Assessment of the antifungal susceptibility of Malassezia pachydermatis in various media using a CLSI protocol

The microdilution antifungal method (CLSI BMD, M27-A3) was used for testing the antifungal susceptibility of Malassezia species. However, optimal broth media that allow sufficient growth of M. pachydermatitis and produce reliable and reproducible MICs using the CLSI BMD protocol are yet to be established. In this study, the susceptibility of M. pachydermatis isolates to ketoconazole (KTZ), itraconazole (ITZ) and fluconazole (FLZ) was evaluated in vitro by the CLSI BMD test using Christensen's urea broth (CUB) and mRPMI 1640 containing lipid supplementation, Sabouraud dextrose broth with 1% tween 80 (SDB), and Dixon broth (DXB). A FLZ-resistant M. pachydermatis was generated in vitro and tested under the same conditions. A good growth of M. pachydermatis incubated for 48 and 72h, respectively, was observed in CUB, SDB and DXB and not in mRPMI 1640 (p<0.001). No statistically significant differences were detected between the MIC values registered after 48h and 72h incubation. ITZ displayed lower MIC values than KTZ and FLZ regardless of the media employed. A large number of FLZ-resistant Malassezia strains (86.6%) was observed using DXB. A MIC>64mg/L was observed only when the FLZ-resistant M. pachydermatis isolate was tested in SDB. Based on the results obtained herein, culture in SDB, stock inoculum suspensions of 1-5×10(6)CFU/ml, and an incubation time of 48h are proposed as optimal conditions for the evaluation of the in vitro antifungal susceptibility of M. pachydermatis using a modified CLSI BMD protocol.     

Comparative evaluation of a commercial microdilution method and a conventional disk diffusion method for determination of antibiograms of gram-positive cocci

A commercial broth microdilution system for testing the antimicrobial susceptibility of gram-positive cocci was compared with the standardized disk agar-diffusion method by testing 254 clinical strains of staphylococci and streptococci using both methods. A total of 2,794 parallel determinations were made with 92.3% complete agreement between the 2 methods; of the discrepancies encountered, 3.0% were minor, 2.5% were major, and 2.1% were very major. The results indicate that the commercial microdilution system may provide a reliable quantitative method for antimicrobial susceptibility testing of clinical isolates from animals.     

Determination of minimum inhibitory and minimum bactericidal concentrations of tiamulin against field isolates of Actinobacillus pleuropneumoniae

Tiamulin activity was measured against 19 UK field isolates of Actinobacillus pleuropneumoniae collected between 2003 and 2009 and the type strain ATCC 27090 as a control, with the intention of comparing broth with serum as growth media. Broth microdilution MIC/MBC tests were performed in accordance with the Clinical and Laboratory Standards Institute (CLSI) guideline M31-A3, in 'Veterinary Fastidious Medium' (VFM) (supplemented Mueller-Hinton broth at pH 7.3) and in 100% swine serum. For improved precision, a modified, overlapping doubling-dilution series was used (tiamulin concentration range 0.3-72 μg/ml). The MBC was reported as the lowest concentration producing a 99.9% reduction in bacterial density in the sub-cultured well contents, relative to the starting inoculum. The mean MBC/MIC ratio for tiamulin against A. pleuropneumoniae in VFM was low (1.74:1), even though tiamulin is classed as a bacteriostatic drug. Only three of the 19 isolates and the reference strain grew in 100% serum and their MICs were higher than those determined in VFM. It is postulated that this difference was due to differences in pH of the matrices or binding of tiamulin to serum proteins or a combination of both factors.     

Layout proposal for a microtitre plate to be used in routine antimicrobial susceptibility testing of bacteria from infections of dogs and cats

The determination of minimum inhibitory concentrations by broth microdilution is recommended as method of choice for susceptibility testing of veterinary bacterial pathogens. Accordingly, broth microdilution is used in veterinary routine diagnostic laboratories at a progressive rate. To reduce the costs of susceptibility testing, it is reasonable to develop widely accepted uniform microtitre plate layouts that are produced in large quantities. Such microtitre plate layouts have already been developed and published for the susceptibility testing of pathogens from food-producing animals. However, a microtitre plate layout, especially designed for the testing of bacteria from dogs and cats, should be available, too. The choice of the antimicrobial agents or combinations of antimicrobial agents to be included in a suitable layout should be based on the following criteria: (1) the approval and availability of an antimicrobial agent or combination of agents, (2) known cross-resistances, and (3) availability of approved clinical breakpoints. The latter point is of particular importance for the choice of the numbers of concentrations per antimicrobial agent tested and the range of test concentrations. Taking into account these aspects, a science-based layout proposal for microtitre plates, which are suitable for routine testing of bacteria from dogs and cats, is presented and discussed.     

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.     

Isolation of antifungal-resistant Candida from the blowholes of captive dolphins

In this study, we isolated eight strains of Candida albicans from the blowhole air cultures of eight dolphins (one Pacific white-sided dolphin and seven bottlenose dolphins) housed at the Enoshima Aquarium. The minimum inhibitory concentrations of antifungals for these isolates were determined by conducting E-test and broth microdilution assays using the CLSI M27-A3 protocol antifungal susceptibility testing method. Only one of the eight dolphins from which Candida had been isolated had been treated with amphotericin B (AMB), and four had been treated with itraconazole (ITZ). All isolates were identified as Candida albicans, and all were resistant to both ITZ and voriconazole, though the isolates exhibited susceptibility to AMB and micafungin. Based on our findings, we suspect that the frequency of occurrence of azole-resistant Candida species is increasing in captive dolphins as well as in their aquarium environments.     

Antimicrobial susceptibility of Brachyspira hyodysenteriae isolated from 21 Polish farms

Swine dysentery (SD) is a common disease among pigs worldwide, which contributes to major production losses. Antimicrobial susceptibility testing of B. hyodysenteriae, the etiological agent of SD, is mainly performed by the agar dilution method. This method has certain limitations due to difficulties in interpretation of results. The aim of this study was the analysis of antimicrobial susceptibility of Brachyspira hyodysenteriae (B. hyodysenteriae) Polish field isolates by broth microdilution procedure. The study was performed on 21 isolates of B. hyodysenteriae, collected between January 2006 to December 2010 from cases of swine dysentery. VetMIC Brachyspira panels with antimicrobial agents (tiamulin, valnemulin, doxycycline, lincomycin, tylosin and ampicillin) were used for susceptibility testing of B. hyodysenteriae. The minimal inhibitory concentration (MIC) was determined by the broth dilution procedure. The lowest antimicrobial activity was demonstrated for tylosin and lincomycin, with inhibition of bacterial growth using concentrations > 128 microg/ml and 32 microg/ml, respectively. In the case of doxycycline, the MIC values were < or = 2.0 microg/ml. No decreased susceptibility to tiamulin was found among the Polish isolates and MIC values for this antibiotic did not exceed 1.0 microg/ml. The results of the present study confirmed that Polish B. hyodysenteriae isolates were susceptible to the main antibiotics (tiamulin and valnemulin) used in treatment of swine dysentery. Further studies are necessary to evaluate a possible slow decrease in susceptibility to tiamulin and valnemulin of B. hyodysenteriae strains in Poland.     

Evaluation of the antifungal susceptibility of Malassezia pachydermatis to clotrimazole, miconazole and thiabendazole using a modified CLSI M27-A3 microdilution method

Background – In this study, we evaluated the antifungal susceptibility of Malassezia pachydermatis to clotrimazole (CTZ), miconazole (MCZ), and thiabendazole (TBD), azole derivatives employed in aural formulations labeled for treatment of canine otitis. Methods – The procedure for in vitro testing was based on the indications of the Clinical and Laboratory Standards Institute (CLSI) M27‐A3 microdilution method. A lipid‐enriched medium was employed to enhance the yeast growth (Christensen’s urea broth, with 0.1% Tween 80 and 0.5% Tween 40 as the lipid sources), while the inoculums size corresponded to approximately 1–5 × 10⁵ yeast cells/mL. Microplates were incubated at 37°C and read 48 h after inoculation. Azole MICs inhibiting fungal growth were the lowest drug concentrations that showed an optical density of ≤50% of the (drug‐free) growth control, as assessed by spectrophotometer (630 nm filter). Results – All isolates were inhibited by the three azoles, with different minimum inhibitory concentration (MIC) values. Most isolates were inhibited by drug concentrations of 2–8 (CTZ), 1–4 (MCZ), or 16–32 (TBD) μg/mL. These results are partially in agreement with the findings of previous studies, in which substantially higher/lower MICs were occasionally reported. This is likely because of the different methodologies employed. Such discrepancies may not apply to clinical situations, where the compounds are applied topically. Conclusion and clinical importance – The concept that clinical failure is linked to increased MICs is debatable, because significantly higher concentrations (in most cases at least 1,000 × the MIC) of the antifungals that were included in our study are routinely used in formulated products.     

Antimicrobial susceptibility testing of bacteria isolated from animals: methods for in-vitro susceptibility testing and their suitability with regard to the generation of the most useful data for therapeutic applications

In-vitro susceptibility testing provides valuable informations for choosing the most suitable antimicrobial agent for the control of bacterial infections in animals. Different diffusion and dilution methods, as conducted according to various approved performance standards, can be used to determine the in-vitro susceptibility of bacterial pathogens. In the present article, problems are discussed which arise from the use of different methods and the difficulty to interpret such results. While most approved performance standards were designed for testing of bacteria from human sources, the NCCLS document M31-A2 exclusively focusses on susceptibility testing of bacteria isolated from animals and--in contrast to all other standards--includes veterinary specific breakpoints for a number of antimicrobial agents used in veterinary medicine. Therefore, performance of in-vitro susceptibility testing of veterinary pathogens should follow the recommendations given in the NCCLS document M31-A2. The microdilution method is recommended as the method of choice for susceptibility testing. The result of a microdilution test is given as the minimum inhibitory concentration (MIC). This value provides a quantitative result which precisely indicates the degree of susceptibility of the tested bacterial strain and in return gives the veterinarian a clear guidance whether therapeutic intervention with the antibiotic in question will be successful.     

In vitro susceptibility of Ornithobacterium rhinotracheale to several antimicrobial drugs

As part of the basic characterization of Ornithobacterium rhinotracheale, the minimal inhibitory concentrations of 10 antimicrobial drugs were determined for reference strains and Mexican isolates by a broth microdilution method. For optimal growth of the organisms, a supplemented brain-heart infusion broth was used. The susceptibility of O. rhinotracheale to amoxicillin, enrofloxacin, and oxytetracycline was variable. However, consistent higher minimal inhibitory concentrations values were obtained for gentamicin, fosfomycin, trimethoprim, sulfamethazine, sulfamerazine, sulfaquinoxaline, and sulfachloropyridazine. Obtained results among Mexican isolates indicate a marked antimicrobial drug resistance trend.     

Development of Similar Broth Microdilution Methods to Determine the Antimicrobial Susceptibility of Flavobacterium columnare and F. psychrophilum

Flavobacterium columnare and F. psychrophilum are major fish pathogens that cause diseases that may require antimicrobial therapy. Choice of appropriate treatment is dependent upon determining the antimicrobial susceptibility of isolates. Therefore we optimized methods for broth microdilution testing of F. columnare and F. psychrophilum to facilitate standardizing an antimicrobial susceptibility test. We developed adaptations to make reproducible broth inoculums and confirmed the proper incubation time and media composition. We tested the stability of potential quality-control bacteria and compared test results between different operators. Log phase occurred at 48 h for F. columnare and 72–96 h for F. psychrophilum, confirming the test should be incubated at 28°C for approximately 48 h and at 18°C for approximately 96 h, respectively. The most consistent susceptibility results were achieved with plain, 4-g/L, dilute Mueller–Hinton broth supplemented with dilute calcium and magnesium. Supplementing the broth with horse serum did not improve growth. The quality-control strains, Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658, yielded stable minimal inhibitory concentrations (MIC) against all seven antimicrobials tested after 30 passes at 28°C and 15 passes at 18°C. In comparison tests, most MICs of the isolates agreed 100% within one drug dilution for ampicillin, florfenicol, and oxytetracycline. The agreement was lower with the ormetoprim–sulfdimethoxine combination, but there was at least 75% agreement for all but one isolate. These experiments have provided methods to help standardize antimicrobial susceptibility testing of these nutritionally fastidious aquatic bacteria.

Received June 24, 2015; accepted October 2, 2015.     

Evaluation of a rapid inoculum standardization system for antimicrobial susceptibility testing of bacterial isolates from the bovine mammary gland

A commercially available rapid inoculum standardization system (RISS) was compared to the broth inoculum standardization method (BIS) for antimicrobial susceptibility testing of bacterial isolates from the bovine mammary gland. Overall agreement between RISS and the BIS method was 95.9%. RISS bypasses the 2-8 h incubation period required by BIS, thus providing antimicrobial susceptibility test data in a more timely manner. RISS was determined to be an acceptable alternative method to the BIS method for preparation of standardized inoculum for antimicrobial susceptibility testing of bovine mammary gland isolates.     

宠物源性大肠杆菌耐药性调查

本试验采用微量肉汤稀释法对120株宠物医院分离的大肠杆菌进行头孢噻呋、恩诺沙星等13种抗生素的最小抑菌浓度(MIC)值测定,并利用WHONET 5.4软件对数据进行处理,以了解广州市宠物源性大肠杆菌的耐药情况。通过对其耐药谱型的调查,为指导宠物临床用药和耐药性及耐药性监测提供理论依据。     

Antimicrobial susceptibility of Mycoplasma hyorhinis

A broth microdilution technique was used to determine the antimicrobial susceptibility of 15 field isolates of Mycoplasma hyorhinis to 10 antimicrobial agents, representative of different classes, and contrasting newer agents to existing ones. For the macrolides, the MIC(90) for tylosin and tilmicosin was 1 and 4 microg/ml, respectively, but was > or = 16 microg/ml for erythromycin. Tetracycline, lincomycin and enrofloxacin each had an MIC(90) of 2 microg/ml. The mycoplasma had similar levels of susceptibility to the aminoglycoside and aminocyclictol classes exhibiting an MIC(90) of 4 microg/ml for gentamicin and 2 microg/ml for spectinomycin. The isolates exhibited high MICs to trimethoprim/sulfamethoxazole with an MIC(90) > or = 16/304 microg/ml. In summary, M. hyorhinis isolates from the US had low MICs against a variety of antimicrobials tested, with the exception of erythromycin and trimethoprim/sulfamethoxazole.     

Evaluation of the antimicrobial activity of florfenicol against bacteria isolated from bovine and porcine respiratory disease

Antimicrobial susceptibility of florfenicol (FFC) against 243 bacterial agents isolated in Korea from cattle and pigs with respiratory disease were investigated by agar diffusion and microdilution broth methods following the recommendations provided by the National Committee for Clinical Laboratory Standards. All Actinobacillus pleuropnemoniae, Pasteurella multocida, Mannheimia haemolytica and 98.6% of the Bordetella bronchiseptica isolates were susceptible to FFC, which as significantly more effective than the other antibiotics used in this study. FFC also showed high in vitro antimicrobial activities (MIC(90) < or = 1 microg/ml) against all strains tested with a minimal inhibitory concentration (MIC) determination ranging from 0.12 to 4 microg/ml. No resistant strains of A. pleuropneumoniae, P. multocida and M. haemolytica to FFC have apparently developed since the first introduction of this antibiotics for veterinary use in Korea. The results suggest that FFC is therapeutically valuable in the treatment of primary or complicating bacterial pathogens causing of the bovine and swine respiratory tract.     

Influence of bacteria on Clostridium perfringens infections in young chickens

When monoflora chickens with Lactobacillus acidophilus or Streptococcus faecalis were inoculated with Clostridium perfringens either in broth culture or resuspended in Gifu anaerobic medium broth or supernatant fluid, few or no chickens died. Approximately 50% of germ-free chickens died after inoculation of C. perfringens culture, whereas no conventional birds died after inoculation of broth culture. C. perfringens in the contents of duodenum from germ-free chickens numbered about 10(4) colony-forming units (CFU)/g after inoculation 10(8) CFU broth culture per bird, but in gnotobiotic and conventional chickens this organism decreased or was not detected. When C. perfringens was cultured in intestinal contents collected from germ-free chickens, C. perfringens proliferated but alpha toxin was not detected. These findings indicate that the pathogenicity of C. perfringens was suppressed by L. acidophilus or S. faecalis administered previously or inhibited by normal intestinal flora.     

Comparison of methods for antimicrobial susceptibility testing and MIC values for pleuromutilin drugs for Brachyspira hyodysenteriae isolated in Germany

In Germany treatment of swine dysentery is hampered by Brachyspira hyodysenteriae strains showing elevated MIC values to the few antibiotics licensed. Therefore, susceptibility testing of clinical isolates is an important service to the swine practitioner. This study compares the established agar dilution procedure for antimicrobial susceptibility testing of this fastidious anaerobe to the broth microdilution test newly developed [Anim. Health Res. 2 (2001) 59; Vet. Microbiol. 84 (2002) 123; J. Clin. Microbiol. 41 (2003) 2596]. A total of 221 isolates were examined twice with either test procedure using tiamulin and valnemulin as antibiotics. Both methods gave reproducible results, and the MIC values for the reference strains B. hyodysenteriae B204 and Staphylococcus aureus ATCC 29213 corresponded to previously published data. However, the results for individual strains differed significantly for both tests (P < 0.001) with MIC values being on average one dilution step lower in the broth dilution method. The 221 strains used for comparing test procedures were isolated between 1989 and 2001. An additional 102 strains isolated in 2002 were tested only with the broth dilution procedure. A significant rise in the average MIC value for both pleuromutilins could be demonstrated when comparing earlier isolates to those from 2000 to 2001 (P < 0.05), while in 2002 the average MIC significantly decreased when compared to the value in 2000 (P < 0.05). However, strains with MIC values for tiamulin as high as 8 microg/ml (broth dilution) could still be isolated.     

A guinea pig model of low-dose Mycobacterium bovis aerogenic infection

In order to develop a model of Mycobacterium bovis infection with pathogenetical relevance, a modified version of the Henderson apparatus was used to deliver infectious aerosols directly to the snouts of guinea pigs. Aerosols generated from 10(6), 10(7), 10(8)CFU/ml M. bovis suspensions established disease in every animal, with estimated retained doses of 10, 100, 1000 CFU, respectively. For comparison, other guinea pigs were inoculated with 100 CFU M. bovis intramuscularly (i.m.). Pathology and bacterial colonisation of lungs and spleen varied according to the dose and route of inoculation. Animals inoculated i.m. gave a significant cutaneous tuberculin hypersensitivity reaction earlier after testing than those infected aerogenically. A serological response to M. bovis antigens was detected in all infected animals. Intensity of antigen recognition was dose-dependent and although the range of antigens recognised varied between animals, a 25 kDa antigen present in the cell fraction was serodominant. Thus, a reproducible guinea pig model has been defined that may be suitable for virulence, vaccination, and immunological studies.     

Antibiotic resistance assessment in S. aureus strains isolated from raw sheep’s milk cheese

In vitro activities of 16 antibiotics were tested against 36 Staphylococcus aureus (SA) strains isolated from raw sheep’s milk cheese from six dairies. The minimum inhibitory concentration (MIC) was determined using a broth microdilution method (CLSI). All 36 isolates were analyzed for the presence of the accessory gene regulator gene, agr (I–IV), and genes encoding resistance to methicillin (mecA), erythromycin (ermA), penicillin (blaZ), and vancomycin (vanA-B). The isolates were also analyzed for similarities in pulsed-field gel electrophoresis (PFGE) patterns. SA strains showed resistance to ampicillin (36.1%), penicillin (33.3%), tetracycline (11.1%), and cloxacillin (2.8%) but were susceptible (≥94.4%) to 12 out of 16 tested antimicrobials. The overall susceptibility of the strains to oxacillin, vancomycin, and erythromycin was confirmed by the absence of the mecA, vanA-B, and ermA genes. The PFGE results showed that 32 strains belonged to 10 different clusters (P1–P10) while four strains were untypeable.     

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.