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.

Disk Diffusion Testing for Detection of Methicillin-Resistant Staphylococci: Does Moxalactam Improve upon Cefoxitin?

Disk diffusion testing is widely used to detect methicillin resistance in staphylococci, and cefoxitin is currently considered the best marker for mecA-mediated methicillin resistance. In low-inoculum diffusion testing (colony suspension at 106 CFU/ml), the addition of moxalactam in combination with cefoxitin has been reported to improve on cefoxitin alone for the detection of methicillin-heteroresistant staphylococci. However, moxalactam is absent from EUCAST and CLSI guidelines, which use high-inoculum diffusion testing (colony suspension at 108 CFU/ml), calling into question the potential interest of including moxalactam in their recommendations. The inhibition zone diameters of cefoxitin and moxalactam, alone and in combination, were evaluated for concordance with mecA and mecC positivity in a large collection of clinical Staphylococcus isolates (611 Staphylococcus aureus, Staphylococcus lugdunensis, and Staphylococcus saprophyticus isolates and 307 coagulase-negative staphylococci other than S. lugdunensis and S. saprophyticus isolates, of which 22% and 53% were mecA-positive, respectively) and in 25 mecC-positive S. aureus isolates using high-inoculum diffusion testing. Receiver operating characteristic, sensitivity, and specificity analyses indicated that the detection of mecA- and mecC-positive and negative isolates did not improve with moxalactam, either alone or in combination with cefoxitin, compared to cefoxitin alone. These findings were similar in both the S. aureus/S. lugdunensis/S. saprophyticus group and in the coagulase-negative staphylococci group. Our results do not support the use of moxalactam as an additional marker of methicillin resistance when testing with high-inoculum disk diffusion.

[The activity of moxalactam against Enterobacteriaceae and anaerobia in vitro].

OBJECTIVE: To observe the antibacterial activity of moxalactam against Enterobacteriaceae bacteria and anaerobic bacteria in vitro, and to compare with other antibacterial drugs, for providing experimental basis for the clinical application of moxalactam. METHODS: Minimum inhibitory concentrations (MICs) of moxalactam and other antibacterial agents against 491 Enterobacteriaceae spp. and anaerobic spp.collecting from clinical settings were determined by agar dilution methods and E-test strips according to the Clinical and Laboratory Standards Institute (CLSI)(2014). RESULTS: Moxalactam showed great antibacterial activity to Enterobacteriaceae spp., including ESBLs-producing Escherichia coli, Klebsiella pneumoniae, and Proteus spp., with the MIC(50), MIC(90), and susceptibility rates of 0.25-4 mg/L, 0.5-8 mg/L, and >90%, respectively. The susceptibility rates of Enterobacteriaceae with ESBLs-producing or non-ESBLs-producing to imipenem and meropenem were both higher than 90%. The susceptibility rates of ESBLs-producing Escherichia coli, Klebsiella pneumoniae, and Proteus spp.to piperacillin/tazobactam and cefoperazone/sulbactam were 90%, 68%, 53% and 76%, 66%, 76.6%, respectively, while the susceptibility rates of non-ESBLs-producing Escherichia coli, Klebsiella pneumoniae, and Proteus spp.were all more than 95%. The susceptibility rates of Enterobacter spp. and other Enterobacter to piperacillin/tazobactam were 80%, 80%and that to cefoperazone/sulbactam were 80%, 76.7%, respectively.The MICs range of moxalactam on anaerobic spp.was from ≤0.064 to >256 mg/L, while MIC(50) was 2 mg/L and MIC(90) was 64 mg/L. CONCLUSIONS: Moxalactam showed well activity against ESBLs-producing and non-ESBLs-producing Enterobacteriaceae and anaerobia.

Conformational Change Observed in the Active Site of Class C ß-Lactamase MOX-1 upon Binding to Aztreonam.

We solved the crystal structure of the class C ß-lactamase MOX-1 complexed with the inhibitor aztreonam at 1.9Å resolution. The main-chain oxygen of Ser315 interacts with the amide nitrogen of aztreonam. Surprisingly, compared to that in the structure of free MOX-1, this main-chain carboxyl changes its position significantly upon binding to aztreonam. This result indicates that the interaction between MOX-1 and ß-lactams can be accompanied by conformational changes in the B3 ß-strand main chain.

IMP-51, a novel IMP-type metallo-ß-lactamase with increased doripenem- and meropenem-hydrolyzing activities, in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate.

A meropenem-resistant Pseudomonas aeruginosa isolate was obtained from a patient in a medical setting in Hanoi, Vietnam. The isolate was found to have a novel IMP-type metallo-ß-lactamase, IMP-51, which differed from IMP-7 by an amino acid substitution (Ser262Gly). Escherichia coli expressing blaIMP-51 showed greater resistance to cefoxitin, meropenem, and moxalactam than E. coli expressing blaIMP-7. The amino acid residue at position 262 was located near the active site, proximal to the H263 Zn(II) ligand.

Potent antibacterial activities of latamoxef (moxalactam) against ESBL producing Enterobacteriaceae analyzed by Monte Carlo simulation.

Latamoxef (LMOX, Moxalactam) is one of the beta-lactam antibiotics which is stable against beta-lactamase. In this study, the antibacterial activity of LMOX was investigated, and Monte Carlo simulation was conducted to determine the appropriate dosing regimens of LMOX against extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae. The probability of target attainment (PTA) was analyzed at 40% and 70% of time above minimum inhibitory concentration (MIC) (time above MIC, T(>MIC)) for bacteriostatic and bactericidal effect respectively. All the tested regimens achieved 85% of PTA at 40% of T(>MIC) against ESBL producing Escherichia coli, and all the tested regimens except 1g q12h with 1 hour infusion achieved 85% of PTA at 40% of T(>MIC) against ESBL producing Klebsiella pneumoniae. The effective regimens to achieve 85% of PTA at 70% of T(>MIC )against E. coli were lg ql2h with 4 hours infusion, lg q8h with 1-4 hours infusion, 2g ql2h with 2-4 hours infusion, and lg q6h with 1-4 hours infusion. The effective regimens to achieve 85% of PTA at 70% of T(>MIC) against K. pneumoniae were 1g q8h with 3-4 hours infusion and 1g q6h with 1-4 hours infusion. These results of pharmacokinetics/pharmacodynamics (PK/PD) modeling showed the potent efficacy of LMOX against bacterial infections caused by ESBL producing Enterobacteriaceae.

Estimation of latamoxef (moxalactam) dosage regimens against ß-lactamase-producing Enterobacterales in dogs: a pharmacokinetic and pharmacodynamic analysis using Monte Carlo simulation.

One of the most significant research areas in veterinary medicine is the search for carbapenem substitutes for the treatment of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales (ESBL-E). This study applied a pharmacokinetic/pharmacodynamic (PK/PD) strategy in validating optimal latamoxef (LMX) therapeutic regimens against canine ESBL-E infections. Five dogs were administered a bolus dose of 40 mg/kg LMX intravenously to measure serum drug concentrations and determine PK indices using the noncompartmental model. The highest minimum inhibitory concentration (MIC) with a probability of target attainment ≥90% was used to compute the PK/PD cutoff values for bacteriostatic (time for which the unbound drug concentration was above the MIC [fTAM] ≥ 40%) and bactericidal (fTAM ≥ 70%) effects when administered at 20, 30, 50, and 60 mg/kg, in addition to 40 mg/kg. The cumulative fraction of response (CFR) was determined using the MIC distribution of wild-type ESBL-E in companion animals. The PK/PD cutoff values can be increased by reducing the dosing interval rather than increasing the dose per time. Based on the calculated CFRs for ESBL-producing Escherichia coli and Klebsiella pneumoniae, all LMX regimens in this study and those administered at 30-60 mg/kg every 8 and 6 hr were found to be optimal (CFR ≥ 90%) for exerting bacteriostatic and bactericidal effects, respectively. However, the regimens of 50 and 60 mg/kg every 6 hr may merely exert bacteriostatic effects on ESBL-producing Enterobacter cloacae. Further clinical trials are required to confirm the clinical efficacy of LMX.

A potential substrate binding conformation of ß-lactams and insight into the broad spectrum of NDM-1 activity.

New Delhi metallo-ß-lactamase 1 (NDM-1) is a key enzyme that the pathogen Klebsiella pneumonia uses to hydrolyze almost all ß-lactam antibiotics. It is currently unclear why NDM-1 has a broad spectrum of activity. Docking of the representatives of the ß-lactam families into the active site of NDM-1 is reported here. All the ß-lactams naturally fit the NDM-1 pocket, implying that NDM-1 can accommodate the substrates without dramatic conformation changes. The docking reveals two major binding modes of the ß-lactams, which we tentatively name the S (substrate) and I (inhibitor) conformers. In the S conformers of all the ß-lactams, the amide oxygen and the carboxylic group conservatively interact with two zinc ions, while the substitutions on the fused rings show dramatic differences in their conformations and positions. Since the bridging hydroxide ion/water in the S conformer is at the position for the nucleophilic attack, the S conformation may simulate the true binding of a substrate to NDM-1. The I conformer either blocks or displaces the bridging hydroxide ion/water, such as in the case of aztreonam, and is thus inhibitory. The docking also suggests that substitutions on the ß-lactam ring are required for ß-lactams to bind in the S conformation, and therefore, small ß-lactams such as clavulanic acid would be inhibitors of NDM-1. Finally, our docking shows that moxalactam uses its tyrosyl-carboxylic group to compete with the S conformer and would thus be a poor substrate of NDM-1.

Sensitive and specific phenotypic assay for metallo-beta-lactamase detection in Enterobacteria by use of a moxalactam disk supplemented with EDTA.

Moxalactam is highly hydrolyzed by plasmid-mediated metallo-ß-lactamases (MBLs), whereas it is poorly inactivated by serine-active carbapenemases. This study demonstrated that moxalactam resistance constituted an effective screen for MBL expression in enterobacteria, which could be confirmed, even in low-MBL-producing isolates, by a disk potentiation test using moxalactam and EDTA.

[Comparison of oxacillin, cefoxitin, ceftizoxime, and moxalactam disk diffusion methods for detection of methicillin susceptibility in staphylococci]. / Stafilokoklarda Metisilin Duyarliliginin Belirlenmesinde Oksasilin, Sefoksitin, Seftizoksim ve Moksalaktam Disk Difüzyon Yöntemlerinin Karsilastirilmasi

Penicillin binding protein 2a/2' (PBP2a/PBP2') which is encoded by the mecA gene, is responsible for the methicillin resistance in staphylococci. Detection of methicillin resistance with phenotypic methods is still a problem especially because of heterogenous expression of mecA gene. Although mecA gene determination by polymerase chain reaction is considered as the gold standard method, molecular tests are not easily applied in all routine laboratories. Thus, for the rapid and accurate diagnosis of MRSA strains, easy and practical phenotypic tests are still required. This study was conducted to compare the oxacillin (OX), cefoxitin (CFX), ceftizoxime (CZX), and moxolactam (MOX) susceptibility testing by disk diffusion method for the detection of methicillin resistance in staphylococci. A total of 247 staphylococci (125 Staphylococcus aureus and 122 coagulase-negative staphylococci; CNS) isolated from various clinical specimens (114 wound and soft tissue materials, 51 urine, 48 blood, 30 respiratory tract, and four other samples) of inpatients and outpatients, were included in this study. PBP2a latex agglutination test was used as the reference method for the recognition of methicillin resistance; four antibiotic disks tested and sensitivity, specificity, positive and negative predictive values (PPV and NPV) were determined for each of them. According to PBP2a latex agglutination test 66 (54.1%) of CNS and 53 (42.4%) of S.aureus isolates were found methicillin- resistant. OX and MOX disks detected 113 (63 CNS and 50 S.aureus) methicillin-resistant strain out of 119 PBP2a positive isolates, where CFX and CZX disks detected 110 (60 CNS and 50 S.aureus) of them. Among 128 PBP2a negative isolates, 123 (52 CNS and 71 S.aureus) were detected as susceptible with OX, 127 (55 CNS and 72 S.aureus) with CFX and CZX, 126 (54 CNS and 72 S.aureus) with MOX. According to these results, the sensitivities and specificities of OX, CFX, CZX, and MOX disks were; 95.4% and 92.8%, 90.9% and 98.2%, 90.9% and 98.2%, 95.4% and 96.4%, respectively for CNS and 94.3% and 98.6%, 94.3% and 100%, 94.3% and 100%, 94.3% and 100%, respectively for S.aureus. The difference between sensitivities and specificities of tested antibiotic disks were not found statistically significant. In conclusion, due to the problems in detection of methicillin resistance with phenotypic methods, the use of different mecA gene-inducing antibiotic disks at the same time, and utilization of molecular methods as reference method might be suggested, when a discordance is observed between the antibiotic disks.

E5531, a pure endotoxin antagonist of high potency.

Shock due to Gram-negative bacterial sepsis is a consequence of acute inflammatory response to lipopolysaccharide (LPS) or endotoxin released from bacteria. LPS is a major constituent of the outer membrane of Gram-negative bacteria, and its terminal disaccharide phospholipid (lipid A) portion contains the key structural features responsible for toxic activity. Based on the proposed structure of nontoxic Rhodobacter capsulatus lipid A, a fully stabilized endotoxin antagonist E5531 has been synthesized. In vitro, E5531 demonstrated potent antagonism of LPS-mediated cellular activation in a variety of systems. In vivo, E5531 protected mice from LPS-induced lethality and, in cooperation with an antibiotic, protected mice from a lethal infection of viable Escherichia coli.

Evaluation of new Vitek 2 card and disk diffusion method for determining susceptibility of Staphylococcus aureus to oxacillin.

Detection of methicillin resistance in Staphylococcus aureus is a challenge, especially low-level resistance, which is often misdiagnosed. The aim of this study was to compare the diagnostic accuracies of the automated Vitek 2 system and disk diffusion tests, using cefoxitin and moxalactam, for the detection of methicillin resistance in S. aureus strains. Four sets of genotypically diverse isolates were selected from a national reference collection, including mecA-negative S. aureus isolates (n = 56), hospital-acquired (n = 88) and community-acquired (n = 40) S. aureus isolates, and heterogeneous methicillin-resistant S. aureus isolates (n = 29). Oxacillin susceptibility was tested by the Vitek 2 system with the AST P549 card and by disk diffusion methods using 10, 30, and 60 microg cefoxitin and 30 microg moxalactam. Oxacillin resistance was confirmed by PCR for the mecA gene. The overall sensitivities for oxacillin resistance detection were 97.5% for the Vitek 2 automated system, 98.7% for 60-microg cefoxitin and moxalactam disk diffusion, and 99.6% for 10- and 30-microg cefoxitin disks, respectively. Methicillin-susceptible S. aureus isolates were correctly reported as susceptible by all methods. The median times for methicillin testing were 7 h for the Vitek 2 system versus 24 h for disk diffusion methods. In conclusion, the cefoxitin and moxalactam disk diffusion methods and the Vitek 2 automated system are highly accurate methods for methicillin resistance detection, including a range of representative Belgian methicillin-resistant S. aureus strains and unusual strains exhibiting cryptic or low-level oxacillin resistance.

Influence of cefmenoxime, ceftriaxone, latamoxef, and ceftazidime on the lysis of Klebsiella pneumoniae: a light and electron microscopic study.

The influence of antibiotics on the morphologic alterations of the cell wall of Klebsiella pneumoniae serotype K 17 has been studied by electron microscopy as well as by phase-contrast microscopy. Ceftazidime, cefmenoxime, ceftriaxone, and latamoxef were the antibiotics used. The effect of each concentration between 0.03 and 1,024 mg/liter was investigated on 5 X 10(7) bacteria per milliliter. Two different reactions could be detected: ceftazidime and latamoxef produced at first long forms and suddenly lysis; cefmenoxime and ceftriaxone produced at first long forms, later spherical forms, and then gradually lysis.

Potential of moxalactam and other new antimicrobial agents for bilirubin-albumin displacement in neonates.

The effects of several novel antibiotics on in vitro binding of bilirubin to human serum albumin were investigated. At physiologic bilirubin-albumin ratios and pH values, aztreonam, imipenem, azlocillin, enoxacin and ciprofloxacin did not compete with bilirubin at drug concentrations less than 900 micrograms/mL. Cefoperazone caused an apparent increase in unbound bilirubin only at concentrations greater than 35 microM (330 micrograms/mL). Moxalactam, however, caused a linear increase in unbound bilirubin, greater than that seen with sulfisoxazole, over the entire range of antibiotic concentrations. These results may have implications for the use of these newer antimicrobial agents in neonatal infections.

A survey of temocillin sensitivity of strains resistant to newer beta-lactam antibiotics.

The susceptibility of a total of 2014 Gram-negative clinical isolates (except Pseudomonas aeruginosa) to a number of antibiotics, including temocillin, was investigated in 2 geographically distinct areas. Overall, more strains were sensitive to temocillin than to mezlocillin, piperacillin, amoxycillin plus clavulanic acid, cefotaxime, cephazolin, latamoxef (moxalactam), cefoxitin, or netilmicin. Susceptibility to temocillin of multiple-resistant strains was studied.

In vitro activity of temocillin against clinical isolates.

The minimum inhibitory concentrations of temocillin against more than 1000 clinical isolates were determined by an agar dilution method. Temocillin showed excellent activity against Haemophilus influenzae, pathogenic Neisseria species and Branhamella catarrhalis, including beta-lactamase producing strains, but showed very low activity or was inactive against Gram-positive cocci and Campylobacter, Bacteroides, Acinetobacter and Pseudomonas species. Good activity was obtained against 702 Enterobacteriaceae, including isolates resistant to the other penicillins and first- and second-generation cephalosporins, with 92% of all the strains inhibited at a concentration of 8 mg/L. However, the most striking property of temocillin was its high beta-lactamase stability which resulted in both a very narrow range of MICs within which all the isolates were inhibited, and a small influence of inoculum size on the MICs.

Moxalactam (latamoxef). A review of its antibacterial activity, pharmacokinetic properties and therapeutic use.

Moxalactam (latamoxef) is a new synthetic oxa-beta-lactam antibiotic administered intravenously or intramuscularly. It has a broad spectrum of activity against Gram-positive and Gram-negative aerobic and anaerobic bacteria, is particularly active against Enterobacteriaceae and is resistant to hydrolysis by beta-lactamases. Moxalactam has moderate activity against Pseudomonas aeruginosa, but on the basis of present evidence can not be recommended as sole antibiotic treatment of known or suspected pseudomonal infections. Like the related compounds, the cephalosporins, moxalactam is effective in the treatment of complicated urinary tract infections and lower respiratory tract infections caused by Gram-negative bacilli. As moxalactam is also active against Bacteroides fragilis it has considerable potential in the treatment of intra-abdominal infections in patients with normal immunological mechanisms, as well as in immunocompromised patients, when used alone or in combination with other antibiotics. Likewise, its ready penetration into the diseased central nervous system, its high level of activity against Gram-negative bacilli, and the lack of necessity to monitor drug plasma concentrations, indicate its potential value in the treatment of neonatal Gram-negative bacillary meningitis. Further clinical experience is needed before it can be determined whether moxalactam alone can be used in the treatment of conditions for which the aminoglycosides are drugs of choice, but if established as equally effective, moxalactam has the advantage of being devoid of nephrotoxicity. Bleeding is a potentially serious problem, however, particularly in the elderly, malnourished and in the presence of renal impairment.

Quantitative relationship between sensitivity to beta-lactam antibiotics and beta-lactamase production in gram-negative bacteria--II. Non-steady-state treatment and progress curves.

A non-steady-state model is discussed for the study of the interplay between beta-lactamase activity and outer membrane permeability with slowly hydrolysed beta-lactams. The analysis shows: (1) that the simple, steady-state model presented in the accompanying paper remains valid as long as kcat (i.e. k3 with chromosome-encoded class C beta-lactamases) is larger than 10(-3)/sec (generation time = 20 min or more); (2) that among the beta-lactam antibiotics studied here, the complete, non-steady-state model needs only be used in the case of aztreonam; (3) that the term "trapping" should be replaced by "formation of a covalent acyl-enzyme" and that such a phenomenon only contributes significantly to the resistance when penetration and hydrolysis are very slow and the periplasmic beta-lactamase concentration is very high. Aztreonam seems to be the only compound which fulfils the first two conditions.

Empiric therapy with moxalactam alone in patients with bacteremia.

Moxalactam was administered (20 mg/kg intravenously every 8 hours) as single-drug empiric antimicrobial therapy to 63 patients with bacteremia who were neither neutropenic nor immunosuppressed. Six patients (10%) had microorganisms that were susceptible to moxalactam and resistant to all other antimicrobial agents tested; two patients (3%) had microorganisms that were resistant to moxalactam and other agents tested. Of these 63 patients, 47 (75%) were cured with moxalactam therapy. Nine patients (14%) had breakthrough bacteremia while receiving other antimicrobial therapy and were cured subsequently with moxalactam therapy alone. The two major risk factors for failure of moxalactam therapy were polymicrobial bacteremia and an extrahepatic intra-abdominal source of infection; these two conditions frequently coexisted. Six of nine patients with polymicrobial bacteremia died. Superinfection (one pseudomonal, five enterococcal) was responsible for 6 of the 16 treatment failures. Enterococcal superinfection occurred exclusively among patients who had received relatively prolonged therapy with moxalactam for extrahepatic intra-abdominal infection, especially intraabdominal abscess. These five patients died, and postmortem examination showed that enterococcal superinfection was the major cause of death in all. Mild, reversible adverse reactions associated with use of moxalactam occurred in 14 of the 63 patients (22%). None had clinically overt bleeding. The use of moxalactam alone seems to be safe and effective and a cost-effective alternative empiric antimicrobial therapy for most patients with bacteremia who are not immunosuppressed or neutropenic and who are not at high risk of having Pseudomonas or polymicrobial bacteremia.