Reference Type Strains Research Articles

Improved identification of Gordonia, Rhodococcus and Tsukamurella species by 5'-end 16S rRNA gene sequencing

The identification of fastidious aerobic Actinomycetes such as Gordonia, Rhodococcus, and Tsukamurella has remained a challenge leading to clinically significant misclassifications. This study is intended to examine the feasibility of partial 5'-end 16S rRNA gene sequencing for the identification of Gordonia, Rhodococcus, and Tsukamurella, and defined potential reference sequences for species from each of these genera. The 16S rRNA gene sequence based identification algorithm for species identification was used and enhanced by aligning test sequences with reference sequences from the List of Prokaryotic Names with Standing in Nomenclature. Conventional PCR based 16S rRNA gene sequencing and the alignment of the isolate 16S rRNA gene sequence with reference sequences accurately identified 100% of clinical strains of aerobic Actinomycetes. While partial 16S rRNA gene sequences of reference type strains matched with the 16S rRNA gene sequences of 19 isolates in our data set, another 13 strains demonstrated a degree of polymorphism with a 1-4 bp difference in the regions of difference. 5'-end 606 bp 16S rRNA gene sequencing, coupled with the assignment of well defined reference sequences to clinically relevant species of bacteria, can be a useful strategy for improving the identification of clinically relevant aerobic Actinomycetes.

Characterisation of carbapenem-resistant Acinetobacter baumannii outbreak strains producing OXA-58 in Turkey

The aim of this study was to characterise the molecular epidemiology and mechanisms of carbapenem resistance of nosocomial Acinetobacter baumannii isolates in a new university hospital in Turkey. A total of 145 carbapenem-resistant A. baumannii (CRAB) isolates were collected during the period 2003–2006. All isolates were typed by amplified fragment length polymorphism (AFLP) analysis. AFLP analysis showed three predominant clusters consisting of 72, 20 and 12 clinical strains as well as some smaller clusters and 23 unique strains. The three main clonal AFLP types corresponded to three major antibiotic susceptibility patterns. One environmental isolate was found related to the major outbreak clone. The reference type strains of European clones I, II and III were also typed by AFLP and analysed for clonal similarity. Polymerase chain reaction (PCR) analysis of different carbapenem resistance genes showed that strains from each of the three main clusters as well as 79% of the remaining strains harboured the bla OXA-58 gene. No genes encoding the metallo-β-lactamases GIM-1, SIM-1, SPM-1, IMP-like and VIM-like or the oxacillinases OXA-24-like and OXA-23-like were detected. In conclusion, multiple clones of CRAB strains producing OXA-58-type oxacillinase were responsible for a sustained CRAB outbreak occurring in a hospital in Turkey. These isolates were not associated with A. baumannii strains of the major European clones I, II or III.

Nhp6 Is a Transcriptional Initiation Fidelity Factor for RNA Polymerase III Transcription in Vitro and in Vivo

The binding of the RNA polymerase III (pol III) transcription factor TFIIIC to the box A intragenic promoter element of tRNA genes specifies the placement of TFIIIB on upstream-lying DNA. In turn, TFIIIB recruits pol III to the promoter and specifies transcription initiating 17-19 base pairs upstream of box A. The resolution of the pol III transcription apparatus into recombinant TFIIIB, highly purified TFIIIC, and pol III is accompanied by a loss of precision in specifying where transcription initiation occurs due to heterogeneous placement of TFIIIB. In this paper we show that Nhp6a, an abundant high mobility group B (HMGB) family, non-sequence-specific DNA-binding protein in Saccharomyces cerevisiae restores transcriptional initiation fidelity to this highly purified in vitro system. Restoration of initiation fidelity requires the presence of Nhp6a prior to TFIIIB-DNA complex formation. Chemical nuclease footprinting of TFIIIC- and TFIIIB-TFIIIC-DNA complexes reveals that Nhp6a markedly alters the TFIIIC footprint over box A and reduces the size of the TFIIIB footprint on upstream DNA sequence. Analyses of unprocessed tRNAs from yeast lacking Nhp6a and its closely related paralogue Nhp6b demonstrate that Nhp6 is required for transcriptional initiation fidelity of some but not all tRNA genes, in vivo.

Development of novel oligonucleotide probes for seven Actinomyces species and their utility in supragingival plaque analysis.

The traditional, biochemical and enzymatic methods of identifying Actinomyces species are frequently confounded by the similar phenotypic characteristics shared by the different members of this genus. Therefore, we developed novel species-specific oligonucleotide probes to accurately speciate seven pathogenic Actinomyces species, namely, Actinomyces bovis, A. gerencseriae, A. israelii, A. meyeri, A. naeslundii, A. odontolyticus and A. viscosus. A pair of universal primers and seven 15- to 19-base oligonucleotide probes with a tail of 20 thymidines on the 5' end were developed. The variable regions of 16S ribosomal DNA of 36 strains of Actinomyces belonging to the above species were amplified and labeled with digoxigenin, and an oligonucleotide-DNA hybridization assay was performed to examine the specificity and sensitivity of these probes. All seven, newly developed probes were specific and sensitive, and accurately detected 36 reference and wild type strains belonging to Actinomyces species, without cross-reactions. The probe for A. naeslundii detected all strains belonging to the genospecies 1 (12 strains) and catalase-negative genospecies 2 (four strains); it failed to detect catalase-positive A. naeslundii genospecies 2 (previous A. viscosus serotype II) (two strains). However, the latter strains of catalase-positive A. naeslundii genospecies 2 were correctly detected by the probe developed for A. viscosus. The new probes were then field tested using supragingival plaque samples from 28 healthy preschool children. Whilst A. odontolyticus was detected in almost all samples (96.4%), A. gerencseriae, A. meyeri, catalase-negative A. naeslundii and catalase-positive A. naeslundii genospecies 2 were detected in < 50% samples. We conclude that the developed oligonucleotide probes, complementary to the variable regions of 16S rDNA, would be of potential value for differentiating Actinomyces spp. in clinical samples from the oral cavity and other ecosystems where such species may abound.

Molecular sub-grouping of enterovirus reference and wild type strains into distinct genetic clusters using a simple RFLP assay

RFLP analysis and sequencing of RT-PCR amplicons in previous studies revealed the existence of intra-serotypic variability in the 5′-UTR of human enteroviruses, complicating the use of this method to serotype isolates. During the present study, the available sequences of many enterovirus reference and wild type strains were analysed in an attempt to discover restriction sites that would rapidly and reliably aid the classification of human enteroviruses into specific sub-groups on the basis of their 5′-UTR for diagnostic and/or epidemiological purposes. Despite intratypic genetic variability in the 5′-UTR, the results of the sequence analysis, as well as data from the RFLP analysis of 61 enterovirus reference strains from 60 different serotypes and 123 clinical isolates showed that one restriction endonuclease, HpaII, may contribute to a reliable sub-classification of CAVs and the rest of enteroviruses, on the basis of 5′-UTR, into five genetic groups, which could be particularly useful in clinical and epidemiological studies. Although more sequence data from enterovirus reference and wild type strains may be required for the elaboration of a precise molecular identification system, the more possible genotypic classification into distinct clusters, as shown with the restriction enzyme HpaII, and the determination of the biological significance of this grouping (pathogenesis, epidemiology) might constitute an alternative means of enterovirus identification against conventional classification into distinct serotypes.

A unique morphological feature commonly associated with Bacillus thuringiensis serovar yunnanensis isolates: An electron microscopic observation

When examined with a phase-contrast microscope, all of the 15 Bacillus thuringiensis serovar yunnanensis (H20ab) strains, the reference type strain and the 14 Japanese isolates, produced large rhomboidal inclusions in spore-lacking cells but not in spore-forming cells. No envelopes were associated with inclusions. Inclusion matrix consisted of electron-dense homogeneous substances. The frequency of inclusion-forming cells was <1% of the cell population in each strain. Also, phase-dark spherical bodies, much smaller than rhomboidal inclusions, were often associated with cells in sporulating cultures. Electron microscopic studies with the four selected yunnanensis strains revealed that: (1) the majority of cells produced spores but not crystalline inclusions, (2) formation of crystalline inclusions was limited to the spore-lacking cells, and (3) electron-dense round bodies, much smaller than rhomboidal crystalline inclusions and putatively assigned to poly-β-hydroxybutyrate inclusions, were often observed in both spore-forming cells and inclusion-forming asporogenous cells. The overall results strongly suggest that the formation of crystalline inclusions in asporogenous cells is an H serotype-specific phenotype characteristic of the B. thuringiensis serovar yunnanensis.

A reliable RT-PCR/RFLP assay for the molecular classification of enterovirus reference and wild type strains to either of the two genetic clusters on the basis of 5′-UTR

The available sequence data from a large segment of the 5′-UTR of all enterovirus reference and wild type strains were analysed in an attempt to discover possible restriction sites for reliable, serotypic identification of wild type isolates. No combination of restriction endonucleases, though, was found to produce serotype-, or group-specific haplotypes. Thirteen restriction enzymes were predicted to differentiate between representatives of the two enterovirus genetic clusters on the basis of 5′-UTR. One of these enzymes, BstOI, was tested in practice for the differentiation of 61 enterovirus reference strains from 56 different serotypes and 82 wild type strains which belong to the two genetic clusters on the basis of 5′-UTR. All the representatives of the two clusters were successfully differentiated with the specific restriction enzyme. Consequently, the simple RFLP-based assay presented here could be used as a very rapid and reliable means for the initial determination of whether a clinical isolate of unknown identity belongs to either of the ‘poliovirus-like’, or the ‘CBV-like’ genetic cluster on the basis of 5′-UTR classification of human enteroviruses.

Rapid Species Identification of Coagulase Negative Staphylococci by rRNA Spacer Length Polymorphism Analysis

Objectives: The identification of coagulase-negative staphylococci (CNS) is carried out mainly through conventional methods, commercial identification kits, and molecular biology-based methods. We evaluated the efficiency of PCR analysis of tRNA intergenic spacer length polymorphism (tDNA-ILP) and 16S-23S intergenic spacer length polymorphism (16S-23S ILP), and also that of restriction analysis of 16S-23S intergenic spacer amplification products (RA 16S-23S), for the rapid and accurate identification of CNS species.Methods: The PCR assays of rRNA spacer length polymorphisms were tested with nine reference type strains and 122 clinical isolates. The results were compared with those obtained by conventional methods and the Vitek system for clinical isolates.Results: Seventy-eight (63.9%) of 122 strains were identified as the same species with conventional identification, the Vitek GPI system, tDNA-ILP, and RA 16S-23S. The 16S-23S ILP analysis did not produce distinguishable PCR fragments. The results of tDNA-ILP analysis were in agreement with those of RA 16S-23S to the species level for 98.2% except for unknown patterns (eight isolates).Conclusions: These results indicate that tDNA-ILP and RA 16S-23S analysis are potentially valuable tools for rapid and accurate CNS identification.

Twenty-three years of Klebsiella phage typing: a review of phage typing of 12 clusters of nosocomial infections, and a comparison of phage typing with K serotyping

To review phage typing of 12 clusters of nosocomial Klebsiella infections which occurred between 1974 and 1997, and to compare phage typing and K serotyping. Materials and methods A total of 489 clinical and laboratory Klebsiella isolates were phage typed using 110 different phage preparations and K typed by counter current immunoelectrophoresis against 77 K antisera. A total of 152 phage types (PT) and 82 K types were found. Thirty-six phage types and 14 K types were represented only by the reference type strains. Of the remaining 68 K types, 60 could be subdivided into from two to 10 phage types. Ten out of 12 clusters of nosocomial Klebsiella infections could be verified as outbreaks by phage typing, whereas two clusters were found to be accumulations of sporadic cases. K typing performed retrospectively confirmed these results. In addition, for a subset of 104 epidemiologically unrelated isolates, O typing and pulsed field gel electrophoresis typing data were available. Based on these results the discriminative power of phage typing was found to be comparable with that of K typing, but phage types were less stable and reproducible. In an outbreak situation, phage typing was found to be very useful, although it seems less suitable for long-term surveillance purposes.

Levels of virulence are not determined by genomic lineage of Salmonella enterica serotype Enteritidis strains

Mouse virulence and the ability to adhere to, and invade cultured MDCK cells were investigated in 38 phage type reference strains of Salmonella enterica serotype Enteritidis and correlated with genomic lineage. The genomic lineage of 11 of the strains was determined in the present study; one IS200 and one ribotype pattern that had not been reported previously were observed. Log c.f.u. in the spleen 10 days post intraperitoneal (i.p.) infection with 3x10(3) bacteria (logVC10) varied between 2.9 and 8.7. The reference strains of PT7 and PT23 were found to be semi-rough and were of low virulence. All other strains possessed smooth LPS. Within each of the two major clonal lines, as well as among phage types outside these, both highly virulent and moderate to low virulent strains were present. While all strains of PT1, PT2 and PT8 were highly virulent, low virulent strains were detected in PT4 and PT13. The ability to adhere to, and invade MDCK cells varied between phage types (adherence between 13 and 61% of the inocula and invasion between 4 and 151% of the adherent cells). The results of the cell culture experiments did not correlate with the results of mouse virulence tests. No correlation between clonal lineage and virulence was found within S. Enteritidis. It seems most likely that some strains have lost some of the essential factors enabling this serotype to cause successful systemic infection.

Genomic fingerprinting and development of a dendrogram for Brucella spp. isolated from seals, porpoises, and dolphins.

Genomic DNA from reference strains and biovars of the genus Brucella was analyzed using pulsed-field gel electrophoresis (PFGE). Fingerprints were compared to estimate genetic relatedness among the strains and to obtain information on evolutionary relationships. Electrophoresis of DNA digested with the restriction endonuclease XbaI produced fragment profiles for the reference type strains that distinguished these strains to the level of species. Included in this study were strains isolated from marine mammals. The PFGE profiles from these strains were compared with those obtained from the reference strains and biovars. Isolates from dolphins had similar profiles that were distinct from profiles of Brucella isolates from seals and porpoises. Distance matrix analyses were used to produce a dendrogram. Biovars of B. abortus were clustered together in the dendrogram; similar clusters were shown for biovars of B. melitensis and for biovars of B. suis. Brucella ovis, B. canis, and B. neotomae differed from each other and from B. abortus, B. melitensis, and B. suis. The relationship between B. abortus strain RB51 and other Brucella biovars was compared because this strain has replaced B. abortus strain 19 for use as a live vaccine in cattle and possibly in bison and elk. These results support the current taxonomy of Brucella species and the designation of an additional genomic group(s) of Brucella. The PFGE analysis in conjunction with distance matrix analysis was a useful tool for calculating genetic relatedness among the Brucella species.

Unique activity associated with non-insecticidal Bacillus thuringiensis parasporal inclusions: in vitro cell-killing action on human cancer cells.

Parasporal inclusion proteins from a total of 1744 Bacillus thuringiensis strains, consisting of 1700 Japanese isolates and 44 reference type strains of existing H serovars, were screened for cytocidal activity against human leukaemia T cells and haemolytic activity against sheep erythrocytes. Of 1684 B. thuringiensis strains having no haemolytic activity, 42 exhibited in vitro cytotoxicity against leukaemia T cells. These non-haemolytic but leukaemia cell-toxic strains belonged to several H-serovars including dakota, neoleonensis, shandongiensis, coreanensis and other unidentified serogroups. Purified parasporal inclusions of the three selected strains, designated 84-HS-1-11, 89-T-26-17 and 90-F-45-14, exhibited no haemolytic activity and no insecticidal activity against dipteran and lepidopteran insects, but were highly cytocidal against leukaemia T cells and other human cancer cells, showing different toxicity spectra and varied activity levels. Furthermore, the proteins from 84-HS-1-11 and 89-T-26-17 were able to discriminate between leukaemia and normal T cells, specifically killing the former cells. These findings may lead to the use of B. thuringiensis inclusion proteins for medical purposes.

GENOMIC FINGERPRINTING OF SOME PSEUDOMONAS SYRINGAE PV. PISI STRAINS FROM SICILY

SUMMARY Pseudomonas syringae pv. pisi strains were characterized by pathogenicity tests on eight differential pea genotypes and by two genomic fingerprinting methods: PCR with REP and ERIC primers, and pulsed-field gel electrophoresis (PFGE) of genomic DNA digested by rare-cutting restriction endonucleases. The study comprised reference type strains of all 7 known races and 21 strains obtained from field pea crops in Sicily. PFGE was the most discriminatory method, delineating 6 pulsed-field gel profiles not coinciding with races, whereas REP and ERIC PCR identified 2 profile types. All the P. syringae pv. pisi isolated in Sicily were identified as race 6 on the basis of pathogenicity tests, and their genomic fingerprints by PFGE and REP and ERIC PCR were identical and more closely related to the type strains of races 3 and 6 than to those of the other races. The results are discussed in relation to improvement of detection methods and race-identification protocols.

Characterization of Lactobacillus sake isolates from dry-cured sausages by restriction fragment length polymorphism analysis of the 16S rRNA gene.

Lactobacillus sake strains originally isolated from dry-fermented sausages were characterized by phenotypic and genotypic methods, including DNA-DNA hybridization, restriction fragment length polymorphism (RFLP), and 16S rDNA sequencing analysis, in order to establish their taxonomic position and relation to well defined reference species. Initially, isolates of Lact. sake showing a characteristic phenotype (melibiose-positive, maltose- and arabinose-negative) were identified by DNA-DNA hybridization. Subsequently, RFLP studies using EcoRI and HindIII as restriction enzymes, and cDNA from Escherichia coli or 16S rDNA from Lact. sake strains as probes, showed distinct polymorphism levels. Thus, EcoRI-digested DNA probed with cDNA from E. coli disclosed the presence of a unique cluster for the meat isolates tested, allowing their differentiation from the reference type strain. When HindIII-digested DNA was hybridized with the cDNA probe, strain-specific patterns were obtained, showing a higher discrimination power. Considerable strain differentiation was also observed when EcoRI and HindIII digests were hybridized with 16S rDNA probes. Finally, sequence analysis of the 16S rDNA from one isolate also revealed a certain degree of genetic variability with respect to the reference strain of Lact. sake.

Characterization of field strains of group A bovine rotaviruses by using polymerase chain reaction-generated G and P type-specific cDNA probes.

Dot and Northern blot hybridization assays were used to analyze field strains of group A bovine rotaviruses (BRVs) by using nucleic acid probes representing P and G type specificities. The probes were prepared by polymerase chain reaction amplification of hyperdivergent regions of the cloned VP4 (nucleotides 211 to 686) and VP7 (nucleotides 51 to 392) genes from four serotypically distinct (in P or G types) strains of rotaviruses: NCDV (G6, P1), IND (G6, P5), 69M (G8, P10), and Cr (G10, P11). The P and G type cDNA probes were radiolabeled with [32P]dCTP and hybridized with RNA extracted from reference cell culture-passaged rotavirus strains or the field samples. The field samples were obtained from young diarrheic calves from Ohio, Nebraska, Washington State, and Canada. The cDNA probes were specific for their respective G or P types on the basis of analysis of known P and G type reference strains. The G typing analysis of 102 field samples revealed that 36.3% (37 of 102) were G6, 2.9% (3 of 102) were G8, 12.7% (13 of 102) were G10, and 23.5% (24 of 102) were untypeable. The P typing results for 93 samples indicated that 2.2% (2 of 93) were P1 (NCDV-like), 20.4% (19 of 93) were P5 (UK-like), 9.3% (10 of 93) were P11 (B223-like), and 40.8% (38 of 93) were untypeable. This is the first report of the identification among BRV strains in North America of a G type other than G6 or G10. Our report further confirms that G6, P5 rotaviruses are predominant among the BRV field strains that we examined, and the P types of these strains differ from that of the BRV vaccine strain used in the United States (G6, P1). The large number of untypeable G (23.5%) and P (40.8%) types suggests that other or new P and G types exist among BRV field strains.

Comparison of DNA fingerprinting and serotyping for identification of avian Pasteurella multocida isolates

The DNA fingerprint profiles and serotypes of 63 avian Pasteurella multocida field isolates, 13 attenuated vaccine isolates (propagated from vaccines manufactured by five companies), and 16 somatic reference strains were compared. DNA fingerprinting established the relationship of isolates that could not be distinguished by serotyping. Of the 76 isolates, 28 DNA fingerprint profiles and 12 somatic types were recognized. One isolate was nonreactive with 16 reference somatic and 5 reference capsule-type antisera. Thirty-one field isolates and seven vaccine isolates were identified as capsule type A. Twenty-nine field isolates and six vaccine isolates were nonencapsulated. Three field isolates were capsule type F. Isolates of capsule types B, D, and E were not found. One field isolate, identified as somatic type 7, had a DNA fingerprint identical to that of the somatic reference type 6 profile. Twelve field isolates had profiles identical to the somatic reference type 3 strain profile; 11 of these were identified as somatic type 3, 4, and 1 was identified as somatic type 3. The DNA fingerprint profiles of 50 field isolates and 13 attenuated vaccine isolates did not match profiles of the 16 somatic type reference strains. Twenty-five DNA fingerprint profiles were recognized from 30 of these field isolates. The DNA fingerprint profiles of 20 field isolates and 13 attenuated vaccine isolates were identical. Three somatic types (3; 3,4; and 4,16) were identified from the field isolates, and two somatic types (3 and 3,4) were identified from the attenuated vaccine isolates. DNA fingerprinting is useful for accurate identification and epidemiologic study of P. multocida isolates.

Relationship between group B streptococcal serotypes and cell surface hydrophobicity.

Cell surface hydrophobicities of streptococci of serological group B were determined by the adherence of the bacteria to hexadecane droplets. A significant adherence to hexadecane was observed with the group B streptococcal type reference strains Ib, V, Ic, R and X, but not with those of serotype Ia, II, III and IV. Cultivation of the bacteria in microcapsule-inducing media reduced the hexadecane adherence properties. The adherence to hexadecane was not related to fibrinogen binding properties of the cultures. Screening a large number of group B streptococci isolated from humans and bovines revealed that those with polysaccharide type antigen alone were generally hydrophilic, those with protein antigen alone or with protein antigen in combination with polysaccharide antigen were mostly hydrophobic. Cultivation of the bacteria under microaerobic conditions or after a single mouse passage enhanced microcapsule production and correspondingly reduced the hexadecane adherence values. Treatment of the bacteria by guanidinium chloride or by neuraminidase enhanced the hexadecane adherence. The hydrophobic component on group B streptococcal surface appeared to be partly inactivated by heat or proteolytic treatment of the bacteria.

Typing of coagulase-negative staphylococci by southern hybridization of chromosomal DNA fingerprints using a ribosomal RNA probe

Ribotyping consists of restriction endonuclease fingerprinting of ribosomal RNA (rRNA) genes visualized by Southern hybridization with an rRNA probe. This method was developed and compared with restriction endonuclease fingerprinting of chromosomal DNA for typing coagulase-negative staphylococci. Twenty-five American Type Culture Collection reference type strains and 53 clinical isolates were typed. Both methods clearly distinguished all 15 species of coagulase-negative staphylococci and most individual strains within each species. Except in the case of Staphylococcus warneri, ribotyping was most discriminating with the use of ClaI, one of eight endonucleases tested. HpaI and AvaI were more specific than ClaI for discrimination between strains of Staphylococcus warneri. The patterns produced by ribotyping were much simpler and thus easier to interpret than corresponding chromosomal fingerprints. However, ribotyping was slightly less discriminating. It is concluded that ribotyping offers an alternative method for molecular typing of coagulase-negative staphylococci. The application of both methods needs to be further evaluated in the clinical setting.