Acanthamoeba release compounds which promote growth of Listeria monocytogenes and other bacteria

Listeria monocytogenes can grow as a saphrophyte in diverse habitats, e.g., soil, rivers, lakes, and on decaying plant material. In these environments, the bacteria are frequently exposed to predatory protozoa such as Acanthamoeba. Although L. monocytogenes is a facultative intracellular pathogen it does not infect or survive intracellular in Acanthamoeba castellanii, unlike several other facultative intracellular bacteria. Instead, motile L. monocytogenes can form large aggregates on amoebal cells and are effectively phagocytosed and eventually digested by Acanthamoeba. Here, we demonstrate that non-motile L. monocytogenes represent a less preferred prey in co-cultures with A. castellanii. Moreover, we found that the presence of Acanthamoeba strongly promotes growth of the bacteria in non-nutrient saline, by releasing nutrients or other growth promoters. Thus, the lack of motility and ability to utilize amoebal metabolites may aid to avoid eradication by amoebal predation in low-nutrient environments.     

Isolation and characterization of Listeria monocytogenes from tropical seafood of Kerala, India

Listeria monocytogenes, which is an intracellular pathogen, causes various illnesses in human as well as in animals. The pathogenicity of this organism depends upon the presence of different virulence genes. A total of 324 tropical seafood and fishery environmental samples were screened for L. monocytogenes. The incidence of the human pathogenic species L. monocytogenes was 1.2 % of the samples. Listeria spp. was detected in 32.3, 27.1, and 5 % of fresh, frozen, and dry fish samples, respectively. Listeria innocua was found to be the most prevalent species of Listeria in the tropical seafood and environmental samples of Kerala. Listeria monocytogenes and L. innocua isolates were confirmed by multiplex PCR. L. monocytogenes isolates from the four positive samples showed phosphatidylinositol-specific phospholipase C reaction on Chromocult® Listeria selective agar. Molecular characterization of L. monocytogenes isolates for virulence genes revealed the presence of β-hemolysin (hly), plcA, actA, metalloprotease (mpl), iap and prfA genes in all the isolates recovered from the positive samples.     

MudPIT Profiling Reveals a Link between Anaerobic Metabolism and the Alkaline Adaptive Response of Listeria monocytogenes EGD-e

Listeria monocytogenes is a foodborne human pathogen capable of causing life-threatening disease in susceptible populations. Previous proteomic analysis we performed demonstrated that different strains of L. monocytogenes initiate a stringent response when subjected to alkaline growth conditions. Here, using multidimensional protein identification technology (MudPIT), we show that in L. monocytogenes EGD-e this response involves an energy shift to anaerobic pathways in response to the extracellular pH environment. Importantly we show that this supports a reduction in relative lag time following an abrupt transition to low oxygen tension culture conditions. This has important implications for the packaging of fresh and ready-to-eat foods under reduced oxygen conditions in environments where potential exists for alkaline adaptation.     

In Vivo Transcriptional Profiling of Listeria monocytogenes and Mutagenesis Identify New Virulence Factors Involved in Infection

Listeria monocytogenes is a human intracellular pathogen able to colonize host tissues after ingestion of contaminated food, causing severe invasive infections. In order to gain a better understanding of the nature of host–pathogen interactions, we studied the L. monocytogenes genome expression during mouse infection. In the spleen of infected mice, ≈20% of the Listeria genome is differentially expressed, essentially through gene activation, as compared to exponential growth in rich broth medium. Data presented here show that, during infection, Listeria is in an active multiplication phase, as revealed by the high expression of genes involved in replication, cell division and multiplication. In vivo bacterial growth requires increased expression of genes involved in adaptation of the bacterial metabolism and stress responses, in particular to oxidative stress. Listeria interaction with its host induces cell wall metabolism and surface expression of virulence factors. During infection, L. monocytogenes also activates subversion mechanisms of host defenses, including resistance to cationic peptides, peptidoglycan modifications and release of muramyl peptides. We show that the in vivo differential expression of the Listeria genome is coordinated by a complex regulatory network, with a central role for the PrfA-SigB interplay. In particular, L. monocytogenes up regulates in vivo the two major virulence regulators, PrfA and VirR, and their downstream effectors. Mutagenesis of in vivo induced genes allowed the identification of novel L. monocytogenes virulence factors, including an LPXTG surface protein, suggesting a role for S-layer glycoproteins and for cadmium efflux system in Listeria virulence.     

Comparison of the Prevalences and Diversities of Listeria Species and Listeria monocytogenes in an Urban and a Rural Agricultural Watershed

Foods and related processing environments are commonly contaminated with the pathogenic Listeria monocytogenes. To investigate potential environmental reservoirs of Listeria spp. and L. monocytogenes, surface water and point source pollution samples from an urban and a rural municipal water supply watershed in Nova Scotia, Canada, were examined over 18 months. Presumptive Listeria spp. were cultured from 72 and 35% of rural and urban water samples, respectively, with 24% of the positive samples containing two or three different Listeria spp. The L. innocua (56%) and L. welshimeri (43%) groups were predominant in the rural and urban watersheds, respectively. Analysis by the TaqMan assay showed a significantly (P < 0.05) higher prevalence of L. monocytogenes of 62% versus 17% by the culture-based method. Both methods revealed higher prevalences in the rural watershed and during the fall and winter seasons. Elevated Escherichia coli (≥100 CFU/100 ml) levels were not associated with the pathogen regardless of the detection method. Isolation of Listeria spp. were associated with 70 times higher odds of isolating L. monocytogenes (odds ratio = 70; P < 0.001). Serogroup IIa was predominant (67.7%) among the 285 L. monocytogenes isolates, followed by IVb (16.1%), IIb (15.8%), and IIc (0.4%). L. monocytogenes was detected in cow feces and raw sewage but not in septic tank samples. Pulsotyping of representative water (n = 54) and local human (n = 19) isolates suggested genetic similarities among some environmental and human L. monocytogenes isolates. In conclusion, temperate surface waters contain a diverse Listeria species population and could be a potential reservoir for L. monocytogenes, especially in rural agricultural watersheds.     

Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.     

Characterization of Isolates of Acanthamoeba from the Nasal Mucosa and Cutaneous Lesions of Dogs

Acanthamoeba spp. are free-living amoebae that are ubiquitously distributed in the environment and can cause encephalomyelitis in animals and humans. The factors that contribute to Acanthamoeba infections include parasite biology, genetic diversity, environmental spread, and host susceptibility. The aim of the present study was to characterize isolates of Acanthamoeba from the nasal mucosa and cutaneous lesions of dogs in order to access the occurence and pathogenicity of these organisms in this animal group. We studied 13 isolates of Acanthamoeba confirmed by polymerase chain reaction. They were sequenced, the genotype was determined, and their potential of pathogenicity was evaluated.     

Development of multiple strain competitive index assays for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> using pIMC; a new site-specific integrative vector

Background  

The foodborne, gram-positive pathogen, Listeria monocytogenes, is capable of causing lethal infections in compromised individuals. In the post genomic era of L. monocytogenes research, techniques are required to identify and validate genes involved in the pathogenicity and environmental biology of the organism. The aim here was to develop a widely applicable method to tag L. monocytogenes strains, with a particular emphasis on the development of multiple strain competitive index assays.     

Mathematical Model of Interaction Between Bacteriocin-Producing Lactic Acid Bacteria and Listeria. Part 1: Steady States and Thresholds

Mathematical modeling is an important tool to assessing quantitative conjectures and to answer specific questions. In the modeling, we assume that a competitor represented by a lactic acid bacterium produces antimicrobial compounds (substances that kill microorganisms or inhibit their growth), such as lactic acid and bacteriocins, with some cost to its own growth. Bacteriocins are protein compounds with antimicrobial effect against related species and bacteria such as Listeria monocytogenes, which is foodborne pathogen that cause listeriosis. From the analysis of the model, we found the thresholds which determine the existence of multiple equilibria and we studied their stability, in order to evaluate the interaction between lactic acid bacteria and L. monocytogenes.     

The species-specific mode of action of the antimicrobial peptide subtilosin against Listeria monocytogenes Scott A

Aims: To elucidate the molecular mechanism of action of the antimicrobial peptide subtilosin against the foodborne pathogen Listeria monocytogenes Scott A. Methods and Results: Subtilosin was purified from a culture of Bacillus amyloliquefaciens. The minimal inhibitory concentration of subtilosin against L. monocytogenes Scott A was determined by broth microdilution method. The effect of subtilosin on the transmembrane electrical potential (ΔΨ) and pH gradient (ΔpH), and its ability to induce efflux of intracellular ATP, was investigated. Subtilosin fully inhibited L. monocytogenes growth at a concentration of 19 μg ml^?1. Subtilosin caused a partial depletion of the ΔΨ and had a similar minor effect on the ΔpH. There was no significant efflux of intracellular ATP. Conclusion: Subtilosin likely acts upon L. monocytogenes Scott A by perturbing the lipid bilayer of the cellular membrane and causing intracellular damage, leading to eventual cell death. Subtilosin’s mode of action against L. monocytogenes Scott A differs from the one previously described for another human pathogen, Gardnerella vaginalis. Significance and Impact of the Study: This is the first report on the specific mode of action of subtilosin against L. monocytogenes and the first report of a bacteriocin with a species‐specific mode of action.     

The Presence of the Internalin Gene in Natural Atypically Hemolytic Listeria innocua Strains Suggests Descent from L. monocytogenes

The atypical hemolytic Listeria innocua strains PRL/NW 15B95 and J1-023 were previously shown to contain gene clusters analogous to the pathogenicity island (LIPI-1) present in the related foodborne gram-positive facultative intracellular pathogen Listeria monocytogenes, which causes listeriosis. LIPI-1 includes the hemolysin gene, thus explaining the hemolytic activity of the atypical L. innocua strains. No other L. monocytogenes-specific virulence genes were found to be present. In order to investigate whether any other specific L. monocytogenes genes could be identified, a global approach using a Listeria biodiversity DNA array was applied. According to the hybridization results, the isolates were defined as L. innocua strains containing LIPI-1. Surprisingly, evidence for the presence of the L. monocytogenes-specific inlA gene, previously thought to be absent, was obtained. The inlA gene codes for the InlA protein which enables bacterial entry into some nonprofessional phagocytic cells. PCR and sequence analysis of this region revealed that the flanking genes of the inlA gene at the upstream, 5′-end region were similar to genes found in L. monocytogenes serotype 4b isolates, whereas the organization of the downstream, 3′-end region was similar to that typical of L. innocua. Sequencing of the inlA region identified a small stretch reminiscent of the inlB gene of L. monocytogenes. The presence of two clusters of L. monocytogenes-specific genes makes it unlikely that PRL/NW 15B95 and J1-023 are L. innocua strains altered by horizontal transfer. It is more likely that they are distinct relics of the evolution of L. innocua from an ancestral L. monocytogenes, as postulated by others.     

Two-Component-System Histidine Kinases Involved in Growth of Listeria monocytogenes EGD-e at Low Temperatures

Two-component systems (TCSs) aid bacteria in adapting to a wide variety of stress conditions. While the role of TCS response regulators in the cold tolerance of the psychrotrophic foodborne pathogen Listeria monocytogenes has been demonstrated previously, no comprehensive studies showing the role of TCS histidine kinases of L. monocytogenes at low temperature have been performed. We compared the expression levels of each histidine kinase-encoding gene of L. monocytogenes EGD-e in logarithmic growth phase at 3°C and 37°C, as well as the expression levels 30 min, 3 h, and 7 h after cold shock at 5°C and preceding cold shock (at 37°C). We constructed a deletion mutation in each TCS histidine kinase gene, monitored the growth of the EGD-e wild-type and mutant strains at 3°C and 37°C, and measured the minimum growth temperature of each strain. Two genes, yycG and lisK, proved significant in regard to induced relative expression levels under cold conditions and cold-sensitive mutant phenotypes. Moreover, the ΔresE mutant showed a lower growth rate than that of wild-type EGD-e at 3°C. Eleven other genes showed upregulated gene expression but revealed no cold-sensitive phenotypes. The results show that the histidine kinases encoded by yycG and lisK are important for the growth and adaptation of L. monocytogenes EGD-e at low temperature.     

Modeling of Spatially Referenced Environmental and Meteorological Factors Influencing the Probability of Listeria Species Isolation from Natural Environments

Many pathogens have the ability to survive and multiply in abiotic environments, representing a possible reservoir and source of human and animal exposure. Our objective was to develop a methodological framework to study spatially explicit environmental and meteorological factors affecting the probability of pathogen isolation from a location. Isolation of Listeria spp. from the natural environment was used as a model system. Logistic regression and classification tree methods were applied, and their predictive performances were compared. Analyses revealed that precipitation and occurrence of alternating freezing and thawing temperatures prior to sample collection, loam soil, water storage to a soil depth of 50 cm, slope gradient, and cardinal direction to the north are key predictors for isolation of Listeria spp. from a spatial location. Different combinations of factors affected the probability of isolation of Listeria spp. from the soil, vegetation, and water layers of a location, indicating that the three layers represent different ecological niches for Listeria spp. The predictive power of classification trees was comparable to that of logistic regression. However, the former were easier to interpret, making them more appealing for field applications. Our study demonstrates how the analysis of a pathogen''s spatial distribution improves understanding of the predictors of the pathogen''s presence in a particular location and could be used to propose novel control strategies to reduce human and animal environmental exposure.The transmission cycle of many pathogens involves biotic hosts and abiotic environments. After infection of a host with a pathogen like Listeria monocytogenes, Bacillus anthracis, enterohemorrhagic Escherichia coli, Salmonella spp., or Toxoplasma gondii, large numbers of the pathogen may be shed into the environment where, under favorable conditions, they may survive, multiply, and infect new hosts, including humans (6, 11, 13, 30, 37). It is important to identify spatially explicit environmental and meteorological factors that favor a pathogen''s presence in a particular environmental location. That information could be used to design novel measures to reduce the presence of the pathogen in the environment and prevent exposure and infection of animal and human hosts. For analysis of pathogens'' spatial distribution in the environment, geographic information systems (GIS) integrated with standard statistical and epidemiological methods provide tremendous opportunities (5).Detection of pathogens in environmental samples is usually based on culturing methods without enumeration, resulting in presence/absence data. For such data, a standard statistical approach to predict microbial presence as influenced by covariates would be logistic regression (LR). However, classification trees (CT) have recently been suggested as a powerful yet simple alternative to LR in ecological studies (7, 48). It is therefore of interest to contrast the performance of the CT with that of the standard LR approach in predicting pathogen isolation from a spatial location.The objective of this study was to develop a methodological framework to study spatially explicit determinants affecting the local probability of pathogen isolation by using Listeria spp. as a model system. Specifically, our goals were (i) to examine the effect of environmental and meteorological factors on isolation of Listeria spp. from a spatial location and from soil, vegetation, and water layers of a location and (ii) to compare the predictive performance of LR and CT models. The genus Listeria was chosen as a model system because of the convenience of gathering data (Listeria bacteria are relatively prevalent in the environment; they have been isolated from 28% of sampled locations in the natural environment [38]) and because the genus Listeria includes the human-pathogenic species L. monocytogenes. There are currently six species in the genus Listeria, including two known pathogens (L. monocytogenes and Listeria ivanovii) and four nonpathogens (Listeria innocua, Listeria seeligeri, Listeria welshimeri, and Listeria grayi). L. ivanovii is predominantly an animal pathogen, infecting ruminants, while L. monocytogenes can cause disease (listeriosis) in a wide range of animal species, including humans. In addition, a new Listeria species, “Listeria marthii” sp. nov., has recently been identified (L. M. Graves, L. O. Helsel, A. G. Steigerwalt, R. E. Morey, M. I. Daneshvar, S. E. Roof, R. H. Orsi, E. D. Fortes, S. R. Millilo, H. C. den Bakker, M. Wiedmann, B. Swaminathan, and B. D. Sauders, submitted for publication).