Cytolysin Activity Research Articles

Characteristics of Enterococcus Faecalis in Periodontitis and Secondary Root Canal Infections in Northern Jordanians-A Pilot Study

Background: Enterococcus faecalis has been found to be related to periodontitis and secondary endodontic infections. In this study, we aimed to study samples of E. faecalis to determine their incidence and virulence in Jordanian patients. Methods: A total of 167 samples were collected from patients with periodontitis and secondary endodontic infections. The Kirby-Bauer method was used to determine the antimicrobial susceptibility. Biofilm formation was studied using the microtiter plate assay and congo red agar assay. Polymerase chain reaction (PCR) was used to detect the presence of E. faecalis virulence genes, namely; asa1, gelE, cylA, esp, hyl, efaA, and ace. Gelatinase and cytolysin activity were also tested using phenotypic methods. Results: Twenty-three (13.8%) samples were positive for E. faecalis. The highest resistance rates were observed against ampicillin (87%), followed by penicillin (82.6%), and erythromycin (60.9%). The highest susceptibility was for levofloxacin (100%), followed by gentamycin and chloramphenicol, each with 95.7%. Most isolates were able to produce biofilm (78.3%). Gelatinase and cytolysin activity were detected in 21.7% and 56.5% of isolates, respectively. efaA was significantly associated with asa1 and gelE (P < 0.05), and esp was significantly associated with cylA, gelE, ace, efaA and asa1 ( P < 0.05). In addition, the gelE. gene was significantly associated with gelatinase production (P < 0.01). Conclusion: In conclusion, we have shown that E. faecalis is involved with periodontal disease and secondary root canal infection with several virulence genes detected. Clinical Relevance: Around 13% of the periodontitis and secondary root canal infection patients were positive for E. faecalis and isolates were resistant to commonly used antibiotics.

Dual functions of discoidinolysin, a cholesterol-dependent cytolysin with N-terminal discoidin domain produced from Streptococcus mitis strain Nm-76

ABSTRACT Background Some strains of Streptococcus mitis exhibit β-hemolysis due to the β-hemolytic activity of cholesterol-dependent cytolysin (CDC). Recently, a gene encoding an atypical lectinolysin-related CDC was found in S. mitis strain Nm-76. However, the product of this gene remains uncharacterized. We aimed to characterize this atypical CDC and its molecular functions and contribution to the pathogenicity of S. mitis strain Nm-76. Methods Phylogenetic analysis of the CDC gene was conducted based on the web-deposited information. The molecular characteristics of CDC were investigated using a gene-deletion mutant strain and recombinant proteins expressed in Escherichia coli. Results The gene encoding CDC found in Nm-76 and its homolog are distributed among many S. mitis strains. This CDC is phylogenetically different from other previously characterized CDCs, such as S. mitis-derived human platelet aggregation factor (Sm-hPAF)/lectinolysin and mitilysin. Because this CDC possesses an additional N-terminal domain, including a discoidin motif, it was termed discoidinolysin (DLY). In addition to the preferential lysis of human cells, DLY displayed N-terminal domain-dependent facilitation of human erythrocyte aggregation and intercellular associations between human cells. Conclusion DLY functions as a hemolysin/cytolysin and erythrocyte aggregation/intercellular association molecule. This dual-function DLY could be an additional virulence factor in S. mitis.

Pathogenic potential of Enterococcus faecalis strains isolated from root canals after unsuccessful endodontic treatment.

To evaluate strains of Enterococcus faecalis isolated from endodontic failures cases for (a) presence of virulence genes, namely, gelatinase production (gelE), surface protein (esp), collagen-binding adhesin (ace), cytolysin activator (cylA), E. faecalis antigen A (efaA) and aggregation substance (asa), all by using PCR; (b) biofilm formation capacity; and (c) activity of gelatinase and β-lactamase. Twenty-five strains of E. faecalis were tested. The DNA extracted from these strains was used for identification of virulence genes by PCR and 1% agarose gel. Biofilm formation was performed on polystyrene microplates by using the violet crystal staining method. For assessment of the gelatinase activity, inoculum of pure cultures was deposited in tubes containing gelatin and a nutrient broth, whereas nitrocefin disks were used to assess the β-lactamase action. The virulence genes efaA and cylA were detected in 100% of the strains, whereas gelE was present in 84%, ace in 68%, esp in 56% and asa in 48%. Four strains had no biofilm formation, 17 had poor formation and four had moderate formation. Gelatinase production was observed in three strains and β-lactamase resistance in five strains of E. faecalis. Diverse patterns of virulence gene detection were observed among the E. faecalis strains, with predominance of those capable of forming biofilm. A few strains have been found to hydrolyze gelatin proteins, whereas β-lactamase resistance was detected in different isolates. To understand the influence of virulence factors in E. faecalis on the host heath status.

Typing of Enterococcus spp. strains in 4 hospitals in the Małopolska region in Poland.

In the Małopolska province, the first case of vancomycin resistant enterococci (VRE) occurrence was an outbreak in 2001 caused by strains of the genus E. faecium carrying the vanA operon. The aim of this study is to determine the antimicrobial resistance and the occurrence of virulence determinants among Enterococcus spp. in patients hospitalized in the Małopolska region in 2015. Antimicrobial susceptibility was determined by disc diffusion and the E test. The presence of aminoglycoside and glycopeptide resistance genes and virulence genes (asa1, gelE, cylA, esp, hyl) was investigated using multiplex polymerase chain reaction (PCR). Also, the presence of IS16 was investigated. The activity of gelatinase, cytolysin (hemolysin), and DNase was tested. All E. faecalis were susceptible to ampicillin, vancomycin, teicoplanin, linezolid and tigecycline. All E. faecium strains were susceptible to quinupristin-dalfopristin. Resistance to ampicillin and vancomycin was detected among all E. faecium isolates from hospitals C and D. 87.32% of E. faecium presented high-level aminoglycoside-resistant (HLAR) phenotype, including 78.33% of strains from hospital C and 100% from hospital D. In hospital C (98.3%) and D (96%), resistance to ciprofloxacin, levofloxacin and norfloxacin was observed. Gene esp was detected in all E. faecium isolates and the majority of E. faecium isolates carried hyl (97%). In E. faecalis, different combinations of virulence genes were detected. All analyzed E. faecium strains showed the presence of IS16 insertion element. E. faecalis isolates were more susceptible to antimicrobials than E. faecium, which were largely multidrug-resistant. E. faecalis strains have diverse virulence factors. E. faecium showed a high percentage of hyl and esp genes and the presence of IS16.

A Novel Biomimetic Nanosponge Protects the Retina from the Enterococcus faecalis Cytolysin.

Intraocular infections are a potentially blinding complication of common ocular surgeries and traumatic eye injuries. Bacterial toxins synthesized in the eye can damage intraocular tissue, often resulting in poor visual outcomes. Enteroccocus faecalis causes blinding infections and is responsible for 8 to 17% of postoperative endophthalmitis cases. These infections are increasingly difficult to treat due to the emergence of multidrug-resistant strains. Virulent E.faecalis isolates secrete a pore-forming bicomponent cytolysin that contributes to retinal tissue damage during endophthalmitis. We hypothesized that a biomimetic nanosponge, which mimics erythrocytes, might adsorb subunits of the cytolysin and reduce retinal damage, protecting vision. To test the efficacy of nanosponges in neutralizing the cytolysin in vitro, hemoglobin release assays were performed on culture supernatants from cytolysin-producing E.faecalis with and without preincubation with nanosponges. Treatment with nanosponges for 30min reduced hemolytic activity by ~70%. To determine whether nanosponges could neutralize the cytolysin in vivo, electroretinography was performed on mice 24h after intravitreal injection with cytolysin-containing supernatants treated with nanosponges. Pretreatment of cytolysin-containing supernatants with nanosponges increased the A-wave retention from 12.2% to 65.5% and increased the B-wave retention from 21.0% to 77.0%. Histology revealed that in nanosponge-treated eyes, retinas remained intact and attached, with little to no damage. Rabbit nanosponges were also nontoxic and noninflammatory when injected into mouse eyes. In an experimental murine model of E.faecalis endophthalmitis, injection of nanosponges into the vitreous 6h after infection with a wild-type cytolysin-producing strain increased A-wave retention from 5.9% to 31% and increased B-wave retention from 12.6% to 27.8%. Together, these results demonstrated that biomimetic nanosponges neutralized cytolysin activity and protected the retinas from damage. These results suggest that this novel strategy might also protect eyes from the activities of pore-forming toxins of other virulent ocular bacterial pathogens. IMPORTANCE Endophthalmitis is a serious, potentially blinding infection that can result in vision loss, leaving a patient with only the ability to count fingers, or it may require enucleation of the globe. The incidence of postoperative endophthalmitis has markedly increased over the past 2 decades, paralleling the rise in ocular surgeries and intravitreal therapies. E.faecalis is a leading cause of infection following ocular procedures, and such infections are increasingly difficult to treat due to multidrug resistance. Cytolysin is the primary virulence factor responsible for retinal tissue damage in E.faecalis eye infections. Treatment of these infections with antibiotics alone does not impede ocular damage and loss of visual function. Pore-forming toxins (PFTs) have been established as major virulence factors in endophthalmitis caused by several bacterial species. These facts establish a critical need for a novel therapy to neutralize bacterial PFTs such as cytolysin. Here, we demonstrate that biomimetic nanosponges neutralize cytolysin, protect the retina, preserve vision, and may provide an adjunct detoxification therapy for bacterial infections.

Genotypic characterization of Enterococcus species isolated from the oral cavity and their pattern of antibiotic susceptibility

Background: Enterococci comprise a small share of oral flora and occur as contaminants in food such as meat and cheese. They are commonly encountered in patients with infections of oral tissues such as necrotic pulp, root canals and periodontitis. Objectives: To characterize the genotypes of Enterococcus isolated from patients with oral infection and to determine their antibiotic susceptibility. Methods: We genotypically characterized a number of isolates of Enterococcus species derived from patients with various oral infections. All the isolates were tested for the presence of putative virulence genes; efaA (gene for endocarditis), gelE (gene for gelatinase), ace (gene for collagen binding antigen), asa (gene for aggregation substance), cylA (gene for cytolysin activator), and esp (gene for surface adhesin) of E. faecalis and E. faecium as described in previous studies. Results: E. faecalis dominated in oral infections compared with E. faecium. Isolates were susceptible to most antibiotics (only four were resistant to vancomycin). To our knowledge, the first report from this region. Conclusions: Treatment of enterococcal infections of the oral mucosal and deep oral areas necessitate microbiological diagnosis and identification of resistance strains. Keywords: Antibiotic susceptibility pattern, Enterococcus species, genotypic characterization, oral infections

Screening of the Enterocin-Encoding Genes and Antimicrobial Activity in Enterococcus Species

In the current study, a total of 135 enterococci strains from different sources were screened for the presence of the enterocin-encoding genes entA, entP, entB, entL50A, and entL50B. The enterocin genes were present at different frequencies, with entA occurring the most frequently, followed by entP and entB; entL50A and L50B were not detected. The occurrence of single enterocin genes was higher than the occurrence of multiple enterocin gene combinations. The 80 isolates that harbor at least one enterocin-encoding gene (denoted "Gene(+) strains") were screened for antimicrobial activity. A total of 82.5% of the Gene(+) strains inhibited at least one of the indicator strains, and the isolates harboring multiple enterocin-encoding genes inhibited a larger number of indicator strains than isolates harboring a single gene. The indicator strains that exhibited growth inhibition included Listeria innocua strain CLIP 12612 (ATCC BAA-680), Listeria monocytogenes strain CDC 4555, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, S. aureus ATCC 29213, S. aureus ATCC 6538, Salmonella enteritidis ATCC 13076, Salmonella typhimurium strain UK-1 (ATCC 68169), and Escherichia coli BAC 49LT ETEC. Inhibition due to either bacteriophage lysis or cytolysin activity was excluded. The growth inhibition of antilisterial Gene+ strains was further tested under different culture conditions. Among the culture media formulations, the MRS agar medium supplemented with 2% (w/v) yeast extract was the best solidified medium for enterocin production. Our findings extend the current knowledge of enterocin-producing enterococci, which may have potential applications as biopreservatives in the food industry due to their capability of controlling food spoilage pathogens.

Virulence of Bacteria Colonizing Vascular Bundles in Ischemic Lower Limbs

We documented previously the presence of bacterial flora in vascular bundles, lymphatics, and lymph nodes of ischemic lower limbs amputated because of multifocal atheromatic changes that made them unsuitable for reconstructive surgery and discussed their potential role in tissue destruction. The question arose why bacterial strains inhabiting lower limb skin and considered to be saprophytes become pathogenic once they colonize deep tissues. Bacterial pathogenicity is evoked by activation of multiple virulence factors encoded by groups of genes. We identified virulence genes in bacteria cultured from deep tissue of ischemic legs of 50 patients using a polymerase chain reaction technique. The staphylococcal virulence genes fnbA (fibronectin-binding protein A), cna (collagen adhesin precursor), and ica (intercellular adhesion) were present in bacteria isolated from both arteries and, to a lesser extent, skin. The IS256 gene, whose product is responsible for biofilm formation, was more frequent in bacteria retrieved from the arteries than skin bacteria. Among the virulence genes of Staphylococcus epidermidis encoding autolysin atlE, icaAB (intercellular adhesion), and biofilm insert IS256, only the latter was detected in arterial specimens. Bacteria cultured from the lymphatics did not reveal expression of eta and IS256 in arteries. The Enterococcus faecalis asa 373 (aggregation substance) and cylA (cytolysin activator) frequency was greater in arteries than in skin bacteria, as were the E. faecium cyl A genes. All Pseudomonas aeruginosa virulence genes were present in bacteria cultured from both the skin and arteries. Staphylococci colonizing arterial bundles and transported to tissues via ischemic limb lymphatics expressed virulence genes at greater frequency than did those dwelling on the skin surface. Moreover, enterococci and Pseudomonas isolated from arterial bundles expressed many virulence genes. These findings may add to the understanding of the mechanism of development of destructive changes in lower limb ischemic tissues by the patient's, but not hospital-acquired, bacteria, as well as the generally unsatisfactory results of antibiotic administration in these cases. More aggressive antibiotic therapy targeted at the virulent species should be applied.

Investigation of virulence factors of Enterococcus faecalis strains isolated in secondary/ persistent infections

Objective: More virulent strains may result from the acquisition of genes by genetic exchange, pathogenicity islands in several species encoding toxins, adhesion factors and other factors associated with virulence. The aim of this study was to investigate the prevalence of E. faecalis strains in secondary endodontic/ persistent using endodontic infection by culture and PCR technqiues; and to investigate for the presence of virulence factor genes of gelatinase (gelE), cytolysin activator (Cyla), surface adhesin of Enterococcus (ESP) and collagen adhesin of Enterococcus (ACE). Material and methods: Microbial samples were obtained from 12 teeth with secondary/ persistent endodontic infection showing apical periodontitis. Culture techniques were used including serial dilution, plating, incubation, and biochemical identification. For PCR detection, samples were analyzed using a species-specific primer of the 16S rDNA and the downstream intergenic spacer region. Results: Culture and PCR detected the test species in 3/12 (25%) and 5/12 (41.6%) of teeth, respectively. A total of 38 Enterococcus faecalis strains were isolated and submitted to the virulence factor genes analysis. PCR products consistent with genes encoding surface adhesion (ESP), gelatinase (gelE) and collagen binding antigen (ACE) were found in 26/38 (68%), 31/38 (81%) and 38/38 (100%) of the isolates. The Cytolysin activator (Cyla) gene was not recovered from E. faecalis isolates. Conclusions: In conclusion, the present study revealed by culture and molecular methods revealed a high prevalence of E. faecalis in teeth with secondary/ persistent endodontic infection. Moreover, of a clinical relevance, we found different E. faecalis strains carrying different virulence determinants.KEYWORDS Bacteria; E. faecalis; Root canal, virulence.

Incidence of virulence factors in enterococci from raw and fermented meat and biofilm forming capacity at 25 °C and 37 °C

Twenty-nine Enterococcus strains from raw and fermented meat products were screened for the presence of virulence genes, including those for aggregation substances (asa1 and asa373), cytolysin activator (cylA), collagen binding protein (ace), endocarditis antigen (efaA), enterococcal surface protein (esp) and gelatinase (gelE). Virulence gene occurrence, expression of gelatinase and pheromone aggregation was greater in Enterococcus faecalis than in Enterococcus faecium strains. All E. faecalis and 54% of E. faecium were positive for at least one or more virulence gene. The only strain of Enterococcus gallinarum tested also contained virulence genes. The effect of different growth temperatures (25 and 37°C) on biofilm formation using polystyrene plates was also assessed. Strong biofilm formation occurred at lower than optimum temperature in all three species of enterococci. Neither esp nor gelE was necessary for biofilm formation and this relationship was species rather than strain specific. This study emphasizes the importance of enterococci as a reservoir of virulence genes and the potential for their genetic transfer to human strains following consumption of uncooked or undercooked contaminated meat.

Subcytolytic effects of suilysin on interaction of Streptococcus suis with epithelial cells

Suilysin is a pore-forming cholesterol-dependent cytolysin secreted by Streptococcus suis (S. suis), an important swine and zoonotic pathogen. The role of suilysin in S. suis host–cell interaction is still unclear. We found a higher adherence and invasion rate of an unencapsulated sly-positive strain in comparison to its isogenic sly-negative mutant. Electron microscopy revealed that formation of membrane ruffles accompanying invasion of the sly-positive strain was abolished in the sly-negative mutant. Inhibition experiments showed that the actin cytoskeleton was involved in suilysin-mediated effects. Point-mutation of the domain putatively responsible for macropore-formation resulted in abolished hemolytic and cytolysin activity, but had no effect on S. suis host cell association. Concluding, our results indicate that subcytolytic suilysin promotes S. suis association with epithelial cells.

What planar lipid membranes tell us about the pore-forming activity of cholesterol-dependent cytolysins

Pore-forming toxins are an important group of natural molecules that damage cellular membranes by forming transmembrane pores. They are used by many organisms for attack or defense and similar proteins are employed in the immune system of vertebrates. Various biophysical approaches have been used to understand how these proteins act at the molecular level. One of the most useful, in terms of monitoring pore formation in real time, is a method that employs planar lipid membranes and involves ionic current measurements. Here we highlight the advantages and possibilities that this approach offers and show how it can advance understanding of the pore-forming mechanism and pore properties for one of the most important families of natural toxins, the cholesterol-dependent cytolysins.

Virulence factors and antibiotic susceptibility in enterococci isolated from oral mucosal and deep infections

Objective This study evaluates the presence of virulence factors and antibiotic susceptibility among enterococcal isolates from oral mucosal and deep infections.Methods Forty-three enterococcal strains from oral mucosal lesions and 18 from deep infections were isolated from 830 samples that were sent during 2 years to Oral Microbiology, University of Gothenburg, for analysis. The 61 strains were identified by 16S rDNA, and characterized by the presence of the virulence genes efa A (endocarditis gene), gel E (gelatinase gene), ace (collagen binding antigen gene), asa (aggregation substance gene), cyl A (cytolysin activator gene) and esp (surface adhesin gene), tested for the production of bacteriocins and presence of plasmids. MIC determination was performed using the E-test method against the most commonly used antibiotics in dentistry, for example, penicillin V, amoxicillin and clindamycin. Vancomycin was included in order to detect vancomycin-resistant enterococci (VRE) strains.Results Sixty strains were identified as Enterococcus faecalis and one as Enterococcus faecium. All the virulence genes were detected in more than 93.3% (efa A and esp) of the E. faecalis strains, while the presence of phenotypic characteristics was much lower (gelatinase 10% and hemolysin 16.7%). Forty-six strains produced bacteriocins and one to six plasmids were detected in half of the isolates.Conclusions Enterococcal strains from oral infections had a high virulence capacity, showed bacteriocin production and had numerous plasmids. They were generally susceptible to ampicillins but were resistant to clindamycin, commonly used in dentistry, and no VRE-strain was found.

The occurrence of virulence traits among high-level aminoglycosides resistant Enterococcus isolates obtained from feces of humans, animals, and birds in South Korea

Enterococcus isolates (1500) obtained from the feces of 48 humans, 209 domesticated food animals, and 155 wild geese in South Korea were characterized with respect to species status by PCR analyses and resistance to antibiotics. Of the 1500 strains examined, the majority (n=577) were Enterococcus faecalis from 224 (54.4%) of the samples feces, while 299 were of E. faecium from 125 of the samples (30.3%), 224 were E. hirae from 101 (24.5%) of the samples, 94 were E. casseliflavus from 43 (10.4%) of the samples, and one was E. gallinarum. While 305 isolated from 125 (30.3%) of the samples were unidentified species. Approximately 15, 60, 50, 55, 3, and 40% of samples obtained from beef cattle, chickens, ducks, swine, wild geese, and humans, respectively, yielded Enterococcus isolates that were resistant to high-levels of aminoglycosides (i.e., of gentamicin, kanamycin, and streptomycin, minimum inhibitory concentrations were >1000mg/l). The 180 Enterococcus isolates that showed high levels of resistance to aminoglycoside antibiotics (HLAR) were screened for virulence genes encoding for aggregation substance (agg), cytolysin activator (cylA), gelatinase (gelE) and surface protein (esp). Of those, the gelE gene was found most frequently in chickens and ducks of the HLAR isolates, while 56 E. faecalis and 13 E. faecium HLAR were gelatinase positive and showed hemolysin activity. Multiple antibiotic resistant Enterococcus isolates carrying virulence genes were most frequently isolated from poultry and swine, and were mostly E. faecalis or E. faecium. These findings suggest that restriction of the use of antibiotics in food animal operations in South Korea, especially those involved in poultry and swine production would be desirable.

Mutations Affecting Export and Activity of Cytolysin A from Escherichia coli

Cytolysin A (known as ClyA, HlyE, and SheA) is a cytolytic pore-forming protein toxin found in several Escherichia coli and Salmonella enterica strains. The structure of its water-soluble monomeric form and that of dodecameric ClyA pores is known, but the mechanisms of ClyA export from bacterial cells and of pore assembly are only partially understood. Here we used site-directed mutagenesis to study the importance of different regions of the E. coli ClyA protein for export and activity. The data indicate that ClyA translocation to the periplasm requires several protein segments located closely adjacent to each other in the "tail" domain of the ClyA monomer, namely, the N- and C-terminal regions and the hydrophobic sequence ranging from residues 89 to 101. Deletion of most of the "head" domain of the monomer (residues 181 to 203), on the other hand, did not strongly affect ClyA secretion, suggesting that the tail domain plays a particular role in export. Furthermore, we found that the N-terminal amphipathic helix alphaA1 of ClyA is crucial for the formation and the properties of the transmembrane channel, and hence for hemolytic activity. Several mutations affecting the C-terminal helix alphaG, the "beta-tongue" region in the head domain, or the hydrophobic region in the tail domain of the ClyA monomer strongly impaired the hemolytic activity and reduced the activity toward planar lipid bilayer membranes but did not totally prevent formation of wild-type-like channels in these artificial membranes. The latter regions thus apparently promote membrane interaction without being directly required for pore formation in a lipid bilayer.

Cytolysins, Superantigens, and Pneumonia Due to Community‐Associated Methicillin‐ResistantStaphylococcus aureus

Cytolysins, Superantigens, and Pneumonia Due to Community‐Associated Methicillin‐Resistant<i>Staphylococcus aureus</i>

Isolation of VanB-Type Enterococcus faecalis Strains from Nosocomial Infections: First Report of the Isolation and Identification of the Pheromone-Responsive Plasmids pMG2200, Encoding VanB-Type Vancomycin Resistance and a Bac41-Type Bacteriocin, and pMG2201, Encoding Erythromycin Resistance and Cytolysin (Hly/Bac)

Eighteen identical VanB-type Enterococcus faecalis isolates that were obtained from different hospitalized patients were examined for their drug resistance and plasmid DNAs. Of the 18 strains, 12 strains exhibited resistance to erythromycin (Em), gentamicin (Gm), kanamycin (Km), tetracycline (Tc), and vancomycin (Van) and produced cytolysin (Hly/Bac) and a bacteriocin (Bac) active against E. faecalis strains. Another six of the strains exhibited resistance to Gm, Km, Tc, and Van and produced a bacteriocin. Em and Van resistance was transferred individually to E. faecalis FA2-2 strains at a frequency of about 10(-4) per donor cell by broth mating. The Em-resistant transconjugants and the Van-resistant transconjugants harbored a 65.7-kbp plasmid and a 106-kbp plasmid, respectively. The 106-kbp and 65.7-kbp plasmids isolated from the representative E. faecalis NKH15 strains were designated pMG2200 and pMG2201, respectively. pMG2200 conferred vancomycin resistance and bacteriocin activity on the host strain and responded to the synthetic pheromone cCF10 for pCF10, while pMG2201 conferred erythromycin resistance and cytolysin activity on its host strain and responded to the synthetic pheromone cAD1 for pAD1. The complete DNA sequence of pMG2200 (106,527 bp) showed that the plasmid carried a Tn1549-like element encoding vanB2-type resistance and the Bac41-like bacteriocin genes of pheromone-responsive plasmid pYI14. The plasmid contained the regulatory region found in pheromone-responsive plasmids and encoded the genes prgX and prgQ, which are the key negative regulatory elements for plasmid pCF10. pMG2200 also encoded TraE1, a key positive regulator of plasmid pAD1, indicating that pMG2200 is a naturally occurring chimeric plasmid that has a resulting prgX-prgQ-traE1 genetic organization in the regulatory region of the pheromone response. The functional oriT region and the putative relaxase gene of pMG2200 were identified and found to differ from those of pCF10 and pAD1. The putative relaxase of pMG2200 was classified as a member of the MOB(MG) family, which is found in pheromone-independent plasmid pHTbeta of the pMG1-like plasmids. This is the first report of the isolation and characterization of a pheromone-responsive highly conjugative plasmid encoding vanB resistance.

Irreversible loss of membrane-binding activity of Listeria-derived cytolysins in non-acidic conditions: a distinct difference from allied cytolysins produced by other Gram-positive bacteria

Listeriolysin O (LLO), a member of the cholesterol-dependent cytolysin (CDC) family, is a major virulence factor of Listeria monocytogenes and contributes to bacterial escape from intracellular killing of macrophages. LLO is activated under weakly acidic conditions; however, the molecular mechanism of this pH-dependent expression of cytolytic activity of LLO is poorly understood. In this study, CDCs including LLO, ivanolysin O (ILO), seeligeriolysin O (LSO), pneumolysin (PLY), streptolysin O (SLO) and perfringolysin O (PFO) were prepared as recombinant proteins and examined for their functional changes after treatment under various pH conditions. Haemolytic and membrane cholesterol-binding activities were not affected in PLY, SLO and PFO at any pH examined. By contrast, all the Listeria-derived cytolysins, LLO, ILO and LSO, were active only at an acidic pH and rapidly inactivated under neutral or alkaline conditions. Once inactivated, LLO could not be reactivated even by a downward pH shift. The hydrophobicity of LLO treated at neutral or alkaline pH was increased. These data suggested that the pH-dependent loss of cytolytic activity appeared to be due to irreversible structural changes of domain 4 that resulted in the loss of target membrane cholesterol binding.

Virulence, phenotype and genotype characteristics of endodontic Enterococcus spp.

Enterococci have been implicated in persistent root canal infections but their role in the infection process remains unclear. This study investigated the virulence, phenotype and genotype of 33 endodontic enterococcal isolates. Phenotypic tests were conducted for antibiotic resistance, clumping response to pheromone, and production of gelatinase, hemolysin and bacteriocin. Genotype analysis involved polymerase chain reaction amplification of virulence determinants encoding aggregation substances asa and asa373, cytolysin activator cylA, gelatinase gelE, gelatinase-negative phenotype ef1841/fsrC, adherence factors esp and ace, and endocarditis antigen efaA. Physical DNA characterization involved pulsed-field gel electrophoresis of genomic DNA, and plasmid analysis. Potential virulence traits expressed included production of gelatinase by Enterococcus faecalis (n=23), and response to pheromones in E. faecalis culture filtrate (n=16). Fourteen strains produced bacteriocin. Five strains were resistant to tetracycline and one to gentamicin, whereas all were susceptible to ampicillin, benzylpenicillin, chloramphenicol, erythromycin, fusidic acid, kanamycin, rifampin, streptomycin and vancomycin. Polymerase chain reaction products encoding efaA, ace, and asa were detected in all isolates; esp was detected in 20 isolates, cylA in six isolates, but asa373 was never detected. The gelatinase gene (gelE) was detected in all isolates of E. faecalis (n=31) but not in Enterococcus faecium (n=2); a 23.9 kb deletion sequence corresponding to the gelatinase-negative phenotype was detected in six of the eight E. faecalis isolates that did not produce gelatinase. Pulsed-field gel electrophoresis and plasmid analyses revealed genetic polymorphism with clonal types evident. Plasmid DNA was detected in 25 strains, with up to four plasmids per strain and a similar (5.1 kb) plasmid occurring in 16 isolates. Phenotypic and genotypic evidence of potential virulence factors were identified in endodontic Enterococcus spp., specifically production of gelatinase and response to pheromones.

Differences in gamma interferon production induced by listeriolysin O and ivanolysin O result in different levels of protective immunity in mice infected with Listeria monocytogenes and Listeria ivanovii.

Two pathogenic species in the genus Listeria, Listeria monocytogenes and Listeria ivanovii, are characterized by the production of hemolysins belonging to cholesterol-dependent cytolysins, listeriolysin O (LLO) and ivanolysin O (ILO), respectively. LLO, produced by L. monocytogenes, is able to induce gamma interferon (IFN-gamma) production and contributes to the generation of Th1-dependent protective immunity. On the other hand, nothing is known about the role of ILO, produced by L. ivanovii, in this regard. In this study, we immunized mice with 0.1 50% lethal dose (LD(50)) of L. monocytogenes and L. ivanovii. Protective immunity against a challenge with 10 LD(50) was generated in mice infected with L. monocytogenes, whereas L. ivanovii infection did not induce protection. After immunization, the level of IFN-gamma in serum samples was increased in mice given L. monocytogenes but not in those given L. ivanovii. To determine the IFN-gamma-inducing activity of cytolysins, recombinant protein was constructed. Recombinant ILO exhibited significantly lower IFN-gamma-inducing activity than LLO. By comparing the IFN-gamma-inducing activity of a chimera incorporating LLO and ILO, it was found that domains 1 to 3 of LLO were critical for IFN-gamma-inducing activity while the counterpart in ILO was unable to induce cytokine production. These results suggested that the weak ability of ILO to induce IFN-gamma production is responsible for the failure of L. ivanovii to generate effective protective immunity.