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New Delhi metallo-β-lactamase-1 (NDM-1) is a major determinant of antibiotic resistance responsible for hydrolyzing β-lactam antibiotics, including carbapenems, in Klebsiella pneumoniae. This study aimed to find potential phytocompound inhibitors of NDM-1 from three medicinal plants, Andrographis paniculata, Gymnema sylvestre, and Plumbago auriculata, by using an in silico computational approach. The NDM-1 three-dimensional structure was obtained from the Protein Data Bank, and phytocompound structures were collected from the IMPPAT database. AutoDock Vina has been used for performing molecular docking, followed by visualization and interaction analysis using AutoDock Tools 1.5.7. Pharmacokinetic and toxicity screening (ADMET and ProTox-III) was performed to assess drug likeness and safety. Docking results revealed different inhibitory potentials among screened phytocompounds. Alpha-Amyrin from Plumbago auriculata shows the highest binding affinity (-8.9 kcal/mol), followed by Beta-Amyrin from Gymnema sylvestre and Plumbago auriculata (-8.8 kcal/mol). All selected phytocompounds showed favourable ADMET profiles and low predicted toxicity, with Beta-Amyrin, Alpha-Amyrin, Daucosterol, and Resiniferonol showing the safest toxicity scores. These findings suggest that selected phytocompounds, particularly Alpha-Amyrin and Beta-Amyrin, may provide promising lead compounds for developing NDM-1 enzyme inhibitors. Despite this, further in vitro and in vivo validation is required to confirm their therapeutic potential.

Abstract New Delhi metallo-β-lactamase-1 (NDM-1) has been known to hydrolyze nearly all β-lactam antibiotics, leading to a multidrug-resistant state. Hence, it is important to study its structure and function in relation to controlling infections caused by such resistant bacterial strains. Mutagenesis is one of the approaches used to explore it. No study has been performed to explore the role of non-active site residues in the enzyme activity. This study includes mutations of three non-active site residues to comprehend its structure and function simultaneously. Three non-active site laboratory mutants of NDM-1 were generated by site-directed mutagenesis. The minimum inhibitory concentrations of cefotaxime, cefoxitin, imipenem and meropenem were reduced by up to 4-fold for these mutants compared with wild-type. The hydrolytic activity of mutants was also found to be reduced. Mutants showed a significant change in secondary structure compared with wild-type, as determined by CD spectrophotometry. The catalytic properties and stability of these mutants were found to be reduced. Hence, it revealed an imperative role of non-active site residues in the enzymatic activity of NDM-1.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in Egyptian hospitals has been reported. However, the genetic basis and analysis of the plasmids associated with carbapenem-resistant hypervirulent K. pneumoniae (CR-HvKP) in Egypt have not been presented. Therefore, we attempted to decipher the plasmid sequences that are responsible for transferring the determinants of carbapenem resistance, particularly blaNDM-1 and blaKPC-2. Out of 34 K. pneumoniae isolates collected from two tertiary hospitals in Egypt, 31 were CRKP. Whole-genome sequencing revealed that our isolates were related to 13 different sequence types (STs). The most prevalent ST was ST101, followed by ST383 and ST11. Among the CRKP isolates, one isolate named EBSI036 has been reassessed by Nanopore sequencing. Genetic environment analysis showed that EBSI036 carried 20 antibiotic resistance genes and was identified as a CR-HvKP strain: it harbored four plasmids, namely, pEBSI036-1-NDM-VIR, pEBSI036-2-KPC, pEBSI036-3, and pEBSI036-4. The two carbapenemase genes blaNDM-1 and blaKPC-2 were located on plasmids pEBSI036-1-NDM-VIR and pEBSI036-2-KPC, respectively. The IncFIB:IncHI1B hybrid plasmid pEBSI036-1-NDM-VIR also carried some virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA). Thus, we set out in this study to analyze in depth the genetic basis of the pEBSI036-1-NDM-VIR and pEBSI036-2-KPC plasmids. We report a high-risk clone ST11 KL47 serotype of a CR-HvKP strain isolated from the blood of a 60-year-old hospitalized female patient from the intensive care unit (ICU) in a tertiary care hospital in Egypt, which showed the cohabitation of a novel hybrid plasmid coharboring the blaNDM-1 and virulence genes and a blaKPC-2-carrying plasmid. IMPORTANCE CRKP has been registered in the critical priority tier by the World Health Organization and has become a significant menace to public health. The emergence of CR-HvKP is of great concern in terms of both disease and treatment. In-depth analysis of the carbapenemase-encoding and virulence plasmids may provide insight into ongoing recombination and evolution of virulence and multidrug resistance in K. pneumoniae. Thus, this study serves to alert contagious disease clinicians to the presence of hypervirulence in CRKP isolates in Egyptian hospitals.

A protracted outbreak of New Delhi metallo-β-lactamase (NDM)– producing carbapenem-resistant Klebsiella pneumoniae started in Tuscany, Italy, in November 2018 and continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug-resistant, NDM-producing K. pneumoniae isolates cultured over a 20-mo period from 76 patients at several healthcare facilities in southeast Tuscany. All isolates belonged to high-risk clone ST-147 and were typically nonsusceptible to all first-line antibiotics. Albeit sporadic, resistances to colistin, tigecycline, and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST-147 isolates circulating in Tuscany were monophyletic and highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates), and shared a recent ancestor with clinical isolates from the Middle East. While the bla NDM-1 gene was carried by an IncFIB-type plasmid, our investigations revealed that the ST-147 lineage from Italy also acquired a hybrid IncFIB/IncHIB–type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST-11 and ST-307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of an archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats.

Chih-Cheng Lai,1 Chi-Chung Chen,2,3 Ying-Chen Lu,3 Hung-Jui Chen,4 Bo-An Su,4 Tzu-Chieh Weng,4 Yu-Hsin Chiu,5 Han-Siong Toh,4 Chun-Cheng Zhang,4 Shyh-Ren Chiang,4 Yin-Ching Chuang,2,5 Hung-Jen Tang2,4,6 1Department of Intensive Care Medicine, Chi Mei Medical Center, Liouying, Tainan, Taiwan; 2Department of Medical Research, Chi Mei Medical Center, Tainan, Taiwan; 3Department of Food Science, National Chiayi University, Chiayi, Taiwan; 4Department of Medicine, Chi Mei Medical Center, Tainan, Taiwan; 5Department of Internal Medicine, Chi Mei Medical Center, Liouying, Tainan, Taiwan; 6Department of Health and Nutrition, Chia Nan University of Pharmacy and Science, Tainan, Taiwan Abstract: New-Delhi metallo-β-lactamase1 (NDM-1) Enterobacteriaceae are increasing worldwide. Herein, we describe a single patient who carried three unusual NDM-1 carbapenem-resistant Enterobacteriaceae - Enterobacter cloacae (E. cloacae) yielded from a urine specimen and Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) from stool specimens. For E. cloacae, its bla NDM-1-encoding plasmid was pKP04NDM with a size of ~54 kb replicons with an IncN backbone. For K. pneumoniae, its bla NDM-1-encoding plasmid was pNDM-BTR with a size of ~59.6 kb and belonged to IncN. For E. coli, its main bla NDM-1-encoding plasmid was pIMP-HK1500, and the NDM-1 gene was obtained from a part of pNDM-BTR (8439 bp). These three clinical strains are reported for the first time and are assumed to be imported from mainland China to Taiwan. The three different plasmids were never reported in K. pneumoniae, E. coli, and Citrobacter spp before. Owing to their associated multidrug resistance, appropriate measures of periodic, targeted surveillance, and development of new antimicrobial agents are urgently needed.Keywords: carbapenem-resistant Enterobacteriaceae, NDM-1, K. pneumoniae, E. coli, Citrobacter, plasmid

Purpose: Compared with non-carbapenemase producing carbapenem-resistant Enterobacterales (non-CP-CRE), carbapenemase- producing carbapenem-resistant Enterobacterales (CP-CRE) are associated with considerable mortality. However, given that the patients are treated with various therapeutic options, it remains unclear whether differences in types of carbapenemase genes yield different mortality rates. Therefore, this study aims to identify carbapenemase genes and identify whether clinical outcomes differ according to the prevalence of genotype and phenotype of carbapenemase among Enterobacterales clinical isolated. Patients and Methods: A retrospective cohort study was performed to determine whether types of carbapenemase genes have an impact on clinical outcomes. Carbapenem-resistant clinical isolates were collected at a tertiary care university hospital in Songkhla, Thailand, between June 2018 and February 2020. Demographic and microbiological data such as antimicrobial susceptibility, carbapenemase genes, and overall mortality were evaluated. Results: A total of 121 Enterobacterales clinical isolated were evaluated. The [bla.sub.NDM-1] gene was detected in 44% of the isolates, followed by [bla.sub.OXA-48] (28%) and [bla.sub.NDM-1/OXA-48] (28%). NDM-1- or NDM-1/OXA-48- producing isolates were more likely to require meropenem MICs of [greater than or equal to] 16 mg/L, while OXA-48-producing isolates were more likely to require meropenem MICs of <16 mg/L. The patients with NDM-1 or NDM-1/OXA-48 had a higher 14 days mortality rate than those with OXA-48 after treating with carbapenem-containing regimens (P-value 0.001) or colistin-containing regimens (P-value < 0.001). Conclusion: Our findings suggest that the mortality for CP-CRE infection in patients with NDM-1 or NDM-1/OXA-48 was higher than the mortality in those with OXA-48, which It seems that the type of carbapenemase gene may affect meropenem MIC levels. Hence, in treatment decisions involving the use of either carbapenem-containing regiment or colistin-containing regiment in patients with CP-CRE infection, especially those in the NDM-1 and NDM-1/OXA-48 groups, the patient symptoms should be closely monitored. Keywords: carbapenemase, carbapenem resistance Enterobacterales, NDM-1, OXA-48, NDM-1/OXA-48

Klebsiella pneumoniae is a serious human pathogen associated with resistance to multiple antibiotics and high mortality. K. variicola and K. quasipneumoniae are closely related organisms that are generally considered to be less-virulent opportunistic pathogens. We used a large, comprehensive, population-based strain collection and whole-genome sequencing to investigate infections caused by these organisms in our hospital system. We discovered that K. variicola and K. quasipneumoniae isolates are often misidentified as K. pneumoniae by routine clinical microbiology diagnostics and frequently cause severe life-threatening infections similar to K. pneumoniae . The presence of KPC in K. variicola and K. quasipneumoniae strains as well as NDM-1 metallo-beta-lactamase in one K. variicola strain is particularly concerning because these genes confer resistance to many different beta-lactam antibiotics. The sharing of plasmids, as well as evidence of homologous recombination, between these three species of Klebsiella is cause for additional concern. Klebsiella pneumoniae is a major threat to public health, causing significant morbidity and mortality worldwide. The emergence of highly drug-resistant strains is particularly concerning. There has been a recognition and division of Klebsiella pneumoniae into three distinct phylogenetic groups: Klebsiella pneumoniae , Klebsiella variicola , and Klebsiella quasipneumoniae . K. variicola and K. quasipneumoniae have often been described as opportunistic pathogens that have less virulence in humans than K. pneumoniae does. We recently sequenced the genomes of 1,777 extended-spectrum-beta-lactamase (ESBL)-producing K. pneumoniae isolates recovered from human infections and discovered that 28 strains were phylogenetically related to K . variicola and K. quasipneumoniae . Whole-genome sequencing of 95 additional non-ESBL-producing K. pneumoniae isolates recovered from patients found 12 K. quasipneumoniae strains. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) analysis initially identified all patient isolates as K. pneumoniae , suggesting a potential pitfall in conventional clinical microbiology laboratory identification methods. Whole-genome sequence analysis revealed extensive sharing of core gene content and plasmid replicons among the Klebsiella species. For the first time, strains of both K. variicola and K. quasipneumoniae were found to carry the Klebsiella pneumoniae carbapenemase (KPC) gene, while another K. variicola strain was found to carry the New Delhi metallo-beta-lactamase 1 (NDM-1) gene. K. variicola and K. quasipneumoniae infections were not less virulent than K. pneumoniae infections, as assessed by in-hospital mortality and infection type. We also discovered evidence of homologous recombination in one K. variicola strain, as well as one strain from a novel Klebsiella species, which challenge the current understanding of interrelationships between clades of Klebsiella . IMPORTANCE Klebsiella pneumoniae is a serious human pathogen associated with resistance to multiple antibiotics and high mortality. K. variicola and K. quasipneumoniae are closely related organisms that are generally considered to be less-virulent opportunistic pathogens. We used a large, comprehensive, population-based strain collection and whole-genome sequencing to investigate infections caused by these organisms in our hospital system. We discovered that K. variicola and K. quasipneumoniae isolates are often misidentified as K. pneumoniae by routine clinical microbiology diagnostics and frequently cause severe life-threatening infections similar to K. pneumoniae . The presence of KPC in K. variicola and K. quasipneumoniae strains as well as NDM-1 metallo-beta-lactamase in one K. variicola strain is particularly concerning because these genes confer resistance to many different beta-lactam antibiotics. The sharing of plasmids, as well as evidence of homologous recombination, between these three species of Klebsiella is cause for additional concern.

Molecular analysis of carbapenem-resistant genes in , an emerging pathogen, is less commonly reported from Nepal. In this study we determined the antibiotic susceptibility profile and genetic mechanism of carbapenem resistance in clinical isolates of were isolated from various clinical specimens and identified based on Gram staining, biochemical tests, and PCR amplification of organism specific 16S rRNA and genes. The antibiotic susceptibility testing was performed using disc diffusion and E-test method. Multiplex PCR assays were used to detect the following β-lactamase genes: four class D carbapenem hydrolyzing oxacillinases ( , , and ). Uniplex PCRs were used to detect three class B metallo-β-lactamases genes ( , and ), class C cephalosporin resistance genes ( ), aminoglycoside resistance gene ( ), and IS of all isolates. Insertion sequence IS among NDM-1 positive strains was detected. Clonal relatedness of all isolates were analyzed using repetitive sequence-based PCR (rep-PCR). Of total 44 analyzed isolates, 97.7% (  = 43) were carbapenem-resistant (CR-AB) and 97.7% (  = 43) were multidrug resistant (MDR-AB). One isolate was detected to be extremely drug resistant (XDR-AB). All the isolates were fully susceptible to colistin (MICs < 2 μg/ml). The gene was detected in all isolates, while was detected in 6 isolates (13.6%). Insertion sequence, IS was detected in all of positive isolates. IS was detected in all positive strains. The and genes were detected in 90.1 and 40.1%, respectively. The rep-PCR of all isolates represented 7 different genotypes. We found high prevalence of CR-AB and MDR-AB with gene in a tertiary care hospital in Nepal. Systemic network surveillance should be established for monitoring and controlling the spread of these resistant strains.