Implications of new legislation (US FSMA) and guidelines (EC) on the establishment of management systems for agricultural water.

This report summarizes key messages related to agricultural water quality as discussed by an ad hoc panel at the 1st International Symposium of Food Safety in Santiago, Chile. Participating representatives of the academia, industry and government of diverse geographical backgrounds and the audience discussed topics such as (1) implications of the US Food Safety Modernization Act (FSMA: www.fda.gov/Food/GuidanceRegulation/FSMA/ucm277706.htm) on the Agricultural Water Quality, (2) comparisons between MPN and CFU in analyzing water quality, (3) alternatives to fecal indicator bacteria (FIB) to be used as indicators to evaluate water quality, and (4) vegetative buffers as an alternative to reduce pathogen loads in agricultural surface waters. Panelists identified the following key messages for each topic discussed that are related to agricultural water quality: (1) the FSMA regulation and the new guidance document elaborated by the EC are highly relevant as they provide a definition of agricultural water and specific criteria for different water uses and circumstances; (2) FSMA supports modification from MPN to CFU; (3) Growers require more alternatives for treatment of agricultural water; (4) Vegetative buffers are a potential practical and feasible alternative for agriculture producers to reduce the pathogen and fecal pollution loads of in their agricultural waters.

The Pathogen-annotated Tracking Resource Network (PATRN) system: a web-based resource to aid food safety, regulatory science, and investigations of foodborne pathogens and disease.

Investigation of foodborne diseases requires the capture and analysis of time-sensitive information on microbial pathogens that is derived from multiple analytical methods and sources. The web-based Pathogen-annotated Tracking Resource Network (PATRN) system (www.patrn.net) was developed to address the data aggregation, analysis, and communication needs important to the global food safety community for the investigation of foodborne disease. PATRN incorporates a standard vocabulary for describing isolate metadata and provides a representational schema for a prototypic data exchange standard using a novel data loading wizard for aggregation of assay and attribution information. PATRN currently houses expert-curated, high-quality "foundational datasets" consisting of published experimental results from conventional assays and next generation analysis platforms for isolates of Escherichia coli, Listeria monocytogenes, and Salmonella, Shigella, Vibrio and Cronobacter species. A suite of computational tools for data mining, clustering, and graphical representation is available. Within PATRN, the public curated data repository is complemented by a secure private workspace for user-driven analyses, and for sharing data among collaborators. To demonstrate the data curation, loading wizard features, and analytical capabilities of PATRN, three use-case scenarios are presented. Use-case scenario one is a comparison of the distribution and prevalence of plasmid-encoded virulence factor genes among 249 Cronobacter strains with similar attributes to that of nine Cronobacter isolates from recent cases obtained between March and October, 2010-2011. To highlight PATRN's data management and trend finding tools, analysis of datasets, stored in PATRN as part of an ongoing surveillance project to identify the predominant molecular serogroups among Cronobacter sakazakii isolates observed in the USA is shown. Use-case scenario two demonstrates the secure workspace available for private users to upload and analyze sensitive data, and for collating cross-platform datasets to identify and validate congruent datapoints. SNP datasets from WGS assemblies and pan-genome microarrays are analyzed in a combinatorial fashion to determine relatedness of 33 Salmonella enterica strains to six strains collected as part of an outbreak investigation. Use-case scenario three utilizes published surveillance results that describe the incidence and sources of O157:H7 E. coli isolates associated with a produce pre-harvest surveillance study that occurred during 2002-2006. In summary, PATRN is a web-based integrated platform containing tools for the management, analysis and visualization of data about foodborne pathogens.

Cpa, the outer membrane protease of Cronobacter sakazakii, activates plasminogen and mediates resistance to serum bactericidal activity.

Cronobacter spp. are emerging neonatal pathogens in humans, associated with outbreaks of meningitis and sepsis. To cause disease, they must survive in blood and invade the central nervous system by penetrating the blood-brain barrier. C. sakazakii BAA-894 possesses an ~131-kb plasmid (pESA3) that encodes an outer membrane protease (Cpa) that has significant identity to proteins that belong to the Pla subfamily of omptins. Members of this subfamily of proteins degrade a number of serum proteins, including circulating complement, providing protection from the complement-dependent serum killing. Moreover, proteins of the Pla subfamily can cause uncontrolled plasmin activity by converting plasminogen to plasmin and inactivating the plasmin inhibitor α2-antiplasmin (α2-AP). These reactions enhance the spread and invasion of bacteria in the host. In this study, we found that an isogenic cpa mutant showed reduced resistance to serum in comparison to its parent C. sakazakii BAA-894 strain. Overexpression of Cpa in C. sakazakii or Escherichia coli DH5α showed that Cpa proteolytically cleaved complement components C3, C3a, and C4b. Furthermore, a strain of C. sakazakii overexpressing Cpa caused a rapid activation of plasminogen and inactivation of α2-AP. These results strongly suggest that Cpa may be an important virulence factor involved in serum resistance, as well as in the spread and invasion of C. sakazakii.

Distinct T cell populations distinguish chronic myeloid leukaemia cells from lymphocytes in the same individual: a model for separating GVHD from GVL reactions.

Donor lymphocyte responses to minor histocompatibility antigen (mHA) differences are involved in allo-responses between HLA matched pairs causing GVHD and graft-versus-leukaemia (GVL). Since some mHA are tissue-restricted, GVHD and GVL responses may be separable. We studied donor lymphocyte responses to patients with CML in a series of 10 HLA-matched sibling and 10 unrelated donor-recipient pairs comparing proliferation to recipient PHA blasts and CML cells and attempting to selectively deplete responses to PHA blasts in vitro. Responses in counts per min (c.p.m) to CML cells and PHA blasts were, respectively, 2809 +/- 2205 (SD) and 7376 +/- 1877 in related and 12,107 +/- 7191 and 26,136 +/- 22,479 in unrelated pairs. Autologous responses to PHA blasts were significantly lower (mean 779 +/- 735) (p < 0.001). Results correlated with clinical outcome: higher responses to recipient cells correlated with transplant-related death (p = 0.02 for CML and p = 0.06 for PHA blasts). Higher responses to CML correlated with GVHD grade > or = II (p = 0.025). Donor lymphocytes exposed to recipient PHA blasts for 5 days and treated with a ricin-conjugated anti-CD25 antibody retained over 75% of their response to CML but < 10% to PHA blasts. Similarly, depletion of response to CML but not to PHA blasts occurred when CML was the primary challenge. These results indicate that distinct populations of donor T cells respond to recipient leukaemic and non-leukaemic cells, and provide the basis for a clinically applicable technique to selectively deplete donor GVHD reacting cells while conserving GVL.

Cell density dependent acid sensitivity in stationary phase cultures of enterohemorrhagic Escherichia coli O157:H7.

Escherichia coli O157:H7, the causative agent of hemorrhagic colitis and hemolytic uremic syndrome, can survive in a highly acidic environment. The acid resistance of this organism, as measured by its ability to survive in low pH, depended on the density of the cells present during the assay. At low cell densities (

Identification and enumeration of virulent Listeria strains.

Recent outbreaks and a continuous increase in cases of listeriosis underscore the need for rapid, sensitive and reliable techniques to detect Listeria. Of the species of Listeria, only L. monocytogenes has been found to be associated with human infections. One factor which definitely contributes to its pathogenicity is the presence of hemolysins, although L. ivanovii and L. seeligeri also elaborate hemolysins. Based upon cloned hemolysin genes, we have developed DNA probes specifically for detecting L. monocytogenes. The technique combines growth of bacterial colonies on selective agar plates and DNA hybridization of these colonies on a solid matrix. This technique permits identification and enumeration, and the entire procedure can be completed in 3-4 days. Our method was found to be suitable for identifying and enumerating this organism in various foods, the main vehicle of human infection. Advantages and disadvantages of this technique are discussed and compared with other existing techniques.

Recurrent focal segmental glomerulosclerosis after renal transplantation.

In a young adult male with chronic renal failure from focal segmental glomerulosclerosis, massive proteinuria appeared within two weeks after transplantation. Although allograft biopsy at eight weeks was normal, a second biopsy eight months after transplant showed focal segmental glomerulosclerosis again. Sixteen months after transplantation maintenance hemodialysis had to be restarted.

Factors controlling acid tolerance of Listeria monocytogenes: effects of nisin and other ionophores.

The acid tolerance of a Listeria monocytogenes serotype 4b strain was studied by measuring its ability to survive at an acidic pH at 37 degrees C. The acid tolerance of L. monocytogenes was much lower than those of Escherichia coli O157:H7 and Shigella flexneri strains. This observation suggested a higher infective dose for L. monocytogenes than E. coli O157:H7 and Shigella. The susceptibility of L. monocytogenes to acidic pH was dependent upon growth medium pH and growth phase of the culture. Nisin and some other ionophores reduced the acid tolerance of both stationary-phase and log-phase cultures of L. monocytogenes. These studies indicated that nisin might be a useful candidate for controlling acid tolerance of L. monocytogenes.

Acid tolerance of enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) strains were tested for their ability to survive in acid pH at 37 degrees C. No loss of viability was observed in an O157:H7 EHEC strain (ATCC 43895) at pH levels of 3.0 and 2.5 for at least 5 h. The level of acid tolerance of most EHEC isolates was very high, similar to that of Shigella flexneri strains. The acid tolerance was dependent on the growth phase and pH of the growth medium.

Mechanisms of recombination by the RecBC and the RecF pathways following conjugation in Escherichia coli K12.

The recombinational processes directed by the RecBC and the RecF pathways following conjugation in E. coli have been compared. The viable recombinant products of the RecF pathway show a higher incidence of mismatch correction, higher percentage of heterogeneous clones produced by single ex-conjugants and a much slower rate of integration and segregation compared to the RecBC pathway. There are reasons to suspect that the product of recB and recC genes may be necessary for conversion of the single stranded donor DNA in the zygote to double stranded DNA. Theoretical considerations suggest that an exchange involving only one strand of DNA may be a much slower process, with more stringent homology requirement for the entire exchanged segment, than a double strand exchange of a comparable length; the latter should be much faster, with stringent homology requirements for only the terminal regions of the exchanged segments. It is suggested that the RecF pathway mainly mediates replacement of relatively long stretches of single strands of recipient DNA by the corresponding strands of donor DNA while the RecBC pathway mediates exchange of mostly double stranded DNA between the donor and the recipient; in addition, the RecBC pathway may also catalyze the integration of very small segments of single strands of the donor DNA. A model based on the above basic hypothesis is described. It is further suggested that the enzymes exonucleaseV and exonucleaseI Control the relative yields of the recombinants produced by the two pathways by regulating the supply of the donor substrates required by these pathways; the former diverts the potential substrate of the RecF pathway (single stranded DNA) to the duplex substrates of the RecBC pathway while the latter destroys the substrates of the RecF pathway, especially in absence of exonucleaseV.

Reduced transposition in rho mutants of Escherichia coli K-12.

Substantially reduced frequencies of transposition for the transposons Tn5 and Tn9 and the insertion sequences IS1 and IS5 were observed in several rho mutants of Escherichia coli K-12 compared with those observed in their isogenic wild-type counterparts. The lower transposition frequencies could be due to decreased supercoiling of DNA, to altered expression of required genes, or to aberrant transcription of transposon or target DNA resulting from the lack of transcription termination at Rho-sensitive sites in rho mutants.

Salt tolerance of lactose-grown Vibrio parahaemolyticus carrying Escherichia coli lac genes.

Lac- strains of Vibrio parahaemolyticus were converted to Lac+ on receiving a hybrid plasmid containing the lactose utilization genes of Escherichia coli K-12. A V. parahaemolyticus strain containing this hybrid plasmid exhibited optimal growth rates on glucose and other carbon sources in the presence of 0.2 to 0.4 M NaCl. Growth of the same strain on lactose was inhibited at similar concentrations of NaCl. The altered growth rate responses in lactose medium appeared to be attributable to effects of NaCl on the activity of lactose permease, and possibly on that of beta-galactosidase, rather than on the levels of these enzymes in V. parahaemolyticus cells.

Effects of glucose, growth temperature, and pH on listeriolysin O production in Listeria monocytogenes.

Expression of listeriolysin O of Listeria monocytogenes as a function of different growth conditions was studied by performing a direct hemolysin assay, immunoblotting experiments, and an enzyme-linked immunosorbent assay. Expression of listeriolysin O was reduced at a lower growth temperatures (26 degrees C) and at higher glucose concentrations (> or = 0.3%) in the growth media. The effect of glucose appeared to be due to a change in the pH of the growth media.

Identification and enumeration of beta-hemolytic Listeria monocytogenes in naturally contaminated dairy products.

A DNA probe was used to identify hemolytic Listeria monocytogenes in naturally contaminated dairy products: unpasteurized milk, ricotta cheese, and imported semisoft cheeses. Of 34 milk samples, 12 were suspected to contain hemolytic L. monocytogenes; 1 contained greater than 6000 viable organisms/g. The ricotta cheese, although temperature-abused, had a titer of 3.6 x 10(6) beta-hemolytic L. monocytogenes cells/g, whereas the semisoft cheeses reached a maximum of 5.6 x 10(6) cells/g. Pure cultures of L. monocytogenes isolated from both types of cheese were found positive by the CAMP test and the DNA probe.

A new pleiotropic mutation causing defective carbohydrate uptake in Escherichia coli K-12: isolation, mapping, and preliminary characterization.

A new pleiotropic mutation, designated cup-1 (for carbohydrate uptake), which impairs the ability of Escherichia coli cells to grow on a large number of phosphotransferase system (PTS) and non-PTS carbohydrates by blocking their entry into the cells, has been isolated, partially characterized, and mapped. The mutants grew poorly even on rich and glucose minimal media. Fast-growing revertants rapidly accumulated in cultures grown on either of the above two media and made stable maintenance of the mutation difficult. Several extragenic suppressor mutations that permitted cup cells to grow on specific single sugars or groups of sugars have been isolated. One such suppressor, which enabled cup cells to grow as well on glycerol minimal medium as their wild-type parent, has been helpful in stably maintaining these cells in this medium. cup-1 has been mapped to 97 min on the standard E. coli map. It cotransduced with a transposon Tn10 inserted clockwise to it and (very weakly) with uxuA. Surprisingly, it failed to cotransduce with pyrB, argI, or valS, three markers located nearby but counterclockwise to it. In F' merodiploids, cup-1 was dominant over its cup+ allele. Cyclic AMP permitted growth of cup-1 cells on some sugars but not all. Apparently, reduced cyclic AMP level and therefore noninduction of several sugar operons is one but not the only effect of cup.

Genetic and physical characterization of proBA genes of the marine bacterium Vibrio parahaemolyticus.

Intracellular proline pools have been implicated in the halotolerance of many organisms. To examine this relationship in a moderately halotolerant marine bacterium, Vibrio parahaemolyticus, proline biosynthesis genes were cloned in various plasmids. Some genetic and structural properties of those genes were examined. Subcloning showed that about 3.1 kilobases of V. parahaemolyticus DNA could complement proA and proB but not proC mutations of Escherichia coli. The same fragment would also complement some Pro- mutants of V. parahaemolyticus. Gamma-delta insertion mutagenesis of this subcloned fragment indicated that proB and proA genes of V. parahaemolyticus might be transcribed from different promoters. Two other genes, phoE and gpt, which map closely to the proBA genes in E. coli, were also found to be in close proximity to the proBA genes of V. parahaemolyticus.

Application of a Synthetic Listeriolysin O Gene Probe to the Identification of ß-Hemolytic Listeria monocytogenes in Retail Ground Beef.

The synthetic gene probe is a 20 mer oligonucleotide, derived from listeriolysin O gene sequence of Listeria monocytogenes and shown to be specific for strains of this organism. This probe was used in a DNA-colony hybridization assay to evaluate its suitability in detecting (ß-hemolytic L. monocytogenes in ground beef. Thirty-six ground beef samples were plated onto three media: Trypticase soy agar with 0.6% yeast extract, lithium chloride-phenylethanol-moxalactam agar and Martin's agar, both directly and after selective enrichment in Food and Drug Administration broth. Of the 118 gram-positive and catalase-positive isolates selected from the plates, only 24 gave detectable hybridization signal with the probe. CAMP-test and standard biochemical tests also revealed that only these 24 probe positive isolates were (ß-hemolytic L. monocytogenes . Of the 36 samples of ground beef, 6 were positive for Listeria spp., out of which 4 were L. monocytogenes .

Detection of chemicals that stimulate Tn9 transposition in Escherichia coli K12.

A spot test has been developed for detecting substances that enhance the transposition of Tn9 in Escherichia coli. Phage lambda::Tn9-infected cells were plated on chloramphenicol media and a drop of the test substance was placed at the center of the plate. Following incubation, chloramphenicol-resistant colonies appeared due to the transposition of Tn9 to the bacterial chromosome. By comparing the test plate and a control plate with respect to the number and distribution of colonies, the effect of the test compound can be evaluated. Out of over 100 compounds tested, acetate, two detergents (Brij 58 and Nonidet P40) and dimethylsulfoxide were found to enhance transposition 3-20 fold. Acetate was also found to enhance the transposition of Tn5 and Tn10. The stimulating effect of Brij 58 was lost when palmitic acid was added with the Brij 58. The nature of these substances, which we refer to as "transposagens", suggests an involvement of lipid or membrane in the transposition process.

Detection of hemolytic Listeria monocytogenes by using DNA colony hybridization.

A fragment of about 500 base pairs of the beta-hemolysin gene from Listeria monocytogenes was used to screen different bacterial strains by DNA colony hybridization. The cells in the colonies were lysed by microwaves in the presence of sodium hydroxide. Of 52 different strains of Listeria species screened, only the DNA from beta-hemolytic (CAMP-positive) strains of L. monocytogenes hybridized with this probe.

Shuttle cloning vectors for the marine bacterium Vibrio parahaemolyticus.

Two cosmid cloning vectors containing lambda cos sequences and a 42-base-pair multipurpose cloning sequence were constructed. pAD22 also contains a 1.4-kilobase TRP-ARS fragment from Saccharomyces cerevisiae. These cosmids transformed Escherichia coli and S. cerevisiae cells and could be mobilized into Vibrio parahaemolyticus strains with a conjugative plasmid, pRK2013. The cosmid pAD22 was genetically and structurally stable during passage through V. parahaemolyticus and E. coli strains.