Antimicrobial resistance heterogeneity among multidrug-resistant Gram-negative pathogens: Phenotypic, genotypic, and proteomic analysis.

Microbes evolve rapidly by modifying their genomes through mutations or through the horizontal acquisition of mobile genetic elements (MGEs) linked with fitness traits such as antimicrobial resistance (AMR), virulence, and metabolic functions. We conducted a multicentric study in India and collected different clinical samples for decoding the genome sequences of bacterial pathogens associated with sepsis, urinary tract infections, and respiratory infections to understand the functional potency associated with AMR and its dynamics. Genomic analysis identified several acquired AMR genes (ARGs) that have a pathogen-specific signature. We observed that blaCTX-M-15, blaCMY-42, blaNDM-5, and aadA(2) were prevalent in Escherichia coli, and blaTEM-1B, blaOXA-232, blaNDM-1, rmtB, and rmtC were dominant in Klebsiella pneumoniae. In contrast, Pseudomonas aeruginosa and Acinetobacter baumannii harbored blaVEB, blaVIM-2, aph(3'), strA/B, blaOXA-23, aph(3') variants, and amrA, respectively. Regardless of the type of ARG, the MGEs linked with ARGs were also pathogen-specific. The sequence type of these pathogens was identified as high-risk international clones, with only a few lineages being predominant and region-specific. Whole-cell proteome analysis of extensively drug-resistant K. pneumoniae, A. baumannii, E. coli, and P. aeruginosa strains revealed differential abundances of resistance-associated proteins in the presence and absence of different classes of antibiotics. The pathogen-specific resistance signatures and differential abundance of AMR-associated proteins identified in this study should add value to AMR diagnostics and the choice of appropriate drug combinations for successful antimicrobial therapy.

Characterization of an ST38 carbapenem-resistant and highly virulent Escherichia coli carrying conjugatively transferable ColV virulence-resistance and blaNDM-5-positive resistance plasmids.

OBJECTIVES: To characterize an Escherichia coli strain causing bloodstream infection encoding both high-virulence and carbapenem-resistance phenotypes. METHODS: Antimicrobial susceptibility testing, WGS and bioinformatics analysis were performed to characterize strain E1. The function of the ColV plasmid was investigated by the Galleria mellonella infection model, serum killing and macrophage killing assays. The fitness effect of the ColV plasmid was tested by growth curve, plasmid stability tests and the in vitro competition assay. The conjugation assay was performed to test the transferability of the ColV and blaNDM-5-carrying plasmids. RESULTS: E. coli E1 from bloodstream infection was MDR and highly virulent in the G. mellonella infection model. It belonged to phylogroup D, ST38 and serotype O7:H8. E1 carried a conjugatively transferable IncI1-type blaNDM-5-positive plasmid, which conferred carbapenem resistance, a conjugative IncFIB/FII-type ColV plasmid encoding an array of virulence-associated genes and antibiotic resistance genes blaTEM-1B, strAB and sul2, and seven other plasmids. Co-transfer of the ColV plasmid and the blaNDM-5-positive plasmid was observed. The ColV virulence-resistance hybrid plasmid contributed to the virulence, resistance to serum killing, and macrophage phagocytosis in E. coli E1. The carriage of this ColV plasmid did not constitute an in vitro fitness burden to strain E1 but caused fitness costs to E. coli strain EC600. CONCLUSIONS: The emergence of such a highly virulent and resistant strain with conjugative blaNDM-5-positive and ColV plasmids posed a significant threat to public health. Implementation of control measures is needed to prevent such strains from further disseminating in hospital settings and the community.

Neisseria gonorrhoeae antimicrobial resistance patterns and associated risk factors in women of childbearing potential in northwestern Ethiopia.

BACKGROUNDS: Neisseria gonorrhoeae causes gonorrhea and poses public health problems, including antimicrobial resistance. Current data on gonorrhea in prenatal participants in the study area are required. Thus, we aimed to identify gonorrhea prevalence, antimicrobial resistance, and risk factors among antenatal care clinic visitors in northwestern Ethiopia. METHODS: A cross-sectional study was conducted from March to August 2022 at the University of Gondar Comprehensive Specialized Hospital. We recruited 278 study participants using convenient sampling techniques. Sociodemographic, clinical and behavioral risk factors were recorded using pre-tested questionnaires. Endocervical swabs were collected by a physician, transported to the microbiology laboratory, immediately inoculated into modified Thayer-Martin medium, and it was incubated at 37 °C for 24-48 hours. Gram staining and biochemical tests were used to identify the organism. AMR testing was performed using disc diffusion and E-test methods. Data were entered in EPI-info version 7 and exported and analyzed in SPSS version 26. A p-value ≤0.05 was considered as statistically significant. Results were presented in words, tables and figure. RESULTS: Of 278 subjects enrolled, majority (44.6%) were 26-35 years, with a mean age of 29.9 (SD = ±7.2) years, 69.4% were urban residents, and 70.5% were married. Twenty-one (7.6%) participants had gonorrhea. Overall antimicrobial resistance ranged from 19 to 100%. High resistant to tetracycline (100%) and penicillin (85.7%) were observed by both tests. Ciprofloxacin resistance was 52.4% by disc diffusion and 85.7% by E-test. By E-test, all isolates were sensitive to ceftriaxone, cefixime, azithromycin and spectinomycin; however, 7 (33.3%), 9 (42.9%), 9 (42.9%) and 5 (23.8%) isolates showed resistant to these antibiotics with disk method. Prevalence of beta-lactamase producing Neisseria gonorrhoeae was 85.7%. Alcohol consumption (p = 0.032), condom-free sexual practice (p = 0.010), multiple sexual partners (p < 0.001), pelvic pain (p = 0.018), and dysuria (p = 0.021) revealed increased risk of infection. CONCLUSIONS: Compared with many previous studies in Ethiopia, we found high prevalence, antimicrobial resistance, and beta-lactamase-positive isolates. Multiple sexual partners, alcohol consumption, not using condom, pelvic pain and dysuria were predictors of this infection. Continuous large-scale monitoring of pathogen is essential for its prevention and control.

Opening of a cryptic pocket in ß-lactamase increases penicillinase activity.

Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in ß-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol-labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across ß-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM ß-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR chemical exchange saturation transfer experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket's functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.

Effect of Nk-lysin peptides on bacterial growth, MIC, antimicrobial resistance, and viral activities.

NK-lysins from chicken, bovine and human are used as antiviral and antibacterial agents. Gram-negative and gram-positive microorganisms, including Streptococcus pyogenes, Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, Klebsiella oxytoca, Shigella sonnei, Klebsiella pneumoniae and Salmonella typhimurium, are susceptible to NK-lysin treatment. The presence of dominant TEM-1 gene was noted in all untreated and treated bacteria, while TOHO-1 gene was absent in all bacteria. Importantly, ß-lactamase genes CTX-M-1, CTX-M-8, and CTX-M-9 genes were detected in untreated bacterial strains; however, none of these were found in any bacterial strains following treatment with NK-lysin peptides. NK-lysin peptides are also used to test for inhibition of infectivity, which ranged from 50 to 90% depending on NK-lysin species. Chicken, bo vine and human NK-lysin peptides are demonstrated herein to have antibacterial activity and antiviral activity against Rotavirus (strain SA-11). On the basis of the comparison between these peptides, potent antiviral activity of bovine NK-lysin against Rotavirus (strain SA-11) is particularly evident, inhibiting infection by up to 90%. However, growth was also significantly inhibited by chicken and human NK-lysin peptides, restricted by 80 and 50%, respectively. This study provided a novel treatment using NK-lysin peptides to inhibit expression of ß-lactamase genes in ß-lactam antibiotic-resistant bacterial infections.

The in vitro effect of new combinations of carbapenem-ß-lactamase inhibitors for Mycobacterium abscessus.

As new treatment alternatives for Mycobacterium abscessus complex (MABC) are urgently needed, we determined the minimum inhibitory concentrations (MICs) for novel carbapenem combinations, including imipenem-relebactam and tebipenem-avibactam against 98 MABC isolates by broth microdilution. The MIC50 was reduced from 16 to 8 mg/L by adding relebactam to imipenem, while the addition of avibactam to tebipenem showed a more pronounced reduction from 256 to 16 mg/L, representing a promising non-toxic, oral treatment option for further exploration.

An improved PKPD modeling approach to characterize the pharmacodynamic interaction over time between ceftazidime/avibactam and colistin from in vitro time-kill experiments against multidrug-resistant Klebsiella pneumoniae isolates.

In contrast to the checkerboard method, bactericidal experiments [time-kill curves (TKCs)] allow an assessment of pharmacodynamic (PD) interactions over time. However, TKCs in combination pose interpretation problems. The objective of this study was to characterize the PD interaction over time between ceftazidime/avibactam (CZA) and colistin (CST) using TKC against four multidrug-resistant Klebsiella pneumoniae susceptible to both antibiotics and expressing a widespread carbapenemase determinant KPC-3. In vitro TKCs were performed and analyzed using pharmacokinetic/pharmacodynamic (PKPD) modeling. The general pharmacodynamic interaction model was used to characterize PD interactions between drugs. The 95% confidence intervals (95%CIs) of the expected additivity and of the observed interaction were built using parametric bootstraps and compared to evaluate the in vitro PD interaction over time. Further simulations were conducted to investigate the effect of the combination at varying concentrations typically observed in patients. Regrowth was observed in TKCs at high concentrations of drugs alone [from 4 to 32× minimum inhibitory concentrations (MIC)], while the combination systematically prevented the regrowth at concentrations close to the MIC. Significant synergy or antagonism were observed under specific conditions but overall 95%CIs overlapped widely over time indicating an additive interaction between antibiotics. Moreover, simulations of typical PK profile at standard dosages indicated that the interaction should be additive in clinical conditions. The nature of the PD interaction varied with time and concentration in TKC. Against the four K. pneumoniae isolates, the bactericidal effect of CZA + CST combination was predicted to be additive and to prevent the emergence of resistance at clinical concentrations.

Case Commentary: When Voldemort Meets Sauron: Treatment Considerations for Emerging Dual-Carbapenemase-Producing Extensively Drug-Resistant Pseudomonas aeruginosa.

Infections caused by extensively drug-resistant Pseudomonas aeruginosa are difficult to treat due to limited effective treatment options. In this issue, a patient with a corneal infection caused by a Verona integron-encoded metallo-ß-lactamase (VIM)- and Guiana extended-spectrum ß-lactamase (GES)-coproducing P. aeruginosa strain associated with the recent artificial tears-related outbreak in the United States is described. This resistance genotype/phenotype further compromises therapeutic options, and this report provides insights into diagnostic and treatment approaches for clinicians dealing with infections due to this highly resistant P. aeruginosa.

Characterization of Pseudomonas aeruginosa resistance to ceftolozane-tazobactam due to ampC and/or ampD mutations observed during treatment using semi-mechanistic PKPD modeling.

A double ampC (AmpCG183D) and ampD (AmpDH157Y) genes mutations have been identified by whole genome sequencing in a Pseudomonas aeruginosa (PaS) that became resistant (PaR) in a patient treated by ceftolozane/tazobactam (C/T). To precisely characterize the respective contributions of these mutations on the decreased susceptibility to C/T and on the parallel increased susceptibility to imipenem (IMI), mutants were generated by homologous recombination in PAO1 reference strain (PAO1- AmpCG183D, PAO1-AmpDH157Y, PAO1-AmpCG183D/AmpDH157Y) and in PaR (PaR-AmpCPaS/AmpDPaS). Sequential time-kill curve experiments were conducted on all strains and analyzed by semi-mechanistic PKPD modeling. A PKPD model with adaptation successfully described the data, allowing discrimination between initial and time-related (adaptive resistance) effects of mutations. With PAO1 and mutant-derived strains, initial EC50 values increased by 1.4, 4.1, and 29-fold after AmpCG183D , AmpDH157Y and AmpCG183D/AmpDH157Y mutations, respectively. EC50 values were increased by 320, 12.4, and 55-fold at the end of the 2 nd experiment. EC50 of PAO1-AmpCG183D/AmpDH157Y was higher than that of single mutants at any time of the experiments. Within the PaR clinical background, reversal of AmpCG183D, and AmpDH157Y mutations led to an important decrease of EC50 value, from 80.5 mg/L to 6.77 mg/L for PaR and PaR-AmpCPaS/AmpDPaS, respectively. The effect of mutations on IMI susceptibility mainly showed that the AmpCG183D mutation prevented the emergence of adaptive resistance. The model successfully described the separate and combined effect of AmpCG183D and AmpDH157Y mutations against C/T and IMI, allowing discrimination and quantification of the initial and time-related effects of mutations. This method could be reproduced in clinical strains to decipher complex resistance mechanisms.

Effectiveness of cefmetazole versus meropenem for invasive urinary tract infections caused by extended-spectrum ß-lactamase-producing Escherichia coli.

Cefmetazole is active against extended-spectrum ß-lactamase-producing Escherichia coli (ESBLEC) and is a potential candidate for carbapenem-sparing therapy. This multicenter, observational study included patients hospitalized for invasive urinary tract infection due to ESBLEC between March 2020 and November 2021 at 10 facilities in Japan, for whom either cefmetazole or meropenem was initiated as a definitive therapy within 96 h of culture collection and continued for at least 3 d. Outcomes included clinical and microbiological effectiveness, recurrence within 28 d, and all-cause mortality (14 d, 30 d, in-hospital). Outcomes were adjusted for the inverse probability of propensity scores for receiving cefmetazole or meropenem. Eighty-one and forty-six patients were included in the cefmetazole and meropenem groups, respectively. Bacteremia accounted for 43% of the cefmetazole group, and 59% of the meropenem group. The crude clinical effectiveness, 14 d, 30 d, and in-hospital mortality for patients in the cefmetazole and meropenem groups were 96.1% vs 90.9%, 0% vs 2.3%, 0% vs 12.5%, and 2.6% vs 13.3%, respectively. After propensity score adjustment, clinical effectiveness, the risk of in-hospital mortality, and the risk of recurrence were similar between the two groups (P = 0.54, P = 0.10, and P = 0.79, respectively). In all cases with available data (cefmetazole : n = 61, meropenem : n = 22), both drugs were microbiologically effective. In all isolates, bla CTX-M was detected as the extended-spectrum ß-lactamase gene. The predominant CTX-M subtype was CTX-M-27 (47.6%). Cefmetazole showed clinical and bacteriological effectiveness comparable to meropenem against invasive urinary tract infection due to ESBLECs.

The Effect of ß-Lactam Antibiotics on the Evolution of Ceftazidime/Avibactam and Cefiderocol Resistance in KPC-Producing Klebsiella pneumoniae.

In this study, we aimed to clarify the evolutionary trajectory of a Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) population during ß-lactam antibiotic therapy. Five KPC-Kp isolates were collected from a single patient. Whole-genome sequencing and a comparative genomics analysis were performed on the isolates and all blaKPC-2-containing plasmids to predict the population evolution process. Growth competition and experimental evolution assays were conducted to reconstruct the evolutionary trajectory of the KPC-Kp population in vitro. Five KPC-Kp isolates (KPJCL-1 to KPJCL-5) were highly homologous, and all harbor an IncFII blaKPC-containing plasmid (pJCL-1 to pJCL-5). Although the genetic structures of these plasmids were almost identical, distinct copy numbers of the blaKPC-2 gene were detected. A single copy of blaKPC-2 was presented in pJCL-1, pJCL-2, and pJCL-5, two copies of blaKPC (blaKPC-2 and blaKPC-33) were presented in pJCL-3, and three copies of blaKPC-2 were presented in pJCL-4. The blaKPC-33-harboring KPJCL-3 isolate presented resistance to ceftazidime-avibactam and cefiderocol. The blaKPC-2 multicopy strain KPJCL-4 had an elevated ceftazidime-avibactam MIC. The patient had been exposed to ceftazidime, meropenem, and moxalactam, after which KPJCL-3 and KPJCL-4 were isolated, which both showed a significant competitive advantage under antimicrobial pressure in vitro. Experimental evolution assays revealed that blaKPC-2 multicopy-containing cells were increased in the original single-copy blaKPC-2-harboring KPJCL-2 population under selection with ceftazidime, meropenem, or moxalactam, generating a low-level ceftazidime-avibactam resistance phenotype. Moreover, blaKPC-2 mutants with a G532T substitution, G820 to C825 duplication, G532A substitution, G721 to G726 deletion, and A802 to C816 duplication increased in the blaKPC-2 multicopy-containing KPJCL-4 population, generating high-level ceftazidime-avibactam resistance and reduced cefiderocol susceptibility. Ceftazidime-avibactam and cefiderocol resistance can be selected by ß-lactam antibiotics other than ceftazidime-avibactam. Notably, blaKPC-2 gene amplification and mutation are important in KPC-Kp evolution under antibiotic selection.

PCBP1 protects bladder cancer cells from mitochondria injury and ferroptosis by inducing LACTB mRNA degradation.

Although Poly C Binding Protein 1 (PCBP1) affects cellular ferroptosis and mitochondrial dysfunction, the mechanisms by which PCBP1 regulates bladder cancer (BC) cell functions are unknown. In this study, two BC cell lines (T24 and UMUC3) were treated with different doses of ferroptosis inducer erastin to analyze the effect of PCBP1. Online databases (RPISeq and CatRAPID) were used to predict the possible direct interaction between PCBP1 protein and serine ß-lactamase-like protein (LACTB) mRNA, which was further validated via RNA pull-down, RNA immunoprecipitation, and luciferase reporter assays. Mitochondria injury and ferroptosis were evaluated using CCK-8 assay, TUNEL staining, flow cytometry, corresponding kits, and JC-1 staining. In vivo experiments were conducted using tumor xenograft models. Quantitative reverse-transcription polymerase chain reaction was used to detect transcript expression levels, while protein levels were analyzed using western blot and immunohistochemistry. PCBP1 expression was significantly upregulated in BC tissues and cell lines. Also, PCBP1 knockdown increased erastin-mediated ferroptosis in T24 and UMUC3 cells, while PCBP1 overexpression decreased erastin-mediated ferroptosis in T24 and UMUC3 cells. Mechanistic results showed that LACTB mRNA is a novel PCBP1-binding transcript. LACTB upregulation promoted erastin-induced ferroptosis and mitochondrial dysfunction. Furthermore, LACTB overexpression reversed PCBP1-mediated ferroptosis protection, including decreased ROS and enhanced mitochondrial function, which were further alleviated after phosphatidylserine decarboxylase (PISD) overexpression. Moreover, PCBP1 silencing significantly enhanced tumor inhibition effect of sulfasalazine in xenograft mice transplanted with T24 and UMUC3 cells, leading to LACTB upregulation and PISD downregulation. In conclusion, PCBP1 protects BC cells against mitochondria injury and ferroptosis via LACTB/PISD axis.

Development of an optimized and practical pharmacokinetics/pharmacodynamics analysis method for aztreonam/nacubactam against carbapenemase-producing K. pneumoniae.

BACKGROUND: Nacubactam, a new ß-lactamase inhibitor with antibacterial activity, is being developed as a single drug to be co-administered with cefepime or aztreonam. However, determining pharmacokinetics/pharmacodynamics (PK/PD) parameters in ß-lactam/ß-lactamase inhibitor combinations remains challenging. We aimed to establish a practical PK/PD analysis method for aztreonam/nacubactam that incorporates instantaneous MIC (MICi). METHODS: Based on chequerboard MIC measurements, MICi of aztreonam against carbapenemase-producing Klebsiella pneumoniae in the presence of nacubactam was simulated. RESULTS: The mean change in the bacterial count of thigh-infected mice in an in vivo PD study was plotted based on %fT>MICi and analysed using the inhibitory effect sigmoid Imax model. fT>MICi calculated from the PK experiments showed a high correlation with the in vivo bactericidal effect, suggesting that fT>MICi is the optimal PK/PD parameter for aztreonam/nacubactam. The target values of fT>MICi achieving growth inhibition, 1 log10 kill and 2 log10 kill, were 22, 38% and 75%, respectively. CONCLUSIONS: The PK/PD analysis method proposed in this study is promising for determining practical PK/PD parameters in combination therapy. In addition, this is the first report of aztreonam/nacubactam showing a potent in vivo therapeutic effect against NDM-producing K. pneumoniae.

Efficacy of ceftazidime/avibactam in various combinations for the treatment of experimental osteomyelitis in rabbits caused by OXA-48-/ESBL-producing Escherichia coli.

BACKGROUND: While the treatment of ESBL-producing Enterobacterales osteomyelitis relies on carbapenems, the optimal regimen for OXA48 types remains unclear. We evaluated the efficacy of ceftazidime/avibactam in different combinations in an experimental model of OXA-48-/ESBL-producing Escherichia coli osteomyelitis. METHODS: E. coli pACYC184 is a clinical strain harbouring blaOXA-48 and blaCTX-M-15 inserts, with 'increased exposure susceptibility' to imipenem (MIC, 2 mg/L), gentamicin (MIC, 0.5 mg/L), colistin (MIC, 0.25 mg/L), ceftazidime/avibactam (MIC, 0.094 mg/L) and fosfomycin (MIC, 1 mg/L), and resistance to ceftazidime (MIC, 16 mg/L). Osteomyelitis was induced in rabbits by tibial injection of 2 × 108 cfu of OXA-48/ESBL E. coli. Treatment started 14 days later for 7 days in six groups: (1) control, (2) colistin 150.000 IU/kg subcutaneously (SC) q8h, (3) ceftazidime/avibactam 100/25 mg/kg SC q8h, (4) ceftazidime/avibactam + colistin, (5) ceftazidime/avibactam + fosfomycin 150 mg/kg SC q12h, (6) ceftazidime/avibactam + gentamicin 15 mg/kg intramuscularly (IM) q24h. Treatment was evaluated at Day 24 according to bone cultures. RESULTS: In vitro, time-kill curves of ceftazidime/avibactam in combination showed a synergistic effect. In vivo, compared with controls, rabbits treated with colistin alone had similar bone bacterial density (P = 0.50), whereas ceftazidime/avibactam alone or in combinations significantly decreased bone bacterial densities (P = 0.004 and P < 0.0002, respectively). Bone sterilization was achieved using ceftazidime/avibactam in combination with colistin (91%) or fosfomycin (100%) or gentamicin (100%) (P < 0.0001), whereas single therapies were not different from controls. No ceftazidime/avibactam-resistant strains emerged in rabbits treated, regardless of the combination. CONCLUSIONS: In our model of E. coli OXA-48/ESBL osteomyelitis, ceftazidime/avibactam in combination was more effective than any single therapy, whatever the companion drug used (gentamicin or colistin or fosfomycin).

Instant detection of extended-spectrum ß-lactamase-producing bacteria from the urine of patients using infrared spectroscopy combined with machine learning.

Antibiotics are considered the most effective treatment against bacterial infections. However, most bacteria have already developed resistance to a broad spectrum of commonly used antibiotics, mainly due to their uncontrolled use. Extended-spectrum beta-lactamase (ESBL)-producing bacteria are an essential class of multidrug-resistant (MDR) bacteria. It is of extreme urgency to develop a method that can detect ESBL-producing bacteria rapidly for the effective treatment of patients with bacterial infectious diseases. Fourier transform infrared (FTIR) microscopy is a sensitive method that can rapidly detect cellular molecular changes. In this study, we examined the potential of FTIR spectroscopy-based machine learning algorithms for the rapid detection of ESBL-producing bacteria obtained directly from a patient's urine. Using 591 ESBL-producing and 1658 non-ESBL-producing samples of Escherichia coli (E. coli) and Klebsiella pneumoniae, our results show that the FTIR spectroscopy-based machine learning approach can identify ESBL-producing bacteria within 40 minutes from receiving a patient's urine sample, with a success rate of 80%.

Baicalein Inhibits Plasmid-Mediated Horizontal Transmission of the blaKPC Multidrug Resistance Gene from Klebsiella pneumoniae to Escherichia coli.

Carbapenem-resistant bacterial infections pose an urgent threat to public health worldwide. Horizontal transmission of the ß-lacatamase Klebsiella pneumoniae carbapenemase (blaKPC) multidrug resistance gene is a major mechanism for global dissemination of carbapenem resistance. Here, we investigated the effects of baicalein, an active ingredient of a Chinese herbal medicine, on plasmid-mediated horizontal transmission of blaKPC from a meropenem-resistant K. pneumoniae strain (JZ2157) to a meropenem-sensitive Escherichia coli strain (E600). Baicalein showed no direct effects on the growth of JZ2157 or E600. Co-cultivation of JZ2157 and E600 caused the spread of meropenem resistance from JZ2157 to E600. Baicalein at 40 and 400 µg/mL significantly inhibited the spread of meropenem resistance. Co-cultivation also resulted in plasmid-mediated transmission of blaKPC from JZ2157 to E600, which was inhibited by baicalein. Therefore, baicalein may be used in clinical practice to prevent or contain outbreaks of carbapenem-resistant infections by inhibiting the horizontal transfer of resistance genes across bacteria species.

Synergistic Antimicrobial Activity of Eugenol in Combination with Fosfomycin to Combat Escherichia coli and Potential Effect on Plasmid-Mediated Fosfomycin Resistance Genes.

The presence of multidrug-resistant pathogenic microorganisms makes it challenging to cure bacterial illnesses. Syzygium aromaticum has been used for medicinal purposes since ancient times. The objective of this study was to investigate the potential synergistic effect of the combination of Eugenol and Fosfomycin against clinically Uropathogenic Escherichia coli (UPEC) and their possible co-treatment as well as their contribution to plasmid-mediated Fosfomycin resistance (fosA3 and fosA4) genes using molecular assays. Eugenol was extracted from clove (Syzygium aromaticum) plants using steam distillation by Clevenger and analyzed by high-performance liquid chromatography (HPLC). UPEC accounted for 63.6 % of all isolates. Specifically, 99.3 % of the UPEC isolates exhibited resistance to multiple types of antibiotics [multidrug-resistant (MDR)]. The MIC for Eugenol was 1.25-5 µg/mL, and Fosfomycin was 512-1024 µg/mL, while the MBC for Eugenol was 5-10 µg/mL and Fosfomycin was 2048 µg/mL. The synergistic effects were considerable, with 1/4 MIC of Eugenol resulting in 1/8 MIC Fosfomycin. Eugenol inhibited most of the UPEC isolates at 4-8 hours, Fosfomycin at 8-12 hours, and co-treatment at 4-8 hours. The fosA3 and fosA4 genes were detected in 5.7 % and 2.9 % of the isolates, respectively. The results showed variable gene expression changes in response to the different treatments.

[In vitro activity of ß-lactamase inhibitors avibanvctam and relebactam in combination with ß-lactams against multidrug-resistant Mycobacterium tuberculosis and mutations of resistance genes].

Objective: To evaluate the activity of six ß-lactams in combination with three ß-lactamase inhibitors against mycobacterium tuberculosis(MTB) in vitro. Methods: A total of 105 multidrug-resistant tuberculosis (MDR-TB) strains from different regions of Henan province from January to September 2020 were included in this study. Drug activity of six ß-lactams (biapenem, meropenem, imipenem, doripenem, ertapenem and tebipenem) alone or in combination with ß-lactamase inhibitors (clavulanic acid, avibactam and relebactam) was examined by minimum inhibitory concentration method (MICs) against 105 clinical isolates. Mutations of blaC, ldtmt1 and ldtmt2 were analyzed by PCR and DNA sequencing. Chi-square test was used to compare the antimicrobial activities of different ß-lactam drugs. Results: Out of the ß-lactams used herein, tebipenem was the most effective against MDR-TB and had an MIC50 value of 8 mg/L(χ2=123.70,P=0.001). Besides, after the addition of ß-lactamase inhibitors, the MICs of most ß-lactam drugs were reduced more evidently in the presence of avibactam and relebactam compared to clavulanic acid.Especially, relebactam decreased both the MIC50 and MIC90 of telbipenem by 16-fold, and diluted the MIC of 23 (21.90%) and 41 (39.04%) isolatesby 32-fold and 16-fold.In addition, a total of 13.33% (14/105) of isolates harbored mutations in the blaC gene, with three different nucleotide substitutions: AGT333AGG, AAC638ACC and ATC786ATT. For the strains with Ser111Arg and Asn213Thr substitution in BlaC, the MIC values of the meropenem-clavulanate combination were reduced compared with a synonymous single nucleotide polymorphism (SNP) group. Conclusions: Both avibactam and relebactam had better synergistic effects on ß-lactams than clavulanic acid. The combination of tebipenem and relebactam showed the most potent activity against MDR-TB isolates. In addition, the Ser111Arg and Asn213Thr substitution of BlaC may be associated with an increased susceptibility of MDR-TB isolates to meropenem in the presence of clavulanate.

Modulation of the Specificity of Carbapenems and Diazabicyclooctanes for Selective Activity against Mycobacterium tuberculosis.

Treatment of multidrug-resistant tuberculosis with combinations of carbapenems and ß-lactamase inhibitors carries risks for dysbiosis and for the development of resistances in the intestinal microbiota. Using Escherichia coli producing carbapenemase KPC-2 as a model, we show that carbapenems can be modified to obtain drugs that are inactive against E. coli but retain antitubercular activity. Furthermore, functionalization of the diazabicyclooctanes scaffold provided drugs that did not effectively inactivate KPC-2 but retained activity against Mycobacterium tuberculosis targets.

Evolutionary Trajectories toward High-Level ß-Lactam/ß-Lactamase Inhibitor Resistance in the Presence of Multiple ß-Lactamases.

ß-Lactam antibiotics are the first choice for the treatment of most bacterial infections. However, the increased prevalence of ß-lactamases, in particular extended-spectrum ß-lactamases, in pathogenic bacteria has severely limited the possibility of using ß-lactam treatments. Combining ß-lactam antibiotics with ß-lactamase inhibitors can restore treatment efficacy by negating the effect of the ß-lactamase and has become increasingly important against infections caused by ß-lactamase-producing strains. Not surprisingly, bacteria with resistance to even these combinations have been found in patients. Studies on the development of bacterial resistance to ß-lactam/ß-lactamase inhibitor combinations have focused mainly on the effects of single, chromosomal or plasmid-borne, ß-lactamases. However, clinical isolates often carry more than one ß-lactamase in addition to multiple other resistance genes. Here, we investigate how the evolutionary trajectories of the development of resistance to three commonly used ß-lactam/ß-lactamase inhibitor combinations, ampicillin-sulbactam, piperacillin-tazobactam, and ceftazidime-avibactam, were affected by the presence of three common ß-lactamases, TEM-1, CTX-M-15, and OXA-1. First-step resistance was due mainly to extensive gene amplifications of one or several of the ß-lactamase genes where the amplification pattern directly depended on the respective drug combination. Amplifications also served as a stepping-stone for high-level resistance in combination with additional mutations that reduced drug influx or mutations in the ß-lactamase gene blaCTX-M-15. This illustrates that the evolutionary trajectories of resistance to ß-lactam/ß-lactamase inhibitor combinations are strongly influenced by the frequent and transient nature of gene amplifications and how the presence of multiple ß-lactamases shapes the evolution to higher-level resistance.