Sensitive In Situ Monitoring of a Recombinant Bioluminescent Yersinia enterocolitica Reporter Mutant in Real Time on Camembert Cheese

Bioluminescent mutants of Yersinia enterocolitica were generated by transposon mutagenesis using a promoterless, complete lux operon (luxCDABE) derived from Photorhabdus luminescens, and their production of light in the cheese environment was monitored. Mutant B94, which had the lux cassette inserted into an open reading frame of unknown function was used for direct monitoring of Y. enterocolitica cells on cheeses stored at 10°C by quantifying bioluminescence using a photon-counting, intensified charge-coupled device camera. The detection limit on cheese was 200 CFU/cm². Bioluminescence of the reporter mutant was significantly regulated by its environment (NaCl, temperature, and cheese), as well as by growth phase, via the promoter the lux operon had acquired upon transposition. At low temperatures, mutant B94 did not exhibit the often-reported decrease of photon emission in older cells. It was not necessary to include either antibiotics or aldehyde in the food matrix in order to gain quantitative, reproducible bioluminescence data. As far as we know, this is the first time a pathogen has been monitored in situ, in real time, in a “real-product” status, and at a low temperature.     

Competitive reporter monitored amplification (CMA)--quantification of molecular targets by real time monitoring of competitive reporter hybridization

Background

State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests.

Methodology and Principal Findings

The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique.

Conclusions and Significance

The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests.     

Studies on a haemolytic substance in Yersinia enterocolitica

Yersinia enterocolitica serotype O3 was found to produce a haemolytic substance which could be released from the bacterial cells by sonic disintegration. The substance was non-dialysable, thermolabile, antigenic, and sensitive to trypsin. Chromatographic studies indicated a high molecular weight. Erythrocytes from different mammalian species differed in sensitivity to the haemolytic substance. Y. enterocolitica serotypes O8 and O9 produced no haemolytic substance.     

Association of Yersinia enterocolitica with the manufacture of cheese and occurrence in pasteurized milk.

Raw milk in southern Ontario frequently contains Yersinia enterocolitica. The potential for transmission of this organism by cheese manufactured from unpasteurized milk was evaluated by examination of milk and cheese curd samples from cheese manufacturing plants and finished cheddar and Italian cheeses. The incidence of Y. enterocolitica was lower in cheese curd samples (9.2%) than in raw milk (18.2%). Most of the curd samples showed a positive phosphatase test, indicating production from raw milk. One curd sample yielded Y. enterocolitica after 4 weeks of storage at 4 degrees C but was negative after 8 weeks. All samples of cheddar and Italian cheeses, most of which showed a positive phosphatase test, were negative for Y. enterocolitica. One out of 265 samples (0.4%) of pasteurized fluid dairy products contained Y. enterocolitica.     

A note on Yersinia enterocolitica in a swine farm watershed

Swine faeces from three pig farms in the La Crosse River watershed near La Crosse, Wisconsin, were sampled for Yersinia enterocolitica ; 19 presumptive isolates were recovered and biochemically confirmed as Y. enterocolitica. Simultaneously, during a 2mD2 cm rainfall, the confluences of runoff water flowing from the swine holding pens and of nearby streams also were sampled; a single isolate was obtained from one holding pen runoff-stream confluence. Biochemical analysis showed that the water isolate was a biotype identical with that of a swine isolate from the adjacent farm. These results demonstrate one possible mechanism for the introduction of Y. enterocolitica into water supplies; faecal material from swine, a suspected natural reservoir of the bacterium, is transported via runoff water to streams.     

A note on Yersinia enterocolitica in a swine farm watershed

Swine faeces from three pig farms in the La Crosse River watershed near La Crosse, Wisconsin, were sampled for Yersinia enterocolitica; 19 presumptive isolates were recovered and biochemically confirmed as Y. enterocolitica. Simultaneously, during a 2.2 cm rainfall, the confluences of runoff water flowing from the swine holding pens and of nearby streams were also sampled; a single isolate was obtained from one holding pen runoff-stream confluence. Biochemical analysis showed that the water isolate was a biotype identical with that of a swine isolate from the adjacent farm. These results demonstrate one possible mechanism for the introduction of Y. enterocolitica into water supplies; faecal material from swine, a suspected natural reservoir of the bacterium, is transported via runoff water to streams.     

Some observations in freeze-drying of recombinant bioluminescent Escherichia coli for toxicity monitoring

A recombinant bioluminescent bacteria, containing a fabA::luxCDABE fusion gene, has been used to characterize freeze-drying methods, which may be conveniently used as a tool for the development of a portable biosensor. Through residual water, viability, biosensing activity and scanning electron microscopy analyses, the characteristics that four cryoprotectants, trehalose, sucrose, sorbitol, and mannitol, conferred on freeze-dried samples were elucidated, including the morphology, water content and activity of the cells. It was found that trehalose showed the best freeze-drying efficiency among the tested cryoprotectants and it might have a specific capacity limitation in protection of the cells during the freeze step. Humidity might result in damage to the cells, according to the viability, when exposed to air during storage, while the water remaining post freeze-drying showed good correlation with damage to the freeze-dried cells when under air-tight storage conditions. The results with other recombinant bioluminescent bacteria indicated that these findings might be general features of the freeze-drying processes.     

In vitro and in vivo expression studies of yopE from Yersinia enterocolitica using the gfp reporter gene

The Yersinia outer protein YopE belongs to the translocated effector proteins of pathogenic yersiniae. We constructed various truncated yopE genes fused to gfp (encoding the green fluorescent protein) to study yopE gene expression and YopE-GFP translocation of Y. enterocolitica in cell culture and mouse infection models. The hybrid gene fusions were co-expressed in Y. enterocolitica (i) on a low-copy plasmid in the presence of the virulence plasmid pYV08 (in trans configuration) and (ii) after co-integration by homologous recombination of a yopE-gfp-carrying suicide plasmid into pYV08 (co-integrate configuration). After 30min of infection of HEp-2 cell monolayers, extracellularly located yersiniae began to emit green fluorescence after excitation. In contrast, internalized bacteria were weakly fluorescent. Translocation of YopE-GFP into HEp-2 cells by attached yersiniae was visualized by optical sectioning of fluorescent HEp-2 cells using confocal laser scanning microscopy and was confirmed by immunoprecipitation of cytosolic YopE-GFP from selectively solubilized HEp-2 cells. The co-translocation of other Yops was not significantly impaired by YopE-GFP as shown by YopH/YopE-mediated suppression of the oxidative burst of infected neutrophils. The time course of yopE-gfp expression (in trans as well as in the co-integrate configuration) in the HEp-2 cell infection model as well as after in vitro induction was studied using a highly sensitive CCD camera and a flow cytometer. Similar results were obtained with a YopE-LUC (firefly luciferase) protein fusion as reporter. After intraperitoneal, intravenous and orogastrical infection of Balb/c mice with the recombinant yersiniae strains, green fluorescing bacteria could be visualized microscopically in the peritoneum, the spleen, the liver and in the Peyer's patches. However, only weakly fluorescent yersiniae were observed in the intestinal lumen. These results were quantified by flow cytometric measurements. The application of gfp as a reporter gene turned out to be promising for the study of protein translocation by protein type III secretion systems and differential virulence gene expression in vivo.     

Yersinia enterocolitica type III secretion chaperone SycD: recombinant expression, purification and characterization of a homodimer

Yersinia species pathogenic to human benefit from a protein transport machinery, a type three secretion system (T3SS), which enables the bacteria to inject effector proteins into host cells. Several of the transport substrates of the Yersinia T3SS, called Yops (Yersinia outer proteins), are assisted by specific chaperones (Syc for specific Yop chaperone) prior to transport. Yersinia enterocolitica SycD (LcrH in Yersinia pestis and Yersinia pseudotuberculosis) is a chaperone dedicated to the assistance of the translocator proteins YopB and YopD, which are assumed to form a pore in the host cell membrane. In an attempt to make SycD amenable to structural investigations we recombinantly expressed SycD with a hexahistidine tag in Escherichia coli. Combining immobilized nickel affinity chromatography and gel filtration we obtained purified SycD with an exceptional yield of 120mg per liter of culture and homogeneity above 95%. Analytical gel filtration and cross-linking experiments revealed the formation of homodimers in solution. Secondary structure analysis based on circular dichroism suggests that SycD is mainly composed of alpha-helical elements. To prove functionality of purified SycD previously suggested interactions of SycD with Yop secretion protein M2 (YscM2), and low calcium response protein V (LcrV), respectively, were reinvestigated.     

Piglets are a source of pathogenic Yersinia enterocolitica on fattening-pig farms

To study the origin and spread of Yersinia enterocolitica among pigs, fecal and blood samples were repeatedly taken on a fattening farm. A few piglets were found to be already infected on breeding farms. After the piglets were mixed, the infection spread through the whole unit. Eventually, all the pigs excreted the pathogen.     

Inhibition of Listeria monocytogenes by piscicolin 126 in milk and Camembert cheese manufactured with a thermophilic starter

The effect of bacteriocin, piscicolin 126, on the growth of Listeria monocytogenes and cheese starter bacteria was investigated in milk and in Camembert cheese manufactured from milk challenged with 10(2) cfu ml(-1) L. monocytogenes. In milk incubated at 30 degrees C, piscicolin 126 added in the range of 512-2,048 AU ml(-1) effectively inhibited growth of L. monocytogenes for more than 20 d when challenged with approximately 10(2) cfu ml(-1) L. monocytogenes. At higher challenge levels (10(4) and 10(6) cfu ml(-1)), piscicolin 126 reduced the viable count of L. monocytogenes by 4-5 log units immediately after addition of the bacteriocin; however, growth of Listeria occurred within 24 h. The minimum inhibitory concentration (MIC) of piscicolin 126 against lactic acid cheese starter bacteria was generally greater than 204,800 AU ml(-1) , and the viable count and acid production of these starter cultures in milk were not affected by the addition of 2,048 AU ml(-1) piscicolin 126. Camembert cheeses made from milk challenged with L. monocytogenes and with added piscicolin 126 showed a viable count of L. monocytogenes 3-4 log units lower than those without piscicolin 126. Inactivation of piscicolin 126 by proteolytic enzymes from cheese starter bacteria and mould together with the emergence of piscicolin 126-resistant isolates was responsible for the recovery of L. monocytogenes in the cheeses during ripening.     

Survey on the Incidence of Yersinia enterocolitica Infection in Canada

Data pertaining to 278 cultures of Yersinia enterocolitica isolated in Canada are summarized in this paper. Of this amount, 256 were isolated from humans, whereas 22 were of nonhuman sources. The typing of these cultures is presented together with their geographical location. Y. enterocolitica serotype O:3 biotype 4, phage type 9b, was practically the only serotype present in the Province of Quebec. This serotype O:3 was also predominant in Ontario, followed by serotypes O:5,27 and O:6,30; other serotypes were seldom isolated. In the central and western areas of Canada, Y. enterocolitica was occasionally isolated; the strains were indole-positive, serotypes O:5,27, O:8, and O:4,32.     

Real-time monitoring of Escherichia coli O157:H7 adherence to beef carcass surface tissues with a bioluminescent reporter

A method for studying bacteria that are attached to carcass surfaces would eliminate the need for exogenous sampling and would facilitate understanding the interaction of potential human food-borne pathogens with food animal tissue surfaces. We describe such a method in which we used a bioluminescent reporter strain of Escherichia coli O157:H7 that was constructed by transformation with plasmid pCGLS1, an expression vector that contains a complete bacterial luciferase (lux) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain, and adherent bacteria were visualized in real time by using a sensitive photon-counting camera to obtain in situ images. The reporter strain was found to luminesce from the tissue surfaces whether it was inoculated as a suspension in buffer or as a suspension in a bovine fecal slurry. With this method, areas of tissues inoculated with the reporter strain could be studied without obtaining, excising, homogenizing, and culturing multiple samples from the tissue surface. Use of the complete lux operon as the bioluminescent reporter eliminated the need to add exogenous substrate. This allowed detection and quantitation of bacterial inocula and rapid evaluation of adherence of a potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, the attachment duration varied with different carcass surface types. On average, the percent retention of bioluminescent signal from the reporter strain was higher on lean fascia-covered tissue (54%) than on adipose fascia-covered tissue (18%) following water washing of the tissues. Bioluminescence and culture-derived viable bacterial counts were highly correlated (r2 = 0.98). Real-time assessment of microbial attachment to this complex menstruum should facilitate evaluation of carcass decontamination procedures and mechanistic studies of microbial contamination of beef carcass tissues.     

Studies on pathogenicity of Yersinia enterocolitica in mice with diabetes mellitus

In this study, we investigated the colonizing ability as well as the association of Yersinia enterocolitica serotype 0:9 to epithelial cells of the intestinal tract, Peyer's patches, mesenteric lymph nodes, liver, spleen and lungs in Alloxan-induced diabetes mellitus in mice and controls. The results showed that: (a) in diabetic mice the Y. enterocolitica colonizing values were in range of 10(6.5)-10(8.25) CFU/g of feces; (b) maximum colonizing values were found in distal ileum and Peyer's patches and lower in colon; (c) the infection was progressive with dissemination of bacteria in the liver, spleen and lung; (d) in control (non-diabetic) mice, the colonizing values were 10-100 times lower than those found in the diabetic batch; (e) the main histopathological changes noticed, namely ileitis, mesenteric lymphadenitis and septicemia, were presumably induced by high bacterial load in the liver, spleen and lung leading to a septic course of infection as well as toxic effects of heat-stable enterotoxins of Y. enterocolitica (Yst). The results were confirmed by electron microscopy observations. Summing up, these results demonstrate that diabetic mice were more susceptible to Y. enterocolitica cells than normal mice.     

Novel pathogenetic mechanism in a clinical isolate of Yersinia enterocolitica KU14

Yersinia enterocolitica induces a broad range of gastrointestinal syndromes, including acute enteritis. We previously reported that the clinical isolate, Y. enterocolitica KU14, which lacks pYV, was still capable of causing clinical infection. The present study demonstrated that KU14 did not trigger the death of macrophages in vitro, unlike WA-314 (ATCC51871, which harbors the pYV virulence plasmid). However, the intracellular growth of KU14 in the macrophages was greater than that of WA-C (ATCC51872, a non-plasmid harboring the derivative pYV plasmid). Treatment with a cholesterol-binding drug (beta-cyclodextrin) that affected lipid rafts resulted in a dramatic reduction in the intracellular growth of KU14. These data clearly indicate that the enhanced intracellular growth of KU14 is related to lipid raft-mediated infection.     

Isolation of Yersinia enterocolitica from Milk and a Dairy Farm in Australia

Forty-eight isolates obtained from pasteurized milk and cream, raw milk, and various sites on a dairy farm were identified as Yersinia enterocolitica biotype 1 but were unusual in their ability to ferment lactose. The majority belonged to serotype 5a. Serotypes 5b, 6, 13/15 and 23/15 were also found. A few isolates were not typable. There is no evidence that strains isolated in this study are pathogenic for humans. Their presence in pasteurized products and growth at refrigeration temperatures are, however, cause for concern.     

The Ngal reporter mouse detects the response of the kidney to injury in real time

Many proteins have been proposed to act as surrogate markers of organ damage, yet for many candidates the essential biomarker characteristics that link the protein to the injured organ have not yet been described. We generated an Ngal reporter mouse by inserting a double-fusion reporter gene encoding luciferase-2 and mCherry (Luc2-mC) into the Ngal (Lcn2) locus. The Ngal-Luc2-mC reporter accurately recapitulated the endogenous message and illuminated injuries in vivo in real time. In the kidney, Ngal-Luc2-mC imaging showed a sensitive, rapid, dose-dependent, reversible, and organ- and cell-specific relationship with tubular stress, which correlated with the level of urinary Ngal (uNgal). Unexpectedly, specific cells of the distal nephron were the source of uNgal. Cells isolated from Ngal-Luc2-mC mice also revealed both the onset and the resolution of the injury, and the actions of NF-κB inhibitors and antibiotics during infection. Thus, imaging of Ngal-Luc2-mC mice and cells identified injurious and reparative agents that affect kidney damage.     

Pleiotropic effects of a Yersinia enterocolitica ompR mutation on adherent-invasive abilities and biofilm formation

The OmpR regulator positively influences flagella synthesis and negatively regulates invasin expression in Yersinia enterocolitica. To determine the physiological consequences of this inverse regulation, we analyzed the effect of the ompR mutation on the ability of Y. enterocolitica Ye9 (serotype O9, biotype 2) to adhere to and invade human epithelial HEp-2 cells and to form biofilms. Cell culture assays with ompR, flhDC and inv mutant strains, which vary in their motility and invasin expression, confirmed the important contribution of flagella to the adherent-invasive abilities of Y. enterocolitica Ye9. However, the loss of motility in the ompR strain was apparently not responsible for its low adhesion ability. When the nonmotile phenotype of the ompR mutant was artificially eliminated, an elevated level of invasion, exceeding that of the wild-type strain, was observed. Confocal laser microscopy demonstrated a decrease in the biofilm formation ability of the ompR strain that was only partially correlated with its loss of motility. These data provide evidence that OmpR promotes biofilm formation in this particular strain of Y. enterocolitica, although additional OmpR-dependent factors are also required. In addition, our findings suggest that OmpR-dependent regulation of biofilm formation could be an additional aspect of OmpR regulatory function.     

The effect of carbon dioxide on the growth of Yersinia enterocolitica in a simulated milk medium

Carbon dioxide (30 mmol/l) was shown to inhibit the growth of four strains of Yersinia enterocolitica grown separately in a simulated milk medium at 7°C and 60 rev/min for 4 d. This indicates that addition of CO₂ to refrigerated raw milk supplies is likely to be a safe process with respect to that organism.