Residence Time-Extended Nanoparticles by Magnetic Field Improve the Eradication Efficiency of Helicobacter pylori.

Helicobacter pylori infection is one of the leading causes of several gastroduodenal diseases, such as gastritis, peptic ulcer, and gastric cancer. In fact, H. pylori eradication provides a preventive effect against the incidence of gastric cancer. Amoxicillin is a commonly used antibiotic for H. pylori eradication. However, due to its easy degradation by gastric acid, it is necessary to administer it in a large dosage and to combine it with other antibiotics. This complexity and the strong side effects of H. pylori eradication therapy often lead to treatment failure. In this study, the chitosan/poly (acrylic acid) particles co-loaded with superparamagnetic iron oxide nanoparticles and amoxicillin (SPIO/AMO@PAA/CHI) are used as drug nano-carriers for H. pylori eradication therapy. In vitro and in vivo results show that the designed SPIO/AMO@PAA/CHI nanoparticles are biocompatible and could retain the biofilm inhibition and the bactericidal effect of amoxicillin against H. pylori. Moreover, the mucoadhesive property of chitosan allows SPIO/AMO@PAA/CHI nanoparticles to adhere to the gastric mucus layer and rapidly pass through the mucus layer after exposure to a magnetic field. When PAA is added, it competes with amoxicillin for chitosan, so that amoxicillin is quickly and continuously released between the mucus layer and the gastric epithelium and directly acts on H. pylori. Consequently, the use of this nano-carrier can extend the drug residence time in the stomach, reducing the drug dose and treatment period of H. pylori eradication therapy.

Effect of the Association and Evaluation of the Induction to Adaptation of the (+)-α-pinene with Commercial Antimicrobials against Strains of Escherichia coli.

BACKGROUND: The increasing and inappropriate use of antibiotics has increased the number of multidrug-resistant microorganisms to these drugs, causing the emergence of infections that are difficult to control and manage by health professionals. As an alternative to combat these pathogens, some monoterpenes have harmful effects on the bacterial cell membrane, showing themselves as an alternative in combating microorganisms. Therefore, the positive enantiomer α -pinene becomes an alternative to fight bacteria, since it was able to inhibit the growth of the species Escherichia coli ATCC 25922, demonstrating the possibility of its use as an isolated antimicrobial or associated with other drugs. AIMS: The aim of this study is to evaluate the sensitivity profile of E. coli ATCC 25922 strain against clinical antimicrobials associated with (+) -α-pinene and how it behaves after successive exposures to subinhibitory concentrations of the phytochemicals. METHODS: The minimum inhibitory concentration (MIC) was determined using the microdilution method. The study of the modulating effect of (+) -α-pinene on the activity of antibiotics for clinical use in strains of E. coli and the analysis of the strain's adaptation to the monoterpene were tested using the adapted disk-diffusion method. RESULTS: The results demonstrate that the association of monoterpene with the antimicrobials ceftazidime, amoxicillin, cefepime, cefoxitin and amikacin is positive since it leads to the potentiation of the antibiotic effect of these compounds. It was observed that the monoterpene was able to induce crossresistance only for antimicrobials: cefuroxime, ceftazidime, cefepime and chloramphenicol. CONCLUSION: It is necessary to obtain more concrete data for the safe use of these combinations, paying attention to the existence of some type of existing toxicity reaction related to the herbal medicine and to understand the resistance mechanisms acquired by the microorganism.

Smart drug delivery against Helicobacter pylori: pectin-coated, mucoadhesive liposomes with antiadhesive activity and antibiotic cargo.

The first step in the development of Helicobacter pylori pathogenicity is the receptor-mediated adhesion to the gastric epithelium. Inhibition of outer membrane proteins of H. pylori (e.g. BabA) by antiadhesive drugs will contribute to reduced recolonization and infection. Pectin from apple inhibits the BabA and LPS-mediated adhesion of H. pylori to human stomach cells. Pectin-coated liposomes with encapsulated amoxicillin were characterized for polydispersity, zeta potential, encapsulation efficiency, stability, and amoxicillin release. Coated liposomes did not influence the viability of AGS and HT29-MTX cells up to 100 µg/mL but exert cytotoxicity against H. pylori at 10 µg/mL. Pectin-coating of liposomes provoked direct interaction and subsequent binding of the particles to surface structures of H. pylori, and interaction with mucus from porcine stomach and mucus secreted by HT29-MTX cells. Laser scanning microscopy of H. pylori and AGS cells together with liposomes indicated co-aggregation. The mucoadhesive effect seems interesting as stomach cells are covered by a mucus layer. H. pylori is able to penetrate and cross the mucin rapidly to reach pH-neutral epithelium to escape the acidic environment, followed by interaction with epithelial cells. In summary, all experimental evidence is consistent with a specific interaction of pectin-coated liposomes with mucins and surface structures of H. pylori. As the coated liposomes show mucoadhesion to the negatively charged mucins, docking to stomach mucin, mucus penetration, and recognition of and adhesion to H. pylori, they can be considered a novel type of multifunctional drug carriers for local antibiotic therapy against H. pylori. KEY POINTS: • Smart, multifunctional mucoadhesive liposomes • Specific targeting against BabA/LPS of Helicobacter pylori • Inhibition of bacterial adhesion of H. pylori to human host cells • Release of antibiotic cargo.

Tuning down the environmental interests of organoclays for emerging pollutants: Pharmaceuticals in presence of electrolytes.

The impact of electrolytes on the adsorption of emerging pollutants: pharmaceuticals onto layered materials: a raw clay mineral and its nonionic and cationic organoclay derivatives was studied. The selected pharmaceuticals: amoxicillin, norfloxacin, sulfamethoxazole, metoprolol, carbamazepine, and trimethoprim show different electric charges: zwitterionic, anionic, cationic and neutral and hydrophobic character (different LogP). Without any salts, the set of complementary data obtained by UV and infrared spectroscopies, X-ray diffraction points out the importance of the electric charge which represents a key parameter in both the spontaneity and feasibility of the adsorption. In contrast, the hydrophobicity of the analytes plays a minor role but determines the magnitude of the adsorbed amount of pharmaceuticals onto organoclays. With a dual hydrophilic and hydrophobic behavior, nonionic organoclay appears to be the most polyvalent material for the removal of the pharmaceuticals. In the presence of electrolytes (NaCl at a concentration of 1 × 10-2 mol L-1), both nonionic and cationic organoclays show a decrease of their efficiencies, whereas the adsorption is particularly enhanced for Na-Mt except for the cationic species (trimethoprim and metoprolol). Thus, in realistic experimental conditions close to those of natural effluents, raw clay mineral appears as the most appropriate sorbent for the studied pharmaceuticals while it raises the question of the usefulness of organoclays in water remediation strategy.

Pig liver esterases PLE1 and PLE6: heterologous expression, hydrolysis of common antibiotics and pharmacological consequences.

Carboxylesterases, historically referred as non-specific esterases, are ubiquitous hydrolases with high catalytic efficiency. Without exceptions, all mammalian species studied contain multiple forms of carboxylesterases. While having been widely studied in humans and experimental animals, these enzymes remain to be characterized in farm animals. In this study, we showed that pig liver esterase 1 (PLE1) and pig liver esterase 6 (PLE6) were highly active toward amoxicillin (AMO) and ampicillin (AMP), two major antibiotics that are widely used in food-supplements. Mass-spectrometric analysis established that the hydrolysis occurred at the ß-lactam amide bond and the hydrolysis drastically decreased or completely eliminated the antibacterial activity. Furthermore, hydrolytic activity and proteomic analysis suggested that trace PLEs existed in pig plasma and contributed little to the hydrolysis of AMO and AMP. These results suggested that carboxylesterases-based hydrolysis determines the therapeutic intensity of these and related antibiotics and the magnitude of the determination occurs in a species-dependent manner.

Effect of Drug Combination on Omeprazole Metabolism by Cytochrome P450 2C19 in Helicobacter pylori Eradication Therapy.

Helicobacter pylori (H. pylori) infection is common and can result in gastric and duodenal ulcers, and in some cases, gastric lymphoma and cancer. Omeprazole (OMP)-in combination with clarithromycin (CLR), amoxicillin (AMX), tinidazole (TND), or metronidazole (MET)-is used in double or triple combination therapy for eradication of H. pylori. However, the roles of the drugs other than OMP are not clearly understood. Therefore, in the present study, we aimed to investigate any effects of these drugs on OMP metabolism by wild-type CYP2C19 using spectroscopy and enzyme kinetics. The dissociation constants (Kd) for CYP2C19 with OMP, CLR, AMX, TND, and MET were 8.6, 126, 156, 174, and 249 µM, respectively. The intrinsic clearance of OMP was determined to be 355 mL/min/µmol of CYP2C19. Metabolism of OMP was significantly inhibited by 69, 66, 28, and 40% in the presence of CLR, TND, AMX, and MET, respectively. Moreover, the combination of CLR and TND resulted in 76% inhibition of OMP metabolism, while the combination of AMX and MET resulted in 48% inhibition of OMP metabolism. Both combinations of drugs not only have antibacterial effects, but also enhance the effect of OMP by inhibiting its metabolism by CYP2C19. These results indicate that drug-drug interactions of co-administered drugs can cause complex effects, providing a basis for OMP dose adjustment when used in combination therapy for H. pylori eradication.

Overcoming stability challenges during continuous intravenous administration of high-dose amoxicillin using portable elastomeric pumps.

While treatment of serious infectious diseases may require high-dose amoxicillin, continuous infusion may be limited by lack of knowledge regarding the chemical stability of the drug. Therefore, we have performed a comprehensive study so as to determine the chemical stability of high-dose amoxicillin solutions conducive to safe and effective continuous intravenous administration using portable elastomeric pumps. First, amoxicillin solubility in water was assessed within the range of 25 to 300 mg/mL. Then, amoxicillin solutions were prepared at different concentrations (25, 50, 125, 250 mg/mL) and stored in different conditions (5±2°C, 25±1°C, 30±1°C and 37±1°C) to investigate the influence of concentration and temperature on the chemical stability of amoxicillin. Finally, its stability was assessed under optimized conditions using a fully validated HPLC-UV stability-indicating method. Degradation products of amoxicillin were investigated by accurate mass determination using high-resolution mass spectrometry. Amoxicillin displayed limited water solubility requiring reconstitution at concentrations below or equal to 150 mg/mL. Amoxicillin degradation were time, temperature as well as concentration-dependent, resulting in short-term stability, in particular at high concentrations. Four degradation products of amoxicillin have been identified. Among them, amoxicilloic acid and diketopiperazine amoxicillin are at risk of allergic reaction and may accumulate in the patient. Optimized conditions allowing for continuous infusion of high-dose amoxicillin has been determined: amoxicillin should be reconstituted at 25 mg/mL and stored up to 12 hours at room temperature (22 ± 4°C) or up to 24 hours between 4 and 8°C.

Fabrication of leaf extract mediated bismuth oxybromide/oxyiodide (BiOBrxI1-x) photocatalysts with tunable band gap and enhanced optical absorption for degradation of organic pollutants.

The use of Azadirachta indica (A.I.) leaf extract to synthesize green photocatalysts for efficient separation of photogenerated charges has been a promising way to enhance the photocatalytic activity. Herein, we report the synthesis of green bismuth oxybromide/oxyiodide composites (G-BiOBrxI1-x) using A.I. leaf extract with effective size control, high specific surface area, and porosity. The A.I. leaf extract also acted as an excellent sensitizer that boosted the optical window of the G-BiOBrxI1-x photocatalysts. The as-prepared G-BiOBrxI1-x photocatalysts possessed three-dimensional (3-D) nanoplates like structure with successive modulation of the band gaps from 2.28 eV to 1.98 eV by varying the bromine/iodine (Br/I) ratio. Furthermore, the photocatalytic activity of the G-BiOBrxI1-x samples was measured and compared with the bismuth oxybromide/oxyiodide composite (C-BiOBr0.5I0.5) synthesized via conventional hydrolysis route (without the leaf extract). The G-BiOBrxI1-x photocatalysts degraded higher percentage of methyl orange (MO) and amoxicillin (AMX) than C-BiOBr0.5I0.5 under visible light irradiation. The superior photocatalytic efficiency was attributed to the multiple heterojunctions developed between BiOBr, BiOI, and electron-accepting π-conjugated system offered by leaf extract constituents, thereby facilitating the charge transfer process and effective separation of photogenerated charges.

Surface restructuring of red mud to produce FeO x (OH) y sites and mesopores for the efficient complexation/adsorption of ß-lactam antibiotics.

In this work, iron oxide in the red mud (RM) waste was restructured to produce mesopores with surface [FeO x (OH) y ] sites for the efficient complexation/adsorption of ß-lactam antibiotics. Red mud composed mainly by hematite was restructured by an acid/base process followed by a thermal treatment at 150-450 °C (MRM150, MRM200, MRM300, and MRM450) and fully characterized by Mössbauer, XRD, FTIR, BET, SEM, CHN, and thermogravimetric analyses. The characterization data showed a highly dispersed Fe3+ oxyhydroxy phase, which was thermally dehydrated to a mesoporous α-Fe2O3 with surface areas in the range of 141-206 m2 g-1. These materials showed high efficiencies (21-29 mg g-1) for the adsorption of ß-lactam antibiotics, amoxicillin, cephalexin, and ceftriaxone, and the data was better fitted by the Langmuir model isotherm (R 2 = 0.9993) with monolayer adsorption capacity of ca. 39 mg g-1 for amoxicillin. Experiments such as competitive adsorption in the presence of phosphate and H2O2 decomposition suggested that the ß-lactamic antibiotics might be interacting with surface [FeO x (OH) y ] species by a complexation process. Moreover, the OH/Fe ratio, BET surface area and porosity indicated that this complexation is occurring especially on [FeO x (OH) y ]surf sites contained in the mesopore space.

Floating mucoadhesive alginate beads of amoxicillin trihydrate: A facile approach for H. pylori eradication.

This study investigates the design of sunflower oil entrapped floating and mucoadhesive beads of amoxicillin trihydrate using sodium alginate and hydroxypropyl methylcellulose as matrix polymers and chitosan as coating polymer to localize the antibiotic at the stomach site against Helicobacter pylori. Beads prepared by ionotropic gellation technique were evaluated for different physicochemical, in-vitro and in-vivo properties. Beads of all batches were floated for >24h with a maximum lag time of 46.3±3.2s. Scanning electron microscopy revealed that the beads were spherical in shape with few oil filled channels distributed throughout the surfaces and small pocket structures inside the matrix confirming oil entrapment. Prepared beads showed good mucoadhesiveness of 75.7±3.0% to 85.0±5.5%. The drug release profile was best fitted to Higuchi model with non fickian driven mechanism. The optimized batch showed 100% Helicobacter pylori growth inhibition in 15h in in-vitro culture. Furthermore, X-ray study in rabbit stomach confirmed the gastric retention of optimized formulation. The results exhibited that formulated beads may be preferred to localize the antibiotic in the gastric region to allow more availability of antibiotic at gastric mucus layer acting on Helicobacter pylori, thereby improving the therapeutic efficacy.

Nanodiamond-Gutta Percha Composite Biomaterials for Root Canal Therapy.

Root canal therapy (RCT) represents a standard of treatment that addresses infected pulp tissue in teeth and protects against future infection. RCT involves removing dental pulp comprising blood vessels and nerve tissue, decontaminating residually infected tissue through biomechanical instrumentation, and root canal obturation using a filler material to replace the space that was previously composed of dental pulp. Gutta percha (GP) is typically used as the filler material, as it is malleable, inert, and biocompatible. While filling the root canal space with GP is the standard of care for endodontic therapies, it has exhibited limitations including leakage, root canal reinfection, and poor mechanical properties. To address these challenges, clinicians have explored the use of alternative root filling materials other than GP. Among the classes of materials that are being explored as novel endodontic therapy platforms, nanodiamonds (NDs) may offer unique advantages due to their favorable properties, particularly for dental applications. These include versatile faceted surface chemistry, biocompatibility, and their role in improving mechanical properties, among others. This study developed a ND-embedded GP (NDGP) that was functionalized with amoxicillin, a broad-spectrum antibiotic commonly used for endodontic infection. Comprehensive materials characterization confirmed improved mechanical properties of NDGP over unmodified GP. In addition, digital radiography and microcomputed tomography imaging demonstrated that obturation of root canals with NDGP could be achieved using clinically relevant techniques. Furthermore, bacterial growth inhibition assays confirmed drug functionality of NDGP functionalized with amoxicillin. This study demonstrates a promising path toward NDGP implementation in future endodontic therapy for improved treatment outcomes.

Mass spectrometric strategies for the identification and characterization of human serum albumin covalently adducted by amoxicillin: ex vivo studies.

This study addresses the detection and characterization of the modification of human serum albumin (HSA) by amoxicillin (AX) in ex vivo samples from healthy subjects under oral amoxicillin administration (acute intake of 1 g every 8 h for 48 h). To reach this goal, we used an analytical strategy based on targeted and untargeted mass spectrometric approaches. Plasma samples withdrawn before AX oral intake represented the negative control samples to test the method selectivity, whereas HSA incubated in vitro with AX was the positive control. Different MS strategies were developed, particularly (1) multiple reaction monitoring (MRM) and precursor ion scan (PIS) using a HPLC system coupled to a triple quadrupole MS analyzer and (2) a dedicated data-dependent scan and a customized targeted MS/MS analysis carried out using a nano-LC system coupled to a high-resolution MS system (LTQ Orbitrap XL). Lys 190 was identified as the only modification site of HSA in the ex vivo samples. The AX adduct was identified and fully characterized by complementary targeted approaches based on triple quadrupole (MRM mode) and orbitrap (SIC mode) mass analyzers. The SIC mode also permitted the relative amount of AX-adducted HSA to be measured, ranging from 1 to 2% (6-12 µM) at 24 and 48 h after the oral intake. No adduct in any ex vivo sample was identified by the untargeted methods (PIS and data-dependent scan mode analysis). The results on one hand indicate that MS, in particular high-resolution MS, analysis represents a suitable analytical tool for the identification/characterization of covalently modified proteins/peptides; on the other hand, they give deeper insight into AX-induced protein haptenation, which is required to better understand the mechanisms involved in AX-elicited allergic reactions.

Development of bilayer floating tablet of amoxicillin and Aloe vera gel powder for treatment of gastric ulcers.

Usual treatment for Helicobacter pylori-induced peptic ulcer includes a 'triple therapy' consisting of two antibiotics (amoxicillin and clarithromycin) and a proton pump inhibitor (omeprazole). The objective of this project work was defined with a view to retain the drug in stomach for better antiulcer activity and substituting one of the synthetic drugs in this therapy with a herbal alternative. Hence, aim of the present work was to design and develop a bilayer floating tablet of amoxicillin and Aloe vera gel powder for the treatment of peptic ulcer. A. vera gel powder is used for its cytoprotective action. Bilayer floating tablets were prepared by applying direct compression technique. The proportion of sodium bicarbonate and citric acid was adjusted to get the least possible lag time with good matrix integrity and total floating time. Polymer concentration was adjusted to get the maximum release in 8 h. The formulation was developed using hydroxypropyl methyl cellulose (HPMC) K4M and HPMC K100M in a ratio of 85:15 along with 1:4 ratio of effervescent agents was found to give floating lag time of less than 1 min with total floating time of more than 8 h and 97.0% drug release in 8 h. In vivo study in rats meets the requirement of antiulcer activity for bilayer tablet in comparison to single amoxicillin as standard.

A two pulse drug delivery system for amoxicillin: an attempt to counter the scourge of bacterial resistance against antibiotics.

Bearing in mind the present scenario of the increasing biological tolerance of bacteria against antibiotics, a time controlled two pulse dosage form of amoxicillin was developed. The compression coating inlay tablet approach was used to deliver the drug in two pulses to different parts of the GIT after a well defined lag time between the two releases. This was made possible by formulating a core containing one of the two drug fractions (intended to be delivered as the second pulse), which was spray coated with a suspension of ethyl cellulose and a hydrophilic but water insoluble agent as a pore former (microcrystalline cellulose). Coating of up to 5% (m/m) was applied over the core tablet, giving a corresponding lag of 3, 5, 7 and 12 h. Increasing the level of coating led to retardation of the water uptake capacity of the core, leading to prolongation of the lag time. Microcrystalline cellulose was used as a hydrophilic but water insoluble porosity modifier in the barrier layer, varying the concentration of which had a significant effect on shortening or prolongation of the lag time. This coated system was further partially compression coated with the remaining drug fraction (to be released as the first immediate release pulse) with a disintegrant, giving a final tablet. The core tablet and the final two pulse inlay tablet were further investigated for their in vitro performance.

Tracing origins of complex pharmaceutical preparations using surface desorption atmospheric pressure chemical ionization mass spectrometry.

A novel strategy to trace the origins of commercial pharmaceutical products has been developed based on the direct chemical profiling of the pharmaceutical products by surface desorption atmospheric pressure chemical ionization mass spectrometry (DAPCI-MS). Besides the unambiguous identification of active drug components, various compounds present in the matrixes are simultaneously detected without sample pretreatment, providing valuable information for drug quality control and origin differentiation. Four sources of commercial amoxicillin products made by different manufacturers have been successfully differentiated. This strategy has been extended to secerning six sources of Liuwei Dihuang Teapills, which are herbal medicine preparations with extremely complex matrixes. The photolysis status of chemical drug products and the inferior natural herd medicine products prepared with different processes (e.g., extra heating) were also screened using the method reported here. The limit of detection achieved in the MS/MS experiments was estimated to be 1 ng/g for amoxicillin inside the capsule product. Our experimental data demonstrate that DAPCI-MS is a useful tool for rapid pharmaceutical analysis, showing promising perspectives for tracking the entire pharmaceutical supply chain to prevent counterfeit intrusions.

Penicillin derivatives induce chemical structure-dependent root development, and application for plant transformation.

We investigated five penicillin derivatives that are popularly used for transformation experiments with Agrobacterium rhizogenes-penicillin G, carbenicillin, ampicillin, amoxicillin and cephalexin-for their effects on the growth and morphology of Beta vulgaris, Capsicum annuum and Glehnia littoralis roots. Attention was given to the relationship between their chemical structures and functions. Ampicillin was found to stimulate root elongation but inhibit root branching, whereas carbenicillin inhibited root elongation but promoted root branching. Root cultures were also exposed to hydrolyzed products of these antibiotics-i.e. phenylmalonic acid (PM), phenylglycine and 6-aminopenicillanic acid (6-APA): PM inhibited root elongation the most, while root elongation was supported best by 6-APA. These results indicate that both the side chains and the major component of penicillin derivatives affect root development and that the nature of the side chains is responsible for the responses. Ampicillin but not carbenicillin was used in subsequent experiments described herein to eliminate bacteria and to support root growth of transformants of the recalcitrant plants.

Fine structural recognition specificities of IgE antibodies distinguishing amoxicilloyl and amoxicillanyl determinants in allergic subjects.

IgE antibodies in the sera of subjects allergic to beta-lactam antibiotics detect a spectrum of specificities ranging from side-chain groups to an entire penicillin or cephalosporin molecule. In addition to such structural heterogeneity of allergenic determinants, IgE antibodies in the sera of different allergic subjects show heterogeneous recognition responses. Detailed immunochemical studies were carried out on the sera of penicillin-allergic subjects that showed selective and unexpected reactions with the frequently prescribed penicillin, amoxicillin. Antibodies from one subject reacted only with the amoxicilloyl determinant while IgE from another subject showed multiple reactivity with penicilloyl and penicillanyl determinants of different penicillins but not with the amoxicilloyl determinant. Quantitative hapten inhibition studies revealed that the combining sites of the former antibodies were complementary to amoxicillin in a form that permits binding to the hydroxyaminobenzyl side-chain and the thiazolidine ring carboxyl. These conditions are satisfied with the drug in the '-oyl' but not in the '-anyl' form which involves linkage through the 2-carboxyl of the thiazolidine ring. With the second serum, adsorption studies showed that the wide-ranging reactivity of IgE was due to a single population of antibodies that detected a common specificity on the different penicillins. Combining site studies revealed clear recognition of the benzyl portion of the side-chain of benzylpenicilloyl, benzylpenicillanyl, ampicilloyl, ampicillanyl and amoxicillanyl determinants when free antibody access to the side-chain was possible but little or no recognition of the ring hydroxyl of amoxicillin. Such uninhibited access may not occur, however, when amoxicillin is conjugated in the '-oyl' form since opening the beta-lactam ring allows increased flexibility and rotation of the molecule and the possibility of close association of the hydroxyaminobenzyl side-chain of amoxicillin with the linked peptide carrier. In such close steric association, H-bonding involving the ring hydroxyl and amino acids of the carrier may prevent antibody access to the side-chain region of the amoxicilloyl determinant.

Broth microdilution testing of Haemophilus influenzae with haemophilus test medium versus lysed horse blood broth. Canadian Haemophilus Study Group.

Broth microdilution testing of 702 community-acquired isolates of Haemophilus influenzae from across Canada was performed with both Mueller-Hinton broth supplemented with 3% lysed horse blood broth (LHB) (BBL Microbiology Systems, Cockeysville, Md.) and haemophilus test medium (HTM). The prevalence of beta-lactamase production was found to be 26% with no regional variation. MICs determined with LHB tended to be higher than those with HTM, but interpretive errors due to these differences were observed only rarely with trimethoprim-sulfamethoxazole (n = 5), cefaclor (n = 8), and cefamandole (n = 3). The interobserver variability in MIC determinations was found to be greater when LHB was used than when HTM was used. There was no difference in intraobserver variability between the two medium formulations. beta-Lactamase-positive isolates developed false resistance to amoxicillin-clavulanate 2 weeks after microdilution panels of both types of medium were stored at -20 degrees C but not when panels were stored at -70 degrees C. In conclusion, this study supports the use of HTM rather than LHB for sensitivity testing of H. influenzae because of its lower rate of interobserver variability and its ability to support the growth of these organisms, which is comparable to that of LHB.