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Annals of Clinical Microbiology and... Nov 2023This study aimed to illustrate the status of carbapenem-resistant Enterobacterales (CRE) infections in a Chinese tertiary hospital and to investigate the role of outer...
BACKGROUND
This study aimed to illustrate the status of carbapenem-resistant Enterobacterales (CRE) infections in a Chinese tertiary hospital and to investigate the role of outer membrane vesicles (OMVs) in antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP).
METHODS
The data of CRE infections was collected from laboratory records, and the CRE isolates from two distinct periods (2015/07 to 2017/07 and 2020/04 to 2021/04) were enrolled to detect the carbapenemase genes by polymerase chain reaction (PCR). Multilocus sequence typing (MLST) was used to analyze the molecular characterization of CRKP. The conjugation assay was performed to verify the transmission of the antibiotic resistance plasmid. The OMVs of CRKP were isolated with a method combining an electrophoretic technique with a 300 kDa cut-off dialysis bag. The protein components in CRKP OMVs were analyzed by liquid chromatography tandem-mass spectrometry (LC-MS/MS), and the meropenem-hydrolyzing bioactivity of KPC in CRKP OMVs was determined with different treatments in vitro.
RESULTS
A total of 178 CRE isolates, including 100 isolates from 2015/07 to 2017/07 and 78 isolates from 2020/04 to 2021/04, were collected for the detection of carbapenemase genes. We found that the carbapenemase gene blaKPC was the most prevalent, followed by blaNDM. By MLST, we found that sequence type (ST) 11 CRKP (96.1%) was the leading type during 2015/07 to 2017/07 and that the ST15 CRKP increased to 46.2% in the late period of 2020/04 to 2021/04. The diameters of Klebsiella pneumoniae OMVs ranged from 100 to 200 nm, and by proteomics analysis the most proteins from OMVs belonged to the "enzyme" group. The KPC enzyme was found in the OMVs from CRKP, and the OMVs could protect inside KPC from proteinase K digestion. Moreover, the KPC enzymes within OMVs, which could be released after Triton X-100 treatment, could hydrolyze meropenem.
CONCLUSIONS
CRE has increasingly caused infections in hospitals, and blaKPC-positive CRKP infections have constituted a major proportion of infections in the past decade. The OMVs play a critical role in antibiotic resistance in CRKP.
Topics: Humans; Meropenem; Klebsiella pneumoniae; Multilocus Sequence Typing; Chromatography, Liquid; Klebsiella Infections; Tandem Mass Spectrometry; beta-Lactamases; Carbapenems; Carbapenem-Resistant Enterobacteriaceae; Drug Resistance, Microbial; Tertiary Care Centers; Anti-Bacterial Agents; Microbial Sensitivity Tests
PubMed: 37919721
DOI: 10.1186/s12941-023-00645-4 -
ACS Infectious Diseases Jan 2024Carbapenem-resistant (CRAB) strains are prevalent worldwide and represent a major threat to public health. However, treatment options for infections caused by CRAB are...
Carbapenem-resistant (CRAB) strains are prevalent worldwide and represent a major threat to public health. However, treatment options for infections caused by CRAB are very limited as they are resistant to most of the commonly used antibiotics. Consequently, understanding the mechanisms underlying carbapenem resistance and restoring bacterial susceptibility to carbapenems hold immense importance. The present study used gas chromatography-mass spectrometry (GC-MS)-based metabolomics to investigate the metabolic mechanisms of antibiotic resistance in clinically isolated CRAB. Inactivation of the pyruvate cycle and purine metabolism is the most typical characteristic of CRAB. The CRAB exhibited a reduction in the activity of enzymes involved in the pyruvate cycle, proton motive force, and ATP levels. This decline in central carbon metabolism resulted in a decrease in the metabolic flux of the α-ketoglutarate-glutamate-glutamine pathway toward purine metabolism, ultimately leading to a decline in adenine nucleotide interconversion. Exogenous adenosine monophosphate (AMP) and adenosine triphosphate (ATP) enhance the killing efficacy of Meropenem against CRAB. The combination of ATP and Meropenem also has a synergistic effect on eliminating CRAB persisters and the biofilm, as well as protecting mice against peritonitis-sepsis. This study presents a novel therapeutic modality to treat infections caused by CRAB based on the metabolism reprogramming strategy.
Topics: Animals; Mice; Meropenem; Acinetobacter baumannii; Acinetobacter Infections; Microbial Sensitivity Tests; Carbapenems; Adenosine Triphosphate; Pyruvates; Purines
PubMed: 37991817
DOI: 10.1021/acsinfecdis.3c00480 -
F1000Research 2022Carbapenems are the treatment of choice for multidrug-resistant (MDR) and extensively drug-resistant (XDR) infections, but the emergence of...
Carbapenems are the treatment of choice for multidrug-resistant (MDR) and extensively drug-resistant (XDR) infections, but the emergence of carbapenem-resistant (CRAB) has rendered it ineffective in the vast majority of cases. Combination therapy has grown in popularity over the last decade; this study aims to analyze growth kinetics after exposure to meropenem and ampicillin-sulbactam compared with meropenem and amikacin antibiotic combinations in clinically relevant concentrations. This experimental laboratory study was conducted on the ATCC 19606 isolate and three clinical isolates that were intermediate or resistant to tested antibiotics. Meropenem and ampicillin-sulbactam, as well as meropenem and amikacin, were tested at four different concentrations against isolates. Turbidity measurements were taken at predetermined time points of 0, 1, 2, 4, 6, 8, and 24 hours following exposure; bacterial concentration was enumerated using the agar plate method, with the results plotted in a time-kill curve. A bactericidal effect was achieved in isolates that were intermediate to ampicillin-sulbactam and resistant to meropenem after the administration of meropenem and ampicillin-sulbactam combination with a concentration of 4 µg/ml and 16/8 µg/ml, respectively. The combination of meropenem and ampicillin-sulbactam demonstrated bacteriostatic activity against isolates that were resistant to both antibiotics. Isolates treated with resistant antibiotics showed an increased growth rate compared to the growth control. The combination of meropenem and ampicillin-sulbactam could be a promising combination therapy in treating CRAB infections. The mechanism and degree of antibiotic resistance in the isolates affect the efficacy of antibiotic combinations; further research is needed to corroborate the findings of this study.
Topics: Humans; Meropenem; Acinetobacter baumannii; Amikacin; Kinetics; Anti-Bacterial Agents; Carbapenems; Acinetobacter Infections
PubMed: 36531260
DOI: 10.12688/f1000research.122221.2 -
Microbiology Spectrum Feb 2024Carbapenem-resistant (CRKP) is an important multidrug resistance (MDR) pathogen that threatens human health and is the main source of hospital-acquired infection. Outer...
Carbapenem-resistant (CRKP) is an important multidrug resistance (MDR) pathogen that threatens human health and is the main source of hospital-acquired infection. Outer membrane vesicles (OMVs) are extracellular vesicles derived from Gram-negative bacteria and contain materials involved in bacterial survival and pathogenesis. They also contribute to cellular communication to nearby or distant recipient cells and influence their functions and phenotypes. In this study, we sought to understand the mechanism of bacterial response to meropenem pressure and explore the relationship between pathogenic proteins and the high pathogenicity of bacteria. We performed whole-genome PacBio sequencing on a clinical CRKP strain, and its OMVs were characterized using nanoparticle tracking analysis, transmission electron microscopy, and proteomic analysis. Thousands of vesicle proteins have been identified in mass spectrometry-based high-throughput proteomics analyses of OMVs. Protein functionality analysis showed that the OMVs were predominantly involved in metabolic, intracellular compartments, nucleic acid binding, survival, defense, and antibiotic resistance, such as Chromosome partition protein MukB, 3-methyl-2-oxobutanoate hydroxymethyltransferase, methionine-tRNA ligase, Heat shock protein 60 family chaperone GroEL, and Gamma-glutamyl phosphate reductase. Additionally, a protein-protein interaction network demonstrated that OMVs from meropenem-treated showed the highest connectivity in DNA polymerase I, phenylalanine-tRNA ligase beta subunit, DNA-directed RNA polymerase subunit beta, methionine-tRNA ligase, DNA-directed RNA polymerase subunit beta, and DNA-directed RNA polymerase subunit alpha. The OMVs proteome expression profile indicates increased secretion of stress proteins released from meropenem-treated , which provides clues for revealing the biogenesis and pathophysiological functions of Gram-negative bacteria OMVs. The significant differentially expressed proteins identified in this study are of great significance for exploring effective control strategies for CRKP infection.IMPORTANCEMeropenem is one of the main antibiotics used in the clinical treatment of carbapenem-resistant (CRKP). This study demonstrated that some important metabolic changes occurred in meropenem-induced CRKP-outer membrane vesicles (OMVs), The OMVs proteome expression profile indicates increased secretion of stress proteins released from meropenem-induced . Furthermore, this is the first study to discuss the protein-protein interaction network of the OMVs released by CRKP, especially under antibiotic stress.
Topics: Humans; Meropenem; Klebsiella pneumoniae; Proteome; Proteomics; Methionine-tRNA Ligase; Anti-Bacterial Agents; Heat-Shock Proteins; DNA-Directed RNA Polymerases; Klebsiella Infections; Microbial Sensitivity Tests
PubMed: 38236023
DOI: 10.1128/spectrum.02917-23 -
The Journal of Applied Laboratory... Sep 2021The class of antibiotics known as β-lactams are a commonly used due to their effectiveness and safety. Therapeutic drug monitoring has been proposed but requires an...
BACKGROUND
The class of antibiotics known as β-lactams are a commonly used due to their effectiveness and safety. Therapeutic drug monitoring has been proposed but requires an accurate assay along with well-characterized preanalytic stability, as β-lactams are known to be relatively unstable.
METHODS
A high-throughput LC-MS/MS assay validation and stability study was performed for cefepime, meropenem, and piperacillin and tazobactam in serum. Patient samples, standards, and QCs were crashed with acetonitrile containing internal standard. Following centrifugation, an aliquot of the supernatant was diluted with clinical laboratory reagent water and analyzed by LC-MS/MS.
RESULTS
The assay showed linearity between 0.5 and 60 µg/mL for each analyte. The intra- and interassay reproducibility at 3 different concentrations (approximately 2, 25, and 40 µg/mL) was <5% for each analyte. Accuracy studies for each analyte were compared using linear regression and demonstrated: slope = 1.0 ± 0.1; r2 ≥ 0.980; and y intercept 95% CI that included zero. Minimal ion suppression or enhancement was observed, and no significant carryover was observed up to 500 µg/mL of each analyte. Stability studies demonstrated significant loss in serum for each analyte at ambient and refrigerated temperatures (2-8 °C) and at -20 °C over days or weeks. In contrast, when stored at -80 °C, no significant loss was observed.
CONCLUSIONS
The LC-MS/MS assay showed acceptable performance characteristics for quantitation of β-lactams. With well-characterized stability, this assay can be used with residual specimens for pharmacokinetic modeling, which may lead to individualized dosing and improved patient care.
Topics: Cefepime; Chromatography, Liquid; Humans; Meropenem; Piperacillin; Reproducibility of Results; Tandem Mass Spectrometry; Tazobactam
PubMed: 34086904
DOI: 10.1093/jalm/jfab036 -
and activity of cefiderocol against spp. and complex, including carbapenem-non-susceptible isolates.Antimicrobial Agents and Chemotherapy Dec 2023spp. and complex (Bcc) are rare but diverse opportunistic pathogens associated with serious infections, which are often multidrug resistant. This study compared the...
spp. and complex (Bcc) are rare but diverse opportunistic pathogens associated with serious infections, which are often multidrug resistant. This study compared the antibacterial activity of the siderophore antibiotic cefiderocol against spp. and Bcc isolates with that of other approved antibacterial drugs, including ceftazidime-avibactam, ciprofloxacin, colistin, imipenem-relebactam, and meropenem-vaborbactam. Isolates were collected in the SIDERO multinational surveillance program. Among 334 spp. isolates [76.6% from respiratory tract infections (RTIs)], cefiderocol had minimum inhibitory concentration (MIC) of 0.06/0.5 µg/mL overall and 0.5/4 µg/mL against 52 (15.6%) carbapenem-non-susceptible (Carb-NS) isolates. Eleven (3.3%) spp. isolates overall and 6 (11.5%) Carb-NS isolates were not susceptible to cefiderocol. Among 425 Bcc isolates (73.4% from RTIs), cefiderocol had MIC of ≤0.03/0.5 µg/mL overall and ≤0.03/1 µg/mL against 184 (43.3%) Carb-NS isolates. Twenty-two (5.2%) Bcc isolates overall and 13 (7.1%) Carb-NS isolates were not susceptible to cefiderocol. Cumulative MIC distributions showed cefiderocol to be the most active of the agents tested against both spp. and Bcc. In a neutropenic murine lung infection model and a humanized pharmacokinetic immunocompetent rat lung infection model, cefiderocol showed significant bactericidal activity against two meropenem-resistant strains compared with untreated controls ( < 0.05) and vehicle-treated controls ( < 0.05), respectively. Meropenem, piperacillin-tazobactam, ceftazidime, and ciprofloxacin comparators showed no significant activity in these models. The results suggest that cefiderocol could be a possible treatment option for RTIs caused by spp. and Bcc.
Topics: Rats; Animals; Mice; Cefiderocol; Meropenem; Carbapenems; Cephalosporins; Burkholderia cepacia complex; Achromobacter; Drug Resistance, Multiple, Bacterial; Anti-Bacterial Agents; Ceftazidime; Respiratory Tract Infections; Ciprofloxacin; Microbial Sensitivity Tests
PubMed: 37971240
DOI: 10.1128/aac.00346-23 -
Diagnostic Microbiology and Infectious... Aug 2023Antibiotic resistance surveillance may be essential to identify patterns of antibiotic resistance and guide treatment choices. Therefore, this systematic review and... (Meta-Analysis)
Meta-Analysis Review
Antibiotic resistance surveillance may be essential to identify patterns of antibiotic resistance and guide treatment choices. Therefore, this systematic review and meta-analysis aimed to evaluate amikacin resistance and susceptibility in children with extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-PE). From inception to September 5, 2022, relevant studies were searched via PubMed, Embase, Cochrane Library, and Web of Science databases. A network meta-analysis was conducted to explore the sequencing of resistance rates in amikacin and other antibiotics. Totally, 26 studies with 2582 clusters of bacterial isolates were included. The resistance rate of amikacin in children with ESBL-PE was 10.1%, higher than the resistance rate of tigecycline (0.0%), ertapenem (0.4%), meropenem (0.7%), and imipenem (3.0%). For the drug susceptibility rate in children with ESBL-PE, the susceptibility rate of amikacin (89.7%) was lower than tigecycline (99.6%), imipenem (96.8%), meropenem (97.3%), and ertapenem (95.6%). Amikacin showed a low drug resistance and a high drug resistance in children with ESBL-PE infection, making it a good option for the treatment of the infection caused by ESBL-PE.
Topics: Child; Humans; Amikacin; Ertapenem; Meropenem; Tigecycline; Escherichia coli; Klebsiella pneumoniae; Anti-Bacterial Agents; Imipenem; beta-Lactamases; Drug Resistance; Microbial Sensitivity Tests
PubMed: 37290259
DOI: 10.1016/j.diagmicrobio.2023.115956 -
Antimicrobial Agents and Chemotherapy Nov 2021Carbapenemase-producing pose an increasing medical threat. Combination therapy is often used for severe infections; however, there is little evidence supporting the...
Carbapenemase-producing pose an increasing medical threat. Combination therapy is often used for severe infections; however, there is little evidence supporting the optimal selection of drugs. This study aimed to determine the effects of polymyxin B combinations against carbapenemase-producing Escherichia coli. The interactions of polymyxin B in combination with aztreonam, meropenem, minocycline or rifampin against 20 clinical isolates of NDM and OXA-48-group-producing E. coli were evaluated using time-lapse microscopy; 24-h samples were spotted on plates with and without 4× MIC polymyxin B for viable counts. Whole-genome sequencing was applied to identify resistance genes and mutations. Finally, potential associations between combination effects and bacterial genotypes were assessed using Fisher's exact test. Synergistic and bactericidal effects were observed with polymyxin B and minocycline against 11/20 strains and with polymyxin B and rifampin against 9/20 strains. The combinations of polymyxin B and aztreonam or meropenem showed synergy against 2/20 strains. Negligible resistance development against polymyxin B was detected. Synergy with polymyxin B and minocycline was associated with genes involved in efflux (presence of , wild-type , and the mutation H44Q) and lipopolysaccharide synthesis ( C27Y, mutations, and L323S). Synergy with polymyxin B and rifampin was associated with sequence variations in , which plays a role in lipid A modification. Polymyxin B in combination with minocycline or rifampin frequently showed positive interactions against NDM- and OXA-48-group-producing E. coli. Synergy was associated with genes encoding efflux and components of the bacterial outer membrane.
Topics: Aztreonam; Bacterial Proteins; Escherichia coli; Klebsiella pneumoniae; Meropenem; Microbial Sensitivity Tests; Minocycline; Polymyxin B; Rifampin; beta-Lactamases
PubMed: 34516251
DOI: 10.1128/AAC.01065-21 -
PloS One 2018The aim of this study was to analyze the metabolome of several Klebsiella pneumoniae strains characterized by different resistance patterns. A total of 59 bacterial...
The aim of this study was to analyze the metabolome of several Klebsiella pneumoniae strains characterized by different resistance patterns. A total of 59 bacterial strains (27 carbapenemase-negative and 32 carbapenemase-positive) were included and their metabolic features were assessed in basal conditions. Moreover, 8 isolates (4 wild-type and 4 KPC-producers) were randomly selected to evaluate the impact of sub-lethal concentrations of meropenem on bacterial metabolism. The metabolomic analysis was performed by 1H-NMR spectroscopy both on filtered supernatants and cell lysates. A total of 40 and 20 molecules were quantified in the intracellular and the extracellular metabolome, respectively. While in basal conditions only five metabolites showed significant differences between carbapenemase-positive and negative strains, the use of meropenem had a profound impact on the whole bacterial metabolism. In the intracellular compartment, a reduction of different overflow metabolites and organic acids (e.g. formate, acetate, isobutyrate) was noticed, whereas, in the extracellular metabolome, the levels of several organic acids (e.g. succinate, acetate, formate, lactate) and amino acids (aspartate, threonine, lysine, alanine) were modified by meropenem stimulation. Interestingly, carbapenemase-positive and negative strains reacted differently to meropenem in terms of number and type of perturbed metabolites. In wild-type strains, meropenem had great impact on the metabolic pathways related to methane metabolism and alanine, aspartate and glutamate metabolism, whereas in KPC-producers the effect was predominant on pyruvate metabolism. The knowledge about the bacterial metabolic profiles could help to set up innovative diagnostic methods and new antimicrobial strategies to fight the global crisis against carbapenemase-positive K. pneumoniae.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Humans; Klebsiella Infections; Klebsiella pneumoniae; Meropenem; Microbial Sensitivity Tests; beta-Lactamases
PubMed: 30440048
DOI: 10.1371/journal.pone.0207478 -
Mikrobiyoloji Bulteni Jul 2023The rate of extensively drug-resistant and pan-resistant gram-negative rods isolated as infectious agents is increasing around the world and in Türkiye. One of the...
The rate of extensively drug-resistant and pan-resistant gram-negative rods isolated as infectious agents is increasing around the world and in Türkiye. One of the important options in the treatment of these infections is the combined use of antibiotics. Therefore, the aim of this study was to investigate the in vitro effect of meropenem/colistin and meropenem/fosfomycin combinations on carbapenem-resistant gram-negative bacilli isolated as infectious agents. Escherichia coli (n= 6), Klebsiella pneumoniae (n= 10), Pseudomonas aeruginosa (n= 5), and Acinetobacter baumannii (n= 6) isolates were recovered from blood and tracheal aspirate samples of patients hospitalized in our hospital's intensive care unit were included in the study. In the first stage of the combination study, minimal inhibitory concentrations (MIC) were investigated by broth microdilution for meropenem and colistin, and agar dilution methods for fosfomycin. In the second stage of the study, synergy, partial synergy, indifference, and antagonistic effects were investigated with the checkerboard method for the meropenem/colistin combination and the agar dilution method for the meropenem/fosfomycin combination. The checkerboard results were interpreted as follows: fractional inhibitory concentration index (FICI) values ≤ 0.5 synergy, < 0.5-≤ 1 partial synergy, > 1-≤ 4 indifference and FIC values of > 4 antagonism. MIC values obtained in the study were interpreted according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Of the 27 isolates studied with the broth microdilution method, 63% were found to be colistin-resistant and 37% susceptible. The MIC values of fosfomycin against Enterobacterales group bacteria were found to be in the range of 2-2048 mg/L. Two of the six E.coli isolates and nine of the 10 K.pneumoniae isolates were found to be resistant to fosfomycin (IV). The MIC values of ≥ 128 mg/L were found in all 11 non-fermentative gram-negative rods with intrinsic resistance to fosfomycin. In the combination of meropenem/ colistin, synergy and partial synergy were observed in 11 (40.7%) of 27 isolates, an indifference effect was observed in 13 (48.2%), and antagonistic effects were observed in three (11.1%) of the isolates. The synergy and partial synergy effects of this combination were 37.5% for Enterobacterales group bacteria, 50% for E.coli, and 30% for K.pneumoniae. Regarding the 11 non-fermentative gram-negative rods included in the study, 83.3% synergy and partial synergy was found in A.baumannii for the meropenem/colistin combination, while no synergy and partial synergistic effect was found in P.aeruginosa. Meropenem/fosfomycin synergy and partial synergy effects were 83.3% (5/6) for E.coli, 100% (8/8) for K.pneumoniae, 100% (6/6) for A.baumannii, and 25% (1/4) for P.aeruginosa. In all of the isolates studied, meropenem/fosfomycin combination was found to be more effective than the meropenem/colistin combination. It would be meaningful to support these data obtained in vitro with clinical efficacy results to be obtained as a result of the application of antibiotics in vivo, taking into account the pharmacokinetic and pharmacodynamic properties of the antibiotics used in this study.
Topics: Humans; Meropenem; Colistin; Fosfomycin; Agar; Drug Synergism; Anti-Bacterial Agents; Gram-Negative Bacteria; Microbial Sensitivity Tests
PubMed: 37462301
DOI: 10.5578/mb.20239930