Gut symbionts alleviate MASH through a secondary bile acid biosynthetic pathway.

The gut microbiota has been found to play an important role in the progression of metabolic dysfunction-associated steatohepatitis (MASH), but the mechanisms have not been established. Here, by developing a click-chemistry-based enrichment strategy, we identified several microbial-derived bile acids, including the previously uncharacterized 3-succinylated cholic acid (3-sucCA), which is negatively correlated with liver damage in patients with liver-tissue-biopsy-proven metabolic dysfunction-associated fatty liver disease (MAFLD). By screening human bacterial isolates, we identified Bacteroides uniformis strains as effective producers of 3-sucCA both in vitro and in vivo. By activity-based protein purification and identification, we identified an enzyme annotated as ß-lactamase in B. uniformis responsible for 3-sucCA biosynthesis. Furthermore, we found that 3-sucCA is a lumen-restricted metabolite and alleviates MASH by promoting the growth of Akkermansia muciniphila. Together, our data offer new insights into the gut microbiota-liver axis that may be leveraged to augment the management of MASH.

OXCT1 functions as a succinyltransferase, contributing to hepatocellular carcinoma via succinylating LACTB.

Metabolic reprogramming is an important feature of cancers that has been closely linked to post-translational protein modification (PTM). Lysine succinylation is a recently identified PTM involved in regulating protein functions, whereas its regulatory mechanism and possible roles in tumor progression remain unclear. Here, we show that OXCT1, an enzyme catalyzing ketone body oxidation, functions as a lysine succinyltransferase to contribute to tumor progression. Mechanistically, we find that OXCT1 functions as a succinyltransferase, with residue G424 essential for this activity. We also identified serine beta-lactamase-like protein (LACTB) as a main target of OXCT1-mediated succinylation. Extensive succinylation of LACTB K284 inhibits its proteolytic activity, resulting in increased mitochondrial membrane potential and respiration, ultimately leading to hepatocellular carcinoma (HCC) progression. In summary, this study establishes lysine succinyltransferase function of OXCT1 and highlights a link between HCC prognosis and LACTB K284 succinylation, suggesting a potentially valuable biomarker and therapeutic target for further development.

In situ targeted base editing of bacteria in the mouse gut.

Microbiome research is now demonstrating a growing number of bacterial strains and genes that affect our health1. Although CRISPR-derived tools have shown great success in editing disease-driving genes in human cells2, we currently lack the tools to achieve comparable success for bacterial targets in situ. Here we engineer a phage-derived particle to deliver a base editor and modify Escherichia coli colonizing the mouse gut. Editing of a ß-lactamase gene in a model E. coli strain resulted in a median editing efficiency of 93% of the target bacterial population with a single dose. Edited bacteria were stably maintained in the mouse gut for at least 42 days following treatment. This was achieved using a non-replicative DNA vector, preventing maintenance and dissemination of the payload. We then leveraged this approach to edit several genes of therapeutic relevance in E. coli and Klebsiella pneumoniae strains in vitro and demonstrate in situ editing of a gene involved in the production of curli in a pathogenic E. coli strain. Our work demonstrates the feasibility of modifying bacteria directly in the gut, offering a new avenue to investigate the function of bacterial genes and opening the door to the design of new microbiome-targeted therapies.

Structure of a B12-dependent radical SAM enzyme in carbapenem biosynthesis.

Carbapenems are antibiotics of last resort in the clinic. Owing to their potency and broad-spectrum activity, they are an important part of the antibiotic arsenal. The vital role of carbapenems is exemplified by the approval acquired by Merck from the US Food and Drug Administration (FDA) for the use of an imipenem combination therapy to treat the increased levels of hospital-acquired and ventilator-associated bacterial pneumonia that have occurred during the COVID-19 pandemic1. The C6 hydroxyethyl side chain distinguishes the clinically used carbapenems from the other classes of ß-lactam antibiotics and is responsible for their low susceptibility to inactivation by occluding water from the ß-lactamase active site2. The construction of the C6 hydroxyethyl side chain is mediated by cobalamin- or B12-dependent radical S-adenosylmethionine (SAM) enzymes3. These radical SAM methylases (RSMTs) assemble the alkyl backbone by sequential methylation reactions, and thereby underlie the therapeutic usefulness of clinically used carbapenems. Here we present X-ray crystal structures of TokK, a B12-dependent RSMT that catalyses three-sequential methylations during the biosynthesis of asparenomycin A. These structures, which contain the two metallocofactors of the enzyme and were determined in the presence and absence of a carbapenam substrate, provide a visualization of a B12-dependent RSMT that uses the radical mechanism that is shared by most of these enzymes. The structures provide insight into the stereochemistry of initial C6 methylation and suggest that substrate positioning governs the rate of each methylation event.

sRNA expression profile of KPC-2-producing carbapenem-resistant Klebsiella pneumoniae: Functional role of sRNA51.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has significant challenges to human health and clinical treatment, with KPC-2-producing CRKP being the predominant epidemic strain. Therefore, there is an urgent need to identify new therapeutic targets and strategies. Non-coding small RNA (sRNA) is a post-transcriptional regulator of genes involved in important biological processes in bacteria and represents an emerging therapeutic strategy for antibiotic-resistant bacteria. In this study, we analyzed the transcription profile of KPC-2-producing CRKP using RNA-seq. Of the 4693 known genes detected, the expression of 307 genes was significantly different from that of carbapenem-sensitive Klebsiella pneumoniae (CSKP), including 133 up-regulated and 174 down-regulated genes. Both the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly related to metabolism. In addition, we identified the sRNA expression profile of KPC-2-producing CRKP for the first time and detected 115 sRNAs, including 112 newly discovered sRNAs. Compared to CSKP, 43 sRNAs were differentially expressed in KPC-2-producing CRKP, including 39 up-regulated and 4 down-regulated sRNAs. We chose sRNA51, the most significantly differentially expressed sRNA in KPC-2-producing CRKP, as our research subject. By constructing sRNA51-overexpressing KPC-2-producing CRKP strains, we found that sRNA51 overexpression down-regulated the expression of acrA and alleviated resistance to meropenem and ertapenem in KPC-2-producing CRKP, while overexpression of acrA in sRNA51-overexpressing strains restored the reduction of resistance. Therefore, we speculated that sRNA51 could affect the resistance of KPC-2-producing CRKP by inhibiting acrA expression and affecting the formation of efflux pumps. This provides a new approach for developing antibiotic adjuvants to restore the sensitivity of CRKP.

Molecular responses during bacterial filamentation reveal inhibition methods of drug-resistant bacteria.

Bacterial antimicrobial resistance (AMR) is among the most significant challenges to current human society. Exposing bacteria to antibiotics can activate their self-saving responses, e.g., filamentation, leading to the development of bacterial AMR. Understanding the molecular changes during the self-saving responses can reveal new inhibition methods of drug-resistant bacteria. Herein, we used an online microfluidics mass spectrometry system for real-time characterization of metabolic changes of bacteria during filamentation under the stimulus of antibiotics. Significant pathways, e.g., nucleotide metabolism and coenzyme A biosynthesis, correlated to the filamentation of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-E. coli) were identified. A cyclic dinucleotide, c-di-GMP, which is derived from nucleotide metabolism and reported closely related to bacterial resistance and tolerance, was observed significantly up-regulated during the bacterial filamentation. By using a chemical inhibitor, ebselen, to inhibit diguanylate cyclases which catalyzes the synthesis of c-di-GMP, the minimum inhibitory concentration of ceftriaxone against ESBL-E. coli was significantly decreased. This inhibitory effect was also verified with other ESBL-E. coli strains and other beta-lactam antibiotics, i.e., ampicillin. A mutant strain of ESBL-E. coli by knocking out the dgcM gene was used to demonstrate that the inhibition of the antibiotic resistance to beta-lactams by ebselen was mediated through the inhibition of the diguanylate cyclase DgcM and the modulation of c-di-GMP levels. Our study uncovers the molecular changes during bacterial filamentation and proposes a method to inhibit antibiotic-resistant bacteria by combining traditional antibiotics and chemical inhibitors against the enzymes involved in bacterial self-saving responses.

Klebsiella pneumoniae carbapenemase variant 44 acquires ceftazidime-avibactam resistance by altering the conformation of active-site loops.

Klebsiella pneumoniae carbapenemase 2 (KPC-2) is an important source of drug resistance as it can hydrolyze and inactivate virtually all ß-lactam antibiotics. KPC-2 is potently inhibited by avibactam via formation of a reversible carbamyl linkage of the inhibitor with the catalytic serine of the enzyme. However, the use of avibactam in combination with ceftazidime (CAZ-AVI) has led to the emergence of CAZ-AVI-resistant variants of KPC-2 in clinical settings. One such variant, KPC-44, bears a 15 amino acid duplication in one of the active-site loops (270-loop). Here, we show that the KPC-44 variant exhibits higher catalytic efficiency in hydrolyzing ceftazidime, lower efficiency toward imipenem and meropenem, and a similar efficiency in hydrolyzing ampicillin, than the WT KPC-2 enzyme. In addition, the KPC-44 variant enzyme exhibits 12-fold lower AVI carbamylation efficiency than the KPC-2 enzyme. An X-ray crystal structure of KPC-44 showed that the 15 amino acid duplication results in an extended and partially disordered 270-loop and also changes the conformation of the adjacent 240-loop, which in turn has altered interactions with the active-site omega loop. Furthermore, a structure of KPC-44 with avibactam revealed that formation of the covalent complex results in further disorder in the 270-loop, suggesting that rearrangement of the 270-loop of KPC-44 facilitates AVI carbamylation. These results suggest that the duplication of 15 amino acids in the KPC-44 enzyme leads to resistance to CAZ-AVI by modulating the stability and conformation of the 270-, 240-, and omega-loops.

Atypical Mycobacterium abscessus BlaRI Ortholog Mediates Regulation of Energy Metabolism but Not ß-Lactam Resistance.

Mycobacterium abscessus (Mab) is highly drug resistant, and understanding regulation of antibiotic resistance is critical to future antibiotic development. Regulatory mechanisms controlling Mab's ß-lactamase (BlaMab) that mediates ß-lactam resistance remain unknown. S. aureus encodes a prototypical protease-mediated two-component system BlaRI regulating the ß-lactamase BlaZ. BlaR binds extracellular ß-lactams, activating an intracellular peptidase domain which cleaves BlaI to derepress blaZ. Mycobacterium tuberculosis (Mtb) encodes homologs of BlaRI (which we will denote as BlaIR to reflect the inverted gene order in mycobacteria) that regulate not only the Mtb ß-lactamase, blaC, but also additional genes related to respiration. We identified orthologs of blaIRMtb in Mab and hypothesized that they regulate blaMab. Surprisingly, neither deletion of blaIRMab nor overexpression of only blaIMab altered blaMab expression or ß-lactam susceptibility. However, BlaIMab did bind to conserved motifs upstream of several Mab genes involved in respiration, yielding a putative regulon that partially overlapped with BlaIMtb. Prompted by evidence that respiration inhibitors including clofazimine induce the BlaI regulon in Mtb, we found that clofazimine triggers induction of blaIRMab and its downstream regulon. Highlighting an important role for BlaIRMab in adapting to disruptions in energy metabolism, constitutive repression of the BlaIMab regulon rendered Mab highly susceptible to clofazimine. In addition to our unexpected findings that BlaIRMab does not regulate ß-lactam resistance, this study highlights the novel role of mycobacterial BlaRI-type regulators in regulating electron transport and respiration.

In-cell kinetic stability is an essential trait in metallo-ß-lactamase evolution.

Protein stability is an essential property for biological function. In contrast to the vast knowledge on protein stability in vitro, little is known about the factors governing in-cell stability. Here we show that the metallo-ß-lactamase (MBL) New Delhi MBL-1 (NDM-1) is a kinetically unstable protein on metal restriction that has evolved by acquiring different biochemical traits that optimize its in-cell stability. The nonmetalated (apo) NDM-1 is degraded by the periplasmic protease Prc that recognizes its partially unstructured C-terminal domain. Zn(II) binding renders the protein refractory to degradation by quenching the flexibility of this region. Membrane anchoring makes apo-NDM-1 less accessible to Prc and protects it from DegP, a cellular protease degrading misfolded, nonmetalated NDM-1 precursors. NDM variants accumulate substitutions at the C terminus that quench its flexibility, enhancing their kinetic stability and bypassing proteolysis. These observations link MBL-mediated resistance with the essential periplasmic metabolism, highlighting the importance of the cellular protein homeostasis.

Global distribution and genomic characteristics of carbapenemase-producing Escherichia coli among humans, 2005-2023.

Carbapenem-resistant Escherichia coli (CREC) has become a major public health problem worldwide. To date, there is a limited understanding of the global distribution of CREC. In this study, we performed a comprehensive genomic analysis of 7, 731 CRECs of human origin collected from different countries worldwide between 2005 and 2023. Our results showed that these CRECs were distributed in 75 countries, mainly from the United States (17.49%), China (14.88%), and the United Kingdom (14.73%). Eight carbapenemases were identified among the CRECs analyzed, including KPC, IMP, NDM, VIM, OXA, FRI, GES, and IMI. NDM was the most predominant carbapenemase (52.15%), followed by OXA (30.09%) and KPC (14.72%). Notably, all CRECs carried multiple antibiotic resistance genes (ARGs), with 178 isolates carrying mcr-1 and 9 isolates carrying tet(X). The CREC isolates were classified into 465 known sequence types (STs), with ST167 being the most common (11.5%). Correlation analysis demonstrated the significant role of mobile genetic elements in facilitating the transfer of carbapenem resistance genes. Furthermore, some CRECs from different countries showed high genetic similarity, suggesting clonal transmission exists. According to the GWAS results, the genetic difference of blaNDM-positive CRECs from China were mainly enriched in bacterial Type IV secretion system pathways compared with those from the United Kingdom and the United States. Therefore, continuous global surveillance of CRECs is imperative in the future.

Klebsiella pneumoniae carbapenemase variants: the new threat to global public health.

Klebsiella pneumoniae carbapenemase (KPC) variants, which refer to the substitution, insertion, or deletion of amino acid sequence compared to wild blaKPC type, have reduced utility of ceftazidime-avibactam (CZA), a pioneer antimicrobial agent in treating carbapenem-resistant Enterobacterales infections. So far, more than 150 blaKPC variants have been reported worldwide, and most of the new variants were discovered in the past 3 years, which calls for public alarm. The KPC variant protein enhances the affinity to ceftazidime and weakens the affinity to avibactam by changing the KPC structure, thereby mediating bacterial resistance to CZA. At present, there are still no guidelines or expert consensus to make recommendations for the diagnosis and treatment of infections caused by KPC variants. In addition, meropenem-vaborbactam, imipenem-relebactam, and other new ß-lactam-ß-lactamase inhibitor combinations have little discussion on KPC variants. This review aims to discuss the clinical characteristics, risk factors, epidemiological characteristics, antimicrobial susceptibility profiles, methods for detecting blaKPC variants, treatment options, and future perspectives of blaKPC variants worldwide to alert this new great public health threat.

What Contributes to the Minimum Inhibitory Concentration? Beyond ß-Lactamase Gene Detection in Klebsiella pneumoniae.

BACKGROUND: Klebsiella pneumoniae is capable of resistance to ß-lactam antibiotics through expression of ß-lactamases (both chromosomal and plasmid-encoded) and downregulation of outer membrane porins. However, the extent to which these mechanisms interplay in a resistant phenotype is not well understood. The purpose of this study was to determine the extent to which ß-lactamases and outer membrane porins affected ß-lactam resistance. METHODS: Minimum inhibitory concentrations (MICs) to ß-lactams and inhibitor combinations were determined by agar dilution or Etest. Outer membrane porin production was evaluated by Western blot of outer membrane fractions. ß-lactamase carriage was determined by whole genome sequencing and expression evaluated by real-time reverse-transcription polymerase chain reaction. RESULTS: Plasmid-encoded ß--lactamases were important for cefotaxime and ceftazidime resistance. Elevated expression of chromosomal SHV was important for ceftolozane-tazobactam resistance. Loss of outer membrane porins was predictive of meropenem resistance. Extended-spectrum ß-lactamases and plasmid-encoded AmpCs (pAmpCs) in addition to porin loss were sufficient to confer resistance to the third-generation cephalosporins, piperacillin-tazobactam, ceftolozane-tazobactam, and meropenem. pAmpCs (CMY-2 and DHA) alone conferred resistance to piperacillin-tazobactam. CONCLUSIONS: Detection of a resistance gene by whole genome sequencing was not sufficient to predict resistance to all antibiotics tested. Some ß-lactam resistance was dependent on the expression of both plasmid-encoded and chromosomal ß-lactamases and loss of porins.

Development of Inhibitory Compounds for Metallo-beta-lactamase through Computational Design and Crystallographic Analysis.

Metallo-ß-lactamases (MBL) deactivate ß-lactam antibiotics through a catalytic reaction caused by two zinc ions at the active center. Since MBLs deteriorate a wide range of antibiotics, they are dangerous factors for bacterial multidrug resistance. In this work, organic synthesis, computational design, and crystal structure analysis were performed to obtain potent MBL inhibitors based on a previously identified hit compound. The hit compound comprised 3,4-dihydro-2(1H)-quinolinone linked with a phenyl-ether-methyl group via a thiazole ring. In the first step, the thiazole ring was replaced with a tertiary amine to avoid the planar structure. In the second step, we virtually modified the compound by keeping the quinolinone backbone. Every modified compound was bound to a kind of MBL, imipenemase-1 (IMP-1), and the binding pose was optimized by a molecular mechanics calculation. The binding scores were evaluated for the respective optimized binding poses. Given the predicted binding poses and calculated binding scores, candidate compounds were determined for organic syntheses. The inhibitory activities of the synthesized compounds were measured by an in vitro assay for two kinds of MBLs, IMP-1 and New Delhi metallo-ß-lactamase (NDM-1). A quinolinone connected with an amine bound with methyl-phenyl-ether-propyl and cyclohexyl-ethyl showed a 50% inhibitory concentration of 4.8 µM. An X-ray crystal analysis clarified the binding structure of a synthesized compound to IMP-1. The δ-lactam ring of quinolinone was hydrolyzed, and the generated carboxyl group was coordinated with zinc ions. The findings on the chemical structure and binding pose are expected to be a base for developing MBL inhibitors.

Mutagenesis and structural analysis reveal the CTX-M ß-lactamase active site is optimized for cephalosporin catalysis and drug resistance.

CTX-M ß-lactamases are a widespread source of resistance to ß-lactam antibiotics in Gram-negative bacteria. These enzymes readily hydrolyze penicillins and cephalosporins, including oxyimino-cephalosporins such as cefotaxime. To investigate the preference of CTX-M enzymes for cephalosporins, we examined eleven active-site residues in the CTX-M-14 ß-lactamase model system by alanine mutagenesis to assess the contribution of the residues to catalysis and specificity for the hydrolysis of the penicillin, ampicillin, and the cephalosporins cephalothin and cefotaxime. Key active site residues for class A ß-lactamases, including Lys73, Ser130, Asn132, Lys234, Thr216, and Thr235, contribute significantly to substrate binding and catalysis of penicillin and cephalosporin substrates in that alanine substitutions decrease both kcat and kcat/KM. A second group of residues, including Asn104, Tyr105, Asn106, Thr215, and Thr216, contribute only to substrate binding, with the substitutions decreasing only kcat/KM. Importantly, calculating the average effect of a substitution across the 11 active-site residues shows that the most significant impact is on cefotaxime hydrolysis while ampicillin hydrolysis is least affected, suggesting the active site is highly optimized for cefotaxime catalysis. Furthermore, we determined X-ray crystal structures for the apo-enzymes of the mutants N106A, S130A, N132A, N170A, T215A, and T235A. Surprisingly, in the structures of some mutants, particularly N106A and T235A, the changes in structure propagate from the site of substitution to other regions of the active site, suggesting that the impact of substitutions is due to more widespread changes in structure and illustrating the interconnected nature of the active site.

The Natural History of Carbapenemase-Producing Enterobacterales: Progression From Carriage of Various Carbapenemases to Bloodstream Infection.

BACKGROUND: Little is known about the risk of progression from carbapenemase-producing Enterobacterales (CPE) carriage to CPE bloodstream infection (BSI) outside of high-risk settings. We aimed to determine the incidence of CPE BSI among CPE carriers and to assess whether the incidence differs by carbapenemase, species, and setting. METHODS: We conducted a nationwide population-based retrospective cohort study using national databases. The cohort consisted of all patients in Israel with CPE detected by screening from 1 January 2020 to 10 October 2022. We calculated the cumulative incidence of CPE BSI within 1 year among CPE carriers. We used a competing-risks model with BSI as the outcome and death as the competing risk. RESULTS: The study included 6828 CPE carriers. The cumulative incidence of CPE BSI was 2.4% (95% confidence interval [CI], 2.1-2.8). Compared with Klebsiella pneumoniae carbapenemase (KPC), the subhazard of BSI was lower for New Delhi metallo-ß-lactamase (NDM) (adjusted subhazard ratio [aSHR], 0.72; 95% CI, .49-1.05) and oxacillinase-48-like (OXA-48-like) (aSHR, 0.60; 95% CI, .32-1.12) but these differences did not reach statistical significance. Compared with K. pneumoniae, the subhazard of BSI was lower for carriers of carbapenemase-producing Escherichia coli (aSHR, 0.33; 95% CI, .21-.52). The subhazard of BSI was higher among patients with CPE carriage first detected in intensive care units (aSHR, 2.10; 95% CI, 1.27-3.49) or oncology/hematology wards (aSHR, 3.95; 95% CI, 2.51-6.22) compared with medical wards. CONCLUSIONS: The risk of CPE BSI among CPE carriers is lower than previously reported in studies that focused on high-risk patients and settings. The risk of BSI differs significantly by bacterial species and setting, but not by carbapenemase.

Non-KPC Attributes of Newer ß-lactam/ß-lactamase Inhibitors, Part 1: Enterobacterales and Pseudomonas aeruginosa.

Gram-negative antibiotic resistance continues to grow as a global problem due to the evolution and spread of ß-lactamases. The early ß-lactamase inhibitors (BLIs) are characterized by spectra limited to class A ß-lactamases and ineffective against carbapenemases and most extended spectrum ß-lactamases. In order to address this therapeutic need, newer BLIs were developed with the goal of treating carbapenemase producing, carbapenem resistant organisms (CRO), specifically targeting the Klebsiella pneumoniae carbapenemase (KPC). These BL/BLI combination drugs, avibactam/avibactam, meropenem/vaborbactam, and imipenem/relebactam, have proven to be indispensable tools in this effort. However, non-KPC mechanisms of resistance are rising in prevalence and increasingly challenging to treat. It is critical for clinicians to understand the unique spectra of these BL/BLIs with respect to non-KPC CRO. In Part 1of this 2-part series, we describe the non-KPC attributes of the newer BL/BLIs with a focus on utility against Enterobacterales and Pseudomonas aeruginosa.

Mutation Rate of AmpC ß-Lactamase-Producing Enterobacterales and Treatment in Clinical Practice: A Word of Caution.

In a retrospective multicenter study of 575 patients with bloodstream infections or pneumonia due to wild-type AmpC ß-lactamase-producing Enterobacterales, species with low in vitro mutation rates for AmpC derepression were associated with fewer treatment failures due to AmpC overproduction (adjusted hazard ratio, 0.5 [95% CI, .2-.9]). However, compared to cefepime/carbapenems, using third-generation cephalosporins as definitive therapy remained associated with this adverse outcome (15% vs 1%).

The Growing Threat of NDM-Producing Escherichia coli With Penicillin-Binding Protein 3 Mutations in the United States-Is There a Potential Role for Durlobactam?

We report identification of 5 patients with infections caused by NDM-5-producing Escherichia coli harboring PBP3 mutations that showed reduced susceptibility to aztreonam-avibactam and cefiderocol. Durlobactam, a novel diazabicyclooctane ß-lactamase inhibitor, demonstrated minimum inhibitory concentrations ranging from 0.5 to 2 µg/mL supporting future investigations into a potential role in clinical management.

Sequential Treatment Failure With Aztreonam-Ceftazidime-Avibactam Followed by Cefiderocol Due to Preexisting and Acquired Mechanisms in a New Delhi Metallo-ß-lactamase-Producing Escherichia coli Causing Fatal Bloodstream Infection.

We report a fatal case of New Delhi metallo-ß-lactamase (NDM)-producing Escherichia coli in a bacteremic patient with sequential failure of aztreonam plus ceftazidime-avibactam followed by cefiderocol. Acquired resistance was documented phenotypically and mediated through preexisting and acquired mutations. This case highlights the need to rethink optimal treatment for NDM-producing organisms.

A preliminary exploration on the mechanism of the carbapenem-resistance transformation of Serratia marcescens in vivo.

BACKGROUND: The infection of carbapenem-resistant organisms was a huge threat to human health due to their global spread. Dealing with a carbapenem-resistant Serratia marcescens (CRSM) infection poses a significant challenge in clinical settings. This study aims to provide insights into strategies for controlling CRSM infection by exploring the transformation mechanism of carbapenem-resistance. METHODS: We used whole genome sequencing (WGS) to investigate the mechanism of carbapenem resistance in 14 S. marcescens isolates in vivo. The expression level of related genes and the minimum inhibitory concentration of meropenem (MICMEM) were also evaluated to confirm the mechanism of carbapenem resistance. RESULTS: Seven groups of S. marcescens, each consisting of two strains, were collected from a hospital and displayed a shift in MICMEM from low to high levels. Homology analysis revealed that the isolates in five groups were significantly different from the remaining two. WGS and experimental evidence indicated that four groups of strains developed carbapenem resistance by acquiring the blaKPC (obtaining group), while two groups (persisting group) increased the expression level of the blaKPC. In contrast, isolates in the last group (missing group) did not carry the blaKPC. All strains possessed multiple ß-lactamase genes, including blaCTX-M-14, blaSRT-1, and blaSRT-2. However, only in the missing group, the carbapenem-resistant strain lost an outer membrane protein-encoding gene, leading to increased blaCTX-M-14 expression compared to the carbapenem-susceptible strain. CONCLUSION: The study findings suggest that S. marcescens strains developed diverse carbapenem resistance in vivo through the evolution of drug resistance, rather than through clone replacement. We hypothesize that carbapenem resistance in S. marcescens was due to certain clonal types with a distinct mechanism.