Molecular analysis of dominant species in Listeria monocytogenes-positive biofilms in the drains of food processing facilities.

Listeria monocytogenes exhibits symbiotic codependence with the dominant commensal bacteria, which may help it avoid being removed or inactivated by disinfectants in local environments. In this study, we investigated L. monocytogenes-positive biofilms at food production facilities, and the dominant bacterial species of the biofilms were identified to determine the properties of the microbiological background. For this purpose, the ISO 11290 method was used for the detection and isolation of L. monocytogenes, and the species were further identified based on 16S rRNA and hly genes. 16S rRNA gene-based cloning, terminal restriction fragment length polymorphism, and denaturing gradient gel electrophoresis were combined to evaluate the dominant bacteria of the drain biofilms. Out of 100 drain samples, 8 were naturally contaminated with L. monocytogenes. Three molecular methods consistently showed that Pseudomonas psychrophila, Pseudomonas sp., and Klebsiella oxytoca were dominant species in 3Q, 5Q, and 6Q samples; Aeromonas hydrophila and Klebsiella sp. were significantly dominant in 1-2, 1-3, and 3-2 samples; A. hydrophila and K. oxytoca were dominant in the 2-3 sample; and A. hydrophila and Pseudomonas sp. were prominent in the 3-3 sample. Different biofilms from the same plant shared common bands, suggesting that similar bacteria can be found and can be dominant in different biofilms. This study provides a better understanding of the dominant compositions in these bacterial communities. Further studies to determine the mechanism of co-culture with L. monocytogenes will be of critical importance in predicting effective disinfection strategies.

Sensitive colorimetric detection of Listeria monocytogenes based on isothermal gene amplification and unmodified gold nanoparticles.

Listeria monocytogenes (L. monocytogenes), one of most problematic food-borne bacteria, is mainly transmitted through the food chain and may cause listeriosis. Therefore, the development of rapid and sensitive L. monocytogenes detection technique has become an urgent task. In this study, we proposed a method using hyperbranching rolling circle amplification (HRCA) combined with gold nanoparticle (GNP) based colorimetric strategy to offer an isothermal, highly sensitive and specific assay for the detection of L. monocytogenes. First, a linear padlock probe targeting a specific sequence in the hly gene was designed and followed with a ligation by Taq DNA ligase. After ligation, further amplification by HRCA with a thiolated primer and an unlabeled primer is performed. The resulting thiolated HRCA products were then captured onto GNP surface and made GNP more salt-tolerant. Detection of the bacteria can be achieved by a facilitated GNP based colorimetric testing using naked eyes. Through this approach, as low as 100 aM synthetic hly gene targets and about 75 copies of L. monocytogenes can be detected. The specificity is evaluated by distinguishing target L. monocytogenes from other bacteria. The artificial contaminated food samples were also detected for its potential applications in real food detection. This method described here is ideal for bacteria detection due to its simplicity and high sensitivity.

The regulator HlyU, the repeat-in-toxin gene rtxA1, and their roles in the pathogenesis of Vibrio vulnificus infections.

HlyU is a master regulator that plays an essential role in the virulence of the human pathogen Vibrio vulnificus. One of the most noteworthy characteristics of HlyU regulation in this organism is its positive control of the expression of the repeat-in-toxin (RtxA1) gene, one of the most important virulence factors accounting for the fulminating and damaging nature of V. vulnificus infections. In this work, we reviewed the latest studies of RtxA1 in this bacterium and highlight the mechanism of gene regulation of rtxA1 expression by HlyU under a broader gene regulatory network.

DNA sequence and analysis of a 90.1-kb plasmid in Shiga toxin-producing Escherichia coli (STEC) O145:NM 83-75.

Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O145 are important emerging food-borne pathogens responsible for sporadic cases and outbreaks of hemorrhagic colitis and hemolytic uremic syndrome. A large plasmid carried by STEC O145:NM strain 83-75 and named pO145-NM was sequenced, and the genes were annotated. pO145-NM is 90,103bp in size and carries 89 open reading frames. Four genes/regions in pO145-NM encode for STEC virulence factors, including toxB (protein involved in adherence), espP (a serine protease), katP (catalase peroxidase), and the hly (hemolysin) gene cluster. These genes have also been identified in large virulence plasmids found in other STEC serogroups, including O26, O157, O111, and O103. pO145-NM carries the espPα subtype that is associated with STEC strains that cause more severe disease. Phylogenetic analyses of HlyB, EspP, and ToxB in various STEC strains showed a high degree of similarity of these proteins in E. coli serotypes O145:NM, O26:H11/H-, O111:NM/H-, and O157:H7 potentially placing these STEC into a related group.

Listeria monocytogenes: a rare complication of ventriculoperitoneal shunt in children.

We report a case of ventriculoperitoneal (VP) shunt infection in a 3-year-old boy caused by the food-borne pathogen Listeria monocytogenes, subsequent to acute peritonitis. This unusual presentation of central nervous system (CNS) listeriosis underlines the ability of the bacteria to form and survive within biofilms on indwelling medical devices. Bacterial persistence may lead to treatment failure and spreading. We highlight the helpfulness of specific quantitative real-time PCR for the hly gene (PCR-hly) for the diagnosis and follow-up of such infections in detecting bacterial persistence within medical devices despite effective antibiotic treatment. Only the surgical replacement of the VP shunt will resolve the infection.

Frequency of virulence-associated genes in Enterococccus faecalis isolated in Kuwait hospitals.

OBJECTIVE: The objective of this study was to investigate the carriage of 6 virulence-associated genes in Enterococcus faecalis isolates obtained from patients in 8 hospitals in Kuwait. MATERIALS AND METHODS: In total, 466 E. faecalis isolates were obtained from 313 urine samples, 68 wound swabs, 36 blood samples, 25 rectal swabs, 12 high vaginal swabs and 12 miscellaneous sources. Genes for gelatinase(gelE),aggregation substance (aggA), hemolysin activation factor (cylA), enhanced expression of pheromone (eep), enterococcal surface protein (esp), and E. faecalis endocarditis antigen A (efaA) were detected in PCR assays. RESULTS: Of 466 isolates, 423 (90.8%) were positive for 1 and up to 5 genes. However, none of the genes was detected in all of the isolates. The prevalence of the individual genes was eep: 31.9%; esp: 31.5%; gelE: 28.5%; efaA: 27.9%; aggA: 23.4%, and cylA: 18.5%. Of the 423 positive isolates, 148 (34.9%) were positive for 2 genes and 52 (12.3%), 15 (3.5%) and 5 (0.9%) isolates were positive for 3, 4 and 5 virulence genes, respectively. The efaA and esp combination was detected in isolates from all clinical sources. CONCLUSION: The study showed a high prevalence of virulence genes in E. faecalis isolated in Kuwait hospitals. The absence of a dominant gene in all of the isolates suggests that infections by E. faecalis may require the involvement of multiple virulence factors.

Production of haemolysins by strains of the Actinobacillus minor/"porcitonsillarum" complex.

Actinobacillus minor and "Actinobacillus porcitonsillarum" are distinguished by their haemolytic activities, the latter organism being haemolytic and the former, non-haemolytic. Analysis of a whole genome shotgun sequence, however, revealed that A. minor strain 202, like "A. porcitonsillarum", possesses a haemolysin-encoding apxII operon. The purpose of this study was therefore to investigate haemolysin production by this organism and also by three additional members of the A. minor/"porcitonsillarum" complex, strains 33PN and 7ATS and A. minor strain NM305(T). Primers based on sequences within the apxII genes of strain 202 allowed the amplification of appropriately sized fragments from DNA from strain 33PN suggesting that this organism also possesses an apxII operon. Analysis of a whole genome shotgun sequence failed to reveal any trace of an apxII operon in strain NM305(T) and attempts to amplify apxII genes from DNA from strain 7ATS also failed. Strains 202 and 33PN, and surprisingly, the type strain of A. minor and strain 7ATS, were all found to be haemolysin-positive as growth media from cultures of these organisms could promote the lysis of erythrocytes in suspension. The erythrocyte specificities of the haemolysins produced by strains 202 and 33PN indicated that the haemolytic activities exhibited by these organisms were due to ApxII. In keeping with the apparent lack of apxII genes in strains NM305(T) and 7ATS, the haemolysins produced by these organisms were not erythrocyte-specific and with both organisms, haemolytic activity appeared to be due to a combination of heat-stable and heat-labile components. The identities of these components, however, remain unknown.

A naturally occurring mutation K220T in the pleiotropic activator PrfA of Listeria monocytogenes results in a loss of virulence due to decreasing DNA-binding affinity.

The sequencing of prfA, encoding the transcriptional regulator of virulence genes, in 26 low-virulence field Listeria monocytogenes strains showed that eight strains exhibited the same single amino-acid substitution: PrfAK220T. These strains exhibited no expression of PrfA-regulated proteins and thus no virulence. This substitution inactivated PrfA, since expression of the PrfAK220T mutant gene in an EGDDeltaprfA strain did not restore the haemolytic and phosphatidylcholine phospholipase C activities, in contrast to the wild-type prfA gene. The substitution of the lysine at position 220 occurred in the helix alphaH. However, the data showed that the PrfAK220T protein is dimerized just as well as its wild-type counterpart, but does not bind to PrfA-boxes. PrfAK220T did not form a PrfA-DNA complex in electrophoretic mobility shift assays, but low concentrations of CI complexes (PrfAK220T-RNA polymerase-DNA complex) were formed by adding RNA polymerase, suggesting that PrfA interacted with RNA polymerase in solution in the absence of DNA. Formation of some transcriptionally active complexes was confirmed by in vitro runoff transcription assays and quantitative RT-PCR. Crystallographic analyses described the structure of native PrfA and highlighted the key role of allosteric changes in the activity of PrfA and especially the role of the Lys220 in the conformation of the helix-turn-helix (HTH) motif.

Eye infections due to Listeria monocytogenes in three cows and one horse.

A retrospective study was conducted to determine case histories, microbiological characteristics, and molecular subtypes associated with Listeria monocytogenes infections of the eye in large animals. For selected cases, environmental L. monocytogenes contamination patterns on case farms were also evaluated to probe for potential sources and spread of listerial eye infections. Records of 170 L. monocytogenes isolates from animal infections were reviewed to determine the fraction of isolates associated with eye infections (conjunctivitis, keratitis, and uveitis) of animals and to gather information on the clinical history of these cases. Overall, 4 of 170 Listeria monocytogenes isolates were associated with eye infections; 3 of these had occurred in cows and 1 in a horse. Molecular subtyping (by EcoRI ribotying) showed that 4 different L. monocytogenes subtypes were isolated from these 4 cases; the same ribotypes had previously been found among invasive animal listeriosis infections. Although a variety of L. monocytogenes subtypes were isolated from environmental sources, on 1 farm, the same ribotype associated with the eye infection was also isolated from a fecal sample of a healthy animal and from a soil sample. The data reported in this study further suggest that L. monocytogenes can be a cause of eye infections in several animal species. Listerial eye infections do not seem to require specific pathogen-related virulence characteristics but rather seem to be a function of environmental or host factors, such as direct exposure of the eyes of susceptible animals to high numbers of the pathogen. Although listerial eye infections are rarely diagnosed because of its ubiquitous nature, L. monocytogenes may have to be considered more commonly as a causative agent of eye infections in ruminants and horses.

Comparative genetic characterization of Listeria monocytogenes isolates from human and animal listeriosis cases.

Listeria monocytogenes isolates from human sporadic and epidemic cases (n=119) and from animal cases (n=76) were characterized by automated ribotyping and PCR-restriction fragment length polymorphism (PCR-RFLP) typing of the virulence genes actA and hly. This combination of typing methods differentiated 39 distinctive strains, each reflecting a unique combination of ribotypes, hly and actA alleles. Simpson's index of discrimination indicated a high discriminatory ability of ribotyping for both animal (0.867) and human isolates (0.857), which was further increased by the addition of hly and actA typing (0.916 and 0.904, respectively). Ribotype and hly allele data were further used to group isolates into three genetically distinct lineages. Each lineage is composed of several ribotype fragment subsets, each of which contains multiple ribotypes characterized by common ribotype fragments. To determine whether certain clones of L. monocytogenes show indications for unique pathogenic potential or host specificity, frequency distributions for five genetic characteristics (i.e. lineage, ribotype, ribotype fragment subset and hly and actA allele) were calculated for isolates from animal cases, human epidemic cases and human sporadic cases. Lineage III isolates were found less frequently in human cases (1 of 119 isolates) than in animal cases (8 of 76 isolates; P=0.003). These results suggest the possibility of host specificity for non-primate mammals among lineage III strains. In addition, lineage I strains were found more frequently among human cases than among animal cases (P<0.001). Among the eight hly alleles observed, hly allele 1 was more common among human isolates as compared to animal isolates (P=0.002). We also identified one ribotype (DUP-1030) which was significantly more common among animal isolates (P=0.005) and one ribotype (DUP-1038; lineage I) which was significantly more common among human epidemic isolates as compared to human sporadic isolates (P<0.001). These findings confirm the presence of clonal groups of L. monocytogenes, which appear to be characterized by unique virulence or host specificity patterns. This study also establishes baseline data describing the genetic diversity of human and animal L. monocytogenes isolates which can be utilized in future surveillance programmes to track the emergence of new strains.

Listeria monocytogenes-infected human dendritic cells: uptake and host cell response.

Dendritic cells (DCs) are potent antigen-presenting cells and play a crucial role in initiation and modulation of specific immune responses. Various pathogens are able to persist inside DCs. However, internalization of the gram-positive bacterium Listeria monocytogenes into human DCs has not yet been shown. In the present study, we demonstrate that human monocyte-derived immature DCs can efficiently phagocytose L. monocytogenes. This uptake is independent of listerial adhesion factors internalin A and internalin B but requires cytoskeletal motion and factors present in human plasma. A major portion of internalized bacteria is found in membrane-bound phagosomes and is rarely free in the cytosol, as shown by transmission electron microscopy and by using an L. monocytogenes strain expressing green fluorescent protein when in the host cell cytosol. The infection caused maturation of the immature DCs into mature DCs displaying high levels of CD83, CD25, major histocompatibility complex class II, and the CD86 costimulator molecule. This effect appeared to be largely mediated by listerial lipoteichoic acid. Although L. monocytogenes infection is known to induce death in other cell types, infection of human DCs was found to induce necrotic but not apoptotic death in fewer than 20% of DCs. Therefore, the ability of DCs to act as effective antigen-presenting cells for listerial immunity is probably enhanced by their resistance to cell death, as well as their ability to rapidly differentiate into mature, immunostimulatory DCs upon encountering bacteria.

PrfA mediates specific binding of RNA polymerase of Listeria monocytogenes to PrfA-dependent virulence gene promoters resulting in a transcriptionally active complex.

There is accumulating evidence that the coordinate transcription of the virulence genes in Listeria monocytogenes constitutes a very complex regulation mechanism which might require other factors in addition to PrfA. We previously described an unknown proteinaceous component from crude bacterial cell extracts, which, together with PrfA, formed a specific complex (CI) in electrophoretic mobility shift assays (EMSA) with an hly promoter probe. Here we identify the RNA polymerase (RNAP) of L. monocytogenes as an essential component of the CI complex. Addition of purified RNAP plus PrfA to the hly promoter probe allowed reconstitution of a complex migrating at the same height as CI. By using EMSA and DNaseI footprint experiments it could be shown that PrfA leads to an enhanced and specific binding of RNAP. Transcriptional activity of RNAP in vitro, using the actA promoter, was strictly dependent on PrfA.

Regulation of the Enterococcus faecalis pAD1-related sex pheromone response: analyses of traD expression and its role in controlling conjugation functions.

The Enterococcus faecalis haemolysin plasmid pAD1 (60 kb) confers a conjugative mating response to an octapeptide sex pheromone (cAD1) secreted by plasmid-free strains. The response involves two plasmid-borne regulatory determinants: traE1, whose product positively regulates all or most conjugation-related structural genes; and traA, whose product negatively regulates traE1 by controlling transcriptional readthrough of an upstream termination site (TTS1/TTS2). TraA binds to the promoter region of iad, which encodes a pheromone-inhibitor peptide, iAD1; and TTS1/TTS2 tightly terminates transcription arriving from this promoter during the uninduced state. A determinant, traD, appearing to encode a small peptide (23 amino acids), is located just downstream of iad and is in the opposite orientation. Transcripts of traD were identified and found to be present at a relatively high level in cells not expressing conjugation functions; the amount of RNA was greatly reduced, however, upon induction of the pheromone response. The decrease in traD RNA was not a consequence of the induced activity of TraE1, as it also occurred in a traE1 insertion mutant. A mutation in traD that would eliminate translation but that did not affect transcription had no apparent effect on the cell phenotype, indicating that RNA was likely to be the functional product. This was consistent with our finding that synthesis of traD RNA containing the translational defect was able to complement, in trans, a temperature-sensitive traD mutation. Thus, transcription of the traD determinant is significantly involved in downregulation of the pAD1 pheromone response.

Listeria monocytogenes virulence factors that stimulate endothelial cells.

Listeria monocytogenes infection of endothelial cells upregulates surface expression of adhesion molecules and stimulates neutrophil adhesion to infected cell monolayers. The experiments presented here tested the roles of specific bacterial virulence factors as triggers for this inflammatory phenotype and function. Human umbilical vein endothelial cell (HUVEC) monolayers were infected with wild-type L. monocytogenes or L. monocytogenes mutants; then surface expression of E-selectin and neutrophil adhesion were measured. The results showed that delta hly and prfA mutants were the most crippled, requiring 100-fold more mutant bacteria than wild-type bacteria for analogous stimulation. By comparison, L. monocytogenes mutants with deletions of actA, inlA, inlB, inlAB, plcA, and plcB resembled their parent strains, and a delta plcA delta plcB mutant displayed decreased intracellular growth rate but only a minor decrease in stimulation of E-selectin or neutrophil adhesion. Other experiments showed that cytochalasin D-treated HUVEC monolayers bound bacteria, but internalization and increased surface E-selectin and intercellular adhesion molecule-1 expression were profoundly inhibited. However, cytochalasin D had no effect on the HUVEC response to stimulation with lipopolysaccharide or tumor necrosis factor alpha. These data suggest that listeriolysin O production by infecting L. monocytogenes contributes to increased expression of surface E-selectin and intercellular adhesion molecule-1, but neither it nor intracellular replication are directly responsible for this event. Nonetheless it is possible that listeriolysin O potentiates the effect(s) of an other molecule(s) that directly triggers this response. Additionally, cellular invasion by L. monocytogenes appears to be critical for initiating the HUVEC response, potentially by providing a signal which results in upregulation of the necessary bacterial genes.

Heterogeneric conjugal transfer of the pheromone-responsive plasmid pIP964 (IncHlyI) of Enterococcus faecalis in the apparent absence of pheromone induction.

Erythromycin-resistant derivatives of the pheromone-responsive plasmid pIP964 from Enterococcus faecalis were constructed to study its host range. This was done by inserting the integrative vector pAT112 and the related replicon pTCR1 harboring oriR of the broad host range plasmid pAM beta 1 into the hemolysin-bacteriocin operon of pIP964, to give pTCR2 and pTCR3, respectively. Plasmid pTCR2 was transferred by filter matings from E. faecalis to Enterococcus faecium and Listeria monocytogenes at frequencies of 2 x 10(-7) and 5 x 10(-7) per donor, respectively, in the apparent absence of pheromone induction and cellular aggregation. In these hosts, pTCR2 remained intact as a self-replicating element and maintained its transfer capabilities. Plasmid pTCR3, but not pTCR2, was transferred at similar frequencies from E. faecalis to Lactococcus lactis and Streptococcus agalactiae. Thus, the transfer system of pIP964 possesses a broader host-range than its replication system.

Fur (ferric uptake regulation) protein interaction with target DNA: comparison of gel retardation, footprinting and electron microscopy analyses.

Fur-DNA interactions were analyzed within the regulatory regions of aerobactin and hemolysin operons by a combination of biochemical and ultrastructural methods. Cartography of the Fur binding sites, carried out from electron micrographs, agreed with the data obtained by DNase I footprinting. Visualization of the complexes confirmed the specificity and metal-dependence of Fur binding and demonstrated that the protein polymerizes on its binding sites. Such a polymerization could be involved in the repression process of the bacterial regulator.

[Genetic determination and the spectrum of the hemolytic activity of Vibrio cholerae: their significance in the complex of biovar-specific traits]. / Geneticheskoe determinirovanie i spektr gemoliticheskoi aktivnosti Vibrio cholerae: znachenie v komplekse biovarospetsificheskikh priznakov.

A collection of 363 V. cholerae strains isolated from different sources were studied by the spectrum of their hemolytic activity in combination with biovar-associated properties. The strains were analyzed for the presence of the cholera toxin (CT) gene (vct) and the hemolysin gene (hly) with the use of the CT probe and a previously cloned 6.56 kb fragment of V. eltor DNA coding the synthesis of hemolysin. The study revealed that all V. cholerae strains had the hly gene irrespective of the spectrum of their hemolytic activity, biovar, 01 agglutinability and the presence of the vct gene in their genome, while other species of the genus Vibrio and related groups contained no hly gene sequence. The results of the comparative study of the hemolytic activity of V. cholerae and V. eltor are discussed.

Characterization of the traC determinant of the Enterococcus faecalis hemolysin-bacteriocin plasmid pAD1: binding of sex pheromone.

pAD1, a conjugative, 60-kb, hemolysin-bacteriocin plasmid in Enterococcus faecalis, encodes a mating response to a small peptide sex pheromone, cAD1, secreted by potential recipient bacteria. A gene, traC, encoding a 60.7-kDa protein with a typical amino terminal signal peptide, was identified within a region that appears to encode a product that binds to exogenous pheromone. A cloned segment of DNA containing traC resulted in specific binding of cells to synthetic cAD1. The putative traC product has strong similarity to a product of the E. faecalis plasmid pCF10 as well as oligopeptide binding proteins of Escherichia coli, Salmonella typhimurium, and Bacillus subtilis.

[Distribution and transcription of determinants homologous to the alpha-hemolysin (hlY) gene in the genomes of HLY- and HLY+ staphylococci]. / Raspredelenie i transkriptsiia determinant, gomologichnykh genu alpha-gemolizina (hlY), v genomakh HLY- i HLY+ stafilokokkov.

The molecular organization of the alpha-hemolysin gene from Staphylococcus aureus strain O15 has been studied. Hybridization of the DNA from this strain with the [32P]-labeled cloned fragment containing alpha-hemolysin gene has shown the studied gene to be unique in the genome of the strain. Sequences homologous to the gene were not found to be dispersed along the genome of Staphylococcus aureus O15. The presence of alpha-hemolysin gene in some Staphylococcus epidermidis strains has been monitored. The strains 1413, 303 have been demonstrated to contain no gene for alpha-hemolysin. The hemolysin genes were found in the genomes of the strains 159, 169, 180 by DNA hybridization technique. In the genome of the strain 180 the long region including the right end of alpha-hemolysin gene is deleted. Hybridization with the net RNA of these strains shows the absence of transcription of intact as well as deleted alpha-hemolysin genes.

Secretion of the Bordetella pertussis adenylate cyclase from Escherichia coli containing the hemolysin operon.

The extracellular calmodulin-sensitive adenylate cyclase produced by Bordetella pertussis is synthesized as a 215-kDa precursor. This polypeptide is transported to the outer membrane of the bacteria where it is proteolytically processed to a 45-kDa catalytic subunit which is released into the culture supernatant [Masure, H.R., & Storm, D.R. (1989) biochemistry 28, 438-442]. The gene encoding this enzyme, cyaA, is part of the cya operon that also includes the genes cyaB, cyaD, and cyaE. A comparison of the predicted amino acid sequences encoded by cyaA, cyaB, and cyaD with the amino acid sequences encoded by hlyA, hlyB, and hlyD genes from the hemolysin (hly) operon from Escherichia coli shows a large degree of sequence similarity [Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A., & Danchin, A. (1988) EMBO J. 7, 3997-4004]. Complementation studies have shown that HlyB and HlyD are responsible for the secretion of HlyA (hemolysin) from E. coli. The signal sequence responsible for secretion of hemolysin has been shown to reside in its C-terminal 27 amino acids. Similarly, CyaB, CyaD, and CyaE are required for the secretion of CyaA from Bordetella pertussis. We placed the cyaA gene and a truncated cyaA gene that lacks the nucleotides that code for a putative C-terminal secretory signal sequence under the control of the lac promoter in the plasmid pUC-19. These plasmids were transformed into strains of E. coli which contained the hly operon. The truncated cyaA gene product, lacking the putative signal sequence, was not secreted but accumulated inside the cell.(ABSTRACT TRUNCATED AT 250 WORDS)