Exploring the genetic determinants underlying the differential production of an inducible chromosomal cephalosporinase - BlaB in Yersinia enterocolitica biotypes 1A, 1B, 2 and 4.

Yersinia enterocolitica is an enteric bacterium which can cause severe gastroenteritis. Beta-lactams are the most widely used antibiotics against Y. enterocolitica. Y. enterocolitica produces two chromosomal ß-lactamases, BlaA and BlaB. BlaB is an Ambler Class C inducible broad spectrum cephlaosporinase which showed differential enzyme activity in different biotypes of Y. enterocolitica. The expression of blaB is mainly regulated by ampR- the transcriptional regulator and, ampD - which helps in peptidoglycan recycling. The aim of this study was to identify and characterize genetic determinants underlying differential enzyme activity of BlaB in Y. enterocolitica biotypes 1 A, IB, 2 and 4. Thus, ampR, blaB and ampD were PCR-amplified and modeled in silico. The intercistronic region containing promoters of ampR and blaB was also investigated. Our results indicated that blaB was more inducible in biotypes 2 and 4, than in biotypes 1 A and 1B. Superimposition of in silico modeled proteins suggested that variations in amino acid sequences of AmpR, BlaB and AmpD were not responsible for hyper-production of BlaB in biotypes 2 and 4. Analysis of promoter regions of ampR and blaB revealed variations at -30, -37 and -58 positions from blaB transcription start site. Studies on relative expression levels of blaB in different biotypes by qRT-PCR indicated that nucleotide variations at these positions might contribute to a higher enzyme activity of BlaB in biotypes 2 and 4. However, this is a preliminary study and further studies including more strains of each biotype are required to strengthen our findings. Nevertheless, to the best of our knowledge, this is the first study which has investigated the genetic determinants underlying differential inducible production of BlaB in different biotypes of Y. enterocolitica.

A new analytical platform based on field-flow fractionation and olfactory sensor to improve the detection of viable and non-viable bacteria in food.

An integrated sensing system is presented for the first time, where a metal oxide semiconductor sensor-based electronic olfactory system (MOS array), employed for pathogen bacteria identification based on their volatile organic compound (VOC) characterisation, is assisted by a preliminary separative technique based on gravitational field-flow fractionation (GrFFF). In the integrated system, a preliminary step using GrFFF fractionation of a complex sample provided bacteria-enriched fractions readily available for subsequent MOS array analysis. The MOS array signals were then analysed employing a chemometric approach using principal components analysis (PCA) for a first-data exploration, followed by linear discriminant analysis (LDA) as a classification tool, using the PCA scores as input variables. The ability of the GrFFF-MOS system to distinguish between viable and non-viable cells of the same strain was demonstrated for the first time, yielding 100 % ability of correct prediction. The integrated system was also applied as a proof of concept for multianalyte purposes, for the detection of two bacterial strains (Escherichia coli O157:H7 and Yersinia enterocolitica) simultaneously present in artificially contaminated milk samples, obtaining a 100 % ability of correct prediction. Acquired results show that GrFFF band slicing before MOS array analysis can significantly increase reliability and reproducibility of pathogen bacteria identification based on their VOC production, simplifying the analytical procedure and largely eliminating sample matrix effects. The developed GrFFF-MOS integrated system can be considered a simple straightforward approach for pathogen bacteria identification directly from their food matrix. Graphical abstract An integrated sensing system is presented for pathogen bacteria identification in food, in which field-flow fractionation is exploited to prepare enriched cell fractions prior to their analysis by electronic olfactory system analysis.

CsrA impacts survival of Yersinia enterocolitica by affecting a myriad of physiological activities.

BACKGROUND: A previous study identified a Yersinia enterocolitica transposon mutant, GY448, that was unable to export the flagellar type three secretion system (T3SS)-dependent phospholipase, YplA. This strain was also deficient for motility and unable to form colonies on Lauria-Bertani agar medium. Preliminary analysis suggested it carried a mutation in csrA. CsrA in Escherichia coli is an RNA-binding protein that is involved in specific post-transcriptional regulation of a myriad of physiological activities. This study investigated how CsrA affects expression of the flagellar regulatory cascade that controls YplA export and motility. It also explored the effect of csrA mutation on Y. enterocolitica in response to conditions that cue physiological changes important for growth in environments found both in nature and the laboratory. RESULTS: The precise location of the transposon insertion in GMY448 was mapped within csrA. Genetic complementation restored disruptions in motility and the YplA export phenotype (Yex), which confirmed this mutation disrupted CsrA function. Mutation of csrA affected expression of yplA and flagellar genes involved in flagellar T3SS dependent export and motility by altering expression of the master regulators flhDC. Mutation of csrA also resulted in increased sensitivity of Y. enterocolitica to various osmolytes, temperatures and antibiotics. CONCLUSIONS: The results of this study reveal unique aspects of how CsrA functions in Y. enterocolitica to control its physiology. This provides perspective on how the Csr system is susceptible to adaptation to particular environments and bacterial lifestyles.

Activity of a bacterial cell envelope stress response is controlled by the interaction of a protein binding domain with different partners.

The bacterial phage shock protein (Psp) system is a highly conserved cell envelope stress response required for virulence in Yersinia enterocolitica and Salmonella enterica. In non-inducing conditions the transcription factor PspF is inhibited by an interaction with PspA. In contrast, PspA associates with the cytoplasmic membrane proteins PspBC during inducing conditions. This has led to the proposal that PspBC exists in an OFF state, which cannot recruit PspA, or an ON state, which can. However, nothing was known about the difference between these two states. Here, we provide evidence that it is the C-terminal domain of Y. enterocolitica PspC (PspC(CT)) that interacts directly with PspA, both in vivo and in vitro. Site-specific photocross-linking revealed that this interaction occurred only during Psp-inducing conditions in vivo. Importantly, we have also discovered that PspC(CT) can interact with the C-terminal domain of PspB (PspC(CT)·PspB(CT)). However, the PspC(CT)·PspB(CT) and PspC(CT)·PspA interactions were mutually exclusive in vitro. Furthermore, in vivo, PspC(CT) contacted PspB(CT) in the OFF state, whereas it contacted PspA in the ON state. These findings provide the first description of the previously proposed PspBC OFF and ON states and reveal that the regulatory switch is centered on a PspC(CT) partner-switching mechanism.

Yersinia enterocolitica palearctica serobiotype O:3/4--a successful group of emerging zoonotic pathogens.

BACKGROUND: High-pathogenic Y. enterocolitica ssp. enterocolitica caused several human outbreaks in Northern America. In contrast, low pathogenic Y. enterocolitica ssp. palearctica serobiotype O:3/4 is responsible for sporadic cases worldwide with asymptomatic pigs being the main source of infection. Genomes of three Y. enterocolitica ssp. palearctica serobiotype O:3/4 human isolates (including the completely sequenced Y11 German DSMZ type strain) were compared to the high-pathogenic Y. enterocolitica ssp. enterocolitica 8081 O:8/1B to address the peculiarities of the O:3/4 group. RESULTS: Most high-pathogenicity-associated determinants of Y. enterocolitica ssp. enterocolitica (like the High-Pathogenicity Island, yts1 type 2 and ysa type 3 secretion systems) are absent in Y. enterocolitica ssp. palearctica serobiotype O:3/4 genomes. On the other hand they possess alternative putative virulence and fitness factors, such as a different ysp type 3 secretion system, an RtxA-like and insecticidal toxins, and a N-acetyl-galactosamine (GalNAc) PTS system (aga-operon). Horizontal acquisition of two prophages and a tRNA-Asn-associated GIYep-01 genomic island might also influence the Y. enterocolitica ssp. palearctica serobiotype O:3/4 pathoadaptation. We demonstrated recombination activity of the PhiYep-3 prophage and the GIYep-01 island and the ability of the aga-operon to support the growth of the Y. enterocolitica ssp. enterocolitica O:8/1B on GalNAc. CONCLUSIONS: Y. enterocolitica ssp. palearctica serobiotype O:3/4 experienced a shift to an alternative patchwork of virulence and fitness determinants that might play a significant role in its host pathoadaptation and successful worldwide dissemination.

Transport of selected bacterial pathogens in agricultural soil and quartz sand.

The protection of groundwater supplies from microbial contamination necessitates a solid understanding of the key factors controlling the migration and retention of pathogenic organisms through the subsurface environment. The transport behavior of five waterborne pathogens was examined using laboratory-scale columns packed with clean quartz at two solution ionic strengths (10 mM and 30 mM). Escherichia coli O157:H7 and Yersinia enterocolitica were selected as representative Gram-negative pathogens, Enterococcus faecalis was selected as a representative Gram-positive organism, and two cyanobacteria (Microcystis aeruginosa and Anabaena flos-aquae) were also studied. The five organisms exhibit differing attachment efficiencies to the quartz sand. The surface (zeta) potential of the microorganisms was characterized over a broad range of pH values (2-8) at two ionic strengths (10 mM and 30 mM). These measurements are used to evaluate the observed attachment behavior within the context of the DLVO theory of colloidal stability. To better understand the possible link between bacterial transport in model quartz sand systems and natural soil matrices, additional experiments were conducted with two of the selected organisms using columns packed with loamy sand obtained from an agricultural field. This investigation highlights the need for further characterization of waterborne pathogen surface properties and transport behavior over a broader range of environmentally relevant conditions.

Hybrid gravitational field-flow fractionation using immunofunctionalized walls for integrated bioanalytical devices.

In this work, the biospecific recognition antigen-antibody reaction was implemented in gravitational field-flow fractionation (GrFFF), a flow-assisted separation technique for micron-sized particles, in order to realize a hybrid immunomodulated GrFFF system in which two different principles are combined to achieve highly versatile fractionation. Micron-sized polystyrene beads coated with horseradish peroxidase (HRP) were used as a model sample, and anti-HRP antibodies were immobilized on the accumulation wall of the GrFFF channel. Ultrasensitive chemiluminescence imaging was employed to visualize the beads during elution and to optimize experimental conditions. The same principle was then applied to real biological samples composed by Yersinia enterocolitica and Escherichia coli cells. Results show the possibility to modify the elution of selected sample components and even to retain them into the channel. The hybrid immunomodulated GrFFF system is a step towards the development of a module that could be integrated in a lab-on-a-chip-based point-of-care testing device which includes sample pre-analytical cleanup and analysis.

Role of oxygen tension on the transport and retention of two pathogenic bacteria in saturated porous media.

To examine the influence of variations in the dissolved oxygen (DO) concentration on pathogen mobility, laboratory-scale filtration experiments were performed using the enterohemorrhagic strain Escherichia coli O157:H7 and the enteroinvasive organism Yersinia enterocolitica. Cells were incubated either in the absence (anaerobic) or in the presence (aerobic) of oxygen to understand how these two growth conditions may affect bacterial transport and retention in water-saturated granular porous media. The influence of DO during growth is found to be organism dependent, whereby E. coli O157:H7 exhibits decreased transport potential when grown in the presence of 02 and Y. enterocolitica exhibits greater transport when grown aerobically. To understand the influence of DO changes during cell acclimation and transport, bacteria were resuspended and acclimated in either oxygen-depleted (low DO) or oxygen-rich (saturated DO) electrolytes prior to conduction of filtration experiments. The effect of DO on bacterial transport and retention is shown to be dependent on the antecedent growth conditions and on the organism studied. Measurements of the cell surface charge, shape, and size reveal some variability when the oxygen tension is changed during bacterial growth or acclimation and are linked to the observed bacterial transport behavior.

[New accumulation medium for isolation of bacteria of the genus Yersinia].

Liquid nutrient peptone-potassium accumulation medium (PPAM) has been designed to isolate Yersinia when samples taken from patients, animals, and environmental objects are examined. PPAM contains enzymatic peptone and disubstituted potassium phosphate. PPAM ensures a rapid Yersinia and accordingly early (days 2-3) isolation of at least 23.4% of Yersinia cultures, suppresses the growth of the associating flora (under the conditions of model experiments by 2-3 orders against one on the buffer-casein-yeast medium); it is easy-to-prepare and reduces the cost of a study significantly (by 8 times). The composition of PPAM is patented.

Global analysis of tolerance to secretin-induced stress in Yersinia enterocolitica suggests that the phage-shock-protein system may be a remarkably self-contained stress response.

The phage-shock-protein (Psp) system is essential for Yersinia enterocolitica virulence. Mislocalized secretins induce psp gene expression, and kill psp null strains. We used transposon mutagenesis to investigate whether other genes are required to tolerate secretin-induced stress. Our motivation included the possibility of identifying signal transducers required to activate psp gene expression. Besides Psp, only defects in the RpoE system and the TrkA potassium transporter caused secretin sensitivity. These mutations did not cause the same specific/severe sensitivity as defects in the Psp system, nor did they affect psp gene expression. The Escherichia coli Psp system was reported to be induced via the ArcB redox sensor and to activate anaerobic metabolism. Our screen did not identify arcB, or any genes involved in anaerobic metabolism/regulation. Therefore, we investigated the role of ArcB in Y. enterocolitica and E. coli. ArcB was not required for secretin-dependent induction of psp gene expression. Furthermore, microarray analysis uncovered a restricted transcriptional response to prolonged secretin stress in Y. enterocolitica. Taken together, these data do not support the proposal that the Psp system is induced via ArcB and activates anaerobic metabolism. Rather, they suggest that Psp proteins may sense an inducing trigger and mediate their physiological output(s) directly.

Yersinia enterocolitica infection of mice reveals clonal invasion and abscess formation.

Yersinia enterocolitica is a common cause of food-borne gastrointestinal disease leading to self-limiting diarrhea and mesenteric lymphadenitis. Occasionally, focal abscess formation in the livers and spleens of certain predisposed patients (those with iron overload states such as hemochromatosis) is observed. In the mouse oral infection model, yersiniae produce a similar disease involving the replication of yersiniae in the small intestine, the invasion of Peyer's patches, and dissemination to the liver and spleen. In these tissues and organs, yersiniae are known to replicate predominantly extracellularly and to form microcolonies. By infecting mice orally with a mixture of equal amounts of green- and red-fluorescing yersiniae (yersiniae expressing green or red fluorescent protein), we were able to show for the first time that yersiniae produce exclusively monoclonal microcolonies in Peyer's patches, the liver, and the spleen, indicating that a single bacterium is sufficient to induce microcolony and microabscess formation in vivo. Furthermore, we present evidence for the clonal invasion of Peyer's patches from the small intestine. The finding that only very few yersiniae are required to establish microcolonies in Peyer's patches is due to both Yersinia-specific and host-specific factors. We demonstrate that yersiniae growing in the small intestinal lumen show strongly reduced levels of invasin, the most important factor for the early invasion of Peyer's patches. Furthermore, we show that the host severely restricts sequential microcolony formation in previously infected Peyer's patches.

Regulatory network of lipopolysaccharide O-antigen biosynthesis in Yersinia enterocolitica includes cell envelope-dependent signals.

Lipopolysaccharide (LPS) is a glycolipid present in the outer membrane of all Gram-negative bacteria, and it is one of the signature molecules recognized by the receptors of the innate immune system. In addition to its lipid A portion (the endotoxin), its O-chain polysaccharide (the O-antigen) plays a critical role in the bacterium-host interplay and, in a number of bacterial pathogens, it is a virulence factor. We present evidence that, in Yersinia enterocolitica serotype O:8, a complex signalling network regulates O-antigen expression in response to temperature. Northern blotting and reporter fusion analyses indicated that temperature regulates the O-antigen expression at the transcriptional level. Promoter cloning showed that the O-antigen gene cluster contains two transcriptional units under the control of promoters P(wb1) and P(wb2). The activity of both promoters is under temperature regulation and is repressed in bacteria grown at 37 degrees C. We demonstrate that the RosA/RosB efflux pump/potassium antiporter system and Wzz, the O-antigen chain length determinant, are indirectly involved in the regulation mainly affecting the activity of promoter P(wb2). The rosAB transcription, under the control of P(ros), is activated at 37 degrees C, and P(wb2) is repressed through the signals generated by the RosAB system activation, i.e. decreased [K+] and increased [H+]. The wzz transcription is under the control of P(wb2), and we show that, at 37 degrees C, overexpression of Wzz downregulates slightly the P(wb1) and P(wb2) activities and more strongly the P(ros) activity, with the net result that more O-antigen is produced. Finally, we demonstrate that overexpression of Wzz causes membrane stress that activates the CpxAR two-component signal transduction system.

Differential stimulation of helper and cytotoxic T cells by dendritic cells after infection by Yersinia enterocolitica in vitro.

Dendritic cells (DC) are antigen-presenting cells crucial for initiating immune responses like sensitization of T cells to foreign antigens. We have previously shown that infection of DC by enteropathogenic Yersinia enterocolitica in vitro leads to a transient suppression in the immunostimulatory capacity for autologous enriched total T cells. In this study, we found that killed Yersinia could replace live bacteria in this aspect, and that yersinial lipopolysaccharide (LPS)-antigen could be detected intracellularly over a time course of 8 days. A suppressive effect on T cell proliferation after stimulation with Yersinia-infected compared to uninfected DC was seen for CD4+ T cells isolated by immunomagnetic separation techniques over the whole time course of 8 days, whereas CD8+ T cells followed to exhibit a suppressed proliferation rate starting on day 5 post infection till the end of the time course. In contrast, enriched total T cells stimulated by Yersinia-infected DC showed weaker proliferation till day 6 post infection compared to stimulation by uninfected DC, but not thereafter. Mixing of purified CD4+ and CD8+ T cells at day 8 post infection could reconstitute the effect seen for enriched total T cells. Thus, helper in concert with cytotoxic T cells might contribute to the immune responses, that are necessary for control of Yersinia-infections.

Effects of tamoxifen, melatonin, coenzyme Q10, and L-carnitine supplementation on bacterial growth in the presence of mycotoxins.

The involvement of toxic oxygen intermediates in the bacteriostatic effects of mycotoxins (T-2 toxin, deoxynivalenol, ochratoxin A, aflatoxin B1, and fumonisin B1) was investigated by producing bacterial growth curves using turbidimetry assays in the presence and absence of oxygen radical-scavenging substances. The strains used in this study included Escherichia coli (FT 101), Streptococcus agalactiae (FT 311, FT 313, FT 315), Staphylococcus aureus (FT 192), Yersinia enterocolitica (FT 430), Salmonella infantis (FT 431), Erysipelothrix rhusiopathiae (FT 432), Lactobacillus plantarum (FT234) and Lactobacillus casei (FT 232). Tamoxifen, melatonin, l-carnitine and coenzyme Q10 were used as radical scavengers against oxygen toxicity to the strains studied. Tamoxifen was the most effective in inhibiting bacterial growth when used at a high concentration, whereas melatonin and l-carnitine were less effective. A combination of l-carnitine and coenzyme Q10 provided better protection against oxygen toxicity caused by the mycotoxins growth than they did individually. It was concluded that oxygen radicals are involved in the killing of bacteria and that there is endogenous formation of toxic oxygen products by mycotoxins. The objective of this study was to determine whether the antioxidants were able to counteract the toxic effects of the mycotoxins. The data obtained indicate that bacterial growth can be inhibited especially by T-2 toxin, aflatoxin B1 and ochratoxin A and that this effect can be partially counteracted by antioxidants such as coenzyme Q10 plus l-carnitine.

[An analysis of the mechanisms of interpopulation interaction of Yersinia with Tetrahymena pyriformis infusorians at the cellular and subcellular levels]. / Analiz mekhanizmov mezhpopuliatsionnogo vzaimodeistviia iersinii s infuzoriiami Tetrahymena pyriformis na kletochnom i subkletochnom urovniakh.

The mechanisms of interaction between the populations of Yersinia and T. pyriformis have been analyzed on the cellular and subcellular levels. As shown in this investigation, Yersinia, when phagocytized by T. pyriformis, may undergo morphological changes, remain unchanged and also multiply, destroying the host cell in the process.

Rapid enumeration of viable bacteria by image analysis.

A direct viable counting method for enumerating viable bacteria was modified and made compatible with image analysis. A comparison was made between viable cell counts determined by the spread plate method and direct viable counts obtained using epifluorescence microscopy either manually or by automatic image analysis. Cultures of Escherichia coli, Salmonella typhimurium, Vibrio cholerae, Yersinia enterocolitica and Pseudomonas aeruginosa were incubated at 35 degrees C in a dilute nutrient medium containing nalidixic acid. Filtered samples were stained for epifluorescence microscopy and analysed manually as well as by image analysis. Cells enlarged after incubation were considered viable. The viable cell counts determined using image analysis were higher than those obtained by either the direct manual count of viable cells or spread plate methods. The volume of sample filtered or the number of cells in the original sample did not influence the efficiency of the method. However, the optimal concentration of nalidixic acid (2.5-20 micrograms ml-1) and length of incubation (4-8 h) varied with the culture tested. The results of this study showed that under optimal conditions, the modification of the direct viable count method in combination with image analysis microscopy provided an efficient and quantitative technique for counting viable bacteria in a short time.

Characteristics of the yellow pigment from a strain of Yersinia enterocolitica biovar 1.

Yersinia enterocolitica 195A14J, a milk-isolated strain of biovar 1, produces a non-diffusible yellow pigment and forms star-shaped colonies when grown on egg-yolk agar at 28 degrees C. Solubility properties and in situ Raman spectrum of the pigment support evidence that it is not a carotenoid, although it contains a 9 (+/- 1) double-bond polyenic chain. Pigmentless variants, also star-shaped, appeared with a frequency of ca.10(-3) when bacteria were grown at 38 degrees C. Agarose gel electrophoresis of DNA extracted from the pigmented strain revealed the presence of a 42-kb plasmid which was lost in pigmentless variants.

Changes in virulence of waterborne enteropathogens with chlorine injury.

We designed experiments to assess the effect of chlorine injury on the virulence of waterborne enteropathogens. Higher chlorine doses (0.9 to 1.5 mg/liter) were necessary to produce injured Yersinia enterocolitica, Salmonella typhimurium, and Shigella spp. than to produce injured enterotoxigenic Escherichia coli or coliform bacteria (0.25 to 0.5 mg/liter) in the test system used; 50% lethal dose experiments in which mice were used showed that injured Y. enterocolitica cells were 20 times less virulent than uninjured control cells (3,300 and 160 CFU, respectively). This decrease in virulence was not related to reduced attachment to Henle 407 intestinal epithelial cells, but could be related to a loss of HeLa cell invasiveness. In contrast, injured S. typhimurium and enterotoxigenic E. coli cells lost their ability to attach to Henle cells. These data show that some enteropathogens and coliform bacteria differ in their sensitivities to chlorine injury and that the virulence determinants affected by chlorine may vary from one pathogen to another.