Synergistic effect of secondary metabolites isolated from Pestalotiopsis sp. FKR-0115 in overcoming ß-lactam resistance in MRSA.

Six aromatic secondary metabolites, pestalone (1), emodin (2), phomopsilactone (3), pestalachlorides B (4), C (5), and D (6), were isolated from Pestalotiopsis sp. FKR-0115, a filamentous fungus collected from white moulds growing on dead branches in Minami Daito Island. The efficacy of these secondary metabolites against methicillin-resistant Staphylococcus aureus (MRSA) with and without meropenem (ß-lactam antibiotic) was evaluated using the paper disc method and broth microdilution method. The chemical structures of the isolated compounds (1-6) were characterised using spectroscopic methods, including nuclear magnetic resonance and mass spectrometry. All six isolated compounds exhibited synergistic activity with meropenem against MRSA. Among the six secondary metabolites, pestalone (1) overcame bacterial resistance in MRSA to the greatest extent.

Implication of Mn-cofactored superoxide dismutase in the tolerance of swarmer Pseudomonas aeruginosa to polymixin, ciprofloxacin and meropenem antibiotics.

The protective role of superoxide dismutase (Sod) against oxidative stress, resulting from the common antibiotic pathway of action, has been studied in the wild type and mutant strains of swarmer Pseudomonas aeruginosa, lacking Cytosolic Mn-Sod (sodM), Fe-Sod (sodB) or both Sods (sodMB).Our results showed that inactivation of sodB genes leads to significant motility defects and tolerance to meropenem. This resistance is correlated with a greater membrane unsaturation as well as an effective intervention of Mn-Sod isoform, in antibiotic tolerance.Moreover, loss of Mn-Sod in sodM mutant, leads to polymixin intolerance and is correlated with membrane unsaturation. Effectivelty, sodM mutant showed an enhanced swarming motility and a conserved rhamnolipid production. Whereas, in the double mutant sodMB, ciprofloxacin tolerance would be linked to an increase in the percentage of saturated fatty acids in the membrane, even in the absence of superoxide dismutase activity.The overall results showed that Mn-Sod has a protective role in the tolerance to antibiotics, in swarmer P.aeruginosa strain. It has been further shown that Sod intervention in antibiotic tolerance is through change in membrane fatty acid composition.

Vaborbactam increases meropenem susceptibility in Pseudomonas aeruginosa clinical isolates displaying MexXY and AmpC upregulation.

To evaluate the resistance mechanisms among Pseudomonas aeruginosa clinical isolates exhibiting meropenem (MEM) MIC values higher than meropenem-vaborbactam (MEV). P. aeruginosa clinical isolates collected in US hospitals from 2014 to 2019 were susceptibility tested. Whole-genome and transcriptome sequencing were performed. Results were analyzed for strain typing, acquired ß-lactamases, and mutations in chromosomal genes; gene expression was measured for known ß-lactam resistance contributors. Results were compared to a control group of 10 P. aeruginosa isolates displaying MIC values at 8 mg/L for meropenem ± vaborbactam (MEM = MEV). Out of 88 isolates displaying MEM > MEV, 33 (37.5%) isolates had reproducibly lower MIC values for meropenem-vaborbactam compared to meropenem when retested. The expression of mexX, mexY, mexZ, and ampC was significantly greater among a higher percentage of the MEM > MEV isolates. Furthermore, the association of mexXY and ampC overexpression was detected in 17/33 MEM > MEV isolates and only 1/10 MEM = MEV isolate. In addition, the Pseudomonas-derived cephalosporinase amino acid substitution R79Q was detected among 33.3% of the isolates displaying MEM > MEV, and none of the isolates displayed MEM = MEV. Other resistance mechanisms were not observed or were equally observed in both groups. In rare cases, vaborbactam plays a role in lowering the meropenem MIC values in P. aeruginosa clinical isolates likely due to the inhibition of the AmpC gene that was overexpressed in the presence of upregulation of MexXY with or without alterations in the AmpC gene. IMPORTANCE Pseudomonas aeruginosa isolates are intrinsically resistant to multiple antimicrobial agents and meropenem is an important therapeutic option to treat infections caused by this organism. Meropenem-vaborbactam activity is similar to that of meropenem alone against P. aeruginosa isolates. Isolates belonging to this species that display lower meropenem-vaborbactam compared to meropenem are rare. We initiated this study to understand the resistance mechanisms that could lead to lower meropenem-vaborbactam MIC values when compared to meropenem alone. We documented that isolates displaying lower meropenem-vaborbactam exhibited overexpression of MexXY and AmpC. In addition, isolates displaying the R79Q PDC (AmpC) mutation were more likely to display lower meropenem-vaborbactam when compared to isolates displaying the same MIC values for these agents.

Neutrophil extracellular trap inhibition improves survival in neonatal mouse infectious peritonitis.

BACKGROUND: Treatment of neonatal peritonitis and sepsis is challenging. Following infection, neutrophils elaborate neutrophil extracellular traps (NETs)-extracellular lattices of decondensed chromatin decorated with antimicrobial proteins. NETs, however, can augment pathogenic inflammation causing collateral damage. We hypothesized that NET inhibition would improve survival in experimental neonatal infectious peritonitis. METHODS: We induced peritonitis in 7 to 10-day-old mice by intraperitoneal injection with cecal slurry. We targeted NETs by treating mice with neonatal NET-Inhibitory Factor (nNIF), an endogenous NET-inhibitor; Cl-amidine, a PAD4 inhibitor; DNase I, a NET degrading enzyme, or meropenem (an antibiotic). We determined peritoneal NET and cytokine levels and circulating platelet-neutrophil aggregates. Survival from peritonitis was followed for 6 days. RESULTS: nNIF, Cl-amidine, and DNase I decreased peritoneal NET formation and inflammatory cytokine levels at 24 h compared to controls. nNIF, Cl-amidine, and DNase I decreased circulating platelet-neutrophil aggregates, and NET-targeting treatments significantly increased survival from infectious peritonitis compared to controls. Finally, nNIF administration significantly improved survival in mice treated with sub-optimal doses of meropenem even when treatment was delayed until 2 h after peritonitis induction. CONCLUSIONS: NET inhibition improves survival in experimental neonatal infectious peritonitis, suggesting that NETs participate pathogenically in neonatal peritonitis and sepsis. IMPACT: 1. Neutrophil extracellular trap formation participates pathogenically in experimental neonatal infectious peritonitis. 2. NET-targeting strategies improve outcomes in a translational model of neonatal infectious peritonitis. 3. NET inhibition represents a potential target for drug development in neonatal sepsis and infectious peritonitis.

Anti-Fungal Potential of Structurally Diverse FDA-Approved Therapeutics Targeting Secreted Aspartyl Proteinase (SAP) of Candida albicans: an In Silico Drug Repurposing Approach.

In recent years, candidiasis attains major clinical importance due to its unique pathogenic strategy, which distinguishes it from other nosocomial infections. Secreted aspartyl proteinases (SAPs) is a hydrolytic enzyme secreted by Candida species that mediate versatile biological activity including hyphal formation, adherence, biofilm formation, phenotypic adaptation, etc. Emerging clinical evidence strongly suggested that conventional anti-fungal agent's are often prone to high level of resistance upon repeated exposure. Drug repurposing is an ideal strategy that shall impose the additional clinical benefits of the already approved molecules. Hence, through this realistic pathway, the potential of the suitable lead candidates will be explored in order to prolong the life span of existing molecules thereby need for newer therapeutics shall be avoided. The main aim of the present investigation is to determine the enzyme inhibitory potential of certain FDA-approved antibiotics and to validate its efficacy against the virulent enzyme secreted aspartyl proteinase (SAP) of Candida albicans via the AutoDock simulation program. The outcome of in silico dynamic simulations depicts that the drugs such as gentamicin, clindamycin, meropenem, metronidazole, and aztreonam emphasize superior binding affinity in terms of demonstrating considerable interaction with the core catalytic residues (Asp 32, Asp86, Asp 218, Gly220, Thr 221, and Thr 222). Data further indicates that the drug gentamicin exhibited best binding affinity of - 14.16 kcal/mol followed by meropenem (- 9.20 kcal/mol), clindamycin (- 9.00 kcal/mol), ciprofloxacin (- 8.95 kcal/mol), and imipenem (- 8.00 kcal/mol). In conclusion, repurposed antibiotics like gentamicin, clindamycin, meropenem, metronidazole, and aztreonam shall be considered an alternate drug of choice for the clinical management of drug resistant candida infections in the near future.

Zinc oxide nanoparticles impact the expression of the genes involved in toxin-antitoxin systems in multidrug-resistant Acinetobacter baumannii.

The aim of this study was to investigate the effect of zinc oxide nanoparticles (ZnO-NPs) on the expression of genes involved in toxin-antitoxin (TA) systems in multidrug-resistant (MDR) Acinetobacter baumannii. Seventy clinical isolates of A. baumannii were collected from variuos clinical samples. Antimicrobial susceptibility test was determined by disk diffusion. Type II TA system-related genes including GNAT, XRE-like, hipA, hipB, hicA, hicB were screened using polymerase chain reaction (PCR). ZnO-NPs prepared and characterized by field emission scanning electron microscopy and X-ray diffraction. MIC of ZnO-NPs of A. baumannii isolates was performed using the microdilution method. The expression of type II TA systems-related genes were assessed with and without exposure to ZnO-NPs using real-time PCR. The highest rate of resistance and sensitivity was observed against cefepime (77.14%), and ampicillin/sulbactam (42.85%), respectively. All A. baumannii isolates were considered as MDR. In this study, three TA loci were identified for A. baumannii including GNAT/XRE-like, HicA/HicB, and HipA/HipB and their prevalence was 100%, 42%, and 27.1%, respectively. There was no significant relationship between the prevalence of these systems and the origin of A. baumannii. Our data showed significant correlations between the presence of HicA/HicB system and resistance to ceftazidime, meropenem, imipenem, and cefepime (p < 0.05), and the presence of HipA/HipB system and resistance to ceftazidime, meropenem, imipenem, and cefepime (p < 0.05). In presence of ZnO-NPs, the expression of all studied genes decreased. GNAT and hicB showed the highest and lowest expression changes by 2.4 folds (p < 0.001) and 1.3 folds (p < 0.05), respectively. This study demonstrates the promising potential of nanoparticles to impact the expression of the genes involved in TA Systems. So, the application of ZnO-NPs may be helpful to design target-based strategies towards MDRs pathogens for empowered clinical applications by microbiologists and nanotechnologists.

Efficient Suppression of Natural Plasmid-Borne Gene Expression in Carbapenem-Resistant Klebsiella pneumoniae Using a Compact CRISPR Interference System.

There is an urgent need for efficient tools for genetic manipulation to assess plasmid function in clinical drug-resistant bacterial strains. To address this need, we developed an all-in-one CRISPR interference (CRISPRi) system that easily inhibited the gene expression of a natural multidrug-resistant plasmid in an sequence type 23 (ST23) Klebsiella pneumoniae isolate. We established an integrative CRISPRi system plasmid, pdCas9gRNA, harboring a dcas9 gene and a single guide RNA (sgRNA) unit under the control of anhydrotetracycline-induced and J23119 promoters, respectively, using a one-step cloning method. This system can repress the single resistance gene blaNDM-1, with a >1,000-fold reduction in the meropenem MIC, or simultaneously silence the resistance genes blaNDM-1 and blaSHV-12, with a 16-fold and 8-fold respective reduction in the meropenem and aztreonam MIC on a large natural multidrug-resistant pNK01067-NDM-1 plasmid in an ST23 K. pneumoniae isolate. Furthermore, an sgRNA targeting the blaNDM-1 promoter region can silence the entire blaNDM-1-bleMBL-trpF operon, confirming the existence of the operon. We also used this tool to knock down the multicopy resistance gene blaKPC-2 in pathogenic Escherichia coli, increasing the susceptibility to meropenem. In a word, the all-in-one CRISPRi system can be used for efficient interrogation of indigenous plasmid-borne gene functions, providing a rapid, easy genetic manipulation tool for clinical K. pneumoniae isolates.

Intrinsic Antibacterial Activity of Xeruborbactam In Vitro: Assessing Spectrum and Mode of Action.

Xeruborbactam (formerly QPX7728) is a cyclic boronate inhibitor of numerous serine and metallo-beta-lactamases. At concentrations generally higher than those required for beta-lactamase inhibition, xeruborbactam has direct antibacterial activity against some Gram-negative bacteria, with MIC50/MIC90 values of 16/32 µg/mL and 16/64 µg/mL against carbapenem-resistant Enterobacterales and carbapenem-resistant Acinetobacter baumannii, respectively (the MIC50/MIC90 values against Pseudomonas aeruginosa are >64 µg/mL). In Klebsiella pneumoniae, inactivation of OmpK36 alone or in combination with OmpK35 resulted in 2- to 4-fold increases in the xeruborbactam MIC. In A. baumannii and P. aeruginosa, AdeIJK and MexAB-OprM, respectively, affected xeruborbactam's antibacterial potency (the MICs were 4- to 16-fold higher in efflux-proficient strains). In Escherichia coli and K. pneumoniae, the 50% inhibitory concentrations (IC50s) of xeruborbactam's binding to penicillin-binding proteins (PBPs) PBP1a/PBP1b, PBP2, and PBP3 were in the 40 to 70 µM range; in A. baumannii, xeruborbactam bound to PBP1a, PBP2, and PBP3 with IC50s of 1.4 µM, 23 µM, and 140 µM, respectively. Treating K. pneumoniae and P. aeruginosa with xeruborbactam at 1× and 2× MIC resulted in changes of cellular morphology similar to those observed with meropenem; the morphological changes observed after treatment of A. baumannii were consistent with inhibition of multiple PBPs but were unique to xeruborbactam compared to the results for control beta-lactams. No single-step xeruborbactam resistance mutants were obtained after selection at 4× MIC of xeruborbactam using wild-type strains of E. coli, K. pneumoniae, and A. baumannii; mutations selected at 2× MIC in K. pneumoniae did not affect antibiotic potentiation by xeruborbactam through beta-lactamase inhibition. Consistent with inhibition of PBPs, xeruborbactam enhanced the potencies of beta-lactam antibiotics even against strains that lacked beta-lactamase. In a large panel of KPC-producing clinical isolates, the MIC90 values of meropenem tested with xeruborbactam (8 µg/mL) were at least 4-fold lower than those in combination with vaborbactam at 64 µg/mL, the concentration of vaborbactam that is associated with complete inhibition of KPC. The additional enhancement of the potency of beta-lactam antibiotics beyond beta-lactamase inhibition may contribute to the potentiation of beta-lactam antibiotics by xeruborbactam.

Interactions Between Meropenem and Renal Drug Transporters.

BACKGROUND: Meropenem is a carbapenem antibiotic and is commonly used with other antibiotics for the treatment of bacterial infections. It is primarily eliminated renally by glomerular filtration and renal tubular secretion. OBJECTIVE: This study aimed to evaluate the roles of renal uptake and efflux transporters in the excretion of meropenem and potential drug interactions mediated by renal drug transporters. METHODS: Uptake and inhibition studies were conducted in human embryonic kidney 293 cells stably transfected with Organic Anion Transporter (OAT) 1, OAT3, Multidrug and Toxin Extrusion Protein (MATE) 1, and MATE2K, as well as membrane vesicles containing breast cancer resistance-related protein (BCRP), multidrug resistance protein 1 (MDR1), and Multidrug Resistance-associated Protein 2 (MRP2). Probenecid and piperacillin were used to assess potential drug interactions with meropenem in rats. RESULTS: We observed that meropenem was a low-affinity substrate of OAT1/3 and had a weak inhibitory effect on OAT1/3 and MATE2K. BCRP, MDR1, MRP2, MATE1, and MATE2K could not mediate renal excretion of meropenem. Moreover, meropenem was not an inhibitor of BCRP, MDR1, MRP2, or MATE1. Among five tested antibiotics, moderate inhibition on OAT3-mediated meropenem uptake was observed for linezolid (IC50 value was 69.2 µM), weak inhibition was observed for piperacillin, benzylpenicillin, and tazobactam (IC50 values were 282.2, 308.0 and 668.1 µM, respectively), and no inhibition was observed for sulbactam. Although piperacillin had a relatively high drug-drug interaction index (ratio of maximal unbound plasma concentration to IC50 was 1.42) in vitro, no meaningful impact was reported on the pharmacokinetics of meropenem in rats. CONCLUSION: Our results indicated that clinically significant interactions between meropenem and these five antibiotics are low.

Penicillin-binding proteins (PBPs) determine antibiotic action in Langmuir monolayers as nanoarchitectonics mimetic membranes of methicillin-resistant Staphylococcus aureus.

The membrane of methicillin-resistant Staphylococcus aureus (MRSA) contains penicillin-binding proteins (PBPs) in the phospholipidic bilayer, with the protein PBP2a being linked with the resistance mechanism. In this work we confirm the role of PBP2a with molecular-level information obtained with Langmuir monolayers as cell membrane models. The MRSA cell membrane was mimicked with a mixed monolayer of dipalmitoyl phosphatidyl glycerol (DPPG) and cardiolipin (CL), also incorporating PBP2a. The surface pressure-area isotherms and the Brewster angle microscopy (BAM) images for these mixed monolayers were significantly affected by the antibiotic meropenem, which is PBP2a inhibitor. The meropenem effects were associated with the presence of PBP2a, as they were absent in the Langmuir monolayers without PBP2a. The relevance of PBP2a was confirmed with results where the antibiotic methicillin, known to be unsuitable to kill MRSA, had the same effects on mixed DPPG/CL and DPPG/CL-PBP2a monolayers since it prevented PBP2a from incorporating in the monolayer. The biological implication of the findings presented here is that a successful antibiotic against MRSA should be able to interact with PBP2a, but in the membrane.

TP0586532, a non-hydroxamate LpxC inhibitor, reduces LPS release and IL-6 production both in vitro and in vivo.

UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is an essential enzyme in the biosynthesis of Lipid A, an active component of lipopolysaccharide (LPS), from UDP-3-O-acyl-N-acetylglicosamine. LPS is a major component of the cell surface of Gram-negative bacteria. LPS is known to be one of causative factors of sepsis and has been associated with high mortality in septic shock. TP0586532 is a novel non-hydroxamate LpxC enzyme inhibitor. In this study, we examined the inhibitory effect of TP0586532 on the LPS release from Klebsiella pneumoniae both in vitro and in vivo. Our results confirmed the inhibitory effect of TP0586532 on LPS release from the pathogenic bacterial species. On the other hand, meropenem and ciprofloxacin increase the level of LPS release. Furthermore, the effects of TP0586532 on LPS release and interleukin (IL)-6 production in the lung were determined using a murine model of pneumonia caused by K. pneumoniae. As observed in the in vitro study, TP0586532 showed the marked inhibitory effect on LPS release in the lungs, whereas meropenem- and ciprofloxacin-treated mice showed higher levels of LPS release and IL-6 production in the lungs as compared to those in the lungs of vehicle-treated mice. Moreover, TP0586532 used in combination with meropenem and ciprofloxacin attenuated the LPS release and IL-6 production induced by meropenem and ciprofloxacin in the lung. These results indicate that the inhibitory effect of TP0586532 on LPS release from pathogenic bacteria might be of benefit in patients with sepsis.

Exploring the Role of L10 Loop in New Delhi Metallo-ß-lactamase (NDM-1): Kinetic and Dynamic Studies.

Four NDM-1 mutants (L218T, L221T, L269H and L221T/Y229W) were generated in order to investigate the role of leucines positioned in L10 loop. A detailed kinetic analysis stated that these amino acid substitutions modified the hydrolytic profile of NDM-1 against some ß-lactams. Significant reduction of kcat values of L218T and L221T for carbapenems, cefazolin, cefoxitin and cefepime was observed. The stability of the NDM-1 and its mutants was explored by thermofluor assay in real-time PCR. The determination of TmB and TmD demonstrated that NDM-1 and L218T were the most stable enzymes. Molecular dynamic studies were performed to justify the differences observed in the kinetic behavior of the mutants. In particular, L218T fluctuated more than NDM-1 in L10, whereas L221T would seem to cause a drift between residues 75 and 125. L221T/Y229W double mutant exhibited a decrease in the flexibility with respect to L221T, explaining enzyme activity improvement towards some ß-lactams. Distances between Zn1-Zn2 and Zn1-OH- or Zn2-OH- remained unaffected in all systems analysed. Significant changes were found between Zn1/Zn2 and first sphere coordination residues.

Impact of cumulative fluid balance on the pharmacokinetics of extended infusion meropenem in critically ill patients with sepsis.

BACKGROUND: Meropenem dosing for septic critically patients is difficult due to pathophysiological changes associated with sepsis as well as supportive symptomatic therapies. A prospective single-center study assessed whether fluid retention alters meropenem pharmacokinetics and the achievement of the pharmacokinetic/pharmacodynamic (PK/PD) targets for efficacy. METHODS: Twenty-five septic ICU patients (19 m, 6f) aged 32-86 years with the mean APACHE II score of 20.2 (range 11-33), suffering mainly from perioperative intra-abdominal or respiratory infections and septic shock (n = 18), were investigated over three days after the start of extended 3-h i.v. infusions of meropenem q8h. Urinary creatinine clearance (CLcr) and cumulative fluid balance (CFB) were measured daily. Plasma meropenem was measured, and Bayesian estimates of PK parameters were calculated. RESULTS: Eleven patients (9 with peritonitis) were classified as fluid overload (FO) based on a positive day 1 CFB of more than 10% body weight. Compared to NoFO patients (n = 14, 11 with pneumonia), the FO patients had a lower meropenem clearance (CLme 8.5 ± 3.2 vs 11.5 ± 3.5 L/h), higher volume of distribution (V1 14.9 ± 3.5 vs 13.5 ± 4.1 L) and longer half-life (t1/2 1.4 ± 0.63 vs 0.92 ± 0.54 h) (p < 0.05). Over three days, the CFB of the FO patients decreased (11.7 ± 3.3 vs 6.7 ± 4.3 L, p < 0.05) and the PK parameters reached the values comparable with NoFO patients (CLme 12.4 ± 3.8 vs 11.5 ± 2.0 L/h, V1 13.7 ± 2.0 vs 14.0 ± 5.1 L, t1/2 0.81 ± 0.23 vs 0.87 ± 0.40 h). The CLcr and Cockroft-Gault CLcr were stable in time and comparable. The correlation with CLme was weak to moderate (CLcr, day 3 CGCLcr) or absent (day 1 and 2 CGCLcr). Dosing with 2 g meropenem q8h ensured adequate concentrations to treat infections with sensitive pathogens (MIC 2 mg/L). The proportion of pre-dose concentrations exceeding the MIC 8 mg/L and the fraction time with a target-exceeding concentration were higher in the FO group (day 1-3 f Cmin > MIC: 67 vs 27%, p < 0.001; day 1%f T > MIC: 79 ± 17 vs 58 ± 17, p < 0.05). CONCLUSIONS: These findings emphasize the importance of TDM and a cautious approach to augmented maintenance dosing of meropenem to patients with FO infected with less susceptible pathogens, if guided by population covariate relationships between CLme and creatinine clearance.

Evaluation of the simplified carbapenem inactivation method as a phenotypic detection method for carbapenemase-producing Enterobacterales.

Carbapenemase-producing Enterobacterales (CPE) have become a global health concern. Current molecular detection methods require special equipment and reagents. Thus, there is an urgent need for a highly sensitive, specific, and simple method for phenotypic detection of CPE in clinical microbiology laboratories. A simplified carbapenem inactivation method (sCIM) was recently reported. However, its utility for CPE detection has not been sufficiently evaluated to date. We evaluated the sCIM and compared it with the modified CIM (mCIM), using 133 CPE strains (producing IMP, 92; NDM, 11; NDM and OXA-48-like, 1; KPC, 13; OXA-48-like, 12; GES-24, 3; Nmc-A, 1) and 82 non-CPE strains (extended spectrum ß-lactamase, 61; AmpC, 21). The sCIM was conducted by loading bacteria onto imipenem and meropenem disks. When imipenem disks with a 1+ bacterial load were used, the sensitivity and specificity of the sCIM were 97.0% and 100%, and those of the mCIM were 97.0% and 96.3%, respectively. The specificity of the sCIM decreased to 57.3% when the bacterial load on imipenem disks was increased to 2+. In contrast, when meropenem disks with a 1+ bacterial load were used, the sCIM had a lower sensitivity (78.2%) and an equivalent specificity (100%). When meropenem disks with a bacterial load of 2+ were used, the sensitivity and specificity of the sCIM increased to 96.2% and 93.9%, respectively. The diameter of the inhibition zone on meropenem disks was larger than that on imipenem disks, and the sCIM was less sensitive when meropenem disks were used. In addition, sCIM detection rates when using meropenem disks were particularly low for OXA-48-like producers (bacterial load 1+, 0/12; bacterial load 2+, 10/12). Our results indicate that the sensitivity and specificity of the sCIM was dependent on the bacterial load and that large bacterial loads led to false positives for AmpC and extended spectrum ß-lactamase producers. Thus, the sCIM has high sensitivity and specificity for appropriate bacterial loads when imipenem disks are used.

Establishing a quantitative index of meropenem hydrolysis for the detection of KPC- and NDM-producing bacteria by MALDI-TOF MS.

BACKGROUND: Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS), commonly used for microorganism identification, can also be applied for the detection of carbapenemase-producing bacteria by the evaluation of carbapenem hydrolysis. Since KPC- and NDM-producing bacteria are related to high mortality rates, diagnostic assays for its detection are essential. The aim of this study was to develop and evaluate a method to establish a quantitative measure (hydrolysis index - HI) to detect meropenem hydrolysis by MLADI-TOF MS. METHODS: blaKPC and blaNDM positive and negative Klebsiella pneumoniae isolates and Escherichia coli ATCC 25922 (control) were incubated in a meropenem solution for 2 h. Protein extraction from these suspensions were submitted to MALDI-TOF MS analysis. The intensity of peaks at 384 m/z and 379 m/z of each isolate were used to establish the HI as follows: HI = (Peak intensity384 Test / Peak intensity379 Test) / (Peak intensity384 Control / Peak intensity379 Control). Receiver Operating Characteristic curve was used to determine a cutoff value to differentiate carbapenemase-producing from carbapenemase non-producing bacteria. RESULTS: As all carbapenemase-producing K. pneumoniae presented HI ≤0.55 and all carbapenemase non-producing isolates presented a HI ≥0.57, the index of 0.56 was established as a cutoff value to differentiate carbapenemase (KPC and NDM) producing and non-producing bacteria.

Perfusate adsorption during ex vivo lung perfusion improves early post-transplant lung function.

OBJECTIVE: Improvement in ex vivo lung perfusion protocols could increase the number of donors available for transplantation and protect the lungs from primary graft dysfunction. We hypothesize that perfusate adsorption during ex vivo lung perfusion reconditions the allograft to ischemia-reperfusion injury after lung transplantation. METHODS: Donor pig lungs were preserved for 24 hours at 4°C, followed by 6 hours of ex vivo lung perfusion according to the Toronto protocol. The perfusate was additionally adsorbed through a CytoSorb adsorber (CytoSorbents, Berlin, Germany) in the treatment group, whereas control lungs were perfused according to the standard protocol (n = 5, each). Ex vivo lung perfusion physiology and biochemistry were monitored. Upon completion of ex vivo lung perfusion, a left single lung transplantation was performed. Oxygenation function and lung mechanics were assessed during a 4-hour reperfusion period. The inflammatory response was determined during ex vivo lung perfusion and reperfusion. RESULTS: The cytokine concentrations in the perfusate were markedly lower with the adsorber, resulting in improved ex vivo lung perfusion physiology and biochemistry during the 6-hour perfusion period. Post-transplant dynamic lung compliance was markedly better during the 4-hour reperfusion period in the treatment group. Isolated allograft oxygenation function and dynamic compliance continued to be superior in the adsorber group at the end of reperfusion, accompanied by a markedly decreased local inflammatory response. CONCLUSIONS: Implementation of an additional cytokine adsorber has refined the standard ex vivo lung perfusion protocol. Furthermore, cytokine removal during ex vivo lung perfusion improved immediate post-transplant graft function together with a less intense inflammatory response to reperfusion in pigs. Further studies are warranted to understand the beneficial effects of perfusate adsorption during ex vivo lung perfusion in the clinical setting.

KPC-2 ß-lactamase enables carbapenem antibiotic resistance through fast deacylation of the covalent intermediate.

Serine active-site ß-lactamases hydrolyze ß-lactam antibiotics through the formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most ß-lactamases owing to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared with the common TEM-1 ß-lactamase. Furthermore, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase ß-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 ß-lactamase.

Comparative Evaluation of Five Assays for Detection of Carbapenemases with a Proposed Scheme for Their Precise Application.

The escalating problem of the dissemination of carbapenemase-producing bacteria (CPB) has gained worldwide attention. The prompt diagnosis of CPB and precise identification of carbapenemases are imperative to enable specific antibiotic therapy and control the spread of these bacteria. The present study was designed to assess the performance of five important assays for the detection of carbapenemases. The modified carbapenem inactivation method (mCIM), CARBA-5, GeneXpert Carba-R, BD MAX Check-Points CPO, and GeneFields CPE assays were evaluated with an international collection of 159 bacterial isolates, including 93 CPB and 66 non-CPB isolates. The overall accuracy/sensitivity/specificity for carbapenemase detection were 100% (95% CI, 97.7%-100%)/100% (95% CI, 96.1%-100%)/100% (95% CI, 94.6%-100%) for mCIM, 98.7% (95% CI, 95.5%-99.9%)/97.9% (95% CI, 92.5%-99.7%)/100% (95% CI, 94.6%-100%) for CARBA-5, 96.9% (95% CI, 92.8%-99%)/95.7% (95% CI, 89.4%-98.8%)/98.5% (95% CI, 91.8%-99.9%) for GeneXpert Carba-R, 94.3% (95% CI, 89.5%-97.4%)/90.3% (95% CI, 82.4%-95.5%)/100% (95% CI, 94.6%-100%) for BD MAX Check-Points CPO, and 86.2% (95% CI, 79.8%-91.1%)/77.4% (95% CI, 67.6%-85.5%)/98.5% (95% CI, 91.8%-100%) for GeneFields CPE. Interestingly, mCIM and CARBA-5 assays showed 100% accuracy/sensitivity/specificity for detection of the target genes. Furthermore, all the other assays showed comparable high accuracy (96.9% to 100%), sensitivity (100%), and specificity (96.4% to 100%) for the detection of the target genes. On the basis of these results, a new scheme was proposed for their efficient application. These results confirmed the high sensitivity of the evaluated assays, and the proposed scheme is reliable and improves the overall sensitivity and specificity of the assays.

Adjustment of Modified Carbapenem Inactivation Method Conditions for Rapid Detection of Carbapenemase-Producing Acinetobacter baumannii.

BACKGROUND: The existing modified carbapenem inactivation methods (mCIMs) recommended by the CLSI for detecting carbapenemase production have not been applicable for Acinetobacter baumannii. We evaluated the influence of matrices used in mCIMs and CIMTris on the stability of the disks for detecting carbapenemase producers and suggested optimal mCIM conditions for detecting carbapenemase-producing A. baumannii. METHODS: Seventy-three A. baumannii isolates characterized for antimicrobial susceptibility and carbapenemase encoding genes were tested for carbapenemase production using mCIM and CIMTris. The influence of the matrices (Tryptic soy broth [TSB] and Tris-HCl) used in these methods on the stability of the meropenem (MEM) disk was also evaluated. The mCIM conditions were adjusted to enhance screening sensitivity and specificity for detecting carbapenemase-producing A. baumannii. RESULTS: The matrices had an impact on the stability of the MEM disk after the incubation period (two or four hrs). TSB nutrient broth is an appropriate matrix for mCIM compared with Tris-HCl pH 7.6, which leads to the loss of MEM activity in CIMTris. The sensitivity and the specificity of the optimal mCIM were both 100%. CONCLUSIONS: We established optimal mCIM conditions for simple, accurate, and reproducible detection of carbapenemase-producing A. baumannii.

Meropenem, Cefepime, and Piperacillin Protein Binding in Patient Samples.

BACKGROUND: The mortality rate of patients with a drug-resistant bacterial infection is high, as are the associated treatment costs. To overcome these issues, optimization of the available therapeutic options is required. Beta-lactams are time-dependent antibiotics and their efficacy is determined by the amount of time the free concentration remains above the minimum inhibitory concentration. Therefore, the aim of this study was to assess the extent and variability of protein binding for meropenem, cefepime, and piperacillin. METHODS: Plasma samples for the analysis of meropenem, cefepime, and piperacillin were collected from patients admitted to a tertiary care hospital as part of the standard care. The bound and unbound drug fractions in the samples were separated by ultrafiltration. Validated liquid chromatography-tandem mass spectrometry assays were used to quantify the total and free plasma concentrations, and the protein binding was determined. RESULTS: Samples from 95 patients were analyzed. The median (range) age of patients was 56 years (17-87) and the median (range) body mass index was 25.7 kg/m (14.7-74.2). Approximately 59% of the patients were men. The median (range) unbound fraction (fu) was 62.5% (41.6-99.1) for meropenem, 61.4% (51.6-99.2) for cefepime, and 48.3% (39.4-71.3) for piperacillin. In the bivariate analysis, as the total meropenem concentration increased, the fu increased (r = 0.37, P = 0.045). A decrease in piperacillin fu was observed as the albumin concentration increased (r = -0.56, P = 0.005). CONCLUSIONS: The average fu values were lower than those reported in the literature. There was also a large variability in fu; hence, it should be considered when managing patients administered with these drugs through direct measurements of free drug concentrations.