Improved PCR detection sensitivity of Erwinia carotovora subsp. atroseptica in potato tuber peel extract by prior enrichment on a selective medium

A simple and sensitive method was developed to replace the need for complex and laborious DNA extraction to remove inhibitory substances in potato tuber peel extract before detection of Erwinia carotovora subsp. atroseptica (Eca) by PCR. Eca was enriched by a factor of 10⁵ when peel extract was inoculated onto a selective medium, CVP, and incubated at 27°C for 24 h. Bacterial micro-colonies which developed were suspended in 500 μl of water and the bacteria diluted in water 100-fold, or 10-fold followed by washing by centrifugation, before PCR testing. The sensitivity of detection obtained with the former was ca 10¹–10² cells ml⁻¹ and with the latter ca 10¹ cells ml⁻¹, when different numbers of streptomycin-resistant Eca strain were added to peel extract from three Eca-free potato cultivars. The method was validated and the sensitivity confirmed relative to two different commonly used Eca detection methods using naturally contaminated tubers.     

Reduction of Brochothrix thermosphacta on beef surfaces following immobilization of nisin in calcium alginate gels

Lean and adipose beef carcass tissues inoculated with Brochothrix thermosphacta (BT) (approx. 4.50 log₁₀ cfu cm⁻²) were left untreated (U) or treated with 100 μg ml⁻¹ nisin (N), calcium alginate (A) or 100 μg ml⁻¹ nisin immobilized in a calcium alginate gel (AN). Tissue samples were refrigerated after treatments and bacterial populations and nisin activity were determined at 0, 1, 2 and 7 d. U, A and N treatments of lean and adipose tissues did not suppress bacterial growth (>6 log₁₀ cfu cm⁻² by day 7) while treatments of lean and adipose tissues with AN suppressed bacteria (>2.42 log₁₀ cfu cm⁻² by day 7). Bacteriocin titres from both tissues were higher in AN vs N samples after the 7 d incubation. This study demonstrates that immobilization of nisin in a gel may be a more effective delivery system of a bacteriocin to the carcass surface than direct application.     

Survival of free and alginate-encapsulated Pseudomonas aeruginosa UG2Lr in soil treated with disinfectants

Pseudomonas aeruginosa UG2Lr, a rifampicin-resistant strain possessing the luxAB on a chromosomal Tn5 insert, was inoculated into soil microcosms as either free cells or encapsulated in dry alginate beads. A 100-fold increase in cell number g^-1 dry soil was observed in microcosms inoculated with alginate-encapsulated UG2Lr after 3 weeks incubation at 22°C compared to microcosms inoculated with free cells. After 98 d, microcosms inoculated with free UG2Lr cells contained 10⁴ cfu g^-1 dry soil compared to 10⁷ cfu g^-1 dry soil in microcosms inoculated with alginate-encapsulated UG2Lr cells. The effects of disinfectants on both the free and alginate-encapsulated UG2Lr cells were also examined. 1·0% (w/g dry soil) calcium hypochlorite, formaldehyde and Spectrum Clear Bath, were added to microcosms each week for 4 weeks. Formaldehyde killed both free and alginate-encapsulated UG2Lr cells within 14 d after only two amendments. Calcium hypochlorite reduced free UG2Lr cell numbers 10-fold 2 d after initial application; however, the introduced population recovered and was unaffected by subsequent treatments at 7, 14 and 21 d. Alginate-encapsulated UG2Lr cells were not affected by calcium hypochlorite treatment. Spectrum Clear Bath did not kill either free or alginate-encapsulated UG2Lr cells in soil. Alginate encapsulation improved survival of introduced bacteria in soil except in the presence of formaldehyde. Killing genetically-engineered bacteria in soil may be difficult unless a powerful disinfectant such as formaldehyde is used or the genetically-engineered micro-organism is allowed to become non-viable over time.     

The development of a conductimetric assay for automated detection of metabolically active soft rot Erwinia spp. in potato tuber peel extracts

Four media were tested for their ability to detect the soft rot potato pathogens Erwinia chrysanthemi (Ech) and Erwinia carotovora ssp. atroseptica (Eca) in potato tubers by means of automated conductance measurements. The specificity of the conductimetric assays was determined by testing a set of different Erwinia spp. and potato-associated saprophytes, including the genera Pseudomonas, Bacillus, Enterobacter and Flavobacterium. All bacteria tested produced conductance responses in Special Peptone Yeast Extract, whereas in minimal medium with L-asparagine only Erwinia spp. and Pseudomonas spp. were able to generate large conductance responses. In minimal medium supplemented with glucose and trimethylamine- N -oxide only Enterobacteriaceae, Erwinia spp. included, generated conductance responses, while with pectate as sole carbon source only Erwinia spp. produced distinct conductance responses. The pectate medium proved to be particularly useful for specific automated conductimetric detection of Erwinia spp. in potato peel extracts. Within 48 h, the detection threshold of the conductimetric assay for Eca varied between 10² and 10³ cfu per ml peel extract at both incubation temperatures of 20° and 26°C. Ech was detected at concentrations of 10⁴–10⁵ or 10³–10⁴ cfu ml^-1 at 20° and 26°C, respectively. To eliminate 'false'-positive reactions in conductimetry caused by Erwinia carotovora ssp. carotovora , results of the conductance measurements have to be confirmed by other techniques, like serology or DNA assays.     

Effectiveness of a steam-vacuum sanitizer for reducing Escherichia coli O157:H7 inoculated to beef carcass surface tissue

A steam-vacuum sanitizer reduced aerobic plate counts associated with bovine faecal contamination from 5.5 log₁₀ cfu cm⁻² to 3.0 ± 0.21 log₁₀ cfu cm⁻² on beef carcass short plates. The same beef carcass short plates inoculated wiht 7.6 ± 0.09 log₁₀ cfu cm⁻² Escherichia coli O157: H7 in faeces, yielded an average residual level of E. coli O157: H7 of 2.1 ± 0.21 log₁₀ cfu cm⁻² after steam-vacuum treatments. This study demonstrates the effectiveness of a steam-vacuum sanitizer for removing E. coli O157: H7 from beef carcasses.     

Changes in cis-zeatin and cis-zeatin riboside levels and biological activity during potato tuber dormancy

The effects of postharvest storage duration and temperature on endogenous cis -zeatin ( cis -Z) and cis -zeatin riboside ( cis -ZR) levels in potato ( Solanum tuberosum L.) tubers were determined in relation to tuber bud dormancy. The tubers used in these studies were completely dormant for at least 81 days of storage. Thereafter, tuber bud dormancy diminished gradually and after 165 days of postharvest storage, the tubers were completely non-dormant. Immediately after harvest, endogenous levels of cis- Z and cis -ZR were approximately 25 pmol (g fresh weight)⁻¹ and 8 pmol (g fresh weight)⁻¹, respectively. In tubers exiting dormancy but stored at a growth-inhibiting temperature (3°C), endogenous levels of cis -Z rose over threefold after 25 days of storage and remained elevated for the duration of the study. Levels of cis -ZR remained essentially constant during this same period. In tubers transferred to a growth permissive temperature (20°C) prior to use, the rise in endogenous cis -Z was less dramatic and more protracted; increasing twofold after 53 days of storage. No change in cis -Z riboside content was observed in these tubers during this period. Dose-response studies using either cis -Z or trans -Z demonstrated a time-dependent increase in cytokinin sensitivity during postharvest storage. Immediately after harvest, dormant tubers were insensitive to both zeatin isomers. Thereafter, tubers exhibited a dose-dependent increase in premature sprouting following injection with either cytokinin isomer. After injection into dormant tubers, cis -[8-¹⁴C]-zeatin was metabolized primarily to adenine/adenosine and cis -Z riboside. Seven days after injection, less than 10% of the recovered radioactivity was associated with trans -ZR. These results are consistent with a role for endogenous cis -Z (and its derivatives) in the regulation of potato tuber dormancy.     

WLIP, a lipodepsipeptide of Pseudomonas 'reactans', as inhibitor of the symptoms of the brown blotch disease of Agaricus bisporus

A cell-free crude extract containing the white line inducing principle (WLIP), a lipodepsipeptide produced by Pseudomonas 'reactans' , could inhibit browning of mushrooms caused by Pseudomonas tolaasii . Mushrooms inoculated with Ps. tolaasii at concentrations of 2·7 × 10⁶ cfu ml⁻¹ or higher showed the symptoms of the disease after 2 d of incubation. Mushroom caps treated with various concentrations of a crude WLIP preparation, and later inoculated with bacterial concentrations higher than the threshold value, did not develop the symptoms of the disease. One milligram of a crude WLIP preparation could block 50% of the symptoms caused by 1·2 × 10⁷ cfu. The inhibition of browning was effective when incubating at low temperatures for 4 d. A suspension containing 1·6 mg ml⁻¹ of pure WLIP was also able to inhibit the symptoms of brown blotch disease induced by 7·6 × 10⁶ cfu ml⁻¹ of Ps. tolaasii .     

Survival of bacterial enteric pathogens in traditional fermented foods

The survival of strains of bacterial enteric pathogens was investigated in two traditional fermented foods (mahewu and sour porridge) and in unfermented porridge. The foods were inoculated with cell suspensions of Salmonella, Shigella, Campylobacter, Aeromonas species and pathogenic Escherichia coli which had a final concentration of 10⁶-10⁷ cfu/ml of food. None of the strains of Aeromonas and Campylobacter were detected in mahewu and sour porridge 20 min after inoculation. The salmonellas were not found 4 h after inoculation in either fermented foods but the shigellas and pathogenic E. coli strains were more tolerant to the low pH of the fermented foods. Some of the shigellas and pathogenic E. coli strains survived for 24 h after inoculation but showed a sharp decrease in numbers. All the strains of the enteric pathogens survived for 24 h in the unfermented porridge and increased in the numbers except for campylobacters, the numbers of which declined. These results suggest that the traditional fermented foods have bacteriostatic and bactericidal properties and are unlikely to play a major role in the transmission of bacterial enteric pathogens.     

Factors affecting the growth of Listeria monocytogenes on minimally processed fresh endive

The influence of various factors on the fate of Listeria monocytogenes on cut leaves of broad-leaved endive has been studied. Factors considered were temperature, characteristics of the leaves (age, quantity and quality of the epiphytic microflora) and characteristics of the L. monocytogenes inoculum (concentration, strain). The increases in numbers of L. monocytogenes were lower than those of the aerobic mesophilic microflora at 3°, 6°, 10° and 20°C. Doubling times of the populations of L. monocytogenes were in the same order of magnitude as those of aerobic bacteria at 10° and 20°C, but longer at 3° and 6°C. There were positive significant correlations between growth of L. monocytogenes and populations of aerobic bacteria, and between growth of L. monocytogenes and extent of spoilage on the leaves.
Of 225 bacteria isolated from the leaves, 84% were identified as fluorescent pseudomonads; there was no difference in the species isolated from leaves that showed a low growth of L. monocytogenes and leaves that showed a high growth of L. monocytogenes. Populations of L. monocytogenes increased faster during the first 2 and 4 d of storage at 10°C on leaves inoculated with 10–10³ cfu g^-1 than on leaves inoculated with about 10⁵ cfu g^-1, but the population reached after 7 d was lower. The behaviour of L. monocytogenes was similar among the three strains tested.     

The use of a surface adhesion immunofluorescent (SAIF) method for the rapid detection of Yersinia enterocolitica serotype O:3 in meat

This study examined the attachment kinetics of Yersinia enterocolitica serotype O:3 to determine the optimum conditions for its isolation from meat enrichment systems using a novel surface adhesion technique. Minced beef was inoculated with Y. enterocolitica at an initial level of 10 cfu g⁻¹ and incubated at 25 °C in an enrichment broth. Yersinia was recovered from enriched samples on polycarbonate membranes by surface adhesion and enumerated using immunofluorescence microscopy. The surface adhesion immunofluorescence technique (SAIF) had a minimum detection limit of approximately 4·0–4·5 log₁₀ cfu ml⁻¹ and provided good correlation between the estimation of the numbers of Yersinia in the enrichment broth derived from plate counts on Yersinia Selective agar (CIN) and those determined by SAIF ( r ² ₌ 0·94; rsd ₌ ± 0·21). A derived regression equation of the SAIF count vs plate counts was used to predict Y. enterocolitica numbers in spiked meat samples stored at 0 °C for up to 20 d. The numbers as predicted by the SAIF method showed good correlation with counts derived by plating techniques ( r ² ₌ 0·78; rsd ₌ ± 0·42). The application of the SAIF technique for the rapid detection of Y. enterocolitica serotype O:3 from meat is discussed.     

A competitive PCR-based method for the detection and quantification of Erwinia carotovora subsp. atroseptica On potato tubers

A PCR-based method was developed for the simultaneous detection and quantification of the potato pathogen Erwinia carotovora subsp. atroseptica (Eca) on potato tubers. The method incorporates a competitor PCR template cloned into Escherichia coli in vector pGEM-T (E. coli 4R l/l). Predetermined numbers of E. coli 4R were added to potato peel extract, either pre-inoculated with Eca or from naturally contaminated tubers, and Eca numbers estimated by comparing the ratio of products generated from Eca target DNA and competitor template DNA following PCR. Estimates of Eca numbers were consistent with counts obtained on crystal violet pectate medium and immunofluorescence colony staining. Unlike these methods, however, the PCR-based method is not affected by the presence of other erwinias and saprophytes and is able to detect all serogroups of Eca. Based on this method, a key was produced relating product ratios, obtained following PCR from contaminated tuber stocks, to the likelihood of blackleg disease incidence. This is the first quantitative PCR-based detection method described for Eca and is the first for any bacterial plant pathogen to incorporate a DNA extraction control.     

Serological response in rabbits to Listeria monocytogenes after oral or intragastric inoculation

Abstract The serological response in rabbits against Listeria monocytogenes after oral or intragastric inoculation was investigated. Both the number of sero-positive and the average serum titres were hgher in animals inoculated by the oral route. This difference was especially marked in rabbits inoculated with the lower dose (1×10³ colony-forming units (cfu)), which developed a strong serological response (average serum titre of 1280 after 4 inoculations) in most of the inoculated animals (80%), without any clinical signs. The implication of these results in the epidemiology of listeriosis is discussed.     

Survival of Escherichia coli O157 : H7 in yoghurt during preparation and storage at 4°C

Cow's milk was inoculated with ca 10³ and 10⁷ cfu ml⁻¹ Escherichia coli O157 : H7. After fermentation at 42°C for 0–5 h, the yoghurt was stored at 4°C. Two kinds of yoghurt were used : traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum . After 7 d E. coli O157 : H7 decreased from 3·52 to 2·72 log₁₀ cfu ml⁻¹ and from 7·08 to 5·32 log₁₀ cfu ml⁻¹ in TY, and from 3·49 to 2·73 log₁₀ cfu ml⁻¹ and from 7·38 to 5·41 log₁₀ cfu ml⁻¹ in BY. The pH values of yoghurt dropped from 6·6 to 4·5 and 4·4 in TY (for low and high pathogen inocula, respectively), and from 6·6 to 4·6 and 4·5 in BY (for low and high pathogen inocula, respectively).     

Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture andripening of Manchego cheese

The inhibitory effect of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4, on Listeria monocytogenes strains Ohio and Scott A during themanufacture and ripening of Manchego cheese was investigated. Raw ewe's milk wasinoculated with ca 10⁵ cfu ml⁻¹ of L.monocytogenes and with 1% of a commercial lactic starter, 1% of an Ent. faecalis INIA 4 culture, or 1% of each culture. Manchego cheeses were manufactured according tousual procedures. Listeria monocytogenes Ohio counts decreased by 3 log units after8 h and by 6 log units after 7 d in cheese made from milk inoculated with Ent. faecalis INIA 4 or with both cultures, whereas no inhibition was recorded after 60 d in cheese made frommilk inoculated with commercial lactic starter. Listeria monocytogenes Scott A wasnot inhibited by enterocin 4 during cheese manufacture, but decreases of 1 log unit after 7 d andof 2 log units after 60 d were achieved in cheese made from milk inoculated with bothcommercial lactic starter and Ent. faecalis INIA 4.     

Genotypic differences in the uptake and distribution of radiocaesium in plants

The ability of endophytic bacteria to influence Erwinia carotovora var. atroseptica (Eca) growth and disease development was examined in potatoes. Bacterial populations isolated from within the tubers of five potato (Solanum tuberosum L.) cultivars (Kennebec, Butte, Green Mountain, Russet Burbank and Sebago) showed antibiosis toward Eca in an in vitro assay. Sebago was host to the highest percentage of bacterial isolates inhibiting Eca growth in vitro (49.5%), followed by Green Mountain (33.3%), Kennebec (29.3%), Russet Burbank (12.9%) and Butte (1.8%). Of these, Curtobacterium luteum was the most common species. Few endophytic bacteria from Butte were inhibitory to Erwinia; all were from Pantoea agglomerans. Significantly higher populations of Erwinia-inhibiting bacteria were recovered from Kennebec (1.89 × 10⁶ cfu fresh weight tuber tissue) as compared to the other cultivars; the lowest populations were recovered from Butte (0.01 × 10⁶ cfu per g fresh weight tuber tissue). Published levels of cultivar disease resistance to blackleg did not correspond to actual bacterial soft rot development (induced by Eca) in an in vivo (tuber) assay. However, bacterial soft rot development was negatively correlated with the density of tuber populations of endophytic bacteria found able to inhibit Eca growth in vitro (R=−0.879, p=0.05).     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

Changes in mucus cell numbers in the epidermis of the Atlantic salmon at the onset of smoltification

A sharp decrease to <50% in the numbers of mucus cells mm⁻² of epidermis was observed in the skin of Atlantic salmon Salmo salar at the beginning of the smoltification period in 1997 and 1999. Average numbers decreased in 1997 from 662 mm⁻² on 6 March to 275 mm⁻² on 20 March and in 1999 from 828 mm⁻² on 3 March to 388 mm² on 24 March. A second smaller decrease was observed towards the end of smoltification in both years.     

Water-relation parameters of giant and normal cells of Capsicum annuum pericarp

Abstract. Measurements of the water-relation parameters of the giant subepidermal cells (volume, V = 0.119 to 1.658 mm³; = 0.53±0.35 mm³, SD, n = 23) and the smaller mesocarp parenchyma cells ( V = 0.10 to 0.79×10⁻³ mm³; = 0.36±0.27×10⁻³ mm³, SD, n = 6) of the inner pericarp surface of Capsicum annuum L. were made using the Jülich pressure probe. The volumetric elastic modulus ɛ for the large cells was between 1.5 and 27 MPa for a pressure range of 0.09 to 0.41 MPa. For the small cells ɛ was 0.1 to 0.6 MPa for a pressure range of 0.22 to 0.39 MPa. The turgor pressure P , the half-time of water exchange T _1/2, and the hydraulic conductivity L _p were as follows, with SD and number of replicates: large cells, P = 0.27±0.06 MPa (23), T _1/2=2.7±2.2 s (46), L _p=5.8±3.7 pm s⁻¹ Pa⁻ (46); small cells, P = 0.33±0.07 MPa (6), T _1/2= 33±10s (12), L _p=0.21±0.07 pm s⁻¹ Pa⁻¹ (12). The determination of these basic water-relation parameters is considered as a prerequisite for future ecotoxicological and phytopathological studies. The differences between the large and the small cells are discussed in relation to a desirable biophysical definition of succulence. Further, for the large cells a pressure and volume dependence of ɛ was demonstrated.     

Catalase inhibition alters suberization and wound healing in potato (Solanum tuberosum) tubers

In response to wounding, potato ( Solanum tuberosum L.) tubers generate hydrogen peroxide (H₂O₂) in association with suberization, a critical phase of the wound-healing process. In the present study, the effect of aminotriazole (AT), a catalase (CAT, EC 1.11.1.6) inhibitor, on cut tubers was investigated using fresh weight (FW) loss and pathogen attack symptoms as indicators of wound-healing efficiency. Seven days after treatment, AT-treated tuber halves lost more FW and developed infection signs compared with the controls. Thiourea, another CAT inhibitor, as well as exogenous H₂O₂ treatments induced the same effects as AT suggesting that the alteration of the wound healing may be caused by CAT inhibition and the resulting accumulation of H₂O₂. Using transgenic tubers, FW losses 1 week after wounding were either higher (CAT repression) or lower (CAT overexpression) than those of the wild-type. When tuber halves were allowed to wound heal for different periods before treatment, AT had no effect on the progress of their wound healing if wound-healed for at least 3 days. This implies that AT may affect early wound-healing-related events, especially those occurring before or during suberization. A time-course analysis of the effects of AT treatment on wounded tuber tissues revealed that AT prevented the deposition of the polyphenolic domain of suberin in association with CAT inhibition and H₂O₂ accumulation. These data are important in identifying factors that may be required to regulate suberization and contribute to a better understanding of this critical process to hasten its rate and limit wound-related losses in stored potato tubers.