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.

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.

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.

Oxacillin Resistance and Antimicrobial Susceptibility Profile of Staphylococcus saprophyticus and Other Staphylococci Isolated from Patients with Urinary Tract Infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular identification and antimicrobial susceptibility of Nocardia spp. isolated from bovine mastitis in Brazil

Nocardia spp. infections can cause severe damage to the mammary gland due to suppurative pyogranulomatous lesions and lack of clinical cure in response to conventional antimicrobial therapy. Although Nocardia infections are considered relatively uncommon in cows, there has been an apparent worldwide increase in the incidence of bovine mastitis caused by Nocardia spp, perhaps due to environmental transmission of this ubiquitous pathogen. The objectives of present study were to determine: (i) species distribution of 80 Nocardia isolates involved in bovine mastitis (based on molecular methods); and (ii) antimicrobial susceptibility pattern of all isolates from three geographical areas in Brazil. In this study, Nocardia nova (80%) was the most frequently isolated species, followed by Nocardia farcinica (9%). Additionally, Nocardia puris, Nocardia cyriacigeorgica, Nocardia veterana, Nocardia africana, and Nocardia arthritidis were detected using 16S rRNA sequencing. This is apparently the first report of N. puris, N. veterana, N. cyriacigeorgica, N. arthritidis and N. africana in association with bovine mastitis. Based on the disk diffusion test, isolates were most frequently resistant to cloxacillin (75%), ampicillin (55%) and cefoperazone (47%), whereas few Nocardia spp. were resistant to amikacin, cefuroxime or gentamicin. © 2013 Elsevier B.V. All rights reserved.

Exogenous bacterial osteomyelitis in 52 dogs: a retrospective study of etiology and in vitro antimicrobial susceptibility profile (2000-2013)

Background: Most clinical cases of osteomyelitis in dogs involve infectious agents, especially bacteria and fungi. The characterization of these microorganisms may aid in the prevention and treatment of disease.Objective: The aim of this study was to evaluate retrospectively microbiological cultures and in vitro antimicrobial susceptibility profile of isolates from 52 cases of bacterial osteomyelitis in long bones of dogs over 2000-2013. In 78% of the cases injuries were caused by a motor vehicle accident, but there were a few cases of dog bites (17%) and ascending infection due to pododermatitis (5%).Animals and methods: The isolated microorganisms were identified based on conventional phenotypic methods. In vitro disk diffusion test was performed using 30 different antimicrobials.Results: The isolates were obtained from femur (28%), humerus (16%), tibia (31%), and radius/ulna (25%). Among 52 cases, culture was positive in 88% of cases. Thirteen genus of different species of microorganisms were isolated. The most common microorganisms isolated were Staphylococcus spp. and Escherichia coli followed by Streptococcus spp., enteric bacteria, Corynebacterium sp. and anaerobic bacteria. In 42% of cases cultures were mixed. The most effective drugs against isolated bacteria were amoxicillin and clavulanate potassium (79%) followed by ceftriaxone (69%). High-resistance rates were documented against azithromycin (80%), penicillin (59%), and clindamycin (59%).Conclusions: The present study highlights diverse etiologic agents in cases of infectious bacterial osteomyelitis, with predominance of Staphylococcus genus, and reinforces the importance of obtaining cultures and susceptibility profiles given the high rates of antimicrobial resistance.

Candida species distribution and antifungal susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing and new vs. old Clinical and Laboratory Standards Institute clinical breakpoints: a 6-year prospective candidaemia survey from the fungal infection network of Switzerland

We analyzed the species distribution of Candida blood isolates (CBIs), prospectively collected between 2004 and 2009 within FUNGINOS, and compared their antifungal susceptibility according to clinical breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in 2013, and the Clinical and Laboratory Standards Institute (CLSI) in 2008 (old CLSI breakpoints) and 2012 (new CLSI breakpoints). CBIs were tested for susceptiblity to fluconazole, voriconazole and caspofungin by microtitre broth dilution (Sensititre® YeastOne™ test panel). Of 1090 CBIs, 675 (61.9%) were C. albicans, 191 (17.5%) C. glabrata, 64 (5.9%) C. tropicalis, 59 (5.4%) C. parapsilosis, 33 (3%) C. dubliniensis, 22 (2%) C. krusei and 46 (4.2%) rare Candida species. Independently of the breakpoints applied, C. albicans was almost uniformly (>98%) susceptible to all three antifungal agents. In contrast, the proportions of fluconazole- and voriconazole-susceptible C. tropicalis and F-susceptible C. parapsilosis were lower according to EUCAST/new CLSI breakpoints than to the old CLSI breakpoints. For caspofungin, non-susceptibility occurred mainly in C. krusei (63.3%) and C. glabrata (9.4%). Nine isolates (five C. tropicalis, three C. albicans and one C. parapsilosis) were cross-resistant to azoles according to EUCAST breakpoints, compared with three isolates (two C. albicans and one C. tropicalis) according to new and two (2 C. albicans) according to old CLSI breakpoints. Four species (C. albicans, C. glabrata, C. tropicalis and C. parapsilosis) represented >90% of all CBIs. In vitro resistance to fluconazole, voriconazole and caspofungin was rare among C. albicans, but an increase of non-susceptibile isolates was observed among C. tropicalis/C. parapsilosis for the azoles and C. glabrata/C. krusei for caspofungin according to EUCAST and new CLSI breakpoints compared with old CLSI breakpoints.

Serotype distribution and antimicrobial susceptibility of group B streptococci in pregnant women: results from a Swiss tertiary centre.

OBJECTIVE To evaluate the rates of penicillin, clindamycin and erythromycin resistance and the serotype distribution among isolates of group B streptococcus (GBS) obtained from pregnant women at the University Hospital of Bern in Switzerland. METHODS We prospectively collected screening samples for GBS colonisation at the University Women's Hospital Bern, Switzerland, between March 2009 and August 2010. We included 364 GBS isolates collected from vaginal, cervical or vaginal-perianal swabs at any gestation time. The minimal inhibitory concentrations for penicillin, clindamycin and erythromycin were established using Etest with 24 hours of incubation, and inducible clindamycin resistance was tested with double disk diffusion tests. Serotyping was done with a rapid latex agglutination test or, if not conclusive, with polymerase chain-reaction (PCR) testing. We looked for significant associations between resistance patterns, age groups, serotype and ethnicity. RESULTS All isolates were susceptible to penicillin. Resistance rates were 14.5% for erythromycin and 8.2% for clindamycin. Of 364 isolates, 5.8% were susceptible to clindamycin but not to erythromycin, although demonstrating inducible clindamycin resistance. Hence, the final reported clindamycin resistance rate was 14%. Serotype III was the most frequent serotype (29%), followed by V (25%) and Ia (19%). Serotype V was associated with erythromycin resistance (p = 0.0007). In comparison with all other ethnicities, patients from Asia showed a higher proportion of erythromycin and clindamycin resistance (p = 0.018). No significant association between resistance patterns and age groups was found. CONCLUSION In pregnant women with GBS colonisation, penicillin is the antibiotic of choice for intrapartum prophylaxis to prevent neonatal early-onset GBS sepsis. In women with penicillin allergy and at high risk for anaphylactic reaction, clindamycin may be an alternative. The resistance rate for clindamycin at our institution was 14%; therefore, susceptibility must be tested before administration.

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.

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.

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.