Emergence of silent NDM-1 carbapenemase gene in carbapenem-susceptible Klebsiella pneumoniae: Clinical implications and epidemiological insights.

The global dissemination of carbapenemase genes, particularly blaNDM-1, poses a significant threat to public health. While research has mainly focused on strains with phenotypic resistance, the impact of silent resistance genes has been largely overlooked. This study documents the first instance of silent blaNDM-1 in a cluster of clonally related carbapenem-susceptible K. pneumoniae strains from a single patient. Despite initial effectiveness of carbapenem therapy, the patient experienced four recurrent lung infections over five months, indicating persistent K. pneumoniae infection. Genomic sequencing revealed all strains harbored blaNDM-1 on the epidemic IncX3 plasmid. A deletion within the upstream promoter region (PISAba125) of blaNDM-1 hindered its expression, resulting in phenotypic susceptibility to carbapenems. However, in vitro bactericidal assays and a mouse infection model showed that K. pneumoniae strains with silent blaNDM-1 exhibited significant tolerance to carbapenem-mediated killing. These findings demonstrate that silent blaNDM-1 can mediate both phenotypic susceptibility and antibiotic tolerance. In silico analysis of 1986 blaNDM sequences showed that 1956 (98.5%) retained the original promoter PISAba125. Given that previous genomic sequencing typically targets carbapenem-resistant strains, accurately assessing the prevalence of silent blaNDM remains challenging. This study highlights the hidden threat of silent resistance genes to clinical antimicrobial therapy and calls for enhanced clinical awareness and laboratory detection.

Relative inhibitory activities of the broad-spectrum ß-lactamase inhibitor taniborbactam against metallo-ß-lactamases.

Taniborbactam (TAN) is a novel broad-spectrum ß-lactamase inhibitor with significant activity against subclass B1 metallo-ß-lactamases (MBLs). Here, we showed that TAN exhibited an overall excellent activity against B1 MBLs including most NDM- and VIM-like as well as SPM-1, GIM-1, and DIM-1 enzymes, but not against SIM-1. Noteworthy, VIM-1-like enzymes (particularly VIM-83) were less inhibited by TAN than VIM-2-like. Like NDM-9, NDM-30 (also differing from NDM-1 by a single amino acid substitution) was resistant to TAN.

Structural basis of metallo-ß-lactamase resistance to taniborbactam.

The design of inhibitors against metallo-ß-lactamases (MBLs), the largest family of carbapenemases, has been a strategic goal in designing novel antimicrobial therapies. In this regard, the development of bicyclic boronates, such as taniborbactam (TAN) and xeruborbactam, is a major achievement that may help in overcoming the threat of MBL-producing and carbapenem-resistant Gram-negative pathogens. Of concern, a recent report has shown that New Delhi MBL-9 (NDM-9) escapes the inhibitory action of TAN by a single amino acid substitution with respect to New Delhi MBL-1 (NDM-1), the most widely disseminated MBL. Here, we report a docking and computational analysis that identifies that "escape variants" against TAN can arise by disruption of the electrostatic interaction of negative charges in the active site loops of MBLs with the N-(2-aminoethyl)cyclohexylamine side chain of TAN. These changes result in non-productive binding modes of TAN that preclude reaction with the MBLs, a phenomenon that is not restricted to NDM-9. This analysis demonstrates that single amino acid substitutions in non-essential residues in MBL loops can unexpectedly elicit resistance to TAN.

Relative inhibitory activities of the broad-spectrum ß-lactamase inhibitor xeruborbactam in comparison with taniborbactam against metallo-ß-lactamases produced in Escherichia coli and Pseudomonas aeruginosa.

Xeruborbactam is a newly developed ß-lactamase inhibitor designed for metallo-ß-lactamases (MBLs). This study assessed the relative inhibitory properties of this novel inhibitor in comparison with another MBL inhibitor, namely taniborbactam (TAN), against a wide range of acquired MBL produced either in Escherichia coli or Pseudomonas aeruginosa. As observed with taniborbactam, the combination of xeruborbactam (XER) with ß-lactams, namely, ceftazidime, cefepime and meropenem, led to significantly decreased MIC values for a wide range of B1-type MBL-producing E. coli, including most recombinant strains producing NDM, VIM, IMP, GIM-1, and DIM-1 enzymes. Noteworthily, while TAN-based combinations significantly reduced MIC values of ß-lactams for MBL-producing P. aeruginosa recombinant strains, those with XER were much less effective. We showed that this latter feature was related to the MexAB-OprM efflux pump significantly impacting MIC values when testing XER-based combinations in P. aeruginosa. The relative inhibitory concentrations (IC50 values) were similar for XER and TAN against NDM and VIM enzymes. Noteworthily, XER was effective against NDM-9, NDM-30, VIM-83, and most of IMP enzymes, although those latter enzymes were considered resistant to TAN. However, no significant inhibition was observed with XER against IMP-10, SPM-1, and SIM-1 as well as the representative subclass B2 and B3 enzymes, PFM-1 and AIM-1. The determination of the constant inhibition (Ki) of XER revealed a much higher value against IMP-10 than against NDM-1, VIM-2, and IMP-1. Hence, IMP-10 that differs from IMP-1 by a single amino-acid substitution (Val67Phe) can, therefore, be considered resistant to XER.

The metallo-ß-lactamases strike back: emergence of taniborbactam escape variants.

Metallo-ß-lactamases (MBLs) have evolved relatively rapidly to become an international public health threat. There are no clinically available ß-lactamase inhibitors with activity against MBLs. This may change with the introduction of cefepime-taniborbactam. Herein, we review three manuscripts (S. I. Drusin, C. Le Terrier, L. Poirel, R. A. Bonomo, et al., Antimicrob Agents Chemother 68:e01168-23, 2024, https://doi.org/10.1128/aac.01168-23; C. Le Terrier, C. Viguier, P. Nordmann, A. J. Vila, and L. Poirel, Antimicrob Agents Chemother 68:e00991-23, 2024, https://doi.org/10.1128/aac.00991-23; D. Ono, M. F. Mojica, C. R. Bethel, Y. Ishii, et al., Antimicrob Agents Chemother 68:e01332-23, 2024, https://doi.org/10.1128/aac.01332-23) in which investigators describe elegant experiments to explore MBL/taniborbactam interactions and modifications to MBLs, in response, to reduce the affinity of taniborbactam. Challenges with MBL inhibition will not disappear; rather, they will evolve commensurate with advancements in medicinal chemistry.

Structural role of K224 in taniborbactam inhibition of NDM-1.

Taniborbactam (TAN; VNRX-5133) is a novel bicyclic boronic acid ß-lactamase inhibitor (BLI) being developed in combination with cefepime (FEP). TAN inhibits both serine and some metallo-ß-lactamases. Previously, the substitution R228L in VIM-24 was shown to increase activity against oxyimino-cephalosporins like FEP and ceftazidime (CAZ). We hypothesized that substitutions at K224, the homologous position in NDM-1, could impact FEP/TAN resistance. To evaluate this, a library of codon-optimized NDM K224X clones for minimum inhibitory concentration (MIC) measurements was constructed; steady-state kinetics and molecular docking simulations were next performed. Surprisingly, our investigation revealed that the addition of TAN restored FEP susceptibility only for NDM-1, as the MICs for the other 19 K224X variants remained comparable to those of FEP alone. Moreover, compared to NDM-1, all K224X variants displayed significantly lower MICs for imipenem, tebipenem, and cefiderocol (32-, 133-, and 33-fold lower, respectively). In contrast, susceptibility to CAZ was mostly unaffected. Kinetic assays with the K224I variant, the only variant with hydrolytic activity to FEP comparable to NDM-1, confirmed that the inhibitory capacity of TAN was modestly compromised (IC50 0.01 µM vs 0.14 µM for NDM-1). Lastly, structural modeling and docking simulations of TAN in NDM-1 and in the K224I variant revealed that the hydrogen bond between TAN's carboxylate with K224 is essential for the productive binding of TAN to the NDM-1 active site. In addition to the report of NDM-9 (E149K) as FEP/TAN resistant, this study demonstrates the fundamental role of single amino acid substitutions in the inhibition of NDM-1 by TAN.

Citrobacter spp. and Enterobacter spp. as reservoirs of carbapenemase blaNDM and blaKPC resistance genes in hospital wastewater.

Antibiotic resistance has emerged as a global threat to public health, generating a growing interest in investigating the presence of antibiotic-resistant bacteria in environments influenced by anthropogenic activities. Wastewater treatment plants in hospital serve as significant reservoirs of antimicrobial-resistant bacteria, where a favorable environment is established, promoting the proliferation and transfer of resistance genes among different bacterial species. In our study, we isolated a total of 243 strains from 5 hospital wastewater sites in Mexico, belonging to 21 distinct Gram-negative bacterial species. The presence of ß-lactamase was detected in 46.9% (114/243) of the isolates, which belonging to the Enterobacteriaceae family. We identified a total of 169 ß-lactamase genes; blaTEM in 33.1%, blaCTX-M in 25.4%, blaKPC in 25.4%, blaNDM 8.8%, blaSHV in 5.3%, and blaOXA-48 in 1.1% distributed in 12 different bacteria species. Among the 114 of the isolates, 50.8% were found to harbor at least one carbapenemase and were discharged into the environment. The carbapenemase blaKPC was found in six Citrobacter spp. and E. coli, while blaNDM was detected in two distinct Enterobacter spp. and E. coli. Notably, blaNDM-1 was identified in a 110 Kb IncFII conjugative plasmid in E. cloacae, E. xiangfangensis, and E. coli within the same hospital wastewater. In conclusion, hospital wastewater showed the presence of Enterobacteriaceae carrying a high frequency of carbapenemase blaKPC and blaNDM. We propose that hospital wastewater serves as reservoirs for resistance mechanism within bacterial communities and creates an optimal environment for the exchange of this resistance mechanism among different bacterial strains. IMPORTANCE: The significance of this study lies in its findings regarding the prevalence and diversity of antibiotic-resistant bacteria and genes identified in hospital wastewater in Mexico. The research underscores the urgent need for enhanced surveillance and prevention strategies to tackle the escalating challenge of antibiotic resistance, particularly evident through the elevated frequencies of carbapenemase genes such as blaKPC and blaNDM within the Enterobacteriaceae family. Moreover, the identification of these resistance genes on conjugative plasmids highlights the potential for widespread transmission via horizontal gene transfer. Understanding the mechanisms of antibiotic resistance in hospital wastewater is crucial for developing targeted interventions aimed at reducing transmission, thereby safeguarding public health and preserving the efficacy of antimicrobial therapies.

Unveiling the genetic architecture and transmission dynamics of a novel multidrug-resistant plasmid harboring blaNDM-5 in E. Coli ST167: implications for antibiotic resistance management.

BACKGROUND: The emergence of multidrug-resistant (MDR) Escherichia coli strains poses significant challenges in clinical settings, particularly when these strains harbor New Delhi metallo-ß-lactamase (NDM) gene, which confer resistance to carbapenems, a critical class of last-resort antibiotics. This study investigates the genetic characteristics and implications of a novel blaNDM-5-carrying plasmid pNDM-5-0083 isolated from an E. coli strain GZ04-0083 from clinical specimen in Zhongshan, China. RESULTS: Phenotypic and genotypic evaluations confirmed that the E. coli ST167 strain GZ04-0083 is a multidrug-resistant organism, showing resistance to diverse classes of antibiotics including ß-lactams, carbapenems, fluoroquinolones, aminoglycosides, and sulfonamides, while maintaining susceptibility to monobactams. Investigations involving S1 pulsed-field gel electrophoresis, Southern blot analysis, and conjugation experiments, alongside genomic sequencing, confirmed the presence of the blaNDM-5 gene within a 146-kb IncFIB plasmid pNDM-5-0083. This evidence underscores a significant risk for the horizontal transfer of resistance genes among bacterial populations. Detailed annotations of genetic elements-such as resistance genes, transposons, and insertion sequences-and comparative BLAST analyses with other blaNDM-5-carrying plasmids, revealed a unique architectural configuration in the pNDM-5-0083. The MDR region of this plasmid shares a conserved gene arrangement (repA-IS15DIV-blaNDM-5-bleMBL-IS91-suI2-aadA2-dfrA12) with three previously reported plasmids, indicating a potential for dynamic genetic recombination and evolution within the MDR region. Additionally, the integration of virulence factors, including the iro and sit gene clusters and enolase, into its genetic architecture poses further therapeutic challenges by enhancing the strain's pathogenicity through improved host tissue colonization, immune evasion, and increased infection severity. CONCLUSIONS: The detailed identification and characterization of pNDM-5-0083 enhance our understanding of the mechanisms facilitating the spread of carbapenem resistance. This study illuminates the intricate interplay among various genetic elements within the novel blaNDM-5-carrying plasmid, which are crucial for the stability and mobility of resistance genes across bacterial populations. These insights highlight the urgent need for ongoing surveillance and the development of effective strategies to curb the proliferation of antibiotic resistance.

ST218 Klebsiella pneumoniae became a high-risk clone for multidrug resistance and hypervirulence.

BACKGROUND: The occurrence of multidrug-resistant and hypervirulent Klebsiella pneumoniae (MDR-hvKp) worldwide poses a great challenge for public health. Few studies have focused on ST218 MDR-hvKp. METHODS: Retrospective genomic surveillance was conducted at the Peking University Third Hospital from 2017 and clinical information was obtained. To understand genomic and microbiological characteristics, antimicrobial susceptibility testing, plasmid conjugation and stability, biofilm formation, serum killing, growth curves and whole-genome sequencing were performed. We also assessed the clinical and microbiological characteristics of ST218 compared with ST23. RESULTS: A total of eleven ST218 Kp isolates were included. The most common infection type was lower respiratory tract infection (72.7%, 8/11) in our hospital, whereas ST23 hvKp (72.7%, 8/11) was closely associated with bloodstream infection. Notably, nosocomial infections caused by ST218 (54.5%, 6/11) was slightly higher than ST23 (36.4%, 4/11). All of the ST218 and ST23 strains presented with the virulence genes combination of iucA + iroB + peg344 + rmpA + rmpA2. Interestingly, the virulence score of ST218 was lower than ST23, whereas one ST218 strain (pPEKP3107) exhibited resistance to carbapenems, cephalosporins, ß-lactamase/inhibitors and quinolones and harbored an ~ 59-kb IncN type MDR plasmid carrying resistance genes including blaNDM-1, dfrA14 and qnrS1. Importantly, blaNDM-1 and qnrS1 were flanked with IS26 located within the plasmid that could successfully transfer into E. coli J53. Additionally, PEKP2044 harbored an ~ 41-kb resistance plasmid located within tetA indicating resistance to doxycycline. CONCLUSION: The emergence of blaNDM-1 revealed that there is great potential for ST218 Kp to become a high-risk clone for MDR-hvKp, indicating the urgent need for enhanced genomic surveillance.

Genetic characteristics of chromosomally integrated carbapenemase gene (blaNDM-1) in isolates of Proteus mirabilis.

OBJECTIVE: This study aims to conduct an in-depth genomic analysis of a carbapenem-resistant Proteus mirabilis strain to uncover the distribution and mechanisms of its resistance genes. METHODS: The research primarily utilized whole-genome sequencing to analyze the genome of the Proteus mirabilis strain. Additionally, antibiotic susceptibility tests were conducted to evaluate the strain's sensitivity to various antibiotics, and related case information was collected to analyze the clinical distribution characteristics of the resistant strain. RESULTS: Study on bacterial strain WF3430 from a tetanus and pneumonia patient reveals resistance to multiple antibiotics due to extensive use. Whole-genome sequencing exposes a 4,045,480 bp chromosome carrying 29 antibiotic resistance genes. Two multidrug-resistant (MDR) gene regions, resembling Tn6577 and Tn6589, were identified (MDR Region 1: 64.83 Kb, MDR Region 2: 85.64 Kbp). These regions, consist of integrative and conjugative elements (ICE) structures, highlight the intricate multidrug resistance in clinical settings. CONCLUSION: This study found that a CR-PMI strain exhibits a unique mechanism for acquiring antimicrobial resistance genes, such as blaNDM-1, located on the chromosome instead of plasmids. According to the results, there is increasing complexity in the mechanisms of horizontal transmission of resistance, necessitating a comprehensive understanding and implementation of targeted control measures in both hospital and community settings.

Epidemiological and molecular study of Providencia rettgeri outbreak at a university hospital during the COVID-19 reference center.

In 2014, Brazil detected New Delhi metallo-ß-lactamase (NDM)-producing Enterobacterales from a Providencia rettgeri isolate obtained through surveillance swabs in the Southern region. Subsequently, various species have reported several NDM enzymes. However, comprehensive data on the current epidemiology of NDM-producing P. rettgeri in Brazil remains limited. This study, aimed to provide a detailed characterization of the phenotypic, genotypic, and epidemiological profile of clinical isolates of P. rettgeri NDM. From April 2020 to December 2022, 18 carbapenem-resistant P. rettgeri strains, previously identified using Vitek2®, were isolated at the University Hospital of Londrina. Resistance and virulence genes were assessed through genetic analysis using ERIC PCR and NextSeq (Illumina) sequencing. Statistical analysis was conducted using SPSS version 2.0. Genomic analysis confirmed the presence of ß-lactamase blaNDM-1 and blaOXA-1. All isolates showed the presence of the NDM encoding gene and genetic similarity above 90% between isolates. Clinical parameters of patients infected with P. rettgeri exhibited significant association with mechanical ventilation, prior use of carbapenems, and polymyxins. We also report a significant association between P. rettgeri infection and death outcome. This study characterizes NDM-1 metallo-ß-lactmases isolates, among P. rettgeri isolates from patients at the University Hospital (HU), during the COVID-19 pandemic. The emergence of this novel resistance mechanism among P. rettgeri poses a significant challenge, limiting the therapeutic options for infections in our hospital.

Emergence and genomic chion of Proteus mirabilis harboring blaNDM-1 in Korean companion dogs.

Proteus mirabilis is a commensal bacterium dwelling in the gastrointestinal (GI) tract of humans and animals. Although New Delhi metallo-ß-lactamase 1 (NDM-1) producing P. mirabilis is emerging as a threat, its epidemiology in our society remains largely unknown. LHPm1, the first P. mirabilis isolate harboring NDM-1, was detected from a companion dog that resides with a human owner. The whole-genome study revealed 20 different antimicrobial resistance (AMR) genes against various classes of antimicrobial agents, which corresponded to the MIC results. Genomic regions, including MDR genes, were identified with multiple variations and visualized in a comparative manner. In the whole-genome epidemiological analysis, multiple phylogroups were identified, revealing the genetic relationship of LHPm1 with other P. mirabilis strains carrying various AMR genes. These genetic findings offer comprehensive insights into NDM-1-producing P. mirabilis, underscoring the need for urgent control measures and surveillance programs using a "one health approach".

Inhibitors against New Delhi metallo-betalactamase-1 (NDM-1) and its variants endemic in Indian settings along with the laboratory functional gain mutant of NDM-1.

PURPOSE: The emergence of NDM-1 producing bacteria has become common in both hospital and community settings, but no inhibitor has yet been available for clinical treatment. Hence, demanding the urgent need of NDM-1 inhibitors, we initiated to screen broad spectrum inhibitors against NDM natural variants and laboratory mutant. METHODS: We used docking and molecular dynamics simulations, in silico pharmacokinetic investigations, and density functional theory calculation to characterize molecules. Furthermore, an in vitro study, including MIC, kinetics, and fluorescence study were carried out to confirm the efficacies of the selected compounds. RESULTS: According to the findings of the computational studies, three compounds were effective against NDM variants. Fourfold reduction in MIC of imipenem and meropenem was observed when combined with inhibitors (D2573, D2148, and D63) against blaNDM-1, blaNDM-4, blaNDM-6, and blaNDM-1Q123A, while twofold reduction in MIC of imipenem and meropenem was observed against blaNDM-5 and blaNDM-7. Similarly in the presence of inhibitors (D2573, D2148, and D63) the efficiency of nitrocefin hydrolysis by NDM-4, NDM-6, and Q123A decreases to much more extent as compared to NDM-5 and NDM-7. These results showed that the efficacy of these broad spectrum inhibitors decreases with increasing resistance of NDM variants. CONCLUSION: This is the first time inhibitors were tested against different NDM natural variants which are endemic in Indian settings. Moreover, a functional gain laboratory mutant was also checked for their efficacies. We may propose these molecules for the pre-clinical trial to further translate.

Clinical and molecular characteristics of Klebsiella pneumoniae infection in a tertiary general hospital of Wuhan, China.

OBJECTIVES: The aim of this study was to investigate the clinical and molecular characteristics of Klebsiella pneumoniae infection from a tertiary general hospital in Wuhan, China. METHODS: From December 2019 to August 2022, 311 non-duplicate isolates of K. pneumoniae were collected from a tertiary hospital in Wuhan. These comprised 140 carbapenem-resistant K. pneumoniae (CRKP) isolates and 171 carbapenem-susceptible K. pneumoniae (CSKP) isolates. The clinical characteristics of patients with K. pneumoniae infection were retrospectively collected. Polymerase chain reaction (PCR) assays were used to identify the main carbapenem resistance genes, virulence genes and multi-locus sequence typing (MLST) profiles of the isolates, and the Galleria mellonella infection model was used to determine their virulence phenotypes. RESULTS: Independent risk factors for CRKP infection were hypertension, neurological disorders, being admitted to the intensive care unit (ICU) and prior use of antibiotics. Patient with CRKP infection had higher mortality than those with CSKP infection (23.6% vs 14.0%, P < 0.05). One hundred and two sequence types (STs) were identified among the K. pneumoniae isolates, and the most prevalent ST type was ST11 (112/311, 36.0%). All of the ST11 isolates were CRKP. Among the 112 ST11 isolates, 105 (93.8%) harboured the carbapenem resistance gene blaKPC-2 (ST11-KPC-2), and of these isolates, 78 (74.3%, 78/105) contained all of the four virulence genes, namely rmpA, rmpA2, iroN and iucA, suggesting that these genes were widespread among the isolates responsible for K. pneumoniae infections. CONCLUSION: In this study, ST11-KPC-2 was responsible for most of the K. pneumoniae infection cases. Carbapenem resistance rather than the co-occurrence of the virulence genes rmpA, rmpA2, iroN and iucA was associated with K. pneumoniae infection-related mortality during hospitalisation. Furthermore, a high proportion of ST11-KPC-2 isolates carried all of the four virulence genes.

Genomic epidemiology and molecular characteristics of blaNDM-1-positive carbapenem-resistant Pseudomonas aeruginosa belonging to international high-risk clone ST773 in the Gauteng region, South Africa.

PURPOSE: The emergence of carbapenem-resistant P. aeruginosa (CRPA) harbouring acquired carbapenemase genes (blaVIM, blaIMP and blaNDM) has become a global public health threat. Three CRPA isolates included in the study had an extensively drug-resistant phenotype with susceptibility to colistin only and were positive for the blaNDM-1 gene. The current study aimed to investigate the genomic epidemiology and molecular characteristics of the blaNDM-1-positive CRPA isolates collected from the Gauteng region, South Africa. METHODS: Short read whole genome sequencing (WGS) was performed to determine sequence types (STs), genetic relatedness, resistome, virulome and the genetic environment of the blaNDM-1 gene. RESULTS: The WGS and phylogenetic analyses revealed that the study isolates belonged to an international high-risk clone ST773 and belonged to the same clade with eight blaNDM-1-positive ST773 isolates from Hungary, India, Nigeria, South Korea and USA. The study isolates harboured a wide repertoire of intrinsic and acquired antibiotic resistance genes (ARGs) related with mobile genetic elements, porins and efflux pumps, as well as virulence factor genes. The clade-specific ARGs (blaNDM-1, floR2/cmlA9, rmtB4, tetG) were found in a putative integrative and conjugative element (ICE) region similar to ICE6660-like. CONCLUSION: As ICE carrying the blaNDM-1 gene can easily spread to other P. aeruginosa isolates and other Gram-negative bacteria, the findings in this study highlight the need for appropriate management strategies and active surveillance of CRPA isolates in the Gauteng region, South Africa.

Successful treatment of sino-pulmonary infection & skull base osteomyelitis caused by New Delhi metallo-ß-lactamase-producing Pseudomonas aeruginosa in a renal transplant recipient by using an investigational antibiotic cefepime/zidebactam (WCK 5222).

A case of sino-pulmonary infection with skull base osteomyelitis due to XDR-Pseudomonas aeruginosa in renal transplant recipient was successfully treated with investigational antibiotic, cefepime/zidebactam (WCK 5222). This case highlights challenges in managing XDR-pseudomonal infection where source control was infeasible, antibiotic options were extremely limited and individualized dose adjustments were needed.

Emergence of a clinical Salmonella enterica serovar 1,4,[5], 12: i:-isolate, ST3606, in China with susceptibility decrease to ceftazidime-avibactam carrying a novel blaCTX-M-261 variant and a blaNDM-5.

PURPOSE: The detection rate of Salmonella enterica serovar 1,4,[5], 12: i: - (S. 1,4,[5], 12: i: -) has increased as the most common serotype globally. A S. 1,4,[5], 12: i: - strain named ST3606 (sequence type 34), isolated from a fecal specimen of a child with acute diarrhea hospitalized in a tertiary hospital in China, was firstly reported to be resistant to carbapenem and ceftazidime-avibactam. The aim of this study was to characterize the whole-genome sequence of S. 1,4,[5], 12: i: - isolate, ST3606, and explore its antibiotic resistance genes and their genetic environments. METHODS: The genomic DNA of S. 1,4,[5], 12: i: - ST3606 was extracted and performed with single-molecule real-time sequencing. Resistance genes, plasmid replicon type, mobile elements, and multilocus sequence types (STs) of ST3606 were identified by ResFinder 3.2, PlasmidFinder, OriTfinder database, ISfinder database, and MLST 2.0, respectively. The conjugation experiment was utilized to evaluate the conjugation frequency of pST3606-2. Protein expression and enzyme kinetics experiments of CTX-M were performed to analyze hydrolytic activity of a novel CTX-M-261 enzyme toward several antibiotics. RESULTS: Single-molecule real-time sequencing revealed the coexistence of a 109-kb IncI1-Iα plasmid pST3606-1 and a 70.5-kb IncFII plasmid pST3606-2. The isolate carried resistance genes, including blaNDM-5, sul1, qacE, aadA2, and dfrA12 in pST3606-1, blaTEM-1B, aac(3)-lld, and blaCTX-M-261, a novel blaCTX-M-1 family member, in pST3606-2, and aac(6')-Iaa in chromosome. The blaCTX-M-261 was derived from blaCTX-M-55 by a single-nucleotide mutation 751G>A leading to amino acid substitution of Val for Met at position 251 (Val251Met), which conferred CTX-M increasing resistance to ceftazidime verified by antibiotics susceptibility testing of transconjugants carrying pST3606-2 and steady-state kinetic parameters of CTX-M-261. pST3606-1 is an IncI1-α incompatibility type that shares homology with plasmids of pC-F-164_A-OXA140, pE-T654-NDM-5, p_dm760b_NDM-5, and p_dmcr749c_NDM-5. The conjugation experiment demonstrated that pST3606-2 was successfully transferred to the Escherichia coli recipient C600 with four modules of OriTfinder. CONCLUSION: Plasmid-mediated horizontal transfer plays an important role in blaNDM-5 and blaCTX-M-261 dissemination, which increases the threat to public health due to the resistance to most ß-lactam antibiotics. This is the first report of blaCTX-M-261 and blaNDM-5 in S. 1,4,[5], 12: i: -. The work provides insights into the enzymatic function and demonstrates the ongoing evolution of CTX-M enzymes and confirms urgency to control resistance of S. 1,4,[5], 12: i: -.

Ceftazidime-avibactam alone or in combination with Aztreonam versus Polymyxins in the management of carbapenem-Resistant Klebsiella pneumoniae nosocomial Infections (CAPRI study): a retrospective cohort study from South India.

INTRODUCTION: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections commonly cause hospital-acquired infections. The study aimed to compare the outcomes of CRKP infections between patients receiving ceftazidime avibactam +/- aztreonam and polymyxins in a hospital setting with a high prevalence of New Delhi Metallo Beta Lactamase production. METHODS: We conducted a retrospective cohort study from January 2020 to September 2022 in critically ill adult patients admitted to a non-COVID-19 medical intensive care unit with CRKP infection. The patients were followed up for a total of 30 days or death, whichever was later. RESULTS: Of a total of 106 patients included in the study, 65 patients received polymyxins and 41 patients received ceftazidime-avibactam +/- aztreonam. Higher 30-day mortality was noted in the polymyxin group (56.9% vs. 29.2%, P = 0.005). The mean time to event (mortality) in ceftazidime-avibactam +/- aztreonam was 23.9 + 1.5 days which was significantly higher compared to polymyxins (17.9 + 1.2 days, p = 0.006). On Cox regression analysis, after adjusting for the covariates, the hazard ratio for time to event with the use of polymyxin was 2.02 (95% CI: 1.03-3.9). CONCLUSION: Ceftazidime-avibactam + aztreonam is possibly associated with better clinical outcomes in patients infected with CRKP.

Genomic characterization of New Delhi metallo-beta-lactamase-producing species of Morganellaceae, Yersiniaceae, and Enterobacteriaceae (other than Klebsiella) from Brazil over 2013-2022.

Over the last decade, New Delhi metallo-beta-lactamase (NDM) carbapenemase has silently spread in Brazil. In this study, we analyzed a large collection of Enterobacterales other than Klebsiella spp. received in our reference laboratory between 2013 and 2022. A total of 32 clinical isolates displaying different pulsed-field gel electrophoresis profiles, and represented by 11 species in the families Enterobacteriaceae (Citrobacter freundii, Citrobacter portucalensis, Enterobacter hormaechei, and Escherichia coli), Morganellaceae (Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia rettgeri, Providencia stuartii, and Raoultella ornithinolytica), and Yersiniaceae (Serratia marcescens) had their whole genomes sequenced and further analyzed. Antimicrobial susceptibility was determined by disk diffusion, except for polymyxin B, assessed by broth microdilution. The blaNDM-1 allele was predominant (n = 29), but blaNDM-5 was identified in an E. coli specimen with a novel ST, and the blaNDM-7 allele was found in E. hormaechei ST45 and E. coli ST1049. Polymyxin was active against all but one Enterobacteriaceae isolate: an mcr-1-producing E. coli presenting minimal inhibitory concentration (4 mg/L). Isolates producing extended-spectrum ß-lactamases were common: cefotaximase from Munich (CTX-M)-15 (n = 10), CTX-M-2 (n = 4), and CTX-M-8 (n = 3) were detected, and the mcr-1-producing E. coli was found to co-produce both CTX-M-8 and CTX-M-55 ß-lactamases. The mcr-9 gene was found in 5/8 E. hormaechei isolates, distributed in four different sequence types, all of them presenting susceptibility to polymyxin. This study showed that NDM-producing Enterobacterales other than Klebsiella are already spread in Brazil, in diversified species, and cocarrying important resistance genes. Prompt detection and effective implementation of measures to prevent further spread are mandatory for mitigating the dissemination of NDM carbapenemase in hospital settings and preserving the already limited antimicrobial therapy options.

Aurones and derivatives as promising New Delhi metallo-ß-lactamase (NDM-1) inhibitors.

Bacterial resistance is undoubtedly one of the main public health concerns especially with the emergence of metallo-ß-lactamases (MBLs) able to hydrolytically inactivate ß-lactam antibiotics. Currently, there are no inhibitors of MBLs in clinical use to rescue antibiotic action and the New Delhi metallo-ß-lactamase-1 (NDM-1) is still considered as one of the most relevant targets for inhibitor development. Following a fragment-based strategy to find new NDM-1 inhibitors, we identified aurone as a promising scaffold. A series of 60 derivatives were then evaluated and two of them were identified as promising inhibitors with Ki values as low as 1.7 and 2.5 µM. Moreover, these two most active compounds were able to potentiate meropenem in in vitro antimicrobial susceptibility assays. The molecular modelling provided insights about their likely interactions with the active site of NDM-1, thus enabling further improvement in the structure of this new inhibitor family.