Genetic structure of Neisseria meningitidis populations in relation to serogroup, serotype, and outer membrane protein pattern.

The genetic structure of populations of Neisseria meningitidis was examined by an analysis of electrophoretically demonstrable allelic variation at 15 genes encoding enzymes in 650 isolates of eight serogroups (A, B, C, W135, X, Y, Z, and 29E) and 38 nonserogroupable isolates. A total of 331 distinctive multilocus genotypes (electrophoretic types, ETs) was identified, among which mean genetic diversity per locus (H = 0.547) was greater than in Escherichia coli and other bacterial species thus far studied. The intercontinental distribution of some ETs and the recovery of organisms of identical genotype over periods of many years strongly suggest that the genetic structure of N. meningitidis is basically clonal as a consequence of low rates of recombination of chromosomal genes. Variation among strains in serogroup, serotype, and the electrophoretic pattern of the major outer membrane proteins has little relationship to the complex structure of populations revealed by enzyme electrophoresis, which involves 14 major lineages of clones diverging from one another at genetic distances greater than 0.50. Genetic diversity among ETs of isolates of the same serogroup was, on average, 84% of that in the total sample. Clones of serogroup A were unusual in being genotypically less heterogeneous than those of other serogroups and in forming a single phylogenetic group. Isolates of the same serotype or outer membrane protein pattern were also highly heterogeneous; on average, 87 and 97%, respectively, of the total species diversity was represented by ETs of the same serotype or outer membrane protein.     

Genetic relatedness amongst intestinal spirochaetes isolated from rate and brids.

Multilocus enzyme electrophoresis was used to determine genetic relationships amongst 32 intestinal sprrochaetes ( Serpulina spp.) isolated from rats (17), rheas (7), chickens (4), ducks (2), a swan (1) and a flamingo (1). The strains were divided into 20 electrophoretic types (ETs), with a mean genetic diversity per locus of 0.62. The results were compared with those previously published for procine intestinal spirochaetes. One strain from a healthy rat, and three rhea strains which were recovered from cases of necrotizing typhlitis, were grouped in the same ETs as certain procine strains of Serpulina hydysenteriae . The rhea strains could be differentiated from these by pulsed-field gel electrophoresis. Fifteen of the rat strains could be differentiated from these by pulsed-field gel electrophoresis. Fifteen of the rat strains were genetically and phenotypically closely related. In contrast the avian strains were genetically more heterogeneous, with pathogenic isolates located in three different genetic groups.     

The panmictic nature of Neisseria meningitidis serogroup B during a period of endemic disease in Canada

Three hundred and one (301) strains of Neisseria meningitidis serogroup B, isolated from patients with meningococcal disease during the years 1994-1996, were subjected to multilocus enzyme electrophoresis, serotyping, and serosubtyping. Based on the analyses of 14 enzyme loci, 177 electrophoretic types (ETs) were identified. Of these, 136 were represented by single isolates and 41 were represented by multiple isolates (range 2-31). The mean genetic diversity for isolates was 0.444 and for ETs was 0.440. The index of association (I(A)) between loci was 0.530 +/- 0.08 for isolates and 0.256 +/- 0.10 for ETs. Cluster analysis revealed the presence of 39 lineages each represented by a single ET or clusters of ETs. The most common serotypes were 4, 15, and 14 and accounted for 84 (28.0%), 53 (17.6%), and 32 (10.6%) of the isolates, respectively, and were dispersed amongst 46 ETs (1-122), 35 ETs (3-165), and 26 ETs (18-76), respectively. The 109 (36.6%) nontypable (NT) isolates were amongst 74 ETs (6-177). The mean genetic diversity for serotypes 4, 15, and 14 and NT isolates was 0.368, 0.371, 0.343, and 0.442, respectively, and for ETs was 0.363, 0.354, 0.397, and 0.440, respectively. Combinations of serotypes and serosubtypes (number of isolates) that occurred most frequently were 4:P1.14 (17), 14:P1.16 (16), NT:P1.16 (16), 15:P1.16 (13), and NT:P1.13 (13). The majority of group B disease in Canada during 1994-1996 was caused by meningococci of considerable genetic diversity, and reflects a situation of endemic disease. However, the results also indicate that organisms belonging to the ET-5 complex, which has been responsible for outbreaks of group B disease globally for several decades, have been introduced into the country.     

Reference collection of strains of the Salmonella typhimurium complex from natural populations

A collection of 72 reference strains of the Salmonella typhimurium complex of clones recovered from a variety of hosts and environmental sources in diverse geographic locations has been established for use in studies of variation in natural populations. Included are strains of the serovars S. typhimurium, S. saintpaul, S. heidelberg, S. paratyphi B (including variety java) and S. muenchen. The strains, which have been characterized by enzyme electrophoresis for allelic variation in 24 chromosomal structural genes and represent 48 distinctive multilocus genotypes (electrophoretic types or ETs), exemplify the full range of genotypic variation in the S. typhimurium complex. Evolutionary genetic relationships among the ETs are indicated in a phylogenetic tree generated by the neighbour-joining method from a matrix of Nei's standard genetic distance.     

Failure of a diagnostic monoclonal immunofluorescent reagent to detect Legionella pneumophila in environmental samples.

Three commercial diagnostic fluorescein-labeled antibodies, one monoclonal and two polyclonal, were compared to evaluate their abilities to detect Legionella pneumophila in environmental samples. The monoclonal conjugate failed to detect L. pneumophila in the 12 environmental samples studied by direct immunofluorescence. In contrast, the two polyclonal conjugates detected L. pneumophila in all 12 samples by both direct and indirect immunofluorescence. However, isolates recovered by culture from the 12 samples demonstrated equal immunofluorescence with all three conjugates. The reason for the failure of the monoclonal antibody to detect L. pneumophila in the environmental samples remains unknown. Laboratories considering the use of the monoclonal conjugate to screen environmental samples for L. pneumophila should be aware of this finding.     

Failure of a diagnostic monoclonal immunofluorescent reagent to detect Legionella pneumophila in environmental samples

Three commercial diagnostic fluorescein-labeled antibodies, one monoclonal and two polyclonal, were compared to evaluate their abilities to detect Legionella pneumophila in environmental samples. The monoclonal conjugate failed to detect L. pneumophila in the 12 environmental samples studied by direct immunofluorescence. In contrast, the two polyclonal conjugates detected L. pneumophila in all 12 samples by both direct and indirect immunofluorescence. However, isolates recovered by culture from the 12 samples demonstrated equal immunofluorescence with all three conjugates. The reason for the failure of the monoclonal antibody to detect L. pneumophila in the environmental samples remains unknown. Laboratories considering the use of the monoclonal conjugate to screen environmental samples for L. pneumophila should be aware of this finding.     

Multilocus genotypes determined by enzyme electrophoresis of Neisseria meningitidis isolated from patients with systemic disease and from healthy carriers

Variation in nine enzymes in 152 isolates of Neisseria meningitidis from Norway (118 from blood or cerebrospinal fluid of patients with systemic disease and 34 from the pharynx of healthy carriers) was analysed by starch-gel electrophoresis. All nine enzymes were polymorphic and the number of allozymes (electromorphs) identified per locus ranged from 3 to 12, with a mean of 6.1. Among the 152 isolates, 55 unique combinations of electromorphs (electrophoretic types, ETs) were distinguished. Twenty ETs were represented among the carrier isolates and 37 among the systemic isolates; hence, only two ETs were found in both groups of isolates. ET-5 was identified 67 times among the 118 systemic isolates (58%), indicating an association of this ET with invasiveness; ET-5 was also the most common type among the carrier isolates (18%). Genetic similarity between ETs was analysed by pairwise comparison of all 55 ETs with respect to the number of electromorphs by which they differed. No evidence of a general genetic difference between carrier and case isolates was found. Two well-defined clusters of ETs were observed, each including one of the two most common ETs identified among the systemic isolates (ET-5 and ET-37), together with isolates differing from them only at one or two loci. All isolates of ET-5 and ET-37, as well as their closely related variants defined by the similarity matrix, were resistant to sulphonamide, independent of their antigenic characteristics and isolation site. The extensive allozyme variation among isolates of the same serogroup demonstrated the limited value of serogrouping as an epidemiological tool. All but one isolate of serotype 15:P1.16 were electrophoretically similar, as were all the 2a:P1.2 isolates. The 15:P1.15 isolates, however, were genetically heterogeneous. The distribution of alleles in genotypes identified among the systemic isolates indicated that genetic recombination may occur in natural populations of N. meningitidis.     

Genetic Structure and Symbiotic Characteristics of a Bradyrhizobium Population Recovered from a Pasture Soil

We examined the genetic structure and symbiotic characteristics of Bradyrhizobium isolates recovered from four legume species (Lupinus albus [white lupine], Lupinus angustifolius [blue lupine], Ornithopus compressus [yellow serradella], and Macroptilium atropurpureum [sirato]) grown in an Oregon soil. We established that multilocus enzyme electrophoresis (MLEE) can provide insights into the genetic relatedness among Bradyrhizobium strains by showing a positive correlation (r² = ≥0.90) between the relatedness of Bradyrhizobium japonicum strains determined by MLEE at 13 enzyme loci and that determined by other workers using either DNA-DNA hybridization or DNA sequence divergence estimates. MLEE identified 17 electrophoretic types (ETs) among 95 Bradyrhizobium isolates recovered from the four hosts. Although the overall genetic diversity among the ETs (H = 0.69) is one of the largest measured to date in a local population of any soilborne bacterial species, there was no evidence of multilocus structure (linkage disequilibrium) within the population. The majority of the isolates (73%) were represented by two closely related ETs (2 and 3) which dominated the root nodules of white lupine, serradella, and siratro. In contrast, ET1 dominated nodules of blue lupine. Although representative isolates from all of the 17 ETs nodulated siratro, white lupine, blue lupine, and big trefoil (Lotus pedunculatus), they were either completely ineffective or poorly effective at fixing nitrogen on these hosts. Despite the widespread use of serradella as a surrogate host for lupine-nodulating bradyrhizobia, 7 of the 17 ETs did not nodulate this host, and the remaining 10 ETs were ineffective at fixing nitrogen.     

Genetic Structure of Acetobacter diazotrophicus Populations and Identification of a New Genetically Distant Group

A total of 55 isolates of Acetobacter diazotrophicus recovered from diverse sucrose-rich host plants and from mealybugs associated with sugarcane plants were characterized by the electrophoretic mobilities of 12 metabolic enzymes. We identified seven different electrophoretic types (ETs), six of which are closely related within a genetic distance of 0.195 and exhibit high DNA-DNA homology. The seventh ET was largely divergent, separated at a genetic distance of 0.53, and had only 54% DNA homology to the reference strain. Strains corresponding to ET 7 could represent a distinct nitrogen-fixing species of the genus Acetobacter. More genetic diversity was found in isolates from Brazil than in those from Mexico, probably due to the very different crop nitrogen fertilization levels used.     

Genetic characterization of Legionella pneumophila serogroup 1 associated with respiratory disease in Australia.

Techniques were developed for genetic characterization of Legionella pneumophila serogroup 1 by using restriction fragment length polymorphism analysis. Allozyme analysis provided an index of the discrimination achieved by restriction fragment length polymorphism. Isolates from human cases of legionellosis were examined by both methods, and their profiles were compared with reference strains of L. pneumophila serogroup 1 obtained from the American Type Culture Collection. Eighteen distinct clones were evident among the isolates examined. Both methods could be used to trace the source of an outbreak of legionellosis caused by L. pneumophila serogroup 1.     

Genetic characterization of Legionella pneumophila serogroup 1 associated with respiratory disease in Australia.

Techniques were developed for genetic characterization of Legionella pneumophila serogroup 1 by using restriction fragment length polymorphism analysis. Allozyme analysis provided an index of the discrimination achieved by restriction fragment length polymorphism. Isolates from human cases of legionellosis were examined by both methods, and their profiles were compared with reference strains of L. pneumophila serogroup 1 obtained from the American Type Culture Collection. Eighteen distinct clones were evident among the isolates examined. Both methods could be used to trace the source of an outbreak of legionellosis caused by L. pneumophila serogroup 1.     

Characterization of urease-positive thermophilic Campylobacter subspecies by multilocus enzyme electrophoresis typing

Thirty-one urease-positive thermophilic Campylobacter (UPTC) isolates, including three reference strains (NCTC12892, NCTC12895 and NCTC12896), and three Campylobacter lari isolates, which were isolated from several countries and sources, were compared genotypically by using multilocus enzyme electrophoresis (MLEE). We examined allelic variation around seven enzyme loci, including the adenylate kinase, alkaline phosphatase, catalase, fumarase, malic enzyme, malate dehydrogenase, and L-phenylalanyl-L-leucine peptidase loci. MLEE typing revealed the presence of 23 different electrophoretic types (ETs) among the 31 UPTC isolates, and 14 isolates shared six electrophoretic profiles. Three different ETs were identified for the three C. lari isolates examined, and no ETs were shared by UPTC and C. lari isolates. Quantitative analyses were subsequently performed by using allelic variation data, and the results demonstrated that the mean genetic diversity was 0.655. In conclusion, MLEE demonstrated that the UPTC isolates examined are genetically hypervariable and form a cluster separate from the C. lari cluster.     

Distribution of Legionella spp. in hydrothermal areas in continental Portugal and the island of São Miguel, Azores.

Nineteen aquatic environment sites from three hydrothermal areas on continental Portugal and one area on the island of São Miguel, Azores, were examined for the recovery of Legionella spp. Physicochemical and bacteriological parameters were also determined for each site. Water temperatures varied between 22 and 67.5 degrees C, although the majority had temperatures above 40 degrees C; the pH varied between 5.5 and 9.2. The number of Legionella spp. recovered varied between 5.0 x 10(2) and 2.3 x 10(6) CFU/liter. A total of 288 isolates from 14 sites were identified by indirect immunofluorescence assay. The majority of the isolates belonged to Legionella pneumophila (74.3%), of which most belong to serogroup 1, but the relative proportion of L. pneumophila serogroups varied considerably. L. pneumophila serogroup 1 constituted 96.2% of the isolates in area 2 from central Portugal, but no isolates of this serogroup were recovered from São Miguel, where serogroup 6 strains were the predominant isolates. Ninety-six percent of the L. pneumophila serogroup 1 isolates belonged to monoclonal antibody subgroups OLDA and Bellingham. Other species identified were L. bozemanii serogroup 2, L. dumoffii, L. micdadei, L. moravica, L. oakridgensis, L. sainticrucis, and L. sainthelensi. Two undescribed species, which react by indirect immunofluorescence assay to antisera to "L. londoniensis" and "L. nautarum" and a group of isolates with strong cross-reaction to L. cincinnatiensis/L. sainticrucis/L. longbeachae by indirect immunofluorescence assay were also recovered. The latter were the only isolates recovered from area 3, in east central Portugal, over a period of 1 year.     

Genetic diversity and relationships among isolates of Rhizobium leguminosarum biovar phaseoli

Fifty-one isolates of Rhizobium leguminosarum biovar phaseoli from various geographic and ecological sources, largely in Mexico, were characterized by the electrophoretic mobilities of 15 metabolic enzymes, and 46 distinctive multilocus genotypes (electrophoretic types [ETs]) were distinguished on the basis of allele profiles at the enzyme loci. Mean genetic diversity per enzyme locus among the 46 ETs was 0.691, the highest value yet recorded for any species of bacterium. The occurrence of strong nonrandom associations of alleles over loci suggested a basically clonal population structure, reflecting infrequent recombination of chromosomal genes. Multilocus genotypic diversity was unusually high, with the most strongly differentiated pairs of ETs having distinctive alleles at all 15 loci and major clusters of ETs diverging at genetic distances as large as 0.89. This great diversity in the chromosomal genome raises the possibility that R. leguminosarum biovar phaseoli is a polyphyletic assemblage of strains. As other workers have suggested, the inclusion of all strains capable of nodulating beans in a single biovar or species is genetically unrealistic and taxonomically misleading. A biologically meaningful classification of Rhizobium spp. should be based on assessment of variation in the chromosomal genome rather than on phenotypic characters, especially those mediated for the most part or wholly by plasmid-borne genes, such as host relationships.     

Urogenital, maternal and neonatal isolates of Haemophilus influenzae: identification of unusually virulent serologically non-typable clone families and evidence for a new Haemophilus species

A collection of 117 strains of Haemophilus influenzae, including 112 non-typable isolates recovered predominantly in the USA and France from genital, obstetric and neonatal sources, was characterized by the electrophoretic mobilities of 10 metabolic enzymes. Eighty-six distinctive multilocus chromosomal genotypes (electrophoretic types, ETs) were distinguished on the basis of allele profiles at the enzyme loci. Isolates of five allied biotype IV ETs were highly divergent from all other strains and hybridization of chromosomal DNA revealed that they undoubtedly represent a previously unrecognized species of Haemophilus. Isolates representing these ETs were recovered predominantly from obstetric infections and serious neonatal diseases and apparently possess specific tropism for the genital tract. Strains of these five ETs were present in samples from both the USA and France, but only in the USA did they cause bacteraemia and meningitis, an occurrence which probably reflects differences in patient management between the two countries. Although strains assigned to H. influenzae (sensu stricto) were strongly polymorphic in multilocus enzyme genotype, 69% of isolates recovered from patients with meningitis and/or septicaemia were assigned to only two clone families, a result suggesting that some serologically nontypable strains of H. influenzae originating from the genital tract are unusually virulent.     

Distribution of Legionella spp. in hydrothermal areas in continental Portugal and the island of S?o Miguel, Azores.

Nineteen aquatic environment sites from three hydrothermal areas on continental Portugal and one area on the island of S?o Miguel, Azores, were examined for the recovery of Legionella spp. Physicochemical and bacteriological parameters were also determined for each site. Water temperatures varied between 22 and 67.5 degrees C, although the majority had temperatures above 40 degrees C; the pH varied between 5.5 and 9.2. The number of Legionella spp. recovered varied between 5.0 x 10(2) and 2.3 x 10(6) CFU/liter. A total of 288 isolates from 14 sites were identified by indirect immunofluorescence assay. The majority of the isolates belonged to Legionella pneumophila (74.3%), of which most belong to serogroup 1, but the relative proportion of L. pneumophila serogroups varied considerably. L. pneumophila serogroup 1 constituted 96.2% of the isolates in area 2 from central Portugal, but no isolates of this serogroup were recovered from S?o Miguel, where serogroup 6 strains were the predominant isolates. Ninety-six percent of the L. pneumophila serogroup 1 isolates belonged to monoclonal antibody subgroups OLDA and Bellingham. Other species identified were L. bozemanii serogroup 2, L. dumoffii, L. micdadei, L. moravica, L. oakridgensis, L. sainticrucis, and L. sainthelensi. Two undescribed species, which react by indirect immunofluorescence assay to antisera to "L. londoniensis" and "L. nautarum" and a group of isolates with strong cross-reaction to L. cincinnatiensis/L. sainticrucis/L. longbeachae by indirect immunofluorescence assay were also recovered. The latter were the only isolates recovered from area 3, in east central Portugal, over a period of 1 year.     

Analysis of clinical and food-borne isolates of Listeria monocytogenes in the United States by multilocus enzyme electrophoresis and application of the method to epidemiologic investigations

To investigate the microbiology and epidemiology of the 1,700 sporadic cases of listeriosis that occur annually in the United States, we developed a multilocus enzyme electrophoresis (MEE) typing system for Listeria monocytogenes. We studied 390 isolates by MEE. Eighty-two electrophoretic types (ETs) were defined. Two distinct clusters of ETs, ET group A (ETGA) and ET group B (ETGB), separated at a genetic distance of 0.440, were identified. Strains of ETGB were associated with perinatal listeriosis (P = 0.03). All strains of H antigen type a were in ETGA, while all strains of H antigen type b were in ETGB. Among 328 clinical isolates from cases of literiosis, 55 ETs of L. monocytogenes were defined. Thirty-four ETs were identified among 62 isolates from food products. The mean number of strains per ET (5.2) was significantly higher among clinical isolates than among food-borne isolates. Examination of isolates from outbreaks further documented the link between cases and contaminated food products. In one investigation, we found 11 different ETs, ruling out a single common source as a cause of that outbreak. By examining a large number of isolates collected over a specified time in diverse geographic locations in the United States, we have begun to establish a baseline for the study of the epidemiology of listeriosis by MEE.     

Analysis of clinical and food-borne isolates of Listeria monocytogenes in the United States by multilocus enzyme electrophoresis and application of the method to epidemiologic investigations.

To investigate the microbiology and epidemiology of the 1,700 sporadic cases of listeriosis that occur annually in the United States, we developed a multilocus enzyme electrophoresis (MEE) typing system for Listeria monocytogenes. We studied 390 isolates by MEE. Eighty-two electrophoretic types (ETs) were defined. Two distinct clusters of ETs, ET group A (ETGA) and ET group B (ETGB), separated at a genetic distance of 0.440, were identified. Strains of ETGB were associated with perinatal listeriosis (P = 0.03). All strains of H antigen type a were in ETGA, while all strains of H antigen type b were in ETGB. Among 328 clinical isolates from cases of literiosis, 55 ETs of L. monocytogenes were defined. Thirty-four ETs were identified among 62 isolates from food products. The mean number of strains per ET (5.2) was significantly higher among clinical isolates than among food-borne isolates. Examination of isolates from outbreaks further documented the link between cases and contaminated food products. In one investigation, we found 11 different ETs, ruling out a single common source as a cause of that outbreak. By examining a large number of isolates collected over a specified time in diverse geographic locations in the United States, we have begun to establish a baseline for the study of the epidemiology of listeriosis by MEE.     

Diversity and genetic structure of a natural population of Rhizobium leguminosarum bv. trifolii isolated from Trifolium subterraneum L.

A collection of 121 isolates of Rhizobium leguminosarum biovar (bv.) trifolii was obtained from root nodules of Trifolium subterraneum L. (subclover) plants growing in an established pasture. The collection consisted of a single isolate from each of 18 plants sampled from seven microplots. The following year, a further 28 and 27 isolates were collected from the first and seventh sampling points, respectively. Analysis of restriction fragment length polymorphisms (RFLPs) of both chromosomal and Sym (symbiotic) plasmid DNA and multilocus enzyme electrophoresis (MLEE) were used to assess the diversity, genetic relationships and structure of this population. Symbiotic effectiveness tests were used to examine the symbiotic phenotype of each isolate collected in the first year. Analysis of RFLPs of the first year isolates revealed 13 chromosomal types and 25 Sym plasmid types. Similar Sym plasmid types were grouped into 14 families containing 1–6 members. No new chromosomal types and six new Sym plasmid types were detected in the second year. The symbiotic effectiveness of the first year isolates of the same Sym plasmid type was similar. Significant differences in symbiotic effectiveness were detected between different Sym plasmid types in the same plasmid family. Representative isolates of each chromosomal type Sym plasmid type identified in the first year were analysed using multilocus enzyme electrophoresis. Mean genetic diversity per locus was high (0.559). Enzyme electrophoresis revealed 17 electrophoretic types (ETs). Ouster analysis of the enzyme data revealed large genetic diversity amongst the ETs. Strong linkage disequilibrium was observed for the population as a whole, i.e. clonal population structure, but significantly less disequilibrium was observed among a cluster of ETs suggesting that recombination occurred between ETs within the cluster. Our results revealed that a population of naturally occurring isolates of Rhizobium leguminosarum bv. trifolii can be genetically diverse and support the possibility that recombination plays a role in generating new genotypes.     

Genetic Diversity of Escherichia coli Isolated from Urban Rivers and Beach Water

Repetitive element anchored PCR was used to evaluate the genetic profiles of Escherichia coli isolated from surface water contaminated with urban stormwater, sanitary sewage, and gull feces to determine if strains found in environmental samples reflect the strain composition of E. coli obtained from host sources. Overall, there was less diversity in isolates collected from river and beach sites than with isolates obtained from human and nonhuman sources. Unique strain types comprised 28.8, 29.2, and 15.0% of the isolate data sets recovered from stormwater, river water, and beach water, respectively. In contrast, 50.4% of gull isolates and 41.2% of sewage isolates were unique strain types. River water, which is expected to contain E. coli strains from many diffuse sources of nonpoint source pollution, contained strains most closely associated with other river water isolates that were collected at different sites or on different days. However, river sites impacted by sewage discharge had approximately 20% more strains similar to sewage isolates than did sites impacted by stormwater alone. Beach sites with known gull fecal contamination contained E. coli most similar to other beach isolates rather than gull isolates collected at these same sites, indicating underrepresentation of possible gull strains. These results suggest large numbers of strains are needed to represent contributing host sources within a geographical location. Additionally, environmental survival may influence the composition of strains that can be recovered from contaminated waters. Understanding the ecology of indicator bacteria is important when interpreting fecal pollution assessments and developing source detection methodology.