Division of Listeria monocytogenes serovar 1/2a strains into two groups by PCR and restriction enzyme analysis

Altogether, 100 strains of Listeria monocytogenes serovar 1/2a isolated from humans, animals, food, and the environment were typed by a combination of PCR and restriction enzyme analysis (REA). A PCR product of 2,916 bp, containing the downstream end of the gene inlA (955 bp), the space between inlA and inlB (85 bp), and 1,876 bp of the gene inlB, was cleaved with the enzyme AluI, and the fragments generated were separated by gel electrophoresis. By this method two different cleavage patterns were obtained. Seventy of the 100 strains shared one restriction profile, and the remaining 30 strains shared the second one. No relation was found between the types differentiated by PCR-REA and the origins of the strains.     

Restriction enzyme analysis of Listeria monocytogenes strains associated with food-borne epidemics.

Listeria monocytogenes (serotype 4b) has caused four major food-borne epidemics in North America. In this study, L. monocytogenes isolates from the Nova Scotia (Canada), Boston (Mass.), and Los Angeles (Calif.) outbreaks were examined by restriction enzyme analysis with the endonuclease HhaI. Human isolates (n = 32) from the 1981 Canadian outbreak were compared with a strain recovered from coleslaw, which was epidemiologically incriminated as the vehicle of infection. After HhaI digestion, 29 of 32 isolates exhibited the restriction enzyme pattern of the reference coleslaw isolate. The restriction enzyme patterns of the nine clinical isolates from the 1983 Massachusetts outbreak were identical to each other but differed from those of raw milk isolates recovered from sources supplying the pasteurizer. Isolates (n = 48) from the 1985 California outbreak were evaluated. The restriction enzyme patterns of the L. monocytogenes isolates from humans and from the suspect cheese samples were identical to those of four of five cheese factory environmental isolates. Isolates from each of these outbreaks exhibited a restriction enzyme pattern that was characteristic of that outbreak. The case with which restriction enzyme analysis can be applied to all serotypes of L. monocytogenes argues for its use in the epidemiology of L. monocytogenes.     

Restriction enzyme analysis of Listeria monocytogenes strains associated with food-borne epidemics

Listeria monocytogenes (serotype 4b) has caused four major food-borne epidemics in North America. In this study, L. monocytogenes isolates from the Nova Scotia (Canada), Boston (Mass.), and Los Angeles (Calif.) outbreaks were examined by restriction enzyme analysis with the endonuclease HhaI. Human isolates (n = 32) from the 1981 Canadian outbreak were compared with a strain recovered from coleslaw, which was epidemiologically incriminated as the vehicle of infection. After HhaI digestion, 29 of 32 isolates exhibited the restriction enzyme pattern of the reference coleslaw isolate. The restriction enzyme patterns of the nine clinical isolates from the 1983 Massachusetts outbreak were identical to each other but differed from those of raw milk isolates recovered from sources supplying the pasteurizer. Isolates (n = 48) from the 1985 California outbreak were evaluated. The restriction enzyme patterns of the L. monocytogenes isolates from humans and from the suspect cheese samples were identical to those of four of five cheese factory environmental isolates. Isolates from each of these outbreaks exhibited a restriction enzyme pattern that was characteristic of that outbreak. The case with which restriction enzyme analysis can be applied to all serotypes of L. monocytogenes argues for its use in the epidemiology of L. monocytogenes.     

Genome sequence of Listeria monocytogenes 07PF0776, a cardiotropic serovar 4b strain

Listeria monocytogenes is a food-borne bacterial pathogen commonly associated with serious invasive infections of the central nervous system or of the developing fetus. We present the genome sequence of Listeria monocytogenes 07PF0776, a serovar 4b isolate from a human myocardial abscess that exhibits enhanced invasion of cardiac tissue.     

Division of Listeria monocytogenes Serovar 1/2a Strains into Two Groups by PCR and Restriction Enzyme Analysis

Altogether, 100 strains of Listeria monocytogenes serovar 1/2a isolated from humans, animals, food, and the environment were typed by a combination of PCR and restriction enzyme analysis (REA). A PCR product of 2,916 bp, containing the downstream end of the gene inlA (955 bp), the space between inlA and inlB (85 bp), and 1,876 bp of the gene inlB, was cleaved with the enzyme AluI, and the fragments generated were separated by gel electrophoresis. By this method two different cleavage patterns were obtained. Seventy of the 100 strains shared one restriction profile, and the remaining 30 strains shared the second one. No relation was found between the types differentiated by PCR-REA and the origins of the strains.     

Atypical Listeria monocytogenes serotype 4b strains harboring a lineage II-specific gene cassette

Listeria monocytogenes is the etiological agent of listeriosis, a severe food-borne illness. The population of L. monocytogenes is divided into four lineages (I to IV), and serotype 4b in lineage I has been involved in numerous outbreaks. Several serotype 4b epidemic-associated clonal groups (ECI, -II, and -Ia) have been identified. In this study, we characterized a panel of strains of serotype 4b that produced atypical results with a serotype-specific multiplex PCR and possessed the lmo0734 to lmo0739 gene cassette that had been thought to be specific to lineage II. The cassette was harbored in a genomically syntenic locus in these isolates and in lineage II strains. Three distinct clonal groups (groups 1 to 3) were identified among these isolates based on single-nucleotide polymorphism-based multilocus genotyping (MLGT) and DNA hybridization data. Groups 1 and 2 had MLGT haplotypes previously encountered among clinical isolates and were composed of clinical isolates from multiple states in the United States. In contrast, group 3 consisted of clinical and environmental isolates solely from North Carolina and exhibited a novel haplotype. In addition, all group 3 isolates had DNA that was resistant to MboI, suggesting methylation of adenines at GATC sites. Sequence analysis of the lmo0734 to lmo0739 gene cassette from two strains (group 1 and group 3) revealed that the genes were highly conserved (>99% identity). The data suggest relatively recent horizontal gene transfer from lineage II L. monocytogenes into L. monocytogenes serotype 4b and subsequent dissemination among at least three distinct clonal groups of L. monocytogenes serotype 4b, one of which exhibits restrictions in regional distribution.     

Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes

This is the first report of generalized transduction in the gram-positive, food-borne pathogen Listeria monocytogenes. Bacteriophages were isolated from the environment and from lysogens, or were obtained from other laboratories. Of the 59 bacteriophages tested, 34 proved to be capable of transduction. We exploited the ability of L. monocytogenes to grow at room temperature and isolated bacteriophages that were incapable of growth at 37 degrees C. Transductions at this temperature therefore eliminated transductant killing and lysogeny, as did inclusion of citrate and the use of a low multiplicity of infection. Transducing bacteriophages were found for each of the well-characterized L. monocytogenes strains: EGD, 10403, Mack (serotype1/2a), L028 (serotype 1/2c), Scott A (serotype 4b) and strains from the Jalisco and Halifax, Nova Scotia outbreaks (serotype 4b). P35 (phiLMUP35) is a particularly useful generalized transducing bacteriophage with a wide host range (75% of all serotype 1/2 strains tested). Its disadvantages are that it is small and transduction is relatively infrequent. U153(phiCU-SI153/95) is larger than P35 and transduction frequency increased 100-fold, but it has a very narrow host range. We demonstrated interstrain transduction and used transduction to test linkage between transposon insertions and mutant phenotypes in a variety of strains.     

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.     

PCR技术检测单核细胞增生李斯特氏菌研究进展

单核细胞增生李斯特氏菌(Listeria monocytogenes)是危害公共卫生和食品行业的一种重要人畜共患病致病菌.PCR技术是近年来被广泛地运用到食源性致病菌快速检测的分子生物学方法之一.就PCR技术检测单核细胞增生李斯特氏菌中的特异性鉴别基因、模板DNA制备等关键因素及多重PCR、巢式PCR、反转录PCR与定量PCR等主要技术进行了综述.     

Real-time PCR assay to differentiate Listeriolysin S-positive and -negative strains of Listeria monocytogenes

Due to the severity of the food-borne infection listeriosis, strict legislation governs the detectable and permissible limits at which Listeria monocytogenes is permitted in foods. These requirements, coupled with the ubiquitous nature of L. monocytogenes strains and the potential for epidemic outbreaks, mean that the pathogen can devastate affected sectors of the food industry. Although almost all L. monocytogenes strains have the potential to cause listeriosis, those implicated in the vast majority of epidemics belong to a subset of strains belonging to evolutionary lineage I. It has been established that a significant proportion of these strains, including those implicated in the majority of outbreaks, produce an additional hemolysin, designated listeriolysin S (LLS), which may be responsible for the enhanced virulence of these strains. In order to ultimately establish this definitively, it is important to first be able to rapidly discriminate between LLS-positive and -negative strains. Here, after essential genes within the LLS-encoding cluster, Listeria pathogenicity island 3, were identified by deletion mutagenesis, a real-time PCR assay which targets one such gene, llsX, was developed as a means of identifying LLS-positive L. monocytogenes. The specificity of the assay was validated against a panel of 40 L. monocytogenes strains (20 of which were LLS positive) and 25 strains representative of other Listeria species. Furthermore, 1 CFU of an LLS-positive strain per 25 g/ml of spiked foods was detected in less than 30 h when the assay was coupled with culture enrichment. The detection limit of this assay was 10 genome equivalents.     

Detection of Phanerochaete chrysosporium in soil by PCR and restriction enzyme analysis.

A nonradioactive method to detect Phanerochaete chrysosporium grown in a soil matrix was developed. This method involved DNA extraction, PCR amplification, and restriction enzyme analysis. Amplification of ligninase H8 DNA from pure cultures of P. chrysosporium was not as sensitive as amplification of the internal transcribed spacer (ITS) of the highly repetitive nuclear ribosomal DNA. Amplified ITS DNA was digested with restriction enzymes for analysis. The restriction enzyme pattern of PCR-amplified ITS DNA of P. chrysosporium was unique compared with those of unrelated fungi. Two strains of Phanerochaete chrysosporium and two strains of Phanerochaete sordida were indistinguishable by restriction enzyme analysis, while a third strain of P. chrysosporium had an unique pattern. These results were confirmed by sequence information and indicate that species designations of Phanerochaete spp. should be reexamined. The restriction enzyme pattern of DNA extracted and PCR amplified from P. chrysosporium grown in soil was identical to that from P. chrysosporium grown in pure culture. The ITS sequence was detected in 14 ng of the 100 micrograms of total DNA extracted from 1 g of soil.     

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.     

Genome sequence of the nonpathogenic Listeria monocytogenes serovar 4a strain M7

This report presents the complete and annotated genome sequence of the naturally nonpathogenic Listeria monocytogenes serovar 4a strain M7, isolated from cow's milk in Zhejiang province, China.     

Differentiation of Listeria monocytogenes and Listeria innocua by 16S rRNA genes and intraspecies discrimination of Listeria monocytogenes strains by random amplified polymorphic DNA polymorphisms.

Differences in the 16S rRNA genes (16S rDNA) which can be used to discriminate Listeria monocytogenes from Listeria innocua have been detected. The 16S rDNA were amplified by polymerase chain reaction with a set of oligonucleotide primers which flank a 1.5-kb fragment. Sequence differences were observed in the V2 region of the 16S rDNA both between L. monocytogenes Scott A and L. innocua and between different L. monocytogenes serotypes. Although L. monocytogenes SLCC2371 had the same V2 region sequence as L. innocua, the two species were different within the V9 region at nucleotides 1259 and 1292, in agreement with previous studies (R.-F. Wang, W.-W. Cao, and M.G. Johnson, Appl. Environ. Microbiol. 57:3666-3670, 1991). Intraspecies discrimination of L. monocytogenes strains was achieved by using the patterns generated by random amplified polymorphic DNA primers. Although some distinction can be made within the L. monocytogenes species by their 16S rDNA sequence, a far greater discrimination within species could be made by generating random amplified polymorphic DNA patterns from chromosomal DNA. By using a number of 10-bp primers, unique patterns for each isolate which in all cases examined differentiate between various L. monocytogenes serotypes, even though they may have the same 16S rRNA sequences, could be generated.     

Quantitative detection of Listeria monocytogenes in biofilms by real-time PCR

A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 x 10(2) CFU/cm2.     

Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 10² CFU/cm².     

Galactosylated wall teichoic acid,but not lipoteichoic acid,retains InlB on the surface of serovar 4b Listeria monocytogenes

Listeria monocytogenes is a Gram-positive, intracellular pathogen harboring the surface-associated virulence factor InlB, which enables entry into certain host cells. Structurally diverse wall teichoic acids (WTAs), which can also be differentially glycosylated, determine the antigenic basis of the various Listeria serovars. WTAs have many physiological functions; they can serve as receptors for bacteriophages, and provide a substrate for binding of surface proteins such as InlB. In contrast, the membrane-anchored lipoteichoic acids (LTAs) do not show significant variation and do not contribute to serovar determination. It was previously demonstrated that surface-associated InlB non-covalently adheres to both WTA and LTA, mediating its retention on the cell wall. Here, we demonstrate that in a highly virulent serovar 4b strain, two genes gtlB and gttB are responsible for galactosylation of LTA and WTA respectively. We evaluated the InlB surface retention in mutants lacking each of these two genes, and found that only galactosylated WTA is required for InlB surface presentation and function, cellular invasiveness and phage adsorption, while galactosylated LTA plays no role thereof. Our findings demonstrate that a simple pathogen-defining serovar antigen, that mediates bacteriophage susceptibility, is necessary and sufficient to sustain the function of an important virulence factor.