Recurrent emergence of Klebsiella pneumoniae carbapenem resistance mediated by an inhibitory ompK36 mRNA secondary structure.

Outer membrane porins in Gram-negative bacteria facilitate antibiotic influx. In Klebsiella pneumoniae, modifications in the porin OmpK36 are implicated in increasing resistance to carbapenems. An analysis of large K. pneumoniae genome collections, encompassing major healthcare-associated clones, revealed the recurrent emergence of a synonymous cytosine-to-thymine transition at position 25 (25c > t) in ompK36. We show that the 25c > t transition increases carbapenem resistance through depletion of OmpK36 from the outer membrane. The mutation attenuates K. pneumoniae in a murine pneumonia model, which accounts for its limited clonal expansion observed by phylogenetic analysis. However, in the context of carbapenem treatment, the 25c > t transition tips the balance toward treatment failure, thus accounting for its recurrent emergence. Mechanistically, the 25c > t transition mediates an intramolecular messenger RNA (mRNA) interaction between a uracil encoded by 25t and the first adenine within the Shine-Dalgarno sequence. This specific interaction leads to the formation of an RNA stem structure, which obscures the ribosomal binding site thus disrupting translation. While mutations reducing OmpK36 expression via transcriptional silencing are known, we uniquely demonstrate the repeated selection of a synonymous ompK36 mutation mediating translational suppression in response to antibiotic pressure.

In vitro and in vivo activities of KSP-1007, a broad-spectrum inhibitor of serine- and metallo-ß-lactamases, in combination with meropenem against carbapenem-resistant Gram-negative bacteria.

KSP-1007 is a novel bicyclic boronate-based broad-spectrum ß-lactamase inhibitor and is being developed in combination with meropenem (MEM) for the treatment of infections caused by carbapenem-resistant Gram-negative bacteria, a global health concern, and here, we describe its characteristics. KSP-1007 exhibited low apparent inhibition constant (Ki app) values against all classes of ß-lactamase, including imipenemase types and oxacillinase types from Acinetobacter baumannii. Against 207 Enterobacterales and 55 A. baumannii, including carbapenemase producers, KSP-1007 at fixed concentrations of 4, 8, and 16 µg/mL dose-dependently potentiated the in vitro activity of MEM in broth microdilution MIC testing. The MIC90 of MEM/KSP-1007 at 8 µg/mL against Enterobacterales was lower than those of MEM/vaborbactam, ceftazidime/avibactam, imipenem/relebactam, and colistin and similar to those of aztreonam/avibactam, cefiderocol, and tigecycline. The in vitro activity of MEM/KSP-1007 at ≥4 µg/mL against Enterobacterales harboring metallo-ß-lactamase was superior to that of cefepime/taniborbactam. MEM/KSP-1007 showed excellent activity against Escherichia coli with PBP3 mutations and New Delhi metallo-ß-lactamase compared to aztreonam/avibactam, cefepime/taniborbactam, and cefiderocol. MEM/KSP-1007 at 8 µg/mL showed greater efficacy against A. baumannii than these comparators except for cefiderocol, tigecycline, and colistin. A 2-fold reduction in MEM MIC against 96 Pseudomonas aeruginosa was observed in combination with KSP-1007. MEM/KSP-1007 demonstrated bactericidal activity against carbapenemase-producing Enterobacterales, A. baumannii, and P. aeruginosa based on minimum bactericidal concentration/MIC ratios of ≤4. KSP-1007 enhanced the in vivo activity of MEM against carbapenemase-producing Enterobacterales, A. baumannii, and P. aeruginosa in murine systemic, complicated urinary tract, and thigh infection models. Collectively, MEM/KSP-1007 has a good profile for treating carbapenem-resistant Gram-negative bacterial infections.

Ethambutol and meropenem/clavulanate synergy promotes enhanced extracellular and intracellular killing of Mycobacterium tuberculosis.

Increasing evidence supports the repositioning of beta-lactams for tuberculosis (TB) therapy, but further research on their interaction with conventional anti-TB agents is still warranted. Moreover, the complex cell envelope of Mycobacterium tuberculosis (Mtb) may pose an additional obstacle to beta-lactam diffusion. In this context, we aimed to identify synergies between beta-lactams and anti-TB drugs ethambutol (EMB) and isoniazid (INH) by assessing antimicrobial effects, intracellular activity, and immune responses. Checkerboard assays with H37Rv and eight clinical isolates, including four drug-resistant strains, exposed that only treatments containing EMB and beta-lactams achieved synergistic effects. Meanwhile, the standard EMB and INH association failed to produce any synergy. In Mtb-infected THP-1 macrophages, combinations of EMB with increasing meropenem (MEM) concentrations consistently displayed superior killing activities over the individual antibiotics. Flow cytometry with BODIPY FL vancomycin, which binds directly to the peptidoglycan (PG), confirmed an increased exposure of this layer after co-treatment. This was reinforced by the high IL-1ß secretion levels found in infected macrophages after incubation with MEM concentrations above 5 mg/L, indicating an exposure of the host innate response sensors to pathogen-associated molecular patterns in the PG. Our findings show that the proposed impaired access of beta-lactams to periplasmic transpeptidases is counteracted by concomitant administration with EMB. The efficiency of this combination may be attributed to the synchronized inhibition of arabinogalactan and PG synthesis, two key cell wall components. Given that beta-lactams exhibit a time-dependent bactericidal activity, a more effective pathogen recognition and killing prompted by this association may be highly beneficial to optimize TB regimens containing carbapenems.IMPORTANCEAddressing drug-resistant tuberculosis with existing therapies is challenging and the treatment success rate is lower when compared to drug-susceptible infection. This study demonstrates that pairing beta-lactams with ethambutol (EMB) significantly improves their efficacy against Mycobacterium tuberculosis (Mtb). The presence of EMB enhances beta-lactam access through the cell wall, which may translate into a prolonged contact between the drug and its targets at a concentration that effectively kills the pathogen. Importantly, we showed that the effects of the EMB and meropenem (MEM)/clavulanate combination were maintained intracellularly. These results are of high significance considering that the time above the minimum inhibitory concentration is the main determinant of beta-lactam efficacy. Moreover, a correlation was established between incubation with higher MEM concentrations during macrophage infection and increased IL-1ß secretion. This finding unveils a previously overlooked aspect of carbapenem repurposing against tuberculosis, as certain Mtb strains suppress the secretion of this key pro-inflammatory cytokine to evade host surveillance.

Role of amino acid 159 in carbapenem and temocillin hydrolysis of OXA-933, a novel OXA-48 variant.

OXA-48 has rapidly disseminated worldwide and become one of the most common carbapenemases in many countries with more than 45 variants reported with, in some cases, significant differences in their hydrolysis profiles. The R214 residue, located in the ß5-ß6 loop, is crucial for the carbapenemase activity, as it stabilizes carbapenems in the active site and maintains the shape of the active site through interactions with D159. In this study, we have characterized a novel variant of OXA-48, OXA-933 with a single D159N change. To evaluate the importance of this residue, point mutations were generated (D159A, D159G, D159K, and D159W), kinetic parameters of OXA-933, OXA-48 D159G, and OXA-48 D159K were determined and compared to those of OXA-48 and OXA-244. The blaOXA-933 gene was borne on Tn2208, a 2,696-bp composite transposon made of two IS1 elements surrounded by 9 bp target site duplications and inserted into a non-self-transmissible plasmid pOXA-933 of 7,872 bp in size. Minimal inhibitory concentration values of E. coli expressing the blaOXA-933 gene or of its point mutant derivatives were lower for carbapenems (except for D159G) as compared to those expressing the blaOXA-48 gene. Steady-state kinetic parameters revealed lower catalytic efficiencies for expanded spectrum cephalosporins and carbapenems. A detailed structural analysis confirmed the crucial role of D159 in shaping the active site of OXA-48 enzymes by interacting with R214. Our work further illustrates the remarkable propensity of OXA-48-like carbapenemases to evolve through mutations at positions outside the ß5-ß6 loop, but interacting with key residues of it.

Comprehensive stability analysis of 13 ß-lactams and ß-lactamase inhibitors in in vitro media, and novel supplement dosing strategy to mitigate thermal drug degradation.

Non-clinical antibiotic development relies on in vitro susceptibility and infection model studies. Validating the achievement of the targeted drug concentrations is essential to avoid under-estimation of drug effects and over-estimation of resistance emergence. While certain ß-lactams (e.g., imipenem) and ß-lactamase inhibitors (BLIs; clavulanic acid) are believed to be relatively unstable, limited tangible data on their stability in commonly used in vitro media are known. We aimed to determine the thermal stability of 10 ß-lactams and 3 BLIs via LC-MS/MS in cation-adjusted Mueller Hinton broth at 25 and 36°C as well as agar at 4 and 37°C, and in water at -20, 4, and 25°C. Supplement dosing algorithms were developed to achieve broth concentrations close to their target over 24 h. During incubation in broth (pH 7.25)/agar, degradation half-lives were 16.9/21.8 h for imipenem, 20.7/31.6 h for biapenem, 29.0 h for clavulanic acid (studied in broth only), 23.1/71.6 h for cefsulodin, 40.6/57.9 h for doripenem, 46.5/64.6 h for meropenem, 50.8/97.7 h for cefepime, 61.5/99.5 h for piperacillin, and >120 h for all other compounds. Broth stability decreased at higher pH. All drugs were ≥90% stable for 72 h in agar at 4°C. Degradation half-lives in water at 25°C were >200 h for all drugs except imipenem (14.7 h, at 1,000 mg/L) and doripenem (59.5 h). One imipenem supplement dose allowed concentrations to stay within ±31% of their target concentration. This study provides comprehensive stability data on ß-lactams and BLIs in relevant in vitro media using LC-MS/MS. Future studies are warranted applying these data to antimicrobial susceptibility testing and assessing the impact of ß-lactamase-related degradation.

Emergence of heteroresistance to carbapenems in Gram-negative clinical isolates from two Egyptian hospitals.

BACKGROUND: Antimicrobial resistance is a global concern, linking bacterial genotype and phenotype. However, variability in antibiotic susceptibility within bacterial populations can lead to misclassification. Heteroresistance exemplifies this, where isolates have subpopulations less susceptible than the main population. This study explores heteroresistance in Gram-negative bacteria, distinguishing between carbapenem-sensitive isolates and stable heteroresistant isolates (SHIs). METHODS: A total of 151 Gram-negative clinical isolates including Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii and Proteus mirabilis from various sources were included. Heteroresistant isolates and their stability were detected by disc-diffusion technique while genotypic analysis was carried out by PCR and efflux activity was assessed by ethidium bromide (EtBr)-agar cartwheel method. RESULTS: A total of 51 heteroresistant subpopulations were detected, producing 16 SHIs upon stability-detection. Amplified resistance genes and EtBr-agar cartwheel method showed a significant difference between resistant subpopulations and their corresponding-sensitive main populations. CONCLUSION: Genotypic analysis confirmed that genetic mutation can lead to resistance development although the main populations were sensitive, thereby leading to treatment failure. This is a neglected issue which should be highly considered for better treatment outcomes.

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.

Molecular analysis of metallo-beta-lactamase-producing Pseudomonas aeruginosa in Switzerland 2022-2023.

OBJECTIVES: The occurrence of metallo-beta-lactamase-producing Pseudomonas aeruginosa (MBL-PA) isolates is increasing globally, including in Switzerland. The aim of this study was to characterise, phenotypically and genotypically, the MBL-PA isolates submitted to the Swiss National Reference Center for Emerging Antibiotic Resistance (NARA) reference laboratory over a 12-month period from July 2022 to July 2023. METHODS: Thirty-nine non-duplicate MBL-PA Isolates were submitted to NARA over the study period from across Switzerland. Susceptibility was determined by broth microdilution according to EUCAST methodology. Whole-genome sequencing was performed on 34 isolates. Sequence types (STs) and resistance genes were ascertained using the Centre for Genomic Epidemiology platform. MBL genes, blaNDM-1, blaIMP-1, and blaVIM-2, were cloned into vector pUCP24 and transformed into P. aeruginosa PA14. RESULTS: The most prevalent MBL types identified in this study were VIM (21/39; 53.8%) followed by NDM (11/39; 28.2%), IMP (6/39; 15.4%), and a single isolate produced both VIM and NDM enzymes. WGS identified 13 different STs types among the 39 isolates. They all exhibited resistance to cephalosporins, carbapenems, and the beta-lactam-beta-lactamase inhibitor combinations, ceftolozane-tazobactam, ceftazidime-avibactam, imipenem-relebactam, and meropenem-vaborbactam, and 8 isolates were cefiderocol (FDC) resistant. Recombinant P. aeruginosa strains producing blaNDM-1, blaIMP-1, and blaVIM-2 exhibited FDC MICs of 16, 8, and 1 mg/L, respectively. CONCLUSIONS: This study showed that the MBL-PA in Switzerland could be attributed to the wide dissemination of high-risk clones that accounted for most isolates in this study. Although FDC resistance was only found in 8 isolates, MBL carriage was shown to be a major contributor to this phenotype.

Ceftazidime-avibactam combination therapy versus monotherapy for treating carbapenem-resistant gram-negative infection: a systemic review and meta-analysis.

BACKGROUND: This meta-analysis was conducted to compare the efficacy of ceftazidime-avibactam combination therapy with that of monotherapy in the treatment of carbapenem-resistant Gram-negative bacterial (CR-GNB). METHODS: A literature search of PubMed, Embase, the Cochrane Library, and ClinicalTrials.gov was conducted until September 1, 2023. Only studies that compared CZA combination therapy with monotherapy for CR-GNB infections were included. RESULTS: A total of 25 studies (23 retrospective observational studies and 2 prospective studies) involving 2676 patients were included. There was no significant difference in 30-day mortality between the study group receiving combination therapy and the control group receiving monotherapy (risk ratio [RR] 0.91; 95% confidence interval [CI] 0.71-1.18). In addition, no significant differences were observed between the study and the control group in terms of in-hospital mortality (RR 1.00; 95% CI 0.79-1.27), 14-day mortality (RR 1.54; 95% CI 0.24-9.91), 90-day mortality (RR 1.18; 95% CI 0.62-2.22), and clinical cure rate (RR 0.95; 95% CI 0.84-1.08). However, the combination group had a borderline higher microbiological eradication rate than the control group (RR 1.15; 95% CI 1.00-1.32). CONCLUSIONS: Compared to monotherapy, CZA combination therapy did not yield additional clinical benefits. However, combination therapy may be associated with favorable microbiological outcomes.

Empirical meropenem versus piperacillin/tazobactam for adult patients with sepsis (EMPRESS) trial: Protocol.

BACKGROUND: Piperacillin/tazobactam may be associated with less favourable outcomes than carbapenems in patients with severe bacterial infections, but the certainty of evidence is low. METHODS: The Empirical Meropenem versus Piperacillin/Tazobactam for Adult Patients with Sepsis (EMPRESS) trial is an investigator-initiated, international, parallel-group, randomised, open-label, adaptive clinical trial with an integrated feasibility phase. We will randomise adult, critically ill patients with sepsis to empirical treatment with meropenem or piperacillin/tazobactam for up to 30 days. The primary outcome is 30-day all-cause mortality. The secondary outcomes are serious adverse reactions within 30 days; isolation precautions due to resistant bacteria within 30 days; days alive without life support and days alive and out of hospital within 30 and 90 days; 90- and 180-day all-cause mortality and 180-day health-related quality of life. EMPRESS will use Bayesian statistical models with weak to somewhat sceptical neutral priors. Adaptive analyses will be conducted after follow-up of the primary outcome for the first 400 participants concludes and after every 300 subsequent participants, with adaptive stopping for superiority/inferiority and practical equivalence (absolute risk difference <2.5%-points) and response-adaptive randomisation. The expected sample sizes in scenarios with no, small or large differences are 5189, 5859 and 2570 participants, with maximum 14,000 participants and ≥99% probability of conclusiveness across all scenarios. CONCLUSIONS: EMPRESS will compare the effects of empirical meropenem against piperacillin/tazobactam in adult, critically ill patients with sepsis. Due to the pragmatic, adaptive design with high probability of conclusiveness, the trial results are expected to directly inform clinical practice.

Comparative effectiveness of cefmetazole versus carbapenems and piperacillin/tazobactam as initial therapy for bacteremic acute cholangitis: A retrospective study.

INTRODUCTION: Carbapenems and piperacillin/tazobactam (PIPC/TAZ) are commonly used as the initial therapy to treat extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales in acute cholangitis. However, the overuse of these antibiotics contributes to the spread of antimicrobial resistance. Cefmetazole (CMZ) is stable to hydrolysis by ESBLs, so it may be an alternative to carbapenems and PIPC/TAZ. However, the effectiveness of CMZ compared with that of carbapenems and PIPC/TAZ as the initial therapy for acute cholangitis is unknown. METHODS: We conducted a retrospective cohort study at a university hospital between April 1, 2014, and December 31, 2022. Patients with bacteremic acute cholangitis who received CMZ, carbapenems, or PIPC/TAZ as the initial therapy were included. The patients were divided into a CMZ group and a carbapenems or PIPC/TAZ (CP) group to compare patient outcomes. RESULTS: A total of 99 patients (54 in the CMZ group and 45 in the CP group) were analyzed. The baseline characteristics of the patients were similar and 30-day mortality did not differ between groups (4% vs. 7%, P = 0.66). However, the CMZ group had a shorter length of stay (LOS) (8 days vs. 15 days, P < 0.001) and lower mean antibiotic cost (98.92 USD vs. 269.49 USD, P < 0.001) than the CP group. CONCLUSIONS: In bacteremic acute cholangitis, initial therapy with CMZ may contribute to a shorter LOS and lower antibiotic costs than treatment with carbapenems and PIPC/TAZ, without worsening patient outcomes.

Antimicrobial Use and Carbapenem-Resistant Enterobacterales in Korea: A Nationwide Case-Control Study With Propensity Score Matching.

BACKGROUND: Nationwide research on the association between carbapenem-resistant Enterobacterales (CREs) and antibiotic use is limited. METHODS: This nested case-control study analyzed Korean National Health Insurance claims data from April 2017 to April 2019. Based on the occurrence of CRE, hospitalized patients aged ≥ 18 years were classified into CRE (cases) and control groups. Propensity scores based on age, sex, modified Charlson comorbidity score, insurance type, long-term care facility, intensive care unit stay, and acquisition of vancomycin-resistant Enterococci were used to match the case and control groups (1:3). RESULTS: After matching, the study included 6,476 participants (1,619 cases and 4,857 controls). Multivariable logistic regression analysis revealed that the utilization of broad-spectrum antibiotics, such as piperacillin/tazobactam (adjusted odds ratio [aOR], 2.178; 95% confidence interval [CI], 1.829-2.594), third/fourth generation cephalosporins (aOR, 1.764; 95% CI, 1.514-2.056), and carbapenems (aOR, 1.775; 95% CI, 1.454-2.165), as well as the presence of comorbidities (diabetes [aOR, 1.237; 95% CI, 1.061-1.443], hemiplegia or paraplegia [aOR, 1.370; 95% CI, 1.119-1.679], kidney disease [aOR, 1.312; 95% CI, 1.105-1.559], and liver disease [aOR, 1.431; 95% CI, 1.073-1.908]), were significantly associated with the development of CRE. Additionally, the CRE group had higher mortality (8.33 vs. 3.32 incidence rate per 100 person-months, P < 0.001) and a total cost of healthcare utilization per person-month (15,325,491 ± 23,587,378 vs. 5,263,373 ± 14,070,118 KRW, P < 0.001) than the control group. CONCLUSION: The utilization of broad-spectrum antibiotics and the presence of comorbidities are associated with increasing development of CRE. This study emphasizes the importance of antimicrobial stewardship in reducing broad-spectrum antibiotic use and CRE disease burden in Korea.

Carbapenem-Resistant Klebsiella pneumoniae in Large Public Acute-Care Healthcare System, New York, New York, USA, 2016-2022.

Controlling the spread of carbapenem-resistant Enterobacterales is a global priority. Using National Healthcare Safety Network data, we characterized the changing epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) in a large public health system in New York, New York, USA. During 2016-2020, CRKP cases declined; however, during 2021-June 2022, a notable increase occurred. Of 509 cases, 262 (51%) were considered community-onset, including 149 in patients who were living at home. Of 182 isolates with proven or presumptive (ceftazidime/avibactam susceptible) enzymes, 143 were serine carbapenemases; most confirmed cases were K. pneumoniae carbapenemase. The remaining 39 cases were proven or presumptive metallo-ß-lactamases; all confirmed cases were New Delhi metallo-ß-lactamases. After 2020, a marked increase occurred in the percentage of isolates possessing metallo-ß-lactamases. Most patients with metallo-ß-lactamases originated from long-term care facilities. An aggressive and universal program involving surveillance and isolation will be needed to control the spread of CRKP in the city of New York.

Multiplicity of blaKPC Genes and pKpQIL Plasmid Plasticity in the Development of Ceftazidime-Avibactam and Meropenem Coresistance in Klebsiella pneumoniae Sequence Type 307.

In 2021, Klebsiella pneumoniae sequence type 307 (ST307) strains causing pulmonary and bloodstream infections identified in a hospital in Rome, Italy, reached high levels of resistance to ceftazidime-avibactam (CZA). One of these strains reached high levels of resistance to both CZA and carbapenems and carried two copies of blaKPC-3 and one copy of blaKPC-31 located on plasmid pKpQIL. The genomes and plasmids of CZA-resistant ST307 strains were analyzed to identify the molecular mechanisms leading to the evolution of resistance and compared with ST307 genomes at local and global levels. A complex pattern of multiple plasmids in rearranged configurations, coresident within the CZA-carbapenem-resistant K. pneumoniae strain, was observed. Characterization of these plasmids revealed recombination and segregation events explaining why K. pneumoniae isolates from the same patient had different antibiotic resistance profiles. This study illustrates the intense genetic plasticity occurring in ST307, one of the most worldwide-diffused K. pneumoniae high-risk clones.

Genetic and enzymatic characterization of two novel blaNDM-36, -37 variants in Escherichia coli strains.

The widespread of different NDM variants in clinical Enterobacterales isolates poses a serious public health concern, which requires continuous monitoring. In this study, three E. coli strains carrying two novel blaNDM variants of blaNDM-36, -37 were identified from a patient with refractory urinary tract infection (UTI) in China. We conducted antimicrobial susceptibility testing (AST), enzyme kinetics analysis, conjugation experiment, whole-genome sequencing (WGS), and bioinformatics analysis to characterize the blaNDM-36, -37 enzymes and their carrying strains. The blaNDM-36, -37 harboring E. coli isolates belonged to ST227, O9:H10 serotype and exhibited intermediate or resistance to all ß-lactams tested except aztreonam and aztreonam/avibactam. The genes of blaNDM-36, -37 were located on a conjugative IncHI2-type plasmid. NDM-37 differed from NDM-5 by a single amino acid substitution (His261Tyr). NDM-36 differed from NDM-37 by an additional missense mutation (Ala233Val). NDM-36 had increased hydrolytic activity toward ampicillin and cefotaxime relative to NDM-37 and NDM-5, while NDM-37 and NDM-36 had lower catalytic activity toward imipenem but higher activity against meropenem in comparison to NDM-5. This is the first report of co-occurrence of two novel blaNDM variants in E. coli isolated from the same patient. The work provides insights into the enzymatic function and demonstrates the ongoing evolution of NDM enzymes.

Clinical Outcomes and Treatment Strategies in Patients With Non-Carbapenemase-producing Carbapenem-Resistant Versus Carbapenem-Susceptible Enterobacterales Infections.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are difficult to treat and can cause significant morbidity and mortality, however most data reflect carbapenemase-producing infections. OBJECTIVE: Our objective was to evaluate clinical outcomes of non-carbapenemase-producing CRE (nCP-CRE) compared with carbapenem-susceptible Enterobacterales (CSE) infections. METHODS: This was a retrospective, multicenter, observational study (January 1, 2018 to December 31, 2020). The primary outcome was clinical success at 30 days with secondary outcomes, including clinical success at 90 days, clinical success based on treatment for nCP-CRE, persistent bacteremia, intensive care unit (ICU) admission, length of stay, and rate of Clostridioides difficile or multidrug resistant infections. RESULTS: The final analysis included 211 patients: 142 (67%) with CSE and 69 (33%) with nCP-CRE infections. Prior carbapenem exposure was more common with nCP-CRE (15% vs 4%, P = 0.01). Clinical success at 30 days was similar between groups (77% vs 74%, P = 0.73). There were no differences in secondary outcomes. There was an overall low use of carbapenems (empiric 6%, definitive 7%). Most nCP-CRE infections were treated with a monotherapy carbapenem-sparing regimen (empiric 88%, definitive 90%). Limitations include the retrospective design and the high rate of urinary infections. CONCLUSION AND RELEVANCE: Our study found no difference in clinical outcomes between nCP-CRE and CSE infections. Application of this study with future studies would help in determining optimal regimens for these infections.

Piperacillin/tazobactam versus carbapenems in patients with severe bacterial infections: A systematic review with meta-analysis.

BACKGROUND: Piperacillin/tazobactam or meropenem are often used to treat patients with severe bacterial infections. We aimed to compare the desirable and undesirable effects of empirical and/or definitive piperacillin/tazobactam versus carbapenems in patients with severe bacterial infections. METHODS: We searched PubMed, Embase, CENTRAL, Epistemonikos, and trial registers for randomised clinical trials of empirical and/or definitive piperacillin/tazobactam versus carbapenems in adult patients with severe bacterial infection (i.e., any bacterial infection requiring hospitalisation). The primary outcome was all-cause short-term mortality within 90 days. Secondary outcomes were all-cause long-term mortality, adverse events, quality of life, days alive without or duration of life support, secondary infections, selection of fungi or resistant bacteria, and days alive and out of hospital or hospital length of stay. We calculated relative risks (RRs) using random effects and fixed effect meta-analyses along with trial sequential analyses. RESULTS: We included 31 trials (n = 8790 patients) with overall high risk of bias. The RR for all-cause short-term mortality was 1.16 (95% confidence interval [CI]: 0.94-1.43, low certainty evidence), for adverse events 1.00 (98% CI: 0.96-1.04, moderate certainty evidence), for secondary infections 1.13 (98% CI: 0.76-1.68, very low certainty evidence), and for selection of fungi or resistant bacteria 1.61 (98% CI: 0.89-2.89, very low certainty evidence). There were no or limited data for the remaining outcomes. CONCLUSIONS: Based on very low or low certainty evidence, piperacillin/tazobactam may be associated with less favourable outcomes in patients with severe bacterial infections as compared with carbapenems, but the information size for a robust conclusion has not been reached.

Determination of valproic acid and its six metabolites in human serum using LC-MS/MS and application to interaction with carbapenems in epileptic patients.

Valproic acid (VPA) is a classic medication for several types of epilepsy and mood disorders, and some of its effectiveness and toxicity is associated with metabolites. Although many reports have reported the drug-drug interactions of VPA, no study has focused on the influence of carbapenems (CBPMs) on VPA's active metabolites. An LC-MS/MS method for determining VPA and its six metabolites (3-hydroxy valproic acid, 4-hydroxy valproic acid, 2-propyl-2-pentenoic acid, 2-propyl-4-pentenoic acid, 3-keto valproic acid, and 2-propylglutaric acid) in human serum was established and applied to evaluate the drug-drug interaction with CBPMs in epileptic patients. The stable isotope valproic acid-d6 was used as an internal standard. Analytes in serum samples (50 µl) were isolated using a Kinetex C18 column (3 × 100 mm, 2.6 µm) and detected via negative electrospray ionization after protein precipitation. It was linear (r > 0.99) over the calibration range for different analytes. The accuracy was 91.44-110.92%, and the precision was less than 9.98%. The matrix effect, recovery, and stability met the acceptance criteria. According to the data collected from 150 epileptic patients, the concentration-dose ratio for VPA and its metabolites decreased with CBPM polytherapy. This method is simple and rapid with great accuracy and precision. It is suitable for routine clinical analysis of VPA and its metabolites in human serum.

Method Development for Determination of Doripenem in Human Plasma via Capillary Electrophoresis Coupled with Field-Enhanced Sample Stacking and Sweeping.

In this study, we established a novel capillary electrophoresis method for monitoring the concentration of doripenem in human plasma. As a time-dependent antibiotic, doripenem maximizes its antibacterial effects and minimizes the potential for antibiotic resistance through careful therapeutic drug monitoring. Two online preconcentration techniques, field-enhanced sample stacking (FESS) and sweeping, were coupled to enhance the detection sensitivity. Briefly, an uncoated fused silica capillary (40 cm × 50 µm i.d) was rinsed with a high conductivity buffer (HCB) composed of 150 mM phosphate buffer (NaH2PO4, pH 2.5) and 20% methanol. A large sample plug prepared in a low-conductivity phosphate buffer (50 mM NaH2PO4, pH 2.5) was then hydrodynamically injected (5 psi, 80 s) into the capillary. Under an applied voltage of -30 kV, the analyte was accumulated at the FESS boundary and swept by the negatively charged micelles toward the UV detector. Plasma samples were pretreated by solid-phase extraction (SPE) to eliminate endogenous interferences. The validation results demonstrated a high coefficient of determination (r2 > 0.9995) for the regression curve with impressive precision and accuracy: relative standard deviation (RSD) <5.86% and relative error <4.63%. The limit of detection (LOD, S/N = 3) for doripenem was determined to be 0.4 µg/mL. Compared to the conventional micellar electrokinetic chromatography method, our developed method achieved a sensitivity enhancement of up to 488-fold for doripenem. Furthermore, the newly developed method successfully quantified doripenem concentrations in plasma samples obtained from patients accepting doripenem regimens, proving its application potential in the clinical realm.

Satisfactory In Vitro Activity of Ceftolozane-Tazobactam against Carbapenem-Resistant Pseudomonas aeruginosa But Not against Klebsiella pneumoniae Isolates.

Background: Gram-negative rods are one of the most commonly isolated bacteria within human infections. These microorganisms are typically opportunistic pathogens that pose a serious threat to public health due to the possibility of transmission in the human population. Resistance to carbapenems is one of the most important antimicrobial resistance mechanisms amongst them. The aim of this study was to evaluate ceftolozane-tazobactam in vitro activity against carbapenem-resistant Pseudomonas aeruginosa and Klebsiella pneumoniae clinical strains. Information on the antimicrobial activity of this antimicrobial against Gram-negative rods was also supplemented with a brief review of the relevant literature. Methods: The research involved 316 strains of Gram-negative rods: P. aeruginosa-206 and K. pneumoniae-110. Results: Of the tested strains, 86.0% P. aeruginosa and 30.0% K. pneumoniae remained susceptible to ceftolozane-tazobactam. Conclusions: Therefore, ceftolozane-tazobactam might be a good option in the treatment of infections caused by carbapenem-resistant P. aeruginosa strains, including those in ICU patients. Meanwhile, due to dissemination of ESBLs among K. pneumoniae strains, infections with this etiology should not be treated with the ceftolozane-tazobactam combination.