Antibiotic resistance in Lactobacillus reuteri and Lactobacillus plantarum

Lactic acid bacteria (LAB) may act as reservoirs of antibiotic resistance genes that can be transferred via the food chain or within the gastrointestinal tract to pathogenic bacteria. This thesis provides data required for assessing the potential risk of using antibiotic resistant strains of the LAB species Lactobacillus reuteri and Lactobacillus plantarum as food processing aids or probiotics. Knowledge of the distributions of antibiotic minimum inhibitory concentrations (MICs) for a species is needed when using a phenotypic method to differentiate strains with acquired resistance from susceptible strains or strains with intrinsic resistance. Controlled and standardised conditions are required for antibiotic susceptibility testing of LAB, as demonstrated here during evaluation of the Etest and broth microdilution MIC determination methods used. Inoculum size and incubation time were varied during broth microdilution testing of the susceptibilities of 35 LAB strains to six antibiotics. An increase in either parameter resulted in elevated MICs for all species. Antibiotic susceptibility profiles were determined for 56 L. reuteri and 121 L. plantarum strains that differed by source and spatial and temporal origin. MIC data obtained with the Etest and the broth microdilution methods corresponded well with each other. All L. plantarum strains were susceptible to ampicillin, gentamicin, erythromycin and clindamycin, and intrinsically resistant to streptomycin. Acquired resistance to tetracycline was associated with plasmid-bound tet(M). Lactobacillus reuteri strains had acquired resistance to tetracycline (n=28), ampicillin (n=14), erythromycin/clindamycin (n=6) and chloramphenicol (n=1). This resistance was attributed to mutational pbp genes for ampicillin and to added tet(W), erm and cat(TC) genes for the antibiotics inhibiting protein synthesis. Genetic relatedness was observed among L. reuteri strains with high MICs for both ampicillin and tetracycline and among strains with high MICs for both erythromycin and clindamycin. The majority of the antibiotic resistant L. reuteri strains carried the resistance genes on plasmids. Traits of putative transfer machineries adjacent to both plasmid- and chromosome-located resistance genes were demonstrated. Lactobacillus reuteri as a donor of resistance genes in the human gut was investigated by studying the transferability of the tetracycline resistance gene tet(W) to faecal enterococci, bifidobacteria and lactobacilli. No gene transfer was demonstrated under the conditions tested

Cryptosporidium in drinking water - Risk assessment

Two waterborne outbreaks in Sweden due to the parasite Cryptosporidium hominis with 47 000 cases motivated this risk assessment. Risk management questions were focused on relationship between occurrence of oocysts in raw water and risk of illness via drinking water. Limited raw water concentration data were available. Thus, all data were used to describe variations in Cryptosporidium concentrations in simulated typical “Swedish” surface raw water using three distributions. Differences in risk using the different distributions were comparatively small, and indicated that independent of distribution a greater mean concentration and spread around the mean resulted in a higher risk and maximum exposure, respectively. It was not possible to determine which distribution was best. Other gaps included the proportion of oocysts that are infectious and dose-response relationships. Exponential and Beta-Poisson dose-response models were compared and resulted in risk estimates that differed by a factor of 10 to 100. Question 1: What is the probability of illness given variable concentrations and treatment efficiencies in the water treatment plant? Oocyst exposure was simulated for 30 000 consumers supplied by a water treatment plant having three treatment barriers; coagulation/flocculation, slow sand filtration and UV-disinfection. Consumers were exposed to oocysts only at exceptionally high oocyst concentrations, >10 000/10 L raw water but not at concentrations <1 000 oocyst/10 L. Q2: The question concerned different aspects of monitoring of raw water/drinking water and associated uncertainties in estimated risks. Q3: Compare risk associated with surface and ground water raw water sources? Q4: Risk and extreme events? Events such as heavy rains or flooding may increase oocyst concentration in raw water 5-100 times which may lead to an increased risk of Cryptosporidiosis

Effects of Inoculum Size and Incubation Time on Broth Microdilution Susceptibility Testing of Lactic Acid Bacteria

Inoculum size and incubation time were varied during broth microdilution testing of the susceptibilities of 35 strains of lactic acid bacteria to six antibiotics. An increase in either parameter resulted in elevated MICs for all species. An inoculum of 3 × 10(5) CFU/ml is recommended to assess the antibiotic susceptibilities of these bacteria by using broth microdilution

Limited Dissemination of Extended-Spectrum β-Lactamase– and Plasmid-Encoded AmpC–Producing Escherichia coli from Food and Farm Animals, Sweden

Extended-spectrum β-lactamase (ESBL)– and plasmid-encoded ampC (pAmpC)–producing Enterobacteriaceae might spread from farm animals to humans through food. However, most studies have been limited in number of isolates tested and areas studied. We examined genetic relatedness of 716 isolates from 4,854 samples collected from humans, farm animals, and foods in Sweden to determine whether foods and farm animals might act as reservoirs and dissemination routes for ESBL/pAmpC-producing Escherichia coli. Results showed that clonal spread to humans appears unlikely. However, we found limited dissemination of genes encoding ESBL/pAmpC and plasmids carrying these genes from foods and farm animals to healthy humans and patients. Poultry and chicken meat might be a reservoir and dissemination route to humans. Although we found no evidence of clonal spread of ESBL/pAmpC-producing E. coli from farm animals or foods to humans, ESBL/pAmpC-producing E. coli with identical genes and plasmids were present in farm animals, foods, and humans

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