Pathogen Sources Estimation and Scenario Analysis Using the Soil and Water Assessment Tool (SWAT)

Concerns over water quality in Ireland have increased in recent years, in part due to the more frequent contamination of drinking water by pathogens such as Escherichia coli and Cryptosporidium. The objective of this study was to assess the use of SWAT for pathogen source estimation and to analyze the effects of various source scenarios on pathogen outputs in Irish catchments. Two agricultural catchments in Ireland susceptible to pathogen contamination of source water were the center of the SWAT model development with the primary focus on levels of E. coli in surface water. Model simulations used site and source specific information which was analyzed considering the total E. coli count for the simulation period (Fergus: January 2005–October 2006; Kilshanvey: January 2006–July 2007). Pathogen source estimation identified point sources as the most significant contributors to E. coli output with direct deposition the primary contributor (95%) in Kilshanvey and wastewater treatment plant outflow (89%) the main contributor in the Fergus catchment. A scenario analysis evaluated possible situations that may occur in study locations. The analysis indicated that restriction of livestock access to water sources and improved wastewater treatment would represent effective methods of improving water quality in both catchments.     

Synthesis and preliminary biological screening of sterol analogues as new antifungal agents against plant pathogens

In this paper we report the synthesis of a new family of sterol analogues that have two amidic bonds on the side chain. These azasterols were obtained by a straightforward procedure including an Ugi condensation that allows the facile attachment of a polyfunctionalized side chain into the steroidal framework.Some of the new compounds showed an interesting inhibitory effect on the growth of two pathogenic fungi involved in plant diseases.     

Plasmid deoxyribonucleic acid in strains of Bacillus sphaericus and in Bacillus moritai

Bacillus moritai and six strains of Bacillus sphaericus pathogenic to dipteran larvae were examined for the presence of covalently closed circular (CCC) DNA. The plasmid profiles of the bacteria were analyzed using a cleared lysate electrophoresis technique. Four of the six strains of B. sphaericus examined contained CCC DNA. Strain SSII-1 contained two plasmids (pKA1, pKA2) having molecular weights of about 8.4 and 2.0 megadaltons (MDa). Strains 1404 and 1881 each contained one plasmid, pKA3 and pKA4, respectively. pKA3 had a molecular weight of about 8.2 MDa. pKA4 had a relatively large plasmid with a molecular weight of about 33.5 MDa. Strain K contained five size classes of CCC DNA. The plasmids pKA5, pKA6, pKA7, pKA8, and pKA9 had molecular weights of about 11.4, 10.9, 7.4, 7.0, and 6.4 MDa, respectively. Strains 1593-4 and 1691 were plasmidless and could not be distinguished from each other based on their plasmid profiles. B. moritai ATCC 21042 contained two size classes of CCC duplex DNA; pRF100 had a molecular weight of about 4.6 MDa and pRF101 had a molecular weight of about 2.1 MDa. No phenotype association with any of the isolated plasmids has been determined.     

AtPTR3, a wound-induced peptide transporter needed for defence against virulent bacterial pathogens in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Mutation in the wound-induced peptide transporter gene AtPTR3 (At5g46050) of Arabidopsis thaliana has been shown to affect germination on media containing a high salt concentration. The heterologous expression in yeast was utilized to verify that the AtPTR3 protein transports di-and tripeptides. The T-DNA insert in the Atptr3-1 mutant in the Arabidopsis ecotype C24 revealed two T-DNA copies, the whole vector sequence, and the gus marker gene inserted in the second intron of the AtPTR3 gene. An almost identical insertion site was found in the Atptr3-2 mutant of the Col-0 ecotype. The AtPTR3 expression was shown to be regulated by several signalling compounds, most clearly by salicylic acid (SA), but also methyl jasmonate (MeJA) and abscisic acid. Real-time PCR experiments suggested that the wound-induction of the AtPTR3 gene was abolished in the SA and JA signalling mutants. The Atptr3 mutant plants had increased susceptibility to virulent pathogenic bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tomato, and produced more reactive oxygen species when grown on media containing paraquat or rose bengal. Public microarray data suggest that the AtPTR3 expression was induced by Pseudomonas elicitors and by avirulent P. syringae pathovars and type III secretion mutants. This was verified experimentally for the hrpA mutant with real-time PCR. These results suggest that AtPTR3 is one of the defence-related genes whose expression is reduced by virulent bacterium by type III dependent fashion. Our results suggest that AtPTR3 protects the plant against biotic and abiotic stresses.     

Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus

AIMS: The aims of this study present were to assess and to evaluate in vitro the abilities of commercial probiotic strains derived from fermented milk products and related sources currently marketed in European countries, to inhibit, compete and displace the adhesion of selected potential pathogens to immobilized human mucus. METHODS AND RESULTS: The adhesion was assessed by measuring the radioactivity of bacteria adhered to the human mucus. We tested 12 probiotic strains against eight selected pathogens. All strains tested were able to adhere to mucus. All probiotic strains tested were able to inhibit and displace (P<0.05) the adhesion of Bacteroides, Clostridium, Staphylococcus and Enterobacter. In addition, the abilities to inhibit and to displace adhered pathogens depended on both the probiotic and the pathogen strains tested suggesting that several complementary mechanisms are implied in the processes. CONCLUSIONS: Our results indicate the need for a case-by-case assessment in order to select strains with the ability to inhibit or displace a specific pathogen. Probiotics could be useful to correct deviations observed in intestinal microbiota associated with specific diseases and also, to prevent pathogen infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The competitive exclusion properties of probiotics as well as their ability to displace and inhibit pathogens are the most importance for therapeutic manipulation of the enteric microbiota. The application of such strategies could contribute to expand the beneficial properties on human health against pathogen infection.     

Bioinformatic analysis of the TonB protein family

TonB is a protein prevalent in a large number of Gram-negative bacteria that is believed to be responsible for the energy transduction component in the import of ferric iron complexes and vitamin B₁₂ across the outer membrane. We have analyzed all the TonB proteins that are currently contained in the Entrez database and have identified nine different clusters based on its conserved 90-residue C-terminal domain amino acid sequence. The vast majority of the proteins contained a single predicted cytoplasmic transmembrane domain; however, nine of the TonB proteins encompass a ∼290 amino acid N-terminal extension homologous to the MecR1 protein, which is composed of three additional predicted transmembrane helices. The periplasmic linker region, which is located between the N-terminal domain and the C-terminal domain, is extremely variable both in length (22–283 amino acids) and in proline content, indicating that a Pro-rich domain is not a required feature for all TonB proteins. The secondary structure of the C-terminal domain is found to be well preserved across all families, with the most variable region being between the second α-helix and the third β-strand of the antiparallel β-sheet. The fourth β-strand found in the solution structure of the Escherichia coli TonB C-terminal domain is not a well conserved feature in TonB proteins in most of the clusters. Interestingly, several of the TonB proteins contained two C-terminal domains in series. This analysis provides a framework for future structure-function studies of TonB, and it draws attention to the unusual features of several TonB proteins. Byron C. H. Chu and R. Sean Peacock contributed equally to this work.     

Identification of sources of microbial pathogens on cantaloupe rinds from pre-harvest to post-harvest operations

Foodborne illness outbreaks involving cantaloupes have increased dramatically in the past 15 years in the United States and other countries. The need for the identification of the microbial sources that contaminate cantaloupe rinds has been raised by various investigators. This study was undertaken to identify the agricultural, industrial and human sources of microbial contamination from the pre- to post-harvest operations of cantaloupes grown at ten different farms in southern Texas. Results indicate that irrigation water contained a wide range of microorganisms that could cause human illness and were able to survive on the rind of cantaloupes before, at, and after harvesting. Fungi, total aerobic bacteria and total coliform bacteria were not completely eliminated by chlorinated water in the disinfection tanks of the six packinghouses under investigation. There were significant (P < 0.05) reductions on rind populations of fungi and total aerobic bacteria as well as drastic reductions in total coliform bacteria on the rinds after the disinfection and rinsing steps in all packing facilities. There was no evidence of the presence of Escherichia coli O157:H7 on packed cantaloupes across packinghouses. Less than a geometric mean of 1 c.f.u. cm⁻² of salmonellae were detected on the surface of packed cantaloupes in two of the packinghouses, and approximately ten times more salmonellae were found on the packed fruit processed in the remaining packinghouses. A similar trend was observed with listeriae. Results suggested that microbial loads originating from river water may survive on the rind or may re-infest cantaloupes after the disinfection and rinsing process at the packinghouses. Disinfection techniques and aseptic handling of cantaloupes at the packing facilities need a closer evaluation to ensure a safe product.     

Antimicrobial activity of various cationic molecules on foodborne pathogens

Antibacterial effects of various arginine- and lysine-rich polycationic proteins and polymers were evaluated by broth and solid dilution assay on a range of foodborne pathogens, Gram-positive and Gram-negative bacteria. The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) of α-poly-l-lysine (poly-lys), α-poly-l-arginine (poly-arg) and protamines from herring sperm (clupeine sulphate) and salmon sperm (salmine sulphate) were determined on Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium, Shigella sonnei, Escherichia coli O157:H7 and Pseudomonas aeruginosa. All these molecules showed antibacterial activity on all strains with different MIC and MBC values. The molecular mechanisms underlying the effect of α-poly-l-arginine might be related to the entrance of the molecule into the cell. In fact α-poly-l-arginine labelled with 7-Diethylamino coumarin-3-carboxylic acid, succinimidyl ester (DEAC,SE) showed ability to permeate the cell membrane of B. cereus and E. coli O157:H7.     

Microbiological quality of water and dialysate in a haemodialysis unit in Ponta Grossa-PR, Brazil

AIMS: The objective of the study was to determine the microbiological quality of samples of water and dialysate in a haemodialysis unit. METHODS AND RESULTS: Seventy-two samples each of water and dialysate were collected during November 2003 to April 2004. The following microbiological analyses were performed: test for total and faecal coliforms, which produced negative results for all the samples; counts of total heterotrophic bacteria, where three samples of water and two of dialysate showed levels higher than those permitted by national standards; and endotoxin assay, which revealed high quantities only in samples of water that preceded reverse osmosis. Nonfermenting Gram-negative bacteria were identified in 54 samples of dialysate and in 26 samples of water. The test for adhesion to an inert surface showed that various bacteria were capable of forming biofilms. Twenty-seven per cent of the bacteria were resistant to sodium hypochlorite at 500 ppm for 10-min contact time. Sixty per cent of the isolates were resistant to three or more antibiotics. CONCLUSIONS: Water and dialysate can be a source of infection for patients who need haemodialysis. SIGNIFICANCE AND IMPACT OF THE STUDY: An adequate system for water treatment, disinfection of the haemodialysis system and microbiological monitoring of the water and dialysate are necessary to reduce bacteraemia and pyrogenia outbreaks.     

Combination of a urease inhibitor and a plant essential oil to control coliform bacteria, odour production and ammonia loss from cattle waste

AIM: To evaluate urea hydrolysis, volatile fatty acid (VFA) production (odour) and coliforms in cattle waste slurries after a urease inhibitor N-(n-butyl) thiophosphoric triamide (NBPT) and a plant oil component (thymol) were added. METHODS AND RESULTS: Faeces from cattle fed a diet of 70% corn silage and 30% alfalfa haylage, urine and distilled water in the ratio 50 : 35 : 15 were blended at high speed for 1 min. Triplicate aliquots of 750 ml were amended with NBPT plus or minus thymol and reblended for 1 min, and were poured into 1.6 l wide-mouth jars covered 90% with a lid. After 56 days, thymol (2000 mg kg(-1) waste) in combination with NBPT (80 mg kg(-1) waste) retained 5.2 g of an initial 9.2 g of urea in cattle waste slurries, compared with less than 1 g of urea retained when NBPT was the only additive (P < 0.05). Another experiment using excreta from cattle fed 76.25% high moisture corn, 19.25% corn silage and a 4.5% supplement, blended at a low speed, gave a similar response with urea hydrolysis; and the two treatments, thymol alone and thymol in combination with NBPT, reduced VFA production (P < 0.01) and eliminated all coliform bacteria by day 1. A third experiment indicated coliforms disappeared in the no addition treatment after 8 days; however, they were viable at 6.6 x 10(4) CFU g(-1) waste beyond 35 days in the NBPT treatment. CONCLUSIONS: Thymol supplements the effect of NBPT by increasing the inhibitory period for hydrolysis of urea in cattle waste slurries and nitrogen retention in the waste. SIGNIFICANCE AND IMPACT OF THE STUDY: Thymol and NBPT offer the potential to reduce odour and pathogens in cattle manure, and increase the fertilizer value.