A144 and A155 versus A118 (ST 404/ND) without antimicrobial pressure suggested a higher ability of GC1 to grow over a clone with sporadic behavior which explains, from an ecological perspective, the global achievement of this successful pandemic clone in the hospital habitat. Together, these data suggest an essential role of still unknown properties of "mobile" and "sedentary" accessory genome that is preserved over time under different antibiotic or stress conditions. [ABSTRACT FROM AUTHOR]

Escherichia coli ST410 (Ec -ST410) is an emerging, multidrug-resistant clone. Recent investigations of its global epidemiology and evolution have been based almost exclusively on isolates from Europe and North America. It is unclear whether Southeast Asian-origin Ec- ST410 (SEA- Ec -ST410) belong to these same clones or represent regionally disseminated variants. Antimicrobial resistance mechanisms among SEA- Ec -ST410 were characterised, and whether they belonged to regional variants was investigated by contextualising them within a global collection. Seven Ec -ST410 were identified among a recent collection of expanded-spectrum cephalosporin-resistant E. coli recovered from 91 healthy women (stool) and 26 infected patients (blood and urine) living in Phnom Penh, Cambodia. Nine additional Ec -ST410 genomes were identified from Thailand (n = 7) and Vietnam (n = 2) through EnteroBase and PubMed searches. The assembled genomes were characterised and a SNP-based phylogenetic tree was created comparing these 16 SEA -Ec -ST410 with a previously published Ec -ST410 collection, primarily sourced from Europe (97/128) and North America (24/128). SEA -Ec -ST410 belonged to several distinct branches within previously described clonal clades. SEA -Ec -ST410 within the B3/ H 24Rx sublineage encoded bla CTX-M-55 (8/12) and F18:A-:B1 plasmid replicons (6/12), neither of which were detected among other Ec -ST410 belonging to this clade. Three of four SEA -Ec -ST410 within the B4/ H 24RxC sublineage lacked both bla OXA-181 and an IncX3 plasmid replicon that were harboured by 97% and 100% of all other Ec -ST410 in this sublineage (n = 64), respectively. In conclusion, Ec -ST410 are present in Southeast Asia following multiple introductions. The unique pattern of antimicrobial resistance elements harboured by SEA -Ec -ST410 suggests independent circulation in the region. [ABSTRACT FROM AUTHOR]

ABSTRACT Escherichia coli sequence type 410 (ST410) has been reported worldwide as an extraintestinal pathogen associated with resistance to fluoroquinolones, third-generation cephalosporins, and carbapenems. In the present study, we investigated national epidemiology of ST410 E. coli isolates from Danish patients. Furthermore, E. coli ST410 was investigated in a global context to provide further insight into the acquisition of the carbapenemase genes blaOXA-181 and blaNDM-5 of this successful lineage. From 127 whole-genome-sequenced isolates, we reconstructed an evolutionary framework of E. coli ST410 which portrays the antimicrobial-resistant clades B2/H24R, B3/H24Rx, and B4/H24RxC. The B2/H24R and B3/H24Rx clades emerged around 1987, concurrently with the C1/H30R and C2/H30Rx clades in E. coli ST131. B3/H24Rx appears to have evolved by the acquisition of the extended-spectrum β-lactamase (ESBL)-encoding gene blaCTX-M-15 and an IncFII plasmid, encoding IncFIA and IncFIB. Around 2003, the carbapenem-resistant clade B4/H24RxC emerged when ST410 acquired an IncX3 plasmid carrying a blaOXA-181 carbapenemase gene. Around 2014, the clade B4/H24RxC acquired a second carbapenemase gene, blaNDM-5, on a conserved IncFII plasmid. From an epidemiological investigation of 49 E. coli ST410 isolates from Danish patients, we identified five possible regional outbreaks, of which one outbreak involved nine patients with blaOXA-181- and blaNDM-5-carrying B4/H24RxC isolates. The accumulated multidrug resistance in E. coli ST410 over the past two decades, together with its proven potential of transmission between patients, poses a high risk in clinical settings, and thus, E. coli ST410 should be considered a lineage with emerging “high-risk” clones, which should be monitored closely in the future. IMPORTANCE Extraintestinal pathogenic Escherichia coli (ExPEC) is the main cause of urinary tract infections and septicemia. Significant attention has been given to the ExPEC sequence type ST131, which has been categorized as a “high-risk” clone. High-risk clones are globally distributed clones associated with various antimicrobial resistance determinants, ease of transmission, persistence in hosts, and effective transmission between hosts. The high-risk clones have enhanced pathogenicity and cause severe and/or recurrent infections. We show that clones of the E. coli ST410 lineage persist and/or cause recurrent infections in humans, including bloodstream infections. We found evidence of ST410 being a highly resistant globally distributed lineage, capable of patient-to-patient transmission causing hospital outbreaks. Our analysis suggests that the ST410 lineage should be classified with the potential to cause new high-risk clones. Thus, with the clonal expansion over the past decades and increased antimicrobial resistance to last-resort treatment options, ST410 needs to be monitored prospectively.

The dissemination of carbapenemase-producing Escherichia coli, although still at low level, should be continuously monitored. OXA-244 is emerging in Europe, mainly in E. coli . In Italy, this carbapenemase was reported from an environmental river sample in 2019. We report clinical isolates of OXA-244-producing ST131 E. coli in four patients admitted to an acute care hospital in Pavia, Italy. The association of this difficult-to-detect determinant with a globally circulating high-risk clone, ST131 E. coli, is of clinical relevance.

Silver is used extensively in both hospitals and outpatient clinics as a disinfectant coating agent on various devices. Resistance to silver was recently reported as an emerging problem in Enterobacteriaceae. Multidrug-resistant high-risk clones of Klebsiella pneumoniae are common causes of serious healthcare-associated infections worldwide posing a serious threat to patients. In this study, we investigated the capacity of both high-risk (CG14/15 and CG258) and minor clone strains of K. pneumoniae to develop resistance to silver. Resistance was induced in vitro in silver-susceptible but otherwise multidrug-resistant clinical isolates. Genetic alterations in the silver-resistant derivative strains with regard to the silver-susceptible isolates were investigated by whole-genome sequencing. The transferability of high-level resistance to silver was also tested. We demonstrated that the high-level resistance to silver can quickly evolve as a consequence of a single-point mutation either in the cusS gene of the chromosomally encoded CusCFBARS efflux system and/or in the silS gene of the plasmid-encoded Copper Homeostasis and Silver Resistance Island (CHASRI) coding also for a metallic efflux. The minimal inhibitory concentrations (MICs) of the strains increased from 4 mg/L (23.5 μM) AgNO3 to >8,500 mg/L (>50,000 μM) AgNO3 during induction. Harboring the CHASRI proved an important selective asset for K. pneumoniae when exposed to silver. Successful conjugation experiments using Escherichia coli K12 J5-3Rif as recipient showed that high-level silver resistance can transmit between strains of high-risk clones of K. pneumoniae (ST15 and ST11) and isolates from additional species of Enterobacteriaceae. The lack of fitness cost associated with the carriage of the CHASRI in a silver-free environment and the presence of the RelEB toxin–antitoxin system on the conjugative plasmids could advance the dissemination of silver resistance. Our results show that multidrug-resistant high-risk clones of K. pneumoniae are capable of evolving and transmitting high-level resistance to silver. This observation should warrant a more judicious use of silver coated-devices to prevent the extensive dissemination of silver resistance. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is an important opportunistic pathogen in hospitals, responsible for various infections that are difficult to treat due to intrinsic and acquired antibiotic resistance. Here, 20 epidemiologically unrelated strains isolated from patients in a general hospital over a time period of two decades were analyzed using whole genome sequencing. The genomes were compared in order to assess the presence of a predominant clone or sequence type (ST). No clonal structure was identified, but core genome-based single nucleotide polymorphism (SNP) analysis distinguished two major, previously identified phylogenetic groups. Interestingly, most of the older strains isolated between 1994 and 1998 harbored exoU , encoding a cytotoxic phospholipase. In contrast, most strains isolated between 2011 and 2016 were exoU -negative and phylogenetically very distinct from the older strains, suggesting a population shift of nosocomial P. aeruginosa over time. Three out of 20 strains were ST235 strains, a global high-risk clonal lineage; these carried several additional resistance determinants including aac(6’)Ib-cr encoding an aminoglycoside N-acetyltransferase that confers resistance to fluoroquinolones. Core genome comparison with ST235 strains from other parts of the world showed that the three strains clustered together with other Brazilian/Argentinean isolates. Despite this regional relatedness, the individuality of each of the three ST235 strains was revealed by core genome-based SNPs and the presence of genomic islands in the accessory genome. Similarly, strain-specific characteristics were detected for the remaining strains, indicative of individual evolutionary histories and elevated genome plasticity. [ABSTRACT FROM AUTHOR]

Virulence plasmids are associated with hypervirulent types of Klebsiella pneumoniae, which generally do not carry antibiotic resistance genes. In contrast, nosocomial isolates are often associated with resistance, but rarely with virulence plasmids. Here, we describe virulence plasmids in nosocomial isolates of “high-risk” clones of sequence types (STs) 15, 48, 101, 147 and 383 carrying carbapenemase genes. The whole genome sequences were determined by long-read nanopore sequencing. The 12 isolates all contained hybrid plasmids containing both resistance and virulence genes. All carried rmpA/rmpA2 and the aerobactin cluster, with the virulence plasmids of two of three representatives of ST383 carrying blaNDM-5 and seventeen other resistance genes. Representatives of ST48 and ST15 had virulence plasmid-associated genes distributed between two plasmids, both containing antibiotic resistance genes. Representatives of ST101 were remarkable in all sharing virulence plasmids in which iucC and terAWXYZ were missing and iucB and iucD truncated. The combination of resistance and virulence in plasmids of high-risk clones is extremely worrying. Virulence plasmids were often notably consistent within a lineage, even in the absence of epidemiological links, suggesting they are not moving between types. However, there was a common segment containing multiple resistance genes in virulence plasmids of representatives of both STs 48 and 383.

Objectives: We aim to investigate, as a first insight, the presence and rates of high-risk Escherichia coli ST131 clone in Istanbul and evaluate antimicrobial resistance and CTX-M-15 production of ST131 and non-ST131 isolates. The use of MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry) to detect E. coli ST131 clone is also evaluated. Materials and Methods: A total of 203 extended-spectrum beta-lactamase (ESBL)-producing urinary isolates from a training hospital in Istanbul were investigated. Detection of E. coli ST131 was done by MALDI-TOF MS and real-time PCR melting curve analysis. The presence of CTX-M and CTX-M-15 beta-lactamases was investigated by PCR and sequence analysis. Results: Of the 203 isolates, 81 (39.9%) and 75 (36.9%) isolates were identified as ST131 clone by PCR and MALDI-TOF MS, respectively. Resistance to ciprofloxacin was significantly higher among ST131 isolates. A total of 169 (83.5%) isolates produced CTX-M beta-lactamase, of which 72 (43%) were CTX-M-15. The production of CTX-M and CTX-M-15 were significantly higher among ST131 isolates. Conclusions: We have demonstrated, for the first time, high rates of ST131 clone among ESBL-producing E. coli isolates in Istanbul, a region with high rates of resistance to third-generation cephalosporins and fluoroquinolones. Further investigation of this high-risk clone and its contribution to high antimicrobial resistance in Turkey is essential. MALDI-TOF MS is a useful tool for detection of high-risk clones and associated resistance patterns, simultaneous to bacterial identification. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa high-risk clones pose severe threats to public health. Here, we characterize the imipenem/relebactam (IR) resistance mechanisms in P. aeruginosa high-risk clones sequence type 235 (ST235) and ST463 in China. Minimum inhibitory concentrations (MICs) were determined, and Illumina short-read sequencing was performed for 1,168 clinical carbapenem-resistant P. aeruginosa (CRPA) isolates. The gene copy number and expression level were analyzed by Illumina sequencing depth and reverse transcription-quantitative PCR, respectively. Resistance conferred by bla GES-5 was evaluated by cloning experiments. ST463 and ST235 accounted for 9.8% (115/1,168) and 4.5% (53/1,168) of total isolates, respectively, and showed high frequencies of extensively drug-resistant and difficult-to-treat resistant phenotypes. The overall IR-resistant rate in CRPA was 21.0% (245/1,168). However, the IR resistance rate was 81.7% (94/115) in ST463-PA and 52.8% (28/53) in ST235-PA. Of the ST463 isolates, 92.2% (106/115) were Klebsiella pneumoniae carbapenemase-producing P. aeruginosa (KPC-PA), and all 94 IR-resistant ST463-PA produced KPC-2. Compared to IR-susceptible ST463 KPC-2-PA, IR-resistant ST463 KPC-2-PA exhibited significantly higher bla KPC-2 copy numbers and expression levels. In ST463 KPC-2-PA, 16 mg/L relebactam resulted in additional fourfold reductions in imipenem MIC 50/90 values compared to 4 mg/L relebactam. In ST235, 1.9% (1/53) carried bla IMP carbapenemase and 54.7% (29/53) carried bla GES carbapenemase. Other than the IMP producer, all 27 IR-resistant ST235-PA produced GES-5. Cloning experiments revealed that imipenem resistance in bla GES-5 -carrying PAO1 transformants was generally unaffected by relebactam. In conclusion, IR-resistant CRPA isolates in China were mainly distributed in P. aeruginosa high-risk clones ST463 and ST235. The major underlying IR resistance mechanisms were bla KPC-2 overexpression and bla GES-5 carriage., Competing Interests: The authors declare no conflict of interest.

BackgroundSince 2021, an emergence of New Delhi metallo-β-lactamase (NDM)-14-producing Klebsiella pneumoniae has been identified in France. This variant with increased carbapenemase activity was not previously detected in Enterobacterales.AimWe investigated the rapid dissemination of NDM-14 producers among patients in hospitals in France.MethodsAll NDM-14-producing non-duplicate clinical isolates identified in France until June 2022 (n = 37) were analysed by whole genome sequencing. The phylogeny of NDM-14-producers among all K. pneumoniae sequence type (ST) 147 reported in France since 2014 (n = 431) was performed. Antimicrobial susceptibility testing, conjugation experiments, clonal relationship and molecular clock analysis were performed.ResultsThe 37 NDM-14 producers recovered in France until 2022 belonged to K. pneumoniae ST147. The dissemination of NDM-14-producing K. pneumoniae was linked to a single clone, likely imported from Morocco and responsible for several outbreaks in France. The gene bla NDM-14 was harboured on a 54 kilobase non-conjugative IncFIB plasmid that shared high homology with a known bla NDM-1 -carrying plasmid. Using Bayesian analysis, we estimated that the NDM-14-producing K. pneumoniae ST147 clone appeared in 2020. The evolutionary rate of this clone was estimated to 5.61 single nucleotide polymorphisms per genome per year. The NDM-14 producers were highly resistant to all antimicrobials tested except to colistin, cefiderocol (minimum inhibitory concentration 2 mg/L) and the combination of aztreonam/avibactam.ConclusionHighly resistant NDM-14 producing K. pneumoniae can rapidly spread in healthcare settings. Surveillance and thorough investigations of hospital outbreaks are critical to evaluate and limit the dissemination of this clone.