Isolation of antifungal-resistant yeasts from bulk milk in Japan

We isolated ascomycetous yeasts including Candida species, that originally belonged to the genus Candida, from bulk milk in the Aichi area of Japan, and determined the minimum inhibitory concentrations (MICs) of antifungal drugs on these isolates by conducting E-tests. We isolated 7 human pathogenic species (14 isolates) from 14 bulk milk samples: 5 Candida species of yeasts, and 2 Candida-related species. Two isolates of C. albicans and C. inconspicua were resistant to fluconazole (MIC >32 mg/l). One isolate of C. krusei was resistant to both azoles (fluconazole: >256 mg/ml and itraconazole: 4 mg/l). One isolate of C. catenulata might be resistant to amphotericin B (>32 mg/l).     

In vitro susceptibility patterns of Aspergillus and Fusarium species isolated from equine ulcerative keratomycosis cases in the midwestern and southern United States with inclusion of the new antifungal agent voriconazole

Objective  To evaluate and compare the in vitro susceptibility of Aspergillus and Fusarium spp. isolated from horses with ulcerative keratomycosis, address regional differences in equine keratomycosis isolates, and provide susceptibility data to update prior studies.
Animal studied  Fourteen horses with ulcerative keratomycosis.
Procedures  Banked fungal isolates from equine corneal ulcers (eight Aspergillus spp. and six Fusarium spp.) were identified at The University of Texas Health Science Center at San Antonio. In vitro minimum inhibitory concentration and susceptibility to natamycin, fluconazole, itraconazole, voriconazole, ketoconazole, and miconazole were determined for each isolate.
Results  Fungi were significantly more susceptible to voriconazole than to natamycin, itraconazole, fluconazole, and ketoconazole, but miconazole susceptibility did not differ significantly from voriconazole. Aspergillus spp. were most susceptible to voriconazole, miconazole, and itraconazole, which were significantly better to fluconazole and ketoconazole. Fusarium spp. susceptibility was greatest to natamycin and voriconazole and lowest to itraconazole and ketoconazole. Fusarium spp. were significantly less susceptible to itraconazole and ketoconazole compared to natamycin. No significant differences in susceptibility were found when isolates from Florida were compared with isolates from other states.
Conclusions and clinical relevance  Based on in vitro evidence, voriconazole appears to be the most effective antifungal for initial treatment of equine keratomycosis in the midwestern and southern United States. Results are comparable with previous studies in that isolated fungi from equine keratomycosis cases showed consistently poor susceptibility to fluconazole. Organisms isolated in different geographic locations of the midwestern and southern United States appeared to have similar patterns of antifungal susceptibility.     

Equine Endometrial Tissue Concentration of Fluconazole Following Oral Administration

The objective of this study was to determine the plasma and endometrial tissue concentrations of orally administered fluconazole and to determine if these tissue levels surpassed the minimum inhibitory concentration (MIC) for Candida spp. organisms in the reproductive tract of the mare. Mares from study 1 (n = 9) were administered a single oral loading dose of 14 mg/kg fluconazole. Plasma and endometrial tissue samples were collected before fluconazole administration and for 24 hours after the loading dose. Study 2 mares (n = 3), a subset of study 1, were administered the loading dose, followed by maintenance doses of 5 mg/kg every 24 hours for 6 days. Plasma and biopsy samples were collected for 48 hours after the last maintenance dose. High pressure liquid chromatography-tandem mass spectrometry was used to determine the concentration of fluconazole in all samples. The mean plasma and endometrial fluconazole levels 24 hours after the loading dose were 9.53 ± 0.824 μg/mL (mean ± standard deviation) and 11.3 ± 2.38 μg/g, respectively. Fluconazole levels in plasma and endometrial tissue 24 hours after the last maintenance dose were 7.82 ± 1.81 μg/mL and 7.23 ± 3.86 μg/g, respectively. Oral fluconazole administered as a 14-mg/kg loading dose and a 5-mg/kg maintenance dose every 24 hours will result in endometrial tissue levels near the accepted MIC values for most Candida spp. and surpass the MIC for Candida albicans in the reproductive tract of the mare. Consequently, this dosage regimen could be considered for the treatment of infectious endometritis caused by susceptible fungal organisms of Candida spp. in the mare.     

PCR-AGE, automated and manual methods to identify Candida strains from veterinary sources: A comparative approach

The increasing incidence of candidiasis has drawn the attention of scientists and clinicians attempting to improve methods of studying Candida yeasts. PCR amplification followed by agarose gel electrophoresis (PCR-AGE) and the manual method (morphological characteristics, biochemical profiles and culturing on CHROMagar-Candida) and VITEK 2 automated method were used to test a total of 30 fungal strains from dog sources. The strains were obtained from cases of dermatitis, otitis externa and from the ears, oral mucosa, vaginal mucosa, prepuce and perianal region of clinically normal dogs. After identification as Candida yeasts by the manual method, the strains were analyzed using both VITEK and PCR-AGE methods. Isolates of C. parapsilosis ATCC 22019, C. krusei ATCC 6258 and C. albicans ATCC 10231 were included as controls. The universal primers ITS1, ITS3 and ITS4 were used in two independent PCR reactions. Of 30 yeast isolates, 3 isolates (Saccharomyces cerevisiae, C. rugosa and C. parapsilosis) that were incompletely identified by the manual method were identified with the PCR-AGE and VITEK methods. The results revealed a 96.7% and 86.7% concurrent identification between the PCR-AGE and VITEK methods versus the manual method, respectively. PCR-AGE showed a greater level of concordance with the manual method, besides being faster and more sensitive than the other methods examined, and is therefore indicated for routine diagnostic testing of Candida spp. strains from veterinary sources.     

Prevalences of resistance to seven antimicrobials among fecal Escherichia coli of swine on thirty-four farrow-to-finish farms in Ontario, Canada

Fecal specimens were composited and a hydrophobic-grid membrane-filter method was used to measure antimicrobial resistance to ampicillin 16 μg/ml, carbadox 30 μg/ml, gentamicin 4 μ/ml, nitrofurantoin 32 μg/ml, spectinomycin 16 μg/ml, sulfisoxazole 32 μg/ml and tetracycline 8 μg/ml among 8119 Escherichia coli isolates from 68 fecal samples collected on 34 farrow-to-finish swine farms marketing over 500 hogs/yr. The overall prevalences of resistance to antimicrobials among these isolates were: ampicillin 29%, carbadox 3.5%, gentamicin 0.6%, nitrofurantoin 27%, spectinomycin 28%, sulfasoxizole 38% and tetracycline 71%. Thirty to seventy-six per cent of the variations in prevalences were explained by between-farm differences.     

Detection of mutations in the gyrA gene and class I integron from quinolone-resistant Salmonella enterica serovar Choleraesuis isolates in Taiwan

The quinolone resistance-determining regions (QRDRs) of the gyrA gene of quinolone-resistant Salmonella enterica serovar Choleraesuis isolates were sequenced. Four types of point mutation, Ser-83-to-Phe (TCC→TTC), Ser-83-to-Tyr (TCC→TAC), Asp-87-to-Gly (GAC→GGC), and Asp-87-to-Asn (GAC→AAC), were found. PCR-RFLP and MAS-touch down PCR were performed on fifty swine clinical isolates of S. enterica serovar Choleraesuis (Nal^R) collected during 1997–2002. The analysis indicated seven isolates with point mutations in codon 83, 13 with point mutations in codon 87, and 30 with double mutations in both codons 83 and 87. The MICs of enrofloxacin of the isolates with a single mutation in codon 83 or 87 were <2 μg/ml, while the MICs of the isolates with double mutations in both codon 83 and 87 ranged from 2 to 64 μg/ml. A class I integron comprised of dhfr, orfF and aad2 was also identified in both human and swine S. enterica serovar Choleraesuis isolates. These results indicate that PCR-RFLP and MAS-touchdown PCR assays can be used for surveillance of gyrA gene mutations, which are important for fluoroquinolone resistance in Salmonella. Isolates with double mutations in gyrA codons 83 and 87 are the major type of quinolone-resistant Salmonella isolated from swine in Taiwan. A surveillance system may be applied to the swine industry to monitor the emergence of fluoroquinolone and/or multi-drug-resistant S. enterica serovar Choleraesuis in Taiwan.     

Phenotypic characterization and in vitro antifungal sensitivity of Candida spp. and Malassezia pachydermatis strains from dogs

Yeasts of the genera Candida and Malassezia can be found as commensal microorganisms in animals. The main species of importance in veterinary medicine are Malassezia pachydermatis and Candida albicans. The objectives of this study were to conduct a phenotypic characterization and to evaluate the in vitro antifungal sensitivity of strains of C. albicans (n=5), C. tropicalis (n=3) and M. pachydermatis (n=32) isolated from dogs. The phenotyping was based on macro and micromorphological features as well as biochemical analysis. The techniques of microdilution in broth and dilution in agar were used to evaluate the in vitro sensitivity of Candida spp. and M. pachydermatis, respectively. The tested drugs were ketoconazole (KTC), itraconazole (ITC), fluconazole (FLC) and amphotericin B (AMB). The morphological analysis of the strains of Candida spp. and M. pachydermatis did not show any noteworthy alterations when compared to standard strains. On the other hand, in the biochemical tests, 34.4% of the strains of M. pachydermatis were negative for the urease test. Four strains of C. albicans were resistant to FLC with a minimum inhibitory concentration (MIC) >64microg/mL and all were resistant to KTC and ITC (MIC>16microg/mL). The MIC for two strains of C. tropicalis were >16microg/mL for KTC and ITC, and >64microg/mL for FLC. It is worth highlighting that all of the strains tested were sensitive to AMB with the MIC varying from 0.25-1.0microg/mL. All strains of M. pachydermatis were sensitive to ITC with a minimum fungistatic concentration (MFC) 0.0075microg/mL. The MIC for 29 strains was the same (MFC0.0075microg/mL) for KTC. The MFCs for FLC varied from 1 to 16microg/mL, and for AMB, the MFC interval was 0.125-8microg/mL. There were no alterations in the classic phenotypic features of the strains of Candida spp. and M. pachydermatis isolated from dogs but, unlike M. pachydermatis, Candida spp. were much more resistant to azole antifungal agents.     

Some pharmacokinetic indices of oral fluconazole administration to koalas (Phascolarctos cinereus) infected with cryptococcosis

Three asymptomatic koalas serologically positive for cryptococcosis and two symptomatic koalas were treated with 10 mg/kg fluconazole orally, twice daily for at least 2 weeks. The median plasma C_max and AUC_0‐8 h for asymptomatic animals were 0.9 μg/mL and 4.9 μg/mL·h, respectively; and for symptomatic animals 3.2 μg/mL and 17.3 μg/mL·h, respectively. An additional symptomatic koala was treated with fluconazole (10 mg/kg twice daily) and a subcutaneous amphotericin B infusion twice weekly. After 2 weeks the fluconazole C_max was 3.7 μg/mL and the AUC_0‐8 h was 25.8 μg/mL*h. An additional three koalas were treated with fluconazole 15 mg/kg twice daily for at least 2 weeks, with the same subcutaneous amphotericin protocol co‐administered to two of these koalas (C_max: 5.0 μg/mL; mean AUC_0‐8 h: 18.1 μg/mL*h). For all koalas, the fluconazole plasma C_max failed to reach the MIC₉₀ (16 μg/mL) to inhibit C. gattii. Fluconazole administered orally at either 10 or 15 mg/kg twice daily in conjunction with amphotericin is unlikely to attain therapeutic plasma concentrations. Suggestions to improve treatment of systemic cryptococcosis include testing pathogen susceptibility to fluconazole, monitoring plasma fluconazole concentrations, and administration of 20–25 mg/kg fluconazole orally, twice daily, with an amphotericin subcutaneous infusion twice weekly.     

Molecular screening and risk factors of enterotoxigenic Escherichia coli and Salmonella spp. in diarrheic neonatal calves in Egypt

The aim of the present study was to carry out molecular epidemiological investigation on enterotoxigenic Escherichia coli (ETEC) K99 and Salmonella spp. in diarrheic neonatal calves. Fecal samples were obtained from 220 diarrheic calves at 9 farms related to four governorates in central and northern Egypt. E. coli and Salmonella spp. isolates were examined for E. coli K99 and Salmonella spp. using PCR. ETEC K99 was recovered from 20 (10.36 %) out of 193 isolates, whereas Salmonella spp. was recovered from nine calves (4.09%).Multivariable logistic regression was used to evaluate the risk factors associated with both infections. ETEC K99 was significantly affected by age (P < 0.01; OR: 1.812; CI 95%: 0.566–1.769), colostrum feeding practice (P < 0.01; OR: 5.525; CI 95%: 2.025–15.076), rotavirus infection (P < 0.001; OR: 2.220; CI 95%: 0.273–1.251), vaccination of pregnant dams with combined vaccine against rotavirus, coronavirus and E. coli (K99) (P < 0.001; OR: 4.753; CI 95%: 2.124–10.641), and vitamin E and selenium administration to the pregnant dam (P < 0.01; OR: 3.933; CI 95%: 0.703–1.248).Infection with Salmonella spp. was found to be significantly affected by the animal age (P < 0.05; OR: 0.376; CI 95%: 0.511–1.369), Hygiene (P < 0.05; OR: 0.628; CI 95%: 1.729–5.612), and region (P < 0. 01; OR: 0.970; CI 95%: 0.841–1.624).The results of the present study indicate the importance of PCR as rapid, effective and reliable tool for screening of ETEC and Salmonella spp. when confronted with cases of undifferentiated calf diarrhea. Moreover, identification of the risk factors associated with the spreading of bacteria causing diarrhea may be helpful for construction of suitable methods for prevention and control.     

Biofilm-forming associated genotypic and phenotypic characteristics of Staphylococcus spp. isolated from animals and air

Biological monitoring is performed to detect and analyze microorganisms that have continuously made an effort to survive in the environment. Of such microorganisms, Staphylococcus spp. is considered a common cause of nosocomial and environmental infections., Microbial Surface Components Recognizing Adhesive Matrix Molecules (MSCRAMMs) are required for the adhesion and biofilm formation of Staphylococci. Thirty-six and thirty-five Staphylococci isolated from animals and air, respectively, were analyzed. Biofilm formation and ten MSCRAMM genes were investigated using Congo red agar, tissue culture plate methods, and PCR. Airborne isolates were shown to have higher adherence and stronger biofilm formation than those from animals. The prevalence of MSCRAMM genes from air isolates was also higher than those from animals. Of the genes, eno was mainly associated with biofilm formation in both animals and airborne isolates (P < 0.05). Moreover, the rate of airborne isolates harboring the eno gene was higher than in animal isolates. These results indicated that analysis of MSCRAMM genes with a phenotypic assay might be a helpful bacterial control system for the environment.     

Distribution of IS900 restriction fragment length polymorphism types among animal Mycobacterium avium subsp. paratuberculosis isolates from Argentina and Europe

Sixty-one Mycobacterium avium subsp. paratuberculosis isolates from cattle and deer from the Buenos Aires province, an important livestock region in Argentina, were typed by restriction fragment length polymorphisms (RFLP) analysis based on IS900. Four different RFLP patterns (designated ‘A’, ‘B’, ‘C’ and ‘E’) were identified in BstEII digests of genomic DNA. The most frequently observed type, pattern ‘A’, was found in 46 isolates (75%). The second, pattern ‘E’, included 8 isolates (13%), while the third, pattern ‘B’, included 6 isolates (10%). Pattern ‘C’ was found for only one isolate. All of the deer isolates were classified as pattern ‘A’, while cattle isolates represented all four RFLP patterns. Twenty-one isolates representing the four different BstEII-RFLP patterns were digested with PstI. Twenty isolates showed identical PstI-RFLP pattern. BstEII-RFLP patterns from Argentine cattle and deer were compared with patterns found in cattle, goat, deer, rabbit, and human isolates from Europe. The most common pattern in Argentina, pattern ‘A’, was identical to a less frequently occurring pattern R9 (C17) from Europe. The other Argentine patterns ‘B’, ‘C’ and ‘E’, were not found in the Europe. These results indicate that the distribution of M. avium subsp. paratuberculosis genotypes in the Buenos Aires province of Argentina is different from that found in Europe.     

Trace micro-nutrients may affect susceptibility to bovine tuberculosis in cattle

Bovine tuberculosis (bTB) is a continuing problem in British herds. Micro-nutrients are important for the maintenance of well-functioning immune system. The aim of this study was to determine whether the selenium, copper and vitamin B12 status of cattle was associated with Mycobacterium bovis (M. bovis) infection. Between 2002 and 2005, 200 cattle (43% dairy, mean age 4.6 years), reactors according to the standard interpretation of the tuberculin test, and 200 in-contacts (41% dairy, mean age 4.4 years) non-reactors, which had been in contact with cattle with bTB, were selected from herds in England and Wales. Levels of the seleno enzyme glutathione peroxidase (GSHPx), copper and vitamin B12 were measured in blood. Confirmation of bTB infection was made by bacteriological culture and histopathology following a detailed postmortem. Levels of selenium and copper were also measured in a random sample of 63 livers. bTB was confirmed by culture/histology in 23/200 (11.5%) of in-contacts and 110/200 (55%) of reactors. In blood drawn at recruitment, GSHPx was lower in cattle with confirmed bTB compared to other cattle (geometric means 59.7 u/mL versus 78.9 u/mL red blood cells (RBC), p < 0.01). Vitamin B12 was similar (geometric means 161.5 pmol/L versus 165.5 pmol/L, p = 0.62) and copper was similar (geometric means 14.4 μmol/L versus 14.1 μmol/L, p = 0.55). In logistic regression models including all micro-nutrients simultaneously and controlling for age, sex, animal production class, herd size, number of reactors, postmortem laboratory and seasonal trends, lower levels of GSHPx (adjusted OR 0.42, 95% CI 0.21–0.81 per 100 u/mL RBC, p = 0.01) and higher levels of copper (adjusted OR 1.69, 95% CI 1.21–2.36 per 5 μmol/L, p < 0.01) were associated with an increased risk of confirmed bTB but there was no association with vitamin B12. There was evidence for a stronger association between confirmed bTB and GSHPx in in-contacts (adjusted OR 0.21, 95% CI 0.06–0.79 per 100 u/mL RBC) compared to reactors (adjusted OR 0.50, 95% CI 0.21–1.23 per 100 u/mL RBC) (p = 0.08 for interaction). Lower liver copper was associated with a higher risk of confirmed bTB (adjusted OR 0.15, 95% CI 0.02–1.0 per 5000 μmol/kg dry mass, p = 0.05) but there was no association between liver selenium and bTB. Trace micro-nutrient status may affect susceptibility to M. bovis infection in cattle. Further studies are needed.     

Antimicrobial susceptibility of porcine Brachyspira hyodysenteriae and Brachyspira pilosicoli isolated in Sweden between 1990 and 2010

Background

The anaerobic spirochetes Brachyspira hyodysenteriae and Brachyspira pilosicoli cause diarrheal diseases in pigs. Their fastidious nature has hampered standardization of methods for antimicrobial susceptibility testing. For monitoring of antimicrobial susceptibility wild type cutoff values are needed to define where the wild type distribution of MICs ends and no approved cutoffs are available for Brachyspira spp. In this study antimicrobial susceptibility data for both species (in total 906 isolates) were compiled and analyzed and wild type cut off values for B. hyodysenteriae proposed.

Methods

The MICs of tiamulin, valnemulin, tylosin, tylvalosin, doxycycline and lincomycin were determined by broth dilution in brain heart infusion broth supplemented with 10% fetal calf serum.

Results

The compiled MICs from the broth dilution tests of the B. hyodysenteriae type strain, B78^T (ATCC® 27164^T), showed that the method yields reproducible results. In an international perspective the frequencies of isolates with decreased antimicrobial susceptibility were low among both B. hyodysenteriae and B. pilosicoli. However, in B. pilosicoli a constant level of 10-15% isolates with tiamulin MICs >4 μg/ml was detected between 2002 and 2010 and in B. hyodysenteriae a gradual increase in tiamulin MICs was seen between 1990 and 2003 although this increase has ceased during the last years. The wild type cutoff values proposed for B. hyodysenteriae are: tiamulin >0.25 μg/ml, valnemulin >0.125 μg/ml, tylosin >16 μg/ml, tylvalosin >1 μg/ml, lincomycin >1 μg/ml and doxycycline >0.5 μg/ml.

Conclusions

The broth dilution method used in this study has over the years generated tightly grouped MIC populations for the field isolates and reproducible results for the control strain B78^T and is therefore a suitable antimicrobial susceptibility test method for monitoring of Brachyspira spp. Here we propose wild type cutoff values for six antimicrobial agents for B. hyodysenteriae tested by broth dilution based on MIC distributions and the current knowledge on mechanisms of resistance in this species. There are few studies on antimicrobial resistance mechanisms and MIC distributions in B. pilosicoli but to some extent the cutoff values proposed for B. hyodysenteriae may be applicable also for monitoring of antimicrobial susceptibility in B. pilosicoli.     

In vitro antiviral activity of chestnut and quebracho woods extracts against avian reovirus and metapneumovirus

Field evidences have suggested that a natural extract, containing tannins, could be effective against poultry enteric viral infections. Moreover previous studies have shown that vegetable tannins can have antiviral activity against human viruses. Based on this knowledge three different Chestnut (Castanea spp.) wood extracts and one Quebracho (Schinopsis spp.) wood extract, all containing tannins and currently used in the animal feed industry, were tested for in vitro antiviral activity against avian reovirus (ARV) and avian metapneumovirus (AMPV). The MTT assay was used to evaluate the 50% cytotoxic compounds concentration (CC₅₀) on Vero cells. The antiviral properties were tested before and after the adsorption of the viruses to Vero cells. Antiviral activities were expressed as IC₅₀ (concentration required to inhibit 50% of viral cytopathic effect). CC₅₀s of tested compounds were >200 μg/ml. All compounds had an extracellular antiviral effect against both ARV and AMPV with IC₅₀ values ranging from 25 to 66 μg/ml. Quebracho extract had also evident intracellular anti-ARV activity (IC₅₀ 24 μg/ml). These preliminary results suggest that the examined vegetable extracts might be good candidates in the control of some avian virus infections. Nevertheless further in vivo experiments are required to confirm these findings.     

P‐17 In vitro activity of posaconazole and other antifungals against Malasseziapachydermatis isolated from dogs

The aim of this study was to determine the in vitro antifungal activity of several antifungal drugs (posaconazole, nystatin, miconazole and clotrimazole) against Malassezia pachydermatis with microdilution and agar dilution techniques. Malassezia pachydermatis isolates were obtained from the skin and ears of dogs. Tests on solid media were performed using 25‐well Petri dishes (2 mL/well containing Sabouraud's dextrose agar and diluted antifungal drug) inoculated with 5 μL suspensions of M. pachydermatis. Microtitre broth dilution used 96‐well microtitre plates containing Sabourauds dextrose broth and appropriate dilutions of antifungal drugs, inoculated with 10 μL standard suspensions of M. pachydermatis. Plates were inoculated in duplicate and incubated at 30°C for 5 days and growth assessed. The four antifungal drugs were tested in 10 dilutions (4.0‐0.007 μg/mL for posaconazole, and 32‐‐0.06 μg/mL for clotrimazole, miconazole and nystatin). Results obtained for 83 strains of M. pachydermatis and a control reference strain (CBS 1879) exhibited the same pattern. Results of the MIC between microtitre and agar methodologies showed no significant differences (≤ 2‐fold) across all drugs. For both solid and liquid methods, posaconazole was the most effective antifungal drug of the four tested with MIC₉₀ of 1–2 μg/mL for posaconazole, 16–32 μg/mL for clotrimazole, and ≥ 32 μg/mL for miconazole and nystatin. Funding: Schering‐Plough.     

In vitro sensitivity of the amphibian pathogen Batrachochytrium dendrobatidis to antifungal therapeutics

Chytridiomycosis, a skin disease caused by Batrachochytrium dendrobatidis, has caused amphibian declines worldwide. Amphibians can be treated by percutaneous application of antimicrobials, but knowledge of in vitro susceptibility is lacking. Using a modified broth microdilution method, we describe the in vitro sensitivity of two Australian isolates of B. dendrobatidis to six antimicrobial agents. Growth inhibition was observed, by measurement of optical density, with all agents. Minimum inhibitory concentrations (µg/ml; isolate 1/2) were – voriconazole 0.016/0.008; itraconazole 0.032/0.016; terbinafine 0.063/0.063; fluconazole 0.31/0.31; chloramphenicol 12.5/12.5; amphotericin B 12.5/6.25. Killing effects on zoospores were assessed by observing motility. Amphotericin B and terbinafine killed zoospores within 5 and 30 min depending on concentration, but other antimicrobials were not effective at the highest concentrations tested (100 µg/ml). This knowledge will help in drug selection and treatment optimization. As terbinafine was potent and has rapid effects, study of its pharmacokinetics, safety and efficacy is recommended.     

Susceptibility testing of Aspergillus niger strains isolated from poultry to antifungal drugs--a comparative study of the disk diffusion, broth microdilution (M 38-A) and Etest methods

The aim of this study was to determine the sensitivity of Aspergillus niger strains isolated from birds to available antifungal drugs using different in vitro assays--classical disk diffusion, Etest and broth microdilution NCCLS/CLSI M 38-A. The study material consisted of about 2.000 swabs and samples from different species of birds. A. niger (n=10) was accounted for 6.81% of the total pool of strains isolated. Determinations were made for 13 antifungal drugs using the disk diffusion method. The A. niger exhibited high susceptibility to enilconazole, terbinafine, voriconazole, tioconazole and ketoconazole, low susceptibility to clotrimazole, miconazole and nystatin, and resistance to amphotericin B, itraconazole, pimaricin, fluconazole and 5-fluorocytosine. Minimum inhibitory concentration (MIC) was determined for 9 antifungal drugs using the micromethod of duplicate serial dilutions in a liquid medium. A. niger strains were most susceptible to enilconazole and voriconazole. MIC ranged from 0.0625 to 0.5 microg/ml for enilconazole, with MIC90-0.5 microg/ml and MIC50-0.125 microg/ml. The corresponding values for voriconazole were 0.25-1 microg/ml, 1 microg/ml and 0.5 microg/ml. MIC for amphotericin B and terbinafine ranged from 0.5 to 4 microg/ml, while the values for the remaining drugs were highly varied. MIC was measured by the gradient diffusion method using Etest for 5 antifungal drugs: amphotericin B, fluconazole, itraconazole, ketoconazole and voriconazole. By far the highest susceptibility was obtained in the case of voriconazole, with MIC ranging from 0.0625 to 1 microg/ml. MIC for amphotericin B ranged from 0.25 to 4 microg/ml, for itraconazole and ketoconazole ranging from 0.5 to 16 microg/ml. Methods available for this purpose are not always applicable in field conditions. The present results indicate that the Etest technique, due to its high percentage of agreement with the M 38-A microdilution method, should find application in medical and veterinary practice.     

In vitro susceptibility patterns of fungi associated with keratomycosis in horses of the northeastern United States: 68 cases (1987-2006)

OBJECTIVE: To determine in vitro susceptibility patterns of fungi associated with keratomycosis in horses in the northeastern United States and compare those patterns with results of studies from other geographic regions. DESIGN: Retrospective case series. ANIMALS: 68 horses with keratomycosis. PROCEDURES: Medical records of horses with a clinical diagnosis of keratomycosis, positive results of corneal fungal cultures, and susceptibility data were reviewed from the years 1987 to 2006. Fungal identification and in vitro antifungal susceptibility test results were recorded. The percentage of susceptible isolates was compared among antifungals for all isolates together and for the most common genera individually. RESULTS: 74 fungal isolates from 68 horses that met inclusion criteria were identified. Aspergillus, Candida, and Fusarium spp were the most frequent isolates. Grouped isolates had the highest percentage of susceptibility to nystatin (87.7%), natamycin (87.5%), and clotrimazole (80.6%). Grouped isolates had the lowest percentage of susceptibility to fluconazole (15.8%) and miconazole (27.5%). Aspergillus spp (> or = 81.0%) were most susceptible to nystatin, clotrimazole, itraconazole, and natamycin. Candida spp (100%) were most susceptible to ketaconazole, natamycin, and nystatin. Fusarium spp (100%) were only consistently susceptible to natamycin. CONCLUSIONS AND CLINICAL RELEVANCE: On the basis of in vitro susceptibility testing, nystatin, natamycin, or clotrimazole is recommended for initial topical treatment of keratomycosis in horses from the northeastern United States. Contrary to results of studies of ocular fungal isolates of horses from other regions, Candida spp were identified more frequently and miconazole had lower in vitro efficacy in the present study.     

Prevalence of vancomycin-resistant Enterococcus faecium (VREF) in pig faeces from slaughterhouses in Spain

A study to estimate the prevalence of vancomycin-resistant Enterococcus faecium in faecal samples from pigs at slaughterhouses in Spain was carried out between November 1998 and January 1999 with 900 samples taken from four abattoirs representing 9.7% of all pig slaughtered in 1998. Using a selective enrichment broth with vancomycin (8 μg/ml), 64 samples (7.1%; 95% CI: 5.5, 9.0%) had E. faecium vancomycin-resistant strains that showed minimal inhibitory concentrations of 256 μg/ml (62 strains) and 512 μg/ml (two strains). Results by farm showed that 43 of the 240 pig farms represented in the sampling had at least one faecal sample with vancomycin-resistant E. faecium.     

First isolation and identification of Elizabethkingia meningoseptica from cultured tiger frog, Rana tigerina rugulosa

Elizabethkingia meningoseptica has been recognised as an occasional but serious opportunistic bacterial pathogen to human beings. Recently, it was frequently isolated from tiger frog, Rana tigerina rugulosa, with cataract disease, which is the most common disease of unknown aetiology of frogs in Hainan, China. The purpose of this study was to identify and characterise the bacterial strains isolated from the recent outbreaks of cataract disease in farmed tiger frog in Hainan, China, and to evaluate their pathogenicity to the frog and their sensitivity to 20 chemotherapeutic agents.The 16S rRNA gene sequences of strains W0701 (1478 bp), W0702 (1477 bp) and W0703 (1478 bp) showed 98.6–98.7% similarity with the sequence of E. meningoseptica type strain (ATCC 13253) and 99.9–100% similarity with that of E. meningoseptica NTU 870424-IL. Six strains (W0701–W0706) were selected to represent 24 isolates retrieved from six moribund frogs. The morphological, physiological and biochemical characteristics of the six representative isolates were consistent with those of E. meningoseptica strains. The organisms were only susceptible to vancomycin and moderately susceptible to cefoperazone among the 20 investigated chemotherapeutic agents. Virulence test with strain W0702 was conducted and pathogenicity (by intramuscular injection) was demonstrated in the tiger frog. In conclusion, 24 isolates obtained from frogs with cataract disease were the E. meningoseptica strains highly pathogenic to tiger frog, and this is the first report of E. meningoseptica as a pathogen for tiger frog.