Antimicrobial susceptibility testing of Brachyspira intermedia and Brachyspira pilosicoli isolates from Australian chickens

Susceptibilities of predominantly Australian isolates of the pathogenic intestinal spirochaetes Brachyspira intermedia (n=25) and Brachyspira pilosicoli (n=17) from chickens were tested in agar dilution against four concentrations each of the antimicrobials tiamulin, lincomycin, tylosin, metronidazole, tetracycline and ampicillin. Based on available minimum inhibitory concentration (MIC) breakpoint values for Brachyspira hyodysenteriae or other Gram-negative enteric veterinary pathogens, isolates of both species generally were susceptible to tiamulin, lincomycin, metronidazole and tetracycline. Although not classed as resistant, four isolates of B. intermedia had an elevated MIC range for tiamulin (1 to 4 mg/l), 11 isolates of B. intermedia and five of B. pilosicoli had an elevated MIC range for lincomycin (10 to 50 mg/l), one isolate of B. pilosicoli had an elevated MIC range for tetracycline (10 to 20 mg/l), and one isolate of B. intermedia and five of B. pilosicoli had an elevated MIC range for ampicillin (10 to 50 mg/l). A clear lack of susceptibility to tylosin (MIC >4 mg/l) was seen in 11 isolates each of B. intermedia and B. pilosicoli, and to ampicillin (MIC >32 mg/l) in two isolates of B. pilosicoli. These data suggest that some resistance to common antimicrobials exists among intestinal spirochetes obtained from laying hens and supports the need of MIC data for clinical isolates before any treatment is considered.

Use of a proposed antimicrobial susceptibility testing method for Haemophilus parasuis

The aim of this study was to examine the antimicrobial susceptibility of 97 Haemophilus parasuis cultured from Australian pigs. As there is no existing standard antimicrobial susceptibility technique available for H. parasuis, methods utilising the supplemented media, BA/SN for disc diffusion and test medium broth (TMB) for a microdilution technique, were initially evaluated with the reference strains recommended by the Clinical and Laboratory Standards Institute. The results of the media evaluation suggested that BA/SN and TMB can be used as suitable media for susceptibility testing of H. parasuis. The proposed microdilution technique was then used with 97 H. parasuis isolates and nine antimicrobial agents. The study found that Australian isolates showed elevated minimum inhibitory concentrations (MICs) for ampicillin (1%), penicillin (2%), erythromycin (7%), tulathromycin (9%), tilmicosin (22%), tetracycline (31%) and trimethoprim-sulfamethoxazole (40%). This study has described potential antimicrobial susceptibility methods for H. parasuis and has detected a low percentage of Australian H. parasuis isolates with elevated antimicrobial MICs.

Antimicrobial susceptibility of Histophilus somni isolated from clinically affected cattle in Australia

This study investigated antimicrobial resistance traits, clonal relationships and epidemiology of Histophilus somni isolated from clinically affected cattle in Queensland and New South Wales, Australia. Isolates (n = 53) were subjected to antimicrobial susceptibility testing against six antimicrobial agents (ceftiofur, enrofloxacin, florfenicol, tetracycline, tilmicosin and tulathromycin) using disc diffusion and minimum inhibitory concentration (MIC) assays. Clonal relationships were assessed using repetitive sequence PCR and descriptive epidemiological analysis was performed. The H. somni isolates appeared to be geographically clonal, with 27/53 (47%) isolates grouping in one cluster from one Australian state. On the basis of disc diffusion, 34/53 (64%) isolates were susceptible to all antimicrobial agents tested; there was intermediate susceptibility to tulathromycin in 12 isolates, tilmicosin in seven isolates and resistance to tilmicosin in one isolate. Using MIC, all but one isolate was susceptible to all antimicrobial agents tested; the non-susceptible isolate was resistant to tetracycline, but this MIC result could not be compared to disc diffusion, since there are no interpretative guidelines for disc diffusion for H. somni against tetracycline. In this study, there was little evidence of antimicrobial resistance in H. somni isolates from Australian cattle. Disc diffusion susceptibility testing results were comparable to MIC results for most antimicrobial agents tested; however, results for isolates with intermediate susceptibility or resistance to tilmicosin and tulathromycin on disc diffusion should be interpreted with caution in the absence of MIC results.

Variation in the antimicrobial susceptibility of actinobacillus pleuropneumoniae isolates in a pig, within a batch of pigs, and among batches of pigs from one farm

Antimicrobial resistance in bacterial porcine respiratory pathogens has been shown to exist in many countries. However, little is known about the variability in antimicrobial susceptibility within a population of a single bacterial respiratory pathogen on a pig farm. This study examined the antimicrobial susceptibility of Actinobacillus pleuropneumoniae using multiple isolates within a pig and across the pigs in three different slaughter batches. Initially, the isolates from the three batches were identified, serotyped, and subsample genotyped. All the 367 isolates were identified as A. pleuropneumoniae serovar 1, and only a single genetic profile was detected in the 74 examined isolates. The susceptibility of the 367 isolates of A. pleuropneumoniae to ampicillin, tetracycline and tilmicosin was determined by a disc diffusion technique. For tilmicosin, the three batches were found to consist of a mix of susceptible and resistant isolates. The zone diameters of the three antimicrobials varied considerably among isolates in the second sampling. In addition, the second sampling provided statistically significant evidence of bimodal populations in terms of zone diameters for both tilmicosin and ampicillin. The results support the hypothesis that the antimicrobial susceptibility of one population of a porcine respiratory pathogen can vary within a batch of pigs on a farm.

Comparative evaluation of 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)2H-tetrazolium, monosodium salt (WST-8) rapid colorimetric assays for antimicrobial susceptibility testing of staphylococci and ESBL-producing clinical isolates

A new generation of water soluble tetrazolium salts have recently become available and in this study we compared a colorimetric assay developed using one of these salts, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), with a previously developed 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide(XTT) colorimetric assay to determine which agent is most suitable for use as a colorimetric indicator in susceptibility testing. The MICs of 6 antibiotics were determined for 33 staphylococci using both colorimetric assays and compared with those obtained using the British Society for Antimicrobial Chemotherapy reference broth microdilution method. Absolute categorical agreement between the reference and test methods ranged from 79% (cefuroxime) to 100% (vancomycin) for both assays. No minor or major errors occurred using either assay with very major errors ranging from zero (vancomycin) to seven (cefuroxime). Analysis of the distribution of differences in the 1092 dilution MIC results revealed overall agreement, within the accuracy limits of the standard test ( 1 1092 dilution), using the XTT and WST-8 assays of 98% and 88%, respectively. Further studies on 31 ESBL-producing isolates were performed using the XTT method with absolute categorical agreement ranging from 87% (nitrofurantoin) to 100% (ofloxacin and meropenem). No errors were noted for either ofloxacin or meropenem with overall agreement of 91%. The data suggests that XTT is more reliable and accurate than WST-8 for use in a rapid antimicrobial susceptibility test. (c) 2007 Elsevier B.V. All rights reserved.

Rapid colorimetric assay for antimicrobial susceptibility testing of Pseudomonas aeruginosa

A colorimetric assay based on the reduction of a tetrazolium salt {2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT)} for rapidly determining the susceptibility of Pseudomonas aeruginosa isolates to bactericidal antibiotics is described. There was excellent agreement between the tobramycin and ofloxacin MICs determined after 5 h using the XTT assay and after 18 h using conventional methods. The data suggests that an XTT-based assay could provide a useful method for rapidly determining the susceptibility of P. aeruginosa to bactericidal antibiotics.

Comparative evaluation of 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8) rapid colorimetric assays for antimicrobial susceptibility testing of staphylococci and ESBL-producing clinical isolates

A new generation of water soluble tetrazolium salts have recently become available and in this study we compared a colorimetric assay developed using one of these salts, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), with a previously developed 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay to determine which agent is most suitable for use as a colorimetric indicator in susceptibility testing. The MICs of 6 antibiotics were determined for 33 staphylococci using both colorimetric assays and compared with those obtained using the British Society for Antimicrobial Chemotherapy reference broth microdilution method. Absolute categorical agreement between the reference and test methods ranged from 79% (cefuroxime) to 100% (vancomycin) for both assays. No minor or major errors occurred using either assay with very major errors ranging from zero (vancomycin) to seven (cefuroxime). Analysis of the distribution of differences in the log2 dilution MIC results revealed overall agreement, within the accuracy limits of the standard test (± 1 log2 dilution), using the XTT and WST-8 assays of 98% and 88%, respectively. Further studies on 31 ESBL-producing isolates were performed using the XTT method with absolute categorical agreement ranging from 87% (nitrofurantoin) to 100% (ofloxacin and meropenem). No errors were noted for either ofloxacin or meropenem with overall agreement of 91%. The data suggests that XTT is more reliable and accurate than WST-8 for use in a rapid antimicrobial susceptibility test.