Genetic Markers Unique to Listeria monocytogenes Serotype 4b Differentiate Epidemic Clone II (Hot Dog Outbreak Strains) from Other Lineages

A small number of closely related strains of Listeria monocytogenes serotype 4b, designated epidemic clone I (ECI), have been implicated in numerous outbreaks of food-borne listeriosis described during the past two decades in Europe and North America. In 1998 to 1999, a multistate outbreak traced to contaminated hot dogs involved a different strain type of serotype 4b, with genetic fingerprints rarely encountered before. In spite of the profound economic and public health impact of this outbreak, the implicated bacteria (designated epidemic clone II [ECII]) have remained poorly characterized genetically, and nucleotide sequences specific for these strains have not been reported. Using genome sequence information, PCR, and Southern blots, we identified DNA fragments which appeared to be either absent or markedly divergent in the hot dog outbreak strains but conserved among other serotype 4b strains. PCR with primers derived from these fragments as well as Southern blots with the amplicons as probes readily differentiated ECII from other serotype 4b strains. The serotype 4b-specific region harboring these fragments was adjacent to inlA, which encodes a well-characterized virulence determinant. The findings suggest that ECII strains have undergone divergence in portions of a serotype-specific region that is conserved in other serotype 4b strains. Although the mechanisms that drive this divergence remain to be identified, DNA-based tools from this region can facilitate the detection and further characterization of strains belonging to this lineage.     

DNA fragments from regions involved in surface antigen expression specifically identify Listeria monocytogenes serovar 4 and a subset thereof: cluster IIB (serotypes 4b, 4d, and 4e).

Listeria monocytogenes serotype 4b has frequently been implicated in sporadic as well as epidemic listeriosis. On the basis of pulsed-field fingerprinting, serotype 4b strains, along with strains of serotypes 4d and 4e, constitute one genomic cluster (IIB). We have identified two genomic regions essential for the expression of surface antigens which previously were shown to be specific to cluster IIB strains. A DNA probe of 1.1 kb derived from one of the regions (probe 1) hybridized only with strains of serotypes 4b, 4d, and 4e in Southern blots and dot blots. A different DNA probe of 0.3 kb (probe 2), derived from the other region, hybridized with all serovar 4 strains (serotypes 4b, 4a, 4c, 4d, and 4e). All other L. monocytogenes serotypes were negative with probe 1 or 2. Use of probe 1 in Southern blots of EcoRI-digested genomic DNA revealed a restriction fragment length polymorphism in serotype 4b strains, with the hybridizing EcoRI fragments being 4.5 kb (strains of the epidemic clone) and either 4.5 or 5.0 kb (all other serotype 4b strains). Although the probes hybridized with a special group of Listeria innocua strains which also expressed the surface antigens, the latter could be readily distinguished by the size of the hybridizing EcoRI fragment with probe 1 (ca. 2.2 kb). These data suggest that the combined use of these probes with L. monocytogenes can readily and specifically identify cluster IIB strains as well as the entire serovar 4 complex.     

Restriction fragment length polymorphisms detected with novel DNA probes differentiate among diverse lineages of serogroup 4 Listeria monocytogenes and identify four distinct lineages in serotype 4b.

Listeria monocytogenes of serotype 4b has been implicated in numerous outbreaks of food-borne listeriosis and in ca. 40% of sporadic cases. Strains of this serotype appear to be relatively homogeneous genetically, and molecular markers specific for distinct serotype 4b lineages have not been frequently identified. Here we show that DNA fragments derived from the putative mannitol permease locus of Listeria monocytogenes had an unexpectedly high potential to differentiate among different strains of serotype 4b when used as probes in Southern blotting of EcoRI-digested genomic DNA, yielding four distinct restriction fragment length polymorphism (RFLP) patterns. Strains of two epidemic-associated lineages, including the major epidemic clone implicated in several outbreaks in Europe and North America, had distinct RFLPs which differed from those of all other serotype 4b strains that we screened but which were encountered among strains of serotypes 1/2b and 3b. In addition, three serogroup 4 lineages were found to have unique RFLPs that were not encountered among any other L. monocytogenes strains. One was an unusual lineage of serotype 4b, and the other two were members of the serotype 4a and 4c group. The observed polymorphisms may reflect evolutionary relationships among lineages of L. monocytogenes and may facilitate detection and population genetic analysis of specific lineages.     

Identification and Characterization of Nucleotide Sequence Differences in Three Virulence-Associated Genes of Listeria monocytogenes Strains Representing Clinically Important Serotypes

Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium that causes invasive, often fatal, disease in susceptible hosts. As a foodborne pathogen, the bacterium has emerged as a significant public health problem and has caused several epidemics in the United States and Europe. Three serotypes (1/2a, 1/2b, 4b) of L. monocytogenes are responsible for nearly 95% of all reported cases of human listeriosis. L. monocytogenes serotype 4b has caused all well-characterized foodborne epidemic outbreaks in North America and Europe between 1981 and 1993. However, most of the genetic studies to characterize virulence factors of L. monocytogenes have been done by using serotypes 1/2a and 1/2c. In this investigation, we examined three virulence-associated genes (hly encoding listeriolysin, plcA encoding phosphotidylinositol-specific phospholipase C, and inlA encoding internalin) of two serotype 4b and two serotype 1/2b strains. We chose these virulence-associated genes on the basis of published sequence differences among strains from Listeria subgroups containing serotypes 1/2a and 1/2c versus 4b, respectively. They correspond to sequence homologies that include very highly conserved (hlyA), highly conserved (plcA) and mostly conserved (inlA). We found by using nucleotide sequence analysis of the hly, plcA, and inlA genes, the two L. monocytogenes strains (including a strain associated with a foodborne disease outbreak in California in 1985) in this study, two serotype 1/2b strains from a study that we recently reported, and other similar published data for serotypes 1/2a, 1/2c, and 4b, had a high degree of sequence conservation at the gene and protein levels for all three genes. However, the sequences for the hly gene of L. monocytogenes strains of serotypes 1/2b and 4b were more closely related to each other and showed significant divergence from serotypes 1/2a and 1/2c. A unique nonsynonymous mutation was found in the hly gene of L. monocytogenes isolates that were associated with the 1985 California outbreak and were the epidemic phage type. When 158 L. monocytogenes isolates from the collection at the Centers for Disease Control and Prevention were screened, the mutation was found only in one other strain that had been isolated in California 3 years before the epidemic. Although the California epidemic clone was lactose negative, other L. monocytogenes serotype 4b isolates that were lactose negative did not possess the unique mutation observed in that epidemic clone. Received: 18 June 1997 / Accepted: 4 December 1997     

Restriction Fragment Length Polymorphisms Detected with Novel DNA Probes Differentiate among Diverse Lineages of Serogroup 4 Listeria monocytogenes and Identify Four Distinct Lineages in Serotype 4b

Listeria monocytogenes of serotype 4b has been implicated in numerous outbreaks of food-borne listeriosis and in ca. 40% of sporadic cases. Strains of this serotype appear to be relatively homogeneous genetically, and molecular markers specific for distinct serotype 4b lineages have not been frequently identified. Here we show that DNA fragments derived from the putative mannitol permease locus of Listeria monocytogenes had an unexpectedly high potential to differentiate among different strains of serotype 4b when used as probes in Southern blotting of EcoRI-digested genomic DNA, yielding four distinct restriction fragment length polymorphism (RFLP) patterns. Strains of two epidemic-associated lineages, including the major epidemic clone implicated in several outbreaks in Europe and North America, had distinct RFLPs which differed from those of all other serotype 4b strains that we screened but which were encountered among strains of serotypes 1/2b and 3b. In addition, three serogroup 4 lineages were found to have unique RFLPs that were not encountered among any other L. monocytogenes strains. One was an unusual lineage of serotype 4b, and the other two were members of the serotype 4a and 4c group. The observed polymorphisms may reflect evolutionary relationships among lineages of L. monocytogenes and may facilitate detection and population genetic analysis of specific lineages.     

Temperature-Dependent Phage Resistance of Listeria monocytogenes Epidemic Clone II

Listeria monocytogenes epidemic clone II (ECII) has been responsible for two multistate outbreaks in the United States in 1998-1999 and in 2002, in which contaminated ready-to-eat meat products (hot dogs and turkey deli meats, respectively) were implicated. However, ecological adaptations of ECII strains in the food-processing plant environment remain unidentified. In this study, we found that broad-host-range phages, including phages isolated from the processing plant environment, produced plaques on ECII strains grown at 37°C but not when the bacteria were grown at lower temperatures (30°C or below). ECII strains grown at lower temperatures were resistant to phage regardless of the temperature during infection and subsequent incubation. In contrast, the phage susceptibility of all other tested strains of serotype 4b (including epidemic clone I) and of strains of other serotypes and Listeria species was independent of the growth temperature of the bacteria. This temperature-dependent phage susceptibility of ECII bacteria was consistently observed with all surveyed ECII strains from outbreaks or from processing plants, regardless of the presence or absence of cadmium resistance plasmids. Phages adsorbed similarly on ECII bacteria grown at 25°C and at 37°C, suggesting that resistance of ECII strains grown at 25°C was not due to failure of the phage to adsorb. Even though the underlying mechanisms remain to be elucidated, temperature-dependent phage resistance may represent an important ecological adaptation of L. monocytogenes ECII in processed, cold-stored foods and in the processing plant environment, where relatively low temperatures prevail.Listeria monocytogenes is responsible for an estimated 2,500 cases of serious food-borne illness (listeriosis) and 500 deaths annually in the United States. It affects primarily pregnant women, newborns, the elderly, and adults with weakened immune systems. L. monocytogenes is frequently found in the environment and can grow at low temperatures, thus representing a serious hazard for cold-stored, ready-to-eat foods (18, 31).Two multistate outbreaks of listeriosis in the United States, in 1998-1999 and in 2002, respectively, were caused by contaminated ready-to-eat meats (hot dogs and turkey deli meats, respectively) contaminated by serotype 4b strains that represented a novel clonal group, designated epidemic clone II (ECII) (3, 4). ECII strains have distinct genotypes as determined by pulsed-field gel electrophoresis and various other subtyping tools, and harbor unique genetic markers (6, 8, 11, 19, 34). The genome sequencing of one of the isolates (L. monocytogenes H7858) from the 1998-1999 outbreak revealed the presence of a plasmid of ca. 80 kb (pLM80), which harbored genes mediating resistance to the heavy metal cadmium as well as genes conferring resistance to the quaternary ammonium disinfectant benzalkonium chloride (10, 29).Listeria phages (listeriaphage) have long been used for subtyping purposes (33), and extensive research has focused on the genomic characterization (2, 24, 26, 35), transducing potential (14), and biotechnological applications of selected phages (25). In addition, applications of listeriaphage as biocontrol agents in foods and the processing plant environment have been investigated (12, 15, 22). However, limited information exists on phages from processing plant environments and on the impact of environmental conditions on susceptibility of L. monocytogenes strains representing the major epidemic-associated clonal groups to such phages. We have found that strains harboring ECII-specific genetic markers can indeed be recovered from the environment of turkey-processing plants (9). Furthermore, environmental samples from such processing plants yielded phages with broad host range, which were able to infect L. monocytogenes strains of various serotypes, and different Listeria species (20). In this study, we describe the impact of growth temperature on susceptibility of L. monocytogenes ECII strains to phages, including phages isolated from turkey-processing plant environmental samples.     

Host-mediated modification of Sau3AI restriction in Listeria monocytogenes: prevalence in epidemic-associated strains.

Most major food-related outbreaks of listeriosis have been traced to a cluster of genetically related strains of serovar 4b (epidemic clone). In spite of numerous searches, distinct bacteriologic or virulence-related features unique to these strains have eluded identification, although a restriction fragment length polymorphism (RFLP) characteristic of the epidemic clone has previously been described (W. Zheng and S. Kathariou, Appl. Environ. Microbiol. 61:4310-4314, 1995). We found that DNAs from 75 strains which were derived from three separate outbreaks and which had the epidemic clone-specific RFLP were also invariably resistant to digestion by Sau3AI and other restriction endonucleases sensitive to cytosine methylation at 5' GATC 3' sites. This modification of Sau3AI restriction was host mediated, as it did not persist when DNA was cloned and propagated in Escherichia coli, and was uncommon among other Listeria strains. Epidemic-associated strains with this modification were resistant to infection by phage propagated in a serotype 4b strain which was not known to be involved in an epidemic and which lacked the epidemic clone-specific RFLP. Screening for susceptibility to MboI digestion revealed that these epidemic strains lacked methylation of adenines at GATC sites. This type of modification was rare among Listeria strains and was found in only three (of eight screened) strains of serovar 1/2b, possibly representing one clonal lineage.     

Differentiation of epidemic-associated strains of Listeria monocytogenes by restriction fragment length polymorphism in a gene region essential for growth at low temperatures (4 degrees C).

The growth of Listeria monocytogenes in food stored in the cold has often been implicated in outbreaks of listeriosis. Many subtyping schemes have suggested that epidemic-associated strains belong to a unique genetic group. It has not yet been possible, however, to identify molecular or bacteriologic markers unique to epidemic-associated strains. Recently we cloned three genes of L. monocytogenes, ltrA, ltrB, and ltrC, which are essential for growth at low temperatures (4 degrees C). The use of a 1.2-kb PstI fragment derived from ltrB as a probe in Southern blots of HindIII-digested DNA revealed three hybridization patterns: the first (a 5.0-kb band) was observed in strains of serotypes 4b, 1/2b, and 3b; the second (a 3.1-kb band) was seen in strains of serotypes 1/2a, 3a, 1/2c, and 3c; and the third (a 9.5-kb band) was characteristic of epidemic-associated serotype 4b strains. These and other data suggest that probes derived from this gene region that is essential for growth at low temperatures can be useful molecular tools for the subtyping of strains implicated in food-borne listeriosis.     

Southern blotting analyses of strains ofCampylobacter fetus using the conserved region ofsapA

Chromosomal DNA of 27 strains of Campylobacter fetus was analyzed by Southern blotting with a probe of the conserved region of sapA. The probe hybridized with 23 strains that produced type A lipopolysaccharide. These strains had more than six sapA homologs. In Southern blots of SalI-digested chromosomal DNA separated by pulsed-field gel electrophoresis, one fragment from 19 strains and two fragments from 4 strains hybridized. These data indicate that multiple sapA homologs are localized to a limited region on the chromosomal DNA of C. fetus and thus suggest the possibility of developing a typing system using this method. Received: 28 June 1995 / Accepted: 19 September 1995     

Whole Genome Sequence Analysis Using JSpecies Tool Establishes Clonal Relationships between Listeria monocytogenes Strains from Epidemiologically Unrelated Listeriosis Outbreaks

In an effort to build a comprehensive genomic approach to food safety challenges, the FDA has implemented a whole genome sequencing effort, GenomeTrakr, which involves the sequencing and analysis of genomes of foodborne pathogens. As a part of this effort, we routinely sequence whole genomes of Listeria monocytogenes (Lm) isolates associated with human listeriosis outbreaks, as well as those isolated through other sources. To rapidly establish genetic relatedness of these genomes, we evaluated tetranucleotide frequency analysis via the JSpecies program to provide a cursory analysis of strain relatedness. The JSpecies tetranucleotide (tetra) analysis plots standardized (z-score) tetramer word frequencies of two strains against each other and uses linear regression analysis to determine similarity (r²). This tool was able to validate the close relationships between outbreak related strains from four different outbreaks. Included in this study was the analysis of Lm strains isolated during the recent caramel apple outbreak and stone fruit incident in 2014. We identified that many of the isolates from these two outbreaks shared a common 4b variant (4bV) serotype, also designated as IVb-v1, using a qPCR protocol developed in our laboratory. The 4bV serotype is characterized by the presence of a 6.3 Kb DNA segment normally found in serotype 1/2a, 3a, 1/2c and 3c strains but not in serotype 4b or 1/2b strains. We decided to compare these strains at a genomic level using the JSpecies Tetra tool. Specifically, we compared several 4bV and 4b isolates and identified a high level of similarity between the stone fruit and apple 4bV strains, but not the 4b strains co-identified in the caramel apple outbreak or other 4b or 4bV strains in our collection. This finding was further substantiated by a SNP-based analysis. Additionally, we were able to identify close relatedness between isolates from clinical cases from 1993–1994 and a single case from 2011 as well as links between two isolates from over 30 years ago. The identification of these potential links shows that JSpecies Tetra analysis can be a useful tool in rapidly assessing genetic relatedness of Lm isolates during outbreak investigations and for comparing historical isolates. Our analyses led to the identification of a highly related clonal group involved in two separate outbreaks, stone fruit and caramel apple, and suggests the possibility of a new genotype that may be better adapted for certain foods and/or environment.     

Epidemic clone I-specific genetic markers in strains of Listeria monocytogenes serotype 4b from foods

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.     

Conservation of Genomic Localization and Sequence Content of Sau3AI-Like Restriction-Modification Gene Cassettes among Listeria monocytogenes Epidemic Clone I and Selected Strains of Serotype 1/2a

Listeria monocytogenes is a food-borne pathogen with a clonal population structure and apparently limited gene flow between strains of different lineages. Strains of epidemic clone I (ECI) have been responsible for numerous outbreaks and invariably have DNA that is resistant to digestion by Sau3AI, suggesting methylation of cytosine at GATC sites. A putative restriction-modification (RM) gene cassette has been identified in the genome of the ECI strain F2365 and all other tested ECI strains but is absent from other strains of the same serotype (4b). Homologous RM cassettes have not been reported among L. monocytogenes isolates of other serotypes. Furthermore, conclusive evidence for the involvement of this RM cassette in the Sau3AI resistance phenotype of ECI strains has been lacking. In this study, we describe a highly conserved RM cassette in certain strains of serotypes 1/2a and 4a that have Sau3AI-resistant DNA. In these strains the RM cassette was in the same genomic location as in the ECI reference strain F2365. The cassette included a gene encoding a putative recombinase, suggesting insertion via site-specific recombination. Deletion of the RM cassette in the ECI strain F2365 and the serotype 1/2a strain A7 rendered the DNA of both strains susceptible to Sau3AI digestion, providing conclusive evidence that the cassette includes a gene required for methylation of cytosine at GATC sites in both strains. The findings suggest that, in addition to its presence in ECI strains, this RM cassette and the accompanying genomic DNA methylation is also encountered among selected strains of other lineages.Listeria monocytogenes is a Gram-positive, facultative intracellular food-borne pathogen capable of causing severe disease (listeriosis) in animals and humans. Listeriosis most often affects pregnant women and their fetuses, neonates, the elderly, and immunocompromised individuals. The disease is predominantly transmitted via the consumption of contaminated foods and has a ca. 20% fatality rate (12, 27). Application of numerous genotyping methods has consistently shown that the organism has a clonal population structure with three major phylogenetic lineages: lineage I consists of strains of serotypes 1/2b, 3b, and 4b, while those of serotypes 1/2a, 1/2c, 3a, and 3c are clustered in lineage II; strains of serotypes 4a and 4c, along with certain serotype 4b strains, constitute lineage III (37, 38).Most epidemics of human listeriosis have involved a small number of closely related strains (epidemic clones), predominantly of serotype 4b (7, 35). The earliest identified clone, epidemic clone I (ECI), has been responsible for several major outbreaks in Europe and North America. In addition, strains of this clonal group are frequently encountered in sporadic illness (10, 28, 29). ECI strains have also been found to comprise a significant portion of the serotype 4b strains from foods and from the environments of food processing plants (10, 11, 40).Genomic DNA of ECI strains has been long known to resist digestion with Sau3AI, suggesting methylation of cytosine at GATC sites (41). Genome sequencing of the ECI strain F2365, implicated in the 1985 California outbreak of listeriosis, revealed a putative restriction-modification (RM) gene cassette with specificity for GATC sites (25). This RM cassette was harbored by all tested serotype 4b strains with Sau3AI-resistant DNA and was absent from those with DNA that could be digested with Sau3AI (40). These findings were in agreement with previous evidence that a fragment of the putative methyltransferase gene was specific to ECI and absent from other strains (14).In spite of extensive documentation for the presence of this putative RM cassette in ECI strains, and its apparent absence among other serotype 4b strains, limited information is available about the possible presence of the cassette among other lineages of L. monocytogenes. Furthermore, conclusive evidence for involvement of the cassette in the resistance of the DNA of ECI strains to Sau3AI digestion has been lacking. In this study, we investigated a panel of food-derived serotype 1/2a strains with Sau3AI-resistant DNA and characterized the genetic content and genomic localization of the RM cassette harbored by these strains. Furthermore, we employed deletion mutagenesis to assess the involvement of the RM cassette in Sau3AI resistance of the DNA of the ECI strain F2365, as well as of a serotype 1/2a strain harboring the cassette.     

Population Structure of Listeria monocytogenes Serotype 4b Isolates from Sporadic Human Listeriosis Cases in the United States from 2003 to 2008

Listeria monocytogenes can cause severe food-borne disease (listeriosis). Numerous outbreaks have involved three serotype 4b epidemic clones (ECs): ECI, ECII, and ECIa. However, little is known about the population structure of L. monocytogenes serotype 4b from sporadic listeriosis in the United States, even though most cases of human listeriosis are in fact sporadic. Here we analyzed 136 serotype 4b isolates from sporadic cases in the United States, 2003 to 2008, utilizing multiple tools including multilocus genotyping, pulsed-field gel electrophoresis, and sequence analysis of the inlAB locus. ECI, ECII, and ECIa were frequently encountered (32, 17, and 7%, respectively). However, annually 30 to 68% of isolates were outside these ECs, and several novel clonal groups were identified. An estimated 33 and 17% of the isolates, mostly among the ECs, were resistant to cadmium and arsenic, respectively, but resistance to benzalkonium chloride was uncommon (3%) among the sporadic isolates. The frequency of clonal groups fluctuated within the 6-year study period, without consistent trends. However, on several occasions, temporal clusters of isolates with indistinguishable genotypes were detected, suggesting the possibility of hidden multistate outbreaks. Our analysis suggests a complex population structure of serotype 4b L. monocytogenes from sporadic disease, with important contributions by ECs and several novel clonal groups. Continuous monitoring will be needed to assess long-term trends in clonality patterns and population structure of L. monocytogenes from sporadic listeriosis.     

我国新分离ECHO30病毒VP1序列分析

测定了引起2003年苏北地区无菌性脑膜炎暴发流行的病因病毒FDJS03分离株的VP1基因序列,并与国外同型流行毒株做比较,以了解本流行株的分子生物学特点及遗传变异规律。随机选取FDJS03分离毒株中的4株,用肠道病毒、VP1序列的特异性引物008／011进行RT-PCR,扩增产物经凝胶纯化后测序。将序列输入GenBank,用BLAgr程序进行核苷酸和氨基酸序列比对;选取32株不同地区不同年代的ECHO30分离毒株,在PHYLIP3．573C和TREE-PUZZLE5．0中构建进化树,比较它们完整VP1序列(876nt)的进化关系。核苷酸和氨基酸同源性比较结果证明：4株分离病毒均为ECHO30。进化树分析显示：本次FDJS03分离株与欧美20世纪70、80年代流行株亲缘关系最近,但自成一簇,与国外毒株仍然有地区差别。ECHO30的VP1基因进化有时间效应,但存在地区差异。本次流行的病原可能是单一基因型的ECHO30病毒。     

DNA homology of surface protein antigen A gene in mutans streptococci

1. A recombinant plasmid, pYA724, containing an 8.45 kb DNA fragment encoding surface protein antigen A (spaA) from Streptococcus sobrinus 6715 was used to examine the DNA homology of the spaA gene with chromosomal DNA of various mutans streptococci strains. 2. Restriction endonuclease BamHI-digested pYA724 DNA was radio-labeled by nick-translation, and a DNA-DNA hybridization experiment was carried out. pYA724 DNA hybridized with chromosomal DNA of serotypes a, c, d, e, f and g strains, but not with b by dot DNA-hybridization and Southern blot DNA hybridization. 3. Chromosomal DNAs were isolated from several serotype c Streptococcus mutans strains, digested with BamHI, and analyzed by Southern blot DNA hybridization. pYA724 DNA hybridized with different sizes and numbers of BamHI-digested DNA fragments of the chromosomal DNAs. 4. These data indicated that all mutans streptococci strains except serotype b have DNA homologous with the spaA gene, although within the same serotype strain the spaA gene has a diversity of arrangement within the chromosome.     

Phylogenetic analysis of viral haemorrhagic septicaemia virus (VHSV) isolates from France (1971-1999)

The nucleotide sequences of a specific region of the glycoprotein gene were compared among 63 strains of viral haemorrhagic septicaemia virus (VHSV) isolated from fish in France between 1971 and 1999. The analysis was performed on a region corresponding to amino acids 238 to 331 of the glycoprotein gene, also designated the V2 region and previously shown to accumulate most of the mutations. The sequences of many VHSV isolates were found to be identical or very conserved. An isolate, designated L59X, obtained from elver in the Loire estuary, depicted a higher degree of divergence compared to the other French isolates. The deduced amino-acid sequences were analysed together with the results of neutralisation tests performed using monoclonal antibody 168m4 specific to serotype 1. Non-neutralised VHSV strains had mutations in the region corresponding to the previously described 168m4 epitope. Phylogenetic analysis showed that all the VHSV isolates studied, except L59X, belong to genotype I, previously described as containing VHSV strains isolated from continental Europe. Most of the VHSV isolates studied were found to be genetically related to one of the previously described VHSV strains representative of the major serotypes. Isolate L59X, which was the only French marine strain studied, was found to belong to genotype II, previously shown to encompass the VHSV strains isolated from the British Isles coastal waters. Overall there was a good correlation between the geographical origin of the studied isolates and their genetic characteristics.     

Epidemic Clone I-Specific Genetic Markers in Strains of Listeria monocytogenes Serotype 4b from Foods

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.