Establishment of an active laboratory-based surveillance for bacterial meningitis in Croatia and molecular characterization of Neisseria meningitidis isolates causing meningococcal disease that were collected in the year 2000, the first year of activity

In 2000, 23 Neisseria meningitidis (meningococcal [Men]) isolates were collected in Croatia through an active laboratory-based surveillance for bacterial meningitis (17 Men serogroup B [MenB], 4 MenC, 1 MenW135, and 1 nongroupable isolate). Molecular characterization revealed a substantial level of diversity with only six isolates belonging to electrophoretic type 5 (ET-5) and ET-37 hypervirulent complexes.     

Intraspecific 16S rRNA gene diversity among clinical isolates of Neisseria species

In the present work, nearly the entire 16S rRNA gene sequences of 46 clinical samples of Neisseria spp. were determined, and the aligned sequences were analyzed to investigate the diversity of 16S rRNA genes in each commensal Neisseria species. Two 16S rRNA types were identified in two Neisseria sicca strains, three 16S rRNA types in five Neisseria macacae strains, fourteen 16S rRNA types in twenty Neisseria flavescens isolates, and fourteen 16S rRNA types in nineteen Neisseria mucosa isolates. The number of nucleotides that were different between 16S rRNA sequences within specie ranged from 1 to 15. We found high intraspecific sequence variation in 16S rRNA genes of Neisseria spp. strains.     

Distinct genotypes of human and canine isolates of Campylobacter upsaliensis determined by 16S rRNA gene typing and plasmid profiling.

The utility of combined 16S rRNA (rrs) gene restriction fragment length polymorphism and plasmid profiles to differentiate between and within Campylobacter upsaliensis of human and canine origin was examined. Fourteen distinct rrs gene restriction fragment length polymorphs consisting of bands sized between 1.9 and 4.8 kb were observed. The copy number of the 16S rRNA gene was three in most strains of C. upsaliensis. Plasmids were found in almost 60% of the strains; ranging in size from 1.5 to 100 kb, they gave 15 distinct plasmid profiles. All isolates from humans contained one or more plasmids, as did strains isolated from dogs with sporadic diarrhea. The two commonest 16S ribotypes were divided into eight and nine subgroups by plasmid profiling. The genotyping of canine isolates from three veterinary surveys detected both multiple infections and reinfection of dogs. Except for one, each of the isolates from humans constituted a single and unique 16S ribotype, and these more frequently carried plasmids than did canine strains. Ribotypes of human strains were not found among canine isolates. These results suggest that host-specific genotypic differences may exist among strains of C. upsaliensis, for example, intraspecific clones or clone complexes pathogenic for humans.     

Irish strains of Neisseria meningitidis: characterisation using multilocus sequence typing

A total of 56 Neisseria meningitidis strains are analysed using multilocus sequence typing (MLST). Twenty-nine distinct sequence types (STs) were identified, eight of which were new. Four known hypervirulent clones--ST-11 (electrophoretic type [ET]-37) complex, ST-44 complex (lineage 3), ST-32 (ET-5) complex and ST-8 complex (cluster A4)--were identified by MLST in 35 disease-associated and four carrier strains. Two other clones (ST-22 complex and ST-269 complex) were identified in nine disease-associated and one carrier strain. The remaining strains were heterogeneous. Additional sequencing within the FumC gene further distinguished the ET-15 clone within the ST-11 (ET-37) clonal complex. This resolution of isolates into genetic clones by MLST enhances the more traditional techniques of serotyping and serosubtyping. The data obtained established that hyperendemic meningococcal disease in Ireland could be attributed to strains belonging to four major hypervirulent clones, all of which account for elevated levels of disease worldwide. The extra information provided by MLST will be used to study the population structure and epidemiology of N. meningitidis and will allow a comparison of Irish strains with those circulating globally.     

Outer membrane protein patterns mark clones of Escherichia coli O2 and O78 strains that cause avian septicemia.

Major outer membrane proteins were isolated from 36 Escherichia coli strains representing six common clones of the O2 and O78 serogroups implicated in avian colisepticemia. Clonal relationships among isolates were inferred from an analysis of polymorphism at 20 enzyme-encoding loci detected by multilocus enzyme electrophoresis. For isolates of these clones, there was a high concordance (greater than 90%) between identity in multilocus genotype and major outer membrane protein patterns. The results indicate that major outer membrane protein patterns discriminate among the genetically different clonal groups that constitute the heterogeneous O2 and O78 serogroups associated with avian disease.     

High level of sequence diversity in the 16S rRNA genes of Haemophilus influenzae isolates is useful for molecular subtyping

A molecular typing method based on the 16S rRNA sequence diversity was developed for Haemophilus influenzae isolates. A total of 330 H. influenzae isolates were analyzed, representing a diverse collection of U.S. isolates. We found a high level of 16S rRNA sequence heterogeneity (up to 2.73%) and observed an exclusive correlation between 16S types and serotypes (a to f); no 16S type was found in more than one serotype. Similarly, no multilocus sequence typing (MLST) sequence type (ST) was found in more than one serotype. Our 16S typing and MLST results are in agreement with those of previous studies showing that serotypable H. influenzae isolates behave as highly clonal populations and emphasize the lack of clonality of nontypable (NT) H. influenzae isolates. There was not a 1:1 correlation between 16S types and STs, but all H. influenzae serotypable isolates clustered similarly. This correlation was not observed for NT H. influenzae; the two methods clustered NT H. influenzae isolates differently. 16S rRNA gene sequencing alone provides a level of discrimination similar to that obtained with the analysis of seven genes for MLST. We demonstrated that 16S typing is an additional and complementary approach to MLST, particularly for NT H. influenzae isolates, and is potentially useful for outbreak investigation.     

Necessity of quality-controlled 16S rRNA gene sequence databases: identifying nontuberculous Mycobacterium species

The use of the 16S rRNA gene for identification of nontuberculous mycobacteria (NTM) provides a faster and better ability to accurately identify them in addition to contributing significantly in the discovery of new species. Despite their associated problems, many rely on the use of public sequence databases for sequence comparisons. To best evaluate the taxonomic status of NTM species submitted to our reference laboratory, we have created a 16S rRNA sequence database by sequencing 121 American Type Culture Collection strains encompassing 92 species of mycobacteria, and have also included chosen unique mycobacterial sequences from public sequence repositories. In addition, the Ribosomal Differentiation of Medical Microorganisms (RIDOM) service has made freely available on the Internet mycobacterial identification by 16S rRNA analysis. We have evaluated 122 clinical NTM species using our database, comparing >1,400 bp of the 16S gene, and the RIDOM database, comparing approximately 440 bp. The breakdown of analysis was as follows: 61 strains had a sequence with 100% similarity to the type strain of an established species, 19 strains showed a 1- to 5-bp divergence from an established species, 11 strains had sequences corresponding to uncharacterized strain sequences in public databases, and 31 strains represented unique sequences. Our experience with analysis of the 16S rRNA gene of patient strains has shown that clear-cut results are not the rule. As many clinical, research, and environmental laboratories currently employ 16S-based identification of bacteria, including mycobacteria, a freely available quality-controlled database such as that provided by RIDOM is essential to accurately identify species or detect true sequence variations leading to the discovery of new species.     

Yersinia enterocolitica 16S rRNA gene types belong to the same genospecies but form three homology groups

The species Yersinia (Y.) enterocolitica consists of biochemically and serologically heterogeneous strains. A vernacular nomenclature divides these strains in 'European' and 'American' bioserotypes. We investigated six strains of each group by DNA-DNA hybridization, determination of G + C mol% content and sequence alignment studies. Based on different DNA-DNA hybridization values and the 16S rRNA gene sequences a division into two Yersinia enterocolitica subspecies is justified. We propose the names Yersinia enterocolitica subsp. enterocolitica for strains belonging to the 16S rRNA gene type represented by the Type strain ATCC 9610 and Yersinia enterocolitica subsp. palearctica for strains belonging to the 16S rRNA gene type of strain Y11 (DSMZ13030).     

Genotypic heterogeneity of Pasteurella gallinarum as shown by ribotyping and 16S rRNA sequencing

Forty-five strains mainly isolated from chickens in Zimbabwe and Denmark, two pig and three rat isolates all identified as Pasteurella gallinarum by conventional phenotypic tests were characterized by ribotyping, and selected strains were subsequently analysed by 16S rRNA gene sequencing. High genotypic diversity was observed, the number of ribotypes totalling 24. A major group of 47 isolates including the type strain of P. gallinarum clustered at 56% similarity and included 21 ribotypes. Ribotyping showed that some genotypes of P. gallinarum seem to be globally distributed. The three isolates from rodents did not share even a single common ribotype fragment with strains from birds and the pig isolates. Two avian isolates from Denmark and Zimbabwe and the pig strain showed from 97.6 to 99.8% 16S rRNA sequence similarity with the type strain of P. gallinarum and with type strains of Pasteurella volantium and Pasteurella avium . Two rat strains showed 98.6% 16S rRNA gene sequence similarity with each other, but were only related with P. gallinarum at 93% similarity. These isolates showed the highest similarity with [ Actinobacillus ] muris at 96.4 to 95.0% similarity. We suggest that conventional identification of P. gallinarum consequently should consider the source of isolation to obtain a correct diagnosis, and that isolation from animals other than fowl should be confirmed by genotypic analysis such as 16S rRNA gene sequence comparison.     

Species-specific identification of Leptospiraceae by 16S rRNA gene sequencing

The genus Leptospira is classified into 13 named species and 4 genomospecies based upon DNA-DNA reassociation studies. Phenotypic tests are unable to distinguish between species of Leptospira, and there is a need for a simplified molecular approach to the identification of leptospires. 16S rRNA gene sequences are potentially useful for species identification of Leptospira, but there are a large number of sequences of various lengths and quality in the public databases. 16S rRNA gene sequences of near full length and bidirectional high redundancy were determined for all type strains of the species of the Leptospiraceae. Three clades were identified within the genus Leptospira, composed of pathogenic species, nonpathogenic species, and another clade of undetermined pathogenicity with intermediate 16S rRNA gene sequence relatedness. All type strains could be identified by 16S rRNA gene sequences, but within both pathogenic and nonpathogenic clades as few as two or three base pairs separated some species. Sequences within the nonpathogenic clade were more similar, and in most cases < or =10 bp distinguished these species. These sequences provide a reference standard for identification of Leptospira species and confirm previously established relationships within the genus. 16S rRNA gene sequencing is a powerful method for identification in the clinical laboratory and offers a simplified approach to the identification of Leptospira species.     

Species-specific identification of campylobacters by partial 16S rRNA gene sequencing

Species-specific identification of campylobacters is problematic, primarily due to the absence of suitable biochemical assays and the existence of atypical strains. 16S rRNA gene (16S rDNA)-based identification of bacteria offers a possible alternative when phenotypic tests fail. Therefore, we evaluated the reliability of 16S rDNA sequencing for the species-specific identification of campylobacters. Sequence analyses were performed by using almost 94% of the complete 16S rRNA genes of 135 phenotypically characterized Campylobacter strains, including all known taxa of this genus. It was shown that 16S rDNA analysis enables specific identification of most Campylobacter species. The exception was a lack of discrimination among the taxa Campylobacter jejuni and C. coli and atypical C. lari strains, which shared identical or nearly identical 16S rDNA sequences. Subsequently, it was investigated whether partial 16S rDNA sequences are sufficient to determine species identity. Sequence alignments led to the identification of four 16S rDNA regions with high degrees of interspecies variation but with highly conserved sequence patterns within the respective species. A simple protocol based on the analysis of these sequence patterns was developed, which enabled the unambiguous identification of the majority of Campylobacter species. We recommend 16S rDNA sequence analysis as an effective, rapid procedure for the specific identification of campylobacters.     

Identification of nocardia species by restriction endonuclease analysis of an amplified portion of the 16S rRNA gene

Identification of clinical isolates of Nocardia to the species level is important for defining the spectrum of disease produced by each species and for predicting antimicrobial susceptibility. We evaluated the usefulness of PCR amplification of a portion of the Nocardia 16S rRNA gene and subsequent restriction endonuclease analysis (REA) for species identification. Unique restriction fragment length polymorphism (RFLP) patterns were found for Nocardia sp. type strains (except for the N. asteroides type strain) and representative isolates of the drug pattern types of Nocardia asteroides (except for N. asteroides drug pattern type IV, which gave inconsistent amplification). A variant RFLP pattern for Nocardia nova was also observed. Twenty-eight clinical isolates were evaluated both by traditional biochemical identification and by amplification and REA of portions of the 16S rRNA gene and the 65-kDa heat shock protein (HSP) gene. There was complete agreement among the three methods on identification of 24 of these isolates. One isolate gave a 16S rRNA RFLP pattern consistent with the biochemical identification but was not identifiable by its HSP gene RFLP patterns. Three isolates gave 16S rRNA RFLP patterns which were inconsistent with the identification obtained by both biochemical tests and HSP gene RFLP; sequence analysis suggested that two of these isolates may belong to undefined species. The PCR and REA technique described appears useful both for the identification of clinical isolates of Nocardia and for the detection of new or unusual species.     

Sequence polymorphism of the 16S rRNA gene of Vibrio vulnificus is a possible indicator of strain virulence

Vibrio vulnificus exhibits considerable strain-to-strain variation in virulence. Attempts to associate phenotypic or genotypic characteristics with strain virulence have been largely unsuccessful. Based on a 17-nucleotide difference throughout the sequence of the small subunit 16S rRNA gene, there are two major groups of V. vulnificus designated types A and B. In a survey of the 16S rRNA genotype in 67 V. vulnificus human clinical and nonclinical strains, we determined that the majority of nonclinical isolates are type A (31 of 33) and that there is a statistically significant association between the type B genotype and human clinical strains (26 of 34).     

Use of 16S rRNA gene sequencing for rapid confirmatory identification of Brucella isolates

Members of the genus Brucella are categorized as biothreat agents and pose a hazard for both humans and animals. Current identification methods rely on biochemical tests that may require up to 7 days for results. We sequenced the 16S rRNA genes of 65 Brucella strains along with 17 related strains likely to present a differential diagnostic challenge. All Brucella 16S rRNA gene sequences were determined to be identical and were clearly different from the 17 related strains, suggesting that 16S rRNA gene sequencing is a reliable tool for rapid genus-level identification of Brucella spp. and their differentiation from closely related organisms.     

鲍曼不动杆菌armA基因的分布与耐药性的研究

目的 调查我院鲍曼不动杆菌中16S rRNA甲基化基因armA的分布以及与鲍曼不动杆菌耐药谱的关系,并初步探讨其在分子流行病学分析中的作用.方法 收集72株鲍曼不动杆菌,采用K-B法对鲍曼不动杆菌进行药物敏感试验,后采用PCR筛选鲍曼不动杆菌的16S rRNA甲基化基因armA,并利用随机扩增多态性DNA法(RAPD)技术进行基因分型.统计各鲍曼不动杆菌菌株对多种氨基糖苷类药物的药敏结果,并分析基因型与耐药性的关系.结果 根据PCR产物片段大小,72株鲍曼不动杆菌共有armA基因阳性菌株20株(27.8%).含有armA基因型鲍曼不动杆菌菌株对庆大霉素、妥布霉素和阿米卡星的耐药率均为90%;随机扩增多态性DNA法显示20株armA基因阳性的鲍曼不动杆菌主要分为7型,A型为优势克隆株.结论 产16S rRNA甲基化基因armA的鲍曼不动杆菌菌株可对多种氨基糖苷类抗生素高水平耐药.同一克隆菌株在病房内和病房间的传播为我院armA基因传播的主要方式.     

Use of 16S rRNA, 23S rRNA, and gyrB gene sequence analysis to determine phylogenetic relationships of Bacillus cereus group microorganisms

In order to determine if variations in rRNA sequence could be used for discrimination of the members of the Bacillus cereus group, we analyzed 183 16S rRNA and 74 23S rRNA sequences for all species in the B. cereus group. We also analyzed 30 gyrB sequences for B. cereus group strains with published 16S rRNA sequences. Our findings indicated that the three most common species of the B. cereus group, B. cereus, Bacillus thuringiensis, and Bacillus mycoides, were each heterogeneous in all three gene sequences, while all analyzed strains of Bacillus anthracis were found to be homogeneous. Based on analysis of 16S and 23S rRNA sequence variations, the microorganisms within the B. cereus group were divided into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, and these seven subgroups were further organized into two distinct clusters. This classification of the B. cereus group conflicts with current taxonomic groupings, which are based on phenotypic traits. The presence of B. cereus strains in six of the seven subgroups and the presence of B. thuringiensis strains in three of the subgroups do not support the proposed unification of B. cereus and B. thuringiensis into one species. Analysis of the available phenotypic data for the strains included in this study revealed phenotypic traits that may be characteristic of several of the subgroups. Finally, our results demonstrated that rRNA and gyrB sequences may be used for discriminating B. anthracis from other microorganisms in the B. cereus group.     

Genetic variation of the ompA and 16S rRNA genes of Riemerella anatipestifer.

The genetic diversity of the 16S rRNA and ompA genes of Riemerella anatipestifer was investigated. A 16S rRNA gene-based PCR was able to amplify all 18 Taiwanese strains and 10 reference strains. The identity of 16S rRNA sequence of these strains and seven other sequences retrieved from GenBank was 95.0-100.0%. The percentage identity of the ompA sequence of the 15 Taiwanese strains and eight reference strains amplified in this study and two other sequences retrieved from GenBank was 88.1-100.0%. Phylogenetic analysis based on the 16S rRNA gene showed that all the R. anatipestifer strains fell into a single cluster. It is concluded that the 16S rRNA gene-based PCR is suitable for the screening of R. anatipestifer infections. Phylogenetic analysis of the ompA of R. anatipestifer resulted in three different clusters, while seven clusters were found when the derived amino acid sequence was the basis of analysis. No apparent cluster was found using the criteria of host, isolate serotype, the year or location of isolation.     

Molecular epidemiology of Ornithobacterium rhinotracheale.

Ornithobacterium rhinotracheale is a recently described gram-negative rod-shaped bacterium associated with respiratory tract infections in poultry. In order to determine the molecular epidemiology of this bacterium, we characterized 55 O. rhinotracheale isolates from eight countries on four continents by multilocus enzyme electrophoresis (MLEE), repetitive sequence based-PCR (rep-PCR), and 16S rRNA gene sequencing. MLEE discriminated the O. rhinotracheale isolates into six electrophoretic types (ETs), of which only three ETs were recovered from domesticated poultry. The 16S rRNA gene sequence and rep-PCR analyses confirmed the results obtained by MLEE and indicated limited heterogeneity among isolates of O. rhinotracheale recovered from poultry. Taken together, the results of our analysis demonstrate that the majority of O. rhinotracheale isolates recovered from domesticated poultry throughout the world are represented by a small group of closely related clones and suggest that the bacterium was recently introduced to domesticated poultry from wild bird populations.     

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.     

Use of 16S rRNA gene sequencing for rapid identification and differentiation of Burkholderia pseudomallei and B. mallei

Burkholderia pseudomallei and B. mallei, the causative agents of melioidosis and glanders, respectively, are designated category B biothreat agents. Current methods for identifying these organisms rely on their phenotypic characteristics and an extensive set of biochemical reactions. We evaluated the use of 16S rRNA gene sequencing to rapidly identify these two species and differentiate them from each other as well as from closely related species and genera such as Pandoraea spp., Ralstonia spp., Burkholderia gladioli, Burkholderia cepacia, Burkholderia thailandensis, and Pseudomonas aeruginosa. We sequenced the 1.5-kb 16S rRNA gene of 56 B. pseudomallei and 23 B. mallei isolates selected to represent a wide range of temporal, geographic, and origin diversity. Among all 79 isolates, a total of 11 16S types were found based on eight positions of difference. Nine 16S types were identified in B. pseudomallei isolates based on six positions of difference, with differences ranging from 0.5 to 1.5 bp. Twenty-two of 23 B. mallei isolates showed 16S rRNA gene sequence identity and were designated 16S type 10, whereas the remaining isolate was designated type 11. This report provides a basis for rapidly identifying and differentiating B. pseudomallei and B. mallei by molecular methods.