Intra- and Interlaboratory Performances of Two Commercial Antimicrobial Susceptibility Testing Methods for Bifidobacteria and Nonenterococcal Lactic Acid Bacteria

In a small-scale harmonization study involving nine laboratories in eight European countries, the intra- and interlaboratory performances of two commercially available systems, i.e., the VetMIC microplate system and Etest, for antimicrobial susceptibility testing of nonenterococcal lactic acid bacteria (NELAB) and bifidobacteria were analyzed. In addition, one laboratory also performed standard broth microdilution as a reference method. MICs of tetracycline, erythromycin, ampicillin, gentamicin, clindamycin, and streptomycin for the type strains of 25 species of NELAB and bifidobacteria and MICs of vancomycin for a selection of relevant taxa were determined. The previously described lactic acid bacterium susceptibility test medium (LSM) and related mixed-medium formulations, all including Iso-Sensitest broth as a basic component, were used as test media. The overall agreement of median MIC ranges ± 1 log₂ dilution determined by the VetMIC and Etest methods with the median MICs determined by the reference method was very good for tetracycline, ampicillin, and streptomycin (92.3 to 100%) but low for erythromycin (19.5 to 30.7%) and clindamycin (50.0 to 80.8%). There was a consensus among the participating laboratories that VetMIC was preferred over Etest because of its lower cost, better growth support, and more uniform criteria for MIC end point reading. With the range for acceptable intralaboratory reproducibility being defined as the median MIC ± 1 log₂ dilution, VetMIC results (with 69.2% of all data sets in the acceptable range) were shown to display greater reproducibility than Etest results (with 58.8% of all data sets in the acceptable range). Also at the interlaboratory level, the proportion of MIC values obtained with VetMIC that belonged to the complete agreement category (60.0%) was higher than the proportion of such values obtained with Etest (47.0%), which indicates a higher degree of interlaboratory reproducibility for the former method. Apart from some agent-specific effects, the majority of VetMIC and Etest replicate data sets were situated within a 1- to 2-log₂ dilution range, suggesting that the two methods can be considered to be equivalent for recognizing resistance phenotypes. This multicenter study has further validated the standard use of LSM and related mixed-medium formulations with commercially available systems and formed the basis for the ongoing development of the ISO 10932/IDF 223 standard for susceptibility testing of NELAB and bifidobacteria.Because of their distinctive fermentative, functional, and potentially health-promoting properties, bifidobacteria and nonenterococcal lactic acid bacteria (NELAB) such as lactobacilli, lactococci, and Streptococcus thermophilus are intensively used in the food industry as starter cultures, adjunct cultures, and probiotics (29). Although the majority of NELAB and Bifidobacterium species are food-grade organisms, the large-scale application and deliberate introduction of such cultures into the food chain has opened the debate over whether or not criteria that document their safety for human and animal use should be defined (35). Despite the overall low pathogenic potential of these organisms, several studies have indicated that especially NELAB can act as reservoirs of potentially transferable antimicrobial resistance genes (1, 10, 15, 31). In the field of probiotics, the absence of acquired resistance traits has been recommended as a safety criterion in the selection of new commercial culture probiotic strains for human use (12, 27, 28, 33).Due mainly to the limited clinical relevance of NELAB and bifidobacteria, the development and optimization of methods for antimicrobial susceptibility testing of these organisms have long been underappreciated. Moreover, the fact that many of these organisms have specific nutritional and atmospheric requirements for growth does not allow uniform use of standardized susceptibility test media such as Mueller-Hinton broth and Iso-Sensitest (IST) broth. There are indications that de Man, Rogosa, and Sharpe (MRS) medium, which is commonly used as a growth medium for most of these organisms, may exhibit antagonistic effects with supplemental antimicrobials in susceptibility testing (13). To address the apparent limitations of using single media, a mixed formulation of IST broth (90%, vol/vol) and MRS broth (10%, vol/vol) referred to as lactic acid bacterium susceptibility test medium (LSM) was developed recently (16). This new formulation has proven to support the growth of a wide taxonomic range of lactobacilli and Bifidobacterium spp. (when supplemented with 0.03% cysteine) and minimizes potential antagonism between medium components and tested antimicrobials. So far, LSM and related mixed-medium formulations, all containing IST broth as the basic component, have been successfully used to determine MICs for members of the genera Bifidobacterium, Lactobacillus, Lactococcus, Pediococcus, and Streptococcus by microdilution and Etest methods (2, 5, 7, 8, 17, 18, 20-24, 32).In the near future, it is expected that the increased use of LSM as the standard medium for susceptibility testing of NELAB and bifidobacteria will produce a large amount of MIC data, enabling the definition of epidemiological cutoffs (ECOFFS). As proposed by the European Committee on Antimicrobial Susceptibility Testing (EUCAST [http://www.eucast.org/]), ECOFFS provide an objective basis to differentiate wild-type organisms, which lack acquired and mutational resistance mechanisms, from non-wild-type members of the same species that contain one or more mechanisms conferring antimicrobial resistance. For this purpose, evaluation of the internal and external quality assurance procedures for reference methods and commercial systems for susceptibility testing is absolutely crucial for the correct interpretation of these ECOFFS. Several harmonization studies of susceptibility testing of clinical (14, 30), veterinary (26, 34), and aquatic (11, 25) organisms have provided important insights into the reproducibility of results from standardized methods at the intra- and/or interlaboratory level. Although such studies would also be highly valuable to all with an interest in evidence-based biosafety assessments of NELAB and bifidobacteria for human and animal use, the performances of susceptibility test methods using LSM within and across laboratories have to our knowledge not been evaluated.Within the framework of an international research project, the European Union Assessment and Critical Evaluation of Antibiotic Resistance Transferability in Food Chain (EU-ACE-ART), a small-scale harmonization study involving nine laboratories in eight European countries was conducted. The study set out to examine the performances of two commercial susceptibility test methods widely used throughout the EU-ACE-ART project, i.e., the VetMIC system (broth microdilution) and Etest (agar diffusion), at the intra- and interlaboratory levels. For this purpose, MICs of six antimicrobials for the type strains of 25 NELAB and Bifidobacterium species and MICs of vancomycin for a selection of relevant taxa were determined using LSM and related mixed-medium formulations as test media.     

Leisure-Time Physical Activity and the Metabolic Syndrome in the Finnish Diabetes Prevention Study

OBJECTIVE

To assess the effects of leisure-time physical activity (LTPA) and resistance training on metabolic syndrome (MetS) and its components in a post hoc analysis of the Finnish Diabetes Prevention Study, a randomized controlled lifestyle counseling trial.

RESEARCH DESIGN AND METHODS

A cohort of 486 middle-aged overweight men and women with impaired glucose tolerance were followed for an average of 4.1 years. The intervention and control groups were combined in the analyses. LTPA was assessed by questionnaires, dietary intake by food records, and features of the MetS by anthropometric and biochemical measures annually. Resistance training sessions were documented for 137 participants.

RESULTS

Increased moderate-to-vigorous LTPA, even after adjustments for changes in dietary intakes of total and saturated fat, fiber, and energy, and change in BMI was associated with a greater likelihood for resolution (29.7 vs. 19.1%; P = 0.004 in the upper versus lower third of change) and a lesser likelihood for development (23.5 vs. 44.7%; P = 0.041) of the MetS. Of the components of the MetS, the increase in moderate-to-vigorous LTPA was associated most strongly with improvement of glycemia. Among the 137 participants who participated in resistance training, MetS components were favorable in individuals who were in the upper third of participation rate (median 51 times/year) compared with individuals in the lowest third (median 8.5 times/year).

CONCLUSIONS

Increased moderate-to-vigorous LTPA was associated with a decreased likelihood of developing the MetS and an increased likelihood of its resolution in individuals at high risk for type 2 diabetes.The metabolic syndrome (MetS) is a constellation of interrelated metabolic risk factors, including abdominal obesity, insulin resistance, hyperglycemia, dyslipidemia, and elevated blood pressure, often accompanied by a prothrombotic and proinflammatory state (1,2). The underlying pathophysiology of the MetS is unclear, but both insulin resistance and abdominal obesity are considered main components (1,2). The MetS increases the risk of both type 2 diabetes (3) and cardiovascular disease (4,5).Recent recommendations for the prevention and treatment of the MetS and its components promote increased physical activity (including aerobic and resistance exercise), a healthy diet, and weight loss (2,6–8). In lifestyle interventions trials, the incidence of type 2 diabetes has been reduced by more than half in individuals with impaired glucose tolerance, and the prevalence of the MetS has also been decreased (9,10). In the Finnish Diabetes Prevention Study (DPS), increased moderate-to-vigorous leisure-time physical activity (LTPA) was strongly associated with a lower risk of type 2 diabetes, independently of dietary changes and weight loss (11).Some prospective epidemiological studies and uncontrolled trials have suggested that increased moderate-to-vigorous exercise decreases the incidence or prevalence of the MetS (8,12,13). However, data on the role of changes in LTPA in the prevention and treatment of the MetS in long-term studies are limited. Therefore, we conducted a post hoc analysis of the Finnish DPS. Our hypothesis was that the change in LTPA and participation in resistance training would be associated with the change in the MetS and its components.     

Recombinant Flagellin A Proteins from Borrelia burgdorferi Sensu Stricto, B. afzelii, and B. garinii in Serodiagnosis of Lyme Borreliosis

Genes for flagellin A (FlaA) proteins from European borrelial strains of Borrelia burgdorferi sensu stricto, B. afzelii, and B. garinii were cloned and sequenced. An identity of 92 to 93% was observed in the flaA sequences of the different species. Polyhistidine-tagged recombinant FlaA (rFlaA) proteins were produced in Escherichia coli and used as antigens in Western blotting (WB) and enzyme-linked immunosorbent assay (ELISA). In immunoglobulin G (IgG) WB, 71% (10 of 14) of the sera from neuroborreliosis and 86% (12 of 14) of those from Lyme arthritis patients reacted with one to three rFlaAs. In IgG ELISA, 74% (14 of 19) and 79% (15 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive. The immunoreactivity in local European patient sera was stronger against rFlaA from B. garinii and B. afzelii than against rFlaA from B. burgdorferi sensu stricto. Neither IgG nor IgM ELISA was sensitive in the serodiagnosis of erythema migrans. Serum samples from patients with syphilis and systemic lupus erythematosus showed mild cross-reactivity in IgG tests. Sera from Yersinia enterocolitica or beta-hemolytic Streptococcus infections showed only occasional responses. With IgM ELISA, 58% (11 of 19) and 37% (7 of 19) of patients with neuroborreliosis and arthritis, respectively, were positive. Cross-reactive antibodies to FlaA, especially in serum samples from patients with rheumatoid factor positivity and Epstein-Barr virus infection, reduced the specificity of IgM serodiagnosis. Therefore, rFlaA seems to have a limited role for IgM serodiagnosis, yet rFlaA might be useful in the IgG serodiagnosis of disseminated Lyme borreliosis.     

Tear fluid concentration of mmp–8 is elevated in non—allergic eosinophilic conjunctivitis and correlates with conjunctival inflammatory cell infiltration

Background  To investigate tear fluid concentration of matrix metalloproteinase 8 (MMP–8) and its relation to conjunctival inflammatory cell infiltration in persistent non—allergic eosinophilic conjunctivitis (NAEC). Methods  Two groups were included: 26 consecutive adult patients with NAEC (conjunctival eosinophils at least 1+ [1-10 eosinophils/slide], skin prick test [SPT] to common allergens negative), and 26 asymptomatic adult persons (no conjunctival eosinophils, SPT negative). MMP–8 tear fluid concentrations were determined by immunofluorometric assay, and conjunctival brush cytology samples from NAEC patients were used for MMP–8 immunocytochemistry. Gelatin zymography was used to illustrate proteolytic activity within the tear fluid samples. Results  The mean MMP–8 concentration was significantly higher among NAEC patients (214.3 ± 327.7 μg/l) than among healthy persons (50.4 ± 62.3 μg/l, P < 0.0001). In the NAEC patients, tear fluid MMP–8 correlated with the numbers of conjunctival neutrophils (r = 0.66, P = 0.0002) as well as with goblet cells and columnar epithelial cells (r = 0.54 for both, P = 0.045), but not with the lymphocyte numbers (r = -0.36, P = 0.0741). By immunocytology, MMP–8 protein could also be detected in vivo in the inflammatory cell population within the conjunctiva. Zymography revealed that proteolysis was significantly higher in the NAEC group, and activated enzymes were practically found only in the NAEC group. Conclusions  The results showed that NAEC is an inflammatory condition characterized by increased tear fluid MMP–8 levels, probably derived from both inflammatory and structural conjunctival cells. The increased proteolytic activity in NAEC patients may indicate risk of conjunctival structural changes (remodeling).     

Myocardial perfusion quantitation with 15O-labelled water PET: high reproducibility of the new cardiac analysis software (Carimas™)

Purpose

Carimas? (Cardiac Image Analysis System) is a new software package developed at the Turku PET Centre for the quantitation of PET studies of the heart with a broad range of tracers. The goal of this study was to assess the reproducibility of results the package provides for myocardial perfusion (MP) quantitation using ¹⁵O-labelled water.

Methods

Four observers with various levels of experience in nuclear medicine independently analysed 20 MP studies (10 rest flow: “rest”, 10 adenosine-induced hyperaemia: “stress”). Each study was analysed twice. The linear mixed model for repeated measures was fitted to the data to calculate intraclass correlation coefficients (ICC), differences between the repeats (the intraobserver differences) and differences between the observers (the interobserver differences). Also, Pearson correlation coefficients (r) were calculated and Bland-Altman plots were drawn. The reproducibility of MP was assessed on global, regional and segmental levels. Thereafter, this analysis was applied in 48 consecutive clinical patients with suspected coronary heart disease (CHD).

Results

For the experienced observer the Pearson r for all segments was 0.974 at rest and 0.978 at stress (p?<?0.0001), and the repeatability coefficients were 0.145 ml/g per min (15.5% of the average) and 0.389 ml/g per min (14.9%), correspondingly. The ICC reflected very good overall reproducibility. The intraobserver and interobserver differences were small, and the difference between the most and the least experienced observers at stress was 8.5% for the global MP. The clinical accuracy of the perfusion in the detection of CHD was excellent (positive predictive value 91% and negative predictive value 88%) against invasive angiography.

Conclusion

The results demonstrate high reproducibility of myocardial perfusion quantitation with ¹⁵O-labelled water PET using Carimas?. The results support the feasibility of robust analysis and good clinical accuracy.