Search

Methicillin-resistant Staphylococcus aureus (MRSA) is among the leading causes of nosocomial infections and forms biofilms, which are difficult to eradicate because of their increasing resistance to antimicrobial agents. This is especially true for pre-existing biofilms. The current study focused on evaluating the efficacy of three β-lactam drugs, meropenem, piperacillin, and tazobactam, alone and in combination against the MRSA biofilms. When used individually, none of the drugs exhibited significant antibacterial activity against MRSA in a planktonic state. At the same time, the combination of meropenem, piperacillin, and tazobactam showed a 41.7 and 41.3% reduction in planktonic bacterial cell growth, respectively. These drugs were further assessed for biofilm inhibition and removal. The combination of meropenem, piperacillin, and tazobactam caused 44.3% biofilm inhibition, while the rest of the combinations did not show any significant effects. Results also revealed that piperacillin and tazobactam exhibited the best synergy against the pre-formed biofilm of MRSA, with 46% removal. However, adding meropenem to the piperacillin and tazobactam combination showed a slightly reduced activity towards the pre-formed biofilm of MRSA and removed 38.7% of it. Although the mechanism of synergism is not fully understood, our findings suggest that these three β-lactam drugs can be used in combination as very effective therapeutic agents for the treatment of pre-existing MRSA biofilms. The in vivo experiments on the antibiofilm activity of these drugs will pave the way for applying such synergistic combinations to clinics.

The present study evaluated the antibacterial activity and the synergy of the sanguisorbigenin (SGB) from the dried root of Sanguisorba officinalis L. combined with β‐lactam antibiotics against methicillin‐resistant Staphylococcus aureus. A total of six strains of reference strain and clinical isolates were used to determine the antibacterial activity using a broth microdilution assay, and the synergistic effects were determined using a checkerboard assay. To analyse the mechanism of synergy, we conducted the level of penicillin‐binding protein 2a by western blot. In addition, quantitative RT‐PCR was performed to analyse the mecA gene expression. The minimal inhibitory concentration values of SGB against six strains of S. aureus were in the range of 12·5–50 μg ml−1, and there were synergy, or partial synergy effects when SGB was combined with antibiotics. Furthermore, when treated with SGB, the level of penicillin‐binding protein 2a and the expression of the mecA gene was reduced significantly. In conclusion, this study demonstrated that SGB is a potential natural antibacterial agent against methicillin‐resistant S. aureus that represents a considerable burden on the healthcare system worldwide, and may an exceptionally modulator of β‐lactam antibiotics., Significance and Impact of the Study: Methicillin‐resistant Staphylococcus aureus is the pathogen represents a considerable burden on the healthcare system worldwide, and resistance to nearly all β‐lactam antibiotics. For the first time, we evaluated the antibacterial activity and the synergy of the sanguisorbigenin (SGB) combined with β‐lactam antibiotics against methicillin‐resistant S. aureus. The result indicated that SGB has a potent antibacterial effect and could effectively reverse the sense of β‐lactam antibiotics by inhibiting mecA expression and decrease the PBP2a expression. This study has a certain reference value for the development of novel antibacterial agents and antibiotic modulators.

The interaction between tumor surface-expressed PDL1 and immune cell PD1 for the evasion of antitumor immunity is well established and is targeted by FDA-approved anti-PDL1 and anti-PD1 antibodies. Nonetheless, recent studies highlight the immunopathogenicity of tumor-intrinsic PDL1 signals that can contribute to the resistance to targeted small molecules, cytotoxic chemotherapy, and αPD1 immunotherapy. As genetic PDL1 depletion is not currently clinically tractable, we screened FDA-approved drugs to identify those that significantly deplete tumor PDL1. Among the candidates, we identified the β-lactam cephalosporin antibiotic cefepime as a tumor PDL1-depleting drug (PDD) that increases tumor DNA damage and sensitivity to DNA-damaging agents in vitro in distinct aggressive mouse and human cancer lines, including glioblastoma multiforme, ovarian cancer, bladder cancer, and melanoma. Cefepime reduced tumor PDL1 post-translationally through ubiquitination, improved DNA-damaging-agent treatment efficacy in vivo in immune-deficient and -proficient mice, activated immunogenic tumor STING signals, and phenocopied specific genetic PDL1 depletion effects. The β-lactam ring and its antibiotic properties did not appear contributory to PDL1 depletion or to these treatment effects, and the related cephalosporin ceftazidime produced similar effects. Our findings highlight the rapidly translated potential for PDDs to inhibit tumor-intrinsic PDL1 signals and improve DNA-damaging agents and immunotherapy efficacy.

Organic anion transporters OAT1 and OAT3 play a key role in elimination of most β-lactam antibiotics. Since nonsteroidal anti-inflammatory drugs, antivirals, antitumor agents, and some other drugs are also substrates of OAT1/3, this enables drug-drug interaction (DDI). The aim of the study was to analyze scientific literature to determine the likelihood and significance of β-lactam antibiotic DDI mediated by organic anion transporters, as well as potential for predicting it. In clinical practice, inhibition of β-lactam antibiotic elimination is used to increase systemic exposition and reduce the cost of antibiotic therapy. OAT inhibitors (cilastatin, betamipron) are used in combination drugs to reduce nephrotoxicity of carbapenems. On the other hand, an increase in the concentration of β-lactams due to OAT inhibition may lead to adverse drug reactions. Therefore, the European Medicines Agency and the Food and Drug Administration recommendations for the development of new drugs state that in the case of significant renal excretion (≥25%) it is necessary to investigate OAT1/3 transportin vitroand calculate inhibition constantKiand/or half maximal inhibitory concentrationIC50for predicting DDI. One of the main problems is the variability ofKi andIC50 values between laboratories, which requires the development of general recommendations for different transporters as regards methods of determination of these parameters.

The increasing emergence of bacteria producing β-lactamases enzymes (BLEs), able to inactivate the available β-lactam antibiotics (BLAs), causing the hydrolytic opening of their β-lactam ring, is one of the global major warnings. According to Ambler classification, BLEs are grouped in serine-BLEs (SBLEs) of class A, C, and D, and metal-BLEs (MBLEs) of class B. A current strategy to restore no longer functioning BLAs consists of associating them to β-lactamase enzymes inhibitors (BLEsIs), which, interacting with BLEs, prevent them hydrolyzing to the associated antibiotic. Worryingly, the inhibitors that are clinically approved are very few and inhibit only most of class A and C SBLEs, leaving several class D and all MBLEs of class B untouched. Numerous non-clinically approved new molecules are in development, which have shown broad and ultra-broad spectrum of action, some of them also being active on the New Delhi metal-β-lactamase-1 (NDM-1), which can hydrolyze all available BLAs except for aztreonam. To not duplicate the existing review concerning this topic, we have herein examined BLEsIs by a chemistry approach. To this end, we have reviewed both the long-established synthesis adopted to prepare the old BLEsIs, those proposed to achieve the BLEsIs that are newly approved, and those recently reported to prepare the most relevant molecules yet in development, which have shown high potency, providing for each synthesis the related reaction scheme.

β-Lactam antibiotics are most commonly used in the critically ill, but their effective dosing is challenging and may result in sub-therapeutic concentrations that can lead to therapy failure and even promote antimicrobial resistance. In this study, we present the analytical tool enabling specific and sensitive determination of the sole biologically active fraction of piperacillin and imipenem in biological material from the critically ill. Thin-film microextraction sampling technique, followed by rapid liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis, was optimized and validated for the quantitative determination of antibiotics in blood and bronchoalveolar lavage (BAL) specimens collected from intensive care unit (ICU) patients suffering from ventilation-associated pneumonia (n = 18 and n = 9, respectively). The method was optimized and proved to meet the criteria of US Food and Drug Administration (FDA) guidelines for bioanalytical method validation. Highly selective, sensitive, accurate and precise analysis by means of thin-film microextraction–LC-MS/MS, which is not affected by matrix-related factors, was successfully applied in clinical settings, revealing poor penetration of piperacillin and imipenem from blood into BAL fluid (reflecting the site of bacterial infection), nonlinearity in antibiotic binding to plasma-proteins and drug-specific dependence on creatinine clearance. This work demonstrates that only a small fraction of biologically active antibiotics reach the site of infection, providing clinicians with a high-throughput analytical tool for future studies on personalized therapeutic drug monitoring when tailoring the dosing strategy to an individual patient.

This research aimed to investigate the presence and transferability of the extended-spectrum β-lactamase resistance genes to identify the genetic context of multi-drug resistant (MDR) loci in two Escherichia coli plasmids from livestock and poultry breeding environment. MICs were determined by broth microdilution. A total of 137 E. coli resistant to extended-spectrum β-lactam antibiotics were screened for the presence of the ESBL genes by PCR. Only two E. coli out of 206 strains produced carbapenemases, including strain 11011 that produced enzyme A, and strain 417957 that produced enzyme B. The genes were blaKPC and blaNDM, respectively. The plasmids containing blaCTX–M were conjugatable, and the plasmids containing carbapenem resistance gene were not conjugatable. Six extended-spectrum β-lactamase resistance genes were detected in this research, including blaTEM, blaCTX–M, blaSHV, blaOAX–1, blaKPC, and blaNDM, and the detection rates were 94.89% (130/137), 92.7% (127/137), 24.81% (34/137), 20.43% (28/137), 0.72% (1/137), and 0.72% (1/137), respectively. Two conjugative lncFII multi-resistance plasmids carrying blaCTX–M, p11011-fosA and p417957-CTXM, were sequenced and analyzed. Both conjugative plasmids were larger than 100 kb and contained three accessory modules, including MDR region. The MDR region of the two plasmids contained many antibiotic resistance genes, including blaCTX–M, mph (A), dfrA17, aadA5, sul1, etc. After transfer, both the transconjugants displayed elevated MICs of the respective antimicrobial agents. A large number of resistance genes clusters in specific regions may contribute to the MDR profile of the strains. The presence of mobile genetic elements at the boundaries can possibly facilitate transfer among Enterobacteriaceae through inter-replicon gene transfer. Our study provides beta-lactam resistance profile of bacteria, reveals the prevalence of β-lactamase resistance genes in livestock and poultry breeding environment in Zhejiang Province, and enriches the research on IncFII plasmids containing blaCTX–M.

Staphylococcus aureus is a leading cause of bacterial infections world-wide. Staphylococcal infections are preferentially treated with β-lactam antibiotics, however, methicillin-resistant S. aureus (MRSA) strains have acquired resistance to this superior class of antibiotics. We have developed a growth-based, high-throughput screening approach that directly identifies cell wall synthesis inhibitors capable of reversing β-lactam resistance in MRSA. The screen is based on the finding that S. aureus mutants lacking the ClpX chaperone grow very poorly at 30°C unless specific steps in teichoic acid synthesis or penicillin binding protein (PBP) activity are inhibited. This property allowed us to exploit the S. aureus clpX mutant as a unique screening tool to rapidly identify biologically active compounds that target cell wall synthesis. We tested a library of ∼50,000 small chemical compounds and searched for compounds that inhibited growth of the wild type while stimulating growth of the clpX mutant. Fifty-eight compounds met these screening criteria, and preliminary tests of 10 compounds identified seven compounds that reverse β-lactam resistance of MRSA as expected for inhibitors of teichoic acid synthesis. The hit compounds are therefore promising candidates for further development as novel combination agents to restore β-lactam efficacy against MRSA.

Pyrazolo[3,4-d]pyrimidines represent an important class of heterocyclic compounds well-known for their anticancer activity exerted by the inhibition of eukaryotic protein kinases. Recently, pyrazolo[3,4-d]pyrimidines have become increasingly attractive for their potential antimicrobial properties. Here, we explored the activity of a library of in-house pyrazolo[3,4-d]pyrimidines, targeting human protein kinases, against Staphylococcus aureus and Escherichia coli and their interaction with ampicillin and kanamycin, representing important classes of clinically used antibiotics. Our results represent a first step towards the potential application of dual active pyrazolo[3,4-d]pyrimidine kinase inhibitors in the prevention and treatment of bacterial infections in cancer patients.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has posed a serious threat to public health, therefore, the development of new antibacterial drugs is imperative. Bisdemethoxycurcumin (BDMC) is a curcumin analog that exists in nature and possesses extensive pharmacological actions. This review focuses on investigating the antibacterial activity of BDMC alone or in combination with three antibiotics against MRSA. We determined the minimal inhibitory concentration of BDMC, with a broth microdilution assay, and the value against all six strains was 7.8 μg/mL. The synergistic effect of BDMC combined with the antibiotics was determined using a checkerboard dilution test and a time–kill curve assay. The results showed that the antimicrobial effect of BDMC combined with antibiotics was superior to treatment with that of a single agent alone. We examined the antibacterial activity of BDMC in the presence of a membrane-permeabilizing agent and an ATPase-inhibiting agent, respectively. In addition, we analyzed the mecA transcription gene and the penicillin-binding protein 2a (PBP2a) level of MRSA treated with BDMC by quantitative RT-PCR or Western blot assay. The gene transcription and the protein level were significantly inhibited. This study demonstrated that BDMC has potent antibacterial activity, and proved that BDMC may be a potential natural modulator of antibiotics.

Enterobacter cloacae complex (ECC), one of the most common opportunistic pathogens causing multiple infections in human, is resistant to β-lactam antibiotics mainly due to its highly expressed chromosomal AmpC β-lactamase. It seems that regulation of chromosomal AmpC β-lactamase is associated with peptidoglycan recycling. However, underlying mechanisms are still poorly understood. In this study, we confirmed that NagZ, a glycoside hydrolase participating in peptidoglycan recycling in Gram-negative bacteria, plays a crucial role in developing resistance of E. cloacae (EC) to β-lactam antibiotics by promoting expression of chromosomal AmpC β-lactamase. Our data shows that NagZ was significantly up-regulated in resistant EC (resistant to at least one type of the third or fourth generation cephalosporins) compared to susceptible EC (susceptible to all types of the third and fourth generation cephalosporins). Similarly, the expression and β-lactamase activity of ampC were markedly enhanced in resistant EC. Moreover, ectopic expression of nagZ enhanced ampC expression and resistance to β-lactam antibiotics in susceptible EC. To further understand functions of NagZ in β-lactam resistance, nagZ-knockout EC model (ΔnagZ EC) was constructed by homologous recombination. Conversely, ampC mRNA and protein levels were down-regulated, and resistance to β-lactam antibiotics was attenuated in ΔnagZ EC, while specific complementation of nagZ was able to rescue ampC expression and resistance in ΔnagZ EC. More interestingly, NagZ and its hydrolyzates 1,6-anhydromuropeptides (anhMurNAc) could induce the expression of other target genes of AmpR (a global transcriptional factor), which suggested that the promotion of AmpC by NagZ is mediated AmpR activated by anhMurNAc in EC. In conclusion, these findings provide new elements for a better understanding of resistance in EC, which is crucial for the identification of novel potential drug targets.

BackgroundIn Gram-negative species, β-lactam antibiotics target penicillin binding proteins (PBPs) resulting in morphological alterations of bacterial cells. Observations of antibiotic-induced cell morphology changes can rapidly and accurately differentiate drug susceptible from resistant bacterial strains; however, resistant cells do not always remain unchanged. Burkholderia pseudomallei is a Gram-negative, biothreat pathogen and the causative agent of melioidosis, an often fatal infectious disease for humans.ResultsHere, we identified β-lactam targets in B. pseudomallei by in silico analysis. Ten genes encoding putative PBPs, including PBP-1, PBP-2, PBP-3 and PBP-6, were detected in the genomes of susceptible and resistant strains. Real-time, live-cell imaging of B. pseudomallei strains demonstrated dynamic morphological changes in broth containing clinically relevant β-lactam antibiotics. At sub-inhibitory concentrations of ceftazidime (CAZ), amoxicillin-clavulanic acid (AMC), and imipenem (IPM), filamentation, varying in length and proportion, was an initial response of the multidrug-resistant strain Bp1651 in exponential phase. However, a dominant morphotype reemerged during stationary phase that resembled cells unexposed to antibiotics. Similar morphology dynamics were observed for AMC-resistant strains, MSHR1655 and 724644, when exposed to sub-inhibitory concentrations of AMC. For all B. pseudomallei strains evaluated, increased exposure time and exposure to increased concentrations of AMC at and above minimal inhibitory concentrations (MICs) in broth resulted in cell morphology shifts from filaments to spheroplasts and/or cell lysis. B. pseudomallei morphology changes were more consistent in IPM. Spheroplast formation followed by cell lysis was observed for all strains in broth containing IPM at concentrations greater than or equal to MICs, however, the time to cell lysis was variable. Length of B. pseudomallei cells was strain-, drug- and drug concentration-dependent.ConclusionsBoth resistant and susceptible B. pseudomallei strains exhibited filamentation during early exposure to AMC and CAZ at concentrations used to interpret susceptibility (based on CLSI guidelines). While developing a rapid β-lactam antimicrobial susceptibility test based on cell-shape alone requires more extensive analyses, optical microscopy detected B. pseudomallei growth attributes that lend insight into antibiotic response and antibacterial mechanisms of action.

Bacterial persistence is one of the major causes of antibiotic treatment failure and the step stone for antibiotic resistance. However, the mechanism by which persisters arise has not been well understood. Maintaining a dormant state to prevent antibiotics from taking effect is believed to be the fundamental mechanistic basis, and persisters normally maintain an intact cellular structure. Here we examined the morphologies of persisters in Acinetobacter baumannii survived from the treatment by three major classes of antibiotics (i.e. β-lactam, aminoglycoside, and fluoroquinolone) with microcopy and found that a fraction of enlarged spherical bacteria constitutes a major sub-population of bacterial survivors from β-lactam antibiotic treatment, whereas survivors from the treatment of aminoglycoside and fluoroquinolone were less changed morphologically. Further studies showed that these spherical bacteria had completely lost their cell wall structures but could survive without any osmoprotective reagent. The spherical bacteria were not the viable-but-non-culturable cells and they could revive upon the removal of β-lactam antibiotics. Importantly, these non-walled spherical bacteria also persisted during antibiotic therapy in vivo using Galleria mellonella as the infection model. Additionally, the combinational treatment on A. baumannii by β-lactam and membrane-targeting antibiotic significantly enhanced the killing efficacy. Our results indicate that in addition to the dormant, structure intact persisters, the non-wall spherical bacterium is another important type of persister in A. baumannii. The finding suggests that targeting the bacterial cell membrane during β-lactam chemotherapy could enhance therapeutic efficacy on A. baumannii infection, which might also help to reduce the resistance development of A. baumannii.

Green tea-derived galloylated catechins have weak direct antibacterial activity against both Gram-positive and Gram-negative bacterial pathogens and are able to phenotypically transform, at moderate concentrations, methicillin-resistant Staphylococcus aureus (MRSA) clonal pathogens from full β-lactam resistance (minimum inhibitory concentration 256–512 mg/L) to complete susceptibility (~1 mg/L). Reversible conversion to susceptibility follows intercalation of these compounds into the bacterial cytoplasmic membrane, eliciting dispersal of the proteins associated with continued cell wall peptidoglycan synthesis in the presence of β-lactam antibiotics. The molecules penetrate deep within the hydrophobic core of the lipid palisade to force a reconfiguration of cytoplasmic membrane architecture. The catechin gallate-induced staphylococcal phenotype is complex, reflecting perturbation of an essential bacterial organelle, and includes prevention and inhibition of biofilm formation, disruption of secretion of virulence-related proteins, dissipation of halotolerance, cell wall thickening and cell aggregation and poor separation of daughter cells during cell division. These features are associated with the reduction of capacity of potential pathogens to cause lethal, difficult-to-treat infections and could, in combination with β-lactam agents that have lost therapeutic efficacy due to the emergence of antibiotic resistance, form the basis of a new approach to the treatment of staphylococcal infections.

In antibiotics, &beta, lactam is one kind of major concern acknowledged as an unavoidable contaminant in milk. Thus, a facile and sensitive method is essential for rapid &beta, lactam antibiotics detection. In our work, a specific electrochemical receptor sensor based on the graphene/thionine (GO/TH) composite was established. The mechanism of the electrochemical receptor sensor was a direct competitive inhibition of the binding of horseradish peroxidase-labeled ampicillin (HRP-AMP) to the mutant BlaR-CTD protein by free &beta, lactam antibiotics. Then, horseradish peroxidase (HRP) catalyzed the hydrolysis of the substrate hydrogen peroxide (H2O2), which produced an electrochemical signal. Under optimal experimental conditions, this method could quantitatively detect cefquinome from 0.1 to 8 &mu, g L&minus, 1 and with the limit of detection (LOD) of 0.16 &mu, 1, much lower than the maximum residue limit (MRL) of 5 &mu, 1 set by the European Union. In addition, the LOD of spiked milk samples with cefalexin, cefquinoxime, cefotafur, penicillin G and ampicillin were 14.88 &mu, 1, 2.46 &mu, 1, 17.16 &mu, 1, 0.06 &mu, 1, 0.21 &mu, 1 and the limits of quantitation (LOQ) were 36.09 &mu, 1, 5.40 &mu, 1, 41.45 &mu, 1, 0.13 &mu, 1, 0.42 &mu, 1, respectively. The sensor showed a favorable recovery of 84.89&ndash, 102.44%. Moreover, the electrochemical receptor sensor was successfully applied to assay &beta, lactam antibiotics in milk, which showed good correlation with the results obtained from liquid chromatography-tandem mass spectrometry (LC-MS/MS).

To determine whether target concentration non-attainment can be anticipated in critically ill patients prior to initiating empiric β-lactam antibiotic therapy based on readily available clinical factors. Retrospective review of consecutive patients treated with piperacillin or meropenem and who underwent therapeutic drug monitoring (TDM) at St Vincent’s Hospital (Sydney, Australia)between January 2013 and December 2015 was performed. Predefined subgroups were patients who received continuous renal replacement therapy (CRRT) and those who did not (non-CRRT). Potential risk factors were evaluated by correlation with β-lactam antibiotic trough concentrations (Cₘᵢₙ) lower than or equal to targeted minimum inhibitory concentration (MIC). Only the first drug concentration after initiation of the antibiotic treatment was included to reflect empirical dose selection. A total of n =249 patients (piperacillin, n = 169; meropenem, n = 80) were investigated. For non-CRRT patients (n = 210), multivariate analysis demonstrated the following: male gender (p = 0.006); younger age (p = 0.015); prescribed daily antibiotic dose less than 1.5 times the product information recommendations (p = 0.004); lack of positive microbiology (p = 0.006); lower overall illness severity (p = 0.005); and estimated glomerular filtration rate (eGFR) ≥ 90 mL/min/1.73 m2 (p < 0.001), to be associated with Cₘᵢₙ ≤ MIC. No predictor variable was found to be significantly associated with Cₘᵢₙ ≤ MIC for the CRRT cohort. Evaluating the risk of target concentration non-attainment using simple clinical factors is possible at the bedside for non-CRRT patients prior to empiric antibiotic initiation. Clinicians should be wary of selecting doses based on the product information especially when treating younger male patients with apparently ‘normal’ renal function. Refereed/Peer-reviewed

The surveillance data on antibiotic resistance of Haemophilus influenzae have shown that strains with non-enzymatic resistance to β-lactam antibiotics have been on the rise in the Czech Republic over the last decade. This type of resistance is more difficult to detect than β-lactamase production. Analysis of 228 H. influenzae strains revealed that isolates with non-enzymatic resistance to β-lactams due to mutations in the ftsI gene could be reliably demonstrated by single run testing of susceptibility to amoxicillin/clavulanic acid (sensitivity of detection is 84.6%), cefuroxime (92.6%), ampicillin and penicillin (both 95.7%). Thirty-seven different amino acid substitution combinations were detected in the PBP3 protein at 23 positions (V329I, D350N, S357N, A368T, M377I, S385T, A388V, L389F, P393L, A437S, I449V, G490E, I491V, R501L, A502S, A502T, A502V, V511A, R517H, I519L, N526K, A530S, and T532S). The most common combination (35%) of amino acid substitutions was the combination D350N, M377I, A502V, N526K. Epidemiological typing does not indicate a clonal spread of a particular MLST type. Altogether there has been detected 74 STs. The most prevalent ST 1034 was associated mainly with a combination D350N, M377I, A502V, N526K. Clonal analysis revealed six clonal complexes (CCs) with the founder found, eight CCs without founder and 33 singletons.

The occurrence of antibiotic resistance bacteria has become a major threat to public health. We have recently discovered a transcriptional activator that belongs to MarR family, EstR, and an esterase B (EstB) with a newly proposed de-arenethiolase activity from Sphingobium sp. SM42. De-arenethiolase activity involves the removal of the small aromatic side chain of cephalosporin antibiotics as an excellent leaving group by the enzymatic CS bond cleavage. Here, we report the regulation of estB through EstR as an activator in response to a third generation cephalosporin, cefoperazone, antibiotic. Cefoperazone induced the expression of estB in wild type Sphingobium sp., but not in the estR knockout strain, and the induction was restored in the complemented strain. Moreover, we revealed the importance of EstB localization in periplasm. Since EsB has the ability to inactivate selected β-lactam antibiotics in vitro, it is possible that the enzyme works at the periplasmic space of Gram negative bacteria similar to β-lactamases. EstB was genetically engineered by incorporating NlpA binding motif, or OmpA signal sequence, or SpyTag-SpyCatcher to the estB gene to mobilize it to different compartments of periplasm; inner membrane, outer membrane, and periplasmic space, respectively. Surprisingly, we found that Sphingobium sp. SM42 and E. coli expressing EstB at the periplasm were more sensitive to cefoperazone. The possible drug enhancement mechanism by enzyme was proposed. This work might lead to a novel strategy to tackle antibiotic resistance problem.

Owing to the thermal instability and low affinity of BlaR-CTD to some β-lactams, the receptor assay based on BlaR-CTD is limited in the detection of abundant variety of drugs and the result is often unstable. In this study, the three-dimensional structure of BlaR-CTD from Bacillus licheniformis ATCC14580 was constructed by homologous modeling based on the crystal structure of BlaR-CTD from B. licheniformis 749/I, and the binding sites of this protein to 40 β-lactams were also obtained by molecular docking. To improve the stability and affinity of the protein, 23 mutant proteins were designed based on docking and homologous alignment results as well as by inserting disulfide bond and building the salt bridge. The mutation was rationality evaluated by SIFT and PloyPhen2 software. The heterologous expressed and purified mutant proteins were then subjected to the activity and stability assay. It was shown that among all mutant proteins, I188K/S19C/G24C, A138E/R50C/Q147C and S190Y/E183C/I188K respectively exhibited a higher affinity to 33, 22 and 21 β-lactams than the wild-type protein, while I188K/S19C/G24C exhibited the best stability. This may due to that the conformation of the active site in mutant protein I188K/S19C/G24C changed, and the random coli in the surface of protein activity increased. Our study suggests a possible structure-function relationship on the stability and affinity of BlaR-CTD, which provides new insights into protein rational design study and lays a solid foundation for establishing the receptor-based screening assay for the detection of β-lactam residues. Electronic supplementary material The online version of this article (10.1186/s13036-019-0157-4) contains supplementary material, which is available to authorized users.

The complex spatial structure and the heterogeneity within biofilms lead to the emergence of specific social behaviors. However, the impact of resistant mutants within bacterial communities is still mostly unknown. Thus, we determined whether antibiotic resistant mutants display selfish or altruistic behaviors in mixed Pseudomonas aeruginosa biofilms exposed to antibiotics. ECFP-tagged P. aeruginosa strain PAO1 and its EYFP-tagged derivatives hyperproducing the β-lactamase AmpC or the efflux pump MexAB-OprM were used to develop single or mixed biofilms. Mature biofilms were challenged with different concentrations of β-lactams to monitor biofilm structural dynamics, using confocal laser scanning microscopy (CLSM), and population dynamics, through enumeration of viable cells. While exposure of single wild-type PAO1 biofilms to β-lactams lead to a major reduction in bacterial load, it had little effect on biofilms formed by the resistant mutants. However, the most reveling finding was that bacterial load of wild-type PAO1 was significantly increased when growing in mixed biofilms compared to single biofilms. In agreement with CFU enumeration data, CLSM images revealed the amplification of the resistant mutants and their protection of susceptible populations. These findings show that mutants expressing diverse resistance mechanisms, including β-lactamases, but also, as evidenced for the first time, efflux pumps, protect the whole biofilm community, preserving susceptible populations from the effect of antibiotics. Thus, these results are a step forward to understanding antibiotic resistance dynamics in biofilms, as well as the population biology of bacterial pathogens in chronic infections, where the coexistence of susceptible and resistant variants is a hallmark.

Introduction: The objective of this study was to determine the current profile of bacteria responsible for the infection of the mammary gland and to assess their sensitivity to selected β-lactam antibiotics. Material and Methods: The study was conducted on 119 (n = 119) dairy cows of the Polish Black-White breed aged 4 to 10 years with inflammation of the mammary gland. The cows came from different farms: smallholder farms and large dairy cattle farms in the Lublin and Bialystok Provinces. Before the process of collection of milk samples, the teats were cleaned and immersed in a liquid disinfectant. The first streams were collected into containers which were subsequently utilised. Afterwards, 2-4 mL of milk or secretions was milked into sterile disposable tubes. The milk samples were collected into plastic bottles and kept in a cooler with ice until transportation to the laboratory. Tests of resistance to β-lactam antibiotics were performed by disc diffusion. Results: Contagious and environmental bacteria were isolated from all dairy barns. In the group of contagious bacteria, the presence of typical pathogens responsible for the mammary gland infections, i.e. Staph. aureus, Str. agalactiae, and C. bovis, was detected. A relatively broad group of the isolates was formed by environmental bacteria responsible for inflammation of the mammary gland: Str. dysgalactiae, Str. uberis, Staph. chromogenes, Staph. hyicus, Staph. warneri, and E. coli. Among the environmental organisms, streptococci constituted the largest percentage (23%), followed by staphylococci (13.2%), and E. coli (8.8%). The largest group of infectious pathogens comprised Str. agalactiae (29.6%) and Staph. aureus (20.8%). Conclusion: Our investigation of the current profile of the isolates responsible for mastitis in the Lublin and Bialystok Provinces showed that environmental bacteria are the major cause of the disease. In view of the substantially varying degrees of sensitivity of the microorganisms isolated from cases of mastitis to β-lactam antibiotics, each therapeutic treatment should be preceded by susceptibility testing.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has posed a serious threat to public health, therefore, the development of new antibacterial drugs is imperative. Bisdemethoxycurcumin (BDMC) is a curcumin analog that exists in nature and possesses extensive pharmacological actions. This review focuses on investigating the antibacterial activity of BDMC alone or in combination with three antibiotics against MRSA. We determined the minimal inhibitory concentration of BDMC, with a broth microdilution assay, and the value against all six strains was 7.8 &mu, g/mL. The synergistic effect of BDMC combined with the antibiotics was determined using a checkerboard dilution test and a time&ndash, kill curve assay. The results showed that the antimicrobial effect of BDMC combined with antibiotics was superior to treatment with that of a single agent alone. We examined the antibacterial activity of BDMC in the presence of a membrane-permeabilizing agent and an ATPase-inhibiting agent, respectively. In addition, we analyzed the mecA transcription gene and the penicillin-binding protein 2a (PBP2a) level of MRSA treated with BDMC by quantitative RT-PCR or Western blot assay. The gene transcription and the protein level were significantly inhibited. This study demonstrated that BDMC has potent antibacterial activity, and proved that BDMC may be a potential natural modulator of antibiotics.

A carbapenem-resistant S. marcescens isolate was recovered from a patient with an inflammed pacemaker inplantation pocket from a Cardiac Surgery ward in a Hungarian University Hospital. Phenotypic tests and polymerase chain reaction (PCR) confirmed a very rare gene responsible for production of a carbapenemase (blaVIM-4), which was further characterized by Sanger-sequencing. The characterization of this S. marcescens strain emphasizes the ongoing emergence of novel or rare carbapenemases. Strains expressing a weak carbapenemase like this strain might go unrecognized by routine diagnostics due to low minimum inhibitory concentrations (MICs) for the bacterial strains producing such enzymes., Microbiology; Bacteria; Microorganism; Public health; Infectious disease; Pharmacology; Antibiotic resistance; β-lactam antibiotics, Metallo-β-lactamases; Serratia marcescens; Sequencing.

Bacterial resistance to present antibiotics is emerging at a high pace that makes the development of new treatments a must. At the same time, the development of novel antibiotics for resistant bacteria is a slow-paced process. Amid the massive need for new drug treatments to combat resistance, time and effort preserving approaches, like the prodrug approach, are most needed. Prodrugs are pharmacologically inactive entities of active drugs that undergo biotransformation before eliciting their pharmacological effects. A prodrug strategy can be used to revive drugs discarded due to a lack of appropriate pharmacokinetic and drug-like properties, or high host toxicity. A special advantage of the use of the prodrug approach in the era of bacterial resistance is targeting resistant bacteria by developing prodrugs that require bacterium-specific enzymes to release the active drug. In this article, we review the up-to-date implementation of prodrugs to develop medications that are active against drug-resistant bacteria.

Bacterial resistance to β-lactams, the most commonly used class of antibiotics, poses a global challenge. This resistance is caused by the production of bacterial enzymes that are termed β-lactamases (βLs). The evolution of serine-class A β-lactamases from penicillin-binding proteins (PBPs) is related to the formation of the Ω-loop at the entrance to the enzyme’s active site. In this loop, the Glu166 residue plays a key role in the two-step catalytic cycle of hydrolysis. This residue in TEM–type β-lactamases, together with Asn170, is involved in the formation of a hydrogen bonding network with a water molecule, leading to the deacylation of the acyl–enzyme complex and the hydrolysis of the β-lactam ring of the antibiotic. The activity exhibited by the Ω-loop is attributed to the positioning of its N-terminal residues near the catalytically important residues of the active site. The structure of the Ω-loop of TEM-type β-lactamases is characterized by low mutability, a stable topology, and structural flexibility. All of the revealed features of the Ω-loop, as well as the mechanisms related to its involvement in catalysis, make it a potential target for novel allosteric inhibitors of β-lactamases.

Background Antibiotic residues are being constantly identified in environmental waters at low concentration. Growing concern has been expressed over the adverse environmental and human health effects even at low concentration. Hence, it is crucial to develop a multi-residues analytical method for antibiotics to generate a considerable dataset which are necessary in the assessment of aquatic toxicity of environmental waters for aquatic organisms and human health. This work aimed to develop a reliable and sensitive multi-residue method based on high performance liquid chromatography coupled with quadrupole-linear ion trap tandem mass spectrometry (HPLC-MS-MS). The method was optimized and validated for simultaneous determination of four classes of antibiotics including, β-lactam, macrolide, fluoroquinolone and nitro-imidazole in treated, ground and surface water matrices. Methods In order to optimize the solid phase extraction process, main parameters influencing the extraction process including, pH, the volume of elution solvent and the amount of Na4EDTA were evaluated. The optimization of extraction process was carried out by response surface methodology using central composite design. Analysis of variance was performed for nine target antibiotics using response surface methodology. Results The extraction recoveries were found to be sensitive to the independent variables of pH, the volume of elution solvent and the amount of Na4EDTA. The extraction process was pH-dependent and pH was a significant model term in the extraction process of all target antibiotics. Method validation was performed in optimum operation conditions in which the recoveries were obtained in the range of 50–117% for seven antibiotics in spiked treated and ground water samples and for six antibiotics in spiked river water samples. Method validation parameters in terms of method detection limit were obtained in the range of 1–10 ng/L in treated water, 0.8–10 ng/L in the ground water and 0.8–25 ng/L in river water, linearity varied from 0.95 to 0.99 and repeatability in term of relative standard deviation values was achieved less than 10% with the exception for metronidazole and ceftriaxone. The developed method was applied to the analysis of target antibiotics in treated, ground and surface water samples. Conclusions Target antibiotics were analyzed in different water matrices including treated, ground and river water. Seven out of nine antibiotics were detected in Kan River and Firozabad Ditch water samples, although none of them were detected in treated water and ground water samples. Electronic supplementary material The online version of this article (10.1186/s40201-017-0282-2) contains supplementary material, which is available to authorized users.

The aim of the present research was to develop a single use, simple but highly sensitive amperometric immunosensor for penicillin G and other β-lactam antibiotics based on a “competitive assay”. The immunosensor developed uses an amperometric electrode for hydrogen peroxide as transducer and the peroxidase enzyme as marker. The results demonstrate the full validity of this immunosensor method which was optimized by comparing two different competitive operating formats. LOD was of the order of 10 −10 M. The immunosensor developed displayed low selectivity toward all β-lactam antibiotics and higher selectivity toward other classes of non-β-lactam antibiotics. In addition the K aff value (about 10 8 M −1 ) was evaluated. Lastly, the immunosensor was used to test a β-lactam antibiotic “pool” and to recover penicillin G in common real matrices such as river waste water, obtaining good recoveries.

Prediction and control of crystal size distributions, a prerequisite for production of consistent crystalline material in the pharmaceutical industry, requires knowledge of potential non-idealities of crystal growth. Ampicillin is one such medicine consumed in crystal form (ampicillin trihydrate). Typically it is assumed that all crystals of the same chemical and geometric type grow at the same rate, however a distribution of growth rates is often observed experimentally. In this study, ampicillin produced enzymatically is crystallized and a distribution of growth rates is observed as individual crystals are monitored by microscopy. Most studies of growth rate dispersion use complex flow apparatuses to maintain a constant supersaturation or imprecise measurements of size distributions to reconstruct growth rate dispersions. In this study, the controllable enzyme reaction enables the same information to be gathered from fewer, less complicated experiments. The growth rates of individual ampicillin trihydrate crystals were found to be normally distributed, with each crystal having an intrinsic growth rate that is constant in time. Differences in the individual crystals, such as different number and arrangement of dislocations and surface morphology, best explain the observed growth rates. There is a critical supersaturation below which growth is not observed, thought to be caused by reactants adsorbing to the crystal surface and pinning advancing growth steps. The distribution of critical supersaturation also suggests that individual crystals&rsquo, surface morphologies cause a distribution of growth rates.

Upper respiratory tract infection (URTI) is an acute infection which involves the upper respiratory tract: nose, sinuses, tonsils and pharynx. URT infections are caused mainly by pathogenic bacteria like Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus. Conventionally, β-lactam antibiotics are used to treat URT infections. Penicillin binding proteins (PBPs) catalyze the cell wall synthesis in bacteria. β-Lactam antibiotics like Penicillin, Cephalosporins, Carbapenems and Monobactams inhibit bacterial cell wall synthesis by binding with PBPs. Pathogenic bacteria have efficiently evolved to resist these β-lactam antibiotics. New generation antibiotics are capable of inhibiting the action of PBP due to its new and peculiar structure. New generation antibiotics and Penicillin derivatives are selected in this study and virtually compared on the basis of interaction studies. 3-Dimensional (3D) interaction studies between Lactivicin, Cefuroxime, Cefadroxil, Ceftaroline, Ceftobiprole and Penicillin derivatives with PBPs of the above-mentioned bacteria are carried out. The aim of this study was to suggest a potent new generation molecule for further modification to increase the efficacy of the drug for the URTI.

Antibiotic resistance is one of the most serious public health problems. Among bacterial resistance, β-lactam antibiotic resistance is the most prevailing and threatening area. Antibiotic resistance is thought to originate in antibiotic-producing bacteria such as Streptomyces. In this review, β-lactamases and penicillin-binding proteins (PBPs) in Streptomyces are explored mainly by phylogenetic analyses from the viewpoint of self-resistance. Although PBPs are more important than β-lactamases in self-resistance, phylogenetically diverse β-lactamases exist in Streptomyces. While class A β-lactamases are mostly detected in their enzyme activity, over two to five times more classes B and C β-lactamase genes are identified at the whole genomic level. These genes can subsequently be transferred to pathogenic bacteria. As for PBPs, two pairs of low affinity PBPs protect Streptomyces from the attack of self-producing and other environmental β-lactam antibiotics. PBPs with PASTA domains are detectable only in class A PBPs in Actinobacteria with the exception of Streptomyces. None of the Streptomyces has PBPs with PASTA domains. However, one of class B PBPs without PASTA domain and a serine/threonine protein kinase with four PASTA domains are located in adjacent positions in most Streptomyces. These class B type PBPs are involved in the spore wall synthesizing complex and probably in self-resistance. Lastly, this paper emphasizes that the resistance mechanisms in Streptomyces are very hard to deal with, despite great efforts in finding new antibiotics.

Metallo-β-lactamases (MBLs) catalyse the hydrolysis of almost all β-lactam antibacterials including the latest generation carbapenems and are a growing worldwide clinical problem. It is proposed that MBLs employ one or two zinc ion cofactors in vivo. Isolated MBLs are reported to use transition metal ions other than zinc, including copper, cadmium and manganese, with iron ions being a notable exception. We report kinetic and biophysical studies with the di-iron(II)-substituted metallo-β-lactamase II from Bacillus cereus (di-Fe(II) BcII) and the clinically relevant B1 subclass Verona integron-encoded metallo-β-lactamase 2 (di-Fe(II) VIM-2). The results reveal that MBLs can employ ferrous iron in catalysis, but with altered kinetic and inhibition profiles compared to the zinc enzymes. A crystal structure of di-Fe(II) BcII reveals only small overall changes in the active site compared to the di-Zn(II) enzyme including retention of the di-metal bridging water; however, the positions of the metal ions are altered in the di-Fe(II) compared to the di-Zn(II) structure. Stopped-flow analyses reveal that the mechanism of nitrocefin hydrolysis by both di-Fe(II) BcII and di-Fe(II) VIM-2 is altered compared to the di-Zn(II) enzymes. Notably, given that the MBLs are the subject of current medicinal chemistry efforts, the results raise the possibility the Fe(II)-substituted MBLs may be of clinical relevance under conditions of low zinc availability, and reveal potential variation in inhibitor activity against the differently metallated MBLs., Graphical abstract Metallo-β-lactamases (MBLs) catalyse the hydrolysis of almost all β-lactam antibiotics and are a growing clinical problem. It is proposed that MBLs employ one or two zinc ion cofactors. We report that MBLs can employ ferrous iron, but with altered kinetic and inhibition profiles compared to the zinc enzymes.Image 1, Highlights • Metallo-β-lactamases (MBLs) can employ ferrous iron to hydrolyse β-lactam antibiotics. • Pre-steady state kinetics reveals altered mechanistic profiles of iron-substituted MBLs. • Di-Fe(II) MBL from Bacillus cereus shows similar active site geometry to di-Zn(II) MBLs. • Zinc(II)- and iron(II)-bound MBLs can exhibit different susceptibilities to inhibitors.

Transcription factors are key regulatory proteins in all living beings. In fungi, transcription factors include either broad-domain regulatory proteins that affect the expression of many genes involved in biosynthetic processes, or proteins encoded by cluster-associated (also called pathway-specific) regulatory genes. Belonging to the most interesting transcription factors in fungi are binuclear zinc finger proteins. In addition to the transcription factors in the strict sense, other proteins play a very important role in the expression of genes for primary and secondary metabolism in fungi, such as winged helix regulators, the LaeA protein and the velvet complex. LaeA appears to be involved in heterochromatin reorganization, and the velvet complex proteins, which are nuclear factors that associate with LaeA, also have a determining role in both differentiation (sporulation) and secondary metabolite biosynthesis. The genes involved in the biosynthesis of β-lactam antibiotics are well known and serve as an excellent model to understand the transcriptional control of genes involved in the biosynthesis of secondary metabolites. Interaction between different regulatory proteins in the promoter regions may represent a cross-talk regulation between different gene clusters.

The relentless emergence and spread of antimicrobial resistance warrant an increased awareness of the problem and improved coordination and standardisation of surveillance systems, as well as resistance control strategies. Moreover, this underscores the urgent need for new antibiotics active against the emerging resistant pathogens. Ceftobiprole is a new β-lactam molecule engineered for bactericidal activity against methicillin-resistant staphylococci that also exhibits an extended broad spectrum of activity covering the most clinically important Gram-positive (methicillin-susceptible staphylococci, penicillin-susceptible and -resistant pneumococci, other streptococci and ampicillin-susceptible enterococci) and Gram-negative (Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter spp., Haemophilus spp. and Moraxella spp.) pathogens. Results from studies with experimental infections and from clinical trials support a role for ceftobiprole in treating complicated skin and skin structure infections caused by Gram-positive pathogens, including methicillin-resistant staphylococci, and suggest a potential role for this drug in treating other types of serious infection caused by Gram-positive and Gram-negative pathogens.

Staphylococcus aureus is a major human and veterinary pathogen worldwide. Methicillin-resistant S. aureus (MRSA) poses a significant and enduring problem to the treatment of infection by such strains. Resistance is usually conferred by the acquisition of a nonnative gene encoding a penicillin-binding protein (PBP2a), with significantly lower affinity for β-lactams. This resistance allows cell-wall biosynthesis, the target of β-lactams, to continue even in the presence of typically inhibitory concentrations of antibiotic. PBP2a is encoded by the mecA gene, which is carried on a distinct mobile genetic element (SCCmec), the expression of which is controlled through a proteolytic signal transduction pathway comprising a sensor protein (MecR1) and a repressor (MecI). Many of the molecular and biochemical mechanisms underlying methicillin resistance in S. aureus have been elucidated, including regulatory events and the structure of key proteins. Here we review recent advances in this area.

金沢大学薬学部, 金沢大学大学院自然科学研究科分子作用学, A comprehensive list of drug transporters has recently become available as a result of extensive genome analysis. Membrane transporters play important roles in determining the pharmacokinetic aspects of intestinal absorption, tissue distribution, and the urinary and biliary excretions of a wide variety of therapeutic drugs. The identification and characterization of transporters responsible for the transfer of nutrients and xenobiotics, including drugs, is expected to provide a scientific basis for understanding drug disposition, as well as the molecular mechanisms of drug-drug/drug-food/drug-protein interactions and inter-individual/inter-species differences. This review focuses on the influence of transporters on the pharmacokinetics of β-lactam antibiotics, new quinolones, and other antimicrobial agents, as well as focusing on the drug-drug interactions associated with transporter-mediated uptake from the small intestine and transporter-mediated elimination from the kidney and liver. © 2006 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases.

The extensive production and usage of antibiotics have led to an increasing occurrence of antibiotic residuals in various aquatic compartments, presenting a significant threat to both ecosystem and human health. This study investigated the degradation of selected β-lactam antibiotics (penicillins: ampicillin, penicillin V, and piperacillin; cephalosporin: cephalothin) by UV-254 nm activated H2O2 and S2O82− photochemical processes. The UV irradiation alone resulted in various degrees of direct photolysis of the antibiotics; while the addition of the oxidants improved significantly the removal efficiency. The steady- state radical concentrations were estimated, revealing a non-negligible contribution of hydroxyl radicals in the UV/S2O82− system. Mineralization of the β-lactams could be achieved at high UV fluence, with a slow formation of SO42− and a much lower elimination of total organic carbon (TOC). The transformation mechanisms were also investigated showing the main reaction pathways of hydroxylation (+16 Da) at the aromatic ring and/or the sulfur atom, hydrolysis (+18 Da) at the β-lactam ring and decarboxylation (–44 Da) for the three penicillins. Oxidation of amine group was also observed for ampicillin. This study suggests that UV/H2O2 and UV/S2O82− advanced oxidation processes (AOPs) are capable of degrading β-lactam antibiotics decreasing consequently the antibiotic activity of treated waters.

Background Ampicillin-resistant S. aureus (ARSA) now poses a serious problem for hospitalized patients, and their care providers. Plant-derived antibacterial that can reverse the resistance to well-tried agents which have lost their original effectiveness are the research objectives of far reaching importance. To this aim, the present study investigated antibacterial and synergistic activities of Stephania suberosa extracts (SSE) against ARSA when used singly and in combination with ampicillin. Results The majority chemical compounds of SSE were alkaloid (526.27 ± 47.27 mg/1 g of dried extract). The Minimum inhibitory concentration (MICs) for ampicillin and SSE against all ARSA strains were >512 μg/ml and 4 mg/ml, respectively. Checkerboard assay revealed synergistic activity in the combination of ampicillin (0.15 μg/ml) and SSE (2 mg/ml) at fractional inhibitory concentration index (FICI)

Antibiotics have had a profound impact on human health and belong to one of the largest-selling classes of drugs worldwide. Introduced into industrial production only some half century ago, these miracle drugs have been the main contributors to the recent increase in human life expectancy. However, the accelerated emergence of bacteria that are resistant to multiple antibiotic types now appears as the most serious threat to continuing success in the treatment of infectious diseases. Recent advances in our knowledge of the structures and mechanisms of enzymes in the biosynthetic pathways of penicillins and cephalosporins, amongst the most important antibiotics in current use, have identified a common structural core together with common iron- and cosubstrate-binding motifs. The diversity in the catalytic specificities of these oxygenases using very similar structural platforms suggests that altering the substrate and product specificities of these enzymes should be possible in the laboratory. This opens up new avenues for industrial production and medical utilisation.

The number of clinical specimens containing β-lactam-resistant Pseudomonas aeruginosa isolates is increasing. However, whether resistance is associated with reduced fitness is still uncertain in clinical Pseudomonas aeruginosa isolates. In this study, we aimed to determine whether β-lactam resistance conferred a fitness cost in Pseudomonas aeruginosa. Growth rate, extracellular slime production, elastase activity, proteolytic activity, LasA protease activity, biofilm formation, and pairwise in vitro competition experiments were investigated in a collection of 11 isogenic, β-lactam-susceptible and -resistant (≥8-fold increase in minimum inhibitory concentration (MIC)) pairs of P. aeruginosa clinical isolates; each pair was recovered from a different patient treated with β-lactam antibiotics. All β-lactam-resistant Pseudomonas aeruginosa isolates showed a significant reduction in elastase activity. In addition, 90.9% (10/11) of β-lactam-resistant Pseudomonas aeruginosa isolates were associated with markedly lower growth rate and proteolytic activity, and 81.8% (9/11) of β-lactam-resistant Pseudomonas aeruginosa isolates had less extracellular slime production, compared to susceptible isolates. Meanwhile, LasA protease activity and biofilm formation ability were variable among isolates. Pairwise in vitro competition experiments showed that 72.7% (8/11) of β- lactam-susceptible strains could outgrow resistant strains. In conclusion, resistance development with β-lactam exposure confers a fitness cost, resulting in a decreased invasion potential, while the effect on viability varied. Thus, the potential for the dissemination of β-lactam-resistant Pseudomonas aeruginosa clinical isolates should not be underestimated.

The aim of this work was to determine the concentrations and properties of free radicals in piperacillin, ampicillin, and crystalline penicillin after gamma irradiation. The radicals were studied by electron paramagnetic resonance (EPR) spectroscopy using an X-band spectrometer (9.3 GHz). Gamma irradiation was performed at a dose of 25 kGy. One- and two-exponential functions were fitted to the experimental data, in order to assess the influence of the antibiotics’ storage time on the measured EPR lines. After gamma irradiation, complex EPR lines were recorded confirming the presence of a large number of free radicals formed during the irradiation. For all tested antibiotics, concentrations of free radicals and parameters of EPR spectra changed with storage time. The results obtained demonstrate that concentration of free radicals and other spectroscopic parameters can be used to select the optimal parameters of radiation sterilization of β-lactam antibiotics. The most important parameters are the constants τ (τ 1(A),(I) and τ 2(A),(I)) and K (K 0(A),(I), K 1(A),(I), K 2(A),(I)) of the exponential functions that describe free radicals decay during samples storage.

Introduction Several reports have shown marked heterogeneity of antibiotic pharmacokinetics (PK) in patients admitted to ICUs, which might potentially affect outcomes. Therefore, the pharmacodynamic (PD) parameter of the efficacy of β-lactam antibiotics, that is, the time that its concentration is above the bacteria minimal inhibitory concentration (T > MIC), cannot be safely extrapolated from data derived from the PK of healthy volunteers. Methods We performed a full review of published studies addressing the PK of intravenous β-lactam antibiotics given to infected ICU patients. Study selection comprised a comprehensive bibliographic search of the PubMed database and bibliographic references in relevant reviews from January 1966 to December 2010. We selected only English-language articles reporting studies addressing β-lactam antibiotics that had been described in at least five previously published studies. Studies of the PK of patients undergoing renal replacement therapy were excluded. Results A total of 57 studies addressing six different β-lactam antibiotics (meropenem, imipenem, piperacillin, cefpirome, cefepime and ceftazidime) were selected. Significant PK heterogeneity was noted, with a broad, more than twofold variation both of volume of distribution and of drug clearance (Cl). The correlation of antibiotic Cl with creatinine clearance was usually reported. Consequently, in ICU patients, β-lactam antibiotic half-life and T > MIC were virtually unpredictable, especially in those patients with normal renal function. A better PD profile was usually obtained by prolonged or even continuous infusion. Tissue penetration was also found to be compromised in critically ill patients with septic shock. Conclusions The PK of β-lactam antibiotics are heterogeneous and largely unpredictable in ICU patients. Consequently, the dosing of antibiotics should be supported by PK concepts, including data derived from studies of the PK of ICU patients and therapeutic drug monitoring.

The phenotype and functional characteristics of skin-infiltrating lymphocytes in β-lactam antibiotic-induced vesiculobullous exanthems were studied in vivo and in vitro . Immunohistochemical analysis demonstrated that CD8+ T lymphocytes were the predominant epidermal T-cell subset in these reactions. Epidermal T lymphocytes were isolated and expanded for in vitro studies. Fluorescence-activated cell sorter analysis showed the majority of epidermal T cells to be CD3+, T-cell receptor α/β+, CD4−, CD8+, and HLA-DR+, which correlated with the predominance of epidermal CD8+ T lymphocytes found in situ . Three CD8+ epidermal T-cell clones derived from cutaneous lesions proliferated in response to penicillin-pulsed autologous antigen-presenting cells but not allogeneic antigen-presenting cells, indicating that those clones were antigen and major histocompatibility complex specific. All T-cell clones produced significant amounts of interleukin-2, interferon-gamma, and granulocyte-macrophage colony-stimulating factor. Additionally, the T-cell clones displayed cytotoxicity against epidermal cells in lectin-mediated cytotoxicity and against B-cell lines in T-cell receptor-triggered cytotoxicity. These data demonstrate the presence of epidermal drug-specific CD8+ T cells in bullous drug reactions. Because these CD8+ T cells have a cytotoxic potential, they may contribute to the necrosis of keratinocytes associated with drug-induced blister formation.

A collection of 15 carbapenem-resistance Acinetobacter baumannii clinical isolates was analysed on two tertiary hospitals in Mexico. The OXA-51 was identified in all isolates, followed by OXA-239 and OXA-58; OXA-239 is described as a new OXA-23-like allele. These carbapenemases were identified on four clonal groups, distributed between two neighbouring hospitals. Acinetobacter baumannii is poorly studied in Mexico; this situation urges the implementation of strategies to prevent its dissemination.

International audience; The effects of cefotaxime and EDTA on the reducing activity of Escherichia coli and Staphylococcus aureus cultures growing in the presence of lipoic acid (LA) were investigated by potential-time measurements. The potentiometric responses of E. coli cultures exposed to EDTA indicated enhanced transmembrane transport of LA as a consequence of the outer membrane permeabilization by the chelator, whereas EDTA exerted no effect on the reducing activity of S. aureus cultures. In the same way, cefotaxime stimulated the reducing activity of E. coli, but not that of S. aureus. These results suggest that cefotaxime, and, more generally, a great variety of β-lactam antibiotics, are able to permeabilize the outer membrane of Gram-negative bacteria.

Methicillin-resistant Staphylococcus aureus (MRSA) strains are important nosocomial pathogens worldwide and now are also of growing importance in community-acquired infection. Their resistance depends upon a supplementary peptidoglycan transpeptidase, PBP2' (PBP-2a), which continues to function when normal PBPs have been inactivated by beta-lactams. PBP2' is encoded by the mecA gene, which is carried by the staphylococcal cassette chromosome, a large and somewhat variable DNA insert of uncertain origin. PBP2' does not wholly lack affinity for beta-lactams, but its affinity for available analogues is very weak. In principle, it should be possible to re-engineer beta-lactams to bind PBP2' strongly, and the desirability of this approach is self-evident: no other antibiotic class has a record equal to the beta-lactams for safety and efficacy. Moreover, there is consensus that beta-lactams are inherently more efficacious than vancomycin against infections due to susceptible staphylococci. In practice, finding viable PBP2'-active beta-lactams has proved difficult and the catalogue of near-misses extends back to the 1980s. At last, however, one cephalosporin with high affinity for PBP2'--ceftobiprole--is entering phase III trials. Ceftobiprole inhibits MRSA at 1-2 mg/L under standard conditions. Even when mecA/PBP2' was induced strongly, ceftobiprole MICs for MRSA only reached 4 mg/L, a clinically attainable concentration. A phase II trial in skin and skin structure infection recorded cures by ceftobiprole in 4/4 MRSA infections, and results of the phase III trials are awaited with great interest.

Background: New Delhi metallo-β-lactamase-1 (NDM-1)-producing Gram-negative bacteria are today's major worldwide health concern. The enzyme NDM-1 provides bacterial resistance by its hydrolytic activity against the β-lactam ring of antibiotics. Inhibition of NDM-1 may prevent the hydrolysis of β-lactam ring of the antibiotics, and therefore, plays an important role against antibacterial resistance. Materials and Methods: Here we made an attempt to design suitable inhibitors against NDM-1 from different natural antibacterial compounds using molecular docking approach. Results: We observed that natural compounds such as Nimbolide and Isomargololone are showing an appreciable IC50 value as well as significant binding energy value for NDM-1. We further observed these compounds showing better affinity to NDM-1 on comparison with 14 β-lactam antibiotics. Conclusion: Finally, our study provides a platform for the development of a potent inhibitor of NDM-1, which may be considered as a potential drug candidate against bacterial resistance.