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Aims: The antibacterial activity of red propolis extract (RPE) and brown propolis extracts (BPE) and the synergistic effect of RPE with cefoxitin (CEFO), imipenem (IMI), and ertapenem (ERTA) was evaluated in vitro against methicillin-resistant Staphylococcus aureus (MRSA) strains., Methods and Results: MRSA ATCC 33591, community-associated (CA-MRSA) USA300, and four clinical isolates were used. A broth microdilution assay was performed to obtain inhibitory and bactericidal concentrations of BPE, RPE, CEFO, IMI, and ERTA. RPE in combination with CEFO, IMI, and ERTA was evaluated on the formation or eradication of biofilm. The bacterial relative membrane conductivity of the strains was assessed after RPE and combinations exposition. Surface/binding computational analyzes between RPE compounds and penicillin binding protein 2a (PBP2a) were performed. BPE samples had no activity against MRSA (MICs 3.2-5 g l-1; MBCs 10-15 g l-1), so the subsequent assays were carried out only with RPE and antimicrobials. RPE exerted a bacteriostatic action (MICs 0.0156-0.125 g l-1; MBCs 0.5-2 g l-1) but the combinations with IMI and ERTA showed the highest inhibition, as observed in the time-kill curve. However, the FICI index showed synergism (≥0.5) only to RPE + IMI. This combination was the most effective in inhibiting the biofilm and showed the highest values of membrane conductivity. Computational predictions indicated that RPE constituents may interact with PBP2a., Conclusion: RPE and RPE + IMI exerted an antibacterial and antibiofilm activity on MRSA strains probably due to membrane/wall damage and interactions with PBP2a., (© The Author(s) 2022. Published by Oxford University Press on behalf of Applied Microbiology International.)

Klebsiella pneumoniae is an opportunistic bacterium that causes many infections, including septicemia, pneumonia, urinary tract infection, and liver abscesses. There are many mechanisms for antibiotic resistance and K. pneumonia is considered a multidrug-resistant pathogen. This study aimed to find the correlation between the susceptibility of K. pneumonia to certain antibiotics with the porin-related resistance and pumps mechanisms. In total, two genes that are responsible for porin formation were considered in the current study OmpK-35 gene and OmpK-36 gene, in addition to other four genes ( CfiaS , CfiaL , MFS , and MdtK genes) related to an efflux pump mechanism of antibiotic resistance. The bacterial resistance was investigated towards five cephalosporins (Cefazolin, Cefoxitin, Ceftazidime, Ceftriaxone, and Cefepime) and two carbapenems (imipenem and ertapenem). Clinical samples, including blood, swabs, and urine, consisting of 20 specimens for each group, were collected from patients who attended three hospitals in Baghdad. The VITEK-2 system and genetic tests (polymerase chain reaction and sequencing) of bacterial isolates were applied to confirm the diagnosis of K. pneumoniae and detect the antibiotic sensitivity profile. The results showed that 51 (85%) and 15 (25%) of the total 60 isolates had positive results for OmpK-35 and Omp-K36 genes, respectively. The MFS and MdtK genes were observed (70-88.3%) in cephalosporin-resistant isolates of K. pneumoniae. There were no significant variations of bacterial resistance genes of antibiotics within the specimen groups. It was concluded that the bacterial resistance of the selected antibiotics was elevated markedly with the loss of the OmpK-36 gene with a high expression of MFS and MdtK genes and a slight minimal occurrence in the new generation of carbapenems. The best antimicrobial agent was ertapenem with a percentage of 0% of resistance in all bacterial isolates., Competing Interests: The authors declare that they have no conflict of interest.

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 β-lactams. Significant reduction of k cat 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 T m B and T m D 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 β-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.

While carbapenem resistance in Gram-negative bacteria is mainly due to the production of efficient carbapenemases, β-lactamases with a narrower spectrum may also contribute to resistance when combined with additional mechanisms. OXA-10-type class D β-lactamases, previously shown to be weak carbapenemases, could represent such a case. In this study, two novel OXA-10 variants were identified as the sole carbapenem-hydrolyzing enzymes in meropenem-resistant enterobacteria isolated from hospital wastewater and found by next-generation sequencing to express additional β-lactam resistance mechanisms. The new variants, OXA-655 and OXA-656, were carried by two related IncQ1 broad-host-range plasmids. Compared to the sequence of OXA-10, they both harbored a Thr26Met substitution, with OXA-655 also bearing a leucine instead of a valine in position 117 of the SAV catalytic motif. Susceptibility profiling of laboratory strains replicating the natural bla OXA plasmids and of recombinant clones expressing OXA-10 and the novel variants in an isogenic background indicated that OXA-655 is a more efficient carbapenemase. The carbapenemase activity of OXA-655 is due to the Val117Leu substitution, as shown by steady-state kinetic experiments, where the k cat of meropenem hydrolysis was increased 4-fold. In contrast, OXA-655 had no activity toward oxyimino-β-lactams, while its catalytic efficiency against oxacillin was significantly reduced. Moreover, the Val117Leu variant was more efficient against temocillin and cefoxitin. Molecular dynamics indicated that Val117Leu affects the position 117-Leu155 interaction, leading to structural shifts in the active site that may alter carbapenem alignment. The evolutionary potential of OXA-10 enzymes toward carbapenem hydrolysis combined with their spread by promiscuous plasmids indicates that they may pose a future clinical threat., (Copyright © 2018 American Society for Microbiology.)

Objectives: Limited treatment options available for Mycobacterium abscessus infections include the parenteral β-lactam antibiotics cefoxitin and imipenem, which show moderate in vitro activity. Other β-lactam antibiotics (except meropenem) have no considerable in vitro activity, due to their rapid hydrolysis by a broad-spectrum β-lactamase (Bla_Mab). We here addressed the impact of β-lactamase production and β-lactam in vitro stability on M. abscessus MIC results and determined the epidemiological cut-off (ECOFF) values of cefoxitin, imipenem and meropenem., Methods: By LC high-resolution MS (LC-HRMS), we assessed the in vitro stability of cefoxitin, imipenem and meropenem. M. abscessus ATCC 19977 strain and its isogenic blaMab deletion mutant were used for MIC testing. Based on MIC distributions for M. abscessus clinical strains, we determined ECOFFs of cefoxitin, imipenem and meropenem., Results: A functional Bla_Mab increased MICs of penicillins, ceftriaxone and meropenem. LC-HRMS data showed significant degradation of cefoxitin, imipenem and meropenem during standard antibiotic susceptibility testing procedures. MIC, MIC50 and ECOFF values of cefoxitin, imipenem and meropenem are influenced by incubation time., Conclusions: The results of our study support administration of imipenem, meropenem and cefoxitin, for treatment of patients infected with M. abscessus. Our findings on in vitro instability of imipenem, meropenem and cefoxitin explain the problematic correlation between in vitro susceptibility and in vivo activity of these antibiotics and question the clinical utility of susceptibility testing of these chemotherapeutic agents., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

TEM-1 beta-lactamases can accurately catalyze the hydrolysis of the beta-lactam rings in beta-lactam antibiotics, which make beta-lactam antibiotics lose its activity, and the prerequisite for the hydrolysis procedure in the binding interaction of TEM-1 beta-lactamases with beta-lactam antibiotics is the beta-lactam rings in beta-lactam antibiotics. Therefore, the binding of TEM-1 beta-lactamase to three beta-lactam antibiotics including penicillin G, cefalexin as well as cefoxitin was explored here by frontal affinity chromatography in combination with fluorescence spectra, adsorption and thermodynamic data in the temperature range of 278-288K under simulated physiological conditions. The results showed that all the binding of TEM-1 beta-lactamase to the three antibiotics were spontaneously exothermic processes with the binding constants of 8.718×10 3 , 6.624×10 3 and 2.244×10 3 (mol/L), respectively at 288K. All the TEM-1 beta-lactamases were immobilized on the surface of the stationary phase in the mode of monolayer and there existed only one type of binding sites on them. Each TEM-1 beta-lactamase bound with only one beta-lactam antibiotic and hydrogen bond interaction and Van der Waals force were the main forces between them. This work provided an insight into the binding interactions between TEM-1 beta-lactamases and beta-lactam antibiotics, which may be beneficial for the designing and developing of new substrates resistant to TEM-1 beta-lactamases., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We evaluated the performance of an immunochromatographic assay (PBP2a Culture Colony Test - Alere™), detecting protein-binding penicillin 2a on staphylococci primary isolates in only 6minutes. The assay is highly sensitive for the direct detection of MRSA on various culture media whereas it requires cefoxitin induction for methicillin-resistant coagulase-negative staphylococci., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Class C β-lactamases poorly hydrolyze cephamycins (e.g., cefoxitin, cefotetan, and moxalactam). In the past 2 decades, a new family of plasmid-based AmpC β-lactamases conferring resistance to cefoxitin, the FOX family, has grown to include nine unique members descended from the Aeromonas caviae chromosomal AmpC. To understand the basis for the unique cephamycinase activity in the FOX family, we determined the first X-ray crystal structures of FOX-4, apo enzyme and the acyl-enzyme with its namesake compound, cefoxitin, using the Y150F deacylation-deficient variant. Notably, recombinant expression of N-terminally tagged FOX-4 also yielded an inactive adenylylated enzyme form not previously observed in β-lactamases. The posttranslational modification (PTM), which occurs on the active site Ser64, would not seem to provide a selective advantage, yet might present an opportunity for the design of novel antibacterial drugs. Substantial ligand-induced changes in the enzyme are seen in the acyl-enzyme complex, particularly the R2 loop and helix H10 (P289 to N297), with movement of F293 by 10.3 Å. Taken together, this study provides the first picture of this highly proficient class C cephamycinase, uncovers a novel PTM, and suggests a possible cephamycin resistance mechanism involving repositioning of the substrate due to the presence of S153P, N289P, and N346I substitutions in the ligand binding pocket., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Objectives: Two natural variants of ADC-type β-lactamases of Acinetobacter spp., ADC-1 and ADC-5, differ by nine mutations in their protein sequence. ADC-5 hydrolyses cefoxitin better than ADC-1 and the opposite is true for ceftazidime. We produced single and combined mutations in ADC-5 and characterized the variants microbiologically and biochemically to determine which amino acid residues are involved in the hydrolysis of β-lactam antibiotics in this family of β-lactamases., Methods: Site-directed mutagenesis, with blaADC-5 as a source of DNA, was used to generate nine single mutated and three combined mutated enzymes. The proteins (wild-type and derivatives) were then expressed in isogenic conditions in Escherichia coli. MICs of β-lactams were determined using Etest strips. ADC-1, ADC-5, ADC-5-P167S and ADC-5-P167S/D242G/Q163K/G342R were also purified and the kinetic parameters determined for ceftazidime, cefoxitin, cefalotin and ampicillin., Results: Single mutations did not significantly convert the hydrolysis spectrum of the ADC-5 enzyme into that of the ADC-1 enzyme, although among all studied mutants only the quadruple mutant (ADC-5-P167S/D242G/Q163K/G342R) displayed microbiological and biochemical properties consistent with those of ADC-1., Conclusions: Although some single mutations are known to affect cefepime hydrolysis in ADC-type β-lactamases, little is known about ceftazidime and cefoxitin hydrolysis in this family of β-lactamases. Hydrolysis of these antibiotics appears to be positively and negatively affected, respectively, by the Q163K, P167S, D242G and G342R amino acid replacements., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

An understanding of antibiotic hydrolysis rates is important for predicting their environmental persistence. Hydrolysis rates and Arrhenius constants were determined as a function of pH and temperature for three common β-lactam antibiotics, ampicillin, cefalotin, and cefoxitin. Antibiotic hydrolysis rates at pH4-9 at 25 °C, 50 °C, and 60 °C were quantified, and degradation products were identified. The three antibiotics hydrolyzed under ambient conditions (pH7 and 25 °C); half-lives ranged from 5.3 to 27 d. Base-catalyzed hydrolysis rates were significantly greater than acid-catalyzed and neutral pH hydrolysis rates. Hydrolysis rates increased 2.5- to 3.9-fold for a 10 °C increase in temperature. Based on the degradation product masses found, the likely functional groups that underwent hydrolysis were lactam, ester, carbamate, and amide moieties. Many of the proposed products resulting from the hydrolysis of ampicillin, cefalotin, and cefoxitin likely have reduced antimicrobial activity because many products contained a hydrated lactam ring. The results of this research demonstrate that β-lactam antibiotics hydrolyze under ambient pH and temperature conditions. Degradation of β-lactam antibiotics will likely occur over several weeks in most surface waters and over several days in more alkaline systems., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cycloserine-cefoxitin fructose agar (CCFA), CCFA with horse blood and taurocholate (CCFA-HT), and cycloserine-cefoxitin mannitol broth with taurocholate and lysozyme (CCMB-TAL) were compared for recovery of Clostridium difficile from 120 stool specimens. Compared to CCFA, CCFA-HT enhanced C. difficile growth and improved recovery by 4%. In a separate study, 9% (8/91) of stool samples previously C. difficile negative on plate medium were C. difficile positive when cultured in CCMB-TAL.

Objective: To compare the sensitivity and specificity of different phenotypic methods for detection of Amp C betalactamase producing bacteria., Study Design: Analytical study., Place and Duration of Study: Department of Microbiology, Army Medical College / National University of Sciences and Technology (NUST), Islamabad, Pakistan, from June 2010 to December 2010., Methodology: A total of 150 clinical isolates were screened for presence of Amp C beta-lactamase by using the cefoxitin disc. The confirmatory methods evaluated were inhibitor based assay (boronic acid), Amp C disc test and Amp C Etest. Three dimensional enzyme extract assay was used as the reference method for determining the sensitivity and specificity., Results: Among the total isolates tested, 62.8% bacteria showed the presence of Amp C beta-lactamase by standard three dimensional enzyme extract assay. Among the three methods compared, boronic acid disk test found out to be highly sensitive (88%) and specific (92%) for the detection of Amp C beta-lactamase producing bacteria., Conclusion: Detection of Amp C production is crucial in order to establish the antibiotic therapy and to attain the favourable clinical outcomes. Implementation of simple tests like boronic acid disk tests in the laboratories will help to alleviate the spread of Amp C beta-lactamase harboring organisms.

Class C cephalosporinases are a growing threat, and clinical inhibitors of these enzymes are currently unavailable. Previous studies have explored the role of Asn152 in the Escherichia coli AmpC and P99 enzymes and have suggested that interactions between C-6' or C-7' substituents on penicillins or cephalosporins and Asn152 are important in determining substrate specificity and enzymatic stability. We sought to characterize the role of Asn152 in the clinically important CMY-2 cephalosporinase with substrates and inhibitors. Mutagenesis of CMY-2 at position 152 yields functional mutants (N152G, -S, and -T) that exhibit improved penicillinase activity and retain cephamycinase activity. We also tested whether the position 152 substitutions would affect the inactivation kinetics of tazobactam, a class A β-lactamase inhibitor with in vitro activity against CMY-2. Using standard assays, we showed that the N152G, -S, and -T variants possessed increased catalytic activity against cefoxitin compared to the wild type. The 50% inhibitory concentration (IC50) for tazobactam improved dramatically, with an 18-fold reduction for the N152S mutant due to higher rates of enzyme inactivation. Modeling studies have shown active-site expansion due to interactions between Y150 and S152 in the apoenzyme and the Michaelis-Menten complex with tazobactam. Substitutions at N152 might become clinically important as new class C β-lactamase inhibitors are developed.

Introduction: The occurrence of multiple β-lactamases among bacteria only limits the therapeutic options but also poses a challenge. A study using boronic acid (BA), an AmpC enzyme inhibitor, was designed to detect the combined expression of AmpC β-lactamases and extended-spectrum β-lactamases (ESBLs) in bacterial isolates further different phenotypic methods are compared to detect ESBL and AmpC., Methods: A total of 259 clinical isolates of Enterobacteriaceae were isolated and screened for ESBL production by (i) CLSI double-disk diffusion method (ii) cefepime- clavulanic acid method (iii) boronic disk potentiation method. AmpC production was detected using cefoxitin alone and in combination with boronic acid and confirmation was done by three dimensional disk methods. Isolates were also subjected to detailed antibiotic susceptibility test., Results: Among 259 isolates, 20.46% were coproducers of ESBL and AmpC, 26.45% were ESBL and 5.40% were AmpC. All of the 53 AmpC and ESBL coproducers were accurately detected by boronic acid disk potentiation method., Conclusion: The BA disk test using Clinical and Laboratory Standards Institute methodology is simple and very efficient method that accurately detects the isolates that harbor both AmpCs and ESBLs.

Pure bacterial cultures were isolated from diseased snakeheads, Channa maculata (Lacepède), suffering high mortality in a farm in Zhongshan, southern China. Three isolates, namely ZS20100725, ZS20100725-1 and ZS20100725-2, were identified as Aeromonas schubertii. All the isolates showed high 16S rRNA sequence similarities with A. schubertii. The isolates exhibited strong virulence to snakeheads in experimental challenges with LD(50) ranging between 1.4 × 10(4) and 6.4 × 10(6) CFU g(-1). Two of the isolates were positive for haemolysin, elastase, lipase and lecithinase by phenotypic determination, which was further confirmed by PCR amplification of the haemolysin and elastase genes. In sterile liquid medium, the best growth conditions of strain ZS20100725 were 30 °C, pH 7 and 0.5% salinity (w/v). Antibiotic susceptibility tests showed that strain ZS20100725 was susceptible to cefoxitin, cefoperazone and chloramphenicol. Furthermore, histopathology of diseased snakeheads infected with A. schubertii showed necrosis and congestion in liver, kidney and spleen and also damage to the cardiac muscle, intestine and gills., (© 2012 Blackwell Publishing Ltd.)

Objectives: The characterization of Bacteroides strains with regard to the cfxA gene, the MTn4555 mobilizable transposon, the role of penicillin-binding proteins (PBPs) and heterogeneous cefoxitin resistance., Methods: Eighty-four randomly selected and 11 heterogeneously or highly cefoxitin-resistant Bacteroides isolates were included. Agar dilution and Etest methods were used for the determination of cefoxitin MICs. PCR experiments and nucleotide sequencing were used to detect the cfxA gene and the molecular features of MTn4555. Cefoxitin-binding experiments to determine its affinity (IC(50)) for PBPs and cefoxitinase assays were also applied. Southern blotting was used to determine the copy number of the cfxA genes., Results: Sixteen strains from the random collection proved to be positive for cfxA, and the MIC distribution for the cfxA-negative and -positive strains did not display a clear separation. The majority of the cfxA-positive strains in this collection harboured a 1.2 kb common region at the 3' end of MTn4555. This region encoded an open reading frame that exhibited homology to abortive phage infection proteins (AbiD). The cfxA genes were transferable only at low frequencies in conjugation experiments. In PBP affinity studies, the PBP-A and PBP3 species were largely insensitive to cefoxitin, whereas the other PBP species were affected at very low concentrations. Seven of the heterogeneously resistant strains were positive for cfxA and most of them had mutations in the regulatory regions of cfxA., Conclusions: Major and minor roles for Bacteroides fragilis PBPs and the CfxA cefoxitinase, respectively, were inferred. The role of the newly recognized abiD may be to control the copy number of cfxA.

Penicillin-binding proteins (PBPs) are the molecular targets for the widely used β-lactam class of antibiotics, but how these compounds act at the molecular level is not fully understood. We have determined crystal structures of Escherichia coli PBP 5 as covalent complexes with imipenem, cloxacillin, and cefoxitin. These antibiotics exhibit very different second-order rates of acylation for the enzyme. In all three structures, there is excellent electron density for the central portion of the β-lactam, but weak or absent density for the R1 or R2 side chains. Areas of contact between the antibiotics and PBP 5 do not correlate with the rates of acylation. The same is true for conformational changes, because although a shift of a loop leading to an electrostatic interaction between Arg248 and the β-lactam carboxylate, which occurs completely with cefoxitin and partially with imipenem and is absent with cloxacillin, is consistent with the different rates of acylation, mutagenesis of Arg248 decreased the level of cefoxitin acylation only 2-fold. Together, these data suggest that structures of postcovalent complexes of PBP 5 are unlikely to be useful vehicles for the design of new covalent inhibitors of PBPs. Finally, superimposition of the imipenem-acylated complex with PBP 5 in complex with a boronic acid peptidomimetic shows that the position corresponding to the hydrolytic water molecule is occluded by the ring nitrogen of the β-lactam. Because the ring nitrogen occupies a similar position in all three complexes, this supports the hypothesis that deacylation is blocked by the continued presence of the leaving group after opening of the β-lactam ring.

Objectives: A natural variant of the AmpC enzyme from Escherichia coli HKY28 with a tripeptide deletion (Gly-286/Ser-287/Asp-288) was recently described. The isolate produced an inhibitor-sensitive AmpC beta-lactamase variant that also conferred higher than usual levels of resistance to ceftazidime in the E. coli host. To demonstrate whether this is true in other class C beta-lactamase enzymes, we deleted the equivalent tripeptide in the FOX-4 plasmid-mediated class C beta-lactamase., Methods: By site-directed mutagenesis, we deleted the tripeptide Gly-306/Asn-307/Ser-308 of FOX-4, thus generating FOX-4(DeltaGNS). The enzymes (FOX-4 wild-type and DeltaGNS) were purified and kinetic parameters (kcat, Km, kcat/Km) as well as IC50 values of several beta-lactams were assessed. Modelling studies were also performed., Results: FOX-4(DeltaGNS) did not increase the catalytic efficiency towards ceftazidime, although it conferred a slight increase in the susceptibility to beta-lactamase inhibitors. There was also a noteworthy decrease in the cefoxitin MIC with the FOX-4(DeltaGNS) mutant (from 512 to 16 mg/L) as well as a 10-fold decrease in kcat/Km towards imipenem, which revealed specific structural features., Conclusions: Although deletions in the R2-loop are able to extend the substrate spectrum of class C enzymes, the present results do not confirm this hypothesis in FOX-4. The FOX-4 R2 site would already be wide enough to accommodate antibiotic molecules, and thus any amino acid replacement or deletion at this location would not affect the hydrolytic efficiency towards beta-lactams and would have a less drastic effect on the susceptibility to beta-lactamase inhibitors.

CTX-M enzymes are an emerging group of extended spectrum beta-lactamases (ESBLs) that hydrolyze not only the penicillins but also the first-, second-, and third-generation cephalosporins. Although they have become the most frequently observed ESBLs in certain areas, there are few effective inhibitors and relatively little is known about their detailed mechanism. Here we describe the X-ray crystal structures of CTX-M enzymes in complex with different transition-state analogues and beta-lactam inhibitors, representing the enzyme as it progresses from its acylation transition state to its acyl enzyme complex to the deacylation transition state. As the enzyme moves along this reaction coordinate, two key catalytic residues, Lys73 and Glu166, change conformations, tracking the state of the reaction. Unexpectedly, the acyl enzyme complex with the beta-lactam inhibitor cefoxitin still has the catalytic water bound; this water had been predicted to be displaced by the unusual 7alpha-methoxy of the inhibitor. Instead, the 7alpha-group appears to inhibit by preventing the formation of the deacylation transition state through steric hindrance. From an inhibitor design standpoint, we note that the best of the reversible inhibitors, a ceftazidime-like boronic acid compound, binds to CTX-M-16 with a K(i) value of 4 nM. When used together in cell culture, this inhibitor reversed cefotaxime resistance in CTX-M-producing bacteria. The structure of its complex with CTX-M enzyme and the structural view of the reaction coordinate described here provide templates for inhibitor design and intervention to combat this family of antibiotic resistance enzymes.

Penicillin-binding proteins (PBPs) catalyze the final stages of bacterial cell wall biosynthesis. PBPs form stable covalent complexes with beta-lactam antibiotics, leading to PBP inactivation and ultimately cell death. To understand more clearly how PBPs recognize beta-lactam antibiotics, it is important to know their energies of interaction. Because beta-lactam antibiotics bind covalently to PBPs, these energies are difficult to measure through binding equilibria. However, the noncovalent interaction energies between beta-lactam antibiotics and a PBP can be determined through reversible denaturation of enzyme-antibiotic complexes. Escherichia coli PBP 5, a D-alanine carboxypeptidase, was reversibly denatured by temperature in an apparently two-state manner with a temperature of melting (T(m)) of 48.5 degrees C and a van't Hoff enthalpy of unfolding (H(VH)) of 193 kcal/mole. The binding of the beta-lactam antibiotics cefoxitin, cloxacillin, moxalactam, and imipenem all stabilized the enzyme significantly, with T(m) values as high as +4.6 degrees C (a noncovalent interaction energy of +2.7 kcal/mole). Interestingly, the noncovalent interaction energies of these ligands did not correlate with their second-order acylation rate constants (k(2)/K'). These rate constants indicate the potency of a covalent inhibitor, but they appear to have little to do with interactions within covalent complexes, which is the state of the enzyme often used for structure-based inhibitor design.

An Escherichia coli strain resistant to a broad spectrum of beta-lactams, including cephamycins, was isolated from a patient suffering from urinary tract infection. A resistance plasmid (pMVP-7) was transferred from the clinical isolate to an Escherichia coli recipient. Both strains produce a cefoxitin-hydrolyzing beta-lactamase focusing at pI 6.7. The phenotype was similar to that of a Klebsiella pneumoniae strain producing cephamycinase FOX-1, so primers were selected from the FOX-1 sequence to amplify the bla gene of the transconjugant. The PCR product obtained was sequenced. The percentage of identity of the deduced amino acid sequence with sequences of other AmpC-type beta-lactamases was 96.9% with FOX-1, 74.9% with CMY-1, and 67.7% with MOX-1. This new plasmid-mediated enzyme is most closely related to FOX-1 (11 amino acid exchanges). We therefore propose the designation FOX-2.

The catalytic pathway of class A beta-lactamases involves an acyl-enzyme intermediate where the substrate is ester-linked to the Ser-70 residue. Glu-166 and Lys-73 have been proposed as candidates for the role of general base in the activation of the serine OH group. The replacement of Glu-166 by an asparagine in the TEM-1 and by a histidine in the Streptomyces albus G beta-lactamases yielded enzymes forming stable acyl-enzymes with beta-lactam antibiotics. Although acylation of the modified proteins by benzylpenicillin remained relatively fast, it was significantly impaired when compared to that observed with the wild-type enzyme. Moreover, the E166N substitution resulted in a spectacular modification of the substrate profile much larger than that described for other mutations of Omega-loop residues. Molecular modeling studies indicate that the displacement of the catalytic water molecule can be related to this observation. These results confirm the crucial roles of Glu-166 and of the "catalytic" water molecule in both the acylation and the deacylation processes.

Penicillin-binding protein (PBP) 2 is the major PBP of five that have been identified in susceptible strains of Staphylococcus aureus. Beta-lactam antibiotic binding to PBP 2 is important for the antibacterial effect. Antibiotic binding to PBP 2 in strain 209P was examined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in competition assays using [3H]penicillin as the radiolabel. Clavulanic acid, which is specifically bound by PBP 2, and cefaclor, which is specific for PBP 3, were studied. Cefaclor, which alone appeared not to bind PBP 2, in combination inhibited PBP 2 binding of clavulanic acid. By varying the temperature during radiolabeling with [3H]penicillin in cefaclor competition assays and in direct radiolabeling assays with [3H]cefaclor, it was shown that cefaclor was bound by PBP 2 with high affinity (50% inhibitory concentration, less than or equal to 0.1 microgram/ml) and that the apparent low-affinity binding (50% inhibitory concentration, greater than 10 micrograms/ml) in competition assays performed at 37 degrees C was due to rapid deacylation. Two penicillin-binding peptides of PBP 2 also were identified in fluorographs of PBPs separated by nonequilibrium pH gradient gel and two-dimensional electrophoresis. Rapid deacylation for some antibiotics and the presence of two penicillin-binding peptides are two properties of PBP 2 that should be considered when correlating results of binding assays with effects of beta-lactam antibiotics on S. aureus.

The susceptibility of the new cephamycin antibiotic, cefminox, to hydrolysis by various types of beta-lactamase produced by organisms of the Bacteroides fragilis group was compared with that of cefoxitin. Several enzymes were able to achieve complete hydrolysis of both drugs during overnight incubation, but no marked difference between the rates of inactivation of cefminox and cefoxitin was observed. Susceptibility of the cephamycins to crude extracts of beta-lactamases from the test strains of Bacteroides spp. did not correlate with the results of conventional minimum inhibitory concentration titrations. Possible reasons for these discrepancies are discussed.

Protein binding, serum kinetics and minimum inhibitory concentrations (MICs) for Staphylococcus aureus were determined for cefoxitin, cefazolin, ceftazidime and ceftriaxone in the rabbit. MICs of cefazolin and cefoxitin were also measured for Escherichia coli. Varying concentrations of the bacteria were administered intradermally to create areas of cellulitis, which were quantified as mean erythematous areas (EAs). Despite large differences in protein binding of the antibiotics (range 12-88%) and antibiotic dosing to allow serum concentrations to drop below the respective MICs, there was no statistical difference in the mean EAs of the animals after bacterial challenge. Antibiotic protein binding did not alter the course of cellulitis nor correlate with bacterial MIC in this model.

The ability of phenothiazines to enhance the rectal absorption of sodium cefoxitin and gentamicin sulphate from aqueous formulations was examined in rats. In the absence of absorption-promoting adjuvants, sodium cefoxitin and gentamicin sulphate bioavailabilities from the rectal compartment were less than 5% of the corresponding intravenous administration. In aqueous microenemas containing 20 mg ml-1 phenothiazine, sodium cefoxitin bioavailability increased to 16-62%, while gentamicin sulphate bioavailability increased to 74-146%. The absorption-promoting potential of chlorpromazine and perphenazine was concentration-dependent, with significant increases in gentamicin sulphate absorption occurring with 1 mg ml-1 chlorpromazine or 2.5 mg ml-1 perphenazine. Maximal gentamicin sulphate bioavailability and serum concentrations were achieved with 10 mg ml-1 chlorpromazine or 20 mg ml-1 perphenazine. The findings indicate that the phenothiazines, which are well absorbed rectally, also significantly enhance the rectal absorption of water-soluble, poorly absorbed compounds.

The pharmacokinetics of cefoxitin were studied in 6 healthy volunteers and in 5 patients with pleural effusion of varied etiologies. The antibiotic was administered in a multiple dosage regimen of 30 mg/kg every 8 hours. Cefoxitin concentrations in serum and pleural fluid were determined after administration of the third dose corresponding to the levels of steady-state in both fluids. The elimination half-life of the antibiotic from the pleural fluid comes to 3.51 +/- 1.15 h, which is significantly greater than the serum half-life, (t 1/2 = 1.42 +/- 0.51 h). This kind of multiple dosage regimen ensures therapeutic levels of cefoxitin in the pleural fluid during the entire treatment.

We have surveyed the susceptibility of 1,575 clinical isolates of the Bacteroides fragilis group of organisms to cefoxitin and eight other antimicrobial agents. Eleven isolates, 0.7% of the total, were highly cefoxitin resistant and had minimum inhibitory concentrations of greater than or equal to 64 micrograms/ml. These isolates were also resistant to other beta-lactam antibiotics. Of 11 isolates, 4 were able to inactivate cefoxitin in broth cultures, as measured by microbiological and high-pressure liquid chromatography assays. Two distinct patterns of cefoxitin breakdown products were detected by high-pressure liquid chromatography analysis. The beta-lactamase inhibitors clavulanic acid and sulbactam failed to show synergism with cefoxitin. These data demonstrate that members of the B. fragilis group have acquired a novel resistance mechanism enabling them to inactivate cefoxitin.

In our segmental pancreatic transplantation technique the pancreatic juice is temporarily diverted to the exterior via a pancreatic duct catheter. This permits studies on pure pancreatic juice to be carried out. In 11 such patients we studied the penetration of clindamycin, cefoxitin, and piperacillin into pancreatic juice. These three antibiotics all have good effect against the bacteria commonly isolated during pancreatic infections. Simultaneous blood and pancreatic juice samples were collected immediately before drug administration and at 30, 60, 90, and 120 minutes and 3, 4, 5, 6, and 8 hours after administration. The concentration of clindamycin in pancreatic juice was 34% of that in serum and exceeded the minimum inhibitory concentration for most bacteria associated with pancreatic infections. In spite of adequate serum concentrations of cefoxitin and piperacillin, the concentrations in pancreatic juice were only 8% and 5%, respectively, and did not exceed the minimum inhibitory concentration for the relevant bacteria. In view of these findings, clindamycin seems to be preferable in the treatment of pancreatic infections.

Cefoxitin was administered in a dose of 2 g intravenously to 17 patients scheduled for cholecystectomy. The concentrations of this agent in serum and gallbladder bile were measured simultaneously upon opening of the abdominal cavity. Concentrations of cefoxitin in excess of 10 microgram/ml were present in the gallbladder bile of 12 of the 13 patients from samples obtained between 35 and 165 min after cefoxitin infusion. Cystic or common duct obstruction did not preclude entry of the drug into gallbladder bile.

The pharmacokinetics of cefoxitin were studied in nine patients with pleural effusion of varied etiologies. All patients received a single intravenous bolus of 30 mg/kg. Cefoxitin levels were determined simultaneously in plasma and pleural fluid by means of a microbiologic plate diffusion method. The antibiotic follows a two-compartment open kinetic model. In the pleural fluid, maximum concentrations of cefoxitin of 19.72 +/- 9.72 microgram/ml were reached two hours after administration. The fraction of the antibiotic that reaches the pleural fluid represents 0.22% to 4.03% of the dose administered. The disappearance constant of the antibiotic from the pleural fluid is significantly smaller (Kd = 0.15 +/- 0.03 hours-1) than the elimination constant determined from the plasma levels (K13 = 2.27 +/- 0.90 hours-1). Cefoxitin was always found in antibacterial concentration in the pleural fluid for a considerable period of time.

The penetration of four cephalosporins into tissue fluid obtained with a skinabrasion technique was studied. Two oral antibiotics, cephalexin and CGP 9000, had similar pharmacology and penetrated the fluid in equivalent amounts. Cefuroxime (33% protein-bound) and cefoxitin (65-79% proteinbound) attained levels in tissue fluid which were very similar to their blood levels. The level of antibiotic in the tissues seems to be closely related to the blood level and is not adversely affected by protein binding.

To prevent infection in 40 patients who underwent vaginal or abdominal hysterectomy, each patient received a single 15-mg/kg dose of either cefoxitin or cefamandole by rapid (5-minute) intravenous injection before the operation. Samples of serum, myometrium, endometrium, ovaries, and tubes were obtained at various intervals after injection of the antibiotic and were assayed for cephalosporin concentration. Maximum tissue concentrations of 30 micrograms/g of both drugs were detected approximately 30 minutes after the dose, with levels dropping below 3 micrograms/g in approximately 2 hours. Although both antibiotics achieved closely similar concentrations in serum and tissues, the ratios of these levels to their usual mean minimum inhibitory concentrations for Bacteroides fragilis were appreciably higher for cefoxitin than for cefamandole. These pharmacokinetic observations support the current dosage recommendations for the use of cefoxitin in treating and preventing gynecologic infections, as well as the recommendation that it be administered shortly before the operation to maximize tissue levels during the perioperative period.

The ability of ceftizoxime to penetrate the outer membrane was compared with those of five other new cephalosporins: cefotiam, cefuroxime, cefotaxime, cefmetazole and cefoxitin, using a clinical isolate of Enterobacter cloacae as a test strain. Estimation of permeability was performed by a method utilizing the inhibitory activities of the cephalosporins against beta-lactamase located in the periplasm. Of the cephalosporins tested, both ceftizoxime and cefmetazole gave remarkably high concentrations in the periplasm, several times higher than those of cefotaxime and cefoxitin and ten or more times higher than those of cefuroxime and cefotiam. The approximate permeability coefficient of ceftizoxime was also several times higher than those of cefotiam and cefmetazole and over ten times higher than those of cefoxitin, cefuroxime and cefotaxime.

Cefoxitin is a useful new chephamycin antibiotic available for IM of IV administration. It is a bactericidal beta-lactam antibiotic indicated for treatment of serious infections caused by a wide spectrum of gram-negative aerobic and anaerobic bacteria. Specific indications include: 1) polymicrobial infections (aerobic gram-negative rods and anaerobic bacteria); 2) nosocomial cephalothin-resistant gram-negative bacillary infections; 3) penicillin-resistant staphylococcal infections, and 4) anaerobic infections (pelvic, intraabdominal). Cefoxitin can be used as a single drug alternate to a two-drug antibiotic regimen (such as a cephalosporin-aminoglycoside combination) in polymicrobic infections of the pelvis, abdomen, skin, bones, or muscles. Cefoxitin is a significant drug in the armamentarium against anaerobic bacteria, particularly Bacteroides species. Since cefoxitin is highly resistant to staphylococcal beta-lactamase, it is effective against penicillin-resistant staphylococci. Cefoxitin has been effective in treating serious nosocomial infections caused by resistant aerobic gram-negative rods and anaerobic bacteria.

Fifteen patients less than 2 months old with bacterial infections caused by pathogens known or presumed to be sensitive to cefoxitin were studied. Cefoxitin was administered as an i.v. bolus injection over 15 min, every 8 h for 6 to 12 days, to a total daily dosage of 90 mg/kg. In 14 patients cefoxitin therapy resulted in eradication of the pathogen and in recovery from clinical signs of infection. Only one patient did not respond to cefoxitin therapy. No adverse clinical or haematological effects definitely caused by cefoxitin were observed. Plasma and urine samples collected after the first dose were assayed for cefoxitin by HPLC. Pharmacokinetic data indicated larger apparent volume of distribution (0.5 1/kg), a smaller plasma clearance (0.27 1/h/kg) and a longer half-life (1.43 h) than in adults. The plasma half-life was inversely correlated (p less than 0.05) to the postnatal age of the patients. Cefoxitin may be safely used in infants with infections caused by susceptible pathogens.

Cefoxitin, a poor substrate of the RTEM beta-lactamase (penicillin amido-beta-lactam hydrolase, EC 3.5.2.6), induces a reversible change in the conformation of the enzyme. The change is manifested in gradual loss of catalytic activity and increased susceptibility to proteolytic inactivation. It is prevented by antibodies, which stabilize the native conformation. By contrast, divalent cations, which have no effect on the native enzyme, delay recovery from the cefoxitin-induced state, presumably by reacting with sites made accessible in the partly unfolded enzyme. Prolonged exposure to excess of cefoxitin causes a similar delay. The kinetic evidence, namely, the initial burst of consumption of cefoxitin and the subsequent gradual recovery of activity with better substrates, appears to be consistent with acylation of the active site by cefoxitin followed by a slower deacylation step [Fisher et al. (1980) Biochemistry 19, 2895-2901]. However, additional evidence leads us to conclude that the kinetics observed reflect deformation of the active site, rather than its blockage, by cefoxitin. Of most significance is the transient change in specificity, i. e. a preferential interaction of the recovering enzyme with substrates which are closest in structure to cefoxitin.

The pharmacokinetics of cefoxitin was studied in patients with renal impairment during haemofiltration and in the intervening periods after administration of 30 and 15 mg/kg of the drug, respectively. Different pharmacokinetic patterns were established during haemofiltration and in the interim period, with average elimination half-lives of 11.85 +/- 4.3 and 3.41 +/- 0.6 h, respectively. The average fraction of the cefoxitin dose eliminated in haemofiltration was 0.62 +/- 0.11, more than that established in haemodialysis. In patients with terminal renal impairment undergoing haemofiltration every 48 h, a dose of 15 or 30 mg/kg is recommended at the start and at the end of each haemofiltration session.

Control measurements of blood flow through and cefoxitin extraction ratios across heart and lungs, liver, kidneys and gut were made under steady-state conditions in awake unrestrained sheep. The studies then were repeated with the same animals anaesthetized with 1.5% halothane or whilst undergoing high thoracic (to approximately T4) subarachnoid blockade with amethocaine. In the control-drug studies, it was shown that cefoxitin was cleared by the kidneys and that the cefoxitin infusion produced no significant changes in haemodynamics or in the kinetics of iodohippurate (the marker substance used for determining renal blood flow). Under general anaesthesia, there were significant reductions in renal blood flow (to 61% of the mean control value), in iodohippurate renal extraction ratio and clearance (to, respectively, 64 and 38% of the mean control values) and in cefoxitin renal extraction ratio and clearance (to, respectively, 48 and 23% of the mean control values). Under spinal anaesthesia there were no significant changes in haemodynamics or in cefoxitin or iodohippurate extraction ratios or clearances.

An experimental model, where fibrin clots were inserted subcutaneously in rabbits, was adapted to study the in vivo efficacy of two cephalosporins against Bacteroides fragilis. The respective MIC's of cefamandole and cefoxitin against the microorganism were 16 microgram/ml and 1 microgram/ml. The clots were infected with 10(7) B. fragilis. Groups of seven animals received an intravenous bolus injection (100 mg/kg) of either drug. The serum levels of both drugs were similar to those seen in humans. The peak concentrations of cefamandole (40 microgram/mg) in the clots were found to be ten times higher than those of cefoxitin (4 microgram/mg). The log number of colony forming units in the clots averaged 7.5 at 0 h. At 6 hours, this number reached 8 in the untreated animals, 1.5 after cefoxitin, and 1.7 after cefamandole. The apparent in vitro superiority of cefoxitin against B. fragilis could not be demonstrated in vivo. This discrepancy between in vitro and in vivo data can be explained by the high degree of penetrance of cefamandole into the infected fibrin loci. In this animal system, both cefoxitin and cefamandole had similar in vivo activity against B. fragilis.

The in vitro activity of cefoxitin, 3-carbamolyloxymethyl-7-alpha-methoxy-7[2-(2-thienyl)acetamido]-3-cephem-4-carboyxlic acid, was investigated. Activity against gram-positive organisms was less than that of cephalothin and cephloridine. It was highly active against gram-negative bacilli, with activity against Escherichia coli, Proteus mirabilis, and Klebsiella pneumoniae equal to that of currently available cephalosporins. In addition, it was active against certain Enterobacter strains, Serratia marcescens, indole-positive Proteae and Herellea. The strains of these latter bacteria were strains susceptible to carbenicillin and ticarcillin. Pseudomonas aeruginosa and other Pseudomonas species were resistant. Changes in pH, inoculum size, and type of growth medium had no significant effect on the activity of the antibiotic. Cefoxitin was highly resistant to hydrolysis by various types of gram-negative beta-lactamases. The precise role of resistance to beta-lactamase hydrolysis varied from strain to strain. Bacterial resistance to cefoxitin was not necessarily related to hydrolysis of the antibiotic. However, the resistance of cefoxitin to hydrolysis did contribute to its activity. Cefoxitin could function as an inducer of beta-lactamase activity and effectively bound to purified beta-lactamases.

To characterize the effect of orally administered probenecid on the pharmacokinetics of cefoxitin in healthy male volunteers, we administered to one group of six subjects 2 g of cefoxitin by intravenous (i.v.) bolus either alone, with 1 g of probenecid concomitantly, or when 1 g of probenecid was administered 1 h previously by using a crossover design. Likewise, we administered to a second group of six subjects 2 g of cefoxitin intramuscularly (i.m.) together with 1 and 2 g of probenecid. Probenecid increased the mean terminal half-life and the area under the serum cefoxitin concentration-time curve (AUC0-24) and decreased renal clearance, but did not alter the volume of the central compartment or the total urinary recovery of i.v.-administered cefoxitin; pretreatment with probenecid produced a greater increase in cefoxitin AUC0-24 and a constant decrease in renal clearance compared to concomitant probenecid. The AUC0-24 after i.m.-administered cefoxitin was greater after 2 g than 1 g of probenecid; the AUC0-24 after i.v.-and i.m.-administered cefoxitin was similar after 1 g of probenecid was given concomitantly. Cefoxitin AUC0-24 was increased further when 1 g of probenecid was given before i.v.-administered cefoxitin or when 2 g of probenecid was given with i.m.-administered cefoxitin. The effect of probenecid was related to both timing and dose.

Intravenous doses of 0.5, 1, and 2 g cephalothin and cefoxitin, a semi-synthetic cephamycin antibiotic highly resistant to bacterial cephalosporinase, were infused over a period of 3 minutes into 18 normal adult males by a randomized, crossover design. Serum and urine data on cefoxitin best fit a two-compartment open model. Serum concentrations following cefoxitin were higher and more prolonged and urine recoveries higher than those following equal doses of cephalothin. The terminal serum half-life of cefoxitin was longer at all dose levels. Renal clearance of cephalothin-like activity exceeded that of cefoxitin, which may possess dose-dependent kinetics. Whereas cephalothin has been reported to metabolize by greater than 35% to the less active desacetyl form, cefoxitin was metabolized by 0.1 to 6% to the descarbamyl form in individual subjects.

We determined the intraoperative serum and wound-muscle concentrations of cefazolin and cefoxitin in 40 patients who were undergoing cholecystectomies. The study employed an open-label design in which all of the patients randomly received cefazolin sodium (20 mg/kg) or cefoxitin sodium (30 mg/kg) intravenously while the patient was in the ward ("on call") or with the induction of anesthesia. Multiple blood and wound-muscle samples were collected intraoperatively and assayed for their cephalosporin concentrations. Considerable differences in intraoperative serum and tissue concentrations between antibiotics were apparent; there were usually higher levels of cefazolin. In all of the patients who received cefazolin sodium, the antimicrobial was detectable in wound tissue at wound closure, while it was detectable in 86% and 38% of patients who received cefoxitin sodium with anesthesia and on call, respectively. Because cefoxitin has a much shorter elimination half-life than cefazolin it seems prudent to administer the agent as close to the start of the operation as possible, and readminister the agent every two to three hours until the wound is closed. For cefazolin, on-call administration appears to be acceptable, with readministration not required for at least four hours.

Thirty-six patients received an intravenous bolus injection of 2 g cefoxitin over 5 min at various times before abdominal or vaginal hysterectomy. Cefoxitin levels in myometrium and salpinges were substantially higher than those in endometrium and almost as high as those in serum during the first two hours following the i. v. administration of the drug. Tissue concentrations of 8 micrograms/g could be maintained for two hours. The cefoxitin tissue concentrations attained in myometrium, endometrium and salpinges justify controlled prospective clinical studies on the prevention of postoperative wound infections with this antibiotic.

The pharmacokinetics of cefoxitin was studied in 6 healthy volunteers and in 5 patients with a pleural effusion after administration of a single dose of 30 mg/kg i.v. infusion. The serum and pleural fluid concentrations of cefoxitin were determined microbiologically. The elimination half-life of the antibiotic from pleural fluid in all cases was 2-3 fold longer than from serum, which shows a difference between the kinetic elimination processes of the antibiotic from the two fluids. The slow elimination of cefoxitin from pleural fluid facilitates its accumulation in this compartment during a multiple dosage regimen.