Search

Carbapenem-resistant Klebsiella pneumoniae (CRKP), a superbug that can be difficult or impossible to treat, has become a worldwide problem. This study presents the first report of a CRKP strain carrying a plasmid co-harbouring bla NDM-1 , bla KPC-2 , and tet(A) and the subsequent analysis of its genomic features. Isolation and identification of bacteria, antimicrobial susceptibility test, whole genome sequencing, and conjugation experiments assay were conducted in clinical epidemiological investigations and plasmid genetic characterisation analysis. A total of 116 strains of bacteria were isolated from patients with bloodstream infections (BSI) between 2018 and 2023. A total of 89.66% of the isolates were carbapenem-resistant Enterobacteriaceae (CRE), with the majority (75/116) being CRKP. Among these, a novel plasmid co-harbouring bla NDM-1 , bla KPC-2 , and tet(A) simultaneously was found in CRKP46, and the three genes mediated conjugation by IS 26 , IS Aba125 , and IS 26 , respectively. This plasmid conferred carbapenem resistance to E. coli J53 after conjugative transfer, which was 2 times greater than that of CRKP46. The present study identified the occurrence of a rare plasmid co-harbouring bla NDM-1 , bla KPC-2 , and tet(A) , and the spread of these genes was mediated by the corresponding mobile elements. The increased carbapenem resistance created by this novel plasmid challenges public health security and poses a potential threat to human health; therefore, it deserves attention. [ABSTRACT FROM AUTHOR]

• HvKP prevalence in hospital-acquired infections is notably rising, accompanied by worrisome antibiotic sensitivity decline. • Early identification of hvKP is crucial for timely infection control. • Combining genetic markers and virulence phenotype assays is essential for accurate hvKP assessment. • Exploring alternative treatment strategies, such as phage therapy, is necessary to address escalating hvKP antibiotic resistance. Hypervirulent Klebsiella pneumoniae (hvKP) typically causes severe invasive infections affecting multiple sites in healthy individuals. In the past, hvKP was characterized by a hypermucoviscosity phenotype, susceptibility to antimicrobial agents, and its tendency to cause invasive infections in healthy individuals within the community. However, there has been an alarming increase in reports of multidrug-resistant hvKP, particularly carbapenem-resistant strains, causing nosocomial infections in critically ill or immunocompromised patients. This presents a significant challenge for clinical treatment. Early identification of hvKP is crucial for timely infection control. Notably, identifying hvKP has become confusing due to its prevalence in nosocomial settings and the limited predictive specificity of the hypermucoviscosity phenotype. Novel virulence predictors for hvKP have been discovered through animal models or machine learning algorithms, while standardization of identification criteria is still necessary. Timely source control and antibiotic therapy have been widely employed for the treatment of hvKP infections. Additionally, phage therapy is a promising alternative approach due to escalating antibiotic resistance. In summary, this narrative review highlights the latest research progress in the development, virulence factors, identification, epidemiology of hvKP, and treatment options available for hvKP infection. [Display omitted] [ABSTRACT FROM AUTHOR]

• AN2718 exhibits a strong inhibitory effect on KPC-2. • AN2718 has a synergistic effect in combination with meropenem, restoring the antimicrobial ability of meropenem in vitro and in vivo. • The combination of AN2718 and meropenem is effective on KPC-2 mutants that are resistant to ceftazidime-avibactam, such as KPC-33. • AN2178 has a high degree of safety for clinical use. The global public health threat of antibiotic resistance continues to escalate, and necessitates the implementation of urgent measures to expand the arsenal of antimicrobial drugs. This study identified a benzoxaborane compound, namely 5-chloro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2178), which can inhibit the catalytic activity of the Klebsiella pneumoniae carbapenemase (KPC-2) enzyme effectively. The efficacy of AN2718 as an inhibitor for the KPC-2 enzyme was verified through various assays, including enzyme activity assays and isothermal titration calorimetry. Results of multiple biochemical assays, minimum inhibitory concentration assays and time-killing assays also showed that binding of AN2718 to KPC-2 enabled restoration of the bactericidal effect of meropenem. The survival rate of mice infected with carbapenem-resistant, high-virulence strains increased significantly upon treatment with AN2718. Most importantly, the meropenem and AN2718 combination was effective on KPC-2 mutations such as KPC-33, which evolved clinically and exhibited resistance to ceftazidime-avibactam after clinical use for a couple of years. Comprehensive safety tests both in vitro and in vivo, such as cytotoxicity, haemolytic activity and cytochrome P450 inhibition assays, demonstrated that AN2718 was safe for clinical use. These promising data indicate that AN2718 has high potential for approval for the treatment of drug resistant-bacterial infections, including those caused by ceftazidime-avibactam-resistant strains. AN2718 can be regarded as a valuable addition to the current antimicrobial armamentarium, and a promising tool to combat antimicrobial resistance. [Display omitted] [ABSTRACT FROM AUTHOR]

• The novel bla KPC variants confer ceftazidime-avibactam resistance and carbapenem susceptibility. • bla KPC variants exhibited adaptive advantage over wild type in vitro. • The IS 26 -flanked translocatable unit contributes to the bla KPC amplification. • IS 26 -insertion could influence the distribution of resistance genes and ability of self-conjugation. More and more ceftazidime-avibactam-resistant KPC-producing Klebsiella pneumoniae have been reported with its widespread use, and the detection rate of KPC variants has increased dramatically. However, the evolutionary mechanism and fitness effects during KPC mutation remained unknown. Here, we report the complex in vivo evolutionary trajectories of two novel KPC variants, KPC-155 (L169P/GT242A) and KPC-185 (D179Y/GT242A), from K. pneumoniae in the same patient. The novel variants were shown to confer ceftazidime-avibactam resistance but restore carbapenem susceptibility based on the results of plasmid transformation assays, cloning experiments, and enzyme kinetic measurements. In vitro, competition experiments highlighted the adaptive advantage conferred by strains carrying these KPC variants, which could lead to the rapid spread of these ceftazidime-avibactam-resistant strains. The growth curve indicated that bla KPC-185 had better growth conditions at lower avibactam concentration compared to bla KPC-155 , which was consistent with ceftazidime-avibactam use in vivo. In addition, replicative transposition of the IS 26 -flanked translocatable unit (IS 26 -IS Kpn6 - bla KPC -IS Kpn27 -IS 26) also contributes to the bla KPC amplification and formation of two copies (bla KPC-2 and bla KPC-185), conferring both carbapenem and ceftazidime-avibactam resistance. However, strains with double copies showed reduced competitive advantage and configuration stability. The comparative plasmid analysis of IS 26 group (IS 26 - bla KPC -IS 26) and Tn1721 group (Tn 1721 - bla KPC -IS 26) revealed that IS 26 -insertion could influence the distribution of resistance genes and ability of self-conjugation. The dynamic changes in bla KPC configuration highlight the need for consistent monitoring including antimicrobial susceptibility testing and determination of bla KPC subtypes – during clinical treatment, especially when ceftazidime-avibactam is administered. [Display omitted] [ABSTRACT FROM AUTHOR]

• We employed machine learning on 3928 K. pneumoniae isolates, yielding stable models with AUCs > 0.9 for various antibiotics from both assembly-based data and short read-based data. • Our model introduced a novel "species-specific kmer" strategy which significantly improved plasmid-derived ARG-species attribution to an average accuracy of 96.67%. • A retrospective clinical case study involving 63 cases showed that GenseqAMR could lead to changes in clinical treatment for 24 (38.10%) cases, with 95.83% (23/24) of these changes deemed beneficial. The study aimed to develop a genotypic antimicrobial resistance testing method for Klebsiella pneumoniae using metagenomic sequencing data. We utilized Lasso regression on assembled genomes to identify genetic resistance determinants for six antibiotics (Gentamicin, Tobramycin, Imipenem, Meropenem, Ceftazidime, Trimethoprim/Sulfamethoxazole). The genetic features were weighted, grouped into clusters to establish classifier models. Origin species of detected antibiotic resistant gene (ARG) was determined by novel strategy integrating "possible species," "gene copy number calculation" and "species-specific kmers." The performance of the method was evaluated on retrospective case studies. Our study employed machine learning on 3928 K. pneumoniae isolates, yielding stable models with AUCs > 0.9 for various antibiotics. GenseqAMR, a read-based software, exhibited high accuracy (AUC 0.926–0.956) for short-read datasets. The integration of a species-specific kmer strategy significantly improved ARG-species attribution to an average accuracy of 96.67%. In a retrospective study of 191 K. pneumoniae -positive clinical specimens (0.68–93.39% genome coverage), GenseqAMR predicted 84.23% of AST results on average. It demonstrated 88.76–96.26% accuracy for resistance prediction, offering genotypic AST results with a shorter turnaround time (mean ± SD: 18.34 ± 0.87 hours) than traditional culture-based AST (60.15 ± 21.58 hours). Furthermore, a retrospective clinical case study involving 63 cases showed that GenseqAMR could lead to changes in clinical treatment for 24 (38.10%) cases, with 95.83% (23/24) of these changes deemed beneficial. In conclusion, GenseqAMR is a promising tool for quick and accurate AMR prediction in Klebsiella pneumoniae , with the potential to improve patient outcomes through timely adjustments in antibiotic treatment. Rapid inference of antibiotic resistance and susceptibility for Klebsiella pneumoniae by clinical shotgun metagenomic sequencing.Conclusion: We provide a promising tool, GenseqAMR, for quick and accurate mNGS-AST prediction in Klebsiella pneumoniae, with the potential to improve patient outcomes through timely adjustments in antibiotic treatment. [Display omitted]. [ABSTRACT FROM AUTHOR]

• We identified interspecies IncX3_NDM-5 plasmid transfer and bla KPC mutation under ceftazidime-avibactam pressure in vivo. • The IncX3_NDM-5 plasmid showed strong transmissibility and stability. • We simulated in vitro what happens in vivo using conjugation, single-step selection, and competition experiments. • Further plasmid analysis revealed the diverse origin and genetic structure of plasmid carring bla NDM-5. The rapid dissemination of carbapenem-resistant Enterobacterales (CRE) especially carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a great threat to global public health. Ceftazidime-avibactam, a novel β-lactam/β-lactamase inhibitor combination, has been widely used due to its excellent antibacterial activity against KPC-producing K. pneumoniae. However, several resistance mechanisms have been reported since its use. Here, we conducted a series of in vitro experiments to reveal and demonstrate the dynamic evolution of ceftazidime-avibactam resistance including interspecies IncX3_NDM-5 plasmid transfer between Enterobacter cloacae and K. pneumoniae and bla KPC mutation from bla KPC-2 to bla KPC-33. Through the analysis of conjugation frequency and fitness cost, the IncX3_NDM-5 plasmid in this study showed strong transmissibility and stability in E. coli EC600 and clinical strain K. pneumoniae 5298 as recipient strain. With increasing ceftazidime-avibactam concentration, the conjugation frequency remained at 10−3–10−5, while the mutation frequency of K. pneumoniae 5298 was 10−6–10−8 at the same concentration. Further plasmid analysis (the IncX3_NDM plasmid from this study and other 658 plasmids from the NCBI database) revealed the diverse origin and genetic structure of bla NDM-5 carrying plasmids. E. coli (42.9%), China (43.9%), IncX3 (66.6%) are the most common strains, regions, and Inc types respectively. By analysing of genetic environment detected in IncX3 plasmids, the dominant structures (168/258, 65.1%) were identified: IS Kox3 -IS 26 - bla NDM-5 -IS 5 -IS Aba125 -Tn 3000 -Tn 3. In additon, several structural variations were found in the core gene structure. In conclusion, the high fitness and transmissibility of the IncX3_NDM-5 plasmids were noteworthy. More importantly, the diverse ceftazidime-avibactam resistance mechanisms including bla NDM-5 tranfer and bla KPC-2 mutation highlighted the importance of the continuous monitoring of antimicrobial susceptibility and carbapenemases subtype during ceftazidime-avibactam treatment. [Display omitted] [ABSTRACT FROM AUTHOR]

• bla NDM-1 + genomes harbor a broader geographical distribution and richer ST diversity than bla KPC-2 +. • bla NDM-1 + genomes display difference with bla KPC-2 + genomes in mobile gene elements. • The dissemination of bla NDM-1 depend mainly on horizontal gene transfer, whereas bla KPC-2 is linked to clonal spread. bla NDM-1 and bla KPC-2 are responsible for the global increase in carbapenem-resistant Klebsiella pneumoniae , posing a great challenge to public health. However, the impact of phylogenetic factors on the dissemination of bla NDM-1 and bla KPC-2 is not yet fully understood. This study established a global dataset of 4051 bla NDM-1 + and 10,223 bla KPC-2 + K. pneumoniae genomes, and compared their transmission modes on a global scale. The results showed that bla NDM-1 + K. pneumoniae genomes exhibited a broader geographical distribution and higher sequence type (ST) richness than bla KPC-2 + genomes, indicating higher transmissibility of the bla NDM-1 gene. Furthermore, bla NDM-1 + genomes displayed significant differences in ST lineage, antibiotic resistance gene composition, virulence gene composition and genetic environments compared with bla KPC-2 + genomes, suggesting distinct dissemination mechanisms. bla NDM-1 + genomes were predominantly associated with ST147 and ST16, whereas bla KPC-2 + genomes were mainly found in ST11 and ST258. Significantly different accessory genes were identified between bla NDM-1 + and bla KPC-2 + genomes. The preference for bla KPC-2 distribution across certain countries, ST lineages and genetic environments underscores vertical spread as the primary mechanism driving the expansion of bla KPC-2. In contrast, bla NDM-1 + genomes did not display such a strong preference, confirming that the dissemination of bla NDM-1 mainly depends on horizontal gene transfer. Overall, this study demonstrates different phylogenetic drivers for the dissemination of bla NDM-1 and bla KPC-2 , providing new insights into their global transmission dynamics. [Display omitted] [ABSTRACT FROM AUTHOR]

• Treatment of multidrug-resistant Gram-negative bacteria is increasingly complex. • ESCMID and IDSA guidelines defined optimal strategies when robust data were available. • However, these guidelines could not address more complex infections due to scarcity of literature. • This 'practical approach' paper aimed to provide guidance for these unmet needs. The emergence of multidrug-resistant Gram-negative bacilli and the development of new antibiotics have complicated the selection of optimal regimens. International guidelines are valuable tools, but are limited by the scarcity of high-quality randomized trials in many situations. A panel of experts from the French and Italian Societies of Infectious Diseases aimed to address unresolved issues in clinical practice based on their experience, an updated literature review and open discussions. The panel reached consensus for the following 'first choices': (i) cefepime for ventilator-acquired pneumonia due to AmpC β-lactamase-producing Enterobacterales; (ii) the β-lactam/β-lactamase inhibitor combination most active in vitro , or cefiderocol combined with fosfomycin, and aerosolized colistin or aminoglycosides, for severe pneumonia due to Pseudomonas aeruginosa resistant to ceftolozane-tazobactam; (iii) high-dose piperacillin-tazobactam (including loading dose and continuous infusion) for complicated urinary tract infections (cUTIs) caused by extended-spectrum β-lactamase-producing Enterobacterales with piperacillin-tazobactam minimum inhibitory concentration (MIC) ≤8 mg/L; (iv) high-dose cefepime for cUTIs due to AmpC β-lactamase-producing Enterobacterales other than Enterobacter spp. if cefepime MIC ≤2 mg/L; (v) ceftolozane-tazobactam or ceftazidime-avibactam plus metronidazole for intra-abdominal infections (IAIs) due to third-generation cephalosporin-resistant Enterobacterales; (vi) ceftazidime-avibactam plus aztreonam plus metronidazole for IAIs due to metallo-β-lactamase-producing Enterobacterales; (vii) ampicillin-sulbactam plus colistin for bloodstream infections (BSIs) caused by carbapenem-resistant Acinetobacter baumannii ; (viii) meropenem-vaborbactam for BSIs caused by Klebsiella pneumoniae carbapenemase-producing Enterobacterales; and (ix) ceftazidime-avibactam plus fosfomycin for neurological infections caused by carbapenem-resistant P. aeruginosa. These expert choices were based on the necessary balance between antimicrobial stewardship principles and the need to provide optimal treatment for individual patients in each situation. [Display omitted] [ABSTRACT FROM AUTHOR]

• Aminoglycoside resistance is abundant in Enterobacterales. • Apramycin covers 99% of more than 15 000 blood culture isolates. • Apramycin has potent in vivo efficacy against carbapenem- and aminoglycoside-resistant isolates. The aminoglycoside apramycin has been proposed as a drug candidate for the treatment of critical Gram-negative systemic infections. However, the potential of apramycin in the treatment of drug-resistant bloodstream infections (BSIs) has not yet been assessed. The resistance gene annotations of 40 888 blood-culture isolates were analysed. In vitro profiling of apramycin comprised cell-free translation assays, broth microdilution, and frequency of resistance determination. The efficacy of apramycin was studied in a mouse peritonitis model for a total of nine Escherichia coli and Klebsiella pneumoniae isolates. Genotypic aminoglycoside resistance was identified in 87.8% of all 6973 carbapenem-resistant Enterobacterales blood-culture isolates, colistin resistance was shown in 46.4% and apramycin in 2.1%. Apramycin activity against methylated ribosomes was > 100-fold higher than that for other aminoglycosides. Frequencies of resistance were < 10-9 at 8 × minimum inhibitory concentration (MIC). Tentative epidemiological cut-offs (TECOFFs) were determined as 8 µg/mL for E. coli and 4 µg/mL for K. pneumoniae. A single dose of 5 to 13 mg/kg resulted in a 1-log colony-forming unit (CFU) reduction in the blood and peritoneum. Two doses of 80 mg/kg resulted in an exposure that resembles the AUC observed for a single 30 mg/kg dose in humans and led to complete eradication of carbapenem- and aminoglycoside-resistant bacteraemia. Encouraging coverage and potent in vivo efficacy against a selection of highly drug-resistant Enterobacterales isolates in the mouse peritonitis model warrants the conduct of clinical studies to validate apramycin as a drug candidate for the prophylaxis and treatment of BSI. [Display omitted] [ABSTRACT FROM AUTHOR]

• Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses an immense threat in paediatric intensive care unit patients. • KPC variant is the main reason leading to treatment failure of KPC-producing K. pneumoniae. • KPC-33-producing K. pneumoniae was first reported in children worldwide. • Ceftazidime-avibactam in combination with aztreonam as salvage therapy for ST11 KPC-33-producing K. pneumoniae Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses immense threats to the health of infected patients worldwide, especially children. This study reports the infection caused by CRKP in a paediatric intensive care unit (PICU) child and its drug-resistant mutation during the treatment. Twelve Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains were isolated from the child. Broth microdilution method, plasmid transformation assay, and whole genome sequencing (WGS) were performed to investigate the antimicrobial susceptibility, resistance mechanisms, and genetic structural features of CRKPs. The results showed that 12 strains were highly resistant to most available antimicrobial agents. Among them, K. pneumoniae FD11 and K. pneumoniae FD12 were resistant to ceftazidime-avibactam (CZA, MIC >64 mg/L) and restored the carbapenem susceptibility (Imipenem, MIC =0.25 mg/L; Meropenem, MIC =2 mg/L). The patient improved after treatment with CZA in combination with aztreonam. Plasmid transformation assay demonstrated that the bla KPC-33 -positive transformant increased MICs of CZA by at least 33-fold and 8-fold compared with the recipient Escherichia coli DH5α and bla KPC-2 -positive transformants. WGS analysis revealed that all strains belonged to the ST11-KL64 type and showed highly homologous (3–26 single nucleotide polymorphisms [SNPs]). A single base mutation (G532T) of bla KPC-2 resulted in a tyrosine to aspartic acid substitution at Ambler amino acid position 179 (D179Y), which conferred CZA resistance in K. pneumoniae. This is the first report of a drug-resistant mutation evolving into bla KPC-33 during the treatment of bla KPC-2 -positive CRKP in paediatric-infected patients. It advises clinicians that routine sequential antimicrobial susceptibility testing and KPC genotyping are critical during CZA therapy in children infected with CRKP. [Display omitted] [ABSTRACT FROM AUTHOR]

• During the treatment of ceftazidime-avibactam, bla KPC-2 mutated to bla KPC-44 and bla KPC-71 with resistance to ceftazidime-avibactam. • bla KPC -bearing plasmid transferred between Serratia marcescens and Klebsiella pneumoniae. • The evolution of bla KPC impacted the activity of Klebsiella pneumoniae carbapenemase towards carbapenem and avibactam. • The evolution of bla KPC may destroyed the conjugation efficiency and stability of plasmids. bla KPC -carrying Enterobacterales have post great challenges to global healthcare systems. In this study, we reported the evolution and spread of bla KPC between Serratia marcescens and Klebsiella pneumoniae. Four S. marcescens and one K. pneumoniae strains were isolated from the sputum samples of the patient. Antimicrobial susceptibility tests and whole genome sequencing were performed to investigate the phenotype & genotype of strains. Conjugation assays, cloning experiment and kinetic parameters measuring were performed to explore the spread and antimicrobial resistance mechanisms. The evolution and transmission of bla KPC-2 occurred during the treatment of ceftazidime-avibactam and trimethoprim-sulfamethoxazole. Analysis of the antimicrobial susceptibility and genetic profiles of the clinical strains showed that bla KPC-2 evolved into bla KPC-71 and bla KPC-44 , together with resistance to ceftazidime-avibactam and carbapenems susceptibility recovery under antimicrobial pressure. Cloning and expression of bla KPC-44 & bla KPC-71 in E. coli DH5α showed that KPC-44 and KPC-71 resulted in a 64∼128-fold increase in the MIC value for ceftazidime-avibactam. Meanwhile, the kinetic assays also showed that the enzyme activity of KPC-44 and KPC-71 towards carbapenems was destroyed and couldn't be inhibited by avibactam. Based on the conjugation assay and whole genome sequence analyses, we provided evolutionary insights into the transmission pathway trace of bla KPC -bearing plasmids between S. marcescens and K. pneumoniae. Mixed-species co-infection is one of the risk factors leading to the spread of plasmids carrying carbapenem-resistant genes, and increased surveillance of multidrug-resistant Enterobacterales is urgently needed. [Display omitted] [ABSTRACT FROM AUTHOR]

• CAZ-AVI therapy is associated with a lower 30-day and 90-day mortality rate than those receiving other antibiotic regimens. • CAZ-AVI therapy is associated with a higher clinical improvement and microbiological cure rate than those receiving other antibiotic regimens. • The CAZ-AVI therapy is a promising option for treating infections in patients with or without SOT. Ceftazidime-avibactam (CAZ-AVI) is a new option to treat KPC- and OXA-48 carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. However, clinical evidence is limited regarding its use in treating CRKP infections, especially in solid organ transplantation (SOT) recipients. In this study, we assessed the efficacy of CAZ-AVI in treating CRKP infections in both the general population and the SOT recipients in comparison with other antibiotic regimens. This is a single-centre retrospective cohort study of patients admitted between January 1, 2018 and June 30, 2021 with the diagnosis of CRKP infections receiving either CAZ-AVI or other regimens ≥ 72 hours and clinical outcomes were analysed. Of 200 patients with CRKP infections, 67 received CAZ-AVI, 133 received other regimens, and 50 were SOT recipients. In the SOT cohort, 30 patients received CAZ-AVI, and 20 received other regimens. The overall 30-day mortality was 38% in the SOT cohort. Compared with patients receiving other regimens, CAZ-AVI therapy resulted in lower 30-day mortality (23.3% vs. 60%, P = 0.014) and 90-day mortality (35.7% vs. 86.7%, P = 0.003), higher clinical cure (93.3% vs. 40%, P < 0.001) and microbiological clearance. Similar promising results of CAZ-AVI were also shown in the whole population cohort. Moreover, clinical outcomes of SOT recipients receiving CAZ-AVI were not inferior to those without SOT. CAZ-AVI therapy was associated with better clinical outcomes in CRKP infections in both the general population and SOT recipients. Considering the limitations of the present study, well-conducted RCTs are still warranted to confirm these findings. [ABSTRACT FROM AUTHOR]

• India: a densely populated country with evolving antimicrobial stewardship. • 2010-20: increased antibiotic resistance of Gram-negative ESKAPE in India. • 2010-20: increased resistance against [β-lactams/β-lactamase inhibitors] in India. • India: required intensified antimicrobial stewardship to curb ESKAPE resistance. • India: urgent need for continuous monitoring and documenting antimicrobial resistance. ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) constitute a threat to humans worldwide. India is now the most populous country. The goal was to investigate the evolution of the rates of antimicrobial resistance in ESKAPE pathogens across India over the 2010-20 decade. The data (89 studies) were retrieved from the Medline PubMed repository using specific keywords. The study of 20 177 ESKAPE isolates showed that A. baumannii isolates were the most represented (35.9%, n = 7238), followed by P. aeruginosa (25.3%, n = 5113), K. pneumoniae (19.5%, n = 3934), S. aureus (16.3%, n = 3286), E. faecium (2.6%, n = 517) and Enterobacter spp. (0.4%, n = 89). A notable increase in the resistance rates to antimicrobial agents occurred over the 2010-20 decade. The most important levels of resistance were observed in 2016-20 for A. baumannii (90% of resistance to the amoxicillin-clavulanate combination) and K. pneumoniae (81.6% of resistance to gentamycin). The rise in β-lactamase activities was correlated with an increase in the positivity of Gram-negative isolates for β-lactamase genes. This review highlighted that, in contrast to developed countries that kept resistance levels under control, a considerable increase in resistance to various classes of antibiotics occurred in ESKAPE pathogens in India over the 2010-2020 decade. [ABSTRACT FROM AUTHOR]

• COVID-19 pandemic highlighted the continuing threat of antimicrobial resistance to global health and economic development. • A study to compare the spread of multidrug-resistant organisms (MDROs) in the pre-pandemic and pandemic periods. • Increased risk of isolation of Staphylococcus aureus, Acinetobacter baumannii , and Klebsiella pneumoniae in 2020 and 2021. • Age, DRG-weight, and length of stay were associated with a higher risk of MDRO isolation. • New insights into the impact of the COVID-19 pandemic on MDRO isolation have important implications for infection control. The COVID-19 pandemic has further highlighted the continuing threat of antimicrobial resistance (AMR) to global health and economic development. In the last two decades, AMR has raised increasing concern, with an estimated 4.95 million deaths globally due to bacterial AMR in 2019 alone. The aim of this study was to analyse the impact of the pandemic on the spread of multidrug-resistant organisms (MDROs) using data from the Hospital "P. Giaccone" in Palermo, comparing pre-pandemic and pandemic periods. This observational study involved adult patients who were discharged from the hospital between 01 January 2018 and 31 December 2021. Hospital Discharge Cards were linked with microbiological laboratory reports to assess MDRO isolations. SARS-CoV-2 positivity during hospitalisation was evaluated using the National Institute of Health surveillance system. A total of 58 427 hospitalisations were evaluated in this study. Half the patients were aged over 65 years (N=26 984) and most admissions were in the medical area (N=31 716). During the hospitalisation period, there were 2681 patients (5%) with MDROs isolations, and 946 patients (2%) were positive for SARS-CoV-2. Multivariable analyses showed that during 2020 and 2021, there was a significantly increased risk of isolation of Staphylococcus aureus, Acinetobacter baumannii , and Klebsiella pneumoniae. Age, weight of the Diagnosis-Related Group (DRG), wards with higher intensity of care, and length-of-stay were associated with a higher risk of MDRO isolation. This study provides new insights into the impact of the COVID-19 pandemic on MDRO isolation and has important implications for infection control and prevention efforts in healthcare facilities. Age, DRG-weight, and longer hospital stays further increased the risk of MDRO isolation. Thus, it is imperative to improve and follow hospital protocols to prevent healthcare-associated infections. [ABSTRACT FROM AUTHOR]

• An antibiotic resistance research framework advocating the use of PK/PD approaches. • Leveraging low-cost, high-yield preclinical studies for model-informed drug optimisation. • Combination therapy against PMB-susceptible and PMB-resistant CRKP isolates. Over the last decade, there has been a growing appreciation for the use of in vitro and in vivo infection models to generate robust and informative nonclinical PK/PD data to accelerate the clinical translation of treatment regimens. The objective of this study was to develop a model-based "learn and confirm" approach to help with the design of combination regimens using in vitro infection models to optimise the clinical utility of existing antibiotics. Static concentration time-kill studies were used to evaluate the PD activity of polymyxin B (PMB) and meropenem against two carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates; BAA2146 (PMB-susceptible) and BRKP67 (PMB-resistant). A mechanism-based model (MBM) was developed to quantify the joint activity of PMB and meropenem. In silico simulations were used to predict the time-course of bacterial killing using clinically-relevant PK exposure profiles. The predictive accuracy of the model was further evaluated by validating the model predictions using a one-compartment PK/PD in vitro dynamic infection model (IVDIM). The MBM captured the reduction in bacterial burden and regrowth well in both the BAA2146 and BRKP67 isolate (R2 = 0.900 and 0.940, respectively). The bacterial killing and regrowth predicted by the MBM were consistent with observations in the IVDIM: sustained activity against BAA2146 and complete regrowth of the BRKP67 isolate. Differences observed in PD activity suggest that additional dose optimisation might be beneficial in PMB-resistant isolates. The model-based approach presented here demonstrates the utility of the MBM as a translational tool from static to dynamic in vitro systems to effectively perform model-informed drug optimisation. [ABSTRACT FROM AUTHOR]

• A new approach for dynamic analysis of population structure of large numbers of genomes was developed. • Global distribution patterns of different types of Klebsiella pneumoniae strains were depicted. • Carbapenem-resistance and hypervirulence convergence are spreading and stabilising. Klebsiella pneumoniae is an important clinical bacterial pathogen that has hypervirulent and multidrug-resistant variants. Uniform Manifold Approximation and Projection (UMAP) was used to cluster genomes of 16 797 K. pneumoniae strains collected, based on core genome distance, in over 100 countries during the period 1937 to 2021. A total of 60 high-density genetic clusters of strains representing the major epidemic strains were identified among these strains. Using UMAP bedding, the relationship between genetic cluster, capsular polysaccharide (KL) types and sequence type (ST) of the strains was clearly demonstrated, with some important STs, such as ST11 and ST258, found to contain multiple clusters. Strains within the same cluster often exhibited significant diverse features, such as originating from different areas and being isolated in different years, as well as carriage of different resistance and virulence genes. These data enable the routes of evolution of the globally prevalent K. pneumoniae strains to be traced. Alarmingly, carbapenem-resistant K. pneumoniae strains accounted for 51.7% of the test strains and worldwide transmission was observed. Carbapenem-resistant and hypervirulent K. pneumoniae strains are mainly reported in China; however, these strains are increasingly reported in other parts of the world. Also identified in this study were several key genetic loci that facilitate development of a new K. pneumoniae typing method to differentiate between high- and low-risk strains. In particular, the acrR, ompK35 and hha genes were predicted to play a key role in expression of the resistance and virulence phenotypes. [Display omitted] [ABSTRACT FROM AUTHOR]

• Co-infection of ST11 and ST147 induced by Klebsiella pneumoniae was reported in a patient. • Microbiota dynamics were measured using metagenomic next-generation sequencing (mNGS). • KPC-2 and NDM-1 accounting for carbapenem resistance were detected in CR-Kp. • Plasmid-borne hypervirulence determinants indicate the intermediate to form CR-hvKp. The increasing emergence of hypervirulent Klebsiella pneumoniae (hv-Kp) and carbapenem-resistant K. pneumoniae (CR-Kp) is a serious and substantial public health problem. The use of the last resort antimicrobials, tigecycline and polymyxin to combat infections is complicated by the expanding repertoire of newly-identified CR-hvKp. The transmission and co-occurrence of the corresponding antimicrobial resistance and virulence determinants are largely unknown. The aim of this study was to investigate the dissemination and dynamics of CR-Kp and its antibiotic resistance in a hospitalised patient. Metagenomic next-generation sequencing (mNGS) was conducted for different specimens collected from an elderly male hospitalised patient. CR-Kp strains were examined using antibiotic susceptibility and string testing. Antimicrobial and virulence genes were annotated using whole-genome sequencing (WGS). A clinical case of a patient infected with a variety of CR-Kp isolates was reported. The co-occurrence of KPC-2 and NDM-1 in the patient was revealed. The CR-Kp isolates, such as BALF2, and Sputum T1 and T3, were classified into ST11 and ST147, respectively. The genetic signature (iuc operon) of hypervirulence was identified in strain T1, although string testing indicated its intermediate virulence. In this study, multiple infections of CR-Kp isolates were revealed by mNGS, and their dissemination was attributed to plasmid variations, mgrB inactivation and integrative conjugative elements (ICEs). Furthermore, the finding indicated one likely convergence to form CR-hvKp, different from acquisition of carbapenem-resistance determinants in hvKp. A combination of mNGS and WGS is beneficial for clinical diagnosis and anti-infection therapy, and facilitates a better understanding of genetic variants conferring antimicrobial and virulence properties. [ABSTRACT FROM AUTHOR]

• Osmin has a shorter sequence than melittin and exhibits significant antibacterial activity, anti-biofilm effects, and low toxicity. • Osmin displayed a bactericidal mechanism that rapidly disrupted bacterial membranes. • Osmin was relatively more stable than melittin under the influence of various environmental factors. • Carbapenem-resistant klebsiella pneumoniae (CRKP) did not show any signs of drug resistance development to Osmin even after 30 subcultures in Osmin-containing medium. • Osmin reduced bacterial growth and the expression of pro-inflammatory cytokines and fibrosis-related genes in CRKP-infected mice, demonstrating its potential as an alternative treatment for drug-resistant K. pneumoniae infection. The emergence of multidrug-resistant (MDR) Klebsiella pneumoniae strains causes severe problems in the treatment of bacterial infections owing to limited treatment options. Especially, carbapenem-resistant Klebsiella pneumoniae (CRKP) is rapidly spreading worldwide and is emerging as a new cause of drug-resistant healthcare-associated infections. CRKP also has been announced by the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO) as one of the most pressing antibiotic resistance threats. Antimicrobial peptides (AMPs) are drawing considerable attention as ideal antibiotic alternative candidates to combat MDR bacterial infections. In a previous study, Osmin is composed of 17 amino acids and is isolated from solitary bee (Osmia rufa) venom. Herein, we evaluated the potential of Osmin to be used against drug-resistant K. pneumoniae as an alternative to conventional antibiotics. Osmin exhibited significant antimicrobial and anti-biofilm activity and lower toxicity than melittin, a well-known bee venom peptide. Additionally, we confirmed that it possesses a bactericidal mechanism that rapidly destroys bacterial membranes. Osmin was relatively more stable than melittin under the influence of various environmental factors and unlike conventional antibiotics, it exhibited a low bacterial resistance risk. During in vivo tests, Osmin reduced bacterial growth and the expression of pro-inflammatory cytokines and fibrosis-related genes in mice with CRKP-induced sepsis. Overall, our results indicate a high potential for Osmin to be used as a valuable therapeutic agent against drug-resistant K. pneumoniae infections. [Display omitted] [ABSTRACT FROM AUTHOR]

• Intestinal MDR-GNB (CRE and ESBL) colonisation is prevalent in patients with haematological malignancies. • Prior exposure to carbapenem, tigecycline or penicillin, as well as AML, neutropenia and chemotherapy, are risk factors for CRE colonisation. • Prior antibiotic exposure is a risk factor for ESBL colonisation. Patients with haematological malignancies (HM patients) are at high risk of infections caused by multidrug-resistant Gram-negative bacteria (MDR-GNB). MDR-GNB intestinal colonisation is associated with MDR-GNB infections. The aim of this systematic review and meta-analysis on HM patients was to pool the prevalence of and risk factors for intestinal colonisation by MDR-GNB, including carbapenem-resistant Enterobacterales (CRE) and extended-spectrum β-lactamase (ESBL)-producing Enterobacterales, reported in previous studies. This study was conducted according to the protocol registered in PROSPERO (CRD42022374425). PubMed, Embase, Web of Science, Ovid MEDLINE(R) ALL and Cochrane Library were searched from inception to 25 October 2022. Observational studies reporting CRE and/or ESBL intestinal colonisation in HM patients were included. Subgroup analyses were conducted by study region. A total of 21 402 HM patients from 32 studies were analysed. The pooled CRE and ESBL colonisation rates were 21.7% [95% confidence interval (95%CI) 18.7–24.8] and 19.2% (95%CI 13.9–24.5), respectively. Prior exposure to tigecycline [odds ratio (OR) 3.99, 95%CI 2.08–7.68], carbapenem (OR 1.84, 95%CI 1.13-2.97) or penicillin (OR 1.72, 95%CI 1.05–2.83), as well as chemotherapy (OR 2.45, 95%CI 1.05–5.73), neutropenia (OR 1.88, 95%CI 1.08–3.26) and acute myeloid leukaemia (AML; OR 1.86, 95%CI 1.33–2.61), were risk factors for CRE colonisation in HM patients. Prior antibiotic exposure was a risk factor for ESBL colonisation in HM patients (OR 4.90, 95%CI 2.76–8.70). This study shows the high prevalence of MDR-GNB (CRE and ESBL) colonisation in HM patients and explains associated factors for the colonisation. The results provide evidence for MDR-GNB infection control in HM management. [ABSTRACT FROM AUTHOR]

• A novel Klebsiella pneumoniae carbapenemase (KPC) variant, KPC-109, was detected in a clinical isolate of K. pneumoniae. • KPC-109 carried a six-amino-acid insertion (KYNKDD) in the 270 loop. • KPC-109 affected susceptibility to avibactam-based β-lactam combinations and cefiderocol. • KPC-109 exhibited a trade-off in resistance conferred to carbapenems. • The co-presence of porin alterations resulted in carbapenem resistance in KPC-109 producers. To investigate a ceftazidime/avibactam (CZA)-resistant Klebsiella pneumoniae (NE368), isolated from a patient exposed to CZA, expressing a novel K. pneumoniae carbapenemase (KPC)-3 variant (KPC-109). Antimicrobial susceptibility testing was performed by reference broth microdilution. Whole-genome sequencing (WGS) analysis of NE368 was performed combining a short- and long-reads approach (Illumina and Oxford Nanopore Technologies). Functional characterization of KPC-109 was performed to investigate the impact of KPC-109 production on the β-lactam resistance phenotype of various Escherichia coli and Klebsiella pneumoniae strains, including derivatives of K. pneumoniae with OmpK35 and OmpK36 porin alterations. Horizontal transfer of the KPC-109-encoding plasmid was investigated by conjugation and transformation experiments. K. pneumoniae NE368 was isolated from a patient after repeated CZA exposure, and showed resistance to CZA, fluoroquinolones, piperacillin/tazobactam, expanded-spectrum cephalosporins, amikacin, carbapenems and cefiderocol. WGS revealed the presence of a large chimeric plasmid of original structure (pKPN-NE368), encoding a novel 270-loop mutated KPC-3 variant (KPC-109; ins_270_KYNKDD). KPC-109 production mediated resistance/decreased susceptibility to avibactam-based combinations (with ceftazidime, cefepime and aztreonam) and cefiderocol, with a trade-off on carbapenem resistance. However, in the presence of porin alterations commonly encountered in high-risk clonal lineages of K. pneumoniae , KPC-109 was also able to confer clinical-level resistance to carbapenems. Resistance of NE368 to cefiderocol was likely contributed by KPC-109 production acting in concert with a mutated EnvZ sensor kinase. The KPC-109-encoding plasmid did not appear to be conjugative. These findings expand current knowledge about the diversity of emerging KPC enzyme variants with 270-loop alterations that can be encountered in the clinical setting. [ABSTRACT FROM AUTHOR]

• The roles of colistin resistance-regulating two-component regulatory system (crrAB) were explored. • The crrAB-positive Klebsiella pneumoniae isolates showed higher survival rates in the early stages of exposure to high colistin concentration. • Contribution of crrAB to virulence was identified by serum resistance assay and Galleria mellonella larvae survival assay. • crrAB would provide advantages for Klebsiella pneumoniae survival by increasing the final growth yield and initial survival against colistin. Alternation of the colistin resistance–regulating two-component regulatory system (crrAB) is a colistin-resistance mechanism in Klebsiella pneumoniae (K. pneumoniae), but its role in bacteria is not fully understood. Twelve colistin-susceptible K. pneumoniae clinical isolates were included in this study: six crrAB -positive and six crrAB -negative. We deleted the crrAB genes from two crrAB -positive isolates and complemented them. We measured the growth yields by determining growth curves in lysogeny broth and minimal media with or without Fe2+. In vitro selection rates for colistin resistance were determined by exposure to colistin, and survival rates against high concentrations of colistin (20 mg/L) at the early stage of growth (20 min) were investigated. Virulence was determined using a serum bactericidal assay and Galleria mellonella larval infection. The presence of crrAB was not associated with colistin resistance and did not increase the in vitro selection rate of colistin resistance after exposure. The growth yield of crrAB -positive isolates was higher in lysogeny broth media and increased when Fe2+ was added to minimal media. The crrAB -positive isolates showed higher survival rates in the early stages of exposure to high colistin concentrations. Decreased serum resistance was identified in the crrAB -deleted mutants. More G. mellonella larvae survived when infected by crrAB -deleted mutants, and higher survival rates of bacteria were identified within the larvae infected with wild-type than crrAB -deletant isolates. Conclusion: Through rapid response to external signals, crrAB would provide advantages for K. pneumoniae survival by increasing the final growth yield and initial survival against colistin treatment. This may partly contribute to the bacterial virulence. [ABSTRACT FROM AUTHOR]

• COL-R K. pneumoniae was converted to be COL-S after TGC exposure. • Induced TGC-R mutants showed increased expression level of ramA. • COL-R K. pneumoniae was eliminated by low concentration of COL with TGC. This study investigated the effect of tigecycline exposure on susceptibility of colistin-resistant Klebsiella pneumoniae isolates to colistin and explored the possibility of antibiotic combination at low concentrations to treat colistin-resistant K. pneumoniae isolates. Twelve tigecycline-resistant (TIR) mutants were induced in vitro from wild-type, colistin-resistant, and tigecycline-susceptible K. pneumoniae isolates. Antibiotic susceptibility was determined using the broth microdilution method. The deduced amino acid alterations were identified for genes associated with colistin resistance, lipid A biosynthesis, and tigecycline resistance. Expression levels of genes were compared between wild-type stains and TIR mutants using quantitative real-time polymerase chain reaction (PCR). Lipid A modification was explored using MALDI-TOF mass spectrometry. Time-killing assay was performed to assess the efficiency of combination therapy using low concentrations of colistin and tigecycline. All TIR mutants except one were converted to be susceptible to colistin. These TIR mutants had mutations in the ramR gene and increased expression levels of ramA. Three genes associated with lipid A biosynthesis, lpxC, lpxL , and lpxO , were also overexpressed in TIR mutants, although no mutation was observed. Additional polysaccharides found in colistin-resistant, wild-type strains were modified in TIR mutants. Colistin-resistant K. pneumoniae strains were eliminated in vitro by combining tigecycline and colistin at 2 mg/L. In this study, we found that tigecycline exposure resulted in reduced resistance of colistin-resistant K. pneumoniae to colistin. Such an effect was mediated by regulation of lipid A modification involving ramA and lpx genes. Conclusion: Because of such reduced resistance, a combination of colistin and tigecycline in low concentrations could effectively eradicate colistin-resistant K. pneumoniae strains. [ABSTRACT FROM AUTHOR]

Bacterial multi-drug resistance has become a concern worldwide, especially after the emergence of carbapenemases. Adjuvants with antibacterial potentiation activity can resensitise drug-resistant strains to carbapenems. However, only a few adjuvants with antibacterial potentiation activity are currently available in clinical practice. Here, we first docked the library containing more than 30,000 small molecules to carbapenemases including Klebsiella pneumoniae carbapenemase 2 (KPC-2) and New Delhi metallo-β-lactamase-5 (NDM-5), through in silico virtual screening to obtain lead compounds against carbapenemase-producing Enterobacterales. Meanwhile, the in vitro antibacterial potentiation assays revealed that ibandronate, azacytidine, ribostamycin sulfate and cidofovir exhibited synergistic or additive activity in the presence of meropenem, with good biocompatibility based on red blood cell hemolysis and cell viability tests. Furthermore, the combination of meropenem and azacytidine showed high efficacy in a mouse sepsis model infected with an NDM-5-producing clinical strain, with a 100% survival rate, decreased bacterial burden and alleviated pathological deterioration. These results suggest that the virtual screening is a promising strategy to identify new antibiotic adjuvants targeting carbapenemase-producing Enterobacterales., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

• Long-term-care hospitals are important sources of extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae (KP) infection. • Recent use of urinary catheters and antibiotics are risk factors for ESBL-producing KP. • CTX-M type ESBL genes are prevalent in community-onset KP throughout South Korea. • The most prevalent sequence type among KP isolates was ST23 in South Korea. Although it is essential to know the particular causes of antibiotic-resistant infections in the community, there is lack of evidence regarding risk factors for community-onset extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL-KP) bacteraemia in South Korea. As such, this study aimed to identify risk factors for community-onset ESBL-KP bacteraemia. From May 2016 to April 2017, patients with community-onset KP bloodstream infection (BSI) (n = 408) from six sentinel hospitals participating in the Global Antimicrobial Surveillance System in South Korea were included in this study. Risk factors of ESBL-KP BSI were assessed. Polymerase chain reaction and sequencing to identify genes encoding ESBLs and multi-locus sequence typing were performed. Of the 408 patients with community-onset KP BSI, 70 (17%) had ESBL-KP BSI. Admission to a long-term-care hospital within the previous 3 months [odds ratio (OR) 5.7, 95% confidence interval (CI) 2.1–15.6; P = 0.001], previous use of trimethoprim/sulfamethoxazole (TMP/SMT; OR 11.5, 95% CI 2.7–48.6; P = 0.001) or extended-spectrum cephalosporin (OR 2.2, 95% CI 1.2–3.9; P = 0.01), and previous use of a urinary catheter (OR 2.3, 95% CI 1.1–4.5; P = 0.02) were independent risk factors for community-onset ESBL-KP BSI. ESBL-KP isolates most frequently carried the CTX-M-1 group ESBL (74%, n = 52). The most prevalent sequence type (ST) among the ESBL-KP isolates was ST48 (14%, n = 10). Among non-ESBL-KP isolates, ST23 was most prevalent (21%, n = 70). Previous admission to a long-term-care hospital, use of a urinary catheter and use of TMP/SMT or extended-spectrum cephalosporin within the previous 3 months were identified as risk factors for community-onset ESBL-KP BSI. Strict antibiotic stewardship and infection control measures are needed for long-term-care hospitals. [ABSTRACT FROM AUTHOR]

Accurate assessment of colistin susceptibility is crucial with the increasing number of multidrug-resistant Gram-negative bacteria and simultaneously increasing colistin resistance. Both EUCAST and CLSI recommend broth microdilution (BMD) to determine colistin susceptibility, however it is cumbersome and growth-dependent. In this study, a rapid flow cytometry method (FASTinovⓇ) to determine colistin susceptibility directly from positive blood cultures (BCs) was evaluated. BCs were spiked with 204 Gram-negative bacilli (137 Enterobacterales, 35 Pseudomonas spp. and 32 Acinetobacter baumannii) at a concentration of 2 × 103 cells/bottle, inoculated with human donor blood and incubated until flagged positive. As quality control strains, two susceptible (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) and two resistant (colistin-resistant mcr-1 -positive E. coli NCTC 13846 and Serratia marcescens ATCC 14756) were used. Bacteria were extracted according to assay instructions and were incubated for 1 h at 37 °C with 2 and 4 mg/L colistin and a fluorescent dye, previously optimised. Cells were analysed on CytoFLEX (Beckman Coulter) and AccuriTM C6 Plus (BD Biosciences) flow cytometers. Colistin susceptibility results were automatically provided by BioFAST software (FASTinovⓇ) and compared with those obtained with standard BMD. Overall categorical agreement between this new flow cytometry method and BMD was 99.0%. No very major errors were detected as well as no discrepancies between both flow cytometers. Here we describe a rapid and accurate assay for colistin susceptibility directly from positive BCs with a turnaround time of 2 h versus 48 h required for BMD. This method represents an accurate alternative to standard BMD. [ABSTRACT FROM AUTHOR]

• Colistin-resistant Klebsiella pneumoniae emerged in two hospitals in Rio de Janeiro state, Brazil. • Novel mutations in genes related to colistin resistance. • Diversity of antimicrobial resistance encoding genes and plasmids. Klebsiella pneumoniae is an important human pathogen, able to accumulate and disseminate a variety of antimicrobial resistance genes. Resistance to colistin, one of the last therapeutic options for multi-drug-resistant bacteria, has been reported increasingly. Colistin-resistant K. pneumoniae (ColRKp) emerged in two hospitals in Rio de Janeiro state, Brazil in 2016. The aim of this study was to investigate if these ColRKp isolates were clonally related when compared between hospitals, to identify the molecular mechanisms of colistin resistance, and to describe other antimicrobial resistance genes carried by isolates. Twenty-three isolates were successively recovered, and the whole-genome sequence was analysed for 10, each of a different pulsed-field gel electrophoresis (PFGE) type. Although some PFGE clusters were found, none of them included isolates from both hospitals. Half of the isolates were assigned to CC258, three to ST152 and two to ST15. One isolate was pandrug resistant, one was extensively drug resistant, and the others were multi-drug resistant. Colistin resistance was related to mutations in mgrB, pmrB, phoQ and crrB. Eleven new mutations were found in these genes, including two nucleotide deletions in mgrB. All isolates were carbapenem resistant, and seven were associated with carbapenemase carriage (bla KPC-2 in six isolates and bla OXA-370 in one isolate). All isolates had a bla CTX-M , and two had a 16S ribosomal RNA methyltransferase encoding gene (armA and rmtB). ColRKp were composed of epidemic clones, but cross-dissemination between hospitals was not detected. Colistin resistance emerged with several novel mutations amid highly resistant strains, further restricting the number of drugs available and leading to pandrug resistance. [ABSTRACT FROM AUTHOR]

• Nitroreductase (NR) activity levels vary considerably between Klebsiella pneumoniae isolates. • High NR activity correlates with susceptibility to nitrofurantoin. • Epidemic strains, such as from ST258 and ST11, are almost all resistant to nitrofurantoin. • Design of novel nitro-containing drugs could be useful for treating infections. To understand the potential utility of novel nitroreductase (NR)-activated prodrugs, NR enzyme activity was assessed in clinical Klebsiella pneumoniae isolates using a NR-activated fluorescent probe. NR activity was constant throughout the bacterial growth cycle, but individual K. pneumoniae isolates exhibited a wide range of NR activity levels. The genes of major NR enzymes (nfsA and nfnB) showed a number of sequence variants. Aside from a C-terminal extension of NfnB , which may be responsible for lower NR activity in specific isolates, the genetic differences did not explain the variation in activity. Analysis of important clinical strains (ST11, ST258, ST14 and ST101) showed significant variation in NR activity between isolates within the same sequence type despite conservation of nfsA / nfnB sequences. Addition of methyl viologen (MV), a known activator of soxRS , caused a significant increase in NR activity for all strains, with proportionally larger increases in activity seen for strains with low uninduced NR levels. Real-time PCR on selected strains following exposure to MV showed upregulation of soxS (15–32-fold) and nfsA (5–22-fold) in all strains tested. Expression of nfnB was upregulated 2–5-fold in 4/6 strains tested. High levels of NR activity in the absence of MV activation correlated with nitrofurantoin susceptibility. These data provide evidence that NR gene mutations and regulatory pathways influence NR activity in K. pneumoniae isolates and this is likely to impact treatment efficacy with novel nitro-containing drugs or prodrugs. [ABSTRACT FROM AUTHOR]

• Fosfomycin and nitrofurantoin are common antibiotics for urinary tract infection. • Pharmacodynamic data are crucial for therapy optimisation and prevention of antimicrobial resistance. • Urinary antibacterial activity was investigated in urine samples from healthy volunteers. • Bactericidal effect of fosfomycin is stronger against E. coli compared with K. pneumoniae. • There is discrepancy between nitrofurantoin's ex vivo and clinical bactericidal effects. Given emerging uropathogen resistance to more recent antibiotics, old antibiotics used for uncomplicated urinary tract infection (UTI) warrant re-examination. In this study, the urinary antibacterial activities of fosfomycin and nitrofurantoin were investigated by determining the urinary inhibitory titre and urinary bactericidal titre against uropathogens in urine samples from female volunteers following administration of single-dose fosfomycin (3 g) or nitrofurantoin (50 mg q6h or 100 mg q8h). Urine samples were collected over 48 h (fosfomycin) or 6 or 8 h (nitrofurantoin), with drug levels quantified with every void. Fosfomycin concentrations ranged from <0.75 mg/L [lower limit of quantification (LLOQ)] to 5729.9 mg/L and nitrofurantoin concentrations ranged from <4 mg/L (LLOQ) to 176.3 mg/L (50 mg q6h) or 209.4 mg/L (100 mg q8h). There was discrepancy in the response to fosfomycin between Escherichia coli and Klebsiella pneumoniae , with fosfomycin displaying strong bactericidal activity for 48 h against E. coli but moderate bactericidal activity for 18 h against K. pneumoniae. This effect was not related to the strain's baseline minimum inhibitory concentration but rather to the presence of a resistant subpopulation. Maximum titres of nitrofurantoin were obtained during the first 2 h, but no antibacterial effect was found in most samples regardless of the dose. In the rare samples in which antibacterial activity was detectable, titres were comparable for both species tested. These findings confirm doubts regarding fosfomycin administration in UTIs caused by K. pneumoniae and reveal a discrepancy between nitrofurantoin's measurable ex vivo activity and its clinical effect over multiple dosing intervals. [ABSTRACT FROM AUTHOR]

• Carbapenem-resistant Klebsiella pneumoniae (CRKp) infections are associated with high mortality. • The INCREMENT-CPE score (ICS) allows a categorisation of mortality risk. • External validation of the ICS for all-cause 30-day mortality is needed. • Colistin resistance was not clearly associated with increased mortality. External validation of the INCREMENT-CPE risk score (ICS) for 30-day all-cause mortality is needed. There is also scarce information about whether colistin resistance influences the prognosis of carbapenem-resistant Klebsiella pneumoniae (CRKp) bacteraemia. In this study, the ability of ICS to predict all-cause mortality in the KAPECOR cohort was calculated using the area under the receiver operating characteristic (AUROC) curve. The association of colistin resistance with mortality was studied. The ICS showed an AUROC curve of 0.77 (95% CI 0.68–0.86). A cut-off of 8 points showed 96.8% sensitivity and 50.7% specificity. Mortality of low-risk patients was not different in those treated with monotherapy versus combination therapy. However, mortality of high-risk patients treated with combination therapy (37.8%) was significantly lower than in those treated with monotherapy (68.4%) (P = 0.008). To study the prognostic significance of colistin resistance, 83 selected cases of bacteraemia due to colistin-susceptible CRKp were obtained from the INCREMENT cohort for comparison. Colistin resistance could not be shown to be associated with higher mortality in either the high-risk ICS group [adjusted odds ratio (aOR) = 1.56, 95% CI 0.69–3.33; P = 0.29] or in 37 ICS-matched pairs (aOR = 1.38, 95% CI 0.55–3.42; P = 0.49), or in a sensitivity analysis including only KPC isolates (aOR = 1.81, 95% CI 0.73–4.57; P = 0.20), but the precision of estimates was low. These results validate ICS for all-cause mortality and to optimise targeted therapy for CRKp bacteraemia. Colistin resistance was not clearly associated with increased mortality. [ABSTRACT FROM AUTHOR]

• Carbapenemase-producing Enterobacteriaceae (CPE) cause invasive infections, including prosthetic joint infections (PJIs). • Evaluation of the efficacy of colistin-impregnated cement spacer in a rabbit model of CPE-PJI. • In vitro, colistin elution achieved local concentrations of 20 mg/L around the colistin-impregnated cement spacer. • Colistin-loaded cement spacer + systemic colistin ± meropenem were the most active in terms of bactericidal activity. Carbapenemase-producing Enterobacteriaceae (CPE) are emerging multidrug-resistant bacteria responsible for invasive infections, including prosthetic joint infections (PJIs). Local administration of colistin may provide bactericidal concentrations in situ. This study evaluated the efficacy of a colistin-impregnated cement spacer, alone and in combination with systemic antibiotics, in a rabbit model of CPE-PJI. Elution of 3 MIU of colistimethate sodium (CMS) in 40 g of poly(methyl methacrylate) cement was studied in vitro. In vivo, 5 × 108 CFU of KPC-producing Klebsiella pneumoniae (colistin and meropenem MICs of 1 mg/L and 4 mg/L, respectively) were injected close to a prosthetic knee. Surgical debridement and prosthesis removal were performed 7 days later, and rabbits were assigned to six treatment groups (11–13 rabbits each): drug-free spacer; colistin-loaded spacer; colistin intramuscular (i.m.); colistin i.m. + colistin spacer; colistin i.m. + meropenem subcutaneous (s.c.); and colistin i.m. + meropenem s.c. + colistin spacer. Systemic treatment was administered at doses targeting pharmacokinetics in humans, and rabbits were euthanised 7 days later to evaluate bacterial counts in infected bones. In vitro, CMS elution was low (<0.1% at 24 h) but reached a local concentration of ≥20 mg/L (>20 × MIC). In vivo, combinations of local and systemic colistin, with or without meropenem, were the only regimens superior to the control group (P ≤ 0.05) in terms of viable bacterial counts and the proportion of rabbits with sterile bone, with no emergence of colistin-resistant strains. Colistin-loaded cement spacer in combination with systemic antibiotics were the most effective regimens in this CPE-PJI model. [ABSTRACT FROM AUTHOR]

• Deformation of normal bone architecture usually appears after 2 weeks of infection. • Osteomyelitis caused by CPE is aggressive according to bone modification scales. • Achieving bone sterilisation promotes a lower degree of bone deformation. • Bone modifications under antibiotics is as a valuable proxy to predict outcome. Natural history and treatment of bone infections caused by carbapenemase-producing Enterobacterales (CPE) are poorly defined. We evaluated the effect of treatment on the progression of subacute osteomyelitis in a rabbit model. Two isolates were used: a KPC-producing Klebsiella pneumoniae and an Escherichia coli harbouring bla OXA-48 and bla CTX-M15 inserts, both susceptible to gentamicin, colistin, fosfomycin, and ceftazidime-avibactam. Osteomyelitis was induced in rabbits by tibial injection of 2 × 108 colony-forming units/mL. Antibiotics were started 14 d later, for 7 d, in 6 groups of 12 rabbits. Three days after treatment completion (D24), rabbits were euthanised and bones were cultured. Bone marrow and bone architecture macroscopic changes were evaluated through analysis of pictures by investigators unaware of the rabbit treatment group and microbiological outcome, using scales ranging from 0 (normal) to 3 (severe lesions) depending on modifications. Bone marrow modifications induced by local infection were similar between prematurely deceased animals and non-sterilised animals (P = 0.14) but differed significantly from animals that achieved bone sterilisation after treatment (P = 0.04). Conversely, when comparing bone deformity, rabbits who died early (n = 13) had similar bone architecture as those achieving bone sterilisation (P = 0.12), as opposed to those not sterilised after treatment (P = 0.04). After a multivariate logistic regression, bone marrow scale ≤2 was associated with bone sterilisation (P < 0.001), and bone architecture scale ≤2 was associated with bone sterilisation (adjusted odds ratio = 2.7; 95% confidence interval 1.14–6.37) and KPC infection (adjusted odds ratio = 5.1; 95% confidence interval 2.17–12.13). Effective antibacterial treatment reduces bone architecture distortion and bone marrow changes. These variables may be used as proxy for bone sterilisation. [ABSTRACT FROM AUTHOR]

• Combination of the Rapid Sepsityper Kit and a machine learning (ML)-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) approach for the rapid prediction of methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Klebsiella pneumoniae (CRKP) from positive blood culture bottles. • In total, 47 S. aureus isolates comprising 406 MALDI-TOF MS files and 132 K. pneumoniae isolates comprising 1249 MALDI-TOF MS files were evaluated. • Accuracy, area under the curve, and F1 score for MRSA/MSSA were 0.875, 0.898 and 0.904, respectively; these values for CRKP/CSKP were 0.766, 0.828 and 0.795, respectively. • The novel ML-based MALDI-TOF MS approach enables rapid identification of MRSA and CRKP from flagged blood cultures within 1 h. This enables earlier initiation of targeted antimicrobial therapy, reducing deaths due to sepsis. This study investigated combination of the Rapid Sepsityper Kit and a machine learning (ML)-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) approach for rapid prediction of methicillin-resistant Staphylococcus aureus (MRSA) and carbapenem-resistant Klebsiella pneumoniae (CRKP) from positive blood culture bottles. The study involved 461 patients with monomicrobial bloodstream infections. Species identification was performed using the conventional MALDI-TOF MS Biotyper system and the Rapid Sepsityper protocol. The data underwent preprocessing steps, and ML models were trained using preprocessed MALDI-TOF data and corresponding labels. The interpretability of the model was enhanced using SHapely Additive exPlanations values to identify significant features. In total, 44 S. aureus isolates comprising 406 MALDI-TOF MS files and 126 K. pneumoniae isolates comprising 1249 MALDI-TOF MS files were evaluated. This study demonstrated the feasibility of predicting MRSA among S. aureus and CRKP among K. pneumoniae isolates using MALDI-TOF MS and Sepsityper. Accuracy, area under the receiver operating characteristic curve, and F1 score for MRSA/methicillin-susceptible S. aureus were 0.875, 0.898 and 0.904, respectively; for CRKP/carbapenem-susceptible K. pneumoniae , these values were 0.766, 0.828 and 0.795, respectively. In conclusion, the novel ML-based MALDI-TOF MS approach enables rapid identification of MRSA and CRKP from flagged blood cultures within 1 h. This enables earlier initiation of targeted antimicrobial therapy, reducing deaths due to sepsis. The favourable performance and reduced turnaround time of this method suggest its potential as a rapid detection strategy in clinical microbiology laboratories, ultimately improving patient outcomes. [ABSTRACT FROM AUTHOR]

• First description of extended-spectrum β-lactamase CTX-M-15 in Haemophilus parainfluenzae. • H. parainfluenzae is a potential reservoir of resistance genes. • Increasing prevalence of urogenital infections caused by H. parainfluenzae. • H. parainfluenzae may be a causative agent of sexually-transmitted infections (STIs). • H. parainfluenzae might transmit resistance determinants to other STI-associated pathogens. Haemophilus parainfluenzae is a commensal organism with rising numbers of multidrug-resistant (MDR) strains. This pathogen is of increasing clinical relevance in urogenital infection. The aim of this work was to identify and characterise the molecular mechanisms of resistance associated with four cephalosporin-resistant H. parainfluenzae strains collected from patients with urethritis. Antimicrobial resistance was determined by microdilution following European Committee on Antimicrobial Susceptibility Testing criteria. Strains were then analysed by whole-genome sequencing to determine clonal relationship and the molecular basis of antimicrobial resistance. Finally, a phylogenetic analysis was performed on all urogenital MDR strains of H. parainfluenzae previously isolated in our hospital. All strains were resistant to β-lactams, macrolides, tetracycline, fluoroquinolones, chloramphenicol, cotrimoxazole, and aminoglycosides. The resistance profile was compatible with the presence of an extended-spectrum β-lactamase (ESBL). Whole-genome sequencing detected bla CTX-M-15 that conferred high minimum inhibitory concentrations to cephalosporins in two novel integrative and conjugative elements (ICE HpaHUB6 and ICE HpaHUB7) that also harboured a bla TEM-1 β-lactamase. This study shows a novel bla CTX-M-15 ESBL carried in an integrative conjugative element in four extensively drug-resistant H. parainfluenzae strains. This resistance determinant could be transmitted to other sexually transmitted pathogens and this is a cause for concern. [ABSTRACT FROM AUTHOR]

• Polymyxin B combinations were tested against carbapenemase-producing Klebsiella pneumoniae. • Synergy was frequently observed with fosfomycin, minocycline, and rifampicin. • Wildtype mgrB was associated with synergy of polymyxin B and aztreonam. • Absence of arr-2 / arr-3 was associated with synergy of polymyxin B and rifampicin. • Bacterial genetic determinants of combination effects warrant further investigation. Combination therapy can enhance the activity of available antibiotics against multidrug-resistant Gram-negative bacteria. This study assessed the effects of polymyxin B combinations against carbapenemase-producing Klebsiella pneumoniae (K. pneumoniae). Twenty clinical K. pneumoniae strains producing NDM-1 (n = 8), OXA-48-like (n = 10), or both NDM-1 and OXA-48-like (n = 2) carbapenemases were used. Whole-genome sequencing was applied to detect resistance genes (e.g. encoding antibiotic-degrading enzymes) and sequence alterations influencing permeability or efflux. The activity of polymyxin B in combination with aztreonam, fosfomycin, meropenem, minocycline, or rifampicin was investigated in 24-hour time-lapse microscopy experiments. Endpoint samples were spotted on plates with and without polymyxin B at 4 x MIC to assess resistance development. Finally, associations between synergy and bacterial genetic traits were explored. Synergistic and bactericidal effects were observed with polymyxin B in combination with all other antibiotics: aztreonam (11 of 20 strains), fosfomycin (16 of 20), meropenem (10 of 20), minocycline (18 of 20), and rifampicin (15 of 20). Synergy was found with polymyxin B in combination with fosfomycin, minocycline, or rifampicin against all nine polymyxin-resistant strains. Wildtype mgrB was associated with polymyxin B and aztreonam synergy (P = 0.0499). An absence of arr-2 and arr-3 was associated with synergy of polymyxin B and rifampicin (P = 0.0260). Emergence of populations with reduced polymyxin B susceptibility was most frequently observed with aztreonam and meropenem. Combinations of polymyxin B and minocycline or rifampicin were most active against the tested NDM-1 and OXA-48-like-producing K. pneumoniae. Biologically plausible genotype-phenotype associations were found. Such information might accelerate the search for promising combinations and guide individualised treatment. [ABSTRACT FROM AUTHOR]

• First report of bla NDM-1 / bla NDM-4 and bla OXA-181 co-harbouring strains in Taiwan • Discovery of a novel bla NDM-1 + bla OXA-181 hybrid plasmid • An outbreak of bla NDM-4 + bla OXA-181 co-carriage with enhanced carbapenem resistance • The emergence of KL51 carbapenem-resistant Klebsiella pneumoniae The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) is related to the transmission of carbapenemase genes. Strains carrying more than one carbapenemase with a broadened spectrum of antibiotic resistance have been detected, which is concerning. Although bla KPC -encoding ST11-KL47/KL64 strains are dominant, other clones are emerging. This study investigated 137 CRKP from patients' blood samples in Taiwan. Polymerase chain reaction (PCR) was used to identify carbapenemase genes and capsular (KL) types. Most strains (56%, 77/137) possessed bla KPC alone; however, 12% (17/137) carried bla NDM + bla OXA-48-like and these strains showed high resistance to imipenem and meropenem. Strains carrying bla NDM + bla OXA-48-like predominantly belonged to KL51 (n =15), followed by KL64 (n =1) and KL47 (n =1). Whole-genome sequencing of one KL51 strain indicated that bla NDM-4 and bla OXA-181 are carried on two different plasmids. PCR was performed using specific primers located in these plasmids, and all bla NDM + bla OXA-48-like -encoding strains except the KL64 strain were considered to carry the two abovementioned plasmids. Genome analysis for the KL64 strain revealed that bla NDM-1 and bla OXA-181 are encoded in one plasmid. Notably, the KL51 bla OXA-181 plasmid shared high sequence similarity with the KL64 bla NDM-1 + bla OXA-181 plasmid, except the KL64 plasmid comprised a 15,040-bp insertion encoding bla NDM-1. The data revealed KL51 as a predominant KL type carrying bla NDM-4 + bla OXA-181 , and identified a novel plasmid carrying bla NDM-1 + bla OXA-181 , highlighting the spread of specific plasmids and clones of CRKP in Taiwan. [ABSTRACT FROM AUTHOR]

• The rise of carbapenemase-producing Enterobacterales (CPE) is concerning due to high mortality and limited treatment options. • KPC producer (or CP- K. pneumoniae) colonisation, multiple CPE species colonisation, chronic kidney disease and haematological malignancy were risk factors for CPE bacteraemia in rectal CPE carriers. • When CPE carriers developed Enterobacterales bacteraemia, non-CP Enterobacterales were more frequent than CPE (63.6% vs. 36.4%). • CPE colonisation at multiple sites, KPC coloniser, recent intraabdominal surgery and a shorter duration from colonisation to bacteraemia (< 30 days) were independently associated with CPE bacteraemia compared with non-CP Enterobacterales bacteraemia. Due to high mortality and limited treatment options, the rise in carbapenemase-producing Enterobacterales (CPE) has become a major concern. This study aimed to evaluate the incidence and characteristics of subsequent CPE bacteraemia in rectal CPE carriers and investigate the risk factors for CPE bacteraemia compared with non-carbapenemase-producing (non-CP) Enterobacterales bacteraemia. A retrospective analysis was conducted on adult patients who were confirmed to have CPE colonisation by stool surveillance culture at a tertiary hospital from January 2018 to February 2022. All episodes of Enterobacterales bacteraemia up to 6 months after CPE colonisation were identified. Of 1174 patients identified as rectal CPE carriers, 69 (5.8%; 95% CI 4.6–7.3%) experienced subsequent CPE bacteraemia during the 6 months after the diagnosis of CPE colonisation. Colonisation by a Klebsiella pneumoniae carbapenemase (KPC) producer (or CP- K. pneumoniae), colonisation by multiple CPE species, chronic kidney disease and haematological malignancy were independently associated with CPE bacteraemia in CPE carriers. When CPE carriers developed Enterobacterales bacteraemia, the causative agent was more frequently non-CP Enterobacterales than CPE (63.6% vs. 36.4%). Among these patients, colonisation with a KPC producer, CPE colonisation at multiple sites, shorter duration from colonisation to bacteraemia (< 30 days) and recent intraabdominal surgery were independent risk factors for CPE bacteraemia rather than non-CP Enterobacterales bacteraemia. In CPE carriers, non-CP Enterobacterales were more often responsible for bacteraemia than CPE. Empirical antibiotic therapy for CPE should be considered when sepsis is suspected in a CPE carrier with risk factors for CPE bacteraemia. [ABSTRACT FROM AUTHOR]

• A meta-analysis on treatment benefits of community-acquired pneumonia was done. • Inclusion of macrolides decreased 30-d mortality. • Meta-analysis focused on pneumonia resolution. • The rate of resolution was increased with the addition of macrolides. • The macrolide benefit was shown for studies coming from under-reported countries. • Clinical efficacy was evident against the most prevalent pathogens. This meta-analysis examined the effect of macrolides on resolution of community-acquired pneumonia (CAP) and interpretation of clinical benefit according to microbiology; emphasis is given to data under-reported countries (URCs). This meta-analysis included 47 publications published between 1994 and 2022. Publications were analysed for 30-d mortality (58 759 patients) and resolution of CAP (6465 patients). A separate meta-analysis was done for the prevalence of respiratory pathogens in URCs. Mortality after 30 d was reduced by the addition of macrolides (odds ratio [OR] 0.65, 95% confidence interval [CI] 0.51–0.82). The OR for CAP resolution when macrolides were added to the treatment regimen was 1.23 (95% CI 1.00–1.52). In the CAP resolution analysis, the most prevalent pathogen was Streptococcus pneumoniae (12.68%; 95% CI 9.36–16.95%). Analysis of the pathogen epidemiology from the URCs included 12 publications. The most prevalent pathogens were S. pneumoniae (24.91%) and Klebsiella pneumoniae (12.90%). The addition of macrolides to the treatment regimen led to 35% relative decrease of 30-d mortality and to 23% relative increase in resolution of CAP. [ABSTRACT FROM AUTHOR]

• Imipenem/relebactam and ceftazidime/avibactam resistance was evaluated in K. pneumoniae. • Resistance to imipenem/relebactam and ceftazidime/avibactam develops rapidly in all clones. • Imipenem/relebactam resistance involves OmpK36 disruption and KPC modification. • KPC variants selected with imipenem/relebactam show resistance to relebactam inhibition. • Development of imipenem/relebactam resistance imposes high fitness costs. In order to inform and anticipate potential strategies aimed at combating KPC-producing Klebsiella pneumoniae infections, we analysed imipenem/relebactam and ceftazidime/avibactam single-step mutant frequencies, resistance development trajectories, differentially selected resistance mechanisms and their associated fitness cost using four representative high-risk K. pneumoniae clones. Mutant frequencies and mutant preventive concentrations were determined using agar plates containing incremental concentrations of β-lactam/β-lactamase inhibitor. Resistance dynamics were determined through incubation for 7 days in 10 mL MH tubes containing incremental concentrations of each antibiotic combination up to their 64 × baseline MIC. Two colonies per strain from each experiment were characterized by antimicrobial susceptibility testing, whole genome sequencing and competitive growth assays (to determine in vitro fitness). KPC variants associated with imipenem/relebactam resistance were characterized by cloning and biochemical experiments, atomic models and molecular dynamics simulation studies. Imipenem/relebactam prevented the emergence of single-step resistance mutants at lower concentrations than ceftazidime/avibactam. In three of the four strains evaluated, imipenem/relebactam resistance development emerged more rapidly, and in the ST512/KPC-3 clone reached higher levels compared to baseline MICs than for ceftazidime/avibactam. Lineages evolved in the presence of ceftazidime/avibactam showed KPC substitutions associated with high-level ceftazidime/avibactam resistance, increased imipenem/relebactam susceptibility and low fitness costs. Lineages that evolved in the presence of imipenem/relebactam showed OmpK36 disruption, KPC modifications (S106L, N132S, L167R) and strain-specific substitutions associated with imipenem/relebactam resistance and high fitness costs. Imipenem/relebactam-selected KPC derivatives demonstrated enhanced relebactam resistance through important changes affecting relebactam recognition and positioning. Our findings anticipate potential resistance mechanisms affecting imipenem/relebactam during treatment of KPC-producing K. pneumoniae infections. [ABSTRACT FROM AUTHOR]

• Phenolics showed bacterial anti-virulence potential by modulating autoinducers. • Pomegranate extract showed the highest potential as quorum quencher at low doses. • Grape extract showed a noted inhibition against bacterial biofilms and motility. • Persimmon extract exhibited strong growth inhibition against Klebsiella pneumoniae. • Persimmon and pomegranate extracts caused bacterial membrane damage after exposure. The goal of this study was to determine the effects of phenolic extracts from grape (GrPE), pomegranate (PoPE), and persimmon (PePE) by-products on bacterial virulence activities such as biofilms, motility, energy-dependent efflux pumps, and β-lactamase activity, which are modulated primarily by quorum sensing (QS), defining their potential applications. The microdilution method was used to determine the minimum inhibitory concentration (MIC) and sub-inhibitory concentrations (SICs) of the extracts against reference pathogenic bacteria. The antibacterial mode of action was determined by labelling bacterial cells in in vivo cell-tracking experiments. Antibiograms showed that PoPE inhibited bacteria at lower concentrations, and PePE had a stronger effect against Klebsiella pneumoniae. Both extracts caused significant cell membrane damage (CMD), whereas GrPE did not. At SICs, all extracts showed anti-QS activity, especially PePE, which inhibited violacein and pyocyanin production at 1/128 × MIC. Additionally, QS autoinducers found in Chromobacterium violaceum and Pseudomonas aeruginosa were modulated by the extracts; PePE showed the highest modulation. Antibiofilm assays revealed that GrPE, at MIC and 2 × MIC, acted as a potent antibiofilm agent against biofilms of Pseudomonas putida, Bacillus cereus , and Staphylococcus aureus , which was related to disruption of swarming motility by GrPE. All extracts, especially PoPE, exerted a potent effect against the activation of efflux pumps of P. aeruginosa as well as β-lactamase activity in K. pneumoniae. Results suggest that the anti-virulence potential of the extracts may be related to their effect as extracellular autoinducer modulators. This study allowed to define potential applications of these extracts. [Display omitted] [ABSTRACT FROM AUTHOR]

• The 30-day mortality rate following carbapenem-resistant Klebsiella pneumoniae bloodstream infection was 39.4% in neonates. • The 30-day mortality rate was 43.2% in the non-neonatal population. • There were variations in risk factors for mortality in neonates and older children. • A higher Pitt score was an independent risk factor for 30-day mortality in neonates. • Appropriate targeted antimicrobial treatment was a protective factor in non-neonates. Carbapenem-resistant Klebsiella pneumoniae (CRKP) is increasingly being identified in children, but data on the clinical outcomes in this population are limited. This study aimed to characterise the risk factors for 30-day mortality with CRKP bloodstream infection (BSI) in children. A retrospective study was performed from January 2018 to December 2021 at the First Affiliated Hospital of Zhengzhou University. Patients aged < 18 years and with CRKP BSI were included. Multivariable Cox and logistic regression were performed to determine risk factors for death and the development of septic shock following CRKP infection, respectively. This study identified 33 neonates aged 0–4 weeks and 37 older children. The 30-day mortality rate was 39.4% in neonates and 43.2% in older children. In the neonatal population, a higher Pitt bacteremia score (HR 1.694; 95% CI 1.313–2.186; P < 0.001) was an independent risk factor for 30-day mortality. In the non-neonatal population, higher platelet count (HR 0.990; 95% CI 0.982–0.998; P = 0.010), the use of carbapenems (HR 0.212; 95% CI 0.064–0.702; P = 0.011) and appropriately targeted antimicrobial treatment (HR 0.327; 95% CI 0.111–0.969; P = 0.044) were associated with decreased 30-day mortality. Monocyte count < 0.1 × 109 cells/L (OR 3.615; 95% CI 1.165–11.444; P = 0.026) and a higher Pitt bacteremia score (OR 1.330; 95% CI 1.048–1.688; P = 0.019) were identified as risk factors for the development of septic shock. Carbapenem-resistant Klebsiella pneumoniae BSI was associated with high mortality in children. Appropriate antimicrobial treatment is important to improve survival, but more work is needed to assess the efficacy of specific treatment regimens in children. [ABSTRACT FROM AUTHOR]

• The level of persister formation was reduced in ptsH knockout strain compared to WT. • The ptsH affects the level of cAMP in Klebsiella pneumoniae to promote persister formation. • The ptsH mutant can increase the accumulation of ROS and decrease the antioxidant capacity. Klebsiella pneumoniae is one of the most common opportunistic pathogens causing hospital- and community-acquired infections. Antibiotic resistance in K. pneumoniae has emerged as a major clinical and public health threat. Persisters are specific antibiotic-tolerant bacterial cells. Studies on the mechanism underlying their formation mechanism and growth status are scarce. Therefore, it is urgent to explore the key genes and signalling pathways involved in the formation and recovery process of K. pneumoniae persisters to enhance the understanding and develop relevant treatment strategies. In this study, we treated K. pneumoniae with a lethal concentration of levofloxacin. It resulted in a distinct plateau of surviving levofloxacin-tolerant persisters. Subsequently, we obtained bacterial samples at five different time points during the formation and recovery of K. pneumoniae persisters to perform transcriptome analysis. ptsH gene was observed to be upregulated during the formation of persisters, and down-regulated during the recovery of the persisters. Further, we used CRISPR-Cas9 to construct Δ ptsH, the ptsH -knockout K. pneumoniae strain, and to investigate the effect of ptsH on the persister formation. We observed that ptsH can promote the formation of persisters, reduce accumulation of reactive oxygen species, and enhance antioxidant capacity by reducing cyclic adenosine monophosphate (cAMP) levels. To the best of our knowledge, this is the first study to report that ptsH plays a vital role in forming K. pneumoniae persisters. This study provided important insights to further explore the mechanism underlying the formation of K. pneumoniae persisters and provided a potential target for treating infection with K. pneumoniae persisters. [Display omitted] [ABSTRACT FROM AUTHOR]

• Multicentre surveillance of antimicrobial susceptibility among clinically important Gram-negative bacteria (GNB) in Taiwan. • Increasing carbapenem resistance among clinically important GNB, especially among nosocomial isolates. • bla KPC , especially the bla KPC-2 variant, was detected in 48.4% of carbapenem-resistant Klebsiella pneumoniae isolates. • Low prevalence of the mcr-1 gene. • mcr-1 was detected in different Enterobacteriaceae species, raising concern of potential dissemination. Multicentre surveillance of antimicrobial susceptibility of clinically important Gram-negative bacteria (GNB) from 16 Taiwanese hospitals was performed. Escherichia coli (n = 398), Klebsiella pneumoniae (n = 346), Pseudomonas aeruginosa (n = 252) and Acinetobacter baumannii complex (ABC) (n = 188) bloodstream isolates, non-typhoidal Salmonella (n = 230) and Shigella flexneri (n = 18) from various sources were collected. Antimicrobial MICs were determined using broth microdilution. Genes encoding K. pneumoniae carbapenemases (KPCs), New Delhi metallo-β-lactamases (NDMs), Verona integron-encoded metallo-β-lactamase (VIM), OXA-48-like carbapenemase (OXA-48) as well as mcr-1–5 genes were detected by molecular methods. Rates of carbapenem non-susceptibility were 2.8%, 9.0%, 0.4%, 0%, 10.3% and 48.8% for E. coli, K. pneumoniae, Salmonella, Shigella, P. aeruginosa and ABC, respectively. For carbapenemases, one (0.3%) E. coli harboured bla NDM-1. Fifteen (4.3%), two (0.6%) and two (0.6%) K. pneumoniae contained bla KPC , bla OXA-48 and bla VIM , respectively. Two (0.5%) E. coli and fourteen (4.0%) K. pneumoniae were non-wild-type according to the colistin MIC. Among Enterobacteriaceae with a colistin MIC ≥ 2 mg/L, mcr-1 was detected in one E. coli , two K. pneumoniae and three Salmonella spp. All three mcr-1 -positive Salmonella isolates were collected from community-acquired infections; none of the six mcr-1 -positive Enterobacteriaceae were carbapenem-resistant. Carbapenem resistance has increased among clinically important GNB, especially among hospital-acquired infections. bla KPC , especially the bla KPC-2 variant, was detected in approximately one-half of the carbapenem-resistant K. pneumoniae isolates in this study. Although resistance rates to colistin remained low among Enterobacteriaceae, the finding of mcr-1 from different species raises concern of potential dissemination. [ABSTRACT FROM AUTHOR]

• Piperacillin/tazobactam (TZP) exhibits a semi-log concentration–effect relationship. • Time dependence is maintained for highly resistant Enterobacteriaceae. • The pharmacodynamic target does not change for highly resistant organisms. • The f T >MIC is 49.85% for TZP for resistant Enterobacteriaceae. Urinary tract infections caused by multidrug-resistant Enterobacteriaceae are a growing burden worldwide. Recent studies of urinary pharmacokinetics described high piperacillin/tazobactam (TZP) concentrations in urine, but it is unknown whether this results in treatment efficacy. This study investigated the pharmacodynamics of TZP in a static in vitro model for Enterobacteriaceae to determine the concentration–effect relationship and ultimately the required free (unbound) time above the minimum inhibitory concentration (f T >MIC) required for bacterial killing. The static simulation model investigated TZP f T >MIC between 0% and 100%. Resistant Escherichia coli and Klebsiella pneumoniae isolates with piperacillin/tazobactam MICs of 4096/512, 1024/128 and 128/16 mg/L were investigated; two of the three organisms were carbapenemase-producers. Clinical efficacy was determined as a 3-log reduction over the dosing interval by comparing interval growth with controls. TZP was observed to exhibit time dependence for all organisms. The f T >MIC was determined to be 37.5%, 37.5% and 50% for MICs of 4096/512, 1024/128 and 128/16 mg/L, respectively. Linear regression identified the overall target to be 49.85 ± 16.9% f T >MIC. In conclusion, bactericidal activity against TZP-resistant Enterobacteriaceae occurred at 49.85 ± 16.9% f T >MIC. This suggests that highly resistant urinary organisms, including carbapenemase-producers, with MICs up to 4096/512 mg/L could be treated with TZP. Further investigations are required to elucidate urinary breakpoints and to explore the impact of different resistance mechanisms. Image, graphical abstract [ABSTRACT FROM AUTHOR]