Strains Of Other Species Research Articles

Sialidase of Streptococcus intermedius: a putative virulence factor modifying sugar chains

A sialidase gene of Streptococcus intermedius was cloned. It was most similar to nanA, a major sialidase gene in Streptococcus pneumoniae, and was expressed in Escherichia coli. Since the gene-knockout S. intermedius strain lost detectable sialidase activity, the gene might code, either solely or mainly, the glycosidase in the bacterial genome. Polymerase chain reaction using the primers for the nanA homologue in S. intermedius (described as nanA below) showed that this sialidase gene was commonly distributed within the isolates of S. intermedius, but not found in the strains of other species among the anginosus group. In biofilm formation assay under cultivation with mucin, the nanA-deleted S. intermedius maintained the amount of biofilm for 72 hr, while that of the parent strain decreased during incubation from 24 to 72 hr. Since sialidase activity in the parent strain increased during that time period, sialidase might contribute to the degradation of biofilm under sialic acid-rich conditions. When S. intermedius was added into the HepG2 hepatoma culture, the calculated disassociation constant (K(d)) of EDTA-releasable bacterial adhesion to the cells was higher in the nanA-deleted strain than in the parent. Furthermore, the rate constant, assuming endocytosis of the bacterium mediated by ASGP-R in HepG2 cells, seemed to be increased by sialidase pretreatment of the bacterial cells before addition to the cell culture. According to the results, modification of sugar chains by sialidase on the bacterial surface and in the surrounding environment might influence both bacterial interaction and host-bacterial interaction in S. intermedius.

Paramecium sexaurelia – Intra-Specific Polymorphism and Relationships with other Paramecium aurelia spp., Revealed by Cytochrome b Sequence Data

Genetic distances among strains of P. sexaurelia were compared by sequencing the mitochondrial cytochrome b gene. The relationships of P. sexaurelia with representative strains of other species of the P. aurelia complex and related species, i.e., P. schewiakoffi, P. jenningsi and P. multimicronucleatum were evaluated. All investigated P. sexaurelia and P. dodecaurelia strains grouped together with high support. This P. sexaurelia/P. dodecaurelia cluster was furthermore composed of three distinct, strongly supported subgroups. Two of these groups contained both P. sexaurelia and P. dodecaurelia strains, suggesting that these species are not monophyletic. The third branch was composed of strains from Sevilla, Spain and was deeply separated (12-14% p-distance) from the other P. sexaurelia/P. dodecaurelia clades. This illustrates the urgent need for further work and more intense sampling of these "rare" species, in order to understand the status and the relationships of P. sexaurelia and P. dodecaurelia within the P. aurelia species complex. We recommend that general investigations on the speciation process be conducted within and between species of the P. aurelia complex due to the high genetic variation combined with observations that for some of the species possible cryptic speciation may have occurred. This is emphasized by data showing that for some species within this complex, species status may be less static and more dynamic than originally thought.

Variovorax ginsengisoli sp. nov., a denitrifying bacterium isolated from soil of a ginseng field

A Gram-negative, aerobic or facultatively anaerobic, non-spore-forming, motile, rod-shaped bacterium (strain Gsoil 3165(T)) was isolated from soil of a ginseng field in Pocheon, South Korea. Its taxonomic position was determined by using a polyphasic approach. On the basis of 16S rRNA gene sequence analysis, strain Gsoil 3165(T) was shown to belong to the family Comamonadaceae, class Betaproteobacteria, and was related most closely to the type strains of Variovorax boronicumulans (98.9 % similarity), Variovorax paradoxus (98.3 %), Variovorax soli (98.2 %) and Variovorax dokdonensis (96.6 %). Levels of 16S rRNA gene sequence similarity between strain Gsoil 3165(T) and the type strains of other species in the family Comamonadaceae were less than 97.0 %. The G+C content of the genomic DNA of strain Gsoil 3165(T) was 66 mol%. Phenotypic and chemotaxonomic data (Q-8 as the major ubiquinone; C(16 : 0) and C(17 : 0) cyclo as major fatty acids) supported the affiliation of strain Gsoil 3165(T) to the genus Variovorax. The results of physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain Gsoil 3165(T) from recognized Variovorax species. Gsoil 3165(T) is therefore considered to represent a novel species of the genus Variovorax, for which the name Variovorax ginsengisoli sp. nov. is proposed. The type strain is Gsoil 3165(T) (=KCTC 12583(T) =LMG 23392(T)).

Nocardioides daedukensis sp. nov., a halotolerant bacterium isolated from soil

A Gram-positive, non-motile and rod- or coccoid-shaped bacterial strain, MDN22(T), was isolated from a soil sample from Korea. Strain MDN22(T) grew optimally at pH 7.0-8.0, at 30 degrees C and in the presence of 0-0.5 % (w/v) NaCl. Phylogenetic analyses based on 16S rRNA gene sequences showed that strain MDN22(T) was phylogenetically most closely related to the genera Nocardioides and Marmoricola. In the neighbour-joining phylogenetic tree, strain MDN22(T) was most closely related to Nocardioides jensenii KCTC 9134(T), with which it exhibited 98.3 % 16S rRNA gene sequence similarity. The strain exhibited 93.1-96.9 % and 95.3-95.9 % 16S rRNA gene sequence similarities to the type strains of other species of the genera Nocardioides and Marmoricola, respectively. The chemotaxonomic properties of strain MDN22(T) were consistent with those of the genus Nocardioides; the cell-wall peptidoglycan type was based on ll-2,6-diaminopimelic acid, the predominant menaquinone was MK-8(H(4)) and the major fatty acids were iso-C(16 : 0) and C(17 : 1). The DNA G+C content was 68.7 mol%. DNA-DNA relatedness data and differential phenotypic properties suggested that strain MDN22(T) could be differentiated from N. jensenii and Nocardioides dubius. On the basis of the data obtained, strain MDN22(T) is considered to represent a novel species of the genus Nocardioides, for which the name Nocardioides daedukensis sp. nov., is proposed. The type strain is MDN22(T) (=KCTC 19601(T)=CCUG 57505(T)).

Kocuria halotolerans sp. nov., an actinobacterium isolated from a saline soil in China

A Gram-positive actinobacterium, designated strain YIM 90716(T), was isolated from a saline soil sample collected from Ganjiahu Suosuo Forest National Nature Reserve in Xinjiang Province, north-west China. The new isolate contained lysine, glutamic acid and alanine with peptidoglycan type Lys-Ala(3) (variation A3alpha). The major phospholipids were phosphatidylglycerol and diphosphatidylglycerol. The predominant menaqinone was MK-7(H(2)). The major fatty acids were anteiso-C(15 : 0), iso-C(16 : 0) and anteiso-C(17 : 0). The DNA G+C content of strain YIM 90716(T) was 68.0 mol%. Chemotaxonomic properties supported the affiliation of strain YIM 90716(T) to the genus Kocuria. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the organism was related most closely to Kocuria kristinae DSM 20032(T) (96.8 % similarity) and showed lower levels of 16S rRNA gene similarity (<96.5 %) with the type strains of other species of the genus Kocuria. The results of fatty acid analysis and physiological and biochemical tests allowed the genotypic and phenotypic differentiation of strain YIM 90716(T) from its closest relatives. On the basis of data from the present polyphasic study, strain YIM 90716(T) is considered to represent a novel species of the genus Kocuria, for which the name Kocuria halotolerans sp. nov. is proposed. The type strain is YIM 90716(T) (=DSM 18442(T)=KCTC 19172(T)=CCTCC AB 206069(T)).

Oceanicola pacificus sp. nov., isolated from a deep-sea pyrene-degrading consortium

A taxonomic study was carried out on a Gram-negative, rod-shaped, non-motile, halophilic bacterium, designated strain W11-2B(T), which was isolated from a pyrene-degrading consortium that was enriched from sediment from the Pacific Ocean. Growth was observed at salinities of 0.5-10 % and at temperatures of 10-41 degrees C. Strain W11-2B(T) was unable to degrade Tween 80 or gelatin. 16S rRNA gene sequence comparisons showed that strain W11-2B(T) was related most closely to Oceanicola nanhaiensis SS011B1-20(T) (95.8 % similarity) and Oceanicola batsensis HTCC2597(T) (95.7 %); levels of 16S rRNA gene sequence similarity between strain W11-2B(T) and the type strains of other species tested were below 95.2 %. The dominant fatty acids of strain W11-2B(T) were C(18 : 1)omega7c (32.1 % of the total), C(19 : 0) cyclo (20.9 %), C(18 : 1)omega7c 11-methyl (19.5 %), C(18 : 0) (7.3 %), C(17 : 0) (6.6 %) and C(16 : 0) (3.8 %). The G+C content of the chromosomal DNA was 64.6 mol%. The above data were in good agreement with those of members of the genus Oceanicola. Based on morphology, physiology, fatty acid composition and 16S rRNA gene sequence data, strain W11-2B(T) is considered to represent a novel species of the genus Oceanicola, for which the name Oceanicola pacificus sp. nov. is proposed. The type strain is W11-2B(T) (=CCTCC AB 208224(T)=LMG 24619(T)=MCCC 1A01034(T)).

Pelagibius litoralis gen. nov., sp. nov., a marine bacterium in the family Rhodospirillaceae isolated from coastal seawater

A Gram-negative, strictly aerobic, slightly curved rod-shaped bacterial strain, designated CL-UU02(T), was isolated from coastal seawater off the east coast of Korea. 16S rRNA gene sequence analysis revealed a clear affiliation of this novel strain with the family Rhodospirillaceae. Strain CL-UU02(T) formed a robust cluster with the type strains of species of the genus Rhodovibrio at 16S rRNA gene sequence similarity levels of 89.9-90.4 %. Strain CL-UU02(T) shared no more than 89 % 16S rRNA gene sequence similarity with the type strains of other species in the family Rhodospirillaceae. Strain CL-UU02(T) was able to grow in the presence of 2-6 % sea salts, and grew optimally at 28-30 degrees C and pH 7-8. The DNA G+C content of strain CL-UU02(T) was 66.3 mol%. On the basis of phylogenetic analyses and chemotaxonomic and physiological data, strain CL-UU02(T) is considered to represent a novel species of a new genus in the family Rhodospirillaceae, for which the name Pelagibius litoralis gen. nov., sp. nov. is proposed. The type strain of Pelagibius litoralis is CL-UU02(T) (=KCCM 42323(T)=JCM 15426(T)).

Secreted sialidase activity of canine mycoplasmas

Through a survey of the phylogenetic distribution of sialidase among mycoplasmas, we detected activity secreted by the type strains of 3 of 11 species frequently or first isolated from dogs. The specific activity of washed cells of the type strains of Mycoplasma canis, Mycoplasma cynos, and Mycoplasma molare ranged from 5.2 ± 0.8 × 10 −6 to 1.1 ± 0.3 × 10 −5 enzymatic units per colony-forming unit (U/CFU). Cells of M. molare strain H542 T had twice the specific activity ( P < 0.05) of M. canis strain PG14 T or M. cynos strain H831 T. Significant differences in sialidase activity existed among nine clinical isolates of M. canis, ranging from not detectable to 2.1 ± 0.1 × 10 −5 U/CFU. The type strains of other species previously isolated from dogs ( Mycoplasma arginini, Mycoplasma bovigenitalium, Mycoplasma edwardii, Mycoplasma felis, Mycoplasma gatae, Mycoplasma maculosum, Mycoplasma opalescens, and Mycoplasma spumans) did not exhibit either secreted or cell-associated sialidase activity. Neither specific nor degenerate PCR primers complementary to the three known mycoplasmal sialidase alleles were able to amplify orthologs in M. canis, M. cynos, or M. molare, further evidence that the secreted sialidase of those species is distinct from the strictly cell-associated sialidases of Mycoplasma alligatoris, Mycoplasma synoviae, and Mycoplasma gallisepticum. This is the first report of a well-known bacterial virulence factor whose expression varies among strains of certain Mycoplasma species that infect dogs.

Multilocus sequence typing confirms synonymy but highlights differences betweenCandida albicansandCandida stellatoidea

We used multi-locus sequence typing (MLST) to investigate 35 yeast isolates representing the two genome-sequenced strains plus the type strain of Candida albicans, four isolates originally identified as Candida stellatoidea type I and 28 representing type strains of other species now regarded as synonymous with C. albicans. DNA from all 32 C. albicans synonyms readily formed PCR products with the C. albicans MLST primer sets. Their sequences placed all of them within the existing C. albicans clade structure, represented by 1516 isolates. One isolate, originally received as Mycotorula sinensis, was resistant to flucytosine, but no other unusual susceptibilities were found to polyene, azole or echinocandin antifungal agents. The four isolates of C. stellatoidea type I coclustered with two other sucrose-negative isolates, originally identified as examples of Candida africana, in a group of strains highly distinct from the majority of C. albicans. Our results not only confirm the synonymity of all the isolates with C. albicans but also confirm an obvious genotypic difference in the case of C. stellatoidea type I.

Oligonucleotide Array-Based Identification of Species in the Acinetobacter calcoaceticus-A. baumannii Complex in Isolates from Blood Cultures and Antimicrobial Susceptibility Testing of the Isolates

Acinetobacter calcoaceticus, A. baumannii, Acinetobacter genomic species (gen. sp.) 3, and Acinetobacter gen. sp. 13TU, which are included in the A. calcoaceticus-A. baumannii complex, are difficult to distinguish by phenotypic methods. An array with six oligonucleotide probes based on the 16S-23S rRNA gene intergenic spacer (ITS) region was developed to differentiate species in the A. calcoaceticus-A. baumannii complex. Validation of the array with a reference collection of 52 strains of the A. calcoaceticus-A. baumannii complex and 137 strains of other species resulted in an identification sensitivity and specificity of 100%. By using the array, the species distribution of 291 isolates of the A. calcoaceticus-A. baumannii complex from patients with bacteremia were determined to be A. baumannii (221 strains [75.9%]), Acinetobacter gen. sp. 3 (67 strains [23.0%]), Acinetobacter gen. sp. 13TU (2 strains [0.7%]), and unidentified Acinetobacter sp. (1 strain [0.3%]). The identification accuracy of the array for 12 randomly selected isolates from patients with bacteremia was further confirmed by sequence analyses of the ITS region and the 16S rRNA gene. Antimicrobial susceptibility testing of the 291 isolates from patients with bacteremia revealed that A. baumannii strains were less susceptible to antimicrobial agents than Acinetobacter gen. sp. 3. All Acinetobacter gen. sp. 3 strains were susceptible to ampicillin-sulbactam, imipenem, and meropenem; but only 67.4%, 90%, and 86% of the A. baumannii strains were susceptible to ampicillin-sulbactam, imipenem, and meropenem, respectively. The observed significant variations in antimicrobial susceptibility among different species in the A. calcoaceticus-A. baumannii complex emphasize that the differentiation of species within the complex is relevant from a clinical-epidemiological point of view.

Genetic characterization of Brazilian strains of Aspergillus flavus using DNA markers

The Aspergillus genus belongs to a filamentous fungal group characterized by wide dispersion in the environment. Some species are associated with diseases, especially in immunocompromised patients, while others are of economical importance due to aflatoxin production or biotechnological applications. Its species identification is nowadays performed by traditional techniques combined with molecular markers, resulting in a higher efficiency of isolate characterization. In the present study, internal transcribed spacer, inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) molecular markers were used, with the aim of genetically characterizing strains of Aspergillus flavus and strains of other species of the A. flavus group. High genetic diversity was revealed by RAPD and by ISSR, in which the use of the (GACA)4 primer yielded a higher diversity than with the (GTG)5 primer, although the latter showed a characteristic banding profile for each species. These data were used to create a similarity matrix for the construction of dendrograms by means of the UPGMA method. The ISSR and RAPD profiles showed that among the strains previously identificated as A. flavus, one should be A. oryzae, one A. parasiticus and two A. tamarii. On the other hand, a strain previously identified as A. parasiticus should be A. flavus. All these strains were retested by traditional methods and their new species identification was confirmed. These results strongly support the need for using molecular markers as an auxiliary tool in differentiating fungal species and strains.

Development of a Simple Latex Agglutination Assay for Detection of Shiga Toxin-Producing Escherichia coli (STEC) by Using Polyclonal Antibody against STEC

Rabbit antiserum raised against the whole-cell antigen of Shiga toxin-producing Escherichia coli (STEC) strain VT3 (stx1+ stx2+ eae+) was repeatedly adsorbed with heat-killed cells of different non-STEC strains and other enteric bacteria. Thus, the antiserum obtained was designated VT3 antiserum. VT3 antiserum reacted with intimin type gamma. We assessed the reactivity of VT3 antiserum to whole-cell lysates of 87 strains of E. coli and other enteric bacteria by immunoblotting. The antiserum recognized the 97-kDa protein in whole-cell lysate from strain VT3, and 36 (83.7%) of the 43 STEC strains were positive for the STEC antigen. None of the non-STEC strains or strains of other species examined tested positive by immunoblotting. Based on this result, we developed a latex agglutination assay for the detection of STEC strains. Thirty-five (81.4%) of the 43 STEC strains tested positive for the STEC antigen by the latex agglutination assay. One (3.3%) of the 30 non-STEC strains and none of the strains of the other enteric bacteria included in this study tested positive by the latex agglutination assay. The corresponding specificity of the latex agglutination assay was approximately 98%. Results of this study showed the production of STEC antiserum and the generation of a simple, cost-effective, sensitive, and specific latex agglutination assay for establishing an etiological diagnosis of STEC.

Ochrobactrum oryzae sp. nov., an endophytic bacterial species isolated from deep-water rice in India

A non-pigmented, motile, Gram-negative bacterium designated MTCC 4195(T) was isolated from surface-sterilized seeds and plant tissue from deep-water rice (Oryza sativa) cultivated in Suraha Tal Lake in northern India. This isolate was shown to reinfect and colonize deep-water rice endophytically. The highest level of 16S rRNA sequence similarity (96.8 %) to strain MTCC 4195(T) was shown by Ochrobactrum gallinifaecis DSM 15295(T). Strain MTCC 4195(T) utilized gamma-hydroxybutyric acid, adonitol, d-glucosaminic acid and arabinose as carbon sources, but failed to use gentiobiose or citrate. The cell-wall fatty acids of strain MTCC 4195(T) were characterized by the presence of a relatively large proportion of C(18 : 1)omega7c and a relative small proportion of C(16 : 0) in comparison with Ochrobactrum species. DNA-DNA relatedness studies showed less than 52 % binding with the DNAs of type strains of other species of the genus Ochrobactrum. On the basis of phenotypic and genotypic characteristics and the results of 16S rRNA gene sequence analysis, the novel species Ochrobactrum oryzae sp. nov. is proposed, with MTCC 4195(T) (=DSM 17471(T)) as the type strain.

Application of real-time PCR for quantitative detection ofVibrio parahaemolyticusfrom seafood in eastern China

Vibrio parahaemolyticus is recognized as a leading human food-borne pathogen. A TaqMan PCR assay based on the gyrase B gene (gyrB) sequence of V. parahaemolyticus was developed for quantitative detection of V. parahaemolyticus in seafood. The study involving 27 V. parahaemolyticus and 10 strains of other species indicated that the real-time PCR test was highly specific. The sensitivity of the assay was approximately a single CFU per PCR in pure culture and six to eight CFU per PCR in spiked raw oyster, respectively. Real-time PCR values of artificially inoculated oyster homogenates correlated well with plate counts determined using culture methods. A total of 300 seafood samples were analyzed and 78 (26%) of these samples were positive for V. parahaemolyticus using a conventional culture method and 97 (32.3%) using the real-time PCR assay. All culture-positive samples were PCR-positive. However, 19 samples positive by PCR were culture-negative. The results show that retail seafood is commonly contaminated with V. parahaemolyticus in harvest season in eastern China. These data also indicate that real-time PCR can provide sensitive species-specific detection and quantification of V. parahaemolyticus in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.

Molecular diagnosis of ochratoxinogenicAspergillus species

Toxigenic and non-toxigenic black aspergilli belonging to theAspergillus niger aggregate and toA. carbonarius were compared to each other and to strains of other species by DNA fingerprinting. AFLPs showed a clear separation ofA. niger andA. carbonarius. However, no clear correlation between the genetic similarity of the strains and the ability to produce ochratoxin A (OTA) was detected. Based on AFLP, marker sequences were chosen for the construction of SCAR-PCR primers for the detection ofA. carbonarius. A similar approach was used forA. ochraceus, another fungus of concern regarding ochratoxin A contamination of coffee. Cluster analysis ofA. ochraceus isolates mainly from Brazilian coffee showed a very close genetic similarity. Three species specific primer pairs were developed and one of these was used for the PCR and realtime PCR (RT-PCR) based detection of the mould in green coffee.A. ochraceus was specifically and rapidly detected and quantified in green coffee. A positive correlation between the amount of DNA and OTA content was established.

A LightCycler real-time PCR hybridization probe assay for detecting food-borne thermophilic Campylobacter

In a previous study, we reported the performance of a PCR assay amplifying 287-bp of the 16S rRNA gene of thermo-tolerant Campylobacter ( C. jejuni, C. lari, C. coli) through an international ring-trial involving 12 participating laboratories. Based on the validated set of primers, a LightCycler real-time PCR assay (LC-PCR), which used fluorescent hybridization probes was developed. The test incorporated an internal amplification control co-amplified with the 16S rRNA gene of Campylobacter to monitor potential PCR inhibitors and ensure successful amplifications. The specificity study involving 39 Campylobacter and nine strains of other species indicated that the LC-PCR test was highly specific, giving cross-reactivity with only one strain of C. upsaliensis (CCUG19559). The sensitivity of the LC-PCR assay, evaluated in 32 spiked poultry-rinse or pork carcass-swab samples, was determined at 10 CFU/ml carcass-rinse. The prevalence of samples positive for thermo-tolerant Campylobacter was 58.8% in 68 naturally contaminated poultry rinse samples tested by LC-PCR and the data were in good concordance with those of bacteriological method. The Ct values of the three replicates obtained for each sample tested in three different runs demonstrate that the LC-PCR was highly reproducible and afford a powerful tool for rapid detection of the thermo-tolerant Campylobacter strains.

Evolution and conservation of microsatellite markers for Leishmania tropica

Sixteen polymorphic microsatellite markers were developed for phylogenetic analysis of Leishmania tropica. The phylogenetic tests done demonstrated that they do provide a powerful tool for epidemiological studies. They were also tested for their ability to differentiate strains of other species of Leishmania, confirming that microsatellite markers developed for one leishmanial species cannot generally be used for other leishmanial species. In addition to length variation, a high degree of allelic heterozygosity was seen among the strains investigated, suggestive of sexual recombination within the species L. tropica.

Effect of temperature on growth and antagonistic activity of Trichoderma spp against Macrophomina phaseolina

Studies were carried out on the influence of temperature on growth, survival and antagonistic performance of various strains of Trichoderma spp. against groundnut dry root-rot pathogen Macrophomina phaseolina (Tassi.) Goid. Different isolates of Trichoderma viride, T. harzianum, T. longibrachiatum, T. Hamatum, T. koningii and T. pseudokoningii were employed at various temperatures, viz., 15, 20, 25, 30, 35, 40 and 45°C for studying its cultural behaviour and antagonistic ability. Increased growth of the pathogen ( M. phaseolina ), and growth, sporulation and biomass production of the fungal antagonist ( Trichoderma spp.) were observed between 25 and 35°C. Antagonistic activity of Trichoderma spp. against M. phaseolina was decreased with increase in temperature except for T. pseudokoningii , which showed maximum inhibition at 35°C. Survival of Trichoderma spp. on seed coat was maximum at lower temperature (15°C). At all the temperature regimes T. harzianum strain Th-5 has shown higher suppression of the root-rot pathogen, better growth and survival than strains of other species.

Usefulness of a two-step PCR procedure for detection and identification of enterotoxigenic staphylococci of bacterial isolates and food samples

A two-step PCR procedure for detection of genes encoding Staphylococcus aureus thermonuclease ( nuc) and enterotoxins ( se s ) was developed. For this, the reaction conditions for multiplex-PCR assays were optimized and generic and specific s e-primers designed. In a step detection of enterotoxigenic staphylococci using a set of primers targeting nuc, and group-generic se[ a, d, e, j] and se[ b, c] sequences, is accomplished. The generated amplicons are 468-, 670- and 540-bp long, respectively. In the second step aliquots of the 670- and/or 540-bp amplicons are analysed by nested-PCR using the following sets of primers: the forward se[ adej]—together with the reverse sea, sed, see and sej, and/or the forward se[ bc]—plus reverse seb and sec. The six se-genes can be distinguished since each of them yield an amplicon of different size. The procedure was validated by using 163 strains of S. aureus, 15 strains of other species, and 31 cake samples; and also by comparison of the obtained results with conventional-PCR-assays and immunoassays (SET-RPLA). The first PCR-step showed that all S. aureus strains, but none of the strains representing other species, were nuc-positive and 52 of them, and only them, were se-positive. Twelve out of the 31 cake samples were probed to be nuc-positive, but only eight were se-positive, and enterotoxigenic S. aureus were collected from them. The second step confirmed that all se-positive strains carried one or more se-genes. A positive correlation between the presence of se-genes by multiplex-PCR, conventional-PCR and SET-RPLA was found. This procedure allows it to be extended to include other se-genes.

Identification of opcA gene in Neisseriapolysaccharea: interspecies diversity of Opc protein family

The gene encoding the outer membrane adhesin/invasin protein OpcA was previously described in the genomes of two pathogenic Neisseria species, N. meningitidis (Nm) and N. gonorrhoeae (Ng). In order to understand the presence or absence of opcA in nonpathogenic Neisseria species, 13 strains of N. polysaccharea (Np), four strains of N. lactamica, three strains of N. subflava and nine strains of other species were examined by DNA hybridization, polymerase chain reaction (PCR) and nucleotide sequencing. The opcA gene was found in two Np strains (85322 and 89357). The Np-opcA gene is a novel member of this gene family with 93% homology to Ng-opcA. Comparison of opcA-surrounding regions among eight Neisseria strains revealed five types of genetic organization at the opcA locus in Neisseria, which result from insertion or deletion of genetic elements at the upstream region of opcA. Comparison of the deduced peptide sequences from two Np strains, two representative Ng strains, two representative Nm strains and 13 Nm sequence variants demonstrates interspecies diversity of the OpcA protein family with conserved transmembrane regions and species-specific polymorphism at the surface-exposed loops and periplasmic turns. Reverse transcription–PCR analysis and Northern blotting showed that Np-opcA was transcribable. From an alignment of the Np-OpcA and Ng-OpcA sequences against the three-dimensional crystal structure of Nm-OpcA we conclude that there is no obvious structural reason why these proteins would not be able to form stable, folded, outer membrane proteins. The data presented here provide additional information for understanding the distribution, variation and expression of opcA in Neisseria.