tRNA Intergenic Length Polymorphism Analysis Research Articles

Identification and genotyping of bacteria from paired vaginal and rectal samples from pregnant women indicates similarity between vaginal and rectal microflora

BackgroundThe vaginal microflora is important for maintaining vaginal health and preventing infections of the reproductive tract. The rectum has been suggested as the major source for the colonisation of the vaginal econiche.MethodsTo establish whether the rectum can serve as a possible bacterial reservoir for colonisation of the vaginal econiche, we cultured vaginal and rectal specimens from pregnant women at 35-37 weeks of gestation, identified the isolates to the species level with tRNA intergenic length polymorphism analysis (tDNA-PCR) and genotyped the isolates for those subjects from which the same species was isolated simultaneously vaginally and rectally, by RAPD-analysis.One vaginal and one rectal swab were collected from a total of each of 132 pregnant women at 35-37 weeks of gestation. Swabs were cultured on Columbia CNA agar and MRS agar. For each subject 4 colonies were selected for each of both sites, i.e. 8 colonies in total.ResultsAmong the 844 isolates that could be identified by tDNA-PCR, a total of 63 bacterial species were present, 9 (14%) only vaginally, 26 (41%) only rectally, and 28 (44%) in both vagina and rectum. A total of 121 (91.6%) of 132 vaginal samples and 51 (38.6%) of 132 rectal samples were positive for lactobacilli. L. crispatus was the most frequently isolated Lactobacillus species from the vagina (40% of the subjects were positive), followed by L. jensenii (32%), L. gasseri (30%) and L. iners (11%). L. gasseri was the most frequently isolated Lactobacillus species from the rectum (15%), followed by L. jensenii (12%), L. crispatus (11%) and L. iners (2%).A total of 47 pregnant women carried the same species vaginally and rectally. This resulted in 50 vaginal/rectal pairs of the same species, for a total of eight different species. For 34 of the 50 species pairs (68%), isolates with the same genotype were present vaginally and rectally and a high level of genotypic diversity within species per subject was also established.ConclusionIt can be concluded that there is a certain degree of correspondence between the vaginal and rectal microflora, not only with regard to species composition but also with regard to strain identity between vaginal and rectal isolates.These results support the hypothesis that the rectal microflora serves as a reservoir for colonisation of the vaginal econiche.

Arcanobacterium pyogenes spondylodiscitis in a veterinary surgeon: A plea for cooperation between medical and veterinary microbiologists in identification of causal agents of zoonotic infections

cholesterolemia. His medications were160 mg of acetylsalicylic acid per day,75 mg of clodipogrel per day, and 40 mgof pravastatin per day. The patient hadextensive contact with cattle and swineand did not wear gloves. He regularlysuffered from excoriations on bothhands. Since treatment with oral sodiumdiclofenac did not relieve his back pain,he consulted an orthopedic surgeon andwas treated with a series of 10 epiduralinfiltrations of methylprednisone, aswell as oral sodium diclofenac. Bloodanalysis ordered by his general practi-tioner revealed anemia with a hemo-globin level of 10.5 g/dl (normal range; 14 to18 g/dl), a white blood cell countof 8,000/µl (normal range; 4,000 to9,000/µl) with 78.4% polymorphonu-clear cells, an elevated erythrocyte sedi-mentation rate (ESR), and an increasedC-reactive protein (CRP) level of 145mg/l (normal range; 0.02 to 7 mg/l).Screening for autoimmune antibodieswas negative. The patient was HLAB27 negative. Conventional X raysshowed spondylodiscitis of the L3-L4interspace. Nuclear magnetic resonanceimaging showed significant destructionof lumbar vertebrae L3 and L4 withsome minor abscess formation in theintervertebral disk, in addition to lim-ited epidural invasion. Transthoracicechocardiography excluded endocardi-tis. Transcutaneous puncture of the lum-bar joint L3-L4 yielded a bloody fluidwith streptococcus-like gram-positivebacteria seen on Gram-stained smear.Culture on horse blood agar grew beta-hemolytic colonies reacting with aLancefield group G antigen reagent(Pro-Lab Diagnostics, Neston, Wirral,United Kingdom), which erroneouslyled to further processing of the isolateas a streptococcus. Molecular identifi-cation at the Laboratory of VeterinaryBacteriology and Mycology (Faculty ofVeterinary Medicine, Ghent University),by using tRNA intergenic length poly-morphism analysis (tDNA-PCR) com-bined with capillary electrophoresis (1)characterized the organism as

Description of Wautersiella falsenii gen. nov., sp. nov., to accommodate clinical isolates phenotypically resembling members of the genera Chryseobacterium and Empedobacter

A total of 26 isolates of non-fermenting, Gram-negative rods, obtained between 1980 and 2004 by various clinical laboratories in Belgium, with phenotypic characteristics resembling those of members of the genera Chryseobacterium and Empedobacter (indole-positive) and a biochemical profile resembling that of CDC group II-h, but urease-positive, were collected at the Université Catholique de Louvain Microbiology Laboratory, Belgium. The 16S rRNA gene sequences were determined for most of the isolates and showed 94-95 % similarity with the type strain of Empedobacter brevis as the closest relative, indicating that these isolates might belong to a separate genus. Furthermore, the 16S rRNA gene sequences of the isolates were similar, but two clusters (genomovars) could be distinguished. The sequence similarities were 99.5-100 % for the 14 isolates of genomovar 1 and 99.4-100 % for the 12 isolates of genomovar 2. The similarity between the two clusters was 98.3-99.5 %. The presence of two clearly different groups was corroborated by using tRNA intergenic length polymorphism analysis, which also enabled differentiation of the novel species from all other species studied thus far using this technique. DNA-DNA hybridization results excluded a close relatedness to Empedobacter brevis. The DNA G+C contents of the reference strains of genomovars 1 and 2 were 33.8+/-0.4 and 34.4+/-0.2 mol%, respectively. The name Wautersiella falsenii gen. nov., sp. nov., is proposed for this group, comprising two closely related genomovars. The type strain of the species and reference strain for genomovar 1 is NF 993(T) (=CCUG 51536(T)=CIP 108861(T)), which was isolated from a surgical wound. The reference strain for genomovar 2 is NF 770 (=CCUG 51537=CIP 108860), which was isolated from blood.

Identification of enterococcal, streptococcal and Weissella species in the faecal flora of individually owned dogs

To improve the limited information on the composition of the faecal Gram-positive coccal flora of healthy dogs by the use of a molecular identification method. Faecal swabs were collected for the selective isolation of Gram-positive coccal strains. Colonies with enterococcal- and streptococcal-like morphology were identified by tRNA intergenic length polymorphism analysis (tDNA-PCR). Fourteen known species belonging to three genera (Enterococcus, Streptococcus and Weissella) and one alleged new enterococcal species were found. The faecal flora of dogs comprises an unusually broad diversity of culturable Gram-positive coccal species with Enterococcus faecalis being most frequently present followed by not less than six other species of about equal importance. Many human- and animal-associated enterococci and streptococci are also present in dog faeces together with the largely uncharacterized Weissella cibaria and a group of strains resembling Enterococcus dispar, but representing a distinct and hitherto unknown species. Phenotypic characteristics of the latter two species were determined and the test results were compared with the species descriptions of W. cibaria and E. dispar respectively.

A comparative evaluation of phenotypic and molecular methods in the identification of members of the Staphylococcus sciuri group

Members of the Staphylococcus sciuri group ( S. sciuri, S. lentus, and S. vitulinus) are coagulase-negative, novobiocin-resistant staphylococci that could be distinguished from other staphylococci on the basis of positive oxidase activity. In the present study, a scheme based on conventional methods and utilization of various carbohydrates was evaluated for the identification of oxidase-positive staphylococci, and validated using two molecular techniques. Of the 173 oxidase-positive staphylococcal tested strains, 161 were identified as S. sciuri, 9 as S. lentus, 2 as S. vitulinus, and one as S. fleurettii by our scheme. The level of agreement with tRNA intergenic length polymorphism analysis (tDNA-PCR) was high (97.5–100% correlation). The accuracy and ease of use of this protocol suggest that it may be useful and valuable in microbiological laboratories for the identification of members of this group.

Enterococcus saccharominimus sp. nov., from dairy products

Four isolates, which were obtained from Belgian, Moroccan and Romanian dairy products, constituted a homogeneous but unidentified taxon after screening with whole-cell protein fingerprinting. Complete 16S rRNA gene sequence analysis classified representative strains in the genus Enterococcus. Highest sequence similarities of 98.6 and 98.0 % were obtained with the species Enterococcus sulfureus and Enterococcus saccharolyticus, respectively. Growth characteristics, biochemical features, tRNA intergenic length polymorphism analysis, DNA-DNA hybridization and DNA G+C contents of selected strains demonstrated that they represent a single, novel Enterococcus species. It differs phenotypically from other enterococci in characteristics commonly considered as typical of this genus: no growth in 6.5 % NaCl or 0.4 % sodium azide, and no acid production from a wide range of carbohydrates. The name Enterococcus saccharominimus sp. nov. is proposed for this novel species; the type strain (LMG 21727(T)=CCM 7220(T)) was isolated from contaminated pasteurized cow's milk.

Streptococcus minor sp. nov., from faecal samples and tonsils of domestic animals.

Nine isolates, which were obtained from tonsils, anal swabs and faeces of dogs and from tonsils of a cat and a calf, constituted a homogeneous but unidentified taxon after screening with tRNA intergenic length polymorphism analysis and whole-cell protein fingerprinting. 16S rDNA sequence analysis classified representative strains in the genus Streptococcus. Highest sequence similarity (95.9 %) was obtained with Streptococcus ovis. Growth characteristics, biochemical features, DNA-DNA hybridization and DNA G+C contents of selected strains demonstrated that they represent a single, novel streptococcal species. The name Streptococcus minor sp. nov. is proposed for the novel species; the type strain (ON59(T)=LMG 21734(T)=CCUG 47487(T)) was isolated from a dog tonsil.

Characterization of yellow-pigmented and motile enterococci isolated from intestines of the garden snail Helix aspersa.

Enterococci associated with garden snails (Helix aspersa) were studied in order to obtain reliable species identification and characterization. Twelve yellow-pigmented and motile enterococci, isolated from the intestines of garden snails, were phenotypically close to Enterococcus casseliflavus, but they showed certain unusual biochemical characteristics. tRNA intergenic length polymorphism analysis (tDNA-PCR) divided all strains studied into two groups, in full agreement with biochemical test results. 16S rDNA sequencing, DNA base composition analysis and DNA-DNA hybridization results showed unambiguously that the enterococci studied belonged to the species Ent. casseliflavus. The representative strains of described ecovars were deposited in the Czech Collection of Microorganisms (CCM) as Ent. casseliflavus CCM 4868, 4869, 4870 and 4871. Enterococcus casseliflavus associated with garden snails can be subdivided into groups. Enterococcus casseliflavus differs from other enterococcal species in that it is typically associated with plants, soil, water and invertebrate animals. The different groups that can be found in these widely occurring bacteria are possibly source-specific ecovars, as exemplified by the Ent. casseliflavus inhabiting the intestines of snails.