DPYD genotype-guided dose individualisation of fluoropyrimidine therapy in patients with cancer: a prospective safety analysis.

BACKGROUND: Fluoropyrimidine treatment can result in severe toxicity in up to 30% of patients and is often the result of reduced activity of the key metabolic enzyme dihydropyrimidine dehydrogenase (DPD), mostly caused by genetic variants in the gene encoding DPD (DPYD). We assessed the effect of prospective screening for the four most relevant DPYD variants (DPYD*2A [rs3918290, c.1905+1G>A, IVS14+1G>A], c.2846A>T [rs67376798, D949V], c.1679T>G [rs55886062, DPYD*13, I560S], and c.1236G>A [rs56038477, E412E, in haplotype B3]) on patient safety and subsequent DPYD genotype-guided dose individualisation in daily clinical care. METHODS: In this prospective, multicentre, safety analysis in 17 hospitals in the Netherlands, the study population consisted of adult patients (≥18 years) with cancer who were intended to start on a fluoropyrimidine-based anticancer therapy (capecitabine or fluorouracil as single agent or in combination with other chemotherapeutic agents or radiotherapy). Patients with all tumour types for which fluoropyrimidine-based therapy was considered in their best interest were eligible. We did prospective genotyping for DPYD*2A, c.2846A>T, c.1679T>G, and c.1236G>A. Heterozygous DPYD variant allele carriers received an initial dose reduction of 25% (c.2846A>T and c.1236G>A) or 50% (DPYD*2A and c.1679T>G), and DPYD wild-type patients were treated according to the current standard of care. The primary endpoint of the study was the frequency of severe (National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03 grade ≥3) overall fluoropyrimidine-related toxicity across the entire treatment duration. We compared toxicity incidence between DPYD variant allele carriers and DPYD wild-type patients on an intention-to-treat basis, and relative risks (RRs) for severe toxicity were compared between the current study and a historical cohort of DPYD variant allele carriers treated with full dose fluoropyrimidine-based therapy (derived from a previously published meta-analysis). This trial is registered with ClinicalTrials.gov, number NCT02324452, and is complete. FINDINGS: Between April 30, 2015, and Dec 21, 2017, we enrolled 1181 patients. 78 patients were considered non-evaluable, because they were retrospectively identified as not meeting inclusion criteria, did not start fluoropyrimidine-based treatment, or were homozygous or compound heterozygous DPYD variant allele carriers. Of 1103 evaluable patients, 85 (8%) were heterozygous DPYD variant allele carriers, and 1018 (92%) were DPYD wild-type patients. Overall, fluoropyrimidine-related severe toxicity was higher in DPYD variant carriers (33 [39%] of 85 patients) than in wild-type patients (231 [23%] of 1018 patients; p=0·0013). The RR for severe fluoropyrimidine-related toxicity was 1·31 (95% CI 0·63-2·73) for genotype-guided dosing compared with 2·87 (2·14-3·86) in the historical cohort for DPYD*2A carriers, no toxicity compared with 4·30 (2·10-8·80) in c.1679T>G carriers, 2·00 (1·19-3·34) compared with 3·11 (2·25-4·28) for c.2846A>T carriers, and 1·69 (1·18-2·42) compared with 1·72 (1·22-2·42) for c.1236G>A carriers. INTERPRETATION: Prospective DPYD genotyping was feasible in routine clinical practice, and DPYD genotype-based dose reductions improved patient safety of fluoropyrimidine treatment. For DPYD*2A and c.1679T>G carriers, a 50% initial dose reduction was adequate. For c.1236G>A and c.2846A>T carriers, a larger dose reduction of 50% (instead of 25%) requires investigation. Since fluoropyrimidines are among the most commonly used anticancer agents, these findings suggest that implementation of DPYD genotype-guided individualised dosing should be a new standard of care. FUNDING: Dutch Cancer Society.

Discovering novel germline genetic variants linked to severe fluoropyrimidine-related toxicity in- and outside DPYD.

BACKGROUND: The Alpe-DPD study (NCT02324452) demonstrated that prospective genotyping and dose-individualization using four alleles in DPYD (DPYD*2A/rs3918290, c.1236G > A/rs75017182, c.2846A > T/rs67376798 and c.1679 T > G/rs56038477) can mitigate the risk of severe fluoropyrimidine toxicity. However, this could not prevent all toxicities. The goal of this study was to identify additional genetic variants, both inside and outside DPYD, that may contribute to fluoropyrimidine toxicity. METHODS: Biospecimens and data from the Alpe-DPD study were used. Exon sequencing was performed to identify risk variants inside DPYD. In silico and in vitro analyses were used to classify DPYD variants. A genome-wide association study (GWAS) with severe fluoropyrimidine-related toxicity was performed to identify variants outside DPYD. Association with severe toxicity was assessed using matched-pair analyses for the exon sequencing and logistic, Cox, and ordinal regression analyses for GWAS. RESULTS: Twenty-four non-synonymous, frameshift, and splice site DPYD variants were detected in ten of 986 patients. Seven of these variants (c.1670C > T, c.1913 T > C, c.1925 T > C, c.506delC, c.731A > C, c.1740 + 1G > T, c.763 - 2A > G) were predicted to be deleterious. The carriers of either of these variants showed a trend towards a 2.14-fold (95% CI, 0.41-11.3, P = 0.388) increased risk of severe toxicity compared to matched controls (N = 30). After GWAS of 942 patients, no individual single nucleotide polymorphisms achieved genome-wide significance (P ≤ 5 × 10-8), however, five variants were suggestive of association (P < 5 × 10-6) with severe toxicity. CONCLUSIONS: Results from DPYD exon sequencing and GWAS analysis did not identify additional genetic variants associated with severe toxicity, which suggests that testing for single markers at a population level currently has limited clinical value. Identifying additional variants on an individual level is still promising to explain fluoropyrimidine-related severe toxicity. In addition, studies with larger samples sizes, in more diverse cohorts are needed to identify potential clinically relevant genetic variants related to severe fluoropyrimidine toxicity.

Spectrum of artificial light at night drives impact of a diurnal species in insect food web.

Artificial light at night (ALAN) has become a profound form of global anthropogenic environmental change differing in from natural light regimes in intensity, duration, distribution and spectra. It is clear that ALAN impacts individual organisms, however, population level effects, particularly of spectral changes, remain poorly understood. Here we exposed experimental multigenerational aphid-parasitoid communities in the field to seven different light spectra at night ranging from 385 to 630 nm and compared responses to a natural day-night light regime. We found that while aphid population growth was initially unaffected by ALAN, parasitoid efficiency declined under most ALAN spectra, leading to reduced top-down control and higher aphid densities. These results differ from those previously found for white light, showing a strong impact on species' daytime performance. This highlights the importance of ALAN spectra when considering their environmental impact. ALAN can have large impacts on the wider ecological community by influencing diurnal species.

The ecological and evolutionary implications of merging different types of networks.

Interactions among species drive the ecological and evolutionary processes in ecological communities. These interactions are effectively key components of biodiversity. Studies that use a network approach to study the structure and dynamics of communities of interacting species have revealed many patterns and associated processes. Historically these studies were restricted to trophic interactions, although network approaches are now used to study a wide range of interactions, including for example the reproductive mutualisms. However, each interaction type remains studied largely in isolation from others. Merging the various interaction types within a single integrative framework is necessary if we want to further our understanding of the ecological and evolutionary dynamics of communities. Dividing the networks up is a methodological convenience as in the field the networks occur together in space and time and will be linked by shared species. Herein, we outline a conceptual framework for studying networks composed of more than one type of interaction, highlighting key questions and research areas that would benefit from their study.

Aphid-parasitoid community structure on genetically modified wheat.

Since the introduction of genetically modified (GM) plants, one of the main concerns has been their potential effect on non-target insects. Many studies have looked at GM plant effects on single non-target herbivore species or on simple herbivore-natural enemy food chains. Agro-ecosystems, however, are characterized by numerous insect species which are involved in complex interactions, forming food webs. In this study, we looked at transgenic disease-resistant wheat (Triticum aestivum) and its effect on aphid-parasitoid food webs. We hypothesized that the GM of the wheat lines directly or indirectly affect aphids and that these effects cascade up to change the structure of the associated food webs. Over 2 years, we studied different experimental wheat lines under semi-field conditions. We constructed quantitative food webs to compare their properties on GM lines with the properties on corresponding non-transgenic controls. We found significant effects of the different wheat lines on insect community structure up to the fourth trophic level. However, the observed effects were inconsistent between study years and the variation between wheat varieties was as big as between GM plants and their controls. This suggests that the impact of our powdery mildew-resistant GM wheat plants on food web structure may be negligible and potential ecological effects on non-target insects limited.

Methotrexate plus or minus cetuximab as first-line treatment in a recurrent or metastatic (R/M) squamous cell carcinoma population of the head and neck (SCCHN), unfit for cisplatin combination treatment, a phase Ib-randomized phase II study Commence.

BACKGROUND: Methotrexate in recurrent or metastatic (R/M) squamous cell carcinoma of the head and neck (SCCHN) has limited progression-free survival (PFS) benefit. We hypothesized that adding cetuximab to methotrexate improves PFS. METHODS: In the phase-Ib-study, patients with R/M SCCHN received methotrexate and cetuximab as first-line treatment. The primary objective was feasibility. In the phase-II-study patients were randomized to this combination or methotrexate alone (2:1). The primary endpoint was PFS. Secondary endpoints were overall survival (OS), toxicity, and quality of life (QoL). RESULTS: In six patients in the phase-Ib-study, no dose limiting toxicities were observed. In the phase II study, 30 patients received the combination and 15 patients methotrexate. In the phase-II-study median PFS was 4.5 months in the combination group vs 2.0 months in the methotrexate group (HR 0.37; P = .002). OS, toxicity, and QoL were not significantly different. CONCLUSION: Cetuximab with methotrexate improved PFS without increased toxicity in R/M SCCHN-patients.

A cost analysis of upfront DPYD genotype-guided dose individualisation in fluoropyrimidine-based anticancer therapy.

BACKGROUND: Fluoropyrimidine therapy including capecitabine or 5-fluorouracil can result in severe treatment-related toxicity in up to 30% of patients. Toxicity is often related to reduced activity of dihydropyrimidine dehydrogenase, the main metabolic fluoropyrimidine enzyme, primarily caused by genetic DPYD polymorphisms. In a large prospective study, it was concluded that upfront DPYD-guided dose individualisation is able to improve safety of fluoropyrimidine-based therapy. In our current analysis, we evaluated whether this strategy is cost saving. METHODS: A cost-minimisation analysis from a health-care payer perspective was performed as part of the prospective clinical trial (NCT02324452) in which patients prior to start of fluoropyrimidine-based therapy were screened for the DPYD variants DPYD*2A, c.2846A>T, c.1679T>G and c.1236G>A and received an initial dose reduction of 25% (c.2846A>T, c.1236G>A) or 50% (DPYD*2A, c.1679T>G). Data on treatment, toxicity, hospitalisation and other toxicity-related interventions were collected. The model compared prospective screening for these DPYD variants with no DPYD screening. One-way and probabilistic sensitivity analyses were also performed. RESULTS: Expected total costs of the screening strategy were €2599 per patient compared with €2650 for non-screening, resulting in a net cost saving of €51 per patient. Results of the probabilistic sensitivity and one-way sensitivity analysis demonstrated that the screening strategy was very likely to be cost saving or worst case cost-neutral. CONCLUSIONS: Upfront DPYD-guided dose individualisation, improving patient safety, is cost saving or cost-neutral but is not expected to yield additional costs. These results endorse implementing DPYD screening before start of fluoropyrimidine treatment as standard of care.

Comparison of poultry exudate and carcass rinse sampling methods for the recovery of Campylobacter spp. subtypes demonstrates unique subtypes recovered from exudate.

The carcass rinse procedure is a method commonly used for the detection of Campylobacter spp. on processed poultry products. Alternatively, carcass exudate (weep or drip), a viscous fluid comprised of blood and water that leaks into packaging, can also be sampled. It is unknown however if direct carcass rinse or exudate/weep can be utilized to preferentially recover different Campylobacter spp. subtypes. If there is a difference in subtypes recovered, the Campylobacter spp. subtypes from carcass rinse analysis may not be indicative of consumer exposure, as the exudate is the fluid to which consumers are potentially exposed to due to kitchen cross-contamination. Experiments were conducted to determine if there are differences in recovery of Campylobacter spp. subtypes between the two methodologies. The experiment was performed in triplicate using three flocks located on different farms. For each flock, 50 fecal samples were obtained on the farm, 25 carcass rinses during pre-chill processing, 25 carcass rinses during post-chill processing, and 50 samples from exudate from carcasses stored at 4 degrees C (25 after 2-day storage and 25 after 6-day storage). Each sample type was cultured for Campylobacter spp. Isolates recovered from positive samples were subtyped using flaA SVR (flagellin A-short variable region) DNA sequence typing and compared for relatedness. The data demonstrated that multiple subtypes of Campylobacter jejuni were present in a flock, and that subtypes present in a flock during production were also present on the final processed product. Subtypes recovered by the two recovery methodologies were similar based on flaA SVR classification. Combining the totals from all 3 flocks a total of 10 flaA SVR subtypes were recovered from post-chill carcass rinses and 9 subtypes recovered from 6-day exudate samples.

Effect of chemical sanitizers with and without ultrasonication on Listeria monocytogenes as a biofilm within polyvinyl chloride drain pipes.

As part of a biofilm in a floor drain, Listeria monocytogenes is exceedingly difficult to eradicate with standard sanitizing protocols. The objective of these studies was to test the use of ultrasonication to break up biofilm architecture and allow chemical sanitizers to contact cells directly. L. monocytogenes biofilms were created in model polyvinyl chloride drain pipes. Chemical sanitizers (quaternary ammonium, peroxide, or chlorine) were applied to the drain pipes with and without a 30-s ultrasonication treatment. Controls using sterile water were included for comparison. L. monocytogenes cells were enumerated from the liquid in the drain and the inside wall surface of the pipe. All chemicals lowered numbers of planktonic cells from 6.6 log CFU/ml in the water control to < 100 CFU/ml. Attached cells were also affected by the chemical sanitizers. Approximately 6.0 log CFU/cm2 of the inner wall surface was detected in water control pipes, and ultrasonication did not lower these numbers. With or without ultrasonication, the peroxide-based sanitizer was effective for reducing the numbers of attached L. monocytogenes cells, resulting in approximately 2.0 log CFU/cm2. Both the chlorine- and quaternary ammonium-based sanitizers reduced the number of attached L. monocytogenes cells to a lesser degree, resulting in 4.2 to 4.4 log CFU/cm2. However, addition of ultrasonication improved the performance of both these sanitizers, causing a further reduction to 3.1 and 2.9 CFU/ cm2 for quaternary ammonium- and chlorine-based chemicals, respectively. These results indicate that a peroxide-based sanitizer alone can be very effective against biofilm L. monocytogenes in drain pipes, and the addition of ultrasonication can improve the effectiveness of chlorine or quaternary ammonium sanitizers.

Antimicrobial susceptibility of Clostridium difficile isolated from food animals on farms.

Clostridium difficile is commonly associated with a spectrum of disease in humans referred to as C. difficile-associated disease (CDAD) and use of antimicrobials is considered a risk factor for development of disease in humans. C. difficile can also inhabit healthy food animals and transmission to humans is possible. As a result of the complexity and cost of testing, C. difficile is rarely tested for antimicrobial susceptibility. A total of 376 C. difficile strains (94 each from swine and dairy feces, and 188 from beef cattle feces) were isolated from healthy food animals on farms during studies conducted by the National Animal Health Monitoring System. Using the Etest (AB Biodisk, Solna, Sweden), samples were tested for susceptibility to nine antimicrobials implicated as risk factors for CDAD (linezolid, amoxicillin-clavulanic acid, ampicillin, clindamycin, erythromycin, levofloxacin, metronidazole, rifampicin, and vancomycin). Vancomycin was active against all isolates of C. difficile (MIC90=3.0µg/ml) while almost all isolates (n=369; 98.1%) were resistant to levofloxacin. With the exception of vancomycin, resistance varied by animal species as follows: linezolid (8.5% resistance among swine versus 2.1 and 1.1% resistance among dairy and beef, respectively), clindamycin (56.4% resistance among swine versus 80% and 90.9% resistance among dairy and beef, respectively), and rifampicin (2.1% and 0% resistance among swine and dairy cattle isolates, respectively versus 14.3% resistance among beef isolates). Regardless of species, multiple drug resistance was observed most often to combinations of clindamycin and levofloxacin (n=195; 51.9%) and ampicillin, clindamycin and levofloxacin (n=41; 10.9%). The reason for the variability of resistance between animal species is unknown and requires further research.

Distribution of Listeria monocytogenes subtypes within a poultry further processing plant.

Samples from environmental sites and raw product in a chicken further processing plant were collected every 6 weeks for 12 months. Each sample site was examined before and after a complete production shift. All samples were examined for the presence of Listeria monocytogenes, which was detected in floor drains on the raw product side of the plant preoperation and in drains on both raw and cooked sides following 8 h of processing operation. L. monocytogenes also was detected in raw product and once in fully cooked product but never on cooked product contact surfaces. One hundred sixty-one isolates were collected from 75 positive samples. All isolates were subtyped using a sequence-based method, and 14 unique subtypes were detected through the course of the study. Four of these types were found repeatedly and appeared to be resident in the plant. Three of the four resident strains were detected on raw product at some point during the year-long study, suggesting that raw product may be one source of L. monocytogenes in the processing plant environment. These data highlight the need for research to investigate why some types of L. monocytogenes persist in a processing plant environment but others do not.

Painful ophthalmoplegia from metastatic nonproducing parathyroid carcinoma: case study and review of the literature.

Parathyroid carcinoma is an uncommon malignancy. Of the fewer than 400 cases reported, most have been cases of producing parathyroid carcinoma with accompanying hypercalcemia. Only 13 patients with nonproducing parathyroid carcinoma have been described. Nine of these 13 patients had metastatic disease. We report a patient with i.c. metastasis. Distal metastases of producing parathyroid carcinoma are treated surgically to prolong survival and prevent complications of hyperparathyroidism and hypercalcemia. One half of the patients with producing parathyroid carcinoma die within 5 years, mostly because of the complications of hypercalcemia. Nonproducing parathyroid carcinoma compares unfavorably with producing parathyroid carcinoma in terms of tumor progression and prognosis. Few data on choice of therapy in nonproducing parathyroid carcinoma are available. We treated our patient with a combination of radiotherapy and chemotherapy. Treatment was followed by an unexpectedly prolonged survival of 31 months after diagnosis of metastatic disease.

Influence of surfactant hydrophobicity on the detachment of Escherichia coli O157:H7 from lettuce.

The influence of surfactant hydrophobicity on detachment of Escherichia coli O157:H7 from lettuce was determined. Lettuce pieces inoculated with the pathogen were rinsed with Tween and Span surfactants of different hydrophobicity. Of the Tweens, only Tween 85, the Tween with the lowest hydrophile/lipophile balance (HLB), significantly detached the pathogen from lettuce surface. Span 85 (the surfactant with the lowest HLB studied) exhibited the greatest ability among surfactants tested to detach cells from lettuce. This surfactant removed cells attached to the leaf cuticle but not to the cut edge, and caused no detectable reduction in viability of cells remaining on the lettuce. Treatment with Span 85 did not detach cells when they were allowed to attach in the presence of calcium ions. The combination of NaCl/NaHCO3 (pH 10) and Span 85 did not detach cells possibly due to reduced hydrophobicity of the Span at this pH. This study suggests that surfactants of low HLB disrupt hydrophobic interactions between E. coli O157:H7 and the lettuce surface but cannot cause release of cells adhering to hydrophilic structures such as cut or damaged tissue.

Attachment of Escherichia coli O157:H7 grown in tryptic soy broth and nutrient broth to apple and lettuce surfaces as related to cell hydrophobicity, surface charge, and capsule production.

This study investigated the effect of growth in tryptic soy broth (TSB) and nutrient broth (NB) on the ability Escherichia coli O157:H7 to attach to lettuce and apple surfaces. In addition, cell surface hydrophobicity, charge and capsule production were determined on cells grown in these media. Cells grown in NB attached less to lettuce and apple surfaces than did those grown in TSB. TSB, but not NB, supported capsule production by E. coli O157:H7. Cells grown in TSB were more hydrophilic than those grown in NB. No difference was found in the electrokinetic properties of cells grown in these media. Electrostatic and hydrophobic interactions and surface proteins did not appear to play an important role in the attachment of E. coli O157:H7 to these surfaces. Of the factors studied, only capsule production was associated with attachment ability.

Rapid detection of Escherichia coli O157:H7 using immuno-magnetic flow cytometry in ground beef, apple juice, and milk.

A rapid method combining flow cytometry and immunomagnetic bead separation (IMFC) was effective for detecting low populations of Escherichia coli O157:H7 inoculated into ground beef, apple juice, and raw milk samples. Modified buffered peptone was most effective for enrichment of E. coli O157:H7 in ground beef. Using IMFC in combination with a 6-h enrichment, as few as four E. coli O157:H7 cells/g of ground beef were detected in as little as a 7-h total analysis time. Our results demonstrate the possibility of using flow cytometry and immunomagnetic bead separation to detect low concentrations of E. coli O157:H7 in ground beef, apple juice and milk.

Factors affecting capsule size and production by lactic acid bacteria used as dairy starter cultures.

The effects of sugar substrates on capsule size and production by some capsule-forming nonropy and ropy dairy starter cultures were studied. Test sugars (glucose, lactose, galactose, or sucrose) were used as a sole carbohydrate source and the presence of a capsule and its size were determined by using confocal scanning laser microscopy. Nonropy strains produced maximum capsule size when grown in milk. Strains that did not produce capsules in milk did not produce them in any other growth medium. Specific sugars required for capsule production were strain-dependent. Increasing lactose content of Elliker broth from 0.5 to 5% or adding whey protein or casein digest produced larger capsules. Whey protein concentrate stimulated production of larger capsules than did casamino acids or casitone. Some Streptococcus thermophilus strains produced capsules when grown on galactose only. Nonropy strains of Lactobacillus delbrueckii subsp. bulgaricus produced capsules on lactose, but not on glucose. A ropy strain of Lactobacillus delbrueckii subsp. bulgaricus produced a constant capsule size regardless of the growth medium. The ability of some strains of Streptococcus thermophilus to use galactose in capsule production could reduce browning of mozzarella cheese during baking by removing a source of reducing sugar. Media that do not support capsule production may improve cell harvesting.

Penetration of Escherichia coli O157:H7 into lettuce tissues as affected by inoculum size and temperature and the effect of chlorine treatment on cell viability.

Penetration of Escherichia coli O157:H7 into iceberg lettuce tissues and the effect of chlorine treatment on cell viability were evaluated. Attachment of different inoculum levels (10(9), 10(8), and 10(7) CFU/ml) was examined by determining the number of cells at the surface and the cut edge of lettuce leaves (2 by 2 cm). E. coli O157:H7 attached preferentially to cut edges at all inoculum levels, with greater attachment per cm2 of lettuce at higher inoculum levels. A longer attachment time allowed more cells to attach at both sites. Immunostaining with a fluorescein isothiocyanate-labeled antibody revealed that cells penetrated into lettuce leaves from cut edges. Cells showed greater penetration when lettuce was held at 4 degrees C compared with 7, 25, or 37 degrees C and were detected at an average of 73.5 +/- 16.0 microm below the surfaces of cut tissues. Penetrating cells were mostly found at the junction of lettuce cells. The viability of attached cells after treatment with 200 mg/liter (200 ppm) of free chlorine for 5 min was examined by plating on tryptic soy agar and by a nalidixic acid elongation method. Although chlorine treatment caused significant reduction in attachment (0.7- and 1.0-log reduction at surfaces and cut edges, respectively), cells remained attached at high numbers (7.9 and 8.1 log CFU/cm2 at surfaces and cut edges, respectively). Elongated cells were observed in stomata and within the tissues of the lettuce, indicating they were protected from contact with chlorine.

Attachment of Escherichia coli O157:H7 to lettuce leaf surface and bacterial viability in response to chlorine treatment as demonstrated by using confocal scanning laser microscopy.

Confocal scanning laser microscopy was used to observe the location of Escherichia coli O157:H7 on and within lettuce leaves. Sections of leaves (ca. 0.5 by 0.5 cm) were inoculated by submersion in a suspension of E. coli O157:H7 (ca. 10(7) to 10(8) CFU/ml) overnight at 7 degrees C. Fluorescein isothiocyanate-labeled antibody was used to visualize the attached bacteria. E. coli O157:H7 was found attached to the surface, trichomes, stomata, and cut edges. Three-dimensional volume reconstruction of interior portions of leaves showed that E. coli O157:H7 was entrapped 20 to 100 microm below the surface in stomata and cut edges. Agar plate culturing and microscopic observation indicated that E. coli O157:H7 preferentially attached to cut edges, as opposed to the intact leaf surface. Dual staining with fluorescein isothiocyanate-labeled antibody and propidium iodide was used to determine viability of cells on artificially contaminated lettuce leaves after treatment with 20 mg/liter chlorine solution for 5 min. Many live cells were found in stomata and on cut edges following chlorine treatment. E. coli O157:H7 did not preferentially adhere to biofilm produced by Pseudomonas fluorescens on the leaf surface. In contrast to E. coli O157:H7, Pseudomonas adhered to and grew mainly on the intact leaf surface rather than on the cut edges.

Thermal inactivation of Mycobacterium paratuberculosis in milk.

Thermal inactivation of Mycobacterium paratuberculosis, a suspected human pathogen, was determined in ultrahigh-temperature whole milk. Three strains of M. paratuberculosis were examined for survival at temperatures from 55 to 75 degrees C using a submerged glass capillary tube method. Clumped and declumped suspensions of the cultures were used to determine the rate of heat inactivation and survival at pasteurization temperatures. Methods for declumping M. paratuberculosis included the use of glass beads, vortexing, and passing the cells through a 26-gauge needle. The latter procedure was found to be superior over other methods and did not affect the viability of cells. Capillary tubes filled with milk containing 4 x 10(6) to 3 x 10(7) CFU/ml were heated at temperatures ranging from 55 to 75 degrees C. At 55 degrees C, minimal thermal inactivation was observed for clumped and declumped cells. At 58 degrees C, thermal inactivation ranging from 0.3 to 0.7 log reduction was observed for both clumped and declumped suspensions. D values at 60 degrees C ranged from 8.6 to 11 min and 8.2 to 14.1 min for clumped and declumped cells, respectively. At 63 degrees C, the D values ranged from 2.7 to 2.9 and 1.6 to 2.5 min for clumped and declumped cells, respectively. Survival of M. paratuberculosis at initial levels ranging from 44 to 10(5) CFU/ml at pasteurization treatment (63 degrees C for 30 min and 72 degrees C for 15 s) was also determined. No survivors were observed after incubating plates for up to 4 months on Middlebrook 7H11 agar and up to 2 months on Herrold's egg yolk medium. The sensitivity of the plating method was 1 CFU/250 microliters. These results demonstrate that low levels of M. paratuberculosis, as might be found in raw milk, will not survive pasteurization treatments.