Studies on enmetazobactam clarify mechanisms of widely used ß-lactamase inhibitors.

ß-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine ß-lactamase (SBL)-catalyzed hydrolysis. The scope of ß-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone­derived acyl­enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl­enzyme complexes.

How Clavulanic Acid Inhibits Serine ß-Lactamases.

Clavulanic acid is a medicinally important inhibitor of serine ß-lactamases (SBLs). We report studies on the mechanisms by which clavulanic acid inhibits representative Ambler class A (TEM-116), C (Escherichia coli AmpC), and D (OXA-10) SBLs using denaturing and non-denaturing mass spectrometry (MS). Similarly to observations with penam sulfones, most of the results support a mechanism involving acyl enzyme complex formation, followed by oxazolidine ring opening without efficient subsequent scaffold fragmentation (at pH 7.5). This observation contrasts with previous MS studies, which identified clavulanic acid scaffold fragmented species as the predominant SBL bound products. In all the SBLs studied here, fragmentation was promoted by acidic conditions, which are commonly used in LC­MS analyses. Slow fragmentation was, however, observed under neutral conditions with TEM-116 on prolonged reaction with clavulanic acid. Although our results imply clavulanic acid scaffold fragmentation is likely not crucial for SBL inhibition in vivo, development of inhibitors that fragment to give stable covalent complexes is of interest.

Pretargeted PET Imaging with a TCO-Conjugated Anti-CD44v6 Chimeric mAb U36 and [89Zr]Zr-DFO-PEG5-Tz.

The recent advances in the production of engineered antibodies have facilitated the development and application of tailored, target-specific antibodies. Positron emission tomography (PET) of these antibody-based drug candidates can help to better understand their in vivo behavior. In this study, we report an in vivo proof-of-concept pretargeted immuno-PET study where we compare a pretargeting vs targeted approach using a new 89Zr-labeled tetrazine as a bio-orthogonal ligand in an inverse electron demand Diels-Alder (IEDDA) in vivo click reaction. A CD44v6-selective chimeric monoclonal U36 was selected as the targeting antibody because it has potential in immuno-PET imaging of head-and-neck squamous cell carcinoma (HNSCC). Zirconium-89 (t1/2 = 78.41 h) was selected as the radionuclide of choice to be able to make a head-to-head comparison of the pretargeted and targeted approaches. [89Zr]Zr-DFO-PEG5-Tz ([89Zr]Zr-3) was synthesized and used in pretargeted PET imaging of HNSCC xenografts (VU-SCC-OE) at 24 and 48 h after administration of a trans-cyclooctene (TCO)-functionalized U36. The pretargeted approach resulted in lower absolute tumor uptake than the targeted approach (1.5 ± 0.2 vs 17.1 ± 3.0% ID/g at 72 h p.i. U36) but with comparable tumor-to-non-target tissue ratios and significantly lower absorbed doses. In conclusion, anti-CD44v6 monoclonal antibody U36 was successfully used for 89Zr-immuno-PET imaging of HNSCC xenograft tumors using both a targeted and pretargeted approach. The results not only support the utility of the pretargeted approach in immuno-PET imaging but also demonstrate the challenges in achieving optimal in vivo IEDDA reaction efficiencies in relation to antibody pharmacokinetics.

Resistance profiles to antifungal agents in Candida albicans isolated from human oral cavities: systematic review and meta-analysis.

AIM: To identify the antifungal susceptibility profile of Candida spp. isolated from the human oral cavity was assessed with meta-analyses of observational studies that collected samples from the oral cavity of human subjects. MATERIAL AND METHODS: Isolated Candida albicans tested by E-test®; disk diffusion test; microdilution and macrodilution; Sensititre YeastOne; and/or FungiTest. Search strategies were conducted on the MEDLINE, Embase, CINAHL, Dentistry, and Oral Sciences, Central, Scopus, and LILACS databases, and gray literature sources. Articles were initially screened by title and then their abstracts. Articles that met the conditions for inclusion were read in full, followed by data extraction. A descriptive analysis was conducted of each study, and the data were tabulated. A first meta-analysis was conducted to assess the resistance of antifungals regardless of systemic comorbidities. An additional stratified analysis was conducted by systemic comorbidity groups for the outcome "resistance" to the antifungals. RESULTS: When not grouping Candida albicans isolates by systemic conditions, the lowest resistance rates to the antifungals tested were observed for amphotericin B, nystatin, flucytosine, and caspofungin. In contrast, the highest resistance rates were observed for miconazole and econazole. There was a high degree of heterogeneity and low resistance in general in all analyses, except for the "several associated comorbidities" group, which had high resistance rates. CONCLUSIONS: Clinical C. albicans isolates had low antifungal resistance. CLINICAL RELEVANCE: The presence of concomitant systemic comorbidities appears to be an essential factor that should be considered when evaluating resistance to antifungals for oral isolates.

Structural Investigations of the Inhibition of Escherichia coli AmpC ß-Lactamase by Diazabicyclooctanes.

ß-Lactam antibiotics are presently the most important treatments for infections by pathogenic Escherichia coli, but their use is increasingly compromised by ß-lactamases, including the chromosomally encoded class C AmpC serine-ß-lactamases (SBLs). The diazabicyclooctane (DBO) avibactam is a potent AmpC inhibitor; the clinical success of avibactam combined with ceftazidime has stimulated efforts to optimize the DBO core. We report kinetic and structural studies, including four high-resolution crystal structures, concerning inhibition of the AmpC serine-ß-lactamase from E. coli (AmpC EC ) by clinically relevant DBO-based inhibitors: avibactam, relebactam, nacubactam, and zidebactam. Kinetic analyses and mass spectrometry-based assays were used to study their mechanisms of AmpC EC inhibition. The results reveal that, under our assay conditions, zidebactam manifests increased potency (apparent inhibition constant [Kiapp], 0.69 µM) against AmpC EC compared to that of the other DBOs (Kiapp = 5.0 to 7.4 µM) due to an ∼10-fold accelerated carbamoylation rate. However, zidebactam also has an accelerated off-rate, and with sufficient preincubation time, all the DBOs manifest similar potencies. Crystallographic analyses indicate a greater conformational freedom of the AmpC EC -zidebactam carbamoyl complex compared to those for the other DBOs. The results suggest the carbamoyl complex lifetime should be a consideration in development of DBO-based SBL inhibitors for the clinically important class C SBLs.

Molecular Basis of Class A ß-Lactamase Inhibition by Relebactam.

ß-Lactamase production is the major ß-lactam resistance mechanism in Gram-negative bacteria. ß-Lactamase inhibitors (BLIs) efficacious against serine ß-lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI, is in phase 3 clinical trials in combination with imipenem for the treatment of infections by multidrug-resistant Enterobacteriaceae We show that relebactam inhibits five clinically important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e., the extended-spectrum ß-lactamases L2 (inhibition constant 3 µM) and CTX-M-15 (21 µM) and the carbapenemases KPC-2, -3, and -4 (1 to 5 µM). Against purified class A SBLs, relebactam is an inferior inhibitor compared with the clinically approved DBO avibactam (9- to 120-fold differences in half maximal inhibitory concentration [IC50]). MIC assays indicate relebactam potentiates ß-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae, with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3, and KPC-4 reveal its C2-linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity than that of avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3, and -4. This comprehensive comparison of relebactam binding across five clinically important class A SBLs will inform the design of future DBOs, with the aim of improving clinical efficacy of BLI-ß-lactam combinations.

High-throughput screen with the l,d-transpeptidase LdtMt2 of Mycobacterium tuberculosis reveals novel classes of covalently reacting inhibitors.

Disruption of bacterial cell wall biosynthesis in Mycobacterium tuberculosis is a promising target for treating tuberculosis. The l,d-transpeptidase LdtMt2, which is responsible for the formation of 3 → 3 cross-links in the cell wall peptidoglycan, has been identified as essential for M. tuberculosis virulence. We optimised a high-throughput assay for LdtMt2, and screened a targeted library of ∼10 000 electrophilic compounds. Potent inhibitor classes were identified, including established (e.g., ß-lactams) and unexplored covalently reacting electrophilic groups (e.g., cyanamides). Protein-observed mass spectrometric studies reveal most classes to react covalently and irreversibly with the LdtMt2 catalytic cysteine (Cys354). Crystallographic analyses of seven representative inhibitors reveal induced fit involving a loop enclosing the LdtMt2 active site. Several of the identified compounds have a bactericidal effect on M. tuberculosis within macrophages, one with an MIC50 value of ∼1 µM. The results provide leads for the development of new covalently reaction inhibitors of LdtMt2 and other nucleophilic cysteine enzymes.

High-fat diet effect on periapical lesions and hepatic enzymatic antioxidant in rats.

AIMS: To evaluate the effects of a high-fat diet (HFD) on the progression of apical periodontitis (AP), local inflammation, systemic antioxidant status, and blood lipid profile in rats. MAIN METHODS: Sixteen male Wistar rats were fed a standard diet (SD) or a HFD. At the sixth experimental week, the pulp chambers of the mandibular first molars were exposed to develop AP. A glucose tolerance test was performed the week before euthanasia. At the tenth experimental week, the animals were euthanized and the livers were collected to estimate catalase (CAT) and reduced glutathione (GSH) levels. Blood was acquired for biochemical analysis. The size of AP was estimated from radiographs and described as AP size-to-body weight ratio; inflammatory grade of AP was determined by histological analysis. KEY FINDINGS: At the end of the experimental period, the rats fed the HFD had 30% less weight (P < 0.0001) and higher blood glucose levels after 30 min of sucrose intake (P < 0.05) than those fed the SD. Animals from the HFD group had lower levels of CAT (P < 0.01), but the same was not observed in the GSH levels. Plasma insulin and total cholesterol were not affected by the diet. The rats fed the HFD presented greater AP than those fed the SD (P < 0.05). However, the local inflammatory infiltrate was similar in both groups. SIGNIFICANCE: The alterations promoted by the consumption of a HFD were not only observed systemically, but also locally, producing greater AP in rats than a SD.

An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography.

Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.

X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis.

Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.

Bicyclic Boronates as Potent Inhibitors of AmpC, the Class C ß-Lactamase from Escherichia coli.

Resistance to ß-lactam antibacterials, importantly via production of ß-lactamases, threatens their widespread use. Bicyclic boronates show promise as clinically useful, dual-action inhibitors of both serine- (SBL) and metallo- (MBL) ß-lactamases. In combination with cefepime, the bicyclic boronate taniborbactam is in phase 3 clinical trials for treatment of complicated urinary tract infections. We report kinetic and crystallographic studies on the inhibition of AmpC, the class C ß­lactamase from Escherichia coli, by bicyclic boronates, including taniborbactam, with different C-3 side chains. The combined studies reveal that an acylamino side chain is not essential for potent AmpC inhibition by active site binding bicyclic boronates. The tricyclic form of taniborbactam was observed bound to the surface of crystalline AmpC, but not at the active site, where the bicyclic form was observed. Structural comparisons reveal insights into why active site binding of a tricyclic form has been observed with the NDM-1 MBL, but not with other studied ß-lactamases. Together with reported studies on the structural basis of inhibition of class A, B and D ß­lactamases, our data support the proposal that bicyclic boronates are broad-spectrum ß­lactamase inhibitors that work by mimicking a high energy 'tetrahedral' intermediate. These results suggest further SAR guided development could improve the breadth of clinically useful ß-lactamase inhibition.

Anaerobic fixed-target serial crystallography.

Cryogenic X-ray diffraction is a powerful tool for crystallographic studies on enzymes including oxygenases and oxidases. Amongst the benefits that cryo-conditions (usually employing a nitro-gen cryo-stream at 100 K) enable, is data collection of di-oxy-gen-sensitive samples. Although not strictly anaerobic, at low temperatures the vitreous ice conditions severely restrict O2 diffusion into and/or through the protein crystal. Cryo-conditions limit chemical reactivity, including reactions that require significant conformational changes. By contrast, data collection at room temperature imposes fewer restrictions on diffusion and reactivity; room-temperature serial methods are thus becoming common at synchrotrons and XFELs. However, maintaining an anaerobic environment for di-oxy-gen-dependent enzymes has not been explored for serial room-temperature data collection at synchrotron light sources. This work describes a methodology that employs an adaptation of the 'sheet-on-sheet' sample mount, which is suitable for the low-dose room-temperature data collection of anaerobic samples at synchrotron light sources. The method is characterized by easy sample preparation in an anaerobic glovebox, gentle handling of crystals, low sample consumption and preservation of a localized anaerobic environment over the timescale of the experiment (<5 min). The utility of the method is highlighted by studies with three X-ray-radiation-sensitive Fe(II)-containing model enzymes: the 2-oxoglutarate-dependent l-arginine hy-droxy-lase VioC and the DNA repair enzyme AlkB, as well as the oxidase isopenicillin N synthase (IPNS), which is involved in the biosynthesis of all penicillin and cephalosporin antibiotics.

Will morphing boron-based inhibitors beat the ß-lactamases?

The ß-lactams remain the most important antibacterials, but their use is increasingly compromised by resistance, importantly by ß-lactamases. Although ß-lactam and non-ß-lactam inhibitors forming stable acyl-enzyme complexes with nucleophilic serine ß-lactamases (SBLs) are widely used, these are increasingly susceptible to evolved SBLs and do not inhibit metallo-ß-lactamases (MBLs). Boronic acids and boronate esters, especially cyclic ones, can potently inhibit both SBLs and MBLs. Vaborbactam, a monocyclic boronate, is approved for clinical use, but its ß-lactamase coverage is limited. Bicyclic boronates rapidly react with SBLs and MBLs forming stable enzyme-inhibitor complexes that mimic the common anionic high-energy tetrahedral intermediates in SBL/MBL catalysis, as revealed by crystallography. The ability of boronic acids to 'morph' between sp2 and sp3 hybridisation states may help enable potent inhibition. There is limited structure-activity relationship information on the (bi)cyclic boronate inhibitors compared to ß-lactams, hence scope for creativity towards new boron-based ß-lactamase inhibitors/antibacterials.

Targeting the Mycobacterium tuberculosis transpeptidase LdtMt2 with cysteine-reactive inhibitors including ebselen.

The l,d-transpeptidases (Ldts) are promising antibiotic targets for treating tuberculosis. We report screening of cysteine-reactive inhibitors against LdtMt2 from Mycobacterium tuberculosis. Structural studies on LdtMt2 with potent inhibitor ebselen reveal opening of the benzisoselenazolone ring by a nucleophilic cysteine, forming a complex involving extensive hydrophobic interactions with a substrate-binding loop.

Bicyclic Boronate VNRX-5133 Inhibits Metallo- and Serine-ß-Lactamases.

The bicyclic boronate VNRX-5133 (taniborbactam) is a new type of ß-lactamase inhibitor in clinical development. We report that VNRX-5133 inhibits serine-ß-lactamases (SBLs) and some clinically important metallo-ß-lactamases (MBLs), including NDM-1 and VIM-1/2. VNRX-5133 activity against IMP-1 and tested B2/B3 MBLs was lower/not observed. Crystallography reveals how VNRX-5133 binds to the class D SBL OXA-10 and MBL NDM-1. The crystallographic results highlight the ability of bicyclic boronates to inhibit SBLs and MBLs via binding of a tetrahedral (sp3) boron species. The structures imply conserved binding of the bicyclic core with SBLs/MBLs. With NDM-1, by crystallography, we observed an unanticipated VNRX-5133 binding mode involving cyclization of its acylamino oxygen onto the boron of the bicyclic core. Different side-chain binding modes for bicyclic boronates for SBLs and MBLs imply scope for side-chain optimization. The results further support the "high-energy-intermediate" analogue approach for broad-spectrum ß-lactamase inhibitor development and highlight the ability of boron inhibitors to interchange between different hybridization states/binding modes.

Studies on the Interaction of the Histone Demethylase KDM5B with Tricarboxylic Acid Cycle Intermediates.

Methylation of lysine-4 of histone H3 (H3K4men) is an important regulatory factor in eukaryotic transcription. Removal of the transcriptionally activating H3K4 methylation is catalyzed by histone demethylases, including the Jumonji C (JmjC) KDM5 subfamily. The JmjC KDMs are Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenases, some of which are associated with cancer. Altered levels of tricarboxylic acid (TCA) cycle intermediates and the associated metabolites D- and L-2-hydroxyglutarate (2HG) can cause changes in chromatin methylation status. We report comprehensive biochemical, structural and cellular studies on the interaction of TCA cycle intermediates with KDM5B, which is a current medicinal chemistry target for cancer. The tested TCA intermediates were poor or moderate KDM5B inhibitors, except for oxaloacetate and succinate, which were shown to compete for binding with 2OG. D- and L-2HG were moderate inhibitors at levels that might be relevant in cancer cells bearing isocitrate dehydrogenase mutations. Crystallographic analyses with succinate, fumarate, L-malate, oxaloacetate, pyruvate and D- and L-2HG support the kinetic studies showing competition with 2OG. An unexpected binding mode for oxaloacetate was observed in which it coordinates the active site metal via its C-4 carboxylate rather than the C-1 carboxylate/C-2 keto groups. Studies employing immunofluorescence antibody-based assays reveal no changes in H3K4me3 levels in cells ectopically overexpressing KDM5B in response to dosing with TCA cycle metabolite pro-drug esters, suggesting that the high levels of cellular 2OG may preclude inhibition. The combined results reveal the potential for KDM5B inhibition by TCA cycle intermediates, but suggest that in cells, such inhibition will normally be effectively competed by 2OG.

Influence of Endodontic Treatment and Retreatment on the Fatigue Failure Load, Numbers of Cycles for Failure, and Survival Rates of Human Canine Teeth.

INTRODUCTION: This study evaluated the effects of endodontic treatment and retreatment on the fatigue failure load, numbers of cycles for failure, and survival rates of canine teeth. METHODS: Sixty extracted canine teeth, each with a single root canal, were selected and randomly divided into 4 groups (n = 15): untreated, teeth without endodontic intervention; prepared, teeth subjected only to rotary instrumentation; filled, teeth receiving complete endodontic treatment; and retreated, teeth retreated endodontically. After the different endodontic interventions, the specimens were subjected to fatigue testing by the stepwise method: 200 N (× 5000 load pulses), 300 N, 400 N, 500 N, 600 N, 800 N, and 900 N at a maximum of 30,000 load pulses each or the occurrence of fracture. Data from load to failure and numbers of cycles for fracture were recorded and subjected to Kaplan-Meier and Log Rank tests (P < .05), in addition to Weibull analysis. The fractures of the specimens were classified as repairable or catastrophic. RESULTS: The retreated, filled, and untreated groups presented statistically significantly higher fatigue failure loads and numbers of cycles for failure than did the prepared group. Weibull analysis showed no statistically significant difference among the treatments for characteristic load to failure and characteristic number of cycles for failure, although, for number of cycles, a higher Weibull modulus was observed in filled and retreated conditions. The predominant mode of failure was catastrophic. CONCLUSION: Teeth subjected to complete endodontic treatment and retreatment behaved similarly in terms of fatigue failure load and number of cycles to failure when compared with untreated teeth.

Incidence of Dentinal Defects and Vertical Root Fractures after Endodontic Retreatment and Mechanical Cycling.

INTRODUCTION: The aim of this study was to evaluate the incidence of dentinal defects and vertical root fractures (VRFs) after endodontic retreatment and mechanical cycling (MC). METHODS AND MATERIALS: Two hundred mandibular premolars were selected. Forty teeth were left unprepared (control group). The remaining 160 root canals were prepared with ProTaper instruments and filled by using two different techniques [eighty with lateral compaction (LC) and eighty with single-cone (SC)]. Forty canals from each group (LC and SC) received no further treatment. The remaining eighty teeth were divided into two groups (LCR and SCR) (n=40) in order to undergo the removal of the root filling, re-preparation and refilling with lateral compaction and single-cone, respectively. All of the teeth were subjected to MC (1000000 cycles, 130 N, 2.2 Hz and 37°C). The roots were sectioned at 3, 6 and 9 mm from the apex and observed under 20× magnification. The defects were classified as: no defect, VRF and other defects. Statistical analysis was performed using the Fisher's Exact test and the Chi-Squared tests (α=0.05). RESULTS: MC alone did not promote any other defects or VRFs. Experimental groups presented higher dentinal defects than the control group (P=0.021). Retreatment groups did not present a higher amount of dentinal defects than the groups that were subjected to the first treatment (P>0.05). CONCLUSION: Endodontic treatment and retreatment, regardless of the filling technique and MC, did not influence the occurrence of dentinal defects or VRFs in the human premolars.

Resistance profiles to antimicrobial agents in bacteria isolated from acute endodontic infections: systematic review and meta-analysis.

Infected root canal or acute apical abscess exudates can harbour several species, including Fusobacterium, Porphyromonas, Prevotella, Parvimonas, Streptococcus, Treponema, Olsenella and not-yet cultivable species. A systematic review and meta-analysis was performed to assess resistance rates to antimicrobial agents in clinical studies that isolated bacteria from acute endodontic infections. Electronic databases and the grey literature were searched up to May 2015. Clinical studies in humans evaluating the antimicrobial resistance of primary acute endodontic infection isolates were included. PRISMA guidelines were followed. A random-effect meta-analysis was employed. The outcome was described as the pooled resistance rates for each antimicrobial agent. Heterogeneity and sensitivity analyses were performed. Subgroup analyses were conducted based upon report or not of the use of antibiotics prior to sampling as an exclusion factor (subgroups A and B, respectively). Data from seven studies were extracted. Resistance rates for 15 different antimicrobial agents were evaluated (range, 3.5-40.0%). Lower resistance rates were observed for amoxicillin/clavulanic acid and amoxicillin; higher resistance rates were detected for tetracycline. Resistance rates varied according to previous use of an antimicrobial agent as demonstrated by the subgroup analyses. Heterogeneity was observed for the resistance profiles of penicillin G in subgroup A and for amoxicillin, clindamycin, metronidazole and tetracycline in subgroup B. Sensitivity analyses demonstrated that resistance rates changed for metronidazole, clindamycin, tetracycline and amoxicillin. These findings suggest that clinical isolates had low resistance to ß-lactams. Further well-designed studies are needed to clarify whether the differences in susceptibility among the antimicrobial agents may influence clinical responses to treatment.

Efeito in vitro da laserterapia e da terapia fotodinâmica na redução de bactérias presentes em canais radiculares / In vitro effect of lasertherapy and photodynamic therapy in bacterial reduction presents in root canals

Este estudo tem por objetivo verificar in vitro o efeito bactericida da laserterapia e da terapia fotodinâmica com laser de baixa potência (660 nm e 808 nm) em bactérias presentes nos canais radiculares. Métodos: foram preparadas 60 placas de Petri com bactérias: 20 placas com Enterococcus faecalis, 20 placas com Staphylococcus aureus e 20 com Pseudomonas aeruginosa. Aleatoriamente, dividiu-se cada grupo em 10 subgrupos (duas placas cada): três subgrupos tratados com laserterapia 660 nm em doses de 150, 225 e 300J/ cm², três subgrupos tratados com terapia fotodinâmica (azul de metileno 0,2% e laser 660 nm) em doses de 150, 225 e 300J/cm²; um subgrupo tratado com laserterapia 808 nm na dose de 225J/cm², um subgrupo com terapia fotodinâmica e laser 808 nm, em dose 225J/cm²; um subgrupo tratado apenas com fotossensibilizante (FS), e um não tratado (controle). Os tratados com laserterapia e terapia fotodinâmica foram irradiados uma única vez e incubados por 24 horas. Os últimos dois não receberam irradiação. As culturas foram analisadas visualmente para verificação do halo de inibição. Nos grupos submetidos somente à laserterapia, para o grupo FS e para o grupo controle, não foram observados halos de inibição, já onde houve aplicação da TFD, tanto com L1 quanto com L2, observaram-se halos de inibição em todas as espécies bacterianas estudadas. Conclui-se que a laserterapia, não produziu efeitos bactericidas e/ou bacteriostáticos, enquanto a terapia fotodinâmica nos dois comprimentos de onda produziu halos significativos de inibição de crescimento nas três bactérias do estudo.(AU)

This study aims to verify in vitro the bactericidal effect of laser therapy and photodynamic therapy with low power laser (660 nm and 808 nm), in bacteria present in the root canals.Methods: 60 Petri dishes were prepared with bacteria: 20 plates with Enterococcus faecalis, 20 plates with Staphylococcus aureus and 20 with Pseudomonas aeruginosa. At random, each group was divided into 10 subgroups (two plates each): three subgroups treated with 660nm laser therapy at doses of 150, 225 and 300J / cm², three subgroups treated with photodynamic therapy, (0.2% methylene blue and laser 660nm) in doses of 150, 225 and 300J / cm²; a subgroup treated with 808nm laser therapy at a dose of 225J / cm², a subgroup with (photodynamic therapy and 808nm laser) at a dose of 225J / cm²; a subgroup treated only with photosensitizer(FS), and an untreated (control). Those treated with laser therapy and photodynamic therapy were irradiated only once and incubated for 24 hours. The last two received no radiation. The cultures were analyzed visually to check the inhibition zone. In the groups submitted to laser therapy only, for the FS group and for the Control group, no inhibition halos were observed, since PDT was applied, with both L1 and L2, inhibition halos were observed in all studied bacterial species. It was concluded that laser therapy did not produce bactericidal and / or bacteriostatic effects, while photodynamic therapy at both wavelengths produced significant growth inhibition halos in the three studied bacteria.(AU)