Molecular analysis of multiple isolates of the major serotypes of group B streptococci

Serotyping of clinical isolates is a widely used technique for epidemiologic study of group B streptococcal infections. However, serotyping cannot definitively determine epidemiologically related or unrelated isolates. We investigated the use of restriction endonuclease analysis (REA) with both conventional agarose gel electrophoresis (AGE) and pulsed-field gel electrophoresis (PFGE) in 50 isolates of the major serotypes of group B streptococci. Single digestion with HindIII and HaeIII and double digestion with HindIII and then EcoRI were used for conventional AGE, and digestion with SmaI was used for PFGE. The molecular profile of one strain was compared with those of the strains within the same serotype as well as with the profiles from strains of different serotypes. Among 10 type Ia, Ia/alpha, Ia/alpha+beta, and Ia/R1 isolates and depending on the restriction enzyme used, we found between five and six REA patterns by conventional AGE and seven by PFGE; among 4 type Ib/alpha+beta isolates we found 2 to 4 REA patterns by conventional AGE and 4 by PFGE; among 21 type II, II/alpha, II/beta, II/alpha+beta, and II/R4 isolates, we found 11 REA patterns by both AGE and PFGE; and among 14 type III, III/R1, and III/R4 isolates, we found from 7 to 12 different REA patterns by AGE and 10 by PFGE. In total, among 13 serotypes and one nontypeable strain, we found 29 to 31 REA patterns by conventional AGE and 33 by PFGE. A particular REA pattern within a serotype was different from the patterns found in the other serotypes, suggesting that REA analysis by using conventional AGE or PFGE is a sensitive method for analyzing genetic relatedness and diversity in group B streptococci and has potential value in molecular epidemiologic studies.     

Biodiversity of bradyrhizobia nodulating Lupinus spp

The genetic structure of Bradyrhizobium isolates recovered from three Lupinus species (Lupinus campestris, Lupinus montanus, and Lupinus exaltatus) grown in Mexico was examined. Among 41 Bradyrhizobium isolates, 18 electrophoretic types (ETs) were distinguished by multilocus enzyme electrophoresis of five metabolic enzymes. The mean genetic diversity, 0.64, indicated that there was great genetic diversity in the population sampled. Most isolates (63%) fell into two closely related clusters (clusters I and II) and were the types most frequently isolated from the root nodules of L. montanus and L. campestris. ET cluster III isolates were frequent nodule occupants of L. exaltatus. The isolates also were assigned to three main groups by using Curie point pyrolysis mass spectrometry. In general, the multilocus enzyme electrophoretic data and pyrolysis mass spectrometric data agreed. We determined the 16S rRNA sequences of representative Lupinus isolates and of Bradyrhizobium japonicum USDA 6T and found that the lupine isolates were highly related to the B. japonicum type strain, although not all B. japonicum type strains (subcultures maintained in different bacterial collections) had identical small-subunit rRNA.     

Epidemiological typing of isolates from an outbreak of infection with multidrug-resistant Enterobacter cloacae by repetitive extragenic palindromic unit b1-primed PCR and pulsed-field gel electrophoresis

An outbreak of multidrug-resistant Enterobacter cloacae infection lasted for 4 months in a neonatal intensive care unit (NICU). Forty-six isolates from the NICU and 20 epidemiologically unrelated strains were characterized by pulsed-field gel electrophoresis (PFGE) and repetitive extragenic palindromic unit b1-primed PCR (REPUb1-PCR) typing. The PFGE patterns after XbaI restriction of the bacterial DNA were analyzed by computer software (Gelcompar) using the UPGMA (unweighted pair group method with arithmetic averages) clustering method and the Dice coefficient. The 46 isolates from the NICU were classified by PFGE typing into five clusters: A (further classified into 7 subtypes, A1 to A7), B, C, D, and E. This outbreak was attributed to multiple genetically related strains of cluster A which had a similarity of 85.8% +/- 4.6%. The minor band differences among strains of cluster A were probably due to minor genetic mutations. The type A1 and A3 strains were isolated from the clinical specimens of patients and hands of nurses. It was probable that these outbreak strains were transmitted among patients via the hands of personnel. REPUb1-PCR typing of the 46 isolates also demonstrated five types, in agreement with results obtained by the PFGE technique, but could not detect the minor mutations among the cluster A strains. Twenty epidemiologically unrelated strains were well distinguished by both PFGE and REPUb1-PCR typing. We conclude that PFGE is a highly discriminatory but time-consuming method for epidemiological typing of E. cloacae and that REPUb1-PCR is a more rapid method with good reproducibility and discriminatory power comparable to that of PFGE.     

Flagellin gene variation between clinical and environmental isolates of Burkholderia pseudomallei contrasts with the invariance among clinical isolates

The flagellin gene sequence from a clinical isolate of Burkholderia pseudomallei was used to design oligonucleotide primers for PCR/RFLP analysis of flagellin gene variation among clinical and environmental isolates of B. pseudomallei. Genes from four clinical and six environmental isolates were amplified and compared by RFLP. The clinical isolates were indistinguishable, but variation was detected among some of the environmental isolates. Sequence analysis of flagellin gene amplified products demonstrated high levels of conservation amongst the flagellin genes of clinical isolates (>99% similarity), compared to the variation observed between the clinical isolates and one of the environmental isolates (<90% similarity). Genomic comparisons with pulsed-field gel electrophoresis (PFGE) revealed differences between the relationships inferred by flagellin genotyping and PFGE, suggesting that a combination of molecular methods may be useful for the subtyping of B. pseudomallei strains.     

An evaluation of intergenic rRNA gene sequence length polymorphism analysis for the identification of Legionella species

There are currently more than 40 species of Legionella and the identification of most of these by standard methods is technically difficult. The aim of this study was to assess the suitability of a previously published PCR-based method of identifying Legionella spp. Intergenic 16S-23S rDNA spacer regions were amplified with primers complementary to conserved regions of the rRNA genes. Following electrophoretic separation of the products, data analyses were performed with the Taxotron software package. Computer-assisted analysis (with an empirically derived error tolerance of 3%) could differentiate only 26 of the 43 strains (representing 43 species), with the remaining 17 species clustering into four groups (group I, comprising 10 species; group II, three species; group III, two species and group IV, two species). Analysis of well-characterised 'non-type' strains of some Legionella spp. (e.g., from type culture collections) resulted in patterns distinct from the corresponding type strain in most cases. Furthermore, recent isolates (identified by conventional methods) were identified by this PCR method to the presumed correct species (or species group) in only a minority of cases. Well characterised strains and recent isolates of Legionella showed heterogeneity within many species. This intra-species variation severely limits the usefulness of the method for the identification of isolates. However, this property may be useful for epidemiological typing within such species.     

Demonstration of antigenic and genotypic variation in Orientia tsutsugamushi which were isolated in Japan, and their classification into type and subtype

A total of 40 strains of Orientia tsutsugamushi (34 isolates from patients and trombiculid mites in Japan, and 6 prototype strains of antigenic variants) were examined for classification based on the reactivities with type-specific monoclonal antibodies in indirect immunofluorescence tests, and on the restriction fragment length polymorphism of a polymerase chain reaction (PCR)-amplified 56-kilodalton type-specific antigenic protein gene. By these methods, several antigenic and genotypic variants were found among the strains, and these variants were classified into types and further into subtypes. These results suggest that there are many variants in O. tsutsugamushi, and the methods used here seem to be useful for the systematic classification of the numerous variants. A strain which may be a new type distinguishable from those identified previously was also found in this study. Furthermore, variety in the degree of pathogenicity in mice related to type and/or subtype classification were observed.     

Evolutionary characteristics of influenza B virus since its first isolation in 1940: dynamic circulation of deletion and insertion mechanism

New antigenic variants of B/Yamagata/16/88-like lineage which appeared in the season of 1997 as a minor strain tended to predominate in the following season. Also, we could observe for the first time, three peaks of activity caused by H3N2 virus and two variants of B influenza virus. Antigenic and phylogenetic analyses revealed that B/Victoria/2/87-like variants appeared again in Japan in 1997 after a nine-year absence. Influenza B viruses evolved into three major lineages, including the earliest strain (I), B/Yamagata/16/88-like variants (II), which comprised of three sublineages (II-(i), II-(ii), II-(iii)), and B/Victoria/2/87-like variants (III). Evolution of influenza B virus hemagglutinin was apparently distinguishable from that of influenza A virus, showing a systematic mechanism of nucleotide deletion and insertion. This phenomenon was observed to be closely related to evolutionary pathways of I, II-(i), II-(ii), II-(iii) and III lineages. It was noteworthy to reveal that the nucleotide deletion and insertion mechanism of influenza B virus completed one cycle over a fifty-year period, and that a three nucleotide deletion was again observed in 1997 strains belonging to lineage II-(iii). It was evident that amino acid substitutions accompanying nucleotide insertions were highly conserved.     

Bartonella clarridgeiae, a newly recognized zoonotic pathogen causing inoculation papules, fever, and lymphadenopathy (cat scratch disease)

Shortly after adopting a 6-week-old cat, a veterinarian was bitten on the left index finger. Within 3 weeks, he developed headache, fever, and left axillary lymphadenopathy. Initial blood cultures from the cat and veterinarian were sterile. Repeat cultures from the cat grew Bartonella-like organisms with lophotrichous flagella. Sera from the veterinarian were not reactive against Bartonella henselae, B. quintana, or B. elizabethae antigens but were seroreactive (reciprocal titer, 1,024) against the feline isolate. Sequential serum samples from the cat were reactive against antigens of B. henselae (titer, 1,024), B. quintana (titer, 128), and the feline isolate (titer, 2,048). Phenotypic and genotypic characterization of this and six additional feline isolates, including microscopic evaluation, biochemical analysis, 16S rRNA gene sequencing, DNA-DNA hybridization, and PCR-restriction fragment length polymorphism of the 16S gene, 16S-23S intergenic spacer region, and citrate synthase gene identified the isolates as B. clarridgeiae. This is the first report of cat scratch disease associated with B. clarridgeiae.     

Three distinct genotypes within Candida parapsilosis from clinical sources

Three genetically distinct groups of Candida parapsilosis were detected among clinical isolates. These were distinguishable on the basis of isoenzyme profiles and DNA sequences of internally transcribed spacer (ITS) sequences flanking the 5.8S RNA gene. In an investigation of 45 strains, including 32 clinical isolates from Texas, C. parapsilosis group I composed the majority of the common clinical isolates. The type strain of C. parapsilosis was a member of this group. The 10 group II isolates were indistinguishable from group I strains when tested with the API 20C kit. The two group III isolates differed from those in groups I and II by being D-xylitol positive by the API 20C kit; however, isolates in all groups assimilated D-xylitol from broth. Isoenzyme profiles excluded the close relationship of any of these groups to Lodderomyces elongisporus, which is a teleomorphic yeast that has a physiological profile similar to that of C. parapsilosis. Although there were insignificant differences in the ITS2 rDNA sequences, comparisons of the ITS1 sequences revealed several differences. A sequence analysis of ITS1 in which missing bases were counted as mismatches showed the following similarities: group I versus group II, 87.7%; group I versus group III, 82.1%; group II versus group III, 84.5%. Also, the activity of secreted proteinase showed differences among the three groups, with many group I isolates having moderate to high activity. The degree of susceptibility to antifungal agents, amphotericin B, ketoconazole, and 5-fluorocytosine, could not be used to determine an isolate's group.     

Homologous protection but lack of heterologous-protection by various species and types of Bartonella in specific pathogen-free cats

Cat-scratch disease (CSD) is caused by Bartonella henselae, and possibly by B. clarridgeiae. In immuno-compromised persons, B. henselae is one of the agents causing bacillary angiomatosis. Domestic cats are the main reservoir of these bacteria, which are transmitted primarily from cat to cat by fleas. Possible strategies to prevent the spread of infection among cats are to eliminate flea infestation or to prophylactically immunize cats. In order to develop an appropriate vaccine, it is important to determine if cats become resistant to re-infection by the same strain or various types or species of Bartonella. In a series of experiments, 21 SPF cats were experimentally infected by the intradermal route with 10(5)-10(10) colony-forming units/ml of either B. henselae type II (17 cats), or a new strain 'Humboldt' isolated from a mountain lion (4 cats). The cats were bled weekly to every other week for determination of bacteremia and specific antibody production. After they cleared their infection, they were challenged by a homologous or heterologous strain of Bartonella: 10 cats were challenged with B. henselae type II, three cats with B. henselae type I, four cats with B. clarridgeiae and four cats with the 'Humboldt' strain. Seven of these cats received a third inoculum dose resulting in three cats sequentially infected with sequence B. henselae type II/B. henselae type II/'Humboldt', two cats with sequence B. henselae type II/'Humboldt'/B. clarridgeiae, and two cats with the sequence 'Humboldt'/B. henselae type II/'Humboldt'. All cats challenged with a homologous strain remained abacteremic after challenge and had an increased IgG antibody titer. All cats challenged with either a different Bartonella species or type became bacteremic. The few cats receiving a third inoculum with a strain homologous to the initial strain remained abacteremicafter that challenge. All cats infected with B. clarridgeiae suffered relapsing bacteremia compared to only 36% of the B. henselae infected cats and 22% of the 'Humboldt'-infected cats (p=0.008). The duration of bacteremia was significantly longer in B. henselae primary-infected cats (mean: 34 weeks) than B. henselae heterologously challenged cats (mean: 9 weeks) (p=0.014). These data clearly indicate the lack of cross-protection between B. henselae and B. clarridgeiae and furthermore, indicate the lack of protection between B. henselae types I and II, and a wildlife isolate. A vaccine strategy for CSD prevention in domestic cats will require a multivalent vaccine approach.     

Epidemiology of an unusually prolonged outbreak of typhoid fever in Terrassa, Spain

An unusually prolonged outbreak of typhoid fever, from 1988 to 1994, in Terrassa (Barcelona, Spain), was caused by a casual food handler who was a carrier. The pattern of this outbreak suggested intermittent low-level exposure to Salmonella typhi. We found 70 patients with S. typhi infections, 52 of whom were available for study. Medical records were reviewed and patients were interviewed with use of a standard questionnaire. Phage typing and pulsed-field gel electrophoresis (PFGE) for strain subtyping were used to confirm the epidemiological data. The 27 outbreak strains shared the same phage type and the same PFGE pattern. Four sporadic strains shared the same phage type as the outbreak strain. PFGE was found to be useful for differentiating strains for epidemiological purposes.     

Evaluation of intraspecies genetic variation within the 16S rRNA gene of Mycoplasma hominis and detection by polymerase chain reaction

Mycoplasma hominis is a heterogeneous species with DNA-DNA hybridization values ranging from 51 to 100%. We report here the sequencing of the 16S rRNA gene of a strain (183) that greatly differs from the type strain (PG21) of this species. Comparison of 16S rDNA sequences from these two strains showed limited differences, indicating that the two strains belong to the same rRNA species complex. Using these nucleotide sequence data, we established a rapid method for the detection of M. hominis by using polymerase chain reaction. This method was shown to be sensitive and specific when tested with reference strains and clinical isolates.     

Molecular analysis of Salmonella enteritidis by pulsed-field gel electrophoresis and ribotyping

A total of 61 isolates of Salmonella enteritidis were analyzed by the techniques of pulsed-field gel electrophoresis (PFGE) and ribotyping. Twenty-three of the isolates were from Zurich, Switzerland, and 38 isolates were from the University Hospital, Kuala Lumpur, Malaysia. Five of the Malaysian isolates were hospital-related outbreak strains and were shown to be indistinguishable by PFGE analysis following digestion with three different restriction endonucleases, XbaI (5'-TCTAGA-3'), SpeI (5'-ACTAGT-3'), and AvrII (5'-CCTAGG-3'). The PFGE pattern of an isolate from a suspected carrier staff nurse was found to be identical to those of the hospital outbreak isolates. These isolates were also indistinguishable by ribotyping with SmaI and SphI. The same single PFGE pattern was also detected in 29 of 32 sporadic isolates of S. enteritidis. Four closely related ribotypes were detected among these 29 isolates. Similarly, outbreak-related strains from Switzerland showed close genetic identity by PFGE and ribotyping. Strains obtained from poultry showed more variations in their PFGE patterns and ribotypes, although the patterns were still closely related. In addition, SphI ribotypes A and D among the Swiss strains correlated with phage types 4 and 8, respectively. No correlation of phage types with PFGE pattern was noted. Both PFGE and ribotyping indicate that the S. enteritidis strains circulating in Malaysia and Switzerland are very similar and may be clonally related. Comparison of the PFGE patterns with the ribotypes for 23 Swiss and 16 Malaysian isolates showed that there was a 69% concordance in the grouping of isolates. We conclude that the close genetic similarity observed between epidemiologically unrelated and outbreak-related isolates of S. enteritidis suggests that both PFGE and ribotyping are of limited value in the epidemiological analysis of these particular isolates, possibly because of the highly clonal nature of pathogenic strains of S. enteritidis.     

Differentiation of bifidobacteria by use of pulsed-field gel electrophoresis and polymerase chain reaction

Several different genomic fingerprints can be obtained from various commercially-important species of Bifidobacterium using pulsed-field gel electrophoresis (PFGE) following digestion of DNA with XbaI and SpeI. Four different genomic finger printings were discernible for reference strains of Bifidobacterium animalis, five for B. bifidum, three for B. breve, five for B. infantis and three for B. longum. Standard commercially-available industrial strains of B. animalis are identical to the reference strain ATCC 27536, previously isolated from chicken feces. There was more genomic heterogeneity among industrial strains of B. longum, in that only one gave profiles similar to the type strain of this species (ATCC 15707). The other 14 commercially-available strains of B. longum (mainly isolated from Japanese commercial preparations) were divided into four new molecular types based on their PFGE patterns. The PFGE method indicated that only five distinct strains of B. longum and one strain of B. animalis are used in commercial preparations. Additionally, the use of polymerase chain reaction amplification of portions of 16S rDNA provides a highly specific technique to discriminate between the species B. breve, B. infantis and B. longum.     

Molecular identification and epidemiology of animal mycoplasmas

Molecular genetic techniques show a high potential for rapid and accurate identification of Mycoplasma species isolated from animals. An important field of application for such methods is the differentiation of species and/or subspecies which are phenotypically closely related, but which show significant differences in epidemiological impact. This need is particularly important for the mycoplasmas of the "mycoides group", which are phenotypically and phylogenetically very closely related. Molecular typing techniques based on 16S rRNA genes give straightforward phylogenetic answers on the species level. For more refined methods of subtyping at the subspecies level, the use of defined genes characteristic of certain Mycoplasma species or clusters is recommended. Genetic fingerprinting, especially insertion sequence typing has proved to be a valuable tool for subtyping and strain identification in particular of vaccine strains and for epidemiological investigations.     

Molecular diagnosis of cat scratch disease: a two-step approach

Amplification of Bartonella henselae DNA has been proposed as a diagnostic test for cat scratch disease (CSD). The sensitivities of the following three PCR assays were compared. PCR/rRNA with universal primers amplifies part of the 16S rRNA gene, followed by hybridization with a specific B. henselae probe; PCR/CS and PCR/HSP amplify portions of the gltA and the htrA genes, respectively, each followed by restriction fragment length polymorphism analysis. The threshold of detection of B. henselae DNA in pus was 10(-4), 10(-3), and 10(-2) ng for PCR/rRNA, PCR/CS, and PCR/HSP, respectively. By these three assays, B. henselae DNA was detected in 100, 94, and 69% of 32 pus and lymph node specimens from CSD patients, respectively. The similar sensitivities of the PCR/rRNA and the PCR/CS assays for detecting B. henselae DNA in clinical specimens are in contrast to the 10-fold difference in sensitivities in favor of PCR/rRNA demonstrated with purified B. henselae DNA in sterile pus, suggesting that in the majority of cases, the bacterial load in clinical specimens is large enough to be identified by the PCR/CS assay. A two-step approach is suggested to achieve maximal sensitivity for detecting B. henselae in clinical specimens: initial testing by PCR/CS (which does not require hybridization), followed by PCR/rRNA with PCR/CS-negative specimens when CSD is strongly suspected.     

Decreased capacity for type-specific-antigen synthesis accounts for high prevalence of nontypeable strains of group B streptococci in Mexico

The low incidence of group B streptococcal (GBS) invasive neonatal disease in Mexico has been attributed to the low prevalence of serotype III strains, a major serotype in developed countries. In addition, nontypeable strains account for 12% of the isolates in Mexico and < 1% of the isolates in the United States. In this study, 57 GBS isolates (28 nontypeable by the Lancefield procedure) from carrier and infected neonates and women from Mexico were also examined for the presence of type-specific antigen by an enzymatic procedure using N-acetylmuramidase digestion of the cell wall to release soluble type-specific antigen. Of the 28 nontypeable strains from Mexico, 23 were typeable by the enzyme extraction procedure, with serotype III being the predominant serotype in invasive disease. These results suggest that nontypeable isolates of GBS should be further examined by the enzymatic extraction procedure to determine the presence of type-specific antigen. Furthermore, these limited results suggest that serotype III is likely a major serotype in invasive disease also in Mexico.     

Identification of atypical strains of Staphylococcus epidermidis by use of molecular tools

Four atypical coagulase-negative staphylococcal (CNS) isolates from clinical sources were compared with Staphylococcus epidermidis strains by ribotyping. The ribotypes of the four strains shared close rDNA restriction profiles with those of the S. epidermidis strains used. The DNA sequence encoding 16S rRNA demonstrated 99.9% homology with S. epidermidis. S1 nuclease experiments showed that these atypical strains formed a homogeneous genomic group. DNA-DNA homologies between the S. epidermidis type strain CCM 2124 and the four CNS isolates ranged from 70 to 89%. The guanine-plus-cytosine content of the deoxyribonucleic acid of the four strains ranged from 31 to 32 mol%.