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In vitro susceptibility of arcobacter butzleri to six antimicrobial drugs

Arch. Med. Vet. XXXVI, Nº 2, 2004
In vitro susceptibility of Arcobacter butzleri to six antimicrobial drugs*
Susceptibilidad in vitro de Arcobacter butzleri a seis drogas antimicrobianas L. OTTH, T.M., M. WILSON, T.M., R. CANCINO, T.M., H. FERNANDEZ1, T.M., D. SC. Instituto de Microbiología Clínica, Universidad Austral de Chile, PO BOX 567. Valdivia. Chile. Se determinaron los patrones de susceptibilidad de 50 cepas de A. butzleri mediante el método del E-test. Ninguna cepa fue resistente a gentamicina y tetraciclina, pero, hubo cepas resistentes a eritromicina (2%) y ciprofloxacina (2%). Además, el 90 y el 98% de las cepas fueron resistentes a ampicilina y cloramfenicol respectivamente. Solamente dos de las 45 cepas ampicilina-resistentes fueron productoras de ß-lactamasa. Palabras clave: Arcobacter butzleri, susceptibilidad antimicrobiana, bacterias emergentes, E-test, ß-lactamasa Key words: Arcobacter butzleri, antibiotic susceptibility, emerging bacteria, E-test, ß-lactamase. contaminated water (Jacob et al., 1998, Mansfield and Forsythe, 2000). The genus Arcobacter belongs to the family Some studies have been carried out in indus­ Campylobacteraceae, class Proteobacteria, sub­ trialized countries in order to establish the sus­ class Gracillicutes and comprises four species ceptibility of Arcobacter to several antimicrobial Arcobacter butzleri, A. cryaerophilus, A. drugs. As the antibiotic resistance of Arcobacter nitrofigilis and A. skirrowii, formerly known as strains from developing countries is not known, aerotolerant Campylobacter-like organisms the aim of this study was to assess the suscepti­ bility and resistance patterns of A. butzleri strains The first isolates were obtained by Ellis et al. (1977) from aborted bovine fetuses. Further stud­ies related these microorganisms with mastitis and abortion in the bovine, ovine, equine and porcine species (Logan et al., 1982; Vandamme 2000). A total of 50 strains of A. butzleri isolated Of the four described species, only A. from cattle (5), pelicans (8) duck feces (2) mus­ cryaerophilus and A. butzleri have been isolated sels (17), chicken livers for human consumption from human beings, and are associated with bac­ (8) and river water (10) were examined. All the teremia and diarrhea. A. butzleri has been iso­ strains were isolated using the enrichment me­ lated from patients with endocarditis, peritonitis dium of de Boer et al. (1996), incubated aerobi­ and appendicitis. Both are considered as emerg­ cally at 26ºC for 48 h. They were then plated on ing foodborne pathogens that could be acquired the medium of Atabay and Corry (1998) and in­ by consuming mussels, poultry meat, offal and cubated as described above. All the strains were identified phenotypically using the standard tests Financial Support: Grant FONDECYT 1030245. L. OTTH, M. WILSON, R. CANCINO, H. FERNANDEZ Susceptibility to ampicillin, ciprofloxacin, complemented with a disc diffusion test for de­ chloramphenicol, erythromycin, gentamicin and tecting ampicillin-sulbactam susceptibility. tetracycline was assessed using the E-test method The results of the E-test are shown in table 1 (AB Biodisk, Solna, Sweden). This method has and are expressed as the Minimal Inhibitory Con­ been used previously in our laboratory for test­ centrations 50 and 90 (MIC and MIC ), corre­ ing Campylobacter jejuni (Fernández et al., sponding to the antibiotic concentration that in­ 2000). In brief, several colonies of each strain, hibits at least the 50 or 90% of the strains respec­ obtained from a fresh culture on a blood agar plate, were suspended in 5 ml of Mueller-Hinton All the strains were susceptible to gentami­ broth to a turbidity equal to 0.5 MacFarland stan­ cin and tetracycline. The lowest MIC and MIC dard. The suspensions were inoculated with ster­ values were obtained with gentamicin and ile swabs onto 150 mm diameter Mueller-Hinton ciprofloxacin. The latter, together with erythro­ agar plates supplemented with 5% sheep blood. mycin, showed the lowest percentages of resis­ The agar surfaces were allowed to dry, and six E-test strips were applied to each plate. Plates were incubated aerobically at 26ºC for 48 h and obtained with ampicillin and chloramphenicol inhibitory concentrations were read at the point which also showed high percentages of resistance where the elliptical zone of inhibition intersected the E-test strip. Staphylococcus aureus ATCC All the strains were susceptible to gentami­ 25923, Escherichia coli ATCC 25922 and a C. cin and tetracycline. With regard to erythromy­ jejuni isolate of known susceptibility/resistance cin and ciprofloxacin we found that 98% of the were used as control strains. The susceptibility strains were susceptible to both antibiotics with criteria were those defined for C. jejuni by the one strain resistant to erythromycin and another National Committee for Clinical Laboratory Stan­ to ciprofloxacin. These results are similar to those dards (National Committee for Clinical Labora­ reported by Atabay and Aydin (2001). They found that all 39 strains of A. butzleri studied by the disk diffusion method were susceptible to tet­ racycline, to the aminoglycoside tobramicin and to the quinolones danofloxacin and enrofloxacin. All the ampicillin-resistant strains (45) were Harrass et al. (1998) reported that two out of 89 tested for ß-lactamase using the chromogenic A. butzleri strains were resistant to tetracycline cephalosporin method (DIFCO Laboratories) and another two showed intermediate resistance TABLE 1. MIC , MIC
and percentages of resistance obtained for 50 strains of A. butzleri. y porcentajes de resistencia obtenidos en 50 cepas de A. butzleri. ARCOBACTER BUTZLERI, SUSCEPTIBILIDAD ANTIMICROBIANA, BACTERIAS EMERGENTES, E-TEST, ß-LACTAMASA to erythromycin, also using the disc diffusion of this test to determine beta lactamase produc- method. Kiehlbauch et al. (1992), using the broth tion in species of the genus Arcobacter has not microdilution method, found that aminoglyco- been described before, we repeated these tests sides, quinolones and tetracyclines were most with a disc diffusion test, using ampicillin and active in vitro against 64 strains of C. butzleri ampicillin-sulbactam discs. Both strains were resistant to ampicillin but susceptible to the com- The other two antibiotics included in our study bination ampicillin-sulbactam. As sulbactam is were ampicillin and chloramphenicol with five a beta lactamase inhibitor we conclude that in strains susceptible to the former and only one to these two strains the ampicillin resistance mecha- the latter. These results are in agreement with nism could be mediated by beta lactamase pro- those reported by other authors for ampicillin (Kiehchelbauch et al., 1992; Harrass et al., 1998; Finally, we conclude that, in general, our Atabay and Aydin, 2001). However, some dif- strains are susceptible to gentamicin, tetracycline, ferences have been observed with relation to erythromycin and ciprofloxacin. They have high chloramphenicol. While Kiehlbauch et al. (1992) resistance to ampicillin and chloramphenicol.
reported 100% resistance, Harrass et al. (1998) Further studies should be conducted in order to found 11.2% of strains to be resistant, but all the establish the resistance mechanisms in A. butzleri strains studied by Atabay and Aydin (2001) were as well as to explain the geographical differences susceptible to this antibiotic. Such differences observed in their susceptibility to these antimi- could be due to local differences, probably re- lated to the use of this antibiotic. In Chile,chloramphenicol is the drug of choice in the treat- ment of typhoid fever which, declining in inci-dence, remains endemic here (Fica et al., 2001).
The susceptibility patterns of 50 A. butzleri strains All the ampicillin resistant strains of A. to six antimicrobial agents were determined using the butzleri (45) were tested for beta lactamase pro- E-test method. No strain was found to be resistant to duction. Table 2 shows the results obtained with gentamicin and tetracycline, but two different strains(2%) were resistant to erythromycin and ciproflo- the chromogenic cepahlosporin test and the disc xacin. Ninety and 98% of the strains were resistant to diffusion susceptibility test for ampicillin- ampicillin and chloramphenicol, respectively. Only sulbactam. Only two strains were able to produce two of the 45 ampicillin resistant strains were able to ß-lactamase, giving a positive chromogenic cepaholosporin test. However, because the use TABLE 2. ß-Lactamase production and suscep-
ATABAY, I., J. CORRY. 1998. Evaluation of a new tibility to ampicillin-sulbactam in 45 A. butzleri
Arcobacter enrichment medium and comparison ampicillin resistant strains.
with two media developed for enrichment of Cam- Producción de ß-lactamasa y susceptibilidad a ampicilina-
pylobacter spp. Int. J. Food. Microbiol. 41: 53-58.
sulbactam en 45 cepas de A. butzleri ampicilina resistentes.
ATABAY, H.I., F. AYDIN. 2001. Susceptibility of Arcobacter butzleri isolates to 23 antimicrobial agents. Lett. Appl. Microbiol. 33:430-433.
DE BOER, E., J.J. TILBURG, D.L. WOODWARD, ß-lactamase
medium for the isolation of Arcobacter from meats.
Lett. Appl. Microbiol. 23: 64-66.
ELLIS, W.A., S.D. NELLY, J.J. O’BRIEN, H.W.
FERGUSON, J. HANNA. 1977. Isolation of Spi- N: number of positive strains; n: number of ampicillin resis- rillum/Vibrio-like organisms from bovine fetuses.
L. OTTH, M. WILSON, R. CANCINO, H. FERNANDEZ KIEHLBAUCH, J.A., C.N. BAKER, I.K. WACHS- ZALEZ. 2000. Antimicrobial Susceptibility of MUTH. 1992. In vitro susceptibilities of Campylobacter jejuni subsp. jejuni assessed by aerotolerant Campylobacter isolates to 22 anti- E-test and double dilution agar method in South- microbial agents. Antimicrob. Agents Chemother. ern Chile. Mem. Inst. Oswaldo Cruz 95: 247-249.
LOGAN, E.F., S.D. NEIL, D.P. MACKIE. 1982.
Mastitis in dairy cows associated with an 2001. Cambios epidemiológicos de las salmo- aerotolerant Campylobacter. Vet. Rec. 110: nelosis en Chile: desde Salmonella tiphy a Sal- monella enteritidis. Rev. Chil. Infectol. 18: MANSFIELD, L.P., S.J. FORSYTHE. 2000.
Arcobacter butzleri, A. skirrowii and A.
HARRASS, B., S. SCHWARZ, S. WENZEL. 1998.
cryaerophilus – Potential emerging human patho- Identification and characterization of Arcobacter gens. Rev. Med. Microbiol. 11: 161-170.
isolates from broilers by biochemical tests, anti- microbial resistance patterns and plasmid analy- sis. Zbl. Vet. Med (B) 45: 87-94.
Performance standards for antimicrobial disk sus- ceptibility tests. NCCLS document M100-S13 W.M. JOHNSON. 1998. Isolation of Arcobacter butzleri in raw water and drinking water treat- VANDAMME, P. Taxonomy of the Family Cam- ment plants in Germany. Zbl. Hyg. Umweltmed. pylobacteraceae. 2000. IN: Nachamkin, I., Blaser, M.J. (Eds) Campylobacter 2nd Edition, ASMPress, Washington, pp. 3-26.

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