Efficacy of chlorine and heat treatment in killing Salmonella stanley inoculated onto alfalfa seeds and growth and survival of the pathogen during sprouting and storage.

The efficacy of chlorine and hot water treatments in killing Salmonella stanley inoculated onto alfalfa seeds was determined. Treatment of seeds containing 10(2) to 10(3) CFU/g in 100-micrograms/ml active chlorine solution for 5 or 10 min caused a significant (P < or = 0.05) reduction in population, and treatment in 290-micrograms/ml chlorine solution resulted in a significant reduction compared with treatment in 100 micrograms of chlorine per ml. However, concentrations of chlorine of up to 1,010 micrograms/ml failed to result in further significant reductions. Treatment of seeds containing 10(1) to 10(2) CFU of S. stanley per g for 5 min in a solution containing 2,040 micrograms of chlorine per ml reduced the population to undetectable levels (< 1 CFU/g). Treatment of seeds in water for 5 or 10 min at 54 degrees C caused a significant reduction in the S. stanley population, and treatment at > or = 57 degrees C reduced populations to < or = 1 CFU/g. However, treatment at > or = 54 degrees C for 10 min caused a substantial reduction in viability of the seeds. Treatment at 57 or 60 degrees C for 5 min appears to be effective in killing S. stanley without substantially decreasing germinability of seeds. Storage of seeds for 8 to 9 weeks at 8 and 21 degrees C resulted in reductions in populations of S. stanley of about 1 log10 and 2 log10 CFU/g, respectively. The behavior of S. stanley on seeds during soaking germination, sprouting, and refrigerated storage of sprouts was determined. An initial population of 3.29 log10 CFU/g increased slightly during 6 h of soaking, by about 10(3) CFU/g during a 24-h germination period, and by an additional 10 CFU/g during a 72-h sprouting stage. A population of 10(7) CFU/g of mature alfalfa sprouts was detected throughout a subsequent 10-day storage period at 5 degrees C. These studies indicate that while populations of S. stanley can be greatly reduced, elimination of this organism from alfalfa seeds may not be reliably achieved with traditional disinfection procedures. If S. stanley is present on seeds at the initiation of the sprout production process, populations exceeding 10(7) CFU/g can develop and survive on mature sprouts exposed to handling practices used in commercial production and marketing.     

Curvularia haloperoxidase: antimicrobial activity and potential application as a surface disinfectant

A presumed antimicrobial enzyme system, the Curvularia haloperoxidase system, was examined with the aim of evaluating its potential as a sanitizing agent. In the presence of hydrogen peroxide, Curvularia haloperoxidase facilitates the oxidation of halides, such as chloride, bromide, and iodide, to antimicrobial compounds. The Curvularia haloperoxidase system caused several-log-unit reductions in counts of bacteria (Pseudomonas spp., Escherichia coli, Serratia marcescens, Aeromonas salmonicida, Shewanella putrefaciens, Staphylococcus epidermidis, and Listeria monocytogenes), yeasts (Candida sp. and Rhodotorula sp.), and filamentous fungi (Aspergillus niger, Aspergillus tubigensis, Aspergillus versicolor, Fusarium oxysporum, Penicillium chrysogenum, and Penicillium paxilli) cultured in suspension. Also, bacteria adhering to the surfaces of contact lenses were killed. The numbers of S. marcescens and S. epidermidis cells adhering to contact lenses were reduced from 4.0 and 4.9 log CFU to 1.2 and 2.7 log CFU, respectively, after treatment with the Curvularia haloperoxidase system. The killing effect of the Curvularia haloperoxidase system was rapid, and 10(6) CFU of E. coli cells/ml were eliminated within 10 min of treatment. Furthermore, the antimicrobial effect was short lived, causing no antibacterial effect against E. coli 10 min after the system was mixed. Bovine serum albumin (1%) and alginate (1%) inhibited the antimicrobial activity of the Curvularia haloperoxidase system, whereas glucose and Tween 20 did not affect its activity. In conclusion, the Curvularia haloperoxidase system is an effective sanitizing system and has the potential for a vast range of applications, for instance, for disinfection of contact lenses or medical devices.     

Enhanced microbial biomass assay using mutant luciferase resistant to benzalkonium chloride

In a biomass assay based on adenosine 5(')-triphosphate (ATP) bioluminescence, extracellular ATP is removed; then intracellular ATP is extracted from the microorganism by an ATP extractant and subsequently reacted with luciferase. To provide a highly sensitive assay, the concentration of benzalkonium chloride (BAC) in the ATP extractant was optimized by using a mutant luciferase resistant to BAC. The use of 0.2% BAC, which was acceptable for the luciferase, simultaneously achieved the maximum extraction of intracellular ATP from microorganisms and the inactivation of the ATP-eliminating enzymes for removal of extracellular ATP. The detection limit (blank+3 SD) for ATP was 1.8x10(-14)M (1.8x10(-18)mol/assay) in the presence of the ATP extractant with coefficients of variation of 0.7 to 6.3%. The reagent system coupled with the ATP-eliminating enzymes allowed for the detection of 93 colony-forming units (CFU)/ml of Escherichia coli ATCC 25922, 170CFU/ml of Pseudomonas aeruginosa ATCC 27853, 170CFU/ml of Proteus mirabilis ATCC 29906, 68CFU/ml of Staphylococcus aureus ATCC 25923, and 7.7CFU/ml of Bacillus subtilis ATCC 6051. The yeast cell of Saccharomyces cerevisiae IFO 10217 could be detected at 1CFU/ml. With 54 kinds of microorganisms, the average ATP extraction efficiency compared to the trichloroacetic acid extraction method was 81.0% in 24 strains among gram-negative bacteria, 99.4% in 13 strains among gram-positive bacteria, and 97.0% in 17 strains among yeast. The ATP contents of the gram-negative bacteria, gram-positive bacteria, and yeasts ranged from 0.40 to 2.70x10(-18)mol/CFU (mean=1.5x10(-18)mol/CFU), from 0.41 to 16.7x10(-18)mol/CFU (mean=5.5x10(-18)mol/CFU), and from 0.714 to 54.6x10(-16)mol/CFU (mean=8.00x10(-16)mol/CFU), respectively.     

Pilot-scale production and liquid formulation of Rhodotorula minuta, a potential biocontrol agent of mango anthracnose

AIMS: To develop a pilot-plant fermentation process for the production of the yeast Rhodotorula minuta, to be used as a biocontrol agent of mango anthracnose, using a low-cost culture medium. To develop a stable liquid formulation that preserve high viability of the yeast stored at 4 degrees C. METHODS AND RESULTS: Keeping constant the volumetric power input, a fermentation process was scaled-up from shake flasks to a 100 l bioreactor. Preharvest applications of the yeast resulted in postharvest anthracnose severity equal or lower than that observed with a chemical fungicide. Glycerol was added to the formulation as water activity reducer and xanthan gum as a viscosity-enhancing agent. Yeast initial concentration of 10(10) CFU ml(-1) resulted in 4-5 orders of magnitude decrease after 1 month of storage at 4 degrees C, whereas when it was formulated at 10(9) CFU ml(-1), the decrease was of two orders of magnitude in 6 months. CONCLUSIONS: The fermentation process was successfully scaled-up using a low-cost culture medium. Postharvest anthracnose severity could be considerably reduced using this yeast. Formulating the yeast at 10(9) CFU ml(-1) and adding glycerol (20%) and xanthan (5 g l(-1)) avoided both contamination and yeast sedimentation and it was able to preserve up to 10(7) CFU ml(-1) after 6 months at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The yeast R. minuta is reported as a novel antagonistic micro-organism against the pathogen Colletotrichum gloeosporioides. Pilot plant production of this yeast allowed us to conduct field tests in commercial orchards during three harvest seasons. Yeast suspensions applied to mango trees reduced the fruit anthracnose severity in levels similar or better than chemical fungicides.     

Comparative methodology for the detection and differentiation of circulating microfilariae of Dirofilaria immitis in the dog

The sensitivities of the Knott's test (four 20-microl sediment aliquots), quantitative buffy coat capillary tube method (QBC tube, 111 microl of whole blood) and direct blood smear (DBS, 20 microl of whole blood) were evaluated for the detection of microfilaraemia in dogs. Undiluted whole blood samples taken from 70 Dirofilaria immitis antigen-positive dogs and 10 serially diluted microfilaraemic blood samples at concentrations of 400, 200, 100, 50, 25 and 12 microfilariae (mff) ml(-1) were examined. For filarial speciation, the buffy coat of QBC tubes was mixed with one drop of methylene blue-formalin solution and examined as a direct smear. In 52/70 microfilaraemic blood samples, the number of mff ranged from 12 to 321987 ml(-1) (median: 3199 ml(-1)). The diagnostic sensitivity of the Knott's test, QBC tube method and DBS in undiluted blood samples attained the 100%, 98% and 92.3% levels, respectively. Eighteen dogs tested amicrofilaraemic by all three methods. At concentrations of 400 mff ml(-1), a 100% sensitivity was found by all three methods, while at 200 mff ml(-1) the Knott's test, QBC tube and DBS were 100%, 100% and 90% sensitive, respectively. The relevant figures at 100 mff ml(-1) were 100%, 100% and 80%, at 50 mff ml(-1) 100%, 100% and 50%, at 25 mff ml(-1) 100%, 100% and 10% and at 12 mff ml(-1) 80%, 50% and 10%. At 50 and 25 mff ml(-1), the DBS was less sensitive compared to the other two methods, while at 12 mff ml(-1), only to the Knott's test. A significant correlation was found between the QBC tube method and Knott's test regarding mff speciation. Therefore, the QBC method may be considered a reliable alternative to the Knott's test for both the detection and speciation of mff in the dog.     

Identification of differentially expressed genes in parasitic phase Miamiensis avidus (Ciliophora: Scuticociliatia) using suppression subtractive hybridization

A multiplex (m-)PCR-based protocol was designed for the simultaneous detection of the main marine bacterial pathogens in Chilean salmon farms: Streptococcus phocae, Aeromonas salmonicida, Vibrio anguillarum and Piscirickettsia salmonis. Each of the 4 oligonucleotide primer pairs exclusively amplified the target gene of the specific bacterial pathogen. The detection limit of the m-PCR using purified total bacterial DNA was 50 pg microl(-1) for V anguillarum, 500 fg microl(-1) for P. salmonis, and 5 pg microl(-1) for S. phocae and A. salmonicida. This corresponded to average limits in the m-PCR sensitivity of 3.69 x 10(5) CFU ml(-1) of V anguillarum, 1.26 x 10(4) CFU m(-1) of S. phocae, and 5.33 x 10(4) CFU ml(-1) of A. salmonicida, while the detection limits for the spiked fish tissues, regardless of the sample (spleen, kidney, liver or muscle) were 2.64 +/- 0.54 x 10(7) CFU g(-1) for V. anguillarum, 9.03 +/- 1.84 x 10(5) CFU g(-1) for S. phocae, 3.8 +/- 0.78 x 10(3) CFU mg(-1) for A. salmonicida and 100 P. salmonis cells. However, high amounts of DNA from 3 bacterial species had a reduction of -1 log-unit on the amplification sensitivity of S. phocae or A. salmonicida when these were present in lower concentration in the multiplex reaction. The assay described in this study is a rapid, sensitive and efficient tool to detect the presence of S. phocae, A. salmonicida, V. anguillarum and P. salmonis simultaneously from pure cultures and tissues from clinically diseased fish. Therefore, it may be a useful alternative to culture-based methods for the diagnosis of infections in fish obtained from Chilean salmon farms.     

Reverse regulation of soluble receptor for advanced glycation end products and proinflammatory factor resistin and S100A12 in Kawasaki disease

Introduction

Kawasaki disease (KD), an acute febrile disease, characterized by systemic vasculitis, predominantly affects infants and children under 5 years of age. Coronary artery lesions (CALs) are its most critical complication, and the etiology remains unknown yet. In order to explore the value of resistin, S100A12 and soluble receptor for advanced glycation end products (sRAGE) in the pathophysiology of KD, we studied the serum levels of resistin, S100A12 and sRAGE in different stages of KD.

Methods

Serum levels of resistin, S100A12 and sRAGE were measured by enzyme-linked immunosorbent assay (ELISA) method in 15 healthy children and 40 KD patients at acute, afebrile and subacute stage.

Results

The resistin and S100A12 levels, including the ratio of resistin to sRAGE and S100A12 to sRAGE increased significantly in the acute stage, and decreased progressively in the afebrile and subacute stage. However, the sRAGE levels decreased significantly in the acute stage, and increased progressively in the afebrile and subacute stage. In the acute, afebrile and subacute stage, the resistin levels were higher in intravenous immunoglobulin (IVIG) non-responders (0.64 ± 0.30, 0.48 ± 0.35, 0.28 ± 0.19, × 10² ng/ml) than in IVIG responders (0.35 ± 0.24, 0.21 ± 0.19, 0.12 ± 0.05, × 10² ng/ml). In the acute and subacute stage, the S100A12 levels were higher in IVIG non-responders (7.92 ± 2.61, 4.98 ± 4.75, × 10² ng/ml) than in IVIG responders (5.05 ± 3.22, 2.35 ± 2.26, × 10² ng/ml). In the afebrile and subacute stage, the sRAGE levels were lower in IVIG non-responders (3.51 ± 2.64, 3.65 ± 3.27, × 10² pg/ml) than in IVIG responders (6.00 ± 2.78, 7.19 ± 2.88, × 10² pg/ml). The resistin levels were positively correlated with S100A12 levels. The sRAGE levels were negatively related with S100A12 and resistin levels.

Conclusions

Resistin, S100A12 and sRAGE are involved in the pathophysiology of KD.     

Monitoring of clinical and laboratory data in two cases of imported Lassa fever

During 2000, four cases of fatal Lassa fever were imported from Africa to Europe. In two patients, consecutive serum samples were available for monitoring of virus load and cytokine levels in addition to standard laboratory data. Both patients had non-specific early clinical symptoms including high fever. Patient 1 developed multi-organ failure and died of hemorrhagic shock on day 15 of illness, while patient 2 died of respiratory failure due to aspiration without hemorrhage on day 16. Ribavirin was administered to both patients beginning only on day 11. High serum aspartate aminotransferase and lactate dehydrogenase (LDH) levels were remarkable in both patients. Patient 1 had an initial virus load of 10(6) S RNA copies/ml as measured by real-time RT-PCR. Viremia increased steadily and reached a plateau of approximately 10(8)-10(9) copies/ml 4 days before death, while IFN-gamma and TNF-alpha rose to extremely high levels only shortly before death. In contrast, in patient 2 the virus load decreased from 10(7) to 10(6) copies/ml during the late stage of illness which was paralleled by a decrease in the IFN-gamma and TNF-alpha levels. The IL-10 level increased when specific IgM and IgG appeared. These data suggest that a high virus load and high levels of pro-inflammatory cytokines in the late stage of Lassa fever play an important role in the pathogenesis of hemorrhage, multi-organ failure, and shock in Lassa fever.     

Inactivation of Staphylococcus aureus in raw milk cheese by combinations of high-pressure treatments and bacteriocin-producing lactic acid bacteria

AIMS: To investigate the combined effect of high-pressure treatments (HPT) and milk inoculation with bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Staphylococcus aureus during ripening of raw milk cheese. METHODS AND RESULTS: Cheeses were manufactured from raw milk artificially contaminated with S. aureus at ca 5 log CFU ml(-1), a commercial starter culture and one of seven strains of BP-LAB, added as adjuncts at 0.1%. HPT of cheeses were performed on days 2 or 50 at 300 MPa (10 degrees C, 10 min) or 500 MPa (10 degrees C, 5 min). On day 3, S. aureus counts were 6.46 log CFU g(-1) in control cheese. Milk inoculation with different BP-LAB lowered S. aureus counts on day 3 when compared with control cheese by up to 0.46 log CFU g(-1), HPT at 300 MPa on day 2 by 0.45 log CFU g(-1) and HPT at 500 MPa on day 2 by 2.43 log CFU g(-1). Combinations of BP-LAB with HPT at 300 and 500 MPa on day 2 lowered S. aureus counts on day 3 by up to 1.02 and 4.00 log CFU g(-1) respectively. CONCLUSIONS: The combined effect of milk inoculation with some of the BP-LAB tested and HPT of cheese on S. aureus inactivation was synergistic. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of HPT at lower pressures with BP-LAB inoculation is a feasible system to improve cheese safety in case of deleterious effects on cheese quality caused by HPT at higher pressures.     

Rectal carriage of enterohemorrhagic Escherichia coli O157 in slaughtered cattle

Escherichia coli O157:H7 is an important cause of diarrhea, hemorrhagic colitis, and potentially fatal human illness. Cattle are considered a primary reservoir of infection, and recent experimental evidence has indicated that the terminal rectum is the principal site of bacterial carriage. To test this finding in naturally colonized animals, intact rectum samples from 267 cattle in 24 separate lots were obtained immediately after slaughter, and fecal material and mucosal surfaces were cultured for E. coli O157 by direct and enrichment methods. Two locations, 1 and 15 cm proximal to the recto-anal junction, were tested. In total, 35 animals were positive for E. coli O157 at at least one of the sites and 232 animals were negative as determined by all tests. The frequency of isolation and the numbers of E. coli O157 cells were higher at the site closer to the recto-anal junction, confirming our previous experimental findings. We defined low- and high-level carriers as animals with E. coli O157 levels of <1 x 10(3) CFU g(-1) or <1 x 10(3) CFU ml(-1) and animals with E. coli O157 levels of > or =1 x 10(3) CFU g(-1) or > or =1 x 10(3) CFU ml(-1) in feces or tissues, respectively. High-level carriage was detected in 3.7% of the animals (95% confidence interval, 1.8 to 6.8%), and carriage on the mucosal surface of the terminal rectum was associated with high-level fecal excretion. In summary, our results support previous work demonstrating that the mucosal epithelium in the bovine terminal rectum is an important site for E. coli O157 carriage in cattle. The data also support the hypothesis that high-level fecal shedding (> or =1 x 10(3) CFU g of feces(-1)) of enterohemorrhagic E. coli O157 results from colonization of this site.     

Immunomagnetic separation of scum-forming bacteria using polyclonal antibody that recognizes mycolic acids

Mycolic acid-containing bacteria (mycolata) are thought to be involved in scum formation in aeration basins of activated sludge plants due to their ability to produce biosurfactants and their cell surface hydrophobicity. To isolate these bacteria, immunomagnetic separation (IMS) using an anti-mycolic acid polyclonal antibody was investigated. IMS that targeted Gordonia amarae SC1 exhibited a 100% recovery at 5x10(3) CFU ml(-1). At cell concentration of 7.8x10(6) CFU ml(-1), the recovery was lowered, but 80% of cells were still captured. Effect of bead concentrations on the recovery of SC1 at 10(6) CFU ml(-1) was examined. The results showed that addition of more than 6-7x10(6) beads for 1x10(6) CFU reached a maximum recovery (83%). Furthermore, the IMS procedure optimized with SC1 cells was tested with another mycolata. The results suggested that variation of the recovery for each mycolata is dependent on the specificity of the polyclonal antibody and that mycolata which are recognized by the antibody can be recovered by this procedure.     

Different bacterial lipopolysaccharides as toxicants and stressors in the shrimp Palaemon elegans

This study compares the in vivo haemocytic response of shrimp, Palaemon elegans (Rathke) to different types of LPS injection. In particular it investigates to what degree and speed the haemocytopenia varies between LPSs from different sources. It further compares the tolerated doses of different LPSs in these animals and finds substantial differences in the various toxicity types. The work then relates this to blood glucose levels and stress-linked variations in glycaemic status. The order of LPS decreasing toxicity determined by LD50 at 96 h was: Salmonella enteritidis, Serratia marcescens, Pseudomonas aeruginosa 10, Escherichia coli K-235 and E. coli 0111:B4. Eyestalkless animals were more sensitive to LPS. The effects of injected LPS on circulating total blood cell count (THC) was tested. The results show that LPS caused a decrease in THC 8 h after injection and then the THC returned to the initial level and this effect depended on the LPS tested. E. coli K-235 was the most effective in causing haemocytopenia followed by E. coli 0111:B4, S. enteritidis, S. marcescens, and P. aeruginosa 10. Moreover, LPS-induced increases in the blood glucose level and the time and dose related curves of response obtained depended on the type of LPS tested. E. coli K-235 LPS was again the most effective in elevating blood glucose followed by E. coli 0111:B4, S. marcescens, S. enteritidis and then P. aeruginosa 10. No significant hyperglycaemia was induced in eyestalkless animals. An inverse order relationship between toxicity (LD50) and stress responses (hyperglycaemia and THC decrease) may suggest a defensive and adaptive role of the latter in occasional septicaemia.     

Efficacy assessment of Candida oleophila (strain O) and Pichia anomala (strain K) against major postharvest diseases of citrus fruits in Morocco

Two yeasts, Candida oleophila (strain O) and Pichia anomala (strain K), were previously selected for their antagonistic activity against postharvest diseases on apples and pears. The objective of the study was to determine the efficacy of both antagonistic yeast's against wound postharvest pathogens of citrus fruits. The efficacy of both strains (applied at 10(5), 10(6) and 10(8) CFU/ml) was assessed against Penicillium digitatum and P. italicum inoculated after one hour (at a concentration of 10(5), 10(6) and 10(7) spores/ml) on citrus varieties 'clementine' and 'valencia-late'. Fruits were incubated for one week at 24 degrees C before measurement of lesion diameter. The protective levels were positively correlated with high concentration of antagonist and low concentration of pathogen. Highest protective levels (from 73 to 100%) were detected with the application of strain O or strain K at 10(8) CFU/ml whatever the pathogen (applied at 10(5) spores/ml) and the citrus variety. The antagonistic activity of both strains was also dependent on the incubation period before pathogen Inoculation. The protective level increased with time between application of the antagonist and inoculation of fungal spores. Whatever the yeast strain (10(8) CFU/ml). the protective level exceed 70% when wounded oranges were inoculated with P. digitatum or P. italicum (both at 10(6) spores/ml) 12 hours after yeast treatment. These protective levels reached 100% when the incubation period separating the antagonist application and the pathogenic inoculation was 24 hours. On the other hand, high protective levels (< 80%) were also observed against the sour rot decay on citrus variety 'clementine' caused by Geotrichum candidum inoculated at concentration of 10(6) spores/ml when strain O or strain K were applied at 10(8) CFU/ml 24 hours before pathogen. All these results support the potential practical application of both strains against major postharvest pathogens on citrus.     

Validation of a Clostridium endospore viability assay and analysis of Greenland ices and Atacama Desert soils

A microscopy-based endospore viability assay (micro-EVA) capable of enumerating germinable Clostridium endospores (GCEs) in less than 30 min has been validated and employed to determine GCE concentrations in Greenland ices and Atacama Desert soils. Inoculation onto agarose doped with Tb(3+) and d-alanine triggers Clostridium spore germination and the concomitant release of ～10(8) molecules of dipicolinic acid (DPA) per endospore, which, under pulsed UV excitation, enables enumeration of resultant green Tb(3+)-DPA luminescent spots as GCEs with time-gated luminescence microscopy. The intensity time courses of the luminescent spots were characteristic of stage I Clostridium spore germination dynamics. Micro-EVA was validated against traditional CFU cultivation from 0 to 1,000 total endospores/ml (i.e., phase-bright bodies/ml), yielding 56.4% ± 1.5% GCEs and 43.0% ± 1.0% CFU. We also show that d-alanine serves as a Clostridium-specific germinant (three species tested) that inhibits Bacillus germination of spores (five species tested) in that endospore concentration regime. Finally, GCE concentrations in Greenland ice cores and Atacama Desert soils were determined with micro-EVA, yielding 1 to 2 GCEs/ml of Greenland ice (versus <1 CFU/ml after 6 months of incubation) and 66 to 157 GCEs/g of Atacama Desert soil (versus 40 CFU/g soil).     

Marine bacteria cause false-positive results in the Colilert-18 rapid identification test for Escherichia coli in Florida waters

The Colilert-18 system for enumeration of total coliforms and Escherichia coli is approved by the U.S. Environmental Protection Agency for use in drinking water analysis and is also used by various agencies and research studies for enumeration of indicator organisms in fresh and saline waters. During monitoring of Pinellas County, Fla., marine waters, estimates of E. coli numbers (by Colilert-18) frequently exceeded fecal coliform counts (by membrane filtration) by 1 to 3 orders of magnitude. Samples from freshwater sites did not display similar discrepancies. Fecal coliforms, including E. coli, could be cultured from 100% of yellow fluorescent wells (denoting E. coli-positive results) inoculated with freshwater samples but could be cultured from only 17.1% of the "positive" wells inoculated with marine samples. Ortho-nitrophenyl-beta-D-galactopyranoside (ONPG)-positive or 4-methylumbelliferyl-beta-D-glucuronide (MUG)-positive noncoliform bacteria were readily cultured from Colilert-18 test wells inoculated with marine samples. Filtered cell-free seawater did not cause false positives. Coculture preparations of as few as 5 CFU of Vibrio cholerae (ONPG positive) and Providencia sp. (MUG positive) ml(-1) inoculated into Colilert-18 caused false-positive E. coli results. Salinity conditions influenced coculture results, as the concentration of coculture inoculum required to cause false positives in most wells increased from about 5 CFU ml(-1) in seawater diluted 1:10 with freshwater to approximately equal to 5,000 CFU ml(-1) in seawater diluted 1:20 with freshwater. Estimated E. coli numbers in various marine water samples processed at the 1:10 dilution ranged from 10 to 7,270 CFU.100 ml(-1), while E. coli numbers in the same samples processed at the 1:20 dilution did not exceed 40 CFU.100 ml(-1). The lower estimates of E. coli numbers corresponded well with fecal coliform counts by membrane filtration. This study indicates that assessment of E. coli in subtropical marine waters by Colilert-18 is not accurate when the recommended 1:10 sample dilution is used. The results suggest that greater dilution may diminish the false-positive problem, but further study of this possibility is recommended.     

Detection of Salmonella enterica serovar Typhi (S. Typhi) by selective amplification of invA, viaB, fliC-d and prt genes by polymerase chain reaction in mutiplex format

AIMS: Development of a PCR assay that can target multiple genes for rapid detection of Salmonella enterica serovar Typhi (S. Typhi) from water and food samples. METHODS AND RESULTS: PCR primers for invasion, O, H and Vi antigen genes, invA, prt, fliC-d and viaB were designed and used for the rapid detection of S. Typhi by multiplex PCR. Internal amplification control, which co-amplified with prt primers, was also included in the assay. The results showed that all cultures of Salmonella were accurately identified by the assay with no nonspecific amplification in other cultures. The assay had 100% detection probability when a cell suspension of 10(4) CFU ml(-1) (500 CFU per reaction) was used. Salmonella Typhi bacteria were artificially inoculated in the water and food (milk and meat rinse) samples and detected by mPCR after overnight pre-enrichment in buffered peptone water. No Salmonella bacteria could be detected from water samples collected from the field by mPCR or standard culture method. CONCLUSIONS: The developed mPCR assay provides specific detection of S. Typhi. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid methods for detection of S. Typhi from complex environmental matrices are almost nonexistent. The mPCR assay reported in this study can be useful to identify S. Typhi bacteria in field environmental samples.     

Selection and characterization of mannanase-producing bacteria useful for the formation of prebiotic manno-oligosaccharides from copra meal

In order to select bacterial strains effectively secreting mannanase activity for the production of prebiotic mannooligosaccharides, a two-step screening procedure was performed. Enriched cultures from isolation medium containing copra meal were primary screened on an isolation agar medium containing 1% locust bean gum (LBG), which resulted in 48 mannanase-producing bacterial isolates with significant clearing zones on the mannan-containing agar. However, only nine isolates showed appreciable mannanase activities against copra meal in their culture supernatants (0.054–0.185 U/mg of protein) as determined in a standard assay based on the detection of reducing sugars released from this substrate. The isolates CW2-3 and ST1-1 displayed the highest activity against LBG and copra meal, respectively. Copra mannan hydrolysates that were obtained by using crude mannanase from these nine isolates were further used for a secondary screening towards a growth-enhancing activity on Lactobacillus reuteri and inhibitory activity against Escherichia coli as well as Salmonella Enteritidis, resulting in 0.09–2.15 log CFU/ml enhancing activity and low inhibitory activity of 0.46–1.78 log CFU/ml as well as 0.37–1.72 log CFU/ml, respectively. The hydrolysate of CW2-3 mannanase showed the highest enhancing activity of 2.15 log CFU/ml while isolate ST1-1 was most effective with respect to growth inhibition against E. coli E010 and S. Enteritidis S003 with 0.76 and 1.61 log CFU/ml, respectively. Based on morphological, physical, biochemical and genetics properties, isolates CW2-3 and ST1-1 were identified as Klebsiella oxytoca and Acinetobacter sp., respectively. Crude mannanase activity from these two strains was characterized preliminarily. The pH optima of mannanase activity from Klebsiella oxytoca CW2-3 and Acinetobacter sp. ST1-1 were 7 and 6, respectively. The enzymes were stable at 4°C over a pH range of 3–6 and 3–10, respectively.     

Comparison of Pathogen DNA Isolation Methods from Large Volumes of Whole Blood to Improve Molecular Diagnosis of Bloodstream Infections

For patients suffering from bloodstream infections (BSI) molecular diagnostics from whole blood holds promise to provide fast and adequate treatment. However, this approach is hampered by the need of large blood volumes. Three methods for pathogen DNA isolation from whole blood were compared, i.e. an enzymatic method (MolYsis, 1–5 ml), the novel non-enzymatic procedure (Polaris, 1–5 ml), and a method that does not entail removal of human DNA (Triton-Tris-EDTA EasyMAG, 200 µl). These methods were evaluated by processing blood spiked with 0–1000 CFU/ml of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Downstream detection was performed with real-time PCR assays. Polaris and MolYsis processing followed by real-time PCRs enabled pathogen detection at clinically relevant concentrations of 1–10 CFU/ml blood. By increasing sample volumes, concurrent lower cycle threshold (Ct) values were obtained at clinically relevant pathogen concentrations, demonstrating the benefit of using larger blood volumes. A 100% detection rate at a concentration of 10 CFU/ml for all tested pathogens was obtained with the Polaris enrichment, whereas comparatively lower detection rates were measured for MolYsis (50–67%) and EasyMAG (58–79%). For the samples with a concentration of 1 CFU/ml Polaris resulted in most optimal detection rates of 70–75% (MolYsis 17–50% and TTE-EasyMAG 20–36%). The Polaris method was more reproducible, less labour intensive, and faster (45 minutes (including Qiagen DNA extraction) vs. 2 hours (MolYsis)). In conclusion, Polaris and MolYsis enrichment followed by DNA isolation and real-time PCR enables reliable and sensitive detection of bacteria and fungi from 5 ml blood. With Polaris results are available within 3 hours, showing potential for improved BSI diagnostics.