Showing papers on "Typing published in 2002"

GP5+/6+ PCR followed by Reverse Line Blot Analysis Enables Rapid and High-Throughput Identification of Human Papillomavirus Genotypes

Abstract: In this study, we developed a simple and fast typing procedure for 37 mucosotropic human papillomavirus (HPV) types using a nonradioactive reverse line blotting (RLB) procedure for general primer (GP5+/6+) PCR products. This system has the advantages not only that in a simple format, up to 42 PCR products can be simultaneously typed per membrane per day, but also that after stripping, the membranes can be easily rehybridized at least 15 times without a loss of signal. RLB appeared highly specific, and its sensitivity was identical to that of conventional typing performed with type-specific oligonucleotide probes in an enzyme immunoassay (EIA). The performance of RLB typing was evaluated with samples of HPV-positive cervical scrapings (n = 196) and biopsies of cervical premalignant lesions (n = 100). The distribution of HPV genotypes detected in these samples was in line with the distribution expected on the basis of literature data. In addition, RLB and EIA typing procedures were compared for the typing of high-risk HPV types in GP5+/6+ PCR products of 210 cervical scrapings from high-risk HPV-positive women who participated in a population-based screening program. The typing procedures had an excellent overall agreement rate of 96.5% (kappa value, 0.77). RLB was successful in detecting multiple HPV infections as well as single infections. In conclusion, the GP5+/6+ PCR-RLB procedure appeared to be a reliable and simple approach that may be of great value for large epidemiological studies, population-based cervical cancer screening programs, and vaccination trials that require high-throughput HPV typing.

Multilocus Sequence Typing for Characterization of Clinical and Environmental Salmonella Strains

Abstract: Multilocus sequence typing (MLST) based on the 16S RNA, pduF, glnA, and manB genes was developed for Salmonella, and its discriminatory ability was compared to those of pulsed-field gel electrophoresis (PFGE) and serotyping. PFGE differentiated several strains undifferentiable by serotyping, and 78 distinct PFGE types were identified among 231 Salmonella isolates grouped into 22 serotypes and 12 strains of undetermined serotype. The strains of several PFGE types were further differentiated by MLST, which suggests that the discriminatory ability of MLST for the typing of Salmonella is better than that of serotyping and/or PFGE typing. manB-based sequence typing identified two distinct genetic clusters containing 32 of 54 (59%) clinical isolates whose manB gene sequences were analyzed. The G+C contents and Splitstree analysis of the manB, glnA, and pduF genes of Salmonella indicated that the genes differ in their evolutionary origins and that recombination played a significant role in their evolution.

Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis Isolates with Low Copy Numbers of IS6110 by Using Mycobacterial Interspersed Repetitive Units

Abstract: A study set of 180 Mycobacterium tuberculosis and Mycobacterium bovis isolates having low copy numbers of IS6110 were genotyped using the recently introduced method based on the variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR). The results were compared with results of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The isolates were collected in Michigan from 1996 to 1999 as part of a project to genotype all isolates from new cases of tuberculosis in the state. Twelve MIRU loci were amplified, and the amplicons were analyzed by agarose gel electrophoresis to determine the copy number at each MIRU locus. MIRU-VNTR produced more distinct patterns (80 patterns) than did IS6110 RFLP (58 patterns), as would be expected in this study set. Spoligotyping identified 59 patterns. No single method defined all unique isolates, and the combination of all three typing methods generated 112 distinct patterns identifying 90 unique isolates and 90 isolates in 22 clusters. The results confirm the potential utility of MIRU-VNTR typing and show that typing with multiple methods is required to attain maximum specificity.

Comparison of Staphylococcus aureus Isolates from Bovine and Human Skin, Milking Equipment, and Bovine Milk by Phage Typing, Pulsed-Field Gel Electrophoresis, and Binary Typing

Abstract: Staphylococcus aureus isolates (n = 225) from bovine teat skin, human skin, milking equipment, and bovine milk were fingerprinted by pulsed-field gel electrophoresis (PFGE). Strains were compared to assess the role of skin and milking equipment as sources of S. aureus mastitis. PFGE of SmaI-digested genomic DNA identified 24 main types and 17 subtypes among isolates from 43 herds and discriminated between isolates from bovine teat skin and milk. Earlier, phage typing (L. K. Fox, M. Gershmann, D. D. Hancock, and C. T. Hutton, Cornell Vet. 81:183-193, 1991) had failed to discriminate between isolates from skin and milk. Skin isolates from humans belonged to the same pulsotypes as skin isolates from cows. Milking equipment harbored strains from skin as well as strains from milk. We conclude that S. aureus strains from skin and from milk can both be transmitted via the milking machine, but that skin strains are not an important source of intramammary S. aureus infections in dairy cows. A subset of 142 isolates was characterized by binary typing with DNA probes developed for typing of human S. aureus. Typeability and overall concordance with epidemiological data were lower for binary typing than for PFGE while discriminatory powers were similar. Within several PFGE types, binary typing discriminated between main types and subtypes and between isolates from different herds or sources. Thus, binary typing is not suitable as replacement for PFGE but may be useful in combination with PFGE to refine strain differentiation.

Development of variable-number tandem repeat typing of Mycobacterium bovis: comparison of results with those obtained by using existing exact tandem repeats and spoligotyping.

Abstract: Various genetic markers have been exploited for fingerprinting the Mycobacterium tuberculosis complex (MTBC) in molecular epidemiological studies, mainly through identifying restriction fragment length polymorphisms (RFLP). In large-scale studies, RFLP typing has practical processing and analysis limitations; therefore, attempts have been made to move towards PCR-based typing techniques. Spoligotyping (spacer oligotyping) and, more recently, variable-number tandem repeat (VNTR) typing have provided PCR-derived typing techniques. This study describes the identification and characterization of novel VNTR loci, consisting of tandem repeats in the size range of 53 to 59 bp in the MTBC, and their assessment as typing tools in 47 Mycobacterium bovis field isolates and nine MTBC strains. Spoligotyping and the previously described set of exact tandem repeats (ETRs) (R. Frothingham and W. A. Meeker-O'Connell, Microbiology 144:1189-1196, 1998) were also applied to the same panel of isolates. The allelic diversity of the individual VNTR loci was calculated, and a comparison of the novel VNTRs was made against the results obtained by spoligotyping and the existing set of ETRs. Eleven unique spoligotypes were discriminated in the panel of 47 M. bovis isolates. Greater resolution was obtained through the combination of the most-discriminating VNTRs from both sets. Considerable discrimination was achieved, with the 47 M. bovis isolates resolved into 14 unique profiles, while all nine MTBC isolates were uniquely differentiated. The novel VNTR markers described increased the discrimination possible in strain typing of M. bovis, with the added benefit of an intuitive digital nomenclature, with the allele copy number of the individual VNTRs providing a profile. VNTR typing was shown to be a valuable technique with great potential for further development and application to epidemiological tracing of tuberculosis transmissions.

Genomic Localization of a T Serotype Locus to a Recombinatorial Zone Encoding Extracellular Matrix-Binding Proteins in Streptococcus pyogenes

Abstract: Streptococcus pyogenes is an important bacterial pathogen afflicting humans. A striking feature is its extraordinary biological diversity, evident in the wide range of diseases it can cause and the antigenic heterogeneity present on its surface. The T antigens form the basis of a major serological typing scheme that is often used as an alternative or supplement to M typing. Unlike M typing, the genetic basis for T typing is poorly understood. In this report, the tee6 gene is localized to a position ≈3.3 kb downstream from prtF1 (or sfbI), which encodes the Fn-binding protein, protein F, a key virulence factor. Comparison of this portion of the genome with those of four additional strains reveals the presence of genes encoding a collagen-binding protein (Cpa) and a second Fn-binding protein (PrtF2 or PfbpI). This chromosomal region—here designated the FCT region—is ≈11 to 16 kb in length and is flanked at both ends by long stretches of highly conserved sequence. For each of the five strains, the FCT region contains a unique combination of semiconserved loci, indicative of extensive intergenomic recombination. The data provide evidence that the highly recombinatorial FCT region of the S. pyogenes genome is under strong selection for change in response to the host environment.

Assessment of the Genetic Diversity among Arcobacters Isolated from Poultry Products by Using Two PCR-Based Typing Methods

Abstract: In this study, enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and randomly amplified polymorphic DNA PCR (RAPD-PCR) were optimized for characterization of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. In addition, a simple and rapid DNA extraction method was tested for use in both typing procedures. Both methods had satisfactory typeability and discriminatory power, but the fingerprints generated with ERIC-PCR were more reproducible and complex than those obtained with RAPD-PCR. The use of nondiluted boiled cell suspensions as DNA templates was found to be very useful in ERIC-PCR. Characterization of large numbers of Arcobacter isolates is therefore preferably performed by the ERIC-PCR procedure. Isolates for which almost identical ERIC fingerprints are generated may subsequently be characterized by RAPD-PCR, although adjustment and standardization of the amount of the DNA template are necessary. In the second part of this study, the genotypic diversity of arcobacters present on broiler carcasses was assessed by using both typing methods. A total of 228 cultures from 24 samples were examined after direct isolation and enrichment. The isolates were identified by using a multiplex PCR as A. butzleri (n = 182) and A. cryaerophilus (n = 46). A total of 131 types (91 A. butzleri types and 40 A. cryaerophilus types) were discerned without discordance between the two typing techniques. The analysis of the poultry isolates showed that poultry products may harbor not only more than one species but also multiple genotypes. All genotypes were confined to one poultry sample, and only three genotypes were found after simultaneous enrichment and direct isolation. These results demonstrate that different outcomes can be obtained in epidemiological studies depending on the isolation procedure used and the number of isolates characterized.

Determining the genetic structure of the natural population of Staphylococcus aureus: A comparison of multilocus sequence typing with pulsed-field gel electrophoresis, randomly amplified polymorphic DNA analysis, and phage typing

Abstract: We used a sample of Staphylococcus aureus strains that are carried by humans and that are representative of the natural population of S. aureus strains in order to assess the value of multilocus sequence typing (MLST), pulsed-field gel electrophoresis, randomly amplified polymorphic DNA analysis, and phage typing as epidemiological tools. Only MLST was able to define clonal complexes unambiguously. All DNA-based typing approaches achieved a high degree of agreement, implying phylogenetic concordance, but predicted epidemiological associations with variable accuracy.

Specific identification and molecular typing analysis of Lactobacillus johnsonii by using PCR-based methods and pulsed-field gel electrophoresis

Abstract: A fast and reliable Multiplex-PCR assay was established to identify the species Lactobacillus johnsonii. Two opposing rRNA gene-targeted primers have been designed for this specific PCR detection. Specificity was verified with DNA samples isolated from different lactic acid bacteria. Out of 47 Lactobacillus strains isolated from different environments, 16 were identified as L. johnsonii by PCR. The same set of strains was investigated with five alternative molecular typing methods: enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), repetitive extragenic palindromic PCR (REP-PCR), amplified fragment length polymorphism, single triplicate arbitrarily primed PCR, and pulsed-field gel electrophoresis in order to compare the discriminatory power of these methods. The reported data strongly support the highly significant heterogeneity among all L. johnsonii isolates, potentially linked to their origin of isolation. The use of species-specific primers as well as rapid and highly powerful PCR-based molecular typing tools (namely ERIC- and REP-PCR techniques) should be respectively envisaged for identifying, differentiating and monitoring L. johnsonii strains from various environmental samples, for product monitoring, for species tracing in clinical studies as well as bacterial profiling of various microecological or gastrointestinal environments.

Molecular Typing of Selected Enterococcus faecalis Isolates: Pilot Study Using Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis

Abstract: The present study compared the recently developed multilocus sequence typing (MLST) approach with a well-established molecular typing technique, pulsed-field gel electrophoresis (PFGE), for subspecies differentiation of Enterococcus faecalis isolates. We sequenced intragenic regions of three E. faecalis antigen-encoding genes (ace, encoding a collagen and laminin adhesin; efaA, encoding an endocarditis antigen; and salA, encoding a cell wall associated antigen) and one housekeeping gene (pyrC) of 22 E. faecalis isolates chosen largely for their temporal and geographical diversity, but also including some outbreak isolates. MLST analysis of polymorphic regions of these four genes identified 13 distinct sequence types (STs) with different allelic profiles; the composite sequences generated from the four sequenced gene fragments of individual isolates showed 98.3 to 100% identity among the 22 isolates. We also found that the allelic profiles from two sequences, ace and salA, were sufficient to distinguish all 13 STs of this study. The 13 STs corresponded to 12 different PFGE types, with one previously designated PFGE clone (a widespread U.S. clone of β-lactamase-producing isolates) being classified into two highly related STs which differed at 2 of 2,894 bases, both in the same allele. MLST also confirmed the clonal relationships among the isolates of two other PFGE clonal groups, including vancomycin resistant isolates. Thus, this pilot study with representative E. faecalis isolates suggests that, similar to PFGE, the sequence-based typing method may be useful for differentiating isolates of E. faecalis to the subspecies level in addition to identifying outbreak isolates.

Estimation of genetic risk for type 1 diabetes.

Abstract: The most important gene loci defining risk of type 1 diabetes mellitus (T1DM) are located within the HLA gene region. HLA-DQ molecules are of primary importance but HLA-DR gene products modify the risk conferred by HLA-DQ. The risk associated with an HLA genotype is defined by the particular combination of susceptible and protective alleles. The highest risk is associated with a combination of two different risk haplotypes (7% risk to develop T1DM in Finland) whereas protective genotypes covering 69% of population have a risk of less than 0.2%). The complicated analysis of HLA genotypes is simplified by strong linkage disequilibrium between HLA-DRB1, -DQA1 and -DQB1 loci. In many cases one can deduce the alleles of other loci based on determination of the alleles in one locus. Differences between various populations in the frequency of marker alleles and in the linkages between them has to be taken into account. We have developed PCR based typing methods that utilize blood spot samples, microtiter plate format and lanthanide labeled oligonucleotide probes to define HLA-DQ and -DR alleles relevant for T1DM risk. Typing is run stepwise so that after initial HLA-DQB1 typing only those samples will be further analyzed in which -DQA1 or -DRB1 typing is informative and expected to contribute to the risk estimation. This method has been used to screen more than 50,000 newborn infants in Finland over a time period of 6 years, and it has been able to identify most children who have developed T1D during the follow-up period. The efficiency of the procedure has also been tested in Finnish and Greek populations. © 2002 Wiley-Liss, Inc.

Clustering of Saccharomyces boulardii strains within the species S. cerevisiae using molecular typing techniques

Abstract: Aims: This study was undertaken to characterize and differentiate therapeutically relevant Saccharomyces yeasts. Among the isolates were so-called Saccharomyces boulardii strains, which are considered as probiotic agents, but whose taxonomic assignment is controversial. Moreover, the discriminative power of the applied molecular typing techniques should be evaluated. Methods and Results: Genotyping was performed using species-specific polymerase chain reaction (PCR), randomly amplified polymorphic DNA-PCR, restriction fragment length polymorphism analysis of rDNA spacer regions and pulsed-field gel electrophoresis. Species-specific PCR assigned all of the product isolates to the species S. cerevisiae. By combining the other techniques, all isolates could be discriminated. Moreover, it could be demonstrated that probiotic S. boulardii strains form a separate cluster located within the species. Conclusions: With the exception of species-specific PCR, all of the applied methodologies were suitable for subspecies typing and indicated a close relationship between the probiotic strains. Significance and Impact of the Study: The methods applied in this study are considered powerful tools for quality control of therapeutically relevant yeasts. It is of crucial importance, especially regarding S. boulardii yeasts, to verify the identity of the correct strain, since the beneficial properties are considered to be strain-specific.

DNA‐based typing of blood groups for the management of multiply‐transfused sickle cell disease patients

Abstract: BACKGROUND: The usefulness of DNA genotyping for RBC antigens as a tool for the management of multiply-transfused patients with sickle cell disease (SCD) to overcome the limitations of hemagglutination assays was evaluated. STUDY DESIGN AND METHODS: Blood samples from 40 multiply-transfused SCD patients were studied by hemagglutination and by PCR-RFLP for antigens or genes in the Rh (D, C/c, E/e), Kell, Kidd, and Duffy systems. RESULTS: Discrepancies were found between hemagglutination and DNA typing test results in six patients: two were discrepant in Rh typing (one was D− by hemagglutination and RhD by DNA, and one was E+e− and RhEe by DNA), two were discrepant in Duffy typing [both were Fy(a+b−) and Fyb/Fyb by DNA], and four were discrepant in Kidd typing [Jk(a+b+) and Jkb/Jkb by DNA; two of these samples were also discrepant in Duffy]. Stored segments from blood units that had been recently transfused to these six recipients were phenotyped, confirming that the transfused RBCs were the source of the discrepancy between genotype and phenotype. CONCLUSION: DNA typing of blood groups by PCR-RFLP in peripheral blood WBCs contributes to the management of transfusions in SCD patients by allowing a more accurate selection of donor units.

Molecular epidemiology of Pseudomonas aeruginosa.

Abstract: Pseudomonas aeruginosa is a serious opportunistic pathogen in certain compromised hosts, such as those with cystic fibrosis, thermal burns and cancer. It also causes less severe noninvasive disease, such as otitis externa and hot tub folliculitis, in normal hosts. P. aeruginosa is phenotypically very unstable, particularly in patients with chronic infection. Phenotypic typing techniques are useful for understanding the epidemiology of acute infections, but they are limited by their discriminatory power and by their inability to group isolates that are phenotypically unrelated but genetically homologous. Molecular typing techniques, developed over the past decade, are highly discriminatory and are useful for typing strains from patients with chronic infection where the bacterial phenotype is unstable; this is particularly true in cystic fibrosis, where patients often are infected with the same strain for several decades, but the bacteria undergo phenotypic alteration. Molecular typing techniques, which have proven useful in typing P. aeruginosa for epidemiological purposes, include pulsed field gel electrophoresis, restriction fragment length polymorphic DNA analysis, random amplified polymorphic DNA analysis, repetitive extrapalindromic PCR analysis, and multilocus sequence typing. These methods are generally only available in specialized laboratories, but they should be used when data from phenotypic typing analysis are ambiguous or when phenotypic methods are unreliable, such as in cystic fibrosis.

Typing of Pasteurella multocida Isolated from Pigs with and without Porcine Dermatitis and Nephropathy Syndrome

Abstract: Porcine dermatitis and nephropathy syndrome (PDNS) is a sporadic, usually fatal disease of growing and finishing pigs that has been recognized in many pig-producing countries. Pasteurella multocida strains isolated from 15 pigs with PDNS and 51 pigs without PDNS were characterized by capsule and somatic antigen typing, random amplified polymorphic DNA (RAP-D) typing, and restriction analysis of genomic DNA using pulsed-field gel electrophoresis (PFGE). While capsular, somatic, and RAP-D typing did not discriminate PDNS isolates from non-PDNS isolates, all of the isolates from PDNS cases showed an identical ApaI PFGE restriction pattern. This pattern was also found in a high proportion (36%) of P. multocida strains isolated from non-PDNS cases. Isolation of a single variant of P. multocida from tissues of pigs with PDNS warrants further investigation into the possible role of these bacteria in the etiology of the disease.

Characterization of Salmonella Serovars by PCR-Single-Strand Conformation Polymorphism Analysis

Abstract: PCR-restriction fragment length polymorphism (PCR-RFLP) and PCR-single-strand conformation polymorphism (PCR-SSCP) analyses were carried out on the 1.6-kb groEL gene from 41 strains of 10 different Salmonella serovars. Three HaeIII RFLP profiles were recognized, but no discrimination between the serovars could be achieved by this technique. However, PCR-SSCP analysis of the groEL genes of various Salmonella serovars produced 14 SSCP profiles, indicating the potential of this technique to differentiate different Salmonella serovars (interserovar differentiation). Moreover, PCR-SSCP could differentiate strains within a subset of serovars (intraserovar discrimination), as three SSCP profiles were produced for the 11 Salmonella enterica serovar Enteritidis strains, and two SSCP profiles were generated for the 7 S. enterica serovar Infantis and five S. enterica serovar Newport strains. PCR-SSCP has the potential to complement classical typing methods such as serotyping and phage typing for the typing of Salmonella serovars due to its rapidity, simplicity, and typeability.

Nucleotide sequence-based typing of bacteria and the impact of automation.

Abstract: DNA-based typing methods are increasingly important for the characterisation of bacteria. They are used to monitor the epidemiology of pathogens with public health significance and also to help understand the evolution and population biology of bacteria. However, these methods require accuracy and reproducibility and are often of a high-throughput nature. Laboratory automation is therefore the key to the successful implementation of such methods. This review describes the impact of automation on DNA-based typing methods, particularly multi-locus sequence typing (MLST), and the method components that can be automated.

Detection and typing of respiratory adenoviruses in a single-tube multiplex polymerase chain reaction.

Abstract: A multiplex polymerase chain reaction (PCR) assay that is capable of detecting and typing six serotypes of respiratory adenovirus (Ad) was developed, using multiple sets of type-specific primers. The detection of each different serotype depended on distinguishing different numbers and sizes of amplification products on agarose gels following PCR. The multiplex PCR was tested with 26 clinical Ad isolates and other respiratory viruses including influenza viruses, parainfluenza viruses, and respiratory syncytial viruses as well as respiratory bacterial pathogens such as Chlamydia pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, and Mycoplasma pneumoniae. The multiplex PCR for the detection and typing of Ads gave an excellent correlation with the results by conventional typing with type-specific antisera. This assay may serve as a rapid means of confirming Ad with simultaneous serotype identification of the isolates. It will also have relevance as an adjunctive tool to conventional serotyping for diagnostic and epidemiological purposes.

Sequencing-based typing of HLA-B*51 alleles and the significant association of HLA-B*5101 and -B*5108 with Behçet's disease in Greek patients.

Abstract: Behcet's disease (BD) is widely known to be strongly associated with human leukocyte antigen (HLA) B51 in many different ethnic groupsRecently, HLA-B51 allele typing of Greek BD patients was performed to study the distribution of B*5101-B*5107 alleles in this Greek population, the B51 antigen strongly associated with BD was found to be predominantly encoded by allele B*5101 As it is now known that the B51 antigen can be encoded by 21 alleles, B*5101-B*5121, we performed HLA-B*51 allele genotyping among 58 Greek patients with BD After serological HLA typing, typing of HLA-B*51 alleles was performed using the polymerase chain reaction-sequencing-based typing (PCR-SBT) method The frequency of the B51 antigen was found to be significantly higher in the patient group as compared with the control group (759% of patients vs 220% of controls In the genotyping of B51 alleles, 34 out of 44 B51-positive patients possessed B*5101, 13 out of the 44 carried B*5108 In contrast, all of the 9 B51-positive normal controls carried B*5101 This study revealed a strong association between Greeks with BD, both B*5101, B*5108, provided important insights into the molecular mechanism underlying the association between HLA status, this disease

Comparison of molecular and immunological typing of isolates of Rice yellow mottle virus.

Abstract: Isolates of Rice yellow mottle virus (RYMV) were typed at the molecular level through the sequences of the open reading frame (ORF) 4 (coding for the coat protein) and ORF1 (coding for the movement protein), and serologically by means of polyclonal and monoclonal antibodies The overall patterns of diversity shown by molecular and serological analyses were similar: East-African isolates differed from West-African ones, and the West-African isolates from forest differed from the savannah ones Each major strain had a different serological profile However, molecular typing was more discriminating than immunological typing since several sequence variants belonged to the same serotype In rare instances, there were explainable discrepancies between molecular and serological typing Two amino acids at positions 115 (alanine vs threonine) and 191 (valine vs threonine) consistently discriminated between the major serotypes These positions were located in antigenic sites as revealed by Spot-scan method and were recognised by discriminating monoclonal antibodies One shared epitope, lying within a conserved region, may be responsible for the cross-reactivity between RYMV isolates A rationale for the correlation between molecular and immunological typing of RYMV and other sobemoviruses is proposed

Sequence typing confirms that campylobacter jejuni strains associated with guillain‐barré and miller‐fisher syndromes are of diverse genetic lineage, serotype, and flagella type

Abstract: Guillain-Barre syndrome (GBS) and Miller-Fisher syndrome (MFS) are correlated with prior infection by Campylobacter jejuni in up to 40% of cases. Nucleotide sequence-based typing of 25 C. jejuni isolates associated with neuropathy permitted robust comparisons with equivalent data from approximately 800 C. jejuni isolates not associated with neuropathy. A total of 13 genetic lineages and 20 flaA short variable region nucleotide sequences were present among the 25 isolates. A minority of isolates (4 of 25) had the flaA short variable region nucleotide sequences that were previously proposed as a marker for GBS-associated isolates. These 4 isolates probably represented the Penner serotype 19 lineage, which has been proposed to have an association with GBS.

Typing of human and bovine staphylococcus aureus by rapd-pcr and ribotyping-pcr

Abstract: Aims: To investigate genetic diversity among Staphylococcus aureus and to delineate the geographical distribution of the strains found. Methods and Results: RAPD-PCR and ribotyping-PCR were employed for the characterization of Staph. aureus isolates from bovine and nosocomial origin. Among the strains, five to nine groups were distinguished by RAPD-PCR, depending on which primer was used, while ribotyping-PCR distinguished seven ribotypes. Conclusions, and Significance and Impact of the Study: These results demonstrate the genetic heterogeneity of the strains studied, and the large dissemination of some clones throughout different regions and hosts, findings that may allow the monitoring of Staph. aureus infections.

Multilocus sequence typing of Neisseria meningitidis directly from cerebrospinal fluid.

Abstract: MLST typing of Neisseria meningitidis directly from clinical material was introduced in the National Reference Laboratory for Meningococcal Infections in Prague. Four cerebrospinal fluid samples were obtained from patients with suspected meningococcal invasive disease. In all samples, all classical laboratory methods gave negative results and the only positive method was PCR, which revealed Neisseria meningitidis group C (two specimens) and group B (two specimens), respectively. MLST performed directly from cerebrospinal fluid revealed that the strains causing the two group C infections were of sequence type (ST) 11, while the two group B infections were characterized as ST-32 and ST-33 respectively. Multi-locus sequence typing of meningococci directly from clinical material offers the opportunity to improve further the surveillance of meningococcal disease and has now been introduced into the routine portfolio of tests employed at the national reference laboratory of the Czech Republic.