Rapid detection of Salmonella enterica with primers specific for iroB.

The iroB gene of Salmonella enterica is absent from the chromosome of the related organism Escherichia coli. We determined the distribution of this gene among 150 bacterial isolates, representing 51 serotypes of different Salmonella species and subspecies and 8 other bacterial species which are frequent contaminants during routine enrichment procedures by Southern hybridization. An iroB-specific DNA probe detected homologous sequences in all strains of S. enterica, including serotypes of S. enterica subsp. enterica (I), salamae (II), diarizonae (IIIb), and houtenae (IV). No hybridization signal was obtained with strains of Salmonella bongori or other bacterial species. In contrast, hybridization with a DNA probe specific for purD, a purine biosynthesis gene, detected homologs in all bacterial species tested. Primers specific for iroB were used to amplify this gene from 197 bacterial isolates by PCR. The iroB gene could be PCR amplified from S. enterica subsp. enterica (I), salamae (II), diarizonae (IIIb), houtenae (IV), arizonae (IIIa), and indica (VI), but not from S. bongori or other bacterial species. Thus, PCR amplification of iroB can be used to distinguish between S. enterica and other bacterial species, including S. bongori. A combination of preenrichment in buffered peptone water supplemented with ferrioxamine E and amplification of iroB by magnetic immuno-PCR allowed detection of S. enterica in albumen within 24 h. In conclusion, PCR amplification of iroB is a new sensitive and selective method which has the potential to rapidly detect S. enterica serotypes.     

Clonal diversity in Haemophilus pleuropneumoniae.

Genetic diversity among 135 isolates of nine serotypes of Haemophilus pleuropneumoniae recovered from pigs with pleuropneumonia or other invasive diseases in 14 countries was estimated by multilocus enzyme electrophoresis, which detects allelic variation in structural genes. Thirty-two multilocus genotypes (electrophoretic types [ETs]) were distinguished on the basis of allele profiles at 15 enzyme loci, and 36 distinctive combinations of ET and serotype were identified. The recovery of isolates with identical properties in widely separated geographic regions and over a 20-year period indicated that the population structure of H. pleuropneumoniae is clonal. Isolates of the same ET generally shared the same serotype and electrophoretic pattern of the outer membrane proteins, but some ETs were represented by isolates of several different serotypes, outer membrane protein patterns, or both. On average, the genetic diversity among ETs of the same serotype was 56% of the total genetic diversity in the species. Isolates of serotype 1, which are unusually pathogenic, belong to a distinctive group of clones that are closely related to clones marked by serotype 9.     

Genetic structure of population of Bacillus cereus and B. thuringiensis isolates associated with periodontitis and other human infections

The genetic diversity and relationships among 35 Bacillus cereus and Bacillus thuringiensis isolates recovered from marginal and apical periodontitis in humans and from various other human infections were investigated using multilocus enzyme electrophoresis. The strains were isolated in Norway, except for three strains isolated from periodontitis patients in Brazil. The genetic diversity of these strains was compared to that of 30 isolates from dairies in Norway and Finland. Allelic variation in 13 structural gene loci encoding metabolic enzymes was analyzed. Twelve of the 13 loci were polymorphic, and 48 unique electrophoretic types (ETs) were identified, representing multilocus genotypes. The mean genetic diversity among the 48 genotypes was 0.508. The genetic diversity of each source group of isolates varied from 0.241 (periodontal infection) to 0.534 (dairy). Cluster analysis revealed two major groups separated at a genetic distance of greater than 0.6. One cluster, ETs 1 to 13, included solely isolates from dairies, while the other cluster, ETs 14 to 49, included all of the human isolates as well as isolates from dairies in Norway and Finland. The isolates were serotyped using antiflagellar antiserum. A total of 14 distinct serotypes were observed. However, little association between serotyping and genotyping was seen. Most of the strains were also analyzed with pulsed-field gel electrophoresis, showing the presence of extrachromosomal DNA in the size range of 15 to 600 kb. Our results indicate a high degree of heterogeneity among dairy strains. In contrast, strains isolated from humans had their genotypes in one cluster. Most strains from patients with periodontitis belonged to a single lineage, suggesting that specific clones of B. cereus and B. thuringiensis are associated with oral infections.     

Molecular analysis of ampicillin-resistant sporadic Salmonella typhi and Salmonella paratyphi B clinical isolates

Objective: To study the mechanisms of antibiotic resistance in Salmonella typhi and Salmonella paratyphi B clinical isolates, and the clonality of resistant strains.
Method: Antibiotic susceptibility was tested by disk-agar diffusion. Conjugation experiments and plasmid analysis by agarose gel electrophoresis after Eco RI digestion were followed by hybridization to a digoxigenin-labeled TEM-type β-lactamase probe. DNA fingerprints were obtained by pulsed-field gel electrophoresis of Xba I-digested chromosomal DNA.
Results: Three S. typhi isolates (7% of the isolates studied), of which one was ampicillin resistant and the other two multiresistant (ampicillin, chloramphenicol, tetracycline, sulfamethoxazole/trimethoprim and streptomycin), and two ampicillin-resistant S. paratyphi B isolates (25% of the isolates studied) were further evaluated. A 34-MDa conjugative plasmid, previously isolated from Salmonella enteritidis , conferred ampicillin resistance. A 100-MDa conjugative plasmid encoded resistance to chloramphenicol, tetracycline and sulfamethoxazole/trimethoprim, as well as ampicillin. Chromosomal fingerprinting revealed two distinct resistant strains for each serovar which were different from a matched set of sensitive S. typhi strains.
Conclusions: Two conjugative, TEM-type β-lactamase-encoding plasmids conferred ampicillin resistance to S. typhi and S. paratyphi B. The 34-MDa plasmid was identical to that previously characterized from S. enteritidis , while the 100-MDa plasmid also encoded resistance to chloramphenicol, tetracycline and sulfamethoxazole/trimethoprim. Resistant isolates did not belong to a single clone but rather represented distinct strains.     

Characterization of isolates of Mycobacterium avium serotypes 4 and 8 from patients with AIDS by multilocus enzyme electrophoresis.

Isolates of Mycobacterium avium serotypes 4 and 8 originating from patients with AIDS in New York City, Los Angeles, or San Francisco were further characterized by multilocus enzyme electrophoresis. Reference strains used to produce typing antisera were also examined. Thirty-one electrophoretic types (ETs) were found among 58 isolates of serotype 4, while 10 ETs were identified among 21 isolates of serotype 8. One major ET was found within each serotype, and these two ETs were closely related, separated by a genetic distance of only 0.05. Six ETs were found in more than one city. In four cases, isolates of serotypes 4 and 8 shared the same ET. Multilocus enzyme electrophoresis in combination with serotyping should be helpful in locating the specific infection sources of these commonly isolated opportunistic pathogens.     

Persistent endemicity of Salmonella bongori 48:z(35):--in Southern Italy: molecular characterization of human,animal, and environmental isolates

From 1984 to 1999, we collected 31 isolates of the rare serovar Salmonella bongori 48:z(35):- in southern Italy. Twenty-four of the isolates were from cases of acute enteritis in humans. Pulsed-field gel electrophoresis analysis showed that all but one of our isolates were at least 80% similar. Our findings suggest that genetically related S. bongori 48:z(35):- strains are endemically circulating in southern Italy.     

Comparison of ribotyping and multilocus enzyme electrophoresis for subtyping of Listeria monocytogenes isolates.

Ribotyping was compared with multilocus enzyme electrophoresis (MEE) for subtyping 305 Listeria monocytogenes isolates from clinical and nonclinical sources. For ribotyping, EcoRI-restricted genomic DNA fragments of L. monocytogenes strains were separated by agarose gel electrophoresis, and Southern blots were probed with a cloned Escherichia coli rrnB operon (plasmid pKK3535) labeled with digoxigenin. The L. monocytogenes isolates were divided into 28 distinct ribotypes, while MEE analysis divided the same isolates into 78 electrophoretic types (ETs). On the basis of their ribotype profiles, the strains were divided into two subgroups. The ribotype alpha (RT alpha) subgroup contained serotypes 1/2a, 1/2c, and 3a, and the ribotype beta (RT beta) subgroup contained serotypes 1/2b, 3b, 4b, and 4ab. This division is in complete agreement with MEE analysis, which divides the species into two subgroups (ET groups A and B), with the same serotype distribution in each subgroup. Overall, MEE was more discriminating than ribotyping. However, in several instances ribotyping discriminated between isolates within the same ET. Ribotyping was more discriminating for serotypes 1/2a, 1/2c, and 3a (Simpson's Index for Diversity [DI] = 0.81) than for serotypes 1/2b and 4b (DI = 0.76). A substantial proportion (69%) of serotype 1/2b and 4b strains clustered in five ETs and five ribotypes. These data suggest that ribotyping and MEE do not provide adequate discrimination between strains of serotypes 1/2b and 4b. Methods such as pulsed-field gel electrophoresis and random amplified polymorphic DNA analysis should be explored for further discrimination of strains of these serotypes.     

Analysis of Salmonella typhi isolates from Southeast Asia by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) revealed that multiple genetic variants of Salmonella typhi are simultaneously present in Southeast Asia and are associated with sporadic cases of typhoid fever and occasional outbreaks. Comparative analysis of PFGE patterns also suggested that considerable genetic diversity exists among S. typhi strains and that some PFGE patterns are shared between isolates obtained from Malaysia, Indonesia, and Thailand, implying movement of these strains within these regions of Southeast Asia, where they are endemic.     

Clonal analysis of the Actinobacillus pleuropneumoniae population in a geographically restricted area by multilocus enzyme electrophoresis.

The genetic diversity among 250 isolates of Actinobacillus pleuropneumoniae from lungs of pigs with pleuropneumonia and from tonsils of apparently healthy pigs at slaughter was estimated by multilocus enzyme electrophoresis. The Danish strains were derived from both specific-pathogen-free and conventional herds. Sixty-six percent of the isolates belonged to three electrophoretic types (ETs) of a total of 37 ETs detected. While five biotype 2 isolates constituted a separate ET closely related to biotype 1 isolates, the type strain of the species (Shope 4074) belonged to its own ET, with a genetic distance of 0.30 from its nearest neighbor. Isolates of serotypes traditionally considered to have less pathogenic potential (serotypes 6, 10, and 12) from herds with acute outbreaks of pleuropneumonia belonged to the same ETs as isolates from apparently healthy pigs, suggesting that factors such as cross immunity and management may lead to divergent clinical results. Isolates from four herds harboring more than one serotype showed distinct profiles between the serotypes, indicating no or only limited chromosomal recombination among clones. Isolates from tonsils belonged to the same ET as isolates from lungs. The same ET was isolated from widely different parts of the world. Evidence from this study indicates that multilocus enzyme electrophoresis may be a valuable tool for the epidemiological analysis of A. pleuropneumoniae.     

Chromosomal DNA, iron-transport systems, outer membrane proteins, and enterotoxin (heat labile) production in Salmonella typhi strains.

We examined a representative collection of Salmonella typhi strains from Chile, Peru, Mexico, India, and England for the presence of several properties. All strains had a conserved pattern of outer membrane proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The electrophoresis profiles of chromosomal DNA digested with EcoRI and PstI restriction enzymes were similar for all the strains. A conserved pattern of hybridization was observed when digested chromosomal DNA was hybridized with DNA probes for the 36-kilodalton porin, enterobactin synthesis, and enterobactin receptor genes. All the strains produced enterobactin but not aerobactin in bioassays. None of the strains produced heat-labile toxin, as measured by an enzyme-linked immunosorbent assay. Colony and Southern hybridizations with DNA probes for aerobactin synthesis and its receptor and heat-labile toxin genes were negative. These results indicate that S. typhi strains from different origins have similar phenotypic and genetic properties and, as has been suggested, constitute a clone.     

Characterization of Streptococcus agalactiae strains by multilocus enzyme genotype and serotype: identification of multiple virulent clone families that cause invasive neonatal disease.

The chromosomal genotypes of 277 isolates of 16 serotypes of Streptococcus agalactiae were characterized by analysis of electrophoretically demonstrable allele profiles at 12 metabolic enzyme loci. The collection comprised the type strain and 276 strains recovered from French symptomatic and asymptomatic subjects. Sixty-one distinctive electrophoretic types (ETs), representing multilocus clonal genotypes, were identified. Cluster analysis of the ETs revealed two primary phylogenetic divisions separated by a genetic distance of 0.62, Division I contained 67 isolates which could be assigned to 13 ETs. Twenty-seven of these isolates were from samples of cerebrospinal fluid (CSF) from neonatal meningitis patients. Two ETs, separated by a genetic distance of 0.217, contained 26 of these 27 isolates. Division II contained 210 isolates, of which 27 were isolated from CSF. This division was more polymorphic and included 48 ETs. Spanning a genetic distance of 0.3, three clusters and one ET were identified within this group. Twenty-four of 27 strains isolated from CSF belonged to one cluster, and 19 of them belonged to two adjacent ETs with a genetic distance of 0.083. Fifty-five of the 68 serotype Ia strains and 24 of the 26 serotype Ib strains were each confined to one of the evolutionary lineages, and 85 of the 86 strains which carried protein antigen c belonged to phylogenetic division II. Most of the type III organisms were assigned to two clone families. The characteristics of this French population argue for the existence of particular groups of strains responsible for neonatal meningitis and demonstrate that serotyping can supply information about the genetic distribution of strains.     

Genetic relationships of penicillin-susceptible and -resistant Streptococcus pneumoniae strains isolated on different continents.

Sixty-six strains of Streptococcus pneumoniae isolated in different parts of the world, 46 resistant and 22 susceptible to penicillin, were subdivided by multilocus enzyme electrophoresis into 28 distinct electrophoretic types (ETs). The ETs to which penicillin-susceptible strains were assigned differed from those containing resistant isolates of the same serotype. Five common clones could be recognized among the penicillin-resistant bacteria by combining the ETs, the antigenic properties of penicillin-binding proteins PBP 1a and 2b, and the tetracycline and chloramphenicol resistance profiles. Two clones were found in Finland and were associated with capsular serotypes 6B and 23F, respectively. Two clones were from Spain (type 6B and 9V, respectively). The fifth clone was isolated in South Africa and in Spain and contained both serotype 23F isolates and one type 19F strain. The other resistant strains were represented by rare isolates distributed among 12 other ETs, confirming that resistance to penicillin has evolved by multiple branches. Because capsular type was mixed in several ETs, the results also demonstrate that it may vary among very closely related pneumococci.     

Clonal relationships and variation in virulence among Escherichia coli strains of avian origin

Average genetic relatedness among 44 Escherichia coli strains of serotypes O1, O2, and O78 isolated mainly from birds with colibacillosis or swollen-head syndrome from France or Saudi Arabia was estimated based on allelic variation detected by multilocus enzyme electrophoresis. For 20 enzyme-encoding loci, we resolved 2.8 alleles per locus and distinguished 17 electrophoretic types (ETs) that were used to mark naturally occurring cell lineages or clones. On average, ETs differed at 37% of their loci. Forty-eight percent of the isolates represent three ETs, two of which belong to previously defined complexes of clones identified in avian disease in North America and Europe. Virulence of strains, assessed in experimental infections of day-old chicks, showed little variation among isolates of a clone, but was significantly variable among isolates of different clone complexes. These findings add support to the evidence that a majority of avian isolates that cause colibacillosis belong to a few cosmopolitan pathogenic clones and indicate a substantial between-clone component of pathogenicity.     

Quantitative analysis and partial characterization of cytotoxin production by Salmonella strains.

The pathogenesis of the wide-spectrum human disease caused by Salmonella species is poorly understood. Cytotoxin production by other enteric pathogens has been increasingly investigated recently, and data are accumulating regarding the role of cytotoxins in enteric infections and hemolytic uremic syndrome. We studied the cytotoxic activity of 131 Salmonella strains of the major serotypes, including 94 strains of Salmonella enteritidis, 12 strains of Salmonella typhi, and 25 strains of Salmonella choleraesuis. Cytotoxicity was quantitatively determined in sonic extracts by a [3H]thymidine-labeled HeLa cell assay. All Salmonella strains examined showed some degree of cytotoxic activity. The geometric means +/- standard deviations of the amounts of cytotoxin produced (50% cytotoxic dose per milligram of bacterial protein) were 27 +/- 2 for S. typhi, 65 +/- 2 for S. enteritidis, and 117 +/- 2 for S. choleraesuis. Analysis of variance showed that the differences in cytotoxin production by the three species were significant (P less than 0.001). No significant differences were found between stool isolates and invasive strains of the same species. Neutralization studies showed that the cytotoxins produced by all Salmonella strains were immunologically distinct from Shiga toxin and the closely related Shiga-like toxins produced by Escherichia coli. DNA hybridization studies with DNA probes for Shiga-like toxins of types I and II showed no hybridization. In each species the cytotoxin was heat labile and sensitive to trypsin treatment, which indicated that its active component was probably protein in nature. Upon ultrafiltration with Amicon membranes and gel filtration chromatography, cytotoxic activity was found in the molecular weight range of 56,000 to 78,000. Our findings indicate that salmonellae produce cytotoxin(s) that may play a role in the manifestations of the various species.     

Outer membrane protein profiles and multilocus enzyme electrophoresis analysis for differentiation of clinical isolates of Proteus mirabilis and Proteus vulgaris.

Outer membrane protein (MP) profiles and multilocus enzyme electrophoresis (MEE) analysis were used as tools for differentiating clinical isolates of Proteus spp. Fourteen distinct MP profiles were established by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis in 54 clinical isolates of Proteus spp. (44 strains identified as P. mirabilis and 10 strains identified as P. vulgaris). Forty-one isolates of P. mirabilis and eight isolates of P. vulgaris were grouped within six and three MP profiles, respectively. The remaining P. mirabilis and P. vulgaris isolates had unique profiles. MEE analysis was used to further discriminate among the strains belonging to the same MP groups. Thirty-five distinct electrophoretic types (ETs) were identified among P. mirabilis isolates. The isolates of P. mirabilis from the four most common MP groups were subgrouped into 30 ETs. All of the P. vulgaris strains had unique ETs. The results suggest that upon biochemical classification of Proteus isolates as P. mirabilis or P. vulgaris, further differentiation among strains of the same species can be obtained by the initial determination of MP profiles followed by MEE analysis of strains with identical MPs.     

Multilocus enzyme electrophoresis analysis of the Mycobacterium avium complex and other mycobacteria.

Multilocus enzyme electrophoresis analysis was used to evaluate the Mycobacterium avium complex (MAC), M. paratuberculosis, and nine other mycobacterial species. The average number of alleles per locus was 2.8 for the 35 MAC and 2 M. paratuberculosis strains which represented 24 electrophoretic types (ETs) and two distinct groups. The M. avium group was resolved into 17 ETs and contained the M. paratuberculosis ET. The M. intracellulare group consisted of six ETs. There was complete agreement between Gen-Probe identification and group placement by multilocus enzyme electrophoresis. The mean genetic diversity per locus for the 24 MAC ETs was 0.38. This procedure subdivided some serovars and, if implemented, should prove to be a powerful epidemiologic tool for the MAC. Eleven additional ETs were formed after the data for the other mycobacterial species were pooled with those for the MAC.     

Population structure of Australian isolates of Streptococcus suis.

The genetic diversity of 109 isolates of Streptococcus suis, which were recovered mainly from Australian pigs, was examined by multilocus enzyme electrophoresis. The collection was genetically diverse. Sixty-five electrophoretic types (ETs) were recognized, with a mean genetic diversity per enzyme locus of 0.512, or 0.431 when the number of isolates in each ET was considered. Serotype diversity varied, being greatest for isolates of capsular serotype 15 (0.364), and then diminishing in the order of serotypes 9, 1, 4, 1/2, 2, 7, and 3 (0.120). On average, isolates from these eight serotypes represented 4.13 separate clonal groups per serotype. This diversity indicated that serotyping of S. suis for subspecific differentiation is not a reliable technique for identifying specific strains and is not a good predictor of the genetic background of a given isolate. No tendency for isolates recovered from healthy pigs to be genetically distinct from those from diseases animals was found, nor were there consistent differences between isolates recovered from animals with different disease syndromes (meningitis, pneumonia, and septicemia). Danish reference strains of serotypes 1, 2, and 7 each belonged to one of the same clonal groupings of these types found in Australia, but Danish strains of serotypes 3, 4, 6, and 8 and a strain of serotype 1 from the United Kingdom were each genetically distinct from the Australian isolates. Generally, isolates in the same ET belonged to the same serotype, but one ET contained isolates of types 6 and 6/16, and three were made up of isolates of types 2 and 1/2. One isolate of serotype 2, which was recovered from a human with meningitis, belonged to the same ET as two isolates of serotype 2 that were recovered from pigs. The human infection was therefore likely to have been zoonotic.     

Molecular analysis of isolates of Salmonella typhi obtained from patients with fatal and nonfatal typhoid fever.

Molecular characterization of a total of 52 human isolates of Salmonella typhi from Papua New Guinea was performed by using pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with three restriction endonucleases, XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3'), and SpeI (5'-ACTAGT-3'). Of the 52 isolates tested, 11 were obtained from patients with fatal typhoid fever and 41 were obtained from patients with nonfatal disease. The 52 isolates showed limited genetic diversity as evidenced by only three different PFGE patterns detected following digestion with XbaI (patterns X1 to X3; F [coefficient of similarity] = 0.86 to 1.0), four patterns detected following digestion with AvrII (patterns A1 to A4; F =0.78 to 1.0), and two patterns detected following digestion with SpeI (patterns S1 and S2; F = 0.97 to 1.0). Of the 52 isolates, 37 were phage typed, and all belonged to phage type D2. All 11 isolates obtained from patients with fatal typhoid fever were identical (F = 1.0) and possessed the PFGE pattern combination X1S1A1, whereas the 41 isolates from patients with nonfatal typhoid fever had various PFGE pattern combinations, the most common being X2S1A2 (39%), X1S1A1 (24%), and X1S1A2 (15%). Thus, all the isolates from patients with the fatal disease had the X1 and A1 patterns, whereas the majority of the isolates from patients with nonfatal typhoid fever possessed the X2 and A2 patterns. The data suggest that there is an association among strains of S. typhi between genotype, as assessed by PFGE patterns, and the capability to cause fatal illness. Analysis of blood and fecal isolates of S. typhi from the same patient also indicated that some genetic changes occur in vivo during the course of infection.     

Population genetic analysis of Borrelia burgdorferi isolates by multilocus enzyme electrophoresis.

Fifty Borellia burgdorferi strains isolated from humans and ticks in Europe and the United States were analyzed by multilocus enzyme electrophoresis. Eleven genetic loci were characterized on the basis of the electrophoretic mobilities of their products. Ten loci were polymorphic. The average number of alleles per locus was 5.9, with a mean genetic diversity of 0.673 among electrophoretic types (ETs). The strains were grouped into 35 ETs constituting three main divisions (I, II, and III) separated at a genetic distance greater than 0.75. Divisions I, II, and III contained 13, 6, and 16 ETs, respectively. These findings, together with previous data from DNA hybridization and restriction enzyme analysis of rRNA genes, suggest that divisions I, II, and III may represent three distinct genomic species. All three divisions contained human clinical ETs. However, in division I, which includes the ET of the type strain of B. burgdorferi, the human pathogenic ETs constituted a single clone. The ETs of division I were from west-central Europe and the United States, whereas divisions II and III contained ETs from west-central and northern Europe but not from the United States. Finally, our data show that the genetic structure of B. burgdorferi populations is clonal.     

Genetic diversity in relation to serotype in Escherichia coli.

The extent of chromosomal-gene diversity among 261 isolates of Escherichia coli sharing single O, K, or H antigens and various combinations thereof was estimated by multilocus enzyme electrophoresis, which detects allelic variation in structural genes. The results of this study indicate that the genetic diversity among isolates sharing single antigenic determinants can approach or equal that observed among randomly chosen strains; that the magnitude of the diversity varies among antigens; and that the genetic diversity is reduced, but not eliminated, among isolates sharing two antigenic determinants. With one exception, isolates of the same O:K:H serotype were of identical or closely related electrophoretic types (ETs). Isolates of the same ET generally shared the same combination of antigenic determinants, but some ETs included isolates of different serotypes. The implications of these findings for epidemiological research and the clone hypothesis of population structure are discussed, and possible evolutionary mechanisms causing antigenic divergence and convergence are considered.