COSMETICS EUROPE: GUIDELINES ON THE MANAGEMENT OF UNDESIRABLE Table of Contents: Section I – Introduction 1. Introduction 2. Definition of terms Section II – Undesirable events 1. Management 1.1. 1.2. Registration - Opening a case file 1.3. Case information and documentation
Ijms.sums.ac.irVol 38, No 3, September 2013 Effect of Medium pH on Antibiotic Activity
against Syrian Brucella spp. Isolates
Ayman Al-Mariri, PhD; Mazen Safi, PhD Abstract
Background: Brucellosis is an endemic zoonosis in Syria,
affecting large numbers of animals. There are an increasing number of cases in humans. Brucella is a facultative intracellular pathogen, a small, non-motile, Gram-negative coccobacillus, which causes abortion in domestic animals and a febrile illness Methods: One hundred isolates collected from different Syrian
regions were confirmed to be Brucella melitensis by biochemical tests. The minimum inhibitory concentration (MIC) of 6 antibiotics, alone and in combination, was determined at pH 7.0 Results: Ciprofloxacin and sparfloxacin were the most effective
antibiotics tested at either pH value. In contrast, rifampicin had low activity and streptomycin was ineffective at either pH value. A combination of rifampicin-doxycycline revealed the highest synergistic activity at both test pH values (against 19/24 and 17/24 isolates, respectively) in vitro. Antagonistic activities were observed using a ciprofloxacin-streptomycin combination (against 9/24 and 13/24 isolates, respectively) as well as a ciprofloxacin- tetracycline combination (against 6/24 and 9/24 isolates, respectively). No differences were observed at both test pH values, when combining a Quinolone with rifampicin or doxycycline. Conclusion: Combination of a Quinolone with doxycycline
demonstrated good in vitro activity against B. melitensis. Further in vivo studies are necessary to support this suggestion.
Please cite this article as: Al-Mariri A, Safi M. Effect of Medium pH on Antibiotic Activity against Syrian Brucella spp. Isolates. Iran J Med Sci. 2013;38(3):248-254.
Keywords Antibacterial Antibiotics Brucella Zoonotic
Department of Molecular Biology Brucellosis is a zoonotic disease with worldwide distribution, but it and Biotechnology, Atomic Energy is most frequent in the Mediterranean basin and South America.1 Commission, Damascus, Syria Because the bacteria are intracellular, successful treatment requires antibiotics with good cellular penetration. Different regimens have Ayman Al-Mariri, PhD; been universally applied in clinical practice.2 The most recent Department of Molecular Biology and recommendation by the World Health Organization (WHO) for the treatment of acute brucellosis in adults suggested a dose of 600 Atomic Energy Commission, P.O. Box 6091, Damascus, Syria to 900 mg rifampicin and 200 mg doxycycline daily for a minimum Tel: +963 11 213580
of 6 weeks.3 Empirically, it has been suggested that a previous Fax: +963 11 6112289
regimen of streptomycin in combination with oral tetracycline results in fewer relapses than a rifampicin-doxycycline combination.4,5 In Received: 1 March 2012 Revised: 10 July 2012 addition, rifampicin monotherapy is the main recommended option Accepted: 15 July 2012 for treatment during pregnancy, and a combination of rifampicin with Iran J Med Sci September 2013; Vol 38 No 3 Medium pH effect on antibiotic activity Trimethoprim-Sulfamethoxazole is the suggested safety cabinet was used. During the work, the regimen for children.6,7 Triple-antibiotic combinations laboratory workers were wearing impermeable have been found to be of value in some cases of protective clothes, gloves, and a face mask. brucel a endocarditis, meningitis, and spondylitis.8-10 Quinolone treatment has been shown to be a Minimum Inhibitory Concentration Determination therapeutic alternative, and various combinations at Different pH Values that incorporate ciprofloxacin and ofloxacin have In order to estimate the antibiotics susceptibility, been tried clinically, yielding similar efficacy to that the well broth microdilution method was utilized of the classic regimens.11 Only in vitro observations with 96-well plates (TPP, Switzerland). The exist for Moxifloxacin and Levofloxacin.12 antibiotics (i.e. doxycycline [Sigma, St. Louis, MO, Fluoroquinolones and newer Macrolides have good USA], rifampicin [Sigma], tetracycline [Sigma], anti-brucel osis activity in vitro13-15 and reach high streptomycin [Sigma], ciprofloxacin [Bayer, intracellular concentrations, but their in vitro activity Istanbul, Turkey], and sparfloxacin [Sigma] were may predict efficacy poorly because Brucella diluted twofold in Brucella broth® (Acumedia, survive in compartments that are inaccessible or Michigan, USA) and adjusted to pH 7.0 and pH hostile to antimicrobial activity. These include the 5.0. The wells were inoculated with 106 CFU of the phagolysosomes of macrophages, where the pH bacteria (in a 0.2-ml final volume). The incubation may be as low as 5. In consequence, specialized period was 48 h at 37°C. The lowest concentration agents that are able to penetrate the macrophages that completely inhibited visual growth was and function within their cytoplasm are required for recorded and interpreted as the minimum the treatment of brucellosis.16 Acidity impairs the inhibitory concentration (MIC). MIC testing was activity of Quinolones and Macrolides.
performed according to the recommendations of The aim of this study was to evaluate, in vitro, the Clinical Laboratory Standards (CLSI).18 The the effect of medium acidity on the activities of range of the concentrations assayed for each some antibiotics, alone and in combination, against antibiotic was 0.125 to 128 μg/ml. Escherichia coli some Syrian Brucella melitensis isolates collected ATCC 25922 and Staphylococcus aureus ATCC from different provinces. The single antibiotics 25923 served as controls. were doxycycline, rifampicin, tetracycline, streptomycin, ciprofloxacin, and sparfloxacin, Antibiotic Combination Studies whereas the antibiotic combinations were Twenty-four of the 100 Brucella isolates (six rifampicin-tetracycline, rifampicin-doxycycline, isolates from each region) were randomly chosen to evaluate the antibiotic combination effects. Checkerboard titrations were used at pH 5.0 and pH 7.0 in the same conditions to assess streptomycin, and ciprofloxacin-sparfloxacin. the MICs and to evaluate the activities of the 9 above-mentioned antibiotic combinations. Strains Materials and Methods
showing synergy, a marked additive effect, or antagonism were retested using the broth Microorganisms and Growth Conditions dilution method, with each well containing the One hundred B. melitensis isolates were final antibiotic concentration used in the plates. collected prospectively between 2004 and 2007 In this checkerboard test, the sum of the fractional from bovine and ovine milk from different Syrian inhibitory concentration (∑ FIC) was calculated as provinces. These provinces were divided into described previously.19,20 The ∑ FIC was classified four regions, as fol ows: Northern (including as follows: synergistic≤0.75; additive from 0.75 Al-Hasakah, Deer-Alzour, Al-Rakah, and Aleppo to 1; indifferent from 1 to 2; and antagonistic≥2.
Provinces); Central (including Edleb, Hamaa, and Homs Provinces); Coastal (including Tartous and Lattakia Provinces); and Southern All the analyses were conducted with version 4.0 (including Al-Quonaitra, Daraa, Al-Souaida, of GraphPad Prism. Fisher's exact test was used to Damascus, and Damascus rural Provinces). make a comparison between the susceptible and Bacteria were isolated from the milk cultures at non-susceptible isolates toward each antibiotic at the Immunology/Microbiology Laboratory, Atomic pH 5.0 and pH 7.0. A P value≤0.05 was considered Energy Commission of Syria (AECS).17 They were statistically significant. identified to the species level via conventional methods (the requirement for CO for growth, production of H S, urease production, sensitivity to thionine and basic fuchsin, and agglutination Table 1 demonstrates that, under the conditions with specific antiserum). A class II biological of our study, ciprofloxacin and sparfloxacin were Iran J Med Sci September 2013; Vol 38 No 3 Al-Mariri A, Safi M Table 1: Effect of medium pH levels on MIC
and MIC and the susceptibility percentage (Susc.%) of some antibiotics against B. melitensis isolates collected from different Syrian regions Regions
(µg/ml) 0.125-1 50% (15) 54% (16) 7% (2) 90% (18) 95% (19) 90% (18) 90% (18) 55% (11) 55% (11) 10% (2) 57% (17) 90% (27) 90% (27) 90% (27) MIC: Minimum inhibitory concentration; Susc.%: Percentage of susceptible isolates in each region; (N): Number of susceptible isolates in each region the most effective individual antibiotics against and 99% at pH 7.0, respectively. Fifty-one isolates B. melitensis from any Syrian region (Northern, were resistant to rifampicin at both pH conditions Central, Coastal, and Southern), with the MICs (particularly the isolates from the Northern (n=28) ranging from 0.125 μg/ml to 8 μg/ml. Doxycycline and and Coastal (n=18) regions), whereas 39 and 27 tetracycline were less effective than ciprofloxacin or isolates were resistant to tetracycline at pH 5.0 sparfloxacin, with the MICs ranging from 0.5 μg/ and pH 7.0, respectively. No significant differences ml to 16 μg/ml for the former and from 0.25 μg/ were observed regarding each individual antibiotic ml to 16 μg/ml for the latter; however, they were between pH 5.0 and pH 7.0, with the exception of less effective against the Brucella isolates from the effect of tetracycline against the Southern region the Coastal region. Rifampicin had the lowest isolates, where the susceptibility was decreased activity against Brucella from the Northern and the at pH 5.0 compared with that at pH 7.0 (17 vs. 27 Coastal regions; the MICs ranged from 32 μg/ml to isolates; P<0.0007). Finally, 100% of the isolates 64 μg/ml at both pH values. Table 1 also reveals were resistant to streptomycin. that the overal susceptibility rates of ciprofloxacin, Figures 1 and 2 present the data on the doxycycline, and sparfloxacin against all the isolates effects of the antibiotic combinations at pH 7.0 were 97%, 92%, and 98% at pH 5.0; and 98%, 94%, and pH 5.0, respectively, on 24 selected Brucella Figure 1: This is a representation of the activity of the antibiotic combinations at pH 7.0. R: Rifampicin; T: Tetracycline; D:
Doxycycline; C: Ciprofloxacin; S: Sparfloxacin; ST: Streptomycin; Anta: Antagonism; Ind: Indifference; Add: Additive; Syn: Synergy Iran J Med Sci September 2013; Vol 38 No 3 Medium pH effect on antibiotic activity Figure 2: This figures il ustrates the activity of the antibiotic combinations at pH 5.0. R: Rifampicin; T: Tetracycline; D: Doxycycline;
C: Ciprofloxacin; S: Sparfloxacin; ST: Streptomycin; Anta: Antagonism; Ind: Indifference; Add: Additive; Syn: Synergy isolates. The rifampicin-doxycycline combination Akova et al.20 studied the combination of showed a synergistic activity against 19 and ofloxacin-rifampicin against 20 isolates at pH 7.0 17 isolates at pH 7.0 and pH 5.0, respectively. and 5.0 and found antagonism in 17 isolates and The ciprofloxacin-doxycycline, ciprofloxacin- indifference in 3 isolates at pH 7.0. In contrast, at sparfloxacin, and rifampicin-sparfloxacin pH 5.0, this combination exhibited antagonism, combinations were indifferent against 20, 22, and indifference, additive effects, and synergy in 7, 8, 17 isolates at pH 7.0, respectively; and against 1, and 4 isolates, respectively. The combination of 20, 22, and 13 isolates at pH 5.0, respectively. rifampicin-doxycycline was found to be the most The rifampicin-tetracycline and rifampicin- streptomycin combinations showed additive On the hand, and in their efforts to evaluate activities against 12 and 7 isolates at pH 7.0; and the susceptibility of B. melitensis against several against 12 and 5 isolates at pH 5.0, respectively. antibiotics, Qadri et al.24 reported cross-resistance However, the ciprofloxacin-streptomycin of B. melitensis isolates to all Quinolones noted and ciprofloxacin-tetracycline combinations after therapy with ciprofloxacin. A good activity demonstrated antagonistic activity against 9 and of ciprofloxacin has been reported in many in 6 Brucella isolates at pH 7.0; and against 13 and vitro studies.25,26 In addition, Baykam et al.27 in a 9 isolates at pH 5.0, respectively. study performed in Turkey and Dimitrov et al.28 in a study performed in AL Kuwait28 found that all Discussion
their isolates were susceptible to ciprofloxacin, but 9.6% and 8% of the isolates were resistant Brucella spp. infect macrophages replicating within to rifampicin in vitro, respectively.
the phagolysosomes at a pH of 5.0.16 Theoretical y, In our study, we detected no differences antibiotics that are able to penetrate the regarding the individual antibiotic activity when phagolysosomal compartment and function under we tested ciprofloxacin (MIC acidic conditions could be used as monotherapy ml at both pH levels) or sparfloxacin (MIC for the treatment of Brucella. However, in practice, 0.125-4 μg/ml at pH 7.0, and 0.25-4 μg/ml at neither doxycycline nor rifampicin (both of which pH 5.0) against the Brucella isolates from any meet these criteria) is effective as a monotherapeutic Syrian region at either pH value. At pH 5.0, agent.1,14 Garcia-Rodriguez et al.21 found a two to the tetracycline activity was reduced in the fourfold decrease in the activity of Quinolones Central region isolates and its susceptibility in against Brucella at a pH of 5.0 compared to a pH the Southern region was decreased at pH 5.0 of 7.0. In their study, all the Quinolones exhibited compared with that at pH 7.0 (P<0.0007). The very low bactericidal activities against the 21 rifampicin activity was very low in the Coastal and tested strains, regardless of the pH, with a MBC the Northern regions at both pH levels (MIC (minimal bactericidal concentration) of ≥8 mg/liter. In 32-64 μg/ml). In addition, rifampicin-resistant addition, Akova et al.20 revealed that only rifampicin isolates were observed in these two regions (18 and doxycycline retained sufficient activity against and 28 resistant isolates, respectively). However, Brucella at a pH of 5.0, in contrast to the other tested one of the most unexpected results in this study antibiotics. The authors showed that the rifampicin was the very poor activity of streptomycin against activity increased two to eightfold at the acidic pH. all the Brucella isolates (MIC >128 μg/ml), Antibiotic combination studies have revealed which has not been published previously.20,22 an absence of synergism between Quinolones We suggest that this resistance to streptomycin and other antibiotics against B. melitensis.22,23 could have been developed as a result of the Iran J Med Sci September 2013; Vol 38 No 3 Al-Mariri A, Safi M aggressive administration of this antibiotic in the opted for, care should be taken because the treatment for all causes of bovine udder infection consumption of Quinolone alone can probably cause the relapse of Brucella disease. Then, Moreover, in another study performed in our when it is used instead of rifampicin, doxycycline laboratory, we found that the MIC should be applied simultaneously.
16 μg/ml for ofloxacin and 0.125-8 μg/ml for Further and more specific studies, in vivo, Levofloxacin, indicating the good activity of these are recommended to determine the efficacy of two antibiotics against Syrian Brucella isolates these Quinolones in the treatment of brucellosis (data not shown).
infections. If rifampicin could be replaced by No antagonism was seen with the rifampicin- ciprofloxacin and sparfloxacin, then rifampicin doxycycline or rifampicin-tetracycline combinations use could be restricted solely to the treatment of at both pH conditions, while antagonism was tuberculosis, which is regarded as a big chal enge clear when the ciprofloxacin-tetracycline and ciprofloxacin-streptomycin combinations were assessed. In addition, antagonism increased Acknowledgment
at pH 5.0 compared to pH 7.0 when rifampicin- ciprofloxacin and particularly rifampicin-sparfloxacin The authors would like to thank the Director General combinations were used. No synergic or additive of the AECS and the Head of the Molecular Biology effects were observed when we applied the new and Biotechnology Department for their support.
combinations at both pH conditions, whereas the rifampicin-doxycycline combination was the most Conflict of interest: None declared.
synergistic at both pH degrees.
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Determination of Quantal Dose-Response Characteristics in Phenotypic Assays using Supervised Classification Daniel Asarnow1, and Rahul Singh1* 1Department of Computer Science, San Francisco State University *Corresponding author R. Singh, [email protected] Abstract We have designed and implemented a fully automatic, high-throughput screen against the causative parasite of the devastating illness, schistosomiasis, using computer vision and machine learning. The computer vision component segments (individually recognizes and delineates) schistosomula in bright-field micrographs, including touching and partially overlapping parasites. A learning model employing support vector machines to identify schistosomula which differ significantly from controls. Classification is performed in a high-dimensional feature space, the dimensions of which correspond to measurements of appearance, shape and texture. Because variation between different populations of schistosomula unavoidably creates different baselines for different experiments, classification is conducted in two stages: one in which putatively "normal" parasites are identified within each control image and used to derive an estimated control centroid, and one in which all parasites are classified as "normal" or "degenerate" on the basis of tuples composed of a given parasite's feature vector and corresponding control centroid. Finally, a continuous measurement of the phenotypic response to a particular experimental condition (such as the concentration of a certain drug) is produced using the notion of quantal response, or the proportion of individuals which differ from controls in a particular subpopulation. The learning model is demonstrated to be highly effective, and for test data has an accuracy of 0.89. Dose-response curves produced for four compounds (fluvastatin, niclosamide, praziquantel and simvastatin) with the automated method are also tightly correlated to those produced by human experts, with high statistical significance. Correlation values are > 0.97 in all cases with p-values << 10-3.