Influence of Bile Acids on Clindamycin Hydrochloride Skin Permeability: In Vitro and In Silico Preliminary Study.

BACKGROUND AND OBJECTIVE: Topical clindamycin formulations are widely used in clinical practice, but poor bioavailability and restricted skin penetration considerably limit their therapeutic efficacy. Penetration enhancement represents a promising and rational strategy to overcome the drawbacks of conventional topical pharmaceutical formulations. We aim to assess the influence of cholic acid (CA) and deoxycholic acid (DCA) on the permeability of clindamycin hydrochloride by performing the in vitro skin parallel artificial membrane permeability assay (skin-PAMPA) at two relevant pH values (5.5 and 6.5) and the interactions of tested substances with skin ATP-binding cassette (ABC) transporters in silico. METHODS: After the incubation period, the clindamycin hydrochloride concentrations in both compartments were determined spectrophotometrically, and the apparent permeability coefficients (Papp) were calculated. Vienna LiverTox web service was used to predict the interactions of clindamycin and bile acids with potential drug transporters located in human skin. RESULTS: Both CA and DCA at the highest studied concentration of 100 µM in the tested solutions increased the skin-PAMPA membrane permeability of clindamycin hydrochloride. This effect was more pronounced for CA and at a higher studied pH value of 6.5, which is characteristic of most dermatological indications treated with topical clindamycin preparations. Clindamycin transport may also be mediated by ABC transporters located in skin and facilitated in the presence of bile acids. CONCLUSIONS: The results of this study provide a solid foundation for further research directed at the improvement of topical formulations using bile acids as penetration-enhancing excipients, as well as the therapeutic efficacy of clindamycin hydrochloride.

A method for tritiation of iboxamycin permits measurement of its ribosomal binding.

Hydrogen-tritium exchange is widely employed for radioisotopic labeling of molecules of biological interest but typically involves the metal-promoted exchange of sp2-hybridized carbon-hydrogen bonds, a strategy that is not directly applicable to the antibiotic iboxamycin, which possesses no such bonds. We show that ruthenium-induced 2'-epimerization of 2'-epi-iboxamycin in HTO (200 mCi) of low specific activity (10 Ci/g, 180 mCi/mmol) at 80 °C for 18 h affords after purification tritium-labeled iboxamycin (3.55 µCi) with a specific activity of 53 mCi/mmol. Iboxamycin displayed an apparent inhibition constant (Ki, app) of 41 ± 30 nM towards Escherichia coli ribosomes, binding approximately 70-fold more tightly than the antibiotic clindamycin (Ki, app = 2.7 ± 1.1 µM).

Selective accumulation of pharmaceutical residues from 6 different soils by plants: a comparative study on onion, radish, and spinach.

The accumulation of six pharmaceuticals of different therapeutic uses has been thoroughly investigated and compared between onion, spinach, and radish plants grown in six soil types. While neutral molecules (e.g., carbamazepine (CAR) and some of its metabolites) were efficiently accumulated and easily translocated to the plant leaves (onion > radish > spinach), the same for ionic (both anionic and cationic) molecules seems to be minor to moderate. The maximum accumulation of CAR crosses 38,000 (onion), 42,000 (radish), and 7000 (spinach) ng g-1 (dry weight) respectively, in which the most majority of them happened within the plant leaves. Among the metabolites, the accumulation of carbamazepine 10,11-epoxide (EPC - a primary CAR metabolite) was approximately 19,000 (onion), 7000 (radish), and 6000 (spinach) ng g-1 (dry weight) respectively. This trend was considerably similar even when all these pharmaceuticals applied together. The accumulation of most other molecules (e.g., citalopram, clindamycin, clindamycin sulfoxide, fexofenadine, irbesartan, and sulfamethoxazole) was restricted to plant roots, except for certain cases (e.g., clindamycin and clindamycin sulfoxide in onion leaves). Our results clearly demonstrated the potential role of this accumulation process on the entrance of pharmaceuticals/metabolites into the food chain, which eventually becomes a threat to associated living biota.

Anti-Fungal Potential of Structurally Diverse FDA-Approved Therapeutics Targeting Secreted Aspartyl Proteinase (SAP) of Candida albicans: an In Silico Drug Repurposing Approach.

In recent years, candidiasis attains major clinical importance due to its unique pathogenic strategy, which distinguishes it from other nosocomial infections. Secreted aspartyl proteinases (SAPs) is a hydrolytic enzyme secreted by Candida species that mediate versatile biological activity including hyphal formation, adherence, biofilm formation, phenotypic adaptation, etc. Emerging clinical evidence strongly suggested that conventional anti-fungal agent's are often prone to high level of resistance upon repeated exposure. Drug repurposing is an ideal strategy that shall impose the additional clinical benefits of the already approved molecules. Hence, through this realistic pathway, the potential of the suitable lead candidates will be explored in order to prolong the life span of existing molecules thereby need for newer therapeutics shall be avoided. The main aim of the present investigation is to determine the enzyme inhibitory potential of certain FDA-approved antibiotics and to validate its efficacy against the virulent enzyme secreted aspartyl proteinase (SAP) of Candida albicans via the AutoDock simulation program. The outcome of in silico dynamic simulations depicts that the drugs such as gentamicin, clindamycin, meropenem, metronidazole, and aztreonam emphasize superior binding affinity in terms of demonstrating considerable interaction with the core catalytic residues (Asp 32, Asp86, Asp 218, Gly220, Thr 221, and Thr 222). Data further indicates that the drug gentamicin exhibited best binding affinity of - 14.16 kcal/mol followed by meropenem (- 9.20 kcal/mol), clindamycin (- 9.00 kcal/mol), ciprofloxacin (- 8.95 kcal/mol), and imipenem (- 8.00 kcal/mol). In conclusion, repurposed antibiotics like gentamicin, clindamycin, meropenem, metronidazole, and aztreonam shall be considered an alternate drug of choice for the clinical management of drug resistant candida infections in the near future.

Evodiamine Enhanced the Anti-Inflammation Effect of Clindamycin in the BEAS-2B Cells Infected with H5N1 and Pneumoniae D39 Through CREB-C/EBPß Signaling Pathway.

Pneumonia is a pulmonary disease among children. Evodiamine, a traditional Chinese medicine, is known for anti-inflammatory effect. This study aimed to investigate the impact of evodiamine on severe pneumonia-like cells and the underlying mechanism involved. H5N1 and pneumoniae D39 was used to induce severe pneumonia-like conditions in BEAS-2B cells. The cell viability in BEAS-2B cells after treatments with 0, 20, 40, 60, 80, and 100 µM evodiamine was examined using MTT assays. The protein concentrations of inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1ß, and Toll-like receptors (TLRs) were measured by enzyme-linked immunosorbent assay methods and the protein and mRNA changes in C/EBPß/CREB were measured using Real Time-quantitative polymerase chain reaction and Western blot methods. Our results revealed that Evodiamine significantly decreased TNF-α, IL-6, and IL-1ß in BEAS-2B cells. Moreover, evodiamine markedly reduced TLR2,3,4 protein expression and the phosphorylated protein of C/EBPß and CREB. Besides, evodiamine combined with clindamycin exerted more significant effects than clindamycin alone. Taken together, our results demonstrated that evodiamine enhanced the anti-inflammation effect of clindamycin in the BEAS-2B cells infected with H5N1 and pneumoniae D39 through CREB-C/EBPß signaling pathway.

Molecular dynamics simulations suggest why the A2058G mutation in 23S RNA results in bacterial resistance against clindamycin.

Clindamycin, a lincosamide antibiotic, binds to 23S ribosomal RNA and inhibits protein synthesis. The A2058G mutation in 23S RNA results in bacterial resistance to clindamycin. To understand the influence of this mutation on short-range interactions of clindamycin with 23S RNA, we carried out full-atom molecular dynamics simulations of a ribosome fragment containing clindamycin binding site. We compared the dynamical behavior of this fragment simulated with and without the A2058G mutation. Molecular dynamics simulations suggest that clindamycin in the native ribosomal binding site is more internally flexible than in the A2058G mutant. Only in the native ribosome fragment did we observe intramolecular conformational change of clindamycin around its C7-N1-C10-C11 dihedral. In the mutant, G2058 makes more stable hydrogen bonds with clindamycin hindering its conformational freedom in the ribosome-bound state. Clindamycin binding site is located in the entrance to the tunnel through which the newly synthesized polypeptide leaves the ribosome. We observed that in the native ribosome fragment, clindamycin blocks the passage in the tunnel entrance, whereas in the mutated fragment the aperture is undisturbed due to a different mode of binding of clindamycin in the mutant. Restricted conformational freedom of clindamycin in a position not blocking the tunnel entrance in the A2058G mutant could explain the molecular mechanism of bacterial resistance against clindamycin occurring in this mutant.

Development and validation of a simple chromatographic method for simultaneous determination of clindamycin phosphate and rifampicin in skin permeation studies.

Rifampicin (RIF) and clindamycin phosphate (CDM) are the main drugs currently used in combination to treat severe infectious diseases in hair follicles. This work describes a simple, rapid and sensitive method for simultaneous analysis of RIF and CDM in the different skin layers using high performance liquid chromatography (HPLC). The efficient chromatographic separation of CDM and RIF was succeeded using a C18 column (150 mm x 4.6 mm, 5 µm) with gradient elution using a mobile phase composed of 0.01 M phosphoric acid and methanol at a flow rate of 1 mL min-1. Determinations were performed using UV-vis detector at 200 nm and 238 nm for CDM and RIF, respectively. The method was precise, accurate and linear (r2 > 0.999) with regression curve in the concentration range from 0.5 to 20.0 µg mL-1 and recovery rates from the skin layers higher than 85%. The retention times for CDM and RIF were approximately 7.4 and 12.2 min, respectively. The presence of skin components did not interfere with the analysis. The validated method was therefore appropriate for quantification of both CDM and RIF and thus may be feasible to be used in skin permeation studies.

Fate of Pirlimycin and Antibiotic-Resistant Fecal Coliforms in Field Plots Amended with Dairy Manure or Compost during Vegetable Cultivation.

Identification of agricultural practices that mitigate the environmental dissemination of antibiotics is a key need in reducing the prevalence of antibiotic-resistant bacteria of human health concern. Here, we aimed to compare the effects of crop (lettuce [ L.] or radish [ L.]), soil amendment type (inorganic fertilizer, raw dairy manure, composted dairy manure, or no amendment), and prior antibiotic use history (no antibiotics during previous lactation cycles vs. manure mixed from cows administered pirlimycin or cephapirin) of manure-derived amendments on the incidence of culturable antibiotic-resistant fecal coliforms in agricultural soils through a controlled field-plot experiment. Antibiotic-resistant culturable fecal coliforms were recoverable from soils across all treatments immediately after application, although persistence throughout the experiment varied by antibiotic class and time. The magnitude of observed coliform counts differed by soil amendment type. Compost-amended soils had the highest levels of cephalosporin-resistant fecal coliforms, regardless of whether the cows from which the manure was derived were administered antibiotics. Samples from control plots or those treated with inorganic fertilizer trended toward lower counts of resistant coliforms, although these differences were not statistically significant. No statistical differences were observed between soils that grew leafy (lettuce) versus rooted (radish) crops. Only pirlimycin was detectable past amendment application in raw manure-amended soils, dissipating 12 to 25% by Day 28. Consequently, no quantifiable correlations between coliform count and antibiotic magnitude could be identified. This study demonstrates that antibiotic-resistant fecal coliforms can become elevated in soils receiving manure-derived amendments, but that a variety of factors likely contribute to their long-term persistence under typical field conditions.

Assessing Antibiotic Permeability of Gram-Negative Bacteria via Nanofluidics.

While antibiotic resistance is increasing rapidly, drug discovery has proven to be extremely difficult. Antibiotic resistance transforms some bacterial infections into deadly medical conditions. A significant challenge in antibiotic discovery is designing potent molecules that enter Gram-negative bacteria and also avoid active efflux mechanisms. Critical analysis in rational drug design has been hindered by the lack of effective analytical tools to analyze the bacterial membrane permeability of small molecules. We design, fabricate, and characterize the nanofluidic device that actively loads more than 200 single bacterial cells in a nanochannel array. We demonstrate a gigaohm seal between the nanochannel walls and the loaded bacteria, restricting small molecule transport to only occur through the bacterial cell envelope. Quantitation of clindamycin translocation through wild-type and efflux-deficient (ΔtolC) Escherichia coli strains via nanofluidic-interfaced liquid chromatography mass spectrometry shows higher levels of translocation for wild-type E. coli than for an efflux-deficient strain. We believe that the assessment of compound permeability in Gram-negative bacteria via the nanofluidic analysis platform will be an impactful tool for compound permeation and efflux studies in bacteria to assist rational antibiotic design.

Natural antimicrobials subtilosin and lauramide arginine ethyl ester synergize with conventional antibiotics clindamycin and metronidazole against biofilms of Gardnerella vaginalis but not against biofilms of healthy vaginal lactobacilli.

The purpose of this study was to evaluate the ability of clindamycin and metronidazole to synergize with natural antimicrobials against biofilms of bacterial vaginosis (BV)-associated Gardnerella vaginalis. Minimum bactericidal concentrations for biofilm cells (MBCs-B) were determined for each antimicrobial. The MBCs-B of lauramide arginine ethyl ester (LAE), subtilosin, clindamycin and metronidazole were 50, 69.5, 20 and 500 µg mL(-1), respectively. A checkerboard assay and isobologram were used to analyze the type of interactions between these antimicrobials. The combination of metronidazole with natural antimicrobials did not inhibit planktonic lactobacilli. Clindamycin with either LAE or with subtilosin was inhibitory for planktonic but not for biofilm-associated lactobacilli. All tested antimicrobial combinations were inhibitory for BV-associated Mobiluncus curtisii and Peptostreptococcus anaerobius. LAE and subtilosin synergized with clindamycin and metronidazole against biofilms of G. vaginalis but not biofilm-associated vaginal lactobacilli. The biofilms of BV-associated pathogens can be controlled by synergistically acting combinations of conventional antibiotics and natural antimicrobials which will help better management of current antibiotics, especially considering robust bacterial resistance. Our findings create a foundation for a new strategy in the effective control of vaginal infections.

Multi-spectral characterization & effect of metal ions on the binding of bovine serum albumin upon interaction with a lincosamide antibiotic drug, clindamycin phosphate.

The interaction of clindamycin phosphate (CP) with bovine serum albumin (BSA) is studied by using fluorescence spectra, UV-visible absorption, synchronous fluorescence spectra (SFS), CD, 3D fluorescence spectra and lifetime measurements under simulated physiological conditions. CP effectively quenched intrinsic fluorescence of BSA. The binding constants KA values are 2.540×10(5), 4.960×10(5), 7.207×10(5) L mol(-1), the number of binding sites n and corresponding thermodynamic parameters ΔG(o), ΔH(o) and ΔS(o) between CP and BSA were calculated at different temperatures. The interaction between CP and BSA occurs through dynamic quenching and the effect of CP on the conformation of BSA was also analyzed using SFS. The average binding distance r between the donor (BSA) and acceptor (CP) was determined based on Förster's theory. The results of fluorescence spectra, UV-vis absorption spectra and SFS show that the secondary structure of the protein has been changed in the presence of CP.

First clinical isolate in Europe of clindamycin-resistant group B Streptococcus mediated by the lnu(B) gene.

We characterize the mechanisms implicated in an unusual phenotype of resistance to macrolides-lincosamides (no halos of inhibition around clindamycin and lincomycin discs, and a 15 mm halo around erythromycin disc) in a Streptococcus agalactiae isolate recovered in Spain. The presence of macrolide or lincosamide resistance genes [erm(A), erm(B), erm(C), erm(T), mef(A), mrs(A), lnu(A), lnu(B), lsa(B), lsa(C) and vga(C)] was investigated by PCR and sequencing. The strain showed a resistant phenotype to erythromycin and clindamycin (MIC = 2 mg/L and MIC = 8 mg/L, respectively) and the presence of lnu(B) and mef(A) genes was demonstrated. Clinical microbiology laboratories should be aware of this unusual phenotype due to the association of two mechanisms mediated by lnu(B) and mef(A) genes. This constitute, to our knowledge, the first report of lnu(B) in S. agalactiae in human isolates in Europe.

A SAM-dependent methyltransferase cotranscribed with arsenate reductase alters resistance to peptidyl transferase center-binding antibiotics in Azospirillum brasilense Sp7.

The genome of Azospirillum brasilense harbors a gene encoding S-adenosylmethionine-dependent methyltransferase, which is located downstream of an arsenate reductase gene. Both genes are cotranscribed and translationally coupled. When they were cloned and expressed individually in an arsenate-sensitive strain of Escherichia coli, arsenate reductase conferred tolerance to arsenate; however, methyltransferase failed to do so. Sequence analysis revealed that methyltransferase was more closely related to a PrmB-type N5-glutamine methyltransferase than to the arsenate detoxifying methyltransferase ArsM. Insertional inactivation of prmB gene in A. brasilense resulted in an increased sensitivity to chloramphenicol and resistance to tiamulin and clindamycin, which are known to bind at the peptidyl transferase center (PTC) in the ribosome. These observations suggested that the inability of prmB:km mutant to methylate L3 protein might alter hydrophobicity in the antibiotic-binding pocket of the PTC, which might affect the binding of chloramphenicol, clindamycin, and tiamulin differentially. This is the first report showing the role of PrmB-type N5-glutamine methyltransferases in conferring resistance to tiamulin and clindamycin in any bacterium.

Determination of clindamycin and its metabolite clindamycin sulfoxide in diverse sewage samples.

In a research project on risk management of harmful substances in water cycles, clindamycin and 12 further antibiotics were determined in different sewage samples. In contrast to other antibiotics, an increase of the clindamycin concentration in the final effluent in comparison to the influent of the sewage treatment plant (STP) was observed. A back transformation from the main metabolite clindamycin sulfoxide to clindamycin during the denitrification process has been discussed. Therefore, the concentration of this metabolite was measured additionally. Clindamycin sulfoxide was stable in the STP and the assumption of back transformation of the metabolite to clindamycin was confuted. To explain the increasing clindamycin concentration in the STP, the ratio of clindamycin sulfoxide to clindamycin was observed. The ratio increased in dry spells with concentrated samples and with long dwell time in the sewer system. A short hydraulic retention in waste water system and diluted samples in periods of extreme rainfall lead to a lower ratio of clindamycin sulfoxide to clindamycin concentration. A plausible explanation of this behavior could be that clindamycin was adsorbed strongly to a component of the sewage during this long residence time and in the STP, clindamycin was released. In the common sample preparation in the lab, clindamycin was not released. Measurements of clindamycin and clindamycin sulfoxide in the influent and effluent of STP is advised for sewage monitoring.

Clindamycin binding to ribosomes revisited: foot printing and computational detection of two binding sites within the peptidyl transferase center.

Clindamycin is a semi-synthetic lincosamide, active against most Gram-positive bacteria and some protozoa. It binds to the 50S ribosomal subunit and inhibits early peptide chain elongation. By kinetic analysis it has been shown that clindamycin (I) competitively interacts with the A-site of translating ribosomes (C) to form the encounter complex CI, which then slowly isomerizes to a tighter complex, termed C*I. As the final complex is capable of synthesizing peptide bonds with decreased velocity, it was assumed that in C*I complex the drug is fixed near the P-site of the ribosome. In the present study, two series of chemical foot printing experiments were carried out. In the first series, clindamycin and ribosomal complex C were incubated for 1 s and then DMS or kethoxal was added (CI probing). In the second series, complex C was preincubated with clindamycin for 1 min before the addition of DMS or kethoxal (C*I probing). It was found that clindamycin in CI complex protects A2451 and A2602 from chemical probing, both located within the A-site of the catalytic center. In contrast, it strongly protects G2505 in C*I complex, which is a discrete foot print of peptidyl-tRNA bound to the P-site. In both CI and C*I complexes, clindamycin also protects nucleotides A2058 and A2059, located next to the entrance of the exit-tunnel where the nascent peptide leaves the ribosome. Polyamines negatively affect the protection of G2505, but favor the protection of A2451 and A2602 nucleotides. Structure modeling confirms the kinetic and chemical foot printing results and suggests that clindamycin mode of action is more complex than a simple competitive inhibition of peptide bond formation.

Interaction of dihydrofolate reductase and aminoglycoside adenyltransferase enzyme from Klebsiella pneumoniae multidrug resistant strain DF12SA with clindamycin: a molecular modelling and docking study.

Klebsiella pneumoniae strain DF12SA (HQ114261) was isolated from diabetic foot wounds. The strain showed resistance against ampicillin, kanamycin, gentamicin, streptomycin, spectinomycin, trimethoprim, tetracycline, meropenem, amikacin, piperacillin/tazobactam, augmentin, co-trimoxazole, carbapenems, penicillins and cefoperazone, and was sensitive to clindamycin. Molecular characterization of the multidrug-resistance phenotype revealed the presence of a class 1 integron containing two genes, a dihydrofolate reductase (DHFR) (PF00186), which confers resistance to trimethoprim; and aminoglycoside adenyltransferase (AadA) (PF01909), which confers resistance to streptomycin and spectinomycin. A class 1 integron in K. pneumoniae containing these two genes was present in eight (18.18%) out of 44 different diabetic foot ulcer (DFU) patients. Hence, there is a need to develop therapeutics that inhibit growth of multidrug resistant K. pneumoniae in DFU patients and still achieve amputation control. Am attempt was made to create a 3D model and find a suitable inhibitor using an in silico study. Rational drug design/testing requires crystal structures for DHFR and AadA. However, the structures of DHFR and AadA from K. pneumoniae are not available. Modelling was performed using Swiss Model Server and Discovery Studio 3.1. The PDBSum server was used to check stereo chemical properties using Ramachandran plot analysis of modeled structures. Clindamycin was found to be suitable inhibitor of DHFR and AadA. A DockingServer based on Autodock & Mopac was used for docking calculations. The amino acid residues Ser(32), Ile(46), Glu(53), Gln(54), Phe(57), Thr(72), Met(76), Val(78), Leu(79), Ser(122), Tyr(128), Ile(151) in case of DHFR and Phe(34), Asp(60), Arg(63), Gln(64), Leu(68), Glu(87), Thr(89), Val(90) for AadA were found to be responsible for positioning clindamycin into the active site. The study identifies amino acid residues crucial to 'DHFR and AadA -drug' and 'DHFR and AadA -inhibitor' interactions that might be useful in the ongoing search for a versatile DHFR and AadA -inhibitor.

Antibiotics that bind to the A site of the large ribosomal subunit can induce mRNA translocation.

In the absence of elongation factor EF-G, ribosomes undergo spontaneous, thermally driven fluctuation between the pre-translocation (classical) and intermediate (hybrid) states of translocation. These fluctuations do not result in productive mRNA translocation. Extending previous findings that the antibiotic sparsomycin induces translocation, we identify additional peptidyl transferase inhibitors that trigger productive mRNA translocation. We find that antibiotics that bind the peptidyl transferase A site induce mRNA translocation, whereas those that do not occupy the A site fail to induce translocation. Using single-molecule FRET, we show that translocation-inducing antibiotics do not accelerate intersubunit rotation, but act solely by converting the intrinsic, thermally driven dynamics of the ribosome into translocation. Our results support the idea that the ribosome is a Brownian ratchet machine, whose intrinsic dynamics can be rectified into unidirectional translocation by ligand binding.

Poly(D,L-lactide-co-glycolide)/hydroxyapatite core-shell nanospheres. Part 3: properties of hydroxyapatite nano-rods and investigation of a distribution of the drug within the composite.

A step-by-step analysis of the formation and the drug loading of the poly(D,L-lactide-co-glycolide)/hydroxyapatite (PLGA/HAp) composite was carried out in a perspective of the following parameters: the structure, the morphology and the adsorption/desorption properties of the composite's bioceramic part. The authors demonstrated the importance of the material's capacity to form a fine dispersion of solid HAp particles, as an initial step, for the further loading of the drug and for the formation of the core-shell structures. The nanometer-sized rods of HAp have the capacity of ensuring a rapid adsorption and a controlled desorption of the drug from their surface, and they can act as a nucleating site for the formation of polymeric cores. Each component of this material was labeled with fluorescence dye, which enabled an insight into the distribution of the components in the core-shells that were obtained as the final outcome. Such an analysis showed a high level of uniformity among the cores enclosed within polymeric shells. From a practical perspective, the labeling of each component of the composite can be regarded as an additional functionality of the material: labeling can enable us to monitor its action during the healing process. This ability to be easily detected is expected to enhance the procedure for the controlled delivery of antibiotics after their local implantation of carriers loaded with the antibiotic and to provide more careful control over this process.

Collaborative evaluation of an erythromycin-clindamycin combination well for detection of inducible clindamycin resistance in beta-hemolytic streptococci by use of the CLSI broth microdilution method.

Constitutive or inducible clindamycin resistance can occur in beta-hemolytic streptococci due to the presence of an erm gene. The Clinical and Laboratory Standards Institute (CLSI) has recommended a disk approximation test (D-zone test) with erythromycin and clindamycin disks and a single-well broth test combining erythromycin and clindamycin for detection of inducible clindamycin resistance in staphylococci, but only a disk approximation test for the beta-hemolytic streptococci. This collaborative study assessed two different erythromycin and clindamycin concentration combinations in single wells (1 µg/ml + 0.25 µg/ml [erythromycin plus clindamycin] and 1 µg/ml + 0.5 µg/ml) with three different brands of Mueller-Hinton broth supplemented with 3% lysed horse blood for testing of frozen panels prepared for this study. All labs performed the D-zone test as described by the CLSI. A total of 155 nonduplicate streptococcal isolates (50 group A, 48 group B, 28 group C, and 29 group G isolates) were tested; 99 isolates showed inducible resistance by the D-zone test. There were some differences noted based upon the test medium. The sensitivity of the erythromycin plus clindamycin combination of 1 µg/ml + 0.25 µg/ml was 91 to 100%, while the sensitivity of the combination of 1 µg/ml + 0.5 µg/ml was 95 to 100%. Specificity overall was 98%. The slightly higher sensitivity of the combination of 1 µg/ml + 0.5 µg/ml is recommended. This study has demonstrated that a single-well microdilution test incorporating erythromycin and clindamycin in combination is a sensitive and specific indicator of inducible clindamycin resistance and could be included in routine test panels.

Pharmacokinetics of clindamycin administered orally to pigeons.

To determine the plasma concentration of clindamycin in pigeons after oral administration, 12 rock pigeons (Columba livia) were used in a 2-phase study. In the first phase, 8 pigeons received clindamycin by gavage at 100 mg/kg as a single dose. Blood samples were collected at 0, 0.25, 0.5, 1, 2, 3, 4, and 6 hours, and the plasma was separated, frozen, and subsequently analyzed by liquid chromatography-mass spectrometry for clindamycin and its active metabolites, N-demethylclindamycin (NCLD) and clindamycin sulfoxide. Clindamycin was rapidly absorbed with plasma concentrations peaking at 0.5 hours at 1.43 microg/mL. The terminal half-life (t(1/2)) was 1.25 hours, and the mean residence time was 2.49 hours. N-demethylclindamycin was detected in 7 of 8 birds (88%), whereas clindamycin sulfoxide was not found in any samples. In phase 2, clindamycin was administered to 3 birds by gavage at 100 mg/ kg q6h for 5 doses. Mean peak plasma concentrations were 2.46 and 0.64 microg/mL, with trough concentrations of 0.11 and 0.44 microg/mL for clindamycin and NCLD, respectively. No adverse effects were observed in any birds. Based on an additive antimicrobial effect of NCLD with clindamycin, an oral dosage of 100 mg/kg q6h in pigeons should reach effective plasma concentrations against common susceptible pathogens. If dose proportionality exists, lower doses and longer intervals likely produce subtherapeutic concentrations to treat systemic infections. How well birds would tolerate an extended oral dose regimen, how frequently birds fail to produce the active metabolite critical for an additive effect, and the application of these results to other avian species require further study.