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 log(2) 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 log(2) 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-log(2) 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.

Diversity of lactic acid bacteria from modified atmosphere packaged sliced cooked meat products at sell-by date assessed by PCR-denaturing gradient gel electrophoresis.

The predominant lactic acid bacteria (LAB) microbiota associated with three types of modified atmosphere packaged (MAP) sliced cooked meat products (i.e. ham, turkey and chicken) was analyzed at sell-by date using a combination of culturing and molecular population fingerprinting. Likewise routine analyses during industrial MAP production, meat samples were plated on the general heterotrophic Plate Count Agar (PCA) and on the LAB-specific de Man, Rogosa, Sharpe (MRS) agar under different temperature and atmosphere conditions. Subsequently, community DNA extracts were prepared from culturable bacterial fractions harvested from both media and used for PCR targeting the V3 hyper-variable region of the 16S rRNA gene followed by denaturing gradient gel electrophoresis (DGGE) of PCR amplicons (PCR-DGGE). Irrespective of aerobic or anaerobic incubation conditions, V3-16S rDNA DGGE fingerprints of culturable fractions from PCA and MRS medium displayed a high level of similarity indicating that LAB constituted the most dominant group in the culturable bacterial community. Comparison of DGGE profiles of fractions grown at 20, 28 or 37 degrees C indicated that part of the culturable community consisted of psychrotrophs. Four DGGE bands were common among cooked ham, turkey and chicken products, suggesting that these represent the microbiota circulating in the plant where all three MAP product types were sliced and packaged. Based on band sequencing and band position analysis using LAB reference strains, these four bands could be assigned to Lactobacillus sakei and/or the closely related Lactobacillus fuchuensis, Lactobacillus curvatus, Carnobacterium divergens and Leuconostoc carnosum. In conclusion, the PCR-DGGE approach described in this study allows to discriminate, identify and monitor core and occasional LAB microbiota of MAP sliced cooked meat products and provides valuable complementary information to the current plating procedures routinely used in industrial plants.

Phenotypic and molecular assessment of antimicrobial resistance in Lactobacillus paracasei strains of food origin.

Antimicrobial resistance data in food-associated lactic acid bacteria (LAB) such as lactobacilli are mostly based on nonstandardized methodologies and/or have been obtained for only a limited number of strains. This susceptibility study included a diverse collection of 115 isolates mainly of food origin originally identified as Lactobacillus paracasei or Lactobacillus casei. Upon reidentification and removal of potential replicate isolates using repetitive DNA element PCR fingerprinting, 65 genotypically unique L. paracasei strains and the L. casei type strain were selected for broth microdilution and Etest assays using the LAB susceptibility test medium. In both methodologies, strains appeared uniformly susceptible to ampicillin and clindamycin but exhibited natural resistance to streptomycin and gentamicin. Three L. paracasei strains from cheese displayed acquired resistance to tetracycline (MIC > or = 32 microg/ml) and/or to erythromycin (MIC >16 microg/ml), which was linked to the presence of a tet(M) or tet(W) gene and/or an erm(B) gene, respectively. Partial sequencing revealed that the tet(M) genes found in two of these strains belonged to two tet(M) sequence homology groups previously found in enterococci. Collectively, phenotypic and genotypic data allowed us to propose tentative epidemiological cutoffs for L. paracasei and L. casei for differentiating susceptible strains from those strains harboring one or more acquired resistance factors.

Accuracy of species identity of commercial bacterial cultures intended for probiotic or nutritional use.

Independent studies have indicated that the microbiological composition of several commercial probiotic products does not correspond to the product label information. The present study set out to investigate to what extent these problems may be due to the use of misidentified cultures at the onset of production. For this purpose, 213 cultures of lactic acid bacteria (LAB) and propionibacteria intended for probiotic or nutritional use were collected from 26 manufacturers of probiotic products, three international culture collections and one research institute. The accuracy of the taxonomic identity provided by the strain depositor was assessed through a polyphasic approach based on validated and standardized identification methods including fluorescent amplified fragment length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, protein profiling and partial 16S rDNA sequencing. The majority of the cultures were received as members of the genera Lactobacillus (57%) and Bifidobacterium (22%); however, propionibacteria, enterococci, Lactococcus lactis (subsp. lactis), Streptococcus thermophilus and pediococci were also obtained. Upon reidentification, 46 cases of misidentification at the genus level (n=19) or species level (n=27) were recorded, including 34 commercial probiotic cultures deposited by 10 different companies. The finding that more than 28% of the commercial cultures intended for human and/or animal probiotic use were misidentified at the genus or species level suggests that many cases of probiotic product mislabeling originate from the incorporation of incorrectly identified strains. A large number of these discrepancies could be related to the use of methods with limited taxonomic resolution (e.g., API strips) or that are unsuitable for reliable identification up to species level (e.g., pulsed-field gel electrophoresis and randomly amplified polymorphic DNA analysis). The current study has again highlighted that reliable identification of LAB and propionibacteria requires molecular methods with a high taxonomic resolution that are linked to up-to-date identification libraries.

Detection and characterization of tet(M) in tetracycline-resistant Listeria strains from human and food-processing origins in Belgium and France.

In the present study, three Listeria monocytogenes strains and one Listeria innocua strain out of a collection of 241 Listeria isolates from human and food-processing sources were found to display resistance to tetracycline (TC) due to the presence of the tet(M) gene. Through sequence analysis, it was shown that tet(M) genes in two of the isolates belong to sequence homology group (SHG) II, a group comprising chromosomally encoded tet(M) genes previously found in Staphylococcus aureus and in lactobacilli. The tet(M) genes of the two other L. monocytogenes strains were associated with a member of the Tn916-Tn1545 family of conjugative transposons and were closely related to SHG III, which harbours enterococcal tet(M) genes associated with Tn916. One of these transposon-containing strains was able to transfer the tet(M) gene to Enterococcus faecalis recipient strain JH2-2. Collectively, these sequence and conjugation data indicate that the acquisition of tet(M) by Listeria strains may be triggered by successive transfers between other Gram-positive organisms.

Human vaginal Lactobacillus rhamnosus harbor mutation in 23S rRNA associated with erythromycin resistance.

Little is known about the diversity and distribution of resistance determinants in human commensal bacteria. The aim of this study was to determine the molecular mechanism responsible for high-level erythromycin resistance among five human vaginal Lactobacillus rhamnosus isolates. PCR screening for the presence of ermA, ermB and ermC methylase genes revealed no determinants responsible for detected erythromycin resistance. Therefore, sequences of 23S rRNA genes from L. rhamnosus strains were studied by PCR-RFLP analysis and sequencing of 23S rRNA genes. According to the results, in all erythromycin-resistant L. rhamnosus strains, the presence of a A-->G transition mutation at position 2058 was discovered. Additionally, the isolates exhibited heterozygosity for the A2058/G2058 mutation among 23S rRNA gene copies. Presumably, the greatest number of mutated 23S rRNA operons was observed for the L. rhamnosus BGHV1' strain that also had the highest MIC for erythromycin (MIC=2048 microg mL(-1)). This study reports the presence of transition mutations in the V region of 23S rRNA genes that most probably account for high-level erythromycin resistance observed for the first time in human vaginal lactobacilli.

Alloscardovia omnicolens gen. nov., sp. nov., from human clinical samples.

The taxonomic position of 12 isolates tentatively assigned to the genus Bifidobacterium on the basis of a limited phenotypic characterization was examined. The isolates were collected between 1978 and 2005 in Belgium, Sweden and Norway, and originated from various human clinical samples, including urine, blood, urethra, oral cavity, tonsil, and abscesses of lung and aortic valve. On the basis of band number and clustering analysis, repetitive DNA element-based PCR fingerprinting using the BOXA1R and (GTG)(5) primers indicated that the clinical isolates represented a taxon probably not belonging to the genus Bifidobacterium. Analysis of 16S rRNA gene sequence similarities revealed that the isolates were most closely affiliated to Parascardovia denticolens LMG 18312(T) (93.0-93.2 %), Scardovia inopinata LMG 18313(T) (92.9-93.1 %) and other members of the Bifidobacteriaceae, indicating that the isolates belong to a novel genus within that family. This observation was further substantiated by the results of partial sequencing of the heat-shock protein 60 gene (hsp60) and determination of the DNA G+C contents (47.3-48.3 mol%). Members of the novel taxon can be phenotypically distinguished from S. inopinata, P. denticolens and Gardnerella vaginalis by the ability to grow on agar under aerobic conditions and on the basis of positive reactions for acid production from L-arabinose, raffinose, salicin and D-xylose. Unambiguous phenotypic differentiation from Aeriscardovia aeriphila and Bifidobacterium species may be difficult, so phenotypic analyses should be complemented by molecular methods. The values for DNA-DNA binding among four members of the novel genus were in the range of 89-100 %, indicating that the strains should be considered as a single novel species of a novel genus, for which the name Alloscardovia omnicolens gen. nov., sp. nov. is proposed. The type strain of Alloscardovia omnicolens is CCUG 31649(T) (=LMG 23792(T)).

Genetic basis of tetracycline and minocycline resistance in potentially probiotic Lactobacillus plantarum strain CCUG 43738.

The potentially probiotic strain Lactobacillus plantarum CCUG 43738, which displayed atypical phenotypic resistance to tetracycline (MIC, 512 microg/ml) and minocycline (MIC, 256 microg/ml), was found to contain a tet(S) gene located on a plasmid of approximately 14 kb. Plasmid curing with novobiocin eliminated this plasmid and restored the tetracycline-susceptible phenotype of the host strain.

Molecular diversity and characterization of tetracycline-resistant Staphylococcus aureus isolates from a poultry processing plant.

DNA fingerprinting and molecular characterization showed that the tetracycline-resistant Staphylococcus aureus population of a South African poultry processing plant comprised one or possibly several tet(K)-containing endemic clones that contaminated chicken and machinery surfaces at all sampled processing stages. The tet(K) gene was transferable by filter mating to S. aureus recipient 80CR5 and was located on a pT181-like plasmid.

Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food.

In the present study, a collection of 187 Enterococcus food isolates mainly originating from European cheeses were studied for the phenotypic and genotypic assessment of tetracycline (TC) resistance. A total of 45 isolates (24%) encompassing the species Enterococcus faecalis (n = 33), E. durans (n = 7), E. faecium (n = 3), E. casseliflavus (n = 1), and E. gallinarum (n = 1) displayed phenotypic resistance to TC with MIC ranges of 16 to 256 microg/ml. Eight of these strains exhibited multiresistance to TC, erythromycin, and chloramphenicol. By PCR detection, TC resistance could be linked to the presence of the tet(M) (n = 43), tet(L) (n = 16), and tet(S) (n = 1) genes. In 15 isolates, including all of those for which the MIC was 256 micro g/ml, both tet(M) and tet(L) were found. Furthermore, all tet(M)-containing enterococci also harbored a member of the Tn916-Tn1545 conjugative transposon family, of which 12 erythromycin-resistant isolates also contained the erm(B) gene. Filter mating experiments revealed that 10 E. faecalis isolates, 3 E. durans isolates, and 1 E. faecium isolate could transfer either tet(M), tet(L), or both of these genes to E. faecalis recipient strain JH2-2. In most cases in which only tet(M) was transferred, no detectable plasmids were acquired by JH2-2 but instead all transconjugants contained a member of the Tn916-Tn1545 family. Sequencing analysis of PCR amplicons and evolutionary modeling showed that a subset of the transferable tet(M) genes belonged to four sequence homology groups (SHGs) showing an internal homology of > or = 99.6%. Two of these SHGs contained tet(M) mosaic structures previously found in Tn916 elements and on Lactobacillus and Neisseria plasmids, respectively, whereas the other two SHGs probably represent new phylogenetic lineages of this gene.