Two novel type II restriction-modification systems occupying genomically equivalent locations on the chromosomes of Listeria monocytogenes strains

Listeria monocytogenes is responsible for the potentially life-threatening food-borne disease listeriosis. One epidemic-associated clonal group of L. monocytogenes, epidemic clone I (ECI), harbors a Sau3AI-like restriction-modification (RM) system also present in the same genomic region in certain strains of other lineages. In this study, we identified and characterized two other, novel type II RM systems, LmoJ2 and LmoJ3, at this same locus. LmoJ2 and LmoJ3 appeared to recognize GCWGC (W = A or T) and GCNGC, respectively. Both RM systems consisted of genes with GC content below the genome average and were in the same genomic region in strains of different serotypes and lineages, suggesting site-specific horizontal gene transfer. Genomic DNA from the LmoJ2 and LmoJ3 strains grown at various temperatures (4 to 42°C) was resistant to digestion with restriction enzymes recognizing GCWGC or GCNGC, indicating that the methyltransferases were expressed under these conditions. Phages propagated in an LmoJ2-harboring strain exhibited moderately increased infectivity for this strain at 4 and 8°C but not at higher temperatures, while phages propagated in an LmoJ3 strain had dramatically increased infectivity for this strain at all temperatures. Among the sequenced Listeria phages, lytic phages possessed significantly fewer recognition sites for these RM systems than lysogenic phages, suggesting that in lytic phages sequence content evolved toward reduced susceptibility to such RM systems. The ability of LmoJ2 and LmoJ3 to protect against phages may affect the efficiency of phages as biocontrol agents for L. monocytogenes strains harboring these RM systems.     

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.     

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.     

Characterization of lytic enzyme open reading frame 9 (ORF9) derived from Enterococcus faecalis bacteriophage phiEF24C

In bacteriophage (phage) therapy against Gram-positive bacteria, such as Staphylococcus aureus, Listeria monocytogenes, and Enterococcus faecalis, members of a genus of SPO1-like viruses are typically employed because of their extreme virulence and broad host spectrum. Phage φEF24C, which is a SPO1-like virus infecting E. faecalis, has previously been characterized as a therapeutic phage candidate. In addition to the phage itself, phage endolysin is also recognized as an effective antimicrobial agent. In this study, a putative endolysin gene (orf9) of E. faecalis phage φEF24C was analyzed in silico, and its activity was characterized using the recombinant form. First, bioinformatics analysis predicted that the open reading frame 9 (ORF9) protein is N-acetylmuramoyl-l-alanine amidase. Second, bacteriolytic and bactericidal activities of ORF9 against E. faecalis were confirmed by zymography, decrease of peptidoglycan turbidity, decrease of the viable count, and morphological analysis of ORF9-treated cells. Third, ORF9 did not appear to require Zn(2+) ions for its activity, contrary to the bioinformatics prediction of a Zn(2+) ion requirement. Fourth, the lytic spectrum was from 97.1% (34 out of 35 strains, including vancomycin-resistant strains) of E. faecalis strains to 60% (6 out of 10 strains) of Enterococcus faecium strains. Fifth, N-acetylmuramoyl-l-alanine amidase activity of ORF9 was confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and the subsequent MALDI-postsource decay (PSD) analyses. Finally, functional analysis using N- or C-terminally deleted ORF9 mutants suggested that a complete ORF9 molecule is essential for its activity. These results suggested that ORF9 is an endolysin of phage φEF24C and can be a therapeutic alternative to antibiotics.     

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.     

Sequence analysis and characterization of plasmids from Streptococcus thermophilus

The nucleotide sequences of eight plasmids isolated from seven Streptococcus thermophilus strains have been determined. Plasmids pSt04, pER1-1, and pJ34 are related and replicate via a rolling circle mechanism. Plasmid pJ34 encodes for a replication initiation protein (RepA) and a small polypeptide with unknown function. Plasmids pSt04 and pER1-1 carry in addition to repA genes coding for small heat shock proteins (sHsp). Expression of these proteins is induced at elevated temperatures or low pH and increases the thermo- and acid resistance. Plasmids pER1-2 and pSt22-2 show identical sequences with five putative open reading frames (ORFs). The gene products of ORF1 and ORF4 reveal some similarities to transposon encoded proteins of Bacillus subtilis and Tn916. ORF1 of plasmid pSt106 encodes a protein similar to resolvases of different Gram-positive bacteria. Integrity of ORF2 and 3, encoding a putative DNA primase and a replication protein, is essential for replication. ORF1 to 3 of plasmid pSt08, which are organized in a tricistronic operon, encode a RepA protein, an adenosine-specific methyltransferase, and a type II restriction endonuclease. Another type II restriction-modification (R/M) system is encoded on plasmid pSt0 which is highly similar to those encoded on lactococcal plasmid pHW393 and B. subtilis plasmid pXH13. Plasmid-free derivatives of strains St0 and St08 show increased phage sensitivity, indicating that in the wild-type strains the R/M systems are functionally expressed. Recombinant plasmids based on the replicons of plasmids pSt04, pJ34, pSt106, pSt08, and pSt0, are able to replicate in Lactococcus lactis and B. subtilis, respectively, whereas constructs carrying pER1-2 only replicate in S. thermophilus.     

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.     

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.     

An efficient and reproducible method for transformation of genetically recalcitrant bifidobacteria

The epidemic community-associated methicillin-resistant clone Staphylococcus aureus USA300 is a major source of skin and soft tissue infections and involves strains with a diverse set of resistance genes. In this study, we report efficient transduction of penicillinase and tetracycline resistance plasmids by bacteriophages φ80α and φJB between clinical isolates belonging to the USA300 clone. High transduction frequencies (10(-5) - 10(-6) CFU/PFU) were observed using phages propagated on donor strains as well as prophages induced from donors by ultraviolet light. Quantitative real-time PCR was employed to detect penicillinase plasmids in transducing phage particles and determine the ratio of transducing particles in phage lysates to infectious phage particles (determined as approximately 1 : 1700). Successful transfer of plasmids between strains in USA300 clone proves transduction is an effective mechanism for spreading plasmids within the clone. Such events contribute to its evolution and to emergence of new multiple drug-resistant strains of this successful clone.     

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.     

comK prophage junction fragments as markers for Listeria monocytogenes genotypes unique to individual meat and poultry processing plants and a model for rapid niche-specific adaptation, biofilm formation, and persistence

Different strains of Listeria monocytogenes are well known to persist in individual food processing plants and to contaminate foods for many years; however, the specific genotypic and phenotypic mechanisms responsible for persistence of these unique strains remain largely unknown. Based on sequences in comK prophage junction fragments, different strains of epidemic clones (ECs), which included ECII, ECIII, and ECV, were identified and shown to be specific to individual meat and poultry processing plants. The comK prophage-containing strains showed significantly higher cell densities after incubation at 30°C for 48 h on meat and poultry food-conditioning films than did strains lacking the comK prophage (P < 0.05). Overall, the type of strain, the type of conditioning film, and the interaction between the two were all highly significant (P < 0.001). Recombination analysis indicated that the comK prophage junction fragments in these strains had evolved due to extensive recombination. Based on the results of the present study, we propose a novel model in which the concept of defective comK prophage was replaced with the rapid adaptation island (RAI). Genes within the RAI were recharacterized as "adaptons," as these genes may allow L. monocytogenes to rapidly adapt to different food processing facilities and foods. If confirmed, the model presented would help explain Listeria's rapid niche adaptation, biofilm formation, persistence, and subsequent transmission to foods. Also, comK prophage junction fragment sequences may permit accurate tracking of persistent strains back to and within individual food processing operations and thus allow the design of more effective intervention strategies to reduce contamination and enhance food safety.     

Highly discriminatory typing method for Listeria monocytogenes using polymorphic tandem repeat regions

Tandem repeats (TR), which are repetitive nucleotide sequences in DNA, are polymorphic both in repeat number and sequence. In this study, we developed a new typing method, multilocus TR sequence analysis (MLTSA), for the foodborne pathogen Listeria monocytogenes using sequence polymorphisms in three tandem repeat regions. The obtained dendrogram clustered L. monocytogenes strains of lineage I and lineage II separately, and formed three groups within the lineage I cluster, each of which included one of the three major L. monocytogenes epidemic clones (ECI, ECIa, and ECII). These results were consistent with a previously established virulence-gene-based MLST method. In comparison, our method grouped some epidemiologically related isolates together, which virulence-gene-based MLST did not. Moreover, our method, using three tandem repeat regions, showed a higher discriminatory power than the MLST method, which uses six virulence gene regions. This MLTSA approach using sequence polymorphisms in TR regions could be a useful tool in the epidemiological study of L. monocytogenes.     

Recipient capacity of clinical strains of staphylococci belonging to different phage groups]

The recipient capacity of the strains of Staph. epidermidis and Staph. areus belonging to different phage groups, as well as the possibility of epidemic distribution of the erythromycin resistance marker among the clinical staphyloccal strains on using the defective phage obtained from strain 8325 P IIde was studied. The defective phage P IIde may be the source of epidemic distribution of the drug resistance among the competent strains of Staph. aureus. All erythromycin sensitive strains of Staph. aureus lysed by the phages of groups I and III proved to be competent recipients of the erythromycin resistance marker. The strains of Staph. aureus of phage group II and phage type 80/81, as well as the strains of Staph. epidermidis were not competent recipients under our experimental conditions. It was not possible to transfer the high level of erythromycin resistance (1000 gamma/ml) on transduction to the strains of phage group I with a relatively low level of resistance to this antibiotic (20-50 gamma/ml.     

Analysis of epidemic and sporadic strains of Listeria monocytogenes by pyrolysis mass spectrometry

Twenty-six cultures of Listeria monocytogenes , serovar 4b, including 10 from a food-borne outbreak in Switzerland and sporadic patient and food isolates from both Switzerland and the UK, were compared by pyrolysis mass spectrometry (PMS). This clustered all of the Swiss epidemic isolates with four other isolates indistinguishable from the Swiss strain by phage typing, a phage non-typable isolate from a Swiss patient not known to be part of the epidemic and two strains (both from Switzerland) of a different phage type. The eight strains excluded from the PMS-derived cluster were all either known to be unrelated to the epidemic, or of a phage type distinct from the epidemic strain, or both.