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Microbiology Spectrum Oct 2022Reports of Gram-negative bacteria harboring multiple carbapenemase genes have increased in South America, leading to an urgent need for appropriate microbiological...
Reports of Gram-negative bacteria harboring multiple carbapenemase genes have increased in South America, leading to an urgent need for appropriate microbiological diagnosis. We evaluated phenotypic methods for detecting Klebsiella pneumoniae carbapenemase 2 (KPC-2) and New Delhi metallo-β-lactamase-1 (NDM-1) coexpression in members of the K. pneumoniae complex (i.e., K. pneumoniae, K. quasipneumoniae, and K. variicola) isolated from human and animal hosts, based on inhibition of ceftazidime-avibactam (CZA) and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, or avibactam (AVI). While the presence of and genes was confirmed by whole-genome sequencing, PCR, and/or GeneXpert, coexpression was successfully detected based on the following: (i) a ≥5-mm increase in the zone diameter of ATM (30 µg) disks plus AVI (4 or 20 µg) and ≥4-mm and ≥10-mm increases in the zone diameters for "CZA 50" (30 µg ceftazidime [CAZ] and 20 µg AVI) and "CZA 14" (10 µg CAZ and 4 µg AVI) disks, respectively, when we added DPA (1 mg/disk) or EDTA (5 mM) in a combined disk test (CDT); (ii) a positive ghost zone (synergism) between ATM (30 µg) and CZA 50 disks and between CZA 50 and DPA (1 mg) disks, using the double-disk synergy test (DDST) at a disk-disk distance of 2.5 cm; (iii) ≥3-fold MIC reductions of ATM and CZA in the presence of AVI (4 µg/mL), DPA (500 µg/mL), or EDTA (320 µg/mL); and (iv) immunochromatography. Although our results demonstrated that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex, additional studies are necessary to confirm the accuracy of these methodologies by testing other Gram-negative bacterial species and other KPC and NDM variants coexpressed by WHO critical priority pathogens detected worldwide. Alerts regarding the emergence and increase of combinations of carbapenemases in in Latin America and the Caribbean have recently been issued by PAHO and WHO, emphasizing the importance of appropriate microbiological diagnosis and the effective and articulated implementation of infection prevention and control programs. In this study, we evaluated methods based on inhibition of ceftazidime (CAZ), ceftazidime-avibactam (CZA), and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, and avibactam (AVI) inhibitors for the identification of KPC-2- and NDM-1-coexpression in members of the K. pneumoniae complex recovered from human and animal hosts. Our results demonstrate that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex.
Topics: Animals; Humans; Ceftazidime; Klebsiella pneumoniae; Aztreonam; Klebsiella Infections; Klebsiella; Edetic Acid; Microbial Sensitivity Tests; Anti-Bacterial Agents; beta-Lactamases; Bacterial Proteins
PubMed: 35980188
DOI: 10.1128/spectrum.01159-22 -
Microbiology Spectrum Feb 2023The frequent resistance associated with β-lactam antibiotics and the high frequency of mutations in β-lactamases constitute a major clinical challenge that can no...
The frequent resistance associated with β-lactam antibiotics and the high frequency of mutations in β-lactamases constitute a major clinical challenge that can no longer be ignored. Andrographolide (AP), a natural active compound, has been shown to restore susceptibility to β-lactam antibiotics. Fluorescence quenching and molecular simulation showed that AP quenched the intrinsic fluorescence of β-lactamase BlaZ and stably bound to the residues in the catalytic cavity of BlaZ. Of note, AP was found to reduce the stability of the cell wall (CW) in methicillin-resistant Staphylococcus aureus (MRSA), and in combination with penicillin G (PEN), it significantly induced CW roughness and dispersion and even caused its disintegration, while the same concentration of PEN did not. In addition, transcriptome sequencing revealed that AP induced a significant stress response and increased peptidoglycan (PG) synthesis but disrupted its cross-linking, and it repressed the expression of critical genes such as , , and . We also validated these findings by quantitative reverse transcription-PCR (qRT-PCR). Association analysis using the GEO database showed that the alterations caused by AP were similar to those caused by mutations in the gene. In summary, AP was able to restore the susceptibility of MRSA to β-lactam antibiotics, mainly by inhibiting the β-lactamase BlaZ, by downregulating the expression of critical resistance genes such as and , and by disrupting CW homeostasis. In addition, restoration of susceptibility to antibiotics could be achieved by inhibiting the global regulator SarA, providing an effective solution to alleviate the problem of bacterial resistance. Increasingly, alternatives to antibiotics are being used to mitigate the rapid onset and development of bacterial resistance, and the combination of natural compounds with traditional antibiotics has become an effective therapeutic strategy. Therefore, we attempted to discover more mechanisms to restore susceptibility and effective dosing strategies. Andrographolide (AP), as a natural active ingredient, can mediate recovery of susceptibility of MRSA to β-lactam antibiotics. AP bound stably to the β-lactamase BlaZ and impaired its hydrolytic activity. Notably, AP was able to downregulate the expression of critical resistance genes such as , , and . Meanwhile, it disrupted the CW cross-linking and homeostasis, while the same concentration of penicillin could not. The multiple inhibitory effect of AP resensitizes intrinsically resistant bacteria to β-lactam antibiotics, effectively prolonging the use cycle of these antibiotics and providing an effective solution to reduce the dosage of antibiotics and providing a theoretical reference for the prevention and control of MRSA.
Topics: Methicillin-Resistant Staphylococcus aureus; Bacterial Proteins; Microbial Sensitivity Tests; Anti-Bacterial Agents; beta-Lactamases; Penicillins; Monobactams
PubMed: 36602386
DOI: 10.1128/spectrum.02978-22 -
Scientific Reports Dec 2022The study's goal was to develop a spore-based paper strip biosensor for detecting ß-lactam antibiotics in milk using the enzyme induction principle. A new spore-based...
The study's goal was to develop a spore-based paper strip biosensor for detecting ß-lactam antibiotics in milk using the enzyme induction principle. A new spore-based paper strip biosensor has been developed after important operating parameters such as spore volume, substrate volume, exposure time and temperature, and incubation time and temperature were optimised. The limit of detection for various ß-lactam antibiotics, including amoxicillin, penicillin, ampicillin, carbenicillin, cloxacillin, nafcillin, oxacillin, cephalothin, cefalexin, cefoxitin, cefazolin, and cefuroxime, was determined in milk with detection sensitivity of 1 ppb, 2 ppb, 2 ppb, 10 ppb, 10 ppb, 10 ppb, 20 ppb, 10 ppb 1000 ppb, 10 ppb 300 ppb and 100 ppb, respectively. It was also tested with other contaminants such non-ß-lactam antibiotics, pesticides, aflatoxin, heavy metals, and other chemical contaminants, and no interference was found, indicating that the created biosensor had a low rate of false positive and negative results. In comparison to the AOAC-approved CHARM-ROSA ß-lactam strip test, which identified 7 raw milk and zero pasteurised milk samples positive for ß-lactam antibiotics, the sensor was further analysed and verified using 200 raw milk and 105 pasteurised milk samples. This indicates a perfect match between our biosensor and the AOAC-approved CHARM-ROSA ß-lactam strip test. The developed spore-based paper strip biosensors are expected to be useful in the rapid and cost-effective detection of ß-lactam antibiotic residues in milk samples at the dairy farm, reception dock, and production units, respectively.
Topics: Animals; Lactams; beta-Lactams; Milk; Anti-Bacterial Agents; Monobactams; Spores
PubMed: 36536009
DOI: 10.1038/s41598-022-26466-7 -
Microbiology Spectrum Dec 2022As multidrug and pan-resistance among Enterobacterales continue to increase, there is an urgent need for more therapeutic options to treat these infections. New...
As multidrug and pan-resistance among Enterobacterales continue to increase, there is an urgent need for more therapeutic options to treat these infections. New β-lactam and β-lactam inhibitor (BLI) combinations have a broad spectrum of activity, but those currently approved do not provide coverage against isolates harboring metallo-β-lactamases (MBL). Aztreonam (ATM) and avibactam (AVI) in combination (ATM/AVI; AVI at 4 μg/mL fixed concentration) provides a similarly broad range of activity while maintaining activity against MBL-producing isolates. The in vitro susceptibility testing of ATM/AVI by standard methods was evaluated during development. This study investigated the impact of nonstandard testing conditions on the activity of ATM/AVI as observed during broth microdilution testing as well as the equivalency between agar dilution and broth microdilution MIC values when testing a diverse panel of Enterobacterales ( = 201). Nonstandard test conditions evaluated included inoculum density, atmosphere of incubation, media pH, varied medium cation concentrations, incubation time, varied serum concentrations, testing in pooled urine instead of media, addition of blood to the media, and the presence of surfactant. Generally, apart from low pH and high inoculum density, nonstandard testing parameters did not affect ATM/AVI broth microdilution MIC values. Correlation of MIC values obtained by agar dilution and broth microdilution resulted in an essential agreement of 97.0% for all tested Enterobacterales. Variation of standard testing conditions had little impact on broth microdilution MIC values for ATM/AVI. The correlation between broth microdilution and agar dilution MICs suggests both methods are reliable for determination of ATM/AVI MIC values. Increasing antibiotic resistance and emergence of pan-resistant isolates threaten the ability to control infections and to provide many other medical interventions such as surgery and chemotherapy, among others. New therapies are required to control emerging resistance mechanisms, including the increase in metallo-β-lactamases. Some new antibiotic combinations provide coverage against highly resistant isolates but are unable to target organisms that produce metallo-β-lactamases. Aztreonam in combination with avibactam provides a broad spectrum of activity against highly resistant isolates that also targets metallo-β-lactamase-producing organisms. An important part of drug development is the ability for clinical labs to determine the susceptibility of isolates to the antimicrobial. This manuscript investigates the in vitro susceptibility testing of aztreonam/avibactam with nonstandard testing conditions and a correlation study between broth microdilution and agar dilution against clinical isolates encoding a variety of resistance mechanisms. Overall, aztreonam/avibactam was generally unaffected by changes in testing conditions and showed strong agar/broth correlation.
Topics: Aztreonam; Agar; Enterobacteriaceae; Anti-Bacterial Agents; beta-Lactamases; Gammaproteobacteria; Microbial Sensitivity Tests
PubMed: 36342288
DOI: 10.1128/spectrum.03601-22 -
Therapeutic Advances in Respiratory... Feb 2015Chronic airway infection in cystic fibrosis (CF) is a main cause of the increased morbidity and mortality found with this disease. The most common cause of Gram-negative... (Review)
Review
Chronic airway infection in cystic fibrosis (CF) is a main cause of the increased morbidity and mortality found with this disease. The most common cause of Gram-negative infection is Pseudomonas aeruginosa. The introduction of inhaled antibiotics has changed the lives of affected patients and the clinical outcome of this infection; this article focuses on the use of inhaled antibiotics in chronic P. aeruginosa infection in CF, and specifically on studies including the use of inhaled aztreonam lysine in P. aeruginosa infection. Studies were identified using PubMed and ClinicalTrials.gov, searching for ‘inhaled aztreonam’ and ‘cystic fibrosis’. Inhaled aztreonam is an important new treatment option for chronic P. aeruginosa infection in CF. Long-term studies have shown that the drug is safe and superior to inhaled tobramycin in these specific infections.
Topics: Administration, Inhalation; Anti-Bacterial Agents; Aztreonam; Cystic Fibrosis; Humans; Pseudomonas Infections; Pseudomonas aeruginosa; Recurrence; Respiratory Tract Infections; Treatment Outcome
PubMed: 25471692
DOI: 10.1177/1753465814561624 -
Annals of Clinical Microbiology and... Jul 2023Infections in critically-ill patients caused by extensively-drug-resistant (XDR)-Pseudomonas aeruginosa are challenging to manage due to paucity of effective treatment...
Compassionate use of a novel β-lactam enhancer-based investigational antibiotic cefepime/zidebactam (WCK 5222) for the treatment of extensively-drug-resistant NDM-expressing Pseudomonas aeruginosa infection in an intra-abdominal infection-induced sepsis patient: a case report.
Infections in critically-ill patients caused by extensively-drug-resistant (XDR)-Pseudomonas aeruginosa are challenging to manage due to paucity of effective treatment options. Cefepime/zidebactam, which is currently in global Phase 3 clinical development (Clinical Trials Identifier: NCT04979806, registered on July 28, 2021) is a novel mechanism of action based β-lactam/ β-lactam-enhancer combination with a promising activity against a broad-range of Gram-negative pathogens including XDR P. aeruginosa. We present a case report of an intra-abdominal infection-induced sepsis patient infected with XDR P. aeruginosa and successfully treated with cefepime/zidebactam under compassionate use. The 50 year old female patient with past-history of bariatric surgery and recent elective abdominoplasty and liposuction developed secondary pneumonia and failed a prolonged course of polymyxins. The organism repeatedly isolated from the patient was a New-Delhi metallo β-lactamase-producing XDR P. aeruginosa resistant to ceftazidime/avibactam, imipenem/relebactam and ceftolozane/tazobactam, susceptible only to cefepime/zidebactam. As polymyxins failed to rescue the patient, cefepime/zidebactam was administered under compassionate grounds leading to discharge of patient in stable condition. The present case highlights the prevailing precarious scenario of antimicrobial resistance and the need for novel antibiotics to tackle infections caused by XDR phenotype pathogens.
Topics: Humans; Cefepime; Pseudomonas Infections; Compassionate Use Trials; Cephalosporins; Anti-Bacterial Agents; Monobactams; Pseudomonas aeruginosa; beta-Lactamases; Sepsis; Intraabdominal Infections; Polymyxins; Microbial Sensitivity Tests
PubMed: 37408075
DOI: 10.1186/s12941-023-00606-x -
Scientific Reports Feb 2024Antimicrobial resistance has emerged as one of the leading public health threats of the twenty-first century. Gram-negative pathogens have been a major contributor to...
Antimicrobial resistance has emerged as one of the leading public health threats of the twenty-first century. Gram-negative pathogens have been a major contributor to the declining efficacy of antibiotics through both acquired resistance and tolerance. In this study, a pan-drug resistant (PDR), NDM-1 and CTX-M-15 co-producing isolate of K. pneumoniae, CDC Nevada, (Kp Nevada) was exposed to the clinical combination of aztreonam + ceftazidime/avibactam (ATM/CAZ/AVI) to overcome metallo-β-lactamases. Unexpectedly, the β-lactam combination resulted in long filamentous cell formation induced by PBP3 inhibition over 168 h in the hollow fiber infection model experiments with eventual reversion of the total population upon drug removal. However, the addition of imipenem to the two drug β-lactam combination was highly synergistic with suppression of all drug resistant subpopulations over 5 days. Scanning electron microscopy and fluorescence microscopy for all imipenem combinations in time kill studies suggested a role for imipenem in suppression of long filamentous persisters, via the formation of metabolically active spheroplasts. To complement the imaging studies, salient transcriptomic changes were quantified using RT-PCR and novel cassette assay evaluated β-lactam permeability. This showed significant upregulation of both spheroplast protein Y (SPY), a periplasmic chaperone protein that has been shown to be related to spheroplast formation, and penicillin binding proteins (PBP1, PBP2, PBP3) for all combinations involving imipenem. However, with aztreonam alone, pbp1, pbp3 and spy remained unchanged while pbp2 levels were downregulated by > 25%. Imipenem displayed 207-fold higher permeability as compared with aztreonam (mean permeability coefficient of 17,200 nm/s). Although the clinical combination of aztreonam/avibactam and ceftazidime has been proposed as an important treatment of MBL Gram-negatives, we report the first occurrence of long filamentous persister formation. To our knowledge, this is the first study that defines novel β-lactam combinations involving imipenem via maximal suppression of filamentous persisters to combat PDR CDC Nevada K. pneumoniae.
Topics: Ceftazidime; Klebsiella pneumoniae; Aztreonam; Anti-Bacterial Agents; Imipenem; beta-Lactamases; Drug Combinations; Microbial Sensitivity Tests; Azabicyclo Compounds
PubMed: 38326428
DOI: 10.1038/s41598-024-53130-z -
Molecules (Basel, Switzerland) Nov 2022Cross contamination of β-lactams is one of the highest risks for patients using pharmaceutical products. Penicillin and some non-penicillin β-lactams may cause...
Cross contamination of β-lactams is one of the highest risks for patients using pharmaceutical products. Penicillin and some non-penicillin β-lactams may cause potentially life-threatening allergic reactions. The trace detection of β-lactam antibiotics in cleaning rinse solutions of common reactors and manufacturing aids in pharmaceutical facilities is very crucial. Therefore, the common facilities adopt sophisticated cleaning procedures and develop analytical methods to assess traces of these compounds in rinsed solutions. For this, a highly sensitive and reproducible ultra-performance liquid chromatography with triple quadrupole mass spectrometry (UHPLC-MS/MS) method was developed for the analysis of Cephapirin and Ceftiofur. As per the FDA guidelines described in FDA-2011-D-0104, the contamination of these β-lactam antibiotics must be regulated. The analysis was performed on an XBridge C18 column with 100 mm length, 4.6 mm diameter, and 3.5 µm particle size at an oven temperature of about 40 °C. The mobile phase was composed of 0.15% formic acid in water and acetonitrile as mobile phases A and B, and a flow rate was set to 0.6 mL/min. The method was validated for Cephapirin and Ceftiofur. The quantification precision and accuracy were determined to be the lowest limit of detection 0.15 parts per billion (ppb) and the lowest limit of quantification 0.4 ppb. This method was linear in the range of 0.4 to 1.5 ppb with the determination of coefficient (R2 > 0.99). This sensitive and fast method was fit-for-purpose for detecting and quantifying trace amounts of β-lactam contamination, monitoring cross contamination in facility surface cleaning, and determining the acceptable level of limits for regulatory purposes.
Topics: Humans; Cephapirin; Tandem Mass Spectrometry; Anti-Bacterial Agents; Reproducibility of Results; Chromatography, Liquid; beta-Lactams; Monobactams; Penicillins
PubMed: 36432023
DOI: 10.3390/molecules27227920 -
Antimicrobial Agents and Chemotherapy May 2021Metallo-β-lactamases (MBLs) result in resistance to nearly all β-lactam antimicrobial agents, as determined by currently employed susceptibility testing methods.... (Review)
Review
Metallo-β-lactamases (MBLs) result in resistance to nearly all β-lactam antimicrobial agents, as determined by currently employed susceptibility testing methods. However, recently reported data demonstrate that variable and supraphysiologic zinc concentrations in conventional susceptibility testing media compared with physiologic (bioactive) zinc concentrations may be mediating discordant - MBL resistance. While treatment outcomes in patients appear suggestive of this discordance, these limited data are confounded by comorbidities and combination therapy. To that end, the goal of this review is to evaluate the extent of β-lactam activity against MBL-harboring in published animal infection model studies and provide contemporary considerations to facilitate the optimization of current antimicrobials and development of novel therapeutics.
Topics: Animals; Anti-Bacterial Agents; Humans; Monobactams; beta-Lactamase Inhibitors; beta-Lactamases; beta-Lactams
PubMed: 33782001
DOI: 10.1128/AAC.02271-20 -
The Journal of Antimicrobial... Mar 2020To examine the in vitro selection of aztreonam/avibactam resistance among MBL-producing Klebsiella pneumoniae and to understand the mechanism of increased resistance.
OBJECTIVES
To examine the in vitro selection of aztreonam/avibactam resistance among MBL-producing Klebsiella pneumoniae and to understand the mechanism of increased resistance.
METHODS
The MICs of aztreonam were determined with and without avibactam (4 mg/L) using a broth microdilution method. Single-step and multi-step mutant selection was conducted on five MBL-producing K. pneumoniae strains, including two dual carbapenemase producers. Genomic sequencing and gene cloning were performed to investigate the mechanism of increased resistance.
RESULTS
We examined the MICs for 68 MBL-producing K. pneumoniae isolates, including 13 dual carbapenemase producers. Compared with aztreonam alone, the addition of avibactam (4 mg/L) reduced the MICs for all isolates by >128-fold, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively. One NDM-1-, OXA-48-, CTX-M-15- and CMY-16-positive ST101 K. pneumoniae strain was selected to be resistant to aztreonam/avibactam, with a >16-fold increase in MIC (>128 mg/L). WGS revealed that the resistant mutants lost the blaNDM-1 gene, but acquired amino acid substitutions in CMY-16 (Tyr150Ser and Asn346His). Construction of blaCMY-16 mutants confirmed that the substitutions (Tyr150Ser and Asn346His) were primarily responsible for the decreased susceptibility to aztreonam/avibactam. In addition, transfer of blaCMY-16 mutant (Tyr150Ser and Asn346His) plasmid constructs into certain clinical carbapenemase-producing isolates demonstrated >64-fold increased MICs of aztreonam/avibactam and aztreonam/avibactam/ceftazidime.
CONCLUSIONS
Aztreonam in combination with avibactam showed potent in vitro activity against MBL-producing K. pneumoniae. However, our study suggested the likelihood of aztreonam/avibactam resistance among MBL- and AmpC-co-producing strains and clinical practice should beware of the possibility of the emerging resistance.
Topics: Anti-Bacterial Agents; Azabicyclo Compounds; Aztreonam; Bacterial Proteins; Ceftazidime; Drug Combinations; Klebsiella pneumoniae; Microbial Sensitivity Tests; beta-Lactamases
PubMed: 31722380
DOI: 10.1093/jac/dkz468